Effect of vitamin B-12 deprivation on the rates of synthesis and degradation of rat liver fatty acid synthetase

To characterize the basis for the increased hepatic fatty acid synthetase activity in vitamin B-12 deprivation, the content and rates of synthesis and degradation for the enzyme were determined. Animals were in a dietary steady state on normal chow or a B-12-deprived diet; animals on the latter diet were further divided into a “supplemented” group given B-12 and those “B-12-deprived.” The B-12-deprived animals had tissue B-12 depletion. Both total and specific activity of fatty acid synthetase were increased in the B-12-deprived animals, and this was due to increased enzyme protein. Rates of synthesis and degradation were studied in each group after a pulse of 20 μCi of l-[U-¹⁴C]leucine. Radioactivity was determined in the immunoprecipitate of the purified enzyme. Relative rates of synthesis in the B-12-deprived group were increased 8.8-fold over the normal and 3.6-fold over the B-12-supplemented group. Degradation of hepatic fatty acid synthetase was 63 hr ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$) in the normal and 65 hr in the B-12-supplemented group. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ in the B-12-deprived group was 35 hr. Degradation rates for the soluble protein pool were not affected by B-12 deprivation. The rate constant for synthesis of hepatic fatty acid synthetase in the B-12-deprived group was 14-fold that of the normal and 6-fold that of the B-12-supplemented animals. Thus, although vitamin B-12 deprivation results in increased rate of degradation of fatty acid synthetase, enzyme synthesis is markedly increased yielding a severalfold net increase in synthetase content and activity.     

Effects of estrogens on aryl hydrocarbon hydroxylase mediated binding of benzo(a) pyrene metabolites to DNA in vitro

$\text{In vivo}$ and $\text{in vitro}$ studies were carried out to determine the effects of estradiol and other steroid hormones on aryl hydrocarbon hydroxylase-mediated binding of benzo(a)pyrene metabolites to DNA. Injection of female $\text{C57}\text{B1}\text{6J}$ mice with 0.2 mg or 2 mg of estradiol 24 hours prior to, during and 24 hours after injection of 3-methylcholanthrene resulted in a significant decrease in the capacity of hepatic microsomes from these animals to mediate the binding of benzo(a)pyrene metabolites to DNA when compared to microsomes from animals receiving 3 methylcholanthrene treatment only. Binding of benzo(a) pyrene metabolites was inhibited between 22 and 50%, depending on the dose of estradiol used. The enzyme and cytochrome components of the aryl hydrocarbon hydroxylase multienzymic complex were not affected by either estradiol treatment. The data suggests that estradiol inhibits aryl hydrocarbon hydroxylase mediated binding of benzo(a)pyrene metabolites to DNA by activity as a non-competitive inhibitor of aryl hydrocarbon hydroxylase activity.     

Morphological analysis and fate map of the intersexual genital disc of the mutant doublesex-dominant in Drosophila melanogaster

The organization of the genital disc in $\text{XX;dsx}{}^{\mathrm{D}}\text{+}$ animals, which have elements of both male and female genitalia, has been analyzed. The intersexual disc contains three major regions, which were isolated by fragmentation. After metamorphosis in host larvae, these regions produced the male genitalia, the female genitalia, and the analia, respectively. Thus, in contrast to the wild-type male and female constitution, the intersexual genetic constitution of $\text{XX;dsx}{}^{\mathrm{D}}\text{+}$ animals allows both genital primordia to develop and to differentiate adult structures. A fate map of the intersexual disc is presented. Observations made on the morphology of the genital disc and its derivatives in $\text{XX;dsx}{}^{\mathrm{D}}\text{+}$ animals and other “intersexual” genetic constitutions are compared and discussed in terms of the development of the sexual dimorphism.     

Isolation of coccidioides immitis from bat guano and preliminary findings on laboratory infectivity of bats with Coccidioides immitis

$\text{Coccidioides}$$\text{immits}$ has been isolated from the guano of bats obtained beneath bat roosting places deep within deserted mine tunnels. Certain species of bats have been demonstrated to be susceptible to experimental infection by $\text{C.}$$\text{immitis}$. There is, however, a difference in the laboratory susceptibility between the species studied ($\text{Antrozous}$$\text{pallidus}$ and $\text{Macrotus}$$\text{californicus}$). $\text{Antrozous}$$\text{pallidus}$ requires 8 times more arthrospores (400 as compared to 50) than does $\text{Macrotus}$$\text{californicus}$ to produce a defineable infection. Our cultures for $\text{C.}$$\text{immitis}$ in the tissue of $\text{Antrozous}$ produced colonies of the bacterium $\text{Serratia}$$\text{marcescens}$. Because these tissues were handled under sterile conditions the presence of this red pigment (prodigiosin) producing bacterium was thought not the result of contamination. Prodigiosin suggests a possible mechanism for the apparent greatly reduced susceptibility to $\text{C}$. $\text{immitis}$ found in the laboratory population of $\text{Antrozous}$. Skin and serological tests on bats were negative as were cultures of guano from experimentally infected bats and mice. It is speculated that infected dying bats that fall to the floor beneath their roosting sites may contaminate the guano. We of course realize that other animals (we have found $\text{Neotoma}$ and $\text{Bassariscus}$ rarely in these tunnels) may have tracked the fungus into these deep tunnel sites.     

Modification of hepatic microsomal oxidative drug metabolism in rats by the opiate maintenance drugs acetylmethadol, propoxyphene, and methadone.

The formation of cytochrome P-450 “metabolic intermediate” complexes $\text{in}$$\text{vivo}$ occurred with acetylmethadol and propoxyphene, but not methadone in both naive and phenobarbital-induced animals. The $\text{in}$$\text{vivo}$ formation correlated with the relative ability of these three compounds to form metabolic intermediate complexes and inhibit mixed-function oxidation reactions $\text{in vitro}$.     

Influence of protease inhibitors and energy metabolism on intracellular protein breakdown in starving Escherichia coli

It has been reported that certain inhibitors of serine proteases block intracellular protein breakdown in $\text{E. coli}$ subjected to nutritional deprivation. We show here that the protease inhibitors p-toluene sulfonyl fluoride and pentamidine isethionate inhibit protein breakdown in $\text{E. coli}$ deprived of glucose, but not in bacteria starved for inorganic phosphate or ammonia. Furthermore, we find that the protease inhibitors cause a drastic decline in cellular ATP levels when glucose is omitted from the incubation medium. It is concluded that these protease inhibitors influence protein breakdown by interfering with cellular energy production, rather than by interacting with a specific serine protease.     

The effect of visible light on the regeneration of rhodopsin

Both $\text{in}\text{vitro}$ and $\text{in}\text{vivo}$, increased exposure to visible light decreases the regenerability of the visual pigment. Isolated opsin irradiated with increasing periods of white light decreased in pigment formation yields on combination with 9- or 11-$\text{cis}$ retinal. The yield of regeneration of the visual pigment extracted from albino rats depended on the amount of light to which the animal had been exposed. Animals exposed to normal room light demonstrated lower regeneration yields than dark-reared animals, but these yields increased on dark adaption. Opsin from animals exposed to sunlamps did not regenerate any pigment. On dark adaption, the pigment yields increased but the opsin level remained below that for the control group.     

Polysome level and stability in stringent E. Coli strains under aminoacyl-tRNA deprivation

Under aminoacyl-tRNA deprivation polysome level is reduced to a different degree in various stringent $\text{E. Coli}$ strains. Polysomes which are maintained in such deprived cells undergo continual turnover. The higher polysome level measured in certain strains seems to be due to a higher ability of these strains to assemble polysomes rather than to a lower rate of polysome decay.     

The phosphorylation of troponin B by phosphorylase b kinase in skeletal muscle of mice carrying the phosphorylase b kinase deficiency gene

In skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene there is < 1% of the normal activity to convert phosphorylase $\text{b}$ to $\text{a}$ in the presence of Ca⁺⁺, Mg⁺⁺, and ATP (1). Correspondingly, there is < 1% of the normal activity to phosphorylate phosphorylase $\text{b}$. Nevertheless, under the same conditions, these extracts catalyze the phosphorylation of troponin at a rate 57% of normal. Phosphorylase $\text{b}$ converting activity can be sedimented from skeletal muscle of control mice by centrifugation. This fraction isolated from I strain skeletal muscle extracts phosphorylates troponin at a rate 29–39% of the control. EGTA¹ (15 mM) inhibits troponin phosphorylation by 50–60% in this fraction from both strains. The EGTA inhibition is reversed by 15 mM Ca⁺⁺. Thus the phosphorylase $\text{b}$ kinase in skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene can phosphorylate troponin B, although it shows little or no activity with phosphorylase as a substrate. This observation is consistent with the normal muscle contractility of I strain animals.     

Pulmonary cell response to bacterial challenge in mutant mice with some hereditary alterations resembling cystic fibrosis

Animal models make it possible to perform studies that normally can not be done in human beings. In the present work, the cellular response pattern of the lungs both to the environment and to a challenger strain of bacteria were analyzed in two mouse mutations-cribriform degeneration ($\text{cri}$) and motheaten (me)-. The mice were submitted to an infective aerosol containing $\text{Staphylococcus aureus}$ and were killed either immediately or 4 h after exposure; pulmonary washings were performed in these animals as well as in non-infected controls. The results showed neither quantitative differences in the total cell count nor in cell viability between the different groups. However, there were qualitative changes in the differential cell content characterized by a neutrophilic response in $\text{cri}$/$\text{cri}$ mice and a giant cell response in $\text{me}$/$\text{me}$ mice. Since most of the cystic fibrosis patients suffer from chronic lung infection, with a persistently high proportion of neutrophils, these findings point to the $\text{cri}$/$\text{cri}$ mouse as a promising animal model for cystic fibrosis studies.     

Seasonal and temperature-related changes in mitochondrial membranes associated with torpor in the mammalian hibernator Spermophilus richardsonii

Seasonal variations in the thermal response of liver mitochondrial membranes from Richardson's ground squirrels (Spermophilus richardsonii) were determined by measuring succinate-cytochrome $\text{c}$ reductase activity and spin label motion over a temperature range of 2 °C to 35 °C. For seven summer animals from the field the Arrhenius-type plots for enzyme activity and spin label motion were biphasic indicating a transition in structure and function at $\text{22 + 2.3°C}\text{and}\text{23 ± 1.9°C}$, respectively; typical of homeothermic mammals. For 12 winter animals maintained at 19°C, the transition in structure and function was lowered to $\text{12 ± 1.1°C}$ and $\text{13 ± 1.4°C}$, respectively. The transition for 5 of 11 winter animals which were kept at 4°C and maintained normal activity and body temperature was similar to animals maintained at 19°C, while for the other six the transition was further lowered to less than 4°C. The transition for seven winter animals which were in deep hibernation was less than 4°C. The results for liver mitochondria show that lowering of the transition in membrane structure and function occurs as a two-stage process of about 10 deg. C for each stage and that the lowering is a requisite for hibernation rather than a response to the low-body temperatures experienced during hibernation.     

Sensitivity of rat brain adenylate cyclase to activation by calcium and ethanol after chronic exposure to ethanol

Rats were fed ethanol (Lieber-DeCarli diet) for three weeks. Stimulation of cerebellar adenylate cyclase by calcium was measured in control (pair-fed), chronic-alcohol and alcohol-withdrawn animals. No differences in the sensitivity or maximal stimulation of this enzyme were observed among these groups. Ethanol $\text{in}$,$\text{vitro}$ (1%) stimulated brain adenylate cyclase approximately 50% in the presence or absence of calcium. Chronic alcohol exposure $\text{in}$,$\text{vivo}$ did not alter the sensitivity of adenylate cyclase to stimulation by alcohol $\text{in}$,$\text{vitro}$.     

Interactions of cadmium and zinc in cultured rat embryos

Rat embryos were cultured in serum taken from animals dosed with cadmium, or serum with cadmium added $\text{in}$$\text{vitro}$ in the presence or absence of additional zinc. Embryos explanted at day ten and grown in serum taken from animals sooner than 4 h after dosing had a reduced DNA content after 24 h culture. In one-hour serum, the yolk sac had become thick and brittle. Zinc ameliorated the effects but had no stimulatory effect on post eight-hour serum when serum zinc levels were at their lowest. The hypothesis that cadmium induces a maternal zinc deficiency sufficient to cause teratogenic changes could not be sustained. Embryos explanted at nine days were much more susceptible to cadmium added $\text{in}$$\text{vitro}$ than ten-day embryos. The principal anomaly, apart from a reduced DNA content, was a thickening of the yolk sac similar to that seen in embryos grown in serum taken from animals one hour after cadmium dosing. Addition of zinc to the medium prevented both of these effects. The suggestion is made that the cadmium-induced dysgenesis of the yolk sac precludes appropriate embryonic nutrition.     

Suppression of lordosis in guinea pigs by ethamoxy-triphetol (MER-25) given at long intervals (34-46 hr) after estradiol benzoate treatment

Ovariectomized guinea pigs were given estradiol benzoate (EB) followed 40 hr later by progesterone (P). Behavioral testing commenced 1 hr after P injection and continued at hourly intervals for 8 hr. This treatment activated lordosis in almost 100% of animals. Administration of the antiestrogen MER-25 (75 mg/kg body wt per injection) between 2 hr before and 6 hr after EB treatment did not cause a significant decline in proportion of animals displaying lordosis, but did cause a decrease in length of time the lordosis position was held (maximum lordosis, sec). In contrast, $\text{13}\text{14}$ animals given MER-25 at 2 hr before and 2 hr after P and $\text{8}\text{10}$ animals given MER-25 simultaneously with and 2 hr after P, failed to show lordosis. Administration of supplementary EB at around the time of P injection, partially alleviated these behavior-blocking effects of MER-25. When MER-25 was given 2–6 hr after administration of P there was a significant decrease in duration of heat (hr). These results suggest that in addition to its early “triggering” effects, estrogen has important “maintenance” effects which determine the character of heat in guinea pigs. Continued presence of estrogen in the nervous system may be a requirement for the facilitatory actions of P on sexual behavior in guinea pigs, but such a requirement may not exist in other rodents such as rats.     

Studies on the activity of brown adipose tissue in suckling,pre-obese, obob mice

The properties and activity of brown adipose tissue have been investigated in suckling, pre-obese, $\text{ob}\text{ob}$ mice in order to determine whether decreased thermogenesis in the tissue precedes the development of obesity in this mutant. At 14 days of age there was no difference between the $\text{ob}\text{ob}$ and normal animals in the total amount of interscapular brown adipose tissue, and the DNA content, protein content, and cytochrome oxidase activity of the tissue were similar in the two groups of mice. Respiration rates of brown adipose tissue mitochondria in the presence of albumin were, however, greater in the normal than the $\text{ob}\text{ob}$ animals, although after the addition of GDP to recouple the mitochondria there was no difference between the two groups. The mitochondrial membrane potential, measured with [³H]methyltriphenylphosphonium, was less affected by exogenous GDP in $\text{ob}\text{ob}$ mice than in normal animals. GDP binding to brown adipose tissue mitochondria, an index of the proton conductance pathway, was much greater in normal than in $\text{ob}\text{ob}$ mice at both 10 and 14 days of age; the decreased GDP binding in the mutant animals was found to result from a reduction in the number of binding sites. It is concluded that brown adipose tissue mitochondria of pre-obese $\text{ob}\text{ob}$ mice are more tightly coupled than those of normal siblings, and that the activity of the ‘thermogenic’ proton conductance pathway is lower in the mutant animals. A decrease in thermogenesis in brown adipose tissue is therefore an early event in the development of the $\text{ob}\text{ob}$ mouse and precedes the appearance of obesity.     

Glutathione peroxidase,glutathione reductase,and thiobarbituric acid-reactive products in muscles of chickens and mice with genetic muscular dystrophy

The specific activities of glutathione peroxidase and glutathione reductase, and the level of thiobarbituric acid-reactive products in muscles from chickens and mice with genetic muscular dystrophy were increased over those of control animals. These findings indicate the occurrence of lipid peroxidation and the activation of an $\text{in}$$\text{vivo}$ lipid peroxidation protective system in these dystrophic animals. A partial explanation for some of the metabolic changes that occur in dystrophic species is provided by this work.     

Fructose 2,6-bisphosphate levels are elevated in livers of genetically obese mice

Fructose 2,6-bisphosphate levels in freeze-clamped livers of $\text{C57BL}\text{6J}$$\text{ob}\text{ob}$ mice were 6-fold higher than the level in their lean (+/?) littermates. Overnight starvation reduced the hepatic level of this unique sugar diphosphate to 0.2 nmol/g in both the obese and lean mice. The elevated level in the obese mouse is consistent with the hyperinsulinemia of these animals.     

Ascorbic acid modulation of splenic cell cyclic GMP metabolism.

Chronic ascorbate deprivation of guinea pigs decreased splenic cell cyclic GMP levels (80%); ascorbate (1 mM) addition to these cells $\text{in vitro}$ restored the cellular concentration to control levels. Splenic cells from non-scorbutic animals also exhibited increases in cyclic GMP levels in response to exogenous ascorbate whereas thiol reducing agents diminished cellular cyclic GMP concentration. Agents that inhibit the propagation of free radicals prevented this cellular effect of ascorbate while agents known to interfere with or promote H₂O₂ production had no effect. Guanylate cyclase activity in cell lysates increased after treatment of intact cells with ascorbate; dithiothreitol reversed this effect. Ascorbate also enhanced guanylate cyclase activity in cell lysates. The results suggest that oxidizing equivalents in the form of the monoanionic free radical of ascorbate alter cyclic GMP metabolism in these cells by activating guanylate cyclase via a mechanism involving oxidation of a cyclase-related component.