Immunochemical cross reactivity of the antibody elicited against L-histidine decarboxylase purified from the whole bodies of fetal rats with the enzyme from rat brain

Rat cytosolic glutathione S-transferases catalyzed the conjugation of phenethyl chloride and phenethyl bromide with glutathione. The reaction proceeded with a high degree of stereoselectivity. The glutathione conjugate possessing the (R,S,S)- absolute configuration was formed in major quantities from the racemic substrates. The use of the enantiomers of the phenethyl chloride substrates indicated that the (S)-phenethyl chloride was conjugated in preference to the (R)-enantiomer. The conjugation proceeded with inversion of configuration at the benzylic carbon consistent with an SN₂-type mechanism. The stereoselectivity was greater for phenethyl chloride than for phenethyl bromide. Varying the substrate or enzyme concentration had no effect upon the observed stereoselectivity. The diastereomeric glutathione conjugates were separated by high performance liquid chromatography. These findings represent the first demonstration of the substrate stereoselectivity of the glutathione S-transferases.     

Hepatic heme metabolism in selenium-deficient rats: effect of phenobarbital.

Hepatic heme metabolism was examined in selenium (Se)-deficient and Se-adequate (control) rats. Administration of phenobarbital stimulated heme synthesis in the liver in both Se-deficient and Se-adequate rats. In contrast to these results, phenobarbital increased microsomal heme oxygenase (MHO) activity six- to eightfold in Se-deficient but not control rats. These data suggest that the previously reported abnormalities of cytochrome P-450 induction in Se-deficient rats are related to increased degradation of hepatic heme.     

The effect of choline deficiency on the activity of a phosphatidylcholine-requiring enzyme: Activity and properties of UDP-glucuronyltransferase in choline-deficient rats

The effect of choline deficiency on the kinetic properties of the microsomal enzyme UDP-glucuronyltransferase (EC2.4.1.17) was investigated in rats. Animals fed choline-deficient diets, as compared with animals fed a choline-replete diet or standard laboratory chow, showed almost a three-fold increase in enzyme activity when the enzyme was assayed at physiological concentrations of UDP-glucuronic acid (0.25 mM). The increase in activity appeared to be due to an enhanced affinity of the enzyme for UDP-glucuronic acid rather than to an increase in the amount of enzyme. These data indicate that the kinetic properties of tightly bound membrane enzymes are altered by a dietary change that is known to cause liver disease in the rat.     

Increased chemiluminescence and superoxide production in the liver of chronically ethanol-treated rats

Rats fed ethanol (1.74 +/- 0.12 g/day/100 g body wt for 12 weeks) showed a 45% increased microsomal production of O-2 (2.23 +/- 0.14 nmol/min/mg protein) and a 28% increased content of endoplasmic reticulum protein (26.8 +/- 1.4 mg/g liver). This could lead, at substrate saturation, to a 86% increased cytosolic production of O-2 which is not compensated by cytosolic superoxide dismutase levels that remain normal. It is claimed that this unbalance between O-2 production and superoxide dismutase leads to a peroxidative stress in agreement with the 54% increased spontaneous liver chemiluminescence (37 +/- 2 cps/cm2) measured in the ethanol-treated rats. Hydroperoxide-induced chemiluminescence was 57, 43, and 28% higher, respectively, in homogenates, mitochondria, and microsomes isolated from ethanol-treated rats as compared with controls. Vitamins E and A were more effective inhibitors of the hydroperoxide-stimulated chemiluminescence in the liver homogenates from ethanol-treated rats as compared with the effect on the homogenates from control animals. The results are consistent with a peroxidative stress in chronic alcoholism leading to increased lipoperoxidation and decreased levels of antioxidants.     

The effect of PGF2a on in vivo cerebral arteriolar diameter in cats and rats

We compared the effect of tropical application of PGF_2a on cerebral arterioles in cats and rats equipped with an acutely implanted cranial window. Arterial diameter was measured using a microscope and image splitting device. PGF_2a in a concentration ranging from 10^?7 to 10^?5 M had no effect on large (≥ 100 μm) or small (< 100 μm) cat pial arterioles, but induced a dose dependent constriction of rat pial arterioles with a maximum constriction fo 76% of control diameter. Dilation of cat large cerebral arterioles by topically applied PGE₂ was not affected by simultaneous application of PGF_2a and PGE₂ induced dilation of small arterioles was decreased 3% by PGF_2a. While we and others have previosuly shown that both cat and rat brain can synthesize PGF_2a, it appears that PGF_2a is not likely to normally be a major modulator of cerebral arteriolar resistance in all species.     

Kinetics of inhibition of ethanol metabolism in rats and the rate-limiting role of alcohol dehydrogenase

If liver alcohol dehydrogenase were rate-limiting in ethanol metabolism, inhibitors of the enzyme should inhibit the metabolism with the same type of kinetics and the same kinetic constants in vitro and in vivo. Against varied concentrations of ethanol, 4-methylpyrazole is a competitive inhibitor of purified rat liver alcohol dehydrogenase (Kis = 0.11 microM, in 83 mM potassium phosphate and 40 mM KCl buffer, pH 7.3, 37 degrees C) and is competitive in rats (with Kis = 1.4 mumol/kg). Isobutyramide is essentially an uncompetitive inhibitor of purified enzyme (Kii = 0.33 mM) and of metabolism in vivo (Kii = 1.0 mmol/kg). Low concentrations of both inhibitors decreased the rate of metabolism as a direct function of their concentrations. Qualitatively, therefore, alcohol dehydrogenase activity appears to be a major rate-limiting factor in ethanol metabolism. Quantitatively, however, the constants may not agree because of distribution in the animal or metabolism of the inhibitors. At saturating concentrations of inhibitors, ethanol is eliminated by inhibitor-insensitive pathways, at about 10% of the total rate at a dose of ethanol of 10 mmol/kg. Uncompetitive inhibitors of alcohol dehydrogenase should be especially useful for inhibiting the metabolism of alcohols since they are effective even at saturating levels of alcohol, in contrast to competitive inhibitors, whose action is overcome by saturation with alcohol.     

Insulinotropic effect of the tumor promoter teleocidin in isolated pancreatic islets

A tumor promoter teleocidin induced insulin secretion from isolated pancreatic islets in a concentration-dependent manner. The teleocidin-induced secretion was inhibited by p-bromophenacyl bromide, nordihydroguaiaretic acid, 3-amino-1-(3-trifluoromethylphenyl)-2-pyrazoline and 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1,4-benzoquinone, but not by indomethacin. Insulinotropic concentrations of teleocidin stimulated 6-keto-prostaglandin F1 alpha release from pancreatic islets. These results suggest that phospholipase A2 activation and lipoxygenase product(s) are involved in the mechanism of teleocidin-induced insulin secretion.     

Nutritional effect of possible intermediates of phytosterol dealkylation in the silkworm, Bombyx mori

Several possible substrates and intermediates of phytosterol dealkylation were tested for their ability to support growth and development in the silkworm, $\text{Bombyx mori}$. These compounds were classified into “effective” (fully substitutive for cholesterol), “partially effective” (partially supporting growth) and “ineffective” These results are discussed in relation to the mechanism of phytosterol dealkylation in insects.     

Dietary tyrosine suppresses the rise in plasma corticosterone following acute stress in rats

Acute, uncontrollable stress increases norepinephrine (NE) turnover in the rat's brain (depleting NE) and diminishes the animal's subsequent tendency to explore a novel environment. Pre-treatment with tyrosine can reverse these adverse effects of stress, presumably by preventing the depletion of NE in the hypothalamus. Numerous studies suggest that NE inhibits the release of adrenocorticotropic hormone (ACTH) by suppressing corticotropic releasing factor (CRF) secretion in the hypothalamus. In the present study, we found that pre-treatment with supplemental tyrosine not only prevented the behavioral depression and hypothalamic NE depletion observed after an acute stress, but also suppressed the rise in plasma corticosterone. These results support a role for brain NE in stress-induced corticosterone secretion and demonstrate that supplemental tyrosine can protect against several adverse consequences of such stress.     

Sexual behavior in male rats treated with estrogen in combination with dihydrotestosterone

Male rats castrated at 30 days of age were treated with estradiol benzoate (dose range: 0.05–50 μg EB for 26 days) and dihydrotestosterone (1 mg DHT for 36 days) as adults. The combined EB and DHT treatments resulted in display of male sexual behavior which did not differ from the behavior shown by intact untreated males or castrated, testosterone propionate (1 mg TP for 26 days) treated males. EB alone or DHT alone were relatively ineffective in activating male behavior in castrated males.     

Fluorescence characterization of the low pH-induced change in diphtheria toxin conformation: effect of salt

When injected into rats, a certain amount of mannitol is taken up by the liver and is associated with sedimentable structures. Isopycnic centrifugation in a sucrose gradient shows that a large part of mannitol is present in mitochondria, what remains is located in the lysosomes. The hypotonic release of mannitol present in organelles shows that the polyol is shared between mitochondria and lysosomes. The trapping of mannitol in lysosomes could result from the heterophagic or autophagic function of the lysosomes; the mechanism of its accumulation in mitochondria is still unexplained.     

Structure of membrane-associated and solubilized uterine adenylate cyclase. Evidence against activation through dissociable subunit interactions in the lipid bilayer

Adenylate cyclase was extracted from the rat uterus with Lubrol PX in a form which remained soluble following centrifugation for 60 min at 100,000g. The soluble enzyme was stimulated by both Mn+2 and by guanyl-5'-yl-imidodiphosphate (Gpp(NH)p), indicating that both the catalytic subunit (C) and the guanyl nucleotide-binding coupling factor (N) had been extracted. Catalytic activity was bound by a GTP-affinity resin only under conditions which resulted in irreversible activation of the native (particulate) form of the enzyme and could be eluted under acidic conditions shown to reverse the activated state. The S020,w of the soluble enzyme in both its activated and unactivated state was determined by linear sucrose gradient centrifugation. Activation by prolonged treatment with Gpp(NH)p did not alter the S020,w of the enzyme whether treatment was carried out before or after solubilization. The chaotrope LiBr (0.4 M) reduced the S020,w of the soluble enzyme but its smaller size was still not altered by activation with Gpp(NH)p. These results indicate that most adenylate cyclase activity in uterine membranes exists as a preformed complex between the catalytic subunit and the coupling factor: NC. The existence of this complex explains some of the temperature-dependent properties previously described for this form of the enzyme and suggests that dissociable interactions between the subunits do not play a role in the activation of C by guanyl nucleotides.     

The effect of deoxymannojirimycin on the processing of the influenza viral glycoproteins

Deoxymannojirimycin (dMM) was tested as an inhibitor of the processing of the oligosaccharide portion of viral and cellular N-linked glycoproteins. The NWS strain of influenza virus was grown in MDCK cells in the presence of various amounts of dMM, and the glycoproteins were labeled by the addition of 2-[3H]mannose to the medium. At levels of 10 micrograms/ml dMM or higher, most of the viral glycopeptides became susceptible to digestion by endoglucosaminidase H, and the liberated oligosaccharide migrated mostly like a Hexose9GlcNAc on a calibrated column of Bio-Gel P-4. This oligosaccharide was characterized as a typical Man9GlcNAc by a variety of chemical and enzymatic procedures. Deoxymannojirimycin gave rise to similar oligosaccharide structures in the cellular glycoproteins. In both the viral and the cellular glycoproteins, this inhibitor caused a significant increase in the amount of [3H]mannose present in the glycoproteins. Deoxymannojirimycin did not inhibit the incorporation of [3H]leucine into protein in MDCK cells, nor did it affect the yield or infectivity of NWS virus particles. However, its effect on mannose incorporation into lipid-linked saccharides depended on the incubation time, the virus strain, and the cell line. Thus, high concentrations of dMM showed some inhibition of mannose incorporation into lipid-linked oligosaccharides with the NWS strain in a 3-h incubation, but no inhibition was observed after 48 h of incubation. On the other hand, the PR8 strain was much more sensitive to dMM inhibition, and mannose incorporation into lipid-linked oligosaccharides was strongly inhibited when the virus was raised in chick embryo cells, but less inhibition was observed when this virus was grown in MDCK cells. Nevertheless, in these cases also, the major oligosaccharide structure in the glycoproteins was the Man9GlcNAc2 species.     

Alterations in central neurotransmitter receptor binding sites following estradiol implantation in female rats

The subcutaneous implantation of an estradiol pellet (10 mg) into female rats induced a hypophyseal hyperplasia with hyperprolactinaemia. Examination of neurotransmitter receptors in the hippocampus, striatum and cerebral cortex one month after the implantation revealed that estrogenization was associated with: an increased density of ³H-domperidone binding sites (D₂ receptors) in the striatum and reduced numbers of ³H-serotonin high affinity sites (5-HT₁ receptors) in the hippocampus and of ³H-muscimol binding sites (GABA receptors) in the hippocampus, striatum and cerebral cortex. In contrast, the characteristics of ³H-spiperone binding to 5-HT₂ receptors (in the cerebral cortex) and those of ³H-flunitrazepam binding to benzodiazepine sites (in the three brain regions examined) were not significantly different in estrogenized and in control female rats. However, the enhancing effect of GABA on ³H-flunitrazepam binding was markedly reduced in brain membranes from estrogenized animals. The respective roles of estradiol and prolactin in mediating these changes in neurotransmitter receptors are discussed notably with regard to the regional heterogeneity of estradiol binding capacity in the rat brain.     

Reye's syndrome: the effect of patient serum on mitochondrial respiration in vitro

Serum from patients with Reye's Syndrome stimulated state 4 respiration in isolated rat liver mitochondria. Inhibitors of specific mitochondrial functions were tested as potential antagonists of the stimulatory effect of RS serum. Oligomycin, ruthenium red, rotenone and antimycin A were all ineffective in preventing the increase in state 4 respiration, but KCN completely abolished all respiratory activity in the presence of RS serum. We conclude that the putative serum factor stimulates respiration by directly or indirectly interacting with the electron transport chain at a point beyond site II.     

Germ-line dependence of the maroon-like maternal effect in Drosophila

We have used pole cell transplantations to construct germ-line mosaics for maroon-like (mal), a maternal effect mutation in Drosophila. Such mosaics allow one to determine the cell type in which a gene is active. We find that the maroon-like maternal effect is (1) autonomous to the germ line and (2) dose sensitive in germ-line mosaics. Aldehyde oxidase activity is used as a histological probe to investigate the tissue and temporal distribution of mal⁺ activity in the developing ovary. The adult ovary shows mal⁺ activity in the germ line at all discernible stages of oogenesis but no activity is observed in the mesodermally derived follicle cells. Differential mal⁺ activity is observed even in the ovary of the third-instar larvae.     

The effect of sulfhydryl group reagents on the permeation of mitochondrial aspartate aminotransferase into mitochondria in vitro.

When mitochondria are incubated with radioactively labeled mitochondrial aspartate aminotransferase (EC 2.6.1.1), the enzyme is taken up into the organelles. Mersalyl and p-hydroxymercuriphenyl sulfonic acid, but not N-ethylmaleimide or ethacrynic acid, decrease the extent of this uptake. Inhibition of the uptake by low concentrations of mercurial reagents is due to blockage of a single sulfhydryl group per monomer of the enzyme. Blockage of mitochondrial thiols does not inhibit uptake of the enzyme. A single sulfhydryl group out of a total of six per monomer of the native enzyme reacts with 5,5′-dithiobis-(2-nitrobenzoic acid). This is the same sulfhydryl group that reacts with low levels of mercurial reagents with consequent inhibition of uptake of the enzyme into mitochondria but without effect on the catalytic activity. N-Ethylmaleimide does not react with this group. N-Ethylmaleimide reacts with a different sulfhydryl group with concomitant decrease in enzymic activity but with no effect on uptake of the enzyme into mitochondria. High levels of mercurial reagents similarly decrease enzymic activity. Unlike the effect on uptake into mitochondria, the inhibition by mercurial reagents of enzymic activity is not reversed by treatment with cysteine. The significance of these observations with respect to the mechanism of uptake of aspartate aminotransferase into mitochondria is discussed, and comparisons are made between the reactivities of sulfhydryl groups in rat liver aspartate aminotransferase and in the enzymes from other animals.