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At least 217 records · Page 12Linked to original sources

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↗

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↗

PubMed1982

Studies on the kinetic properties and subcellular localization of adenine nucleotide phosphatases in peripheral blood lymphocytes from control subjects and patients with common variable primary hypogammaglobulinaemia.

Specific assays for 5'-nucleotidase, adenosine diphosphatase (ADPase) and Mg2+-dependent adenosine triphosphatase (Mg2+-ATPase) have been optimized for human lymphocytes and their subcellular localizations determined by sucrose density gradient centrifugation. 5'-Nucleotidase was localized solely to the plasma membrane of the lymphocyte. ADPase activity has been shown to have a dual localization to the plasma membrane and mitochondria, whilst Mg2+-ATPase was mainly located in the mitochondria. No [Na+,K+] activated Mg2+-dependent ATPase could be measured in these cells. We have confirmed the striking decrease in the specific activity of 5'-nucleotidase activity in lymphocytes from patients with common variable primary hypogammaglobulinaemia. In contrast, the specific activities of ADPase and Mg2+-ATPase showed no alteration in lymphocytes from the patient group when compared to controls. Thus the deficiency of ecto-5'-nucleotidase in the lymphocytes of patients with hypogammaglobulinaemia is a highly selective defect in purine metabolism.

5'-Nucleotidase↗

PubMed1995

Subcellular localization of gadolinium injected as soluble salt in rats: a microanalytical study.

The rare earth gadolinium (Gd) is used in modern industry. Solubilized DTPA Gd and DOTA Gd complexes are used as contrast media in nuclear magnetic resonance imaging. In order to determine the subcellular localization of Gd, rats were injected intraperitoneally with Gd nitrate. Two microanalytic methods, ion microanalysis and electron microprobe, enabled the distribution and the intracellular localization of Gd to be determined in the liver, spleen, bone marrow, kidneys and lung. The results showed: a) a punctual distribution of Gd in the tissues (liver, spleen, bone marrow and lung) as observed by ion microscopy; b) a selective concentration of Gd in the lysosomes of macrophages of the liver (hepatocytes), spleen (macrophages), bone marrow (macrophages) and lung (phagocyte cells), as determined by electron probe X-ray microanalysis. In all these sites the Gd is associated to phosphorus. Results are compared to those found for other rare earths and metal elements.

Animals↗

PubMed1990

Developmental changes of synapsin I subcellular localization in rat cerebellar neurons.

Synapsin I, one of the major synaptic proteins, is thought to associate with synaptic vesicles and to play a regulatory role in neurotransmitter release. In mature neurons, it is concentrated almost exclusively in presynaptic nerve endings. Here, we studied the subcellular localization of synapsin I during the development of rat cerebellar cortices by immunocytochemistry, using anti-synapsin I antibodies and found that during the development of rat cerebellar cortices it tentatively exists in the dendritic growth cones of immature internal granule cells and in the axonal growth cones of mossy fibers as well as mature presynaptic endings. Also, we found that synapsin I, in the axonal and dendritic growth cones does not necessarily associate with vesicles, but rather with fuzzy filamentous structures in the cytoplasm. In search of the structure of synapsin I in vivo, we employed the quick-freeze, deep-etch technique after immunogold labeling. Synapsin I seems to thereby connect synaptic vesicles or anchor them to cytoskeletons in presynaptic endings.

Animals↗

PubMed1987

Influence of conversion of penicillin G into a basic derivative on its accumulation and subcellular localization in cultured macrophages.

beta-Lactam antibiotics do not accumulate in phagocytes, probably because of their acidic character. We therefore synthesized a basic derivative of penicillin G, namely, 14C-labeled N-(3-dimethylamino-propyl)benzylpenicillinamide (ABP), and studied its uptake and subcellular localization in J774 macrophages compared with that of 14C-labeled penicillin G. Whereas the intracellular concentration (Ci) of penicillin G remained lower than its extracellular concentration (Ce), ABP reached a Ci/Ce ratio of 4 to 5. Moreover, approximately 50% of intracellular ABP was found associated with lysosomes after isopycnic centrifugation of cell homogenates in isoosmotic Percoll or hyperosmotic sucrose gradients. The behavior of ABP was thus partly consistent with the model of de Duve et al. (C. de Duve, T. de Barsy, B. Poole, A. Trovet, P. Tulkens, and A. Van Hoof, Biochem. Pharmacol. 23:2495-2531, 1974), in which they described the intralysosomal accumulation of weak organic bases in lysosomes. Although ABP is microbiologically inactive, our results show that beta-lactam antibiotics can be driven into cells by appropriate modification. Further efforts therefore may be warranted in the design of active compounds or prodrugs that may prove useful in the chemotherapy of intracellular infections.

Animals↗

PubMed1996

Granulocytic protein p25 is a DNA-binding subunit of protein M(r) = 50,000: subcellular localization, cell and species specificity.

We have previously reported the presence and isolation of the novel protein M(r) = 25,000 (p25) from human granulocytes. In this study, the protein p25 was characterized by its: (a) ability to bind DNA, (b) subunit association, (c) partial protein sequencing, (d) subcellular localization, (e) cellular and species specificity and (f) stability in the presence of released granulocytic proteinases. For the detection of p25 in various extracts, fractions and types of human or animal hematopoietic cells, SDS-PAGE/Western blotting and immunohistochemical staining were used. The protein p25 was subjected to N-terminal amino acid sequence analysis. Protein p25-DNA interactions were monitored using Southwestern blotting. Selective inhibition of granulocytic proteinases was performed. Granulocytic protein p25 was found to be a product of oxidative cleavage of disulfide bridges in the p50 dimer. It was shown that neither protein p50 nor the p25 subunit is a degradation product of a protein of higher molecular weight. The N-terminal amino acid sequence of p25 was: RLNYNKPHAA. Binding capacity for double stranded DNA without significant sequence specificity was revealed and nuclear localization of some fraction of p50 dimer was established. The data concerning the cell and species specificity demonstrated that the protein is expressed only in normal human granulocytes. In summary, protein p25 originates from splitting of the p50 dimer. This subunit shows no identity with proteins already sequenced. DNA-binding of p25 is not sequence specific. It is concluded that the protein p50 is localized in the nuclei and cytoplasmic granules of mature human polymorphonuclear leukocytes or granulocytes of species high on the evolutionary tree. The functions of this protein remain to be determined.

Amino Acid Sequence↗

PubMed1993

Platelet multielemental composition, lability, and subcellular localization.

Diagnostic X-ray spectrometry (DXS), based on X-ray fluorescence, was used to quantitate directly the multiple elemental composition of washed, intact human platelets (n = 16), with the following results: K = 3.08 +/- 1.00 mg/g, Ca = 1.18 +/- 0.29 mg/g, Zn = 35 +/- 9 micrograms/g. These values show that washed platelets contain significant pools of K, Ca, and Zn, the latter some 30-60-fold higher than plasma levels. Dialysis of whole platelets against cation exchange resin (Chelex-100) did not extract Ca(II) and Zn(II) sequestered within whole cells. To identify the subcellular locale of the elements, platelet lysate was subjected to 30-70% sucrose gradient ultracentrifugation and subcellular enriched fractions were obtained. Fractions were analyzed by DXS (for elements), electron microscopy (for dense granules), and subcellular markers fibrinogen and von Willebrand factor. In contrast to Ca and K, which accumulate in the dense granules and the cytoplasm, respectively, Zn appears to be distributed in the alpha-granules (40%) and the cytoplasm (60%). The subcellular distribution of Zn(II) is discussed within the context of the sensitivity of platelet response to the availability of Zn(II) and the platelet release reactions following stimulation.

Absorption↗

PubMed1995

Cell cycle-related shifts in subcellular localization of BCR: association with mitotic chromosomes and with heterochromatin.

The disruption of the BCR gene and its juxtaposition to and consequent activation of the ABL gene has been implicated as the critical molecular defect in Philadelphia chromosome-positive leukemias. The normal BCR protein is a multifunctional molecule with domains that suggest its participation in phosphokinase and GTP-binding pathways. Taken together with its localization to the cytoplasm of uncycled cells, it is therefore presumed to be involved in cytoplasmic signaling. By performing a double aphidicolin block for cell cycle synchronization, we currently demonstrate that the subcellular localization of BCR shifts from being largely cytoplasmic in interphase cells to being predominantly perichromosomal in mitosis. Furthermore, with the use of immunogold labeling and electron microscopy, association of BCR with DNA, in particular heterochromatin, can be demonstrated even in quiescent cells. Results were similar in cell lines of lymphoid or myeloid origin. These observations suggest a role for BCR in the phosphokinase interactions linked to condensed chromatin, a network previously implicated in cell cycle regulation.

Cell Compartmentation↗

PubMed1991

Domains of the human androgen receptor involved in steroid binding, transcriptional activation, and subcellular localization.

A series of human androgen receptor (AR) deletion mutants was constructed to study the relationship between the structural domains and their different functions in the AR protein. Human AR mutants were expressed in COS-1 and HeLa cells to investigate hormone binding, transcriptional activation, and subcellular localization. The wild-type human AR (AR 1-910) was expressed as a 110- to 112-kDa doublet, as revealed on immunoblots. All mutant AR proteins also migrated as doublets, except for one. This AR has a deletion from amino acid residues 51-211 and migrated as a single protein band, possibly due to altered posttranslational modification. The AR steroid-binding domain is encoded by approximately 250 amino acid residues in the C-terminal end. Deletions in this domain as well as truncation of the last 12 C-terminal amino acid residues abolished hormone binding. Cotransfection studies in HeLa cells showed that transcriptional activation of an androgen-regulated reporter gene construct was induced by the wild-type human AR. Mutational analysis revealed two regions in the N-terminal part, encoded by amino acid residues 51-211 and 244-360, to be essential for this transcriptional activation. Deletion of the hormone-binding domain yielded a constitutively active AR protein, indicating that in the absence of hormone this domain displays an inhibitory function. In the presence of its ligand, the wild-type AR was located in the cell nucleus. In the absence of androgens the receptor was mainly nuclear, but cytoplasmic localization was observed as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization.

Proclotting enzyme is an intracellular serine protease zymogen closely associated with an endotoxin-sensitive hemolymph coagulation system in limulus. Its active form, clotting enzyme, catalyzes conversion of coagulogen to insoluble coagulin gel. We present here the cDNA and amino acid sequences, disulfide locations, and subcellular localization of proclotting enzyme. The isolated cDNA for proclotting enzyme consists of 1,501 base pairs. The open reading frame of 1,125 base pairs encodes a sequence comprising 29 amino acid residues of prepro-sequence and 346 residues of the mature protein with a molecular mass of 38,194 Da. Three potential glycosylation sites for N-linked carbohydrate chains were confirmed to be glycosylated. Moreover, the zymogen contains six O-linked carbohydrate chains in the amino-terminal light chain generated after activation. The cleavage site that accompanies activation catalyzed by trypsin-like active factor B, proved to be an Arg-Ile bond. The resulting carboxyl-terminal heavy chain is composed of a typical serine protease domain, with a sequence similar to that of human coagulation factor XIa (34.5%) or factor Xa (34.1%). The light chain has a unique disulfide-knotted domain which shows no significant homology with any other known proteins. Thus, this proclotting enzyme has a mammalian serine protease domain and a structural domain not heretofore identified in coagulation and complement factors. Immunohistochemical studies showed that the proclotting enzyme is localized in large granules of hemocytes.

Amino Acid Sequence↗

PubMed1996

Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast.

The green fluorescent protein (GFP) from the jellyfish Aequorea victoria has attracted much attention as a tool to study a number of biological processes. This study describes the use of GFP as a vital reporter molecule for localization and expression studies in Saccharomyces cerevisiae. Construction of GFP expression vectors which allow N- or C-terminal fusion of the gfp gene to a gene of interest allowed the generation of fusion proteins whose subcellular localization was followed by fluorescence microscopy in living yeast cells. Analysis of three unknown open reading frames obtained from the budding yeast chromosome XIV resulted in distinct staining patterns, allowing prediction of the cellular localization of these unknown proteins. Furthermore, GFP was used to construct a gene replacement cassette which, after homologous integration into the genomic locus, placed the gfp gene behind a promoter of interest. The amount of GFP produced from this promoter was then quantified in living yeast cells by flow cytometry. With this novel replacement cassette a gene of interest can be deleted and at the same time its expression level studied under various growth conditions. The experiments presented here suggest that GFP represents a convenient fluorescent marker for localization studies as well as gene expression studies in budding yeast. Systematic studies of a large number of genes should benefit from such assays.

Animals↗

PubMed1991

Subcellular localization of secretogranin II and synaptophysin by immunoelectron microscopy in differentiated hypothalamic neurons in culture.

Secretogranin II (SgII), a tyrosine-sulfated secretory protein, is a widespread component of endocrine and neuronal cells. In the present study we used mouse hypothalamic neurons differentiated in culture and studied the subcellular localization of SgII by two methods, i.e., by the use of immunoperoxidase or immunogold electron microscopy. By immunoperoxidase labeling, SgII was mainly detected in the matrix of large dense-core vesicles (LDCVs). In addition, usually in nerve terminals containing LDCVs, peroxidase reaction product was also found in association with the membrane of small synaptic vesicles (SSVs). By immunogold labeling, SgII was detected only in the matrix of LDCVs. We also compared the localization of SgII and synaptophysin (SY), an integral membrane protein of SSVs, by double labeling, using a combination of pre-embedding immunogold and -peroxidase techniques for SgII and SY, respectively. In perikarya, SgII-positive LDCVs were observed in the vicinity of the Golgi complex and scattered in the cytoplasm. In contrast, SY labeling was restricted to electron-translucent vesicles and tubular membranes in the Golgi area. Moreover, membrane structures positive for both SgII and SY were not found either in the Golgi zone or in other regions of the cytoplasm. In synaptic boutons, immunolabeling of LDCVs and SSVs with anti-SgII and anti-SY, respectively, was mutually exclusive. In summary, within the limitation of the methods used, our data are consistent with the notion that SgII and SY are segregated from each other on exit from the trans-Golgi network, than follow two distinct membrane traffic pathways, and that the presence of SgII on the membrane of some SSVs is due to endocytosis.

Animals↗

PubMed1995

The two isoforms of mouse terminal deoxynucleotidyl transferase differ in both the ability to add N regions and subcellular localization.

Two alternatively spliced terminal deoxynucleotidyl transferase transcripts, TdTS and TdTL which code respectively for proteins of 509 and 529 amino acids have been previously identified in the mouse thymus. Here we show that the same two transcripts are also present in B lineage cells from bone marrow. In addition we demonstrate that the corresponding 20 amino acid insertion found near the carboxy-terminal end of TdTL significantly alters the function of the enzyme. In contrast to TdTS, TdTL does not catalyse N region insertions at the recombination junction of a V(D)J site-specific recombination substrate. In an attempt to explain the lack of N region insertions we have characterized the different parameters which distinguish the two isoforms of TdT. Examination of transfected cell extracts revealed a reduced capacity of TdTL to add nucleotides to the 3' end of DNA, consistent with a lower terminal transferase activity. Furthermore, the half-life of the TdTL protein in these cells is 2-fold shorter than that of TdTS. Finally, despite the fact that TdTL has the same nuclear localization signal as TdTS, the cellular localization of the two isoforms was strikingly different. In contrast to nuclear TdTS, TdTL was found exclusively in the cytoplasm. All these characteristics could contribute to the functional difference between the two isoforms of TdT. However, the subcellular localization of TdTL on its own can account for its inability to add N regions.

Alternative Splicing↗

PubMed1995

Induction and subcellular localization of two major stress proteins in response to copper in the fathead minnow Pimephales promelas.

In the present study we characterize the stress response induced by copper in the fathead minnow, Pimephales promelas. The fathead minnow epithelial cell line ATCC CCL 42 was used to examine the induced synthesis and subcellular localization of the two major stress proteins, stress 70 and cpn60. Western blot analysis demonstrated increased stress70 in cells exposed to 400 and 500 microM Cu. Two-dimensional analysis revealed three isoforms of stress70, one of 70 kDa and two of 72 kDa, at the highest Cu concentration. Chaperonin60 abundance did not change over the same range of Cu concentrations. Indirect immunofluorescence microscopy revealed that stress70 localized in the cytoplasm, particularly in the paranuclear region. Chaperonin60 was localized in mitochondria. Further, when we examined the stress response elicited by Cu in fathead minnow larvae in vivo, we found that Cu induced the stress response at nominal Cu concentrations that were more than an order of magnitude lower that in the cell culture. This disparity between the concentration of Cu, which induced the stress response in cells in culture and in vivo, may be the result of differences in Cu complexation that alter its availability, uptake and toxicity.

Animals↗

PubMed1991

Subcellular localization, partial purification, and characterization of a dynorphin processing endopeptidase from bovine pituitary.

An enzyme capable of cleaving dynorphin B-29 to dynorphin B-13 is present in bovine pituitary, with 40- to 50-fold higher specific activity in the posterior and intermediate lobes than in the anterior lobe. Subcellular fractionation of bovine neurointermediate pituitary shows that this enzyme is present in the peptide-containing secretory vesicles. The enzyme has been purified 2,800-fold from whole bovine pituitaries using ion-exchange and gel filtration chromatography. Purified dynorphin-converting enzyme has a neutral pH optimum, and is subsantially inhibited by the thiol-protease inhibitor p-chloromercuriphenylsulfonic acid, but not by serine or metalloprotease inhibitors. The purified enzyme processes dynorphin B-29 at Arg14, producing both dynorphin B-14 and dynorphin B-13 in a 5:1 ratio. No other cleavages are observed, suggesting that the activity is free from other proteases and is specific for single Arg sequences. Purified enzyme also processes dynorphin A-17 at the single Arg cleavage site, generating both dynorphin A-8 and A-9 in a 7:1 ratio. The tissue distribution, subcellular localization, and substrate specificity of this enzyme are consistent with a physiological role in the processing of dynorphin B-29 and dynorphin A-17, and possibly other peptides, at single Arg residues.

4-Chloromercuribenzenesulfonate↗

PubMed1989

Subcellular localization of the nodD gene product in Rhizobium leguminosarum.

In Rhizobium strains the transcription of symbiosis plasmid-localized nod genes, except nodD, is induced by plant flavonoids and requires the nodD gene product. In order to localize NodD protein in R. leguminosarum, a NodD protein-specific antiserum was raised against a lacZ'-'nodD gene fusion product. Using these antibodies, we determined that the NodD protein is located exclusively in the cytoplasmic membrane of wild-type R. leguminosarum biovar viciae cells. This localization is independent of the presence of inducers. In a Rhizobium strain that overproduced the NodD protein, the protein was present both in the cytoplasmic membrane and the cytosol, indicating an influence of the protein abundance on its ultimate subcellular localization. It was estimated that 20 to 80 molecules of NodD protein were present per wild-type Rhizobium cell. A model which combines the localization and the DNA-binding properties of the NodD protein as well as the observed association of flavonoids with the cytoplasmic membrane is discussed.

Bacterial Proteins↗