RESUMO
Background: Mesenchymal Stem/Stromal Cells (MSCs) from the decidua parietalis (DPMSCs) of human term placenta express several molecules with important biological and immunological properties. DPMSCs induce natural killer cell expression of inflammatory receptors and their cytotoxic activity against cancer cells. These properties make DPMSCs promising therapeutical agent for cancer. The successful development of MSCs as an anti-cancer therapeutic cells rely on their ability to function in a hostile inflammatory and oxidative stress cancer environment. Here, we studied the effects of conditioned medium obtained from the culture of breast cancer cells (CMMDA-231) on the functional and phenotypic properties of DPMSCs. Methods: DPMSCs were cultured with CMMDA-231 and important functions of DPMSCs were measured. The effect of CMMDA-231 on DPMSC expression of several genes with different functions was also evaluated. Results: DPMSCs were able to function in response to CMMDA-231, but with reduced proliferative and adhesive potentials. Preconditioning of DPMSCs with CMMDA-231 enhanced their adhesion while reducing their invasion. In addition, CMMDA-231 modulated DPMSC expression of many genes with various functional (i.e., proliferation, adhesion, and invasion) properties. DPMSCs also showed increased expression of genes with anti-cancer property. Conclusion: These data show the ability of DPMSCs to survive and function in cancer environment. In addition, preconditioning of DPMSCs with CMMDA-231 enhanced their anti-cancer properties and thus demonstrating their potential as an anti-cancer therapeutic agent. However, future studies are essential to reveal the mechanism underlying the effects of MDA-231 on DPMSC functional activities and also to confirm the anti-cancer therapeutic potential of DPMSCs.
Assuntos
Proliferação de Células/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultivo Condicionados/química , Decídua/citologia , Feminino , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Neoplasias/metabolismo , Neoplasias/patologia , GravidezRESUMO
BACKGROUND: Mesenchymal stem cells derived from the chorionic villi of human placentae (pMSCs) produce a unique array of mediators that regulate the essential cellular functions of their target cells. These properties make pMSCs attractive candidates for cell-based therapy. Here, we examined the effects of culturing human natural killer (NK) cells with pMSCs on NK cell functions. METHODS: pMSCs were cultured with IL-2-activated and non-activated NK cells. NK cell proliferation and cytolytic activities were monitored. NK cell expression of receptors mediating their cytolytic activity against pMSCs, and the mechanisms underlying this effect on pMSCs, were also investigated. RESULTS: Our findings show that IL-2-activated NK cells, but not freshly isolated NK cells, efficiently lyse pMSCs and that this response might involve the activating NK cell receptor CD69. Interestingly, although pMSCs expressed HLA class I molecules, they were nevertheless lysed by NK cells, suggesting that HLA class I antigens do not play a significant role in protecting pMSCs from NK cell cytolytic activity. Co-culturing NK cells with pMSCs also inhibited NK cell expression of receptors, including CD69, NKpG2D, CD94, and NKp30, although these co-cultured NK cells were not inhibited in lysing cancer cells in vitro. Importantly, co-cultured NK cells significantly increased their production of molecules with anti-tumor effects. CONCLUSIONS: These findings suggest that pMSCs might have potential applications in cancer therapy.
Assuntos
Vilosidades Coriônicas/metabolismo , Células Matadoras Naturais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Diferenciação Celular , Proliferação de Células , HumanosRESUMO
BACKGROUND: Human decidua basalis mesenchymal stem/multipotent stromal cells (DBMSCs) inhibit endothelial cell activation by inflammation induced by monocytes. This property makes them a promising candidate for cell-based therapy to treat inflammatory diseases, such as atherosclerosis. This study was performed to examine the ability of DBMSCs to protect endothelial cell functions from the damaging effects resulting from exposure to oxidatively stress environment induced by H2O2 and monocytes. METHODS: DBMSCs were co-cultured with endothelial cells isolated from human umbilical cord veins in the presence of H2O2 and monocytes, and various functions of endothelial cell were then determined. The effect of DBMSCs on monocyte adhesion to endothelial cells in the presence of H2O2 was also examined. In addition, the effect of DBMSCs on HUVEC gene expression under the influence of H2O2 was also determined. RESULTS: DBMSCs reversed the effect of H2O2 on endothelial cell functions. In addition, DBMSCs reduced monocyte adhesion to endothelial cells and also reduced the stimulatory effect of monocytes on endothelial cell proliferation in the presence of H2O2. Moreover, DBMSCs modified the expression of many genes mediating important endothelial cell functions. Finally, DBMSCs increased the activities of glutathione and thioredoxin reductases in H2O2-treated endothelial cells. CONCLUSIONS: We conclude that DBMSCs have potential for therapeutic application in inflammatory diseases, such as atherosclerosis by protecting endothelial cells from oxidative stress damage. However, more studies are needed to elucidate this further.
Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Peróxido de Hidrogênio/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Monócitos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Adulto , Antígenos CD/genética , Antígenos CD/metabolismo , Biomarcadores/metabolismo , Adesão Celular/efeitos dos fármacos , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados/farmacologia , Decídua/citologia , Decídua/metabolismo , Feminino , Expressão Gênica , Glutationa Redutase/genética , Glutationa Redutase/metabolismo , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Monócitos/citologia , Gravidez , Tiorredoxina Dissulfeto Redutase/genética , Tiorredoxina Dissulfeto Redutase/metabolismo , Cordão Umbilical/citologia , Cordão Umbilical/metabolismoRESUMO
BACKGROUND: Mesenchymal stem/stromal cells derived from chorionic villi of human term placentae (pMSCs) protect human endothelial cells from injury induced by hydrogen peroxide (H2O2). In diabetes, elevated levels of glucose (hyperglycaemia) induce H2O2 production, which causes the endothelial dysfunction that underlies the enhanced immune responses and adverse complications associated with diabetes, which leads to thrombosis and atherosclerosis. In this study, we examined the ability of pMSCs to protect endothelial cell functions from the negative impact of high level of glucose. METHODS: pMSCs isolated from the chorionic villi of human term placentae were cultured with endothelial cells isolated from human umbilical cord veins in the presence of glucose. Endothelial cell functions were then determined. The effect of pMSCs on gene expression in glucose-treated endothelial cells was also determined. RESULTS: pMSCs reversed the effect of glucose on key endothelial cell functions including proliferation, migration, angiogenesis, and permeability. In addition, pMSCs altered the expression of many genes that mediate important endothelial cell functions including survival, apoptosis, adhesion, permeability, and angiogenesis. CONCLUSIONS: This is the first comprehensive study to provide evidence that pMSCs protect endothelial cells from glucose-induced damage. Therefore, pMSCs have potential therapeutic value as a stem cell-based therapy to repair glucose-induced vascular injury and prevent the adverse complications associated with diabetes and cardiovascular disease. However, further studies are necessary to reveal more detailed aspects of the mechanism of action of pMSCs on glucose-induced endothelial damage in vitro and in vivo.
Assuntos
Meios de Cultivo Condicionados/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/farmacologia , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Apoptose/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Quimiocinas/genética , Quimiocinas/metabolismo , Vilosidades Coriônicas/metabolismo , Técnicas de Cocultura , Colágeno/química , Colágeno/genética , Colágeno/metabolismo , Combinação de Medicamentos , Endotelinas/genética , Endotelinas/metabolismo , Feminino , Glucose/antagonistas & inibidores , Células Endoteliais da Veia Umbilical Humana/citologia , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Interleucinas/genética , Interleucinas/metabolismo , Laminina/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Células-Tronco Mesenquimais/citologia , Gravidez , Proteoglicanas/química , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Transdução de Sinais , Células THP-1 , Cordão Umbilical/citologia , Cordão Umbilical/metabolismoRESUMO
Stem cell-based therapies rely on stem cell ability to repair in an oxidative stress environment. Preconditioning of mesenchymal stem cells (MSCs) to a stress environment has beneficial effects on their ability to repair injured tissues. We previously reported that MSCs from the decidua basalis (DBMSCs) of human placenta have many important cellular functions that make them potentially useful for cell-based therapies. Here, we studied the effect of DBMSC preconditioning to a stress environment. DBMSCs were exposed to various concentrations of hydrogen peroxide (H2O2), and their functions were then assessed. DBMSC expression of immune molecules after preconditioning was also determined. DBMSC preconditioning with H2O2 enhanced their proliferation, colonogenicity, adhesion, and migration. In addition, DBMSCs regardless of H2O2 treatment displayed antiangiogenic activity. H2O2 preconditioning also increased DBMSC expression of genes that promote cellular functions and decreased the expression of genes, which have opposite effect on their functions. Preconditioning also reduced DBMSC expression of IL-1ß, but had no effects on the expression of other immune molecules that promote proliferation, adhesion, and migration. These data show that DBMSCs resist a toxic environment, which adds to their potential as a candidate stem cell type for treating various diseases in hostile environments.
RESUMO
BACKGROUND: Human decidua parietalis mesenchymal stem/multipotent stromal cells (DPMSCs) have unique phenotypic and functional properties that make them promising candidates for cell-based therapy. Here, we investigated DPMSC interaction with natural killer (NK) cells, and the effects of this interaction on NK cell phenotypic characteristics and functional activities. METHODS: DPMSCs isolated from the decidua parietalis of human fetal membranes were cultured with interleukin (IL)-2-activated and IL-2-unactivated NK cells isolated from healthy human peripheral blood. NK cell proliferation and cytolytic activities were then examined using functional assays. NK cell expression of receptors mediating the cytolytic activity against DPMSCs, and the mechanism underlying this effect on DPMSCs, were also examined using flow cytometry and light microscopy, respectively. RESULTS: DPMSCs stimulated IL-2-induced proliferation of resting NK cells and the proliferation of activated NK cells. Moreover, IL-2-activated NK cells, but not freshly isolated NK cells, efficiently lysed DPMSCs. The induction of this NK cell cytolytic activity against DPMSCs was mediated by the activating NK cell receptors NKG2D, CD69, NKp30, and NKp44. However, DPMSCs showed a direct induction of NK cell cytolytic activity through CD69. We also found that DPMSCs expressed the ligands for these activating NK cell receptors including Nectin-2, ULBP-2, MICA, and MICB. Although DPMSCs expressed HLA class I molecules, they were susceptible to lysis by NK cells, suggesting that HLA class I antigens do not play a significant role in NK cell cytolytic action. In addition, DPMSCs did not inhibit NK cell cytolytic activity against cancer cells. Importantly, DPMSCs significantly increased NK expression of inflammatory molecules with anticancer activities. CONCLUSIONS: We conclude that DPMSCs have potential for therapeutic application in cancer therapy, but not in transplantation or immunological diseases.
Assuntos
Decídua/metabolismo , Células Matadoras Naturais/imunologia , Células-Tronco Mesenquimais/imunologia , Feminino , Humanos , MasculinoRESUMO
Mesenchymal stem cell (MSC) therapies for the treatment of diseases associated with inflammation and oxidative stress employ primarily bone marrow MSCs (BMMSCs) and other MSC types such as MSC from the chorionic villi of human term placentae (pMSCs). These MSCs are not derived from microenvironments associated with inflammation and oxidative stress, unlike MSCs from the decidua basalis of the human term placenta (DBMSCs). DBMSCs were isolated and then extensively characterized. Differentiation of DBMSCs into three mesenchymal lineages (adipocytes, osteocytes, and chondrocytes) was performed. Real-time polymerase chain reaction (PCR) and flow cytometry techniques were also used to characterize the gene and protein expression profiles of DBMSCs, respectively. In addition, sandwich enzyme-linked immunosorbent assay (ELISA) was performed to detect proteins secreted by DBMSCs. Finally, the migration and proliferation abilities of DBMSCs were also determined. DBMSCs were positive for MSC markers and HLA-ABC. DBMSCs were negative for hematopoietic and endothelial markers, costimulatory molecules, and HLA-DR. Functionally, DBMSCs differentiated into three mesenchymal lineages, proliferated, and migrated in response to a number of stimuli. Most importantly, these cells express and secrete a distinct combination of cytokines, growth factors, and immune molecules that reflect their unique microenvironment. Therefore, DBMSCs could be attractive, alternative candidates for MSC-based therapies that treat diseases associated with inflammation and oxidative stress.
RESUMO
BACKGROUND: Calcium-dependent signaling mechanisms play a critical role in platelet activation. Unlike calcium-activated protease and kinase, the contribution of calcium-activated protein serine/threonine phosphatase in platelet activation is poorly understood. OBJECTIVE: To assess the role of catalytic subunit of protein phosphatase 2B (PP2B) or calcineurin in platelet function. RESULTS: Here, we showed that an increase in PP2B activity was associated with agonist-induced activation of human and murine platelets. Pharmacological inhibitors of the catalytic subunit of protein phosphatase 2B (PP2B-A) such as cyclosporine A or tacrolimus (FK506) potentiated aggregation of human platelets. Murine platelets lacking the ß isoform of PP2B-A (PP2B-Aß(-/-) ) displayed increased aggregation with low doses of agonist concentrations. Loss of PP2B-Aß did not affect agonist-induced integrin αII b ß3 inside-out signaling, but increased basal Src activation and outside-in αII b ß3 signaling to p38 mitogen-activated protein kinase (MAPK), with a concomitant enhancement in platelet spreading on immobilized fibrinogen and greater fibrin clot retraction. Fibrinogen-induced increased p38 activation in PP2B-Aß(-/-) platelets were blocked by Src inhibitor. Both PP2B-Aß(-/-) platelets and PP2B-Aß-depleted human embryonal kidney 293 αII b ß3 cells displayed increased adhesion to immobilized fibrinogen. Filamin A, an actin crosslinking phosphoprotein that is known to associate with ß3 , was dephosphorylated on Ser(2152) in fibrinogen-adhered wild-type but not in PP2B-Aß(-/-) platelets. In a FeCl3 injury thrombosis model, PP2B-Aß(-/-) mice showed decreased time to occlusion in the carotid artery. CONCLUSION: These observations indicate that PP2B-Aß by suppressing outside-in αII b ß3 integrin signaling limits platelet response to vascular injury.
Assuntos
Plaquetas/citologia , Calcineurina/metabolismo , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/metabolismo , Animais , Artérias Carótidas/metabolismo , Domínio Catalítico , Adesão Celular , Cloretos/química , Feminino , Compostos Férricos/química , Fibrinogênio/metabolismo , Citometria de Fluxo , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Fosforilação , Agregação Plaquetária , Isoformas de Proteínas , Transdução de Sinais , Trombose/fisiopatologiaRESUMO
BACKGROUND: Secretion of Weibel-Palade body (WPB) contents is regulated, in part, by the phosphorylation of proteins that constitute the endothelial exocytotic machinery. In comparison to protein kinases, a role for protein phosphatases in regulating endothelial exocytosis is undefined. OBJECTIVE AND METHOD: In this study, we investigated the role of protein phosphatase 2B (PP2B) in the process of endothelial exocytosis using pharmacological and gene knockdown approaches. RESULTS: We show that inhibition of protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus or a cell-permeable PP2B autoinhibitory peptide promotes the secretion of ultralarge von Willebrand factor (ULVWF) from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs mediate platelet tethering. In support of a role for PP2B in von Willebrand factor (VWF) secretion, the catalytic subunit of PP2B interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, is increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis. Furthermore, the plasma VWF antigen level is also enhanced in CsA-treated mice, and small interfering RNA-mediated knockdown of the alpha and beta isoforms of the PP2B-A subunit in HUVECs enhanced VWF secretion. CONCLUSIONS: These observations suggest that CsA promotes VWF release, in part by inhibition of PP2B activity, and are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans.
Assuntos
Inibidores de Calcineurina , Endotélio Vascular/metabolismo , Fator de von Willebrand/metabolismo , Animais , Sequência de Bases , Calcineurina/genética , Células Cultivadas , Primers do DNA , Endotélio Vascular/citologia , Inibidores Enzimáticos/farmacologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
c-Jun N-terminal kinase (JNK) has been reported to either potentiate or inhibit oncogenesis, depending upon the cellular context, but its role in lung neoplasia is unclear. Here we sought to define the role of JNK in lung neoplasia by examining evidence of JNK phosphorylation in non-small-cell lung cancer (NSCLC) biopsy samples and by using genetic and pharmacologic approaches to modulate JNK expression and activity in cultured cells. Immunohistochemical staining for JNK phosphorylation was detected in 114 (45%) of 252 NSCLC biopsy samples and was predominantly nuclear, providing evidence of JNK activation in a subset of NSCLC cases. Introduction of a doxycycline-inducible, constitutively active, mutant mitogen-activated protein kinase kinase 4 (MKK4) into the human bronchial epithelial cell lines BEAS-2B and HB56B increased the cells' proliferation, migration, invasion and clonogenicity. Depletion of JNK in MKK4 mutant-transformed BEAS-2B cells by introduction of JNK1/2 short hairpin RNA reversed the transformed phenotype, indicating that JNK activation is oncogenic and MKK4 confers neoplastic properties in these cells. The proliferation of NSCLC cell lines HCC827 and H2009, in which JNK and its substrate c-Jun are constitutively phosphorylated, was inhibited by SP600125, a JNK kinase inhibitor. We conclude that JNK is activated in a subset of NSCLC biopsy samples and promotes oncogenesis in the bronchial epithelium, suggesting that strategies to inhibit the JNK pathway should be considered for the prevention and treatment of NSCLC.
Assuntos
Brônquios/metabolismo , Carcinoma Pulmonar de Células não Pequenas/enzimologia , Transformação Celular Neoplásica , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Brônquios/citologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Movimento Celular , Proliferação de Células , Células Cultivadas , Ativação Enzimática , Células Epiteliais/metabolismo , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Neoplasias Pulmonares/enzimologia , Neoplasias Pulmonares/patologia , MAP Quinase Quinase 4/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-jun/genética , Transdução de SinaisRESUMO
Melanoma antigens (MAGE) are thought to induce a tumor-specific immune response and to be potential therapeutical targets for cancer immunotherapy. We have earlier identified the cDNA of feline melanoma antigen 1 (fMAGE-1), but its product was not characterized in detail. We have expressed the recombinant fMAGE-1 protein and have generated monoclonal antibodies (mAbs) against it, to identify the native fMAGE-1 protein in feline lymphoma cell lines and tumor tissues. The fMAGE-1 protein was found to be approximately 39 kDa in molecular mass on sodium dodecyl-sulphate-polycrylamide gel electrophoresis (SDS-PAGE), and it was found to be located in the cytoplasm of the cells by immunofluorescence. Immunoblotting analysis detected the fMAGE-1 gene product in the fMAGE-1-mRNA-positive cells, but not in the fMAGE-1-mRNA-negative cells. An interesting finding of the present study was the distribution of the fMAGE-1 protein, which was found to have a spindle-like distribution, with filaments twining around the nucleus, suggesting that the fMAGE-1 protein may be associated with or form some cytoplasmic filaments. This type of finding is so far the first report of its kind, and to the best of our knowledge it has not been reported in either human or mouse MAGE proteins until now. It most probably implies the major diversity of the MAGE family genes.
Assuntos
Anticorpos Monoclonais/imunologia , Proteínas de Neoplasias/imunologia , Animais , Antígenos de Neoplasias , Gatos , Linhagem Celular , Técnica Indireta de Fluorescência para Anticorpo/métodos , Glutationa Transferase/metabolismo , Humanos , Antígenos Específicos de Melanoma , Proteínas de Neoplasias/análise , Proteínas de Neoplasias/genética , Fases de Leitura Aberta , Sensibilidade e EspecificidadeRESUMO
Melanoma antigens (MAGE), thought to induce tumor-specific immune responses, are used as potential therapeutical targets for cancer immunotherapy. We hereby report the cloning and sequencing of MAGE cDNA clone, called feline MAGE-2 (fMAGE-2), obtained from a lymphoma cell line. fMAGE-2 cDNA is 1535 base pairs (bp) in length and contains an open reading frame (ORF) of 1131 bp encoding a protein of 376 amino acids. The predicted amino acid sequence shows 45%, 32-42%, 44-47%, and 33% homology with feline MAGE-1, human MAGE-A, human MAGE-B, and human MAGE-C proteins, respectively. mRNA transcripts of fMAGE-2 were detected by RT-PCR in some feline tumors, as well as in testis of adult cat, but not in other normal tissues, indicating that the expression pattern of fMAGE-2 is similar to that of the human MAGE family genes in tumors and normal tissues.
Assuntos
Linfoma/imunologia , Proteínas de Neoplasias/genética , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias , Sequência de Bases , Gatos , Clonagem Molecular , Antígenos Específicos de Melanoma , Dados de Sequência Molecular , Proteínas de Neoplasias/análise , Filogenia , Análise de Sequência de DNA , Células Tumorais CultivadasRESUMO
Melanoma antigens (MAGE) are regarded as inducing tumor-specific immune response and thought to be potential therapeutical agents for cancer immunotherapy. We hereby report the cloning of feline MAGE cDNA obtained from a lymphoma cell line derived from cat malignant lymphoma, and its expression pattern in tumor and normal tissues. The cDNA encoding the MAGE is 1668 base pairs (bp) in length, and contains an open reading frame (ORF) of 936 bp encoding a protein of 311 amino acids. The predicted amino acid sequence has 29-46% of homology with other MAGE proteins from human and mouse. mRNA transcripts for the feline MAGE were detected in certain tumors, but not in adult cat normal tissues except in testis, by reverse transcription polymerase chain reaction (RT-PCR) analysis. This indicates that the expression pattern of feline MAGE mRNA is similar to those of other MAGE family genes in tumors and normal tissues.
Assuntos
Antígenos de Neoplasias/genética , Doenças do Gato/imunologia , Linfoma/veterinária , Proteínas de Neoplasias/genética , Sequência de Aminoácidos , Animais , Antígenos de Neoplasias/biossíntese , Sequência de Bases , Southern Blotting , Doenças do Gato/genética , Gatos , Clonagem Molecular , DNA de Neoplasias/química , Regulação Neoplásica da Expressão Gênica/imunologia , Linfoma/genética , Linfoma/imunologia , Antígenos Específicos de Melanoma , Dados de Sequência Molecular , Proteínas de Neoplasias/biossíntese , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Técnica de Amplificação ao Acaso de DNA Polimórfico/veterinária , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
Serum amyloid A (SAA) has been characterized as an inflammatory marker in many species. In this study, we have developed and characterized monoclonal antibodies (MAbs) against feline SAA (fSAA) derived from culture hybridomas. These hybridomas were produced from the fusion of Balb/c-derived myeloma s/p20-Ag14 and splenocytes from mice immunized with purified recombinant feline SAA (rfSAA). Six hybridomas secreting MAbs, M2, M5, M7, M8, M13, and M15, were selected and subcloned on the basis of their specificity to rfSAA by enzyme-linked immunoabsorbent assay (ELISA), and confirmed based on their specificity to rf-SAA by immunoblot analysis. Out of six clones, two clones (M5 and M7) showed higher reactivity with rf-SAA, and were selected for further analysis of ELISA additivity and Western blot cross-reactivity tests. As a result, M5 and M7 clones recognized the same or excessively near epitopes on rfSAA and reacted with rfSAA, fSAA and equine recombinant SAA, but showed no reaction with human recombinant SAA. Because of their specificity, these MAbs may be usefully applied in studying the measurement of SAA concentration in cat serum.
Assuntos
Anticorpos Monoclonais/imunologia , Proteína Amiloide A Sérica/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Especificidade de Anticorpos , Gatos , Reações Cruzadas/imunologia , Epitopos , Hibridomas , Camundongos , Dados de Sequência Molecular , Alinhamento de SequênciaRESUMO
T-cells express CD28 and CTLA-4, and through binding to their shared ligands (CD80/CD86) on antigen presenting cells, provide a potent co-stimulatory signal for T-cell activation and proliferation. To investigate the role of CD28 in canine immune system, we hereby report the molecular cloning and sequencing of the full-length complementary DNA (cDNA) coding for canine CD28, from pokeweed mitogen stimulated canine peripheral blood lymphocytes. The cloned cDNA contains an open reading frame of 663 nucleotides, encoding for a polypeptide of 221 amino acids. The amino acid sequence of the canine CD28 showed 91.9, 80, and 79.6% similarities with those of the cat, cattle, and human counterparts, respectively. Five sequence motifs of TATT or ATTTA involved in the regulation of gene expression by influencing mRNA stability are found in the 3' untranslated region. The hexapeptide motif (MYPPPY), five cysteine residues, a potential N-glycosylation site and a cytoplasmic phosphatidylinositol 3-kinase binding site in canine CD28 molecule are completely conserved in canine CTLA-4. The availability of full length canine CD28 will provide a useful molecule for studying its role in dog immune system.
Assuntos
Antígenos CD28/química , Antígenos CD28/genética , Imunoconjugados , Abatacepte , Sequência de Aminoácidos , Animais , Antígenos CD , Antígenos de Diferenciação/química , Antígenos de Diferenciação/metabolismo , Sequência de Bases , Sítios de Ligação , Antígeno CTLA-4 , Clonagem Molecular , Cisteína/análise , Cães , Dados de Sequência Molecular , Fases de Leitura Aberta , Fosfatidilinositol 3-Quinases/metabolismo , Ligação Proteica , RNA Mensageiro/análiseRESUMO
Tektins are a group of proteins that form filamentous polymers in the walls of ciliary microtubules. The cloning of canine cDNA encoding tektin, was carried out and identified from the testis of beagle dog. Canine tektin cDNA is 1,523 bp in length, has an open reading frame of 1,281 bp nucleotides encoding a protein of 426 deduced amino acids. The predicted amino acid sequence has 77% and 33-50% of homology with the murine tektin and the sea urchin tektins. The amino acid sequence RPNVELCRD and four cysteine residues were conserved in the dog, mouse and sea urchin, suggesting the functional significance of this protein domain and the amino acid residues in the tektin proteins.
Assuntos
Cães/genética , Proteínas dos Microtúbulos/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Camundongos , Dados de Sequência MolecularRESUMO
Melanoma antigens (MAGE) are regarded to induce tumour-specific immune response and thought to be potential therapeutical agents for cancer immunotherapy. We hereby report the canine MAGE cDNA cloned from the testis of a beagle dog. Canine MAGE cDNA is 1,455 base pair (bp) nucleotides in length, and contains an open reading frame (ORF) of 1,137 bp nucleotides encoding a protein of 378 amino acids. The predicted amino acid sequence has 22-49% of homology with other MAGE proteins. mRNA transcripts of canine MAGE were detected only in the melanoma and testis and not in other normal tissues of adult dog by reverse transcriptase-polymerase chain reaction (RT-PCR), indicating that the expression pattern of canine MAGE mRNA is similar to that of the MAGE family genes in tumor and normal tissues.
Assuntos
Melanoma/imunologia , Proteínas de Neoplasias/genética , Animais , Sequência de Bases , Clonagem Molecular , DNA Complementar , Cães , Masculino , Melanoma/genética , Dados de Sequência Molecular , Proteínas de Neoplasias/imunologia , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Linfócitos T Citotóxicos/imunologia , Testículo/imunologiaAssuntos
Antígenos de Diferenciação/genética , DNA Complementar/genética , Cães/genética , Cães/imunologia , Imunoconjugados , Abatacepte , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Antígenos CD , Antígenos de Diferenciação/química , Sequência de Bases , Antígeno CTLA-4 , Clonagem Molecular , Sequência Conservada/genética , Glicosilação , Humanos , Dados de Sequência Molecular , Sinais Direcionadores de Proteínas/genética , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
The serum amyloid A (SAA) protein is a characteristic and sensitive acute phase reactant in all vertebrates investigated. We molecularly cloned the equine cDNA encoding SAA from the liver of a healthy horse by polymerase chain reaction (PCR). The cloned cDNA is 480 bases in length, and contains an open reading frame (ORF) of 387 nucleotides encoding a precursor SAA protein of 128 amino acids. The precursor of horse SAA seems to have an 18-residue signal peptide and differs from the reported amino acid sequences of the horse SAA by substitution of valine at residue 81. It shows high homology with SAA amino acid sequence of other species such as dog (80.6%), mink (77.5%), human (76.9%) and duck (71.9%). An insertion of eight amino acids at residues between 85 and 92, as compared to human SAA, has also been found in horse SAA. The availability of the equine SAA cDNA will provide a useful reagent for studying its role in diseased horses.
Assuntos
DNA Complementar/química , Proteína Amiloide A Sérica/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Cães , Cavalos , Humanos , Dados de Sequência Molecular , Proteína Amiloide A Sérica/químicaRESUMO
Cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) is a CD28 homologue which down-modulate T cell responses rather than augment them. To investigate its biological role in feline immune system, we cloned and sequenced full-length feline CTLA-4 (fCTLA-4) cDNA by RT-PCR from pokeweed mitogen stimulated peripheral blood lymphocytes. The fCTLA-4 contains an open reading frame of 669 nucleotides, coding for a polypeptide of 223 amino acids. The predicted fCTLA-4 amino acids sequence shows the homology of 86.6%, 87.0%, and 76.2% with human, bovine, and murine molecules respectively. The hexapeptide motif (MYPPPY) within the extra-cellular domain of CTLA-4 molecule, which is believed to be responsible for interaction with the B7 family members, is completely conserved in all the species.