Calcium ionophore A23187 action on cardiac myocytes is accompanied by enhanced production of reactive oxygen species

We show that rat neonatal cardiac myocytes exposed to 1 micromol/l of the calcium ionophore A23187 respond with an enhanced production of reactive oxygen species (ROS). This dose is not cytotoxic to the myocytes. A higher concentration (10 micromol/l) evokes less ROS production and is significantly cytotoxic 24 h after exposure, but not immediately after removal of the A23187, when ROS are measured. Both cell death and the decrease in mitochondrial potential are only partially sensitive to MPT inhibitor cyclosporin A. Experiments performed to elucidate the sources of ROS included use of the nitric oxide synthase (NOS) inhibitor L-NAME; NOS involvement was excluded. Experiments with the oxidative phosphorylation uncoupler CCCP revealed that mitochondria are at least partially responsible for the observed effect. Further studies with cyclooxygenase (COX) and lipoxygenase (LOX) inhibitors (indomethacin and MK886, respectively) showed that these enzymes could also be sources of ROS when the calcium level is elevated. Their effect appeared to be independent of phospholipase A(2) inhibition, suggesting that COX and LOX stimulation is not due to elevated substrate (arachidonic acid) concentration but rather to a direct effect of calcium.     

Nitric oxide and platelet energy metabolism

This study was undertaken to determine whether nitric oxide (NO) can affect platelet responses through the inhibition of energy production. It was found that NO donors: S-nitroso-N-acetylpenicyllamine, SNAP, (5-50 microM) and sodium nitroprusside, SNP, (5-100 microM) inhibited collagen- and ADP-induced aggregation of porcine platelets. The corresponding IC50 values for SNAP and SNP varied from 5 to 30 microM and from 9 to 75 microM, respectively. Collagen- and thrombin-induced platelet secretion was inhibited by SNAP (IC50 = 50 microM) and by SNP (IC50 = 100 microM). SNAP (20-100 microM), SNP (10-200 microM) and collagen (20 microg/ml) stimulated glycolysis in intact platelets. The degree of glycolysis stimulation exerted by NO donors was similar to that produced by respiratory chain inhibitors (cyanide and antimycin A) or uncouplers (2,4-dinitrophenol). Neither the NO donors nor the respiratory chain blockers affected glycolysis in platelet homogenate. SNAP (20-100 microM) and SNP (50-200 microM) inhibited oxygen consumption by platelets. The effect of SNP and SNAP on glycolysis and respiration was not reduced by 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, a selective inhibitor of NO-stimulated guanylate cyclase. SNAP (5-100 microM) and SNP (10-300 microM) inhibited the activity of platelet cytochrome oxidase and had no effect on NADH:ubiquinone oxidoreductase and succinate dehydrogenase. Blocking of the mitochondrial energy production by antimycin A slightly affected collagen-evoked aggregation and strongly inhibited platelet secretion. The results indicate that: 1) in porcine platelets NO is able to diminish mitochondrial energy production through the inhibition of cytochrome oxidase, 2) the inhibitory effect of NO on platelet secretion (but not aggregation) can be attributed to the reduction of mitochondrial energy production.     

Nuclear overexpression of NAD(P)H:quinone oxidoreductase 1 in Chinese hamster ovary cells increases the cytotoxicity of mitomycin C under aerobic and hypoxic conditions

The effects of the subcellular localization of overexpressed bioreductive enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) on the activity of the antineoplastic agent mitomycin C (MC) under aerobic and hypoxic conditions were examined. Chinese hamster ovary (CHO-K1/dhfr(-)) cells were transfected with NQO1 cDNA to produce cells that overexpressed NQO1 activity in the nucleus (148-fold) or the cytosol (163-fold) over the constitutive level of the enzyme in parental cells. Subcellular localization of the enzyme was confirmed using antibody-assisted immunofluorescence. Nuclear localization of transfected NQO1 activity increased the cytotoxicity of MC over that produced by overexpression in the cytosol under both aerobic and hypoxic conditions, with greater cytotoxicity being produced under hypoxia. The greater cytotoxicity of nuclear localized NQO1 was not attributable to greater metabolic activation of MC but instead was the result of activation of the drug in close proximity to its target, nuclear DNA. A positive relationship existed between the degree of MC-induced cytotoxicity and the number of MC-DNA adducts produced. The findings indicate that activation of MC proximal to nuclear DNA by the nuclear localization of transfected NQO1 increases the cytotoxic effects of MC regardless of the degree of oxygenation and support the concept that the mechanism of action of MC involves alkylation of DNA.     

Biosynthesis of NAAG by an enzyme-mediated process in rat central nervous system neurons and glia

The peptide transmitter N-acetylaspartylglutamate (NAAG) is present in millimolar concentrations in mammalian spinal cord. Data from the rat peripheral nervous system suggest that this peptide is synthesized enzymatically, a process that would be unique for mammalian neuropeptides. To test this hypothesis in the mammalian CNS, rat spinal cords were acutely isolated and used to study the incorporation of radiolabeled amino acids into NAAG. Consistent with the action of a NAAG synthetase, inhibition of protein synthesis did not affect radiolabel incorporation into NAAG. Depolarization of spinal cords stimulated incorporation of radiolabel. Biosynthesis of NAAG by cortical astrocytes in cell culture was demonstrated by tracing incorporation of [3H]-glutamate by astrocytes. In the first test of the hypothesis that NAA is an immediate precursor in NAAG biosynthesis, [3H]-NAA was incorporated into NAAG by isolated spinal cords and by cell cultures of cortical astrocytes. Data from cerebellar neurons and glia in primary culture confirmed the predominance of neuronal synthesis and glial uptake of NAA, leading to the hypothesis that while neurons synthesize NAA for NAAG biosynthesis, glia may take it up from the extracellular space. However, cortical astrocytes in serum-free low-density cell culture incorporated [3H]-aspartate into NAAG, a result indicating that under some conditions these cells may also synthesize NAA. Pre-incubation of isolated spinal cords and cultures of rat cortical astrocytes with unlabeled NAA increased [3H]-glutamate incorporation into NAAG. In contrast, [3H]-glutamine incorporation in spinal cord was not stimulated by unlabeled NAA. These results are consistent with the glutamate-glutamine cycle greatly favoring uptake of glutamine into neurons and glutamate by glia and suggest that NAA availability may be rate-limiting in the synthesis of NAAG by glia under some conditions.     

Evaluation of selected alcoholic antiseptics activity against clinical bacterial strains

The aim of this study was to evaluate antimicrobial activity of selected alcoholic antiseptics against clinical strains, which possessed in majority a high level of drug resistance: MRSA (7), MSSA (3), E. coli: (9): strains producing ESBL (4), P. aeruginosa: (4), E. cloacae: (3), K. pneumoniae: (3). These strains were defined by MIC value, using antibiotic agar dilution method according to NCCLS. Fourteen alcoholic antiseptics were used in this study. Beside alcohol, they contained other active substances like iodine, hydrogen peroxide, chlorhexidine. Some additional agents were included for easier application, such as: gelling, moisturizing, aromatic or coloring substances. The objective of this study was also to determine the dependence of bactericidal activity on preparations (concentration). Product undiluted and diluted two and four times in water was analyzed according to prEN 12054 standard; 30 seconds and 1 minute contact time was used. The obtained data indicate that all tested undiluted antiseptics possessed bactericidal activity described by producers. However antiseptics (dilution leads to decrease and even loss of bactericidal activity. Two-times dilution of gel almost completely inactivated the product. Antimicrobial activity after 30 seconds of contact time was not affected by presence of additional agents in the tested antiseptics.     

Syn- and anti-conformations of 5′-deoxy- and 5′-O-methyl-uridine 2′,3′-cyclic monophosphate

Two uridine 2′,3′-cyclic monophosphate (cUMP) derivatives, 5′-deoxy (DcUMP) and 5′-O-methyl (McUMP), were studied by means of quantum chemical methods. Aqueous solvent effects were estimated based on the isodensity-surface polarized-continuum model (IPCM). Gas phase calculations revealed only slight energy differences between the syn- and anti-conformers of both compounds: the relative energies of the syn-structure are −0.9 and 0.2 kcal mol^-1 for DcUMP and McUMP, respectively. According to the results from the IPCM calculations, however, both syn-conformers become about 14 kcal mol^-1 more stable in aqueous solution than their corresponding anti-structures. Additionally, the effects of a countercation and protonation on DcUMP were studied, revealing that the syn-structure is also favored over the anti-one for these systems.     

The effect of aromatase inhibitor, fadrozole, on sGnRHa stimulated LH secretion in goldfish (Carassius auratus) and common carp (Cyprinus carpio)

The aromatase inhibitor, fadrozole, was applied to common carp and goldfish in order to examine its ability to increase the spontaneous and sGnRHa stimulated LH secretion. First, trials in goldfish in 2003 showed fadrozole's moderate ability to potentiate sGnRHa-stimulated LH secretion. However, this ability was much weaker than that obtained with dopamine antagonist, pimozide. There was no ovulation in fadrozole-treated fish. Several experiments on goldfish and carp during the two consecutive years with the different treatment regimes and doses of fadrozole did not confirm the optimistic results obtained in 2003. This shows that fadrozole is unable to replace the antidopaminergic drugs being used in fisheries practice.     

Enhancement of DNA uptake in FUT8‐deleted CHO cells for transient production of afucosylated antibodies

Removal of the core α1,6 fucose from the glycans in the Fc region of IgG1 antibodies has been demonstrated to improve antibody‐dependent cellular cytotoxicity (ADCC) activity. In order to produce afucosylated antibodies using transient transfection, a FUT8 knockout (FUT8KO) cell line was generated in a CHO host cell line using the zinc finger nuclease technology. Transient transfection of DNA into mammalian cells using the cationic polymer, polyethylenimine (PEI), is commonly used for rapid generation of recombinant proteins. FUT8KO cells evaluated in PEI transfections yielded lower titers than parental CHO WT cells. FACS and HPLC analyses revealed that the FUT8KO cells had lower cell surface heparan sulfate (HS) levels than CHO WT. Removal of cell surface HS resulted in reduced uptake of PEI‐transfected DNA (PEI:DNA) and lower transfection titers suggesting that PEI:DNA relies on HS for binding and cellular entry. The absence of cell surface HS did not severely impact transfections performed with cationic liposomes. We undertook two approaches to improve transient production of afucosylated antibodies. First, we evaluated transfection of FUT8KO cells with cationic liposomes, which were observed to be less dependent on HS levels for uptake. Transfection of FUT8KO cells using the cationic liposome, DMRIE‐C, produced similar titers to CHO WT in both shake flask and large‐scale 10 L bioreactors. The second approach was to engineer a cell line overexpressing exostosin‐1 (EXT1), an enzyme responsible for HS chain elongation, to increase HS content. EXT1‐FUT8KO and CHO WT cells produced comparable levels of antibody from PEI transfections. Biotechnol. Bioeng. 2010;106: 751–763. © 2010 Wiley Periodicals, Inc.