Lomofungin inhibition of allophanate hydrolase synthesis in Saccharomyces cerevisiae

Summary The RNA polymerase inhibitor, lomofungin has been used to determine the half life of specific synthetic capacities (invertase and -glucosidase) as well as that for gross protein synthesis. In both cases the studies conclude that cognate messenger RNAs decay with a half life of approximately 20 minutes. This antibiotic has been used to determine the half life of allophanate hydrolase specific synthetic capacity. We find that it decays with a half life of about three minutes; a value that agrees with the decay rates of allophanate hydrolase synthetic capacity following removal of inducer. These observations argue that mRNA may be metabolized by two separate routes in Saccharomyces.     

Sequence of molecular events involved in induction of allophanate hydrolase.

Addition of urea to an uninduced culture of Saccharomyces at 22 C results in appearance of allophanate hydrolase activity after a lag of 12 min. We have previously demonstrated that both ribonucleic acid (RNA) and protein synthesis are needed for this induction to occur. To elucidate the time intervals occupied by known processes involved in induction, temperature-sensitive mutants defective in messenger RNA transport from nucleus to cytoplasm (rna1) and in protein synthesis initiation (prt1) were employed along with an RNA polymerase inhibitor in experiments that measure cumulative synthetic capacity to produce allophanate hydrolase. These measurements identify the time within the lag period at which each of the above processes is completed. We observed that RNA synthesis, rna1 gene product function, and protein synthesis initiation are completed at 1 to 1.5, 4, and 9 to 10 min, respectively.     

Molecular events associated with induction of arginase in Saccharomyces cerevisiae.

Arginase, the enzyme responsible for arginine degradation in Saccharomyces cerevisiae, is an inducible protein whose inhibition of ornithine carbamoyl-transferase has been studied extensively. Mutant strains defective in the normal regulation of arginase production have also been isolated. However, in spite of these studies, the macromolecular biosynthetic events involved in production of arginase remain obscure. We have, therefore, studied the requirements of arginase induction. We observed that: (i) 4 min elapsed between the addition of inducer (homoarginine) and the appearance of arginase activity at 30 degrees C; (ii) induction required ribonucleic acid synthesis and a functional rna1 gene product; and (iii) production of arginase-specific synthetic capacity occurred in the absence of protein synthesis but could be expressed only when protein synthesis was not inhibited. Termination of induction by inducer removal, addition of the ribonucleic acid synthesis inhibitor lomofungin, or resuspension of a culture of organisms containing temperature-sensitive rna1 gene products in a medium at 35 degrees C resulted in loss of ability for continued arginase synthesis with half-lives of 5.5, 3.8, and 4.5 min, respectively. These and other recently published data suggest that a variety of inducible or repressible proteins responding rapidly to the environment may be derived from labile synthetic capacities, whereas constitutively produced proteins needed continuously throughout the cell cycle may be derived from synthetic capacities that are significantly more stable.     

Cell culture density as a modulator of collagenase expression in normal human fibroblast cultures

The effect of various stages of normal cell growth on human fibroblast collagenase found in the culture medium was studied, so that the regulatory mechanisms of synthesis, secretion and activity of the enzyme could be established. Specific activity of collagenase increased 6- to 10-fold shortly after confluence was reached when compared with low density levels and decreased in post-confluent cultures, suggesting that synthesis and/or release of the enzyme changes with culture density. To assess this possibility, culture medium was examined for immunoreactive collagenase protein by radioimmunoassay. After confluence was reached, immunoreactive collagenase had increased approx. 2-fold, indicating greater secretion, and probably synthesis, of the enzyme. However, the increase in specific activity of the enzyme observed shortly after confluence was greater than could be accounted for by an increase in immunoreactive enzyme protein. As a result of the disproportionate increase in collagenase activity, the collagenase activity per unit immunoreactive protein was also found to be greatest shortly after confluence and decreased in post-confluent cultures. This density-associated modulation of collagenase expression could be reproduced by initiating the cultures at high density after subculture. Expression of collagenase activity was dependent upon intact protein synthetic mechanisms, since cultures maintained in the presence of cycloheximide failed to secrete collagenase into the culture medium.     

Glucocorticoid-dependent uteroglobin synthesis and uteroglobulin mRNA levels in rabbit lung explants cultured in vitro

In rabbit lung explants cultured in vitro in a synthetic medium, the synthesis of the protein uteroglobin decayed progressively becoming virtually undetectable between 24-48 h of culture. Addition of glucocorticoids to the medium maintained the synthesis of uteroglobin. This glucocorticoid effect was dose-dependent with optima at about 0.1 microM and 1 microM for dexamethasone and cortisol respectively. Estradiol, progesterone, triiodothyronine, insulin or 10% calf serum added to the medium were ineffective in maintaining uteroglobin synthesis. Actinomycin D (10 micrograms/ml) added to the medium inhibited the effect of cortisol on uteroglobin synthesis. After 24 h of culture, both the relative levels of uteroglobin mRNA, measured by molecular hybridization, and uteroglobin synthesis were correlatively higher (up to 10-fold) in glucocorticoid-treated than in control explants.     

Environmental pH modulation of collagenase in normal human fibroblast cultures

Normal human skin fibroblast cultures have been used to assess the effects of relatively minor changes in environmental pH on collagenase, a major extracellular gene product. Collagenase accumulation in the culture medium, measured both as enzyme activity and immunoreactive material, was 2- to 10-fold greater at pH 7.6–8.2 than at pH 6.8–7.2. The pH-associated increase in collagenase was parallel by an increase in general protein synthesis. Nevertheless, prototypic lysosomal and cytoplasmic enzymes changed very little under identical culturing conditions. Although substantial intracellular protein degradation occurred at all pH values, the small differences either in general protein degradation or in specific collagenase degradation in the medium were of insufficient magnitude to account for the increased accumulation of collagenase.     

Activation of Protein Synthesis upon Dilution of an Arachis Cell Culture from the Stationary Phase

When a stationary phase cell culture of Arachis hypogaea L. is diluted into fresh media, there occurs a 10-fold increase in the rate of protein synthesis. The kinetics of the activation of amino acid-incorporating capacity show a lag of 10 to 15 minutes with maximal activity reached at 2 hours after dilution. The activation of protein synthesis is oxygen-dependent and is accompanied by a 2- to 4-fold increase in polyribosome content, as well as by a 3- to 4-fold increase in the rate of mRNA synthesis. Ribosomal function, as ascertained by determination of ribosomal transit time, is about 2.5 times more efficient in 2-hour diluted cultures as in cells immediately after dilution. These observations indicate that a very early response in the transition of plant cell cultures from the stationary state is an increased capacity for protein synthesis. At a molecular level, this increase in protein synthetic capacity is due in part to an increased mobilization of mRNA into polyribosomes and in part to a more efficient ribosomal translational capacity.     

Effects of progestin antagonists, glucocorticoids and estrogen on progesterone-induced protein secreted by rabbit endometrial stromal cells in culture

Progesterone enhances the synthesis of a 42 kDa protein secreted by rabbit endometrial stromal cells in primary culture. The duration of that response, the effects of estrogen and the inhibitory ability of antiprogestin steroid analogs, RU486, ZK98.299 and ZK98.734, were tested. Although there was a progressive decrease in the amount of the 42 kDa protein synthesized during a 6-day culture period, progesterone stimulated its rate of synthesis greater than 2-fold throughout that period. The addition of estrogen did not prevent the progressive decrease in the amount of the protein synthesized, nor did it enhance the progesterone effect when the culture medium contained phenol red. Estrogen alone did slightly induce 42 kDa protein synthesis by cells grown in phenol red-free medium, and the progesterone response was accentuated to the same degree. When present in a concentration that was 100-fold that of the progesterone, RU486, ZK98.299 and ZK98.734 each abolished stimulation. This antagonistic effect was overcome by addition of an equimolar concentration of progesterone. Deoxycorticosterone (DOC) also stimulated 42 kDa protein synthesis. The antiprogestins blocked this stimulatory effect, even when both steroids were in equimolar concentrations. There was no difference in the ability of ZK98.299 or ZK98.734 to block DOC stimulation, even though ZK98.734 exhibits no antiglucocorticoid activity [J. Steroid Biochem. 25 (1986) 835]. Therefore, it is likely that the DOC effect is mediated by the progesterone receptor system. These studies indicate that enhanced synthesis of the 42 kDa protein represents a progesterone receptor mediated event and that the cell culture system described can be used as a bioassay for determination of antiprogestin activity.     

Functional capacities and the adenylate energy charge in Escherichia coli under conditions of nutritional stress.

Functional capacities in Escherichia coli cells starved for glucose were examined by comparing protein synthesis, utilization of new substrates, and maintenance of viability with the adenylate energy charge of the culture. When growth ceased because of glucose exhaustion in an E. coli culture, the energy charge dropped from 0.90 to about 0.80. During this time, the viable-cell count and the capacity for protein synthesis and for induction of new enzymes were maintained only if other substrates were available in the medium. The culture could be maintained for many hours without growth or death if glucose was added slowly; the energy charge in this case stabilized at about 0.80. A consistent transient decrease in the energy charge to around 0.80, accompanied by a decrease in protein synthesis, was also observed during the adaptation from glucose to other substrates during diauxic growth on glucose and glycerol or lactose.     

Allophanate hydrolase, not urease, functions in bacterial cyanuric acid metabolism

Growth substrates containing an s-triazine ring are typically metabolized by bacteria to liberate 3 mol of ammonia via the intermediate cyanuric acid. Over a 25-year period, a number of original research papers and reviews have stated that cyanuric acid is metabolized in two steps to the 2-nitrogen intermediate urea. In the present study, allophanate, not urea, was shown to be the 2-nitrogen intermediate in cyanuric acid metabolism in all the bacteria examined. Six different experimental results supported this conclusion: (i) synthetic allophanate was shown to readily decarboxylate to form urea under acidic extraction and chromatography conditions used in previous studies; (ii) alkaline extraction methods were used to stabilize and detect allophanate in bacteria actively metabolizing cyanuric acid; (iii) the kinetic course of allophanate formation and disappearance was consistent with its being an intermediate in cyanuric acid metabolism, and no urea was observed in those experiments; (iv) protein extracts from cells grown on cyanuric acid contained allophanate hydrolase activity; (v) genes encoding the enzymes AtzE and AtzF, which produce and hydrolyze allophanate, respectively, were found in several cyanuric acid-metabolizing bacteria; and (vi) TrzF, an AtzF homolog found in Enterobacter cloacae strain 99, was cloned, expressed in Escherichia coli, and shown to have allophanate hydrolase activity. In addition, we have observed that there are a large number of genes homologous to atzF and trzF distributed in phylogenetically distinct bacteria. In total, the data indicate that s-triazine metabolism in a broad class of bacteria proceeds through allophanate via allophanate hydrolase, rather than through urea using urease.     

Culture of honeybee organs: Development of a new medium and the importance of tracheation

Summary A culture system for honeybeen fat body and ovary was developed that supported optimal levels of protein synthesis by the explanted tissues. Abdominal body wall preparations of honeybee workers and queens, with adhering fat body, and ovaries of egg-laying queens were incubated in a culture medium designed to match honeybee hemolymph composition as closely as possible. Incorporation of [³H]eucine into soluble tissue proteins was measured. The new medium makes possible rates of tracer incorporation into fat body proteins that are up to three times higher than other media tested. When the tracheal system of the organs was let intact and open to the air during incubation, protein synthesis increased 17-fold (fat body) or 15-fold (ovary) as compared to preparations without open tracheas. After explantation into the medium, labeled proteins were synthesized at a highly variable rate for 10 h, probably due to wound response, and at a constant rate for the next 60 h. In contrast, ovarian protein synthesis occurred at a constant rate for at least 20 h and showed no wound response. The rate of tracer incorporation into fat body proteins was 3.2 times greater in tissues from the queen. This culture system is therefore suitable for a variety of investigations in honeybeen development and reproduction. These studies were supported by grants from the Deutsche Forschungsgemeinschaft, a Senior Scientist Award from the Alexander v. Humboldt Foundation for H. H. Hagedorn, and a fellowship from the Deutscher Akademischer Austauschdienst for H. H. Kaatz.     

Stimulation of ornithine decarboxylase synthesis and its control by polyamines in regenerating rat liver and cultured rat hepatoma cells.

Ornithine decarboxylase has been induced in log phase hepatoma cells grown in suspension culture. Induction with N6, O2'-dibutyryl cyclic adenosine 3':5'-monophosphate produced a 4-fold increase in enzyme activity by 3 hours which was followed by a return to base levels by 6 hours. Induction with dexamethasone, a potent synthetic glucocorticoid, exhibited a slow steady rate of increase in enzyme activity, reaching a plateau level of approximately 5- to 6-fold stimulation by about 12 hours. Induced cell and regenerating rat liver ornithine decarboxylase were shown to be indistinguishable by titration with antibody monospecific to the latter and by heat stability. L-[14C]Leucine incorporation into immunoprecipitable enzyme protein after induction in vitro or partial hepatectomy showed an increase which, when coupled with the increase in enzymatic activity, indicated de novo synthesis of enzyme protein. Physiological concentrations of the naturally occurring polyamines, spermidine and spermine, abolish cyclic AMP induction whereas they have no effect on dexamethasone induction. Both inductions were abolished by cycloheximide; in contrast, inhibition by actinomycin D was complete for dexamethasone induction and only partial with respect to cyclic AMP induction. The different time pattern of induction seen with cyclic AMP and dexamethasone, the partial inhibition of the cyclic AMP induction seen with actinomycin D, as well as the absence of inhibition of the dexamethasone induction by polyamines, indicate that these inducers might affect different aspects of the control of the same enzyme.     

The induction of allophanate lyase during the vegetative cell cycle in light-synchronized cultures of Chlamydomonas reinhardi.

Allophanate lyase can be induced by urea or acetamide 20-40-fold within 4 h in NH4 + -deprived cultures of Chlamydomonas reinhardi. In light-synchronized cultures, allophanate lyase induction appeared to be limited to the light phase of the cell cycle, provided that culture samples were induced under ongoing illumination conditions (i.e. light induction of light phase cells and dark induction of dark phase cells). However, when culture samples were induced under constant light conditions this cell cycle pattern was abolished. Light was found to be required for allophanate lyase induction and this was shown to be due, in part, to the light requirement for inducer uptake. The relationship between allophanate lyase induction and gametogenesis is discussed.     

Stimulation of Synaptosomal γ-Aminobutyric Acid Synthesis by Glutamate and Glutamine

gamma-Aminobutyric acid (GABA) synthesis was studied in rat brain synaptosomes by measuring the increase of GABA level in the presence of the GABA-transaminase inhibitor gabaculine. The basal rate of synaptosomal GABA synthesis in glucose-containing medium (25.9 nmol/h/mg of protein) was only 3% of the maximal activity of glutamate decarboxylase (GAD; 804 +/- 83 nmol/h/mg of protein), a result indicating that synaptosomal GAD operates at only a small fraction of its catalytic capacity. Synaptosomal GABA synthesis was stimulated more than threefold by adding 500 microM glutamine. Glutamate also stimulated GABA synthesis, but the effect was smaller (1.5-fold). These results indicate that synaptosomal GAD is not saturated by endogenous levels of its substrate, glutamate, and account for part of the unused catalytic capacity. The greater stimulation of GABA synthesis by glutamine indicates that the GAD-containing compartment is more accessible to extrasynaptosomal glutamine than glutamate. The strong stimulation by glutamine also shows that the rates of uptake of glutamine and its conversion to glutamate can be sufficiently rapid to support GABA synthesis in nerve terminals. Synaptosomes carried out a slow net synthesis of aspartate in glucose-containing medium (7.7 nmol/h/mg of protein). Aspartate synthesis was strongly stimulated by glutamate and glutamine, but in this case the stimulation by glutamate was greater. Thus, the larger part of synaptosomal aspartate synthesis occurs in a different compartment than does GABA synthesis.     

Progesterone induces a secretory protein in cultured rabbit endometrial stromal cells

Secretion of newly synthesized proteins by rabbit endometrial stromal cells in culture was studied. Progesterone (P) stimulated the synthesis and secretion of a protein with a mol. wt of approx 62 K and a pI of 6.5-7.0. Induction of 62 K protein synthesis was dose dependent; addition of 10 nM P to primary cultures caused a 2-fold or greater increase in the amount of this protein in the medium while addition of 1 microM P resulted in a 4.3-fold increase. Synthesis of this 62 K protein was not induced by estradiol, dexamethasone, or testosterone, nor was progesterone stimulation of the protein modified by estradiol. Thus, induction appears to be specific for a progesterone receptor mediated effect. Synthesis and secretion of this protein in the culture system described is potentially useful as a progestin bioassay.     

Regulation of purine de novo synthesis in cultured human fibroblasts: the role of P-ribose-PP.

Procedures for assaying the rate of purine de novo synthesis in cultured fibroblast cells have been compared. These were (i) the incorporation of [(14)C]-glycine or [(14)C]formate in alpha-N-formylglycinamide ribonucleotide (an intermediate in the purine synthetic pathway) and (ii) the incorporation of [(14)C]-formate into newly synthesised cellular purines and purines excreted by the cell into the medium. Fibroblast cells, derived from patients with a deficiency of hypoxanthine phosphoribosyltransferase (HPRT-) (EC 2.4.2.8) and increased rates of purine de novo synthesis, were compared with fibroblasts from healthy subjects (HPRT+). Fetal calf serum, which was used to supplement the assay and cell growth medium, was found to contain sufficient quantities of the purine base hypoxanthine to inhibit purine de novo synthesis in HPRT+ cells. This inhibition was the basis of differentiation between HPRT- and HPRT+ cells. In the absence of added purine base, both cell types had similar capacities for purine de novo synthesis. This result contrasts with the increased rates of purine de novo synthesis reported for a number of human HPRT- cells in culture but conforms recent studies made on human HPRT- lymphoblast cells. The intracellular concentration and utilisation of 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP), a substrate and potential controlling factor for purine de novo synthesis, were determined in HPRT- and HPRT+ cells. The rate of utilisation of P-Rib-PP in the salvage of free purine bases was far greater than that in purine de novo synthesis. Although HPRT- cells had a 3-fold increase in P-Rib-PP content, the rate of P-Rib-PP generation was similar to HPRT+ cells. Thus, in fibroblasts, the concentration of P-Rib-PP appears to be critical in the control of de novo purine synthesis and its preferential utilisation in the HPRT reaction limits its availability for purine de novo synthesis. In vivo, HPRT+ cells, in contrast to HPRT- cells, may be operating purine de novo synthesis at a reduced rate because of their ability to reutilise hypoxanthine.     

Protein synthetic patterns during differentiation of imaginal discs in vitro.

Leg, wing, and eye-antennal imaginal discs of Drosophila melanogaster were obtained by dissection from late-third instar larvae and cultured in vitro in Schneider's medium containing β-ecdysone. Differentiation of adult structures was obtained in more than 90% of all cultures. Differentiation was somewhat slower than normal in vivo development, but synchronous, repeatable, and reasonably completed structures were obtained. Our initial efforts at analyzing the molecular events of imaginal disc differentiation in culture have been to study the protein synthetic pattern which occurs throughout the culture period. Discs were pulse labeled with [³⁵S]methionine, and the proteins were separated by SDS-gel electrophoresis. Analysis of the synthetic pattern was done by autoradiography of these gels using X-ray film. In all three disc types, pronounced changes in protein synthetic patterns occurred throughout the culture period. These changes appeared to be under strict temporal control. Although disc-specific differences could be seen, a comparison of the three discs types revealed a striking similarity in the changes which occurred in the patterns of protein synthesis during the 5-day culture period. In general, the protein synthetic patterns of different imaginal discs at the same period during differentiation showed greater similarities than the patterns of a single disc type at different periods. These results are consistent with a view of differentiation as a tightly controlled program of gene activation and deactivation operating throughout the differentiation process.     

A reexamination of the effects of creatine on muscle protein synthesis in tissue culture

Experiments designed to test the hypothesis that intracellular creatine level regulates the synthesis of muscle specific proteins have failed to demonstrate any creatine regulatory effect. Manipulation of the extracellular creatine in culture medium over a 5,700-fold range (1.3- 7.4 mM) was successful in altering intracellular total creatine by only a factor of 20 (1.4-42 mg creatine/mg protein), an indication that muscle cells are able to regulate intracellular creatine levels over a wide range of external creatine concentrations. Alterations of cell creatine had no effect on either total protein synthesis or synthesis of myosin heavy chain. Methods were perfected to measure total creatine, and incorporation of [3H]leucine into total protein and purified myosin heavy chain from the same culture dish to avoid the possibility of variation between dishes. The creatine analog 1- carboxymethyl-2-iminohexahydropyrimidine (CMIP) previously reported to stimulate myosin synthesis in culture was found to depress creatine accumulation by cells and depressed total protein synthesis and synthesis of myosin heavy chain. This inhibitory action of CMIP is consistent with the reported competitive inhibition of creatine kinase and presumed interference with energy metabolism.