RESUMEN
Essentials Factor (F)VIII with an intermediate-length B-domain showed higher levels in murine gene therapy. FVIII with different B-domain lengths were analysed. FVIII variants with B-domains between 186 and 240 amino acids (aa) have extended half-life in mice. Reduced cell binding of FVIII with a 237aa B-domain may explain the extended half-life. SUMMARY: Background Factor VIII consists of the A1-domain, A2-domain, B-domain, A3-domain, C1-domain, and C2-domain. FVIII with an intermediate-length B-domain of 226 amino acids (aa) has previously been evaluated in murine gene therapy studies. Objective To characterize FVIII with intermediate-length B-domains in vitro and in vivo in F8-knockout (KO) mice. Methods and results FVIII molecules with B-domains of 186-240aa had longer half-lives in F8-KO mice than FVIII molecules with shorter or longer B-domains. FVIII with a B-domain containing the 225 N-terminal aa fused to the 12 C-terminal aa of the wild-type B-domain (FVIII-237) had a 1.6-fold extended half-life in F8-KO mice as compared with FVIII with a 21aa B-domain (FVIII-21). The in vitro and in vivo activity of FVIII-237 were comparable to those of FVIII-21, as was binding to von Willebrand factor. Cell binding to LDL receptor-related protein 1 (LRP-1)-expressing cells was markedly reduced for FVIII-237 as compared with FVIII-21, whereas the affinity for LRP-1 was not reduced in surface plasmon resonance (SPR) studies. FVIII-21 cell binding and internalization could be inhibited by a fragment consisting of the 226 N-terminal aa of the FVIII B-domain, and SPR analysis suggested that this B-domain fragment might bind with weak affinity to FVIII-21. Conclusion Reduced cell binding of FVIII-237 might explain the observed extended half-life in F8-KO mice. This may contribute to the increased FVIII levels measured in murine gene therapy studies using FVIII constructs with similar B-domain lengths.
Asunto(s)
Coagulantes/farmacocinética , Factor VIII/farmacocinética , Hemofilia A/tratamiento farmacológico , Animales , Línea Celular , Coagulantes/sangre , Modelos Animales de Enfermedad , Factor VIII/genética , Técnicas de Inactivación de Genes , Semivida , Hemofilia A/sangre , Proteína 1 Relacionada con Receptor de Lipoproteína de Baja Densidad/metabolismo , Ratones Noqueados , Unión Proteica , Dominios Proteicos , Proteínas Recombinantes/farmacocinéticaRESUMEN
N8, a new recombinant factor VIII (rFVIII) compound developed for the treatment of haemophilia A, is produced in Chinese hamster ovary (CHO) cells and formulated without human- or animal-derived materials. The aim of the present study was to compare the pharmacokinetics (PK) and the procoagulant effect, measured by ex vivo whole blood clot formation, of N8 and a commercial rFVIII in a cross-over study in haemophilia A dogs. N8 and Advate® (100 IU kg⻹) were administered intravenously to three haemophilia A dogs. Blood was sampled between 0 and 120 h postdose and FVIII:C analysed. PK parameters maximum plasma concentration, area under the curve, half-life (t(½)), clearance, mean residence time (MRT) and volume of distribution and incremental recovery were calculated. Whole blood clotting time (WBCT) and thromboelastography (TEG®) were used to determine the haemostatic potential. No adverse reactions were observed with N8 or Advate ®. N8 and Advate® exhibited similar PK parameters, with t(½) 7.7-11 h and MRT 11-14 h. Both rFVIII compounds corrected the prolonged WBCT (> 48 min) to the range of normal dogs (8-12 min), i.e. N8 to 7.5-10.5 min and Advate® to 7.5-11.5 min. N8 and Advate® also normalized the whole blood clot formation according to TEG®. The native whole blood clotting assays (WBCT, TEG®) appeared to be more sensitive to low concentrations of FVIII than assays in citrated plasma samples. In conclusion, comparison of N8 and Advate ® in haemophilia A dogs revealed similar safety, similar PK and similar effects in whole blood clot formation assays.
Asunto(s)
Coagulación Sanguínea/efectos de los fármacos , Factor VIII/farmacocinética , Hemofilia A/sangre , Proteínas Recombinantes/farmacocinética , Animales , Estudios Cruzados , Enfermedades de los Perros/sangre , Enfermedades de los Perros/tratamiento farmacológico , Perros , Evaluación Preclínica de Medicamentos/métodos , Femenino , Semivida , Hemofilia A/tratamiento farmacológico , Hemofilia A/inmunología , Hemofilia A/veterinaria , Hemostasis/efectos de los fármacos , Inyecciones Intravenosas , Masculino , Tasa de Depuración MetabólicaRESUMEN
SUMMARY: A new recombinant factor VIII (FVIII), N8, has been produced in Chinese hamster ovary (CHO) cells. The molecule consists of a heavy chain of 88 kDa including a 21 amino acid residue truncated B-domain and a light chain of 79 kDa. The two chains are held together by non-covalent interactions. The four-step purification includes capture, affinity purification using a monoclonal recombinant antibody, anion exchange chromatography and gel filtration. The specific clotting activity of N8 was 8800-9800 IU mg(-1). Sequence and mass spectrometry analysis revealed two variants of the light chain, corresponding to two alternative N-terminal sequences also known from plasma FVIII. Two variants of the heavy chain are present in the purified product, namely with and without the B-domain linker attached. This linker is removed upon thrombin activation of N8 rendering an activated FVIII (FVIIIa) molecule similar to plasma FVIIIa. All six known tyrosine sulphations of FVIII were confirmed in N8. Two N-linked glycosylations are present in the A3 and C1 domain of the light chain and two in the A1 domain of the heavy chain. The majority of the N-linked glycans are sialylated bi-antennary structures. An O-glycosylation site is present in the B-domain linker region. This site was glycosylated with a doubly sialylated GalNAc-Gal structure in approximately 65% of the product. In conclusion, the present data show that N8 is a pure and well-characterized FVIII product with biochemical properties that equal other FVIII products.
Asunto(s)
Factor VIII/química , Secuencia de Aminoácidos , Animales , Western Blotting , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Cricetinae , Factor VIII/aislamiento & purificación , Glicoproteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Tripsina/metabolismoRESUMEN
Peptides of the trefoil factor family (TFF) are expressed along the gastro-intestinal tract. They protect mucous epithelia from damage and contribute to mucosal repair, which is essential for preventing inflammation. Moreover, it has been suggested that TFF2 and TFF3, in particular, play a role in regulating immune responses. Depending on their activation status, dendritic cells (DC) can initiate either tolerance or immunity. This study, by comparing LPS-induced maturation of mTFF3-treated DC and non-treated DC, investigated whether murine TFF3 directly regulated DC function. mTFF3-treated DC and non-treated DC did not differ phenotypically or functionally. Both populations expressed, both before and after LPS-stimulation, similar levels of co-stimulatory molecules and cytokines, and were both efficient stimulators of T-cells. Our results suggest that mTFF3 does not govern immune responses on the level of DC function.
Asunto(s)
Células Dendríticas/inmunología , Lipopolisacáridos/metabolismo , Mucinas/metabolismo , Animales , Diferenciación Celular/inmunología , Ciclooxigenasa 2/biosíntesis , Citocinas/biosíntesis , Células Dendríticas/citología , Células Dendríticas/metabolismo , Femenino , Inmunidad Mucosa , Activación de Linfocitos/inmunología , Ratones , Mucinas/inmunología , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Linfocitos T/metabolismo , Factor Trefoil-3RESUMEN
BACKGROUND AND PURPOSE: Trefoil factors (TFFs) secreted by mucus-producing cells are essential for the defence of the gastrointestinal mucosa. TFFs probably influence the viscoelastic properties of mucus, but this has not been demonstrated in vivo. We therefore studied the gastric secretion of systemically administered TFF2 and TFF3, and their influence on the viscosity of the secretions. EXPERIMENTAL APPROACH: Mice and rats under general anaesthesia were injected intravenously with human (h) TFF2, hTFF3 (5 mg kg(-1) to mice and 25 mg kg(-1) to rats), murine (m) (125)I-TFF3, or (125)I-hTFF3 (300,000 cpm, mice only). The appearance of TFFs in the gastric mucosa and luminal secretions was analysed by autoradiography, gamma-counting, and ELISA, and the viscosity by rheometry. KEY RESULTS: (125)I-mTFF3 and (125)I-hTFF3 were taken up by secretory cells of the gastrointestinal tract and detected at the gastric mucosal surface 15 min after injection. Stressing the stomach by carbachol (3.5 microg kg(-1)) and pyloric ligation significantly increased the uptake. Injected hTFF2, hTFF3, and mTFF3 were retrieved from the gastric contents after 4 h. In rats, an approximately seven-fold increase in the viscosity was detected after injection of TFF2 compared to the controls, whereas TFF3 did not increase the viscosity. In mice, TFF2 increased the viscosity approximately 4-fold. CONCLUSIONS: These data indicate that systemically administered TFFs are transferred to the gastric lumen in a biologically active form.
Asunto(s)
Mucosa Gástrica/metabolismo , Contenido Digestivo/efectos de los fármacos , Péptidos/farmacología , Animales , Autorradiografía , Ensayo de Inmunoadsorción Enzimática , Femenino , Inmunohistoquímica , Hibridación in Situ , Inyecciones Intravenosas , Ratones , Ratones Endogámicos C57BL , Péptidos/metabolismo , Ratas , Ratas Wistar , Distribución Tisular , Factor Trefoil-2 , Viscosidad/efectos de los fármacosRESUMEN
Trefoil factor family (TFF) peptides have many in vivo and in vitro effects on restitution, wound healing, apoptosis, cell motility, adhesion and vectorial ion pumping, amongst others. (125)I-TFF peptides bind to cell membranes with classical saturable ability. It would be surprising if there were not TFF-protein interactions that would explain these actions, but to date no convincing TFF-binding partner has been shown which unambiguously takes part in any of these functions. Nevertheless, several TFF-binding proteins exist, including the small intestinal CRP-ductin (muclin), which binds TFF2, and the recently described gastric foveolar proteins TFIZ1 (TFF1-binding) and blottin (TFF2-binding), any of which may yet interact in novel ways to elicit TFF-mediated events. This review describes the expression and, where known, the functions of such proteins.
Asunto(s)
Péptidos/metabolismo , Proteínas/metabolismo , Animales , Proteínas de Unión al Calcio , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN , Humanos , Mucinas/metabolismo , Péptidos/genética , Unión Proteica , Proteínas/genética , Factor Trefoil-1 , Factor Trefoil-2 , Proteínas Supresoras de TumorRESUMEN
The present review will include the mammalian trefoil factors, TFF1, TFF2 and TFF3. It will summarise the amino acid sequences from different species, their posttranslational modifications and their structures determined by X-ray analysis and nuclear magnetic resonance studies. Trefoil factors all have a well-defined, structurally conserved trefoil domain. The trefoil domain consists of 42 or 43 amino acid residues and contains 6 cysteine residues that form disulphide bonds in a 1-5, 2-4 and 3-6 configuration. By the establishment of an additional intra-molecular disulphide bond at the C-terminal end, TFF1 and TFF3 form homodimers or heterodimers. This dimer formation of TFF1 and TFF3 will be discussed, and the possible implications for biological activity will be reviewed. The physicochemical characteristics including protease stability of trefoil factors will be summarised. The biological implications of different molecular forms of trefoil factors and their interaction with mucins will be discussed together with other functional insights.
Asunto(s)
Péptidos/química , Péptidos/fisiología , Secuencia de Aminoácidos , Animales , Humanos , Datos de Secuencia Molecular , Conformación Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Homología de Secuencia de Aminoácido , Factor Trefoil-2RESUMEN
BACKGROUND: The trefoil factors (TFF1-3) are cysteine-rich peptides expressed in the gastrointestinal tract where they play a critical role in mucosal protection and repair. The expression is up-regulated at sites of ulceration in various chronic inflammatory diseases. Recently, we presented an ELISA method for measurement of TFF3. The aims of the present study were to develop and evaluate ELISAs for the other two known human trefoil peptides, TFF1 and TFF2, and to carry out a cross-sectional study on serum TFF levels in patients with inflammatory bowel disease (IBD). METHODS: The TFF1-ELISA was based on two polyclonal rabbit antibodies and the TFF2-ELISA on a monoclonal mouse antibody and a polyclonal rabbit antibody. RhTFF1 and 2 were employed to prepare the calibrators. TFF1-3 were assayed in serum from IBD patients (n=41) and controls (n=13). RESULTS: The TFF1- (TFF2-) ELISA had a detection limit of 3 pmol/L (6 pmol/L) and an analytical imprecision (CV(A)) of 7.0-8.8 for mean concentrations of 24-120 pmol/L (6.1-8.0 for mean concentrations of 17-77 pmol/L). The central reference intervals (n=300) were 140-1400 pmol/L (37-190 pmol/L). There was no variation with age and menstrual cycle. Food intake reduced concentrations of TFF1 by approximately 15%, but did not influence concentrations of TFF2. TFF1 and TFF3 were increased in serum from IBD patients. CONCLUSIONS: We have developed assays for measuring TFF1 and TFF2. Finding increased TFF concentrations in serum from IBD patients suggests that measurements of trefoil peptides may be of clinical relevance in IBD.
Asunto(s)
Inmunoensayo/métodos , Enfermedades Inflamatorias del Intestino/sangre , Mucinas/sangre , Proteínas Musculares/sangre , Péptidos/sangre , Proteínas/análisis , Adolescente , Adulto , Anciano , Envejecimiento/sangre , Ensayo de Inmunoadsorción Enzimática , Femenino , Salud , Humanos , Masculino , Persona de Mediana Edad , Mucinas/inmunología , Proteínas Musculares/inmunología , Péptidos/inmunología , Proteínas/inmunología , Sensibilidad y Especificidad , Factor Trefoil-1 , Factor Trefoil-2 , Factor Trefoil-3 , Proteínas Supresoras de TumorRESUMEN
Peptides of the trefoil factor family (TFF1, TFF2 and TFF3) are co-secreted with mucus in most organ systems and are believed to interact with mucins to produce high-viscosity, stable gel complexes. We have previously demonstrated that cells in the GI tract possess binding sites to TFF2 and that injected TFF2 ends up in the mucus layer. In the present study, tissue binding and metabolism of parenterally administered human TFF1 and TFF3 in rats were described and compared to the immunohistochemical localization of the TFF peptides. 125I-TFF1 monomer and 125I-TFF3 mono- and dimer were given intravenously to female Wistar rats. The tissue distribution was assessed by gamma counting of organ samples and by autoradiography of histological sections. The degradation of 125I-TFF3 was studied by means of trichloracetic acid (TCA) precipitation and the saturability of the binding by administration of excess unlabelled peptide. The TFF peptides were localized in histologic sections from the GI tract by immunohistochemistry. Injected TFF3 dimer (12%) was taken up by the GI tract. At autoradiography, grains were localized to the same cells that were immunoreactive to TFF2. The binding could be displaced by excess TFF3. Similar binding was observed for the TFF1 and TFF3 monomers apart from binding in the stomach, where the uptake was only 15% in comparison to the dimer. There was no specific binding outside the GI tract and no binding to TFF1 or TFF3 immunoreactive cells. In conclusion, the TFF2-binding cells in the gastrointestinal tract seem to have basolateral, receptor-like activity to all three TFF peptides. The mucous neck cells of the stomach predominantly take up TFFs with two trefoil domains, indicating a different receptor-like activity in the stomach compared to the rest of the GI tract.
Asunto(s)
Sistema Digestivo/metabolismo , Inhibidores de Crecimiento/metabolismo , Mucinas/metabolismo , Proteínas Musculares/metabolismo , Neuropéptidos , Fragmentos de Péptidos/farmacología , Péptidos/metabolismo , Proteínas/metabolismo , Animales , Células Cultivadas , Estrógenos/metabolismo , Femenino , Inhibidores de Crecimiento/administración & dosificación , Humanos , Técnicas para Inmunoenzimas , Inyecciones Intravenosas , Radioisótopos de Yodo , Mucinas/administración & dosificación , Proteínas Musculares/administración & dosificación , Proteínas/administración & dosificación , Ratas , Ratas Wistar , Distribución Tisular , Factor Trefoil-1 , Factor Trefoil-2 , Factor Trefoil-3 , Proteínas Supresoras de TumorRESUMEN
BACKGROUND: Trefoil peptides (TFFs) are expressed and secreted in a tissue-specific manner in the gastrointestinal tract. Evidence of coexpression of trefoil peptides and mucins has been demonstrated in most mucus-producing cells in the gastrointestinal tract. The expression of trefoil peptides is up-regulated in gastric ulceration and colitis. It is believed that TFF peptides interact with mucin to increase viscosity but this has never been confirmed. The aims of the present study were to elucidate the direct effect of trefoil peptides on mucus gel formation. MATERIALS AND METHODS: The viscosity of mucin solutions was measured by means of a rotational rheometer after adding three mammalian trefoil peptides: TFF1, TFF2, and TFF3. RESULTS: Adding TFF2 (0.3%) to the mucin solutions (8%) resulted in more than a factor 10 increase in viscosity and elasticity, and the mucin solution was transformed into a gel-like structure with serpentine-like complexes between the mucin and TFF2. The dimer form of TFF3 also increased viscosity but resulted in a spider's web-like structure. The monomer forms of TFF1 and TFF3 had very little effect on the viscosity and elasticity of the mucin solutions. CONCLUSIONS: The addition of TFF2 to mucin solutions results in significantly increased viscosity and elasticity, under which the mucin solutions are transformed into a gel-like state. The ability of some trefoil peptides to catalyse the formation of stable mucin complexes may be one of the ways by which these peptides exert their protective and healing functions.
Asunto(s)
Sustancias de Crecimiento/farmacología , Mucinas/fisiología , Proteínas Musculares , Neuropéptidos , Péptidos/farmacología , Proteínas , Animales , Bovinos , Elasticidad/efectos de los fármacos , Geles , Sustancias de Crecimiento/análisis , Humanos , Inmunohistoquímica , Técnicas In Vitro , Péptidos/análisis , Reología , Esputo , Porcinos , Factor Trefoil-1 , Factor Trefoil-2 , Factor Trefoil-3 , Proteínas Supresoras de Tumor , Viscosidad/efectos de los fármacosRESUMEN
Several CART (cocaine- and amphetamine-regulated transcript) peptides have been identified in the brain. One peptide, rlCART 55-102, has been previously characterized in locomotor and feeding assays in rodents. The present study characterized the locomotor-stimulating effect of several additional CART peptides after intra-VTA administration in rats. The results confirm earlier findings that intra-VTA administration of rlCART 55-102 dose-dependently increased locomotor activity. Intra-VTA administration of rlCART 62-102 increased activity comparable with that produced by rlCART 55-102. However, intra-VTA administration of rsCART 10-89 and rlCART 55-59 did not increase motor activity. These results suggest that the portion of rlCART 55-102 responsible for increasing motor activity must reside within the portion of the protein also encompassed by rlCART 61-102, as the initial 5-amino acid sequence of rlCART 55-102 did not increase motor activity. In summary, rlCART 55-102 and rlCART 62-102 are behaviorally active in this locomotor assay, while rsCART 10-89 and rlCART 55-59 are not.
Asunto(s)
Actividad Motora/efectos de los fármacos , Proteínas del Tejido Nervioso/farmacología , Neurotransmisores/farmacología , Animales , Conducta Alimentaria/efectos de los fármacos , Inyecciones , Masculino , Proteínas del Tejido Nervioso/administración & dosificación , Proteínas del Tejido Nervioso/química , Neurotransmisores/administración & dosificación , Neurotransmisores/química , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Ratas , Ratas Sprague-Dawley , Relación Estructura-Actividad , Área Tegmental Ventral/efectos de los fármacos , Área Tegmental Ventral/fisiologíaRESUMEN
A process termed "restitution" enables rapid repair of the respiratory epithelium by migration of neighbouring cells. Mucin-associated TFF-peptides (formerly P-domain peptides or trefoil factors) are typical motogens enhancing migration of cells in various in vitro models mimicking restitution of the intestine. The human bronchial epithelial cell line BEAS-2B was used as a model system of airway restitution. The motogenic activities of recombinant human TFF2 as well as porcine TFF2 were demonstrated by in vitro wound healing assays of BEAS-2B cells. TFF2 did not induce phosphorylation of the epidermal growth factor (EGF) receptor. EGF was capable of enhancing the motogenic effect of human TFF2 at a concentration of 3 x 10(-10) M whereas EGF itself (i.e., in the absence of TFF2) did not stimulate migration at this low concentration. Furthermore, TFF2 as well as monomeric and dimeric forms of TFF3 enhanced migration of BEAS-2B cells in Boyden chambers. Motogenic activity of TFF2 was also shown for normal human bronchial epithelial (NHBE) cells in Boyden chambers. These results suggest that TFF-peptides act as motogens in the human respiratory epithelium triggering rapid repair of damaged mucosa in the course of airway diseases such as asthma.
Asunto(s)
Bronquios/citología , Sustancias de Crecimiento/farmacología , Mucinas , Proteínas Musculares , Neuropéptidos , Péptidos/farmacología , Proteínas/farmacología , Mucosa Respiratoria/citología , Bronquios/efectos de los fármacos , Técnicas de Cultivo de Célula/métodos , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/efectos de los fármacos , Receptores ErbB/metabolismo , Humanos , Mucosa Respiratoria/efectos de los fármacos , Factor Trefoil-2 , Factor Trefoil-3 , Heridas y Lesiones/patologíaRESUMEN
The secreted proteins intestinal trefoil factor (ITF, 59 residues), pS2 (60 residues), and spasmolytic polypeptide (SP, 106 residues) form a small family of trefoil domain-containing mammalian cell motility factors, which are essential for the maintenance of all mucous-coated epithelial surfaces. We have used 1H NMR spectroscopy to determine the high-resolution structure of human ITF, which has allowed detailed structural comparisons with the other trefoil cell motility factors. The conformation of residues 10-53 of hITF is determined to high precision, but the structure of the N- and C-terrminal residues is poorly defined by the NMR data, which is probably indicative of significant mobility. The core of the trefoil domain in hITF consists of a two-stranded antiparallel beta-sheet (Cys 36 to Asp 39 and Trp 47 to Lys 50), which is capped by an irregular loop and forms a central hairpin (loop 3). The beta-sheet is preceded by a short alpha-helix (Lys 29 to Arg 34), with the majority of the remainder of the domain contained in two loops formed from His 25 to Pro 28 (loop 2) and Ala 12 to Arg 18 (loop 1), which lie on either side of the central hairpin. The region formed by the surface of loop 2, the cleft between loop 2 and loop 3, and the adjacent face of loop 3 has previously been proposed to form the functional site of trefoil domains. Detailed comparisons of the backbone conformations and surface features of the family of trefoil cell motility factors (porcine SP, pS2, and hITF) have identified significant structural and electrostatic differences in the loop 2/loop 3 regions, which suggest that each trefoil protein has a specific target or group of target molecules.
Asunto(s)
Sustancias de Crecimiento/química , Mucinas , Proteínas Musculares , Neuropéptidos , Péptidos/química , Secuencia de Aminoácidos , Humanos , Intestinos/química , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Estructura Terciaria de Proteína , Alineación de Secuencia , Factor Trefoil-2 , Factor Trefoil-3RESUMEN
Recombinant activated coagulation factor VII (rFVIIa) (NovoSeven) was developed for treatment of bleeding in hemophilia patients with inhibitors (antibodies) against factors VIII or IX. rFVIIa initiates the coagulation cascade by binding to tissue factor at the site of injury and causes the formation of sufficient amounts of thrombin to trigger coagulation. Patients with a variety of other coagulation deficiencies than hemophilia characterized by an impaired thrombin generation and life-threatening bleeding have been reported as successfully treated with rFVIIa. Data are now entered into clinical registries established to further monitor this experimental treatment with NovoSeven. rFVIIa is produced free of any added human protein. The amino acid sequence of rFVIIa is identical to plasma-derived FVIIa (pdFVIIa). Posttranslational modifications (i.e., gamma-carboxylations, N- and O-glycosylations) are qualitatively identical in pdFVIIa and rFVIIa although some quantitative differences exist. The activities of rFVIIa and pdFVIIa are indistinguishable. Manufacturing of rFVIIa involves expression in baby hamster kidney (BHK) cells followed by purification, including three ion-exchange and one immunoaffinity chromatography steps. The last anion-exchange chromatography step ensures completion of the autoactivation of recombinant factor VII (rFVII) to rFVIIa. This review describes the mechanism of action, characterization, manufacturing, and preclinical and current clinical evidence for the efficacy and safety of rFVIIa.
Asunto(s)
Factor VII/química , Factor VII/farmacología , Microbiología Industrial/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Animales , Ensayos Clínicos como Asunto , Clonación Molecular , Factor VII/uso terapéutico , Factor VIIa , Humanos , Estructura Molecular , Proteínas Recombinantes/uso terapéutico , Transformación GenéticaRESUMEN
The trefoil factor TFF3 is a peptide predominantly produced by mucus-secreting cells in the small and large intestines. It has been implicated in intestinal protection and repair. The mechanisms that govern TFF3 secretion are poorly understood. The aim of this study was, therefore, to evaluate the influence of neurotransmitters, hormonal peptides and mediators of inflammation on the release of TFF3. For this purpose, an isolated vascularly perfused rat colon preparation was used. After a bolus administration of 1 ml isotonic saline into the lumen, TFF3 secretion was induced by a 30-min intra-arterial infusion of the compounds to be tested. TFF3 was evaluated in the luminal effluent using a newly developed radioimmunoassay. TFF3 was barely detected in crude luminal samples. In contrast, dithiothreitol (DTT) treatment of the effluent revealed TFF3 immunoreactivity, which amounted to about 0.3 pmol min(-1) cm(-1) in the basal state. Gel chromatography of DTT-treated luminal samples revealed a single peak that co-eluted with the monomeric form of TFF3. TFF3 was not detected in the portal effluent. Bethanechol (10(-6)-10(-4) M), vasoactive intestinal peptide (VIP, 10(-8)-10(-7) M) or bombesin (10(-8)-10(-7) M) induced a dose-dependent release of TFF3. In contrast, substance P evoked a modest release of TFF3, whereas calcitonin gene-related peptide (CGRP), somatostatin, neurotensin or peptide YY (PYY) did not modify TFF3 secretion. The degranulator compound bromolasalocid, 16,16-dimethyl PGE2 (dmPGE2) or interleukin-1-beta (IL-1-beta) also evoked a marked release of TFF3. In conclusion, TFF3 in the colonic effluent is present in a complex. This association presumably involves a disulfide bond. Additionally, the present results suggest a role for enteric nervous system and resident immune cells in mediation of colonic TFF3 secretion.
Asunto(s)
Colon/metabolismo , Lasalocido/análogos & derivados , Mucinas , Proteínas Musculares , Neuropéptidos/farmacología , Proteínas/metabolismo , 16,16-Dimetilprostaglandina E2/administración & dosificación , 16,16-Dimetilprostaglandina E2/farmacología , Animales , Betanecol/administración & dosificación , Betanecol/farmacología , Bombesina/administración & dosificación , Bombesina/farmacología , Colon/irrigación sanguínea , Colon/efectos de los fármacos , Infusiones Intraarteriales , Interleucina-1/administración & dosificación , Interleucina-1/farmacología , Lasalocido/administración & dosificación , Lasalocido/farmacología , Masculino , Neuropéptidos/administración & dosificación , Neurotransmisores/administración & dosificación , Neurotransmisores/farmacología , Péptidos , Perfusión , Proteínas/inmunología , Radioinmunoensayo , Ratas , Ratas Wistar , Factor Trefoil-3 , Péptido Intestinal Vasoactivo/administración & dosificación , Péptido Intestinal Vasoactivo/farmacologíaRESUMEN
Cocaine and amphetamine regulated transcript (CART) peptide has been shown to be an anorectic peptide that inhibits both normal and starvation-induced feeding and completely blocks the feeding response induced by neuropeptide Y and regulated by leptin in the hypothalamus. The C-terminal part containing the three disulfide bridges CART(48-89) is the biologically active part of the molecule affecting food intake. The solution structure of the active part of CART has a fold equivalent to other functionally distinct small proteins. CART consists mainly of turns and loops spanned by a compact framework composed by a few small stretches of antiparallel beta-sheet common to cystine knots.
Asunto(s)
Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/fisiología , Pliegue de Proteína , Secuencia de Aminoácidos , Cistina/química , Disulfuros/química , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , SolucionesRESUMEN
We have investigated the possible functional relationships between cellular invasion pathways induced by trefoil factors (TFFs), src, and the cyclooxygenases COX-1 and COX-2. Pharmacological inhibitors of the Rho small GTPase (C3 exoenzyme), phospholipase C (U-73122), cyclooxygenases (SC-560, NS-398), and the thromboxane A2 receptor (TXA2-R) antagonist SQ-295 completely abolished invasion induced by intestinal trefoil factor, pS2, and src in kidney and colonic epithelial cells MDCKts.src and PCmsrc. In contrast, invasion was induced by the TXA2-R mimetic U-46619, constitutively activated forms of the heterotrimeric G-proteins Galphaq (AGalphaq), Galpha12, Galpha13 (AGalpha12/13), which are signaling elements downstream of TXA2-R. Ectopic overexpression of pS2 cDNA and protein in MDCKts.src-pS2 cells and human colorectal cancer cells HCT8/S11-pS2 initiate distinct invasion signals that are Rho independent and COX and TXA2-R dependent. We detected a marked induction of COX-2 protein and accumulation of the stable PGH2/TXA2 metabolite TXB2 in the conditioned medium from cells transformed by src. This led to activation of the TXA2-R-dependent invasion pathway, which is monitored via a Rho- and Galpha12/Galpha13-independent mechanism using the Galphaq/PKC signaling cascade. These findings identify a new intracrine/paracrine loop that can be monitored by TFFs and src in inflammatory diseases and progression of colorectal cancers.
Asunto(s)
Sustancias de Crecimiento/farmacología , Mucinas , Proteínas Musculares , Neuropéptidos , Péptidos/farmacología , Prostaglandina-Endoperóxido Sintasas/metabolismo , Proteínas Proto-Oncogénicas pp60(c-src)/farmacología , Receptores de Tromboxanos/metabolismo , Línea Celular Transformada , Células Cultivadas , Medios de Cultivo Condicionados , Ciclooxigenasa 1 , Ciclooxigenasa 2 , ADN Complementario/genética , Proteínas de Unión al ADN/metabolismo , GTP Fosfohidrolasas/metabolismo , Subunidades alfa de la Proteína de Unión al GTP G12-G13 , Subunidades alfa de la Proteína de Unión al GTP Gq-G11 , Proteínas de Unión al GTP Heterotriméricas/metabolismo , Humanos , Mucosa Intestinal/citología , Mucosa Intestinal/enzimología , Isoenzimas/metabolismo , Riñón/citología , Riñón/enzimología , Proteínas de la Membrana , Invasividad Neoplásica , Proteínas/genética , Proteínas/farmacología , Transducción de Señal , Transfección , Factor Trefoil-1 , Factor Trefoil-2 , Factor Trefoil-3 , Células Tumorales Cultivadas , Proteínas Supresoras de Tumor , Fosfolipasas de Tipo C/metabolismoRESUMEN
Trefoil factors (TFFs) are protease-resistant peptides that promote epithelial cell migration and mucosal restitution during inflammatory conditions and wound healing in the gastrointestinal tract. To date, the molecular mechanism of TFFs action and their possible role in tumor progression are unclear. In the present study, we observed that premalignant human colonic PC/AA/C1 and canine kidney MDCK epithelial cells are not competent to invade collagen gels in response to exogenously added TFFs (pS2, spasmolytic polypeptide, and intestinal trefoil factor). In contrast, activated src and RhoA exert permissive induction of invasion by the TFFs that produce similar parallel dose-response curves in src-transformed MDCKts.src and PCmsrc cells (EC50=20-40 nM). Cell scattering is also induced by TFFs in MDCKts.src cells. Stable expression of the pS2 cDNA promotes constitutive invasiveness in MDCKts.src-pS2 cells and human colonic HCT8/S11-pS2 cells established from a sporadic tumor. Furthermore, we found that TFF-mediated cellular invasion is dependent of several signaling pathways implicated in cell transformation and survival, including phosphoinositide PI3'-kinase, phospholipase C, protein kinase C, and the rapamycin target TOR. Constitutive and intense expression of pS2 was revealed by Western blot analyses and immunohistochemistry in human colorectal tumors and their adjacent control mucosa during the neoplastic progression, from the adenoma to the liver metastases. Our studies indicated that TFFs can be involved in cell scattering and tumor invasion via autocrine loops and may serve as potential targets in the control of colon cancer progression.
Asunto(s)
Neoplasias Colorrectales/patología , Genes src , Sustancias de Crecimiento/farmacología , Mucinas , Proteínas Musculares , Invasividad Neoplásica , Neuropéptidos , Péptidos/farmacología , Proteína de Unión al GTP rhoA/fisiología , Animales , Línea Celular Transformada , Movimiento Celular , Colágeno , Neoplasias del Colon , Perros , Inhibidores Enzimáticos/farmacología , Humanos , Mucosa Intestinal/patología , Mucosa Intestinal/fisiopatología , Riñón , Lesiones Precancerosas , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Factor Trefoil-2 , Factor Trefoil-3 , Urotelio , Proteína de Unión al GTP rhoA/genéticaRESUMEN
The recombinant protein human trefoil factor 1 (hTFF1), formerly called hpS2, has been produced for the first time in a yeast-based expression in Pichia pastoris. hTFF1 was secreted in large amounts in the extracellular medium of P. pastoris under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter. The fermentation broth containing hTFF1 was concentrated by tangential flow filtration prior to purification by anion- and cation-exchange chromatography, followed by preparative high-performance liquid chromatography. The resulting hTFF1 was found to be intact by Western blot analysis. Further analysis revealed mainly the presence of the monomeric form of the hTFF1 peptide. Finally, in vitro, the recombinant hTFF1 was shown to decrease proliferation of the HCT116 cancer cells.
Asunto(s)
Proteínas/genética , Proteínas/aislamiento & purificación , Secuencia de Aminoácidos , División Celular/efectos de los fármacos , Línea Celular , Cromatografía Líquida de Alta Presión/métodos , Cromatografía por Intercambio Iónico/métodos , Clonación Molecular/métodos , Escherichia coli , Fermentación , Humanos , Espectrometría de Masas , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Pichia/crecimiento & desarrollo , Reacción en Cadena de la Polimerasa , Proteínas/química , Proteínas/farmacología , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Factor Trefoil-1 , Células Tumorales Cultivadas , Proteínas Supresoras de TumorRESUMEN
On the basis of histamine release from rat peritoneal mast cells, an octadecapeptide was isolated from the skin extract of the Northern Leopard frog (Rana pipiens). This peptide was purified to homogeneity using reversed-phase high performance liquid chromatography and found to have the following primary structure by Edman degradation and pyridylethylation: LVRGCWTKSYPPKPCFVR, in which Cys(5) and Cys(15) are disulfide bridged. The peptide was named peptide leucine-arginine (pLR), reflecting the N- and C-terminal residues. Molecular modeling predicted that pLR possessed a rigid tertiary loop structure with flexible end regions. pLR was synthesized and elicited rapid, noncytolytic histamine release that had a 2-fold greater potency when compared with one of the most active histamine-liberating peptides, namely melittin. pLR was able to permeabilize negatively charged unilamellar lipid vesicles but not neutral vesicles, a finding that was consistent with its nonhemolytic action. pLR inhibited the early development of granulocyte macrophage colonies from bone marrow stem cells but did not induce apoptosis of the end stage granulocytes, i.e. mature neutrophils. pLR therefore displays biological activity with both granulopoietic progenitor cells and mast cells and thus represents a novel bioactive peptide from frog skin.