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At least 595 records · Page 33Linked to original sources
PubMed2003

The PmSOD1 gene of the protistan parasite Perkinsus marinus complements the sod2Delta mutant of Saccharomyces cerevisiae, and directs an iron superoxide dismutase to mitochondria.

The facultative intracellular oyster parasite, Perkinsus marinus, taxonomically related to both dinoflagellates and apicomplexan parasites, possesses at least two distinct genes (PmSOD1 and PmSOD2) predicted to encode iron-containing superoxide dismutases (Fe-SOD). The present study demonstrates that PmSOD1 complements a Saccharomyces cerevisiae mutant lacking the mitochondrial manganese-containing SOD (Mn-SOD), whereas PmSOD2 complements an Escherichia coli mutant lacking genes for cytosolic SOD activities. Mitochondria isolated from complemented yeast contain an SOD activity susceptible to inhibition by hydrogen peroxide, but resistant to cyanide, both characteristics of Fe-SODs. In cultured P. marinus trophozoites, indirect immunofluorescence using anti-PmSOD1 antibodies shows colocalization of PmSOD1 product with the mitochondrial marker MitoTracker Red. Further analysis of the leader sequence of the predicted PmSOD1 product revealed similarities to a mitochondrial targeting domain, an unusual observation for Fe-SODs, which are typically localized in the cytoplasm. These results suggest that PmSOD1 encodes a mitochondrial Fe-SOD, which may contribute to P. marinus resistance to exogenous oxidative damage in host phagocytes. The present study constitutes the first report of an endogenous Fe-SOD that is directed to the mitochondria, and suggests that mitochondria targeting sequences have been conserved among diverse branches of the eukaryotes, including the early protista. It also illustrates the potential of complementation-based approaches for further gene discovery and characterization in P. marinus.

Amino Acid Sequence↗
PubMed1999

Characterization of a novel nucleolar protein that transiently associates with the condensed chromosomes in mitotic cells.

We report the isolation and characterization of a murine gene encoding a conserved mammalian nucleolar protein. The protein, called Tsg118, has a predicted molecular mass of 59.4 kDa and a high content of basic amino acids. A homologous human gene was localized to chromosome 16p12.3. The Tsg118 protein is predominantly expressed in proliferating somatic cells and in male germ cells. Indirect immunofluorescence microscopy analysis using an affinity-purified anti-Tsg118 serum shows colocalization of Tsg118 and a known nucleolar protein, fibrillarin, to the dense fibrillar component of the nucleolus. The nucleolar localization of the Tsg118 protein appears to be temporally restricted to the interphase stages of the somatic cell cycle and to the meiotic phase of spermatogenesis. We find that the Tsg118 protein localizes to the nucleolus in both proliferating and serum-starved cells. Interestingly, as the nucleolar signal disappears in mitotic cells, the Tsg118 protein instead becomes associated with the surface of the condensed chromosomes.

Amino Acid Sequence↗
PubMed2003

The human Sir2 ortholog, SIRT2, is an NAD+-dependent tubulin deacetylase.

The silent information regulator 2 protein (Sir2p) of Saccharomyces cerevisiae is an NAD-dependent histone deacetylase that plays a critical role in transcriptional silencing. Here, we report that a human ortholog of Sir2p, sirtuin type 2 (SIRT2), is a predominantly cytoplasmic protein that colocalizes with microtubules. SIRT2 deacetylates lysine-40 of alpha-tubulin both in vitro and in vivo. Knockdown of SIRT2 via siRNA results in tubulin hyperacetylation. SIRT2 colocalizes and interacts in vivo with HDAC6, another tubulin deacetylase. Enzymatic analysis of recombinant SIRT2 in comparison to a yeast homolog of Sir2 protein (Hst2p) shows a striking preference of SIRT2 for acetylated tubulin peptide as a substrate relative to acetylated histone H3 peptide. These observations establish SIRT2 as a bona fide tubulin deacetylase.

Cell Line↗
PubMed2002

Promyelocytic leukemia protein PML inhibits Nur77-mediated transcription through specific functional interactions.

The promyelocytic leukemia protein PML is a tumor and growth suppressor and plays an important role in a multiple pathways of apoptosis and regulation of cell cycle progression. Our previous studies and others also documented a role of PML in transcriptional regulation through its association with transcription coactivator CBP and transcription corepressor HDAC. Here, we showed that PML is a potent transcriptional repressor of Nur77, an orphan receptor and a member of the steroid receptor superfamily of proteins. We found that PML represses Nur77-mediated transactivation through a physical and functional interaction between the two proteins. PML interacts with Nur-77 in vitro in a GST-pull down assay and in vivo by coimmunoprecipitation assay. PML/Nur77 colocalized in vivo in a double immunofluorescent staining and confocal microscopic analysis. Our study further showed that the coiled-coil domain of PML interacts with the DNA-binding domain of Nur77 (amino acids 267-332). Electrophoretic mobility shift assay demonstrated that PML interferes with Nur77 DNA binding in a dose-dependent manner. This study indicates that PML interacts with the DNA-binding domain of Nur77 and represses transcription by preventing it from binding to the target promoter. This study supports a role of PML/Nur77 interaction in regulating cell growth and apoptosis.

Apoptosis↗
PubMed2001

Evidence for distinct membrane traffic pathways to melanosomes and lysosomes in melanocytes.

We report here morphologic and biochemical evidence that melanosomes are distinct from lysosomes. Immunofluorescence analysis revealed that TRP-1, a melanosomal membrane protein, did not colocalize with lysosomal membrane proteins LAMP1 and LGP85 in melan-a cells. Wortmannin treatment of melanocytes enhanced the distinct compartmentalization of these melanosomal/lysosomal membrane proteins by the swelling of the endosomal-lysosomal systems. The heavily melanized melanosomes did not have an altered shape, which suggests a lesser degree of membrane dynamics of stage IV melanosomes. Terminal lysosomes loaded with TR-dextran are also distinct from melanosomes, thus indicating that melanosomes are isolated from the endocytic pathway that is a representative route to lysosomes. Because AP-3 mutation leads to mistargeting of both melanosome and lysosome membrane proteins, we propose that there is a late sorting step for melanosomes and lysosomes in melanocytes after AP-3 sorting.

Androstadienes↗
PubMed1999

Single stress induces long-lasting elevations in vasopressin mRNA levels in CRF hypophysiotrophic neurones, but repeated stress is required to modify AVP immunoreactivity.

Repeated stress is known to induce an increased vasopressin (AVP) expression in paraventricular corticotrophin-releasing factor (CRF) neurones which is supposed to enhance the ACTH-releasing capacity of these cells. To test the hypothesis that a single stress is sufficient to produce these changes, we used quantitative in-situ hybridization analysis to measure steady state CRF and AVP mRNA. Moreover the colocalized AVP and CRF immunoreactive sites were assessed in the dense core vesicle compartment of CRF axon terminals in the external zone of the median eminence with quantitative immunoelectron microscopy. Acute immobilization produced a significant increase in the average AVP and CRF mRNA levels (145% and 65%, respectively, above control values) in the medial parvocellular subdivisions of the paraventricular nucleus (PVN), and these changes persisted for over 4 days after stress. In contrast to these changes in AVP mRNA levels, there were no concomitant changes in AVP immunostaining in CRF terminals and axons during the 4-day period. However, when immobilization stress was repeated daily, the number of CRF terminals containing AVP increased progressively. Moreover, the ratio of AVP and CRF immunoreactivity in the dense core vesicle compartment was increased. Taken together, these results provide evidence that single stress experience can cause long-lasting changes in AVP and CRF mRNA steady state expression that is not apparently accompanied by changes in peptide levels. They also suggest that repeated stress is required for developing progressive shifts in the neurohormone storage pattern of these neurones.

Animals↗
PubMed2002

Molecular misreading of the ubiquitin B gene and hepatic mallory body formation.

BACKGROUND & AIMS: Molecular misreading of the ubiquitin B gene has been documented in the cerebral cortex of patients with Alzheimer's disease and Down syndrome. This novel process consists of the unfaithful conversion of genomic information into aberrant transcripts and its subsequent translation into +1 proteins. METHODS: Because Mallory bodies (MBs) also contain ubiquitinated proteins, we stained 11 autopsied and 6 biopsied MB-containing livers from patients with steatohepatitis with an antibody to ubiquitin(+1) to look for the presence of mutant (ubiquitin(+1)) protein. Antibodies to wild-type ubiquitin were used to document the presence of MBs in all cases. RESULTS: Ubiquitin(+1) immunoreactivity was detected in all MB-containing livers with steatohepatitis; no ubiquitin(+1) immunoreactivity was found in 13 MB-free liver controls. A subpopulation (about one third of the MBs) of the MB-containing hepatocytes in autopsied livers showed ubiquitin(+1) immunoreactivity (i.e., ubiquitin and ubiquitin(+1) colocalized in MBs). MB-containing liver biopsy specimens showed colocalization of ubiquitin and ubiquitin(+1) in every MB. Western blot analysis showed an ubiquitin(+1) band of 11 kilodaltons. Molecular misreading of the ubiquitin B gene (DeltaGU) was shown in one of the livers, which contained numerous MBs using an expression cloning strategy. CONCLUSIONS: The results showed that molecular misreading of the ubiquitin B gene occurred in hepatocytes in virtually all of the MB-containing livers tested. Ubiquitin(+1) protein was only found within the MBs and therefore may act by interfering with the degradation of the MBs because ubiquitin(+1) may inhibit proteolytic function of the proteasome.

Adult↗
PubMed1993

DNA topoisomerase II alpha is the major chromosome protein recognized by the mitotic phosphoprotein antibody MPM-2.

We have determined that the major mitotic phosphoprotein in chromosomes recognized by the antiphosphoprotein antibody MPM-2 is the 170-kDa isoform of topoisomerase II (topo II), the isoform predominant in proliferating cells. As a prerequisite to making this discovery, it was necessary to develop protocols to protect chromosomal proteins from dephosphorylation during cell extraction and chromosome isolation procedures. Immunofluorescence analysis of the large chromosomes prepared from Indian Muntjac cells revealed colocalization of MPM-2 and anti-topo II antibodies to the chromosomal centromeres and to the axial regions of the chromosomal arms. For biochemical fractionation studies, large quantities of chromosomes from the P388D1 mouse lymphocyte cell line were isolated and treated to remove DNA and histone proteins. Immunoblot and immunoprecipitation experiments with this chromosome scaffold fraction identified the major MPM-2-reactive phosphoprotein to be DNA topo II. Using a panel of anti-peptide antibodies specific to the isoforms of topo II, we determined that the major phosphoprotein recognized by MPM-2 is the 170-kDa isoform of topo II, topo II alpha. The 180-kDa isoform, topo II beta, present in the isolated chromosomes in much smaller quantities, is also recognized by MPM-2. The mitotic phosphorylation of the topo II proteins may be critical for proper chromosome condensation and segregation.

Animals↗
PubMed2000

SM-20 is a novel mitochondrial protein that causes caspase-dependent cell death in nerve growth factor-dependent neurons.

Sympathetic neurons undergo protein synthesis-dependent apoptosis when deprived of nerve growth factor (NGF). Expression of SM-20 is up-regulated in NGF-deprived sympathetic neurons, and ectopic SM-20 is sufficient to promote neuronal death in the presence of NGF. We now report that SM-20 is a mitochondrial protein that promotes cell death through a caspase-dependent mechanism. SM-20 immunofluorescence was present in the cytoplasm in a punctate pattern that colocalized with cytochrome oxidase I and with mitochondria-selective dyes. Analysis of SM-20/dihydrofolate reductase fusion proteins revealed that the first 25 amino acids of SM-20 contain a functional mitochondrial targeting sequence. An amino-terminal truncated form of SM-20 was not restricted to mitochondria but instead localized throughout the cytosol and nucleus. Nevertheless, the truncated SM-20 retained the ability to induce neuronal death, similar to the wild type protein. SM-20-induced death was accompanied by caspase-3 activation and was blocked by a general caspase inhibitor. Additionally, overexpression of SM-20, under conditions where cell death is blocked by a general caspase inhibitor, did not result in widespread release of cytochrome c from mitochondria. These results indicate that SM-20 is a novel mitochondrial protein that may be an important mediator of neurotrophin-withdrawal-mediated cell death.

3T3 Cells↗
PubMed2004

Vitamin D-interacting protein 205 (DRIP205) coactivation of estrogen receptor alpha (ERalpha) involves multiple domains of both proteins.

Vitamin D-interacting protein 205 (DRIP205) is a mediator complex protein that anchors the complex to the estrogen receptor (ER) and other nuclear receptors (NRs). In ZR-75 breast cancer cells treated with 17beta-estradiol (E2) and transfected with a construct containing three tandem estrogen responsive elements (pERE(3)), DRIP205 coactivates ERalpha-mediated transactivation. DRIP205Delta587-636 is a DRIP205 mutant in which both NR boxes within amino acids 587-636 have been deleted and, in parallel transfection studies, DRIP205Delta587-636 also coactivates ERalpha. Moreover, both wild-type and variant DRIP205 also colocalize with ERalpha in the nuclei of transfected cells. Extensive deletion analysis of DRIP205 shows that multiple domains of this protein play a role in coactivation of ERalpha and in interactions with ERalpha. Coactivation of ERalpha by DRIP205 does not require NR boxes, and variants with deletion of N-terminal (amino acids 1-639) and C-terminal (amino acids 576-1566) significantly coactivate ERalpha. DRIP205 resembles p160 coactivators that also interact with multiple regions of ERalpha; however, unlike p160 coactivators, DRIP205 coactivation of ERalpha does not require NR boxes.

Animals↗
PubMed2005

A new heterocyclic glucoside from the fruits of Xanthium pungens.

A new thiazinedione glucoside, 7-(beta-D-glucopyranosyloxymethyl)-8,8-dimethyl-4,8-dihydrobenzo[1,4]thiazine-3,5-dione (1) and named xanthiside, was isolated from the methanol extract of the fruits of Xanthium pungens. Its structure was determined by spectroscopic techniques including, IR, CIMS, high-resolution-EIMS, and extensive 400 MHz one- and two-dimensional NMR-analysis (1H, 13C-NMR, DEPT, 1H-1H COSY, HMQC, COLOC, and NOE experiments).

Chemistry Techniques, Analytical↗
PubMed1998

Monoclonal antibody FC-5.01, directed against CD63 antigen, is internalized into cytoplasmic vesicles in the IIB-BR-G human breast cancer cell line.

Monoclonal antibody (MAb) FC-5.01, raised against the undifferentiated breast cancer cell line IIB-BR-G, has been recently shown to react with CD63. The antigen (Ag) recognized by MAb FC-5.01 is expressed in plasma membranes of IIB-BR-G and other neoplastic cells, as well as in activated platelets and endothelial cells, as detected by indirect immunofluorescence performed at 4 degrees C on live cells. In permeabilized cells, MAb FC-5.01 colocalizes with acridine orange in acidic vesicles (lysosomal/endosomal compartment). Scatchard plot analysis performed on IB-BR-G cells demonstrated a 1.4+/-0.4 x 10(7) M(-1) affinity constant and 2.1 x 10(6) antigenic sites per cell. MAb FC-5.01 is not able to mediate C fixation or ADCC toward CD63+ cells, but the FC-5.01-CD63 complex is efficiently internalized into cytoplasmic vesicles, as shown by an acid wash immunofluorescence assay. Cellular catabolism of the antibody bound by IIB-BR-G cells was studied using [125I]-FC-5.01. At 18 h, >70% of the radioactivity was present in the supernatant as degraded fragments (TCA-soluble). After internalization, rapid Ag re-expression could be demonstrated in IIB-BR-G cells. MAb FC-5.01 diminished migration of CD63+ cells in a Boyden chamber assay. Some of the above-mentioned properties would enable the use of MAb FC-5.01 as a vehicle to target different compounds inside CD63+ cells.

Antibodies, Monoclonal↗
PubMed2006

Vascular tenascin-C regulates cardiac endothelial phenotype and neovascularization.

Microenvironmental cues mediate postnatal neovascularization via modulation of endothelial cell and bone marrow-derived endothelial progenitor cell (EPC) activity. Numerous signals regulate the activity of both of these cell types in response to vascular injury, which suggests that parallel mechanisms regulate angiogenesis in the vascular beds of both the heart and bone marrow. To identify mediators of such shared pathways, in vivo bone marrow/cardiac phage display biopanning was performed and led to the identification of tenascin-C as a candidate protein. Functionally, tenascin-C inhibits cardiac endothelial cell spreading and enhances migration in response to angiogenic growth factors. Analysis of human coronary thrombi revealed tenascin-C protein expression colocalized with the endothelial cell/EPC marker Tie-2 in intrathrombi vascular channels. Immunostains in the rodent heart demonstrated that tenascin-C also colocalizes with EPCs homing to sites of cardiac angiogenic induction. To determine the importance of tenascin-C in cardiac neovascularization, we used an established cardiac transplantation model and showed that unlike wild-type mice, tenascin-C-/- mice fail to vascularize cardiac allografts. This demonstrates for the first time that tenascin-C is essential for postnatal cardiac angiogenic function. Together, our data highlight the role of tenascin-C as a microenvironmental regulator of cardiac endothelial/EPC activity.

Aging↗
PubMed2000

Presenilin I expression in yeast lowers secretion of the amyloid precursor protein.

Presenilin (PS) mutations are associated with early-onset Alzheimer's disease and PS proteins are involved with gamma-secretase cleavage of the amyloid precursor protein, APP. We have shown previously that alpha-, beta- and gamma-secretase cleavages of APP are conserved in Pichia pastoris. Here, we report co-expression of APP and PSI in P. pastoris and show by immunoelectron microscopy colocalization of these two proteins in expanded endoplasmic reticulum. Western blot analysis indicates a drastic reduction of both alpha- and beta-secretase products. A relative increase in beta-secretase product derived from immature APP is also observed, pointing to a beta-secretase activity of P. pastoris associated with the early secretory pathway.

Amyloid Precursor Protein Secretases↗
PubMed1991

Expression of intercellular adhesion molecule-1 (ICAM-1) in the skin of patients with systemic scleroderma.

The expression and tissue distribution of intercellular adhesion molecule-1 (ICAM-1) in skin biopsies from 12 patients with systemic (SSc) and localized (LS) scleroderma was studied and compared to the biopsies from patients with lupus erythematosus (LE) and normal individuals. In normal human skin ICAM-1 expression was restricted to the vascular endothelium, infiltrating mononuclear cells (MNC), and to few individual keratinocytes. In the inflammatory stage of SSc, however, the expression of ICAM-1 was dramatically increased at the site of MNC infiltrates and could also be detected on fibroblast-like cells lying well apart from these infiltrates in the deep dermis. In contrast, in LS ICAM-1 was expressed mainly at the sites of MNC infiltrates. In LE ICAM-1 expression was confined to the keratinocytes, endothelial cells, and mononuclear cells in the upper parts of the dermis. Analysis of serial tissue sections from patients with SSc demonstrated also colocalization of staining of ICAM-1 around blood vessels with LFA-1-positive lymphocytes. Increased expression of ICAM-1 in the dermis of patients with SSc may represent an important mechanism by which MNC become localized and retained at a site of connective tissue inflammation, leading to the activation of fibroblasts.

Adult↗
PubMed2000

Effect of lipoteichoic acid on the uptake of Streptococcus pyogenes by HEp-2 cells.

Lipoteichoic acid (LTA) is thought to play a role in the interactions between Streptococcus pyogenes and host cells. We have examined the effect of exogenous LTA on the adherence and entry of S. pyogenes JRS4 strain into HEp-2 epithelial cells. LTA markedly inhibited bacterial entry in a concentration-dependent manner, up to 250 microg ml(-1). In contrast, LTA had only a slight inhibitory effect on adherence. LTA also inhibited the entry but not adherence of Salmonella typhimurium strain into HEp-2 cells. Binding experiments showed a dose-dependent binding of LTA to cells up to 10 microg ml(-1). Confocal laser microscopy imaging and analysis revealed that LTA was internalized by the epithelial cells and colocalized with F-actin. These results might imply that, following binding, exogenous LTA enters HEp-2 cells and exerts a cytotoxic effect that interferes with bacterial internalization. A possible target for LTA activity might be the actin cytoskeleton, which is known to be essential for bacterial uptake.

Bacterial Adhesion↗
PubMed2004

Adenosine A2A receptor and dopamine D3 receptor interactions: evidence of functional A2A/D3 heteromeric complexes.

Adenosine A(2A) and dopamine D(2) receptors have been shown previously to form heteromeric complexes and interact at the level of agonist binding, G protein coupling, and trafficking. Because dopamine D(2) and D(3) receptors show a high degree of sequence homology, A(2A) and D(3) receptors may also interact in a similar manner. The present studies with confocal microscopy showed that A(2A)-yellow fluorescent protein (YFP) and D(3)-green fluorescent protein 2 (GFP2) receptors colocalize in the plasma membrane. Furthermore, fluorescence resonance energy transfer (FRET) analysis demonstrated that A(2A)-YFP and D(3)-GFP2 receptors give a positive FRET efficiency and are thereby likely to exist as heteromeric A(2A)/D(3) receptor complexes. Saturation experiments with [(3)H]dopamine demonstrated that the A(2A) receptor agonist 4-[2-[[6-amino-9(N-ethyl-beta-d-ribofuranuronaminoamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid (CGS-21680) reduced the affinity of the high-affinity agonist binding state of the D(3) receptor for [(3)H]dopamine. The A(2A) and D(2A) receptors seem to interact also at the level of G protein coupling, because the adenosine A(2A) receptor agonist CGS-21680 fully counteracted the D(3) receptor-mediated inhibition of a forskolin-mediated increase in cAMP levels. Taken together, when coexpressed in the same neuron, A(2A) and D(3) receptors seem to form A(2A)/D(3) heteromeric receptor complexes in which A(2A) receptors antagonistically modulate both the affinity and the signaling of the D(3) receptors. D(3) receptor is one of the therapeutic targets for treatment of schizophrenia, and therefore, the A(2A)/D(3) receptor interactions could provide an alternative antischizophrenic treatment.

Adenosine↗
PubMed2003

Demonstration of a functional apical sodium hydrogen exchanger in isolated rat gastric glands.

Previous studies have shown that gastric glands express at least sodium-hydrogen exchanger (NHE) isoforms 1-4. Our aim was to study NHE-3 localization in rat parietal cells and to investigate the functional activity of an apical membrane NHE-3 isoform in parietal cells of rats. Western blot analysis and immunohistochemistry showed expression of NHE-3 in rat stomach colocalizing the protein in parietal cells together with the beta-subunit of the H(+)-K(+)-ATPase. Functional studies in luminally perfused gastric glands demonstrated the presence of an apical NHE isoform sensitive to low concentrations of 5-ethylisopropyl amiloride (EIPA). Intracellular pH measurements in parietal cells conducted in omeprazole-pretreated superfused gastric glands showed an Na+-dependent proton extrusion pathway that was inhibited both by low concentrations of EIPA and by the NHE-3 specific inhibitor S3226. This pathway for proton extrusion had a higher activity in resting glands and was inhibited on stimulation of histamine-induced H(+)-K(+)-ATPase proton extrusion. We conclude that the NHE-3 isoform located on the apical membrane of parietal cells offers an additional pathway for proton secretion under resting conditions. Furthermore, the gastric NHE-3 appears to work under resting conditions and inactivates during periods of H(+)-K(+)-ATPase activity.

Amiloride↗