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At least 397 records · Page 22Linked to original sources
PubMed2004

Recombinant prolylcarboxypeptidase activates plasma prekallikrein.

The serine protease prolylcarboxypeptidase (PRCP), isolated from human umbilical vein endothelial cells (HUVECs), is a plasma prekallikrein (PK) activator. PRCP cDNA was cloned in pMT/BIP/V5-HIS-C, transfected into Schneider insect (S2) cells, and purified from serum-free media. Full-length recombinant PRCP (rPRCP) activates PK when bound to high-molecular-weight kininogen (HK). Recombinant PRCP is inhibited by leupeptin, angiotensin II, bradykinin, anti-PRCP, diisopropyl-fluorophosphonate (DFP), phenylmethylsulfonyl fluoride (PMSF), and Z-Pro-Proaldehyde-dimethyl acetate, but not by 1 mM EDTA (ethylenediaminetetraacetic acid), bradykinin 1-5, or angiotensin 1-7. Corn trypsin inhibitor binds to prekallikrein to prevent rPRCP activation, but it does not directly inhibit the active site of either enzyme. Unlike factor XIIa, the ability of rPRCP to activate PK is blocked by angiotensin II, not by neutralizing antibody to factor XIIa. PRCP antigen is detected on HUVEC membranes using flow cytometry and laser scanning confocal microscopy. PRCP antigen does not colocalize with LAMP1 on nonpermeabilized HUVECs, but it partially colocalizes in permeabilized cells. PRCP colocalizes with all the HK receptors, gC1qR, uPAR, and cytokeratin 1 antigen, on nonpermeabilized HUVECs. PRCP activity and antigen expression on cultured HUVECs are blocked by a morpholino antisense oligonucleotide. These investigations indicate that rPRCP is functionally identical to isolated HUVEC PRCP and is a major HUVEC membrane-expressed, PK-activating enzyme detected in the intravascular compartment.

Animals↗
PubMed2005

Actin cytoskeleton of rabbit intestinal cells is a target for potent marine phycotoxins.

Biotoxins produced by harmful marine microalgae (phycotoxins) can be accumulated into seafood, representing a great risk for public health. Some of these phycotoxins are responsible for a variety of gastrointestinal disturbances; however, the relationship between their mechanism of action and toxicity in intestinal cells is still unknown. The actin cytoskeleton is an important and highly complicated structure in intestinal cells, and on that basis our aim has been to investigate the effect of representative phycotoxins on the enterocyte cytoskeleton. We have quantified for the first time the loss of enterocyte microfilament network induced by each toxin and recorded fluorescence images using a laser-scanning cytometer and confocal microscopy. Our data show that pectenotoxin-6, maitotoxin, palytoxin and ostreocin-D cause a significant reduction in the actin cytoskeleton. In addition, we found that the potency of maitotoxin, palytoxin and ostreocin-D to damage filamentous actin is related to Ca(2+) influx in enterocytes. Those results identify the cytoskeleton as an early target for the toxic effect of those toxins.

Acrylamides↗
PubMed2000

Integrin alpha(2)beta(1) (VLA-2) is a principal receptor used by neutrophils for locomotion in extravascular tissue.

Cell adhesion molecules are critically involved in the multistep process of leukocyte recruitment in inflammation. The specific receptors used by polymorphonuclear leukocytes (PMN) for locomotion in extravascular tissue have as yet not been identified. By means of immunofluorescence flow cytometry and laser scanning confocal microscopy, this study demonstrated that surface expression of the alpha(2)beta(1) (VLA-2) integrin, though absent on blood PMN, is induced in extravasated PMN collected from human skin blister chambers, and rat PMN accumulated in the peritoneal cavity after chemotactic stimulation. Intravital time-lapse videomicroscopy was used to investigate chemoattractant-induced PMN locomotion in the rat mesentery in vivo. Local administration of function-blocking monoclonal antibody or peptide recognizing the alpha(2)beta(1) integrin reduced PMN migration velocity in the extravascular tissue by 73% +/- 3% and 70% +/- 10%, respectively (means +/- SD). The distance f-met-leu-phe peptide (fMLP)-stimulated human PMN migrated in a collagen gel in vitro was markedly reduced by treatment with anti-alpha(2) mAbs or peptide, whereas no effect was observed with antibodies or peptides recognizing the alpha(4)beta(1) or alpha(5)beta(1) integrins. Further evidence for a critical role of expression of alpha(2)beta(1) integrin in PMN locomotion in extravascular tissue was obtained in the mouse air pouch model of acute inflammation where chemoattractant-induced PMN recruitment was substantially inhibited by local anti-alpha(2) mAb treatment. Thus, expression of alpha(2)beta(1) integrin on extravasated PMN has been identified and a novel role of this receptor in regulating the extravascular phase of leukocyte trafficking in inflammation has been formulated. (Blood. 2000;95:1804-1809)

Adult↗
PubMed2001

[A study on the direct binding of LPS to endothelium and the characteristics of endothelial injury].

OBJECTIVE: To explore the characteristics of the combination of LPS to the endothelium and endothelial injury. METHODS: Myocardial microvascular endothelial cells (MMECs) were isolated and cultured to adhering state. Flow cytometry, laser confocal scanning microscope (LCSM) and image analysis were employed to evaluate the effects of different concentrations of LPS and different culturing time with the same concentration of LPS on the adhered MMECs RESULTS: (1) The OD values of MMECs increased progressively along with the prolongation of culturing time of MMECs with LPS and reached top level at 30 min. In addition, the increase of OD values was dependent on time. (2) The number of positive MMECs and the OD values increased after MMECs was cultured for 2 hours with LPS at concentrations ranging from 0.03125 similar 2.0000 g/L. The OD value reached peak level when LPS concentration was 0.2500 g/L, which implied that this concentration of LPS (0.25 g/L) was the optimal one for the combination of LPS with MMECs, and there was concentration saturation. (3) LPS could enter into the cytoplasm and nucleus of MMECs. (4) LPS could induce nucleic translocation and denucleation. CONCLUSION: (1) LPS could combine MMECs and enter into the nucleus without the presence of serum. (2) LPS could directly damage MMECs.

Animals↗
PubMed2000

[The Role of Calcium Ion in Apotopsis of HL-60 Cells Induced by VP-16]

To study the significance of calcium in the apoptosis of HL-60 cells induced by VP-16, the technology of flow cytometry, confocal laser scanning microscopy and Western blot were used. The results showed that VP-16 could induced the apoptosis of HL-60 cells and transient increase of intracellular calcium concentration; EGTA [ethylene glycol-bis(2-aminoethyl)-N,N,N',N'-tetraacetic acid], that could combine the extracellular calcium, did not prevent the apoptosis of HL-60 cells. BAPTA-AM [1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxy-methyl) ester], however, a chelating agent of intracellular calcium ions, could prevent apoptosis and the release of cytochrome C from HL-60 cells. It was concluded that the calcium plays an important role in apoptosis and the release of cytochrome C.

Journal Article↗
PubMed2004

[Nuclei and nucleolar organizing regions in chromosomes of lymphocytes on different stages of periodic disease].

By scanning cytomorphometry a cytological study was first performed on the behavior of nuclei and nucleolar organizing regions (NOR) in chromosomes of peripheral blood lymphocytes of healthy men and of patients with periodic disease (familial Mediterranean fever, FMF) on different stages of development, including its complication with amyloidosis. The volume and total surface of nuclei, the sum total volume and sum total surface of NOR, the mean number of NOR for one nucleus and distribution of nuclei according to NOR number were measured. It is shown that the parameters of nuclei and NOR for patients with FMF on all stages clearly and trustworthy differ from those for healthy men. They are sufficiently informative, can be successfully used in clinical practice and even serve as an early diagnostic test for amyloidosis complication.

Amyloidosis↗
PubMedDate not supplied

Videomicrofluorometry on living cells and discriminant factorial analysis to study cell cycle distributions.

After a rapid overview of the approaches used to study cell cycle, a fluorescent digital imaging microscopy method is proposed. This method is improved by a factorial analysis relying on the evaluation of several parameters recorded on each living cell. Single lympho-blastoid living cells are labeled with three fluorescent markers: Hoechst 33342 for nuclear DNA, Rhodamine 123 for mitochondria and Nile Red for plasma membrane. For each cell, morphological and functional information parameters are obtained. A typological analysis is used to separate control cells into four groups: G0-G1, S, G2+M and polyploid cells Gn. These control cells define a learning population used to analyze untreated and adriamycine treated cells as supplementary individuals in a discriminant factorial analysis. Such an approach allows to accurately evidence the change of the values of some cellular parameters.

Benzimidazoles↗
PubMed2005

Pharmacodynamic analysis of target inhibition and endothelial cell death in tumors treated with the vascular endothelial growth factor receptor antagonists SU5416 or SU6668.

PURPOSE: To determine the effects of small molecule inhibitors of vascular endothelial growth factor receptor (VEGFR)-2 (SU5416 and SU6668) on receptor phosphorylation in tumor xenografts and in paired tumor biopsies obtained in three clinical trials in patients with advanced solid malignancies. EXPERIMENTAL DESIGN: The dose-dependent effects of SU6668 on angiogenesis and tumor growth were investigated in orthotopic L3.6pl pancreatic tumors. Excisional or 18G core biopsies were obtained from patients before and after therapy with SU5416 or SU6668. Laser scanning cytometry-mediated analysis was used to quantify levels of phosphorylated and total VEGFRs and platelet-derived growth factor receptors (PDGFR), tumor microvessel densities, vessel sizes, and endothelial and tumor cell apoptosis. RESULTS: Significant inhibition of tumor microvessel density and growth and increased apoptosis were observed at SU6668 maximum tolerated dose (100 mg/kg) in L3.6pl xenografts. At 6 hours post therapy, SU6668 reduced VEGFR and PDGFR phosphorylation in the tumors by 50% and 92%, respectively, but levels rebounded beyond the baselines by 24 hours. Levels of phosphorylated VEGFR-2 and PDGFR also decreased significantly ( approximately 50%) 6 hours after therapy in 1 of 6 primary human tumors treated with SU6668, but these effects were not associated with increased apoptosis. A significant increase in endothelial cell apoptosis was observed in one tumor exposed to SU5416 and was associated with an increase in vessel size, but these changes occurred without an increase in tumor cell death. CONCLUSIONS: SU5416 and SU6668 displayed biological activity in xenografts. However, neither drug produced marked biological activity in primary patient tumors.

Adult↗
PubMed2005

Cathepsins and BID are involved in the molecular switch between apoptosis and autophagy in breast cancer MCF-7 cells exposed to camptothecin.

The details of molecular switching points between apoptosis and autophagy in tumor cells have still not been fully elucidated. This study focused on the role of cathepsin B and its substrate, BID as molecular links between apoptosis and autophagy in human breast cancer MCF-7 cells exposed to camptothecin. Apoptosis occurred rapidly with a peak in 60 min after drug administration, whereas autophagy developed at a much slower rate with continuous progression during 24 h of cell exposure to the drug. CPT induced very rapid activation of cathepsin B. Inhibition of cathepsins by E64d prevented CPT-induced BAX and BID aggregation on mitochondria and reduced significantly reduced apoptotic cell number. The above effects were accompanied by an increase in autophagosome formation, measured by expression of MAP I LC3. BID knock down resulted in strong suppression of CPT-induced apoptosis and a shift of cell death towards autophagy, manifesting with an increase of Beclin 1 and MAP I LC3 cellular content.

Antineoplastic Agents, Phytogenic↗
PubMed2005

Three-dimensional visualization and quantitative analysis of cervical cell nuclei with confocal laser scanning microscopy.

OBJECTIVE: To develop a method for the acquisition and processing of 3-dimensional images based on confocal laser scanning microscopy for the purpose of 3-dimensional visualization and quantitative analysis of cell nuclei. STUDY DESIGN: A contour-based surface rendering method was used, and volume rendering was implemented according to the basic volume rendering pipeline. To extract quantitative features, a 3-dimensional labeling method based on slice information was used. After applying the labeling algorithm, the measurements for 3-dimensional quantitative analysis of nuclei were extracted: nuclear volume, surface area and spherical shape factor. We compared the 3-dimensional features of normal and abnormal cervical cell nuclei. RESULTS: Comparison of the size of 3-dimensional cervical cell nuclei between normal and abnormal revealed a statistically significant difference. The proposed method could overcome the limitation inherent in 2-dimensional analysis and could become a way of improving the accuracy and reproducibility of quantification of cell nuclei. CONCLUSION: Three-dimensional visualization and quantification of cell nuclei provide valuable medical information that can lead to a more objective diagnosis.

Algorithms↗
PubMed2005

5E, 8Z, 11Z, 14Z-eicosatetraenoic acid, a novel trans isomer of arachidonic acid, causes G1 phase arrest and induces apoptosis of HL-60 cells.

Trans arachidonic acid isomers (trans-AA) constitute a new group of trans fatty acids (trans-FA) generated in vivo via endogenous cis-trans isomerization stimulated by the NO2 radical. Because both NO2 and trans-FA have been implicated as causative factors in cancer, we studied the effect of the trans-AA isomers on proliferation and viability of human promyelocytic (HL-60) cells. The four trans arachidonic (trans-AA) acid isomers synthesized by us have been presently tested with respect to their competence to affect the proliferation and viability of human promyeolocytic HL-60 cells in culture. The data demonstrate that one of the isomers, 5,6-trans-AA, showed distinct activity by targeting cell progression through the cell cycle and inducing apoptosis. The effects were time- and concentration-dependent: the cytostatic effect of 5E-AA was observed at 10 microM following 72 h of treatment. This effect was manifested as a perturbation of cell progression through G1 phase, indicating the 'on' activation of the G1 checkpoint as evidenced by the flow- and laser scanning-cytometry techniques. Apoptotic cells were identified by comparison of their morphology, DNA fragmentation, caspase activation and collapse of mitochondrial potential with control cells. These observations suggested that 5E-AA induced a mitochondrial pathway of apoptosis. There was no evidence of cell-cycle phase specificity in induction of apoptosis by 5E-AA, as the cells showing highly fragmented DNA or caspase-3 activation were distributed in all phases of the cycle. The data suggest that 5E-AA may have at least two targets: one that is cell-cycle specific and associated with the observed arrest in the G1 phase and another, unrelated to the cell cycle, which is responsible for triggering apoptosis indiscriminately, regardless of cycle phase I.

Apoptosis↗
PubMed2005

[Laser DNA-flow cytofluorometry in the differential diagnosis and prognosis of the cervix uteri cancer].

Laser DNA-flow cytofluorometry (DNA-FC) was used to examine 102 primary patients with morphologically verified pathology of the epithelium of the cervix uteri. There was a significant reduction in the count of cells in the cellular cycle phase (CCP) G0/1 and its increase in the CCP S and G2 + M, as well as a rose in the cell proliferation index (PI) in actually invasive (n=45) and microinvasive cancer of the cervix uteri (CCU) (n=21) as compared with the baseline values (n=8), CIN I-II (n=7), and CIN III (21 patients with severe dysplasia and cancer in situ; their DNA-FC parameters were close and significantly indistinguishable). With unfavorable clinical and morphological factors of CCU prognosis (age over 50 years, postmenopause, stages II-III, high-grade tumor, more than 3 mm stromal invasion, more than 4 cm in size, lymphovascular invasion in the regional lymph nodes, the endophytic form of growth, and a primary focus in the endocervix), aneuploid tumors were more significantly frequently found. The poor CCU prognostic factors significantly decreasing total and relapse-free 4-year survival included tumors of aneuploid type, those containing greater than 40% of aneuploid cells, those containing less than 70% of tumor cells in CCP G0/1, those having 10% of tumor cells or more in CCP S or more than 30% cell PI. While predicting CCU, the DNA index (DNAI) and PI with 0.09 and 0.05 informative value coefficients, respectively are the most significant DNA-FC parameters (after Shenon). By using them, the risk of progressive disease may be predicted with 67.9% probability. When DNAI and PI are used in combination with the most significant clinical and morphological factors of CCU, the likelihood of the latter increases up to 89.3%.

Aneuploidy↗
PubMed1995

Overexpression of human cyclin A advances entry into S phase.

Cyclin A is a cell cycle regulatory protein that functions in mitotic and S-phase control in mammalian cells. Using a genomic construction corresponding to the human cyclin A gene under the control of its own promoter, we have established stable transfectants overexpressing cyclin A protein. Experiments assisted by laser scanning image cytometry showed that this overexpression begins from late G1 phase onwards and is therefore cell cycle-regulated in this model. We demonstrated that this overexpression advances entry into S phase, leading to a contraction of the overall cell generation time. These results provide evidence that cyclin A can be a rate-limiting factor with respect to the control of the transition to S phase in mammalian cells.

Cyclins↗
PubMed1997

Dual effects of LPS antibodies on cellular uptake of LPS and LPS-induced proinflammatory functions.

Human phagocytes recognize bacterial LPS (endotoxin) through membrane CD14 (mCD14), a proinflammatory LPS receptor. This study tested the hypothesis that anti-LPS Abs neutralize endotoxin by blocking cellular uptake through mCD14. Ab-associated changes in the uptake and cellular distribution of FITC-LPS were assessed by flow cytometry and laser scanning confocal microscopy in human CD14-transfected Chinese hamster ovary fibroblasts (CHO-CD14 cells) and human peripheral blood monocytes. LPS core- and O-side chain-specific mAbs inhibited mCD14-mediated LPS uptake by both cell types in the presence of serum. O-side chain-specific mAb concurrently enhanced complement-dependent LPS uptake by monocytes through complement receptor-1 (CR1) and uptake by CHO-CD14 cells involving another heat-labile serum factor(s) and cell-associated recognition molecule(s). Core-specific mAb inhibited mCD14-mediated uptake of homologous and heterologous LPS, while producing less concurrent enhancement of non-mCD14-mediated LPS uptake. The modulation by anti-LPS mAbs of mCD14-mediated LPS uptake was associated with inhibition of LPS-induced nuclear factor-kappaB (NF-kappaB) translocation and TNF-alpha secretion in CHO-CD14 cells and monocytes, respectively, while mAb enhancement of non-mCD14-mediated LPS uptake stimulated these activities. LPS-specific Abs thus mediate anti-inflammatory and proinflammatory functions, respectively, by preventing target cell uptake of LPS through mCD14 and augmenting uptake through CR1 or other cell receptors.

Adult↗
PubMed1998

Application of confocal scanning laser microscopy to 3-D DNA image cytometry of prostatic lesions.

OBJECTIVE: To explore the potential of three-dimensional (3-D) image cytometry for the measurement of DNA content in prostatic specimens using confocal scanning laser microscopy (CSLM) and 3-D image analysis. STUDY DESIGN: Thick tissue slices (100 microns), stained for DNA with chromomycin A3, from four patients with benign hyperplasia, prostatic intraepithelial neoplasia (PIN), well- and poorly differentiated adenocarcinoma of the prostate, were studied. Two different blocks from the same slice were studied for each case. Cell nuclei were segmented automatically. DNA content and nuclear volume were measured. RESULTS: DNA histograms showed a single peak in the diploid range for the hyperplasia and PIN cases. For the case of well-differentiated carcinoma, two peaks were observed, one in the diploid range and one in the tetraploid range. The two peaks were observed on two different blocks of the same tissue slice. Poorly differentiated carcinoma was characterized by an aneuploid distribution. For the cases of PIN and carcinoma, we observed a considerable variation in nuclear volume. CONCLUSION: The results indicate the potential of 3-D image cytometry for the measurement of DNA content in prostatic specimens while preserving tissue architecture.

Cell Nucleus↗