Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Trypan Blue”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 757 records · Page 42Linked to original sources
PubMed2003

Induction of necrosis and DNA fragmentation during hypothermic preservation of hepatocytes in UW, HTK, and Celsior solutions.

Donor cells can be preserved in University of Wisconsin (UW), histidine-tryptophan-ketoglutarate (HTK), or Celsior solution. However, differences in efficacy and mode of action in preventing hypothermia-induced cell injury have not been unequivocally clarified. Therefore, we investigated and compared necrotic and apoptotic cell death of freshly isolated primary porcine hepatocytes after hypothermic preservation in UW, HTK, and Celsior solutions and subsequent normothermic culturing. Hepatocytes were isolated from porcine livers, divided in fractions, and hypothermically (4 degrees C) stored in phosphate-buffered saline (PBS), UW, HTK, or Celsior solution. Cell necrosis and apoptosis were assessed after 24- and 48-h hypothermic storage and after 24-h normothermic culturing following the hypothermic preservation periods. Necrosis was assessed by trypan blue exclusion, lactate dehydrogenase (LDH) release, and mitochondrial 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) reduction. Apoptosis was assessed by the induction of histone-associated DNA fragments and cellular caspase-3 activity. Trypan blue exclusion, LDH release, and MTT reduction of hypothermically preserved hepatocytes showed a decrease in cell viability of more than 50% during the first 24 h of hypothermic preservation. Cell viability was further decreased after 48-h preservation. DNA fragmentation was slightly enhanced in hepatocytes after preservation in all solutions, but caspase-3 activity was not significantly increased in these cells. Normothermic culturing of hypothermically preserved cells further decreased cell viability as assessed by LDH release and MTT reduction. Normothermic culturing of hypothermically preserved hepatocytes induced DNA fragmentation, but caspase-3 activity was not hanced in these cells. Trypan blue exclusion, LDH leakage, and MTT reduction demonstrated the highest cell viability after storage in Celsior, and DNA fragmentation was the lowest in cells that had been stored in PBS and UW solutions. None of the preservation solutions tested in this study was capable of adequately preventing cell death of isolated porcine hepatocytes after 24-h hypothermic preservation and subsequent 24-h normothermic culturing. Culturing of isolated and hypothermically preserved hepatocytes induces DNA fragmentation, but does not lead to caspase-3 activation. With respect to necrosis and DNA fragmentation of hypothermically preserved cells, UW and Celsior were superior to PBS and HTK solutions in this model of isolated porcine hepatocyte preservation.

Adenosine↗
PubMed1969

The inhibitory effect of convulsant agents on the enzyme in brain which inactivates nerveside.

1. An enzyme which can be extracted from brain inactivates nerveside in the optimum pH range 5.8-7.0.2. The polybasic acids trypan blue and its analogue trypan red, bromphenol blue and its analogue bromthymol blue at concentrations of 0.22 mM and ethylenediaminetetra-acetic acid (EDTA) at a concentration of 1 mM are strong inhibitors of the enzyme.3. Penicillin which is a monobasic carboxylic acid also inhibits the enzyme but only if concentrations as high as 3.6 mM are used. The antibiotic streptomycin which is a basic substance does not inhibit the enzyme.4. Caffeine at a concentration of 7.2 mM only weakly inhibits the enzyme.5. Chymotrypsin and wheat germ acid phosphatase also inactivate nerveside at pH 5.9 and are inhibited by the acidic dyes and penicillin. EDTA inhibits wheat germ phosphatase but activates chymotrypsin.6. Inactivation of nerveside by the brain enzyme and by wheat germ phosphatase is different from the action of chymotrypsin. Nerveside solutions incubated with chymotrypsin completely lose all biological activity whereas if incubation is carried out with either the brain enzyme or wheat germ acid phosphatase a residual biological activity remains even when the concentration of these two enzymes is increased. This residual biological activity is due to a peptide as it is destroyed by chymotrypsin.7. The manner in which nerveside is inactivated by the brain enzyme is uncertain as the preparation of the latter contained phosphodiesterase and protease activities which were similarly inhibited by the acid dyes, penicillin and EDTA.8. Pentylenetetrazole, picrotoxin, strychnine and tetanus toxin do not inhibit the brain enzyme.9. The nerveside-inactivating enzyme is not identical with the Substance P-inactivating enzyme in brain as the former is inhibited by EDTA while the latter is not.

Acid Phosphatase↗
PubMed1993

Effect of extracellular ions and modulators of calcium transport on survival of tert-butyl hydroperoxide exposed cardiac myocytes.

OBJECTIVE: The aim was to investigate the effects of extracellular ions and agents that modify calcium translocating pathways on oxidative stress induced cell injury. METHODS: Survival of cardiac myocytes exposed to tert-butyl hydroperoxide (t-BHP, 0.2 to 2 mM) was estimated by their ability to maintain rod shaped morphology and exclude trypan blue, and by the release of lactate dehydrogenase (LDH). Mean life time (mu) of a myocyte was analysed in accordance with the Weibull distribution. RESULTS: In pCa 3.0 Ringer's solution, t-BHP exposed myocytes underwent time and concentration dependent hypercontracture. The fraction of rod shaped cells excluding trypan blue correlated with LDH release. The lifetime of a cell was increased upon reduction of [Ca]o. Myocytes exposed to t-BHP in pCa 7.0 or 8.0 Ringer's solution attained a rigor state and became permeable to trypan blue. t-BHP-induced hypercontracture was delayed on increasing [K]o or [Na]o. Reduction of [Na]o to 67.5 mM, but not to 13.5 mM, accelerated t-BHP induced hypercontracture. Early reduction of [Ca]o to a pCa of 8.0 had no protective effect if 1 mM calcium was reintroduced after half the duration of exposure. Verapamil (10 microM), nifedipine (1 microM), butanedione monoxamine (20 mM), and caffeine (10 mM) accelerated t-BHP-induced loss of rod shaped morphology. Ryanodine (1 microM), Trolox-C (1 microM), and butylated hydroxytoluene (BHT, 500 microM) had no effect on survival of t-BHP exposed myocytes. Survival was prolonged upon preincubation of the myocytes with BAPTA-AM or desferrioxamine or when the cells were exposed to t-BHP in the presence of La (1 mM) or N-propyl gallate (10 microM). CONCLUSIONS: Ferrous catalysed free radical reactions initiated by t-BHP lead to hypercontracture or rigor shortening of myocytes, depending upon [Ca]o. It is unlikely that t-BHP-induced hypercontracture is mediated by Ca influx through L- or T-type Ca channels or due to release of Ca from intracellular stores. Activation of Ca influx via Na-Ca exchange and/or increase in the passive membrane permeability to Ca is consistent with the observations reported. Myocyte rigor observed upon attenuation of Ca overload suggests contribution of Ca-independent processes.

Animals↗
PubMedDate not supplied

[Method of determining the area of nonperfused myocardium during coronary-occlusion ischemia using trypan blue-stained microspheres].

The authors developed a method for measurement of the area of the non-perfused myocardium with the aid of stained latex microspheres in rats with coronaro-occlusion ischemia followed and not followed by reperfusion. The method can be used for studying the therapeutic effect of the drugs in rats with coronaro-occlusion myocardial ischemia.

Animals↗
PubMed1976

Hormonal control of pinocytosis in the uterine epithelium of the rat.

Ovariectomized rats were treated with oestradiol-17 beta and/or progesterone to mimic the hormonal parameters inducing uterine sensitivity for implantation. The degree of pinocytosis of trypan blue and ferritin in the endometrial cells was examined. Significant epithelial pinocytosis of trypan blue occurred after a 3-day treatment of progesterone, and uptake was independently increased by priming with oestrogen and by oestradiol given on the 3rd day of progesterone treatment. Progesterone treatment caused uptake of ferritin by the epithelial cells; in control animals epithelial and stromal cells were involved. Oestrogen priming enhanced ferritin absorption, while 'nidatory' oestrogen had no effect. Oestradiol given alone completely blocked pinocytosis of both intraluminally injected substances.

Animals↗
PubMed1990

Observations on the cooling and cryopreservation of pig oocytes at the germinal vesicle stage.

This study examined the viability of pig oocytes at the germinal vesicle stage following cooling or cryopreservation. Cumulus-intact oocytes (n = 641) were collected from slaughterhouse pig ovaries and used in two experiments. In Exp. I the viability of 1) control, 2) cryoprotectant control (CC, 1.5 M glycerol/.5 M sucrose), 3) cooled (0 degrees C) and 4) cryopreserved (-196 degrees C) oocytes was assessed after no incubation or a 24-h incubation. Survivability was judged by morphological appearance, trypan blue exclusion and fluorescein diacetate staining. Survival rate of control oocytes (90%; based primarily on morphological appearance of the cumulus) incubated 0 h was greater (P less than .05) than that of all other groups, whereas survival rate of -196 degrees C oocytes (57%) was less (P less than .05) than that of all other groups. However, vital staining of 0 degrees C and -196 degrees C oocytes showed 0% survival rate as evidenced by trypan blue uptake and lack of fluorescence. The cumulus cells surrounding oocytes that were stored at 0 degrees C or -196 degrees C survived freezing as evidenced by trypan blue exclusion and intense fluorescence. Similar differences among treatment groups were found for oocytes incubated 24 h. Exp. 2 examined the temperature at which oocytes became sensitive to cooling. Oocyte death occurred when oocytes were cooled to 15 degrees C or lower. These results demonstrate that pig oocytes at the germinal vesicle stage did not survive cooling to 15 degrees C or below. When assessing the viability of cryopreserved cumulus enclosed oocytes it is important to use vital stains in conjunction with morphological appearance.

Animals↗
PubMed2002

Comparison of the effectiveness of ET-Kyoto with Euro-Collins and University of Wisconsin solutions in cold renal storage.

BACKGROUND: Previously, we developed a new organ preserving solution, ET-Kyoto solution (ETKS), and showed that it is effective for lung and skin cold storage. Our high sodium-low potassium solution containing trehalose and gluconate is chemically stable at room temperature. In this study, the efficacy of ETKS for renal cold storage compared with Euro-Collins solution (ECS) and University of Wisconsin solution (UWS) was investigated. METHODS: A preflush of Krebs-Henseleit buffer was used before cold storage because ECS and UWS, but not ETKS, failed to be distributed thoroughly into each renal segment when directly flushed, as determined by staining with trypan blue. The kidneys were stored at 4 degrees C in each solution after the preflush and were reperfused 24 hr later for 120 min with 37 degrees C Krebs-Henseleit buffer containing albumin. For physiological evaluation, perfusion flow rates, creatinine clearance, and fractional sodium reabsorption were evaluated. Histological examination also was performed to determine tubular, glomerular, and interstitial changes. Moreover, distribution of perfusate at the beginning of reperfusion was assessed by using trypan blue. RESULTS: The kidneys preserved in ETKS and UWS showed better physiological function and less histological damage than those preserved in ECS. Furthermore, at the beginning of reperfusion using trypan blue, almost all renal segments were stained in the ETKS and UWS groups, whereas they were partially stained in the ECS group. CONCLUSIONS: These results indicate that this simple and chemically stable solution, ETKS, could be a promising substitute for UWS.

Absorption↗
PubMed1991

Effects of sodium valproate and 4en-valproic acid on isolated hepatocytes of guinea pigs.

The effects of valproic acid (VPA) (100 to 2,000 micrograms/ml) and its metabolite 4en-valproic acid (4enVPA) (2 to 2,000 micrograms/ml) on plasma membrane permeability of isolated hepatocytes of guinea pigs were studied. The ability of hepatocytes to exclude trypan blue was decreased significantly (p less than 0.05) by VPA at concentrations higher than 100 micrograms/ml. The leakage of intracellular enzymes, lactate dehydrogenase (LDH), glutamic oxaloacetic transaminase (GOT), glutamic pyruvic transaminase (GPT) and gamma-glutamyl transpeptidase (GGT), were not significantly increased by VPA within therapeutic concentrations (100-200 micrograms/ml). However, GOT and GPT leakage were significantly increased (p less than 0.05) by VPA, 500 micrograms/ml, but tended to decrease with further increases in VPA concentration. The enzyme leakage or trypan blue exclusion was not significantly altered by 10 micrograms/ml 4enVPA unless phenytoin (PT) (20 micrograms/ml) or phenobarbital (PB) (20 micrograms/ml) was presented. Higher concentrations of 4enVPA significantly decreased the trypan blue exclusion as well as increased GOT and GPT leakage of hepatocytes. Under scanning electron microscopic examination some hepatocytes at therapeutic concentrations of VPA or 4enVPA showed blebs on the membrane. Higher drug concentrations caused serious membrane damage such as pits and pores. These results suggest that there may be at least two mechanisms for the hepatotoxic effect of VPA. One may occur at therapeutic concentrations of VPA, resulting in increases in membrane permeability and decreases in membrane tension of hepatocytes. The other mechanism may occur at high concentrations of VPA or 4enVPA, resulting in substantial damage to the hepatocyte membrane.

Animals↗
PubMed1996

Ligation of CD69 induces apoptosis and cell death in human eosinophils cultured with granulocyte-macrophage colony-stimulating factor.

Peripheral blood (PB) eosinophils rapidly undergo apoptosis and cell death in vitro unless cultured in the presence of cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) in which their survival is prolonged for up to 10 days. CD69 is a type II membrane antigen expressed by cytokine-activated, but not freshly isolated, PB human eosinophils. We have examined the effect of ligation of CD69 by specific monoclonal antibody (MoAb) on the viability of human eosinophils cultured with recombinant human (rh)GM-CSF. Eosinophils were purified by immunomagnetic selection and cultured with GM-CSF (10(-10) mol/L). Eighteen hours after the start of culture, a panel of CD69 MoAb or controls (anti-CR3 or isotype-matched control MoAb) were added. Viability was assessed by trypan blue exclusion and apoptosis by morphologic assessment, DNA laddering, and flow cytometric analysis of eosinophil red autofluorescence. Up to 50% of the eosinophils had undergone apoptosis 48 hours after addition of anti-CD69 MoAb compared with less than 10% apoptosis for CR3 or the isotype matched control. The majority of apoptotic eosinophils excluded trypan blue at 48 hours post CD69 ligation. More apoptotic eosinophils were observed at later time-points and this was associated with loss of viability. At 120 hours post-addition of the anti-CD69 MoAb MLR3, 24% +/- 10.6% eosinophils were viable compared with 84% +/- 3.4% for the CR3 control (P < .001). A F(ab)2 fragment of CD69 MoAb P8, also induced apoptosis in GM-CSF cultured eosinophils. A more rapid induction of eosinophil apoptosis was obtained with CD69 MoAb immobilized via their Fc portions on protein-A coated plastic 96 well plates. Ligation of CD69 or CR3 resulted in the release of comparable quantities of eosinophil peroxidase at 48 hours post-ligation. These levels of EPO were consistent with the viability of these cells at 48 hours as assessed by exclusion of trypan blue. Finally, a neutralizing MoAb to TGF beta 1 had no effect on CD69-dependent apoptosis induction nor were there detectable quantities of TGF beta 1 in supernatants from GM-CSF--cultured eosinophils ligated with CD69 or control MoAb. These results suggest that eosinophils cultured with GM-CSF can be induced to undergo apoptosis as a result of cell signalling mediated by perturbation of CD69. This may represent an important physiologic mechanism for eosinophil removal in vivo.

Antigens, CD↗
PubMed2000

Investigation of functional and morphological integrity of freshly isolated and cryopreserved human hepatocytes.

There is a pressing need for alternative therapeutic methods effective in the treatment of patients with liver insufficiency. Isolated human hepatocytes may be a viable alternative or adjunct to orthotopic liver transplantation in such patients. The purpose of this study was to evaluate the viability and functional integrity of freshly isolated and cryopreserved human hepatocytes, in preparation for a multi-center human hepatocyte transplantation trial. We are currently processing transplant-grade human parenchymal liver cells from nondiseased human livers that are obtained through a network of organ procurement organizations (OPOs). Thus far, sixteen hepatocyte transplants have been performed using hepatocytes processed by our methods. At the time of referral all specimens were deemed unsuitable for transplantation due to anatomical anomalies, high fat content, medical history, etc. Hepatocytes were isolated from encapsulated liver sections by a modified two-step perfusion technique. Isolated cells were cryopreserved and stored in liquid nitrogen for one to twelve months. The total yield of freshly isolated hepatocytes averaged 3.7x10(7) cells per gram of wet tissue. Based on trypan blue exclusion, fresh preparations contained an average of 85% viable hepatocytes vs. 70% in cryopreserved samples. The plating efficiencies of cells seeded immediately after isolation ranged from 87% to 98%, while those of cryopreserved/thawed cells were markedly lower. Flow cytometry analysis of cells labeled with 5,6-carboxyfluorescein diacetate succinimidyl ester (CFSE) showed that there was no significant difference in viability compared with trypan blue staining. Both freshly isolated hepatocytes and those recovered from cryopreservation showed typical and intact morphology as demonstrated by light and electron microscopy. The product of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reaction was always expressed more intensely in cultures of freshly isolated hepatocytes. Measurements of lactate dehydrogenase (LDH) leakage were inversely correlated with trypan blue exclusion and CFSE labeling. Energy status, evaluated by the intracellular ATP concentration measurements, and various liver-specific functions such as urea synthesis and metabolism of 7-ethoxycoumarin were maintained both in fresh and cryopreserved/thawed hepatocytes. However, the activities were expressed at different levels in thawed cells. These data illustrate the importance and feasibility of human hepatocyte banking. In addition, it is clear that further refinements in the methods of hepatocyte isolation and cryopreservation are needed to utilize more fully these valuable cells in the clinic.

Journal Article↗
PubMed1998

Osteoblast-derived survival factors protect PC-3 human prostate cancer cells from adriamycin apoptosis.

OBJECTIVES: Hormone-independent and cytotoxic drug-resistant tumor growth in osteoblastic metastases defines poor survival in patients with advanced prostate cancer. Therefore, we analyzed the ability of human osteoblast-like cells (MG-63 cells) and MG-63 conditioned media (MG-63 CM) to protect PC-3 human prostate cancer cells from adriamycin cytotoxicity in vitro. METHODS: Adriamycin cytotoxicity was assessed in MG-63 osteoblast-like and PC-3 prostate cancer monolayer and three-dimensional collagen coculture systems using the DNA content and trypan blue exclusion assays, analysis of indexes of cell cycle by flow cytometry, determination of DNA fragmentation on simple agarose gel and terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay, and immunocytochemistry. RESULTS: Adriamycin (100 nM) arrested both the PC-3 and MG-63 cells at the G2/M phase in the cell cycle but induced apoptosis only in PC-3 cells, as assessed by flow cytometry, trypan blue exclusion, and agarose gel. Optimal doses of MG-63 CM (50 microg/mL), insulin-like growth factor I (50 ng/mL), and transforming growth factor-beta-1 (25 ng/mL), as determined by DNA content assay, partially neutralized the adriamycin cytotoxicity of PC-3 cells detected by flow cytometry and trypan blue exclusion. In addition, MG-63 cells rescued PC-3 cells from adriamycin apoptosis in the three-dimensional type I collagen gel coculture system, as analyzed by TUNEL assay. CONCLUSIONS: These data suggest that osteoblast-like cells and osteoblast-derived growth factors can optimize survival of metastatic prostate cancer cells, thereby helping to develop cytotoxic drug-resistant growth in vitro.

Biological Factors↗
PubMed1996

The effect of preservation temperature and period on resuscitation of the ischemically damaged canine pancreas during preservation by the two-layer (University of Wisconsin solution/perfluorochemical) method.

We have shown that 24 to 48 hour-preservation by the two-layer (University of Wisconsin solution (UW)/perfluorochemical (PFC)) method at 4 degrees C resuscitates a canine pancreas subjected to 90 min of warm ischemia. However, it is necessary to shorten preservation period for resuscitation of the ischemically damaged pancreas in a clinical simultaneous pancreas-kidney transplantation. The purpose of this study was to clarify the effect of preservation temperature and period on the resuscitation of the ischemically damaged pancreas during preservation by the two-layer method. First of all, we examined the possibility of resuscitation of the ischemically damaged pancreas during short-term preservation by the two-layer method. After 90 minutes of warm ischemia, canine pancreases were preserved by the two-layer method at 4, 20, or 37 degrees C. In control group, the pancreas graft was autotransplanted without preservation. Graft viability was judged by graft survival after autotransplantation. Pancreas grafts subjected to 90 min of warm ischemia were not viable (0/5). At 4 degrees C, 5 to 12 hr-preservation did not resuscitate the grafts (0/3 and 0/3 respectively). At 20 degrees C, 3 and 5 hr-preservation resuscitated the grafts (3/5 and 5/5 respectively), although 1 and 8 hr-preservation were not successful (0/3 and 0/3 respectively). At 37 degrees C, all the grafts were not resuscitated irrespective of preservation period. It was clear that the ischemically damaged pancreas was resuscitated during shot-term preservation by the two-layer method only at 20 degrees C. Secondly, we measured tissue adenine nucleotide levels by high performance liquid chromatography and pancreatic tissue perfusions using H2 clearance technique on reperfusion and examined the viability of vascular endothelium by nuclear trypan blue staining to make clear the necessary conditions for resuscitation of the ischemically damaged pancreas at 20 degrees C. ATP tissue levels in one hr-preserved grafts were 2.55 +/- 0.38 mumol/g dry weight and were significantly lower compared with the levels in 5 and 8 hr-preserved grafts, 9.40 +/- 2.09 (P < 0.01) and 7.37 +/- 1.06 mumol/g dry weight (P < 0.01) respectively. On the other hand, nuclear trypan blue uptakes of endothelial cells in 8 hr-preserved grafts were 37.6 +/- 11.6% and were significantly higher than 1 hr- and 5 hr-preserved grafts 5.6 +/- 4.5 (P < 0.01) and 5.0 +/- 3.0% (P < 0.01) respectively. As a consequence, pancreatic tissue perfusions in 8 hr-preserved grafts, 31.0 +/- 3.5 ml/min/g, were significantly lower than 1 hr- and 5 hr-preserved graft 72.0 +/- 11.6 (P < 0.01), 63.9 +/- 13.3 ml/min/g (P < 0.01) respectively. However, thromboxane A2 synthesis inhibitor (OKY046 0.1 mM/L) decreased the percentage of trypan blue uptake (8.2 +/- 3.6%) without interfering ATP synthesis (8.44 +/- 0.92 mumol/g dry weight) in 8 hr-preserved pancreas and tissue perfusions after reperfusion were dramatically improved (99.6 +/- 11.8 ml/min/g). As a result the ischemically damaged pancreas was resuscitated (4/5, 80%). It was suggested that 1 hr-preservation was not enough to synthesize ATP, which was essential to repair damaged cells, although vascular endothelial cells were maintained. Eight hr-preservation incurs endothelial cell damage although ATP tissue levels were maintained and consequently microcirculation was disturbed at reperfusion but OKY046 protects endothelial cells against preservation/reperfusion injury. As a consequence, the ischemically damaged pancreas was resuscitated. We conclude that short-term (3 to 8 hr) preservation at 20 degrees C by the two-layer method with OKY046 accelerates ATP synthesis, which is essential for repairing damaged cells and protects microvascular endothelial cells. This makes it possible to resuscitate the canine pancreas graft subjected to 90 min warm ischemia. This method holds promise for pancreas-kidney transplantation from cardiac arrest donors.

Animals↗
PubMed1995

Ascorbate-induced free radical toxicity to isolated islet cells.

Ascorbate is known to be cytotoxic by generating free oxygen radicals, a property shared with the diabetogenic drug alloxan. Some reports indicate that also ascorbate may be diabetogenic. In this study the cytotoxicity of ascorbate on isolated mouse islet cells has been investigated. Ascorbate (0.5-2.0 mmol/l) induced a concentration-dependent increase of trypan blue uptake by the cells, indicating an increase of membrane permeability to the dye. Trypan blue uptake induced by 2.0 mmol/l ascorbate was inhibited by concomitant incubation of the cells with 200 mg/l superoxide dismutase, 200 mg/l catalase, 3.0 mmol/l cytochrome-c or 50 mumol diethylenetriaminepentacetic acid (DTPA), but not by 50 mmol/l D-mannitol. The results indicate that ascorbate is cytotoxic to islet cells by metal-catalysed free radical generation.

Alloxan↗
PubMed1996

Radiation inactivation of human prostate cancer cells: the role of apoptosis.

Radiation-induced apoptosis detected by gel electrophoresis was measured in cells of three human prostate carcinoma cell lines (TSU, PC-3 and DU-145) and compared to their intrinsic radiosensitivities as measured by clonogenic assays. The intrinsic radiosensitivities of each cell line were defined by their alpha and beta coefficients and their surviving fraction at 2 Gy, derived from complete survival curves. The temporal expression and kinetics of radiation-induced apoptosis for DU-145 cells, the human prostate carcinoma cell line which expressed the highest rate of radiation-induced apoptosis, was characterized further by differential sedimentation and the immunofluorescence assay (Apoptag) which was specific for 3'-OH ends in cellular DNA. Cell viability was measured microscopically with trypan blue staining. Cell survival after various doses was computer-fitted to either a simple linear or a linear-quadratic equation. Twenty-four hours after a 10-Gy dose of 137Cs gamma rays, DNA fragmentation to nucleosome multimers was strongly expressed in only DU-145 cells. In this cell line, when centrifugation at 12,000g for 10 min was used to separate fragmented from large molecular weight DNA, the proportion of DNA in the supernatant increased to a maximum of approximately 17% of the total by 10-12 h after radiation treatment. Cell death 24 h after irradiation measured by trypan blue exclusion assays followed single-hit kinetics up to 80 Gy. The proportion of cells which were labeled with Apoptag displayed single-hit kinetics and yielded the same inactivation coefficient as measured by trypan blue. Together, these data indicate that the rapid (24 h) inactivation of irradiated DU-145 cells results from apoptosis and accounts for about 5% of the single-hit killing measured by clonogenic assay. Temporal studies of radiation-induced killing of DU-145 cells distinguished this rapid mechanism of cell death from the major mechanism (72-144 h). These may correlate with apoptosis and proliferative cell death, respectively. Of the three prostate cancer cell lines investigated, only DU-145 cells displayed significant levels of radiation-induced DNA fragmentation and rapid cell death, with characteristics of apoptosis. This mechanism of cell death was complete by 24 h after irradiation and was well separated in time from the death of cells by the major mechanisms which occurred after 72 h, and accounted for about 5% of cell inactivation by a single-hit mechanism.

Apoptosis↗
PubMed1988

Differentiation of phase I and variant strains of Bordetella pertussis on Congo red media.

The addition of Congo red, Trypan blue or haemin to the growth medium allowed the differentiation of phase-I and variant strains of Bordetella pertussis. Phase-I strains produced red (CR+), blue or dark brown colonies on a modified cyclodextrin solid medium containing Congo red, Trypan blue or haemin, respectively, whereas variant (Vir- and phase IV) strains grew as pale (CR-) colonies. Spontaneous CR- variants were isolated and characterised and had a phenotype like that of Vir- or phenotypically modulated, C-mode strains in that they did not produce the haemolysin, haemagglutinin(s), histamine-sensitising factor (pertussis toxin), heat-labile toxin and two major envelope polypeptides associated with phase-I strains. Two such variants had reduced virulence for mice. CR+ strains, when grown on a high nicotinic acid medium to induce modulation, gave CR- colonies. Thus the CR+ phenotype is a characteristic of phase-I B. pertussis and its expression appears to be controlled in a manner similar to that of other phase I-related factors. CR- variants of B. parapertussis and B. bronchiseptica were also deficient in these factors. Four isolates of B. avium were CR-.

Bordetella pertussis↗
PubMed1992

Interactions of 1,10-phenanthroline and its copper complex with Ehrlich cells.

Mechanistic details of the interaction of 1,10-phenanthroline and its copper complex with Ehrlich ascites tumor cells were examined, using inhibition of cell proliferation, DNA breakage, and increased membrane permeability as indices of cellular damage. The metal chelating agent, 1,10-phenanthroline (OP), the 1:0.5 complex of 1,10-phenanthroline and CuCl2 [(OP)2Cu], and CuCl2 inhibited growth of Ehrlich ascites tumor cell monolayers during 48-h treatments by 50% at about 3.5, 2, and 70 nmol/10(5) cells/mL, respectively. (OP)2Cu at 10 nmol/10(5) cells also enhanced uptake of trypan blue dye during 6 h of treatment, while dye uptake in OP- and CuCl2-treated cells remained similar to controls. DNA breakage, measured by DNA alkaline elution, was produced during 1-h treatments with (OP)2Cu at drug/cell ratios similar to those producing growth inhibition. Copper uptake was similar for both (OP)2Cu and CuCl2. Electron spin resonance (ESR) spectroscopy suggested that cellular ligands bind copper added as (OP)2Cu or CuCl2 and then undergo time-dependent reductions of Cu(II) to Cu(I) for both forms. Inhibition of (OP)2Cu-induced single-strand scission and trypan blue uptake by scavengers of activated oxygen is consistent with participation of superoxide and H2O2 in both processes. In contrast, superoxide dismutase (SOD) did not reduce the magnitude of the fraction of cellular DNA appearing in lysis fractions prior to alkaline elution of (OP)2Cu-treated cells. Dimethyl sulfoxide (DMSO) inhibited uptake of trypan blue dye but did not inhibit DNA strand scission produced by (OP)2Cu. Thus, multiple mechanisms for generation of oxidative damage occur in (OP)2Cu-treated cells. Growth inhibition produced by OP or (OP)2Cu, as well as the low levels of strand scission produced by OP, was not reversed by scavengers.

Animals↗
PubMed2001

Melanoma differentiation associated gene-7 (mda-7): a novel anti-tumor gene for cancer gene therapy.

BACKGROUND: The mda-7 gene (melanoma differentiation associated gene-7) is a novel tumor suppressor gene. The anti-proliferative activity of MDA-7 has been previously reported. In this report, we analyze the anti-tumor efficacy of Ad-mda7 in a broad spectrum of cancer lines. MATERIALS AND METHODS: Ad-mda7-transduced cancer or normal cell lines were assayed for cell proliferation (tritiated thymidine incorporation assay, Alamar blue assay, and trypan-blue exclusion assay), apoptosis (TUNEL, and Annexin V staining visualized by fluorescent microscopy or FACs analysis), and cell cycle regulation (Propidium Iodide staining and FACs analysis). RESULTS: Ad-mda7 treatment of tumor cells resulted in growth inhibition and apoptosis in a temporal and dose-dependent manner. The anti-tumor effects were independent of the genomic status of p53, RB, p16, ras, bax, and caspase 3 in these cells. In addition, normal cell lines did not show inhibition of proliferation or apoptotic response to Ad-mda7. Moreover, Ad-mda7-transduced cancer cells secreted a soluble form of MDA-7 protein. Thus, Ad-mda7 may represent a novel gene-therapeutic agent for the treatment of a variety of cancers. CONCLUSIONS: The potent and selective killing activity of Ad-mda7 in cancer cells but not in normal cells makes this vector a potential candidate for cancer gene therapy.

Adenoviridae↗
PubMed1989

Gossypol-induced free radical toxicity to isolated islet cells.

1. The cytotoxicity of the polyphenolic potential male antifertility agent gossypol was investigated on isolated mouse islets cells. 2. Gossypol shared many properties with the diabetogenic agent alloxan. 3. Gossypol (0.1-1.0 mmol/l) induced a concentration-dependent increase of Trypan Blue uptake by the cells, indicating an increase of membrane permeability to the dye. 4. Trypan Blue uptake induced by 0.5 mmol/l gossypol was inhibited by concomitant incubation of the cells with enzymatic (200 mg/l superoxide dismutase, 200 mg/l catalase, 3 mmol/l cytochrome-c), or low-molecular weight (50 mmol/l D-mannitol) scavengers of oxygen radicals, and the metal chelator diethylenetriaminepentacetic acid (DTPA) (50 mumol/l). 5. The results support the hypothesis that gossypol is B-cytotoxic by generation of noxious free radicals and that when proposing gossypol as a male antifertility agent, studies to exclude gossypol as a diabetogenic agent should first be performed in vivo.

Animals↗