Enhanced innate immune parameters in Labeo rohita (Ham.) following oral administration of Bacillus subtilis

The present study assessed the use of Bacillus subtilis in fish as a probiotic. The bacterium was administered orally at three different doses 0.5 x 10(7) (T(2)), 1 x 10(7) (T(3)), 1.5 x 10(7) (T(4)) cfu/g feed to Labeo rohita for two weeks. The positive control group (T(1)) and negative control group (T(5,)) were fed feed without B. subtilis for the same period. On the 15th day blood and serum were sampled to determine respiratory burst activity (NBT assay), differential leukocyte counts (DLC) and serum bactericidal activity. Fishes were challenged intraperitoneally with Aeromonas hydrophila O:18 after two weeks in the treatment groups (T(2), T(3) and T(4)) and also in the positive control group(T(1)), while the negative control group (T(5)) was challenged with phosphate buffered saline (PBS, pH 7.2) only. The respiratory burst activity and DLC were assessed on the 3rd day post-challenge. B. subtilis treated fish showed significantly higher (P<0.05) respiratory burst activity and bactericidal activity during the pre-challenge compared with the control groups. The highest respiratory burst activity (0.37+/-0.03) and serum bactericidal activity were recorded in the group (T(4)) fed feed containing B. subtilis at 1.5 x 10(7)cfu/g feed. Granulocyte numbers were significantly higher (P<0.05) in treatment groups in comparison to the control in both the pre- and post-challenge periods. The result suggests that B. subtilis can enhance certain innate immune responses in rohu.     

Molecular cloning and characterization of Brugia malayi hexokinase

5' EST from filarial gene database has been subjected to 3' rapid amplification of cDNA ends (RACE), semi-nested PCR and PCR to obtain full-length cDNA of Brugia malayi. Full-length hexokinase gene was obtained from cDNA using gene specific primers. The elicited PCR product was cloned, sequenced and expressed as an active enzyme in Escherichia coli. Sequence analysis of B. malayi hexokinase (BmHk) revealed 59% identity with nematode Caenorhabditis elegans but low similarity with all other available hexokinases including human. BmHk, an apparent tetramer with subunit molecular mass of 72 kDa, was able to phosphorylate glucose, fructose, mannose, maltose and galactose. The Km values for glucose, fructose and ATP were found to be 0.035+/-0.005, 75+/-0.3 and 1.09+/-0.5 mM respectively. BmHk was strongly inhibited by ADP, glucosamine, N-acetyl glucosamine and mannoheptulose. The recombinant enzyme was found to be activated by glucose-6-phosphate. ADP exhibited noncompetitive inhibition with the substrate glucose (Ki=0.55 mM) while, mixed type of inhibition was observed with inorganic pyrophosphate (PPi) when ATP was used as substrate (Ki=9.92 microM). The enzyme activity is highly dependent on maintenance of free sulfhydryl groups. CD analysis indicated that BmHk is composed of 37% alpha-helices and 26% beta-sheets. The observed differences in kinetic properties of BmHk as compared to host enzyme may facilitate designing of specific inhibitors against BmHk.     

Oxidative stresses induced by glycoxidized human or bovine serum albumin on human monocytes

Oxidative stress and protein modifications are frequently observed in numerous disease states. Albumin, the major circulating protein in blood, can undergo increased glycoxidation in diabetes. Protein glycoxidation can lead to the formation of advanced glycoxidation end products, which induce various deleterious effects on cells. Herein, we report the effect of glucose or methylglyoxal-induced oxidative modifications on BSA or HSA protein structures and on THP1 monocyte physiology. The occurrence of oxidative modifications was found to be enhanced in glycoxidized BSA and HSA, after determination of their free thiol group content, relative electrophoretic migration, carbonyl content, and antioxidant activities. Cells treated with glycoxidized albumin exhibited an overgeneration of intracellular reactive oxygen species, impairments in proteasomal activities, enhancements in RAGE expression, and an accumulation of carbonylated proteins. These novel observations made in the presence of a range of modified BSA and HSA facilitate the comparison of the glycoxidation extent of albumin with the oxidative stress induced in cultured monocytes. Finally, this study reconfirms the influence of experimental conditions in which AGEs are generated and the concentration levels in experiments designed to mimic pathological conditions.     

Effects of different factors on immature embryo culture, PLBs differentiation and rapid mass multiplication of Coelogyne suaveolens (Lindl.) Hook

In vitro mass production of C. suaveolens (Lindl.) Hook, an endangered orchid with its snowy white flowers having horticultural potential was accomplished through immature seed culture, and subsequent plant regeneration. The developmental stage of the immature seeds and nutrient media significantly influenced the germination frequency. Seeds at 13 months after pollination cultured on 3% sucrose containing Murashige and Skoog (MS) medium with 9 microM alpha-naphthaleneacetic acid (NAA), and 15% coconut water exhibited 93% germination after 40 days of culture. Upon subculture, the germinated shoots on MS medium with 9 microM BA, 6 microM NAA, 3% casein hydrolysate and 0.1% activated charcoal (AC) yielded >12 shoots per shoot or bud. Addition of AC favoured the enlargement of pseudobulbs and better rooting. The plantlets transferred to community potting mix after in vitro hardening (8-10 wk) displayed 85% survival.     

The binding of auxin to the Arabidopsis auxin influx transporter AUX1

The cellular import of the hormone auxin is a fundamental requirement for the generation of auxin gradients that control a multitude of plant developmental processes. The AUX/LAX family of auxin importers, exemplified by AUX1 from Arabidopsis (Arabidopsis thaliana), has been shown to mediate auxin import when expressed heterologously. The quantitative nature of the interaction between AUX1 and its transport substrate indole-3-acetic acid (IAA) is incompletely understood, and we sought to address this in the present investigation. We expressed AUX1 to high levels in a baculovirus expression system and prepared membrane fragments from baculovirus-infected insect cells. These membranes proved suitable for determination of the binding of IAA to AUX1 and enabled us to determine a K(d) of 2.6 mum, comparable with estimates for the K(m) for IAA transport. The efficacy of a number of auxin analogues and auxin transport inhibitors to displace IAA binding from AUX1 has also been determined and can be rationalized in terms of their physiological effects. Determination of the parameters describing the initial interaction between a plant transporter and its hormone ligand provides novel quantitative data for modeling auxin fluxes.     

Role of NADH/NAD+ transport activity and glycogen store on skeletal muscle energy metabolism during exercise: in silico studies

Skeletal muscle can maintain ATP concentration constant during the transition from rest to exercise, whereas metabolic reaction rates may increase substantially. Among the key regulatory factors of skeletal muscle energy metabolism during exercise, the dynamics of cytosolic and mitochondrial NADH and NAD+ have not been characterized. To quantify these regulatory factors, we have developed a physiologically based computational model of skeletal muscle energy metabolism. This model integrates transport and reaction fluxes in distinct capillary, cytosolic, and mitochondrial domains and investigates the roles of mitochondrial NADH/NAD+ transport (shuttling) activity and muscle glycogen concentration (stores) during moderate intensity exercise (60% maximal O2 consumption). The underlying hypothesis is that the cytosolic redox state (NADH/NAD+) is much more sensitive to a metabolic disturbance in contracting skeletal muscle than the mitochondrial redox state. This hypothesis was tested by simulating the dynamic metabolic responses of skeletal muscle to exercise while altering the transport rate of reducing equivalents (NADH and NAD+) between cytosol and mitochondria and muscle glycogen stores. Simulations with optimal parameter estimates showed good agreement with the available experimental data from muscle biopsies in human subjects. Compared with these simulations, a 20% increase (or approximately 20% decrease) in mitochondrial NADH/NAD+ shuttling activity led to an approximately 70% decrease (or approximately 3-fold increase) in cytosolic redox state and an approximately 35% decrease (or approximately 25% increase) in muscle lactate level. Doubling (or halving) muscle glycogen concentration resulted in an approximately 50% increase (or approximately 35% decrease) in cytosolic redox state and an approximately 30% increase (or approximately 25% decrease) in muscle lactate concentration. In both cases, changes in mitochondrial redox state were minimal. In conclusion, the model simulations of exercise response are consistent with the hypothesis that mitochondrial NADH/NAD+ shuttling activity and muscle glycogen stores affect primarily the cytosolic redox state. Furthermore, muscle lactate production is regulated primarily by the cytosolic redox state.     

Surveillance of vibriophages reveals their role as biomonitoring agents in Kolkata

Cholera is a public health threat in all developing countries. Kolkata, a city in eastern India, is an endemic zone for cholera. During the course of a comprehensive investigation on the distribution of phages of Vibrio cholerae O1 and O139 in freshwater bodies in Kolkata, we were able to isolate the phages of V. cholerae O1 and O139. Vibrio cholerae O1 phages were found at all the sites and exhibited a distinct seasonal cycle, with a primary peak (13.6–17.2 PFU mL⁻¹) during monsoon (June to August) in both 2006 and 2007. Vibrio cholerae O139 phages were present in the environment and were predominant during monsoon in the year 2006, except for late winter and early summer from February to April. In contrast, in the year 2007, the O139 phages could be isolated only during July to December, with the highest counts of 12.0 PFU mL⁻¹ determined in August. The multiplex PCR results showed that 90 samples were positive for wbe of V. cholerae O1, 32 samples for O139 ( wbf ) and 18 samples for both. This study shows that surveillance of vibriophages indicates the presence of V. cholerae O1 and O139 in water bodies in and around Kolkata and could therefore serve as a powerful biomonitoring agent.     

Stringent response in Vibrio cholerae: genetic analysis of spoT gene function and identification of a novel (p)ppGpp synthetase gene

RelA and SpoT of Gram-negative organisms critically regulate cellular levels of (p)ppGpp. Here, we have dissected the spoT gene function of the cholera pathogen Vibrio cholerae by extensive genetic analysis. Unlike Escherichia coli , V. cholerae Δ relA Δ spoT cells accumulated (p)ppGpp upon fatty acid or glucose starvation. The result strongly suggests RelA-SpoT-independent (p)ppGpp synthesis in V. cholerae . By repeated subculturing of a V. cholerae Δ relA Δ spoT mutant, a suppressor strain with (p)ppGpp⁰ phenotype was isolated. Bioinformatics analysis of V. cholerae whole genome sequence allowed identification of a hypothetical gene ( VC1224 ), which codes for a small protein (∼29 kDa) with a (p)ppGpp synthetase domain and the gene is highly conserved in vibrios; hence it has been named relV . Using E. coli Δ relA or Δ relA Δ spoT mutant we showed that relV indeed codes for a novel (p)ppGpp synthetase. Further analysis indicated that relV gene of the suppressor strain carries a point mutation at nucleotide position 676 of its coding region (Δ relA Δ spoT relV676 ), which seems to be responsible for the (p)ppGpp⁰ phenotype. Analysis of a V. cholerae Δ relA Δ spoT Δ relV triple mutant confirmed that apart from canonical relA and spoT genes, relV is a novel gene in V. cholerae responsible for (p)ppGpp synthesis.     

Changes in Phenolics, Polyphenol Oxidase and its Isoenzyme Patterns in Relation to Resistance in Taro against Phytophthora colocasiae

The effect of Phytophthora leaf blight disease, caused by Phytophthora colocasiae Raciborski, on the accumulation of phenolics and polyphenol oxidase (PPO) activity in ex vitro plants was studied in three resistant (DP‐25, Duradim and Jhankri) and one susceptible (N‐118) genotypes of taro [Colocasia esculenta (L). Schott]. The inoculation of taro leaves with P. colocasiae spores resulted in a quantitative change in both biochemical parameters and induction of PPO isoforms in resistant genotypes. The amount of phenolics was increased owing to blight by 68.02%, 58.87%, 52.67% and 11.50% in DP‐25, Duradim, Jhankri and N‐118, respectively. The per cent increase in PPO under stress over non‐stress condition was also highest in DP‐25 (49.14%) followed by Duradim (41.56%), Jhankri (40.55%) and N‐118 (17.08%). The resistant genotypes showed higher activity of PPO as compared with susceptible ones, which was reflected through its banding pattern in isoenzyme analysis, detecting four different isoforms. The intensity of the bands was higher in the resistant genotypes than in susceptible N‐118. The appearance of high intensity bands and/or reduction in the intensity of particular isoform(s) in the zymograms of all the three resistant taro genotypes studied, led to the apparent conclusion of linking PPO isoenzyme expression with blight resistance in taro. The blight incidence (per cent leaf infection and leaf area infection) was lower in the resistant genotypes than in susceptible, N‐118. The yield reduction owing to blight was below 20% in DP‐25, Jhankri and Duradim, while the same was more than 40% in N‐118. The phenolics and PPO activity was negatively correlated with disease incidence and yield reduction owing to blight. Based on the results of disease incidence, biochemical contents and yield, the pattern of stress tolerance was DP‐25 > Duradim > Jhankri > N‐118. The studied parameters, i.e. phenolics and PPO could be used as biochemical markers for leaf blight stress tolerance studies in taro.     

Synthesis and SAR of 4-substituted-2-aminopyrimidines as novel c-Jun N-terminal kinase (JNK) inhibitors

The development of a series of novel 4-substituted-2-aminopyrimidines as inhibitors of c-Jun N-terminal kinases is described. The synthesis, in vitro inhibitory values for JNK1, and the in vitro inhibitory value for a c-Jun cellular assay are discussed. Optimization of microsomal clearance led to the identification of 9c, whose kinase selectivity is reported.