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At least 1,153 records · Page 64Linked to original sources
PubMed2004

Aging and subcellular localization of m2 muscarinic autoreceptor in basalocortical neurons in vivo.

By using immunohistochemical approaches at the light and electron microscopic levels, we have shown that aging modifies the subcellular distribution of the m2 muscarinic autoreceptor (m2R) differentially at somato-dendritic postsynaptic sites and at axonal presynaptic sites in cholinergic basalocortical neurons, in vivo. In cholinergic perikarya and dendrites of the nucleus basalis magnocellularis (NBM), aging is associated with a decrease of the density of m2R at the plasma membrane and in the cytoplasm, suggesting a decrease of the total number of m2R in the somato-dendritic field. In contrast, the number of substance P receptors per somato-dendritic surface was not affected. In the frontal cortex (FC), we have shown a decrease of cytoplasmic m2R density also leading to a decrease of the number of m2R per surface of varicosities but with no change of the density of m2R at the membrane. Our results suggest that the decrease of m2R in the somato-dendritic field of the NBM, but not a modification of the number of presynaptic m2 autoreceptors at the plasma membrane in the FC, could contribute to the decrease of the efficacy of cholinergic transmission observed with aging in the rat.

Age Factors↗
PubMed1984

Subcellular localization of hexokinase in pea leaves. Evidence for the predominance of a mitochondrially bound form.

Hexokinase (ATP: D-hexose-6-phosphotransferase, EC 2.7.1.1) activity was determined in subcellular fractions prepared from pea (Pisum sativum) leaf homogenates. About 60% of the total detectable activity of hexokinase was found associated with a particulate fraction consisting essentially of mitochondria and chloroplasts and free of cytosol contamination. The hexokinase specific activity of the particulate fraction was 2-fold higher than that of the homogenate and about 4-fold higher than that of the cytosol. Using a specially designed isokinetic-isopycnic sucrose density gradient centrifugation method, the distribution of hexokinase activity correlated with that of the mitochondrial marker (cytochrome oxidase) and not with that of the chloroplast membrane marker ( chlorophyll ) or that of the cytosol marker (phosphoenolpyruvate carboxylase). Thus, the hexokinase/mitochondria ratio was close to 1.0 along the entire gradient, while the hexokinase/chloroplast ratio varied over a 10-fold range. The results strongly suggest that hexokinase is predominantly bound to mitochondria of pea leaves, and that pea leaf chloroplasts are essentially devoid of any specifically associated hexokinase activity. This work provides the first demonstration of a specific association of hexokinase with mitochondria from photosynthetic tissues of higher plants.

Centrifugation, Isopycnic↗
PubMed2002

Species-specific subcellular localization of RPGR and RPGRIP isoforms: implications for the phenotypic variability of congenital retinopathies among species.

The retinitis pigmentosa GTPase regulator (RPGR) is encoded by the X-linked RP3 locus, which upon genetic lesions leads to neurodegeneration of photoreceptors and blindness. The findings that RPGR specifically and directly interacts in vivo and in vitro with retina-specific RPGR-interacting protein 1 (RPGRIP) and that human mutations in RPGR uncouple its interaction with RPGRIP provided the first clue for the retina-specific pathogenesis of X-linked RP3. Recently, mutations in RPGRIP were found to lead to the retinal dystrophy, Leber congenital amaurosis. However, mouse models null for RPGR had, surprisingly, a very mild phenotype compared with those observed in XlRP3-affected humans and dogs. Moreover, recent reports are seemingly in disagreement on the localization of RPGR and RPGRIP in photoreceptors. These discrepancies were compounded with the finding of RPGR mutations leading exclusively to X-linked cone dystrophy. To resolve these discrepancies and to gain further insight into the pathology associated with RPGR- and RPGRIP-allied retinopathies, we now show, using several isoform-specific antibodies, that RPGR and RPGRIP isoforms are distributed and co-localized at restricted foci throughout the outer segments of human and bovine, but not mice rod photoreceptors. In humans, they also localize in cone outer segments. RPGRIP is also expressed in other neurons such as amacrine cells. Thus, the data lend support to the existence of species-specific subcellular processes governing the function and/or organization of the photoreceptor outer segment as reflected by the species-specific localization of RPGR and RPGRIP protein isoforms in this compartment, and provide a rationale for the disparity of phenotypes among species and in the human.

Amino Acid Sequence↗
PubMed1998

Subcellular localization of mRNA in neuronal cells. Contributions of high-resolution in situ hybridization techniques.

The development of technologies for high-resolution nucleic acid localization in cells and tissues has contributed significantly to our understanding of transcriptional and translational regulation in eukaryotic cells. These methods include nonisotopic in situ hybridization methods for light and electron microscopy, and fluorescent tagging for the study of nucleic acid behavior in living cells. In situ hybridization to detect messenger RNA has led to the discovery that individual transcripts may be selectively targeted to particular subcellular domains. In the nervous system, certain species of mRNA have been localized in distal processes in nerve cells and glia. Direct visualization of mRNA and its interactions with subcellular features, such as synaptic specializations, cytoskeletal elements, and nuclear pores, have been achieved. Of particular interest is the presence of mRNA and ribosomes in dendrites, beneath synaptic contacts, suggesting the possibility of synaptic regulation of protein synthesis. The following article will describe the application of high-resolution in situ hybridization and live imaging techniques to the study of mRNA targeting in neurons.

Animals↗
PubMed1999

Postembedding immunogold labelling reveals subcellular localization and pathway-specific enrichment of phosphate activated glutaminase in rat cerebellum.

Phosphate activated glutaminase is a key enzyme in glutamate synthesis. Here we have employed a quantitative and high-resolution immunogold procedure to analyse the cellular and subcellular expression of this enzyme in the cerebellar cortex. Three main issues were addressed. First, is phosphate activated glutaminase exclusively or predominantly a mitochondrial enzyme, as biochemical data suggest? Second, to what extent is the mitochondrial content of glutaminase dependent on cell type and transmitter identity? Third, can individual neurons maintain a subcellular segregation of mitochondria with different glutaminase content? An attempt was also made to disclose the intramitochondrial localization of glutaminase, and to correlate the content of this enzyme with that of glutamate and glutamine in the same mitochondria (by use of triple labelling). Antisera to the N- and C-termini of glutaminase revealed strong labelling of the putatively glutamatergic mossy fibre terminals. The vast majority of gold particles (approximately 96%) was associated with the mitochondria. Equally high labelling intensities were found in mitochondria of perikarya and dendrites in the pontine nuclei, a major source of mossy fibres. The level of glutaminase immunoreactivity in parallel and climbing fibres (which like the mossy fibres are thought to use glutamate as transmitter) was only about 20% of that in mossy fibres, and similar to that in non-glutamatergic neurons (Purkinje and Golgi cells). Glial cell mitochondria were devoid of specific glutaminase labelling and revealed a much lower glutamate:glutamine ratio than did the mitochondria of mossy fibres. As to the submitochondrial localization of glutaminase, immunogold particles were often found to be aligned with the cristae, suggesting an association of the enzyme with the inner mitochondrial membrane. However, the existence of a glutaminase pool in the mitochondrial matrix could not be excluded. The outer mitochondrial membrane was unlabelled. The present study provides quantitative evidence for a substantial heterogeneity in the mitochondrial content of glutaminase. This heterogeneity applies not only to neurons with different transmitter signatures, but also to different categories of glutamatergic pathways. In terms of the routes involved, the synthesis of transmitter glutamate may be less uniform than previously expected.

Animals↗
PubMed1975

Subcellular localization and properties of mouse adrenal C19-steroid 5beta-reductase.

The localization and some characteristics of mouse adrenal C19-steroid 5 beta-reductase were determined by the incubation of subcellular fractions of mouse adrenal tissue with [7 alpha-3H]androst-4-ene-3,17-dione. This enzyme was present only in the soluble fraction and was NADPH-dependent, although a small activity in the presence of NADH was also detected. The soluble fraction also contained 3alpha-, 3beta- and a small amount of 17 beta-hydroxy steroid dehydrogenase. These and other steroid-metabolizing enzymes present in the remaining subcelluar fractions are also described briefly. To measure 5 beta-androstane-3,17-dione production by the mouse adrenal soluble fraction, all 5 beta products first had to be oxidized to 5 beta-androstane-3,17-dione, and the recovery of radio-activity between the substrate androst-4-ene-3,17-dione and product 5 beta-androstane-3,17-dione of 96.1 +/-3.2% validated this technique. C19-steroid 5 beta-reductase has a pH optimum of 6.5 and at low substrate concentrations the Km and Vmax. for 5 beta reduction of [7 alpha-3H]androst-4-ene-ene-3,17-dione was 2.22 times 10(-6) "/- 0.48 times 10(-6) M and 450+/- 53 pmol/min per mg of protein respectively. At high substrate concentration, inhibition of the reaction occurred, which was shown to be due to increasing product concentration.

Adrenal Glands↗
PubMed1985

Subcellular localization and some properties of the enzymes hydrolysing inositol polyphosphates in rat liver.

The hydrolysis of inositol [32P]trisphosphate (IP3) and inositol [32P]bisphosphate (IP2) has been examined in subcellular fractions of rat liver. IP3 was degraded by an enzyme located in the plasma membrane which did not degrade IP2. Cytosolic fractions were found to degrade both IP2 and IP3. The IP3 phosphatase activity of liver plasma membranes displayed a neutral pH optimum, was Mg2+ dependent and was not inhibited by high concentrations of Li+. Half-maximal activity of the enzymes hydrolysing IP3 and IP2 was obtained with substrate concentrations in the range 1-2 microM. The significance of these observations to the proposed Ca2+-mobilizing role of IP3 is discussed.

Animals↗
PubMed1980

Subcellular localization of increased incorporation of [3H]fucose following passive avoidance learning in the chick.

The effect of training on a passive avoidance test on incorporation of [3H]fucose into subcellular fractions of the anterior forebrain roof of 1-day-old chicks was determined. Isotope was injected intraperitoneally, and birds tested and killed after 3 h. There was an increase in incorporation in trained compared with untrained control birds in the synaptic membrane (19% elevation, P less than 0.02) and the mitochondrial (14% elevation, P less than 0.05) fractions. Following intracranial injection, increased incorporation in trained birds was seen only in the synaptic membrane fraction (39% elevation, P less than 0.01); this increase in incorporation was not limited to any particular electrophoretically separated glycopeptides, but occurred in all 9 detected peaks.

Animals↗
PubMed2006

Subcellular localization and possible functions of gamma-glutamyltransferase in the radish (Raphanus sativus L.) plant.

Previously we reported the purification of soluble gamma-glutamyltransferases (GGTs) from radish cotyledon. Subcellular fractionation of radish cells revealed that soluble GGT is a vacuolar enzyme. Acivicin, a GGT inhibitor, mediated the in vivo catabolism inhibition of the glutathione S-conjugate generated from endogenous glutathione and exogenously supplied monochlorobimane. Thus soluble GGT is possibly involved in the catabolism of glutathione S-conjugates.

Glutathione↗
PubMedDate not supplied

Cellular and subcellular localization of an octadecaneuropeptide derived from diazepam binding inhibitor: immunohistochemical studies in the rat brain.

Immunocytochemical methods, both light and electron microscopic, were used to identify the cellular and subcellular locations of octadecaneuropeptide-like immunoreactivity (ODN-LI) in rat brains serially sectioned in total. ODN-LI includes a newly discovered family of rat brain neuropeptides that are processing products of a common endogenous neuropeptide precursor, diazepam binding inhibitor (DBI). The members of this neuropeptide family have been shown to displace benzodiazepines and beta carbolines from their specific recognition sites located on the allosteric modulatory centers of GABAA receptors. We have previously examined the distribution of DBI-LI in rat brain. The anti-ODN antiserum used in this study does not cross-react with rat DBI, and thus allows a distinct analysis of ODN-LI as opposed to DBI-LI, in rat brain. Neuronal perikarya with ODN-LI were located in many brain nuclei, such as the pontine n., reticular thalamic n., subgeniculate n., supraoptic n. and suprachiasmatic n., and also in brain areas such as cerebral and cerebellar cortex, hippocampus, inferior colliculus, olfactory bulb and subiculum. In addition to perikaryal labelling, a punctate or diffuse immunostaining with ODN antibodies was detected in many brain regions such as cerebellum, hippocampus, amygdaloid area, olfactory tubercle, some of the deep cerebellar nuclei and some circumventricular organs. At the electron microscopic level ODN-LI was identified in neuronal perikarya, processes and terminals. In the axon terminals, ODN-LI appears to be associated with synaptic vesicles. Whenever ODN-LI was detected within neurons, DBI-LI was also found in identical cells. In addition to neurons, DBI-LI was found in glia or glial-like cells, while ODN-LI was not found in these cells. Our findings are consistent with the hypothesis that ODN may be a neuron-specific processing product of DBI and that ODN-like peptides may act as putative endogenous allosteric modulators of various GABAA receptor subtypes.

Animals↗
PubMed2000

Subcellular localization of the Agrobacterium tumefaciens T-DNA transport pore proteins: VirB8 is essential for the assembly of the transport pore.

Agrobacterium tumefaciens transforms plants by transferring DNA to the plant cell nucleus. The VirB membrane proteins are postulated to form a pore for the transport of the DNA across the bacterial membranes. Immunofluorescence and immunoelectron microscopy were used to study the transport pore complex. Three likely components of the transport pore, VirB8, VirB9 and VirB10, localized primarily to the inner membrane, outer membrane and periplasm respectively. A significant amount of VirB10 was also found associated with the outer membrane. When expressed alone VirB9 and VirB10 were randomly distributed along the cell membrane. Subcellular location of both proteins changed dramatically in the presence of the other VirB proteins. Both proteins localized to fewer sites and most of the gold particles representing protein molecules were found in clusters suggesting that the two proteins are in a protein complex. VirB8, on the other hand, localized to clusters even in the absence of the other VirB proteins. To investigate the role of VirB8 in the formation of VirB9 and VirB10 protein complexes, we studied the effect of deletion of virB8 on the subcellular location of VirB9 and VirB10. In a virB8 deletion mutant both proteins were distributed randomly on the cell membrane indicating that VirB8 is essential for complex assembly.

Agrobacterium tumefaciens↗
PubMed1989

Subcellular localization of cyclo-oxygenase-prostaglandin E2 synthetase complex in goat vesicular gland by catalytic activity analysis.

The catalytic activity of the cyclo-oxygenase prostaglandin E2 synthetase complex in subcellular organelles of goat vesicular gland was determined. The enzyme activity was found to be located mostly in the rough endoplasmic reticulum and partly in the nuclear membrane; comparatively very little activity could be detected in the smooth endoplasmic reticulum. There was no detectable activity of the enzyme in the plasma membrane.

Animals↗
PubMed2001

Differential subcellular localization of caspase family proteins in the adult rat brain.

The distribution of the caspase family (caspase-2, -3, -6, -7, -8, -9, -10) was assessed using immunochemical detection of subcellular fractions of 8-week-old rat brain tissues. The present study demonstrated that the relative protein level of caspase-2, -3, -6, -8 and -10 was highest in the soluble cytosolic fraction, while that for caspase-9 was highest in the nucleus. We also found that caspase-3 and -6 were present at high levels and caspase-2, -8 and -9 at moderate levels in the nerve endings fraction as well as in the soluble cytosolic fraction. These results suggest that rat brain caspases are differentially expressed in the subcellular fractions of the rat brain, and that caspases not only contribute to the regulation of neuronal death, but also to synaptic plasticity.

Animals↗
PubMed1982

The subcellular localization of DNA components from Cyanophora paradoxa, a flagellate containing endosymbiotic cyanelles.

Cyanophora paradoxa, a unicellular flagellate, contains cyanelles which are supposed to be cyanobacterial origin. DNA was isolated from subcellular fractions and separated according to density components in CsC1 density gradients. The main DNA component, comprising more than 90% of the total DNA, has a buoyant density of 1.724 g X cm-3. Several subsfractions in the range from 1.718 g X cm-3 to 1.735 g X cm-3 are contained in this component. This DNA of high complexity was considered to be host nuclear DNA. The DNA from the endosymbiotic cyanelles, which were isolated, treated with DNase, and purified by sucrose density gradient centrifugation exhibited a buoyant density of 1.692 g X cm-3 in one strain and 1.695 g X cm-3 in a second strain. Both cyanelle DNAs (cyDNA) have a complexity of approximately 126 X 10(3) base pairs and comprise about 5% of the total cellular DNA content. Two additional DNA components of low complexity were isolated from crude cyanelle pellets obtained without DNase treatment. The larger of these, approximately 48 X 10(3) base pairs in size, had a density of approximately 1.688 g X cm-3. The second component, about 15 X 10(3) base pairs in size, banded in the density range between 1.710 g X cm-3 and 1.720 g X cm-3. The latter is associated with nuclear DNA. The 48 X 10(3)-base-pair component was located in the cytosol and could be obtained after CsC1/ethidium bromide density gradient centrifugation at the position of covalently closed circular DNA. Both these components amounted to approximately 0.5-1% of total DNA. A further DNA component with a complexity of more than 150 X 10(3) base pairs, enriched in fractions where mitochondria are expected, was not characterized further. The density was intermediate between cyDNA and nuclear DNA (1.710-1.720 g X cm-3) and it amounted to 1-2% of the total DNA. Our results indicate that the DNA from cyanelles, believed to be endosymbiotic cyanobacteria, is not more complex than higher plant chloroplast DNAs.

Centrifugation, Density Gradient↗
PubMed1983

Arabinan synthase and xylan synthase activities of Phaseolus vulgaris. Subcellular localization and possible mechanism of action.

Membrane fractions from bean hypocotyl or suspension cultures incorporated arabinose from UDP-beta-L-arabinose into arabinan and xylose from UDP-alpha-D-xylose in vitro; the level of each activity was dependent on the state of differentiation of the cells. These activities may be due to single transglycosylases, since no lipid or proteinaceous intermediate acceptors were found in either case. Subcellular fractionation studies showed that enzyme activity in vitro was localized in both Golgi-derived membranes and endoplasmic reticulum in similar amounts. However, incorporation into the polymers in vivo in suspension culture cells incubated with [1-3H]arabinose was considerably greater in the Golgi-derived membranes. Thus, although these enzymes may be translated and inserted at the level of the endoplasmic reticulum, their activities are under other levels of control, so that most of the activity in vivo is confined to the Golgi apparatus. Initiation of glycosylation in the endoplasmic activity may, however, occur.

Arabinose↗
PubMed1979

Melanotropic peptides: presence in brain of normal and hypophysectomized rats, and subcellularly localized in synaptosomes.

A major and several minor trichloroacetic acid (TCA) soluble, bioassayable melanotropic peptides, as well as bioreactive and immunoreactive alpha-MSH have been found in the hypothalamus, olfactory bulb and cerebral cortex of normal and hypophysectomized rats. By employing subcellular fractionation procedures it was demonstrated that alpha-MSH and the major TCA soluble melanotropic peptide (MMPB) were localized in synaptosomes and were released by hypoosmotic shock. The analysis of MMPB by electrophoretic and chromatographic procedures reveales that it is not ACTH4-10, ACTH1-10, ACTH1-24, NAcACTH1-10 or alpha-MSH. MMPB was found to cross-react with an antiserum specific for the Lys-Pro-Val NH2 sequence in alpha-MSH, indicating that this C-terminal sequence of alpha-MSH may be present in its structure. MMPB was also shown to differ electrophoretically from the two major TCA soluble melanotropic peptides found in the neurointermediate lobe of the pituitary. In view of their synaptosomal localizations MMPB and alpha-MSH may play a role in synaptic function in the nervous system.

Adrenocorticotropic Hormone↗
PubMed1977

Monoamine oxidase in rat reticulocytes: subcellular localization and identification of isoenzymes.

After "chemically induced reticulocytosis" in rats by treatment with acetyl-phenylhydrazide, monoamine oxidase (MAO) activities were determined in erythrocyte preparations of these animals. Studies on subcellular fractions obtained by differential centrifugation showed that the enzyme activity of rat reticulocytes is a classical mitochondrial MAO. The patterns of inhibition produced by clorgyline (A-type MAO), deprenil (B-type MAO) and pargyline or tranylcypromine (both types of MAO) in reticulocytes were determined in vitro using tryptamine as a substrate for both types of MAO and phenylethylamine as a substrate for the B-type. The results indicate that both A-type (approximately 75%) and B-type (approximately 25%) MAO are present in rat reticulocytes; while tryptamine was mainly deaminated by the A-type enzyme, both types of MAO were shown to contribute to the deamination of phenylethylamine. These findings were confirmed in investigations on the thermostabilities of the tryptamine and phenylethylamine deaminating activities of rat reticulocyte MAO.

Animals↗