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)

We report a patient who relapsed in a patella and knee joint after allogeneic bone marrow transplantation (BMT) for Ph chromosome-positive acute lymphoblastic leukemia. The patient complained of pain and swelling of knee joint 14 months post-BMT. Fluid from the knee joint included leukemic cells consistent with the immunophenotype of blasts prior to BMT and also revealed the bcr/abl transcript by reverse-transcriptase polymerase chain reaction. Magnetic resonance imaging demonstrated an abnormal signal in the patella. Radiotherapy to the localized extramedullary lesion was successful and no bone marrow relapse has been detected cytologically and cytogenetically to date. This case suggests that the physician should be aware of unusual relapse sites of leukemia post-BMT.
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PMID:Isolated extramedullary relapse in knee joint after allogeneic bone marrow transplantation for Ph ALL. 948 61

The inv(16)(p13q22) and t(16;16)(p13;q22) cytogenetic abnormalities occur commonly in acute myeloid leukemia (AML), typically associated with French-American-British (FAB) AML-M4Eo subtype. Reverse transcriptase-polymerase chain reaction (RT-PCR) techniques have been recently developed to detect the presence of several variants of the resultant CBFB-MYH11 fusion gene that encodes a CBFbeta-smooth muscle myosin heavy chain (SMMHC) fusion protein. We have now determined the clinical use of a polyclonal antibody [anti-inv(16) Ab] directed against a junctional epitope of the most common type of CBFbeta-SMMHC fusion protein (type A), which is present in 90% of inv(16)/t(16;16) AML cases. Using flow cytometry, reproducible methods were developed for detection of CBFbeta-SMMHC proteins in permeabilized cells; flow cytometric results were then correlated with cytogenetics and RT-PCR detection methods. In an analysis of 42 leukemia cases with various cytogenetic abnormalities and several normal controls, the anti-inv(16) Ab specifically detected all 23 cases that were cytogenetically positive for inv(16) or t(16;16), including a single AML case that was RT-PCR-negative. In addition to detecting all type A fusions, the anti-inv(16) Ab also unexpectedly identified the type C and type D CBFbeta-SMMHC fusion proteins. Molecular characterization of one RT-PCR-positive and Ab-positive t(16;16) case with a non-type A product showed a novel previously unreported CBFB-MYH11 fusion (CBFB nt 455-MYH11 nt 1893). Flow cytometric results were analyzed using the Kolmogorov-Smirnov statistic D-value and the median value for positive samples was 0.65 (range, 0.35 to 0.77) versus 0.07 (range, -0.21 to 0.18) in the negative group (P < .0001). The overall concordance between cytogenetics and RT-PCR was 97%, whereas the concordance between flow cytometry and cytogenetics was 100%. Thus, using the anti-inv(16) Ab, all cytogenetically positive and RT-PCR-positive AML cases with inv(16) or t(16;16) could be rapidly identified. This study demonstrates the use of this antibody as an investigational tool in inv(16)/t(16;16) AML and suggests that the development of such reagents may have potential clinical diagnostic use.
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PMID:Characterization and use of an antibody detecting the CBFbeta-SMMHC fusion protein in inv(16)/t(16;16)-associated acute myeloid leukemias. 949 Jun 70

While there is considerable evidence for phosphate (Pi) reabsorption in the distal tubule, Pi transport and its regulation have not been well characterized in this segment of the nephron. In the present study, we examined Na+-dependent Pi transport in immortalized mouse distal convoluted tubule (MDCT) cells. Pi uptake by MDCT cells is Na+-dependent and, under initial rate conditions, is inhibited by phosphonoformic acid (41 +/- 3% of control), a competitive inhibitor of Na+-Pi cotransport. The transport system has a high affinity for Pi (Km = 0.46 mM) and is stimulated by lowering the extracellular pH from 7.4 to 6.4 and inhibited by raising the pH from 7.4 to 8.4. Exposure to Pi-free medium for 21 h increased Na+-Pi cotransport from 2.1 to 5.5 nmol/mg of protein/5 minutes (p < 0.05) while parathyroid hormone, forskolin, and phorbol 12-myristate 13-acetate failed to alter Pi uptake in MDCT cells. Reverse transcriptase polymerase chain reaction of MDCT cell RNA provided evidence for the expression of the Npt1 but not the Npt2 Na+-Pi cotransporter gene. However, preincubation of MDCT cells with Npt1 antisense oligonucleotide led to only 20% inhibition of Na+-Pi cotransport, suggesting that other Na+-Pi cotransporters are operative in MDCT cells. Indeed, we showed, by ribonuclease protection assay, that MDCT cells express the ubiquitous cell surface receptors for gibbon ape leukemia virus (Glvr-1) and amphoteric murine retrovirus (Ram-1) that also function as Na+-Pi cotransporters. In summary, we demonstrate that the pH dependence and regulation of Na+-Pi cotransport in MDCT cells is distinct from that in the proximal tubule and suggest that different gene products mediate Na+-Pi cotransport in the proximal and distal segments of the nephron.
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PMID:Na+ -phosphate cotransport in mouse distal convoluted tubule cells: evidence for Glvr-1 and Ram-1 gene expression. 955 59

We examined the MLL genomic translocation breakpoint in acute myeloid leukemia of infant twins. Southern blot analysis in both cases showed two identical MLL gene rearrangements indicating chromosomal translocation. The rearrangements were detectable in the second twin before signs of clinical disease and the intensity relative to the normal fragment indicated that the translocation was not constitutional. Fluorescence in situ hybridization with an MLL-specific probe and karyotype analyses suggested t(11;22)(q23;q11. 2) disrupting MLL. Known 5' sequence from MLL but unknown 3' sequence from chromosome band 22q11.2 formed the breakpoint junction on the der(11) chromosome. We used panhandle variant PCR to clone the translocation breakpoint. By ligating a single-stranded oligonucleotide that was homologous to known 5' MLL genomic sequence to the 5' ends of BamHI-digested DNA through a bridging oligonucleotide, we formed the stem-loop template for panhandle variant PCR which yielded products of 3.9 kb. The MLL genomic breakpoint was in intron 7. The sequence of the partner DNA from band 22q11.2 was identical to the hCDCrel (human cell division cycle related) gene that maps to the region commonly deleted in DiGeorge and velocardiofacial syndromes. Both MLL and hCDCrel contained homologous CT, TTTGTG, and GAA sequences within a few base pairs of their respective breakpoints, which may have been important in uniting these two genes by translocation. Reverse transcriptase-PCR amplified an in-frame fusion of MLL exon 7 to hCDCrel exon 3, indicating that an MLL-hCDCrel chimeric mRNA had been transcribed. Panhandle variant PCR is a powerful strategy for cloning translocation breakpoints where the partner gene is undetermined. This application of the method identified a region of chromosome band 22q11.2 involved in both leukemia and a constitutional disorder.
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PMID:t(11;22)(q23;q11.2) In acute myeloid leukemia of infant twins fuses MLL with hCDCrel, a cell division cycle gene in the genomic region of deletion in DiGeorge and velocardiofacial syndromes. 960 Sep 80

Fas (APO-1/CD95) is a cell surface receptor that mediates apoptosis when it reacts with Fas ligand (FasL) or Fas antibody. In this study, we analyzed Fas and FasL expression in normal esophageal mucosa and esophageal squamous cell carcinomas. Reverse transcriptase-PCR revealed that Fas, soluble Fas, and FasL were expressed in all eight esophageal squamous carcinoma cell lines analyzed. Furthermore, it was demonstrated that FasL expressed in esophageal carcinoma cells is functional because coculture experiments using FasL-expressing TE-15 esophageal carcinoma cells resulted in apoptosis of Jurkat T leukemia cells, which are sensitive to Fas-mediated apoptosis. Immunohistochemistry of Fas and FasL showed that they are constitutively expressed in normal esophageal mucosa, FasL being predominantly in the basal and suprabasal layers, whereas Fas is in more differentiated layers, i.e., rows of polyhedral cells of the intermediate layers and squamous cells forming the outer layers. In 18 of 19 invasive esophageal squamous cell carcinomas, FasL expression was found in >50% of tumor cells. In contrast, most tumors (15 of 19, 79%) either showed no Fas expression or showed expression in <5% of tumor cells. These alterations were already detected in dysplasia and carcinoma in situ. These results suggest that up-regulation of FasL and down-regulation of Fas expression are early and frequent events associated with the evolution of esophageal squamous cell carcinomas.
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PMID:Up-regulation of Fas (APO-1/CD95) ligand and down-regulation of Fas expression in human esophageal cancer. 960 41

Reverse transcriptase (RT) plays a critical role in retrovirus replication, directing the synthesis of a double- stranded DNA copy of the viral RNA genome. We have previously described a mutant RT of the Moloney murine leukemia virus in which F155 was replaced by valine, and we demonstrated that this substitution allowed the enzyme to incorporate ribonucleotides to form RNA while still retaining its normal ability to incorporate deoxyribonucleotides to form DNA. When introduced into the viral genome, this mutation rendered the virus incapable of replication. Characterization of the mutant virus revealed that the enzyme was still active and able to synthesize minus-strand strong stop DNA and some longer products but failed to make full-length minus-strand DNA. We propose that the failure of the enzyme to complete DNA synthesis in vivo resulted from its ability to incorporate ribonucleotides into the products, which served as inhibitors for DNA synthesis. We also tested seven other amino acid residues for their abilities to substitute for F155 in virus replication; of these, only tyrosine could support virus replication. In an attempt to select for second-site suppressor mutations, the F155V mutant was subjected to random mutagenesis and was used as a parent for the isolation of revertant viruses. Two independent revertants were found to have changed the valine residue at position 155 back to the wild- type phenylalanine. These results suggest that an aromatic ring at this position is important for virus replication.
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PMID:Replication defect of moloney murine leukemia virus with a mutant reverse transcriptase that can incorporate ribonucleotides and deoxyribonucleotides. 962 Oct 52

Reverse transcriptase plays an essential role in the early steps of the replicative cycle of retroviruses. Because of the resistance against nucleoside analogue inhibitors such as 3'-azido-2',3'-dideoxythymidine, the importance of the investigation of non-nucleoside analogue inhibitors is increasing. We have investigated the influence of trifluoperazine (TFP--a species of phenothiazines) and its newly prepared TFP-metal complexes (TFP-VO(IV), TFP-Cu(II), TFP-Ni(II), TFP-Pd(II), TFP-Sn(IV)). The compounds were tested on Moloney murine leukemia virus reverse transcriptase assay. The inhibitory effect of metal complexes was higher than that of TFP. TFP-VO(IV) showed higher effectiveness compared the added effect of parent tricyclic chemical and metal. Therefore we concluded that the improved biological action depends on the formation of metal complexes. This phenothiazine and its metal coordination complexes could become a new non-nucleoside analogue group of compounds inhibiting the retrovirus replication.
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PMID:Trifluoperazine and its metal complexes inhibit the Moloney leukemia virus reverse transcriptase. 967 43

Reverse transcriptase is an essential retroviral enzyme that uses RNA- and DNA-directed DNA polymerase activities as well as an RNaseH activity to synthesize a double-stranded DNA copy of the single-stranded RNA genome. In an effort to obtain high-resolution structural information regarding the polymerase active site of reverse transcriptase, we have pursued studies on a catalytic fragment from Moloney murine leukemia virus reverse transcriptase. DNA encoding the catalytic fragment, defined originally by limited proteolytic digestion, has been cloned, and the protein has been expressed and purified from Escherichia coli. The fragment obtained by limited proteolytic digestion and the bacterially expressed fragnment retain polymerase activity. Crystallization studies involving nucleic acid complexes with a catalytic fragment from both sources are reported, including variables screened to improve crystals and cryocooling. Three crystal forms of catalytic fragment-nucleic acid complexes have been characterized, which all contain at least two protein molecules in the asymmetric unit. As isolated, the catalytic fragment is monomeric. This analysis indicates that the enzyme dimerizes in the presence of nucleic acid.
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PMID:Cloning, expression, and purification of a catalytic fragment of Moloney murine leukemia virus reverse transcriptase: crystallization of nucleic acid complexes. 968 90

Reverse transcriptase (RT) is a modular enzyme carrying polymerase and ribonuclease H (RNase H) activities in separable domains. Retroviral replication requires both of these activities. The RNase H domain is responsible for hydrolysis of the RNA portion of RNA x DNA hybrids, and this activity requires the presence of divalent cations (Mg2+ or Mn2+) that bind its active site. This domain is a part of a large family of homologous RNase H enzymes of which the RNase HI protein from Escherichia coli is the best characterized. Although the isolated RNase H domain from human immunodeficiency virus RT is inactive, the Moloney murine leukemia virus (MMLV) domain is active in the absence of the polymerase domain, making functional studies more accessible. Using circular dichroism spectroscopy, we characterized the stability and folding of two different fragments of MMLV RT that retain RNase H activity. The smaller fragment corresponding to the 157 C-terminal residues of RT is predominantly unfolded in the absence of divalent cations, but folding can be induced by the addition of metal. The larger fragment corresponding to the 175 C-terminal residues, however, is stably folded in the absence of metal. Thus, an 18 residue N-terminal extension outside the region homologous to E. coli RNase HI is important for the structural stability of the RNase H domain of MMLV RT. Therefore, this region should be considered part of the RNase H domain.
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PMID:Folding the ribonuclease H domain of Moloney murine leukemia virus reverse transcriptase requires metal binding or a short N-terminal extension. 974 51

We have previously reported that intravenous administration of splenocytes prepared from mice in the early stages of pregnancy promoted embryo implantation in pseudopregnant mice. Since a T-lymphocyte-rich, but not a monocyte-rich preparation from splenocytes enhanced embryo implantation, similar effects of thymocytes from non-pregnant mice on implantation were examined in this study. Thymocytes were prepared from immature 21 day old ICR female mice and the supernatant of a thymocyte suspension (Th-sup) was used as the control. Thymocytes or Th-sup were injected into the caudal vein of recipient mice on pseudopregnancy day 2, and blastocysts were transferred into the endometrial lumen. The implantation rates per recipient were significantly higher in the thymocyte-treated group. ICR mice were then oophorectomized on pseudopregnancy day 3. After 3-day progesterone supplementation, blastocysts were transferred with intravenous injection of thymocytes or Th-sup. Under progesterone supplementation, successful implantations were observed in the thymocyte-treated group, but not in the Th-sup-treated group. Reverse transcriptase-polymerase chain reaction analysis revealed that mRNA expression of leukaemia inhibitory factor in the uterus was induced by thymocyte administration, but not by Th-sup. Thymocytes were divided into two populations, CD4(+/-)CD8(-) group and CD4(-)CD8(+/-) group, by separation columns. On pseudopregnancy day 2, the separated thymocytes in each group or their supernatant were injected into the endometrial stroma of the recipient mice, and blastocysts were transferred into the endometrial lumen. The administration of CD4(+/-) CD8(-) lymphocytes significantly promoted implantation rates, but no effect was observed in the CD4(-) CD8(+/-) group. These findings showed that thymocytes, especially CD4-positive lymphocytes, facilitate embryo implantation, probably by regulating endometrial differentiation.
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PMID:Administration of thymocytes derived from non-pregnant mice induces an endometrial receptive stage and leukaemia inhibitory factor expression in the uterus. 980 51


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