RESUMEN
Dynamic attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy at both solutions and coatings of a semicrystalline silk material derived from Bombyx mori was applied to monitor the ß-sheet conformation, which is known to correlate with silk protein crystallinity. The secondary structure-sensitive Amide I band was analyzed. Two silk protein samples were studied: native-based silk buffer fibroin (NSF) was extracted from silk glands and regenerated silk fibroin (RSF) was extracted from degummed cocoons. Solutions of both NSF and RSF at 2 mg/mL featured low initial ß-sheet contents of 5-12%, which further increased to 47-53% after 24 h. RSF and NSF solutions at 23 mg/mL also featured low initial ß-sheet contents of 9-10%, which yet only slightly increased to 16-17% after 24 h. Coatings deposited from RSF solutions showed high surface integrity (Q > 99%) after rinsing in mineralized water, enabling interfacial drug delivery applications. RSF coatings were post-treated with either formic acid (FA) or pure methanol (MeOH) vapor to showcase inducibility of crystalline domains in RSF coatings. Such coatings were loaded with the model antibiotic drugs tetracycline (TCL) and streptomycin (STRP), and the sustained release of TCL was followed in contact with (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) (HEPES) buffer. RSF/TCL coatings post-treated with formic acid (FA) vapor followed by methanol (MeOH) vapor showed a significantly lower (52%) initial burst of rather hydrophobic TCL compared to untreated RSF/TCL coatings (72%), while no such significant release difference was observed for hydrophilic STRP. This was rationalized by a specific interaction between nonpolar TCL and hydrophobic crystalline RSF domains.
Asunto(s)
Bombyx , Fibroínas , Animales , Bombyx/química , Espectroscopía Infrarroja por Transformada de Fourier , Fibroínas/química , Conformación Proteica en Lámina beta , Liberación de Fármacos , Seda/química , Soluciones , Materiales Biocompatibles Revestidos/química , Tetraciclina/químicaRESUMEN
The degeneration of the intervertebral disc (IVD) within the spinal column represents a major pain source for many patients. Biological restoration or repair of the IVD using "compressive-force-resistant" and at the same time "cytocompatible" materials would be desirable over current purely mechanical solutions, such as spinal fusion or IVD implants. This review provides an overview of recent research on the repair of the inner (nucleus pulposus = NP) and the outer (annulus fibrous = AF) parts of the IVD tissue. Many studies have addressed NP repair using hydrogel-like materials. However, only a few studies have so far focused on AF repair. As the AF possesses an extremely low self-healing capacity and special attention to shear-force resistance is essential, special repair designs are required. In our review, we stated the challenges in IVD repair and highlighted the use of composite materials such as silk biomaterials and fibrin cross-linked reinforced hydrogels. We elaborated on the origin of silk and its many in tissue engineering. Furthermore, techniques such as electrospinning and 3D printing technologies allow the fabrication of versatile and functionalised 3D scaffolds. We summarised the research that has been conducted in the field of regenerative medicine over the recent years, with a special focus on the potential application and the potential of combining silk and reinforced - and thus mechanically tailored - hydrogels for IVD repair.
Asunto(s)
Materiales Biocompatibles/farmacología , Hidrogeles/farmacología , Degeneración del Disco Intervertebral/tratamiento farmacológico , Disco Intervertebral/efectos de los fármacos , Seda/farmacología , Animales , Humanos , Disco Intervertebral/patología , Disco Intervertebral/fisiopatología , Degeneración del Disco Intervertebral/fisiopatología , Regeneración/efectos de los fármacosRESUMEN
Chronic wounds represent a serious problem in daily medical routine requiring improved wound care. Silk of the domesticated silkworm (Bombyx mori) has been used to form a variety of biomaterials for medical applications. We genetically engineered B. mori to produce silk functionalized with growth factors to promote wound healing in vitro. In this study FGF-, EGF-, KGF-, PDGF- or VEGF-functionalized silk membranes were compared to native B. mori silk membranes without growth factors for their ability to support wound healing in vitro. All silk membranes were cytocompatible and supported macrophage secretion of neutrophil recruiting factor CXCL1 and monocyte chemoattractant protein 1 (MCP-1). VEGF-functionalized silk significantly outperformed other growth factor-functionalized silk membranes, but not native silk in angiogenesis assays. In addition, EGF- and VEGF-functionalized silk membranes slightly enhanced macrophage adhesion compared to silk without growth factors. In wound healing assays in vitro (reduction of wound lesion), dermal equivalents showed a higher wound healing capacity when covered with EGF-, FGF- or VEGF-functionalized silk membranes compared to native, KGF- or PDGF-functionalized silk membranes. Keratinocyte migration and growth is overstimulated by KGF- and VEGF-functionalized silk membranes. In conclusion, growth factor-functionalized silk membranes prepared from genetically engineered silk worm glands are promising wound dressings for future wound healing therapies.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/administración & dosificación , Seda , Cicatrización de Heridas/efectos de los fármacos , Animales , Animales Modificados Genéticamente , Materiales Biocompatibles/química , Bombyx/genética , Línea Celular , Humanos , Técnicas In Vitro , Péptidos y Proteínas de Señalización Intercelular/genética , Macrófagos/efectos de los fármacos , Macrófagos/fisiología , Ensayo de Materiales , Ratones , Neovascularización Fisiológica/efectos de los fármacos , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/genética , Seda/química , Piel/efectos de los fármacos , Piel/lesiones , Andamios del Tejido , Cicatrización de Heridas/fisiologíaRESUMEN
Mesenchymal stem cells (MSC) are precursor cells of mesodermal tissue and, because of their trophic phenotype, they are known to play beneficial roles in wound healing. In addition, various tissue engineering strategies are based on MSC/biomaterial constructs. As the isolation and expansion of MSCs is a long-term process, a major goal is to develop an endogenous stem cell recruitment system that circumvents all ex vivo steps generally used for tissue engineering. Therefore collagen and silk fibroin were loaded with hepatocyte growth factor (HGF), a chemoattractant for MSCs. Collagen was mixed with HGF during polymerization, while silk fibroin and HGF were produced as fusion proteins by transgenic silkworms. To demonstrate release of active HGF, enzyme-linked immunosorbent assay, in vitro migration assays and animal studies were performed to demonstrate MSC migration in vivo, followed by detailed examinations of the immunological effects of the biomaterials. Hepatocyte growth factor was released burst-like, both from silk fibroin and collagen during the first 8 h and gradually for up to 168 h in vitro. Directed migration in vitro was demonstrated when MSCs were exposed to HGF. In vivo, HGF-loaded collagen and silk fibroin were tolerated as subcutaneous implants. In addition, it was proved that endogenous MSCs were recruited from the local environment. These results show for the first time recruitment of endogenous MSCs to HGF-loaded collagen (fast degradable) and silk fibroin scaffolds (long-term degradable) in vitro and in vivo. This knowledge could be applied to make off-the-shelf, readily available constructs for use in patients with chronic wound or burns. Copyright © 2016 John Wiley & Sons, Ltd.
Asunto(s)
Materiales Biocompatibles , Movimiento Celular/efectos de los fármacos , Factor de Crecimiento de Hepatocito , Células Madre Mesenquimatosas/metabolismo , Heridas y Lesiones/terapia , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacocinética , Materiales Biocompatibles/farmacología , Implantes de Medicamentos/química , Implantes de Medicamentos/farmacocinética , Implantes de Medicamentos/farmacología , Femenino , Fibroínas/química , Fibroínas/farmacocinética , Fibroínas/farmacología , Factor de Crecimiento de Hepatocito/química , Factor de Crecimiento de Hepatocito/farmacocinética , Factor de Crecimiento de Hepatocito/farmacología , Humanos , Masculino , Células Madre Mesenquimatosas/patología , Heridas y Lesiones/metabolismo , Heridas y Lesiones/patologíaRESUMEN
Histidine-rich glycoprotein (HRG) is a serum protein belonging to the cystatin superfamily. HRG may play a regulatory role in hemostasis and innate immunity. However, this role is uncertain because of a lack of rigorous testing in an animal model. We generated mice lacking the translation start point of exon 1 of the Hrg gene, effectively resulting in a null mutation (Hrg-/-). The mice were viable and fertile but had no HRG in their blood. Antithrombin activity in the plasma of Hrg-/- mice was higher than in the plasma of heterozygous Hrg+/- or wild-type Hrg+/+ mice. The prothrombin time was shorter in Hrg-/- mice than in Hrg+/- and Hrg+/+ mice. Bleeding time after tail tip amputation in Hrg-/- mice was shorter than in Hrg+/+ mice. The spontaneous fibrinolytic activity in clotted blood of Hrg-/- mice was higher than in Hrg+/+ mice. These findings suggest that HRG plays a role as both an anticoagulant and an antifibrinolytic modifier, and may regulate platelet function in vivo.
Asunto(s)
Coagulación Sanguínea , Proteínas/genética , Proteínas/fisiología , Animales , Tiempo de Sangría , Plaquetas/fisiología , Southern Blotting , Clonación Molecular , Exones , Fibrinólisis , Vectores Genéticos , Genotipo , Heterocigoto , Ratones , Ratones Transgénicos , Modelos Genéticos , Datos de Secuencia Molecular , Unión Proteica , Piel/metabolismo , Células Madre , Cicatrización de HeridasRESUMEN
OBJECTIVES: The goal of this study was to test the hypothesis that moderate hypothermia during cardiopulmonary bypass (CPB) provides myocardial protection by enhancing intra-myocardial anti-inflammatory cytokine balance. BACKGROUND: Moderate hypothermia during experimental CPB stimulates production of interleukin-10 (IL10) and blunts release of tumor necrosis factor-alpha (TNFalpha). METHODS: Twelve young pigs were assigned to a temperature (T degrees ) regimen during CPB: moderate hypothermia (T degrees : 28 degrees C; n = 6) and normothermia (T degrees : 37 degrees C; n = 6). Intra-myocardial TNFalpha- and IL10-messenger RNA were detected by competitive reverse transcriptase polymerase chain reaction and quantification of cytokine synthesis by Western blot. Levels of cardiac troponin I (cTnI) in cardiac lymph and in arterial and coronary venous blood were examined during and after CPB. Myocardial cell damage was assessed by histologic and ultrastructural anomalies of tissue probes taken 6 h after CPB. RESULTS: Synthesis of IL10 was significantly higher, while that of TNFalpha was significantly lower, in pigs that were in moderate hypothermia during surgery than in the others. In contrast with normothermia, moderate hypothermia was also associated with significantly lower cumulative cardiac lymphatic flow during and after CPB, significantly lower lymphatic cTnI concentrations after CPB, significantly lower percentages of myocardial cell necrosis and a significantly lower score of ultrastructural anomalies of myocardial cells. While the percentage of apoptotic cells was not different between groups, the apoptosis/necrosis ratio tended to be higher in animals that were in moderate hypothermia during surgery. In all animals, TNFalpha synthesis correlated positively while IL10 production correlated negatively with necrosis and total cell death, respectively. CONCLUSIONS: Our results suggest that moderate hypothermia during CPB provides myocardial protection by enhancing intra-myocardial anti-inflammatory cytokine balance.
Asunto(s)
Puente Cardiopulmonar , Hipotermia Inducida , Miocardio/patología , Animales , Apoptosis , Muerte Celular , Femenino , Hemodinámica , Etiquetado Corte-Fin in Situ , Interleucina-10/biosíntesis , Miocardio/metabolismo , Porcinos , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The diverse biological effects of the hormone somatostatin are mediated by five genetic different receptor subtypes (sst1-sst5), which belong to the superfamily of G-protein coupled receptors with seven transmembrane domains. The sst2 subtype is unique among the somatostatin receptors in its structure, since it is expressed in two protein variants which differ within their carboxy-terminal ends, generated by alternative splicing. Within the 5' untranslated region of the gene two introns separate three transcriptional units with distinct promoters. Due to the latter feature, the sst2 gene is also unique among all somatostatin receptor genes regarding its transcriptional regulation. The three alternative promoters are tissue specifically active and show alternative responsiveness to extracellular signals. The second sst2 promoter is important for expression of the gene in tissues where somatostatin has essential physiological functions, such as brain, pituitary and gastrointestinal tissues. Furthermore, it contains cis-acting regulatory elements involved in the transcriptional response to elevated cyclic AMP levels and glucocorticoids.
Asunto(s)
Regulación de la Expresión Génica , Regiones Promotoras Genéticas/fisiología , Receptores de Somatostatina/genética , Animales , Secuencia de Bases , AMP Cíclico/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Glucocorticoides/metabolismo , Ratones , Sitios de Empalme de ARN/genética , Receptores de Somatostatina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Activación Transcripcional/fisiologíaRESUMEN
The aim of this study was to examine the effects of the phorbol ester O-tetradecanoylphorbol 13-acetate (TPA) and forskolin on delta-opioid receptor gene transcription. Treatment of NG108-15 cells with TPA (100 nM) for 48 h increased delta-opioid receptor mRNA levels, whereas different concentrations of forskolin induced a transient down-regulation of mRNA 5 h after treatment, followed by increased mRNA levels after 48 h. Reporter gene assays in transiently transfected NG108-15 cells in combination with electrophoretic mobility shift assays indicate that the increase of delta-opioid receptor mRNA after stimulation with TPA is mediated by transcription factor AP-1, which binds 355 bp upstream of the start codon within the gene promoter. The forskolin-induced mRNA increase is mediated neither by a cyclic AMP-response element nor indirectly by AP-1 up-regulation. Reporter gene assays, mutational analysis, and electrophoretic mobility shift assays revealed that delta-opioid receptor gene regulation by forskolin is mediated by transcription factor AP-2, which binds to an element 157 bp upstream of the start codon.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Receptores Opioides delta/genética , Factor de Transcripción AP-1/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Anciano , Animales , Carcinógenos/farmacología , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/fisiología , Prueba de Complementación Genética , Glioma , Humanos , Ratones , Inhibidores de Fosfodiesterasa/farmacología , Regiones Promotoras Genéticas/efectos de los fármacos , Regiones Promotoras Genéticas/fisiología , Proteína Quinasa C/metabolismo , ARN Mensajero/metabolismo , Receptor Cross-Talk/fisiología , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-2 , Células Tumorales CultivadasRESUMEN
Prodynorphin, the precursor of the dynorphin opioid peptides, has been shown to play an important role in several aspects of human diseases and complex traits, e.g., drug abuse, epilepsy, and mood disorders. The objective of this study was to identify polymorphisms in the 5' control region of the human prodynorphin gene and to relate these polymorphisms to prodynorphin gene expression. Within the core promoter region, a 68-bp sequence was found to occur as a polymorphic element, either singular or as tandemly repeated element two, three, or four times. This 68-bp repeat element contains an AP-1 transcription factor binding site as demonstrated by electrophoretic mobility shift assay. Reporter gene assays were performed and provided evidence for allele dependent different promoter activity. Dynorphin was found to be involved in many pathophysiological processes so that the described prodynorphin alleles may correlate with the occurrence of several diseases, for example, drug addiction. However, prodynorphin allelic distributions were not significantly different in heroin addicts and control subjects.
Asunto(s)
Alelos , Encefalinas/genética , Expresión Génica , Variación Genética/fisiología , Regiones Promotoras Genéticas/genética , Precursores de Proteínas/genética , Secuencia de Bases/genética , Sitios de Unión/genética , Línea Celular , Cloranfenicol O-Acetiltransferasa/metabolismo , ADN/genética , Frecuencia de los Genes , Dependencia de Heroína/genética , Humanos , Reacción en Cadena de la Polimerasa , Valores de Referencia , Secuencias Repetidas en Tándem , Acetato de Tetradecanoilforbol/farmacología , Factor de Transcripción AP-1/metabolismo , TransfecciónRESUMEN
Somatostatin is a regulatory peptide with important inhibitory functions. Its effects are mediated by five receptors, sst1-sst5. Previous studies revealed that sst2 contains three alternative, tissue specific promoters. Here, molecular mechanisms for the regulation of sst2 transcription by glucocorticoids were investigated. Reporter gene assays in NG 108-15 cells and electrophoretic mobility shift assays revealed that a glucocorticoid responsive element at position -1044 on the second promoter mediates dexamethasone induction. These findings, the existence of a cAMP response element and the tissue dependent activity (brain, pituitary and gastrointestinal tissues) indicate the importance of the second sst2 gene promoter.
Asunto(s)
Regulación de la Expresión Génica , Glucocorticoides/fisiología , Receptores de Somatostatina/genética , Animales , Ratones , Regiones Promotoras Genéticas , Receptores de Glucocorticoides/metabolismo , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
Important physiological actions of somatostatin are mediated by the somatostatin receptor type 2. Its transcription is regulated by three tissue specific, alternative promoters. It is known that the mRNA of the somatostatin receptor type 2 gene is induced by cAMP, but little is known about the mechanisms underlying this regulation. We have identified and characterized a cAMP responsive element located at nucleotide -162 on the second promoter of the gene consisting of the classical palindromic octameric sequence 5'-TGACGTCA-3'. Using transient expression of reporter gene deletion constructs in NGC108-15 cells the necessity of the intact element for forskolin-induced reporter gene activity was demonstrated. The first and the third promoter are not responsive to forskolin, nor did any promoter respond to the phorbol ester PMA. Electrophoretic mobility shift assays in combination with competition experiments suggest the interaction of the promoter element with the cAMP responsive element binding protein.
Asunto(s)
AMP Cíclico/fisiología , Regulación de la Expresión Génica , Regiones Promotoras Genéticas/genética , Receptores de Somatostatina/genética , Elementos de Respuesta/genética , Animales , Secuencia de Bases , Unión Competitiva , Línea Celular , Colforsina/farmacología , Secuencia de Consenso , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , ADN/metabolismo , Genes Reporteros , Ratones , Proteínas Nucleares/metabolismo , Oligodesoxirribonucleótidos/metabolismo , Eliminación de Secuencia , Acetato de Tetradecanoilforbol/farmacología , TransfecciónRESUMEN
We have cloned the 5' upstream regulatory region of the mouse somatostatin receptor 2 gene. Its genomic organization is novel among all somatostatin receptor genes. It contains two previously unrecognized exons, separated by introns larger than 25 kb, and three tissue and cell specific alternative promoters. The first promoter in front of exon 1 is active only in AtT-20 tumor cells. The second promoter, located 5' to exon 2, is used in brain, pituitary, adrenals, pancreas, NG 108-15 and AtT-20 cells. Furthermore, it contains putative DNA elements for regulation by glucocorticoids, estradiol and cAMP. A third promoter, located in exon 3, is additionally used in lung, kidney and spleen.
Asunto(s)
Empalme Alternativo , Regulación de la Expresión Génica , Regiones Promotoras Genéticas , Receptores de Somatostatina/biosíntesis , Receptores de Somatostatina/genética , Transcripción Genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Cartilla de ADN , Exones , Intrones , Ratones , Datos de Secuencia Molecular , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/biosíntesis , Células Tumorales CultivadasRESUMEN
An acute, double-blind study was carried out in 8 healthy male volunteers to investigate any effect of a new antispasmodic, pinaverium bromide, compared with placebo on the lower oesophageal sphincter. Manometric measurements showed no significant differences in resting pressures either after placebo or a therapeutic dose (200 mg) of pinaverium bromide, suggesting that the active drug does not cause any impairment of function of the lower oesophageal sphincter.
Asunto(s)
Unión Esofagogástrica/efectos de los fármacos , Morfolinas/farmacología , Adulto , Método Doble Ciego , Humanos , Masculino , ParasimpatolíticosRESUMEN
14 patients suffering from diaethylpentenamide-intoxication were treated in the medical School Hannover, in the period from January 1 to June 6, 1978. 7 of the patients had to be treated in the intensive care unit, and of these 5 received haemoperfusion therapy. The indications for haemoperfusion were severe clinical symptoms and a plasma diaethylpentenamide-level in excess 285 mu mol/l. The duration of coma was significantly less for patients treated by haemoperfusions than for those without haemoperfusion. Although preparations containing diaethylpentenamide are only available through prescription in Germany now, it can be expected that these drugs will continue to be used in cases of suicidal intent. Haemoperfusion seems to be the most effective therapy of diaethylpentenamide-intoxication.