Using laser scanning confocal microscopy as a guide for electron microscopic study: a simple method for correlation of light and electron microscopy.
AuthorsSun XJ, Tolbert LP, Hildebrand JG
JournalJ Histochem Cytochem
PubMed ID7868862
'Anatomic study of synaptic connections in the nervous system is laborious and difficult, especially when neurons are large or have fine branches embedded among many other processes. Although electron microscopy provides a powerful tool for such study, the correlation of light microscopic appearance and electron microscopic detail is very time-consuming. ... More
Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle.
AuthorsNixon GF, Mignery GA, Somlyo AV
JournalJ Muscle Res Cell Motil
PubMed ID7706424
'Although agonist stimulation leads to an increase in inositol 1,4,5-trisphosphate (InsP3) and decreased calcium in peripherally and centrally located sarcoplasmic reticulum in smooth muscle, the distribution of InsP3 receptors is unknown. InsP3 receptor and the calcium binding protein, calsequestrin were localized by immunolabelling in a tonic and a phasic smooth ... More
Localization of core spindle pole body (SPB) components during SPB duplication in Saccharomyces cerevisiae.
AuthorsAdams IR, Kilmartin JV
JournalJ Cell Biol
PubMed ID10330408
'We have examined the process of spindle pole body (SPB) duplication in Saccharomyces cerevisiae by electron microscopy and found several stages. These include the assembly, probably from the satellite, of a large plaque-like structure, the duplication plaque, on the cytoplasmic face of the half-bridge and its insertion into the nuclear ... More
Identification by differential display of a hypertonicity-inducible inward rectifier potassium channel highly expressed in chloride cells.
AuthorsSuzuki Y, Itakura M, Kashiwagi M, Nakamura N, Matsuki T, Sakuta H, Naito N, Takano K, Fujita T, Hirose S
JournalJ Biol Chem
PubMed ID10196230
'By using differential mRNA display to monitor the molecular alterations associated with adaptation of euryhaline eels to different salinities, we identified a cDNA fragment strongly induced in seawater eel gills. Cloning of a full-length cDNA and its expression in COS-7 cells indicated that the clone codes for an inward rectifier ... More
Subcellular localization of SV2 and other secretory vesicle components in PC12 cells by an efficient method of preembedding EM immunocytochemistry for cell cultures.
AuthorsTanner VA, Ploug T, Tao-Cheng JH
JournalJ Histochem Cytochem
PubMed ID8985140
'We demonstrated the subcellular localization of SV2, a transmembrane protein associated with neuroendocrine secretory vesicles, in NGF-treated PC12 cells by preembedding EM immunocytochemistry (ICC), using a small gold probe followed by silver enhancement. The use of a multiwell chamber slide substantially improved the efficiency of the preembedding EM ICC procedures ... More
Immunolocalization of a Drosophila phosphatidylinositol transfer protein (rdgB) in normal and rdgA mutant photoreceptor cells with special reference to the subrhabdomeric cisternae.
AuthorsSuzuki E, Hirosawa K
JournalJ Electron Microsc (Tokyo)
PubMed ID7996077
'Distribution of rdgB protein, which was recently shown to be a Drosophila phosphatidylinositol transfer protein, was studied in the photoreceptor cells of compound eyes of normal and rdgAPC47 mutant of Drosophila melanogaster by immunoelectron microscopy using (1) pre-embedding HRP staining, (2) pre-embedding NANOGOLD labeling followed by siliver enhancement, (3) and ... More
New frontiers in gold labeling.
AuthorsHainfeld JF, Powell RD
JournalJ Histochem Cytochem
PubMed ID10727288
'Recent advances in gold technology have led to probes with improved properties and performance for cell biologists: higher labeling density, better sensitivity, and greater penetration into tissues. Gold clusters, such as the 1.4-nm Nanogold, are gold compounds that can be covalently linked to Fab'' antibody fragments, making small and stable ... More
Subsynaptic segregation of metabotropic and ionotropic glutamate receptors as revealed by immunogold localization.
AuthorsNusser Z, Mulvihill E, Streit P, Somogyi P
JournalNeuroscience
PubMed ID7969918
'Glutamate is a major neurotransmitter in the brain that acts both through fast ionotropic receptors and through slower metabotropic receptors coupled to G proteins. Both receptors are present throughout the somatodendritic domain of neurons as shown by immunohistochemical and patch clamp recording studies. Immunogold labelling revealed a concentration of metabotropic ... More
A practical technique to postfix nanogold-immunolabeled specimens with osmium and to embed them in Epon for electron microscopy.
AuthorsSawada H, Esaki M
JournalJ Histochem Cytochem
PubMed ID10727291
'Nanogold is a tiny gold probe, freely diffusible in cells and tissues, and is suitable for pre-embedding immunohistochemistry. However, it is necessary to develop Nanogold to a larger size so that it can be observed by conventional transmission electron microscopy. Silver enhancement is usually used for visualizing Nanogold, but the ... More
ERG30, a VAP-33-related protein, functions in protein transport mediated by COPI vesicles.
AuthorsSoussan L, Burakov D, Daniels MP, Toister-Achituv M, Porat A, Yarden Y, Elazar Z
JournalJ Cell Biol
PubMed ID10427086
'Intracellular transport of newly synthesized and mature proteins via vesicles is controlled by a large group of proteins. Here we describe a ubiquitous rat protein-endoplasmic reticulum (ER) and Golgi 30-kD protein (ERG30)-which shares structural characteristics with VAP-33, a 33-kD protein from Aplysia californica which was shown to interact with the ... More
Expression of guanylin in "pars tuberalis-specific cells" and gonadotrophs of rat adenohypophysis.
AuthorsD'Este L, Kulaksiz H, Rausch U, Vaccaro R, Wenger T, Tokunaga Y, Renda TG, Cetin Y
JournalProc Natl Acad Sci U S A
PubMed ID10655496
'The intestinal peptide guanylin regulates the electrolyte/water transport in the gastrointestinal epithelium by paracrine/luminocrine mechanisms. Because guanylin also circulates in the blood, we investigated the rat hypothalamo-pituitary region for expression and cellular localization of this peptide. Reverse transcriptase-PCR analyses with guanylin-specific primers revealed expression of the peptide in the pars ... More
A polypeptide bound by the chaperonin groEL is localized within a central cavity.
AuthorsBraig K, Simon M, Furuya F, Hainfeld JF, Horwich AL
JournalProc Natl Acad Sci U S A
PubMed ID8097882
'Chaperonins are oligomeric protein complexes that play an essential role in the cell, mediating ATP-dependent polypeptide chain folding in a variety of cellular compartments. They appear to bind early folding intermediates, preventing their aggregation; in the presence of MgATP and a cochaperonin, bound polypeptides are released in a stepwise manner, ... More
High-resolution immunocytochemical labeling of replicas with ultrasmall gold.
AuthorsTakizawa T
JournalJ Histochem Cytochem
PubMed ID10082759
'The use of 10-15-nm gold probes in freeze-fracture immunocytochemistry sometimes results in poor immunogold labeling. Replica sites are labeled with only one or two gold particles, making it unlikely that the labeling depicts the true distribution of antigen. In this study, the feasibility of using ultrasmall ( approximately 1.4-nm) gold ... More
Ultrastructural localization of the vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) receptor-2 (FLK-1, KDR) in normal mouse kidney and in the hyperpermeable vessels induced by VPF/VEGF-expressing tumors and adenoviral vectors.
AuthorsFeng D, Nagy JA, Brekken RA, Pettersson A, Manseau EJ, Pyne K, Mulligan R, Thorpe PE, Dvorak HF, Dvorak AM
JournalJ Histochem Cytochem
PubMed ID10727296
'Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) interacts with two high-affinity tyrosine kinase receptors, VEGFR-1 and VEGFR-2, to increase microvascular permeability and induce angiogenesis. Both receptors are selectively expressed by vascular endothelial cells and are strikingly increased in tumor vessels. We used a specific antibody to localize VEGFR-2 (FLK-1, KDR) ... More
Group I metabotropic glutamate receptors at GABAergic synapses in monkeys.
AuthorsHanson JE, Smith Y
JournalJ Neurosci
PubMed ID10414977
'Recent data showed that group I metabotropic glutamate receptors (mGluRs) are located perisynaptic to the postsynaptic specializations of asymmetric glutamatergic synapses in the cerebellum and hippocampus in rats. In the present study, we used immunogold labeling to elucidate the subsynaptic localization of group I mGluRs (mGluR1a and mGluR5) in the ... More
Vitronectin in the cytoplasm of Leydig cells in the rat testis.
AuthorsSawada H, Sugawara I, Kitami A, Hayashi M
JournalBiol Reprod
PubMed ID8837997
'Vitronectin is a multifunctional protein present in serum and in extracellular matrices, but its localization has not been fully elucidated. In the present study, immunoblotting with anti-rat vitronectin antibody showed that rat testes contained vitronectin in both Triton-soluble and -insoluble fractions. Immunofluorescence microscopy demonstrated that this immunoreactivity was localized mainly ... More
Nuclear and neuropil aggregates in Huntington's disease: relationship to neuropathology.
AuthorsGutekunst CA, Li SH, Yi H, Mulroy JS, Kuemmerle S, Jones R, Rye D, Ferrante RJ, Hersch SM, Li XJ
JournalJ Neurosci
PubMed ID10087066
'The data we report in this study concern the types, location, numbers, forms, and composition of microscopic huntingtin aggregates in brain tissues from humans with different grades of Huntington''s disease (HD). We have developed a fusion protein antibody against the first 256 amino acids that preferentially recognizes aggregated huntingtin and ... More
Salient features of synaptic organisation in the cerebral cortex.
AuthorsSomogyi P, Tamás G, Lujan R, Buhl EH
JournalBrain Res Brain Res Rev
PubMed ID9651498
'The neuronal and synaptic organisation of the cerebral cortex appears exceedingly complex, and the definition of a basic cortical circuit in terms of defined classes of cells and connections is necessary to facilitate progress of its analysis. During the last two decades quantitative studies of the synaptic connectivity of identified ... More
Differential synaptic localization of two major gamma-aminobutyric acid type A receptor alpha subunits on hippocampal pyramidal cells.
AuthorsNusser Z, Sieghart W, Benke D, Fritschy JM, Somogyi P
JournalProc Natl Acad Sci U S A
PubMed ID8876241
'Hippocampal pyramidal cells, receiving domain specific GABAergic inputs, express up to 10 different subunits of the gamma-aminobutyric acid type A (GABAA) receptor, but only 3 different subunits are needed to form a functional pentameric channel. We have tested the hypothesis that some subunits are selectively located at subsets of GABAergic ... More
Target-cell-specific concentration of a metabotropic glutamate receptor in the presynaptic active zone.
AuthorsShigemoto R, Kulik A, Roberts JD, Ohishi H, Nusser Z, Kaneko T, Somogyi P
JournalNature
PubMed ID8632825
'The probability of synaptic neurotransmitter release from nerve terminals is regulated by presynaptic receptors responding to transmitters released from the same nerve terminal or from terminals of other neurons. The release of glutamate, the major excitatory neurotransmitter, is suppressed by presynaptic autoreceptors. Here we show that a metabotropic glutamate receptor ... More
Organization of AMPA receptor subunits at a glutamate synapse: a quantitative immunogold analysis of hair cell synapses in the rat organ of Corti.
AuthorsMatsubara A, Laake JH, Davanger S, Usami S, Ottersen OP
JournalJ Neurosci
PubMed ID8699256
'Sensitive and high-resolution immunocytochemical procedures were used to investigate the spatial organization of AMPA receptor subunits (GluR1-4) at the synapse between the inner hair cells and the afferent dendrites in the rat organ of Corti. This is a synapse with special functional properties and with a presynaptic dense body that ... More
Domains of axin involved in protein-protein interactions, Wnt pathway inhibition, and intracellular localization.
AuthorsFagotto F, Jho E, Zeng L, Kurth T, Joos T, Kaufmann C, Costantini F
JournalJ Cell Biol
PubMed ID10330403
'Axin was identified as a regulator of embryonic axis induction in vertebrates that inhibits the Wnt signal transduction pathway. Epistasis experiments in frog embryos indicated that Axin functioned downstream of glycogen synthase kinase 3beta (GSK3beta) and upstream of beta-catenin, and subsequent studies showed that Axin is part of a complex ... More
Glutamatergic synapses on oligodendrocyte precursor cells in the hippocampus.
AuthorsBergles DE, Roberts JD, Somogyi P, Jahr CE
JournalNature
PubMed ID10821275
'Fast excitatory neurotransmission in the central nervous system occurs at specialized synaptic junctions between neurons, where a high concentration of glutamate directly activates receptor channels. Low-affinity AMPA (alpha-amino-3-hydroxy-5-methyl isoxazole propionic acid) and kainate glutamate receptors are also expressed by some glial cells, including oligodendrocyte precursor cells (OPCs). However, the conditions ... More
Chromogranin A, an "on/off" switch controlling dense-core secretory granule biogenesis.
AuthorsKim T, Tao-Cheng JH, Eiden LE, Loh YP
JournalCell
PubMed ID11525735
'We present evidence that regulation of dense-core secretory granule biogenesis and hormone secretion in endocrine cells is dependent on chromogranin A (CGA). Downregulation of CGA expression in a neuroendocrine cell line, PC12, by antisense RNAs led to profound loss of dense-core secretory granules, impairment of regulated secretion of a transfected ... More
Tech.Sight. Worth its weight in gold.
AuthorsBendayan M
JournalScience
PubMed ID11233453
Ultra-small gold particles and silver enhancement as a detection system in immunolabeling and in situ hybridization experiments.
AuthorsHumbel BM, Sibon OC, Stierhof YD, Schwarz H
JournalJ Histochem Cytochem
PubMed ID7608530
Silver-enhanced immunogold labeling of calcium-ATPase in sarcoplasmic reticulum of skeletal muscle.
AuthorsKrenács T, Dux L
JournalJ Histochem Cytochem
PubMed ID8014481
Confocal epipolarization microscopy of gold probes in plant cells and protoplasts.
AuthorsGriffing LR, Villanueva MA, Taylor J, Moon S
JournalMethods Cell Biol
PubMed ID8531749
Immunogold labeling in scanning electron microscopy.
AuthorsHermann R, Walther P, Müller M
JournalHistochem Cell Biol
PubMed ID8858365
Enhanced labeling efficiency using ultrasmall immunogold probes: immunocytochemistry.
AuthorsRobinson JM, Takizawa T, Vandré DD
JournalJ Histochem Cytochem
PubMed ID10727290
Detection of antigen-antibody interactions in immunocytochemistry relies on a reporter system. The most commonly employed reporter systems used are fluorochromes, enzymes, and particulate probes. This article considers the advantages and disadvantages associated with ultrasmall immunogold particles as the reporter system in immunocytochemical applications. ... More
Labeling with nanogold and undecagold: techniques and results.
AuthorsHainfeld JF
JournalScanning Microsc Suppl
PubMed ID9601549
A significant new development in gold labeling for microscopy has been achieved through the use of gold cluster compounds that are covalently attached to antibodies or other probe molecules. These unique gold probes are smaller than most colloidal gold conjugates and exhibit improved penetration into tissues, higher labeling densities, and ... More
Use of nanogold followed by silver enhancement and gold toning for preembedding immunolocalization in osmium-fixed, Epon-embedded tissues.
AuthorsSawada H, Esaki M
JournalJ Electron Microsc (Tokyo)
PubMed ID7722428
A reliable, sensitive, high-resolution method with good structural visualization for preembedding immunoelectron microscopy was proposed. The present technique involves the following processes. Immunolabeling of cryostat sections with primary antibodies and with nanogolds, silver intensification for visualization of nanogold secondary antibodies, gold-toning for stabilization of silver shells, and osmium postfixation and ... More
Localization of Rabphilin-3A on the synaptic vesicle.
AuthorsMizoguchi A, Yano Y, Hamaguchi H, Yanagida H, Ide C, Zahraoui A, Shirataki H, Sasaki T, Takai Y
JournalBiochem Biophys Res Commun
PubMed ID8060298
Rabphilin-3A is a putative target protein for Rab3A small GTP-binding protein which is implicated in neurotransmitter release. Rabphilin-3A is expressed mainly in brain, but its subcellular localization remains to be clarified. Immunohistochemical analysis has revealed that Rabphilin-3A is most abundant in the synaptic area of the rat cerebellum, retina, and ... More
Electron microscopy of alpha 2-macroglobulin with a thiol ester bound ligand.
AuthorsBoisset N, Pochon F, Chwetzoff S, Barray M, Delain E, Lamy J
JournalJ Struct Biol
PubMed ID1282356
In order to covalently bind the hydrolyzed thiol ester groups of the human alpha 2-macroglobulin (alpha 2M) transformed by methylamine, the phospholipase A2 (PLA2), a small enzyme (M(r) = 13,000) from Naja nigricollis snake venom was activated by succinimidyl 4-(maleimidomethyl)cyclohexane-1-carboxylate (SMCC). Average images determined from electron micrographs of the methylamine-transformed ... More
A 1.4-nm gold cluster covalently attached to antibodies improves immunolabeling.
AuthorsHainfeld JF, Furuya FR
JournalJ Histochem Cytochem
PubMed ID1552162
A large gold cluster (Au1.4nm) was covalently coupled to IgG and Fab' fragments. Its gold core is 1.4 nm in diameter and the Fab'-Au1.4nm immunoconjugate is the smallest gold immunoprobe that can be seen directly in the conventional electron microscope. It is useful in high-resolution immunolabeling, providing a resolution of ... More
The metabotropic glutamate receptor (mGluR1 alpha) is concentrated at perisynaptic membrane of neuronal subpopulations as detected by immunogold reaction.
AuthorsBaude A, Nusser Z, Roberts JD, Mulvihill E, McIlhinney RA, Somogyi P
JournalNeuron
PubMed ID8104433
An antiserum to mGluR1 alpha labeled a 160 kd protein in immunoblots of membranes derived from rat brain or cells transfected with mGluR1 alpha. Immunoreactivity for mGluR1 alpha was present in discrete subpopulations of neurons. The GABAergic neurons of the cerebellar cortex were strongly immunoreactive; only some Golgi cells were ... More
Immunogold labelling of neuroendocrine peptides with special reference to antibody specificity and multiple staining techniques.
AuthorsLarsson LI
JournalHistochem Cell Biol
PubMed ID8858369
Immunogold methods have been very important for research on the neuroendocrine system. The compatibility of immunogold probes with optimal contrasting for electron microscopy has made localizations of neuroendocrine peptides to different subtypes of secretory organelles possible and, currently, methods using covalent attachment of nanogold particles to antibodies and neuropeptide ligands ... More
A covalent fluorescent-gold immunoprobe: simultaneous detection of a pre-mRNA splicing factor by light and electron microscopy.
Immunoprobes that combine a fluorescent label with a 1.4-nm gold cluster compound have been prepared by covalent conjugation with polyclonal antibody Fab' fragments. These new immunoconjugates allow the collection of two complementary sets of data, from fluorescence and electron microscopy, from a single labeling experiment. We find that incorporation of ... More
Peculiar distribution of fodrin in fat-storing cells.
AuthorsAoki T, Hagiwara H, Fujimoto T
JournalExp Cell Res
PubMed ID9260899
Fat-storing cells (FSCs) show unique morphology containing many lipid droplets in the cytoplasm. In this study, we found that a membrane skeletal protein, fodrin, shows peculiar distribution in FSCs of rat liver. By immunofluorescence microscopy of FSCs in culture, intense labeling for fodrin was seen as coarse filaments in the ... More
Ultrastructural localization of gustducin immunoreactivity in microvilli of type II taste cells in the rat.
Gustducin is a transducin-like G protein (guanine nucleotide-binding protein) that is expressed in taste bud cells. Gustducin is believed to be involved in bitter and possibly sweet taste transduction. In the present study, we demonstrate that a subset of type II cells displays immunoreactivity to antisera directed against gustducin in ... More
Signal enhancement at the electron microscopic level using Nanogold and gold-based autometallography.
AuthorsWeipoltshammer K, Schöfer C, Almeder M, Wachtler F
JournalHistochem Cell Biol
PubMed ID11201611
Immunoelectron microscopy using ultrasmall gold markers is a very sensitive method to detect molecules at high resolution. In order to discriminate the gold particles in the electron microscope, enlargement of gold particles is necessary. So far, mostly silver ions were used for deposition onto the surface of gold grains. In ... More
Localization of a metabotropic glutamate receptor, mGluR7, in axon terminals of presumed nociceptive, primary afferent fibers in the superficial layers of the spinal dorsal horn: an electron microscope study in the rat.
AuthorsLi H, Ohishi H, Kinoshita A, Shigemoto R, Nomura S, Mizuno N
JournalNeurosci Lett
PubMed ID9080455
It was examined electron microscopically in the rat if a metabotropic glutamate receptor, mGluR7, might be localized in axon terminals of nociceptive, primary afferent fibers in laminae I and II of the spinal dorsal horn. Nociceptive nature of axon terminals showing mGluR7-like immunoreactivity (mGluR7-LI) was indicated by binding to the ... More
Cloning and characterization of a novel membrane-associated antigenic protein of Helicobacter pylori.
AuthorsYoshida M, Wakatsuki Y, Kobayashi Y, Itoh T, Murakami K, Mizoguchi A, Usui T, Chiba T, Kita T
JournalInfect Immun
PubMed ID9864228
Infection by Helicobacter pylori, a noninvasive bacterium, induces chronic leukocyte infiltration in the stomach by still largely unknown molecular mechanisms. We investigated the possibility that a membrane protein of H. pylori induces an inflammatory reaction in the subepithelial tissue of the stomach. By generating an expression library of H. pylori ... More
Differences in synaptic GABA(A) receptor number underlie variation in GABA mini amplitude.
AuthorsNusser Z, Cull-Candy S, Farrant M
JournalNeuron
PubMed ID9331359
In many neurons, responses to individual quanta of transmitter exhibit large variations in amplitude. The origin of this variability, although central to our understanding of synaptic transmission and plasticity, remains controversial. To examine the relationship between quantal amplitude and postsynaptic receptor number, we adopted a novel approach, combining patch-clamp recording ... More
The use of electron microscopic immunocytochemistry with silver-enhanced 1.4-nm gold particles to localize GAD in the cerebellar nuclei.
AuthorsGilerovitch HG, Bishop GA, King JS, Burry RW
JournalJ Histochem Cytochem
PubMed ID7868863
Silver enhancement of small gold particles can be used with pre-embedding immunocytochemistry to analyze the distribution of label over cell organelles. We have developed a method that improves tissue morphology, has good penetration of reagents, and allows greater control of silver enhancement of 1.4-nm gold. In this study we analyzed ... More
Use of 1.4-nm immunogold particles for immunocytochemistry on ultra-thin cryosections.
AuthorsTakizawa T, Robinson JM
JournalJ Histochem Cytochem
PubMed ID7983362
We present a new application for the use of small immunogold particles (approximately 1.4-nm diameter) for ultrastructural immunocytochemistry. These small gold particles have been used on ultra-thin cryosections in conjunction with a silver enhancement procedure that does not degrade ultrastructural detail. We have used the human neutrophil as a model ... More
Combined fluorescent and gold immunoprobes: reagents and methods for correlative light and electron microscopy.
AuthorsPowell RD, Halsey CM, Hainfeld JF
JournalMicrosc Res Tech
PubMed ID9712158
Immunoprobes which incorporate both a fluorescent label and a 1.4 nm gold cluster compound were prepared by covalent conjugation to Fab' antibody fragments of the Nanogold cluster label followed by a fluorescent moiety. These new immunoconjugates allow the collection of two complementary sets of data, from fluorescence and electron microscopy, ... More
Preparation, use, and enlargement of ultrasmall gold particles in immunoelectron microscopy.
AuthorsBaschong W, Stierhof YD
JournalMicrosc Res Tech
PubMed ID9712164
The introduction of ultrasmall (approximately 1-3 nm) colloidal gold markers in immunoelectron microscopy (IEM) in 1989 has considerably improved the sensitivity of this marker system. Ultrasmall gold markers have opened the field of pre-embedding labeling studies to gold markers without the need of harsh permeabilizing steps. They are recommended for ... More
Immunogold silver staining for light microscopy.
AuthorsLackie PM
JournalHistochem Cell Biol
PubMed ID8858363
The immunogold silver staining method (IGSS) is widely used as a sensitive and specific immunohistochemical visualisation technique. IGSS involves the specific deposition of metallic silver at the site of immunogold labelling and provides a means of visualisation at low magnification by light or electron microscopy. Silver developers for IGSS rapidly ... More
Cellular defects and altered gene expression in PC12 cells stably expressing mutant huntingtin.
AuthorsLi SH, Cheng AL, Li H, Li XJ
JournalJ Neurosci
PubMed ID10377328
Expanded polyglutamine tracts cause huntingtin and other proteins to accumulate and aggregate in neuronal nuclei. Whether the intranuclear aggregation or localization of a polyglutamine protein initiates cellular pathology remains controversial. We established stably transfected pheochromocytoma PC12 cells that express the N-terminal fragment of huntingtin containing 20 (20Q) or 150 (150Q) ... More
The mouse SKD1, a homologue of yeast Vps4p, is required for normal endosomal trafficking and morphology in mammalian cells.
AuthorsYoshimori T, Yamagata F, Yamamoto A, Mizushima N, Kabeya Y, Nara A, Miwako I, Ohashi M, Ohsumi M, Ohsumi Y
JournalMol Biol Cell
PubMed ID10679028
The mouse SKD1 is an AAA-type ATPase homologous to the yeast Vps4p implicated in transport from endosomes to the vacuole. To elucidate a possible role of SKD1 in mammalian endocytosis, we generated a mutant SKD1, harboring a mutation (E235Q) that is equivalent to the dominant negative mutation (E233Q) in Vps4p. ... More
Relative densities of synaptic and extrasynaptic GABAA receptors on cerebellar granule cells as determined by a quantitative immunogold method.
AuthorsNusser Z, Roberts JD, Baude A, Richards JG, Somogyi P
JournalJ Neurosci
PubMed ID7722639
Ion channels gated by the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) are thought to be located in synaptic junctions, but they have also been found throughout the somatodendritic membrane of neurons independent of synapses. To test whether synaptic junctions are enriched in GABAA receptors, and to determine the relative densities of ... More
Insoluble gamma-tubulin-containing structures are anchored to the apical network of intermediate filaments in polarized CACO-2 epithelial cells.
AuthorsSalas PJ
JournalJ Cell Biol
PubMed ID10444072
We have previously shown that a thin ( approximately 1 microm) layer of intermediate filaments located beneath the apical membrane of a variety of simple epithelial cells participates in the organization of apical microfilaments and microtubules. Here, I confirmed the apical distribution of gamma-tubulin-containing structures (potential microtubule-organizing centers) in CACO-2 ... More
Water channel protein AQP3 is present in epithelia exposed to the environment of possible water loss.
AuthorsMatsuzaki T, Suzuki T, Koyama H, Tanaka S, Takata K
JournalJ Histochem Cytochem
PubMed ID10490456
Aquaporins (AQPs) are membrane water channel proteins expressed in various tissues in the body. We surveyed the immunolocalization of AQP3, an isoform of the AQP family, in rat epithelial tissues. AQP3 was localized to many epithelial cells in the urinary, digestive, and respiratory tracts and in the skin. In the ... More
Sites of rDNA transcription are widely dispersed through the nucleolus in Pisum sativum and can comprise single genes.
AuthorsThompson WF, Beven AF, Wells B, Shaw PJ
JournalPlant J
PubMed ID9351243
Incorporation by RNA polymerases of BrUTP into both plant root tissue and isolated plant nuclei as a method for localization of the sites of transcription has been used. In this paper pea root tissue was used, and under the conditions employed, nearly all the incorporation occurs in the nucleolus, and ... More
Preparation of plasmid DNA in transfection complexes for fluorescence and electron spectroscopic imaging.
AuthorsMalecki M
JournalScanning Microsc Suppl
PubMed ID9601525
The aim of this project was to develop procedures necessary to study mechanisms of receptor mediated gene transfer by means of integrated microscopy. Plasmid DNA was incorporated into a transfection complex consisting of poly(L)lysine and transferrin to which the nuclear localization signal was conjugated. This complex was presented to cultured ... More
Ultrastructural localization of beta-actin and amphoterin mRNA in cultured cells: application of tyramide signal amplification and comparison of detection methods.
AuthorsPunnonen EL, Fages C, Wartiovaara J, Rauvala H
JournalJ Histochem Cytochem
PubMed ID9857217
We describe a nonradioactive preembedding in situ hybridization protocol using digoxigenin-labeled RNA probes and tyramide signal amplification to increase the sensitivity of detection. The protocol is sensitive enough for electron microscopic localization of endogenous messenger RNAs encoding beta-actin and amphoterin. Three visualization methods were compared: diaminobenzidine enhanced by nickel, Nanogold ... More
The alpha 6 subunit of the GABAA receptor is concentrated in both inhibitory and excitatory synapses on cerebellar granule cells.
AuthorsNusser Z, Sieghart W, Stephenson FA, Somogyi P
JournalJ Neurosci
PubMed ID8613776
Although three distinct subunits seem to be sufficient to form a functional pentameric GABAA receptor channel, cerebellar granule cells express nRNA for nine subunits. They receive GABAergic input from a relatively homogenous population of Golgi cells. It is not known whether all subunits are distributed similarly on the surface of ... More
Immunoelectron microscopic localization of phosphoproteins associated with the mitotic spindle.
AuthorsVandré DD, Burry RW
JournalJ Histochem Cytochem
PubMed ID1453002
We examined the immunogold staining of microtubules and microtubule organizing centers using an improved silver-enhancement reagent for small (1-1.4 nm) gold-conjugated secondary antibodies. First, the staining properties of different commercial preparations of gold-labeled antibodies were compared for sample penetration, label uniformity, and labeling density, and Nanogold 1.4-nm gold-conjugated F(ab') was ... More
Binding of amyloid beta protein to the 20 S proteasome.
AuthorsGregori L, Hainfeld JF, Simon MN, Goldgaber D
JournalJ Biol Chem
PubMed ID8995227
Neurodegenerative disorders of aging are characterized by the intraneuronal accumulation of ubiquitin conjugates into tangles and inclusions. Ubiquitin conjugates are degraded by cellular particles known as proteasomes. We have previously shown that amyloid beta protein (Abeta) inhibits proteasomal activity and thereby blocks ubiquitin conjugate degradation. In the present studies, we ... More
Dendritic and postsynaptic protein synthetic machinery.
AuthorsGardiol A, Racca C, Triller A
JournalJ Neurosci
PubMed ID9870948
There is a growing body of evidence that local protein synthesis beneath synapses may provide a novel mechanism underlying plastic phenomena. In vivo and in vitro biochemical data show that dendrites can perform translation and glycosylation. Using antibodies directed against the eukaryotic protein synthetic machinery, we sought to identify the ... More
In vivo localization of DNA sequences and visualization of large-scale chromatin organization using lac operator/repressor recognition.
AuthorsRobinett CC, Straight A, Li G, Willhelm C, Sudlow G, Murray A, Belmont AS
JournalJ Cell Biol
PubMed ID8991083
We report a new method for in situ localization of DNA sequences that allows excellent preservation of nuclear and chromosomal ultrastructure and direct, in vivo observations. 256 direct repeats of the lac operator were added to vector constructs used for transfection and served as a tag for labeling by lac ... More
Physiological evidence for the discrimination of L-arginine from structural analogues by the zebrafish olfactory system.
AuthorsLipschitz DL, Michel WC
JournalJ Neurophysiol
PubMed ID10601449
Although it is generally assumed that fish are capable of discriminating amino acid odorants on the basis of differences in side-chain structure, less is known about their ability to discriminate amino acids with modifications to alpha-carboxyl and alpha-amino groups. In this study, the ability of the zebrafish olfactory system to ... More
Regulation of the subcellular distribution of m4 muscarinic acetylcholine receptors in striatal neurons in vivo by the cholinergic environment: evidence for regulation of cell surface receptors by endogenous and exogenous stimulation.
AuthorsBernard V, Levey AI, Bloch B
JournalJ Neurosci
PubMed ID10575021
Our aim was to determine how the cholinergic environment influences, in vivo, the membrane abundance and the intracellular trafficking of the muscarinic receptor m4 (m4R). Immunohistochemistry at light and electron microscopic level was used to detect the subcellular localization of m4R in several populations of striatal cholinoceptive neurons, including cholinergic ... More
Immunocytochemistry of extracellular matrix components in the rat seminiferous tubule: electron microscopic localization with improved methodology.
AuthorsYazama F, Esaki M, Sawada H
JournalAnat Rec
PubMed ID9143667
BACKGROUND: Cell-cell interactions between Sertoli, myoid, Leydig, and germ cells are thought to be essential for spermatogenesis. These cells interact with each other through the extracellular matrices (ECMs) of the testicular lamina propria, which thus may have an important function in spermatogenesis. For an understanding of the role of ECMs ... More
Cellular, subcellular, and subsynaptic distribution of AMPA-type glutamate receptor subunits in the neostriatum of the rat.
AuthorsBernard V, Somogyi P, Bolam JP
JournalJ Neurosci
PubMed ID8987803
Glutamate released in the basal ganglia is involved in the expression of clinical symptoms of neurodegenerative diseases like Parkinson's or Huntington's. Neostriatal neurons are the targets of glutamatergic inputs derived from the cortex and the thalamus acting via AMPA-type as well as other glutamate receptors. To determine the location of ... More
Differential plasma membrane distribution of metabotropic glutamate receptors mGluR1 alpha, mGluR2 and mGluR5, relative to neurotransmitter release sites.
AuthorsLuján R, Roberts JD, Shigemoto R, Ohishi H, Somogyi P
JournalJ Chem Neuroanat
PubMed ID9412905
Two group I metabotropic glutamate receptor subtypes, mGluR1 and mGluR5, have been reported to occur in highest concentration in an annulus surrounding the edge of the postsynaptic membrane specialisation. In order to determine whether such a distribution is uniform amongst postsynaptic mGluRs, their distribution was compared quantitatively by a pre-embedding ... More
Synaptic target selectivity and input of GABAergic basket and bistratified interneurons in the CA1 area of the rat hippocampus.
AuthorsHalasy K, Buhl EH, Lörinczi Z, Tamás G, Somogyi P
JournalHippocampus
PubMed ID8841829
To assess the position of interneurons in the hippocampal network, fast spiking cells were recorded intracellularly in vitro and filled with biocytin. Sixteen non-principal cells were selected on the basis of 1) cell bodies located in the pyramidal layer and in the middle of the slice, 2) extensive labeling of ... More
Immunocytochemical localization of the alpha 1 and beta 2/3 subunits of the GABAA receptor in relation to specific GABAergic synapses in the dentate gyrus.
AuthorsNusser Z, Roberts JD, Baude A, Richards JG, Sieghart W, Somogyi P
JournalEur J Neurosci
PubMed ID7620614
Dentate granule cells receive spatially segregated GABAergic innervation from at least five types of local circuit neurons, and express mRNA for at least 11 subunits of the GABAA receptor. At most two to four different subunits are required to make a functional pentamer, raising the possibility that cells have on ... More
High-resolution immunogold localization of AMPA type glutamate receptor subunits at synaptic and non-synaptic sites in rat hippocampus.
AuthorsBaude A, Nusser Z, Molnár E, McIlhinney RA, Somogyi P
JournalNeuroscience
PubMed ID8848093
The cellular and subcellular localization of the GluRA, GluRB/C and GluRD subunits of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) type glutamate receptor was determined in the rat hippocampus using polyclonal antipeptide antibodies in immunoperoxidase and immunogold procedures. For the localization of the GluRD subunit a new polyclonal antiserum was developed using the C-terminal ... More
Altered membrane proteins and permeability correlate with cardiac dysfunction in cardiomyopathic hamsters.
AuthorsIkeda Y, Martone M, Gu Y, Hoshijima M, Thor A, Oh SS, Peterson KL, Ross J
JournalAm J Physiol Heart Circ Physiol
PubMed ID10749734
A mutation in the delta-sarcoglycan (SG) gene with absence of delta-SG protein in the heart has been identified in the BIO14.6 cardiomyopathic (CM) hamster, but how the defective gene leads to myocardial degeneration and dysfunction is unknown. We correlated left ventricular (LV) function with increased sarcolemmal membrane permeability and investigated ... More
A novel helicase-type protein in the nucleolus: protein NOH61.
AuthorsZirwes RF, Eilbracht J, Kneissel S, Schmidt-Zachmann MS
JournalMol Biol Cell
PubMed ID10749921
We report the identification, cDNA cloning, and molecular characterization of a novel, constitutive nucleolar protein. The cDNA-deduced amino acid sequence of the human protein defines a polypeptide of a calculated mass of 61.5 kDa and an isoelectric point of 9.9. Inspection of the primary sequence disclosed that the protein is ... More
Segregation of different GABAA receptors to synaptic and extrasynaptic membranes of cerebellar granule cells.
AuthorsNusser Z, Sieghart W, Somogyi P
JournalJ Neurosci
PubMed ID9464994
Two types of GABAA receptor-mediated inhibition (phasic and tonic) have been described in cerebellar granule cells, although these cells receive GABAergic input only from a single cell type, the Golgi cell. In adult rats, granule cells express six GABAA receptor subunits abundantly (alpha1, alpha6, beta2, beta3, gamma2, and delta), which ... More
Signal amplification in immunohistochemistry at the light microscopic level using biotinylated tyramide and nanogold-silver staining.
AuthorsKöhler A, Lauritzen B, Van Noorden CJ
JournalJ Histochem Cytochem
PubMed ID10858270
Signal amplification techniques greatly enhance the sensitivity of immunohistochemical (IHC) and in situ hybridization (ISH) methods. In particular, catalyzed signal amplification (CSA) using labeled tyramide or Nanogold-silver staining is an important signal amplification tool. We have applied a combination of both techniques, as has been introduced for ISH, for a ... More
Clinical relevance of HPV 16/18 testing methods in cervical squamous cell carcinoma.
AuthorsGraf AH, Cheung AL, Hauser-Kornberger C, Dandachi N, Tubbs RR, Dietze O, Hacker GW
JournalAppl Immunohistochem Mol Morphol
PubMed ID11127922
Three different in situ hybridization (ISH) methods were compared for their clinical relevance and suitability in detecting human papillomavirus (HPV) 16/18 in 55 cases of squamous cell carcinoma (SCC) of the uterine cervix. After the initial biopsy, surgery, and/or radiation therapy, patients were followed for 5 to 8 years. A ... More
The K+ channel, Kv2.1, is apposed to astrocytic processes and is associated with inhibitory postsynaptic membranes in hippocampal and cortical principal neurons and inhibitory interneurons.
AuthorsDu J, Tao-Cheng JH, Zerfas P, McBain CJ
JournalNeuroscience
PubMed ID9522360
A variety of voltage-gated ion channels are expressed on principal cell dendrites and have been proposed to play a pivotal role in the regulation of dendritic excitability. Previous studies at the light microscopic level demonstrated that the K+ channel subunit Kv2.1 expression was polarized to the cell soma and dendrites ... More
Silver enhancement of gold antibody probes in pre-embedding electron microscopic immunocytochemistry.
AuthorsBurry RW, Vandré DD, Hayes DM
JournalJ Histochem Cytochem
PubMed ID1453003
In pre-embedding EM immunocytochemistry with gold probes, the gold must be small enough to penetrate through cell membranes treated with mild detergents. Antibodies labeled with small gold probes (1-1.4 nm) are too small to be resolved in thin sections but can be seen if they are silver-enhanced after the gold ... More
Adhesion-induced receptor segregation and adhesion plaque formation: A model membrane study.
AuthorsKloboucek A, Behrisch A, Faix J, Sackmann E
JournalBiophys J
PubMed ID10512849
A model system to study the control of cell adhesion by receptor-mediated specific forces, universal interactions, and membrane elasticity is established. The plasma membrane is mimicked by reconstitution of homophilic receptor proteins into solid supported membranes and, together with lipopolymers, into giant vesicles with the polymers forming an artificial glycocalix. ... More
Perisynaptic location of metabotropic glutamate receptors mGluR1 and mGluR5 on dendrites and dendritic spines in the rat hippocampus.
AuthorsLujan R, Nusser Z, Roberts JD, Shigemoto R, Somogyi P
JournalEur J Neurosci
PubMed ID8758956
Ionotropic and metabotropic (mGluR1a) glutamate receptors were reported to be segregated from each other within the postsynaptic membrane at individual synapses. In order to establish whether this pattern of distribution applies to the hippocampal principal cells and to other postsynaptic metabotropic glutamate receptors, the mGluR1a/b/c and mGluR4 subtypes were localized ... More