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)

The 47-kDa lipoprotein is an abundant integral membrane protein and dominant immunogen of Treponema pallidum subsp. pallidum. Previous DNA sequencing of the 47-kDa-lipoprotein gene did not reveal consensus features representative of other bacterial lipoprotein genes; this prompted further analyses with emphasis on elucidation of the N terminus of the molecule. To assist in localizing start signals for the protein, the transcription initiation site for the 47-kDa-antigen gene was determined. RNA isolated from both T. pallidum and recombinant Escherichia coli expressing the 47-kDa antigen was used as a template in reverse transcriptase primer extension. Upon analysis of cDNA products, transcription initiation was localized to one nucleotide in T. pallidum and to two adjacent nucleotides in E. coli. When various primers were used in DNA sequencing reactions for these analyses, a previously undetected nucleotide (G) was found in the purported 5' untranslated region; this altered the upstream reading frame to reveal plausible sites for ribosome binding (GGAGG), translation initiation (GTG start codon), and signal peptidase II processing (Val-Val-Gly-Cys). Discounting acylation, the molecular weight of the mature polypeptide is 45,756 (approximately 46,600 with acylation). To derive nonacylated 47-kDa antigen for further structure-function studies, the 47-kDa-antigen gene was subcloned without acylation signals as a genetic construct encoding a glutathione S-transferase fusion protein; following cleavage with thrombin, the nonacylated 47-kDa protein was hydrophilic rather than amphiphilic but retained its antigenicity when tested against 116 human serum samples from patients with various stages of syphilis.
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PMID:Analysis of the N-terminal region of the 47-kilodalton integral membrane lipoprotein of Treponema pallidum. 137 97

Human umbilical vein endothelial cells have recently been shown to respond to C5a with increases in intracellular Ca2+, production of D-myo-inositol 1,4,5-triphosphate and superoxide anion generation. In the current studies, C5a had been found to cause in a time- and dose-dependent manner rapid expression of endothelial P-selectin, secretion of von Willebrand factor, and adhesiveness for human neutrophils. The effects of C5a in P-selectin expression and adhesiveness of neutrophils were similar to the effects of histamine and thrombin on endothelial cells. The adhesiveness of C5a-stimulated endothelium for neutrophils was blocked by anti-P-selectin, but not by antibodies to intercellular adhesion molecule 1, E-selectin, or CD18. A cell-based ELISA technique has confirmed upregulation of P-selectin in endothelial cells exposed to C5a. Binding of C5a to endothelial cells has been demonstrated, with molecules bound being approximately 10% of those binding to neutrophils. By a reverse transcriptase-PCR technique, endothelial cells have been shown to contain mRNA for the C5a receptor. These data suggest that C5a may be an important inflammatory mediator for the early adhesive interactions between neutrophils and endothelial cells in the acute inflammatory response.
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PMID:C5a-induced expression of P-selectin in endothelial cells. 752 84

Human proteinase inhibitor 6 (PI-6) is a recently described protein belonging to the serine proteinase inhibitor (serpin) superfamily. Sequence similarity suggests that PI-6 most resembles the ovalbumin (ov) serpins which include plasminogen activator inhibitor-2, the squamous cell carcinoma antigen, monocyte/neutrophil elastase inhibitor, and maspin. Although these proteins are associated with carcinomas and inflammation, they appear to have diverse functions and little is known of their physiological roles. In this study we have characterized cDNA and genomic clones encoding mouse PI-6 in order to analyze the localization, structure, and expression of the gene. The reactive center residues (Arg-Cys) are conserved in the mouse molecule, and recombinant mouse PI-6 was shown to bind thrombin, indicating that it has similar inhibitory properties to its human counterpart. Using reverse transcriptase-polymerase chain reaction assays on RNA isolated from 15-day-old embryos and adult mice, we have shown that mouse PI-6 expression is developmentally regulated, and that, unlike human PI-6, it is absent from the placenta. The mouse homologue of the human PI-6 gene has been designated Spi3 and was mapped to chromosome 13 between the Pl1 and ctla2 alpha genes. It spans 20 kilobases, consists of 7 exons and 6 introns, and contains a TATA motif 24 nucleotides upstream of the transcriptional start site. A 680-base pair DNA fragment containing this motif and 31 nucleotides of the 5'-untranslated region of the structural gene directed transcription of a bacterial cat gene, demonstrating the presence of a functional promoter. The PI-6 gene lacks an intron present in the ovalbumin and PAI-2 genes; otherwise it is identical in terms of the numbers, position, and phasing of the intron/exon boundaries. These results suggest that PI-6 and the ov-serpin genes have diverged and do not belong to the same subgroup.
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PMID:Gene structure, chromosomal localization, and expression of the murine homologue of human proteinase inhibitor 6 (PI-6) suggests divergence of PI-6 from the ovalbumin serpins. 760 71

The effects of thrombin, D-phenylalanyl-L-propyl-L-arginine chloromethyl ketone (PPACK)-inhibited thrombin, and thrombin receptor agonist peptide, SFLLRNPNDKYEPF (SFLL, a portion of the receptor unmasked after thrombin cleavage), on the expression of tissue factor (TF) and thrombomodulin by human saphenous vein endothelial cells (HSVECs) in culture were studied. Unstimulated cells contained very low amounts of TF mRNA as measured by the reverse transcriptase-PCR method. Thrombin treatment increased TF mRNA to 8.0 +/- 1.9 (n = 3) times the control level. The increase was detectable within 2 h and declined to near basal level by 6 h. Induction of TF mRNA was not blocked by cycloheximide, treatment with cycloheximide alone also increased TF mRNA levels, and thrombin in combination with cycloheximide further enhanced the accumulation of TF mRNA. Thrombin caused a 14.5 +/- 1.5-fold (n = 5) increase in TF activity on the surface of HSVECs and a 20.5 +/- 1.4-fold (mean +/- S.D., n = 2) increase in the extracellular matrix. The thrombin-induced effects on TF synthesis could be fully reproduced by the thrombin receptor agonist peptide, SFLL, whereas PPACK-inhibited thrombin did not influence TF expression. Thrombin increased thrombomodulin mRNA to 190 +/- 39% (n = 5) of control levels, whereas PPACK-inhibited thrombin or SFLL did not influence thrombomodulin mRNA levels. In contrast, surface-bound thrombomodulin cofactor activity and thrombomodulin antigen in the cell lysates did not change over 24 h of incubation with thrombin. However, thrombin caused a 2-fold increase in thrombomodulin antigen released into the conditioned medium, and immunoelectron microscopy of HSVECs also demonstrated the presence of thrombomodulin vesicles close to the luminal cell surface in thrombin-treated cultures. The Western blot pattern thrombomodulin in the conditioned medium of untreated and thrombin-treated cells was found to be similar, and soluble thrombomodulin occurred mainly as fragments of the cell-associated form. We conclude that the transcriptional control by thrombin causes an increase in both TF and thrombomodulin mRNA. The increase in TF mRNA levels is also paralleled by an increase in surface expression, is dependent on the proteolytic activity of thrombin, and is mediated by the same receptor as the recently cloned thrombin receptor in platelets. Up-regulation of thrombomodulin mRNA levels by thrombin is distinct from this pathway and is associated with unchanged expression on the cell surface.
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PMID:Thrombin regulates tissue factor and thrombomodulin mRNA levels and activities in human saphenous vein endothelial cells by distinct mechanisms. 767

Endothelin-1, a potent vasoconstrictor of cerebral vessels, is produced by rat primary astrocytes and is subject to autostimulatory regulation in these cells. In this study we examined the effect of thrombin on astrocytic endothelins and report that endothelin-1 is released into the culture fluid in response to thrombin treatment. However, increased production of endothelin-1 is not accompanied by a concomitant increase in steady-state levels of endothelin-1 mRNA as assessed by reverse transcriptase-polymerase chain reaction, even though thrombin stimulation leads to increased inositolphospholipid turnover and activation of the nuclear factor AP1. Thus, astrocytic production of endothelin-1 may be mainly post-transcriptionally regulated in response to thrombin stimulation. In addition, two endothelin receptor genes (ET(A) and ETB) were found to be transcribed simultaneously in primary astrocyte cultures, and both thrombin and endothelin-1 stimulation result in a distinct temporary decrease in ET(A) mRNA. These studies suggest a role for thrombin in the regulation of brain perfusion through astrocytic endothelin-1 expression.
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PMID:Thrombin is a regulator of astrocytic endothelin-1. 767 2

Molecular genetic studies have revealed that the human hepatitis B viral (HBV) Pol protein, a polypeptide of about 94 kDa, contains four domains. These are the 5'-terminal protein, spacer, RNA reverse transcriptase/DNA polymerase, and RNase H, respectively, from the amino (N) to carboxy (C) terminus. No evidence indicates as yet the involvement of a specific protease in cleaving the Pol protein or the presence of protease-cutting sites in the Pol protein. An in vitro-translated Pol protein was shown to be cleaved by purified thrombin but not in the presence of its inhibitor, hirudin. Two thrombin-cutting sites, spanning 194 amino acids, were then deduced by thrombin digestion of Pol protein with various lengths of C-terminal deletion. These two putative cutting sites, one located in the spacer region and the other in the beginning of the polymerase region, were found to be conserved at similar positions in the Pol protein of all hepadnaviruses. By using a novel method called the LacZ localization assay (LLA), it was demonstrated that a tripartite fusion protein containing the nucleus localization sequence (NLS) of SV40 large T Ag, the putative thrombin cutting sequence (Ile-Arg-Ile-Pro-Arg320-Thr) of HBV Pol protein and the full length beta-galactosidase of E. coli, exhibited a lower percentage (approximately 53%) of targeting into the nucleus of transfected hepatoma cells when compared with a similar tripartite protein containing a single mutation (Arg320 residue into Trp320) of HBV Pol protein (approximately 78%) or with a bipartite protein of SV40 NLS-beta-galactosidase (approximately 90%). These results indicate that the putative thrombin-cutting site in the spacer region of HBV Pol protein could be cleaved by a cellular protease resulting in the separation of NLS sequence from the beta-galactosidase and rendering a lower frequency of X-gal staining in the nucleus.
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PMID:Demonstration of the presence of protease-cutting site in the spacer of hepatitis B viral Pol protein. 773 Apr 38

The endothelial cell (EC) urokinase receptor plays an important role in the localization and receptor-mediated activation of EC-bound plasminogen and hence surface-localized fibrinolysis. Thrombin induced a rapid (< 5 minute), time- (0 to 30 minutes) and dose- (0.1 to 8 U/mL) dependent decrease in the specific binding of 125I-labeled two-chain urokinase-type plasminogen activator (tcu-PA) or diisopropylfluoro-phosphate-tcu-PA to urokinase-type plasminogen activator receptor (u-PAR) in cultured ECs from various sources (range, 21% to 50%). The thrombin receptor activation peptide but not control peptide showed a similar but reduced decrease in the specific binding of 125I-labeled tcu-PA to u-PAR. Incubation of thrombin-treated cultures (10 to 12 hours) in complete medium restored 125I-labeled tcu-PA ligand binding to normal levels. u-PAR mRNA levels rapidly (1 hour) increased and peaked 10 to 12 hours after thrombin treatment as analyzed by reverse transcriptase-polymerase chain reaction. Decreased thrombin-induced 125I-labeled tcu-PA binding correlated with the time-dependent decrease in surface-localized plasmin generation, as measured by the direct activation of 125I-labeled Glu-plasminogen and quantification of the 20-kD light chains of 125I-labeled plasmin. After incubation with thrombin, plasmin generation was decreased 50% to 56% (125 to 152 fmol/3 to 3.5 x 10(4) cells). Isolation of metabolically labeled 35S-labeled u-PAR from the media of thrombin and phospholipase C-treated human aortic cultures yielded approximately 10- and approximately 12-fold more 55-kD M(r) and approximately 6-fold more 35-kD M(r) 35S-labeled u-PAR forms than control cultures, respectively. The u-PAR antigen forms (M(r), 54 kD) and the glycosyl-phosphatidylinositol-anchored protein CD59 (M(r), 20 kD) were also simultaneously identified by immunoprecipitation in the media of thrombin-treated cultures. This suggests that thrombin may release u-PAR and decrease u-PA ligand binding through a common pathway involving phospholipase C. These results establish a novel interrelation between thrombin and EC fibrinolysis and suggest that thrombin may also have an additional regulatory role in the net expression of surface-localized EC fibrinolytic activity.
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PMID:Thrombin decreases the urokinase receptor and surface-localized fibrinolysis in cultured endothelial cells. 774 51

The cDNA encoding the C-terminal nucleotide-binding domain (NBD2) from mouse P-glycoprotein involved in multidrug resistance was obtained from adrenal cell mRNA and amplified by reverse transcriptase polymerase chain reaction. NBD2 was highly overexpressed in Escherichia coli in fusion with glutathione S-transferase and could be purified after efficient thrombin cleavage. Both fused and purified NBD2 bound TNP (2',3'-O-(2,4,6-trinitrophenyl))- derivatives of nucleotides with high affinity. TNP-ATP or TNP-ADP binding at micromolar concentrations produced a characteristic blue-shifted enhancement of extrinsic fluorescence and was specifically prevented or chased by ATP or ADP at millimolar concentrations. A similar affinity binding was monitored by quenching of intrinsic fluorescence. The spectrum of fusion protein, containing 5 tryptophan residues, was maximally quenched at 328 nm upon interaction with TNP-nucleotides. TNP-GTP exhibited a lower affinity than TNP-ATP but produced a higher maximal quenching (44% instead of 28%). The intrinsic fluorescence of purified NBD2, containing a single tryptophan residue, exhibited a narrow spectrum with a maximum at 328 nm characteristic of a hydrophobic tryptophan environment. A high quenching was observed upon nucleotide interaction with similar affinity. The results put forward a functional role for the tryptophan-containing sequence of P-glycoprotein NBD2 that was not detected up to now.
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PMID:Overexpression and purification of the carboxyl-terminal nucleotide-binding domain from mouse P-glycoprotein. Strategic location of a tryptophan residue. 791 13

We addressed the balance between thrombin and its serpin protease nexin I (PNI) after sciatic nerve injury in the mouse. Prothrombin levels increased twofold 24 h after nerve crush, as measured by a specific chromogenic assay, and peaked at day 3. Thrombin activity also increased 2-4 days after injury in distal sciatic nerve segments. Nerve RNA analysis using reverse transcriptase--polymerase chain reaction (RT-PCR) assay confirmed that prothrombin was synthesized locally. We also monitored PNI levels in these injured nerve samples by complex formation with an 125I-labeled target protease and found peak activity occurring later, 6-9 days after the thrombin induction. These data indicate that nerve injury first induces the synthesis of prothrombin, which is subsequently converted to active thrombin. Nerve crush-induced thrombin is followed by the generation of functionally active PNI and may be directly responsible for its induction. By immunocytochemistry with anti-PNI antibody, we found that activated Schwann cells were the source of induced PNI. These results support the concept that the balance between serine proteases and their serpins is dysregulated during nerve injury and suggests a role for its reestablishment in nerve damage repair.
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PMID:Neural thrombin and protease nexin I kinetics after murine peripheral nerve injury. 886 30

Thrombin's potent effects on astrocytes are mediated by a specific receptor and inhibited by a serpin, protease nexin I (PNI). Thrombomodulin (TM), a membrane protein that forms complexes with thrombin, changing its enzymatic specificity, has not been studied in astrocytes. In primary astrocyte cultures, using Western blotting and immunocytochemistry, we found a 70 kDa TM band and TM localized to the surface with an anti-mouse TM monoclonal antibody. By reverse transcriptase coupled with polymerase chain reaction (RT-PCR), we found the correct sequence for mouse TM mRNA in astrocytes. Finally, we documented calcium-dependent activation of protein C by a thrombin:TM complex with thrombin added to the astrocytes. These results indicate the presence of functionally active TM at the astrocyte surface and add support to a role for thrombin signaling in the nervous system.
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PMID:Novel expression and localization of active thrombomodulin on the surface of mouse brain astrocytes. 906 32


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