Effects of methyl parathion (o,o-dimethyl o-4-nitrophenyl phosphorothioate) on rat sperm morphology and sperm count, but not fertility, are associated with decreased ascorbic acid level in the testis

Methyl parathion (MP; o,o-dimethyl o-4-nitrophenyl phosphorothioate) is an organophosphorous pesticide used world wide to spray agricultural crops. The present study was aimed to investigate the genotoxic and cytotoxic effects on male germ cells and their possible relation with testicular ascorbic acid levels. Adult male Wistar rats (n=5/group) received MP at 0, 0.5, or 1 mg/kg (experiments 1 and 2) for 12 days and 0, 0.75 or 1.5 mg/kg (experiment 3) for 25 days (i.p.) everyday at intervals of 24 h. The epididymal sperm count, sperm abnormalities and testicular ascorbic acid levels (by 2,4-dinitrophenyl hydrazine method) were estimated on days 130, 77 and 17 following the last exposure in experiments 1, 2, and 3, respectively. Virgin untreated female rats were mated with treated males from experiments 2 and 3 for a week effective from day 35 to 41 after the first treatment, and fertility indices were measured after the birth of pups. Sperm count was decreased in experiments 2 and 3 (P<0.01), and in all three experiments, the abnormal sperms increased (P<0.001). Concomitantly, the ascorbic acid levels decreased in the testis (P<0.05-0.001; one-way ANOVA and Bonferroni's post hoc test). The body weights of offspring of treated males did not show significant changes from those of the controls, although there were some decreases observed. MP reduced the lactation index in experiment 2 (P<0.001; Chi-square test). The number of pups/parent along with fertility indices showed some numerical decrease but without any statistical significance. The present findings suggest that MP is a weak genotoxic and cytotoxic agent in the rat exposed to human exposure dose-levels, and that these effects, except the fertility are well correlated with decreased ascorbic acid level in the testis. Furthermore, MP-induced changes in the germ cells do not have any significant effects on F1 generation.     

Prediction and characterization of T-cell epitopes for epitope vaccine design from outer membrane protein of Neisseria meningitidis serogroup B

Neisseria meningitidis serogroup B (MC58) is a leading cause of meningitis and septicaemia, principally infects the infants and adolescents. No vaccine is available for the prevention of these infections because the serogroup B capsular polysaccharide is unable to stimulate an immune response, due to its similarity with polysialic acid. To overcome these obstacles, we proposed to develop a peptide based epitope vaccine from outer membrane protein contained in outer membrane vesicles (OMV) based on our computational analysis. In OMV a total of 236 proteins were identified, only 15 (6.4%) of which were predicted to be located in outer membrane. The major requirement is the identification and selection of T-cell epitopes that act as a vaccine target. We have selected 13 out of 15 outer membrane proteins from OMV proteins. Due to similarity of the fkpA and omp85 with the human FKBP2 and SAMM50 protein, we removed these two sequences from the analysis as their presence in the vaccine is likely to elicit an autoimmune response. ProPred and ProPred1 were used to predict promiscuous helper T Lymphocytes (HTL) and cytotoxic T Lymphocytes (CTL) epitopes and MHCPred for their binding affinity in N. meningitidis serogroup B (MC58), respectively. Binding peptides (epitopes) are distinguished from nonbinding peptides in properties such as amino acid preference on the basis of amino acid composition. By using this dataset, we compared physico-chemical and structural properties at amino acid level through amino acid composition, computed from ProtParam server. Results indicate that porA, porB, opc, rmpM, mtrE and nspA are more suitable vaccine candidates. The predicted peptides are expected to be useful in the design of multi-epitope vaccines without compromising the human population coverage.     

The effects of increasing swelling and anionic charges on the enzymatic hydrolysis of organosolv-pretreated softwoods at low enzyme loadings

Organosolv‐pretreated Lodgepole pine substrates were physically and chemically treated to increase their hydrophilicity and swelling as these are two substrate attributes which have been shown to improve cellulolytic hydrolysis. Surprisingly, mechanical treatment of the organosolv‐treated substrates by PFI‐mill refining did not significantly increase hydrolysis yields despite decreases in particle size and crystallinity and increases in swelling. However, sulfonation of the substrate did, significantly, increase enzymatic hydrolysis at loadings of both 5 and 2.5 FPU g⁻¹ cellulose (from 80% to 95% and from 35% to 80%, respectively). In addition, sulfonation resulted in an increase in the amount of free enzymes detected during the course of hydrolysis to a maximum of 80% after 72 h. This suggested that the beneficial effects of sulfonation were primarily due to a decrease in the non‐specific binding of the cellulases to the lignin. Biotechnol. Bioeng. 2011; 108:1549–1558. © 2011 Wiley Periodicals, Inc.     

Impact of rapid urbanization on the rates of infection by Vibrio cholerae O1 and enterotoxigenic Escherichia coli in Dhaka, Bangladesh

Background

In Bangladesh, increases in cholera epidemics are being documented with a greater incidence and severity. The aim of this prospective study was to identify the prevalence and importance of V. cholerae O1 and enterotoxigenic Escherichia coli (ETEC) as causal agents of severe diarrhea in a high diarrhea prone urban area in Dhaka city.

Methodology

Systematic surveillance was carried out on all diarrheal patients admitted from Mirpur between March 2008 to February 2010 at the ICDDR, B hospital. Stool or rectal swabs were collected from every third diarrheal patient for microbiological evaluation.

Principal Findings

Of diarrheal patients attending the hospital from Mirpur, 41% suffered from severe dehydration with 39% requiring intravenous rehydration therapy. More diarrheal patients were above five years of age (64%) than those below five years of age (36%). About 60% of the patients above five years of age had severe dehydration compared with only 9% of patients under five years of age. The most prevalent pathogen isolated was Vibrio cholerae O1 (23%) followed by ETEC (11%). About 8% of cholera infection was seen in infants with the youngest children being one month of age while in the case of ETEC the rate was 11%. Of the isolated ETEC strains, the enterotoxin type were almost equally distributed; ST accounted for 31% of strains; LT/ST for 38% and LT for 31%.

Conclusion

V. cholerae O1 is the major bacterial pathogen and a cause of severe cholera disease in 23% of patients from Mirpur. This represents a socioeconomic group that best reflects the major areas of high cholera burden in the country. Vaccines that can target such high risk groups in the country and the region will hopefully be able to reduce the disease morbidity and the transmission of pathogens that impact the life and health of people.     

Arsenic, cadmium and neuron specific enolase (ENO2, γ-enolase) expression in breast cancer

Background

Neuron specific enolase (ENO2, γ-enolase) has been used as a biomarker to help identify neuroendocrine differentiation in breast cancer. The goal of the present study was to determine if ENO2 expression in the breast epithelial cell is influenced by the environmental pollutants, arsenite and cadmium. Acute and chronic exposure of MCF-10A cells to As⁺³ and Cd⁺² sufficient to allow colony formation in soft agar, was used to determine if ENO2 expression was altered by these pollutants.

Results

It was shown that both As⁺³ and Cd⁺² exposure caused significant increases in ENO2 expression under conditions of both acute and chronic exposure. In contrast, ENO1, the major glycolytic enolase in non-muscle and neuronal cells, was largely unaffected by exposure to either As⁺³ or Cd⁺². Localization studies showed that ENO2 in the MCF-10A cells transformed by As⁺³ or Cd⁺² had both a cytoplasmic and nuclear localization. In contrast, ENO1 was localized to the cytoplasm. ENO2 localized to the cytoplasm was found to co-localized with ENO1.

Conclusion

The results are the first to show that ENO2 expression in breast epithelial cells is induced by acute and chronic exposure to As⁺³ or Cd⁺². The findings also suggest a possible link between As⁺³ and Cd⁺² exposure and neuroendocrine differentiation in tumors. Overall, the results suggest that ENO2 might be developed as a biomarker indicating acute and/or chronic environmental exposure of the breast epithelial cell to As⁺³ and Cd⁺².     

Interaction with a kinesin-2 tail propels choline acetyltransferase flow towards synapse

Bulk flow constitutes a substantial part of the slow transport of soluble proteins in axons. Though the underlying mechanism is unclear, evidences indicate that intermittent, kinesin-based movement of large protein-aggregates aids this process. Choline acetyltransferase (ChAT), a soluble enzyme catalyzing acetylcholine synthesis, propagates toward the synapse at an intermediate, slow rate. The presynaptic enrichment of ChAT requires heterotrimeric kinesin-2, comprising KLP64D, KLP68D and DmKAP, in Drosophila. Here, we show that the bulk flow of a recombinant Green Fluorescent Protein-tagged ChAT (GFP::ChAT), in Drosophila axons, lacks particulate features. It occurs for a brief period during the larval stages. In addition, both the endogenous ChAT and GFP::ChAT directly bind to the KLP64D tail, which is essential for the GFP::ChAT entry and anterograde flow in axon. These evidences suggest that a direct interaction with motor proteins could regulate the bulk flow of soluble proteins, and thus establish their asymmetric distribution.     

Faster Protein Splicing with the Nostoc punctiforme DnaE Intein Using Non-native Extein Residues

Inteins are naturally occurring intervening sequences that catalyze a protein splicing reaction resulting in intein excision and concatenation of the flanking polypeptides (exteins) with a native peptide bond. Inteins display a diversity of catalytic mechanisms within a highly conserved fold that is shared with hedgehog autoprocessing proteins. The unusual chemistry of inteins has afforded powerful biotechnology tools for controlling enzyme function upon splicing and allowing peptides of different origins to be coupled in a specific, time-defined manner. The extein sequences immediately flanking the intein affect splicing and can be defined as the intein substrate. Because of the enormous potential complexity of all possible flanking sequences, studying intein substrate specificity has been difficult. Therefore, we developed a genetic selection for splicing-dependent kanamycin resistance with no significant bias when six amino acids that immediately flanked the intein insertion site were randomized. We applied this selection to examine the sequence space of residues flanking the Nostoc punctiforme Npu DnaE intein and found that this intein efficiently splices a much wider range of sequences than previously thought, with little N-extein specificity and only two important C-extein positions. The novel selected extein sequences were sufficient to promote splicing in three unrelated proteins, confirming the generalizable nature of the specificity data and defining new potential insertion sites for any target. Kinetic analysis showed splicing rates with the selected exteins that were as fast or faster than the native extein, refuting past assumptions that the naturally selected flanking extein sequences are optimal for splicing.