Lucigenin chemiluminescence assay as an adjunctive tool for assessment of various stages of inflammation: A study of quiescent inflammatory cells

A simple, fast, precise and biologically relevant toxicity assay for screening cytotoxicity of minerals would have distinct advantages due to its cost benefits and relative savings in time. Furthermore, a bioassay to differentiate acute and chronic in vivo pulmonary reactions could have potential value as predictors of fibrogenicity and pathogenicity. In this study we examined the potential use of lucigenin as a probe to evaluate the correlation between chemiluminescence (CL) generated by alveolar macrophages with the known cytotoxicity and patho genicity by conventional bioassays. In this study, we used small doses of dust (20 microg) to minimize cellular overload and to maintain homeostasis. Crystalline silica a highly fibrogenic dust was used as positive control and results are compared with those for bentonite, kaolin and talc. Among the three minerals compared with silica, bentonite was more reactive (27%) in CL assay and declined sharply compared to other minerals. This sudden decline in bentonite CL is caused by cytotoxicity leading to cell death. CL-induced by talc was comparable to silica and declines slowly. Kaolin on the other hand produced relatively a weaker (25%) CL compared to silica. Our data using relatively low doses of dust suggest that the CL assay may have a better predictive value in cytotoxicity evaluations compared to conventional toxicity assays.     

Allantoin,the oxidation product of uric acid is present in chicken and turkey plasma

Urate oxidase is not present in birds yet allantoin, a product of this enzyme, has been measured in birds. Studies were designed to compare the concentrations of plasma purine derivatives in chickens and turkeys fed inosine-supplemented diets. The first study consisted of 12 male chicks that were fed diets supplemented with 0.6 mol inosine or hypoxanthine per kilogram diet from 3- to 6-week-old. Study 2 consisted of 12 turkey poults (toms) fed inosine-supplemented diets (0.7 mol/kg) from 6- to 8-week-old. Plasma allantoin and oxypurines concentrations were measured weekly using high performance liquid chromatography. Plasma uric acid (PUA) in chickens fed inosine-supplemented diets increased from 0.31 to 1.34 mM (P<0.05) at the end of week 2. In turkeys, those fed control diet had 0.17 mM PUA concentration compared to 0.3 mM in those fed the inosine diet at week 2 (P<0.05). Allantoin concentration increased in chickens from week 1 to 2 while a decrease was observed in turkeys (P<0.005) for both treatments. These data show that allantoin is present in turkey and chicken plasma. The presence of allantoin in avian plasma is consistent with uric acid acting as an antioxidant in these species.     

The methyl donor S-Adenosylmethionine inhibits active demethylation of DNA: a candidate novel mechanism for the pharmacological effects of S-Adenosylmethionine

S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA.     

Camptothecin-induced apoptosis in non-small cell lung cancer is independent of cyclooxygenase expression

Recent observations show a positive correlation between the expression of cyclooxygenase (COX), especially COX-2), and cancer development. Here we tested the hypothesis that expression of COX-2 could influence apoptosis in lung cancer cell lines. To address this question, we determined the effects of camptothecin-induced apoptosis on three lung cancer cell lines which over express COX-1 (CORL23), COX-2 (MOR-P) and neither isoform (H-460), and determine if these effects were prostaglandin mediated. We also compared the effects of non-selective and isoenzyme selective COX-2 inhibitors on camptothecin-induced apoptosis in these three cell lines. Camptothecin induced apoptosis in all three cell lines independently of COX-1 or COX-2 expression. Indomethacin, a non-selective COX inhibitor and NS398, a selective COX-2 inhibitor had no effect on camptothecin-induced apoptosis at concentrations that abolished prostaglandin production. In conclusion, these finding suggest that the COX pathway is not involved in camptothecin-induced apoptosis of non-small cell lung cancer cell lines.     

Digestive proteases of blood-feeding nematodes

Blood-feeding parasites employ a battery of proteolytic enzymes to digest the contents of their bloodmeal. Host haemoglobin is a major substrate for these proteases and, therefore, a driving force in the evolution of parasite-derived proteolytic enzymes. This review will focus on the digestive proteases of the major blood-feeding nematodes - hookworms (Ancylostoma spp. and Necator americanus) and the ruminant parasite, Haemonchus contortus - but also compares and contrasts these proteases with recent findings from schistosomes and malaria parasites. Haematophagous nematodes express proteases of different mechanistic classes in their intestines, many of which have proven or putative roles in degradation of haemoglobin and other proteins involved in nutrition. Moreover, the fine specificity of the relationships between digestive proteases and their substrate proteins provides a new molecular paradigm for understanding host-parasite co-evolution. Numerous laboratories are actively investigating these molecules as antiparasite vaccine targets.     

Progress in the development of a recombinant vaccine for human hookworm disease: the Human Hookworm Vaccine Initiative

Hookworm infection is one of the most important parasitic infections of humans, possibly outranked only by malaria as a cause of misery and suffering. An estimated 1.2 billion people are infected with hookworm in areas of rural poverty in the tropics and subtropics. Epidemiological data collected in China, Southeast Asia and Brazil indicate that, unlike other soil-transmitted helminth infections, the highest hookworm burdens typically occur in adult populations, including the elderly. Emerging data on the host cellular immune responses of chronically infected populations suggest that hookworms induce a state of host anergy and immune hyporesponsiveness. These features account for the high rates of hookworm reinfection following treatment with anthelminthic drugs and therefore, the failure of anthelminthics to control hookworm. Despite the inability of the human host to develop naturally acquired immune responses to hookworm, there is evidence for the feasibility of developing a vaccine based on the successes of immunising laboratory animals with either attenuated larval vaccines or antigens extracted from the alimentary canal of adult blood-feeding stages. The major antigens associated with each of these larval and adult hookworm vaccines have been cloned and expressed in prokaryotic and eukaryotic systems. However, only eukaryotic expression systems (e.g., yeast, baculovirus, and insect cells) produce recombinant proteins that immunologically resemble the corresponding native antigens. A challenge for vaccinologists is to formulate selected eukaryotic antigens with appropriate adjuvants in order to elicit high antibody titres. In some cases, antigen-specific IgE responses are required to mediate protection. Another challenge will be to produce anti-hookworm vaccine antigens at high yield low cost suitable for immunising large impoverished populations living in the developing nations of the tropics.     

Arabinogalactan proteins in embryogenic and non-embryogenic callus cultures of Euphorbia pulcherrima

The arabinogalactan protein (AGP) fractions of embryogenic and non-embryogenic callus lines of Euphorbia pulcherrima Willd. ex. Klotzsch were analysed over a cultivation period of 9 weeks using the β -glucosyl Yariv reagent and an anti-AGP antibody (LM2). The amount of AGPs detected with the Yariv reagent increased in embryogenic cultures during the development of somatic embryos. The embryogenic and non-embryogenic callus contained different sets of AGPs characterized with the Yariv reagent and the LM2 monoclonal antibody. AGPs recognized by LM2 are localized primarily in the protodermal cells of globular somatic embryos. The development of somatic embryos of E. pulcherrima appears to be associated with the presence of particular AGPs.     

Spatial regulation of pectic polysaccharides in relation to pit fields in cell walls of tomato fruit pericarp

Scanning electron microscopic examination of intact tomato (Lycopersicon esculentum) pericarp and isolated pericarp cell walls revealed pit fields and associated radiating ridges on the inner face of cell walls. In regions of the cell wall away from pit fields, equivalent ridges occurred in parallel arrays. Treatment of isolated cell walls with a calcium chelator resulted in the loss of these ridges, indicating that they contain homogalacturonan-rich pectic polysaccharides. Immunolabeling procedures confirmed that pit fields and associated radiating ridges contained homogalacturonan. Epitopes of the side chains of pectic polysaccharides were not located in the same regions as homogalacturonan and were spatially regulated in relation to pit fields. A (1-->4)-beta-galactan epitope was absent from cell walls in regions of pit fields. A (1-->5)-alpha-arabinan epitope occurred most abundantly at the inner face of cell walls in regions surrounding the pit fields.     

Inhibition of the SHV-1 beta-lactamase by sulfones: crystallographic observation of two reaction intermediates with tazobactam

Two species resulting from the reaction of the SHV-1 class A beta-lactamase with the sulfone inhibitor tazobactam have been trapped at 100 K and mapped by X-ray crystallography at 2.0 A resolution. An acyclic form of tazobactam is covalently bonded to the catalytic Ser70 side chain, and a second species, a five-atom vinyl carboxylic acid fragment of tazobactam, is bonded to Ser130. It is proposed that the electron density map of the crystal is a composite picture of two complexes, each with only a single bound species. It is estimated that the two complexes exist in the crystal in approximately equal populations. Results are discussed in relation to the mechanism-based inhibition of class A beta-lactamases by the similar inhibitors sulbactam and clavulanic acid.