Mycobacterium tuberculosis encodes a YhhN family membrane protein with lysoplasmalogenase activity that protects against toxic host lysolipids

The pathogen Mycobacterium tuberculosis (M.tb) resides in human macrophages, wherein it exploits host lipids for survival. However, little is known about the interaction between M.tb and macrophage plasmalogens, a subclass of glycerophospholipids with a vinyl ether bond at the sn-1 position of the glycerol backbone. Lysoplasmalogens, produced from plasmalogens by hydrolysis at the sn-2 carbon by phospholipase A₂, are potentially toxic but can be broken down by host lysoplasmalogenase, an integral membrane protein of the YhhN family that hydrolyzes the vinyl ether bond to release a fatty aldehyde and glycerophospho-ethanolamine or glycerophospho-choline. Curiously, M.tb encodes its own YhhN protein (MtbYhhN), despite having no endogenous plasmalogens. To understand the purpose of this protein, the gene for MtbYhhN (Rv1401) was cloned and expressed in Mycobacterium smegmatis (M.smeg). We found the partially purified protein exhibited abundant lysoplasmalogenase activity specific for lysoplasmenylethanolamine or lysoplasmenylcholine (pLPC) (V_max∼15.5 μmol/min/mg; K_m∼83 μM). Based on cell density, we determined that lysoplasmenylethanolamine, pLPC, lysophosphatidylcholine, and lysophosphatidylethanolamine were not toxic to M.smeg cells, but pLPC and LPC were highly toxic to M.smeg spheroplasts, which are cell wall–deficient mycobacterial forms. Importantly, spheroplasts prepared from M.smeg cells overexpressing MtbYhhN were protected from membrane disruption/lysis by pLPC, which was rapidly depleted from the media. Finally, we found that overexpression of full-length MtbYhhN in M.smeg increased its survival within human macrophages by 2.6-fold compared to vector controls. These data support the hypothesis that MtbYhhN protein confers a growth advantage for mycobacteria in macrophages by cleaving toxic host pLPC into potentially energy-producing products.     

Influence of hexaconazole, carbofuran and ethion on soil microflora and dehydrogenase activities in soil and intact cell

Total microbial count was highly affected (up to 61% at 1000 micrograms level) in presence of hexaconazole and persisted up to 21 days. Bacteria were more susceptible than actinomycetes. Carbofuran and ethion were moderately toxic to soil microflora. Inhibitory effects of all the three pesticides gradually decreased after 21 days as was evident by increase in total microbial count except in carbofuran. GDH activity in soil was also affected initially (up to 14 days) by all the three pesticides (60.3% in hexaconazole at 1000 micrograms level) and inhibition gradually decreased to zero except in carbofuran (15-20% toxicity persisted up to 35 days). GDH and LDH activity in presence of hexaconazole was strongly affected in intact cells of some standard culture of bacteria like Rhizobium sp. (host Dolichos sp., 32.1 and 72.5%), Bacillus subtilis Cohn (86.75 and 76.5%), Azotobacter sp. (36.9 and 55.4%) and B. sphaericus (67.6% GDH) respectively. Carbofuran inhibited the enzyme activity in B. subtilis (55.55 and 35.3%) and to some extent in B. sphaericus. Ethion moderately inhibited LDH activity in Rhodococcus sp. AK1 (17.1 and 33.3%), Rhizobium (27.6% LDH), E. coli HB 101 (34.2% LDH) as evidenced by formazan formation. From the result, it might be concluded that among the above three pesticides tested hexaconazole strongly inhibited the dehydrogenase system in bacteria including nitrogen fixing bacteria of soil and thus may affect soil fertility. It was concluded that hexaconazole was more toxic than ethion to dehydrogenase enzymes.     

Variation of toxigenic Vibrio cholerae O1 in the aquatic environment of Bangladesh and its correlation with the clinical strains

The diversity of toxigenic V. cholerae O1 in the aquatic environment of Bangladesh is not known. A total of 18 environmental and 18 clinical strains of toxigenic V. cholerae O1 were isolated simultaneously from four different geographical areas and tested for variation by the pulsed-field gel electrophoresis method. Environmental strains showed diversified profiles and one of the profiles was common to some environmental strains and most clinical strains. It appears that one clone has an advantage over others to cause disease. These findings suggest that the study of the molecular ecology of V. cholerae O1 in relation to its environmental reservoir is important in identifying virulent strains that cause disease.     

Phosphorylation of plasma membrane aquaporin regulates temperature-dependent opening of tulip petals

The opening and closing of tulip petals was reproduced in the dark by changing the temperature from 5 degrees C to 20 degrees C for opening and 20 degrees C to 5 degrees C for closing. The opening process was accompanied by (3)H(2)O transport through the stem from the incubation medium to the petals. A Ca(2+)-channel blocker and a Ca(2+)-chelator inhibited petal opening and (3)H(2)O transport. Several proteins in the isolated plasma membrane fraction were phosphorylated in the presence of 25 micro M Ca(2+) at 20 degrees C. The 31-kDa protein that was phosphorylated, was suggested immunologically as the putative plasma membrane aquaporin (PM-AQP). This phosphorylated PM-AQP clearly reacted with the anti-phospho-Ser. In-gel assay revealed the presence of a 45-kDa Ca(2+)-dependent protein kinase in the isolated plasma membrane. Phosphorylation of the putative PM-AQP was thought to activate the water channel composed of PM-AQP. Dephosphorylation of the phosphorylated PM-AQP was also observed during petal closing at 5 degrees C, suggesting the inactivation of the water channel.     

PhyA gene product of Aspergillus ficuum and Peniophora lycii produces dissimilar phytases

PhyA gene products of Aspergillus ficuum (AF) and Peniophora lycii (PL) as expressed in industrial strains of Aspergillus niger and Aspergillus oryzae, respectively, were purified to homogeneity and then characterized for both physical and biochemical properties. The PL phytase is 26 amino acid residues shorter than the AF phytase. Dynamic light scattering studies indicate that the active AF phytase is a monomer while the PL phytase is a dimer. While both of the phytases retained four identical glycosylatable Asn residues, unique glycosylation sites, six for PL and seven for AF phytase, were observed. Global alignment of both the phytases has shown 38% sequence homology between the two proteins. At 58 degrees C and pH 5.0, the PL phytase gave a specific activity of 22,000 nKat/mg as opposed to about 3000 nKat/mg for AF phytase. However, the AF phytase is more thermostable than its counterpart PL phytase at 65 degrees C. Also, AF phytase is more stable at pH 7.5 than the PL phytase. The two phytases differed in K(m) for phytate, K(i) for myo-inositol hexasulfate (MIHS), and pH optima profile. Despite similarities in the active site sequences, the two phytases show remarkable differences in turnover number, pH optima profile, stability at higher temperature, and alkaline pH. These biochemical differences indicate that phytases from ascomycete and basidiomycete fungi may have evolved to degrade phytate in different environments.     

Computerized automated morphometric assay including frequency estimation of pentachlorophenol induced nuclear anomalies (micronucleus) in catfish Heteropneustes fossilis

An in vivo study of the effects of pentachlorophenol was carried out with a pre-acclimatized fish species, Heteropneustes fossilis, using four sub-lethal concentrations, 0.1, 0.2, 0.3 and 0.4 ppm, and three sampling times, 48, 72 and 96 h. Cytogenetic preparations were stained by the haematoxylin-eosin technique. The incidence of micronuclei was scored by a manual and an automated method. Small-sized micronuclei appeared in the cytoplasm in addition to the main nucleus. The frequency of micronucleated erythrocytes peaked at 4 days (96 h) exposure. The percentage of single micronuclei increased with longer exposures. The Mann-Whitney U test showed all micronuclei frequencies were significantly different from control (P<0.05). No statistical difference was observed between scores obtained by the manual and automated methods. A linear relationship between the percentage of micronucleated erythrocytes and dose was confirmed at all levels. Computer image analysis of morphological variations of erythrocytes indicated a 1:5 ratio of micronuclei and main nucleus accompanied by a reduction in cell volume by 600 dot units. Pentachlorophenol-mediated genotoxicity was confirmed in this fish for the first time. Possible consequences of genotoxicity and cytotoxicity are discussed.     

Natural versus adaptive regulatory T cells

The regulation of immune responses to self-antigens is a complex process that involves maintaining self-tolerance while retaining the capacity to mount robust immune responses against invading microorganisms. Over the past few years, many new insights into this process have been gained, leading to the re-emergence of the idea that regulatory T (T(Reg)) cells are a central mechanism of immune regulation. These insights have raised fundamental questions concerning what constitutes a T(Reg) cell, where they develop and what signals maintain T(Reg)-cell populations in a functional state. Here, we propose the existence of two subsets of CD4+ T(Reg) cells--natural and adaptive--that differ in terms of their development, specificity, mechanism of action and dependence on T-cell receptor and co-stimulatory signalling.     

Child Centred Approach to Climate Change and Health Adaptation through Schools in Bangladesh: A Cluster Randomised Intervention Trial

Background

Bangladesh is one of the most vulnerable countries to climate change. People are getting educated at different levels on how to deal with potential impacts. One such educational mode was the preparation of a school manual, for high school students on climate change and health protection endorsed by the National Curriculum and Textbook Board, which is based on a 2008 World Health Organization manual. The objective of this study was to test the effectiveness of the manual in increasing the knowledge level of the school children about climate change and health adaptation.

Methods

This cluster randomized intervention trial involved 60 schools throughout Bangladesh, with 3293 secondary school students participating. School upazilas (sub-districts) were randomised into intervention and control groups, and two schools from each upazila were randomly selected. All year seven students from both groups of schools sat for a pre-test of 30 short questions of binary response. A total of 1515 students from 30 intervention schools received the intervention through classroom training based on the school manual and 1778 students of the 30 control schools did not get the manual but a leaflet on climate change and health issues. Six months later, a post-intervention test of the same questionnaire used in the pre-test was performed at both intervention and control schools. The pre and post test scores were analysed along with the demographic data by using random effects model.

Results

None of the various school level and student level variables were significantly different between the control and intervention group. However, the intervention group had a 17.42% (95% CI: 14.45 to 20.38, P = <0.001) higher score in the post-test after adjusting for pre-test score and other covariates in a multi-level linear regression model.

Conclusions

These results suggest that school-based intervention for climate change and health adaptation is effective for increasing the knowledge level of school children on this topic.     

Site-directed mutagenesis of disulfide bridges in Aspergillus niger NRRL 3135 phytase (PhyA), their expression in Pichia pastoris and catalytic characterization

Earlier studies have established the importance of five disulfide bridges (DBs) in Aspergillus niger phytase. In this study, the relative importance of each of the individual disulfide bridge is determined by its removal by site-directed mutagenesis of specific cysteines in the cloned A. niger phyA gene. Individually, these mutant phytases were expressed in a Pichia expression system and their product purified and characterized. The removal of disulfide bridge 2 yielded a mutant phytase with a complete loss of catalytic activity. The other disulfide mutants displayed a broad array of altered catalytic properties including a lower optimum temperature from 58°C to 53°C for bridge number 1, 37°C for bridge number 3 and 4, and 42°C for bridge number 5. The pH versus activity profile was also modified in the DB mutants. The pH profile of the wild-type phytase was modified by the DB mutations. In bridge number 1, 3, and 4, the second peak at pH 2.5 was abolished, and in bridge number 5, the peak at pH 5.0 was abolished completely leaving only the pH 2.5. While the K _m was not affected drastically, the turnover number was lowered significantly in bridge number 3, 4, and 5.     

AtPTR3, a wound-induced peptide transporter needed for defence against virulent bacterial pathogens in <Emphasis Type="Italic">Arabidopsis</Emphasis>

Mutation in the wound-induced peptide transporter gene AtPTR3 (At5g46050) of Arabidopsis thaliana has been shown to affect germination on media containing a high salt concentration. The heterologous expression in yeast was utilized to verify that the AtPTR3 protein transports di-and tripeptides. The T-DNA insert in the Atptr3-1 mutant in the Arabidopsis ecotype C24 revealed two T-DNA copies, the whole vector sequence, and the gus marker gene inserted in the second intron of the AtPTR3 gene. An almost identical insertion site was found in the Atptr3-2 mutant of the Col-0 ecotype. The AtPTR3 expression was shown to be regulated by several signalling compounds, most clearly by salicylic acid (SA), but also methyl jasmonate (MeJA) and abscisic acid. Real-time PCR experiments suggested that the wound-induction of the AtPTR3 gene was abolished in the SA and JA signalling mutants. The Atptr3 mutant plants had increased susceptibility to virulent pathogenic bacteria Erwinia carotovora subsp. carotovora and Pseudomonas syringae pv. tomato, and produced more reactive oxygen species when grown on media containing paraquat or rose bengal. Public microarray data suggest that the AtPTR3 expression was induced by Pseudomonas elicitors and by avirulent P. syringae pathovars and type III secretion mutants. This was verified experimentally for the hrpA mutant with real-time PCR. These results suggest that AtPTR3 is one of the defence-related genes whose expression is reduced by virulent bacterium by type III dependent fashion. Our results suggest that AtPTR3 protects the plant against biotic and abiotic stresses.