Changes in NO bioavailability regulate cardiac O2 consumption: control by intramitochondrial SOD2 and intracellular myoglobin

The aim of this study was to investigate the significance of two intracellular scavengers of nitric oxide (NO): 1) superoxide dismutase (SOD) (SOD2) to scavenge intramitochondrial superoxide anion, and 2) cytosolic myoglobin (Mb) in the regulation of tissue O2 consumption. O2 consumption was measured in vitro using a Clark-type O2 electrode. SOD heterozygous mice (SODHZ) (n = 13) and SOD wild-type (SODWT) (n = 5) mice were used. Bradykinin (BK, 10-4 mol/l) reduced O2 consumption by 15% +/- 1 in hearts of SODHZ mice, which was significantly different from SODWT (reduced by 24 +/- 0.4%). Tiron significantly increased the inhibition of O2 consumption by BK in male mice from 15 +/- 1% (n = 13) to 29 +/- 1.2% (n = 4) at 10-4 mol/l concentration (P < 0.05). The effect of carbachol was similar to BK. S-nitroso-N-acetyl penicillamine (SNAP, 10-4 mol/l) reduced O2 consumption by 39 +/- 1.3% in hearts of SODHZ mice, which was not significantly different from SODWT. But at 10-7 mol/l, SNAP caused significantly less inhibition of O2 consumption in SODHZ mice. Mb knockout (MbKO; Mb wild-type n = 6) and (MbWT) mice (n = 6) were also used. Kidney cortex was studied as the negative control because it does not contain Mb. BK (10-4 mol/l) reduced O2 consumption by 32 +/- 2, 29 +/- 1, and 26 +/- 1% in the heart, skeletal muscle, and kidney of MbKO mice, which was also not significantly different from MbWT. SNAP (10-4 mol/l) reduced O2 consumption by 39 +/- 3, 42 +/- 4, and 46 +/- 2% in the heart, skeletal muscle, and kidney of MbKO mice, which was also not significantly different from MbWT. NG-nitro-l-arginine methyl ester (P < 0.05) inhibited the reduction in O2 consumption induced by BK in the MbKO mouse heart (15 +/- 1%), skeletal muscle (17 +/- 1%), and kidney (17 +/- 1%) as in the MbWT mice. These results suggest that the role of Mb as an intracellular NO scavenger is small, and the increase in mitochondrial superoxide in SODHZ mice may cause a decrease NO bioavailability and alter the control of myocardial O2 consumption by NO.     

Monoclonal antibodies to surface glycoconjugates in Chlamydomonas eugametos recognize strain-specific O-methyl sugars

Monoclonal antibodies are described that are directed against cell surface components of the unicellular green alga Chlamydomonas eugametos. These antibodies recognize strain-specific epitopes which occur at the surface of vegetative and gametic cells. Two different groups of epitopes are distinguished that are never detectable together in one clonal cell culture. Evidence is presented showing that the antigenicity of cell surface molecules is a consequence of the presence of particular O-methylated sugars. Monoclonal antibodies reacting with one group of epitopes were studied in more detail, and immunoprecipitation and Western-blot studies showed that these epitopes can be arranged into four classes. The use of these monoclonal antibodies as strain-specific markers in light- and electron-microscopical techniques is illustrated.Abbreviations ELISA enzyme-linked immunosorbent assay - mt ^+/- mating type plus or minus - PAS periodic acid Schiff - Mab monoclonal antibody - PBS phosphate-buffered saline     

Concomitant analysis of cambial abscisic acid and cambial growth activity in poplar

Endogenous levels of cambial region abscisic acid (ABA) were quantified by immunoassay and assessed together with cambial growth activity in poplar (Populus nigra L. × P. maximowiczii Henry, clone Kamabuchi) over the course of a growing season. The level of cambial region ABA increased from spring to late-summer but decreased sharply in autumn. Cambial growth activity, measured as the radial number of undifferentiated cambial cells and enlarging xylem cells, also increased from spring to summer and decreased sharply in autumn, indicating the onset of cambial dormancy. Exogenous ABA, applied laterally to poplar stems at two times within the growing season, enhanced cambial growth activity, as the radial number of undifferentiated cambial cells increased in ABA-treated trees subsequent to the two application times. Xylem cell development was also affected by exogenous ABA as fibre length increased significantly in ABA-treated trees at both application times. The positive correlation of cambial region ABA and cambial growth activity as well as the positive effects of exogenous ABA application thereon sheds new light on the role of this hormonal growth regulator.     

Production of IL-1 receptor antagonist by human in vitro-derived macrophages. Effects of lipopolysaccharide and granulocyte-macrophage colony-stimulating factor.

The objective of these experiments was to evaluate the production of IL-1ra, a specific receptor antagonist of IL-1, by human in vitro-derived macrophages, a model for differentiated macrophages. IL-1ra protein levels in supernatants and lysates of cultured cells were determined by a specific ELISA. Relative steady-state IL-1ra mRNA levels were measured using a specific cDNA probe. Human monocytes were differentiated by 6 days culture in either medium or granulocyte-macrophage colony-stimulating factor (GM-CSF), after which the effects of subsequent LPS and/or GM-CSF on the production of IL-1ra were evaluated. In vitro-derived macrophages cultured in medium for 6 days constitutively produced IL-1ra protein during the 24-h period of the 7th day in culture. The constitutive production of IL-1ra by medium-aged cells correlated with low steady-state IL-1ra mRNA levels determined over this same time period. In contrast, cells cultured for 6 days in GM-CSF synthesized significantly increased levels of IL-1ra protein during the 7th day in culture but the secreted levels remained unchanged. Cells differentiated in GM-CSF displayed enhanced steady-state levels of IL-1ra mRNA in comparison with cells aged in medium. Stimulation of in vitro-derived macrophages aged for 6 days in medium or in GM-CSF, with LPS or adherent IgG, did not result in increased levels of IL-1ra protein production in comparison with non-LPS stimulated cells. The IL-1ra protein detected in the supernatants of cells differentiated in GM-CSF was biologically active in the IL-1-augmented murine thymocyte proliferation assay. By Western blot analysis, the IL-1ra protein in the in vitro-derived macrophage supernatants was predominantly the 22- to 24-kDa glycosylated species, whereas the lysates contained additional lower molecular weight forms. These results suggest that as monocytes differentiate in vitro into macrophages, they constitutively produce IL-1ra protein and that this production is enhanced by the continuous presence of GM-CSF.     

Identification of fatty acid translocase on human skeletal muscle mitochondrial membranes: essential role in fatty acid oxidation

Fatty acid translocase (FAT/CD36) is a transport protein with a high affinity for long-chain fatty acids (LCFA). It was recently identified on rat skeletal muscle mitochondrial membranes and found to be required for palmitate uptake and oxidation. Our aim was to identify the presence and elucidate the role of FAT/CD36 on human skeletal muscle mitochondrial membranes. We demonstrate that FAT/CD36 is present in highly purified human skeletal mitochondria. Blocking of human muscle mitochondrial FAT/CD36 with the specific inhibitor sulfo-N-succimidyl-oleate (SSO) decreased palmitate oxidation in a dose-dependent manner. At maximal SSO concentrations (200 muM) palmitate oxidation was decreased by 95% (P<0.01), suggesting an important role for FAT/CD36 in LCFA transport across the mitochondrial membranes. SSO treatment of mitochondria did not affect mitochondrial octanoate oxidation and had no effect on maximal and submaximal carnitine palmitoyltransferase I (CPT I) activity. However, SSO treatment did inhibit palmitoylcarnitine oxidation by 92% (P<0.001), suggesting that FAT/CD36 may be playing a role downstream of CPT I activity, possibly in the transfer of palmitoylcarnitine from CPT I to carnitine-acylcarnitine translocase. These data provide new insight regarding human skeletal muscle mitochondrial fatty acid (FA) transport, and suggest that FAT/CD36 could be involved in the cellular and mitochondrial adaptations resulting in improved and/or impaired states of FA oxidation.