| 8243830 |
Ala1048-->Asp mutation in the kinase domain of insulin receptor causes defective kinase activity and insulin resistance |
10.2337/diab.42.12.1837. |
Diabetes |
Ala1048-->Asp mutation in the kinase domain of insulin receptor causes defective kinase activity and insulin resistance
Abstract
- We identified a heterozygous missense mutation that substituted aspartic acid (GAC) for alanine (GCC) at codon 1048 of the insulin receptor gene in a patient who displayed typical symptoms of Type A syndrome of insulin resistance. The proband's mother and younger brother were also found to be heterozygous for the mutation. We constructed the identified mutant insulin receptor cDNA by site-directed mutagenesis, transfected the mutant cDNA into COS 7 cells, and found that kinase activity of the mutant insulin receptors was markedly impaired. Ala1048 is located in the kinase domain of the insulin receptor beta-subunit and is conserved in most of protein-tyrosine kinases. Besides, neighboring Glu1047 is invariant in all protein kinases and is thought to be involved in interaction with ATP. Photoaffinity labeling of the mutant insulin receptor with ATP analogue, 8-azido (alpha-32P)ATP was not influenced by the mutation, suggesting that the mutation did not inhibit ATP binding but possibly interfered with subsequent phosphoryl transfer. Insulin-stimulated phosphorylation of exogenous substrate by partially purified insulin receptors prepared from COS 7 cells that were cotransfected with wild-type and mutant insulin receptor cDNAs was markedly impaired, whereas autophosphorylation was decreased by approximately 50% of wild-type receptors. These results indicated that the identified heterozygous substitution of Asp for Ala1048 in insulin receptor was responsible for insulin resistance of this patient.
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| 8247543 |
A survey of protein tyrosine kinase mRNAs expressed in normal human melanocytes. |
10.1021/bi00212a019 |
Oncogene |
A survey of protein tyrosine kinase mRNAs expressed in normal human melanocytes.
Abstract
- We have used the reverse transcription-polymerase chain reaction to survey the repertoire of protein tyrosine kinases expressed in cultured normal human melanocytes, a differentiated cell type derived from the neural crest. We identified 25 different tyrosine kinase cDNAs among a total of 608 protein tyrosinase kinase-related cDNAs analyzed. Six encode receptor tyrosine kinases for known ligands, several of which have been implicated in controlling melanocyte proliferation in vitro. Two others encode apparent receptor tyrosine kinases for unknown ligands. Four encode known non-receptor tyrosine kinases and five encode previously identified anonymous protein tyrosine kinases. Of the eight other melanocyte-associated protein tyrosine kinases, most or all appear to be novel. These 25 protein tyrosine kinase genes exhibit distinct patterns of expression in cultured human melanocytes, human erythroleukemia cells, and a variety of normal human tissues. We mapped 16 of the corresponding protein tyrosine kinase genes to specific human chromosomes, identifying a total of 19 human genetic loci, some of which may constitute candidate genes for genetic disorders of mammalian development.
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| 8251938 |
Changes in interactions in complexes of hirudin derivatives and human alpha-thrombin due to different crystal forms |
10.1002/pro.5560021009. |
Protein Sci |
Changes in interactions in complexes of hirudin derivatives and human alpha-thrombin due to different crystal forms
Abstract
- The three-dimensional structures of D-Phe-Pro-Arg-chloromethyl ketone-inhibited thrombin in complex with Tyr-63-sulfated hirudin (ternary complex) and of thrombin in complex with the bifunctional inhibitor D-Phe-Pro-Arg-Pro-(Gly)4-hirudin (CGP 50,856, binary complex) have been determined by X-ray crystallography in crystal forms different from those described by Skrzypczak-Jankun et al. (Skrzypczak-Jankun, E., Carperos, V.E., Ravichandran, K.G., & Tulinsky, A., 1991, J. Mol. Biol. 221, 1379-1393). In both complexes, the interactions of the C-terminal hirudin segments of the inhibitors binding to the fibrinogen-binding exosite of thrombin are clearly established, including residues 60-64, which are disordered in the earlier crystal form. The interactions of the sulfate group of Tyr-63 in the ternary complex structure explain why natural sulfated hirudin binds with a 10-fold lower K(i) than the desulfated recombinant material. In this new crystal form, the autolysis loop of thrombin (residues 146-150), which is disordered in the earlier crystal form, is ordered due to crystal contacts. Interactions between the C-terminal fragment of hirudin and thrombin are not influenced by crystal contacts in this new crystal form, in contrast to the earlier form. In the bifunctional inhibitor-thrombin complex, the peptide bond between Arg-Pro (P1-P1') seems to be cleaved.
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| 8257133 |
Identification of the satA gene encoding a streptogramin A acetyltransferase in Enterococcus faecium BM4145. |
10.1128/aac.37.10.2119 |
Antimicrob. Agents Chemother. |
Identification of the satA gene encoding a streptogramin A acetyltransferase in Enterococcus faecium BM4145.
Abstract
- Enterococcus faecium BM4145, a clinical isolate from urine, was resistant to streptogramin group A antibiotics by inactivation. The strain harbored a plasmid containing a gene, satA, responsible for this resistance; this gene was cloned and sequenced. It encoded SatA, a protein deduced to be 23,634 Da in mass and homologous with a new family of chloramphenicol acetyltransferases described in Agrobacterium tumefaciens, Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus. The similarity of SatA to other acetyltransferases, LacA (thiogalactoside acetyltransferase) and CysE (serine acetyltransferase) from E. coli, and to two putative acetyltransferases, NodL from Rhizobium leguminosarum and Urf1 from E. coli, was also observed in a region considered to be the enzyme's active site. Acetylation experiments indicated that acetyl coenzyme A was necessary for SatA activity and that a single acetylated derivative of pristinamycin IIA was produced. Other members of the streptogramin A group such as virginiamycin M and RP54476 were also substrates for the enzyme. We conclude that resistance to the streptogramin A group of antibiotics in E. faecium BM4145 is due to acetylation by an enzyme related to the novel chloramphenicol acetyltransferase family.
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| 8257688 |
Characterization of human placental insulin-like growth factor-I/insulin hybrid receptors by protein microsequencing and purification. |
10.1021/bi00212a019 |
Biochemistry |
Characterization of human placental insulin-like growth factor-I/insulin hybrid receptors by protein microsequencing and purification.
Abstract
- Protein microsequencing of human placental IGF-I receptors purified by immunoaffinity chromatography using IGF-I receptor specific monoclonal antibody revealed amino acid sequences of both IGF-I and insulin receptors. Since this finding indicated the presence of IGF-I/insulin receptor hybrids, hybrid receptors were further purified by immunoaffinity chromatography using insulin receptor specific monoclonal antibody. The molecular size of the nonreduced hybrid receptor was approximately 350K, indicating that the IGF-I and insulin receptor alpha beta halves were disulfide-linked. The ratio of IGF/insulin binding activity of purified hybrid receptors was approximately 25 when measured using tracer amounts of radioactive ligands. 125I-IGF binding to these receptors was inhibited by IGF-I and insulin with IC50s of approximately 2 and approximately 1000 nM, respectively. 125I-Insulin binding to these receptors was similarly inhibited by IGF-I and insulin with IC50 of approximately 3 nM. Autophosphorylation and kinase activities of the hybrid receptor were stimulated by IGF-I more effectively than insulin in a dose-dependent manner. Thus, the present studies indicate that hybrid receptors purified from human placenta have the functional properties of an IGF-I receptor.
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| 8262059 |
Mutation of Tyr697, a GRB2-binding site, and Tyr721, a PI 3-kinase binding site, abrogates signal transduction by the murine CSF-1 receptor expressed in Rat-2 fibroblasts |
10.1002/j.1460-2075.1993.tb06211.x. |
EMBO J |
Mutation of Tyr697, a GRB2-binding site, and Tyr721, a PI 3-kinase binding site, abrogates signal transduction by the murine CSF-1 receptor expressed in Rat-2 fibroblasts
Abstract
- The receptor for the myeloid cell growth factor colony stimulating factor 1 (CSF-1) is a protein tyrosine kinase that is closely related to the PDGF receptor. Ligand binding results in kinase activation and autophosphorylation. Three autophosphorylation sites, Tyr697, Tyr706 and Tyr721, have been mapped to the kinase insert domain. Deletion of the entire kinase insert domain completely abrogates signal transduction by the CSF-1 receptor expressed in Rat-2 fibroblasts. To investigate the function of individual phosphorylation sites present in the CSF-1 receptor kinase insert domain, a number of phosphorylation site mutants were expressed in Rat-2 fibroblasts. Mutation of either Tyr697 or Tyr721 compromised signal transduction by the CSF-1 receptor. A mutant receptor, in which both Tyr697 and Tyr721 were replaced by phenylalanine, has lost all ability to induce changes in morphology or to increase cell growth rate in response to CSF-1. Tyr721 has been identified recently as the binding site for PI 3-kinase. Here we report that GRB2 associates with the CSF-1 receptor upon ligand binding. The phosphorylation on tyrosine of SHC and several other GRB2-associated proteins increased upon stimulation with CSF-1. Tyr697 was identified as a binding site for GRB2. We suggest that PI 3-kinase, GRB2 and some of the GRB2-associated proteins could play an important role in signal transduction by the CSF-1 receptor.
|
| 8270499 |
Isolation of RP 71955, a new anti-HIV-1 peptide secondary metabolite |
10.7164/antibiotics.46.1756. |
J Antibiot (Tokyo) |
Isolation of RP 71955, a new anti-HIV-1 peptide secondary metabolite
Abstract
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| 8276809 |
Direct activation of the phosphatidylinositol 3'-kinase by the insulin receptor. |
10.1016/s0021-9258(17)42304-0 |
J. Biol. Chem. |
Direct activation of the phosphatidylinositol 3'-kinase by the insulin receptor.
Abstract
- We have previously shown that phosphatidylinositol (PtdIns) 3'-kinase is activated by the binding of proteins or peptides containing the phosphorylated motif Y(P)XXM. In the present study, we examine interactions between PtdIns 3'-kinase and the human insulin receptor, which contains a C-terminal phosphorylation site in the sequence Y1322THM. Partially purified insulin receptors bound tightly to bacterial fusion proteins containing the N- or C-terminal SH2 domains from PtdIns 3'-kinase regulatory subunit (p85). In contrast, a mutant insulin receptor, truncated by 43 amino acids at the C terminus (IR delta CT), bound poorly to the SH2 domains; these mutant receptors have normal kinase activity but lack the Y1322THM motif. Similarly, incubation with wild-type receptors increased the activity of immunopurified PtdIns 3'-kinase, whereas incubation with IR delta CT receptors did not affect PtdIns 3'-kinase activity. Activation of PtdIns 3'-kinase by the wild-type receptor was mimicked by a tyrosyl phosphopeptide derived from the insulin receptor C terminus and containing the Y1322THM motif; non-phosphorylated peptide did not affect activity. Thus, the insulin receptor C terminus activates PtdIns 3'-kinase in vitro by binding to the SH2 domains of the 85-kDa regulatory subunit. These data support the hypothesis that binding of tyrosyl-phosphorylated receptors to p85 SH2 domains is a general mechanism for PtdIns 3'-kinase activation, and they suggest that direct interactions between the insulin receptor and PtdIns 3'-kinase may provide an alternative pathway for the activation of this enzyme by insulin.
|
| 8280748 |
Cellular protein receptors of maculosin, a host specific phytotoxin of spotted knapweed (Centaurea maculosa L.) |
10.1016/0304-4165(94)90090-6. |
Biochim Biophys Acta |
Cellular protein receptors of maculosin, a host specific phytotoxin of spotted knapweed (Centaurea maculosa L.)
Abstract
- Maculosin (the diketopiperazine, cyclo (L-Pro-L-Tyr)) is a host specific phytotoxin produced by Alternaria alternata on spotted knapweed (Centaurea maculosa L.). Receptors for this phytotoxin have been isolated from spotted knapweed. Knapweed leaves possess most of the maculosin-binding activity in the cytosolic fraction. However, activity was also observed in the whole membrane fraction of the leaf. The binding component of the cytosolic fraction was identified as a protein(s) because of its heat-lability and sensitivity to proteases. A 16-fold purification of a toxin-binding protein was carried out by ammonium sulfate fractionation, and Sephadex G-200, and maculosin-affinity column chromatography. The affinity column was prepared with epoxy activated Sepharose 6B to which the phenolic group of maculosin was attached. The receptor was estimated to contain more than one binding protein by native and SDS-PAGE. At least one of the maculosin-binding proteins was identified as ribulose-1,5-biphosphate carboxylase (RuBPcase).
|
| 8281568 |
Molecular conformation of patellamide A, a cytotoxic cyclic peptide from the ascidian Lissoclinum patella, by X-ray crystal analysis |
10.1248/cpb.41.1686. |
Chem Pharm Bull (Tokyo) |
Molecular conformation of patellamide A, a cytotoxic cyclic peptide from the ascidian Lissoclinum patella, by X-ray crystal analysis
Abstract
- As part of a series of investigations into the conformational stability of a C2-symmetric or related cyclic peptide isolated from the ascidian Lissoclinum patella, the molecular conformation of patellamide A, the chemical structure of which deviates slightly for C2-symmetry, was determined by X-ray crystal analysis. Patellamide A took on a saddle-shaped rectangular form and wrapped around the water and methanol solvents. This conformation which is very similar to that of C2-symmetric ascidiacyclamide would be proposed as a possible candidate for biologically "active" conformation.
|
| 8286361 |
Solution structure of RP 71955, a new 21 amino acid tricyclic peptide active against HIV-1 virus |
10.1021/bi00167a006. |
Biochemistry |
Solution structure of RP 71955, a new 21 amino acid tricyclic peptide active against HIV-1 virus
Abstract
- The structure of RP 71955, a new tricyclic 21 amino acid peptide active against human immunodeficiency virus 1, was determined. Its amino acid composition was inferred from the results of fast atom bombardment mass spectrometry, nuclear magnetic resonance, Raman spectroscopy, and amino acid analysis. Its sequence could not be determined classically, using Edman degradation, given the lack of a free terminal NH2. It was deduced from the interpretation of interresidue nuclear Overhauser effects and confirmed by the sequencing of peptides obtained by limited chemical hydrolysis. It was found to be CLGIGSCNDFAGCGYAVVCFW. An internal amide bond between the NH2 of C1 and the gamma-COOH of D9 was observed, as well as two disulfide bridges, one between C1 and C13 and one between C7 and C19. The three-dimensional structure of RP 71955 was determined from nuclear magnetic resonance derived constraints using distance geometry, restrained molecular dynamics, nuclear Overhauser effect back calculation, and an iterative refinement using a full relaxation matrix approach. Analogies between the structure of RP 71955 and some functional domains of gp41, the transmembrane protein of human immunodeficiency virus 1, suggest hypotheses concerning the mode of action of RP 71955.
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| 8288049 |
Prevalence of mutations in the insulin receptor gene in subjects with features of the type A syndrome of insulin resistance |
10.2337/diab.43.2.247. |
Diabetes |
Prevalence of mutations in the insulin receptor gene in subjects with features of the type A syndrome of insulin resistance
Abstract
- Mutations of the insulin receptor gene are a cause of the type A syndrome of extreme insulin resistance. This study assessed the prevalence of such mutations in women with clinical features of the type A syndrome including ovarian hyperandrogenism, moderate-to-severe degrees of insulin resistance, and acanthosis nigricans. We studied 22 unrelated women with insulin resistance (fasting insulin > 300 pM [50 microU/ml] and/or peak during an oral glucose tolerance test (OGTT) > 1,800 pM [300 microU/ml]), acanthosis nigricans, and the polycystic ovary syndrome (hyperandrogenemia, oligoamenorrhea, and hirsutism). Two insulin-resistant probands with congenital generalized lipodystrophy and one male proband with severe insulin resistance also were included in the study. Southern blotting experiments were performed to exclude gross gene deletions, insertions, or rearrangements. Exons 2-22 of the insulin receptor gene were polymerase chain reaction (PCR) amplified from genomic DNA and screened for nucleotide variation using single-strand conformation polymorphism (SSCP). No nucleotide variation between study subjects was detected in exons 4-6, 10-12, 15, 16, 18, 19, or 21. Sequencing of amplified DNA revealed that SSCP variants in exons 2, 3, 8, 9, and 17 corresponded to known silent polymorphisms within the coding region. Variants in exons 2, 9, 13, and 14 were caused by novel silent polymorphisms; variants in exons 7 and 22 were caused by nucleotide substitutions in flanking introns. One proband was found to have a heterozygous point mutation in exon 20 (CGG-->CAG, Arg1174-->Gln) that involves the intracellular receptor beta-subunit.(ABSTRACT TRUNCATED AT 250 WORDS)
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| 8292046 |
Annetocin: an oxytocin-related peptide isolated from the earthworm, Eisenia foetida |
10.1006/bbrc.1994.1055. |
Biochem Biophys Res Commun |
Annetocin: an oxytocin-related peptide isolated from the earthworm, Eisenia foetida
Abstract
- An oxytocin-vasopressin-related peptide, Cys-Phe-Val-Arg-Asn-Cys-Pro-Thr-Gly-NH2, was isolated from the lumbricid earthworm, Eisenia foetida and termed annectocin. Annetocin potentiated not only spontaneous contractions of the gut but also pulsatory contractions and bladder-shaking movement of the nephridia. Annetocin may be involved in osmoregulation of the animal through nephridial function.
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| 8298129 |
Isolation and characterization of a TATA-less promoter for the human beta 3 integrin gene. |
10.1101/gr.2596504 |
Blood |
Isolation and characterization of a TATA-less promoter for the human beta 3 integrin gene.
Abstract
- Proper expression of the human platelet fibrinogen receptor is necessary for the maintenance of normal hemostasis. This receptor is formed by the heterodimer alpha IIb beta 3, a prototypic member of the integrin family of adhesive molecules. beta 3 is also expressed in other tissues with alpha v as the vitronectin receptor. It was not possible to study the basis for tissue-specific expression of this gene, because the beta 3 gene promoter had not been isolated previously. We have now isolated a 6.0-kb human genomic DNA fragment containing 2.0 kb of sequence 5' to the beta 3 ATG start codon. This clone also contains sequence encoding the signal peptide of the immature beta 3 protein and 3.0 kb of 3' intronic sequence. Primer extension and RNase protection studies of poly A+ RNA from a human erythroleukemia (HEL) cell line indicated a major transcription start site 30 bp upstream of the ATG start codon. In an orientation-dependent manner, a 584-bp fragment 5' to the start codon promotes expression of the chloramphenicol acetyl transferase (CAT) reporter gene in K562 cells. CAT expression from this beta 3 promoter is fivefold above expression from a "promoter-less" control CAT construct. This beta 3 promoter lacks TATA and CAAT cis-acting elements, but there are two Sp1 sites flanking the transcription start site. Other potential transcription factor binding sites are also identified. Phorbol esters (TPA), which increase beta 3 transcription in K562 cells, stimulated transcription from the 584-bp 5' beta 3 region. The isolation of this beta 3 promoter region should permit a more detailed analysis of its transcriptional regulation.
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| 8307686 |
Molecular structures of two crystalline forms of the cyclic heptapeptide antibiotic ternatin, cyclo-beta-OH-D-Leu-D-Ile-(NMe)Ala-(NMe)Leu-Leu-(NMe)Ala-D-(NMe)Ala- |
10.1111/j.1399-3011.1993.tb00362.x. |
Int J Pept Protein Res |
Molecular structures of two crystalline forms of the cyclic heptapeptide antibiotic ternatin, cyclo-beta-OH-D-Leu-D-Ile-(NMe)Ala-(NMe)Leu-Leu-(NMe)Ala-D-(NMe)Ala-
Abstract
- The crystal structures of two solvated forms of ternatin, cyclo-beta-OH-D-Leu-D-Ile-(NMe)Ala-(NMe)Leu-Leu-(NMe)Ala-D-(NMe)Ala- are reported. The first crystallizes with two molecules of peptide and one of dioxane in the asymmetric unit: P2(1)2(1)2(1), a = 11.563(1), b = 21.863(2), c = 36.330(4) A. The second crystallizes with two molecules of peptide and one of water in the asymmetric unit: P2(1)2(1)2(1), a = 14.067(2), b = 16.695(1), c = 36.824(6) A. N-Methylation of four of the seven residues of ternatin appears to reduce the number of low-energy conformations the molecule can assume. The same H-bonded macrocyclic ring conformation is adopted by the backbone of each of the four molecules observed here. All the amino-acid side chains, with the exception of D-Ile2, have similar orientations in each of the four conformers. The heptapeptide macrocycle is characterized by: (i) a cis peptide between (NMe)Ala3 and (NMe)Leu4, (ii) a type II beta-bend, involving residues Leu5-(NMe)Ala6-D-(NMe)Ala7-beta-OH-D-Leu1, stabilized by two H-bonds, N1-->O5 and N5-->O1, between Leu5 and beta-OH-D-Leu1 residues, (iii) a third intramolecular H-bond, observed in each of the four molecules, between the hydroxyl group of beta-OH-D-Leu1 and the carbonyl oxygen of D-Ile2.
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