Role of NAD(P)H:Quinone Oxidoreductase Encoded by drgA Gene in Reduction of Exogenous Quinones in Cyanobacterium Synechocystis sp. PCC 6803 Cells

Insertion mutant Ins2 of the cyanobacterium Synechocystis sp. PCC 6803, lacking NAD(P)H:quinone oxidoreductase (NQR) encoded by drgA gene, was characterized by higher sensitivity to quinone-type inhibitors (menadione and plumbagin) than wild type (WT) cells. In photoautotrophically grown cyanobacterial cells more than 60% of NADPH:quinone-reductase activity, as well as all NADPH:dinoseb-reductase activity, was associated with the function of NQR. NQR activity was observed only in soluble fraction of cyanobacterial cells, but not in membrane fraction. The effects of menadione and menadiol on the reduction of Photosystem I reaction center (P700(+)) after its photooxidation in the presence of DCMU were studied using the EPR spectroscopy. The addition of menadione increased the rate of P700(+) reduction in WT cells, whereas in Ins2 mutant the reduction of P700(+) was strongly inhibited. In the presence of menadiol the reduction of P700(+) was accelerated both in WT and Ins2 mutant cells. These data suggest that NQR protects the cyanobacterial cells from the toxic effect of exogenous quinones by their reduction to hydroquinones. These data may also indicate the probable functional homology of Synechocystis sp. PCC 6803 NQR with mammalian and plant NAD(P)H:quinone oxidoreductases (DT-diaphorases).     

Flora Europaea Notulae Systematicae ad Floram Europaeam spectantes No. 20 Corrigenda et Addenda

Following the publication of the last of the series of Flora Europaea Notulae, No. 20 in the Botanical Journal of the Linnean Society , 76: 297–384 (1978), a number of additions or alterations have been drawn to our attention. These are published in continuation.     

Kinetic study of the pH-dependence of maximal rate of Ca-ATP hydrolysis by myosin]

Curves of V pH-dependence for Ca ATPase of myosin and heavy meromyosin are demonstrated to be well modelled with theoretical curves for the case of proton dissociation at three groups of enzyme-substrate complex with the loss of the activity at some intermediate ionization stage. Variation of pK values for these three groups and the degree of inhibition for intermediate forms of enzyme-substrate complex are found to be sufficient to reproduce main varieties of described in the literature and obtained in this work multiformity of pH-dependence curves of different nucleoside triphosphates hydrolysis by both native and modified enzymes. Calculated pK values and modification data suggest a significant importance of the dissociation of two imidazole groups ("activating" and "inhibitory") and cisteine sulhydryl group for the catalytic activity of myosin. Inhibition of ATPase activity by increasing of KCl concentrations is found to be due first of all to a shift in pK values of "inhibitory" imidazole and sulhydryl groups.     

Elimination from bone marrow of a supplementary population participating in the proliferation of pluripotent hematopoietic stem cells using mouse brain antiserum

Changes in the number of spleen exo-colonies and post-radiation repopulation of hematopoietic organs were studied in recipients upon injection of bone marrow treated with anti-brain serum (ABS) with and without thymocytes on days 9-14. It was shown that on days 9-11 colony formation in mice injected bone marrow treated with ABS was much lower than the control level. However, by day 14 the number of colonies increased drastically as compared to the control. Thymocyte supplementation normalized colony formation at any time of observation. Similar pattern is noted in post-radiation repopulation of spleen and bone marrow of mice injected bone marrow pretreated with ABS with or without thymocytes. It is assumed that ABS inactivates bone marrow cells participating in the regulation of CFUs proliferation.     

Study of the structure of the histidine decarboxylase of Micrococcus sp. n. by the method of circular dichroism]

Ring dichroism spectra (RD) of histidine decarboxylase (HDC) from Micrococcus sp. n. at the regions of peptide bonds (200-240 nm) and aromatic amino acids (250-300 nm) absorption are studied. The treatment of RD spectra according to methods of Greenfield-Fasman, Saksena-Vetlaufer and Mayer permits to conclude that at the pH range within 4-8 the content of ordered structures of alpha-helix type comprises 20%, that of beta-structure type-40%, while the rest 40% are represented with polypeptide chain in a disordered globular state. When pH is varied from 1 to 12, the content of alpha-helices decreases from 17 to 5%. There are two distinct dichroic bands in the spectrum of aromatic chromophores absorption (at 270 and 290 nm), the former containing tirosine, tryptophane and phenylalanine residues and the latter being induced with triptophane residues. The study of HDC RD spectra at the regions of peptide bonds and aromatic acids absorption at different temperatures has shown that a part of triptophane, tyrosine and phenylalanine residues is in an ordered structure of the alpha-helix type. The HDC undergoes irreversible changes under heating to 70 degrees and in 8 M urea. 5 M guanidine chloride eliminates the ordered HDC structure, while sodium dodecylsulphate at concentrations up to 1% does not affect the enzyme structure.     

pH-dependence characteristics of Ca-ATPase activity of heavy meromyosin with modified SH-groups]

Study of pH-dependence of Ca-ATPase activity of heavy meromyosin (HMM) at low and high ionic strength showed essential differences in the modifying effect of two sulfhydryl reagents, p-CMB and silver. Silver ions in conditions studied independently on pH and KCl concentration produce an inhibition of ATP hydrolysis by myosin and HMM, the shape of the pH-dependence curve remaining similar to that of the native enzyme up to 40% of blocking free sulfhydryl groups. At the same degree of binding of sulfhydryl groups with p-CMB at 0,5 M KCl the pH-dependence curve due to activation at neutral pH changes it's shape and becomes similar to that for dissociation of two ionizable groups (at neutral and alkaline regions). In contrast to this, a low or zero concentrations of KCl no activation was observed for the enzyme with 40-50% of SH-Groups modified by p-CMB and Ca-ATPase in this case seemed to be independent of pH. The data obtained suggest that SH-Groups are not included into the active site of myosin, and the activating effect observed for some sulfhydryl reagents, is due to conformational changes and it can be the result of the penetrance of the organic part of the reagent molecule into hydrophobic region of the protein.     

Novel mechanism regulating endothelial permeability via T-cadherin-dependent VE-cadherin phosphorylation and clathrin-mediated endocytosis

T-cadherin is a unique member of the cadherin superfamily of adhesion molecules. In contrast to “classical” cadherins, T-cadherin lacks transmembrane and cytoplasmic domains and is anchored to the cell membrane via a glycosilphosphoinositol moiety. T-cadherin is predominantly expressed in cardiovascular system. Clinical and biochemical studies evidence that expression of T-cadherin increases in post-angioplasty restenosis and atherosclerotic lesions—conditions associated with endothelial dysfunction and pathological expression of adhesion molecules. Here, we provide data suggesting a new signaling mechanism by which T-cadherin regulates endothelial permeability. T-cadherin overexpression leads to VE-cadherin phosphorylation on Y731 (β-catenin-binding site), VE-cadherin clathrin-dependent endocytosis and its degradation in lysosomes. Moreover, T-cadherin overexpression results in activation of Rho GTPases signaling and actin stress fiber formation. Thus, T-cadherin up-regulation is involved in degradation of a key endothelial adhesion molecule, VE-cadherin, resulting in the disruption of endothelial barrier function. Our results point to the role of T-cadherin in regulation of endothelial permeability and its possible engagement in endothelial dysfunction.