Influence of cetylpyridinium chloride on the ultrastructural appearance of sulphated glycosaminoglycans in human colonic mucosa

The effect of adding cetylpyridinium chloride to the fixative on the preservation of sulphated glycosaminoglycans (SGs) was studied in human normal colonic mucosa. SGs were visualized at the ultrastructural level through the application of Spicer's High Iron Diamine (HID) technique followed by a post-fixation with potassium ferrocyanide-reduced osmium tetroxide. SGs were mainly localized in basement membranes of epithelium and capillary wall and along collagen fibers. The morphology of the reactive sites depended on the presence of cetylpyridinium chloride, SGs being granular in absence of the salt and more or less elongated when cetylpyridinium chloride was added to the fixative. We suggest that the use of cetylpyridinium chloride during fixation may help to preserve SG molecule at the ultrastructural level.     

The effect of dietary imbalances on the activation of benzo[a]pyrene by the metabolizing enzymes from rat liver

Male Sprague-Dawley rats (70-80 g) were fed ad libitum a standard control diet (22% casein, 5% lard), or a high lipid diet (30% lard) or a low protein diet (6% casein) or a standard diet containing 50 ppm phenoclor DP6. After 6 weeks on these diets, the cytochrome P-450 microsomal content, the benzo[a]pyrene monooxygenase (BaP-MO) and the epoxide hydrolase (EH) were assayed. The formation of mutagenic B(a)P metabolites which covalently bind with DNA was compared. The activity of BaP-MO and of EH were increased by the high lipid diet (+27% and 106% respectively) and by the phenoclor DP6 treatment (+63% and 400% respectively), compared to the standard diet. In animals fed a low protein diet the BaP-MO was decreased (-34%) and the EH activity was strongly increased (+262%) compared to those fed a standard diet. All experimental diets increased both the activation of BaP to metabolites able to bind DNA and the mutagenicity of BaP versus TA98 Salmonella typhimurium strain. It was concluded that dietary imbalances can be considered as a factor in chemical carcinogenesis.     

Significant linkage on chromosome 10p in families with bulimia nervosa

Bulimia nervosa (BN) is strongly familial, and additive genetic effects appear to contribute substantially to the observed familiality. In turn, behavioral components of BN, such as self-induced vomiting, are reliably measured and heritable. To identify regions of the genome harboring genetic variants conferring susceptibility to BN, we conducted a linkage analysis of multiplex families with eating disorders that were identified through a proband with BN. Linkage analysis of the entire sample of 308 families yielded a double peak, with the highest nonparametric multipoint maximum LOD score (MLS), of 2.92, on chromosome 10. Given the high heritability of self-induced vomiting and the reliability with which it can be measured, we performed linkage analysis in a subset (n=133) of families in which at least two affected relatives reported a symptom pattern that included self-induced vomiting. The highest MLS (3.39) observed was on chromosome 10, between markers D10S1430 and D10S1423. These results provide evidence of the presence of a susceptibility locus for BN on chromosome 10p. Using simulations, we demonstrate that both of these scores, 2.92 and 3.39, meet the widely accepted criterion for genomewide significance. Another region on 14q meets the criterion for genomewide suggestive linkage, with MLSs of 1.97 (full sample) and 1.75 (subset) at 62 centimorgans from p-ter.     

Structural genomics of Mycobacterium tuberculosis: a preliminary report of progress at UCLA

The growing list of fully sequenced genomes, combined with innovations in the fields of structural biology and bioinformatics, provides a synergy for the discovery of new drug targets. With this background, the TB Structural Genomics Consortium has been formed. This international consortium is comprised of laboratories from 31 universities and institutes in 13 countries. The goal of the consortium is to determine the structures of over 400 potential drug targets from the genome of Mycobacterium tuberculosis and analyze their structures in the context of functional information. We summarize the efforts of the UCLA consortium members. Potential drug targets were selected using a variety of bioinformatics methods and screened for certain physical and species-specific properties to yield a starting group of protein targets for structure determination. Target determination methods include protein phylogenetic profiles and Rosetta Stone methods, and the use of related biochemical pathways to select genes linked to essential prokaryotic genes. Criteria imposed on target selection included potential protein solubility, protein or domain size, and targets that lack homologs in eukaryotic organisms. In addition, some protein targets were chosen that are specific to M. tuberculosis, such as PE and PPE domains. Thus far, the UCLA group has cloned 263 targets, expressed 171 proteins and purified 40 proteins, which are currently in crystallization trials. Our efforts have yielded 13 crystals and eight structures. Seven structures are summarized here. Four of the structures are secreted proteins: antigen 85B; MPT 63, which is one of the three major secreted proteins of M. tuberculosis; a thioredoxin derivative Rv2878c; and potentially secreted glutamate synthetase. We also report the structures of three proteins that are potentially essential to the survival of M. tuberculosis: a protein involved in the folate biosynthetic pathway (Rv3607c); a protein involved in the biosynthesis of vitamin B5 (Rv3602c); and a pyrophosphatase, Rv2697c. Our approach to the M. tuberculosis structural genomics project will yield information for drug design and vaccine production against tuberculosis. In addition, this study will provide further insights into the mechanisms of mycobacterial pathogenesis.     

Understanding the transition states of phosphodiester bond cleavage: insights from heavy atom isotope effects

The nucleotides of DNA and RNA are joined by phosphodiester linkages whose synthesis and hydrolysis are catalyzed by numerous essential enzymes. Two prominent mechanisms have been proposed for RNA and protein enzyme catalyzed cleavage of phosphodiester bonds in RNA: (a) intramolecular nucleophilic attack by the 2'-hydroxyl group adjacent to the reactive phosphate; and (b) intermolecular nucleophilic attack by hydroxide, or other oxyanion. The general features of these two mechanisms have been established by physical organic chemical analyses; however, a more detailed understanding of the transition states of these reactions is emerging from recent kinetic isotope effect (KIE) studies. The recent data show interesting differences between the chemical mechanisms and transition state structures of the inter- and intramolecular reactions, as well as provide information on the impact of metal ion, acid, and base catalysis on these mechanisms. Importantly, recent nonenzymatic model studies show that interactions with divalent metal ions, an important feature of many phosphodiesterase active sites, can influence both the mechanism and transition state structure of nonenzymatic phosphodiester cleavage. Such detailed investigations are important because they mimic catalytic strategies employed by both RNA and protein phosphodiesterases, and so set the stage for explorations of enzyme-catalyzed transition states. Application of KIE analyses for this class of enzymes is just beginning, and several important technical challenges remain to be overcome. Nonetheless, such studies hold great promise since they will provide novel insights into the role of metal ions and other active site interactions.     

Calcium response after stimulation by substance P of U373 MG cells: inhibition of store-operated calcium entry by protein kinase C

In this paper we investigate the Ca2+ response after Substance P (SP) stimulation of U373 MG cells. SP is a tachykinin and physiologically acts as a neurotransmitter and neuromodulator in the nervous system, but pathologically triggers malignant glial cells, such as U373 MG, to release cytokines and increase proliferation rate.In this paper we show that SP increases the proliferation rate of U373 MG cells and the intracellular Ca2+ concentration by mobilizing Ca2+ only from thapsigargin-sensitive stores. In fact, Ca2+ entry through store-operated calcium entry (SOCE) channels, which was observed after thapsigargin treatment, was not detected after stimulation by SP. The inhibition of SOCE after SP stimulation must be mediated by protein kinase C (PKC), because it was not observed in the presence of calphostin C (an inhibitor of PKC). Moreover, stimulation by SP-induced membrane potential hyperpolarization. Our results are consistent with the following sequence of events: (i) SP interacts with NK(1) receptors; (ii) fast homologous receptor desensitization occurs; (iii) reuptake by endoplasmic reticulum Ca(2+)-ATPase quantitatively overwhelms the extrusion by plasma membrane Ca2+-ATPase. These results have two important consequences. In U373 MG cells the SOCE does not contribute to the Ca2+ response after SP, and is not necessarily involved in promoting cell proliferation.