The binding of porphyrin cytochrome c to yeast cytochrome c peroxidase. A fluorescence study of the number of sites and their sensitivity to salt

Porphyrin c, the iron-free derivative of cytochrome c, is a reasonably good model for cytochrome c binding to cytochrome c peroxidase (CcP). It binds with the same stoichiometry but only one-quarter as tightly as cytochrome c. CcP (resting, FeIII) and CcP X CN can both bind up to two molecules of porphyrin c. The binding of the first porphyrin c is tight (kd = 1 X 10(-9) M, pH 6, ionic strength mu = 0, 4 degrees C) and results in quenching of the porphyrin c fluorescence. The binding is sensitive to ionic strength. The binding of the second porphyrin c is looser (Kd unknown) and does not result in quenching of the porphyrin fluorescence. The binding of porphyrin c to the cyano form and the resting forms of CcP cannot be distinguished by our methods. ES is the first acceptor of electrons from c(II) and can bind at least two molecules of porphyrin c. The binding of the first porphyrin c is extremely tight, results in substantial quenching and is insensitive to ionic strength. The binding of porphyrin c to the loose site (Kd = 2 X 10(-9) M, pH 6, 4 degrees C, mu = 0) results, unlike the resting and cyano forms, in quenching of fluorescence of the second porphyrin c. The binding of the second porphyrin c to ES is sensitive to ionic strength. The calculated distances between porphyrin c and the hemes of CcP(FeIII) and ES are approximately 2.5 nm.     

Reversible activation of the neutrophil superoxide generating system by hexachlorocyclohexane: correlation with effects on a subcellular superoxide-generating fraction

gamma-Hexachlorocyclohexane was found to exert profound effects on the phosphatidylinositol cycle, cytosolic calcium level, and the respiratory burst of human neutrophils. Exposure of neutrophils prelabelled with 32P to 4 X 10(-4) M gamma-hexachlorocyclohexane almost tripled radioactivity in phosphatidic acid and correspondingly decreased radioactivity in phosphatidylinositol 4,5 bisphosphate. Under similar conditions, gamma-hexachlorocyclohexane evoked the generation of superoxide at a rate of over 11 nmol/min/10(6) cells and more than doubled cytosolic-free calcium concentration as monitored by Quin-2 fluorescence. Because intermediates of the phosphatidylinositol cycle, via increases in available calcium levels or activated protein kinase C, are considered potential second messengers for activation of the NADPH-dependent O-2-generating system, we compared neutrophil responses to gamma-hexachlorocyclohexane with responses to phorbol myristate acetate, an activator of protein kinase C with well known effects on neutrophils. Like phorbol myristate acetate, gamma-hexachlorocyclohexane induced neutrophil degranulation but was not an effective chemotactic stimulus. The ability of gamma-hexachlorocyclohexane to induce a pattern of oxidative activation in neutrophil cytoplasts similar to that in intact cells indicated that concurrent degranulation was not required for sustained O-2 generation in response to this agent. When neutrophils or neutrophil cytoplasts exposed to gamma-hexachlorocyclohexane were centrifuged and resuspended in stimulus-free medium, O-2 generation ceased entirely but could be reinitiated by addition of the same stimulus. This finding was in contrast to the continued O-2 production by phorbol myristate acetate-stimulated neutrophils similarly washed and resuspended in stimulus-free medium. Unlike subcellular fractions of phorbol myristate acetate-stimulated neutrophils, corresponding fractions prepared from gamma-hexachlorocyclohexane-stimulated neutrophils contained almost no detectable NADPH-dependent O-2-generating activity. Subcellular oxidase activity was not recovered when cells and membrane fractions were continuously exposed to gamma-hexachlorocyclohexane during disruption and fractionation after cell stimulation, nor could it be induced by the addition of the stimulus to the subcellular fractions. Thus, the stimulus dependence of continuous neutrophil superoxide release evoked by gamma-hexachlorocyclohexane does not merely reflect a physical interaction of the agonist with the enzyme system involved.(ABSTRACT TRUNCATED AT 400 WORDS)     

A comparison of the efficiency of melatonin treatments in advancing oestrus in ewes

Early oestrous cycles were induced in adult, maiden, 18-month-old Suffolk-cross ewes, maintained from birth in natural photoperiod by the following treatments applied from mid-June: subcutaneous implantation of melatonin (1 g) in Silastic packets, daily, oral, melatonin administration (3 mg/ewe) at 15:30 h, an artificial photoperiod of 8L:16D (lights on 07:30 h). Ovarian cycles began 5-10 weeks before those of control ewes maintained in a natural photoperiod. In contrast, the onset of ovarian cycles in ewes given s.c. implants of melatonin (1 g) in April, and a further group in May, was highly variable, and not significantly different from that of the control ewes. Plasma melatonin profiles in sheep with implants showed a night-time rise super-imposed on a constant level, which was itself within the physiological night-time range. Implant-derived melatonin declined with time but remained at or above physiological night-time levels for at least 3 1/2 months. These results indicate that melatonin implants in June, but not in April or May, advance onset of oestrus in the non-lactating, adult ewe. The effects of melatonin implants in June on onset of ovarian cycles were indistinguishable from those of melatonin feeding or artificial short photoperiod initiated at this time of year.     

Treatment of dialysis membranes for simultaneous dialysis and concentration

Dialysis membranes used for simultaneous dialysis-concentration required pretreatment to remove uv-absorbing compounds leached from the membranes and to reduce the absorption of protein to the membranes. This was accomplished with sodium carbonate and ethanol or with "sulfur-removal solutions." Protein determinations were made with a micro-Bradford protein reaction and with uv absorbance at 280 nm. Soluble membrane components contributed to aberrant uv spectra and altered the ratio of 280/260-nm absorbance. Simultaneous dialysis and concentration in the micro protein dialyzer-concentrator apparatus, combining aspects of thin-layer dialysis and ultrafiltration, resulted in rapid removal of salts from the protein solutions. Prior treatment of membranes reduced uncertainties in retentate recoveries, eliminated uv-absorbing components of membranes, and improved recoveries of protein.     

Isolation of pituitary fibroblast growth factor by fast protein liquid chromatography (FPLC): partial chemical and biological characterization

Bovine pituitary fibroblast growth factor has been purified 222,000-fold to homogeneity by a combination of differential salt extraction, gel filtration, and ion exchange chromatography on Mono S column. Pituitary FGF is a single-chain polypeptide with an apparent molecular mass of 15,800 and an isoelectric point of 9.6. It is highly active in triggering the proliferation of bovine and human vascular endothelial cell [half-maximal stimulation at 23-40 pg/ml (1.5-2.6 pM) and saturation between 140 and 280 pg/ml (9.3-18.6 pM)]. It displays a similar activity on bovine vascular smooth muscle cells, corneal endothelial cells, granulosa and adrenal cortex cells, and rabbit costal chondrocytes.     

Effect of intravenous infusion of insulin in diabetics with acute myocardial infarction.

Diabetes mellitus is associated with a high mortality after myocardial infarction. To see whether this may be decreased by improved diabetic control the effect of an insulin infusion regimen was studied in patients with acute myocardial infarction. From April 1982 to April 1983, 33 diabetics were admitted with acute myocardial infarction. Those being treated with diet alone or oral hypoglycaemic drugs continued with this unless control was poor, when they were changed to a "sliding scale" regimen of subcutaneous insulin injections thrice daily. Those already receiving insulin were maintained on thrice daily subcutaneous injections. From April 1983 to April 1984, 29 diabetics had acute myocardial infarction. Those receiving treatment with oral hypoglycaemic drugs or insulin were changed to continuous intravenous infusion of insulin, the aim being to maintain the blood glucose concentration at 4-7 mmol/I (72-126 mg/100 ml). Those being treated with diet alone continued with this if blood glucose concentrations were acceptable. Total mortality fell from 42% in the first year to 17% in the second (p less than 0.05). Over the same period mortality among non-diabetic patients with myocardial infarction did not change significantly. There was a significant fall in cardiac arrhythmias (expressed as the percentage of patients in whom arrhythmias were recorded) from 42% to 17% (p less than 0.05). The most significant fall in the incidence of complications occurred in those who had been receiving oral hypoglycaemic drugs on entry to the study (87% to 50%, p less than 0.05).     

Whole-cell antigens of members of the sulfate-reducing genus Desulfovibrio

Antisera have been developed against the wholecell antigens of Desulfovibrio africanus Benghazi and Walvis Bay, D. vulgaris Hildenborough, D. salexigens British Guiana, D. gigas, and D. desulfuricans Essex 6. An enzymelinked immunoadsorption assay (ELISA) was developed to measure the reaction of these antisera with the homologous and heterologous antigens. The ELISA method demonstrated a reaction between pre-immune sera and cells of D. africanus, D. gigas and D. desulfuricans, suggesting the presence of a lectin-like substance on these cell surfaces. Extensive cross-reactions were seen between the antisera and heterologous cells, suggesting the sharing of a number of surface antigens amongst the Desulfovibrio. However, the pattern of these cross-reactions was different from that observed for an ELISA reaction developed for the cytochrome c₃ from various Desulfovibrio.Abbreviation ELISA enzyme-linked immunoadsorption assay     

Demonstration of a proteolytic activity on intracellular origin in Aeromonas hydrophila LP 50

Aeromonas hydrophila LP 50, isolated from packaged pasteurized milk, was grown in glucose-polypeptone medium at 30 degrees C. The proteolytic activity of A. hydrophila LP 50, optimum at the stationary phase of growth, is attributed to extracellular or membrane protease; no intracellular proteolytic activity was shown.     

The effect of grazing intensity on phosphorus spiralling in autotropic streams

The effect of grazing on primary productivity and phosphorus cycling in autotrophic streams was studied using the snail Goniobasis clavaeformes. Snails were added to each of three replicate laboratory stream channels, receiving once-through flow of groundwater, in densities of 2.1, 3.0, and 4.2 g ash free dry mass (AFDM)/m². A fourth channel received no snails and served as an ungrazed control. Presence of snail grazers resulted in a large reduction in aufwuchs biomass, primary productivity, and biotic phosphorus uptake; a modest reduction in fine particulate organic matter (FPOM); and an increase in the fraction of stream particulate organic matter (POM) exported as seston. Although primary production and aufwuchs biomass continued to decline with increasing snail density, phosphorus uptake increased. This increased phosphorus uptake is attributed to abiotic sorption to inorganic surfaces exposed as a result of efficient removal of aufwuchs at high snail densities. Although snail densities were chosen to bracket the density measured in a natural stream, the experimental densities may result in considerably higher grazing pressure on aufwuchs due to the absence of alternate food sources (e.g., coarse particulate organic matter) usually found in natural streams. Presence of snail grazers increased the spiralling length of phosphorus, primarily by reducing aufwuchs biomass and consequently reducing uptake of phosphorus from the water. Presence of snails also increased downstream transport velocity of phosphorus bound to organic particles. These results follow the patterns predicted in a previous theoretical analysis for mildly phosphorus-limited streams.