High resolution magic angle spinning NMR spectroscopy used to investigate the ability of drugs to bind to synthetic melanin

Iodobenzamides are known to possess an affinity for melanoma tissue dependent on tumor pigmentation. In order to investigate the molecular interactions of drugs with melanin in vitro, a synthetic pigment swelled in deuterium buffer at physiological pH was used. The spectra of various mixtures of each Iodobenzamide (BZ) with melanin were studied at 25 degrees C by NMR under MAS conditions. The drug which interacts with the pigment exhibits linewidths greater than those observed for the free drug in solution. Line-broadening of the resonance occurred for the N-methyl group of acetylcholine or N-ethyl and aromatic groups of BZ. However, linewidths associated with methanol or hippuric acid were less altered by the presence of melanin. These observations indicate the specificity of the interaction between some drug moieties and the sites of melanin. From the concentration dependence of line-broadening, the apparent equilibrium dissociation constant (K(d)) of drug interaction with melanin was approached. It seems that the residual concentration-dependent line-broadening is caused by perturbations of ligand exchange between free and bound states and by differences in magnetic susceptibility present in the sample at the pigment-interacting drug moiety interface. Taken together, these results demonstrate the utility of this technique for investigating binding drugs.     

Postexercise nutrient intake timing in humans is critical to recovery of leg glucose and protein homeostasis

Although the importance of postexercise nutrient ingestion timing has been investigated for glycogen metabolism, little is known about similar effects for protein dynamics. Each subject (n = 10) was studied twice, with the same oral supplement (10 g protein, 8 g carbohydrate, 3 g fat) being administered either immediately (EARLY) or 3 h (LATE) after 60 min of moderate-intensity exercise. Leg blood flow and circulating concentrations of glucose, amino acids, and insulin were similar for EARLY and LATE. Leg glucose uptake and whole body glucose utilization (D-[6,6-2H(2)]glucose) were stimulated threefold and 44%, respectively, for EARLY vs. LATE. Although essential and nonessential amino acids were taken up by the leg in EARLY, they were released in LATE. Although proteolysis was unaffected, leg (L-[ring-2H(5)]phenylalanine) and whole body (L-[1-13C]leucine) protein synthesis were elevated threefold and 12%, respectively, for EARLY vs. LATE, resulting in a net gain of leg and whole body protein. Therefore, similar to carbohydrate homeostasis, EARLY postexercise ingestion of a nutrient supplement enhances accretion of whole body and leg protein, suggesting a common mechanism of exercise-induced insulin action.     

Processing of vitamin A and E in the human gastrointestinal tract

We aimed to provide basic data on the processing of vitamin A and E in the human gastrointestinal tract and to assess whether the size of emulsion fat globules affects the bioavailability of these vitamins. Eight healthy men received intragastrically two lipid formulas differing in their fat-globule median diameter (0.7 vs. 10. 1 microm. Formulas provided 28 mg vitamin A as retinyl palmitate and 440 mg vitamin E as all-rac alpha-tocopherol. Vitamins were measured in gastric and duodenal aspirates, as well as in chylomicrons, during the postprandial period. The gastric emptying rate of lipids and vitamin A and E was similar. The free retinol/total vitamin A ratio was not significantly modified in the stomach, whereas it was dramatically increased in the duodenum. The proportion of ingested lipid and vitamins was very similar in the duodenal content. The chylomicron response of lipids and vitamins was not significantly different between the two emulsions. Our main conclusions are as follows: 1) there is no significant metabolism of vitamin A and E in the human stomach, 2) the enzyme(s) present in the duodenal lumen is significantly involved in the hydrolysis of retinyl esters, and 3) the size of emulsion fat globules has no major effect on the overall absorption of vitamin A and E.     

Effect of transforming growth factor beta on fibroblasts in three-dimensional lattice cultures

Human skin fibroblasts were cultivated in three-dimensional fibrin or collagen lattices, under retracting or non-retracting conditions, and the influence of transforming growth factor beta (TGF beta) was tested. TGF beta stimulated the synthesis of non-collagen protein and of collagen in all the systems. However, only in non-retracting fibrin lattices did it restore a level of protein synthesis comparable to that found in monolayers. The effects of TGF beta greatly depended on the type of substratum and on the presence or absence of retraction.     

DNA-Methylation Patterns in Trisomy 21 Using Cells from Monozygotic Twins

DNA methylation is essential in mammalian development. We have hypothesized that methylation differences induced by trisomy 21 (T21) contribute to the phenotypic characteristics and heterogeneity in Down syndrome (DS). In order to determine the methylation differences in T21 without interference of the interindividual genomic variation, we have used fetal skin fibroblasts from monozygotic (MZ) twins discordant for T21. We also used skin fibroblasts from MZ twins concordant for T21, normal MZ twins without T21, and unrelated normal and T21 individuals. Reduced Representation Bisulfite Sequencing (RRBS) revealed 35 differentially methylated promoter regions (DMRs) (Absolute methylation differences = 25%, FDR < 0.001) in MZ twins discordant for T21 that have also been observed in comparison between unrelated normal and T21 individuals. The identified DMRs are enriched for genes involved in embryonic organ morphogenesis (FDR = 1.60 e -03) and include genes of the HOXB and HOXD clusters. These DMRs are maintained in iPS cells generated from this twin pair and are correlated with the gene expression changes. We have also observed an increase in DNA methylation level in the T21 methylome compared to the normal euploid methylome. This observation is concordant with the up regulation of DNA methyltransferase enzymes (DNMT3B and DNMT3L) and down regulation of DNA demethylation enzymes (TET2 and TET3) observed in the iPSC of the T21 versus normal twin. Altogether, the results of this study highlight the epigenetic effects of the extra chromosome 21 in T21 on loci outside of this chromosome that are relevant to DS associated phenotypes.