Cells In Logarithmic Phase Research Articles

Isolation and properties of beta-glucosidase from Ruminococcus albus.

An enzyme active against p-nitrophenyl-beta-D-glucoside was purified from logarithmic-phase cells of Ruminococcus albus cultivated in a medium containing ball-milled cellulose. The purification yielded homogeneous enzyme after an approximately 520-fold increase in specific activity and a 9% yield. The enzyme was identified as a beta-glucosidase because it can hydrolyze cellobiose and cellooligosaccharides to glucose from the nonreducing ends.

Modifying factors of the cellular concentration of photolyase molecules in Saccharomyces cerevisiae--II. Effects of preillumination with light flashes.

Abstract The effects of preillumination with photoreactivating light flashes before UV‐irradiation on the number of photoreactivable complexes consisting of UV‐induced DNA‐damages and active photolyase molecules (NPREact), on the fluence decrements, ΔDPRE, that are obtained from two UV‐survival curves without and with 1 flash photoreactivation and proportional to NPREact were determined in haploid Saccharomyces cerevisiae cells. ΔDPRE increased by preillumination from 0.115 Jm–2 to 0.460 Jm–2 and from 0.376 Jm–2 to 0.494 Jm–2 in cells in logarithmic growth phase and in stationary growth phase, respectively. ΔPRF in log‐cells that were preilluminated before and after resuspension in buffer at 40°C for 60 min was larger than ΔDPRE in log‐cells preilluminated only after resuspension in buffer.

Variability in expression of a cell surface determinant on Candida albicans as evidenced by an agglutinating monoclonal antibody.

The expression of a surface immunodeterminant of Candida albicans was investigated with an agglutinating immunoglobulin M monoclonal antibody. The 96 strains of C. albicans tested, of which 76% were recent clinical isolates, were capable of expressing the antigen. The antigen was also produced by strains of Candida tropicalis and Torulopsis glabrata, but not by other yeast species. Expression of the surface immunodeterminant in C. albicans varied as a function of growth as indicated by agglutinin reactions and indirect immunofluorescence tests. When yeast cells were tested with the agglutinin, three patterns of reactivity were observed. In general, cells in the early logarithmic phase were less reactive than cells in the mid-logarithmic phase. Antigen expression, as determined by agglutinin reactivity, was also influenced by the nutritional composition of the growth medium, and in general, cells from broth cultures were usually more reactive than cells grown on solid media. The antigen was solubilized from the cell surface of C. albicans by hot 1 M NaCl. These water-soluble extracts were capable of binding antibody, and a single precipitin band formed when soluble antigen was reacted with the monoclonal antibody in an Ouchterlony double-diffusion test. Whole cell preparations and hot NaCl extracts from yeast strains which did not agglutinate when mixed with the antibody also did not absorb the agglutinin from solution. These data indicate that the expression of surface antigens on C. albicans is a dynamic process which may be influenced by a number of environmental factors. The use of monoclonal antibodies may allow characterization of surface antigens presented to the host during candidiasis.

Anaphase-telophase analysis of chromosomal damage induced by chemicals.

Three main aspects involved in the chemical induction of anaphase-telophase aberrations in the first mitosis after treatment were analyzed: 1) the relationship between the frequency of anaphase-telophase aberrations and the time of fixation after treatment; 2) the dose-response relationships; and 3) the proliferative rate of cells exposed to chemicals which interact with DNA by different mechanisms. Experiments were carried out using Chinese hamster ovary (CHO) cells. The compounds examined were adriamycin (ADR) and mitomycin C (MMC). The frequency of cells with chromatin bridges or with lagging chromosomes as well as the mitotic index was determined in each experiment. The results obtained showed that 1) chromatin bridges and lagging chromosomes are apparently induced during the S period of the previous interphase; 2) the increase in the cytotoxicity index (inferred from the mitotic index) and the frequency of cells with chromatin bridges and lagging chromosomes were proportional to the treatment lapse and to the dose employed; and 3) the effect of ADR on cell growth differs from the effect of MMC. While ADR decreased the mitotic activity of cells in logarithmic growth phase, MMC induced mitotic delay. In accordance with these results, the occurrence of chromatin bridges in anaphase-telophase could be explained by the induction of chromosome stickiness and, to a lesser extent, by the induction of exchange-type aberrations. On the other hand, lagging chromosomes seem to be the result of chromatid or chromosome breaks because the lagging chromosomes observed were primarily, if not all, fragments and not whole chromosomes. Our evaluation of the anaphase-telophase test indicates that it is very sensitive method for the detection of chemical clastogens, but other factors, such as mitotic depression, must be taken into account to avoid false-negative results.

Phospholipids ofRhizobium meliloti andAgrobacterium tumefaciens: Lack of effect of Ti plasmid

AbstractWe have studied the phospholipid composition ofRhizobium meliloti strains which do or do not contain the large, tumor‐inducing (Ti) plasmid ofAgrobacterium tumefaciens. The major phospholipids of stationary phase cells were phosphatidylethanolamine (PE) (22%), phosphatidyl‐N‐methylethanolamine (22%), phosphatidylcholine (PC) (27%), phosphatidylglycerol (11.4%), and cardiolipin (11%); as average percent of lipid phosphorus. Phosphatidyl‐N,N‐dimethylethanolamine (3.7%) was also present. The proportions of PE were higher, and PC lower, in logarithmic phase cells. No significant differences were seen in the proportions of phospholipids in strains with or without the Ti plasmid. Qualitative examination of the phospholipids ofA. tumefaciens with or without the Ti plasmid similarly revealed no differences.

Role of DNA and bacteriophage in Campylobacter auto-agglutination.

Auto-agglutinated and non-agglutinated cells of Campylobacter jejuni and C. coli were examined by transmission electronmicroscopy in phosphotungstate negative stain. Agglutination was induced by three factors (1) extracellular DNA, (2) an aggregated protein, probably a bacteriophage precursor, and (3) free phage-tail sheaths. Auto-agglutinated cells were often "leaky," with a mantle of adhering DNA. About 80% of the auto-agglutinated cells could be resuspended after treatment with DNAase. Flagella were loosely embedded in protein aggregates, especially in phage-infected cultures. They were clumped in a side-by-side arrangement by free phage-tail sheaths. These findings suggest that auto-agglutination could be minimised in suspensions of organisms intended for use in agglutination tests by harvesting early logarithmic-phase cells containing no more than a low phage population. The most common C. jejuni phage had a contractile tail, a head diameter of 60-70 nm, and an overall length of 180-210 nm. A phage isolated from C. jejuni strain 1590 was morphologically identical with C. coli phage.

Escherichia coli B membrane stability related to cell growth phase : Measurement of temperature dependent physical state change of the membrane over a wide range

Escherichia coli B cytoplasmic and outer membrane from cells in different growth phases showed different chemical compositions. In freezing, logarithmic phase cells showed a marked permeability increase in the outer as well as the cytoplasmic membrane. Whereas, in the stationary phase cells no such change in membrane permeability was observed. Cooling of phospholipids and lipopolysaccharides with trans-parinaric acid showed a distinct fluorescence increase from room temperature to far below 0°C. In the outer membrane the fluorescence similar to that of lipopolysaccharides was shown. The outer membranes of cells in different growth phases showed similar temperature-dependent fluorescence changes. The cytoplasmic membrane exhibited a temperature-dependent fluorescence similar to that of the phospholipids. The onset temperature of the increase in fluorescence differed with the cells at different growth phases. The presence of EDTA and MgCl 2 modified the fluorescence changes in the membranes from cells in logarithmic phase. Whereas, in the membranes from cells in stationary phase no such effect was observed. These results suggest that the organizational stability of the membranes from cells in stationary phase is a fundamental basis of the membrane's resistance to the freezing damage.

Enhancement of ionic currents through voltage-gated channels in the mouse oocyte after fertilization.

1. The changes of voltage-gated ion channels in the mouse oocyte after fertilization were investigated under voltage clamp.2. About 60 min after introduction of sperm suspension into the fertilization medium, the amplitude of inward current through Ca(2+)-channels increased, which occurred at anaphase during the second meiotic division. The peak amplitude of the maximum inward current per unit membrane capacity of the oocytes at metaphase was 20+/-3 muA/muF in 50 mM-Sr medium. It was 28+/-8 muA/muF at anaphase, and 32+/-5 muA/muF at telophase. The kinetic properties as well as selectivity among Ca, Sr and Mn ions were not altered after fertilization.3. The outward surge current which was found at the higher membrane potential over +50 mV also increased in amplitude after fertilization, simultaneously with the increase in amplitude of inward current through Ca(2+)-channels. The means and the standard deviations of the surge current per unit membrane capacity at 120 mV were 31+/-8 muA/muF at metaphase, and 48+/-7 muA/muF at telophase. The kinetic properties of the outward surge current were not altered after fertilization.4. Application of colcemid (10(-7) mole/l.) or cytochalasin B (2 x 10(-5) mole/l.) did not prevent the increase in amplitude of both inward current through Ca channels and the outward surge current.5. The membrane currents in N-18 mouse neuroblastoma cells in logarithmic growth phase were examined under voltage clamp. The N-18 neuroblastoma cells possessed the Ca inward current and the delayed outward current. The kinetic properties and the steady-state inactivation of Ca(2+)-channels in N-18 neuroblastoma cells were compared with those in mouse oocytes. It was concluded that they could be regarded as identical between the mouse oocyte and the N-18 neuroblastoma cell.

Carbon-13 nuclear magnetic resonance studies of acetate metabolism in intact cells of Rhodopseudomonas sphaeroides

13C-nuclear magnetic resonance was used to study the metabolism of [2- 13C]acetate in suspensions of Rhodopseudomonas sphaeroides. In the dark, in logarithmic-phase cells the 13C label appeared first in butyrate C-2 and C-4 and subsequently in glutamate C-4 and succinate C-2 and C-3. In the light, synthesis of poly(β-hydroxybutyrate) (PHB) takes place. Butyrate synthesis seems to be independent of PHB synthesis or degradation activity. Starved, logarithmic-phase cells also show massive synthesis of PHB in the dark. Stationary-phase cells incorporate 13C predominantly into glutamate and succinate. No significant butyrate biosynthesis can be detected in the dark or during illumination. The incorporation of label in PHB is very slow in these cells and most probably originates from exchange of 12C for 13C into PHB. This might indicate slow turnover without net synthesis of the polymer occurring under these conditions. The results are discussed in relation to the redox state and the availability of metabolic energy for biosynthetic reactions in the dark and during illumination of cell suspensions of Rps. sphaeroides.

Glucose transport in nongrowing yeast

The inducible, nonenergy-requiring glucose transport system of the yeast Kluyveromyces lactis is inactivated upon starving cells of glucose by (1) transferring logarithmic phase glucose-grown cells to synthetic medium containing a nonglycolytic carbon source, and (2) upon transition of logarithmic phase glucose-grown cells to stationary phase. The steady-state accumulation of nonmetabolizeable 6-deoxyglucose and the apparent K m of transport of 6-deoxyglucose is the same in stationary phase cells and in logarithmic phase cells. The rate of transport is lower in the nongrowing cells. Restoration of activity requires energy and protein synthesis as well as inducer.

Cytochrome c oxidase in cytochrome c oxidase-deficient mutant strains of Neurospora crassa.

Three mutant strains of Neurospora crassa, previously characterized as cytochrome c oxidase deficient, were found to possess this respiratory enzyme. The mitochondrial cytochrome contents of logarithmic phase cells of wild type (74A) and the respiration-deficient strains mi-3, cya-4-23, and cya-5-34 were investigated with low temperature difference spectrophotometry; two of the strains were found to possess cytochrome a. Cytochrome c oxidase catalytic activity measurements performed on isolated mitochondria showed that all three mutant strains contained significant amounts of active enzyme which was subsequently found to be active in vivo. However, activity of the cyanide-insensitive, alternate electron transport pathway was responsible for most of the oxygen consumed by the logarithmic phase cells of these mutant strains. Electrophoretic analysis of the polypeptide subunits of cytochrome c oxidase radiolabeled in vivo during logarithmic phase growth showed that, in contrast to previous reports, the respiration-deficient strains appeared to contain all seven subunits of the enzyme.

High performance liquid chromatographic analysis of catecholamines in growing and non-growing Tetrahymena pyriformis

Tetrahymena pyriformis, strains NT-1 and W, harvested in logarithmic (growing) and stationary (non-growing) phases, were found by high-performance liquid chromatography to contain considerable quantities of dopamine. In addition, small amounts of epinephrine and norepinephrine were detected. Logarithmic-phase strain NT-1 cells contained 249±44 pg dopamine/10 6 cells compared to 477±42 pg/10 6 cells for logarithmic-phase strain W cells for logarithmic-phase strain W cells. The dopamine content of stationary-phase cells was approximately half the value of the logarithmic-phase cells. There was a significant amount of dopamine in the growth medium from stationary-phase cultures and, to a lesser extent, logarithmic-phase cells.

A method for the immobilization of living Tetrahymena

The surfaces of plastic (polystyrene) Petri dishes from several suppliers were discovered to have the useful property of immobilizing cells of the ciliate Tetrahymena thermophilia upon contact in nutrient-free buffer (10 mM Tris, pH 7.4). The procedure works with cells in both logarithmic and stationary growth phase, so long as they are first transferred to nutrient-free buffer, and then added to dishes already containing buffer to a depth of 2–10 mm. Dish surfaces specially treated for tissue cultures are unsuitable for this purpose. Cells can be released from the dish surfaces by the simple addition of growth medium (1% proteose peptone). Immobilized cells are fully competent to complete conjugation or cell division. The technique offers promise for facilitating experiments requiring microinjection, microsurgery, or simply detailed observation of living protozoan cells.

Phosphorylation of chromosomal proteins from mammary cell lines.

Phosphorylation of histone and none-histone chromosomal proteins in two clonal mouse mammary cell lines having low (V-14) and high (T-19) tumorigenicity was investigated. Logarithmic phase cells were incubated in medium containing 125 muCi/ml[32P]-orthophosphate. Cell nuclei were isolated, chromosomal proteins (histones and non-histones) extracted and their amino acid composition, protein/DNA mass ratios, 32P-uptake and gel electrophoretic patterns examined. Significant differences in the histone and NHC-protein phosphorylation of these two cell lines were found. Comparison of radioactivity profiles of electrophoretically separated T-19 and V-14 NHC-proteins showed that 60% of the T-19 bands in the mol. wt range of 160,000 to 15,000 daltons had increased [32P]-orthophosphate uptake. Differences in histone radiolabelling between V-14 and T-19 were confined to the H1, H3 and H4 classes. [32P]-ATP-pool measurements of V-14 and T-19 at 30, 60 and 120 min of incubation were similar, hence the difference in 32P-uptake was not due to ATP-pool fluctuations. Results of studies on growth rate, growth potential and the possibility that comparisons were made between cells at different stages of the growth cycle indicated that these variables did not account for the higher phosphorylation of T-19 chromosomal proteins. The increased [32P]-phosphate uptake into T-19 cell protein was correlated with an elevated content of NHC and histone proteins and pointed towards a correlation between the degree of phosphorylation and the high tumorigenicity of this cell line.

7,12-Dimethylbenz[<italic>a</italic>]anthracene-Induced DNA Binding and Repair Synthesis in Susceptible and Nonsusceptible Mammary Epithelial Cells in Culture<xref ref-type="fn" rid="FN2">2</xref>

The effect of age and parity on the binding of 7,12-dimethybenz[a]anthracene (DMBA) to DNA and the repair of DMBA-damaged DNA have been demonstrated in logarithmic phase and confluent mammary epithelial cell cultures from young virgin (YV), old virgin (OV), and parous (P) noninbred and inbred Sprague-Dawley rats. Over a dose range of 0.1-0.4 micrograms DMBA/ml, DNA binding was 1.5-to 2.0-fold higher in YV cells than in OV or P cells. In addition, a steeper slope of the dose-response curve was obtained with YV cells, suggesting a greater susceptibility of YV cells to DMBA. Excision repair was determined by measuring, in the presence of hydroxyurea and 5-bromodeoxyuridine, tritiated thymidine incorporation into DNA during the repair process. At high doses od DMBA (0.5-2.0 micrograms/ml), excision repair in YV cells was 1.5 times higher than in OV cells and 2 times higher than in P cells. However, with lower DMBA doses (less than 0.5 micrograms/ml) similar levels of repair were obtained in all 3 groups of rats. Since binding to DNA is higher in YV cells at these low DMBA doses, ti is apparent that OV and P cells exhibit a greater DNA repair per unit damage. These results, therefore, suggest that age and parity not only lower the binding of DMBA to mammary epithelial cell DNA but also increase the efficiency of DNA repair processes, which may explain the lower susceptibility of OV and P rats to DMBA-induced mammary carcinogenesis.

Growth of Vibrio alginolyticus: Interacting effects on pH, temperature, salt concentration, and incubation time

Summary The growth of five strains of Vibrio alginolyticus, using standard inoculum cells in the late logarithmic growth phase, was tested in Trypticase-Soy-broth with 0.5, 3, and 6% sodium chloride concentration (0.5, 3, 6 TSB) adjusted at pH values ranging from 4.5 to 12 in 0.5 unit. The individual growth as well as the mean growth levels for the five strains were recorded after 2, 4, 7, and 14 days incubated at 5, 10, 20, 37, and 42 °C, respectively. V. alginolyticus strains showed most favourable growth rate in a 3% NaCl solution, while the growth in a 6% solution was lower and the lowest growth was found in the 0.5% solution. Testing the influence of low temperature the strains showed that at 10 °C and 5 °C, respectively four days and seven days elapsed before signs of growth appeared, while high temperature (42 °C) produced regular growth (except with 0.5 TSB). Under favourable temperature and salt concentration V. alginolyticus showed acceptable growth at pH values ranging from 4.5–5 to 10.5–11. After two weeks incubation at 5 °C growth could be registered. The marine strain gave sharp and accurate results in contrast to the international type cultures.

Ultrastructure of the green flagellate Dunaliella tertiolecta (Chlorophyceae, Volvocales) with comparative notes on three other species

The ultrastructure of Dunaliella tertiolecta, a naked, halotolerant unicellular green alga, is described and compared with ultrastructural features reported previously for three other species: D. bioculata, D. primolecta and D. salina. Most internal cell features are chlamydomonad in structure and arrangement. Conspicuous changes occur in D. tertiolecta between logarithmic and stationary growth phases. Vacuoles are more abundant and vacuolar inclusions have a different appearance in stationary as opposed to logarithmic phase cells. Thylakoids of the chloroplast are densely stacked in eight to ten layers in stationary phase cells, but are in stacks of two to four in logarithmic phase. Cytoplasmic and chloroplastic lipids, including eyespot granules, appear to increase with age of the cell. During logarithmic phase the endoplasmic reticulum shows greater proliferation. In cells subjected to increasing osmotic stress, large masses of dense-staining, tubular material is seen protruding and sometimes separating from the plasmalemma. Thylakoid stacking is more pronounced in cells introduced into high salt concentration than in normal cells.

Ampicillin resistance in Neisseria denitrificans: studies on ampicillin-sensitive enzymes.

The beta-lactam sensitivities of enzymes involved in peptidoglycan synthesis were examined in two strains of Neisseria denitrificans having widely disparate degrees of ampicillin sensitivity. One strain of n. denitrificans was 400 times more resistant to ampicillin than the other; the former is known to have altered penicillin-binding proteins. No differences in the levels of sensitivities of total peptidoglycan synthesis as measured by the incorporation of glutamate into peptidoglycan, or of D-alanine carboxypeptidase, were observed between the two strains. However, the rate of glutamate incorporation into peptidoglycan by logarithmic growth phase cells was somewhat less for the ampicillin-resistant cells than for the parent cells.

Growth conditions and heat resistance of Citrobacter freundii.

The influence of the growth medium and the growth temperature on the heat resistance of Citrobacter freundii has been established. Logarithmic growth phase cells grown on rich media have a higher heat resistance than cells of the same phase grown on minimal media. This finding was independent of type of carbon source in the growth medium, but the kind of carbon source has a definite influence on the heat resistance. Logarithmic phase cells grown at 37 degrees C are much more heat stable than cells grown at 20 or 41 degrees C. Stationary growth phase cells are much more heat resistance than logarithmic phase cells, whereas Mg2+ - or glucose-starved cells are even slightly more heat stable than stationary phase cells.

Regulation of self-recognition by T-cell hybrids

Somatic cell hybrids were prepared between BW 5147, an AKR T lymphoma, and purified T cells from three sources: spleen cells exposed to sheep red blood cells, lymph node cells from mice sensitized to ovalbumin, and spleen cells of mice injected with azobenzenearsonate-IgG. Hybrid lines expressed constitutive markers of both parents which include H-2 antigens and the isoenzymes glucose phosphate isomerase and isocitrate dehydrogenase. Furthermore, they expressed both parental alleles of Thy 1, a differentiation antigen. Many of the hybrid lines formed rosettes with mouse erythrocytes. T-cell hybrids did not bind human or chicken red blood cells, though they did rosette with sheep erythrocytes to the same extent as with mouse red cells. We interpret the latter reaction as due to recognition of shared antigens by the murine T cells. This form of self-recognition is influenced by culture conditions and is expressed optimally by cells in late logarithmic phase of growth.