Cells In Exponential Growth Research Articles

Use of fluorescent staining and flow cytometry for monitoring physiological changes in solventogenic clostridia

Physiological changes in populations of Clostridium beijerinckii and Clostridium tetanomorphum were monitored by fluorescence staining and flow cytometry. To estimate the number of metabolically active cells in exponential growth, a combination of the dyes propidium iodide and carboxy fluorescein diacetate appeared to be a good choice for both species. During stationary phase, these stains did not reflect physiological changes sufficiently and therefore additional labeling with bis-(1,3-dibutylbarbituric acid) trimethineoxonol was applied. Results of fluorescence staining in solventogenic batch fermentations were compared with substrate-use data, the concentration of key metabolites and growth curves. We demonstrate that measurements by all methods were mutually compatible.

Heterogeneity of HDAC3 expression and its modulation by valproic acid in glioblastoma cells.

e13038 Background: Glioblastoma (gbm) is a devastating malignancy and a therapeutic challenge. Our group and others have observed long-term gbm survivors treated with valproic acid (VPA), a histone deacetylase (HDAC) inhibitor. While in vitro studies of HDAC inhibitors have been promising, clinical trials and retrospective analyses have been disappointing. We hypothesize that HDAC3 may be a more specific target or treatment marker for gbm. Since limited data is available pertaining to HDAC3 and its modulation in gbm, our goals were to: 1) develop methods for determining HDAC3 expression and localization in gbm cells, 2) study HDAC3 heterogeneity among different gbm cell lines, including one in low passage, and 3) test the effect of VPA treatment on HDAC3 expression of gbm cells in vitro. Methods: Three established gbm cell lines (U-87, U-251 and E297), were cultured using standard technique. HeLa cells were used as the positive HDAC3 control. Cells were stained using 2 different antibodies, and analyzed using immunofluoresence microscopy (IFM), and Western blotting (with β-actin standards). IgG was used for negative isotype controls. Cell counts, viability, and H&E staining for changes in morphology were also recorded. Lysates for Western blots were made from whole cells, nuclei, and cytoplasmic preparations. Band quantification was performed using the ImageJ software. Cells in exponential growth were treated for 48 hours with 10 mM VPA, and compared to untreated cells. Results: Untreated cells studied for HDAC3 using IFM showed nuclear and some cytoplasmic staining in all 4 lines, which varied according to cell type and morphology. Western blot analysis confirmed the variability of HDAC3 expression seen by IFM. The effect of VPA treatment on gbm HDAC3 expression (cytoplasmic and nuclear) was seen to be highly variable, ranging from marked down-modulation in some gbm cells, to minimal effect in others. Conclusions: These data provide the groundwork for studying HDAC3 levels in patients undergoing therapy with HDAC inhibitors. The effect of VPA on the HDAC3 expression of gbm cells is heterogeneous; individualized assessment of such effect will be necessary in delineating any subset of patients that may respond to VPA treatment.

Cell density-dependent swimming patterns of Alexandrium fundyense early stationary phase cells

AME Aquatic Microbial Ecology Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsSpecials AME 68:251-258 (2013) - DOI: https://doi.org/10.3354/ame01617 Cell density-dependent swimming patterns of Alexandrium fundyense early stationary phase cells Agneta Persson1,3, Barry C. Smith2,* 1Department of Biological and Environmental Sciences, G?teborg University, Box 461, 40530 G?teborg, Sweden 2National Oceanic and Atmospheric Administration, National Marine Fisheries Service, Northeast Fisheries Science Center, Milford Laboratory, Milford, Connecticut 06460, USA 3Present address: Smedjebacksv?gen 13, 77190 Ludvika, Sweden *Corresponding author. Email: barry.smith@noaa.gov ABSTRACT: Different life-history stages of Alexandrium fundyense have different swimming behaviors and show different responses to water movement. Early stationary phase cells assemble in bioconvection patterns along the water surface and as stripes in the water, while cells in exponential growth do not. We studied the swimming behavior of early stationary phase A. fundyense cells, both on the individual level and on the population level. Cells assembled in spots in shallow Petri dishes, and were studied using an inverted microscope. We analyzed 53 videos of cells at different distances from the center of accumulated spots of cells with the program CellTrak for swimming behavior of individual cells. The closer the cells were to the center of spots, the faster they swam (>600 ?m s-1 in the center of spots compared to ca. 300 ?m s-1 outside) and the more often they changed direction (>1400 degrees s-1 in the center compared to <400 degrees s-1 outside). On a population level, the behavior of spots of assembled cells was studied using time-lapse photography. The spots entrained more and more cells as they grew and fused with each other; the closer the spots came to each other, the faster they moved until they fused. We suggest that chemical attraction between gametes causes the observed behavior. KEY WORDS: Alexandrium fundyense · Swimming pattern · Stationary phase · Gamete · Cell density Full text in pdf format Supplementary material PreviousNextCite this article as: Persson A, Smith BC (2013) Cell density-dependent swimming patterns of Alexandrium fundyense early stationary phase cells. Aquat Microb Ecol 68:251-258. https://doi.org/10.3354/ame01617 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in AME Vol. 68, No. 3. Online publication date: March 18, 2013 Print ISSN: 0948-3055; Online ISSN: 1616-1564 Copyright © 2013 Inter-Research.

Proteomic analysis of spontaneous mutants of Lactococcus lactis: Involvement of GAPDH and arginine deiminase pathway in H2O2 resistance

Lactococcus lactis, one of the most commonly used dairy starters, is often subjected to oxidative stress in cheese manufacturing. A comparative proteomic analysis was performed to identify the molecular modifications responsible for the robustness of three spontaneous H(2)O(2)-resistant (SpOx) strains. In the parental strain, glyceraldehyde-3-phosphate deshydrogenase (GAPDH) activity is ensured by GapB and the second GAPDH GapA is not produced in standard growth conditions. We showed that GapA was overproduced in the highly resistant SpOx2 and SpOx3 mutants. Its overproduction in the MG1363 strain led to an increased H(2)O(2) resistance of exponential growing cells. Upon H(2)O(2) exposure, GapB was fully inactivated by oxidation in the parental strain. In SpOx mutants, it partly remained in the reduced form sustaining partially GAPDH activity. The analysis of gapA disruption in these SpOx strains indicated that additional unraveled mechanisms likely contribute to the resistance phenotype. In the SpOx1 mutant, the arginine deiminase pathway was found to be upregulated and disruption of arcA or arcB genes abolished H(2)O(2) resistance. We concluded that arginine consumption was directly responsible for the SpOx1 phenotype. Finally, these results suggest that sustaining energy supply is a major way of leading to oxidative stress resistance in L. lactis.

Abstract 3447: Tetrac (tetraiidothyroacetic acid) decreases in vitro x-ray survival of a human medullary thyroid cancer cell line under oxic and hypoxic conditions

Abstract In 2009 we showed that 2 µM tetrac given for 1 hour at 37oC prior to 250 kVP x-irradiation sensitized GL261 murine brain tumor cells. Radiosensitization was accomplished by increasing the alpha component in the linear-quadratic equation of cell survival (Cell Cycle, 8:2586-91). In 2011, we showed that tetrac also sensitized U87MG human brain tumor cells to 250 kVp x-rays (Cell Cycle, 10: 352-57). Again radiosensitization was accomplished by an increase in the αx-ray component. Tetrac also inhibited the repair of DNA double strand breaks in U87MG cells. However, tetrac which binds to the αvβ3 integrin receptor and inhibitssignaling from the αvβ3 receptor, penetrates the cell and has a thyromimetic effect which offsets radiosensitization. In this regard, we synthesized a nanoparticle tetrac formulation termed Nano-T. Nano-T also binds to the αvβ3 receptor but does notpenetrate the cell. Nano-T causes radiosensitization in vitro in H522 lung cancer cells, producing an added benefit compared to tetrac. We have also shown that daily treatment of in vivo human-MTC (h-MTC) solid tumors in mice with Nano-T for 20 days produced significant growth inhibition. It is our intent to combine Nano-T with ionizing radiation in vivo, and we have begun by studying the effects of tetrac on h-MTC cells in vitro. These h-MTC cells are show an in vitro cell culture doubling time of 96.2 hours and a clonogenicity of 3.16%. We irradiated cells in exponential growth with 250 kVp X-rays. Below we give the LQ survival parameters, and the surviving fraction at 2 Gy (SF2). Table 1. Initial Results of 250 kVp Single Dose X-irradiation on h-MTC CellsIn Vitro without and with Tetrac ≤ α SF2 Tetrac Oxygen (Gy-1 x 10-1) (Gy-2 x 10-2) Enhancment Enhancement Factor Factor Without Tetrac Oxic Cells - 0.033 0.0256 0.901 2.55 Hypoxic Cells 0.030 0.0023 0.922 With Tetrac Oxic Cells 0.472 0.0495 0.330 2.84 2.60 Hypoxic Cells 0.013 0.0256 0.803 2.84 First, h-MTC cells irradiated as oxic or hypoxic, exponentially growing cells are very radioresistant suggesting a large capacity to repair radiation damage. Second, tetrac sensitized both oxic and hypoxic h-MTC cells. These characteristics speak to our proposed Nano-T in treatment of h-MTC tumors in vivo. Nano-T should increase the radiosensitivity of both oxic and hypoxic cells and should also decrease the repair of radiation damage in conventional fractionated radiation therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3447. doi:1538-7445.AM2012-3447

Interaction between Escherichia coli DNA polymerase IV and single-stranded DNA-binding protein is required for DNA synthesis on SSB-coated DNA

DNA polymerase IV (Pol IV) is one of three translesion polymerases in Escherichia coli. A mass spectrometry study revealed that single-stranded DNA-binding protein (SSB) in lysates prepared from exponentially-growing cells has a strong affinity for column-immobilized Pol IV. We found that purified SSB binds directly to Pol IV in a pull-down assay, whereas SSBΔC8, a mutant protein lacking the C-terminal tail, failed to interact with Pol IV. These results show that the interaction between Pol IV and SSB is mediated by the C-terminal tail of SSB. When polymerase activity was tested on an SSBΔC8-coated template, we observed a strong inhibition of Pol IV activity. Competition experiments using a synthetic peptide containing the amino acid sequence of SSB tail revealed that the chain-elongating capacity of Pol IV was greatly impaired when the interaction between Pol IV and SSB tail was inhibited. These results demonstrate that Pol IV requires the interaction with the C-terminal tail of SSB to replicate DNA efficiently when the template ssDNA is covered with SSB. We speculate that at the primer/template junction, Pol IV interacts with the tail of the nearest SSB tetramer on the template, and that this interaction allows the polymerase to travel along the template while disassembling SSB.

1,3-Propanediol dehydrogenases in Lactobacillus reuteri: impact on central metabolism and 3-hydroxypropionaldehyde production

BackgroundLactobacillus reuteri metabolizes glycerol to 3-hydroxypropionaldehyde (3-HPA) and further to 1,3-propanediol (1,3-PDO), the latter step catalysed by a propanediol dehydrogenase (PDH). The last step in this pathway regenerates NAD+ and enables therefore the energetically more favourable production of acetate over ethanol during growth on glucose.ResultsA search throughout the genome of L. reuteri DSM 20016 revealed two putative PDHs encoded by ORFs lr_0030 and lr_1734. ORF lr_1734 is situated in the pdu operon encoding the glycerol conversion machinery and therefore likely involved in 1,3-PDO formation. ORF lr_0030 has not been associated with PDH-activity so far. To elucidate the role of these two PDHs, gene deletion mutant strains were constructed. Growth behaviour on glucose was comparable between the wild type and both mutant strains. However, on glucose + glycerol, the exponential growth rate of Δlr_0030 was lower compared to the wild type and the lr_1734 mutant. Furthermore, glycerol addition resulted in decreased ethanol production in the wild type and Δlr_1734, but not in Δlr_0030. PDH activity measurements using 3-HPA as a substrate revealed lower activity of Δlr_0030 extracts from exponential growing cells compared to wild type and Δlr_1734 extracts.During biotechnological 3-HPA production using non-growing cells, the ratio 3-HPA to 1,3-PDO was approximately 7 in the wild type and Δlr_0030, whereas this ratio was 12.5 in the mutant Δlr_1734.ConclusionThe enzyme encoded by lr_0030 plays a pivotal role in 3-HPA conversion in exponential growing L. reuteri cells. The enzyme encoded by lr_1734 is active during 3-HPA production by non-growing cells and this enzyme is a useful target to enhance 3-HPA production and minimize formation of the by-product 1,3-PDO.

Lethal and mutagenic interactions between γ-rays, cisplatin and etoposide at the cellular and molecular levels

Purpose: We analysed the lethal and mutagenic interactions between γ-rays, cisplatin (Pt) and etoposide (E), three agents used in tumour chemoradiotherapy. Corresponding results at cellular and molecular levels could provide additional elements on involved mechanisms and, on antitumour activity and toxicity in combined cancer treatments.Materials and methods: The yeast Saccharomyces cerevisiae SC7K(lys2–3) (auxotrophic for lysine) was used as eukaryotic model. Exponential growing cells were exposed to the mentioned agents, as single and combined treatments. Lethal and mutation interaction equations were determined as a function of doses according to quantitative models. DNA double-strand breaks were evaluated immediately after treatments, through pulsed-field electrophoresis and laser densitometry.Results: All three agents induced significant mutant frequency. The γ +Pt + E combination determined maximal lethal and mutagenic synergism, followed by γ + Pt and γ + E combinations. Meanwhile, Pt + E combination showed lethal additivity and very low mutagenic synergism. Pt + E double combination determined moderate DNA degradation. DNA degradation after γ-exposure, was similar to that of γ + Pt, γ + E and γ + Pt + E combinations.Conclusions: Synergistic lethal and mutagenic interactions indicate crosstalk between non-homologous end joining, homologous recombination and postreplicative repair pathways. Pt + E additivity indicate independence of involved repair pathways. Furthermore, the quantification of interactive events may be an additional suitable tool in tumour therapy planning.

Carcinogenic heavy metals replace Ca 2+ for DNA binding and annealing activities of mono-ubiquitinated annexin A1 homodimer

Mono-ubiquitinated annexin A1 was purified from rat liver nuclei. The homodimer form of mono-ubiquitinated annexin A1 was able to unwind dsDNA in a Mg 2+- and ATP-dependent manner, and to anneal ssDNA in a Ca 2+-dependent manner. Phospholipids decreased the concentration of Ca 2+ required for maximal annealing activity. Heavy metals such as As 3+, Cr 6+, Pb 2+ and Cd 2+ substituted for Ca 2+ in the ssDNA binding and annealing activities of annexin A1. While these metals inhibited the unwinding of dsDNA by nuclear annexin A1 in the presence of Mg 2+ and ATP, they enhanced dsDNA-dependent ATPase activity of annexin A1. Heavy metals may have produced dsDNA, a substrate for the DNA unwinding reaction, via the DNA annealing reaction. DNA synthesomes were isolated from L5178Y tk(+/−) mouse lymphoma cells in exponential growth, and were found to contain helicase activities. The As 3+- or Cr 6+-induced increases in ssDNA binding activity of DNA synthesomes were reduced by a mono-specific anti-annexin A1 antibody, but not by anti-Ig antibody. Anti-annexin A1 antibody also blocked the inhibitory and stimulatory effects of As 3+ or Cr 6+ towards DNA unwinding and annealing activities of DNA synthesomes. Based on these observations, it can be concluded that the effects of heavy metals on DNA annealing and unwinding activities are mediated, at least in substantial part, through actions of the mono-ubiquitinated annexin A1 homodimer.

A tool for enhancement and scoring of DNA repair foci

Upon induction of DNA double-strand breaks (DSBs), Mre11 and Rad50 proteins of the Mre11 DNA repair complex accumulate at the sites of DSBs and form discrete nuclear foci. Precision in scoring of Mre11/Rad50-containing foci depends upon detection of those foci, some of which have a fluorescence staining intensity that is too close to the fluorescence staining intensity of the remaining Mre11 and Rad50 proteins that have not been incorporated into foci. Human U-1 melanoma cells in exponential growth were irradiated with various doses of X-rays (0-12 Gy) to induce the formation of repair foci. Four hours after irradiation, cells were simultaneously labeled for Mre11 and Rad50 proteins, using a two-color immunofluorescence staining technique. Laser scanning confocal microscopy was used to collect the composite images of randomly selected cell nuclei. Intensity correlation analysis (ICA) of equally intense fluorescence signals from Mre11 and Rad50 proteins was performed to obtain the regions with correlated pixels. ICA permitted enhanced detection of low level fluorescence of Mre11/Rad50 foci ("hidden" foci) that can be barely detected upon imaging of only one protein. For example, while imaging of only one protein (either Mre11 or Rad50) in the nucleus of a 6 Gy-irradiated cell revealed 9 foci, imaging of two proteins with ICA revealed 11 foci. ICA permitted an evaluation of the dose dependence of nuclear foci in cells irradiated with various doses of X-rays, with focus formation increasing up to a dose of 6 Gy. Our data accumulated using two-color immunofluorescence staining of Mre11 and Rad50 proteins and ICA of these two target proteins provide a basis for enhanced detection and accuracy in the scoring of DNA repair foci.

Cooperative action of NC2 and Mot1p to regulate TATA-binding protein function across the genome

Promoter recognition by TATA-binding protein (TBP) is an essential step in the initiation of RNA polymerase II (pol II) mediated transcription. Genetic and biochemical studies in yeast have shown that Mot1p and NC2 play important roles in inhibiting TBP activity. To understand how TBP activity is regulated in a genome-wide manner, we profiled the binding of TBP, NC2, Mot1p, TFIID, SAGA, and pol II across the yeast genome using chromatin immunoprecipitation (ChIP)-chip for cells in exponential growth and during reprogramming of transcription. We find that TBP, NC2, and Mot1p colocalize at transcriptionally active pol II core promoters. Relative binding of NC2alpha and Mot1p is higher at TATA promoters, whereas NC2beta has a preference for TATA-less promoters. In line with the ChIP-chip data, we isolated a stable TBP-NC2-Mot1p-DNA complex from chromatin extracts. ATP hydrolysis releases NC2 and DNA from the Mot1p-TBP complex. In vivo experiments indicate that promoter dissociation of TBP and NC2 is highly dynamic, which is dependent on Mot1p function. Based on these results, we propose that NC2 and Mot1p cooperate to dynamically restrict TBP activity on transcribed promoters.

Changing medium and passaging cell lines

Cell lines are widely used in biomedical research. This protocol describes the methods used routinely to change the medium and passage the cells. Medium changes keep the cells healthy by providing fresh nutrients, while cell passage or splitting is required to maintain cells in exponential growth. Despite the simplicity of the methods used, each cell line has idiosyncracies. Whether working with one or several cell lines, there is no substitute for knowledge of their needs, including the range of phenotypes and growth patterns under different physical and nutrient conditions. Given the necessary care and attention, most cell lines are easy to maintain and grow.

Evaluation of XIAP as a Molecular Target To Increase Sensitivity to Imatinib.

Imatinib, a potent and selective inhibitor of BCR-ABL, has emerged as an effective drug for targeted therapy of CML. However, resistance to this drug has been arising, mainly due to an overexpression of BCR-ABL, to mutations in the tyrosine-kinase domain of BCR-ABL or to an overexpression of the multidrug resistance protein (Pgp). Cytotoxic drugs induce cellular apoptosis and resistance to these drugs is often due to resistance to apoptosis. Cancer cells may escape apoptosis due to an overexpression of anti-apoptotic proteins such as XIAP, which binds caspases 3, 7 and 9 and inhibits their action.Downregulation of XIAP expression has been documented to increase sensitivity of several cell lines to some conventional drugs. In the current study we investigated if downregulation of XIAP expression, following treatment with XIAP-siRNAs, would increase sensitivity to Imatinib in: i) a sensitive CML cell line (K562), when compared to treatment with control siRNAs and ii) a resistant cell line which overexpresses Pgp (K562Dox), when compared to treatment with Pgp-siRNAs or with control siRNAs. To confirm if the siRNAs were capable of downregulating the expression of their targets, Western Blots were carried out at 24, 48 and 72h after transfection. The cells had been: i) treated with medium only (Blank), ii) transfected with a control-siRNAs, iii) transfected with siRNAs for XIAP and iv) tranfected with siRNAs for Pgp (in the K562Dox cell line only). It was verified that the XIAP-siRNAs decreased XIAP protein levels in both cell lines 48h following transfection and Pgp-siRNAs decreased Pgp protein levels in the resistant cell line (K562Dox) 24h following transfection, when compared to the control-siRNA treatments. To further investigate if the downregulation of XIAP expression (and Pgp in the resistant cell line) caused sensitization of cells to1μM Imatinib, Imatinib was added to the cells previously transfected with siRNAs (for P-gp or XIAP or control siRNAs) and to cells in exponential growth (Blank cells). Viable cell number was counted by the Trypan Blue exclusion assay and apoptosis was verified with the TUNEL assay. Results of viable cell numbers from several experiments indicate that Imatinib treatment on its own, as expected, decreased more the viable cell number of the sensitive cell line than of the resistant cell line (from 100% in the Blank cells to 63 % in the treated K562 cells and to 86 % in the treated K562Dox cells). Furthermore, downregulation of XIAP expression on its own decreased the viable cell number of both cell lines (from 100% in the Blank cells to 62% in the K562 XIAP-siRNA treated cells and to 80% in the K562Dox XIAP-siRNA treated cells). Concomitant treatment with both XIAP-siRNAs and Imatinib further reduced the number of viable cells to 36% in the K562 cells and to 57% in the K562Dox cells. In the resistant cell line, downregulation of Pgp expression on its own reduced the viable cell number from 100% in Blank cells to 84% in the Pgp-siRNA treated cells. Concomitant treatment with Pgp-siRNAs and Imatinib further reduced the viable cell number to 60%. The reduction of viable cell number coincided with an increase in apoptosis. It was concluded that downregulation of XIAP expression may be a good approach to enhance sensitivity to Imatinib, even in the case of existing Pgp overexpression.

Overcoming Resistance to Imatinib Mesylate in a CML Cell Line by Downregulation of P-gp with siRNAs.

Imatinib Mesylate (Gleevec, formely STI571 from NOVARTIS) is a highly selective drug, capable of inhibiting the BCR-ABL tyrosine kinase which is responsible for CML. However, resistance to this drug has been documented, mainly due to an overexpression of the BCR-ABL protein, to mutations in the tyrosine-kinase domain of BCR-ABL itself or to an overexpression of the multidrug resistance protein 1 (P-gp). In the current study we investigated if downregulation of P-gp expression with siRNAs, in a cell line resistant to Imatinib which overexpressed P-gp (K562Dox), would allow to overcome resistance to this drug. In order to optimise transfection of the siRNAs, we used a fluorochrome labelled control-siRNA and fluorescence microscopy to evaluate the % of uptake of the siRNA under different transfection conditions. The optimised conditions (200 nM siRNA using the jetSi™ transfection reagent) allowed us to obtain uptake of siRNAs in 53% of cells. In order to confirm if the siRNAs were capable of downregulating the expression of P-gp we carried out, 24h after transfection, Real-Time Quantitative PCR studies and Western Blots of cells: i) treated with medium only (Blank), ii) transfected with the siRNAs suspension buffer, iii) transfected with a control siRNA (CRNAi), iv) transfected with two different siRNAs for P-gp (MDR-FE or MDR-CR). We verified that in cells treated with the MDR-CR siRNA there was a reduction in the P-gp mRNA expression of 26%, when compared to the CRNAi. Furthermore, when cells were treated with the MDR-FE or MDR-CR siRNAs, protein levels were also reduced by 33% and 57% respectively, when compared to the CRNAi. It was then verified if this downregulation of MDR expression caused sensitization of cells to Imatinib, in comparison to control cells, using both viability (Trypan Blue) and apoptosis (TUNEL) assays. For this purpose, Imatinib Mesylate was added to the cells previously transfected with siRNAs (for P-gp or control siRNAs) and to cells in exponential growth (Blank cells). Results showed that there was an enhancement of the effect of Imatinib in cells previously transfected with both P-gp siRNAs (MDR-FE or MDR-CR). This increase in the effect of Imatinib was due to an increase in cellular apoptosis. Indeed, the % of apoptotic cells increased from 3 % in cells treated with solvent to 7 % in cells treated with 1 μM Imatinib. It was concluded that downregulation of P-gp protein expression by RNAi technology permitted sensitisation of CML cells to Imatinib, suggesting that P-gp inhibition may be considered as an adjuvant therapy in cases of Imatinib resistance mediated by this mechanism.

Effect of growth phase on the Escherichia coli response to ultraviolet-A radiation: influence of conditioned media, hydrogen peroxide and acetate

The results reported herein indicate that the ultraviolet-A (UVA) radiation-induced effects in Escherichia coli depend on its growth phase. Stationary-phase cells recover faster from a sub-lethal UVA exposure and have a higher resistance to lethal effect of the radiation than exponential growing cells. Although pre-incubation in spent medium supernatant increased the resistance of log-phase cells to lethal UVA effects, this pre-treatment considerably prolonged the duration of the radioinduced sub-lethal growth delay. The aim of the present study was to investigate the effect exerted by the E. coli conditioned media and evaluate the influence of nutritional stress, hydrogen peroxide and acetate. Pre-incubated in conditioned medium, cells in exponential growth phase were irradiated and the induced effects were compared with those found when catalase, high culture densities and acetate were employed. Unexpectedly, the duration of the growth delay in cells submitted to these treatments was shortened in comparison with control cells incubated in conditioned medium with no modifications. Lengthening of the growth delay was mimicked when exponentially growing cells were incubated in fresh medium supplied with 5 μM H 2O 2. The effects of spent medium on wild type and rpoS mutant strains were similar, indicating that this response is independent of RpoS controlled functions. We assumed that an oxidative component of the spent medium, probably H 2O 2, could be involved in the observed phenomenon. This effect is specific of E. coli and independent of rpoS.

Gompertzian growth pattern correlated with phenotypic organization of colon carcinoma, malignant glioma and non-small cell lung carcinoma cell lines.

In the current study we present a Gompertzian model for cell growth as a function of cell phenotype using six human tumour cell lines (A-549, NCI-H596, NCI-H520, HT-29, SW-620 and U-251). Monolayer cells in exponential growth at various densities were quantified over a week by sulforhodamine B staining assay to produce cell-growth curves. A Gompertz equation was fitted to experimental data to obtain, for each cell line, three empirical growth parameters (initial cell density, cell-growth rate and carrying capacity - the maximal cell density). A cell-shape parameter named deformation coefficient D (a morphological relationship among spreading and confluent cells) was established and compared by regression analysis with the relative growth rate parameter K described by the Gompertz equation. We have found that coefficient D is directly proportional to the growth parameter K. The fit curve significantly matches the empirical data (P < 0.05), with a correlation coefficient of 0.9152. Therefore, a transformed Gompertzian growth function was obtained accordingly to D. The degree of correlation between the Gompertzian growth parameter and the coefficient D allows a new interpretation of the growth parameter K on the basis of morphological measurements of a set of tumour cell types, supporting the idea that cell-growth kinetics can be modulated by phenotypic organization of attached cells.

Glyceraldehyde-3-phosphate dehydrogenase, a glycolytic enzyme present in the periplasm of Aeromonas hydrophila.

This is the first report describing the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as a protein associated with the cell envelope of a gram-negative bacterium (Aeromonas hydrophila). Dose-dependent GAPDH activity was detected in whole bacterial cells from exponentially growing cultures, indicating that an active form of GAPDH is located outside the plasma membrane. This activity represents roughly 10-20% of total cell activity, and it is not reduced by pretreatment of the cells with trypsin. Assays with soluble GAPDH indicate that the activity measured in intact cells does not originate by rebinding to intact cells of cytosolic enzyme released following cell lysis. GAPDH activity levels detected in intact cells varied during the growth phase. The relationship between GAPDH activity and cell culture density was not linear, showing this activity as a major peak in the late-logarithmic phase (A600 = 1.1-1.3), and a decrease when cells entered the stationary phase. The late exponential growing cells showed a GAPDH activity 3 to 4-fold higher than early growing or stationary cells. No activity was detected in culture supernatants. Enzymatic and Western-immunoblotting analysis of subcellular fractions (cytosol, whole and outer membranes, and periplasm) showed that GAPDH is located in the cytosol, as expected, and also in the periplasm. These results place the periplasmic GAPDH of A. hydrophila into the family of multifunctional microbial cell wall-associated GAPDHs which retain their catalytic activity.

Cell-cell interactions influence resistance and survival of Salmonella serotype Typhimurium to environmental stress.

The aim of this work was to study the effects of prolonged nutrient stress on survival, cell interactions and resistance to inimical processes in Salmonella serotype Typhimurium. Salmonella Typhimurium cells were subjected to prolonged incubation in the stationary phase of growth and the properties of starved cells (old) were investigated with reference to those of exponentially-growing cells (young). Competition experiments between old and young cells revealed cell-cell interactions that influenced stationary phase survival and response of the bacterium to heat stress. During prolonged incubation of cells, cycles of resistance and sensitivity to heat stress were identified. Competition experiments between old and young cells revealed that the resistance of young cells to heat increased to levels more like those of stationary phase cells than growing cells. The presence of old cells influenced the phenotype of young cells, possibly by means of cell-cell interactions. There was no evidence for the involvement of any extracellularly-produced factors in this phenomenon, but a requirement that the old competitor cells be viable could be demonstrated. It is proposed that the complex interactions within stationary phase cultures of Salm. Typhimurium may be due to cycles of mutation in concert with an as yet undefined interaction between old cells and growing ones. This study provides evidence for active and diverse responses to nutrient stress within populations of Salm. Typhimurium that promote survival and that may be important for the success of this bacterium as a pathogen.

Differential gene expression in γ-irradiated BALB/3T3 fibroblasts under the influence of 3-aminobenzamide, an inhibitior of parp enzyme

3-Aminobenzamide (3AB) is an inhibitor of poly (ADP-ribose) polymerase (PARP), an enzyme implicated in the maintenance of genomic integrity, which is activated in response to radiation-induced DNA strand breaks. cDNA macroarray membranes containing 1536 clones were used to characterize the gene expression profiles displayed by mouse BALB/3T3 fibroblasts (A31 cell line) in response to ionizing irradiation alone or in combination with 3AB. A31 cells in exponential growth were pre-treated with 3AB 4 mM 1 h before γ-irradiation (4 Gy), remaining in culture during 6 h until harvesting time. A31 cells treated with 3AB alone presented a down-regulation in genes involved in protein processing and cell cycle control, while an up-regulation of genes involved in apoptosis and related to DNA/RNA synthesis and repair was verified. A31 cells irradiated with 4 Gy displayed 41 genes differentially expressed, being detected a down-regulation of genes involved in protein processing and apoptosis, and genes controlling the cell cycle. Concomitantly, another set of genes for protein processing and related to DNA/RNA synthesis and repair were found to be up-regulated. A positive or negative interaction effect between 3AB and radiation was verified for 29 known genes. While the combined treatment induced a synergistic effect on the expression of LCK proto-oncogene and several genes related to protein synthesis/processing, a negative interaction effect was found for the expression of genes related to cytoskeleton and extracellular matrix assembly (SATB1 and Anexin III), cell cycle control (tyrosine kinase), and genes participating in DNA/RNA synthesis and repair (RNA helicase, FLAP endonuclease-1, DNA-3 glycosylase methyladenine, splicing factor SC35 and Soh1). The present data open the possibility to investigate the direct participation of specific genes, or gene products acting in concert in the mechanism underlying the cell response to radiation-induced DNA damage under the influence of PARP inhibitor.

Transcription of Schizosaccharomyces pombe thioltransferase-1 in response to stress conditions.

Thioltransferase, also known as glutaredoxin, is an enzyme that catalyzes the reduction of a variety of disulfide compounds. In Schizosaccharomyces pombe, two thioltransferases were reported and the cDNA of one of the thioltransferases (thioltransferase-1) was cloned. Using a Northern blot assay, we investigated the thioltransferase transcription in response to various stress conditions. When the culture was shifted to a high temperature, the thioltransferase transcription was not significantly changed compared to the unshifted 30 degrees culture. Treatment of zinc chloride to exponentially-growing cells remarkably increased the thioltransferase transcription, whereas the treatment of mercury chloride greatly reduced the transcription. Treatment of hydrogen peroxide and cadmium chloride caused no significant effects on the transcription of the thioltransferase. These results suggest that the transcription of thioltransferase-1 in S. pombe is induced in response to metal stress that is caused by zinc chloride, but not in response to heat stress or oxidative stress that is caused by hydrogen peroxide.