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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Reverse transcriptase activity was measured by incorporation of dUMP linked to digoxigenin into a suitable template-primer molecule. Incorporation was monitored by using peroxidase-conjugated Fab fragments directed against digoxigenin. The standard assay measuring incorporation of radiolabeled nucleotides into acid-precipitable material was compared with this new immunochemical assay with regard to its usefulness for testing inhibitors of reverse transcriptase.
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PMID:Measurement of HIV-1 reverse transcriptase by a nonradioactive assay system. 128 11

Small numbers of CD34+ primitive hematopoietic progenitors are found in normal human peripheral blood. These cells differentiate to myeloid or lymphoid lineage under the influence of different growth factors. We investigated the effects of IL5 and other growth factors on the production of eosinophils from peripheral blood CD34+ cells. CD34+ cells were enriched from normal donors by apheresis and positive selection using an affinity column and plated in agarose with different combinations of cytokines. At 14 days of growth a triple stain technique was used to identify eosinophil, monocyte, and neutrophil colonies. IL5 alone did not support colony growth from CD34+ cells. In contrast, GM-CSF and IL3 alone or together without added IL5 supported the generation of more than 50% pure eosinophil colonies. Addition of IL5 did not change the total number of colonies, but increased the fraction of pure eosinophil colonies to over 70%. Addition of G-CSF reduced the percentage of eosinophil colonies and increased the percentage of neutrophil colonies. Under the best conditions for eosinophil colony growth (IL3+GM-CSF+IL5), the addition of interferon-alpha or bacterial lipopolysaccharide inhibited colony growth by 51 and 58%, respectively. Addition of interferon-gamma, tumor necrosis factor-alpha, or dexamethasone had no effect on eosinophil colonies. Since IL5 alone did not support colony growth from CD34+ cells, we determined when IL5-responsive cells appeared in culture. Cells were grown initially with IL3 + GM-CSF in suspension, washed, and plated in agarose with IL5 alone. Only when progenitors were grown at least 3 days could IL5 serve as the single growth factor supporting pure eosinophil colony growth (47 colonies/10(4) cells plated at Day 3 and 134 colonies/10(4) cells at Day 7). We used neutralizing anti-IL5 antibodies to demonstrate that this late acting IL5 growth effect was specific, and that differentiation of eosinophils in the presence of IL3 + GM-CSF was IL5 independent. Using reverse-transcriptase polymerase chain reaction, the mRNA encoding the eosinophil-specific protein eosinophil peroxidase (EPO) was not detected in Day 0 CD34+ cells, but was demonstrated by Day 3 of culture. We conclude that within 3 days of culture, peripheral blood CD34+ cells can become committed to the eosinophil lineage as demonstrated by responsiveness to IL5 and production of EPO transcripts.
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PMID:Modulation of growth and differentiation of eosinophils from human peripheral blood CD34+ cells by IL5 and other growth factors. 753 Nov 18

A 78 year old female was found to have pancytopenia in February 1991. Bone marrow was normocellular with 11.7% blasts and showed dysmegakaryopoietic changes. A diagnosis of MDS (RAEB) was made and she was treated with transfusions and ubenimex. Leukemic transformation was noted in July. On Admission in October 1991, her laboratory examinations revealed the following: WBC 38,900/microliters with 93% blast, Hb 8.0 g/dl, Plt 2.1 x 10(4)/microliters, a hypercellular bone marrow with 74% blasts which were negative for myeloperoxidase (MPO) by light microscopy, but were positive by electron microscopy. Surface marker for CD13 was positive. These findings corresponded to M0 of the FAB subtype. Chromosome analysis revealed Ph1 chromosome with 46XX, t (9;22) (q34;q11) in 3 of 3 cells examined, Southern analysis showed the rearrangement of the break point cluster region (bcr). Reverse transcriptase polymerase chain reaction technique demonstrated the presence of major bcr/abl mRNA. She was treated with transfusions and methyl-prednisolone. Her blast counts declined and Ph1 chromosome was only positive in 1 of 12 metaphases examined. She died of pneumonia in December 1991. Eleven cases with MDS showing Ph1 chromosome have previously been reported. The observations indicate that Ph1 chromosome positive acute leukemias were heterogenous in nature.
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PMID:[RAEB transformed into AML (M0) showing Ph1 chromosome and rearrangement of major cluster region]. 825 8

There is evidence suggesting the importance of the interleukin-1 receptor type I (IL-1Rtl) and interleukin-1 beta (IL-1 beta) as mediator in local intercellular interactions in endometrial tissue and embryonic implantation. To complete our understanding of the entire endometrial IL-1 system in humans, we have investigated the immunohistochemical distribution of IL-1 receptor antagonist (IL-1ra) in the human endometrium throughout the menstrual cycle. We have also identified the forms of IL-1ra present in human endometrial cells. Immunoreactive IL-1ra was found in both cryostat and paraffin-embedded sections of human endometrium using the alkaline phosphatase-peroxidase (A-P) method with two different IL-1ra antibodies. IL-1ra was present throughout the entire menstrual cycle, located primarily in the endometrial epithelium. However, IL-1ra staining was significantly higher during follicular phase in comparison with early and mid-late luteal phases. Reverse transcriptase polymerase chain reaction of cultured stromal and glandular cells showed that these cells express the intracellular form of IL-1ra mRNA (icIL-1ra). Our results demonstrate the regulated presence of the icIL-1ra in the human endometrium. This finding supports a possible autocrine-paracrine role for the IL-1 system in the human endometrium and embryonic implantation.
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PMID:Immunohistochemical localization, identification and regulation of the interleukin-1 receptor antagonist in the human endometrium. 853 Jun 93

Methods for isolation and cultivation of CD34+ peripheral blood progenitor cells (PBPCs) have facilitated their use in autologous transplantation and as potential targets for gene therapy. In this work, we present the possibility of using these isolated cells to study lineage-specific hematopoietic differentiation. We have shown that differentiating PBPCs faithfully replicate transcriptional events that occur during maturation of the eosinophil lineage; messenger RNAs encoding the five eosinophil granule proteins were detected by reverse-transcriptase polymerase chain reaction (RT-PCR) after 2-3 days of cytokine-stimulated growth. Only three of the five proteins were detected by immunofluorescence staining after 14 days of cytokine-stimulated growth; the percentage of Charcot-Leyden crystal protein (CLC)-containing cells (16-18%) exceeded that of eosinophil peroxidase (EPO)-containing cells (7-8%), which in turn exceeded that of eosinophil-derived neurotoxin (EDN)-containing cells (2-4%). While the electrophoretic mobilities of both CLC and EPO synthesized by differentiating PBPCs were similar to those of their normal counterparts, immunoreactive EDN was found to be heterogeneous and of higher molecular weight that EDN found in mature eosinophils. It is not clear whether our results, which show progressive, but incomplete, differentiation of PBPCs into eosinophils, reflect a lack of knowledge as to what factors are essential for complete differentiation in vitro or relate to the inherent capacity of PBPCs to differentiate along this lineage.
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PMID:Characterization of eosinophils generated in vitro from CD34+ peripheral blood progenitor cells. 869 47

The distribution of myeloid lineage-associated cytokine receptors and lysosomal proteins was analyzed in human CD34+ cord blood cell (CB) subsets at different stages of myeloid commitment by reverse-transcriptase polymerase chain reaction (RT-PCR). The highly specific granulomonocyte-associated lysosomal proteins myeloperoxidase (MPO) and lysozyme (LZ), as well as the transcription factor PU.1, were already detectable in the most immature CD34+Thy-1+ subset. Messenger RNA (mRNA) levels for the granulocyte-colony stimulating factor (G-CSF) receptor, granulocyte-macrophage (GM)-CSF receptor alpha subunit and tumor necrosis factor (TNF) receptors I (p55) and II (p75) were also detected in this subset in addition to c-kit and flt-3, receptors known to be expressed on progenitor cells. By contrast, the monocyte-macrophage colony stimulating factor (M-CSF) receptor was largely absent at this stage and in the CD34+Thy-1-CD45RA- subsets. The M-CSF receptor was first detectable in the myeloid-committed CD34+Thy-l-CD45RA+ subset. All other molecules studied were found to be expressed at this stage of differentiation. Different cocktails of the identified ligands were added to sorted CD34+Thy-1+ single cells. Low proliferative capacity was observed after 1 week in culture in the presence of stem cell factor (SCF) + Flt-3 ligand (FL) + G-CSF. Addition of GM-CSF to this basic cocktail consistently increased the clonogenic capacity of single CD34+Thy-1+ cells, and this effect was further enhanced (up to 72.3 +/- 4.3% on day 7) by the inclusion of TNF-alpha. In conclusion, the presence of myeloid-associated growth factor receptor transcripts in CD34+ CB subsets does not discriminate the various stages of differentiation, with the exception of the M-CSF receptor. In addition, we show that TNF-alpha is a potent costimulatory factor of the very immature CD34+Thy-1+ CB subset.
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PMID:Analysis of myeloid-associated genes in human hematopoietic progenitor cells. 932 52

Marrow stromal cells mediate the effect of 1alpha,25-dihydroxyvitamin D3 on formation of osteoclast-like cells from undifferentiated hematopoetic precursors in bone marrow. Induction by the vitamin D hormone of multinucleated, calcitonin receptor- and tartrate-resistant acid phosphatase-positive cells in primary mouse bone marrow culture can be modulated by other members of the steroid/thyroid hormone family, such as triiodothyronine, which has a positive effect, as well as 17beta-estradiol and 5alpha-dihydrotestosterone, which both act as inhibitors of osteoclastogenesis. In an attempt to relate these effects of the steroid/thyroid hormones to the presence of their respective nuclear receptors, we studied expression of the vitamin D receptor (VDR), estrogen receptor (ER)-alpha and -beta, thyroid hormone receptor (TR)-alpha and -beta, and androgen receptor (AR) in total bone marrow as well as primary marrow stromal cell cultures. By using reverse-transcriptase-polymerase chain reaction, in both cases amplification products were obtained, which were identified by multiple restriction fragment length analysis as transcripts from mRNA specific for the ligand-binding domains of the VDR, ER-alpha, ER-beta, TR-alpha, TR-beta, and AR. Specific immunostaining by indirect peroxidase labeling revealed that among the various cell types present in bone marrow, the steroid/ thyroid hormone receptors are abundant particularly in marrow stromal cells. In another series of experiments, we extended our survey on receptor expression also to stromal/osteoblastic cell lines. At the mRNA level, the complete repertoire of steroid/thyroid hormone receptors was present in preadipocytic ST2 cells as well as in osteoblastic MC3T3-E1 cells. By immunocytochemical staining of the latter, it became apparent that single cells exhibit wide variations in intensity of specific signals for all the receptors investigated, so that, notably in contrast to primary stromal cells and ST2 cells, MC3T3-E1 display a mosaic pattern of receptor protein expression.
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PMID:Expression of the vitamin D receptor, of estrogen and thyroid hormone receptor alpha- and beta-isoforms, and of the androgen receptor in cultures of native mouse bone marrow and of stromal/osteoblastic cells. 1032 6

A cDNA clone encoding the Prx7 peroxidase from barley (Hordeum vulgare L.) predicted a 341-amino acid protein with a molecular weight of 36,515. N- and C-terminal putative signal peptides were present, suggesting a vacuolar location of the peroxidase. Immunoblotting and reverse-transcriptase polymerase chain reaction showed that the Prx7 protein and mRNA accumulated abundantly in barley coleoptiles and in leaf epidermis inoculated with powdery mildew fungus (Blumeria graminis). Two isoperoxidases with isoelectric points of 9.3 and 7.3 (P9.3 and P7.3, respectively) were purified to homogeneity from barley coleoptiles. P9.3 and P7.3 had Reinheitszahl values of 3.31 and 2.85 and specific activities (with 2,2'-azino-di-[3-ethyl-benzothiazoline-6-sulfonic acid], pH 5.5, as the substrate) of 11 and 79 units/mg, respectively. N-terminal amino acid sequencing and matrix-assisted laser desorption/ionization time-of-flight mass-spectrometry peptide analysis identified the P9. 3 peroxidase activity as due to Prx7. Tissue and subcellular accumulation of Prx7 was studied using activity-stained isoelectric focusing gels and immunoblotting. The peroxidase activity due to Prx7 accumulated in barley leaves 24 h after inoculation with powdery mildew spores or by wounding of epidermal cells. Prx7 accumulated predominantly in the epidermis, apparently in the vacuole, and appeared to be the only pathogen-induced vacuolar peroxidase expressed in barley tissues. The data presented here suggest that Prx7 is responsible for the biosynthesis of antifungal compounds known as hordatines, which accumulate abundantly in barley coleoptiles.
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PMID:Barley coleoptile peroxidases. Purification, molecular cloning, and induction by pathogens. 1036 1

Intercellular adhesion molecule-1 (ICAM-1) plays an important role in the pathogenesis of either human and experimental myocardial ischaemia. Tacrolimus, formerly known as FK506, has been previously shown to display cardioprotective effects on experimental ischaemia/reperfusion-induced myocardial damage. This study investigated whether cardioprotection induced by tacrolimus in myocardial ischaemia-reperfusion (MI/R) injury might be due to inhibition of the nuclear factor kappa B (NF- kappaB) that in turn causes reduced cardiac ICAM-1 expression and blunted polymorphonuclear leukocyte accumulation. Anaesthetized rats were subjected to total occlusion (45 min) of the left main coronary artery followed by 5 h reperfusion (MI/R). Sham myocardial ischaemia-reperfusion rats (Sham MI/R) were used as controls. Myocardial necrosis, myocardial myeloperoxidase activity, serum creatine kinase (CK) activity, cardiac mRNA for ICAM-1 reverse-transcriptase polymerase chain reaction, the inhibitory protein of NF- kappaB I kappaB alpha (Western blot analysis) in the myocardium-at-risk, and left ventricle d P/d t(max)were evaluated. Myocardial ischaemia plus reperfusion in untreated rats produced marked myocardial necrosis, increased serum CK activity and myeloperoxidase activity (MPO, a marker of leukocyte accumulation) both in the area at risk and in the necrotic area, and reduced the left ventricle dP/d t(max). Furthermore, inhibitory protein I kappaB alpha levels decreased, and cardiac mRNA for ICAM-1 increased, after 0.5 and 5 h of reperfusion, respectively. Administration of tacrolimus (25, 50 and 100microg/kg as an i.v. infusion 5 min after reperfusion) lowered myocardial necrosis and myeloperoxidase activity in the area at risk and in necrotic area, decreased serum CK activity, increased left ventricle dP/d t(max), reduced the loss the of inhibitory protein I kappaB alpha and blunted the message for ICAM-1. The present data suggest that tacrolimus blocks the early activation of the transcription factor NF- kappaB, suppresses ICAM-1 gene activation, reduces leukocyte accumulation and protects against myocardial ischaemia-reperfusion injury.
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PMID:Tacrolimus limits polymorphonuclear leucocyte accumulation and protects against myocardial ischaemia- reperfusion injury. 1073 42

Cholinergic muscarinic inputs to subfornical organ (SFO) neurones in rats were studied using histochemical, molecular-biological and electrophysiological techniques. Neurones in the medial septum and the diagonal band (MS-DBB) were retrogradely labelled by a tracer wheat germ agglutinin-conjugated horseradish peroxidase-colloidal gold complex injected into the SFO. Some in the MS-DBB were double-labelled by choline acetyltransferase (ChAT) antibody. Many ChAT-immunoreactive fibres were observed in the SFO. M3 muscarinic receptor subtype-like immunoreactivity, detected using a polyclonal antiserum, was observed in the SFO. In slice preparations, muscarine induced inward currents in a dose-related manner. The inward currents were suppressed by the relatively M3 muscarinic receptor selective antagonist 4-diphenylacetoxy-N-methylpiredine methiodide. In the whole-cell current mode, muscarine depolarized the membrane with increased frequency of action potentials. Reverse transcriptase-polymerase chain reaction showed the presence of M2-M5 receptor mRNA in the SFO tissues. These results suggest that the SFO receives cholinergic muscarinic synaptic inputs from the MS-DBB. Acetylcholine postsynaptically activates and depolarizes neurones in the SFO partly through specific muscarinic receptors, including M3 receptor subtypes.
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PMID:Activation of muscarinic receptors in rat subfornical organ neurones. 1283 38


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