Effect of the oral hypoglycemic agent 2-tetradecylglycidic acid on fatty acid oxidation in suckling rats in vivo and in perfused liver

The hypoglycemic agent, 2-tetradecylglycidic acid (TDGA), administered in vivo lowered the concentration of plasma glucose and ketone bodies but raised the concentration of liver and plasma triglycerides in 10-day-old suckling rats. Phospholipid and cholesterol content of the plasma and liver were unaffected by drug treatment. TDGA inhibited the in vivo oxidation of [1-¹⁴C]palmitate but not that of [1-¹⁴C]decanoate. In suckling rat liver perfusion, TDGA totally inhibited ketone body formation from palmitate and depressed ketone body production from decanoate by 20%. Liver ATP and ADP content in the presence of TDGA decreased although this was probably a reflection of the increased triglyceride content of the liver since the $\text{ATP}\text{ADP}$ was the same as control livers. The results are discussed in relation to the diet and to the inhibition of carnitine acyl transferase in suckling rats.     

Inhibition of macromolecular synthesis in tumors by L-1-tosylamido-2-phenylethyl chloromethyl ketone.

L-1-tosylamido-2-phenylethyl chloromethyl ketone was observed to inhibit the incorporation of [³H] amino acids into protein and [³H] thymidine incorporation into DNA in Novikoff hepatoma ascites cells $\text{in vitro}$ Similar effects were seen with several Morris hepatomas and a transplanted colon tumor in rats, and were accompanied by decreased uptake of isotope into acid soluble tissue fractions. Under the same conditions, there was no significant inhibition in regenerating liver and there was an increased uptake of [³H] amino acids in the livers of normal and tumor bearing rats.     

Demonstration of [3H] diazepam binding to benzodiazepine receptors in vivo.

Benzodiazepine receptors were labeled with [³H] diazepam following intravenous injection in rats. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ to rat forebrain membranes was displaceable by co-injection of clonazepam or the pharmacologically active enantiomers of two benzodiazepines, B9 and B10, but was not displaced by equal doses of the pharmacologically in-active enantiomers. Binding of [³H] diazepam $\text{in}$$\text{vivo}$ was bserved in kidney, liver, and abdominal muscle, but was not stereospecifically diplaced in any peripheral tissue studied. The regional distribution of benzodiazepine receptors in brain was uneven, with specific [³H] diazepam binding being highest in the cerebral cortex and lowest in the ponsmedulla. Preliminary studies of the subcellular distribution of [³H] diazepam binding demonstrated highest specific binding to synaptosomal membranes. These data demonstrate the feasibility of labeling benzodiazepine receptors in rat brain $\text{in}$$\text{vivo}$.     

The in vivo incorporation of mannose, retinol and mevalonic acid into phospholipids of hamster liver

Hamsters were injected intraperitoneally with [¹⁴C]mannose, [¹⁴C]retinol and [³H]mevalonic acid. The livers were removed, extracted with chloroform-methanol and the lipids chromatographed on DEAE-cellulose and silicic acid. The hamster liver lipid contained a component which could be labelled with mannose and mevalonic acid. The properties of this compound were in accord with it being dolichyl-mannosyl-phosphate, a possible lipid intermediate required for the biosynthesis of some glycoproteins. [¹⁴C]Retinol and [¹⁴C] mannose were incorporated into another phospholipid which was labile to mild alkali conditions commonly used for the preparation of dolichyl-mannosyl-phosphate. The retinol labelled compound had similar properties to $\text{in vitro}$ prepared mannosyl-retinyl-phosphate.     

Aflatoxin B1-2,3-oxide: evidence for its formation in rat liver in vivo and by human liver microsomes in vitro

Injection of [³H]aflatoxin B₁ into rats yielded covalently bound derivatives in hepatic DNA, rRNA, and protein. Mild acid hydrolysis of the DNA and rRNA adducts formed a derivative indistinguishable from 2,3-dihydro-2,3-dihydroxy-aflatoxin B₁. The data indicate that approximately 60% of the nucleic acid adducts were derived from reactions $\text{in vivo}$ with aflatoxin B₁-2,3-oxide. Acid hydrolysis of rRNA-[³Haflatoxin B₁ adduct formed by human liver microsomes $\text{in vitro}$ also liberated the dihydrodiol in significant amount. The 2,3-oxide of aflatoxin B₁ is a probable ultimate carcinogenic metabolite.     

Liver microsomal DT diaphorase: noninvolvement in hydroxylation of benzo(a)pyrene.

A highly purified reconstituted system isolated from the microsomes of 3-methylcholanthrene-treated rats consisting of cytochrome P-448, NADPH-cytochrome $\text{c}$ reductase and synthetic dilauroyl phosphatidylcholine had no DT diaphorase activity, but hydroxylated benzo[a]pyrene at a faster rate than microsomes from 3-methylcholanthrene-treated rats. DT diaphorase purified from liver microsomes of 3-methylcholanthrene-treated rats when added to this reconstituted system did not stimulate or inhibit benzo[a]pyrene hydroxylation, nor could it replace or NADPH-cytochrome $\text{c}$ reductase in supporting the reaction. We therefore conclude that microsomal DT diaphorase is not involved in microsomal hydroxylation of benzo[a]pyrene to its phenolic products despite the observation that both DT diaphorase activity and the hydroxylation of benzo[a]pyrene are induced by 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin     

Enhanced activity of tRNA-pseudouridine synthetase in Yoshida ascites sarcoma.

Five days after transplantation of Yoshida ascites sarcoma cells into a rat, specific activity of tRNA-pseudouridine synthetase was extremely high in the supernatant of tumor cells and moderately high in the tumor-bearing rat liver compared with normal rat liver. Enzyme assay was performed at 37°C by determining the release of tritium from heterogeneous [³H] tRNA extracted from $\text{E. coli}$ B grown in the presence of [5,6-³H]-uracil and resulting in the increased ratio of the amount of pseudouridine to uridine residues in [³H] tRNA. Neither [5-³H]-uridine, [5,6-³H]-UTP, nor [5,6-³H]-poly U released tritium in the present assay conditions.     

Displacement of palmitate from albumin by chlorophenoxyisobutyrate.

The effect of chlorophenoxyisobutyrate, a hypolipidemic drug that decreases plasma free fatty acids, triglycerides, and cholesterol, on the partitioning of [¹⁴C]-palmitate between hexane and bovine serum albumin was studied at 37°. In this system, hexane served as a hydrophobic trap for free fatty acids displaced from BSA by chlorophenoxyisobutyrate, allowing less than 0.3% to remain in the aqueous phase. As the concentration of chlorophenoxy isobutyrate was raised from 0.4 to 3.2 mM, there was a progressive displacement of palmitate from the [¹⁴C]-palmitate-BSA complex into hexane, the magnitude being dependent on the initial $\text{V}$ value (moles palmitate bound/mole BSA). Beginning with [¹⁴C]-palmitate in hexane, chlorophenoxyisobutyrate (2 mM) decreased the moles palmitate bound/mole of BSA by 16% at $\text{V}$ = 0.2, and 34% at $\text{V}$ = 3.0.     

The effect of vitamin B12 inhibition in vivo: Impaired folate polyglutamate biosynthesis indicating that 5-methyltetrahydropteroylglutamate is not its usual substrate

Using 5-[¹⁴C] methyltetrahydropteroylglutamate and [³H] pteroylglutamate it was found that vitamin B₁₂ dependent methyltransferase can be inhibited $\text{in vivo}$ in mice by nitrous oxide. Examination of the liver [³H] folates showed as expected that both controls and nitrous oxide treated mice had synthesised the reduced 5-methyl form. However, the controls had converted [³H] almost exclusively into a polyglutamate with virtually no monoglutamate present, while nitrous oxide treated mice had failed to add glutamates to over half of the incorporated folate. Thus $\text{in vivo}$ demethylation may be necessary prior to polyglutamate biosynthesis and act as a method of controlling folate accumulation, or be used to create a concentration gradient with the circulating folate.     

Acetylcholine stimulates hydrolysis of 32 P-labeled phosphatidic acid in guinea pig synaptosomes

[³H]-inositol or [³H]-arachidonate was injected intracerebrally into guinea pigs. Labeled nerve endings were incubated with Ach¹ or CCh, both of which stimulate labeling of PhA and PhI from ³²P_i by > 100% and 70% respectively. Their addition did not affect the $\text{in}$$\text{vivo}$ labeled phosphatidyl-[³H]-inositol or [³H]-arachidonyl-diglyceride and -PhI. Enhanced hydrolysis of [³H]-inositol-PhiP and -PhIP₂ in the presence of ACh, CCh or choline was not reversed by atropine. In a two-step experiment, PhA was labeled with ³²P_i, and DNP was added to block further $\text{γ-[}{}^{32}\text{P]-ATP}$ formation. Addition of ACh stimulated an atropine-sensitive $\text{decrease}$ in [³²P]-PhA.     

Thiol reactivity and the molecular individuality of α- and β-adrenoreceptors in rat liver plasma membranes

Whether or not α- and β-adrenoreceptors are non-identical binding sites on the same protein is still an open question. We investigated the effects of sulfhydryl reagents and dithiothreitol on the binding of [³H]dihydroalprenolol and [³H]dihydroergocryptine to β- and α-adrenoreceptors of rat liver plasma membranes. Dithiothreitol inhibited the binding of [³H]dihydroalprenolol to the β-adrenoreceptor, whereas it had no effect on the specific binding of [³H]dihydroergocryptine to the α-adrenoreceptor. In contrast, mersalyl, a mercurial SH reagent, readily blocked the α-adrenoreceptor and, although to a lesser extent, the β-adrenoreceptor. The interaction of mersalyl with the α-adrenoreceptors was almost instantaneous. In contrast, under the same experimental conditions, the inactivation of the β-adrenoreceptors was much slower ($\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{:7}\text{min}$). Finally, a marked difference in the accessibility of the SH groups to mersalyl was observed between the α- and β-adrenoceptors. The presence of 15 μM (?)-epinephrine or 1.5 μM phentolamine was sufficient to prevent the blockade of the α-adrenoreceptor by mersalyl, but inactivation of the β-adrenoreceptor by mersalyl was not modified by 500 μM (?)-epinephrine and was only slightly decreased by 50 μM (?)-propranolol. Thus, the α- and β-adrenoreceptors from rat liver plasma membranes exhibited biochemical differences which may be interpreted in favor of their molecular individuality.     

Effect of experimental diabetes and insulin onphosphatidyl-inositol synthesis in rat sciatic nerve.

The rate of incorporation of [2-³H]myo-inositol into phosphatidylinositol was found to be significantly decreased in sciatic nerve from both alloxan and streptozotocin-diabetic rats. The rates of incorporation into phospholipid of tritiated serine and ethanolamine were unchanged while choline showed an upward trend in sciatic nerve from alloxan-diabetic rats. Insulin added $\text{in}\text{vitro}$ significantly increased [2-³H]myo-inositol incorporation into phospholipids by normal rat sciatic nerve; only small changes were recorded with high concentrations of glucose, and galactose. The results are discussed in relation to the physiological functions of phosphatidylinositol and the role of free myo-inositol in the regulation of cellular processes.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

A study of the relationships between carbon tetrachloride-induced lipid peroxidation and liver damage in rats pretreated with vitamin E

The relationships between the peroxidation of musomal lipids and the early liver damage have been investigated in rats pretreated with progressively higher doses of α-tocopherol (vit. E) and intoxicated with various amounts of carbon tetrachloride.Pretreatment of rats with vit. E at 25 $\text{mg}\text{100g}$ body wt. has minor effects on both the peroxidation of musomal lipids and the liver triglyceride accumulation in rats poisoned with CC1₄ at 250 $\text{μl}\text{100g}$ body wt. However, a decrease of the peroxidative reaction and of the liver steatosis occurs when the rats are pretreated with progressively higher doses of vit.E. A close correlation exists between the two phenomena, when the intoxication is accomplished with CC1₄ at 250 $\text{μ1}\text{100 g}$ body wt. Also, the musomal concentration of α-tocopherol is strictly correlated to both the decrease of musomal lipoperoxidation and the decrease of liver triglyceride accumulation. The CCl₄-induced impairment of musomal glucose-6-phosphatase and the incorporation of ¹⁴C from ¹⁴CC1₄ into liver musomal lipids are not affected by vit. E pretreatment.The extent of musomal lipoperoxidation is not correlated to the liver triglyceride accumulation when vit. E-pretreated rats are given CC1₄ at 25 or 2.5 $\text{μ1}\text{100 g}$ body wt. However, a correlation between lipoperoxidation and liver steatosis occurs when non-pretreated rats are challenged with the three different doses of the halogenated hydrocarbon.     

Glucagon and epinephrine-stimulated phospholipid methylation in hepatic microsomes

Phospholipid methylation by hepatic microsomes was measured following glucagon or epinephrine administration either to intact rats or to the isolated perfused liver. Both hormones stimulated the methylation measured as the incorporation of S-adenosyl-L-[methyl-³H]methionine into phospholipids. The labeled products were identified by thin layer chromatography and most of the counts were found to be incorporated into phosphatidylcholine. The stimulatory effects of the hormones were evident already 5 minutes following hormone administration both in $\text{in vivo}$ and in $\text{in vitro}$. The observed stimulation of the methylation process by glucagon and epinephrine might be related to the previously reported stimulatory effect of these hormones on the microsomal Ca²⁺-ATPase, and indicate that methylation process(es) might mediate some of the effects of these hormones.     

Differential phosphorylation of histone H1 subfractions in vivo.

A study was made of the phosphorylation of chromatographically purified histone H1 subfractions from the liver of premetamorphic tadpoles ($\text{Rana}\text{catesbeiana}$). Two H1 subfractions were obtained which differed in terms of net incorporation of [³²P]phosphate $\text{in}\text{vivo}$. Analysis of N-bromosuccinimide cleavage products further revealed that the two subfractions also differed in the relative distribution of [³²P]phosphate in N- and C-terminal regions of the molecule. Incorporation of [³²P]phosphate into both regions of the molecule occurred virtually exclusively in serine residues.     

Androgen glucuronides: I. Direct formation in rat accessory sex organs

A simple one-step procedure is described on the isolation of androgen glucuronides from various rat tissues. This procedure uses polyacrylamide gel electrophoresis, and permits a quantitative isolation of a single band containing the total androgen glucuronides without the contamination of free androgens and androgen sulfates. This procedure was used to determine the ability of various tissues of the rat to form androgen glucuronides directly when they were incubated with 1,2-[³H]-testosterone (0.1 μM) $\text{in}$$\text{vitro}$. Of eleven organs studied, only the accessory sex organs (ventral prostate, seminal vesicle, and coagulating gland), liver, and kidney were capable of forming androgen glucuronides. At the end of a one-hour incubation period, approximately 1% of the total radiolabeled steroids in the prostatic tissue minces were in the form of glucuronide conjugates. The predominant androgen glucuronide formed in the accessory sex organs was 5α-androstane-3α,17β-diol 17β-d-glucuronide. This is in contrast to the rat liver and kidney in which testosterone glucuronide was the predominant conjugate.A similar amount of labeled glucuronide conjugates was formed from either [³H]-testosterone, [³H]-dihydrotestosterone or [³H]-androstenedione, whereas negligible amount of steroid conjugates was formed from [³H]-cortisol. The formation of androgen glucuronides requires metabolically active tissues; furthermore, the conjugation process was inhibited by the antiandrogen, cyproterone acetate, or by metabolic inhibitors, such as oligomycin or N-ethylmaleimide.     

Deuterium magnetic resonance study of the interaction between chondroitin sulfate and human plasma low density lipoproteins

[²H]Chondroitin sulfate was prepared by partial $\text{N}$-deacetylation of chondroitin sulfate ($\text{via}$ hydrazinolysis) followed by treatment with [²H₆]acetic anhydride. ²H NMR spectra of [²H]chondroitin sulfate in the presence of human plasma low density lipoprotein provide evidence for a soluble complex stoichiometry of 3 (and possibly 2) lipoproteins per polysaccharide molecule, and allow a rough estimation of the dissociation constant K_d.     

The synthesis of freezing-point-depressing protein of the winter flounder Pseudopleuronectusamericanus in Xenpuslaevis oocytes

The serum of the winter flounder $\text{Pseudopleuronectus}$$\text{americanus}$ contains a freezing-point-depressing protein of a molecular weight approximately 10,000 with 60% alanine in its composition. When injected into Xenopus o?cyte, a 6–10 S, poly A-rich RNA preparation isolated from the fish liver polysomes stimulated 3–4 fold the incorporation of [³H] alanine into 10% trichloroacetic acid-soluble, non-dialysable proteins. Analysis of the protein fractions showed a translation product similar in molecular weight and electrophoretic mobility to flounder freezing-point-depressing protein. These observations indicated that the 6–10 S RNA from the flounder contained mRNA for the synthesis of flounder's freezing-point-depressing protein.