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PubMed1979

Subcellular localization of the eosinophil chemotactic factor (ECF) and its inactivator in human polymorphonuclear leucocytes (PMN).

An eosinophil chemotactic factor (ECF) of low MW can be released from human polymorphonuclear leucocytes (PMN) on stimulation with the Ca-ionophore, arachidonic acid and during phagocytosis. After a rapid rise of ECF activity in the supernatant a steep fall of in its activity occurred at the later times of secretion suggesting a mechanism of ECF inactivation. ECF obtained at the later times of secretion represents a stable biological activity and does not decrease on further incubation. In addition, intact PMN and ECF combined do not lead to its inactivation, while incubation of homogenized PMN with ECF decreased its activity. These data suggest the presence of an inactivator for ECF within human PMN. The purpose of the study was to localize ECF and its inactivator within human PMN. After cell disruption, differential and equilibrium gradient centrifugation, subcellular components of human PMN can be obtained which reveal eosinophilotactic (ECF) or ECF-inactivating activity. ECF activity can be recovered (in a structurally bound state) from the microsomal fraction of unstimulated and stimulated PMNs, while another portion is obtainable as a soluble, low mol. wt ECF. The PMN-derived ECF inactivator can be recovered from the peroxidase positive (azurophilic) granules and has a mol. wt of 60,000 and less. We suggest that low mol. wt ECF is derived from the plasma membrane of PMN which can be inactivated by components of the azurophilic granules. The mechanism of inactivation is still unresolved.

Arachidonic Acids↗

PubMed1999

Subcellular localization of the Galphai3 protein and G alpha interacting protein, two proteins involved in the control of macroautophagy in human colon cancer HT-29 cells.

Autophagic sequestration is controlled by the Galphai3 protein in human colon cancer HT-29 cells. Immunofluorescence and subcellular fractionation studies showed that the Galphai3 protein is preferentially associated with Golgi membranes but co-localization was also observed with the endoplasmic reticulum (ER) membrane. The Galphai2 protein, which is not involved in the control of autophagic sequestration, is associated with the plasma membrane. Transfection of chimaeric Galphai proteins (Galphai3/2, Galphai2/3) containing the N- and C-terminal parts of the relevant Galphai demonstrated that the C-terminal part of the Galphai3 protein, by governing its membrane localization [de Almeida, Holtzman, Peters, Ercolani, Ausiello and Stow (1994) J. Cell Sci. 107, 507-515], is important in the control of macroautophagic sequestration. G alpha interacting protein (GAIP),which stimulates the GTPase activity of the Galphai3 protein and favours macroautophagic sequestration in HT-29 cells,was shown, by immunofluorescence studies using confocal microscopy, to be confined to the cytoplasm. The cytoplasmic distribution of GAIP only partially overlaps with that of the Galphai3 protein. However, the presence of the two proteins on Golgi and ER membranes was confirmed by subcellular fractionation. These results point to the importance of the cytoplasmic localization of the Galphai3 protein and GAIP in controlling autophagic sequestration in HT-29 cells.

Autophagy↗

PubMed1992

Subcellular localization of bFGF in human retinal pigment epithelium in vitro.

Basic fibroblast growth factor is a polypeptide mitogen with potential biological roles in angiogenesis, differentiation, and the survival of neurons. To study the expression and subcellular distribution of basic fibroblast growth factor in human retinal pigment epithelium in vitro, affinity-purified antipeptide antibodies were generated against a 15 amino acid sequence in the amino-terminus of this growth factor. Analysis of the cross reactivity and specificity of the affinity-purified antibodies demonstrated no ability to recognize acidic fibroblast growth factor and the ability to label specifically the major known forms of basic fibroblast growth factor in whole-cell lysates of retinal pigment epithelium in vitro. Examination of paraformaldehyde- or glutaraldehyde-fixed pigment epithelium at the light and electron microscopic levels revealed prominent localization of basic fibroblast growth factor to the nucleus and nucleolus. In cells fixed with organic reagents, prominent cytoplasmic staining was noted in addition to the nuclear staining seen in aldehyde fixed cells. Investigation of subcellular fractions by Western blot analysis indicated cytosolic as well as nuclear localization of the basic fibroblast growth factor. These analyses, however, demonstrated that the higher molecular weight forms of basic fibroblast growth factor predominate in the nucleus.

Antibody Specificity↗

PubMed2005

Subcellular localization suggests novel functions for prolyl endopeptidase in protein secretion.

For a long time, prolyl endopeptidase (PEP) was believed to inactivate neuropeptides in the extracellular space. However, reports on the intracellular activity of PEP suggest additional, as yet unidentified, physiological functions for this enzyme. Here, we demonstrate using biochemical methods of subcellular fractionation, immunocytochemical double-labelling procedures and localization of PEP-enhanced green fluorescent protein fusion proteins that PEP is mainly localized to the perinuclear space, and is associated with the microtubulin cytoskeleton in human neuroblastoma and glioma cell lines. Disassembly of the microtubules by nocodazole treatment disrupts both the fibrillar tubulin and PEP labelling. Furthermore, in a two-hybrid screen, PEP was identified as binding partner of tubulin. These findings indicate novel functions for PEP in axonal transport and/or protein secretion. Indeed, a metabolic labelling approach revealed that both PEP inhibition and PEP antisense mRNA expression result in enhanced peptide/protein secretion from human U-343 glioma cells. Because disturbances in intracellular transport and protein secretion mechanisms are associated with a number of ageing-associated neurodegenerative diseases, cell-permeable PEP inhibitors may be useful for the application in a variety of related clinical conditions.

Alanine↗

PubMed1996

Presynaptic and postsynaptic subcellular localization of substance P receptor immunoreactivity in the neostriatum of the rat and rhesus monkey (Macaca mulatta).

The substance P receptor (SPR) gene is expressed at high levels in basal ganglia, but the paucity of information about localization of the encoded receptor protein has limited our understanding of this peptide's involvement in cellular and subcellular mechanisms in this region. Morphological evidence in the rodent striatum indicates that SPRs are expressed in postsynaptic neuronal elements, while pharmacological studies suggest the existence of presynaptic SPRs in this structure. We have examined the issue of subcellular distribution of this receptor protein in rat and primate neostriatal tissue, employing an antiserum raised against SPR. Electron microscopic analysis revealed that SPR immunoreactivity is present in presynaptic and postsynaptic neuronal elements in both species. In agreement with earlier studies, SPR immunoreactivity was found predominantly in perikarya and dendrites of a small subset of striatal neurons, the large and medium-sized aspiny interneurons. In addition, a small but significant proportion of the immunoreaction product was localized in presynaptic profiles, both in axons and axon terminals. The majority of SPR immunoreactive boutons formed asymmetric synapses with dendrites and dendritic spines. The association of SPRs with asymmetric synapses provides a morphological substrate for peptidergic modulation of excitatory neurotransmission of extrastriatal origin. A minor proportion of immunolabeled axons established symmetric synaptic junctions with unlabeled dendrites. The presence of SPRs in these synapses suggests a presynaptic peptidergic modulation of intrinsic striatal transmitter systems. The observations in this study also indicate that SPR mediates a complex combination of postsynaptic and presynaptic effects on acetylcholine release in the mammalian striatum.

Afferent Pathways↗

PubMed1993

Subcellular localization of prostaglandin endoperoxide synthase-2 in murine 3T3 cells.

Polyclonal antisera specific for prostaglandin endoperoxide (PGH) synthases-1 and -2 were used to determine the subcellular locations of each PGH synthase isozyme in detergent-permeabilized mouse 3T3 fibroblasts by indirect immunocytofluorescence. Antiserum to PGH synthase-1 demonstrated a mottled pattern of cytoplasmic and perinuclear staining of both serum-starved and serum-stimulated 3T3 cells. This pattern of staining is consistent with the results of earlier studies which demonstrated that PGH synthase-1 is associated with the endoplasmic reticulum and nuclear envelope of these cells. As expected, antibodies directed against a peptide unique to PGH synthase-2 failed to stain serum-starved cells, which lack appreciable levels of this second form of the enzyme. However, serum-stimulated 3T3 cells, which do express PGH synthase-2, showed the same pattern of staining with PGH synthase-2 antibodies as was observed with anti-PGH synthase-1 serum--mottled cytoplasmic staining and perinuclear staining. We conclude that the subcellular location of PGH synthase-2 is the same as PGH synthase-1 in murine 3T3 cells. Thus, the notable differences in the primary amino acid sequence--the signal peptide and the additional 18 amino acid C-terminal segment in PGH synthase-2--do not cause a change in localization. Colocalization of PGH synthases-1 and -2 implies that the source of arachidonate substrate, the site of PGH2 and prostanoid formation, and the mechanism of product transport from the inside to the outside of the cell are the same for these isozymes.

3T3 Cells↗

PubMed1993

Subcellular localization and antiviral activity of carminic acid/poly r(A-U) combinations.

Carminic acid (CAR) enhances the antiviral activity of poly r(A-U) twelve-fold without increasing interferon induction, inactivating the vesicular stomatitis virus or inducing host cell cytotoxicity. Phase contrast photomicrographs of human foreskin fibroblasts (HSF) incubated with CAR alone, poly r(A-U) alone or with a CAR/poly r(A-U) combination illustrate that the CAR/poly r(A-U) combinations display altered subcellular distribution with the CAR being localized in the nucleoli and chromatin. Phase contrast and fluorescence photomicrographs of adriamycin (ADR)-treated and ADR/poly r(A-U)-treated HSF cells corroborate these findings. These results suggest that modulation of one or more nucleolar processes may be responsible for the enhanced antiviral activity.

Antiviral Agents↗

PubMed1984

Subcellular localization of the PGE2 synthesis activity in mouse resident peritoneal macrophages.

The aim of this work was to establish, on a quantitative basis, the subcellular distribution of the enzyme system that converts arachidonic acid into prostaglandin (PG) E2 in mouse resident peritoneal (MRP) macrophages. Kinetic studies were conducted on cell-free extracts derived from cells cultivated for 1 d, using [1-14C]arachidonic acid as substrate and measuring the label in PGE2 after extraction and thin layer chromatography. The activity was synergistically enhanced by L-adrenaline and reduced glutathione, inhibited by indomethacin, and linearly related to the concentration of the cell-free extract. It was labile at 0 degrees C in the medium used for homogenization and fractionation of the cells (half-life less than 2 h). Addition of catalase (0.15 mg/ml) to the suspension medium increased the initial activity (by congruent to 70%) and the stability (half-life congruent to 6 h) of the enzyme in cytoplasmic extracts. It enabled us to establish the density distribution after isopycnic centrifugation in a linear gradient of sucrose. The sample centrifuged consisted of untreated cytoplasmic extracts, or cytoplasmic extracts treated with digitonin and Na pyrophosphate. Comparison of the centrifugation behavior of PGE2 synthesis activity with that of various enzymes used as reference for the major subcellular entities has revealed that PGE2 synthesis fairly fits the density profile of sulfatase C in each case. The conclusion is that at least the rate-limiting reaction in the conversion of arachidonic acid into PGE2 is catalyzed by an enzyme associated with the endoplasmic reticulum.

Animals↗

PubMed1987

NADPH oxidase of human neutrophils. Subcellular localization and characterization of an arachidonate-activatable superoxide-generating system.

The superoxide-forming NADPH oxidase of human neutrophils was studied in subcellular fractions of unstimulated cells. Purified neutrophils were disrupted by nitrogen cavitation and separated on Percoll density gradients into four fractions: alpha, azurophil granules; beta, mostly specific granules; gamma, plasma membrane, and cytosol. NADPH-dependent O2-. formation by these fractions was quantitated as the rate of superoxide dismutase-inhibitable reduction of ferricytochrome c. In the presence of cytosol, NADPH, and either arachidonic acid (optimum 90 microM) or sodium dodecyl sulfate (optimum 160 microM), 70-75% of the oxidase was in the beta fraction and about 25% was in the gamma fraction. A similar distribution was found for cytochrome b559 and FAD, two putative components of the oxidase. The reaction rates observed with arachidonic acid activation were sufficient to account for 25-75% of the O2-. generated by intact neutrophils. The properties of the beta and gamma enzymes were similar and closely resembled those of the oxidase in intact neutrophils or disrupted prestimulated cells. These included resistance to azide and cyanide, a pH optimum of 7.4, and a preference for NADPH (Km approximately 40-45 microM) rather than NADH (Km approximately 2.5 mM) as the electron donor. The combination of beta and gamma fractions displayed additive activity. The activatable oxidase required Mg2+ but not Ca2+. ATP was required for maximum reaction rates. When beta and gamma membranes were preincubated with cytosol and arachidonic acid in the presence of millimolar Mg2+ and then ultracentrifuged membrane-bound O2-. -forming activity was recovered in the pellet and the enzyme required only NADPH (i.e. no cytosol, arachidonic acid, or Mg2+) for expression of activity. These data suggest that cytosol contains a Mg2+-dependent oxidase-activating factor. Molecular sieve chromatography of cytosol indicated a single peak of activity (i.e. ability to activate O2-. generation by beta and/or gamma fraction) eluting with molecules of about 10,000 daltons.

Arachidonic Acid↗

PubMed1998

Subcellular localization of tyrosine hydroxylase (TH) gene transcripts: new insights into the pattern of TH gene expression in the locus coeruleus under pharmacological stimulation.

To document the subcellular compartmentalization of the tyrosine hydroxylase (TH) primary (heterogeneous nuclear, hnRNA) and processed mRNA transcripts in vivo, we have studied the expression of the TH gene in the locus coeruleus of the rat brain in basal conditions and under pharmacological stimulation by reserpine. Using exon-specific probes and electron microscopic in situ hybridization in reserpine-treated animals, we have demonstrated that the TH mRNA is localized both in the perikaryal cytoplasm (in association with domains of the endoplasmic reticulum) and in the nucleus (in large and smaller aggregates). By comparing hybridization patterns with intron- and exon-specific probes, we have shown that the two main foci of nuclear TH RNA labeling correspond to the primary (hnRNA) transcript, while accessory secondary tracks or dots represent mature transcript devoid of introns. Finally, our data indicate that the pattern of expression of the TH gene is heterogeneous within the locus coeruleus neuron population. Under basal conditions, many neurons exhibit no detectable intron signal, although all locus coeruleus neurones express the TH gene. Other neurons display high intron labeling, and obviously express the gene at a much higher level. Upon reserpine stimulation, the number of neurons displaying detectable intron signals, as well as the intensity of the intron signal per neuron progressively increases during the first 24 h, suggesting that TH expressing neurons are progressively recruited for higher expression of this gene as the stimulation progresses.

Animals↗

PubMedDate not supplied

Subcellular localization of specific mRNAs and their protein products in Purkinje cells by combined fluorescence in situ hybridization and immunocytochemistry.

In this study we have investigated the subcellular distribution of two mRNAs coding for the Purkinje cell-specific proteins, calbindin D28K and L7 (L7/pcp-2). Whereas calbindin mRNA was found to be in the cell body only, L7 transcripts could be detected within the molecular layer, corresponding to Purkinje cell dendrites. We have now combined a highly sensitive fluorescence-based in situ hybridization protocol with immunofluorescence in conjunction with confocal optical sectioning to analyze the precise localization of these mRNAs in individual Purkinje neurons. We show that L7 mRNA is localized in clusters within the proximal and distal branches of dendrites, but also in the proximal part of Purkinje cell axons. In contrast, calbindin transcripts are restricted to the axonal pole of the perikaryon. Purkinje cells grown in primary cultures reveal similar mRNA distribution patterns for the two transcripts. Thus, the mechanism underlying localization of mRNA within Purkinje cells seems to function in a cell-intrinsic manner, guiding specific transcripts, such as L7 mRNA, to neuronal processes while restricting others, such as calbindin mRNA, to the perikaryon.

Alkaline Phosphatase↗

PubMed2003

Subcellular localization of the voltage-dependent potassium channel Kv3.1b in postnatal and adult rat medial nucleus of the trapezoid body.

A pre-embedding immunocytochemical method was used to study the subcellular distribution of the voltage-dependent potassium channel Kv3.1b in the medial nucleus of the trapezoid body (MNTB) in developing and adult rat. The main finding was the localization of the channel in specific membrane compartments of the calyces of Held and principal globular neurons. Thus, at postnatal day (P) 9 immunoparticles were densely localized in plasma membranes of globular cell bodies and their main dendrites. At P16, a strong Kv3.1b labeling was still observed in these globular cell compartments, but the most remarkable feature was the presence of immunoparticles in synaptic terminal membranes of the calyces of Held. However, the presynaptic and postsynaptic specializations of the calyx of Held-globular cell synapses were virtually devoid of immunoparticles. This same subcellular distribution of Kv3.1b was seen in adult, with membranes of calycine terminals more uniformly labeled. The developmental profile of Kv3.1b expression in MNTB coincides with the functional maturation of the calyx of Held-principal globular neuron synapse. The presence of the channel in this system is crucial for the high-frequency synaptic transmission of auditory signals.

Aging↗

PubMed1999

Regulated expression and subcellular localization of syndecan heparan sulfate proteoglycans and the syndecan-binding protein CASK/LIN-2 during rat brain development.

The syndecan family of cell surface heparan sulfate proteoglycans interacts via their cytoplasmic C-terminal tail with the PDZ domain of CASK/LIN-2, a membrane-associated guanylate kinase homolog. The syndecan-CASK interaction may be involved in intercellular signaling and/or cell adhesion. Here we show that syndecan-1 to syndecan-4 have distinctive mRNA distributions in adult rat brain by in situ hybridization, with syndecan-2 and -3 being the major syndecans expressed in neurons of the forebrain. At the protein level, syndecan-2 and -3 are differentially localized within neurons; syndecan-3 is concentrated in axons, whereas syndecan-2 is localized in synapses. The synaptic accumulation of syndecan-2 occurs late in synapse development. CASK is a cytoplasmic-binding partner for syndecans, and its subcellular distribution changes strikingly during development, shifting from a primarily axonal distribution in the first 2 postnatal weeks to a somatodendritic distribution in adult brain. This change in CASK distribution correlates temporally and spatially with the expression patterns of syndecan-3 and -2, consistent with the association of both of these syndecans with CASK in vivo. In support of this, we were able to coimmunoprecipitate a complex of CASK and syndecan-3 from brain extracts. Our results indicate that specific syndecans are differentially expressed in various cell types of the brain and are targeted to distinct subcellular compartments in neurons, where they may serve specialized functions. Moreover, CASK is appropriately expressed and localized to interact with both syndecan-2 and -3 in different compartments of the neuron throughout postnatal development.

Aging↗

PubMed1996

Subcellular localization and analysis of apparent 180-kDa and 220-kDa proteins of the breast cancer susceptibility gene, BRCA1.

The breast cancer susceptibility gene BRCA1 encodes an 1863-amino acid protein that acts as a tumor suppressor. The biochemical function of BRCA1 is unknown, and there are conflicting results describing its subcellular location. We have identified a 220-kDa protein, which is reactive with three antibodies raised against the amino- and carboxyl-terminal regions of BRCA1. Immunoflourescence staining with an antibody to the carboxyl terminus of BRCA1 localized the protein to the nucleus of breast, ovarian, and cervical carcinoma-derived cell lines. A similar result was observed by biochemical subcellular fractionation that indicated that the 220-kDa protein was localized primarily to the nucleus of cell lines established from breast carcinomas. In addition to the 220-kDa protein, one antibody, C-20, also recognized a 180-kDa protein in MDA-MB-468 total cell lysates that was not detected by the other two antibodies. Several observations suggest the 180-kDa protein is the epidermal growth factor (EGF) receptor: (i) C-20 reacted avidly with a 180-kDa protein immunoprecipitated by an antibody to the EGF receptor; (ii) an EGF receptor antibody detected a 180-kDa protein immunoprecipitated by C-20; (iii) the affinity purified EGF receptor was both immunoprecipitated and detected on immunoblots by the C-20 antibody but not another BRCA1 antibody; (iv) similar phosphopeptide maps were generated from the EGF receptor and the 180-kDa protein immunoprecipitated by C-20, and this peptide map was distinct from the 220-kDa phosphoprotein; and (v) the C-20 immunizing peptide bears sequence identity to the EGF receptor. These results indicate that BRCA1 is a 220-kDa nuclear protein and that the 180-kDa protein reported previously may be unrelated to BRCA1.

Antibodies↗

PubMed1987

Subcellular localization of leucine aminotransferase and alpha-hydroxyacid dehydrogenase in Trypanosoma cruzi.

Homogenates of Trypanosoma cruzi epimastigotes (Tulahuén strain) show L-leucine aminotransferase activity (EC 2.6.1.6). Subcellular distribution of this enzyme and of alpha-hydroxyacid dehydrogenase, enzymes which share a common substrate/product (alpha-ketoisocaproate), has been studied by means of differential centrifugation, digitonin treatment of entire parasites, isopycnic centrifugation and determination of latency of enzymes in the large granule fraction. The results indicate that both enzymes have a dual localization, in the cytosol and in the mitochondrion, probably in the matrix. On the basis of this location, it is proposed that they operate in a shuttle system transferring reducing equivalents between the cytosol and the mitochondrion.

Alcohol Oxidoreductases↗

PubMed2000

Biochemical characterization and subcellular localization of the sterol C-24(28) reductase, erg4p, from the yeast saccharomyces cerevisiae.

The yeast ERG4 gene encodes sterol C-24(28) reductase which catalyzes the final step in the biosynthesis of ergosterol. Deletion of ERG4 resulted in a complete lack of ergosterol and accumulation of the precursor ergosta-5,7,22,24(28)-tetraen-3beta-ol. An erg4 mutant strain exhibited pleiotropic defects such as hypersensitivity to divalent cations and a number of drugs such as cycloheximide, miconazole, 4-nitroquinoline, fluconazole, and sodium dodecyl sulfate. Similar to erg6 mutants, erg4 mutants are sensitive to the Golgi-destabilizing drug brefeldin A. Enzyme activity measurements with isolated subcellular fractions revealed that Erg4p is localized to the endoplasmic reticulum. This view was confirmed in vivo by fluorescence microscopy of a strain expressing a functional fusion of Erg4p to enhanced green fluorescent protein. We conclude that ergosterol biosynthesis is completed in the endoplasmic reticulum, and the final product is supplied from there to its membranous destinations.

Mutagenesis↗

PubMed1984

Subcellular localization of proteins encoded by oncogenes of avian myeloblastosis virus and avian leukemia virus E26 and by chicken c-myb gene.

Analysis of the subcellular location of the proteins encoded by the oncogenes of avian myeloblastosis virus and avian leukemia virus E26 ( p45v -myb and p135gag -myb-ets, respectively) and by the chicken c-myb gene ( p75c -myb) shows that all three proteins are located in the nucleus. In AMV-infected (but not transformed) chicken fibroblasts p45v -myb also resides in the nucleus, indicating that a nuclear location of p45v -myb in these cells is not sufficient to achieve transformation. In AMV-transformed myeloblasts a small fraction of p45v -myb occupies an additional site in the perinuclear region of the cytoplasm. If the myeloblasts are caused to differentiate to macrophages, most of p45v -myb is found in the cytoplasm. This redistribution of p45v -myb within the cell may be responsible for reversion of the transformed phenotype.

Animals↗

PubMed1977

[Enzymatic character and subcellular localization of the transformation reaction of 18 hydroxycorticosterone to aldosterone].

The authors study in vitro the conversion of 18 hydroxycorticosterone into aldosterone in duck adrenal subcellular fractions. The enzymatic nature of this conversion is studied by classical enzymologic tests. All results are in favor of an enzymatic conversion and this enzyme is strictly located in the mitochondrial fraction. Results concerning this localisation are expressed as specific activity and relative specific activity. Preliminary results on further experimental data point out that this conversion depends on the energetic state of mitochondria. Complete electron transfer blokage inhibits this transformation whereas respiratory chain blokage with a NADPH generating system and ATP supplementation does not seem to produce any inhibition. Oligomycine and guanidine stimulation suggest that ATP or its precursors may be involved in this reaction.

18-Hydroxycorticosterone↗