A recombinant mimetics of the HIV-1 gp41 prehairpin fusion intermediate fused with human IgG Fc fragment elicits neutralizing antibody response in the vaccinated mice

HIV-1 gp41 prehairpin fusion intermediate (PFI) composed of three N-terminal heptad repeats (NHR) plays a crucial role in viral fusion and entry and represents an attractive target for anti-HIV therapeutics (e.g., enfuvirtide) and vaccines. In present study, we constructed and expressed two recombinant gp41 PFI mimetics, designated N46Fd and N46FdFc. N46Fd consists of N46 (residues 536-581) in gp41 NHR and foldon (Fd), a trimerization motif. N46FdFc is composed of N46Fd fused with human IgG Fc fragment as an immunoenhancer. We immunized mice with N46 peptide, N46Fd and N46FdFc, respectively, and found that only N46FdFc elicited neutralizing antibody response in mice against infection by HIV-1 strains IIIB (clade B, X4), 92US657 (clade B, R5), and 94UG103 (clade A, X4R5). Anti-N46FdFc antibodies inhibited PIE7 binding to PFI, blocked gp41 six-helix bundle formation, and suppressed HIV-1 mediated cell-cell fusion. These findings provide an important clue for developing recombinant gp41 PFI mimetics-based HIV vaccines.     

Cyclic adenosine monophosphate induces plasminogen activator inhibitor-1 expression in human mast cells

Plaminogen activator inhibitor-1 (PAI-1), the key physiological inhibitor of the plasmin fibrinolytic system, plays important roles in the pathogenesis of asthma. Mast cells (MCs) are crucial effector cells and a major source of PAI-1 for asthma. Cyclic adenosine monophosphate (cAMP) is the important regulator of MCs; however, its effects on PAI-1 expression in MCs remain unknown. We reported cAMP/protein kinase A pathway positively regulates PAI-1 expression through cAMP-response element binding protein binding to hypoxia response element-1 at −158 to −153 bp of human PAI-1 promoter in human MCs. Moreover, cAMP synergistically augments PAI-1 expression with ionomycin- or IgE receptor cross-linking-mediated stimulation.     

Phosphorylation of the protein kinase A catalytic subunit is induced by cyclic AMP deficiency and physiological stresses in the fission yeast, Schizosaccharomyces pombe

In the fission yeast, Schizosaccharomyces pombe, cyclic AMP (cAMP)-dependent protein kinase (PKA) is not essential for viability under normal culturing conditions, making this organism attractive for investigating mechanisms of PKA regulation. Here we show that S. pombe cells carrying a deletion in the adenylate cyclase gene, cyr1, express markedly higher levels of the PKA catalytic subunit, Pka1, than wild type cells. Significantly, in cyr1Δ cells, but not wild type cells, a substantial proportion of Pka1 protein is hyperphosphorylated. Pka1 hyperphosphorylation is strongly induced in cyr1Δ cells, and to varying degrees in wild type cells, by both glucose starvation and stationary phase stresses, which are associated with reduced cAMP-dependent PKA activity, and by KCl stress, the cellular adaptation to which is dependent on PKA activity. Interestingly, hyperphosphorylation of Pka1 was not detected in either cyr1⁺ or cyr1Δ S. pombe strains carrying a deletion in the PKA regulatory subunit gene, cgs1, under any of the tested conditions. Our results demonstrate the existence of a cAMP-independent mechanism of PKA catalytic subunit phosphorylation, which we propose could serve as a mechanism for inducing or maintaining specific PKA functions under conditions in which its cAMP-dependent activity is downregulated.     

Whole cell‐catalyzed transesterification of waste vegetable oil

Enzymatic transesterification of waste cooking oil, comprising fats, oil and grease (FOG), to produce fatty acid methyl esters (FAME) i.e. biodiesel, was investigated using a novel strain of the fungus Aspergillus niger, immobilized as a whole‐cell biocatalyst. Response surface methodology (RSM), with a five‐level‐three‐factor central composite rotatable design, was used to optimize the reaction and analyze the relationship of reaction variables and their coinfluent on the response i.e. FAME yield. Independent variables that affect the transesterification reaction include temperature, feedstock water content and enzyme amount. Using RSM, a second‐order polynomial equation was derived for FAME yield using multiple regression analysis. The second‐order polynomial regression model was highly significant (P<0.001) in predicting the actual relationship between the response and the variables, where a linear relationship was apparent between observed and predicted values (R²=0.9651). In addition, the predicted determination coefficient q² i.e. 0.7723, also proved that the model has a high predictive ability. The validation experiments, under optimized conditions, showed that the predicted value of maximum FAME yield (i.e. 91.3%) was in close agreement with the experimental value (i.e. 91.8%).     

Malaria: an evaluation of the current state of research on pathogenesis and antimalarial drugs

This brief review provides an overview of the pathogenesis of malaria, the human immune response to malaria, current methodology for malaria diagnosis, and current antimalarial drug regimens. The review also provides a critical evaluation of the research directions in the areas of drug design and vaccine design.     

Paraquat-induced oxidative stress response during amphibian early embryonic development

In addition to the endogenous production of reactive oxygen species (ROS) as a result of normal development, amphibian external development often forces embryos to deal with oxidative stress-producing agents present in the environment. Embryos should therefore develop protective systems to reduce ROS toxicity and achieve successful development. The present work was aimed to characterize the effects produced by the widespread-used ROS-generator pesticide Paraquat during early embryonic development in the toad Chaunus arenarum, as well as to get insights into the defense response elicited by amphibian embryos. The approach consisted in generating a sharp and brief oxidative stress condition early during embryonic development to stimulate the cellular mechanisms involved in ROS-antioxidant response. Results revealed that Paraquat-treatment reduced the ability of embryos to develop normally, leading to arrests of development and severe malformations such as tail abnormalities, abdominal edema, reduced head development and curved dorsal structures. Although Paraquat effects were morphologically evident from gastrula stage on, alterations such as chromatin condensation were observed even at blastula stage by histological examinations. Regarding detoxifying enzymes, a significant induction of Mn-superoxide dismutase activity was detected at stages beyond gastrula in embryos surviving Paraquat treatment, suggesting a major role of this enzyme in the antioxidant response during early embryonic development.     

Nitrofen induces apoptosis independently of retinaldehyde dehydrogenase (RALDH) inhibition

BACKGROUND: Nitrofen is a diphenyl ether that induces congenital diaphragmatic hernia (CDH) in rodents. Its mechanism of action has been hypothesized as inhibition of the retinaldehyde dehydrogenase (RALDH) enzymes with consequent reduced retinoic acid signaling. METHODS: To determine if nitrofen inhibits RALDH enzymes, a reporter gene construct containing a retinoic acid response‐element (RARE) was transfected into HEK‐293 cells and treated with varying concentrations of nitrofen in the presence of retinaldehyde (retinal). Cell death was characterized by caspace‐cleavage microplate assays and terminal deoxynucleotidyl transferase dUTP nick end‐labeling (TUNEL) assays. Ex vivo analyses of cell viability were characterized in fetal rat lung explants using Live/Dead staining. Cell proliferation and apoptosis were assessed using fluorescent immunohistochemistry with phosphorylated histone and activated caspase antibodies on explant tissues. Nile red staining was used to identify intracellular lipid droplets. RESULTS: Nitrofen‐induced dose‐dependent declines in RARE‐reporter gene expression. However, similar reductions were observed in control‐reporter constructs suggesting that nitrofen compromised cell viability. These observed declines in cell viability resulted from increased cell death and were confirmed using two independent assays. Ex vivo analyses showed that mesenchymal cells were particularly susceptible to nitrofen‐induced apoptosis while epithelial cell proliferation was dramatically reduced in fetal rat lung explants. Nitrofen treatment of these explants also showed profound lipid redistribution, primarily to phagocytes. CONCLUSIONS: The observed declines in nitrofen‐associated retinoic acid signaling appear to be independent of RALDH inhibition and likely result from nitrofen induced cell death/apoptosis. These results support a cellular apoptotic mechanism of CDH development, independent of RALDH inhibition. Birth Defects Res (Part B) 89:223–232, 2010. © 2010 Wiley‐Liss, Inc.     

Recent findings in pulsed light disinfection

Nonthermal disinfection technologies are gaining increasing interest in the field of minimally processed food in order to improve the microbial safety or to extend the shelf life. Especially fresh‐cut produce or meat and fish products are vulnerable to microbial spoilage, but, due to their sensitivity, they require gentle preservation measures. The application of intense light pulses of a broad spectral range comprising ultraviolet, visible and near infrared irradiation is currently investigated as a potentially suitable technology to reduce microbial loads on different food surfaces or in beverages. Considerable research has been performed within the last two decades, in which the impact of various process parameters or microbial responses as well as the suitability of pulsed light (PL) for food applications has been examined. This review summarizes the outcome of the latest studies dealing with the treatment of various foods including the impact of PL on food properties as well as recent findings about the microbicidal action and relevant process parameters.     

The feeding rates ofPleurobrachia (ctenophora) andSagitta (chaetognatha), with notes on the potential seasonal role of planktonic predators in the dynamics of north sea zooplankton communities

The zooplankton off the north-east coast of England has been the subject of a number of studies focusing on its productivity. It has also been shown to be representative of the zooplankton of much of the western North Sea. The community contains a number of predatory species, three of which are widely described as ‘voracious’, the ctenophorePleurobrachia pileus, the chaetognathSagitta elegans and the hyperiid amphipodThemisto compressa (≡ Parathemisto gaudichaudi). This study investigates the role of these planktonic predators in this community, with special reference to the seasonal changes in predation pressure. The functional response ofPleurobrachia pileus feeding onAcartia was determined from laboratory experiments. It was found to be linear at prey densities typical of UK coastal waters, although the linear relationship appeared to break down at low and high prey densities. Feeding rate data forSagitta elegans were obtained from gut content analysis and published laboratory derived estimates of digestion time. Of the 1,789 individuals examined 198 (11.1%) had food remains in the gut. A linear relationship betweenSagitta length and prey size was established and the daily feeding rate ofSagitta elegans was estimated to be 0.4 prey items d⁻¹ ind⁻¹. For comparative purposes, the proportion of the copepod standing stock removed bySagitta elegans, Pleurobrachia pileus andThemisto gaudichaudi was estimated for each month of the year. From this model it was shown thatThemisto applied the most predation pressure, andSagitta elegans applied the least predation pressure of the three planktonic predators considered. The impact ofPleurobrachia will be to a large extent offset due to its peak of seasonal abundance coinciding with the zooplankton peak in the summer.