A liquid chromatography-tandem mass spectrometry method for measurement of N-modified phosphatidylethanolamines

N-Acyl phosphatidylethanolamines (NAPEs) are synthesised in response to stress in a variety of organisms from bacteria to humans. More recently, nonenzymatic modification of the ethanolamine headgroup of phosphatidylethanolamine (PE) by various aldehydes, including levuglandins/isoketals (which are γ-ketoaldehydes [γKAs] derived from arachidonic acid), has also been demonstrated. The levels of these various N-modified PEs formed during stress and their biological significance remain to be fully characterized. Such studies require an accurate, facile, and cost-effective method for quantifying N-modified PEs. Previously, NAPE and some of the nonenzymatically N-modified PE species have been quantified by mass spectrometry after hydrolysis to their constituent N-acylethanolamine by enzymatic hydrolysis, most typically with Streptomyces chromofuscus phospholipase D. However, enzymatic hydrolysis is not cost-effective for routine analysis of a large number of samples, and hydrolytic efficiency may vary for different N-modified PEs, making quantitation more difficult. Therefore, we sought a robust and inexpensive chemical hydrolysis approach. Methylamine (CH₃NH₂)-mediated deacylation has previously been used in headgroup analysis of phosphatidylinositol phosphates. Therefore, we developed an accurate assay for NAPEs and γKA-PEs using CH₃NH₂-mediated deacylation and quantitation of the resulting glycerophospho-N-modified ethanolamines by liquid chromatography-tandem mass spectrometry.     

Characterization of an anion-exchange porous polypropylene hollow fiber membrane for immobilization of ABL lipase

Hollow fiber membrane offers the advantage to integrate catalytic conversion, product separation and catalyst recovery into a single separation process compared to conventional systems. Polypropylene (PP) hollow fiber membrane is a chemically inert and stable membrane with high potential for enzyme immobilization. The surface properties of polypropylene have been modified by radiation induced graft polymerization. Samples were prepared by grafting of glycidylmethacrylate (GMA) using gamma radiation, at different monomer concentrations and irradiation dose. The resulting epoxy was converted into a diethylamino group as an anion-exchange medium to bind the lipase molecules. Surface properties of the grafted and amine treated samples were characterized using atomic force microscopy (AFM), scanning electron microscopy (SEM) and contact angle measurements. AFM revealed higher surface roughness for grafted samples than that of virgin polymer. SEM micrographs illustrated that the porous network was retained at high degree of grafting. Contact angle measurements showed excellent wetting properties with water for the grafted and amine treated membranes. Thermal properties were studied using differential scanning calorimeter (DSC) and thermogravimetic analysis (TGA). It was observed that grafting occurred mainly in the amorphous region of the membranes. Activity and operational stability of ABL lipase, isolated from Arthobacter sp. were assayed after immobilizing it to the modified PP hollow fiber. Immobilized lipase retained 20U/g activity after ten hydrolysis cycles and 68% residual activity after 12 weeks of storage.     

Intraspecific variation in group size in the blackbuck antelope: the roles of habitat structure and forage at different spatial scales

The main ecological factors that are hypothesized to explain the striking variation in the size of social groups among large herbivores are habitat structure, predation, and forage abundance and distribution; however, their relative roles in wild populations are not well understood. I combined analyses of ecological correlates of spatial variation in group size with analyses of individual behaviour in groups of different sizes to investigate factors maintaining variation in group size in an Indian antelope, the blackbuck Antilope cervicapra. I measured group size, habitat structure, forage, and the occurrence of predators in ten blackbuck populations, and, at a smaller spatial scale, within an intensively studied population. To examine the processes by which these ecological factors influence group size, I used behavioural observations and an experiment to estimate the shape of the relationship between group size and potential costs and benefits to individuals. Group size varied extensively both among and within populations. Analyses of spatial variation in group size suggested that both forage and habitat structure influence group size: large-scale, among-population variation in group size was primarily related to habitat structure, while small-scale, within-population variation was most closely related to forage abundance. Analyses of individual behaviour suggested that larger groups incur greater travel costs while foraging. However, individuals in larger groups appeared to experience greater benefits, namely the earlier detection of a “predator”, a reduction in vigilance, and an increase in the time spent feeding. Overall, these findings suggest that individuals in groups experience a trade-off between predation-related benefits and costs arising from feeding competition. Habitat structure and forage likely influence the nature of this trade-off; thus, variation in these ecological factors may maintain variation in group size. The role of predation pressure and other factors in explaining the remaining variation needs further exploration. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Fas-associated death domain-containing protein-mediated antiviral innate immune signaling involves the regulation of Irf7

The induction of type I (alphabeta) IFN following virus infection is necessary for the stimulation of effective antiviral host defense. In fibroblasts, a subset of primary genes (including those encoding IFN-beta and IFN-alpha4) are induced directly by intracellular dsRNA generated by the virus during its replication. These primary type I IFNs induce expression of IFN regulatory factor (IRF)-7, required for production of a second cascade of IFN-alpha subtypes and the further establishment of a complete antiviral state. Previously, we had reported on a role for Fas-associated death domain-containing protein (FADD) in the control of TLR-independent innate immune responses to virus infection. Our data in this study demonstrate that FADD is not only required for efficient primary gene induction, but is also essential for induction of Irf7 and effective expression of secondary IFN-alphas and other antiviral genes. Ectopic overexpression of IRF-7 partially rescued dsRNA responsiveness and IFN-alpha production, and a constitutively active variant of IRF-7 displayed normal activity in Fadd(-/-) murine embryonic fibroblasts. MC159, a FADD-interacting viral protein encoded by the molluscum contagiosum poxvirus was found to inhibit dsRNA-activated signaling events upstream of IRF-7. These data indicate that FADD's antiviral activity involves regulation of IRF-7-dependent production of IFN-alpha subtypes and consequent induction of secondary antiviral genes.     

Theaflavins induced apoptosis of LNCaP cells is mediated through induction of p53, down-regulation of NF-kappa B and mitogen-activated protein kinases pathways

Prostate cancer (PCA), the most frequently diagnosed malignancy in men, represents an excellent candidate disease for chemoprevention studies because of its particularly long latency period, high rate of mortality and morbidity. Infusion of black tea and its polyphenolic constituents have been shown to possess antineoplastic effects in androgen dependent PCA in both in vivo and in vitro models including transgenic animals. In the present study, we report that black tea polyphenol, Theaflavins (TF)-induced apoptosis in human prostate carcinoma, LNCaP cells is mediated via modulation of two related pathways: up-regulation of p53 and down-regulation of NF-kappa B activity, causing a change in the ratio of pro-and antiapoptotic proteins leading to apoptosis. The altered expression of Bcl-2 family member proteins triggered the release of cytochrome-C and activation of initiator capsase 9 followed by activation of effector caspase 3. Furthermore, TF also affected the protein expression of mitogen activated protein kinases (MAPK) pathways. Our results demonstrated that TF treatment resulted in down-regulation of phospho-extracellular signal-regulated protein kinase (Erk1/2) and phospho-p38 MAPK expressions. We conclude that TF induces apoptosis in LNCaP cells by shifting the balance between pro-and antiapoptotic proteins and down-regulation of cell survival pathways leading to apoptosis. Further extending this work, we also showed that TF induces apoptosis in androgen independent PCA cell line, PC-3 through caspases and MAPKs mediated pathways. Thus, effect of TF on PCA cell lines seems to be irrespective of their androgen status.     

Phosphopantetheine adenylyltransferase from Escherichia coli: investigation of the kinetic mechanism and role in regulation of coenzyme A biosynthesis

Phosphopantetheine adenylyltransferase (PPAT) from Escherichia coli is an essential hexameric enzyme that catalyzes the penultimate step in coenzyme A (CoA) biosynthesis and is a target for antibacterial drug discovery. The enzyme utilizes Mg-ATP and phosphopantetheine (PhP) to generate dephospho-CoA (dPCoA) and pyrophosphate. When overexpressed in E. coli, PPAT copurifies with tightly bound CoA, suggesting a feedback inhibitory role for this cofactor. Using an enzyme-coupled assay for the forward-direction reaction (dPCoA-generating) and isothermal titration calorimetry, we investigated the steady-state kinetics and ligand binding properties of PPAT. All substrates and products bind the free enzyme, and product inhibition studies are consistent with a random bi-bi kinetic mechanism. CoA inhibits PPAT and is competitive with ATP, PhP, and dPCoA. Previously published structures of PPAT crystallized at pH 5.0 show half-the-sites reactivity for PhP and dPCoA and full occupancy by ATP and CoA. Ligand-binding studies at pH 8.0 show that ATP, PhP, dPCoA, and CoA occupy all six monomers of the PPAT hexamer, although CoA exhibits two thermodynamically distinct binding modes. These results suggest that the half-the-sites reactivity observed in PPAT crystal structures may be pH dependent. In light of previous studies on the regulation of CoA biosynthesis, the PPAT kinetic and ligand binding data suggest that intracellular PhP concentrations modulate the distribution of PPAT monomers between high- and low-affinity CoA binding modes. This model is consistent with PPAT serving as a “backup” regulator of pathway flux relative to pantothenate kinase.