Search

The three human α adrenergic receptor subtypes, α_1a, α_1b, and α_1c, have been cloned, expressed in COS-7 cells, and characterized pharmacologically. Competition binding studies of several adrenergic ligands with the three human receptor subtypes reveals a distinct pharmacological profile for each receptor subtype. RNase protection analysis demonsrates that the three receptor subtypes have different patterns of distribution in human tissue. The availability of the three human α₁ receptor subtypes will facilitate the development of subtype-selective α₁ antagonists for a variety of therapeutic indications.Copyright 1994, 1999 Academic Press, Inc.

Four structural analogs of human insulin-like growth factor I (hIGF-I) have been prepared by site-directed mutagenesis of a synthetic IGF-I gene and subsequent expression and purification of the mutant protein from the conditioned media of transformed yeast. [Phe-1,Val1,Asn2, Gln3,His4,Ser8, His9,Glu12,Tyr15,Leu16]IGF-I (B-chain mutant), in which the first 16 amino acids of hIGF-I were replaced with the first 17 amino acids of the B-chain of insulin, has greater than 1,000-, 100-, and 2-fold reduced potency for human serum binding proteins, the rat liver type 2 IGF receptor, and the human placental type 1 IGF receptor, respectively. The B-chain mutant also has 4-fold increased affinity for the human placental insulin receptor. [Gln3,Ala4]IGF-I has 4-fold reduced affinity for human serum binding proteins, but is equipotent to hIGF-I at the types 1 and 2 IGF and insulin receptors. [Tyr15,Leu16]IGF-I has 4-fold reduced affinity for human serum binding proteins and 10-fold increased affinity for the insulin receptor. This peptide is also equipotent to hIGF-I at the types 1 and 2 IGF receptors. The peptide in which these four-point mutations are combined, [Gln3,Ala4,Tyr15,Leu16]IGF-I, has 600-fold reduced affinity for the serum binding proteins. This peptide has 10-fold increased potency for the insulin receptor, but is equipotent to hIGF-I at the types 1 and 2 IGF receptors. All four of these mutants stimulate DNA synthesis in the rat vascular smooth muscle cell line A10 with potencies reflecting their potency at the type 1 IGF receptor. These studies identify some of the domains of hIGF-I which are responsible for maintaining high affinity binding with the serum binding protein and the type 2 IGF receptor. In addition, these peptides will be useful in defining the role of the type 2 IGF receptor and serum binding proteins in the physiological actions of hIGF-I.

The Escherichia coli dnaC protein is not absolutely required in vivo for bacteriophage phiX174 parental replicative-form synthesis (Kranias and Dumas, 1974). However, when rifampin is present at a concentration that inhibits DNA-dependent RNA polymerase, phiX174 parental replicative-form synthesis is dependent on the dnaC protein activity. We conclude that E. coli DNA-dependent RNA polymerase can substitute for the dnaC protein in phiX174 parental replicative-form DNA synthesis, presumably in its initiation. The implications of this result with respect to the in vitro synthesis of the complementary strand of phiX174 DNA are discussed.

A synthetic gene encoding human insulin-like growth factor I (hIGF-I) was assembled and inserted into an expression vector containing the cytomegalovirus immediate early (CMV-IE) transcriptional regulatory region and portions of the bovine growth hormone gene. The recombinant plasmid encodes a 97 amino acid fusion protein containing the first 27 amino acids of the bovine growth hormone precursor and the 70 amino acids of hIGF-I. This plasmid, when transiently introduced into cultured mouse fibroblasts, directs synthesis of the fusion protein, subsequent proteolytic removal of the bovine growth hormone signal peptide, and secretion of hIGF-I into the culture medium. Conditioned medium from transfected cells inhibits binding of 125I-labeled IGF-I to type I IGF receptors on human placental membranes and to acid-stable human serum carrier proteins. The recombinant hIGF-I produced is biologically active, as monitored by the stimulation of DNA synthesis in vascular smooth muscle cells.

One of the two forms of DNA polymerase alpha from ovaries of the frog Xenopus laevis catalyzed ribonucleoside triphosphate-dependent DNA synthesis on single-stranded circular fd phage DNA templates. DNA synthesis was dependent on ATP and added template. CTP, GTP, and UTP stimulated DNA synthesis but were not required and could not substitute for ATP. DNA synthesis was not inhibited by alpha-amanitin. Neither poly(dT) nor double-stranded DNA served as template. Analysis of [32P]-dTMP-labeled product by neutral and alkaline agarose gel electrophoresis showed that 0.1- to 1-kilobase DNA fragments (average size of approximately equal to 0.25 kilobase) were synthesized. The fragments were not covalently linked to the template. Either [alpha-32P]NMP, [gamma-32P]ATP, or [gamma-32P]GTP were incorporated also into the product. Analysis of the product after hydrolysis by KOH, alkaline phosphatase, or bacteriophage T4 3' leads to 5' exonuclease showed the presence of a small oligoribonucleotide primer at the 5' end of the newly synthesized DNA. NTP-dependent DNA-synthesizing activity copurified on six columns and cosedimented during glycerol gradient centrifugation with one form of DNA polymerase alpha activity but not with the other form. These results suggest that DNA primase activity is associated with one of the two forms of X. laevis DNA polymerase alpha.

The neuropeptide Y family of peptides, which includes neuropeptide Y (NPY), peptide YY (PYY), and pancreatic polypeptide (PP), are found in the central and peripheral nervous system and display a wide array of biological activities. These actions are believed to be mediated through pharmacologically distinct G protein-coupled receptors, and, to date, three members of the NPY receptor family have been cloned. In this study we describe the cloning and expression of a novel NPY receptor from mouse genomic DNA. This receptor, designated NPY Y5, shares 60% amino acid identity to the murine NPY Y1 receptor. The pharmacology of this novel receptor resembles that of the NPY Y1 receptor and is distinct from that described for the NPY Y2, Y3, and Y4 receptors. In situ hybridization of mouse brain sections reveals expression of this receptor within discrete regions of the hypothalamus including the suprachiasmatic nucleus, anterior hypothalamus, bed nucleus stria terminalis, and the ventromedial nucleus with no localization apparent elsewhere in the brain.

Glioma-derived vascular endothelial cell growth factor (GD-VEGF) is a 46-kDa dimeric glycoprotein mitogen with apparently greater specificity for vascular endothelial cells than the well-characterized fibroblast growth factors. The GD-VEGF cDNA sequence encodes a 190-amino acid residue subunit that is converted, by removal of an amino-terminal hydrophobic secretory leader sequence, to the mature 164-residue subunit characterized by direct amino acid sequencing. The GD-VEGF homodimeric subunit is homologous to the platelet-derived growth factor A and B chains and its oncogene homologue v-sis.

Galanin, a 29-30 amino acid neuropeptide, is found in the central and peripheral nervous systems and displays several important physiological activities. The actions are believed to be mediated through distinct G protein-coupled receptors. To date, two galanin receptor subtypes have been cloned. In this report, we describe the cloning and expression of a cDNA encoding a novel galanin receptor (GalR3). The receptor has 370 amino acids and shares 36 and 54% homology with the rat GalR1 and GalR2 receptors. 125I-Porcine galanin binds the rat GalR3 receptor expressed in COS-7 cells with high affinity (Kd = 0.6 nM) and could be displaced by galanin and galanin fragments and galanin-chimeric peptides. The pharmacological profile of this novel receptor is distinct from those of GalR1 and GalR2, revealing different pharmacophores within galanin for the three galanin receptor subtypes. Northern blot analysis showed expression in heart, spleen, and testis. Unlike GalR1 and GalR2, no expression of GalR3 was detectable in the brain, suggesting that GalR3 may mediate some of the peripheral functions of galanin.

Viral and complementary strand circular DNA molecules were isolated from intracellular bacteriophage f1 replicative-form DNA. Soluble protein extracts of Escherichia coli were used to examine the initiation of DNA synthesis on these DNA templates. The initiation of DNA synthesis on f1 viral strand DNA was catalyzed by E. coli DNA-dependent RNA polymerase, as was initiation of f1 viral strand DNA isolated from mature phage particles. The site of initiation was the same as that used in vivo. In contrast, no de novo initiation of DNA synthesis was detected on f1 complementary strand DNA. Control experiments demonstrated that the E. coli dnaB, dnaC, and dnaG initiation proteins were active under the conditions employed. The results suggest that the viral strand of the f1 replicative-form DNA molecule carries the same DNA synthesis initiation site as the viral strand packaged in mature phage, whereas the complementary strand of the replicative-form DNA molecule carries no site for de novo primer synthesis. These in vitro observations are consistent with the simple rolling circle model for f1 DNA replication in vivo proposed by Horiuchi and Zinder.

Polyomavirus minichromosomes were isolated and fractionated as described previously (B. B. Gourlie, M. R. Krauss, A. J. Buckler-White, R. M. Benbow, and V. Pigiet, J. Virol. 38:805-814, 1981). Specific assays for DNA topoisomerase II and DNA ligase activity were carried out on each fraction. The enzymatic activity in each fraction was determined by quantitative electron microscopy and compared with the number of replicative intermediate and total polyomavirus DNA molecules in each fraction. DNA topoisomerase II activity cosedimented with polyomavirus replicative intermediate minichromosomes. DNA ligase activity cosedimented with mature polyomavirus minichromosomes.

We have used site-directed mutagenesis of a synthetic gene for insulin-like growth factor (IGF) I to prepare three analogs in which specific residues in the A region are replaced with the corresponding residues in the A chain of insulin. The analogs are [Ile41, Glu45, Gln46, Thr49, Ser50, Ile51, Ser53, Tyr55, Gln56]IGF I (A chain mutant), in which residue 41 is changed from threonine to isoleucine and residues 42 to 56 of the A region are replaced, [Thr49, Ser50, Ile51]IGF I, and [Tyr55, Gln56]IGF I. These analogs are all equipotent to IGF I at the type 1 IGF receptor in human placental membranes, and in stimulating the incorporation of [3H]thymidine into DNA in the rat vascular smooth muscle cell line A10. However, the A chain mutant and [Thr49, Ser50, Ile51]IGF I have greater than 20-fold lower relative affinity for the type 2 IGF receptor of rat liver membranes, respectively. In contrast, [Tyr55, Gln56]IGF I has 7-fold higher affinity than IGF I for the type 2 IGF receptor. Residues 49, 50, and 51 in IGF I are Phe-Arg-Ser and are strictly conserved in IGF II. Residues 55 and 56 of IGF I and the corresponding residues in IGF II are Arg-Arg and Ala-Leu, respectively. Thus, the presence of the charged residues at these positions in IGF I appears to be responsible, in part, for the lower affinity of IGF I for the type 2 IGF receptor. In addition to the alterations in affinity for the type 2 IGF receptor, the A chain mutant has a 7-fold increase in affinity for insulin receptors, and [Thr49, Ser50, Ile51]IGF I has a 4-fold lower affinity for acid-stable human serum binding protein. These data strongly suggest that specific determinants in the A region of IGF I are important for maintaining binding to the type 2 IGF receptor, and that these determinants are different from those required for maintaining high affinity for the type 1 IGF receptor.

We have produced and characterized the binding properties of three structural analogs of human insulin-like growth factor I (hIGF-I). These analogs are [1–62]hIGF-I, an analog lacking the carboxyl-terminal 8-amino acid D region of hIGF-I; [1–27,Gly4,38–70]hIGF-I, an analog in which residues 28–37 of the C region of hIGF-I are replaced by a 4-reside glycine bridge; and [1–27,Gly4,38–62]hIGF-I, an analog with the C region glycine replacement and a D region deletion. The removal of the D region of hIGF-I has little effect on binding to the type 1 and type 2 insulin-like growth factor (IGF) receptors. [1–62]hIGF-I has 2-fold higher affinity for the insulin receptor and 4-fold higher affinity for IGF serum-binding proteins. The replacement of the C region of hIGF-I with a four-glycine span results in a 30-fold loss of affinity for the type 1 IGF receptor. However this analog has near normal affinity for the type 2 IGF receptor, the insulin receptor, and IGF serum-binding proteins. Incorporating the C region glycine replacement and the D region deletion into one analog does not affect binding to either the type 2 receptor or to IGF serum-binding proteins. As predicted from the single deletion analogs [1–27,Gly4,38–-62]hIGF-I has reduced affinity for the type 1 IGF receptor (∼40-fold) and increased affinity for the insulin receptor (5-fold). These data indicate that determinants in the C region of hIGF-I are involved in maintaining high affinity binding to the type 1 IGF receptor and that neither the C region nor the D region are required for high affinity binding to the type 2 IGF receptor or to IGF serum-binding proteins.

A functional cDNA clone coding for the human prostaglandin E receptor EP1 subtype has been isolated from a human erythroleukemia cell cDNA library probed by low-stringency hybridization using a polymerase chain reaction fragment of the human thromboxane receptor. The human EP1 receptor is comprised of 402 amino acids with a predicted molecular mass of 41,858 and has the topography common to all G-protein-coupled receptors with seven predicted transmembrane spanning domains. Prostaglandin (PG) E2 challenge of Xenopus oocytes injected with EP1 cDNA resulted in an increase in intracellular Ca2+. In addition, the rank order of potency for prostaglandins in competition for [3H]PGE2 specific binding to membranes prepared from EP1 cDNA transfected COS cells was PGE2 > PGE1 > PGF2 alpha > PGD2. Furthermore, the EP1 receptor-selective antagonists AH 6809 and SC19220 were more potent than the EP2 receptor-selective agonist butaprost in these competition binding assays. In summary, therefore, we have cloned the human EP1 receptor subtype which is functionally coupled to an increase in intracellular Ca2+.

Vascular endothelial growth factor (VEGF) is a potent and selective mitogen for endothelial cells that is angiogenic in vivo and induced by hypoxia. A homologous protein, placenta growth factor (PlGF), is also reported to be mitogenic for endothelial cells in culture. The rat GS-9L glioma cell line produces not only VEGF homodimers but also PlGF homodimers and a novel heterodimer composed of VEGF and PlGF subunits. All three dimeric forms were purified to apparent homogeneity, and their structures and mitogenic activities were compared. VEGF.PlGF heterodimers are vascular endothelial cell mitogens nearly as potent as VEGF homodimers. Therefore, some of the biological activities attributed to VEGF homodimers might be mediated by VEGF.PlGF heterodimers. In contrast, pure PlGF homodimers are mitogenic for endothelial cells only at high, possibly non-physiologic concentrations; thus the biological relevance of their mitogenic activity for these cells is not obvious. However, the existence of not only homodimers but also heterodimers clearly extends the similarity between the VEGF/PlGF and the homologous platelet-derived growth factor systems.

Structural analogs of recombinant human insulin-like growth factor-I (IGF-I), with alterations to each of the B, C, A, and D domains, have been tested for their ability to form binary complexes with IGF-binding protein-3 (IGFBP-3) and ternary complexes with IGFBP-3 and the acid-labile subunit (alpha-subunit). Two functionally distinct regions of IGF-I have been identified. The first, involving residues 3 and 4 and the alpha-helix between residues 8 and 18 of the B-domain, as well as residues 49-51 in the A-domain, appears important for IGFBP-3 binding, such that substitution of these residues results in decreased binary complex available for alpha-subunit binding. The second region, distal to the IGFBP-3-binding epitope and primarily involving the D-domain and B-domain near residue 24, with some involvement of the C-domain, appears slightly inhibitory to binary complex formation, such that analogs with a truncated D-domain or with a Gly4 bridge substituted for the C-domain show enhanced binding to IGFBP-3. However, binary complexes formed from these analogs bind the alpha-subunit with reduced affinity, the effect being most marked when substitution of the C-domain, or replacement of Tyr24, is superimposed on D-domain truncation. It is concluded that although the alpha-subunit does not itself bind IGF-I, its interaction with IGFBP-3 in the ternary complex is dependent on structural determinants on IGF-I distal to the IGFBP-3 binding domain.

The ability of insulin to modulate glucose metabolism is impaired in insulin resistant ob/ob mice. It has been shown that insulin-like growth factor I stimulates the uptake and metabolism of glucose in muscle through the insulin-like growth factor receptor not the insulin receptor. Thus, we have compared the abilities of insulin-like growth factor I and insulin to stimulate the in vivo incorporation of [14C]-glucose into glycogen in the diaphragm of ob/ob mice and their lean littermates. The animals used in these studies were 12–14 weeks old and the serum insulin levels of the ob/ob mice were 16-fold higher than in their lean littermates. There were no differences in the serum levels of glucose or insulin-like growth factor I. Both insulin and insulin-like growth factor I stimulate the incorporation of [14C]-glucose into glycogen in lean mice. Significant stimulation occurs at doses as low as 1 μg/kg of either peptide. The effective doses of insulin and insulin-like growth factor I are quite similar, which indicates that the effect of insulin-like growth factor I is mediated by the insulin-like growth factor receptor and not the insulin receptor. In contrast, greater than 100 μg/kg of insulin-like growth factor I is required to stimulate [14C]-glucose incorporation into glycogen in the diaphragm of ob/ob mice. Thus, ob/ob mice are resistant to the action of both insulin and insulin-like growth factor I. In contrast to the decrease in the number of insulin receptors which occurs in ob/ob mice, there is no significant difference in the number of type 1 insulin-like growth factor receptors or in their affinity for insulin-like growth factor I in muscle membranes prepared from lean and ob/ob mice. In addition, the ability of insulin-like growth factor I to stimulate the catalysis of Val5-angiotensin II phosphorylation by the partially purified muscle type 1 insulin-like growth factor receptor is not decreased in ob/ob mice as compared with their lean littermates. These data indicate that the loss in sensitivity of the ob/ob mouse of both insulin and insulin-like growth factor I is most likely mediated by a post-receptor defect in metabolism and not by receptor down-regulation or desensitisation.

This study concerns the deleterious effects of secondary bile salts, tauro-deoxycholate (TDCh)and deoxycholate (DCh), particularly on small intestinal macromolecular absorption. Rat jejunal segments (30-40 cm)were perfused in vivo with an isotonic NaCl-glucose solution (pH6.9)containing 0.5 g% of horseradish peroxidase (HRP)a macromolecular tracer,with 5mM TDCh or DCh or without bile salts (C). After 30 and 60 min serum HRP levels and cytochemical HRP localization were determined. Both TDCh and DCh induced a greater absorption of HRP than C: at 30 min TDCh .157± .005, DCh .439 ±.080 vs C .082 ± .010, and at 60 min TDCh .381 ± .061, DCh 1.001 ± .138 vs C .114 ± .011 (data=u/ml means ± SEM, p<.001). The most striking absorption of HRP was with DCh. Under light and electron microscopy HRP was demonstrable in the intercellular spaces between enterocytes, in the basement membranes, and capillaries of the lamina propria of the jejunum. In contrast, in C, HRP was confined to the microvillar brush border and endocytotic structures of enterocytes. DCh also produced ultrastructural damage including swollen mitochondria and vacuolization of membrane systems whereas TDCh did not alter the intestinal epithelium. These results indicate that high concentrations of secondary bile salts enhance macromolecular absorption and induce functional damage to the jejunal epithelium. Deconjugated bile salts are the most injurious.

The effect of cholestyramine(Ch)on cAMP-mediated jejunal Na+secretion associated with increased luminal free fatty acids(FFA) and deconjugated bile salts(DBS)during bacterial colonization of the small bowel was studied.Male Wistar rats(70-90 gm) were injected with mecamylamine HCl for 3 days, to induce bacterial proliferation in the upper small bowel(MC);controls received NaCl(C). Half of each group were fed Ch 4% in the diet.Supernatants of small intestinal contents were assayed for FFA and DBS;jejunal mucosa was examined for cAMP and ATPases;jejunal Na+transport was measured by in vivo perfusion with Krebs-Henseleit buffer.MC rats not fed Ch had higher luminal concentrations of FFA and DBS as compared to C(FFA:.21±.03 vs .13±.003 mEq/l;p<.02. DBS: 1.11 vs .23 mM).They also had elevated cAMP(34.14±6.17 vs 10.7±2.73 pmol/mg protein;p<.01)and a marked Na+efflux into the jejunum(secretion of 101.07±33.5 vs absorption of 90.15±14.9 µEq/min/cm;p<.001). Feeding Ch to MC rats inhibited these effects:FFA and DBS were lower than in C and CAMP did not differ from C.While Na+efflux was noted in MC rats,Na+absorption did occur in MC rats fed Ch (38.14±21.6 µEq/min/cm;p<.001).The ATPases were similar in all groups. The data indicate that the cAMP-mediated jejunal Na secretion and the increased luminal FFA and DBS during small intestinal bacterial colonization is inhibited by oral Ch,which binds these products and renders them innocuous.

The complexes (I) and (II) are obtained by refluxing 2:1 molar mixtures of the ligands and Co halides in triethyl orthoformate‐ethylacetate for one week.

Bile salt deconjugation may occur in the jejunun under conditions of fecal colonic overgrowth of the small intestine.We studied the effects of conjugated and deconjugated bile salts on rat jejunal absorption of a macromolecular tracer,horseradish peroxidase(HRP), in an in vivo perfusion system.At a 0.5mM perfusion concentration, only the deconjugated bile salts,cholate and deoxycholate,produced a significant increase in HRP absorption into serum.At this level the bile salts did not produce evidence of morphological damage to villi or absorptive epithelial cell organelles.At a 0.5mm concentration of the conjugated salt,taurocholate,HRP absorption was indistinguishable from that seen in bile salt free preparations.At a higher 5mM concentration,both the conjugated and deconjugated salts increased jejunal HRP absorption into serum over that seen in bile salt free preparations;this absorption was most marked with the deconjugated salts.At a 5mM level,the deconjugated salts also damage cellular organelles,including mitochondria and Golgi apparatus.Our observations suggest that an alteration in the tigh junctional barrier to macromolecular absorption may play a role in some of the enhanced HRP absorption seen in these experiments with low concentrations of DCh.A more extensive cellular damage leading to enhanced HRP transport may occur with higher concentrations of bile salts.