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© 2008 American Society for Nutrition

Nutrient Physiology, Metabolism, and Nutrient-Nutrient Interactions

Arginine Stimulates cdx2-Transformed Intestinal Epithelial Cell Migration via a Mechanism Requiring Both Nitric Oxide and Phosphorylation of p70 S6 Kinase1,2

J. Marc Rhoads3,4,*, Yuying Liu3, Xiaomei Niu4, Sankar Surendran3 and Guoyao Wu5

3 Department of Pediatrics, University of Texas Health Science Center, Houston, TX 77030; 4 Ochsner Clinic Foundation, Research Institute, Department of Pediatrics, Section of Pediatric Gastroenterology, New Orleans, LA 70121; and 5 Department of Animal Science and Faculty of Nutrition, Texas A&M University, College Station, TX 77843

* To whom correspondence should be addressed. E-mail: j.marc.rhoads{at}uth.tmc.edu.


   ABSTRACT
TOP
 ABSTRACT
Introduction
 Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
In intestinal cells, arginine (Arg) is 1 of the 2 most potent amino acid activators of p70s6k, a key regulator of 5'- terminal oligopyrimidine mRNA translation, a necessary condition for increased cell migration. To investigate the mechanism of response to Arg, we used the rat crypt cell line cdx2-transformed IEC-6 cells (cdx2-IEC) and measured cell migration, immunocytochemical analysis of p70s6k activation in response to Arg, and production of nitric oxide (NO). When treated with Arg, cdx2-IEC increased in phosphorylation on Thr-389 of p70s6k (pp70s6k) compared with control (P < 0.01). Phospho-Thr-421/Ser-424-p70s6k was located in the nucleus shortly after Arg treatment. Arg enhanced pp70s6k, cell migration (55% wound coverage), and NO production. In comparison, the branched-chain amino acid leucine (Leu) activated pp70s6k, was a weaker stimulator of migration (23% coverage), and did not increase NO. A total of 25 µmol/L DETA-NONOate (DETA/NO) did not significantly enhance phosphorylation of p70s6k but enhanced the rate of cell migration by ~25%. Wound coverage with Leu plus DETA/NO (25 µmol/L) was greater than coverage with DETA/NO alone (P < 0.01). These and our previous studies lead to a model in which Arg must stimulate both pp70s6k (in the nucleus) and NO release to enhance intestinal epithelial cell migration, which may be relevant to diseases that involve intestinal villous injury.


   Introduction
TOP
 ABSTRACT
 Introduction
Materials and Methods
 Results
 Discussion
 LITERATURE CITED
 
Arginine (Arg) is a recognized enhancer of wound healing (1,2) and of muscle protein synthesis in vivo. Arg also stimulates intestinal cell migration and ex vivo intestinal protein synthesis, while significantly enhancing mammalian target of rapamycin (Rapa)6 (mTOR) signaling (3,4). Previous studies showed that mTOR shuttles to and from the nucleus during cellular activation (5,6). Targeted knockout of p70s6k in mice produces mutants that are globally smaller than normal (5). Our investigations of Rapa inhibition in intestinal cells suggested that p70s6k is an important mediator of Arg-stimulated migration. However, Rapa may have inhibitory effects on other effectors distinct from mTOR; for example, Arg reduces levels of cyclin D1 and p21 in tumor cells (7).

Amino acids have considerable clinical relevance as both activators of cellular kinases in the intestine (8) and as therapeutic agents. Arg has been investigated as a component of an oral rehydration solution to enhance intestinal absorption and villus recovery after injury (9,10). Arg also is the physiological substrate for nitric oxide (NO) synthesis, an important substrate for collagen synthesis and polyamine biosynthesis (11–13). One study showed the promise of Arg as a prophylaxis against necrotizing enterocolitis in newborn premature infants (9). However, long-term overproduction of NO released from Arg has been shown to enhance apoptosis by activating the caspase family proteases, producing the release of mitochondrial cytochrome c (13).

The current studies were designed to further test the hypothesis that Arg acts to enhance epithelial cell migration via NO generation and phosphorylation of p70s6k (pp70s6k) signaling. We further aimed to determine the intracellular site(s) of activation of p70s6k during Arg stimulation of cultured intestinal epithelial cells.


   Materials and Methods
TOP
 ABSTRACT
 Introduction
 Materials and Methods
Results
 Discussion
 LITERATURE CITED
 
    Materials. Anti-p70s6k, phospho-p70s6k (Thr-421/Ser-424), phospho-p70s6k (Thr-389) antibodies, and phospho-ribosomal protein S6 (prpS6) antibody were from Cell Signaling. L-Arginine, leucine, DETA-NONOate (DETA/NO), and Rapa were purchased from Sigma. Affinity-purified goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate was from Bio-Rad. Alexa Fluor donkey anti-rabbit IgG anti-goat IgG, 4',6-diamidino-2-phenylindole (DAPI), and prolong gold antifade reagent were all from Molecular Probes. Enhanced chemiluminescence plus Western blotting detection system (ECL Plus) was purchased from GE Bio-Sciences.

    Cells. Cdx2-IEC, a transformed rat crypt IEC-6 cell line, was obtained from Dr. J-Y. Wang (University of Maryland, Baltimore, MD). Cdx2-IEC cells were maintained as described (14) at 37°C in a humidified incubator with 5% CO2 in DMEM containing 5% (v:v) fetal bovine serum (FBS), 0.5% (v:v) ITS +1 liquid media supplement, 0.1 million units/L penicillin, 100 mg/L streptomycin, and 4 mmol/L sopropylthio-β-D-galactoside, which served as an inducer. In some studies, cells were grown in a medium that contained concentrations of amino acids resembling those of serum, called basal medium Eagle (BME). In other experiments, cells were treated with Hank's balanced salt solution (HBSS) (14).

    Phospho-p70s6k immunoblot analysis. Levels of phosphorylated p70s6k were measured as previously described (14). Briefly, cells were seeded in 6-well plates. After reaching 80% confluence, cells were made quiescent overnight and treated with 4 mmol/L Arg, 4 mmol/L Leu, 25 µmol/L DETA/NO, and Leu plus DETA/NO for 30 min. In another set of experiments, cells were starved of amino acids and serum in HBSS for 30 min and then treated with twice the normal concentration of amino acids (2xAA), glutamine (Gln), glutamate (Glu), phenylalanine (Phe), Arg, Leu, DETA/NO, proline (Pro), and BME as control, respectively, for 30 min. Cells were lysed and 30 µg of protein was separated on 7.5% SDS-PAGE and membranes were blocked with 5% nonfat dry milk and successively incubated in buffers containing anti-phospho-p70s6k (1: 1000 dilution) overnight at 4°C and goat anti-rabbit IgG (H+L) horseradish peroxidase conjugate (1: 5000 dilution) for 1 h. Bands were detected with ECL Plus. Membranes were reprobed with β-actin antibody and processed as described above. The semiquantitative data were obtained using Kodak 1 D image analysis software. Phosphorylated p70s6k measurements were normalized to β-actin immunoreactivity.

    Immunocytochemistry. Localization of p70s6k, pp70s6k, and prp6s in cells after treatment was detected by using immunofluorescence as previously described (14). In short, cdx2-IEC cells were grown on glass cover slips in 24-well plates. Cells were serum-starved in BME or HBSS for 24 h after reaching 80% confluence. The cells were treated with 4 mmol/L Arg for 30 min or pretreated with 50 nmol/L of Rapa for 30 min followed by addition of Arg. Then, cells were fixed in cold acetone and nonspecific binding was blocked with 5% normal donkey serum followed by incubation for 90 min with primary antibodies at a 1:200 dilution. Slides were washed 3 times for 5 min each with PBS and then labeled with 1:500 dilutions of donkey Alexa-Fluor 568 anti-goat IgG and/or Alexa-Fluor 488 anti-rabbit IgG for 30 min at room temperature with light shielding. The slides were then stained with DAPI at a 1:5000 dilution in PBS for 2 min and subsequently mounted with prolong gold antifade reagent. The fluorescent images were captured using a Zeiss deconvolution microscope equipped with Slidebook (Intelligent Imaging Innovations) software under a 63x oil immersion objective.

    Cell migration analysis. Cells were plated in 96-well plates with flat bottoms at a density of 1.5 x 104 cells/well. After attaching, cells were made quiescent overnight by serum starvation with BME. Cell wounding was performed using a 96-well floating-pin transfer device with a pin diameter of 1.58 mm coming to a flat point at the tip, with a diameter of 0.4 mm (VP-408FH, V&P Scientific). The pin array was placed in the top corner of a well, pushed down into the plate to engage all pins, and then pulled toward the user (15). After wounding, cells were treated with 4 mmol/L Arg, 4 mmol/L Leu, DETA/NO alone at 2.5, 25, or 100 µmol/L and a combination of Leu and DETA/NO for 12 h. We used 2.5% (v:v) FBS as positive migration control and cells left in BME were the untreated control. Cells were fixed with 3.7% formaldehyde after removal of the media and the wound areas were photographed using BD Pathway 800 Bioimager (BD Biosciences). We measured the uncovered area using Imaging Tool software. The area of cell migration was normalized for that of cells in BME by dividing the covered area of treated cells by the mean value of cells in BME. Thus, 100% was defined as complete wound coverage and 1% as the covered area of cells in BME.

    Determination of NO by HPLC. Cells were seeded in 6-well plates and made quiescent overnight after reaching confluence. Cells were treated with 1 or 4 mmol/L Leu and 4 mmol/L Arg with or without razor wounding for 24 h. Razor wounding was accomplished as described in (3) with a single razor cut applied to an 80% cell monolayer in 6-well plastic dishes. Nitrite and nitrate, oxidation products of NO, were determined using HPLC, as described previously (16). Briefly, nitrate in culture medium was reduced to nitrite by nitrate reductase in the presence of NADPH. The total amount of nitrite then reacted with 2,3-diaminonaphthalene to form 2,3-naphthotriazole, which was detected at a 375-nm excitation wavelength and 415-nm emission wavelength.

    Statistical analysis. Statistic analysis was performed with 1-way ANOVA using Prizm 4.0 (GraphPad Software). For comparison of multiple groups with a control group, we used Dunnett's multiple comparison test. For comparison of multiple groups with each other, we used Tukey's multiple comparison test. We considered a P-value of <0.05 significant for all analyses.


   Results
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
Discussion
 LITERATURE CITED
 
    Phosphorylation of p70s6k. We performed Western blotting to determine whether cdx2-IEC cellular p70s6k phosphorylation is activated by Arg, Leu, NO donor, and other amino acids. Treatment of the cells with Arg or Leu induced phosphorylation of p70s6k on Thr-389. The level of relative phosphorylation increased compared with untreated cells (BME; P < 0.05 by 1-way ANOVA and Dunnett's multiple comparison test) (Fig. 1). Although DETA/NO alone had no effects on activation of phosphorylation of p70s6k, the combination of Leu and DETA/NO produced a significant increase of pp70s6k compared with untreated cells (P < 0.05) (Fig. 1). The results also demonstrated that Arg, Leu, and Leu plus DETA/NO were similar in their efficacy to increase p70s6k phosphorylation.


Figure 1
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  		FIGURE 1  Levels of phosphorylated p70s6k on Thr-389 and β-actin in cdx2-IEC cells treated with 4 mmol/L Arg, 4 mmol/L Leu, 25 umol/L DETA/NO, or the combination of Leu and DETA/NO for 30 min. For each blot, the ratio of phosphorylated p70s6k:actin for cells in BME was defined as 1. The upper panel is a typical blot. In the lower panel, densitometric values are means ± SEM, n = 3. Asterisks indicate different from control BME: **P < 0.01; *P < 0.05.

 
Previous studies showed powerful effects of glutamine and glutamate on intestinal cell proliferation and gut repair (17,18). We compared different amino acids with respect to p70s6k phosphorylation on Thr-421/Ser-424 in cdx2-IEC. While medium containing 2xAA stimulated phosphorylation of p70s6k, Leu and Arg individually added at 4 mmoL/L were as effective as 2xAA, with a 100% increase in p70s6k phosphorylation compared with BME control (Fig. 2). The intestinal metabolic fuel glutamine, which is also mitogenic, and its metabolic product glutamate were ineffective. Proline and phenylalanine were also ineffective. These findings suggest that among amino acids, Arg and Leu have unique effects on the mTOR/p70s6k pathway.


Figure 2
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  		FIGURE 2  Immunoblot of phosphorylated p70s6k on Thr-421/Ser-424 and β-actin in cdx2-IEC cells starved of amino acids and serum in HBSS for 30 min followed by treatment with 4 mmol/L amino acids. A representative of 2 blots probed with anti-phospho-p70s6k or anti-β-actin is shown. β-Actin was used as a loading control.

 
    Localization of activation of p70s6k. To localize the subcellular site of activation of p70s6k, we used double-staining, with 2 antibodies to pp70s6k and immunocytochemical analysis. We have previously suggested that Arg treatment of cdx2-IEC induced phosphorylation of nuclear p70s6k, using an antibody to phospho-Thr-421/Ser-424, which recognizes phosphorylation in the autoinhibitory domain of p70s6k (5,6). In the current study, we compared localization of an antibody to total p70s6k (Fig. 3A, red) to that of Thr-421/Ser-424 phosphorylated p70s6k (Fig. 3B, green). Double staining demonstrated antibody to total p70s6k was found primarily in the cytosol, while Thr-421/Ser-424 phosphorylated p70s6k mainly in the nucleus and at the periphery of the cell (Fig. 3).


Figure 3
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  		FIGURE 3  Immunocytochemical localization of unphosphorylated p70s6k (A), phosphorylated p70s6k (B), and composite (C) in cdx2-IEC cells after Arg treatment. Antibodies to p70s6k yield a red color and phospho-p70s6k yield a green color. Composite photomicrograph emphasizes the localization of pp70s6k to the cell periphery, presumably at sites of cell adhesion to the glass, viewed at 63x.

 
    Cdx2-IEC migration. To determine whether Leu, which activates p70s6k, or a NO donor stimulates cdx2-transformed intestinal cell migration, we compared the effect on cdx2-IEC migration of Leu and DETA/NO with that of Arg (Fig. 4). We used 96-well plates with uniform scrape wounding. Leu, like Arg, significantly enhanced cell migration, with migrating epithelial cells covering 31.6 ± 5.3% of the wounded surface at 12 h compared with 1% for BME (P < 0.01) and 72.4 ± 1.9% for FBS. We tested whether NO itself would be adequate to stimulate cell migration. DETA/NO by itself and 2.5–100 µmol/L stimulated migration by 24 ± 2% (P < 0.01). However, wound coverage with all concentrations of DETA/NO and Leu were less than coverage by Arg (P < 0.01). Wound coverage with Leu plus DETA/NO (25 µmol/L) was greater than coverage with DETA/NO alone (P < 0.01). Leu plus DETA/NO (25 µmol/L) treatment produced surface area coverage that did not significantly differ from that of Arg (area covered, 45.6 ± 3.9% of the denuded surface). We found that the highest concentration of DETA/NO (100 µmol/L) plus Leu had deleterious effects compared with Leu by itself (P < 0.01; Fig. 4B).


Figure 4
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  		FIGURE 4  Photomicrograph of the uncovered surface (A) and the percentage of covered area (B) in migrating wounded cdx2-IEC cells after 12-h treatments. The area of covered surface is expressed as the surface area of wound coverage in treated cells divided by mean coverage in cells bathed in BME. One-hundred percent was defined as complete wound coverage and 1% as the covered area of cells in BME. Values are means ± SEM, n = 4–6 observations with 3 repetitions for each different cell passage. Asterisks indicate different from control BME: *P < 0.01, **P < 0.001. +Not different from Arg, P > 0.05; §different from Leu, P < 0.01.

 
    Activation of rpS6. To determine whether activation of p70s6k resulted in downstream signaling to rpS6, we performed immunocytochemical staining with an antibody to phospho-rpS6 in cultured cdx2-IEC (Fig. 5). Cells had little rpS6 when incubated in HBSS, but the addition of Arg (4 mmol/L) or amino acid-containing BME enhanced cytosolic phosphorylation. The addition of Arg to BME caused powerful stimulation of phosphorylation of rpS6 and adding Rapa to HBSS with Arg reversed the Arg stimulation of rpS6.


Figure 5
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  		FIGURE 5  Immunocytochemical appearance of phosphorylated rpS6 in cdx2-IEC cells with and without Arg and Rapa. Representatives of 2 observations are shown. Antibodies to prpS6 yielded a green color, DAPI staining for nuclei is blue, viewed at 63x.

 
    NO production. We postulated that NO production is not enhanced by cells treated with Leu. The addition of Leu at 1 and 4 mmol/L to the culture medium for 24 h did not affect NO synthesis by the intestinal cells (data not shown). Concentrations of nitrite and nitrate were similar in control monolayers (16.9 ± 0.84 µmol/L) and razor-cut monolayers (16.6 ± 0.75 µmol/L). Arg at the concentration of 4 mmol/L increased nitrite and nitrate levels (23.4 ± 0.90 µmol/L) (P < 0.05) compared with control.

We concluded from these observations that both NO generation and p70s6k activation by amino acids are stimuli that are mechanistically related to amino acid-stimulated intestinal cell migration. Whereas Arg produces both the above stimulatory effects, Leu must be added to a NO donor to similarly affect intestinal cell migration.


   Discussion
TOP
 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
LITERATURE CITED
 
Migration of intestinal epithelial cells represents a model for villus repair from mucosal ulceration secondary to a variety of insults, including viral infection, necrotizing enterocolitis, radiation or chemotherapy damage, Crohn disease, and nonsteroidal antiinflammatory drug treatment. Inhibition of cell migration results in increased bowel permeability and can contribute to multiorgan system failure (19). Therefore, treatments for patients with bowel damage are aimed at improving cell migration. These include transforming growth factor-β, epidermal growth factor, hepatocyte growth factor, trefoil growth factors, insulin-like growth factor-1, serum, and Arg (20,21). In our previous studies, Rapa, an inhibitor of mTOR, also severely inhibited cell migration (14). In humans, adverse events associated with Rapa include mucositis and skin rashes (22,23).

The 2 key translational regulators in mammalian cells are p70s6k and 4E-BP1, which are regulated in parallel by mTOR. Our studies focused on p70s6k, because our cells demonstrated weak immunoreactivity to commercial 4E-BP1 antibodies. Our findings agreed with those of Ban et al. (24), who also reported that Arg and Leu are the 2 best stimulators of p70s6k phosphorylation in intestinal cells. The present studies further support the hypothesis that NO plays a key role in migration. Facilitation of cell migration by 2.5, 25, and 100 µmol/L DETA/NO in our study but the reported inhibition of pp70s6k and cell migration by concentrations from 50 to 250 µmol/L in a previous study (25) cannot be readily reconciled at this time. Our experimental techniques differed: we used cdx2-IEC cells (26), whereas Cetin et al. (25) used parental IEC-6 cells. We chose cdx2-IEC-6 because it has features resembling an "immortalized villus cell line" and has shown very reproducible results for both cell migration and p70s6k studies. We previously used a line from piglet jejunum called IPEC-J2, which when compared with cdx2-IEC, showed an identical dose response of cell migration to Arg (3), but the IPEC-J2 cell line is difficult to maintain in culture. The caudal-related transcription factors cdx1 and cdx2 have been studied by Silberg et al. (27), whose findings indicated that cdx1 is expressed mainly in the crypt whereas cdx2 is expressed in the villus. Cdx2 expression induces development of multilamellar structures, microvilli on the cell surface, and sucrase isomaltase expression (28). Furthermore, forced expression of the Cdx2 gene in undifferentiated intestinal crypt cells induces the development of a differentiated phenotype and a 4-fold increase in the rate of cell migration (26).

One additional factor that may have resulted in a different impact of Arg compared with NO on migration is that we used a shorter time-period of cell migration (12 h) that may have resulted in briefer NO exposure and perhaps less risk of cellular apoptosis (25). We previously showed that NO synthase (NOS) inhibitors blocked Arg-stimulated intestinal cell migration; we also showed that inducible iNOS was induced at the leading edge of lamellipodia (3). Arg stimulation of migration could be via NO produced by endothelial NOS and/or iNOS.

NO is generated at the site of lamellipodia and has been postulated to enhance scalar motion as a precursor or initiator of vectorial motion (29). As an early step in the intestinal epithelial response to infection (cryptosporidiosis), the intestinal villi have a rapid induction of iNOS in the villus tips. In the infected intestine, NO may function to damage pathogenic parasites and also to generate migration-promoting NO at the site of restitution (30). However, there have been concerns that excessive NO produced by immune and epithelial cells can promote inflammation during necrotizing enterocolitis and in inflammatory bowel disease (31,32). As mentioned, enterocytes treated with 50–250 µmol/L DETA/NO for 20 h inhibited an in vitro assay of cell migration (25). Our studies suggest that NO regulation of cell migration is dose and time dependent.

We postulated that Leu would stimulate pp70s6k and cell migration. However, athough Leu stimulated pp70s6k, it did not induce NO production and was a less effective stimulator of cell migration compared with Arg. Thus, in intestinal cells, NO synthesis appears to be independent of pp70s6k activation. We previously reported that glutamine (Gln) stimulated cell migration by 46% in cultured intestinal cells compared with Arg stimulation, which was 67% (3). Gln could enhance migration by mechanisms other than p70s6k stimulation; e.g. by enhancing the activation of extracellular-related kinases (33). However, in studies of leukocyte transmigration, Gln was recently shown to inhibit cell motility at high concentrations (34).

When we investigated the intracellular site of signaling, we were initially surprised to see a component of the ribosomal translational apparatus (p70s6k) in the nucleus. However, the coiled body has been increasingly recognized as a nuclear site for mRNA processing (35). Recently, SKAR (S6K1 Aly/REF-like substrate) has been identified as a nuclear protein that binds to and is phosphorylated by p70s6k. SKAR binds to RNA and has been proposed to facilitate mRNA splicing and nuclear export (36). It was beyond the scope of the current studies to determine whether SKAR increases in the nucleus after Arg treatment, but future studies should investigate this potential mechanism for p70s6k translocation.

Concentrations of free Arg in the milk of most mammals (including humans and pigs) are relatively low (~50–200 µmol/L) (30). However, because of hydrolysis of dietary protein by digestive enzymes and the synthesis of Arg by intestinal cells, concentrations of Arg in the luminal fluid of the piglet small intestine are 2–3 mmol/L at 1 h after suckling (12). Our studies are important in demonstrating that a concentration of Arg approximately equal to luminal concentration is necessary for maximal bowel healing. They indicate that the low levels in serum are inadequate. In hosts that are not consuming milk or other proteins, Arg, therefore, could be limiting.

Compelling evidence shows that endogenous synthesis of Arg via the small intestinal epithelial cells plays a crucial role in maintaining its homeostasis in milk-fed neonates (37). Notably, there is a particularly high requirement for Arg by neonates because of its abundance in tissue proteins and its active utilization by multiple pathways. Thus, either dietary supplementation with Arg or activation of intestinal Arg synthesis by N-carbamoylglutamate markedly enhances protein accretion in neonatal pigs and intestinal protein synthesis during the neonatal period (38,39).

We hypothesize that Arg is deficient in preterm neonates because of the underdevelopment of its synthetic pathways in the small intestine coupled with its inadequate provision from diet. Collectively, our studies and others cited indicate that an adequate concentration of Arg is critical for optimal intestinal protein synthesis, cell migration, and intestinal integrity.


   ACKNOWLEDGMENTS
 
We thank Drs. Andrew P. Morris and James R. Broughman from the Laboratory of Epithelial Polarity and Ion Channel Trafficking, Department of Integrative Biology and Pharmacology, UT Medical School at Houston, for assisting in development of the cell wounding assay and use of the BD Pathway 800 Bioimager for photography.


   FOOTNOTES
 
1 Supported by the University of Texas School of Medicine, Department of Pediatrics, Ochsner Clinic Foundation. Back

2 Author disclosures: J. M. Rhoads, Y. Liu, X. Niu, S. Surendran, and G. Wu, no conflicts of interest. Back

6 Abbreviations used: 2xAA, twice the normal concentration of amino acids; BME, basal medium Eagle; cdx2-IEC, cdx2-transformed IEC-6; DAPI, 4',6-diamidino-2-phenylindole; DETA/NO, DETA-NONOate; FBS, fetal bovine serum; HBSS, Hank's balanced salt solution; mTOR, mammalian target of rapamycin; NO, nitric oxide; NOS, nitric oxide synthase; p70s6k, p70 S6 kinase; pp70s6k, phosphorylated p70 S6 kinase; phospho-rpS6, phospho-ribosomal protein S6; Rapa, rapamycin; rpS6, ribosomal protein S6. Back

Manuscript received 18 March 2008. Initial review completed 28 April 2008. Revision accepted 18 June 2008.


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 ABSTRACT
 Introduction
 Materials and Methods
 Results
 Discussion
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