Molecular Vision 1999; 5:36
Iontophoresis of lysophosphatidic acid into rabbit cornea induces HSV-1 reactivation: Evidence that neuronal signaling changes after infection
Rex E. Martin,1 Jeannette M. Loutsch,2 Hildegardo H. Garza, Jr.,3 Daniel J. Boedeker,1 James M. Hill2
1Department of Cell Biology and Oklahoma Center for Neurosciences, University of Oklahoma College of Medicine, Oklahoma City, OK; 2LSU Eye Center and Neuroscience Center, Louisiana State University Medical Center School of Medicine, New Orleans, LA; 3Department of Biology, Texas A & M University, Kingsville, TX
Purpose: Lysophosphatidic acid induces neurite retraction; it is also present in tears and aqueous humor. We determined whether lysophosphatidic acid induces HSV-1 reactivation in latently infected rabbits and whether the nerve growth associated protein GAP-43 undergoes posttranslational modification during the course of HSV-1 infection. Methods: Rabbits were infected with HSV-1 and acute infection was documented by slit lamp examination. Corneas of latently infected rabbits were treated with lysophosphatidic acid or lysophosphatidylserine (structurally similar but lack- ing biological potency). For application to the cornea, these compounds were impregnated into collagen shields, applied as topical drops, or iontophoresed. In another experiment, corneas of latently infected rabbits were either untreated or treated iontophoretically with lysophosphatidic acid, lysophosphatidylserine, or saline. Ocular swabs detected shedding of infectious virus. Western blot and immunoprecipitation identified GAP-43 in corneal extracts and densitometry of silver-stained isoelectric focusing gels measured changes in GAP-43 isoform abundance. Results: Iontophoresis of lysophosphatidic acid induced HSV-1 shedding more frequently than lysophosphatidylserine or saline. Viral shedding induced by collagen shield and topical drop administration was low and not significantly different for lysophosphatidic acid and lysophosphatidylserine. Five discrete GAP-43 isoforms predominated in the IEF gels. Most abundant were the pI 4.7 band in uninfected cornea and the pI 5.05 band in latently-infected cornea. Compared to latently-infected cornea, there was no significant change in isoform abundance 1 h after lysophosphatidic acid iontophore- sis, but 24 and 72 h later, the pI 5.05 band was diminished. Conclusions: Lysophosphatidic acid can induce HSV-1 reactivation and changes in GAP-43 pI suggest that posttransla- tional modifications, possibly related to phosphorylation and ADP-ribosylation, are occurring during HSV-1 latency and after LPA is iontophoretically applied to the cornea. How lysophosphatidic acid-induced signaling, HSV-1 reactivation, and GAP-43 pI are related remains to be determined.
Most clinically significant ocular herpes simplex virus When phosphorylated in the appropriate site by protein ki- type-1 (HSV-1) infections result from the reactivation of la- nase C, GAP-43 interacts with the cytoskeleton to potentiate tent virus [1]. Systemic stresses or physical traumas that dis- actin polymerization and neurite outgrowth [12]. GAP-43 rupt the corneal nerves induce HSV-1 reactivation [2]. Our expression is induced by nerve growth factor [13,14]. This intent is to learn how to impair or prevent HSV-1 reactivation finding, plus the fact that systemic nerve growth factor deple- by characterizing the biochemical changes that occur in neu- tion induces HSV-1 reactivation [15], suggests that neuronal rons during infection. As a marker of these biochemical systems that potentiate neurite elongation are involved in the changes, we have chosen to study a nerve growth associated establishment and maintenance of HSV-1 latency as well as protein called GAP-43 [3] and we have shown that the con- the switch from the latent to the lytic phase. To test this con- centration of GAP-43 increases in corneal nerves during HSV- cept, we hypothesized that if the latent HSV-1 in corneal nerves 1 latency [4,5]. A specific role for GAP-43 in HSV-1 infec- can be reactivated by physical trauma or systemic stress, then tion has not been established. substances that perturb neurite homeostasis in in vitro culture GAP-43 participates in the remodeling and regeneration could also induce latent HSV-1 to reactivate in vivo. of synaptic terminals; it is also implicated in the constant re- Lysophosphatidic acid (LPA) was selected to perturb neu- modeling of nerve endings that occurs in the densely inner- ronal homeostasis because it is present in fluids that bathe the vated cornea [3-7]. GAP-43 can be palmitoylated or ADP- cornea and it induces neurites to retract in culture [16,17]. ribosylated and has at least four phosphorylation sites [8-11]. LPA is a negatively charged, naturally occurring, receptor bind- ing, lysophospholipid that is synthesized from phosphatidic
acid by phospholipase A2 [18-20]. In non-pathologic condi- Correspondence to: Rex E. Martin, Ph.D., Room 517, 940 Stanton tions, LPA is present at concentrations of 2-20 µM in serum, 1 Young Boulevard, Department of Cell Biology and Oklahoma Cen- µM in lacrimal gland fluid, and 0.2 µM in aqueous humor ter for Neurosciences, University of Oklahoma College of Medicine, [16,21]. In cryogenically-injured cornea, the LPA concentra- Oklahoma City, OK, 73104; Phone: (405) 271-2377, ext 229; FAX: tion increases 3-6 fold in aqueous humor but not in tear fluid (405) 271-3548; email: [email protected] Molecular Vision 1999; 5:36
[16]. The details of signaling events that relate to LPA-recep- lysophosphatidylserine, applied to the corneal surface, and left tor binding and to posttranslational changes in GAP-43 are in place for 8 h. Iontophoretic drug delivery was performed unknown. A direct connection between LPA and GAP-43 sig- once daily for 3 consecutive days as previously described [24]. naling is also unknown, but a compelling connection is sug- In a second experiment, LPA, lysophosphatidylserine, or sa- gested because LPA stimulates neurite retraction and GAP-43 line was iontophoresed once daily in 10 eyes each for 3 con- supports neurite elongation [3,13,14,17]. Moreover, the LPA secutive days as in experiment 1; eight eyes were monitored receptor functions through membrane-bound and soluble G as untreated controls. proteins [18,20] and GAP-43 is associated with G proteins Corneal Proteins: Following previously described pro- [22,23], and ADP-ribosylated [9,10]. cedures [5], corneas were dissected and immediately frozen We delivered LPA and another negatively charged in liquid nitrogen. The frozen tissue from individual corneas lysophospholipid, lysophosphatidylserine, to the corneas. To were ground to a powder in a liquid nitrogen-cooled mortar determine the optimal way to deliver these compounds, topi- and pestle. The powder from each cornea was resuspended in cal drops, collagen shields, or iontophoresis were tested. We 15 volumes of homogenization buffer, which consisted of 20 then determined frequency of HSV-1 reactivation after each mM Tris-HCl (pH 7.5) containing 320 mM sucrose, 10 mM procedure. We observed that the pI of GAP-43 changed dur- ethylene glycol-bis [β-aminoethylether], 2 mM ing HSV-1 infection and after LPA treatment. ethylenediaminetetraacetic acid (EDTA), 2 mM dithiothreitol, 0.05% leupeptin, and 0.2 mM phenylmethylsulfonyl fluoride. METHODS The suspension was centrifuged at 2000 x g for 10 min at 4 °C Virus and Rabbits: HSV-1 strain McKrae was propagated on to remove the collagenous stromal matrix. The pellet was primary rabbit kidney cells and titered on African Green Mon- resuspended with sonication in five volumes of ice-cold ho- key cells (CV-1). The virus was aliquoted and stored at -70 mogenization buffer and centrifuged to remove the collagen °C. New Zealand White rabbits (2-3 kg) were inoculated bi- and maximize extraction of non-collagenous proteins. Pro- laterally by applying 2 x 105 plaque forming units of HSV-1 tein concentrations were determined using Bio-Rad (Rich- in a 25-µl suspension into the cul-de-sac of each eye. The eye mond, CA) protein assay reagent and bovine serum albumin was closed and massaged for 30 seconds. The corneas were as a standard. All corneal protein procedures were done at 4 not scarified. Slit-lamp examination and ocular swabbing were °C. done 3 days after inoculation to identify epithelial lesions and GAP-43 Identification and Densitometry: Three differ- to verify a productive infection, respectively. HSV-1 was pre- ent approaches were used to identify discrete GAP-43 isoforms sumed to be latent when slit-lamp examination indicated no in rabbit corneal extracts (Figure 1). Two of these approaches corneal defects and tear film swabs contained no infectious used pI 4-6 horizontal IEF gels and an anti-GAP-43 mono- virus [24]. clonal antibody that binds GAP-43 irrespective of its phos- The reactivation studies were performed 5-6 weeks after phorylation state [13]. In the third approach, proteins migrat- virus inoculation. All eyes underwent membranectomies 1 ing at 43 kDa in sodium dodecyl sulfate (SDS)-polyacryla- week prior to treatment. A sample of the tear film from each mide gels were electroeluted from the gel, concentrated, and eye of each rabbit was collected on Dacron swabs once daily then vertically electrophoresed in precast pI 3-7 IEF gels for 7 days after the start of treatment. The swabs were placed (Novex, San Diego, CA). All procedures were done at room in tubes containing monolayers of primary rabbit kidney cells temperature. and the cells were monitored for the development of cyto- Figure 1, IP/IEF, shows the first approach taken to iden- pathic effect indicative of infectious virus. Rabbits used in tify GAP-43 in IEF gels. GAP-43 was immunoprecipitated studies of reactivation were sacrificed seven days after initia- with staphylococcal protein G-coated agarose beads (Hyclone tion of the first treatment. For densitometric analysis of GAP- Laboratories Inc., Logan, UT) and monoclonal antibody 91E12 43 isoforms, corneas from latently infected rabbits were taken (Boehringer Mannheim, Indianapolis, IN) [25,26]. The ex- 1, 24, and 72 h after iontophoretic LPA treatment. The rabbits tracts were first cleared of proteins that non-specifically bound were handled and maintained in accordance with the tenets of protein G-coated agarose beads and then incubated overnight the Association for Research in Vision and Ophthalmology with gentle stirring and monoclonal antibody/protein G-coated Resolution on the Use of Animals in Research. agarose beads. The resulting immune complex was collected Treatments: LPA and lysophosphatidylserine were solu- by centrifugation at 300 x g for 5 min. The supernatant was bilized in deionized water at a final concentration of 40 µM discarded and the immune complex was washed free of impu- (Sigma Chemical Co., St. Louis, MO). Lysophosphatidylserine rities with three cycles of centrifugation and resuspension in was used as a control for the non-specific effects of LPA be- phosphate-buffered saline containing 5 mM EDTA and 1% cause it is structurally similar to LPA but lacks biological po- IGEPAL CA-630 (NP-40, Sigma Chemical Co.). The immune tency. The phospholipids were applied to both corneas of la- complex was dissociated with brief sonication in 2 M urea tently infected rabbits in topical drops (8 eyes), in collagen and centrifuged at 300 x g. Proteins in the resulting superna- shields (8 eyes), or by iontophoresis (8 eyes). Three different tant were dialyzed against water, concentrated by lyophiliza- methods were used to apply the phospholipids in the first ex- tion, resuspended in water, and applied to horizontal IEF gels periment. Topical drops were applied every 30 minutes for 9 with a 4-6 pI gradient (pI 3-10 and pI 4-6 ampholytes mixed hours. Collagen shields (Bio-Cor, Bausch & Lomb; at a ratio of 1:4). These 0.8 mm-thick gels were prefocused in Clearwater, FL) were saturated with LPA or a Biophoresisª apparatus (Bio-Rad) for 15 min at 400 V (con- Molecular Vision 1999; 5:36
TABLE 1. HSV-1 SHEDDING AFTER ADMINISTRATION OF LYSOPHOSPHATIDIC ACID (LPA) OR LYSOPHOSPHATIDYLSERINE (LPS) BY THREE METHODS
Frequency ------Average Figure 1. Three methods of GAP-43 identification. In the first method, Eyes Swabs Duration ------Shedding** proteins were immunoprecipitated (IP) with monoclonal antibody Treatment +/Total % p* +/Total % p* (Days) ------91E12 and applied to isoelectric focusing (IEF) gels (IP/IEF). In the Iontophoresis, LPA 6/8 75 - 18/56 32 - 2.25 second method, proteins were applied to IEF gels and transferred to Iontophoresis, LPS 3/8 37 0.315 6/56 11 <0.010 0.75 nitrocellulose membranes for Western blotting with monoclonal an- Collagen shield, LPA 3/8 37 - 6/56 11 - 0.75 tibody 91E12 and chemiluminescent detection (W). In the third Collagen shield, LPS 2/8 25 1.000 3/56 5 0.489 0.38 method, proteins were electroeluted from the 43 kDa region of so- Topical drops, LPA 2/8 25 - 4/56 7 - 0.50 Topical drops, LPS 1/8 13 1.000 2/56 4 0.679 0.25 dium dodecyl sulfate polyacrylamide gels, applied to IEF gels, and silver stained (EE). The proteins of interest had isoelectric points of *P values obtained by two-tailed Fisher's exact test. Comparisons are between LPA 5.05, 4.95, 4.80, 4.70, and 4.50. In some preparations, two bands and LPS administration by each method. focused near pI 4.70 and 4.50. The isoelectric points of standard **Total number of positive swabs per total number of eyes proteins (STD) are noted at the left and those of GAP-43 isoforms are noted at the right. LPA iontophoresis, a significantly higher frequency of shed- fied [28]. The most basic band had a pI of approximately 5.05 ding (positive swabs) was seen after LPA treatment (p<0.001 and the most acidic had a pI of 4.5. In some samples, doublet for all; Table 2). In terms of eyes positive for infectious virus, bands existed at pI 4.7 and 4.5 after immunoprecipitation (Fig- the frequency was significantly greater for LPA treatment, com- ure 1, IP/IEF). The consistent focusing of the GAP-43 isoforms pared with saline (p=0.020) and no treatment (p=0.003), but in IEF gels facilitated densitometric quantitation. The percent not in comparison with lysophosphatidylserine treatment contribution of each band (A, B, C, D, and E) to their total (p=0.057). The average duration of shedding was markedly was measured densitometrically (Figure 2). When doublet greater for LPA-treated eyes than for any of the other groups bands were evident at pI 4.7 (Figure 2), they were considered (2.90 days for LPA, 1.10 days for lysophosphatidylserine, 0.4 one band in these calculations. The results shown in Figure 2 days for saline and 0.12 days for no treatment; Table 2). indicated that there were statistically significant differences In both of these experiments, iontophoresis of LPA in- in the relative abundance of GAP-43 isoforms between duced shedding of infectious virus in at least one eye of every uninfected (UI), latently-infected (LAT), and LPA-treated la- rabbit tested (Table 1 and Table 2). The results demonstrate tently-infected rabbits (LPA). that HSV-1 reactivation and shedding are induced in the rab- In uninfected, untreated rabbits (Figure 2, UI), the most bit when a compound known to perturb neural homeostasis basic GAP-43 isoform, band A (pI 5.05) represented 19% of (LPA) is iontophoresed into the cornea. the five GAP-43 isoforms while band D (pI 4.7) represented Corneal Proteins: GAP-43 is abundant in neurites of the 33% of the total GAP-43. Comparing uninfected rabbits (Fig- cornea [4,5]. It is an ADP-ribosylated and multiply-phospho- ure 2, UI) to latently-infected rabbits (Figure 2, LAT), the abun- rylated protein with isoelectric points ranging from 4.3 to 5.2 dance of band C (pI 4.8) was increased in the latently-infected [9,10,28,29]. To correlate changes in neuronal homeostasis rabbits and band D (pI 4.7) was less abundant. The statisti- with the course of HSV-1 infection, IEF gels were used to cally significant increase in band C and decrease in band D, evaluate changes in the endogenous pI of GAP-43. Irrespec- relative to uninfected rabbits, was maintained in the LPA- tive of the experimental approach that was used to find GAP- treated rabbit corneas suggesting that the GAP-43 isoforms in 43 in IEF gels, protein bands with isoelectric points in the latently infected corneas were collectively less acidic (modi- reported range for GAP-43 (GAP-43 isoforms) were identi- fied). One hour after LPA was iontophoretically applied to latently-infected cornea there were no statistically significant differences in GAP-43 isoform abundance, but after 24 and 72 h, the LPA-treated corneas contained less band A (pI 5.05) (Figure 2, LPA 12 h & LPA 72 h). This decrease in band A abundance following LPA treatment demonstrates an increase in the collective acidity (posttranslational modification) of GAP-43 after LPA iontophoresis.
DISCUSSION The effects of HSV-1 latency on neurons and the effects of host neurons on latent HSV-1 are not understood. Until we demonstrated that GAP-43 expression increased in the trigemi- nal ganglia and corneas of rabbits latently infected with HSV- 1 [5], it was believed that latent HSV-1 infections had no pro- longed effect on the host’s neuronal proteins. We show here that when LPA is administered iontophoretically, latent HSV- 1 can be induced to reactivate. Moreover, we show that when Figure 2. Relative abundance of rabbit corneal GAP-43 isoforms fo- HSV-1 infects corneal neurons, the pI of the nerve growth cusing at pI 5.05, 4.95, 4.80, 4.70, and 4.50. The pI 4.7 GAP-43 associated protein GAP-43, changes. isoform (band D) was most abundant in uninfected rabbits (UI). Comparing UI rabbits with the other groups, band C (pI 4.80) was more abundant and band D (pI 4.70) was decreased in all infected conditions (*). After lysophosphatidic acid (LPA) iontophoresis, the TABLE 2. HSV-1 SHEDDING AFTER IONTOPHORESIS OF collective posttranslational modification of GAP-43 in latently-in- LYSOPHOSPHATIDIC ACID (LPA), LYSOPHOSPHATIDYLSERINE (LPS), fected rabbits was increased because the relative abundance of the OR SALINE most basic GAP-43 isoform (band A, pI 5.05) was decreased at 24 h Frequency and 72 h (**). Beneath each group, bands (A-E) are shown in a gel ------Average Eyes Swabs Duration exemplary of those used for densitometric analysis. Values are den------Shedding** Treatment +/Total % p* +/Total % p* (Days) sitometric averages ± standard error of four separate determinations ------
(corneas). One-way ANOVA with post-hoc Newman-Keuls tests LPA Iontophoresis 9/10 90 - 29/70 41 - 2.90 show significant differences between treatment groups (p<0.05). The LPS Iontophoresis 4/10 40 0.057 11/70 16 <0.001 1.10 NaCl Iontophoresis 3/10 30 0.020 4/70 6 <0.001 0.40 (*) indicates significant differences between uninfected corneas (UI) None 1/8 13 0.003 1/56 1 <0.001 0.12 and all other experimental groups. The (**) indicates significant *P values obtained by two-tailed Fisher's exact test. All comparisons are with differences between the latently-infected corneas (LAT) and the LPA- LPA iontophoresis. treated, latently-infected corneas. **Total number of positive swabs per total number of eyes Molecular Vision 1999; 5:36
In determining the best way to deliver LPA we demon- sisted [5]. This increased GAP-43 abundance during HSV-1 strated that significant reactivation of latent HSV-1 occurred latency was not expected; it suggested that neuronal homeo- when LPA was iontophoresed into the cornea. This result is stasis was changed by HSV-1 infection. Our current study attributed to the anatomy of the cornea and rapid metabolism further supports the hypothesis that HSV-1 infection changes of LPA by endogenous lysophospholipases [31,32]. The cor- neuronal homeostasis because the abundance of a pI 4.8 GAP- neal nerves pass through the stroma superficial to the endot- 43 isoform was increased and that of a pI 4.7 isoform was helium and the sensory terminals are located in epithelium decreased when the rabbits were latently infected with HSV- [32]. LPA in the tear film (lacrimal gland fluid), in collagen 1. These findings demonstrate that in addition to the increased shields, or in topical drops could make direct contact with the expression of GAP-43 in latently infected rabbits [5], there corneal nerve endings but it would be unlikely to diffuse into are also biochemical differences in the corneal nerves of la- the epithelium or stroma without being metabolized. How- tently infected rabbits that likely involve the kinase(s) and/or ever, iontophoresis driving LPA though the epithelium and into phosphatase(s) that act on GAP-43 [33,34]. Moreover, the the stroma would facilitate LPA binding to receptors on the abundance of a pI 5.05 GAP-43 isoform was decreased after nerve fiber (in addition to those on the nerve ending) before it latently-infected corneas were treated with LPA. This change could be metabolized [31]. Even though LPA concentrations was not noted 1 hour after LPA iontophoresis but it was evi- increase after cryogenic injury [16], we do not know whether dent 24 and 72 h later. The altered pI of GAP-43 at 24 and 72 they ever reach 40 µM (40 times that of normal lacrimal gland h after LPA iontophoresis could be due to three things: LPA- fluid and 200 times that of normal aqueous humor), but these stimulated signaling, reactivation of latent HSV-1, or simply experiments do demonstrate that HSV-1 can be reactivated by the iontophoretic treatment of rabbit corneas. The latter possi- a new and different pathway. Perhaps an understanding of bility seems unlikely because saline iontophoresis did not in- LPA receptor-mediated signaling will provide more clues to duce significant HSV-1 reactivation and 1 h after LPA treat- the mechanisms that regulate HSV-1 latency. ment there was no difference in the pI of GAP-43 between GAP-43 has isoelectric points ranging from 4.3 to 5.2 This latently-infected corneas and LPA-treated latently-infected range can be attributed to phosphorylation or ADP ribosylation corneas (Figure 2). More research will be necessary to dem- [9,10,28,29]. Proteins that are phosphorylated or ADP- onstrate that neuronal signaling mechanisms affecting GAP- ribosylated typically appear as a single band in SDS-polyacry- 43 posttranslational modification mediate either LPA signal- lamide gels but they are resolved into multiple bands in IEF ling or HSV-1 infection. gels because these posttranslational modifications increase the The nerve growth factor receptor [15], the glucocorticoid acidity of the protein and decrease its pI. When GAP-43 was receptor/response element [35], and the cAMP response ele- immunoprecipitated from rabbit corneal extracts and electro- ment [36] have been implicated in HSV-1 reactivation. We phoresed in SDS-polyacrylamide gels, it migrated as a single demonstrate that LPA, a lipid present in lacrimal gland fluid band at 43 kDa (data not shown). However, when these ex- and aqueous humor [16], also stimulates HSV-1 reactivation tracts were electrophoresed in IEF gels, multiple distinct GAP- and shedding. Significantly, another positively charged 43 isoforms were identified (Figure 1). The differing pI of lysophospholipid, lysophosphatidylserine, has minimal effect these isoforms is likely due to phosphorylation and/or ADP on reactivation. This specificity of LPA in the induction of ribosylation. Edgar et al. [11], showed that there are four (or reactivation suggests that it could act through a receptor-me- more) sites for endogenous GAP-43 phosphorylation. If there diated event [18,20]. In neurons of the cerebral cortex, LPA was no ADP-ribosylation, the five bands seen in Figure 1 and binds a 7-transmembrane receptor that activates two different Figure 2, therefore, could reflect increasing (successive) phos- signaling cascades. One cascade operates through a pertussis phorylation of GAP-43, with the most basic band (pI 5.05) toxin-sensitive Gi G-protein and the other cascade, which is being dephosphorylated GAP-43 and the most acidic band pertussis toxin-insensitive, operates through the small GTP- being GAP-43 with four phosphorylations. Finally, either of binding protein Rho [20]. Further experiments are necessary these posttranslational modifications could induce conforma- to demonstrate if either pathway is active in HSV-1-infected tional changes that “cover-up” or “expose” charged amino neurons. acids and change GAP-43 pI. Whether the changes in pI re- We propose that the signal transduction cascades mediat- ported in Figure 2 reflect changes in GAP-43 phosphoryla- ing GAP-43 expression and posttranslational modification are tion, ADP ribosylation, or protein confirmation, the changing intertwined with those mediating the course of recurrent HSV- abundance of specific GAP-43 isoforms is indicative of 1 disease. We make this statement because GAP-43 posttrans- changes in neuronal signal transduction. These results sup- lational modification and expression change as HSV-1 becomes port the hypothesis that substances that perturb neurite ho- latent [5] and because neuronal stimulators (nerve growth fac- meostasis also induce latent HSV-1 to reactivate. tor, LPA, and glucocorticoids) manifest changes in cell sig- In our previous experiments, increased concentrations of naling that affect GAP-43, HSV-1 reactivation and neurite GAP-43 were observed while the corneal nerves were recov- elaboration [3,5,13,14,33-35]. Moreover, the reversible ering from the lytic phase of infection This result was expected palmitoylation of GAP-43 mediates its association with mem- because GAP-43 concentrations are high when neurites are branes or its presence in the cytoplasm [37] and correspond- growing or regenerating or when terminals remodel ingly, there are data confirming that GAP-43 interacts with [3,4,13,14]. We also found that 154 days post inoculation (dur- both membrane and soluble G-proteins. In fibroblasts trans- ing viral latency), the increased concentration of GAP-43 per- fected with GAP-43, the membrane-bound protein stabilizes Molecular Vision 1999; 5:36
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