RESUMO
In this study, we identified ecdysteroidogenic enzymes in the cabbage armyworm, Mamestra brassicae, and demonstrated reduced expression of these genes during diapause. Some insects employ a temporary developmental arrest, diapause, to survive in severe environments. The titres of the moulting hormone ecdysteroid were reduced in diapause pupae of M. brassicae; therefore, ecdysteroidogenesis might be suppressed by a diapause-specific mechanism. To clarify expression changes of ecdysteroidogenic enzyme genes during diapause in M. brassicae, we first identified the genes for seven ecdysteroidogenic enzymes: Neverland, Non-molting glossy (Nm-g), CYP307A1 (Spook), CYP306A1 (Phantom), CYP302A1 (Disembodied), CYP315A1 (Shadow) and CYP314A1 (Shade). Enzymatic assays using heterologous expression in Drosophila Schneider 2 (S2) cells and analysis of mRNA distribution indicated that the identified genes were ecdysteroidogenic enzymes of M. brassicae. Expression levels of these ecdysteroidogenic enzyme genes were compared between prothoracic glands in different pupal stages throughout diapause. Immediately after pupation, diapause-destined pupae showed similar expression levels of ecdysteroidogenic enzyme genes to those of nondiapause pupae. All of these genes showed reduced gene expression after diapause initiation. Expression was immediately increased in diapause-destined pupae at the postdiapause quiescence phase. These results indicate that reduced expression of ecdysteroidogenic enzyme genes suppresses ecdysteroidogenesis and maintains developmental arrest during diapause.
Assuntos
Diapausa de Inseto , Ecdisteroides/biossíntese , Mariposas/enzimologia , Animais , Linhagem Celular , Feminino , Expressão Gênica , Masculino , Mariposas/genética , Pupa/enzimologiaRESUMO
WHAT IS KNOWN AND OBJECTIVE: The pharmacokinetic-pharmacodynamic parameter that best predicts the efficacy of vancomycin is the ratio of the area under the concentration versus time curve (AUC) to the minimum inhibitory concentration (MIC). A 24-h AUC (AUC24 )/MIC ratio ≥ 400 was recommended in an American consensus review, but vancomycin treatment occasionally fails despite maintenance of AUC24 /MIC ≥ 400. We evaluated the association between clinical efficacy of vancomycin and two novel pharmacokinetic parameters, the 'area under the trough level' (AUTL) and the 'area above the trough level' (AATL), in hospitalized elderly patients with methicillin-resistant Staphylococcus aureus (MRSA) pneumonia. METHODS: The subjects were hospitalized elderly patients who were administered vancomycin for treatment of MRSA pneumonia between 2006 and 2012 at Sasebo Chuo Hospital (Nagasaki, Japan). Pharmacokinetic parameters of vancomycin were estimated for each patient by Bayesian analysis using population pharmacokinetic parameters for Japanese patients. Based on the patient-specific parameters thus obtained, AUC24 values were calculated as the vancomycin dosage divided by vancomycin clearance. AUTL was calculated as the trough serum concentration multiplied by 24 h, whereas AATL was calculated by subtracting AUTL from AUC24 . RESULTS AND DISCUSSION: Logistic regression analysis demonstrated that efficacy of vancomycin was more strongly associated with AUTL than AUC24 . The optimal cut-off value of AUTL was 331 µgâh/mL, which means that the optimal cut-off value of the trough serum concentration was 13·8 µg/mL. WHAT IS NEW AND CONCLUSION: Efficacy of vancomycin was associated with AUTL, a novel pharmacokinetic parameter. Determining the target AUTL or trough concentration may enhance the efficacy of vancomycin therapy in elderly patients with MRSA pneumonia. Given that nephrotoxicity may increase with a Ctrough in excess of 15 µg/mL, this level should ideally not be exceeded.
Assuntos
Antibacterianos/uso terapêutico , Infecção Hospitalar/tratamento farmacológico , Pneumonia Bacteriana/tratamento farmacológico , Vancomicina/uso terapêutico , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/administração & dosagem , Antibacterianos/farmacocinética , Área Sob a Curva , Teorema de Bayes , Infecção Hospitalar/microbiologia , Feminino , Hospitalização , Humanos , Japão , Modelos Logísticos , Masculino , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Testes de Sensibilidade Microbiana , Pneumonia Bacteriana/microbiologia , Infecções Estafilocócicas/tratamento farmacológico , Infecções Estafilocócicas/microbiologia , Resultado do Tratamento , Vancomicina/administração & dosagem , Vancomicina/farmacocinéticaRESUMO
T cell receptor-alpha mutant mice (TCR-alpha-/-), created by gene targeting of the TCR-alpha gene in embryonic stem cells, spontaneously develop inflammatory bowel disease (IBD) resembling human ulcerative colitis. Since gut-associated lymphoid tissue is likely to play an important role in the development of chronic intestinal inflammation, we examined the changes in the appendix lymphoid follicle (ALF) and Peyer's patches (PP) in these mice. We found the structure of the ALF to be remarkably similar to that of the PP in the small intestine; in both instances, lymphoid follicles covered by surface epithelium (dome-formation) were found. The amount of proliferation in the lymphoid follicles of the appendix estimated by in vivo incorporation of 5-bromo-2'deoxyuridine was more than two times that of PP in TCR-alpha-/- mice. ELISPOT assay showed an increase of IgA, IgG1, and IgG2a, but not IgM-secreting B cells in ALF of TCR-alpha-/- mice compared to TCR-alpha+/- control mice. Furthermore, TCR-alpha-/- mice revealed an increase of autoantibody-producing B cells against the cytoskeletal protein tropomyosin in ALF as compared to PP. When TCR-alpha-/- mice underwent appendectomy at a young age (3-5 wk), the number of mesenteric lymph nodes cells at 6-7 mo were markedly less than in the sham-operated TCR-alpha-/- mice. Furthermore, appendectomy at 1 mo of age suppressed the development of IBD, with only 3.3% of these mice developing IBD in the 6-7-mo period of observation. In contrast, approximately 80% of controls, including the sham-operated TCR-alpha-/- mice, developed IBD during this period. These results suggest that ALF, rather than PP, is the priming site of cells involved in the disease process and plays an important role in the development of IBD in TCR-alpha-/- mice.
Assuntos
Apêndice/fisiopatologia , Doenças Inflamatórias Intestinais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/imunologia , Animais , Apendicectomia , Autoanticorpos/biossíntese , Linfócitos B/imunologia , Divisão Celular , Imunidade nas Mucosas , Imunofenotipagem , Mucosa Intestinal/citologia , Antígenos Comuns de Leucócito/análise , Camundongos , Camundongos Knockout , Nódulos Linfáticos Agregados/citologia , Nódulos Linfáticos Agregados/imunologia , Receptores de IgE/análise , Linfócitos T/imunologiaRESUMO
The role of antibodies (Abs) in the development of chronic colitis in T cell receptor (TCR)-alpha-/- mice was explored by creating double mutant mice (TCR-alpha-/- x immunoglobulin (Ig)mu-/-), which lack B cells. TCR-alpha-/- x Ig mu-/- mice spontaneously developed colitis at an earlier age, and the colitis was more severe than in TCR-alpha-/- mice. Colitis was induced in recombination-activating gene-1 (RAG-1-/-) mice by the transfer of mesenteric lymph node (MLN) cells from TCR-alpha-/- x Ig mu-/- mice. When purified B cells from TCR-alpha-/- mice were mixed with MLN cells before cell transfer, colitis did not develop in RAG-1-/- mice. Administration of the purified Ig from TCR-alpha-/- mice and a mixture of monoclonal autoAbs reactive with colonic epithelial cells led to attenuation of colitis in TCR-alpha-/- x Ig mu-/- mice. Apoptotic cells were increased in the colon, MLN, and spleen of TCR-alpha-/- x Ig mu-/- mice as compared to Ig mu-/- mice and TCR-alpha-/- mice. Administration of the purified Ig from TCR-alpha-/- mice into TCR-alpha-/- x Ig mu-/- mice led to decrease in the number of apoptotic cells. These findings suggest that although B cells are not required for the initiation of colitis, B cells and Igs (autoAbs) can suppress colitis, presumably by affecting the clearance of apoptotic cells.
Assuntos
Subpopulações de Linfócitos B/imunologia , Colite/imunologia , Colite/prevenção & controle , Tolerância Imunológica , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Transferência Adotiva , Animais , Autoanticorpos/fisiologia , Autoantígenos/biossíntese , Autoantígenos/sangue , Subpopulações de Linfócitos B/patologia , Doença Crônica , Colite/genética , Colite/patologia , Genes RAG-1/imunologia , Cadeias Pesadas de Imunoglobulinas/genética , Cadeias mu de Imunoglobulina/genética , Transfusão de Linfócitos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Spontaneous inflammatory bowel disease (IBD) resembling human ulcerative colitis develops in mice mutant for the T cell receptor alpha gene (TCR-alpha-/-). TCR-alpha-/- mice lack TCR-alpha/beta+ cells but contain TCR-gamma/delta+ cells and a small population of a unique CD4+, TCR-alpha-/beta+(low) cells. Since all the immunoglobulin (Ig) classes are present in these mice, help to B cells must be provided by cells other than TCR-alpha/beta+ cells. In the present study, we found serum levels of IgG1 and IgG2 to be markedly increased in TCR-alpha-/- mice with IBD as compared to TCR-alpha-/- mice without IBD or TCR-alpha+/- controls. An increase in IgG1-, IgG2a- and IgA- but not IgM-secreting mesenteric lymph node (MLN) B cells was detected in TCR-alpha-/- mutant mice. There was also a marked increase in MLN B cells secreting autoantibody (IgG) to tropomyosin, a cytoskeletal protein. Examination of the hyperplastic MLN showed a marked increase in the number of B, TCR-delta+, and CD4+ TCR-alpha-/beta+ cells, similar to the cell population observed at the site of colonic inflammation. Analysis of spontaneous cytokine production by MLN cells using an enzyme-linked immunospot assay, immunohistochemistry, and reverse transcription/polymerase chain reaction showed a decrease of interleukin 2 (IL-2) but a marked increase of IL-4 and interferon gamma (IFN-gamma) production in TCR-alpha-/- mice with IBD as compared to TCR-alpha-/- mice without IBD and TCR alpha+/- control mice. Both TCR-alpha-/beta+ and TCR-delta+ cells were found to be capable of producing IL-4; IFN-gamma was produced mostly by non-T cells, many of which were shown to be CD3- NK 1.1+ cells. We propose that the cytokine imbalance present in these mice results in expansion of B cells, production and switching of autoantibodies to IgG2 subclass, and development of IBD. It is possible that the unusual CD4+ TCR-alpha-/beta+ population and expanded TCR-gamma/delta+ population present in TCR-alpha-/- mice plays a central role in this abnormal immune response.
Assuntos
Autoanticorpos/biossíntese , Citocinas/biossíntese , Doenças Inflamatórias Intestinais/genética , Doenças Inflamatórias Intestinais/imunologia , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Linfócitos T/imunologia , Animais , Doenças Autoimunes/genética , Linfócitos B/imunologia , Células Cultivadas , Colite Ulcerativa/imunologia , Citocinas/análise , Primers do DNA , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Humanos , Isotipos de Imunoglobulinas/análise , Isotipos de Imunoglobulinas/biossíntese , Imunofenotipagem , Mucosa Intestinal/imunologia , Camundongos , Camundongos Mutantes , Reação em Cadeia da Polimerase , Subpopulações de Linfócitos T/imunologiaRESUMO
The balance between pro and antiinflammatory cytokines secreted by T cells regulates both the initiation and perpetuation of inflammatory bowel diseases (IBD). In particular, the balance between interferon (IFN)-gamma/interleukin (IL)-4 and transforming growth factor (TGF)-beta activity controls chronic intestinal inflammation. However, the molecular pathways that evoke these responses are not well understood. Here, we describe a critical role for the transcription factor T-bet in controlling the mucosal cytokine balance and clinical disease. We studied the expression and function of T-bet in patients with IBD and in mucosal T cells in various T helper (Th)1- and Th2-mediated animal models of chronic intestinal inflammation by taking advantage of mice that lack T-bet and retroviral transduction techniques, respectively. Whereas retroviral transduction of T-bet in CD62L(+) CD4(+) T cells exacerbated colitis in reconstituted SCID mice, T-bet-deficient T cells failed to induce colitis in adoptive transfer experiments suggesting that overexpression of T-bet is essential and sufficient to promote Th1-mediated colitis in vivo. Furthermore, T-bet-deficient CD62L(-) CD4(+) T cells showed enhanced protective functions in Th1-mediated colitis and exhibited increased TGF-beta signaling suggesting that a T-bet driven pathway of T cell activation controls the intestinal balance between IFN-gamma/IL-4 and TGF-beta responses and the development of chronic intestinal inflammation in T cell-mediated colitis. Furthermore, TGF-beta was found to suppress T-bet expression suggesting a reciprocal relationship between TGF-beta and T-bet in mucosal T cells. In summary, our data suggest a key regulatory role of T-bet in the pathogenesis of T cell-mediated colitis. Specific targeting of this pathway may be a promising novel approach for the treatment of patients with Crohn's disease and other autoimmune diseases mediated by Th1 T lymphocytes.
Assuntos
Colite/imunologia , Doença de Crohn/imunologia , Regulação da Expressão Gênica/imunologia , Linfócitos T/imunologia , Fatores de Transcrição/imunologia , Adulto , Animais , Sequência de Bases , Linfócitos T CD4-Positivos/imunologia , Citocinas/genética , Primers do DNA , Modelos Animais de Doenças , Feminino , Técnicas de Transferência de Genes , Genes RAG-1 , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Imunidade nas Mucosas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos SCID , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Baço/imunologia , Proteínas com Domínio T , Subpopulações de Linfócitos T/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Fatores de Transcrição/genéticaRESUMO
BACKGROUND/OBJECTIVE: There have been many studies of therapeutic drug monitoring (TDM) of vancomycin (VCM) based on Bayesian analysis, but there have been no reports of the accuracy of prediction based on Bayesian-estimated patient-specific parameters. This study was conducted to compare the accuracy of prediction based on the population pharmacokinetic (PPK) method and Bayesian-estimated parameters. METHOD: The subjects were 22 patients who were treated with VCM for MRSA infection and whose blood was sampled twice or more during the administration period. The concentrations between the blood samples were predicted based on the concentrations in the first blood samples based on the PPK method using mean parameters for the Japanese population and Bayesian-estimated patient-specific pharmacokinetic parameters. The mean prediction error (ME), mean absolute error (MAE) and root mean squared error (RMSE) were compared to examine the accuracy of prediction based on Bayesian-estimated patient-specific parameters. RESULTS AND DISCUSSION: The mean measured VCM concentration was 10·43±5·19 µg/mL, whereas the mean concentration predicted based on the PPK method was 8·52±4·34 µg/mL, with an ME of -1·91, MAE of 2·93 and RMSE of 3·21. The mean concentration predicted based on patient-specific parameters was 9·62±4·95 µg/mL with ME of -0·81, MAE of 1·38 and RMSE of 1·74. The ME and MAE for the concentrations predicted using patient-specific parameters were smaller compared with those predicted using the PPK method (P=0·0471 and 0·0003, respectively), indicating superior prediction with a significant difference between approaches. CONCLUSION: Prediction using Bayesian estimates of patient-specific parameters was better than by the PPK method. However, when using patient-specific parameters it is still necessary to fully understand the clinical status of the patient and frequently determine VCM concentrations.
Assuntos
Antibacterianos/sangue , Antibacterianos/farmacocinética , Monitoramento de Medicamentos , Staphylococcus aureus Resistente à Meticilina , Vancomicina/sangue , Vancomicina/farmacocinética , Adulto , Idoso , Idoso de 80 Anos ou mais , Teorema de Bayes , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infecções Estafilocócicas/sangue , Infecções Estafilocócicas/tratamento farmacológico , Adulto JovemRESUMO
We recently isolated a novel actin filament (F-actin)-binding protein, afadin, that has two isoforms, l- and s-afadins. l-Afadin is ubiquitously expressed and specifically localized at zonula adherens (ZA) in epithelial cells and at cell-cell adherens junction (AJ) in nonepithelial cells, whereas s-afadin is abundantly expressed in neural tissue. l-Afadin has one PDZ domain, three proline-rich regions, and one F-actin-binding domain, whereas s-afadin lacks the third proline-rich region and the F-actin-binding domain. To understand the molecular mechanism of the specific localization of l-afadin at ZA in epithelial cells and at cell-cell AJ in nonepithelial cells, we attempted here to identify an l-afadin-binding protein(s) and isolated a protein, named ponsin. Ponsin had many splicing variants and the primary structures of two of them were determined. Both the two variants had three Src homology 3 (SH3) domains and turned out to be splicing variants of SH3P12. The third proline-rich region of l-afadin bound to the region of ponsin containing the second and third SH3 domains. Ponsin was ubiquitously expressed and localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells. Ponsin furthermore directly bound vinculin, an F-actin-binding protein localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells. Vinculin has one proline-rich region where two proline-rich sequences are located. The proline-rich region bound to the region of ponsin containing the first and second SH3 domains. l-Afadin and vinculin bound to ponsin in a competitive manner and these three proteins hardly formed a ternary complex. These results indicate that ponsin is an l-afadin- and vinculin-binding protein localized at ZA in epithelial cells, at cell-cell AJ in nonepithelial cells, and at cell-matrix AJ in both types of cells.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Vinculina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células COS , Membrana Celular/metabolismo , DNA Complementar , Cinesinas , Camundongos , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/isolamento & purificação , Microscopia de Fluorescência , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Miosinas , Ligação Proteica , Homologia de Sequência de Aminoácidos , Células Tumorais CultivadasRESUMO
We purified from rat brain a novel actin filament (F-actin)-binding protein of approximately 180 kD (p180), which was specifically expressed in neural tissue. We named p180 neurabin (neural tissue-specific F-actin- binding protein). We moreover cloned the cDNA of neurabin from a rat brain cDNA library and characterized native and recombinant proteins. Neurabin was a protein of 1,095 amino acids with a calculated molecular mass of 122,729. Neurabin had one F-actin-binding domain at the NH2-terminal region, one PSD-95, DlgA, ZO-1-like domain at the middle region, a domain known to interact with transmembrane proteins, and domains predicted to form coiled-coil structures at the COOH-terminal region. Neurabin bound along the sides of F-actin and showed F-actin-cross-linking activity. Immunofluorescence microscopic analysis revealed that neurabin was highly concentrated in the synapse of the developed neurons. Neurabin was also concentrated in the lamellipodia of the growth cone during the development of neurons. Moreover, a study on suppression of endogenous neurabin in primary cultured rat hippocampal neurons by treatment with an antisense oligonucleotide showed that neurabin was involved in the neurite formation. Neurabin is a candidate for key molecules in the synapse formation and function.
Assuntos
Actinas/metabolismo , Proteínas dos Microfilamentos/fisiologia , Proteínas do Tecido Nervoso/fisiologia , Neuritos/ultraestrutura , Sequência de Aminoácidos , Animais , Células Cultivadas , Clonagem Molecular , DNA Complementar/genética , Hipocampo/metabolismo , Dados de Sequência Molecular , Oligonucleotídeos Antissenso , Ratos , Distribuição TecidualRESUMO
The dynamic rearrangement of cell-cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell-cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell-cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell-cell contacts and found that AF-6 accumulated at the cell-cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell-cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell-cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell-cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell-cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.
Assuntos
Células Epiteliais/fisiologia , Cinesinas/fisiologia , Proteínas de Membrana/fisiologia , Miosinas/fisiologia , Fosfoproteínas/fisiologia , Junções Íntimas/fisiologia , Proteínas ras/fisiologia , Animais , Cálcio/fisiologia , Bovinos , Comunicação Celular , Linhagem Celular , Proteínas do Citoesqueleto/metabolismo , Cães , Células Epiteliais/metabolismo , Fibroblastos , Intestinos , Rim , Cinesinas/genética , Cinesinas/metabolismo , Cinesinas/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Camundongos , Microscopia Imunoeletrônica , Miosinas/genética , Miosinas/metabolismo , Miosinas/ultraestrutura , Ocludina , Células PC12 , Fosfoproteínas/metabolismo , Fosfoproteínas/ultraestrutura , Ratos , Proteínas Recombinantes/metabolismo , Junções Íntimas/metabolismo , Junções Íntimas/ultraestrutura , Proteína da Zônula de Oclusão-1 , alfa Catenina , Proteínas ras/metabolismoRESUMO
We have isolated a novel actin filament-binding protein, named afadin, localized at cadherin-based cell-cell adherens junctions (AJs) in various tissues and cell lines. Afadin has one PDZ domain, three proline-rich regions, and one actin filament-binding domain. We found here that afadin directly interacted with a family of the immunoglobulin superfamily, which was isolated originally as the poliovirus receptor-related protein (PRR) family consisting of PRR1 and -2, and has been identified recently to be the alphaherpes virus receptor. PRR has a COOH-terminal consensus motif to which the PDZ domain of afadin binds. PRR and afadin were colocalized at cadherin-based cell-cell AJs in various tissues and cell lines. In E-cadherin-expressing EL cells, PRR was recruited to cadherin-based cell-cell AJs through interaction with afadin. PRR showed Ca2+-independent cell-cell adhesion activity. These results indicate that PRR is a cell-cell adhesion molecule of the immunoglobulin superfamily which is recruited to cadherin-based cell-cell AJs through interaction with afadin. We rename PRR as nectin (taken from the Latin word "necto" meaning "to connect").
Assuntos
Caderinas/metabolismo , Moléculas de Adesão Celular/genética , Junções Intercelulares/metabolismo , Proteínas dos Microfilamentos/metabolismo , Receptores do Fator de Necrose Tumoral , Receptores Virais , Processamento Alternativo/genética , Sequência de Aminoácidos , Animais , Células COS/química , Células COS/metabolismo , Cálcio/metabolismo , Moléculas de Adesão Celular/química , Moléculas de Adesão Celular/metabolismo , Agregação Celular/fisiologia , Células Epiteliais/química , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Junções Intercelulares/química , Junções Intercelulares/ultraestrutura , Cinesinas , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Proteínas dos Microfilamentos/química , Microscopia Eletrônica , Miocárdio/química , Miocárdio/citologia , Miocárdio/metabolismo , Miosinas , Nectinas , Estrutura Terciária de Proteína , Coelhos , Membro 14 de Receptores do Fator de Necrose Tumoral , Vinculina/metabolismoRESUMO
Afadin is an actin filament-binding protein that binds to nectin, an immunoglobulin-like cell adhesion molecule, and is colocalized with nectin at cadherin-based cell-cell adherens junctions (AJs). To explore the function of afadin in cell-cell adhesion during embryogenesis, we generated afadin(-/-) mice and embryonic stem cells. In wild-type mice at embryonic days 6.5-8.5, afadin was highly expressed in the embryonic ectoderm and the mesoderm, but hardly detected in the extraembryonic regions such as the visceral endoderm. Afadin(-/-) mice showed developmental defects at stages during and after gastrulation, including disorganization of the ectoderm, impaired migration of the mesoderm, and loss of somites and other structures derived from both the ectoderm and the mesoderm. Cystic embryoid bodies derived from afadin(-/-) embryonic stem cells showed normal organization of the endoderm but disorganization of the ectoderm. Cell-cell AJs and tight junctions were improperly organized in the ectoderm of afadin(-/-) mice and embryoid bodies. These results indicate that afadin is highly expressed in the ectoderm- derived cells during embryogenesis and plays a key role in proper organization of AJs and tight junctions of the highly expressing cells, which is essential for proper tissue morphogenesis.
Assuntos
Polaridade Celular , Embrião de Mamíferos/citologia , Células Epiteliais/citologia , Proteínas dos Microfilamentos/metabolismo , Junções Íntimas/metabolismo , Actinas/metabolismo , Animais , Encéfalo/metabolismo , Caderinas , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Embrião de Mamíferos/metabolismo , Células Epiteliais/metabolismo , Feminino , Gástrula/citologia , Gástrula/metabolismo , Deleção de Genes , Genótipo , Camadas Germinativas/citologia , Camadas Germinativas/metabolismo , Cinesinas , Masculino , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/genética , Morfogênese , Miosinas , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
A novel actin filament (F-actin)-binding protein with a molecular mass of approximately 205 kD (p205), which was concentrated at cadherin-based cell-to-cell adherens junction (AJ), was isolated and characterized. p205 was purified from rat brain and its cDNA was cloned from a rat brain cDNA library. p205 was a protein of 1,829 amino acids (aa) with a calculated molecular mass of 207,667 kD. p205 had one F-actin-binding domain at 1,631-1,829 aa residues and one PDZ domain at 1,016- 1,100 aa residues, a domain known to interact with transmembrane proteins. p205 was copurified from rat brain with another protein with a molecular mass of 190 kD (p190). p190 was a protein of 1,663 aa with a calculated molecular mass of 188,971 kD. p190 was a splicing variant of p205 having one PDZ domain at 1,009-1,093 aa residues but lacking the F-actin-binding domain. Homology search analysis revealed that the aa sequence of p190 showed 90% identity over the entire sequence with the product of the AF-6 gene, which was found to be fused to the ALL-1 gene, known to be involved in acute leukemia. p190 is likely to be a rat counterpart of human AF-6 protein. p205 bound along the sides of F-actin but hardly showed the F-actin-cross-linking activity. Northern and Western blot analyses showed that p205 was ubiquitously expressed in all the rat tissues examined, whereas p190 was specifically expressed in brain. Immunofluorescence and immunoelectron microscopic studies revealed that p205 was concentrated at cadherin-based cell-to-cell AJ of various tissues. We named p205 l-afadin (a large splicing variant of AF-6 protein localized at adherens junction) and p190 s-afadin (a small splicing variant of l-afadin). These results suggest that l-afadin serves as a linker of the actin cytoskeleton to the plasma membrane at cell-to-cell AJ.
Assuntos
Encéfalo/metabolismo , Adesão Celular , Junções Intercelulares/metabolismo , Proteínas dos Microfilamentos/análise , Proteínas dos Microfilamentos/biossíntese , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos , Química Encefálica , Caderinas/análise , Mapeamento Cromossômico , Clonagem Molecular , DNA Complementar , Feto , Humanos , Junções Intercelulares/ultraestrutura , Cinesinas , Camundongos , Proteínas dos Microfilamentos/química , Dados de Sequência Molecular , Peso Molecular , Miosinas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Mapeamento de Peptídeos , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Baço/metabolismo , Vinculina/análiseRESUMO
Three molecular forms of prothoracicotropic hormone were isolated from the head of the adult silkworm, Bombyx mori, and the amino acid sequence of 19 amino acid residues in the amino terminus of these prothoracicotropic hormones was determined. These residues exhibit significant homology with insulin and insulin-like growth factors.
RESUMO
Prothoracicotropic hormone (PTTH), a brain secretory polypeptide of insects, stimulates the prothoracic glands to produce and release ecdysone, the steroid essential to insect development. The complementary DNAs encoding PTTH of the silkmoth Bombyx mori were cloned and characterized, and the complete amino acid sequence was deduced. The data indicated that PTTH is first synthesized as a 224-amino acid polypeptide precursor containing three proteolytic cleavage signals. The carboxyl-terminal component (109 amino acids) that follows the last cleavage signal represents one PTTH subunit. Two PTTH subunits are linked together by disulfide bonds, before or after cleavage from prepro-PTTH, to form a homodimeric PTTH. When introduced into Escherichia coli cells, the complementary DNA directed the expression of an active substance that was functionally indistinguishable from natural PTTH. In situ hybridization showed the localization of the prepro-PTTH mRNA to two dorsolateral neurosecretory cells of the Bombyx brain.
Assuntos
Bombyx/genética , Hormônios de Inseto/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Bombyx/fisiologia , Clonagem Molecular , DNA/genética , Expressão Gênica , Dados de Sequência Molecular , Sistemas Neurossecretores/fisiologia , Hibridização de Ácido Nucleico , Precursores de Proteínas/genéticaRESUMO
Dilated cardiomyopathy is a severe pathology of the heart with poorly understood etiology. Disruption of the gene encoding the negative immunoregulatory receptor PD-1 in BALB/c mice, but not in BALB/c RAG-2-/- mice, caused dilated cardiomyopathy with severely impaired contraction and sudden death by congestive heart failure. Affected hearts showed diffuse deposition of immunoglobulin G (IgG) on the surface of cardiomyocytes. All of the affected PD-1-/- mice exhibited high-titer circulating IgG autoantibodies reactive to a 33-kilodalton protein expressed specifically on the surface of cardiomyocytes. These results indicate that PD-1 may be an important factor contributing to the prevention of autoimmune diseases.
Assuntos
Antígenos de Superfície/fisiologia , Autoanticorpos/análise , Doenças Autoimunes/imunologia , Cardiomiopatia Dilatada/imunologia , Miocárdio/imunologia , Animais , Antígenos de Superfície/genética , Proteínas Reguladoras de Apoptose , Autoanticorpos/sangue , Autoantígenos/química , Autoantígenos/imunologia , Doenças Autoimunes/patologia , Doenças Autoimunes/fisiopatologia , Cardiomiopatia Dilatada/patologia , Cardiomiopatia Dilatada/fisiopatologia , Complemento C3/análise , Ecocardiografia , Insuficiência Cardíaca/etiologia , Imunoglobulina G/análise , Imunoglobulina G/sangue , Proteínas de Membrana/química , Proteínas de Membrana/imunologia , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Miocárdio/patologia , Receptor de Morte Celular Programada 1RESUMO
Allergic asthma is caused by the aberrant expansion in the lung of T helper cells that produce type 2 (TH2) cytokines and is characterized by infiltration of eosinophils and bronchial hyperreactivity. This disease is often triggered by mast cells activated by immunoglobulin E (IgE)-mediated allergic challenge. Activated mast cells release various chemical mediators, including prostaglandin D2 (PGD2), whose role in allergic asthma has now been investigated by the generation of mice deficient in the PGD receptor (DP). Sensitization and aerosol challenge of the homozygous mutant (DP-/-) mice with ovalbumin (OVA) induced increases in the serum concentration of IgE similar to those in wild-type mice subjected to this model of asthma. However, the concentrations of TH2 cytokines and the extent of lymphocyte accumulation in the lung of OVA-challenged DP-/- mice were greatly reduced compared with those in wild-type animals. Moreover, DP-/- mice showed only marginal infiltration of eosinophils and failed to develop airway hyperreactivity. Thus, PGD2 functions as a mast cell-derived mediator to trigger asthmatic responses.
Assuntos
Asma/fisiopatologia , Prostaglandina D2/fisiologia , Receptores Imunológicos , Receptores de Prostaglandina/fisiologia , Alérgenos/imunologia , Animais , Asma/imunologia , Asma/metabolismo , Asma/patologia , Hiper-Reatividade Brônquica , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Cruzamentos Genéticos , Feminino , Marcação de Genes , Humanos , Imunoglobulina E/sangue , Interferon gama/metabolismo , Interleucinas/metabolismo , Pulmão/imunologia , Pulmão/metabolismo , Pulmão/patologia , Linfócitos/imunologia , Masculino , Mastócitos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Muco/metabolismo , Ovalbumina/imunologia , Prostaglandina D2/metabolismo , Receptores de Prostaglandina/genética , Receptores de Prostaglandina/metabolismo , Mucosa Respiratória/metabolismoRESUMO
OBJECTIVE: Renal function was estimated in 129 elderly patients with methicillin-resistant Staphylococcus aureus (MRSA) who were treated with vancomycin (VCM). The estimation was performed by substituting serum creatinine (SCR) measured enzymatically and a value converted using the Jaffe method into the Cockcroft-Gault and Modification of Diet in Renal Disease (MDRD) equations. The serum trough level was predicted from three estimates of renal function by the population mean (PM) and Bayesian methods and the predictability was assessed. METHODS: Two-compartment model-based Japanese population parameters for VCM were used, and the mean prediction error (ME) and root mean squared error (RMSE) were calculated as indices of bias and accuracy, respectively, for predictions by the PM and Bayesian methods. RESULTS: The PM method gave the highest correlation with the measured value using the estimate of renal function obtained by substituting the Jaffe-converted SCR into the Cockcroft-Gault equation. There was no positive or negative bias in the ME and the value was significantly smaller than for other predicted data (P < 0.05). RMSE was also the smallest, indicating that this method increases the predictability of the serum VCM trough level. While, ME showed a negative bias for all values predicted by the Bayesian method, both the ME and RMSE were very small. CONCLUSION: In the application of the PM method for VCM treatment of elderly patients with MRSA, substitution of SCR based on the Jaffe method into the Cockcroft-Gault equation increases the predictability of the serum VCM trough level. The Bayesian method predicted the serum VCM trough level with high accuracy using any of the estimates of renal function.
Assuntos
Antibacterianos/farmacocinética , Modelos Biológicos , Infecções Estafilocócicas/tratamento farmacológico , Vancomicina/farmacocinética , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Teorema de Bayes , Creatinina/sangue , Feminino , Humanos , Japão , Nefropatias/fisiopatologia , Testes de Função Renal , Masculino , Staphylococcus aureus Resistente à Meticilina/isolamento & purificação , Infecções Estafilocócicas/microbiologia , Distribuição Tecidual , Vancomicina/uso terapêuticoRESUMO
Syntaxin-1 is a component of the synaptic vesicle docking and/or membrane fusion soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) complex (7S and 20S complexes) in nerve terminals. Syntaxin-1 also forms a heterodimer with Munc18/n-Sec1/rbSec1 in a complex that is distinct from the 7S and 20S complexes. In this report, we identify a novel syntaxin-1-binding protein, tomosyn, that is capable of dissociating Munc18 from syntaxin-1 and forming a novel 10S complex with syntaxin-1, soluble N-etyhlmaleimide-sensitive factor attachment (SNAP) 25, and synaptotagmin. The 130 kDa isoform of tomosyn is specifically expressed in brain, where its distribution partly overlaps with that of syntaxin-1 in nerve terminals. High level expression of either syntaxin-1 or tomosyn results in a specific reduction in Ca2+-dependent exocytosis from PC12 cells. These results suggest that tomosyn is an important component in the neurotransmitter release process where it may stimulate SNARE complex formation.