Ca2+ and Mg2+-enhanced reduction of arsenazo III to its anion free radical metabolite and generation of superoxide anion by an outer mitochondrial membrane azoreductase

At the concentrations usually employed as a Ca2+ indicator, arsenazo III underwent a one-electron reduction by rat liver mitochondria to produce an azo anion radical as demonstrated by electron-spin resonance spectroscopy. Either NADH or NADPH could serve as a source of reducing equivalents for the production of this free radical by intact rat liver mitochondria. Under aerobic conditions, addition of arsenazo III to rat liver mitochondria produced an increase in electron flow from NAD(P)H to molecular oxygen, generating superoxide anion. NAD(P)H generated from endogenous mitochondrial NAD(P)+ by intramitochondrial reactions could not be used for the NAD(P)H azoreductase reaction unless the mitochondria were solubilized by detergent or anaerobiosis. In addition, NAD(P)H azoreductase activity was higher in the crude outer mitochondrial membrane fraction than in mitoplasts and intact mitochondria. The steady-state concentration of the azo anion radical and the arsenazo III-stimulated cyanide-insensitive oxygen consumption were enhanced by calcium and magnesium, suggesting that, in addition to an enhanced azo anion radical-stabilization by complexation with the metal ions, enhanced reduction of arsenazo III also occurred. Accordingly, addition of cations to crude outer mitochondrial membrane preparations increased arsenazo III-stimulated cyanide-insensitive O2 consumption, H2O2 formation, and NAD(P)H oxidation. Antipyrylazo III was much less effective than arsenazo III in increasing superoxide anion formation by rat liver mitochondria and gave a much weaker electron spin resonance spectrum of an azo anion radical. These results provide direct evidence of an azoreductase activity associated with the outer mitochondrial membrane and of a stimulation of arsenazo III reduction by cations.     

NADPH-generating system: Influence on microsomal mono-oxygenase stability during incubation for the liver-microsomal assay with rat and mouse S9 fractions

Activity levels of 7-ethoxycoumarin O-deethylase (ED), aminopyrine N-demethylase (APD), p-nitroanisoleO-demethylase (p-NAD) and glucose-6-phosphate dehydrogenase (G-6-PDH) were determined in incubation mixtures for the liver-microsomal assay (LMA) at time 0 and after 1 and 2 h incubation under conditions for mutagenic assay. The experiments were performed with S9 liver fractions from mice (induced with Na-phenobarbital and β-naphthoflavone) and rats (induced with Aroclor 1254) with and without G-6-PDH in the incubation mixtures.

In the absence of G-6-PDH the activities were significantly lower at time 0 in the mouse. The pattern of stability, however, was similar for the activities, with an increase of stability after 1 and 2 h of pre-incubation (an exception for p-NAD).

Only ED activity showed a similar behaviour in the rat. No differences were present for APD and p-NAD activities at time 0 in the rat, but the enzyme stabilities were significantly decreased after 2 h of incubation (about 15% and 10% for APD and p-NAD respectively) in the absence of G-6-PDH.

At time 0, the amounts of G-6-PDH differed between mouse and rat fractions; however, during the incubations for LMA they decreased by about 57% and 53% for the two species, respectively. In addition to the above biochemical results, the presence of exogenous G-6-PDH in the incubations for the mutagenic assay, significantly increased the mitotic gene conversion and mitotic crossing-over of dimethylnitrosamine (DMN) and AR2MNFN (a nitroimidazo[2,1-b]thiazole) in the D₇ strain of Saccharomyces cerevisiae.     

Isovolumetric and isobaric rabbit tracheal contraction in vitro

Isovolumetric and isobaric tracheal smooth muscle (TSM) contraction were studied in vitro in a preparation of the whole rabbit trachea. Eight tracheae from New Zealand White rabbits were excised and mounted at a fixed length in an organ bath. Electrical field stimulation (EFS) was performed in isovolumetric and isobaric conditions at varying transmural pressures (TMP). Supramaximal stimulation with methacholine was done at 0 TMP. Active change in pressure (delta P) with EFS showed a peak at 3.1 +/- 1.06 cmH2O TMP during inflation and at 4.1 +/- 1.18 cmH2O TMP during deflation (mean +/- SE). Active delta P decreased at higher or lower TMP. Active change in volume with EFS showed a peak at 3.2 +/- 1.26 cmH2O TMP during inflation and at 1.8 +/- 0.98 cmH2O TMP during deflation. A decrease in response was also observed at higher and lower TMP. From these data, we concluded that TSM is at optimal length (Lmax) at TMP of 2-3 cmH2O. Maximal TSM shortening with supramaximal stimulation with methacholine was 32% Lmax. This figure is considerably smaller than the 80% shortening found in unloaded strips of TSM. We conclude that rabbit TSM length is close to Lmax at TMP similar to those found at functional residual capacity and that the loads that the muscle has to overcome probably contribute to the limited shortening observed in situ.     

Quantitative measurement of smooth muscle shortening in isolated pig trachea

Matched porcine tracheal rings were exposed to theophylline and increasing doses of carbachol in Krebs solution. Histological sections of each ring were traced and each of the following dimensions measured: the external perimeter (Pe) and external area (Ae) defined by the outer border of smooth muscle and inner surface of cartilage, and the internal perimeter (Pi) and internal area (Ai) defined by the luminal surface of the epithelium and the muscle length (L) along its outer border. Absolute wall area (WA = Ae - Ai) and relative wall area (PW = WA/Ae) were calculated. Carbachol-treated tracheal ring dimensions were compared with those of their matched theophylline-treated rings. In tracheal rings with intact cartilage, maximal smooth muscle shortening of 44% was achieved with 10(-2) M carbachol. In tracheal rings in which anterior and posterior segments of cartilage were excised, the trachealis muscle passively shortened by 20% and maximal shortening (10(-3) M carbachol) was 57%. Although Ai decreased with maximal smooth muscle shortening, there were no changes in the length of Pi or in WA. These data show that the cartilage in the porcine trachea exerts both a preload that passively stretches the trachealis muscle and an afterload that limits maximal smooth muscle shortening.     

A comparative study of salivary secretion by parotid and mandibular glands of anaesthetized Capra hircus: effect of pilocarpine

A study was made of basal secretion and the effect of the infusion of pilocarpine on the flow and composition of saliva in the parotid and mandibular glands of the anaesthetized lactating goat. In the parotid gland there was a basal flow (1.6 +/- 0.29 microliter/min) which was not present in the mandibular gland. There is a statistically significant dose-effect relationship between pilocarpine and salivary flow in both glands. Salival composition and its variation with respect to the flow of saliva did not conform to either of the two glands to an exclusive monogastric or ruminant model.     

Seasonal variation of nitrogenous compounds in the xylem sap of Citrus

Samples of tracheal sap of Citrus sinensis (L.) Osbeck cv. Washington Navel were taken from field trees throughout the year and the nitrogen composition of the sap was determined. The nitrogenous fraction of the sap was composed mainly of free amino acids (92–97% of total nitrogen) and nitrates throughout the year. Proline was the most abundant amino acid during almost the entire cycle, and its concentration was especially high during the autumn and winter period. Nevertheless, a significant part (40–60%) of the total organic nitrogen was transported as arginine. Total nitrogen as well as amino acids and nitrates were maximal at spring flush. At spring flush and summer flush there was also a diversification of α-amino nitrogen among different amino acids. During the spring flush, nitrates, asparagine and γ-aminobutyric acid in the xylem sap seemed to have a radicular origin, whereas glutamic acid and arginine were released from the surrounding parenchyma. The results suggest a metabolic transformation in the wood parenchyma of nitrogenous compounds coming from the roots (including reduction of nitrates) and a turnover of different nitrogen metabolites between the xylem and surrounding cells.     

Immunological properties of monoclonal antibodies specific for meningococcal polysaccharides: the protective capacity of IgM antibodies specific for polysaccharide group B

Two IgM monoclonal antibodies, MB32 and MB34 specific for meningococcal polysaccharide group B have been raised. Both were detectable by radioimmunoassay and agglutination, but only MB34 was effective in counter immunoelectrophoresis and complement fixation. MB34 was also far more potent than MB32 when tested for passive protection of mice infected with either Neisseria meningitidis group B or Escherichia coli K1. These data demonstrate that group B-specific antibodies do play a protective role in mice infected with these bacteria.     

Protein kinase from Mucor rouxii

Summary Cyclic AMP binding to Mucor rouxii protein kinase holoenzyme and free regulatory subunit was measured by the classical membrane filtration technique and by equilibrium dialysis. The results obtained demonstrate that the filtration method can be used without loss of any cyclic AMP binding site. Both methods unambiguously demonstrate that the number of molecules of cyclic AMP bound to the holoenzyme are half of those bound to the regulatory subunit. This result suggests that unshielding of new cyclic AMP binding sites occurs upon dissociation of the ternary complex holoenzyme-cyclic AMP.