| 8314008 |
Molecular scanning of the insulin receptor gene in syndromes of insulin resistance |
10.2337/diab.43.3.357. |
Diabetes |
Molecular scanning of the insulin receptor gene in syndromes of insulin resistance
Abstract
- Using the molecular scanning technique of single-stranded conformational polymorphism (SSCP), we have examined the exons encoding the insulin receptor gene in 26 patients with syndromes of insulin resistance. We found 27 variant sequences, 4 of which were mutations that altered an amino acid. One patient with the Rabson-Mendenhall syndrome was homozygous for a mutation in the extracellular alpha-subunit (Ser to Leu323), one type A insulin-resistant patient was heterozygous for Pro to Leu1178, and another type A insulin-resistant patient was heterozygous for a mutation in the COOH-terminus of the receptor (Arg to Gln1351). The previously reported, and probably functionally insignificant, variant Val to Met985 was detected in one patient. No missense or nonsense insulin receptor mutations were found in any patients whose insulin resistance was associated with gross obesity, lipoatrophy, or acromegaloid features. No missense or nonsense mutations were found in subjects with polycystic ovary syndrome or Syndrome X. Putting these findings in the context of other work in this field, we conclude that subjects with leprechaunism or Rabson-Mendenhall syndrome have a high probability of having a missense or nonsense insulin receptor mutation. Nonobese, nondysmorphic, severely insulin-resistant females with hirsutism, acanthosis nigricans, and menstrual disturbance (type A phenotype) have an intermediate probability of having this type of insulin receptor mutation. Although insulin receptor mutations have been occasionally described in other phenotypes of insulin resistance, the frequency of point mutations in the exons of the insulin receptor gene in patients with those phenotypes appears to be low.
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| 8318831 |
Separation of human pancreatic carboxypeptidase A isoenzymes by high performance liquid chromatography |
10.1002/bmc.1130070308. |
Biomed Chromatogr |
Separation of human pancreatic carboxypeptidase A isoenzymes by high performance liquid chromatography
Abstract
- Human pancreatic carboxypeptidase A, which was isolated from a pool of necrobiotic pancreae, crystallized spontaneously and appeared homogenous in sodium dodecylsulphate polyacrylamide gel electrophoresis. Reversed phase high performance liquid chromatography of the dissolved crystals, however, revealed the presence of two distinct isoenzymes, which were shown by aminoterminal sequence analysis to be only 61% homologous in their 31 amino terminal amino acids. On the other hand, amino terminal sequences of the isoenzymes were found to be 79% and 87% homologous with CAP1 and CPA2 of the rat, respectively. Thus, the presence of two distinct pancreatic carboxypeptidase A isoenzymes could be clearly demonstrated for the first time in human tissue.
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| 8322286 |
Inhibition of thrombin and other trypsin-like serine proteinases by cyclotheonamide A |
10.1016/0049-3848(93)90158-k. |
Thromb Res |
Inhibition of thrombin and other trypsin-like serine proteinases by cyclotheonamide A
Abstract
- Cyclotheonamide A (CA), a cyclic peptide isolated from the marine sponge of the genus Theonella was shown to be a slow-binding inhibitor of several trypsin-like serine proteinases. Values of 4.6 x 10(4), 4.8 x 10(4), 9.3 x 10(3), 2.1 x 10(3) and 2.7 x 10(2) M-1 s-1 were determined for the second-order rate constants for formation of CA complexes with thrombin, trypsin, plasmin, 2-chain t-PA and factor Xa, respectively. The equilibrium constant (Ki) was measured for dissociation of CA from the CA complex with human thrombin (Ki = 1.0 nM), bovine trypsin (Ki = 0.2 nM), human plasmin (Ki = 12 nM), human factor Xa (Ki = 50 nM) and human 2-chain tissue plasminogen activator (t-PA) (Ki = 40 nM). CA produces dose dependent increases in clotting time assays. The clotting time in the thrombin time, activated partial thromboplastin time and prothrombin time assays, were doubled by 1.5, 0.9 and 48 microM CA, respectively. A model for the binding of CA to the active site of thrombin is proposed.
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| 8326490 |
A syndrome of insulin resistance resembling leprechaunism in five sibs of consanguineous parents |
10.1136/jmg.30.6.470. |
J Med Genet |
A syndrome of insulin resistance resembling leprechaunism in five sibs of consanguineous parents
Abstract
- Leprechaunism is a rare autosomal recessive disorder associated with extreme insulin resistance with paradoxical hypo-glycaemia. It is characterised by prenatal and postnatal growth retardation, reduced subcutaneous tissue, coarse features, acanthosis nigricans, enlarged genitalia, and death in the first year of life. Defects in both the insulin receptor and postreceptor steps of the insulin action pathway have been reported. At the molecular level, several mutations have been described. The patients reported here are from a Yemeni family with a syndrome of insulin resistance similar to leprechaunism in which the parents are second cousins and five of their eight children are affected. However, the phenotypes seem to be less severe than the classical leprechaunism previously described. All the children are alive (oldest 11 years), there is normal subcutaneous tissue, and a normal growth pattern in some of them. It may be that this is a milder type of leprechaunism with a better prognosis, perhaps caused by a different type of mutation from those previously described.
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| 8343587 |
Crystal structures of two cyclic pseudopentapeptides containing psiCH2S and psiCH2SO backbone surrogates |
10.1002/bip.360330712. |
Biopolymers |
Crystal structures of two cyclic pseudopentapeptides containing psiCH2S and psiCH2SO backbone surrogates
Abstract
- The solid state conformations of cyclo Gly-Pro psiCH2S Gly-D-Phe-Pro and cyclo Gly-Pro psiCH2-(S)-SOGly-D-Phe-Pro have been characterized by X-ray diffraction analysis. Crystals of the sulfide trihydrate are orthorhombic, P2(1)2(1)2(1), with a = 10.156(3) A, b = 11.704(3) A, c = 21.913(4) A, and Z = 4. Crystals of the sulfoxide are monoclinic, P2(1) with a = 10.662(1) A, b = 8.552(3) A, c = 12.947(2) A, beta = 94.28(2), and Z = 2. Unlike their all-amide parent, which adopts an all-trans backbone conformation and a type II beta-turn encompassing Gly-Pro-Gly-D-Phe, both of these peptides contain a cis Gly1-Pro2 bond and form a novel turn structure, i.e., a type II' beta-turn consisting of Gly-D-Phe-Pro-Gly. The turn structure in each of these peptides is stabilized by an intramolecular H bond between the carbonyl oxygen of Gly1 and the amide proton of D-Phe4. In the cyclic sulfoxide, the sulfinyl group is not involved in H bonding despite its strong potential as a hydrogen-bond acceptor. The crystal structure made it possible to establish the absolute configuration of the sulfinyl group in this peptide. The two crystal structures also helped identify a type II' beta-turn in the DMSO-d6 solution conformers of these peptides."
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| 8357254 |
Isolation and identification of eight microcystins from thirteen Oscillatoria agardhii strains and structure of a new microcystin |
10.1128/aem.59.7.2204-2209.1993. |
Appl Environ Microbiol |
Isolation and identification of eight microcystins from thirteen Oscillatoria agardhii strains and structure of a new microcystin
Abstract
- Microcystins (cyclic heptapeptide hepatotoxins), isolated from 13 freshwater Oscillatoria agardhii strains from eight different Finnish lakes by high-performance liquid chromatography, were characterized by amino acid analysis, fast atom bombardment mass spectrometry (FABMS), and tandem FABMS (FABMS/collisionary-induced dissociation/MS). All strains produced two to five different microcystins. In total, eight different compounds, of which five were known microcystins, were isolated. The known compounds identified were D-Asp3MCYST (microcystin)-LR, Dha7MCYST-LR, D-Asp3MCYST-RR, Dha7MCYST-RR, and D-Asp3,Dha7MCYST-RR. This is the first time that isolation of these toxins from Oscillatoria spp., with the exception of D-Asp3MCYST-RR, has been reported. Three of the strains produced a new microcystin, and the structure was assigned as D-Asp3,Mser7MCYST-RR. The structures of two new microcystins, produced as minor components by one Oscillatoria strain, could not be determined because of the small amounts isolated from the cells. Four strains produced Dha7MCYST-RR as the main toxin, but D-Asp3MCYST-RR was clearly the most abundant and most frequently occurring toxin among these isolates of O. agardhii.
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| 8367461 |
Molecular basis for the inhibition of human alpha-thrombin by the macrocyclic peptide cyclotheonamide A |
10.1073/pnas.90.17.8048. |
Proc Natl Acad Sci U S A |
Molecular basis for the inhibition of human alpha-thrombin by the macrocyclic peptide cyclotheonamide A
Abstract
- The macrocyclic peptide cyclotheonamide A (CtA), isolated from the marine sponge Theonella sp., represents an unusual class of serine protease inhibitor. A complex of this inhibitor with human alpha-thrombin, a protease central to the bioregulation of thrombosis and hemostasis, was studied by x-ray crystallography. This work (2.3-A resolution) confirms the structure of CtA and reveals intimate details about its molecular recognition within the enzyme active site. Interactions due to the "Pro-Arg motif" (Arg occupancy of the S1 specificity pocket; formation of a hydrogen-bonded two-strand antiparallel beta-sheet with Ser214-Gly216) and the alpha-keto amide group of CtA are primarily responsible for binding to thrombin, with the alpha-keto amide serving as a transition-state analogue. A special interaction with the "insertion loop" of thrombin (Tyr60A-Thr60I) is manifested through engagement of the hydroxyphenyl group of CtA with Trp60D as part of an "aromatic stacking chain." Biochemical inhibition data (Ki values at 37 degrees C) were obtained for CtA with thrombin and a diverse collection of serine proteases. Thus, CtA is just a moderate inhibitor of human alpha-thrombin (Ki = 0.18 microM) but a potent inhibitor of trypsin (Ki = 0.023 microM) and streptokinase (Ki = 0.035 microM). The relative lack of potency of CtA as a thrombin inhibitor is discussed with respect to certain structural features of the enzyme complex. We also report the total synthesis of CtA, by a convergent 2 + 3 fragment-condensation approach, to serve the preparation of cyclotheonamide analogues for structure-function studies.
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| 8370638 |
Formation of pyroglutamylglutamine (or asparagine) diketopiperazine in 'non-classical' conditions: a side reaction in peptide synthesis |
10.1111/j.1399-3011.1993.tb00343.x. |
Int J Pept Protein Res |
Formation of pyroglutamylglutamine (or asparagine) diketopiperazine in 'non-classical' conditions: a side reaction in peptide synthesis
Abstract
- Formation of by-products pyroglutamylglutamine diketopiperazine (5a) or pyroglutamylasparagine diketopiperazine (5b) were observed during the condensation of pyroglutamic acid active ester with C-protected glutaminyl(or asparaginyl)-proline derivatives. Successful synthesis of Glp-Gln(or Asn)-Pro-derivatives is possible after coupling of Glp-Gln(or Asn)-OH with proline-derivatives.
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| 8373420 |
Cloning, functional expression and pharmacological characterization of a fourth (hSSTR4) and a fifth (hSSTR5) human somatostatin receptor subtype. |
10.1006/bbrc.1993.2122 |
Biochem. Biophys. Res. Commun. |
Cloning, functional expression and pharmacological characterization of a fourth (hSSTR4) and a fifth (hSSTR5) human somatostatin receptor subtype.
Abstract
- Somatostatin exerts diverse effects in various tissues upon binding its specific membrane receptors. Recently, we have cloned three different somatostatin receptor subtypes. Here we report the sequence and functional expression of a fourth and a fifth human somatostatin receptor subtype, termed hSSTR4 and hSSTR5, respectively. The hSSTR4 encodes a protein of 388 amino acids and the hSSTR5 is a protein of 364 amino acids. There is 42-60% identity among the amino acid sequences of the five human somatostatin receptor subtypes identified to date. RNA blotting studies reveal that the hSSTR4 is expressed as a single transcript of 4.8 kb in MIA PaCa-2 cells, a cell line derived from human pancreatic cancer while the hSSTR5 is undetectable in the tissues examined. The hSSTR4 and hSSTR5 transiently expressed in COS1 cells exhibit specific binding to somatostatin-14 with IC50 values of 1.6 and 0.16 nM, respectively. We also have characterized the binding affinity of various somatostatin analogues to the hSSTR4 and hSSTR5. The rank of the potency of the analogues are: somatostatin-14 = somatostatin-28 >> RC-160 >> SMS201-995 for the hSSTR4 and somatostatin-28 > somatostatin-14 >> RC-160 > SMS201-995 for the hSSTR5. These
Results suggest that diverse actions of somatostatin are mediated by at least five somatostatin receptor subtypes with potentially different function.
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| 8381596 |
Comparison of receptors for Escherichia coli heat-stable enterotoxin: novel receptor present in IEC-6 cells. |
10.1152/ajpgi.1993.264.1.g172 |
Am. J. Physiol. |
Comparison of receptors for Escherichia coli heat-stable enterotoxin: novel receptor present in IEC-6 cells.
Abstract
- Enterotoxigenic Escherichia coli elaborate a heat-stable enterotoxin that causes diarrhea in humans and animals. The primary event in the diarrheal cascade is the binding of this enterotoxin to specific receptors on enterocytes and activation of guanylyl cyclase. Two intestinal cell lines, Caco-2 and IEC-6, were tested for the presence of these receptors. Although both cell lines exhibited specific binding, only the Caco-2 cell line responded to heat-stable enterotoxin with increased guanylyl cyclase activity. Cloning and expression studies confirmed that the receptor present in Caco-2 cells is a homologue of guanylyl cyclase C, a known transmembrane heat-stable enterotoxin receptor. Expression of the receptor in differentiating Caco-2 cells increases with cell maturation, indicating that these cells are a suitable model for future studies. However, Northern and polymerase chain reaction analyses demonstrated that guanylyl cyclase C is not expressed in IEC-6 cells, strongly suggesting the presence of a novel heat-stable enterotoxin receptor that is not coupled to guanylyl cyclase activity.
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| 8384323 |
Amino-aromatic interaction between histidine 197 of the neurokinin-1 receptor and CP 96345 |
10.1038/362350a0. |
Nature |
Amino-aromatic interaction between histidine 197 of the neurokinin-1 receptor and CP 96345
Abstract
- Substance P is a peptide neurotransmitter that binds to the neurokinin-1 receptor and is involved in pain transmission and neurogenic inflammation. Recently, a non-peptide substance P antagonist (CP 96345) has been shown to be effective in animal models of pain and inflammation. An understanding of the molecular interactions responsible for ligand binding will be critical for the development of more specific and selective antagonists for the neurokinin-1 receptor and for the discovery of new antagonists for related G-protein-coupled receptors. Here we report that histidine at position 197 in the fifth transmembrane helix of the human neurokinin-1 receptor binds specifically to CP 96345 but not to peptide agonists. Substitution of His 197 by different amino acids and analysis of structural analogues of antagonists suggest that His 197 is involved in an amino-aromatic interaction with the benzhydryl moiety of CP 96345.
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| 8384598 |
Immunomodulation of human T cell responses with receptor selective enkephalins |
10.1016/0165-2478(93)90144-q. |
Immunol Lett |
Immunomodulation of human T cell responses with receptor selective enkephalins
Abstract
- The delta-opioid receptor selective 2-D-penicillamine-5-D-penicillamine enkephalin (DPDPE) and the mu receptor selective Tyr-D-Orn-Phe-Asp-NH2 (TOPA) were found respectively, to have marked immunostimulant and immunosuppressant activities in both normal subjects and patients suffering from leprosy and tuberculosis. Antigen specific lymphoproliferation and numbers of rosette forming T cells were significantly (P < 0.05) enhanced on in vitro treatment with Met-enkephalin. This was further increased (P < 0.001) in the presence of the delta selective DPDPE. In contrast, treatment with mu selective TOPA inhibited lymphoproliferation substantially (P < 0.01) and rosette formation to a lesser extent.
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| 8386508 |
Multiple gene transcripts of the somatostatin receptor SSTR2: tissue selective distribution and cAMP regulation. |
10.1006/bbrc.1993.1412 |
Biochem. Biophys. Res. Commun. |
Multiple gene transcripts of the somatostatin receptor SSTR2: tissue selective distribution and cAMP regulation.
Abstract
- The rodent SSTR2 mRNA has been reported to be alternatively spliced to generate long (SSTR2A) and short (SSTR2B) receptor isoforms which differ in sequence at their C-terminal regulatory domains. By extending the 3' nucleotide sequence of the human gene (hSSTR2) we show highly conserved intron/exon boundaries suggesting that hSSTR2 is also capable of generating spliced variants. mRNA blots of rat tissues reveal 2 transcripts of 2.8 and 2.3 kb that are differentially expressed in brain regions and multiple peripheral organs. The 2.3 kb mRNA is preferentially expressed in pituitary tumor cells (AtT-20 mouse, GH3 rat, human prolactinoma, human somatotroph adenoma), but not in rat or human insulinoma cells. This transcript shows 4 fold induction by forskolin in AtT-20 cells suggesting cAMP dependent control of SSTR2 gene expression. The abundant expression of SSTR2 gene, the occurrence of 2 isoforms and evidence of extensive regulation at both gene and peptide levels, suggests that SSTR2 is the principal SST-14 selective subtype.
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| 8388384 |
Insulin stimulates association of insulin receptor substrate-1 with the protein abundant Src homology/growth factor receptor-bound protein 2 |
None |
J Biol Chem |
Insulin stimulates association of insulin receptor substrate-1 with the protein abundant Src homology/growth factor receptor-bound protein 2
Abstract
- Insulin activates the ras proto-oncogene product p21ras (Ras) by stimulating conversion of the inactive GDP-bound form of Ras to the active GTP-bound form. The protein ASH (for abundant Src homology) (Matuoka, K., Shibata, M., Yamakawa, A., and Takenawa, T. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 9015-9019) is composed of one Src homology (SH)2 and two SH3 domains and highly homologous to the Caenorhabditis elegans protein sem-5 that couples a tyrosine kinase to a Ras protein. We have studied an interaction of ASH with insulin-stimulated tyrosine-phosphorylated proteins in Chinese hamster ovary cells overexpressing human insulin receptors (CHO-HIR cells). In an anti-ASH (alpha ASH) immunoprecipitates, we detected a 170-kDa phosphoprotein that was recognized by an anti-phosphotyrosine antibody and an anti-insulin receptor substrate 1 antibody (alpha IRS-1) from the insulin-stimulated [32P]orthophosphate-labeled CHO-HIR cells. We failed to detect the tyrosine phosphorylation of the protein ASH. These data suggested that insulin stimulates IRS-1.ASH complex formation in intact cells. Incubation of an ASH fusion protein with the lysates of insulin-stimulated CHO-HIR cells revealed that the fusion protein of ASH was able to bind the tyrosine-phosphorylated 170-kDa protein that was recognized by alpha IRS-1. We also demonstrated that fusion protein of ASH was able to bind the fusion protein of tyrosine-phosphorylated IRS-1 fragments, suggesting that ASH is able to bind tyrosine-phosphorylated IRS-1 directly. These data suggest that IRS-1.ASH complex formation may play a role in coupling the insulin receptor kinase to a Ras signaling pathway. Furthermore, we observed an insulin-stimulated phosphatidylinositol (PI) 3-kinase activity in alpha ASH immunoprecipitates, suggesting the formation of an ASH.IRS-1.PI 3-kinase complex. This complex formation was detected as early as 10 s after insulin stimulation in intact CHO-HIR cells. This is the first report that supports the notion that IRS-1 binds several signal transducing molecules containing SH2 domains, thus serves as an SH2 docking protein that transduces insulin's signal multidirectionally.
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| 8388389 |
Antibodies to the extracellular receptor domain restore the hormone-insensitive kinase and conformation of the mutant insulin receptor valine 382 |
None |
J Biol Chem |
Antibodies to the extracellular receptor domain restore the hormone-insensitive kinase and conformation of the mutant insulin receptor valine 382
Abstract
- A mutation substituting a valine for phenylalanine at residue 382 in the insulin receptor alpha-subunit has been found in two sisters with a genetic form of extreme insulin resistance. This receptor mutation impairs the ability of the hormone to activate autophosphorylation of solubilized receptors and phosphorylation of substrates (Accili, D., Mosthaf, L., Ullrich, A., and Taylor, S. I. (1991) J. Biol. Chem. 266, 434-439). We have previously demonstrated that in native receptors insulin induces a conformational change in the receptor beta-subunit, which is thought to be necessary for receptor activation (Baron, V., Gautier, N., Komoriya, A., Hainaut, P., Scimeca, J. C., Mervic, M., Lavielle, S., Dolais-Kitabgi, J., and Van Obberghen, E. (1990) Biochemistry 29, 4634-4641). Hence, it was thought that a defect in this conformational change might explain the functional defect of the mutant receptor. This appears to be the case, as we demonstrate here that the mutant receptor is locked in its inactive configuration. However, we found two monoclonal antibodies, directed to the extracellular domain, which are capable of restoring the mutant receptor kinase activity. The activation of the mutant receptor was accompanied by restoration of conformational changes in the beta-subunit C terminus. From these data, we draw the two following conclusions. (i) A causal link exists between receptor kinase activation and the occurrence of conformational changes. (ii) Ligands other than insulin, such as antibodies, which perturb the extracellular domain, can function as alternative ways to restore the mutant receptor kinase.
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