Log-phase Cultures Research Articles

Activation of Rad53 kinase in response to DNA damage and its effect in modulating phosphorylation of the lagging strand DNA polymerase.

The Saccharomyces cerevisiae Rad53 protein kinase is required for the execution of checkpoint arrest at multiple stages of the cell cycle. We found that Rad53 autophosphorylation activity depends on in trans phosphorylation mediated by Mec1 and does not require physical association with other proteins. Uncoupling in trans phosphorylation from autophosphorylation using a rad53 kinase-defective mutant results in a dominant-negative checkpoint defect. Activation of Rad53 in response to DNA damage in G(1) requires the Rad9, Mec3, Ddc1, Rad17 and Rad24 checkpoint factors, while this dependence is greatly reduced in S phase cells. Furthermore, during recovery from checkpoint activation, Rad53 activity decreases through a process that does not require protein synthesis. We also found that Rad53 modulates the lagging strand replication apparatus by controlling phosphorylation of the DNA polymerase alpha-primase complex in response to intra-S DNA damage.

Competition of various beta-lactam antibiotics for the major penicillin-binding proteins of Helicobacter pylori: antibacterial activity and effects on bacterial morphology.

The penicillin-binding proteins (PBPs) of helical (log-phase) Helicobacter pylori ATCC 43579 were identified by using biotinylated ampicillin. The major PBPs had apparent molecular masses of 47, 60, 63, and 66 kDa; an additional minor PBP of 95 to 100 kDa was also detected. The relative affinities of various beta-lactams for these PBPs were tested by competitive-binding assays. Only PBP63 appeared to be significantly bound to each of the competing antibiotics, whereas PBP66 strongly bound mezlocillin, oxacillin, amoxicillin, and ceftriaxone. Whereas most of the beta-lactams significantly bound two or more PBPs, aztreonam specifically targeted PBP63. The influence of sub-MICs of these beta-lactams on the morphologies of log-phase H. pylori was observed at both the phase-contrast and transmission electron microscopy levels. Each of the eight beta-lactams examined induced blebbing and sphere formation, whereas aztreonam was the only antibiotic studied which induced pronounced filamentation in H. pylori. Finally, studies comparing the PBPs of helical (log-phase) cultures with those of coccoid (7-, 14-, and 21-day-old) cultures of H. pylori revealed that the major PBPs at 60 and 63 kDa seen in the helical form were almost undetectable in the coccoid forms, whereas PBP66 remained the major PBP in the coccoid forms, although somewhat reduced in level compared to the helical form. PBP47 was present in both forms at approximately equal concentrations. These studies thus identified the major PBPs in both helical and coccoid forms of H. pylori and compared the relative affinities of seven different beta-lactams for the PBPs in the helical forms and their effects on bacterial morphology.

Nutrient deprivation and the subsequent survival of biocontrol Pseudomonas fluorescens CHA0 in soil

The effects of deprivation of multiple nutrients or of selected single nutrients (C, S, N, or P) for 7 d in vitro on the subsequent persistence of the biocontrol agent Pseudomonas fluorescens CHA0 in natural soil were investigated. Experiments were carried out with the spontaneous rifampicin-resistant mutant CHA0-Rif and the strain was monitored in vitro and in soil using colony counts (on plates containing rifampicin), Kogure’s direct viable counts and total cell counts (by immunofluorescence microscopy). Single nutrient or multiple nutrient deprivation in vitro did not affect the colony-forming ability of CHA0-Rif cells. However, cell length of the strain was smaller in all nutrient deprivation treatments but one (P deprivation) when compared with cells from log-phase cultures. Once introduced into soil, CHA0-Rif cells from log-phase cultures persisted up to 14 d as culturable cells, and their population numbers (10 8 CFU (g soil) −1) had not declined. The strain was recovered at lower cell numbers at subsequent samplings, regardless of the method used for cell counts, and at 48 d about 90% of the cells had lost both their ability to respond to Kogure’s viability test and to form a colony on plate. Cells of CHA0-Rif deprived of a single nutrient persisted similarly to log-phase cells once introduced into soil. In contrast, deprivation of CHA0-Rif cells for multiple nutrients prior to their introduction into soil resulted in the early occurrence (i.e. within hours of soil inoculation) of cells that had lost their colony-forming ability and that did not respond to Kogure’s viability test. This suggests that the lack of a single nutrient (other than C, S, N or P) or deprivation of a combination of several nutrients, under in vitro conditions, had a negative effect on the subsequent survival of CHA0-Rif in soil.

Mutational analysis of ComS: evidence for the interaction of ComS and MecA in the regulation of competence development in Bacillus subtilis.

The development of Bacillus subtilis genetic competence is a highly regulated adaptive response to stationary-phase stress. A key step in competence development is the activation of the transcriptional regulator ComK, which is required for the expression of genes encoding the products that function in DNA uptake. In log-phase cultures, ComK is trapped in a complex composed of MecA and ClpC, in which it is rendered inactive. The comS gene, contained within the srf operon, is induced in response to high culture cell density and nutritional stress. Its product functions to release active ComK from the complex, allowing ComK to stimulate the transcription initiation of its own gene as well as that of the late competence operons. Western analysis showed that ComS accumulates to maximal levels between T3 and T4, mirroring the pattern of competence cell development and late competence gene expression. Experiments to examine the target of ComS activity in vitro showed that ComS binds to MecA. This is further supported by coimmunoprecipitation using anti-MecA antiserum. To clarify the role of ComS in competence regulation, a system for evaluating the effect of comS and mutant derivatives on the expression of comG, one of the late competence operons, was constructed. comS mutations, created by alanine-scanning mutagenesis, that significantly reduced comG-lacZ expression were clustered within two regions, one at the N-terminus and the other at the C-terminus of ComS. ComSI13 --> A and ComSW43 --> A were selected for further analysis as representative mutants for both regions required for ComS activity. We observed that ComSI13 --> A showed significantly reduced affinity for MecA, whereas ComSW43 --> A showed near normal binding affinity for MecA. The results show that binding to MecA is critical for ComS function, but do not rule out the possibility that ComS possesses other activities.

Heat shock-induced thermotolerance inListeria monocytogenes13-249 is dependent on growth phase, pH and lactic acid

The effect of heat shock on the thermotolerance ofListeria monocytogenes13-249 was compared as a function of growth phase (log and late stationary phase), pH (5.4, 5.8, 6.2) and heating medium (broth vs minced beef). Lactic acid was added to tryptic phosphate broth (TPB+) to simulate the level found in beef. Furthermore, the specific effect of lactic acid was investigated, comparing results obtained in broth with and without lactic acid with pH adjusted to 5.4 and 7.0. In late stationary cultures, heat shock produced only marginal differences in thermotolerance in TPB+and beef regardless of pH. For log phase cultures, the increase in heat shock induced thermotolerance (HSIT) depended on pH in both TPB+and beef with gradually larger increases at rising pH. At the highest pH tested (pH 7.050 in TPB+and pH 6.2 in beef) the increase in HSIT was 5.7-fold and 2.4-fold, respectively. These increases were highly significant (P< 0.001). If no lactic acid was present, the HSIT increased at both pH 5.4 for 7.0 for log phase cultures. This indicates that the presence of lactic acid in naturally occurring levels will have a specific effect on the response ofL. monocytogenesto heat shock. The D60-values ofL. monocytogenes13-249 were generally higher (2—6-fold) in minced beef than in TPB+.

The yeast non-Mendelian factor [ETA+] is a variant of [PSI+], a prion-like form of release factor eRF3.

The yeast non-Mendelian factor [ETA+] is lethal in the presence of certain mutations in the SUP35 and SUP45 genes, which code for the translational release factors eRF3 and eRF1, respectively. One such mutation, sup35-2, is now shown to contain a UAG stop codon prior to the essential region of the gene. The non-Mendelian inheritance of [ETA+] is reminiscent of the yeast [PSI+] element, which is due to a self-propagating conformation of Sup35p. Here we show that [ETA+] and [PSI+] share many characteristics. Indeed, like [PSI+], the maintenance of [ETA+] requires the N-terminal region of Sup35p and depends on an appropriate level of the chaperone protein Hsp104. Moreover, [ETA+] can be induced de novo by excess Sup35p, and [ETA+] cells have a weak nonsense suppressor phenotype characteristic of weak [PSI+]. We conclude that [ETA+] is actually a weak, unstable variant of [PSI+]. We find that although some Sup35p aggregates in [ETA+] cells, more Sup35p remains soluble in [ETA+] cells than in isogenic strong [PSI+] cells. Our data suggest that the amount of soluble Sup35p determines the strength of translational nonsense suppression associated with different [PSI+] variants.

Differentiation of Lactococcus lactis subspecies lactis and subspecies cremoris strains by their adaptive response to stresses.

Lactococcus lactis subspecies lactis (L. lactis ssp. lactis) and Lactococcus lactis subspecies cremoris (L. lactis ssp. cremoris) were investigated in respect to their response to acid, bile-salt and freezing stresses. First, the sublethal and lethal levels of each stress were determined for both subspecies. For acid stress, the levels were pH 4.5 and 2.5, respectively, for L. lactis ssp. lactis, and pH 5.0 and 3.0, respectively, for L. lactis ssp. cremoris. For bile-salt stress, the levels were 0.03 and 0.1%, respectively, for L. lactis ssp. lactis, and 0.01 and 0.04%, respectively, for L. lactis ssp. cremoris. For freezing stress, 10 degrees C was used as the sublethal temperature and -20 degrees C was used as the lethal temperature for both subspecies. To evaluate the effect of each stress at log phase, a log-phase culture was challenged directly with the appropriate lethal level (control culture) and a second log-phase culture was pre-exposed to the appropriate sublethal level prior to testing survival under normally lethal conditions (test culture). Some, if not most, of the cells were killed in the control cultures for all three stresses. However, in the test cultures, the viability was significantly improved for all of the L. lactis ssp. lactis strains tested, but not for the L. lactis ssp. cremoris strains. It appears, therefore, that L. lactis ssp. lactis is capable of displaying adaptive response to stresses, whereas L. lactis ssp. cremoris seems to lack this phenotype or the response is much weaker in this subspecies. The effect of each stress on stationary-phase cultures was also investigated. Unlike the log-phase cultures, the stationary-phase cultures of both subspecies, challenged directly with the lethal levels, were highly resistant to each of the three stresses tested.

Differentiation of Lactococcus lactis subspecies lactis and subspecies cremoris strains by their adaptive response to stresses

Lactococcus lactis subspecies lactis ( L. lactis ssp. lactis) and Lactococcus lactis subspecies cremoris ( L. lactis ssp. cremoris) were investigated in respect to their response to acid, bile-salt and freezing stresses. First, the sublethal and lethal levels of each stress were determined for both subspecies. For acid stress, the levels were pH 4.5 and 2.5, respectively, for L. lactis ssp. lactis, and pH 5.0 and 3.0, respectively, for L. lactis ssp. cremoris. For bile-salt stress, the levels were 0.03 and 0.1%, respectively, for L. lactis ssp. lactis, and 0.01 and 0.04%, respectively, for L. lactis ssp. cremoris. For freezing stress, 10°C was used as the sublethal temperature and −20°C was used as the lethal temperature for both subspecies. To evaluate the effect of each stress at log phase, a log-phase culture was challenged directly with the appropriate lethal level (control culture) and a second log-phase culture was pre-exposed to the appropriate sublethal level prior to testing survival under normally lethal conditions (test culture). Some, if not most, of the cells were killed in the control cultures for all three stresses. However, in the test cultures, the viability was significantly improved for all of the L. lactis ssp. lactis strains tested, but not for the L. lactis ssp. cremoris strains. It appears, therefore, that L. lactis ssp. lactis is capable of displaying adaptive response to stresses, whereas L. lactis ssp. cremoris seems to lack this phenotype or the response is much weaker in this subspecies. The effect of each stress on stationary-phase cultures was also investigated. Unlike the log-phase cultures, the stationary-phase cultures of both subspecies, challenged directly with the lethal levels, were highly resistant to each of the three stresses tested.

Transcription of two sigma 70 homologue genes, sigA and sigB, in stationary-phase Mycobacterium tuberculosis.

The sigA and sigB genes of Mycobacterium tuberculosis encode two sigma 70-like sigma factors of RNA polymerase. While transcription of the sigA gene is growth rate independent, sigB transcription is increased during entry into stationary phase. The sigA gene transcription is unresponsive to environmental stress but that of sigB is very responsive, more so in stationary-phase growth than in log-phase cultures. These data suggest that SigA is a primary sigma factor which, like sigma70, controls the transcription of the housekeeping type of promoters. In contrast, SigB, although showing some overlap in function with SigA, is more like the alternative sigma factor, sigmaS, which controls the transcription of the gearbox type of promoters. Primer extension analysis identified the RNA start sites for both genes as 129 nucleotides upstream to the GTG start codon of sigA and 27 nucleotides from the ATG start codon of sigB. The -10 promoter of sigA but not that of sigB was similar to the sigma70 promoter. The half-life of the sigA transcript was very long, and this is likely to play an important part in its regulation. In contrast, the half-life of the sigB transcript was short, about 2 min. These results demonstrate that the sigB gene may control the regulons of stationary phase and general stress resistance, while sigA may be involved in the housekeeping regulons.

Rapid plasmid DNA isolation from Lactococcus lactis using overnight cultures.

A simple and quick method has been developed to isolate plasmid DNA from Lactococcus lactis using overnight or stationary-phase cultures which therefore eliminates the need for subculturing for generating log-phase cultures that are necessary with existing methods. The new method was effective in isolating plasmids, from 1.4 to 64 kb, from the three subspecies of Lactococcus lactis. The resultant DNA was of high yield and purity and therefore no additional purification steps were required for down-stream molecular procedures.

Lactic acid bacteria growth promoters from Spirulina platensis

Spirulina has been used for many years as human food because of its high protein content and nutritional value. Some strains also produce bioactive substances that may inhibit or promote microbial growth. Lactococcus lactis, Streptococcus thermophilus, Lactobacillus casei, Lactobacillus acidophilus, and Lactobacillus bulgaricus were grown in rich media, MRS and RM, as well as in minimal saline medium with and without addition of extracellular products obtained from a late log phase culture of Spirulina platensis in Zarrouk medium. In both MRS and RM media, the extracellular products significantly promote the growth of the lactic acid bacteria assayed. This stimulatory effect was observed in media with pH adjusted to 5.3, 6.3 and 7.0. No effect was observed in minimal saline medium.

Sugar transport and glut transporter expression in a variety of human immunodeficiency virus-1 (HIV-1) chronically infected target cell lines

In this study, sugar transport and the cellular content of the human Glut 1 and 3 glucose transporters were ascertained in uninfected and chronically HIV-infected Jurkat and H9 cell lines (T-cell lines) and U937 cells (a promonocytic cell line). Sugar transport was determined by monitoring 2-deoxy glucose uptake (2DG) and glut transporter content was determined by Western analysis. Although `acute' HIV infection of H9 cells led to increased cellular transport activity and Glut3 transporter content, chronic HIV infection exhibited no significant differences in sugar transport in any of the cell types investigated whether log or stationary phase cultures were employed. When uninfected and chronically HIV-infected cell lines were compared, all cell lines expressed the Glut 1 transporter, however, significant differences in Glut 1 transporter content were not observed. The Glut 3 transporter which could only be detected in the H9 cell line exhibited no differences in Glut 3 content in uninfected or chronically HIV-infected cells (2.1±0.6 versus 3.8±2.1×10 −3 arbitrary units/μg protein). A trend towards lower amino acid uptake was seen in the chronically HIV-infected cells but this was not significantly different from uninfected cell cultures. The data indicate that: (1) glucose transport and the Glut 1 and 3 transporters are not increased in cells chronically infected with HIV-1 and (2) the expression of the Glut3 sugar transporters is not the same in all target cells.

Radiosensitizing potential of gemcitabine (2′,2′-difluoro-2′-deoxycytidine) within the cell cycle in vitro

Purpose: Gemcitabine (2′,2′-difluorodeoxycytidine; dFdCyd) is a new deoxycitidine analog which exhibits substantial activity against solid tumors and radiosensitizing properties in vitro. To examine cell cycle–specific effects of a combined treatment with gemcitabine and radiation, the in vitro clonogenic survival of two different cell lines was measured for cells from log-phase culture, G1 and S-phase cells. Methods and Materials: Chinese hamster (V79) and human colon carcinoma (Widr) cells were exposed to different radiation doses and for different points of time relative to gemcitabine treatment (2 h). Experiments were also carried out with different cell-cycle populations obtained after mitotic selection (V79) or after serum stimulation of plateau-phase cells (Widr). The resulting survival curves were analyzed according to the LQ model, and mean inactivation doses (MID) and the cell cycle–specific enhancement ratios (ER) were calculated from the survival curve parameters. Results: Effectiveness of combined treatment of log-phase cells was greatest when cells were irradiated at the end of the gemcitabine exposure [ER: 1.28 (V79), 1.24 (Widr)]. For later times after the removal of the drug, radiosensitization declined, approaching independent toxicity. From the time course of interactive-type damage decay half-life values of 75 min (V79) and 92 min (Widr) were derived. Gemcitabine did not radiosensitize G1 Widr cells or V79 cells from the G1/S border, but substantial radiosensitization was observed for the S-phase cell preparations [ER: 1.45 (V79-lateS), 1.57 (Widr)]. Conclusions: Treatment of cells with gemcitabine immediately before irradiation eliminates, or at least greatly reduces, the variation in radiosensitivity during the cell cycle that is manifested by radioresistance during S phase. This reversal of S-phase radioresistance could imply that gemcitabine interferes with the potentially lethal damage repair/fixation pathway. Other approaches have been taken to overcome S-phase radioresistance, such as hyperthermia or densely ionizing radiation, and combined treatments with dFdCyd could prove of value to complement such efforts.

Growth and processing conditions affecting acid tolerance inEscherichia coli O157:H7

The impact of growth conditions (anaerobiosis, growth phase, NaCl, pH, and temperature) on the development of acid tolerance in Escherichia coli O157:H7 was investigated directly (DpH1.15) and indirectly by monitoring the specific activity of acid phosphatase. Anaerobic growth of O157:H7 strain 43895 in synthetic rumen fluid resulted in earlier development of acid tolerance than aerobic growth. However, stationary-phase cells of both aerobic and anaerobic cultures had an equivalent degree of acid tolerance that was greater than that achieved in log-phase cultures grown anaerobically. These results are consistent with the growth-phase regulation of acid tolerance by the stationary-phase sigma factors38. The addition of NaCl (1%) also enhanced acid tolerance of log-phase but not stationary-phase cells of strain 43895. Growth temperature influenced the acid tolerance with progressively greater DpH1.15values obtained at 15, 25, and 37°1C, in both log and stationary phase. Therefore, the influence of temperature on the subsequent survival and acid tolerance of E. coli O157:H7 strain 43895 in ground beef was evaluated. Numbers of strain 43895 decreased c. 1.14 log10cfu g-1in inoculated ground beef stored at 4°1C, whereas numbers remained essentially unchanged during storage at -20°C. While pre-incubation at 15°1C for 4 h prior to storage at 4 or -20°C did not influence survival, the acid tolerance of E. coli O157:H7 survivors was significantly decreased (P<0.10001). These results indicate that the processing temperature can influence acid tolerance in E. coli O157:H7.

Particulate dimethyl sulphoxide in seawater:production by microplankton

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 167:291-296 (1998) - doi:10.3354/meps167291 Particulate dimethyl sulphoxide in seawater: production by microplankton Rafel Simó*, Angela D. Hatton**, Gillian Malin, Peter S. Liss School of Environmental Sciences, University of East Anglia, Norwich NR4 7TJ, United Kingdom *Present address: Institut de Ciències del Mar (CSIC), Pg Don Joan de Borbó s/n, E-08039 Barcelona, Catalonia, Spain. E-mail: rsimo@icm.csic.es **Present address: Dunstaffnage Marine Laboratory, Oban, Argyll PA34 4AD, United Kingdom Dimethyl sulphoxide (DMSO) represents a major pool of dissolved dimethylated sulphur in seawater. However, the origin and fate of this compound in the marine environment, and its role in the biogeochemical cycle of dimethyl sulphide (DMS), remain unclear. The only established route for the formation of DMSO in oxygenated seawater is photochemical oxidation of DMS. It is not known whether significant biotic production pathways exist. In a study of methylated sulphur speciation in coastal North Sea waters and cultures of marine unicellular algae, we measured pools of particulate DMSO (DMSO p) at nanomolar and micromolar concentrations, respectively. Analyses of size-fractionated seawater particulates and incubation experiments provided evidence that DMSO p was associated with microplanktonic organisms. Log-phase cultures of Amphidinium carterae and Emilianiahuxleyi exhibited intracellular dimethylsulphoniopropionate (DMSP) to DMSO molar ratios of 25 and 8, respectively. Our results strongly suggest the existence of biological production and release of DMSO in eukaryotic microplankton. Dimethyl sulphoxide · Phytoplankton · Dimethylsulphoniopropionate · Dimethyl sulphide · Amphidinium carterae Full text in pdf format PreviousNextExport citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 167. Publication date: June 18, 1998 Print ISSN:0171-8630; Online ISSN:1616-1599 Copyright © 1998 Inter-Research.

The Role of the Variable Region in Tet Repressor for Inducibility by Tetracycline

A set of deletions and substitutions to alanine was introduced into the loop separating helices alpha8 and alpha9 of Tn10 Tet repressor (TetR). This region appears as an unstructured loop in the crystal structure of the TetR(D).([Mg-tc]+)2 complex and is the only internal segment of variable length in an alignment of Tet repressors from seven different resistance determinants. In vivo analysis of 10 mutants shows that this loop is important for inducibility by tetracycline (tc), whereas DNA binding is not or only marginally affected. All deletions have an induction-deficient TetRS phenotype, but the corresponding substitutions do not or only slightly affect inducibility. The purified mutant TetR proteins have a reduced affinity for tc in vitro that correlates with their lack of inducibility. The association rate of [Mg-tc]+ to the TetR mutants is enhanced. Since none of the mutated residues contacts tc directly in the crystal structure, we propose that the length of the loop is important for the structural transition between a closed, tc binding and an open, operator binding conformation of TetR. We propose that the deletions in the loop shift the equilibrium between both forms toward the open, operator binding conformation.

Quantitative Reverse Transcriptase Polymerase Chain Reaction for Measuring theN-Methylpurine–DNA Glycosylase mRNA Level in Rodent Cells

A modified quantitative reverse transcriptase polymerase chain reaction (QRT-PCR) procedure was developed for measuring mRNA concentration, in rodent cells, of theN-methylpurine–DNA glycosylase (MPG), a ubiquitous DNA repair protein responsible for the removal ofN-alkylpurines and ethenoadducts of adenine, guanine, and cytosine from DNA. The method, applicable for quantitation of any mRNA, is based on the standard approach of comparing the relative amounts of PCR products of the experimental mRNA and a known amount of an exogenous reference RNA which is nearly identical to the experimental RNA. However, unlike in the earlier procedures in which deletion or insertion sequences were added to the reference RNA template, which may affect the efficiency of PCR but are needed to generate different size PCR products, experimental and reference RNAs yield PCR products of the same size in the new method. However, prior digestion withEcoRI allows separation of the two products because a uniqueEcoRI site was created in the reference RNA vector by point mutations. The QRT-PCR procedure is particularly useful for studying expression of the MPG gene whose mRNA level is very low and difficult to quantitate by Northern blot analysis. The number of MPG mRNA molecules/cell in late log-phase cultures varied from about 6 to 30 in several rodent lines. The SSV-NRK rat cell line has 6 ± 0.2 molecules/cell, while mouse NIH3T3 cells have about 30 ± 1 molecules/cell. If the mRNA level is indicative of the level of the active MPG enzyme, these results may imply a variation in the capacity of various lines to remove the cytotoxic and mutagenic adducts from DNA.

Heat-shock response in Schizosaccharomyces pombe cells lacking cyclic AMP-dependent phosphorylation.

Heat sensitivity at 48 degrees C was determined in log-phase cultures of control and pka1-disrupted cells of the fission yeast Schizosaccharomyces pombe grown at 25 degrees C. Cells devoid of protein kinase A exhibited a considerable heat-shock resistance as compared to control cells. Addition of cAMP to control cells prompted a further decrease in viability during heat shock. This effect was not observed with pka1-disrupted cells, suggesting that cAMP-dependent phosphorylation is involved in modulation of the heat-shock response. When control or pka1-disrupted cells were grown at 25 degrees C and then shifted to 37 degrees C they acquired thermo-tolerance to a subsequent treatment at 48 degrees C both in the absence and in the presence of exogenous cAMP. Inhibition of protein synthesis during the adaptive treatment did not block the development of thermo-tolerance. However, the arrest in translation significantly prevented trehalose accumulation in control cells but only slightly affected trehalose increase in pka1-disrupted cells. These data indicate that heat resistance may be established in growing cells of S. pombe by at least two independent post-translational mechanisms: a decrease in cAMP-dependent protein phosphorylation and a hitherto unknown process which may be independent of trehalose accumulation.

Optimum temperatures for the growth and protease production of Aeromonas hydrophila.

An invesigation on optimal temperature for the growth and protease production of a mesophilic bacterium, Aeromonas hydrophila was carried out. The studied 12 strains of fish origin were collected and stored at -80deg;C. Optimal growth temperature was determined by a temperature gradient incubator using TYE medium, a suitable medium for protease production. The protease production was estimated by the Bio-Rad DC protein assay with broth culture supernatant at around stationary phase. Casein (Hammarsten quality) was used as a protein substrate. There was no considerable amount of protease within log phase culture. Although the optimum temperatures for growth and protease production were variable with the strain, optimal temperatures for protease production were always lower than those for the growth. Peak growth was favoured at 34.5±1.0deg;C, while the protease production at 27.6±4.9deg;C. The differences among the peaks of protease production and growth were 1 to 15deg;C.

DNA synthesis and cell proliferation in C6 glioma and primary glial cells exposed to a 836.55 MHz modulated radiofrequency field.

We have tested the hypothesis that modulated radiofrequency (RF) fields may act as a tumor-promoting agent by altering DNA synthesis, leading to increased cell proliferation. In vitro tissue cultures of transformed and normal rat glial cells were exposed to an 836.55 MHz, packet-modulated RF field at three power densities: 0.09, 0.9, and 9 mW/cm2, resulting in specific absorption rates (SARs) ranging from 0.15 to 59 muW/g. TEM-mode transmission-line cells were powered by a prototype time-domain multiple-access (TDMA) transmitter that conforms to the North American digital cellular telephone standard. One sham and one energized TEM cell were placed in standard incubators maintained at 37 degrees C and 5% CO2. DNA synthesis experiments at 0.59-59 muW/g SAR were performed on log-phase and serum-starved semiquiescent cultures after 24 h exposure. Cell growth at 0.15-15 muW/g SAR was determined by cell counts of log-phase cultures on days 0, 1, 5, 7, 9, 12, and 14 of a 2 week protocol. Results from the DNA synthesis assays differed for the two cell types. Sham-exposed and RF-exposed cultures of primary rat glial cells showed no significant differences for either log-phase or serum-starved condition. C6 glioma cells exposed to RF at 5.9 muW/g SAR (0.9 mW/cm2) exhibited small (20-40%) significant increases in 38% of [3H]thymidine incorporation experiments. Growth curves of sham and RF-exposed cultures showed no differences in either normal or transformed glial cells at any of the power densities tested. Cell doubling times of C6 glioma cells [sham (21.9 +/- 1.4 h) vs. field (22.7 +/- 3.2 h)] also demonstrated no significant differences that could be attributed to altered DNA synthesis rates. Under these conditions, this modulated RF field did not increase cell proliferation of normal or transformed cultures of glial origin.