Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Propidium”

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 451 records · Page 25Linked to original sources
PubMed2000

Effect of saikosaponin, a triterpene saponin, on apoptosis in lymphocytes: association with c-myc, p53, and bcl-2 mRNA.

1. The mechanisms involved in the apoptotic effect of saikosaponin-d, a triterpene saponin from Bupleurum falcatum L., were studied in human CEM lymphocytes and compared with those of dexamethasone (3 x 10(-7) M). 2. Saikosaponin-d (10(-8) to 10(-5) M) inhibited the serum-stimulated [(3)H]-thymidine incorporation in a concentration-dependent manner. Dexamethasone also inhibited serum-stimulated [(3)H]-thymidine incorporation. 3. Cell viability was unaffected by saikosaponin-d until 10(-5) - 10(-4) M. Dexamethasone significantly reduced the number of viable cells. 4. Following saikosaponin-d (10(-5) - 10(-4) M) treatment, flow cytometry analysis of propidium iodide-stained cells showed a significant increase in the percentage of cells in the apoptotic region. Dexamethasone also significantly increased the percentage of apoptotic cells. The supravital exposure to propidium iodide and annexin V labelling demonstrated that saikosaponin-d (10(-5) - 10(-4) M) induced apoptosis as well as necrosis. 5. The apoptotic effect of saikosaponin-d (3 x 10(-6) - 10(-4) M) was also demonstrated by TUNEL analysis and DNA laddering. The percentage of apoptotic cells induced by saikosaponin-d (3 x 10(-6) - 10(-5) M) was unaffected by the presence of Z-VAD-FMK, indicating that saikosaponin-d-induced apoptosis may not be mediated by caspase activity. However, the percentage of apoptotic cells induced by dexamethasone was significantly reduced by the presence of Z-VAD-FMK. 6. Levels of c-myc, p53, and bcl-2 mRNA were analysed by the reverse transcription-polymerase chain reaction. Levels of c-myc and p53 mRNA were significantly increased, while the level of bcl-2 mRNA was decreased, by saikosaponin-d (10(-5) M) treatment. Dexamethasone did not significantly change the expression of these genes. 7. It is suggested that the apoptotic effect of saikosaponin-d may be partly mediated by increases in c-myc and p53 mRNA levels accompanied by a decrease in bcl-2 mRNA level.

Amino Acid Chloromethyl Ketones↗
PubMed2000

Use of flow cytometry to monitor cell damage and predict fermentation activity of dried yeasts.

Viable dried yeast is used as an inoculum for many fermentations in the baking and wine industries. The fermentative activity of yeast in bread dough or grape must is a critical parameter of process efficiency. Here, it is shown that fluorescent stains and flow cytometry can be used in concert to predict the abilities of populations of dried bakers' and wine yeasts to ferment after rehydration. Fluorescent dyes that stain cells only if they have damaged membrane potential (oxonol) or have increased membrane permeability (propidium iodide) were used to analyse, by flow cytometry, populations of rehydrated yeasts. A strong relationship (r2 = 0.99) was found between the percentages of populations staining with the oxonol and the degree of cell membrane damage as measured by the more traditional method of leakage of intracellular compounds. There were also were good negative relationships (r2 > or = 0.83) between fermentation by rehydrated bakers' or wine dry yeasts and percentage of populations staining with either oxonol or propidium iodide. Fluorescent staining with flow cytometry confirmed that factors such as vigour of dried yeast mixing in water, soaking before stirring, rehydration in water or fermentation medium and temperature of rehydration have profound effects on subsequent yeast vitality. These experiments indicate the potential of flow cytometry as a rapid means of predicting the fermentation performance of dried bakers' and wine yeasts.

Colony Count, Microbial↗
PubMed2002

The effect of LPS on neutrophils from patients with high risk of type 1 diabetes mellitus in relation to IL-8, IL-10 and IL-12 production and apoptosis in vitro.

Innate immunity includes neutrophil inflammatory function, tissue destruction and regulatory cytokine production. Programmed cell death (apoptosis) is postulated to be a key mechanism for neutrophil elimination during inflammation. The aim of the present study was to evaluate the neutrophil apoptosis in relation to IL-8, IL-10 and IL-12 production in vitro by neutrophils of patients suffering from diabetes mellitus (DM)1 and the first-degree relatives of patients with DM1. The early stage of neutrophils apoptosis was assessed morphologically, and the later stage by DNA-binding dye propidium iodide, both after treatment with lipopolysaccharide (LPS), insulin or anti-CD95 antibody (Ab) as stimulators. CD16 (FcgammaRIII) receptor expression was also evaluated. Production of IL-8, IL-10, and IL-12 cytokine was evaluated in supernatant after neutrophil incubation for 21 h in culture medium alone, in medium in the presence of LPS, insulin or anti-CD95 antibody (Ab). Cytokine concentrations were measured by enzyme-linked immunosorbent assay (ELISA) method using commercially available kits. Our study demonstrates that LPS inhibits the early stage of apoptosis (as evaluated morphologically) of healthy donors' neutrophils. The LPS-dependent early apoptosis inhibition of neutrophil of patients with DM1 or in prediabetics was decreased in comparison with control. The later stage of apoptosis of neutrophils treated in vitro with anti-CD95 Ab of patients suffering from DM1 was decreased in comparison with prediabetics and healthy donors (propidium iodide (PI) staining). LPS-induced production of anti-apoptotic cytokines IL-8, IL-10 by neutrophils of prediabetic and patients with DM1 was increased. The formyl-methionyl-leucyl-phenylalanine (fMLP)-induced proapoptotic reactive oxygen intermediates (ROI) production was significantly higher in DM1 patients. We have concluded that neutrophils from prediabetic and diabetic patients demonstrated the misbalance in anti-apoptotic IL-8 and IL-10 cytokine and proapoptotic ROI production. LPS-dependent IL-12 overproduction by neutrophils is responsible for the switch in T helper Th1/Th2 balance to Th1 and in this way may participate in inflammation and autoimmune DM1 progression.

Adolescent↗
PubMed1999

Acetylcholinesterase and inhibitors: effects upon normal and regenerating nerves of the rat.

In peripheral nerves, the function of acetylcholinesterase (AChE) is not related to hydrolysis of acetylcholine. To test for a trophic role, AChE or its inhibitors were administered locally to normal and regenerating nerves of rats. In the normal nerve, neither AChE nor serum albumin affected the cytological pattern of the nerve. BW284c51, a specific inhibitor of AChE, resulted in demyelination, proliferation of Schwann cells and sprouting of axons after 5-7 days. Edrophonium or propidium, other specific inhibitors of AChE, did so to a much lesser extent. Vehicle, and iso-OMPA (inhibitor of pseudocholinesterases) did not affect the cytology of the nerve. Elongation of regenerating axons was evaluated at day 3 post-crush. Native AChE applied distal to the crush reduced the elongation of regenerating axons (- 36%), while serum albumin, heated AChE and filtered AChE did not. BW284c51, edrophonium or propidium enhanced the axonal elongation (33%) when they were administered for 2 days before, but not after, the crush. Iso-OMPA or vehicle administered before or after the crush were not effective. Thus, AChE reduces elongation of regenerating axons, while inhibition of AChE enhances elongation and affects the cytology of the normal nerve as well. We propose that AChE has a trophic role in mammalian peripheral nerves.

Acetylcholinesterase↗
PubMed2002

Apoptosis in the cortex of the developing mouse kidney.

Published levels of apoptosis in developing rat kidney (approximately 2.5%) seem large for a tissue with no obvious need for continual cell death. This paper examines the levels and patterns of apoptosis and mitosis in the cortical region of the developing metanephros of the mouse, the standard mammalian model embryo. Using confocal microscopy on specimens stained with propidium iodide to highlight nuclear morphology, optical sections of wholemount kidneys to a depth of approximately 50 microm were analysed and mitotic, apoptotic and interphase nuclei counted in the various compartments. Of the approximately 200 000 cells examined over E11.5-16.5, 2-3% were mitotic, confirming observations based on cryosections; the mitotic index peaked at E14.5, dropping to approximately 0.5% by P14. The mean apoptotic index during this period was 0.28%; this figure from wholemounts was approximately 10% of that earlier reported in cryosectioned rat kidneys. One possible explanation for the difference is that cryosectioning turns out to create small nuclear fragments that can stain strongly with propidium. Such fragments are not seen in wholemounts and do not stain with TUNEL. Wholemount mouse E11.5 tails and E16.5 lungs were also analysed and both their mitotic and their apoptotic indexes were similar to those in wholemount developing kidneys. These results show that the level of apoptosis in wholemount embryonic mouse kidney cortex is far less than previously reported in cryosectioned rat embryonic kidneys, and typical of that in other mouse embryonic tissues whose development seems not to require apoptosis.

Animals↗
PubMed1998

Externalization of phosphatidylserine may not be an early signal of apoptosis in neuronal cells, but only the phosphatidylserine-displaying apoptotic cells are phagocytosed by microglia.

Earlier reports on nonneural cells have shown that the normally inner plasma membrane lipid, phosphatidylserine (PS), flip-flops out during the early stages of apoptosis, whereas DNA laddering and plasma membrane permeabilization occur during the late stages. In this study, the applicability of these parameters to CNS-derived neuronal cells was tested using hippocampal HN2-5, cells that undergo apoptosis under anoxia. Because such insults on unsynchronized cells, e.g., undifferentiated HN2-5 cells, result in both early and late apoptotic cells, we mechanically separated these cells into three fractions containing (a) cells that had completely detached during anoxia, (b) cells that remained weakly attached to the tissue culture dish and, once detached by trituration in serum-containing medium, did not reattach, and (c) cells that reattached in 2-3 h. Fractions a and b contained cells that showed pronounced DNA laddering, whereas cells in fraction c did not show any DNA laddering. Double staining with fluorescein isothiocyanate-annexin V (which binds to PS) and propidium iodide (which stains the DNA in cells with a permeable cell membrane) revealed that all cells in fraction a had a permeable cell membrane (propidium iodide-positive) and PS molecules in the outer leaflet of the plasma membrane (fluorescein isothiocyanate-annexin V-positive). By contrast, fractions b and c contained cells with no externalized PS molecules. Cells in fractions a-c also showed, respectively, 50-, 21-, and 5.5-fold higher caspase-3 (CPP32) activity than that in healthy control cells. All these results show that fraction a contained late apoptotic cells, which also had the highest CPP32 activity; cells in fraction b were at an intermediate stage, when DNA laddering had already occurred; and fraction c contained very early apoptotic cells, in which no DNA laddering had yet occurred. Therefore, in the neuronal HN2-5 cells, externalization of PS occurs only during the final stages of apoptosis when the cells have completely lost their adhesion properties. Further experiments showed that ameboid microglial cells isolated from neonatal mouse brain phagocytosed only the cells in fraction a. These results show that in CNS-derived HN2-5 cells, (a) PS externalization is a late apoptotic event and is concomitant with a complete loss of surface adhesion of the apoptotic cells and (b) PS externalization is crucial for microglial recognition and phagocytosis of the apoptotic HN2-5 cells. Thus, PS externalization could be causally linked to the final detachment of apoptotic neuronal cells, which in turn prepares them for rapid phagocytosis by microglia.

Amoeba↗
PubMed1999

Effect of recombinant human erythropoietin on endothelial cell apoptosis.

BACKGROUND: Recombinant human erythropoietin (rHuEPO) induces endothelial cell growth and angiogenesis in vitro. The mechanisms are unknown. Because an increase in endothelial cell survival could play a role in this process, we examined the effect of rHuEPO on lipopolysaccharide (LPS)-induced apoptosis in bovine pulmonary artery endothelial cells (BPAECs). METHODS: Four groups of cells were studied. The first group was preincubated in serum-free medium followed by treatment with LPS. The second group was preincubated with rHuEPO followed by LPS. The third group was treated with only rHuEPO. Control cells were cultured in the absence of rHuEPO and LPS. Apoptosis was determined by flow cytometric DNA analysis, propidium iodide staining, cellular DNA fragmentation by ELISA, and gel electrophoresis. RESULTS: LPS-treated cells showed an increase in hypodiploid DNA (36.4 +/- 6.1%) compared with controls (9.8 +/- 3.3%, P < 0.001). Preincubation with rHuEPO decreased this effect to 14.7 +/- 5.1% (P < 0.001). Apoptosis determined by propidium iodide was observed in 33 +/- 8% of LPS-treated cells, but in only 9 +/- 3% of cells preincubated with rHuEPO cells (P < 0.001). Similarly, DNA fragmentation was decreased in rHuEPO pretreated cells compared with LPS alone (0.155 OD +/- 0.02 vs. 0.538 +/- 0.09 OD, P < 0.001). DNA breakdown was observed in only LPS-treated cells. CONCLUSIONS: These results suggest that rHuEPO prevents LPS-induced apoptosis in endothelial cells. This protective effect could be an important factor in the action of rHuEPO on vascular endothelium.

Animals↗
PubMed1999

Palmitate induces structural alterations in nuclei of cardiomyocytes.

The objective of this study was to examine the hypothesis that the alterations of cardiac nuclei, that has been noted in some cardiomyopathies, can be produced by palmitate, a saturated fatty acid present in high circulating concentrations in patients with conditions associated with a high probability of developing cardiomyopathy. Cardiomyocytes isolated from embryonic chick ventricle were maintained in culture for 72 h and then treated with palmitate, 100 microM for 24 h. Cells were stained with acridine orange or Giemsa and examined microscopically. Cell size and nuclear size were examined by forward light scatter during flow cytometry. Cells were permeabilized and their nuclei were stained with propidium iodide and examined by flow cytometry on populations of 10,000 cells. Cardiomyocytes treated with palmitate displayed changes in nuclear appearance as nuclei were larger, relative to cell size, with more intense acridine orange staining in a peripheral location. Nucleoli were often disrupted. Palmitate produced a significant (P < 0.001) and 17% increase in nuclear size compared to untreated cells using flow cytometry analysing forward light scatter to estimate nuclear and whole cells size. There were no significant changes in the size of the whole cell and ratio of nucleus to whole cell was significantly (P < 0.01) increased compared to control cells. Fluorescent activating cell sorting analysis of propidium iodide stained nuclei demonstrated that the nuclear enlargement was not due to cell mitosis as the proportion of nuclei in Go/G1, S or M was not changed by palmitate. In summary, these studies identify that palmitate can induce structural abnormalities of cardiomyocytes nuclei by producing increased nuclear size and nucleolar destruction.

Animals↗
PubMed2002

Inhibitory effect of lidocaine on T cells from patients with allergic asthma.

BACKGROUND: Nebulized lidocaine enables dosages of oral corticosteroids to be tapered in the treatment of severe asthma. OBJECTIVE: We sought to investigate the effect of lidocaine on T cells from patients with allergic asthma. METHODS: PBMCs and CD4(+) T cells were isolated from 6 patients with asthma and house dust mite allergy. PBMCs were cultured with lidocaine for up to 7 days and then stained with propidium iodide to evaluate the involvement of apoptosis. In addition, the viability of CD4(+) T cells when cultured with lidocaine was investigated. Effects of lidocaine on proliferative response, mRNA expression, and protein production of IL-5 and IFN-gamma by PBMCs were investigated after stimulation with Dermatophagoides farinae, purified protein derivative, and phorbol 12-myristate 13-acetate plus calcium ionophore. The effects of lidocaine on the proliferative response of steroid-insensitive PBMCs from 6 nonallergic donors induced by preculture with IL-2 and IL-4 were also investigated. RESULTS: No significant increase in the staining of PBMCs with propidium iodide was observed in the presence of 100 micromol/L lidocaine. The viability of CD4(+) T cells was not significantly affected by culture with lidocaine at this concentration. However, lidocaine inhibited, in a dose-dependent manner, the proliferative response and mRNA expression and protein production of IL-5 and IFN-gamma of PBMCs stimulated with D farinae, purified protein derivatives, or phorbol 12-myristate 13-acetate plus calcium ionophore. Preincubation of PBMCs with IL-2 and IL-4 significantly decreased the inhibitory effects of both corticosteroids and lidocaine compared with that after preincubation with medium alone. CONCLUSION: Lidocaine has immunoregulatory effects on T cells. Therefore, lidocaine might be studied as an anti-inflammatory agent in the treatment of severe steroid-dependent asthma.

Allergens↗
PubMed2002

Endothelial cell apoptosis is induced by fetal plasma from pregnancy with umbilical placental vascular disease.

OBJECTIVE: Vascular disease in the umbilical placental circulation that is detected by umbilical artery Doppler study is associated with adverse fetal outcome. Endothelial cell activation and platelet consumption are features of this pathologic condition. We postulated that this was due to the local release of factors that cause endothelial cell injury and that these would spill into the fetal circulation. To test this hypothesis, we examined for the presence in fetal plasma of factors that induced endothelial cell apoptosis in pregnancies that were complicated by umbilical placental vascular disease. STUDY DESIGN: Isolated and cultured human umbilical vein endothelial cells were exposed to fetal plasma from the 3 fetal groups: normal pregnancy (n = 32 patients), pregnancy with umbilical placental vascular disease that was identified by an abnormal umbilical artery Doppler study (n = 38 patients), and pregnancy with maternal preeclampsia and normal umbilical artery Doppler study (n = 16 patients). Early apoptosis can be recognized by a loss of plasma membrane asymmetry with membrane uptake of annexin V. This was measured with annexin V and propidium iodide staining by fluorescent-activated cell scanning. Cells that underwent early apoptosis stained positive for annexin V and negative for propidium iodide (in contrast with cells that underwent necrosis). Cytosolic proteolytic activity was also measured. The lysates from endothelial cells that were stimulated by fetal plasma from umbilical placental vascular disease were tested for caspase-3 and caspase-8 activities by a fluorescent assay with spectrofluorophotometry. RESULTS: The percentage of endothelial cells that underwent apoptosis was significantly higher (P <.05) when stimulated with fetal plasma from pregnancies with umbilical placental vascular disease (17.71% +/- 1.31%) than with fetal plasma from normal pregnancies (9.76% +/- 0.87%). In the presence of maternal preeclampsia with normal umbilical artery Doppler study, the percent of apoptotic cells (11.31% +/- 1.59%) was similar to that of the normal group. In the group with abnormal umbilical artery Doppler study, there was no difference between pregnancies with preeclampsia (n = 17 pregnancies) and without preeclampsia (n = 21 pregnancies). The protease activity of caspase-3 was significantly enhanced in the group with umbilical placental vascular disease compared with normal pregnancy (0.79 +/- 0.06 vs 0.45 +/- 0.08 microMol/L). However, no difference in caspase-8 activity was detected (0.66 +/- 0.05 vs 0.56 +/- 0.05 microMol/L). CONCLUSION: Endothelial cell apoptosis is a feature of umbilical placental vascular disease. Our study demonstrates the presence of factors in the fetal plasma that caused endothelial cells to undergo early apoptosis. This increased apoptosis was only seen in the presence of placental vascular disease and was independent of the presence or absence of maternal preeclampsia. Our results indicate that programmed endothelial cell death occurs in the fetal circulation as a part of the injury that is associated with the development of umbilical placental vascular disease. The caspase-3, rather than caspase-8, signal transduction pathway appears to be involved in the mediation of endothelial cell apoptosis that was detected in our study.

Adult↗
PubMed2003

Apoptosis induced by environmental stresses and amphotericin B in Candida albicans.

New antifungal agents are urgently required to combat life-threatening infections caused by opportunistic fungal pathogens like Candida albicans. The manipulation of endogenous fungal programmed cell death responses could provide a basis for future therapies. Here we assess the physiology of death in C. albicans in response to environmental stresses (acetic acid and hydrogen peroxide) and an antifungal agent (amphotericin B). Exposure of C. albicans to 40-60 mM acetic acid, 5-10 mM hydrogen peroxide, or 4-8 microg.ml-1 amphotericin B produced cellular changes reminiscent of mammalian apoptosis. Nonviable cells that excluded propidium iodide displayed the apoptotic marker phosphatidylserine (as shown by annexin-V-FITC labeling), were terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive (indicating nuclease-mediated double-strand DNA breakage), and produced reactive oxygen species. Ultrastructural changes in apoptotic cells included chromatin condensation and margination, separation of the nuclear envelope, and nuclear fragmentation. C. albicans cells treated at higher doses of these compounds showed cellular changes characteristic of necrosis. Necrotic cells displayed reduced TUNEL staining, a lack of surface phosphatidylserine, limited reactive oxygen species production, and an inability to exclude propidium iodide. Necrotic cells lacked defined nuclei and showed extensive intracellular vacuolization. Apoptosis in C. albicans was associated with an accumulation of cells in the G2/M phase of the cell cycle, and under some apoptosis-inducing conditions, significant proportions of yeast cells switched to hyphal growth before dying. This is a demonstration of apoptosis in a medically important fungal pathogen.

Acetic Acid↗
PubMed1977

Transient inhibition of initiation of S-phase associated with dimethyl sulfoxide induction of murine erythroleukemia cells to erythroid differentiation.

The murine erythroleukemia cell (MELC) line in suspension culture can be induced to differentiate to erythroid cells by various compounds, including dimethyl sulfoxide (Me2SO). Analysis of the cell cycle, during differentiation induced by Me2SO, using thymidine incorporation, thymidine labeling index, and relative DNA content per cell as measured by flow microfluorometry, demonstrates a transient inhibition of entry of cells into S-phase of the cell cycle which is detected as early as 5 hr and is maximal about 20 hr after beginning of nonsynchronous cultures. Furthermore, in the presence of Me2SO there is restricted binding of the intercalating dye propidium iodide to chromatin from MELC in G1 phase of the cell cycle, as early as 10 hr of culture. This restricted binding of propidium iodide to chromatin is observed in MELC cultured with other inducing agents, such as butyric acid and dimethyl-acetamide, but is not detected with an Me2SO-resistant cell line cultured with Me2SO.

Cell Differentiation↗
PubMed1990

Gangliosides normalize distorted single-cell intracellular free Ca2+ dynamics after toxic doses of glutamate in cerebellar granule cells.

Glutamate-induced delayed neurotoxicity after abusive and paroxismal activation of its receptors has been proposed to depend upon a sustained increase in intracellular free Ca2+ [( Ca2+]i). To elucidate the temporal and causal relationship between glutamate-induced changes in [Ca2+]i and neuronal death, we simultaneously studied the dynamics of [Ca2+]i changes in single neurons with the acetoxymethyl ester of fura-2 and the cell viability by imaging the nuclear penetration of propidium iodide. The main difference between toxic (50 microM) and nontoxic (5 microM) doses of glutamate is the lack of regulation in [Ca2+]i 20 min after glutamate is removed. This protracted rise in [Ca2+]i in a single cell is correlated with (r = 0.87, P less than 0.01, Spearman's test), and consequently predictive of, the time of appearance of neuronal death, as measured by propidium iodide fluorescence. In addition, the glutamate receptor antagonists dibenzocyclohepteneimine (MK-801) and 3,3-(2-carboxypiperazine-4-yl)propyl 1-phosphate reduce the acute increase of [Ca2+]i induced by glutamate but fail to revert the protracted increase of [Ca2+]i, elicited by toxic doses of glutamate. In contrast, the ganglioside GM1 and the semisynthetic lysoGM1 with N-acetylsphingosine (LIGA-4) and lysoGM1 with N-dichloroacetylsphingosine (LIGA-20) failed to change the immediate rise of [Ca2+]i elicited by glutamate but prevented the protracted increase in [Ca2+]i after toxic doses of glutamate. Voltage-dependent Ca2+ channel blockers (nifedipine, etc.) did not change the initial or protracted responses to glutamate.

Animals↗
PubMed2002

Cell death and mechanoprotection by filamin a in connective tissues after challenge by applied tensile forces.

Cells in mechanically challenged environments must cope with high amplitude forces to maintain cell viability and tissue homeostasis. Currently, force-induced cell death and the identity of mechanoprotective factors are not defined. We examined death in cultured periodontal fibroblasts, connective tissue cells that are exposed to heavy applied forces in vivo. Static tensile forces (0.48 piconewtons/microm2 cell area) were applied through magnetite beads coated with collagen or bovine serum albumin. There was a time-dependent increase of the percentage of propidium iodide-permeable cells in force-loaded cultures incubated with collagen but not bovine serum albumin beads, indicating a role for integrins. Cells exhibited reduced mitochondrial membrane potential, increased caspase-3 activation, nuclear condensation, terminal deoxynucleotidyl transferase nick end labeling staining, and detachment from the culture dish. The caspase-3 inhibitor acetyl-Asp-Glu-Val-Asp-aldehyde reduced detachment 3-fold. There was a rapid (<10-s) decrease in plasma membrane potential after force application, which, in filamin A-deficient melanoma cells, contributed to irreversible cell depolarization. In fibroblast cultures, cells with increased permeability to propidium iodide exhibited approximately 2-fold less filamin A content than impermeable cells. Fibroblasts transfected with antisense filamin A constructs or with filamin A constructs without an actin-binding domain exhibited 2-3-fold increased proportions of dead cells relative to controls. We conclude that high amplitude forces delivered through integrins can promote apoptosis in a proportion of cells and that filamin A confers mechanoprotection by preventing membrane depolarization.

Actins↗
PubMed1996

RRR-alpha-tocopheryl succinate induces apoptosis in avian retrovirus-transformed lymphoid cells.

The RRR-alpha-tocopheryl succinate form of vitamin E [vitamin E succinate (VES)] inhibits the proliferation of avian reticuloendotheliosis virus-transformed RECC-UTC4-1 (C4-1) lymphoblastoid cells in a dose-dependent manner, blocks the cells in the G2/M cell cycle phase, and induces the cells to undergo apoptosis. Apoptosis was documented by demonstrating changes that are characteristic of this type of cell death, including morphological analyses of chromatin condensation by 4',6-diamidine-2'-phenylindole dihydrochloride (DAPI) staining using scanning confocal and traditional fluorescent microscopy; flow cytometry analyses of propidium iodide-labeled DNA showing fragmented DNA as a pre-G1 peak; two-color flow cytometry analyses of intact cells labeled first by the TUNEL procedure (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick-end-labeled DNA stained with fluorescein isothiocyanate-labeled avidin) and then by propidium iodide demonstrating fragmented DNA; and electrophoresis of DNA showing a DNA ladder created by internucleosomal DNA fragmentation. The percentage of apoptotic cells was determined by DAPI staining and showed 11%, 27%, and 49% of cells to be apoptotic after treatment with 10 micrograms/ml VES for one, two, and three days, respectively. Analyses of mRNA levels of genes that have been implicated in the apoptotic process, namely, bcl-2, c-myc, and c-jun, revealed no change in bcl-2, decreases in c-myc mRNA levels after 36 hours of treatment, and increases in c-jun mRNA levels within four hours after treatment. Western immunoblotting analyses of protein levels for the transcription factors c-Myc and c-Jun showed normal levels of c-Myc at early time points and decreased levels at 24 and 48 hours after treatment. c-Jun increased as early as 6 hours after treatment and returned to lower (yet still elevated over control) levels by 48 hours. To determine possible functional consequences of increased c-Jun expression, gel electrophoretic mobility assays were conducted that showed increased AP-1 binding at 24 and 48 hours after treatment. These data show that VES induces apoptosis in reticuloendotheliosis virus-transformed lymphoid cells and suggest that decreases of c-Myc protein and increases of c-Jun protein and DNA binding capacity may be playing a role in VES-mediated events leading to apoptosis in this cell type.

Animals↗
PubMed2005

Retinal toxicity of commercial tissue plasminogen activator is mediated by the induction of nitric oxide in the mouse retinal primary cells.

PURPOSE: Tissue plasminogen activator (tPA) is an efficient thrombolytic agent, but the dose-dependent retinal toxicity of intravitreal injection of commercial tPA (containing L-arginine) has been reported. Here, we sought to investigate the mechanism of tPA-induced cell death in mouse retinal cell cultures and the role of nitric oxide (NO). METHODS: Primary retinal cell cultures were maintained using glial conditioned medium (GCM) solution. Mouse retinal cell death was observed by using Hoechst-propidium iodide staining. Mouse retinal cell death was also measured by lactate dehydrogenase (LDH) assay. The formation of NO was measured using Griess reagent. RESULTS: tPA-induced cell death was detected in mouse retinal cell cultures by Hoechst-propidium iodide staining or LDH assay. L-arginine seems to be the major factor in retinal toxicity of commercial tPA (containing L-arginine). The formation of NO was markedly increased in mouse retinal cell cultures treated with tPA (containing L-arginine) or L-arginine. NO inhibitor reduced the cell death induced by commercially available tPA or L-arginine. CONCLUSIONS: This study suggests that l-arginine from commercial tPA (containing L-arginine) induces the majority of cell death in mouse retinal cell cultures and that its cytotoxicity may depend on the induction of NO.

Animals↗
PubMed2002

Prolongation of human neutrophil survival by low-level mercury via inhibition of spontaneous apoptosis.

Low levels of organic and inorganic mercury compounds have been reported previously to induce cell death by apoptosis in human peripheral blood mononuclear cells (MNC). but little is known about their potential effects on the viability and death of polymorphonuclear neutrophils (PMN). In contrast to MNC, PMN are known to undergo readily spontaneous apoptosis both in vivo and in vitro. Therefore, it was hypothesized that PMN may differ from MNC in their reactions to low mercury levels. The effects of methylmercuric chloride (MeHgCl) and mercuric chloride (HgCl2) were evaluated in concentration-response and time-course studies on human PMN viability and on their modes of cell death after in vitro incubation at 37 degrees C. Cell death by apoptosis or necrosis was assessed by annexin V-fluorescein isothiocyanate binding to externalized phosphatidylserine in conjunction with propidium iodide, and flow cytometry analysis. Morphologic counting of pyknotic nuclei and the fluorescence properties of the DNA-binding dye Hoechst 33342 in combination with propidium iodide were used to further confirm apoptotic cell death and to characterize the sequence of Hg-induced cell death. Results show that low concentrations of MeHgCl (1-7.5 microM) that were cytotoxic to MNC actually inhibited PMN spontaneous apoptosis. Low-level HgCl, reproduced the anti-apoptotic effects of MeHgCl on PMN, but to a lower extent. Higher concentrations of MeHgCl and HgCl2 were necrogenic to PMN, but MeHgCl was about an order of magnitude more toxic, and discrete differences were observed in the modalities of cell death induced by both species. These data reveal for the first time that (1) low levels of organic and inorganic mercury species protect human PMN from cell death via inhibition of spontaneous apoptosis, and (2) PMN are more resistant than MNC to mercury-induced cytotoxicity. Since delayed apoptosis and increased resistance to toxicant-induced cell death may lead to excessive accumulation of senescent PMN, evidence indicates that findings of this study may have implications for mercury-induced autoimmunity and inflammation.

Adult↗
PubMed2001

Application of an optical immersion-gel in a flow cytometer with horizontally oriented objective.

In certain flow cytometry systems, it is desirable to use immersion optics to obtain optimum fluorescence yield. This is important when propidium iodide and other DNA fluorochromes are used that have weaker fluorescence emission compared to DAPI, when a lamp is used instead of a laser and when the DNA concentrations are low. Our Partec PA II with a horizontally oriented objective and a vertically oriented flow chamber precludes using a liquid immersion medium. The problem was solved using an optical gel with appropriate characteristics. This gel is commercially available and commonly used for connecting glass fiber cables, but has never been used for microscopy before. Compared to the manufacturer's objective (40 x, aperture 0.8), the fluorescence yield was improved approximately four-fold using the optical gel and a 40 x glycerol objective (aperture 1.25). This innovation widens the applicability of flow cytometers with horizontally oriented objectives and vertical flow chambers. We expect it to facilitate the use of propidium iodide as a DNA stain, especially when interspecific genome size comparisons are to be done and base ratio dependent bias must be avoided.

Cell Nucleus↗