Biomedical subjects

Dmitri V Gnatenko

Publications and source records attributed to Dmitri V Gnatenko.

5 recordsLinked to original sources

PubMed2004

Expression of protease activated receptor 3 (PAR3) is upregulated by induction of megakaryocyte phenotype in human erythroleukemia (HEL) cells.

OBJECTIVE: Two major protease-activated receptors (PARs), PAR1 and PAR4, are involved in the activation of human platelets by thrombin. A third, PAR3, is preferentially expressed by tissues of hematopoietic origin and megakaryocytes. Although PAR3 is also a thrombin substrate, its low-level expression on human platelets suggests a function distinct from that of PAR1, the major receptor involved in thrombin-mediated platelet activation. We studied the expression of PARs during megakaryocyte differentiation of human erythroleukemia (HEL) cells in order to determine the role of PAR3 in megakaryocytopoiesis. METHODS: HEL cells exposed to phorbol 12-myristate 13-acetate (PMA) to induce megakaryocyte differentiation were examined by light microscopy and flow cytometry (DNA ploidy, surface expression of PAR1, PAR3, GPIIb-IIIa). Northern blot, RT-PCR, and quantitative RT-PCR were used to evaluate the expression of PARs 1, 3, and 4 mRNA. HEL cells were also exposed to thrombin and thrombopoietin (TPO). RESULTS: In baseline studies, unstimulated HEL cells were found to express comparable levels of PAR1 and PAR3 by Northern blot. Minimal expression of PAR4 was detected by RT-PCR, but not by Northern analysis. Exposure to PMA, but not thrombin or TPO, resulted in megakaryocytic differentiation as evident by increased cell size and nuclear complexity, increased ploidy, and enhanced expression of GPIIb-IIIa, a specific marker of megakaryocytes/platelets. PMA-stimulated HEL cells showed enhanced PAR3 cell-surface expression (approximately threefold increase by day 2) by flow cytometry. In contrast, there was no change in cell-surface PAR1 expression. Northern blot analysis (approximately 10-fold) and quantitative RT-PCR (approximately threefold) confirmed the upregulation of PAR3 mRNA expression (by 24 hours) in cells exposed to PMA. This did not occur with exposure to TPO. CONCLUSION: These data demonstrate increased expression of PAR3 mRNA and protein in HEL cells undergoing megakaryocytic maturation following PMA exposure, suggesting a developmental role for PAR3. Furthermore, regulation of PAR3 expression appears to be specifically coupled to the protein kinase C system, but independent of the Ras/Raf/MAP kinase pathway.

Cell Differentiation↗

PubMed2004

Platelet transcriptome: the application of microarray analysis to platelets.

Human blood platelets are intimately involved in the regulation of thrombosis, inflammation, and wound repair. These cells retain megakaryocyte-derived cytoplasmic mRNA and functionally intact protein translational capabilities, although very little is known about normal or pathological mRNA profiles. Microarray analysis has demonstrated a clear and reproducible molecular signature unique to platelets. There is a relative paucity of expressed transcripts compared with those found in other eukaryotic cells, most likely related to mRNA decay in these anucleate cells. In contrast, a complementary methodology for transcript profiling (serial analysis of gene expression [SAGE]) demonstrates that 89% of tags represent mitochondrial (mt) transcripts (enriched in 16S and 12S ribosomal RNAs), presumably related to persistent mt-transcription in the absence of nuclear-derived transcripts. The abundance of nonmitochondrial SAGE tags parallels relative expression for the most abundant transcripts as determined by microarray analysis, establishing the concordance of both techniques for platelet profiling. These observations establish the validity of transcript analysis as a tool for identifying novel platelet genes that may regulate normal and pathologic platelet (and/or megakaryocyte) functions. The potential application of platelet-specific microarrays in scientific and clinical settings related to platelet production, cardiovascular, and cerebrovascular diseases is reviewed.

Animals↗

PubMed2004

Expression of therapeutic levels of factor VIII in hemophilia A mice using a novel adeno/adeno-associated hybrid virus.

We have generated an E1a/E1b/E3-deleted adeno/adeno-associated (Ad/AAV) hybrid virus driven by a small nuclear RNA (pHU1-1) promoter for expression of a B domain-deleted (Thr761-Asn1639) factor VIII transgene (FVIIIDelta761-1639). Productive replication of Ad/AAV/FVIIIDelta761-1639 in AAV rep-expressing cells resulted in generation of monomeric and dimeric mini-adenoviral (mAd) replicative forms that retained the AAV integration elements (mAd/FVIIIDelta761-1639). In vitro studies using Ad/AAV/FVIIIDelta761-1639 generated approximately 2-logs greater FVIII activity than mAd/FVIIIDelta761-1639. To determine its capacity for in vivo excision and/or genomic integration, Ad/AAV/FVIIIDelta761-1639 was injected by tail vein into three groups of hemophilia A mice (2 x 10(11) vp [n = 3]; 4 x 10(11) vp [n = 3]; 8 x 10(11) vp [n = 3]), with clear concentration-dependent increase in FVIII activity (range 160-510 mU/ml; plasma activity 16%-51% of normal). Peak activity was seen by Day (D) 5, with slow return to baseline by D28 (0.1-0.9% activity); in only 3/9 mice was loss of FVIII activity associated with development of anti-FVIII antibodies. Quantitative-PCR using genomic DNA isolated from D28 liver, spleen, heart, lungs, and kidney demonstrated the highest concentration in liver (approximately 10 genomes/cell), with little to no organ toxicity at early (D5 or 6) or late (D28) post-infusion time points. There was no evidence for spontaneous transgene excision or genomic integration in vivo as evaluated by quantitative PCR and genomic blotting. These data establish (i) the feasibility and applicability of developing high-titer Ad/AAV hybrid viruses for FVIII delivery using a small cellular promoter, (ii) the potential utility of this virus for generation of "gutted" monomeric and dimeric mAD/FVIII retaining AAV integration elements, and (iii) that the development of strategies for regulated Rep68/78 co-expression may provide a novel approach for excision, integration, and long-term FVIII transgene expression.

Adenoviridae↗

PubMed2002

Transcript profiling of human platelets using microarray and serial analysis of gene expression.

Human platelets are anucleate blood cells that retain cytoplasmic mRNA and maintain functionally intact protein translational capabilities. We have adapted complementary techniques of microarray and serial analysis of gene expression (SAGE) for genetic profiling of highly purified human blood platelets. Microarray analysis using the Affymetrix HG-U95Av2 approximately 12 600-probe set maximally identified the expression of 2147 (range, 13%-17%) platelet-expressed transcripts, with approximately 22% collectively involved in metabolism and receptor/signaling, and an overrepresentation of genes with unassigned function (32%). In contrast, a modified SAGE protocol using the Type IIS restriction enzyme MmeI (generating 21-base pair [bp] or 22-bp tags) demonstrated that 89% of tags represented mitochondrial (mt) transcripts (enriched in 16S and 12S ribosomal RNAs), presumably related to persistent mt-transcription in the absence of nuclear-derived transcripts. The frequency of non-mt SAGE tags paralleled average difference values (relative expression) for the most "abundant" transcripts as determined by microarray analysis, establishing the concordance of both techniques for platelet profiling. Quantitative reverse transcription-polymerase chain reaction (PCR) confirmed the highest frequency of mt-derived transcripts, along with the mRNAs for neurogranin (NGN, a protein kinase C substrate) and the complement lysis inhibitor clusterin among the top 5 most abundant transcripts. For confirmatory characterization, immunoblots and flow cytometric analyses were performed, establishing abundant cell-surface expression of clusterin and intracellular expression of NGN. These observations demonstrate a strong correlation between high transcript abundance and protein expression, and they establish the validity of transcript analysis as a tool for identifying novel platelet proteins that may regulate normal and pathologic platelet (and/or megakaryocyte) functions.

Base Sequence↗

PubMed2002

Genomic signature tags (GSTs): a system for profiling genomic DNA.

Genomic signature tags (GSTs) are the products of a method we have developed for identifying and quantitatively analyzing genomic DNAs. The DNA is initially fragmented with a type II restriction enzyme. An oligonucleotide adaptor containing a recognition site for MmeI, a type IIS restriction enzyme, is then used to release 21-bp tags from fixed positions in the DNA relative to the sites recognized by the fragmenting enzyme. These tags are PCR-amplified, purified, concatenated, and then cloned and sequenced. The tag sequences and abundances are used to create a high-resolution GST sequence profile of the genomic DNA. GSTs are shown to be long enough for use as oligonucleotide primers to amplify adjacent segments of the DNA, which can then be sequenced to provide additional nucleotide information or used as probes to identify specific clones in metagenomic libraries. GST analysis of the 4.7-Mb Yersinia pestis EV766 genome using BamHI as the fragmenting enzyme and NlaIII as the tagging enzyme validated the precision of our approach. The GST profile predicts that this strain has several changes relative to the archetype CO92 strain, including deletion of a 57-kb region of the chromosome known to be an unstable pathogenicity island.

Binding Sites↗