Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Laser Scanning Cytometry”

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 613 records · Page 34Linked to original sources
PubMed2006

Expression modification of uncoupling proteins and MnSOD in retinal endothelial cells and pericytes induced by high glucose: the role of reactive oxygen species in diabetic retinopathy.

Uncoupling proteins (UCPs) are mitochondrial transporters present in the inner membrane of mitochondria. They belong to the family of anion mitochondrial carriers. UCPs could act as proton carriers activated by metabolites and create a shunt between complexes of the respiratory chain and ATP synthase. The increased leakiness of the mitochondrial inner membrane to protons may be to minimize superoxide production by limiting the maximum Deltamu(H+). The purpose of this study was to detect UCP expression in retinal capillary cells and their modification in high levels of glucose. The role of reactive oxygen species (ROS) of mitochondria and UCPs in pathogenesis of diabetic retinopathy was investigated. Bovine retinal capillary endothelial cells and pericytes were cultured with selective culture media, respectively. Passage cells were cultured in three different glucose concentrations (5, 23, 30 mM) until passage four. ROS changes in mitochondria of these cells in different glucose concentrations were detected with scanning laser confocal microscopy (SLCM). The mitochondria membrane potential (Deltapsi), cell death rate and apoptosis rate were measured with flowing cytometry. UCP expression in retinal capillary cells was detected by immunocytochemistry. Expression and modification of MnSOD and uncoupling proteins (UCPs) in different concentrations of glucose were detected by means of semi-quantitative RT-PCR. ROS in mitochondria of both endothelial cells and pericytes increased as the glucose concentration of media increased. Deltapsi and cell death rate of endothelial cells increased also. ROS was correlated to Deltapsi and cell death rate positively in endothelial cells. No difference in Deltapsi and cell death rate among different glucose levels was found in pericytes. Apoptosis rate of endothelial cells and pericytes in high glucose levels was higher than that in lower glucose levels. UCP1 and UCP2 were expressed in cultured retinal capillary cells whereas UCP3 was not. At high levels of glucose, expression of UCP1, UCP2 and MnSOD increased to accommodate ROS production compensatively. The compensative mechanism disappeared when glucose concentration was too high (30 mM). The results of this study showed that increasing mitochondrial ROS could be induced by high glucose concentration. Those proteins related to antioxidation mechanism, such as MnSOD and UCPs, could exert compensative action to a certain extent. This compensative action was insufficient when the glucose concentration was too high.

Animals↗
PubMed1998

P-Glycoprotein (P-gp) mediated efflux in Caco-2 cell monolayers: the influence of culturing conditions and drug exposure on P-gp expression levels.

The influence of cell culture conditions and previous drug exposure on P-glycoprotein (P-gp) expression levels in Caco-2 cells was determined. In this study, the expression of P-gp is demonstrated (i) visually by confocal laser scanning microscopy (CLSM), (ii) functionally by transport studies with substrates of the efflux pump, and (iii) quantitatively by flow cytometry (FCM) analysis using specific monoclonal antibodies (anti P-gp MRK 16 as an external antibody and P-GlycoCheck C219 as an internal antibody). Trypsinization of the cells after reaching confluence led to a decrease of P-gp expression levels, while trypsinization before reaching confluence led to an increase after long-term cultivation. Culturing the cells on polycarbonate filters did not elicit a significant change of P-gp expression over time in culture, whereas in plastic flasks (polystyrene) a decrease was detected. Using CLSM a strong fluorescence on the apical side of Caco-2 cell monolayers was observed, as a result of incubation with MRK 16 as primary and IgG Cy5 as secondary antibody. Previous drug exposure of the cells showed that verapamil, celiprolol, and vinblastine induced the P-gp expression, while metkephamid (MKA) decreased the P-gp expression level as compared to the control. Permeation studies consolidated the theory that P-gp is expressed in the Caco-2 cells examined. For talinolol and MKA, a higher transport from basolateral to apical side than from apical to basolateral could be measured. Incubation of the cell monolayer with MRK 16 reduced the secretion process to the apical side, but did not influence [3H]mannitol flux. Caco-2 cells seem to be a suitable cell line model for P-gp-mediated secretion studies. However, the variability of the P-gp expression requires careful control when this model is to be used in quantitative structure/secretion studies.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2004

Effects of ox-LDL on number and activity of circulating endothelial progenitor cells.

BACKGROUND: Endothelial dysfunction is thought to play a crucial role in the pathogenesis of atherosclerosis induced by ox-LDL. Recently, a variety of evidence suggested that endothelial progenitor cells (EPCs) participated in neovascularization and reendothelialization. However, effects of ox-LDL on EPCs number and activity are ill understood. METHODS: Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with ox-LDL (to make a series of final concentrations: 25 microg/mL, 50 microg/mL, 100 microg/mL, 200 microg/mL), native LDL (100 microg/mL) or vehicle control for the respective time points (6 h, 12 h, 24 h and 48 h). EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPCs were further documented by demonstrating the expression of KDR, VEGFR-2 and AC133 with flow cytometry. Proliferation, migration and in vitro vasculogenesis activity of EPCs were assayed by MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. RESULTS: Incubation of isolated human EPCs with ox-LDL decreased the number of EPCs in concentration-dependent manner, maximum at 200 microg/mL (approximately 70% reduction, P < 0.001). In time-course experiments performed with an ox-LDL concentration of 100 microg/mL, decrease of EPCs number became apparent at 12 hours and reached the maximum at 24 hours (approximately 50% reduction, P < 0.01). In addition, ox-LDL dose and time dependently impaired EPC proliferative, migratory, adhesive and in vitro vasculogenesis capacity. CONCLUSION: The results of the present study defined a novel mechanism of action of ox-LDL: the reduction of EPCs with decreased functional activity.

Adult↗
PubMed2004

Effects of Ginkgo biloba extract on number and activity of endothelial progenitor cells from peripheral blood.

The aim of this study is to investigate whether Ginkgo biloba extract can augment endothelial progenitor cells numbers, and promote the cells' proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. Total mononuclear cells were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days culture, attached cells were stimulated with Ginkgo biloba extract (to make a series of final concentrations: 10 mg/L, 25 mg/L, and 50 mg/L) or vehicle control for the respective time points (6 hours, 12 hours, 24 hours, and 48 h). Endothelial progenitor cells were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. They were further documented by demonstrating the expression of KDR, VEGFR-2, and AC133 with flow cytometry. Endothelial progenitor cells proliferation, migration, and in vitro vasculogenesis activity were assayed with 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay, modified Boyden chamber assay, and in vitro vasculogenesis kit, respectively. Endothelial progenitor cells adhesion assay was performed by replating those on fibronectin-coated dishes, and then counting adherent cells. Incubation of isolated human mononuclear cells with Ginkgo biloba extract dose- and time-dependently increased the number of endothelial progenitor cells, maximum at 25 mg/L, 24 hours (approximately 1-fold increase, P < 0.01). In addition, Ginkgo biloba extract also dose- and time-dependently promoted endothelial progenitor cells proliferative, migratory, adhesive, and in vitro vasculogenesis capacity. The results of the present study defined a novel functional effect of Ginkgo biloba extract: the augmentation of endothelial progenitor cells with enhanced functional activity.

Blood↗
PubMed2004

Effects of nicotine on the number and activity of circulating endothelial progenitor cells.

Recently, some studies have shown that nicotine increased neovascularization, which involves endothelial progenitor cells (EPCs). The effects of nicotine on EPCs are still unclear at present. Therefore, the authors investigated whether nicotine had influences on EPC number and activity. The EPCs were stimulated with nicotine (to make a series of final concentrations: 10(-12) mol/L, 10(-10) mol/L, 10(-8) mol/L, 10(-6) mol/L, 10(-4) mol/L) or vehicle control for the respective time points(12, 18, 24, 32, and 48 hours). The EPCs were characterized as adherent cells double positive for DiLDL uptake and lectin binding by direct fluorescent staining under a laser-scanning confocal microscope. They were further documented by demonstrating the expression of KDR, VEGFR-2, and AC133 with flow cytometry. The EPC proliferation, migration, and in vitro vasculogenesis activity were assayed with the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay; the modified Boyden chamber assay; and the in vitro vasculogenesis kit, respectively. The EPC adhesion assay was performed by replating those on fibronectin-coated dishes and then counting the adherent cells. As a result, nicotine dose dependently increased the EPC number and the proliferative, migratory, adhesive, and in vitro vasculogenesis capacity at nicotine concentrations of 10(-12) to 10(-8) mol/L. The peak effects on EPCs were observed at concentrations of nicotine 10(-8) mol/L, similar to those in the blood of smokers. In addition, nicotine (10(-8) mol/L) time dependently increased the EPC number and activity. However, cytotoxicity was seen at higher nicotine concentrations (> 10(-6) mol/L). In conclusion, nicotine had complex effects on EPCs: nicotine might induce the augmentation of EPCs with enhanced functional activity at relatively low concentrations. However, cytotoxicity was seen at higher nicotine concentrations.

Cell Adhesion↗
PubMed2005

Effects of extracellular iron concentration on calcium absorption and relationship between Ca2+ and cell apoptosis in Caco-2 cells.

AIM: To determine the method of growing small intestinal epithelial cells in short-term primary culture and to investigate the effect of extracellular iron concentration ([Fe3+]) on calcium absorption and the relationship between the rising intracellular calcium concentration ([Ca2+]i) and cell apoptosis in human intestinal epithelial Caco-2 cells. METHODS: Primary culture was used for growing small intestinal epithelial cells. [Ca2+]i was detected by a confocal laser scanning microscope. The changes in [Ca2+]i were represented by fluorescence intensity (FI). The apoptosis was evaluated by flow cytometry. RESULTS: Isolation of epithelial cells and preservation of its three-dimensional integrity were achieved using the digestion technique of a mixture of collagenase XI and dispase I. Purification of the epithelial cells was facilitated by using a simple differential sedimentation method. The results showed that proliferation of normal gut epithelium in vitro was initially dependent upon the maintenance of structural integrity of the tissue. If 0.25% trypsin was used for digestion, the cells were severely damaged and very difficult to stick to the Petri dish for growing. The Fe3+ chelating agent desferrioxamine (100, 200 and 300 micromol/L) increased the FI of Caco-2 cells from 27.50+/-13.18 (control, n=150) to 35.71+/-13.99 (n=150, P<0.01), 72.19+/-35.40 (n=150, P<0.01) and 211.34+/-29.03 (n=150, P<0.01) in a concentration-dependent manner. There was a significant decrease in the FI of Caco-2 cells treated by ferric ammonium citrate (FAC, a Fe3+ donor; 10, 50 and 100 micromol/L). The FI value of Caco-2 cells treated by FAC was 185.85+/-33.77 (n=150, P<0.01), 122.73+/-58.47 (n=150, P<0.01), and 53.29+/-19.82 (n=150, P<0.01), respectively, suggesting that calcium absorption was influenced by [Fe3+]. Calcium ionophore A23187 (0.1, 1.0 and 10 micromol/L) increased the FI of Caco-2 cells from 40.45+/-13.95 (control, n=150) to 45.19+/-21.95 (n=150, P<0.01), 89.87+/-43.29 (n=150, P<0.01) and 104.64+/-51.07 (n=150, P<0.01) in a concentration-dependent manner. The positive apoptotic cell number of the Caco-2 cells after being treated with A23187 increased from 0.32% to 0.69%, 0.90% and 1.10%, indicating that the increase in the positive apoptotic cell number was positively correlated with [Ca2+]i. CONCLUSION: Ca2+ absorbability is increased with the decrease of extracellular iron concentration Fe3+ and hindered with the increase of Fe3+ consistence out of them. Furthermore, increase of [Ca2+]i can induce apoptosis in Caco-2 cells.

Apoptosis↗
PubMed2004

Statins contribute to enhancement of the number and the function of endothelial progenitor cells from peripheral blood.

The aim of the present study was to investigate whether fluvastatin augments the number of endothelial progenitor cells (EPCs), and promotes EPCs proliferation, migration and adhesion. Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation. The cells were then plated on fibronectin-coated culture dishes. After being cultured for 7 d, the attached cells were stimulated with fluvastatin (final concentrations: 0.01, 0.1, 1, 10 micromol/L), simvastatin (1 micromol/L) or a vehicle for the respective time points (6, 12, 24 and 48 h). EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPCs were further documented by demonstrating the expression of KDR, VEGFR-2 and AC133 with flow cytometry. EPCs proliferation, migration and in vitro vasculogenesis activity were assayed by MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating it on fibronectin-coated dishes, and the adherent cells were then counted. In addition, we also studied EPCs culture assay of peripheral blood from fluvastatin-treated animals in vivo. Incubation of isolated human MNCs with fluvastatin dose- and time-dependently increased the number of EPCs, while reached the maximum 24 h after the administration at 1 micromol/L, (2.5-fold increase, P<0.05). Moreover, treatment of rats with fluvastatins elevated the number of EPCs (3-fold increase, P<0.05), thus extending the in vitro data. In addition, fluvastatin also promoted EPC proliferation, migration, adhesion and in vitro vasculogenesis in a concentration-dependent manner. The effects of fluvastatin on EPCs were compared with those of simvastatin at the same concentration (1 micromol/L), with a result of no statistical difference. The results of the present study define a novel mechanism of the action of statins: the augmentation of EPCs with enhanced functional activity.

Cell Adhesion↗
PubMed2004

[Effects of Ginkgo biloba extract on number and activity of endothelial progenitor cells from peripheral blood].

AIM: To investigate whether Ginkgo biloba extract can augment endothelial progenitor cell (EPC) number, and promote EPC proliferation, migration and adhesion. METHODS: Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation, and then the cells were plated on fibronectin-coated culture dishes. After 7 days of culture, attached cells were stimulated with Ginkgo biloba extract (10, 25 and 50 mg x L(-1)) or vehicle control for the respective time points (6, 12, 24 and 48 h). EPC were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPC were further documented by demonstrating the expression of CD34, VEGFR-2 and AC133 with flow cytometry. EPC proliferation, migration and in vitro vasculogenesis activity were assayed with MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating MNCs on fibronectin-coated dishes, and then counting adherent cells. RESULTS: Incubation of isolated human MNCs with Ginkgo biloba extract increased the number of EPC, maximum at 25 mg x L(-1), 24 hours (approximately 1-fold increase, P < 0.01). In addition, Ginkgo biloba extract promotes EPC proliferative, migratory, adhesive and in vitro vasculogenesis capacity. CONCLUSION: Ginkgo biloba may promote EPC augmentation and enhance its functional activity.

Cell Adhesion↗
PubMed2005

[Effects of angiotensin II on peripheral blood endothelial progenitor cells].

OBJECTIVE: To investigate the effect of Angiotensin II (AngII) on the quantity proliferation, migration and adhesion of endothelial progenitor cells (EPCs). METHODS: Total mononuclear cells (MNCs) were isolated from peripheral blood by Ficoll density gradient centrifugation and then the cells were plated on fibronectin-coated culture dishes. After 7 days of culture, several groups of attached cells were incubated with AngII (to make a series of concentrations: 10(-3) mol/L, 10(-5) mol/L, 10(-7) mol/L, 10(-9) mol/L) or vehicle control for the respective time points (6, 12, 24 h and 48 h). In other groups of EPCs, valsartan (1 x 10(-5) mol/L) and AngII were added to the culture medium together.EPCs were characterized as adherent cells double positive for DiLDL-uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope. EPCs were further documented by demonstrating the expression of KDR, VEGFR-2 and AC133 with flow cytometry. EPCs proliferation, migration and in vitro vasculogenesis activity were assayed with MTT assay, modified Boyden chamber assay and in vitro vasculogenesis kit, respectively. EPCs adhesion assay was performed by replating MNCs on fibronectin-coated dishes. RESULTS: Incubation of isolated human MNCs with AngII increased the number of EPCs, with a maximum at 10(-3) mol/L after 24 hours (P < 0.01). In addition, AngII promotes EPCs proliferative, migratory, adhesive and in vitro vasculogenetic capacity. The effect of AngII was blocked by pretreatment of valsartan. CONCLUSIONS: It is suggested that angiotensin II may promote EPCs augmentation and enhance its functional activity through angiotensin receptor.

Angiotensin II↗
PubMed1993

[Prognostic relevance of cytometric methods for tumors of the urinary tract and prostate].

Cytometry means quantification of various cellular parameter using different instruments like computer based image analysers, flow cytometers or laser-scan microscopes. Using the more time consuming computer based image analysis, different parameters of possible prognostic validity for tumors of the urothelium or prostate may be quantified, like cellular structures, DNA-ploidy, chromosomal aberrations (interphase cytogenetics) AgNORs and a variety of functional antigens like proliferation markers, hormone receptors or oncogene products. The advantage of this method is that measurements can be performed on selected, morphologically classified cells. Using flow cytometers a representative number of tumor cells can be measured in a short time. With laser-scan microscopes oncogene amplification and chromosomal aberrations may be measured using fluorescence DNA in situ-hybridization. Of all cytometric parameters obtainable at present, only DNA ploidy is of clinical relevance in transitional cell carcinomas as it can predict the probability of tumor recurrence, -progression and of survival time with high accuracy. Similarly, at this time, only DNA measurements are of clinical relevance in prostatic cancer, as they can predict tumor progression, death from cancer, survival probability and sensitivity to hormonal treatment. No other grading parameter is of comparable prognostic significance in prostate cancer.

Chromosome Aberrations↗
PubMed2001

Quantitation and visualization of ultraviolet-induced DNA damage using specific antibodies: application to pigment cell biology.

The major types of DNA damage induced by sunlight in the skin are DNA photoproducts, such as cyclobutane pyrimidine dimers (CPDs), (6-4)photoproducts (6-4PPs) and Dewar isomers of 6-4PPs. A sensitive method for quantitating and visualizing each type of DNA photoproduct induced by biologically relevant doses of ultraviolet (UV) or sunlight is essential to characterize DNA photoproducts and their biological effects. We have established monoclonal antibodies specific for CPDs, 6-4PPs or Dewar isomers. Those antibodies allow one to quantitate photoproducts in DNA purified from cultured cells or from the skin epidermis using an enzyme-linked immunosorbent assay. One can also use those specific antibodies with in situ laser cytometry to visualize and measure DNA photoproducts in cultured cells or in the skin, using indirect immunofluorescence and a laser-scanning confocal microscope. This latter method allows us to reconstruct three-dimensional images of nuclei containing DNA photoproducts and to simultaneously examine DNA photoproducts and histology in multilayered epidermis. Using those techniques, one can determine the induction and repair of these three distinct types of DNA photoproducts in cultured cells and in the skin exposed to sublethal or suberythematous doses of UV or solar simulated radiation. As examples of the utility of these techniques and antibodies, we describe the DNA repair kinetics following irradiation of human cell nuclei and the photoprotective effect of melanin against DNA photoproducts in cultured pigmented cells and in human epidermis.

Animals↗
PubMed1999

Cellular penetration of fluorescently labeled superoxide dismutases of various origins.

BACKGROUND: Using fluorescently labeled superoxide dismutase (SOD) and flow cytometry, we have shown previously that the enzyme CuZn SOD (EC 1.15.1.1) from bovine erythrocytes binds rapidly to the cell surface with slow uptake into the cell during the following hours. The degree of labeling was most important for monocytes in comparison to other blood cells (erythrocytes, lymphocytes, and neutrophils) and fibroblasts. In agreement with the flow-cytometric findings, the inhibition of superoxide production was more important for SOD-pretreated monocytes than for neutrophils, as demonstrated with the cytochrome c reduction assay. It was thus of interest to confirm the observed differences between monocytes and neutrophils with confocal laser microscopy, study in greater detail the kinetics of binding, penetration, and intracellular localization of the enzyme, and compare the results obtained with bovine CuZn SOD with those from SODs of other origins and carrying different active sites. MATERIALS AND METHODS: Recombinant human (rh), bovine, and equine CuZn SODs, as well as rh and E. coli Mn SODs, were studied before use with respect to specific activity and purity (HPLC, SDS-PAGE electrophoresis). Fluorescein isothiocyanate was covalently conjugated to the various SODs for study with high-resolution confocal scanning laser microscopy. Superoxide production by monocytes and neutrophils was measured with the cytochrome c assay. RESULTS: As expected from our experiments with flow cytometry, only rare neutrophils were labeled with FITC-SOD, even with the longest incubation time of 3 hr and the highest dose of 1500 units/ml. In addition, they showed a localized fluorescence pattern that was quite different from the diffuse punctate fluorescence pattern of monocytes. Lymphocytes were not labeled at all. The rapid binding to the cellular surface of monocytes was confirmed, and even after 5 min of preincubation, FITC-SOD was found on a small percentage of monocytes. This was correlated with a reduction in superoxide release after phorbolmyristate acetate (PMA) stimulation by 40%. An interesting finding was the perinuclear accumulation of the penetrated SOD after the longest pretreatment of 3 hr, suggesting a barrier against further progression. Indeed, through confocal microscopy we were able to exclude any fluorescence at the nuclear level. While the fluorescence labeling patterns and the kinetics of penetration were quite similar for bovine, equine, and rh CuZn SOD, the Mn SODs showed poor labeling, correlated with a weak inhibitory effect on cytochrome c reduction, which was not statistically significant. CONCLUSIONS: The rapid binding of native CuZn SODs on the surface of monocytes, leading to reduced superoxide release by these cells, explains the observation that beneficial effects of injected SOD lasted for months despite rapid clearance of the enzyme from the bloodstream, according to pharmacodynamic studies. The preferential binding to monocytes, in contrast to neutrophils, may play a role in chronic inflammatory diseases in which the monocytes are in an activated state. The differences in binding capacity between CuZn SODs and Mn SODs, correlated with different inhibitory effects of superoxide production by monocytes, may also have therapeutic significance.

Animals↗
PubMed1994

Substance P and calcitonin gene-related peptide modulate leukocyte infiltration to mouse skin during allergic contact dermatitis.

The effects of the neuropeptides substance P (SP) and calcitonin gene-related peptide (CGRP) on leukocyte infiltration during allergic contact dermatitis (ACD) in mice were studied. Concomitant topical application of SP or CGRP with the allergen oxazolone resulted in enhanced leukocyte recruitment at the sites of challenge. Immunohistochemical studies revealed that the numbers of T-helper (L3T4+) and cytotoxic (Lyt-2) lymphocytes and infiltrating macrophages (BM8+) were increased. In addition, ICAM-1 and MHC class II molecule expression by these cells was enhanced after neuropeptide application. Analysis by confocal laser scanning microscopy revealed an increase in the immunoreactivity for SP and CGRP in nerve fibres during the course of ACD. Flow cytometry studies showed that SP and CGRP did not upregulate expression of the adhesion molecules ICAM-1 and VCAM-1 by murine endothelial cell lines in vitro. This suggests that increased infiltration of leukocytes during ACD is not a consequence of direct neuropeptide-promoted upregulation of endothelial adhesion molecules in vivo. In conclusion, our observations provide evidence for a modulatory role of neuropeptides in the pathogenesis of ACD.

Animals↗
PubMed2002

Protective effect of verbascoside on 1-methyl-4-phenylpyridinium ion-induced neurotoxicity in PC12 cells.

The neuroprotective effects of verbascoside, one of phenylpropanoid glucoside isolated from the Chinese herbal medicine Buddleja officinalis Maxim, on 1-methyl-4-phenylpyridinium ion (MPP(+)) induced apoptosis and oxidative stress in PC12 neuronal cells were investigated. Treatment of PC12 cells with MPP(+) for 48 h induced apoptotic death as determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry, the activation of caspase-3 measured by the caspase-3 activity assay kit, the reduction in mitochondrial membrane potential with laser scanning confocal microscopy and the increase in the extracellular hydrogen peroxide level. Simultaneous treatment with verbascoside markedly attenuated MPP(+)-induced apoptotic death, increased extracellular hydrogen peroxide level, the activation of caspase-3 and the collapse of mitochondrial membrane potential. These results strongly indicate that verbascoside may provide a useful therapeutic strategy for the treatment of oxidative stress-induced neurodegenerative disease such as Parkinson's disease.

1-Methyl-4-phenylpyridinium↗
PubMed2000

TNF-alpha modulates the differentiation induced by butyrate in the HT-29 human colon adenocarcinoma cell line.

The aim of this study was to determine whether and how tumour necrosis factor alpha (TNF-alpha) modulates butyrate effects. After the treatment of human colon adenocarcinoma HT-29 cells with sodium butyrate (NaBt), TNF-alpha or with their combinations we detected cell cycle (flow cytometry), cell proliferation (amidoblack and MTT assays), the amount of dead (floating) and apoptotic cells (flow cytometry and fluorescence microscopy), and the level of differentiation by alkaline phosphatase (ALP) activity (spectrophotometry), relative F-actin content (confocal laser scanning microscopy analysis) and E-cadherin expression (Western blot analysis). Both TNF-alpha and NaBt decreased cell growth in a dose-dependent manner. After combined treatment of the cells with both agents used, either none or additive effects were observed as compared with NaBt treatment alone. The level of dead and apoptotic cells was dose-dependently increased after this combined treatment. In contrast, TNF-alpha suppressed ALP activity and F-actin accumulation induced by NaBt. The results suggest that TNF-alpha does not influence significantly the antiproliferative effects of NaBt but, contrary to its potentiation of apoptosis, it markedly reduces NaBt-induced differentiation of HT-29 colon adenocarcinoma cells.

Alkaline Phosphatase↗
PubMedDate not supplied

Biological cells as templates for hollow microcapsules.

Microcapsules in the micrometer size range with walls of nanometer thickness are of both scientific and technological interest, since they can be employed as micro- and nano-containers. Liposomes represent one example, yet their general use is hampered due to limited stability and a low permeability for polar molecules. Microcapsules formed from polyelectrolytes offer some improvement, since they are permeable to small polar molecules and resistant to chemical and physical influences. Both types of closed films are, however, limited by their spherical shape which precludes producing capsules with anisotropic properties. Biological cells possess a wide variety of shapes and sizes, and, thus, using them as templates would allow the production of capsules with a wide range of morphologies. In the present study, human red blood cells (RBC) as well as Escherichia coli bacteria were used; these cells were fixed by glutardialdehyde prior to layer-by-layer (LbL) adsorption of polyelectrolytes. The growth of the layers was verified by electrophoresis and flow cytometry, with morphology investigated by atomic force and electron microscopy; the dissolution process of the biological template was followed by confocal laser scanning microscopy. The resulting microcapsules are exact copies of the biological template, exhibit elastic properties, and have permeabilities which can be controlled by experimental parameters; this method for microcapsule fabrication, thus, offers an important new approach for this area of biotechnology.

Biotechnology↗
PubMed2006

Effects of astragaloside IV on pathogenesis of metabolic syndrome in vitro.

AIM: To investigate the diverse pharmacological actions of astragaloside IV from the perspective of metabolic syndrome, and to investigate the effect of the drug on the pathogenesis of metabolic syndrome. METHODS: Adipogenesis was used as an indicator of the effect of astragaloside IV on preadipocyte differentiation, and was measured by using an oil red O assay. Glucose uptake was determined by measuring the transport of [2-(3)H]-deoxyglucose into the cells. The concentrations of peroxisome proliferator-activated receptor-gamma (PPARgamma) and aP2 mRNA were determined by using reverse transcription-polymerase chain reaction. Apoptosis and viability loss of endothelial cells were detected by using flow cytometry and the WST-1 assay, respectively. Intracellular free Ca2+ was labeled with Fluo-3 AM and measured by using a laser scanning confocal microscope. RESULTS: Astragaloside IV can significantly potentiate insulin-induced preadipocyte differentiation at concentrations of 3, 10, and 30 microg/mL, improve high glucose-induced insulin resistance in adipocytes at a concentration of 30 microg/mL, and prevent tumor necrosis factor (TNF)-alpha-induced apoptosis and viability loss at concentrations of 10 and 30 microg/mL, and 30 microg/mL, respectively, in endothelial cells. Furthermore, we found that these effects were partly due to the promotion of PPARgamma expression and to the inhibition of abnormal TNF-alpha-induced intracellular free Ca(2+) accumulation in endothelial cells. CONCLUSION: The diverse pharmacological actions of astragaloside IV can all be linked to metabolic syndrome pathogenesis. Our study provides a new insight into the mechanism by which astragaloside IV exerts its effect.

Adipocytes↗
PubMed2005

[Effect of glycoprotein alpha II bA2334C mutation on the biosynthesis and transportation of alpha II bbeta3 complex].

OBJECTIVE: To study the effect of glycoprotein (GP) alpha II bA2334C mutation on the biosynthesis and expression of alpha II bbeta3 complex. METHODS: The GP alpha II bA2334C eukaryotic expression plasmid pc3.1-2334M2b was constructed. Chinese hamster ovary (CHO) cells were transfected with the plasmid with or without integrin beta3 expression plasmid pc3.1-3a. The whole expression of alpha II bA2334C was confirmed by Western blot and the membrane expression was analyzed by flow cytometry. A newly constructed alpha II bA2334C GFP fusion protein expressing plasmid was used to determine its subcellular localization by laser confocal scanning microscopy. RESULTS: Expression of the mutant protein, alpha II bA2334C, in the transfected CHO cells was confirmed by Western blot with a lower rate of the mature type than the wild type control. The expression on membrane was only 25% of the normal. Subcellular localization analysis showed that alpha II bA2334C GFP was able to be expressed in CHO cells and could be transported from endoplasmic reticulum to Golgi apparatus. CONCLUSIONS: The mutant alpha II bA2334C can be synthesized in CHO cells and form alpha II bbeta3 complex. However, only a small fraction of the premature alpha II bA2334C can be transported to Golgi apparatus and transformed to mature alpha II b. The possible pathogenesis of this type II thrombasthenia may be that the misfolded alpha II bA2334C is partially degraded in the endoplasmic reticulum causing lower expression of alpha II bbeta3 complex on the membrane and resulting in impared function of platelets than normal alpha II b.

Animals↗