Glyoxalase-I activity and cell cycle regulation in yeast

The status of glyoxalase-I was explored in exponentially growing and G₁ arrested temperature sensitive (ts) cell division cycle (cdc) mutants of Saccharomyces cerevisiae. It was observed that the specific activity of this enzyme was correlated with overall growth status. The activity was high in actively growing cells and was low in G₁ arrested cells. Specific activities of glyoxalase-I were also low in G₁ arrested prolonged stationary phase (PSP) cells of S. cerevisiae and Candida albicans. The activity of glyoxalase-I recovered when G₁ arrested S. cerevisiae (ts) cells were allowed to regrow under permissive conditions. Results demonstrate that although glyoxalase-I activity is a good indicator of cell growth status, it is not involved in cell cycle regulation of this eukaryotic organism.     

Catalytic activity and EPR signals of DPNH dehydrogenase in relation to the acquisition and loss of piericidin sensitivity and of coupling site 1

The events accompanying the development of piericidin sensitivity and of energy coupling site 1 during the transition of Candida utilis cells from the log phase to the late stationary phase of growth have been investigated. There is a large increase in DPNH dehydrogenase (DPNHD) activity, and major increases in the EPR signals of iron-sulfur centers 1, 2, and 3 of the enzyme, as measured at 13°K. The increase in DPNHD activity, however, does not reflect the increased development of the same enzyme as is present in the log phase, for the enzyme being synthesized in the stationary phase is different from that present in the log phase, as judged by juglone reductase and DPNH oxidase activities, which declined during the transition, and by stability, kinetics, and in the type of EPR signal present, which are different in the log and stationary phases. On catabolite repression of stationary phase cells the converse occurs: the specific activities of juglone reductase and DPNH oxidase rise, DPNHD activity and EPR signals corresponding to centers 1 and 2 disappear. This process of catabolite repression by ethanol is prevented by cycloheximide.     

REGULATION OF TYROSINE HYDROXYLASE ACTIVITY IN MOUSE NEUROBLASTOMA CLONE N1E-115

Abstract— Mouse neuroblastoma (clone N1E-115) cells in the logarithmic growth phase were incubated for 12 days. From early log phase to late stationary phase, the specific activity of tyrosine hydroxylase (EC 1.14.3a) increased greater than 30-fold. The increase in tyrosine hydroxylase per cell and per dish was 12- and 2700-fold, respectively. When cell division was stopped by removing serum or by adding 0.1 m m -5-fluorodeoxyuridine and 0.1 m m -uridine, the enzyme activity was also found to increase. These results show that tyrosine hydroxylase is regulated in neuroblastoma clone N1E-115.     

Hydroxylation of geraniol and nerol by a monooxygenase from Vinca rosea

A microsomal mixed function oxidase isolated from V. rosea seedlings was shown to catalyze the hydroxylation of the monoterpene alcohols, geraniol and nerol, to their corresponding 10-hydroxy derivatives. Hydroxylase activity was dependent upon NADPH and oxygen and was associated with the 100,000 X g pellet which exhibited a characteristic reduced P-450-CO binding spectra. Light reversible inhibition by CO as well as differential sensitivity to other inhibitors established the hydroxylase as a cytochrome P-450 type. Cis-trans isomerase activity was not observed in this preparation. Both geraniol and nerol were shown to be hydroxylated almost exclusively at the C-10 methyl group.     

Lipase production byCandida rugosa: Fermentation behaviour

Summary Extracellular lipase production byCandida rugosa growth has been studied. The main growth parameters, and the lipase activity in the culture broth were determined in order to identify the maximum of enzyme activity.The effect of lipidic material and size and growth phase of the inoculum on enzymatic production have been studied. Maximum extracellular lipase activity was associated with an increase in enzyme production when the number of viable cells started to decrease.     

DOPAMINERGIC PROPERTIES OF A SOMATIC CELL HYBRID LINE OF MOUSE NEUROBLASTOMA X SYMPATHETIC GANGLION CELLS

Abstract— Studies were made on the regulation of dopamine metabolism in a cell line derived by hydridization of a non-tyrosine-hydroxylase-containing line of murine neuroblastoma cells with a neur-onally-enriched population of murine embryonic sympathetic ganglion cells. Hybrid subclones with tyrosine hydroxylase activity were selected by exposure to tyrosine-free medium. The cells also exhibited DOPA decarboxylase activity and the subclone (named T28) with the highest specific activities of both enzymes was further characterized. The hybrid T28 line did not contain dopamine-β-hydroxylase activity. The specific activity of tyrosine hydroxylase as well as of DOPA decarboxylase increased significantly in T28 cultures when the cells entered the stationary phase of growth. Both of these enzymes were also induced after several days of exposure to 1 m m -dibutyryl cyclic AMP in culture medium containing either 5% or 0.8% serum. However, maintenance in medium containing 0.8% serum alone, which inhibited cell multiplication, did not induce either enzyme. The dopamine content of T28 cells was also regulated as a function of cell density. High density (stationary phase) cultures of T28 cells contained about 300 pmol dopamine per mg protein and at least half of this endogenous amine appeared to he stored in vesicles or granules (as judged by depletion with reserpine or α-methyl- m -tyramine). The T28 and other neuronal hybrid lines appear to be useful model systems for neuro-chemical studies.     

Induction of enzymes of phytoalexin synthesis in cultured soybean cells by an elicitor from Phytophthora megasperma f. sp. glycinea

The glucan elicitor from cell walls of the fungal pathogen, Phytophthora megasperma f. sp. glycinea, induced rapid but transient increases in enzyme activities of general phenylpropanoid metabolism (phenylalanine ammonia-lyase and 4-coumarate: CoA ligase) and of the flavonoid pathway (chalcone synthase) in cell suspension cultures of soybean (Glycine max). After transferring cells into fresh medium, two peaks of inducibility for the enzymes by elicitor were observed, one shortly after transfer (stage I), and one at the end of the linear growth phase (stage II). Only one of the two isoenzymes of 4-coumarate: CoA ligase (isoenzyme 2), for which a specific involvement in flavonoid biosynthesis has been postulated, was affected by the elicitor. For two of the induced enzymes, phenylalanine ammonia-lyase and chalcone synthase, the changes in activity at stage I were shown to be preceded by large changes in their rates of synthesis, as determined by in vivo labelling with [³⁵S] methionine and immunoprecipitation.Abbreviations Pmg Phytophthora megasperma f. sp. glycinea - glyceollin is a term used to designate the 3 isomers which accumulate in challenged soybean tissue (Moesta and Grisebach 1981b)     

Production of active human interferon-β inE. coli II. Optimal condition for induced synthesis by 3,β-indoleacrylic acid

Summary The maximum level of human interferon- activity was expressed under the control of theE. coli tryptophan promoter whenE. coli cells were induced at late logarithmic growth phase by 3,-indoleacrylic acid (IAA). The level is one order of magnitude higher than that obtained when the cells were induced at early logarithmic or stationary phase. When IAA was subsequently further added, the decrease in the activity observed at a latter period of fermentation was suppressed.     

Extracellular accumulation of high specific-activity peroxidase by cell suspension cultures of cowpea

Cell suspension cultures of cowpea (Vigna sp.) were able to produce extracellular peroxidase. Different growth regulator concentrations induced different peroxidase activity in callus. The crude extracellular medium after four weeks of culture showed higher (6 times) specific peroxidase activity and higher thermo stability than commercial horse-radish peroxidase. The commercial production of peroxidase enzyme from cowpea by utilizing plant cell cultures is discussed.     

Energy conservation in malolactic fermentation by Lactobacillus plantarum and Lactobacillus sake

A comparably poor growth medium containing 0.1% yeast extract as sole non-defined constituent was developed which allowed good reproducible growth of lactic acid bacteria. Of seven different strains of lactic acid bacteria tested, only Lactobacillus plantarum and Lactobacillus sake were found to catalyze stoichiometric conversion of l-malate to l-lactate and CO₂ concomitant with growth. The specific growth yield of malate fermentation to lactate at pH 5.0 was 2.0 g and 3.7 g per mol with L. plantarum and L. sake, respectively. Growth in batch cultures depended linearly on the malate concentration provided. Malate was decarboxylated nearly exclusively by the cytoplasmically localized malo-lactic enzyme. No other C₄-dicarboxylic acid-decarboxylating enzyme activity could be detected at significant activity in cell-free extracts. In pH-controlled continuous cultures, L. plantarum grew well with glucose as substrate, but not with malate. Addition of lactate to continuous cultures metabolizing glucose or malate decreased cell yields significantly. These results indicate that malo-lactic fermentation by these bacteria can be coupled with energy conservation, and that membrane energetization and ATP synthesis through this metabolic activity are due to malate uptake and/or lactate excretion rather than to an ion-translocating decarboxylase enzyme.     

Studies on the distribution and regulation of microbial keto ester reductases

In a limited screening 65 microorganisms were tested with regard to their ability to reduce keto acids or esters of different chain length and position of the keto group with NADH or NADPH as coenzymes. Twenty-seven organisms exhibited reductase activity. Among these, Candida parapsilosis and Rhodococcus erythropolis have been chosen for further investigation. The keto ester reductases of both C. parapsilosis and R. erythropolis prefer NADH as coenzyme and show higher activity towards keto esters than keto acids. The keto ester reductase production of C. parapsilosis during growth passed a maximum in the late exponential phase, decreased and reaches a plateau in the stationary phase. In contrast, the specific activity of the keto ester reductase of R. erythropolis did not decrease in the stationary growth phase. The enzyme of C. parapsilosis was inducible by a keto ester when growing on glycerol as the sole carbon source. Furthermore, the enzyme of C. parapsilosis was subject to catabolite repression. When C. parapsilosis and R. erythropolis were cultivated on n-alcane the specific activity of their keto ester reductases was enhanced about seven- and eightfold, respectively, compared to growth on glucose. This leads to the assumption that, while growing on n-alcane, a degradation product is formed in both strains that induces the production of the keto ester reductase. Correspondence to: M.-R. Kula     

Plant cell immobilization in a dual hollow fiber bioreactor

Summary Lithospermum erythrorhizon was immobilized in a dual hollow fiber bioreactor (DHFBR) to maintain high cell density and to operate continuously. The cells grew well and its dry biomass density was 325 g/L of the void volume for the cell growth. Volumetric and specific productivities of phenolics were 221 mg/L.day and 0.68 mg/g.dry wt.day, respectively, which are 58 and 2 times of those of shake flask cultures.     

Effects of organism type and growth conditions on cell disruption by impingement

Data for disruption of C. utilis, S. cerevisiae and B. subtilis cells by impingement of a high velocity jet of suspended cells against a stationary surface are compared. Differences between organisms were observed, but there were no general differences found between yeast and bacteria. In addition, growth conditions were found to have an effect on disruption with cells grown at a high specific growth rate easier to disrupt than cells grown at a low rate.Nomenclature a exponent of pressure (dimensionless) - D dilution rate (h^\s-1) - K dimensional rate constant (Pa ^\s-) - N number of passes (dimensionless) - P operating pressure (Pa) - R fraction of cells disrupted (dimensionless) - u_m maximum specific growth rate (h^\s-1)     

Glucose isomerase activity of streptomyces kanamyceticus

Summary Streptomyces kanamyceticus produces a significant level of intracellular glucose isomerase when grown in submerged culture. The optimum temperature for enzyme activity is 90°C, but the optimum pH is changed by the kinds of buffer solution used. The activity is higher at pH 7.0–9.5. Treatment of cells with cetyl trimethyl ammonium bromide extracts almost the same amount of the enzyme as ultrasonic treatment. The selection of the method of treatment for enzyme extraction depends, however, on the nature of cell growth in synthetic or complex medium.     

Prolyl hydroxylase activity in L-929 fibroblasts incubated with and without ascorbate

An improved procedure was used to assay prolyl hydroxylase activity in both early-log and late-log L-929 fibroblasts grown on plastic surfaces. When 40 μg/ml of ascorbate was added to early-log phase cultures, the rate of hydroxy-[¹⁴C] proline synthesis increased 2-fold within 4 h, but there was no change in prolyl hydroxylase activity per cell. The results indicated therefore that ascorbate did not “activate” prolyl hydroxylase in the sense of converting inactive enzyme protein to active enzyme protein. Instead ascorbate appeared to increase hydroxyproline synthesis in early-log L-929 fibroblasts because the prolyl hydroxylase reaction in such cells was limited by the availability of ascorbate or a similar cofactor. When 40 μg/ml of ascorbate was added to late-log phase cultures, there was essentially no effect on the rate of hydroxyl[¹⁴C]-proline synthesis or prolyl hydroxylase activity. The late-log phase cells, however, contained three times more enzyme activity and about two times more immuno-reactive enzyme protein than early-log phase cells. In addition, the rate of protein synthesis per cell in late-log phase cells was only one-tenth the rate in early-log phase cells. The results suggested that as the cells grew to confluency, collagen polypeptides were more completely hydroxylated in part because the rate of polypeptide synthesis decreased and at the same time prolyl hydroxylase activity per cell increased. The results appear to provide an alternate explanation for previous observations on the effects of ascorbate and “crowding” on hydroxy[roline synthesis in cultures of L-929 fibroblasts.     

A Deuterium Effect in Insect Attraction

Brevibacterium flavum cells obtained from different growth phases were immobilized with κ-carrageenan and the stability of the fumarase activity was investigated. The stability of fumarase activity of the immobilized preparation of cells of the stationary growth phase was highest. The highest stability of the immobilized cells seemed to be correlated to the high stability of fumarase activity in free cells of the stationary phase. High rigidity of the cell wall and membrane of B. flavum cells of the stationary phase and firm binding of fumarase protein to the cell membrane were suggested from several lines of evidence obtained on treatment of the cells with lysozyme and detergents or sonication of the cells. Electronmicrographs showed that the cells of the stationary phase retained the original shape after repeated batch reactions. Solubilized fumarase prepared from cells of the stationary phase showed the highest stability. Experiments using the partially purified enzyme strongly suggested the existence of fumarase-stabilizing components in the cells.     

Isolation and characteristics of <Emphasis Type="Italic">Bacillus intermedius</Emphasis> glutamyl endopeptidase secreted by a recombinant strain of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> at various growth phases

The recombinant strain of Bacillus subtilis bearing B. intermedius glutamyl endopeptidase gene in multicopy plasmid Δ58.21 secretes the enzyme to the medium at the phase of slowing of growth and the stationary growth phase with accumulation maxima at 24 and 48 h. Enzyme samples were isolated from the culture liquid after 24 and 48 h of culturing of and were purified up to homogeneity by ion exchange chromatography on carboxymethyl cellulose and HPLC on a MonoS column. The molecular weight of the corresponding proteins was 29 kDa. Both preparations had identical structure, but differed in affinity to the specific substrate Z-Glu-pNA. The effects of Ca²⁺ ions and specific low-molecular and protein inhibitors on the activity of the enzyme corresponding to various growth phases has been studied.     

The association between prolyl hydroxylase metabolism and cell growth in cultured L-929 fibroblasts

Prolyl 4-hydroxylase (EC 1.14.11.2) is a key enzyme in collagen biosynthesis, its active form is a tetramer (alpha 2 beta 2). In L-929 fibroblasts in the log phase of culture there is a low level of active enzyme. When the cell culture reaches confluency, prolyl hydroxylase activity in cells increases by a process that requires de novo RNA and protein synthesis. The same result may be achieved by crowding the cells (replating log phase cells at the density of stationary phase cells). In the work reported here we further examined induction of the enzyme. RNA synthesis necessary for enzyme induction is complete 6 h after "crowding" while protein synthesis requires 12 h. Thymidine (0.2-0.5 mM) added to log phase cells will also cause enzyme induction to the level found in "crowded" or resting cells. We also looked at the decay of the enzyme activity after subculture. This occurs rapidly (enzyme half-life is 1-2 h) and is concurrent with the re-entry of resting cells into cell cycle; however, thymidine added at the time of subculture to block DNA synthesis does not prevent the loss of prolyl hydroxylase activity. These results suggest that when cells are not engaged in propagation, they begin to synthesize luxury proteins such as prolyl hydroxylase. However, the loss of prolyl hydroxylase during subculture is probably not a direct consequence of DNA synthesis.