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PubMed1975

Effect of hypotonicity on cyclic adenosine monophosphate formation and action in vasopressin target cells.

We have shown previously that overhydration of toads renders their urinary bladders less responsive to the antidiuretic action of vasopressin (AVP). The present study investigates the relationship between osmotic swelling of vasopressin target cells and their sensitivity to AVP and dibutyryl cyclic adenosine monophosphate (db-cAMP). Conditions which engender osmotic swelling of toad bladder epithelial cells, such as immersing bladders on both surfaces in hypotonic Ringer's fluid or subjecting them to a net mucosal-to-serosal volume flux, markedly inhibited the effectiveness of db-cAMP in raising bladder permeability to water. This inhibitory phenomenon was seen both with serosal and mucosal applications of the nucleotide. Examination of isolated epithelial cells by phase contrast microscopy showed them to behave as osmometers, doubling their volume as the effective osmolality of the incubation medium was halved. AVP was found to increase the total content of cAMP about 3.5-fold both in the swollen and the normal cells, so that the actual concentration of cAMP may have diminished as the cell volume increased. Consistent with this suggestion was the observation that increasing exogenous db-cAMP abolished, in part, the inhibitory effects of hypotonicity. These observations indicate that homeostasis of body fluids in the toad depends in part upon the osmotic regulation of anti-diuretic homone action, and that intracellular cAMP may participate in coupling changes in cell volume to the altered state of responsiveness of the vasopressin target cell.

Animals↗

PubMed1968

Respiration and mitochondrial content in single neurons of the supraoptic nucleus. A correlative study in osmotic stress.

The study was undertaken to investigate the possible correlation of total volume of mitochondria per cell with the rate of succinate oxidation in isolated nerve cell bodies, after various functional stresses in the experimental animals. Significant cytological effects were found in the nerve cells of the supraoptic nucleus in rats which had been thirsting for 4-12 days or had been given 2% sodium chloride solution as a substitute for drinking water for a few weeks. Quantitation of mitochondria was done from electron micrographs. The cell volumes were calculated from sections of Epon-embedded tissue under phase-contrast microscopy. Succinate oxidation was measured on groups of 10 nerve cells with the microdiver technique. As a result of either thirst or sodium chloride load, the volume of mitochondria per nerve cell more than doubled. The rate of succinate oxidation was not changed after the rats had been thirsting but was enhanced by over 100% after they had drunk sodium chloride. A linear relationship was found for the amount of mitochondria versus respiration in the supraoptic neurons for all experimental groups except the thirsting animals. The mitochondria in the supraoptic neurons from thirsting animals were of the same size or smaller than those in controls, whereas in animals given sodium chloride solution the mitochondria were considerably enlarged. The observed effects were specific for the supraoptic nucleus.

Animals↗

PubMed2005

Glioma expansion in collagen I matrices: analyzing collagen concentration-dependent growth and motility patterns.

We study the growth and invasion of glioblastoma multiforme (GBM) in three-dimensional collagen I matrices of varying collagen concentration. Phase-contrast microscopy studies of the entire GBM system show that invasiveness at early times is limited by available collagen fibers. At early times, high collagen concentration correlates with more effective invasion. Conversely, high collagen concentration correlates with inhibition in the growth of the central portion of GBM, the multicellular tumor spheroid. Analysis of confocal reflectance images of the collagen matrices quantifies how the collagen matrices differ as a function of concentration. Studying invasion on the length scale of individual invading cells with a combination of confocal and coherent anti-Stokes Raman scattering microscopy reveals that the invasive GBM cells rely heavily on cell-matrix interactions during invasion and remodeling.

Cell Line, Tumor↗

PubMed1984

[Urinary sediment in acute renal insufficiency. Diagnostic and prognostic value of phase-contrast microscopic examination].

Living cells from the urinary tract can be examined by phase-contrast microscopy in a pellet obtained by centrifugation of 10 ml of freshly voided urine. Once these cells have been identified and classified according to their sources, their respective proportions can be evaluated, thus providing some information on the renal structures affected. Urine sediment examination was performed in 60 cases of acute renal failure in order to determine the relationship between the abnormalities encountered and the clinical or histological diagnosis. An abnormal sediment was always associated with parenchymal acute renal failure. Cellular debris and casts were abundant in acute tubular necrosis and less numerous in toxic acute renal failure than in failure resulting from shock. The finding of deformed erythrocytes was strongly suggestive of glomerular nephropathy, a diagnosis which was confirmed by renal biopsy in almost every case.

Acute Kidney Injury↗

PubMedDate not supplied

Culture and characterization of rat middle-ear epithelium.

This study was performed to design a method for the culture of rat middle-ear epithelium and to apply the method to investigate the characteristics of this epithelium. Culture of explants of middle-ear epithelium in the presence of the epidermal growth factor was successful, whereas serial cultivation required 3T3 feeder cells in addition to the epidermal growth factor. Cultured middle-ear epithelium was studied by phase-contrast microscopy, transmission and scanning electron microscopy, and combined light and scanning electron microscopy (LM/SEM). These techniques showed similarity between the cultured and the natural middle-ear epithelium. Explants and outgrowths showed both flat polygonal and ciliated epithelial cells. In serial cultivation, however, only the first of these cell types was observed. Frequently, a single primary cilium was found on the cell surface. Transmission electron microscopy showed cross-linked envelopes whose formation was promoted by ionophore X537A. Cytokeratin was demonstrated by immunoblotting, immunofluorescence, and immunoperoxidase methods, using an anti-cytokeratin monoclonal antibody. The model described here permits study of the differentiation of middle-ear epithelium in vitro and may be of future value for the study of chronic middle-ear diseases.

Animals↗

PubMed1998

Diisopropylglutathione ester protects A549 cells from the cytotoxic effects of sulphur mustard.

The A549 cell line was used to assess the ability of diisopropylglutathione (DIPE) to protect against a 100 microM challenge dose of sulphur mustard (HD) using gentian violet (GV), thiazolyl blue (MTT) and neutral red (NR) assays as indicators of cell culture viability. As part of a continuing study of the efficacy of protective nucleophiles as candidate treatments for HD poisoning, several different combinations of protectant and HD were used to determine the optimal means of protecting A549 cells from the effects of HD. It was found that DIPE (4 mM) could protect cells against the effects of HD though for optimal effect, DIPE had to be present at the time of HD challenge. Cultures protected with DIPE were up to 2.9-fold more viable than HD exposed cells 48 h after HD challenge when using the GV, MTT and NR assays to assess viability. Observations by phase contrast microscopy of GV stained cultures confirmed these findings. Pretreating A549 cultures with DIPE for 1 h followed by its removal prior to HD challenge did maintain cell viability, though at a relatively low level (only up to 1.4-fold more viable than HD only exposed cells). DIPE was also able to protect HD exposed A549 cultures when added to cell cultures at intervals of up to 12 to 15 min after the initial HD exposure, though viability tended to decrease over this period, so that at 1 h, addition of DIPE did not maintain the viability of the cultures. This is the first such report of the anti-HD protectant properties of DIPE in A549 cells. It is concluded that the protection observed against HD is probably largely due to extracellular inactivation of HD by DIPE.

Cell Line↗

PubMed1996

A new wound healing model of retinal pigment epithelial cells in sheet culture.

PURPOSE: To evaluate some RPE cell functions, such as wound healing, in a preparation more similar to in situ conditions, we developed a method to obtain and culture retinal pigment epithelial (RPE) cells as a sheet. And we assessed the effects of fetal bovine serum (FBS) on the rate of RPE wound healing. METHODS: We prepared RPE sheet cultures by incubating rat eyes in 0.1% proteinase K for 13 min, peeling away the neural retina-RPE complex, and then incubating the tissue for 1 h to promote spontaneous separation of the RPE sheet from the retina. After several days of incubation, the cultured sheets of RPE cells were examined by phase-contrast microscopy, scanning and transmission electron microscopy and immunocytochemistry. We made round defects 1 mm in diameter in cultured RPE sheets and estimated the rate of wound closure in media with different concentrations of FBS (0 to 10%). RESULTS: The RPE cells cultured in sheets retained their in situ features, including microvilli, tight junctions and gap junctions, and the distribution of actin and cytokeratin filaments. A wound was noted to close with restoration of a polygonal configuration. The rate of wound closure depended on serum concentration in the culture medium; when supplemented with 10% fetal bovine serum, wound closure was complete in approximately 40 h. CONCLUSIONS: The RPE sheet-culture technique we developed thus provides a suitable model for studying such RPE cell functions as wound healing or phagocytosis.

Animals↗

PubMed1995

Anticoagulant-induced pseudothrombocytopenia and pseudoleucocytosis.

Pseudothrombocytopenia (PTP) is the phenomenon of falsely low platelet counts due to in vitro platelet clumping in the presence of platelet autoantibodies and anticoagulants. We assessed anticoagulant-dependence, time course of platelet counts and impact of different counter devices on the phenomenon. Blood of 10 persons with previously recognized pronounced EDTA-dependent PTP was collected into 7 different anticoagulants and counted after different intervals in parallel in a Coulter T540 and a Coulter STKS counter and by phase contrast microscopy. With the Coulter T540 model PTP was most pronounced in blood samples anticoagulated with EDTA, Na-oxalate or Na-citrate. In the STKS counter EDTA, heparin and oxalate presented as the worst anticoagulants. The time course of platelet counts was significantly different between the two counters. Our results demonstrate that PTP is not restricted to EDTA, but is also present with other anticoagulants. In contrast, pseudoleucocytosis was observed only in EDTA-anticoagulated blood in the Coulter T540 device. We investigated the expression of platelet integrins and activation antigens on platelets of persons with anticoagulant-dependent PTP and in healthy controls without PTP. In the presence of EDTA the expression of GpIIb/IIIa was significantly reduced in the PTP subjects compared to control. Activation antigens CD62, CD63 and thrombospondin-antigen were upregulated in the presence of EDTA. These alterations in the expression of platelet antigens could also be induced on platelets of normal donors by incubation with sera of PTP subjects and EDTA.(ABSTRACT TRUNCATED AT 250 WORDS)

Anticoagulants↗

PubMed2002

Biochemical effects and growth inhibition in MCF-7 cells caused by novel sulphonamido oxa-polyamine derivatives.

The novel polyamine derivatives sulphonamido oxa-spermine (oxa-Spm) and sulphonamido oxa-spermidine (oxa-Spd) exhibited rapid cytotoxic action towards MCF-7 human breast cancer cells with IC50 values of 4.35 and 6.47 pM, respectively, after 24-h drug exposure. Neither compound is a substrate of serum amine oxidase. Both oxa-Spm and oxa-Spd caused cell shrinkage, as determined by phase-contrast microscopy. After incubation with 10 microM of either compound for 8 h, the cells underwent chromatin condensation and nuclear fragmentation. However, no clear DNA ladder was obtained by electrophoresis. The sulphonamido oxa-polyamine derivatives and especially oxa-Spd enhanced the activity of polyamine oxidase (PAO), an enzyme capable of oxidising N1-acetylated spermine and spermidine to spermidine and putrescine, respectively, generating cytotoxic H2O2 and 3-acetamidopropanal as by-products. The intracellular polyamine content was only marginally reduced in response to drug treatment. In conclusion, our data show that these novel sulphonamido oxa-polyamine derivatives possess high cytotoxic activity against MCF-7 cells and indicate that induction of PAO may mediate their cytotoxicity via apoptosis.

Amine Oxidase (Copper-Containing)↗

PubMed1991

Mineralized nodule formation in rat bone marrow stromal cell culture without beta-glycerophosphate.

Rat bone marrow stromal cells were cultured in the presence of 10 nM dexamethasone and various concentrations of beta-glycerophosphate. At day 12-15, some nodules consisting of polygonal cells were formed in all culture conditions, and these nodules were mineralized 2-3 days later. beta-Glycerophosphate significantly enhanced nodule formation at concentrations of not less than 5 mM. The mineralized nodules formed in the absence of beta-glycerophosphate were examined using phase-contrast microscopy, undemineralized and demineralized tissue histology, histochemistry for alkaline phosphatase, immunohistochemistry for type I, II, and III collagen, energy dispersive X-ray microanalysis, electron diffraction, and Fourier transform infrared spectroscopy (FT-IR). Mineralized nodules had histological characteristic similar to bone. Cells associated with nodules exhibited high alkaline phosphatase activity, and extracellular matrix of the nodules predominantly consisted of type I collagen. X-Ray microanalysis showed the presence of Ca and P in the mineralized area, and electron diffraction pattern showed the mineral to have apatite crystal structure. Moreover FT-IR indicated that the mineral was a mixture of hydroxyapatite and carbonateapatite. From these observations, it is concluded that the mineralized nodules formed in our culture system are truly bone-like.

Alkaline Phosphatase↗

PubMed2003

Isolation of a putative keratocyte activating factor from the corneal stroma.

Fetal bovine serum has commonly been used to expand the population of keratocytes in culture. Tissue extracts, however, have also been used to grow other cell types. We prepared a DMEM/F12 extract of corneal stroma and compared the growth and morphology of collagenase-isolated keratocytes cultured in DMEM/F12, or DMEM/F12 containing either stromal extract or fetal bovine serum. Cell proliferation was measured by 3H-thymidine and BrdU incorporation as well as by DNA quantitation. The extract was fractionated by gel filtration. Cell morphology was assessed by phase-contrast microscopy. Culture in both extract and serum stimulated keratocytes to proliferate, but keratocytes cultured in the extract grew more slowly due to a longer cell cycle and to a lower final density because of greater sensitivity to contact inhibition. Keratocytes cultured in serum became fibroblastic while those cultured in extract retained the dendritic morphology of quiescent keratocytes. The stimulating factors in the corneal extract were more sensitive to heat inactivation and of higher molecular weight than the stimulating factors in serum. These results indicate that the mitogenic activity in extract and serum are different and that the phenotypes resulting from growth in serum and extract are also different. Keratocytes cultured at low cell densities in the corneal extract may mimic keratocyte activation, an initial and crucial event for keratocytes during the corneal wound healing process.

Animals↗

PubMed2002

Electrophysiological recordings of patterned rat brain stem slice neurons.

Dissociated neuronal cultures on substrates patterned with extracellular matrix (ECM) proteins have yielded much information in the past. However, although the culture of brain slices has many advantages over dissociated neuronal cultures, its feasibility on patterned substrates has not been demonstrated to date. In the present study, neuronal outgrowth from brain stem slices onto homogeneous control substrates, and onto laminin structures of grid- and line-shape was achieved. Cultures were evaluated by means of phase contrast microscopy, antibody staining, and patch-clamp measurements. Only patterns with line sizes of more than 4 microm yielded satisfactory neuronal outgrowth. The size of the nodes in the pattern influenced the nodal compliance of the spreading cells and the amount of unstructured overgrowth. Best grid patterns were 4 microm lines and 10 microm nodes, best line patterns were 4 microm lines and 20 microm nodes. On patterned substrates, average sodium and potassium currents were reduced by approximately 50% compared to controls, whereas area-normalized ion-currents were in the same order of magnitude. This indicates that as a consequence of the pattern-enforced geometrical confinement, neurons tend to have a smaller surface. In addition, neurons on patterned substrates were rapidly covered with glial overgrowth. This was shown by antibody staining.

Animals↗

PubMed1974

Calcium-induced extrusion of secretory granules (exocytosis) in mast cells exposed to 48-80 or the ionophores A-23187 and X-537A.

Isolated peritoneal mast cells from rats were observed, by phase contrast microscopy, to extrude secretory granules when exposed to 48/80 or the ionophores A-23187 and X-537A, which are known to facilitate transmembrane fluxes of calcium. These effects were abolished when the cells were treated with EDTA and suspended in a Ca-free environment. Ca-deprived cells exposed to any one of the three drugs promptly extruded granules when calcium, but not magnesium, was added to the incubation medium. Such Ca-evoked or Ca-dependent responses persisted when Na was omitted from the incubation medium and replaced with sucrose, choline, or K. The responses thus seem independent of possible shifts in the alkali metal ions. The results are considered support for the view that Ca influx mediates stimulus-secretion coupling and does so by initiating exocytosis.

Animals↗

PubMed2003

In vitro toxicity of netilmicin and ofloxacin on corneal epithelial cells.

PURPOSE: To evaluate the in vitro cytotoxic effect of the aminoglycoside antibiotic netilmicin on rabbit corneal epithelial cells (SIRC) compared with ofloxacin, a commonly used fluoroquinolone ocular antibiotic. METHODS: SIRC cell cultures were incubated for 8 to 72 h in the presence and absence of netilmicin (1.5, 3, and 6 mg/mL) and equal concentrations of ofloxacin. Cell viability in treated and untreated SIRC cells was measured by both neutral red and MTT colorimetric assays at 8, 24, and 72 h, whereas changes in cell morphology were examined at 8, 24, 48, and 72 hours by the use of phase-contrast microscopy. RESULTS: Netilmicin, at all tested concentrations, failed to alter SIRC cell viability or morphology. In contrast, all concentrations of ofloxacin caused statistically significant dose- and time-dependent reductions in cell viability even after 8 h. After 72 h there was complete loss of cell viability. Morphologic examination of SIRC cells after 8 h of incubation with ofloxacin revealed that the fluoroquinolone antibiotic, at all concentrations, produced large numbers of dead cells, compromised intercellular contacts, and altered general morphology. After 48 h the cell monolayer was observed to be completely destroyed. CONCLUSION: Netilmicin, at the concentrations used, is an antibiotic devoid of obvious cellular toxicity and may also be considered as a suitable first-choice drug in the treatment of those pathologies that compromise the integrity of the ocular surface.

Animals↗

PubMed1990

Tissue culture of feline normal mammary gland.

To study the growth and morphology of feline mammary epithelial cells in vitro, normal feline mammary tissues as well as cells collected from feline milk were cultured and examined using phase-contrast microscopy and immunohistochemistry. The method employed was a modification of Hiratsuka et al., and was found to be useful for the culture of dissociated feline mammary tissue. Small polygonal cells and large spindle-shaped cells that were positively stained with anti-keratin and anti-actin antibodies formed colonies with a pavement-like structure in culture of mid-pregnant mammary gland. Cultures of involuting mammary gland were composed mainly of small spindle-shaped cells, which were negative for keratin immunostaining and thought to be fibroblasts. Myofibroblasts, which participate in feline mammary carcinogenesis, and show keratin negative, actin positive, were not encountered in the present culture system of normal mammary gland. The cells from milk formed small colonies, but did not grow to reach confluent monolayer.

Animals↗

PubMed1985

[Myocardial infarct of effort with normal coronaries. Study of in vivo platelet activation].

Platelet activation may play a part in causing myocardium infarction with angiographically normal coronary arteries. We investigated this possibility by performing ergometric stress testing in a series of 9 patients (Group A) who had suffered myocardial infarction after a violent effort with angiographically documented coronary insufficiency responsible for a stable effort angina (Group B) and 11 healthy subjects (Group C). Blood samples were taken separately before exercise, at the peak of exercise, and during the recovery period. Platelet morphology, a sensitive indication of the degree of platelet activation, was studied by phase contrast microscopy after immediate fixation of the blood. The percentage of non-discoidal platelets presenting with one or several spicules was measured. At the same time, the plasma concentrations of platelet factor 4 (PF4) and beta-thromboglobulin (beta-TG) were measured. At rest, there was no difference in platelet morphology or specific platelet proteins between the 3 groups. At the peak effort, there was a significant increase of the number of morphologically modified platelets in Groups A and B but not in healthy subjects. This platelet activation could not be linked to the presence of myocardial ischaemia because it was found both in patients with a negative maximal exercise stress test (Group A). Finally, no increase of the plasma concentrations of the platelet protein was observed in any of the groups.

Adult↗

PubMed1979

Microscopic observations on the filopodia of Entamoeba histolytica.

Living Entamoeba histolytica trophozoites were examined by phase-contrast microscopy. Intact critical point dried trophozoites were examined by transmission electron microscopy at an accelerating voltage of 1000 kV (HVEM) and by scanning electron microscopy (SEM). Half and quarter micrometer thick sections of epoxy-embedded trophozoites were examined by HVEM. Many of the trophozoites of 2 strains examined had surface filopodia, 1 to over 100 micrometers in length. The cytoplasm of filopodia was continuous with the cytoplasm and bounded by surface plasmalemma bearing a glycocalyx. Structures called "surface-active lysosomes with trigger," "dendritic plasmalemmal extensions," and "extra-amebic vesicles" by previous investigators probably represent portions of filopodia demonstrated in the present study. Filopodia appear to be of frequent normal occurrence in E. histolytica and may function in: (a) endocytosis or pinocytosis; (b) exocytosis; (c) attachment to substratum; (d) penetration of tissue; (e) release of cytotoxic substances; or (f) contact cytolysis of host cells.

Animals↗

PubMed2005

Amniotic membrane maintains the phenotype of rabbit retinal pigment epithelial cells in culture.

The success of surgical removal of choroidal neovascularisation followed by transplantation of autologous retinal pigment epithelial cells (RPE) for age-related macular degeneration (ARMD) may be limited by damage in Bruch's membrane. We investigated whether amniotic membrane (AM) might be used as an alternative basement membrane-containing matrix to support RPE growth and differentiation. Primary RPE plastic cultures were established from freshly enucleated Dutch belted rabbit eyes in DMEM/F12 containing 0.1 mM Ca(++) and 10% dialysed FBS. Upon subconfluence, cells were subcultured at 5000-9000 cells cm(-2) in the above-mentioned culture medium on intact AM (iAM), epithelially denuded AM (dAM) or plastic. After confluence, the Ca(++) concentration in the medium was increased to 1.8 mm for 4 weeks. Growth and morphology were monitored by phase contrast microscopy, and the phenotype by immunostaining with antibodies against cytokeratin 18, tight junction protein ZO-1, and RPE65 protein, and by transepithelial resistance (TER) measurement. Immunostaining to cytokeratin 18 confirmed the epithelial origin of isolated cells in both primary culture and subcultures. Compared to plastic cultures, RPE increased pigmentation within 24 hr after seeding on AM, with iAM being more pronounced than dAM. RPE adopted a hexagonal epithelial phenotype with more organised pigmentation, strong expression of ZO-1 and RPE65, and a significantly higher TER 4 weeks after Ca(++) switch on dAM. Our results indicate that AM may be used as a basement membrane-containing matrix to maintain RPE phenotype in vitro, and may facilitate subsequent transplantation to treat ARMD.

Amnion↗