Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In nitrinergic signal transduction, nitrogen oxide (NO) synthases (NOS) (EC 1.14.23) catalyze the conversion of L-arginine to L-citrulline and NO, which in turn activates soluble guanylyl cyclase. Macrophages were reported to contain a single isoform of NOS (type II, soluble, Ca(2+)-independent, 130-kDa) and only upon activation of the cells by interferon-gamma (INF) and lipopolysaccharides (LPS). By a mechanism involving L-type Ca2+ channels, calmodulin, and serine proteases, INF/LPS also induce a cytotoxic activation of macrophages. In RAW264.7 macrophages, NO release was detected upon activation of the cells by INF/LPS but also, although at a 20-fold lower level, in control cells. The latter constitutive NOS activity and NO release were Ca2+ dependent and were decreased in INF/LPS-activated RAW264.7 cells or with increasing passage number. RAW264.7 cells did not express soluble guanylyl cyclase, suggesting other target molecules for NO. In INF/LPS-activated cells, NOS activities and NO release were Ca2+ independent (type II) and coinduced with NADPH-diaphorase activities both in the soluble and in the particulate fractions. The NOS-II activities corresponded to a 130-kDa protein, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which was not recognized in a protein immunoblot with anti-NOS-I antibody. The serine protease inhibitor tosyl-lysyl chloromethyl ketone abolished the induction of NOS-II by INF/LPS, by depleting intracellular thiol pools and interfering with protein synthesis. Induction of NOS-II by INF/LPS was transcriptionally based and, for maximal enzyme activity, required increased intracellular tetrahydrobiopterin levels, intracellular Ca2+ mobilization, and activation of non-L-type Ca2+ channels but, unlike the induction of macrophage-mediated cytotoxicity, neither L-type-Ca2+ channels nor calmodulin.
Mol Pharmacol 1992 Apr
PMID:Regulation and subcellular location of nitrogen oxide synthases in RAW264.7 macrophages. 137 97

Two monoclonal antibodies, designated 1F8 (IgG1) and 5B10 (IgG1), have been produced in mice against native human type III collagen. These antibodies were highly type and species specific, recognizing the triple helical domain of type III as tested by ELISA. Immunofluorescence studies using each of these antibodies resulted in a fibrous staining pattern in human skin dermis. Immunogold electron microscopy resulted in a periodic distribution of gold particulates along banded collagen fibrils. Assuming that the total contour length of pepsin digested type III collagen is 300 nm, measurements of antibody-antigen complexes visualized by rotary shadowing revealed that each antibody bound at the same two sites: one approximately at the middle of the helix (153 nm from the N-terminus), the other at a site one-quarter the triple helical length from the N-terminus (75 nm). That the one-quarter binding site was closest to the N-terminus was determined by antibody incubation following tadpole collagenase treatment, which results in a larger, N-terminus containing fragment (binding antibody) and a smaller C-terminus containing fragment (not binding antibody). Located at each antibody binding epitope is a sequence of 10 amino acids: Gly-Ala-Hyp-Gly-Leu-Arg-Gly-Gly-Ala-Gly. Renatured cyanogen bromide-cleaved(CB)-peptides, CB4 and CB8, containing these repeated sequences reacted with each antibody, whereas other renatured type III CB-peptides were unreactive as determined by Western blotting analysis and ELISA. This was further confirmed by inhibition tests using a 10 residue synthetic peptide of identical sequence, which yielded 20-30% inhibition of antibody binding to native type III collagen at 4 degrees C. However, no inhibition was noted at higher temperature. These results indicate that both monoclonal antibodies recognize a specific helical conformation of 10 or slightly fewer residues in the three identical polypeptide chains comprising type III collagen.
Mol Immunol 1992 Jun
PMID:Repeated helical epitopes of defined amino acid sequence in human type III collagen identified by monoclonal antibodies. 137 14

Lipopolysaccharide (LPS), either alone or in combination with cytokines, induces nitric oxide (NO) synthase activity in cells that normally release little or no NO. In arterial smooth muscle cells and various macrophage cell lines, NO synthase activity is induced after several hours of incubation with LPS. In brain, NADPH-dependent diaphorase activity has been associated with constitutive NO synthase. Here we show that incubation of rat aorta or cultured macrophages with LPS causes a time-dependent induction of NO synthase. The NO synthase activity in both rat aorta and macrophages was calcium independent and inhibited by NG-monomethyl-L-arginine and NG-nitro-L-arginine. We also found that LPS caused a time-dependent induction in NADPH-dependent diaphorase activity in both rat aorta and cultured macrophages. The diaphorase activity was mainly NADPH dependent and NADH independent. NO synthase activity and NADPH-diaphorase activity in crude cytosol from LPS-treated macrophages were found to co-purify, using 2',5'-ADP-Sepharose followed by Superose-6 gel permeation chromatography.
Mol Pharmacol 1992 Jun
PMID:Induction of NADPH-dependent diaphorase and nitric oxide synthase activity in aortic smooth muscle and cultured macrophages. 137 28

An efficient expression system in Escherichia coli for several biologically active insulin-like growth factor-I (IGF-I) fusion peptide analogues is described. These novel IGF-I fusion protein analogues have properties that make them very useful reagents in the investigation of IGF-I action. The analogues comprise an IGF-I sequence and the first 11 amino acids of methionyl porcine growth hormone (pGH) and include [Met1]-pGH(1-11)-Val-Asn-IGF-I, which contains the authentic IGF-I sequence, and two analogues, [Met1]-pGH(1-11)-Val-Asn-[Gly3]-IGF-I and [Met1]-pGH(1-11)-Val-Asn-[Arg3]-IGF-I, where Glu-3 in the human IGF-I sequence has been replaced by Gly or Arg respectively. The three peptides are referred to as Long IGF-I, Long [Gly3]-IGF-I or Long [Arg3]-IGF-I depending on the IGF-I sequence present. Production of the purified fusion peptides was aided by folding the reduced and denatured fusion peptide sequence under conditions that gave very high yields of biologically active product. Introduction of a hydrophobic N-terminal extension peptide appears to facilitate the correct folding of the IGF-I analogues compared with that obtained previously when folding normal-length IGFs. The biological activities of the IGF-I fusion peptides were compared with authentic IGF-I and the truncated analogue, des(1-3)IGF-I. In L6 rat myoblasts, all the analogues were more potent than authentic IGF-I in their abilities to stimulate protein and DNA synthesis and inhibit protein breakdown. In H35 hepatoma cells, where the IGFs act through the insulin receptor, the Long IGF-I analogues maintained a similar potency relative to IGF-I as was observed in the L6 myoblasts. The order of biological potency in cell lines secreting IGF-binding proteins (IGFBPs) into the medium was Long [Arg3]-IGF-I-des(1-3)IGF-I greater than Long [Gly3]-IGF-I greater than Long IGF-I greater than IGF-I. In chicken embryo fibroblasts, a cell line that does not secrete detectable IGFBPs into the medium, Long [Arg3]-IGF-I, was less potent than IGF-I. Investigation of receptor and IGFBP association by these analogues reinforced our previous findings that N-terminal analogues of IGF-I show increased biological potency due to changes in the degree of their IGFBP interactions.
J Mol Endocrinol 1992 Jun
PMID:Novel recombinant fusion protein analogues of insulin-like growth factor (IGF)-I indicate the relative importance of IGF-binding protein and receptor binding for enhanced biological potency. 137 42

A comprehensive mutational analysis was used to analyze the side-chains on human growth hormone (hGH) important for binding 21 different anti-hGH mouse monoclonal antibodies (MAbs) whose equivalent concentrations for 50% binding (EC50) ranged from approximately 10(7) to 3 x 10(10) M-1. A combination of homolog- and alanine-scanning mutagenesis coupled with a robot-aided enzyme-linked immunosorbent assay were used to create high resolution "functional epitopes" for each MAb. Every functional epitope mapped to at least two polypeptide segments of hGH that were close together in the folded protein to form a patch. Although these patches sometimes overlapped, each was different indicating no two MAbs bound identically to hGH. The MAbs bound to determinants in loops and helices that were generally most accessible to a 9 A radius probe. Only a few side-chains dominated each functional epitope and these tended to be Arg greater than Pro greater than Glu approximately Asp approximately Phe approximately Ile (Ala, Cys and Trp were not tested). Our studies indicate that most of the accessible surface of hGH is potentially antigenic in the mouse and suggest that functional epitopes are dominated by fewer side-chains than may be in the contact epitope.
J Mol Biol 1992 Aug 05
PMID:High resolution functional analysis of antibody-antigen interactions. 138 May 63

We have isolated an ovine insulin-like growth factor-binding protein-2 (IGFBP-2) cDNA from an adult sheep cDNA library, to determine the structure of ovine IGFBP-2 and to examine the pattern of IGFBP-2 gene expression in adult sheep tissues. This cDNA had 81, 96 and 87% identity with the rat, bovine and human sequences respectively. The deduced amino acid sequence of the ovine IGFBP-2 showed 86, 95 and 85% homology with the rat, bovine and human peptide sequences respectively. The ovine IGFBP-2 cDNA encoded a precursor protein of 317 amino acids which comprised a 33 residue hydrophobic leader sequence and a 284 residue, 30.9 kDa, mature peptide. The 18 cysteine residues, which are a characteristic feature of IGFBPs, were conserved. Also, an Arg-Gly-Asp (RGD) sequence near the C terminus was present. A single transcript of approximately 1.5 kb was expressed in abundance in selected tissues of an adult sheep, namely liver, kidney, adrenal, pituitary and choroid plexus. Southern blot analysis of ovine genomic DNA with the cDNA probe demonstrated that IGFBP-2 is encoded by a single gene. These findings indicate that the ovine IGFBP-2 protein is similar to that in other species and that, in the adult, the mRNA is expressed only in selected tissues.
J Mol Endocrinol 1992 Aug
PMID:The characterization and expression of ovine insulin-like growth factor-binding protein-2. 138 Nov 82

SH2 (src homology region 2) domains are implicated in protein-protein interactions involved in signal transduction pathways. Isolated SH2 domains bind proteins that are tyrosine phosphorylated. A novel, phosphotyrosine-independent binding interaction between BCR, the Philadelphia chromosome breakpoint cluster region gene product, and the SH2 domain of its translocation partner c-ABL has recently been reported. We have examined the ability of additional SH2 domains to bind phosphotyrosine-free BCR and compared this with their ability to bind tyrosine-phosphorylated c-ABL 1b. Of 11 individual SH2 domains examined, 8 exhibited relatively high affinity for c-ABL 1b, whereas only 4 exhibited relatively high affinity for BCR. Binding of tyrosine-phosphorylated c-ABL 1b by the relatively high-affinity ABL and ARG SH2 domains was quantitatively analyzed, and equilibrium dissociation constants for both interactions were estimated to be in the range of 5 x 10(-7) M. The ABL SH2 domain exhibited relatively high affinity for phosphotyrosine-free BCR as well; however, this interaction appears to be about two orders of magnitude weaker than binding of tyrosine-phosphorylated c-ABL 1b. The ARG SH2 domain exhibited relatively weak affinity for BCR and was determined to bind about 10-fold less strongly than the ABL SH2 domain. The ABL and ARG SH2 domains differ by only 10 of 91 amino acids, and the substitution of ABL-specific amino acids into either the amino- or carboxy-terminal half of the ARG SH2 domain was found to increase its affinity for BCR. We discuss these results in terms of a model which has been proposed for peptide binding by class I histocompatibility glycoproteins.
Mol Cell Biol 1992 Nov
PMID:A limited set of SH2 domains binds BCR through a high-affinity phosphotyrosine-independent interaction. 138 90

In the current study, we describe cytokine and Escherichia coli lipopolysaccharide (LPS) induction of nitric oxide (NO) synthase mRNA levels in cultured smooth muscle from rat pulmonary artery (RPASM). Exposure of RPASM to interleukin-1 beta, interferon-gamma, or LPS alone did not significantly affect NO synthesis, as determined by nitrite concentrations in media. Exposure to tumor necrosis factor-alpha caused a modest (2x) increase in nitrite production. In contrast, exposure to a combination of the above three cytokines and LPS caused a large increase in NO synthesis. Exposure of RPASM to this combination caused an increase in mRNA levels of NO synthase (as described by Northern blot analysis with 32P-cDNA probe to an inducible form of NO synthase present in murine macrophages) that was apparent as early as 4 h. Expression of the induced gene product after exposure to the cytokine and LPS mixture was evident by significant increases in nitrite production at 12 h. Production of nitrite was completely abolished in the presence of NG-monomethyl-L-arginine (NMA), and this inhibition was reversible by the addition of excess L-arginine. NO synthase mRNA levels were not affected by NMA. The nitrite production induced by the combination of cytokines and LPS was abolished by pretreating cells with cycloheximide. These data indicate that a combination of cytokines and LPS affect expression of the gene for the inducible form of NO synthase in cultured RPASM.
Am J Respir Cell Mol Biol 1992 Nov
PMID:Cytokines and lipopolysaccharide induce nitric oxide synthase in cultured rat pulmonary artery smooth muscle. 138 80

We studied the effects of aerosolized DL-2-mercaptomethyl-3-guanidino-ethylthiopropanoic acid (MGTA) (10(-4) M, 90 breaths), a specific inhibitor of carboxypeptidase B-type enzymes, on changes in total pulmonary resistance (RL) induced by aerosolized capsaicin (10(-7) to 10(-4) M; 10 breaths at each concentration) and vagus nerve stimulation (5 V, 5 ms, for 20 s at frequencies varying from 2 to 10 Hz) in anesthetized, atropinized, and ventilated guinea pigs. We also studied the effect of aerosolized MGTA on the bronchoconstrictor response to either aerosolized substance P, neurokinin A (10(-7) to 10(-4) M; 10 breaths at each concentration), and carbachol (10(-5) to 2 x 10(-4) M; 10 breaths at each concentration) or to i.v. administration of neurokinin A (10(-11) to 10(-8) mol/kg), bradykinin (10(-10) to 10(-7) mol/kg), and histamine (10(-8) to 10(-6) mol/kg). Although aerosolized MGTA caused no change in basal RL (P > 0.5), it did potentiate the noncholinergic bronchoconstrictor response to capsaicin (n = 5; P < 0.001) as well as to vagus nerve stimulation (n = 5; P = 0.001). In contrast, MGTA did not potentiate the bronchoconstrictor response to either aerosolized substance P, neurokinin A, and carbachol or to i.v. administration of neurokinin A, histamine, and bradykinin. Carboxypeptidase activity cleaving C-terminal arginine or lysine was found in the membrane preparations of trachea and lung from guinea pigs. The membrane-bound carboxypeptidase activity was maximal at pH 7.0 and was enhanced by the presence of CoCl2 (1 mM) in both the tracheal and lung tissue.(ABSTRACT TRUNCATED AT 250 WORDS)
Am J Respir Cell Mol Biol 1992 Nov
PMID:Carboxypeptidase M-like enzyme modulates the noncholinergic bronchoconstrictor response in guinea pig. 138 81

Amino acids in the serine proteinase inhibitor eglin c important for its inhibitory specificity and activity have been investigated by site-directed mutagenesis. The specificity of eglin c could be changed from elastase to trypsin inhibition by the point mutation Leu45----Arg (L45R) in position P1 [nomenclature according to Schechter and Berger (1967) Biochem. Biophys. Res. Commun. 27, 157-162]. Model building studies based on the crystal structure of mutant L45R [Heinz et al. (1991) J. Mol. Biol. 217, 353-371] were used to rationalize this specificity change. Surprisingly, the double mutant L45R/D46S was found to be a substrate of trypsin and various other serine proteinases. Multidimensional NMR studies show that wild-type eglin c and the double mutant have virtually identical conformations. In the double mutant L45R/D46S, however, the N-H bond vector of the scissile peptide bond shows a much higher mobility, indicating that the internal rigidity of the binding loop is significantly weakened due to the loss or destabilization of the internal hydrogen bond of the P1' residue. Mutant T44P was constructed to examine the role of a proline in position P2, which is frequently found in serine proteinase inhibitors [Laskowski and Kato (1980) Annu. Rev. Biochem. 49, 593-626]. The mutant remains a potent elastase inhibitor but no longer inhibits subtilisin, which could be explained by model building. Both Arg51 and Arg53, located in the core of the molecule and participating in the hydrogen bonding network with residues in the binding loop to maintain rigidity around the scissile bond, were individually replaced with the shorter but equally charged amino acid lysine. Both mutants showed a decrease in their inhibitory potential. The crystal structure of mutant R53K revealed the loss of two hydrogen bonds between the core and the binding loop of the inhibitor, which are partially restored by a solvent molecule, leading to a decrease in inhibition of elastase by 2 orders of magnitude.
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PMID:Changing the inhibitory specificity and function of the proteinase inhibitor eglin c by site-directed mutagenesis: functional and structural investigation. 139 Jun 62


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