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At least 235 records · Page 13Linked to original sources

PubMed1983

The subcellular localization of ubiquinone in human neutrophils.

Ubiquinone-10 has recently been proposed as a component of the microbicidal oxidase system of neutrophil leukocytes [Crawford & Schneider (1982) J. Biol. Chem. 257, 6662-6668]. We have determined the subcellular localization of ubiquinone and could detect it only in the mitochondrial fractions. It was not enriched in phagolysosomes which were enriched in O2-. generating activity and cytochrome b-245. It is proposed that ubiquinone is unlikely to play a direct role in the electron transport chain located in the plasma membrane which functions to produce microbicidal oxygen radicals.

Centrifugation, Density Gradient↗

PubMed1983

The brain angiotensin system: subcellular localization and interferences with converting enzyme inhibitors.

Renin is stored in synaptosomes of rat brain, separately from cathepsin D and intraneuronal angiotensin II (ANG II) has been demonstrated with the electron-microscope. Although the subcellular localization of other components of the renin-angiotensin system (RAS) have still to be investigated, these data suggest possible intracellular synthesis of ANG II in the brain. Brain ANG II is biochemically identical to the plasma peptide and corresponds to (IIe) 5-ANG II. The peptide level is unchanged after bilateral nephrectomy, and angiotensin I (ANG I) accumulation is observed in nephrectomized animals following brain angiotensin converting enzyme blockade. The significantly greater accumulation of ANG I and reduction of ANG II in stroke prone spontaneously hypertensive Wistar-Kyoto rats (WKY) indicates a higher synthesis and turnover rate of ANG II in SHR. Most converting enzyme inhibitors (CEI) penetrate the brain after chronic oral treatment. Part of their blood pressure lowering action may therefore be explained by an inhibition of the brain RAS.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1982

An improved method for the subcellular localization of calcium using a modification of the antimonate precipitation technique.

A new variation of the antimonate precipitation technique, employing tannic acid in the primary aldehyde-antimonate fixative, is described for use in the subcellular localization of calcium in various tissues. Chelation studies and electron microscopic, X-ray microanalytical studies of antimonate precipitates in etiolated oat tissues indicate that calcium is the major cation localized using the present experimental protocol. Preservation of ultrastructural morphology in these tissues is greatly improved over that observed in tissues fixed with conventional antimonate-aldehyde or antimonate-osmium fixatives. The regularity and reproducibility of tissue precipitate patterns suggests that 1) penetration of the tissue by the fixative, and subsequent precipitation of calcium, is rapid and uniform and 2) ion displacement during sample preparation is negligible. Calcium appears to be immobilized efficiently in situ, with greater than 90% 45Ca retention in radiolabeled tissues prepared for electron microscopy. Quantitative aspects of calcium precipitation by antimonate in 45Ca-labeled CaCl2 solutions were examined over a wide range of calcium concentrations. Precipitation was essentially linear over the expected range of biological concentrations of calcium. Furthermore, the 3:1 antimonate to calcium ratio estimated for test tube precipitates was also established for Sb/Ca in tissue precipitates analyzed using energy dispersive x-ray microanalytical (EDX) techniques. These observations suggest that the present technique is potentially useful in the semiquantitative estimation of tissue calcium levels.

Animals↗

PubMed1992

Subcellular localization of prostaglandin E2 receptors in the gastric mucosa.

Gastric mucosal PG E2 receptors are the common antisecretory working point of all prostanoid types and may also be involved in "protective" effects. We investigated the subcellular localization of these receptors, as measured by displaceable 3H-PG E2 binding, and identified different organelles by monitoring the activities of specific marker enzymes. Porcine mucosal homogenates were subdivided by differential centrifugation into fractions P1 (1000 x g), P2 (20,000 x g), P3 (300,000 x g) and the supernatant S1. P3 was further fractionated over a series of sucrose step gradients. Mitochondria and lysosomes were enriched in P2 (maximum specific activities of cytochrome-c-oxidase of beta-glucosidase, beta-glucuronidase, beta-galactosidase, respectively). Plasma membranes (alkaline phosphatase, gamma-glutamyl-transpeptidase, 5-nucleotidase), tubulovesicles (H+/K(+)-ATPase) and rough endoplasmic reticulum (NADPH-cytochrome-c-reductase) were mainly found in P3, which also contained the majority of 3H-PG E2 binding sites. In contrast, prostanoid binding was barely detectable in S1. Density fractionation of P3 revealed that 3H-PG E2 binding sites shared a similar sedimentation profile with plasma membranes and tubulovesicular markers. No or negative correlation was found with lysosomes, rough endoplasmic reticulum and mitochondria. We conclude that mucosal PG E2 receptors are predominantly located at the cell surface. This supports the view that prostanoids inhibit gastric secretion through membrane receptors, but gives no clue for intracellular "protective" working points.

Animals↗

PubMed1982

Subcellular localization of the 52,000 molecular weight major postsynaptic density protein.

We have recently reported that in isolated synaptic junctions, the quantity of the major post-synaptic density protein (mPSDp, Mr = 52,000) increases approximately twenty-fold during the third and fourth weeks of postnatal development. In the study that follows, systematic analyses were carried out to determine the subcellular localization of this prominent synaptic protein in adult brain and non-neuronal tissues. Subcellular fractionation and SDS-gel electrophoresis were used to isolate various tissue components and identify proteins that possessed molecular weights similar to that of the mPSDp. To unambiguously verify the molecular identity of all proteins suspected of being the mPSDp, two-dimensional peptide fingerprinting was carried out. In addition, the different subcellular fractions were examined for the presence of structures morphologically resembling the postsynaptic density. The mPSDp was found only in fractions containing identifiable asymmetric synaptic structures and/or postsynaptic densities. This protein was not found in non-neuronal tissues or any other fraction in which there was not a demonstrable presence of postsynaptic densities. This work strongly indicates that the major PSD protein is a molecular 'marker' specific to asymmetric synapses in the mammalian forebrain.

Animals↗

PubMed1982

Subcellular localization of phosphoenolpyruvate carboxykinase in the trypanosomatids Trypanosoma cruzi and Crithidia fasciculata.

Particulate fractions obtained from Trypanosoma cruzi and Crithidia fasciculata by different procedures were subjected to isopycnic centrifugation in sucrose gradients, in order to determine the subcellular localization of phosphoenolpyruvate carboxykinase (PEPCK) in both organisms, and of malic enzyme (ME) I in T. cruzi. The more clear-cut results were obtained with T. cruzi by breaking the cells by grinding in a mortar with silicon carbide and using a gradient from 0.4 to 2.0 M sucrose, whereas with C. fasciculata, the best procedure was disruption of the cells by digitonin treatment and potter homogenization and use of a gradient from 1.1 to 2.0 M sucrose. PEPCK banded together with the glycosomal marker hexokinase in both organisms; there was a clear separation from the mitochondrial markers, oligomycin-sensitive Mg2+-APTase and citrate synthase. PEPCK showed a latency of 24% in the enriched 'glycosoma' fraction of T. cruzi. ME I from T. cruzi, on the other hand, banded together with the mitochondrial markers. These results indicate that PEPCK and ME are present in different subcellular compartments, a fact significant for the prevention of a futile cycle between C4-dicarboxylic acids and C3-monocarboxylic acids, which might take place if both enzymes functioned in the same compartment.

Animals↗

PubMed1987

Modification, processing, and subcellular localization in Escherichia coli of the pCloDF13-encoded bacteriocin release protein fused to the mature portion of beta-lactamase.

A fusion between the pCloDF13-derived bacteriocin release protein and beta-lactamase was constructed to investigate the subcellular localization and posttranslational modification of the bacteriocin release protein in Escherichia coli. The signal sequence and 25 of the 28 amino acid residues of the mature bacteriocin release protein were fused to the mature portion of beta-lactamase. The hybrid protein (Mr, 31,588) was expressed in minicells and whole cells and possessed full beta-lactamase activity. Immunoblotting of subcellular fractions revealed that the hybrid protein is present in both the cytoplasmic and outer membranes of E. coli. Radioactive labeling experiments in the presence or absence of globomycin showed that the hybrid protein is modified with a diglyceride and fatty acids and is processed by signal peptidase II, as is the murein lipoprotein. The results indicated that the pCloDF13-encoded bacteriocin release protein is a lipoprotein which is associated with both membranes of E. coli cells.

Bacterial Proteins↗

PubMed1994

Subcellular localization of peripheral benzodiazepine receptors on human leukocytes.

BACKGROUND: The peripheral-type benzodiazepine receptor (PBR) was initially identified in many peripheral tissues and in some blood cells. Drugs that bind with high affinity to PBRs have previously been described as having immunomodulating properties. The number of PBRs varies according to the cell population considered. The aim of this study was to study the localization of PBRs in two human leukocyte populations, T4-lymphocytes, and monocytes. EXPERIMENTAL DESIGN: Both cell populations were purified by negative immunoselection in order to keep only the physiologically accessible sites on the viable cells. Mitochondria were quantified by electron microscopy and flow cytometric analysis. Subcellular localization was then studied after PBR photoaffinity labeling using electron microscopic ultrastructural autoradiography. RESULTS: We have shown that monocytes contain twice as many mitochondria as lymphocytes. We have also shown that the global labeling of monocytes by ultrastructural autoradiography is actually higher than that of lymphocytes and the labeling of monocyte mitochondria is higher than that of lymphocyte mitochondria. In addition, the distribution of subcellular labeling indicates that there are different populations of mitochondria in one cell, i.e., labeled and unlabeled, and that the percentage of labeled mitochondria is greater in monocytes. These results are consistent with those obtained in previous binding studies. Finally, over 50% of receptors are localized in cell compartments devoid of visible mitochondria. CONCLUSIONS: The subcellular distribution of the PBR shows that this receptor could have other physiologic functions towards immune cells than a function associated with mitochondria.

Autoradiography↗

PubMed1998

Intrarenal and subcellular localization of rat CLC5.

Dent's disease, an inherited disorder characterized by hypercalciuria, nephrolithiasis, nephrocalcinosis, rickets, low-molecular-weight proteinuria, Fanconi's syndrome, and renal failure, is caused by mutations in the renal chloride channel, CLC5. The normal role of CLC5 is unknown. We have investigated the intrarenal and subcellular localization of CLC5 in rat kidney by in situ hybridization and immunohistochemistry. By in situ hybridization, CLC5 mRNA was detected predominantly in cortical medullary ray and outer medullary tubule epithelial cells. Polyclonal antiserum was generated against a CLC5 fusion protein, affinity purified, and immunoadsorbed against CLC3 and CLC4 to yield a CLC5 isoform-specific antiserum. By immunohistochemistry, CLC5 protein was localized to the intracellular domain of tubular epithelial cells in the S3 segment of the proximal tubule and the medullary thick ascending limb. By subcellular membrane fractionation and flow cytometry, CLC5 expression was found in outer medullary endosomes. These findings are consistent with a model in which CLC5 encodes an endosomal chloride channel that facilitates acidification and trafficking of renal epithelial endosomes.

Animals↗

PubMed1998

Disruption of a putative SH3 domain and the proline-rich motifs in the 53-kDa substrate of the insulin receptor kinase does not alter its subcellular localization or ability to serve as a substrate.

The recently identified 53-kDa substrate of the insulin receptor family was further characterized in several retroviral-generated stable cell lines overexpressing the wild type and various mutant forms of the protein. To facilitate the study of its subcellular localization in NIH3T3 cells overexpressing insulin receptor, a myc epitope-tag was added to the carboxy terminus of the 53-kDa protein. Like the endogenous protein in Chinese hamster ovary cells, the expressed myc-tagged 53-kDa protein was found partially in the particulate fraction and was tyrosine phosphorylated in insulin-stimulated cells. Immunofluorescence studies showed for the first time that a fraction of the 53-kDa protein was localized to the plasma membrane. Confocal microscopy of cells double-labeled with antibodies to the insulin receptor and the myc epitope showed the two proteins co-localize at the plasma membrane at the level of light microscopy. Further analyses of the protein sequence of the 53-kDa substrate revealed the presence of a putative SH3 domain and two proline-rich regions, putative binding sites for SH3 and WW domains. Disruption of these three motifs by the introduction of previously characterized point mutations did not affect the membrane localization of the 53-kDa protein, its ability to serve as substrate of the insulin receptor, or its colocalization with the insulin receptor, suggesting these domains are not important in the subcellular targeting of the protein and instead may function in the interaction with subsequent signaling proteins.

3T3 Cells↗

PubMed1979

Re-examination of the subcellular localization of thyroxine 5'-deiodination in rat liver.

We describe the existence of at least two thyroxine 5'-deiodinases in rat liver. They co-fractionate with NADPH-cytochrome c reductase, the marker enzyme for membranes of the endoplasmic reticulum. Subcellular-localization studies of the most active microsomal thyroxine 5'-deiodinase were performed under substrate saturation and at optimal pH 6.8. This enzyme was a Km(app.) of about 3 microM-thyroxine and a Vmax. of about 8 ng of tri-iodothyronine/min per mg of protein. Our study confirms in part the earlier reports of microsomal localization of thyroxine 5'-deiodination. However, this process is not mediated by only a single enzyme.

Animals↗

PubMed1996

Cellular and subcellular localization of the vasopressin- regulated urea transporter in rat kidney.

The renal urea transporter (RUT) is responsible for urea accumulation in the renal medulla, and consequently plays a central role in the urinary concentrating mechanism. To study its cellular and subcellular localization, we prepared affinity-purified, peptide-derived polyclonal antibodies against rat RUT based on the cloned cDNA sequence. Immunoblots using membrane fractions from rat renal inner medulla revealed a solitary 97-kDa band. Immunocytochemistry demonstrated RUT labeling of the apical and subapical regions of inner medullary collecting duct (IMCD) cells, with no labeling of outer medullary or cortical collecting ducts. Immunoelectron microscopy directly demonstrated labeling of the apical plasma membrane and of subapical intracellular vesicles of IMCD cells, but no labeling of the basolateral plasma membrane. Immunoblots demonstrated RUT labeling in both plasma membrane and intracellular vesicle-enriched membrane fractions from inner medulla, a subcellular distribution similar to that of the vasopressin-regulated water channel, aquaporin-2. In the outer medulla, RUT labeling was seen in terminal portions of short-loop descending thin limbs. Aside from IMCD and descending thin limbs, no other structures were labeled in the kidney. These results suggest that: (i) the RUT provides the apical pathway for rapid, vasopressin-regulated urea transport in the IMCD, (ii) collecting duct urea transport may be increased by vasopressin by stimulation of trafficking of RUT-containing vesicles to the apical plasma membrane, and (iii) the rat urea transporter may provide a pathway for urea entry into the descending limbs of short-loop nephrons.

Animals↗

PubMed1996

Subcellular localization of the type 2 11beta-hydroxysteroid dehydrogenase. A green fluorescent protein study.

11beta-Hydroxysteroid dehydrogenase (11beta-HSD) is thought to confer aldosterone specificity to mineralocorticoid target cells by protecting the inherently non-selective mineralocorticoid receptor (MR) from occupancy by endogenous glucocorticoids. Recently, we characterized a novel isoform of 11beta-HSD in aldosterone target cells, which has high affinity for its substrate, is unidirectional, and prefers NAD as cofactor. In this study we utilized a green fluorescent protein (GFP) technique to determine the subcellular localization of this isoform, 11beta-HSD2. We generated a chimeric gene encoding the full-length rabbit 11beta-HSD2 and, fused to its C terminus, the coding sequence of GFP. This construct was stably transfected into CHO cells. The enzymatic characteristics of the expressed 11beta-HSD2/GFP fusion protein were undistinguishable from those of the native enzyme: high affinity for corticosterone (KM 8-10 nM), NAD dependence, and lack of reductase activity. The intracellular location of the recombinant protein was determined by fluorescence microscopy. 11beta-HSD2-associated fluorescence was observed as a reticular network over the cytoplasm and nuclear envelope, whereas the plasma membrane and the nucleus were negative, suggesting endoplasmic reticulum (ER) localization. Staining of CHO cells expressing 11beta-HSD2/GFP with established subcellular organelle markers revealed a colocalization of 11beta-HSD2/GFP only with ER markers and tubulin. To examine the orientation of 11beta-HSD2 within the ER, we selectively permeabilized CHO cells and stained them with an anti-GFP antibody. Fluorescence microscopy indicated that the C-terminal region of 11beta-HSD2 is on the cytoplasmic surface of the ER membrane, since it was accessible to the GFP antibody. This conclusion was confirmed by trypsin treatment of permeabilized cells followed by Western blotting. The C-terminal region of 11beta-HSD2 was accessible to trypsin, indicating that it is on the cytoplasmic side of the ER membrane. These results indicate that 11beta-HSD2 is localized exclusively to the ER. Since 11beta-HSD2 does not contain any known ER retrieval signal, experiments are currently under way to determine what structural motifs are responsible for its ER localization.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1993

Subcellular localization of immunoreactive oxytocin within thymic epithelial cells of the male mouse.

Immunoreactive oxytocin is expressed by thymic epithelial cells, which share properties with neuroendocrine cells. In order to investigate the assumed paracrine secretion of oxytocin, we studied the subcellular localization of immunoreactive oxytocin within thymic tissue and cultured thymic epithelial cells of the male mouse. Three types of immunoreactive cells were distinguished with the electron microscope. Immunoreactive oxytocin was found to be restricted to the cytoplasm by the use of pre- and postembedding methods. Some epithelial cells, especially in the cortex, showed a pronounced labelling of vesicular membranes and membrane tubules of the endoplasmic reticulum. In some cells, keratin filaments were associated with the electron-dense stain. Under culture conditions immunoreactive cells of different shapes were found, all displaying similar patterns of labelling. The contents of different types of vacuoles were only rarely labelled. A special class of immunoreactive exocytotic vesicles could not be identified. Thus, our results do not support neuroendocrine secretion of oxytocin via vesicles of thymic epithelial cells but offer alternative modes of secretion.

Animals↗

PubMed1993

Subcellular localization of alpha-tubulin and opsin mRNA in the goldfish retina using digoxigenin-labeled cRNA probes detected by alkaline phosphatase and HRP histochemistry.

This paper describes a method for non-radioactive in situ hybridization providing subcellular localization of mRNA in 3 microns cryosections. We used two alternative colorimetric reactions to detect digoxigenin-labeled cRNA probes: alkaline phosphatase and HRP (horseradish peroxidase). With some probes the signal with the alkaline phosphatase reaction was intense, and diffusion of the reaction product was noticeable. Using HRP-conjugated antibodies improved the resolution but decreased the sensitivity of the signal. Photoamplification of the HRP reaction product increased the contrast and improved the sensitivity of the technique.

Alkaline Phosphatase↗

PubMed1992

Identification and subcellular localization of the Q gene product of visna virus.

The genome of the sheep visna lentivirus contains an open reading frame, Q, which has a coding potential of 230 amino acid residues. This paper reports the identification and the subcellular localization of the Q ORF-encoded protein detected in lysates of visna virus-infected sheep choroid plexus cells. Sera from sheep either experimentally or naturally infected with visna virus reacted with the bacterially synthesized Q protein indicating that the in vivo expressed Q product is immunogenic. Antibodies raised against a synthetic N-terminal peptide, reacted with either the bacterial Q or the in vitro translated Q protein as well as with the Q protein expressed during cellular infection. This 29 kDa protein is detectable late in the lytic viral cycle, i.e., 72 hr postinfection, and this expression correlates with the late transcription of its 4.8-kb mRNA. These results provide evidence for the first time that the Q ORF is a late gene of visna virus and that the Q protein is located in the cytosol compartment, without evidence of accumulation at the cell membrane, or in cell-free virion particles.

Animals↗

PubMed1984

Accumulation, subcellular localization and release of propranolol from synaptosomes of rat cerebral cortex.

Propranolol is accumulated at several adrenergic neuroeffector junctions after chronic oral administration in the dog, and is released subsequently during sympathetic nerve stimulation. In the present study, the accumulation, subcellular localization and release of propranolol was examined in rat cortical synaptosomes. Synaptosomal propranolol accumulation was rapid and attained equilibrium within 1 min. Propranolol uptake increased in a nonlinear manner with increasing drug concentration in the medium, but could not be fully saturated over the concentration range studied (10(-7) - 10(-3) M). Uptake was unaffected by cocaine or ouabain and showed no stereoselectivity. Subsynaptosomal fractionation of propranolol-loaded synaptosomes revealed that the drug was concentrated principally in fractions enriched in synaptic plasma membranes and synaptic storage vesicles. Exposure of propranolol-loaded synaptosomes to elevated potassium evoked a concentration-dependent increase in propranolol overflow, which was not seen in mitochondrial fractions, myelin fractions or in freeze-thawed synaptosomal preparations. Veratridine was also effective in promoting propranolol overflow in a concentration-dependent manner. The increase in propranolol overflow induced by elevated potassium was significantly reduced, but not completely inhibited, in a calcium-free, ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid-supplemented medium. These results indicate that propranolol may be accumulated by neuronal tissue and stored at sites from which release may occur in response to depolarizing stimuli. The data further suggest that propranolol release in the synaptosome preparation may occur by both calcium-dependent and calcium-independent processes.

Animals↗

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↗