Inhibition of nitric oxide synthase activity in cerebral cortical synaptosomes by nitric oxide donors: Evidence for feedback autoregulation

Despite evidence which supports a neurotransmitter-like role for nitric oxide (NO) in the CNS, relatively little is known regarding mechanisms which control NO formation within CNS neurons. In this study, isolated nerve endings (synaptosomes) from rat cerebral cortex were used to ascertain whether NO can autoregulate its own formation within neurons through feedback inhibition of the NO biosynthetic enzyme nitric oxide synthase (NOS). Under the conditions described here, N-nitro-l-arginine methyl ester-sensitive conversion ofl-[³H]arginine intol-[³H]citrulline (i.e., NOS activity) was found to be highly calcium-dependent and strongly inhibited (up to 60 percent) by NO donors, including sodium nitroprusside, hydroxylamine and nitroglycerin. The inhibitory effect of sodium nitroprusside was concentration-dependent (IC₅₀100 M) and prevented by the NO scavenger oxyhemoglobin.l-Citrulline, the other major end-product from NOS, had no apparent effect on synaptosomal NOS activity. Taken together, these results indicate that neuronal NOS can be inhibited by NO released from exogenous donors and, therefore, may be subject to end-product feedback inhibition by NO that is formed locally within neurons or released from proximal cells.     

Cyanopeptolin S, a sulfate-containing depsipeptide from a water bloom of Microcystis sp.

Abstract A new sulfated, cyclic depsipeptide, called cyanopeptolin S, from Microcystis sp. was isolated from a water bloom in the Auensee/Leipzig (Germany). The depsipeptide had a relative molecular mass of 925 and contained l-arginine, l-threonine, l-isoleucine, N-methyl-l-phenylalanine, a l-glutamic acid-δ-aldehyde ring system and a sulfated d-configurated glyceric acid as a side chain. The structure was elucidated by means of two-dimensional ¹H and ¹³C nuclear magnetic resonance spectroscopy, fast atom bombardment mass spectroscopy, Fourier transformed infrared spectroscopy and combined gas-liquid chromatography/mass spectrometry. Cyanopeptolin S inhibited trypsin with an IC₅₀≤ 0.2 μg ml⁻¹.     

Peroxidation of lipids and growth inhibition induced by UV-B irradiation

Cotyledons excised from dark-grown seedlings of cucumber (Cucumis sativus L.) were cultured in vitro under UV radiation at different wavelengths, obtained by passage of light through cut-off filters with different transmittance properties. Growth and the synthesis of chlorophyll (Chl) in cotyledons were inhibited and malondialdehyde was accumulated upon irradiation at wavelengths below 320 nm. Exogenous application of scavengers of free radicals reversed the growth inhibition induced by UV-B. Measurement of the fluorescence of Chl a suggested that electron transfer in photosystems was affected by UV-B irradiation. On the basis of these results, the involvement is postulated of active species of oxygen in damages to thylakoid membranes and the growth inhibition that are induced by UV-B irradiation.Abbreviations Chl chlorophyll - F_m maximal fluorescence (dark) - F_m maximal fluorescence (light) - F_v variable fluorescence (dark) - F_v variable fluorescence (light) - MDA malondialdehyde - O₂ Superoxide radical - PS photosystem - q_N non-photochemical quenching of fluorescence - q_P photochemical quenching of fluorescence - UV-B_BE biologically effective UV-B radiation - WL(T = 0.5) wavelength at which 50% transmittance occurs     

N-acetyl-β-D-glucopyranosylamine: A potent T-state inhibitor of glycogen phosphorylase. A comparison with α-D-glucose

Structure-based drug design has led to the discovery of a number of glucose analogue inhibitors of glycogen phosphorylase that have an increased affinity compared to α-D-glucose (K_i = 1.7 mM). The best inhibitor in the class of N-acyl derivatives of β-D-glucopyranosylamine, N-acetyl-β-D-glucopyranosylamine (1-GlcNAc), has been characterized by kinetic, ultracentrifugation, and crystallographic studies. 1-GlcNAc acts as a competitive inhibitor for both the b (K_i = 32 μM) and the α (K_i = 35 μM) forms of the enzyme with respect to glucose 1-phosphate and in synergism with caffeine, mimicking the binding of glucose. Sedimentation velocity experiments demonstrated that 1-GlcNAc was able to induce dissociation of tetrameric phosphorylase α and stabilization of the dimeric T-state conformation. Co-crystals of the phosphorylase b-1-GlcNAc-IMP complex were grown in space group P4₃2₁2, with native-like unit cell dimensions, and the complex structure has been refined to give a crystallographic R factor of 18.1%, for data between 8 and 2.3 Å resolution. 1-GlcNAc binds tightly at the catalytic site of T-state phosphorylase b at approximately the same position as that of α-D-glucose. The ligand can be accommodated in the catalytic site with very little change in the protein structure and stabilizes the T-state conformation of the 280s loop by making several favorable contacts to Asn 284 of this loop. Structural comparisons show that the T-state phosphorylase b-1-GlcNAc-IMP complex structure is overall similar to the T-state phosphorylase b-α-D-glucose complex structure. The structure of the 1-GlcNAc complex provides a rational for the biochemical properties of the inhibitor.     

Proferrioxamine synthesis in Erwinia amylovora in response to precursor or hydroxylysine feeding: metabolic profiling with liquid chromatography-electrospray mass spectrometry

Metabolic profiling by capillary liquid chromatography-electrospray mass spectrometry was used to monitor shifts in the proferrioxamine profiles of Erwinia amylovora in response to externally supplied potential proferrioxamine precursors, selected stable-isotope-labeled precursors and atypical precursors. Based on the qualitative and quantitative shifts in the proferrioxamine profiles, lysine and arginine are unambiguous, and agmatine, ornithine, diaminobutyric acid and the corresponding C_3–5 diamines are highly likely precursors for proferrioxamine biosynthesis in E. amylovora. 5-Hydroxylysine (Hyl), a recently discovered growth inhibitor for E. amylovora, suppresses proferrioxamine production. The Hyl-induced growth inhibition can be reversed by basic amino acids. The basic amino acids also partly restore proferrioxamine synthesis.Part 12 in the series Metabolites of Erwinia, for Parts 10 and 11 see Feistner (1994d) and Feistner (1995b), respectively. Presented, in part, at ALEX '93. San Francisco. October 5–7. 1993, and at the 42nd ASMS Conference. Chicago. May 29–June 3, 1994.     

The effect of manganese oxides and manganese ion on growth and siderophore production by Azotobacter vinelandii

The addition of manganese oxides to iron-limited medium promoted the formation of the pyoverdin siderophore azotobactin by Azotobacter vinelandii. When active-MnO₂ was used, there was greatly decreased iron uptake into the cells, hyperproduction of azotobactin and the abiotic, chemical destruction or adsorbtion of the catechol siderophores azotochelin and aminochelin by this strong oxidizing agent. Although the iron content of the cells was the same as iron-limited cells, the growth of cells in medium with active-MnO₂ was increased 1.5- to 2.5-fold over iron-limited controls. This growth promotion was not caused by iron contaminating the oxide or by manganese solubilized from the oxide. Soluble 0.05–4 mm Mn²⁺ inhibited the growth of iron-limited cells and had a minimal effect on iron uptake, but caused hyperproduction of azotobactin. This later effect was caused by the inhibition of soluble ferric reductase, in a manner identical to that previously observed for Zn²⁺. These results suggest that active-MnO₂ may interfere with a surface-localized iron uptake site, possibly another ferric reductase. The reason for the growth promotion by active-MnO₂ remains unknown, but is most likely related to decreased oxygen toxicity.     

Steady-state kinetics of the overall oxidative phosphorylation reaction in heart mitochondria. Determination of the coupling relationships between the respiratory reactions and miscellaneous observations concerning rate-limiting steps

The linear sequence of steps involved in the oxidation of extramitochondrial succinate by O₂ in bovine heart mitochondria was examined by a steady-state kinetic method to determine whether or not freely diffusible intermediates occur between the various inhibitor-sensitive steps. The kinetic method is based on the facts (1) that if two inhibitor-sensitive steps within a sequence are linked by a freely diffusible intermediate, inhibition of one will make the other less rate limiting in the overall reaction and thus will increase the amount of inhibitor of the other step required for half-maximal inhibition of the overall reaction, and (2) that if the two steps are not linked in this manner, inhibition of one will make the other more rate limiting and thus will decrease the amount of inhibitor of the other required for half-maximal inhibition. These two types of coupling relationships between steps were designated as sequential and fixed, respectively. The results indicate the existence of freely diffusible intermediates (sequential coupling relationships) between the succinate transport and succinate dehydrogenase reactions, between the succinate dehydrogenase and cytochromebc ₁ reactions, and between the cytochromesbc ₁ andaa ₃ reactions. Uncoupling respiration from phosphorylation results in the coupling relationship between thebc ₁ andaa ₃ reactions becoming partially fixed. This change is accompanied by marked decreases in the degrees to which thebc ₁ andaa ₃ reactions limit the overall reaction and appears to account for the large uncoupler-induced releases of inhibition at the levels of thebc ₁ andaa ₃ reactions observed previously by others. It is suggested that cytochromec is the freely diffusible intermediate between thebc ₁ andaa ₃ reactions and that the uncoupler-induced changes occur as a result of formation of functional and highly efficient supercomplexes between cytochromec and the cytochromesbc ₁ andaa ₃ complexes.     

Inhibition of methanogenesis and its reversal during biogas formation from cattle manure

The composition of volatile fatty acids in the biogas digester based on cattle manure as substrate and stabilised at 25°C showed that it contained 87–88% branched chain fatty acids, comprising of isobutyric and isovaleric acids, in comparison to 38 % observed in the digester operating at 35°C. Mixed cellulolytic cultures equilibrated at 25°C (C-25) and 35‡C (C-35) showed similar properties, but rates of hydrolysis were three times higher than that observed in a standard biogas digester. The proportion of isobutyric and isovaleric were drastically reduced when C-25 was grown with glucose or filter paper as substrates. The volatile fatty acids recovered from C-25 (at 25°C) inhibited growth of methanogens on acetate, whereas that from C-35 was not inhibitory. The inhibitory effects were due to the branched chain fatty acids and were observed with isobutyric acid at concentrations as low as 50 ppm. Addition of another micro-organismRhodotorula selected for growth on isobutyric completely reversed this inhibition. Results indicate that the aceticlastic methanogens are very sensitive to inhibition by branched chain fatty acids and reduction in methane formation in biogas digester at lower temperature may be due to this effect.