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Am J Physiol Endocrinol Metab 289: E710-E715, 2005. First published May 31, 2005; doi:10.1152/ajpendo.00155.2005
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AMP-activated protein kinase kinase activity and phosphorylation of AMP-activated protein kinase in contracting muscle of sedentary and endurance-trained rats

Denise Hurst, Eric B. Taylor, Troy D. Cline, Lyle J. Greenwood, Cori L. Compton, Jeremy D. Lamb, and William W. Winder

Department of Physiology and Developmental Biology, Brigham Young University, Provo, Utah

Submitted 7 April 2005 ; accepted in final form 17 May 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was designed to examine activity of AMP-activated protein kinase kinase (AMPKK) in muscles from nontrained and endurance-trained rats. Rats were trained 5 days/wk, 2 h/day for 8 wk at a final intensity of 32 m/min up a 15% grade with 30-s sprints at 53 m/min every 10 min. Gastrocnemius muscles were stimulated in situ in trained and nontrained rats for 5 min at frequencies of 0.4/s and 1/s. Gastrocnemius LKB1 protein, a putative component of the AMPKK complex (LKB1, STRAD, and MO25), increased approximately twofold in response to training. Phosphorylation of AMP-activated protein kinase (AMPK) determined by Western blot and AMPK activity of immunoprecipitates (both isoforms) was increased at both stimulation rates in both trained and nontrained muscles. AMPKK activity was 73% lower in resuspended polyethylene glycol precipitates of muscle extracts from the trained compared with nontrained rats. AMPKK activity did not increase in either trained or nontrained muscle in response to electrical stimulation, even though phospho-AMPK did increase. These results suggest that AMPKK is activated during electrical stimulation of both trained and nontrained muscle by mechanisms other than covalent modification.

AMP-activated protein kinase; adenosine 5'-triphosphate; metabolism; skeletal muscle; serine/threonine kinase 11

AMP-ACTIVATED PROTEIN KINASE (AMPK) has been characterized in skeletal muscle as an energy-sensing/signaling protein (34, 35). This heterotrimeric protein kinase is activated in response to muscle contraction, which produces the increase in 5'-AMP and decrease in creatine phosphate (PCr) (16, 23, 33, 36). In response, the activated AMPK phosphorylates other proteins (some yet unidentified) involved in the enhancement of fatty acid oxidation and glucose uptake, and in control of protein synthesis and gene regulation (2, 6, 7, 13, 15, 20, 21, 30, 35–37).

The activation of AMPK is dependent on phosphorylation of the Thr172 residue of the {alpha}-subunit by enzymes collectively referred to as AMPK kinase (AMPKK) (10, 18, 29). In liver and in cells in culture, the protein complex responsible for most of the AMPKK activity appears to be the kinase LKB1 with regulating proteins STRAD and MO25 (12, 27, 38). The upstream kinase has not yet been fully characterized in skeletal muscle. Recently, LKB1 protein, MO25 protein, and STRAD mRNA have been found to be expressed in skeletal muscle (25, 32). LKB1 and MO25 proteins increase markedly in skeletal muscle in response to endurance training (32). Deficiency of LKB1 has now been reported to eliminate phosphorylation and activation of AMPK induced by 5-aminoimidazole-4-carboxamide riboside (AICAR), phenformin, or muscle contraction (26). This report provides strong evidence that the major upstream kinase in skeletal muscle includes LKB1 as a component. This laboratory and others have reported an enhancement of phosphorylation of AMPK in the Thr172 position in response to muscle contraction (4, 22, 23). Thus AMPKK becomes more active by an unknown mechanism in phosphorylating AMPK as the muscle contracts. In these experiments we have attempted to determine in nontrained and endurance-trained muscle whether the AMPKK is activated in a way that can be detected by in vitro assays. That is, if AMPKK is activated by phosphorylation, other covalent modification, or by stable association with other proteins, this activation should be detectable in extracts of the muscle. If it is activated by only allosteric mechanisms or by transient association with other proteins, activity should not be increased in AMPKK fractions extracted from the contracted muscle. We hypothesized that AMPKK is activated by phosphorylation and that contraction will result in an increase in AMPKK activity in extracts from muscle that has been stimulated to contract. Furthermore, because activation of muscle AMPK is attenuated in exercising endurance-trained rats compared with nontrained rats, we hypothesized that AMPKK activity would be lower in muscles of endurance-trained rats.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal care. All procedures were approved by the Institutional Animal Care and Use Committee of Brigham Young University. Male Sprague-Dawley rats (Sasco, Wilmington, MA) were housed in a temperature-controlled (21–22°C) room with a 12:12-h light-dark cycle. Rats were assigned to either a training or control group. Training rats were fed standard rat chow (Harlan Teklad rodent diet) and water ad libitum. Control rats were food restricted to maintain body weight similar to training rats. Body weights at the end of the study were 356 ± 13 g for trained rats and 353 ± 13 g for control rats. Rats were trained on a motor-driven rodent treadmill 5 days/wk for 10–11 wk up a 15% grade in a room maintained at 16–17°C. Initially, rats completed two 45-min exercise bouts/day at 16 m/min. Training progressed to a single 120-min exercise bout at 32 m/min with 30-s sprints at 53 m/min every 10 min for the final 4–5 wk. Rats were anesthetized 18–24 h after the last training bout by intraperitoneal injection of pentobarbital sodium (50 mg/kg body wt). Food consumption was 23 ± 1 g for control rats and 21 ± 1 g for trained rats the night before collection of tissues. These values are not statistically different from each other (P < 0.05).

In a separate experiment, nontrained rats were first accustomed to running on the treadmill 5 min/day for 5 days. Tissues were collected from rats anesthetized at rest by intravenous (jugular catheter) injection of pentobarbital sodium or anesthetized after running at 21 m/min up a 15% grade for 90 min. Only the red quadriceps was analyzed for AMPKK activity.

In situ stimulation of the gastrocnemius muscle. On the day of the experiment, rats were anesthetized with pentobarbital sodium (50 mg/kg body wt). They were kept anesthetized at surgical level by injection of additional anesthetic for ≥45 min before surgery. The purpose of this delay was to allow any increase in AMPK because of the handling procedure to return to baseline values before the beginning of the experiment. The tibial nerve was then exposed using blunt dissection. Gastrocnemius muscles were collected at rest or after stimulation via the tibial nerve with single pulses of 10 ms duration and 10 V for 5 min at a frequency of 0.4/s or 1/s. At the end of the stimulation, the gastrocnemius was clamp frozen between stainless steel clamps at liquid nitrogen temperature and then stored at –95°C until analyzed.

Analysis. These muscles were ground to powder under liquid nitrogen and homogenized in a buffer (1 g muscle powder + 9 ml buffer) containing 200 mM mannitol, 50 mM NaF, 100 mM Tris, 1 mM EDTA, 10 mM {beta}-mercaptoethanol, pH 7.5, and proteolytic enzyme inhibitors (8 trypsin-inactivating U/l aprotonin, 1 mg/l leupeptin, and 1 mg/l antitrypsin). Acetyl-CoA carboxylase (ACC) for activity measurements was isolated from this homogenate as described previously (36). ACC activity was determined at a citrate concentration of 0.5 mM as described previously (36). A second homogenate (1:9) was also prepared in 50 mM Tris·HCl, 250 mM mannitol, 50 mM NaF, 5 mM sodium pyrophosphate, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, pH 7.4, and proteolytic enzyme inhibitors 1 mM benzamidine, 0.1 mM phenylmethylsulfonyl fluoride (PMSF), and 5 µg/ml soybean trypsin inhibitor (SBTI). DTT, benzamidine, PMSF, and SBTI were added to the buffer immediately before tissue homogenization. The Western blots for LKB1, phospho-AMPK, and phospho-ACC were performed on the 1,200-g supernatant of this homogenate as described previously (32) using immunoaffinity-purified primary antibodies (Cell Signaling Technology, Beverly, MA). The dilutions of primary antibodies for the Western blots were 1:1,000. Samples from each stimulation frequency were run on the same gel and blot. After densitometric scanning and quantitation, intensities of all spots were normalized for exposure.

AMPKK was also isolated from this homogenate by precipitation with polyethylene glycol (PEG). After the initial 1,200-g centrifugation, PEG precipitations were performed by adding 0.32 ml of 25% PEG 6000/ml muscle homogenates (Calbiochem) to yield a PEG concentration of 6% (wt/vol). After being mixed and allowed to stand for 15 min on ice, homogenates were centrifuged at 30,000 g for 15 min. PEG was added to the 6% PEG supernatant to bring PEG concentration to 10% (wt/vol). Homogenates with 10% PEG were incubated on ice for 30 min and then centrifuged at 30,000 g for 15 min. Pellets were resuspended in of the original homogenate volume and frozen at –95°C prior to assays.

The {alpha}1- and {alpha}2-AMPK activities were determined on immunoprecipitates by using commercially prepared (Affinity Bioreagents, Golden, CO) affinity-purified antibodies to the peptides TSPPDSFLDDHHLTR ({alpha}1) and MDDSAMHIPPGLKPH ({alpha}2) conjugated to keyhole limpet hemocyanin at the NH2 terminus via a cysteine residue. The immunoprecipitation and AMPK activity measurements were done by methods described by Hardie et al. (11) with the exceptions that the immunoprecipitation was overnight and the AMPK assay was on resuspended immunoprecipitate in the medium described previously (23).

The AMPKK activity of resuspended PEG precipitates from muscle was assayed by the activation of a standardized preparation of bacterially expressed AMPK{alpha}1312 (9, 31, 32). Activation of {alpha}1312 was measured by 32P incorporation from [{gamma}-32P]ATP into AMARA peptide. Resuspended PEG precipitates (2 µl) were incubated for 20 min (30°C) with AMPKK assay buffer (4 µl; 100 mM HEPES, 200 mM NaCl, 20% glycerol, 2 mM EDTA, 12.5 mM MgCl2, 0.5 mM AMP, 0.5 mM ATP, 2.0 mM DTT, pH 7.0) and AMPK{alpha}1312 in storage buffer (4 µl of 50 mM Tris·HCl, 250 mM mannitol, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.02% Brij-35, 10% vol/vol glycerol, pH 7.4). Phosphorylation buffer (15 µl of 40 mM HEPES, 80 mM NaCl, 8% glycerol, 0.8 mM EDTA, 0.8 mM DTT, 5 mM MgCl2, 0.2 mM AMP, 0.2 mM ATP, 0.33 mM AMARA peptide, 0.133 µCi/µl [{gamma}-32P]ATP, pH 7.0) was added, and after a 10-min incubation, 15 µl was spotted on a 1-cm2 piece of P81 filter paper (Whatman, Tewksbury, MA). Filter papers were washed 6 x 5 min in 100 ml of 1% phosphoric acid rinsed with acetone, dried, and counted for 10 min in 3 ml of Ecolite (ICN, Irvine, CA).

A perchloric acid extract (100 mg powder/ml 6% perchloric acid) was also made of the frozen muscle powder for determination of phospocreatine (PCr) (14), ATP (14), and lactate (8). Glycogen content of the frozen powder was also measured using an enzymatic method (24). Citrate synthase activity was measured by the method of Srere (28)

Results are expressed as means ± SE. Statistical comparison of treatment groups was performed using Student's t-test or analysis of variance. Post hoc comparisons were performed using Fisher's protected least significant difference test. Number Cruncher statistical software (Kaysville, UT) was utilized for the statistical analysis. Differences between means were considered statistically significant when P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
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DISCUSSION
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Citrate synthase was measured in the gastrocnemius as an index of training. Citrate synthase activity in muscle of endurance-trained rats was increased to 257% of values in the sedentary rats (77 ± 4 vs. 30 ± 3 µmol·g–1min–1, P < 0.001). Western blots demonstrated that LKB1 also was markedly increased in response to the training (Fig. 1).



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  		Fig. 1. LKB1 in nontrained (NT) and trained (T) gastrocnemius muscle. Values are means ± SE, n = 9. *Significantly different from nontrained, P < 0.05.

 
Muscle stimulation for 5 min via the tibial nerve resulted in significant increases in lactic acid and decreases in PCr and glycogen content (Fig. 2). Lactate content was significantly lower in the trained gastrocnemius only at a stimulation frequency of 1/s. PCr decreased significantly at both stimulation frequencies. Although a trend was noted for PCr to be lower in the nontrained muscle, this difference was not significant. Glycogen was significantly higher in the trained muscle at both stimulation frequencies. A statistically significant drop in glycogen was observed in the nontrained muscle at both stimulation frequencies, but only at a stimulation rate of 1/s in the trained muscle (P < 0.05). ATP content of the muscle was significantly decreased in both trained and nontrained rats at a stimulation frequency of 1/s. No statistically significant differences were noted between trained and nontrained muscles at rest or at either stimulation frequency.



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  		Fig. 2. Muscle ATP, creatine phosphate (PCr), lactate, and glycogen from nontrained and trained rats. Values are means ± SE; n = 9. *Significantly different from Rest; {dagger}significantly different from nontrained at same stimulation rate, P < 0.05.

 
The activity of the muscle AMPK precipitated with the antibody against both the {alpha}1- and {alpha}2-subunits was increased significantly (P < 0.05) in response to stimulation at a frequency of 0.4/s or at 1/s, but no statistically significant differences were noted between the nontrained and trained rats (Fig. 3). ACC activity was determined on resuspended ammonium sulfate precipitates from resting and stimulated muscles (Fig. 4). The activity at 0.5 mM citrate was decreased significantly (P < 0.05) in response to stimulation at 0.4/s and 1/s. No difference was observed between the nontrained and trained muscle. Phospho-ACC determined by Western blot was slightly higher in the trained muscle in response to stimulation at 0.4/s, but not at 1/s (Fig. 4).



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  		Fig. 3. AMP-activated protein kinase (AMPK) activity in resting and stimulated gastrocnemius muscles from nontrained and trained rats. Values are means ± SE; n = 9. *Significantly different from Rest, P < 0.05. No differences were detected between trained and nontrained muscle.

 


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  		Fig. 4. Phospho-acetyl-CoA carboxylase (P-ACC) and ACC activity in resting and stimulated muscle from nontrained and trained rats. Values are means ± SE; n = 9. *Significantly different from Rest, P < 0.05; {dagger}significantly different from nontrained at same stimulation rate, P < 0.05.

 
Phospho-AMPK, quantitated with a specific antibody raised against the phosphorylation site of AMPK was significantly increased at both stimulation rates (Fig. 5). At a stimulation rate of 1/s, phospho-AMPK tended to be lower in trained than in nontrained muscle (P < 0.14). This difference was not statistically significant using a post hoc test after ANOVA, but it was significant (P < 0.05) if just those two groups were compared.



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  		Fig. 5. Phospho-Thr172 of AMPK in resting and stimulated gastrocnemius muscles from nontrained and trained rats. Values are means ± SE; n = 9. *Significantly different from Rest, P < 0.05.

 
AMPKK activity was quantitated on resuspended PEG precipitates of the muscle homogenates and on the 1,200-g supernatant from the crude homogenate. AMPKK activity was not increased in response to stimulation in either the nontrained or trained muscle (Fig. 6). The activity of AMPKK, quantitated by measuring the increase in activity of the truncated {alpha}-subunit, was considerably lower in these extracts of the trained compared with the nontrained muscles. AMPKK activity was also decreased in whole homogenates (Fig. 6). The activity of AMPKK was significantly lower after stimulation in the supernatant from the crude homogenates of the nontrained stimulated muscles compared with the nontrained resting muscles. No such trend was noted in the homogenates from trained muscle. Interestingly, in these same homogenates, it was apparent that phosphorylation of AMPK was markedly increased in response to electrical stimulation of the muscle, yet no change or even a decrease was noted in AMPKK activity. When nontrained rats were run for 90 min on the treadmill, the AMPKK activity in the red region of the quadriceps was not significantly changed (Rest = 4.1 ± 0.3 vs. Run = 4.4 ± 0.6 nmol·g–1·min–1, n = 3/group).



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  		Fig. 6. AMP-activated protein kinase kinase (AMPKK) activity in resting and stimulated gastrocnemius muscles from nontrained and trained rats. Top: activity as measured in the 1,200-g supernatant. Bottom: activity measured in resuspended polyethylene glycol (PEG) precipitates. Values are means ± SE; n = 7–9. *Significantly different from nontrained at same stimulation rate, P < 0.05; {dagger}significantly different from nontrained rest, P < 0.05.

 
Western blots were also done for LKB1 and MO25 on the resuspended PEG precipitates used for AMPKK quantitation. The same relationships were seen in the PEG precipitates as in the crude 1,200-g supernatant. Both LKB1 and MO25 were higher in the PEG precipitates of nontrained than in trained muscle. Relative values for LKB1 were 1.00 ± 0.11 for nontrained resting muscle and 1.75 ± 0.23 for resting trained muscle (P < 0.05). Relative values for MO25 in resuspended PEG precipitates were 1.00 ± 0.05 for nontrained and 1.75 ± 0.23 for trained (P < 0.05). The results on LKB1 and on AMPKK activity are apparently inconsistent with our previous report, where we reported no difference in LKB1 in resuspended PEG precipitates of nontrained and trained red quadriceps (32) and no difference in AMPKK activity. We did use a higher detergent concentration (1 vs. 0.5%) in the homogenizing buffer for the present experiments. We obtained a more complete extraction of the LKB1 in the nontrained muscle with the higher detergent concentration.


   DISCUSSION
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
It is now well established that muscle contraction induced by either electrical stimulation or exercise results in activation of AMPK. Several studies have now demonstrated increased phosphorylation of AMPK (as indicated using specific phospho-AMPK antibody) in response to muscle contraction (19, 22, 23, 39). This demonstrates that the upstream kinase becomes more active in phosphorylating AMPK in response to changes that take place in the muscle in response to the contraction. The mechanism of the contraction-induced activation is not clearly defined. Several possibilities were hypothesized to be responsible for this activation. First, it is possible that the AMPKK was phosphorylated and activated by an upstream kinase, a covalent form of regulation. Second, it is possible that the AMPKK is activated by allosteric mechanisms by molecules or ions that increase in response to muscle contraction. A third possibility is that contraction induces association of proteins, which in combination have AMPKK activity. A fourth possibility is that regulation of the AMPKK is at the substrate level; that is, contraction-induced changes in conformation of the AMPK allow AMPKK to bind and phosphorylate AMPK. Finally, a reduction in activity of phosphatases that dephosphorylate the Thr172 site of AMPK could result in an increase in the phosphorylation state without a detectable change in AMPKK activity. Previous reports indicate that AMPK becomes a better substrate for dephosphorylation and inactivation by phosphatases in response to increases in 5'-AMP concentration (10).

In the present study, when AMPKK activity was measured in extracts from resting and contracting muscle using phosphorylation and activation of the truncated {alpha}-subunit, we were unable to demonstrate an increase in activity in the contracting muscle. This was true in both trained and nontrained muscle, where phospho-AMPK increased in response to contraction. If the mechanism of the increased AMPK phosphorylation in response to contraction were because of phosphorylation/activation of AMPKK, it seems reasonable that the activation should persist into the homogenate and resuspended PEG precipitates, similar to the increase in activity of AMPK. The homogenizing media contained phosphatase inhibitors expected to prevent dephosphorylation during the time of preparation of the homogenates. Because an increase in AMPKK was not observed, we consider it quite unlikely that phosphorylation of AMPKK was the means of activation and therefore must seek other mechanisms to explain the increase in phospho-AMPK that occurs with contraction.

Although several studies report enhancement of phospho-AMPK content of muscle in response to contraction, relatively few have measured AMPKK activity. AMPKK activity can be quantitated using the truncated {alpha}-subunit of AMPK, measuring the increase in phosphorylation of either SAMS peptide or AMARA peptide. Using this method, Chen et al. (4) reported an increase in muscle AMPKK activity during long-term exercise in human subjects. Subjects cycled for 20 min each at 40, 59, and 79% of maximum oxygen consumption. An ~50% increase in AMPKK activity was noted in crude extracts of muscle biopsies taken after the 59 and 79% work periods, but not after the 40% work period (4). The extent of AMPK phosphorylation was not reported in that study. In rats run on the treadmill for as long as 90 min, we have been unable to see any increase in AMPKK activity. The only other study is on electrically stimulated muscle, where no change in LKB1 activity (measured by phosphorylation of LKB1-tide) was observed in response to contraction (25). The reason for the discrepancies between different studies is not clear at this time, but it is possible that species differences may exist in the mechanism of activation of AMPKK.

Although the LKB1/STRAD/MO25 complex has clearly been shown to be the major AMPKK in liver extracts, it is unclear what proportion of the AMPKK activity of skeletal muscle is due to this complex. LKB1 and MO25 protein have been demonstrated to be present in skeletal muscle extracts, along with mRNA (32). Both of these proteins increased markedly with endurance training. STRAD mRNA is also present (32). However, some dissociation has been noted between LKB1 protein content and AMPKK activity. In the present study, LKB1 protein content of the gastrocnemius muscle increased in response to endurance training, but AMPKK activity was consistently decreased both in PEG precipitates and in supernatants of crude homogenates. Previous studies have shown the kinase activity of LKB1 to be dependent on the presence of STRAD (3, 12). Although the STRAD mRNA in red quadriceps does not change with endurance training, it is still possible that the STRAD protein may be lower, thus accounting for the decline in AMPKK activity, even in the face of increases in LKB1. This decline in AMPKK activity appears to be a result of the chronic training stimulus rather than because of a single bout of exercise. Activity does not decrease during a single 90-min bout of exercise. However, as seen in the assays on 1,200-g supernatants, the AMPKK activity did appear to be lower after 5 min of stimulation of the muscle at both stimulation frequencies.

Another possible reason for the dissociation observed between LKB1 and AMPKK activity may be that LKB1 might not be the principal AMPKK in skeletal muscle. Another AMPKK has been identified in heart muscle extracts that appears to be independent of immunodetectable LKB1, using an antibody against the COOH terminus (1). However, a very recent report has provided evidence from the conditional LKB1 knockout that a deficiency in LKB1 results in marked reduction in AMPK phosphorylation and activation (26), thus providing strong evidence indicating that the principal AMPKK of muscle includes LKB1.

Previous studies in rats clearly demonstrated that the increase in AMPK activity in response to exercise is attenuated in endurance-trained muscle compared with nontrained muscle (5). The decline in ACC activity and the decline in malonyl-CoA was reported to be less in muscle of trained rats compared with nontrained rats during prolonged bouts of exercise (17). Although trends were noted in some of the assays for lower AMPK activity and ACC activity in the trained muscle of the present study, no consistent and statistically significant difference was noted. The results clearly demonstrate, however, that when phosphocreatine concentrations decline to similar levels in nontrained and trained muscle in response to electrical stimulation, similar increases in AMPK activity and phospho-AMPK also are seen. Another point to consider is that the gastrocnemius muscle consists of both types IIA and IIB fibers. The training effects in the IIA fibers may have been diluted by the presence of the IIB fibers, which have lower levels of mitochondrial oxidative enzymes.

In conclusion, endurance training was found to increase LKB1 protein expression but to decrease AMPKK activity. AMPKK activity was not increased in either nontrained or trained gastrocnemius muscle in response to 5-min stimulation at a frequency of 0.4/s and 1/s. It was clear, however, that phospho-AMPK was markedly increased in the stimulated muscles of both nontrained and trained rats. This implies that the AMPKK of muscle becomes more active during contraction by mechanisms other than covalent modification. Other possibilities include enhancement of association of essential subunits, allosteric activation by yet-undefined modulators, and change in conformation of the AMPK, resulting in more efficient phosphorylation by the constitutively active AMPKK. Finally, the lower AMPK activity previously reported in trained muscle during exercise may be because of the lower AMPKK activity.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
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 GRANTS
REFERENCES
 
This work was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-41438.


   FOOTNOTES
 

Address for reprint requests and other correspondence: W. W. Winder, 545 WIDB, Brigham Young University, Provo, UT 84602 (email: william_winder{at}byu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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