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Flow Cytometric Analysis (flow + cytometric_analysis)
Selected AbstractsDecreased expression of ,2 integrin in fibroblasts isolated from cyclosporin A- induced gingival overgrowth in ratsJOURNAL OF PERIODONTAL RESEARCH, Issue 5 2003Masatoshi Kataoka Objectives:, Cyclosporin A (CsA), an immunosuppressive agent, induces fibrous gingival overgrowth through reduction of collagen phagocytosis by fibroblasts. Distinct receptors are involved in the binding of collagen to fibroblasts in collagen phagocytosis, and ,2,1 integrin serves as a specific receptor for type I collagen on fibroblasts. To elucidate the role of ,2,1 integrin in CsA-induced gingival overgrowth, we investigated collagen phagocytosis and ,2,1 integrin expression in rat gingival fibroblasts. Materials and methods:, Fibroblats were isolated from gingiva of rats fed a powdered diet containing or lacking CsA for 30 d. Flow cytometric analysis were performed to measure the collagen phagocytosis and the ,2 integrin expression in fibroblasts. Furthermore, total RNAs were isolated from fibroblasts, and the reverse transcriptase-polymerase chain reaction was employed to investigate the mRNA levels of ,2 integrin. Results:,In vitro collagen phagocytosis assay revealed that CsA-treated and control fibroblasts contained a mean of 13.5% and 36.1% phagocytic cells, respectively. CsA-treated fibroblasts had 28% lower expression of ,2 integrin than that of control. and mRNA expression of ,2 integrin in CsA-treated fibroblasts was apparently lower than in the controls, but the mRNA expression of ,1 integrin was not affected. Conclusion:, These findings suggest that one etiological factor of gingival overgrowth may be inhibition of collagen phagocytosis by reducing ,2 integrin expression in gingival fibroblasts. [source] Expression of receptor activator of nuclear factor-,B ligand by B cells in response to oral bacteriaMOLECULAR ORAL MICROBIOLOGY, Issue 3 2009X. Han Introduction:, We investigated receptor activator of nuclear factor-,B ligand (RANKL) expression by B lymphocytes during early and late aspects of the immune response to Aggregatibacter actinomycetemcomitans, a gram-negative, anaerobic bacterium associated with aggressive periodontal disease. Methods:, Expression of messenger RNA transcripts (tumor necrosis factor-,, Toll-like receptors 4 and 9, interleukins 4 and 10, and RANKL) involved in early (1-day) and late (10-day) responses in cultured rat splenocytes was examined by reverse transcription,polymerase chain reaction (RT-PCR). The immune cell distribution (T, B, and natural killer cells and macrophages) in cultured rat splenocytes and RANKL expression in B cells were determined by flow cytometric analyses. B-cell capacity for induction of osteoclast differentiation was evaluated by coculture with RAW 264.7 cells followed by a tartrate-resistant acid phosphatase (TRAP) activity assay. Results:, The expression levels of interleukins 4 and 10 in cultured cells were not changed in the presence of A. actinomycetemcomitans until cultured for 3 days, and peaked after 7 days. After culture for 10 days, the percentages of B and T cells, the overall RANKL messenger RNA transcripts, and the percentage of RANKL-expressing immunoglobulin G-positive cells were significantly increased in the presence of A. actinomycetemcomitans. These increases were considerably greater in cells isolated from A. actinomycetemcomitans -immunized animals than from non-immunized animals. RAW 264.7 cells demonstrated significantly increased TRAP activity when cocultured with B cells from A. actinomycetemcomitans -immunized animals. The addition of human osteoprotegerin-Fc to the culture significantly diminished such increases. Conclusion:, This study suggests that B-lymphocyte involvement in the immune response to A. actinomycetemcomitans through upregulation of RANKL expression potentially contribute to bone resorption in periodontal disease. [source] Comparison between propidium iodide and 7-amino-actinomycin-D for viability assessment during flow cytometric analyses of the human sperm acrosomeANDROLOGIA, Issue 1 2010N. Falzone Summary Evaluation of the acrosome reaction can shed light on the fertilising competence of spermatozoa. To eliminate false-positive results when evaluating the acrosome status of human sperm cells, two viability probes propidium iodide (PI) and 7-amino-actinomycin D (7-AAD) were compared for their ability to stain nonviable cells post-fixation and permeabilisation. Both the mean fluorescence and % dead cells differed significantly with time (P < 0.0001). Unlike PI, 7-AAD did not leach from cells and fluorescence remained stable for up to 4 h. Furthermore, 7-AAD proved to be a proficient marker to exclude dead sperm cells during flow cytometric evaluation of ionophore-induced acrosome reaction. [source] Acquired but reversible loss of erythrocyte complement receptor 1 (CR1, CD35) and its longitudinal alteration in patients with severe acute respiratory syndromeCLINICAL & EXPERIMENTAL IMMUNOLOGY, Issue 1 2005F. S. Wang Summary This longitudinal study investigates the change of erythrocyte complement receptor (E-CR1) expression in patients with severe acute respiratory syndrome (SARS). Circulating E-CR1 expression was semiquantified by flow cytometric analyses in 54 SARS patients and in 212 healthy individuals as a control. Since E-CR1 expression is influenced by the genetic polymorphisms in the CR1 gene, a major genetic polymorphism located within intron 27 of the CR1 gene was simultaneously analysed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). The results showed that the expression level of E-CR1 (referred to as net fluorescence intensity values, NFI) was statistically correlated with the relevant genetic genotypes among the Chinese population including the healthy individuals (NFI: 5·14 ± 0·82, 3·57 ± 0·66 and 2·67 ± 0·32 for HH, HL and LL genotypes, respectively) and SARS patients (NFI: 3·52 ± 0·91 and 2·63 ± 0·70 for HH and HL genotypes, respectively). Interestingly, the expression density of E-CR1 was found to fall significantly during the initiation and progressive phases (weeks 1 and 2 after the disease onset) and gradually returned close to normal through their whole convalescent phase (beginning from weeks 2 or 3 to weeks 7 or 8) in SARS patients irrespective CR1 genotype. In conclusion, our findings, at least, suggest that E-CR1 is likely involved in immune pathogenesis of SARS disease. [source] Perinatal development of the rat kidney: Apoptosis and epidermal growth factorCONGENITAL ANOMALIES, Issue 3 2003Toshiya Okada ABSTRACT, Localization of apoptotic cells in the kidney of perinatal rats was examined by the terminal deoxynucleotidyl transferase,mediated d,UTP,biotin nick end labeling (TUNEL) method and electron microscopy. Perinatal changes in the percentage of kidney cells with DNA fragmentation were determined by flow cytometric analysis. Through observation of two successive sections, the relationship between the localization of the epidermal growth factor receptor (EGFR) positive cells and TUNEL positive cells in the kidney was determined. From fetal day 18 to neonatal day 5, TUNEL positive cells were noted in immature glomeruli, collecting ducts and interstitium. Electron microscopically, chromatin condensed nuclei and apoptotic bodies were seen in the same tissue component as the TUNEL positive cells. The percentage of DNA fragmented cells significantly increased from fetal days 18 to 20 and significantly decreased from fetal days 20 to 22, while they still remained low in the neonatal period. The TUNEL positive cells in immature glomeruli and collecting ducts were not reactive to the EGFR antibody. The TUNEL positive cells were not observed in the proximal tubular cells, which were positive to EGFR antibody. These results indicate that apoptotic cells are present in the kidney throughout the perinatal period in the rat and that EGF plays an important role in perinatal development of the rat kidney. [source] Novel application of flow cytometry: Determination of muscle fiber types and protein levels in whole murine skeletal muscles and heartCYTOSKELETON, Issue 12 2007Connie Jackaman Abstract Conventional methods for measuring proteins within muscle samples such as immunohistochemistry and western blot analysis can be time consuming, labor intensive and subject to sampling errors. We have developed flow cytometry techniques to detect proteins in whole murine heart and skeletal muscle. Flow cytometry and immunohistochemistry were performed on quadriceps and soleus muscles from male C57BL/6J, BALB/c, CBA and mdx mice. Proteins including actins, myosins, tropomyosin and ,-actinin were detected via single staining flow cytometric analysis. This correlated with immunohistochemistry using the same antibodies. Muscle fiber types could be determined by dual labeled flow cytometry for skeletal muscle actin and different myosins. This showed similar results to immunohistochemistry for I, IIA and IIB myosins. Flow cytometry of heart samples from C57BL/6J and BALB/c mice dual labeled with cardiac and skeletal muscle actin antibodies demonstrated the known increase in skeletal actin protein in BALB/c hearts. The membrane-associated proteins ,-sarcoglycan and dystrophin could be detected in C57BL/6J mice, but were decreased or absent in mdx mice. With the ability to label whole muscle samples simultaneously with multiple antibodies, flow cytometry may have advantages over conventional methods for certain applications, including assessing the efficacy of potential therapies for muscle diseases. Cell Motil. Cytoskeleton 2007. © 2007 Wiley-Liss, Inc. [source] Interaction of the alpha-helical H6 peptide from the pro-apoptotic protein tBid with cardiolipinFEBS JOURNAL, Issue 21 2009Patrice X. Petit BH3 interacting domain death agonist (Bid), a pro-apoptotic member of the Bcl-2 family of proteins, is activated through cleavage by caspase-8. The active C-terminal fragment of Bid (tBid) translocates to the mitochondria where it interacts with cardiolipins at contact sites and induces the release of cytochrome c by a mechanism that is not yet fully understood. It has been shown that the alpha-helices ,H6 and ,H7 (which create the hairpin-forming domain of tBid) mediate the insertion of Bid into mitochondrial membranes and are essential for the cytochrome c -releasing activity. In the present study, we focused on the interaction between the ,H6 and the mitochondrial membrane. By the use of single-cell electropermeabilization associated with flow cytometric analysis of intact cells, we demonstrated that H6 is able to induce cell death when used in the micromolar range. We also studied the interactions of the ,H6 with artificial monolayers. We showed that the presence of negatively charged cardiolipins greatly enhances the insertion of ,H6 into the phospholipid monolayer. The modification of two charged amino acid residues in ,H6 abolished its insertion and also its in vivo effects. Furthermore, the negative values of the excess areas of mixing indicate that attractive interactions between cardiolipins and ,H6 occur in the mixed monolayers. Fluorescence microscopy observations revealed that ,H6 significantly disrupts cardiolipin packing and stabilizes the fluid lipid phase. These results suggest that cardiolipins at the contact sites between the two mitochondrial membranes could mediate the binding of tBid via ,H6. [source] Internalization of tenecin 3 by a fungal cellular process is essential for its fungicidal effect on Candida albicansFEBS JOURNAL, Issue 16 2001Dae-Hee Kim Tenecin 3 is a glycine-rich, antifungal protein of 78 residues isolated from the insect Tenebrio molitor larva. As an initial step towards understanding the antifungal mechanism of tenecin 3, we examined how this protein interacts with the pathogenic fungus Candida albicans to exert its antifungal action. Tenecin 3 did not induce the release of a fluorescent dye trapped in the artificial membrane vesicles and it did not perturb the membrane potential of C. albicans by the initial interaction. Fluorescence confocal microscopy and flow cytometric analysis revealed that tenecin 3 is rapidly internalized into the cytoplasmic space in energy-dependent and temperature-dependent manners. This internalization is also dependent on the ionic environment and cellular metabolic states. These results suggest that the internalization of tenecin 3 into the cytoplasm of C. albicans is mediated by a fungal cellular process. The internalized tenecin 3 is dispersed in the cytoplasm, and the loss of cell viability occurs after this internalization. [source] The surface-associated elongation factor Tu is concealed for antibody binding on viable pneumococci and meningococciFEMS IMMUNOLOGY & MEDICAL MICROBIOLOGY, Issue 2 2008Jan Kolberg Abstract Proteome analyses revealed that elongation factor-Tu (EF-Tu) is associated with cytoplasmic membranes of Gram-positive bacteria and outer membranes of Gram-negative bacteria. It is still debatable whether EF-Tu is located on the external side or the internal side of the membranes. Here, we have generated two new monoclonal antibodies (mAbs) and polyclonal rabbit antibodies against pneumococcal EF-Tu. These antibodies were used to investigate the amount of surface-exposed EF-Tu on viable bacteria using a flow cytometric analysis. The control antibodies recognizing the pneumococcal surface protein A and phosphorylcholine showed a significant binding to viable pneumococci. In contrast, anti-EF-Tu antibodies did not recognize pneumococcal EF-Tu. However, heat killing of pneumococci lacking capsular polysaccharides resulted in specific antibody binding to EF-Tu and, moreover, increased the exposure of recognized phosphorylcholine epitopes. Similarly, our EF-Tu-specific antibodies did not recognize EF-Tu of viable Neisseria meningitidis. However, pretreatment of meningococci with ethanol resulted in specific antibody binding to EF-Tu on outer membranes. Importantly, these treatments did not destroy the membrane integrity as analysed with control mAbs directed against cytoplasmic proteins. In conclusion, our flow cytrometric assays emphasize the importance of using viable bacteria and not heat-killed or ethanol-treated bacteria for surface-localization experiments of proteins, because these treatments modulate the cytoplasmic and outer membranes of bacteria and the binding results may not reflect the situation under physiological conditions. [source] Near-diploid and near-triploid human sporadic colorectal adenocarcinomas differ for KRAS2 and TP53 mutational statusGENES, CHROMOSOMES AND CANCER, Issue 2 2003Walter Giaretti Mutations of the KRAS2 protoncogene and inactivation of the TP53 oncosuppressor gene have been suggested to contribute to chromosomal instability (CIN) and aneuploidy in colorectal cancer (CRC). Previous work has also shown that the degree of DNA ploidy [DNA index (DI)], as obtained by flow cytometry in CRC, is non-randomly distributed and, in particular, that DI near-diploid and near-triploid values are well separated by a low-probability valley region. At present, it is not known whether a relationship exists between DI and the mutational status of KRAS2 and TP53. Multiple samples obtained from 35 human sporadic CRCs have been used to provide nuclei suspensions for flow cytometric analysis and sorting of specific DI subpopulations. Sorted nuclei were then used to analyze the high-microsatellite-instability (MSI-H) phenotype and the mutation spectrum of the KRAS2 and TP53 genes. A single MSI-H case was detected. There were 6 DNA diploid (DI = 1) and 29 aneuploid (DI , 1) CRCs, with the DI aneuploid cases non-randomly subdivided in 9 near-diploid (DI , 1 and DI , 1.4), 8 near-triploid (1.4 < DI < 1.6), and 12 high-aneuploid (DI , 1.6) cases. Proximal CRCs were more often DNA diploid and near-diploid than distal ones, and Dukes' C cases were more commonly high-aneuploid than Dukes' B. Moreover, the incidence of mutations of the KRAS2 and TP53 genes was lowest among the DNA near-triploid subpopulations and highest among the near-diploid ones. We suggest that DNA near-diploid and near-triploid subpopulations in human sporadic CRC reflect different genetic mechanisms of CIN and have a potentially different clinical behavior. © 2003 Wiley-Liss, Inc. [source] Can flow cytometrically determined DNA ploidy and S-phase fraction predict regional metastasis in squamous cell carcinoma of the oral cavity?HEAD & NECK: JOURNAL FOR THE SCIENCES & SPECIALTIES OF THE HEAD AND NECK, Issue 2 2002Ryoichi Oya DDS Abstract Background The value of flow cytometric analysis of DNA ploidy and S-phase fraction (SPF) as an indicator of regional metastasis in oral cancer is currently being debated. Intratumoral heterogeneity makes this problem complex. Methods Intratumoral DNA ploidy heterogeneity and intratumoral SPF variation were examined using multiple specimens from 31 surgically resected specimens taken from patients with oral cancer without preoperative therapy. Flow cytometric analysis of single biopsy specimens from 79 patients with oral cancer was also undertaken to ascertain their value as indicators of regional metastasis. Results Forty-five percent (14 of 31) of tumors showed intratumoral ploidy heterogeneity. Intratumoral SPF variation in the 31 tumors ranged from 0.2% to 6.9% (mean, 3.3%). Multivariate analysis showed that a SPF greater than 27% was the most important parameter for predicting regional metastasis. Conclusions DNA ploidy is heterogeneous within a tumor, whereas SPF is relatively stable and can be correlated with regional metastasis in oral cancer. © 2002 John Wiley & Sons, Inc. [source] Patients with bisphosphonates-associated osteonecrosis of the jaw have reduced circulating endothelial cellsHEMATOLOGICAL ONCOLOGY, Issue 4 2007A Allegra Abstract Osteonecrosis of the jaws (ONJ) associated with the use of bisphosphonates is a newly described entity. To elucidate the mechanism leading to ONJ and to test the hypothesis that in patients with ONJ the bisphosphonates may interfere with endothelial cell proliferation, using flow cytometric analysis we evaluated the number of circulating endothelial progenitor cells (EPCs) and circulating endothelial cells (CECs) in eight patients with bisphosphonate treatment and osteonecrosis, eight multiple myeloma (MM) patients with bisphosphonates treatment without ONJ and five normal subjects. MM patients showed an increase of CD34+ cells with respect the control subjects and ONJ subjects. EPCs and CECs were higher in MM patients compared to controls and ONJ patients. ONJ patients showed a decrease of EPCs compared to control subjects while CECs were similar to the controls group. Our results seem to show the possibility that bisphosphonates could have a antiangiogenic effect and a suppressive effect on CECs of patients with ONJ. Copyright © 2007 John Wiley & Sons, Ltd. [source] Phenotypic analysis of peripheral CD4+ CD8+ T cells in the ratIMMUNOLOGY, Issue 2 2000E. Kenny Summary Among peripheral T cells, the expression of CD4 and CD8 is almost mutually exclusive. However, here we show, using flow cytometric analysis, that ex vivo approximately 6% of rat T cells stained for both CD4 and CD8. These double positive cells were also detected by confocal microscopy. Only around 50% of double positive cells expressed the CD8, chain, the remaining cells expressed the CD8, chain alone. Double positive cells were blast-like with a phenotype, distinct from that of either CD4 or CD8 single positive cells, suggestive of an activated state. Previous reports of double positive T cells have also suggested that coexpression of CD4 and CD8 is linked to the activation state of the cell. There was an indication that priming animals with a hapten-carrier complex increased the ratio of CD8,,,:,,, expressing double positive T cells, although we did not detect an increase in the frequency of double positive T cells following priming. We also show that the frequency of double positive cells was reduced following thymectomy and with age. In conclusion, these studies show that peripheral T cells expressing both CD4 and CD8 can be detected in the rat and that they are phenotypically distinct from CD4 and CD8 single positive T cells. [source] Expression of multiple human endogenous retrovirus surface envelope proteins in ovarian cancerINTERNATIONAL JOURNAL OF CANCER, Issue 1 2007Feng Wang-Johanning Abstract Individual classes of human endogenous retrovirus (HERV) genes and proteins are expressed in cancer, but expression of more than one type of HERV is rare. We report here the expression of multiple HERV genes and proteins in ovarian cell lines and tissues. Expression of HERV-K env mRNA was greater in ovarian epithelial tumors than in normal ovarian tissues (N = 254). The expression of this protein on the surface and in the cytoplasm of ovarian cancer cells was confirmed using anti-HERV-K specific antibody by flow cytometric analysis. The frequency of expression of HERV-K env protein in multitissue microarrays (N = 641) was determined by immunohistochemistry and a significant correlation with tumor histotype was found. A significantly increased expression of HERV-K was observed in tumors with low malignant potential and low grade, relative to expression in normal ovarian tissues. The increase in expression of HERV-K env protein took place in a stepwise fashion in serous papillary adenocarcinoma. Interestingly, we found that other classes of HERV env mRNAs, including ERV3 and HERV-E, are expressed in the same ovarian cancer tissues that expressed HERV-K. Furthermore, anti-HERV antibodies including anti-ERV3 (30%), anti-HERV-E (40%) and anti-HERV-K (55%) were detected in patients with ovarian cancer, but not in normal female controls. HERV env proteins are frequently transcribed and translated in ovarian epithelial tumors, and multiple HERV families are detectable in ovarian cancer. HERV env proteins, and especially those expressed on the cell surface, may serve as novel tumor targets for detection, diagnosis and immunotherapy of ovarian cancer. © 2006 Wiley-Liss, Inc. [source] Osteoclast Differentiation by RANKL Requires NF-,B-Mediated Downregulation of Cyclin-Dependent Kinase 6 (Cdk6),JOURNAL OF BONE AND MINERAL RESEARCH, Issue 7 2004Toru Ogasawara Abstract This study investigated the involvement of cell cycle factors in RANKL-induced osteoclast differentiation. Among the G1 cell cycle factors, Cdk6 was found to be a key molecule in determining the differentiation rate of osteoclasts as a downstream effector of the NF-,B signaling. Introduction: A temporal arrest in the G1 phase of the cell cycle is a prerequisite for cell differentiation, making it possible that cell cycle factors regulate not only the proliferation but also the differentiation of cells. This study investigated cell cycle factors that critically influence differentiation of the murine monocytic RAW264.7 cells to osteoclasts induced by RANKL. Materials and Methods: Growth-arrested RAW cells were stimulated with serum in the presence or absence of soluble RANKL (100 ng/ml). Expressions of the G1 cell cycle factors cyclin D1, D2, D3, E, cyclin-dependent kinase (Cdk) 2, 4, 6, and Cdk inhibitors (p18 and p27) were determined by Western blot analysis. Involvement of NF-,B and c- jun N-terminal kinase (JNK) pathways was examined by overexpressing dominant negative mutants of the I,B kinase 2 (IKKDN) gene and mitogen-activated protein kinase kinase 7 (MKK7DN) gene, respectively, using the adenovirus vectors. To determine the direct effect of Cdk6 on osteoclast differentiation, stable clones of RAW cells transfected with Cdk6 cDNA were established. Osteoclast differentiation was determined by TRACP staining, and cell cycle regulation was determined by BrdU uptake and flow cytometric analysis. Results and Conclusion: Among the cell cycle factors examined, the Cdk6 level was downregulated by RANKL synchronously with the appearance of multinucleated osteoclasts. Inhibition of the NF-,B pathway by IKKDN overexpression, but not that of the JNK pathway by MKK7DN overexpression, caused the decreases in both Cdk6 downregulation and osteoclastogenesis by RANKL. RAW cells overexpressing Cdk6 resist RANKL-induced osteoclastogenesis; however, cell cycle regulation was not affected by the levels of Cdk6 overexpression, suggesting that the inhibitory effect of Cdk6 on osteoclast differentiation was not exerted through cell cycle regulation. These results indicate that Cdk6 is a critical regulator of RANKL-induced osteoclast differentiation and that its NF-,B-mediated downregulation is essential for efficient osteoclast differentiation. [source] Novel quinolone CHM-1 induces apoptosis and inhibits metastasis in a human osterogenic sarcoma cell lineJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 12 2009Shu-Chun Hsu Abstract Novel 2-phenyl-4-quinolone compounds have potent cytotoxic effects on different human cancer cell lines. In this study, we examined anticancer activity and mechanisms of 20-fluoro-6,7-methylenedioxy-2-phenyl-4-quinolone (CHM-1) in human osterogenic sarcoma U-2 OS cells. CHM-1-induced apoptosis was determined by flow cytometric analysis, DAPI staining, Comet assay, and caspase inhibitors. CHM-1-inhibited cell migration and invasion was assessed by a wound healing assay, gelatin zymography, and a Transwell assay. The mechanisms of CHM-1 effects on apoptosis and metastasis signaling pathways were studied using Western blotting and gene expression. CHM-1 induced G2/M arrest and apoptosis at an IC50 (3 µM) in U-2 OS cells and caspase-3, -8, and -9 were activated. Caspase inhibitors increased cell viability after exposure to CHM-1. CHM-1-induced apoptosis was associated with enhanced ROS generation, DNA damage, decreased ,,m levels, and promotion of mitochondrial cytochrome c release. CHM-1 stimulated mRNA expression of caspase-3, -8, and -9, AIF, and Endo G. In addition, CHM-1 inhibited cell metastasis at a low concentration (<3 µM). CHM-1 inhibited the cell metastasis through the inhibition of MMP-2, -7, and -9. CHM-1 also decreased the levels of MAPK signaling pathways before leading to the inhibition of MMPs. In summary, CHM-1 is a potent inducer of apoptosis, which plays a role in the anticancer activity of CHM-1. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27:1637,1644, 2009 [source] Paclitaxel induces apoptosis via caspase-3 activation in human osteogenic sarcoma cells (U-2 OS)JOURNAL OF ORTHOPAEDIC RESEARCH, Issue 5 2005K.-H. Lu Abstract Paclitaxel has been found to exhibit cytotoxic and antitumor activity. There is little information regarding the mechanisms of apoptotic-inducing effect of paclitaxel on human osteogenic sarcoma U-2 OS cells. Several key regulatory proteins are involved in the initiation of apoptosis. Caspase-3 plays a direct role in proteolytic cleavage of cellular proteins responsible for progression to apoptosis. We examined the effect of paclitaxel on the cell cycle arrest and apoptosis in U-2 OS cells using flow cytometric analysis and Western blotting. We also measured the inhibition of paclitaxel-induced apoptosis and the caspase-3 activity by the broad-spectrum caspase inhibitor z-VAD-fmk on U-2 OS cells. The increased levels of casapse-3 were also confirmed by cDNA microarray. Our observations were: (1) paclitaxel treatment resulted in G2/M-cycle arrest in U-2 OS cells; (2) time and dose dependent apoptosis of U-2 OS cells was induced by paclitaxel; (3) in U-2 OS cells, z-VAD-fmk blocked the paclitaxel-induced apoptosis and caspase-3 activation. These results suggest that paclitaxel-induced G2/M-cycle arrest of the G2/M phase and apoptosis via a caspase-3 pathway in U-2 OS cells. © 2005 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. [source] Statin-induced apoptosis linked with membrane farnesylated Ras small G protein depletion, rather than geranylated Rho proteinJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 11 2005Sumio Matzno Rhabdomyolysis is a severe adverse effect of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins). This myopathy is strongly enhanced by the combination with statins and fibrates, another hypolipidaemic agent. We have evaluated the initial step of statin-induced apoptosis by the detection of membrane flip-flop using flow cytometric analysis. L6 rat myoblasts were treated with various statins (atorvastatin (3 ,m), cerivastatin (3 ,m), fluvastatin (3 ,m), pravastatin (3 mm), or simvastatin (3 ,m)) for 2, 4 or 6 h followed by reacting with FITC-conjugated annexin V for the detection of initial apoptosis signal (flip-flop). Various statin-treated myoblasts were significantly stained with FITC-annexin V at 6 h, whereas they were not detected at 2 h. Moreover, immunoblot analysis indicated that when the cells were treated with cerivastatin (3 ,m), membrane-associated Ras protein was activated and detached until 6 h, resulting in cell death through the consequent activation of caspase-8. On the other hand, since cytosolic Ras activation did not activate, there is still an unknown mechanism in statin-related Ras depletion. In conclusion, statin-induced apoptosis in muscular tissue was directly initiated by the farnesyl-anchored Ras protein depletion from cell membrane with subsequent apoptosis. [source] SYMBIODINIUM (PYRRHOPHYTA) GENOME SIZES (DNA CONTENT) ARE SMALLEST AMONG DINOFLAGELLATES,JOURNAL OF PHYCOLOGY, Issue 4 2005Todd C. LaJeunesse Using flow cytometric analysis of fluorescence, we measured the genome sizes of 18 cultured "free-living" species and 29 Symbiodinium spp. isolates cultured from stony corals, gorgonians, anemones, jellyfish, and giant clams. Genome size directly correlated with cell size, as documented previously for most eukaryotic cell lines. Among the smallest of dinoflagellates, Symbiodinium spp. (6,15 ,m) possessed the lowest DNA content that we measured (1.5,4.8 pg·cell,1). Bloom-forming or potentially harmful species in the genera Alexandrium, Karenia, Pfiesteria, and Prorocentrum possessed genomes approximately 2 to 50 times larger in size. A phylogenetic analysis indicated that genome/cell size has apparently increased and decreased repeatedly during the evolution of dinoflagellates. In contrast, genome sizes were relatively consistent across distantly and closely related Symbiodinium spp. This may be the product of intracellular host habitats imposing strong selective pressures that have restricted symbiont size. [source] Intravenous administration of melatonin reduces the intracerebral cellular inflammatory response following transient focal cerebral ischemia in ratsJOURNAL OF PINEAL RESEARCH, Issue 3 2007Ming-Yang Lee Abstract:, We have previously shown that exogenous melatonin improves the preservation of the blood,brain barrier (BBB) and neurovascular unit following cerebral ischemia,reperfusion. Recent evidence indicates that postischemic microglial activation exaggerates the damage to the BBB. Herein, we explored whether melatonin mitigates the cellular inflammatory response after transient focal cerebral ischemia for 90 min in rats. Melatonin (5 mg/kg) or vehicle was given intravenously at reperfusion onset. Immunohistochemistry and flow cytometric analysis were used to evaluate the cellular inflammatory response at 48 hr after reperfusion. Relative to controls, melatonin-treated animals did not have significantly changed systemic cellular inflammatory responses in the bloodstream (P > 0.05). Melatonin, however, significantly decreased the cellular inflammatory response by 41% (P < 0.001) in the ischemic hemisphere. Specifically, melatonin effectively decreased the extent of neutrophil emigration (Ly6G-positive/CD45-positive) and macrophage/activated microglial infiltration (CD11b-positive/CD45-positive) by 51% (P < 0.01) and 66% (P < 0.01), respectively, but did not significantly alter the population composition of T lymphocyte (CD3-positive/CD45-positive; P > 0.05). This melatonin-mediated decrease in the cellular inflammatory response was accompanied by both reduced brain infarction and improved neurobehavioral outcome by 43% (P < 0.001) and 50% (P < 0.001), respectively. Thus, intravenous administration of melatonin upon reperfusion effectively decreased the emigration of circulatory neutrophils and macrophages/monocytes into the injured brain and inhibited focal microglial activation following cerebral ischemia,reperfusion. The finding demonstrates melatonin's inhibitory ability against the cellular inflammatory response after cerebral ischemia,reperfusion, and further supports its pleuripotent neuroprotective actions suited either as a monotherapy or an add-on to the thrombolytic therapy for ischemic stroke patients. [source] Alcohol Exposure Impairs Myeloid Dendritic Cell Function in Rhesus MacaquesALCOHOLISM, Issue 9 2009Robert W. Siggins Background:, Alcohol intoxication suppresses both the innate and adaptive immunities. Dendritic cells (DCs) are the major cell type bridging the innate and acquired immune responses. At the present time, the effects of alcohol on DC development in hematopoietic tissues and the functional activities of DCs are incompletely elucidated. This study investigated the impact of chronic alcohol exposure on the alteration of hematopoietic precursor cell and DC populations in the bone marrow and peripheral blood of rhesus macaques. Methods:, Rhesus macaques were administered alcohol or isocaloric sucrose daily for a period of 3 months through surgically implanted gastric catheters. Peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs) were isolated for flow cytometric analysis after 3 months. Monocytes were cultured with human IL-4 (10 ng/ml) and GM-CSF (50 ng/ml) in the absence and presence of alcohol (50 mM). On day 6 of the culture, a cocktail of stimulants including IL-1, (18 ng), IL-6 (1800 U), TNF-, (18 ng), and PGE2 (1.8 ,g) were added to the designated wells for transformation of immature dendritic cells (iDCs) to mature myeloid DCs. The cells were analyzed on day 8 by flow cytometry for expression of DC costimulatory molecule expression. Results:, EtOH-treated animals had significantly lower numbers of myeloid DCs (lineage-HLA-DR+CD11c+CD123,) in both the PBMCs and BMCs compared to controls (5,654 ± 1,273/106 vs. 2,353 ± 660/106 PBMCs and 503 ± 34 vs. 195 ± 44/106 BMCs). Under culture conditions, the number of lineage-HLA-DR+CD83+ cells was low in control wells (0.38 ± 0.08%). Alcohol inhibited the increase in the number of lineage-HLA-DR+CD83+ cells in iDC wells (2.30 ± 0.79% vs. 5.73 ± 1.40%). Alcohol also inhibited the increase in the number of lineage-HLA-DR+CD83+ cells in mature DC wells (1.23 ± 0.15% vs. 4.13 ± 0.62%). Conclusions:, Chronic EtOH decreases the bone marrow and circulating pools of myeloid DCs. Additionally, EtOH suppresses costimulatory molecule CD83 expression during DC transformation, which may attenuate the ability of DCs to initiate T-cell expansion. [source] Immunological Detection of in Vitro Formed Phosphatidylethanol,An Alcohol Biomarker,With Monoclonal AntibodiesALCOHOLISM, Issue 6 2008Antti E. Nissinen Background:, Phosphatidylethanol (PEth) is a promising new marker for detecting long-term alcohol abuse with excellent sensitivity and specificity. Current methods are based on the high performance liquid chromatography,mass spectrometric method and therefore require high levels of expertise and expensive instrumentation. This study was designed to generate PEth-specific monoclonal antibodies for PEth immunoassay development. Methods:, C57/BL6 mice were immunized with PEth in 3 different carriers, mouse serum albumin, mouse high-density lipoproteins, and human low-density lipoprotein (LDL). Mouse splenocytes were fused with a mouse myeloma cell line using the hybridoma technique. Mouse IgM-producing cell lines were selected by limiting dilutions. Binding characteristics of the anti-PEth antibodies were studied using luminometric immunoassays and sequence analysis of the variable region mRNA sequences of the antibodies. Produced antibodies were purified by chromatographic methods. PEth was detected with these antibodies in fluorescence immunoassay and flow cytometric analysis. Results:, We generated monoclonal cell lines (2B1 and 2E9) that produce IgM antibodies binding specifically to PEth but not to structurally or chemically similar phospholipids, such as phosphatidylcholine, phosphatidic acid, and cardiolipin. We show here that these anti-PEth antibodies can be used to detect PEth in a fluorescent PEth assay and FACS analysis of human red blood cell samples spiked with PEth. Conclusions:, The present study shows that PEth-specific monoclonal antibodies can be generated using traditional hybridoma technique. Immunogenicity of PEth was enhanced using human LDL as an immunization carrier. The generated monoclonal anti-PEth antibodies, 2B1 and 2E9 bind to PEth in fluid phase and in biological membranes. [source] Usefulness of a flow cytometric analysis of intraplatelet vasodilator-stimulated phosphoprotein phosphorylation for the detection of patients with genetic defects of the platelet P2Y12 receptor for ADPJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2010M. L. ZIGHETTI No abstract is available for this article. [source] A novel flow cytometric analysis for platelet activation on immobilized von Willebrand factor or fibrillar collagenJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 2 2003S. Kao Summary., Under flow conditions, platelets adhere singly or in small aggregates on von Willebrand factor (VWF)-coated surfaces, but form large aggregates on immobilized fibrillar collagen. We developed a novel flow cytometric analysis to study the mechanisms underlying these distinct platelet deposition patterns. Flow cytometry was used to measure platelet activation after platelet adherence onto microspheres coated with either VWF or collagen fibrils. Two representative indices were calculated to quantify activated GpIIb,IIIa and P-selectin expression on adherent platelets. The signaling pathways responsible for platelet activation after interacting with fibrillar collagen were elucidated using various inhibitors. An in vitro endothelial cell wound model was also used to study the roles of VWF and fibrillar collagen in platelet deposition onto subendothelial matrixes. The adherent platelets on fibrillar collagen express more activated GpIIb,IIIa and P-selectin than those on VWF. Activation of GpIIb,IIIa and expression of P-selectin after platelet interaction with collagen occur via different intracellular signaling pathways; however, Ca2+ released from intracellular pools is common to both phenomena. Platelets were deposited singly or formed small aggregates on the endothelial cell wounded area, and this deposition pattern was dependent on VWF molecules secreted by endothelial cells and the absence of subendothelial collagen fibrils. As less activated GpIIb,IIIa and P-selectin are expressed after platelets interact with immobilized VWF alone, subsequent flowing platelet recruitment is minimal. Collagen fibrils, however, can activate adherent platelets sufficiently to promote the formation of large platelet aggregates. [source] Quantitative assessment of chondrocyte viability after laser mediated reshaping: A novel application of flow cytometryLASERS IN SURGERY AND MEDICINE, Issue 1 2003Alexandre Rasouli BS Abstract Background and Objectives Lasers can be used to reshape cartilage by accelerating mechanical stress relaxation. In this study, fluorescent differential cell viability staining and flow cytometry were used to determine chondrocyte viability following laser heating. Study Design/Materials and Methods Porcine septal cartilages were irradiated with an Nd:YAG laser (,,= 1.32 ,m, 25 W/cm2) while surface temperature, stress relaxation, and diffuse reflectance were recorded. Each slab received one, two, or three laser exposures (respective exposure times of 6.7, 7.2, 10 seconds). Irradiated samples were then divided into two groups analyzed immediately and at 5 days following laser exposure. Chondrocytes were isolated following serial enzymatic digestion, and stained using SYTO®/DEAD RedÔ (Molecular Probes, Eugene, OR). A flow cytometer was then used to detect differential cell fluorescence; size; granularity; and the number of live cells, dead cells, and post-irradiation debris in each treatment population. Results Nearly 60% of chondrocytes from reshaped cartilage samples isolated shortly after one irradiation, were viable while non-irradiated controls were 100% viable. Specimens irradiated two or three times demonstrated increasing amounts of cellular debris along with a reduction in chondrocyte viability: 31 and 16% after two and three exposures, respectively. In those samples maintained in culture medium and assayed 5 days after irradiation, viability was reduced by 28,88%, with the least amount of deterioration in untreated and singly irradiated samples. Conclusions Functional fluorescent dyes combined with flow cytometric analysis successfully determines the effect of laser irradiation on the viability of reshaped cartilage. Lasers Surg. Med. 32:3,9,2003. © 2003 Wiley-Liss, Inc. [source] Differential apoptosis by gallotannin in human colon cancer cells with distinct p53 statusMOLECULAR CARCINOGENESIS, Issue 3 2007Sahar Al-Ayyoubi Abstract Gallotannin (GT), a plant polyphenol, has shown anticarcinogenic activities in several animal models including colon cancer. In our previous study, we showed that GT inhibits 1,2-dimethylhydrazine-induced colonic aberrant crypt foci and tumors in Balb/c mice, thus supporting a role for GT as a chemopreventive agent in colon cancer. However, at the molecular level, GT's mechanism of chemoprevention is still unclear. In this study, we aim at identifying GT's potential molecular mechanisms of action in in vitro studies. We show that GT differentially inhibits the growth of two isogenic HCT-116 (p53+/+, p53,/,) human colon cancer cells versus normal human intestinal epithelial cells (FHs 74Int). DNA flow cytometric analysis showed that GT induced S-phase arrest in both HCT-116 cell lines. Cell-cycle arrest in p53 (+/+) cells was associated with an increase in p53 protein levels and p21 transcript and protein levels. The inhibition of cell-cycle progression of HCT-116 p53 (+/+) cells by GT correlated with a reduction in the protein levels of cyclin D1, pRb, and the Bax/Bcl-2 ratio. Although GT did not induce apoptosis in p53 (+/+) cells, a significant induction of apoptosis was observed in p53 (,/,) cells as shown by TUNEL staining and flow cytometry analysis. Apoptosis induction in p53 (,/,) cells was associated with a significant increase in Bax/Bcl-2 protein levels. Our results demonstrate that GT inhibits the growth of HCT-116 colon cancer cells in a p53-independent manner but exhibits differential sensitivity to apoptosis induction in HCT-116 cells with distinct p53 status. © 2006 Wiley-Liss, Inc. [source] Cooperative inhibitory effect of ZD1839 (Iressa) in combination with 17-AAG on glioma cell growth,MOLECULAR CARCINOGENESIS, Issue 5 2006Daniel R. Premkumar Abstract ZD1839 ("Iressa") is an orally active, selective epidermal growth factor (EGF) receptor-tyrosine kinase inhibitor. We evaluated the antitumor activity of ZD1839 in combination with HSP90 antagonist, 17-AAG in malignant human glioma cell lines. ZD1839 independently produced a dose-dependent inhibition of cellular proliferation in glioma cells grown in culture with time- and dose-dependent accumulation of cells in G1 phase of the cell cycle on flow cytometric analysis, although the concentrations required for optimal efficacy were at or above the limits of clinically achievable levels. Because the heat shock protein (HSP) is involved in the conformational maturation of a number of signaling proteins critical to the proliferation of malignant glioma cells, we hypothesized that the HSP90 inhibitor 17-AAG would potentiate ZD 1839-mediated glioma cytotoxicity by decreasing the activation status of EGF receptor, as well as downregulating the levels of other relevant signaling effectors. We, therefore, examined the effects of ZD1839 and 17-AAG, alone and in combination, on signal transduction and apoptosis in a series of malignant glioma cell lines. Simultaneous exposure to these inhibitors significantly induced cell death and quantitative analysis revealed that interaction between ZD1839 and 17-AAG-induced cytotoxicity was synergistic, leading to a pronounced increase in active caspase-3 and PARP cleavage. No significant growth inhibition or caspase activation was seen in control cells. The enhanced cytotoxicity of this combination was associated with diminished Akt activation and a significant downregulation of EGFR receptor, Raf-1 and mitogen activated protein kinase (MAPK). Cells exposed to 17-AAG and ZD1839 displayed a significant reduction in cell cycle regulatory proteins, such as CDK4 and CDK6. Taken together, these findings suggest that ZD1839, an EGF receptor tyrosine kinase inhibitor, plays a critical role in regulating the apoptotic response to 17-AAG and that multi-site targeting of growth signaling and cell survival pathways could provide a potent strategy to treat patients with malignant gliomas. © 2006 Wiley-Liss, Inc. [source] Immunohistochemical estimation of cell cycle entry and phase distribution in astrocytomas: applications in diagnostic neuropathologyNEUROPATHOLOGY & APPLIED NEUROBIOLOGY, Issue 5 2005Ian S. Scott An immunohistochemical method for assessing cell cycle phase distribution in neurosurgical biopsies would enable such data to be incorporated into diagnostic algorithms for the estimation of prognosis and response to adjuvant chemotherapy in glial neoplasms, without the requirement for flow cytometric analysis. Paraffin-embedded sections of intracerebral gliomas (n = 48), consisting of diffuse astrocytoma (n = 9), anaplastic astrocytoma (n = 8) and glioblastoma (n = 31), were analysed by immunohistochemistry using markers of cell cycle entry, Mcm-2 and Ki67, and putative markers of cell cycle phase, cyclins D1 (G1-phase), cyclin A (S-phase), cyclin B1 (G2-phase) and phosphohistone H3 (Mitosis). Double labelling confocal microscopy confirmed that the phase markers were infrequently coexpressed. Cell cycle estimations by immunohistochemistry were corroborated by flow cytometric analysis. There was a significant increase in Mcm-2 (P < 0.0001), Ki67 (P < 0.0001), cyclin A (P < 0.0001) and cyclin B1 (P = 0.002) expression with increasing grade from diffuse astrocytoma through anaplastic astrocytoma to glioblastoma, suggesting that any of these four markers has potential as a marker of tumour grade. In a subset of glioblastomas (n = 16) for which accurate clinical follow-up data were available, there was a suggestion that the cyclin A:Mcm-2 labelling fraction might predict a relatively favourable response to radical radiotherapy. These provisional findings, however, require confirmation by a larger study. We conclude that it is feasible to obtain detailed cell cycle data by immunohistochemical analysis of tissue biopsies. Such information may facilitate tumour grading and may enable information of prognostic value to be obtained in the routine diagnostic laboratory. [source] Hematopoietic and immune recovery after allogeneic peripheral blood stem cell transplantation and bone marrow transplantation in a pediatric populationPEDIATRIC TRANSPLANTATION, Issue 4 2002Yoshihisa Nagatoshi Abstract: To compare the hematopoietic and immune recoveries after allogeneic transplantation with different cell sources, we analyzed the recovery patterns of blood components after allogeneic peripheral blood stem cell transplantation (PBSCT) in comparison with that after allogeneic bone marrow transplantation (BMT) in a pediatric population. Sixteen patients received PBSCT, and 24 received BMT between January, 1995 and March, 2000. The patients had acute lymphoblastic leukemia (ALL; n = 22), acute myelogenous leukemia (AML; n = 8), myelodysplastic syndrome (MDS; n = 3), or other diseases (n = 7). The median ages of patients in the PBSCT and BMT groups were 9 yr and 6 yr, respectively. Cyclosporin A (CsA) plus methotrexate or methylprednisolone was used as a graft-vs.-host disease (GvHD) prophylaxis regimen in the PBSCT group, whereas CsA alone or methotrexate alone was used in the BMT group. Circulating lymphocyte numbers and subpopulations determined by flow cytometric analysis were used as markers of immune recovery. In the PBSCT group, the median number of harvested CD34+ cells was 7.25 (range: 1.3,27.6) × 106/kg of the recipient's body weight, while the median number of harvested nucleated cells was 4.7 (range: 3.7,10.5) × 108/kg. All of the patients were engrafted. Myeloid engraftment occurred sooner after PBSCT than after BMT (median number of days to achieve absolute neutrophil counts (ANC) > 0.5 × 109/L; 11 and 15, respectively; p < 0.0001) and similar results were found for platelet engraftment (median number of days to achieve a platelet count of > 20 × 109/L; 12 and 21, respectively; p = 0.004). On the other hand, after PBSCT the absolute numbers of total circulating lymphocytes and lymphocyte subpopulations were not significantly different from those after BMT. The incidence of acute GvHD after PBSCT was the same as that after BMT, while chronic GvHD developed more frequently after PBSCT than after BMT (p = 0.005). In a pediatric population, the indications for PBSCT and BMT should be based on these findings in addition to regard for the donor's safety. [source] Association Between the Photodynamic Loss of Bcl-2 and the Sensitivity to Apoptosis Caused by Phthalocyanine Photodynamic Therapy,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2003Jitsuo Usuda ABSTRACT We have reported that photodynamic therapy (PDT) using the photosensitizer phthalocyanine (Pc) 4 and red light damages the antiapoptotic protein Bcl-2. Recently, using transient transfection of Bcl-2 deletion mutants, we identified the membrane anchorage domains of Bcl-2 as necessary to form the photosensitive target. However, it is not clear how Bcl-2 photodamage sensitizes cells to Pc 4-PDT,induced apoptosis, whether overall cell killing is also sensitized or how upregulation of Bcl-2 in tumors might make them more or less responsive to Pc 4-PDT. In this study we report on MCF-7c3 cells (human breast cancer cells expressing stably transfected procaspase-3) overexpressing wild-type Bcl-2 or certain deletion mutants in either a transient or a stable mode. By flow cytometric analysis of transiently transfected cells, we found that wild-type Bcl-2, Bcl-2,33-54 and Bcl-2,37-63 (each of which can be photodamaged) protected cells from apoptosis caused by Pc 4-PDT. In contrast, Bcl-2,210-239, which lacks the C-terminal transmembrane domain and cannot be photodamaged, afforded no protection. We then evaluated the PDT sensitivity of transfected cell lines stably overexpressing high levels of wild-type Bcl-2 or one of the Bcl-2 mutants. Overexpression of wild-type Bcl-2, Bcl-2,33-54 or Bcl-2,37-63 resulted in relative resistance of cells to Pc 4-PDT, as assessed by morphological apoptosis or loss of clonogenicity. Furthermore, overexpression of Bcl-2 also inhibited the activation-associated conformational change of the proapoptotic protein Bax, and higher doses of Pc 4 and light were required to activate Bax in cells expressing high levels of Bcl-2. Many advanced cancer cells have elevated amounts of Bcl-2. Our results show that increasing the dose of Pc 4-PDT can overcome the resistance afforded by either Bcl-2 or the two mutants. PDT regimens that photodamage Bcl-2 lead to activation of Bax, induction of apoptosis and elimination of the otherwise resistant tumor cells. [source] | |||||||||||||||||||||||||||||||||||||