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Results for “Subcellular localization”

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PubMed1984

A simple method to determine whole cell uptake of radiolabelled oestrogen and progesterone and their subcellular localization in breast cancer cell lines in monolayer culture.

Specific uptake of tritiated 17 beta-oestradiol and R5020, a synthetic progestin, in breast cancer cell lines ( MCF7 and T47D) growing in monolayer culture in multiwell plates has been shown. Binding characteristics, calculated by Scatchard analysis, indicate the presence of steroid receptors of similar affinities and capacities to those already obtained with broken cell preparations. Lysis of the cells by treatment with a hypotonic buffer reveals the subcellular localization of the receptors so the method can be used to study receptor dynamics such as nuclear translocation and processing. Cell growth can be measured by DNA determination directly in the multiwell plates. Thus, the method provides a convenient way of studying the effects of steroid hormones (or any antihormone or chemotherapeutic agent) on growth and receptor content of breast cancer cells in monolayer culture.

Breast Neoplasms↗

PubMed1984

Subcellular localization and tissue distribution of sialic acid-forming enzymes. N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase.

The activities of N-acetylneuraminate 9-phosphate synthase and N-acetylneuraminate 9-phosphatase, the two enzymes involved in the final steps of the biosynthetic pathway of N-acetylneuraminic acid, were measured with the substrates N-acetyl[14C]mannosamine 6-phosphate and N-acetyl[14C]neuraminic acid 9-phosphate respectively. Subcellular localization studies in rat liver indicated that both enzymes are localized in the cytosolic fraction after homogenization in sucrose medium. To test the possibility of misinterpretation due to the hydrolysis of N-acetylneuraminic acid 9-phosphate by non-specific phosphatases, the hydrolysis of various phosphate esters by the cytosolic fraction was tested. Only p-nitrophenyl phosphate was hydrolysed; however, competition studies with N-acetylneuraminic acid 9-phosphate and p-nitrophenyl phosphate indicated that two different enzymes were involved and that no competition existed between the two substrates. In various other rat tissues N-acetylneuraminate-9-phosphate synthase and N-acetylneuraminate 9-phosphatase activities were detected, suggesting that N-acetylmannosamine 6-phosphate is a general precursor for N-acetylneuraminic acid biosynthesis in all the tissues studied.

Animals↗

PubMed1990

Conversion of alkylacetylglycerol to platelet-activating factor in HL-60 cells and subcellular localization of the mediator.

A human promyelocytic leukemia (HL-60) cell line was used to investigate the conversion of 1-alkyl-2-acetyl-sn-glycerol (alkylacetyl-G) to platelet-activating factor (PAF; 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by intact cells and in subcellular fractions in order to examine the fate of PAF synthesized de novo. Lipid extracts obtained from undifferentiated HL-60 cells incubated with [3H]alkylacetyl-G contained 2-4% of the label as [3H]PAF; several related metabolites were also detected. The yield of [3H]PAF could be dramatically increased by pretreating the cells with either oleic acid, an activator of CTP:phosphocholine cytidylyltransferase, or phenylmethylsulfonyl fluoride, an inhibitor of PAF acetylhydrolase. These results, together with a kinetic study of [3H]alkylacetyl-G metabolism, indicate the sequential participation of a cholinephosphotransferase for the conversion of [3H]-alkylacetyl-G to PAF and acetylhydrolase and transacylase activities in the remodeling pathway that metabolize the newly formed [3H]PAF to 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3-phosphocholine. The dithiothreitol-insensitive cholinephosphotransferase activity capable of converting alkylacetyl-G to PAF was localized in subcellular fractions that contain CDP-choline:1,2-dioleoyl-sn-glycerol cholinephosphotransferase (dithiothreitol-sensitive), as well as marker enzyme activities for the endoplasmic reticulum and Golgi membranes. Subcellular localization analyses also indicated that the majority of newly formed [3H]PAF and a large portion of its deacetylated metabolite were associated with the plasma membrane-containing fractions, whereas most of the 1-[3H]alkyl-2-acyl(long chain)-sn-glycero-3- phosphocholine was present in the intracellular organelles. Incubations of HL-60 cells with exogenous [3H]PAF produced a similar subcellular distribution of metabolites. Very little (less than 10%) of the [3H]PAF produced from [3H]alkylacetyl-G was released from intact cells under a variety of incubation conditions but 50% of the de novo-derived mediator was recovered in the medium of cells that were permeabilized with saponin. Our results indicate that PAF is rapidly translocated from its intracellular site of enzymatic synthesis to the plasma membrane where it is apparently sequestered in a pool that is not accessible to extracellular acceptors in contact with intact cells.

Cell Line↗

PubMed1988

Subcellular localization and dynamics of components of the respiratory burst oxidase.

Membrane and cytosolic factors cooperate to generate NADPH-oxidase. The study of the syndrome of NADPH-oxidase deficiencies, chronic granulomatous disease, has enabled the identification of two membrane factors: a flavin adenine dinucleotide flavoprotein and a b cytochrome. The nature of the cytosolic components is still unknown, but a 47-kD protein, whose phosphorylation occurs in parallel with the generation of a respiratory burst in intact cells, seems to be one of the cytosolic factors. The subcellular localization of the membrane-bound NADPH-oxidase components has been studied in neutrophils: In unstimulated cells, only a minute fraction of the NADPH-oxidase components is localized in the plasma membrane, whereas approximately 80% is localized in the membrane of the specific granules and the majority of the rest is in a newly described membrane-bound compartment, the secretory granules, identified by latent alkaline phosphatase. During stimulation, these NADPH-oxidase components are translocated to the plasma membrane as a result of fusion of granule membrane with plasma membrane. Only the NADPH-oxidase components present in the plasma membrane are incorporated in the respiratory burst oxidase generated in intact cells.

Animals↗

PubMed1990

Subcellular localization of rev-gene product in visna virus-infected cells.

The 1.4-kb mRNA of visna lentivirus is expressed early during the lytic infection of sheep choroid plexus cell cultures. It encodes for visna early gene 1 (VEG1) product, since renamed rev gene product (or Rev), based on significant amino acid sequence homologies between this protein and the proteins of simian immunodeficiency virus of macaque and human immunodeficiency virus type 2. In this report, we examined the subcellular localization and time course appearance of the Rev protein in visna virus-infected cells. Immunoprecipitation assays of [35S]methionine-labeled cell lysates with antisera raised against the Rev protein revealed a polypeptide of 19 kDa (p19rev). This protein was predominant early in the viral replication cycle and accumulated preferentially in the cytoplasmic/membrane fraction of infected cells. Indirect immunofluorescence staining of infected cells confirmed the cytoplasmic location of visna Rev protein and could reveal in some stained cells a higher concentration of Rev at the cellular plasma membrane. The regulating protein, still present late in the viral lytic cycle, is packaged into mature viral particles along with the structural gag and env gene products.

Animals↗

PubMed1992

Surfactant protein D: subcellular localization in nonciliated bronchiolar epithelial cells.

Surfactant protein D (SP-D, CP4) is a collagenous surfactant-associated carbohydrate binding protein that was initially characterized as a biosynthetic product of type II pneumocytes. Immunoperoxidase studies of formaldehyde solution-fixed and paraffin-embedded rat lung demonstrated staining for SP-D in the cytoplasm of a subpopulation of bronchiolar epithelial cells as well as type II cells. Accordingly, immunogold-labeling techniques were used to further examine the cellular distribution and subcellular localization of SP-D in the small airways. Lung tissues were fixed with 0.5% glutaraldehyde-3% paraformaldehyde and embedded in LR White resin. Sections were reacted with affinity purified polyclonal antibodies to SP-D, and sites of antibody binding were demonstrated using a biotinylated secondary antibody-streptavidin-gold detection system. Anti-SP-D selectively decorated secretory compartments of nonciliated bronchiolar cells (Clara cells) with strong and specific labeling of apical electron-dense secretory granules. Almost all of the granules in nonciliated columnar cells were labeled; however, labeling was typically nonuniform, with preferential decoration of the periphery of the granule. The largest numbers of immunoreactive epithelial cells were observed in the distal membranous bronchioles, with progressively smaller numbers of cells in more proximal bronchioles. There was no detectable labeling of cells lining the large cartilagenous airways or trachea. These studies provide evidence that SP-D is a secretory product of nonciliated bronchiolar cells. We suggest that Clara cell-derived SP-D is a component of bronchiolar lining material, consistent with our hypothesis that SP-D contributes to surfactant metabolism and/or host defense within small airways.

Animals↗

PubMed1985

Subcellular localization of [3H]-nitrendipine binding sites in guinea-pig ileal smooth muscle.

The binding of [3H]-nitrendipine was studied in microsomal fractions isolated from guinea-pig ileal smooth muscle. Only one class of specific binding sites was detected, with a KD of 0.4 nM. For various dihydropyridine derivatives, including the stereoisomers of nimodipine and the 'Ca agonist' Bay K 8644, the potency for inhibition of [3H]-nitrendipine binding correlated well with the reported pharmacological potency in smooth muscle preparations. To establish the subcellular localization of [3H]-nitrendipine binding sites, untreated and digitonin-treated microsomal fractions were subfractionated by isopycnic density gradient centrifugation. The density distribution of [3H]-nitrendipine binding was markedly shifted by digitonin towards higher densities, as were the distributions of 5'-nucleotidase and [3H]-ouabain binding, whereas the distributions of NADPH:cytochrome c reductase and NADH:cytochrome c reductase were hardly modified by digitonin. It is concluded that most, if not all, [3H]-nitrendipine binding sites in guinea-pig ileal smooth muscle are present in the plasma membrane, in agreement with the postulated mode of action of dihydropyridines as inhibitors of plasmalemmal Ca channels.

5'-Nucleotidase↗

PubMed1991

Subcellular localization and immunological detection of proteins encoded by the vir locus of Bordetella pertussis.

The DNA sequence of the central regulatory locus vir of Bordetella pertussis predicts that three gene products, BvgA, BvgB, and BvgC, are encoded. Features of the predicted primary structures of these proteins and their homology to other two-component systems suggest that BvgA is located in the cytoplasm, BvgB is located in the periplasm, and BvgC spans the inner membrane. We have used gene fusions to the phoA and lacZ genes of Escherichia coli to investigate the subcellular localization and membrane topology of these proteins. PhoA fusion proteins were also purified and used to raise antibodies that allowed visualization of the vir-encoded polypeptides by Western immunoblotting. Our results have largely confirmed the predictions of the DNA sequence, with the exception that BvgB and BvgC were found to constitute one larger protein that was homologous to the sensor class of two-component systems. We propose that this protein be named BvgS (for sensor) and that its gene be named bvgS.

Amino Acid Sequence↗

PubMed1975

Identification, subcellular localization, and estrogen regulation of peroxidase in 7,12-dimethylbenz(a)anthracene-induced rat mammary tumors.

An estrogen-induced, intensely staining peroxidase 3,3-diaminobenzidine-positive reaction product is found to be characteristic of hormone-dependent, 7,12-dimenthylbenz(a)anthracene-induced mammary tumors of the rat. This product is demonstrated in thick sections of such tumors from intact or estrogen-treated castrate rats but is not seen in tumors that are in regression due to castration or estrogen deprivation. It is, furthermore, absent from tumors whose growth is unaffected by castration. The subcellular localization of this enzyme activity is restricted mainly to the nuclear envelope and cisternae of the granular endoplasmic reticulum in addition to secretory granules. This provides the first evidence for a criterion that would allow differentiation of hormone-dependent and hormone-independent mammary cancer on histological sections and, as such, may have considerable potential as an aid in the classification of human breast cancer.

Animals↗

PubMed1990

Subcellular localization of hematoporphyrin derivative in bladder tumor cells in culture.

Mitochondria have been implicated as a primary subcellular site of porphyrin localization and photodestruction. However, other organelles including the cell membrane, lysosomes and nucleus have been shown to be damaged by hematoporphyrin derivative (HpD) photosensitized destruction as well. In this study we attempted to follow the translocation of the fluorescent components of HpD in human bladder tumor cells (MGH-U1) in culture to determine whether specific subcellular localization occurs over time. Following a 30 min exposure to HpD the cellular fluorescence was examined immediately and 1, 2, 4, and 24 h after HpD removal using fluorescence microscopy and an interactive laser cytometer. The in vitro translocation of dye appeared to be fairly rapid with fluorescence present at the cell membrane and later (1-2 h) within a perinuclear area of the cytoplasm. To determine whether HpD had become concentrated into a specific subcellular organelle, these fluorescence distribution patterns were compared with fluorescent marker dyes specific for mitochondria, endoplasmic reticulum and other membranous organelles. The HpD fluorescence did not appear to be as discrete as the dyes specific for mitochondria or endoplasmic reticulum but appeared similar to the diffuse cytomembrane stain. Finally, the interaction between the fluorescent components of HpD and the cellular constituents was evaluated using a "fluorescence redistribution after photobleaching" technique. The results indicated that the mean lateral diffusion for HpD in MGH-U1 cells was 1.05 x 10(-8) cm2/s, a rate closer to that of lipid diffusion (10(-8)) than that of protein diffusion (10(-10)).(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Line↗

PubMed1991

Purification, characterization, and subcellular localization of an acid phosphatase from black mustard cell-suspension cultures: comparison with phosphoenolpyruvate phosphatase.

An acid phosphatase from Brassica nigra (black mustard) leaf petiole cell-suspension cultures has been purified 1633-fold to a final specific activity of 1225 (mumols orthophosphate produced/min)/mg protein and near homogeneity. The native protein was a glycosylated monomer having a molecular mass of 60 kDa and a pI of 4.5. The enzyme displayed a broad pH optimum of about pH 5.6 and was heat stable. The final preparation hydrolyzed a wide variety of phosphate esters. The highest specificity constants were obtained with 3-phosphoglycerate, 2,3-diphosphoglycerate, PPi, and phosphoenolpyruvate (PEP). The enzyme was activated 1.4-fold by 4 mM Mg2+ or Mn2+, but was strongly inhibited by Mo, Pi, F, and several phosphorylated compounds. Subcellular localization experiments revealed that this nonspecific acid phosphatase is probably a secreted enzyme, localized in the cell wall. By contrast, B. nigra PEP phosphatase appeared to be localized in the cell vacuole. Peptide mapping via CNBr fragmentation was employed to investigate the structural relatedness of the two phosphatases. Their respective CNBr cleavage patterns were dissimilar, suggesting that B. nigra acid and PEP phosphatases are distinct polypeptides. Putative metabolic functions of these two phosphatases are discussed in relation to the biochemical adaptations of B. nigra cell-suspension cultures to nutritional phosphate deprivation.

Acid Phosphatase↗

PubMed1978

Hepatic alpha-adrenergic receptors. Identification and subcellular localization using [3H]dihydroergocryptine.

Recently, several workers have shown that adrenergic control of hepatic carbohydrate metabolism has the characteristics of an alpha-receptor-mediated process. Using the rat liver membrane preparation of Neville (Neville, D. (1968) Biochim. Biophys. Acta 154, 540-552), alpha-adrenergic receptors have been identified using the ligand [3H]dihydroergocryptine. The receptors are saturable and of high affinity. Scatchard analysis yields a KD of 1.8 nM with 1.7 +/- 0.55 pmol of sites/mg of protein. Competition of dihydroergocryptine binding with various pharmacologic agents yields the typical (alpha-adrenergic potency series: (-)-epinephrine greater than (-)-norepinephrine greater than (-)-isoproterenol. (-)-Isomers are more potent than (+)-isomers. The alpha-blocker phentolamine is 3.4 orders of magnitude more potent than the beta-blocker propranolol. To determine subcellular localization of alpha-adrenergic receptors, livers were fractionated into a crude homogenate, a 1500 X g pellet, and the purified membrane preparation used previously for binding. Specific dihydroergocryptine binding, ouabain-inhibitable (Na,K)-ATPase, and F--stimulated adenylate cyclase activities, were followed in these fractions. Specific binding was enriched, relative to that in the crude homogenate, 2.88-fold in the pellet and 6.28-fold in the membranes. Similarly, (Na,K)-ATPase acticity was enriched 2.6-fold in the pellet and 7.1-fold in the membranes while adenylate cyclase activity was enriched 2.9-fold in the pellet and 3.5-fold in the membranes. It is concluded that hepatic alpha-adrenergic receptors are likely concentrated in the plasma membranes.

Adenylyl Cyclases↗

PubMed1989

Protamine 3'-untranslated sequences regulate temporal translational control and subcellular localization of growth hormone in spermatids of transgenic mice.

Although the mouse protamine 1 gene (mP1) is first transcribed in round spermatids, its mRNA is not translated until about 1 week later in elongating spermatids. To determine what mP1 sequences are important for its transcriptional and translational regulation, we have constructed fusions between mP1 and the human growth hormone (hGH) structural gene and analyzed their expression in transgenic mice. We show that mP1 sequences 5' to the start of transcription are sufficient to confer spermatid-specific expression on the hGH gene. We also show that 156 nucleotides of mP1 3'-untranslated sequence is sufficient to confer mP1-like translational regulation on the hGH mRNA. Interestingly, the subcellular localization of hGH was dependent on the time during spermiogenesis that it was made. Synthesis of hGH in early round spermatids resulted in localization in the acrosome, whereas synthesis in late elongating spermatids resulted in intracellular, but not acrosomal, localization.

Acrosome↗

PubMed1987

Subcellular localization of sulfated glucuronic acid-containing glycolipids reacting with anti-myelin-associated glycoprotein antibody.

Peripheral nerve glycolipids, with which anti-myelin-associated glycoprotein (MAG) antibodies from patients with demyelinating neuropathy and plasma cell dyscrasia cross-react, proved to be novel glycosphingolipids containing a sulfated glucuronyl residue. Consequently, there has been much interest in the immunological role that these sulfated glucuronyl-glycosphingolipids (SGGLs) may play in the pathogenesis of this disorder. For the determination of the distribution of these glycolipids in various nervous tissues and, thereby, the elucidation of their pathogenicity, a quantitative immunostaining-TLC method for their detection has been devised. Using this method, we demonstrated that these glycolipids were distributed in greatly different amounts in the peripheral nerves from human, bovine, chicken, rat, and rabbit. Subcellular localization studies of bovine peripheral nerve also demonstrated that they were enriched in the axolemma-enriched fraction and present in glial-related membranes in lower concentrations. In addition, these glycolipids were present in bovine dura mater and transformed rat Schwann cells. These biochemical results suggest that not only myelin but also axons could be involved as targets of the anti-MAG antibody in macroglobulinemia neuropathy, and it may also be necessary to examine anti-SGGL activity in patients with axonal neuropathy associated with plasma cell dyscrasia.

Animals↗

PubMed1978

Biosynthesis and subcellular localization of histamine in H-110 lymphoma.

The histidine decarboxylase activity has been studied in the H-110 Lymphoma implanted in Balb-c mice. The enzymatic activity has been determined using DL-Histidine 1-14C by measuring the I--14CO2 liberated during the incubation. It has been found that the histidine decarboxylase activity in the tumor is the highest of the studied tissues. The pretreatment for 5 days with two cytostatic drugs, cyclophosphamide and 5-fluoruracile, determined a decrease of the enzymatic activity in the tumor of 33 and 54 per cent, respectively. This effect has not been observed in the lung. The pretreatment of the animals during 10 days with 0.1 mg per kg body of histamine, which in other experimental tumors induces an increase of the endogenous content of histamine, produces a decrease of the tumoral histidine decarboxylase activity. The histamine-14C uptake was not modified by this pretreatment. The subcellular localization of the radioactivity after pretreatment with histamine-14C evidenced that the nuclear fraction of the tumor contained between 2 and 10 times the radioactivity of other tissues of the same animal.

Animals↗

PubMed1989

Differential expression and subcellular localization for subunits of cAMP-dependent protein kinase during ram spermatogenesis.

The expression of mRNAs for the RI alpha, RII alpha, and C alpha subunits of cAMP-dependent protein kinase has been studied in different ram germ cells. The sizes of the specific RI alpha, RII alpha, and C alpha mRNAs, observed in germ cells were 1.6, 2.0, and 2.6 kb, respectively. RI alpha and C alpha mRNAs were mainly expressed in primary spermatocytes. A postmeiotic expression predominating in early spermatids was unique to RII alpha mRNA. The location of RI, RII alpha, and C subunits in well-defined organelles of ram spermatids and epididymal sperm was assessed by immunogold electron microscopy. In spermatids, RI, RII alpha, and C were essentially present in the forming acrosome and, to a lesser extent, in the nucleus. During sperm epididymal maturation, the protein kinases disappeared from the acrosome and were detected in a variety of sperm functional areas, such as the tip of the acrosome, the motility apparatus, and the membrane network. The present study on subunits of cAMP-dependent protein kinase supports the concept that specific functions are attached to the different subunits in that it shows differential expression and differential subcellular localization in germ cells.

Animals↗

PubMed1990

Functional domains of the HIV-1 rev gene required for trans-regulation and subcellular localization.

The rev gene of human immunodeficiency virus type 1 (HIV-1) encodes a 116 amino acid nuclear regulatory protein (Rev) that increases the cytoplasmic expression of viral mRNAs containing the Rev response element (RRE) and coding for the structural proteins, Gag and Env. To identify the functional domains of Rev, amino acid deletion and chain termination mutations were introduced in the Rev coding region. The ability of these mutants to increase the cytoplasmic expression of a Rev-test plasmid (pSV-AR), containing the RRE cloned into the 3' noncoding region of the CAT gene in plasmid pSV2CAT, was examined in transient expression assays in HeLa cells. Our results indicate that three distinct regions mapping within the N-terminal 98 amino acids of Rev are essential for its activity. The subcellular localization of the various Rev proteins was examined in COS cells by indirect immunofluorescence. Rev was found to localize predominantly in the nucleolus of transfected cells. All mutant Rev proteins, with the exception of a deletion mutant (rev delta 41-44) lacking four Arg residues of a highly basic domain, were found to localize in the nucleolus. Mutant rev delta 41-44 exhibited weak diffuse fluorescence in the nucleus with a tendency to accumulate in the cytoplasm. A 15 amino acid region encompassing this basic domain (38-52) when fused to the Escherichia coli beta-galactosidase gene efficiently directed the fusion gene product to the nucleus and nucleolus, suggesting a role for this domain in the nucleolar localization of Rev.

Amino Acid Sequence↗

PubMed1992

Differential expression and subcellular localization of secretogranin II and synaptophysin during early development of mouse hypothalamic neurons in culture.

Mature neurons contain two distinct regulated secretory pathways, characterized electron microscopically by so-called large dense core vesicles and small synaptic vesicles, respectively. Each vesicle type is characterized by vesicle-specific proteins, such as the granins (chromogranins/secretogranins) for the matrix of large dense core vesicles and synaptophysin for the membrane of small synaptic vesicles. So far, no data exist on the biogenesis of these two distinct vesicle types during neuronal development. We have used secretogranin II and synaptophysin as markers for the biogenesis of these two vesicle types during the development of mouse hypothalamic neurons in culture, using immunocytochemistry and biochemical analyses. By immunofluorescence, we found that secretogranin II appears as early as synaptophysin, but in a subset of neurons only, and with different subcellular localizations. It was observed in cytoplasmic areas where little or no synaptophysin immunofluorescence was detected, such as lamellipodia, emerging neurites and growth cones. At later stages, the proportion of secretogranin II-containing varicosities remained steady whereas that of synaptophysin-containing varicosities increased dramatically. By quantitative analysis we found that the level of expression of synaptophysin increased several-fold during synaptogenesis whereas that of secretogranin II decreased. These data suggest that large dense core vesicles and small synaptic vesicles can be formed separately and expressed at different levels. They provide evidence for a differential biogenesis of these two distinct vesicle types.

Animals↗