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PubMed2006

[Effect of 2, 5-hexanedione on calcium homeostasis of motor neuron].

OBJECTIVE: To explore the mechanism of cytotoxic effect of 2, 5-hexanedione (2, 5-HD) on motor neuron. METHODS: Vsc4.1 (a cell line from motor neuron) was incubated with a series concentration of 2, 5-HD. The cell viability, Ca(2+) Mg(2+) ATPase and Na(+)K(+) ATPase were detected. Laser scanning confocal microscope (LSCM) was used for detecting intracellular calcium level. The average calcium level in VSC4.1 was measured by flow cytometry. RESULTS: The cell viability was decreased when Vsc4.1 cells were treated with 2, 5-HD at the dosage of 2.5, 5.0, 7.5, 10, 15 and 20 mmol/L for 24 hours. Compared with the control group the activity of Ca(2+) Mg(2+) ATPase was decreased to 70.02%, 77.44% and 47.47% respectively; the activity of Na(+)K(+) ATPase was decreased to 82.07%, 72.45% and 50.71%. The difference was significant. Intracellular free calcium of VSC4.1 cell was increased rapidly within 10 s and then recovered within 40 seconds when it was exposed to 33.5 mmol/L 2, 5-HD. An increase in intracellular calcium was observed when the VSC4.1 was treated with 33.5 mmol/L 2, 5-HD. The peak of intracellular calcium level occurred ten minutes later. CONCLUSION: The disturbance of calcium homeostasis may be involved in the mechanisms of neurotoxicity of 2, 5-HD.

Animals↗
PubMed2000

Cadmium- and chromium-induced oxidative stress, DNA damage, and apoptotic cell death in cultured human chronic myelogenous leukemic K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells.

Sodium dichromate [Cr(VI)] and cadmium chloride [Cd(II)] are both cytotoxic and mutagenic. This study examined the toxic and apoptotic potentials of these two cations on three cell types in vitro, namely, human chronic myelogenous leukemic (CML) K562 cells, promyelocytic leukemic HL-60 cells, and normal human peripheral blood mononuclear cells. The cells were incubated with 0-100 microM concentrations of the two cations for 0, 24, or 48 hours at 37 degrees C. Both Cr(VI) and Cd(II) induced changes in intracellular oxidized states of cells, which were detected using laser scanning confocal microscopy. Cell cycle modulation and apoptosis of the K562 cells by Cr(VI) and Cd(II) were determined by flow cytometry. Significant decreases in the G2/M phase were observed in the Cr(VI) and Cd(II) treated CML cells compared with untreated cells. At 12.5 microM, Cr(VI) induced greater apoptosis in K562 cells as compared with Cd(II). In the K562 cells, 2.2- and 3.0-fold increases in DNA fragmentation were observed following incubation with 12.5 and 25 microM Cr(VI), respectively, and 1.2- and 1.7-fold increases in DNA fragmentation were observed with Cd(II). Furthermore, approximately 2.7- and 4.9-fold increases in cytochrome c reduction were observed following incubation with 12.5 and 25 microM Cr(VI), respectively, and 1.6- and 3.3-fold increases in cytochrome c reduction were observed with Cd(II), demonstrating enhanced production of superoxide anion. Approximately 3.1 to 6-fold increases in hydroxyl radical production were observed following incubation of the K562 cells with these cations at 12.5 and 25 microM concentrations. These results in K562 cells were compared with promyelocytic leukemic HL-60 cells and normal human peripheral blood mononuclear cells. More pronounced effects were observed on K562 and HL-60 cells, and much lesser effects were observed on normal human peripheral blood mononuclear cells. The results demonstrate that both cations are toxic, producing oxidative tissue damage and apoptosis. Furthermore, more drastic effects were observed on K562 and HL-60 cells as compared with normal human peripheral blood mononuclear cells.

Apoptosis↗
PubMed1995

Early postoperative enteral nutrition with arginine-omega-3 fatty acids and ribonucleic acid-supplemented diet versus placebo in cancer patients: an immunologic evaluation of Impact.

OBJECTIVE: To evaluate the effect of early postoperative feeding with a nutritionally complete enteral diet supplemented with the nutrients arginine, ribonucleic acid (RNA), and omega-3 fatty acids on the immune function in patients undergoing surgery for upper gastrointestinal (GI) malignancies. DESIGN: Prospective, randomized, placebo-controlled, double-blind study. SETTING: Surgical intensive care unit (ICU) in a German university hospital. PATIENTS: Forty-two consecutive patients receiving an enteral diet via needle catheter jejunostomy after GI surgery for cancer. INTERVENTIONS: Patients were randomized to receive either the arginine, RNA, and omega-3 fatty acids supplemented diet or an isocaloric and isonitrogenous placebo diet. Early enteral nutrition was started on postoperative day 1 in the surgical ICU with 20 mL/hr and progressed to the optimal goal of 80 mL/hr by postoperative day 5. MEASUREMENTS AND MAIN RESULTS: Clinical examination and adverse GI symptoms were recorded on a daily basis. Body weight was determined twice weekly. Immunoglobulin concentrations were determined by laser nephelometry. Interferon-gamma concentrations were measured with a modified enzyme-linked immunosorbent assay method. Fluorescence-activated cell scan flow cytometry was performed to analyze B cells, T lymphocytes and their subsets. Clinical patient characteristics and mean caloric intake were similar between the two groups and both formulas were well tolerated. The number of T lymphocytes and their subsets, helper T cells (CD4) and activated T cells (CD3, HLA-DR), were significantly higher in the supplemented diet group on postoperative days 10 and 16 (p < .05). Mean interferon-gamma concentration after phytohemagglutinin stimulation was higher in the supplemented diet group on postoperative day 16. In the supplemented diet group, mean immunoglobulin M concentrations were significantly higher on postoperative day 10 and mean immunoglobulin G concentrations were higher on postoperative day 16 (p < .05) compared with the results in the placebo group. B-lymphocyte indices were significantly higher in the supplemented vs. the placebo diet group on postoperative days 7 and 10 (p < .05). CONCLUSIONS: Supplementation of enteral diet with arginine, RNA, and omega-3 fatty acids in the early postoperative time period improves postoperative immunologic responses and helps to overcome more rapidly the immunologic depression after surgical trauma.

Aged↗
PubMed2004

[Translocation of orphan receptor TR3 from nuclei to mitochondria induced by staurosporine].

BACKGROUND & OBJECTIVE: Staurosporine (STS), a non- specific protein kinase inhibitor, can extensively induce cell apoptosis. Orphan receptor TR3, an immediate-early response gene, belongs to the steroid/thyroid receptor superfamily. After stimulated by apoptosis-inducing agents, TR3 is expressed rapidly and translocates from nuclei to mitochondria,which finally induces cell apoptosis. This study was to investigate the mechanism by which STS induces apoptosis of gastric cancer cell line BGC-823,and its correlation with translocation of TR3 in BGC-823 cells. METHODS: Fluorescent microscopy was used to observe apoptotic morphology of BGC-823 cells after treated with STS for 3 and 24 h and stained by DAPI, and determine cell apoptotic rate. Laser scan confocal microscopy and Western blot were performed to investigate translocation of orphan receptor TR3 and the release of cytochrome c. Flow cytometry and confocal microscopy were used to examine mitochondrial membrane potential. RESULTS: After treated for 3 h,and 24 h,apoptosis rates of BGC-823 cells in STS-treatment groups were higher than those of cells in control groups (15.7% vs. 4.0%, and 39.4% vs. 4.7%). After BGC-823 cells exposed to STS for 3 h,TR3 translocated from nuclei to mitochondria, mitochondrial membrane potential was decreased, and cytochrome c released from mitochondria to the cytoplasm. CONCLUSION: TR3, in response to STS, translocates from nuclei to cytoplasm, where it targets to mitochondria to induce cytochrome c release, finally results in apoptosis of gastric cancer cells.

Active Transport, Cell Nucleus↗
PubMed2005

[Effects of bradykinin on the proliferation, apoptosis and differentiation of human keratinocytes].

OBJECTIVE: To investigate the effects of bradykinin (BK) on the proliferation, apoptosis and differentiation of human keratinocyte (HKC) and the underlying mechanisms. METHODS: HKCs were cultured together with 1 x 10(-4) - 1 x 10(-9) mol/L of BK. With methyl thiotetrazole (MTT) and trypan blue staining it was shown that the BK in dose of 1 x 10(-4) mol/L possessed most powerful inhibitory effect, and the survival rate of HKC was 69.3%. Therefore, BK was employed in the dose of 1 x 10(-4) mol/L in the following studies. When the growth of HKCs reached the logarithmic phase, BK in the concentration of 1 x 10(-4) mol/L was added, and it was categorized as the test group (E). HKCs without BK served as the control group (C). The cell cycle and apoptosis were detected by flow cytometry after being cultured for 24 and 48 hours. The change in intracellular calcium [Ca(2+)](i) was determined by means of laser scanning confocal microscopy with calcium fluorescence probe Fluo-3/AM technique. The expression of HKC differentiation labeling protein keratin10 (K10) and involucrin were detected with Strept Avidin-Biotin Complex (SABC) immunocytochemical assay. RESULTS: The cell ratio in G0/G1 phase in E group increased by 34.57% while in S phase decreased by 58.91% in reference to that in C group. The G1/S phase switching of HKCs was obviously inhibited by BK, and apoptosis was stimulated (apoptotic rate of 15.34% in E group vs 5.60% in C group, P < 0.05). The [Ca(2+)](i) increased transiently in HKCs by 163.0% in E group after 3 minutes of BK activation and decreased thereafter in reference to that in C group. The K10 expression in HKC was down-regulated in E group with positive cell rate of 2.20%, which was lower than that of C group (6.89%, P < 0.05). CONCLUSION: The cell cycle process of HKC could be inhibited by high concentration of BK with increased apoptosis and an increase in [Ca(2+)](i), which might be the mechanism of inhibition of growth of HKC in vitro. Furthermore, the epithelial regeneration and HKC differentiation can also be inhibited by BK.

Apoptosis↗
PubMed2005

[Effects of histamine on proliferation, apoptosis and differentiation of human keratinocytes].

OBJECTIVE: To investigate the effects of histamine on the proliferation, apoptosis and differentiation of human keratinocytes (HKC) and the mechanisms. METHODS: The effect of histamine on the growth of HKC in vitro was examined by MTT assay and trypan blue exclusion assay. Cell cycle analysis and early apoptosis analysis by double staining with Annexin V-FITC and PI were carried out by flow cytometry. DNA ladder assay was performed for the detection of cell apoptosis. HKC free calcium concentration ([Ca(2+)](i)) was measured by laser scanning confocal microscopy in combination with calcium fluorescence probe Fluo-3/AM. HKC differentiation markers keratin 10 (K10) and involucrin was detected by streptavidinbiotin complex immunocytochemical assay. RESULTS: Histamine at high concentration inhibited the proliferation of HKC with cell viability ratio of 65.6% at 1 x 10(-4) mol/L, while histamine at low concentrations promoted proliferation of HKC with the cell viability ratio of 130.7% at 1 x 10(-8) mol/L. Histamine at 1 x 10(-4) mol/L altered cell cycle distribution of HKC with an increase in G0/G1-phase cell population to 30.97%, a decrease in S-phase population to 73.81% and inhibition of G1/S switching. Histamine at 1 x 10(-4) mol/L induced obvious apoptosis of HKC with early apoptosis ratio of 18.64% as compared with the control (5.60%, P<0.05). Histamine 1 x 10(-4) mol/L induced an increase of HKC [Ca(2+)](i) up to 58.9% and cimetidine (an H(2) receptor antagonist) decreased HKC [Ca(2+)](i) down to 25.4%. Histamine at this concentration down-regulated the expressions of K10 and involucrin of HKC but these changes were not significantly different from those of the control (P>0.05). CONCLUSIONS: Histamine at high concentrations inhibits cell cycle progress of HKC, mediates cell apoptosis and induces the increase of [Ca(2+)](i), which might be a partial explanation for growth arrest of HKC elicited by histamine. Histamine may regulate epidermal tissue turnover under physiological conditions, whereas under pathological circumstances of the skin as in trauma and inflammation, histamine at high concentrations may inhibit the regeneration of epidermis and the differentiation of HKC.

Apoptosis↗
PubMed2005

Gene delivery with low molecular weight linear polyethylenimines.

BACKGROUND: Linear polyethylenimine (LPEI) with a molecular weight (MW) of 22 kDa has been described as having a superior ability to induce gene transfer compared to its branched form. However, the transfection efficiency of the polymer cannot be enhanced beyond a certain limit due to cytotoxicity. We explored the potential of utilizing LPEIs with MWs ranging from 1.0 to 9.5 kDa to overcome this limitation. METHODS: Polyplexes of plasmid DNA encoding for the enhanced green fluorescent protein (EGFP) and various LPEIs were compared concerning their transfection efficiency and cytotoxicity in CHO-K1 and HeLa cells by flow cytometry. The involvement of endolysosomes in LPEI-mediated gene transfer was investigated by applying the proton pump inhibitor bafilomycin A1 and the lysosomotropic agent sucrose. Confocal laser scanning microscopy was applied to assess the size and shape of polyplexes under cell culture conditions, to detect their endolysosomal localization and to observe their translocation to the nucleus. RESULTS: The transfection efficiency could be altered by varying the MW and the amount of the polymer available for polyplex formation. The highest transfection efficiency (about 44%), i.e. the fraction of EGFP-positive cells, was obtained with LPEI 5.6 kDa, while the cytotoxicity remained low. The colocalization of polyplexes and endolysosomes was observed, and it appeared that the larger polyplexes escaped from the acidic organelles particularly quickly. For LPEI 5.0 and 9.0 kDa, the number of cells and nuclei that had taken up DNA after 6 hours was similar, as determined by flow cytometry. CONCLUSIONS: Our study suggests that LPEIs with low MWs are promising candidates for non-viral gene delivery, because they are more efficient and substantially less toxic than their higher MW counterparts.

Animals↗
PubMed2003

Flow cytometric measurement of fluorescence (Förster) resonance energy transfer from cyan fluorescent protein to yellow fluorescent protein using single-laser excitation at 458 nm.

BACKGROUND: Use of distinct green fluorescent protein (GFP) variants permits the study of protein-protein interactions and colocalization in viable transfected cells by fluorescence (Förster) resonance energy transfer (FRET). Flow cytometry is a sensitive method to detect FRET. However, the typical dual-laser methods used in flow cytometric FRET assays are not generally applicable because they require a specialized krypton ultraviolet (UV) laser. The purpose of this work was to develop a flow cytometric method to detect FRET between cyan fluorescent protein (CFP; donor) and yellow fluorescent protein (YFP; acceptor) by using the 458-nm excitation from a single tunable argon-ion laser. METHODS: FUSE-binding protein (FBP) interacting repressor (FIR) and FBP are c-myc transcription factors and are known to interact physically. To examine their interaction within viable cells, FIR and the binding motif of FBP, the FBP central domain (FBPcd), were fused with CFP and YFP, respectively, and this pair of fluorescently-tagged proteins was used to detect FRET in vivo. Cells transfected with expression plasmids encoding a CFP-FIR fusion protein and YFP as a negative control, a CFP-YFP fusion protein as a positive control, or CFP-FIR and YFP-FBPcd fusion proteins were examined for FRET after excitation with a 458-nm line from a tunable argon-ion laser. FRET was measured as the ratio of YFP:CFP emission or as YFP emission at 564-606 nm. Conventional FRET using the 413-nm UV line from a krypton laser was examined for comparison. Fluorescence signals were separated with a customized optical filter configuration using 530-nm shortpass, 500-nm longpass, and 560-nm shortpass dichroics in addition to 488/30 nm (CFP), 530/30 nm (YFP), and 585/42 nm (FRET) bandpass filters. Further, a laser-scanning confocal microscopic photobleach technique was used to document that FRET occurred by showing that the intensity of donor CFP fluorescence increased after its acceptor YFP was photobleached. Steady-state spectrofluorometry was used to confirm and validate the results detected by flow cytometry. RESULTS: Upon excitation with the 458-nm line of the argon-ion laser, the enhancement of the acceptor YFP signal and the decrease of the CFP signal were easily detected in cells transfected with the CFP-YFP construct or CFP-FIR and YFP-FBPcd. Similarly, FRET was detected under these conditions when the YFP emission was assessed at 564-606 nm. A strong correlation was observed between the increase in the YFP:CFP ratio and the YFP emission detected at 564-606 nm, consistent with the conclusion that FRET was detected comparably by both methods. A conventional flow cytometric krypton UV-laser technique was also used to confirm that FRET occurred with the CFP-YFP fusion protein and from CFP-FIR --> YFP-FBPcd. FRET also was confirmed by a confocal photobleaching technique, in which donor CFP intensity was enhanced after its acceptor YFP was photobleached. The flow cytometric and confocal microscopic results were confirmed by spectrofluorometry. CONCLUSION: These results demonstrated the feasibility of flow cytometric detection of FRET signals from CFP to YFP by excitation with the 458-nm line from the tunable argon-ion laser. The method was as efficient as excitation with the krypton UV laser and therefore should make FRET a more generally available flow cytometric technique.

Argon↗
PubMed2006

[Effects of fuzheng yiliu granules on apoptotic rate and mitochondrial membrane potential of hepatocellular carcinoma cell line H22 from mice].

OBJECTIVE: To evaluate the effects of Fuzheng Yiliu Granules (FZYLG) on apoptotic rate and mitochondrial membrane potential (Delta psi m) of hepatocellular carcinoma cell line H22 from mice. METHODS: Forty-eight mice inoculated with H22 cells were randomly divided into four groups: untreated group, cyclophosphamide-treated group, high-dose FZYLG-treated group and low-dose FZYLG-treated group. After 14 days of corresponding treatment, H22 cells in each group were stained with propidium iodide, and the apoptotic rates were detected by flow cytometry (FCM). The rhodamine 123 was used as a fluorescence probe to label the H22 cells, and the fluorescence intensities were observed with laser scanning confocal microscope. The fluorescence intensity of H22 cells indicated the Delta psi m of H22 cells. RESULTS: FZYLG could significantly increase the apoptotic rate while reduce the Delta psi m of H22 cells from mice as compared with those in the untreated group. CONCLUSION: The antitumor effects of FZYLR on H22 cells from mice are related to decreasing the Delta psi m and then inducing the apoptosis of the H22 cells.

Animals↗
PubMed1995

Quantification and Characterization of Phagocytosis in the Soil Amoeba Acanthamoeba castellanii by Flow Cytometry.

Phagocytosis in the common grazing soil amoeba Acanthamoeba castellanii was characterized by flow cytometry. Uptake of fluorescently labelled latex microbeads by cells was quantified by appropriate setting of thresholds on light scatter channels and, subsequently, on fluorescence histograms. Confocal laser scanning microscopy was used to verify the effectiveness of sodium azide as a control for distinguishing between cell surface binding and internalization of beads. It was found that binding of beads at the cell surface was complete within 5 min and 80% of cells had beads associated with them after 10 min. However, the total number of phagocytosed beads continued to rise up to 2 h. The prolonged increase in numbers of beads phagocytosed was due to cell populations containing increasing numbers of beads peaking at increasing time intervals from the onset of phagocytosis. Fine adjustment of thresholds on light scatter channels was used to fractionate cells according to cell volume (cell cycle stage). Phagocytotic activity was approximately threefold higher in the largest (oldest) than in the smallest (newly divided) cells of A. castellanii and showed some evidence of periodicity. At no stage in the cell cycle did phagocytosis cease. Binding and phagocytosis of beads were also markedly influenced by culture age and rate of rotary agitation of cell suspensions. Saturation of phagocytosis (per cell) at increasing bead or decreasing cell concentrations occurred at bead/cell ratios exceeding 10:1. This was probably a result of a limitation of the vacuolar uptake system of A. castellanii, as no saturation of bead binding was evident. The advantages of flow cytometry for characterization of phagocytosis at the single-cell level in heterogeneous protozoal populations and the significance of the present results are discussed.

Journal Article↗
PubMed2003

Laser scanning detection of FISH-labelled Escherichia coli from water samples.

The development of rapid and accurate methods for the detection and quantification of bacteria without cultivation is of increasing importance for water monitoring. The aim of this study was to develop a solid phase cytometry detection method for DVC-FISH labelled Escherichia coli cells. In order to allow Stomatic detection with ChemScan RDI, the fluorescein-tyramide was combined with an oligonucleotide probe directly labelled with horseradish peroxidase to increase the fluorescence intensity. The method developed was tested for the enumeration of pure cultures, for GAC-filtered and drinking water samples. The method, which appeared to be equivalent to the culture method, was less sensitive than the DVC-FISH method followed by microscopic analysis. Research is underway to further optimise the labelling conditions.

Automation↗
PubMed2005

In vitro growth inhibition of human colonic tumor cells by Verapamil.

AIM: To investigate the effects and mechanisms of Verapamil on cultured human colonic tumor (HCT) cells. METHODS: HCT cells were treated with different concentrations of Verapamil, and their proliferation was examined by MTT assay. The areas of sub-diploid peak were measured by flow cytometry, and the DNA ladder was found by agarose gel electrophoresis. The characteristic changes in morphology were observed under light microscopy. The cell nuclei (propidium iodide labeled, PI-labeled) and cellular distribution and concentration of calcium (Fluo-3-labeled) were studied by using laser confocal scanning microscope. RESULTS: The proliferation of HCT cells was inhibited by different concentrations of Verapamil. With the increase in concentration of Verapamil, the percent of G0-G1 phase cells in HCT cells increased and that of S phase cells decreased. After treating with different concentrations of Verapamil, flow cytometry showed that HCT cells were enlarged in areas of sub-diploid in a dose-dependent manner. Gel electrophoresis results displayed a typical DNA ladder. On staining with Wrights-Giemsa, the typical cellular apoptosis morphologic changes were also observed. PI-labeled cell nuclei were found markedly changed. In addition, we inspected that the 100 micromol/L Verapamil could increase the intracellular calcium ion concentration [Ca(2+)](i) in HCT cells. CONCLUSION: Verapamil can inhibit proliferation of HCT cells via inducing cell apoptosis.

Apoptosis↗
PubMed2004

Number and activity of endothelial progenitor cells from peripheral blood in patients with hypercholesterolaemia.

Hypercholesterolaemia contributes to atherosclerosis and coronary artery diseases by inducing endothelial cell injury and dysfunction. Recent studies have provided increasing evidence that EPCs (endothelial progenitor cells) participate in ongoing endothelial repair and postnatal neovascularization. However, the changes in EPCs in patients with hypercholesterolaemia have not been elucidated to date. Therefore we investigated the number and functional activity of EPCs in patients with hypercholesterolemia. Total MNCs (mononuclear cells) were isolated from 20 patients with hypercholesterolaemia and 20 matched control subjects. EPCs were characterized as adherent cells double-positive for DiI-LDL (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanide percholate-labelled low-density lipoprotein) uptake and lectin binding by direct fluorescent staining under a laser scanning confocal microscope, and were characterized further by demonstrating the expression of KDR (kinase insert domain-containing receptor), CD34 and AC133 by flow cytometry. Proliferation, migration and in vitro vasculogenesis activity of EPCs were assayed using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assay, modified Boyden chamber assay and an in vitro vasculogenesis kit respectively. EPC adhesion assay was performed by replating cells on fibronectin-coated dishes and then counting the adherent cells. As a result, the number of EPCs was significantly reduced in patients with hypercholes-terolaemia compared with that in control subjects (41.8 +/- 8.7 compared with 64.5 +/- 16.6 EPCs/x 200 field respectively; P < 0.05). The number of EPCs was inversely correlated with total cholesterol (r = -0.659, P < 0.001) and LDL-cholesterol (r = -0.611, P < 0.001) levels. In addition, the functional activities of isolated EPCs, such as proliferative, migratory, adhesive and in vitro vasculogenesis capacity, were also impaired. In conclusion, the results of the present study may state a novel pathophysiological mechanism of hypercholesterolaemia: the reduction of EPCs with decreased functional activity.

Aged↗
PubMed1998

Lectin-mediated drug targeting: quantification of binding and internalization of Wheat germ agglutinin and Solanum tuberosum lectin using Caco-2 and HT-29 cells.

For the potential use of Wheat germ agglutinin (WGA) and Solanum tuberosum lectin (STL) as auxiliary excipients for targeting drugs to colonocytes, the number of Caco-2 and HT-29-bound lectins was determined by fluorimetry using fluorescein-labelled derivatives of the N-acetylglucosamine-specific lectins. After 1 h of incubation, the WGA-binding capacity of 5 x 10(4) Caco-2 cells was 26.9 +/- 0.5 pmol at 4 degrees C and 27.2 +/- 1.0 pmol at 37 degrees C respectively. In comparison, 19.5 +/- 2.9 pmol (37 degrees C) and 16.7 +/- 0.9 pmol (4 degrees C) WGA were bound within 1 h to 5 x 10(4) HT-29 cells referring to about 80% of the total amount of WGA bound within 4 h of incubation. In contrast, binding of STL to the colon carcinoma cell lines was independent of incubation times and temperatures tested exhibiting a binding rate of 8.4 +/- 0.6 pmol (HT-29) and 9.9 +/- 0.8 pmol (Caco-2) STL/5 x 10(4) cells. As determined by flow cytometry, non-specific binding is lower than 1.0% (WGA) and 3.4% (STL). Uptake and intracellular accumulation of the lectins were investigated by confocal laser scanning microscopy at 4 degrees C and 37 degrees C respectively. A decrease of initially membrane-bound lectins concurrent with increasing cytoplasmic enrichment by time was observed by digital cell image analysis. Due to specific and numerically sufficient adhesion as well as internalization, WGA and STL are anticipated as targeting tools in lectin-mediated drug delivery systems.

Acetylglucosamine↗
PubMed1995

Analytical methods for evaluation on whole cells of human papillomavirus infection.

Analytical methods for evaluation on whole cells of human papillomavirus infection. Human papillomavirus (HPV) infection is currently identified by the presence of viral DNA using molecular biology. As in situ hybridization is valuable for HPV-DNA detection mainly with non-isotopic probes, we evaluated the sensitivity of various techniques, using as models three cell lines containing different copy numbers of HPV DNA/cell (CaSki with 600 copies of HPV 16, SiHa with 1-2 copies of HPV 16, HeLa with 10-50 copies of HPV 18). Epifluorescence microscopy and flow cytometry allowed detection of 600 copies in CaSki cells; in addition, cell fixation was found to influence the fluorescent intensity. Several procedures were assayed to increase the sensitivity of in situ hybridization. The use of biotinylated HPV-16 oligonucleotides as probes was not effective, because only CaSki cells were positive. After amplification of HPV-16 or -18 DNA sequences with polymerase chain reaction (PCR) on whole cells in suspension and hybridization with plasmid probes, fluorescent hybridization spots were found in CaSki and HeLa cells by both epifluorescence microscopy and flow cytometry. The various procedures applied for revelation of DNA-DNA hybrids (use of phycoerythrin or cyanine instead of fluorescein, Pinkel's 3-step amplified system of fluorescein) did not enhance the sensitivity of in situ hybridization. HPV DNA was very effectively detected by cell examination under a laser-scanning confocal microscope, since 1-2 copies of HPV 16 were observed in SiHa cells without previous PCR amplification. Thus, the efficacy of in situ hybridization for HPV detection may be conditioned by different factors. Laser-scanning microscopy represents an alternative to the use of PCR amplification. These techniques are potentially useful to study single genes.

DNA Probes, HPV↗
PubMed1996

Molar quantification by flow cytometry of fatty acid binding to cells using dipyrrometheneboron difluoride derivatives.

Fatty acid analogs of a dipyrrometheneboron difluoride fluorophore (BDY-FA) have recently been developed. Relative to other fluorescent fatty acids, some of these have the advantages of excitation and emission spectra similar to those of fluorescein and of high quantum yield, which permits their use in conventional argon laser cytometry or microscopy. For the cytofluorimetric quantification of BDY-FA analogs, expressed as molecules bound per cell, we have compared the fluorescence of BDY-dodecanoic acid (BDY-C12) with that of fluorescein. Fluorescent beads with different amounts of bound fluorescein were used to calibrate a flow cytometer in order to correlate the fluorescence intensity with the number of fluorescein molecules per bead. In addition, starting from the basic equation defining the relationship between fluorescence and concentration, we have derived another equation which makes it possible to establish, for a given fluorescence, the relative molar concentration of both fluorochromes and, consequently, to express the fluorescence intensity emitted by the BDY-FA as the equivalent number of BDY-FA molecules. As an example of the potential application of this procedure, the time-course and concentration-dependent binding of BDY-C12 to quiescent and mitogen-activated human lymphocytes and to cultured human T-lymphoma cells have been studied. The method described is of general interest as it can also be applied to the flow cytometric or laser scanning microscopic quantification of other fluorescent dyes.

Boron Compounds↗
PubMed1994

[Damage to tumor cells by photodynamic therapy].

Photodynamic therapy (PDT) is a new, promising method in the treatment of cancer. To gain insights into PDT-mediated tumour destruction we studied the influence of treatment with Photofrin and laser light on changes in cell volume and cell viability. A-Mel-3 tumour cells were subjected to Photofrin or illumination with laser light, or a combination of both (PDT). Cell volume was measured by flow cytometry and cell viability by the trypan blue exclusion test for up to 60 min after PDT and the respective controls. In addition, scanning and transmission electron microscopy were performed. Tumour cells incubated in concentrations of 0.75, 1.5 and 3.0 micrograms Photofrin/ml revealed a rapid increase in cell volume to 117%, 207% and 235% 30 min after PDT and to 147%, 210% and 199% 60 min after PDT. Cell viability with 1.5 and 3.0 micrograms Photofrin/ml and laser light was reduced to 83% and 44% at 30 min after PDT and to 38% and 17% 60 min after PDT. At Photofrin concentrations of 1.5 micrograms/ml and exposure to laser light scanning electron microscopy revealed extreme loss of microvilli and formation of blebs on the cellular surface. Transmission electron microscopy showed swollen mitochondria and ruptures of the cell membrane. This study demonstrates that PDT induces a significant time-dependent and dose-related increase in tumour cell volume. We suggest that the PDT-induced swelling of tumour cells contributes to the increase of interstitial fluid pressure and to impairment of microvascular perfusion of tumours.

Cell Division↗