Biomedical subjects

M H Ellisman

Publications and source records attributed to M H Ellisman.

At least 91 records · Page 5Linked to original sources

PubMed1994

In vivo analysis of the stability and transport of nuclear poly(A)+ RNA.

We have studied the distribution of poly(A)+ RNA in the mammalian cell nucleus and its transport through nuclear pores by fluorescence and electron microscopic in situ hybridization. Poly(A)+ RNA was detected in the nucleus as a speckled pattern which includes interchromatin granule clusters and perichromatin fibrils. When cells are fractionated by detergent and salt extraction as well as DNase I digestion, the majority of the nuclear poly(A)+ RNA was found to remain associated with the nonchromatin RNP-enriched fraction of the nucleus. After inhibition of RNA polymerase II transcription for 5-10 h, a stable population of poly(A)+ RNA remained in the nucleus and was reorganized into fewer and larger interchromatin granule clusters along with pre-mRNA splicing factors. This stable population of nuclear RNA may play an important role in nuclear function. Furthermore, we have observed that, in actively transcribing cells, the regions of poly(A)+ RNA which reached the nuclear pore complexes appeared as narrow concentrations of RNA suggesting a limited or directed pathway of movement. All of the observed nuclear pores contained poly(A)+ RNA staining suggesting that they are all capable of exporting RNA. In addition, we have directly visualized, for the first time in mammalian cells, the transport of poly(A)+ RNA through the nuclear pore complexes.

Animals↗

PubMed1994

Fluorescence photooxidation with eosin: a method for high resolution immunolocalization and in situ hybridization detection for light and electron microscopy.

A simple method is described for high-resolution light and electron microscopic immunolocalization of proteins in cells and tissues by immunofluorescence and subsequent photooxidation of diaminobenzidine tetrahydrochloride into an insoluble osmiophilic polymer. By using eosin as the fluorescent marker, a substantial improvement in sensitivity is achieved in the photooxidation process over other conventional fluorescent compounds. The technique allows for precise correlative immunolocalization studies on the same sample using fluorescence, transmitted light and electron microscopy. Furthermore, because eosin is smaller in size than other conventional markers, this method results in improved penetration of labeling reagents compared to gold or enzyme based procedures. The improved penetration allows for three-dimensional immunolocalization using high voltage electron microscopy. Fluorescence photooxidation can also be used for high resolution light and electron microscopic localization of specific nucleic acid sequences by in situ hybridization utilizing biotinylated probes followed by an eosin-streptavidin conjugate.

Animals↗

PubMed1994

A 28 kDa sarcolemmal antigen in kidney principal cell basolateral membranes: relationship to orthogonal arrays and MIP26.

Two recently cloned water channels, CHIP28 and WCH-CD, are homologous to MIP26, an integral membrane channel-forming protein found in lens fiber plasma membranes. CHIP28 is found in basolateral and apical plasma membranes of kidney proximal tubules and thin descending limbs of Henle, whereas WCH-CD is apically located in collecting duct principal cells. So far, the putative water channel that may be responsible for the high constitutive permeability of principal cell basolateral membranes has not been identified. Interestingly, freeze-fracture electron microscopy has shown that characteristic orthogonal arrays of intramembrane particles (OAPs) are found on the basolateral plasma membranes of collecting duct principal cells, and that morphologically identical OAPs present in lens fiber cell plasma membranes contain the protein MIP26. Similar OAPs have also been detected on plasma membranes of other cell types including gastric parietal cells, astroglial cells and skeletal muscle fibers. By indirect immunofluorescence, western blotting and northern blotting, MIP26 was found only in lens fibers. In addition, functional studies on reconstituted and oocyte-expressed MIP26 excluded the possibility that MIP26 might be a basolateral water channel in the kidney. However, a polyclonal antibody raised against skeletal muscle sarcolemmal vesicles, which are enriched in OAPs, produced an intense staining of principal cell basolateral plasma membranes in kidney collecting duct and immunoprecipitated a 28 kDa protein from kidney papilla. The immunoprecipitated protein from papilla was not recognized by anti-CHIP28 or anti-MIP26 antibodies, indicating that principal cell basolateral membranes contain a novel member of the CHIP/MIP family. Because this antibody also stained brain astrocyte end feet, which are enriched in OAPs, it is possible that the 28 kDa protein is related to these structures. We conclude that OAPs probably contain related but distinct proteins that may have different membrane channel functions in different cell types.

Animals↗

PubMed1994

TTX-sensitive dendritic sodium channels underlie oscillatory discharge in a vertebrate sensory neuron.

Immunocytochemical and electrophysiological techniques were used to localize TTX-sensitive sodium channels (NaChs) over the soma-dendritic axis of basilar and nonbasilar pyramidal cells of the electrosensory lateral line lobe (ELL) of weakly electric fish (Apteronotus leptorhynchus). Dense NaCh-like immunolabel was detected on the membranes of basilar and nonbasilar pyramidal cell somata. Punctate regions of immunolabel (approximately 15 microns) were separated by nonlabeled expanses of membrane over the entire extent of basal dendrites. Similar punctate immunolabel was observed over the apical dendrites, and frequently on membranes of afferent parallel fiber boutons in the distal apical dendritic region. Intracellular recordings from pyramidal cell somata or proximal apical dendrites (75-200 microns) were obtained using an in vitro ELL slice preparation. TTX-sensitive potentials were identified by focal pressure ejection of TTX. Somatic recordings demonstrated both TTX-sensitive fast spike discharge and a slow prepotential; similar but lower amplitude potentials were recorded in apical dendrites. Dendritic spikes were composed of at least two active components triggered by a fast prepotential (FPP) generated by the somatic spike. TTX-sensitive spikes propagated in a retrograde fashion over at least the proximal 200 microns of the apical dendrites, as determined by the conduction of an antidromic population spike and focal TTX ejections. Somatic spikes were followed by a depolarizing afterpotential (DAP) that was similar in duration and refractory period to that of proximal dendritic spikes. During repetitive spike discharge, the DAP could increase in amplitude and attain somatic spike threshold, generating a high-frequency spike doublet and a subsequent hyperpolarization that terminated spike discharge. Repetition of this process gave rise to an oscillatory burst discharge (2-6 spikes/burst) with a frequency of 40-80 Hz. Both the DAP and oscillatory discharge were selectively blocked by TTX ejections restricted to the proximal apical dendritic region. The present study demonstrates an immunolocalization of NaChs over somatic and dendritic membranes of a vertebrate sensory neuron that correlates with the distribution of TTX-sensitive potentials. The interaction of somatic and dendritic action potentials is further shown to underlie an oscillatory discharge believed to be important in electrosensory processing.

Animals↗

PubMed1993

Distribution of ryanodine receptors in the chicken central nervous system.

The ryanodine receptor (RR), an intracellular calcium release channel, has been identified in the nervous system but its contributions to neuronal function are unknown. We have utilized immunohistochemical techniques to establish the distribution of RRs in the central nervous system (CNS) of the chick as a step toward elucidating the function of RRs in this system. RR immunoreactivity is observed throughout the brain, most prominently in large neurons. The strongest immunoreactivity is found in cerebellar Purkinje neurons, but nuclei in the motor, visual and vestibular systems are also intensely labeled, and immunoreactive neurons are observed the olfactory bulb and the hippocampus. In these neurons, labeling is prominent in cell bodies, dendrites and axons, but is not observed in the dendritic spines or in plasma membranes. The neuronal RRs bind [3H]ryanodine with high affinity and this activity is regulated by calcium, caffeine, MgCl2/ATP and ionic strength. Multiple forms of the RRs are found in the chicken CNS. Immunoprecipitation and localization studies using RR isoform specific monoclonal antibodies reveal major differences in their distribution. The predominant species in the cerebellum is similar to the skeletal muscle isoform while there is a lower level of expression of either the cardiac or beta skeletal isoforms. In the remainder of the brain, the predominant isoform is similar to the cardiac or beta skeletal muscle isoforms. The broad distribution of RRs in the CNS suggests that calcium release events mediated by these proteins may have a functional role in a diverse array of neurons. Moreover within the populations of neurons expressing RR's, the presence of specific RR isoforms may correlate with specialization in the calcium release events mediated by these proteins.

Animals↗

PubMed1993

Failure to make normal alpha ryanodine receptor is an early event associated with the crooked neck dwarf (cn) mutation in chicken.

We have investigated the molecular basis of the Crooked Neck Dwarf (cn) mutation in embryonic chickens. Using biochemical and pharmacological techniques we are unable to detect normal alpha ryanodine receptor (RyR) protein in intact cn/cn skeletal muscle. Extremely low levels of alpha RyR immunoreactivity can be observed in mutant muscles, but the distribution of this staining differs from that in normal muscle and colocalizes with the rough endoplasmic reticulum immunoglobulin binding protein, BiP. This suggests the existence of an abnormal alpha RyR protein in mutant muscle. In day E12 cn/cn muscle the levels of RyR mRNA are reduced by approximately 80%, while the levels of other muscle proteins, including the alpha 1 subunit of the dihydropyridine receptor, the SR Ca(2+)-ATPase, calsequestrin, and glyceraldehyde-3-phosphate dehydrogenase, and their associated mRNAs are essentially normal in cn/cn muscle. There is also a failure to express alpha RyR in cn/cn cerebellar Purkinje neurons. Expression of the beta RyR, a second RyR isoform, is not initiated in normal skeletal muscle until day E18. In cn/cn skeletal muscle significant muscle degeneration has occurred by this time and the beta RyR is found at low levels in only a subset of fibers suggesting the reduced levels of this isoform are a secondary consequence of the mutation. The cardiac RyR isoform is found in cn/cn cardiac muscle, which contracts in a vigorous manner. In summary, a failure to make normal alpha RyR receptor appears to be an event closely associated with the cn mutation and one which may be largely responsible for development of the cn/cn phenotype in embryonic skeletal muscle.

Animals↗

PubMed1993

Crooked neck dwarf (cn) mutant chicken skeletal muscle cells in low density primary cultures fail to express normal alpha ryanodine receptor and exhibit a partial mutant phenotype.

The Crooked Neck Dwarf (cn) mutation in chickens causes marked changes in intact embryonic skeletal muscle. We have investigated whether the cn/cn phenotype develops in vitro, and if cultured muscle cells are suitable for studies of this mutation. The properties of cn/cn muscle cells maintained in low density primary cultures (6.25 x 10(3) cells/cm2) are described in this report. In normal muscle cells, the alpha ryanodine receptor (RyR) isoform appears prior to, and at greater levels than, the beta RyR, and is detected in mononucleated myocytes. The beta RyR isoform appears within 24 hr after the initiation of myotube formation, which is earlier than anticipated from studies with intact embryonic muscle. Normal alpha RyR protein is not detected in cultured cn/cn muscle cells, whereas the beta RyR, the alpha 1-subunit of the dihydropyridine receptor, the sarcoplasmic reticulum Ca(2+)-ATPase, and calsequestrin are expressed at comparable levels in normal and mutant muscle cells. Calcium transients elicited by electrical stimulation, acetylcholine, and caffeine are similar in normal and cn/cn cultured myotubes and are blocked by ryanodine in both cell types. In addition, comparable L- and T-type calcium currents are observed in normal and mutant muscle cells, suggesting that both the alpha 1-subunit of the dihydropyridine receptor and the beta RyR in mutant muscle cells are functional. Normal and cn/cn muscle cells proliferate and form myotubes in a similar manner. These latter events do not appear to depend on sarcoplasmic reticulum calcium release, as they also occur in normal muscle cells in which calcium release is prevented by chronic treatment with 100 microM ryanodine. Both cn/cn and ryanodine-treated normal muscle cells exhibit morphological changes similar to those observed in intact cn/cn skeletal muscle. Thus, the mutant phenotype observed in ovo is partially expressed under low density culture conditions, and neither beta RyR protein nor its function appear to be capable of preventing the associated changes.

Animals↗

PubMed1993

Ultrastructure of the glomus cells in the carotid body of chronically hypoxic rats: with special reference to the similarity of amphibian glomus cells.

The ultrastructural characteristics of the glomus cells in the rat carotid body exposed to extremely long-term hypoxia (10-12 weeks) were investigated. The glomus cells could be classified into four distinct types according to the shape of dense-cored vesicles in the glomus cell cytoplasm: 1) small vesicle cells (SVCs, 50 nm in mean diameter), 2) large vesicle cells (LVCs, 80 nm in mean diameter), 3) dilated eccentric vesicle cells (EVCs, 400-800 nm in diameter), and 4) mixed vesicle cells (MVCs, large and eccentric vesicles). Many clusters of glomus cells were found to contain all four categories of cell types. The appearance of EVCs was a unique and common characteristic of glomus cells in this long-term hypoxia model. We also noted other ultrastructural features with chronic hypoxia which are characteristic of the amphibian carotid labyrinth glomus cells: 1) incomplete covering of glomus cells with the supporting cell missing over a wide area, 2) long thin cytoplasmic projections in the intervascular stroma, and 3) intimate apposition of the glomus cells and pericytes (g-p connection), endothelial cells (g-e connection), plasma cells, and fibrocytes. Because arterial PO2 is generally low in amphibia, these may be general features of hypoxic adaptation and facilitate both uptake of oxygen from blood and release of catecholamine into the blood. The g-p and g-e connections may take part in the regulation of the microcirculation in the enlarged carotid body.

Amphibians↗

PubMed1993

High-sensitivity lens-coupled slow-scan CCD camera for transmission electron microscopy.

A lens-coupled slow-scan CCD camera has been built for transmission electron microscopy (TEM) applications. In this design, a leaded glass window, which is coated with a 20 microns layer of red P20 phosphor, is mounted on the bottom of the microscope. A lens assembly and mirror prism, located outside the microscope vacuum below the leaded glass, relays the image onto a back-thinned 1kx1k charge-coupled device (CCD) detector. This CCD is electronically cooled to below -30 degrees C during operation. It is found that X-ray irradiation, generally found to be annoying in fiber-optically coupled CCD cameras, is completely eliminated by this configuration. The collection efficiency of this system, although not as high as some of the fiber-optically coupled CCD cameras, is high enough to achieve single-electron sensitivity under a high-gain mode.

Equipment Design↗

PubMed1993

Telemicroscopy.

The feasibility of operating an electron microscope from a remote location using a high-speed computer network has been investigated. A 400 kV electron microscope was modified so that most microscope parameters and stage movement (x, y, z and tilt), can be controlled by a local host computer. The host computer also controls acquisition of digital images from the microscope from either a TV or a slow-scan CCD camera. A computer located at a remote site may send commands to the host computer via a fiber optic network to receive digital images and information concerning the status of microscope. Such a system has been demonstrated at the SIGGRAPH 1992 conference in Chicago and the Supercomputing 92 conference in Minneapolis, where we remotely collected three-dimensional image data sets using the electron microscope in San Diego. This approach, referred to as "telemicroscopy", promises to make possible live microscopy teleconferencing and thereby increase the accessibility and effective usage of the limited number of specialized microscopy resources.

Computer Communication Networks↗

PubMed1993

Performance characteristics of radioluminescent fiber optics as electron scintillators.

Terbium-activated radioluminescent fiber-optic face plates have been studied for their suitability as electron scintillators. The energy conversion efficiency of such material is determined to be about 2.5% in the electron energy range of 100-400 keV tested. The resolution is approximately 40 microns at electron energy of 100 keV, as measured by the rise width of an edge. The main advantage of such a device is the potential simplification of CCD camera designs, since it functions both as a scintillator as well as a light guide so that it can be coupled direct to a CCD detector.

Animals↗

PubMed1993

Three-dimensional visualization of the smooth endoplasmic reticulum in Purkinje cell dendrites.

The three-dimensional organization of the smooth endoplasmic reticulum (SER) in Purkinje cell dendrites in the chick cerebellum was investigated to assess the connectivity between its various components. Three-dimensional reconstructions of the SER within portions of Purkinje cell dendrites were performed from serial sections through the cerebellar molecular layer. In addition, semithick and thick sections (0.25-1 micron) of chick cerebellum in which the endomembrane system had been selectively stained were examined with an intermediate high-voltage electron microscope. Three-dimensional information was extracted from thick sections using stereo pairs and tomographic reconstructions from single axis tilt series. In contrast to its discontinuous appearance in thin sections, in both the three-dimensional reconstructions and selectively stained thick sections the endoplasmic reticulum formed a highly interconnected network of tubules and cisterns extending throughout the dendritic shaft and into the spines. Several distinct morphological domains of endoplasmic reticulum were noted, including the hypolemmal cisternae, the endomembranes associated with the dendritic spines, and the tubular and cisternal endoplasmic reticulum in the dendritic shaft. In older chicks (aged 2 weeks), stacks of endoplasmic reticulum were also noted within the dendritic shaft. All elements of the SER within the dendritic shaft and spine appeared to be continuous with one another. The results of this study strongly suggest that the endoplasmic reticulum forms a complicated network that may be part of a single endomembrane system within Purkinje cell dendrite.

Aging↗

PubMed1992

Immunohistochemical localization of ryanodine binding proteins in the central nervous system of gymnotiform fish.

The ryanodine receptor, an integral membrane protein of the sarcoplasmic reticulum in muscle, embodies a high conductance channel permeable to calcium ions. Recent studies have identified ryanodine-binding proteins in avian and mammalian central nervous systems. These neuronal ryanodine receptors appear to function as Ca2+ channels which may gate the release of Ca2+ from caffeine-sensitive intracellular pools in neurons. In the present investigation, we employed monoclonal antibodies against ryanodine-binding proteins of avian muscle cells to the brain of weakly electric gymnotiform fish. Immunoprecipitation and Western blot analysis revealed two isoforms in the fish brain, with molecular weights comparable to those of avian and fish muscle ryanodine-binding proteins. By employing immunohistochemical techniques, we mapped these proteins in fish brain. Ryanodine receptor-like immunoreactivity was found in nerve cell bodies as well as dendrites and axonal processes. The ryanodine-binding protein is distributed throughout the neuraxis in specific cell types of the gymnotiform brain. In the telencephalon, immunoreactive cells were found in the glomerular layer of the olfactory bulb, in the supracommissural subdivision of the ventral telencephalon, and in the intermediate rostral subdivision of the ventral telencephalon. In the diencephalon, immunoreactive cells or fibers were observed in the nucleus prethalamicus and the habenula, within the nucleus at the base of the optic tract and the adjacent dorsal tegmental nucleus, the pretectal nuclei A and B, and the nucleus electrosensorius. In addition, immunopositive cells were seen in several nuclei of the hypothalamus, with the inferior and lateral subdivision of the nucleus recessus lateralis displaying the highest concentration of neurons. In the mesencephalon, the optic tectum contained the greatest number of immunopositive cells. In the rhombencephalon, labelling was seen in the nucleus of the lateral valvula, central gray, lateral tegmental nucleus, in boundary cells of the nucleus praeminentialis, efferent octavolateral nucleus, an area adjacent to the medial edge of the lateral reticular nucleus, nucleus medialis, and electrosensory lateral line lobe. As in avian brain, cerebellar Purkinje cells were positive for ryanodine-binding protein, although only subsets of Purkinje cells were labelled.

Animals↗

PubMed1992

Region-specific expression of a K+ channel gene in brain.

Northern blot analysis and in situ hybridization studies reveal the highly localized expression in rat brain of transcripts from a gene (KShIIIA) encoding components for voltage-gated K+ channels. KShIIIA expression is particularly prominent throughout the dorsal thalamus. The expression of KShIIIA is compared to that of a closely related gene, here called NGK2-KV4. These two genes encode transcripts that induce currents in Xenopus oocytes that are as of yet indistinguishable, but they show very different patterns of expression in rat brain. NGK2-KV4 transcripts are particularly abundant in the cerebellar cortex, where KShIIIA expression is very weak. These results demonstrate the existence of cell-type-specific K+ channel components and suggest that one reason for the unusually large diversity of K+ channel proteins is the presence of subtypes that participate in specific brain functions.

Amino Acid Sequence↗

PubMed1992

Distribution and ontogeny of parvalbumin immunoreactivity in the chicken retina.

The distribution of parvalbumin-like immunoreactivity was studied in the embryonic and postnatal chicken retina. In post-hatched chickens, parvalbumin-like immunoreactivity was confined to amacrine cells. Three distinct subpopulations were identifiable based upon soma position and level of dendritic arborization in the inner plexiform layer. The primary dendrites from parvalbumin-immunoreactive amacrine cells descended vertically into the inner plexiform layer and eventually branched to give rise to a laminarly arrayed plexus in sublamina I, sublamina V and, to a lesser extent, at the boundary between sublaminae III and IV. Parvalbumin-like immunoreactive amacrine cells projecting to sublamina I of the inner plexiform layer were consistently monostratified. Some, but not all, contributed thick fibers to sublamina I that could be followed for long distances across the retina and were generally not radially organized. The parvalbumin-like immunoreactive cells that projected to sublamina V gave rise to a primary dendrite from which three to five fibers branched radially. Collateral branches of these same primary dendrites gave rise to the parvalbumin-like immunoreactive plexus at the interface between sublaminae III and IV. In prenatal chickens, parvalbumin-like immunoreactivity was not detected until embryonic day 14. At this time it appeared as a faint band at the inner nuclear layer-inner plexiform layer boundary in the central retina. By embryonic day 18 the intensity of immunoreactivity and the complexity of the arborizations of the parvalbumin-like immunoreactive dendrites approached that seen in the post-hatched chicken. In the chicken retina, parvalbumin-like immunoreactivity was displayed by morphologically distinct subpopulations of amacrine cells suggesting that these amacrine cells may subserve diverse functions.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Programs for visualization in three-dimensional microscopy.

Three-dimensional data representing biological structures can be derived using several methods, including serial section reconstruction, optical sectioning, and tomography. The investigation, comprehension, and communication of structural relationships to others is greatly facilitated by computer-based visualization procedures. We describe SYNU, a suite of programs developed for interactive investigation of three-dimensional structure and for the production of high-quality three-dimensional images and animations. We illustrate the capabilities of SYNU in applications to biological data obtained by confocal light microscopy, serial section, and high-resolution electron microscopy from investigations at the cellular, subcellular, and molecular levels.

Actin Cytoskeleton↗

PubMed1992

Cek5, a membrane receptor-type tyrosine kinase, is in neurons of the embryonic and postnatal avian brain.

Cek5 is a recently identified receptor-type tyrosine kinase of the Eph subclass that is nearly ubiquitously expressed during embryonic development (Pasquale, 1991). Cek5 is predominantly expressed in the avian CNS throughout development, and high levels remain apparent in adult neurons. By means of immunofluorescence microscopy and high-resolution immunoelectron microscopy, Cek5 was found to be expressed in many regions of the chicken brain at various developmental stages, most notably in the hippocampus and cerebellum. The highest concentration of Cek5 was observed in the molecular layer of the cerebellum, associated within the axons of mature granule cells (parallel fibers) and with the cell bodies of immature granule cells. In the axons of parallel fibers, Cek5 was concentrated in the fasciculated nonsynaptic portions. This localization, together with the "adhesion" motifs present in the Cek5 extracellular region suggest that Cek5 may interact with other cell surface-associated molecules and be involved in the growth, guidance, and/or bundling of certain unmyelinated axonal processes. Alternatively (or in addition), Cek5 may represent the receptor for a neurotrophic substance, similar to several other neuronal transmembrane tyrosine kinases.

Animals↗

PubMed1991

Three-dimensional fine structure of cytoskeletal-membrane interactions at nodes of Ranvier.

Cytoskeleton-membrane-extracellular matrix interactions at the node of Ranvier were examined in both central and peripheral axons by combining three different methods for tissue preparation with three different electron microscopic techniques for imaging supramolecular structure. Conventional and three-dimensional high voltage electron microscopy of thin and semithick sections of tissues stained en bloc with ferric chloride revealed the presence of transcellular structures across the nodal gap traversing the paranodal glial-axonal junction. These structures penetrate both axonal and glial membranes and are further traced to the cortical axoplasm. This observation was verified by an examination of similar regions in rapidly-frozen freeze-substituted fresh axons. The filamentous nature of these structures, their focal attachment to the external true surface of the nodal and paranodal axolemma and their association with membrane particles were visualized in deep etch rotary-shadow replicas. At the node, both extracellular gap-crossing filaments and membrane-cytoskeletal linkers in the nodal axoplasm are joined to one of the prominent membrane particles of the nodal axolemma. At the paranodal axo-glial junction, the anchoring site of these membrane-cytoskeleton linkers are found on the linear arrays of 16 nm particles. Thus, cytoplasmic filaments and extracellular filaments or bridge structures are involved in the membrane-cytoskeletal interaction at the node and paranode. Some of these membrane particles are known to play a role in ionic conductances known to occur at this site. An additional role in cell adhesion or maintenance of the membrane specialization of this functionally important site of axolemma is now indicated.

Animals↗