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Results for “Subcellular localization”

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PubMed1995

Subcellular localization and kinetic properties of aromatase activity in rat brain.

The conversion of testosterone to estradiol is catalyzed by cytochrome P450 aromatase. In situ aromatization is required for the full expression of the effects of testosterone in the brain. This study examined the subcellular distribution and reaction kinetics of aromatase in the adult rat brain. Preoptic area, hypothalamus and amygdala were homogenized in isotonic sucrose buffered with potassium phosphate. Tissue homogenates were fractionated by ultracentrifugation. Aromatase activity was measured using a previously validated 3H2O assay. Marker enzymes were measured to identify organelles in the different subcellular fractions. Aromatase activity in all 3 tissues was enriched 10-fold in microsomes, but not in other subcellular fractions. The addition of either a NADPH-generating system or 1 mM NADPH to the reaction mixture stimulated aromatase activity in all subcellular fractions, whereas NADH was only minimally effective. In general, substrate affinity constants were equivalent in all brain areas and subcellular fractions (approximately 10 nM) suggesting that one predominant catalytic form of the enzyme is present in the rat brain. One week after castration, aromatase activity was significantly reduced in all subcellular fractions of preoptic area and in the whole homogenate and microsomal fraction of the hypothalamus. Castration did not significantly alter aromatase activity in any subcellular compartment of amygdala. To more critically evaluate its subcellular localization, aromatase activity was measured in purified synaptosomes. Aromatase activity was not enriched in these preparations suggesting that it is not substantially associated with nerve terminals in rat brain.

Amygdala↗

PubMed1999

Use of SIMS microscopy and electron probe X-ray microanalysis to study the subcellular localization of aluminium in Vicia faba roots cells.

Received January 4, 1999; Accepted March 25, 1999 Secondary ion mass spectrometry (SIMS), electron probe X-ray microanalysis (EPMA) and transmission electron microscopy (TEM) were used to study the tissular distribution and subcellular localization of aluminium (Al) precipitate in roots of Viciafaba. The broad bean plant, grown in nitrate solution with 193 microM Al3+ at pH 4.8, for 15 days showed Al deposits in the roots. Al accumulation was not detected in the stems nor in the leaves. Al was found mainly localized on the root's surfaces and within the cell walls of the cortical cells. Al signal was not detected in the vascular tissues. Two weeks exposure to Al caused ultrastructural changes in cortical cells and sometimes a complete disruption of these cells. Deposition of Al in form of insoluble complexes associated with phosphorus, appeared as electron opaque materials in the vacuoles of disrupted cortex cells and in the intercellular inclusions. The leaves turned yellowish at the end of 15 days exposure. The use of electron microprobe, to investigate the same tissues as the ones investigated by SIMS, provided complementary results on aluminium allocation.

Aluminum↗

PubMed1997

Subcellular localization of arachidonate 12-lipoxygenase and morphological effect of its overexpression on murine keratinocytes.

Arachidonate 12-lipoxygenase enzyme oxygenates the position 12 of arachidonic acid and produces 12-hydroperoxy-arachidonic acid. Mouse keratinocytes were transiently transfected with an expression vector of human platelet 12-lipoxygenase cDNA. The cells were homogenized, and the subcellular localization of the enzyme was examined by differential centrifugation. The 12-lipoxygenase activity was detected predominantly in the particulate fractions. In contrast, immunoelectron microscopy detected the enzyme mainly in the cytoplasm of the transfected cell, but not in the nucleus, subcellular organelles or plasma membrane. To explain the discrepancy between these findings, we performed an electron-microscopic examination of the 176000 g pellet of the keratinocyte homogenate. The pellet contained mainly insoluble proteins such as keratin but not membrane structures such as the plasma membrane. Thus, it is possible that the enzyme was localized originally in the cytoplasm of the keratinocyte, and found in the particulate fractions due to its association with insoluble proteins during fractionation procedures. Unique structural changes were observed in the transfected keratinocytes. The nucleus had very scant karyoplasm and coarse fibrillary structures. When the keratinocytes were transfected with a mutant 12-lipoxygenase cDNA or a vector without 12-lipoxygenase cDNA, these structural changes were not observed.

Animals↗

PubMed1993

Glycoprotein III (clusterin, sulfated glycoprotein 2) in endocrine, nervous, and other tissues: immunochemical characterization, subcellular localization, and regulation of biosynthesis.

Specific antisera were raised against the A and B chains of glycoprotein III. Immunoblotting revealed that in adrenal medulla both chains migrate very closely together in two-dimensional electrophoresis. Both chains with slightly differing molecular sizes are found in several endocrine tissues and in brain, kidney, liver, and serum. The mRNA has an analogous widespread distribution. In primary cultures of chromaffin cells the level of message becomes significantly increased by treatment with histamine or 12-O-tetradecanoylphorbol 13-acetate/forskolin. However, the increase is small when compared with that of secretogranin II. The subcellular localization of glycoprotein III in endocrine organs and in the posterior pituitary was investigated by subcellular fractionation and immunoelectron microscopy. Glycoprotein III was found to be confined to the large dense-core vesicles of these organs. For a discussion of the function of glycoprotein III, its localization in these organelles has to be taken into account.

Animals↗

PubMed1989

Subcellular localization of iodinated thyroid tubulin.

Subcellular fractions enriched in mitochondria, plasma membranes, microsomes and Golgi apparatus were obtained from thyroid glands of rats injected with I125. Autoradiography of SDS-polyacrylamide gels revealed the presence of a number of radiolabelled proteins in each membrane fraction. One polypeptide, with the same electrophoretic mobility as brain tubulin, was found in all fractions except the plasma membranes and was immunoprecipitated with commercial anti-tubulin monoclonal antibodies. Hydrolysis of Asp-Pro linkages of I125 labelled tubulin with formic acid indicated that there were iodination sites in both the carboxy terminal one third and the amino terminal two thirds of the molecule. These results, together with the absence of iodinated tubulin from the cytosolic fraction, are consistent with the idea that a population of thyroid membrane tubulin is iodinated at multiple sites either just before or after insertion into intracellular membranes where it may act as an anchorage point for microtubule-membrane interactions.

Animals↗

PubMed1993

Developmental pattern and subcellular localization of parvalbumin in the rat tooth germ.

The EF-hand calcium-binding protein parvalbumin has been extensively studied in nerve and muscle cells. Its possible role in biomineralization during tooth development was here investigated by determining its subcellular localization by immunogold cytochemistry. The developmental sequences of amelogenesis and dentinogenesis were studied in rat molars, and in continuously growing rat incisors. The findings confirm that parvalbumin is a nuclear and a cytosolic protein, not associated with any particular intracellular organelle. Epithelial and mesenchymal undifferentiated cells contained no specific parvalbumin immunolabelling. In differentiated ameloblasts, secretory-pole (Tomes' process) formation was associated with a proximal-distal gradient of parvalbumin labelling. But after the Tomes' process had formed, parvalbumin was evenly distributed throughout the cell. The parvalbumin contents of ruffle-ended and smooth-ended ameloblasts appeared to be very different. Differentiated odontoblasts were less heavily labelled than ameloblasts, and the label was restricted to the cell body during the whole of dentinogenesis. These data suggest that parvalbumin could contribute to membrane plasticity during differentiation, as shown during dendritic growth in the nervous cells. Moreover, as may occur in excitable cells, parvalbumin could buffer calcium specifically in the cells producing mineralized enamel and dentine during the later stages of tooth development.

Ameloblasts↗

PubMed1997

Subcellular localization and translocation of protein kinase C isoforms zeta and epsilon in human peripheral blood lymphocytes.

The calcium-independent members of the protein kinase C (PKC) family may play a significant role in T cell function. We have characterized the subcellular localization and redistribution of calcium-independent kinase C activity and of two specific members of this family (zeta and epsilon) in response to activation of human peripheral blood lymphocytes with phorbol myristate acetate (PMA) or through the TCR-CD3 complex. Both PMA and OKT3, an antibody against the TCR-associated CD3 complex, induce an increase in membrane and cytoskeletal activity with a concomitant decrease in cytosolic activity. By Western blot analysis, PKC epsilon is present in resting cytosol and membrane fractions, and is detected in the membrane following activation with PMA and in both the membrane and cytoskeleton following OKT3 activation. By contrast, PKC zeta is progressively lost from the cytoskeleton following activation with anti-CD3. Immunocytochemistry reveals distinct redistribution patterns for these enzymes in response to activation through anti-CD3 and by PMA. These findings demonstrate that signaling through the CD3 complex induces significant changes in calcium-independent PKC activity and in the intracellular distribution of specific isoenzymes, and support a role for specific functions for individual isoenzymes in T cell activation. Lastly, changes in the cytoskeletal distribution of these isoenzymes suggest a potential role in the modulation of cell structure in response to activation.

Biological Transport, Active↗

PubMed1997

Effect of cell type on the subcellular localization of the thyrotropin-releasing hormone receptor.

The localization of an epitope-tagged receptor for thyrotropin-releasing hormone (TRH) expressed in different cell contexts was studied with immunofluorescence microscopy. In pituitary lactotrophs, which normally express TRH receptors, and in AtT20 pituitary corticotrophs, TRH receptor immunoreactivity was primarily confined to the plasma membrane. In HEK 293 and COS7 cells, TRH receptors were predominantly intracellular. In transiently transfected COS7 cells, the TRH receptor colocalized with endoplasmic reticulum and Golgi markers. The pattern of TRH receptor immunofluorescence was the same over a wide range of receptor expression in transiently transfected COS7 cells, and all cell lines bound similar amounts of 3H- and rhodamine-labeled TRH analogs, suggesting that cell-specific differences in TRH receptor localization were not simply the result of overexpression. In all cell contexts, TRH receptors on the plasma membrane underwent extensive ligand-driven endocytosis. Inhibitors of glycosylation did not alter the subcellular distribution of receptors. In HEK 293 cells expressing the transfected TRH receptor, protein synthesis inhibitors caused translocation of intracellular receptors to the cell surface, as shown by a marked increase in cell surface immunofluorescence and [3H][N3-methyl-His2]TRH binding. These results demonstrate that the subcellular localization of the TRH receptor depends on the cell context in which it is expressed and that intracellular receptors are capable of translocation to the plasma membrane.

Amino Acid Sequence↗

PubMed1980

Subcellular localization of long-chain alcohol dehydrogenase and aldehyde dehydrogenase in n-alkane-grown Candida tropicalis.

Long-chain alcohol dehydrogenase and long-chain aldehyde dehydrogenase were induced in the cells of Candida tropicalis grown on n-alkanes. Subcellular localization of these dehydrogenases, together with that of acyl-CoA synthetase and glycerol-3-phosphate acyltransferase, was studied in terms of the metabolism of fatty acids derived from n-alkane substrates. Both long-chain alcohol and aldehyde dehydrogenases distributed in the fractions of microsomes, mitochondria and peroxisomes obtained from the alkane-grown cells of C. tropicalis. Acyl-CoA synthetase was also located in these three fractions. Glycerol-3-phosphate acyltransferase was found in microsomes and mitochondria, in contrast to fatty acid beta-oxidation system localized exclusively in peroxisomes. Similar results of the enzyme localization were also obtained with C. lipolytica grown on n-alkanes. These results suggest strongly that microsomal and mitochondrial dehydrogenases provide long-chain fatty acids to be utilized for lipid synthesis, whereas those in peroxisomes supply fatty acids to be degraded via beta-oxidation to yield energy and cell constituents.

Acetate-CoA Ligase↗

PubMed1990

Subcellular localization of GABAA/benzodiazepine receptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus.

The monoclonal antibody bd-17, which recognizes the beta 2 and beta 3-subunits of GABAA/benzodiazepine receptors, was used to determine the cellular and subcellular localization of receptor-like immunoreactivity in the superficial gray layer of the rat superior colliculus. In numerous dendrites, very strong immunostaining was present in the cytoplasm and on the postsynaptic dendritic membrane of synaptic junctions. The extrasynaptic portion of the dendritic membrane also very often showed [beta 2 + beta 3]-like immunoreactivity. However, due to methodological limitations, it could not be stated with certainty whether presynaptic beta 2- and beta 3-subunits of GABAA/benzodiazepine receptors actually occur in this mesencephalic visual structure. In conclusion, these results strongly suggest that synaptic and non-synaptic GABAA/benzodiazepine receptors are present in the superficial gray layer of the rat superior colliculus. These receptors may modulate neuronal cell activity in different ways, depending on their location.

Animals↗

PubMed2005

Subcellular localization of mycobacteria in tissues and detection of lipid antigens in organelles using cryo-techniques for light and electron microscopy.

The survival of intracellular pathogens within a host is determined by microbial evasion, which can be partially attributed to their subcellular trafficking strategies. Microscopic techniques have become increasingly important in understanding the cell biology of microbial infections. These recently developed techniques can be used for the subcellular localization of antigens not only in cultured cells but also within tissues such as Mycobacterium tuberculosis in lung and Mycobacterium leprae in skin. High-resolution immunofluorescence microscopy can be used in combination with cryo-immunogold electron microscopy using consecutive cryo-sections on the same tissue block forming a direct connection between the two microscopy techniques. The detection of mycobacterial lipid antigens in situ at an ultrastructural level is currently a challenge, but new modifications can be used to address this. These methods might be of interest to microbiologists and cell biologists who study host-pathogen interactions.

Antigens, Bacterial↗

PubMed1994

Multiple subcellular localization of bcl-2: detection in nuclear outer membrane, endoplasmic reticulum membrane, and mitochondrial membranes.

The use of biochemical fractionation, immunofluorescence laser-scanning confocal microscopy, and immunoelectron microscopy with mouse anti-human bcl-2 monoclonal antibody to analyze the subcellular localization of the bcl-2 gene product revealed the protein prominently in the nuclear envelope, endoplasmic reticulum membrane, and mitochondrial membranes. Electron microscopy at high magnification more precisely localized bcl-2 to the nuclear outer membrane as confirmed by the biochemical fractionation, as well as to mitochondrial outer and, to a lesser degree, inner membrane. This multisite membrane distribution of bcl-2 suggests an important role for this protein in several different membrane compartments.

B-Lymphocytes↗

PubMed1997

A 55-kDa endonuclease of mammalian mitochondria: comparison of its subcellular localization and endonucleolytic properties with those of endonuclease G.

A novel endonuclease of 55-kDa was found in rat liver mitochondria by a zymographic assay, in addition to the 29 kDa enzyme that is well-known as endonuclease G (Endo G). Subcellular localization of these enzymes in rat liver cells was examined by biochemical fractionation. Endo G was located in both nuclei and mitochondria as has been previously reported, while the 55-kDa enzyme was only detected in the mitochondrial fraction. The levels of the endonucleases in the mitochondria varied greatly among the rat organs, and the activity in the heart was about 30 times higher than that in the liver. The 55-kDa enzyme and Endo G were extracted from bovine heart mitochondria with 0.4 M NaCl. During purification the 55-kDa enzyme and Endo G were copurified because of their similar chromatographic behavior, so they were separated by gel filtration or electrophoresis in the presence of SDS and the proteins were then renatured. The nucleolytic properties of the 55-kDa enzyme resembled those of Endo G and other known mitochondrial nucleases. The enzyme degraded single-stranded DNA more rapidly than duplex DNA at a weak alkaline pH1 requiring Mg2+ or Mn2+ but not Ca2+ or Zn2+. Nicks generated by the enzyme had 5'-P and 3'-OH ends. The 55-kDa enzyme, like Endo G, displayed an unusually strong preference to nick within a (dG)n.(dC)n tract.

Animals↗

PubMed2006

Subcellular localization of CIAPIN1.

Cytokine-induced apoptosis inhibitor 1 (CIAPIN1) is a newly identified anti-apoptotic molecule. Our previous studies have demonstrated that CIAPIN1 is ubiquitously expressed in normal fetal and adult human tissues and confers multidrug resistance in gastric cancer cells, possibly by upregulating the expression of multidrug resistance gene 1 and multidrug resistance-related protein 1. However, fundamental biological functions of CIAPIN1 have not been elucidated. In this study, we first predicted the subcellular localization of CIAPIN1 with bioinformatic approaches and then characterized the intracellular localization of CIAPIN1 in both human and mouse cells by a combination of techniques including (a)immunohistochemistry and immunofluorescence, (b) His-tagged CIAPIN1 expression, and (c)subcellular fractionation and analysis of CIAPIN1 in the fractions by Western blotting. All methods produced consistent results; CIAPIN1 was localized in both the cytoplasm and the nucleus and was accumulated in the nucleolus. Bioinformatic prediction disclosed a putative nuclear localization signal and a putative nuclear export signal within both human and mouse CIAPIN1. These findings suggest that CIAPIN1 may undergo a cytoplasm-nucleus-nucleolus translocation.

Animals↗

PubMed2006

Cellular and subcellular localization of alpha-1 adrenoceptors in the rat visual cortex.

Noradrenaline is thought to play modulatory roles in a number of physiological, behavioral, and cellular processes. Although many of these modulatory effects are mediated through alpha-1 adrenoceptors, basic knowledge of the cellular and subcellular distributions of these receptors is limited. We investigated the laminar distribution pattern of alpha-1 adrenoceptors in rat visual cortex, using immunohistochemistry at both light and electron microscopic levels. Affinity-purified anti-alpha-1 antibody was confirmed to react only with a single band of about 70-80 kDa in total proteins prepared from rat visual cortex. Alpha-1 adrenoceptors were widely distributed though all cortical layers, but relatively high in density in layers I, II/III, and V. Immunoreactivity was observed in both neuronal perikarya and processes including apical dendrites. In double-labeling experiments with anti-microtubule-associated protein 2, anti-neurofilament, anti-glial fibrillary acidic protein, anti-glutamic acid decarboxylase 65/67, anti-2-3-cyclic nucleotide 3-phosphodiesterase, and anti-tyrosine hydroxylase antibodies, alpha-1 adrenoceptors were found mainly in dendrites and somata of microtubule-associated protein 2-immunopositive neurons. About 20% of alpha-1 adrenoceptors were in GABAergic neurons. A small number of alpha-1 adrenoceptors were also distributed in axons of excitatory neurons, astrocytes, oligodendrocytes and noradrenergic fibers. Using an immunoelectron microscopic technique, numerous regions of alpha-1 adrenoceptor immunoreactivity were found in cell somata, on membranes of dendrites, and in postsynaptic regions. Moreover, a small number of immunoreaction products were also detected in axons and presynaptic sites. These findings provide the first quantitative evidence regarding the cellular and subcellular localization of alpha-1 adrenoceptor immunoreactivity in visual cortex. Moreover, the ultrastructural distribution of alpha-1 adrenoceptor immunoreactivity suggests that alpha-1 adrenoceptors are transported mainly into dendrites and that they exert effects at postsynaptic sites of neurons.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗

PubMed1998

Cloning of human PEX cDNA. Expression, subcellular localization, and endopeptidase activity.

Mutations in the PEX gene are responsible for X-linked hypophosphatemic rickets. To gain insight into the role of PEX in normal physiology we have cloned the human full-length cDNA and studied its tissue expression, subcellular localization, and peptidase activity. We show that the cDNA encodes a 749-amino acid protein structurally related to a family of neutral endopeptidases that include neprilysin as prototype. By Northern blot analysis, the size of the full-length PEX transcript is 6.5 kilobases. PEX expression, as determined by semi-quantitative polymerase chain reaction, is high in bone and in tumor tissue associated with the paraneoplastic syndrome of renal phosphate wasting. PEX is glycosylated in the presence of canine microsomal membranes and partitions exclusively in the detergent phase from Triton X-114 extractions of transiently transfected COS cells. Immunofluorescence studies in A293 cells expressing PEX tagged with a c-myc epitope show a predominant cell-surface location for the protein with its COOH-terminal domain in the extracellular compartment, substantiating the assumption that PEX, like other members of the neutral endopeptidase family, is a type II integral membrane glycoprotein. Cell membranes from cultured COS cells transiently expressing PEX efficiently degrade exogenously added parathyroid hormone-derived peptides, demonstrating for the first time that recombinant PEX can function as an endopeptidase. PEX peptidase activity may provide a convenient target for pharmacological intervention in states of altered phosphate homeostasis and in metabolic bone diseases.

Adult↗

PubMed2004

Subcellular localization and secretion of activity-dependent neuroprotective protein in astrocytes.

Activity-dependent neuroprotective protein (ADNP, approximately 123562.8 Da), is synthesized in astrocytes and expression of ADNP mRNA is regulated by the neuroprotective peptide vasoactive intestinal peptide (VIP). The gene that encodes ADNP is conserved in human, rat and mouse, and contains a homeobox domain profile that includes a nuclear-export signal and a nuclear-localization signal. ADNP is essential for embryonic brain development, and NAP, an eight-amino acid peptide that is derived from ADNP, confers potent neuroprotection. Here, we investigate the subcellular localization of ADNP through cell fractionation, gel electrophoresis, immunoblotting and immunocytochemistry using alpha-CNAP, an antibody directed to the neuroprotective NAP fragment that constitutes part of an N-terminal epitope of ADNP. Recombinant ADNP was used as a competitive ligand to measure antibody specificity. ADNP-like immunoreactivity was found in the nuclear cell fraction of astrocytes and in the cytoplasm. In the cytoplasm, ADNP-like immunoreactivity colocalized with tubulin-like immunoreactivity and with microtubular structures, but not with actin microfilaments. Because microtubules are key components of developing neurons and brain, possible interaction between tubulin and ADNP might indicate a functional correlate to the role of ADNP in the brain. In addition, ADNP-like immunoreactivity in the extracellular milieu of astrocytes increased by approximately 1.4 fold after incubation of the astrocytes with VIP. VIP is known to cause astrocytes to secrete neuroprotective/neurotrophic factors, and we suggest that ADNP constitutes part of this VIP-stimulated protective milieu.

Journal Article↗

PubMed2006

Subcellular localization and membrane topology of the melon ethylene receptor CmERS1.

Ethylene receptors are multispanning membrane proteins that negatively regulate ethylene responses via the formation of a signaling complex with downstream elements. To better understand their biochemical functions, we investigated the membrane topology and subcellular localization of CmERS1, a melon (Cucumis melo) ethylene receptor that has three putative transmembrane domains at the N terminus. Analyses using membrane fractionation and green fluorescent protein imaging approaches indicate that CmERS1 is predominantly associated with the endoplasmic reticulum (ER) membrane. Detergent treatments of melon microsomes showed that the receptor protein is integrally bound to the ER membrane. A protease protection assay and N-glycosylation analysis were used to determine membrane topology. The results indicate that CmERS1 spans the membrane three times, with its N terminus facing the luminal space and the large C-terminal portion lying on the cytosolic side of the ER membrane. This orientation provides a platform for interaction with the cytosolic signaling elements. The three N-terminal transmembrane segments were found to function as topogenic sequences to determine the final topology. High conservation of these topogenic sequences in all ethylene receptor homologs identified thus far suggests that these proteins may share the same membrane topology.

Amino Acid Sequence↗