RESUMO
Peptides are used for diagnostics, therapeutics, and as antimicrobial agents. Most peptides are produced by chemical synthesis, but recombinant production has recently become an attractive alternative due to the advantages of high titers, less toxic waste and correct folding of tertiary structure. Somatostatin-28 is a peptide hormone that regulates the endocrine system, cell proliferation and inhibits the release of numerous secondary hormones in human body. It is composed of 28 amino acids and has one disulfide bond, which makes it to an optimal model peptide for a whole downstream purification process. We produced the peptide in the periplasm of E. coli using the CASPON™ technology, an affinity fusion technology system that enables high soluble expression of recombinant proteins and cleaves the fusion tag with a circularly permuted human caspase-2. Furthermore, purification of the products is straight forward using an established platform process. Two different case studies for downstream purification are presented, starting with either hydrochloric acid or polyethyleneimine as an extraction aid. After release of affinity-tagged somatostatin-28 out of E. coli's periplasm, several purification steps were performed, delivering a pure peptide solution after the final polishing step. The process was monitored by reversed-phase high-performance liquid chromatography as well as mass spectrometry to determine the yield and correct disulfide bond formation. Monitoring of impurities like host cell proteins, DNA and endotoxins after each downstream unit confirmed effective removal for both purification pathways.
Assuntos
Escherichia coli , Ácido Clorídrico , Polietilenoimina , Somatostatina , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Somatostatina/química , Somatostatina/genética , Somatostatina/isolamento & purificação , Ácido Clorídrico/química , Polietilenoimina/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/biossínteseRESUMO
Structural studies of human peptide hormone somatostatin 14 (SS14) require high amounts of isotopically labelled SS14 to be produced. Here we report a method for effective production of isotopically labelled SS14. SS14 was expressed as a fusion protein with thioredoxin in Escherichia coli. Co-expression of a longer polypeptide product lowered the yield of the target peptide and complicated its purification. The side product contained the N-terminal 6His-tag together with the thioredoxin fusion partner and the specific enzymatic cleavage site-containing linker followed by an unknown peptide starting with the first 7N-terminal amino acid residues of SS14, as revealed by the Edman degradation. The combination of DNA sequence analysis, the Edman degradation, and high-resolution mass spectrometry allowed to identify the amino acid sequence of the unknown peptide. The appearance of the side product was attributed to inefficient termination of mRNA translation. The stop codon and its downstream sequence optimization allowed eliminating the side product synthesis. The optimized expression system, purification protocol, and post-translational modification procedure yielded 1.5mg of SS14 per liter of minimal medium. Nearly 99% incorporation of (13)C and (15)N isotopes was achieved, as demonstrated by high-resolution mass spectrometry.
Assuntos
Somatostatina/isolamento & purificação , Isótopos de Carbono , Códon de Terminação/genética , Escherichia coli/metabolismo , Humanos , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Isótopos de Nitrogênio , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Somatostatina/biossínteseRESUMO
Somatostatin/growth hormone-inhibiting hormone is the peptide that inhibits secretion of somatotropin/growth hormone. Solid-phase synthesis methods are being currently used to produce somatostatin. Recombinant peptide synthesis is widely described for the production of small proteins and peptides; however, the production at industrial scale of peptides for biopharmaceutical applications is limited for economic reasons. Here, we propose the use of a new pGB-SMT plasmid to produce Somatostatin, as a C-terminal fusion protein with a Kluyveromyces lactis ß-galactosidase fragment. To facilitate removal of that fragment by CNBr cleavage, a methionine residue was introduced at the N-terminal of the hormone peptide. The use of this construction enables an IPTG-free expression system. The suitability of this procedure has been assessed in a 15 l scale-up experiment yielding almost 300 mg, with purity >99 % and it is being implemented for commercial scale. The plasmid pGB-SMT here described is an alternative option for a cheap and high expression of other short peptide hormones.
Assuntos
Escherichia coli/genética , Vetores Genéticos , Somatostatina/biossíntese , Somatostatina/isolamento & purificação , Cromatografia de Fase Reversa , Clonagem Molecular , Escherichia coli/metabolismo , Expressão Gênica , Humanos , Metionina/metabolismo , Plasmídeos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Somatostatina/genética , beta-Galactosidase/genética , beta-Galactosidase/metabolismoRESUMO
Somatostatin is a natural inhibitor of growth hormone, and its analogues are clinically used for the therapy of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndrome. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins, and Pichia pastoris was used as an expression system. Three fusion proteins, (somatostatin (SS)14)2-human serum albumin (HSA), (SS14)3-HSA, and HSA-(SS14)3, were constructed with different fusion copies of somatostatin-14 and fusion orientations. The expression level of (SS14)3-HSA and HSA-(SS14)3 was much lower than (SS14)2-HSA due to the additional fusion of the somatostatin-14 molecule. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard of somatostatin-14, all three fusion proteins were able to inhibit growth hormone secretion in the blood, with (SS14)2-HSA being the most effective one. On the whole, (SS14)2-HSA was the most effective protein in both production level and bioactivity, and increasing the number of small protein copies fused to HSA may not be a suitable method to improve the protein bioactivity.
Assuntos
Escherichia coli/fisiologia , Pichia/fisiologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Albumina Sérica/metabolismo , Somatostatina/biossíntese , Humanos , Albumina Sérica/genética , Somatostatina/genética , Somatostatina/isolamento & purificação , Transfecção/métodosRESUMO
The chronic use of nicotine, the main psychoactive ingredient of tobacco smoking, alters diverse physiological processes and consequently generates physical dependence. To understand the impact of chronic nicotine on neuropeptides, which are potential molecules associated with dependence, we conducted qualitative and quantitative neuropeptidomics on the rat dorsal striatum, an important brain region implicated in the preoccupation/craving phase of drug dependence. We used extensive LC-FT-MS/MS analyses for neuropeptide identification and LC-FT-MS in conjunction with stable isotope addition for relative quantification. The treatment with chronic nicotine for 3 months led to moderate changes in the levels of endogenous dorsal striatum peptides. Five enkephalin opioid peptides were up-regulated, although no change was observed for dynorphin peptides. Specially, nicotine altered levels of nine non-opioid peptides derived from precursors, including somatostatin and cerebellin, which potentially modulate neurotransmitter release and energy metabolism. This broad but selective impact on the multiple peptidergic systems suggests that apart from the opioid peptides, several other peptidergic systems are involved in the preoccupation/craving phase of drug dependence. Our finding permits future evaluation of the neurochemical circuits modulated by chronic nicotine exposure and provides a number of novel molecules that could serve as potential therapeutic targets for treating drug dependence.
Assuntos
Corpo Estriado/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Neuropeptídeos/metabolismo , Nicotina/administração & dosagem , Tabagismo/metabolismo , Administração Oral , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Doença Crônica , Corpo Estriado/metabolismo , Corpo Estriado/fisiopatologia , Dinorfinas/genética , Dinorfinas/isolamento & purificação , Dinorfinas/metabolismo , Encefalinas/genética , Encefalinas/isolamento & purificação , Encefalinas/metabolismo , Marcação por Isótopo , Masculino , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/isolamento & purificação , Proteínas do Tecido Nervoso/metabolismo , Neuropeptídeos/genética , Neuropeptídeos/isolamento & purificação , Precursores de Proteínas/genética , Precursores de Proteínas/isolamento & purificação , Precursores de Proteínas/metabolismo , Proteoma/genética , Proteoma/metabolismo , Ratos , Ratos Long-Evans , Somatostatina/genética , Somatostatina/isolamento & purificação , Somatostatina/metabolismo , Espectrometria de Massas em Tandem , Tabagismo/genética , Tabagismo/fisiopatologiaRESUMO
For iodination ((125/127)I) of tyrosine-containing peptides, chloramin-T, Pre-Coated Iodo-Gen(®) tubes and Iodo-Beads(®) (Pierce) are commonly used for in vitro radioligand investigations and there have been reliant vendors hereof for decades. However, commercial availability of these radio-iodinated peptides is decreasing. For continuation of our research in this field we investigated and optimized (radio-)iodination of somatostatin analogues. In literature, radioiodination using here described somatostatin analogues and iodination techniques are described separately. Here we present an overview, including High Performance Liquid Chromatography (HPLC) separation and characterisation by mass spectrometry, to obtain mono- and di-iodinated analogues. Reaction kinetics of (125/127)I iodinated somatostatin analogues were investigated as function of reaction time and concentration of reactants, including somatostatin analogues, iodine and oxidizing agent. To our knowledge, for the here described somatostatin analogues, no (127)I iodination and optimization are described. (Radio-)iodinated somatostatin analogues could be preserved with a >90% radiochemical purity for 1 month after reversed phase HPLC-purification.
Assuntos
Halogenação , Neoplasias/diagnóstico , Compostos Radiofarmacêuticos/química , Somatostatina/análogos & derivados , Quelantes/química , Cromatografia Líquida de Alta Pressão , Diagnóstico por Imagem , Humanos , Radioisótopos do Iodo , Marcação por Isótopo , Neoplasias/diagnóstico por imagem , Estabilidade Proteica , Cintilografia , Compostos Radiofarmacêuticos/isolamento & purificação , Somatostatina/isolamento & purificaçãoRESUMO
The (99m)Tc-labeled agent, ((99m)TcO)depreotide, has received regulatory approval in the United States and Europe for use in the detection of cancer. It is essential to establish a simple and reliable method of direct radiolabeling of (99m)Tc-depreotide and to investigate its specific receptor binding properties with human non small cell lung cancer (NSCLC) A549 cell in vitro. So we made some researches as follow: Depreotide was labeled with (99m)Tc using SnCl2 as a reductant. Labeling efficiencies at different pH values and temperatures were compared. Radioreceptor assay was used to observe the uptake kinetics, stagnation and retention half time of (99m)Tc-depreotide in A549 cells. As the results of the investigation ,many facts is shown below: The labeling rate of pH 6.0 group was higher than that of pH 5.0 and pH7.0 groups. The labeling rate decreased when temperature increased from 15 °C to 50 °C. The uptake rate increased with rising temperature, and the maximum uptake was observed at 60 min at 37 °C. The cleaning curves were similar at different temperatures, and the half cleaning time at 37 °C was 48 min. The results showed that the optimal conditions for labeling depreotide with (99m)Tc was found to be below 15 °C at a pH lower than 6.0. Furthermore, at 37 °C, (99m)Tc-depreotid may have the potential as an ideal imaging agent for somatostatin receptors.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/diagnóstico por imagem , Neoplasias Pulmonares/diagnóstico por imagem , Compostos de Organotecnécio/isolamento & purificação , Compostos Radiofarmacêuticos/isolamento & purificação , Somatostatina/análogos & derivados , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Estabilidade de Medicamentos , Humanos , Concentração de Íons de Hidrogênio , Neoplasias Pulmonares/metabolismo , Compostos de Organotecnécio/síntese química , Cintilografia , Compostos Radiofarmacêuticos/síntese química , Receptores de Somatostatina/metabolismo , Somatostatina/síntese química , Somatostatina/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , TemperaturaRESUMO
The early and later eluting [(99m)TcO]depreotide products on RP-HPLC were confirmed to be the anti and syn diastereomers, respectively, based on proton NMR and circular dichroism spectroscopy. NMR provided evidence of a folded, conformationally constrained structure for the syn diastereomer. The syn diastereomer is predominant (anti/syn approximately 10:90) in the [(99m)TcO]depreotide preparation and shows a slightly higher affinity (IC50 = 0.15 nM) for the somatostatin receptor than the anti diastereomer (IC50 = 0.89 nM). Both diastereomers showed higher binding affinities than the free peptide (IC(50) = 7.4 nM). Biodistribution studies in AR42J tumor xenograft nude mice also showed higher tumor uptake for syn [(99m)TcO]depreotide (6.58% ID/g) than for the anti [(99m)TcO]depreotide (3.38% ID/g). Despite the differences in biological efficacy, the favorable binding affinity, tumor uptake, and tumor-to-background ratio results for both diastereomeric species predict that both are effective for imaging somatostatin receptor-positive tumors in vivo.
Assuntos
Neoplasias/diagnóstico por imagem , Compostos de Organotecnécio/isolamento & purificação , Compostos Radiofarmacêuticos/isolamento & purificação , Receptores de Somatostatina/metabolismo , Somatostatina/análogos & derivados , Animais , Linhagem Celular Tumoral , Dicroísmo Circular , Feminino , Marcação por Isótopo , Espectroscopia de Ressonância Magnética , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neoplasias/metabolismo , Compostos de Organotecnécio/química , Compostos de Organotecnécio/farmacocinética , Neoplasias Pancreáticas , Ensaio Radioligante , Cintilografia , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Somatostatina/química , Somatostatina/isolamento & purificação , Somatostatina/farmacocinética , Estereoisomerismo , Distribuição TecidualRESUMO
68Ge/68Ga generators provide cyclotron-independent access to positron emission tomography (PET) radiopharmaceuticals. We describe a system which allows the safe and efficient handling of 68Ge/68Ga generator eluates for labelling of DOTA-derivatised peptide ligands. The system comprises concentration and purification of the 68Ga eluate as well as labelling and purification steps for peptides, and can be used with different 68Ge/68Ga generator types. The suitability and efficiency were tested with two different DOTA-derivatised somatostatin derivatives and a DOTA-derivatised bombesin derivative. Amounts of 10-20 nmol of the peptides were sufficient and resulted in labelling yields of 50% for all peptides. The built-in safety precautions have proven to be appropriate in allowing use of the method for routine clinical applications. The system was set up and operated in a "hot lab" by personnel with no previous experience in the preparation of PET radiopharmaceuticals.
Assuntos
Radioisótopos de Gálio/química , Compostos Heterocíclicos com 1 Anel/química , Marcação por Isótopo/instrumentação , Marcação por Isótopo/métodos , Peptídeos/química , Compostos Radiofarmacêuticos/química , Bombesina/análogos & derivados , Bombesina/isolamento & purificação , Radioisótopos de Gálio/isolamento & purificação , Compostos Heterocíclicos com 1 Anel/síntese química , Compostos Heterocíclicos com 1 Anel/isolamento & purificação , Ligantes , Peptídeos/síntese química , Peptídeos/isolamento & purificação , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/isolamento & purificação , Somatostatina/análogos & derivados , Somatostatina/isolamento & purificação , Manejo de Espécimes/instrumentação , Manejo de Espécimes/métodosRESUMO
Although the existence of two somatostatin variants (SS1 and SS2) has now been demonstrated in the brain of mammals, amphibians, and fish, only one isoform of somatostatin (SS1) has been characterized to date in the brain of birds. Here we report cloning of the cDNA encoding a 101-amino-acid protein (PSS2) that encompasses the somatostatin variant [Pro(2)]somatostatin-14 (SS2) at its C-terminus. Sequence analysis indicated that chicken PSS2 is more closely related to fish PSS2 than to mammalian cortistatin precursors. Northern blot analysis showed that the chicken PSS1 gene is expressed in the central nervous system (CNS) and in the pancreas, whereas the PSS2 gene is expressed only in the CNS and not in peripheral organs. In situ hybridization histochemistry revealed that, in the chicken brain, PSS1 mRNA is more widely distributed than PSS2 mRNA. In particular, PSS1 mRNA expression was found in the hippocampus, the hyperstriatum, the preoptic area, the ventricular hypothalamic nuclei, the optic tectum, and several nuclei of the mesencephalon and rhombencephalon. In contrast, the distribution of PSS2 mRNA was restricted to a few regions of the brain, including the paraolfactory lobe, the paleostriatum, and some nuclei of the mesencephalon and rhombencephalon. The fact that the PSS1 and PSS2 genes are differently expressed in the brain and in peripheral organs indicates that, in chicken, the two somatostatin variants likely exert distinct functions. In particular, the observation that PSS1 mRNA, but not PSS2 mRNA, occurs in the preoptic area and in the ventral hypothalamic nuclei suggests that, of the two somatostatin isoforms, only SS1 acts as a hypophysiotropic factor.
Assuntos
Encéfalo/metabolismo , Galinhas/genética , DNA Complementar/isolamento & purificação , Precursores de RNA/metabolismo , Somatostatina/genética , Processamento Alternativo , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Feminino , Expressão Gênica , Dados de Sequência Molecular , Precursores de Proteínas/genética , Precursores de Proteínas/isolamento & purificação , Homologia de Sequência , Somatostatina/classificação , Somatostatina/isolamento & purificação , Distribuição TecidualRESUMO
This study was undertaken to confirm the presence of CCK receptor subtypes in calf pancreas and establish their cellular localization. Using specific antibodies against CCKA and CCKB receptors, somatostatin, glucagon and insulin, we were able to confirm by Western blot the presence of both CCK receptor protein subtypes in the calf pancreas as a 80-85-kDa CCKA receptor and 40-45-kDa CCKB receptor. By immunofluorescence, the CCKB receptor colocalizes with the islets' somatostatin delta cells, confirming what was previously shown in other species, as well as on ductal cells. We could not reproduce in the calf its colocalization with glucagon alpha cells as observed in human and rat. Any specific localization of CCKA receptors with our multiple antibodies failed. Our observation that the CCKB receptor subtype is specifically localized on pancreatic delta cells as well as on ductal cells lets us support the hypothesis that in this species, CCK could be involved in somatostatin metabolism as well as hydrelatic secretion; its effect on enzyme secretion would be indirect.
Assuntos
Bovinos/metabolismo , Pâncreas/metabolismo , Receptores da Colecistocinina/metabolismo , Animais , Western Blotting , Imunofluorescência , Glucagon/isolamento & purificação , Imuno-Histoquímica , Insulina/isolamento & purificação , Ilhotas Pancreáticas/metabolismo , Queratinas/isolamento & purificação , Masculino , Pâncreas/citologia , Receptores da Colecistocinina/classificação , Somatostatina/isolamento & purificaçãoRESUMO
The sequence of somatostatin-14 (SS1) has been strongly preserved throughout the evolution of vertebrates from agnathans to mammals. In Acipenseridae (sturgeons), two isoforms of somatostatin have been characterized to date: somatostatin-14 has been identified from the gastrointestinal tract of the pallid sturgeon Scaphirhynchus albus and [Pro(2)]somatostatin-14 has been identified from the pituitary of the Russian sturgeon Acipenser gueldenstaedti. In the present study, we report the cloning of two distinct somatostatin cDNAs from the brain of the sturgeon Acipenser transmontanus. One of the cDNAs encodes a 116-amino acid protein (PSS1) that contains the SS1 sequence at its C-terminal extremity and, thus, is clearly orthologous to other vertebrate PSS1. The other cDNA encodes a 111-amino acid protein that contains the somatostatin variant [Pro(2)]somatostatin-14 at its C-terminal extremity. This second precursor exhibits more than 67% identity with the recently characterized lungfish PSS2 and goldfish PSS2. Reverse transcriptase-polymerase chain reaction analysis revealed that PSS1 is expressed in the central nervous system, the pancreas and the gut, whereas PSS2 is found in the central nervous system but not in the digestive system. In situ hybridization histochemistry showed that the PSS1 and PSS2 genes are differently expressed in numerous regions of the sturgeon brain. Interestingly, PSS1 and PSS2 mRNAs are present in the hypothalamus suggesting that, in sturgeon, both SS1 and SS2 may play hypophysiotropic functions. The PSS2 mRNA but not the PSS1 mRNA was found in the intermediate lobe of the pituitary. The present data demonstrate that two somatostatin genes are expressed in the sturgeon brain: one precursor generates somatostatin-14 and the other one gives rise to a [Pro(2)]somatostatin-14 variant, which is orthologous to goldfish, lungfish, and frog SS2.
Assuntos
Herança Multifatorial/genética , RNA Mensageiro/isolamento & purificação , Somatostatina/biossíntese , Somatostatina/genética , Sequência de Aminoácidos/genética , Animais , Química Encefálica/genética , Clonagem Molecular/métodos , Feminino , Peixes , Regulação da Expressão Gênica , Masculino , Dados de Sequência Molecular , RNA Mensageiro/biossíntese , Somatostatina/isolamento & purificaçãoRESUMO
Lanreotide was labelled with 188Re obtained from 188W/188Re generator, using stannous ion as reducing agent, ascorbic acid as stabilizers and hydroxy ethylidene bisphosphonate (HEDP) as intermediary ligand at different molar ratios, pH and incubation times. Best yields (>95%) were obtained using molar ratios SnF2/lanreotide, ascorbic/lanreotide and HEDP/lanreotide of 40, 12 and 260, respectively, pH 1-2 with an incubation at 100 degrees C for 30 min. Quality control evaluation and stability of the radiolabel compound was done by the following selected methods: chromatography in Whatman 3 MM with MEK and NaCl 0.15 M as solvents, ITLC-SG with ethanol-HCl 0.01N (90:10); reverse phase extraction cartridge (Sep-pak C18, Waters Associated) and RP-HPLC with radiometric and UV detection (220 nm) using MCH-5 n-capp column with linear gradient from 90% H2O (TFA 0.1%): 10% ACN (TFA 0.1%) up to 10% H2O (TFA 0.1%):90% ACN (TFA 0.1%) in 30 min, at flow 1 ml/min. Biodistribution in normal mice showed that 188Re-lanreotide is excreted mainly through the hepatobiliary system: more than 70% I.D. is present in gallbladder and intestines at 2 hr post injection. The stability of the 188Re-peptide bond by cysteine challenge test at 37 degrees C, during 2 and 24 hr of incubation time, reveals that approximately 300 and 100 molar ratio cys/peptide is required to displace 50% of the 188Re from the complex. In vitro stability of 188Re-lanreotide at room temperature (Rt) was demonstrated during 24 hr Future works must be done in order to investigate its binding capacity to somatostatin receptors.
Assuntos
Peptídeos Cíclicos/isolamento & purificação , Radioisótopos/isolamento & purificação , Compostos Radiofarmacêuticos/isolamento & purificação , Rênio/isolamento & purificação , Somatostatina/análogos & derivados , Somatostatina/isolamento & purificação , Animais , Camundongos , Peptídeos Cíclicos/farmacocinética , Peptídeos Cíclicos/normas , Controle de Qualidade , Radioisótopos/farmacocinética , Radioisótopos/normas , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/normas , Receptores de Somatostatina/metabolismo , Rênio/farmacocinética , Rênio/normas , Somatostatina/farmacocinética , Somatostatina/normas , Distribuição TecidualRESUMO
Lanreotide, a synthetic octapeptide analog of a native hormone somatostatin, was labeled with a commonly available, inexpensive radionuclide 99mTc. Labeling was accomplished by reduction of the cysteine bridge, which provided sulfhydryl groups for chelation with 99mTc. Stannous chloride was used as reducing agent, while tartrate acted as transchelating agent. Lanreotide (100 microg), stannous chloride dihydrate (100 microg) and tartaric acid (64 microg) were dissolved in acetate/acetic acid buffer (pH 2.8). After overnight (approximately 18 h) incubation, approximately 444 MBq (12 mCi) 99mTc was added and kept in boiling water for 30 min. More than 97% labeling efficiency was confirmed by RP-HPLC, ITLC-SG and C18 cartridge analysis. Radiolabeling results in one major peak when analyzed by reverse-phase HPLC. The stability of the 99mTc-peptide bond was evaluated by cysteine challenge studies.
Assuntos
Peptídeos Cíclicos/síntese química , Compostos Radiofarmacêuticos/síntese química , Somatostatina/síntese química , Cromatografia Líquida de Alta Pressão , Cisteína , Humanos , Peptídeos Cíclicos/isolamento & purificação , Compostos Radiofarmacêuticos/isolamento & purificação , Somatostatina/análogos & derivados , Somatostatina/isolamento & purificação , Tecnécio/isolamento & purificaçãoRESUMO
Insulin, glucagon, somatostatin-14, and three structurally related molecular forms of peptide tyrosine-tyrosine (PYY) were isolated from an extract of the combined pancreas and gastrointestinal tract of the pallid sturgeon, Scaphirhynchus albus. Pallid sturgeon insulin was identical to insulin from the Russian sturgeon, Acipenser guldenstaedti, and to insulin-2 from the paddlefish, Polyodon spathula, and was approximately twofold less potent than human insulin in inhibiting the binding of [3-[(125)I] iodotyrosine-A14] human insulin to the soluble human insulin receptor. The sturgeon glucagon (HSQGMFTNDY(10)-SKYLEEKLAQ(20) EFVEWLKNGK(30)S), like the two paddlefish glucagons, contains 31 rather than 29 amino acid residues, indicative of an anomalous pathway of posttranslational processing of proglucagon. Pallid sturgeon somatostatin, identical to human somatostatin-14, was also isolated in a second molecular form containing an oxidized tryptophan residue, but [Pro(2)]somatostatin-14, previously isolated from the pituitary of A. guldenstaedti, was not identified. Sturgeon PYY (FPPKPEHPGD(10)DAPAEDVAKY(20)YTALRHYINL(30) ITRQRY.HN(2)) was also isolated in variant forms containing the substitutions (Phe(1) --> Ala) and (Ala(18) --> Val), indicative of at least two gene duplications occurring within the Acipenseriformes lineage. The amino acid sequences of the pallidsturgeon PYY peptides are appreciably different from the proposed "ancestral" PYY sequence that has otherwise been very strongly conserved among the actinopterygian and elasmobranch fish.
Assuntos
Dipeptídeos/isolamento & purificação , Peixes , Glucagon/isolamento & purificação , Insulina/isolamento & purificação , Somatostatina/isolamento & purificação , Sequência de Aminoácidos , Animais , Ligação Competitiva , Cromatografia em Gel , Cromatografia Líquida de Alta Pressão , Sistema Digestório/química , Dipeptídeos/química , Dipeptídeos/metabolismo , Glucagon/química , Glucagon/metabolismo , Humanos , Insulina/química , Insulina/metabolismo , Dados de Sequência Molecular , Pâncreas/química , Receptor de Insulina/metabolismo , Homologia de Sequência , Somatostatina/química , Somatostatina/metabolismoRESUMO
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.
Assuntos
Apoptose/fisiologia , Ingestão de Alimentos/fisiologia , Mucosa Gástrica/fisiologia , Óxido Nítrico Sintase/metabolismo , Antro Pilórico/fisiologia , Animais , Feminino , Gastrinas/isolamento & purificação , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , NG-Nitroarginina Metil Éster/farmacologia , Óxido Nítrico Sintase/antagonistas & inibidores , Ratos , Ratos Wistar , Somatostatina/isolamento & purificação , InaniçãoRESUMO
A rapid method for the simultaneous assay of 7 peptide mixture, including angiotensin I, II, III, substance P, neurokinin, somatostatin and neurotensin, by high performance capillary electrophoresis has been established. The nature, pH and concentration of buffer, running voltage, detection wavelength, injection time and the effective length of amino-coated capillary were defined with the results of experiment. With 50 mmol/L ammonium acetate (pH 4.5) as running buffer and siphonage injection for 10 seconds, the measurements were carried out at 25 degrees C and 10 kV running voltage [(-)-->(+)] applied to a 57 cm x 75 microns i.d. (50 cm effective length) amino-coated capillary. The 7 peptide mixture was determined by a UV detector at 214 nm. The total time for separation and determination was within 8 min. The recoveries ranged from 95% to 98% with RSD from 2.9% to 4.2%. It has been found that the 75 microns i.d. capillary has higher sensitivity than 50 microns, but its efficiency and Rs were worse.
Assuntos
Angiotensina I/isolamento & purificação , Eletroforese Capilar/métodos , Peptídeos/isolamento & purificação , Substância P/isolamento & purificação , Angiotensina II/isolamento & purificação , Angiotensina III/isolamento & purificação , Neurocinina A/isolamento & purificação , Somatostatina/isolamento & purificaçãoRESUMO
The fruit-eating teleost fish, the pacu Piaractus mesopotamicus (Characiformes, Characidae) is classified along with the carp and the catfish in the superorder Ostariophysi. The pacu is able to survive and grow in captive conditions feeding exclusively on carbohydrates. Hormonal polypeptides in an extract of pacu Brockmann bodies were purified to homogeneity by reversed phase HPLC and their primary structures determined by automated Edman degradation. Pacu insulin contains only two substitutions, Glu-->Asp at A15 and Thr-->Ser at B24 (corresponding to B22 in mammalian insulins) compared with carp insulin. The B-chains of both insulins contain a dipeptide extension to the N-terminus and a deletion of the C-terminal residue compared with human insulin. Pacu glucagon differs from catfish glucagon by a single substitution at position 17 (Arg-->Gln. The primary structure of the 34 amino acid residue glucagon-like peptide (GLP) differs from catfish GLP only at positions 12 (Ser-->Ala) and 33 (Pro-->Gln). In common with other teleost species, the pacu expresses two somatostatin genes. Somatostatin-14, derived from preprosomatostatin-I (PSS-I), is identical to mammalian/catfish somatostatin-14. Although pacu somatostatin-II was not identified in this study, a peptide was purified that shows 67% sequence identity with residues (1-58) of catfish preprosomatostatin-II (PSS-II). This relatively high degree of sequence similarity contrasts with the fact that catfish PSS-II shows virtually no sequence identity with the corresponding PSS-II from anglerfish (Acanthopterygii) and trout (Protoacanthopterygii). A comparison of the primary structures of the islet hormones suggest that amino acid sequences may have been better conserved within the Ostariophysi than in other groups of the taxon Euteleostei that have been studied.
Assuntos
Peixes/metabolismo , Glucagon/química , Glucagon/isolamento & purificação , Insulina/química , Insulina/isolamento & purificação , Precursores de Proteínas/química , Precursores de Proteínas/isolamento & purificação , Somatostatina/química , Somatostatina/isolamento & purificação , Sequência de Aminoácidos , Aminoácidos/análise , Animais , Cromatografia Líquida de Alta Pressão , Feminino , Masculino , Dados de Sequência Molecular , Proglucagon , Homologia de Sequência de Aminoácidos , Fatores de TempoRESUMO
In recent years, peptides and proteins have received much attention as drug candidates. For many polypeptides, particularly hormones, it is desirable to release the drug continuously at a controlled rate over a period of weeks or even months, and thus a controlled release system is needed. Polylactic acid (PLA) is a biocompatible and biodegradable material with wide utility for many applications, including the design of controlled release systems for pharmaceutical agents. Pharmaceutical development of these delivery systems presents new problems in the area of stability assessment, especially for peptide drugs. In this study, we aimed to investigate the influence of different steps, during the manufacturing of an implant, on peptide stability in the polymeric matrix. Polylactic acid implants containing vapreotide, a somatostatin analogue, were prepared by extrusion. The effects of time, extrusion and temperature on the peptide stability were studied. The influence of various gamma sterilization doses, as well as the conditions under which the implants were irradiated, were also investigated. Peptide stability in the polymeric matrix was evaluated at various temperatures and at various time intervals up to 9 months.
Assuntos
Implantes Absorvíveis , Antineoplásicos/química , Raios gama , Peptídeos/química , Somatostatina/análogos & derivados , Antineoplásicos/síntese química , Antineoplásicos/isolamento & purificação , Preparações de Ação Retardada , Relação Dose-Resposta a Droga , Contaminação de Medicamentos , Sistemas de Liberação de Medicamentos , Polímeros/química , Somatostatina/síntese química , Somatostatina/química , Somatostatina/isolamento & purificação , Temperatura , Fatores de TempoRESUMO
Current views on Agnathan phylogeny favor the hypothesis that the genera of holarctic lampreys belong to a single family (Petromyzontidae) and form an interrelated progression in which Petromyzon is near to Ichthomyzon at the base of the phylogenetic tree and Lampetra is the most derived. A stock similar to that of contemporary Ichthomyzon is considered to have given rise to the southern hemisphere lamprey Geotria australis, the sole member of the Geotriidae. In the present study, two molecular forms of glucagon were isolated from an extract of G. australis intestine that differed in structure by six amino acid residues. One form shows two amino acid substitutions (Leu14 --> Met and Ala29 --> Ser) compared with the single molecular form of glucagon isolated from the sea lamprey Petromyzon marinus and the second form shows three substitutions (Asp15 --> Glu, Ser16 --> Ala, Ile24 --> Thr) compared with the single glucagon isolated from the river lamprey Lampetra fluviatilis. As Petromyzon and Lampetra glucagons differ by six amino acid residues, the data suggest that a duplication of the glucagon gene occurred prior to or early in lamprey evolution. Although both genes are strongly expressed in G. australis, the expression of one gene predominates in P. marinus while that of the other gene predominates in L. fluviatilis. Previous work has shown that, in the islet organ of G. australis, preprosomatostatin is processed almost exclusively to somatostatin-33. However, the present study demonstrates that somatostatin-14 is the major molecular form in G. australis intestine with somatostatin-33 present only as a minor component. This result demonstrates a tissue-dependent pathway of posttranslational processing of preprosomatostatin in the Geotria enteropancreatic system.