Heme peptide as a model substance for halogenomethane activation and heme modification.

Octa-heme peptide (CHP) obtained from Candida krusei cytochrome c was tested for suicidal activation of halogenomethanes. Under anaerobic conditions, CHP was kept in the reduced state in the presence of NADPH and NADPH-cytochrome P-450 reductase. Addition of CBrCl3 to the reduced CHP caused spectral changes such as rapid disappearance of alpha and beta bands and gradual decrease in the gamma-peak height, accompanied by oxidation of NADPH. Heme content of the reaction mixture, determined as pyridine hemochrome, also decreased NADPH dependently. CCl4 was less effective than CBrCl3, while CHCl3 had almost no effect. N-tert-butyl-alpha-phenylnitrone (PBN) suppressed the CBrCl3-induced heme damage, and resulted in the formation of radical adduct .PBN-CCl3 as evidenced by ESR spectroscopy. Radical formation was also observed with CCl4. The CHP damage induced by CBrCl3 was also accompanied by the release of Br- about 11-12-times molar excess of CHP, whereas the release of CHCl3 was about 20% that of Br-.FD-MS assay of the product of CHP reaction suggested that 10 trichloromethyl radicals bonded with CHP. Thus, CBrCl3 undergoes single-electron reduction in the presence of reduced CHP to trichloromethyl radicals, which covalently bind to CHP molecules. Heme peptide may be a useful tool in the study of mechanisms involved in the destruction of cytochrome P-450 by halogenomethanes.     

Temperature conditions enhancing resting egg production of the euryhaline rotifer Brachionus plicatilis O. F. Müller (Kamiura strain)

We examined the effect of low temperature treatment(12°C), followed by transfer to highertemperature (25°C), on resting egg formation ofthe rotifer Brachionus plicatilis, Kamiurastrain. This strain has been mass cultured as livefeed at Kamiura Station (Japan Sea FarmingAssociation) for 9 years at 20°C without theappearance of sexual reproductive stages.After preculture in 20 l of 27 seawater at 12°C for 0, 10, 20, and 30 days,rotifers were inoculated into 0.5 l mass cultures andcultured at 25°C for 7–9 days. The inoculationdensities were changed from 20 to 400 ind. ml^–1,depending on mixis rate. Condensed and frozen Nannochloropsis oculata was fed to rotifers at thefeeding rate of 0.14 µg (dry weight)rotifer^–1day^–1. The control was cultured at12°C for the entire 36 day experiment. No mixisappeared and no resting eggs were produced when thelow temperature treatment was 0 or 10 days. However,mixis rates reached 50-60% after 20 or 30 days ofexposure to 12°C. The number of resting eggsproduced in these treatments reached 25,500 about 13 times higher than the control. Our resultssuggest that low temperature stimulated mictic femaleproduction and the transfer to the high temperatureaccelerated resting egg formation. This method may beuseful for producing resting eggs of rotifer strainsthat lack sexual reproduction in the common culturecondition at larval rearing facilities.     

Exogenously Hypercholesterolemic Rats, Compared with Their Progenitor Sprague-Dawley Rats, Have Altered mRNAs for Cholesterol 7α-Hydroxylase and Low-Density-Lipoprotein Receptor and Activities of Cholesterol 7α-Hydroxylase and Acyl-CoA:Cholesterol Acyltransferase in the Liver in Response to Dietary Cholesterol

Exogenously hypercholesterolemic (ExHC) rats promptly increase serum cholesterol concentration in response to dietary cholesterol. To examine underlying mechanism(s) for this susceptibility, responses of mRNAs for cholesterol metabolism-related proteins and their activities in the liver to dietary cholesterol were compared between ExHC rats and their progenitor Sprague-Dawley rats. ExHC rats slightly decreased the abundance of low-density-lipoprotein (LDL) receptor mRNA in response to dietary cholesterol, although the amount of LDL receptor was not influenced. The abundance of cholesterol 7α-hydroxylase mRNA and the enzyme activity in response to dietary cholesterol were greater in ExHC rats, but the fecal excretion of bile acid was comparable between the strain. Dietary cholesterol-dependent elevation of acyl-CoA:cholesterol acyltransferase activity was greater in ExHC rats. The concentration of liver triacylglycerols was markedly lower in ExHC rats. These results suggest that ExHC rats may increase serum cholesterol by increasing hepatic secretion of cholesteryl ester-rich particles.     

Exogenous hypercholesterolemic rats, compared with their progenitor, Sprague-Dawley rats, promptly alter cholesterol metabolism in the liver and secrete cholesterol-rich particles in response to dietary cholesterol

Early responses of cholesterol metabolism to dietary cholesterol were compared between exogenous hypercholesterolemic (ExHC) and Sprague-Dawley rats. Both strains had a similar radioactivity of [¹⁴C]cholesterol in the serum half a day after the oral administration, but thereafter the radioactivity disappeared slowly in ExHC rats. ExHC rats promptly altered in response to the dietary cholesterol, activities of cholesterol 7α-hydroxylase and cholesterol synthesis in the liver and fecal excretion of bile acids derived from [¹⁴C]cholesterol administered orally. Lymphatic transport for 24 hr of [¹⁴C]cholesterol was similar between the strains. Triton administration resulted in a marked accumulation of cholesterol in serum d > 1.006 g/ml lipoproteins in ExHC rats; in addition, the formation of cholesteryl esters from [¹⁴C]oleic acid intravenously infused was greater in ExHC rats. These results indicate that ExHC rats increase serum cholesterol in response to exogenous cholesterol by decreasing the liver uptake and enhancing the secretion in the liver.     

Alteration of Antiviral Signalling by Single Nucleotide Polymorphisms (SNPs) of Mitochondrial Antiviral Signalling Protein (MAVS)

Genetic variation is associated with diseases. As a type of genetic variation occurring with certain regularity and frequency, the single nucleotide polymorphism (SNP) is attracting more and more attention because of its great value for research and real-life application. Mitochondrial antiviral signalling protein (MAVS) acts as a common adaptor molecule for retinoic acid-inducible gene-I (RIG-I)-like receptors (RLRs), which can recognize foreign RNA, including viral RNA, leading to the induction of type I interferons (IFNs). Therefore, MAVS is thought to be a crucial molecule in antiviral innate immunity. We speculated that genetic variation of MAVS may result in susceptibility to infectious diseases. To assess the risk of viral infection based on MAVS variation, we tested the effects of twelve non-synonymous MAVS coding-region SNPs from the National Center for Biotechnology Information (NCBI) database that result in amino acid substitutions. We found that five of these SNPs exhibited functional alterations. Additionally, four resulted in an inhibitory immune response, and one had the opposite effect. In total, 1,032 human genomic samples obtained from a mass examination were genotyped at these five SNPs. However, no homozygous or heterozygous variation was detected. We hypothesized that these five SNPs are not present in the Japanese population and that such MAVS variations may result in serious immune diseases.     

Effect of phosphatidylethanolamine and its constituent base on the metabolism of linoleic acid in rat liver

Dietary phosphatidylethanolamine (PE) contributes the circulatory and hepatic free-ethanolamine in rats (Ikeda et al. (1987) Biochim. Biophys. Acta 921, 245). A role for circulatory ethanolamine has not been defined; however, our recent studies have shown that exogenous ethanolamine influences cholesterol and linoleic acid metabolism in rats (Imaizumi et al. (1983) J. Nutr. 113, 2403). In order to understand the role of dietary PE the effects of PE and its base on the hepatic metabolism of linoleic acid were investigated in vivo and in primary cultured hepatocytes in rats. Dietary PE increased the plasmic level of ethanolamine from 37 to 52 microM and decreased the ratio of arachidonate to linoleate in hepatic phospholipids. Activity of hepatic delta 6-desaturase decreased in rats given PE and the desaturation of [14C]linoleate in the cultured hepatocytes decreased by the addition of ethanolamine. Secretion [14C]linoleate labeled very-low-density lipoprotein from the cultured hepatocytes decreased by the addition of ethanolamine. Dietary PE caused an increased formation of CO2 from [14C]acetate by liver slices, and ethanolamine added to the hepatocytes caused an increased oxidation of [14C]linoleate and a suppression of fatty acid synthesis from [3H]serine. These results suggest that ethanolamine derived from the dietary PE plays a regulatory role in the linoleate metabolism in the liver.     

Initial lesions in the mouse brain induced by intraperitoneal injection of hypertonic saline

A dose of 30 ml/kg of 8.5% solution of sodium chloride was intraperitoneally injected into ddY mice and time course observations were conducted. Colloidal carbon was infused 6 hr after injection and distribution of carbon in the brain was observed. A transient increase in hematocrit value, serum osmotic pressure and ascites was observed 1 hr arter injection. Distribution of colloidal carbon in the brain showed less penetration of the basal ganglia. Histopathological examination of the brain showed degeneration of pyramidal cells in hippocampus 6 hr after injection. Electron microscopical examination revealed intracytoplasmic microvacuoles, swollen mitochondria and dilated cisternae in rough-surfaced endoplasmic reticulum of pyramidal cells. It was suggested that serum viscosity was increased by rapid transport of water from blood into the peritoneal cavity or urine, due to the high osmotic pressure of hypertonic saline injected, resulting in a decrease in blood flow and ischemic changes in the brain.     

Electron spin resonance study on peroxidase- and oxidase-reactions of horse radish peroxidase and methemoglobin.

The intermediate free radicals generated from phenols, naphthols and benzoate, in the peroxidase- and oxidase-reactions of horse radish peroxidase and in the peroxidase-reaction of methemoglobin, were studied by electron spin resonance spectroscopy.The difference between the peroxidase- and oxidase-reactions of HRP are demonstrated, i.e., the ferro horse radish peroxidase-O₂ system attacks both phenols and benzoate yielding unidentified radicals, which may be hydroxy-cyclohexadienyl radicals, while the horse radish peroxidase-H₂O₂ system reacts only with phenols and naphthols producing the phenoxy-and naphthoxy-radicals.Phenoxy-radical formation from phenols, in the reactions horse radish peroxidase-H₂O₂ and methemoglobin-H₂O₂, occurs independently of the molecular sizes of phenols but dependently on their redox-potentials.On the basis of kinetic studies on methemoglobin-H₂O₂ system, the existence of a reactive intermediate complex between methemoglobin and H₂O₂ is proposed, which may be similar to compound-I or -II of horse radish peroxidase and which further degenerates to MetHb radical. The oxidation of phenols and naphthols takes place outside of the hemepocket of methemoglobin.     

Characterization of ribonucleic acids from the venom glands of Crotalus durissus terrificus (Ophidia, Reptilia) after manual extraction of the venom. Studies on template activity and base composition

RNA synthesis in the venom glands of Crotalus durissus terrificus was stimulated by the manual extraction of the venom (milking). RNA was extracted from venom glands activated by milking and fractionated by centrifugation through sucrose density gradients. Template activity for protein synthesis and base composition of the RNA fractions were studied. RNA fractions that sediment between 18S and 4S had the highest template activity. The base composition analysis indicated that the 28S and 18S rRNA have a C+G content of 65.4 and 58% respectively. The ;melting' temperature (T(m)) of DNA in 0.15m-NaCl-0.015m-trisodium citrate, pH7.0, was 85 degrees C, corresponding to a C+G content of 38%. The base ratio of the RNA fractions that showed a high template activity was intermediate between that of rRNA and homologous DNA. The possible role of these fractions in the synthesis of the two main toxins (crotoxin and crotamine) of the South American rattlesnake's venom is discussed.