Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .     

Regulation of Rac1 and Reactive Oxygen Species Production in Response to Infection of Gastrointestinal Epithelia

Generation of reactive oxygen species (ROS) during infection is an immediate host defense leading to microbial killing. APE1 is a multifunctional protein induced by ROS and after induction, protects against ROS-mediated DNA damage. Rac1 and NAPDH oxidase (Nox1) are important contributors of ROS generation following infection and associated with gastrointestinal epithelial injury. The purpose of this study was to determine if APE1 regulates the function of Rac1 and Nox1 during oxidative stress. Gastric or colonic epithelial cells (wild-type or with suppressed APE1) were infected with Helicobacter pylori or Salmonella enterica and assessed for Rac1 and NADPH oxidase-dependent superoxide production. Rac1 and APE1 interactions were measured by co-immunoprecipitation, confocal microscopy and proximity ligation assay (PLA) in cell lines or in biopsy specimens. Significantly greater levels of ROS were produced by APE1-deficient human gastric and colonic cell lines and primary gastric epithelial cells compared to control cells after infection with either gastric or enteric pathogens. H. pylori activated Rac1 and Nox1 in all cell types, but activation was higher in APE1 suppressed cells. APE1 overexpression decreased H. pylori-induced ROS generation, Rac1 activation, and Nox1 expression. We determined that the effects of APE1 were mediated through its N-terminal lysine residues interacting with Rac1, leading to inhibition of Nox1 expression and ROS generation. APE1 is a negative regulator of oxidative stress in the gastrointestinal epithelium during bacterial infection by modulating Rac1 and Nox1. Our results implicate APE1 in novel molecular interactions that regulate early stress responses elicited by microbial infections.     

Vitamin B12-induced alterations in activities of α-glycerophosphate dehydrogenase and malic enzyme in brain of singi fish, Heteropneustes fossilis (Bloch)

Different doses of vitamin B₁₂ (0.25, 0.5, 1, 2 and 4 μg/g, injected intraperitoneally for three consecutive days) altered the activities of mitochondrial-α-glycerophosphate dehydrogenase (α-GPD) and NADP-dependent cytosolic malic enzyme (ME) in the brain of singi fish. The α-GPD activity increased at doses of 0.5, 1, 2 and 4 μg/g vitamin B₁₂. A dose of 0.5 μg/g vitamin B₁₂ induced less activity than higher doses. ME activity increased with 1, 2 and 4 μg/g of vitamin B₁₂/g. The mitochondrial and cytosolic protein content remained unchanged after vitamin B₁₂ administration. Cycloheximide treatment inhibited the vitamin B₁₂-induced increase in α-GPD and ME activity. Thus, vitamin B₁₂ is involved in the induction of some enzymes in fish brain.     

A novel plasma membrane-bound thioredoxin from soybean

Two thioredoxin cDNAs from soybean were isolated by screening an expression library using an anti-(plasma membrane) serum. The nucleotide sequences of the two cDNAs were found to be 89% identical. The polypeptides encoded by the two cDNAs, designated TRX1 and TRX2, contain a disulfide active site, as found in other thioredoxins. TRX1 was expressed as a fusion protein in Escherichia coli and shown to possess thiol-disufide interchange activity. Unlike other eukaryotic thioredoxins, these two soybean thioredoxins contain a putative transmembrane domain in their N-terminal regions. To determine subcellular location, the TRX1 was fused with a reporter epitope at its C-terminus and expressed in transgenic tobacco plants. The fusion protein was co-purified with plasma membrane markers 1,3-glucan synthase and vanadate-sensitive ATPase, indicating the plasma membrane location of TRX1. When the reporter epitope was inserted between the start codon and the transmembrane domain in the N-terminus, the fusion protein was found in the soluble fraction, possibly due to disruption of the transmembrane domain by the highly hydrophilic epitope sequence. Taken together, our results demonstrate that soybean TRX1 is a plasma membrane-bound thioredoxin, which is most likely anchored to the membrane through the N-terminal transmembrane domain. It is known that plant plasma membranes contain various proteins with thiol-disulfide interchange activity. The soybean thioredoxins reported here are the first group of such proteins to be characterized at the molecular level. However, the biological function of the plasma membrane-bound thioredoxin remains to be determined.     

Lactobacillus acidophilus Alleviates Platelet-Activating Factor-Induced Inflammatory Responses in Human Intestinal Epithelial Cells

Probiotics have been used as alternative prevention and therapy modalities in intestinal inflammatory disorders including inflammatory bowel diseases (IBD) and necrotizing enterocolitis (NEC). Pathophysiology of IBD and NEC includes the production of diverse lipid mediators, including platelet-activating factor (PAF) that mediate inflammatory responses in the disease. PAF is known to activate NF-κB, however, the mechanisms of PAF-induced inflammation are not fully defined. We have recently described a novel PAF-triggered pathway of NF-κB activation and IL-8 production in intestinal epithelial cells (IECs), requiring the pivotal role of the adaptor protein Bcl10 and its interactions with CARMA3 and MALT1. The current studies examined the potential role of the probiotic Lactobacillus acidophilus in reversing the PAF-induced, Bcl10-dependent NF-κB activation and IL-8 production in IECs. PAF treatment (5 µM×24 h) of NCM460 and Caco-2 cells significantly increased nuclear p65 NF-κB levels and IL-8 secretion (2-3-fold, P<0.05), compared to control, which were blocked by pretreatment of the cells for 6 h with L. acidophilus (LA) or its culture supernatant (CS), followed by continued treatments with PAF for 24 h. LA-CS also attenuated PAF-induced increase in Bcl10 mRNA and protein levels and Bcl10 promoter activity. LA-CS did not alter PAF-induced interaction of Bcl10 with CARMA3, but attenuated Bcl10 interaction with MALT1 and also PAF-induced ubiquitination of IKKγ. Efficacy of bacteria-free CS of LA in counteracting PAF-induced inflammatory cascade suggests that soluble factor(s) in the CS of LA mediate these effects. These results define a novel mechanism by which probiotics counteract PAF-induced inflammation in IECs.     

Peptidyl transferase activity of tRNA: a quantum chemical study

The mechanism of protein synthesis is still unknown due to inability to detect the so-called enzyme "peptidyl transferase" even after elucidation of high-resolution crystal structure of ribosome. We have recently shown by model building and semi-empirical energy calculation that the tRNA molecule at P-site of ribosome may act as peptidyl transferase (Das et al. (1999) J. Theor. Biol. 200, 193-205). We proposed that the tetrahedral intermediate formed from nucleophylic attack of CO of P-site amino-acylated tRNA by NH2 of A-site amino-acylated tRNA is converted to a six-member ring intermediate by conformational change. This ring intermediate produces a free tRNA and a tRNA covalently linked to a peptide. However, energy of the six-member ring intermediate was calculated to be quite high. We show here that the energy values of all the reactants, intermediates and products are within the expected range when they are calculated using high level ab initio quantum chemical methods.     

Purification and characterization of protease from a newly isolated Rhizopus oryzae

A newly isolated Rhizopus oryzae was found to exhibit some unusual phenomenon of secreting alkaline protease which was purified and characterized. The molecular weight was determined to be 28,600 dalton in gel electrophoresis. The enzyme is stable in the pH range from 3 to 11 and most active at pH 8. The temperature optimum of this thermostable biocatalyst is at 60 °C. The enzyme is sensitive to metal chelators, most of the metal ions (excepting a few monovalent cations) and inhibitor like PMSF. This indicates that the protease of isolated Rhizopus oryzae falls under alkaline serine group.     

Polymicrobial Infection with Major Periodontal Pathogens Induced Periodontal Disease and Aortic Atherosclerosis in Hyperlipidemic ApoEnull Mice

Periodontal disease (PD) and atherosclerosis are both polymicrobial and multifactorial and although observational studies supported the association, the causative relationship between these two diseases is not yet established. Polymicrobial infection-induced periodontal disease is postulated to accelerate atherosclerotic plaque growth by enhancing atherosclerotic risk factors of orally infected Apolipoprotein E deficient (ApoE^null) mice. At 16 weeks of infection, samples of blood, mandible, maxilla, aorta, heart, spleen, and liver were collected, analyzed for bacterial genomic DNA, immune response, inflammation, alveolar bone loss, serum inflammatory marker, atherosclerosis risk factors, and aortic atherosclerosis. PCR analysis of polymicrobial-infected (Porphyromonas gingivalis [P. gingivalis], Treponema denticola [T. denticola], and Tannerella forsythia [T. forsythia]) mice resulted in detection of bacterial genomic DNA in oral plaque samples indicating colonization of the oral cavity by all three species. Fluorescent in situ hybridization detected P. gingivalis and T. denticola within gingival tissues of infected mice and morphometric analysis showed an increase in palatal alveolar bone loss (p<0.0001) and intrabony defects suggesting development of periodontal disease in this model. Polymicrobial-infected mice also showed an increase in aortic plaque area (p<0.05) with macrophage accumulation, enhanced serum amyloid A, and increased serum cholesterol and triglycerides. A systemic infection was indicated by the detection of bacterial genomic DNA in the aorta and liver of infected mice and elevated levels of bacterial specific IgG antibodies (p<0.0001). This study was a unique effort to understand the effects of a polymicrobial infection with P. gingivalis, T. denticola and T. forsythia on periodontal disease and associated atherosclerosis in ApoE^null mice.     

Role of gestation delay in a plankton-fish model under stochastic fluctuations

The present paper studies a minimal prey-predator model in the context of marine plankton interaction together with predation by planktivorous fish. The time lag required for gestation of the predator is incorporated and the resulting delayed model is analyzed for stability and bifurcation phenomena. A stochastic extension of the model is considered by perturbing the growth process of phytoplankton using colored noise process known to be more appropriate for the marine environment. The stochastic models with and without gestation delay are analyzed for stability aspects and a threshold value of gestation delay is obtained; this threshold is then compared with that of the deterministic model.