Biomedical subjects

Jerzy Ostrowski

Publications and source records attributed to Jerzy Ostrowski.

At least 19 recordsLinked to original sources

PubMed2006

Three clinical variants of gastroesophageal reflux disease form two distinct gene expression signatures.

It has been proposed recently that gastroesophageal reflux disease (GERD) patients may be categorized into three distinct groups exhibiting non-erosive reflux disease (NERD), erosive reflux disease (ERD), and Barrett's esophagus (BE). Measurement of relative gene expression levels was undertaken to identify distinct molecular subclasses in different variants of gastroesophageal disease. The measurements were made with Affymetrix U133A 2.0 GeneChips and RNA isolated from mucosal samples of normal squamous esophageal epithelium from 24, 28, and 26 patients with NERD, ERD and BE, respectively. Statistical testing of microarray data showed that gene expression profiles are discriminative for BE and NERD, but not for combinations of BE and ERD or NERD and ERD. In addition, women developing NERD exhibited transcriptional patterns that differed from those of men with BE. In clustering analyses, we did not observe correlations between sex and assignment of gene expression profile of ERD patients to either the NERD or the BE group. Although the biological significance of the identified genes remains uncertain, we hypothesize that GERD is a monophyletic disease that develops with the onset of gastroesophageal reflux and represents two main molecular classes, which may result in different progressions to inflammatory process within esophageal epithelium modulated by sexual dimorphism. While normal epithelium samples from NERD and BE patients are molecularly homogeneous, esophageal mucosa from ERD patients is molecularly similar to either NERD or BE. These findings may be useful for defining molecular markers which could predict potential progression to Barrett's metaplasia among patients with reflux disease.

Adult↗

PubMed2006

Helicobacter pylori protein oxidation influences the colonization process.

Dsb proteins control the formation and rearrangement of disulfide bonds during the folding of membrane and exported proteins. Here we examined the role of DsbI protein in Helicobacter pylori pathogenesis and demonstrated that a dsbI mutant impaired in disulfide bond formation revealed a greatly reduced ability to colonize mice gastric mucosa.

Animals↗

PubMed2006

Up-regulation of human PNPase mRNA by beta-interferon has no effect on protein level in melanoma cell lines.

Human mitochondrial polynucleotide phosphorylase (hPNPase) is an exoribonuclease localized in mitochondria. The exact physiological function of this enzyme is unknown. Recent studies have revealed the existence of a relationship between induction of hPNPase mRNA and both cellular senescence and growth arrest of melanoma cells following beta-interferon treatment. The aim of this study was to verify whether the augmented hPNPase mRNA level results in increase of the protein level. In several cell lines established from five metastatic melanoma patients we did not find any such correlation. However, an elevated level of hPNPase protein was observed in interferon-induced HeLa and Jurkat cells. This increase was correlated with a slight shortening of poly(A) tails of mitochondrial ND3 transcript.

Base Sequence↗

PubMed2006

The diverse involvement of heterogeneous nuclear ribonucleoprotein K in mitochondrial response to insulin.

Heterogeneous nuclear ribonucleoprotein K (hnRNP K protein) is an RNA/DNA-binding protein that acts in several compartments, including mitochondria. It integrates cellular signaling cascades with multiple processes of gene expression mechanisms. Our studies demonstrate that: (1) insulin activates the import of hnRNP K protein into mitochondria in vitro and in vivo; (2) overexpression of hnRNP K protein modulates insulin-activated mitochondrial gene expression; and (3) insulin treatment stimulates binding of hnRNP K protein to mitochondrial DNA. Based on these and our previously reported results we conclude that hnRNP K protein may be a mediator of mitochondrial response to insulin.

Animals↗

PubMed2006

Mitochondria-associated satellite I RNA binds to hnRNP K protein.

hnRNP K protein, which localizes to the nucleus, cytoplasm and mitochondria, is involved in the various cellular processes that compose gene expression. We used a SAGE-based assay to profile RNAs associated with hnRNP K protein in rat mitochondria. RNA was isolated from mitoplasts obtained from highly purified and RNase-treated mitochondria. Total RNA and RNA associated with hnRNP K protein were then used as input material for generating two SAGE libraries. Mitochondrion-derived tags isolated from the total mitoplast RNA library represented 86.3%, while those isolated from the library constructed from RNA associated with hnRNP K protein represented only 28.2% of selected tags. Thus, an unexpected number of nuclear-encoded RNAs were purified from mitochondria. Many of these transcripts were co-purified with hnRNP K protein, and high levels of nuclear-encoded RNAs co-immunoprecipitating with K protein corresponded to elevated hnRNP K protein levels of the organelle. The most abundant RNAs that were co-purified with hnRNP K protein represented transcripts originating from satellite I DNA. While satellite I RNA levels were higher in the nucleus and cytoplasm than in mitochondria, the most abundant binding of satellite I transcripts to hnRNP K protein was found in mitochondria. The role of satellite I RNA in mitochondria remains to be elucidated.

Animals↗

PubMed2006

Casein kinases phosphorylate multiple residues spanning the entire hnRNP K length.

Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) is an RNA/DNA-binding protein involved in many processes that regulate gene expression. K protein's pleiotropic action reflects the diversity of its molecular interactions. Many of these interactions have been shown to be regulated by phosphorylation. K protein contains more than seventy potential phosphorylation sites. We used an integrated approach of mass spectrometry and computer analysis to explore patterns of K protein phosphorylation. We found that in vitro a single kinase can phosphorylate K protein on multiple sites spanning the entire length of the protein, including residues contained within the RNA/DNA-binding domains. 2-D gel electrophoresis of K protein purified from cells identified 5-8 spots. Mass spectrometry of K protein isolated from proliferating cells and from cells under oxidative stress revealed the same pattern of phosphopeptides. The structural implications of phosphorylation are discussed.

Amino Acid Sequence↗

PubMed2006

Landscape of the hnRNP K protein-protein interactome.

The heterogeneous nuclear ribonucleoprotein K is an ancient RNA/DNA-binding protein that is involved in multiple processes that compose gene expression. The pleiotropic action of K protein reflects its ability to interact with different classes of factors, interactions that are regulated by extracellular signals. We used affinity purification and MS to better define the repertoire of K protein partners. We identified a large number of new K protein partners, some typically found in subcellular compartments, such as plasma membrane, where K protein has not previously been seen. Electron microscopy showed K protein in the nucleus, cytoplasm, mitochondria, and in vicinity of plasma membrane. These observations greatly expanded the view of the landscape of K protein-protein interaction and provide new opportunities to explore signal transduction and gene expression in several subcellular compartments.

Animals↗

PubMed2005

Helicobacter pylori VacA cytotoxin interacts with fibronectin and alters HeLa cell adhesion and cytoskeletal organization in vitro.

Helicobacter pylori vacuolating cytotoxin VacA causes multiple effects on epithelial cell function and morphology, but the effects of VacA on signal transduction pathways and the cytoskeleton have not been investigated in detail. In this study, we analyzed the effects of native VacA on HeLa and AGS cell adhesion to fibronectin and laminin under serum-free conditions. Confocal microscopic examination revealed increased number of cells with rounded morphology and inhibition of actin fiber formation, in the presence of VacA. VacA binds to fibronectin in vitro in a dose-dependent manner. This interaction was partly inhibited by a peptide containing an arginine-glycine-aspartic acid motif. The adhesion of HeLa cells to fibronectin, but not to laminin, was decreased in the presence of VacA. Thus, VacA may interact with fibronectin and influence integrin receptor-induced cell signaling and cytoskeleton-dependent cell functions.

Bacterial Proteins↗

PubMed2005

Increased mitochondrial gene expression during L6 cell myogenesis is accelerated by insulin.

Insulin is the most potent anabolic hormone. The greatest sensitivity to insulin is exhibited by muscle, liver and adipose cells. To study links between insulin and mitochondrial function over the course of cellular quiescence and differentiation, we quantified mitochondrial RNA and DNA in L6 myoblasts and HTC-IR hepatocytes cultured under low-serum conditions in the presence of insulin. The expression of the whole set of mitochondrial genes was determined using reverse transcriptase (RT)-real time PCR. Cell proliferation was assayed by the incorporation of (3)H-thymidine and myoblast differentiation was analyzed by morphological and biochemical markers of myogenesis. Low growth factor concentration in medium decreased proliferation of both cell types and induced differentiation of myoblasts. The expression of all mitochondrial genes decreased in quiescent hepatocytes whereas it increased in quiescent differentiated myotubes, as compared with proliferating cells, similarly to reflecting the expression of the insulin receptor gene, both in myoblasts and hepatocytes. The kinetics of mitochondrial RNA levels were similar to the expression patterns of two nuclear genes, subunit e of mitochondrial ATP-synthase and uncoupling protein-2; however, they did not reflect changes in mitochondrial DNA content. Insulin accelerated myogenesis and expression of both mitochondrial and nuclear genes in differentiated myotubes but not in quiescent hepatocytes. Our studies prove that myogenesis may require the orchestrated transcriptional activation of both mitochondrial and nuclear genes and provide additional evidence confirming the regulatory impact of insulin on the function of muscle mitochondria.

Animals↗

PubMed2005

The binding activity of yeast RNAs to yeast Hek2p and mammalian hnRNP K proteins, determined using the three-hybrid system.

K homology (KH) domains are scaffolds for the binding of RNAs by the heterogeneous nuclear ribonucleoprotein (hnRNP) K protein and its yeast ortholog, Hek2p. KH domains are remarkably conserved between mammals and yeast. To assess the binding activity for yeast RNA of the two proteins, we used full-length K protein and Hek2p as baits in the yeast three-hybrid system. All the unique RNA sequences bound by Hek2p and all but two bound by K protein represented different fragments of only two transcripts, encoded by the 18S and 25S ribosomal RNA genes. Most of them were transcribed from the antisense strand. The RNA-binding activity of K protein was significantly higher than that of Hek2p. These results and those from our previously published reports demonstrate that the specificity of target RNA recognition by both the K protein and Hek2p depends on both RNA-specific sequences and the structure of the protein. Both mammalian K protein and its yeast ortholog may be involved in the regulation of gene expression.

Animals↗

PubMed2004

Cooperative binding of the hnRNP K three KH domains to mRNA targets.

The heterogeneous nuclear ribonucleoprotein (hnRNP) K homology (KH) domain is an evolutionarily conserved module that binds short ribonucleotide sequences. KH domains most often are present in multiple copies per protein. In vitro studies of hnRNP K and other KH domain bearing proteins have yielded conflicting results regarding the relative contribution of each KH domain to the binding of target RNAs. To assess this RNA-binding we used full-length hnRNP K, its fragments and the yeast ortholog as baits in the yeast three-hybrid system. The results demonstrate that in this heterologous in vivo system, the three KH domains bind RNA synergistically and that a single KH domain, in comparison, binds RNA weakly.

Base Sequence↗

PubMed2004

Heterogeneous nuclear ribonucleoprotein K enhances insulin-induced expression of mitochondrial UCP2 protein.

The uncoupling protein 2, UCP2, is a member of a family of inner mitochondrial membrane ion carriers involved in a host of metabolic processes. UCP2 protein is encoded by nuclear genome, but the protein is found exclusively in the mitochondria. The heterogeneous nuclear ribonucleoprotein K (hnRNPK) is an RNA-binding protein involved in many processes that compose gene expression, including mRNA processing and translation. The yeast three-hybrid screen revealed K protein bound to ucp2 mRNA through sites located in the 3'-untranslated region of the transcript. ucp2 mRNA-K protein complexes were associated with polysome-coated mitochondria. Expression of exogenous K protein augmented the insulin-induced mitochondrial level of UCP2 protein that was not accompanied by a corresponding increase in ucp2 mRNA. These results suggest the insulin stimulates translation of ucp2 mRNA in a process that involves K protein.

3' Untranslated Regions↗

PubMed2004

Characterization of hnRNP K protein-RNA interactions.

The heterogeneous nuclear ribonucleoprotein K protein is an RNA-binding protein found in several subcellular compartments where it is thought to be involved in signaling multiple processes that compose gene expression. K protein contains three K homology (KH) domains that mediate RNA-binding. We used a serial analysis of gene expression (SAGE)-based strategy, yeast three-hybrid screen, RNA pull-down assays and computational analysis to characterize K protein-associated RNAs. We demonstrate that K protein interacts with many sense and antisense nuclear and mitochondrial transcripts through both direct and indirect binding. The highly specific direct binding of transcripts to K protein is mediated by a consensus sequence comprising three C-rich patches. Structural analysis suggests a three-prong interaction model whereby each of the three KH domains binds one of the C-rich patches. Genome-wide and yeast three-hybrid clone analysis revealed that these sequences are located preferentially in the 3' untranslated regions, which are known to regulate mRNA translation and processing.

Animals↗

PubMed2004

hnRNP K: one protein multiple processes.

Since its original identification as a component of the heterogeneous nuclear ribonucleoprotein (hnRNP) complex, K protein has been found not only in the nucleus but also in the cytoplasm and mitochondria and is implicated in chromatin remodeling, transcription, splicing and translation processes. K protein contains multiple modules that, on one hand, bind kinases while, on the other hand, recruit chromatin, transcription, splicing and translation factors. Moreover, the K- protein-mediated interactions are regulated by signaling cascades. These observations are consistent with K protein acting as a docking platform to integrate signaling cascades by facilitating cross-talk between kinases and factors that mediate nucleic-acid-directed processes. Comparison of K across species reveals that it is an essential factor in metazoans, but not in yeast. Although some of the K protein interactions and functions are conserved in eukaryotes from yeast to man, the mammalian protein seems to play a wider role. The greater diversity of mammalian K protein interactions and function may reflect gain of novel docking sites and expansion evolutionary of gene expression networks.

Alternative Splicing↗

PubMed2004

Protective effect of vaccination with DNA of the H. Pylori genomic library in experimentally infected mice.

Immunologically mediated protection against H. pylori infection is an attractive alternative to antibiotic treatment. We compared the efficacy of conventional protein vaccination with that of genetic vaccination against experimental infection with H. pylori in mice. For oral immunization, we used the recombinant peptide of an antigenic fragment of UreB (rUreB) or H. pylori-whole cell lysate antigens, and for genetic immunization, we used recombinant pcDNA and pSec plasmids inserted with the fragment of ureB or DNA of the H. pylori genomic library. Mice were challenged with the mouse stomach-adapted H. pylori Sidney Strain. The detection of gastric bacterial colonization was performed by real-time PCR of a 26-kDa Helicobacter-specific gene, and the presence of serum H. pylori-specific antibodies was determined using direct ELISA assay. The most effective treatment appeared to be oral vaccination with rUreB and either intramuscular or intradermal vaccination with DNA of the H. pylori genomic library. Intradermal genetic vaccination with genomic library DNA significantly increased the IgG antibody response. Our study revealed acceptable efficacies of genetic vaccination with DNA of the H. pylori genomic library.

Animals↗

PubMed2004

["Burning mouth syndrome"--is not a symptom of gastro-oesophageal reflux disease].

Burning mouth syndrome (BMS) is a predominantly oral condition characterized by the occurrence of a chronic burning that commonly involves the anterior tongue, painful sensation, dryness and taste alterations. The syndrome is reported more often in women, usually without any oral mucosal signs and laboratory abnormalities. Its etiopathogenesis remains poorly understood, and there is no consensus on diagnostic criteria and treatment strategies. Tongue burning is though to be also one of a non-oesophageal symptom of gastro-oesophageal reflux disease. As reported below, although this symptom may well be diagnostically misleading, careful diagnosis based on clinical signs may distinguish patients with BMS from those with reflux disease, and successful management of burning mouth is often enables.

Burning Mouth Syndrome↗

PubMed2003

Members of the poly (rC) binding protein family stimulate the activity of the c-myc internal ribosome entry segment in vitro and in vivo.

The 5' untranslated region of the proto-oncogene c-myc contains an internal ribosome entry segment and c-Myc translation can be initiated by cap-independent as well as cap-dependent mechanisms. In contrast to the process of cap-dependent initiation, the trans-acting factor requirements for cellular internal ribosome entry are poorly understood. Here, we show that members of the poly (rC) binding protein family, poly (rC) binding protein 1 (PCBP1), poly (rC) binding protein 2 (PCBP2) and hnRNPK were able to activate the IRES in vitro up to threefold when added in combination with upstream of N-ras and unr-interacting protein. The interactions of PCBP1, PCBP2 and hnRNPK with c-myc-IRES-RNA were shown to be specific by ultraviolet crosslinking analysis and electrophoretic mobility shift assays, while immunoprecipitation of the three proteins using specific antibodies followed by reverse transcriptase-polymerase chain reaction showed that they were able to bind c-myc mRNA. c-myc-IRES-mediated translation from the reporter vector was stimulated by cotransfection of plasmids encoding PCBP1, PCBP2 and hnRNPK. Interestingly, the mutated version of the c-myc IRES that is prevalent in patients with multiple myeloma bound hnRNPK more efficiently in vitro and was stimulated by hnRNPK to a greater extent in vivo.

Binding Sites↗

PubMed2003

Transient recruitment of the hnRNP K protein to inducibly transcribed gene loci.

The heterogeneous nuclear ribonucleoprotein K protein is an RNA- and DNA-binding protein implicated in the regulation of multiple processes that comprise gene expression. We used chromatin immunoprecipitation (ChIP) assays to explore K protein interactions with serum-inducible, constitutively expressed and untranscribed gene loci in vivo. In the rat HTC-IR hepatoma cell line, serum treatment induced transient increases in the mRNA levels of two immediate-early genes, egr-1 and c-myc. ChIP analysis showed that the induction of egr-1 and c-myc genes was associated with a transient recruitment of K protein to multiple sites within each of these loci, including the promoter and transcribed regions. In contrast, recruitment of K protein to the constitutively transcribed beta-actin locus and to randomly chosen non-transcribed loci was far weaker. In rat mesangial cells, c-myc was constitutively expressed while egr-1 remained serum responsive. In these cells, ChIP analysis showed serum-induced recruitment to the inducible egr-1 but not to the c-myc locus. Pre-treatment with the transcription inhibitor actinomycin D blocked the inducible but not the constitutive binding of K protein to these loci. Taken together, the results of this study suggest that the transient recruitment of K protein to serum-responsive loci depends on the inducible transcription of these immediate-early genes.

Animals↗