RESUMEN
AIMS: Demineralised bone matrix (DBM) is rarely used for the local delivery of prophylactic antibiotics. Our aim, in this study, was to show that a graft with a bioactive glass and DBM combination, which is currently available for clinical use, can be loaded with tobramycin and release levels of antibiotic greater than the minimum inhibitory concentration for Staphylococcus aureus without interfering with the bone healing properties of the graft, thus protecting the graft and surrounding tissues from infection. MATERIALS AND METHODS: Antibiotic was loaded into a graft and subsequently evaluated for drug elution kinetics and the inhibition of bacterial growth. A rat femoral condylar plug model was used to determine the effect of the graft, loaded with antibiotic, on bone healing. RESULTS: We found that tobramycin loaded into a graft composed of bioglass and DBM eluted antibiotic above the minimum inhibitory concentration for three days in vitro. It was also found that the antibiotic loaded into the graft produced no adverse effects on the bone healing properties of the DBM at a lower level of antibiotic. CONCLUSION: This antibiotic-loaded bone void filler may represent a promising option for the delivery of local antibiotics in orthopaedic surgery. Cite this article: Bone Joint J 2016;98-B:1126-31.
Asunto(s)
Antibacterianos/administración & dosificación , Curación de Fractura/efectos de los fármacos , Infecciones Estafilocócicas/prevención & control , Tobramicina/administración & dosificación , Animales , Antibacterianos/farmacología , Técnica de Desmineralización de Huesos , Trasplante Óseo/métodos , Vías de Administración de Medicamentos , Fracturas del Fémur/fisiopatología , Fémur/cirugía , Pruebas de Sensibilidad Microbiana , Ratas Desnudas , Staphylococcus aureus , Tobramicina/farmacologíaRESUMEN
The ability of the alpha4 integrin counterligands vascular cell adhesion molecule (VCAM)-1 or mucosal addressin (MAd)CAM-1 to support eosinophil rolling or firm adhesion under conditions of physiologic flow has not been delineated. Using a parallel plate flow chamber in vitro and intravital microscopy in vivo, we demonstrate that eosinophil rolling and adhesion on VCAM-1 is mediated by both alpha4beta1 and alpha4beta7 integrins. Eosinophils rolled equally efficiently on both VCAM-1 2 domain and VCAM-1 7 domain, suggesting that the N-terminal 2 domains of VCAM-1 are sufficient to support eosinophil rolling under conditions of flow. Furthermore, activation of the eosinophil beta1 integrin with monoclonal antibody (mAb) 8A2 resulted in both resistance to shear stress-induced detachment from VCAM-1 in vitro and in stable arrest of rolling eosinophils on interleukin (IL)-1beta-stimulated venules in vivo. Eosinophils rolled less efficiently on MAdCAM-1- than on VCAM-1-coated coverslips under conditions of flow. However, eosinophils firmly adhered as efficiently to MAdCAM-1 as to VCAM-1. Overall, these results demonstrate that both VCAM-1 and MAdCAM-1 can support eosinophil firm adhesion under conditions of flow. In contrast, VCAM-1 is significantly more efficient than MAdCAM-1 in supporting eosinophil rolling under conditions of flow. (Blood. 2000;95:592-601)
Asunto(s)
Eosinófilos/fisiología , Inmunoglobulinas/farmacología , Cadenas beta de Integrinas , Mucoproteínas/farmacología , Molécula 1 de Adhesión Celular Vascular/farmacología , Anticuerpos Monoclonales/farmacología , Antígenos CD/sangre , Antígenos CD/inmunología , Asma/sangre , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Moléculas de Adhesión Celular , Movimiento Celular/efectos de los fármacos , Movimiento Celular/fisiología , Eosinófilos/efectos de los fármacos , Eosinófilos/inmunología , Humanos , Técnicas In Vitro , Integrina alfa4 , Integrina beta1/sangre , Integrina beta1/inmunología , Integrinas/sangre , Integrinas/inmunología , Proteínas Recombinantes/farmacología , Estrés MecánicoRESUMEN
Soluble vascular cell adhesion molecule-1 (sVCAM-1) is generated during inflammation and can alter lymphocyte functions. The authors report that the binding of sVCAM-1 to alpha4 integrin-bearing cells is a dynamically regulated, active cellular process. Binding of recombinant sVCAM-1 to alpha4 integrins on peripheral blood mononuclear cells was cell-type specific. Circulating CD16+ NK cells constitutively bound sVCAM-1 with high affinity, whereas a subpopulation of T-lymphocytes, primarily CD45RO+ (memory), bound sVCAM-1 only after phorbol ester stimulation. sVCAM-1 binding to homogenous stable cell lines was also cell-type specific, and required active cellular processes because it was blocked by the inhibition of ATP synthesis and by Fas-induced apoptosis. Indeed, the loss of high-affinity VCAM-1 binding was an early event in apoptosis. Furthermore, an H-Ras/Raf-initiated signaling pathway also suppressed sVCAM-1 binding to alpha4beta1 integrins. Collectively, these results showed that the capacity of alpha4 integrins to bind VCAM-1 is actively regulated and that this regulation may control alpha4 integrin-dependent cellular functions. (Blood. 2000;95:602-609)
Asunto(s)
Antígenos CD/fisiología , Linfocitos T/fisiología , Molécula 1 de Adhesión Celular Vascular/metabolismo , Animales , Apoptosis/inmunología , Humanos , Integrina alfa4 , Integrina alfa4beta1 , Integrinas/fisiología , Células Jurkat , Leucemia Basofílica Aguda , Activación de Linfocitos , Ratas , Complejo Receptor-CD3 del Antígeno de Linfocito T/fisiología , Receptores Mensajeros de Linfocitos/fisiología , Proteínas Recombinantes de Fusión/metabolismo , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/fisiología , Linfocitos T/citología , Linfocitos T/inmunología , Células Tumorales Cultivadas , Células U937 , Receptor fas/fisiologíaRESUMEN
Eosinophil adhesion to vascular cell adhesion molecule-1 (VCAM-1) is important for cellular recruitment into allergic inflammatory sites. To determine whether eosinophil adhesion to VCAM-1 affects cell function, leukotriene C4 (LTC4) was measured. Human eosinophils were incubated with platelet-activating factor (PAF) in the presence or absence of soluble VCAM-Fc fusion protein (sVCAM-Fc) or immobilized VCAM-Fc. sVCAM-Fc induced a concentration-dependent increase in LTC4 secretion, which was dependent on the presence of PAF and not blocked by cyclic peptides shown to inhibit alpha4beta1-dependent adhesion. Likewise, soluble ICAM-Fc induced a concentration-dependent LTC4 secretion. LTC4 secretion was induced by the calcium ionophore, A23187, and the combination of sVCAM-Fc and A23187 had synergistic properties. It is interesting to note that Mn2+ or anti-beta1 monoclonal antibody, TS2/16, inhibited LTC4 secretion induced by sVCAM-Fc and PAF. Eosinophil adhesion to VCAM-Fc or interleukin-1 beta-stimulated endothelial cells did not induce LTC4 secretion. These data suggest that sVCAM-Fc-induced LTC4 secretion depends on distinct signals from those of eosinophil adhesion.
Asunto(s)
Eosinófilos/efectos de los fármacos , Eosinófilos/metabolismo , Fragmentos Fc de Inmunoglobulinas/farmacología , Leucotrieno C4/metabolismo , Factor de Activación Plaquetaria/farmacología , Molécula 1 de Adhesión Celular Vascular/farmacología , Secuencia de Aminoácidos , Animales , Calcimicina/farmacología , Adhesión Celular/fisiología , Sinergismo Farmacológico , Eosinófilos/fisiología , Humanos , Fragmentos Fc de Inmunoglobulinas/genética , Ionóforos/farmacología , Ratones , Datos de Secuencia Molecular , Péptidos Cíclicos/farmacología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/farmacología , Estimulación Química , Molécula 1 de Adhesión Celular Vascular/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/inmunologíaRESUMEN
Monocyte chemoattractant protein-1 (MCP-1) is a member of the beta chemokine family which acts through specific seven transmembrane receptors to recruit monocytes, basophils, and T lymphocytes to sites of inflammation. To identify regions of the human MCP-1 protein which are important for its biological activity, we have synthesized domain-specific peptides and tested their ability to antagonize MCP-1 binding and chemotaxis in THP-1 cells. We have found that an intercysteine first loop peptide encompassing amino acids 13-35 inhibits MCP-1 binding and chemotactic activity, while peptides representing the amino-terminus (amino acids 1-10), second loop (amino acids 37-51), and carboxy-terminus (amino acids 56-71) of MCP-1 have no effect. In addition, we have found that cyclization of the first loop peptide by disulfide linkage and blocking the C-terminus of the peptide by amidation increases the activity of this peptide to block MCP-1 binding and chemotaxis. In order to specifically identify amino acid residues within the first loop that are crucial for MCP-1 functional activity, we have substituted alanine for tyrosine (Y13A) or arginine (R18A) in MCP-1 recombinant proteins. While baculovirus produced wild type and R18A MCP-1 proteins are indistinguishable in their ability to induce THP-1 chemotaxis and show modest effects in binding activity compared to commercially available recombinant MCP-1 protein, the Y13A point mutation causes a dramatic loss in function. The identification of functional domains of MCP-1 will assist in the design of MCP-1 receptor antagonists which may be clinically beneficial in a number of inflammatory diseases.
Asunto(s)
Quimiocina CCL2/química , Quimiotaxis de Leucocito/fisiología , Péptidos/farmacología , Secuencia de Aminoácidos , Arginina/fisiología , Unión Competitiva , Línea Celular , Quimiocina CCL2/genética , Humanos , Datos de Secuencia Molecular , Monocitos/citología , Monocitos/metabolismo , Mutagénesis Sitio-Dirigida , Mutación , Péptidos/síntesis química , Péptidos Cíclicos/síntesis química , Péptidos Cíclicos/farmacología , Proteínas Recombinantes de Fusión , Tirosina/fisiologíaRESUMEN
Human monocyte chemoattractant protein-1 (MCP-1) is expressed by a variety of cell types in response to various stimuli. MCP-1 expressed by the endothelium plays an important role in cell migration and activation. MCP-1 is a major chemoattractant for monocytes, T lymphocytes, and basophils. In the present study, we present evidence that the proteasome complex is involved in mediating the interleukin (IL)-1beta induction of MCP-1 in endothelial cells. We present evidence that a proteasome inhibitor, N-acetyl-leucinyl-leucinyl-norleucinal (norLeu), and the protease inhibitor tosyl-Phe-chloromethylketone (TPCK) block IL-1beta induction of MCP-1 protein expression. norLeu and TPCK also blocked IL-1beta-induced MCP-1 promoter-driven reporter gene expression as well as nuclear factor (NF)-kappaB-mediated reporter gene expression. The effects of norLeu were due to its inhibition of the proteasome rather than calpain, because other calpain inhibitors had no effect on MCP-1 expression. In contrast to TPCK, which blocked NF-kappaB translocation to the nucleus, norLeu had no effect on NF-kappaB nuclear translocation or IL-1beta-induced phosphorylation of p65. This study demonstrates that the proteasome pathway is involved in IL-1beta-induced MCP-1 gene expression in human endothelial cells.
Asunto(s)
Quimiocina CCL2/genética , Cisteína Endopeptidasas/metabolismo , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica , Interleucina-1/fisiología , Complejos Multienzimáticos/metabolismo , Inhibidores de Serina Proteinasa/farmacología , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Humanos , Leupeptinas/farmacología , FN-kappa B/metabolismo , Complejo de la Endopetidasa Proteasomal , Clorometilcetona de Tosilfenilalanila/farmacologíaRESUMEN
P130cas is a dominant tyrosine phosphorylated protein in v-src-and v-crk-transformed cells. Tyrosine phosphorylation also occurs in response to integrin-mediated cell adhesion. P130cas has a unique structure with multiple SH2 and SH3 binding sites, which makes it a candidate docking protein that might be involved in several signal transduction pathways. Little is known about how p130cas itself is regulated. In this report we present evidence that tyrosine phosphorylated p130cas was rapidly dephosphorylated in several lymphatic cell lines after treatment with calyculin A, a serine/threonine phosphatase inhibitor. A similar result was obtained with okadaic acid, but higher concentrations and longer incubation times were required. Constitutive phosphorylation as well as receptor-cross linking-induced p130cas phosphorylation was inhibited. Furthermore, the p130cas-Crk association was disrupted by treatment of cells with calyculin A. However, the p130cas-Lyn association was not affected. These results suggest that calyculin A specifically affects SH2 domain-mediated protein-protein interactions and that Lyn does not bind to a susceptible SH2 domain. Furthermore, the data presented is consistent with the existence of a calyculin A-sensitive phosphatase or tyrosine kinase that may be a critical regulator of p130cas tyrosine phosphorylation.
Asunto(s)
Oxazoles/farmacología , Fosfoproteínas/metabolismo , Proteínas Quinasas , Proteínas , Proteínas Protozoarias , Agregación Celular , Proteína Sustrato Asociada a CrK , Inhibidores Enzimáticos/farmacología , Humanos , Toxinas Marinas , Ácido Ocadaico/farmacología , Fosforilación , Fosfotirosina/metabolismo , Unión Proteica , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-crk , Proteína p130 Similar a la del Retinoblastoma , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Familia-src Quinasas/metabolismoRESUMEN
Chemokines are potent mediators of cell migration and activation and therefore play an essential role in early events of inflammation. In conjunction with cell adhesion molecules, chemokines help to localize cells to a specific site and enhance the inflammatory reaction at the site. Clinically, elevated levels of chemokines have been found in a variety of inflammatory diseases. The prototype C-C chemokine is monocyte chemoattractant protein-1 (MCP-1) which is synthesized by variety of cell types including endothelial cells in response to a variety of stimuli. MCP-1 is a major chemoattractant for monocytes, T lymphocytes, and basophils. In the present study, we investigated the factors involved in cytokine-induced MCP-1 gene expression in human endothelial cells. We present evidence that the nuclear factor (NF)-kappa B-like binding site and the AP-1 binding site located 90 and 68 base pairs upstream of the transcriptional start site, respectively, are required for maximal induction of the human MCP-1 promoter by interleukin-(IL)-1 beta. Site-directed mutagenesis or deletion of the NF-kappa B-like site decreased the cytokine-induced activity of the promoter. Site-directed mutagenesis of the AP-1 binding site also decreased the cytokine-induced activity of the promoter. We show that the NF-kappa B-like site located at-90 in the MCP-1 promoter binds to the p50/p65 heterodimer of the NF-kappa B/Rel family in IL-1 beta-stimulated human endothelial cells. Overexpression of p65 results in the transactivation of the MCP-1 promoter as well. The data presented in this study suggest that cytokine-induced MCP-1 gene expression in human endothelial cells depends on the cooperative action of NF-kappa B and AP-1.
Asunto(s)
Quimiocina CCL2/biosíntesis , Quimiocina CCL2/genética , Citocinas/fisiología , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica/inmunología , FN-kappa B/fisiología , Factor de Transcripción AP-1/fisiología , Secuencia de Bases , Células Cultivadas , Dimerización , Humanos , Interleucina-1/fisiología , Datos de Secuencia Molecular , FN-kappa B/biosíntesis , FN-kappa B/metabolismo , Subunidad p50 de NF-kappa B , Regiones Promotoras Genéticas/fisiología , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas c-rel , Factor de Transcripción ReIA , Factores de Transcripción/biosíntesis , Factor de Necrosis Tumoral alfa/fisiología , Venas Umbilicales/citologíaRESUMEN
Inflammation is characterized by the recruitment of leukocytes and their subsequent migration from the vasculature into the tissue, where they often cause severe damage. Endothelial cells play a major role in this cascade by expressing cell surface adhesion molecules, such as VCAM-1 and ICAM-1, and chemokines, in response to cytokines. Many of these genes are under the control of inflammatory response transcription factors such as nuclear factor (NF)-kappa B. In this study, we examined the effects of 5-lipoxygenase inhibitors (nordihydroguaiaretic acid and AA861) on IL-1 beta-induced VCAM-1 gene expression in HUVECs. We demonstrated that 5-lipoxygenase inhibitors, but not cyclooxygenase inhibitors, block IL-1 beta-induced VCAM-1 cell surface expression and promoter activity. In transiently transfected HUVECs, NF-kappa B-dependent gene expression was inhibited by 5-lipoxygenase inhibitors. These inhibitors did not block IL-1 beta-induced nuclear translocation of NF-kappa B, inhibitor of kappa B-alpha proteolytic degradation, or significantly reduce phosphorylation of p65. These studies indicate that inhibition of 5-lipoxygenase blocks cytokine-induced VCAM-1 gene expression by reducing the functional activity of NF-kappa B/Rel proteins in HUVECs.
Asunto(s)
Endotelio Vascular/inmunología , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1/farmacología , Inhibidores de la Lipooxigenasa , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Molécula 1 de Adhesión Celular Vascular/genética , Benzoquinonas/farmacología , Transporte Biológico/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/genética , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Humanos , Inhibidores de la Lipooxigenasa/farmacología , Masoprocol/farmacología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Fosforilación/efectos de los fármacos , Regiones Promotoras Genéticas/efectos de los fármacos , Transcripción Genética/efectos de los fármacos , Molécula 1 de Adhesión Celular Vascular/efectos de los fármacosRESUMEN
Adherence of monocytes to endothelial cells and subsequently to basement membrane represents the initial steps in monocyte migration from the vasculature to the interstitium. We investigated the role of adhesion to endothelial cells and basement membrane in the induction of the cytokine IL-1beta. We demonstrated that mRNA for IL-1beta is induced in adherent THP-1 cells, but not in a matrix-specific manner. Adherence to fibrinogen, however, causes an increase in mRNA for IL-1beta. A background level of IL-1beta mRNA induction was observed in cells adherent to all matrices, including the non-specific human serum albumin substrate, as compared to non-adherent cells cultured in teflon troughs. In addition, antibodies to CD11a, CD11b, beta1 integrin, VLA4, (alpha)v(beta)3 (VNR), and ICAM-1 did not induce significant IL-1beta mRNA when THP-1 cells were adherent to those immunoglobulins. THP-1 cells adherent to immune complexes of anti-CD11a, anti-CD11b, anti-VLA4, anti-VNR, and anti-ICAM-1 showed greater mRNA induction than cells adherent to primary antibodies alone. THP-1 cells adherent to non-specific immune complexes gave the highest level of mRNA induction. Secretion of IL-1beta protein, measured by ELISA at 24 h, was greatest when cells were adherent to immobilized immune complexes or to fibrinogen. Our results demonstrate that a general adherence-induced increase in IL-1beta gene expression is greatly enhanced by the presence of immune complex.
Asunto(s)
Complejo Antígeno-Anticuerpo/metabolismo , Adhesión Celular/fisiología , Regulación de la Expresión Génica , Interleucina-1/metabolismo , Monocitos/fisiología , Receptores de IgG/metabolismo , Antígenos CD/metabolismo , Antígenos CD18/metabolismo , Línea Celular , Fibrinógeno/metabolismo , Integrina beta1/metabolismo , Integrina beta3 , Interleucina-1/genética , Interleucina-8/biosíntesis , Interleucina-8/genética , Ligandos , Glicoproteínas de Membrana Plaquetaria/metabolismo , Unión Proteica , ARN Mensajero/análisis , Transducción de SeñalRESUMEN
The NF-kappaB/Rel family of transcription factors regulates the inducible expression of many genes in activated human monocytes and endothelial cells. In this study, we examined the molecular mechanism by which agents that elevate intracellular cAMP inhibit the expression of the tumor necrosis factor alpha (TNFalpha), tissue factor, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1 genes. Both forskolin and dibutyryl cAMP, which elevate intracellular cAMP by independent mechanisms, inhibited TNFalpha and tissue factor expression at the level of transcription. Induction of NF-kappaB-dependent gene expression in transiently transfected human monocytic THP-1 cells and human umbilical vein endothelial cells was inhibited by elevated cAMP and by overexpression of the catalytic subunit of protein kinase A (PKA). Elevated cAMP did not prevent nuclear translocation of p50/p65 and c-Rel/p65 heterodimers, decrease nuclear translocation of p65, or significantly modify TNFalpha-induced phosphorylation of p65. Functional studies demonstrated that transcriptional activation of a plasmid containing multimerized kappaB sites by p65 was inhibited by agents that elevate cAMP and by overexpression of the catalytic subunit of PKA. This study indicates that activation of PKA reduces the induction of a distinct set of genes in monocytes and endothelial cells by inhibiting NF-kappaB-mediated transcription.
Asunto(s)
Endotelio Vascular/metabolismo , Monocitos/metabolismo , FN-kappa B/metabolismo , Factor de Necrosis Tumoral alfa/fisiología , Transporte Biológico , Compartimento Celular , Núcleo Celular/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/fisiología , Selectina E/genética , Expresión Génica/efectos de los fármacos , Humanos , Lipopolisacáridos/farmacología , Regiones Promotoras Genéticas , ARN Mensajero/genética , Tromboplastina/genética , Transcripción Genética , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
At sites of inflammation, endothelial cells play a major role in defining the types of leukocytes that are recruited to a specific area. This is accomplished, at least in part, through the cytokine induction of cell surface adhesion molecules, including vascular cell adhesion molecule 1 (VCAM-1). We investigated the role of phosphatidylcholine-specific phospholipase C in the induction of VCAM-1 gene expression by interleukin-1 beta. D609, a phosphatidylcholine-specific phospholipase C inhibitor, reduced VCAM-1 cell surface expression and VCAM-1 promoter activity in human endothelial cells in a dose-dependent manner. D609 did not affect nuclear translocation of nuclear factor-kappa B but inhibited nuclear factor-kappa B-mediated transcription. The results of this study indicate that phosphatidylcholine-specific phospholipase C is required for activation of nuclear factor-kappa B and cytokine induction of VCAM-1 gene expression in endothelial cells.
Asunto(s)
Hidrocarburos Aromáticos con Puentes/farmacología , Endotelio Vascular/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1/farmacología , Tionas/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , Molécula 1 de Adhesión Celular Vascular/genética , Secuencia de Bases , Células Cultivadas , Endotelio Vascular/metabolismo , Humanos , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Norbornanos , Regiones Promotoras Genéticas , Tiocarbamatos , Fosfolipasas de Tipo C/fisiologíaRESUMEN
Endothelial cells play a major role in recruiting leukocytes to sites of inflammation. This is accomplished, at least in part, by up-regulation of cell surface adhesion molecules, including VCAM-1 and ICAM-1, in response to cytokines. In this report, we investigated the role of the proteasome complex in mediating the interleukin (IL)- 1 beta induction of VCAM-1 and ICAM-1 gene expression in human endothelial cells. We present evidence that a proteasome inhibitor, n-acetyl-leucinyl-leucinyl-norleucinal (norLEU), as well as specific protease inhibitors, n-tosyl-Lys-chloromethylketone and N-tosyl-Phe-chloromethylketone, blocked IL-1 beta induction of VCAM-1 and ICAM-1 promoter-driven reporter gene expression in stably transfected endothelial cells. These inhibitors also blocked cytokine induced cell surface expression of VCAM-1 and ICAM-1 by human umbilical vein endothelial cells. As expected, the protease inhibitors blocked the activation of nuclear factor (NF)-kappa B in response to IL-1 beta stimulation. In contrast, norLEU did not prevent IL-1 beta-induced nuclear translocation of NF-kappa B. The effects of norLEU were specific because it did not inhibit the IL-1 beta induction of plasminogen activator inhibitor type 1 gene expression. This study demonstrates that inhibition of the proteolytic activity of the proteasome blocks IL-1 beta induction of VCAM-1 and ICAM-1 gene expression in human endothelial cells.
Asunto(s)
Calpaína/fisiología , Núcleo Celular/metabolismo , Cisteína Endopeptidasas/fisiología , Endotelio Vascular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Molécula 1 de Adhesión Intercelular/biosíntesis , Leupeptinas/farmacología , Complejos Multienzimáticos/fisiología , FN-kappa B/metabolismo , Clorometilcetona Tosilisina/farmacología , Clorometilcetona de Tosilfenilalanila/farmacología , Molécula 1 de Adhesión Celular Vascular/biosíntesis , Secuencia de Bases , Transporte Biológico/efectos de los fármacos , Calpaína/antagonistas & inhibidores , Células Cultivadas , Endotelio Vascular/efectos de los fármacos , Humanos , Molécula 1 de Adhesión Intercelular/genética , Interleucina-1/farmacología , Datos de Secuencia Molecular , Inhibidor 1 de Activador Plasminogénico/biosíntesis , Inhibidor 1 de Activador Plasminogénico/genética , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Proteínas Recombinantes de Fusión/metabolismo , Transfección , Molécula 1 de Adhesión Celular Vascular/genéticaRESUMEN
Vascular endothelial cells respond to cytokines such as IL-1 beta or TNF-alpha by undergoing a number of functional alterations. Among these alterations is the induction of cell surface adhesion molecules, including VCAM-1. In this report, we investigated the effects of a 3-alkoxybenzo[beta]thiophene-2-carboxamide (BZT) on the cytokine induction of VCAM-1 expression and activation of the transcription factor NF-kappa B in human endothelial cells. BZT blocked the IL-1 beta induced cell surface expression of VCAM-1 in human endothelial cells but did not prevent nuclear translocation of NF-kappa B. This study demonstrates that BZT is a potent inhibitor of VCAM-1 expression in human endothelial cells.
Asunto(s)
Endotelio Vascular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Interleucina-1/farmacología , FN-kappa B/metabolismo , Tiofenos/farmacología , Molécula 1 de Adhesión Celular Vascular/genética , Secuencia de Bases , Transporte Biológico , Núcleo Celular/metabolismo , Células Cultivadas , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Humanos , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , ARN Mensajero/análisis , Tiofenos/síntesis química , Clorometilcetona de Tosilfenilalanila/farmacología , Venas UmbilicalesRESUMEN
Mouse lymphoma cells of the L5178Y TK + /-3.7.2C line were exposed to varying concentrations of the anti-schistosomal drug hycanthone methanesulfonate. The trifluorothymidine (TFT)-resistant cells fell into two classes based on colony size. Southern blot analyses were performed using NcoI-digested DNA from a number of large and small mutant colonies from each treatment group. Two different restriction fragment banding patterns were identified in these analyses, those colonies that contained the 6.4 kb NcoI restriction fragment and those that did not. A total of 471 mutant colonies were analyzed and 84.5% (398) of these colonies did not exhibit the 6.4 kb fragment. There did not appear to be a hycanthone methanesulfonate dose response effect in the number of colonies that did not contain the 6.4 kb fragment among the treated groups. In addition, 82% (154 out of 188) of spontaneous mutants did not contain the 6.4 kb fragment. The results imply that greater than 80% of all spontaneous mutations found in the mouse lymphoma assay regardless of colony size do not contain the 6.4 kb fragment and each may be considered to be a large scale mutation. In addition, greater than 80% of the hycanthone induced mutations in the mouse lymphoma assay do not contain the 6.4 kb fragment and thus may be considered to be a large scale mutation.
Asunto(s)
Hicantona/análogos & derivados , Linfoma/genética , Mutágenos/toxicidad , Timidina Quinasa/genética , Animales , División Celular/efectos de los fármacos , Hicantona/toxicidad , Linfoma/enzimología , Linfoma/patología , Ratones , Mutación , Células Tumorales CultivadasRESUMEN
The integrin supergene family includes receptors for a variety of extracellular matrix as well as cell surface proteins. Integrin alpha 4 has been shown to play an important role in leukocyte adhesion and extravasation during immune and inflammatory reactions. One recognition sequence known to interact with alpha 4 is the Leu-Asp-Val (LDV) site contained in the connecting segment 1 region of fibronectin. Here we present evidence that shows that a conformationally restricted RGD-containing peptide is a potent inhibitor of cell adhesion mediated by alpha 4 beta 1, a receptor not convincingly documented to interact with RGD peptides. This peptide, 1-adamantaneacetyl-Cys-Gly-Arg-Gly-Asp-Ser-Pro-Cys (disulfide bridge between residues 1-8), blocks Jurkat cell adhesion to connecting segment 1-containing peptides as well as cell adhesion to cytokine-activated endothelial cells. Adhesion of Jurkat cells to either vascular cell adhesion molecule-expressing cells or recombinant vascular cell adhesion molecule-coated plates was likewise inhibited by this peptide. Furthermore, alpha 4 beta 1 can bind directly to a cyclic RGD peptide immobilized to Sepharose. Integrins, alpha 5 beta 1, alpha v beta 3, alpha IIb/beta IIIa, alpha 2 beta 1, alpha v beta 1, alpha v beta 5, alpha v beta 6, and alpha 3 beta 1, have been shown to recognize the Arg-Gly-Asp (RGD) sequence present in a variety of extracellular matrix proteins, and our data support the addition of alpha 4 beta 1 to this group. Further studies using molecular modeling of such cyclic RGD peptides could help in the design of more potent peptides or nonpeptide mimetics that could effectively block alpha 4-mediated activity and have potential application in a number of inflammatory diseases.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Adhesión Celular/efectos de los fármacos , Fibronectinas/metabolismo , Integrinas/fisiología , Fragmentos de Péptidos/metabolismo , Péptidos Cíclicos/farmacología , Secuencia de Aminoácidos , Anticuerpos/farmacología , Moléculas de Adhesión Celular/efectos de los fármacos , Línea Celular , Cromatografía de Afinidad , Relación Dosis-Respuesta a Droga , Proteínas de la Matriz Extracelular/metabolismo , Fibronectinas/antagonistas & inhibidores , Humanos , Integrina alfa4beta1 , Integrinas/antagonistas & inhibidores , Cinética , Datos de Secuencia Molecular , Familia de Multigenes , Fragmentos de Péptidos/antagonistas & inhibidores , Péptidos/síntesis química , Péptidos/farmacología , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Transfección , Células Tumorales Cultivadas , Molécula 1 de Adhesión Celular VascularRESUMEN
A recent recombination event within the mouse alpha-globin gene region was discovered in the recombinant inbred mouse strain AKXL-7. Polymorphisms (RFLPs) that distinguish the AKR and C57L strains were used to determine the parental origins of segments of the AKXL-7 Chromosome 11. Southern blot analysis revealed that the left and right AKXL-7 flanking regions originated from different parental chromosomes, indicating that a recombination event had occurred within the locus complex during the generation of this RI strain. Further analysis using an intergenic region probe narrowed the region of crossover to approximately 5.2 kb, located between the tandem adult alpha-globin genes. Additional mouse strains were studied concurrently to identify RFLPs that may be informative for defining evolutionary relationships and serve as markers to discriminate among different inbred strains as well as different mouse species.
Asunto(s)
Globinas/genética , Polimorfismo de Longitud del Fragmento de Restricción , Recombinación Genética , Animales , Evolución Biológica , Southern Blotting , Ratones , Ratones Endogámicos AKR , Ratones Endogámicos C57BL , Especificidad de la EspecieRESUMEN
Inflammation, metastasis and ischemia are processes that require lymphocyte or leukocyte cell recognition and adherence to endothelial counter receptors such as ICAM-1. Mapping the sites of interaction of ICAM-1 with LFA-1, the receptor for ICAM-1 on lymphocytes, may lead to the design of novel inhibitors of inflammation or metastasis. To this end, recombinant soluble ICAM-1 cDNA was engineered into the baculovirus expression system, which is capable of expressing large amounts of proteins. These constructs were designed to contain a protein leader sequence so that the transfected insect cells would secrete the recombinant polypeptide into the culture media for ease of isolation. We engineered four constructs of ICAM-1 into the baculovirus system and obtained relatively high expression of two soluble forms of ICAM-1, a two domain and a five domain form. These truncated proteins were isolated and shown to promote adherence of HL-60 cells and Molt-4 cells. These recombinant soluble proteins also inhibited cell adherence to purified intact ICAM-1 isolated from K562 cells.
Asunto(s)
Baculoviridae/genética , Moléculas de Adhesión Celular/genética , Adhesión Celular/efectos de los fármacos , Transfección , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Moléculas de Adhesión Celular/biosíntesis , Moléculas de Adhesión Celular/aislamiento & purificación , Línea Celular , Clonación Molecular , Escherichia coli/genética , Vectores Genéticos , Humanos , Insectos , Molécula 1 de Adhesión Intercelular , Datos de Secuencia Molecular , Oligodesoxirribonucleótidos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacologíaRESUMEN
Ethylene dibromide (1,2-dibromoethane; EDB) was tested for the induction of dominant lethal and electrophoretically-detectable specific-locus mutations in the germ cells of DBA/2J male mice. Males were treated with a single intraperitoneal injection of 100 mg/kg EDB and mated to two C57BL/6J females. In the dominant lethal assay, matings were carried out to measure the effect of EDB on meiotic and postmeiotic stages; germ cells representing spermatogonial stem cells were analyzed in the electrophoretic specific-locus test. Neither of these germ cell tests produced any evidence that EDB is a germ cell mutagen. It appears from these data and those reported in the literature that EDB, a genotoxic carcinogen that affects male fertility in some mammalian species, is not mutagenic in the germ cells of the male mouse.
Asunto(s)
Dibromuro de Etileno/toxicidad , Animales , Fertilidad/efectos de los fármacos , Genes Dominantes , Genes Letales , Masculino , Ratones , Pruebas de MutagenicidadRESUMEN
The cytoplasmic malic enzyme (Mod-1) catalyzes the oxidative decarboxylation of malate: malate + NADP+----pyruvate + CO2 + NADPH + H+. Using a cDNA clone of Mod-1 as a probe, two new DNA markers not at the Mod-1 locus (restriction fragment length polymorphisms, RFLP) were detected by Southern blot analysis that showed extensive homology to Mod-1 sequences. Linkage of each restriction fragment length polymorphism to loci other than Mod-1 was assessed using the BXD (C57BL/6J x DBA/2J) recombinant inbred strains and confirmed by backcrosses. One polymorphic site, designated D9Rti1, was found to be closely linked to the phosphoglucomutase (Pgm-3) locus on Chromosome 9. The other hybridization site, designated D1Rti2, was closely linked to the isocitrate dehydrogenase (Idh-1) locus on Chromosome 1. The data presented imply that Mod-1 homologous sequences are tightly linked to three different metabolic enzymes.