Chemical synthesis of (22E)-3 alpha,6 beta,7 beta-trihydroxy-5 beta-chol-22-en-24-oic acid and its taurine and glycine conjugates: a major bile acid in the rat

A method for the synthesis of Delta(22)-beta-muricholic acid (Delta(22)-beta-MCA), (22E)-3 alpha,6 beta,7 beta-trihydroxy-5 beta-chol-22-en-24-oic acid, and its taurine and glycine conjugates (Delta(22)-beta-muricholyltaurine and Delta(22)-beta-muricholylglycine) is described. The key intermediate, 3 alpha,6 beta,7 beta-triformyloxy-23,24-dinor-5 beta-cholan-22-al, was prepared from beta-muricholic acid (beta-MCA) via the 24-nor-22-ene and 24-nor-22,23-diol derivatives. Wittig reaction of the aldehyde with (carbomethoxymethylene) triphenylphosphorane and subsequent hydrolysis gave (unconjugated) Delta(22)-beta-MCA. Condensation reaction of the unconjugated acid with taurine or glycine methyl ester using diethylphosphorocyanide yielded the naturally occurring taurine or glycine conjugate (N-acylamidate) of Delta(22)-beta-MCA. These synthetic reference compounds are now available for investigation of the metabolism of beta-MCA by bacterial and hepatic enzymes in the rat and should also be useful as substrates for reductive deuteration or tritiation to give the 22,23-(2)H or (3)H-beta-MCA.     

Changes in the expression of small intestine extracellular matrix proteins in streptozotocin-induced diabetic rats

Diabetes mellitus is characterized by anatomical and functional alterations of the intestinal tract. However, the aetiology of these disturbances remains unclear. The aim of the present work was to investigate the effects of diabetes on the expression of laminin-1 and fibronectin in the small intestine of Streptozotocin (STZ)-induced diabetic rats. The Western immunoblotting of the extracts from the small intestine revealed that experimental diabetes resulted in a marked increase in the intensity of the bands corresponding to laminin-1 and fibronectin. Immunohistochemical studies demonstrated a strong labelling to these two extracellular matrix (ECM) proteins in the small intestine of diabetic rats, mainly localized in the smooth muscle layer. These results occur together with a thickening of the basement membrane (BM) of the smooth muscle cells, demonstrated by transmission electron microscopy (TEM). We propose that the accumulation of ECM proteins in the smooth muscle layer may be an effect mediated by hyperglycaemia, since insulin treatment of diabetic rats reversed this accumulation. These results could provide information on the potential role of the ECM in the intestine, an organ which is known to exhibit important alterations in diabetes.     

Identification of a novel conjugate in human urine: bile acid acyl galactosides

We report a novel conjugate, bile acid acyl galactosides, which exist in the urine of healthy volunteers. To identify the two unknown peaks obtained in urine specimens from healthy subjects, the specimens were subjected to solid phase extraction and then to liquid chromatographic separation. The eluate corresponding to the unknown peaks on the chromatogram was collected. Following alkaline hydrolysis and liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometric (MS) analysis, cholic acid (CA) and deoxycholic acid (DCA) were identified as liberated bile acids. When a portion of the alkaline hydrolyzate was subjected to a derivatization reaction with 1-phenyl-3-methyl-5-pyrazolone, a derivative of galactose was detected by LC/ESI-MS. Finally, the liquid chromatographic and mass spectrometric properties of these unknown compounds in urine specimens were compared to those of authentic specimens and the structures were confirmed as CA 24-galactoside and DCA 24-galactoside. These results strongly imply that bile acid 24-galactosides, a novel conjugate, were synthesized in the human body.     

More than royal food - <Emphasis Type="Italic">Major royal jelly protein</Emphasis> genes in sexuals and workers of the honeybee <Emphasis Type="Italic">Apis mellifera</Emphasis>

Background

In the honeybee Apis mellifera, female larvae destined to become a queen are fed with royal jelly, a secretion of the hypopharyngeal glands of young nurse bees that rear the brood. The protein moiety of royal jelly comprises mostly major royal jelly proteins (MRJPs) of which the coding genes (mrjp1-9) have been identified on chromosome 11 in the honeybee’s genome.

Results

We determined the expression of mrjp1-9 among the honeybee worker caste (nurses, foragers) and the sexuals (queens (unmated, mated) and drones) in various body parts (head, thorax, abdomen). Specific mrjp expression was not only found in brood rearing nurse bees, but also in foragers and the sexuals.

Conclusions

The expression of mrjp1 to 7 is characteristic for the heads of worker bees, with an elevated expression of mrjp1-4 and 7 in nurse bees compared to foragers. Mrjp5 and 6 were higher in foragers compared to nurses suggesting functions in addition to those of brood food proteins. Furthermore, the expression of mrjp9 was high in the heads, thoraces and abdomen of almost all female bees, suggesting a function irrespective of body section. This completely different expression profile suggests mrjp9 to code for the most ancestral major royal jelly protein of the honeybee.

     

Prevention of LLC-PK(1) cell overgrowth in a bioartificial renal tubule device using a MEK inhibitor, U0126

A common approach to construct a bioartificial renal tubule system is to utilize renal tubular cells seeded in porous polymer membrane hollow fibers. We have reported that overgrowth of renal tubular cells was not beneficial for the transport and reabsorption functions of bioartificial tubules. Therefore, long-term maintenance of a confluent monolayer of cells in hollow fibers is essential and technically challenging. In this study, we examined whether MEK inhibitor, U0126, could maintain the monolayer of Lewis-lung cancer porcine kidney 1 (LLC-PK(1)) cells on polystyrene plates and in a dialysis module housing hollow fibers made of ethylene vinyl alcohol (EVAL). We also evaluated the leakage of urea nitrogen (UN) and creatinine (Cr) through the cell-lined hollow fibers, and reabsorption of glucose and sodium by the cells, comparing the U0126-treated cells with nontreated cells in the module. Treatment with 50micromol l(-1) U0126 prevented the overgrowth of cells cultured on polystyrene plates. Moreover, U0126-treatment reduced the leakage of UN, and increased the reabsorption of electrolytes in 65cm(2) modules. Scanning electron microscopy revealed that it also prevented the overconfluence of cells in modules. Therefore, application of U0126 is a potentially effective method to improve the performance of the device.     

The interplay of processivity, substrate inhibition and a secondary substrate binding site of an insect exo-beta-1,3-glucanase

Abracris flavolineata midgut contains a processive exo-beta-glucanase (ALAM) with lytic activity against Saccharomyces cerevisiae, which was purified (yield, 18%; enrichment, 37 fold; specific activity, 1.89 U/mg). ALAM hydrolyses fungal cells or callose from the diet. ALAM (45 kDa; pI 5.5; pH optimum 6) major products with 0.6 mM laminarin as substrate are beta-glucose (61%) and laminaribiose (39%). Kinetic data obtained with laminaridextrins and methylumbelliferyl glucoside suggest that ALAM has an active site with at least six subsites. The best fitting of kinetic data to theoretical curves is obtained using a model where one laminarin molecule binds first to a high-affinity accessory site, causing active site exposure, followed by the transference of the substrate to the active site. The two-binding-site model is supported by results from chemical modifications of amino acid residues and by ALAM action in MUbetaGlu plus laminarin. Low laminarin concentrations increase the modification of His, Tyr and Asp or Glu residues and MUbetaGlu hydrolysis, whereas high concentrations abolish modification and inhibit MUbetaGlu hydrolysis. Our data indicate that processivity results from consecutive transferences of substrate between accessory and active site and that substrate inhibition arises when both sites are occupied by substrate molecules abolishing processivity.     

Maternal zinc deficiency impairs brain nestin expression in prenatal and postnatal mice

INTRODUCTIONZinc is essential for normal brain development,evidenced by the fact that zinc deficiency in lactating mothers is characterized by a high incidence ofneuroanatomical maiformatinns and functional abnormalities in suckling offspring[1-3]. By colltrast,relatively little is known about the relationship be{tween maternal zinc nutrition and fetal brain development[2, 4, 5]. Dvergsten et al[6-81 investigated theeffects of maternal zinc deficiency on postnatal development of the rat ce…     

Hypoglycemic activity of leaf organic extracts from Smallanthus sonchifolius: Constituents of the most active fractions

The aim of the present study was to determine the in vivo hypoglycemic activity of five organic extracts and enhydrin obtained from yacon leaves. The main constituents of the most active fraction were identified. Five organic extracts and pure crystalline enhydrin were administered to normoglycemic, transiently hyperglycemic and streptozotocin (STZ)-diabetic rats. The fasting and post-prandial blood glucose, and serum insulin levels were estimated and an oral glucose tolerance test (OGTT) was performed for the evaluation of hypoglycemic activity and dose optimization of each extract.We found that the methanol, butanol and chloroform extracts showed effective hypoglycemic activity at minimum doses of 50, 10 and 20 mg/kg body weight, respectively, and were selected for further experiments. Oral administration of a single-dose of each extract produced a slight lowering effect in the fasting blood glucose level of normal healthy rats, whereas each extract tempered significantly the hyperglycemic peak after food ingestion. Daily administration of each extract for 8 weeks produced an effective glycemic control in diabetic animals with an increase in the plasma insulin level. Phytochemical analysis of the most active fraction, the butanol extract, showed that caffeic, chlorogenic and three dicaffeoilquinic acids were significant components. Additionally, enhydrin, the major sesquiterpene lactone of yacon leaves, was also effective to reduce post-prandial glucose and useful in the treatment of diabetic animals (minimum dose: 0.8 mg/kg body weight).The results presented here strongly support the notion that the phenolic compounds above as well as enhydrin are important hypoglycemic principles of yacon leaves that could ameliorate the diabetic state.