US 2005.0014697A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2005/0014697 A1 Stamler et al. (43) Pub. Date: Jan. 20, 2005

(54) COMPOSITIONS AND METHODS FOR on Feb. 18, 2004. Provisional application No. 60/550, MODULATING S-NITROSOGLUTATHONE 833, filed on Mar. 4, 2004. REDUCTASE Publication Classification (76) Inventors: Jonathan S. Stamler, Chapel Hill, NC (US); Limin Liu, Durham, NC (US) (51) Int. Cl." ...... A61K 38/18; A61K 31/198 (52) U.S. Cl...... 514/18: 514/565 Correspondence Address: MINTZ, LEVIN COHN FERRIS GLOWSKY & POPEO (57) ABSTRACT 666 THIRDAVENUE Disclosed herein are methods and compositions for modu NEW YORK, NY 10017 (US) lating the levels and/or activity of S-nitroSoglutathione (21) Appl. No.: 10/861,304 reductase (GSNOR) in vivo or in vitro. Specifically dis closed are GSNOR deletion constructs, host cells and non (22) Filed: Jun. 4, 2004 human mammals comprising GSNOR deletions, and meth ods of screening employing GSNOR deletion mutants. Also Related U.S. Application Data Specifically disclosed are reagents and procedures for mea Suring, monitoring, or altering GSNOR levels or activity (as (60) Provisional application No. 60/476,055, filed on Jun. well as nitric oxide and S-nitroSothiol levels) in connection 4, 2003. Provisional application No. 60/545,965, filed with various medical conditions. Patent Application Publication Jan. 20, 2005 Sheet 1 of 27 US 2005/0014697 A1

A Wild-ild-type allelee Ab-SS) s

Targetingtly vector- PGKn xs / B / PGK h N 2. 4 is 9

2. so Fse %2 5 aE. so g 2 4. is S is 40an s. 2 2 35 r 2.% wer they Heart Lung Spleen Thymus y: atweaning F.G. 1 Patent Application Publication Jan. 20, 2005 Sheet 2 of 27 US 2005/0014697 A1

A B C 80 92 10 360 a Se 2. 2 6 X 4 E 40 3 S O 9 E 5 20 g 9 2. is t WT KO WT KO SNO FeNO

D Redblood Cell Pierra

GSeoGye-ND Remi-Abuadr-SNO

Largsgata

Wasecarterestigato Wasodilation during ypgdstagfig demand No deficiency

FIG 2 Patent Application Publication Jan. 20, 2005 Sheet 3 of 27 US 2005/0014697 A1

A 1 B 10 9 or 7 7 w ad o 6 2 2 e C a O 1 2 3 4. 5 6 O 1. 2 3 4. 5 6 Days after LPS Days after LPS C 1 D 1 9 s 2 7 7 3 3 S. O 1 2 3 4. S 6 Days after LPS Days after LPS E 2 2

t

O 2 3 4 5 & 7 Days after CLP FIG. 3 Patent Application Publication Jan. 20, 2005 Sheet 4 of 27 US 2005/0014697 A1

A 40 ++ total B 1200 -- total 000 30 S -- low-mass 'o 2 s 800 asE 2 retS. 600 sE s 400 2 200 O O PBS LPS24 LPS48 PBS LPS24h LPS48h

C 8 D 6 E 4.

s g3 A 5 E3 22 re. 2 9.

O D PBS LPS24h LPS48h LPS 24h LPS48h 0

FIG. 4 Patent Application Publication Jan. 20, 2005 Sheet 5 of 27 US 2005/0014697 A1

- PBS LPS24h LPS48h PBS LPS24h LPS48h

PES is lish PBS LPS24h LPS48h

ps ships

G 140 H2 2 s 100 S. O

a tha O s - - 2. t 2 3 4 5) 8) to PBS PS24h LPS48h SNO (pmol mg) F.G. 5 Patent Application Publication Jan. 20, 2005 Sheet 6 of 27 US 2005/0014697 A1

FIG 6 Patent Application Publication Jan. 20, 2005 Sheet 7 of 27 US 2005/0014697 A1

A 1200 B 40 C D 100 OO) 35 90 30 8 as 800 'o pa s E 25 s to 600 as 20 S. wn E 5 so O 3, 15 8 2. O 10 50 20 e 5 4. O 1 2 3 4 S 6 LPS 24h LPS 48h LPS48h Days after LPS

FIG. 7 Patent Application Publication Jan. 20, 2005 Sheet 8 of 27 US 2005/0014697 A1

5 to

H H at at & 3s

s

ck O C 3. S

bf) b) w b e b vu h O (oosiu? Huo) dugoseqiad) in O Patent Application Publication Jan. 20, 2005 Sheet 9 of 27 US 2005/0014697 A1

a S5 S 33> 2. a 3O s- O2 : :

< > O O N4

t > O Od s CD L

M A A. O NZ

V A A.

C O C O O C O C C O lf C b) O lf) N N y yu Patent Application Publication Jan. 20, 2005 Sheet 10 of 27 US 2005/0014697 A1

9. Ek s Sa

9 O

O CD I

O O N4

O

S. S. 92 S C2 e e g e. Oe Ce Oi sCo seO OOO Oe Cot Oe O (I u?irl) el-TI ITV Patent Application Publication Jan. 20, 2005 Sheet 11 of 27 US 2005/0014697 A1

cysNO (M) O 500 50 5 a GRK2 ------ISO ------B-AR

cysNO (uM)

GRK2 32P-rhodopsin

FIG. 11B

5000 s 20000 L CPM 9.4000 a. 3000 s 10000 2000 t3. 5 1000 8 E O saxx, s O S s s .

c) s e a ge Yala o 2-- o 39 FIG. 12A FIG. 12B

7 as s S s 80 92 x s 2 - SO s 43 g 40 33s a g 2 X cap g 20 2 1 s O : O PBS ISO GSNO ISO+GSNO PBS ISO GSNO SO-GSNO PBS ISO GSNO GSNO+SO

F.G. 13A F.G. 13B FIG. 13C

Patent Application Publication Jan. 20, 2005 Sheet 13 of 27 US 2005/0014697 A1

LOCUS NM 000671 2496 bp mRNA linear DEFINITION Homo sapi ens alcohol dehydrogenase 5 (class III), chi polypeptide (ADH5), mRNA. ACCESSION NM 000671 COMPLETENESS: full length. FEATURES Location/Qualifiers Solice . . 2496 A chromosome="4"

1. . 2496 /gene="ADH5" Anote= "synonyms: FDH, ADHX, ADH-3" misc feature 1. /gene="ADH5" Wnote= "alternative start site; active site" variation complement (4) /replace="T" /replace="C" /db Xref="dbSNP : 1154400" misc feature 83 /gene="ADH5" /note= "alternative start site; active site" Inisc feature 85 /gene="ADH5" /note= "alternative start site; active site" CDS 63 . . .287 /gene="ADH5" /EC number="l. 1. l. 1" /EC number="1.2.1.1" /note= "Alcohol dehydrogenase (class III), chi polypeptide; glutathione-dependent formaldehyde dehydrogenase; go function : formaldehyde dehydrogenase (glutathione) activity (goid 0.004327) evidence TAS) (pinid 84.60164); go function: fatty acid binding Igoid OOO5504) (evidence TAS) pmid 84.60164); go function: electron transporter activity (goid 0005489) evidence TAS) pmid 846O164); go function: alcohol dehydrogenase activity, zinc-dependent (goid OOO4024) evidence IEA) ; go function : zinc ion binding (goid OOO827O) evidence IEA) ; go function: oxidoreductase activity goid OO16491) evidence IEA); go function: alcohol dehydrogenase activity, metal ion-independent goid 0004023) evidence IEA) ; go function: alcohol dehydrogenase activity, iron-dependent goid 0004 025) evidence IEA) ; go process: ethanol oxidation (goid 0006069) (evidence TAS) (pmid 84.60164); go process: alcohol metabolism (goid 0006066 (evidence NR) FIG. 15A Patent Application Publication Jan. 20, 2005 Sheet 14 of 27 US 2005/0014697 A1

/codon start=1 /product = "class III alcohol dehydrogenase 5 chi subunit" /protein id="NP OOO662. 2" /db xref="GI: 11496891" /db xref="GenerD: 128." /db Xref="Locus ID: 128." /db xref="MIM:103710" /translation= "MANEVIKCKAAVAWEAGKPLSIEEIEVAPPKAHEVRIKIIATAV CHTDAYTLSGADPEGCFPVILGHEGAGIVESVGEGVTKLKAGDTVIPLYIPOCGECKF CLNPKTNLCOKIRVTOGKGLMPDGTSRFTCKGKTILHYMGTSTFSEYTVVADISVAKI DPLAPLYKVCILGCGSTGYGAAVNTAKLEPGSWCAVFGLGGWGLAVIMGCKVAGASR IIGVDINKDKFARAKEFGATECINPQDSKPIOEVLIEMTDGGVDYSFECIGNVKVMR AALEACHKGWGVSVVVGVAASGEEIATRPFQLVTGRTWKGTAFGGWKSVESVPKLVSE YMSKKIKVDEFWTHNLSFDEINKAFELMHSGKSIRTVVK I misc feature 63. 1284 /gene="ADH5" /note="KOG0022; Region: Alcohol dehydrogenase, class III Secondary metabolites biosynthesis, transport and catabolism.)" /db xref="CDD: 1782.0" misc feature 559 .56 /gene="ADH5" /note= "important for formaldehyde dehydrogenase activity and activation by fatty acids; binding site" variation complement (359) /replace="T" /replace="C" /db xref="dbSNP: 94.45600." variation complement (475) /replace="T" /replace= "A" /db xref="dbSNP: 94.45599." variation 66. /gene="ADH5" /note= "WARNING: map location ambiguous" /replace="T" /replace="G" /db xref="dbSNP:1050636." variation 900 /gene="ADH5" /replace="T" /replace= "A" /db xref="dbSNP:1050637" variation complement (932) /replace="G" /replace= "A" /db xref="dbSNP:43927 09" variation 153 /gene="ADH5" /replace="T" /replace="C" /db Xref="dbSNP:2229168" FIG. 15B Patent Application Publication Jan. 20, 2005 Sheet 15 of 27 US 2005/0014697 A1

variation 158 /gene="ADH5" /note="WARNING: map location ambiguous" /replace="G" /replace="C" /db xref="dbSNP: 1803039." variation complement (1400) /note= "WARNING: map location ambiguous" /replace="T" /replace="C" /db xref="dbSNP:30883 06" variation 1478 /gene="ADH5" /replace="T" /replace="C" /db xref="dbSNP:12697" variation 504 /gene="ADH5" /replace="T" /replace="C" /db xref="dbSNP: 12106" polyA signal 527 . . 1532 /gene="ADH5" /note= "alternative polyA signal." /evidence=experimental variation 1703 /gene="ADH5" /replace="G" /replace="A" /db xref="dbSNP:1803037" variation 779 /gene="ADH5" /replace="T" /replace="G" /db xref="dbSNP:13832" variation complement (1859) /replace="T" /replace="C" /db xref="dbSNP: 682 7292." variation 2OOO /gene="ADH5" /note="WARNING: map location ambiguous" /replace="T" /replace="C" /db xref="dbSNP:1061187." variation 217. /gene="ADH5" /replace="T" /replace="C" /db xref="dbSNP:1803.038" polyA signal 2376. .238 /gene="ADH5" /note= "alternative polyA signal" /evidence=experimental polyA site 2443 /gene="ADH5" /note= "alternative polyA site" /evidence=experimental FIG. 15C Patent Application Publication Jan. 20, 2005 Sheet 16 of 27 US 2005/0014697 A1

ORIGIN agC9gCaC9C accacagctic gag caccgCC Cttccggttg gagcCattgc aag.ccc.cccc 6. CacgCCCC gC ccccct cqct aggcgct.cgc cacgc.cCatg cctc.cgtogc tgcgc.ggCCC 121 accCCggatg totagoccCCC gCGCCdacca gaatc.cgtga acatggcgaa cgaggittatc 181 aagttgcaagg CtgcagttgC ttgggaggCt ggaaag.cctic totccataga ggagatagag 241 gtggcacccc caaaggct Ca tgaagttcga atcaagatca ttgccactgc ggtttgccac 301 accgatgcct at accCtgag tggagctgat Cctgagggitt gtttitccagt gatcttggga 361 Catgaaggtg Ctggaattgt ggaaagttgtt g9tgagggag ttactaagct galagg.cgggt 42 gacactgtca tCccact teta catcccacag tgttggaga at gcaaattittg totaaat CCt 48 aaala Ctala CC tttgccagaa gatalaga.gtc act Calaggga aaggattaat gccagatggit 54. accagcagat ttacttgcaa aggaaaga Ca attittgcatt acatgggaac cagdacattt 601 totgaataca cagttgttggC tgatatctot gttgctaaaa tagatcCttt agcacctittg 66. tataaagttct gCCttctagg ttgttggcatt toaaccggitt atggtgctgc tgtgaacact 721 gCCaagttgg agcCtggctic tgtttgttgcc gtc.tttgg to tgggaggagt cggattggca 781 gttatcatgg gctgtaaagt ggctggtgct toccggatca ttggtgtgga CatCaataaa 841 gataaatttg Caagggccaa agagtttgga gccactgaat gtattaa.ccc toaggattta 901 agtaaaccca tcCaggaagt gctcattgag atgaccgatg gaggagtgga ctatt CCttt 961 gaatgtattg gtaatgtgaa ggtocatgaga gcagcacttg aggCatgtca Caagggctgg 1021 gg.cgt.ca.gc.g tcgtggttgg agtagctgct tcaggtgaag aaattgccac togtocatt c 1081 Cagctogtala Cagg togcac atggaaaggC actgcc tittg gaggatggala gagtgtagaa 141 agtgtcCCaa agttggtgtc tgaatatatg to Caaaaaga taaaagttga tgaatttgttg l2O1 act Cacaat C tgtc.ttittga tgaaatcaac aaagcc tittg aactgatgca ttctggaaag 126 agcattcgaa Ctgttgtaaa gatttaattic aaaagagalaa ataatgtc.ca to Ctgtcgtg 1321 atgtgatagg agCagCttaa CaggCaggga gaag.cgCCtc Calacct Caca gCctcgtaga 138 gcttcacagc tacticcagaa aatagggitta tgttgttgtcat toatgaatct Ctata at Caa 1441 gga Caaggat aattcagtica tgaacctgtt ttctggatgc toctocacat aaataattgC 1501. tagtttatta aggaatattt talacatalata aaagtaattit ctacatttgt gtggaaattg 1561. tottgttitta tgctgtcatc attgtcacgg tttgttctgcc cattatct tc attctgcaag 1621 ggalaagggala aggalagcagg gCagtggtgg gtgtctgaala CctCagaaac ataacgttga 1681 acttittaagg gtCtcagtCC cc.gttgatta aagaacagat cctago catc agtgaCaaag 1741 ttaat Cagga CCCaagttctg Cttctgttgat attat Cttta agggagg taC tgtgcct togt 1801 toatacctgt aCCCCaaatt cCtaggatgg catctgccitt Caggggg CaC taaaatgitat 1861 tattgaaa.ca gCattctggg Cttaaatagg tg tatgtatg tgttggttgt gactgtacta 1921 tttctagtat agtgaactac a tactgaata to caagttct cagoaccitac ttttgtcaaa 1981 tCttalacatt ttgccacttic gagat Cacat tgccatt cot cc.cctocaag aggtaacaat 2041 tatCcacaat ttgatgttta toattoctgt gttgttgtac ttt cactgtg tatalacCtaa 2101 aCCatCtact Ctttagtact gttittatata tttittaa.gc.c toatacttgc tCattctaca 216 gcttttittca ct cattattg tataattata totgaagctic togttcatta attt tag to c 2221 tgttgtag cag alattcaatta CgggalactaC Catalattitat ctgttcticca gtCcagttga 2281 agg catgaag ttgttgc.ca.g tttctg tatt ataa.cactgt agtggalacat tottctgcat 2341 tgggctCact gCdtgttacC talagacgitat Cacagaataa acacatttag ccttatagac 24 O1 attgccaaat tgcticttcaa agtaaatgtg agtttttgtg aattacatga g tatggaatg 246 gtgttittatt atgactittag tttgcattitt CCtcala

F.G. 15D Patent Application Publication Jan. 20, 2005 Sheet 17 of 27 US 2005/0014697 A1

LOCUS HUMADH5O1 448 bp DNA linear DEFINITION Human alcohol dehydrogenase chi polypeptide (ADH5) gene, exon 1. ACCESSION M8112 REFERENCE l (bases 1 to 448) FEATURES Location/Qualifiers SOCe 1. . 448 RNA 197 . . .370 Wnote= "minor mRNA start site" IRNA 279 . . .370 /note= "major mRNA start site" mRNA 281. . .370 /note= "major mRNA start site" SXO. 359 . . 37 O /gene="ADH5" /note="G00 18-978" /number=1 ORIGIN tttcagtgaa attoccgttc cct caccgtc cacatctgta atttgcaagt cittatctacg 61 ataactgctg taaagttaca C9999aagCC citttcc.cgac aaaaaaaacg agttctgcaa 2 caagtcc.gtc ggattittagc aatgaalacc.g gC9CC9tgca gcct Cg CC99 CgagtgCaCC 18 Ctggaacgca caacttagcg gcacgcacca cagotcgagc accg.ccct tc cggttggagc 24 Cattgcaag C CCCCCC cacg cc.ccgc.cccc Ctc.gctaggc gctc.gc.cacg cc.catgcctic 30 cgt.cgctg.cg cgg.cccaccC Cggatgtcag CCCCCCg.cgc cgaccagaat CCgtgala Cat 361 ggCgaacgag gtagggCCCg ttgagoggag ggccCtgagt cCaagggagg gagtgcatgg 42 Catgggacta ggctgctatc Ctcggggc LOCUS HUMADH502 146 bp DNA linear DEFINITION Human alcohol dehydrogenase chi polypeptide (ADH5) gene, exon 2. ACCESSION M813 FEATURES Location/Qualifiers SOCe 1. .146 intron order (M81112. 1: 371 . . 448, 1 . . 12) /gene="ADH5" /note= "GOO 118-978 /number=1 exO 13 . . 114 /gene="ADH5" /note="G00 118-978 /number=2 ORIGIN l tocccacttic aggttatcaa gtgcaaggct gcagttgctt gggaggctgg aaagcc totC 61 to Catagagg agatagaggt ggCaccCCCa aaggctoatg aagttcgaat Caaggtaatg 121 atacatttaa ggcactggga aaaaaa

//

FIG. 16A Patent Application Publication Jan. 20, 2005 Sheet 18 of 27 US 2005/0014697 A1

LOCUS HUMADH503 383 bp DNA linear : DEFINITION Human alcohol dehydrogenase chi polypeptide (ADH5) gene, exon 3. ACCESSION M814 FEATURES Location/Qualifiers source 1. 383 intron order (M81113.1 : 115 . . 146, 1 . . 26) /gene="ADH5" /note= "GOO 118-978 /number=2 eXO. 27 . . .168 /gene="ADH5"9 /note="G00 118-978 /number=3 ORIGIN cattcgttgt tittaatgtcC tgatagat.ca ttgccactgc ggtttgCCaC accgatgcct 6 a tacCCtgag tggagctgat CCtgagggitt gttitt.ccagt gatCttggga Catgalaggtg 121 Ctggaattgt ggaaagttgtt ggtgagggag t tactaagct galagg.cggg t aaggagaata 181 CttgaaCCaC ttgtttaata attittggctt atticcitatgg ggaaattgtt tttctgataa 241 alacta CCCaC tatt tatgaa tagg tatcat ctaagtagat tgtcaagatt aagttgatto 3 O1 Ctcatctggg aag Cacaaat atttggatat tttitt totCt ctgattittgc agacitactgt 361 ttttgttgaaa cittittctaag tac LOCUS HUMADH504 326 bp DNA linear DEFINITION Human alcohol dehydrogenase chi polypeptide (ADH5) gene, exon 4. ACCESSION M81115 FEATURES Location/Qualifiers SOULC8 1. .. 326 /organism="Homo sapiens" /mol type="genomic DNA" / isolate="620" /db xref="taxon: 96.06" / tissue type="liver" /dev stage="adult" intron order (M81114. 1: 169. .354, 1. . 18) /gene="ADH5" /note= "GOO 118-978 /number=3 SeXO 9. ... 106 /gene="ADH5" /note="GOO 18-978 /number=4 ORIGIN citttgcattt gtttcCaggit gaCactgtca tCCCaCttta CatCCCaCag tgtggagaat 6 gcaaattittg totaaatcct aaalactaaCC tttgc.ca.gala gatalaggtta gtatctttitt 12 atgttcttct taaaatacala gtgctg.cggg ataattalagg aat Cacagag accgaggggit 181 tgaggaggala ttatttaata atttaggttc attaaccCag tcggattaac gttcaaagga 241 CtgagtCCCG aacaaagagt Caagctacct tttalagcatt tCgtggggtg gggggaga CC 3 O1 tttgtagggg gag Catatta Cagalag FIG. 16B Patent Application Publication Jan. 20, 2005 Sheet 19 of 27 US 2005/0014697 A1

LOCUS HUMADH505 934 bp DNA linear DEFINITION Human alcohol dehydrogenase chi polypeptide (ADH5) gene, exons 5-6. ACCESSION M81116 FEATURES Location/Qualifiers SOCe 1.934 intron order (M81115. l: 107 . .325, l. .200) /gene="ADH5" /note="G00 18-978 /number=4 exo 2O. . . 420 /gene="ADH5" /note= "GOO 18-978 /number=5 intron 421. . 571 /gene="ADH5" /note="G00 118-978 /number=5 exon 572. 832 /gene="ADH5" /note="G00 118-978 /number=6 tggatalagat tataaatat C ttatalaattit cctittataag gcattgctgc aaggtgctaa 61 attactaatg aata tatttg aaattgtagt ttacaa Cact tt Cttaatat ttactgg toa 121 ttatttittaa alacatttctt tttcctgtgg gttttaagaa aCCtaatt CC aacttgcctt 181 tettcCtttitt ttittctt tag agt cacticala gggaaaggat taatgccaga tgg taccagc 241 agatt tactt gCalaaggaaa gacaattittg cattacatgg gaaccagcac attttctgaa 30 tacacagttg tggctgatat citctgttgct aaaatagatC Ctttagcacc tittggataala 36 gtctgcct tc taggttgttgg catttcaacc ggittatoggtg ctgctgtgaa cactgccaag 42 gtaagagact gacttgggitt ttttgcttct gcct tctaat ttaatticagt gaaactitt CC 481. tggaatagtg aaattggc.ca atctg. tatga aaccCagtga ttct cittgac totgg tagaa 541 tgagtact tt ataaactitt C tt tactCcta gttggagect ggctotgttt gtgcc.gtctt 6O1 tggtctggga ggagt cggat tggCagttat catcggctgt aaagtggCtg gtgct tcc.cg 661 gatcattggit gtgga catca ataaagataa atttgcaagg gccaaagagt ttggagcCaC 721 tgaatgtatt aac CCtcagg attittagtaa acccatcCag galagtgct Ca ttgagatgac 781 cgatggagga gtggactatt cctttgaatg tattggtaat gtgaaggtoa tggtgagtat 841 gggCtt Catt cct ttt tagt titatggaact gct tttitttg aagtgCagtt ttagattaaa 901 aattgttittg ttttggcaaa gdaaaagttct Ctta

LOCUS HUMADH5O6 307 bp DNA linear DEFINITION Human alcohol dehydrogenase chi polypeptide (ADH5) gene, exon 7. ACCESSION M81117 FEATURES Location/Qualifiers SOULCS . .307 intron order (M81116. 1 : 833. - 934, 1 . . 45) /gene="ADH5" /note= "GOO 18-978 /number=6 exOn 46. ... 181 /gene="ADH5" /note= "GOO 118-978 it /number=7 aaatgcactg actitctgttga tttggtoata totctgtgcc tgcagagagc agcacttgag 61 gCatgtcaca aggg Ctgggg Cgt cagogtC gtggttggag tagctgCttC aggtgaagaa 121 attgccactic gtc.catt coa gCtgg taa.ca ggtc.gcacat ggaaaggCaC tgcc tittgga 81 gg taatticga tggatgagat gactgcattt totct tttgt tagttgcatt ggCagatgtt 241 taatccCagc caccitaattit tttittatatt aacaagaatc ttcatgagtt cCttct catt 301 ataagtt FIG. 16C

Patent Application Publication Jan. 20, 2005 Sheet 21 of 27 US 2005/0014697 A1

ORIGIN tgtgatttct tttaggatgg aagagtg tag aaagtgtc.cc aaagttggtg totgaatata 61 tgtc.caaaaa gataaaagtt gatgaatttg tgacticacaa totgtcttitt gatgaaatca l21 acaaagCCtt tgaactgatg cattctggaa agagg taggC titt citctitta tatatoatag 181 aatgtaataa tgatgttgag tittgagggga tggaga.gtcg aalaalacaca a ttittagttgt 24l Cttaalaggta tCatttaaaa. Cttag tatgt tgct tcc titt tacagcattC gaactgttgt 30 aaagatttaa ttcaaaagag aaaataatgt ccatcctgtc gtgatgttgat aggagcagct 361 taa.caggCag ggagalag C9C CtcCalaccitc acagcctcgt. agagctt CaC agctact.cca 421 gaaaataggg ttatgttgttgt catt catgaa tCtectataat Caaggacaag gataatticag 481 tCatgalacct gttttctgga tgctcc toca Cataaataat tgctagttta ttaaggaata 541 ttittalacata ataaaagtaa titt Ctacatt tgttgttggaaa ttgtcttgtt ttatgctgtc 6O1 at cattgtca cggtttgttct gccCattatc ttcattctgc alagggaaagg gaaaggaagc 661 agggcagtgg tgggtgttctg aaacct caga aacataacgt. tgaacttitta agggtCtcag 721 to cccgttga ttaaagaa.ca gatcCtagcc at Cagtgaca aagttaatca ggaCCCalagt 781. citgcttctgt gatattatct ttalagggagg tactgtgcct tgttcatacc tgtacCC Cala 841 att cotagga tggcatctgc Ctt Caggggg Cactaaaatg tattattgaa acagcattct 901 ggg Cttaaat aggtgtatgt. atgttgttggit tgtgactgta ctatttctag tatagtgaac 961. tacatactga atatocaagt totcagdacc tact tttgtc aaatcttaac attittgccac 1021 titcgagatca cattgccatt CCtcCCCtcc. aagagg taac aattatccac aatttgatgt 1081 ttat catt CC tgtgttgttg tact t t cact gtgtataacc taalaccatct actictittagt 1141 actgttittat at atttittala gcct catact tgct cattct acagotttitt toactCatta 12O1 ttgtataatt atatctgaag citct cogttca ttaattittag tcCtgtg tag Cagaattcaa 1261 ttacgggaac taccatalatt tatctgttct cCagtCcagt tgaaggcatg aagttgttgc 1321 cagtttctgt attatala CaC tgtag toggaa catt Cttctg Cattgggcto actg.cgtgtt 1381 acctaagacg tat cacagaa taalacacatt tagccttata gacattgc.ca aattgct citt 1441 caaagtaaat gtgagtttitt gtgaattaca tgagtatgga atggtgttitt attatgactt 1501 tagtttgcat tittCct cala FIG. 16E Patent Application Publication Jan. 20, 2005 Sheet 22 of 27 US 2005/0014697 A1

NM_007410. Mus musculus alco. . . (gi:31982510) LOCUS NM_007 410 1533 bp mRNA linear DEFINITION Mus musculus alcohol dehydrogenase 5 (class III), chi polypeptide (Adh5), mRNA. ACCESSION NM_007 410 FEATURES Location/Qualifiers SCCS . . 1533 /organism="Mus musculus" /mol type="mRNA" /strain="C57BL/6 J" /db xref="taxon: 10090" /chromosome="3" ele 1. . 1533 /gene= "Adh5" /note= "synonyms: Adh3. Adh-5" /db xref="GeneID: 11532" /db xref="Locus ID: 11532" /db xref="MGI: 87929" CDS 19 . . 1143 /gene= "Adh5" /note= "go function: oxidoreductase activity (goid OO16491) (evidence IEA) ; go function: alcohol dehydrogenase activity, zinc-dependent (goid OOO4024) (evidence IEA); go function : zinc ion binding (goid 0008270) evidence IEA) ; go process: retinoid metabolism (goid 0001523) evidence IMP) pmid 12027900) /codon start=1 A product = "alcohol dehydrogenase 5 (class III), chi polypeptide" A protein id="NP 031436. 2" /db xref="GI: 31982511" /db xref="GenelD: 11532" /db xref="Locus ID: 11532" /db xref="MGI: 87929" /translation="MANQVIRCKAAVAWEAGKPLSIEEIEVAPPKAHEVRIKILATAV CHTDAYTLSGADPEGCFPVILGHEGAGIVESVGEGVTKLKAGDTVIPLYIPOCGECKF CLNPKTNLCQKIRVTQGKGLMPDGTSRFTCKGKSVFHFMGTSTFSEYTV VADISVAKI DPSAPLDKVCLLGCGISTGYGAAVNTAKVEPGSTCAVEGLGGVGLAVMGCKWAGASR IIGIDINKDKFAKAKEFGASECISPQDFSKSIQEVLVEMTDGGVDYSFECIGNVKVMR SALEAAHKGWGVSVVVGVAASGEEISTRPFOLVTGRTWKGTAFGGWKSVESWPKLVSE YMSKKIKVDEFVTGNLSFDQINOAFDLMHSGDSIRTVLKMii

F.G. 17A

Patent Application Publication Jan. 20, 2005 Sheet 26 of 27 US 2005/0014697 A1

1MC5A. Chain A, Ternary . . . (gi : 2894 8616) LOCUS 1MC5. A 374 aa linear DEFINITION Chain A, Ternary Complex Of Human Glutathione-Dependent Formaldehyde Dehydrogenase With S- (Hydroxymethyl)glutathione And Nadh. ACCESSION 1MC5. A FEATURES Location/Qualifiers source 374 /organism- "Homo sapiens" /db xref="taxon: 96.06" Region join (1... 167, 323 . . 374) /region name="Domain 1" /note= "NCBI Domains" SecStr 4 - . 13 /sec str type="sheet" Anote= "strand 1." Sec.St. 2O. .29 /sec str type= "sheet" Anote= "strand 2" SecSt 33 . , 39 /sec str type= "sheet" Wnote= "strand 3" Sec.Ster 4 O. . 43 /sec str type= "sheet" Anote= "strand 4" Het join (bond (45), bond (67), bond (174), bond (174), bond (174), bond (174), bond (174), bond (174) ) /heterogen=" ( ZN, 376 Sec Str 46. .54 /sec str type="helix" /note= "helix 1" Sec.Ster 62 . . 65 /sec str type="sheet" A note= "strand 5." SecStr 68. 73 /sec str type= "sheet" /note= "strand 6." SecStr 74.78 /sec str type="sheet" /note= "strand 7" SecStr 87. 92 /sec str type= "sheet" /note= "strand 8" Het join (bond (97), bond (100), bond (103), bond (111) ) /heterogen=" ( ZN, 375 )." SecStr 14. .. 2 /sec str type="helix" /note= "helix 2" SecStr 29. 133 /sec str type="sheet" /note= "strand 9" Sec.Ster 134 . . 14 O /sec str type="sheet" /note= "strand 10" SeCStr 148 . . .54 /sec str type="sheet" /note= "strand 11" FIG. 19A Patent Application Publication Jan. 20, 2005 Sheet 27 of 27 US 2005/0014697 A1

SecSt 56 . . 160 /sec str type= "sheet" /note= "strand 12" Region 168. . .322 /region name="Domain 2" /note= "NCBI Domains" SecStr 175. 183 /sec str type="helix" /note= "helix 3" SecStr 191. 199 /sec str type= "sheet" /note= "strand 13" SecStr 2O2 . . 214 /sec str type="helix" /note= "helix 4" SecStr 25 . . 224 /sec str type= "sheet" /note= "strand 14" SecStr 229 . .236 /sec str type="helix" Wnote= "helix 5" SecStr 238 . . 243 /sec str type= "sheet" /note= "strand 15" SecStr 25 O. .258 /sec str type="helix" /note= "helix 6" SecSter 263 . . 271 /sec str type="sheet" Wnote= "strand 16" SecStr 272 . . 281 /sec str type="helix" /note= "helix 7" SecStr 286. .295 /sec str type= "sheet" /note= "strand 17" SecSt 31. . .317 /sec str type="sheet" /note= "strand 18" SecSt 328. 337 /sec str type="helix" /note= "helix 8" SecStr 347 .352 /sec stri type= "sheet" /note= "strand 19" SecStr 357. .364 /sec str type="helix" /note= "helix 9" SecStr 369 - 374 /sec str type= "sheet" /note= "strand 20" l manevikcka avaweagkpil Sileeievapp kahevrikii atavchtday tillsgadpegc 61 fpvilghega givesvgegv thulkagdtvi plyipqcgeckfolnpktnl cqkirvitagk 121 glimpdgtsrf tokgktilhy mgtstfseyt v.vadisvaki diplapildkvc illgcgistgy 181 gaavintakle pgsvcav fgl ggvglaving clvagasrii gvidinkdkfa rakefgatec 24l inpqdfskpi qevliemtdg gvdysfe cig nvkvmraale achkgwgvsv v vgvaasgee 301 iatrpfalvt girtwkgtafg gwksvesvpk livseymskki kvdefvthinl sfaeinkafe 361 lmhsgksirt v vki FIG. 19B US 2005/0014697 A1 Jan. 20, 2005

COMPOSITIONS AND METHODS FOR globin O Saturation that are coupled to metabolic demand. MODULATING S-NITROSOGLUTATHONE The mechanism through which the O. content of blood REDUCTASE evokes this response and the basis for its impairment in many diseases, including heart failure, diabetes, and shock, RELATED APPLICATIONS have been major and longstanding questions in vascular physiology. Previous Studies have Suggested that the 0001) This application claims the benefit of U.S. Provi answers reside with hemoglobin's ability to serve as both an sional Application Ser. No. 60/476,055 filed Jun. 4, 2003, O Sensor and O2-responsive transducer of vasodilator activ U.S. Provisional Application Ser. No. 60/545,965 filed Feb. ity. It was later determined that albumin and hemoglobin are 18, 2004, and U.S. Provisional Application Ser. No. 60/550, privileged sites of SNO production. In albumin, both a 833 filed Mar. 4, 2004. All of these applications are herein hydrophobic pocket and bound metals (copper and perhaps incorporated by reference in their entirety. heme) can facilitate S-nitrosylation by NO (Foster MW, et al., 2003, Trends Mol. Med. 9:160-168; Rafikova O, et al., FIELD OF THE INVENTION 2002, Proc. Natl. Acad. Sci. USA99:5913-5918). In contrast, 0002 This invention relates to nitric oxide (NO) biology. hemoglobin (Hb) has several channels through which it can Specifically, this invention relates to the modulation of react with NO, nitrite, or GSNO to produce SNO-Hb (Gow S-nitrosoglutathione reductase (GSNOR) and nitric oxide AJ, et al., 1998, Nature 391: 169-173; Gow AJ, et al., 1999, bioactivity in the regulation of hemodynamic responses. Proc. Natl. Acad. Sci. USA. 96:9027-9032; Luchsinger BP, et al., 2003, Proc. Natl. Acad. Sci. USA 100:461–466; Jia L., BACKGROUND OF THE INVENTION et al., 1996, Nature 380:221-226; Romeo AA, et al., 2003, 0003) Three classes of nitric oxide (NO) synthase (NOS) J. Am. Chem. Soc. 2003; 125:14370-14378). enzymes play important roles in a wide range of cellular 0006 Additional studies indicated that S-nitrosylation of functions and in host defense (Moncada et al., 1991; Nathan blood proteins may be catalyzed by Superoxide dismutase and Xie, 1994). The expression, regulation, and activities of (SOD), ceruloplasmin, and nitrite. In particular, ceruloplas these enzymes have been Studied extensively through both min catalyzes the conversion of NO to GSNO (Inoue K, et genetic and pharmacological approaches. The events down al., 1999, J. Biol. Chem. 274:27069-27075) and NO in stream of NO synthesis are, however, much less well under solution or derived from GSNO is targeted by SOD to stood. It has been reported that both endogenous and eXog cysf393 in hemoglobin rather than heme iron (Gow AJ, et al., enous nitric oxide (NO) react with thiols in proteins such as 1999, Proc. Natl. Acad. Sci. USA 96:9027-9032; Romeo A albumin to form long-lived S-nitrosothiols (SNOs) with A, 2003, J. Am. Chem. Soc. 125:14370-14378). A similar vasodilatory activity (Stamler JS, et al., 1992, Proc. Natl. mechanism (involving SOD and nitrite) has been postulated Acad. Sci. USA. 89:444-448). Also reported has been the to operate in albumin. Numerous laboratories have verified presence of a circulating pool of S-nitroSoalbumin in plasma the presence of SNO albumin, GSNO, and SNO-Hb in blood whose levels were coupled to NOS activity, such that and tissueS of both animals and humans. However, the inhibition of NOS led to a decline in SNO-albumin with amounts that form, the Suitability of various methods for concomitant production of low-mass SNOs (Stamler JS, et assaying various SNOS, and the physiological roles of these al., 1992, Proc. Natl. Acad. Sci. USA 89:7674-7677). It was molecules remain in question. It has been proposed that proposed that SNO-albumin provided a reservoir of NO S-nitrosylation of cysteine thiols constitutes a significant bioactivity that could be utilized in states of NO deficiency, route for transduction of NO bioactivity. S-nitrosylation is and that vasodilation by SNO-albumin was transduced by believed to stabilize and diversify NO-related signals, and the small mass SNOs with which it exists in equilibrium. act as a ubiquitous regulatory modification for a broad spectrum of proteins (Boehning and Snyder, 2003; Foster et 0004 Shortly thereafter, it was determined that a key al., 2003; Stamler et al., 2001). Several lines of evidence low-mass SNO in biological systems is S-nitrosoglutathione Support this proposition. (GSNO; Gaston B, et al., 1993, Proc. Natl. Acad. Sci. USA 1993:90:10957-10961). In contrast to NO, GSNO retains 0007 First, SNO derivatives of peptides and proteins are Smooth muscle relaxant activity in the presence of blood present in most tissues and extracellular fluids under basal hemoglobin, and GSNO acts as a more potent relaxant than conditions (Gaston et al., 1993; Gow et al., 2002; Jaffrey et SNO-proteins. It was then demonstrated the existence of al., 2001; Jia et al., 1996; Kluge et al., 1997; Mannicket al., intraerythrocytic equilibria between NO bound to the thiol 1999; Rodriguez et al., 2003; Stamler et al., 1992). Second, of glutathione and reactive thiols (cysf893) of hemoglobin there are examples of physiological responses that are (Jia L., et al., 1996, Nature 380:221-226), and NO bound to uniquely recapitulated by specific SNOS (De Groote et al., thiols of hemoglobin and membrane-associated band 3 pro 1996; Lipton et al., 2001; Travis et al., 1997). Third, tein (AE1; Pawloski JR, et al., 2001, Nature 409:622-626). researchers have found that S-nitrosylation/denitroySlation The exchange of NO groups between S-nitrosohemoglobin of proteins is dynamically regulated by diverse physiologi (SNO-Hb) and the red blood cell (RBC) membrane was cal Stimuli acroSS a spectrum of cells types and in Vitro shown to be governed by O. tension (PO). Thus, it was systems (Eu et al., 2000; Gaston et al., 1993; Gow et al., found that RBCs dilated blood vessels at low PO (Pawloski 2002; Haendeler et al., 2002; Mannick et al., 1999; Matsu J R, et al., 2001, Nature 409:622-626; McMahon TJ, et al., moto et al., 2003; Matsushita et al., 2003; Rizzo and Piston, 2002, Nat. Med. 8:711-717; Datta B, et al., 2004, Circulation 2003). (in press)); and the production of membrane SNO was 0008 However, investigators lack biochemical or genetic shown to be required for vasodilation. means to distinguish the in vivo activity of SNOs from NO 0005. In peripheral tissues, experiments have demon (or other reactive nitrogen species, RNS). Thus, their exact strated that blood flow is determined by variations in hemo roles and relative importance in various physiological US 2005/0014697 A1 Jan. 20, 2005 responses remain in question. At basal conditions, NOSS teins (Liu et al., 2001). Such precise control over ambient influence arteriolar tone through complex effects on blood levels of GSNO and SNO-proteins raises the possibility that vessels, kidneys, and brain (Ortiz and Garvin, 2003; Stamler, GSNO/GSNOR may play roles in both physiological sig 1999; Stoll et al., 2001). In addition, studies from a number naling and protection against nitrosative StreSS. Indeed, of laboratories have pointed toward the role of red blood GSNO has been implicated in responses ranging from the cells (RBCs), and derived NO bioactivity, in the integrated drive to breathe (Lipton et al., 2001) to regulation of the vascular response that regulates arteriolar resistance (Cirillo cystic fibrosis transmembrane regulator (Zaman et al., 2001) et al., 1992; Gonzalez-Alonso et al., 2002; McMahon et al., and host defense (de Jesus-Berrios et al., 2003). Other 2002). NO itself has not been detected in blood or tissues. studies have found that GSNOR protects yeast cells against This has led to the hypothesis that SNOS contribute to nitrosative stress both in vitro (Liu et al., 2001) and in vivo vascular homeostasis (Foster et al., 2003; Gow et al., 2002). (de Jesus-Berrios et al., 2003). 0009 Inducible NOS (iNOS) can produce higher output 0013 Currently, there is a great need in the art for of NO/RNS and thereby disrupt cellular function (Moncada diagnostics, prophylaxes, ameliorations, and treatments for et al., 1991; Nathan and Xie, 1994). This pathophysiological medical conditions relating to increased NOSynthesis and/or situation, termed nitrosative stress (Hausladen et al., 1996), increased NO bioactivity. There is also a need for compo has been likened to oxidative StreSS caused by reactive sitions and methods for blocking the effects of NO, for oxygen species (ROS) (Hausladen et al., 1996; Hausladen example, on cell death and cell proliferation, particularly, and Stamler, 1999). Studies of Superoxide dismutase, cata Stem cell proliferation, and vascular homeostasis. In addi lase, and peroxidases have provided incontrovertible genetic tion, there is a significant need for compositions and meth evidence for an enzymatic defense against ROS. However, ods for preventing, ameliorating, or reversing other NO the role and mechanism of RNS detoxification in multicel asSociated disorders. lular organisms is unknown. Nonetheless, accumulating evidence points to the existence of a nitrosative StreSS SUMMARY OF THE INVENTION response that subserves NO/SNO homeostasis. In particular, 0014. The invention relates to methods of alleviating or iNOS expression coincides with an increase in S-nitrosy inhibiting the onset of at least one Symptom of a disorder lated proteins, which rapidly reaches a new steady State level asSociated with increased levels of nitric oxide bioactivity (Eu et al., 2000; Marshall and Stamler, 2002). These data comprising: administering to a patient (e.g., a female Suggest that SNOS are being actively degraded. patient) with the disorder a therapeutically effective amount 0.010 Expression of iNOS is strongly induced in Septic of an agent that increases activity or levels of a S-nitroSo shock, a complex syndrome that claims over 100,000 human glutathione reductase and/or decreases levels of SNOS (e.g., lives per year in the United States alone (Feihl et al., 2001). SNO-Hb). In various aspects of the invention, the disorder The role of iNOS in Septic and endotoxic shock has been is a degenerative disorder (e.g., Parkinson's disease, Alzhe probed extensively in mice. Initial analyses of two indepen imer's disease, amyotrophic lateral Sclerosis (ALS)), Stroke, dently generated iNOS-deficient (iNOS-/-) mouse lines did Systemic infection (e.g., bacteremia, Sepsis, neonatal Sepsis, not reveal clear differences in mortality when compared with Septic shock, cardiogenic Shock, endotoxic shock, toxic wild-type controls (Laubach et al., 1995; MacMicking et al., Shock Syndrome, or Systemic inflammatory response Syn 1995). However, more thorough studies of these mice drome), inflammatory disease (e.g., colitis, inflammatory showed that iNOS deficiency actually increased mortality bowel disease, rheumatoid arthritis, Osteoarthritis, psoriatic following lipopolysaccharide (LPS) challenge (Laubach et arthritis, infectious arthritis, ankylosing spondylitis, ten al., 1998; Nicholson et al., 1999). This indicated a protective donitis, burSitis, vasculitis, fibromyalgia, polymyalgia rheu role for iNOS, which was most apparent in females matica, temporal arteritis, giant cell arteritis, polyarteritis, (Laubach et al., 1998). Consistent with these data, the iNOS HIV-associated rheumatic disease Syndromes, Systemic inhibitors 1400W and N-(1-iminoethyl)-L-lysine, either lupus, erythematosus, gout, and pseudogout (calcium pyro have little effect or worsen injury in animal models of phosphate dihydrate crystal deposition disease), hypoten endotoxic shock (Feihl et al., 2001; Ou et al., 1997). Sion (e.g., in connection with anesthesia, dialysis, orthostatic hypotension), proliferative disorders (e.g., cancer or other 0.011 Researchers have recently identified a highly con neoplasms), or another disorder. served S-nitrosoglutathione (GSNO) reductase (GSNOR) (Jensen et al., 1998; Liu et al., 2001). The enzyme is 0015. In accordance with the invention, this agent may classified as an alcohol dehydrogenase (ADH III; also decrease levels of nitric oxide bioactivity or SNOS, or known as glutathione-dependent formaldehyde dehydroge increase nitric oxide/SNO breakdown (e.g., SNO-Hb). In nase) (Uotila and Koivusalo, 1989), but shows much greater Specific aspects, the agent comprises a S-nitroSoglutathione activity toward GSNO than any other substrate (Jensen et reductase polypeptide (e.g., SEQID NO:17-SEQID NO:21) al., 1998; Liu et al., 2001). GSNOR appears to be the major or peptide (e.g., peptide encoded by SEQ ID NO:9-SEQ ID GSNO-metabolizing activity in eukaryotes (Liu et al., NO:14), a S-nitrosoglutathione reductase mimetic (e.g., a 2001). Thus, GSNO can accumulate in extracellular fluids peptide, Small molecule, or anti-idiotype antibody), a vector where GSNOR activity is low or absent (e.g. airway lining for expressing a S-nitroSoglutathione reductase polypeptide (e.g., SEQ ID NO:17-SEQ ID NO:21) or peptide (e.g., fluid) (Gaston et al., 1993). Conversely, GSNO cannot be peptide encoded by SEQ ID NO:9-SEQ ID NO:14), any detected readily inside cells (Eu et al., 2000; Liu et al., fragment, derivative, or modification thereof, or other acti 2001). Vator. In certain aspects, the activating agent is co-admin 0012 Yeast deficient in GSNOR accumulate S-nitrosy istered with one or more inhibitor of nitric oxide synthase lated proteins that are not Substrates of the enzyme. This (e.g., N-3-(aminomethyl)benzyl)acetamidine (1400W); indicates that GSNO exists in equilibrium with SNO-pro N6-(1-Iminoethyl)-L-lysine (L-NIL); monomethyl arginine US 2005/0014697 A1 Jan. 20, 2005

(e.g., for non-specific inhibition); or 7-Nitroindazole (e.g., plasia, neoplasm, or precancer lesions) or another disorder. for inhibition of nNOS in brain tissue), etc.). In a particular The disorders for diagnosis relating to increased levels of embodiment, increased SNOs can be targeted by combina S-nitroSoglutathione reductase and decreased levels of tion therapy with an S-nitroSoglutathione reductase activator SNOS (e.g., SNO-HB) include vascular disorder is heart and a nitric oxide Synthase inhibitor, or by an S-nitroSoglu disease, heart failure, heart attack, hypertension, atheroscle tathione reductase activator alone. rosis, restenosis, asthma, or impotence. The diagnostic 0016. The invention further relates to methods for alle methods of the invention can employ blood, urine, Saliva, or viating or inhibiting the onset of at least one Symptom of a other body fluid or cellular or tissue samples. vascular disorder comprising: administering to a patient 0019. In accordance with the invention, the levels of the Suffering from the disorder a therapeutically effective S-nitroSoglutathione reductase in the biological Sample can amount of an agent that decreases activity or levels of a be determined using an antibody that binds to a S-nitroSo S-nitroSoglutathione reductase and/or increases levels of glutathione reductase antigen and/or an antibody that binds SNOS (e.g., SNO-Hb). In various aspects, the vascular to a SNO antigen. In certain embodiments, the antibody is a disorder is heart disease, heart failure, heart attack, hyper monoclonal antibody and is, optionally, labeled. In other tension, atherosclerosis, restenosis, asthma, or impotence. embodiments, the levels of the S-nitroSoglutathione reduc The agent may comprise an antibody (e.g., monoclonal tase in the biological Sample are determined using a nucleic antibody) or antibody fragment that binds to a S-nitrosoglu acid probe that binds to a S-nitroSoglutathione reductase tathione reductase, an antisense or Small interfering RNA nucleotide sequence (e.g., SEQ ID NO:7-SEQ ID NO:16 or Sequence, a Small molecule, or other inhibitor. In certain a complementary Sequence). In certain embodiments, the aspects, the inhibitory agent is co-administered with a phos probe is a DNA probe and is, optionally, labeled. Alterna phodiesterase inhibitor (e.g., rolipram, cilomilast, roflumi tively, the activity of a S-nitroSoglutathione reductase can be last, Viagra(E) (sildenifil citrate), Cialis(R) (tadala?il), Lev determined by known methods. The levels of SNO in a itra(E) (vardenifil), etc.). In other aspects, the inhibitor is biological Sample (e.g., plasma levels) are preferably deter co-administered with a B-agonist, especially for use with mined by photolysis-chemiluminescence-based methods. heart failure, hypertension, and asthma. Preferably, stable nitrosothiol standards for, e.g., for SNO albumin or SNO-Hb measurements, are used in conjunction 0.017. The invention also relates to methods of diagnosing with Such methods. or monitoring a disorder (or treatment of a disorder) asso ciated with increased levels of nitric oxide bioactivity com 0020. In addition, the invention relates to transgenic prising: (a) measuring levels or activity of a S-nitrosoglu non-human mammals (e.g., mice, rats, etc.) having genomes tathione reductase in a biological Sample from a patient (e.g., that comprise a disruption of the endogenous GSNOR gene, a female patient); (b) comparing the levels or activity of the wherein the disruption comprises the insertion of a Select S-nitroSoglutathione reductase in the biological Sample to able marker Sequence, and wherein the disruption results in levels in a control Sample, and (c) determining if the levels the mouse exhibiting an increase (e.g., intracellular or extra or activity of the S-nitroSoglutathione reductase in the cellular) in nitrosylation compared to a wild-type mouse. In biological sample are lower than the levels of the S-nitroso certain aspects, this increase in nitrosylation results in an glutathione reductase in the control Sample. In other aspects, accumulation of SNOs. The disruption may be a homozy the diagnostic or monitoring method comprises (a) measur gous disruption, for example, that results in a null mutation ing levels of SNOS in a biological Sample from a patient of the endogenous gene encoding S-nitroSoglutathione (e.g., plasma levels); (b) comparing the levels of SNOS in reductase, using the neomycin resistance gene as the Select the biological Sample to levels in a control sample; and (c) able marker. determining if the levels of SNOs in the biological sample 0021. The invention further relates to nucleic acids com are higher than the levels of SNOs in the control sample. prising a GSNOR knockout construct comprising a Select Similar diagnostic and monitoring methods are also encom able marker Sequence flanked by DNA sequences homolo passed for determining increased or deleteriously high levels gous to the endogenous GSNOR gene. Also related are of S-nitroSoglutathione reductase, or decreased or deleteri vectors comprising these nucleic acids and host cells and ously low levels of SNOs. cell lines (e.g., non-human mammal embryonic cell lines) 0.018. In various aspects of the invention, the disorder for comprising these vectors. Additionally related are methods diagnosis relating to increased levels of nitric oxide bioac for identifying an agent for alleviating at least one Symptom tivity is a degenerative disease (e.g., Parkinson's disease, of a Systemic infection or hypotension comprising: (a) Alzheimer's disease, amyotrophic lateral Sclerosis), Stroke, administering a test agent to a GSNOR knockout mouse Systemic infection (e.g., bacteremia, Sepsis, neonatal Sepsis, with a Systemic infection or hypotension, and (b) determin Septic shock, cardiogenic shock, endotoxic shock, toxic ing whether the test agent alleviates a Symptom of the Shock Syndrome, or Systemic inflammatory response Syn Systemic infection or hypotension in the knockout mouse. In drome), inflammatory diseases (e.g., colitis, inflammatory various aspects, the Systemic infection is bacteremia, Sepsis, bowel disease, rheumatoid arthritis, osteoarthritis, psoriatic neonatal Sepsis, Septic Shock, endotoxic Shock, toxic shock arthritis, infectious arthritis, ankylosing spondylitis, ten Syndrome, or Systemic inflammatory response Syndrome, donitis, burSitis, vasculitis, fibromyalgia, polymyalgia rheu while the hypotension is due to anesthesia (e.g., phenobar matica, temporal arteritis, giant cell arteritis, polyarteritis, bitol, ketamine Xylazine, or urethane). The Symptom may be HIV-associated rheumatic disease Syndromes, Systemic an increase in nitrosylation, for example, which results in an lupus, erythematosus, gout, and pseudogout (calcium pyro accumulation of SNOS. phosphate dihydrate crystal deposition disease), hypoten 0022. Other embodiments, objects, aspects, features, and Sion (e.g., associated with anesthesia, dialysis, or orthostatic advantages of the invention will be apparent from the hypotension), proliferative disease (e.g., cancer, tumor, dys accompanying description and claims. US 2005/0014697 A1 Jan. 20, 2005

BRIEF DESCRIPTION OF THE DRAWINGS of GSNOR female mice (filled, n=9) was significantly 0023 FIGS. 1A-1F. Targeted Disruption of the GSNOR lower than that of wild-type controls (open, n=8) following Gene. FIG. 1A: Strategy for targeted disruption of the cecal ligation and puncture (CLP; P<0.03). GSNOR gene. The structures of the targeting vector, wild 0026 FIGS. 4A-4E: Abnormal SNO metabolism in type and disrupted GSNOR alleles are shown. The restric GSNOR Mice. FIG. 4A: Liver S-nitrosothiols in wild tion Sites used for construction of the targeting vector and type and GSNOR mice after intraperitoneal injection of Southern analysis are: B, BamH I; H, Hind III; N, Not I; S, PBS (48 h) or LPS. Levels of SNO in GSNOR mice were Sac I; X, Xba I. Cassettes PGKneo and PGKtk are the determined to be significantly higher than in wild-type Selectable genes neo and tk respectively, under control of the controls at both 24h (P=0.005) and 48 h (P=0.006) after LPS mouse phosphoglycerokinase gene promoter. Double challenge. FIG. 4B: Serum nitrate in wild-type (open) and headed arrows represent expected fragments of wild-type GSNOR' (filled) mice. Nitrate levels in GSNOR mice and disrupted GSNOR alleles in Southern analyses with Sac were significantly higher (P=0.016) than in wild-type con I or Xba I restriction. Neo3se and GSNOR3 as are the PCR trols at 48 h after LPS. FIG. 4C: Serum nitrite in wild-type primers used to detect the disrupted allele. FIG. 1B: South (open) and GSNOR' (filled) mice. FIG. 4D: Elevated ern analysis of genomic DNA from GSNOR-targeted ES ratios of liver SNO to serum nitrate were significantly higher clones. The DNA was digested with Sac I and probed with (P=0.010) at 48 h than 24 h after LPS in GSNOR mice. ex2-3, a cDNA probe specific for exons 2-3 of GSNOR. WT, (Analysis was carried out on mice with significantly wild-type; KO, disrupted allele. FIG. 1C: Southern analysis elevated nitrate levels (>100 uM)). FIG. 4E: The level of of genomic DNA from wild-type (+/+), heterozygous (+/-) liver SNO was significantly higher (P=0.007) in GSNO and GSNOR (-/-) null mice. DNA was digested with Xba (filled) mice than in wild-type (open) controls at 72 h after I and hybridized with ex8-9, a probe specific for exons 8-9. CLP FIG. 1D: GSNOR activity in mouse tails. The data include 0027 FIGS. 5A-5H: Serum Markers of Tissue Injury. the means (ESD) of 2-4 samples. FIG. 1E: GSNOR activi Serum was collected 48 h following control PBS injection ties in various tissues. Protein extracts (500 ug/ml) were and 24 h or 48 h following LPS injection. Data (meaniSE) incubated with 200 uM NADH and 0 or 150 uM GSNO. were obtained from 4-12 wild-type (open) or GSNOR Values were obtained from 3 wild-type (filled) or 2 (filled) mice. Significant pair-wise differences are indicated GSNOR' (open) mice. FIG. 1F. Body weights of 80-day by an asterisk (p<0.015). Markers assayed were: (FIG. 5A) old mice (n=18-29) and litter sizes at weaning (n=16-32). alanine aminotransferase (ALT); (FIG. 5B) aspartate ami Mice from wild-type (open), GSNORO line one (streaked) notransferase (AST); (FIG.5C) creatinine; (FIG. 5D) urea and line two (filled) were raised on a standard mouse diet in nitrogen (BLN); (FIG. 5E) creatine phosphokinase (CPK); the same animal facility. (FIG.5F) amylase; (FIG.5G) lipase. FIG.5H: Correlation 0024 FIGS. 2A-2D: Blood Pressure and S-nitrosothiols between ALT (R =0.85, p<0.01) or AST (R-0.94, p<0.01) in Wild-type compared to GSNOR Mice. FIG. 2A. Mean and liver SNO in six GSNOR mice (48 h after LPS). arterial pressure in anesthetized C57BL/6 (WT) and 0028 FIGS. 6A-6H. Histopathology of LPS-Challenged GSNOR (KO) mice. The data include the means-SE of Mice. Shown are sections of liver (FIGS. 6A-6B), thymus two males and two females in each Strain (i.e., n=4 per (FIGS. 6C-6D), spleen (FIGS. 6E-6F), and mesenteric strain). FIG.2B: Systolic blood pressure in conscious mice. (pancreatic) lymph node (FIGS. 6G-6H) of wild-type Data are the meansitSE of 8 C57BL/6 (4 males) and 12 (FIGS. 6A, 6C,6E and 6G) and GSNOR (FIGS. 6B, 6D, GSNOR (4 males) mice. FIG.2C: Nitrosylation in RBCs 6F and 6H) mice 48 hours after LPS. All the micrographs are from unanesthetized wild-type (open) and GSNOR of the same magnification, and the scale bar in (FIG. 6A) is (filled) mice. SNO-Hb levels in GSNOR mice were 20 lim. N, necrotic hepatocyte, T, tingible body macrophage determined to be significantly higher than in wild-type mice with phagocytosed apoptotic cells. Each micrograph is rep (P<0.05, in 12), whereas iron-nitrosylHb levels were not resentative of three animals. different. FIG. 2D: Schematic showing vasodilation by 0029 FIGS. 7A-7D: iNOS Inhibition Prevents SNO RBC-SNO coupled to hypoxia/metabolic demand by plasma Elevation, Reduces Liver Injury, and Improves Survival of SNOs and vasodilation during NO deficiency states. LPS-Challenged GSNOR Mice. FIGS. 7A-7C: Serum 0025 FIGS. 3A-3E: Increased Mortality from Endotoxic levels of nitrate (FIG. 7A; n=7), liver S-nitrosothiol (FIG. and Septic Shock in GSNOR Mice. FIG.3A: Survival of 7B; n=4) and serum ALT (FIG.7C; n=5) in GSNOR mice GSNOR mice (filled circles, n=69) was significantly that were given 1400W 6 h following LPS injection (filled lower than that of wild-type mice (open circles, n=39) columns). Open columns represent the values obtained in following intraperitoneal injection of LPS(P<0.001). FIG. the absence of 1400W and are reproduced from FIGS. 4A, 3B: Survival of mice from GSNOR' line one (GSNOR' 4B and 5A. FIG. 7D: Survival of LPS-challenged 1, upright triangle; n=37) and line two (GSNOR2, GSNOR mice that received either 1400W (n=12; squares) inverted triangle; n=32) was similar. Both values were or PBS (n=6; diamonds) 6 h following LPS injection. Significantly lower than the wild-type mice (open circles, 0030 FIG. 8 shows the amount of airway resistance n=39) after LPS(P<0.002 for GSNO/1, P-0.004 for treated with increasing amounts of methylcholine (MCh) GSNOR'2). FIG. 3C: Survival of male GSNOR' mice wild-type mice and GSNOR mice treated with ovalbumin (filled circles, n=31) was not significantly lower than wild (OVA) and PBS. type controls (open circles, n=16) after LPS(P=0.12). FIG. 3D: Survival of both female GSNOR1 (upright triangle; 0031 FIG. 9 shows the level of IgE in both wild-type and P-0.01, n=19) and female GSNO/2 (inverted triangle; GSNOR mice after treatment with OVA or PBS. P<0.002, n=19) mice was significantly lower than wild-type 0032 FIG. 10 shows the level of BALF IL-13 in OVA controls (open circles, n=23) after LPS. FIG. 3E: Survival treated GSNOR and wild-type mice. US 2005/0014697 A1 Jan. 20, 2005

0033 FIGS. 11A-11B. Results from GRK studies. FIG. GenBank No. 13431519 corresponds to glutathione-depen 11A. Representative gel from experiments examining the dent formaldehyde dehydrogenase 2 from Schizosaccharo effect of cysNO (500, 50, and 5 uM) on isoproterenol (10 myces pombe; GenBank No. 30697.873 corresponds to oxi uM) stimulated GRK2 mediated receptor phosphorylation doreductase from Arabidopsis thaliana; GenBank No. using purified B-AR reconstituted in Synthetic vesicles and 15238330 corresponds to an alcohol dehydrogenase purified GRK2. FIG. 11B: Representative gel from experi sequence from Arabidopsis thaliana; GenBank No. ments examining the effect of cysNO (5, 50, and 500 uM) 15217715 corresponds to an alcohol dehydrogenase light stimulated GRK2 mediated phosphorylation of rhodop sequence from Arabidopsis thaliana; GenBank No. Sin using purified bovine rod outer Segments and purified 15219884 corresponds to an alcohol dehydrogenase GRK2. Sequence from Arabidopsis thaliana. Conserved domains 0034 FIGS. 12A-12B: Effect of cysNO (A: 500, 50, and are shown in bold. Positions with conservative Substitutions 5uM) and GSNO (B: 500, 50, and 5uM) on purified GRK2 are shown in bold, with italics. mediated in vitro phosphorylation of a Soluble peptide 0041 FIGS. 19A-19B: Secondary Structure Information substrate (RRREEEEESAAA; SEQ ID NO:30) (n=2; for Human GSNOR. Structural information for human *P-0.05). GSNOR (SEQ ID NO:19) was obtained from NCBI Acces 0035 FIGS. 13A-13C: Results of Cardiac Studies. FIG. sion No. 1MC5 A. SecStr designates secondary structure. 13A: Heart weight to body weight ratio (hw:bw, mg:g)(n= 10); FIG. 13B: Cardiac B-AR density (BMax, finol/mg DETAILED DESCRIPTION OF INVENTION protein (n=5); FIG. 13C: Cardiac BARK protein expression levels (n=4), in mice following mini-Osmotic pump implan 0042. Definitions tation and treatment for 7 days with either PBS, isoproter 0043. As used herein, “protein' is used synonymously enol (ISO) (30 mg/kg/day), GSNO (10 mg/kg/day) or a with “polypeptide'. A "purified’ polypeptide, protein, or combination of ISO and GSNO. All data expressed as mean peptide is Substantially free of cellular material or other (+/-SEM) (*P-0.05 versus PBS treated mice, "P-0.05 ver contaminating proteins from the cell, tissue, or cell-free SuS ISO treated mice, unpaired t test). Source from which the amino acid Sequence is obtained, or Substantially free from chemical precursors or other chemi 0036 FIG. 14: Human GSNOR Nucleotide and Amino cals when chemically Synthesized. Acid Sequence Information. Nucleotide (SEQID NO:7) and amino acid (SEQ ID NO:17) sequence information was 0044. The language “substantially free of cellular mate obtained from the National Center for Biotechnology Infor rial” includes preparations of polypeptides or peptides that mation (NCBI; Bethesda, Md.) databases under Accession are Separated from cellular components of the cells from No. M29872. In the nucleotide sequence, the start site and which the amino acid Sequences are isolated or recombi Stop Site are underlined. CDS designates coding Sequence. nantly produced. In one embodiment, the language "Sub Stantially free of cellular material” includes preparations of 0037 FIGS. 15A-15D: Human GSNOR Nucleotide and a polypeptide or peptide having less than about 30% (by dry Amino Acid Sequence Information. Nucleotide (SEQ ID weight) of other proteins (also referred to herein as a NO:8) and amino acid (SEQ ID NO:18) sequence informa “contaminating protein'), more preferably less than about tion was obtained from NCBI databases under Accession 20% of contaminating protein, Still more preferably less than No. NM 000671. In the nucleotide sequence, the start site about 10% of contaminating protein, and most preferably and Stop Site are underlined. CDS designates coding less than about 5% contaminating protein. When a polypep Sequence. SNP designates Single nucleotide polymorphism. tide or peptide is recombinantly produced, it is also prefer 0038 FIGS. 16A-16E: Human GSNOR EXon Sequences. ably Substantially free of culture medium, e.g., culture Nucleotide (SEQ ID NO:9-SEQ ID NO:15, consecutively) medium represents less than about 20%, more preferably and amino acid (SEQ ID NO:19) sequence information was less than about 10%, and most preferably less than about 5% obtained from NCBI databases under Accession Nos. of the Volume of the preparation. M81112-M81118. CDS designates coding sequence. 004.5 The term “antibody” as used herein refers to immu 0039 FIGS. 17A-17B: Mouse GSNOR Nucleotide and noglobulin molecules and immunologically active portions Amino Acid Sequence Information. Nucleotide (SEQ ID of immunoglobulin molecules, e.g., molecules that contain NO:16) and amino acid (SEQ ID NO:20) sequence infor an antigen binding site that specifically binds (immunoreacts mation was obtained from NCBI databases under Accession with) an antigen, Such as a polypeptide or peptide. Such Nos. NM 007410. CDS designates coding sequence. antibodies include, e.g., polyclonal, monoclonal, chimeric, 0040 FIGS. 18A-18E3: Amino Acid Sequence Alignment Single chain, Fab and F(ab')2 fragments, and an Fab expres for Human GSNOR and Homologous or Orthologous Sion library. In specific embodiments, antibodies are gener Sequences. Amino acid Sequence information (SEQ ID ated against human polypeptides, e.g., one or more NO:21-SEQ ID NO:29, consecutively) and sequence align GSNORS. ment was obtained from NCBI Conserved Domain Database 0046) The term “monoclonal antibody” or “monoclonal CD: KOGO022.1, KOGO022. In the alignment, Accession antibody composition', as used herein, refers to a population No.1MC5. A corresponds to human GSNOR; GenBank No. of antibody molecules that contain only one Species of an 113389 corresponds to human alcohol dehydrogenase 6; antigen binding Site capable of immunoreacting with a GenBank No. 174441816 corresponds to a sequence similar particular epitope of a polypeptide or peptide. A monoclonal to human class IV alcohol dehydrogenase; GenBank No. antibody composition thus typically displays a single bind 13432155 corresponds to glutathione-dependent formalde ing affinity for a particular amino acid Sequence with which hyde dehydrogenase 1 from Schizosaccharomyces pombe, it immunoreacts. US 2005/0014697 A1 Jan. 20, 2005

0047 As used herein, “modulate” is meant to refer to an provided by gaseous nitric oxide. Compounds having the increase or decrease the levels of a polypeptide, or to structure X-NO, wherein X is a nitric oxide releasing, increase or decrease the Stability or activity of a polypeptide. delivering or transferring moiety, including any and all Such Thus, an agent can be tested for its ability to activate a compounds which provide nitric oxide to its intended site of polypeptide, or to promote the Synthesis or Stability of a action in a form active for their intended purpose, and Y is polypeptide. 1 or 2. 0048. As used herein, the term “derivative” or “derived” 0056. As used herein, the term “bioactivity” indicates an refers to a chemical Substance that is related Structurally to effect on one or more cellular or extracellular process (e.g., another Substance and theoretically derivable from it, e.g., a via binding, signaling, etc.) which can impact physiological truncated protein or peptide. or pathophysiological processes. 0049. As used herein, the term “region” or “domain”, as 0057 The term “treating” in its various grammatical in protein region or domain, refers to a number of amino forms in relation to the present invention includes prevent acids in a defined area of a parent protein. ing, curing, reversing, attenuating, alleviating, minimizing, 0050 AS used herein, the term “physiological levels' Suppressing or halting at least one deleterious Symptom or refer to a characteristic of or appropriate to an organism’s effect of a disease (disorder) State, disease progression, healthy or normal functioning. AS used herein, the term disease causative agent (e.g., bacteria or viruses), or other “physiologically compatible” refers to a Solution or Sub abnormal condition. stance, for example media, that can be utilized to mimic an 0058 As used herein, “gene therapy” includes both con organism's healthy or normal environment. For in Vivo use, ventional gene therapy where a lasting effect is achieved by the physiological compatible Solution may include pharma a single treatment, and the administration of gene therapeu ceutically acceptable carriers, excipients, adjuvants, Stabi tic agents, which involves the one time or repeated admin lizers, and vehicles. istration of a therapeutically effective DNA or mRNA. 0051 AS utilized herein, the term “pharmaceutically 0059) The phrase “SEQ ID NO:7-SEQ ID NO:16,” and acceptable” means approved by a regulatory agency of the the like, is used herein for convenience, and may refer to Federal or a state government or listed in the U.S. Pharma each SEQID NO individually or more than one SEQ ID NO copoeia or other generally recognized pharmacopoeia for in accordance with the methods of the invention. use in animals and, more particularly, in humans. The term 0060 A “biological sample” for diagnostic testing “carrier” refers to a diluent, adjuvant, excipient, or vehicle includes, but is not limited to, Samples of blood (e.g., Serum, with which the therapeutic is administered and includes, but plasma, or whole blood), urine, Saliva, Sweat, breast milk, is not limited to Such Sterile liquids as water and oils. vaginal Secretions, Semen, hair follicles, skin, teeth, bones, 0052. The terms “cell culture medium” and “culture nails, or other Secretions, body fluids, tissues, or cells. medium” refer to a nutrient Solution used for growing cells that typically provides at least one component from one or 0061 The headings for the Subsequent sections are pro more of the following categories: 1) an energy Source, Vided for organizational purposes only. They are not to be usually in the form of a carbohydrate Such as glucose; 2) all considered limiting. essential amino acids, and usually the basic Set of twenty 0062 Polypeptides amino acids plus cysteine; 3) vitamins and/or other organic compounds required at low concentrations; 4) free fatty 0063. The invention encompasses GSNOR polypeptides acids; and 5) trace elements, where trace elements are (e.g., SEQ ID NO:17-SEQ ID NO:21), peptides (e.g., pep defined as inorganic compounds or naturally-occurring ele tides encoded by SEQ ID NO:9-SEQ ID NO:14), and ments that are typically required at very low concentrations, fragments, variants, modifications, and derivatives thereof. usually in the micromolar range. Such polypeptides or peptides can be made using techniques known in the art. For example, one or more of the polypep 0.053 For mammalian cells, the cell culture medium is tides or peptides can be chemically Synthesized using art generally "Serum free” when the medium is essentially free recognized methods. For example, a peptide Synthesizer can of Serum from any mammalian Source (e.g. fetal bovine be used. See, e.g., Peptide Chemistry, A Practical Textbook, serum (FBS)). By “essentially free” is meant that the cell Bodasinsky, Ed. Springer-Verlag, 1988; Merrifield, Science culture medium comprises between about 0-5% serum, 232:241-247 (1986); Barany, et al., Intl. J. Peptide Protein preferably between about 0-1% serum, and most preferably Res. 30.705-739 (1987); Kent, Ann. Rev. Biochem, 57.957 between about 0-0.1% serum. Advantageously, serum-free 989 (1988), and Kaiser, et al, Science 243:187-198 (1989). “defined' medium can be used, wherein the identity and 0064. Alternatively, GSNOR polypeptides or peptides concentration of each of the components in the medium is can be made by expressing one or more amino acid known (i.e., an undefined component Such as bovine pitu Sequences from a nucleic acid Sequence. Any known nucleic itary extract (BPE) is not present in the culture medium). acids that express the polypeptides or peptides (e.g., human 0.054 As defined herein “specific binding” refers to the or chimerics) can be used, as can vectors and cells express ability of a protein, peptide, or antigen to interact with an ing these polypeptides or peptides. Sequences of human antibody or each other. ORFs and polypeptides are publicly available, e.g. in Gen 0.055 As used here, the term “nitric oxide' encompasses Bank and other databases (see FIGS. 14-19). If desired, the uncharged nitric oxide (NO) and charged nitric oxide spe polypeptides or peptides can be recovered and isolated. cies, particularly including nitroSonium ion (NO") and 0065 Recombinant cells expressing the polypeptide, or a nitroxyl ion (NO). The reactive form of nitric oxide can be fragment or derivative thereof, may be obtained using meth US 2005/0014697 A1 Jan. 20, 2005 ods known in the art, and individual gene products or mosaic virus 35S RNA promoter (see e.g., Garder, et al., fragments may be isolated and analyzed (e.g., as described Nucl. Acids Res. 9:2871 (1981)) and (iii) the promoter of the in Sambrook et al., eds., MOLECULAR CLONING: A photosynthetic enzyme ribulose bisphosphate carboxylase LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor (see e.g., Herrera-Estrella, et al., Nature 310:115-120 Laboratory Press, Cold Spring Harbor, N.Y., 1989; and (1984)). Ausubel, et al., eds., CURRENT PROTOCOLS IN 0071 Promoter/enhancer elements from yeast and other MOLECULAR BIOLOGY, John Wiley & Sons, New York, fungi (e.g., the Gal4 promoter, the alcohol dehydrogenase N.Y., 1993). promoter, the phosphoglycerol kinase promoter, the alkaline 0.066 Assays may be used based upon the physical and/or phosphatase promoter), as well as the following animal functional properties of the polypeptides or peptides. The transcriptional control regions, which possess tissue speci assays can include, e.g., radioactive labeling of one or more ficity and have been used in transgenic animals, may be of the polypeptides, followed by analysis by gel electro utilized in the production of proteins of the present inven phoresis and immunoassay. Polypeptides and peptides may tion. be isolated and purified by standard methods known in the 0072 Other animal transcriptional control sequences art either from natural Sources or recombinant host cells derived from animals include, e.g.: (i) the insulin gene expressing the proteins/peptides. These methods can control region active within pancreatic B-cells (see e.g., include, for example, column chromatography (e.g., ion Hanahan, et al., Nature 315:115-122 (1985)); (ii) the immu eXchange, affinity, gel eXclusion, reverse-phase, high pres noglobulin gene control region active within lymphoid cells Sure, fast protein liquid, etc.), differential centrifugation, (see e.g., Grosschedl, et al., Cell 38:647-658 (1984)); (iii) differential solubility, or similar methods used for the puri the albumin gene control region active within liver (see e.g., fication of proteins. Pinckert, et al., Genes and Devel. 1:268-276 (1987)); (iv) the 0067. In certain aspects of the invention, particular myelin basic protein gene control region active within brain domains of the GSNOR polypeptides can be used. Highly oligodendrocyte cells (see e.g., Readhead, et al., Cell conserved domains in human GSNOR include amino acids 48:703-712 (1987)); and (v) the gonadotrophin-releasing 17-172 and amino acids 193-241, as well as amino acids hormone gene control region active within the hypothalamus 64-80 and amino acids 215-228 (FIGS. 18A-18E). Less (see e.g., Mason, et al., Science 234:1372-1378 (1986)). conserved domains in human GSNOR include amino acids 0073. The vector may include a promoter operably 1-16 and amino acids 172-193, as well as amino acids linked to nucleic acid sequences which encode a GSNOR 242-374 (FIGS. 18A-18B). In other aspects, conservative polypeptide or peptide, one or more origins of replication, variants of these polypeptides or polypeptide domains can and optionally, one or more Selectable markers (e.g., an be used. antibiotic resistance gene). A host cell Strain may be selected 0068 Nucleic acids encoding one or more GSNOR which modulates the expression of polypeptide or peptide polypeptide or peptide, as well as vectors and cells com Sequences, or modifies/processes the expressed Sequences in prising these nucleic acids, are within the Scope of the a desired manner. Moreover, different host cells possess present invention. Host-vector Systems that can be used to characteristic and Specific mechanisms for the translational express the polypeptides or peptides include, e.g.: (i) mam and post-translational processing and modification (e.g., malian cell Systems which are infected with vaccinia virus, glycosylation, phosphorylation, and the like) of expressed adenovirus; (ii) insect cell Systems infected with baculovi polypeptides or peptides. Appropriate cell lines or host rus; (iii) yeast containing yeast vectors, or (iv) bacteria Systems may thus be chosen to ensure the desired modifi transformed with bacteriophage, DNA, plasmid DNA, or cation and processing of the polypeptide or peptide is coSmid DNA. Depending upon the host-vector System uti achieved. For example, protein expression within a bacterial lized, any one of a number of Suitable transcription and System can be used to produce an unglycosylated core translation elements may be used. protein; whereas expression within mammalian cells can be 0069. The expression of the specific polypeptides or used to obtain native glycosylation of a heterologous pro peptides may be controlled by any promoter/enhancer tein. known in the art including, e.g.: (i) the SV40 early promoter 0074) Prokaryotic host cells include gram-negative or (see e.g., Bemoist & Chambon, Nature 290:304-310 gram-positive organisms. Suitable prokaryotic host cells for (1981)); (ii) the promoter contained within the 3'-terminus transformation include, for example, E. coli, Bacillus Sub long terminal repeat of Rous Sarcoma Virus (see e.g., tilis, Salmonella typhimurium, and various other Species Yamamoto, et al., Cell 22:787-797 (1980)); (iii) the Herp within the genera Pseudomonas, Streptomyces, and Staphy esvirus thymidine kinase promoter (see e.g., Wagner, et al., lococcuS. Alternatively, the polypeptides or peptides may be Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)); (iv) the expressed in yeast host cells, preferably from the Saccha regulatory sequences of the metallothionein gene (see e.g., romyces genus (e.g., S. cerevisiae). Other genera of yeast, Brinster, et al., Nature 296:39-42 (1982)), (v) prokaryotic Such as Schizosaccharomyces, Pichia, or Kluyveromyces, expression vectors Such as the f-lactamase promoter (See may also be employed. e.g., Villa-Kamaroff, et al., Proc. Natl. Acad. Sci. USA 0075 Mammalian or insect host cell culture systems may 75:3727-3731 (1978)); (vi) the tac promoter (see e.g., be used to express recombinant polypeptides or peptides. DeBoer, et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)). Baculovirus Systems for production of heterologous proteins 0070 Plant promoter/enhancer sequences within plant in insect cells are well known (see, e.g., Luckow and expression vectors may also be utilized including, e.g.: (i) Summers, Bio/Technology 6:47 (1988)). Established cell the nopaline Synthetase promoter (see e.g., Herrar-Estrella, lines of mammalian origin also may be employed. Examples et al., Nature 303:209-213 (1984)); (ii) the cauliflower of Suitable mammalian host cell lines include, but are not US 2005/0014697 A1 Jan. 20, 2005 limited to, the COS-7 line of monkey kidney cells (ATCC Sequence thereto. Such a fragment generally comprises at CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, least about 14 nucleotides, preferably from about 14 to 30 C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster nucleotides. The design of Such oligonucleotides based upon ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) a cDNA sequence has been previously described (See, e.g., cell lines, and the CV1/EBNA cell line derived from the Stein and Cohen Cancer Res. 48:2659, 1988; van der Krol African green monkey kidney cell line CV1 (ATCCCCL70; et al. BioTechniques 6:958, 1988). These short antisense McMahan et al. EMBO J. 10: 2821, 1991). oligonucleotides can be imported into cells where they act as inhibitors, even where there is low intracellular concentra 0076) Nucleic Acids tions caused by their restricted uptake by the cell membrane 0077. The invention encompasses GSNOR nucleic acids (Zamecnik et al., Proc. Natl. Acad. Sci. USA 83:4143-4146 (e.g., SEQID NO:7-SEQID NO:16 and sequences encoding (1986)). SEQ ID NO:17-SEQ ID NO:21), and fragments, variants, 0082 For use with the methods of the invention, oligo derivatives, and complementary Sequences thereof. nucleotides can include modified Sugar-phosphodiester Sequences of human GSNOR genes and coding Sequences backbones or other Sugar linkages (see, e.g., WO 91/06629). are publicly available, e.g. in GenBank and other databases Such oligonucleotides with Sugar linkages exhibit increased (see FIGS. 14-19). GSNOR nucleic acids can be used, for Stability in Vivo (i.e., are capable of resisting enzymatic example, for hybridization probes, in chromosome and gene degradation) but retain sequence specificity to be able to mapping and in the generation of anti-Sense RNA and DNA, bind to target nucleotide Sequences. In other aspects, oligo Small interfering RNAS, and gene therapy vectors (see, e.g., nucleotides can be covalently linked to organic moieties, U.S. Published Application 2004/0023323). Such nucleic Such as those described in WO90/10048, and other moieties acids are also useful for the preparation of GSNOR polypep that increases affinity of the oligonucleotide for a target tides and by the recombinant techniques previously nucleic acid sequence, Such as poly-(L-lysine). Further still, described. intercalating agents, Such as ellipticine, and alkylating 0078. The full-length sequence of the GSNOR gene, or agents or metal complexes may be attached to Sense or portions thereof, may be used as hybridization probes to antisense oligonucleotides to modify binding Specificities of detect (or determine levels of) GSNOR expression, or to the oligonucleotide for the target nucleotide Sequence. detect variants of GSNOR (e.g., SNPs; see FIG. 17B), or 0083 Oligonucleotides may be introduced into a cell GSNOR nucleic acids from other species. Optionally, the containing the target nucleic acid Sequence by any gene length of the probes will be about 20 to about 50 bases. The transfer method, including, for example, CaPO-mediated hybridization probes may be derived from at least partially DNA transfection, electroporation, or by using gene transfer novel regions of the full length native nucleotide Sequence vectorS Such as Epstein-Barr virus. In a preferred procedure, wherein those regions may be determined without undue an oligonucleotide is inserted into a Suitable retroviral experimentation, or from genomic Sequences including pro vector. A cell containing the target nucleic acid Sequence is moters, enhancer elements, and introns of native Sequence of contacted with the recombinant retroviral vector, either in GSNOR. vivo or ex vivo. Suitable retroviral vectors include, but are 0079. As one example, a screening method may comprise not limited to, those derived from the murine retrovirus isolating the coding region of the GSNOR gene using the M-MuLV, N2 (a retrovirus derived from M-MuLV), or the known DNA sequence to Synthesize a Selected probe of double copy vectors designated DCT5A, DCT5B and about 40 bases. Hybridization probes may be labeled by a DCT5C (see, e.g., WO 90/13641). variety of labels, including radionucleotides such as 'P or 0084 Oligonucleotides also may be introduced into a cell S, or enzymatic labels such as alkaline phosphatase, containing the target nucleotide Sequence by formation of a coupled to the probe (e.g., via avidin/biotin coupling Sys conjugate with a ligand binding molecule (e.g., as in WO tems). Any GSNOR EST sequences may be employed as 91/04753). Suitable ligand binding molecules include, but probes, using the methods disclosed herein. are not limited to, cell Surface receptors, growth factors, 0080. Other useful GSNOR nucleic acids include anti other cytokines, or other ligands that bind to cell Surface Sense or Sense oligonucleotides comprising a Singe-Stranded receptors. Preferably, conjugation of the ligand binding nucleic acid sequence (either RNA or DNA) capable of molecule does not substantially interfere with the ability of binding to target GSNOR mRNA or GSNOR DNA the ligand binding molecule to bind to its cognate ligand(s), Sequences. Binding of oligonucleotides to target nucleic acid or block entry of the oligonucleotide or its conjugated Sequences can be used to form duplexes that block tran version into the cell. Alternatively, an oligonucleotide may Scription or translation of the target Sequence. Oligonucle be introduced into a cell containing the target nucleic acid otide binding may cause enhanced degradation of the Sequence by formation of an oligonucleotide-lipid complex duplexes, premature termination of transcription or transla (see, e.g., WO 90/10448). The oligonucleotide-lipid com tion, or another inhibitory effect. Thus, the oligonucleotides pleX is preferably dissociated within the cell by an endog may be used to decrease expression of a GSNOR polypep enous lipase. tide. For example, an antisense RNA or DNA molecule can directly block the translation of mRNA by hybridizing to 0085 Antisense (or sense) RNA or DNA molecules are targeted mRNA and preventing protein translation, or by generally at least about 5 bases in length, about 10 bases in hybridizing to targeted DNA to form triple-helixes. length, about 15 bases in length, about 20 bases in length, about 25 bases in length, about 30 bases in length, about 35 0081. Such oligonucleotides, according to the present bases in length, about 40 bases in length, about 45 bases in invention, comprise a fragment of GSNOR DNA, e.g., a length, about 50 bases in length, about 55 bases in length, fragment of the coding Sequence or complementary about 60 bases in length, about 65 bases in length, about 70 US 2005/0014697 A1 Jan. 20, 2005 bases in length, about 75 bases in length, about 80 bases in 0090 Nucleic acids which encode GSNOR or its modi length, about 85 bases in length, about 90 bases in length, fied forms can also be used to generate transgenic animals or about 95 bases in length, about 100 bases in length, or more. cell lines, or knockout animals or cell lines. Transgenics and The oligonucleotides can be modified to enhance their knock outs are useful in the development and Screening of uptake, e.g. by Substituting their negatively charged phoS therapeutically useful reagents, as described below. A trans phodiester groups by uncharged groups. genic animal (e.g., a mouse or rat) is an animal having cells that contain a transgene, which was introduced into the 0.086 An oligonucleotide can be designed to be comple animal or an ancestor of the animal at a prenatal, for mentary to a region of a transcript or the gene involved in example, an embryonic Stage. Methods for generating trans transcription (see Lee et al., Nucl. Acids Res., 6:3073 (1979); genic animals, particularly animals. Such as mice or rats, are Cooney et al., Science, 241:456 (1988); Dervan et al., now conventional in the art and are described, for example, Science, 251:1360 (1991); Okano, Neurochem, 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of in U.S. Pat. Nos. 4,736,866 and 4,870,009. Gene Expression CRC Press: Boca Raton, Fla., 1988). To 0091. In one approach, particular cells can be targeted for target a transcript, the 5' coding portion of the GSNOR GSNOR transgene incorporation with tissue-specific polynucleotide Sequence can be used to design an antisense enhancers. Animals that include a copy of a transgene oligonucleotide of from about 10 to 40 base pairs in length. encoding GSNOR introduced into the germ line of the To target the gene, oligodeoxyribonucleotides derived from animal at an embryonic Stage can be used to examine the the translation-initiation site, e.g., between about -10 and effect of increased expression of GSNOR. Such animals can +10 positions of the target gene nucleotide Sequence can be be used as tester animals for reagents thought to confer used. Nucleic acid molecules for triple-helix formation can protection from, for example, pathological conditions asso be using via Hoogsteen base-pairing rules, which generally ciated with its overexpression. In accordance with this facet require sizeable Stretches of purines or pyrimidines on one of the invention, an animal is treated with the reagent and a strand of a duplex. See, e.g., WO 97/33551. reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a 0087. Other useful nucleic acids include ribozymes, potential therapeutic intervention for the pathological con which are enzymatic RNA molecules capable of catalyzing the Specific cleavage of RNA. Ribozymes act by Sequence dition. Specific hybridization to the complementary target RNA, 0092 Alternatively, as demonstrated herein, non-human followed by endonucleolytic cleavage. Specific ribozyme homologs (i.e., orthologs) of GSNOR can be used to con cleavage Sites within a potential RNA target can be identified Struct a knock out animal which has a defective or altered by known techniques (see, e.g., Rossi, Current Biology, gene encoding GSNOR. Knock outs can be produced by 4:469-471 (1994), and WO 97/33551). homologous recombination between the endogenous gene encoding GSNOR and altered genomic DNA encoding 0088. In another approach, interfering RNAS (iRNAs; GSNOR introduced into an embryonic stem cell of the Tijsterman, M., et al., 2002, Annu. Rev. Genet. 36,489-519; animal. For example, cDNA encoding GSNOR can be used Tabara, H., et al., 2002, Cell 109,861-871) can be used to to clone genomic DNA encoding GSNOR in accordance “knock-down” GSNOR expression. iRNAS are double with established techniques. A portion of the genomic DNA stranded molecules that are cleaved in the cell by an RNase encoding GSNOR can be deleted or replaced with another III like enzyme into small (21 to 23 nucleotides) interfering gene, Such as a gene encoding a Selectable marker which can RNAs (siRNAS; Bernstein, E., et al., 2001, Nature 409, be used to monitor integration. 363-366; Ketting, R. F., et al., 2001, Genes Dev 15, 2654 2659; Knight, S. W. and Bass, B. L., 2001, Science 293, 0093. In one approach, a vector includes several kilo 2269-2271; Zamore, P. D., et al., 2000, Cell 101, 25-33). bases of unaltered flanking DNA both at the 5' and 3' ends SiRNAS associate with a large multiprotein complex, the (see, e.g., Thomas and Capecchi, Cell, 51:503 (1987)). The RISC, which unwinds the siRNA to help target the appro vector is introduced into an embryonic stem cell line (e.g., priate mRNA (Martinez, J., et al., 2002, Cell 110,563-574). by electroporation) and cells in which the introduced DNA The siRNA-mRNA hybrid is then cleaved, the siRNA is has homologously recombined with the endogenous DNA released, and the mRNA is degraded by endo- and exonu are selected (see e.g., Li et al., Cell, 69.915 (1992)). The cleases (reviewed in Dillin, 2003, Proc. Natl. Acad. Sci. Selected cells are then injected into a blastocyst of an animal USA, 100: 6289-6291). (e.g., a mouse or rat) to form aggregation chimeras (see e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells. A 0089. In mammalian cells, siRNAS can be added directly to the cells to lead to a Specific depletion of the targeted Practical Approach, E. J. Robertson, ed. (IRL, Oxford, mRNA and consequently the encoded protein product. Such 1987), pp. 113-152). siRNAS can be made synthetically or by use of expression 0094. Achimeric embryo can be implanted into a suitable vectors. SiRNAS can be designed using known methods pseudopregnant female foster animal and the embryo (Elbashir S M, et al., 2001, Nature 411: 494-498) and brought to term to create a knock out animal. Progeny algorithms (see, e.g., Cenix BioScience, Dresden, Ger harboring the homologously recombined DNA in their germ many). In addition, siRNAS and siRNA expression vectors cells can be identified by Standard techniques and used to can be obtained from commercial Sources (see, e.g., breed animals in which all cells of the animal contain the Ambion, Inc., Austin, Tex.; OIAGEN, Inc., Valencia, Calif.; homologously recombined DNA. Knockout animals can be Promega, Madison Wis.; InvivoGen, San Diego, Calif.). characterized for instance, for their ability to defend against Advantageously, siRNAS may be useful for Specifically certain pathological conditions (e.g., LPS challenge) and for targeting a GSNOR transcript, and leaving related Sequences their development of pathological conditions (e.g., hypoten unaffected. sion) due to absence of the GSNOR polypeptide. US 2005/0014697 A1 Jan. 20, 2005

0.095 Nucleic acids encoding GSNOR polypeptides or Hopp and Woods, Proc. Nat. Acad. Sci. USA 78:3824-3828 peptides may also be used in gene therapy. In particular, a (1981); Kyte and Doolittle, J. Mol. Biol. 157:105-142 GSNOR coding sequence can be introduced into cells to (1982). produce a therapeutically effective GSNOR product, for 0100 Various procedures known within the art may be example to replace a defective gene or to increase gene used for the production of polyclonal or monoclonal anti expression. There are a variety of techniques available for bodies. For example, for the production of polyclonal anti introducing nucleic acids into viable cells. The techniques bodies, various Suitable host animals (e.g., rabbit, goat, vary depending upon whether the nucleic acid is transferred mouse or other mammal) may be immunized by injection into cultured cells in vitro, or in vivo in the cells of the with the native polypeptide, or a variant thereof, or a intended host. Techniques suitable for the transfer of nucleic fragment or derivative of the foregoing. An appropriate acid into mammalian cells in vitro include the use of immunogenic preparation can contain, for example, a liposomes, electroporation, microinjection, cell fusion, recombinantly expressed polypeptide. Alternatively, the DEAE-dextran, the calcium phosphate precipitation method, immunogenic polypeptides or peptides may be chemically etc. Synthesized, as previously discussed. 0096. In one preferred method, gene transfer is per 0101 The immunogenic preparation can further include formed in vivo by transfection with viral (typically retrovi an adjuvant. Various adjuvants used to increase the immu ral) vectors and viral coat protein-liposome mediated trans nological response include, e.g., Freund's (complete and fection (Dzau et al., Trends in Biotechnology 11, 205-210 incomplete), mineral gels (e.g., aluminum hydroxide), Sur (1993)). In some situations, it is desirable to provide the face active Substances (e.g., lysolecithin, pluronic polyols, nucleic acid Source with an agent that targets the target cells, polyanions, peptides, oil emulsions, dinitrophenol, etc.), Such as an antibody Specific for a cell Surface membrane human adjuvants such as Bacille Calmette-Guerin and protein or the target cell, a ligand for a receptor on the target Corynebacterium parvum, or similar immunostimulatory cell, etc. Where liposomes are employed, proteins which agents. If desired, the antibody molecules directed against a bind to a cell Surface membrane protein associated with polypeptide or peptide can be isolated from the mammal endocytosis may be used for targeting and/or to facilitate (e.g., from the blood) and further purified by well known uptake, e.g. capsid proteins or fragments thereof tropic for a techniques, Such as protein A chromatography to obtain the particular cell type, antibodies for proteins which undergo IgG fraction. internalization in cycling, proteins that target intracellular localization and enhance intracellular half-life. The tech 0102) Any technique may be used to prepare monoclonal nique of receptor-mediated endocytosis has been previously antibodies directed towards a particular polypeptide or pep described (see, e.g., Wu et al., J. Biol. Chem. 262,4429-4432 tide. For example, continuous cell line cultures may be (1987); and Wagner et al., Proc. Natl. Acad. Sci. USA 87, utilized as in, e.g., hybridoma techniques (see Kohler & 3410-3414 (1990)). For review of gene marking and gene Milstein, Nature 256:495-497 (1975)); trioma techniques; therapy protocols see Anderson et al., Science 256, 808–813 human B cell hybridoma techniques (see Kozbor, et al., (1992). Immunol Today 4:72 (1983)); and EBV hybridoma tech niques to produce human monoclonal antibodies (see, Cole, 0097 Antibodies et al., In: Monoclonal Antibodies and Cancer Therapy, Alan 0098. The invention further encompasses antibodies and R. Liss, Inc., (1985) pp. 77-96). If desired, human mono antibody fragments (such as Fab or F(ab')2 fragments) that clonal antibodies may be prepared by using human hybri bind specifically to a GSNOR polypeptide (e.g., SEQ ID domas (see Cote, et al., Proc. Natl. Acad. Sci. USA 80:2026 NO:17-SEQ ID NO:21) peptide (e.g., peptide encoded by 2030 (1983)) or by transforming human B cells with Epstein SEQ ID NO:9-SEQ ID NO:14), or fragment thereof. An Barr Virus in vitro (see Cole, et al., In: Monoclonal Anti antibody that “specifically binds' is one that recognizes and bodies and Cancer Therapy, Supra). binds to a particular GSNOR amino acid sequence, but 0.103 Methods can be adapted for the construction of Fab which does not Substantially recognize or bind to other expression libraries (see e.g., Huse, et al., Science 246:1275 molecules in a biological Sample. In one approach, a purified 1281 (1989)) to allow rapid and effective identification of polypeptide or a portion, variant, or fragment thereof, can be monoclonal Fab fragments with the desired specificity for used as an immunogen to generate antibodies that Specifi the desired protein or derivatives, fragments, analogs or cally bind the amino acid Sequence using Standard tech homologs thereof. Non-human antibodies can be “human niques for polyclonal and monoclonal antibody preparation. ized” by techniques well known in the art (see e.g., U.S. Pat. No. 5,225,539). Antibody fragments that contain the idio 0099. A full-length polypeptide can be used, if desired. types to a polypeptide or peptide may be produced by Alternatively, antigenic fragments of polypeptides can be techniques known in the art including, e.g.: (i) an F(ab') used as immunogens. In Some embodiments, the antigenic fragment produced by pepsin digestion of an antibody fragment includes at least 6, 8, 10, 15, 20, or 30 or more amino acid residues of a polypeptide. In one embodiment, molecule; (ii) an Fab fragment generated by reducing the epitopes include specific domains of the polypeptide, or are disulfide bridges of an F(ab')2 fragment; (iii) an Fab frag located on the Surface of the polypeptide, e.g., hydrophilic ment generated by the treatment of the antibody molecule regions. If desired, peptides containing antigenic regions can with papain and a reducing agent; and (iv) F fragments. be selected using hydropathy plots showing regions of 0104 Chimeric and humanized monoclonal antibodies hydrophilicity and hydrophobicity. These plots may be gen against the polypeptides or peptides described herein are erated by any method well known in the art, including, for also within the Scope of the invention. Such antibodies can example, the Kyte Doolittle or the Hopp Woods methods, be produced by recombinant DNA techniques known in the either with or without Fourier transformation. See, e.g., art, for example using methods described in PCT Interna US 2005/0014697 A1 Jan. 20, 2005 tional Application No. PCT/US86/02269; European Patent altered expression of polypeptides involved in cell processes Application No. 184,187; European Patent Application No. and pathways can lead to deleterious effects in a Subject. For 171,496; European Patent Application No. 173,494; PCT example, medical conditions that relate to decreased International Publication No. WO 86/01533; U.S. Pat. No. GSNOR levels and increased NO synthesis and/or increased 4,816,567; European Patent Application No. 125,023; Better NO levels include, for example, degenerative diseases (e.g., et al., Science 240:1041-1043 (1988); Liu et al., Proc. Nat. Parkinson's disease, Alzheimer's disease, ALS), Stroke (e.g., Acad. Sci. USA 84:3439-3443 (1987); Liu et al., J. Immunol. ischemic stroke), and proliferative diseases (e.g., neoplasms, 139:3521-3526 (1987); Sun et al., Proc. Nat. Acad. Sci. USA tumors, cancers, dysplasias, and precancerous lesions). 84:214-218 (1987); Nishimura et al., Cancer Res. 47: 999 Medical conditions that relate to increased GSNOR levels 1005 (1987); Wood et al., Nature 314:446-449 (1985); Shaw and decreased SNO levels (e.g., SNO-Hb) include, for et al., J. Natl. Cancer Inst. 80:1553–1559 (1988); Morrison, example, vascular disorderS Such as hypertension (e.g., Science 229:1202-1207 (1985); Oi et al., BioTechniques pulmonary hypertension), heart disease, heart failure, heart 4:214 (1986); U.S. Pat. No. 5.225,539; Jones et al., Nature attack, atherOSclerosis, restenosis, asthma, and impotence. 321:552-525 (1986); Verhoeyan et al., Science 239:1534 Medical conditions that relate to decreased GSNOR levels (1988); and Beidler et al., J. Immunol. 141:4053-4060 and increased SNO levels (e.g., SNO-Hb) include, for (1988). example, tissue injury (e.g., hepatic, renal, muscle, and/or 0105 Methods for the screening of antibodies that pos lymphatic tissue) or death due to Systemic infections Such as SeSS the desired Specificity include, e.g., enzyme-linked bacteremia, Sepsis, Systemic inflammatory response Syn immunosorbent assay (ELISA) and other immunologically drome, neonatal Sepsis, cardiogenic shock, or toxic shock. mediated techniques known within the art. For example, 0110. Other conditions that relate to decreased GSNOR Selection of antibodies that are specific to a particular levels and increased SNO levels (e.g., SNO-Hb) include, for domain of a polypeptide can be facilitated by generation of example, inflammatory disease Such as colitis, inflammatory hybridomas that bind to the polypeptide or fragment thereof, bowel disease, rheumatoid arthritis, Osteoarthritis, psoriatic possessing Such a domain. arthritis, infectious arthritis, ankylosing spondylitis, ten 0106. In certain embodiments of the invention, antibodies donitis, burSitis, vasculitis, fibromyalgia, polymyalgia rheu specific for the GSNOR polypeptides or peptides described matica, temporal arteritis, giant cell arteritis, polyarteritis, herein may be used in various methods, Such as detection or HIV-associated rheumatic disease Syndromes, Systemic inhibition of amino acid Sequences, and identification of lupus, erythematosus, gout, and pseudogout (calcium pyro agents which inhibit these Sequences. Detection can be phosphate dihydrate crystal deposition disease), among oth facilitated by coupling (e.g., physically linking) the antibody ers. In addition, decreased GSNOR levels and increased to a detectable Substance. Examples of detectable Substances SNO levels (e.g., SNO-HB) are associated with hypotension include various enzymes, prosthetic groups, fluorescent during anesthesia, and tissue damage and morbidity due to materials, luminescent materials, bioluminescent materials, Shock (e.g., endotoxic or Septic shock), as shown herein and radioactive materials. Examples of Suitable enzymes below. include horseradish peroxidase, alkaline phosphatase, B-ga 0111 One diagnostic method involves providing a bio lactosidase, or acetylcholinesterase; examples of Suitable logical Sample from a Subject, measuring the levels of prosthetic group complexes include Streptavidin/biotin and GSNORS or SNOs in the sample, and comparing the level to avidin/biotin; examples of Suitable fluorescent materials a reference sample having known GSNOR or SNO levels. A include umbelliferone, fluorescein, fluorescein isothiocyan higher or lower level in the Sample versus the reference ate, rhodamine, dichlorotriazinylamine fluorescein, dansyl indicates altered expression of GSNORS or SNOs. Alterna chloride or phycoerythrin; an example of a luminescent tively, the enzymatic activity of GSNOR can be measured in material includes luminol; examples of bioluminescent any cell of interest. The detection of altered expression or materials include GFP. luciferase, luciferin, and aequorin, activity of a polypeptide can be use to diagnose a given and examples of suitable radioactive material include 'I, disease State, and or used to identify a Subject with a 131I, 35S O 3H. predisposition for a disease State. Any Suitable reference 0107 Polypeptide-specific or peptide-specific antibodies Sample may be employed, but preferably the test Sample and can also be used to isolate amino acid Sequences using the reference Sample are derived from the same medium, e.g. Standard techniques, Such as affinity chromatography or both are blood or urine, etc. The reference sample should be immunoprecipitation. Thus, the antibodies disclosed herein Suitably representative of the level polypeptide expressed in can facilitate the purification of Specific polypeptides or a control population. peptides from cells, as well as recombinantly produced 0112 The invention also provides a kit to determine polypeptides or peptides expressed in host cells. GSNOR or SNO levels or GSNOR activity. In one aspect, the kit comprises one or more antibodies directed to a 0108 Diagnostic Methods and Kits GSNOR polypeptide or peptide, or one or more antibodies 0109 Methods of determining expression and activity directed to a SNO. In another aspect, the kit can contain a levels of a GSNOR polypeptide (e.g., SEQID NO:17-SEQ substrate for a GSNOR enzyme. Such kits can contain, for ID NO:21), peptide (e.g., peptide encoded by SEQ ID example, reaction vessels, reagents for detecting GSNOR or NO:9-SEQ ID NO:14), or fragment thereof in a subject, e.g. SNO in Sample, and reagents for development of detected for diagnostic purposes, are also encompassed by the inven GSNOR or SNO, e.g. a secondary antibody coupled to a tion. In accordance with the invention, diagnostic methods detectable marker. The label incorporated into the anti can be used to predict or establish the onset of a medical polypeptide antibody may include, e.g., a chemiluminescent, condition described herein, or to monitor the progression or enzymatic, fluorescent, calorimetric, or radioactive moiety. Success of treatment of Such condition. It is understood that For detecting GSNOR enzyme activity, the kit can contain US 2005/0014697 A1 Jan. 20, 2005

a colormetric or fluorometric assay for measuring reaction detected by their label. Where the originally non-immobi with a Substrate. As an alternative approach, the kit can lized component does not carry a label, complexing can be include nucleic acid probes for measuring levels of GSNOR detected, for example, by using a labeled antibody that gene expression or gene dosage. The nucleic acid probes Specifically binds to the immobilized complex. may be unlabeled or labeled with a detectable marker. If unlabeled, the nucleic acid probes may be provided in the kit 0119) If the test agent interacts with a GSNOR polypep with labeling reagents. Kits of the present invention may be tide, its interaction with that polypeptide can be assayed by employed in diagnostic and/or clinical Screening assayS. methods well known for detecting protein-protein interac tions. Such assays include traditional approaches, Such as, 0113 Screens for Modulating Agents e.g., croSS-linking, co-immunoprecipitation, and co-purifi cation through gradients or chromatographic columns. In 0114. The invention further encompasses agents (e.g., addition, protein-protein interactions can be monitored by inhibitors/antagonists or activators/agonists) which modu using a yeast-based genetic System, e.g., a two-hybrid SyS late the levels of one or more GSNORS or SNOS, or tem (Fields and Song, Nature (London), 340:245-246 modulate GSNOR activity, and methods for identifying such (1989); Chien et al., Proc. Natl. Acad. Sci. USA, 88: 9578 agents. Screening assays can be designed to identify com 9582 (1991); Chevray and Nathans, Proc. Natl. Acad. Sci. pounds that bind or complex with a GSNOR polypeptide or USA, 89:5789-5793 (1991)). Two-hybrid systems employs peptide, or otherwise alter expression or Stability of the two fusion proteins, one in which the target protein is fused GSNOR transcript or translation product, or interfere with to a DNA-binding domain, and another, in which candidate the interaction of GSNOR with other cellular proteins. binding proteins are fused to the activation domain (e.g., 0115 The screening assays of the invention can include GAL4 binding and activation domains can be used). Cells methods amenable to high-throughput Screening of chemical are transformed with both fusion constructs, and colonies libraries, making them particularly Suitable for identifying containing interacting polypeptides are detected with a chro Small molecule drug candidates. The assays can be per mogenic Substrate for B-galactosidase. A complete kit formed in a variety of formats, including protein-protein (MATCHMAKERTM) for identifying protein-protein inter binding assays, biochemical Screening assays, immunoas actions between two specific proteins using the two-hybrid says, and cell-based assays, which are well characterized in technique is commercially available from CLONTECH. the art. For in vitro Screening, modulating agents can be 0120 Test agents that interfere with the interaction of a identified by, e.g., phage display, GST-pull down, FRET GSNOR polypeptide and other intra- or extracellular com (fluorescence resonance energy transfer), or BIAcore (Sur ponents can be tested by established methods. In one face plasmon resonance, Biacore AB, Uppsala, Sweden) approach, a reaction mixture is prepared containing the analysis. For in Vivo Screening, agents can be identified by, GSNOR gene product and the intra- or extracellular com e.g., yeast two-hybrid analysis, co-immunoprecipitation, co ponent under conditions and for a time allowing for the localization by immunofluorescence, or FRET. interaction and binding of the two products. The reaction is 0116 Modulation of activity (or levels) due to the test run in the absence and in the presence of the test compound. agent, e.g. binding of the agent to the polypeptide, can be In addition, an nonreactive agent may be added to a third determined using art recognized methods. For example, the reaction mixture, to Serve as positive control. The formation polypeptide can be detected using polypeptide-specific anti of a complex in the control reaction(s) but not in the reaction bodies, as described above. Bound agents can alternatively mixture containing the test compound indicates that the test be identified by comparing the relative electrophoretic compound interferes with the interaction of the test com mobility of polypeptides exposed to the test agent to the pound and its reaction partner. mobility of complexes that have not been exposed to the test 0121) To identify inhibitors, the GSNOR polypeptide agent. GSNO reductase activity can be measured by GSNO may be added to a cell along with the test agent, and then dependent NADH consumption as previously described (Liu checked for decreased activity. The gene encoding the agent et al., 2001). SNO levels can be measured by photolysis can be identified by numerous methods known to those of chemiluminescence (Liu et al., 2000b). skill in the art, for example, ligand panning, FACS Sorting, 0117. In one specific embodiment, a binding complex and expression cloning (see, e.g., Coligan et al., Current between a GSNOR polypeptide and test agent is isolated or Protocols in Immun., 1(2): Chapter 5 (1991)). As an alter detected in the reaction mixture. For example, the GSNOR native approach, labeled GSNOR polypeptide can be pho polypeptide or the test agent can be immobilized on a Solid toaffinity-linked with cell membrane or extract preparations phase, e.g., on a microtiter plate, by covalent or non that express the receptor molecule. Cross-linked material covalent attachments. Non-covalent attachment can be can be resolved by PAGE and exposed to X-ray film. The accomplished by coating the Solid Surface with a Solution of labeled complex containing the receptor can be excised, the GSNOR polypeptide and drying. Alternatively, an resolved into peptide fragments, and Subjected to protein immobilized antibody, e.g., a monoclonal antibody, Specific micro-Sequencing. The amino acid Sequence obtained from for the GSNOR polypeptide to be immobilized can be used micro-Sequencing can be used to design a Set of degenerate to anchor it to a Solid Surface. oligonucleotide probes to screen a cDNA library to identify the gene encoding the agent. 0118. The assay can be performed by adding the non immobilized component (e.g., the polypeptide or test agent), 0122 One method of identifying an agent (i.e., an inhibi which may be labeled by a detectable label, to the immo tor) which decreases the levels and/or activity of a GSNOR bilized component on the solid surface. When the reaction is comprises: (a) providing a GSNOR polypeptide or peptide; complete, the non-reacted components can be removed, e.g., (b) contacting the GSNOR polypeptide or peptide with a test by Washing, and complexes anchored on the Surface can be agent; and (c) detecting the presence of an agent that binds US 2005/0014697 A1 Jan. 20, 2005 to the GSNOR polypeptide or peptide, wherein the binding alleviating one or more Symptoms) for medical conditions. agent down-regulates the level and/or activity of the AS non-limiting examples, medical conditions that relate to GSNOR polypeptide or peptide. One method of identifying decreased GSNOR levels and increased NO synthesis and/or an agent (i.e., an activator) which decreases the levels and/or increased NO levels include degenerative diseases (e.g., activity of a GSNOR comprises: (a) providing a GSNOR Parkinson's disease, Alzheimer's disease, ALS), Stroke (e.g., polypeptide or peptide; (b) contacting the GSNOR polypep ischemic stroke), and proliferative diseases (e.g., cancers, tide or peptide with a test agent; and (c) detecting the tumors, dysplasias, and neoplasms). Medical conditions that presence of an agent that binds to the GSNOR polypeptide relate to increased GSNOR levels and decreased SNO levels or peptide, wherein the binding agent up-regulates the level (e.g., SNO-Hb) include, for example, vascular disorders and/or activity of the GSNOR polypeptide or peptide. Such as hypertension (e.g., pulmonary hypertension), heart 0123. In addition, one method of identifying an agent disease, heart failure, heart attack, atherOSclerosis, resteno (i.e., inhibitor) which decreases S-nitrosylation comprises: sis, asthma, and impotence. Medical conditions that relate to (a) culturing a first cell capable of S-nitrosylation in a media decreased GSNOR levels and increased SNO levels (e.g., comprising a test agent; (b) culturing a second cell capable SNO-Hb) include, for example, tissue injury (e.g., liver, of S-nitrosylation in a media without the test agent, wherein kidney, muscle, and or lymph tissue) or death due to the Second cell is similar to the first cell except for lacking Systemic infections Such as bacteremia, Sepsis, Systemic the test agent; and (c) comparing S-nitrosylation in both the inflammatory response Syndrome, neonatal Sepsis, cardio first cell and the second cell wherein the agent which inhibits genic Shock, or toxic Shock. S-nitrosylation is identified when S-nitrosylation is less in 0128. Other conditions that relate to decreased GSNOR the first cell than in the second cell. One method of identi levels and increased SNO levels (e.g., SNO-Hb) include, for fying an agent (i.e., activator) which increases S-nitrosyla example, inflammatory disease Such as colitis, inflammatory tion comprises: (a) culturing a first cell capable of S-nitrosy bowel disease, rheumatoid arthritis, Osteoarthritis, psoriatic lation in a media comprising a test agent; (b) culturing a arthritis, infectious arthritis, ankylosing spondylitis, ten Second cell capable of S-nitrosylation in a media without the donitis, burSitis, vasculitis, fibromyalgia, polymyalgia rheu test agent, wherein the Second cell is similar to the first cell matica, temporal arteritis, giant cell arteritis, polyarteritis, except for lacking the test agent; and (c) comparing S-ni HIV-associated rheumatic disease Syndromes, Systemic trosylation in both the first cell and the second cell wherein lupus, erythematosus, gout, and pseudogout (calcium pyro the agent which increaseS S-nitrosylation is identified when phosphate dihydrate crystal deposition disease). In addition, S-nitrosylation is greater in the first cell than in the second decreased GSNOR levels and increased SNO levels (e.g., cell. SNO-HB) are associated with hypotension (e.g., in associa 0124) Any compound or other molecule (or mixture or tion with anesthesia), and tissue damage and death due to aggregate thereof) can be used as a test agent. In Some Shock (e.g., endotoxic or Septic shock), as shown herein embodiments, the agent can be a Small peptide, or other below. Small molecule produced by combinatorial Synthetic meth 0129. In one aspect, the pharmaceutical composition ods known in the art. In other embodiments, the agent can includes a reagent of the invention, which can be adminis be a Soluble receptor, receptor agonist, antibody, or antibody tered alone or in combination with the Systemic or local fragment. An agent can be a nucleic acid, Such as an co-administration of one or more additional agents. A antisense molecule or interfering RNA molecule which reagent of the invention can include a GSNOR polypeptide binds to a GSNOR transcript or gene Sequence. Agents can (e.g., SEQ ID NO:17-SEQ ID NO:21), peptide (e.g., a be antibodies including, without limitation, poly- and mono peptide encoded by SEQ ID NO:9-SEQ ID NO:14), an clonal antibodies and antibody fragments, Single-chain anti anti-GSNOR antibody or antibody fragment, a GSNOR bodies, anti-idiotypic antibodies, and chimeric or humanized mimetic (e.g., peptide, Small molecule, or anti-idiotype versions of Such antibodies or fragments, as well as human antibody), a GSNOR antisense or iRNA sequence, or frag antibodies and antibody fragments. ment, derivative, or modification thereof, or another 0.125 For use with the invention, an inhibitor may be a GSNOR inhibitor or activator. Additional agents for admin closely related protein, for example, a mutated form of the istration may include preservatives, anti-StreSS medications, GSNOR polypeptide that recognizes one or more substrates phosphodiesterase inhibitors, iNOS inhibitors, B-agonists, but lackS enzymatic activity. An inhibitor can be an antisense and anti-pyrogenics. Suitable phosphodiesterase inhibitors RNA or DNA construct prepared using antisense technology include, but are not limited to, rollipram, cilomilast, roflu (described above). Inhibitors can include small molecules milast, Viagra(E) (sildenifil citrate), Cialis(R (tadala?il), Lev that bind to the substrate binding site or other relevant itra(E) (vardenifil). Suitable f-agonists include, but are not binding site of the GSNOR polypeptide, thereby blocking limited to, isoproterenol, metaproterenol, terbutaline, the normal biological activity. Examples of Small molecules albuterol, bitolterol, ritodrine, dopamine, and dobutamine. include, but are not limited to, Small peptides or peptide-like 0130 Suitable iNOS inhibitors include, but are not lim molecules, preferably Soluble peptides, and Synthetic non ited to, Type II iNOS inhibitors, specific NOS inhibitors, and peptidyl organic or inorganic compounds. These Small mol non-specific NOS inhibitors. Non-limiting examples of NOS ecules can be identified by any one or more of the Screening inhibitors include L-N(6)-(1-iminoethyl)lysine tetrazole assays discussed hereinabove and/or by any other Screening amide (SC-51), aminoguanidine (AG), S-methilisourea techniques which are known to those skilled in the art. (SMT); S-(2-Aminoethyl)isothiourea; 2-Amino-5,6-dihy dro-6-methyl-4H-1,3-thiazine (AMT); L-2-Amino-4-(guan 0.126 Pharmaceutical Compositions idiooxy)butyric acid (L-Canavanine Sulphate); S-Ethyl 0127. The invention further encompasses pharmaceutical isothiourea (EIT): 2-Iminopiperidine, compositions useful as prophylaxes or treatments (e.g., for S-Isopropylisothiourea; and 1,4-phenylenebis(1,2- US 2005/0014697 A1 Jan. 20, 2005

ethanediyl)-diisothiourea (PBIT). Preferred NOS inhibitors the active compound into a sterile vehicle that contains a for use with the invention are N-3-(aminomethyl)benzyl basic dispersion medium and the required other ingredients acetamidine (1400W); N6-(1-Iminoethyl)-L-lysine (L-NIL); from those enumerated above. In the case of Sterile powders monomethyl arginine (e.g., for non-specific inhibition); for the preparation of Sterile injectable Solutions, methods of 7-Nitroindazole (e.g., for inhibition of NNOS in brain tis preparation are vacuum drying and freeze-drying that yields Sue), etc. a powder of the active ingredient plus any additional desired 0131) A pharmaceutical composition of the invention is ingredient from a previously Sterile-filtered Solution thereof. preferably formulated to be compatible with its intended 0135) Oral compositions generally include an inert dilu route of administration. Examples of routes of administra ent or an edible carrier. They can be enclosed in gelatin tion include oral and parenteral, e.g., intravenous, intrader capsules or compressed into tablets. For the purpose of oral mal, Subcutaneous, inhalation, transdermal (topical), trans therapeutic administration, the active compound can be mucosal, and rectal administration. Solutions or Suspensions incorporated with excipients and used in the form of tablets, used for parenteral, intradermal, or Subcutaneous application troches, or capsules. Oral compositions can also be prepared can include the following components: a Sterile diluent Such using a fluid carrier for use as a mouthwash, wherein the as water for injection, Saline Solution, fixed oils, polyethyl compound in the fluid carrier is applied orally and Swished ene glycols, glycerine, propylene glycol or other Synthetic and expectorated or Swallowed. Pharmaceutically compat Solvents, antibacterial agents Such as benzyl alcohol or ible binding agents, and/or adjuvant materials can be methyl parabens, antioxidants Such as ascorbic acid or included as part of the composition. The tablets, pills, Sodium bisulfite, chelating agents Such as ethylenediamine capsules, troches and the like can contain any of the fol tetraacetic acid; bufferS Such as acetates, citrates or phos lowing ingredients, or compounds of a similar nature: a phates, and agents for the adjustment of tonicity Such as binder Such as microcrystalline cellulose, gum tragacanth or Sodium chloride or dextrose. The pH can be adjusted with gelatin; an excipient Such as Starch or lactose, a disintegrat acids or bases, Such as hydrochloric acid or Sodium hydrox ing agent Such as alginic acid, Primogel, or corn Starch; a ide. The parenteral preparation can be enclosed in ampoules, lubricant Such as magnesium Stearate or Sterotes, a glidant disposable Syringes or multiple dose Vials made of glass or Such as colloidal Silicon dioxide, a Sweetening agent Such as plastic. Sucrose or Saccharin; or a flavoring agent Such as pepper mint, methyl Salicylate, or orange flavoring. 0132) Pharmaceutical compositions suitable for inject able use include sterile aqueous Solutions (where water 0.136 For administration by inhalation, the compounds Soluble) or dispersions and Sterile powders for the extem are delivered in the form of an aeroSol Spray from pressured poraneous preparation of Sterile injectable Solutions or dis container or dispenser that contains a Suitable propellant, persion. For intravenous administration, Suitable carriers e.g., a gas Such as carbon dioxide, or a nebulizer. For include physiological Saline, bacteriostatic water, Cremo transmucosal or transdermal administration, penetrants phor ELTM (BASF, Parsippany, N.J.) or phosphate buffered appropriate to the barrier to be permeated are used in the saline (PBS). In all cases, the composition must be sterile formulation. Such penetrants are generally known in the art, and should be fluid to the extent that easy syringability and include, for example, for transmucosal administration, exists. It should be stable under the conditions of manufac detergents, bile Salts, and fusidic acid derivatives. Transmu ture and Storage and should be preserved against the con cosal administration can be accomplished through the use of taminating action of microorganisms. Such as bacteria and nasal Sprays or Suppositories. For transdermal administra fungi. tion, the active reagents are formulated into ointments, Salves, gels, or creams as generally known in the art. The 0133. The carrier can be a solvent or dispersion medium reagents can also be prepared in the form of Suppositories containing, for example, water, ethanol, polyol (for example, (e.g., with conventional Suppository bases Such as cocoa glycerol, propylene glycol, and liquid polyethylene glycol, butter and other glycerides) or retention enemas for rectal and the like), and Suitable mixtures thereof. The proper delivery. fluidity can be maintained, for example, by the use of a coating Such as lecithin, by the maintenance of the required 0.137 In one embodiment, the active reagents are pre particle size in the case of dispersion and by the use of pared with carriers that will protect against rapid elimination Surfactants. Prevention of the action of microorganisms can from the body. For example, a controlled release formulation be achieved by various antibacterial and antifingal agents, can be used, including implants and microencapsulated for example, parabens, chlorobutanol, phenol, ascorbic acid, delivery Systems. Biodegradable, biocompatible polymers thimerosal, and the like. In many cases, it will be preferable can be used, Such as ethylene Vinyl acetate, polyanhydrides, to include isotonic agents, for example, Sugars, polyalcohols polyglycolic acid, collagen, polyorthoesters, and polylactic Such as manitol, Sorbitol, Sodium chloride in the composi acid. Methods for preparation of such formulations will be tion. Prolonged absorption of the injectable compositions apparent to those skilled in the art. The materials can also be can be brought about by including in the composition an obtained commercially from Alza Corporation and Nova agent which delays absorption, for example, aluminum Pharmaceuticals, Inc. Liposomal Suspensions (including monoStearate and gelatin. liposomes targeted to infected cells with monoclonal anti 0134 Sterile injectable solutions can be prepared by bodies to viral antigens) can also be used as pharmaceuti incorporating the active reagent (e.g., polypeptide, peptide, cally acceptable carriers. These can be prepared according to antibody, or antibody fragment) in the required amount in an methods known to those skilled in the art, for example, as appropriate Solvent with one or a combination of ingredients described in U.S. Pat. No. 4,522,811. enumerated above, as required, followed by filtered Steril 0.138. Additionally, Suspensions of the active compounds ization. Generally, dispersions are prepared by incorporating may be prepared as appropriate oily injection Suspensions. US 2005/0014697 A1 Jan. 20, 2005

Suitable lipophilic solvents or vehicles include fatty oils, tidic molecules may also be Synthesized with D-amino acids Such as Sesame oil, or Synthetic fatty acid esters, Such as to increase resistance to enzymatic degradation. In Some ethyl oleate, triglycerides, or liposomes. Non-lipid polyca cases, the composition can be co-administered with one or tionic amino polymerS may also be used for delivery. more Solubilizing agents, preservatives, and permeation Optionally, the Suspension may also include Suitable Stabi enhancing agents. The composition can include a preserva lizers or agents to increase the Solubility of the compounds tive or a carrier Such as proteins, carbohydrates, and com and allow for the preparation of highly concentrated Solu pounds to increase the density of the pharmaceutical com tions. position. The composition can also include isotonic Salts and 0.139. It is especially advantageous to formulate oral or redox-control agents. In addition, the pharmaceutical com parenteral compositions in dosage unit form for ease of positions can be included in a container, pack, or dispenser administration and uniformity of dosage. Dosage unit form together with instructions for administration. as used herein refers to physically discrete units Suited as 0144. In various embodiments of the invention, Suitable unitary dosages for the Subject to be treated; each unit in Vitro or in Vivo assays are performed to determine the containing a predetermined quantity of active reagent cal effect of a specific reagent and whether its administration is culated to produce the desired therapeutic effect in associa indicated for treatment of the affected tissue. Reagents for tion with the required pharmaceutical carrier. The Specifi use in therapy may be tested in Suitable animal model cation for the dosage unit forms of the invention are dictated Systems including, but not limited to rats, mice, chicken, by and directly dependent on the unique characteristics of cows, monkeys, rabbits, and the like, prior to testing in the active reagent and the particular therapeutic effect to be human Subjects. Similarly, for in Vivo testing, any of the achieved, and the limitations inherent in the art of com animal model System known in the art may be used prior to pounding Such an active agent for the treatment of individu administration to human Subjects. als. 0145 Therapeutic Methods 0140 Nucleic acid molecules encoding a proteinaceous 0146 The invention also encompasses methods of pre agent can be inserted into Vectors and used as gene therapy venting or treating (e.g., alleviating one or more Symptoms vectors. Gene therapy vectors can be delivered to a Subject of) medical conditions through use of one or more of the by, for example, intravenous injection, local administration disclosed reagents. A reagent for use with these methods can (see U.S. Pat. No. 5,328,470) or by stereotactic injection include a GSNOR polypeptide (e.g., SEQ ID NO:17-SEQ (see e.g., Chen et al. (1994) PNAS 91:3054-3057). The ID NO:21) or peptide (e.g., peptide encoded by SEQ ID pharmaceutical preparation of the gene therapy vector can NO:9-SEQ ID NO:14), an anti-GSNOR antibody or anti include the gene therapy vector in an acceptable diluent, or body fragment, a GSNOR mimetic (e.g., peptide, Small can comprise a slow release matrix in which the gene molecule, or anti-idiotype antibody), a GSNOR antisense or delivery vehicle is imbedded. Alternatively, where the com iRNA sequence, or a fragment, derivative, or modification plete gene delivery vector can be produced intact from thereof, or another GSNOR inhibitor or activator. As dis recombinant cells, e.g., retroviral or adenoviral vectors, the cussed above, altered levels of GSNORs, NO, and SNOS pharmaceutical preparation can include one or more cells have been implicated in various medical conditions. Thus, that produce the gene delivery System. methods are disclosed for treating or preventing a disease or 0.141. In one embodiment, the reagent is administered in disorder involving altered or unwanted levels of GSNORS, a composition comprising at least 90% pure reagent. Pref NO, and/or SNOS, or GSNOR activity, by administering to erably the reagent is formulated in a medium providing a Subject a therapeutically effective amount of at least one maximum Stability and the least formulation-related Side molecule that modulates the activity or levels thereof. effects. In addition to the reagent, the composition of the 0147 In subjects with deleteriously high levels of invention will typically include one or more protein carrier, GSNOR or GSNOR activity), modulation may be achieved, buffer, isotonic Salt and Stabilizer. In Some instances, the for example, by administering a reagent that disrupts or reagent can be administered by a Surgical procedure down-regulates GSNOR function, or decreases GSNOR implanting a catheter coupled to a pump device. The pump levels (e.g., through decreased production or increased deg device can also be implanted or be extracorporally posi radation or instability). These reagents may include anti tioned. Administration of the reagent can be in intermittent GSNOR antibodies or antibody fragments, GSNOR anti pulses or as a continuous infusion. Sense, iRNA, or Small molecules, or other inhibitors, alone 0142. A reagent can be administered in a manner as to or in combination with other agents (e.g., phosphodiesterase pass through or by-pass the blood-brain barrier. Methods for inhibitors) as described in detail herein. allowing factors to pass through the blood-brain barrier include minimizing the size of the factor, providing hydro 0.148. In subjects with deleteriously low levels of phobic factors which may pass through more easily, conju GSNOR or GSNOR activity (and concomitantly high levels of SNOs and or NO), modulation may be achieved, for gating the protein reagent or other agent to a carrier mol example by administering a reagent that activates or ecule that has a Substantial permeability coefficient acroSS enhances GSNOR function, increases GSNOR levels (e.g., the blood brain barrier (see, e.g., U.S. Pat. No. 5,670.477). through increased production or Stability or decreased deg Alternatively, devices can be used for injection to discrete radation), or decreases SNO or NO levels. These reagents areas of the brain (see, e.g., U.S. Pat. Nos. 6,042,579; may include GSNOR polypeptides or peptides, GSNOR 5,832,932; and 4,692,147). mimetics (e.g., peptides, Small molecules, or anti-idiotype 0143 Modifications can be made to the agents to affect antibodies), GSNOR expression vectors, or other activators, Solubility or clearance of an amino acid Sequence (e.g., alone or in combination with anti-SNO antibodies or anti polypeptide, peptide, antibody, or antibody fragment). Pep body fragments, or NOS inhibitors or NO scavengers. US 2005/0014697 A1 Jan. 20, 2005

0149 Pharmaceutical preparations suitable for adminis ticular, increases in plasma SNO-albumin are associated tration of these reagents are described above. Additional with high blood pressure and predict adverse cardiovascular agents for administration may include preservatives, anti outcome (Massy Z A, et al., J. Am. Soc. Nephrol. stress medications, phosphodiesterase inhibitors, iNOS 2004;15:470-476). New genetic evidence makes it clear that inhibitors, and anti-pyrogenics as described in detail herein. SNOS play essential roles in the vasculature (Liu L, et al., 0150. In one embodiment, the modulatory method of the 2004, Cell 116:617-628). Taken together, these studies Sug invention involves contacting a cell with an agent that gest that SNO-albumin may dispense NO bioactivity in modulates one or more of the activities of GSNOR or NOS states characterized by NO deficiency. They also indicate activity. In another embodiment, the agent Stimulates or that cysteines in albumin and other key blood proteins Such inhibits the activity of the GSNOR or NOS signaling path as hemoglobin represent new therapeutic targets. way. These modulatory methods can be performed in vitro 0155 Inhaled NO increases circulating levels of SNO (e.g., by culturing the cell with the agent) or, alternatively, albumin, but it does not reveal the mechanism by which in Vivo (e.g., by administering the agent to a Subject). AS SNO-albumin is made, where in the circulation it is pro Such, the invention provides methods of treating an indi duced, or how much NO actually takes this path. Inhaled NO vidual afflicted with a disorder, as described above. In one first accumulates in the airways and lung parenchyma in the embodiment, the method involves administering a reagent, form of SNOs and other complexes with proteins, and then or combination of reagents that modulate (e.g., up-regulate leaches into the blood (Simon D I, et al., Proc. Natl. Acad. or down-regulate) GSNOR or NO levels or activity. Sci USA 1996:93:4736-4741; McCarthy T J, et al., Nucl. 0151. As demonstrated herein below, inhibitors of Med. Biol. 1996:23:773-777; McCarthy T J, et al., Nuc. GSNOR may be used as a means to improve B-adrenergic Med. Biol. 1996:23:773-777). Salient features of this pro signaling. In particular, inhibitors of GSNOR alone or in ceSS are not currently known, including the form in which combination with 3-agonists could be used to treat or protect NO bioactivity enters the blood over time and the flux against heart failure, or other vascular disorderS Such as through SNO-albumin. hypertension and asthma. GSNOR inhibitors can be used to 0156. In albumin, both a hydrophobic pocket and bound modulate G protein coupled receptors (GPCRs) by potenti metals (copper and perhaps heme) can facilitate S-nitrosy ating GS G-protein, leading to Smooth muscle relaxation lation by NO, while hemoglobin has several channels (e.g., airway and blood vessels), and by attenuating Gd through which it can react with NO, nitrite, or GSNO to G-protein, and thereby preventing Smooth muscle contrac produce SNO-Hb (FIG. 2D; see above). It is believed that tion (e.g., in airway and blood vessels). hemoglobin out-competes albumin for NO. However, this outcome appears not to be absolute, as the relative yield of 0152 SNO-Based Diagnostics and Therapeutics NO bound to hemoglobin in bioactive form appears 0153. The invention further encompasses methods of inversely proportional to the rate and amount of NO admin diagnosis and treatment based on measurement or alteration, istered and exhibits a plateau at low micromolar levels (Gow respectively, of SNO levels in a patient in accordance with AJ, Stamler JS, Nature 1998;391: 169-173). By comparison, the methods disclosed herein (See, e.g., J. Stamler. Circ. only nanomolar levels are required for vasoregulation. With Res., 2004; 94: 414-417). One physiological benefit of the high amounts of NO administered clinically and by other SNOs as compared to NO is their resistance to inactivation experiments (Ng ESM, et al., Circ. Res. 2004;94:559-565) it by Superoxide (O2). In damaged tissues, increased O can appears that hemoglobin slows the production of SNO by react with NO to produce toxic peroxynitrite. But the lodging NO on the C-hemes and eliminating it as nitrate amounts of peroxynitrite that accrue depend at a minimum (Gow AJ, Stamler JS, Nature 1998;391: 169-173; Gow AJ, on the relative rates of NO/O production: NO2O in fact et al., Proc. Natl. Acad. Sci. USA 1999;96:9027-9032; favors production of SNO (Schrammel A, et al., Biol. Med. Luchsinger B P, et al., Proc. Natl. Acad. Sci. USA. 2003:34:1078-1088). Researchers have demonstrated that 2003;100:461–466; Napoli C, et al., Proc. Natl. Acad. Sci. Superoxide, generated by ischemia/reperfusion (I/R) in USA 2002;99:1689-1694; Kirima K, et al., Am. J. Physiol. mesenteric vessels, facilitates the synthesis of SNO-albumin Heart Circ. Physiol. 2003:285:H589-H596; Gow AJ, et al., (Ng ESM, et al., Circ. Res. 2004:94:559-565). SNO-albumin Proc. Natl. Acad. Sci. USA 1999;96:9027-9032; Romeo AA, is known to protect tissues against I/R-induced damage et al., J. Am. Chem. Soc. 2003; 125:14370-14378; Cannon (Hallstrom S, Circulation. 2002; 105:3032-3038). Thus, RO 3rd, et al., J. Clin. Invest. 2001; 108:279-287). there appears to be a means to exploit Superoxide to preserve NO bioactivity. A remaining problem is that the oxidative O157. In accordance with the present invention, preferred damage caused by I/R impairs NO production. It is therefore diagnostic assays for SNO levels preserve the physiological milieu, and employ Standards that best emulate the mol noteworthy that it has also been shown that the thiols of ecules being measured (see, Stamler, J. S., 2004, Cir. Res. albumin can transport inhaled NO to the gut and subserve 94:414-417). Diagnostic assays for determining plasma lev relaxation of blood vessels (Ng ESM, et al., Circ. Res. els of SNO levels are preferred. Particularly preferred are 2004:94:559-565). photolysis/chemiluminescence-based methods as disclosed 0154 Although relatively high concentrations of SNO herein below (see also J. S. Stamler and M. Feelisch, in albumin are required to increase blood flow, the amounts Methods in Nitric Oxide Research, J. S. Stamler and M. that attenuate vasoconstriction are in the physiological Feelisch, Eds. Wiley, Chichester, UK, 1996, pp. 521-539; range. Furthermore, as evident from the accrual of SNO Stamler, J. S., et al., 1997, Science 276, 2034-2037; Man albumin in Some hypertensive and uremic patients, it is the nick, J. B., et al., 1999, Science 284, 651-654, Buga, G. M., efficiency of NO group release that determines bioactivity et al., 1998, Am. J. Physiol. 275, R1256-R 1264). Also (Tyurin VA, et al., Circ. Res. 2001;88: 1210-1215; Massy Z preferred is the use of Stable nitroSothiol Standards for, e.g., A, et al., J. Am. Soc. Nephrol. 2004;15:470-476). In par SNO-albumin or SNO-Hb measurements, in conjunction US 2005/0014697 A1 Jan. 20, 2005

with Such methods. In this way, the range of SNO bond 0.161 In subjects with deleteriously low levels of SNOs, quantum yields can be covered. In addition, dose depen modulation may be achieved, for example by administering dence and reproducibility of assays can be checked to ensure a reagent (e.g., via intravenous administration) that up against Systematic artifacts. For use with the invention, any regulates SNO levels. This up-regulation may be achieved biological Sample can be used to measure SNO levels, through increasing production or Stability or decreasing although blood Samples are preferred (e.g., Serum, plasma, degradation of SNOS, or by decreasing levels or activity of or whole blood), and plasma samples are particularly pre GSNOR. Exemplary reagents include anti-GSNOR antibod ferred. ies or antibody fragments, GSNOR antisense, iRNA, small molecules, and other GSNOR inhibitors, as well as SNO 0158. In one aspect, the diagnostic or monitoring method activators, alone or in combination with other agents (e.g., of the invention comprises (a) measuring levels of SNOs in phosphodiesterase inhibitors) as described in detail herein. a biological Sample from a patient (e.g., plasma levels); (b) Such methods can be used for medical conditions associated comparing the levels of SNOs in the biological sample to with undesirably low levels of SNOs. As examples, levels in a control Sample, and (c) determining if the levels decreased levels of SNO-Hb are associated with heart fail of SNOs in the biological sample are higher than the levels ure, diabetes, and other conditions (e.g., oxygen deficit of SNOs in the control sample. This method can be used for conditions) as described herein, while decreased levels of diagnosing or monitoring medical conditions (or the efficacy SNO-albumin are associated with renal disease Such as of treatments of medical conditions) associated with uremia and other conditions having defective platelet-ag increased or otherwise deleteriously high levels of SNOs. gregation. For example, increased levels of SNO-Hb are associated with hypotension, Sepsis, and other conditions as described in detail herein, while increased levels of SNO-albumin are EXAMPLES asSociated with hypertension, preeclampsia, and other con 0162 The examples presented herein below describe the ditions with platelet-aggregation. generation of GSNOR-deficient (GSNOR) mice through 0159. In another aspect, the diagnostic or monitoring homologous recombination, and the response of the mice to method comprises (a) measuring levels of SNOs in a bio a nitrosative challenge induced by both LPS and cecal logical Sample from a patient (e.g., plasma levels); (b) ligation-Sepsis. The bacterial endotoxin model of Shock was comparing the levels of SNOs in the biological sample to used in the disclosed experiments, Since alternative models levels in a control sample; and (c) determining if the levels could obscure the elucidation of the specific roles of SNOS of SNOs in the biological sample are lower than the levels in governance of NO bioactivity. Abacterial model of sepsis of SNOs in the control sample. Such method can be used for was also used. The GSNOR-deficient animals exhibited diagnosing or monitoring medical conditions (or the efficacy Substantial increases in whole cell S-nitrosylation, tissue of treatments of medical conditions) associated with damage, and mortality following endotoxic or bacterial decreased or otherwise deleteriously low levels of SNOs. challenge. Further, GSNOR-/- mice showed increased For example, decreased levels of SNO-Hb are associated basal levels of SNOs in red blood cells and were hypotensive with heart failure, diabetes, and other conditions (e.g., under anesthesia. From the disclosed experiments, it was oxygen deficit conditions) as described herein, while determined that GSNOR is indispensable for SNO metabo decreased levels of SNO-albumin are associated with renal lism, for vascular homeostasis, and for Survival in endotoxic disease Such as uremia and other conditions having defective shock. It was further determined that SNOS regulate innate platelet-aggregation. immune and vascular function, and are actively cleared to ameliorate nitrosative StreSS. Accordingly, the results 0160 In accordance with the invention, the disclosed obtained herein have identified nitrosylation of cysteine methods can be used for preventing or treating (e.g., alle thiols as critical mechanism of NO function in both health viating one or more Symptoms of) medical conditions asso and disease. ciated with altered or deleterious levels of SNOs through use of one or more of the disclosed reagents. In Subjects with 0163 The examples are presented in order to more fully increased or deleteriously high levels of SNOs, modulation illustrate the preferred embodiments of the invention. These may be achieved, for example, by administering a reagent examples should in no way be construed as limiting the (e.g., via intravenous administration) that down-regulates Scope of the invention, as defined by the appended claims. SNO levels. This down-regulation may be achieved by decreasing production or increasing degradation or instabil Example 1 ity of SNOS, or by increasing activity or levels of GSNOR. Exemplary reagents include GSNOR polypeptides or pep Experimental Procedures tides, GSNOR mimetics (e.g., peptides, Small molecules, and anti-idiotype antibodies), GSNOR expression vectors, 0.164 Construction of a GSNOR Targeting Vector and other GSNOR activators, as well as anti-SNO antibodies 0.165 For the disclosed experimental procedures, results, or antibody fragments, small molecules, and other SNO and discussion, see also Liu et al., 2004, Cell 116:617-628, inhibitors, alone or in combination with other agents (e.g., which is incorporated herein by reference in its entirety. For NOS inhibitors or NO scavengers) as described in detail the primerS depicted herein, "Se' indicates Sense Strand; “as' herein. As examples, increased levels of SNO-Hb are asso indicates antisense Strand. Abacterial artificial chromosome ciated with hypotension, Sepsis, and other conditions as (BAC) library derived from genomic DNA of mouse strain herein described, while increased levels of SNO-albumin are 129SV/CJ7 (Invitrogen) was screened for the GSNOR gene asSociated with hypertension, preeclampsia, and other con by PCR with primers from exon 8 (MoADHI1001se, ditions with platelet-aggregation. For exceSS SNOS, treat 5'-gatggaagagtgtggagagtg; SEQ ID NO:1) and exon 9 ments can also include infusions of thiols or antioxidants. (MoADH1290 as, 5'-cagtctegattatgcacattcc, SEQ ID NO:2) US 2005/0014697 A1 Jan. 20, 2005

(Foglio and Duester, 1996). Two BAC clones were identified (Farmingdale, N.Y.): alanine aminotransferase (ALT), aspar (36c24 and 91m09), and subjected to restriction mapping tate aminotransferase (AST), creatine phosphokinase and Southern blot analysis with probes ex8-9 and ex2-3. The (CPK), urea nitrogen (BUN), creatinine, amylase, lipase, probes were generated from a mouse ADH III cDNA clone lactate dehydrogenase, alkaline phosphatase, total protein, (ATCC, GenBank accession number AAO08355) by PCR globulin, albumin, calcium, magnesium, Sodium, potassium, with primer pairs for exons 8-9 (MoADH1001 se, chloride, phosphorus, glucose, bilirubin, cholesterol, trig MoADH1290 as) and exons 2-3 (MoADH52se, 5'-gtgatcag lycerides, and OSmolality. gtgtaaggctgc, SEQ ID NO:3; MoADH295 as, 5'-ctgcct tcagctitcgtgac, SEQID NO:4), respectively. A Sac I fragment 0.175. The following parameters were measured with a containing exons 2-4 and a Hind III-BamH I fragment Pentra 60 C+ system of ABX Diagnostics (Montpellier, containing exons 7-9 were isolated from BAC clone 91m09 France): hemoglobin, hematocrit, mean corpuscular volume, and inserted 5' and 3' to the neomycin resistance gene (neo) and counts of erythrocytes, leukocytes, neutrophils, lym in the vector pPNT (Tybulewicz et al., 1991), respectively phocytes, monocytes, eosinophils, and platelets. (FIG. 1A). The resulting GSNOR targeting vector was confirmed by DNA sequencing and linearized by Not I. 0176 Levels of iron-nitrosyl hemoglobin and SNO-he moglobin/SNO-proteins in RBCs were measured by pho 0166 Generation of GSNOR Mice tolysis-chemiluminescence (McMahon et al., 2002). 0167 ES cells derived from 129sv mice were transfected 0177 Serum nitrate and nitrite were measured by capil with the linearized targeting vector and Selected for the lary electrophoresis (CE) (Zunic et al., 1999) with a P/ACE presence of neo and absence of the herpes simplex virus MDQ system (Beckman) and by chemiluminescence (Siev thymidine kinase (tk; Duke transgenic mouse facility). ers NO Analyzer). For CE, sera were diluted (1:10) with Selected ES clones were first screened for homologous water and filtered through a 5 kDa cut-off membrane. recombination by PCR with a neo-derived primer (Neo3'se, Electrophoresis of the filtered Samples and of nitrate and 5'-tctgacgagttcttctgagg, SEQ ID NO:5) and a GSNOR nitrite Standards was carried out in a neutral capillary with primer (GSNOR3'as, 5'-cagttgactgtcaatgaactgg, SEQ ID Tris buffer (100 mM, pH 8.0), and monitored by absorbance NO:6) external to the homologous region in the targeting at 214 nm. Nitrite concentrations are higher when measured vector (FIG. 1A). This PCR reaction produced a 2.7 kb by CE than by chemiluminescence (Zunic et al., 1999), but DNA fragment only in the cells with the targeted disruption. no relative differences between CE and chemiluminescence Recombinant clones were further screened by Southern were observed. analyses of Sac I- and Xba I-digested genomic DNA with probes ex2-3 and ex8-9, respectively. The correctly dis 0178 Histology rupted allele produced a 7.3 kb Sac I and a 1.8 kb Xba I fragment. In contrast, the wild-type allele produced a 5.5 kb 0179 Organs were fixed with phosphate-buffered forma Sac I and a 2.4 kb Xba I fragment (FIG. 1A). lin and embedded in paraffin. Tissue Sections, 5-6 um thick, were stained with hematoxylin and eosin (H&E). The 0.168. Two correctly targeted ES clones with normal Stained Sections were examined by light microscopy by a karyotype were used independently to generate chimeric board certified veterinary pathologist. Apoptosis was mice. These were subsequently bred with C57BL/6 mice to assessed by TUNEL assay. produce F1 heterozygotes. The F1 mice were either mated with each other to produce F2 GSNOR mice or further 0180 LPS Treatment backcrossed with C57BL/6 mice. Two independent GSNORT mouse lines from the two ES clones were 0181 LPS (E. coli, serotype 026:B6, Sigma) at a dosage established after both Seven and ten consecutive backcrosses of 150,000 endotoxin units/g (EU/g) was injected intraperi with C57BL/6 mice. All mice were fed with standard mouse toneally into C57BL/6 and GSNOR mice. The mice were chow and housed in a pathogen-free facility. matched for age (11-12 weeks old), gender, and weight. LPS used for the males was lot number 050K4117 (15 million 0169. GSNOR Activity EU/mg) and LPS used for the females was lot number 0170 GSNO reductase activity was measured by GSNO 101K4080 (3 million EU/mg). Studies were done in 45 dependent NADH consumption as described previously (Liu additional male mice (22 wild-type and 23 GSNOR) administered lot number 101K4080. This ensured that gen et al., 2001). der and strain differences did not result from a batch effect. 0171 Blood Pressure Phosphate-buffered saline (PBS, 20 ul/g) was injected in 0172 Mice aged 6-8 months were anesthetized by a controls. In additional Sets of experiments, LPS-challenged combination of ketamine (70 mg/kg), xylazine (9 mg/kg), GSNOR mice were injected subcutaneously with the and urethane (1 mg/g). Mean arterial pressure was measured iNOS inhibitor 1400W (1 lug/g, Cayman) or PBS (10 ul/g). through a catheter inserted in the right carotid artery. Blood Injections were performed at 6, 24 and 30 hours after LPS, preSSure was also measured in conscious mice by a com or at 24, 42 and 48 hours after LPS. puterized tail-cuff system (Krege et al., 1995). Values shown 0182 Cecal Ligation and Puncture are the means of daily readings on four consecutive dayS. 0183 Female mice aged 3 months were anesthetized with 0173 Blood Chemistry, Cell Counts, Nitrite, Nitrate and ketamine (150 mg/kg) and Xylazine (10 mg/kg). The cecum S-nitrosothiols was ligated below the ileocecal valve, and punctured once 0.174 Blood was obtained by cardiac puncture after ani on the anti-mensenteric border with a 26-gauge needle. After mals were euthanized by CO inhalation. The following Surgery, the mice were Subcutaneously injected with 0.5 ml Serum chemistries were quantified by Antech Diagnostics of normal Saline. US 2005/0014697 A1 Jan. 20, 2005

0184 Septic Shock in Humans 0193 Phenotype 0185. Twelve consecutive adult patients with septic 0194 Heterozygous males and females were bred under pathogen-free conditions. This produced 31 (25%) wild shock in the Duke University Medical Center (DUMC) ICU type, 61 (50%) heterozygous and 30 knockout (25%) mice were enrolled. Septic Shock was defined according to the at weaning. Thus, the inheritance of the wild-type and American College of Cardiology/Society of Critical Care disrupted GSNOR gene followed the expected Mendelian Medicine guidelines (1992). The presence of gram-negative ratio. bacteremia within 72 hours of enrollment was ascertained from the medical records. The control group consisted of 12 healthy volunteers. Radial arterial and central venous blood TABLE 1. samples were collected for analysis of RBC NO content Growth and Reproduction of GSNOR and wild-type mice (McMahon et al., 2002). Informed consent was obtained, and the study was approved by the DUMC Internal Review Body Weight (g)" Board. Genotype Male Female Litter Size C57BL/6 27.9 O.S 21.7 0.4 6.4 O.6 0186 Liver SNO GSNOR / 1 26.8 0.5 22.7 - 0.6 6.2 O.4 0187 Liver homogenates were prepared in lysis buffer GSNOR / 2 26.8 0.5 23.4 - 0.5 6.5 + 0.4 (20 mM Tris-HCl, pH 8.0, 0.5 mM EDTA, 100 uM dieth GSNOR and C57BL/6 mice were raised on a standard mouse diet in the ylenetriamine pentaacetic acid, 0.1% NP-40 and 1 mM same animal facility. phenylmethylsulfonyl fluoride). SNO levels in the total Values are mean E SD of 80-day-old mice (n = 18-29). lysate and in a fraction filtered through a 5 kDa cut-off Values are meant SD at weaning (n = 16-32). ultrafiltration membrane (low-mass SNO) were measured by 0195 The GSNOR-deficient mice did not show a sur photolysis-chemiluminescence (Liu et al., 2000b) and nor vival disadvantage under these conditions. GSNOR mice malized for protein content. reproduced litters with a size and frequency Similar to 0188 Statistical Analysis C57BL/6 mice (FIG. 1F). They developed normally and weighed the same as C57BL/6 mice (FIG.1F). Histological 0189 Survival data on day 6 after LPS treatment were examination of 4 wild-type (2 males, 2 females) and 4 analyzed by both the X test and the Fisher exact test of GSNOR mice (2 males, 2 females) showed no gross contingency tables, with Similar results. Blood pressure, morphological or histological difference between the two SNO levels and serum chemistries were analyzed with the mouse Strains in any of the tissues Studied. This included Student's t-test or with the nonparametric Mann-Whitney brain, heart, lung, liver, kidney, Spleen, thymus, mesenteric teSt. lymph node, Salivary gland, gastrointestinal tract, pancreas, testis, ovary, uterus, and urinary bladder. Blood cell counts Example 2 and serum chemistries were normal in GSNOR mice (see below). Results 0196 Blood Pressure and Basal SNO 0190. Generation of GSNOR Mice 0197) emodynamic responses to SNOS, although the 0191) The GSNOR gene includes nine exons (Foglio and mechanism has not been unexplained (Travis et al., 1997). Duester, 1996); exons 5 and 6 encode most of the coenzyme AS shown herein, blood preSSure was much lower in binding domain of GSNOR (Yang et al., 1997). A targeting GSNOR mice than in the wild-type mice (P<0.001) when vector was constructed with GSNOR genomic DNA. This anesthetized with urethane (FIG. 2A). In contrast, blood was used to replace exons 5 and 6 with a neomycin resis pressure in conscious GSNOR mice did not differ from tance gene (neo) through homologous recombination in the controls (FIG. 2B). mouse (129Sv) embryonic stem (ES) cells (FIG. 1A). 0198 Previous studies have determined that most of the Homologous recombination on both sides flanking the tar NO in blood is found in red blood cells (RBCs) as iron geted region was confirmed in four ES clones. Southern blot nitrosyl hemoglobin and SNO-hemoglobin (Jia et al., 1996; analyses were performed with probes Specific to exons 2-3 Kirima et al., 2003; McMahon et al., 2002; Milsom et al., and exons 8-9, respectively. As further confirmation, PCR 2002). Here it is shown that levels of SNO-Hb (RBC-SNO) was performed to specifically identify the disrupted allele were higher in unanesthetized GSNOR mice than in (FIG. 1B). wild-type mice (P<0.05). Yet, levels of iron nitrosyl hemo globin did not differ between wild-type and GSNOR 0.192 Two mouse lines with the targeted disruption were (FIG. 2C). Thus, the experiments disclosed herein indicate independently generated from two of the ES clones (FIG. that GSNOR deficiency caused increases in basal SNO, and 1C). Southern hybridization with a probe specific to exons predisposed mice to disregulation of blood pressure. Endot 8-9 showed that GSNOR mice included only a single OXin treatment precluded accurate measurement of blood mutant (1.8 kb) fragment that resulted from recombination. preSSure due to diminished and irregular tail blood flow in These mice were backcrossed consecutively to C57BL/6 un-anesthetized mice and low blood pressure in anesthetized mice a total of seven-ten times. GSNO reductase activity CC. was absent in both tail and tissues of GSNOR mice (FIGS. 1D and 1E). The activity in heterozygous 0199 Mortality from Endotoxic Shock (GSNOR") mice was roughly half that in wild-type litter 0200 AS demonstrated herein, LPS-induced shock was mateS. used as a model of nitrosative stress. The dose of LPS for US 2005/0014697 A1 Jan. 20, 2005 producing ~50% mortality in GSNOR mice was estab elevated above baseline (FIG. 4B). The levels of serum lished in initial dose-response studies (FIGS. 3A-3D). In a nitrite (<5,000 dal markers and histopathology indicated that LPS-induced tons; FIG. 4A). Thus, in endotoxic shock, the metabolism of liver damage was much worse in GSNOR mice than in endogenously generated nitroSothiols was Severely impaired wild-type mice. In Sharp contrast to the near complete in the GSNOR-deficient mouse. recovery by wild-type livers, GSNOR' livers showed no Sign of recovery. 0204) The levels of nitrate plus nitrite (NOx) in the circulation have been known to reflect overall NOS activity 0209 The marker of muscle injury, creatine phosphoki in mammals. Here, it was found that basal nitrate levels in nase (CPK), and the markers of kidney dysfunction, urea GSNOR mice did not differ from wild-type mice (FIGS. nitrogen (BUN), and creatinine, all increased Substantially 4B, 4C, and Methods). After treatment with LPS, nitrate and to similar levels in wild-type and GSNOR mice at 24 concentrations in wild-type mice rose at 24 h to the same h after LPS (FIGS. 5C-5E). In wild-type controls, these level as in GSNOR mice, and returned to baseline at 48 activities decreased at 48 h almost to baseline, but levels did h (FIG. 4B). While the nitrate level in GSNOR mice not decline in the GSNOR mice (FIGS. 5C-5E). Thus, decreased at 48 h (~50%, P=0.03), it was still substantially organ dysfunction did not resolve in the GSNOR mice. US 2005/0014697 A1 Jan. 20, 2005

The kidneys and hearts of the wild-type and GSNOR Example 3 mice were grossly normal on histological examination. Discussion 0210 Pancreatic islet cells are known to be highly sus ceptible to NO toxicity in vitro (Liu et al., 2000a). However, 0216) The disclosed experiments demonstrate that: (1) as shown herein, LPS challenge had little effect on the S-nitrosothiols play an essential role in NO biology, influ pancreas in wild-type and GSNOR mice. In particular, encing blood preSSure and related homeostatic functions, Serum levels of both amylase and lipase changed little and contributing to the pathogenesis of endotoxic/septic following LPS treatment (FIGS. 5F-5G), and no histologi shock; (2) NO bioactivity is regulated not only at the level cal abnormalities were detected. of synthesis (i.e., NOS) but also by degradation, in particular by GSNOR; (3) turnover of GSNO influences the level of 0211 A protective role for GSNOR was evident in lym whole cell S-nitrosylation; (4) accumulation of SNOs can phatic tissue (FIGS. 6C-6H). At 24 h after LPS, the two produce a StreSS on the mammalian organism that influences Strains showed a similar amount and pattern of lymphocyte Survival, and in particular, nitrosative StreSS that is identified apoptosis in thymus, Spleen, mesenteric lymph nodes, Pey with GSNO is implicated in disease pathogenesis; (5) er's patches, and other lymphoid tissues. Wild-type lym GSNOR protects mice from excessive declines in blood phatic tissues showed little cell death at 48 h after LPS preSSure under anesthesia, and from tissue injury following (FIGS. 6C, 6E and 6G). In contrast, GSNOR tissues endotoxemia; (6) the systems affected most by GSNOR showed substantial apoptosis (FIGS. 6D, 6F and 6H). deficiency include the liver, immune System and cardiovas Lymphocyte apoptosis in the thymus was extensive, espe cular System. These results signal a fundamental change for cially in cortical regions (FIG. 6D). Further, at 48 h after the current paradigm of NO biology, which centers on the LPS, lymphocyte depletion was more severe in the activity of NOS. The disclosed data also provide genetic GSNOR, thymus than in the wild-type (FIGS. 6C-6D). Support for the importance of redox-based regulation of Thus, GSNOR was required to protect the immune system proteins through modification at cysteine thiols. from endotoxic injury. 0217 GSNOR is Essential for SNO Metabolism 0212 Effect of INOS Inhibition on Tissue Injury and Survival of GSNOR / Mice 0218. According to the disclosed data, it appears that GSNO reductase is not essential for development, growth, 0213 Additional experiments were performed to estab and reproduction of mice. It has been Suggested that normal lish the contribution of nitrosative StreSS to the pathogenesis growth and reproduction of ADH 111-deficient mice of endotoxic shock. LPS-challenged GSNOR mice were requires dietary Supplementation with large amounts of treated with 1400W, a selective iNOS inhibitor. Adminis vitamin A (retinol) (Molotkov et al., 2002). Here, no such tration of 1400W, initiated 6 h following LPS injection, requirement was observed in either of the GSNORT mouse reduced serum nitrate (i.e., NOS activity) by about 50% Strains. The origin of the unusual nutritional requirement (FIG. 7A, P=0.015) and liver injury by about 90% (FIG. reported by Molotkov et al. may lie in the deletion construct 7C, P=0.020). This improvement coincided with a reduction that was used or in the genetic background of the mice. of liver SNO by about 90% (FIG. 7B). Measurements of Serum markers showed that tissue injury was also reduced in 0219. One important discovery disclosed herein is that kidney, pancreas, and muscle (48 h after LPS treatment). GSNOR is crucial for SNO metabolism in animals. Most importantly, the Survival rate of LPS-challenged GSNOR mice accumulated higher amounts of S-nitro GSNOR mice was significantly improved by 1400W Sothiols than wild-type mice despite comparable levels of (FIG. 7D vs. FIG. 3D, P=0.03). By comparison, PBS NOS expression and activity. Levels of SNOs in vivo were (volume control) had little effect (FIG. 7D vs. FIG. 3D). thus determined not only by NOS activities, but also by When administration of 1400W was delayed for 24 h GSNOR. This conclusion was further supported by the following LPS injection, this allowed SNOs to accumulate observed increases in GSNOR mice regarding (1) the to hazardous levels, and the protection conferred by NOS ratio of SNO to iron nitrosyl compounds at basal conditions; inhibition was lost (3 out of 5 female GSNOR mice died). and (2) the ratio of SNO to nitrate or nitrite or both during These data Strongly Suggested that nitrosative StreSS from the course of endotoxic Shock. Inasmuch as measurements iNOS in GSNOR mice mediated tissue damage and of nitrite and nitrate are the Standard means of assessing NO bioactivity in biological Systems, the disclosed results raise increased mortality. interesting questions regarding many previous assumptions. 0214) GSNOR in Septic Shock 0220 GSNO is the only SNO substrate recognized by 0215. The role and function of GSNOR was also inves GSNOR, yet the disclosed data indicate deletion of the tigated in bacterial Septic shock induced by cecal ligation enzyme results in greater increases in SNO-proteins than in and puncture (CLP) (Wichterman et al., 1980), an animal GSNO itself. Similar results were obtained in GSNOR model that resembles the human condition. CLP resulted in deficient yeast (Liu et al., 2001) and in RBCs exposed to significantly higher mortality in GSNOR mice (n=9) than GSNO ex vivo (Jia et al., 1996). This suggests that at least in wild-type mice (n=8) (FIG.3E), whereas a sham control Some key protein SNOs are in equilibrium with GSNO both without puncture did not result in death of either GSNOR under basal and stress conditions (Equation 1, below). In (n=3) or wild-type (n=3) mice. Following CLP, the levels of addition, the equilibrium apparently favors protein SNOs. liver SNOs (FIG. 4E) and marker enzymes were signifi Prompt disposal of GSNO by GSNOR (Equation 2, below), cantly higher in GSNOR than in wild-type mice. Thus, acts to drive the equilibrium towards the denitrosylated GSNOR protects mice against SNO-related morbidity and State. Thus, it appears that glutathione (GSH) cannot effec mortality induced by CLP. tively or fully terminate SNO signaling or protect proteins US 2005/0014697 A1 Jan. 20, 2005 22 from hazardous levels of S-nitrosylation in the absence of of females to Septic shock. This phenomenon is seen in both GSNOR. animals (Laubach et al., 1998; Zellweger et al., 1997) and humans (Oberholzer et al., 2000; Schroder et al., 1998). Previous experiments showed that iNOS protects female Equation 1: mice more than male mice from endotoxemia-induced death (Laubach et al., 1998). The sum of these data suggests that Protein-SNO + GSH R- GSNO + protein the beneficial effect of iNOS is nullified by nitrosative stress Equation 2: in GSNOR animals. Without wishing to be bound by GSNOR theory, it is hypothesized that GSNOR is a genetic determi GSNO - NADH + H - - GEEG + NH4 nant of Sepsis outcome, particularly in female patients, and that the potential benefits of iNOS inhibition in septic patients will relate to GSNOR activity. 0221) GSNOR Protects from Nitrosative Stress in 0227) Hemodynamic Consequence of GSNOR Defi Response to Endotoxin and Bacteria ciency 0222. In the experiments disclosed herein, mice with 0228. Hypotension has been observed as one of the most elevated iNOS activity were subjected to nitrosative stress frequent Side effects of anesthesia, but the basis for patient characterized by elevated levels of S-nitrosylated proteins. susceptibility has not been determined. Here, GSNOR However, the LPS-challenged mice did not suffer detrimen deficient mice were hypotensive when anesthetized in the tal consequences unless protection afforded by GSNOR was absence of LPS challenge. In GSNOR-deficient animals, abolished (GSNOR7). In the absence of GSNOR, the basal SNO levels were increased approximately two-fold in animals exhibited hazardous accumulations of S-nitrosy RBCs. These levels have been known to produce vasodila lated proteins and tissue damage. The finding herein that tion in bioassays (McMahon et al., 2002; Pawloski et al., GSNOR protected lymphatic tissues and liver from apop 2001) and lower blood pressure (or vascular resistance) tosis added Support to the accumulating evidence that death when either RBCs or SNO-Hb (the major RBC SNO) were signaling is regulated by SNOS (Eu et al., 2000; Haendeler infused intravenously (Jia et al., 1996). It was previously et al., 2002; Mannick et al., 1999; Marshall and Stamler, shown that urethane or pentobarbital anesthesia markedly 2002; Matsumoto et al., 2003). Additionally, as shown potentiates the vasorelaxant and hypotensive effects of herein, inhibition of iNOS improved all measures of injury SNOS administered intravenously in rats (Travis et al., acroSS tissues as well as Survival of the animals. Collectively 1997). The disclosed results point to the possibility that these data establish that nitrosative StreSS is a major cause of blood pressure under anesthesia may reflect SNO bioactivity morbidity in GSNOR mice. and may have a genetic basis in GSNOR activity. 0223 GSNOR is one of several factors that mediate 0229 Interestingly, the hypotensive effects of iNOS have resistance to microbial challenge (Cohen, 2002). AS Such, its also been linked to anesthesia. Hypotension has been found role is influenced not only by microbial susceptibility to to be greater in pentobarbital-anesthetized wild-type mice SNOS, but also by its part in protecting immune function challenged with LPS than in iNOS' mice (MacMicking et (FIGS. 6A-6H). This complexity notwithstanding, recent al., 1995). In addition, concentrations of LPS that lowered genetic and chemical evidence Suggests that SNOS are blood preSSure to comparable degrees in anesthetized VS. produced in mice to counter cryptococcal (de Jesus-Berrios conscious iNOS mice, produced far greater hypotension et al., 2003), Salmonella (De Groote et al., 1996) and in anesthetized than conscious wild-type mice (MacMicking tuberculous (MacMicking et al., 1997) infections. The find et al., 1995; Rees et al., 1998). LPS has been known to ings disclosed herein indicate that SNOS are also produced increase levels of SNO-Hb in rodents (Jourd'heuil et al., by the host in additional forms of polymicrobial/gram nega 2000). Here, similar increases were observed in the blood of tive sepsis. Specifically, the protection afforded by GSNOR patients with Sepsis. Collectively, these data Suggest that the was not only observed in the endotoxic model of shock, but increased SNOs derived from iNOS contribute to the also against CLP-induced bacteremia. hypotensive effects of anesthesia in endotoxic animals. The 0224. As demonstrated herein, GSNOR deficiency disclosed data do not exclude the effects of GSNO exerted resulted in elevated hepatic levels of SNOs in the CLP centrally or in the kidney (Ortiz and Garvin, 2003; Stamler, model. In Support of relevance of these mouse models to the 1999; Stoll et al., 2001). However, the results support the human condition, we find that SNO-Hb levels, which are recent discovery that RBCs dilate blood vessels (Gonzalez known to be increased in the blood of endotoxic animals Alonso et al., 2002, Jia et al., 1996; McMahon et al., 2002) (Jourd'heuil et al., 2000), were several-fold higher in the and the proposition that SNOs in RBCs may contribute to a blood of patients with gram-negative sepsis (0.0037+0.0010 hypotensive phenotype. SNO/Hb, n=7) than in healthy controls (0.0010+0.0004 0230. The mechanism(s) by which SNOs are both gen SNO/Hb, n=12; P-0.01). Taken together, these data suggest erated in RBCs and the activity liberated to dilate blood that S-nitrosothiols may play important roles in both the vessels (McMahon et al., 2002; Pawloski et al., 2001) is only amelioration and pathogenesis of endotoxic/septic shock. partly understood. It has been shown that Hb can react with 0225 Gender NO (Gow et al., 1999), nitrite (Luchsinger et al., 2003) or GSNO (Jia et al., 1996; Romeo et al., 2003) to produce 0226 GSNOR deficiency resulted in a 10-fold increase in SNO-Hb, and that vasodilation by RBCs requires transfer of mortality (vs. wild-type) in LPS-challenged female mice, the NO from SNO-Hb to RBC membrane thiols (Pawloski but only a ~2-fold increase in males. The protective effect of et al., 2001). Additional Studies point to a role for plasma GSNOR may therefore contribute to the relative resistance GSNO in dispensing of RBC membrane bioactivity (Lipton US 2005/0014697 A1 Jan. 20, 2005 23 et al., 2001). The disclosed results clearly establish the (RRREEEEESAAA; SEQ ID NO:30). In addition to importance of GSNO/GSNOR in maintaining the levels of decreasing GRK2-mediated receptor phosphorylation, both RBC-SNO in vivo. Moreover, the disclosed experiments cysNO and GSNO significantly inhibited GRK2 mediated show that increases in SNO occur without detectable phosphorylation of the synthetic peptide substrate (FIG. increases in other bioactive NO compounds (iron nitrosylHb 12A-12B). and nitrite). This provides Strong genetic Support for the idea 0235. This data provided compelling whole-cell and in that SNOs can mediate NO bioactivity in blood (Jia et al., vitro evidence in support of the hypothesis that NO/SNO 1996; Stamler et al., 1992) and tissues (Gow et al., 2002; directly decreases GRK2-mediated B-AR phosphorylation Stamler et al., 2001). and provides a likely mechanism of action through S-ni trosylation of GRK2. Without being bound by theory, it was CONCLUSION hypothesized that NO targets cysteine thiol and transition 0231. The disclosed findings underscore the central role metal centers and transduces a panoply of effects, including of S-nitrosothiols in NO biology and disease. Specifically, cGMP-independent effects on many receptors by S-nitrosy the genetic evidence provided in this Study Suggests that lation. In addition to these Studies, a number of in Vivo and GSNO turnover is required not only to prevent accumulation eX Vivo experiments were conducted aimed at elucidating of SNO that predisposes to disease diathesis, but also to the effects of NO on B-AR physiology and on the observed regulate the turnover of SNOs in the context of physiologi defects in B-AR function associated with heart failure. cal signaling (e.g. the dispensing of a messenger to regulate Evidence showing the involvement of S-nitrosothiols was blood pressure). This homeostatic role of GSNO reductase is obtained. reminiscent of that played by Superoxide dismutase (SOD). 0236) Effect of Bioavailable NO/Nitrosothiols on GSNOR affords protection against nitrosative stress and Chronic B-AR Stimulation Induced Cardiac Hypertrophy influences vascular tone in a way that is evocative of SOD and Receptor Down-Regulation protection against oxidative StreSS and regulation of blood 0237 Previous studies have demonstrated B-AR desen pressure (Didion et al., 2002; Nakazono et al., 1991). Thus, Sitization and down-regulation associated with cardiac GSNOR may play additional roles in the regulation of hypertrophy and heart failure. One well established model of critical organ functions. Further, nitrosative StreSS may con experimental cardiac hypertrophy in the mouse involves the tribute broadly to disease pathogenesis, Since Studies in chronic administration of the B-AR agonist, isoproterenol. endotoxemia and bacteremia are paradigmatic of other This treatment leads to a functional uncoupling and down innate immune, inflammatory, degenerative and prolifera regulation of cardiac B-ARS and the development of a tive conditions in which iNOS is implicated. Thus, diseases Significant increase in wall mass. Experiments were per characterized by malfunction in S-nitrosylation represent formed to study the effects of GSNO on the development of new therapeutic opportunities and targets for intervention cardiac hypertrophy and its ability to alter the pattern of (see Liu et al., 2004, Cell 116:617-628). B-AR down-regulation associated with chronic isoproter enol Stimulation. Example 4 0238 Whole cell and membrane receptor/ligand binding was performed to assess functional affinity of receptor for Cardiac Studies ligand and overall receptor density as described previously. 0232) Effect of NO/SNOs on GRK2-Mediated Phospho In particular, whole cell and membrane binding was rylation of the B-AR and a Soluble Peptide Substrate Using assessed for cells treated with multiple concentrations of NO/SNO in the presence and absence of desensitizing Purified Protein in a Reconstituted System conditions (agonist pre-stimulation). The administration of 0233 Previous data provided strong evidence supporting GSNO (delivered via osmotic mini-pump over 2 weeks) had the hypothesis that NO prevented GRK (G protein-coupled no effect on isoproterenol-stimulated changes in heart receptor kinase)-mediated phosphorylation of the f-AR weight to body weight ratio (FIG. 13A), but significantly (adrenergic receptor). Further experiments were required to decreased f-AR down-regulation (FIG. 13B). Previous determine whether NO was acting directly on the receptor or Studies have demonstrated that preserving B-AR function in the GRKS. To elucidate the site of NO action, its effect was heart failure can delay the progression of disease. Thus, tested on GRK2-mediated phosphorylation of the B-AR and these data suggest that GSNO, by virtue of its ability to on a Soluble peptide Substrate using purified proteins in a prevent the down-regulation of cardiac B-ARS, represents a reconstituted System. In these Studies, cySNO significantly novel therapeutic modality for the treatment of heart failure. decreased GRK2-mediated phosphorylation of the purified 0239). These sum of these results indicate that GSNO f3-AR (FIG. 11A). NO bioactivity also significantly elevating agents (i.e. inhibitors of GSNOR) may be used as decreased GRK2-mediated phosphorylation of rhodopsin a means to improve f3-adrenergic signaling. FIGS. 13A-13C from purified bovine rod Outer Segments Suggesting a gen demonstrate that the B-adrenergic agonist isoproterenol eralized mechanism of NO action (FIG. 1B). (ISO), infused for 7 days into mice using a pump, lead to 0234. These data were in agreement with whole cell increases in cardiac weight (FIG. 13A), decreased Badren receptor phosphorylation data and limited the possible site ergic receptor levels (FIG. 13B), and increased activity of NO action to the receptor or the GRK. These experiments fARK (GRK2) expression. In contrast, the combined infu also demonstrated that SNO decreased GRK2 autophospho sion of GSNO with ISO maintained B receptor density (FIG. rylation, Suggesting the direct inhibition of GRK2 by 13B) because it inhibits BARK (GRK2) (FIGS. 12A-12B). nitrosylation. To confirm this, experiments were performed Therefore, inhibitors of GSNOR alone or in combination to examine the capacity of SNOs to inhibit purified GRK2 with 3-agonists could be used to improve heart failure, or mediated phosphorylation of a Synthetic peptide Substrate other vascular disorderS Such as hypertension and asthma. US 2005/0014697 A1 Jan. 20, 2005 24

0240 The details of one or more embodiments of the antagonism of nitric oxide-related cytostasis in Salmo invention have been Set forth in the accompanying descrip nella typhimurium. Science 272, 414-417. tion above. Although any methods and materials Similar or equivalent to those described herein can be used in the 0251 de Jesus-Berrios, M., Liu, L., Nussbaum, J. C., practice or testing of the present invention, the preferred Cox, G. M., Stamler, J. S., and Heitman, J. (2003). methods and materials are now described. Other features, Enzymes that counteract nitrosative StreSS promote objects, and advantages of the invention will be apparent fungal virulence. Curr Biol 13, 1963-1968. from the description and from the claims. 0252) Didion, S. P., Ryan, M. J., Didion, L. A., Fegan, 0241. In the specification and the appended claims, the P. E., Sigmund, C. D., and Faraci, F. M. (2002). Singular forms include plural referents unless the context Increased Superoxide and vascular dysfunction in clearly dictates otherwise. Unless defined otherwise, all CuZnSOD-deficient mice. Circ Res 91, 938-944. technical and Scientific terms used herein have the same 0253 Eu, J. P., Liu, L., Zeng, M., and Stamler, J. S. meaning as commonly understood by one of ordinary skill (2000). An apoptotic model for nitrosative stress. Bio in the art to which this invention belongs. Unless expressly chemistry 39, 1040-1047. Stated otherwise, the techniques employed or contemplated herein are Standard methodologies well known to one of 0254 Feihl, F, Waeber, B., and Liaudet, L. (2001). Is ordinary skill in the art. All patents and publications cited in nitric oxide overproduction the target of choice for the this specification are hereby incorporated by reference management of septic shock?Pharmacol Ther 91, 179 herein, including the previous disclosure provided by U.S. 213. Application Ser. No. 60/476,055 filed Jun. 4, 2003 and U.S. 0255 Foglio, M. H., and Duester, G. (1996). Charac Application Ser. No. 60/545,965 filed Feb. 18, 2004, and terization of the functional gene encoding mouse class U.S. Application Ser. No. 60/550,833 filed Mar. 4, 2004. III alcohol dehydrogenase (glutathione-dependent formaldehyde dehydrogenase) and an unexpressed pro REFERENCES cessed pseudogene with an intact open reading frame. 0242 American College of Chest Physicians/Society Eur J Biochem 237, 496-504. of Critical Care Medicine Consensus Conference: defi 0256 Foster, M. W., McMahon, T.J., and Stamler, J. S. nitions for Sepsis and organ failure and guidelines for (2003). S-nitrosylation in health and disease. Trends the use of innovative therapies in sepsis. (1992). Crit Mol Med 9, 160-168. Care Med 20, 864-874. 0257 Gaston, B., Reilly, J., Drazen, J. M., Fackler, J., 0243 Boehning, D., and Snyder, S. H. (2003). 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SEQUENCE LISTING

<160> NUMBER OF SEQ ID NOS : 31

<21 Oc SEQ ID NO 1 <211 LENGTH 21 <212> TYPE DNA <213> ORGANISM: Artificial <22O > FEATURE OTHER INFORMATION: Oligonucleotide <400 SEQUENCE: 1

gatggaagag togtgagagt g 21

SEQ ID NO 2 LENGTH 22 TYPE DNA ORGANISM: Artificial FEATURE OTHER INFORMATION: Oligonucleotide

<400 SEQUENCE: 2

cagtctdgat tatgcacatt co 22

SEQ ID NO 3 LENGTH 2.0 TYPE DNA ORGANISM: Artificial FEATURE US 2005/0014697 A1 Jan. 20, 2005 28

-continued <223> OTHER INFORMATION: Oligonucleotide <400 SEQUENCE: 3 gtgatcaggt gtaaggctgc 20

<210> SEQ ID NO 4 &2 11s LENGTH 19 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Oligonucleotide <400 SEQUENCE: 4 citgccttcag ctitcgtgac 19

<210 SEQ ID NO 5 <211& LENGTH 21 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Oligonucleotide <400 SEQUENCE: 5 tottgacgag ttcttctgag g 21

<210> SEQ ID NO 6 <211& LENGTH 22 &212> TYPE DNA <213> ORGANISM: Artificial &220s FEATURE <223> OOTHER INFORMATION: Oligonucleotide <400 SEQUENCE: 6 cagttgactg. tcaatgaact gg 22

<21 Oc EQ ID NO 7 <211 ENGTH 1613 YPE DNA RGANISM: Homo sapiens <400 SEQUENCE: 7 ggg catgggc gcggcc acco C ggatgtcag ccc.ccc.gc.gc cqaccagaat cogtgaacat 60 ggcgaacgag gttatcaagt gcaaggctgc agttgcttgg gaggctggala agcct citct c 120 catagaggag atagaggtgg caccoccaaa gqc to atgaa gttcgaatca agatcattgc 18O cactg.cggitt toccacaccg atgccitatac cct gagtgga gct gatcct g agg gttgttt 240 to cagtgatc ttggga catg aaggtgctgg aattgtggaa agtgttggtg agg gagttac 3OO taagctgaag gogggtgaca citgtcatc.cc actttacatc ccacagtgtg gagaatgcaa. 360 attttgtcta aatcctaaaa cita acctttg ccagaagata agagt cactic aagggaaagg 420 attaatgcca gatggtacca gcagatttac ttgcaaagga aag acaattt to cattacat 480 gggalaccago acattttctg aatacacagt totggctgat atctotgttg citaaaataga 540 toctittagca cctttgtata aagttctgcct tctaggttgt ggcatttcaa ccggittatgg 600 tgctgctgtgaac act gcca agttggagcc tdgctctgtt totg.ccgtot ttggtotggg 660 aggagtcgga ttggcagtta totatoggctg taaagtggct ggtgctt.ccc ggatcattgg 720 tgtgga catc aataaagata aatttgcaag ggccaaagag tittggagcca citgaatgitat 78O

US 2005/0014697 A1 Jan. 20, 2005 31

-continued tocc cacttic aggttatcaa gtgcaaggct gcagttgctt go gaggctgg aaag.cct citc 60 to catagagg agatagaggit ggc accocca aaggotcatg aagttcgaat caaggtaatg 120 atacatttaa gocactggga aaaaaa 146

<210> SEQ ID NO 11 &2 11s LENGTH 383 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 11 cattcgttgt tittaatgtcc tdatagat.ca ttgcc actogc ggtttgccac accgatgcct 60 ataccctgag toggagctgat cotgagggitt gttitt coagt gatcttggga catgalaggtg 120 Ctggaattgt ggaaagtgtt ggtgagggag titactaagct galagg.cgggit aaggagaata 18O cittgaaccac ttgtttaata attittggctt attccitatgg ggaaattgtt tittctgataa 240 aact accolac tatttatgaa taggitatcat citaagtag at tdtcaagatt aagttgattic 3OO citcatctggg aag cacaaat atttggatat ttttittctdt citgattittgc agacitactgt 360 ttttgttgaaa cittittctaag tac 383

<210> SEQ ID NO 12 &2 11s LENGTH 326 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 12 citttgcattt gtttcCaggit gacactgtca toccactitta catcc cacag totggagaat 60 gcaaattittg totaaatcct aaaactaacc tittgccagaa gataaggitta gitatctttitt 120 atgttcttct taaaatacaa gtgctg.cggg ataattalagg aatcacagag accgaggggit 18O tgaggaggaa ttatttaata atttaggttc attaa.cccag toggattaac gttcaaagga 240 citgagtcc.cg aacaaagagt caagctacct tittaa.gcatt togtoggggtg gggggag acc 3OO tttgtagggg gag cat atta cagaag 326

<210> SEQ ID NO 13 &2 11s LENGTH 934 &212> TYPE DNA <213> ORGANISM: Homo sapiens <400 SEQUENCE: 13 tggataagat tataaatato ttataaattt cotttataag goattgctgc aaggtgctaa 60 attactaatgaatatatttg aaattgtagt ttacaacact ttcttaatat titactggtoa 120 ttatttittaa aacatttctt tittcctgtgg gttittaagaa acctaattcc aacttgccitt 18O tittcctttitt ttittctittag agt cactcaa gqgaaaggat taatgccaga tiggtaccago 240 agatttacitt gcaaagga aa gacaattittg cattacatgg galaccagdac attittctgaa 3OO tacacagttg togctgatat citctgttgct aaaatagatc ctittago acc tittggataaa 360 gtotgcct to taggttgtgg catttcaa.cc ggittatogto citgctgttgaa cactgccaag 420 gtaagagact g acttgggitt ttittgcttct gccttctaat ttaattcagt gaaacttitcc 480 tggaatagtg aaattggcca atctgitatga aacco agtga ttctottgac totggtagaa 540 tgagtactitt ataaacttitc tittacticcita gttggagcct ggctctgttt gtgcc.gtctt 600

US 2005/0014697 A1 Jan. 20, 2005 34

-continued

<210 SEQ ID NO 17 &2 11s LENGTH 392 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 17 Met Gly Ala Ala Thr Pro Asp Val Ser Pro Pro Arg Arg Pro Glu Ser 1 5 10 15 Val Asn Met Ala Asn. Glu Val Ile Lys Cys Lys Ala Ala Val Ala Trp 2O 25 30 Glu Ala Gly Lys Pro Leu Ser Ile Glu Glu Ile Glu Val Ala Pro Pro 35 40 45 Lys Ala His Glu Val Arg Ile Lys Ile Ile Ala Thr Ala Val Cys His 50 55 60 Thr Asp Ala Tyr Thr Leu Ser Gly Ala Asp Pro Glu Gly Cys Phe Pro 65 70 75 8O Val Ile Leu Gly His Glu Gly Ala Gly Ile Val Glu Ser Val Gly Glu 85 90 95 Gly Val Thir Lys Lieu Lys Ala Gly Asp Thr Val Ile Pro Leu Tyr Ile 100 105 110 Pro Glin Cys Gly Glu Cys Lys Phe Cys Lieu. Asn. Pro Lys Thr Asn Lieu 115 120 125 Cys Gln Lys Ile Arg Val Thr Glin Gly Lys Gly Leu Met Pro Asp Gly 130 135 1 4 0 Thr Ser Arg Phe Thr Cys Lys Gly Lys Thr Ile Leu. His Tyr Met Gly 145 15 O 155 160 Thr Ser Thr Phe Ser Glu Tyr Thr Val Val Ala Asp Ile Ser Val Ala 1.65 170 175 Lys Ile Asp Pro Leu Ala Pro Leu Tyr Lys Val Cys Lieu Lieu Gly Cys 18O 185 19 O Gly Ile Ser Thr Gly Tyr Gly Ala Ala Wall Asn. Thr Ala Lys Lieu Glu 195 200 2O5 Pro Gly Ser Val Cys Ala Val Phe Gly Lieu Gly Gly Val Gly Lieu Ala 210 215 220 Val Ile Met Gly Cys Lys Val Ala Gly Ala Ser Arg Ile Ile Gly Val 225 230 235 240 Asp Ile Asn Lys Asp Llys Phe Ala Arg Ala Lys Glu Phe Gly Ala Thr 245 250 255 Glu Cys Ile Asin Pro Glin Asp Leu Ser Lys Pro Ile Glin Glu Val Lieu 260 265 27 O Ile Glu Met Thr Asp Gly Gly Val Asp Tyr Ser Phe Glu Cys Ile Gly 275 280 285 Asn. Wall Lys Wal Met Arg Ala Ala Lieu Glu Ala Cys His Lys Gly Trip 29 O 295 3OO Gly Val Ser Val Val Val Gly Val Ala Ala Ser Gly Glu Glu Ile Ala 305 310 315 320 Thr Arg Pro Phe Gln Leu Val Thr Gly Arg Thr Trp Lys Gly Thr Ala 325 330 335 Phe Gly Gly Trp Lys Ser Val Glu Ser Val Pro Lys Leu Val Ser Glu 340 345 35 O Tyr Met Ser Lys Lys Ile Lys Val Asp Glu Phe Val Thr His Asn Leu 355 360 365 US 2005/0014697 A1 Jan. 20, 2005 35

-continued

Ser Phe Asp Glu Ile Asn Lys Ala Phe Glu Lieu Met His Ser Gly Lys 370 375 38O Ser Ile Arg Thr Val Val Lys Ile 385 390

<210> SEQ ID NO 18 &2 11s LENGTH 374 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 18 Met Ala Asn. Glu Val Ile Lys Cys Lys Ala Ala Val Ala Trp Glu Ala 1 5 10 15 Gly Lys Pro Leu Ser Ile Glu Glu Ile Glu Val Ala Pro Pro Lys Ala 2O 25 30 His Glu Val Arg Ile Lys Ile Ile Ala Thr Ala Val Cys His Thr Asp 35 40 45 Ala Tyr Thr Leu Ser Gly Ala Asp Pro Glu Gly Cys Phe Pro Val Ile 50 55 60 Leu Gly. His Glu Gly Ala Gly Ile Val Glu Ser Val Gly Glu Gly Val 65 70 75 8O Thr Lys Leu Lys Ala Gly Asp Thr Val Ile Pro Leu Tyr Ile Pro Glin 85 90 95 Cys Gly Glu Cys Lys Phe Cys Lieu. Asn Pro Llys Thr Asn Lieu. Cys Glin 100 105 110 Lys Ile Arg Val Thr Glin Gly Lys Gly Lieu Met Pro Asp Gly Thr Ser 115 120 125 Arg Phe Thr Cys Lys Gly Lys Thr Ile Leu. His Tyr Met Gly Thr Ser 130 135 1 4 0 Thr Phe Ser Glu Tyr Thr Val Val Ala Asp Ile Ser Val Ala Lys Ile 145 15 O 155 160 Asp Pro Leu Ala Pro Leu Tyr Lys Val Cys Lieu Lieu Gly Cys Gly Ile 1.65 170 175 Ser Thr Gly Tyr Gly Ala Ala Val Asn. Thir Ala Lys Lieu Glu Pro Gly 18O 185 19 O Ser Val Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ala Val Ile 195 200 2O5 Met Gly Cys Lys Wall Ala Gly Ala Ser Arg Ile Ile Gly Val Asp Ile 210 215 220 Asn Lys Asp Llys Phe Ala Arg Ala Lys Glu Phe Gly Ala Thr Glu Cys 225 230 235 240 Ile Asin Pro Glin Asp Leu Ser Lys Pro Ile Glin Glu Val Lieu. Ile Glu 245 250 255 Met Thr Asp Gly Gly Val Asp Tyr Ser Phe Glu Cys Ile Gly Asn Val 260 265 27 O Lys Wal Met Arg Ala Ala Leu Glu Ala Cys His Lys Gly Trp Gly Val 275 280 285 Ser Val Val Val Gly Val Ala Ala Ser Gly Glu Glu Ile Ala Thr Arg 29 O 295 3OO Pro Phe Gln Leu Val Thr Gly Arg Thr Trp Lys Gly Thr Ala Phe Gly 305 310 315 320 Gly Trp Llys Ser Val Glu Ser Val Pro Llys Leu Val Ser Glu Tyr Met US 2005/0014697 A1 Jan. 20, 2005 36

-continued

325 330 335 Ser Lys Lys Ile Lys Val Asp Glu Phe Val Thr His Asn Lieu Ser Phe 340 345 35 O Asp Glu Ile Asn Lys Ala Phe Glu Lieu Met His Ser Gly Lys Ser Ile 355 360 365 Arg Thr Val Val Lys Ile 370

<210 SEQ ID NO 19 &2 11s LENGTH 374 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 19 Met Ala Asn. Glu Val Ile Lys Cys Lys Ala Ala Val Ala Trp Glu Ala 1 5 10 15 Gly Lys Pro Leu Ser Ile Glu Glu Ile Glu Val Ala Pro Pro Lys Ala 2O 25 30 His Glu Val Arg Ile Lys Ile Ile Ala Thr Ala Val Cys His Thr Asp 35 40 45 Ala Tyr Thr Leu Ser Gly Ala Asp Pro Glu Gly Cys Phe Pro Val Ile 50 55 60 Leu Gly. His Glu Gly Ala Gly Ile Val Glu Ser Val Gly Glu Gly Val 65 70 75 8O Thr Lys Leu Lys Ala Gly Asp Thr Val Ile Pro Leu Tyr Ile Pro Glin 85 90 95 Cys Gly Glu Cys Lys Phe Cys Lieu. Asn Pro Llys Thr Asn Lieu. Cys Glin 100 105 110 Lys Ile Arg Val Thr Glin Gly Lys Gly Lieu Met Pro Asp Gly Thr Ser 115 120 125 Arg Phe Thr Cys Lys Gly Lys Thr Ile Leu. His Tyr Met Gly Thr Ser 130 135 1 4 0 Thr Phe Ser Glu Tyr Thr Val Val Ala Asp Ile Ser Val Ala Lys Ile 145 15 O 155 160 Asp Pro Leu Ala Pro Leu Asp Llys Val Cys Lieu Lieu Gly Cys Gly Ile 1.65 170 175 Ser Thr Gly Tyr Gly Ala Ala Val Asn. Thir Ala Lys Lieu Glu Pro Gly 18O 185 19 O Ser Val Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ala Val Ile 195 200 2O5 Met Gly Cys Lys Wall Ala Gly Ala Ser Arg Ile Ile Gly Val Asp Ile 210 215 220 Asn Lys Asp Llys Phe Ala Arg Ala Lys Glu Phe Gly Ala Thr Glu Cys 225 230 235 240 Ile Asin Pro Glin Asp Phe Ser Lys Pro Ile Glin Glu Val Lieu. Ile Glu 245 250 255 Met Thr Asp Gly Gly Val Asp Tyr Ser Phe Glu Cys Ile Gly Asn Val 260 265 27 O Lys Wal Met Arg Ala Ala Leu Glu Ala Cys His Lys Gly Trp Gly Val 275 280 285 Ser Val Val Val Gly Val Ala Ala Ser Gly Glu Glu Ile Ala Thr Arg 29 O 295 3OO US 2005/0014697 A1 Jan. 20, 2005 37

-continued Pro Phe Gln Leu Val Thr Gly Arg Thr Trp Lys Gly Thr Ala Phe Gly 305 310 315 320 Gly Trp Llys Ser Val Glu Ser Val Pro Llys Leu Val Ser Glu Tyr Met 325 330 335 Ser Lys Lys Ile Lys Val Asp Glu Phe Val Thr His Asn Lieu Ser Phe 340 345 35 O Asp Glu Ile Asn Lys Ala Phe Glu Lieu Met His Ser Gly Lys Ser Ile 355 360 365 Arg Thr Val Val Lys Ile 370

<210> SEQ ID NO 20 &2 11s LENGTH 374 &212> TYPE PRT <213> ORGANISM: Mus musculus

<400 SEQUENCE: 20 Met Ala Asn Glin Val Ile Arg Cys Lys Ala Ala Val Ala Trp Glu Ala 1 5 10 15 Gly Lys Pro Leu Ser Ile Glu Glu Ile Glu Val Ala Pro Pro Lys Ala 2O 25 30 His Glu Val Arg Ile Lys Ile Leu Ala Thr Ala Val Cys His Thr Asp 35 40 45 Ala Tyr Thr Leu Ser Gly Ala Asp Pro Glu Gly Cys Phe Pro Val Ile 50 55 60 Leu Gly. His Glu Gly Ala Gly Ile Val Glu Ser Val Gly Glu Gly Val 65 70 75 8O Thr Lys Leu Lys Ala Gly Asp Thr Val Ile Pro Leu Tyr Ile Pro Glin 85 90 95 Cys Gly Glu Cys Lys Phe Cys Lieu. Asn Pro Llys Thr Asn Lieu. Cys Glin 100 105 110 Lys Ile Arg Val Thr Glin Gly Lys Gly Lieu Met Pro Asp Gly Thr Ser 115 120 125 Arg Phe Thr Cys Lys Gly Lys Ser Val Phe His Phe Met Gly Thr Ser 130 135 1 4 0 Thr Phe Ser Glu Tyr Thr Val Val Ala Asp Ile Ser Val Ala Lys Ile 145 15 O 155 160 Asp Pro Ser Ala Pro Leu Asp Llys Val Cys Lieu Lieu Gly Cys Gly Ile 1.65 170 175 Ser Thr Gly Tyr Gly Ala Ala Val Asn Thr Ala Lys Val Glu Pro Gly 18O 185 19 O Ser Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ala Val Ile 195 200 2O5 Met Gly Cys Lys Wall Ala Gly Ala Ser Arg Ile Ile Gly Ile Asp Ile 210 215 220 Asn Lys Asp Llys Phe Ala Lys Ala Lys Glu Phe Gly Ala Ser Glu Cys 225 230 235 240 Ile Ser Pro Glin Asp Phe Ser Lys Ser Ile Glin Glu Val Leu Val Glu 245 250 255 Met Thr Asp Gly Gly Val Asp Tyr Ser Phe Glu Cys Ile Gly Asn Val 260 265 27 O Lys Wal Met Arg Ser Ala Leu Glu Ala Ala His Lys Gly Trp Gly Val 275 280 285 US 2005/0014697 A1 Jan. 20, 2005 38

-continued

Ser Val Val Val Gly Val Ala Ala Ser Gly Glu Glu Ile Ser Thr Arg 29 O 295 3OO Pro Phe Gln Leu Val Thr Gly Arg Thr Trp Lys Gly Thr Ala Phe Gly 305 310 315 320 Gly Trp Llys Ser Val Glu Ser Val Pro Llys Leu Val Ser Glu Tyr Met 325 330 335 Ser Lys Lys Ile Lys Val Asp Glu Phe Val Thr Gly Asn Lieu Ser Phe 340 345 35 O Asp Glin Ile Asn Glin Ala Phe Asp Leu Met His Ser Gly Asp Ser Ile 355 360 365 Arg Thr Val Leu Lys Met 370

<210> SEQ ID NO 21 &2 11s LENGTH 374 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 21 Met Ala Asn. Glu Val Ile Lys Cys Lys Ala Ala Val Ala Trp Glu Ala 1 5 10 15 Gly Lys Pro Leu Ser Ile Glu Glu Ile Glu Val Ala Pro Pro Lys Ala 2O 25 30 His Glu Val Arg Ile Lys Ile Ile Ala Thr Ala Val Cys His Thr Asp 35 40 45 Ala Tyr Thr Leu Ser Gly Ala Asp Pro Glu Gly Cys Phe Pro Val Ile 50 55 60 Leu Gly. His Glu Gly Ala Gly Ile Val Glu Ser Val Gly Glu Gly Val 65 70 75 8O Thr Lys Leu Lys Ala Gly Asp Thr Val Ile Pro Leu Tyr Ile Pro Glin 85 90 95 Cys Gly Glu Cys Lys Phe Cys Lieu. Asn Pro Llys Thr Asn Lieu. Cys Glin 100 105 110 Lys Ile Arg Val Thr Glin Gly Lys Gly Lieu Met Pro Asp Gly Thr Ser 115 120 125 Arg Phe Thr Cys Lys Gly Lys Thr Ile Leu. His Tyr Met Gly Thr Ser 130 135 1 4 0 Thr Phe Ser Glu Tyr Thr Val Val Ala Asp Ile Ser Val Ala Lys Ile 145 15 O 155 160 Asp Pro Leu Ala Pro Leu Asp Llys Val Cys Lieu Lieu Gly Cys Gly Ile 1.65 170 175 Ser Thr Gly Tyr Gly Ala Ala Val Asn. Thir Ala Lys Lieu Glu Pro Gly 18O 185 19 O Ser Val Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ala Val Ile 195 200 2O5 Met Gly Cys Lys Wall Ala Gly Ala Ser Arg Ile Ile Gly Val Asp Ile 210 215 220 Asn Lys Asp Llys Phe Ala Arg Ala Lys Glu Phe Gly Ala Thr Glu Cys 225 230 235 240 Ile Asin Pro Glin Asp Phe Ser Lys Pro Ile Glin Glu Val Lieu. Ile Glu 245 250 255 Met Thr Asp Gly Gly Val Asp Tyr Ser Phe Glu Cys Ile Gly Asn Val US 2005/0014697 A1 Jan. 20, 2005 39

-continued

260 265 27 O Lys Wal Met Arg Ala Ala Leu Glu Ala Cys His Lys Gly Trp Gly Val 275 280 285 Ser Val Val Val Gly Val Ala Ala Ser Gly Glu Glu Ile Ala Thr Arg 29 O 295 3OO Pro Phe Gln Leu Val Thr Gly Arg Thr Trp Lys Gly Thr Ala Phe Gly 305 310 315 320 Gly Trp Llys Ser Val Glu Ser Val Pro Llys Leu Val Ser Glu Tyr Met 325 330 335 Ser Lys Lys Ile Lys Val Asp Glu Phe Val Thr His Asn Lieu Ser Phe 340 345 35 O Asp Glu Ile Asn Lys Ala Phe Glu Lieu Met His Ser Gly Lys Ser Ile 355 360 365 Arg Thr Val Val Lys Ile 370

<210> SEQ ID NO 22 &2 11s LENGTH 378 &212> TYPE PRT <213> ORGANISM: Arabidopsis thaliana <400 SEQUENCE: 22 Thr Glu Gly Lys Pro Ile Arg Cys Lys Ala Ala Ile Lieu Arg Lys Ala 1 5 10 15 Gly Glu Pro Leu Val Ile Glu Glu Ile Glin Val Asp Pro Pro Glin Ala 2O 25 30 Tyr Glu Val Arg Ile Lys Ile Lieu. Cys Thir Ser Lieu. Cys His Thr Asp 35 40 45 Val Thr Phe Trp Lys Leu Asp Ser Gly Pro Leu Ala Arg Phe Pro Arg 50 55 60 Ile Leu Gly His Glu Ala Val Gly Val Val Glu Ser Ile Gly Glu Lys 65 70 75 8O Val Asp Gly Phe Lys Glin Gly Asp Val Val Leu Pro Val Phe His Pro 85 90 95 Glin Cys Glu Glu Cys Lys Glu Cys Ile Ser Pro Lys Ser Asn Trp Cys 100 105 110 Thr Lys Tyr Thr Asn Asp Tyr Leu Ser Asn Thr Arg Arg Tyr Gly Met 115 120 125 Thir Ser Arg Phe Lys Asp Ser Arg Gly Glu Asp Ile His His Phe Ile 130 135 1 4 0 Phe Val Ser Ser Phe Thr Glu Tyr Thr Val Val Asp Ile Ala His Leu 145 15 O 155 160 Wall Lys Ile Ser Pro Glu Ile Pro Val Asp Ile Ala Ala Lieu Lleu Ser 1.65 170 175 Cys Ser Val Ala Thr Gly Lieu Gly Ala Ala Trp Llys Val Ala Asp Wal 18O 185 19 O Glu Glu Gly Ser Thr Val Val Ile Phe Gly Leu Gly Ala Val Gly Leu 195 200 2O5 Ala Val Ala Glu Gly Val Arg Lieu Arg Gly Ala Ala Lys Ile Ile Gly 210 215 220 Val Asp Lieu. Asn Pro Ala Lys Phe Glu Ile Gly Lys Arg Phe Gly Ile 225 230 235 240 US 2005/0014697 A1 Jan. 20, 2005 40

-continued Thr Asp Phe Val Asn Pro Ala Leu Cys Gly Glu Lys Thr Ile Ser Glu 245 250 255 Val Ile Arg Glu Met Thr Asp Val Gly Ala Asp Tyr Ser Phe Glu Cys 260 265 27 O Ile Gly Lieu Ala Ser Leu Met Glu Glu Ala Phe Lys Ser Thr Arg Pro 275 280 285 Gly Ser Gly Lys Thr Ile Val Lieu Gly Met Glu Gln Lys Ala Lieu Pro 29 O 295 3OO Ile Ser Lieu Gly Ser Tyr Asp Leu Lieu Arg Gly Arg Thr Val Cys Gly 305 310 315 320 Thr Lieu Phe Gly Gly Lieu Lys Pro Llys Lieu. Asp Ile Pro Ile Leu Val 325 330 335 Asp Arg Tyr Lieu Lys Lys Glu Lieu. Asn Lieu Glu Asp Lieu. Ile Thr His 340 345 35 O Glu Lieu Ser Phe Glu Glu Ile Asn Lys Ala Phe His Lieu Lieu Ala Glu 355 360 365 Gly Asn. Ser Ile Arg Cys Ile Ile Trp Met 370 375

<210> SEQ ID NO 23 &2 11s LENGTH 378 &212> TYPE PRT <213> ORGANISM: Arabidopsis thaliana <400 SEQUENCE: 23 Thr Glin Gly Lys Val Ile Thr Cys Lys Ala Ala Val Ala Trp Gly Ala 1 5 10 15 Gly Glu Pro Leu Val Met Glu Asp Wall Lys Val Asp Pro Pro Glin Arg 2O 25 30 Leu Glu Val Arg Ile Arg Ile Leu Phe Thr Ser Ile Cys His Thr Asp 35 40 45 Leu Ser Ala Trp Lys Gly Glu Asn. Glu Ala Glin Arg Ala Tyr Pro Arg 50 55 60 Ile Leu Gly His Glu Ala Ala Gly Ile Val Glu Ser Val Gly Glu Gly 65 70 75 8O Val Glu Glu Met Met Ala Gly Asp His Val Leu Pro Ile Phe Thr Gly 85 90 95 Glu Cys Gly Asp Cys Arg Val Cys Lys Arg Asp Gly Ala Asn Lieu. Cys 100 105 110 Glu Arg Phe Arg Val Asp Pro Met Lys Llys Val Met Val Thr Asp Gly 115 120 125 Lys Thr Arg Phe Phe Thr Ser Lys Asp Asn Lys Pro Ile Tyr His Phe 130 135 1 4 0 Leu Asn Thr Ser Thr Phe Ser Glu Tyr Thr Val Ile Asp Ser Ala Cys 145 15 O 155 160 Val Lieu Lys Val Asp Pro Leu Phe Pro Leu Glu Lys Ile Ser Lieu Lieu 1.65 170 175 Ser Cys Gly Val Ser Thr Gly Val Gly Ala Ala Trp Asn. Wall Ala Asp 18O 185 19 O Ile Glin Pro Ala Ser Thr Val Ala Ile Phe Gly Leu Gly Ala Val Gly 195 200 2O5 Leu Ala Val Ala Glu Gly Ala Arg Ala Arg Gly Ala Ser Lys Ile Ile 210 215 220 US 2005/0014697 A1 Jan. 20, 2005 41

-continued

Gly Ile Asp Ile Asn Pro Asp Llys Phe Glin Leu Gly Arg Glu Ala Gly 225 230 235 240 Ile Ser Glu Phe Ile Asn Pro Lys Glu Ser Asp Lys Ala Wal His Glu 245 250 255 Arg Val Met Glu Ile Thr Glu Gly Gly Val Glu Tyr Ser Phe Glu Cys 260 265 27 O Ala Gly Ser Ile Glu Ala Leu Arg Glu Ala Phe Leu Ser Thr Asn. Ser 275 280 285 Gly Val Gly Val Thr Val Met Leu Gly Val His Ala Ser Pro Glin Leu 29 O 295 3OO Leu Pro Ile His Pro Met Glu Leu Phe Glin Gly Arg Ser Ile Thr Ala 305 310 315 320 Ser Val Phe Gly Gly Phe Lys Pro Llys Thr Gln Leu Pro Phe Phe Ile 325 330 335 Thr Glin Cys Lieu Glin Gly Lieu Lieu. Asn Lieu. Asp Leu Phe Ile Ser His 340 345 35 O Glin Leu Pro Phe His Asp Ile Asn. Glu Ala Met Glin Lieu Lieu. His Glin 355 360 365 Gly Lys Ala Lieu Arg Cys Lieu Lieu. His Lieu 370 375

<210> SEQ ID NO 24 &2 11s LENGTH 378 &212> TYPE PRT <213> ORGANISM: Arabidopsis thaliana <400 SEQUENCE: 24 Ser Ser His Lys Pro Ile Arg Cys Lys Ala Ala Val Ser Arg Lys Ala 1 5 10 15 Gly Glu Pro Leu Val Met Glu Glu Ile Met Val Ala Pro Pro Glin Pro 2O 25 30 Phe Glu Val Arg Ile Arg Ile Ile Cys Thr Ala Lieu. Cys His Ser Asp 35 40 45 Val Thr Phe Trp Lys Leu Glin Val Pro Pro Ala Cys Phe Pro Arg Ile 50 55 60 Leu Gly. His Glu Ala Ile Gly Val Val Glu Ser Val Gly Glu Asin Val 65 70 75 8O Lys Glu Val Val Glu Gly Asp Thr Val Leu Pro Thr Phe Met Pro Asp 85 90 95 Cys Gly Asp Cys Val Asp Cys Lys Ser His Lys Ser Asn Lieu. Cys Ser 100 105 110 Lys Phe Pro Phe Lys Val Ser Pro Trp Met Pro Arg Tyr Asp Asin Ser 115 120 125 Ser Arg Phe Thr Asp Leu Asn Gly Glu Thr Leu Phe His Phe Leu Asn 130 135 1 4 0 Val Ser Ser Phe Ser Glu Tyr Thr Val Leu Asp Val Ala Asn Val Val 145 15 O 155 160 Lys Ile Asp Ser Ser Ile Pro Pro Ser Arg Ala Cys Lieu Lleu Ser Cys 1.65 170 175 Gly Val Ser Thr Gly Val Gly Ala Ala Trp Glu Thr Ala Lys Val Glu 18O 185 19 O Lys Gly Ser Thr Val Val Ile Phe Gly Leu Gly Ser Ile Gly Leu Ala US 2005/0014697 A1 Jan. 20, 2005 42

-continued

195 200 2O5 Val Ala Glu Gly Ala Arg Lieu. Cys Gly Ala Ser Arg Ile Ile Gly Val 210 215 220 Asp Ile Asin Pro Thr Lys Phe Glin Val Gly Gln Lys Phe Gly Val Thr 225 230 235 240 Glu Phe Val Asn Ser Met Thr Cys Glu Lys Asn Arg Val Ser Glu Val 245 250 255 Ile Asn. Glu Met Thr Asp Gly Gly Ala Asp Tyr Cys Phe Glu Cys Val 260 265 27 O Gly Ser Ser Ser Lieu Val Glin Glu Ala Tyr Ala Cys Cys Arg Glin Gly 275 280 285 Trp Gly Lys Thr Ile Thr Leu Gly Val Asp Llys Pro Gly Ser Glin Ile 29 O 295 3OO Cys Lieu. Asp Ser Phe Asp Wall Lieu. His His Gly Lys Ile Leu Met Gly 305 310 315 320 Ser Lieu Phe Gly Gly Lieu Lys Ala Lys Thr His Ile Pro Ile Leu Lieu 325 330 335 Lys Arg Tyr Lieu Ser Asn. Glu Lieu Glu Lieu. Asp Llys Phe Val Thr His 340 345 35 O Glu Met Lys Phe Glu Glu Ile Asn Asp Ala Phe Glin Lieu Lleu Lieu Glu 355 360 365 Gly Lys Cys Ile Arg Cys Val Leu Trp Met 370 375

<210> SEQ ID NO 25 &2 11s LENGTH 378 &212> TYPE PRT <213> ORGANISM: Arabidopsis thaliana <400 SEQUENCE: 25 His Val Ser Pro Gly Gly Phe Met Arg Gly Ala Val Tyr Arg Glu Pro 1 5 10 15 Asn Llys Pro Leu Thir Ile Glu Glu Phe His Ile Pro Arg Pro Llys Ser 2O 25 30 Asn Glu Ile Lieu. Ile Lys Thr Lys Ala Cys Gly Val Cys His Ser Asp 35 40 45 Leu. His Val Met Lys Gly Glu Ile Pro Phe Ala Ser Pro Cys Val Ile 50 55 60 Gly His Glu Ile Thr Gly Glu Val Val Glu His Gly Pro Leu Thr Asp 65 70 75 8O His Lys Ile Ile Asn Arg Phe Pro Ile Gly Ser Arg Val Val Gly Ala 85 90 95 Phe Ile Met Pro Cys Gly. Thir Cys Ser Tyr Cys Ala Lys Gly His Asp 100 105 110 Asp Lieu. Cys Glu Asp Phe Phe Ala Tyr Asn Arg Ala Lys Gly. Thir Lieu 115 120 125 Tyr Asp Gly Glu Thr Arg Lieu Phe Leu Arg His Asp Asp Ser Pro Wal 130 135 1 4 0 Tyr Met Tyr Ser Met Gly Gly Met Ala Glu Tyr Cys Val Thr Pro Ala 145 15 O 155 160 His Gly Leu Ala Pro Leu Pro Glu Ser Leu Pro Tyr Ser Glu Ser Ala 1.65 170 175 US 2005/0014697 A1 Jan. 20, 2005 43

-continued Ile Leu Gly Cys Ala Val Phe Thr Ala Tyr Gly Ala Met Ala His Ala 18O 185 19 O

Ala Glu Ile Arg Pro Gly Asp Ser Ile Ala Wall Ile Gly Gly Gly 195 200 2O5

Val Gly Ser Ser Cys Leu Glin Ile Ala Arg Ala Phe Gly Ser Asp 210 215 220

Ile Ile Ala Val Asp Val Glin Asp Asp Teu Glin Lys Thr 225 230 235 240

Leu Gly Ala Thr His Ile Val Asn Ala Ala Glu Asp Wall Glu 245 250 255

Arg Ile Arg Glu Ile Thr Gly Gly Met Gly Asp Wall Wall Glu 260 265

Ala Leu Gly Lys Pro Gln Thr Phe Met Glin Thr Teu Ser Wall 275 280 285

Asp Gly Gly Lys Ala Wal Met Ile Gly Telu Ser Glin Ala Gly Ser Wall 29 O 295

Gly Glu Ile Asp Ile Asn Arg Lieu Wall Arg Arg Ile Wall Ile 305 310 315 320

Gly Ser Tyr Gly Gly Arg Ala Arg Glin Asp Teu Pro Wall Wall 325 330 335

Leu Ala Glu Ser Gly Ile Phe Asn Telu Thr Asn Ala Wall Ser Ser 340 345 35 O

Tyr Lys Phe Glu Asp Ala Gly Lys Ala Phe Glin Asp Teu Asn Glu Gly 355 360 365

Lys Ile Val Ser Arg Gly Val Val Glu Ile 370 375

SEQ ID NO 26 LENGTH 200 TYPE PRT ORGANISM: Homo sapiens <400 SEQUENCE: 26

Met Ala Ala Ile Phe Ala His Ile Ser Wall Teu Glin Ala Gly Asn 1 5 10 15

Lys Arg Llys Val Gly Glu Ile Leu Ile Ser Phe Gly Thr Asp 30

Asp Cys Ala Ile Asn Gly Lys Lieu Thr Phe Telu Telu 35 40

Gly His Glu Ala Ala Gly Ile Val Glu Ser Ile Gly Wall Arg 50 55

Lys Wall Lys Pro Gly Asp Llys Val Ile Telu Glin Gly Ser Asp 65 70 75

Pro Cys Lieu. Ile Pro Lys Gly Asn Ile Cys Asn Glin Phe Asn Phe Asp 85 90 95

Gly Ile Cys Glu Thr Met Ala Asp Asp Thr Thr Arg Phe Thr 100 105 110

Arg Llys Pro Ile Tyr Glin Leu Pro Asn Thr Ser Thr Phe Thr Glu 115 120 125

Thr Val Leu Arg Glu Ser Ala Phe Wall Lys Ile Asp Pro Asp Ala Pro 130 135 1 4 0

Leu Glu Lys Val Lys Phe Phe Lieu Thr Cys Thr Wall Phe Gly Telu Gly 145 15 O 155 160 US 2005/0014697 A1 Jan. 20, 2005 44

-continued

Glu Val Gly Leu Phe Ile Ile Met Gly Cys Lys Ala Thr Gly Ala Ser 1.65 170 175 Arg Ile Ile Gly Ile Asp Ile Asn Arg Asn Lys Phe Lys Lys Ala Ile 18O 185 19 O Ser Lieu Gly Ala Ala Glu Cys Tyr 195 200

<210 SEQ ID NO 27 &2 11s LENGTH 366 &212> TYPE PRT <213> ORGANISM: Homo sapiens <400 SEQUENCE: 27 Thir Thr Gly Glin Val Ile Arg Cys Lys Ala Ala Ile Leu Trp Llys Pro 1 5 10 15 Gly Ala Pro Phe Ser Ile Glu Glu Val Glu Val Ala Pro Pro Lys Ala 2O 25 30 Lys Glu Val Arg Ile Lys Val Val Ala Thr Gly Lieu. Cys Gly Thr Glu 35 40 45 Met Lys Wall Leu Gly Ser Lys His Lieu. Asp Leu Lleu Tyr Pro Thir Ile 50 55 60 Leu Gly. His Glu Gly Ala Gly Ile Val Glu Ser Ile Gly Glu Gly Val 65 70 75 8O Ser Thr Val Lys Pro Gly Asp Llys Val Ile Thr Leu Phe Leu Pro Glin 85 90 95 Cys Gly Glu Cys Thr Ser Cys Lieu. Asn. Ser Glu Gly Asn. Phe Cys Ile 100 105 110 Gln Phe Lys Glin Ser Lys Thr Gln Leu Met Ser Asp Gly Thr Ser Arg 115 120 125 Phe Thr Cys Lys Gly Lys Ser Ile Tyr His Phe Gly Asn Thr Ser Thr 130 135 1 4 0 Phe Cys Glu Tyr Thr Val Ile Lys Glu Ile Ser Val Ala Lys Ile Asp 145 15 O 155 160 Ala Val Ala Pro Leu Glu Lys Val Cys Lieu. Ile Ser Cys Gly Phe Ser 1.65 170 175 Thr Gly Phe Gly Ala Ala Ile Asn Thr Ala Lys Val Thr Pro Gly Ser 18O 185 19 O Thr Cys Ala Val Phe Gly Leu Gly Gly Val Gly Leu Ser Val Val Met 195 200 2O5 Gly Cys Lys Ala Ala Gly Ala Ala Arg Ile Ile Gly Val Asp Wall Asn 210 215 220 Lys Glu Lys Phe Lys Lys Ala Glin Glu Lieu Gly Ala Thr Glu Cys Lieu 225 230 235 240 Asn Pro Glin Asp Leu Lys Lys Pro Ile Glin Glu Val Lieu Phe Asp Met 245 250 255 Thr Asp Ala Gly Ile Asp Phe Cys Phe Glu Ala Ile Gly Asn Lieu. Asp 260 265 27 O Val Lieu Ala Ala Ala Lieu Ala Ser Cys Asn. Glu Ser Tyr Gly Val Cys 275 280 285 Val Val Val Gly Val Leu Pro Ala Ser Val Glin Leu Lys Ile Ser Gly 29 O 295 3OO Gln Leu Phe Phe Ser Gly Arg Ser Leu Lys Gly Ser Val Phe Gly Gly US 2005/0014697 A1 Jan. 20, 2005 45

-continued

305 310 315 320 Trp Llys Ser Arg Glin His Ile Pro Llys Lieu Val Ala Asp Tyr Met Ala 325 330 335 Glu Lys Lieu. Asn Lieu. Asp Pro Lieu. Ile Thr His Thr Lieu. Asn Lieu. Asp 340 345 35 O Lys Ile Asn. Glu Ala Val Glu Lieu Met Lys Thr Gly Lys Trp 355 360 365

<210> SEQ ID NO 28 &2 11s LENGTH 373 &212> TYPE PRT <213> ORGANISM: Schizosaccharomyces pombe <400 SEQUENCE: 28 Phe Glu Gly Lys Thr Ile Thr Cys Lys Ala Ala Val Ala Trp Gly Ala 1 5 10 15 Lys Glu Pro Leu Ser Ile Glu Asp Ile Glin Val Ala Pro Pro Lys Ala 2O 25 30 His Glu Val Arg Val Lys Wall Asp Trp Ser Ala Val Cys His Thr Asp 35 40 45 Ala Tyr Thr Leu Ser Gly Val Asp Pro Glu Gly Ala Phe Pro Ile Val 50 55 60 Leu Gly. His Glu Gly Ala Gly Ile Val Glu Ser Ile Gly Glu Gly Val 65 70 75 8O Ile Asin Val Arg Pro Gly Asp His Val Ile Leu Leu Tyr Thr Pro Glu 85 90 95 Cys Lys Glu Cys Lys Phe Cys Arg Ser Gly Lys Thr Asn Lieu. Cys Ser 100 105 110 Lys Ile Arg Glu Thr Glin Gly Arg Gly Lieu Met Pro Asp Gly Thr Ser 115 120 125 Arg Phe Ser Cys Arg Asp Lys Thr Lieu Lieu. His Tyr Met Gly Cys Ser 130 135 1 4 0 Ser Phe Ser Glin Tyr Thr Val Val Ala Asp Ile Ser Leu Val Ala Ile 145 15 O 155 160 Ser His Ser Ala Pro Leu Arg Ser Ile Cys Lieu Lleu Gly Cys Gly Val 1.65 170 175 Thir Thr Gly Phe Gly Ala Val Thr His Ser Ala Lys Val Glu Ser Gly 18O 185 19 O Ser Thr Val Ala Val Val Gly Cys Gly Cys Val Gly Leu Ala Ala Met 195 200 2O5 Glin Gly Ala Val Ala Ala Gly Ala Ser Arg Ile Ile Ala Ile Asp Ile 210 215 220 Asn Ala Asp Lys Glu Val Tyr Ala Lys Llys Phe Gly Ala Thr Asp Phe 225 230 235 240 Ile Asp Ser Ser Lys Wall Lys Asp Leu Val Glin Tyr Val Ile Asp Wall 245 250 255 Thr Asp Gly Gly Val Asp Tyr Ala Phe Asp Cys Thr Gly Asn Val Thr 260 265 27 O Wal Met Glin Glin Glu Lieu Glin Phe Cys His Lys Gly Trp Gly Lys Lieu 275 280 285 Cys Val Ile Gly Val Ala Ala Ala Gly Lys Thr Lieu. Asp Phe Arg Pro 29 O 295 3OO US 2005/0014697 A1 Jan. 20, 2005 46

-continued Phe Telu Wall Wall Thr Gly Arg Glin Wall Telu Gly Ser Ala Phe Gly Gly 305 310 315 320

Wall Gly Arg Ser Glu Teu Pro Asn Phe Wall Asp Glu Tyr Met Glin 325 330 335

Gly His Phe Lys Wall Asp Glu Tyr Ile Thr Asn Glu Glu Pro Telu 340 345 35 O

Asn Ile Asn Lys Ala Phe Asp His Met His Glu Gly Lys Ile Arg 355 360 365

Wall Wall Asp Met 370

SEQ ID NO 29 LENGTH 374 TYPE PRT ORGANISM: Schizosaccharomyces pombe

<400 SEQUENCE: 29

Thr Ala Gly Lys Ile Ile Asn Lys Ala Ala Wall Ala Trp Glin Pro 1 5 10 15

Ala Ala Pro Telu Ser Ile Glu Asn Wall Glin Wall Phe Pro Pro Arg Wall 25 30

His Glu Wall Arg Ile Lys Ile Wall Asn Ser Gly Wall Cys His Thr Asp 35 40 45

Ala Tyr Thr Telu Ser Gly Lys Asp Pro Glu Gly Teu Phe Pro Wall Ile 50 55 60

Teu Gly His Glu Gly Ala Gly Ile Wall Glu Ser Wall Gly Pro Glin Wall 65 70 75

Thr Thr Wall Glin Wall Gly Pro Wall Ile Ala Teu Thr Pro Glu 85 90 95

Thr Cys Lys Phe Cys Ser Gly Lys Thr Asn Telu Gly 100 105 110

Arg Ile Arg Thr Thr Glin Gly Lys Gly Telu Met Pro Asp Gly Thr Ser 115 120 125

Arg Phe Ser Asn Gly Asn Thr Telu Telu His Phe Met Gly Cys Ser 130 135 1 4 0

Thr Phe Ser Glu Tyr Thr Wall Wall Ala Asp Ile Ser Wall Wall Ala Ile 145 15 O 155 160

Glu Telu Ala Pro Teu Asp Ser Wall Cys Teu Teu Gly Gly Ile 1.65 170 175

Thr Thr Gly Tyr Gly Ala Ala Thr Ile Thr Ala Asp Ile Lys Glu Gly 18O 185 19 O

Asp Ser Wall Ala Wall Phe Gly Telu Gly Ser Wall Gly Teu Ala Wall Ile 195 200

Glin Gly Ala Wall Lys Lys Arg Ala Gly Arg Ile Phe Gly Ile Asp Wall 210 215 220

Asn Pro Glu Lys Asn Trp Ala Met Ser Phe Gly Ala Thr Asp Phe 225 230 235 240

Ile Asn Pro Asn Asp Teu Glin Ser Pro Ile Glin Asp Wall Telu Ile His 245 250 255

Glu Thr Asp Gly Gly Teu Asp Trp Thr Phe Asp Thr Gly Asn Wall 260 265 27 O

His Wall Met Arg Ser Ala Teu Glu Ala Cys His Gly Trp Gly Glin 275 280 285 US 2005/0014697 A1 Jan. 20, 2005 47

-continued

Ser Ile Val Ile Gly Val Ala Ala Ala Gly Glin Glu Ile Ser Thr Arg 29 O 295 3OO Pro Phe Gln Leu Val Thr Gly Arg Val Trp Arg Gly Cys Ala Phe Gly 305 310 315 320 Gly Wall Lys Gly Arg Ser Glin Leu Pro Asp Leu Val Lys Glu Tyr Lieu 325 330 335 Asp His Lys Lieu Glu Ile Asp Llys Tyr Ile Thr His Arg Arg Pro Leu 340 345 35 O Lys Glu Ile Asn. Glu Ala Phe Thr Asp Met His Asn Gly Asn. Cys Ile 355 360 365 Lys Thr Val Leu Ser Ile 370

<210 SEQ ID NO 30 <211& LENGTH: 12 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Peptide <400 SEQUENCE: 30 Arg Arg Arg Glu Glu Glu Glu Glu Ser Ala Ala Ala 1 5 10

<210> SEQ ID NO 31 &2 11s LENGTH 375 &212> TYPE PRT <213> ORGANISM: Artificial &220s FEATURE <223> OTHER INFORMATION: Consensus

<400 SEQUENCE: 31 Thr Ala Gly Lys Val Ile Thr Cys Lys Ala Ala Val Ala Trp Glu Ala 1 5 10 15 Gly Lys Pro Leu Val Ile Glu Glu Ile Glu Val Ala Pro Pro Lys Ala 2O 25 30 His Glu Val Arg Ile Lys Ile Leu Ala Thr Gly Val Cys His Thr Asp 35 40 45 Ala Tyr Val Trp Ser Gly Lys Asp Pro Glu Gly Leu Phe Pro Val Ile 50 55 60 Leu Gly. His Glu Ala Ala Gly Ile Val Glu Ser Val Gly Glu Gly Val 65 70 75 8O Thir Thr Val Lys Pro Gly Asp His Val Ile Pro Leu Phe Thr Pro Glin 85 90 95 Cys Gly Glu Cys Lys Phe Cys Lys Ser Pro Llys Thr Asn Lieu. Cys Glu 100 105 110 Lys Phe Arg Ala Asp Asn Gly Lys Gly Gly Met Pro Tyr Asp Gly Thr 115 120 125 Ser Arg Phe Thr Cys Lys Gly Lys Pro Ile Tyr His Phe Met Gly. Thr 130 135 1 4 0 Ser Thr Phe Ser Glu Tyr Thr Val Val Asp Asp Ile Ser Val Ala Lys 145 15 O 155 160 Ile Asp Pro Ser Ala Pro Leu Glu Lys Val Cys Lieu Lleu Gly Cys Gly 1.65 170 175 Val Ser Thr Gly Tyr Gly Ala Ala Trp Asn Thr Ala Lys Val Glu Pro US 2005/0014697 A1 Jan. 20, 2005 48

-continued

18O 185 19 O

Gly Ser Thr Val Ala Val Phe Gly Leu Gly Gly Val Gly Telu Ala Wall 195 200 2O5

Ala Met Gly Ala Lys Ala Ala Gly Ala Ser Arg Ile Ile Gly Val Asp 210 215 220

Ile Asn Pro Asp Lys Phe Glu Lys Ala Glu Phe Gly Ala Thr Glu 225 230 235 240

Phe Ile Asn Pro Lys Asp Teu Lys Pro Ile Glin Glu Wall Ile Ile 245 250 255

Glu Met Thr Asp Gly Gly Wall Asp Tyr Ser Phe Glu Cys Ile Gly Asn 260 265 27 O

Wall Ser Thr Met Arg Ala Ala Leu Glu Ser Cys His Lys Gly Trp Gly 275 280 285

Ser Wal Wall Ile Gly Wall Ala Ala Ala Gly Glin Glu Ile Ser Thr 29 O 295

Arg Pro Phe Glin Teu Wall Thr Gly Thr Trp Gly Ser Ala Phe 305 310 315 320

Gly Gly Phe Ser Lys Ser Asp Ile Pro Lys Leu Wall Lys Asp Tyr 325 330 335

Met Lys Lys Lieu. Asn Lieu Asp Glu Phe Ile Thr His Glu Leu Pro 340 345 35 O

Phe Glu Glu Ile Asn Lys Ala Phe Asp Leu Leu His Glu Gly Ser 355 360 365

Ile Arg Cys Wall Teu Trp Met 370 375

What is claimed is: Sepsis, Septic shock, cardiogenic shock, endotoxic shock, 1. A method of alleviating at least one Symptom of a toxic shock Syndrome, and Systemic inflammatory response disorder associated with increased levels of nitric oxide Syndrome. bioactivity comprising: administering to a patient with the 7. The method of claim 6, wherein the patient is female. disorder a therapeutically effective amount of an agent that 8. The method of claim 1, wherein the agent decreases increases activity or levels of a S-nitroSoglutathione reduc levels of nitric oxide Synthesis. tase, thereby decreasing levels of nitric oxide bioactivity and 9. The method of claim 1, wherein the agent increases alleviating a Symptom of the disorder. levels of nitric oxide breakdown. 2. The method of claim 1, wherein the disorder is selected 10. The method of claim 1, wherein the agent comprises from the group consisting of degenerative disease, shock, a S-nitroSoglutathione reductase polypeptide Selected from Stroke, Systemic infection, inflammatory disease, and pro the group consisting of SEQ ID NO:17-SEQ ID NO:21. 11. The method of claim 1, wherein the agent comprises liferative disorder. a S-nitroSoglutathione reductase peptide of a polypeptide 3. The method of claim 2, wherein the disorder is selected selected from the group consisting of SEQ ID NO:17-SEQ from the group consisting of colitis, inflammatory bowel ID NO:21. disease, rheumatoid arthritis, osteoarthritis, pSoriatic arthri 12. The method of claim 1, wherein the agent comprises tis, infectious arthritis, ankylosing spondylitis, tendonitis, a S-nitroSoglutathione reductase mimetic Selected from the burSitis, Vasculitis, fibromyalgia, polymyalgia rheumatica, group consisting of a peptide, Small molecule, and anti temporal arteritis, giant cell arteritis, polyarteritis, HIV idiotype antibody mimetic. asSociated rheumatic disease Syndromes, Systemic lupus, 13. The method of claim 1, wherein the agent comprises erythematosus, gout, and calcium pyrophosphate dihydrate a vector for expressing a S-nitroSoglutathione reductase crystal deposition disease. polypeptide Selected from the group consisting of SEQ ID 4. The method of claim 2, wherein the disorder is selected NO:17-SEO ID NO:21. from the group consisting of Parkinson's disease, Alzhe 14. The method of claim 1, wherein the agent comprises imer's disease, and amyotrophic lateral Sclerosis. a vector for expressing a S-nitroSoglutathione reductase 5. The method of claim 2, wherein the proliferative peptide of a polypeptide Selected from the group consisting disorder is cancer. of SEO ID NO:17-SEO ID NO:21. 6. The method of claim 2, wherein the disorder is selected 15. The method of claim 1, wherein the agent is co from the group consisting of bacteremia, Sepsis, neonatal administered with an inhibitor of nitric oxide synthase. US 2005/0014697 A1 Jan. 20, 2005 49

16. The method of claim 15, wherein the inhibitor of nitric reductase, thereby decreasing levels of nitric oxide bioac oxide Synthase is Selected from the group consisting of tivity and alleviating a Symptom of hypotension. L-N(6)-(1-iminoethyl)lysine tetrazole-amide (SC-51); ami 30. The method of claim 29, wherein the hypotension is noguanidine (AG); S-methilisourea (SMT); S-(2-Aminoet asSociated with anesthesia, dialysis, and Orthostatic hypoten hyl)isothiourea; 2-Amino-5,6-dihydro-6-methyl-4H-1,3-thi Sion. azine (AMT), L-2-Amino-4-(guanidiooxy)butyric acid (L-Canavanine Sulphate); S-Ethylisothiourea (EIT); 2-Imi 31. The method of claim 29, wherein the agent decreases nopiperidine, S-Isopropylisothiourea; 1,4-phenylenebis(1,2- levels of nitric oxide Synthesis. ethanediyl)-diisothiourea (PBIT); N-3-(aminomethyl)ben 32. The method of claim 29, wherein the agent increases zyl)acetamidine (1400W); N6-(1-Iminoethyl)-L-lysine levels of nitric oxide breakdown. (L-NIL); monomethyl arginine; and 7-Nitroindazole. 33. The method of claim 29, wherein the agent comprises 17. A method of alleviating at least one Symptom of a a S-nitroSoglutathione reductase polypeptide Selected from Systemic infection comprising: administering to a patient the group consisting of SEQ ID NO:17-SEQ ID NO:21. with the infection a therapeutically effective amount of an 34. The method of claim 29, wherein the agent comprises agent that increases activity or levels of a S-nitroSoglu a S-nitroSoglutathione reductase peptide of a polypeptide tathione reductase, thereby decreasing levels of nitric oxide selected from the group consisting of SEQ ID NO:17-SEQ bioactivity and alleviating a Symptom of the infection. ID NO:21. 18. The method of claim 17, wherein the systemic infec 35. The method of claim 29, wherein the agent comprises tion is Selected from the group consisting of bacteremia, a S-nitroSoglutathione reductase mimetic Selected from the Sepsis, neonatal Sepsis, Septic shock, cardiogenic shock, group consisting of a peptide, Small molecule, and anti endotoxic shock, toxic shock Syndrome, and Systemic idiotype antibody mimetic. inflammatory response Syndrome. 36. The method of claim 29, wherein the agent comprises 19. The method of claim 18, wherein the patient is female. a vector for expressing a S-nitroSoglutathione reductase 20. The method of claim 17, wherein the agent decreases polypeptide Selected from the group consisting of SEQ ID levels of nitric oxide Synthesis. NO:17-SEO ID NO:21. 21. The method of claim 17, wherein the agent increases 37. The method of claim 29, wherein the agent comprises levels of nitric oxide breakdown. a vector for expressing a S-nitroSoglutathione reductase 22. The method of claim 17, wherein the agent comprises peptide of a polypeptide Selected from the group consisting a S-nitrosoglutathione reductase polypeptide selected from of SEQ ID NO:17-SEQ ID NO:21. the group consisting of SEQ ID NO:17-SEQ ID NO:21. 38. The method of claim 29, wherein the agent is co 23. The method of claim 17, wherein the agent comprises administered with an inhibitor of nitric oxide synthase. a S-nitroSoglutathione reductase peptide of a polypeptide 39. The method of claim 38, wherein the inhibitor of nitric selected from the group consisting of SEQ ID NO:17-SEQ oxide Synthase is Selected from the group consisting of ID NO:21. L-N(6)-(1-iminoethyl)lysine tetrazole-amide (SC-51); ami noguanidine (AG); S-methilisourea (SMT); S-(2-Aminoet 24. The method of claim 17, wherein the agent comprises hyl)isothiourea; 2-Amino-5,6-dihydro-6-methyl-4H-1,3-thi a S-nitroSoglutathione reductase mimetic Selected from the azine (AMT); L-2-Amino-4-(guanidiooxy)butyric acid group consisting of a peptide, Small molecule, and anti (L-Canavanine Sulphate); S-Ethylisothiourea (EIT); 2-Imi idiotype antibody mimetic. nopiperidine, S-Isopropylisothiourea; 1,4-phenylenebis(1,2- 25. The method of claim 17, wherein the agent comprises ethanediyl)-diisothiourea (PBIT); N-3-(aminomethyl)ben a vector for expressing a S-nitroSoglutathione reductase zyl)acetamidine (1400W); N6-(1-Iminoethyl)-L-lysine polypeptide Selected from the group consisting of SEQ ID (L-NIL); monomethyl arginine; and 7-Nitroindazole. NO:17-SEO ID NO:21. 40. A method of alleviating at least one Symptom of a 26. The method of claim 17, wherein the agent comprises vascular disorder comprising: administering to a patient a vector for expressing a S-nitroSoglutathione reductase Suffering from the disorder a therapeutically effective peptide Selected from the group consisting of SEQ ID amount of an agent that decreases activity or levels of a NO:17-SEO ID NO:21. S-nitroSoglutathione reductase, thereby increasing levels of 27. The method of claim 17, wherein the agent is co S-nitroSothiols and alleviating a Symptom of the disorder. administered with an inhibitor of nitric oxide synthase. 41. The method of claim 40, wherein the disorder is 28. The method of claim 27, wherein the inhibitor of nitric Selected from the group consisting of hypertension, heart oxide Synthase is Selected from the group consisting of failure, pulmonary hypertension, atherosclerosis, restenosis, L-N(6)-(1-iminoethyl)lysine tetrazole-amide (SC-51); ami asthma, and impotence. noguanidine (AG); S-methilisourea (SMT); S-(2-Aminoet hyl)isothiourea; 2-Amino-5,6-dihydro-6-methyl-4H-1,3-thi 42. The method of claim 40, wherein the agent comprises azine (AMT), L-2-Amino-4-(guanidiooxy)butyric acid an antibody or antibody fragment that binds to a S-nitroSo (L-Canavanine Sulphate); S-Ethylisothiourea (EIT); 2-Imi glutathione reductase. nopiperidine, S-Isopropylisothiourea; 1,4-phenylenebis(1,2- 43. The method of claim 42, wherein the agent comprises ethanediyl)-diisothiourea (PBIT); N-3-(aminomethyl)ben a monoclonal antibody. zyl)acetamidine (1400W); N6-(1-Iminoethyl)-L-lysine 44. The method of claim 40, wherein the agent comprises (L-NIL); monomethyl arginine; and 7-Nitroindazole. an antisense or Small interfering RNA sequence. 29. A method of alleviating at least one symptom of 45. The method of claim 40, wherein the agent comprises hypotension comprising: administering to a patient with a Small molecule. hypotension a therapeutically effective amount of an agent 46. The method of claim 40, wherein the agent is co that increases activity or levels of a S-nitroSoglutathione administered with a phosphodiesterase inhibitor. US 2005/0014697 A1 Jan. 20, 2005 50

47. The method of claim 46, wherein the phosphodi paring the levels of the S-nitroSoglutathione reductase in the esterase inhibitor is Selected from the group consisting of biological Sample to levels in a control Sample; and (c) rolipram, cilomilast, roflumilast, Sildenifil citrate, tadalafil, determining if the levels of the S-nitrosoglutathione in the and Vardenifil. biological Sample are altered compared to the levels of the 48. A method of diagnosing a disorder associated with S-nitroSoglutathione in the control Sample, thereby moni increased levels of nitric oxide bioactivity comprising: (a) toring the condition of the patient. measuring levels of a S-nitroSoglutathione reductase in a 64. The method of claim 63, wherein the systemic infec biological Sample from a patient; (b) comparing the levels of tion is Selected from the group consisting of bacteremia, the S-nitroSoglutathione reductase in the biological Sample Sepsis, neonatal Sepsis, Septic shock, cardiogenic shock, to levels in a control Sample; and (c) determining if the endotoxic shock, toxic shock Syndrome, and Systemic levels of the S-nitroSoglutathione in the biological Sample inflammatory response Syndrome. are lower than the levels of the S-nitrosoglutathione in the 65. The method of claim 64, wherein the patient is female. control Sample, thereby diagnosing the disorder. 66. The method of claim 63, wherein the levels of the 49. The method of claim 48, wherein the disorder is S-nitroSoglutathione reductase in the biological Sample are Selected from the group consisting of degenerative disease, determined using an antibody that binds to an antigen vascular disease, shock, Stroke, Systemic infection, and Selected from the group consisting of a S-nitroSoglutathione proliferative disease. reductase antigen and a S-nitroSothiol antigen. 50. The method of claim 49, wherein the disorder is 67. The method of claim 66, wherein the antibody is a Selected from the group consisting of colitis, inflammatory monoclonal antibody. bowel disease, rheumatoid arthritis, osteoarthritis, psoriatic 68. The method of claim 66, wherein the antibody is arthritis, infectious arthritis, ankylosing spondylitis, ten labeled. donitis, burSitis, vasculitis, fibromyalgia, polymyalgia rheu 69. The method of claim 63, wherein the levels of the matica, temporal arteritis, giant cell arteritis, polyarteritis, S-nitroSoglutathione reductase in the biological Sample are HIV-associated rheumatic disease Syndromes, Systemic determined using a nucleic acid probe that binds to a lupus, erythematosus, gout, and calcium pyrophosphate S-nitroSoglutathione reductase nucleotide Sequence. dihydrate crystal deposition disease. 70. The method of claim 69, wherein the nucleic acid 51. The method of claim 49, wherein the disorder is probe is a DNA probe. Selected from the group consisting of Parkinson's disease, 71. The method of claim 69, wherein the nucleic acid Alzheimer's disease, and amyotrophic lateral Sclerosis. probe is labeled. 52. The method of claim 49, wherein the disorder is 72. The method of claim 63, wherein the levels of the CCC. S-nitroSoglutathione reductase in the biological Sample are 53. The method of claim 49, wherein the disorder is determined using an assay for S-nitroSoglutathione reduc Selected from the group consisting of pulmonary hyperten tase enzyme activity. Sion and atherosclerosis. 73. A transgenic non-human mammal whose genome 54. The method of claim 49, wherein the disorder is comprises a disruption of the endogenous GSNOR gene, Selected from the group consisting of bacteremia, Sepsis, wherein the disruption comprises the insertion of a Select neonatal Sepsis, Septic Shock, endotoxic Shock, toxic shock able marker Sequence, and wherein the disruption results in Syndrome, and Systemic inflammatory response Syndrome. the mouse exhibiting an increase in nitrosylation compared 55. The method of claim 54, wherein the patient is female. to a wild-type mouse. 56. The method of claim 48, wherein the levels of the 74. The transgenic non-human mammal of claim 73, S-nitroSoglutathione reductase in the biological Sample are wherein the nitrosylation occurs intracellularly or extracel determined using an antibody that binds to an antigen lularly. Selected from the group consisting of a S-nitroSoglutathione 75. The transgenic non-human mammal of claim 73, reductase antigen and a S-nitroSothiol antigen. wherein the increase in nitrosylation results in an accumu 57. The method of claim 56, wherein the antibody is a lation of S-nitrosothiols. monoclonal antibody. 76. The transgenic non-human mammal of claim 73, 58. The method of claim 56, wherein the antibody is wherein the disruption is a homozygous disruption. labeled. 77. The transgenic non-human mammal of claim 76, 59. The method of claim 48, wherein the levels of the wherein the homozygous disruption results in a null muta S-nitroSoglutathione reductase in the biological Sample are tion of the endogenous gene encoding S-nitroSoglutathione determined using a nucleic acid probe that binds to a reductase. S-nitroSoglutathione reductase nucleotide Sequence. 78. The transgenic non-human mammal of claim 73, 60. The method of claim 59, wherein the nucleic acid wherein the Selectable marker is a neomycin resistance gene. probe is a DNA probe. 79. An isolated nucleic acid comprising a GSNOR knock 61. The method of claim 59, wherein the nucleic acid out construct comprising a Selectable marker Sequence probe is labeled. flanked by DNA sequences homologous to the endogenous 62. The method of claim 48, wherein the levels of the GSNOR gene. S-nitroSoglutathione reductase in the biological Sample are 80. A vector comprising the nucleic acid of claim 79. determined using an assay for S-nitroSoglutathione reduc 81. A mammalian cell line comprising the GSNOR tase enzyme activity. knockout construct of claim 79. 63. A method of monitoring a condition of a patient 82. A non-human mammal embryonic Stem cell line exhibiting one or more Symptoms of a Systemic infection comprising the GSNOR knockout construct of claim 79. comprising: (a) measuring levels of a S-nitrosoglutathione 83. A method for identifying an agent for alleviating at reductase in a biological Sample from the patient; (b) com least one Symptom of a Systemic infection or hypotension US 2005/0014697 A1 Jan. 20, 2005 comprising: (a) administering a test agent to a GSNOR Sepsis, neonatal Sepsis, Septic shock, cardiogenic endotoxic knockout mouse with a Systemic infection or hypotension, Shock, toxic shock Syndrome, and Systemic inflammatory and (b) determining whether the test agent alleviates a response Syndrome. Symptom of the Systemic infection or hypotension in the 87. The method of claim 83, wherein the hypotension is knockout mouse. due to anesthesia. 84. The method of claim 83, wherein the symptom is an increase in nitrosylation. 88. The method of claim 87, wherein the anesthesia is 85. The method of claim 84, wherein the increase in Selected from the group consisting of phenobarbitol, ket nitrosylation results in an accumulation of S-nitroSothiols. amine Xylazine, and urethane. 86. The method of claim 83, wherein the systemic infec tion is Selected from the group consisting of bacteremia,