In vitro effect of the potent poly(ADP-ribose) polymerase (PARP) inhibitor INO-1001 alone and in combination with aspirin, eptifibatide, tirofiban, enoxaparin or alteplase on haemostatic parameters

It has been shown that PARP inhibition is protective in several models of ischemia-reperfusion injury including cardiac, cerebral and renal ones. Due to their ability to reduce myocardial necrosis and to improve myocardial function PARP inhibitors emerged as candidates for treating various cardiovascular diseases including acute myocardial ischemia. Since the pathophysiology of acute ischemic cardiac diseases involves haemostatic impairment and the therapeutic regimen includes antithrombotic drugs, we investigated the effect of the potent poly(ADP-ribose) polymerase (PARP) inhibitor INO-1001 alone and in combination with platelet aggregation inhibitors (aspirin, eptifibatide and tirofiban), unfractionated heparin, low molecular weight heparin (enoxaparin) or the recombinant fibrinolytic drug (alteplase), on various haemostatic parameters in vitro. ADP- and epinephrine-induced platelet aggregation was evaluated by optical aggregometry in the presence or absence of different concentrations of INO-1001, in combination with aspirin, tirofiban, eptifibatide or saline on ten healthy volunteers' platelet rich plasma (PRP). Activated partial thromboplastin time, Anti-Xa activity and euglobulin lysis time were determined in the presence or absence of different concentrations of INO-1001, in combination with sodium heparin, enoxaparin or alteplase, respectively. INO-1001, on its own does not affect the measured platelet, and haemostatic functions, i.e. does not reduce the respective anti-platelet, anti-coagulant and thrombolytic activity of therapeutically relevant concentrations of aspirin, tirofiban, eptifibatide, enoxaparin and alteplase in vitro. INO-1001 enhanced the effects of heparins above therapeutic ranges; the magnitude of this effect was negligible. Consequently, the PARP inhibitor INO-1001 can be safely applied together with the drugs tested.     

Survey of antibiotic resistance in an integrated marine aquaculture system under oxolinic acid treatment

The consequences of antibiotic use in aquatic integrated systems, which are based on trophic interactions between different cultured organisms and physical continuity through water, need to be examined. In this study, fish reared in a prototype marine integrated system were given an oxolinic acid treatment, during and after which the level of resistance to this quinolone antibiotic was monitored among vibrio populations from the digestive tracts of treated fish, co-cultured bivalves and sediments that were isolated on thiosulfate-citrate-bile-sucrose. Oxolinic acid minimum inhibitory concentration distributions obtained from replica plating of thiosulfate-citrate-bile-sucrose plates indicated that a selection towards oxolinic acid resistance had occurred in the intestines of fish under treatment. In contrast, and despite oxolinic acid concentrations higher than minimum inhibitory concentrations of susceptible bacteria, no clear evolution of resistance levels was detected either in bivalves or in sediments.     

Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of sugars, alcohols, and organic acids with transcriptional microarrays

Microorganisms respond to environmental changes by reprogramming their metabolism primarily through altered patterns of gene expression. DNA microarrays provide a tool for exploiting microorganisms as living sensors of their environment. The potential of DNA microarrays to reflect availability of nutrient components during fermentations on complex media was examined by monitoring global gene expression throughout batch cultivation of Escherichia coli MG1655 on Luria-Bertani (LB) medium. Gene expression profiles group into pathways that clearly demonstrate the metabolic changes occurring in the course of fermentation. Functional analysis of the gene expression related to metabolism of sugars, alcohols, and organic acids revealed that E. coli growing on LB medium switches from a sequential mode of substrate utilization to the simultaneous one in the course of the growth. Maltose and maltodextrins are the first of these substrates to support growth. Utilization of these nutrients associated with the highest growth rate of the culture was followed by simultaneous induction of enzymes involved in assimilation of a large group of other carbon sources including D-mannose, melibiose, D-galactose, L-fucose, L-rhamnose, D-mannitol, amino sugars, trehalose, L-arabinose, glycerol, and lactate. Availability of these nutrients to the cells was monitored by induction of corresponding transport and/or catabolic systems specific for each of the compounds.     

Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of amino acids, peptides, and nucleotides with transcriptional microarrays

Analysis of gene expression data related to assimilation and biosynthesis of nitrogen-containing compounds amino acids, peptides, and nucleotides was used to monitor availability of these nutrients to Escherichia coli MG1655 growing on Luria-Bertani medium. The data indicate that free amino acids and nucleotides only transiently support the nitrogen requirement for growth and are no longer available by 3.5 h of fermentation. The resulting shortage of available nitrogen sources induces the Ntr response, which involves induction of the glnALG, glnK-amtB, dppABCDF, and oppABCDF operons as well as the genes coding for outer membrane proteins, porins OmpA and OmpC, and proteases OmpP and OmpT. The increased uptake of peptides facilitated by the products of dppABCDF, oppABCDF, ompA, ompC, ompP, and ompT alleviates nitrogen limitation of the growth.     

Human apoB overexpression and a high-cholesterol diet differently modify the brain APP metabolism in the transgenic mouse model of atherosclerosis

Epidemiological and biochemical data suggest a link between the cholesterol metabolism, the amyloid precursor protein (APP) processing and the increased cerebral beta-amyloid (Abeta) deposition in Alzheimer's disease (AD). The individual and combined effects of a high-cholesterol (HC) diet and the overexpression of the human apoB-100 gene were therefore examined on the cerebral expression and processing of APP in homozygous apoB-100 transgenic mice [Tg (apoB(+/+))], a validated model of atherosclerosis. When fed with 2% cholesterol for 17 weeks, only the wild-type mice exhibited significantly increased APP695 (123%) and APP770 (138%) mRNA levels in the cortex. The HC diet-induced hypercholesterolemia significantly increased the APP isoform levels in the membrane-bound fraction, not only in the wild-type animals (114%), but also in the Tg apoB(+/+) group (171%). The overexpression of human apoB-100 gene by the liver alone reduced the brain APP isoform levels in the membrane-bound fraction (78%), whereas the levels were increased by the combined effect of HC and the overexpression of the human apoB-100 gene (134%). The protein kinase C and beta-secretase protein levels were not altered by the individual or combined effects of these two factors. Our data indicate that the two atherogenic factors, the HC diet and the overexpression of the human apoB-100 gene by the liver, could exert different effects on the processing and expression of APP in the mice brain.     

Fibronectin quantification without antibodies: a bioassay for the detection of the gelatin-captured macromolecule

We have developed a new cell-adhesion-bioassay (CAA) for the quantitative determination of fibronectin in biological fluids. The assay is based on two particular properties of fibronectin: it specifically binds to gelatin with high affinity and simultaneously it can anchor to different surface molecules of a cell. First fibronectin, derived from very different biological fluids, is purified in situ, within the wells of the microtiter plates applied for the assay, using solid surface bound gelatin. After capturing the macromolecule, it is quantified based on its cell adhesive properties. In contrast to ELISA the CAA does not require specific antibodies, and as the Jurkat cells used as indicator cells, seem to recognize fibronectin from different species equally; species specificity of the reagent plays smaller, perhaps negligible, role in the determination of the amount of the macromolecule. The CAA method may not replace fibronectin specific ELISA-s, but using its principle, improved applications, for example a capture EIA for determining fibronectin can easily be envisioned and CAA may serve as a viable alternative for EIA-s when specific antibodies are not available or when relative measurement of not only the soluble but cell surface associated fibronectin is necessary.     

Heterogeneity of non-serotypable Campylobacter jejuni isolates

Several phenotypic and genotypic methods are currently used to identify subtypes of Campylobacter jejuni isolates. Of the phenotypic methods one, i.e. serotyping based on the heat stable antigen, is often hindered by the relatively large number of un-typable (NT) strains. Little is known, however, about the heterogeneity of the group formed by these NT strains. Therefore we serotyped 92 Hungarian, non-outbreak C. jejuni isolates and subjected the 28 NT strains to molecular analysis using PCR-RFLP of the flaA gene and to pulsed-field gel electrophoresis. With both methods the NT strains were classified into several molecular types (17 and 25, respectively), while the number of subgroups based on the results of the two techniques combined was twenty-six. These results indicate that the NT group of strains is extremely heterogeneous in Hungary, and the epidemiological connection between two NT isolates cannot be established or excluded without the use of molecular typing techniques.     

Plasmodium vivax dihydrofolate reductase as a target of sulpha drugs

Sulpha drugs act as competitive inhibitors of p-amino benzoic acid, an intermediate in the de novo folate pathway. Dihydropteroate synthase condenses sulpha drugs into sulpha-dihydropteroate (sulpha-DHP), which competes with dihydrofolate, the dihydrofolate reductase (DHFR) substrate. This designates DHFR as a possible target of sulpha-DHP. We suggest here that Plasmodium vivax DHFR is indeed the in vivo target of sulpha drugs. The wild-type DHFR expressed in Saccharomyces cerevisiae leads to cell growth inhibition, while sensitivity to the drug is exacerbated in the mutants. Contrary to what is observed with sulphanilamide, methotrexate is less effective on P. vivax-DHFR mutants than on wild-type mutant.     

Combining a polar resin and a pseudo-proline to optimize the solid-phase synthesis of a 'difficult sequence'.

This paper describes the optimization of a synthesis of a difficult sequence related to a 12-mer sequence of a Pan DR epitope (PADRE). Elongation was followed by on-line monitoring of the N(alpha)-Fmoc removal adapted for the batch methodology. Studying the intrinsic factors related to the peptide-resin, such as substitution level, resin nature and backbone protecting group, has led to an increase in the elongation yield and purity of the crude peptide. Optimal elongation was obtained by combining a polar resin such as PEGA and a pseudo-proline as the backbone protecting group.