| 2823072 |
Nucleotide sequence of the fhuC and fhuD genes involved in iron (III) hydroxamate transport: domains in FhuC homologous to ATP-binding proteins. |
10.1007/bf00329835 |
Mol. Gen. Genet. |
Nucleotide sequence of the fhuC and fhuD genes involved in iron (III) hydroxamate transport: domains in FhuC homologous to ATP-binding proteins.
Abstract
- The transport of Fe3+ into cells of Escherichia coli occurs via siderophores and the uptake through the outer membrane of three Fe3+-siderophore compounds containing hydroxamate residues requires three specific receptor proteins. In contrast, transport through the cytoplasmic membrane is catalysed by three common proteins encoded by the fhuB, fhuC and fhuD genes. The nucleotide sequence of a DNA fragment containing the fhuC and fhuD genes has been determined: the open reading frame of fhuC contains 795 nucleotides which encode a polypeptide with a molecular weight of 29,255 and the largest open reading frame of the fhuD region comprises 888 nucleotides. However, we propose that translation of fhuD initiates at the fourth potential start codon resulting in a polypeptide with a molecular weight of 28,282. Both proteins are moderately nonpolar and membrane-bound. They lack obvious signal sequences. Segments of the FhuC protein display strong homology to ATP-binding proteins, suggesting a function in Fe3+ uptake similar to the ATP-binding proteins of transport systems that depend on periplasmic proteins. This study completes the nucleotide sequence of the fhu operon which consists of the four genes fhuA fhuC fhuD fhuB arranged in this order on the E. coli chromosome and transcribed from fhuA to fhuB.
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| 2825773 |
Nucleotide sequence of the gene for human prothrombin. |
10.1021/bi00393a033 |
Biochemistry |
Nucleotide sequence of the gene for human prothrombin.
Abstract
- A human genomic DNA library was screened for the gene coding for human prothrombin with a cDNA coding for the human protein. Eighty-one positive lambda phage were identified, and three were chosen for further characterization. These three phage hybridized with 5' and/or 3' probes prepared from the prothrombin cDNA. The complete DNA sequence of 21 kilobases of the human prothrombin gene was determined and included a 4.9-kilobase region that was previously sequenced. The gene for human prothrombin contains 14 exons separated by 13 intervening sequences. The exons range in size from 25 to 315 base pairs, while the introns range from 84 to 9447 base pairs. Ninety percent of the gene is composed of intervening sequence. All the intron splice junctions are consistent with sequences found in other eukaryotic genes, except for the presence of GC rather than GT on the 5' end of intervening sequence L. Thirty copies of Alu repetitive DNA and two copies of partial KpnI repeats were identified in clusters within several of the intervening sequences, and these repeats represent 40% of the DNA sequence of the gene. The size, distribution, and sequence homology of the introns within the gene were then compared to those of the genes for the other vitamin K dependent proteins and several other serine proteases.
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| 2827873 |
In vivo apparent peptide-receptor dissociation rate constants for arginine vasopressin analogs estimated from inhibition of rat pressor responses |
10.1139/y87-329. |
Can J Physiol Pharmacol |
In vivo apparent peptide-receptor dissociation rate constants for arginine vasopressin analogs estimated from inhibition of rat pressor responses
Abstract
- Apparent pressor receptor dissociation rate constants for arginine vasopressin, arginine vasotocin, oxytocin, oxypressin, and 1-deamino, 9-D-alanineamidearginine vasopressin were estimated by the following method. Two minutes after injection of a moderate dose of agonist into urethane-anesthetized rats prepared for recording mean blood pressure, a large dose of inhibitor was injected. Under these conditions, in the first few moments after inhibitor injection, there should be no rebinding of the agonist after it dissociates, because vacant receptors should be immediately occupied by inhibitor. The rate of the blood pressure drop at the initiation of inhibition was calculated and used as an estimate of the dissociation rate of the agonist. Apparent dissociation rate constants thus estimated were 1.1, 1.1, 6.9, 5.8, and 13.9 min-1 for arginine vasopressin, arginine vasotocin, oxytocin, oxypressin, and 1-deamino, 9-D-alanineamidearginine vasopressin, respectively. These rate constants were inversely related to the pressor potencies (435, 250, 5, 3, and 0.7 U/mg, respectively) of these five compounds. Such a relationship is to be expected if decreased potency is in part due to a faster "off" rate from pressor receptors.
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| 2833744 |
cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7- kDa carboxyl-terminal fragment. |
10.1073/pnas.85.8.2543 |
Proc. Natl. Acad. Sci. U.S.A. |
cDNA cloning of human DNA topoisomerase I: catalytic activity of a 67.7- kDa carboxyl-terminal fragment.
Abstract
- cDNA clones encoding human topoisomerase I were isolated from an expression vector library (lambda gt11) screened with autoimmune anti-topoisomerase I serum. One of these clones has been expressed as a fusion protein comprised of a 32-kDa fragment of the bacterial TrpE protein linked to 67.7 kDa of protein encoded by the cDNA. Three lines of evidence indicate that the cloned cDNA encodes topoisomerase I. (i) Proteolysis maps of the fusion protein and human nuclear topoisomerase I are essentially identical. (ii) The fusion protein relaxes supercoiled DNA, an activity that can be immunoprecipitated by anti-topoisomerase I serum. (iii) Sequence analysis has revealed that the longest cDNA clone (3645 base pairs) encodes a protein of 765 amino acids that shares 42% identity with Saccharomyces cerevisiae topoisomerase I. The sequence data also show that the catalytically active 67.7-kDa fragment is comprised of the carboxyl terminus.
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| 2834824 |
Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance |
10.1126/science.2834824. |
Science |
Two mutant alleles of the insulin receptor gene in a patient with extreme insulin resistance
Abstract
- Insulin receptor complementary DNA has been cloned from an insulin-resistant patient with leprechaunism whose receptors exhibited multiple abnormalities in insulin binding. The patient is a compound heterozygote, having inherited two different mutant alleles of the insulin receptor gene. One allele contains a missense mutation encoding the substitution of glutamic acid for lysine at position 460 in the alpha subunit of the receptor. The second allele has a nonsense mutation causing premature chain termination after amino acid 671 in the alpha subunit, thereby deleting both the transmembrane and tyrosine kinase domains of the receptor. Interestingly, the father is heterozygous for this nonsense mutation and exhibits a moderate degree of insulin resistance. This raises the possibility that mutations in the insulin receptor gene may account for the insulin resistance in some patients with non-insulin-dependent diabetes mellitus.
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| 2835685 |
Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings |
10.1038/333276a0. |
Nature |
Prepeptide sequence of epidermin, a ribosomally synthesized antibiotic with four sulphide-rings
Abstract
- The genetic basis for the biosynthesis of large polypeptide antibiotics such as nisin has not been explained so far. We show here that the structural gene epiA encoding the antibiotic epidermin from Staphylococcus epidermidis is located on a 54-kilobase plasmid and codes for a 52-amino-acid prepeptide, which is processed to the tetracyclic 21-peptide amide antibiotic. The mature sequence of epidermin corresponds to the C-terminal 22-peptide segment of pre-epidermin and contains the precursor amino acids Ser, Thr and Cys, from which the unusual amino-acid constituents are derived. The more lipophilic epidermin is cleaved at a hydrophilic turn between Arg-1 and Ile+1 from the N-terminal segment-30 to -1, which probably assumes a partially amphiphilic alpha-helix conformation. We propose that the N-terminus (-30 to -1) plays a cooperative role during modification reactions and prevents toxicity of the mature epidermin to the producing strain before the antibiotic is cleaved off and secreted.
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| 2842060 |
Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity. |
10.1016/s0092-8674(88)80008-4 |
Cell |
Mutation of the insulin receptor at tyrosine 960 inhibits signal transmission but does not affect its tyrosine kinase activity.
Abstract
- Tyrosyl phosphorylation is implicated in the mechanism of insulin action. Mutation of the beta-subunit of the insulin receptor by substitution of tyrosyl residue 960 with phenylalanine had no effect on insulin-stimulated autophosphorylation or phosphotransferase activity of the purified receptor. However, unlike the normal receptor, this mutant was not biologically active in Chinese hamster ovary cells. Furthermore, insulin-stimulated tyrosyl phosphorylation of at least one endogenous substrate (pp185) was increased significantly in cells expressing the normal receptor but was barely detected in cells expressing the mutant. Therefore, beta-subunit autophosphorylation was not sufficient for the insulin response, and a region of the insulin receptor around Tyr-960 may facilitate phosphorylation of cellular substrates required for transmission of the insulin signal.
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| 2845399 |
Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22. |
10.1073/pnas.85.19.7177 |
Proc. Natl. Acad. Sci. U.S.A. |
Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22.
Abstract
- Two overlapping cDNA clones encoding human DNA topoisomerase II were identified by two independent
Methods. In one, a human cDNA library in phage lambda was screened by hybridization with a mixed oligonucleotide probe encoding a stretch of seven amino acids found in yeast and Drosophila DNA topoisomerase II; in the other, a different human cDNA library in a lambda gt11 expression vector was screened for the expression of antigenic determinants that are recognized by rabbit antibodies specific to human DNA topoisomerase II. The entire coding sequences of the human DNA topoisomerase II gene were determined from these and several additional clones, identified through the use of the cloned human TOP2 gene sequences as probes. Hybridization between the cloned sequences and mRNA and genomic DNA indicates that the human enzyme is encoded by a single-copy gene. The location of the gene was mapped to chromosome 17q21-22 by in situ hybridization of a cloned fragment to metaphase chromosomes and by hybridization analysis with a panel of mouse-human hybrid cell lines, each retaining a subset of human chromosomes.
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| 2852955 |
Cyclic lactam analogues of Ac-Nle4alpha-MSH4-11-NH2 |
10.1021/bi00421a029. |
Biochemistry |
Cyclic lactam analogues of Ac-Nle4alpha-MSH4-11-NH2
Abstract
- Two side-chain cyclic lactam analogues of the 4-11 fragment of alpha-melanocyte-stimulating hormone (alpha-MSH), Ac-Nle4,D-Orn5,Glu8alpha-MSH4-11-NH2 and Ac-Nle4,D-Orn5,D-Phe7,Glu8alpha-MSH4-11-NH2, were prepared on p-methylbenzhydrylamine resin by using a combination of N alpha-Boc and N alpha-Fmoc synthetic strategies with diphenyl phosphorazidate mediated cyclization. The melanotropin activities of these two analogues were examined and compared relative to those of alpha-MSH, Ac-Nle4alpha-MSH4-11-NH2, and Ac-Nle4,D-Phe7alpha-MSH4-11-NH2. In the frog (Rana pipiens) skin bioassay, the L-Phe7 17-membered ring cyclic analogue was slightly more potent than the linear Ac-Nle4alpha-MSH4-11-NH2 and exhibited prolonged melanotropic bioactivity (greater than or equal to 4 h). In this same assay, the D-Phe7 cyclic analogue was more than 100-fold less potent than the L-Phe cyclic analogue and was 10,000 times less potent than linear Ac-Nle4,D-Phe7alpha-MSH4-11-NH2. In the lizard skin (Anolis carolinensis) bioassay, the L-Phe7 cyclic analogue was 100-fold less potent than Ac-Nle4alpha-MSH4-11-NH2, while the D-Phe7 cyclic analogue was 10,000-fold less potent than both Ac-Nle4alpha-MSH4-11-NH2 and the D-Phe7 linear derivative Ac-Nle4,D-Phe7alpha-MSH4-11-NH2. The solution conformation of these two cyclic analogues in dimethyl sulfoxide-d6 was examined by 1D and 2D 500-MHz 1H NMR spectroscopy. Our analysis suggests an H bond stabilized C10 (or C13) turn for the D-Phe7 cyclic structure while the L-Phe7 analogue is more conformationally flexible.(ABSTRACT TRUNCATED AT 250 WORDS)
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| 2856554 |
Synthetic peptides bind to high-affinity thrombin receptors and modulate thrombin mitogenesis. |
10.1016/s0021-9258(18)69292-0 |
Pept. Res. |
Synthetic peptides bind to high-affinity thrombin receptors and modulate thrombin mitogenesis.
Abstract
- Initiation of cell proliferation by thrombin requires signals generated by thrombin interaction with specific high-affinity receptors and thrombin enzymic activity. Using synthetic peptides representing various domains of thrombin, we have identified a region adjacent to the proteolytic pocket of thrombin which confers high-affinity binding and generation of mitogenic signals. One peptide, representing residues 508 to 530 of human prothrombin (p508-530), inhibits up to 70% of the specific binding of 125I-alpha-thrombin at concentrations of less than 100 nM, enhances the ability of thrombin to stimulate DNA synthesis and stimulates DNA synthesis in cells treated with 25 ng/ml phorbol myristate acetate (PMA). Thus, this peptide or a portion of this peptide appears to represent the high-affinity receptor binding domain of thrombin. In contrast to the 23 amino acid peptide (p508-530), the tetrapeptide RGDA (p517-520) contained in this region competes for 125I-thrombin binding at concentrations from 100 to 2000 nM, but inhibits rather than stimulates the mitogenic effects of alpha-thrombin. Non-homologous peptides, or fibronectin-specific peptides (such as RGDS or GRGDSP) do not compete for 125I-alpha-thrombin binding and have no effect on thrombin mitogenesis. These studies demonstrate that peptides representing portions of the binding domain of thrombin: i) can generate receptor-occupancy related signals that enhance thrombin mitogenesis and are themselves mitogenic in cells treated with PMA; or ii) in the case of RGDA (which may be too small to generate signals), can act as antagonists, inhibiting the mitogenic effects of thrombin by preventing thrombin-receptor interaction.
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| 2857053 |
Effects of cyclic hexapeptide analog of somatostatin on pancreatic secretion in dogs |
10.3181/00379727-178-41985. |
Proc Soc Exp Biol Med |
Effects of cyclic hexapeptide analog of somatostatin on pancreatic secretion in dogs
Abstract
- The effects of a cyclic hexapeptide analog of somatostatin, cyclo(Pro-Phe-D-Trp-Lys-Thr-Phe) (cyclo-SS), administered intravenously (iv) or instilled into the duodenum (id) on the pancreatic response to endogenous (meal and duodenal acidification) and exogenous (secretin, CCK) stimulants were compared in five dogs with esophageal, gastric, and pancreatic fistulae. Cyclo-SS given iv in graded doses against a constant background stimulation with secretin caused a similar and dose-dependent inhibition of pancreatic HCO3 and protein secretion being about twice as potent as somatostatin-14 (SS-14). Cyclo-SS, whether applied topically to the duodenal mucosa in a dose of 1 microgram/kg or given iv at a dose of 0.5 microgram/kg-hr, resulted in a similar inhibition of pancreatic secretion induced by feeding a meat meal, sham-feeding, duodenal acidification, or infusion of secretin or CCK. The inhibition of pancreatic secretion by cyclo-SS was due in part to direct inhibitory action on the exocrine pancreas as well as to the suppression of the release of secretin, insulin, and pancreatic polypeptide. It is concluded that cyclo-SS is a more potent inhibitor of pancreatic secretion than SS-14 and that it is active when administered both parenterally and intraduodenally.
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| 2859121 |
The human insulin receptor cDNA: the structural basis for hormone- activated transmembrane signalling. |
10.1016/0092-8674(85)90334-4 |
Cell |
The human insulin receptor cDNA: the structural basis for hormone- activated transmembrane signalling.
Abstract
- A cloned approximately 5 kb cDNA (human placenta) contains the coding sequences for the insulin receptor. The nucleotide sequence predicts a 1382 amino acid precursor. The alpha subunit comprises the N-terminal portion of the precursor and contains a striking cysteine-rich "cross-linking" domain. The beta-subunit (the C-terminal portion of the precursor) contains a transmembrane domain and, in the intracellular region, the elements of a tyrosine phosphokinase: an ATP-binding site and a possible tyrosine autophosphorylation site or sites. The overall structure is reminiscent of the EGF receptor; the cross-linking domain of the alpha subunit and several regions of the beta subunit exhibit sequence homology with the EGF receptor. The phosphokinase domain also exhibits homology with some oncogenic proteins that have tyrosine phosphokinase activity, in particular, a striking homology with v-ros. Southern blotting experiments suggest that the coding region spans more than 45 kb. The insulin receptor gene is located on chromosome 19.
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| 2859510 |
New specific radioligand for one subpopulation of brain somatostatin receptors |
10.1016/0024-3205(85)90155-9. |
Life Sci |
New specific radioligand for one subpopulation of brain somatostatin receptors
Abstract
- Cyclic octapeptide analogues of somatostatin (SS) like SMS 201-995 H-(D) Phe-Cys-Phe-(D) Trp-Lys-Thr-Cys-Thr(ol) or its Tyr3-derivative 204-090, displaced 125I-Tyr11-SS 100% from pancreatic membranes but only 62-75% from brain membranes; the remaining sites were displaced by SS. These data indicate that some mini-somatostatins bind to a subpopulation of SS receptors in rat brain. The iodinated Tyr3-derivative (125I-204-090) can be considered a selective radioligand for one rat brain SS receptor subpopulation: It shows saturable and high affinity binding (KD = 0.29 nM; Bmax = 350 fmoles/mg protein) to rat cortex. The pharmacological properties of 125I-204-090 binding sites are similar to those of 125I-Tyr11-SS sites. Distribution of these sites correspond to SS receptor-rich areas such as cortex, hippocampus, striatum, pituitary, pancreatic beta-cell. SS as well as SMS 201-995 bind to these sites with high affinity. The stability and high specific vs non-specific binding ratio makes 204-090 a radioligand of choice to measure this SS receptor subpopulation in CNS but also the SS receptors in pituitary and pancreas.
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| 2859546 |
Localization and characterization of somatostatin-14 and somatostatin-28 receptors in the rat pituitary as studied by slide-mounted frozen sections |
10.1016/0143-4179(85)90129-5. |
Neuropeptides |
Localization and characterization of somatostatin-14 and somatostatin-28 receptors in the rat pituitary as studied by slide-mounted frozen sections
Abstract
- The localization and characterization of somatostatin-14 (SS-14) and somatostatin-28 (SS-28) receptors were studied in the rat pituitary using the iodinated agonists Tyr0, D-Trp8-SS-14 and Leu8, D-Trp22, Tyr25-SS-28 as tracers. Slide-mounted frozen sections were used for the autoradiographic localization and biochemical characterization of somatostatin receptors. In the latter case, counting was performed on scraped off serial sections which contained both anterior and posterior pituitary. Specificity studies demonstrated that either tracer could be displaced with SS-28 and SS-14. A number of unrelated peptides were unable to compete with either tracer. Scatchard analysis of saturation curves gave one-site interactions with KD values of 0.53 +/- 0.08 nM and 0.22 +/- 0.03 nM for the SS-14 and SS-28 iodinated agonists, respectively. Using autoradiography, SS-14 and SS-28 receptors were shown to be distributed in the anterior pituitary only. Using densitometry, displacement and saturation curves could be also obtained.
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| 2861078 |
Relationship between receptor binding and biopotency of somatostatin-14 and somatostatin-28 in mouse pituitary tumor cells |
10.1210/endo-117-1-271. |
Endocrinology |
Relationship between receptor binding and biopotency of somatostatin-14 and somatostatin-28 in mouse pituitary tumor cells
Abstract
- Somatostatin-14 (S-14) acts via specific receptors to inhibit basal as well as hormone- and forskolin-stimulated ACTH secretion in tumor cells (AtT-20/D16-16) of mouse anterior pituitary. In addition S-14 inhibits the stimulated but not basal cAMP accumulation. The potency of somatostatin-28 (S-28) for regulating these processes in these tumor cells has not been reported. In this study we have investigated the relationship between receptor-binding affinities of S-14 and S-28 and their biopotency in these cells. Membrane receptors for S-14 characterized using 125I-Tyr11S-14 as the radioligand maximum binding capacity (Bmax) = 1.28 +/- 0.1 pmol/mg; dissociation constant (Kd) = 1.1 +/- 0.04 nM bound S-28 with 3-fold greater affinity than S-14. Binding sites quantitated using an S-28 analog Leu8, D-Trp22, 125I-Tyr25S-28 as radioligand (Bmax = 1.18 +/- 0.15 pmol/mg; Kd = 0.08 +/- 0.06 nM) also exhibited greater affinity for S-28 than S-14. Forskolin-stimulated cAMP accumulation and ACTH secretion in these cells were inhibited to a greater extent (4- and 9-fold, respectively) by S-28 than S-14. Preincubation of the cells with S-14 and S-28 (10(-7) M) resulted in a marked decrease (36% and 71%, respectively) of S-14 receptor concentration. Coincubation of the cells with both S-14 and S-28 led to 56% decrease in S-14 receptor binding. The responsiveness of the cells to forskolin stimulation of ACTH secretion and cAMP accumulation was significantly enhanced by preincubation with S-14 (10(-7) M) whereas the responsiveness to forskolin was completely abolished by preincubation with S-28. Simultaneous exposure of the cells to both S-14 and S-28 resulted in a partial reversal of the inhibiting effect of S-28 on forskolin-stimulated cAMP accumulation in these cells but did not result in a partial reversal of the inhibitory effect of S-28 on forskolin-stimulated ACTH secretion in these cells. These results demonstrate that S-28 is more potent than S-14 in AtT-20/D16-16 cells, its greater potency arising from its greater affinity for binding to S-14 receptors. The differential effects of these peptides after preincubation on the responsiveness of ACTH secretion and cAMP accumulation in these cells to forskolin stimulation suggests the possibility of existence of distinct S-14 and S-28 receptors, but these could not be identified by direct binding experiments using the S-14 and S-28 analogs employed in the study as radioligands.(ABSTRACT TRUNCATED AT 400 WORDS)
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