Biomedical subjects

R R Shivers

Publications and source records attributed to R R Shivers.

At least 37 records · Page 2Linked to original sources

PubMed1988

The effect of heat shock on primary cultures of brain capillary endothelium: inhibition of assembly of zonulae occludentes and the synthesis of heat-shock proteins.

Subjecting primary cultures of bovine brain microvessel endothelial cells to thermal stress (heat shock) results in: (1) an inhibition of further tight junction assembly, (2) the disappearance and/or disassembly of tight junctions, (3) a 30-fold increase in the number of plasmic fracture (PF)-face intramembrane particles, and (4) the new and/or enhanced synthesis of at least three heat-shock polypeptides (HSPs) with molecular masses of approximately 100,000, 90,000 and 70,000. Endothelial cells which are heat-shocked and allowed to recover at 37 degrees C exhibit, within the first 2 h, a marked depression in the synthesis of HSPs and the new and/or enhanced synthesis of a 47,000 dalton "recovery" polypeptide. In later periods of recovery (2-4 h), the synthesis of this polypeptide is even more pronounced and is accompanied by the new and/or enhanced synthesis of a polypeptide(s) with a molecular mass of 35 to 37,000. The appearance of these "recovery protein(s)" in the endothelial cells is concomitant with a decrease in the number of PF-face intramembrane particles and the resumption of tight junction assembly. Results of this study suggest that some of the HSPs synthesized by thermally-stressed cultures of brain endothelial cells may activate or be directly involved in a mechanism(s) to ensure survival of these cells by decreasing membrane fluidity and stabilizing the plasma membrane of these cells. Moreover, our results also suggest that the recovery of these cells from the stress of heat shock is accompanied by the synthesis of "recovery" proteins which, in some manner, may be directly involved in, or necessary for, rapidly reversing the membrane-stabilizing effect of heat shock by promoting membrane fluidity and the apparent amplified synthesis and assembly and/or reassembly of tight junctions.

Animals↗

PubMed1987

Magnetic resonance imaging temporarily alters blood-brain barrier permeability in the rat.

Exposure to a short (23.2 min) standard clinical magnetic resonance imaging (MRI) procedure elicits a temporary dysfunction of the blood-brain barrier in rats. Monitoring of the increased permeability of rat brain frontal cortex microvessels with the protein tracer horseradish peroxidase and freeze-fracture electron microscopy, revealed an amplified vesicle-mediated transport of tracer across the microvessel endothelium to the albuminal basal lamina and extracellular compartment of the brain parenchyma. Recovery of normal blood-brain function, as evidenced by exclusion of protein tracer from subendothelial basal lamina and neuropil extracellular milieu, was complete 15-30 min following cessation of the MRI exposure. These findings raise the possibility that exposure to clinical MRI procedures may also temporarily alter central blood-brain permeability in human subjects.

Animals↗

PubMed1987

Chemical synapses, particle arrays, pseudo-gap junctions and gap junctions of neurons and glia in the buccal ganglion of Helisoma.

The nervous system of the snail, Helisoma trivolvis, has been utilized for a wide range of studies of neuronal plasticity; however, the ultrastructural features of this tissue were previously unknown. The present study examined the nature of synaptic interactions of neurons and glia and considered several plasma membrane specializations of these cells. The symmetrical pair of buccal ganglia consisted of a ring of unipolar neurons surrounding a central neuropil. The neurons were separated by two morphologically distinct types of glia: type I were most numerous and possessed an electron-dense homogeneous cytoplasm, whereas type II glia were of lower electron density, possessed a heterogeneous cytoplasm, and appeared to be phagocytic. Gap junctions were abundant between glia and were occasionally found between neuronal processes, including those of neurons 19 injected with horseradish peroxidase (HRP). Comparison of neuron and glial gap junction widths (16.4 and 17.6 nm, respectively) in thin sections and their intramembrane particle diameters (13.1 and 13.7 nm, respectively) by freeze fracture, did not elucidate significant differences. A heterogeneous population of putative chemical synapses, similar to those reported in other molluscs, was also observed between axonal collaterals in the neuropil. Additionally, examination of freeze-fractured neuropil revealed rhombic arrays of particles localized on neuronal membranes; these arrays do not appear to form intercellular junctions but may represent postsynaptic receptor sites. Freeze fracture also revealed small, square arrays consisting of 7-9 nm diameter particles on glial membranes which may correspond to pentalaminar membrane contacts (pseudo-gap junctions) seen in thin sections between glia situated around dilated extracellular spaces (lacunae).

Animals↗

PubMed1987

Gap junctions and zonulae occludentes of hepatocytes during biliary atresia in the lamprey.

Gap junctions and zonulae occludentes of hepatocytes were examined in thin sections and freeze-fracture replicas from livers of larval and juvenile adult lampreys and during the phase of metamorphosis when bile ducts and bile canaliculi disappear (biliary atresia). Larvae possess zonulae occludentes at the canaliculi which are composed of one to five (mean = 2.81) junctional strands that provide a bile-blood barrier. Morphometry demonstrates that during biliary atresia the decreases in number of junctional strands and apico-basal depth of the zonulae occludentes are accompanied by an increase in the frequency of gaps or interruptions in the strands and in a breakdown of the bile-blood barrier. The zonulae occludentes completely disappear during metamorphosis and are not found in the adult liver. Gap junctions of the larval liver occupy 1% of the surface of the plasma membrane and have a mean area of 0.167 micron 2 but, following an initial decline in these parameters during early biliary atresia, they rise sharply in later stages of metamorphosis and in adults are 3.2% and 0.502 micron 2, respectively. The events of alteration in junctional morphology during lamprey biliary atresia is in many ways comparable to the changes in gap junctions and zonulae occludentes during experimental and pathological intra- and extrahepatic cholestasis in mammals.

Animals↗

PubMed1987

A freeze-fracture study of muscle fibres infected with Trichinella spiralis.

The muscle fibres of mice containing the infective-stage larvae of the nematode Trichinella spiralis have been studied by means of the freeze-fracturing technique. The larva lies in what appears to be a fluid-filled cavity within the cytoplasm of an altered muscle fibre. There is no membrane separating the cytoplasm of the nurse cell from the cavity surrounding the larva which is therefore truly intracellular, unlike many parasites that reside within a membrane-lined parasitophorous vacuole within the host cell. This altered muscle fibre, known as a nurse cell, lacks myofilaments but does contain extensive cisternae of endoplasmic reticulum; membrane-bound vesicles are budded off from the endoplasmic reticulum and traverse the cytoplasm towards the cavity containing the nematode where they apparently pass into the cavity. It is suggested that the contents of these vesicles are used to sustain the nematode. Attention is drawn to the similarity to giant cells that have been induced by the plant-parasitic nematode Meloidogyne in the roots of host plants and which sustain the nematode. The conversion of the muscle fibre into a nurse cell is probably brought about by the presence of a metabolic sink, the larval nematode, within the cell. This take-over of the control of a metazoan cell by another metazoan organism is most unusual and warrants further study.

Animals↗

PubMed1987

Astrocyte-mediated induction of tight junctions in brain capillary endothelium: an efficient in vitro model.

Fourth passage rat brain capillary endothelial cell cultures, which no longer possess the tight junctions characteristic of this highly specialized component of the blood-brain barrier, were used to study induction of zonulae occludentes in vitro. These cells, when grown in 50% rat brain astrocyte-conditioned medium and 50% alpha-MEM on an endothelial cell matrix-coated substrate (Cedarlane Labs, Hornby, Ont.), possessed numerous, elaborately complex, tight junctions which were identical to those displayed in vivo by intact brain capillary endothelium. Endothelial cells grown in 50% astrocyte-conditioned medium and 50% alpha-MEM on bare plastic or fibronectin-coated substrate, possessed no tight junctions. Results of this study clearly demonstrate the local control of tight junction biogenesis in brain capillary endothelial cells depends on: (1) an astrocyte-produced factor(s), and (2) a 'competent' (cell-produced) extracellular matrix.

Animals↗

PubMed1987

The uptake and storage of iron and lead in cells of the crayfish (Orconectes propinquus) hepatopancreas and antennal gland.

The heavy metals iron and lead are taken up and metabolized in a similar manner by the crayfish hepatopancreas, but only lead appears to enter cells of the antennal gland (green gland). Iron, on the other hand, which apparently is not taken up by the antennal gland cells following systemic injections of low doses (0.05 mg), exerts striking alterations in cell ultrastructure after pericardial injections of massive doses (0.5 mg). Electron microscopic examination and atomic absorption spectrophotometric analyses of the hepatopancreas and antennal glands of iron-injected crayfish revealed that iron was selectively stored in metal-containing vacuoles of R- and F-cells in the hepatopancreatic cells, where it accumulated in concentrations that were toxic to these cells. High doses of iron caused alterations in the ultrastructural morphology of the cells of the antennal glands, although no accumulation of iron was apparent. Lead was similarly stored in metal-containing vacuoles of the cells of the hepatopancreas of lead-injected crayfish, but also accumulated in high concentrations (prior to being excreted) in vacuoles, cytoplasmic bodies and vesicles in the cells of the antennal gland. In contrast, lead in high concentrations was relatively non-toxic to any of these cells, suggesting that crayfish were more efficient in detoxifying and eliminating lead than iron.

Animals↗

PubMed1986

Freeze-fracture analysis of intramembrane particles of erythrocytes from normal and dystrophic hamsters.

The intramembrane particles on plasma membranes of erythrocytes of Syrian hamsters afflicted with hereditary muscular dystrophy were compared to those from normal controls by freeze-fracture analysis. The reduced number of particles in both fracture faces of dystrophic erythrocyte plasmalemmae, as compared to that of control red cells, was found to be highly significant (P = .001). Results of this study therefore, support the concept of a generalized membrane defect (abnormality) in muscular dystrophy.

Animals↗

PubMed1986

Intramembranous organization of lobster excitatory neuromuscular synapses.

The fine structure of identified neuromuscular synapses of the single excitatory axon to the distal accessory flexor muscle in lobster limbs was examined with freeze-fracture and serial thin-section electron microscopy. The latter technique reveals presynaptic dense bars with synaptic vesicles aligned on either side of these bars and often fused to the membrane, suggesting exocytosis and confirming our previous contention that these bars are active zones of transmitter release. The intramembranous organization of these active zones, as revealed in freeze-etched tissue, is a ridge-like elevation of the P-face of the axolemma with a matching trough on the complementary E-face. The ridge on the P-face has rows of large scattered intramembranous particles along the apex and is often bordered by a series of small, circular depressions which are presumed to represent exocytotic vesicles attached to the presynaptic membrane. Complementing these depressions are a few volcano-like protuberances seen occasionally on the E-face membrane. Because such evidence for transmitter release occurred in both stimulated and non-stimulated preparations, it demonstrates that chemical fixatives employing aldehydes induce transmitter release. The postsynaptic receptor sites of these excitatory synapses are characterized by oval-shaped patches of densely packed particles on the E-face, arranged in a random pattern on the sarcolemma. The complementary P-face view exhibits a regular square array of particle imprints or pits.

Aminopyridines↗

PubMed1986

A freeze-fracture study of the body wall of adult, in utero larvae and infective-stage larvae of Trichinella (Nematoda).

The surface layers of the cuticle, the hypodermal membranes and the muscle membranes of the adult, the in utero larvae and the infective-stage larvae of the nematode Trichinella spiralis have been studied by means of the freeze-fracturing technique. The surface of the cuticle of both adults and larvae fractures in ways different from membranes of internal cells. The surface coat on top of the epicuticle is probably the layer that changes antigenically. Reticulate ridges, with associated particles, on the E face of the outer hypodermal membrane of the adult are probably sites of attachment of the hypodermis to the cuticle. Longitudinally arranged ridges, with associated particles, of the outer hypodermal membrane are probably points of attachment to the cuticle in the in utero and infective larvae. Rectilinear arrays of particles are present on the P face of the inner hypodermal membrane and the P face of the muscle membrane adjacent to the hypodermis of adults and larvae and probably play a role in adhesion of the muscle membrane to the hypodermis. Particle-free areas of membrane lie external to the Z bundles of the muscle cell and are similar to the sites of attachment of Z lines in insect muscles.

Animals↗

PubMed1986

Detection of carriers of human Duchenne muscular dystrophy by freeze-fracture analysis of erythrocyte plasmalemmal intramembrane particles.

Critical examination of plasma membrane particles on fracture faces of erythrocyte plasma membranes of human obligate carriers of Duchenne muscular dystrophy, control patients, and a patient afflicted with Duchenne muscular dystrophy revealed a 32% decrease in the number of intramembrane particles in erythrocytes of carriers compared with erythrocytes sampled from control patients. These results support the notion that quantitative analysis of intramembrane particles in freeze-fractured erythrocyte plasma membranes represents a new, rapid, simple, and highly accurate diagnostic tool for detection of carriers of human Duchenne muscular dystrophy.

Cell Membrane↗

PubMed1985

Nonparenchymal liver cells in a vertebrate without bile ducts.

The nonparenchymal portion of the liver of parasitic adult lampreys (Petromyzon marinus L.) consists of endothelial, Kupffer, fibroblast-like, fat-storing, and granulated cells. The fenestrae of endothelial cells are not organized into sieve plates but are of highly variable size and distribution. The dimension of some fenestrae suggests the possible transport of substances of large molecular size. Small numbers of Kupffer cells possess many features of these cells observed in other vertebrates but they do not have worm-like bodies and endogenous peroxidase activity. They are involved in erythrophagocytosis and perhaps the ingestion of other foreign material but they do not store iron. Fat-storing and fibroblast-like cells share many morphological features and may be different expressions of the same cell type. These perisinusoidal cells are rich in organelles suggesting protein synthesis but the fibroblast-like cells lack fat droplets. A cell with a large Golgi apparatus and associated cytoplasmic granules resembles the pit cell described in the liver of a few other vertebrates. The morphology of nonparenchymal cells of the liver in parasitic adult lampreys does not reflect the absence of bile ducts in this organism.

Animals↗

PubMed1985

A model for de novo synthesis and assembly of tight intercellular junctions. Ultrastructural correlates and experimental verification of the model revealed by freeze-fracture.

The structure and function of intercellular tight (occluding) junctions, which constitute the anatomical basis for highly regulated interfaces between tissue compartments such as the blood-testis and blood-brain barriers, are well known. Details of the synthesis and assembly of tight junctions, however, have been difficult to determine primarily because no model for study of these processes has been recognized. Primary cultures of brain capillary endothelial cells are proposed as a model in which events of the synthesis and assembly of tight junctions can be examined by monitoring morphological features of each step in freeze-fracture replicas of the endothelial cell plasma membrane. Examination of replicas of non-confluent monolayers of endothelial cells reveals the following intramembrane structures proposed as 'markers' for the sequential events of synthesis and assembly of zonulae occludentes: development of surface contours consisting of elongate terraces and furrows (valleys) orientated parallel to the axis of cytoplasmic extensions of spreading endothelial cells, appearance of small circular PF face depressions (or volcano-like protrusions on the EF face) that represent cytoplasmic vesicle-plasma membrane fusion sites, which are positioned in linear arrays along the contour furrows, appearance of 13-15 nm intramembrane particles at the perimeter of the vesicle fusion sites, and alignment of these intramembrane particles into the long, parallel, anastomosed strands characteristic of mature tight junctions. These structural features of brain endothelial cells in monolayer culture constitute the morphological expression of: reshaping the cell surface to align future junction-containing regions with those of adjacent cells, delivery and insertion of newly synthesized junctional intramembrane particles into regions of the plasma membrane where tight junctions will form, and aggregation and alignment of tight junction intramembrane particles into the complex interconnected strands of mature zonulae occludentes. The distribution of filipin-sterol complex-free regions on the PF intramembrane fracture face of junction-forming endothelial plasmalemmae corresponds precisely to the furrows, aligned vesicle fusion sites and anastomosed strands of tight junctional elements.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1985

Filipin-cholesterol complexes in plasma membranes and cell junctions of Tenebrio molitor epidermis.

The polyene antibiotic filipin combines with cholesterol in membranes to form complexes that are readily identifiable in the electron microscope. The distribution of filipin-cholesterol (FC) complexes is most easily studied by freeze-fracture. Larval epidermis of Tenebrio molitor (Insecta, Coleoptera) was maintained in vitro for 48 hr, since the electrophysiological properties of the cells are best characterized under these conditions. The cells were fixed in buffered 3.0% glutaraldehyde at RT for 15 min, transferred to fresh fixative containing 1% DMSO and filipin (final concentration; 0.5 mg/ml) for 3 hr RT. Control cells were treated in fixative containing 1% DMSO only. In freeze fracture replicas, FC complexes appear on the plasma membrane as large circular protrusions measuring 26.5 +/- 6.8 nm (x +/- s.d.) n = 50, in diameter and 17.1 +/- 2.8 nm, n = 50, in height and 11.7 +/- 2.6 nm, n = 25, in depth. Protrusions are about two times more frequent on the E face while pits are several times more frequent on the P face. FC complexes are most abundant (greater than 50/mu m2) on the basal membrane surface of the cells but are excluded from regions of hemidesmosomal plaques that anchor the cells to the basal lamina. FC complexes are also abundant on the apical surfaces of the cells where cuticle secretion occurs. In the lateral regions below the junctional belt, FC complexes are less numerous but often appear to increase in frequency in a graded fashion away from the junctional region. The septate junctions are relatively free of FC complexes except in regions where they open to form islands. These islands often contain gap junctions but the FC complexes rarely invade the particle domains of the gap junctions. Single FC complexes were seen in three out of a total of 97 gap junctions. Exposure of the epidermis to 20-hydroxyecdysone for 24 hr in vitro did not induce the appearance of FC complexes within the cell junctions.

Animals↗

PubMed1985

A freeze-fracture study of the digestive tract of the parasitic nematode Trichinella.

Freeze-fracture preparations of the esophagus and intestine of larvae and adults of the nematode Trichinella spiralis illustrate the distribution of intramembranous particles in membranes of a number of cell types, and several specializations were found. Esophageal glands are prominently linked by gap junctions, but gap junctions were not found between intestinal cells. Muscle cells of the esophagus have rectilinear arrays of particles, thought to be points of adherence of the muscles to the esophageal epithelium. Clusters of particles are associated with these arrays and particle-free areas (probably Z bodies) also occur. Intestinal cells have small particles in their microvilli, large particles in the cells' apical membranes, and intermediate size particles, similar to membranes of other cells, in the lateral and basal membranes. Apical smooth septate junctions and tricellular junctions occur between intestinal cells.

Animals↗

PubMed1985

Ultrastructure of the hepatocytes in a vertebrate liver without bile ducts.

Thin sections and freeze fracture replicas were used to study the structure of the hepatocytes of the parasitic adult lamprey (Petromyzon marinus L.). Despite the absence of bile ducts and bile canaliculi, the hepatocytes have some features which resemble those of cells in the livers of other vertebrates. Hepatocytes are characterised by large gap junctions, many cytoplasmic inclusions, and large deposits of iron. The latter is present throughout the cytoplasmic matrix and within large inclusion bodies which may arise through sequestration of parts of the cytoplasm by membrane isolation. There is no evidence for the involvement of hepatocytes in glucose metabolism but their fine structure reflects the production of bile products and the processing of lipoproteins. The accumulation of bile products within cytoplasmic inclusions resembles the situation resulting from biliary atresia or other cholestatic conditions in higher organisms. There is little folding of the plasma membrane facing the perivascular space (of Dissé), perhaps indicating limited involvement of this surface in the transport of bile products. Nerve endings in close apposition to hepatocytes suggest possible nervous control or metabolic function or the presence of sensory receptors in lamprey liver.

Animals↗

PubMed1985

A freeze-fracture paradigm of the mechanism for delivery and insertion of gap junction particles into the plasma membrane.

Formation of intercellular gap junctions between cultured bovine aorta endothelial cells was studied by examination of freeze-fracture replicas of endothelial plasma membranes. Newly divided cells in the endothelial monolayer displayed numerous plaque-like aggregates of intramembrane particles situated on the PF fracture face of the plasmalemma interpreted to represent gap junctions in various stages of assembly. Closely associated with these particle aggregates are numerous small circular PF face depressions (or EF face volcano-like protrusions) that are presumed to result from fusion of cytoplasmic vesicles with the plasma membrane. In most cases, intramembrane particles, identical to those that comprise the developing gap junctions, appear intimately associated with the vesicle fusion sites and with the forming junction particle aggregates; a morphological relationship that strongly suggests the gap junction particles have emanated from the fusion sites. These observations are suggested to constitute morphological evidence for insertion of gap junction intramembrane particles into the plasma membrane by fusion of particle-bearing cytoplasmic vesicle couriers with the plasmalemma.

Animals↗

PubMed1984

Isolated rat brain capillaries possess intact, structurally complex, interendothelial tight junctions; freeze-fracture verification of tight junction integrity.

Populations of isolated brain capillaries have been proposed as useful models for in vitro studies of the blood-brain barrier. Preliminary investigations of barrier properties using such preparations of brain microvessels have suggested that the tight interendothelial junctions (zonulae occludentes) are intact and retain the impermeability to the protein tracer horseradish peroxidase, exhibited by them in vivo. The endothelial junctions of isolated capillaries are therefore assumed to be functionally "tight' in vitro. In order to determine the precise structural organization of these occluding junctions, including an estimate of their tightness (complexity), and to demonstrate a method for simple but precise assessment of junctional integrity, pellets of isolated rat brain capillaries were freeze-fractured and then replicated with platinum and carbon. The freeze-fracture images of interendothelial zonulae occludentes revealed complex arrays of intramembrane ridges and grooves characteristic of tight junctions. Longitudinal fractures of the cellular lining of capillaries exposed vast expanses of interendothelial plasma membrane interfaces and the junctional complexes situated between the cells. From such arrays, the elaborate and complex architecture of the zonulae occludentes could be readily appreciated. Situated on the PF fracture faces are 6-8 parallel ridges which display a high degree of anastomosing between adjacent strands. The EF fracture face contains grooves complementary to the PF face ridges. The zonulae occludentes of these capillary endothelial cells are similar in complexity to those reported in the literature for reptilian brain capillaries and therefore can be presumed "very tight'. This study demonstrates that freeze-fracture of pellets of brain capillaries alleviates sampling problems inherent in whole tissue preparations and, in addition, demonstrates the usefulness of freeze-fracture as a tool to monitor junction structure during in vitro investigation of the blood-brain barrier.

Animals↗