PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “Subcellular localization”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,459 records · Page 81Linked to original sources
PubMed1995

Prostaglandin biosynthesis and subcellular localization of prostaglandin H synthase activity in the lone star tick, Amblyomma americanum.

We report on prostaglandin (PG) biosynthesis in the lone star tick, Amblyomma americanum. In vitro preparations of whole female ticks and internal tissues were competent to biosynthesize four PGs: PGA2/PGB2, PGD2, PGE2, PGF2 alpha. PGA2/PGB2 was the major product under optimal conditions. PG biosynthesis by whole tick and internal tissues were sensitive to incubation conditions including, protein concentration, time, temperature, pH, and presence of a co-factor cocktail composed of reduced glutathione, hydroquinone, and hemoglobin. Under standard assay conditions, 2 mg/ml protein were incubated at pH 8.0 for 2 min at 32 degrees C. PG biosynthesis was inhibited by indomethacin, a potent cyclooxygenase inhibitor in mammalian systems. Internal tissue preparations were fractionated into cytosolic and microsomal preparations by ultracentrifugation. PG biosynthetic activity was detected in both fractions. The subcellular distribution of PG biosynthetic activity in ticks is similar to other invertebrates, but quite different from mammals, in which PG biosynthetic activity is almost exclusively localized in the microsomal fractions. PGH synthase-2 was detected in the microsomal fraction on western blot analysis. These results suggest that the lone star tick is competent to biosynthesize PGs. These compounds may contribute to the success of tick feeding ecology by attenuating the defense responses of vertebrate hosts during lengthy feeding periods.

Animals↗
PubMed1991

Uptake and subcellular localization of tritiated dihydro-microcystin-LR in rat liver.

Microcystin-LR (MC-LR), a cyclic heptapeptide hepatotoxin (mol. wt = 994) produced by the blue-green alga (cyanobacterium), Microcystis aeruginosa, was reduced with tritium labeled sodium borohydride, converted to [3H]-dihydro-microcystin-LR ( [3H]-2HMC-LR), and purified to greater than 99% purity by C-18 reverse-phase high-performance liquid chromatography. The uptake and subcellular distribution of [3H]-2HMC-LR were determined in suspensions of hepatocytes at 0 degrees C and 37 degrees C, or following rifampicin pretreatment, and in perfused rat liver. The remaining cells were homogenized and subfractionated using sucrose gradient centrifugation. Suspensions of 7.5 x 10(6) hepatocytes also were incubated with 10 micrograms/ml of toxin, solubilized in Triton X-100, and ultracentrifuged to pellet the detergent insoluble fraction (containing actin). Isolated rat livers were perfused with media containing [3H]-2HMC-LR and the uptake of radiolabel was determined. Sequential biopsy samples were collected for histologic examination. The remaining liver was homogenized and subcellular fractions prepared. Uptake of radiolabel was rapid in both cell suspension at 37 degrees C and perfused liver; however, uptake in cell suspensions was reduced by about 50% at 0 degrees C and by rifampicin (50 micrograms/ml) pretreatment. Hepatocyte necrosis was observed in isolated perfused livers 45 min after initiation of perfusion with [3H]-2HMC-LR. In both hepatocyte suspensions and perfused livers 65 to 77% of the radiolabel was in the cytosolic fraction. In the hepatocyte suspensions, 13 to 18% of the radiolabel was present in the plasma membrane/nuclear fraction with lesser amounts in the other fractions. Trichloroacetic acid treatment of cytosolic fractions indicated that in hepatocyte suspensions, 50-60% of the radiolabel was bound to cytosolic protein. Studies using the perfused liver confirmed that the majority of the radiolabeled MCLR (78-88%) was bound to cytosolic protein. These data suggest that the uptake of [3H]-2HMC-LR occurs primarily by an energy-dependent transport process involving the rifampicin-sensitive hepatic bile acid carrier and that once inside the hepatocyte, the toxin binds to a cytosolic protein(s).

Animals↗
PubMed1985

Subcellular localization of creatine kinase in Torpedo electrocytes: association with acetylcholine receptor-rich membranes.

Creatine kinase (CK, EC 2.7.3.2) has recently been identified as the intermediate isoelectric point species (pl 6.5-6.8) of the Mr 40,000-43,000 nonreceptor, peripheral v-proteins in Torpedo marmorata acetylcholine receptor-rich membranes (Barrantes, F. J., G. Mieskes, and T. Wallimann, 1983, Proc. Natl. Acad. Sci. USA, 80: 5440-5444). In the present study, this finding is substantiated at the cellular and subcellular level of the T. marmorata electric organ by immunofluorescence and by protein A-gold labeling of either ultrathin cryosections of electrocytes or purified receptor-membrane vesicles that use subunit-specific anti-chicken creatine kinase antibodies. The muscle form of the kinase, on the one hand, is present throughout the entire T. marmorata electrocyte except in the nuclei. The brain form of the kinase, on the other hand, is predominantly located on the ventral, innervated face of the electrocyte, where it is closely associated with both surfaces of the postsynaptic membrane, and secondarily in the synaptic vesicles at the presynaptic terminal. Labeling of the noninnervated dorsal membrane is observed at the invaginated sac system. In the case of purified acetylcholine receptor-rich membranes, antibodies specific for chicken B-CK label only one face of the isolated vesicles. No immunoreaction is observed with anti-chicken M-CK antibodies. A discussion follows on the possible implications of these localizations of creatine kinase in connection with the function of the acetylcholine receptor at the postsynaptic membrane, the Na/K ATPase at the dorsal electrocyte membrane, and the ATP-dependent transmitter release at the nerve ending.

Animals↗
PubMed2006

Fluorescence protease protection of GFP chimeras to reveal protein topology and subcellular localization.

Understanding the cell biology of many proteins requires knowledge of their in vivo topological distribution. Here we describe a new fluorescence-based technique, fluorescence protease protection (FPP), for investigating the topology of proteins and for localizing protein subpopulations within the complex environment of the living cell. In the FPP assay, adapted from biochemical protease protection assays, GFP fusion proteins are used as noninvasive tools to obtain details of protein topology and localization within living cells in a rapid and straightforward manner. To demonstrate the broad applicability of FPP, we used the technique to define the topology of proteins localized to a wide range of organelles including the endoplasmic reticulum (ER), Golgi apparatus, mitochondria, peroxisomes and autophagosomes. The success of the FPP assay in characterizing the topology of the tested proteins within their appropriate compartments suggests this technique has wide applicability in studying protein topology and localization within the cell.

Animals↗
PubMed2005

Tissue distribution, subcellular localization and endocrine disruption patterns induced by Cr and Mn in the crab Ucides cordatus.

The essential trace elements Cr and Mn are toxic at high concentrations and information about low concentration is insufficient in the literature. In polluted mangroves, the crab Ucides cordatus can represent a useful tool to assess information on the potential impact of trace elements like Cr and Mn on the environment, since this species is comestible and thus, commercially negotiated. Therefore, U. cordatus crabs were exposed in vivo to different concentrations of Cr and Mn solved in seawater and had their tissue distribution and subcellular deposits evaluated. The gill, hepatopancreas and muscle concentrations were determined by atomic absorption spectroscopy and the results showed that Cr and Mn presented the highest values in the gills rather than in the hepatopancreas and muscular tissue. Electron microscopy and analytical X-ray microanalysis revealed Cr precipitates on the gill surface, co-localized with epiphyte bacteria. In addition, since Cr and Mn did not equally accumulate in most of the tissues studied, glycemic rate of animals, which received injections of extracts of eyestalks of the contaminated crabs, were measured in order to evaluate whether the studied concentrations of Cr and Mn could produce any metabolic alteration. The results indicated that extracts of the eyestalks of crabs submitted to Cr and Mn salts and injected into normal crabs markedly influenced crustacean hyperglycemic hormone synthesis and/or release. The results are discussed with respect to sensitivity of the employed methods and the possible significance of the concentrations of Cr and Mn in the organisms.

Analysis of Variance↗
PubMed1994

Subcellular localization of Clostridium thermocellum ORF3p, a protein carrying a receptor for the docking sequence borne by the catalytic components of the cellulosome.

The ORF3 gene of Clostridium thermocellum encodes a polypeptide (ORF3p) which contains a receptor domain for the docking sequence borne by the catalytic subunits of the cellulosome and a triplicated domain related to some bacterial cell surface proteins. It was thus surmised that ORF3p is a surface protein. In this study, this hypothesis was confirmed. Subcellular fractionation, Western blotting (immunoblotting), and electron microscopy of immunocytochemically labeled cells indicated that ORF3p produced by C. thermocellum was located in the outer surface layer of the bacterium. This layer appeared to consist of a soft matrix shedding off particulate fragments. Nonsedimenting ORF3p derived from sonicated cells was associated with high-molecular-mass fractions (> 20 MDa), probably corresponding to fragments of the outer cell layer. The same high-molecular-mass fractions also contained the cellulosomal marker CipA. Contrary to CipA, however, ORF3p was not associated with 2- to 4-MDa fractions corresponding to individual cellulosomes, and a significant fraction of ORF3p failed to bind to cellulose. It is proposed that ORF3 and ORF3p be renamed olpA and OlpA, respectively (for outer layer protein).

Antibodies, Bacterial↗
PubMed1993

Multiple defects in the adipocyte glucose transport system cause cellular insulin resistance in gestational diabetes. Heterogeneity in the number and a novel abnormality in subcellular localization of GLUT4 glucose transporters.

Mechanisms causing cellular insulin resistance in gestational diabetes mellitus are not known. We, therefore, studied isolated omental adipocytes obtained during elective cesarean sections in nondiabetic (control) and GDM gravidas. Cellular insulin resistance was attributed to impaired stimulation of glucose transport; compared with control subjects, basal and maximally insulin-stimulated transport rates (per surface area) were reduced 38 and 60% in GDM patients, respectively. To determine underlying mechanisms, we assessed the number, subcellular distribution, and translocation of GLUT4, the predominant insulin-responsive glucose transporter isoform. The cellular content of GLUT4 was decreased by 44% in GDM patients as assessed by immunoblot analysis of total postnuclear membranes. However, GDM patients segregated into two subgroups; half expected profound (76%) cellular depletion of GLUT4 and half had GLUT4 levels in the normal range. Cellular GLUT4 was negatively correlated with adipocyte size in the control subjects and GDM patients with normal GLUT4 (r = 0.60), but fell way below this continuum in GDM patients with low GLUT4, indicating that heterogeneity was not caused by differences in obesity. All GDM. distribution. In basal cells, increased amounts of GLUT4 were detected in membranes fractionating with (such that the plasma membrane GLUT4 level in GDM (such that the plasma membrane GLUT4 level in GDM patients was equal to that observed in insulin-stimulated cells from control subjects). Furthermore, insulin stimulation induced translocation of GLUT4 from low-density microsomes to plasma membranes in control subjects but did not alter subcellular distribution in GDM patients. In other experiments, cellular content of GLUT1 was normal in GDM patients, and GLUT1 did not undergo insulin-mediated recruitment to plasma membranes in either control subjects or GDM patients. A faint signal was detected for GLUT3 only in low-density microsomes and only with one of two different antibodies. In GDM, we conclude that insulin resistance in adipocytes involves impaired stimulation of glucose transport and arises from a heterogeneity of defects intrinsic to the glucose transport effector system. GLUT4 content in adipocytes is profoundly depleted in approximately 50% of GDM patients, whereas all patients are found to exhibit a novel abnormality in GLUT4 subcellular distribution. This latter defect is characterized by accumulation of GLUT4 in membranes cofractionating with plasma membranes and high-density microsomes in basal cells and absence of translocation in response to insulin. The data suggest that abnormalities in cellular traffic or targeting relegate GLUT4 to a membrane compartment from which insulin cannot recruit transporters to the cell surface and have important implications regarding skeletal muscle insulin resistance in GDM and NIDDM.

Adipocytes↗