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PubMedDate not supplied

Human HT-29 colon carcinoma cells: mucin production and tumorigenicity in relation to growth phases.

The main aim of this study was to determine whether changes in mucin production/secretion during the growth phases of a human adenocarcinoma cell line, HT-29, are associated with a different tumorigenic potential. HT-29 cells cultured in DMEM supplemented with 10% FCS were harvested in the exponential and post-confluent phases of growth. Metabolic labeling of the cells was performed using [3H]-glucosamine. Following a 24 hr-incubation period, radioactivity was measured in 1 ml-aliquots of cell cytosol and culture medium. Concurrently, mucin synthesis was assessed by size exclusion chromatography of [3H]-glucosamine-labeled glycoprotein in a Sepharose CL-4B column. Clonigenic assay in soft agar of pre- and post-confluent HT-29 cells was determined by scoring viable colonies according to size and number using phase-contrast microscopy. To assess in vivo tumorigenicity, pre-and post-confluent HT-29 cells (4 x 10(6)) in 0.2 ml DMEM were inoculated into nude mice. Tumor size and volume were recorded for 31 days. H-29 cells grew as multilayers of unpolarized, undifferentiated cells. Colony forming efficiency was similar at all growth stages. A significant increase in mucin synthesis was noted in HT-29 cells harvested in the exponential phase of growth compared to the stationary phase (148.3 +/- 41.9 versus 49.1 +/- 5.0, mean +/- SE, dpm/4 x 10(3) cells, p < 0.05). Mucin secretion was not significantly changed. Tumorigenicity in nude mice was consistently higher when the cells were injected in the exponential phase of growth. On day 31 after cell inoculation the average tumor volume/site was 332.7 mm3 in mice injected with HT-29 cells in log phase compared to 142.7 mm3 (p < 0.01) in animals which received post-confluent cells. Tumor weights were 0.36 g and 0.22 g respectively (p < 0.05). The present results indicate a definitive correlation between growth phases of HT-29 cells and mucin synthesis: mucin production was significantly higher in exponentially growing cells. The cloning efficiency in soft agar, a marker of in vitro tumorigenicity, was similar in HT-29 cells irrespective of the growth stage. A main finding of the present study was the increased in vivo tumorigenicity of colon cancer cells inoculated into athymic mice in the log phase of growth compared to cells harvested at post-confluence. These results are consistent with the view that mucin plays an important contributory role in determining tumorigenicity.

Animals↗
PubMed1999

Direct or referral microscopy of vaginal wet smear for bacterial vaginosis: experience from an STD clinic.

A new wet smear diagnostic criterion for bacterial vaginosis was applied to 124 consecutive female patients attending an STD clinic located in the centre of Copenhagen. Bacterial vaginosis was detected in 54 (44%) women, making bacterial vaginosis the most prevalent pathological condition encountered. A total of 47 (87%) of the women were symptomatic. Concomitant genital infections were found in 13 (24%) of these women, most often as vulvo-vaginal candidiasis. A correct microscopic diagnosis could also be obtained by sending the vaginal smear to the local microbiologist for rehydration and phase contrast microscopy. It is suggested that the previously described vaginal wet smear criteria are used in place of Amsel's criteria for routine diagnosis of bacterial vaginosis.

Adult↗
PubMed1998

Quantitative assessment of leading edge adhesion: reattachment kinetics modulated by injury-derived intracellular calcium predict wound closure rates in endothelial monolayers.

Migrating cells continually develop new substrate attachments at the leading edge (LE) in order to maintain traction for movement. This study evaluates the relationship between LE adhesion and wound closure by modulating injury-derived intracellular free Ca2+ ([Ca2+]i) signaling in endothelial cell (EC) monolayers following scrape-wounding. These data show that brief treatment with increased extracellular Ca2+ ([Ca2+]e) during wounding accelerated wound area closure rates by 50-65%, while brief treatments with calcium influx inhibitors reduced rates by 30-50%. Fura-2 studies in wounded monolayers indicated supranormal [Ca2+]e during wounding increased (by 52%), while influx-inhibitors decreased (by 36%) the percentage of cells exhibiting elevated plateau [Ca2+]i levels. Quantitative time-lapse interference reflection microscopy (IRM) together with indirect alphavbeta3 integrin immunofluorescence was used to measure the effects of 100 microM Gd3+ and 5 mM [Ca2+]e treatment on fractional LE adhesion after wounding. Influx inhibition blocked development of increased injury-derived LE adhesion. Measurements indicated a linear relationship (r2 = 0.99, 0.98) between LE adhesion, development rates (quantified as an association rate constant) and steady state wound closure rates. Changes in filopodial activity, as indicated by phase contrast microscopy, did not correlate with changes in wound closure rates, but an association existed between the percentile peak [Ca2+]i response and the initiation of filopodial activity, suggesting a role for filopodia in mediating Ca2+-sensitive acceleration. Taken together, our data suggest that injury-derived [Ca2+]i signaling may regulate wound closure rates by an adhesion-mediated mechanism.

Animals↗
PubMed1982

Monolayer culture of human endometrium: methods of culture and identification of cell types.

Monolayer cultures can be established from human endometrial tissue after enzymatic dispersal into isolated glands or single cells. Three cell types that have distinct morphology by light and electron microscopy are observed in the resulting primary cultures. One cell type, an elongated spindle cell, is similar in appearance to fibroblasts derived from other tissues. A second cell type forms colonies of tightly cohesive cells, ranging in shape from oval to polygonal. These cells have typical organelles and junctional complexes characteristic of epithelial cells from the endometrium. The third cell type assumes a pavement-like appearance composed of polygonal cells when viewed by phase contrast microscopy, but lacks distinctive ultrastructural features of epithelial cells. These cells in culture resemble the endometrial stromal cell, the predominant cell type of the human endometrium in vivo. The epithelial cell does not survive subculturing but the other two cell types can be passaged through several generations and can be stored in liquid nitrogen and subsequently returned to culture.

Cells, Cultured↗
PubMed1999

Timing of FtsZ assembly in Escherichia coli.

The timing of the appearance of the FtsZ ring at the future site of division in Escherichia coli was determined by in situ immunofluorescence microscopy for two strains grown under steady-state conditions. The strains, B/rA and K-12 MC4100, differ largely in the duration of the D period, the time between termination of DNA replication and cell division. In both strains and under various growth conditions, the assembly of the FtsZ ring was initiated approximately simultaneously with the start of the D period. This is well before nucleoid separation or initiation of constriction as determined by fluorescence and phase-contrast microscopy. The durations of the Z-ring period, the D period, and the period with a visible constriction seem to be correlated under all investigated growth conditions in these strains. These results suggest that (near) termination of DNA replication could provide a signal that initiates the process of cell division.

Animals↗
PubMed1991

Cytosolic Ca2+ deregulation and blebbing after HgCl2 injury to cultured rabbit proximal tubule cells as determined by digital imaging microscopy.

Acute injury to renal proximal tubule cells has previously been shown to result in elevated cytosolic Ca2+ ([Ca2+]i), blebbing, and eventual cell death. In this study, digital imaging fluorescence microscopy was used to evaluate these changes in response to HgCl2 treatment of cultured rabbit proximal tubular cells. Monolayer cells loaded with fura-2 were treated with 10, 50, or 100 microM HgCl2 in both 1.37 mM CaCl2-containing and nominally Ca(2+)-free (less than 5 microM) Hanks' balanced salt solution. [Ca2+]i was estimated by measuring the ratio of fluorescent image pairs (collected at 340- and 380-nm excitation), morphological changes were observed by phase-contrast microscopy, and viability was assessed by trypan blue exclusion. After exposure of cells to 10 microM HgCl2, [Ca2+]i initially increased about 2-fold by 5 min; after 50 or 100 microM HgCl2, [Ca2+]i rapidly rose 2- to 3-fold, peaked at 1-3 min, and then generally decreased slightly. In nominally Ca(2+)-free (less than 5 microM) medium, [Ca2+]i stabilized, but in 1.37 mM Ca(2+)-containing medium, [Ca2+]i continued to slowly rise, often reaching levels of fura-2 saturation. The rate and extent of blebbing and the rate of cell death were increased in the presence of 1.37 mM Ca2+. These results show that sustained elevations of [Ca2+]i precede both cell blebbing and cell death and that when these elevations are limited by removing extracellular Ca2+ the amount of blebbing is reduced and cell viability is prolonged.

Animals↗
PubMed1987

Electron microscopical studies of Strongyloides ratti infective larvae: loss of the surface coat during skin penetration.

Previous indications using radiolabelled larvae that Strongyloides ratti free-living infective larvae lose a surface coat during penetration of the skin were further investigated by transmission electron microscopy of the cuticle of S. ratti infective larvae in the free-living stage, after penetration of mouse skin, and after migration to the lungs. These studies demonstrated the presence of a faint electron-dense surface coat external to the epicuticle on free-living worms which was absent from larvae recovered from the skin and lungs. When free-living infective larvae were incubated in 10% CO2 at 37 C and then examined with phase-contrast microscopy, worms were observed in the process of losing this coat. These observations confirm the hypothesis that S. ratti infective larvae lose a surface coat during penetration of the skin.

Animals↗
PubMed1989

NBD-taurine uptake by alpha-type carbonic anhydrase cells of turtle bladder.

The uptake of the fluorescent anion, N-(7-nitrobenz-2-oxa-1,3 diazol-4-yl)aminoethanosulfonic acid-taurine (NBD-T) was examined in sheets of turtle bladder epithelial cells after either serosal or mucosal exposure to the fluorescent anion. Serosal uptake of NBD-T into a population of epithelial cells was demonstrated in thin epithelial sheets scraped from bladder sacs exposed to low (0.5 mM) concentrations of NBD-T sufficient to cause fluorescence in turtle erythrocytes. In nine sheets NBD-T fluorescence was observed in 14.4 +/- 1.3% of epithelial cells; by phase-contrast microscopy of the same fields, these cells were nongranular or carbonic anhydrase (CA) cells. An additional 8.1 +/- 1.0% of cells was nongranular, but failed to exhibit fluorescence after serosal NBD-T exposure, consistent with a beta-CA cell population. Apical uptake of NBD-T into epithelial cells was demonstrable only at high (5 mM) mucosal concentrations in both thin sheets and superfused bladder segments. In thin sheets, NBD-T fluorescence was observed in 15.8 +/- 0.7% of cells and appeared to have a punctate subapical distribution; phase-contrast of the same fields (n = 46) revealed a nongranular cell population of 21.9 +/- 0.9%. Nongranular cells corresponded closely to CA cells as identified with carboxyfluorescein fluorescence. Hence, apical NBD-T was taken up primarily by alpha-CA cells. In conclusion, NBD-T is transported by alpha-CA cells either across the basolateral membrane with high affinity comparable to that of turtle red cell band 3 protein or across the apical membrane with low affinity via a less-specific mechanism such as endocytosis.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1964

SPORULATION IN PROTOPLASTS AND ITS DEPENDENCE ON PRIOR FORESPORE DEVELOPMENT.

Fitz-James, Philip C. (University of Western Ontario, London, Ontario, Canada). Sporulation in protoplasts and its dependence on prior forespore development. J. Bacteriol. 87:667-675. 1964.-Phase-contrast and electron microscopy were used to decide whether cells from late growth or sporulating cultures could, where converted into protoplasts, complete the process of sporulation. The cell wall was found essential to the development of the forespore. Incomplete forespores formed a satellite protoplast. Completed forespores were enclosed by the sporangial protoplast, and only these or later forms were able to complete sporulation in protoplasts.

Bacillus megaterium↗
PubMed2006

Characterization of physical properties of supported phospholipid membranes using imaging ellipsometry at optical wavelengths.

Subnanometer-scale vertical z-resolution coupled with large lateral area imaging, label-free, noncontact, and in situ advantages make the technique of optical imaging ellipsometry (IE) highly suitable for quantitative characterization of lipid bilayers supported on oxide substrates and submerged in aqueous phases. This article demonstrates the versatility of IE in quantitative characterization of structural and functional properties of supported phospholipid membranes using previously well-characterized examples. These include 1), a single-step determination of bilayer thickness to 0.2 nm accuracy and large-area lateral uniformity using photochemically patterned single 1,2-dimyristoyl-sn-glycero-3-phosphocholine bilayers; 2), hydration-induced spreading kinetics of single-fluid 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine bilayers to illustrate the in situ capability and image acquisition speed; 3), a large-area morphological characterization of phase-separating binary mixtures of 1,2-dilauroyl-sn-glycero-3-phosphocholine and galactosylceramide; and 4), binding of cholera-toxin B subunits to GM1-incorporating bilayers. Additional insights derived from these ellipsometric measurements are also discussed for each of these applications. Agreement with previous studies confirms that IE provides a simple and convenient tool for a routine, quantitative characterization of these membrane properties. Our results also suggest that IE complements more widely used fluorescence and scanning probe microscopies by combining large-area measurements with high vertical resolution without the use of labeled lipids.

Cholera Toxin↗
PubMed1994

Filopodia initiate choices made by sensory neuron growth cones at laminin/fibronectin borders in vitro.

Localized expression of environmental cues is thought to provide directional information to migrating neuronal growth cones by enhancing or suppressing axon outgrowth over limited regions. To investigate how such a mechanism may function in vivo, we observed growth cones of embryonic chick dorsal root ganglion neurons at a substratum border between the extracellular matrix components laminin and fibronectin in vitro using time-lapse phase-contrast and interference reflection microscopy. We found that patterns of laminin and fibronectin could locally promote or suppress the direction of growth cone migration. While migrating on either laminin or fibronectin, at least 79% of growth cones changed their rate and/or direction of outgrowth upon contact with the alternative substratum, in a manner suggesting that growth cones were selecting one substratum over the other. Complex changes in growth cone behavior were initiated by filopodial contact with the alternate substratum, suggesting that filopodia were providing intracellular signals to the growth cone. Using interference reflection microscopy, we have found that selection of a substratum is independent of the degree of close contact to the substratum. We conclude that spatially localized ECM components can direct axon outgrowth by mechanisms based on intracellular signaling through growth cone filopodia.

Animals↗
PubMed1995

In vitro evaluation of donor liver preservation fluids on human hepatocyte function.

Successful liver transplantation depends on adequate preservation of cellular function. We therefore tested the effects of two currently used liver preservation fluids, Euro-Collins (EC) solution and University of Wisconsin (UW) solution, on the viability and some functional activities of hepatocytes isolated from human livers. Cells in primary culture were maintained under hypoxic (95% N2/5% CO2) and hypothermic (4 degrees C) conditions for 24 h, either in EC or UW solution. This treatment did not result in significant hepatocyte damage, as judged by phase contrast microscopy, intracellular LDH release, and the MTT mitochondrial test. However, neutral red uptake indicated that lysosomal functions were slightly affected (35% decrease) when compared to control conditions. At the end of the hypoxia/hypothermia period, hepatocyte monolayers were incubated at 37 degrees C under normoxic conditions for 24 h, in order to simulate the reperfusion of a transplanted liver. Three drugs--midazolam, diazepam, zidovudine--were used as diagnostic substrates to check the metabolic abilities of human hepatocytes replaced in normal conditions. Both phase I (hydroxylation, demethylation) and phase II (glucuronidation) metabolic reactions were affected by the hypoxia/hypothermia shock. Indeed, a 30%-50% decrease in these activities was observed as compared to values obtained in control hepatocytes. No difference could, however, be found at the cellular level regarding the solution used for cold storage. These results suggest that the superiority of UW over EC solution, already reported in clinical practice after transplantation of preserved human livers, was not due to a better preservation of the hepatocytes.

Antimetabolites↗
PubMed2003

Organotypic slices in vitro: repeated, same-cell, high-resolution tracking of nuclear and cytoplasmic fluorescent signals in live, transfected cerebellar neurons by confocal microscopy.

The culture of organotypic slices for the purposes of tracking dynamic cellular events within the same live cell at high resolution, as a function of development in vitro has not been previously reported. The present study was undertaken to define the conditions most suitable for both the in vitro organotypic development of Purkinje neurons in cerebellar slices of neonatal mice, and the repeated visualization of nuclear signals within such cells. Slices of cerebella were maintained on 25 mm diameter, collagen-coated Anodisc membranes, placed in six-well plates and raised to the air-medium interface by use of glass fibre filter supports. This system permits cultures to be repeatedly observed both by phase contrast microscopy and, upon biolistic transfection, by laser confocal microscopy using 40x, 60x, and 100x water-immersion objectives, at high resolution. Upon co-transfection with two plasmids, differentiation of the same transfected Purkinje neurons was followed across in vitro development for periods of up to 10 days. Despite the relative thickness of the slice culture, even small, punctate, nuclear signals, were detectable. The results show that Purkinje neurons in cerebellar slices explanted from postnatal day 2 mice, developed cytotypically, although some were ectopically located. In contrast, Purkinje neurons in slices from postnatal day 6 cerebella developed in an organotypic manner. It is concluded that this culture system serves as an ideal tool for applications in experimental biology where high resolution tracking of cellular signals, over extended time periods, is of interest.

Animals↗
PubMed2006

Atomic force microscopy visualization of poly(urethane urea) microphase rearrangements under aqueous environment.

Polyurethane biomaterials are a critically important class of polymers used in a variety of medical devices. It has been suggested that the good blood compatibility of polyurethanes arises from nanoscale chemical heterogeneities at the surface as a consequence of the microphase separated morphology. In this study, we used tapping mode atomic force microscopy with phase imaging under aqueous conditions to visualize the distribution of the surface microphases for a series of poly(urethane urea) block co-polymers with varying hard segment content. The surfaces were prehydrated for 24 h under a flow of 1 mM phosphate buffer. Topographic images showed the formation of nanometer-sized raised features on the surface, having lateral dimensions of 50-70 nm and heights of 10-15 nm. Phase images, reflecting the local distribution of the mechanical properties under aqueous conditions, were quite different from those obtained in ambient conditions, consistent with water-induced structural reorientation. Images suggest that there is little soft phase material at the polymer surface in the presence of water, while images acquired after dehydration of the samples show that the surface layer remains rich in hard domains, indicating that the films do not return to their original states over the time period studied.

Microscopy, Atomic Force↗
PubMed1992

Quick-freezing of cultured cardiac cells in situ with special attention to the mitochondrial ultrastructure.

A new method has been developed which allows quick-freezing in situ of primary, cardiac cell cultures grown to confluence on gas-permeable membranes (Petriperm dishes). Small pieces of the growth substratum, with rhythmically beating myocardial cells, were slam-frozen, without cryoprotectants, against the surface of a helium-cooled copper block at approximately 16 K. The quality of the cellular cryopreservation, as judged by ultrastructural criteria, was studied in freeze-substituted specimens processed for transmission electron microscopy. The ultrastructure of cryofixed cardiac cells was compared with that of unfrozen, chemically fixed samples. The severity of cryodistortions increased progressively with increasing distance from the point of first impact. Of particular interest were the dramatic alterations of the mitochondrial ultrastructure. The concept that the reticular and the outer mitochondrial membranes are intimately and strongly associated was clearly demonstrated. Optimally frozen material revealed cryopreserved ultrastructure of high quality. The method described appears to offer an ideal model system for correlating the information gained by phase-contrast microscopy of living cell cultures with the ultrastructure of the same samples fixed in situ by chemical or physical techniques. Cryofixation would be particularly useful for studying dynamic cellular processes associated with physiological and pathophysiological conditions, e.g. metabolic inhibition, anoxia and substrate deprivation.

Animals↗
PubMed1991

Mapping trajectories of Pgp-1 membrane protein patches on surfaces of motile fibroblasts reveals a distinct boundary separating capping on the lamella and forward transport on the retracting tail.

Patches of aggregated membrane proteins on motile fibroblasts are transported from the surfaces of the leading lamella to a site just ahead of the nucleus in the phenomenon known as capping. A major cell surface glycoprotein, Pgp-1 (GP80), was tagged with a monoclonal IgG and then aggregated with fluorescent secondary antibodies. Correlated digitized fluorescence and phase-contrast microscopy were used to map the trajectories of fluorescent Pgp-1 patches located in various regions of the cell surface. The response of patches located in lamellar and nonlamellar regions to spontaneous retraction of the trailing cell margin, or tail was examined in detail. During capping, Pgp-1 patches accumulated at a morphologically distinct site on the cell surface, the 'null border', corresponding to the boundary between lamelloplasm and endoplasm and the posterior edge of the dorsal cortical F-actin sheath. Posterior to this site, gradual forward movement of patches accompanied the gradual narrowing phase of the trailing edge retraction that occurs prior to abrupt detachment of the tail, but patches did not actually accumulate at the null border. The rate of forward patch movement was generally greater at positions further behind the boundary. Patch movement correlated approximately with forward organelle movement in the trailing region of the cell. The boundary was also apparent during simultaneous capping and retraction when forward patch transport on the trailing edge and rearward transport of patches across the lamellar surface appeared to converge on the null border. Forward patch transport was strictly confined to regions behind the boundary while retrograde patch transport was confined to the lamellar region ahead of the boundary. Patches are thought to be linked to the cortical cytoskeleton and their transport is discussed in terms of the very different cortical cytoskeletal dynamics occurring in the leading and trailing edges of locomoting cells.

Actins↗
PubMed2000

Robust autofocusing in microscopy.

BACKGROUND: A critical step in automatic microscopy is focusing. This report describes a robust and fast autofocus approach useful for a wide range of microscopic modalities and preparations. METHODS: The focus curve is measured over the complete focal range, reducing the chance that the best focus position is determined by dust or optical artifacts. Convolution with the derivative of a Gaussian smoothing function reduces the effect of noise on the focus curve. The influence of mechanical tolerance is accounted for. RESULTS: The method is shown to be robust in fluorescence, bright-field and phase contrast microscopy, in fixed and living cells, as well as in fixed tissue. The algorithm was able to focus accurately within 2 or 3 s, even under extremely noisy and low contrast imaging conditions. CONCLUSIONS: The proposed method is generally applicable in light microscopy, whenever the image information content is sufficient. The reliability of the autofocus method allows for unattended operation on a large scale.

Animals↗
PubMed1994

Intrafamilial outbreak of astrovirus gastroenteritis in São Paulo, Brazil.

This report describes an intrafamilial outbreak of astrovirus-associated gastroenteritis that occurred among 5 children in a family living in the State of São Paul, Brazil. Astrovirus was detected by direct electron microscopy (DEM) and confirmed by solid phase immune electron microscopy (SPIEM) using astrovirus reference serum (T2-6-89). The rise in titre of astrovirus antibody in patients' sera was observed by IEM using acute and convalescent-phase sera. Combined use of the EM techniques to detect virus in stool and antibody rise in sera led us to conclude that this intrafamilial outbreak might have been caused by astrovirus infection. In Brazil, astrovirus may be a more common agent of gastroenteritis than in currently appreciated.

Antibodies, Viral↗