Interferon-mediated inhibition of prostaglandin synthesis in human mononuclear leukocytes

In this study, the question of whether human leukocyte-derived and fibroblast-derived interferon had an effect on prostaglandin metabolism in human peripheral blood mononuclear cells has been considered. Both leukocyte- and fibroblast-derived interferon were potent inhibitors of mononuclear cell prostaglandin synthesis at low physiological concentrations. Inhibition required a minimum incubation of 1 hr. Interferon had no effect on release of arachidonic acid; synthesis of hydroxy fatty acids was slightly increased.     

Coordinate inhibition of DNA synthesis and thymidylate synthase activity following DNA damage and repair

Two agents, 3-aminobenzamide (3-AB) and beta lapachone, that inhibit repair of mammalian cell DNA damaged by methyl methane sulfonate (MMS), also coordinately blocked both DNA replication (incorporation of 3H-thymidine) and thymidylate synthase (TS) activity. Aphidicolin also inhibited both 3H-TDR incorporation and TS in damaged cells, the former more strongly than the latter, in a manner not coordinated with lethality. It is proposed that the DNA lesions created by MMS and modified by repair inhibit semiconservative DNA synthesis by allosterically interacting with the DNA replication replitase complex, so as to block its overall function and also the activity of TS, one of its enzymes.     

Regulation of collagen synthesis by ascorbic acid. Ascorbic acid increases type I procollagen mRNA

Ascorbic acid specifically stimulates collagen production in cultured human skin fibroblasts, an effect that appears to be independent of its cofactor role in prolyl and lysyl hydroxylation. In order to investigate the level of regulation of ascorbic acid on collagen synthesis, we have translated mRNA in a cell-free system derived from rabbit reticulocytes. Total RNA was prepared from normal human skin fibroblasts and similar fibroblasts which had been exposed to 100 uM ascorbic acid for four days. Ascorbic acid treatment resulted in a twofold stimulation of procollagen mRNA whereas non-collagenous mRNA was unchanged. These results reveal that ascorbic acid has a preferential stimulating effect on type I procollagen mRNA.     

Studies on the mechanism of 5-fluoroorotic acid inhibition of serine dehydratase induction

Administration of glucagon to rats fed a protein-free diet caused a significant induction of the liver enzyme, serine dehydratase. This effect of glucagon is inhibited by the concomitant administration of fluoroorotic acid. This inhibition was enhanced by pretreatment with glucosamine or galactosamine, probably through depletion of the intracellular uridine pools. Although less than a doubling of enzyme activity was observed after glucagon plus fluoroorotic acid administration, the amount of protein precipitable by antisera specifically reactive against serine dehydratase increased 4.5 times. Ouchterlony double-diffusion analysis showed a completely cross-reacting single precipitin band from liver extracts of untreated animals and rats treated with the analog. Analysis of the antigen-antibody complex by Na dodecyl sulfate-gel electrophoresis indicated that a single protein was being immunochemically precipitated from both the glucagon- and glucagon plus fluoroorotic acid-treated rats. In the latter, the precipitated protein had a molecular weight similar to purified serine dehydratase. These results are consistent with the concept that the incorporation of fluoroorotic acid into mRNA results in the synthesis of a protein with characteristics similar to authentic serine dehydratase but without normal enzymatic activity. Other possible mechanisms to explain the production of this abnormal protein are discussed.     

Reversal of immunological tolerance by aclacinomycin through inhibition of suppressor cell activity

The elimination of suppressor cells by aclacinomycin, which could be the mechanism by which immune responses are enhanced after its administration, was studied in mice in which tolerance had been induced by the injection of high doses of sheep red blood cells (SRBC). We observed that tolerance could not be induced in aclacinomycin-treated mice, and that aclacinomycin inhibited the expression of tolerance to SRBC. This drug also diminished the capacity of spleen cells from SRBC-tolerant mice to inhibit the response of normal animals upon adoptive transfer, indicating that suppressor cells had been eliminated from the tolerant spleen cell population. The efficiency of the elimination of suppressor cells for DTH reactions appears greater than that of suppressor cells for plaque-forming cell responses.     

Stress-induced inhibition of sexual behavior: Corticosterone inhibits courtship behaviors of a male amphibian (Taricha granulosa)

When male rough-skinned newts (Taricha granulosa) are exposed to presumptive stressors, the incidence of courtship decreases and plasma corticosterone concentration increases. When sexually active males are injected intraperitoneally with corticosterone (1, 5, 10, 15, 20, or 25 μg), the incidence of courtship decreases rapidly and in proportion to the dose of corticosterone. Intracerebroventricular infusion of synthetic corticotropin-releasing factor (CRF) elevates plasma corticosterone levels and suppresses courtship. When male newts receive an injection of metyrapone, a drug that interferes with corticosterone synthesis, the inhibitory effects of stress or CRF infusion on courtship are reduced. These results support the hypothesis that, in this amphibian, elevated levels of corticosterone associated with exposure to stressful stimuli inhibit sexual behaviors.     

RNA synthesis and coding capacity of polyadenylated and nonpolyadenylated mRNA from cultures of differentiating Drosophila melanogaster myoblasts

We have investigated the synthesis and coding capacity of RNA isolated from cultures of differentiating Drosophila embryonic muscle cells. We find that following muscle cell fusion, the sedimentation profile of newly synthesized polyadenylated RNA becomes somewhat lighter. In vitro translation products analyzed by two-dimensional gel electrophoresis indicate that the coding capacity of translatable myogenic mRNA changes during differentiation. A group of several muscle-specific proteins (including the contractile proteins) is translated only from mRNA isolated after the initiation of fusion. This pattern coincides with proteins synthesized in vivo during differentiation. Additionally, we find that polyadenylated and nonpolyadenylated myogenic mRNA from a given developmental stage in culture have extremely similar coding potentials.     

Genetic toxicology of ascorbic acid

The activation mechanism of emodin, a fungal anthraquinone and constituent of rhubarb, into a direct mutagen to Salmonella typhimurium TA1537 was investigated by using the S9 and microsomes of rat livers. Upon incubating emodin with the hepatic S9 derived from PCB-pretreated rats, this anthraquinone exhibited mutagenicity in the presence of NADPH or NADH, and this enzymatic activation, maximal at pH 7.0 and occurring in the microsomes, was induced by the pretreatment of rats with PCB, 3-methylcholanthrene or phenobarbital and was inhibited by α-naphthoflavone, SKF 525A and carbon monoxide. Thin-layer chromatographic analysis revealed that emodin was biotransformed by the microsomal enzymes into at least 5 quinonoid metabolites, among which one pigment, identified as 2-hydroxyemodin (1,2,3,8-tetrahydroxy-6-methyl-anthraquinone), was proved to be a direct mutagen to the test strain, and the remaining 4 quinonoid metabolites were negative or far less active than this active principle.     

Effects of feeding and lighting stimuli on the synthesis of ornithine aminotransferase and serine dehydratase in rat liver

In a previous study we showed that rats fed ad libitum and maintained on a 12-h light/ 12-h dark cycle demonstrated out-of-phase circadian oscillations in the rates of ornithine aminotransferase and serine dehydratase synthesis. As part of an investigation of the factors regulating both the generation of these cycles and their dissimilarity, this paper ompares the circadian fluctuations in the rates of ornithine aminotransferase and serine dehydratase synthesis measured immunochemically in rats given a single 2-h daily feeding in conjunction with exposure to constant light or a 12-h light/12-h dark cycle. When the 2-hr feeding was administered to rats under constant light, reciprocal circadian oscillations in ornithine aminotransferase and serine dehydratase synthesis were observed regardless of the temporal location of the feeding interval. Ornithine aminotransferase synthesis began to increase after the feeding interval and reached a maximum 12 h later while serine dehydratase showed the opposite response. In rats maintained on both the restricted feeding regimen and a 12-h light/12-h dark cycle, however, retention of synthesis oscillations depended on the temporal location of the restricted feeding interval within the light-dark cycle. Rats fed for 2 h at the beginning of the dark phase exhibited circadian oscillations in serine dehydratase synthesis and a high nonoscillating level of ornithine aminotransferase synthesis, whereas rats fed for 2 h at the beginning of the light phase exhibited circadian oscillations in ornithine aminotransferase synthesis and a low nonoscillating level of serine dehydratase synthesis. These responses suggest the existence of meal-responsive and light-responsive regulators of ornithine aminotransferase and serine dehydratase synthesis.     

Normal level of unscheduled DNA synthesis in Werner''s syndrome fibroblasts in culture

We investigated UV-induced unscheduled DNA synthesis (UDS) in skin fibroblasts from seven unrelated patients with clinically apparent Werner's syndrome (WS). WS cells exhibited greatly abbreviated in vitro lifespans, the extents of which ranged from about 20 to 50% of the normal. However, WS cells in early and senescent phases of growth showed the same quantity of DNA repair following UV exposure as did normal fibroblasts.     

Conformational preferences of dopamine analogues for inhibition of norepinephrine N-methyltransferase. Conformationally defined adrenergic agents. 7

A series of analogues of dopamine (DA) with varying degrees of conformational flexibility have been examined as potential substrates or competitive inhibitors of the enzyme norepinephrine N-methyltransferase (NMT). A conformationally defined (rigid) analogue of the fully extended conformation of DA, 2-amino-6, 7-dihydroxybenzonorbornene hydrobromide ($\text{3}$; 6, 7-D2HX) proved to be a better substrate than the non-catechol parent 2-aminobenzonorbornene ($\text{4}$; 2HX). However, analogues $\text{3}$ and $\text{4}$ displayed equivalent competitive inhibitory activity toward phenylethanolamine (PEA). Neither 6, 7-ADTN (5), a DA analogue in the 2-aminotetralin (2AT) system, nor 6, 7-DTHIQ ($\text{7}$), a DA analogue in the tetrahydroisoquinoline (THIQ) system, showed substrate activity; 6, 7-ADTN was a poorer competitive inhibitor than the parent 2AT but 6, 7-DTHIQ was a better competitive inhibitor than its parent, THIQ ($\text{8}$). A tricyclic conformationally defined analogue $\text{9}$ of 6, 7-ADTN was devoid of either substrate or inhibitory activity. From these results it may be concluded that a fully extended side chain conformation is required for NMT substrate activity, and the better substrate activity for 6, 7-D2HX compared to $\text{4}$ is consistent with a proper catechol orientation for interaction with the norepinephrine (NE) binding site of NMT.     

The regulation of ornithine aminotransferase synthesis by glucagon in the rat.

Ornithine aminotransferase (OAT) from rat liver mitochondria was purified to homogeneity. A monospecific antiserum against the enzyme protein was prepared in rabbits. Immunotitrations were performed on OAT present in crude mitochondrial extracts obtained from the livers and kidneys of rats in several hormonal and dietary states. No evidence was found for the existence of an immunologically reactive but enzymatically inactive form of OAT. The relative rate of enzyme synthesis in vivo was studied by pulselabeling rats with [4, 5-³H]leucine, isolating the enzyme protein by immunoprecipitation, and dissociating the immunoprecipitates on sodium dodecyl sulfate-acrylamide gels. Nine hours after a single subcutaneous injection of a glucagon oil emulsion, a 3-fold increase in OAT activity and a 12-fold increase in the synthetic rate of the enzyme were observed. Serine dehydratase activity increased on a time-course very similar to that of OAT following glucagon injection. These increases occurred only on low (0–12.5%) protein diets. At higher levels of dietary protein (30% and up), no further stimulation of OAT synthesis by glucagon was observed. Administration of actinomycin D within the first 2 h after glucagon injection resulted in an inhibition of OAT induction. When the administration of the antibiotic was delayed until 4 h after glucagon, no inhibition of OAT induction was observed. Glucose repression of the glucagon induction of the enzyme in hepatic mitochondria was demonstrated to be the result of a rapid inhibition of OAT synthesis.     

The amino acid composition of secreted mouse prolactin,growth hormone,and hamster prolactin: The presence of one tryptophan in mouse prolactin

The amino acid compositions and the electrophoretic properties of secreted mouse prolactin (mPRL), mouse growth hormone (mGH), and hamster prolactin (haPRL) were determined. The amino acid compositions of secreted mPRL and haPRL were similar to the compositions of other rodent prolactins, except that secreted mPRL contained only one tryptophan residue rather than the usual two. The composition of secreted mGH was similar to that of rat growth hormone. On 10% alkaline polyacrylamide gels, mPRL, mGH, and haPRL migrated with R_f's of 0.54, 0.21, and 0.69, respectively. The molecular weights of mPRL, mGH, and haPRL, determined by SDS-gel electrophoresis, were 23,000, 21,000, and 22,000, respectively.     

The effects of L-2,4-diaminobutyric acid on the uptake of gamma-aminobutyric acid by a synaptosomal fraction from rat brain

l-2,4-diaminobutyric acid was studied as an inhibitor of gamma-aminobutyric acid uptake by a synaptosomal fraction isolated from rat brain. Competitive inhibition was observed during short-term exposure of the synaptosomal fraction to the inhibitor but noncompetitive inhibition was observed following prolonged exposure. Studies on the mode of action of l-2,4-diaminobutyric acid showed that the synaptosomal fraction was capable of accumulating this compound and that both the uptake and the effectiveness of the inhibitor were sodium-dependent and temperature-sensitive. In addition, the degree of inhibition of gamma-aminobutyric acid uptake was related to the amount of l-2,4-diaminobutyric acid accumulated. It is suggested that the observed noncompetitive inhibition of gamma-aminobutyric acid uptake by l-2,4-diaminobutyric acid is a result of the accumulation of the inhibitor which exerts its effect from within the synaptosomes. Raising the external concentration of gamma-aminobutyric acid to saturating levels did not completely inhibit the accumulation of l-2,4-diaminobutyric acid. Thus, the transport of l-2,4-diaminobutyric acid appears to be mediated, at least in part, by a carrier which is not involved in the transport of gamma-amiuobutyric acid.     

The amino acid sequence of carbonic anhydrase I from the rhesus macaque

The complete amino acid sequence of carbonic anhydrase I (CA I) isolated from the red cells of the rhesus macaque ($\text{Macaca}$$\text{mulatta}$) is presented. This sequence was obtained by aligning peptides derived from various fragmentation procedures with the fully characterized sequence of human CA I. When the peptides of rhesus CA I were ordered in this manner, 13 of the 260 residues were found to differ from the human CA I sequence. The known markedly higher specific esterase activity of rhesus CA I compared to human CA I could not be correlated with any changes in residues postulated to be within 10 Å of the single zinc ion at the active site.     

Questioning of reported evidence for guanosine tetraphosphate synthesis in a ribosome system from mouse embryos.

In 1974, Irr, Kaulenas and Unsworth reported that ppGpp is synthesized by cytosolic ribosomes from mouse embryos and proposed a role for ppGpp in the process of differentiation. This proposal is being challenged because ribosomes of mouse embryos from various stages of development and of mouse embryoid bodies were completely inactive in ppGpp formation.     

Synthesis of RNA containing polyadenylic acid sequences in preimplantation rabbit embryos

Messenger RNA synthesis has been estimated by assaying polyadenylic acid (poly A)-rich sequences in heterogeneous RNA from preimplantation rabbit embryos. Poly A containing RNAs are synthesized at least as early as the 16-cell stage and continue to be made through blastocyst formation and maturation. Sixty to 78% of the heterogeneous polysomal RNA in blastocysts contain poly A sequences. The portion of the heterogeneous RNA containing poly A sequences does not appear to change markedly between cleavage and blastocyst stages of development. Poly Arich sequences are greater than 4 S and consist of at least 84% adenine residues. RNA molecules ranging from 6 S to greater than 28 S contain poly A sequences.