RESUMEN

Accumulation of misfolded proteins in the endoplasmic reticulum (ER) induces the unfolded protein response (UPR), which alleviates protein overload in the secretory pathway. Although the UPR is activated under diverse pathological conditions, its physiological role during development and in adulthood has not been fully elucidated. Binding immunoglobulin protein (BiP) is an ER chaperone, which is central to ER function. We produced knock-in mice expressing a mutant BiP lacking the retrieval sequence to cause a defect in ER function without completely eliminating BiP. In embryonic fibroblasts, the UPR compensated for mutation of BiP. However, neonates expressing mutant BiP suffered respiratory failure due to impaired secretion of pulmonary surfactant by alveolar type II epithelial cells. Expression of surfactant protein (SP)-C was reduced and the lamellar body was malformed, indicating that BiP plays a critical role in the biosynthesis of pulmonary surfactant. Because pulmonary surfactant requires extensive post-translational processing in the secretory pathway, these findings suggest that in secretory cells, such as alveolar type II cells, the UPR is essential for managing the normal physiological ER protein overload that occurs during development. Moreover, failure of this adaptive mechanism may increase pulmonary susceptibility to environmental insults, such as hypoxia and ischemia, ultimately leading to neonatal respiratory failure.

Asunto(s)

Retículo Endoplásmico/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Surfactantes Pulmonares/metabolismo , Animales , Animales Recién Nacidos , Secuencia de Bases , Células Cultivadas , Cartilla de ADN/genética , Chaperón BiP del Retículo Endoplásmico , Péptidos y Proteínas de Señalización Intercelular , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Electrónica , Mutación , Péptidos/metabolismo , Alveolos Pulmonares/metabolismo , Alveolos Pulmonares/patología , Proteína A Asociada a Surfactante Pulmonar/biosíntesis , Proteína C Asociada a Surfactante Pulmonar , ARN Mensajero/genética , ARN Mensajero/metabolismo , Insuficiencia Respiratoria/genética , Insuficiencia Respiratoria/metabolismo , Insuficiencia Respiratoria/patología , Eliminación de Secuencia

RESUMEN

Newly synthesized proteins in the endoplasmic reticulum (ER) must fold and assemble correctly before being transported to their final cellular destination. While some misfolded or partially assembled proteins have been shown to exit the ER, they fail to escape the early secretory system entirely, because they are retrieved from post-ER compartments to the ER. We elucidate a mechanistic basis for this retrieval and characterize its contribution to ER quality control by studying the fate of the unassembled T-cell antigen receptor (TCR) alpha chain. While the steady-state distribution of TCRalpha is in the ER, inhibition of retrograde transport by COPI induces the accumulation of TCRalpha in post-ER compartments, suggesting that TCRalpha is cycling between the ER and post-ER compartments. TCRalpha associates with BiP, a KDEL protein. Disruption of the ligand-binding function of the KDEL receptor releases TCRalpha from the early secretory system to the cell surface, so that TCRalpha is no longer subject to ER degradation. Thus, our findings suggest that retrieval by the KDEL receptor contributes to mechanisms by which the ER monitors newly synthesized proteins for their proper disposal.

Asunto(s)

Retículo Endoplásmico/metabolismo , Receptores de Péptidos/fisiología , Animales , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Receptores de Antígenos de Linfocitos T alfa-beta/metabolismo , Receptores de Péptidos/genética , Receptores de Péptidos/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/fisiología

RESUMEN

ADP-ribosylation factor 1 (ARF1) is a key regulator of transport in the secretory system. Like all small GTPases, deactivation of ARF1 requires a GTPase-activating protein (GAP) that promotes hydrolysis of GTP to GDP on ARF1. Structure-function analysis of a GAP for ARF1 revealed that its activity in vivo requires not only a domain that catalyzes hydrolysis of GTP on ARF1 but also a non-catalytic domain. In this study, we show that the non-catalytic domain of GAP is required for its recruitment from cytosol to membranes and that this domain mediates the interaction of GAP with the transmembrane KDEL receptor. Blocking its interaction with the KDEL receptor leaves the GAP cytosolic and prevents the deactivation in vivo of Golgi-localized ARF1. Thus, these findings suggest that the KDEL receptor plays a critical role in the function of GAP by regulating its recruitment from cytosol to membranes, where it can then act on its membrane-restricted target, the GTP-bound form of ARF1.

Asunto(s)

Proteínas de Unión al GTP/metabolismo , Proteínas/metabolismo , Receptores de Péptidos/metabolismo , Factor 1 de Ribosilacion-ADP , Factores de Ribosilacion-ADP , Animales , Células COS , Dominio Catalítico , Proteínas Activadoras de GTPasa , Células HeLa , Humanos , Cinética , Mutación , Unión Proteica , Receptores de Péptidos/genética

RESUMEN

The small GTP-binding protein ADP-ribosylation factor-1 (ARF1) regulates intracellular transport by modulating the interaction of coat proteins with the Golgi complex. Coat protein association with Golgi membranes requires activated, GTP-bound ARF1, whereas GTP hydrolysis catalyzed by an ARF1-directed GTPase-activating protein (GAP) deactivates ARF1 and results in coat protein dissociation. We have recently cloned a Golgi-associated ARF GAP. Overexpression of GAP was found to result in a phenotype that reflects ARF1 deactivation (Aoe, T., Cukierman, E., Lee, A., Cassel, D., Peters, P. J., and Hsu, V. W. (1997) EMBO J. 16, 7305-7316). In this study, we used this phenotype to define domains in GAP that are required for its function in vivo. As expected, mutations in the amino-terminal part of GAP that were previously found to abolish ARF GAP catalytic activity in vitro abrogated ARF1 deactivation in vivo. Significantly, truncations at the carboxyl-terminal part of GAP that did not affect GAP catalytic activity in vitro also diminished ARF1 deactivation. Thus, a noncatalytic domain is required for GAP activity in vivo. This domain may be involved in the targeting of GAP to the Golgi membrane.

Asunto(s)

Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Factor 1 de Ribosilacion-ADP , Factores de Ribosilacion-ADP , Animales , Sitios de Unión , Células COS , Catálisis , ADN Complementario , Proteínas de Unión al GTP/genética , Aparato de Golgi/fisiología , Guanosina Trifosfato/metabolismo , Guanilato Ciclasa/química , Guanilato Ciclasa/metabolismo , Células HeLa , Humanos , Cinética , Sustancias Macromoleculares , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Transfección

RESUMEN

Intracellular transport is best understood for how proteins are shuttled among different compartments of the secretory pathway by membrane-bound transport carriers. However, it remains unclear whether regulation of this transport is modulated by the transported (cargo) proteins in the lumen of transport pathways. In the early secretory pathways that connect the endoplasmic reticulum (ER) and the Golgi complex, the small GTPase ADP-ribosylation factor 1 (ARF1) recruits a cytosolic coat protein complex named COPI onto membranes as a key step in the formation of transport vesicles. Transport of newly synthesized proteins that leave the ER includes a class of cargo proteins with a sequence motif of KDEL. When these KDEL proteins leave the ER to reach the Golgi complex, they are recognized by their receptor and transported retrograde in COPI-coated vesicles back to the ER. We now demonstrate that stimulation of the KDEL receptor by a KDEL protein enhances an interaction between the KDEL receptor and a GTPase-activating protein for ARF1. As a result, more cytosolic GTPase-activating protein is recruited to membranes to inactivate ARF1. Thus, the KDEL proteins are examples of luminal cargo proteins that regulate transport by activating their receptor. Most likely, this regulation affects retrograde transport from the Golgi complex to the ER, as activated KDEL receptor appears to reside only in retrograde COPI-coated vesicles.

Asunto(s)

Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas/metabolismo , Receptores de Péptidos/metabolismo , Factor 1 de Ribosilacion-ADP , Factores de Ribosilacion-ADP , Transporte Biológico , Compartimento Celular , Membrana Celular/metabolismo , Vesículas Cubiertas/metabolismo , Proteína Coatómero , Retículo Endoplásmico/metabolismo , Proteínas Activadoras de GTPasa , Células HeLa , Humanos , Microscopía Electrónica

RESUMEN

OBJECTIVES: To determine whether ischemia-reperfusion and hypoxia-reoxygenation cause cellular damages and stress responses in an isolated perfused rat liver model. To determine whether the increased synthesis of stress protein messenger RNA reflects cellular injury. DESIGN: Prospective, controlled study. SETTING: Institutional laboratories. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: Isolated rat livers with cell free perfusion were exposed to various periods of ischemia-reperfusion or hypoxia-reoxygenation. MEASUREMENTS AND MAIN RESULTS: We measured hepatic oxygen consumption and alanine aminotransferase leakage from liver during perfusion. We analyzed the gene expression of heat shock protein 70, a major stress protein, of the liver by Northern blotting after perfusion. The expression of heat shock protein 70 messenger RNA augmented as the reperfusion period increased. The expression level after graded ischemia or hypoxia significantly correlated with the calculated hepatic oxygen debt (r2 = .737; p < .001; n = 21), or with the accumulated alanine aminotransferase leakage from the liver (r2 = .509; p < .001; n = 21). CONCLUSIONS: These results suggest that the accumulation of heat shock protein 70 messenger RNA reflects the severity of ischemia-reperfusion and hypoxia-reoxygenation injuries, and that a stress response in reperfusion can be triggered without formed elements of blood.

Asunto(s)

Proteínas de Choque Térmico/fisiología , Hipoxia/terapia , Hígado/metabolismo , Daño por Reperfusión/metabolismo , Alanina Transaminasa/metabolismo , Animales , Regulación de la Expresión Génica , Hipoxia/metabolismo , Hígado/irrigación sanguínea , Hígado/enzimología , Masculino , Consumo de Oxígeno , ARN Mensajero/fisiología , Ratas , Ratas Sprague-Dawley

RESUMEN

The small GTPase ADP-ribosylation factor 1 (ARF1) is a key regulator of intracellular membrane traffic. Regulators of ARF1, its GTPase-activating protein (GAP) and its guanine nucleotide exchange factor have been identified recently. However, it remains uncertain whether these regulators drive the GTPase cycle of ARF1 autonomously or whether their activities can be regulated by other proteins. Here, we demonstrate that the intracellular KDEL receptor, ERD2, self-oligomerizes and interacts with ARF1 GAP, and thereby regulates the recruitment of cytosolic ARF1 GAP to membranes. Because ERD2 overexpression enhances the recruitment of GAP to membranes and results in a phenotype that reflects ARF1 inactivation, our findings suggest that ERD2 regulates ARF1 GAP, and thus regulates ARF1-mediated transport.

Asunto(s)

Proteínas de Unión al GTP/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas/metabolismo , Factor 1 de Ribosilacion-ADP , Factores de Ribosilacion-ADP , Animales , Anticuerpos Monoclonales , Secuencia de Bases , Células COS , Cartilla de ADN , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/biosíntesis , Proteínas Activadoras de GTPasa , Aparato de Golgi/metabolismo , Aparato de Golgi/ultraestructura , Células HeLa , Humanos , Sustancias Macromoleculares , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/química , Ratones , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Reacción en Cadena de la Polimerasa , Receptores de Péptidos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transfección

RESUMEN

One of the important mechanisms of immunosuppression in the tumor-bearing status has been attributed to the down-modulation of the CD3 zeta chain and its associated signaling molecules in T cells. Thus, the mechanism of the disappearance of CD3 zeta was investigated in tumor-bearing mice (TBM). The decrease of CD3 zeta was observed both in the cell lysate and intact cells. Direct interaction of T cells with macrophages from TBM (TBM-macrophages) induced the decrease of CD3 zeta, and depletion of macrophages rapidly restored the CD3 zeta expression. We found that treatment of such macrophages with N-acetylcysteine, known as antioxidant compound, prevented the decrease of CD3 zeta. Consistent with this result, the addition of oxidative reagents such as hydrogen peroxide and diamide induced the decrease of CD3 zeta expression in T cells. Consequently, the loss of CD3 zeta resulted in suppression of the antigen-specific T-cell response. These results demonstrate that oxidative stress by macrophages in tumor-bearing status induces abnormality of the T-cell receptor complex by cell interactions with T cells. Therefore, our findings suggest that oxidative stress contributes to the regulation of the expression and function of the T-cell receptor complex.

Asunto(s)

Neoplasias del Colon/inmunología , Macrófagos/inmunología , Proteínas de la Membrana/biosíntesis , Estrés Oxidativo/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/biosíntesis , Receptores de Antígenos de Linfocitos T/biosíntesis , Linfocitos T/inmunología , Animales , Regulación hacia Abajo , Femenino , Citometría de Flujo , Macrófagos/fisiología , Ratones , Ratones Endogámicos BALB C , Especies Reactivas de Oxígeno , Bazo/inmunología

RESUMEN

Antigen recognition signals by the TCR are transduced through activation motifs present in the cytoplasmic region of CD3 chains. In vitro analysis has suggested that the CD3zeta chain mediates different signals from other CD3 chains. To analyze the in vivo function of CD3zeta-mediated signals for T cell development, mice expressing a mutant CD3zeta chain lacking all the activation motifs were generated by introducing the transgene into zeta-knockout mice. Mature CD4(+) single-positive (SP) thymocytes in these mice were greater in number than in zeta-deficient mice, and the promoted differentiation was indicated by the changes of CD69 and HSA phenotypes. We found that even in the absence of activation motifs in CD3zeta, these mature cells became functional, being able to induce Ca2+ mobilization and proliferation upon stimulation. On the other hand, CD4(-)CD8(-) double-negative (DN) thymocytes, most of which were arrested at the CD44(-)CD25(+) stage similarly to those in zeta-deficient mice, could not be promoted for differentiation into CD4(+)CD8(+) double-positive thymocytes in these mice in spite of the fact that the expression of the transgene in DN thymocytes was higher than that of zeta in wild-type mice. These results demonstrate the preferential dependence of the promotion of development and/or expansion of DN thymocytes rather than mature thymocytes upon the activation signals through the zeta chain and suggest differential requirements of TCR signaling for mature SP and immature DN thymocyte developments in vivo.

Asunto(s)

Complejo CD3/fisiología , Proteínas de la Membrana/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/inmunología , Linfocitos T/citología , Timo/citología , Animales , Complejo CD3/inmunología , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Citometría de Flujo , Proteínas de la Membrana/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Linfocitos T/inmunología , Timo/inmunología

RESUMEN

Recognition of the antigen/MHC complex by the T cell receptor (TCR)-CD3 complex in T cells triggers early activation events such as tyrosine phosphorylation, phosphatidylinositol turnover, intracellular Ca2+ mobilization or activation of protein kinases, and finally exhibits effector functions such as lymphokine secretion by helper T cells or cytotoxicity by killer T cells as late activation events. Several key molecules have been shown to engage in these signaling cascades. In addition to the TCR-CD3 molecules, other surface molecules such as CD28 or LFA-1 contribute to the regulation of T cell activation as a co-stimulator. Growing knowledge about the downstream of antigen recognition is promoting the attempt to modulate the signal transduction by specific drugs, mAbs, altered peptides or cytokines. Further investigations on the molecular mechanism of T cell activation will provide clinical successes to control immune responses.

Asunto(s)

Activación de Linfocitos , Linfocitos T/inmunología , Animales , Complejo CD3/fisiología , Humanos , Linfocinas/metabolismo , Fosforilación , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal , Tirosina/metabolismo

RESUMEN

The mechanism of the structural alterations of the T cell receptor (TCR)-CD3 complex, which appear to be greatly responsible for immunosuppression in the tumor-bearing status, was investigated in tumor-bearing mice. Splenic T cells from tumor-bearing hosts lost the expression of the CD3 zeta chain without being replaced by FcR gamma, despite the normal expression of other components of the TCR complex. Tumor growth induced the accumulation of non-T, non-B cells in the spleen in correlation with the loss of zeta. Those cells were found to be macrophages that were able to induce the loss of zeta, as well as structural changes of CD3 gamma delta epsilon, even in freshly isolated normal T cells by cell contact-dependent interaction. More importantly, macrophages activated with zymosan A+LPS but not residential macrophages were able to induce the similar abnormality of the TCR complex. These results indicate that macrophages in certain activation stages play a crucial role in causing an abnormal TCR complex in tumor-bearing conditions, as well as in regulating the structure of the TCR complex in immune responses.

Asunto(s)

Activación de Macrófagos , Macrófagos/fisiología , Neoplasias Experimentales/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Animales , Proteínas de la Membrana/análisis , Ratones , Ratones Endogámicos BALB C , Receptores de Antígenos de Linfocitos T/análisis

RESUMEN

Structural abnormality of T cell receptor (TCR)-CD3 complex on the cell surface was investigated in peripheral blood lymphocytes (PBL) from 55 cancer patients. In 24 of the 68 tests done on these patients, the CD3 zeta chain was not detected by immunoprecipitation with anti-CD3 epsilon monoclonal antibody (mAb), but was observed with anti-CD3 zeta mAb, suggesting that a high frequency of cancer patients possesses abnormal T cell receptor (TCR) complex in PBL. On the other hand, the total zeta chain was missing in several advanced cases. During follow-up of several patients, the zeta chain became undetectable after two or three months of cancer progression. It appears that immunosuppressive status can be monitored by analyzing the TCR-CD3 complex on the cell surface of PBL.

Asunto(s)

Proteínas de la Membrana/química , Neoplasias/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Receptores de Antígenos de Linfocitos T/química , Progresión de la Enfermedad , Femenino , Neoplasias Gastrointestinales/sangre , Neoplasias Gastrointestinales/inmunología , Neoplasias Gastrointestinales/patología , Humanos , Vigilancia Inmunológica/inmunología , Masculino , Estadificación de Neoplasias , Neoplasias/sangre , Neoplasias/patología , Pruebas de Precipitina , Complejo Receptor-CD3 del Antígeno de Linfocito T/fisiología , Transducción de Señal/fisiología , Relación Estructura-Actividad , Linfocitos T/inmunología

RESUMEN

The TCR complex transduces the antigen recognition signal through common activation motifs present in both CD3 gamma delta epsilon chains and zeta dimers within the complex. We have investigated functional roles of the cytoplasmic domain in zeta and CD3 gamma delta epsilon for T cell activation in early and late responses by comparing the signaling capability of the TCR complexes containing mutant zeta lacking some or all motifs, or eta chain, another zeta family molecule. The results with the mutant zeta lacking all motifs indicated that CD3 gamma delta epsilon can transduce signals to cause early activation events and production of IL-2 upon antigen stimulation in the absence of zeta motifs. However, any one of the zeta motifs was required to respond to Thy-1 stimulation and this requirement cannot be replaced by other CD3 chains. Such zeta motif-dependent responses were also observed in tyrosine phosphorylation of a 90 kDa protein upon TCR stimulation. Furthermore, we found that the C-terminal unique region of the eta chain exhibits inhibitory function in phosphorylation and Ca2+ response upon TCR stimulation as well as IL-2 production upon Thy-1 stimulation. Collectively, the present analyses suggest that two types of signals are induced through the TCR-CD3 complex: (i) the common motif-dependent signals which are mediated equally through zeta dimers and CD3 gamma delta epsilon, and (ii) zeta specific motif-dependent signals. Differences in the cytoplasmic domain of zeta family molecules may modulate the cooperation of these two signals, resulting in alteration of T cell functions.

Asunto(s)

Proteínas de la Membrana/química , Proteínas de la Membrana/inmunología , Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Complejo Receptor-CD3 del Antígeno de Linfocito T/inmunología , Receptores de Antígenos de Linfocitos T/química , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Animales , Secuencia de Bases , Calcio/metabolismo , ADN/genética , Electroforesis en Gel Bidimensional , Fosfatos de Inositol/metabolismo , Interleucina-2/biosíntesis , Activación de Linfocitos/inmunología , Proteínas de la Membrana/genética , Ratones , Datos de Secuencia Molecular , Complejo Receptor-CD3 del Antígeno de Linfocito T/genética , Receptores de Antígenos de Linfocitos T/genética , Transfección

RESUMEN

CD3 zeta and eta chains are components of the T cell antigen receptor (TCR) complex and are transcribed from a common gene by alternative splicing. TCR complexes containing the zeta eta dimer have been thought to mediate different functions than complexes containing the zeta 2 dimer. To analyze the role of eta in the development and function of T cells, we generated eta-deficient mice without affecting zeta by gene targeting in embryonic stem cells. Homozygous mutant embryos developed normally. Unexpectedly, however, these mice exhibited high mortality soon after birth for unknown reason(s). Analysis of surviving homozygous animals revealed that the development and function of T cells were normal in the absence of the eta chain. Recently, the zeta/eta locus was reported to encode a transcription factor, Oct-1, on the opposite DNA strand. Our targeting strategy resulted in modulation of Oct-1 transcription--reduction of the authentic Oct-1 mRNA and induction of aberrant transcripts. Although differences in tissue distribution and DNA binding capacity of Oct-1 between wild-type and eta-deficient mice were not evident from in situ hybridization and gel shift analysis, the high mortality in the eta-deficient strain may well be due to the disturbance of Oct-1 transcription by the mutation in the zeta/eta locus. Such possible complexities have to be taken into account in the interpretation of gene targeting experiments.

Asunto(s)

Complejo CD3/biosíntesis , Complejo CD3/genética , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Línea Celular , Cartilla de ADN , Femenino , Genes Letales , Homocigoto , Factor C1 de la Célula Huésped , Hibridación in Situ , Inositol/metabolismo , Sustancias Macromoleculares , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Datos de Secuencia Molecular , Factor 1 de Transcripción de Unión a Octámeros , Reacción en Cadena de la Polimerasa , Embarazo , Mapeo Restrictivo , Células Madre , Linfocitos T/inmunología

RESUMEN

CD3 zeta is a component of the T cell antigen receptor (TCR) complex and is important for signal transduction. We have established mice selectively lacking CD3 zeta but able to express CD3 eta, a polypeptide produced from the same locus through alternative splicing, using the method of gene targeting in embryonic stem cells. In homozygous mutant mice, the numbers of thymocytes and peripheral T cells were greatly reduced and the expression levels of TCR on these cells were 5-fold lower than those on wild-type cells. By contrast, TCR gamma delta+ intestinal intraepithelial lymphocytes were not obviously affected by the mutation. T cells from homozygous mutants exhibited an impaired proliferative response. These results imply that CD3 zeta has a critical role in the development and signal transduction of T cells in vivo.

Asunto(s)

Complejo CD3/genética , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Animales , Secuencia de Bases , Células Sanguíneas/citología , Células Sanguíneas/inmunología , Homocigoto , Intestinos/citología , Intestinos/embriología , Intestinos/inmunología , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Transducción de Señal , Células Madre , Timo/citología , Timo/embriología , Timo/inmunología

RESUMEN

The structure and function of the TCR-CD3 complex containing a homodimer of the gamma chain of the high affinity receptor for IgE (FcR gamma) (FcR gamma+ TCR) was investigated by transfecting the FcR gamma gene into a CD3 zeta-, CD3 eta-, FcR gamma- T cell line. Introduction of FcR gamma, as well as CD3 zeta, induced a high expression of the TCR-CD3 complex on the cell surface. Transfected FcR gamma formed a homodimer and associated firmly with the TCR alpha beta dimer but only weakly with the CD3 gamma delta epsilon. Stimulation of both FcR gamma and CD3 zeta transfectants by antibodies against TCR or CD3 induced accumulation of inositol phosphates, the Ca2+ response, IL-2 production, and growth inhibition. On the other hand, antigen stimulation of transfectants expressing FcR gamma as well as CD3 zeta induced IL-2 production, but only the latter exhibited the antigen-induced growth inhibition. In vitro kinase assay suggested that the CD3 zeta dimer but not the FcR gamma dimer associates with the Fyn kinase. These results indicate that the FcR gamma homodimer is able to form a functional TCR complex but that the mode of assembly and the signaling function of FcR gamma+ TCR, including its association with tyrosine kinase(s), may differ from the TCR-CD3 complex containing CD3 zeta homodimers (zeta+ TCR). This provides an example which illustrates that different TCR isoforms mediate distinct signals and functions.

Asunto(s)

Complejo Receptor-CD3 del Antígeno de Linfocito T/química , Receptores de IgG/química , Animales , Calcio/metabolismo , Línea Celular , Citometría de Flujo , Fosfatos de Inositol/metabolismo , Interleucina-2/metabolismo , Ratones , Fosfotransferasas/metabolismo , Pruebas de Precipitina , Complejo Receptor-CD3 del Antígeno de Linfocito T/fisiología , Receptores de IgG/fisiología , Transducción de Señal/fisiología , Relación Estructura-Actividad , Transfección

RESUMEN

Specific inactivation of the CD3 zeta or CD3 eta gene was introduced into a murine T cell hybridoma cell line by homologous recombination to elucidate the role of the CD3 zeta chain in the assembly of and signal transduction through the TCR complex. Since CD3 zeta and CD3 eta are alternatively spliced forms from a common gene with the only difference occurring in the last exon, we constructed targeting vectors by introducing a neomycin phosphotransferase gene into the CD3 zeta- or CD3 eta-specific exon to selectively inactivate zeta or eta. Subsequently, clones bearing a mutated allele were established. In spite of the disruption of only a single allele of the CD3 zeta gene in the CD3 zeta-targeted clone, most of the authentic zeta transcripts and zeta proteins disappeared from the cells, resulting in an extreme decrease in cell surface expression of the TCR complex. Consequently, these cells exhibited no antigen response. These defects were compensated by transfecting the CD3 eta gene. These results confirm previous studies on a somatic mutant showing that CD3 zeta has crucial roles in antigen recognition by and signaling through, as well as the expression of, the TCR-CD3 complex. Our results suggest that there is a major transcriptionally active allele for the expression of these genes in this cell line which seems to be susceptible to homologous recombination. In vitro gene targeting, therefore, provides a powerful approach for studying the roles of intracellular molecules.

Asunto(s)

Hibridomas/inmunología , Proteínas de la Membrana/genética , Receptores de Antígenos de Linfocitos T/genética , Linfocitos T/inmunología , Animales , Secuencia de Bases , Línea Celular , Columbidae , Exones , Proteínas de la Membrana/fisiología , Ratones , Datos de Secuencia Molecular , ARN Mensajero/análisis , ARN Mensajero/genética , Complejo Receptor-CD3 del Antígeno de Linfocito T/análisis , Receptores de Antígenos de Linfocitos T/fisiología

RESUMEN

A 79 year old man, with left femoral neck fracture, was scheduled for an elective operation. After admission, severe hyponatremia probably due to diuretics developed. No neurological abnormalities were observed before surgery. He recovered from anesthesia with no problems. But on the 5th postoperative day he showed transient unresponsiveness. Grand mal seizures were also seen after the serum Na level had recovered to around 130 mEq.l-1. This case shows that in the management of severe hyponatremia, the discrimination between acute and chronic hyponatremia seems to be important.

Asunto(s)

Anestesia/métodos , Coma/etiología , Hiponatremia/complicaciones , Complicaciones Posoperatorias , Procedimientos Quirúrgicos Operativos , Anciano , Humanos , Masculino

RESUMEN

The anaesthetic management of a child with Goldenhar's syndrome and upper airway dysmorphology is presented. She had a history of severe dyspnoea due to deterioration of cor pulmonale caused by upper airway obstruction. The patency of the upper airway and oxygenation were evaluated during the perioperative period with respiratory inductive plethysmography (RIP) and pulse oximetry, which did not show severe upper airway obstruction or oxygen saturation below 80 per cent. Tracheal intubation was performed under inhalational anaesthesia with spontaneous breathing. This case suggests that RIP and pulse oximetry may be useful monitoring devices in the anaesthetic management of patients with upper airway problems as in Goldenhar's syndrome.

Asunto(s)

Anestesia por Inhalación , Síndrome de Goldenhar/cirugía , Disostosis Mandibulofacial/cirugía , Trastornos Respiratorios/diagnóstico , Preescolar , Femenino , Humanos , Oximetría , Pletismografía

RESUMEN

Despite its importance, very little has been known on metabolic abnormalities in the patients with postoperative multiple organ failure (POMOF). The present study was undertaken to investigate the metabolic abnormalities and to establish effective nutritional management on these patients. Systemic energy metabolism and hepatocellular mitochondrial function were studied with indirect calorimetry and arterial ketone body ratio measurements on 30 POMOF patients treated between 1984 and 1985. The POMOF patients were hypermetabolic and their hepatic mitochondrial function was impaired. Their caloric intake was depleted for the limitation in administrable water volume due to renal failure. Some of them could not utilize exogenous glucose given by total parenteral nutrition (TPN). To overcome these problems, on 26 POMOF patients treated between 1986 and 1987 continuous hemofiltration (CHF) was applied to remove excess water and ATP-Mg was administered to improve hepatic mitochondrial function during TPN. The sufficiency rate of energy intake (caloric intake/energy expenditure) was improved from 73.3 +/- 14.7% to 92.0 +/- 8.1% with simultaneous CHF and the hepatic mitochondrial function was also improved with ATP-Mg resulting in better utilization of exogenous glucose. These results indicate that CHF and ATP-Mg administration are effective adjunctive treatments in the nutritional management for POMOF.

Asunto(s)

Insuficiencia Multiorgánica/fisiopatología , Adenosina Trifosfato/administración & dosificación , Adenosina Trifosfato/uso terapéutico , Metabolismo Energético , Hemofiltración , Humanos , Complicaciones Posoperatorias