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)

Acute inflammation is characterized by increased production of acute phase proteins in the liver. The induction of the hepatocytic response is primarily mediated through soluble cytokines such as IL-1, IL-6, TNF-alpha, and transforming growth factor beta, which bind to specific cell surface receptors and regulate gene expression of acute-phase proteins. Hepatoma cell lines, such as HepG2, represent a model system for studying acute-phase protein synthesis. HepG2 is induced to produce a variety of acute-phase proteins, including alpha 1-antitrypsin, alpha 1-antichymotrypsin, fibrinogen, alpha 1-acid glycoprotein, and haptoglobin, upon stimulation with cytokines. Analysis of HepG2 by reverse transcriptase PCR indicated that this cell line synthesized mRNA specific for the human C5a receptor (CD88). Flow cytometric analysis of HepG2 cells indicated that these cells bound anti-CD88 Ab, thus confirming our RT-PCR data by demonstrating that these cells also express the C5a receptor. Because C5a has been shown to be a potent mediator of inflammation and HepG2 cells express CD88, we assessed the possibility that C5a was capable of stimulating acute-phase protein synthesis by HepG2 cells. The results indicate that binding of human C5a to CD88 on HepG2 cells resulted in an increased production of alpha 1-antitrypsin- and alpha 1-antichymotrypsin-specific mRNA as assayed by RT-PCR. Analysis of culture supernatants derived from C5a-stimulated HepG2 cells showed an increased production of alpha 1-antitrypsin as measured by solid-phase ELISA. alpha 1-antitrypsin production by HepG2 cells was a direct result of C5a stimulation as evidenced by the fact that anti-C5a receptor Ab inhibited the response. These results suggest that C5a may be an important mediator of APP production in the regulation of the inflammatory response.
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PMID:Expression of functional receptors for human C5a anaphylatoxin (CD88) on the human hepatocellular carcinoma cell line HepG2. Stimulation of acute-phase protein-specific mRNA and protein synthesis by human C5a anaphylatoxin. 754 17

In vitro experiments have suggested that interleukin (IL)-6 may contribute to human immunodeficiency virus (HIV) burden and to immunological abnormalities in HIV-infected patients. We had the opportunity to directly address this question in vivo through the virological and immunological monitoring of HIV-infected patients treated with an anti-IL-6 monoclonal antibody (mAb) for a lymphoma (ANRS 018 trial). Sixteen courses of anti-IL-6 mAb administration, performed in 11 patients, were studied. All patients were at a late stage of HIV infection. The HIV load and the immunological status were determined at the initiation of each course and at its end, 21 days later. The mAb induced no significant change of HIV load, as evaluated by p24 antigenemia, plasma viremia, and quantification of circulating HIV RNA by reverse transcriptase-polymerase chain reaction and branched DNA techniques. The anti-IL-6 mAb also did not affect CD4+, CD8+, and CD19+ circulating cell counts, nor the serum concentrations of sIL-2R and of sCD8. In contrast, the mAb completely abrogated acute-phase reaction, as demonstrated by the normalization of C-reactive protein and fibrinogen circulating levels (p = 0.013 and p = 0.008, respectively). It increased serum albumin concentration. The latter effect was restricted to patients with a spontaneously low albuminemia (p = 0.01). It decreased B-lymphocyte hyperactivity, as reflected by decreased IgG and IgA serum levels (p = 0.008 and p < 0.001, respectively), and by a decreased production of IgG in vitro (p = 0.017). In contrast, the IgM hyperproduction was not affected by the mAb. Therefore, increased IL-6 production in HIV-infected patients at a late stage of the infection may not stimulate HIV replication in vivo, but it may represent a key mechanism contributing to the metabolic and immunological dysbalance of the disease.
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PMID:In vivo role of IL-6 on the viral load and on immunological abnormalities of HIV-infected patients. 852 34

The mechanism by which platelets regulate the function of integrin alphaIIbbeta3 (or GPIIb/IIIa), the platelet fibrinogen receptor, is unknown but may involve the binding of proteins or other factors to integrin cytoplasmic domains. To identify candidate cytoplasmic domain binding proteins, we screened a human fetal liver cDNA library in the yeast two-hybrid system, using the alphaIIb cytoplasmic domain as "bait," and isolated a novel 855-base pair clone. The open reading frame encodes a novel 191-amino acid polypeptide (termed CIB for calcium- and integrin-binding protein) that appears to be specific for the cytoplasmic domain of alphaIIb, since it does not interact with the alphav, alpha2, alpha5, beta1, or beta3 integrin cytoplasmic domains in the yeast two-hybrid system. This protein has sequence homology to two known Ca2+-binding regulatory proteins, calcineurin B (58% similarity) and calmodulin (56% similarity), and has two EF-hand motifs corresponding to the two C-terminal Ca2+ binding domains of these proteins. Moreover, recombinant CIB specifically binds 45Ca2+ in blot overlay assays. Using reverse transcriptase-polymerase chain reaction and Western blot analysis, we detected CIB mRNA and protein ( approximately 25 kDa), respectively, in human platelets. An enzyme-linked immunosorbent assay performed using either immobilized recombinant CIB or monoclonal antibody-captured alphaIIbbeta3 indicates a specific interaction between CIB and intact alphaIIbbeta3. These results suggest that CIB is a candidate regulatory molecule for integrin alphaIIbbeta3.
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PMID:Identification of a novel calcium-binding protein that interacts with the integrin alphaIIb cytoplasmic domain. 903 May 14

Quantitative reverse transcriptase PCR (RT-PCR) is a sensitive method for the measurement of mRNA copy number. However, the methodology has gained a reputation for poor reproducibility, leading to concern over the validity of much of the data generated using this technique. We have developed two variants of quantitative competitive RT-PCR using a synthesized RNA as an internal standard to measure precisely the relative levels of alpha-, beta- and gamma-fibrinogen mRNAs in the four lobes of the rat liver. In the first of these variants we altered only the amount of total RNA in the RT-PCR reaction, keeping the amount of internal standard RNA and the number of PCR cycles constant. In the second variant only the number of PCR cycles was altered, and the amounts of total RNA and standard RNA were kept constant. Both variants of RT-PCR allowed calculation of the number of mRNA copies, which did not differ significantly between the two techniques. Of the two variants, the second gave better reproducibility, and the intra-assay coefficient of variation for this technique was 14% (n = 20). Using these two variants we have shown that there are different numbers of fibrinogen mRNAs in the four liver lobes for each of the three genes (alpha-fibrinogen F = 14.64, P = 0.0003; beta-fibrinogen F = 3.74, P = 0.04; gamma-fibrinogen F = 3.75, P = 0.04). In conclusion, by using two variants of quantitative competitive RT-PCR we have shown that this technique can be used to give reproducible results, and the low intra-assay coefficient of variation suggests that quantitative RT-PCR should be the technique of choice for accurate measurement of mRNA copy number.
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PMID:Two variants of quantitative reverse transcriptase PCR used to show differential expression of alpha-, beta- and gamma-fibrinogen genes in rat liver lobes. 903 65

We have previously described the private or family platelet antigen, Gro(a), which was identified in a case of neonatal alloimmune thrombocytopenia. The Gro(a) antigen was found to be located on the GP IIIa (beta3) subunit of the GP IIb/IIIa complex, the most prominent fibrinogen receptor of platelets. Initial experiments to characterize the Gro(a) antigen at the molecular genetic level were unsuccessful. We therefore decided to use a different strategy to unravel the molecular basis of this antigen. Platelet GP IIIa mRNA of a Gro(a+) and a Gro(a-) donor was amplified with suitable primers in a reverse transcriptase-polymerase chain reaction (RT-PCR) and subjected to single-strand conformational polymorphism (SSCP) analysis. Three regions of the amplified GP IIIa cDNA derived from the Gro(a+) donor showed a different SSCP pattern when compared to that of the Gro(a-) donor. Direct nucleotide sequence analysis of these three segments revealed that two of them contained silent substitutions, A1163C, A1553G and G1565A. The first and the latter changes were described previously. In the third segment a G1996A mutation was found, predicting an arginine --> histidine substitution at position 633 of the mature glycoprotein. PCR-ASRA (allele-specific restriction enzyme analysis) performed on cDNA as well as on genomic DNA with the restriction enzyme MaeIII showed that the His633 form of GPIIIa is restricted to the Gro(a+) phenotype. The observed mutation is three amino acids upstream of the mutation underlying the HPA-8/Sr system (Arg636Cys), suggesting this region of GP IIIa to be susceptible for mutations. Moreover, the presence of a silent mutation and two low-frequency forms of the silent polymorphisms strongly suggests that the G1996A mutation did not occur in a direct ancestral allele.
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PMID:The Arg633His substitution responsible for the private platelet antigen Gro(a) unravelled by SSCP analysis and direct sequencing. 916 97

Small changes in plasma fibrinogen concentration are often clinically important. To detect changes in fibrinogen mRNA level, we have designed a novel quantitative competitive reverse transcriptase (RT) PCR system, based on our recent understanding that an important factor causing imprecision with RT PCR is a change in the initial ratio of target to standard RNA templates during reverse transcription. The system comprises: (1) titration of a fixed amount of standard RNA with four different amounts of total RNA in each tube during cDNA synthesis; (2) amplification of each of the four cDNAs with four consecutive PCR cycles; and (3) quality control reactions in which synthesised quality control RNA (rather than total RNA) is added to the standard RNA under the same RT PCR conditions as the assay reaction. To obtain reproducible data, we suggest three criteria for analysis of RT PCR results. Employing these criteria, reproducibility of RT PCR was markedly improved with an inter-assay CV of 6.5% (n=5). Using this system, we are able to detect reduction in mRNA levels of all fibrinogen genes caused by dietary protein restriction in rats after weaning (alpha 39%, p= 0. 028; beta 55%, p = 0.017; gamma 35 %, p= 0.038), with a parallel reduction of plasma fibrinogen concentration (mean (+/-SD), 157(+/-19) versus 130(+/-14) mg/dl, p=0.006).
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PMID:A novel highly reproducible quantitative competitve RT PCR system. 940 44

Synthesis of a number of rat liver proteins, including albumin, fibrinogen, apolipoprotein AI, and transferrin, is elevated in the nephrotic syndrome (NS). Increased synthesis of these proteins is regulated at the transcriptional level and occurs in the context of increased mRNA encoding each protein. Changes in albumin, fibrinogen, apolipoprotein AI, and transferrin mRNA levels in total cellular RNA isolated from the livers of normal rats and rats with passive Heymann nephritis were measured using a kinetically monitored, reverse transcriptase-initiated PCR (kRT-PCR) assay. The kRT-PCR assay rapidly quantitated changes in rat liver mRNA levels with an accuracy comparable to that of more labor-intensive mRNA quantitation methods. The relative levels of beta-actin, apolipoprotein AI, fibrinogen, and albumin mRNAs were very similar in total cellular RNA isolated from rat liver versus H4C3 hepatocytes in culture, suggesting that the H4C3 hepatocyte is an appropriate model for studying expression of genes encoding proteins secreted by the liver. Taken together, the results demonstrate the feasibility of using the kRT-PCR assay for isolation and characterization of a soluble factor responsible for elevated synthesis of hepatocyte mRNAs associated with the nephrotic syndrome.
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PMID:Rat liver transcript profiling in normal and disease states using a kinetic polymerase chain reaction assay. 948 Jul 87

Italian investigations have shown an association between Chlamydia pneumoniae infection and atherosclerosis. With the use of several diagnostic techniques, including serology, a microimmunofluorescence test, and nucleic acid amplification methods, a temporal association was found between acute C. pneumoniae reinfection and acute myocardial infarction, suggesting that an acute infection superimposed on a chronic or latent infection may trigger the onset of acute myocardial infarction. C. pneumoniae but not Helicobacter pylori or Mycoplasma pneumoniae was found in atherosclerotic plaques of abdominal aortic aneurysms and the carotid artery. A reverse transcriptase-polymerase chain reaction process confirmed the presence of viable C. pneumoniae in carotid atheromas. Nucleic amplification of peripheral blood mononuclear cells may enable the identification of subjects carrying C. pneumoniae in the vascular wall. Macrolide treatment reduced fibrinogen and C-reactive protein plasma levels and C. pneumoniae burden in patients with atherosclerotic diseases.
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PMID:Chlamydia pneumoniae detection in atherosclerotic plaques in Italy. 1102 90

Congenital afibrinogenemia is a rare autosomal recessive disorder characterized by the complete absence of plasma fibrinogen and by a bleeding tendency ranging from mild to moderately severe. Beside a deletion of the almost entire Aalpha-chain gene, only 2 missense mutations in the C-terminal domain of the Bbeta-chain have been very recently described as being associated with afibrinogenemia. We studied a Pakistani patient with unmeasurable plasma levels of functional and immunoreactive fibrinogen. Sequencing of the fibrinogen genes revealed a homozygous G-->A transition at position +5 of intron 1 of the gamma-chain gene. The predicted mutant fibrinogen gamma-chain would contain the signal peptide, followed by a short stretch of aberrant amino acids, preceding a premature stop codon. To demonstrate the causal role of the identified mutation, we prepared expression vectors containing a region of the fibrinogen gamma-chain gene spanning from exon 1 to intron 4 and carrying either a G or an A at position +5 of intron 1. Transient transfection of the mutated plasmid in HeLa cells, followed by RNA extraction and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, allowed us to demonstrate the production of an erroneously spliced messenger RNA (mRNA), retaining intron 1, as shown by direct sequencing. A normal splicing occurred in HeLa cells transfected with the wild-type plasmid. This is the first report of a mutation in the fibrinogen gamma-chain gene causing afibrinogenemia and indicates that, in addition to the Aalpha and Bbeta-chain genes, the gamma-chain gene must also be considered in mutation screening for afibrinogenemia.
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PMID:Afibrinogenemia: first identification of a splicing mutation in the fibrinogen gamma chain gene leading to a major gamma chain truncation. 1100 2

Congenital afibrinogenemia is a rare inherited coagulopathy, characterized by very low or unmeasurable plasma levels of immunoreactive fibrinogen. So far, 25 mutations have been identified in afibrinogenemia, 17 in the Aalpha, 6 in the gamma, and only 2 in the Bbeta fibrinogen-chain genes. Here, 2 afibrinogenemic probands, showing undetectable levels of functional fibrinogen, were screened for causative mutations at the genomic level. Sequence analysis of the 3 fibrinogen genes disclosed 2 novel homozygous mutations in introns 6 and 7 of the Bbeta-chain gene (IVS6 + 13C > T and IVS7 + 1G > T), representing the first Bbeta-chain gene splicing mutations described in afibrinogenemia. The IVS6 + 13C > T mutation predicts the creation of a donor splice site in intron 6, whereas the IVS7 + 1G > T mutation causes the disappearance of the invariant GT dinucleotide of intron 7 donor splice site. To analyze the effect of these mutations, expression plasmids containing Bbeta-chain minigene constructs, either wild-type or mutant, were transfected in HeLa cells. Assessed by semiquantitative analysis of reverse transcriptase-polymerase chain reaction products, the IVS7 + 1G > T mutation resulted in multiple aberrant splicings, while the IVS6 + 13C > T mutation resulted in activation of a new splice site 11 nucleotides downstream of the physiologic one. Both mutations are predicted to determine protein truncations, supporting the importance of the C-terminal domain of the Bbeta chain for fibrinogen assembly and secretion.
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PMID:Congenital afibrinogenemia: first identification of splicing mutations in the fibrinogen Bbeta-chain gene causing activation of cryptic splice sites. 1239 40


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