Colonization factors of diarrheagenicE. coli and their intestinal receptors

WhileEscherichia coli is common as a commensal organism in the distal ileum and colon, the presence of colonization factors (CF) on pathogenic strains ofE. coli facilitates attachment of the organism to intestinal receptor molecules in a species- and tissue-specific fashion. After the initial adherence, colonization occurs, and the involvement of additional virulence determinants leads to illness. EnterotoxigenicE. coli (ETEC) is the most extensively studied of the five categories ofE. coli that cause diarrheal disease, and has the greatest impact on health worldwide. ETEC can be isolated from domestic animals and humans. The biochemistry, genetics, epidemiology, antigenic characteristics, and cell and receptor binding properties of ETEC have been extensively described. Another major category, enteropathogenicE. coli (EPEC), has virulence mechanisms, primarily effacement and cytoskeletal rearrangement of intestinal brush borders, that are distinct from ETEC. An EPEC CF receptor has been purified and characterized as a sialidated transmembrane glycoprotein complex directly attached to actin, thereby associating CF-binding with host-cell response. Three, additional categories ofE. coli diarrheal disease, their colonization factors and their host cell receptors are discussed. It appears that biofilms exist in the intestine in a manner similar to oral bacterial biofilms, and thatE. coli is part of these biofilms as both commensals and pathogens.Abbreviations CF colonization factor - CFA Colonization Factor Antigen - CS coli-surface-associated antigen - EAggEC enteroaggregativeE. coli - ECDD E. coli diarrheal disease - EHEC enterohemorrhagicE. coli - EIEC enteroinvasiveE. coli - EPEC enteropathogenicE. coli - ETEC enterotoxigenicE. coli - Gal galactose - GalNAc N-acetyl galactosamine - LT heat-labile toxin - NeuAc N-acetyl neuraminic acid - PCF Putative colonization factor - RBC red blood cells - SLT Shiga-like toxin - ST heat-stable toxin     

Molecular drift of the bride of sevenless (boss) gene in Drosophila

DNA sequences were determined for three to five alleles of the bride-of- sevenless (boss) gene in each of four species of Drosophila. The product of boss is a transmembrane receptor for a ligand coded by the sevenless gene that triggers differentiation of the R7 photoreceptor cell in the compound eye. Population parameters affecting the rate and pattern of molecular evolution of boss were estimated from the multinomial configurations of nucleotide polymorphisms of synonymous codons. The time of divergence between D. melanogaster and D. simulans was estimated as approximately 1 Myr, that between D. teissieri and D. yakuba as approximately 0.75 Myr, and that between the two pairs of sibling species as approximately 2 Myr. (The boss genes themselves have estimated divergence times approximately 50% greater than the species divergence times.) The effective size of the species was estimated as approximately 5 x 10(6), and the average mutation rate was estimated as 1-2 x 10(-9)/nucleotide/generation. The ratio of amino acid polymorphisms within species to fixed differences between species suggests that approximately 25% of all possible single-step amino acid replacements in the boss gene product may be selectively neutral or nearly neutral. The data also imply that random genetic drift has been responsible for virtually all of the observed differences in the portion of the boss gene analyzed among the four species.      

Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein gene

We have analyzed the conserved regions of the gene coding for the circumsporozoite protein (CSP) in 12 species of Plasmodium, the malaria parasite. The closest evolutionary relative of P. falciparum, the agent of malignant human malaria, is P. reichenowi, a chimpanzee parasite. This is consistent with the hypothesis that P. falciparum is an ancient human parasite, associated with humans since the divergence of the hominids from their closest hominoid relatives. Three other human Plasmodium species are each genetically indistinguishable from species parasitic to nonhuman primates; that is, for the DNA sequences included in our analysis, the differences between species are not greater than the differences between strains of the human species. The human P. malariae is indistinguishable from P. brasilianum, and P. vivax is indistinguishable from P. simium; P. brasilianum and P. simium are parasitic to New World monkeys. The human P. vivax-like is indistinguishable from P. simiovale, a parasite of Old World macaques. We conjecture that P. malariae, P. vivax, and P. vivax-like are evolutionarily recent human parasites, the first two at least acquired only within the last several thousand years, and perhaps within the last few hundred years, after the expansion of human populations in South America following the European colonizations. We estimate the rate of evolution of the conserved regions of the CSP gene as 2.46 x 10(-9) per site per year. The divergence between the P. falciparum and P. reichenowi lineages is accordingly dated 8.9 Myr ago. The divergence between the three lineages leading to the human parasites is very ancient, about 100 Myr old between P. malariae and P. vivax (and P. vivax-like) and about 165 Myr old between P. falciparum and the other two.      

Oligomeric forms of the membrane-bound acetylcholine receptor disclosed upon extraction of the M(r) 43,000 nonreceptor peptide

Oligomeric forms of the acetylcholine receptor are directly visualized by electron microscopy in receptor-rich membranes from torpedo marmorata. The receptor structures are quantitatively correlated with the molecular species so far identified only after detergent solubilization, and further related to the polypeptide composition of the membranes and changes thereof. The structural identification is made possibly by the increased fragility of the membranes after extraction of nonreceptor peptides and their subsequent disruption upon drying onto hydrophilic carbon supports. Receptor particles in native membranes depleted of nonreceptor peptides appear as single units of 7-8 nm, and double and multiple aggregates thereof. Particle doublets having a main-axis diameter of 19 +/- 3 nm predominate in these membranes. Linear aggregates of particles similar to those observed in rotary replicas of quick-frozen fresh electrolytes (Heuser, J.E. and S. R. Salpeter. 1979, J. Cell Biol. 82: 150-173) are also present in the alkaline-extracted membranes. Chemical modifications of the thiol groups shift the distribution of structural species. Dithiothreitol reduction, which renders almost exclusively the 9S, monomeric receptor form, results in the observation of the 7-8 nm particle in isolated form. The proportion of doublets increases in membranes alkylated with N-ethylmaleimide. Treatment with 5,5’-dithiobis-(nitrobenzoic acid) increases the proportion of higher oligomeric species, and particle aggregates (n=oligo) predominate. The nonreceptor v-peptide (doublet of M(r) 43,000) appears to play a role in the receptor monomer-polymer equilibria. Receptor protein and v-peptide co-aggregate upon reduction and reoxidation of native membranes. In membranes protected ab initio with N- ethylmaleimide, only the receptor appears to self-aggregate. The v-peptide cannot be extracted from these alkylated membranes, though it is easily released from normal, subsequently alkylated or reduced membranes. A stabilization of the dimeric species by the nonreceptor v-peptide is suggested by these experiments. Monospecific antibodies against the v-peptide are used in conjunction with rhodamine- labeled anti-bodies in an indirect immunoflourescence assay to map the vectorial exposure of the v-peptide. When intact membranes, v-peptide depleted and “holey” native membranes (treated with 0.3 percent saponin) are compared, maximal labeling is obtained with the latter type of membranes, suggesting a predominantly cytoplasmic exposure of the antigenic determinants of the v-peptide in the membrane. The influence of the v-peptide in the thiol-dependent interconversions of the receptor protein and the putative topography of the peptide are analyzed in the light of the present results.     

Proteomic,metabolic and immunological changes in Biomphalaria glabrata infected with Schistosoma mansoni

Mansonic schistosomiasis is a neglected disease transmitted by Biomphalaria spp. snails. Understanding what happens inside the intermediate host is important to develop more efficient ways of reducing schistosomiasis prevalence. Our purpose was to characterize metabolic and immunological changes in Biomphalaria glabrata 24 h after exposure to Schistosoma mansoni. For this purpose, proteins were extracted from snails’ whole tissue with Tris-Urea buffer and digested with tripsin. Mass spectrometry was performed and analyzed with MaxQuant and Perseus software. Also, the hemolymph of five snails 24 h post exposure was collected, and the numbers of hemocytes, levels of urea, uric acid, nitric oxide, calcium, glycogen and alanine and aspartate aminotransferases activities were assessed. Snails were also dissected for measurement of glycogen content in the cephalopodal region and gonoda-digestive gland complex. Globin domain proteins were found to be up-regulated; also the number of circulating hemocytes was significantly higher after 24 h of exposure to the parasite. NO levels were higher 24 h post exposure. Several proteins associated with energy metabolism were found to be up-regulated. Glycogen analysis showed a significant decrease in the gonad-digestive gland complex glycogen content. We found several proteins which seem to be associated with the host immune response, most of which were up-regulated, however some were down-regulated, which may represent an important clue in understanding B. glabrata – S. mansoni compatibility.     

Increased glycated calmodulin in the submandibular salivary glands of streptozotocin‐induced diabetic rats

Non‐enzymatic glycosylation, a post translational protein modification may be implicated in the diabetes complications. Calmodulin is an important calcium binding protein that complexed with Ca²⁺ may be implicated in salivary gland secretory process. Glycated calmodulin has shown to be less effective in binding calcium. The aim of this study was to determine whether the concentration of glycated‐calmodulin may be elevated in the submandibular salivary glands of streptozotocin‐induced diabetic rats. Diabetes was induced by an intraperitoneal injection of spreptozotocin, and hyperglycemia was confirmed 72 h after injection using a glucosimeter. Thirty days after the induction of diabetes, submandibular salivary glands were used for the analysis of glycated and non‐glycated calmodulin, using a glycogel B columns for separation. Glycated and non‐glycated calmodulin were assayed by an enzymatic method and by ELISA. The overall concentration of CaM (non‐glycated + glycated) in induced diabetic rats was significantly lower than in controls (p < 0.05). The concentration of non‐glycated CaM in controls was significantly higher than in experimental group (p < 0.05), while the concentration of glycated calmodulin between these groups was statistically similar (p > 0.05). Copyright © 2009 John Wiley & Sons, Ltd.     

Mitochondrial and Oxidative Stress Aspects in Hippocampus of Rats Submitted to Dietary n-3 Polyunsaturated Fatty Acid Deficiency After Exposure to Early Stress

Neurochemical Research - Chronic dietary long-chain polyunsaturated fatty acids (PUFAs) deficiency may lead to changes in cortex and hippocampus neuronal membrane phospholipids, and may be linked...     

Finite element analysis of stress distribution in intact and porcelain veneer restored teeth

The aim of this study was to investigate the stress distribution generated in a veneer restoration of an upper central incisor compared to intact teeth using the finite element analysis after applying a lingual buccal loading at the incisal edge. Methods: Two models were developed: one model contained enamel, dentine, cementum, periodontal ligament, cortical and trabecullar bones, and the other model was a veneer restoration; both models were developed using MSC/Nastran software (MacNeal-Schwendler Corporation, Los Angeles, CA, USA) as the pre- and post-processor. A 10-N load was applied at the incisal edge from the lingual to the buccal side to simulate oral conditions in this area (protrusion). Results: Von Mises stresses were then analysed for three different regions: A-B (enamel elements under the veneer or second enamel layer), A'-B' (buccal enamel and/or veneer element layer) and C-D (lingual enamel elements layer). A higher stress mode was observed for both models at the lingual cervical region. Conclusions: The presence of a veneer restoration on the incisors is a good alternative to mimic the behaviour of enamel under protrusion loading conditions. The use of veneers to replace enamel during rehabilitations is recommended.     

Characterization of the TRBP domain required for Dicer interaction and function in RNA interference

Background  

Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain.