Simulation modeling of ligand receptor interactions at non-equilibrium conditions: processing of noisy inputs by ionotropic receptors

The first event in signal transduction at a synapse is the binding of transmitters to receptors. Because of rapidly changing transmitter levels this binding is unlikely to occur at equilibrium. We describe a mathematical approach that models complex receptor interactions in which the timing and amplitude of transmitter release are noisy. We show that exact solutions for simple bimolecular interactions and receptor transitions can be used to model complex reaction schemes by expressing them in sets of difference equations. Results from the difference equation method to describe binding and channel opening at extended time points compare well with standard solutions using ordinary differential equations. Because it is applicable to noisy systems we used the difference method to investigate the information processing capabilities of GABA receptors and predict how pharmacological agents may modify these properties. As previously demonstrated, the response to a single pulse of GABA is prolonged through entry into a desensitized state. During trains of stimuli the signal to noise ratio can change, and even increase progressively, but the overall transmitted fidelity of the signal decreases with increased driving frequency. The GABA modulator chlorpromazine (primarily affects agonist on and off rates) is predicated to increase receptor signal to noise ratio at all frequencies whereas pregnenolone sulfate (affects receptor desensitization) completely inhibits information transfer.     

Hypoxia-induced Activation of Epidermal Growth Factor Receptor (EGFR) Kinase in the Cerebral Cortex of Newborn Piglets: The Role of Nitric Oxide

The present study aims to investigate the mechanism of EGFR kinase activation during hypoxia and tests the hypothesis that hypoxia-induced increased activation of EGFR kinase in the cerebral cortical membrane fraction of newborn piglets is mediated by nitric oxide (NO) derived from neuronal nitric oxide synthase (nNOS). Fifteen newborn piglets were divided into normoxic (Nx, n = 5), hypoxic (Hx, n = 5) and hypoxic-treated with nNOS inhibitor (Hx-nNOSi, n = 5). Hypoxia was induced by an FiO2 of 0.07 for 60 min. nNOS inhibitor I (selectivity >2,500 vs. endothelial NOS, eNOS, and >500 vs. inducible NOS, iNOS) was administered (0.4 mg/kg, i. v.) 30 min prior to hypoxia. EGFR kinase tyrosine phosphorylation at Tyr1173, an index of activation of EGFR kinase, was determined by Western blot analysis using an anti-phospho (pTyr¹¹⁷³)-EGFR kinase antibody. Protein bands were analyzed by imaging densitometry and expressed as absorbance (OD × mm²). EGFR kinase activity was determined radiochemically using immunopurified enzyme. EGFR kinase activity was expressed as pmols/mg protein/hr. Density of phosphor (pTyr¹¹⁷³)-EGFR kinase (OD × mm²) was 60.2 ± 9.8 in Nx, 177.0 ± 26.9 in Hx (P < 0.05 vs. Nx) and 79.9 ± 15.7 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Activity of EGFR kinase (pmoles/mg protein/hr) was 4,603 ± 155 in Nx, 8,493 ± 427 in Hx (P < 0.05 vs. Nx) and 4,516 ± 104 in Hx-nNOSi (P < 0.05 vs. Hx, P = NS vs. Nx). Pretreatment with nNOS inhibitor prevented the hypoxia-induced increased phosphorylation and increased activity of EGFR kinase. We conclude that the mechanism of hypoxia-induced increased activation of EGFR kinase is mediated by nNOS-derived NO.     

Phytochemical and genetic analysis in selected chemotypes of Withania somnifera

The main active components and genetic profile of 15 selected accessions of Withania somnifera Dunal. were analysed. Ethanolic extract of the dried roots/leaves of the plant was concentrated under pressure at 50+/-5 degrees C and was analysed for main compounds (withanolides and withaferin A) by HPLC. All the main components were found to be present in accessions (AGB 002, AGB 009, RSS 009, RSS 033). Correlation of these main components with their genetic factors, was undertaken using AFLP (amplified fragment length polymorphism) markers. Among 64 primers 7 yielded optimum polymorphism. A total of 913 polymorphic peaks were generated using these primers. Jaccard's similarity coefficient indicated that accessions having almost the same active compounds clustered together. The present study demonstrates that AFLP can be successfully used to resolve the correlation of AFLP data with the presence of secondary metabolites.     

Bioreactor studies on the endophytic fungus Entrophospora infrequens for the production of an anticancer alkaloid camptothecin

Twigs (young and old) from Nothapodytes foetida growing in the Jammu and Mahabaleshwar regions in India were used for the isolation of 52 strains of endophytic fungi and were tested for their ability to produce the anticancer alkaloid camptothecin. One of the isolates from the inner bark tissue of the N. foetida plant growing in the Jammu region of J&K state, India, was found to produce detectable quantities of camptothecin and its derivatives when grown in a semi-synthetic liquid medium. Camptothecin was identified by physicochemical analysis and further confirmed by spectroscopic studies. No camptothecin was detected in zero time cultures or in uninoculated culture broth. The maximum yield of camptothecin was 0.575 +/- 0.031 mg/100 g of dry cell mass in 96 h in shake flasks, whereas 4.96 +/- 0.73 mg/100 g of dry mass was recorded in 48 h in a bioreactor.     

Molecular Characterization of Mercury Resistant Bacteria Inhabiting Polluted Water Bodies of Different Geographical Locations in India

Mercury pollution is a major environmental problem that arises as a result of natural processes as well as from anthropogenic sources. In response to toxic mercury compounds, microbes have developed astonishing array of resistance systems to detoxify them. To address this challenge, this study was aimed in screening bacterial isolates for their tolerance against varied concentrations of phenylmercuric acetate. Mercury transformation by bacteria being sensitive to factors such as available carbon source, etc. that affect mer-mediated transformation, screened mercury tolerant bacteria were also studied for their tolerance to different antimicrobials and carbon sources, followed by identification using biochemical as well as 16S rRNA approach. Following identification, gene encoding organomercurial lyase catalyzing protonolytic cleavage of C-Hg bond of organic mercury was amplified using gene specific primers, cloned in pGEMT(?) easy vector and sequenced. Microbe-based approach using organomercurial lyase encoded by merB gene being potentially economic, provides foundation to facilitate genetic manipulation of this environmentally important enzyme to remove high concentrations of obstinate mercury using holistic, multifaceted approach for use in bioremediation through generation of transgenics or as catalyst for use in bioreactors.     

Novel thermostable lipase from <Emphasis Type="Italic">Bacillus circulans</Emphasis> IIIB153: comparison with the mesostable homologue at sequence and structure level

Thermophilic Bacillus circulans IIIB153 isolated from hot springs of North West Himalayas, India, produced an extracellular lipase, which exhibited significant biofilm disruption property on the static biofilm disruption model with a single species of Actinomyces viscosous. The gene encoding the lipase was cloned and overexpressed in Escherichia coli. Recombinant Bacillus circulans lipase (BCL), a monomer with molecular mass of 43 kDa also exhibited significant biofilm disruption activity. The enzyme was optimally active at 60°C, pH 8.5 and retained >70% of its original activity after 1 h incubation at 60°C. 3D structure of BCL developed by homology modeling showed a typical α/β hydrolase fold, a characteristic feature of lipolytic enzymes. Comparison of thermostable BCL with mesostable lipase from Chromobacterium viscosum at the sequence and structure level showed distinct variations in the structural features, with the presence of a high content of proline residues, aromatic amino acids and salt bridges. These features along with the presence of zinc-binding site observed in BCL structure could have a potential role in thermal stability of the enzyme.     

An essential oil and its major constituent isointermedeol induce apoptosis by increased expression of mitochondrial cytochrome c and apical death receptors in human leukaemia HL-60 cells

An essential oil from a lemon grass variety of Cymbopogon flexuosus (CFO) and its major chemical constituent sesquiterpene isointermedeol (ISO) were investigated for their ability to induce apoptosis in human leukaemia HL-60 cells because dysregulation of apoptosis is the hallmark of cancer cells. CFO and ISO inhibited cell proliferation with 48 h IC50 of approximately 30 and 20 microg/ml, respectively. Both induced concentration dependent strong and early apoptosis as measured by various end-points, e.g. annexinV binding, DNA laddering, apoptotic bodies formation and an increase in hypo diploid sub-G0 DNA content during the early 6h period of study. This could be because of early surge in ROS formation with concurrent loss of mitochondrial membrane potential observed. Both CFO and ISO activated apical death receptors TNFR1, DR4 and caspase-8 activity. Simultaneously, both increased the expression of mitochondrial cytochrome c protein with its concomitant release to cytosol leading to caspase-9 activation, suggesting thereby the involvement of both the intrinsic and extrinsic pathways of apoptosis. Further, Bax translocation, and decrease in nuclear NF-kappaB expression predict multi-target effects of the essential oil and ISO while both appeared to follow similar signaling apoptosis pathways. The easy and abundant availability of the oil combined with its suggested mechanism of cytotoxicity make CFO highly useful in the development of anti-cancer therapeutics.     

Isolation and identification of an endophytic strain of Fusarium oxysporum producing podophyllotoxin from Juniperus recurva

Podophyllotoxin, a well-known naturally occurring aryltetralin lignan occurs in few plant species that is used as a precursor for the chemical synthesis of the anticancer drugs like etoposide, teniposide and etopophose phosphate. The availiability of this lignan is becoming increasingly limited because of the scarce occurance of its natural sources and also because synthetic approaches for its production are still commercially unacceptable. This paper reports first time the production of podophyllotoxin by an endophytic fungus Fusarium oxysporum isolated from the medicinal plant Juniperus recurva. Further confirmation and quantification of podophyllotoxin was performed by HPLC, LC-MS, and LC-MS/MS.     

Phenolic constituents and genetic profile of Hypericum perforatum L. from India

The content of hypericins (hypericin and pseudohypericin), hyperforin, and flavonoids (rutin, hyperoside, quercitrin, and quercetin) and genetic profiles of eight accessions of Hypericum perforatum L., collected from different locations in India, have been determined. The secondary metabolite content was determined using a highly selective LC/MS/MS method. Pearson and Spearman's correlation coefficient were used to investigate the relationships between the secondary metabolites and a significant positive correlation was found between hypericin and pseudohypericin contents. Genetic profiling was undertaken using the random amplification of polymorphic DNA (RAPD) and single sequence repeat (SSR) methods. Among the 49 random primers used for the initial screening, only nine yielded polymorphic RAPD profiles. The SSR analysis shows that seven out of the 11 primers were polymorphic. There exists only a partial correlation between the chemical content and genetic profiling data among the accessions under study.