Human esophageal microvascular endothelial cells respond to acidic pH stress by PI3K/AKT and p38 MAPK-regulated induction of Hsp70 and Hsp27

The heat shock response maintains cellular homeostasis following sublethal injury. Heat shock proteins (Hsps) are induced by thermal, oxyradical, and inflammatory stress, and they chaperone denatured intracellular proteins. Hsps also chaperone signal transduction proteins, modulating signaling cascades during repeated stress. Gastroesophageal reflux disease (GERD) affects 7% of the US population, and it is linked to prolonged esophageal acid exposure. GERD is characterized by enhanced and selective leukocyte recruitment from esophageal microvasculature, implying activation of microvascular endothelium. We investigated whether phosphatidylinositol 3-kinase (PI3K)/Akt and MAPK regulate Hsp induction in primary cultures of human esophageal microvascular endothelial cells (HEMEC) in response to acid exposure (pH 4.5). Inhibitors of signaling pathways were used to define the contribution of PI3K/Akt and MAPKs in the heat shock response and following acid exposure. Acid significantly enhanced phosphorylation of Akt and MAPKs in HEMEC as well as inducing Hsp27 and Hsp70. The PI3K inhibitor LY-294002, and Akt small interfering RNA inhibited Akt activation and Hsp70 expression in HEMEC. The p38 MAPK inhibitor (SB-203580) and p38 MAPK siRNA blocked Hsp27 and Hsp70 mRNA induction, suggesting a role for MAPKs in the HEMEC heat shock response. Thus acidic pH exposure protects HEMEC through induction of Hsps and activation of MAPK and PI3 kinase pathway. Acidic exposure increased HEMEC expression of VCAM-1 protein, but not ICAM-1, which may contribute to selective leukocyte (i.e., eosinophil) recruitment in esophagitis. Activation of esophageal endothelial cells exposed to acidic refluxate may contribute to GERD in the setting of a disturbed mucosal squamous epithelial barrier (i.e., erosive esophagitis, peptic ulceration). esophagus; esophagitis; gastroesophageal reflux disease; microvasculature; phosphatidylinositol 3-kinase/Akt; VCAM-1     

Potential of xylanase from thermophilic Bacillus sp. XTR-10 in biobleaching of wood kraft pulp

An extracellular xylanase produced under optimal conditions by a thermophilic strain of Bacillus sp. XTR-10 was evaluated for its potential application in biobleaching of wood kraft pulp. Spectrophotometric analysis showed considerable release of lignin derived compounds and chromophoric material by the xylanase treated pulp samples. Xylanase was found to be effective in the liberation of reducing sugars in the pulp filtrates with increment in enzyme dose and reaction time. Eight hours pretreatment with 40 IU of xylanase/g of dry pulp resulted in 16.2% reduction of kappa number with 25.94% ISO increase in brightness as compared to the control. The same treatment slightly lowered the tensile strength and burst index, however. Enzyme pretreatment of the pulp saved 15% active chlorine charges in single step and 18.7% in multiple steps chemical bleaching with attainment of brightness at the level of the control. These results indicate the potential of enzymatic pretreatment of pulp for reduction in environmental discharge of hazardous waste from the pulp and paper industry.     

Structure of human endothelin-converting enzyme I complexed with phosphoramidon

Endothelin-converting enzyme I (ECE-1) is a mammalian type II integral membrane zinc-containing endopeptidase. ECE-1 catalyzes the final step in the biosynthesis of endothelins in a rate-limiting fashion, through post-translational conversion of the biologically inactive big endothelins. Endothelin-1 overproduction has been implicated in a heterogeneous list of diseases including systemic and pulmonary hypertension, stroke and asthma, cardiac and renal failure. Therefore, ECE-1 is a prime therapeutic target for the regulation of endothelin-1 production in vivo and there is considerable interest in selective inhibitors of this enzyme. Here, we present the crystal structure of the extracellular domain (residues 90-770) of human ECE-1 (C428S) with the generic metalloprotease inhibitor phosphoramidon determined at 2.38 Å resolution. The structure is closely related to that of human NEP, providing essential information for a detailed understanding of ligand-binding, specificity determinants as well as selectivity criteria. Selective inhibitors of ECE-1s should have beneficial effects for the treatment of diseases in which an overproduction of ETs plays a pathogenic role.     

Bioinformatics in molecular immunology laboratories demonstrated: Modeling an anti-CMV scFv antibody

A scFv (single chain variable fragment) antibody clone from anti-CMV (anti-cucumber mosaic virus) was successfully constructed from immunized mouse and the DNA sequence was submitted to GenBank (AY337618 and AY337619). The expression of a 32 kDa recombinant antibody in bacteria was verified using ELISA (enzyme-linked immunoassay) and western blot. However, elucidation of specific anti-CMV scFv function requires detailed and time consuming immuno-assays. Alternatively, useful functional information on anti-CMV scFV antibody can be obtained using available Bioinformatics tools and techniques without performing tedious assays. Here, we use the commonly used Bioinformatics tools and databases such as BLAST (basic local alignment search tool), GenBank, PDB (protein databank), KABAT numbering, SWISS-MODEL and Insight II to gain specific functional insights into anti-CMV scFv.     

A strategy for mitigating avian colibacillosis disease using plant growth promoting rhizobacteria and green synthesized zinc oxide nanoparticles

Avian colibacillosis caused by the zoonotic pathogen Escherichia coli is a common bacterial infection that causes major losses in the poultry sector. Extracts of different medicinal plants and antibiotics have been used against poultry bacterial pathogens. However, overuse of antibiotics and extracts against pathogenic strains leads to the proliferation of multi-drug resistant bacteria. Due to their environmentally friendly nature, nanotechnology and beneficial bacterial strains can be used as effective strategies against poultry infections. Green synthesis of zinc oxide nanoparticles (ZnO-NPs) from Eucalyptus globulus leaves was carried out in this study. Their characterization was done by UV–vis spectroscopy, X-ray diffraction (XRD), and Fourier transmission infrared spectroscopy (FT-IR) which confirmed their synthesis, structure, and size. In vitro, antimicrobial activities of plant leaf extract, ZnO-NPs, and plant growth-promoting rhizobacteria (PGPR) were checked against E. coli using well diffusion as well as disc diffusion method. Results proved that the antimicrobial activity of ZnO-NPs and PGPR strains was more enhanced when compared to eucalyptus leaf extract at 36 h. The maximum relative inhibition shown by ZnO-NPs, PGPR strains and eucalyptus leaf extracts was 88%, 67% and 58%, respectively. The effectiveness of ZnO-NPs was also increased with an increase in particle dose and treatment time. The 90 mg/ml of ZnO-NPs was more effective. PGPR strains from all over the tested strains, Pseudomonas sp. (HY8N) exhibited a strong antagonism against the E. coli strain as compared to other PGPR strains used in this study. However, combined application of PGPR (Pseudomonas sp. (HY8N)) and ZnO-NPs augment antagonistic effects and showed maximum 69% antagonism. The study intends to investigate the binding affinity of ZnO-NPs with the suitable receptor of the bacterial pathogen by in silico methods. The binding site conformations showed that the ligand ZnO binds with conserved binding site of penicillin-binding protein 6 (PBP 6) receptor. According to the interactions, ZnO-NPs form the same interaction pattern with respect to other reported ligands, hence it can play a significant role in the inhibition of PBP 6. This research also found that combining ZnO-NPs with Pseudomonas sp. (HY8N) was a novel and effective technique for treating pathogenic bacteria, including multidrug-resistant bacteria.     

Pregnant Women in and Around Dhaka City: Are Their Children at Risk of Developing Congenital Rubella Syndrome?

Rubella Virus (RUBV) is a common cause of childhood rash and fever in non-immunized populations, and its public health importance relates to teratogenic effects of primary rubella infection in women with early pregnancy. Infection of the fetus may lead to congenital rubella syndrome (CRS). This work aimed to assess the degree of risk associated in acquiring rubella virus infection by the women during pregnancy and developing CRS among their children in Bangladesh. The study population (n = 275) included pregnant mothers (15–38 years) from various socioeconomic backgrounds attending a women health care based hospital. All subjects were personally interviewed, clinically examined and a standardized questionnaire was filled up for each of them. From each participant 3 ml blood was taken and serum was separated. Commercially available ELISA kit was used for the qualitative and quantitative determination of IgM and IgG class antibodies against RUBV in collected serum samples. 209 women were found to contain detectable level of antiRUBV IgG antibodies, but did not possess IgM antibodies against rubella. Only 9% participants were vaccinated previously against rubella virus among the whole antenatal population studied. Ninety-two percent of these vaccinated pregnant women contained serum anti-rubella IgG antibody which was significantly (P = 0.05) higher than that of the nonvaccinated study population (75%). Pregnant women from lower middle and poor socioeconomic class had significantly (P = 0.05) more intra uterine growth retardation (IUGR) of fetus than the upper middle class. 20% of the women of child bearing age examined in this work were not yet exposed to RUBV and at risk of acquiring this virus during pregnancy and subsequently transmitting the virus to the fetus. Our work demonstrates rubella attack rate among antenatal population in Bangladesh as 14.5 in 1000 during pregnancy. A proper and reliable vaccination policy against rubella virus is not yet adopted at the national level in many developing countries including Bangladesh. This work identifies the requirement of detailed study for the identification of intrauterine rubella infection and its related influence on perinatal morbidity and mortality. Thorough epidemiological studies are also considered necessary prior to the development and acceptance of national immunization program against rubella virus in Bangladesh.     

Pre and post cloning characterization of a β-1,4-endoglucanase from <Emphasis Type="Italic">Bacillus</Emphasis> sp.

Consistent with its precloning characterization from the cellulolytic Bacillus sp., β-1,4-endoglucanase purified from the recombinant E. coli exhibited maximum activity at 60°C and pH 7.0. It was highly specific for CMC hydrolysis, with stability up to 70°C and over a pH range of 6.0–8.0. The K _m and V _max values for CMCase activity of the enzyme were 4.1 mg/ml and 25 μmole/ml min⁻¹, respectively. The purified enzyme was a monomer of 65 kDa, as determined by SDS-PAGE. The presence of sucrose and IPTG in fermentation media increased the endoglucanase activity of the recombinant enzyme to 5.2-folds as compared with that of the actual one.     

Relatedness of Vibrio cholerae O1/O139 Isolates from Patients and Their Household Contacts,Determined by Multilocus Variable-Number Tandem-Repeat Analysis

The genetic relatedness of Vibrio cholerae O1/O139 isolates obtained from 100 patients and 146 of their household contacts in Dhaka, Bangladesh, between 2002 and 2005 was assessed by multilocus variable-number tandem-repeat analysis. Isolate genotypes were analyzed at five loci containing tandem repeats. Across the population, as well as within households, isolates with identical genotypes were clustered in time. Isolates from individuals within the same household were more likely to have similar or identical genotypes than were isolates from different households, but even within a household, isolates from different individuals often had different genotypes. When household contacts were sampled regularly for 3 weeks after the illness of the household index patient, isolates with genotypes related to the index patient appeared in contacts, on average, ∼3 days after the index patient, while isolates with unrelated genotypes appeared in contacts ∼6 days after. Limited data revealed that multiple isolates from the same individual collected within days of each other or even from a single stool sample may have identical, similar, or unrelated genotypes as well. Our results demonstrate that genetically related V. cholerae strains cluster in local outbreaks but also suggest that multiple distinct strains of V. cholerae O1 may circulate simultaneously within a household.Vibrio cholerae is the etiologic agent of cholera, a secretory diarrheal disease with a high mortality rate in humans if untreated (25). Serogroups of V. cholerae, a motile, Gram-negative, curved rod, can be defined serologically by the O side chain of the lipopolysaccharide (LPS) component of the outer membrane (9). V. cholerae is found in a variety of forms in aquatic ecosystems (41, 42), and more than 200 different serogroups have been isolated, mostly from environmental sources (45). However, the vast majority of V. cholerae strains that cause the clinical disease cholera belong to serogroup O1 or O139 (37, 42). V. cholerae O1, the historical agent of epidemic and pandemic cholera and the current leading cause of cholera both globally and in Bangladesh (42), is classified into two major biotypes, classical and El Tor (44), and two major serotypes, Ogawa and Inaba (48). The current global pandemic is caused by V. cholerae O1 El Tor. A second pathogenic serogroup, O139, emerged in the Bengal region in 1992 by horizontal transfer of new LPS biosynthesis-encoding genes into the El Tor biotype (1, 4). This new serogroup continues to cocirculate with El Tor V. cholerae O1 serotypes Ogawa and Inaba as a cause of disease in humans, although it accounts for a smaller proportion of all cholera now than in its first years of circulation (16, 20). Recently, comparative genomics has revealed an extensive amount of lateral gene transfer between strains, suggesting that genomic classification may be an alternative to serogrouping for classifying pathogenic V. cholerae strains (11).Toxigenic V. cholerae may be present in environmental sources in regions of endemicity and emerge, often seasonally, to cause cholera in humans (12, 18). Once an outbreak has begun, organisms from one infected individual are more infectious for the next individual, a property termed hyperinfectivity, and these forms may be able to pass directly from human to human through fecal-oral contamination (35). However, because vibrio organisms are difficult to isolate from implicated environmental or domestic water sources (28, 29), little is known about the diversity of V. cholerae in inocula that cause human infection.Established laboratory methods for differentiating V. cholerae strains, apart from serogrouping and serotyping, include rRNA restriction fragment length polymorphism (ribotyping), pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). These methods, however, have a limited capacity to differentiate between pathogenic V. cholerae strains, as clinical isolates are relatively genetically monomorphic. For instance, V. cholerae O1 comprises approximately 30 ribotypes (39); however, only a few ribotypes are common in clinical isolates, ribotypes evolve slowly, and all isolates of a given pathogenic V. cholerae serotype in a local area over a period of multiple years often belong to a single ribotype (8, 14, 17). In a broad sampling of 154 V. cholerae isolates from Bangladesh and worldwide over several decades, only 15 ribotypes were identified, and of these, many were found in nonpathogenic environmental isolates only; only five ribotypes were associated with the V. cholerae O1 El Tor biotype that currently predominates as the cause of clinical disease, while pathogenic isolates of serogroup O139 were indistinguishable from each other by ribotype (19).PFGE, in which restriction endonuclease digestion of genomic DNA generates mutation-sensitive banding patterns, is often more sensitive than ribotyping in detecting strain variation (7, 34, 51) and detects extensive genetic variation within nonpathogenic V. cholerae serogroups (3, 46). However, PFGE types change slowly and are useful primarily for distinguishing between strains in different pandemics or between different continental branches of those pandemics. In an analysis of 180 mostly western-hemisphere isolates (7), PFGE differences had developed from a prior pandemic strain over the 30 years since its arrival in Latin America, but a new strain that had been causing disease for 2 years still had only a single PFGE type across the 64 isolates analyzed. Similarly, in a Japanese study (2), although 19 PFGE types were identified among O1 isolates, the majority of the domestic isolates, along with several imported isolates, belonged to a single PFGE type.Further differentiation between V. cholerae isolates is achievable by MLST, which characterizes isolates by internal DNA sequences in selected housekeeping genes (32). Nevertheless, epidemic strains also cluster tightly in this typing scheme (5, 32) and the method has been useful primarily for determining relationships between nontoxigenic strains (36) or for linking regional outbreaks (which typically appear monoclonal by these methods) with the pandemic strain responsible (5, 33).Although these methods have distinguished major pandemic clones from other nonpathogenic human and environmental isolates of V. cholerae, the near clonality of pathogenic O1 and O139 strains means that established methods may not provide sufficiently robust differentiation of these genetically similar pathogenic strains to answer important epidemiological questions. Therefore, there is a need for other methods that can distinguish among clinical O1 and O139 isolates and track the epidemiology of outbreaks in a restricted geographic area on a shorter time scale.Multilocus variable-number tandem-repeat (VNTR) analysis (MLVA) is one method that may be useful for differentiating between pathogenic V. cholerae O1 and O139 strains that would be indistinguishable by other techniques (15). This method examines short repeating DNA segments at various locations in the genome that can vary in number at each location and uses the number of repeats at each varying locus as a fingerprint to distinguish between isolates.Escherichia coli is the paradigm organism for demonstrating the value of the MLVA method. Noller et al. (38) showed that E. coli O157 isolates that were indistinguishable by MLST could be distinguished to some extent by PFGE but that MLVA distinguished between isolates that had the same PFGE type and did so in a manner consistent with the known epidemiology of the isolates (38a). In addition, machine-scored VNTR assays have been demonstrated to be robust and portable and to discriminate clearly between isolates by using relatively few loci, therefore limiting the effect of compounding genotyping errors (6).For V. cholerae, five VNTR loci have been identified (15), and the initial application of MLVA at those loci has demonstrated distinct populations of clinical isolates of V. cholerae in different geographic regions within Bangladesh and India (23, 47). Predominant isolates in each of two rural Bangladeshi regions varied gradually over a time scale of months to years (47), and isolates collected from India over a 15-year period varied widely, with individual MLVA types clustering in time and place—some with widespread dissemination and others with limited local occurrence only (23). MLVA has also been used to classify hybrid and altered V. cholerae variants and to demonstrate their genetic distance from the pandemic El Tor strain (10). Use of the MLVA method for epidemiologic study of cholera requires that V. cholerae VNTR alleles remain reasonably stable during bacterial replication in patients or in laboratory culture after isolation. Some degree of stability of two of the five loci used in V. cholerae MLVA has been demonstrated previously by serial passage in vitro through four overnight cultures (15). In this study, we used MLVA to examine V. cholerae O1 and O139 isolates obtained from infected patients and their household contacts—including multiple isolates from the same individual and isolates from multiple individuals within the same household—in a large city where cholera is endemic.     

Delineation of exoenzyme S residues that mediate the interaction with 14-3-3 and its biological activity

14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells, which play an important role in a multitude of signaling pathways. 14-3-3 proteins bind to phosphoserine/phosphothreonine motifs in a sequence-specific manner. More than 200 14-3-3 binding partners have been found that are involved in cell cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation. A phosphorylation-independent interaction has been reported to occur between 14-3-3 and a C-terminal domain within exoenzyme S (ExoS), a bacterial ADP-ribosyltransferase toxin from Pseudomonas aeruginosa. In this study, we have investigated the effect of amino acid mutations in this C-terminal domain of ExoS on ADP-ribosyltransferase activity and the 14-3-3 interaction. Our results suggest that leucine-428 of ExoS is the most critical residue for ExoS enzymatic activity, as cytotoxicity analysis reveals that substitution of this leucine significantly weakens the ability of ExoS to mediate cell death. Leucine-428 is also required for the ability of ExoS to modify the eukaryotic endogenous target Ras. Finally, single amino acid substitutions of positions 426-428 reduce the interaction potential of 14-3-3 with ExoS in vitro.