Anion inhibition studies of the α-carbonic anhydrases from Neisseria gonorrhoeae

The bacterial pathogen Neisseria gonorrhoeae encodes for an α-class carbonic anhydrase (CA, EC 4.2.1.1), NgCA, which was investigated for its inhibition with a series of inorganic and organic anions. Perchlorate and hexafluorophosphate did not significantly inhibit NgCA CO₂ hydrase activity, whereas the halides, azide, bicarbonate, carbonate, stannate, perosmate, diphosphate, divanadate, perruthenate, and trifluoromethanesulfonate showed inhibition constants in the range of 1.3–9.6 mM. Anions/small molecules such as cyanate, thiocyanate, nitrite, nitrate, bisulphite, sulphate, hydrogensulfide, phenylboronic acid, phenylarsonic acid, selenate, tellurate, tetraborate, perrhenate, peroxydisulfate, selenocyanate, iminodisulfonate, and fluorosulfonate showed K_Is in the range of 0.15–1.0 mM. The most effective inhibitors detected in this study were sulfamide, sulfamate, trithiocarbonate and N,N-diethyldithiocarbamate, which had K_Is in the range of 5.1–88 µM. These last compounds incorporating the CS₂^- zinc-binding group may be used as leads for developing even more effective NgCA inhibitors in addition to the aromatic/heterocyclic sulphonamides, as this enzyme was recently validated as an antibacterial drug target for obtaining novel antigonococcal agents     

Syngeneic tumor immunizations produce Qa antibodies. Discovery of a new Qa antigen,Qa-6

BALB/cBy (Qa-2^–) mice injected with the syngeneic tumor, ORA I-a (Qa-2⁺), produced antibodies to Qa-2 and a newly discovered antigen, Qa-6. Specific antisera against Qa-6, in the presence of complement, lyses approximately 40% of lymph-node lymphocytes and splenocytes. Strain distribution analyses indicate that Qa-6 is specified by a gene within the TL subregion of the major histocompatibility complex. Thus, Qa-6 is the third member of the Qa/TL subset of cell surface antigens which is anomalously expressed on certain tumor cells. This finding suggests that the Qa and TL molecules may have a unique, functional role on the cell surface.     

Adenovirus vectors activate survival pathways in lung epithelial cells

Airway epithelial cells are often the sites of targeted adenovirus vector delivery. Activation of the host inflammatory response and modulation of signal transduction pathways by adenovirus vectors have been previously documented, including activation of MAP kinases and phosphatidylinositol 3-kinase (PI3-kinase). The effect of activation of these pathways by adenovirus vectors on cell survival has not been examined. Both the PI3-kinase/Akt and ERK/MAP kinase signaling pathways have been linked to cell survival. Akt has been found to play a role in cell survival and apoptosis through its downstream effects on apoptosis-related proteins. Constitutive activation of either PI3-kinase or Akt blocks apoptosis induced by c-Myc, UV radiation, transforming growth factor-beta, Fas, and respiratory syncytial virus infection. We examined the effect of adenovirus vector infection on activation of these prosurvival pathways and its downstream consequences. Airway epithelial cells were transduced with replication-deficient adenoviral vectors containing a nonspecific transgene, green fluorescent protein driven by the cytomegalovirus promoter, or an empty vector with no transgene. They were then exposed to the proapoptotic stimulus actinomycin D plus TNF-alpha, and evidence of apoptosis was evaluated. Compared with the cells treated with actinomycin/TNF alone, the adenovirus vector-infected cells had a 50% reduction in apoptosis. When we examined induction of the prosurvival pathways, ERK and AKT, in the viral vector-infected cells, we found that there was significant activation of both Akt and ERK.     

Inhibition of Mitochondrial Aconitase by Succination in Fumarate Hydratase Deficiency

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Highlights? Fumarate inhibits Aconitase2 activity via succination of critical cysteine residues ? Endogenous Aconitase2 is succinated and inhibited in FH-deficient cells ? Succination occurs in multiple proteins with roles in diverse cellular processes ? Succination can alter metabolism in FH-deficient cells     

Response of native and exotic longhorn beetles to common pheromone components provides partial support for the pheromone-free space hypothesis

Longhorn beetles are among the most important groups of invasive forest insects worldwide. In parallel, they represent one of the most well-studied insect groups in terms of chemical ecology. Longhorn beetle aggregation-sex pheromones are commonly used as trap lures for specific and generic surveillance programs at points of entry and may play a key role in determining the success or failure of exotic species establishment. An exotic species might be more likely to establish in a novel habitat if it relies on a pheromone channel that is different to that of native species active at the same time of year and day, allowing for unhindered mate location (i.e., pheromone-free space hypothesis). In this study, we first tested the attractiveness of single pheromone components (i.e., racemic 3-hydroxyhexan-2-one, racemic 3-hydroxyoctan-2-one, and syn-2,3-hexanediol), and their binary and tertiary combinations, to native and exotic longhorn beetle species in Canada and Italy. Second, we exploited trap catches to determine their seasonal flight activity. Third, we used pheromone-baited “timer traps” to determine longhorn beetle daily flight activity. The response to single pheromones and their combinations was mostly species specific but the combination of more than one pheromone component allowed catch of multiple species simultaneously in Italy. The response of the exotic species to pheromone components, coupled with results on seasonal and daily flight activity, provided partial support for the pheromone-free space hypothesis. This study aids in the understanding of longhorn beetle chemical ecology and confirms that pheromones can play a key role in longhorn beetle invasions.     

The sweet spot for biologics: recent advances in characterization of biotherapeutic glycoproteins

Introduction: Glycosylation is recognized as a Critical Quality Attribute for therapeutic glycoproteins such as monoclonal antibodies, fusion proteins and therapeutic replacement enzymes. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for their discovery, development and quality control. The aim of this review is to highlight relevant and recent advances in analytical technologies for characterization of biotherapeutic glycoproteins.

Areas covered: The review gives an overview of the glycosylation trends of biotherapeutics approved in 2016 and 2017 by FDA. It describes current and novel analytical technologies for characterization of therapeutic glycoproteins and is explored in the context of released glycan, glycopeptide or intact glycoprotein analysis. Ultra performance liquid chromatography, mass spectrometry and capillary electrophoresis technologies are explored in this context.

Expert commentary: There is a need for the biopharmaceutical industry to incorporate novel state of the art analytical technologies into existing and new therapeutic glycoprotein workflows for safer and more efficient biotherapeutics and for the improvement of future biotherapeutic design. Additionally, at present, there is no ‘gold-standard’ approach to address all the regulatory requirements and as such this will involve the use of orthogonal glycoanalytical technologies with a view to gain diagnostic information about the therapeutic glycoprotein.     

Association of ALOX5AP with ischemic stroke: a population-based case-control study

Arachidonate 5-lipoxygenase activating protein (ALOX5AP) has been reported to demonstrate linkage and association with ischemic stroke and myocardial infarction. However, replication studies have been conflicting and to date, a significant proportion of blacks have not been studied. We prospectively recruited cases of ischemic stroke from all 16 hospitals in the Greater Cincinnati/Northern Kentucky region and demographically matched them to stroke-free population-based controls. Single nucleotide polymorphisms (SNPs) were selected based on association with ischemic stroke in prior studies. Allelic, genotypic and haplotypic association testing was performed using HAPLOVIEW. Multiple logistic regression was used to control for the presence of traditional risk factors including hypertension, diabetes, hypercholesterolemia and smoking. A total of 357 cases and 482 controls were genotyped. The SNPs, rs9579646 and rs4769874 were found to be significantly associated at both allelic (P = 0.019 and P < 10⁻⁴, respectively) and genotypic level with ischemic stroke among whites after correction for multiple testing. Haplotype association was identified with ischemic stroke as well as ischemic stroke subtypes among whites. Although an overall haplotype association with ischemic stroke was identified among blacks no evidence of association among individual haplotypes, alleles or genotypes were observed. Allele frequencies for the SNPs examined were markedly different among whites and blacks. In conclusion, we report significant association of variants of ALOX5AP with ischemic stroke and ischemic stroke subtypes among whites. No significant association was identified among blacks.