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:2.7.10.1 (ERK)
95,504 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neutral endopeptidase (NEP; E.C. 3.4.24.11) is a mammalian ectopeptidase identified as the common acute lymphoblastic leukemia antigen (CALLA or CD10). In order to investigate its cellular processing and its role in B lymphocyte differentiation, a fluorescent derivative of the mercapto NEP inhibitor thiorphan, N-[fluoresceinyl]-N'-[1-(6-(3-mercapto-2-benzyl-1-oxopropyl) amino-1-hexyl]thiocarbamide (FTI), has been synthesized. The fluorescent characteristics of fluorescein were conserved in FTI after linkage with the thiol NEP inhibitor. FTI inhibited NEP with an IC50 value of 10 nM and a good selectivity compared to that of aminopeptidase N (greater than 100 microM) and angiotensin converting enzyme (32 microM). The FTI probe was shown to detect membrane-bound NEP using photomicroscopy on cultured cells or flow cytometry techniques. Using NEP-expressing MDCK cells and episcopic fluorescence microscopy, a specific labeling was obtained with 100 nM FTI which was completely displaced by 10 microM HACBOGly, a specific and potent inhibitor of NEP. Therefore, FTI can be considered a suitable tool for following cellular NEP traffic. In flow cytometry, the fluorescent probe FTI, used at concentrations as low as 1 nM with Reh6 cells, could be very useful for detecting NEP/CALLA on lymphoid cells. In addition, the recognition of FTI is independent of tissues and species, a major advantage of inhibitors over monoclonal antibodies.
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PMID:Detection of neutral endopeptidase-24.11/CD10 by flow cytometry and photomicroscopy using a new fluorescent inhibitor. 135 7

Alternatively spliced variants of fibroblast growth factor receptor 1 mRNA are predicted to encode secreted forms and membrane-bound forms of receptors. The predicted amino acid sequences of these receptor variants differ in a portion of the extracellular region. In this study, we characterized the function of one of these splice variants which was predicted by its cDNA to be a secreted FGF receptor. We expressed this secreted form of the human FGFR1 (sFGFR1) in Chinese hamster ovary cells. The sFGFR1 protein oligomerized upon ligand binding. Surprisingly, the sFGFR1 preferentially bound basic FGF over acidic FGF. In cross-linking experiments, the sFGFR1 showed higher binding affinity for basic FGF (Kd approximately 30 nM) than for acidic FGF (Kd greater than 300 nM). These results suggest that this secreted form of FGF receptor has an unusual ligand binding specificity that may be important for its biological role in vivo.
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PMID:A naturally occurring secreted form of fibroblast growth factor (FGF) receptor 1 binds basic FGF in preference over acidic FGF. 137 91

Preleukemia is thought to be a clonal disorder of hemopoietic stem cells. The conversion of a normal cell into a preleukemic and ultimately leukemic state is a multistep process requiring the accumulation of a number of genetic lesions. The myelodysplastic syndromes have become a paradigm for human preleukemia, where nonrandom chromosomal abnormalities, including complete or partial deletions of chromosomes five and seven, trisomy eight and Y chromosome loss suggest specific changes. Of particular significance are 5q deletions, as many genes important in hemopoiesis are located in this region, including the proto-oncogene FMS, which encodes the receptor for the macrophage colony-stimulating factor, CSF-1. Genetic damage such as point mutations in the RAS and FMS genes has been detected in preleukemia patients. The RAS gene family (N, K and H) encodes membrane-bound G proteins, which, like other proto-oncogenes, are components of the intracellular signal transduction pathways controlling mitogenesis and differentiation. The characterization of such lesions may ultimately identify those patients at greatest risk of leukemic transformation.
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PMID:Genetic lesions in preleukemia. 142 Apr 44

Some of the phenothiazines and dibenz[b,f]azepines exert an antiproliferative effect on HEp-2 and rat prolactinoma cells. The same compounds also have effects on different membrane-bound biochemical events, such as H2O2 formation and the peroxide-generated chemiluminescence of polymorphonuclear leukocytes. The superoxide dismutase inhibition by 7,8-dioxochlorpromazine and 6,9-dioxochlorpromazine have some relation to the growth inhibitory action on the growth of HEP-2 and prolactinoma cells in vitro. The antiproliferative effects of phenothiazines were synergized with resistance modifiers like verapamil, omeprazole and tubulozole-C, due to increased drug-influx or decreased drug-efflux and due to possible action on the cytoskeleton.
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PMID:In vitro antiproliferative effects of tricyclic psychopharmaceutical agents and synergism with some resistance modifiers. 156 77

Neutral endopeptidase (EC 3.424.11, NEP) is a membrane-bound zinc-metallopeptidase. The substrate specificity and catalytic activity of NEP resemble those of thermolysin, a bacterial zinc-metalloprotease. Comparison of the primary structure of both enzymes suggests that several amino acids present in the active site of thermolysin are also found in NEP. Using site-directed mutagenesis of the cDNA encoding the NEP sequence, we have already shown that His residues 583 and 587 are two of the three zinc ligands. In order to identify the third zinc ligand, we have substituted Val or Asp for Glu616 or Glu646. Val616 NEP showed the same kinetic parameters as the non-mutated NEP. In contrast, the mutant Val646 NEP was almost completely devoid of catalytic activity and unable to bind the tritiated inhibitor [3H]N-[2(R,S)-3-hydroxyaminocarbonyl-2-benzyl-1-oxypropyl]gl ycine, the binding of which is dependent on the presence of the zinc ion. Replacing Glu for Asp at position 646 conserved the negative charge, and the mutant enzyme exhibited the same Km value as the non-mutated enzyme, but kCat was decreased to less than 3% of the value of the non-mutated enzyme. When compared to the non-mutated enzyme Asp646 NEP showed a higher susceptibility to chelating agents, but bound the tritiated inhibitor with the same affinity. Taken together, these observations strongly suggest that Glu646 of NEP is the third zinc-coordinating residue and is equivalent to Glu166 in thermolysin.
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PMID:Identification of glutamic acid 646 as a zinc-coordinating residue in endopeptidase-24.11. 167 40

Neutral endopeptidase (NEP, also known as enkephalinase, CALLA, or EC 3.4.24.11) is a membrane-bound peptidase present in many different cell types. Previous studies have shown that it modulates the actions of a variety of biologically active peptides on several airway responses. More recent studies have demonstrated that reductions in neutral endopeptidase activity in animal airways is associated with increased responses to exogenously applied and endogenously released peptides. To study the regulation of NEP expression, we used human airway epithelial cells transformed in vitro with an origin-defective SV40 plasmid. Enzymatic activity, measured using [3H-Tyr,D-Ala2]leucine enkephalin, increased with cell density (1.4 ng/10(6) cells at 530 cells/cm2 and 21 ng/10(6) cells at confluence, 400 X 10(3) cells/cm2). In both confluent and nonconfluent cultures, the glucocorticoid budesonide increased neutral endopeptidase activity in time- and concentration-dependent fashions. Maximal increases of 10 ng/10(6) cells greater than control were observed after 6 days of incubation at 10(-7) M budesonide. Dexamethasone also increased NEP, suggesting that the effect is due to glucocorticoid receptor effects. Transcription, as assessed by Northern blot analysis of total cellular RNA, showed that NEP-specific RNAs also increased with increasing concentration of glucocorticoid. We conclude that neutral endopeptidase can be increased by cell growth or density and by glucocorticoids and that the effects of glucocorticoids are mediated by increased NEP gene expression.
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PMID:Glucocorticoids induce neutral endopeptidase in transformed human tracheal epithelial cells. 184 94

The pregnant rat uterus contains a membrane-bound metalloendopeptidase that is biochemically and immunologically similar to kidney enkephalinase (E.C.3.4.24.11). The uterus enzyme readily cleaved specific neutral endopeptidase substrates and oxytocin as well as the synthetic elastase substrate, Suc(Ala)3-pNA, yet did not digest native elastin. Using specific inhibitors, the uterus endopeptidase was identified as a metallopeptidase and not a serine protease, having an absolute requirement for zinc and perhaps calcium for maximal activity. The uterus endopeptidase cross-reacted with polyclonal antiserum to kidney microvillar endopeptidase and a monoclonal antibody to common acute lymphocytic leukemia antigen. Immunohistochemical localization of the enzyme in a 17 day pregnant uterus indicated that the enzyme was localized on the smooth muscle bundles of the myometrium and the endometrial epithelium. Total enzyme activity was 25 times higher in the late-term pregnant uterus (17th day of pregnancy) than in the nonpregnant uterus. Enzyme levels dropped rapidly prior to parturition and within 4 days after delivery the enzyme activity had returned to control levels. Inhibition of NEP in uterine strips with phosphoramidon resulted in a marked potentiation of oxytocin-induced contractions. Our results suggest that the uterine endopeptidase may have an important role in regulating uterine smooth muscle cell contraction during the later stages of pregnancy through its action on oxytocin and perhaps other biologically active peptides.
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PMID:Neutral metalloendopeptidase associated with the smooth muscle cells of pregnant rat uterus. 204 32

The neutral zinc metalloendopeptidase (NEP, EC 3.4.24.11) is an integral membrane protein found in brain tissue, polymorphonuclear leukocytes, and many epithelia. We show here that NEP is expressed on rabbit synovial fibroblasts and on simian virus 40 (SV40) DNA- and H-ras-transformed rabbit mammary epithelial cells. Treatment of these cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) for 24 h decreased expression of NEP mRNA transcripts and decreased the biosynthetically labeled immunoprecipitable NEP antigen. In contrast to its effects on NEP, TPA treatment induced expression of the secreted metalloproteinase collagenase and the tissue inhibitor of metalloproteinases. TPA induced stromelysin, another secreted metalloproteinase, only in the fibroblasts. These data provide evidence that the expression of the membrane-bound NEP is regulated in several cell types.
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PMID:Phorbol diesters regulate expression of the membrane neutral metalloendopeptidase (EC 3.4.24.11) in rabbit synovial fibroblasts and mammary epithelial cells. 254 98

Membrane metallo-endopeptidase (NEP; neutral endopeptidase, kidney-brush-border neutral proteinase, enkephalinase, EC 3.4.24.11) cleaves peptides at the amino side of hydrophobic amino acids. While the enzyme is known to be in organs such as kidney and brain, we found it in human neutrophils. These cells cleaved the NEP substrate glutaryl (Glut)-Ala-Ala-Phe-(4-methoxynaphthylamine) (Glut-Ala-Ala-Phe-MNA) at a rate of 9.5 nmol X hr-1 per 10(6) cells, and phosphoramidon (1 microM) inhibited the hydrolysis by 90%. Intact neutrophils from donors who smoked had NEP activities about twice that of nonsmokers. Subcellular fractionation and sucrose density gradient centrifugation of lysed neutrophils showed that most of the NEP activity was membrane bound. A washed membrane fraction from human neutrophils rapidly cleaved 0.5 mM Glut-Ala-Ala-Phe-MNA (96 nmol X min-1 X mg-1) and the hydrolysis was inhibited by phosphoramidon and by specific antiserum to human renal NEP. The washed membrane fraction also rapidly cleaved 0.1 mM bradykinin (34 nmol X min-1 mg-1) and 0.1 mM fMet-Leu-Phe (49 nmol X min-1 X mg-1). The membrane-bound enzyme cleaved the peptide substrates at the same site as the homogeneous human renal NEP, and phosphoramidon and thiorphan inhibited the hydrolysis. Kinetic studies with pure human renal NEP showed that the chemotactic peptide fMet-Leu-Phe was one of the best biologically active substrates (Km, 59 X 10(-6) M; kcat, 3654 min-1). Immunocytochemistry at the light microscopic level revealed a high concentration of NEP on the cell membrane of neutrophils. This was confirmed with electron microscopy using the immunogold technique on ultrathin cryosections. These studies indicate that NEP in neutrophils may have important functions in inflammation and chemotaxis.
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PMID:Neutral endopeptidase 24.11 in human neutrophils: cleavage of chemotactic peptide. 390 53

To develop a model somatic gene therapy system for diabetes, a human hepatoma cell line (HEP G2) was transfected with a mammalian expression vector carrying the full-length human insulin cDNA. More proinsulin than insulin was released daily by the stably transformed cell line (HEP G2ins). However, on acute stimulation with 5mM 8-Br-cAMP and 10mM theophylline the HEP G2ins cells released predominantly insulin into the medium. The cells did not secrete insulin in response to glucose. Examination of acid-ethanol extracts confirmed insulin was preferentially being stored. Immunohistochemical analysis of the cells also showed (pro)insulin was being stored. Electron microscopy revealed large membrane-bound vacuoles, containing electron-dense material, which were not seen in control cells. Glucokinase activity and albumin secretion of the transfectants were unaltered from the controls. Five-minute pulse-chase labelling of the HEP G2ins cells with 3H-leucine confirmed insulin synthesis in the presence of 20mM glucose and 5mM 8-Br-cAMP. A dose-response curve for insulin synthesis was also generated to increasing concentrations of glucose with a half Vmax of 4.9mM. Our results show that the introduction of insulin cDNA into a human hepatoma cell line results in synthesis, storage and acute regulated insulin release and lend credence to the possibility of engineering a liver cell to secrete insulin acutely in response to physiological stimuli.
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PMID:Functional expression of the human insulin gene in a human hepatoma cell line (HEP G2). 761 54


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