Oncogene (1998) 17, 1617 ± 1623  1998 Stockton Press All rights reserved 0950 ± 9232/98 $12.00 http://www.stockton-press.co.uk/onc The ras-related GTPase rac1 regulates a proliferative pathway selectively utilized by G-protein coupled receptors

Ethan S Burstein1, Diane J Hesterberg1, J Silvio Gutkind2, Mark R Brann1, Erika A Currier1 and Terri L Messier1

1ACADIA Pharmaceuticals Inc., 3911 Sorrento Valley Blvd., San Diego, California 92121; 2Molecular Signaling Unit, Oral and Pharyngeal Cancer Branch, National Institute of Dental Research, National Institutes of Health, 9000 Rockville Pike, Bethesda, Maryland 20892-4330, USA

Ras and rac are each members of the superfamily of GTPases can be constitutively activated by mutations monomeric GTPases and both function as molecular which impair GTPase activity (Bourne et al., 1991; switches to link cell-surface signals to intracellular Lowy and Willumsen, 1993; Barbacid, 1987), while responses. Using a novel assay of cellular proliferation dominant negative alleles can be created by mutations called R-SATTM (Receptor Selection and Ampli®cation that render these proteins unable to bind GTP (Feig Technology), we examined the roles of ras and rac in and Cooper, 1988). The ras proteins regulate a wide mediating the proliferative responses to a variety of cell- variety of cellular processes including growth and surface receptors. Activated, wild-type and dominant- di€erentiation, and constitutively activated ras is negative mutants of rac and ras were tested for their oncogenic. The rac proteins control cytoskeletal e€ects on cellular proliferation either alone or in assembly (Tapon and Hall, 1997) and the exchange combination with receptors. Activated rac (rac Q61L, factors that activate rac are also oncogenic (Hart et al., henceforth rac*) and ras (ras G12V, henceforth ras*) 1994; Horii et al., 1994; Cerione and Zheng 1996) each induced strong proliferative responses. Dominant- implying that like ras, rac also regulates mitogenic negative rac (rac T17N, henceforth rac(7)) dramatically signaling pathways. suppressed proliferative responses to G-protein coupled Three distinct protein phosphorylation cascades receptors (GPCR's) including the m5 muscarinic have been described which mediate intracellular receptor and the a1B adrenergic receptor. In contrast, responses to extracellular signals (Denhardt, 1996), rac(7) had little or no e€ect upon responses to the including the MAP/ERK kinase cascade (Marshall, tyrosine kinase receptor TrkC, and only partially 1995; Davis, 1993) which is preferentially activated by suppressed responses to the Janus kinase (JAK/STAT) mitogens and growth factors and the SAPK/JNK and/ linked granulocyte macrophage colony stimulating factor or p38 kinase cascades which are preferentially (GM-CSF) receptor. Dominant-negative ras (ras T17N, activated by cytokines and cellular stress (Kyriakis et henceforth ras(7)) blocked the proliferative responses to al., 1994; Derijard et al., 1994). The MAP/ERK kinase all of the tested receptors. Compared to rac(7) and cascade is linked to inputs from cell surface receptors ras(7), wild-type rac and ras had only modest e€ects on by ras while rac and rac-related GTPases activate the the tested receptors. Overall these results demonstrate SAPK/JNK pathway (Coso et al., 1995; Minden et al., that rac mediates the proliferative e€ects of G-protein 1995). coupled receptors through a pathway that is distinct from The molecular links between cell surface receptors the proliferative signaling pathway utilized by tyrosine and intracellular kinase cascades is the subject of kinase linked and JAK-linked receptors. intensive research. The pathways utilized by growth factor receptors which have tyrosine kinase activity are Keywords: rac; ras; G-protein coupled receptor; currently the best understood (Schlessinger, 1993). tyrosine kinase linked receptor; JAK/STAT linked Upon ligand binding, the tyrosine kinase linked receptor receptors dimerize, become autophosphorylated, and recruit signaling molecules including adaptor proteins such as Grb2 and Shc. This receptor/adaptor complex can then bind ras exchange factors which catalyze the Introduction exchange of GTP for GDP, leading to activation of ras which in turn activates a variety of cellular e€ectors Rac and ras are each members of the superfamily of including the MAP/ERK kinase cascade. monomeric GTPases (Hall, 1990; 1994). These Cytokine receptors signal using a similar strategy GTPases function as molecular switches which are (Ihle, 1995). Ligand binding causes receptor aggrega- activated upon binding GTP, and become deactivated tion which promotes association of the receptors with by hydrolyzing GTP to GDP. The monomeric intracellular tyrosine kinases called JAKs (Janus kinases). The JAKs can then directly activate a family of transcription factors called STATs (Signal Transdu- cers and Activators of Transcription). Thus, these receptors are also known as JAK/STAT linked Correspondence: ES Burstein receptors. The JAK/STAT receptors induce tyrosine Received 8 December 1997; revised 20 April 1998; accepted 21 April phosphorylation of vav which has high homology to 1998 exchange factors that activate rac-related GTPases. Rac-regulated proliferative pathways ES Burstein et al 1618 The JAK/STAT linked receptors also recruit many of proliferation (Denhardt, 1996). The monomeric the same signaling molecules the tyrosine kinase GTPase ras plays a pivotal role in linking cell surface receptors utilize, and have been shown to activate ras. receptors to intracellular signaling pathways and Compared to the tyrosine-kinase and JAK/STAT recently it has become clear that the ras-related linked receptors, less is known about the signaling GTPase rac performs similar functions. We therefore pathways linking GPCR's to proliferative responses. tested activated, wild-type and dominant negative GPCR's have been shown to stimulate the phosphor- mutants of rac and ras for their e€ects on cellular ylation of both MAPK's and JNK's (Gardener et al., proliferation either alone or in combination with 1993; Coso et al., 1995b), however it is not apparent receptors. As shown in Figure 1, both rac and which pathways are required by GPCR's to generate constitutively activated rac (rac*, see Materials and mitogenic signals, nor is it clear how they are linked to methods) induced signi®cant proliferative responses, kinase cascades. We have developed a high throughput amplifying cells up to threefold over control values. functional assay called R-SAT (Receptor Selection and The response was dose-dependent, and was greater for Ampli®cation Technology, see Burstein et al., 1995) rac* than rac. For comparison, wild-type and activated which measures proliferative responses of cultured versions of the heterotrimeric G-protein Ga13, which is cells. Using R-SAT, we have found that compared to oncogenic (Dhanasekaran and Dermott, 1996; Voyno- other cell surface receptors, G-protein coupled Yasenetskaya et al., 1994) and is thought to regulate receptors are selectively linked by rac to proliferative rac-dependent signaling pathways (Voyno-Yasenets- signaling pathways. kaya et al., 1996; Vara Prasad et al., 1995), were also tested. Ga13* induced a response very similar in magnitude to rac*. As expected ras* induced very Results strong proliferative responses. We next tested the e€ects of co-expressing wild-type G-protein coupled receptors (GPCR's), tyrosine and dominant negative versions of rac and ras upon kinase-linked receptors and JAK/STAT-linked recep- the cellular responses induced by m5 muscarinic tors each activate intracellular kinase cascades, which receptor. As shown in Figure 2, NIH3T3 cells in turn mediate complex cellular responses such as transfected with the m5 muscarinic receptor (Bonner

Figure 1 Stimulation of proliferative responses by G-proteins. NIH3T3 cells were transfected with the indicated amounts of G- Figure 2 E€ects of ras and rac on proliferative responses to the protein, 500 ng of b-galactosidase and sucient control vector to m5 muscarinic receptor. NIH3T3 cells were transfected with 40 bring the total pool to 1 mg DNA. Transfected cells were cultured nanograms of receptor. 500 ng of b-galactosidase, 800 ng of G- and assayed by R-SATTM as described in the Materials and protein and sucient control vector to bring the total pool to methods. Rac*, ras* and Ga13* are constitutively activated by 1.5 mg DNA. Transfected cells were cultured with the indicated mutations (to rac Q61L, ras G12V and Ga13 Q226L respectively) concentrations of carbachol and assayed by R-SATTM as which impair their intrinsic GTPase activities (see Bourne et al., described in Materials and methods. Rac(7) and ras(7) are 1991). Fold stimulation was calculated as the ratio of G-protein each dominant negative mutants which contain the mutation response to control response. Control transfections consisted of b- T17N, rendering these proteins unable to bind GTP (see Feig and gal and control vector Cooper, 1988) Rac-regulated proliferative pathways ES Burstein et al 1619

Figure 3 E€ects of ras and rac on proliferative responses to receptors. NIH3T3 cells were transfected with 40 nanograms of receptor, 500 ng of b-galactosidase, 800 ng of G-protein and sucient control vector to bring the total pool to 1.5 mg DNA. Transfected cells were cultured in the presence of the indicated concentrations of ligand and assayed by R-SATTM as described in the Materials and methods. Open circles, receptor; open squares, receptor + rac; ®lled squares, receptor + rac(7); open triangles, receptor + ras; ®lled triangles, receptor + ras(7). Rac(7) and ras(7) are each dominant negative mutants which contain the mutation T17N, rendering these proteins unable to bind GTP (see Feig and Cooper 1988). (a) TrkC neurotrophin receptor assayed with the indicated concentrations of neurotrophin-3 (NT-3.) (b) Alpha 1B adrenergic receptor assayed with the indicated concentrations of phenylephrine. (c) Granulocyte macrophage colony stimulating factor receptor assayed with the indicated concentrations of granulocyte macrophage colony stimulating factor et al., 1988) and b-galactosidase were ampli®ed in the signi®cantly reduced e€ect on the GM-CSF receptor. presence of the muscarinic agonist carbachol compared Co-expression of ras(7) blocked the proliferative with cells grown in the absence of ligand. Co- responses to all of the tested receptors. Wild-type transfection of m5 with rac(7) markedly reduced the rac had modest inhibitory e€ects on a1B and TrkC proliferative response observed at all tested concentra- and a slight stimulatory e€ect on GM-CSF (see Table tions of carbachol (Figure 2, Table 1). Similar results 1). Co-transfection of ras consistently raised the basal were obtained with ras(7). Co-transfection of m5 with (ligand-free) response, but had little or no e€ect on wild-type rac did not block cellular responses to m5, the ligand-dependent responses of a1B and TrkC, and raised the baseline slightly, possibly due to direct though it paradoxically inhibited responses to GM- stimulation of proliferation by rac itself. In all cases, CSF. the observed reductions in maximum response values To rule out the possibility that the di€erential e€ects were not accompanied by large changes in EC50 values, of rac(7) and ras(7) were due to transfection suggesting the e€ects of rac(7) and ras(7) were distal artifacts, or general e€ects on cellular viability, a1B and not directly at the receptor level. Radioligand and TrkC were co-transfected and assayed with rac, binding studies indicated that co-expression of rac(7) ras, rac(7), ras(7) or control vector (Figure 4). had little or no e€ect on the expression of m5 receptors Virtually identical results were obtained for each (2.9+0.3 fmol/mg for m5 versus 3.5+0.3 fmol/mg for receptor compared to the results when the receptors m5 + rac(7)), thus the rac(7) induced blockade was were tested separately. Again rac(7) markedly not due to decreased expression of m5. inhibited the a1B induced response but had little or A number of other receptors were tested to examine no e€ect upon the TrkC induced response in the whether or not the inhibitory e€ects of rac(7)and presence of their respective ligands. Similarly, the ras(7) were speci®c to G-protein coupled receptors. results obtained with the GM-CSF co-transfected with As shown in Figure 3, NIH3T3 cells transfected with a1B receptor were essentially unchanged compared to either the a1B adrenergic receptor (a GPCR, see the outcome when these receptors were tested Cotecchia et al., 1988), the TrkC neurotrophin separately (data not shown). Thus the selective e€ects receptor (a tyrosine kinase receptor, see Tsoulfas et of rac upon a1B-generated proliferative responses are al., 1993), or the granulocyte/macrophage colony not due to transfection artifacts or a general reduction stimulating factor receptor (GM-CSF, a JAK/STAT of cellular viability. linked receptor, see Areces et al., 1993) were ampli®ed The e€ects of varying the amount of G-protein, 6 to 8-fold in the presence of the appropriate agonist receptor, or ratio of receptor to G-protein were ligands compared with cells grown in the absence of examined to quantify the e€ects of rac and ras upon ligand. Co-expression of rac(7) dramatically sup- receptor signaling (see Table 1). The rac-mediated and pressed the proliferative response to the a1B ras-mediated inhibition of a1B signaling was very adrenergic receptor, similar to results shown above similar across most of the doses tested. In each case, for the m5 receptor. Strikingly, rac(7) had little or no a twofold excess of the GTPase was sucient to e€ect on the response to the TrkC receptor, and a achieve 70% inhibition suggesting that rac and ras do Rac-regulated proliferative pathways ES Burstein et al 1620 Table 1 E€ects of ras and rac on proliferative responses to receptors

Condition G-protein (ng) response % EC50 ratio m5 ± 100 1 m5 + rac 800 117 0.3 m5 + rac (7) 800 12 1 m5 + ras (7) 800 13 2 Alpha1B ± 100 1 Alpha1B+rac 1000 85 1 Alpha1B+rac(7) 50 54 6 Alpha1B+rac(7) 100 29 3 Alpha1B+rac(7) 200 24 4 Alpha1B+rac(7) 400 23 4 Alpha1B+rac(7) 800 15 6 Alpha1B+rac(7) 1000 10 4 Alpha1B+ras 1000 105 2 Alpha1B+ras(7) 50 30 1 Alpha1B+ras(7) 100 30 2 Alpha1B+ras(7) 200 33 4 Alpha1B+ras(7) 400 25 5 Alpha1B+ras(7) 800 17 10 Alpha1B+ras(7) 1000 8 14 TrkC ± 100 1 TrkC+rac 800 54 1 TrkC+rac(7) 800 99 2 TrkC+ras 800 109 1 TrkC+ras(7) 50 74 3 TrkC+ras(7) 100 51 5 TrkC+ras(7) 200 40 4 TrkC+ras(7) 400 17 7 TrkC+ras(7) 800 11 3

TrkC (2 ng) ± 100 1 TrkC (2 ng)+rac(7) 800 97 3 TrkC (2 ng)+ras(7) 800 1 nd

TrkC (5ng) ± 100 1 TrkC (5ng)+rac(7) 800 121 1 TrkC (5ng)+ras(7) 800 2 nd GM-CSF ± 100 1 GM-CSF+rac 800 120 0.7 GM-CSF+rac(7) 800 50 0.7 GM-CSF+ras 800 60 0.01 GM-CSF+ras(7) 800 5 0.1 Note: nd indicates could not be determined. NIH3T3 cells were transfected with forty nanograms of receptor (except where noted), 500 ng of b- galactosidase, the indicated amounts of G-protein and sucient control vector to bring the total pool to 1.5 mg DNA. Transfected cells were cultured and assayed by R-SATTM as described in the Materials and methods. Responses to receptor/G-protein combinations were normalized to the responses of the individual receptors. EC50's of the receptor/G-protein combinations were divided by the EC50 of the individual receptors to derive the EC50 ratio. Values represent means of 2 ± 4 experiments. Rac(7) and ras(7) are each dominant negative mutants which contain the mutation T17N, rendering these proteins unable to bind GTP ( see Feig and Cooper 1988)

not di€er greatly in their abilities to inhibit a1B. The Discussion amount of ras(7) required to achieve 50% inhibition of TrkC was at least twice what was needed to inhibit We have demonstrated that G-protein coupled a1B, although the amount of ras needed to achieve receptors are selectively linked by the ras-related over 80% inhibition was greater for a1B (800 ng) than GTPase rac to proliferative signaling pathways. for TrkC (400 ng). To investigate whether the inability Dominant negative rac selectively inhibits proliferative of rac(7) to inhibit responses to TrkC was because the responses to the a1B adrenergic receptor and the m5 TrkC induced signal was inherently stronger than the muscarinic receptor, which are G-protein coupled a1B induced signal, the amount of TrkC transfected receptors, but does not block proliferative responses was reduced 20-fold and tested in the presence and to the TrkC tyrosine kinase receptor, and only partially absence of a 400-fold excess of rac(7). Under these suppresses responses to the JAK/STAT linked GM- conditions, the TrkC response was reduced to under CSF receptor. Furthermore, rac itself stimulates half the a1B response (data not shown), yet rac(7) still proliferative responses. had little or no e€ect upon TrkC signaling, whereas the The molecular links between G-protein coupled same amount of ras completely suppressed the TrkC receptors and the kinase cascades are not currently mediated signal (Table 1). Thus the selective ability of understood but the proximal step is receptor catalyzed rac(7) to suppress a1B induced signals is most likely exchange of GTP for GDP followed by dissociation of because a1B utilizes a rac-dependent signal transduc- the a- and bg-subunits of G-proteins (Rens-Domiano tion pathway that is distinct from the pathway utilized and Hamm, 1995). a- and bg-subunits each activate by TrkC. e€ector enzymes and each activates proliferative Rac-regulated proliferative pathways ES Burstein et al 1621

Figure 4 Multiplexed assay of the e€ects of ras and rac on proliferative responses to the Alpha 1B adrenergic receptor and the TrkC neurotrophin receptor. NIH3T3 cells were transfected and assayed as described for Figure 2 except that 40 ng of each receptor were co- transfected into the same dish of cells. Cells were trypsinized, divided and aliquoted into the wells of a 96-well rack as described in the Materials and methods and cultured in the presence of the indicated concentrations of either phenylephrine or neurotrophin-3 (NT-3). Control experiments have veri®ed that there is no cross-reactivity of either ligand with either receptor (data not shown) pathways (Clapham and Neer, 1993). The heterotri- (Mansour et al., 1994; Cowley et al., 1994), and Jun is meric G-proteins Gaq, Ga12 and Ga13 are each required for transformation by ras (Smeal et al., 1991), capable of transforming NIH3T3 cells (Dhanasekaran thus it is possible that either MAPK or JNK pathways, and Dermott, 1996; Voyno-Yasenetskaya et al., 1994; or both together are utilized to elicit mitogenic Chan et al., 1993; Xu et al., 1993; Jiang et al., 1993; responses. The ser/thr kinase raf has been implicated Kalinec et al., 1992) and we have previously shown in transformation by m1 receptors (Crespo et al., that Gaq but not Ga12 is responsible for transducing 1994b), suggesting a MAPK mediated proliferative m5-induced proliferative responses in NIH3T3 cells signal. MEKK1, which preferentially activates SEK of (Burstein et al., 1995b; 1997). It has been recently the JNK pathway but is capable of activating MEK shown that GTPase de®cient mutants of Gaq, Ga12 (Yan et al., 1994; Minden et al., 1994) binds to ras and Ga13 activate the JNK pathway (Voyno- (Russell et al., 1995). Thus intersection of these distinct Yasenetskaya et al., 1996; Vara Prasad et al., 1995) pathways may occur at the level of ras or MEKK to and we have found that Gaq, Ga12 and Ga13 can each generate mitogenic responses. Consistent with these induce proliferative responses in R-SATTM (Figure 1 observations that both kinase cascades contribute to and see Burstein et al., 1997). Thus, although bg proliferative responses is the evidence that ras requires subunits have been implicated in MAP kinase pathway rac1 and other rac-related GTPases to fully transform activation (Crespo et al., 1994), the available evidence cells (Khosravi-Far et al., 1995). suggests that the Ga subunit probably transduces It is likely that the dbl-related family of genes, proliferative responses along the signaling pathway including dbl, ost and vav (Cerione and Zheng, 1996) regulated by rac. serve as molecular links immediately upstream of rac1 Both JunK and MAP kinase pathways have been linking it to proliferative signals emanating from G- implicated in transformation of 3T3 cells. The m1 protein coupled receptors. The dbl proteins are muscarinic receptor, which transforms NIH3T3 cells in themselves oncogenic, stimulate JNK activation and response to agonist stimulation (Gutkind et al., 1991), many show exchange factor activity towards the rac- has been shown to activate both the MAP kinase and related GTPases (Hart et al., 1994; Horii et al., 1994; Jun kinase cascades (Coso et al., 1995b), which are Cerione and Zheng, 1996; Denhardt, 1996). Candidate each preferentially regulated by ras and rac, respec- molecules which could act upstream of these exchange tively (Denhardt, 1996; Coso et al., 1995; Minden et factors include the src-like non-receptor tyrosine al., 1995). The closely related m5 muscarinic receptor kinases, Pyk2 and the adaptor molecule shc (Dikic et also transforms via a ras-dependent pathway (Mat- al., 1996; Chen et al., 1996). Deletion of the src-like tingly et al., 1994). Mutationally activated MEK is kinases Lyn, Syk, Btk and Csk abolishes muscarinic sucient for cellular transformation and tumorigenesis receptor activation of the MAP kinase cascade in avian Rac-regulated proliferative pathways ES Burstein et al 1622 B lymphoma cells, while retransfection of the deleted Functional assays genes restores responsiveness (Wan et al., 1996; 1997). The Receptor Selection and Ampli®cation Technology (R- A direct connection between the IgE Fc (Fcepsilon) SATTM) assay is based on protocols previously used to receptor and sequential activation of Syk, Vav, rac1 evaluate induction of foci by ligand-occupied muscarinic and the JNK pathway has recently been demonstrated receptors (Gutkind et al., 1991). In this assay technology, (Teramoto et al., 1997). Potential e€ectors operating ligands select and amplify cells that express functional immediately downstream from rac1 include the p21 receptors. The same cells express a reporter gene, thus the activated kinase (PAK) which is stimulated upon ampli®cation of these cells results in ampli®cation of the binding GTP-loaded rac (Manser et al., 1994). reporter enzyme. R-SAT assays were performed as However numerous other e€ectors may also respond described previously (Burstein et al., 1995) using 2 ± 200 ng receptor, 500 ng pSI-b-galactosidase (Promega, to rac1 (Tapon and Hall, 1997) and other targets Madison WI), 50 ± 1000 ng G-protein, and sucient besides PAK may connect rac1 to JNK activation control vector to balance the total DNA pool. DNA was (Gerwins et al., 1997; Teramoto et al., 1996; Burbelo et transfectedinto26105 cells per well of a six-well plate. rac al., 1995). Q61L, ras G12V and Ga13 Q226L are constitutively In conclusion, we have shown that the ras-related activated by mutations which impair GTPase activity (see GTPase rac1 regulates a proliferative pathway selec- Bourne et al., 1991) and are referred to as rac*, ras* and tively utilized by G-protein coupled receptors. In Ga13*. rac T17N and ras T17N are dominant negative addition, we have shown that tyrosine kinase linked alleles each containing a mutation which abolishes GTP- receptors stimulate cellular proliferation through a binding but preserves GDP-binding (Feig and Cooper, pathway that is distinct from the pathway(s) regulated 1988) and are referred to as rac(7) and ras(7). Previous experiments veri®ed that these constructs are expressed by rac1. Thus rac1 could operate upstream of ras to (Coso et al., 1995). Cells were transfected by calcium link GPCR's to a ras-regulated proliferative pathway precipitation, or using Lipofectamine (Gibco ± BRL) or that tyrosine kinase receptors access downstream of Superfect (Qiagen) according to manufacturers instruc- rac1. Alternatively GPCR's could stimulate two tions. One day after transfection media were changed and parallel pathways controlled by rac1 and ras respec- after another day cells were trypsinized and aliquoted into tively, which each contribute to generating a prolif- the wells of a 96-well plate (50 ml/well). One well of a 6- erative response. Such a scheme would explain the well plate yields enough cells for 24 wells. Cells were partial sensitivity of the GM-CSF receptor to rac- combined with ligands in DMEM supplemented with 2% blockade. The possibility that rac1 operates down- cyto-SF3 synthetic supplement to a ®nal volume of 200 ml/ stream of ras appears less likely because in that case well (Kemp Laboratories). After 4 days in culture b- galactosidase levels were measured using 3.5 m O- one would expect each class of receptor to be sensitive M nitrophenyl-b-D-galactopyranoside (Sigma, St Louis, MO) to rac-blockade as well as ras-blockade. Further work as the b-galactosidase substrate. Plates were read at will be needed to elucidate all of the steps in the 420 nm on a plate -reader (Bio-Tek EL 310 or Molecular proliferative pathways used by G-protein coupled Devices). Data from R-SAT assays were ®tted to the receptors. equation: R=A+B*x/(x+c) where A=minimum response,

B=maximum response and c=EC50, x=concentration of ligand. Curves were generated by least-squares ®ts using the program KaleidaGraphTM (Abelbeck Software). Materials and methods

Cell culture NIH3T3 cells (ATCC no. CRL 1658) were incubated at

378C in a humidi®ed atmosphere (5% CO2) in Dulbecco's modi®ed Eagles medium supplemented with 4500 mg/l Acknowledgements glucose, 4 mML-glutamine, 50 U/ml penicillin G/strepto- We wish to acknowledge F Piu for critically reviewing the mycin (A.B.I.) and 10% calf serum. manuscript.

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