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)

Investigations with chemical inhibitors and with inhibitory antibodies specific for cytochrome P4501A-catalyzed ethoxyresorufin (ethoxyphenoxazone) O-deethylation and 2-acetylaminofluorene (N-2-fluorenylacetamide) ring hydroxylation indicated that cytochrome(s) P450 of the 1A subfamily was functionally expressed in human embryonic hepatic tissues at very early stages (days 50-60) of gestation. Lack of detectable capacity of hepatic microsomal enzymes to catalyze either N-hydroxylation of 2-acetylaminofluorene or O-demethylation of methoxyresorufin indicated that functional cytochrome P4501A2 is expressed minimally or negligibly in human embryonic hepatic tissues. By contrast, profound inhibition of the ring hydroxylation of 2-acetylaminofluorene and of the O-deethylation of ethoxyresorufin by 7,8-benzoflavone as well as by anti-cytochrome P4501A1 antibodies indicated the presence of significant levels of functional cytochrome P4501A1 in hepatic microsomes of human embryos. Using the reverse transcriptase-linked polymerase chain reaction with specific oligonucleotide primers, we also detected significant expression of cytochrome P4501A1 mRNA in human embryonic livers. Polymerase chain reaction amplification, cloning and sequencing of the corresponding cDNA provided evidence that the cytochrome P4501A1 mRNA expressed in human embryonic tissues was identical to that expressed in adult human tissues. The results of the study have important implications in terms of the embryotoxic effects of chemicals that are known to be substrates, inhibitors or inducers of cytochrome P4501A1 and to which pregnant women are exposed.
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PMID:Expression of functional cytochrome P4501A1 in human embryonic hepatic tissues during organogenesis. 788 87

Polymerase chain reaction (PCR)-based amplification of cytomegalovirus (CMV) DNA has been demonstrated to be a sensitive tool for the diagnosis of CMV infection. However, PCR can detect the presence of viral DNA in some specimens from clinically asymptomatic patients. In an attempt to obviate this shortcoming, a reverse transcriptase-PCR-based assay (RT-PCR) was developed to look for CMV immediate-early (IE) mRNA in peripheral blood leukocytes from organ transplant recipients. The results of the PCR- and RT-PCR-based assays for CMV were correlated with clinical symptoms from 21 patients. Absence of circulating IE mRNA was associated with a lack of CMV-associated clinical symptoms in all 14 cases, irrespective of the presence or absence of CMV DNA. In contrast, all 7 RNA-positive samples were associated with CMV disease. Thus, RT-PCR appears to be more predictive than PCR for detection of clinically significant CMV disease in immunosuppressed patients.
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PMID:Circulating immediate-early mRNA in patients with cytomegalovirus infections after solid organ transplantation. 796 23

Placental protein 14 (PP14), an immunosuppressive molecule previously known to be expressed in the female and male reproductive tracts only, was shown to be expressed by hematopoietic cells of the megakaryocytic lineage. Northern blot analysis confirmed the induction specificity of PP14 mRNA in phorbol ester-treated K562 cells. Potent immunosuppressive activity in conditioned medium from phorbol ester-treated K562 cells was attributed to hematopoietic PP14 by anti-PP14 antibody blocking. Immunoprecipitation with anti-PP14 antibodies from conditioned medium revealed two distinct PP14 protein isoforms, designated PP14.1 and PP14.2. Polymerase chain reaction cloning and analysis demonstrated the presence of distinct mRNA counterparts to PP14.1 and PP14.2 that had not been resolved by Northern blot analyses. Hematopoietic PP14.1 mRNA corresponds in size to endometrial PP14 mRNA, whereas the smaller hematopoietic PP14.2 mRNA displays an internal in-frame 66-nucleotide deletion that can be explained by alternative splicing and predicts a 22-amino-acid deletion in the encoded gene product. Both PP14 mRNA isoforms were additionally detected by reverse transcriptase polymerase chain reaction analyses in two human megakaryocytic cell lines and in normal human megakaryocytes and platelets. PP14 mRNA was not detected by reverse transcriptase polymerase chain reaction in a panel of nonhematopoietic, nonendometrial tissues examined. The finding of hematopoietic PP14 within the megakaryocytic lineage provides an additional regulatory link between the coagulation and immune systems in normal and pathological settings.
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PMID:Hematopoietic placental protein 14. An immunosuppressive factor in cells of the megakaryocytic lineage. 799 28

We determined T-cell cytokine profiles in the epidermis, dermis, and blood of cutaneous T-cell lymphoma to differentiate whether unique cytokine profiles were associated with mycosis fungoides (MF) versus Sezary syndrome. Punch biopsy specimens from plaque stage MF (n = 7) were compared to Sezary skin (n = 3) after undergoing rapid heat-saline separation of epidermis from dermis. Normal adult skin (n = 11), neonatal foreskin (n = 4), untreated psoriatic plaques (n = 6), and normal donor peripheral blood leukocytes (n = 3) were studied as controls. Total RNA was extracted from all skin specimens, as well as peripheral blood leukocytes from MF (n = 3) and Sezary patients (n = 7), and was converted to cDNA by reverse transcriptase. Polymerase chain reaction amplification of cDNAs using interleukin 2 (IL-2), IL-4, IL-5, IL-10, and interferon gamma-specific primers was used to differentiate Th1-type responses (IL-2+ and interferon gamma +) from Th2-type responses (IL-4+, IL-5+, and IL-10+). beta-actin specific primers were included as a positive control for mRNA integrity. All MF specimens contained mRNAs for IL-2 and interferon gamma limited to epidermis but not IL-4, IL-5, or IL-10. In contrast, Sezary skin and blood showed a cytokine mRNA pattern dominated by IL-4, IL-5, and IL-10. MF blood showed a pattern similar to normal peripheral blood T cells with mixed detection of all T-helper cell cytokine mRNAs. All psoriasis samples contained mRNAs for IL-2 and interferon gamma in both epidermis and dermis with no IL-4 or IL-10 in either compartment. These findings demonstrate that the cutaneous lesions of MF are characterized by an epidermal Th1-type cytokine profile, whereas both the blood and skin of patients with Sezary syndrome is characterized by a Th2-type profile. This work suggests that differences in cytokine production may be related to the pathophysiology and clinical presentation in cutaneous T-cell lymphoma.
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PMID:Mycosis fungoides exhibits a Th1-type cell-mediated cytokine profile whereas Sezary syndrome expresses a Th2-type profile. 749 Apr 83

The ability of thermostable DNA polymerases to mediate template-dependent DNA synthesis in the presence of phenol has been examined as monitored by amplification of a specific Borrelia burgdorferi rRNA sequence. Tth DNA polymerase displayed the unique property of maintaining both DNA- and RNA-dependent DNA polymerase activities in the presence of 2%-5% (vol/vol) of phenol-saturated PBS buffer. Tth DNA polymerase mediated reverse transcriptase activity was unaffected by phenol-saturated phosphate-buffered saline concentrations as high as 15% (vol/vol). By contrast, Taq DNA Polymerase was inactive under these conditions. The ability to function in the presence of phenol can greatly simplify reverse transcriptase, PCR and reverse transcription-PCR protocols since the phenol-saturated aqueous phase of a phenol partition can be added directly to the reaction mixtures. The simplicity of the procedures described should have applicability to a broad range of basic research, clinical and forensic applications.
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PMID:A distinctive property of Tth DNA polymerase: enzymatic amplification in the presence of phenol. 813 48

Polymerase chain reaction (PCR) involves alternate denaturing and re-annealing of DNA in test samples in the presence of appropriate oligonucleotide primers complementary to opposite strands of the target DNA together with a heat-stable DNA polymerase, Mg2+ and the four nucleotide triphosphates. DNA target segments can be 'amplified' ten-millionfold by 25-35 such cycles. Even greater amplification (approximately 10(12)-fold) with enhanced specificity can be obtained by a second set of amplification cycles using a further pair of 'nested' primers sited within the DNA sequence defined by the original primers. PCR can be applied to the study of the whole range of transfusion-transmitted infections, both plasma and cell associated; RNA viruses can be analyzed if a DNA copy is made from the viral RNA by treatment with reverse transcriptase. In a transfusion context, the retroviruses (HIV-1, HIV-2, HTLV-I, HTLV-II), HCV and HBV have been the viruses most intensively subjected to PCR analysis. The advantages of PCR in this context include its ability to detect virus during the 'window period' or seronegative stages of infections and its value as a marker for viraemia and for the detection of viruses in products made from large pools of plasma. True immunity may also be differentiated from persistent infection in the presence of antibody. Similarly, PCR can overcome problems of diagnosis of acute infection caused by the presence of passively transferred antibody. Detailed strain differentiation is also possible by PCR, in conjunction with sequencing or with the aid of restriction endonucleases.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Polymerase chain reaction and transfusion microbiology. 838 93

Polymerase chain reaction [PCR, reverse transcriptase-PCR (RT-PCR)] has been used to amplify the mRNA subspecies of the plasma membrane calcium pump isoform 1 (PMCA1) in total RNA extracted from hamster tissues. Two primers were synthesized that encompass the site at which a 154-bp exon is included totally (PMCA1a), partially (PMCA1c and d), or completely excluded (PMCA1b) in the carboxy-terminal regulatory region. PCR amplification revealed two bands (PMCA1b and 1c) that are more abundant in various tissues, while Southern hybridization of the samples after PCR amplification has detected two additional mRNA variants corresponding to PMCA1a and 1d. The distribution of these mRNA variants are tissue specific and correlate well with the pump protein distribution patterns on immunoblot. Since these multiple bands on the immunoblot are not derived from proteolysis, it is suggested that they represent the PMCA1 isozymes encoded by these alternatively spliced mRNAs. To our knowledge, this is the first report to show all four alternatively spliced mRNAs that are simultaneously detected in one single RNA sample using PCR technique. Since these isozymes are different in their regulatory domain, their tissue-specific expression may be physiologically important.
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PMID:Use of the polymerase chain reaction for the detection of alternatively spliced mRNAs of plasma membrane calcium pump. 839 Aug 40

For diagnosis of Human Immunodeficiency Virus (HIV) infection by the recently developed Polymerase Chain Reaction (PCR), the two commonly used clinical samples are either the peripheral blood monocytes (PBMC) or the plasma of the infected individuals. In the former instance, DNA is extracted from PBMC. The integrated proviral DNA is then amplified using HIV specific oligonucleotide primers. In the latter instance, RNA is extracted from plasma. This is reverse transcribed in vitro into cDNA by using extraneous reverse transcriptase. This cDNA is then used as a target in PCR experiments with HIV specific primers. In contrast we have recently used DNA directly extracted from plasma of infected individuals. This DNA was used for amplification of HIV genome with primer pairs specific for HIV. An interesting outcome of this study was a model to explain the presence of DNA of HIV in the plasma. We suggest that possibly there is an alternative mode of replication of HIV. Apart from the obligatory integration of the DNA of HIV into the DNA of lymphocytes as provirus, several additional copies of the DNA are also made which remain unintegrated. These probably exist as a housekeeping repertoire of the viral genome. These DNA molecules may be released into the circulation along with the newly formed mature virion particles during the usual course of replication and release of the virus. In our experiments with direct extraction of DNA from plasma, these unintegrated DNA of HIV may act as the target for PCR to give positive signals with HIV specific primers.
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PMID:Alternative mode of replication of human immunodeficiency virus: a hypothesis. 845 60

This study developed a methodology to increase the sensitivity of enteric virus detection in tap water concentrates. Polymerase chain reaction (PCR) detection of virus in reduced volumes of virus-containing water concentrates was successful following removal of PCR inhibitory substances. Poliovirus 1 and coxsackievirus B3 were seeded into 378 l of tap water, concentrated with 1MDS filters, and reconcentrated by organic flocculation. The volume of concentrates was successfully reduced from 25 to 5 ml without loss of virus recovery. PCR detection of virus after treatment of a water concentrate (1.1 x 10(5)-fold concentration) with a Sephadex G-100 plus Chelex-100 column, or Sephadex G-50 plus Chelex-100 column, followed by heat treatment to release viral RNA, was compared with direct phenol-chloroform-isoamyl alcohol (PCI) extraction of viral RNA. The Sephadex G-50 plus Chelex-100 column did not remove inhibitory substances efficiently. The Sephadex G-100 plus Chelex-100 column could remove inhibitory substances, however, 99% of the viruses were also removed by the column. PCI extraction was found to be sufficient to remove inhibitory substances for reverse transcriptase (RT)-seminested PCR with a sensitivity of 0.2 plaque-forming units/10 microliters (0.2 PFU/l tap water).
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PMID:Increased sensitivity of poliovirus detection in tap water concentrates by reverse transcriptase-polymerase chain reaction. 860 95

We have investigated viral breakthrough during a long-term culture of HIV-1-infected cells with the non-nucleoside reverse transcriptase inhibitors (NNRTIs) 6-benzyl-1-ethoxymethyl-5-isopropyluracil (MKC-442), nevirapine and loviride (alpha-APA). When the compounds were examined for their inhibitory effects on HIV-1 (HE strain) replication in MT-4 cells on day 4 after virus infection, the 50% effective concentrations (EC50) of MKC-442, nevirapine and loviride were 9.4, 98 and 21 nM, respectively. After a 48-day culture period, MKC-442, nevirapine and loviride completely inhibited viral breakthrough at concentrations of 1, 5 and 1 microM, respectively. These concentrations were 50-100-fold higher than their EC50 values. When the cells were treated with either MKC-442 (0.04 and 0.2 microM), nevirapine (0.2 and 1 microM) or loviride (0.04 and 0.2 microM) in combination with AZT (0.005 microM), only the combination of 0.2 microM MKC-442 with 0.005 microM AZT could completely inhibit the breakthrough of HIV-1 after a 68-day culture period. Polymerase chain reaction (PCR) analysis revealed that no proviral DNA was detected in the cells treated with this combination. Except for two combinations (0.04 microM MKC-442 + 0.005 microM AZT and 0.04 microM loviride + 0.005 microM AZT), all of the viruses isolated during combination treatments had various amino acid mutations in their reverse transcriptase (RT). These results indicate that the combination treatment with a relatively high dose of MKC-442 and a low dose of AZT may have potential to suppress the emergence of drug resistance during a long-term treatment in vivo and should be further pursued in HIV-1-infected patients.
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PMID:Complete inhibition of viral breakthrough by combination of MKC-442 with AZT during a long-term culture of HIV-1 infected cells. 879 10


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