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PubMed1995

Co-expression of fibroblastic, histiocytic and smooth muscle cell phenotypes on cultured adherent cells derived from human palatine tonsils: a morphological and immunocytochemical study.

Adherent cells derived from human palatine tonsils were isolated and cultivated. Exponentially growing adherent cells (TAC) were observed by phase-contrast microscopy and transmission electron microscopy. Immunocytochemical studies were also performed. TAC were composed of relatively monotonous cells with polygonal or spindle shapes and high proliferative activity. In addition to the development of rough endoplasmic reticulum and lysosomes, the TAC possessed a moderate amount of pinocytotic vesicles and a few microfilaments. All of the TAC strongly expressed fibroblastic markers and partial monocyte/macrophage markers, such as beta-subunit of prolyl 4-hydroxylase (DAKO-fibroblast), lysozyme, anti-alpha-1-antichymotrypsin (alpha ACT), and CD68 (KP-1, EBM/11). It was noted that, as the TAC were cultured for a longer period, they gradually increased the reactivity with the monoclonal antibody PG-M1. Furthermore, the TAC expressed myocytic phenotype, such as alpha-smooth muscle actin (alpha SMA) with various intensity. Moreover, as to extracellular matrix, TAC stained for collagen type I, collagen type III, laminin, and fibronectin. Collagen type IV was weakly positive. The results presented here showed that the TAC expressed three different phenotypes of fibroblasts, histiocytes and smooth muscle cells at the same time. The monoclonal antibody raised against the TAC reacted strongly with the subendothelial pericytes and/or smooth muscle cells in the extrafollicular area in human tonsils. The present results also suggested that the origin of the TAC was probably subendothelial pericytes and/or smooth muscle cells of the microvasculatures in the tonsil.

Adult↗

PubMed1989

Ice crystal patterns in artificial gels of extracellular matrix macromolecules after quick-freezing and freeze-substitution.

Artificial gels, composed of collagen with or without hyaluronate (HA), a glycosaminoglycan (GAG), and chondroitin sulfate (CS), were prepared and quick-frozen for the purpose of studying the influence of composition and concentration on ice patterns. Dilute gels were spread on coverslips, plunged into a slush of 30% isopentane/70% propane (-185 degrees C), freeze-substituted, and examined by phase-contrast microscopy. Ice patterns were revealed as "ice cavities" in the gel after freeze-substitution. Ice morphology in the gels was gel-type-specific, suggesting that composition in dilute gels can influence ice pattern formation. Crystallization patterns reflecting high, intermediate, and low rates of freezing were observed in all gel types. Intermediate freezing in differentiating gel-type-specific ice patterns. Gels which included hyaluronate (HA) and chondroitin sulfate (CS) altered the ice crystal pattern commonly observed in collagen gels. Ice structure in collagen gels consisted predominantly of long, parallel crystals in the herringbone pattern. Ice crystals separated gel into thin, unbranched fibers with a primary spacing of approximately 2 microns. Ice morphology in HA gels formed a mosaic consisting of packets of ice crystals. Contiguous packets were often oriented at right angles to each other. Periodic crossbridges interconnect primary gel fibers of HA gels and interrupt the lengthwise growth of ice crystals. Smooth beads were visible on primary strands in HA gels frozen at intermediate velocities. The addition of CS to collagen gels resulted in formation of randomly oriented ice crystals in gels frozen at intermediate rates. CS has little influence on ice morphology at low freezing velocities. Primary strands in CS gels were decorated with rough-surfaced, osmiophilic aggregates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1976

Geometry of normal mammalian platelets by quantitative microscopic studies.

The shape distributions of normal and hardened human and rabbit erythrocytes and platelets were obtained for edge-on orientations of a few hundred freely rotating cells from analyses of microphotographs obtained similarly as by Ponder(1930, Q. J. Exp. Physiol. 20:29) by phase-contrast microscopy at 800 X magnification. Major average diameters (d) and thicknesses (t) were estimated for both normal and hardened cells, and were used to calculate an average geometric axis ratio, rp = t/d, which increases to unity as cells become more spherical. Our fixation procedure did not alter these shape parameters: rp was unchanged for erythrocytes, with d and t values similar to those reported by Ponder (1930); platelets had d X t = 3.6 +/- 0.7 mum X 0.9 +/- 0.3 mum and 3.1 +/- 0.4 mum X 0.6 +/- 0.3 mum, respectively, for human and rabbit cells, with rp = 0.26 and 0.20, respectively. Agreement in rp was found with data obtained by a novel rheo-optical method which allows for a direct statistical averaging for large populations (greater than 100 X 10(3) cells). Histograms and linear correlation studies were made of the above three parameters (d,t,rp), as well as volume (V), total surface area are (S), and sphericity index (S.I.) calculated for both "prolate ellipsoid" and "disc with rounded edges" models. Results indicate very high linear correlations between rp - t, rp - S. I., and d -S, with high correlations for t - V,d -V and S. Data are in agreement with the few reports in the literature determined by other methods, with the best model for platelets appearing to be an oblate spheroid.

Animals↗

PubMed1996

Adhesion of sickle red blood cells and damage to interleukin-1 beta stimulated endothelial cells under flow in vitro.

Two factors that are hypothesized to contribute to vasoocclusive crises in sickle cell anemia are increased sickle red blood cell-endothelial cell interactions and damage to endothelium. Despite considerable study, the mechanisms by which erythrocyte-endothelial interactions occur and the role of endothelial damage have not yet been fully elucidated. In this report, we demonstrate that adhesion and damage may be related in a model of vasoocclusion in sickle cell anemia. Phase contrast microscopy coupled to digital image processing was used to determine the adhesion of sickle red blood cells to 1-, 4-, and 24-hour interleukin-I beta (IL-1 beta) stimulated endothelial calls in a parallel plate flow chamber. Morphological alterations to activated endothelial cells after the perfusion of sickle erythrocytes were also identified. Pretreatment of monolayers with 50 pg/mL of IL-1 beta for 1, 4, and 24 hours caused approximately 16-fold increases in adhesion of sickle cells to activated endothelium at all time points. Results with an Arginine-glycine aspartic acid (RGD) peptide and monoclonal antibodies indicated a role for three different endothelial cell receptors: alpha v beta 3 after 1 hour of IL-1 beta stimulation; E-selectin after 4 hours of IL-1 beta stimulation; and vascular cell adhesion molecule-1 after prolonged exposure to cytokines. Perfusion of sickle, but not normal, erythrocytes resulted in alteration of endothelial morphology. Approximately 6% to 8% damage was observed on 4- and 24-hour IL-1 beta stimulated endothelial cells after the perfusion of sickle cells. Damage to 24-hour activated endothelial cells showed a positive correlation (r = .899) with the number of adherent sickle erythrocytes.

Amino Acid Sequence↗

PubMed1982

Hormonally induced changes in the cytoskeleton of human thyroid cells in culture.

Serially cultivated thyroid follicular cells are not active in hormone synthesis but retain a thyrotropin-responsive adenylate cyclase. The exposure of such cells to thyrotropin leads to an increase in the concentration of intracellular cAMP and a drastic change in morphology including a total cytoplasmic arborization. The present communication describes these changes at the cytoskeletal level using a cell line derived from a human functioning thyroid adenoma. Phase contrast microscopy showed that the cytoplasmic arborization was preceded by a total disappearance of stress fibers, visible within 20 min of exposure. Small marginal membrane ruffles could also be seen. These morphological changes could also be induced by the addition of dibutyryl cAMP. The action of both thyrotropin and dibutyryl cAMP was potentiated by theophylline. High voltage electron microscopy of whole mounted cells confirmed the loss of stress fibers (microfilament bundles). In addition, thyrotropin treatment led to an uneven redistribution of the cytoplasmic ground substance and to changes in the organization of the microtrabecular lattice. Stereo images demonstrated numerous minute surface ruffles. The thyrotropin-induced arborization was reversible even in the presence of thyrotropin. After 24 h of treatment, cells had flattened and then contained very straight and condensed microfilament bundles. The results thus demonstrate that thyrotropin induces a disintegration of microfilament bundles in human, partially dedifferentiated, follicular cells and that this effect to all appearances is caused by cAMP, the second messenger in thyrotropin action. The relation of this event in partially dedifferentiated cells to the effect of thyrotropin in the intact thyroid gland is unclear. The fact that several other cultured hormone-responsive cells round up or become arborized in conjunction with an increase in cAMP levels implies that cAMP may be a major factor in the disassembly of microfilament bundles in these cells.

Bucladesine↗

PubMed2006

In vitro effects of fluoroquinolone and aminoglycoside antibiotics on human keratocytes.

PURPOSE: The purpose of this study was to assess the cytotoxic effects of the fluoquinolone ofloxacin and the aminoglycoside netilmicin on stromal human keratocytes in vitro. METHODS: Cultured human keratocytes were exposed to various concentrations of ofloxacin or netilmicin (0.16-5.0 mg/mL). Both cell proliferation (MTT assay) and cell morphology (phase-contrast microscopy) were evaluated after 1, 4, 12, and 24 hours of incubation. Measurement of annexin V binding performed in association with the dye exclusion test using propidium iodide (PI) was also performed by FACS analysis after 4 hours of exposure. RESULTS: Both antimicrobials induced dose- and time-dependent morphologic changes in keratocytes, yet the effects of netilmicin were minimal. After 24 hours of exposure, both drugs induced a dose-dependent inhibition of cell proliferation; however, ofloxacin demonstrated significantly more toxic effects than netilmicin (t test for ED50 values, P < 0.0001). Statistical differences between 2 antibiotics start at concentrations above 1.25 mg/mL (ANOVA with post-hoc test, P < 0.01). Expression of the apoptotic marker annexin V was unaffected by antibiotic exposure, whereas the uptake of the necrotic marker PI was increased by ofloxacin (5 mg/mL) but not by netilmicin (ofloxacin versus netilmicin, ANOVA, P < 0.05). CONCLUSIONS: Relative effects of aminoglycosides and fluoroquinolones on stromal keratocytes appear to be different: netilmicin was shown to be significantly less toxic than ofloxacin. This finding is particularly relevant in deciding the optimal antibiotic to be applied in clinical situations in which the epithelium is absent or compromised, as after photorefractive keratectomy, alkali burns, or ulcerative keratitis.

Adult↗

PubMed2005

Cell detection in phase-contrast images used for alpha-particle track-etch dosimetry: a semi-automated approach.

A novel alpha-particle irradiator has recently been developed that provides the ability to characterize cell response. The irradiator is comprised of a collimated, planar alpha-particle source which, from below, irradiates cells cultured on a track-etch material. Cells are imaged using phase-contrast microscopy before and following irradiation to obtain geometric information and survival rates; these can be used with data from alpha-particle track images to assess cell response. A key step in this process is determining cell location within the pre-irradiation images. Although this can be done completely by a human observer, the number of images requiring analysis makes the process time-consuming and tedious. To reduce the potential human error and decrease user interaction time, a semi-automated, computer-aided method of cell detection has been developed. The method employs a two-level adaptive thresholding technique to obtain size and position information about potential cell cytoplasms and nuclei. Proximity and geometry-based thresholds are then used to mark structures as cells. False-positive detections from the automated algorithm are due mostly to imperfections in the track-etch background, camera effects and cellular residue. To correct for these, a human observer reviews all detected structures, discarding false positives. When analysing two randomly selected cell dish image databases, the semi-automated method detected 92-94% of all cells and 94-97% of cells with a well-defined cytoplasm and nucleus while reducing human workload by 32-83%.

Algorithms↗

PubMed1998

Differential cataractogenic potency of TGF-beta1, -beta2, and -beta3 and their expression in the postnatal rat eye.

PURPOSE: Transforming growth factor-beta has been shown to induce cataractous changes in rat lenses. This study assesses the relative cataractogenic potential of TGF-beta1, TGF-beta2, and TGF-beta3 and their expression patterns in the rat eye. METHODS: Lens epithelial explants and whole lenses from weanling rats were cultured with TGF-beta1, TGF-beta2, or TGF-beta3 at concentrations ranging from 0.025 ng/ml to 4 ng/ml for 3 to 5 days. Cataractous changes were monitored daily by phase contrast microscopy and by immunofluorescent detection of cataract markers alpha-smooth muscle actin and type I collagen. Expression of TGF-beta was studied by immunofluorescence and in situ hybridization on eye sections from neonatal and weanling rats. RESULTS: All three isoforms induced morphologic changes in lens epithelial explants and cultured lenses that are typically associated with human subcapsular cataract. Transforming growth factor-beta2 and TGF-beta3 were approximately 10 times more potent than TGF-beta1. All three isoforms were expressed in the eye in spatially distinct but overlapping patterns. Transforming growth factor-beta1 and TGF-beta2 and their mRNA were detected in most ocular tissues, including the lens. Although TGF-beta3 was immunolocalized in lens epithelium and fibers and in other ocular tissues, its mRNA was detected only in the retina and choroid. CONCLUSIONS: All three isoforms of TGF-beta are potentially available to lens cells and have the potential to induce cataractous changes. The results suggest that TGF-beta activity is normally tightly regulated in the eye. Activation of TGF-beta in the lens environment, such as may occur during injury, in wound healing, or in pathologic conditions may contribute to cataractogenesis in vivo.

Actins↗

PubMed2000

Histopathological differences between temporary and permanent threshold shift.

The structural changes associated with noise-induced temporary threshold shift (TTS) were compared to the damage associated with permanent threshold shift (PTS). A within-animal paradigm involving survival-fixation was used to minimize problems with data interpretation from interanimal variability in response to noise. Auditory brainstem response thresholds for clicks and tone pips were determined pre- and 1-2 h post-exposure in 11 chinchillas. The animals were exposed for 24 h to an octave band of noise with a center frequency of 4 kHz and a sound pressure level of 86 dB. Three animals (0/0-day) had both cochleas terminal-fixed 2-3 h post-exposure. Two animals (27/27-day) had threshold shifts determined every other day for 1 week, every week thereafter, and underwent terminal-fixation of both cochleas 27 days after exposure. Six animals (0/n-day) had threshold shifts determined in both ears upon removal from the noise; their left cochlea was then survival-fixed 2-3 h post-exposure. Threshold shifts were determined in their right ear every 2-3 days until their hearing either returned to pre-exposure values or stabilized at a reduced level at which time their right cochlea was terminal-fixed (4-13 days post-exposure). All cochleas were prepared as plastic-embedded flat preparations. Missing hair cells were counted and supporting cells and nerve fibers were evaluated throughout the organ of Corti using phase-contrast microscopy. Post-exposure, all animals had moderate TTSs in their left and right ears which averaged 43 dB for 4-12 kHz. In the 0/0-day animals, the only abnormality which correlated with TTS was a buckling of the pillar bodies. In the 0/n-day animals, their left cochlea (survival-fixed 2-3 h post-exposure) had outer hair cell (OHC) stereocilia which were not embedded in the tectorial membrane in the region of the TTS whereas OHC stereocilia were embedded in the tectorial membrane throughout the cochleas of non-noise-exposed, survival-fixed controls. Three of six right cochleas (terminal-fixed 4-13 days post-exposure) from the 0/n-day animals developed a PTS and two of these cochleas had focal losses of inner and outer hair cells and afferent nerve fibers at the corresponding frequency location. The other cochlea with PTS had buckled pillars in the corresponding frequency region. These results suggest that with moderate levels of noise exposure, buckling of the supporting cells results in an uncoupling of the OHC stereocilia from the tectorial membrane which results in a TTS. The mechanisms resulting in TTS appear to be distinct from those that produce permanent hair cell damage and a PTS.

Acoustic Stimulation↗

PubMedDate not supplied

Cardiomyopathy in vitro.

Phase contrast microscopy of cultured embryonic heart cells showed the beating frequency decreased more rapidly and the regularity the rhythm of of the beating cells was lost sooner in heart cells from cardiomyopathic hamsters than from the control hamsters. Studies of cultured heart cells by differential interference contrast (with Nomarski's prism) and by electron microscopy revealed a significant impediment in the maturation of the sarcomeric units in the diseased animals compared to controls. The incorporation of [14C] leucine into acid-insoluble fractions was studied, and no significant difference in incorporation between the two groups was found. An analysis of polyacrylamide gel electrophoresis revealed the possible existence of a quantitative difference in one of the composing proteins of the erythrocyte membrane between the two groups. The protein kinase activity of ghosts from the control group was more sensitive to cAMP than that from the diseased animals. In addition, the binding of [3H] cAMP to the ghost was almost identical between the two. The morphological and biochemical observations lead one to the plausible supposition that there are some differences in the interaction of the so-called catalytic and regulatory subunits between the two groups and that there is an impairment of the higher arrangement of myofibrils from their building blocks in the diseased hamster. The significance of the existence of abundant corpuscles resembling neurosecretory granules was not established by this study. They may have an etiological significance or they may be related to a disturbed function in the cultured cells of the cardiomyopathic hamster.

Animals↗

PubMed2001

Shape diversity among chick retina Müller cells and their postnatal differentiation.

It is currently believed that in each vertebrate species Müller cells in the central retina constitutes a fairly homogeneous population from the morphologic point of view and that particularly the chick Müller cell attains full shape differentiation at prenatal stages. However, in this study of the chick retina, from day 1 to day 55 of life, we show that there is a large variety of Müller cell shapes and that many of them complete shape differentiation postnatally. We used a cell dissociation method that preserves the whole shape of the Müller cells. Unstained living and unstained fixed cells were studied by phase-contrast microscopy, and fixed cells immunostained for intermediate filaments of the cytoskeleton were studied by fluorescence microscopy. Our results show that (1) Müller cell shapes vary in the origination of the hair of vitread processes, in the shape of the ventricular (outer or apical) process, in the presence or absence of an accessory process, as well as in the number and shape of processes leaving from the ventricular process at the level of the outer nuclear and outer plexiform layers (ONL/OPL); (2) during the first month of life, many Müller cells differentiate the portion of the ventricular process that traverses the ONL, most Müller cells differentiate the ONL/OPL processes, and all Müller cells differentiate the thin short lateral processes leaving from the vitread hair processes at the level of the inner plexiform layer (IPL). The number of cells differing in the shape of the ventricular process and that of cells with and without accessory process were estimated. The spatial relationship between the outer portion of the ventricular process of the Müller cell and the photoreceptor cells was also studied. Our results show that the branching of the ventricular process and the refinement of Müller cell shape is achieved without apparent participation of growth cones. We give a schematic view of how the branching of the ventricular process might take place and propose the size increase of photoreceptor soma as a factor responsible for this branching.

Aging↗

PubMed2001

FGF: an autocrine regulator of human lens cell growth independent of added stimuli.

PURPOSE: Posterior capsule opacification (PCO) arises because of a persistent growth of lens epithelial cells. Cultured human lens cells residing on their native collagen capsule and maintained in serum-free medium actively grow and thus show an intrinsic capacity for regulation. In the present study, the authors investigated the role of the putative FGF autocrine system in human capsular bags. METHODS: Capsular bags were prepared from human donor eyes and maintained in a 5% CO(2) atmosphere at 35 degrees C. On-going observations were by phase-contrast microscopy. Cellular architecture was examined by fluorescence cytochemistry. De novo protein synthesis was determined by the incorporation of 35S-methionine. Basic fibroblast growth factor (FGF) and FGF receptor (R)-1 were detected using enzyme-linked immunosorbent assay (ELISA) and reverse transcription-polymerase chain reaction (RT-PCR) techniques. FGFR-1 inhibition was achieved using the specific antagonist SU5402. RESULTS: Human lens epithelial cells can maintain metabolic activity for more than 1 year in a protein-free medium. Basic FGF was shown to be present in capsular bags throughout culture and also in capsular bags removed from donor eyes that had previously undergone cataract surgery. Furthermore, FGFR-1 was identified. Inhibition of FGFR-1 caused a significant retardation of growth on the posterior capsule. On no occasion did any treated bag reach confluence, whereas all match-paired control samples did. CONCLUSIONS: The results provide evidence that FGF plays an integral role in the long-term survival and growth of human lens epithelial cells, independent of external stimuli. Inhibition of FGFR-1 by specific synthetic molecules, such as SU5402, could provide a potential therapeutic approach to resolving PCO.

Autocrine Communication↗

PubMed1994

Normal human urothelial cells in vitro: proliferation and induction of stratification.

BACKGROUND: The purpose of the work was to establish urothelium as an in vitro model for the study of proliferation, stratification, and differentiation in "complex" epithelia. EXPERIMENTAL DESIGN: Normal human urothelial cells were cultured in a serum-free medium. The effects of epidermal growth factor (EGF), cholera toxin (CT), extracellular calcium and 13-cis-retinoic acid on cell growth, morphology, phenotype, and cytodifferentiation were studied using phase-contrast microscopy and indirect immunofluorescence. Stratification-related changes were additionally analyzed by transmission electron microscopy. RESULTS: Under optimized conditions, long-term cultures were successful in 44 (74.5%) out of 59 specimens. Bacterial infection was the most common cause of failure (9 cases). Primary urothelial cells required an initial plating density of > or = 10(4) cells/cm2 for survival; passaged cells survived much lower plating densities (> or = 2.5 x 10(2) cells/cm2). CT significantly improved cell attachment, but neither CT nor EGF were essential for growth. By contrast, cells failed to proliferate without bovine pituitary extract. In media containing bovine pituitary extract, CT, and EGF, cultures had a mean population doubling time of 14.7 +/- 1.8 hours, maintained a nonstratified phenotype, and expressed the cytokeratin (CK) profile of basal/intermediate urothelium: CK7, CK8, CK17, CK18 and CK19, with variable expression of CK13. CK20 was not expressed in vitro. CK14 and CK16 were also expressed, suggestive of squamous metaplasia in culture, which could be inhibited with 13-cis-retinoic acid. Increasing extracellular calcium from 0.09 to 0.9-4.0 mM slowed cell proliferation, induced stratification and desmosome formation, and increased expression of E-cadherin. High calcium, EGF, CT, and retinoic acid did not induce markers of late/terminal urothelial cytodifferentiation. CONCLUSIONS: We describe a simplified technique for the isolation and long-term culture of human urothelial cells. Urothelial cells in vitro are capable of rapid proliferation and can be induced to form integrated stratifying cell layers in high calcium medium. Stratification-related changes are not necessarily accompanied by urothelial cell maturation and differentiation.

Adolescent↗

PubMed2005

Upregulation of heat shock protein expression by proteasome inhibition: an antiapoptotic mechanism in the lens.

PURPOSE: Studies have shown that proteasome inhibition protects lens epithelial cells (LECs) against interferon (IFN)-gamma-induced apoptosis. The present study was conducted to test the hypothesis that proteasome inhibition can protect lens cells against apoptosis by upregulating heat shock protein (HSP) expression. METHODS: Murine lens epithelial alphaTN4-1 cells were treated with combinations of 100 U/mL IFN-gamma, 10 muM MG132 (proteasome inhibitor), and 100 muM quercetin (HSP inhibitor). mRNA and protein expression were observed by RT-PCR and Western blot analysis, respectively. Caspase activities were measured by using cleavage of colorimetric substrate. Apoptosis was measured by phase-contrast microscopy and flow cytometry. RESULTS: At the mRNA level, the proteasome inhibitor, MG132, caused a >10-fold increase in HSP27 and a small increase (1.2- to 1.6-fold) in alphaB-crystallin but no change in HSP70 or -90. At the protein level, a more than twofold increase in HSP27 and -90, a marked increase in HSP70, but no significant change in alphaB-crystallin, was observed. Downregulation of alphaA-crystallin by MG132 was observed at both the mRNA and protein levels. MG132 caused no significant change in heat shock factor (HSF)-1, but a more than twofold increase in HSF2 and -4 protein expression. MG132 prevented the IFN-gamma-induced increase in caspase-1, -6, and -8 activities. Quercetin decreased MG132-induced expression of HSP27, -70, and -90 by more than 70%, and heat shock factors HSF2 and -4 by more than 65%. Quercetin pretreatment significantly reversed the decrease in caspase-1, -6, and -8 activities and the antiapoptotic effect of MG132 on IFN-gamma-treated LECs. CONCLUSIONS: The antiapoptotic effect of proteasome inhibition of IFN-gamma-induced apoptosis in LECs correlates with increased expression of HSPs and inhibition of caspase activities. Inhibition of HSP expression restores caspase activities and abolishes the antiapoptotic effect of proteasome inhibition, implicating HSPs as mediators of the protective effect of proteasome inhibition.

Animals↗

PubMed1999

Heterogeneity in microparticle formation and exposure of anionic phospholipids at the plasma membrane of single adherent platelets.

Adherent platelets were examined for their ability to form microvesicles and procoagulant sites for thrombin formation. Epifluorescence and phase-contrast microscopy were employed to visualize shape changes, changes in intracellular Ca(2+) levels ([Ca(2+)](i)), vesiculation of the plasma membrane and appearance of anionic phospholipids in the outer leaflet of the plasma membrane, as probed by annexin V binding. In the absence of extracellular Ca(2+) two stable populations of adherent platelets were observed. The majority of the adherent platelets were fully spread and about 10% remained in a non-spread dendritic state. In the presence of extracellular Ca(2+) vesiculation at the surface of spread platelets occurred at a rather slow rate (10% of the platelets after 20 min) concomitantly with an increase in [Ca(2+)](i) and binding of annexin V. However, a small fraction of the adherent platelets ( approximately 1%) responded much faster. Ionomycin-enhanced influx of Ca(2+) in dendritic platelets resulted in a rapid transformation of these platelets into inflated, balloon-shaped, platelets having a diameter of 2.0+/-0.7 microm without notable microvesicle formation. In contrast, fully spread platelets retained their shape but obtained frayed edges as a result of microvesicle formation. Confocal scanning fluorescence microscopy indicated that annexin V bound to very distinct sites at the outer plasma membrane of spread as well as balloon-shaped platelets. Inhibition of platelet calpain activity suppressed ionomycin-enhanced microvesicle formation and ballooning of platelets, but not annexin V binding. These findings indicate that vesiculation and ballooning, but not the exposure of phosphatidylserine at the outer leaflet of the adherent platelet membrane, are associated with cytoskeleton destruction. Altogether, the data suggest a similar relationship between [Ca(2+)](i) and the formation of platelet procoagulant sites as reported for platelets in suspension. However, the present investigations on single adherent platelets reveal for the first time that adhesion and spreading of platelets is not necessarily associated with the appearance of procoagulant sites. Secondly, an unexpected diversity was observed among adherent platelets with respect to sensitivity to Ca(2+)-induced generation of procoagulant sites and Ca(2+)-induced vesiculation of plasma membrane. It is tempting to speculate that this diversity is of importance for the procoagulant response of platelets to a hemostatic challenge elicited by an injured vessel wall.

Anions↗

PubMed2005

The heterogeneous murine corneal stromal cell populations in vitro.

PURPOSE: To demonstrate that the murine corneal stroma is inhabited by heterogeneous cell populations that include cells expressing nestin. METHODS: Collagenase-isolated corneal stroma cells obtained from newborn and adult mice (2nd and 12th postnatal weeks, respectively), were seeded at low (5 cells/mm2), intermediate (50 cells/mm2), and high (500 cells/mm2) densities in DMEM/F12 containing insulin, transferrin, selenium, and 1% nonessential amino acids. Corneal stroma cells cultured at 500 cells/mm2 were treated with 10 ng/mL human recombinant transforming growth factor (TGF)-beta1 for 5 days. Cell morphology and expression of alpha-smooth muscle actin, choline acetyltransferase, CD45, glial fibrillary acidic protein (GFAP), keratocan, nestin, neurofilaments, protein gene product 9.5, tyrosine hydroxylase, and vimentin were examined. RESULTS: Phase-contrast microscopy demonstrated that freshly isolated corneal stromal cells are heterogeneous in morphology and include dendritic, stellate, neuronal, and small polyhedral cells. Immunostaining of primary cultures of 2- and 12-week-old mice, 24 hours after seeding at the intermediate density, showed that 100% of cells expressed vimentin and 97.7% +/- 2.7% expressed keratocan. alpha-Smooth muscle actin was expressed by 0.2% +/- 0.05% of cells in the 2-week-old group and 0.1% +/- 0.07% in 12-week-old group. Neurofilament was expressed by 0.5% +/- 0.03% and 0.7% +/- 0.03% of cells in the 2- and 12-week-old groups, respectively. No cell expressed GFAP or nestin. After 5 days in culture, cells seeded at high density aggregated as clusters that were immunoreactive to nestin in both groups. Cell clusters and migrating cells reacted to pgp 9.5, and migrating cells, but not the cell clusters, reacted to tyrosine hydroxylase. Cell cluster formation and nestin expression were abolished by culturing in the presence of TGF-beta1. CONCLUSIONS: Normal murine corneal stroma contains heterogeneous cell populations including cells with the potential to form clusters and express the progenitor marker nestin. This potential is disrupted by the addition of TGF-beta1 to the culture medium.

Actins↗

PubMed1984

Establishment of glucagon-producing cells by cell hybridization.

Glucagon-producing cell lines were established by fusing pancreatic islet cells of adult hamster and 6-thioguanine-resistant hamster insulinoma cells. Under phase-contrast microscopy, the morphology of cultured hybrid cells was intermediate between those of the parental cells. The hybrid cells contained A-like granules, though few in number, and were stained with anti-glucagon antibody. The mode of chromosome number decreased to 78 or 79 by 3 mo after hybridization in comparison with the expected chromosome number of the heterokaryon of 104, and showed a minute decrease in 4 of 6 cell lines after 6 mo. The population doubling time ranged from 24 to 38 h, while that of parental insulinoma cells was 22.8 h. There was no correlation between the expression of cellular function and the stability of chromosome number or the length of population doubling time. The capacity of glucagon secretion was between that of the parental cells. The glucagon secreted into the medium, as assayed by the glucagon-specific antibody, was 0.6-2.5 ng/10(6) cells for 2 h, which was about 40% of total glucagon-like immunoreactivity secreted. Secretion of glucagon was not affected by high concentration of glucose, was markedly increased by theophylline, and was suppressed by exogenous insulin. All of the hybrid cells produced tumors on transplantation 6 mo after hybridization. The tumor-bearing hamsters exhibited high levels of plasma glucagon and blood glucose as well as a high level of serum insulin.

Adenoma, Islet Cell↗

PubMed1978

Intracellular motility of mitochondria: role of the inner compartment in migration and shape changes of mitochondria in XTH-cells.

Mitochondrial movements have been followed by phase-contrast microscopy in living XTH-cells (Xenopus laevis tadpole-heart cells) in tissue culture. The same organelles have been viewed subsequently in electron micrographs. Locomotion of mitochondria proceeds at velocities up to 100 micrometer/min. Formation of branches of mitochondria and other shape changes may occur with the same speed. Mitochondrial motility can be classified into 4 types: (I) Alternating extension and contraction at the two ends of rod-shaped mitochondria. (2) Lateral branching. (3) Alternate stretching and contraction of arbitrary parts of a mitochondrion amounting to a kind of peristaltic action. (4) Transverse wave propagation along the organelle. Types I to 3 can be reduced to the same underlying principle; they cause locomotion. Formation of mitochondrial extensions is due to elongation of cristae. The observations are discussed in terms of 4 specific proposals. (I) Intracellular locomotion of mitochondria is caused by local enlargements and contractions of the organelles. (2) The shape changes are correlated with alterations in the arrangement of the cristae. (3) Such arrangements are not associated with overall swelling or shrinkage of the mitochondrion; they are local features. (4) Estimates of the time required for rearrangement of the inner compartment amount to less than 0.3 s for single crista arrangements during the fastest shape changes, and less than 1-3 s during slower alterations. This high velocity is in good accord with the hypothesis of energy conservation by conformational events during oxidative phosphorylation.

Animals↗