Inhibition of bacterial alpha-glucosidases by castanospermine in pure cultures and activated sludge

Castanospermine (CAST) is a known and potent inhibitor of various alpha-glucosidases in eukaryotes. In this work, we elucidated whether CAST could also be used for determining bacterial alpha-glucosidase activity, when measured with 4-methylumbelliferyl-alpha- D-glucoside as a substrate, both in a complex bacterial community, in activated sludge and in pure cultures of bacterial isolates. We found that 140 microM CAST inhibited alpha-glucosidase activity by 30% in a pure culture of Pseudomonas stutzeri. The alpha-glucosidase activity in Chryseobacterium gleum was inhibited by 90% at a concentration of 150 microM CAST, whereas the alpha-glucosidase in Paracoccus denitrificans was resistant to the inhibitor. CAST (140 microM) reduced alpha-glucosidase activity in activated sludge by 40%, the respiration rate being reduced by only 12%. No significant inhibition of the respiration rate was observed in Ps. stutzeri or Pa. denitrificans, whereas the respiration rate in C. gleum grown in a medium containing starch was inhibited by 50% with 140 microM CAST. No effect of CAST was observed in C. gleum grown in a complex medium. This indicated that CAST, at the concentration used, did not cause a general negative effect on bacterial activity. The results suggest that the CAST assay may potentially be useful in determining whether alpha-glucosidase activity, starch, poly- and disaccharides contribute appreciably to the overall activity of a bacterial community. However, the assay should not be used for quantitative measurements of such activity.     

Molecular characterization of a ring X chromosome in a male with short stature

We report the molecular characterization of a ring X chromosome that was transmitted from a mother to a male who has short stature and minor dysmorphic features. This represents only the second reported ring X chromosome in a male. The ring is derived from breakage within the Xp pseudoautosomal region (PAR) and just proximal to the Xq PAR. The total amount of deleted material is 700-900 kb DNA and includes six known transcribed genes. Interestingly, SHOX, a gene implicated in short stature, is not deleted from the ring chromosome. Possible pathogenetic explanations for the patient's clinical features include insufficient dosage of deleted genes, a position effect on SHOX expression, and cell death during development because of ring chromosome nondisjunction. The findings are also relevant to observations made of "complete" ring chromosomes.     

Structure-function analysis of human triacylglycerol hydrolase by site-directed mutagenesis: identification of the catalytic triad and a glycosylation site

Triacylglycerol hydrolase is a microsomal enzyme that hydrolyzes stored cytoplasmic triacylglycerol in the liver and participates in the lipolysis/re-esterification cycle during the assembly of very-low-density lipoproteins. The structure-activity relationship of the enzyme was investigated by site-directed mutagenesis and heterologous expression. Expression of human TGH in Escherichia coli yields a protein without enzymatic activity, which suggests that posttranslational processing is necessary for the catalytic activity. Expression in baculovirus-infected Sf-9 cells resulted in correct processing of the N-terminal signal sequence and yielded a catalytically active enzyme. A putative catalytic triad consisting of a nucleophilic serine (S221), glutamic acid (E354), and histidine (H468) was identified. Site-directed mutagenesis of the residues (S221A, E354A, and H468A) yielded a catalytically inactive enzyme. CD spectra of purified mutant proteins were very similar to that of the wild-type enzyme, which suggests that the mutations did not affect folding. Human TGH was glycosylated in the insect cells. Mutagenesis of the putative N-glycosylation site (N79A) yielded an active nonglycosylated enzyme. Deletion of the putative C-terminal endoplasmic reticulum retrieval signal (HIEL) did not result in secretion of the mutant protein. A model of human TGH structure suggested a lipase alpha/beta hydrolase fold with a buried active site and two disulfide bridges (C87-C116 and C274-C285).     

Hair-bearing submental artery island flap for reconstruction of mustache and beard

Loss of mustache and beard in the adult male caused by severe burn, trauma, or tumor resection may cause cosmetic and psychological problems for these patients. Reconstruction of the elements of the face presents difficult and often daunting problems for plastic surgeons. The tissue that will be used for this purpose should have the same characteristics as the facial area, consisting of thin, pliable, hair-bearing tissue with a good color match. There is a very limited amount of donor area that has these characteristics. A hair-bearing submental island flap was used successfully for mustache and beard reconstruction in 11 male patients during the last 5 years. The scar was on the mentum in four cases, right cheek in two cases, right half of the upper lip in two cases, left cheek in one case, left half of the upper lip in one case, and both sides of the upper lip in one case. The submental island flap is supplied by the submental artery, a branch of the facial artery. The maximum flap size was 13 x 6 cm and the minimum size was 6 x 3 cm (average, 10 x 4 cm) in this series. Direct closure was achieved at all donor sites. Patients were followed up for 6 months to 5 years. No major complication was noted other than one case of temporary palsy of the marginal mandibular branch of the facial nerve. The mean postoperative stay was 7 days. Color and texture match were good. Hair growth on the flap was normal, and characteristics of the hair were the same as the intact side of the face in all patients. The submental island flap is safe, rapid, and simple to raise and leaves a well-hidden donor-site scar. The authors believe that the submental artery island flap surpasses the other flaps in reconstruction of the mustache and beard in male patients. Application of the technique and results are discussed in this article.     

Short-term stretch translocates the alpha-1-subunit of the Na pump to plasma membrane

Short-term (2–30 min) cyclic stretch activates the Na pump in cultured aortic smooth muscle cells (ASMCs). This effect of stretch involves the phosphotidylinositol 3-kinase (PI 3-kinase) participation. Presently, we investigated whether this stimulation is the result of translocation of Na⁺,K⁺-ATPase from endosomes to the plasma membrane. ASMCs were stretched 20% for 5 min using the Flexercell Strain Unit. The plasma membrane and endosome fractions were isolated and Western blotted to localize the Na⁺,K⁺-ATPase α-1-subunit protein. Membrane marker enzyme, 5′ nucleotidase activity, and the early and recycling endosome markers Rab4 and Rab11 were used to verify the enrichment of these fractions. Stretch increased Na⁺,K⁺-ATPase α-1 expression in plasma membrane fractions and decreased it in endosomes. PI 3-kinase inhibitors LY294002 and wortmannin blocked the stretch-induced translocation of the Na⁺,K⁺-ATPase α-1-subunit. Rab4 and Rab11 were enriched in the endosomal fraction, whereas 5′ nucleotidase activity was enriched in the plasma membrane fraction. We conclude that stimulation of the Na pump activity by shortterm cyclic stretch is the result, at least in part, of transport of the α-subunit of the enzyme from endosomes to the plasma membrane.     

In vivo and in vitro effects of some plant hormones on rat erythrocyte carbonic anhydrase and glucose-6-phosphate dehydrogenase activities

The present study was undertaken to determine in vivo and in vitro effects of some plant growth regulators on rat erythrocyte carbonic anhydrase (CA) and glucose-6-phosphate dehydrogenase (G6PD) activities. Both in vivo and in vitro, spermidine and kinetin did not affect enzymatic activities of CA and G6PD, whereas putrescine decreased these activities, and abscisic acid increased them. Since plants use such growth regulators, their effects should be considered on mammals consuming them since they may possess important biological effects.     

Conformational preferences in diastereomeric(5S)-methyl-3-(o-aryl)-2,4-oxazolidinediones

The thermally interconvertible diastereomers of the (5S)-methyl-3-(o-aryl)-2,4-oxazolidinediones were synthesized and their conformers studied by (1)H NMR and HPLC. The barriers to rotation about the N-C(aryl) bond were found to be very much solvent dependent. For the o-fluoro oxazolidinedione, difference in barriers to rotation in deuterated methanol and deuterated chloroform amounted to 34 kJ/mol. ortho-Bromo substitution increased the barrier to rotation up to 100 kJ/mol in ethanol, which enabled the analytical separation of the diastereomers and observation of the thermodynamic enrichment of the S-P conformer by HPLC. In CDCl(3) by (1)H NMR, on the other hand, a barrier of only 89 kJ/mol was determined. The S-M and S-P conformers of the diastereomers of o-methyl, alpha-naphthyl and o-iodo derivatives have been assigned by NOESY experiments and the kinetic and thermodynamic constants for the interconversion between the S-M and S-P conformers were determined.