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)

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

On the basis of the previous work by Okuma and Uchino [Blood 54, 1258-1271, 1979], three patients with myeloproliferative disorders were investigated with a special reference to arachidonate 12-lipoxygenase in their platelets. The cytosol of the patients' platelets showed a markedly reduced activity of arachidonic acid oxygenation to 12-hydroperoxy acid. A peroxidase-linked immunoassay for the 12-lipoxygenase demonstrated only 7-12% of the normal level of the enzyme protein in the cytosol fraction of platelets. Furthermore, 12-lipoxygenase mRNA level was determined quantitatively by a reverse transcriptase-polymerase chain reaction with an internal standard cRNA which was synthesized by in vitro transcription of human platelet 12-lipoxygenase cDNA with a 105-bp deletion. The 12-lipoxygenase mRNA content was 4.7 +/- 2.0 (mean +/- S.D.) ng/10(11) platelets in 13 normal subjects. In contrast, the mRNA content was as low as 0.15, 0.11 and 0.10 ng/10(11) platelets in the three patients. Taken together, the 12-lipoxygenase deficiency in these patients was attributable to the decreased 12-lipoxygenase mRNA level and thus the impaired synthesis of the enzyme protein in their platelets.
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PMID:Decreased messenger RNA of arachidonate 12-lipoxygenase in platelets of patients with myeloproliferative disorders. 842 29

A colorimetric reverse transcriptase assay (cRT assay) was developed for quantitative detection of HIV-1. In this format, reverse transcriptase incorporates biotin-labeled dUTP onto oligo-dT primers hybridized to poly A templates. The templates are covalently bound to the surface of microtiter wells. The amount of incorporated biotin-labeled dUTP is measured by binding horseradish peroxidase conjugated streptavidin, washing away unbound peroxidase, adding colorimetric substrate and then reading with a standard colorimetric reader. The sensitivity of the assay is very good. As little as 3 x 10(5) molecules of recombinant HIV-RT can be detected after 20 h of reaction time. Direct comparison using 3 cultured clinical isolates indicates that this level of detection is equivalent to the commercially available p24 antigen capture assay and the HIV-RNA assay based on branched DNA signal amplification. Other retroviruses, such as HIV-2 and feline immunodeficiency virus (FIV), can also be detected in this format. This non-isotopic assay is easy to perform and could provide a convenient and quantitative method for HIV study by monitoring reverse transcriptase, an essential activity in the infection process.
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PMID:Poly A-linked non-isotopic microtiter plate reverse transcriptase assay for sensitive detection of clinical human immunodeficiency virus isolates. 860

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

To obtain evidence of renin-synthesizing cells in the murine coagulating gland (CG), CG renin mRNA was detected by hybridohistochemistry, as well as in vitro reverse transcriptase-polymerase chain reaction (RT-PCR) in intact, castrated and testosterone-treated C57BL/6 mice. Hybridohistochemistry using paraffin sections of the kidneys and the CGs for the detection of renin mRNA was performed with digoxigenin-labelled probes. Some paraffin sections were immunohistochemically stained for renin by the peroxidase-anti-peroxidase method. Total RNA was extracted, incubated by reverse transcriptase, and amplified by PCR. In the kidneys, the immunoreactivity and the positive signals of hybridohistochemistry using an antisense probe were restricted to the same juxtaglomerular cells. In the control and at 7 days after testosterone administration to castrated mice, both renin-immunoreactivity and -hybridoreactivity were expressed by the epithelial cells in the CGs, while, in the CGs of the castrated mice and 3 days after testosterone injection of castrated animals, neither renin-immunoreactivity nor -hybridoreactivity was detected in the epithelial cells. Using RT-PCR, renin mRNA from the mice in the control and 7 days after testosterone injection of castrated was amplified, whereas, in the castrated and the 3 days after testosterone injection of castrated groups, it was not detected. The data presented here provide additional evidence that CG renin is regulated by testosterone.
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PMID:Detection of coagulating gland renin by hybridohistochemistry. 901 Nov 6

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 expression of two closely related peroxidase isogenes, Shpx6a and Shpx6b, of the legume Stylosanthes humilis was studied using isogene-specific reverse transcriptase PCR techniques. Results indicated that transcripts of both genes were rapidly induced following inoculation with the fungal pathogen Colletotrichum gloeosporioides, wounding and treatment with the defense regulator methyl jasmonate (MeJA). In contrast treatment of leaves of S. humilis with abscisic acid (ABA) and salicylic acid (SA) did not induce transcripts of either isogene. A genomic clone containing the Shpx6b gene was isolated and 594 bp of 5' sequence upstream of the translation start was fused in frame to the coding region of the uidA reporter gene and introduced into tobacco. Expression from the Shpx6b promoter in transgenic plants was determined by histochemical staining and quantitative assays of beta-glucuronidase (GUS). In transgenic tobacco, GUS expression was detected in cotyledons, vascular cells of young leaves, anthers, pollen, and the stigma and style. Wounding of the tobacco plants produced very localized GUS staining. Much more extensive staining for GUS was observed following inoculation of tobacco leaves with conidia of the fungal pathogen Cercospora nicotianae and the inoculation of wound sites with mycelium of the Oomycete pathogen Phytophthora parasitica var. nicotianae. Treatment of mature leaves with methyl jasmonate induced GUS activity while treatment with ABA, SA, and H2O2 had no effect. A similar strong induction of GUS activity was measured in young transgenic seedlings germinated on MeJA while some, but much weaker, induction of GUS activity was observed in seedlings treated with SA. The sequence of the promoter contained motifs homologous to putative cis elements in other plant genes responsive to MeJA. The Shpx6b gene is the first plant peroxidase gene shown to be induced by both microbial pathogens and MeJA and its promoter will be useful for investigations of signaling processes during fungal infection and for the expression of foreign gene products at infection sites.
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PMID:A peroxidase gene promoter induced by phytopathogens and methyl jasmonate in transgenic plants. 910 Mar 78


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