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

---

Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Salmonella enterica serovar Typhimurium secretes virulence factors for invasion called Sip proteins or Sips into its hosts through a type III secretion system (T3SS). In the absence of a host, S. enterica induces Sip secretion in response to sucrose or simple salts, such as NaCl. We analyzed induction of host-independent Sip secretion by monitoring protein secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), assembly of needle complexes by electron microscopy, and transcription of virulence regulatory genes by quantitative reverse transcriptase PCR (real-time PCR). SDS-PAGE showed that addition of sucrose or simple salts, such as NaCl, to the growth medium induced Sip secretion without altering flagellar protein secretion, which requires a distinct T3SS. Electron microscopy confirmed that the amount of secreted Sips increased as the number of assembled needle complexes increased. Real-time PCR revealed that added sucrose or NaCl enhanced transcription of hilA, hilC, and hilD, which encode known regulators of Salmonella virulence. However, epistasis analysis implicated HilD and HilA, but not HilC, in the direct pathway from the salt stimulus to the Sip secretion response. Further analyses showed that the BarA/SirA two-component signal transduction pathway, but not the two-component sensor kinase EnvZ, directly activated hilD and hilA transcription and thus Sip secretion in response to either sucrose or NaCl. Finally, real-time PCR showed that salt does not influence transcription of the BarA/SirA-dependent csrB and csrC genes. A model is proposed for the major pathway in which sucrose or salt signals to enhance virulence gene expression.
...
PMID:Signal pathway in salt-activated expression of the Salmonella pathogenicity island 1 type III secretion system in Salmonella enterica serovar Typhimurium. 1844 Oct 68

The insulin-like growth factor (IGF-I) gene (GenBank accession no. AY247412) of Qiantang River triangular bream (Megalobrama terminalis) was cloned for the first time from the liver by reverse transcriptase polymerase chain reaction. The gene was inserted into pMD 18-T vector to construct the recombinant plasmid pMD 18-T/IGF-I. Sequence analysis indicated that the IGF-I cDNA consisted of 486 nucleotides encoding 161 amino acids, which spanned the complete signal peptide, mature peptide (including B, C, A, and D domains), and E-domain. Analysis of the E domain indicated that triangular bream IGF-I gene belonged to the IGF-I Ea-2 subtype. To construct the expression plasmid, the IGF-I gene was subcloned into prokaryotic expressing vector pGEX-4T-1. The recombinant plasmid pGEX-4T-1/IGF-I was transformed into Escherichia coli BL21 (DE3), and the transgene expression was observed after being induced with isopropylthiogalactoside. The results of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting indicated that the recombinant fusion protein had immune activity, and the molecular weight was about 47 kDa. The results of SDS-PAGE and thin-layer scanning showed that the yield of fusion protein had been enlarged with prolonging time. When the time of induced expression was 1, 2, 3, 4, 5, and 6 h, the expression amount was approximately 1.4, 4.3, 8.1, 11.3, 16.3, and 18.8% of total bacterial protein, respectively.
...
PMID:Cloning of Qiantang River triangular bream (Megalobrama terminalis) IGF-I gene and expression of the recombinant pre-IGF-I in Escherichia coli. 1850 8

A mannose/glucose-specific lectin has been purified from Chinese evergreen chinkapin (Castanopsis chinensis) seeds, one of the most popular foods in East Asia. This lectin, designated as CCL, exhibited hemagglutinating activity in mouse and rabbit erythrocytes. It displayed a single band with a molecular mass of 29 kDa in SDS-PAGE and a 120-kDa peak in gel-filtration on Superdex-200. Its hemagglutinating activity was stable in the pH range 6-12 and at temperatures below 60 degrees C. The N-terminal amino acid sequence of CCL differed from those of other lectins in the same family. CCL inhibited the proliferation of HepG2 cells and adult emergence in fruitflies. CCL exhibited mitogenic activity toward mouse splenocytes, and induced nitric oxide production from mouse peritoneal macrophages but was devoid of inhibitory activity toward mycelial growth and HIV-1 reverse transcriptase.
...
PMID:A mannose/glucose-specific lectin from Chinese evergreen chinkapin (Castanopsis chinensis). 1857 Aug 98

Retroviral vectors are powerful tools for the introduction of transgenes into mammalian cells and for long-term gene expression. However, their application is often limited by a rapid loss of bioactivity: retroviruses spontaneously loose activity at 37 degrees C, with a half-life of 4 to 9 h depending on the retrovirus type. We sought to determine which components of the retrovirus are responsible for this loss in bioactivity and to obtain a quantitative characterization of their stability. To this end, we focused on RNA and viral proteins, two major components that we hypothesized may undergo degradation and negatively influence viral infectivity. Reverse transcription PCR (RT-PCR) targeting RNA encoding portions of the viral genome clearly demonstrated time-dependent degradation of RNA which correlated with the loss in viral bioactivity. Circular dichroism spectroscopy, SDS-PAGE and two-dimensional SDS-PAGE analyses of viral proteins did not show any change in secondary structure or evidence of proteolysis. The mechanism underlying the degradation of viral RNA was investigated by site-directed mutagenesis of proteins encoded by the viral genome. Reverse transcriptase and protease mutants exhibited enhanced RNA stability in comparison to wild type recombinant virus, suggesting that the degradation of RNA, and the corresponding virus loss of activity, is mediated by the reverse transcriptase enzyme.
...
PMID:Moloney murine leukemia virus decay mediated by retroviral reverse transcriptase degradation of genomic RNA. 1870 68

To produce the reverse transcriptase of moloney murine leukemia virus (MMLV-RT) through gene recombination, MMLV-rt gene was amplified by polymerase chain reaction (PCR) with specifically designed primers bearing restriction enzyme sites. Five mutation sites increasing the solution of the target protein were introduced through Site-directed mutation. After verification by sequencing, the gene was cloned into the expression vector pET15b to construct the recombinant plasmid pET15b-MMLV-rt. Purified MMLV-RT was obtained by affinity chromatography (Ni3+-NTA beads). Molecular weight and purity of MMLV-RT were analyzed with SDS-PAGE. Enzyme activity was characterized with RT-PCR. We successfully constructed the recombinant plasmid pET15b-MMLV-rt and obtained the MMLV-RT fusion protein with 6His on the N-terminus. Recombinant protein was purified through Ni3+-NTA beads based affinity chromatography, the purity of which was 96%. The Activity of the enzyme was high. MMLV-RT of 96% purity was obtained with the prokaryotic expression technique, which serves as the basis for mass production of this enzyme.
...
PMID:[Prokaryotic expression and purification of moloney murine leukemia virus reverse transcriptase and verification of the activity]. 1872 16

Otosclerosis is a primary bone remodeling disorder of the human otic capsule and is associated with persistent measles virus infection. The human cellular receptor of measles virus is the membrane cofactor protein (MCP, CD46), which has 14 well-described splicing variants. Unique CD46 expression pattern of the otic capsule and the stapes footplate may determine the susceptibility for persistent measles virus infection. A total of 51 surgically removed ankylotic stapes footplates were analyzed by histopathological and molecular biological methods, respectively. Nucleic acids were extracted. Measles virus sequences were detected by nucleoprotein RNA-specific reverse transcriptase polymerase chain reaction (RT-PCR). Alternatively spliced RNA of CD46 isoforms was amplified by RT-PCR; cDNA amplimers were separated by SDS poly-acrylamide gel electrophoresis and were purified from the gel. Complementary DNA of CD46 isoforms was restricted by endonuclease enzymes having CD46-specific recognition sites. The presence of viral RNA was associated exclusively with the histopathological diagnosis of otosclerosis; the stapes specimens with negative measles virus belonged to non-otosclerotic stapes fixations. All specimens (N = 51) were characterized by the consecutive expression of five CD46 variants (c, d, e, f and one shorter unidentified isoform). Histologically confirmed ostosclerotic specimens (N = 21) were characterized by increased expression levels of variant "f" and the unknown isoform. Increased expression levels of these isoforms and special CD46 expression pattern of the human otic capsule might produce modified or pathological intracellular signalization that could create the possibility of persistent measles virus infection.
...
PMID:Restriction analysis of otosclerosis-associated CD46 splicing variants. 1959 33

Influenza A virus matrix protein (M1) is encoded by a spliced mRNA derived from RNA segment 7 and plays an important role in the virus life cycle. In the present study, we extracted the viral genome RNAs from allantoic fluid of 9-day-old embryonated chicken eggs infected with swine influenza A virus (SIV) H3N2 subtype and amplified the SIV M1 gene by reverse transcriptase-polymerase chain reaction using the isloated viral genome RNAs as template. The amplified cDNA was cloned into an expression vector pET-28a (+) (designated pET-28a-M1) and confirmed by DNA sequencing analysis. We then transformed the plasmid pET-28a-M1 into Escherichia coli BL21 strain for heterologous expression. The expression of M1 was induced by 1mM isopropyl-beta-D-thiogalactopyranoside. SDS-PAGE analysis of the induced bacterial cells revealed that the recombinant M1 protein was expressed in high yield level. Next, we purified the expressed recombinant M1 using Ni2+ affinity chromatography and immunized Wistar rat with the purified M1 protein for producing polyclonal antibodies specific for M1. Western blotting analysis showed that the produced antibodies were capable of reacting with M1 protein expressed in Escherichia coli as well as that synthesized in SIV-infected cells. We further cloned the amplified M1 cDNA into a eukaryotic expression plasmid p3xFLAG-CMV-7.1 to construct the recombinant plasmid p3xFLAG-CMV-M1 for expressing M1 in eukaryotic cells. Western blotting analysis revealed that the M1 protein was expressed in p3xFLAG-CMV-M1-transfected Vero cells and recognized by the produced anti-M1 antibodies. Using the produced anti-M1 antibodies, we analyzed the kinetics of M1 protein in the virus-infected cells during influenza virus infection and estimated the possibility of M1 as an indicator of influenza virus replication. The recombinant M1 protein, anti-M1 antibodies and recombinant expression plasmids would provide useful tools for studies of biological function of M1 protein and the basis of SIV replication.
...
PMID:[Cloning and characterization of M1 gene of H3N2 subtype swine influenza virus]. 1967 Jun 34

The HIV reverse transcriptase (RT) is an important antiviral target for the chemotherapy of AIDS because of its key role in virus replication. Nevirapine is a first generation of non-nucleoside reverse transcriptase inhibitors (NNRTIs), which is usually used for the therapy of AIDS. In this study, a high-performance analytical method based on capillary electrophoresis (CE) to investigate interactions between HIV RT and nevirapine was developed. Samples containing HIV RT and nevirapine at various ratios were incubated at 37 degrees C for 45 min and then separated by CE with Tris-acetate buffer at pH 7.3 containing 0.15% SDS. Both qualitative and quantitative characterizations of the binding were determined by CE for the first time. The binding constants of the interactions between HIV RT and nevirapine were calculated as (3.25+/-0.16)x10(4) and (1.25+/-0.07)x10(2) M(-1) by Scatchard analysis. HIV RT and nevirapine have two binding sites. The presented methodology should be generally applicable to study the interactions between HIV RT and nevirapine quantitatively and qualitatively.
...
PMID:Study on the interaction between HIV reverse transcriptase and its non-nucleoside inhibitor nevirapine by capillary electrophoresis. 2045 46

An antiproliferative ribonuclease with a new N-terminal sequence was purified from fruiting bodies of the edible wild mushroom Russula delica in this study. This novel ribonuclease was unadsorbed on DEAE-cellulose, but absorbed on SP-Sepharose and Q-Sepharose. It had a molecular mass of 14 kDa as judged by fast protein liquid chromatography on Superdex 75 and SDS-polyacrylamide gel electrophoresis. Its optimal pH and optimal temperature were pH 5 and 60 degrees , respectively. The ranking of its activity toward various polyhomoribonucleotides was poly C > poly G > poly A > poly U. It could inhibit proliferation of HepG2 and MCF-7 cancer cells with an IC50 value of 8.6 microM and 7.2 microM, respectively. It was devoid of antifungal and HIV-1 reverse transcriptase inhibitory activity.
...
PMID:An antiproliferative ribonuclease from fruiting bodies of the wild mushroom Russula delica. 2046 40

Epidermal mRNA for interleukin 1beta (IL-1beta) has been shown to be increased following exposure of mouse skin to sensitizing compounds. In addition, this early upregulation of IL-1beta was specific for contact sensitizers, while expression of IL-1beta was unaffected by irritants. Langerhans cells are the major source of IL-1beta within the epidermis in the induction phase of skin sensitization. Since the isolation of Langerhans cells from skin biopsies results only in low frequencies, we decided to use dendritic cells (DCs) generated from peripheral blood as Langerhans cell equivalents to investigate the ability of five contact sensitizers and one irritant to induce IL-1beta gene expression in vitro. For our studies we cultivated DCs in serum-free medium supplemented with granulocyte/macrophage-colony stimulation factor (GM-CSF) and interleukin 4 (IL-4). The DCs showed a typical dendritic morphology, a characteristic expression of surface markers and high stimulatory capacity for autologous T cells. 5-day-old DCs were incubated with subtoxic concentrations of the contact sensitizers pentadecyl-catechol, 2,4,6-trinitrobenezene sulfonic acid, 2,4-dinitrofluorobenzene, NiSO(4), K(2)Cr(2)O(7) and the irritant sodium dodecyl sulfate. IL-1beta mRNA expression was detected by using the reverse transcriptase-polymerase chain reaction (RT-PCR) technique and non-radioactive hybridization procedures. For all contact sensitizers, expression of IL-1beta mRNA increased, whereas treatment with the irritant SDS had no significant effect on IL-1beta expression. Thus we developed an in vitro system, which may be useful to evaluate allergic potentials of chemicals and products.
...
PMID:In vitro model for contact sensitization: II. Induction of IL-1beta mRNA in human blood-derived dendritic cells by contact sensitizers. 2065 60


<< Previous 1 2 3 4 5 6 7 8 9 10

---