| 2280779 |
Structural and functional analysis of the insulin receptor promoter. |
10.1210/mend-4-4-647 |
Mol. Endocrinol. |
Structural and functional analysis of the insulin receptor promoter.
Abstract
- The insulin receptor plays a critical role in the maintenance of glucose homeostasis. Regulation of this key function must be under stringent controls. In order to study the regulation of insulin receptor gene expression, we have cloned, sequenced and characterized its promoter. The first exon of the insulin receptor gene is embedded in an unusual segment of DNA composed of Alu repeats. The promoter has the characteristics typical of a housekeeping gene. It is GC-rich and has multiple start sites of transcription. A 574 base pair fragment immediately upstream of the translation initiation site contains promoter activity when transfected into eukaryotic cell lines. Deletion analysis was performed to study promoter function. These studies showed that only 150 base pairs of promoter sequence were necessary for promoter function. This region contains three potential binding sites for the transcription factor, Sp1 and a TC box sequence. Furthermore, the fragment functions equally well in either orientation. We have defined an element in this region with enhancer function for both its homologous and a heterologous promoter. In addition, this region seems to contribute some degree of tissue specificity to insulin receptor gene expression.
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| 2293021 |
Insulin-degrading enzyme: stable expression of the human complementary DNA, characterization of its protein product, and chromosomal mapping of the human and mouse genes. |
10.1210/mend-4-8-1125 |
Mol. Endocrinol. |
Insulin-degrading enzyme: stable expression of the human complementary DNA, characterization of its protein product, and chromosomal mapping of the human and mouse genes.
Abstract
- We have recently described the isolation of a cDNA encoding an enzyme thought to be involved in the degradation of insulin by insulin-responsive tissues. This enzyme, referred to as insulin-degrading enzyme (IDE), is a cytosolic proteinase of 110,000 mol wt which shares structural and functional homology with bacterial protease III. The enzyme may function in the termination of the insulin response. We report here the mapping of the human and mouse IDE genes to human chromosome 10 and mouse chromosome 19, respectively, and evidence for the existence of a single complex IDE gene. We also describe the stable transfection of Chinese hamster ovary cells with a plasmid containing the IDE cDNA under the transcriptional control of the SR alpha promoter. The recombinant protein synthesized by these cells is indistinguishable from the isolated human enzyme in both its size and immunoreactivity and degrades insulin with a specific activity similar to that of the purified proteinase. Overexpression of IDE using this system should allow for a functional test of the role of IDE in insulin action. In addition, expression of various site-directed mutants of IDE will aid in identifying the residues of IDE and protease III that are essential to the activity of this unique family of proteinases.
|
| 2298169 |
Interactions of dopaminergic and peptidergic factors in the control of prolactin release |
10.1210/endo-126-2-728. |
Endocrinology |
Interactions of dopaminergic and peptidergic factors in the control of prolactin release
Abstract
- Oxytocin (OT) has been shown to play a role in the control of physiological PRL release and has been demonstrated to have a direct effect on the pituitary to stimulate PRL secretion. Administration of OT into the third ventricle, however, lowers PRL levels. This reduction could be mediated by either an inhibition of the release of endogenous OT into the hypohysial portal circulation or via an alteration in the release of some other PRL releasing (PRF) or PRL release-inhibiting (PIF) factor. In order to determine if centrally administered OT lowers PRL levels by increasing secretion of dopamine (DA) into the portal circulation, endogenous dopaminergic tone was blocked by injection of the DA antagonist domperidone (DOM). Subcutaneous administration of DOM resulted in elevated PRL levels which could be further augmented by iv infusion of OT (at 0.01 or 0.1 microgram OT/kg.min) or partially, but significantly, reduced by pretreatment with anti-OT antiserum (0.75 ml) indicating that under conditions of DA blockade, OT (which has little PRF activity during conditions of normal dopaminergic tone) can stimulate PRL secretion by a direct pituitary action. Treatment with DOM did not prevent, however, the reduction in PRL levels produced by central administration of OT (2 micrograms). This suggests that the effect of OT to alter PRL secretion when administered into the third ventricle was not mediated via an increase in DA release into the portal circulation. Furthermore, central administration of the OT antagonist CAV-259 (1-deamino-2-D-Trp-4-Val-8-Orn-OT) after DOM treatment resulted in a significant increase in PRL secretion indicating that endogenous levels of OT within the hypothalamus inhibit PRL secretion through a nondopaminergic mechanism. This stimulatory effect of the OT antagonist was not blocked by pretreatment with anti-OT antiserum (iv) which had been demonstrated previously to reduce the PRL surges in lactating mothers and steroid-primed ovariectomized rats, as well as to block the increase in PRL secretion seen after central administration of vasoactive intestinal peptide (VIP). Thus the central effect of OT to alter PRL secretion was probably not due to a change in the release of OT into the portal circulation. Intravenous administration of a VIP antagonist (D-4-Cl-6-Phe-17-Leu-VIP, previously demonstrated to be capable of reducing the PRL surge seen in lactating mothers) into DOM-treated rats does not alter PRL levels but blocks the ability of central administration of the OT antagonist CAV-259 to increase PRL levels under these conditions.(ABSTRACT TRUNCATED AT 400 WORDS)
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| 2307232 |
Further studies on the human pancreatic binary complexes involving procarboxypeptidase A |
10.1016/0014-5793(90)80665-6. |
FEBS Lett |
Further studies on the human pancreatic binary complexes involving procarboxypeptidase A
Abstract
- In contrast to procarboxypeptidase B which has always been reported to be secreted by the pancreas as a monomer, procarboxypeptidase A occurs as a monomer and/or associated to one or two functionally different proteins, depending on the species. Recent studies showed that, in the human pancreatic secretion, procarboxypeptidase A is mainly secreted as a 44 kDa protein involved in at least three different binary complexes. As previously reported, two of these complexes associated procarboxypeptidase A to either a glycosylated truncated protease E or zymogen E. In this paper, we identified proelastase 2 as the partner of procarboxypeptidase A in the third complex, thus reporting for the first time the occurrence of a proelastase 2/procarboxypeptidase A binary complex in vertebrates. Moreover, from N-terminal sequence analyses, the 44 kDa procarboxypeptidase A involved in these complexes was identified as being of the A1 type. Only one type of procarboxypeptidase B, the B1 type, has been detected in the analyzed pancreatic juices, thus emphasizing the previously observed genetic differences between individuals.
|
| 2317495 |
Iturin lipopeptides: interactions of mycosubtilin with lipids in planar membranes and mixed monolayers |
10.1016/0005-2736(90)90006-a. |
Biochim Biophys Acta |
Iturin lipopeptides: interactions of mycosubtilin with lipids in planar membranes and mixed monolayers
Abstract
- The interactions between the antifungal lipopeptide mycosubtilin and lipids are studied. Mycosubtilin increases the ion permeability of planar lipid membranes by forming ion conducting pores. The lifetime of these pores is greatly increased when the membrane contains cholesterol. In mixed monolayers the interaction between mycosubtilin and DMPC leads to the formation of a mycosubtilin/DMPC 1:2 complex non miscible in the excess DMPC monolayer but miscible in the mycosubtilin monolayer. Mycosubtilin and cholesterol interact strongly in monolayers in all proportions and form a mycosubtilin-cholesterol (1:2) complex. These results are analyzed with reference to the overall view of the activity of iturins and the importance of the lipopeptide conformation is outlined.
|
| 2339848 |
Experimental pulmonary eosinophilia in mice by Ascaris suum extract |
10.1164/ajrccm/141.5_Pt_1.1289. |
Am Rev Respir Dis |
Experimental pulmonary eosinophilia in mice by Ascaris suum extract
Abstract
- The transnasal administration of an extract of the parasite Ascaris suum (Asc) to C57BL/6 mice for 3 wk produced marked eosinophilia in the bronchoalveolar lavage (BAL) fluid. This pulmonary eosinophilia was not accompanied by blood eosinophilia. The oral administration of cyclosporin, 50 mg/kg body weight, every other day significantly suppressed the pulmonary eosinophilia. Athymic C57BL/6-nu/nu mice failed to develop pulmonary eosinophilia. These data indicate that pulmonary eosinophilia caused by this parasite extract is T-cell-dependent. Genetically mast-cell-deficient (WB X C57BL/6)F1-W/WV (W/WV) mice developed marked eosinophilia in the BAL, which shows that mast cells are not necessary in the formation of eosinophilia in BAL in this model. C57BL/6, W/WV, and Balb-C mice that developed eosinophilia in the BAL also showed elevated total IgE levels and IgE titers against Asc in the sera. On the other hand, C57BL/6-nu/nu mice and IgE low-responder SJL mice, which developed little eosinophilia, did not show elevated total IgE levels and IgE titers against Asc in the sera. However, oral administration of cyclosporin, 50 mg/kg, which inhibited eosinophilia in the BAL in C57BL/6 mice, did not significantly inhibit the elevation of total IgE or IgE against Asc in the sera. This indicates that serum IgE production is not required for the formation of eosinophilia.
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| 2341395 |
The genomic organization of platelet glycoprotein IIIa. |
10.1016/s0021-9258(19)38928-8 |
J. Biol. Chem. |
The genomic organization of platelet glycoprotein IIIa.
Abstract
- The platelet membrane glycoprotein (GP) IIb/IIIa complex, a member of the integrin family of adhesive receptors involved in cell-cell and cell-matrix interactions, contains binding sites for fibrinogen, von Willebrand factor, fibronectin, and vitronectin. Absence or defects of this receptor result in the platelet bleeding disorder Glanzmann's thrombasthenia. In this report, we describe the isolation of genomic DNA coding for the entire mature GPIIIa protein. Mature GPIIIa is encoded by 14 exons which range in length from 90 to 3618 base pairs, which are contained within an approximately 46-kilobase (kb) stretch of genomic DNA on chromosome 17. All of the exon/intron junctions were found to conform to the consensus splice donor and acceptor sequences. The coding region of the GPIIIa gene is identical with the previously described cDNA sequence except for three silent substitutions. One substitution creates a TaqI site which may be the site of a known GPIIIa polymorphism. A second substitution eliminates a SmaI site. Aside from the start of the first exon described, which begins at the second base of the first codon of the mature protein, there is no correlation between the organization of the exons in this gene and proposed functional domains of the protein based on analysis of the primary amino acid sequence. The less frequently used polyadenylation signal AAATTAAA was present at the 3'-end of the major RNA transcript. Recently, an alternatively processed GPIIIa transcript has been described. We demonstrate that this transcript
Results from nonsplicing of the final intron. The description of the GPIIIa gene organization should be of importance in understanding the evolution of the integrin family of receptors and should be useful in the molecular biology analysis of thrombasthenic patients who have a defect in the GPIIIa gene.
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| 2345548 |
GPIIb and GPIIIa amino acid sequences deduced from human megakaryocyte cDNAs. |
10.1007/bf00422712 |
Mol. Biol. Rep. |
GPIIb and GPIIIa amino acid sequences deduced from human megakaryocyte cDNAs.
Abstract
- Platelet GPIIbIIIa is only synthesized in megakaryocyte or in cell lines with megakaryocytic features. The sequence for GPIIb and GPIIIa have recently been derived from cDNAs obtained from HEL cells. The sequence of these proteins produced by the megakaryocyte, has however, not been determined yet. This study describes full length cDNAs for GPIIb and GPIIIa isolated from megakaryocyte cDNA libraries. The cDNA sequences indicate the presence of nucleotide differences, between the sequence of the GPIIIa cDNAs from HEL cells, endothelial cells and megakaryocytes. One difference was also observed between HEL and megakaryocyte GPIIb at position 633 where a cysteine in the megakaryocyte GPIIb, is replaced by a serine in the HEL sequence. The mRNA species for GPIIb (3.4 kb) and GPIIIa (6.1 kb) were of the same size in HEL cells and human megakaryocytes.
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| 2354877 |
S-deoxo-Abu1,Ile3-amaninamide, an inactive amatoxin analogue |
10.1111/j.1399-3011.1990.tb00947.x. |
Int J Pept Protein Res |
S-deoxo-Abu1,Ile3-amaninamide, an inactive amatoxin analogue
Abstract
- The title compound 3, an amatoxin analogue containing L-alpha-aminobutyric acid instead of L-asparagine in position 1, as in natural toad stool peptides, has been synthesized. It does not inhibit the eukaryotic DNA-dependent RNA polymerase form II (or B) in concentrations up to 10(-4)M, whereas 50% inhibition is exerted in 10(-6)M solution by the corresponding Asn-analogue S-deoxo-Ile3-amaninamide 2. The striking difference seems to be due to a relatively small variation of the conformation recognized by sensitive NMR spectroscopic methods.
|
| 2358424 |
Determination of the disulfide array in sapecin, an antibacterial peptide of Sarcophaga peregrina (flesh fly) |
10.1093/oxfordjournals.jbchem.a123077. |
J Biochem |
Determination of the disulfide array in sapecin, an antibacterial peptide of Sarcophaga peregrina (flesh fly)
Abstract
- Sapecin is a 40-residue peptide containing 6 half-cystine residues. The disulfide structure of sapecin was determined by sequencing cystine-containing peptides obtained by digesting sapecin with thermolysin. Results showed that sapecin has a vortical structure fixed by 3 disulfide bonds between cysteine residues 3 and 30, 16 and 36, and 20 and 38, respectively, and that these disulfide bonds are essential for its antibacterial activity.
|
| 2358465 |
Cyclosporin synthetase. The most complex peptide synthesizing multienzyme polypeptide so far described |
None |
J Biol Chem |
Cyclosporin synthetase. The most complex peptide synthesizing multienzyme polypeptide so far described
Abstract
- Cyclosporin A and its homologues are synthesized by a single multifunctional enzyme from their precursor amino acids. Cyclosporin synthetase is a polypeptide chain with a molecular mass of approximately 800 kDa. In 3% polyacrylamide-sodium dodecyl sulfate gels it shows a single band of approximately 650 kDa, which appears to not be glycosylated. The enzyme could be purified to near-homogeneity in five steps. A 72-fold purification was obtained. All constitutive amino acids of cyclosporins are activated as thioesters via aminoadenylation by the same enzyme. Then N-methylation of the thioester-bound amino acids which are present in methylated form in the cyclosporin molecule takes place, whereby S-adenosyl-L-methionine serves as the methyl group donor. Methyltransferase activity is an integral entity of the enzyme; this could be shown by a photoaffinity labeling method. 4'-Phosphopantetheine is a prosthetic group of cyclosporin synthetase similar to other peptide and depsipeptide synthetases. Cyclosporin synthetase shows cross-reactions with monoclonal antibodies directed against enniatin synthetase."
|
| 2365819 |
Five mutant alleles of the insulin receptor gene in patients with genetic forms of insulin resistance |
10.1172/JCI114693. |
J Clin Invest |
Five mutant alleles of the insulin receptor gene in patients with genetic forms of insulin resistance
Abstract
- The nucleotide sequence was determined for all 22 exons of the insulin receptor gene from three patients with genetic syndromes associated with extreme insulin resistance. In all three patients, insulin resistance was caused by decreased insulin binding to the cell surface. The patient with leprechaunism (leprechaun/Winnipeg) came from a consanguineous pedigree and was homozygous for a missense mutation substituting arginine for His209 in the alpha-subunit of the insulin receptor. The other two patients were both compound heterozygotes with a nonsense mutation in one allele of the insulin receptor gene, and a missense mutation in the other allele. In the patient with the Rabson-Mendenhall syndrome (patient RM-1), the missense mutation substituted lysine for Asn15 in the alpha-subunit. In the patient with type A extreme insulin resistance (patient A-1), the missense mutation substituted serine for Asn462 in the alpha-subunit. Both nonsense mutations markedly reduced the levels of insulin receptor mRNA transcribed from the alleles with the nonsense mutation as compared to the transcripts from the other allele. The reduction in the level of mRNA would be predicted to greatly reduce the rate at which the truncated receptors would be synthesized. Furthermore, the truncated receptors would be severely impaired in their ability to mediate insulin action.
|
| 2369577 |
Hepatocellular uptake of 3H-dihydromicrocystin-LR, a cyclic peptide toxin |
10.1016/0005-2736(90)90190-y. |
Biochim Biophys Acta |
Hepatocellular uptake of 3H-dihydromicrocystin-LR, a cyclic peptide toxin
Abstract
- The cellular uptake of microcystin-LR, a cyclic heptapeptide hepatotoxin from the cyanobacterium Microcystis aeruginosa, was studied by means of a radiolabelled derivative of the toxin. 3H-dihydromicrocystin-LR. The uptake of 3H-dihydromicrocystin-LR was shown to be specific for freshly isolated rat hepatocytes whereas the uptake in the human hepatocarcinoma cell line Hep G2 as well as the mouse fibroblast cell line NIH-3T3, and the human neuroblastoma cell line SH-SY5Y, was negligible. By means of a surface barostat technique it was shown that the membrane penetrating capacity (surface activity) of microcystin-LR was low, indicating that the toxin requires an active uptake mechanism. The hepatocellular uptake of microcystin-LR could be inhibited in the presence of bile acid transport inhibitors such as antamanide (5 microM), sulfobromophthalein (50 microM) and rifampicin (30 microM). The uptake was also reduced in a concentration dependent manner when the hepatocytes were incubated in the presence the bile salts cholate and taurocholate. A complete inhibition of the hepatocellular uptake was achieved by 100 microM of either bile salt. The overall results indicate that the uptake of microcystin-LR is through the multispecific transport system for bile acids. This mechanism of cell entry would explain the previously observed cell specificity and organotropism of microcystin-LR.
|
| 2369893 |
Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition |
10.1002/j.1460-2075.1990.tb07410.x. |
EMBO J |
Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease inhibition
Abstract
- Thrombin is a serine protease that plays a central role in blood coagulation. It is inhibited by hirudin, a polypeptide of 65 amino acids, through the formation of a tight, noncovalent complex. Tetragonal crystals of the complex formed between human alpha-thrombin and recombinant hirudin (variant 1) have been grown and the crystal structure of this complex has been determined to a resolution of 2.95 A. This structure shows that hirudin inhibits thrombin by a previously unobserved mechanism. In contrast to other inhibitors of serine proteases, the specificity of hirudin is not due to interaction with the primary specificity pocket of thrombin, but rather through binding at sites both close to and distant from the active site. The carboxyl tail of hirudin (residues 48-65) wraps around thrombin along the putative fibrinogen secondary binding site. This long groove extends from the active site cleft and is flanked by the thrombin loops 35-39 and 70-80. Hirudin makes a number of ionic and hydrophobic interactions with thrombin in this area. Furthermore hirudin binds with its N-terminal three residues Val, Val, Tyr to the thrombin active site cleft. Val1 occupies the position P2 and Tyr3 approximately the position P3 of the synthetic inhibitor D-Phe-Pro-ArgCH2Cl. Thus the hirudin polypeptide chain runs in a direction opposite to that expected for fibrinogen and that observed for the substrate-like inhibitor D-Phe-Pro-ArgCH2Cl.
|
| 2369896 |
Functionally distinct insulin receptors generated by tissue-specific alternative splicing. |
10.1002/j.1460-2075.1990.tb07416.x |
EMBO J. |
Functionally distinct insulin receptors generated by tissue-specific alternative splicing.
Abstract
- Cloning of the insulin receptor cDNA has earlier revealed the existence of two alternative forms of the receptor differing by the presence or absence of 12 amino acids near the C-terminus of the receptor alpha-subunit. This insert has been shown by others to be encoded by a discrete exon, and alternative splicing of this exon leads to tissue-specific expression of two receptor isoforms. We have studied the functional significance of the receptor isoforms and have confirmed that they are generated by alternative splicing. When cDNAs encoding the two forms of the insulin receptors are expressed in Rat 1 cells, the receptor lacking the insert (HIR-A) has a significantly higher affinity for insulin than the receptor with the insert (HIR-B). This difference in affinity is maintained when insulin binding activity is assayed in solution using detergent solubilized, partially purified receptors. These data, combined with the tissue specificity of HIR-A and HIR-B expression, suggest that alternative splicing may result in the modulation of insulin metabolism or responsiveness by different tissues.
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