Study of the relationship between the free water fraction in dried Candida utilis and the phosphorus content of cells]

The relationship between the free water fraction and the phosphate content in lyophilized yeast cells of Candida utilis was studied. The yeast were cultivated continuously and periodically, their growth rate being limited by phosphates. The decrease in the phosphate pool from 2% to 0.56% was followed by an increase in the fraction of free water by 6.3 times.     

Rate and extent of interconversion of tetrahydrofolate cofactors to dihydrofolate after cessation of dihydrofolate reductase activity in stationary versus log phase L1210 leukemia cells.

Previous studies from this laboratory established that the rapid but partial interconversion of tetrahydrofolate cofactors to dihydrofolate after exposure of L1210 leukemia cells to antifolates cannot be due to direct feedback inhibition of thymidylate synthase by dihydrofolate or any other endogenous folylpolyglutamates when dihydrofolate reductase activity is abolished by antifolates. Rather, the data suggested this preservation of tetrahydrofolate cofactor pools is likely due to a fraction of cellular folates unavailable for oxidation to dihydrofolate. This paper explores the role of cell cycle phase in L1210 leukemia cells in logarithmic versus stationary phase growth as a factor in the rate and extent of tetrahydrofolate cofactor interconversion to dihydrofolate after exposure of cells to the dihydrofolate reductase inhibitor trimetrexate. The S phase fraction was reduced by inoculating L1210 leukemia cells at high density to achieve a stationary state. Flow cytometric analysis of DNA content indicated that log phase cultures were 53.0% S phase; this decreased to 42.1% at 24 h and 24.1% at 48 h in stationary phase cultures. 5-Bromo-2'-deoxyuridine incorporation into DNA decreased 80 and 96%, while [3H]dUrd incorporation into DNA declined 70 and 95% for stationary cultures at 24 and 48 h, respectively, as compared with the log phase rates. Log phase cells interconverted 28.0% of the total pool of radiolabeled folates to dihydrofolate with a half-time of approximately 30 s. Stationary cells at 24 h interconverted 20.4% of the total folate pool with a t1/2 of approximately 3 min, and at 48 h, net interconversion to dihydrofolate decreased further to 12.1% with a t1/2 of approximately 6 min. The decrease in the extent of tetrahydrofolate cofactor interconversion to dihydrofolate in stationary phase cells was directly proportional to the decrease in the S phase fraction determined by total DNA content. This suggests that tetrahydrofolate cofactor depletion occurs only in S phase cells. The much larger drop in [3H]dUrd and 5-bromo-2'-deoxyuridine incorporation into DNA in comparison with the decline in the S phase fraction measured by DNA content along with the reduced rate of tetrahydrofolate cofactor interconversion to dihydrofolate indicates that the rate of DNA synthesis is decreased in S phase cells in stationary cultures. Network thermodynamic simulations suggest that a reduction in the number of S phase cells and their thymidylate synthase catalytic activity would account for the observed decrease in the rate and extent of interconversion of tetrahydrofolate cofactors to dihydrofolate after trimetrexate in stationary phase cultures.(ABSTRACT TRUNCATED AT 400 WORDS)     

Non-haem iron and the dissociation of piericidin A sensitivity from site 1 energy conservation in mitochondria from Torulopsis utilis

1. The aerobic incubation of iron-deficient Torulopsis utilis cells for 12h under non-growing conditions results in the recovery by mitochondria of the previously absent site 1 energy conservation and sensitivity to piericidin A. 2. The recovery of piericidin A sensitivity but not site 1 is prevented by the presence of cycloheximide (100mug/ml) in the medium used for aerobic incubation of the cells. Rotenone sensitivity behaved similarly. 3. Chloramphenicol, erythromycin and tetracycline were without effect on the recovery of site 1 and piericidin A sensitivity. 4. Inclusion of (59)Fe in the growth medium can be used as the basis for a highly sensitive assay for non-haem iron. 5. Iron-limited growth of T. utilis lowers the concentration of both non-haem iron and acid-labile sulphide of submitochondrial particles by over 20-fold compared with the ;normal' situation with iron-supplemented glycerol-limited growth. 6. Increases in the non-haem iron and acid-labile sulphide concentrations of submitochondrial particles occur when site 1 and piericidin A sensitivity are recovered. The increase is approximately halved by the presence of cycloheximide. 7. The non-haem iron of T. utilis submitochondrial particles does not exchange with added iron. 8. Continuous culture of T. utilis at the transition between glycerol- and iron-limitation results in cells where mitochondria possess site 1 energy conservation but lack piericidin A sensitivity. 8. It is concluded, in contrast with widely held views to the opposite, that energy conservation at site 1 does not require electron flow to proceed through a piericidin A- or rotenone-sensitive route. 9. Restriction of the iron supplied to growing T. utilis to a concentration just above that required for growth limitation demonstrates that a 10- to 20-fold decrease of the ;normal' non-haem iron concentration of both cells and mitochondria is without effect on the growth yield per unit of carbon source. Submitochondrial particles prepared from such iron-restricted but otherwise functionally normal cells have a non-haem iron concentration of about 0.5-0.8ng-atom/mg of protein. It is concluded that the concentration of iron-sulphur protein required for normal function by the respiratory chain is close to the concentrations of cytochromes and flavoproteins.     

Purification of protein components of the clostridial glycine reductase system and characterization of protein A as a selenoprotein

A procedure for the isolation in nearly homogeneous form of protein A, a low molecular-weight, acidic, protein component of clostridial glycine reductase, is described. The yield of protein A is high only in early log phase cells of Clostridium sticklandii grown under standard laboratory conditions in a rich tryptone-yeast extract-distilled water medium but, when selenite (1 μm) is added, the levels of protein A remain high throughout the entire log phase of growth. Addition of ⁷⁵Se-labeled selenite to the culture medium results in the highly selective incorporation of radioactive selenium into protein A. The procedure for isolation of protein A results in about a 700-fold enrichment when extracts prepared from cells that actively catalyze glycine reduction are used. However, the catalytic activity of the purified protein varies considerably from preparation to preparation. The molecular weight of protein A, estimated by sucrose density-gradient centrifugation, is approximately 12,000.The other higher molecular-weight components of glycine reductase are associated with the membrane fraction of the cell and are released as soluble proteins by sonic disruption of the membrane. After purification by ion-exchange and molecular sieve chromatography, these components are separated by DEAE-cellulose chromatography into two protein fractions both necessary for glycine reductase activity in protein A-supplemented assays. One of these fractions consists of a major protein component, protein B, also nearly homogeneous as determined by polyacrylamide gel electrophoresis. The other protein fraction still is heterogeneous.     

Synchronous cell differentiation in Caulobacter crescentus.

The growth of a stalked bacterium, Caulobacter crescentus, has been synchronized easily and reproducibly by a new method. When this bacterium is grown to a late log phase in nutrient broth at 30 C with aeration, swarmer cells are accumulated in the culture to 80% of the whole cell population. When this culture is inoculated into fresh pre-warmed broth at twentyfold dilution, it immediately initiates synchronous cell growth. Simultaneously, synchronous cell differentiation is monitored by the susceptibility of the cells to RNA phage infection. The swarmer cells accumulated in the late log phase of growth possess nearly the same susceptibility to RNA phage infection as those in the early log phase of growth while RNA phage-adsorbing capacity is lower in such swarmer cells. It is suggested that the swarmer cells accumulated in the late log phase of growth have lost some pili.     

Synchronous Cell Differentiation in Caulobacter crescentus

The growth of a stalked bacterium, Caulobacter crescentus, has been synchronized easily and reproducibly by a new method. When this bacterium is grown to a late log phase in nutrient broth at 30 C with aeration, swarmer cells are accumulated in the culture to 80% of the whole cell population. When this culture is inoculated into fresh pre-warmed broth at twentyfold dilution, it immediately initiates synchronous cell growth. Simultaneously, synchronous cell differentiation is monitored by the susceptibility of the cells to RNA phage infection. The swarmer cells accumulated in the late log phase of growth possess nearly the same susceptibility to RNA phage infection as those in the early log phase of growth while RNA phage-adsorbing capacity is lower in such swarmer cells. It is suggested that the swarmer cells accumulated in the late log phase of growth have lost some pili.     

Incorporation of [1-14C]-linoleic acid by LLC-WRC256 tumour cells

The incorporation of [(14)C]-linoleic acid (LA) into total lipid fractions was higher in LLC-WRC256 cells from the log phase of growth as compared to those of the plateau phase. LA was mainly incorporated into the phospholipid (PL) fraction of cells during the log phase, whereas in the plateau phase it was mostly taken into cholesterol ester. The proportion of radioactivity was higher in phosphatidylserine of cells from the log phase, whereas in the plateau phase it was higher in phosphatidylcholine. This feature of LA incorporation may be an important factor in determining the proliferative capacity of tumour cells.     

Effect of seawater on Escherichia coli β-galactosidase activity

An investigation of β-galactosidase activity of Escherichia coli strain H10407, under different physiological and environmental conditions, e.g. induced and uninduced osmotic stress, light, etc., was undertaken. In this study E. coli was employed as a model for faecal coliforms in waste water. β-Galactosidase activity was induced by isopropyl-β-D-thiogalactoside (IPTG). Enzyme activity (U cell^-1)/cell for sewage bacteria and for induced E. coli was similar, i.e. log U cell^-1= -8.5 whereas uninduced E. coli yielded log U cell^-1= -12.1. Initial enzyme activity was not dependent on phase of growth of the cell (exponential vs stationary phase) or whether marine or fresh water at the time of initial dilution. However, osmotic change resulted in a decrease in culturable cells, even though enzyme activity remained constant. A significant decrease in the number of culturable bacteria, followed by a decrease in β-galactosidase activity, was observed after exposure of cells to visible light radiation. It is concluded that β-galactosidase enzyme is retained in viable but non-culturable E. coli. Furthermore, β-galactosidase appears to offer a useful and rapid (25 min) measure of the viability of faecal coliforms, and therefore, of the water quality of bathing and shellfishing areas.     

Regulation of mitochondrial membrane assembly in Neurospora crassa. Transient expression of a respiratory mutant phenotype.

Cultures of mutant cni-1, a chromosomal mutant of Neurospora crassa, undergo a marked change in respiratory properties as the age of the culture increases. Early log phase cultures have a high level of respiration that is insensitive to inhibition by cyanide or antimycin A. Late log and stationary phase cultures have reduced rates of respiration. A high percentage of this respiration is inhibited by cyanide. Mitochondria from early log phase cni-1 have an excess of cytochrome c and little or no detectable cytochrome aa3. Mitochondria from late log and stationary phase cultures have levels of c-, b-, and a-type cytochromes that are not significantly different in concentration from those found in wild type cells. The cytochrome aa3 content and the cytochrome oxidase activity of cni-1 mitochondria increase 5- to 10-fold as the age of the culture increases. Mitochondria from early log phase cells of cni-1 synthesize only polypeptides of apparent molecular weights 7,000 to 10,000 and donot synthesize any of the mitochondrial components of cytochrome oxidase. Mitochondria from late log and stationary phase cells synthesize the normal complement of mitochondrial translation products including the mitochondrial components of cytochrome oxidase. The assembly of cytochrome oxidase is likely due to the availability of the mitochondrially synthesized components of the enzyme. The regulation of mitochondrial translation in the cni-1 mutant is independent of the nutrient content of the growth medium and is due to the accumulation or depletion of some component within the cell.     

Changes in the trophic organization of mitotic cycle of Candida utilis after exposure to RNAse from Bacillus intermedius]

The effect of the RNase from Bacillus intermedius on the growth and trophic cycle of Candida utilis was studied. The RNase at concentrations of 0.001-0.01 microgram/ml stimulated yeast growth by 30-40% as compared to the control, reduced the mitotic cycle of the yeast by shortening its G1 phase, and decreased the number of exotrophic cells in the G1 phase to a minimum. It was suggested that RNase is involved in the regulation of the transition of cells from the exo- to endotrophic state.     

Ribosome biosynthesis in Tetrahymena thermophila. III. Regulation of ribosomal RNA degradation in growing and growth arrested cells

We have measured the turnover rate of ribosomal RNA in exponentially growing Tetrahymena thermophila cells, cells entering the plateau phase of growth, and nutrient-deprived (starved) cells. Ribosomal RNA is stable in cells in early log phase growth but it begins to turnover as the cells begin a deceleratory growth phase prior to entering a plateau state. Likewise, rRNA in cells transferred from early log phase growth to a starvation medium begins to be degraded immediately upon starvation. In both cases the degradation of rRNA exhibits biphasic kinetics. A rapid initial exponential degradation with a half time of nine and one-half hours lasting for six hours is followed by a slower exponential degradation with a half-life of 35 hours. When starved cells are transferred to fresh growth medium turnover of rRNA ceases. The evidence presented suggests that the alteration in degradation rate is a regulated process which is most likely independent of the cell cycle.     

Synthesis of native 60S and 40S ribosomal subunits in Yoshida rat ascites hepatoma AH-130 cells: correlation with the rate of cell growth

The 60Sn and 40Sn subunit ribosome synthesis declined significantly in Yoshida rat ascites hepatoma AH-130 cells from the log phase to the plateau phase of the in vivo growth. Two main classes of 40Sn particles with protein/RNA ratios of 1.82 (p2) and 1.20 (p3) and a minor "heavy" one with protein/RNA ratio of 0.96 (p1) could be distinguished reproducibly by their ultraviolet absorption after sucrose zone sedimentation. The p2 particles appeared the dominating class in log phase cells. In plateau phase cells a decrease of p2 and an increase of p3 particles was observed. Under these conditions the p1 particles and the peaks corresponding to 60Sn subunits and to 80S ribosomes were also increased. Newly synthetized 40Sn particles banded in the p3 region of the gradient and p2 particles originated from them. These particles entered into the ribosomal cycle and contained poly(A) RNA. Formation of radioactive 80S couples by subunits entering into the ribosomal cycle was markedly stimulated in log phase cells and almost completely blocked in cells at the plateau phase of growth.     

Variables that regulate production of insulin-like peptide(s) in human leukemia cell line (HL-60)

A human myeloid leukemia cell line (HL-60) produces a peptide or peptides with insulin-like activity which is distinct from insulin or insulin-like growth factors (somatomedins). Factors regulating the production of this peptide (HL-ILP) were explored in the present study. The production of HL-ILP was maximal in the early log phase of cell growth and declined with increasing cell density. Differentiation of HL-60 cells to macrophages, induced by dihydroxyvitamin D3 or phorbol esters, was also associated with a decrease in HL-ILP production. Glucose consumption by the cells in the early log phase was closely related with HL-ILP production, and HL-ILP was found to stimulate glucose consumption by HL-60 cells. Production of HL-ILP was dependent on glucose concentrations in the culture medium and glucose concentrations higher than 1mg/d1 suppressed the release of HL-ILP. These observations are not inconsistent with a hypothesis that HL-ILP is involved in the glucose metabolism of the HL-60 cells that produce this peptide.     

Fractional protein makeup of Candida utilis yeast protoplasts in the process of their growth and development

Candida utilis IBFMY-405 was grown in a synthetic medium with glucose. Cells taken at the logarithmic phase of growth were studied. The cells were treated with the enzyme from Helix pomatia to prepare protoplasts which were separated by differential centrifugation into groups according to their size. Three protein fractions were isolated from each group and the amino acid composition of the proteins was determined. Proteins of the first fraction (cytoplasmic) prevailed in all of the protoplast groups while the content of proteins of the second fraction (intermediate or myosin-like) was the lowest. As the size of protoplasts increased, difference in the quantitative content of proteins from the first and second fractions became less pronounced. The content of proteins of the third fraction was 3.6 and 2.4 times higher in the protoplasts of the medium size than in the largest protoplasts. The amino acid composition of each protein fraction differed quantitatively and qualitatively in all of the protoplast groups.     

The cellular control of growth in cultures of Tetrahymena 2 In fluence of Fe medium and aeration on culture growth and cellular exchange of Ca.

Cultures of Tetrahymena pyriformis were grown exponentially with and without the addition of Fe and with no aeration. During the prestationary growth phase of all the cultures, there was a decrease in the cellular Fe concentration in the water insoluble cell fraction (IS) containing membranes and mitochondria, simultaneous with an increase in the Ca concentration in the water soluble cell fraction (S) containing ribosomes. This has been correlated to an energy deficit in the cells at the transition to the prestationary growth phase. In spite of the ability of Fe-deficient cultures to concentrate Fe, cultures grown in media with low Fe levels soon showed the lowest cellular Fe content. The high Fe levels seen in cultures grown with no aeration may reflect cellular adaptation to a different gaseous tension in the medium. Determinations with 45Ca showed an initial, large and rapid increase in cell radioactivity which was not correlated to cellular metabolism. After this there was differentiated increase due to the metabolic status of the cells. The following sequence was seen in all the cultures: (1) an increase in the exchange of S-and (mostly) IS-Ca at the end of the exponential growth phase, (2) an accumulation of Ca in the S fraction without an increased exchange (the IS-Ca is less exchangeable), and (3) a renewed increase in the exchange of Ca when the concentration is further increased at the end of the prestationary growth phase.     

Changing arabinosylation patterns of wall-bound hydroxyproline in bean cell cultures

The arabinosylation patterns of wall-bound hydroxyproline in Phaseolus vulgaris L. cell suspension cultures were determined by separating free hydroxyproline and hydroxyproline-arabinose oligomers over a Bio-Gel P-2 column. Total hydroxyproline accounted for about 3.3% of wall dry weight during all growth phases of batch-cultured bean cells. The chemical arabinosylation patterns of wall-bound hydroxyproline varied during the lag phase and early log phase of the culture. First, an increase in nonglycosylated hydroxyproline occurred accompanied by a corresponding decrease in hydroxyproline tetra-arabinoside. During the early log phase the reverse happened. In later stages of growth the chemical arabinosylation patterns remained constant. The radiochemical arabinosylation patterns were also determined, after pulselabeling the cultures with [¹⁴C]proline at various times during growth, to be able to distinguish recently incorporated hydroxyproline. The time course of the arabinosylation pattern of this fraction indicated that the initial changes in the chemical pattern were due to the temporary incorporation of less extensively glycosylated hydroxyproline-containing protein into the cell wall.Abbreviations Hyp hydroxyproline - HA_n hydroxyproline arabinoside - with n arabinosyl residues - TFA trifluoroacetic acid     

Increased disruption resistance of Candida utilis grown from survivors of enzymatic lysis combined with high-pressure homogenization

The resistance of Candida utilis (ATCC 9226) to disruption as a result of enzymatic pretreatment combined with high-pressure homogenization was found to increase when the yeast was grown from an inoculum which had previously been subjected to enzymatic pretreatment combined with high-pressure homogenization. The inoculum thus consisted of a mixture of undisrupted, viable cells and non-viable cells. The enzyme preparation employed was Zymolyase, which depolymerizes various components of the cell walls of viable yeast. A Microfluidizer was used for the high-pressure homogenization step. In order to obtain the 'disruption-resistant' cell fraction for use as an inoculum, 'normal' C. utilis was enzymatically pretreated, and subsequently homogenized (herein referred to as Microfluidization) using either three or 10 passes through the Microfluidizer at an operating pressure of 95 MPa. Yeast grown from the survivors of the enzyme/3-pass treatment were found to be somewhat more resistant to disruption by either enzymatic pretreatment alone or to enzymatic pretreatment followed by Microfluidization. Cells grown from enzyme/ 10-pass treated inocula exhibited the highest resistance to disruption. The 'disruption-resistant' fraction exhibited this characteristic through three serial re-cultivations. Possible mechanisms for the increased 'disruption-resistance' of this isolated population of C. utilis are presented.     

Response of Escherichia coli to ethyl methanesulfonate: Influence of growth phase and repair ability on survival and mutagenesis

The effects of altering the cell growth rate (physiological state) and DNA repair capacity (genetic state) on susceptibility to inactivation and mutagenesis by ethyl methanesulfonate (EMS) were studied in 4 strains of E. coli. Logarithmic and stationary phase cells of the polymerase I deficient mutant, P₃₄₇8 polA, a recombination deficient mutant, DZ₄₁₇ recA, and the respective parental strains, W₃₁₁₀pol⁺ and AB₂₅₃ rec⁺, were exposed to EMS and the surviving fraction and mutant frequency determined. At the same EMS concentration both mutants were more susceptible to inactivation than the parental strains. In all 4 strains, log phase cells were more sensitive to inactivation than stationary cells. The surviving fraction of stationary cells exceeded log cells by a factor of 18 for polA, 6 for recA, and about 2 for the parental strains. In all strains, except recA, log phase cells exhibited higher spontaneous mutant frequencies than stationary phase cells. At the same concentration of EMS, survivors of both polA and recA showed more than 10-fold higher induced frequencies than the wild types. However, at the same survival levels the repair deficient mutants exhibited induced mutant frequencies comparable to the repair proficient strains. There was no significant effect of growth phase on EMS induced mutability in recA or the parental strains. In marked contrast, the polymerase I deficient mutant shows both a higher spontaneous frequency and a greater than 10-fold higher EMS induced mutant frequency in log phase cultures compared to stationary phase cultures. Our results support the hypothesis that cellular susceptibility to alkylating agents is influenced by both the genetic capability for repair and the particular physiological state of the cell.     

Factors affecting the heat resistance of Escherichia coli O157 : H7

Escherichia coli O157 : H7 has been reported as being not particularly heat resistant. However, several factors which might increase its heat resistance have been investigated in this study using five strains. Increase in growth temperature to 40 °C, as found in the cow gut, heat-shock at sub-lethal temperatures of 42, 45, 48 and 50 °C, and variable heating rate (1 °C min⁻¹ to 23 °C min⁻¹) had no dramatic effect on heat resistance. Growth phase had a marked impact on heat resistance ; late stationary phase cells were more heat-resistant than were log phase cells. The difference in heat resistance between the two phases of growth became more pronounced when cells were resuspended in fresh nutrient broth ; heat resistance of late stationary phase cells increased dramatically whereas no such effect was observed with log phase cells. The addition of polyphosphates to the heating medium did not increase heat resistance. A reduction in water activity of the heating medium from 0·995 to levels between 0·980 and 0·960 also resulted in a marked increase in heat resistance. This effect was more pronounced under conditions of extremely low water activity created by resuspending late stationary phase cells in sunflower oil. Survivors were detected even after a heat treatment at 60 °C for 1 h or 70 °C for 5 min. It can be confirmed that this serotype has no unusual heat resistance and that the heating environment markedly affects resistance.     

Role of water activity in ethanol fermentations

A separate role for water activity in the conversion of sugars to ethanol by two strains of yeast is identified. During fermentation of both single and mixed sugar substrates, the water activity was shown to remain constant during the logarithmic growth phase. This is despite the changes in concentration of substrates and product, the constancy reflecting the fact that the greater influence of ethanol on the solution activity is counterbalanced, in the early stages of the fermentation, by its low yield. The end of the log phase of growth coincides with the start of a period of gradually decreasing water activity. For the more ethanol-tolerant strain UQM66Y, growth was found to cease at a constant value of water activity while that for the less tolerant strain UQM70Y depended on both ethanol concentration and water activity. It is argued that water activity is a more appropriate variable than ethanol concentration for describing some of the nonspecific inhibitory effects apparent in ethanol fermentations. A straightforward method for the calculation of water activity during such fermentations based on the use of solution osmolality is presented.