Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have applied the recently developed differential display method to extend the molecular characterization of the less mature CD34+CD38lo bone marrow progenitors in comparison with the CD34+CD38hi cells to better understand their functional differences. Immunomagnetic enrichment of CD34+ cells followed by flow cytometry was used to isolate CD34+CD38lo and CD34+CD38hi cells from human organ donor bone marrow. A limited set of the poly A+ RNA sequences present in these two cell populations was amplified by a combination of reverse transcription with an anchored oligo dT-based primer and polymerase chain reaction with the same oligo dT primer and arbitrary decamers. A radioactive tracer allowed these sequences to be displayed as a series of bands on a denaturing polyacrylamide gel. Eight bands were chosen that appeared in multiple displays to represent gene sequences differentially expressed between CD34+CD38hi and CD34+CD38lo cells. Comparison of the sequences with public DNA sequence databases available identified one sequence as myeloperoxidase. Two other clones matched sequence fragments of unknown function, whereas the remaining five are novel sequences not present in existing databases. The relative level of expression of all of the sequences was tested by an independent reverse transcriptase-polymerase chain reaction with sequence-specific oligonucleotide primers. The lower level of myeloperoxidase mRNA in CD34+CD38lo cells was confirmed, as was the higher expression of the novel sequence 345. Sequence 345 expression is highest in CD34+CD38- cells and decreases with increased CD38 expression. It is expressed in negligible amounts in hematopoietic cell lines and other sources of human tissue, suggesting it may have a functional role in normal hematopoiesis.
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PMID:Identification of a novel DNA sequence differentially expressed between normal human CD34+CD38hi and CD34+CD38lo marrow cells. 754 67

The granule proteins are among the most abundant and characteristic proteins of myeloid cells. They are essential for the antimicrobial activity of these cells and they provide important markers for the differentiation stage of the myeloid series and for the diagnosis of myeloid leukemias. In acute promyelocytic leukemia (APL) there is high production of myeloperoxidase, and its cytochemical detection as well as the t(15;17) chromosomal translocation are important markers in the diagnosis of this acute myelogenous disease. The expression of other granule protein genes in APL has not been systematically determined. We have used the reverse transcriptase-polymerase chain reaction (RT-PCR) method to determine the pattern of expression of granule protein genes at the mRNA level in APL cells. We have examined the expression of the primary granule proteins defensin, myeloperoxidase, elastase, and cathepsin G; the secondary granule proteins lactoferrin, collagenase, and transcobalamin; as well as lysozyme, a protein reportedly found in both primary and secondary granules. mRNAs for all of these granule proteins were present in normal bone marrow mononuclear cells. We found that APL cells from three patients contain, in addition to myeloperoxidase mRNA, mRNAs for elastase, cathepsin G, and lysozyme. One patient had faint but detectable lactoferrin mRNA signal, but collagenase and transcobalamin mRNAs were not detectable in this patient. Defensin mRNA was found in one of the three APL patients, and all the primary granule protein mRNAs measured were found to be expressed in the APL cell line NB4. None of the secondary granule protein mRNAs measured were detectable in NB4 cells. After treatment with retinoic acid (RA), which induces neutrophil maturation of these cells, weak induction of lactoferrin and collagenase but not transcobalamin was observed. However, in view of the weak transcobalamin signal observed in normal bone marrow, the absence of transcobalamin in RA-induced NB4 cells must be interpreted with caution. Interestingly, elastase and cathepsin G mRNA disappeared after RA induction, whereas defensin and myeloperoxidase mRNAs remained present. These findings indicate that granule protein mRNAs are regulated separately and differently, and that only minimal expression of secondary granule protein genes can occur in APL cells.
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PMID:Expression of granule protein mRNAs in acute promyelocytic leukemia. 811 51

The enzyme myeloperoxidase (MPO), an important constituent of granulocytes, is used clinically in the diagnosis and classification of acute leukemia. Whereas MPO protein or enzyme activity is detectable in mature granulocytes, MPO RNA is present only in myeloblasts or promyelocytes. Some undifferentiated, biphenotypic, or lymphoblastic leukemias, although lacking MPO enzyme activity, express low levels of MPO RNA, a fact that may be of prognostic and therapeutic importance. However, current methods are inadequate for reliably detecting low levels of MPO RNA in leukemic cells containing few copies of the message. We have developed a new and highly sensitive reverse transcriptase-polymerase chain reaction (RT-PCR) assay for detecting low levels of MPO RNA in peripheral blood specimens of leukemic patients. This report describes the assay, and demonstrates its potential applicability to the diagnosis and classification of acute leukemias.
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PMID:Detection of leukemic blasts in peripheral blood specimens by reverse transcriptase polymerase chain reaction assay for myeloperoxidase mRNA. 811 6

We examined the sensitivity of different myeloperoxidase (MPO) detection methods in leukemia cell lines. To this end the MPO-positive acute promyelocytic leukemia cell line NB-4 was diluted into cell populations of the MPO-negative myeloma cell line MM-1 at different ratios. MPO protein was identified by classical cytochemical staining and by a specific anti-MPO monoclonal antibody in an immunofluorescent reaction. Cytochemical staining detected 1% positive cells among 99% negative cells. Careful, but time-consuming observation enabled the detection of positive cells in even higher dilutions. At least a 10-fold increase in sensitivity was achieved with the immunofluorescent method, as brightly fluorescent cells are more amenable for a screening of slides at lower microscopic magnification than the cytochemically visualized cells. MPO mRNA expression was examined in whole cell populations by Northern blotting (maximal sensitivity 1%), a reverse transcriptase-polymerase chain reaction (RT-PCR) amplification assay (sensitivity 0.1%), and by RT-PCR followed by Southern blotting (sensitivity 0.05%). The high sensitivity of PCR-based techniques is offset by the fact that these methods do not allow for the identification and further characterization of the individual, MPO-positive cells. Thus, methods examining bulk populations require homogeneous cell samples in order to avoid false-positivity stemming from a few residual bystander cells. The five different techniques were used to determine the status of MPO expression in 20 randomly chosen leukemia cell lines of myelomonocytic origin. In 11 cell lines (8 positive and 3 negative) all five tests provided concordant results. Three cell lines were Northern-negative, but RT-PCR-positive and MPO protein-positive suggesting that Northern blot analysis is the least sensitive tool. Six cell lines were devoid of MPO protein, at least according to the methods used here, but trace expression of MPO message was documented by PCR. All five techniques have advantages and drawbacks and must be carefully selected in order to obtain useful data. The detection of MPO is of experimental and clinical importance in the distinction of myeloid from lymphoid leukemias, and in the lineage assignment of apparently biphenotypic or unclassifiable cases.
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PMID:Sensitivity of different methods for the detection of myeloperoxidase in leukemia cells. 830 58

We present a 15-year-old woman with acute myelomonocytic leukemia without marrow eosinophilia, M4 in the FAB classification. She was admitted to our hospital with nausea and headaches. Upon admission, the leukocyte count was 284,000/microliters with 95% leukemic cells. The bone marrow aspirate was hypercellular with 74.8% blasts and 0.2% eosinophils. Leukemic cells were positive for myeloperoxidase and esterase staining. Initially, the karyotype of the bone marrow cells on admission was considered to be normal. However, the PEBP2 beta/MYH11 fusion transcript was detected in the bone marrow mononuclear cells by using the reverse transcriptase-polymerase chain reaction (RT-PCR). Reevaluation of karyotypes showed a t(16;16) (p13;q22) in the bone marrow cells. After achieving complete remission, she was treated with low-dose etoposide. Chromosome analysis showed a normal karyotype and no amplified chimeric transcripts were observed. This case indicates that the molecular analysis of PEBP2 beta and MYH11 genes is a useful tool to detect inv (16) and t(16;16) which were often difficult to find, and that leukemic cells from some cases of M4 without marrow eosinophilia have these chromosome abnormalities.
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PMID:[Detection of PEBP2 beta/MYH11 fusion mRNA in acute myelomonocytic leukemia without marrow eosinophilia]. 877 82

Although acute myeloid leukemias (AMLs) cytochemically negative for myeloperoxidase are now well recognized, myeloid surface antigen-negative AMLs are rare. The morphologic, cytochemical, immunologic, and cytogenetic or molecular features of such cases are described in four adults aged 19 to 60 years. All had AML with maturation (FAB M2) and were myeloperoxidase positive. Immunologic studies showed all to be HLA-DR positive but negative for the CD13, CD14, and CD33 antigens. Two of four were CD34 antigen positive. Cytogenetic studies were performed in three patients, and all demonstrated t(8;21)(q22;q22). In studies using the reverse transcriptase polymerase chain reaction in two patients, including the patient in whom karytypic analysis was not performed, the AML1-ETO fusion product of t(8;21) was identified. These findings suggest an association between the lack of myeloid antigen expression in myeloperoxidase-positive AML and the presence of t(8;21). In addition, the results demonstrate the continued need for cytochemical studies in the evaluation of acute leukemias.
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PMID:Presence of t(8;21)(q22;q22) in myeloperoxidase-positive, myeloid surface antigen-negative acute myeloid leukemia. 1176 84

The t(16;21)(p11;q22) translocation is a non-random chromosomal aberration observed in several types of human acute myeloblastic leukemia (AML), whereas the der(16)t(1;16) and chromosome rearrangements at 12q13 are frequently found in solid tumors. A novel cell line YNH-1 was established from peripheral blood cells of a 46-year-old male with AML (M1) carrying t(16;21) and t(1;16) translocations. YNH-1 has been maintained with a doubling time of 82 h for more than 20 months as a granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) dependent line. Morphologically YNH-1 cells were free-floating immature myeloblasts with lobulated nuclei and vacuoles in the cytoplasm. They were positive for myeloperoxidase but negative for alpha-naphthyl butylate esterase and chloroacetate esterase stainings. In surface marker analysis YNH-1 cells were positive for CD13, CD33 and CD34. Chromosomal analysis showed 46, XY, der(16)t(16;21)(p11;q22)t(1;16) (q12;q13), der(21)t(16;21)(p11;q22), der (6)t(6;12)(q13;q13), der(12)t(6;12)(q21;q13). These translocations were confirmed by fluorescence in situ hybridization (FISH) studies with the ERG-YAC clone and chromosome-specific DNA libraries. Both the FUS/ERG and ERG/FUS chimeric transcripts were identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Thus, YNH-1 could be a useful tool for elucidating the pathophysiology and molecular mechanism in AML with t(16;21),t(1;16) and 12q13 translocations.
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PMID:Establishment of a novel human acute myeloblastic leukemia cell line (YNH-1) with t(16;21), t(1;16) and 12q13 translocations. 909 2

The skeleton has the ability to alter its mass, geometry, and strength in response to mechanical stress. In order to elucidate the molecular mechanisms underlying this phenomenon, differential display reverse transcriptase-polymerase chain reaction (DDRT-PCR) was used to analyze gene expression in endocortical bone of mature female rats. Female Sprague-Dawley rats, approximately 8 months old, received either a sham or bending load using a four-point loading apparatus on the right tibia. RNA was collected at 1 h and 24 h after load was applied, reverse-transcribed into cDNA, and used in DDRT-PCR. Parallel display of samples from sham and loaded bones on a sequencing gel showed several regulated bands. Further analysis of seven of these bands allowed us to isolate two genes that are regulated in response to a loading stimulus. Nucleotide analysis showed that one of the differentially expressed bands shares 99% sequence identity with rat osteopontin (OPN), a noncollagenous bone matrix protein. Northern blot analysis confirms that OPN mRNA expression is increased by nearly 4-fold, at 6 h and 24 h after loading. The second band shares 90% homology with mouse myeloperoxidase (MPO), a bactericidal enzyme found primarily in neutrophils and monocytes. Semiquantitative PCR confirms that MPO expression is decreased 4- to 10-fold, at 1 h and 24 h after loading. Tissue distribution analysis confirmed MPO expression in bone but not in other tissues examined. In vitro analysis showed that MPO expression was not detectable in total RNA from UMR 106 osteoblastic cells or in confluent primary cultures of osteoblasts derived from either rat primary spongiosa or diaphyseal marrow. Database analysis suggests that MPO is expressed by osteocytes. These findings reinforce the association of OPN expression to bone turnover and describes for the first time, decreased expression of MPO during load-induced bone formation. These results suggest a role for both OPN and MPO expression in bone cell function.
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PMID:Analysis of differential gene expression in rat tibia after an osteogenic stimulus in vivo: mechanical loading regulates osteopontin and myeloperoxidase. 951 61

During human development, the liver and marrow both function as hematopoietic organs, but little is known about differences in the production of macrophages and neutrophils by these two organs. We used immunohistochemical stains to quantify the ratio of neutrophils to macrophages within the liver and the marrow of 16 fetuses from 5 to 16 wk postconception. At 5 wk the liver had a ratio of one granulocyte [myeloperoxidase (MPO)-positive cell] to every 9 +/- 5 (X +/- SD) macrophages (KP-1-positive cells). Between 5 and 16 wk, the granulocyte to macrophage ratio in the liver was constant, whereas it changed markedly in the marrow. Before 8 wk no MPO-positive or KP-1-positive cells were observed in bones. At 10 wk, bones still had no MPO-positive cells, but KP-1-positive cells were abundant. At 11-12 wk the granulocyte to macrophage ratio was 1 to 1 +/- 1, but by 13-16 wk it had increased to 8 +/- 3 MPO-positive cells to one KP-1-positive cell. We hypothesized that at 13-16 wk the abundance of MPO-positive cells in the marrow and their scarcity in the liver was the result of production of granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSF-R) in the marrow and their absence in the liver. However, by reverse transcriptase-PCR mRNAs for G-CSF and G-CSF-R were positive in both organs at all gestations, and G-CSF and G-CSF-R proteins (by immunohistochemistry) were also abundant in all liver and marrow specimens. We then hypothesized that progenitors in the fetal liver were intrinsically different from those in the marrow, and were unable to generate clones of neutrophils. However, progenitors from the liver produced neutrophils abundantly in culture. Thus, the explanation is likely related to as yet undescribed environmental differences between the fetal liver and marrow.
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PMID:Hematopoiesis in the liver and marrow of human fetuses at 5 to 16 weeks postconception: quantitative assessment of macrophage and neutrophil populations. 962 87

A high incidence of co-expression of myeloid-associated antigens in infant B-precursor Acute Lymphocytic Leukemia (B-ALL) has been reported, but the significance of this finding is uncertain. To further assess myeloid differentiation and its prognostic significance in this disease, we investigated the frequency of myeloperoxidase (MPO) gene expression in the blast cells from 43 infants with B-ALL registered in a Pediatric Oncology Group (POG) Pilot Study for Treatment of Infant ALL, utilizing a molecular probe for detection of MPO messenger RNA (mRNA) by Northern blot hybridization and a monoclonal antibody to detect MPO-protein by immunohistochemical staining. Sufficient RNA for Northern blot was extracted from 32 bone marrow or blood samples. In two cases, MPO mRNA was determined by a reverse transcriptase-polymerase chain reaction assay and was negative in both cases. MPO-specific transcripts (MPO+) were present in 19 of 34 (56%) samples analyzed. Immunoreactive MPO protein was positive in 13 of the 20 (65%) patients studied. No correlation was found between MPO gene expression and clinical or laboratory features, karyotypic patterns or clinical outcome. The high frequency of MPO gene expression demonstrated in this study suggests that leukemogenic events in many cases of infant B-ALL appear to involve a pluripotent stem cell not yet fully committed to lymphoid differentiation.
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PMID:Myeloperoxidase gene expression in infant leukemia: a Pediatric Oncology Group Study. 963 84


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