Scopolamine Induced Amnesia is Reversed by <Emphasis Type="Italic">Bacopa monniera</Emphasis> Through Participation of Kinase-CREB Pathway

Scopolamine, an anticholinergic drug, is reported to produce amnesia by interference of long term potentiation and has been used for discerning the efficacy of various antiamnesic drugs. The intoxication with anticholinergics and benzodiazepines tend to produce neurodegeneration which cause memory deficits. Our earlier reports have shown the antiamnesic drug, B. monniera to be capable of alleviating diazepam induced memory deficits. We have now tested how scopolamine affects downstream signaling molecules of long term potentiation and if B. monniera can also modulate the scopolamine induced amnesia. We used Morris water maze scale to test the amnesic effect of scopolamine and its reversal by B. monniera. Rota-rod test was used to screen muscle coordination activity of mice before water maze investigations were carried out. The results showed that scopolamine downregulated protein kinase C and iNOS without affecting cAMP, protein kinase A, calmodulin, MAP kinase, nitrite, CREB and pCREB. B. monniera reversed the scopolamine induced amnesia by significantly improving calmodulin and by partially attenuating protein kinase C and pCREB. These observations suggest involvement of calmodulin in evoking antiamnesic effects of B. monniera.     

Structural and functional characterization of HMG-COA reductase from Artemisia annua

Plants synthesize a great variety of isoprenoid products that are required not only for normal growth and development but also for their adaptive responses to environmental challenges. However, despite the remarkable diversity in the structure and function of plant isoprenoids, they all originate from a single metabolic precursor, mevalonic acid. The synthesis of mevalonic acid is catalysed by the enzyme, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG- CoA reductase). The analysis of the amino acid sequence of HMG-CoA reductase from Artemisia annua L. plant showed that it belongs to class I HMG-CoA reductase family. The three dimensional structure of HMG-CoA reductase of Artemisia annua has been generated from amino acid sequence using homology modelling with backbone structure of human HMG-CoA reductase as template. The model was generated using the SWISS MODEL SERVER. The generated 3-D structure of HMG-CoA reductase was evaluated at various web interfaced servers to checks the stereo interfaced quality of the structure in terms of bonds, bond angles, dihedral angles and non-bonded atom-atom distances, structural as well as functional domains etc. The generated model was visualized using the RASMOL. Structural analysis of HMG-CoA reductase from Artemisia annua L. plant hypothesize that the N and C-terminals are positioned in cytosol by the two membrane spanning helices and the C-terminals domain shows similarity to the human HMG-CoA reductase enzyme indicating that they both had potential catalytic similarities.     

Akt-phosphorylated Mitogen-activated Kinase-activating Death Domain Protein (MADD) Inhibits TRAIL-induced Apoptosis by Blocking Fas-associated Death Domain (FADD) Association with Death Receptor 4

MADD plays an essential role in cancer cell survival. Abrogation of endogenous MADD expression results in significant spontaneous apoptosis and enhanced susceptibility to tumor necrosis factor α-related apoptosis-inducing ligand (TRAIL)-induced apoptosis. However, the regulation of MADD function is largely unknown. Here, we demonstrate that endogenous MADD is phosphorylated at three highly conserved sites by Akt, and only the phosphorylated MADD can directly interact with the TRAIL receptor DR4 thereby preventing Fas-associated death domain recruitment. However, in cells susceptible to TRAIL treatment, TRAIL induces a reduction in MADD phosphorylation levels resulting in MADD dissociation from, and Fas-associated death domain association with DR4, which allows death-inducing signaling complex (DISC) formation leading to apoptosis. Thus, the pro-survival function of MADD is dependent upon its phosphorylation by Akt. Because Akt is active in most cancer cells and phosphorylated MADD confers resistance to TRAIL-induced apoptosis, co-targeting Akt-MADD axis is likely to increase efficacy of TRAIL-based therapies.     

Effect of sesamol on radiation-induced cytotoxicity in Swiss albino mice

The radio-protective ability of sesamol (SM) at various doses viz., 0, 10, 25, 40, 50, 70 and 100 mg/kg bw, administered intraperitoneally 30 min prior to 9.5 Gy whole-body γ-irradiation was studied in Swiss albino mice. Radiation toxicity and mortality were observed during a period of 30 days and the percentage mortality was calculated. SM pretreatment with 50 mg/kg bw was found to be the most effective dose in maintaining body weight and in reducing the percentage mortality, while 100 mg/kg bw was found to be more effective in maintaining the spleen index and in stimulation of endogenous spleen colony-forming units. Pretreatment with SM (50 mg/kg bw) in mice irradiated with 15 Gy significantly reduced dead, inflammatory, mitotic and goblet cells in irradiated jejunum. SM at 50 mg/kg bw also increased crypt cells, maintained villus height, and prevented mucosal erosion. Nuclear enlargement in epithelial cells was found less in SM-treated mice compared with the irradiated control. The radiation-induced decrease in endogenous antioxidant enzymes (GSH, GST, catalase) and the increase in lipid peroxidation were also reduced by pretreatment with SM [50 and 100 mg/kg bw] at all monitored post-irradiation intervals. There was no protection at a dose less than 25 mg/kg bw.     

Fractionation of digestive proteinases from Tenebrio molitor (Coleoptera: Tenebrionidae) larvae and role in protein digestion

Tenebrio molitor larval digestive proteinases were purified and characterized by gel filtration chromatography combined with activity electrophoresis. Cysteine proteinases, consisting of at least six distinct activities, were found in three chromatographic peaks in anterior and posterior midgut chromatographies. The major activity in the anterior midgut, peak cys II, consisted of cysteine proteinases with Mm of 23 kDa. The predominant peak in the posterior, cys I, was represented by 38 kDa proteinases. The activities of all cysteine proteinases were maximal in buffers from pH 5.0 to 7.0, with 80% stability at pH values from 4.0 to 7.0. In the conditions of the last third of the midgut, the activity and stability of cysteine proteinases was sharply decreased. Trypsin-like activity included a minor peak of "heavy" trypsins with Mm 59 kDa, located mainly in the anterior midgut. An in vitro study of the initial stages of digestion of the main dietary protein, oat 12S globulin, by anterior midgut proteinases revealed that hydrolysis occurred through the formation of intermediate high-Mm products, similar to those formed during oat seed germination. Cysteine proteinases from the cys III peak and heavy trypsins were capable of only limited proteolysis of the protein, whereas incubation with cys II proteinases resulted in substantial hydrolysis of the globulin.     

Evidence for polyadenylated mRNA in Pseudomonas aeruginosa

In this paper, we report the synthesis of Pseudomonas aeruginosa cDNA in the presence of oligo(dT) primers. Hybridization of oligonucleotide DNA microarrays indicates that under the experimental conditions used, at least 43.7% of the expressed genes from P. aeruginosa PAO1, representing many different functional classes, can be detected by using oligo(dT)-primed cDNAs.     

Site specific integration of FLP recombinase in BHK-21 cell line

A binary system for gene activation and site specific integration based on conditional recombination of transfected sequences mediated by FLP recombinase from yeast was implemented in mammalian cells. In several cell lines, FLP rapidly and precisely recombined copies of its specific target sequences to activate an otherwise silent beta-galactosidase reporter gene. Clones of marked cells were generated by excisional recombination within a chromosomally integrated copy of the silent reporters. These clones exhibited intense blue colour with X-Gal staining solution.     

Insulin receptor substrate 1 regulation of sarco-endoplasmic reticulum calcium ATPase 3 in insulin-secreting beta-cells

We have previously characterized an insulin receptor substrate 1 (IRS-1)-overexpressing beta-cell line. These beta-cells demonstrated elevated fractional insulin secretion and elevated cytosolic Ca(2+) levels compared with wild-type and vector controls. This effect of IRS-1 may be mediated via an interaction with the sarco-endoplasmic reticulum calcium ATPase (SERCA). Here we demonstrate that IRS-1 and IRS-2 localize to an endoplasmic reticulum (ER)-enriched fraction in beta-cells using subcellular fractionation. We also observe co-localization of both IRS-1 and IRS-2 with ER marker proteins using immunofluorescent confocal microscopy. Furthermore, immuno-electron microscopy studies confirm that IRS-1 and SERCA3b localize to vesicles derived from the ER. In Chinese hamster ovary-T (CHO-T) cells transiently transfected with SERCA3b alone or together with IRS-1, SERCA3b co-immunoprecipitates with IRS-1. This interaction is enhanced with insulin treatment. SERCA3b also co-immunoprecipitates with IRS-1 in wild-type and IRS-1-overexpressing beta-cell lines. Ca(2+) uptake in ER-enriched fractions prepared from wild-type and IRS-1-overexpressing cell lines shows no significant difference, indicating that the previously observed decrease in Ca(2+) uptake by IRS-1-overexpressing cells is not the result of a defect in SERCA. Treatment of wild-type beta-cells with thapsigargin, an inhibitor of SERCA, resulted in an increase in glucose-stimulated fractional insulin secretion similar to that observed in IRS-1-overexpressing cells. The colocalization of IRS proteins and SERCA in the ER of beta-cells increases the likelihood that these proteins can interact with one another. Co-immunoprecipitation of IRS-1 and SERCA in CHO-T cells and beta-cells confirms that these proteins do indeed interact directly. Pharmacological inhibition of SERCA in beta-cells results in enhanced secretion of insulin. Taken together, our data suggest that interaction between IRS proteins and SERCA is an important regulatory step in insulin secretion.     

Bitter gourd proteinase inhibitors: potential growth inhibitors of Helicoverpa armigera and Spodoptera litura

Proteinase inhibitors (PIs) from the seeds of bitter gourd (Momordica charantia L.) were identified as strong inhibitors of Helicoverpa armigera gut proteinases (HGP). Biochemical investigations showed that bitter gourd PIs (BGPIs) inhibited more than 80% HGP activity. Electrophoretic analysis revealed the presence of two major proteins (BGPI-1 and-2) and two minor proteins (BGPI-3 and-4) having inhibitory activity against both trypsin and HGP. The major isoforms BGPI-1 and BGPI-2 have molecular mass of 3.5 and 3.0 kDa, respectively. BGPIs inhibited HGP activity of larvae fed on different host plants, on artificial diet with or without added PIs and proteinases excreted in fecal matter. Degradation of BGPI-1 by HGP showed direct correlation with accumulation of BGPI-2-like peptide, which remained stable and active against high concentrations of HGP up to 3 h. Chemical inhibitors of serine proteinases offered partial protection to BGPI-1 from degradation by HGP, suggesting that trypsin and chymotrypsin like proteinases are involved in degradation of BGPI-1. In larval feeding studies, BGPIs were found to retard growth and development of two lepidopteran pests namely Helicoverpa armigera and Spodoptera litura. This is the first report showing that BGPIs mediated inhibition of insect gut proteinases directly affects fertility and fecundity of both H. armigera and S. litura. The results advocate use of BGPIs to introduce insect resistance in otherwise susceptible plants.     

1-Methyl anthraquinones and their biogenetic precursors from Stereospermum personatum

Two novel 1(17)-methyl anthraquinones, sterequinone-A and -D, their biogenetic precursors sterequinone-B, -C, and a new naphthoquinone sterequinone-E along with a known naphthoquinone, sterekunthal-B, have been isolated from the petroleum ether extract of stem bark of Stereospermum personatum. Their structures were established using spectroscopic methods.