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)

Detection of viral nucleic acids in blood samples from cadavers is often difficult because of inhibition of the reverse transcriptase (RT) or polymerase chain reaction (PCR) steps by substances present in the samples. A robust method for the extraction and detection of hepatitis C virus (HCV) RNA from cadaver blood samples by polymerase chain reaction RT-PCR has been developed on the basis of the Qiagen QIAamp DNA mini kit extraction system (Basel, Switzerland). Twenty of 36 samples tested were positive for HCV RNA. Six of the 16 HCV-antibody- and RNA-negative samples contained inhibitors that were successfully removed by pretreatment of samples with the Qiagen AX matrix before extraction.
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PMID:Elimination of false-negative hepatitis C virus RNA results by removal of inhibitors in cadaver-organ donor blood specimens. 1288 7

A 58-year-old man was admitted with generalized lymphadenopathy. On admission, the patient showed polyclonal hyper-gammopathy in a blood examination, positive results in the direct/indirect Coombs test, and an elevated cold agglutinin titer. Autoimmune thrombocytopenia with a high level of platelet-associated immunoglobulin G complicated the patient's condition. An enzyme immunoassay kit for human immunodeficiency virus (HIV) recombinant proteins p24, gp41, and gp36 showed positive results. Western blot analysis showed the presence of antibodies cross-reacting with HIV p24 gag protein. HIV RNA was not detected by means of a reverse transcriptase-polymerase chain reaction assay, so the patient was not an HIV carrier. Angioimmunoblastic T-cell lymphoma (AILT) was diagnosed on the basis of lymph node biopsy specimens. We speculated that in this case some of the numerous subtypes of polyclonal gamma globulin had coincidentally cross-reacted with HIV p24. Cross-reactive phenomena with HIV in patients with systemic lupus erythematosus have been well investigated, but to our knowledge our patient is the first case of such cross-reactivity involving AILT. Physicians should pay close attention to serologic tests to determine whether the patient truly is a viral carrier.
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PMID:Angioimmunoblastic T-cell lymphoma associated with an antibody to human immunodeficiency virus protein. 1295 12

To discuss the false-positive of serological diagnostic testing for coronavirus antibody in patients with systemic lupus erythematosus(SLE), 66 normal individual and 31 SLE with non-SARS patients were detected for SARS-associated coronavirus (SARS-CoV) antibody and RNA by enzymelinked immunosorbent assays(ELISA) and reverse transcriptase-polymerase chain reaction(RT-PCR). The result showed 2/66 cases(3.0%) were positive of SARS-CoV-IgG antibody and 66 cases were negative of SARS-CoV-IgM antibody in the 66 cases healthy controls; in 31 cases with SLE, positive rates of SARS-CoV-IgG and IgM antibody were 58.1% (18/31) and 29% (9/31), respectively, in which 7 cases(22.6%) were positive of both SARS-CoV-IgG and IgM antibody. All samples of positive SARS-CoV-IgG and IgM antibody were negative by RT-PCR. The ELISA kit coated by non-purification antigen may induce the false-positive of SARS-CoV antibody in patients with SLE. This result suggested that the specificity of ELISA tests for SARS was excellent and has low false-positive rates when using SARS-CoV-IgG and IgM antibody tests. A possible cause of false-positive of SARS-CoV-IgG and IgM antibody in SLE patients is coated antigens with SARS-CoV and Vero-E6 cells in ELISA methods.
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PMID:[Analysis of false-positive associated with antibody tests for SARS-CoV in SLE patients]. 1457 97

Quantitative RT(reverse transcriptase) assay was established to detect the reverse transcriptase in plasma of thirty-four Chinese Banna minipig inbred in this work. The protocol was given in the RT kit (Roche), using HIV-1 as the positive control of the kit and supernatant of PK-15 as the PERV positive control respectively. The results show that positive reverse transcriptase reaction can be detected in the plasma of the pigs, but the levels are much lower than that of HIV-1 and lower than that of PERV in supernatant of PK-15.
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PMID:[The quantity analysis of reverse transcriptase in porcine endogenous retrovirus expressed in Banna minipig inbred]. 1471 53

Peroxisome proliferator-activated receptors (PPARs) play an important role in vascular events during progression of atherosclerosis, associated with lipid metabolism, inflammatory response and others. To clarify relationships between the expression of PPARs subtypes and regression, we studied mRNA expression of PPARs subtypes with reverse transcriptase polymerase chain reaction (RT-PCR) and immunohistochemistry, especially in centrally depressed atherosclerotic plaques (depressed plaque) in the aortas of elderly patients, and proposed morphological feature of atherosclerotic regression. Samples were separated from the depressed plaque, atheromatous plaque, and diffuse intimal thickening (DIT) in the aortas of elderly patients at autopsy and they were analyzed for RT-PCR. The depressed plaques obtained were divided into two parts: depressed area and surrounding elevated area. Total RNA was prepared, using the TRIzol Reagent, and was reverse transcribed using random hexamer primers and Thermoscript kit for RT-PCR. Immunohistochemical analysis was processed for mouse anti-PPAR gamma antibody, detected by the ABC method. 1) Decreased foam cells were found in the depressed area than in the surrounding elevated area of the depressed plaque. These foam cells were immunohistochemically positive for HAM56 and strong reactivities for PPAR gamma were found in the nuclei of macrophage-derived foam cells. PPAR gamma was also detected in the nuclei of endothelial cells and smooth muscle cells. 2) Expressions of PPAR alpha and PPAR gamma were found in the depressed plaques with RT-PCR. These expressions were found both in the depressed area and the surrounding elevated area without significant differences. 3) PPAR gamma mRNA expression both in the depressed area and the surrounding elevated area, was greater than in DIT, and was less than in the atheromatous plaque. 4) Expression of PPAR gamma mRNA was intense and increased in the surrounding elevated area than in the depressed area. 5) A significant increase of PPAR gamma expression was found in the atheromatous plaque than in the DIT. 6) There was no significant difference of PPAR alpha mRNA expression between the depressed area and the surrounding elevated area. The depressed plaque has been considered to be a morphological characteristic of regression in recent studies. Expression of mRNA for PPARs was detected in the depressed plaque as well as the atheromatous plaque, furthermore, there were different expressions and intensity of PPARs between the depressed area and the surrounding elevated area of the depressed plaque. These findings suggest that the expression of PPARs may be involved not only in the progression of atherosclerosis but also in regression.
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PMID:[Atherosclerotic regression in the aortas of elderly. (4). Expression of peroxisome proliferator-activated receptors with references of centrally depressed atherosclerotic plaque]. 1499 25

The piezoelectric quartz crystal resonators modified with oligonucleotide probes were used for detection of hepatitis C virus (HCV) in serum. The gold electrodes on either rough or smooth surface crystals were modified with a self-assembled monolayer of cystamine. After activation with glutaraldehyde, either avidin or streptavidin were immobilized and used for attachment of biotinylated DNA probes (four different sequences). Piezoelectric biosensors were used in a flow-through setup for direct monitoring of DNA resulting from the reverse transcriptase-linked polymerase chain reaction (RT-PCR) amplification of the original viral RNA. The samples of patients with hepatitis C were analyzed and the results were compared with the standard RT-PCR procedure (Amplicor test kit of Roche, microwell format with spectrophotometric evaluation). The piezoelectric hybridization assay was completed in 10 min and the same sensing surface was suitable for repeated use.
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PMID:Piezoelectric biosensors for real-time monitoring of hybridization and detection of hepatitis C virus. 1504 Dec 11

Telomeres cap chromosome ends and are pivotal for DNA stability. Deregulation of the telomere stabilising enzyme telomerase in malignancy has implications in diagnosis, prognosis and therapeutics of cancer. Quantification of the expression of the telomerase catalytic subunit, hTERT, using the LightCycler TeloTAGGG hTERT Quantification kit is not optimal for analysis of chronic myeloid leukemia (CML) samples. The internal control, porphobilinogen deaminase (PBGD) is amplified in a separate tube to hTERT and has an unstable genomic localisation of 11q23. Our laboratory thus developed a real-time reverse transcriptase polymerase chain reaction which co-amplifies hTERT and either mitochondrial single-stranded DNA binding protein 1 (ssBP1) or ubiquitin C (UBC).
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PMID:Real-time quantitative RT-PCR for human telomere elongation reverse transcriptase in chronic myeloid leukemia. 1523 74

Follow-up of recurrent differentiated thyroid carcinoma involves the measurement of serum thyroglobulin (Tg). However, Tg autoantibodies are present in a high proportion of thyroid carcinoma patients (up to 25%) and these can interfere with the Tg immunoassays. To overcome this obstacle, investigators have used real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR) to measure Tg mRNA in the blood of patients with differentiated thyroid cancer, with varying degrees of success. In the present study, we demonstrate the first reported use of the PAXgene Blood RNA collection tube and extraction kit method for the preparation of RT-PCR-quality RNA with subsequent deployment of the latter in the development of a specific, sensitive, and reproducible Taqman assay for the detection and quantification of thyroglobulin mRNA. Beta-actin mRNA was also assayed and results are expressed as a ratio of Tg to beta-actin mRNA. The intra-assay coefficient of variations (CVs) for Tg and beta-actin mRNA assay were 27.7% and 25.4%, respectively. Inter-assay CVs were 20.8% and 28.8%, respectively, for the two assays. Tg mRNA was detected in all cancer subjects (n = 42) and healthy individuals (n = 20). Tg mRNA was significantly higher in cancer patients than in the healthy subjects (0.00169 +/- 0.00013 vs. 0.00051 +/- 0.00015; P<0.0001). Fourteen cancer patients had detectable levels of serum Tg, and Tg mRNA levels tended to be higher in these than in cancer subjects with undetectable serum Tg (0.00188 +/- 0.00021 vs. 0.00157 +/- 0.000178; P = 0.08). Circulatory Tg mRNA measurement may serve a useful role in the assessment of thyroid cancer.
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PMID:Real-time quantitative PCR measurement of thyroglobulin mRNA in peripheral blood of thyroid cancer patients and healthy subjects. 1525 54

Diabetic retinopathy is the commonest complication of diabetes and is the biggest single cause of registered blindness in the UK. No biochemical tests exist to determine the precise state and rate of change of the eyes in the diabetic patient. In the present study, using real-time quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we measured mRNA encoding the retina-specific pigment protein rhodopsin (RHO) in the peripheral blood of healthy individuals (n = 20) and diabetic patients (n = 46) with and without retinopathy. Beta-actin mRNA was also assayed and results are expressed as a ratio of RHO to beta-actin mRNA. Peripheral blood was taken by venipucture directly into PAXgene Blood RNA collection tubes and RNA extracted by use of the PAXgene Blood RNA extraction kit, as per the manufacturer's (Qiagen) instructions. Diabetic patients were divided into three groups defined by the severity of retinopathy as assessed by fundoscopy: A, diabetic without retinopathy; B, background retinopathy; and C, preproliferative retinopathy. Medians of the ratios between groups were compared. RHO mRNA was successfully detected and quantified in peripheral blood in all healthy and diabetic groups, with levels shown to be significantly higher in diabetic patients than in healthy controls (2.54 x 10(-5) vs. 1.29 x 10(-5); P = 0.002). Significant differences in RHO mRNA levels were also seen between healthy control subjects and diabetic groups A (2.52 x 10(-5); P = 0.022), B (1.98 x 10(-5); P = 0.028), and C (5.08 x 10(-5); P = 0.002). The results suggest that there is an increase in circulatory RHO mRNA with the severity of diabetic retinopathy.
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PMID:Real-time quantitative PCR measurement of circulatory rhodopsin mRNA in healthy subjects and patients with diabetic retinopathy. 1525 55

In order to elucidate the mechanism of fungal heterokayosis, a wild type strain of Fusarium oxysporum f. sp. vasinfectum was isolated from the cotton field in Anyang, Henan Province. Through single hyphal-tip isolation, a heterokaryon, Ag149, was obtained, and its two different phenotypic segregants, Ag149-I and Ag149-III, were separated from the mutated sectors on colony of the heterokaryon. They have remarkable differences on color of colony, morphology of hypha and pathogenicity. After analyzing by RAPD on their nuclear DNA with 100 random primers, no polymorphic difference was found among them. On going to find the different expressed gene, the mRNA differential display method was performed. Two kinds of reverse transcriptase AMV and MMLV, and a kit which consists of three kinds of 3' terminal anchor primers and eight kinds of 5' terminal arbitrary primers were used in differential display PCR (DD-PCR). Total RNA as template was reverse transcribed into corresponding cDNA by 3' terminal anchor primers, and the cDNA were amplified by polymerase chain reaction with a set of one same 3' anchor primer and one 5' arbitrary primer. The PCR products were then resolved on denaturing polyacylamimide gel, and the cDNA bands were visualized by silver staining. Among the 144 PCR products, 19 differentially expressed cDNA fragments ranged from 300 bp to 700 bp were purified. All of them were ligated to pGEM-T vector respectively for sequencing and Rev-Northern blotting. Two cDNA fragments (G5 and C6) were observed to be positive after Rev-Northern blotting. The C6 was highly expressed in the heterokaryon Ag149 and its segregant Ag149-I. It is 564 bp in length and can be predicted 77 amino acids from the beginning of the 3rd to 233th base, and then searched from GenBank. The amino acid sequence of C6 shared homologies with the 6th subunit of NADH dehydrogenase found in some bacteria, plants and animals at 30% - 70% level. While the G5 was highly expressed in Ag149 and its segregant Ag149-III. It is 432 bp in length and can be predicted 101 amino acids from the beginning of the 2nd to 304th base, and the amino acid sequence is 35% homologies comparing with the tetracycline efflux protein (OtrB) of Streptomyces rimosus. We also checked these two kinds of the pGEM-T vector harboring cDNA fragments (C6 and G5) by Southern blotting with their nuclear DNA and mitochondrial DNA (mtDNA) separately. The positive identification signals only appeared from nuclear DNA,and it addressed that C6 and G5 locate on their nuclear genome. The result indicated that the difference between the heterokaryon and its segregants is distinct on gene transcriptional level. Thus a molecular evidence for the formation of heterokaryon in filamentous fungi was provided.
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PMID:[Transcriptional differences between a heterokaryon and its segregants of Fusarium oxysporum f. sp. vasinfectum]. 1547 7


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