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At least 1,333 records · Page 74Linked to original sources
PubMed2000

Cellular and subcellular localization of necdin in fetal and adult mouse brain.

Necdin is a 325-amino-acid residue protein encoded by a cDNA clone isolated from neurally differentiated embryonal carcinoma cells. Ectopic expression of necdin induces growth arrest of proliferative cells. Necdin binds to major transcription factors E2F1 and p53, suggesting that necdin exerts its functions through the interactions with these cell-cycle-regulating factors. However, information about precise localization of endogenous necdin protein is currently lacking. A rabbit polyclonal antibody was raised against a bacterially expressed recombinant protein of necdin (amino acids 83-325). Immunoblot analysis revealed that necdin protein was expressed almost exclusively in the brain of adult mice. A relative molecular mass of endogenous necdin was estimated at approximately 43,000. In developing mouse brain, necdin was most abundant during fetal and neonatal periods. Necdin was highly enriched in the cytoplasm of hypothalamic neurons in fetal and adult mice. The subcellular fractionation analysis revealed that necdin was concentrated in the cytosol fraction of brain cells. These results suggest that endogenous necdin protein is localized predominantly in the cytoplasm of differentiated neurons and moves into the nucleus under specific conditions.

Age Factors↗
PubMed1976

Platelet antiplasmin: its extrusion during the release reaction, subcellular localization, characterization, and relationship to antiheparin in pig platelets.

Antiplasmin activity was shown to be released from washed pig platelets by thrombin following a time course similar to that of 3H-serotonin. Antiheparin activity (platelet factor 4) appeared to be released by thrombin at a slower rate than 3H-serotonin or antiplasmin activity. Subcellular fractionation of pig platelets showed that the storage site for antiplasmin is probably the dense (amine storage) granules. Antiheparin was distributed among all of the subcellular particulate fractions except the fraction rich in dense granules. Material released from washed pig platelets and concentrated by ZnSO4 precipitation (crude antiheparin) was found to be rich in antiplasmin activity. Gel filtration on Sephadex G-150, DEAE cellulose column chromatography, and polyacrylamide gel electrophoresis showed that pig platelet antiplasmin is a low molecular weight (approximately 30,000 d) material of alpha1-globulin electrophoretic mobility. It was found to be heat labile and also inhibitory to the caseinolytic activity of trypsin but had no effect on the action of thrombin on fibrinogen. These data indicate that platelet antiplasmin is distinct from platelet antiheparin.

Animals↗
PubMed1981

Studies on the kinetic properties and subcellular localization of adenosine diphosphatase activity in human peripheral blood lymphocytes.

Using a recently developed radioassay, the conditions for measuring adenosine diphosphatase (ADPase) activity in human lymphocytes were optimized. Kinetic studies showed that the activity was optimal at pH 8.0 and required 0.2 mM MgCl2. The Km of the enzyme for ADP was 0.03 mM. Analytical subcellular fractionation showed that the ADPase activity was distributed between the plasma membrane and mitochondria. Studies with the non-penetrating inhibitor, diazotized sulphanilic acid, indicate that plasma membrane ADPase activity is located on the external aspect of the cell. Polymorphonuclear leucocytes were found to have about three times the ADPase activity. Lymphocytes, therefore, have the ability to generate AMP on their surface which can be further metabolized to adenosine by ecto-5' nucleotidase. A role for this system is discussed.

Apyrase↗
PubMed1999

Subcellular localization and in vivo identification of the putative movement protein of olive latent virus 2.

The gene encoding the 36.5 kDa ('36K') nonstructural protein located on RNA3 of olive latent virus 2 (OLV-2) was cloned, expressed with the Escherichia coli pGEX-2T system and the purified protein used to raise a polyclonal antiserum. Immunoblot analysis of OLV-2-infected Nicotiana benthamiana plants showed that the 36K protein accumulated in the early stages of infection and was associated with a subcellular fraction enriched in cytoplasmic membranes. In infected cells there were tubular structures, some containing virus-like particles, scattered in the cytoplasm or protruding from or penetrating the cell wall at the plasmodesmata. Immunogold labelling localized the 36K protein in the plasmodesmata of OLV-2-infected cells and showed it to be associated with virus-containing tubules. Leaf trichome cells of N. tabacum plants, transformed with a 36K-green fluorescent protein (GFP) fusion construct, revealed localized fluorescence in the cell walls, possibly due to association of the fusion protein with plasmodesmata. When the same 36K-GFP fusion protein was expressed in N. tabacum protoplasts, long tubular fluorescent structures protruded from the protoplast surface, suggesting that the 36K protein is responsible for tubule induction. The conclusion is drawn that this protein is likely to be the OLV-2 movement protein, mediating cell-to-cell virus movement, and that movement is by a tubule-guided mechanism.

Bromoviridae↗
PubMed1996

Immunocytochemical characterization and subcellular localization of human myristoyl-CoA: protein N-myristoyltransferase in HeLa cells.

Antisera have been raised to three synthetic peptides based on the sequence of human myristoyl-CoA:protein N-myristoyl transferase (NMT) and to the purified enzyme following its expression in Escherichia coli. These antisera have been affinity purified and shown to react both with the E. coli expressed human NMT, and specifically with a protein of molecular weight of 63 kDa in immunoblots of the human cell line HeLa. The affinity purified antibodies have also been used to localize NMT in methanol/acetone permeabilized HeLa cells by immunofluorescent staining. The immunofluorescence showed a diffuse staining pattern throughout the cell, suggesting that the enzyme is predominantly cytosolic. This was confirmed by determining the distribution of NMT activity in different subcellular fractions of HeLa cells. Over 90% of NMT enzymatic activity was released from cell lysates during either hypotonic or isotonic homogenization. However, a small amount of enzymatic activity remained associated with cell membranes, despite extensive washing, and this was confirmed by immunoblot analysis of these membranes for NMT. In comparison, over 99.5% of lactate dehydrogenase activity was released under the same conditions, which suggests that the NMT was genuinely associated with the cell membranes. The membrane-bound enzyme behaved like a peripheral membrane protein. Permeabilization HeLa cells with 50 microM digitonin resulted in the release of 90-93% of lactate dehydrogenase compared to 73-85% of NMT, again suggesting that the majority of the enzyme is cytosolic, but that some may be associated with cell membranes or organelles.

Acyltransferases↗
PubMed2006

Cellular and subcellular localization of PDE10A, a striatum-enriched phosphodiesterase.

PDE10A is a recently identified phosphodiesterase that is highly expressed by the GABAergic medium spiny projection neurons of the mammalian striatum. Inhibition of PDE10A results in striatal activation and behavioral suppression, suggesting that PDE10A inhibitors represent a novel class of antipsychotic agents. In the present studies we further elucidate the localization of this enzyme in striatum of rat and cynomolgus monkey. We find by confocal microscopy that PDE10A-like immunoreactivity is excluded from each class of striatal interneuron. Thus, the enzyme is restricted to the medium spiny neurons. Subcellular fractionation indicates that PDE10A is primarily membrane bound. The protein is present in the synaptosomal fraction but is separated from the postsynaptic density upon solubilization with 0.4% Triton X-100. Immuno-electron microscopy of striatum confirms that PDE10A is most often associated with membranes in dendrites and spines. Immuno-gold particles are observed on the edge of the postsynaptic density but not within this structure. Our studies indicate that PDE10A is associated with post-synaptic membranes of the medium spiny neurons, suggesting that the specialized compartmentation of PDE10A enables the regulation of intracellular signaling from glutamatergic and dopaminergic inputs to these neurons.

Animals↗
PubMed2000

Subcellular localization and speciation of nickel in hyperaccumulator and non-accumulator Thlaspi species.

The ability of Thlaspi goesingense Hálácsy to hyperaccumulate Ni appears to be governed by its extraordinary degree of Ni tolerance. However, the physiological basis of this tolerance mechanism is unknown. We have investigated the role of vacuolar compartmentalization and chelation in this Ni tolerance. A direct comparison of Ni contents of vacuoles from leaves of T. goesingense and from the non-tolerant non-accumulator Thlaspi arvense L. showed that the hyperaccumulator accumulates approximately 2-fold more Ni in the vacuole than the non-accumulator under Ni exposure conditions that were non-toxic to both species. Using x-ray absorption spectroscopy we have been able to determine the likely identity of the compounds involved in chelating Ni within the leaf tissues of the hyperaccumulator and non-accumulator. This revealed that the majority of leaf Ni in the hyperaccumulator was associated with the cell wall, with the remaining Ni being associated with citrate and His, which we interpret as being localized primarily in the vacuolar and cytoplasm, respectively. This distribution of Ni was remarkably similar to that obtained by cell fractionation, supporting the hypothesis that in the hyperaccumulator, intracellular Ni is predominantly localized in the vacuole as a Ni-organic acid complex.

Nickel↗
PubMed2000

Cellular and subcellular localization of peripheral benzodiazepine receptors after trimethyltin neurotoxicity.

The peripheral benzodiazepine receptor (PBR) is currently used as a marker of inflammation and gliosis following brain injury. Previous reports suggest that elevated PBR levels in injured brain tissue are specific to activated microglia and infiltrating macrophages. We have produced hippocampal lesions using the neurotoxicant trimethyltin (TMT) to examine the cellular and subcellular nature of the PBR response. Degenerating, argyrophilic pyramidal neurons were observed in the hippocampus at 2 and 14 days after TMT exposure. Reactive microglia were also evident at both times with a maximal response observed at 14 days, subsiding by 6 weeks. Astrocytosis was observed at 14 days and 6 weeks, but not 2 days, after TMT administration, suggesting that the onset of the astroglia response is delayed, but more persistent, compared with microgliosis. Morphological evidence from [3H]PK11195 microautoradiography and PBR immunohistochemistry indicates that both astrocytes and microglia are capable of expressing high levels of PBR after injury. This was confirmed by double labeling of either Griffonia simplicifolia isolectin B4, a microglial-specific marker, or glial fibrillary acidic protein, an astrocyte-specific protein with PBR fluorescence immunohistochemistry. These results demonstrate that PBR expression is increased after brain injury in both activated microglia and astrocytes. Our findings also provide the first evidence for in situ nuclear localization of PBR in glial cells.

Animals↗