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Results for “Morphological Characterization”

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PubMed1985

Etiopathogenetic aspects of hepatitis A. I. Excretion of hepatitis A virus, biochemistry of liver function, and humoral immune response in patients with hepatitis A on admission to hospital.

The excretion of hepatitis A virus (HAV) in stools from 30 patients with clinically overt hepatitis A infection on the day of their admission to the hospital was determined and compared with the dynamics and values of biochemical indices of hepatocyte injury as well as with the immune response to HAV. Virus was found in 16 out of 30 stools (53%) collected within 1 week after the appearance of clinical symptoms. In sera obtained on the day of hospitalization both IgM and IgA anti-HAV were detected in all of the 30 patients, while IgG anti-HAV were found in 20 (67%). There was a correlation between HAV excretion and increasing SGPT upon admission to hospital, while the level of SGPT or bilirubin as well as presence or absence of IgG anti-HAV did not correlate with excretion of HAV. HAV from stools was characterized morphologically and physicochemically. The majority of particles visualized by immune electron microscopy had electrondense appearance, while electron-lucid particles were only occasionally encountered. Isopycnic banding of HAV in CsCl revealed a broad range of densities with HAV activity. Rebanding of pooled fractions containing HAV revealed peak amounts of the virus in fractions with densities 1.32-1.33 gm/cm3.

Adolescent↗

PubMed1984

Characterization of a subset of oligodendrocytes separated on the basis of selective adherence properties.

A subset of oligodendrocytes (B3,f) was isolated by taking advantage of selective cell-substratum interaction. B3,f cells were characterized morphologically, biochemically, and immunocytochemically. Oligodendrocytes were isolated from 4-to-6-month-old lamb brains by a modified version of our published procedure [Szuchet et al, J Neurosci Methods 3:7-19, 1980]. Freshly isolated cells from band III were plated on plastic culture plates at a concentration of 2 X 10(6) cells/ml. Approximately 40% of the cells attached to the plate under these conditions. The remaining cells formed small floating clusters. We refer to the latter as B3,f oligodendrocytes. After 4 to 5 days, the supernatant containing B3,f cells was removed and centrifuged, and the pellet was resuspended in culture medium and replated on polylysine-coated petri dishes. B3,f oligodendrocytes attached to this surface and extended an intricate network of processes. The purity of the cultures, judged by the number of cells staining with a monoclonal antibody against galactocerebroside was 98-99%. This high degree of cell homogeneity was maintained throughout the life of the cultures. B3,f cells appeared to be highly differentiated and remained so in vitro. This is surmised by the expression of oligodendrocytic characteristic functions such as high levels of CNPase activity typically, 5 microM/min/mgP; high incorporation of H2 35SO4 into sulfatides, an overall lipid metabolism that mimics events associated with myelinogenesis [Szuchet et al, PNAS 80:7019-7023, 1983]; the presence, detected immunocytochemically, of myelin-associated glycoprotein and myelin basic proteins. It is concluded that this culture system offers an opportunity for studying the biology of interfascicular oligodendrocytes and their interaction with neurons and/or astrocytes. It also should open up a way of examining the relevance of oligodendrocyte polymorphism.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1994

D2 dopamine receptor protein location: Golgi impregnation-gold toned and ultrastructural analysis of the rat neostriatum.

The neostriatal distribution of D2 dopamine receptor protein has been assessed using subtype-selective polyclonal antibodies generated against three unique polypeptide sequences of the receptor. The experimental tissues were processed by peroxidase based immunohistochemical procedures for routine light microscopy, Golgi impregnation-gold toned morphological characterization, and correlative light/electron microscopy. The results demonstrated a regional gradient of D2-like dopamine receptor expression in the neostriatum, where lateral portions in the nucleus exhibited more reactive cell bodies than medial portions. D2-like expression was detected in the three populations of neostriatal neurons, i.e., the medium-sized spiny projection neurons, and the medium- and large-sized aspiny interneuron types. Morphometric measurements of labeled neurons verified that medium and large diameter neurons expressed the D2-like receptor subtype. D2-like immunoreactivity was distributed throughout the cytoplasm in dendritic processes, and in presynaptic terminal boutons. Immunoreactivity for the receptor protein was also detected in small, thinly myelinated axons, suggesting the possibilities of anterograde transport of the receptor from cell bodies in the substantia nigra to their neostriatal terminal fields, as well as from local axon collaterals of neostriatal projections neurons. These findings provide evidence of widespread distribution of the D2-like receptor protein in neostriatal neurons, and showed that the presynaptic D2 receptors contain analogous epitopes to the postsynaptic receptor subtype.

Amino Acid Sequence↗

PubMed2002

Topical formulation studies with DEET (N,N-diethyl-3-methylbenzamide) and cyclodextrins.

The purpose of this study was to investigate the utility of cyclodextrins as a safer alternative to the commonly used cosolvent ethanol in topical N,N-diethyl-3-methylbenzamide (DEET) formulations. Ethanol is a known penetration enhancer and would ideally not be used in a formulation containing DEET, a compound known for its potential toxicity. DEET formulations were prepared containing up to 20% w/w gamma-cyclodextrin (GCD) or 30% w/w hydroxypropyl-beta-cyclodextrin (HPBCD). These formulations were evaluated by determining the release rate of DEET from its vehicle through a synthetic membrane into a receiver medium with a high affinity for DEET using a fully automated Franz-diffusion cell system. DEET release was measured by ultraviolet spectrophotometry at 260 nm. Both the traditional Higuchi release model and an alternate repeated measures analysis of variance with nested factors were used to analyze the data. The influence of the cyclodextrins on the volatility of DEET was determined by solid-phase microextraction. Morphological characterization of the formulations was performed by light microscopy. Physical stability of the formulations was evaluated by rheology in an accelerated study performed at 50 degrees C for 28 days. The cyclodextrins caused a decrease in the release of DEET from its vehicle compared with an ethanol-containing formulation. This effect became more pronounced as the amount of cyclodextrin in the formulation was increased. The addition of the cyclodextrins resulted in a cream-like product compared with the ethanol formulation, which was a lotion. The DEET formulations had the following rank order of volatility: ethanol < DEET = GCD < HPBCD. Examination of the formulations by light microscopy indicated that the CD-based formulations produced a smaller and more homogeneous droplet size distribution than the ethanol-based formulations. The accelerated physical stability study indicated that cyclodextrin-based formulations are both technically and commercially feasible. Cyclodextrins are a promising alternative to ethanol in DEET-containing formulations. The reduced release rate of DEET from these formulations suggests that the toxic potential of DEET may be decreased in these products, while its repellency and stability are maintained.

Administration, Topical↗

PubMed1976

Microtubules and actin filaments in teleost visual cone elongation and contraction.

Teleost retinal cones contract in light and elongate in darkness. This paper describes the disposition of microtubules and cytoplasmic filaments in cone cells of 2 species of fish (Haemulon sciurus and Lutjanus griseus). In Haemulon, the neck-like "myoid" region of the cone changes in length from 5 mu to 75 mu. Maximal observed rates of elongation and contraction are comparable to that of chromosome movement in mitosis (2-3 mu/min). Microtubules presumably participate in cone elongation, since numerous longitudinal microtubules are present in the myoid region, and colchicine blocks dark-induced elongation. Myoid shortening, on the other hand, appears to be an active contractile process. Disruption of microtubules in dark-adapted cones does not produce myoid shortening in the absence of light, and light-induced myoid shortening is blocked by cytochalasin-B. Cone cells possess longitudinally-oriented thin filaments which bind myosin subfragment-1 to form arrowhead complexes typical of muscle actin. Myoid thin filaments are clearly observed in negatively stained preparations of isolated cones which have been disrupted with detergent after attachment to grids. These myoid filaments are not, however, generally preserved by conventional fixation, though bundles of thin filaments are preserved in other regions of the cell. Thus, actin filaments are poorly retained by fixation in precisely the region of the cone cell where contraction occurs. Cone cells also possess longitudinally-oriented thick filaments 130-160 A in diameter. That these thick filaments may be myosin is suggested by the presence of side-arms with approximately 150 A periodicity. The linear organization of the contractile apparatus of the retinal cone cell makes this cell a promising model for morphological characterization of the disposition of actin and myosin filaments during contraction in a nonmuscle cell.

Actins↗

PubMed1988

A myasthenic syndrome with congenital paucity of secondary synaptic clefts: CPSC syndrome.

Two cases of a newly recognized myasthenic syndrome were investigated (CPSC syndrome). The course of the disease was observed for periods of 6 and 3 years. In infancy, exacerbations of the symptoms occurred during febrile illness, but thereafter the clinical course was stable and the children appeared to be only slightly handicapped. Biopsies were taken from the intercostal muscle in both patients. Microelectrode studies revealed small Mepp amplitudes. Light microscopy demonstrated predominance of type I fibers and focal type-grouping. There was a lowered cholinesterase activity and frequent branching of preterminal axons. Electron microscopy revealed that there were few, if any, folds of the postsynaptic membrane and that there were no signs of degeneration. Methods for localization of acetylcholine receptors (AChR) revealed a deficiency and altered distribution of AChRs at these postsynaptic membranes and the occurrence of extrajunctional AChRs in some muscle fibers. It is concluded that the syndrome is a clinicopathological entity, characterized morphologically by a congenital paucity of secondary synaptic clefts (CPSC syndrome).

Child↗

PubMed1990

Biliary epithelial cells from the liver of patients with primary biliary cirrhosis: isolation, characterization, and short-term culture.

Since biliary epithelial cells of the middle-sized interlobular bile ducts are targets for lymphocyte-mediated damage in patients with primary biliary cirrhosis (PBC), we have developed a method for isolating and maintaining these cells in short-term tissue culture. Intrahepatic biliary epithelial cells were isolated from small segments of liver removed at the time of transplantation. Cells were separated from a collagenase digest by immunomagnetic separation using Dynabeads coupled to a monoclonal antibody (HEA 125) specific for a biliary epithelial cell surface antigen. The yield was approximately 3 x 10(5) cells/g of liver. The isolated cells were characterized morphologically and ultrastructurally using light and electron microscopy, and immunocytochemically using HEA 125 and anti-cytokeratin, anti-vimentin and anti-asialoglycoprotein receptor antibodies. By these criteria cells were judged to be identical to biliary epithelial cells from normal liver. The cells could be maintained in short-term tissue culture for up to 4 weeks without loss of biliary epithelial cell markers. Availability of interlobular biliary epithelial cells will be of value in future investigations of the pathogenetic mechanisms of PBC.

Bile Ducts, Intrahepatic↗

PubMed1992

Dominantly inherited tubular aggregate myopathy.

We report an unusual familial myopathy characterized morphologically by the presence of large tubular aggregates in all fibre types. Two patients, a father and daughter, presented with slowly progressive proximal weakness, limitation of eye movement, and Achilles tendon contractures. Serum creatine kinase was 5-10 times normal. Light microscopy revealed type I fibre predominance. Basophilic accumulations, which stained intensely with the NADH-TR reaction, were present in both fibre types. Electron microscopy revealed that these consisted of tightly packed parallel tubular arrays. These varied somewhat in their ultrastructural appearance and were classified accordingly as type I, II, and III tubular structures. The tubular aggregates appear to be derived from the sarcoplasmic reticulum. This report further supports the evidence of a distinct clinico-pathological entity of genetic origin.

Adult↗

PubMedDate not supplied

Practical method for high-resolution imaging of polymers by low-voltage scanning electron microscopy.

Morphologic characterization of polymers by scanning electron microscopy (SEM) is often made difficult by their sensitivity to electron beam damage. We describe here a specimen preparation method for the imaging of polymer blends by low-voltage SEM (LV-SEM) that improves their stability in the electron beam and hence facilitates focusing and recording of high magnification images. Its application to nanosized core-shell latexes embedded in a polymethylmethacrylate matrix and semi-crystalline polypropylene/ethylene-propylene rubber blends is discussed.

Journal Article↗

PubMed1988

Ultrastructure of stratum lacunosum-moleculare interneurons of hippocampal CA1 region.

Intracellular recordings were obtained from nonpyramidal neurons (interneurons) in stratum lacunosum-moleculare (L-M) of the CA1 region of guinea pig hippocampal slices. These interneurons had response characteristics that distinguish them from pyramidal cells and other interneuron types: the L-M neurons had relatively broad action potentials with large spike afterhyperpolarizations, high input resistance and little spike-firing adaptation, and low spontaneous activity. Lucifer Yellow (LY) and horseradish peroxidase (HRP) were injected intracellularly into physiologically identified L-M interneurons, and the cells were characterized morphologically using light and electron microscopy. L-M somata were fusiform-shaped (15 x 25 micron), had multiple processes, and were located at the border between stratum (str.) lacunosum-moleculare and str. radiatum. L-M dendrites coursed through str. lacunosum-moleculare and projected into str. radiatum. L-M axons made axodendritic synaptic contacts primarily in str. lacunosum-moleculare and str. radiatum, but also in str. moleculare of the dentate gyrus. These axodendritic synaptic contacts were made onto spiny dendritic processes (presumably pyramidal cell and granule cell dendrites) and onto aspinous dendrites (presumably interneuron dendrites), and appeared to be of the symmetric type (type 2), characteristic of inhibitory synapses. In separate groups of animals, selective lesions were made of afferents to the CA1 and dentate regions of hippocampus, and subsequent degeneration of contacts and L-M interneuron somata and dendrites was examined at the ultrastructural level. Fibers originating from contralateral and ipsilateral CA3 region, and from ipsilateral entorhinal cortex, were found to make synaptic contact onto presumed L-M interneurons. Degenerating terminals appeared to be of the asymmetric type (type 1), characteristic of excitatory synapses. These morphological data are consistent with electrophysiological results showing that L-M interneurons can mediate feedforward inhibition of CA1 pyramidal cells.

Action Potentials↗

PubMedDate not supplied

Selective labeling of zebrafish thrombocytes: quantitation of thrombocyte function and detection during development.

Zebrafish thrombocytes, the nucleated equivalents of mammalian platelets, have been characterized morphologically, but knowledge about their developmental synthesis and biochemistry is limited. Given the increasing use of zebrafish as a genetic model to study hemostasis, it is important to isolate and study the function of zebrafish thrombocytes. Therefore, the objective of this study was to isolate thrombocytes, study their function in vitro, and identify the developmental stage at which they enter circulation. To achieve these goals, we developed a method for the selective labeling of thrombocytes and assayed these cells for activation by known mammalian platelet agonists. In both in vitro incubations of whole blood and blood labeled in vivo with the lipophilic dye DiI-C(18), we found labeling in only a single population of cells. These cells were identified as zebrafish thrombocytes by Wright-Giemsa staining. Using selective DiI-C(18) labeling, we showed the formation of thrombocyte aggregates, filopodia, and lipid rafts in response to platelet agonists. Additionally, we showed that aggregates are labeled by binding FITC-conjugated annexin V to exposed phosphatidylserine on the thrombocyte membrane. Using these fluorescent-labeling methods, we developed the first microquantitative assay for thrombocyte aggregation. With this assay, we provided evidence for the presence of an ADP receptor, P2Y(1), in the zebrafish thrombocytes. To study the developmental stage at which thrombocytes appear, we microinjected DiI-C(18) into the circulation of zebrafish embryos and identified the presence of DiI-C(18)-labeled thrombocytes at the 36 h postfertilization stage. These findings will prove helpful in dissecting the functions of thrombocytes in hemostasis and provide further insight into the role of platelets in thrombosis.

Animals↗

PubMed2002

Fractionation analysis of the endosomal compartment during rat reticulocyte maturation.

A subcellular fractionation procedure was developed to isolate the different endosomal compartments present during reticulocyte maturation. After reticulocyte lysis and removal of excess haemoglobin by gel chromatography, membrane vesicles were separated over a discontinuous sucrose gradient (10-40%). Two fractions were isolated: P1 at the 25-35% sucrose interface and P2 at the 17-25% sucrose interface. These fractions were morphologically characterized by electron microscopy and the distribution of endocytic markers in the fractions was detected by Western blot. Moreover, this fractionation technique was used to study the effect of 3-methyladenine (3-MA), an autophagy inhibitor, on reticulocyte maturation. The presence of 3-MA during in vitro maturation of reticulocytes induced a decrease in exosome secretion, as measured by the amount of transferrin receptor (TfR) released in the extracellular medium. The subcellular fractionation results suggested that multivesicular endosome formation from early endosomes is the step affected by 3-MA.

Adenine↗

PubMed1996

Detection on immunoblot of new proteins from the microsomal fraction recognized by anti-liver-kidney microsome antibodies type 1.

Previous studies have demonstrated that sera from patients with autoimmune hepatitis type 1 contain antibodies which react with proteins other than the endoplasmic reticulum integral membrane protein of apparent Mr 50,000, now known to be a cytochrome P450 of the IID subfamily. Sera from 141 patients found by immunofluorescence to be positive for anti-liver-kidney microsome antibodies type 1, and sera from 50 blood donors used as controls, were analyzed by immunoblotting experiments on rat liver microsomes, microsomal subfractions, and also microsomes subjected to various treatments, as described in the text. These fractions were characterized morphologically by electronic microscopy and biochemically by different enzymatic activities. Five bands were found to be stained more often by the patients' sera than by the controls' and with a statistically significant difference in frequency. These antigenic proteins were located at apparent Mr 62,000, 58,000, 50,000, 40,000, and 35,000. The 50,000 protein was of course more often stained than the others. Antibodies against these antigens belonged essentially to the IgG1 subclass. For some of them, subcellular localization and membrane topography are discussed. Interestingly, the 58,000 protein is not an integral membrane protein.

Adult↗

PubMed1994

Taxol-treated fibroblasts acquire an epithelioid shape and a circular pattern of actin bundles.

The effects of two microtubule-specific drugs, taxol and colcemid, upon the cell shape and cytoskeleton of several types of cultured fibroblastic cells were compared. While colcemid depolymerized completely the whole microtubular system, taxol induced decentralization of this system, leading to formation of numerous free microtubules filling the central cytoplasm. Morphometric determinations of two cell shape parameters, dispersion and elongation (G. Dunn and A. Brown, J. Cell Sci. (1986) 83, 313-340), have shown that, in all the tested cultures, taxol induced significantly larger decreases of average dispersion than colcemid; in addition, most taxol-treated cells, but not colcemid-treated ones, developed circumferential bundles of actin microfilaments instead of straight bundles. These results show that decentralization of the microtubular system, in contrast to its complete depolymerization, leads to the transformation of a polarized "fibroblast-like" cell morphology to an "epithelioid" morphology characterized by the smooth discoid cell shape and a circular actin pattern. Possible mechanisms of this transformation are briefly discussed.

3T3 Cells↗

PubMed1995

Mechanisms of activation and suppression in rat Nb 2 lymphoma cells: a model for interactions between prolactin and the immune system.

Rat Nb 2 lymphoma cells have been widely used to bioassay human growth hormone and many species of prolactin. Because their morphologic characterization suggests a T-cell lineage, Nb 2 cells were examined for their response to the T-cell mitogens concanavalin A, pokeweed mitogen, and phytohemagglutinin P. As expected, a dose-response to rat prolactin was observed; however, attempts to induce proliferation using the conventional T-cell mitogens failed at concentrations normally stimulatory for rat primary lymphocytes. Moreover, when Nb 2 cells were simultaneously incubated with lectin plus a suboptimal concentration of prolactin, a dose-dependent suppression of the stimulatory effects of prolactin was observed with phytohemagglutinin P and pokeweed mitogen, although not with concanavalin A. Culture medium of prolactin-stimulated Nb 2 cells also contained a factor which inhibited normal rat lymphocyte activation by concanavalin A. The factor did not block induction of the IL-2 receptor and proliferation of IL-2-dependent CTLL-2 cells could be restored by exogenous IL-2. Because Nb 2 cells evolved from a lactogen-dependent lymph node tumor, these results may have implications for further understanding the role of pituitary hormones, particularly prolactin, in the immune response to hormone-dependent tumor progression.

Animals↗

PubMed1997

Immunophenotype of human ovarian malignancies (cystadenocarcinomata and mixed müllerian tumor) established in SCID mice.

Human ovarian malignancies from three different patients (histology: two serous cystadenocarcinomata and one mixed Müllerian tumor, homologous type) were successfully serially transplanted intraperitoneally into severe combined immunodeficient (SCID) mice where the tumor cells spread around the peritoneal cavity. If the ascites derived from cystadenocarcinoma cells engrafted in the female genital tract of the SCID mice, they formed cystic tumors resembling remarkably well the original tumors in the patients. Immunohistochemical analysis revealed that the immunophenotype of the patients' original tumor and those grown in SCID mice were similar in the case of the two cystadenocarcinomata; in addition, the marker expression in general was stable during serial transplantation. If distant metastases occurred in the lungs, they immunophenotypically resembled those grown intraperitoneally. In contrast, the cells derived from the mixed Müllerian tumor shifted during serial transplantation from a spindle cell morphology toward a morphology characterized by cuboidal cells. The transition toward a more epithelial phenotype was accompanied by a changed immunophenotype of the tumor cells which became positive for epithelial cell markers such as carcinoembryonic antigens, CA 19-9 and CA 125. Concurrently with this differentiation, the p53 immunophenotype changed from positive to negative, indicating a further mutation in the p53 gene during serial passages.

Animals↗

PubMed2002

Changes in immunoreactivity to calcium-binding proteins in the anterior olfactory nucleus of the rat after neonatal olfactory deprivation.

The effects of olfactory deprivation on the density of neuronal populations expressing the calcium-binding proteins calbindin D-28k, calretinin, and parvalbumin in the anterior olfactory nucleus of the rat were studied immunohistochemically in 60-day-old rats subjected to unilateral naris closure on the day of birth. The neuronal populations were characterized morphologically and topologically, and the density of each cell type was calculated in each subdivision of the anterior olfactory nucleus at seven rostrocaudal levels. Data were gathered into three groups: data from either the ipsilateral or contralateral anterior olfactory nucleus of experimental animals and data from control animals. Statistical analysis indicated that disruption of the normal afferent activity to one olfactory bulb affects the expression of the calcium-binding proteins investigated in the anterior olfactory nucleus, as revealed by variations in the density of certain neuronal populations. The observed effects were very heterogeneous and could not be related to any specific neuronal type, location, or to the expression of a given calcium-binding protein. Nevertheless, as a general rule the most affected neuronal populations were those expressing calbindin D-28k located in the rostral subdivisions. These subdivisions are the latest to develop in mammals and are those that receive the largest amount of inputs from the olfactory bulb.

Animals↗

PubMed1995

Mapping of the gracile axonal dystrophy (gad) gene to a region between D5Mit197 and D5Mit113 on proximal mouse chromosome 5.

The gracile axonal dystrophy (gad) mouse, which shows hereditary sensory ataxia and motor paresis, has been morphologically characterized by the dying back type of axonal degeneration in the nerve terminals of dorsal root ganglion cells and motor neurons. In the present study, using an intraspecific backcross between gad and C57BL/6J mice, the gracile axonal dystrophy (gad) gene was mapped to a region between D5Mit197 and D5Mit113. Estimated distances between gad and D5Mit197 and between gad and D5Mit113 are 0.4 +/- 0.3 and 5.0 +/- 1.0 cM, respectively. The gene order was defined: centromere-D5Mit81-D5Mit233-D5Mit184/D5Mit254- D5Mit256-D5Mit197-gad-D5Mit113-D5Mit7. The mouse map location of the gad locus appears to be in a region homologous to human 4p15-p16. Our present data suggest that the nearest flanking marker D5Mit197 provides a useful anchor for the isolation of the gad gene in a yeast artificial chromosome contig.

Alleles↗