Asunto(s)
Infecciones por Coronavirus/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Fitoterapia , Neumonía Viral/tratamiento farmacológico , Betacoronavirus , COVID-19 , China/epidemiología , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/metabolismo , Humanos , Medicina Tradicional China , Simulación del Acoplamiento Molecular , Pandemias , Neumonía Viral/epidemiología , Neumonía Viral/metabolismo , Mapas de Interacción de Proteínas , SARS-CoV-2 , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/metabolismo , Tratamiento Farmacológico de COVID-19RESUMEN
Hepatic fibrosis is overly exuberant wound healing in which excessive connective tissue builds up in the liver. The treatment of hepatic fibrosis is still difficult and remains a challenge to the clinician. In recent years, the TGF-ß signaling pathway regulator tyrosine kinase Abl has been raised as a new and promising target of hepatic fibrosis therapy. Here, considering that there are numerous drugs and drug-like compounds being approved or under clinical development and experimental investigation, it is expected that some of the existing drugs can be re-exploited as new agents to target Abl with the capability of suppressing hepatic fibrosis. To achieve this, a synthetic protocol that integrated molecular docking, affinity scoring dynamics simulation and free energy analysis was described to systematically profile the inhibitory potency of various drugs and drug-like compounds against the kinase domain of Abl. Consequently, 4 out of 13 tested drug candidates were successfully identified to have high-Abl inhibitory activities. By visually examining the dynamics behavior, structural basis and energetic property of few typical Abl-drug complex cases, a significantly different pattern of non-bonded interactions between the binding of active and inactive drug ligands to Abl receptor was revealed; the former is defined by strong, specific chemical forces, while the latter can only form non-specific hydrophobic contacts with slight atomic collisions.
Asunto(s)
Diseño de Fármacos , Inhibidores Enzimáticos/química , Simulación del Acoplamiento Molecular , Proteínas Proto-Oncogénicas c-abl/química , Proteínas Proto-Oncogénicas c-abl/ultraestructura , Sitios de Unión , Evaluación Preclínica de Medicamentos/métodos , Activación Enzimática , Humanos , Cirrosis Hepática/metabolismo , Cirrosis Hepática/prevención & control , Modelos Químicos , Unión Proteica , Conformación Proteica , Proteínas Proto-Oncogénicas c-abl/antagonistas & inhibidores , Factor de Crecimiento Transformador beta/químicaRESUMEN
The purpose of this study was to develop and validate a screening method based on scintillation probes for the simultaneous evaluation of in vivo growth factor release profiles of multiple implants in the same animal. First, we characterized the scintillation probes in a series of in vitro experiments to optimize the accuracy of the measurement setup. The scintillation probes were found to have a strong geometric dependence and experience saturation effects at high activities. In vitro simulation of 4 subcutaneous limb implants in a rat showed minimal interference of surrounding implants on local measurements at close to parallel positioning of the probes. These characteristics were taken into consideration for the design of the probe setup and in vivo experiment. The measurement setup was then validated in a rat subcutaneous implantation model using 4 different sustained release carriers loaded with (125)I-BMP-2 per animal. The implants were removed after 42 or 84 days of implantation, for comparison of the non-invasive method to ex vivo radioisotope counting. The non-invasive method demonstrated a good correlation with the ex vivo counting method at both time-points of all 4 carriers. Overall, this study showed that scintillation probes could be successfully used for paired measurement of 4 release profiles with minimal interference of the surrounding implants, and may find use as non-invasive screening tools for various drug delivery applications.
Asunto(s)
Implantes Absorbibles , Proteínas Morfogenéticas Óseas , Sistemas de Liberación de Medicamentos/métodos , Fémur , Factor de Crecimiento Transformador beta , Animales , Materiales Biocompatibles/química , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/farmacocinética , Proteínas Morfogenéticas Óseas/farmacología , Preparaciones de Acción Retardada , Evaluación Preclínica de Medicamentos/instrumentación , Evaluación Preclínica de Medicamentos/métodos , Fémur/efectos de los fármacos , Fémur/metabolismo , Cámaras gamma , Humanos , Radioisótopos de Yodo , Masculino , Ratas , Ratas Endogámicas , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacocinética , Proteínas Recombinantes/farmacología , Distribución Tisular , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/farmacocinética , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
The bone morphogenetic proteins (BMPs) constitute a subfamily of the transforming growth factor type beta (TGF-beta) supergene family. BMP-2 plays an important role not only in osteoblast differentiation but also in pattern formation during development. To determine the function of BMP-2 in Pinctada fucata development and hard tissue formation, we isolated a BMP-2 genomic DNA clone and the BMP-2 cDNA. The deduced BMP-2 sequence consisted of 447 amino acids. The BMP-2 gene was composed of three exons. The C-terminal portion (149 amino acids) had 86% and 66% identity to the Crassostrea gigas and the human BMP-2 sequence respectively. The 5' flanking promoter region contained putative glioma transcription factor (Gli) and retinoic acid receptor (RAR) responding elements. The BMP-2 gene was expressed strongly in the inner part of the mantle tissue, corresponding to the nacreous aragonite shell layer. This finding suggests that BMP-2 has a key role in nacreous layer formation.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Moluscos/genética , Animales , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/metabolismo , Clonación Molecular , Cartilla de ADN , ADN Complementario/genética , Biblioteca Genómica , Humanos , Intrones , Moluscos/metabolismo , Músculo Esquelético/fisiología , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/genéticaRESUMEN
An anodic spark deposition process was used for preparation of inorganic, glass-ceramic like conversion coatings. The microstructure of the layers was characterized by surface and solid state techniques such as scanning electron microscopy, electron probe microanalysis and Raman spectroscopy. The porous coatings, typically up to 8 mum thick, consist mainly of titanium oxides and amounts of incorporated electrolyte constituents like Se, Ca or P. Beside nano crystalline anatase phases, a mostly amorphous structure is proposed in which network-forming [PO(4)] tetrahedras and [TiO(6)] octahedras in various degrees of condensation are connected. A drastic modification of the film structure was observed when selenium was incorporated into the glassy oxide structure of the coating. In these cases no nano crystalline phases of titanium oxides or other chemical compounds were detected. First cell culture investigations show a significant improvement of the biological properties. Cell proliferation and TGF-beta-expression of these coatings in comparison with commercial pure titanium (CPT) with native titanium oxide films were examined.
Asunto(s)
Materiales Biocompatibles/química , Materiales Biocompatibles Revestidos/química , Selenio/química , Titanio/química , Proliferación Celular , Electrólitos , Humanos , Ensayo de Materiales , Microscopía Electrónica de Rastreo , Nanopartículas/química , Osteoblastos/metabolismo , Espectrometría Raman , Propiedades de Superficie , Factor de Crecimiento Transformador beta/químicaRESUMEN
Calcium hydroxide (Ca(OH)(2)) has been used extensively to induce dentine regeneration through formation of dentine bridges at sites of pulp exposure after dental tissue injury, however, the biological processes underpinning these events are unclear. We hypothesise that growth factors and other bio-active molecules, sequestered within dentine matrix, may be released by the action of Ca(OH)(2) and signal gene expression in pulp cells, which mediates the changes in cell behaviour observed during regeneration. Powdered sound, human dentine samples were extracted with either 0.02 m Ca(OH)(2), pH 11.7 or 10% EDTA, pH 7.2 ( a control known extractant of bio-active and other ECM molecules from dentine) over a 14-day period. Extracts were compared for non-collagenous protein (NCP) and glycosaminoglycan (GAG) content using dye binding assays and protein compositions were analysed by 1D-polyacrylamide gel electrophoresis (1D-PAGE) and TGF-beta1 ELISA. The effects of extracts on TGF-beta1, Collagen-1alpha and Nestin gene expression were analysed using semi-quantitative RT-PCR in the dental MDPC-23, OD-21 and fibroblastic Swiss 3T3 cell lines following 24h of exposure. Ca(OH)(2) solubilised NCPs and GAGs from the dentine ECM, although with a lower yield than the EDTA solution and with different kinetics. 1D-PAGE analysis demonstrated some differences in profiles for proteins solubilised from dentine by Ca(OH)(2) and EDTA. Both solutions released TGF-beta1 from the dentine with higher concentrations present in the EDTA (1.395 +/- 0.036 ng/mg) versus the Ca(OH)(2) (0.364 +/- 0.012 ng/mg) extract. Notably, both extracts induced similar gene expression profiles in all cell lines. These data provide a rational explanation for the action of Ca(OH)(2) during pulp capping in which the cellular activities involved in dentine bridge formation may be mediated through release of growth factors and other bio-active molecules from the dentine by Ca(OH)(2).
Asunto(s)
Hidróxido de Calcio/química , Dentina/química , Secuencia de Bases , Cartilla de ADN , ADN Complementario , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Expresión Génica , Humanos , Concentración de Iones de Hidrógeno , Proteínas/química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Solubilidad , Factor de Crecimiento Transformador beta/químicaRESUMEN
The chondroitin/dermatan sulfate proteoglycans (CS/DSPGs), biglycan, decorin, and versican play several important roles in extracellular matrix influencing matrix organization, cell proliferation, and recruitment. Moreover, they bind and regulate growth factors in the extracellular matrix. We have previously shown that cultured human lung fibroblasts treated with transforming growth factor-beta (TGF-beta) alone or in combination with epidermal growth factor and platelet-derived growth factor, increase the production of these PGs. In this report, we describe that the structure of their galactosaminoglycan side chains is altered, albeit there is no alteration of polysaccharide length. The findings showed that iduronic acid content is reduced by 50% in decorin and biglycan, whereas 4-O-sulfation is increased 2-fold in versican. To unravel the mechanism behind these changes, the activities of chondroitin C-5 epimerase and of O-sulfotransferases in cellular fractions prepared from fibroblasts were quantitated, and transcript levels of the relevant sulfotransferases were measured by real time polymerase chain reaction (RT-PCR). The C-5 epimerase activity was reduced by 25% in TGF-beta1 treated cells and 50% in fibroblasts treated with the growth factor combination. No change in activity in dermatan 4-O sulfotransferase was observed, and only a minor decrease in dermatan 4-O-sulfotransferase-1 (D4ST-1) mRNA was observed. On the other hand, chondroitin 4-O sulfotransferase activity increased 2-fold upon TGF-beta1 treatment and 3-fold upon treatment with the growth factor combination. This is in agreement with a 2-fold up-regulation of chondroitin-4-O-sulfotransferase 1 (C4ST-1) mRNA, and no changes in chondroitin-4-O-sulfotransferase 2 (C4ST-2) mRNA. Thus, cellular activity and transcript level correlated well with the changes in the structure of the dermatan/chondroitin sulfate chains.
Asunto(s)
Condroitín/química , Citocinas/metabolismo , Dermatán Sulfato/química , Regulación de la Expresión Génica , Factor de Crecimiento Transformador beta/metabolismo , Carbohidrato Epimerasas/química , Línea Celular , Células Cultivadas , Sulfatos de Condroitina/química , Cartilla de ADN/química , ADN Complementario/metabolismo , Decorina , Disacáridos/química , Factor de Crecimiento Epidérmico/metabolismo , Matriz Extracelular/metabolismo , Proteínas de la Matriz Extracelular/química , Fibroblastos/metabolismo , Humanos , Ácido Idurónico/química , Microsomas/metabolismo , Polímeros/química , Polisacáridos/química , Proteoglicanos/química , ARN/química , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sulfotransferasas/química , Sulfotransferasas/metabolismo , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta1 , Regulación hacia ArribaRESUMEN
This study investigated a novel drug delivery system (DDS), consisting of polycaprolactone (PCL) or polycaprolactone 20% tricalcium phosphate (PCL-TCP) biodegradable scaffolds, fibrin Tisseel sealant and recombinant bone morphogenetic protein-2 (rhBMP-2) for bone regeneration. PCL and PCL-TCP-fibrin composites displayed a loading efficiency of 70% and 43%, respectively. Fluorescence and scanning electron microscopy revealed sparse clumps of rhBMP-2 particles, non-uniformly distributed on the rods' surface of PCL-fibrin composites. In contrast, individual rhBMP-2 particles were evident and uniformly distributed on the rods' surface of the PCL-TCP-fibrin composites. PCL-fibrin composites loaded with 10 and 20 microg/ml rhBMP-2 demonstrated a triphasic release profile as quantified by an enzyme-linked immunosorbent assay (ELISA). This consisted of burst releases at 2 h, and days 7 and 16. A biphasic release profile was observed for PCL-TCP-fibrin composites loaded with 10 microg/ml rhBMP-2, consisting of burst releases at 2 h and day 14. PCL-TCP-fibrin composites loaded with 20 microg/ml rhBMP-2 showed a tri-phasic release profile, consisting of burst releases at 2 h, and days 10 and 21. We conclude that the addition of TCP caused a delay in rhBMP-2 release. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and alkaline phosphatase assay verified the stability and bioactivity of eluted rhBMP-2 at all time points.
Asunto(s)
Líquidos Corporales/química , Proteínas Morfogenéticas Óseas/administración & dosificación , Sustitutos de Huesos/administración & dosificación , Fosfatos de Calcio/química , Implantes de Medicamentos/administración & dosificación , Adhesivo de Tejido de Fibrina/administración & dosificación , Poliésteres/química , Factor de Crecimiento Transformador beta/administración & dosificación , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/química , Materiales Biocompatibles Revestidos/administración & dosificación , Materiales Biocompatibles Revestidos/química , Difusión , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos , Implantes de Medicamentos/química , Adhesivo de Tejido de Fibrina/química , Ensayo de Materiales , Factor de Crecimiento Transformador beta/químicaRESUMEN
Bone morphogenetic protein (BMP)-2 has a critical role in bone formation and regeneration. Therefore, the ability to immobilize this molecule in certain matrices may be crucial in bone tissue engineering. Using carbodiimide chemistry, BMP-2 was directly immobilized on silk fibroin films. Whereas human bone marrow stromal cells cultured on unmodified silk fibroin films in the presence of osteogenic stimulants exhibited little if any osteogenesis, the same cells cultured on BMP-2 decorated films in the presence of osteogenic stimulants differentiated into an osteoblastic lineage as assessed by their significantly elevated alkaline phosphatase activity, calcium deposition, and higher transcript levels of collagen type I, bone sialoprotein, osteopontin, osteocalcin, BMP-2, and cbfa1. Using cell culture inserts, it was demonstrated that differentiation was induced by the immobilized protein and not by protein released into the culture medium. Comparison with a similar amount of medium-supplemented BMP-2, where no additional protein was added with medium changes, showed that delivery of BMP-2 immobilized on the biomaterial surface was more efficient than soluble delivery. The results illustrate that BMP-2 covalently coupled on silk biomaterial matrices retains biological function in vitro based on the induction of osteogenic markers in seeded bone marrow stromal cells.
Asunto(s)
Células de la Médula Ósea/efectos de los fármacos , Proteínas Morfogenéticas Óseas/química , Proteínas Morfogenéticas Óseas/farmacología , Diferenciación Celular/efectos de los fármacos , Fibroínas/química , Osteogénesis/efectos de los fármacos , Seda/química , Células del Estroma/efectos de los fármacos , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/farmacología , Fosfatasa Alcalina/metabolismo , Animales , Bombyx , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/administración & dosificación , Tampones (Química) , Calcio/metabolismo , Células Cultivadas , Humanos , Masculino , Soluciones , Células del Estroma/citología , Células del Estroma/metabolismo , Transcripción Genética/efectos de los fármacos , Transcripción Genética/genética , Factor de Crecimiento Transformador beta/administración & dosificaciónRESUMEN
Matrix gamma-carboxyglutamic acid protein (MGP) is a member of the vitamin K-dependent protein family with unique structural and physical properties. MGP has been shown to be an inhibitor of arterial wall and cartilage calcification. One inhibitory mechanism is thought to be binding of bone morphogenetic protein-2. Binding has been shown to be dependent upon the vitamin K-dependent gamma-carboxylation modification of MGP. Since MGP is an insoluble matrix protein, this work has focused on intracellular processing and transport of MGP to become an extracellular binding protein for bone morphogenetic protein-2. Human vascular smooth muscle cells (VSMCs) were infected with an adenovirus carrying the MGP construct, which produced non-gamma-carboxylated MGP and fully gamma-carboxylated MGP. Both forms of MGP were found in the cytosolic and microsomal fractions obtained from the cells by differential centrifugation. The crude microsomal fraction was shown to contain an additional, more acidic Ser-phosphorylated form of MGP believed to be the product of Golgi casein kinase. The data suggest that phosphorylation of MGP dictates different transport routes for MGP in VSMCs. A proteomic approach failed to identify a larger soluble precursor of MGP or an intracellular carrier protein for MGP. Evidence is presented for a receptor-mediated uptake mechanism for fetuin by cultured human VSMCs. Fetuin, shown by mass spectrometry not to contain MGP, was found to be recognized by anti-MGP antibodies. Fetuin uptake and secretion by proliferating and differentiating cells at sites of calcification in the arterial wall may represent an additional protective mechanism against arterial calcification.
Asunto(s)
Proteínas Morfogenéticas Óseas/química , Proteínas de Unión al Calcio/química , Proteínas de la Matriz Extracelular/química , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Factor de Crecimiento Transformador beta/química , Adenoviridae/genética , Arterias/metabolismo , Transporte Biológico , Biotina/química , Biotina/metabolismo , Western Blotting , Proteína Morfogenética Ósea 2 , Proteínas Morfogenéticas Óseas/metabolismo , Caseína Quinasas/metabolismo , Células Cultivadas , Medio de Cultivo Libre de Suero/metabolismo , Citosol/metabolismo , ADN Complementario/metabolismo , Electroforesis en Gel de Poliacrilamida , Endocitosis , Aparato de Golgi/metabolismo , Humanos , Inmunohistoquímica , Inmunoprecipitación , Espectrometría de Masas , Microscopía Confocal , Microsomas/metabolismo , Fosforilación , Transporte de Proteínas , Fracciones Subcelulares/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , alfa-Fetoproteínas/biosíntesis , alfa-Fetoproteínas/química , Proteína Gla de la MatrizRESUMEN
A new hypothesis of amino acid complementarity based on the genetic code periodicity is presented and evaluated on the peptide pairs composed of the fragments of TGF-beta(2) protein (YIGKTPKI and YYIGKTPKIE) and corresponding complementary peptides [IYPLC(Acm)GLY, IIYTLWGLYL, IIYPLC(Acm)GLYL and IIYTLC(Acm)GLYL]. The ESI-MS and CD methods were used for monitoring of the complexation. It was found that heterodimeric structures are formed between the peptides and complementary peptides. No complexation appears in solutions of single components of the systems, nor in solutions containing the mixtures of TGF-beta(2) peptides or complementary peptides. CD measurements suggest that the conformation of peptides needed for complex formation is of the beta-structure type. The binding forces, which stabilize the complexes, consist mainly of hydrophobic interactions.
Asunto(s)
Aminoácidos/química , Péptidos/química , Factor de Crecimiento Transformador beta/química , Aminoácidos/genética , Secuencia de Bases , Dimerización , Datos de Secuencia Molecular , ARN , Espectrometría de Masa por Ionización de Electrospray/métodos , Factor de Crecimiento Transformador beta/genética , Factor de Crecimiento Transformador beta2RESUMEN
Porcine colostrum and milk collected at different stages of lactation were assessed for transforming growth factor-beta (TGF-beta) activity using an epithelial cell bio-assay. A high level of TGF-beta activity was recorded in all colostrum samples after transient acidification treatment, ranging between 126 and 260 ng/ml for samples collected at the time of parturition and 73 ng/ml for the sample collected 12 h after parturition. Without transient acidification treatment TGF-beta activity was detected only in 2 samples collected at the time of parturition (12 ng/ml) and 12 h after parturition (9 ng/ml), respectively. TGF-beta activity was undetectable in milk collected 5 days after parturition. These results suggest that TGF-beta exists mainly in latent form in porcine colostrum and the concentration declines rapidly as lactation proceeds. After enrichment with cation exchange chromatography a low level of TGF-beta activity was detected in porcine milk. Further separation by size exclusion chromatography revealed two molecular mass forms of TGF-beta in both colostrum and milk samples, a major peak of about 80 kD and a minor peak of about 25 kD representing latent and active forms of TGF-beta, respectively. A further experiment showed that the latent form of TGF-beta in colostrum can be activated at pH 3.5 or less. It is speculated that TGF-beta found in the colostrum may play a physiological role in regulating postnatal adaptation of the gastro-intestinal tract in newborns.
Asunto(s)
Calostro/química , Porcinos , Factor de Crecimiento Transformador beta/análisis , Animales , Cromatografía por Intercambio Iónico , Femenino , Concentración de Iones de Hidrógeno , Cinética , Trabajo de Parto/metabolismo , Lactancia , Leche/química , Peso Molecular , Embarazo , Valores de Referencia , Factor de Crecimiento Transformador beta/químicaRESUMEN
Asbestosis is characterized by increased collagen deposition along the walls of terminal respiratory bronchioles that extends into the alveolar ducts and septae. Alveolar macrophages are activated and release growth factors that stimulate mesenchymal cell proliferation and enhanced formation of extracellular matrix. Both insulin-like growth factor-I (IGF-I), and transforming growth factor beta (TGF-beta) regulate cellular growth and promote matrix accumulation and are hypothesized to play important roles in asbestosis. We performed immunohistochemistry using polyclonal antibodies to specific synthetic peptides of the three mammalian isoforms of TGF-beta (TGF-beta 1, -beta 2, -beta 3) and to IGF-I on lungs of sheep treated intratracheally with chrysotile asbestos. All three TGF-beta isoforms were found in bronchial and bronchiolar epithelium, macrophages, and bronchial and vascular smooth muscle in control lungs. The distribution of TGF-beta was increased in these lung constituents as fibrotic lesions developed. Fibrotic lesions additionally demonstrated intense immunostaining of all three TGF-beta isoforms that localized to the extracellular matrix zones with little staining of interstitial cells. In the control sheep lungs, IGF-I staining was detected in bronchial and bronchiolar epithelium, bronchial glands, bronchial and vascular smooth muscle, endothelium, and macrophages. IGF-I immunostaining was detected in macrophages in peribronchial fibrosis and in fibroblasts along the periphery of and within lesions, but not in the extracellular matrix. Metaplastic proliferating epithelium and macrophages were strongly immunoreactive for IGF-I in advanced lesions. Our data demonstrate different immunostaining patterns for IGF-I and TGF-beta in asbestosis, with IGF-I in the cellular periphery and TGF-beta in the extracellular matrix consistent with a complementary role in stimulating interstitial fibroblast proliferation and new collagen deposition in areas of active fibrosis.
Asunto(s)
Asbestos Serpentinas/efectos adversos , Asbestosis/patología , Factor I del Crecimiento Similar a la Insulina/química , Factor de Crecimiento Transformador beta/química , Animales , Asbestos Serpentinas/química , Asbestosis/etiología , Asbestosis/metabolismo , Modelos Animales de Enfermedad , Inmunohistoquímica , Factor I del Crecimiento Similar a la Insulina/fisiología , Fibrosis Pulmonar/etiología , Fibrosis Pulmonar/metabolismo , Fibrosis Pulmonar/patología , Ovinos , Factor de Crecimiento Transformador beta/fisiologíaRESUMEN
Continuing our research on the immunomodulatory activity of the peptides related to the proteins of TGF-beta family, we have synthesized and investigated by Jerne's plaque forming cell (PFC) test and delayed type hypersensitivity (DTH) test the following linear peptides and their cyclic (disulphide-bridged) analogues: Lys-Thr-Pro-Lys-Ile (Ia) and Cys-Lys-Thr-Pro-Lys-Ile-Cys (Ib), Tyr-Ile-Gly-Lys-Thr-Pro (IIIa) and Cys-Tyr-Ile-Gly-Lys-Thr-Pro-Cys (IIIb), Tyr-Ile-Gly-Lys-Thr-Pro-Lys-Ile (IVa) and Cys-Tyr-Ile-Gly-Lys-Thr-Pro-Lys-Ile-Cys (IVb), Tyr-Tyr-Ile-Gly-Lys-Thr-Pro-Lys-Ile-Glu (Va) and Cys-Tyr-Tyr-Ile-Gly-Lys-Thr-Pro-Lys-Ile-Glu-Cys (Vb). It has been found that the insertion of the sequence of Ia into the disulphide bridged Ib generates a product with enhanced immunosuppressive activity. The similar procedure applied to IIIa, IVa and Va produces cyclic peptides devoid of any activity or of a diminished activity in DTH test. The exchange of Pro residue of Ia D-Pro resulted in the analogue IIa with a preserved immunosuppressive activity. The disulphide-bridged peptide IIb, a cyclic analogue of IIa, exhibited the same activity as IIa in both: PFC and DTH tests.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Fragmentos de Péptidos/farmacología , Péptidos Cíclicos/farmacología , Factor de Crecimiento Transformador beta/química , Secuencia de Aminoácidos , Animales , Cistina/química , Técnica de Placa Hemolítica , Ratones , Ratones Endogámicos CBA , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Péptidos Cíclicos/química , Conformación Proteica , Relación Estructura-ActividadRESUMEN
The formation of a non-covalent complex between mature transforming growth factor beta 1 (TGF-beta 1) and its pro region, the beta 1-latency-associated peptide (beta 1-LAP), is important in regulating the activity of this multipotent growth factor. We have overexpressed simian beta 1-LAP in Chinese hamster ovary (CHO) cells to produce a cell line which secretes beta 1-LAP into the culture medium at > 1 mg/l, thus enabling structural studies of complex formation between beta 1-LAP and TGF-beta 1. The simian beta 1-LAP expressed in CHO cells reversed the growth inhibitory effect of exogenous TGF-beta 1 on Mv1Lu (mink lung epithelial) cells and was able to form a cross-linked complex with 125I-TGF-beta 1. Simian beta 1-LAP was purified to homogeneity by a combination of ammonium sulphate precipitation, gel filtration, dye ligand chromatography and anion-exchange chromatography, with a yield of 15%. The purified protein had an apparent molecular mass of 114 kDa as determined by SDS/PAGE, which is greater than that determined for the transient expression of simian beta 1-LAP in COS-1 and for the simian precursor of TGF-beta 1 (pro-TGF-beta 1) in CHO cells, this major difference being due to more extensive glycosylation of beta 1-LAP expressed by this CHO clone. Far-UV CD spectroscopy of simian beta 1-LAP indicates a mostly beta-sheet structure, with extensive structural rearrangements occurring upon formation of the latent complex between TGF-beta 1 and beta 1-LAP.
Asunto(s)
Fragmentos de Péptidos , Precursores de Proteínas , Proteínas/aislamiento & purificación , Proteínas/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Células CHO/metabolismo , Carbohidratos/análisis , División Celular/efectos de los fármacos , Línea Celular , Precipitación Química , Cromatografía por Intercambio Iónico , Dicroismo Circular , Cricetinae , ADN Complementario/genética , Amplificación de Genes , Pulmón/citología , Pulmón/efectos de los fármacos , Manosafosfatos/análisis , Visón , Estructura Secundaria de Proteína , Proteínas/fisiología , Tetrahidrofolato Deshidrogenasa/genética , Factor de Crecimiento Transformador beta/química , Factor de Crecimiento Transformador beta/farmacología , Factor de Crecimiento Transformador beta1RESUMEN
The examination of immunomodulatory properties of oligopeptides derived from two exposed loops (containing thymopentin-like and tuftsin-like sequences, respectively) of the proteins belonging to TGF beta family suggests that the particular species of the TGF beta family should differ distinctly in their influence on the immune response. According to our results obtained from three TGF beta species of mammals, TGF beta 2 should be a strong immunosuppressor, whereas for TGF beta 3 the immunostimulative potency is more probable. TGF beta 1 species would possess both immunosuppressive and immunostimulative potency, residing in two different loops of the protein. The results obtained also suggest that chicken TGF beta 4 should be associated with immunostimulative effects and xenopus TGF beta 5 with immunosuppressive ones.
Asunto(s)
Adyuvantes Inmunológicos/farmacología , Inmunosupresores/farmacología , Oligopéptidos/farmacología , Factor de Crecimiento Transformador beta/química , Adyuvantes Inmunológicos/química , Adyuvantes Inmunológicos/metabolismo , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Técnica de Placa Hemolítica , Inmunosupresores/química , Inmunosupresores/inmunología , Inmunosupresores/metabolismo , Ratones , Datos de Secuencia Molecular , Oligopéptidos/química , Oligopéptidos/inmunología , Oligopéptidos/metabolismo , Fragmentos de Péptidos , Timopentina/química , Factor de Crecimiento Transformador beta/inmunología , Factor de Crecimiento Transformador beta/metabolismo , Tuftsina/químicaRESUMEN
Transforming growth factor-beta 1 (TGF-beta 1) is a homodimeric protein stabilized by a single disulfide bridge between Cys77 on the respective monomers and two paired complementary hydrophobic interfaces between the two subunits. A TGF-beta 1 mutant with Cys77 replaced by serine has been expressed in stably transfected Chinese hamster ovary cells and purified to homogeneity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirms that the sole interchain disulfide bond in TGF-beta 1 has been eliminated. It is 20% as potent as native TGF-beta 1 in the induction of plasminogen activator inhibitor-1 promoter expression in mink lung epithelial cells (Mv1Lu), although it is less than 1% as potent as native TGF-beta 1 in inhibition of growth in the same cell line. The mutant acts as a full agonist in both bioassays. [Ser77]TGF-beta 1 binds to soluble type II receptors and competes with native TGF-beta 1 in sandwich-enzyme-linked immunosorbent assays; however, in Mv1Lu cells, the mutant shows preferential cross-linking to type I rather than type II receptors. [Ser77]TGF-beta 1 is a useful tool for understanding the different ligand-receptor complexes and numerous biological activities of this multifunctional cytokine.
Asunto(s)
Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Factor de Crecimiento Transformador beta/química , Animales , Bioensayo , Células CHO , Línea Celular , Cricetinae , Cisteína/química , Disulfuros/química , Técnicas In Vitro , Ratones , Visón , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes , Serina/química , Relación Estructura-ActividadRESUMEN
The transforming growth factors-beta (TGFs-beta) are multifunctional peptide growth factors that have been localized in neuronal and glial cells of the CNS of mice, rats, and chick embryos. We tested the TGF-beta isoforms 1, 2, and 3 for their protective effects against neuronal degeneration caused by cytotoxic hypoxia or by the excitatory amino acid L-glutamate. A cytotoxic hypoxia was induced in cultured chick embryo telencephalic neurons by adding 1 mM sodium cyanide to the culture medium for a period of 30 min. Treatment with TGF-beta 1 (1-30 ng/ml) led to a statistically significant increase in cell viability, neuronal ATP levels, and protein content of the cultures assessed 72 h after the toxic insult. TGF-beta 3 was able to reduce the cyanide-induced neuronal damage at concentrations of 0.3 and 1 ng/ml, whereas TGF-beta 2 only showed neuroprotective activity at concentrations of 30 and 50 ng/ml. Both pre- and post-treatment with TGF-beta 1 also prevented the degeneration of cultured chick embryo telencephalic neurons that had been exposed to 1 mM L-glutamate in a buffered salt solution for a period of 60 min. Furthermore, TGF-beta 1 (0.3-3 ng/ml), and to a lesser extent TGF-beta 3 (0.1-1 ng/ml), significantly reduced excitotoxic injury of cultured neurons from rat cerebral cortex that had been exposed to serum-free culture medium supplemented with 1 mM L-glutamate. These results demonstrate that the TGFs-beta are able to prevent the degeneration of primary neuronal cultures, which was caused by energy depletion and activation of glutamate receptors, in an isoform-specific manner.