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PubMed2001

Excessive base excision repair of 5-hydroxymethyluracil from DNA induces apoptosis in Chinese hamster V79 cells containing mutant p53.

We have demonstrated previously that the toxicity of 5-hydroxymethyl-2'-deoxyuridine (hmdUrd) to Chinese hamster fibroblasts (V79 cells) results from enzymatic removal of large numbers of hydroxymethyluracil residues from the DNA backbone [Boorstein,R. et al. (1992) Mol. Cell. Biol., 12, 5536-5540]. Here we report that a significant portion of the hmdUrd-induced cell death that is dependent on DNA base excision repair in V79 cells is apoptosis. Incubation of V79 cells with pharmacologically relevant concentrations of hmdUrd resulted in the characteristic changes of apoptosis as measured by gel electrophoresis, flow cytometry and phase contrast microscopy. However, hmdUrd did not induce apoptosis in V79mut1 cells, which are deficient in DNA base excision repair of 5-hydroxymethyluracil (hmUra). Apoptosis was not prevented by addition of 3-aminobenzamide, which inhibits synthesis of poly(ADP-ribose) from NAD, indicating that apoptosis was not the direct consequence of NAD depletion. Pulsed field gel electrophoresis indicated that hmdUrd treatment resulted in high molecular weight (2.2-4.5 Mb) DNA double-strand breaks prior to formation of internucleosomal ladders in V79 cells. Simultaneous measurement of DNA strand breaks with bromodeoxyuridine/terminal deoxynucleotidyl transferase-fluorescein isothiocyanate labeling and of cell cycle distribution indicated that cells with DNA strand breaks accumulated in late S/G(2) and that hmdUrd-treated cells underwent apoptosis after arrest in late S/G(2) phase. Our results indicate that excessive DNA base excision repair results in the generation of high molecular weight DNA double-strand breaks and eventually leads to apoptosis in V79 cells. Thus, delayed apoptosis following DNA damage can be a consequence of excessive DNA repair activity. Immunochemical analysis showed that both V79 and V79mut1 cells contained mutant p53, indicating that apoptosis induced by DNA base excision repair can be independent of p53.

Animals↗

PubMed2006

A study on medium chain length-polyhydroxyalkanoate accumulation in Escherichia coli harbouring phaC1 gene of indigenous Pseudomonas sp. LDC-5.

AIMS: This study is mainly focused on the heterologous expression and accumulation of polyhydroxyalkanoates (PHA) in Escherichia coli. METHODS AND RESULTS: PHA synthase gene (phaC1) from indigenous Pseudomonas sp. LDC-5 was amplified by PCR and cloned in E. coli (Qiagen EZ competent cells). The recombinant E. coli was analysed and confirmed for its expression of phaC1 gene by phase contrast microscopy, Western blot analysis and spectral studies (Fourier-transform infrared spectroscopy). It was further evaluated for its accumulation in different carbon and nitrogen sources. The accumulation of PHA (3.4 g l(-1)) was enhanced in the medium supplemented with glycerol and fish peptone compared to the other carbon and nitrogen sources used in this study. CONCLUSIONS: This study would enable the reduction of cost of PHA production. SIGNIFICANCE AND IMPACT OF THE STUDY: An important part of this study is that E. coli harbouring partial phaC1 gene could accumulate medium chain length PHA significantly. The results demonstrated that the E. coli strain could be a potential candidate for the large-scale production of polymer. The conditions for the higher yield and productivity will be optimized in the next phase using fermentation studies.

Acyltransferases↗

PubMed2004

Human corneal endothelial cell proliferation: potential for use in regenerative medicine.

PURPOSE: To review and update the experience of our laboratory in culturing human corneal endothelial cells (HCEC) from young and older donors. METHODS: Corneas were obtained from National Disease Research Interchange, Philadelphia, PA. Data from the past 3 years were reviewed to develop criteria for selecting donor corneas to be used for endothelial cell culture. Immunocytochemical localization using mAb 9.3.E identified endothelial cells, and Ki67 staining demonstrated actively cycling cells. Cell counts demonstrated the effect of growth-promoting agents on proliferation of cells from young (<30 years old) and older (>50 years old) donors. Phase-contrast microscopy documented morphologic characteristics of cells in primary culture and the effect of growth factors on cell morphology. RESULTS: Exclusion criteria were developed to increase the chance of successful culture of HCEC. Isolation methods to remove Descemet membrane with attached endothelial cells avoided contamination with other corneal cell types. EDTA treatment combined with mechanical disruption facilitated isolation of cells. Culture medium containing FBS, EGF, NGF, and bovine pituitary extract stimulated maximal growth and facilitated normal monolayer formation. Age-related differences were detected in the density of confluent cells in primary culture and in the proliferative response to growth-promoting agents. CONCLUSIONS: Untransformed HCEC can be successfully cultured from the corneas of both young and older donors by using care in the selection of donor material. Care must also be taken in the early phases of endothelial cell isolation to obtain maximal numbers of healthy cells for culture. There appear to be true age-related differences in overall proliferative capacity; however, the relative response to specific growth factors was similar in cells from young and older donors. Results of these studies provide guidelines for successful growth of untransformed HCEC for use in regenerative medicine.

Adolescent↗

PubMed1988

The control of DNA replication in a cell-free extract that recapitulates a basic cell cycle in vitro.

Cell-free extracts prepared from Xenopus eggs support chromosome decondensation and pronuclear formation on demembranated sperm heads. 32P-dCTP pulse-labelling studies demonstrate that DNA synthesis occurs in multiple bursts of 30-40 min in extracts containing pronuclei, each burst being followed by a period of 20-50 min during which no synthesis occurs. Density substitution with bromodeoxyuridine indicates that the synthesis in each burst is semiconservative and results from new initiations, and that, following multiple bursts of synthesis, reinitiation events can occur. Changes in nuclear morphology have been characterized in the extract by phase-contrast microscopy and by fluorescence microscopy following pulse labelling with biotin-11-dUTP and staining with anti-lamin antibodies. Lamin accumulation occurs as DNA decondenses and parallels the acquisition of membrane structures. Biotin-11-dUTP incorporation is first observed in small nuclei having decondensed DNA and an extensive lamina. While DNA synthesis is occurring nuclei remain relatively small, but rapid swelling accompanied by chromosome condensation occurs when biotin incorporation ceases. Nuclear swelling and chromatin condensation is followed by nuclear membrane breakdown, lamin dispersal and chromosome formation. Mitosis lasts for approximately 20 min. Nuclear reassembly is recognized by the appearance of membrane vesicles around small pieces of decondensed DNA, which parallels the appearance of lamin islands within a chromatin mass. These 'islands' incorporate biotin, indicating that DNA synthesis is occurring, and apparently fuse as larger S-phase nuclei are formed. Extensive protein synthesis occurs for at least 4 h in most extracts. This synthesis is required for the initiation of mitotic events and the reinitiation of DNA synthesis.

Animals↗

PubMed1984

Generation of flagella by cultured mouse spermatids.

During the short-term culturing of mouse spermatogenic cells, flagella were generated by round spermatids previously lacking tails. Unseparated germ cells were obtained by enzymatic treatments and round spermatids (greater than 90% pure) were purified by unit gravity sedimentation. As determined by Nomarski or phase-contrast microscopy, no cells had flagella immediately after isolation; flagella were first clearly detected after 6 1/2 h of culture in Eagle's minimal essential medium containing 10% fetal bovine serum and 6 mM lactate. After 24 h, approximately 20% of round spermatids had formed flagella. Multinucleated round spermatids often formed multiple flagella, the number never exceeding the number of nuclei per symplast. Round spermatids were the only spermatogenic cells capable of tail formation. Flagella elongation was blocked by 1 microM demecolcine, an inhibitor of tubulin polymerization. Indirect immunofluorescence localized tubulin in the flagella. As seen by scanning electron microscopy, flagella developed as early as 2 h after culture and continued to elongate over the next 20 h, reaching lengths of at least 19 micron. Transmission electron microscopy demonstrated that flagella formed in culture resembled flagella from Golgi-phase round spermatids in situ; the flagella consisted of "9+2" axonemes lacking other accessory structures such as outer dense fibers and the fibrous sheath. As determined by acridine orange staining of the developing acrosomes, all spermatids that formed flagella in culture were Golgi-phase spermatids. By these criteria, the structures are indeed true flagella, corresponding in appearance to what others have described for early mammalian spermatid flagella in situ. We believe this is the first substantiated report of limited in vitro differentiation by isolated mammalian spermatids.

Animals↗

PubMed1975

Inhibition of formation of microtubular paracrystals in HeLa-S3 cells by neocarzinostatin.

The effect of an antitumor antibiotic, neocarzinostatin (NCS), on the formation of microtubular paracrystals (PC) induced by vinblastine sulfate, 10 mug/ml, in HeLa-S3 cells was examined by phase-contrast microscopy. The pretreatment of HeLa-S3 cells with NCS, 5 to 50 mug/ml, for 4 hr prevented the PC formation, and there was a dose response of NCS to the degree of inhibition. When the same inhibitory effect on PC formation was examined with other antitumor agents at high doses (50 mug/ml), colchicine was found to be one of the most effective agents, like NCS. Puromycin, antimycin, adriamycin, cytochalasin B, and cycloheximide revealed moderate activity, and the other antibiotics, such as mitomycin C, bleomycin, and rifampicin, did not show any effect at all. NCS was a unique antibiotic that inhibited PC formation among inhibitors of DNA synthesis. It was suggested that NCS affects the microfibrillar-microtubular proteins system in vivo, resulting in the inhibitions of organization of spindle fibers from microtubules at the G2 phase in HeLa cells.

Anti-Bacterial Agents↗

PubMed2001

Apoptosis, necrosis, and cell proliferation induced by S-(1,2-dichlorovinyl)-L-cysteine in primary cultures of human proximal tubular cells.

Apoptosis, necrosis, and cell proliferation induced by S-(1,2-dichlorovinyl)-L-cysteine (DCVC), the cysteine conjugate of the environmental and occupational contaminant trichloroethylene, were studied in primary cultures of human proximal tubular (hPT) cells. Cells from male and female donors were incubated with a range of concentrations of DCVC (10 to 1000 microM) for up to 48 h, and assessments of cellular morphology (phase-contrast microscopy), necrosis (lactate dehydrogenase (LDH) release), apoptosis(cell cycle analysis, annexin V staining, and caspase activation), and proliferation (cell cycle analysis and DNA synthesis) were made. Time- and concentration-dependent changes in cellular morphology, including elongation of cell shape, formation of intracellular vesicles, and formation of apoptotic bodies, were observed. Significant increases in LDH release occurred in hPT cells incubated with < or =100 microM DCVC for at least 24 h. hPT cells from males were modestly more sensitive to DCVC than those from females, with maximal LDH release of 78 and 65% in cells from males and females, respectively. Flow cytometry analysis of propidium iodide-stained and DCVC-treated hPT cells showed that apoptosis occurred at markedly lower concentrations (10 microM) and at much earlier incubation times (2 h) than necrosis. A small increase was also noted in the percentage of cells in S-phase after a 4-h treatment with as little as 10 microM DCVC, suggesting that cell proliferation was stimulated. This was supported further by increased DNA synthesis. These results show that DCVC causes apoptosis and enhances cell proliferation in hPT cells at environmentally relevant doses and at earlier time points and lower concentrations than necrosis.

Adult↗

PubMed1984

Human autoantibodies against desmosomes: possible causative factors in pemphigus.

Pemphigus is a human disease that causes extensive blistering of the skin. This blistering is related to a loss of epidermal cell cohesion and is accompanied by circulating autoantibodies that stain epidermal cell surfaces, as shown by immunofluorescence microscopy. One of the major components involved in epidermal cell cohesion is the desmosome. The pathological changes that accompany pemphigus led us to determine whether the autoantibodies are specific for desmosomes. Incubation of cultured mouse keratinocytes in medium containing pemphigus antiserum leads to cell separation at cell-cell contact sites, which possess desmosomes. Tissue sections of mouse skin processed for indirect immunofluorescence, using pemphigus antiserum or a rabbit antiserum directed against components of desmosomes, show similar punctate cell-surface staining patterns within the epidermis. Cultured mouse keratinocytes possessing well-defined intermediate filament bundles (tonofilaments) and desmosomes were processed for double indirect immunofluorescence, using a monoclonal antibody directed against mouse skin keratin and either pemphigus antiserum or the desmosome antiserum. The keratinocytes exhibit a complex system of keratin-containing tonofilaments. Tonofilaments in contacting cells are separated by thin dark bands at the cell surface, which correspond precisely to desmosomal plaques seen by phase-contrast microscopy. These bands specifically stain with both pemphigus antiserum and the desmosome antiserum. Double indirect immunofluorescence of the cultured mouse keratinocytes, using pemphigus antiserum and the desmosome antiserum, reveals that the pemphigus autoantibodies stain the same areas of cell-cell contact as the desmosome antibodies. Our evidence supports the idea that pemphigus blisters form, at least in part, from a specific antibody-induced disruption of desmosomes in the epidermis.

Animals↗

PubMed2002

Telomere and telomerase in the initial stage of immortalization of esophageal epithelial cell.

AIM: To search for the biomarker of cellular immortalization, the telomere length, telomerase activity and its subunits in cultured epithelial cells of human fetal esophagus in the process of immortalization. METHODS: The transgenic cell line of human fetal esophageal epithelium (SHEE) was established with E(6)E(7) genes of human papillomavirus (HPV) type 18 in our laboratory. Morphological phenotype of cultured SHEE cells from the 6th to 30th passages, was examined by phase contrast microscopy, the telomere length was assayed by Southern blot method, and the activity of telomerase was analyzed by telomeric repeat amplification protocol (TRAP). Expressions of subunits of telomerase, hTR and hTERT, were assessed by RT-PCR. DNA content in cell cycle was detected by flow cytometry. The cell apoptosis was examined by electron microscopy (EM) and TUNEL label. RESULTS: SHEE cells from the 6th to 10th passages showed cellular proliferation with a good differentiation. From the 12th to the 16th passages, many senescent and apoptotic cells appeared, and the telomere length sharply shortened from 23kb to 17kb without expression of hTERT and telomerase activity. At the 20th passage, SHEE cells overcame the senescence and apoptosis and restored their proliferative activity with expression of telomerase and hTERT at low levels, but the telomere length shortened continuously to the lowest of 3kb. After the 30th passage cells proliferation was restored by increment of cells at S and G2M phase in the cell cycle and telomerase activity expressed at high levels and with maintenance of telomere length. CONCLUSION: At the early stage of SHEE cells, telomeres are shortened without expression of telomerase and hTERT causing cellular senescence and cell death. From the 20th to the 30th passages, the activation of telomerase and maintenance of telomere length show a progressive process for immortalization of esophageal epithelial cells. The expression of telomerase may constitute a biomarker for detection of immortalization of cells.

Apoptosis↗

PubMed1997

Expression of extracellular matrix proteins in human periodontal ligament cells during mineralization in vitro.

Periodontal regeneration is a complex process that requires coordinated responses from several cell types within the periodontium. It is generally accepted that the periodontal ligament (PDL) has a heterogeneous cell population, where some of the cells may be capable of differentiating into either cementoblasts or osteoblasts. Thus, it has been hypothesized that PDL cells play a role in promoting periodontal regeneration. However, definitive evidence to support this concept is lacking. Previously, we reported that PDL cells induce biomineralization as determined by Von Kossa histochemistry and transmission electron microscopy. To further determine the osteoblast-like properties of PDL cells, human PDL cells were exposed to dexamethasone (DEX) in order to promote an osteoblast phenotype, and then cell activity monitored during mineral nodule formation in vitro. For mineralization studies, cells were cultured in DMEM containing 10% FBS and a) vehicle only; b) ascorbic acid (50 micrograms/ml) and beta-glycerophosphate (10 mM); or c) ascorbic acid, beta-glycerophosphate and DEX (100 nM) for 30 days. In addition, the effects of DEX on PDL cells in non-mineralizing media were determined. Cells were stained weekly to evaluate mineral-like nodules, using the Von Kossa method. Northern blot analyses for mRNA steady state levels for several bone-associated proteins, i.e., osteopontin (OPN), bone sialoprotein (BSP), alkaline phosphatase (ALP), osteocalcin (OCN), alpha 2(1)(type 1) collagen and osteonectin (ON), were performed. DNA levels were also determined during the 30-day mineralization period. Under phase contrast microscopy, PDL cells in non-mineralizing media treated with DEX exhibited a more spindle-shaped morphology when compared with similar cells not exposed to DEX. Mineralizing conditions were required to induce mineral nodule formation. However, in this situation, mineral induction was independent of DEX; and furthermore, DEX-treated cells did not exhibit a different morphological pattern when compared with non-DEX treated cells. Mineral-like nodules were first seen at day 15, in concert with an increase followed by a decrease in expression of type I collagen and ON mRNA in both DEX-treated and non-treated cultures. Using Northern blot analysis for detection of specific proteins, we found that PDL cells did not express OPN, BSP, OCN, or ALP under any of the conditions used in this study. DEX did not alter DNA content in the cultures during the mineralization period. These results confirm that human periodontal ligament cells can be induced to mineralize in vitro and indicate that dexamethasone does not significantly alter the extent and pattern of mineralization.

Alkaline Phosphatase↗

PubMed2004

[Surface architectonics and cytoskeleton of erythrocytes and their modulation by adrenergic agents in patients with bronchial asthma].

AIM: To evaluate erythrocyte surface architectonics and cytoskeleton in bronchial asthma (BA) under modulation of adrenergic agents. MATERIAL AND METHODS: 24 healthy persons, 61 patients with bronchial asthma (BA) were examined. Of them, 28 patients had allergic BA (ABA) and 33 ones had nonallergic BA (NABA). Erythrocyte surface architectonics was studied by phase-contrast microscopy. Fixation in 0.5% solution of glutar aldehyde was used, preparation "squashed drop" was prepared. In all preparations adrenalin hydrochloride and obzidan were used in equimolar final concentrations of 4.10(-5) M. The mean morphologic index of transformation that reflects the shift to stomatocytosis or to echinocytosis was estimated to characterize integrally erythrocyte surface architectonics. The cytoskeleton was studied by the modified method of Chentosov's et al. The integral optic density was evaluated on imaging analyzer of Ista-Videotest company (St-Petersburg). RESULTS: In healthy persons there was a correlation between erythrocyte surface architectonics and cytoskeleton under modulation of adrenergic system. Adrenalin-induced decrease of cytoskeletal proteins correlated with the shift to stomatocytosis. In NABA erythrocyte surface architectonics was characterized by a pronounced shift to stomatocytosis. This shift was accompanied with the most pronounced decrease of cytoskeletal proteins and it reduced in remission. In ABA manifestation of the shift was minimal and did not depend on the phase of the disease. In ABA no changes in erythrocyte surface architectonics in the presence of adrenalin were revealed. In the same conditions a decrease in cytoskeletal proteins was found. CONCLUSION: A correlation was found between erythrocyte surface architectonics and cytoskeleton in healthy persons. In ABA, under modulation of adrenergic system by adrenalin dissociation was revealed between receptor and cytoskeletal mechanisms of cell form induction. This dissociation does not depend on the phase of the disease and is thought to be one of the important postreceptor disorders typical for this variant of the disease.

Adrenergic beta-Antagonists↗

PubMed1983

Analysis of anaphase B in saccharomyces cerevisiae using a monoclonal antibody against yeast tubulin.

In Saccharomyces cerevisiae the mitotic spindle is composed of approximately 10 continuous microtubules, that pass uninterrupted between the 2 spindle pole bodies (SPBs), and up to 34 discontinuous microtubules, 17 of which arise from each SPB and terminate in the nucleoplasm [7, 12]. We have previously shown that during spindle elongation (anaphase B) the number of continuous microtubules decreases rapidly until at a length of 4 microns only 1 remains [8]. Further elongation from 4 to 8 microns is achieved with only a single continuous microtubule. Here we have extended our analysis of spindle elongation by investigating the timing of this event by indirect immunofluorescence microscopy using a monoclonal antibody to yeast tubulin. From the frequency distribution of spindle lengths in exponential phase cultures of the haploid wild type strain A364A, the time in mitosis and the velocity of spindle elongation have been calculated. For the first 55 min of mitosis spindle elongation proceeds at a slow rate, 0.36 microns/min. There is then a marked increase to a maximal value of 4.16 microns/min. Correlated with data from previous ultrastructural studies on the decrease in continuous microtubule number during anaphase B, this 12-fold increase in velocity is shown to occur at the single microtubule stage. These experiments provide further evidence to suggest that the role of the spindle is to regulate the rate of anaphase B which is actively achieved by an, as yet, unknown motive force generating system. Antibody staining has also allowed the terminal phenotypes of 2 diploid cell division cycle (cdc) mutants to be more accurately described. This has shown that, although arrested synchronously as judged by phase contrast microscopy, these strains do in fact exhibit considerable heterogeneity in spindle length.

Anaphase↗

PubMed1966

The regulation of pinocytosis in mouse macrophages. I. Metabolic requirements as defined by the use of inhibitors.

A method is described to study the formation of pinocytic vesicles in cultivated mouse macrophages. Vesicles which arise in the peripheral cytoplasm and are in transit to the centrosphere region are enumerated by the phase-contrast microscopy of glutaraldehyde-fixed cells. Under these conditions there is a prompt reversible response of vesicle formation to calf serum factors in the external environment. The reduction of pinosome formation by a variety of metabolic inhibitors was then studied in a medium containing 50% newborn calf serum. Inhibitors of both glycolysis and respiration reduced vesicle formation to low levels. This influence was most striking with cyanide, antimycin A, and anaerobiosis. This indicates an important contribution of an intact respiratory pathway in pinocytosis. Both 2,4-dinitrophenol and oligomycin inhibited vesicle formation at low concentrations. These data suggest a central role of ATP as an energy source for vesicle formation. Inhibitors of protein synthesis, such as puromycin and p-fluorophenylalanine, produced a prompt reduction in vesicle formation. The action of p-fluorophenylalanine was effectively reversed with L-phenylalanine. Actinomycin D depressed pinocytosis to low levels at concentrations of 0.1 to 0.003 microg/ml. This effect was observed only after a 120 min lag phase. A 10 degrees C reduction in ambient temperature lowered vesicle counts to 30% of control preparations at 37 degrees C.

Animals↗

PubMed1985

Hematopoietic factor production by a cell line (TC-1) derived from adherent murine marrow cells.

An adherent cell line, termed TC-1, has been isolated from long-term liquid culture of murine marrow cells by repeated exposure of the adherent cells to 0.1% trypsin. This is an alkaline phosphatase-positive cell line showing variable staining with acid phosphatase and alpha-naphthyl acetate esterase. On electron microscopy, the cells have moderate amounts of rough endoplasmic reticulum and variable numbers of polyribosomes. Some cells contain large clusters of laked glycogen particles. Intermediate junctions are present between some cells. Conditioned medium from this cell line produced from 384 to 638 units of CSF-1 per milliliter by radioimmunoassay and a CSF-1-dependent synergistic activity, which stimulates giant macrophage colony formation of marrow cells in soft agar. The conditioned medium also stimulates 3H-TdR incorporation by marrow cells in liquid culture and induces secondary adherent cell lines. The growth factor(s) produced by the TC-1 stromal cell line may be important in the regulation of early stages of hematopoietic differentiation. Two subclones, TC-1-C-11 and TC-1-C-3, have been isolated from passage 25 of the TC-1 cells by a penicylinder separation technique. The TC-1-C-11 is phenotypically like the parent TC-1 line and produces macrophage growth factors. The TC-1-C-3 grows as an epithelioid monolayer with visible junctions among adjacent cells under phase contrast microscopy. This subclone produces retrovirus and is capable of providing anchorage support for hematopoietic stem cells. The TC-1 cell line and its subclones may provide models for the control of early stem cell proliferation and differentiation.

Agar↗

PubMed1993

Trophic influences of human and rat amniotic fluid on neural tube-derived rat fetal cells.

Normal human (week 17-20) and rat (E16-17) amniotic fluids were used as culture media for primary cultures of rat fetal (E 16) cortical, mesencephalic and striatal cell dissociates, or astroglial subcultures from the same brain regions. Phase-bright and dark cells were identified under phase contrast microscopy and their cell processes were measured utilizing semi-automated procedures. Subcultured astroglia were immuno-reacted against glial fibrillary acidic protein and fibronectin. Rat and human amniotic fluid allowed survival and growth of neuronal and non-neuronal cells. Human amniotic fluid samples were trophic in variable degrees. Cerebral cortex subcultured astroglia usually expressed a radial-like morphotype. Although charcoal-adsorbed human amniotic fluid was trophic for primary cultures, its ability to sustain neuritic growth depended on its degree of trophism before treatment. Growth of cell processes in neuronal- and glial-like cells in primary cultures was inhibited to different degrees by the addition of antisera towards nerve or epidermal growth factors. It is concluded that amniotic fluid constitutes a trophic medium for astroglia and neurons. Both, nerve and epidermal growth factors appear to be necessary for growth of cell processes in neuronal and glial primary cultures in amniotic fluid. Trophic effect of amniotic fluid on subcultured astroglia did not seem to be diminished by nerve growth factor antiserum. The role of amniotic fluid during the early phases of brain organogenesis is discussed.

Amniotic Fluid↗

PubMed1999

Interferon-gamma induces apoptosis and expression of inflammation-related proteins in Chang conjunctival cells.

PURPOSE: The purpose of this study was to investigate the effect of interferon (IFN)gamma on cell viability, cell growth, and apoptosis and on expression of apoptotic and inflammation-related proteins in epithelial conjunctival cells in vitro. Some aspects of transduction pathways of IFNgamma-induced alterations were also investigated, especially the role of protein kinase C (PKC) and IFNgamma transcriptional factor STAT1. METHODS: A human conjunctival cell line was treated with different concentrations (30 and 300 U/ml) of human recombinant IFNgamma. After 24, 48, and 72 hours of treatment, cell viability and relative cell number were studied with 3-(4,5-dimethylthiazol-2yl)2,5-diphenyl tetrazolium bromide (MTT) and crystal violet colorimetric assays. The apoptotic process was sought by phase-contrast microscopy, 4',6'-diamidino-2-phenylindole dihydrochloride (DAPI) staining, and transmission electron microscopy and was confirmed by DNA electrophoresis and immunoblotting of poly(ADP-ribose) polymerase (PARP). The cell cycle and expression of apoptotic proteins Fas, bax, and p53; of inflammation-related proteins HLA-DR and intercellular adhesion molecule (ICAM)-1; and of IFNgamma signal-transducing factor STAT1 were evaluated by flow cytometry and/or western blot analysis. To investigate PKC-related transduction pathways, two PKC modulators, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and staurosporine, were applied for 3 hours, followed by IFNgamma treatment for 72 hours. Moreover, the effects of PKC depletion were studied after a 24-hour application of TPA, also followed by IFNgamma treatment for 72 hours. Then, Fas, ICAM-1, and HLA-DR expressions were studied by flow cytometry. RESULTS: IFNgamma at 30 U/ml induced no change in cell cycle and in cell viability. Cell viability significantly decreased after 48 hours of treatment with 300 U/ml IFNgamma, associated with cell cycle alterations (decrease in number of cells in the S-M phase), apoptotic chromatin condensation and fragmentation, ladder pattern on DNA electrophoresis assay, and cleavage of PARP. Moreover, IFNgamma-treated cells overexpressed plasma membrane Fas, HLA-DR, and ICAM-1 in a dose- and time-dependent manner, and STAT1 in both nuclear and cytosolic cell fractions. Only 300 U/ml IFNgamma-treated cells overexpressed bax, whereas Bcl-2 and p53 proteins were not modified. HLA-DR and Fas were upregulated after addition of staurosporine or after PKC-depleting treatment and repressed with TPA. Staurosporine, PKC depletion, and TPA all enhanced ICAM-1 expression. CONCLUSIONS: In our model, IFNgamma induced expression of inflammatory molecules and apoptotic mediators, cell growth arrest, and apoptosis of Chang conjunctival cells. Moreover, our results suggest that activation of PKC is not involved in some IFNgamma cellular effects that possibly imply the upregulation and nuclear translocation of STAT1. IFNgamma-induced apoptosis could explain in part the recently reported coexistence of inflammation and programmed cell death in ocular surface inflammatory disorders such as Sjögren's syndrome.

Apoptosis↗

PubMed2006

Morphometric changes in goat sperm heads induced by cryopreservation.

Two experiments were designed to evaluate the effect of cryopreservation on morphometric characteristics of the goat sperm head. To address this question, we evaluated the size of the sperm head in fresh control cells, post-cooling cells after equilibration with the glycerol preservation solution, and post-thawing cells. Assessment was by automated morphometric sperm head analysis (ASMA) using phase-contrast microscopy without staining. In the first experiment, ASMA was performed on heterospermic pooled samples (fresh, post-cooling after equilibration with the glycerol preservation solution and post-thawing): length, width, area and perimeter were measured. In the second experiment, sperm viability was assessed by Hoechst staining and head morphometry was carried out as before, simultaneously during the cryopreservation process, and the head size was identified for both live and dead spermatozoa. The data were analysed by principal component analysis (PCA). The purpose of PCA is to derive a small number of linear combinations (principal components) from a set of variables (length, width, area and perimeter), that retain as much of the information in the original variables as possible. The main findings that have emerged from this study are that (i) a simple procedure has been developed for measuring spermatozoa heads without staining, which minimises the possibility that sperm head dimensions were influenced by procedural artefacts; (ii) the dimensions of goat sperm heads after cryopreservation in skimmed milk-glucose medium were smaller than in fresh sperm, but this was due to the equilibration phase with the cryoprotectant and not to the cryopreservation process itself; and (iii) dead spermatozoa showed smaller heads than live sperm, consequent upon the loss of membrane function. No differences were observed between post-cooling cells after equilibration with the glycerol preservation solution and post-thawing spermatozoa and only minor osmotic differences between them and fresh sperm were observed.

Animals↗

PubMed1982

The synthesis of elastin, collagen, and glycosaminoglycans by high density primary cultures of neonatal rat aortic smooth muscle. An ultrastructural and biochemical study.

Primary cultures of neonatal rat aortic smooth muscle cells inoculated at high densities (1 X 10(6) cells/25 cm2 Falcon flask) with adequate nutrient media and pH control grow rapidly and form multilayers of cells with typical "hill and valley" organization. After 10 days growth insoluble elastin formation could be visualized by phase contrast microscopy as small particles which grew rapidly to become larger irregular refractile aggregates and later coalesced to form larger aggregates and small fibres. With light and electronmicroscopy, elastin was the predominant matrix protein formed, with the "hill regions" of cultures containing abundant elastin aggregates and some collagen. In 2-week-old cultures differentiation could be observed within the cell multilayer. The older deeper cells contained more protein synthesis organelles and myofilaments and were in close association with large often coalescing elastin aggregates; compared to younger more superficial cells which contained more free polyribosomes less myofilaments, and were associated with fewer and small elastin aggregates. In older cultures this differentiation was not apparent; the cells contained many myofilaments, dense bodies, and lysosomes. Elastin aggregates and newly formed elastic fibres were abundant in the matrix. Quantitative analysis of insoluble elastin formation in the cell layer during the 4-week culture period indicated continuous biosynthesis and deposition which paralleled that of desmosine formation. Amino-acid analysis of a hot alkali insoluble residue (regarded as elastin) from 30-day-old cultures gave a profile identical with neonatal rat aortic elastin in vivo. Insoluble collagen formation in the cell layer tended to plateau after the log phase of growth was completed (10 days). Proteoglycans were found predominantly in the supernatant media. Glycosaminoglycan analysis revealed a profile of dermatan sulphate (32%), chondroitin 4-sulphate (43%), keratan and heparan sulphate (30%), with only a trace of hyaluronic acid. This study indicates that primary cultures of neonatal rat aortic smooth muscle cells remain differentiated in culture and have the unique capacity to continue to synthesize and deposit large amounts (mg) of insoluble elastin which aggregate and from elastic fibres in vitro.

Animals↗