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PubMed1981

Immunocytochemical localization of S-100 protein in the brain of adult rat. An ultrastructural study.

The cellular and subcellular distribution of the nervous system-specific S-100 protein has been investigated in the brain of adult rat at the ultrastructural level by the pre-embedding unlabelled antibody PAP method. The protein is found in both fibrous and protoplasmic astrocytes and in the ependymal cells. the neurons, the oligodendrocytes as well as the microglial cells are lacking S-100. The labelled cells show a reaction product diffusely distributed in the cytoplasmic matrix and on specialized membranes, namely plasma membranes, outer mitochondrial membranes and membranes of the endoplasmic reticulum and Golgi apparatus. The astrocytic filaments and the axonemes of the ependymal cilia exhibit a strong immunoreactivity. The reaction product is also present in the nucleoplasm of the astrocytes and ependymal cells but it is absent from the nucleolus and nuclear envelope. This immunocytochemical data on tissue with satisfactory ultrastructural preservation, provides new information on the localization of the S-100 protein, and could contribute to the understanding of the biological role of the protein.

Astrocytes↗
PubMed1995

A method for determining the unitary functional capacity of cloned channels and transporters expressed in Xenopus laevis oocytes.

The Xenopus laevis oocyte is widely used to express exogenous channels and transporters and is well suited for functional measurements including currents, electrolyte and nonelectrolyte fluxes, water permeability and even enzymatic activity. It is difficult, however, to transform functional measurements recorded in whole oocytes into the capacity of a single channel or transporter because their number often cannot be estimated accurately. We describe here a method of estimating the number of exogenously expressed channels and transporters inserted in the plasma membrane of oocytes. The method is based on the facts that the P (protoplasmic) face in water-injected control oocytes exhibit an extremely low density of endogenous particles (212 +/- 48 particles/microns2, mean, SD) and that exogenously expressed channels and transporters increased the density of particles (up to 5,000/microns2) only on the P face. The utility and generality of the method were demonstrated by estimating the "gating charge" per particle of the Na+/glucose cotransporter (SGLT1) and a nonconducting mutant of the Shaker K+ channel proteins, and the single molecule water permeability of CHIP (Channel-like In-tramembrane Protein) and MIP (Major Intrinsic Protein). We estimated a "gating charge" of approximately 3.5 electronic charges for SGLT1 and approximately 9 for the mutant Shaker K+ channel from the ratio of Qmax to density of particles measured on the same oocytes. The "gating charges" were 3-fold larger than the "effective valences" calculated by fitting a Boltzmann equation to the same charge transfer data suggesting that the charge movement in the channel and cotransporter occur in several steps. Single molecule water permeabilities (pfs) of 1.4 x 10(-14) cm3/sec for CHIP and of 1.5 x 10(-16) cm3/sec for MIP were estimated from the ratio of the whole-oocyte water permeability (Pf) to the density of particles. Therefore, MIP is a water transporter in oocytes, albeit approximately 100-fold less effective than CHIP.

Animals↗
PubMed1980

Interlamellar tight junctions of central myelin. II. A freeze fracture and cytochemical study on their arrangement and composition.

The interlamellar tight junctions (ITJ) of central myelin (white matter from the parietal lobe and the medulla oblongata of the rat) were analyzed electron microscopically, making use of a wide range of different preparatory techniques. Freeze-fracture observations indicate that the ITJ are composed of rows of particulate subunits in glutaraldehyde-fixed or formaldehyde-fixed material, and in the unfixed state. The particulate subunits of the ITJ are preferentially associated with the protoplasmic (P) face in the aldehyde-fixed state, and no shift in the binding characteristics of the particles was observed after omission of aldehyde fixation. Tracer studies in conjunction with the dissociated appearance of the junctional globules suggest that the ITJ represent a leaky type of zonula occludens. It is assumed that the ITJ particles represent an "integral-type protein" that preferentially serves as a mechanical device maintaining the structural integrity of the central myelin sheath. By means of cytochemical experiments, the proteinaceous character of the ITJ subunits is established. An attempt is made, based on results from lipid extraction and protein digestion, to define certain cytochemical parameters of the ITJ proteins and to compare them with the current collection of chemically identified proteins of central myelin.

Animals↗
PubMed1980

Light and scanning electron microscopic study of cerebellar cortex of teleost fishes.

The teleostean cerebellar cortex has been studied with respect to its cytoarchitectonic arrangement and intracortical neuronal circuits. Samples of fish cerebellum were fixed either by immersion of vascular perfusion in 5% glutaraldehyde solution and processed for light and scanning electron microscopy. The cerebellar cortex shows four distinct layers: granular; fibrous stratum; Purkinje cell; and molecular layers. In the granular layer, mossy and climbing fiber glomeruli were characterized. The mossy glomerular region appeared as polygonal, round or ovoid clews formed by the convergence of up to 17 dendritic profiles upon a thick mossy fiber branch. The "en passant" nature of mossy fiber-granule cell dendrite synaptic relationship was clearly appreciated. The climbing fibers showed tendril and glomerular collaterals. The latter form thin, elongated glomeruli. Remnants of a neuroglial envelope were observed in the mossy fiber glomeruli but are apparently absent from the climbing fiber glomeruli. The beaded-shape Golgi cell axonal ramifications were observed participating in the formation of both glomerular types. Velate protoplasmic astrocytes and oligodendrocytes were also identified. The fibrous stratum appeared to be formed by compact bundles of thick and think myelinated axons, running horizontally beneath the Purkinje cell layer and apparently belonging to ascending climbing fibers and descending Purkinje cell axons. At the Purkinje cell layer a selective removal of Bergmann glial cells was observed allowing the visualization of the pericellular basket and the pinceaux. Climbing fiber stems and their tendril collaterals were seen on their way to the molecular layer ascending parallel to the Purkinje dendritic ramifications. Stellate neuron processes were found passing through the fan-like arborescence of Purkinje cell dendrites.

Animals↗
PubMed1980

The astrocytes in the retina and optic nerve head of mammals: a special glia for the ganglion cell axons.

The neuroglia in the retina and the intraocular portion of the optic nerve of the monkey and cat has been examined by light and electron microscopy. In the retina two types of macroglial cells can be distinguished: 1) Müller cells, and 2) astrocytes. The bipolar radial glial cells of Müller penetrate the entire thickness of the retina and their basal processes align in the nerve fibre layer to form septa that fasciculate the axons of the ganglion cells. In contrast to the Müller cells, the retinal astrocytes are not homogeneously distributed throughout the retina; their number correlates with the thickness of the nerve fibre layer. The processes of the astrocytes are confined to the ganglion cell layer and to the nerve fibre layer. In the latter, the astrocytic processes run parallel to and between the axons of a given nerve fibre bundle. According to cytological criteria, the retinal astrocytes are protoplasmic. In the intraocular portion of the optic nerve, however, the astrocytes are fibrous and their processes run perpendicular to the axon bundles of the prelaminar portion of the optic nerve. Thus, because of their intimate morphological relationship to axons of the nerve fibre layer and the intraocular portion of the optic nerve, the astrocytes in the eye of the monkey and the cat may be considered as a special glia for the axons of ganglion cells.

Animals↗
PubMed1993

Spirosymplokos deltaeiberi nov. gen., nov. sp.: variable-diameter composite spirochete from microbial mats.

Large (up to 100 micrometers long), loosely coiled, free-living spirochetes with variable diameters (from 0.4 to 3 micrometers in the same cell) were seen at least 40 times between August 1990 and January 1993. These spirochetes were observed in mud water and enrichment media from highly specific habitats in intertidal evaporite flats at three disjunct localities, one in Spain and two in Mexico. All three are sites of commercial saltworks. Associated with Microcoleus chthonoplastes the large spirochetes from Spain display phototaxis and a composite organization. Shorter and smaller-diameter spirochetes are seen inside both healthy and spent periplasm of larger ones. Small spirochetes attached to large ones have been observed live. From two to twelve spirochete protoplasmic cylinders were seen inside a single common outer membrane. A distinctive granulated cytoplasm in which the granules are of similar diameter (20-32 nanometers) to that of the flagella (26 nanometers) was present. Granule diameters were measured in thin section and in negatively-stained whole-mount preparations. Based on their ultrastructure, large size, variable diameter, number of flagella (3 to 6), and phototactic behavior these unique spirochetes are formally named Spirosymplokos deltaeiberi. Under anoxic (or low oxygen) conditions they formed blooms in mixed culture in media selective for spirochetes. Cellobiose was the major carbon source in 80% seawater, the antibiotic rifampicin was added, mat from the original field site was present and tubes were incubated in the light at from 18-31 degrees C. Within 1-2 weeks populations of the large spirochete developed at 25 degrees C but they could not be transferred to fresh medium.

Environmental Microbiology↗
PubMed1986

The human erythrocyte membrane skeleton may be an ionic gel. II. Numerical analyses of cell shapes and shape transformations.

In the first paper in this series (Stokke et al. Eur Biophys J 1986, 13:203-218) we developed the general theory of the mechanochemical properties and the elastic free energy of the protein gel--lipid bilayer membrane model. Here we report on an extensive numerical analysis of the human erythrocyte shapes and shape transformations predicted by this new cell membrane model. We have calculated the total elastic free energy of deformation of four different cell shape classes: disc-shaped cells, cup-shaped cells, crenated cells, and cells with membrane invaginations. We find that which of these shape classes is favoured depends strongly on the spectrin gel osmotic tension, IIGu, and the surface tensions, IIEu and IIPu, of the extracellular and protoplasmic halves of the membrane lipid bilayer, respectively. For constant ratio IIEu/IIPu greater than O large negative or positive values of IIGu favour respectively the crenated and invaginated cell shape classes. For small absolute values of IIGu, IIEu, and IIPu, biconcave or cup-shaped cells are the stable ones. Our numerical analysis shows that the higher the membrane skeleton compressibility is, the smaller are the values of IIGu needed to induce cell shape transformation. We find that the stable and metastable shapes of discocytes and stomatocytes generally depend both on the shape of the stressfree membrane skeleton and the membrane skeleton compressibility.

Elasticity↗
PubMed1994

Surface distribution and partition during freeze-fracture of CD8 antigens on human lymphocytes and on epithelial transfected cells.

Freeze-fracture immunocytochemistry was used to analyse the surface distribution, redistribution induced by antibodies, and partition during freeze-fracture, of CD8 molecules on human T lymphocytes and rat epithelial transfected (FRT-U10) cells. Immunogold labelling of CD8 antigens was uniform over the unfractured cell surfaces of both lymphocytes and epithelial transfected cells. After freeze-fracture, the gold particles were associated with the exoplasmic outer leaflets of the plasma membranes in both cell types. In lymphocytes, incubation with antibodies at 37 degrees C up to 20 min induced patching and capping of the antigens on the unfractured cell surface. After fracture, the patched molecules appeared associated with the protoplasmic inner leaflet of the plasma membranes. Parallel antibody-treatment at 37 degrees C of FRT-U10 cells induced clustering of CD8 molecules but failed to cause further aggregation in larger patches or in caps. After freeze-fracture, the immunolabelling was clustered, but associated with the exoplasmic outer leaflet of the plasma membranes as in untreated cells. The different redistribution induced by antibodies and the different behaviour on fracture of the redistributed molecules in the two cell types may be regulated by CD8 interaction with the cytoskeleton.

Animals↗
PubMed1994

Cytoplasmic inclusions of astrocytic elements of glial tumors: special reference to round granulated body and eosinophilic hyaline droplets.

Round granulated body (RGB) and eosinophilic hyaline droplets (EHDs) have been described as cytoplasmic inclusions of certain astrocytic tumors. In the previous literature, however, these inclusions have been described using various terms or regarded as nosologically the same entity. Light microscopically, RGB appeared as a round discrete body filled with fine uniform granules, while EHDs demonstrated a cluster of bright eosinophilic, round objects of various size. They could be clearly distinguished even by conventional histochemical staining such as the Masson trichrome stain and the phosphotungstic acid hematoxylin preparation. Both RGB and EHDs expressed positive immunoreactions for glial fibrillary acidic protein, several lysosomal markers, and some stress-response proteins. The ultrastructural appearances of these inclusions were distinct, however, one common feature was that they consisted of aggregations of numerous membrane-bound electron-dense bodies. Thus, both inclusions appear to be produced by neoplastic astrocytes and are possibly related to the lysosomal system. We examined the presence of RGB and EHDs in 138 astrocytic tumors. Both inclusions occurred most frequently in pleomorphic xanthoastrocytomas, followed by gangliogliomas and pilocytic astrocytomas. Subependymal giant cell astrocytomas exhibited only RGBs. RGBs and EHDs were not seen in any abundance in glioblastomas, gliosarcomas, fibrillary astrocytomas, protoplasmic astrocytomas, or oligo-astrocytomas. Some glioblastomas, however, showed only EHDs in small numbers. Several anaplastic astrocytomas were associated with a large number of RGBs and/or EHDs, and they revealed only rare mitosis despite marked cellular pleomorphism. Although RGB and EHDs have different morphological features, the presence of these inclusions in abundance may represent either a degenerative change, a long-standing lesion, or an indolent growth of the astrocytic tumors.

Adolescent↗
PubMed1983

A Golgi study of Bergmann glial cells in developing rat cerebellum.

In order to examine the relationship between the Bergmann glial cells and the migrating granule cells, the postnatal development of the Bergmann glial cells in the rat cerebellum was analysed by a rapid Golgi method. In newborn rats where immature Purkinje cells occupied a rather thick zone (about 8 cells thick) between the thin molecular layer and the intermediate zone, immature Bergmann glial cells were recognized by the irregularly contoured somata situated within the deep part of the zone of Purkinje cells and by several perpendicular thin fibers (filiform fibers) which traversed the external granular layer (EGL) to terminate at the pial surface. After day 2 of the postnatal age (PD2), both somata and fibers of Bergmann glial cells showed gradual or fairly abrupt changes. The somata migrated upwards toward the molecular layer on PD2 and on PD4 were situated just beneath the Purkinje cells which had become arranged in a single layer. After PD6 the distance between the pial surface and the somata situated in the Purkinje cell layer and concomitantly the length of the Bergmann glial fibers, progressively increased in accordance with the thickening of the molecular layer. Between PD0 and PD8 the somata were irregularly contoured with short protoplasmic processes extending radially. After PD8 they gradually lost these short processes and became smooth. The Bergmann glial fibers were rather smooth with a few beady enlargements and tiny bud-like excrescences on their surface between PD0 and PD8. On PD12 the bushy expansions, characteristic of matured Bergmann glial fibers, suddenly increased in number on most fibers. After PD12 they continued to augment until PD25, when most fibers were entirely covered with the expansions. The number of fibers issuing from each Bergmann glial cell and entering the EGL increased postnatally reaching a peak on PD8, and then decreased gradually. These changes in the number of Bergmann glial fibers corresponded well with those in the number of external granule cells, suggesting the presence of developmental interactions between these two kinds of cells.

Animals↗
PubMed1992

Vimentin immunoreactivity in normal and pathological human brain tissue.

Vimentin immunoreactivity was examined in brain tissues from non-neurological and various human central nervous system disease cases. In all brain tissues examined, vimentin immunoreactivity was intensely positive in ependymal cells and subpial tissues, and weakly positive in some capillaries and some white matter astrocytes. In affected areas of Alzheimer's disease (AD), Pick's disease, amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS) and cerebral infarction cases, numerous intensely vimentin-immunopositive astrocytes of both protoplasmic and fibrous morphology were demonstrated. A few such astrocytes were also observed in Parkinson's disease and progressive supranuclear palsy. ALS, MS and infarction brains also had numerous, strongly vimentin-positive, round and fat-laden microglia/macrophages. In AD and ALS, a few reactive microglia with irregularly enlarged shapes were vimentin positive. In AD, they were almost exclusively related to senile plaques.

Aged↗
PubMed1994

Rosenthal fibers, eosinophilic inclusions, and anchorage densities with desmosome-like structures in astrocytes in Alzheimer's disease.

Ultrastructural study of the cerebral cortex of nine brains of individuals with Alzheimer's disease (AD) revealed four types of pathological changes of astrocytes. Rosenthal fibers were found in three cases, eosinophilic inclusions in one, anchoraged densities with desmosome-like structures in two, and corpora amylacea in four. In two biopsies, Rosenthal fibers were seen in less than 5% astrocytes, but in a third biopsy with numerous plaques, tangles, and severe neuronal loss, they were present in about 40% of astrocytes. In one case with severe AD pathology and numerous Rosenthal fibers, the cytoplasm of some astrocytes was occupied by inclusions composed of electron-dense granules 3-6 microns in diameter or aggregates of inclusions greater than 12 microns in diameter. Ultrastructurally, they were similar to eosinophilic inclusions observed in Aicardi syndrome and brain malformations. The presence of eosinophilic inclusions in the brain of elderly persons with Alzheimer's disease does not confirm the previous suggestion that this form of astrocyte pathology is typical for protoplasmic astrocytes and developmental brain malformations. Development anchorage densities associated with hemidesmosome-like structures, which reinforce astrocyte cell membranes facing the perivascular space, may reflect adaptation of astrocytes to the complex of changes that occurs in atrophic brain. Morphological changes in astrocytes in areas with numerous plaques and massive infiltration of intercellular space with beta-amyloid fibrils and remnants of neurons and ghost tangles suggest that astrocyte pathology is a late unspecific reaction to the cascade of changes induced by beta-amyloid deposition that causes neuronal degeneration and brain atrophy.

Alzheimer Disease↗
PubMed1991

Three-dimensional model of tight junction fibrils based on freeze-fracture images.

To study the three-dimensional structure of tight junction fibrils, the epithelia of the jejunum and epididymis of adult mice were examined by the freeze-fracture technique in unfixed and in aldehyde-fixed specimens. The fibrils have a stronger affinity for the protoplasmic (P) face of the lipid bilayer in fixed material, and for the external (E) face in unfixed and rapidly frozen material. Therefore we can observe the fibrils both from the outside and inside of the cell. Fibrils appearing on the P-face are smoothly contoured ridges and rows of hemispherical particles, while those appearing on the E-face are exclusively rows of hemispherical particles. Based on these observations, we wish to propose a new fibril model for the tight junction. There are two distinctive types of junctional elements. One type is composed of a smooth and continuous strand in the external view of the cell, but is studded with hemispherical bulgings in its internal view. This type will be referred to as the "continuous type". The other type is bead-like, and will be referred to as the "particle type". The relative proportion of these two types of elements appearing within a tight junction network differs among tissues.

Animals↗
PubMed1984

The perineuronal net in the fastigial nucleus of the rat cerebellum. A Golgi and quantitative study.

The morphological study of the rat fastigial nucleus with the Golgi-Rio Hortega method showed the presence of glial perineuronal nets surrounding the large neurons, but not the small ones. This perineuronal net appeared as a mesh of tenuous glial processes which covers the neuronal perikarya and proximal dendrites. The small alveolate compartments in this mesh seem to correspond to the holes for the synaptic boutons. Our results also indicate that the perineuronal net is derived from interneuronal protoplasmic and velate astrocytes. Using camera lucida drawings of this perineuronal net we have made a quantitative estimation of the size and density of synaptic boutons on these large neurons. The average numerical density of synaptic boutons was about 19 per 100 micron 2 of the neuronal surface, the mean area of the synaptic holes being 2.5 micron 2. Furthermore, the quantitative data evidence that about 52.5% of the neuronal surface is presumably occupied by synaptic boutons whereas the remaining 47.5% is covered by the glial processes of the perineuronal net. Semithin sections prepared from thick Golgi sections were used for the cytological study of the neurons surrounded by this glial pericellular network. The possible functional significance of the perineuronal net in the regulation of synaptic transmission in the fastigial cerebellar nucleus is briefly discussed.

Animals↗
PubMed1989

The structure of rabbit retinal Müller (glial) cells is adapted to the surrounding retinal layers.

Radial glial (Müller) cells of the rabbit retina were studied by various techniques including Golgi impregnation, scanning electron microscopy, horseradish peroxidase application, and staining of enzymatically isolated cells. This combination of methods produced detailed information on the specialized morphology of the Müller cells within the different topographical regions of the retina, and of the Müller cell processes within the various retinal layers. As a general rule, the retinal periphery contains short thick Müller cells with big endfeet, whereas the thick central retina is occupied by long slender cells with small endfeet. Independent of their location within the retina, Müller cell processes were found to be adapted to the structure of the surrounding retinal layers. Within the outer and inner nuclear layers, Müller cell processes (and somata) extend thin cytoplasmic "bubbles" ensheathing the neuronal somata, as do the "velate" astrocytes in the brain. In the plexiform layers, Müller cells extend many fine side branches between the neuropil, comparable to the protoplasmic astrocytes of the brain. In the thick myelinated nerve fibre layer of the central retina the Müller cell processes are rather smooth, similar to those of fibrous astrocytes. It is concluded that the neuronal microenvironment determines the morphology of a given glial process, or even of a part of a glial process running through a specialized neuronal compartment.

Animals↗
PubMed1993

p53 protein expression in central nervous system tumors: an immunohistochemical study with CM1 polyvalent and DO-7 monoclonal antibodies.

Formalin-fixed, paraffin-embedded surgical specimens from 137 primary central nervous system tumors, including 26 astrocytomas (21 fibrillary, 1 protoplasmic, 1 gemistocytic and 3 pilocytic), 26 anaplastic astrocytomas, 9 glioblastomas, 1 gliosarcoma, 8 oligodendrogliomas, 4 ependymomas, 1 anaplastic ependymoma, 2 subependymomas, 3 paragangliomas, and 57 meningiomas, were immunostained with the CM1 polyclonal (pAb) and the DO-7 monoclonal (mAb) antibodies against the p53 protein, using the streptavidin/peroxidase method. In addition, two series of 17 and 9 medulloblastomas were also immunostained with the above pAb and mAb, respectively. p53 protein expression was observed in 7 fibrillary astrocytomas, 17 anaplastic astrocytomas, 5 glioblastomas, 1 gliosarcoma, 1 oligodendroglioma, 1 anaplastic ependymoma, and 4 meningiomas with the CM1 pAb. An additional 10 cases (i.e., 3 anaplastic astrocytomas and 7 meningiomas) were found to be p53 protein positive with the DO-7 mAb. Of the medulloblastomas, 8 (of the 17) and 4 (of the 9) were found to express p53 protein with CM1 pAb and DO-7 mAb, respectively. Our results indicate that p53 protein is expressed in a number of central nervous system neoplasms, and suggest that in astrocytic tumors a possible association may exist between p53 protein expression and tumor progression through increasing histological grades of malignancy.

Antibodies, Monoclonal↗
PubMed1985

Three-dimensional analysis of dendritic spines. III. Glial sheath.

The glial envelope of dendritic spines in the visual and cerebellar cortices was evaluated by analysis of serial sections. Three-dimensional reconstructions of the protoplasmic astrocyte processes were made and the quantitative proportions of the glial cover on dendritic spines were measured and compared. Whereas dendritic spines on spiny branchlets of Purkinje cells are, with the exception of afferent axon terminals, completely covered by the glial sheath (74.44%), dendritic spines of pyramidal cells are only partially covered (28.89%), so that spine stalks and even synaptic clefts frequently lack glial isolation. A new, relatively frequent configuration of subsurface cistern--astrocyte process--dendritic spine is described. A possible functional significance of the differences in the glial ensheathment of dendritic spines in visual and cerebellar cortices is discussed.

Animals↗
PubMed1977

Suppression of cell-mediated immunity in experimental Chagas' disease.

The effect of acute infection with the Tulahuén strain of Trypanosoma cruzi on the cellular immune response in Swiss mice was studied. Mice were immunized with either Freund's complete adjuvant or oxazolone, a skin sensitizing agent, and subsequently skin-tested with either BCG protoplasm or oxazolone to detect delayed hypersensitivity. Depression of the response to these antigens was observed in infected mice during the stage of marked parasitemia. Mice which were responsive to oxazolone before infection lost their ability to respond as the infection progressed. When immunized with live attenuated T. cruzi before infection with virulent organisms, mice developed a greater than normal sensitivity to oxazolone and survived infection. These experiments do not conclude whether immunosuppression due to infection with T. cruzi is directed toward induction or expression of the cell-mediated immune response to the antigens employed.

Animals↗