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PubMed2003

Selective neuromicrovascular endothelial cell death by 8-Iso-prostaglandin F2alpha: possible role in ischemic brain injury.

BACKGROUND AND PURPOSE: Free radical-induced peroxidation is an important factor in the genesis of hypoxic-ischemic encephalopathy, including that of the preterm infant. Isoprostanes are major peroxidation products. Since microvascular dysfunction seems to contribute to ischemic encephalopathies, we studied the cytotoxicity of 8-iso-prostaglandin F2alpha (PGF2alpha) on cerebral microvascular cells. METHODS: Microvascular endothelial, astroglial, and smooth muscle cells from newborn brain were cultured. The cytotoxicity of 8-iso-PGF2alpha on these cells was determined by MTT assays and lactate dehydrogenase (LDH) release, propidium iodide incorporation, and DNA fragmentation (terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling [TUNEL]). In addition, effects of intraventricular injections of 8-iso-PGF2alpha and possible involvement of thromboxane in 8-iso-PGF2alpha-induced cytotoxicity were determined. RESULTS: 8-Iso-PGF2alpha induced time- and concentration-dependent endothelial cell death (EC50=0.1 nmol/L) but exerted little effect on smooth muscle and astroglial cells; endothelial cell death seemed mostly of oncotic nature (propidium iodide incorporation and LDH release). Cell death was associated with increased endothelial thromboxane A2 (TXA2) formation and was prevented by TXA2 synthase inhibitors (CGS12970 and U63557A); TXA2 mimetics U46619 and I-BOP also caused endothelial cell death. Intraventricular injection of 8-iso-PGF2alpha induced periventricular damage, which was attenuated by CGS12970 pretreatment. CONCLUSIONS: These data disclose a novel action of 8-iso-PGF2alpha involving TXA2 in oxidant stress-induced cerebral microvascular injury and brain damage.

Animals↗
PubMed2001

Ischemic and pharmacological preconditioning in Girardi cells and C2C12 myotubes induce mitochondrial uncoupling.

Pharmacological uncoupling of mitochondrial oxidation from phosphorylation promotes preconditioning-like cardioprotection in the isolated rat heart. We hypothesized that modest mitochondrial uncoupling may be a critical cellular event in orchestrating preconditioning. Human-derived Girardi cells and murine C2C12 skeletal myotubes were preconditioned using simulated ischemia, adenosine, and diazoxide. Cell viability after 6 hours of simulated ischemia was measured using lactate dehydrogenase release and propidium iodide uptake. Mitochondrial inner membrane potential (DeltaPsim) was investigated by flow cytometry, cellular ATP by recombinant firefly-luciferase bioluminescence, and cellular oxygen consumption using oximetry. Preconditioning enhanced cell viability with attenuation of lactate dehydrogenase release (>/=30%, P<0.05 versus ischemic controls) and a reduction in propidium iodide uptake by >/=26% versus ischemic controls after simulated ischemia in both cell lines. In Girardi cells, preconditioning induced the following phenotype immediately before index ischemia: (1) decreased DeltaPsim (JC-1: simulated ischemia 90+/-3%, adenosine 82+/-7%, diazoxide 87+/-4%, versus control 100%, P<0.05); (2) attenuation in cellular ATP levels (CTL 0.21+/-0.03 nmol/L ATP/microg protein, simulated ischemia 0.12+/-0.02, adenosine 0.15+/-0.02, diazoxide 0.11+/-0.02, P<0.05); and (3) enhanced cellular oxygen consumption (control 2.3+/-0.1 nmol/L oxygen/min/1x10(6) cells, simulated ischemia 3.1+/-0.1, adenosine 3.1+/-0.3, diazoxide 2.6+/-0.2, P<0.05). Cytoprotection, mitochondrial depolarization, and enhanced oxygen consumption were attenuated by the putative mitochondrial K(ATP)-channel antagonist 5-hydroxydecanoate. The uncoupled phenotype in response to preconditioning was similarly observed in C2C12 myotubes. The present study suggests that modest mitochondrial uncoupling represents a unifying cellular response which may be important in directing preconditioning-mediated cytoprotection.

Adenosine↗
PubMed1993

Mechanisms of hydroxyl free radical-induced cellular injury and calcium overloading in alveolar macrophages.

Excessive production of reactive oxygen radicals by alveolar macrophages is proposed to play an important role in oxidative lung injury. A major product oxygen radical formation is the highly reactive hydroxyl radical (.OH) generated via a biologic Fenton reaction. In addition to its known ability to induce lipid peroxidation, recent studies have suggested that the .OH may exert its cytotoxic effect through the alteration of [Ca2+]i homeostasis. To test this potential mechanism as well as to investigate the relationship between .OH and Ca2+ overloading in cytotoxic injury, isolated rat alveolar macrophages were exposed to externally generated radical system, H2O2 (0.01 to 1 mM) and Fe2+ (1 mM) and their [Ca2+]i levels and cell injury were monitored using quantitative fluorescence microscopy with the aid of the specific Ca2+ indicator, Fura-2, and membrane integrity indicator, propidium iodide. Electron spin resonance measurements using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO) confirmed the production of the .OH radical by this system. Upon the addition of the radicals, the macrophages displayed a rapid initial rise in [Ca2+]i which was followed by a slower but more pronounced [Ca2+]i elevation that reached a level 3 to 5 times higher than the basal level. This process preceded cell death as evident by nuclear propidium iodide fluorescence. Depletion of extracellular Ca2+ inhibited both the [Ca2+]i response and cell injury. Preincubation of the cells with the Ca2+ channel blocker verapamil or .OH radical scavenger mannitol similarly inhibited the [Ca2+]i rise and loss of viability. Firefly luciferase assay of cellular ATP content demonstrated that the alterations in [Ca2+]i following .OH treatment preceded the depletion of ATP.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1976

Rapid staining methods for analysis of deoxyribonucleic acid and protein in mammalian cells.

Quantitative fluorescent staining and analysis of cellular deoxyribonucleic acid (DNA) were accomplished using three groups of reagents having different mechanisms of action for DNA binding. These reagents included (a) the fluorescent antitumor antibiotics mithramycin, chromomycin A3 and olivomycin; (b) the Feulgen reagents acriflavine and flavophosphine N and (c) the intercalating dyes ethidium bromide and propidium iodide. Propidium iodide (PI) was used in combination with fluorescein isothiocyanate (FITC) to stain both cellular DNA and protein, respectively. Multiparameter analysis of PI/FITC-stained cells provided a direct correlation of DNA and protein for cells in all stages of the cell cycle. Nuclear-to-cytoplasmic ratio determinations were also performed on PI/FITC-stained cells by analysis of the time duration of the red (DNA) and green (protein) fluorescence signals from each cell. These staining and analysis techniques provide alternative methods for directly determining the quantitative relationship between cellular DNA and protein and will be extremely useful in investigations where fluctuations of these parameters are of importance for assessing experimental results.

Antibiotics, Antineoplastic↗
PubMed1979

Automatic cell identification and enrichment in lung cancer. II. Acridine orange for cell sorting of sputum.

Fluorescence spectra were obtained from cells from sputum and pleural effusions stained with different fluorescent dyes and fixed by alternate methods. The spectra were referenced to a standard allowing for fluorescence comparisons of unstained and stained cells under various conditions. The metachromasia of acridine orange-stained cells offers nuclear/cytoplasmic differentiation in a single stain; mithramycin and propidium iodide do not. Unstained cells have an appreciable amount of green (546 nm) fluorescence, as does Carbowax in Saccomanno's preservative. Cytoplasm stained with acidine orange also has appreciable green fluorescence. Consequently, cells with much cytoplasm have high total fluorescence. Cytoplasmic fluorescence is negligible with mithramycin or propidium iodide. The metachromasia of acridine orange-stained cells is altered by alcohol and Carbowax levels in fixatives, keeping other factors constant.

Acridine Orange↗
PubMed1979

Spectra of cells in flow cytometry using a vidicon detector.

A TV type vidicon detector was interfaced to a flow cytometer (FCM) to obtain spectra of fluorophores in cells during flow. The normal operations of the FCM are undisturbed. A spectrograph spreads 320 nm of the fluorophore fluorescence emission across the 500 channels of the detector. Spectra of fluorescamine (a surface labeling agent) and of propidium iodide (a nuclear stain) were obtained from Balb 3T3 cells, and the chlorophyll and phycobilin peaks were resolved from flowing blue-green algae in the FCM. Under typical flow conditions, operation of the vidicon in the continuous mode gives for these fluorophores a S/N of several hundred to one in approximately 3 sec. The vidicon was also gated to obtain spectra of single cells and of cells in selected portions of the cell cycle. For example, the spectrum of fluorescamine was obtained from cells in the G1 phase of the growth cycle by using as a gate trigger the FCM discriminator output derived from the propidium iodide signal.

Animals↗
PubMed1991

Quantification of chemotactic response of quiescent and proliferating fibroblasts in Boyden chambers by computer-assisted image analysis.

The chemotactic response of human gingival fibroblasts to platelet-derived growth factor (PDGF) was investigated in 48-well modified Boyden chambers. Results were quantified using computer-assisted image analysis of propidium iodidestained cells and were compared with results obtained using the conventional method of quantification by direct counting. Quantification by image analysis was found to be rapid and accurate, and correlated closely with results obtained by direct counting. Bromodeoxyuridine (BrdU)-labeled proliferating cells were double stained with propidium iodide and fluorescein isothiocyanate (FITC)-conjugated anti-BrdU antiserum. The cycling cells showed a markedly reduced chemotactic response to PDGF, whereas cells in S-phase of the cell cycle did not show any response. This method of quantification of results of Boyden chamber assays is simple and reliable, and allows the use of double labels to investigate the chemotactic response of subpopulations of cells within a heterogeneous population of cells.

Cell Cycle↗
PubMed2003

IGF-1/IGFBP-1 increases blastocyst formation and total blastocyst cell number in mouse embryo culture and facilitates the establishment of a stem-cell line.

BACKGROUND: Apoptosis occurs frequently for blastocysts cultured in vitro, where conditions are suboptimal to those found in the natural environment. Insulin-like growth factor-1 (IGF-1) plays an important role in preventing apoptosis in the early development of the embryo, as well as in the progressive regulation of organ development. We hypothesize that IGF-1 and its dephosphorylated binding protein (IGFBP-1) may be able to improve embryo culture with an associated reduced cell death, and that the resultant increase in the total cell number of the embryo could increase the chances of establishing an embryonic stem-cell line. RESULTS: In vivo fertilized zygotes were cultured in medium containing supplementary IGF-1, or IGFBP-1/IGF-1. The stages of the resultant embryos were evaluated at noon on day five post-hCG injection. The extent of apoptosis and necrosis was evaluated using Annexin V and propidium iodine staining under fluorescent microscopy. The establishment of embryonic stem-cell lines was performed using the hatching blastocysts that were cultured in the presence of IGF-1 or IGFBP-1/IGF-1. The results show that the rate of blastocyst formation in a tissue-culture system in the presence of IGF-1 was 88.7% and IGFBP-1/IGF-1 it was 94.6%, respectively, and that it was significantly greater than the figure for the control group (81.9%). IGFBP-1/IGF-1 also resulted in a higher hatching rate than was the case for the control group (68.8% vs. 48.6% respectively). IGF-1 also increased the number of Annexin V-free and propidium iodine-free blastocysts in culture (86.8% vs. 75.9% respectively). Total cell number of blastocyst in culture was increased by 18.9% for those examples cultured with dephosphorylated IGFBP-1/IGF-1. For subsequent stem-cell culture, the chances of the successful establishment of a stem-cell line was increased for the IGF-1 and IGFBP-1/IGF-1 groups (IGF-1 vs. IGFBP-1/IGF-1 vs. control: 45.8% vs. 59.6% vs. 27.3% respectively). CONCLUSION: IGF-1 or dephosphorylated IGFBP-1/IGF-1 supplement does result in an anti-apoptotic effect for early embryo development in culture, with a subsequent increased total cell number resulting from cell culture. The effect is beneficial for the later establishment of a stem-cell line.

Animals↗
PubMed1993

Growth and cellular proliferation of ovine corpora lutea throughout the estrous cycle.

This study was conducted to determine the rates of growth and cellular proliferation of ovine corpora lutea (CL) throughout the estrous cycle. To determine the cellular labeling index (LI), ewes received an iv injection of bromodeoxyuridine (BrdU) 1 h before death on days 2, 4, 8, 12, or 15 (day 0 = estrus; n = 6-12 ewes/day). At death, CL were weighed, and samples of each were fixed in Carnoy's solution or frozen until analyzed for DNA, protein, and progesterone contents. Nuclear incorporation of BrdU was determined in paraffin-embedded tissue sections by using a primary antibody against BrdU and a fluorescent (fluorescein isothiocyanate-labeled) secondary antibody, and sections were counterstained with propidium iodide (a nuclear stain). The labeling index (BrdU-labeled nuclei as a proportion of propidium iodide-labeled nuclei) of each CL was determined by using dual channel interactive laser cytometry and image analysis. Moreover, BrdU and 3 beta-hydroxysteroid dehydrogenase (a marker for steroidogenic cells) or BrdU and factor VIII (a marker for endothelial cells) were immunolocalized in tissue sections by using double immunohistochemical or dual immunofluorescent staining, respectively. Results demonstrated that cellular proliferation was greatest (LI, 34.1 +/- 2.1%) on day 2 and decreased (P < 0.01) through day 15 (LI, 0.7 +/- 0.1%) of the estrous cycle. The results of the immunohistochemical studies provide evidence that both parenchymal (steroidogenic) and nonparenchymal (e.g. endothelial, fibroblastic) luteal cells proliferated throughout the ovine estrous cycle. Conversely, from days 2-12 of the estrous cycle, fresh weight and DNA content of CL increased linearly (P < 0.01; 8- and 10-fold, respectively), then decreased (P < 0.02) from days 12-15. Ratios of protein/DNA on days 2, 4, and 8 were similar and were greater (P < 0.02) than those on days 12 and 15, which also were similar. These data demonstrate that growth of the ovine CL is extremely rapid, linear from days 2-12, and primarily due to hyperplasia. In addition, the high rate of cellular proliferation is associated primarily with nonsteroidogenic cells, a large proportion of which appear to be endothelial cells. Data such as these will enable us to determine the factors that are important in regulating luteal growth and development in normal and pathological conditions.

3-Hydroxysteroid Dehydrogenases↗
PubMed2006

Methods for determining the proliferation of cells in response to EGFR ligands.

The evaluation of cell proliferation can be accomplished by several methods. The number of cells can be determined directly by counting manually (e.g., hemocytometer) or automatically (e.g., Coulter counter or flow cytometer). The amount of DNA, which reflects the number of cells or the stage of the cell cycle, can be quantified by incorporation of labeled nucleotides (e.g., [3H]-thymidine) or nucleic acid stains (e.g., propidium iodide). Alternatively, the relative metabolic activity, which is correlative with the number of cells, can be determined through the use of metabolic dyes and measurement of the colored metabolites (e.g., MTT and MTS). Each assay has its advantages and limitations. Determining which assay to use will depend on the equipment available, the experimental design, and the questions being addressed. In this chapter we will describe methods for the use of a hemocytometer, [3H]-thymidine incorporation, cell cycle analysis with propidium iodide by flow cytometry, and evaluation of cellular metabolic activity with the MTS reagent.

Cell Adhesion↗
PubMed1999

Sequence of apoptosis and inflammatory necrosis within the formative ovulatory site of sheep follicles.

The aim of this study was to define the temporal and spatial patterns of apoptosis, necrosis and inflammation within preovulatory ovine follicles. A gonadotrophin surge was induced in pro-oestrous ewes by GnRH, and isolated follicles were hemisected into apical and basal segments at 0, 10, 18 and 22 h (the time of ovulatory stigma development) after GnRH. Ovarian surface epithelial and granulosa cells were isolated and assessed by fluorescence microscopy for membrane phosphatidylserine translocation-annexin V (early-stage apoptosis), oligonucleosomal DNA nick endlabelling (advanced apoptosis), and nuclear propidium iodide incorporation (necrotic membrane disruption). Thecal shells were analysed for interstitial blood cells. Preovulatory follicles were also hemisected and subjected to electrophoretic DNA degradation analysis. Annexin V binding and in situ DNA fragmentation among ovarian surface epithelial and granulosa cells along the follicular apex were high 18 and 22 h after GnRH. Propidium iodide staining of apical ovarian surface and granulosa cells was apparent at 22 h. There was a coincident increase within the apical theca as the time of ovulation approached in extravasated leucocytes (18 and 22 h) and erythrocytes (22 h). Apoptotic DNA laddering and necrotic DNA smears within the follicular apex were evident on agarose gels at 18 and 22 h, respectively. In contrast, ovarian surface epithelium not associated with the ovulation site and the basal follicular wall were largely unafflicted. It is suggested that both modalities of cellular death, apoptosis and necrosis (with acute inflammation and vascular injury), contribute progressively to follicular stigma formation and ovarian rupture.

Animals↗
PubMed2000

Endocytosis and transcytosis of an immunoliposome-based brain drug delivery system.

Immunoliposomes conjugated with the OX26 monoclonal antibody to the rat transferrin receptor can be used for brain delivery of small molecules. In the present study the uptake of OX26-immunoliposomes by target cells as well as their transcytosis across the blood-brain barrier was investigated. Microscopy of RG2 rat glioma cells incubated with fluorescence labeled OX26-immunoliposomes revealed intracellular co-localization of liposomal cargo, the liposomal membrane bilayer and the OX26 monoclonal antibody. The distinct particulate staining pattern was indicative for accumulation of OX26-immunoliposomes within endosomal or lysosomal compartments. Prolonged incubations demonstrated endosomal release of the liposomal cargo propidium iodide to the cytoplasm. A maximum of 50% of propidium iodide was released from the endosomal compartment after 24 hours of incubation. Transcytosis was studied using an in vitro model of the blood-brain barrier consisting of immortalized RBE4 rat brain endothelial cells. OX26-immunoliposomes did permeate across the RBE4 cell monolayer and showed a permeability coefficient of P(app) = 1.6 x 10(-5) ml/s. Transport was inhibited at low temperature, by competition with free OX26 or by exchanging the OX26 monoclonal antibody for an unspecific isotype antibody. Transcytosis of OX26-immunolipsomes was confirmed in vivo by the brain perfusion and capillary depletion technique. OX26-immunoliposomes were detected within the post-vascular compartment of brain parenchyma (PS product = 2.4 microl/g/min.) and were not associated with the brain microvasculature.

Animals↗
PubMed2001

Application of differential inflammatory cell count as a tool to monitor udder health.

A flow cytometric technique called differential inflammatory cell count was standardized by staining bovine peripheral blood leukocytes with a combination of DNA binding dyes SYBR green 1 and propidium iodide in water. Leukocytes were also stained with propidium iodide in detergent to determine total cell count. Differential inflammatory cell count assay was evaluated with individual quarter milk samples from 13 cows. Cows were sampled at weekly intervals for 3 wk and assayed for total cell count, mononuclear leukocyte count, and polymorphonuclear leukocyte count. Simultaneously, milk samples were evaluated by the conventional electronic somatic cell count (SCC) technique. Somatic cell count positively correlated with total cell count (r = 0.9), mononuclear leukocyte count (r = 0.8), and polymorphonuclear leukocyte count (r = 0.89). Quarters with SCC > log10 5.4 had a higher total cell count, mononuclear leukocyte count, and polymorphonuclear leukocyte count and were more often culture positive compared with quarters with SCC < log10 5.4. Quarters that were culture positive on all three test occasions had a higher proportion of polymorphonuclear leukocytes (33 to 49%) compared with quarters that were culture negative on all three test occasions (17 to 25%). The findings of this study suggest that differential inflammatory cell count assay has the potential to evolve as a new technique for evaluation of udder health status.

Animals↗
PubMed2006

Evaluation of the overall accuracy of the DeLaval cell counter for somatic cell counts in ovine milk.

The DeLaval cell counter (DCC) is a portable device designed for on-farm somatic cell count (SCC) analysis in bovine milk. This study evaluated the performance of the DCC when analyzing ovine milk. A total of 29 composite ovine milk samples, ranging between 20 x 10(3) and 2,200 x 10(3) cells/mL, were divided into 15 aliquots/milk sample corresponding to 5 SCC methods using 3 types of preservation (unpreserved, azidiol, and bronopol). The SCC methods were the Fossomatic (FSCC), the DCC in undiluted samples, and the DCC in samples diluted 1:1 in 3 different types of diluents (PBS + Triton X-100, PBS + ethidium bromide + Triton X-100, and PBS + propidium iodide + Triton X-100). All analyses were carried out in duplicate. In addition, each sample was analyzed in quadruplicate by the direct microscopic method (DMSCC) using Pyronin Y-methyl green as a stain. Comparison of methods was based on overall accuracy studies (means comparison, repeatability, and regression studies vs. DMSCC and FSCC as reference methods). The DCC methods used to analyze milk samples diluted in staining solution (with ethidium bromide or propidium iodide) showed large coefficients of regression (b = 0.91 to 1.01) and correlation (r > 0.99) when compared with the DMSCC and FSCC methods. In these samples the DCC gave repeatability values (s(r) = 33 to 48 x 10(3) cells/mL) similar to the DMSCC (s(r) = 36 x 10(3) cells/mL), and their log SCC means (5.52 to 5.54) did not differ from the reference value (5.54). However, undiluted samples analyzed by the DCC method showed large standard deviations of repeatability and SCC values lower than those by the DMSCC or FSCC methods, probably because of the high solids content in ovine milk. The type of preservation did not affect the outcomes. Consequently, the DCC was determined to be accurate when analyzing diluted ovine milk based on comparison with the SCC reference methods.

Animals↗
PubMed1994

DNA index and S-phase in primary brain tumors. A comparison between fresh and deparaffinized specimens studied by flow cytometry.

Forty-nine primary intracranial tumors (33 astrocytomas, eight acoustic schwannomas, five oligodendrogliomas, and three miscellaneous tumors) were studied by flow cytometry. Each tumor specimen was divided into two portions: one was studied as an unfixed suspension stained with propidium iodide and the other after formalin fixation and paraffin embedding. The 50-microns sections from the paraffin blocks were deparaffinized, rehydrated, and enzymatically disintegrated, and the cells in suspension were stained with propidium iodide. Flow cytometry of the two portions showed a significant correlation between fresh and fixed specimens regarding the S-phase (r = 0.87) and a very close correlation fo the deoxyribonucleic acid (DNA) index (r = 0.95). When the 33 astrocytomas were analyzed separately, similar results were obtained (r = 0.86 for S-phase and r = 0.93 for DNA index, respectively). This study demonstrated a high correlation between fresh and fixed specimens for DNA ploidy and S-phase both in primary intracranial tumors in general and also in the selected subgroup of astrocytomas.

Adolescent↗
PubMed2000

Method for flow cytometric monitoring of Renibacterium salmoninarum inactivation.

The slow growth of Renibacterium salmoninarum limits the usefulness of culture as a research tool. Development of a 2-color flow cytometric assay to quantify the proportions of live and dead R. salmoninarum in a test population is described. Bacteria were simultaneously stained with fluorescein isothiocyanate-conjugated immunoglobulin and exposed to the exclusion dye propidium iodide. Propidium iodide red fluorescence profiles of control groups of untreated and killed R. salmoninarum were compared with those for bacteria exposed to chlorine. Bacterial inactivation was based on mean red fluorescence intensity, and analyzed by high-red fluorescence intensity (HRFI) and curve subtraction (CS) analyses. When the concentration of R. salmoninarum was 8.65 x 10(6) bacteria ml(-1) and the bacteria exposed to chlorine at 1 mg l(-1) for periods from 1 to 20 min (high-Rs assessment), the mean red fluorescence intensity of the profile for each chlorine-exposure group was higher than that for the untreated control (p < 0.0001). When the concentration of R. salmoninarum was reduced to 1.76 x 10(6) bacteria ml(-1) and exposed to 0.8 mg l(-1) free chlorine level for periods from 20 s to 5 min (reduced-Rs assessment), the mean red fluorescence intensities of the exposure groups were higher than that for the untreated control only when the R. salmoninarum was exposed to chlorine for at least 1 min (p < or = 0.01). On the basis of red fluorescence intensity, the proportion of dead cells generally increased with the duration of chlorine exposure. Whereas the rates of inactivation derived from the HRFI and CS analyses did not correlate with the duration of exposure in the high-Rs assessment (r2 < or = 0.27), there was a correlation between these estimates and the duration of exposure in the reduced-Rs assessment (r2 > or = 0.92). Because of the rapid loss of culturable R. salmoninarum in both assessments following chlorine exposure, neither the duration of exposure nor the inactivation estimates correlated with bacteriological culture (r2 < or = 0.22). In both assessments, there was a correlation between the estimates of inactivation based upon HRFI and CS analyses (r2 > 0.99). These results suggest that flow cytometry can be used as a supplementary or alternative method to bacteriological culture for monitoring the inactivation of R. salmoninarum.

Bacteriological Techniques↗
PubMed2004

Red oil A5 inhibits proliferation and induces apoptosis in pancreatic cancer cells.

AIM: To study the effect of red oil A5 on pancreatic cancer cells and its possible mechanisms. METHODS: Effect of different concentrations of red oil A5 on proliferation of three pancreatic cancer cell lines, AsPC-1, MiaPaCa-2 and S2013, was measured by (3)H-methyl thymidine incorporation. Time-dependent effects of 1:32 000 red oil A5 on proliferation of three pancreatic cancer cell lines, were also measured by (3)H-methyl thymidine incorporation, and Time-course effects of 1:32 000 red oil A5 on cell number. The cells were counted by Z1-Coulter Counter. Flow-cytometric analysis of cellular DNA content in the control and red oil A5 treated AsPC-1, MiaPaCa-2 and S2013 cells, were stained with propidium iodide. TUNEL assay of red oil A5-induced pancreatic cancer cell apoptosis was performed. Western blotting of the cytochrome c protein in AsPC-1, MiaPaCa-2 and S2013 cells treated 24 hours with 1:32 000 red oil A5 was performed. Proteins in cytosolic fraction and in mitochondria fraction were extracted. Proteins extracted from each sample were electrophoresed on SDS-PAGE gels and then were transferred to nitrocellulose membranes. Cytochrome c was identified using a monoclonal cytochrome c antibody. Western blotting of the caspase-3 protein in AsPC-1, MiaPaCa-2 and S2013 cells treated with 1:32 000 red oil A5 for 24 hours was carried out. Proteins in whole cellular lysates were electrophoresed on SDS-PAGE gels and then transferred to nitrocellulose membranes. Caspase-3 was identified using a specific antibody. Western blotting of poly-ADP ribose polymerase (PARP) protein in AsPC-1, MiaPaCa-2 and S2013 cells treated with 1:32 000 red oil A5 for 24 hours was performed. Proteins in whole cellular lysates were separated by electrophoresis on SDS-PAGE gels and then transferred to nitrocellulose membranes. PARP was identified by using a monoclonal antibody. RESULTS: Red oil A5 caused dose- and time-dependent inhibition of pancreatic cancer cell proliferation. Propidium iodide DNA staining showed an increase of the sub-G0/G1 cell population. The DNA fragmentation induced by red oil A5 in these three cell lines was confirmed by the TUNEL assay. Furthermore, Western blotting analysis indicated that cytochrome c was released from mitochondria to cytosol during apoptosis, and caspase-3 was activated following red oil A5 treatment which was measured by procaspase-3 cleavage and PARP cleavage. CONCLUSION: These findings show that red oil A5 has potent anti-proliferative effects on human pancreatic cancer cells with induction of apoptosis in vitro.

Apoptosis↗
PubMed2005

Transfection of apoptosis related gene Fas ligand in human hepatocellular carcinoma cells and its significance in apoptosis.

AIM: To evaluate the expression of apoptosis related gene Fas ligand (FasL) in human hepatocellular carcinoma (HCC) cells HepG2 and its significance in apoptosis. METHODS: Levels of soluble Fas ligand (sFasL) in a group of patients with hepatitis B virus (HBV)-induced chronic hepatitis, HBV-positive liver cirrhosis and HCC were evaluated. In a further study, the recombinant eukaryotic expression plasmid pcDNA3.1hisB-FasL was transfected into HCC cells HepG2 by lipofection, and then soluble FasL was examined in the supernatant of culture cells by EIA, FasL expression in HepG2 cells was detected by immunohistochemistry. After being stained by annexin V and propidium iodine, cells were passed through a flow cytometer and examined by a fluorescence microscope and a laser scanning microscope. RESULTS: The sFasL levels were significantly lower in patients with HCC when compared to the patients with hepatitis or liver cirrhosis. In comparison with untransfected cells, the soluble FasL could be detected in the supernatant of transfected cells. FasL was expressed on the membranes and cytoplasm of transfected cells. The apoptotic cell rate was 36.30% in transfected cells, and was 11.53% in untransfected cells. Moreover, the different stage of apoptotic cells could be distinguished by annexin V and propidium iodine staining. CONCLUSION: Fas ligand is an apoptotic pathway of HCC cells.

Apoptosis↗