Persistent diarrhoea in a 5-month-old baby carrying Vibrio cholerae nonO1/nonO139 producing Haitian cholera toxin.

Cholera toxin (CT) is the principal virulence factor of Vibrio cholerae for fatal cholera diarrhoea. Serogroups O1 and O139 harbour CT and are known to be epidemic strains. The remaining serogroups (nonO1/nonO139) are non-toxigenic and may be associated with mild disease. O1 serogroup emerged with a variant of CT known as Haitian cholera toxin (HCT). The HCT strains are hypervirulent and have been associated with severe cholera outbreaks in India, Western Africa and Haiti. Here, we report the presence of HCT (ctxB7) in a nonO1/nonO139 isolate causing persistent diarrhoea.

Vibrio cholerae O1 Ogawa El Tor strains with the ctxB7 allele driving cholera outbreaks in south-western India in 2012.

Cholera has been a recurrent epidemic disease in human populations for the past 200years. We present herein a comparative characterization of clinical Vibrio cholerae strains isolated from two consecutive cholera outbreaks in 2012 and associated environmental strains from western India. The clinical and toxigenic environmental isolates were identified as hybrid V. cholerae O1, serotype Ogawa, biotype El Tor carrying the variant ctxB7 allele. Partial sequences of SXT integrase from the isolates revealed 100% identity to ICEVchInd5 (Sevagram, India, 1994) and VC1786ICE (Haiti, 2013). The full clonal relationship of the strains established by RAPD, Box PCR, ERIC PCR and MLST (pyrH, recA and rpoA) analyses, and the short time between the two outbreaks, strongly supported that both outbreaks were due to a single strain. The study corroborated that faecal contamination of the potable water supply was the main reason for the first outbreak, which further spread to other areas and resulted in the second outbreak. The study concluded that the circulating El Tor variant strains of epidemic potential in the region can be a serious concern in the future.

Haitian variant ctxB producing Vibrio cholerae O1 with reduced susceptibility to ciprofloxacin is persistent in Yavatmal, Maharashtra, India, after causing a cholera outbreak.

Vibrio cholerae O1 biotype El Tor producing Haitian variant Cholera Toxin (HCT) and showing reduced susceptibility to ciprofloxacin caused a cholera outbreak associated with a high case fatality rate (4.5) in India. HCT-secreting strains responsible for severe cholera epidemics in Orissa (India), Western Africa and Haiti were associated with increased mortality. There is a pressing need for an integrated multidisciplinary approach to combat further spread of newly emerging variant strains. The therapeutic effect of ciprofloxacin was diminished whereas use of doxycycline in moderate to severe cholera patients was found to be effective in outbreak management.

Cold shock domain protein from Philosamia ricini prefers single-stranded nucleic acids binding.

The cold shock proteins are evolutionarily conserved nucleic acid-binding proteins. Their eukaryotic homologs are present as cold shock domain (CSD) in Y-box proteins. CSDs too share striking similarity among different organisms and show nucleic acid binding properties. The purpose of the study was to investigate the preferential binding affinity of CSD protein for nucleic acids in Philosamia ricini. We have cloned and sequenced the first cDNA coding for Y-box protein in P. ricini; the sequence has been deposited in GenBank. Comparative genomics and phylogenetic analytics further confirmed that the deduced amino acid sequence belongs to the CSD protein family. A comparative study employing molecular docking was performed with P. ricini CSD, human CSD, and bacterial cold shock protein with a range of nucleic acid entities. The results indicate that CSD per se exhibits preferential binding affinity for single-stranded RNA and DNA. Possibly, the flanking N- and C-terminal domains are additionally involved in interactions with dsDNA or in conferring extra stability to CSD for improved binding.

Long-term effect of Trigonella foenum graecum and its combination with sodium orthovanadate in preventing histopathological and biochemical abnormalities in diabetic rat ocular tissues.

Trigonella foenum graecum seed powder (TSP) and Sodium Orthovanadate (SOV) have been shown to demonstrate antidiabetic effects by stabilizing glucose homeostasis and carbohydrate metabolism in experimental type-1 diabetes. However their efficacy in controlling histopathological and biochemical abnormalities in ocular tissues associated with diabetic retinopathy is not known. The purpose of this study was to investigate the comparative efficacy of individual as well as combination therapy of TSP and SOV in 8 weeks diabetic rat lens and retina. Retinas and lenses were taken from control, alloxan-induced diabetic rats and diabetic rats treated separately with insulin, 5%TSP, SOV (0.6 mg/ml) and a combined dose of SOV (0.2 mg/ml) and 5%TSP for 60 days. Control and each experimental group had six rats. Alterations in the activities of enzymes HK (hexokinase), AR (aldose reductase), SDH (sorbitol dehydrogenase), G-6-PD (glucose-6-phosphate dehydrogenase), GPx (glutathione peroxidase), GR (glutathione reductase) and levels of metabolites like sorbitol, fructose, glucose, MDA (malondialdehyde) and GSH (reduced glutathione) were measured in the cytosolic fraction of lenses besides measuring blood glucose levels and glycosylated haemoglobin. Histopathological abnormalities were studied in the lens using photomicrography and retina using transmission electron microscopy. Blood glucose, glycosylated haemoglobin levels and polyol pathway enzymes AR and SDH increased significantly causing accumulation of sorbitol and fructose in the diabetic lens and treatment with SOV and TSP significantly (p < 0.05) decreased these to control levels. Similarly, SOV and TSP treatments modulated the activities of HK, G-6-PD, GPx and GR in the rat lens to control values. Ultrastructure of the diabetic retina revealed disintegration of the inner nuclear layer cells with reduction in rough endoplasmic reticulum and swelling of mitochondria in the bipolar cells; and these histopathological events were effectively restored to control state by SOV and TSP treatments. In this study SOV and TSP effectively controlled ocular histopathological and biochemical abnormalities associated with experimental type-1 diabetes, and a combination regimen of low dose of SOV with TSP demonstrated the most significant effect. In conclusion, the potential of SOV and TSP alone or in low dose combination may be considered as promising approaches for the prevention of diabetic retinopathy and other ocular disorders.

Gene targeting by ribozyme against TNF-alpha mRNA inhibits autoimmune arthritis.

Ribozymes are catalytic RNA that bind and cleave specific regions of target RNA. Therefore, protein synthesis by the target RNA may be specifically inhibited by ribozymes. In this study, we have investigated if ribozymes possess therapeutic activity on inflammatory processes in vivo, as judged from effects on an arthritis model. A hammerhead ribozyme against TNF-alpha was designed and its catalytic activity in vitro was verified. The ribozyme was employed in vivo without any delivery system, as the plasmid-based ribozyme was taken up adequately by various tissues in mice by intravenous injection. The ability of the ribozyme to regulate the development of collagen-induced arthritis (CIA), a model largely dependent on TNF-alpha, was investigated. Systemic administration of the ribozyme to mice immunized with collagen type II in CFA significantly reduced the development of CIA. No effect was observed with a catalytically inactive variant of the ribozyme. Furthermore, the ribozyme efficiently blocked cartilage and bone destruction in the joints and ameliorated established CIA. These data demonstrate for the first time that gene targeting by a ribozyme to inactivate TNF-alpha in vivo is highly efficient in suppressing autoimmune arthritis, thus providing proof of concept that it may be used as therapeutic tool for TNF-alpha-dependent chronic inflammatory disorders.

Chemopreventive potential of Azadirachta indica (Neem) leaf extract in murine carcinogenesis model systems.

Numerous laboratory studies reveal that various naturally occurring dietary substances can modify the patho-physiological process of various metabolic disorders and can be an effective preventive strategy for various diseases, including cancer. Indian Neem tree, Azadirachta indica A. Juss. (family: Meliaceae), contains at least 35 biologically active principles and is widely grown all over the tropics. The effect of two different doses (250 and 500 mg per kilogram body weight) of 80% ethanolic extract of the leaves of Azadirachta indica were examined on drug metabolizing Phase-I and Phase-II enzymes, antioxidant enzymes, glutathione content, lactate dehydrogenase, and lipid peroxidation in the liver of 7-week-old Swiss albino mice. Also anticarcinogenic potential of Azadirachta indica leaf extract was studied adopting protocol of benzo(a)pyrene-induced fore-stomach and 7,12-dimethyl benz(a)anthracene (DMBA)-induced skin papillomagenesis. Our primary findings reveal its potential to induce only the Phase-II enzyme activity associated mainly with carcinogen detoxification in liver of mice. The hepatic glutathione S-transferase (P < 0.005) and DT-diaphorase specific activities (P < 0.01) were elevated above basal level. With reference to antioxidant enzymes the investigated doses were effective in increasing the hepatic glutathione reductase (GR), glutathione peroxidase (GPX), superoxide dismutase (SOD) and catalase (CAT) activities significantly (from P < 0.005 to P < 0.001). Reduced glutathione measured as non-protein sulphydryl was found to be significantly elevated in liver (P < 0.005) and in extrahepatic organs (from P < 0.005 to P < 0.001) examined in our study. Glutathione S-transferase (GST) and DT-diaphorase (DTD) showed a dose-dependent increase in extrahepatic organs. Chemopreventive response was measured by the average number of papillomas per mouse, as well as percentage of tumor-bearing animals. There was a significant inhibition of tumor burden, in both the tumor model system studied (from P < 0.005 to P < 0.001). Tumor incidence was also reduced by both the doses of Azadirachta indica extract.

Chemomodulatory efficacy of basil leaf (Ocimum basilicum) on drug metabolizing and antioxidant enzymes, and on carcinogen-induced skin and forestomach papillomagenesis.

Basil or sweet basil (Ocimum basilicum) is cultivated throughout India and is known for its medicinal value. The effects of doses of 200 and 400 mg/kg body weight of hydroalcoholic extract (80% ethanol, 20% water) of the fresh leaves of Ocimum basilicum on xenobiotic metabolizing Phase I and Phase II enzymes, antioxidant enzymes, Glutathione content, Lactate dehydrogenase and lipid peroxidation in the liver of 8-9 weeks old Swiss albino mice were examined. Furthermore, the anticarcinogenic potential of basil leaf extract was studied, using the model of Benzo(a)pyrene-induced forestomach and 7,12 dimethyl benz(a)anthracene (DMBA)-initiated skin papillomagenesis. The hepatic glutathione S-transferase and DT-diaphorase specific activities were elevated above basal level by basil leaf treatment (from p < 0.005 to p < 0.001). Basil leaf extract was very effective in elevating antioxidant enzyme response by increasing significantly the hepatic glutathione reductase (GR) (p < 0.005), superoxide dismutase (SOD) (p < 0.05), and catalase activities (p < 0.005). Reduced glutathione (GSH), the major intracellular antioxidant, showed a significant elevation in the liver (p < 0.005) and also in all the extrahepatic organs (from p < 0.05 to p < 0.005). In the forestomach, kidney and lung, glutathione S-transferase and DT-diaphorase levels were augmented significantly, varying from p < 0.01 to p < 0.001. There were significant decreases in lipid peroxidation and lactate dehydrogenase activity. Chemopreventive response was evident from the reduced tumor burden (the average number of papillomas/mouse, p < 0.005 to p < 0.001), as well as from the reduced percentage of tumor bearing-animals. Basil leaf, as deduced from the results, augmented mainly the Phase II enzyme activity that is associated with detoxification of xenobiotics, while inhibiting the Phase I enzyme activity. There was an induction in antioxidant level that correlates with the significant reduction of lipid peroxidation and lactate dehydrogenase formation. Moreover, Basil leaf extract was highly effective in inhibiting carcinogen-induced tumor incidence in both the tumor models at peri-initiational level.

Modulatory effect of henna leaf (Lawsonia inermis) on drug metabolising phase I and phase II enzymes, antioxidant enzymes, lipid peroxidation and chemically induced skin and forestomach papillomagenesis in mice.

Henna leaf (Lawsonia inermis), commonly known as Mehndi is cultivated throughout India and is a very popular natural dye to color hand and hair. It is an integral part of indigenous culture, and is also known for its medicinal value. The effect of 200 and 400 mg/kg body weight of 80% ethanolic extract of the fresh leaves of Lawsonia inermis were examined on drug metabolizing phase-I and phase-II enzymes, antioxidant enzymes, glutathione content, lactate dehydrogenase and lipid peroxidation in the liver of 7 weeks old Swiss albino mice. Also anticarcinogenic potential of Henna leaf extract was studied adopting the protocol of benzo(a)pyrene induced forestomach and 7,12 dimethylbenz(a)anthracene (DMBA)-initiated and croton oil-promoted skin papillomagenesis. Our primary findings reveal the 'duel-acting' nature of henna leaf as deduced from its potential to induce only the phase-II enzyme activity, associated mainly with carcinogen detoxification in liver of mice and inhibit the phase I enzyme activities. The hepatic glutathione S-transferase and DT-diaphorase specific activities were elevated above basal (p < 0.005) level by Lawsonia inermis extract treatment. With reference to antioxidant enzymes the investigated doses were effective in increasing the hepatic glutathione reductase (GR), superoxide dismutase (SOD) and catalase activities significantly (from p < 0.05 to p < 0.005) at both the dose levels. Reduced glutathione (GSH) measured as non-protein sulphydryl was found to be significantly elevated in liver (p < 0.005) and in all the extrahepatic organs studied (from p < 0.05 to p < 0.005). Among the extrahepatic organs examined (forestomach, kidney and lung) glutathione S-transferase and DT-diaphorase level were increased in a dose independent manner (from p < 0.05 to p < 0.005). Chemopreventive response was measured by the average number of papillomas per mouse (tumor burden) as well as percentage of tumor bearing animals and tumor multiplicity. There was a significant inhibition of tumor burden in both the tumor model systems studied (from p < 0.01 to p < 0.001). Tumor incidence was also reduced by both the doses used in our experiment in both the model systems.

Trigonellafoenum graecum (fenugreek) seed powder improves glucose homeostasis in alloxan diabetic rat tissues by reversing the altered glycolytic, gluconeogenic and lipogenic enzymes.

Trigonella foenum graecum (fenugreek) seed powder has been suggested to have potential antidiabetic effects. The effect of oral administration of Trigonella whole seed powder (5% in the diet) for 21 days on glycolytic, gluconeogenic and NADP-linked lipogenic enzymes were studied in liver and kidney tissues of alloxan-induced diabetic Wistar rats. Diabetic rats were characterised by a 4-fold higher blood glucose level and a 0.7-fold lower body weight compared to normal controls. The activities of the glycolytic enzymes were significantly lower in the diabetic liver and higher in the diabetic kidney. The activities of gluconeogenic enzymes were higher in both liver and kidney during diabetes, however the activities of the lipogenic enzymes were decreased in both tissues during diabetes. Trigonella seed powder treatment to diabetic rats for 21 days brought down the elevated fasting blood glucose levels to control levels. The altered enzyme activities were significantly restored to control values in both the liver and kidney after Trigonella seed powder treatment. The therapeutic role of Trigonella seed powder in type-1 diabetes as exemplified in this study can be attributed to the change of glucose and lipid metabolising enzyme activities to normal values, thus stabilizing glucose homeostasis in the liver and kidney. These biochemical effects exerted by Trigonella seeds make it a possible new therapeutic in type-1 diabetes.

Cell size and heterocyst differentiation in nostocacean blue-green algae.

Cell size variations in three heterocystous blue-green algae, viz. Nostoc muscorum (ISU strain), N. muscorum (UW strain) and N. calcicola (a local isolate) have been surveyed under conditions permissive and non-permissive of heterocyst differentiation. A monomodal distribution pattern was observed for the two strains of N. muscorum under conditions not requiring heterocyst differentiation, while elemental nitrogen grown cultures retained a group of relatively larger cells in addition to this mode. N. calcicola could not be similarly viewed because of the formation of hormogonia-like slender filaments causing heterogeneity. The heterocyst size showed two major peaks in each case, the larger heterocysts being almost twice as voluminous as the smaller ones. Attainment of a critical cell size, and the cell division which follows, may be crucial for making a cell differentiate into a heterocyst.

Histidine sensitive variant of the blue-green alga Nostoc muscorum: response to corepressors of histidine biosynthesis.

A model has been proposed to account for growth inhibition by L-histidine in a variant strain of Nostoc muscorum. This strain has been characterized for its response to 3-amino-1,2,4-triazole and 1,2,4-triazole-3-alanine known to act as false core-pressors of the histidine biosynthesis genes. The histidine sensitive strain retained its sensitivity to triazole alanine while the inhibitory effects of aminotriazole were much reduced indicating a change in regulation of his genes. The probable interactions between nif and his genes in cyanobacteria (blue-green algae) have been discussed.