![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan;
* Department of Applied Biological Sciences, School of Agricultural Sciences, Nagoya University, Nagoya 464-8601, Japan; and
Faculty of Nutritional Sciences, The University of Shizuoka, Shizuoka 422-8526, Japan
1To whom correspondence should be addressed. E-mail: actmori{at}agr.shizuoka.ac.jp.
 |
ABSTRACT |
|---|
 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
KEY WORDS: • settling volume in water • mucins • polystyrene foam • dietary fiber • rats
Multiple mechanisms appear to be involved in producing the wide range of effects associated with the ingestion of dietary fibers. A number of studies focused on the interaction of dietary fibers with some nutrients present in the gut, i.e., modification of lipid and carbohydrate metabolisms by regulation of the absorption rate of these nutrients from the intestinal tract (1–3). However, it is also possible that dietary fibers may interact directly with the mucosa itself through their own physicochemical properties.
Previous studies in rats showed that a purified diet supplemented with water-insoluble dietary fibers [(IDFs)2 ; 10% cellulose or wheat bran] stimulated small intestinal cytokinetics and goblet cell activities (4) and incorporation of radiolabeled tracers into intestinal mucin glycoproteins (5). Recently, Satchithanandam et al. (6) showed that supplementation of 5% citrus fiber in a purified diet produced a significant increase in small intestinal mucin secretion. They attributed this effect to the insoluble fraction (cellulose and lignin) of the citrus fiber preparation because they also observed in that and other experiments (7) that supplementation of wheat bran in the diet increased luminal mucin secretion in the small intestine, but water-soluble guar gum or carrageenan had no effects. More recently, luminal mucin secretion was evaluated using an ELISA technique in rats fed a purified diet supplemented with 20% cellulose, rice bran, or psyllium, but no significant effects were observed in the small intestine (8). Also, using the same technique for mucin analysis, Frankel et al. (9) showed that cellulose feeding had no effects on jejunal mucin secretion in rats fed an elemental diet or administered total parenteral nutrition. Thus, the relation between small intestinal mucin secretion and IDF ingestion is not fully understood and is complex. This is in contrast to the findings of colonic mucin secretion in which the contribution of the bulk-forming property of IDF is apparent (10,11). Differences in experimental conditions as well as methods for mucin determination might lead to different results. However, the fact that the bulk-forming property of IDF was not clearly defined in the previous studies might also compromise the interpretation of the effects of IDF on small intestinal mucin secretion.
The concept of settling volume (SV) is useful to define a bulk-forming property of IDF in the lumen (12). Settling volume is the volume formed by an IDF layer (mL/g IDF) after sedimentation equilibrium is attained in water. In the present study, we attempted to clarify the effects of IDFs on small intestinal mucin secretion from the aspect of their own SV values. For this purpose, we prepared polystyrene foam (PSF) with different expansion ratios in which powders might have SV that differ greatly. PSF does not have any profound effects on the viscosity and fermentability of luminal contents and allows precise analyses solely from the aspect of SV. To extend the findings from experiments with PSF, some naturally occurring IDFs with different SV values were also evaluated for their effects on small intestinal mucin secretion.
Mucin, a viscous gel covering the gastrointestinal mucosa, is an important component that lubricates the epithelial surface and protects it from potential luminal insults. Also, the increased mucin content in the mucosal surface produced by dietary fiber feeding might be related to the rate-limiting diffusion barrier for nutrient absorption in the small intestine (13,14). Therefore, it is important to gain better insight into the mechanism by which IDF stimulate mucin secretion in the small intestine.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Care of animals. Male rats (n = 108) of the Wistar strain (purchased from Shizuoka Laboratory Animal Center) were housed in individual stainless steel cages with wire-screen bottoms in a room with controlled temperature (23 ± 2°C) and lighting (lights on from 0800 to 2000 h). After adaptation to a control diet (Table 1) for at least 3 d, rats weighing 122–129 g were divided into groups of 6 rats on the basis of body weight and given free access to each experimental diet and water. The basic composition of the experimental diets was the same as that of control diet (Table 1). The addition of PSF or dietary fiber to the diet was performed at the expense of an equal amount of cornstarch in the control diet. Body weight and food intakes were recorded each morning before the diet was replenished. The study was approved by the Animal Use Committee of Shizuoka University, Faculty of Agriculture, and the rats were maintained in accordance with the guidelines for the care and use of laboratory animals, Shizuoka University, Faculty of Agriculture.
|
In Expt. 2, rats were fed the control diet or 1 of the diets containing PSF-30, -60, or -90 at 10 g/kg diet for 7 d. After 8 h of food deprivation, rats were refed 3 g of their respective diets within 90 min. The gathering and preparation of luminal contents were as for Expt. 1.
In Expt. 3, after rats were fed the control diet or that diet containing 50 g of PSF-60/kg diet for 7 d, each dietary group was further divided into 2 groups. Half of the rats previously fed the control diet consumed the 5% PSF-60 diet (Control-PSF group), and the other half of rats consumed the same diet (Control-Control group). This was also the case for the rats previously fed the 5% PSF-60 diet, i.e., PSF-Control and PSF-PSF groups. After 8 h of food deprivation, rats were refed 3 g of their respective diets within 90 min. The gathering and preparation of luminal contents were as for Expt. 1. For histologic evaluation, the mid-jejunum and mid-ileum from each of 6 rats in the Control-Control and PSF-PSF groups were removed and placed in 10% buffered formalin.
In Expt. 4, rats were fed the control diet or diets containing cellulose, corn husk, beet fiber, wheat bran, or PSF-60 at 50 g/kg diet for 7 d. After 8 h of food deprivation, rats were refed 3 g of their respective diets within 90 min. The gathering and preparation of luminal contents were as for Expt. 1.
Preparation of mucin fraction. The mucin fraction was isolated by the method of Lien et al. (18) with modifications (19). Briefly, total freeze-dried samples were suspended in sodium chloride solution (0.15 mol/L) containing 0.02 mol sodium azide/L at 4°C. Samples were homogenized for 1 min and immediately centrifuged at 10,000 x g for 30 min to obtain the supernatant. Mucins were recovered as a 60% ethanol precipitate of the supernatant and were finally dissolved in 3.0 mL distilled water for analyses, including SDS/PAGE analysis and determinations of sialic acid, O-linked oligosaccharide chain, and protein as markers of mucin content.
SDS-PAGE analysis of periodic acid-Schiff reagent (PAS)-reactive substances. The mucin fraction prepared from the small intestinal contents was analyzed by SDS-PAGE (3% stacking gel/4% running gel) according to the method of Tytgat et al. (20) with modifications (19). Gels were stained with Coomassie Brilliant Blue for proteins or PAS for sugars (glycoproteins) according to Konad et al. (21). The density of the PAS-stained area was scanned and analyzed using NIH Image (19). Part of the gel was blotted on polyvinylidene difluoride membrane (Immobilon P, Millipore) and used for Western blotting to test the reactivity of peanut agglutinin toward mucins (19). Before testing the reactivity, part of the membrane was treated with 25 mmol sulfuric acid/L at 80°C for 60 min to remove sialic acid attached to the galactose- or N-acetylgalactosamine-moiety within the oligosaccharide chains of mucins.
Sialic acids. Part of the mucin fraction (0.1 mL) was hydrolyzed with 50 mmol sulfuric acid/L for 60 min at 100°C, and sialic acid was determined by the method described previously (19). N-Acetylneuraminic acid was used as a standard.
O-Linked oligosaccharide chains. After an appropriate dilution of the mucin fraction, O-linked oligosaccharide chains were measured using a fluorometric assay (22) that discriminated O-linked glycoproteins (mucin) from N-linked glycoproteins as described by Bovee-Oudenhoven et al. (23). Standard solutions of N-acetylgalactosamine (Sigma) were used to calculate the amount of oligosaccharide chains liberated from mucins during the procedure.
Protein contents. Protein was measured by the method of Lowry et al. (24) using bovine serum albumin as a standard.
Histologic evaluation. Cross-sections (n = 6/rat; 5 µm thick) were prepared from paraffin-embedded samples and stained with PAS counterstained with hematoxylin. Two observers independently analyzed each section on the light microscopic level (Olympus BH2) with a micrometer eyepiece. The following variables were determined: number of crypts/100 µm length of mucosa, crypt length, and number of goblet cells/crypt column (left side of crypt column).
Statistical analyses. Data were analyzed by 1-way or, in Expt. 3, 2-way ANOVA, and post hoc Tukey-Kramer tests. Differences were considered significant at P < 0.05. Data are expressed as means ± SEM. The statistical calculations were carried out using Stat View 5.0 computer software (SAS Institute). Regression analyses were performed using the Stat Cel 2 program (Tokyo Shoseki).
|
RESULTS |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
Graded levels of PSF-60 affect small intestinal mucin content (Expt. 1). A typical pattern of the SDS/PAGE analysis of mucin fraction prepared from the small intestinal contents showed that the PAS-stained area was not stained by Coomassie Brilliant Blue, probably due to a heavy glycosylation of the surface of the polypeptide of mucins (Fig. 1). Part of the gel containing the identical sample was used for Western blotting. After removal of sialic acid with sulfuric acid treatment, peanut agglutinin reacted mainly with the high-molecular-weight band with mobility similar to that of the PAS-stained band, whereas the untreated sample scarcely reacted with peanut agglutinin. This indicates that the PAS-stained band was composed mainly of mucins because proteoglycan does not contain any sialic acid. Accordingly, we measured the PAS-stained band on the SDS/PAGE gel as mucin-like substances.
|
|
|
|
|
|
|
|
|
DISCUSSION |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
When graded levels of PSF-60 (SV = 15) were added to a fiber-free purified (control) diet, all of the mucin markers in the small intestinal contents increased in a dose-dependent manner (Fig. 2). Because the SV of PSF-60 was 15, the total SV of the 10, 30, and 90 g of PSF-60/kg diets were supposed to be 150 (15 x 10), 450 (15 x 30), and 1350 (15 x 90), respectively. When using the total SV as X-dependent and the PAS-reactive substances as Y-dependent, the correlation equation is Y = 110.2 logX + 112.0 (r = 0.995), indicating a saturation phenomenon between 10 and 30 g of PSF-60/kg diet. Therefore, we chose a dietary level of 10 g/kg when the effects of PSF (PSF-30, 60 and 90) with different SV values were examined on the alterations of mucin markers. Figure 3 clearly showed that all of the mucin markers in mucin fractions from the small intestinal contents were positively related mucin glycoproteins such as proteoglycan. However, proteoglycan does not contain any sialic acid, and the oligosaccharide chain of proteoglycan is always linked to the peptide core via an N-glycosidic bond, in contrast to mucins in which oligosaccharide linkage is characterized by an O-glycosidic bond (27). Therefore, PAS-reactive substances, sialic acid, and the O-linked oligosaccharide chain determined here should represent approximate amounts of mucins in the lumen. Because little digestion of mucin occurs before the large intestine (28), an estimate of mucins in the small intestinal contents is necessary to account for the sum of both gastric and small intestinal mucins. However, in our separate experiments, PSF-60 ingestion at 5% in the diet had no effect on the amount of gastric O-linked oligosaccharide chains (control diet, 2.2 ± 0.3 µmol vs. 5% PSF diet, 2.3 ± 0.4 µmol). Accordingly, the present results indicate that the small intestinal mucins were increased in proportion to the SV of dietary fiber sources and PSF fed to rats. These findings were further reinforced by the fact that there was a significant relation between SV values of some naturally occurring IDF and the amount of mucins in the small intestinal contents (Fig. 6).
Takeda and Kiriyama reported that IDFs with a higher SV value ameliorated growth retardation more effectively in rats fed a toxic dose of amaranth (Food & C Red No. 2) and that this beneficial effect might be associated with the transit speed of chyme (gut contents) traveling through the small intestine (12,29). These findings suggested the possibility that the higher amount of mucins in rats fed a diet with IDF might reflect a sort of intestinal stasis of mucins by slowing transit speed. However, Figure 4 showed that small intestinal mucins in rats previously fed the PSF diet for 7 d were significantly greater than in those previously fed the control diet, even when they were refed the control diet on the day of collection of the small intestinal contents. This indicates that the higher amount of mucins in rats previously fed the PSF diet should be ascribed to enhancement of the total capacity for mucin secretion in the small intestinal lumen, but not to mucin stasis by a slowing of transit time. At present, a precise mechanism for the increased mucin secretion is not clear. However, the increased amount of luminal mucins may result from the increased numer of goblet cells or increased rate at which mucins are secreted. Interestingly, Figure 4 also showed that ingestion of a single fiber meal (i.e., Control-PSF group) did not increase intestinal mucin contents compared with the Control-Control group, suggesting that chronic ingestion of IDF is required for stimulation of mucin secretion. Sheldon et al. (30) and Dryden et al. (31) indicated that only chronic, but not a single ingestion of IDF, significantly decreased intestinal glucose absorption in rats fed a diet with 10% cellulose or 30% coarse wheat bran for 5 or 8 wk. They hypothesized that these effects were related to adaptive changes of the mucosal surface. Indeed, the greater numbers of goblet cells in rats fed the 5% PSF diet support this notion (Fig. 5). If this is the case, alteration of mucin quantity in the lumen might be associated in part with the mechanism by which slowing of glucose absorption occurs in rats chronically fed the diet with IDF.
Satchithanandam et al. (7) reported that 10% wheat bran in the diet stimulated small intestinal mucin secretion, whereas 20% rice bran or cellulose did not (8). At present, we do not have a direct explanation for this disparity in the effects on mucin secretion among the IDFs tested. However, we suggest that the differences in SV value between wheat bran and cellulose or rice bran are partially responsible because wheat bran had a greater SV value (8.0 mL/g) than cellulose (3.5 mL/g) in the present study. Indeed, when a direct comparison was made by Student’s t test, there were significant differences in the amount of O-linked oligosaccharide chains in the small intestinal contents between the control diet– and 5% wheat bran diet–fed groups (P = 0.02), but not between the control diet– and 5% cellulose diet–fed groups (Expt. 4). In addition, the mesh size of IDF greatly influences its SV value: SV value decreases as the particle size decreases (29,32), suggesting that an IDF with a smaller size may have a weaker effect on small intestinal mucin secretion. The physiologic importance of particle size and shape of a dietary indigestible component is also seen in whole-gut and orocecal transit times (33). Therefore, we suggest that when the comparative effects of various fractions of IDF are evaluated on mucin secretions, the particle size of IDF and its SV value should be clearly defined in nutrition experiments.
In conclusion, among the various aspects of nutritional functions of IDF, all of the physical properties might be interrelated, but SV seems to be the most important factor for the enhancement of total capacity of mucin secretion in the small intestinal lumen. Moreover, the intensity of small intestinal mucin secretion depends on the composition of previously consumed diets, specifically responding to the SV of indigestible components. However, the mechanism by which bulk formation in the intestinal lumen exerts its stimulatory effect on mucin secretion remains to be examined in future investigations.
|
FOOTNOTES |
|---|
Manuscript received 26 April 2005. Initial review completed 25 May 2005. Revision accepted 5 July 2005.
|
LITERATURE CITED |
|---|
|
TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
1. Jenkins D. J., Wolever T. M., Leeds A. R., Gassull M. A., Haisman P., Dilawari J., Goff D. V., Metz G. L., Alberti K. G. Dietary fibers, fiber analogues, and glucose tolerance: importance of viscosity. Br. Med. J. 1978;1:1392-1394.
2. Marlett J. A. Sites and mechanisms for the hypocholesterolemic actions of soluble dietary fiber sources. Kritchevsky D. Bonfield C. eds. Sites and mechanisms for the hypocholesterolemic actions of soluble dietary fiber sources. Dietary Fiber in Health and Disease. :109-121 Plenum Press New York, NY.
3. Lairon D. Soluble fibers and dietary lipids. Kritchevsky D. Bonfield C. eds. Soluble fibers and dietary lipids. Dietary Fiber in Health and Disease. :99-108 Plenum Press New York, NY.
4. Cassidy M. M., Lightfoot F. G., Grau L. E., Story J. A., Kritchevsky D., Vahouny G. V. Effect of chronic intake of dietary fibers on the ultrastructural topography of rat jejunum and colon: a scanning electron microscopy study. J. Nutr. 1981;34:218-228.
5. Vahouny G. V., Le T., Ifrim I., Satchithanandam S., Cassidy M. M. Stimulation of intestinal cytokinetics and mucin turnover in rats fed wheat bran or cellulose. Am. J. Clin. Nutr. 1985;41:895-900.
6. Satchithanandam S., Vargofcak-Apker M., Calvert R. J., Leeds A. R., Cassidy M. M. Alteration of gastrointestinal mucin by fiber feeding in rats. J. Nutr. 1990;120:1179-1184.
7. Satchithanandam S., Jahangeer S., Cassidy M. M., Floor M. K., Carvert R. J., Leeds A. R., Alabaster O. Quantitative effects of wheat bran feeding on rat intestinal mucin. FASEB J. 1989;3:A1066 (abs.).
8. Satchithanandam S., Klurfeld D. M., Calvert R. J., Cassidy M. M. Effects of dietary fibers on gastrointestinal mucin in rats. Nutr. Res. 1996;16:1163-1177.
9. Frankel W., Zhang W., Singh A., Bain A., Satchithanandam S., Klurfeld D. M., Rombeau J. Fiber: effect on bacterial translocation and intestinal mucin content. World J. Surg. 1995;19:144-149.[Medline]
10. Enss M.-L., Schmidt-Wittig U., Honer K., Kownatzki R., Gartner K. Mechanical challenge causes alterations of rat colonic mucosa and released mucin. Alterations of mucin and mucosa. J. Exp. Anim. Sci. 1994;36:128-140.[Medline]
11. Schmidt-Wittig U., Enss M-L., Coenen M., Gartner K., Hedrich H. J. Response of rat colonic mucosa to a high fiber diet. Ann. Nutr. Metab. 1996;40:343-350.[Medline]
12. Takeda H., Kiriyama S. Correlations between the physical properties of dietary fibers and their protective activities against amaranth toxicity in rats. J. Nutr. 1979;109:388-396.
13. Leeds A. R. Modification of intestinal absorption by dietary fiber and fiber components. Vahouny G. V. Kritchevsky D. eds. Modification of intestinal absorption by dietary fiber and fiber components. Dietary Fiber in Health and Disease. :53-71 Plenum Press New York, NY.
14. Dryden P. A., Jones G. P., Burcher E., Read R.S.D. Effect of chronic ingestion of dietary fiber on the rate of glucose absorption in rats. Nutr. Rep. Int. 1985;31:609-614.
15. Prosky L., Asp N. G., Schweizer T. F., Devries J. W., Furda I. Determination of insoluble, soluble, and total dietary fiber in food and food products: interlaboratory study. J. Assoc. Off. Anal. Chem. 1988;71:1017-1023.[Medline]
16. Middleton H. E., Byers H. G. The settling volume of soils. Soil Sci. 1934;37:15-27.
17. American Institute of Nutrition. Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies. J. Nutr. 1977;107:1340-1348.
18. Lien K. A., McBurney M. I., Beyde B. I., Thomson A.B.R., Sauer W. C. Ileal recovery of nutrients and mucin in humans fed total enteral formulas supplemented with soy fiber. Am. J. Clin. Nutr. 1996;63:584-595.
19. Morita T., Tanabe H., Takahashi K., Sugiyama K. Ingestion of resistant starch protects endotoxin influx from the intestinal tract and reduces D-galactosamine-induced liver injury in rats. J. Gastroenterol. Hepatol. 2004;19:303-313.[Medline]
20. Tytgat K.M.A.J., Buller H. A., Opdam F.J.M., Kim Y. S., Einerhand A.W.C., Dekker J. Biosynthesis of human colonic mucin: MUC2 is the prominent secretory mucin. Gastroenterology. 1994;107:1352-1363.[Medline]
21. Konad G., Offner H., Mellah J. Improved methods for staining of glycoproteins in polyacrylamide gels. Experientia. 1984;40:303-304.
22. Crowther R. S., Wetmore R. F. Fluorometric assay of O-linked glycoproteins by reaction with 2-cyanoacetamide. Anal. Biochem. 1987;163:170-174.[Medline]
23. Bovee-Oudenhoven I.M.J., Termont D.S.M.L., Heidt P. J., Van der Meer R. Increasing the intestinal resistance of rats to the invasive pathogen Salmonella enteritidis: additive effects of dietary lactulose and calcium. Gut. 1997;40:497-504.
24. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 1951;193:265-275.
25. Shimotoyodome A., Meguro S., Hase T., Tokimitsu I., Sakata T. Short chain fatty acids but not lactate and succinate stimulate mucus release in the rat colon. Comp. Biochem. Physiol. 2000;125:525-531.
26. Barcelo A., Claustre J., Moro F., Chayvialle J.-A., Cuber J.-C., Plaisancie P. Mucin secretion is modulated by luminal factors in the isolated vascularly perfused rat colon. Gut. 2000;46:218-224.
27. La Mont J. T. Structure and function of gastrointestinal mucus. Viewp. Dig. Dis. 1985;17:1-4.
28. Hoskins L. C. Mucin degradation by enteric bacteria: ecological aspects and implications for bacterial attachment to the gut mucosa. Boedeker E. C. eds. Mucin degradation by enteric bacteria: ecological aspects and implications for bacterial attachment to the gut mucosa. Attachment of Organisms to the Gut Mucosa. 1984;2:51-67 CRC Press Boca Raton, FL.
29. Kiriyama S. The significance of small intestinal transit speed in the antitoxic activity of dietary fiber. Chen S. C. eds. The significance of small intestinal transit speed in the antitoxic activity of dietary fiber. Proceedings of Kellogg International Symposium on Dietary Fiber. :76-91 Center for Academic Publications Japan Tokyo, Japan.
30. Sheldon E. S., Gary D. L. Effects of dietary fiber on intestinal glucose absorption and glucose tolerance in rats. Gastroenterology. 1980;79:833-836.[Medline]
31. Dryden P. A., Jones G. P., Burcher E., Read R.S.D. Effect of chronic ingestion of dietary fiber on the rate of glucose absorption in rats. Nutr. Rep. Int. 1985;31:609-614.
32. Aritsuka T., Tanaka K., Kiriyama S. Protection of amaranth (Food & C Red No. 2) toxicity by beet dietary fiber and its ability to slow down small intestinal transit speed in rats[in Japanese]. Nippon Nogeikagaku Kaishi. 1989;63:1213-1219.
33. Lewis S. J., Heaton K. W. Roughage revisited: the effect on intestinal function of inert plastic particles of different sizes and shape. Dig. Dis. Sci. 1999;44:744-748.[Medline]
This article has been cited by other articles:
|
|
 |
|
  G. K. Law, R. F. Bertolo, A. Adjiri-Awere, P. B. Pencharz, and R. O. Ball Adequate oral threonine is critical for mucin production and gut function in neonatal piglets Am J Physiol Gastrointest Liver Physiol, May 1, 2007; 292(5): G1293 - G1301. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 T. Morita, H. Tanabe, H. Ito, S. Yuto, T. Matsubara, T. Matsuda, K. Sugiyama, and S. Kiriyama Increased Luminal Mucin Does Not Disturb Glucose or Ovalbumin Absorption in Rats Fed Insoluble Dietary Fiber J. Nutr., October 1, 2006; 136(10): 2486 - 2491. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
F. Guarner Prebiotics and Mucosal Barrier Function J. Nutr., August 1, 2006; 136(8): 2269 - 2269. [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
