Changes in aerobic and anaerobic ATP-synthesizing activities in hypoxic mouse brain

The changes in cerebral metabolism in mice in severe hypoxia were investigated by analyses of changes in the levels of energy metabolites and near-infrared spectrophotometric assessment of the states of hemoglobin and cytochrome oxidase. Under 4.4% O2, the contribution of anaerobic ATP production was at most about 20% of the demand. However, the cerebral ATP level was kept at the control level until about 1 min before death. Pentobarbital anesthesia, which reduced the cerebral rate of metabolism, prolonged the survival time, although anaerobic ATP production still did not support ATP demand. Under these conditions, deoxygenation of hemoglobin and reduction of cytochrome oxidase proceeded rapidly within 1 min. Hemoglobin reached the maximum state of deoxygenation in the middle phase of hypoxia, with no further change until death. However, cytochrome oxidase was reduced slowly with one phase of partial reoxidation due to increase of cerebral blood volume, and reached the completely reduced state at death. From these results it was concluded that the aerobic ATP synthesis, which supplied more than 80% of the cerebral demand, decreased gradually because of limitation of oxygen supply, and that the failure of oxidative phosphorylation to meet demand triggered the decrease in the cellular ATP level that led to death.     

A male-specific renal cytochrome P-450 isozyme(s) responsible for mutagenic activation of 3-methoxy-4-aminoazobenzene in mice

Renal microsomes from male mice (BALB/c, DBA/2 and BALB/c x DBA/2 F1) showed about 10-fold greater activity for mediating mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) toward Salmonella typhimurium TA98 than did the corresponding hepatic microsomes, as compared on the basis of nmol of microsomal cytochrome P-450. On the other hand, female renal microsomes and other extrahepatic microsomes (lung, small intestine and colon) in both sexes of mice showed little or no activity for converting 3-MeO-AAB to mutagen(s). The mutagenic activation of 3-MeO-AAB with the male renal enzyme(s) was definitely inhibited by cytochrome P-450 inhibitors, 7,8-benzoflavone and SKF 525A. All these findings suggest that in mice, there is a male-specific renal 3-MeO-AAB activation enzyme(s), a cytochrome P-450 isozyme(s), which is different, at least in proportion and/or in nature, from hepatic cytochrome P-450 isozymes.     

Iodoacetylated and biotinylated liposomes: effect of spacer length on sulfhydryl ligand binding and avidin precipitability

Because of the sustained interest in liposomes as immunogens and vehicles for drug delivery, the present investigation was designed to reevaluate the iodoacetyl group as a means of binding sulfhydryl-containing substances to liposomes in thioether linkage, and to develop an alternative method by which liposomes with bound ligand can be conveniently and rapidly separated from free ligand. For the purpose of the first goal, we synthesized a homologous series of dimyristoylphosphatidylethanolamine (DMPE) derivatives in which the iodoacetyl (IA) function was separated from the phospholipid amino group by either 0, 1, or 2 aminoethylthioacetyl (AETA) spacers. Results show that liposomes prepared with IA-DMPE can not bind 125I-radiolabeled rabbit IgG which had been thiolated by reaction with S-acetylmercaptosuccinic anhydride. Significant IgG attachment was, however, obtained with liposomes containing either IA-AETA-DMPE or IA-(AETA)2-DMPE, and the amount bound was directly related to spacer length. In contrast, spacer length had no effect on the covalent binding of a low molecular weight hapten, N-dinitrophenylcysteine. Other parameters (incubation time, IgG concentration, density of IA-(AETA)2-DMPE, sulfhydryl inhibitors) were also examined. To achieve the second objective, biotinyl-(AETA)2-DMPE was incorporated into the same liposomal bilayers that contained the iodoacetylated derivatives. Thus, liposomes with bound ligand could be readily precipitated by avidin, and washed free of unreacted IgG by low speed centrifugation. Comparative experiments with liposomes containing biotinyl-DMPE revealed that spacer length also had a pronounced effect on the avidin precipitability of liposomes in the presence of proteins that may be non-covalently absorbed or covalently bound to the model membrane surface.     

Gender-related effects of chronic ethanol ingestion on rat hepatic acyl-CoA:cholesterol acyltransferase

The influence of chronic ethanol ingestion on hepatic acyl-CoA: cholesterol acyltransferase activity was investigated to determine the relationship between alcohol intake and cholesterol ester accumulation. Rats were given nutritionally complete liquid diets supplemented with 6.3% ethanol or an isocaloric equivalent of dextrin-maltose for 5 weeks. During this period, the hepatic acyl-CoA: cholesterol acyltransferase activity of ethanol-fed male rats remained constant, whereas the same activity in pair-fed controls as well as chow-fed rats exhibited a 30% decrease in activity. Unlike alcohol-fed male rats, the hepatic acyl-CoA: cholesterol acyltransferase activity of female rats decreased by approximately 30% by the fifth week of ethanol ingestion. Despite the fact that the gender of the animals led to disparate levels of acyl-CoA: cholesterol acyltransferase activity in response to ethanol ingestion, similar levels of cholesteryl ester accumulation were observed. The altered levels of acyl-CoA: cholesterol acyltransferase activity caused no significant change in the cholesterol concentration, cholesterol/phospholipid ratio, phospholipid fatty acid composition, or the membrane fluidity of the hepatic microsomes. We conclude that the altered hepatic acyl-CoA: cholesterol acyltransferase activity of ethanol-fed female rats cannot be directly responsible for ethanol-induced accumulation of cholesteryl esters.     

Pretreatment of wheat straw for fermentation to methane

The effects of pretreating wheat straw with gamma-ray irradiation, ammonium hydroxide, and sodium hydroxide on methane yield, fermentation rate constant, and loss of feedstock constituents were evaluated using laboratory-scale batch fermentors. Results showed that methane yield increased as pretreatment alkali concentration increased, with the highest yield being 37% over untreated straw for the pretreatment consisting of sodium hydroxide dosage of 34 g OH(-)/kg volatile solids, at 90 degrees C for 1 h. Gamma-ray irradiation had no significant effect on methane yield. Alkaline pretreatment temperatures above 100 degrees C caused a decrease in methane yield. After more than 100 days of fermentation, all of the hemi-cellulose and more than 80% of the cellulose were degraded. The loss in cellulose and hemicellulose accounted for 100% of the volatile solids lost. No consistent effect of pretreatments on batch fermentation rates was noted. Semicontinuous fermentations of straw-manure mixtures confirmed the relative effectiveness of sodium and ammonium-hydroxide pretreatments.     

Spontaneous Release of γ-Aminobutyric Acid Formed from Putrescine and Its Enhanced Ca2+-Dependent Release by High K+ Stimulation in the Brains of Freely Moving Rats

The spontaneous release of [3H] gamma-aminobutyric acid ([3H]GABA) in various areas of rat brain injected with [3H]putrescine was examined using a push-pull perfusion technique. The release in a 25-min perfusate was highest in the caudate-putamen. The effect of high K+ stimulation on the release of [3H]GABA formed from [3H]putrescine was examined in the caudate-putamen. The release was enhanced by high K+ solution in a Ca2+-dependent manner.     

Starvation-induced changes in rat brain corticotropin-releasing factor (CRF) and pituitary-adrenocortical response

Starvation-induced changes in CRF concentration in major brain regions and abnormalities in the pituitary-adrenal axis were examined in rats using rat CRF radioimmunoassay. The CRF concentrations in the hypothalamus and cerebellum were significantly reduced in the completely starved rats, while those in the midbrain, thalamus and neurointermediate lobe of the pituitary were significantly increased in the semi-starved or completely starved rats. No significant changes in the CRF concentrations were found in the pons, medulla oblongata and cerebral cortex. In the completely starved rats, the serum ACTH level was significantly reduced, whereas the serum corticosterone level was markedly elevated. These observations suggest that starvation may stimulate the CRF-ACTH-corticosterone system and that not only hypothalamic CRF but also extrahypothalamic CRF may be discretely related to feeding behavior or starvation. The reduced serum ACTH level in starved rats may be ascribed to the negative feedback effect of the elevated serum corticosterone.     

Growth and Aspartate Kinase Activity in Wheat Cell Suspension Culture: Effects of Lysine Analogs and Aspartate-Derived Amino Acids

The effects of lysine analogs and aspartate-derived amino acidson the growth of wheat cell suspension culture were studied.S-(2-Aminoethyl)-L-cysteine (AEC), -hydroxylysine (DHL) andtrans-lysene caused complete growth inhibition at 1.0 mM. Thegrowth inhibition of lysine analogs were, in the order of decreasingeffectiveness; AECDHL, trans-lysene>oxalysine, homolysineand lysyne. cis-Lysene and methyllysine were not inhibitoryeven at concentrations of 10 mM. Lysine effectively relievedgrowth inhibition induced by the lysine analogs. Lysine plusthreonine showed concerted inhibition, which was relieved bythe addition of methionine. Activity of aspartate kinase extracted from wheat cell suspensionculture was strongly inhibited by L-lysine; 0.75 to 1 mM oflysine was required for half-maximal inhibition. Threonine andmethionine, individually or in combination with lysine, showedno inhibitory effect on the enzyme activity. S-Adenosylmethionine,when added with lysine in equimolar concentrations, enhancedthe feedback inhibition by lysine, lowering the concentrationof lysine for half-maximal inhibition to 0.13 mM. The aspartatekinase isolated from the cells cultured in the presence of 5mM lysine did not differ in regulatory properties from the enzymefrom the cells cultured without lysine. AEC at 5 mM inhibitedthe enzyme activity by 50%. Other lysine analogs were not inhibitoryto the enzyme activity even at 10 mM. Growth inhibition of wheat suspension culture by aspartate-derivedamino acids and lysine analogs were discussed in relation totheir inhibitory effects on aspartate kinase activity. (Received October 25, 1985; Accepted February 26, 1986)     

Hyoscyamine 6beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase, in alkaloid-producing root cultures

Root cultures of various solanaceous plants grow well in vitro and produce large amounts of tropane alkaloids. Enzyme activity that converts hyoscyamine to 6β-hydroxyhyoscyamine is present in cell-free extracts from cultured roots of Hyoscyamus niger L. The enzyme hyoscyamine 6β-hydroxylase was purified 3.3-fold and characterized. The hydroxylation reaction has absolute requirements for hyoscyamine, 2-oxoglutarate, Fe²⁺ ions and molecular oxygen, and ascorbate stimulates this reaction. Only the l-isomer of hyoscyamine serves as a substrate; d-hyoscyamine is nearly inactive. Comparisons were made with a number of root, shoot, and callus cultures of the Atropa, Datura, Duboisia, Hyoscyamus, and Nicotiana species for the presence of the hydroxylase activity. Decarboxylation of 2-oxoglutarate during the conversion reaction was studied using [1-¹⁴C]-2-oxoglutarate. A 1:1 stoichiometry was shown between the hyoscyamine-dependent formation of CO₂ from 2-oxoglutarate and the hydroxylation of hyoscyamine. Therefore, the enzyme can be classified as a 2-oxoglutarate-dependent dioxygenase (EC 1.14.11.-). Both the supply of hyoscyamine and the hydroxylase activity determine the amounts of 6β-hydroxyhyoscyamine and scopolamine produced in alkaloid-producing cultures.