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PubMed1967

Experimental transmission of a murine microsporidian in Swiss mice.

The production of ascitic fluid and splenomegaly on intraperitoneal injection in weanlings was used as a test for microsporidia after introduction by other routes and in other loci. Oral and cerebral administration was followed only by enlarged spleens which reproduced the ascitic response on passage. Microsporidia were demonstrable by phase microscopy in all fluids. Positive findings were also obtained with liver, kidney, brain, lungs, blood, and urine. Intramuscular and intranasal injection were occasionally followed by ascites, but splenomegaly again predominated. The results of contact experiments indicated that the organisms were not readily communicable either in weanlings or nurslings. Relation of the microsporidian to Encephalitozoon cuniculi (Nosema cuniculi Lainson et al.) is discussed.

Animals↗
PubMed2006

Automated method for the isolation of collecting ducts.

The structural and functional heterogeneity of the collecting duct present a tremendous experimental challenge requiring manual microdissection, which is time-consuming, labor intensive, and not amenable to high throughput. To overcome these limitations, we developed a novel approach combining the use of transgenic mice expressing green fluorescent protein (GFP) in the collecting duct with large-particle-based flow cytometry to isolate pure populations of tubular fragments from the whole collecting duct (CD), or inner medullary (IMCD), outer medullary (OMCD), or connecting segment/cortical collecting duct (CNT/CCD). Kidneys were enzymatically dispersed into tubular fragments and sorted based on tubular length and GFP intensity using large-particle-based flow cytometry or a complex object parametric analyzer and sorter (COPAS). A LIVE/DEAD assay demonstrates that the tubules were >90% viable. Tubules were collected as a function of fluorescent intensity and analyzed by epifluorescence and phase microscopy for count accuracy, GFP positivity, average tubule length, and time required to collect 100 tubules. Similarly, mRNA and protein from sorted tubules were analyzed for expression of tubule segment-specific genes using quantitative real-time RT-PCR and immunoblotting. The purity and yield of sorted tubules were related to sort stringency. Four to six replicates of 100 collecting ducts (9.68+/-0.44-14.5+/-0.66 cm or 9.2+/-0.7 mg tubular protein) were routinely obtained from a single mouse in under 1 h. In conclusion, large-particle-based flow cytometry is fast, reproducible, and generates sufficient amounts of highly pure and viable collecting ducts from single or replicate animals for gene expression and proteomic analysis.

Animals↗
PubMed2006

Docosahexaenoic acid promotes photoreceptor differentiation without altering Crx expression.

PURPOSE: The precise molecular cues required for photoreceptor development are still unknown. Pax6 and Crx are essential during early retinal development and for photoreceptor differentiation, respectively. The lipid molecule docosahexaenoic acid (DHA) has also been shown to promote photoreceptor differentiation. Pax6 expression during the early steps in photoreceptor development and whether the mutual contribution of Crx and DHA enhances photoreceptor differentiation were investigated. METHODS: Neuroblast proliferation, Crx, and Pax6 expression were investigated in rat retinas in vivo and in neuronal cultures with or without DHA. BrdU incorporation, nestin and opsin expression, apical differentiation, and axonal outgrowth were determined by phase microscopy and immunochemistry. RESULTS: Pax6 expression occurred in all proliferating retinal neuroblasts in vivo; however, after their last mitotic division, photoreceptors stopped expressing Pax6 and started expressing Crx. In vitro, photoreceptor progenitors also showed a switch from Pax6 to Crx expression immediately after they exited the cell cycle and started differentiation. In contrast, those progenitors differentiating into amacrine neurons continued expressing Pax6 and did not express Crx. Most postmitotic photoreceptors expressing Crx showed little axon development and few of them expressed opsin. The addition of DHA dramatically increased differentiation in Crx-positive photoreceptors, enhancing opsin expression, apical differentiation, and axonal outgrowth, without affecting Crx expression. CONCLUSIONS: The results suggest that Pax6 and Crx expression are mutually exclusive during photoreceptor differentiation. Onset of Crx expression may provide a permissive stage that is essential to initiate photoreceptor differentiation, but additional support of DHA, among other environmental signals, is necessary to accomplish further differentiation.

Animals↗
PubMed1975

Duchenne dystrophy: electron microscopic findings pointing to a basic or early abnormality in the plasma membrane of the muscle fiber.

In seven patients with Duchenne dystrophy, high-resolution phase microscopy demonstrated a population on non-necrotic fibers with one or more focal lesions. The typical lesion was wedge-shaped, with the base resting on the fiber surface. In the electron microscope, the plasma membrane overlying the lesion was either absent or disrupted, while the basement membrane was always preserved. Within the lesion, there were cytoplasmic abnormalities, and in the neighboring fiber region, the myofibrils were usually highly contracted. The structural defect in the plasma membrane suggested that this site was an ineffective cellular barrier. This was confirmed by the frequent ingress of peroxidase-containing extracellular fluid into the lesions. In two control subjects, peroxidase penetration into fibers was seen only rarely and only with other evidence of mechanical injury to the specimen. The findings point to an early and possibly basic abnormality in the plasma membrane of the muscle fiber in Duchenne's dystrophy.

Adult↗
PubMed1987

Co-culture of rat trigeminal ganglion neurons and corneal epithelium.

Corneal epithelium and the trigeminal ganglion neurons which normally innervate the epithelium have been grown in adjacent chambers of a 35 mm tissue culture plate. Dissociated nerve cells from late embryonic rats were plated inside an 8 mm cloning cylinder attached to the center of the culture plate by silicone grease. In 7-10 days neurites extended out of this inner chamber by growing through the grease seal and along parallel scratches in the collagen coating of the tissue culture plate. Once this occurred, pure corneal epithelial explants were isolated from young adult rats and plated in the area surrounding the cloning cylinder, i.e. in the outer chamber. Cultures were monitored regularly with phase microscopy and, at various times, were fixed for ultrastructural examination. Within 24-48 hours of the epithelial plating, there were both individual neurites and bundles of neurites in contact with the epithelium. This interaction increased substantially over the next few days. Growth cones of the neurites could be seen to approach the microvilli-covered surface of the epithelium, travel over the surface and penetrate between the epithelial cells. This tissue culture model of the innervated ocular surface may prove valuable in the study of a variety of ocular conditions or diseases, as well as provide a means to study functional relationships and mechanisms of cellular interaction between neurons and their target cells.

Animals↗
PubMed2001

Cytoskeletal-induced alterations in the adhesion of HT-1080 fibrosarcoma cells to extracellular matrix.

We have investigated the adhesion of the human fibrosarcoma cell line, HT-1080, transfected with glial fibrillary acidic protein (GFAP) to a variety of extracellular matrix macromolecules (ECM) including collagen type IV, laminin, and fibronectin. The GFAP-transfectants demonstrated altered adhesiveness to extracellular matrix substrates when compared to controls. GFAP-positive, heavy metal-induced fibrosarcoma cells were more adherent to plastic and collagen type IV than were the parental or uninduced cells. In contrast, GFAP-positive fibrosarcoma cells were less adherent to laminin- or fibronectin-coated dishes than controls. Time course adhesion studies over 9 days showed that the heavy metal-induced fibrosarcoma cells progressively became more adherent to collagen type IV and less adherent to laminin- or fibronectin-coated dishes than did uninduced cells. However, with the removal of heavy metal from the medium, the HT-1080 fibrosarcoma cells were restored to their original adhesive potential. By phase microscopy, uninduced and induced HT-1080 cells demonstrated different morphological features and remained viable in an anchorage-dependent fashion on collagen type IV as a substrate. By way of contrast, GFAP-induced HT-1080 cells were not particularly viable in monolayer culture and readily detached from laminin as a substrate. The expression of beta1 integrin in GFAP-positive fibrosarcoma cells was decreased following heavy metal induction by Western blot analyses. In contrast, the expression of alpha2 integrin was increased whereas alpha5 integrin was unchanged in HT-1080 cells following the induction of GFAP. Gelatin zymography showed that 72 kDa collagenase was less expressed in GFAP-induced clones than in controls. Our data suggest that the forced expression of the intermediate filament, GFAP, in HT-1080 cells may modulate cell adhesion to different ECM substrates through alterations in expression of integrins.

Blotting, Western↗
PubMed2000

Fas- and interferon gamma-induced apoptosis in Chang conjunctival cells: further investigations.

PURPOSE: Previously interferon (IFN)gamma-induced apoptosis and expression of inflammation-related proteins in a human conjunctival cell line were demonstrated. The aim of this study was to further investigate the mechanisms of IFNgamma-, Fas-, and cycloheximide (CHX)-induced programmed cell death, with special attention to the role of transcriptional factors NF-kappaB and STAT1. METHODS: In a human conjunctival cell line (Chang conjunctival cells) apoptosis was induced with 500 ng/ml anti-Fas antibody (anti-Fas ab) alone (24 or 48 hours) or, as previously reported, with 300 U/ml of human recombinant IFNgamma alone (48 hours). To study the role of IFNgamma on Fas-induced apoptosis, cells were treated first with IFNgamma at 30 U/ml during 24 hours (nontoxic dose), and then anti-Fas ab was applied for 24 hours. Moreover, to study the influence of CHX on Fas- and IFNgamma-induced apoptosis, cells were treated for 24 hours with 300 U/ml IFNgamma together with a nontoxic concentration (1 microg/ml) of CHX, or with 500 ng/ml anti-Fas ab together with 1 microg/ml CHX (24 hours). After treatment, cell viability (neutral red assay), mitochondrial membrane potential (rhodamine 123 assay), chromatin condensation (Hoechst 33342 assay), and the index Hoechst/neutral red were studied by cold light microplate cytometry. The apoptotic process was sought for by contrast phase microscopy and DAPI staining and was confirmed by immunoblotting of PARP. Activation of caspase-3 (CPP32) and caspase-8 were investigated by Western blot analysis. NF-kappaB and STAT DNA-binding activities were studied by electrophoretic mobility shift assays (EMSA). RESULTS: After 24 and 48 hours of treatment with anti-Fas ab alone, 15% to 20% and 30%, respectively, of apoptotic cells were observed. When anti-Fas sera were applied after IFNgamma pretreatment or together with CHX, 50% to 80% of cells demonstrated morphologic characteristics of programmed cell death. Apoptosis was confirmed by a cleavage of PARP and CPP32, by caspase-8 activation, and by an index Hoechst/neutral red greater than one. All these modifications were preceded by a decrease in mitochondrial membrane potential. EMSA revealed that NF-kappaB was activated after IFNgamma and anti-Fas ab treatments and inhibited after CHX treatment. STAT1 was strongly activated after IFNgamma treatment and only in a minor degree after anti-Fas ab treatment. STAT1-binding activity persisted after CHX treatment. CONCLUSIONS: The relative resistance of Chang cells toward Fas-induced apoptosis could be related to the activation of NF-kappaB. IFNgamma-induced programmed cell death preferentially involves the activation of STAT1 that counterbalances NF-kappaB antiapoptotic effects. In fact, Fas-induced apoptosis was potentiated by IFNgamma or CHX treatments. These results suggest that NF-kappaB activation could maintain cell viability as well as participate in IFNgamma-induced inflammatory modifications, whereas STAT1 activation could provide, in this model, a proapoptotic signal.

Antibodies, Monoclonal↗
PubMedDate not supplied

[Pathomorphology of the endocardium and myocardium in the heart injury].

Light, polarization, luminescent microscopy, phase contrast, special staining methods and scanning electron microscopy were used for studying sections and fragments of the endocardium and myocardium from 123 patients who had died of heart contusion and ischemic heart disease and from 50 rats with experimental heart contusion. The morphological classification of heart contusion and differential-diagnostic table of myocardial alterations due to contusion of the heart and ischemic heart disease are proposed to facilitate pathomorphological diagnosis of heart contusion. The classification of pathomorphological signs in heart contusion is presented.

Adolescent↗
PubMedDate not supplied

Evaluation of platelet damage in extracorporeal circuits using a visual platelet morphology method.

Platelet damage in extracorporeal circuits occurs as the result of contact with foreign surfaces, shear stress, gas interface, and other nonphysiologic conditions. A scoring method developed to determine platelet activation was modified for evaluating platelet damage in extracorporeal circuits. This method assigns a numerical score to platelet damage, as assessed by direct visualization with phase microscopy. Various commercially available blood oxygenators were tested by using a modified AAMI/ASAIO in vitro blood trauma protocol. Direct gas contact oxygenators showed a marked decrease in total platelet count and significant platelet damage. Platelet depletion and platelet damage were lower in membrane oxygenators than in direct gas contact oxygenators. Differences in platelet damage were observed between membrane oxygenators. The observed differences between devices demonstrate the influence of materials and hemodynamic design on platelet depletion and damage. The method developed allows quantitative evaluation of platelet damage caused by extracorporeal devices and is a sensitive indicator of lethal and sublethal trauma.

Blood Flow Velocity↗
PubMed1989

Cytosolic ionized calcium and bleb formation after acute cell injury of cultured rabbit renal tubule cells.

Changes in cytosolic calcium ([Ca2+]i) and cell blebbing of cultured rabbit kidney proximal tubule cells were studied in response to injury induced through a variety of mechanisms. [Ca2+]i was measured in Fura 2-loaded cells and blebbing was observed by phase microscopy. The severity of injury was evaluated by electron microscopy and cell killing was estimated by trypan blue dye uptake. The types of injury included interaction with sulfhydryl groups (HgCl2, N-ethylmaleimide, p-chloromercuribenzene sulfonic acid, inhibition of energy metabolism (carbonyl cyanide m-chlorophenylhydrazone, KCN, KCN + iodoacetate) and ion deregulation (ouabain, ionomycin, A23187). The role of extracellular calcium ([Ca2+]e) in injury was also studied. HgCl2, N-ethylmaleimide and ionomycin + [Ca2+]e caused the highest elevations of [Ca2+]i, the most extensive blebbing, and most rapid cell death. P-chloromercuribenzene sulfonic acid treatment resulted in a moderate increase in [Ca2+]i, as well as less extensive blebbing and slower cell death. Ouabain and inhibitors of mitochondrial and cellular energy metabolism caused only a 2-fold increase in [Ca2+]i, a few blebs and delayed cell death. Ionomycin - [Ca2+]e caused a transient elevation of [Ca2+]i, minimal blebbing and very slow cell killing. The increase in [Ca2+]i may result from redistribution of intracellular stores (N-ethylmaleimide, p-chloromercuribenzene sulfonic acid, KCN, carbonyl cyanide m-chlorophenylhydrazone, ionomycin - [Ca2+]e), from influx of extracellular [Ca2+]e (ionomycin + [Ca2+]e, ouabain), or from both redistribution and influx (HgCl2). Therefore, removing [Ca2+]e is protective only in certain types of injury, (HgCl2, ionomycin). Cytoplasmic blebbing was seen with all the types of injury studied and occurred before to cell death. Blebs formed rapidly, enlarged, and sometimes detached with membrane sealing. Our results indicate that cell injury which initiates a 3-fold or greater sustained elevation in [Ca2+]i, resulting from either an influx of [Ca2+]e or by Ca2+ release from intracellular pools, is also associated with abundant bleb formation and rapid cell death.

Animals↗
PubMed1983

Palate cell motility and substrate interaction.

To elucidate the mechanism by which palate shelves reorient during embryogenesis, migration of palate mesenchymal cells has been studied employing various substrates. When palate explants were cultured in a hydrated collagen lattice, it was observed that bipolar spindle-shaped cells migrated out of each explant toward the other. These cells were aligned parallel to each other and to the fibrous tracks that formed. The cells appeared to move along and through the fibrous tracks. Cell migration was dependent on the presence of serum and fibronectin. The fibrous tracks viewed by phase microscopy were sensitive to collagenase. Scanning electron microscopy revealed that the collagen fibers of the hydrated lattice had coalesced into larger bundles. Pretreatment of explants with serotonin stimulated cell migration out of the explant into the hydrated collagen lattice. This effect was specific, since the antagonist methysergide blocked the stimulation produced by serotonin. Employing other substrates, it was noted that palate cells migrating out of double explants toward each other produced large wrinkles in a polysiloxane substratum. Similarly, cultured monolayer cells also produced wrinkles that disappeared as cells rounded up after trypsin treatment. Finally, monolayer cells pulled on and distorted collagen films when cultured on the substrate. It is concluded that migrating palate cells can interact with their substrate producing tractional forces. Serotonin-induced modulation of cell motility and its relationship to palate reorientation are discussed.

Animals↗
PubMed1994

The connection between the nuclei of binucleated hepatocytes: an ultrastructural study.

Hepatocytes commonly have double nuclei and polyploidy. Both increase with age. The relationship of the double nuclei to each other has never been investigated at the ultrastructural level. Using a newly developed extraction and embedding technique we observed that the two nuclei were often linked by connecting DNA strands which span the gap between the two nuclei. The DNA nature of these filaments was determined by digestion with DNase. Artifactual intermolecular disulfide bonds were prevented by the employment of iodoacetamide in the extracting medium. It was further shown that partially fused and/or separated nuclei were observed by phase microscopy of living cultures as well as in fixed liver tissue. The results may indicate that one way in which binucleation occurs is through nuclear separation when the nucleus contains multiples of DNA (polyploid).

Animals↗
PubMed1976

Ultrastructure of lymphocyte tumor cell interaction with localization of cell-bound antibody by ferritin labeling.

Splenic lymphocytes derived from Walker carcinoma-bearing rats were harvested and incubated with Walker carcinoma cells growing in tissue culture. The sequence of events leading to target cell death was studied by phase microscopy and scanning and transmission electron microscopy. The sensitized lymphocytes adhere to the tumor cells by multiple cytoplasmic appendages, but no ultrastructural changes are seen at this interface. After 1 hr these lymphocytes release cytoplasmic components consisting of membrane-lined vesicles, cell membranes, endoplasmic reticulum, and cytoplasmic material. This material adheres closely to the surface of the tumor cells and is subsequently seen within the cytoplasm of the tumor cell. The tumor cells then undergo degenerative changes and cell death occurs in 24 to 36 hr. The lymphocyte-derived material appears to contain immunoglobulin components as determined by specific ferritin labeling.

Animals↗
PubMed1993

Effect of the electret-blood contact time on the adhesion of human blood platelets.

The paper presents the results of research into the time characteristics of adhesion of platelets to the electret surface made from polyethylene terephthalate PET foil of 36 microns thickness. The electrets were formed by using the electron beam method. The method based on the electrostatic induction phenomenon was utilized to measure the electric charge surface density. Evaluation of the time characteristics of the adhesion of blood platelets was performed on the basis of microscopic examination and with the use of the chamber method for determining the number of blood platelets in the phase microscopy. The results obtained have confirmed the influence of the electret effect on PET foil thrombogenesis. The number of spacious aggregates found on the surfaces being analysed did not undergo any changes despite the extension of the electret--blood contact time. The stationary course of the adhesion process as well as the unappreciable amount of blood platelets on the surface have proved that the negative electric charge makes it possible to obtain thrombogenic materials.

Biocompatible Materials↗