Deltamethrin-induced oxidative stress and mitochondrial caspase-dependent signaling pathways in murine splenocytes.

Deltamethrin (DLM) is a well-known pyrethroid insecticide used extensively in pest control. Exposure to DLM has been demonstrated to cause apoptosis in various cells. However, the immunotoxic effects of DLM on mammalian system and its mechanism is still an open question to be explored. To explore these effects, this study has been designed to first observe the interactions of DLM to immune cell receptors and its effects on the immune system. The docking score revealed that DLM has strong binding affinity toward the CD45 and CD28 receptors. In vitro study revealed that DLM induces apoptosis in murine splenocytes in a concentration-dependent manner. The earliest markers of apoptosis such as enhanced reactive oxygen species and caspase 3 activation are evident as early as 1 h by 25 and 50 µM DLM. Western blot analysis demonstrated that p38 MAP kinase and Bax expression is increased in a concentration-dependent manner, whereas Bcl 2 expression is significantly reduced after 3 h of DLM treatment. Glutathione depletion has been also observed at 3 and 6 h by 25 and 50 µM concentration of DLM. Flow cytometry results imply that the fraction of hypodiploid cells has gradually increased with all the concentrations of DLM at 18 h. N-acetyl cysteine effectively reduces the percentage of apoptotic cells, which is increased by DLM. In contrast, buthionine sulfoxamine causes an elevation in the percentage of apoptotic cells. Phenotyping data imply the effect of DLM toxicity in murine splenocytes. In brief, the study demonstrates that DLM causes apoptosis through its interaction with CD45 and CD28 receptors, leading to oxidative stress and activation of the mitochondrial caspase-dependent pathways which ultimately affects the immune functions. This study provides mechanistic information by which DLM causes toxicity in murine splenocytes. © 2014 Wiley Periodicals, Inc. Environ Toxicol 31: 808-819, 2016.

Deltamethrin induced an apoptogenic signalling pathway in murine thymocytes : exploring the molecular mechanism.

Deltamethrin (DLM) is a well-known pyrethroid insecticide; however, the immunotoxic effects of DLM on the mammalian system and its mechanism is still unclear. This study has been designed to first observe the binding affinity of DLM to immune cell receptors and its effects on the immune system. The docking score revealed that DLM has a strong binding affinity towards the CD4 and CD8 receptors. DLM induces apoptosis in murine thymocytes in a concentration-dependent manner. The ear\ly markers of apoptosis such as enhanced reactive oxygen species (ROS) and caspase-3 activation are evident as early as 1 h by 25 and 50 µM DLM. Glutathione (GSH) depletion has also been observed at 1 h by 50 µM DLM concentration. In cell-cycle studies using flow cytometry, the fraction of hypodiploid cells has gradually increased with all the concentrations of DLM at 18 h. The Annexin V binding assay measures the effect of DLM on apoptotic and necrotic cells. The apoptotic cells raised from 18.6% to 35.21% (10-50 µM DLM) at 18 h. N-acetyl cysteine (NAC) effectively reduced the percentage of apoptotic cells which is increased by DLM. In contrast, buthionine sulfoximine (BSO) caused an elevation in the percentage of apoptotic cells. These results demonstrate that caspase activation, ROS activation and GSH act as critical mediators in a DLM-induced apoptogenic signalling pathway in murine thymocytes. In the presence of caspase inhibitor, the percentage of apoptotic cells is partially decreased. Thus, there may be the possibility of some other caspase-independent pathways in DLM-induced apoptosis.

Development of selective and reversible pyrazoline based MAO-B inhibitors: virtual screening, synthesis and biological evaluation.

In an effort to develop selective MAO (monoamine oxidase) B inhibitors, structure based virtual screening was initiated on an in-house library. Top 10 HITS were synthesized and evaluated for MAO (A and B) inhibitory activity, both against human and rat enzymes. All the compounds were found selective, reversible and active in nM range (100 times more potent than selegeline) towards MAO-B. Outstanding co-relation between predicted and experimental K(i) values were observed.

3D-QSAR study of 8-azabicyclo[3.2.1] octane analogs antagonists of cholinergic receptor.

3D-QSAR models of Comparative of Molecular Field Analysis (CoMFA) and Comparative of Molecular Similarities Indices Analysis (CoMSIA) of 20 8-azabicyclo[3.2.1] octane (potent muscarinic receptor blocker) was performed. These benztropine analogs were optimized using ligand based alignment method. The conventional ligand-based 3D-QSAR studies were performed based on the low energy conformations employing database alignment rule. The ligand-based model gave q(2) value 0.819 and 0.810 and r(2) value 0.991 and 0.988 for CoMFA and CoMSIA, respectively, and the predictive ability of the model was validated. Results indicate that the CoMFA and CoMSIA models could be reliable model which may be used in the design of novel muscarinic antagonists as leads.

Serological evidence of antibodies against Chlamydophila abortus in free-ranging yak (Poephagus grunniens) in Arunachal Pradesh, India.

Serum samples were randomly collected from 172 free-ranging yak (Poephagus grunniens, presently Bos grunniens) from six different yak tracts of Arunachal Pradesh, India, and subjected to enzyme-linked immunosorbent assay to detect the presence of specific antibodies against Chlamydophila abortus. The overall prevalence of this disease in yak was 35%. The prevalence of Cp. abortus-specific antibodies was significantly higher in yak cows (41%) than among bulls (25%). The highest prevalence (39%: 95% confidence interval [Cl] = 27, 55) was found in yak between one and three years of age, while the lowest prevalence (20%: 95% CI = 10, 41) was reported in yak below one year of age.

A serological survey of antibodies against bovine herpesvirus-1 in yak (Poephagus grunniens) in Arunachal Pradesh in India.

Serum samples were collected from 254 yak (Poephagus grunniens, presently Bos grunniens) in different yak tracts of India. These samples were then screened by virus neutralisation test (VNT) and avidin-biotin enzyme-linked immunosorbent assay (AB-ELISA) to study the seroprevalence of antibodies against bovine herpesvirus type 1 (BHV-1). The overall seroprevalence in yak was found to be 41% (105) by VNT and AB-ELISA. The sex of the animal, whether it was on a farm or free-ranging and the location of the different yak tracts did not seem to have any effect on seroprevalence. However, seroprevalence was found to increase with the age of the animals, being highest in yak older than three years of age (49%). Yak generally share feeding, watering and grazing areas with other domestic and wild animals and this common ecological niche is thought to be a possible avenue of infection. This is the first time that the seroprevalence of antibodies against BHV-1 has been studied in yak in India.

Anti-diarrheal activity of methanol extract of Litsea polyantha bark in mice.

The anti-diarrheal activity of methanol extract of dried bark and aerial parts of Litsea polyantha (MELP) has been evaluated in mice using different models (castor oil-induced diarrhea and propulsive gut motility in mice). MELP (50, 75, 100 mg/kg, p.o.) significantly (P<0.01) reduced the onset of diarrhea, fecal excretion and also showed a significant (P<0.001) reduction in gastrointestinal motility on charcoal meal test in mice. The results of the study support the folklore use of the plant for diarrheal remedies.

Immunomodulatory role of piperine in deltamethrin induced thymic apoptosis and altered immune functions.

Deltamethrin (DLM), a well-known pyrethroid insecticide, is a potent immunotoxicant. In rodents, it is primarily characterized by marked thymic apoptosis. Mechanism of DLM induced thymic apoptosis in primary murine thymocytes has been recently explored. Oxidative stress and activation of caspase dependent pathways appear to be involved in the DLM induced thymic injury. Thus, for the amelioration of its effect, this study has been designed to first observe the binding affinity of piperine to immune cell receptors and its protective effects on the DLM induced immunotoxicity under in vitro condition. The docking results demonstrated that piperine has good binding affinity towards CD4 and CD8 receptors. In vitro study results have shown that piperine (1, 10 and 50 µg/ml) increased cell viability in a concentration dependent manner. The early activated markers of apoptosis such as enhanced reactive oxygen species (ROS) and caspase-3 activation by DLM was significantly reduced by piperine treatment. GSH depletion induced by DLM has been also restored by piperine treatment. At 18 h, all concentration of piperine (1, 10 and 50 µg/ml) significantly ameliorated the DLM induced apoptosis. Further, DLM induced phenotypic changes were mitigated by the piperine. In addition, piperine also restored the cytokine levels, which were suppressed by DLM treatment. These findings strongly indicate the anti-oxidative, anti-apoptotic and chemo-protective ability of piperine in the DLM induced thymic apoptosis.

Purification and characterisation of a hemolysin with phospholipase C activity from Vibrio cholerae O139.

A hemolysin was purified from a Vibrio cholerae O139 strain which moved as a single protein band of 67 kDa in SDS-PAGE. The hemolysin showed high level of phospholipase C activity. The purified phospholipase C-hemolysin demonstrated enterotoxic activity in rabbit ileal loop, suckling mice and enhanced permeability of rabbit skin. The pI of the purified hemolysin was 6.4. Erythrocytes from rabbit, chicken, guinea pig, sheep and horse were sensitive to the purified hemolysin in decreasing order of intensity. Erythrocytes from human and cow were unaffected by purified hemolysin.

Comparative analysis of cytotoxin, hemolysin, hemagglutinin and exocellular enzymes among clinical and environmental isolates of vibrio cholerae O139 and non-O1, non-O139.

The presence of three major virulence genes toxR, tcpA and ctxA as well as expression of several putative virulence factors were compared in 12 Vibrio cholerae O139 and non-O1,non-O139 strains of clinical and environmental origin. All the strains possessed the gene encoding the regulatory protein TOXR. None of the non-O1, non-O139 strains as well as one of the O139 environmental strains carried the genes for ctxA and tcpA. Statistically significant differences in hemagglutinin and hemolysin production were observed amongst the strains depending on the source of their isolation. Expression of extracellular enzymes such as protease, elastase, neuraminidase, phospholipase A and phospholipase C, however, did not vary significantly from the groups of strains isolated from different sources.

N-acetyl-D-glucosamine-specific lectin purified from Vibrio cholerae 01.

An N-acetyl-D-glucosamine-specific cell associated hemagglutinin (HA) was isolated and purified from a strain of Vibrio cholerae 01 by chitin affinity chromatography followed by separation on Bio Gel P-150. A single stained protein band of 47 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was observed with the purified HA. HA-antisera produced a single precipitin band against the purified HA in an immunodiffusion test without exhibiting any reactivity towards purified lipopolysaccharide (LPS). Purified HA, used as solid-phase antigen in an enzyme-linked immunosorbent assay (ELISA), reacted strongly with HA-antisera but cross-reacted negligibly with antisera raised against purified LPS. Hemagglutinating activity of the purified HA was highly sensitive to N-acetyl-D-glucosamine. The immunogold-labelling method using HA-antisera confirmed the location of the HA on the surface of the bacterial cells. The HA-antisera reacted with a protein component of the homologous outer membrane preparation. A significant inhibition was observed in the adhesive capability of the V. cholerae 01 strain to isolated rabbit intestinal epithelial cells (RIEC) in vitro when the later were pre-treated with the purified HA.

Adhesion and invasion of a mutant Shigella flexneri to an eukaryotic cell line in absence of the 220-kb virulence plasmid.

A Shigella flexneri strain, cured of the large 220-kb virulence plasmid, expresses adhering and invading ability in confluent monolayers of HeLa cells similar to its parent strain. Invasion by both the parent and the cured strains resulted in alteration of the monomeric actin (G) in the total actin pool of HeLa cells. Other indicators of invasive characteristics of virulent Shigella strains such as production of keratoconjunctivitis in guinea pig eye in vivo, Congo red binding and expression of contact hemolysin however, indicated loss of invasive properties in the plasmid cured strain. Further, pretreatment of bacterial cells with para-bromophenacyl bromide (p-BPB), a specific chemical inhibitor of phospholipase A, adversely affected adhesion to and invasion of HeLa cells in vitro, irrespective of the presence of the 220-kb plasmid indicating the possible involvement of the enzyme phospholipase A in the invasion process. Adherence of both the strains to guinea pig colonic epithelial cells (CECs) in vitro was reduced significantly on pretreatment of bacteria or CECs with p-BPB. Expression of exocellular enzymes viz. protease, elastase, phospholipase A and phospholipase C were not related to the large plasmid.

Adhesion of Shigella dysenteriae type 1 and Shigella flexneri to guinea-pig colonic epithelial cells in vitro.

Adhesion of bacteria to guinea-pig colonic epithelial cells in vitro was inhibited by fucose with all the four strains tested (two of Shigella dysenteriae type 1 and two of S. flexneri). N-acetyl neuraminic acid and N-acetyl mannosamine also caused inhibition, suggesting a multiplicity of receptors on the epithelial cell. Congo-red binding of the strains correlated with their adhesive ability, whereas haemagglutination of rabbit erythrocytes by the bacteria did not.

N-acetyl-D-glucosamine specific hemagglutinin receptor of Vibrio cholerae O1 in chicken erythrocyte membranes.

N-Acetyl-D-glucosamine specific cell-associated hemagglutinin (HA)/lectin, previously purified from a strain of Vibrio cholerae O1, had been established as an adhesin molecule of V. cholerae O1 cells. This communication records the isolation and purification of the glycoprotein receptor of the N-acetyl-D-glucosamine specific HA of the V. cholerae O1 strain from chicken erythrocyte membranes. The most salient feature of this study is that the pretreatment of partially purified glycoprotein with purified HA could completely inhibit the hemagglutinating activity of the V. cholerae O1 strain with chicken erythrocytes.

Purification of a mannose/glucose-specific hemagglutinin/lectin from a Vibrio cholerae O1 strain.

A cell-associated mannose/glucose-specific hemagglutinin (MSHA) has been purified from a strain of Vibrio cholerae O1 by chromatography on a chitin column followed by affinity purification on Sephadex G100. The purified protein gave a single stained band of 40 kDa by SDS-PAGE, exhibited high affinity towards D-mannose and D-glucose but was resistant to L-fucose and N-acetyl-D-glucosamine. The purified MSHA was revealed as a globular form of protein under electron microscope. In immunodiffusion tests the purified MSHA produced a single precipitin band against homologous antisera and antisera raised against the whole cell bacteria without any reactivity towards antisera raised against the purified N-acetyl-D-glucosamine-specific lectin of the same bacterial strain. Immunogold labelling confirmed the location of hemagglutinin on the surface of the bacteria. Purified MSHA reacted strongly with sera from convalescent cholera patients in immunodiffusion tests.

Studies on adhesion, haemagglutination and other biological properties of Vibrio cholerae O139.

The adhesive capabilities of eight Vibrio cholerae O139 epidemic strains to isolated rabbit intestinal epithelial cells (RIEC) were observed to be high similar to those observed with a Vibrio cholerae O1 strain isolated from patients. Toxin production by the strains, measured by accumulation of fluid in rabbit ileal loop model, was high and the toxin was lethal as the animal expired within 6 h. Culture filtrates of the strains exhibited the presence of vascular permeability factor which produce induration and necrosis in the adult rabbit and guinea pig skin. All the strains showed high to moderate haemagglutinin titres against chicken erythrocytes and produced El Tor-like haemolysin. SDS-PAGE of the outer membrane preparation of the strains showed the presence of major protein component at 38 kDa region. The lethality of the toxin, high adhesive activity, shifting of the major outer membrane protein band and production of thermolabile haemolysin on Wagatsuma agar were the major variations of these epidemic strains from V. cholerae O1 and V. cholerae non-O1 strains isolated previously.

Purification of a cell-associated hemagglutinin from Shigella dysenteriae type 1.

A cell-associated hemagglutinin (HA) was isolated and purified from a clinical isolate of Shigella dysenteriae type 1 by affinity chromatography on a fetuin-agarose column. The purified hemagglutinin produced a single-stained protein band of around 66 kDa in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). In an immunodiffusion test, HA-antisera produced a single precipitin band against the purified HA without exhibiting any reactivity towards lipopolysaccharide (LPS) of S. dysenteriae type 1 strain. Inhibition of the hemagglutination by the glycoproteins fetuin, asialofetuin and a sugar derivative N-acetyl-neuraminic acid but not by simple sugars, suggested the specific requirement of complex carbohydrate for binding. Electron micrographs of the purified HA revealed a morphology typical of globular protein.

Electrophoretic mobility and immune response of outer membrane proteins of Vibrio cholerae O139.

The outer membrane (OM) protein components of a Vibrio cholerae O1 and four V. cholerae O139 strains, collected from cholera patients, were analysed by SDS-PAGE. A protein of 69 kDa molecular mass was observed only when the OMPs were prepared from strains grown in synthetic broth. As a result of passage in the rabbit ileal loop (RIL), virulence was enhanced, and a protein component around 18 kDa of the V. cholerae O139 OM became the major protein component. On immunoblot analysis with rabbit antiserum against V. cholerae O139 OM, it was shown that, apart from the major protein component of V. cholerae O1 OM of around 45 kDa and that of V. cholerae O139 OM of around 38 kDa, all other minor protein components were cross-reactive between the two serogroups. In immunoblot assays with convalescent sera obtained from V. cholerae O139-infected patients, it was observed that in addition to the lipopolysaccharide (LPS)-induced antibody, only the 38 kDa major protein component elicited considerable levels of antibody in the patient. Minor OM components of 18 kDa were detected in the immunoblot analysis by LPS-directed antibody, however, as the OM proteins are known to be associated with LPS.

Role of cell-associated N-acetyl-D-glucosamine specific haemagglutinin in the adhesion of Vibrio cholerae O1 to rabbit intestinal epithelial cells in vitro.

Previously a N-acetyl-D-glucosamine specific cell-associated haemagglutinin (HA) had been purified from a Vibrio cholerae O1 strain. This study documents the role of this purified HA as an adhesin of V. cholerae O1. A significant inhibition in the adhesion of V. cholerae O1 bacterial cells to isolated rabbit intestinal brush borders (RIBB) was observed when the latter were pretreated with purified HA in ELISA. Antibody raised against purified HA and Fab (IgG) fragment of this serum inhibited adhesion of the bacteria to isolated rabbit intestinal epithelial cells (RIEC). V. cholerae O1 (both Ogawa and Inaba serovars) showed less adherence to isolated RIEC of animals immunised with the purified HA. Patients convalescing from V. cholerae O1 infection showed high ELISA titres against the purified HA indicating that it is expressed in the host during the disease process.

Hematological changes produced in mice by ochratoxin A and citrinin.

The effect of 6 weekly injections of ochratoxin A (5 mg/kg) and citrinin (20 mg/kg) on the following hematological indices of mice was studied: erythrocyte sedimentation rate, hematocrit percentage, mean corpuscular values, red cell indices, platelet count, total count of bone marrow cells, differential count of bone marrow cells, total count of splenic cells, spleen weight, and serum calcium. These mycotoxins resulted in a significant decrease in platelet count and hematocrit values. On the other hand, they increased erythrocyte sedimentation rate, mean corpuscular values and red cell indices. Weight of the spleen and the splenic cell count decreased significantly in ochratoxin A and citrinin treated mice. Total count of bone marrow cells, erythrocyte precursors, leucocyte precursors and megakaryocyte precursors of femoral bone marrow decreased significantly in toxin treated mice. Calcium concentration in the serum of toxin treated mice also was decreased significantly.