RESUMEN
BACKGROUND/AIM: Helicobacter pylori (Hp) infection affects a substantial proportion of the world population and is a major risk factor of gastric cancer (GC). The caveats of common Hp-tests can be evaded by a serological biomarker test (GastroPanel®, Biohit Oyj, Helsinki), the most comprehensive Hp-test on the market. The clinical validation of Helicobacter pylori IgG ELISA of the new-generation GastroPanel® test is reported. The aim of the study is to validate the clinical performance of the Helicobacter pylori IgG ELISA test in diagnosis of biopsy-confirmed Hp-infection in gastroscopy referral patients. PATIENTS AND METHODS: A cohort of 101 patients (mean age=50.1 years) referred for gastroscopy at the outpatient Department of Gastroenterology (SM Clinic, St. Petersburg) were examined by two test versions to validate the new-generation GastroPanel®. All patients were examined by gastroscopy and biopsies, which were stained with Giemsa for specific identification of Hp in the antrum (A) and corpus (C). RESULTS: Biopsy-confirmed Hp-infection was found in 64% of patients, most often confined to antrum. The overall agreement between Hp IgG ELISA and gastric biopsies in Hp-detection was 91% (95%CI=84.1-95.8%). Hp IgG ELISA diagnosed biopsy-confirmed Hp (A&C) with sensitivity (SE) of 92.3%, specificity (SP) of 88.6%, positive predictive value (PPV) of 93.8% and negative predictive value (NPV) of 86.1%, with AUC=0.904 (95%CI=0.842-0.967). In ROC analysis for Hp detection (A&C), Hp IgG ELISA shows AUC=0.978 (95%CI=0.956-1.000). CONCLUSION: The Hp IgG ELISA test successfully concludes the clinical validation process of the new-generation GastroPanel® test, which retains the unrivalled diagnostic performance of all its four biomarkers, extensively documented for the first-generation test in different clinical settings.
Asunto(s)
Anticuerpos Antibacterianos/aislamiento & purificación , Infecciones por Helicobacter/diagnóstico , Helicobacter pylori/aislamiento & purificación , Inmunoglobulina G/aislamiento & purificación , Adolescente , Adulto , Anticuerpos Antibacterianos/genética , Anticuerpos Antibacterianos/inmunología , Biopsia , Ensayo de Inmunoadsorción Enzimática/métodos , Femenino , Gastrinas/genética , Gastrinas/aislamiento & purificación , Gastritis Atrófica/diagnóstico , Gastritis Atrófica/genética , Gastritis Atrófica/microbiología , Gastritis Atrófica/patología , Gastroscopía/métodos , Infecciones por Helicobacter/genética , Infecciones por Helicobacter/microbiología , Infecciones por Helicobacter/patología , Helicobacter pylori/genética , Helicobacter pylori/patogenicidad , Humanos , Inmunoglobulina G/inmunología , Masculino , Persona de Mediana Edad , Pepsinógeno A/genética , Pepsinógeno A/aislamiento & purificación , Pepsinógeno C/genética , Pepsinógeno C/aislamiento & purificación , Derivación y Consulta , Estómago/microbiología , Estómago/patología , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/genética , Neoplasias Gástricas/microbiología , Neoplasias Gástricas/patología , Adulto JovenAsunto(s)
Colecistoquinina/inmunología , Gastrinas/inmunología , Fisiología/historia , Secretina/inmunología , Bioensayo/métodos , Colecistoquinina/aislamiento & purificación , Gastrinas/aislamiento & purificación , Historia del Siglo XX , Humanos , Inmunoensayo/métodos , Secretina/aislamiento & purificación , Reino UnidoRESUMEN
BACKGROUND: Numerous studies have demonstrated that in vitro controlled enzymatic hydrolysis of fish and shellfish proteins leads to bioactive peptides. Ultrafiltration (UF) and/or nanofiltration (NF) can be used to refine hydrolysates and also to fractionate them in order to obtain a peptide population enriched in selected sizes. This study was designed to highlight the impact of controlled UF and NF on the stability of biological activities of an industrial fish protein hydrolysate (FPH) and to understand whether fractionation could improve its content in bioactive peptides. RESULTS: The starting fish protein hydrolysate exhibited a balanced amino acid composition, a reproducible molecular weight (MW) profile, and a low sodium chloride content, allowing the study of its biological activity. Successive fractionation on UF and NF membranes allowed concentration of peptides of selected sizes, without, however, carrying out sharp separations, some MW classes being found in several fractions. Peptides containing Pro, Hyp, Asp and Glu were concentrated in the UF and NF retentates compared to the unfractionated hydrolysate and UF permeate, respectively. Gastrin/cholecystokinin-like peptides were present in the starting FPH, UF and NF fractions, but fractionation did not increase their concentration. In contrast, quantification of calcitonin gene-related peptide (CGRP)-like peptides demonstrated an increase in CGRP-like activities in the UF permeate, relative to the starting FPH. The starting hydrolysate also showed a potent antioxidant and radical scavenging activity, and a moderate angiotensin-converting enzyme (ACE)-1 inhibitory activity, which were not increased by UF and NF fractionation. CONCLUSION: Fractionation of an FPH using membrane separation, with a molecular weight cut-off adapted to the peptide composition, may provide an effective means to concentrate CGRP-like peptides and peptides enriched in selected amino acids. The peptide size distribution observed after UF and NF fractionation demonstrates that it is misleading to characterize the fractions obtained by membrane filtration according to the MW cut-off of the membrane only, as is currently done in the literature.
Asunto(s)
Proteínas de Peces/química , Gastrinas/aislamiento & purificación , Péptidos/aislamiento & purificación , Aminoácidos/aislamiento & purificación , Animales , Antioxidantes/aislamiento & purificación , Antioxidantes/farmacología , Péptido Relacionado con Gen de Calcitonina/aislamiento & purificación , Colecistoquinina/aislamiento & purificación , Productos Pesqueros , Peces , Hidrólisis , Peso Molecular , Péptidos/química , Péptidos/farmacología , Peptidil-Dipeptidasa A/aislamiento & purificación , Peptidil-Dipeptidasa A/farmacología , Ultrafiltración/métodosRESUMEN
Retention behaviour of biological peptides was investigated on a stationary phase bearing an embedded quaternary ammonium group in a C21 alkyl chain by both high-performance liquid chromatography (HPLC) and capillary electrochromatography (CEC). In HPLC experiments, variation of acetonitrile (ACN) content in the mobile phase showed that peptides are mainly separated by RP mechanism. The weak or negative retention factors observed as compared to C18 silica stationary phase suggested the involvement of an electrostatic repulsion phenomenon in acidic conditions. Comparison of HPLC and CEC studies indicated that (i) ion-exclusion phenomenon is more pronounced in HPLC and (ii) higher ACN percentage in mobile phase induce for some peptides an increase of retention in CEC, pointing out the existence of mechanisms of retention other than partitioning mainly involved in chromatographic process. This comparative study demonstrated the critical role of electric field on peptide retention in CEC and supports the solvatation model of hydrolytic pillow proposed by Szumski and Buszewski for CEC using mixed mode stationary phase in CEC.
Asunto(s)
Electrocromatografía Capilar/métodos , Cromatografía Líquida de Alta Presión/métodos , Péptidos/aislamiento & purificación , Angiotensinógeno/aislamiento & purificación , Electrocromatografía Capilar/instrumentación , Cromatografía Líquida de Alta Presión/instrumentación , Eledoisina/aislamiento & purificación , Factor de Crecimiento Epidérmico/aislamiento & purificación , Gastrinas/aislamiento & purificaciónRESUMEN
Gastric acid production is important in intestinal iron absorption. The peptide hormone gastrin exists in both amidated and non-amidated forms, which stimulate and potentiate gastric acid secretion, respectively. Since non-amidated gastrins require ferric ions for biological activity in vitro, this study investigated the connection between iron status and gastrin by measurement of circulating gastrin concentrations in mice and humans with hemochromatosis. Gastrin concentrations are increased in the plasma and gastric mucosa of Hfe(-/-) mice, and in the sera of humans with HFE-related hemochromatosis. The discovery of a relationship between iron status and circulating gastrin concentrations opens a new perspective on the mechanisms of iron homeostasis.
Asunto(s)
Gastrinas/sangre , Hemocromatosis/sangre , Animales , Mucosa Gástrica/química , Gastrinas/análisis , Gastrinas/aislamiento & purificación , Glicina , Hemocromatosis/metabolismo , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Hierro/sangre , Hierro/fisiología , Proteínas de la Membrana/deficiencia , Proteínas de la Membrana/genética , Ratones , Ratones NoqueadosRESUMEN
Amidated and nonamidated progastrin-derived peptides have distinct biological activities that are mediated by a range of receptor subtypes. The objective was to determine the nature of the stored and secreted progastrin-derived peptides and to investigate whether progastrin release is regulated by gastric acidity. Using an antiserum directed to the C terminus of progastrin for identification and to monitor purification, C-terminal flanking peptides (CTFP) of progastrin (prog(76-83), prog(77-83), and prog(78-83) in approximately equivalent amounts) were isolated and identified from extracts of sheep antrum using ion exchange, HPLC, and mass spectrometry. Only trace amounts of full-length progastrin were present. Progastrin CTFP was the predominant progastrin-derived peptide in the antrum [progastrin CTFP/gastrin amide (Gamide) = 3]. Similarly, progastrin CTFP was the major circulating form in the antral (CTFP, 710 +/- 62 pmol/liter; Gamide, 211 +/- 35 pmol/liter) and jugular (CTFP, 308 +/- 16 pmol/liter; gastrin amide, 32 +/- 3 pmol/liter) veins. Alteration of gastric acidity in sheep by iv infusion of a H/K-adenosine triphosphatase inhibitor or somatostatin or by intragastric infusion of HCl demonstrated that the CTFP concentrations changed, although to a lesser extent than the changes in circulating gastrin amide. We conclude that the CTFP of progastrin is the major stored and circulating species of the gastrin gene, and that it is secreted in a regulated fashion rather than constitutively. Because full-length progastrin is bioactive, but is only a minor antral and secreted form, determination of the biological activity of the C-terminal flanking peptides will be important for a complete understanding of gastrin endocrinology.
Asunto(s)
Gastrinas/metabolismo , Fragmentos de Péptidos/metabolismo , Precursores de Proteínas/metabolismo , Antro Pilórico/metabolismo , Anestesia , Animales , Antiulcerosos/farmacología , Cromatografía , Estado de Conciencia , Gastrinas/aislamiento & purificación , Espectrometría de Masas , Omeprazol/farmacología , Fragmentos de Péptidos/aislamiento & purificación , Precursores de Proteínas/aislamiento & purificación , Ovinos , Somatostatina/farmacologíaRESUMEN
To identify the teleost gastrin, CCK/gastrin family genes were isolated from puffer and flounder. The cDNA of puffer gastrin, CCK1 and CCK2 were 678, 752 and 533bp, respectively. Puffer gastrin gene consists of three exons, in contrast, CCKs consist of four exons. Flounder gastrin mRNA (526bp) was expressed in the intestine but not in the brain. It was developed synchronously with the stomach differentiation in the larval stage. The phylogenetic analysis shows that puffer and flounder gastrin classified into the vertebrate gastrin cluster and two types of CCK were probably produced by the genome duplication occurred in teleost phylogeny.
Asunto(s)
Colecistoquinina/genética , Peces/genética , Gastrinas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Colecistoquinina/aislamiento & purificación , Clonación Molecular , ADN Complementario/química , ADN Complementario/genética , Evolución Molecular , Femenino , Lenguado/genética , Gastrinas/aislamiento & purificación , Regulación de la Expresión Génica , Masculino , Datos de Secuencia Molecular , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Tetraodontiformes/genéticaRESUMEN
Evidence is accumulating that gastrin precursors may act as growth factors for the colonic mucosa in vivo. The aims of this study were to prepare recombinant human progastrin(6-80) and to investigate its structure and biological activities in vitro. Human progastrin(6-80) was expressed in Escherichia coli as a glutathione S-transferase fusion protein. After thrombin cleavage progastrin(6-80) was purified by reverse phase high pressure liquid chromatography and characterized by radioimmunoassay, amino acid sequencing, and mass spectrometry. Assays for metal ions by atomic emission spectroscopy revealed the presence of a single tightly bound calcium ion. Progastrin(6-80) at concentrations in the pm to nm range stimulated proliferation of the conditionally transformed mouse colon cell line YAMC. The observations that progastrin(6-80) did not bind to either the cholecystokinin (CCK)-A or the gastrin/CCK-B receptor expressed in COS cells and that antagonists selective for either receptor did not reverse the proliferative effects of progastrin(6-80) suggested that progastrin(6-80) stimulated proliferation independently of either the CCK-A or the gastrin/CCK-B receptor. We conclude that recombinant human progastrin(6-80) is biologically active and contains a single calcium ion. With the exception of the well known zinc-dependent polymerization of insulin and proinsulin, this is the first report of selective, high affinity binding of metal ions to a prohormone.
Asunto(s)
Calcio/metabolismo , Gastrinas/química , Fragmentos de Péptidos/química , Precursores de Proteínas/química , Secuencia de Aminoácidos , Animales , Células COS , División Celular/efectos de los fármacos , Gastrinas/aislamiento & purificación , Gastrinas/fisiología , Humanos , Datos de Secuencia Molecular , Precursores de Proteínas/aislamiento & purificación , Precursores de Proteínas/fisiología , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Sincalida/metabolismoRESUMEN
The turnover of the epithelium of the gastrointestinal tract is regulated by a balance between cell multiplication and cell loss. We examined the effects of starvation on apoptosis in endocrine and other epithelial cells of rat antropyloric mucosa. Apoptosis was determined by the TUNEL reaction combined with immunocytochemical staining for gastrin and somatostatin. Apoptotic cell morphology was determined by bisbenzimide staining for DNA. Both gastrin and somatostatin cells showed a significantly lower apoptotic index than the general epithelium. This agrees with the longer turnover kinetics of gastric endocrine cells. On starvation, the apoptotic index of the general epithelium and of the gastrin but not of the somatostatin, cells increased significantly. This was prevented by the nitric oxide synthase (NOS) inhibitor L-NAME but not by its inactive stereoisomer D-NAME. Immunoreactive neuronal NOS was present in somatostatin cells, in nonendocrine cells predominating in the surface and pit epithelium, and in rare nerve fibers. Endothelial cell NOS was present in vessels, whereas the inducible isoform was barely detectable. Thus, endogenous NOS isoforms participate in regulating antropyloric epithelial apoptosis during starvation. The close paracrine relation between somatostatin cells and gastrin cells suggests that the former regulates apoptosis of the latter through release of NO.
Asunto(s)
Apoptosis/fisiología , Ingestión de Alimentos/fisiología , Mucosa Gástrica/fisiología , Óxido Nítrico Sintasa/metabolismo , Antro Pilórico/fisiología , Animales , Femenino , Gastrinas/aislamiento & purificación , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico Sintasa/antagonistas & inhibidores , Ratas , Ratas Wistar , Somatostatina/aislamiento & purificación , InaniciónRESUMEN
The distribution of cholecystokinin and gastrin-like immunoreactive cell bodies and fibers in the nervous system of 2 annelid worms, Lumbricus terrestris and Eisenia fetida, has been studied by means of immunohistochemistry. The cerebral ganglion contains 170-250, the subesophageal ganglion contains 120-150, and the ventral ganglia contain 50-75 cholecystokinin immunoreactive cells, that represent 8-12%, 8-10% and 4-5% of the total cell number, respectively. The anti-gastrin serum stained 330-360 nerve cells in the cerebral, 32-46 in the subesophageal and 7-25 in the ventral cord ganglia, representing 15-16%, 2-3% and 0.5-2% of the total cell number. Immunopositivity was found with both antisera in the enteric nervous system, where the stomatogastric ganglia and the enteric plexus contain immunoreactive cells and fibers. Immunopositive cells were found in the epithelial and subepithelial cells, as well as in nerve cells innervating the muscular layer of the gastrointestinal tube. Various epidermal sensory cells also displayed strong immunoreactivity. According to our findings and the results of several functional studies, it is suggested that in annelids cholecystokinin- and gastrin-like peptides may be involved in digestive regulation, sensory processes and central integrating processes.
Asunto(s)
Colecistoquinina/aislamiento & purificación , Gastrinas/aislamiento & purificación , Sistema Nervioso/química , Oligoquetos/química , Serotonina/aislamiento & purificación , Animales , Ganglios de Invertebrados/química , Inmunohistoquímica , Sistema Nervioso Periférico/químicaRESUMEN
Gastrin-like immunoreactive peptides were extracted from the gastric antrum of the American alligator (Alligator mississippiensis) and purified by fractionation using C18 Sep-Paks, Sephadex G-50, pH stable C8 reversed-phase HPLC, and C18 reversed-phase HPLC. Three major immunoreactive peaks were purified and found to correspond to 49, 45, and 34 residue peptides by microsequence analysis. The amino acid sequence of the largest peptide was DWLASLSQDQ KHLISKFLPH IYGELAN QEN YWQEDDALHD HDYPGWMDF-amide. The two smaller peptides corresponded to carboxyl-terminal 45 and 34 residue fragments of the 49 residue peptide. The putative proteolysis of the 49 residue peptide to the two shorter peptides occurs at cleavage sites that are unusual in biosynthetic processing. Mass spectral analysis confirmed the molecular weights that were predicted from the amino acid sequences, thus revealing the absence of any post-translational modifications, such as sulfation. Although the three alligator gastrins resemble mammalian cholecystokinin in having a tyrosine residue in the seventh position from the carboxyl terminus, this tyrosine is apparently nonsulfated as in turtle gastrin. When tested by radioreceptor assay, a synthetic replicate of alligator gastrin-49 exhibited a gastrin-like pattern of biological activity on mammalian CCK-A and CCK-B receptors. Comparison of the amino acid sequences of known peptides revealed that alligator gastrin is most similar to turtle gastrin (76% identical), followed by frog gastrin (51% identical), chicken gastrin (49% identical), and human gastrin (12% identical). These similarities closely reflect vertebrate phylogeny and support the hypothesis that functionally distinct gastrins evolved from CCK in early tetrapods. However, gastrin evolved via different mechanisms in the mammalian lineage (mechanism unknown) versus the amphibian and reptilian/avian lineages, in which two different single nucleotide base changes can account for the separate evolution of amphibian gastrin and reptilian/avian gastrin.
Asunto(s)
Caimanes y Cocodrilos , Gastrinas/química , Gastrinas/metabolismo , Receptores de Colecistoquinina/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Gastrinas/aislamiento & purificación , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Antro Pilórico/química , Ensayo de Unión Radioligante , Receptor de Colecistoquinina A , Receptor de Colecistoquinina B , Análisis de Secuencia , Homología de SecuenciaRESUMEN
Cholecystokinins from brain and small intestine of the bullfrog (Rana catesbeiana) and red-eared slider turtle (Pseudomys scripta) were isolated. The purifications were monitored by an antiserum specific for the common C-terminus of mammalian cholecystokinin and gastrin. The peptide structures were identified by sequence analysis of the intact peptides and proteolytic fragments, mass spectrometry, and amino acid analysis. Brain and small intestine of both species contained cholecystokinin-8 and substantial amounts of cholecystokinin-7. Furthermore, the small intestine of both frog and turtle contained a major fraction of the immunoreactive material as large peptides consisting of 69 residues and 70 residues, respectively. The structure for frog cholecystokinin-69 is ASSSAQLKPFQRIDGTSDQKAVIGAMLAKYLQTRKAGSSTGRYAVLPNRPVIDPTHRINDRDYMGWMDF .NH2 and the structure for turtle cholecystokinin-70 is VPSSAGQLKPIQRLDGNVDQKANIGALLAKYLQQARKGPTGRISMMGNRVQNIDPTHRINDRDYMGWMD F.NH2. All the isolated peptides were tyrosine sulfated at the seventh last residue. The peptides are highly similar to each other and to mammalian cholecystokinins (70% mutual identity and more than 50% identity with human cholecystokinin). Thus, they are clearly related to the known mammalian cholecystokinins. Both peptides include the monobasic and dibasic cleavage sites giving rise to cholecystokinins-33, -39, and -58 in mammals. However, only a small amount of turtle cholecystokinin-40 (corresponding to mammalian cholecystokinin-39) was isolated. This confirms that post-translational processing is highly species dependent. Recently, we isolated peptides from frog and turtle antrum. Following their origin they were named gastrins in spite of their C-terminal cholecystokinin-like structure. Thus, two different cholecystokinin/gastrin peptides exist in frog and turtle justifying the choice of two names. This finding of two members of the cholecystokinin/gastrin family in frog shows that the divergence of cholecystokinin and gastrin occurred simultaneously with or earlier than the appearance of amphibia during phylogenesis. Frog cholecystokinin and gastrin show sufficient similarity along the whole sequence to support the notion of a gene duplication of a common ancestor.
Asunto(s)
Evolución Biológica , Encéfalo/metabolismo , Colecistoquinina/genética , Gastrinas/genética , Variación Genética , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Rana catesbeiana/genética , Tortugas/genética , Secuencia de Aminoácidos , Animales , Química Encefálica , Cerebelo/química , Colecistoquinina/química , Colecistoquinina/aislamiento & purificación , Gastrinas/química , Gastrinas/aislamiento & purificación , Mucosa Intestinal/química , Intestino Delgado/química , Espectrometría de Masas , Datos de Secuencia Molecular , Bulbo Olfatorio/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/aislamiento & purificación , Homología de Secuencia de Aminoácido , Colículos Superiores/químicaRESUMEN
Chicken antrum was found to contain 7 nmol/g of carboxyamidated gastrin/CCK-like peptides. The predominant chicken gastrin (so named due to the antral origin) contained 53 amino acid residues: DWPEPPSQEQ QQRFISRFLP HVFAELSDRK GFVQGNGAVE ALHDHFYPDW MDF-NH2. Three smaller (less abundant) forms corresponded to the 30-, 21-, and 7-residue carboxyamidated C-terminal fragments. The major part was sulfated at the tyrosine residue in position seven from the C-terminus. A lower isoelectric point and abrupt termination of the sequencing suggest that some of the peptides had an isoAsp-Gly bond instead of an Asn-Gly bond. The three shorter forms were all derived from the precursor by post-Phe cleavages. This cleavage pattern suggests a processing enzyme specific for bonds between Phe and moderately hydrophobic residues.
Asunto(s)
Pollos/fisiología , Mucosa Gástrica/química , Gastrinas/química , Procesamiento Proteico-Postraduccional , Antro Pilórico/química , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Reacciones Cruzadas , Gastrinas/aislamiento & purificación , Datos de Secuencia Molecular , RadioinmunoensayoRESUMEN
Following the curative resection of a pancreatic gastrinoma in a cat, gastrin peptides were purified from the tissue and sequenced. The sequence of cat gastrinoma G17 (18-34) confirms the previously published sequence. The sequence of cat G34 (1-34) is reported for the first time. The NH2-terminal portion of cat G34 differs from that of dog by having a Q (Gln) for L (Leu) at position 10 from the NH2-terminus.
Asunto(s)
Gastrinoma/veterinaria , Gastrinas/química , Neoplasias Pancreáticas/veterinaria , Secuencia de Aminoácidos , Animales , Gatos , Cromatografía Líquida de Alta Presión , Femenino , Gastrinoma/metabolismo , Gastrinas/aislamiento & purificación , Datos de Secuencia Molecular , Neoplasias Pancreáticas/metabolismo , Alineación de SecuenciaRESUMEN
So far the only CNS neurons found to express gastrin have been hypothalamohypophyseal. Using a library of radioimmunoassays specific for different sequences of porcine progastrin in combination with chromatography and enzyme cleavages, 21 or 24 porcine cerebelli was now found to contain progastrin or its products. Sixteen cerebelli processed progastrin via glycine-extended intermediates to bioactive, carboxyamidated gastrin-17 and -34, half of the gastrins being tyrosine O-sulfated. The mean concentration of carboxyamidated gastrins was 0.8 pmol/g tissue (range less than 0.1-2.1 pmol/g), of glycine-extended intermediates 0.1 pmol/g (range less than 0.1-0.3 pmol/g) and of progastrin 0.4 pmol/g (range less than 0.1-1.0 pmol/g). The results show that the gastrin gene is expressed in more than a single region of the brain.
Asunto(s)
Cerebelo/química , Gastrinas/aislamiento & purificación , Precursores de Proteínas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Cromatografía en Gel , Gastrinas/química , Datos de Secuencia Molecular , Fragmentos de Péptidos/aislamiento & purificación , Conformación Proteica , Precursores de Proteínas/química , Radioinmunoensayo , PorcinosRESUMEN
Binding of 125I-[Nle15]gastrin to albumin purified from porcine serum, from porcine gastric mucosal cytosol, and from bovine serum has been demonstrated by covalent cross-linking and ultracentrifugation. Binding was enhanced in the presence of Zn2+, Ni2+, Cu2+, Co2+, and Cd2+, but not Ca2+, Mg2+, or Mn2+. The best fit to the binding data for bovine serum albumin was obtained with a model assuming two nonequivalent binding sites. The affinity of both sites for gastrin was increased in the presence of 100 microM Zn2+ or Ni2+ ions. The highest association constant observed was 2.3 X 10(5) M-1 in the presence of 100 microM Zn2+ ions. The similarity of the Zn(2+)-dependence of binding for bovine and porcine serum albumins, despite the replacement of His3 by Tyr, suggested that the N-terminal metal ion-binding site was not involved. Although all gastrin affinities were reduced by 50% in the presence of 150 mM NaCl, the Zn(2+)-dependence of binding was retained. We therefore propose that the ternary complex of gastrin, Zn2+ ions, and albumin may play a physiological role in the serum transport of Zn2+ ions and in the uptake of Zn2+ ions from the lumen of the gastrointestinal tract.
Asunto(s)
Gastrinas/metabolismo , Metales/farmacología , Albúmina Sérica/metabolismo , Animales , Sitios de Unión , Cationes Bivalentes/farmacología , Mucosa Gástrica/química , Gastrinas/aislamiento & purificación , Albúmina Sérica Bovina/metabolismo , Porcinos , Ultracentrifugación , Zinc/farmacocinéticaRESUMEN
Four gastrin/cholecystokinin-like peptides (G/CCK) which cross-react with a specific C-terminal gastrin/CCK antiserum have been isolated from the stomach of the marine crustacean Nephrops norvegicus. The molecular weight of the four peptides was estimated between 1000 and 2000 Da by molecular sieving. By radioimmunoassay, the cross-reactivity of these peptides with human gastrin 17-I was found to be around 0.03%. Pure peptidic fractions were recovered after four successive steps of HPLC. Amino-acid analysis suggested a similarity between the four peptides identified which may belong to a new family. A limited homology between the C-terminus of one Nephrops peptide and vertebrate G/CCK was found after sequencing. Two of the peptides exhibited secretagogue effects on crustacean isolated midgut glands. The Nephrops peptides, although structurally distinct from the vertebrate G/CCKs, appear to serve similar biological functions in crustaceans.
Asunto(s)
Colecistoquinina/química , Gastrinas/química , Nephropidae/química , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Colecistoquinina/aislamiento & purificación , Colecistoquinina/farmacología , Cromatografía Líquida de Alta Presión , Endopeptidasas/metabolismo , Gastrinas/aislamiento & purificación , Gastrinas/farmacología , Hidrólisis , Datos de Secuencia Molecular , Peso Molecular , RadioinmunoensayoRESUMEN
Gastrin, the primary hormonal mediator of postprandial gastric acid secretion, is produced from its precursor progastrin by a series of posttranslational processing reactions including dibasic residue cleavage and carboxyl-terminal alpha-amidation. Progastrin contains three dibasic cleavage signals, Arg57Arg58, Lys74Lys75, and Arg94Arg95, that appear to be cleaved differently in different tissues. Differential processing is a potential means by which the production of biologically active peptides may be regulated in a tissue-specific manner. To study these reactions further, we used the pZipNeo SV(X) retroviral vector to express human gastrin cDNA in a heterologous cell line (MTC 6-23) known to be capable of processing other peptide precursors. The psi 2 packaging cell line transfected with the gastrin cDNA-retroviral construct (pSVXgas) produced progastrin, but no substantial amounts of processed amidated gastrin were detected. amounts of processed amidated gastrin were detected. In contrast, MTC 6-23 cells infected with the viral stock obtained from the supernatant of pSVXgas-transfected psi 2 cells produced carboxyl-terminally amidated gastrin in all of its standard molecular forms, including sulfated and nonsulfated forms of tetratriacontagastrin (G-34), heptadecagastrin (G-17), and tetradecagastrin (G-14). These studies indicate that heterologous endocrine cell lines infected with a retroviral-peptide cDNA construct can serve as useful models for peptide hormone posttranslational processing.
Asunto(s)
Gastrinas/genética , Precursores de Proteínas/genética , Secuencia de Aminoácidos , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Gastrinas/análisis , Gastrinas/aislamiento & purificación , Expresión Génica , Vectores Genéticos , Humanos , Técnicas para Inmunoenzimas , Ratones , Datos de Secuencia Molecular , Plásmidos , Poli A/genética , Poli A/aislamiento & purificación , Procesamiento Proteico-Postraduccional , ARN/genética , ARN/aislamiento & purificación , ARN Mensajero , Radioinmunoensayo , Mapeo Restrictivo , Neoplasias de la Tiroides , TransfecciónRESUMEN
The biological activity of natural chicken gastrins isolated from the "antrum"-like region of the chicken gut have been studied on gastric secretion in chickens, turkeys, and rats, pancreatic secretion in turkeys and rats, and gallbladder contraction in chickens and guinea pigs. Natural chicken gastrin was shown to be approximately 85% sulfated on the tyrosine that occurs at position 7 from the COOH-terminus. In both avian and mammalian systems, chicken gastrins were found to be potent stimulants of acid secretion but were virtually inactive as stimulants of pancreatic secretion and gallbladder contraction. Peptides with the COOH-terminal tetrapeptide amide of CCK and a sulfated tyrosine at position 7 from the COOH-terminus are usually potent stimulants of pancreas and gallbladder. However, although chicken gastrin has a CCK-like structure, it has a gastrin-like spectrum of biological actions. A proline immediately adjacent to the sulfated tyrosine may produce a steric effect that lowers the activity of chicken gastrin on pancreas and gallbladder. Evidently, then, the factors that determine specificity of action of CCK and gastrin are different in birds and mammals.
Asunto(s)
Gastrinas/aislamiento & purificación , Secuencia de Aminoácidos , Aminoácidos/análisis , Animales , Bioensayo , Colecistoquinina/genética , Colecistoquinina/farmacología , Cromatografía Líquida de Alta Presión , Vesícula Biliar/efectos de los fármacos , Vesícula Biliar/fisiología , Gastrinas/genética , Gastrinas/farmacología , Técnicas In Vitro , Masculino , Datos de Secuencia Molecular , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Páncreas/efectos de los fármacos , Páncreas/metabolismo , Fragmentos de Péptidos/aislamiento & purificación , Ratas , Ratas Endogámicas , Homología de Secuencia de Ácido Nucleico , Sincalida/farmacología , Relación Estructura-Actividad , Tripsina , PavosRESUMEN
The predominant immunoreactive cholecystokinin (CCK) forms in acid extracts of rat intestine eluted from reverse-phase high-performance liquid chromatography columns in the positions of CCK-8, CCK-33/39, and CCK-58. Control experiments indicated that smaller CCK forms did not arise from artifactual degradation of large CCK forms. Less than 10% of CCK-8 added to and extracted from intestine was recovered in acid; CCK-8 could not be recovered by subsequent alkaline extraction, but subsequent urea extraction yielded approximately 25% of the added peptide. This suggests that CCK binds to proteins during acid extraction and that the preponderance of large CCK forms in acid extracts is not due to inhibition of CCK degradation but results from poor extraction of small CCK forms. No evidence for a CCK-22-like peptide was found in acid or subsequent urea extracts of rat intestine, suggesting CCK posttranslational processing in adult rats is like that in humans and dogs.