Effects of chloramphenicol isomers and erythromycin on enzyme and lipid synthesis induced by oxygen in wild-type and petite yeast

The synthesis of mitochondrial enzymes induced by exposure of anaerobically grown, lipid-depleted Saccharomyces cerevisiae to oxygen is inhibited by d(-)-threo-chloramphenicol and erythromycin. The concentration of these antibiotics required to cause 50% inhibition of this synthesis is less than 1 mm; this is also approximately the concentration required to inhibit by the same amount mitochondrial protein synthesis in situ. The synthesis of unsaturated fatty acids, ergosterol, and phospholipid induced by aeration is inhibited by d(-)-threo-chloramphenicol at high concentrations (12 mm) but is unaffected by erythromycin. l(+)-threo-Chloramphenicol affects neither enzyme nor lipid synthesis and is without effect on mitochondrial protein synthesis in situ. All three compounds inhibit the oxidative activity of isolated mitochondria; the chloramphenicol isomers also inhibit phosphorylation. In a euflavine-derived petite mutant, lacking mitochondrial protein synthesis and respiration, aeration results in the normal development of lipid in the cells, but no synthesis of mitochondrial enzymes. d(-)-threo-Chloramphenicol does not inhibit lipid synthesis in these cells. Thus inhibition of mitochondrial protein synthesis with erythromycin or genetic deletion of mitochondrial protein synthesis results in loss of the capacity to synthesize enzymes during aeration. d(-)-threo-Chloramphenicol, as well as inhibiting induced enzyme formation, inhibits lipid synthesis induced by oxygen. It is unlikely that the latter effect of chloramphenicol is due to inhibition of energy production and transformation, to direct effects on lipid synthesis, or to an inhibition of mitochondrial protein synthesis. It is, however, an effect not shared with the l isomer.     

Preserved protein synthesis in the heart in response to acute fasting and chronic food restriction despite reductions in liver and skeletal muscle

Whole body protein synthesis is reduced during the fed-to-fasted transition and in cases of chronic dietary restriction; however, less is known about tissue-specific alterations. We have assessed the extent to which protein synthesis in cardiac muscle responds to dietary perturbations compared with liver and skeletal muscle by applying a novel (2)H(2)O tracer method to quantify tissue-specific responses of protein synthesis in vivo. We hypothesized that protein synthesis in cardiac muscle would be unaffected by acute fasting or food restriction, whereas protein synthesis in the liver and gastrocnemius muscle would be reduced when there is a protein-energy deficit. We found that, although protein synthesis in liver and gastrocnemius muscle was significantly reduced by acute fasting, there were no changes in protein synthesis in the left ventricle of the heart for either the total protein pool or in isolated mitochondrial or cytosolic compartments. Likewise, a chronic reduction in calorie intake, induced by food restriction, did not affect protein synthesis in the heart, whereas protein synthesis in skeletal muscle and liver was decreased. The later observations are supported by changes in the phosphorylation state of two critical mediators of protein synthesis (4E-BP1 and eIF2alpha) in the respective tissues. We conclude that cardiac protein synthesis is maintained in cases of nutritional perturbations, in strong contrast to liver and gastrocnemius muscle, where protein synthesis is decreased by acute fasting or chronic food restriction.     

Control of proteoglycan biosynthesis. Further studies on the effect of serum on cultured bovine articular cartilage.

Proteoglycan synthesis in explant cultures of adult bovine articular cartilage is stimulated in a dose-dependent manner when the tissue is cultured in the presence of foetal-calf serum. The stimulation of proteoglycan synthesis is paralleled by a similar increase in DNA synthesis; however, when DNA synthesis is inhibited by hydroxyurea the stimulation of proteoglycan synthesis by serum remains essentially the same. The apparent half-life of the pool of proteoglycan core protein precursor was measured in freshly isolated tissue as well as in tissue cultured for 7 days in the presence and in the absence of foetal-calf serum; under all conditions the half-life was the same, suggesting that this value is independent of the net rate of proteoglycan synthesis. In the presence of actinomycin D, an inhibitor of RNA synthesis, there was a difference in the apparent half-life of the available pool of mRNA coding for proteoglycan core protein: 8.5 h for tissue maintained in the presence of serum and 3.8 h for tissue cultured in the absence of serum. It is suggested that proteoglycan synthesis is stimulated by serum factors at the level of DNA-dependent RNA synthesis. Concomitant with an increase in the rate of proteoglycan synthesis induced by the presence of serum in the culture medium, an increase in the concentrations of several glycosyltransferases involved in chondroitin sulphate synthesis was also observed.     

Role of ethylene in phytochrome-induced anthocyanin synthesis

Summary Synthesis of anthocyanin pigments in etiolated cabbage seedlings is influenced by ethylene at concentrations higher than 10 ppb, and etiolated seedlings produce sufficient ethylene to influence their anthocyanin synthesis. When escape of endogenous ethylene from this tissue is enhanced by means of hypobaric treatment, anthocyanin synthesis is accelerated. Stimulation of anthocyanin synthesis by brief red illumination is completely prevented by applied ethylene and indoleacetic acid inhibits anthocyanin synthesis by stimulating ethylene production. Red light reduces endogenous as well as auxin-induced ethylene production and there is a close correlation between light-induced inhibition of ethylene synthesis and stimulation of anthocyanin formation. We suggest that in part photo-induced anthocyanin synthesis is due to a lowered ethylene content in light-treated tissue.     

Biphasic platelet-activating factor synthesis by human monocytes stimulated with IL-1-beta, tumor necrosis factor, or IFN-gamma

The capacity of IL-1-beta, TNF, and IFN-gamma to stimulate platelet-activating factor (PAF) synthesis by human monocytes is examined in our report. All three cytokines induced PAF synthesis in a novel biphasic pattern with peaks of PAF synthesis 1 to 2 and 6 to 8 h after stimulation of the monocytes. In contrast, calcium ionophore A23187 elicited a single peak of early PAF synthesis. PAF in the early peak was largely retained intracellularly whereas PAF in the late peak was largely released into culture fluids. Combinations of cytokines were subadditive or antagonistic in inducing PAF synthesis. Cycloheximide inhibited the late peak of PAF synthesis indicating that protein synthesis is required for synthesis of the phospholipid PAF. Specific antibodies to TNF or IL-1-beta inhibited the late peak of PAF synthesis induced by IFN-gamma indicating that late PAF synthesis is dependent on cytokine synthesis. The quantities of PAF produced by cytokine-activated monocytes are sufficient to activate human monocytes. Thus, these studies suggest that PAF may mediate in part monocyte activation by cytokines.     

Swelling of rat hepatocytes stimulates glycogen synthesis

In hepatocytes from fasted rats, several amino acids are known to stimulate glycogen synthesis via activation of glycogen synthase. The hypothesis that an increase in cell volume resulting from amino acid uptake may be involved in the stimulation of glycogen synthesis is supported by the following observations. 1) The extent of stimulation of glycogen synthesis by both metabolizable and nonmetabolizable amino acids was directly proportional to their ability to increase cell volume, except for proline, which stimulated glycogen synthesis more than could be accounted for by the increase in cell volume. 2) Both cell swelling and stimulation of glycogen synthesis by amino acids were prevented when hepatocytes were incubated in hyperosmotic media containing sucrose or raffinose. 3) Increasing the cell volume by incubating hepatocytes in Na(+)-depleted media in the absence of amino acids also stimulated glycogen synthesis. 4) Stimulation of glycogen synthesis by Na+ depletion was prevented by restoring the normal osmolarity with sucrose, but not with choline chloride which, by itself, stimulated glycogen synthesis and increased the cell volume. It is concluded that stimulation of glycogen synthesis by amino acids is due, at least in part, to an increase in hepatocyte volume resulting from amino acid uptake, and that hepatocyte swelling per se stimulates glycogen synthesis.     

DNA synthesis in rat hepatocytes: Inhibition by a platelet factor and stimulation by an endogenous factor

Primary monolyer cultures of adult rat hepatocytes can be induced to undergo DNA synthesis in serum-free medium in the presence of insulin, glucagon, and epidermal growth factor (three factors). We have found that hepatocyte DNA synthesis is affected not only by an endogenous stimulant produced by the hepatocytes and released into the culture medium. Serum has a strong inhibitory effect on hepatocyte DNA synthesis. Partially purified human platelet extract (“platelet inhibitor”) inhibits the three-factor-induced DNA synthesis in a concenration-dependent manner. Pure βTGF at 0.5 ng/ml as well as HPLC-purified PDGF at 10 ng/ml completely inhibit the three-factor-induced DNA synthesis. Determination of the time required for the presence of the three factors and the platelet inhibitor to exert their effects indicated that the inhibition of DNA synthesis is caused not by competition of the platelet inhibitor with any of the three factors but through an independent pathway. Hepatocyte DNA synthesis is density-dependent and is greater if medium is not changed during the course of an experiment than if medium is changed daily. Hepatocyte-conditioned medium is also affective in stimulating DNA synthesis beyond the level induced by the three factors. These results suggest that an endogenous stimulant for hepatocyte DNA synthesis is produced by the hepatocytes themselves. Our studies demonstrate that hepatocyte DNA synthesis is subject to both stimulatory and inhibitory controls. Unlike the three factors, the endogenous stimulant can overcome the inhibition by the platelet inhibitor, suggesting the importance of these factors in the physiological control of hepatocyte DNA synthesis.     

Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle.

Nitric oxide (NO) synthesis is induced in vascular smooth muscle cells by lipopolysaccharide (LPS) where it appears to mediate a variety of vascular dysfunctions. In some cell types tetrahydrobiopterin (BH4) synthesis has also been found to be induced by cytokines. Because BH4 is a cofactor for NO synthase, we investigated whether BH4 synthesis is required for LPS-induced NO production in rat aortic smooth muscle cells (RASMC). The total biopterin content (BH4 and more oxidized states) of untreated RASMC was below our limit of detection. However, treatment with LPS caused a significant rise in biopterin levels and an induction of NO synthesis; both effects of LPS were markedly potentiated by interferon-gamma. 2,4-Diamino-6-hydroxypyrimidine (DAHP), a selective inhibitor of GTP cyclohydrolase I, the rate-limiting enzyme for de novo BH4 synthesis, completely abolished the elevated biopterin levels induced by LPS. DAHP also caused a concentration-dependent inhibition of LPS-induced NO synthesis. Inhibition of NO synthesis by DAHP was reversed by sepiapterin, an agent which circumvents the inhibition of biopterin synthesis by DAHP by serving as a substrate for BH4 synthesis via the pterin salvage pathway. The reversal by sepiapterin was overcome by methotrexate, an inhibitor of the pterin salvage pathway. Sepiapterin, and to a lesser extent BH4, dose-dependently enhanced LPS-induced NO synthesis, indicating that BH4 concentration limits the rate of NO production by LPS-activated RASMC. Sepiapterin also caused LPS-induced NO synthesis to appear with an abbreviated lag period phase, suggesting that BH4 availability also limits the onset of NO synthesis. In contrast to the stimulation of LPS-induced NO synthesis, observed when sepiapterin was given alone, sepiapterin became a potent inhibitor of NO synthesis in the presence of methotrexate. This is attributable to a direct inhibitory action of sepiapterin on GTP cyclohydrolase I, an activity which is only revealed after blocking the metabolism of sepiapterin to BH4. Further studies with sepiapterin, methotrexate, and N-acetylserotonin (an inhibitor of the BH4 synthetic enzyme, sepiapterin reductase) indicated that the BH4 is synthesized in RASMC predominantly from GTP; however, a lesser amount may derive from pterin salvage. We demonstrate that BH4 synthesis is an absolute requirement for induction of NO synthesis by LPS in vascular smooth muscle. Our findings also suggest that pterin synthesis inhibitors may be useful for the therapy of endotoxin- and cytokine-induced shock.     

Photocontrol of the synthesis of chlorophyll a and phycocyanin in the cyanobacterium Calothrix crustacea Schousboe]

Chlorophyll a and phycocyanin synthesis in the cyanobacterium Calothrix crustacea Schousboe (ecophene Rivularia bullata) have been studied in white light after the application of red and green light pulses. The light quality produces a complementary pattern in the pigment synthesis. Chlorophyll synthesis is stimulated by red light pulses whereas phycocyanin synthesis is by green light pulses. Because the effect of red light on chlorophyll synthesis shows some far-red photoreversibility, the action of phytochrome is proposed. The green light effect on phycocyanin synthesis is only partially reversed by far-red light. This reversion is lost after incubation in white light for two hours. The effect of green light on phycocyanin synthesis could not only be due to phytochrome since theoretically in green light the level of the active form of phytochrome is lower than in red light. Thus, the action of a specific green light photoreceptor is proposed.     

Ribosomal RNA synthesis in newly sliced discs of potato tuber

A burst of ribosomal RNA synthesis is induced in potato tissue by slicing, and continues at a decreasing rate for about 12 hours. Ribosomal RNA synthesis in potato discs is sensitive to puromycin, in contrast to non-ribosomal RNA synthesis. Thus, the influence of puromycin on total RNA synthesis is significant only during the first 12 hours following slicing. The function of RNA made after 12 hours in a puromycin-insensitive manner is unknown. However, it is apparently unrelated to protein synthesis, since it has been shown that total inhibition of RNA synthesis by addition of actinomycin D to potato tissue after 12 hours of aging has no effect upon protein synthesis during the ensuing 12 hours.     

Long-term modulation of type L pyruvate kinase activity in young and mature rats

The regulation of type L pyruvate kinase concentrations in liver of young (35–45 days old) and adult (60–85 days old) rats starved and re-fed a 71% sucrose diet was investigated. Re-feeding is accompanied by an increase in the enzyme level in liver determined kinetically and immunologically. A constant ratio of kinetic activity to immunological activity was observed under all conditions examined, indicating that activity changes are the result of a regulation of synthesis or degradation and not an interconversion between kinetically active and inactive forms of the enzyme. Synthesis of pyruvate kinase was directly examined by using hepatocytes isolated from starved and re-fed rats. A stimulation of pyruvate kinase synthesis is observed on re-feeding. This increase in synthesis of pyruvate kinase is retained by the isolated hepatocyte for up to 7h in the absence of hormonal stimuli. Administration of glucagon (1μm) to the isolated hepatocytes had no influence on synthesis of pyruvate kinase and no evidence for a glucagon-directed degradation of the enzyme was found. Re-feeding the rat was followed by a transient increase in the synthesis of pyruvate kinase. The peak rate of synthesis was observed before a detectable increase in the enzyme concentration. After a rapid synthesis period, a new steady-state level of the enzyme was achieved and synthesis rates declined. The time course and magnitude for the response to the sucrose diet was dependent on the age of the rat. In young rats, an increase in pyruvate kinase synthesis is observed within 6h and peak synthesis occurs at 11h after re-feeding sucrose. The peak synthesis rate for pyruvate kinase for young rats represents approx. 1% of total protein synthesis. With adult rats, increased pyruvate kinase synthesis is not observed for 11h, with peak synthesis occurring at 24h after re-feeding. In the older rats, peak pyruvate kinase synthesis constitutes greater than 4% of total protein synthesis. Continued re-feeding of the adult rat beyond 24h is accompanied by a decline of pyruvate kinase synthesis to approx. 1.5% of total protein synthesis. The concentration of the enzyme, however, does not decline during this period, suggesting that control of pyruvate kinase degradation as well as synthesis occurs.     

Action of phyenethyl alcohol on the synthesis of macromolecules in Escherichia coli

Prevost, C. (University of California, Berkeley), and V. Moses. Action of phenethyl alcohol on the synthesis of macromolecules in Escherichia coli. J. Bacteriol. 91:1446-1452. 1966.-A kinetic study of the effects of various concentrations of phenethyl alcohol on the synthesis of ribonucleic acid (RNA), deoxyribonucleic acid (DNA), protein, and beta-galactosidase in Escherichia coli has confirmed that RNA synthesis, rather than DNA synthesis, is first and most affected by phenethyl alcohol. The presence of inducer did not protect beta-galactosidase synthesis from inhibition by phenethyl alcohol. Little preferential inhibition of beta-galactosidase synthesis was observed; this is in contrast to the severe catabolite repression which results from partial inhibition of total protein synthesis caused by chloramphenicol or starvation for a required amino acid. We found no evidence that messenger RNA synthesis was inhibited to a greater extent than total RNA synthesis.     

Protein synthesis in relation to ripening of pome fruits

Protein synthesis by intact Bartlett pear fruits was studied with ripening as measured by flesh softening, chlorophyll degradation, respiration, ethylene synthesis, and malic enzyme activity. Protein synthesis is required for normal ripening, and the proteins synthesized early in the ripening process are, in fact, enzymes required for ripening. ¹⁴C-Phenylalanine is differentially incorporated into fruit proteins separated by acrylamide gel electrophoresis of pome fruits taken at successive ripening stages. Capacity for malic enzyme synthesis increases during the early stage of ripening. Fruit ripening and ethylene synthesis are inhibited when protein synthesis is blocked by treatment with cycloheximide at the early-climacteric stage. Cycloheximide became less effective as the climacteric developed. Ethylene did not overcome inhibition of ripening by cycloheximide. The respiratory climacteric is not inhibited by cycloheximide. It is concluded that normal ripening of pome fruits is a highly coordinated process of biochemical differentiation involving directed protein synthesis.     

Induction of the nuclear protein cyclin in serum-stimulated quiescent 3T3 cells is independent of DNA synthesis

Inhibition of DNA synthesis and cell proliferation of mouse 3T3 cells by aphidicolin did not affect the expression of cyclin, a nuclear protein whose synthesis correlates with cell proliferation, as determined by quantitative two-dimensional gel electrophoresis analysis. Serum stimulation of quiescent 3T3 cells revealed that cyclin synthesis increases shortly before DNA synthesis. Inhibition of DNA synthesis by aphidicolin in serum-stimulated quiescent cells did not affect the increase of cyclin following stimulation. These results demonstrate that cyclin synthesis is not coupled to DNA synthesis and that it is one of the latest events before DNA replication.     

Differential effects of endotoxin and fibrinogen degradation products (FDPS) on liver synthesis of fibrinogen and albumin: evidence for the involvement of a novel monokine in the stimulation of fibrinogen synthesis induced by FDPS

1. Administration of endotoxin or fibrinogen degradation products (FDPs) in rats increase fibrinogen synthesis comparable to that found during the acute phase response. 2. An increased fibrinogen synthesis is also found in co-cultures of hepatocytes with peripheral blood mononuclear cells upon administration of endotoxin or FDPs, but not in primary cultures of hepatocytes alone. 3. However, the increased synthesis of fibrinogen by FDPs is not accompanied by a decreased albumin synthesis, as in the case of stimulated fibrinogen synthesis induced by endotoxin in vivo and in co-cultures of hepatocytes with peripheral blood mononuclear cells, or induced by monocytic products in vivo and in primary cultures of hepatocytes alone. 4. Since IL-1 and/or IL-6 could not be accounted for the stimulation of fibrinogen synthesis without a decreased albumin synthesis, a novel monokine produced by mononuclear cells upon FDP administration might be involved.     

Differential stimulation by polyamines of phage RNA-directed synthesis of proteins

The effect of polyamines on Q beta and MS2 phage RNA-directed synthesis of three kinds of protein in an Escherichia coli cell-free system has been studied. With both phage RNAs, the degree of stimulation of protein synthesis by spermidine was in the order RNA replicase greater than A protein, while the synthesis of coat protein was not stimulated significantly by spermidine. The synthesis of RNA replicase was stimulated by 1 mM spermidine approx. 8-fold. From the results of Q beta RNA direct alanyl-tRNA and seryl-tRNA binding to ribosomes and initiation dipeptide synthesis, it is suggested that the preferential stimulation of the synthesis of RNA replicase by spermidine is due at least partially to the stimulation of the initiation of RNA replicase synthesis.     

Mitochondrial RNA synthesis in rat liver : Dependence on nuclear and cytoplasmic systems

The synthesis of mitochondrial RNA (mitRNA) in rat liver after inhibition of nuclear genome expression by actinomycin or of cytoplasmic protein synthesis by cycloheximide has been studied. Both drugs cause an inhibition of mitRNA synthesis. The suggestion is made that mitRNA synthesis is under nuclear control through the synthesis of some protein coded for by the nuclear genome.     

Macromolecular synthesis in E. coli, isolated mitochondria and L5178Y cells in the presence of hydroxyurea or formamidoxime

The effects of formamidoxime and hydroxyurea over a 10⁵ concentration range were studied on macromolecular synthesis in E. coli, L5178Y mouse leukemic cells, isolated rat liver mitochondria and isolated rat cerebral cortex mitochondria. In E. coli 2 mg per ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 20% and 17%, DNA synthesis by 91% and 96%, protein synthesis by 54% and 60% and lipopolysaccharide synthesis by 65% and 48%. In L5178Y mouse leukemic cells 2 mg/ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 41% and 24%, DNA synthesis by 90% and 97%, protein synthesis by 59% and 44% and glycoprotein synthesis by 83% and 50%. In isolated rat liver mitochondria 2 mg/ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 43% and 52%, DNA synthesis by 42% and 56% and protein synthesis by 18% and 30%. Glycoprotein synthesis was not affected. In isolated rat cerebral cortex mitochondria 2 mg/ml of formamidoxime and hydroxyurea inhibited, respectively, RNA synthesis by 50% and 44%, DNA synthesis by 59% and 66% and protein synthesis by 48% and 40%. Glycoprotein synthesis again was not affected. Lower concentrations of the drugs produced less inhibition of macromolecular synthesis in each of the systems.