The involvement of biological quinones in the formation of hydroxyl radicals via the Haber-Weiss reaction

The present investigation was made to evaluate biologically relevant quinones as possible catalysts in the generation of hydroxyl radicals from hydrogen peroxide and superoxide radicals. ESR spectra demonstrated that menadione, plastoquinone, and ubiquinone derivatives could all be reduced to their semiquinone forms by electron transfer from superoxide radicals. Reductive homolytic cleavage of H₂O₂ was observed to be dependent upon the presence of semiquinones in the reaction medium. Our data strongly support the concept that quinones normally involved in physiological processes may also play a role as catalysts of the Haber-Weiss reaction in the biological generation of hydroxyl radicals.     

Regulated nitrate transport in the cyanobacterium Anacystis nidulans.

Intracellular accumulation of nitrate, indicative of the operation of an active nitrate transport system, has been measured in intact cells of the cyanobacterium Anacystis nidulans. The ability of the cells to accumulate nitrate was effectively hindered by either ammonium addition or selective inhibition of CO2 fixation by DL-glyceraldehyde, with the effect of either compound being prevented by previously blocking ammonium assimilation. The results support the contention that nitrate utilization in cyanobacteria is regulated at the level of nitrate transport through the concerted action of ammonium assimilation and CO2 fixation.     

Organization of Methanobacterium thermoautotrophicum bacteriophage psi M1 DNA

Summary M1 is a virulent bacteriophage of Methanobacterium thermoautotrophicum strain Marburg. Restriction enzyme analysis of the linear, 30.4 kb phage DNA led to a circular map of the 27.1 kb M1 genome. M1 is thus circularly permuted and exhibits terminal redundancy of approximately 3 kb. Packaging of M1 DNA from a concatemeric precursor initiates at the pac site which was identified at coordinate 4.6 kb on the circular genome map. It proceeds clockwise for at least five packaging rounds. Headful packaging was also shown for M2, a phage variant with a 0.7 kb deletion at coordinate 23.25 on the map.     

Determination of intracellular nitrate.

A sensitive procedure has been developed for the determination of intracellular nitrate. The method includes: (i) preparation of cell lysates in 2 M-H3PO4 after separation of cells from the outer medium by rapid centrifugation through a layer of silicone oil, and (ii) subsequent nitrate analysis by ion-exchange h.p.l.c. with, as mobile phase, a solution containing 50 mM-H3PO4 and 2% (v/v) tetrahydrofuran, adjusted to pH 1.9 with NaOH. The determination of nitrate is subjected to interference by chloride and sulphate when present in the samples at high concentrations. Nitrite also interferes, but it is easily eliminated by treatment of the samples with sulphamic acid. The method has been successfully applied to the study of nitrate transport in the unicellular cyanobacterium Anacystis nidulans.     

A sensitive and continuous fluorometric assay for phospholipase A2 using pyrene-labeled phospholipids in the presence of serum albumin

Phospholipase A2 activity can be determined fluorometrically in the presence of serum albumin using phospholipids labeled at the sn-2-acyl position with 10-pyrenyldecanoic acid. In the water reaction medium 10-pyrene phospholipids form vesicles and the monomer fluorescence of the pyrene is negligible due to pyrene-pyrene interaction. Upon phospholipid hydrolysis 10-pyrenyldecanoic acids are produced and tightly bind to albumin so that a monomer pyrene fluorescence is observed. We obtained an excellent parallelism between hydrolysis determined by a classical extraction method and that followed by direct and continuous spectrofluorometric recording of the monomer emission of pyrene. This assay can measure picomole amounts of phospholipids hydrolyzed per minute so that picogram quantities of phospholipases A2 from pancreas or from venoms can be measured. Phospholipase activity remains proportional to enzyme concentration over three orders of magnitude. The method can be used to quantify the phospholipase A2 activity of crude extracts of low specific activity.     

Immunocytochemical study of parvalbumin fibers and cell bodies in the rat hipothalamus

The distribution of parvalbumin (PA) cell bodies and fibers in the hypothalamus of the rat was studied using a monoclonal antibody and the avidin-biotin-peroxidase method. The densest clusters of immunoreactive perikarya were observed in the nuclei mamillare medialis, arcuatus and dorsomedialis hypothalami, whereas the corpus mamillare lateralis had the lowest density. The densest network of immunoreactive fibers was observed in the corpus mamillare lateralis and nucleus arcuatus. The corpus mamillare medialis contained a moderate number of PA fibers, whereas the nucleus dorsomedialis hypothalami had the lowest density of immunoreactive fibers. In addition, a large number of immunoreactive fibers was found in the tractus opticus and the tractus mamillo-thalamicus. Essentially, the distribution of PA in the rat hypothalamus after using a monoclonal antibody seems to be broader in comparison with previous studies carried out in the same diencephalic region of the rat. The presence of PA in several nuclei of the rat hypothalamus suggests that this protein could be directly or indirectly involved in neuroendocrine, limbic and visual functions.     

Relationship between a 47-kDa cytoplasmic membrane polypeptide and nitrate transport in Anacystis nidulans

The polypeptide composition of cytoplasmic membranes of the cyanobacterium Anacystis nidulans changes in response to variations in the nitrogen source available to the cells, differing specifically in the amount of a polypeptide of 47-kDa molecular mass. Synthesis of the polypeptide and expression of nitrate transport activity are repressed by ammonium. Transfer of ammonium-grown cells to a medium containing nitrate as the sole nitrogen source results in parallel development of the 47-kDa polypeptide and nitrate transport activity of the cells. These results suggest the involvement of the 47-kDa cytoplasmic membrane polypeptide in nitrate transport by A. nidulans.     

Inhibition of macrophage activation by isoquinolinesulfonamides, phenothiazines, and a napthalenesulfonamide

The influence of isoquinolinesulfonamides (H-7 and H-8), phenothiazines(trifluoperazine and fluphenazine), and a naphthalenesulfonamide (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) on stimulated superoxide anion production and phosphatidyl inositol (PI) cycle activity was investigated in the guinea pig alveolar macrophage. All five drugs were able to inhibit superoxide anion production stimulated by n-formyl-nel-leu-phe (FNLP), leukotriene B4 (LTB4), and phorbol-12,13-dibutyrate (PDB). The order of potency was trifluoperazine greater than or equal to fluphenazine greater than H-7 = W-7 greater than H-8. The dose response curves could be shifted to less efficacy by increasing extracellular calcium. By itself, W-7 markedly stimulated 45Ca+2 efflux, fluphenazine and trifluoperazine slightly stimulated 45Ca+2 efflux, while H-7 and H-8 had no effect on 45Ca+2 efflux from macrophages preloaded with 45Ca+2. Consistent with these results, W-7 markedly stimulated PI cycle activity, fluphenazine and trifluoperazine slightly stimulated PI cycle activity, while H-7 and H-8 had no significant effects on PI cycle activity. In addition, W-7 by itself was able to stimulate a weak and short-lived "burst" of superoxide anion production. In order to evaluate whether a site of action of the inhibitors was at protein kinase C and whether protein kinase C was involved in terminating the normally short-lived FNLP- and LTB4-stimulated macrophage activation, fluphenazine and H-7 were used to evaluate the duration of FNLP- and LTB4-stimulated PI cycle activity, at concentrations of the inhibitors that significantly blocked stimulated superoxide anion production. In all cases, FNLP and LTB4 still stimulated PI cycle activity, which still terminated even though protein kinase C was inhibited. These results suggest that all five drugs block protein kinase C, but H-7 was the most specific in its action at protein kinase C, while the phenothiazines and W-7 have multiple sites of action. In addition, these results suggest that protein kinase C may not function to cause the termination of FNLP- and LTB4-stimulated PI cycle activity and subsequent superoxide anion production.