Proteolytic processing of human L-type pyruvate kinase increases its ability to be phosphorylated

Red cell soluble cyclic 3′-5′ AMP-dependent protein kinase phosphorylates more efficient L₄ liver pyruvate kinase or the L_b partially proteolysed form of erythrocyte enzyme than the L′₄ precursor. Affinity of protein kinase for liver L₄ and L′₄ as substrates is similar (10 μM at 0.1 M ATP and 1 μM cyclic AMP), but maximal velocity of the phosphorylation reaction is twice higher with L₄ than L′₄. Thus it appears that proteolytic processing of pyruvate kinase increases its ability to be phosphorylated, in the same way that it increases its allosteric properties.     

Morphogenetic sequences of the rooting process after in vitro culture or Agrobacterium rhizogenes inoculation

Abstract

After a brief review dealing with the factors inducing adventitious rhizogenesis, the morphogenetic patterns of the rooting process induced by in vitro culture or after Agrobacterium rhizogenes inoculations are described. In vitro cultures of Nicotiana tabacum, Datura innoxia and Crepis capillaris leaf explants on Murashige and Skoog's medium supplemented with sucrose and IAA or NAA have been used in order to identify the target cells for direct and indirect rooting. The structural and functional features of the prerhizogenetic cells are described and the ploidy levels of both direct and indirect root meristems are examined by means of DNA microspectrophotometry. Data on the synergistic effects between auxin, sucrose and amino acids (particularly L-ornithine) for the reactivation of the prerhizogenetic cells are also given.

Transformed roots from carrct root discs or pea epicotyls after Agrobacterium rhizogenes inoculation arise indirectly from cambium-like layers differentiated inside a previously formed callus. Numerous auxin-like symptoms at the cell level are detected after the inoculation, suggesting modifications in the endogenous contents of auxin prior to the rooting process. It is also shown that initially non-susceptible cells (fully differentiated tobacco pith) can be induced to become susceptible by in vitro treatments resulting in cell reactivation before inoculation with the bacteria. Further work is in progress to extend these observations to non-susceptible species in order to check, via the occurrence of transformed roots, their ability to react positively to foreign Ri T-DNA.     

Hormonal Contraception and the Risk of HIV Acquisition: An Individual Participant Data Meta-analysis

BackgroundObservational studies of a putative association between hormonal contraception (HC) and HIV acquisition have produced conflicting results. We conducted an individual participant data (IPD) meta-analysis of studies from sub-Saharan Africa to compare the incidence of HIV infection in women using combined oral contraceptives (COCs) or the injectable progestins depot-medroxyprogesterone acetate (DMPA) or norethisterone enanthate (NET-EN) with women not using HC.ConclusionsThis IPD meta-analysis found no evidence that COC or NET-EN use increases women’s risk of HIV but adds to the evidence that DMPA may increase HIV risk, underscoring the need for additional safe and effective contraceptive options for women at high HIV risk. A randomized controlled trial would provide more definitive evidence about the effects of hormonal contraception, particularly DMPA, on HIV risk.     

Modulation of the Pyrococcus abyssi NucS endonuclease activity by replication clamp at functional and structural levels

Pyrococcus abyssi NucS is the founding member of a new family of structure-specific DNA endonucleases that interact with the replication clamp proliferating cell nuclear antigen (PCNA). Using a combination of small angle x-ray scattering and surface plasmon resonance analyses, we demonstrate the formation of a stable complex in solution, in which one molecule of the PabNucS homodimer binds to the outside surface of the PabPCNA homotrimer. Using fluorescent labels, PCNA is shown to increase the binding affinity of NucS toward single-strand/double-strand junctions on 5' and 3' flaps, as well as to modulate the cleavage specificity on the branched DNA structures. Our results indicate that the presence of a single major contact between the PabNucS and PabPCNA proteins, together with the complex-induced DNA bending, facilitate conformational flexibility required for specific cleavage at the single-strand/double-strand DNA junction.     

The Group B Streptococcus NADH oxidase Nox-2 is involved in fatty acid biosynthesis during aerobic growth and contributes to virulence

Numerous Streptococcaceae produce an H2O-forming NADH oxidase, Nox-2, which has been generally implicated in aerobic survival. We examined the roles of Nox-2 in Group B Streptococcus (GBS), a leading agent of neonatal infections. While nox2 inactivation caused an aerobic growth arrest, no improvement was seen by addition of antioxidants to cultures, suggesting that this defect was not due to accumulation of toxic oxygen species. Using several approaches, we show that the observed inability of the nox2 mutant to grow aerobically is mainly due to an underlying defect in fatty acid (FA) biosynthesis: (i) the nox2 aerobic growth defect is fully and rapidly complemented by adding oleic acid to culture medium, and (ii) direct assimilation of this unsaturated FA in both wild type (WT) and nox2 GBS membranes is demonstrated and correlated with mutant growth rescue. We propose that NAD+ depletion in the nox2 mutant results in reduced acetyl-CoA production, which perturbs FA biosynthesis and hence blocks growth in aerobiosis. The nox2 aerobic growth defect was also complemented when GBS respiration metabolism was activated by exogenous haem and menaquinone. The membrane NADH oxidase activity generated by the functional respiratory chain thus compensates the cytoplasmic NADH oxidase deficiency. The nox2 mutant was attenuated for virulence, as assessed in lung, intraperitoneal and intravenous murine infection models. As the nox2 defect seems only to affect aerobic growth of GBS, its reduced virulence supports the suggestion that aerobic conditions and NADH oxidase activities are relevant to the GBS infection process.     

Inhibition of P-glycoprotein-mediated multidrug efflux by aminomethylene and ketomethylene analogs of reversins

Several aminomethylene analogs and a ketomethylene analog of reversins were synthesized in order to evaluate their ability to inhibit P-glycoprotein-mediated drug efflux in K562/R7 human leukemic cells overexpressing P-glycoprotein. These analogs retained good activity compared to cyclosporin A and the original reversins.     

Microwave expedited synthesis of 5-aminocamptothecin analogs: Inhibitors of hypoxia inducible factor HIF-1alpha

A series of 5-aminosubstituted camptothecin analogs were prepared from the corresponding 5-hydroxycamptothecin using microwave irradiation. The analogs were assayed for ability to inhibit the action of hypoxia inducible factors (HIF-1alpha and HIF-2alpha). The 5-fluoroethyl analog showed potent inhibitory activity and is now the focus of ongoing pathway analysis and potential as an antiproliferative agent.     

Fluorometric assay for tissue transglutaminase-mediated transamidation activity

Herein, we report the development of a direct discontinuous fluorometric transamidation assay for determining tissue transglutaminase (TG2) activity. In the assay reaction, TG2 catalyzes the formation of a biotin-fluorophore conjugate, using a fluorescent, high affinity γ-glutamyl donor substrate and a biotinylated amine as a γ-glutamyl acceptor substrate. After the reaction, the conjugate is fixed on streptavidin-coated beads and excess substrate is washed away, allowing the transamidation activity to be quantified by fluorescence measurement. This method was used to detect the activity of as little as 0.6 mU of purified TG2, and can be used for detection of activity from crude cellular lysates. Furthermore, this assay can be used for screening potential inhibitors and synthetic substrates, the latter of which was demonstrated herein.