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PubMed2005

Modulation of Leishmania donovani infection and cell viability by testosterone in bone marrow-derived macrophages: signaling via surface binding sites.

Androgens can increase susceptibility toward numerous parasitic infections as well as modulate apoptosis of immune cells. According to the current view, androgens mediate immune cell activities not only through classical intracellular androgen receptors (AR), but also through membrane receptors on the cell surface. Here, using murine bone marrow-derived macrophages (BMMs), we examined the influence of testosterone on Leishmania donovani infection and cell viability in vitro as well as the possible mechanisms. Our data demonstrated that testosterone directly increased intramacrophage infection by L. donovani. In addition, testosterone decreased cell viability by way of apoptosis, accompanied by increased Fas, FasL, and Caspase-8 expression. However, these effects of testosterone could not be associated with the classical AR in BMMs since AR was not detectable using different experimental techniques. Instead, it was found that testosterone could bind to the surface of BMMs by the use of an impermeable testosterone-BSA-FITC in confocal laser scanning microscopy and flow cytometry. Collectively, our data indicated that the influence of testosterone on L. donovani infection and viability of BMMs was mediated through the binding sites of testosterone on cell surfaces, which provided a novel mode of direct action of testosterone on AR-free BMMs.

Animals↗
PubMed1973

Effect of glutaraldehyde on cell viability, triphenyltetrazolium reduction, oxygen uptake, and beta-galactosidase activity in Escherichia coli.

Acid (pH 5) and alkaline (pH 8.5) glutaraldehyde solutions were compared for their effects on cell viability, oxygen uptake, and beta-galactosidase activities in Escherichia coli. The action of glutaraldehyde at pH 7 on dehydrogenase activity was also studied. Dehydrogenase activity was inhibited at aldehyde concentrations which had little effect on cell viability. In contrast, oxygen uptake and beta-galactosidase activity took place in cells killed by acid or alkaline glutaraldehyde. The effect of glutaraldehyde on dehydrogenase activity and beta-galactosidase activity of disrupted suspensions was also investigated. The dialdehyde was considerably less inhibitory to these enzyme systems than to those of whole cells, and it is thus feasible that the results with whole cells are a consequence of its interaction with, and strengthening of, the outer cell surface, thereby preventing ready access of substrate to enzyme.

Aldehydes↗
PubMedDate not supplied

Comparative chemosensitivity profiles in three human breast cancer cell lines with the ATP-cell viability assay.

In this study the dose-response curves for doxorubicin, pirarubicin, 5-fluorouracil, 4-hydroperoxy-cyclophosphamide and taxol were obtained in three breast cancer cell lines (MCF-7, T47D and BT-20). The ATP cell viability assay was chosen to evaluate the chemosensitivity profiles and was a reproducible, practicable method to assess drug response in breast cancer cell lines. The IC50 values were calculated on the median effect principle and indicated that taxol was the most active drug tested in this study with a mean IC50 value of 0.02 microM. This in vitro effect correlated well with clinical observations in metastatic breast cancer where taxol proved to be a vary active drug. Pirarubicin was the second most active drug tested with an IC50 value 10 times less compared to that of doxorubicin. The results obtained with the ATP cell viability assay are promising, therefore further testing with drug combination chemotherapy and fresh human breast cancer tumor testing are warranted and ongoing.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1999

Characterization of the ssnA gene, which is involved in the decline of cell viability at the beginning of stationary phase in Escherichia coli.

When grown in rich medium, Escherichia coli exhibits a drastic reduction of the number of viable cells at the beginning of stationary phase. The decline of cell viability was retarded by disruption of the ssnA gene, which was identified as a gene subject to RpoS-dependent negative regulation. Moreover, ssnA expression was induced at the time of decline of cell viability at early stationary phase. The viability decline was augmented in the rpoS background, and this augmentation was suppressed by ssnA mutation. Cloning of the ssnA gene in a multicopy plasmid, pBR322, caused small colony formation and slow growth in liquid medium. Cells harboring the ssnA clone showed aberrant morphology that included enlarged and filamentous shapes. The gene product was identified as a 44-kDa soluble protein, but its function could not be deduced by homology searching. From these results, we conclude that ssnA is expressed in response to a phase-specific signal(s) and that its expression level is controlled by RpoS, by a mechanism which may contribute to determination of cell number in the stationary phase.

Amino Acid Sequence↗
PubMed2006

Effect of 5-fluorouracil, Optison and ultrasound on MCF-7 cell viability.

The aim of this study was to analyze cell viability and expression of apoptotic-related signaling proteins in MCF-7 breast cancer cells induced by combinations of ultrasound, the anticancer drug 5-fluorouracil (5-FU) and the ultrasound contrast agent Optison. MCF-7 cells were treated with 5-FU and sonicated at the frequency of 3.0 MHz and intensity of 3.0 W/cm2 for 1 min in the presence of Optison. The cells were analyzed for lactate dehydrogenase (LDH) release (a measure of cytotoxicity) and cell proliferation (by MTT assays). The LDH/MTT ratio was used for assessment of cell death. Expression of the apoptotic-related proteins, Bax and p27kip1, as well as phosphorylated forms of ERK and Akt proteins was assessed by Western blot analysis. We demonstrate that, immediately after treatment, cell death was most dependent on Optison; however, 24 h after treatment, cell death was more dependent on 5-FU. Ultrasound duty cycle increased cell death associated with either Optison or 5-FU. Furthermore, we show that treatment with 5-FU and ultrasound increased the levels of the Bax and p27kip1 proteins, but the addition of Optison appears to suppress apoptotic protein expression.

Albumins↗
PubMedDate not supplied

Assessment of a simple, non-toxic Alamar blue cell survival assay to monitor tomato cell viability.

The Alamar Blue (AB) assay, which incorporates a medox indicator that changes colour or fluorescence in response to metabolic activity, is commonly used to assess quantitatively the viability and/or proliferation of mammalian cells and micro-organisms. In this study the AB assay was adapted for the determination of the viability of plant cells. Cell suspension cultures of tomato, Lycopersicon esculentum, L., with differing viabilities, served as the experimental model for a comparison of the AB assay with the conventional 2,3,5-triphenyltetrazolium chloride (TTC) viability assay. The AB assay showed a sigmoidal relationship between cell viability and AB reduction (as quantified by spectrofluorometry or spectrophotometry), which was similar to that obtained using the TTC assay. Both assays detected a significant reduction in cell viability after 48 h exposure to virulent Ralstonia solanacearum (biovar III), while the TTC assay, in addition, revealed cell proliferation in control cells from 24 to 72 h. The TTC assay detected cell proliferation over a wider range of cell densities, while the AB assay was more rapid and versatile whilst being non-toxic and thus allowing subsequent cell analysis.

Cell Survival↗
PubMedDate not supplied

Growth characteristics of nonmalignant cells in the ATP cell viability assay.

Over the last 5 years, the ATP cell viability assay (ATP-CVA) has been used to study the in vitro response of cell lines and fresh gynecologic human tumors to a variety of antineoplastic agents including chemotherapeutic agents, hormones and biological response modifiers. This assay measures light production as intracellular ATP interacts with the luciferin-luciferase complex. Quantitation of the light produced has been shown to directly correspond with the number of viable cells. A past criticism is that in the ATP-CVA, when applied to fresh tumor tissue, normal cells (fibroblasts, macrophages and lymphocytes) also produce ATP, and if present in sufficient numbers, could lead to errors in chemosensitivity testing results. This study was designed to evaluate the growth characteristics of various benign cells found in fresh tumors. The cells were studied under multiple plating conditions to show the relative increase or decrease of fractional ATP measured at different time points. We found that agar/McCoy underlayer and agarose-coated wells do not permit the growth of nonmalignant cells. In the culture conditions of the ATP-CVA, non-malignant cells do not contribute relevant ATP levels when treated samples are compared to controls on day 6. Therefore, results of the ATP-CVA in fresh tumors should not be affected.

Adenosine Triphosphate↗
PubMed1996

Comparative study of classical, colorimetric and immunologic staining methods for the assessment of tumor cell viability.

The trypan blue exclusion test, the MTT test and an immunostaining test for apoptosis were performed before and after incubation of SW620 human colonic carcinoma cells with different cytotoxic agents (CTAs) in order to assess tumor cell viability and CTA efficacy in vitro. A modified MTT test using light microscopy was also performed. A good correlation was found between the trypan blue assay and the MTT test, as determined by spectrophotometry. There was no 'overestimation' of cell viability as measured by the trypan blue test. The monitoring of formazan formation by light microscopy was feasible, but not very reliable since it did not show a good correlation with findings determined by spectrophotometry. The apoptosis test failed to show good correlation with other tests. Distilled water had no relevant cytotoxic effect, while chlorhexidine cetrimide (HAC 3.5%), chloramine 0.5% and polyvinylpyrrolidone iodine (PVP-I) > or = 0.05% damaged a large majority of cells. PVP-I at a concentration of > or = 5% was found to be the most effective CTA.

Antineoplastic Agents↗
PubMed2002

Evaluation of cell viability by double-staining fluorescence assay.

Various methods are available for the assessment of cell viability. Recently, interest has centered on methods using fluorescent dyes. In this work, we used a double-staining assay, involving the use of rhodamin 123, which stains the mitochondria of viable cells, and propidium iodide, which stains the nuclei of dead cells, to investigate their use in assessing the viability of cells. We added adriamycin to NIH 3T3 cells and double-stained the cells that adhered to the dish and those that were suspended in the culture solution, and observed the results over time. We found that nearly all the adherent cells were stained with rhodamin 123 alone. However, the suspended cells in the control group accounted for most of the double-stained cells, and when adriamycin was added, most were stained with PI alone.

3T3 Cells↗
PubMed2002

Study of mitochondrial membrane potential, reactive oxygen species, DNA fragmentation and cell viability by flow cytometry in human sperm.

BACKGROUND: Sperm cell death appears to be a cause of male infertility. The objective of this study was to determine the most reliable method for the evaluation of sperm quality in semen samples during sperm preparation for IVF. METHODS: Conventional analysis of semen samples was compared with several cytofluorometric methods detecting death-associated changes. Neat semen from infertile patients and sperm prepared by PureSperm gradient were studied by conventional microscopy and analysed for mitochondrial membrane potential (Delta Psi(m)), generation of reactive oxygen species, DNA fragmentation and cell viability. RESULTS: In neat semen, a positive correlation was found between the percentage of Delta Psi(m)(high) sperm cells and standard semen parameters (concentration/motility). Sperm cells depicting Delta Psi(m)(high) and cells with low DNA fragmentation displayed high fertilization rate after IVF. The only changes that could be detected in prepared sperm were changes in Delta Psi(m), with Delta Psi(m)(high) sperm positively correlated with forward motility and also with high fertilization rates after IVF. CONCLUSION: Analysis of mitochondrial membrane potential is the most sensitive test by which to determine sperm quality. These findings promise development of a test that may help to predict successful IVF.

Adult↗
PubMed1982

A postulated mechanism for the coordinate effects of ionophore A23187 on calcium uptake and cell viability in rat thymocytes.

At the physiological concentration of Ca2+, the presumed calcium ionophore A23187 produced dose-related increases in 45Ca uptake by rat thymocytes and decreases in cell viability, effects that displayed a strong linear correlation. In media containing a very low concentration of Ca2+ (7.10(-6) M), in contrast, ionophore A23187 had no specific effect on either 45Ca uptake or cell viability. The calcium-dependent cytotoxicity of ionophore A23187 resembles that of other agents that are not ionophores, but that are known to perturb the plasma membrane. Consequently, we suggest that, in the rat thymocyte, ionophore, ionophore A23187 may not act as a true ionophore, but may perturb the cell membrane, allowing Ca2+ to pass freely through the membrane along its electrochemical gradient.

Animals↗
PubMed1999

Effect of storage temperature on cell viability in cryopreserved canine aortic, pulmonic, mitral, and tricuspid valve homografts.

We determined how long cryopreserved aortic, pulmonic, mitral, and tricuspid valve homografts could be stored in a deep freezer (-80 degrees C) without compromising fibroblast viability. Valves harvested from 20 anesthetized mongrel dogs were grouped into nonfrozen control, frozen and stored in liquid nitrogen (-196 degrees C), and frozen and stored in a deep freezer (-80 degrees C). Frozen groups were divided into subgroups and stored for 2, 4, 8, or 12 weeks. A leaflet of each valve was divided into three fragments, and fibroblast viability was analyzed by flow cytometry. Cell viability was defined as staining by fluorescent diacetate but not by propidium iodide. The viability of untreated control valves from all four sites was about 70%, decreasing to about 50% when treated with low doses of antibiotics. The viability of frozen valves stored in liquid nitrogen was about 45% without a significant difference among storage periods. The viability of valves frozen and stored in a deep freezer was significantly lower than for the liquid nitrogen group at 2 weeks for the mitral valve and at 4 weeks for other valves. These results suggest that homografts can be stored in a deep freezer for up to 2 weeks without deterioration.

Animals↗
PubMed1992

The Schizosaccharomyces pombe rhp3+ gene required for DNA repair and cell viability is functionally interchangeable with the RAD3 gene of Saccharomyces cerevisiae.

The RAD3 gene of Saccharomyces cerevisiae is required for excision repair and is essential for cell viability. RAD3 encoded protein possesses a single stranded DNA-dependent ATPase and DNA and DNA.RNA helicase activities. Mutational studies have indicated a requirement for the RAD3 helicase activities in excision repair. To examine the extent of conservation of structure and function of RAD3 during eukaryotic evolution, we have cloned the RAD3 homolog, rhp3+, from the distantly related yeast Schizosaccharomyces pombe. RAD3 and rhp3+ encoded proteins are highly similar, sharing 67% identical amino acids. We show that like RAD3, rhp3+ is indispensable for excision repair and cell viability, and our studies indicate a requirement of the putative rhp3+ DNA helicase activity in DNA repair. We find that the RAD3 and rhp3+ genes can functionally substitute for one another. The level of complementation provided by the rhp3+ gene in S.cerevisiae rad3 mutants or by the RAD3 gene in S.pombe rhp3 mutants is remarkable in that both the excision repair and viability defects in both yeasts are restored to wild type levels. These observations suggest a parallel evolutionary conservation of other protein components with which RAD3 interacts in mediating its DNA repair and viability functions.

Adenosine Triphosphatases↗
PubMedDate not supplied

Saphenous vein endothelial cell viability: a comparative study of endoscopic and open saphenectomy for coronary artery bypass grafting.

BACKGROUND: The use of endoscopic saphenous vein harvesting (ESVH) for coronary artery bypass grafting (CABG) is growing. This study was done to investigate the extent of endothelial injury in ESVH compared with that of the standard open method (OSVH), and under various physical and chemical preservation factors. METHODS: We endoscopically removed the saphenous vein from 45 consecutive patients undergoing saphenectomy for CABG together with a segment retrieved by the no-touch OSVH method. Vein samples from each group were divided into 8 subgroups of 5 samples each, and incubated in Plasma-Lyte solution with or without papaverine, at distending pressures of 100 or 300 mm Hg, and at either 4 degrees C or 28 degrees C, respectively. A ninth subgroup was preserved at room temperature without pressure or papaverine. The viability of cultured saphenous vein endothelial cells was assessed by counting the number of total cells and deriving the proportion of viable cells, following incubation for 72 hours. RESULTS: The median proportion of viable cells (PVC) showed a slight decline over days 0 to 4 for both harvesting methods. No significant difference existed in the median PVC between the two techniques (day 0: 75%, 72%, P = 0.8; day 1: 66.7%, 66.7%, P = 0.9; day 2: 66.7%, 66.7%, P = 0.3; day 3: 65.3%, 66.7%, P = 0.16, respectively). The mean PVC compared across temperatures of 4 degrees C, 28 degrees C, and room temperature for the ESVH was highly significant, with the highest value being for room temperature (69.5%, 56.4%, 70.3%, respectively, P = 0.0003). Results for the OSVH were not significant. The effect of distension pressure did not vary significantly for 0, 100, and 300 mm Hg for both techniques (70.3%, 63.2% and 63.4%, respectively, P = 0.46 for the ESVH; 66.5%, 68.4%, 67.4%, respectively, P = 0.94 for the OSVH). The addition of papaverine improved PCV slightly for the OSVH only (61.7%, 64.3%, respectively, P = 0.02), whereas that for the ESVH was not significant (67.3%, 72.5%, P = 0.12). CONCLUSION: The effect of ESVH on endothelial cell viability is comparable to that of the OSVH. Among the factors influencing endothelial viability during vein preparation, temperature had a major effect with lower temperatures in the range of 4 degrees C to room temperature being the most favorable one. Mechanical distension and papaverine had unimportant or inconsistent roles. We recommend the ESVH as the procedure of choice for saphenous vein harvesting due to the lower postoperative morbidity, and the lower incubation temperature needed for its better influence on potential graft patency.

Cells, Cultured↗
PubMed2003

Mammalian elongin A is not essential for cell viability but is required for proper cell cycle progression with limited alteration of gene expression.

Elongin A is a transcription elongation factor that increases the overall rate of mRNA chain elongation by RNA polymerase II. To investigate the function of Elongin A in vivo, the two alleles of the Elongin A gene have been disrupted by homologous recombination in murine embryonic stem (ES) cells. The Elongin A-deficient ES cells are viable, but show a slow growth phenotype because they undergo a delayed mitosis. The cDNA microarray and RNase protection assay using the wild-type and Elongin A-deficient ES cells indicate that the expression of only a small subset of genes is affected in the mutant cells. Taken together, our results suggest that Elongin A regulates transcription of a subset but not all of genes and reveal a linkage between Elongin A function and cell cycle progression.

Animals↗
PubMed2000

Effects of NAMI-A and some related ruthenium complexes on cell viability after short exposure of tumor cells.

A series of three ruthenium complexes, i.e. trans-dichlorote-trakisdimethyl-sulfoxide ruthenium(ll) (trans-Ru), imidazolium trans-imidazoletetra-chlororuthenate (ICR) and sodium trans-tetramethylensulfoxideisoquinoline-tetrachlororuthenate (TEQU), were studied in vitro in comparison to NAMI-A, a potent ruthenium-based antimetastasis agent. In vitro challenge of TS/A adenocarcinoma or KB oral carcinoma tumor cells with 10(-4) M concentration for 1 h evidenced the lack of cytotoxicity of NAMI-A, ICR and trans-Ru, the accumulation of cells in the G2/M pre-mitotic cell phase by NAMI-A and the attachment of tumor cells to the plastic substrate was significantly greater for NAMI-A than for ICR. These data stress that in vitro cytotoxicity is not necessary for in vivo activity of ruthenium antitumor complexes: NAMIA, ICR and trans-Ru, are in fact known to be active against murine tumors in the mouse system. Rather, TEQU, the compound free of in vivo activity, was the only one to reduce cell growth of in vitro cultured cells. In conclusion, the data on the effects of NAMI-A on in vitro cultured cells show that the increase of cell adhesion properties and the transient cell cycle arrest in the G2/M phase are much more relevant than the effects on cell properties relevant to cell growth (i.e. on CD44, CD54 or CD71 antigens) for determining in vivo antimetastasis activity.

Adenocarcinoma↗
PubMed2004

Effects of beta-carotene on cell viability and antioxidant status of hepatocytes from chronically ethanol-fed rats.

The purpose of the present study was to evaluate the effects of beta-carotene on the cell viability and antioxidant status of hepatocytes from chronically ethanol-fed rats. Rats in the ethanol group were given an ethanol-containing liquid diet that provided 36 % of total energy as ethanol, while rats in the control group were fed an isoenergetic diet without ethanol. After 4 weeks, hepatocytes were taken out and cultured for 24 h. Hepatocytes from the rats in the control and ethanol groups were cultured in medium without (HC, HE) or with beta-carotene (HC+B, HE+B). The results showed that lactate dehydrogenase leakage was significantly increased in the HE compared with that in the HC group. However, lactate dehydrogenase leakage of the HE+B group was similar to that of the HC group. When compared with the HC group, activities of glutathione peroxidase and catalase in the HE group were significantly decreased by 54 and 31 %, respectively. Catalase activity in the HE+B group was significantly increased by 61 % compared with that in the HE group. However, activities of glutathione reductase and superoxide dismutase showed no difference among the groups. The level of glutathione in the HC+B and HE+B groups was significantly increased to 155 and 143 % compared with those in the HC and HE groups, respectively. The concentration of lipid peroxides showed no difference among the groups. The present results demonstrate that beta-carotene improved the cell viability of hepatocytes, and increased catalase activities and glutathione levels in hepatocytes from chronically ethanol-fed rats.

Animals↗