Catabolite repression of the formation of precursors of electron transfer complexes III and IV in adapting bakers' yeast: dependence upon a mitochondrially translated repressor.

When bakers' yeast cells which had been grown anaerobically in galactose were aerated in the presence of 10% glucose, they showed a 40% decrease in $\text{in}\text{vivo}$ [¹⁴C]-leucine incorporation into a washed mitochondrial membrane fraction compared with cells which had been aerated in a low glucose medium. The observed catabolite repression of membrane protein synthesis was primarily due to a decrease in cytoplasmic translational activity, but this repression was entirely dependent upon concomitant mitochondrial translation. The inductions of reduced coenzyme Q cytochrome $\text{c}$ reductase (complex III) and of cytochrome $\text{c}$ oxidase (complex IV) activities were repressed 30 and 60%, respectively, by aeration of the cells for 8 hours in 10% glucose. The catabolite repression of the formation of these two inner membrane complexes was again shown to be dependent upon concomitant mitochondrial translation. Both the amino acid incorporation and enzyme induction data suggest that catabolite repression of both cytoplasmically and mitochondrially translated mitochondrial membrane proteins is mediated through a mitochondrially translated repressor.     

Role of the mitochondrial protein synthesis is the catabolite repression of the petite-negative yeast K.lactis.

The effect of glucose in two different strains of the petite-negative yeast $\text{K. lactis}$ is studied. The results obtained show that one strain ($\text{K. lactis}\text{CBS 2359}$) is glucose repressible for Glutamate Dehydrogenase and β-Galactosidase, whereas the other one (CBS 2360) is almost completely insensitive. The effect of Erythromycin on expression of catabolite repression in CBS 2359 is also analyzed. The results show that the dependence of catabolite repression on mitochondrial protein synthesis reflect the degree of interaction between the nuclear and mitochondrial compartments.     

Glucose repression of the inducible catabolic pathway for N-acetylglucosamine in yeast.

In order to investigate the mechanism of glucose repression of the N-acetylglucosamine metabolic enzymes in $\text{Candida}\text{albicans}$, an obligatory aerobic yeast, the activities of the following inducible enzymes were assayed: the N-acetylglucosamine uptake, N-acetylglucosamine kinase and glucosamine-6-phosphate deaminase. In the presence of glucose or other sugars e.g. succinate and glycerol, synthesis of these enzymes took place at a normal rate, suggesting that the hexose produces no catabolite repression in this organism. On the contrary, strong inhibition by glucose was observed on the activities of N-acetylglucosamine uptake and deaminase in N-acetylglucosamine-grown cells of $\text{Saccharomyces}\text{cerevisiae}$, a facultative aerobe. From the results, it is concluded that “glucose effect” or catabolite repression is absent in $\text{Candida}\text{albicans}$, a pathogenic strain of yeast.     

Cyclic Adenosine 3′,5′-Monophosphate in Escherichia coli

The concentration of cyclic adenosine 3',5'-monophosphate (c-AMP) in Escherichia coli growing on different sources of carbon was studied. Cultures utilizing a source of carbon that supported growth relatively poorly had consistently higher concentrations of c-AMP than did cultures utilizing sugars that supported rapid growth. This relationship was also observed in strains defective in c-AMP phosphodiesterase and simultaneously resistant to catabolite repression; in such strains the c-AMP concentration was slightly higher for several sources of carbon tested. Cultures continued to synthesize c-AMP and secreted it into the medium, under conditions that brought about an inhibition of the intracellular accumulation of the cyclic nucleotide. Transient repression of the synthesis of beta-galactosidase was not associated with an abrupt decrease in the cellular concentration of c-AMP.     

Synthesis of the E. coli pyruvate dehydrogenase complex: non-dependence on 3'-5'-cyclic AMP

A determination of the level of the pyruvate dehydrogenase complex in a 3′–5′-c-AMP deficient mutant of $\text{E.}$$\text{coli}$ K12 has been carried out. The deficiency has no effect on specific activities for derivatives carrying either the inducible genes for two components of the complex or constitutive mutants. We conclude that synthesis of the complex is not sensitive to catabolite repression.     

Effect of NH3 on c-AMP associated activities and extracellular c-AMP production in Dictyostelium discoideum.

A model of morphogenetic regulation in $\text{Dictyostelium discoideum}$ (1) is based on the assumption that NH₃ inhibits the synthesis and/or release of extracellular 3′,5′-cyclic AMP and that by topographical restriction of c-AMP production to specified zones within the cell aggregate, NH₃ is presumed to set up the conditions for apical dominance and directed morphogenetic movements. This study indicates that: exposure of preaggregative cells to exogenous NH₃ + NH₄⁺ inhibits the acquisition of c-AMP-induced properties associated with aggregation competence (accumulation of specific contact sites required to form EDTA resistant aggregates and the synthesis of extracellular and membrane-bound c-AMP phosphodiesterase); exposure of aggregation competent cells which are actively producing extracellular c-AMP to exogenous NH₃ + NH₄⁺ is followed by the immediate cessation of extracellular c-AMP release. The pH dependence of these effects suggests that the active species is NH₃.     

Adenosine 3', 5'-monophosphate in mycobacteria.

Cyclic adenosine 3′,5′-monophosphate (cyclic AMP) is present in saprophytic fast growing as well as pathogenic and non-pathogenic slow growing mycobacteria. Apparently there does not seem to be any direct relationship between either intra- or extra-cellular cyclic AMP content with the growth rate of the bacteria. Intracellular cyclic AMP content is much higher than that of $\text{E}$. $\text{coli}$ grown on a similar carbon source. Glucose when added to the cells suspended in phosphate buffer lowers the intracellular cyclic AMP content by 6–8 fold.     

Accumulation of 2-deoxyglucose against its concentration gradient in rat adipocytes

Rat adipocytes were incubated at 37°C with 2-deoxy-d-[1-¹⁴C]glucose ([¹⁴C]2dGlc) at various concentrations and the intracellular concentrations of [¹⁴C]2dGlc and deoxy[¹⁴C]glucose phosphate ($\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$) were measured. Using 7 μM extracellular [¹⁴C]2dGlc, the intracellular [¹⁴C]2dGlc concentration approached the extracellular by 5 min in insulin-stimulated cells and by 60 min it exceeded the extracellular concentration by 50-fold. A maximum accumulation ratio of 3.5 was reached by 7 min using 1 mM and a ratio of 1.6 was reached by 1 to 3 min using 10 mM extracellular 2dGlc. The time at which the concentration of intracellular 2dGlc exceeded the extracellular was inversely related to the accumulation of $\text{2}\text{dGlc}\text{P}$. The rate of accumulation of total radioactivity ([¹⁴C]2dGlc plus $\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$ decreased after 20 min using 7 μM extracellular [¹⁴C]2dGlc. This change occurred later at 22°C or in the absence of insulin and sooner at higher concentrations of 2dGlc. Experiments where uptake was stopped by dilution indicated that radioactivity appearing in the medium was [¹⁴C]2dGlc, but radioactivity disappearing from the cells was largerly $\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$. Addition of 10 mM unlabelled 2dGlc or glucose to cells preincubated with 7 μM [¹⁴C]2dGlc resulted in a more rapid loss of accumulated label from the cells, while addition of 10 mM $\text{3-O-}\text{methylglucose}$, a non-metabolizeable sugar analogue with about the same affinity for the transport system as 2dGlc, was without effect. The results show that 2dGlc is accumulated against its concentration gradient. It is suggested that the mechanism involves first, dephosphorylation of $\text{2}\text{dGlc}\text{P}$ and second, the presence of a diffusion barrier between the site of dephosphorylation and the transport site.     

Effects of amino acids and derivatives of cyclic adenosine 3′, 5′-monophosphate on the production of three exoenzymes by Staphylococcus, simulans biovar staphylolyticus

The extracellular protease, endopeptidase, and hexosaminidase produced by $\text{Staphylococcus}\text{,}\text{simulans}\text{biovar}\text{staphylolyticus}$ were neither induced nor repressed by amino acids but required a tryptic digest of casein for their production. Catabolite repression of exoenzyme production by glucose was not affected by exogenous cyclic adenosine 3′, 5′-monophosphate but was partially relieved by di- or monobutyryl derivatives of this compound.     

A partial defect in carbon catabolite repression in mutants of Saccharomyces cerevisiae with reduced hexose phosphyorylation

Summary Mutants of Saccharomyces cerevisiae with reduced glucose phosphorylation were investigated. They were all recessive and belonged to one gene HEX1, mutant designation hex1. Carbon catabolite repression of alpha-glucosidases, invertase and part of the total malate dehydrogenase was reduced. Repression of the glyoxylate cycle enzymes, isocitrate lyase and malate synthetase, as well as that of gluconeogenetic fructose-1, 6-bisphosphatase was normal. A slight effect on repression of succinate: cytochrome c oxidoreductase and respiration was to be detected. The effect on repression by fructose was much less pronounced but still clear. However, there was a paradoxical effect of hexose concentration with higher concentrations repressing less. Maltose was also less repressing in the mutant. Growth on all sugars degraded via the hexose phosphorylation reaction was reduced and more strongly so at higher concentrations. Intracellular concentrations of glucose-6-phosphate, fructose-6-phosphate and fructose-1,6-bisphosphate were largely the same in mutant and wild type. The only striking difference between mutant and wild type was a fourfold higher intracellular glucose concentration in maltose grown mutants cells. The data obtained do not support the contention that carbon catabolite repression of the enzymes studied is triggered by intracellular hexoses or their metabolites alone. They rather suggest that it is some component of the hexose phosphorylating system that contributes to carbon catabolite repression.     

Cyclic adenosine 3':5'-monophosphate levels in normal and transformed cells of higher plants

Cyclic adenosine 3′:5′-monophosphate (cAMP) concentrations were determined for various normal and transformed (crown-gall) plant tissues grown in sterile culture. No significant differences in cAMP concentrations were found between normal and transformed cells of $\text{Vinca rosea, Helianthus annuus}$, and $\text{Nicotiana tabacum}$, unlike the suppressed synthesis observed in transformed cells of mammalian systems. cAMP concentrations of these tissues in culture averaged 135 nanomolar. No correlation was found between cAMP concentrations and tissue culture generation times.     

Studies on the modification of the cellular response to injury. IV. Protective effect of extracellular acidosis against anoxia,thermal, and p-chloromercuribenzene sulfonic acid treatment of isolated rat liver cells

Isolated rat liver cells were exposed $\text{in}$$\text{vitro}$ to ageing in an aerobic condition and to the effects of anoxia, thermal and $\text{p}$-chloromercuribenzene sulfonic (PCMBS) acid treatment, The survival of the cells was studied at normal and acidic pH using vital dye staining, intracellular concentration of potassium and ATP as indicators of viability. In aerobic conditions at pH 7.4, control cells lost their viability within approximately 6 hours. Extracellular acidosis not only prolonged the life span of isolated control hepatocytes against mechanical and other possible stress factors associated with the incubation and the absence of substrates in the medium, but also protected the cells significantly against various other superimposed injurious effects. These observations augment our previous observations on Ehrlich ascites tumore cells and lend further support to the hypothesis that extracellular acidosis, a common phenomenon associated with cell injury, is not harmful to cell survival $\text{in}$$\text{vitro}$ evidently prolongs it. The mechanism(s) behind this effect is not fully understood but it is suggested that extracellular acidosis stabilizes, and thereby protects cellular membrane systems making them more resistant to various deteriorating effects.     

Cyclic 3',5'-adenosine monophosphate stimulates trehalose degradation in baker's yeast

The levels of cyclic 3′,5′-AMP and trehalose, as well as the specific activity of the trehalase have been investigated in cells of baker's yeast ($\text{Saccharomyces cerevisiae}$) during the lag phase preceding growth. During the first few minutes a substantial increase in the intracellular concentration of cyclic 3′,5′-AMP was observed, followed by a 6–8 fold increase in trehalase activity concomitant with the rapid degradation of trehalose. Cell free extracts prepared from resting yeast were shown to contain a cryptic trehalase, which under physiological conditions could be activated by cyclic 3′,5′-AMP to the same degree as $\text{in vivo}$. These observations suggest that in the lag phase of growth, the level of trehalose in baker's yeast is under control of a system, regulated by the level of cyclic 3′,5′-AMP.     

Extracellular arabinases in Aspergillus nidulans: the effect of different cre mutations on enzyme levels

The regulation of the syntheses of two arabinan-degrading extracellular enzymes and several intracellular l-arabinose catabolic enzymes was examined in wild-type and carbon catabolite derepressed mutants of Aspergillus nidulans. α-l-Arabinofuranosidase B, endoarabinase, l-arabinose reductase, l-arabitol dehydrogenase, xylitol dehydrogenase, and l-xylulose reductase were all inducible to varying degrees by l-arabinose and l-arabitol and subject to carbon catabolite repression by d-glucose. With the exception of l-xylulose reductase, all were clearly under the control of creA, a negative-acting wide domain regulatory gene mediating carbon catabolite repression. Measurements of intracellular enzyme activities and of intracellular concentrations of arabitol and xylitol in mycelia grown on d-glucose in the presence of inducer indicated that carbon catabolite repression diminishes, but does not prevent uptake of inducer. Mutations in creA resulted in an apparently, in some instances very marked, elevated inducibility, perhaps reflecting an element of “self” catabolite repression by the inducing substrate. creA mutations also resulted in carbon catabolite derepression to varying degrees. The regulatory effects of a mutation in creB and in creC, two genes whose roles are unclear, but likely to be indirect, were, when observable, more modest. As with previous data showing the effect of creA mutations on structural gene expression, there were striking instances of phenotypic variation amongst creA mutant alleles and this variation followed no discernible pattern, i.e. it was non-hierarchical. This further supports molecular data obtained elsewhere, indicating a direct role for creA in regulating structural gene expression, and extends the range of activities under creA control.     

Evidence against the presence of cyclic AMP and related enzymes in selected strains of Bacteroides fragilis

Cyclic AMP was not detected in whole cells, expended culture medium or culture supernatant fluid of selected strains of $\text{Bacteroides fragilis}$. Adenyl cyclase and c-AMP phosphodiesterase activities were also not detected in cell extracts of $\text{B. fragilis}$. The exogenous addition of dibutyryl-c-AMP or sodium cholate to cultures of $\text{B. fragilis}$ growing on lactose did not significantly affect the specific activity of β-galactosidase measured in cell extracts of this organism. No diauxic growth pattern could be demonstrated in a chemically defined medium containing 5 mM glucose + 28 mM lactose.     

Thymidine accumulation by choroid plexus in vitro

In vitro, the accumulation and release of [methyl-³H]thymidine ([${}^3\text{H}$]thymidine) by the isolated choroid plexus, the anatomical locus of the blood-cerebrospinal fluid barrier, was studied. With concentrations of [${}^3\text{H}$]thymidine in the medium of 1.0 μm (or greater), the choroid plexus accumulated [${}^3\text{H}$]thymidine against a concentration gradient by a process that depended on intracellular energy production but did not depend on intracellular binding or metabolism of the [${}^3\text{H}$]thymidine. This transport process was inhibited (although differentially) by various nucleosides and low temperatures but not by 2-deoxyribose or pyrimidine bases. With concentrations of less than 1.0 μm [${}^3\text{H}$]thymidine in the medium, the choroid plexus accumulated [${}^3\text{H}$]thymidine against a concentration gradient. However, the majority of the [${}^3\text{H}$]thymidine within the choroid plexus was metabolized to [${}^3\text{H}$]thymidine nucleotides at low extracellular [${}^3\text{H}$]thymidine concentrations (3 nm). This accumulation process depended, in large part, on saturable intracellular phosphorylation. Thymidine was the principal form released from choroid plexuses that had been incubated for various times in media containing concentrations of thymidine from 3 to 1.0 mm. The release of thymidine from choroid plexus was depressed by cold temperatures and a very high (2.56 mmol/kg) intracellular thymidine concentration.     

Sodium dependence of maximum flux, JM, and Km of amino acid transport in Ehrlich ascites cells

The Michaelis-Menten parameters, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$ and $\text{K}{}_{\mathrm{m}}$ of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na⁺ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, $\text{J}{}_{\mathrm{<mtext>M</mtext>}}$, decreased with decrease in extracellular Na⁺ for both α-aminoisobutyric acid and phenylalanine but the $\text{K}{}_{\mathrm{m}}$ for α-aminoisobutyric acid increased markedly as the Na⁺ concentration fell whereas the $\text{K}{}_{\mathrm{m}}$ for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na⁺-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na⁺-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na⁺-independent but it has a high $\text{K}{}_{\mathrm{m}}$ and is not additive with the Na⁺-dependent flux.     

Pyridoxal 5' phosphate: a selective inhibitor of oncornaviral DNA polymerases.

Pyridoxal 5′ phosphate at concentrations < 0.5 mM inhibits $\text{polymerization}$ of deoxynucleoside triphosphate catalysed by variety of DNA polymerases isolated from type C RNA tumor viruses, as well as $\text{E.}\text{coli}$, but $\text{does}\text{not}$ affect the polymerase associated RNase H activity. Both phosphate and aldehyde groups of pyridoxal phosphate are essential for the inhibition which appears to be mediated through the reversible Schiff base.     

Glutamine-requiring mutants of Bacillus subtilis.

Two glutamine-requiring (Gln^?) mutants of $\text{Bacillus subtilis}$ SMY were deficient in glutamine synthetase activity $\text{in vitro}$. The Gln^? mutants sporulated poorly unless glutamine was provided at high concentrations. The differential rate of histidase synthesis following induction was 4- to 6-fold higher in the Gln^? mutants than in wild-type cells. In addition, glucose repression of utilization of alternative carbohydrates appeared to be partially relieved in the Gln^? mutants.     

Roles of sodium and potassium ions on p-aminohippurate transport in rabbit kidney slices

This investigation was principally undertaken to test the ionic gradient hypothesis as applied to active $\text{p-}\text{aminohippurate}$ uptake in the rabbit kidney cortical slice preparation. Efflux of $\text{p-}\text{aminohippurate}$ from the slice was shown to be independent of external Na⁺ concentration. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing $\text{p-}\text{aminohippurate}$ increased intracellular concentrations of both Na⁺ and K⁺, and $\text{p-}\text{aminohippurate}$ accumulation occurred. Transferring slices from a low sodium preincubation to a high sodium incubation medium containing ouabain and $\text{p-}\text{aminohippurate}$ resulted in a net increase in intracellular Na⁺ concentration but no $\text{p-}\text{aminohippurate}$ accumulation occurred. Different combinations of preincubation and incubation media gave a high to low array of intracellular Na⁺ concentrations and these directly reflected their respective $\text{p-}\text{aminohippurate}$ uptake. These results suggest that the Na⁺-gradient hypothesis does not adequately explain the transport of organic acids in rabbit kidney. These results also suggest that Na⁺ possibly has an intracellular role through its stimulation of $\text{(Na}{}^{\mathrm{+}}\text{+ K}{}^{\mathrm{+}}\text{)-ATPase}$ channeled to energizing the $\text{p-}\text{aminohippurate}$ accumulative mechanism.