RESUMEN

Linker for activation of T cells (LAT) is critical for the propagation of T-cell signals upon T-cell receptor (TCR) activation. Previous studies demonstrated that substitution of LAT lysines with arginines (2KR LAT) resulted in decreased LAT ubiquitination and elevated T-cell signaling, indicating that LAT ubiquitination is a molecular checkpoint for attenuation of T-cell signaling. To investigate the role of LAT ubiquitination in vivo, we have generated transgenic mice expressing WT and ubiquitin-defective 2KR LAT. On TCR stimulation of T cells from these mice, proximal signaling and cytokine production was elevated in 2KR versus wild-type (WT) LAT mice. Enhanced cytolytic activity as well as T-helper responses were observed on LAT expression, which were further elevated by 2KR LAT expression. Despite greater T-effector function, WT or 2KR LAT expression did not have any effect on clearance of certain pathogens or tumors. Our data support the model that lack of tumor clearance is due to increased differentiation and acquisition of effector phenotype that is associated with suboptimal immunity in an immunotherapy model. Thus, our data further reinforce the role of LAT ubiquitination in TCR signaling and uncovers a novel role for LAT in driving T-cell differentiation.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales/genética , Activación de Linfocitos , Linfocitos/inmunología , Proteínas de la Membrana/genética , Fosfoproteínas/genética , Proteínas Adaptadoras Transductoras de Señales/inmunología , Sustitución de Aminoácidos , Animales , Diferenciación Celular/genética , Activación de Linfocitos/genética , Proteínas de la Membrana/inmunología , Ratones , Ratones Transgénicos , Fosfoproteínas/inmunología , Receptores de Antígenos de Linfocitos T/inmunología , Ubiquitinación

RESUMEN

Listeria monocytogenes, a psychrotrophic foodborne pathogen, is an occasional postprocess contaminant on ready-to-eat meat (RTE) products including frankfurters. Ultraviolet C light (UVC) is an FDA-approved technology for the decontamination of food surfaces. In this study, the ability of UVC to inactivate L. monocytogenes on frankfurters that contained potassium lactate (PL) and sodium diacetate (SDA), either before or after packaging, was investigated. UVC irradiation of frankfurters that were surface-inoculated with L. monocytogenes resulted in a 1.31, 1.49, and 1.93 log reduction at doses of 1, 2, and 4 J/cm(2), respectively. UVC treatment had no effect on frankfurter color or texture at UVC doses up to 4 J/cm(2). Frankfurter meat treated with UVC doses up to 16 J/cm(2) did not increase mutagenesis in bacterial or human cells, either with or without exogenous metabolic activation. UVC treatment of single-layer frankfurter packs at a dose of 2 J/cm(2) resulted in a 0.97 (+/- 0.14) log reduction of L. monocytogenes. Following 8 wk of refrigerated storage L. monocytogenes levels decreased by only 0.65 log in non-UVC-treated frankfurter packs compared with 2.5 log in the UVC-treated packs. Because the numbers of L. monocytogenes associated with contaminations of ready-to-eat meats are typically very low, the use of UVC in combination with potassium lactate and sodium diacetate has the potential to reduce the number of frankfurter recalls and foodborne illness outbreaks.

Asunto(s)

Microbiología de Alimentos , Lactatos/análisis , Listeria monocytogenes/efectos de la radiación , Productos de la Carne/microbiología , Acetato de Sodio/análisis , Rayos Ultravioleta , Animales , Frío , Recuento de Colonia Microbiana , Embalaje de Alimentos , Conservación de Alimentos , Productos de la Carne/análisis , Productos de la Carne/efectos de la radiación , Microscopía Confocal , Microscopía Electrónica de Rastreo , Factores de Tiempo

RESUMEN

Listeria monocytogenes, a psychrotrophic foodborne pathogen, is a recurring postprocess contaminant on ready-to-eat meat (RTE) products including frankfurters. Flash (Steam) Pasteurization (FP) and ultraviolet light (254 nm-UVC) has been shown to reduce levels of L. monocytogenes and L. innocua on frankfurters. In this study, the use of UVC light followed by FP to inactivate L. innocua, a nonpathogenic surrogate for L.monocytogenes, on frankfurters that contained sodium diacetate and potassium lactate (SDA/PL) in a pilot-plant setting was investigated. Application of UVC (1.0 J/cm2), followed by FP (0.75 s steam/121 degrees C) resulted in inactivation of 3.19 log L. innocua, while application of UVC (4.0 J/cm2), followed by FP (3 s steam/121 degrees C) resulted in inactivation of 3.89 log of L. innocua. A refrigerated storage study (8 degrees C) of frankfurters that contained SDA/PL that were treated with UVC followed by FP revealed the growth of L. innocua was inhibited for approximately 8 wk following application of the interventions. The use of UVC in combination with FP had little effect on frankfurter color and texture. The combination of UVC, FP, and SDA/PL was found to be an effective hurdle process for decontamination of frankfurter surfaces.

Asunto(s)

Microbiología de Alimentos , Conservación de Alimentos/métodos , Listeria/crecimiento & desarrollo , Productos de la Carne/microbiología , Vapor , Rayos Ultravioleta , Animales , Frío , Lactatos/análisis , Listeria/efectos de los fármacos , Listeria/efectos de la radiación , Productos de la Carne/análisis , Acetato de Sodio/análisis , Factores de Tiempo

RESUMEN

Listeria monocytogenes, a psychrotrophic foodborne pathogen, is a recurring postprocess contaminant on ready-to-eat meat (RTE) products, including frankfurters. Potassium lactate (PL) and sodium diacetate (SDA) are FDA-approved antimicrobials that inhibit the growth of L. monocytogenes when incorporated into the formulation of fine emulsion sausage. Flash (steam) pasteurization (FP) has been shown to reduce levels of L. monocytogenes, and its surrogate L. innocua, on frankfurter surfaces. The ability of FP to inactivate and prevent the growth of the L. monocytogenes surrogate L. innocua in a pilot plant setting was investigated. FP treatment (1.5 s, 121 degrees C) of single layers of frankfurters that were surface-inoculated with either 5, 4, or 3 log CFU/g of L. innocua immediately before FP (1.5 s, 121 degrees C) resulted in log reductions of 1.97 (+/- 0.11), 2.03 (+/- 0.10), or 2.07 (+/- 0.14), respectively. Inoculum level had no effect on the inactivation of L. innocua. Following 8 wk of refrigerated storage (4 degrees C), L. innocua levels decreased by 0.5 log in non-FP-treated frankfurter packs, while the 2 log reduction of L. innocua was maintained for FP-treated frankfurters. FP (1.5 s, 121 degrees C) had no effect on frankfurter color or texture. Because the numbers of L. monocytogenes associated with contaminations of ready-to-eat meats are typically very low, the use of FP in combination with PL and SDA has the potential to reduce the number of frankfurter recalls and foodborne illness outbreaks.

Asunto(s)

Manipulación de Alimentos/métodos , Conservación de Alimentos/métodos , Conservantes de Alimentos/farmacología , Listeria/crecimiento & desarrollo , Productos de la Carne/microbiología , Animales , Recuento de Colonia Microbiana , Seguridad de Productos para el Consumidor , Microbiología de Alimentos , Humanos , Lactatos/farmacología , Acetato de Sodio/farmacología , Temperatura , Factores de Tiempo

RESUMEN

Food irradiation is a safe and effective method for inactivation of pathogenic bacteria, including Escherichia coli O157:H7, in meat, leafy greens, and complex ready-to-eat foods without affecting food product quality. Determining the radiation dose needed to inactivate E. coli O157:H7 in foods and the validation of new irradiation technologies are often performed through inoculation of model systems or food products with cocktails of the target bacterium, or use of single well-characterized isolates. In this study, the radiation resistance of 4 E. coli strains, 2 DNA repair deficient strains used for cloning and recombinant DNA technology (JM109 and DH5alpha) and 2 strains of serotype O157:H7 (C9490 and ATCC 35150), were determined. The D-10 values for C9490, ATCC 35150, JM109, and DH5alpha stationary phase cells suspended in Butterfield's Phosphate Buffer and irradiated at 4 degrees C were 229 (+/- 9.00), 257 (+/- 7.00), 61.2 (+/- 10.4), and 51.2 (+/- 8.82) Gy, respectively. The results of this study indicate that the extreme radiation sensitivity of JM109 and DH5alpha makes them unsuitable for use as surrogate microorganisms for pathogenic E. coli in the field of food irradiation research. Use of E. coli JM109 and DH5alpha, which carry mutations of the recA and gyrA genes required for efficient DNA repair and replication, is not appropriate for determination of radiation inactivation kinetics and validation of radiation processing equipment.

Asunto(s)

ADN Bacteriano/efectos de la radiación , Escherichia coli/crecimiento & desarrollo , Escherichia coli/efectos de la radiación , Irradiación de Alimentos/métodos , Fosfatos/farmacología , Biotecnología , Recuento de Colonia Microbiana , Seguridad de Productos para el Consumidor , Relación Dosis-Respuesta en la Radiación , Escherichia coli O157/crecimiento & desarrollo , Escherichia coli O157/efectos de la radiación , Irradiación de Alimentos/normas , Microbiología de Alimentos , Rayos gamma , Humanos , Investigación

RESUMEN

This study presents mathematical models that describe the inactivation of Salmonella Enteritidis, Salmonella Typhimurium, and Salmonella Senftenberg suspended in liquid whole egg (LWE) by irradiation followed by heat treatments (IR-H treatments). These models also enable prediction of cell injury in Salmonella after exposure to IR-H. Salmonella viability decreased exponentially (primary model) with heat treating time for all the radiation doses (0, 0.1, 0.3, 0.5, 1.0, and 1.5 kGy) and temperatures investigated (55, 57, and 60 degrees C). Two secondary models that related the D(T) values (time required to eliminate 90% of viable cells at a given temperature) with radiation dose, heating temperature, and recovery medium after treatments were also developed. The developed final equations enabled to establish the process criterion (combinations of irradiation doses, temperature, and heat treatment times) required to achieve a given reduction (performance criterion) in Salmonella spp. suspended in LWE or the cell damage caused by the treatments. Process criteria to obtain the established performance criteria (a 5-log(10) reduction) on any of the investigated Salmonella serovars were determined to be, 57.7 degrees C/3.5 min following 1.5 kGy when treated cells were recovered in tryptic soy agar and 59.3 degrees C/3.5 min following 0.5 kGy when cells were recovered in tryptic soy agar amended with 3% NaCl. Based on our results, current industrial LWE heat treatments (60 degrees C/3.5 min) would inactivate 3 log(10) cycles of the Salmonella population. The results of this study can be applied to engineering design and for the evaluation and optimization of the IR-H process as a new technique to obtain Salmonella-free LWE.

Asunto(s)

Huevos/microbiología , Manipulación de Alimentos/métodos , Irradiación de Alimentos , Conservación de Alimentos/métodos , Calor , Salmonella/crecimiento & desarrollo , Animales , Recuento de Colonia Microbiana , Seguridad de Productos para el Consumidor , Relación Dosis-Respuesta en la Radiación , Contaminación de Alimentos/análisis , Contaminación de Alimentos/prevención & control , Microbiología de Alimentos , Rayos gamma , Humanos , Matemática , Modelos Biológicos , Modelos Teóricos , Valor Predictivo de las Pruebas , Salmonella/efectos de la radiación , Salmonella enteritidis/crecimiento & desarrollo , Salmonella enteritidis/efectos de la radiación , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/efectos de la radiación

RESUMEN

Kidney and pancreas transplantation in 2005 improved in quantity and outcome quality, despite the increasing average age of kidney graft recipients, with 56% aged 50 or older. Geography and ABO blood type contribute to the discrepancy in waiting time among the deceased donor (DD) candidates. Allocation policy changes are decreasing the median times to transplant for pediatric recipients. Overall, 6% more DD kidney transplants were performed in 2005 with slight increases in standard criteria donors (SCD) and expanded criteria donors (ECD). The largest increase (39%) was in donation after cardiac death (DCD) from non-ECD donors. These DCD, non-ECD kidneys had equivalent outcomes to SCD kidneys. 1-, 3- and 5-year unadjusted graft survival was 91%, 80% and 70% for non-ECD-DD transplants, 82%, 68% and 53% for ECD-DD grafts, and 95%, 88% and 80% for living donor kidney transplants. In 2005, 27% of patients were discharged without steroids compared to 3% in 1999. Acute rejection decreased to 11% in 2004. There was a slight increase in the number of simultaneous pancreas-kidney transplants (895), with fewer pancreas after kidney transplants (343 from 419 in 2004), and a stable number of pancreas alone transplants (129). Pancreas underutilization appears to be an ongoing issue.

Asunto(s)

Trasplante de Riñón/estadística & datos numéricos , Trasplante de Páncreas/estadística & datos numéricos , Rechazo de Injerto/tratamiento farmacológico , Rechazo de Injerto/epidemiología , Supervivencia de Injerto , Humanos , Terapia de Inmunosupresión/métodos , Trasplante de Riñón/mortalidad , Trasplante de Riñón/tendencias , Donadores Vivos/estadística & datos numéricos , Trasplante de Páncreas/tendencias , Selección de Paciente , Análisis de Supervivencia , Donantes de Tejidos/estadística & datos numéricos , Estados Unidos

RESUMEN

PURPOSE: Two tripeptide chemoattractants, acetyl-proline-glycine-proline (Ac-PGP) and methyl-proline-glycine-proline (Me-PGP), are the primary triggers for early neutrophil invasion into the alkali-injured cornea. In the present study the effectiveness of a complementary peptide designed to inhibit the PGP chemoattractants (arginine-threonine-arginine [RTR] tetrameric peptide) and an apo A-1 mimicking peptide (5F) was investigated in the alkali-injured rabbit eye. METHODS: (L)-RTR tetramer, (D)-RTR tetramer, and 5F were tested in vitro for their effects on neutrophil polarization. Synthetic 5F was also tested in vitro for its effect on the neutrophil respiratory burst. In the alkali-injured rabbit eye model, the right corneas of 48 rabbits were exposed to 1 N NaOH for 35 seconds. Sixteen animals were randomly assigned to each of three groups: phosphate-buffered saline (PBS) control; 800 microM RTR (dextrorotatory) tetramer in PBS alternating each hour with 1.5 mM RTR (levorotatory) tetramer in PBS; and 12 microM 5F in PBS. One topical drop of each substance was administered hourly (14 times per day) for 33 days. The experiment was continued until day 42 with no additional drops administered. RESULTS: (L)-RTR tetramer and (D)-RTR tetramer inhibited neutrophil polarization activated by the PGP chemoattractants in vitro. Synthetic 5F did not inhibit neutrophil polarization in the presence of Ac-PGP or the respiratory burst of neutrophils in the presence of a metabolic stimulant derived from alkali-degraded corneas. During the entire animal experiment, statistically fewer ulcers occurred in the RTR tetramer group than in the PBS control group (43.8% vs. 87.5%, P = 0.0046). The frequency of ulceration in the 5F group (68.8%) was not significantly different from the PBS control group. CONCLUSIONS: The reduction in the frequency of corneal ulceration by the RTR tetramer possibly resulted from its complementary binding to Ac-PGP and Me-PGP in the cornea shortly after alkali injury, leading to a reduction in the early and late infiltration of neutrophils. RTR tetramer appears to hold enough promise to warrant additional study as a therapeutic drug for the alkali-injured eye.

Asunto(s)

Quemaduras Químicas/prevención & control , Factores Quimiotácticos/antagonistas & inhibidores , Quimiotaxis de Leucocito/efectos de los fármacos , Úlcera de la Córnea/prevención & control , Quemaduras Oculares/inducido químicamente , Neutrófilos/fisiología , Oligopéptidos/uso terapéutico , Prolina/análogos & derivados , Animales , Elementos sin Sentido (Genética)/uso terapéutico , Apolipoproteína A-I/química , Quemaduras Químicas/inmunología , Terapias Complementarias , Córnea/efectos de los fármacos , Córnea/inmunología , Úlcera de la Córnea/inducido químicamente , Úlcera de la Córnea/inmunología , Inmunoensayo de Polarización Fluorescente , Oligopéptidos/antagonistas & inhibidores , Oligopéptidos/síntesis química , Prolina/antagonistas & inhibidores , Conejos , Estallido Respiratorio/efectos de los fármacos , Hidróxido de Sodio

RESUMEN

The integral membrane adapter protein linker for activation of T cells (LAT) performs a critical function in T cell antigen receptor (TCR) signal transduction by coupling the TCR to downstream signaling pathways. After TCR engagement, LAT is tyrosine phosphorylated by ZAP-70 creating docking sites for multiple src homology 2-containing effector proteins. In the Jurkat T cell line, the distal four tyrosines of LAT bind PLCgamma-1, Grb2, and Gads. Mutation of these four tyrosine residues to phenylalanine (4YF) blocked TCR-mediated calcium mobilization, Erk activation, and nuclear factor (NF)-AT activation. In this study, we examined whether these four tyrosine residues were essential for T cell development by generating LAT "knock-in" mutant mice that express the 4YF mutant protein under the control of endogenous LAT regulatory sequences. Significantly, the phenotype of 4YF knock-in mice was identical to LAT(-/)- (null) mice; thymocyte development was arrested at the immature CD4(-)CD8(-) stage and no mature T cells were present. Knock-in mice expressing wild-type LAT protein, generated by a similar strategy, displayed a normal T cell developmental profile. These results demonstrate that the distal four tyrosine residues of LAT are essential for preTCR signaling and T cell development in vivo.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Proteínas de la Membrana , Fosfoproteínas/genética , Fosfoproteínas/inmunología , Linfocitos T/inmunología , Animales , Secuencia de Bases , Proteínas Portadoras/química , Diferenciación Celular , Cartilla de ADN/genética , Activación de Linfocitos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Mutantes , Fenotipo , Fosfoproteínas/química , Fosforilación , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal , Linfocitos T/citología , Linfocitos T/metabolismo , Tirosina/química , Tirosina/genética

RESUMEN

Reconstituted orange juice inoculated with Salmonella Anatum, Salmonella Infantis, Salmonella Newport, or Salmonella Stanley was treated with gamma radiation at 2 degrees C. To determine the relationship between juice antioxidant power and Dgamma (dose required to achieve 90% mortality), juice solids were removed prior to inoculation by centrifugation and/or filtration to create juice preparations of varying turbidity. In unadulterated orange juice, Salmonella Anatum (Dgamma = 0.71 kGy) was significantly more resistant than the other species tested. Salmonella Newport (Dgamma = 0.48 kGy) and Salmonella Infantis (Dgamma = 0.35 kGy) were significantly different, while Salmonella Stanley (Dgamma = 0.38 kGy) was intermediate between the two. Neither the resistance of each isolate nor the pattern of relative resistance among isolates was altered in reduced turbidity juice preparations. Although total antioxidant power was associated with the level of juice solids resuspended in phosphate buffer, antioxidant power was not significantly associated with turbidity in the juice preparations or with Dgamma of any species. The variable resistance to irradiation of the Salmonella isolates suggests this as a more significant factor than turbidity or antioxidant power in designing antimicrobial juice irradiation protocols.

Asunto(s)

Bebidas/microbiología , Citrus/microbiología , Irradiación de Alimentos , Salmonella/efectos de la radiación , Bebidas/efectos de la radiación , Citrus/efectos de la radiación , Relación Dosis-Respuesta en la Radiación , Rayos gamma , Modelos Lineales , Tolerancia a Radiación , Salmonella/crecimiento & desarrollo , Serotipificación

RESUMEN

Current data indicate that CD5 functions as an inhibitor of TCR signal transduction. Consistent with this role, thymocyte selection in TCR transgenic/CD5(-/-) mice is altered in a manner suggestive of enhanced TCR signaling. However, the impact of CD5 deletion on thymocyte selection varies depending on the transgenic TCR analyzed, ranging from a slight to a marked shift from positive toward negative selection. An explanation for the variable effect of CD5 on selection is suggested by the observation that CD5 surface expression is regulated by TCR signal intensity during development and CD5 surface levels on mature thymocytes and T cells parallel the avidity of the positively selecting TCR/MHC/ligand interaction. In this study, we generated mice that overexpress CD5 during thymocyte development (CD5-tg), and then examined the effect of CD5 overexpression or CD5 deletion (CD5(-/-)) on selection of thymocytes that express the same TCR transgenes. The results demonstrate that the effect on thymocyte selection of altering CD5 expression depends on the avidity of the selecting interaction and, consequently, the level of basal (endogenous) CD5 surface expression. Substitution of endogenous CD5 with a transgene encoding a truncated form of the protein failed to rescue the CD5(-/-) phenotype, demonstrating that the cytoplasmic domain of CD5 is required for its inhibitory function. Together, these results indicate that inducible regulation of CD5 surface expression during thymocyte selection functions to fine tune the TCR signaling response.

Asunto(s)

Antígenos CD5/fisiología , Receptores de Antígenos de Linfocitos T/fisiología , Transducción de Señal/inmunología , Animales , Antígenos CD5/biosíntesis , Antígenos CD5/genética , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Membrana Celular/genética , Membrana Celular/inmunología , Membrana Celular/metabolismo , Citoplasma/inmunología , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Femenino , Humanos , Ligandos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Unión Proteica/genética , Unión Proteica/inmunología , Estructura Terciaria de Proteína/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Transducción de Señal/genética , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timo/citología , Timo/inmunología , Timo/metabolismo

RESUMEN

Alkaline hydrolysis of corneal proteins in the alkali-injured eye releases N-acetyl-proline-glycine-proline (Ac-Pro-Gly-Pro-OH) among other peptides. It has been shown that this tripeptide is a neutrophil chemoattractant. Existing data suggest that the release of this peptide is the catalytic event for early neutrophil invasion of the cornea leading to corneal ulcers. In order to design inhibitors of this tripeptide chemoattractant that would block neutrophil invasion and diminish corneal ulcers, we studied the solution properties of this tripeptide by NMR spectroscopy and compared this peptide to Ac-Pro-Gly-OH (a weaker chemoattractant), and to Ac-Pro-OH (inactive). The NMR data were consistent with Ac-Pro-Gly-Pro-OH existing in solution as a mixture of four isomers with different cis and trans conformations about the two X-proline amide bonds. The isomer with two trans conformations (trans-trans) was the most dominant (41%) in aqueous solution. This was followed by the isomers with mixed cis and trans conformations (trans-cis, 26% and cis-trans, 20%). The isomer with two cis conformations (cis-cis) was the least favored (13%). The populations of these isomers were investigated in DMSO and they were similar to those reported in aqueous solutions except that the ordering of the trans-cis and cis-trans isomers were reversed. NMR NH temperature coefficients and nuclear Overhauser effect (NOE) measurements as well as CD spectroscopy were used to demonstrate that the four isomers exist primarily in an extended conformation with little hydrogen bonding. The available (NOE) information was used with molecular dynamics calculations to construct a dominant solution conformation for each isomer of the tripeptide. This information will serve as a model for the design of peptide and nonpeptide inhibitors of the chemoattractant.

Asunto(s)

Factores Quimiotácticos/química , Neutrófilos/fisiología , Oligopéptidos/química , Prolina/química , Álcalis/efectos adversos , Animales , Quimiotaxis de Leucocito , Córnea/química , Lesiones de la Cornea , Lesiones Oculares/inducido químicamente , Humanos , Resonancia Magnética Nuclear Biomolecular , Estereoisomerismo

RESUMEN

The T cell antigen receptor (TCR) and pre-TCR complexes are composed of multiple signal-transducing subunits (CD3 gamma, CD3 delta, CD3 epsilon, and zeta) that each contain one or more copies of a semiconserved functional motif, the immunoreceptor tyrosine-based activation motif (ITAM). Although biochemical studies indicate that individual TCR-ITAMs may bind selectively or with different affinity to various effector molecules, data from other experiments suggest that at least some ITAMs are functionally equivalent. In this study, we examined the role of CD3straightepsilon ITAM-mediated signals in T cell development by genetically reconstituting CD3 epsilon-deficient mice with transgenes encoding either wild-type or ITAM-mutant (signaling defective) forms of the protein. The results demonstrate that signals transduced by CD3 epsilon are not specifically required for T cell maturation but instead contribute quantitatively to TCR signaling in a manner similar to that previously observed for zeta chain. Unexpectedly, analysis of TCR-transgenic/CD3 epsilon-mutant mice reveals a potential role for CD3 epsilon signals in T cell survival.

Asunto(s)

Complejo CD3 , Calcio/metabolismo , Receptores de Antígenos de Linfocitos T/inmunología , Transducción de Señal/inmunología , Linfocitos T/inmunología , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Secuencia Conservada , Citocinas/análisis , Ganglios Linfáticos/inmunología , Activación de Linfocitos , Ratones , Ratones Noqueados , Ratones Transgénicos , Subunidades de Proteína , Receptores de Antígenos de Linfocitos T/deficiencia , Receptores de Antígenos de Linfocitos T/genética , Timo/inmunología , Tirosina

RESUMEN

PURPOSE: We have previously presented evidence that the neutrophil chemoattractant, N-acetyl-proline-glycine-proline (N-acetyl-PGP), triggers the initial polymorphonuclear leukocyte (PMN) invasion into the alkali-injured eye. In this study, sense-antisense methodology was used to develop novel complementary peptides to be potential inhibitors of N-acetyl-PGP. METHODS: The polarization assay was used to measure the potential chemotactic response of PMNs to synthetic N-acetyl-PGP, the ultrafiltered tripeptide chemoattractants obtained from alkali-degraded rabbit corneas, or leukotriene B4 (LTB4). Inhibition was expressed as the peptide concentration producing 50% inhibition (ID50) of polarization. Five complementary peptides were tested as potential inhibitors of N-acetyl-PGP: arginine-threonine-arginine (RTR), RTR-glycine-glycine (RTRGG), RTR dimer, RTR tetramer, and alanine-serine-alanine (ASA) tetramer. In addition, the RTR tetramer and both monomeric peptides (RTR and RTRGG) were separately tested for inhibition of the ultrafiltered tripeptide chemoattractants or LTB4. RESULTS: The complementary RTR tetrameric peptide was a powerful antagonist of N-acetyl-PGP-induced PMN polarization (ID50 of 200 nM). The RTR dimer was much less potent (ID50 of 105 microM). Both monomeric peptides, RTR and RTRGG, were only antagonistic at millimolar concentrations. The ASA tetramer showed no capacity to inhibit N-acetyl-PGP. The RTR tetramer also inhibited PMN activation by the ultrafiltered tripeptide chemoattractants (ID50 of 30 microM) but had no effect on LTB4. CONCLUSIONS: A complementary peptide (RTR) was designed which is an effective inhibitor of the neutrophil chemoattractant, N-acetyl-PGP. The potency of the RTR complementary peptide is dramatically enhanced by tetramerization. Inhibition of N-acetyl-PGP by complementary peptides offers great promise for control of the inflammatory response in the alkali-injured eye.

Asunto(s)

Quemaduras Químicas/metabolismo , Factores Quimiotácticos/antagonistas & inhibidores , Quimiotaxis de Leucocito/efectos de los fármacos , Lesiones de la Cornea , Quemaduras Oculares/inducido químicamente , Neutrófilos/fisiología , Oligopéptidos/antagonistas & inhibidores , Oligopéptidos/farmacología , Prolina/análogos & derivados , Prolina/antagonistas & inhibidores , Animales , Elementos sin Sentido (Genética)/síntesis química , Elementos sin Sentido (Genética)/farmacología , Quemaduras Químicas/tratamiento farmacológico , Factores Quimiotácticos/aislamiento & purificación , Córnea/química , Córnea/efectos de los fármacos , Quemaduras Oculares/metabolismo , Inmunoensayo de Polarización Fluorescente , Humanos , Oligopéptidos/síntesis química , Oligopéptidos/aislamiento & purificación , Prolina/aislamiento & purificación , Conejos , Hidróxido de Sodio

RESUMEN

The linker molecule LAT is a substrate of the tyrosine kinases activated following TCR engagement of T cells. LAT is also expressed in platelets, NK, and mast cells. Although LAT-deficient mice contain normal numbers of mast cells, we found that LAT-deficient mice were resistant to IgE-mediated passive systemic anaphylaxis. LAT-deficient bone marrow-derived mast cells (BMMC) showed normal growth and development. Whereas tyrosine phosphorylation of Fc(epsilon)RI, Syk, and Vav was intact in LAT-deficient BMMCs following Fc(epsilon)RI engagement, tyrosine phosphorylation of SLP-76, PLC-gamma1, and PLC-gamma2 and calcium mobilization were dramatically reduced. LAT-deficient BMMCs also exhibited profound defects in activation of MAPK, degranulation, and cytokine production after Fc(epsilon)RI cross-linking. These results show that LAT plays a critical role in Fc(epsilon)RI-mediated signaling in mast cells.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/inmunología , Mastocitos/inmunología , Fosfoproteínas/inmunología , Receptores de IgE/inmunología , Animales , Proteínas de la Membrana/inmunología , Ratones , Transducción de Señal/inmunología

RESUMEN

Activation of G protein coupled receptors (GPCRs) by binding of ligand is the initial event in diverse cellular signaling pathways. To examine the frequency and diversity of mutations that cause constitutive activation of one particular GPCR, the yeast alpha-factor receptor, we screened libraries of random mutations for constitutive alleles. In initial screens for mutant receptor alleles that exhibit signaling in the absence of added ligand, 14 different point mutations were isolated. All of these 14 mutants could be further activated by alpha-factor. Ten of the mutants also acquired the ability to signal in response to binding of desTrp(1)¿Ala(3)alpha-factor, a peptide that acts as an antagonist toward normal alpha-factor receptors. Of these 10 mutants, at least eight alleles residing in the third, fifth, sixth, and seventh transmembrane segments exhibit bona fide constitutive signaling. The remaining alleles are hypersensitive to alpha-factor rather than constitutive. They can be activated by low concentrations of endogenous alpha-factor present in MATa cells. The strongest constitutively active receptor alleles were recovered multiple times from the mutational libraries, and extensive mutagenesis of certain regions of the alpha-factor receptor did not lead to recovery of any additional constitutive alleles. Thus, only a limited number of mutations is capable of causing constitutive activation of this receptor. Constitutive and hypersensitive signaling by the mutant receptors is partially suppressed by coexpression of normal receptors, consistent with preferential association of the G protein with unactivated receptors.

Asunto(s)

Péptidos/metabolismo , Receptores de Péptidos/genética , Saccharomyces/metabolismo , Factores de Transcripción , Secuencia de Aminoácidos , Proteínas Fúngicas/genética , Proteínas de Unión al GTP/metabolismo , Biblioteca de Genes , Genes Reporteros , Operón Lac , Factor de Apareamiento , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Datos de Secuencia Molecular , Mutagénesis , Péptidos/farmacología , Plásmidos , Mutación Puntual , Unión Proteica , Receptores del Factor de Conjugación , Receptores de Péptidos/metabolismo , Saccharomyces/genética , Transducción de Señal

RESUMEN

Nuclear factor kappa B (NF-kappaB) is a ubiquitous, inducible transcription factor that regulates the initiation and progression of immune and inflammatory stress responses. NF-kappaB activation depends on phosphorylation and degradation of its inhibitor protein, IkappaB, initiated by an IkappaB kinase (IKK) complex. This IKK complex includes a catalytic heterodimer composed of IkappaB kinase 1 (IKK1) and IkappaB kinase 2 (IKK2) as well as a regulatory adaptor subunit, NF-kappaB essential modulator. To better understand the role of IKKs in NF-kappaB activation, we have cloned, expressed, purified, and characterized the physiological isoform, the rhIKK1/rhIKK2 heterodimer. We compared its kinetic properties with those of the homodimers rhIKK1 and rhIKK2 and a constitutively active rhIKK2 (S177E, S181E) mutant. We demonstrate activation of these recombinantly expressed IKKs by phosphorylation during expression in a baculoviral system. The K(m) values for ATP and IkappaBalpha peptide for the rhIKK1/rhIKK2 heterodimer are 0.63 and 0.60 micrometer, respectively, which are comparable to those of the IKK2 homodimer. However, the purified rhIKK1/rhIKK2 heterodimer exhibits the highest catalytic efficiency (k(cat)/K(m)) of 47.50 h(-1) micrometer(-1) using an IkappaBalpha peptide substrate compared with any of the other IKK isoforms, including rhIKK2 (17.44 h(-1) micrometer(-1)), its mutant rhIKK2 (S177E, S181E, 1.18 h(-1) micrometer(-1)), or rhIKK1 (0.02 h(-1) micrometer(-1)). Kinetic analysis also indicates that, although both products of the kinase reaction, ADP and a phosphorylated IkappaBalpha peptide, exhibited competitive inhibitory kinetics, only ADP with the low K(i) of 0.77 micrometer may play a physiological role in regulation of the enzyme activity.

Asunto(s)

Proteínas Serina-Treonina Quinasas/química , Catálisis , Dimerización , Electroforesis en Gel de Poliacrilamida , Humanos , Quinasa I-kappa B , Cinética , Peso Molecular , Proteínas Recombinantes/química

RESUMEN

A novel T cell-specific adaptor protein, RIBP, was identified based on its ability to bind Rlk/Txk in a yeast two-hybrid screen of a mouse T cell lymphoma library. RIBP was also found to interact with a related member of the Tec family of tyrosine kinases, Itk. Expression of RIBP is restricted to T and natural killer cells and is upregulated substantially after T cell activation. RIBP-disrupted knockout mice displayed apparently normal T cell development. However, proliferation of RIBP-deficient T cells in response to T cell receptor (TCR)-mediated activation was significantly impaired. Furthermore, these activated T cells were defective in the production of interleukin (IL)-2 and interferon gamma, but not IL-4. These data suggest that RIBP plays an important role in TCR-mediated signal transduction pathways and that its binding to Itk and Rlk/Txk may regulate T cell differentiation.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Mapeo Cromosómico , Células Asesinas Naturales/inmunología , Activación de Linfocitos , Proteínas Tirosina Quinasas/metabolismo , Linfocitos T/inmunología , Secuencia de Aminoácidos , Animales , Línea Celular , Células Cultivadas , Clonación Molecular , Cruzamientos Genéticos , Biblioteca de Genes , Humanos , Interleucina-2/biosíntesis , Linfoma de Células T , Ratones , Ratones Endogámicos , Ratones Noqueados , Datos de Secuencia Molecular , Muridae , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transfección , Células Tumorales Cultivadas

RESUMEN

In the present study, we have addressed the role of the linker for activation of T cells (LAT) in the regulation of phospholipase Cgamma2 (PLCgamma2) by the platelet collagen receptor glycoprotein VI (GPVI). LAT is tyrosine phosphorylated in human platelets heavily in response to collagen, collagen-related peptide (CRP), and FcgammaRIIA cross-linking but only weakly in response to the G-protein-receptor-coupled agonist thrombin. LAT tyrosine phosphorylation is abolished in CRP-stimulated Syk-deficient mouse platelets, whereas it is not altered in SLP-76-deficient mice or Btk-deficient X-linked agammaglobulinemia (XLA) human platelets. Using mice engineered to lack the adapter LAT, we showed that tyrosine phosphorylation of Syk and Btk in response to CRP was maintained in LAT-deficient platelets whereas phosphorylation of SLP-76 was slightly impaired. In contrast, tyrosine phosphorylation of PLCgamma2 was substantially reduced in LAT-deficient platelets but was not completely inhibited. The reduction in phosphorylation of PLCgamma2 was associated with marked inhibition of formation of phosphatidic acid, a metabolite of 1,2-diacylglycerol, phosphorylation of pleckstrin, a substrate of protein kinase C, and expression of P-selectin in response to CRP, whereas these parameters were not altered in response to thrombin. Activation of the fibrinogen receptor integrin alpha(IIb)beta(3) in response to CRP was also reduced in LAT-deficient platelets but was not completely inhibited. These results demonstrate that LAT tyrosine phosphorylation occurs downstream of Syk and is independent of the adapter SLP-76, and they establish a major role for LAT in the phosphorylation and activation of PLCgamma2, leading to downstream responses such as alpha-granule secretion and activation of integrin alpha(IIb)beta(3). The results further demonstrate that the major pathway of tyrosine phosphorylation of SLP-76 is independent of LAT and that there is a minor, LAT-independent pathway of tyrosine phosphorylation of PLCgamma2. We propose a model in which LAT and SLP-76 are required for PLCgamma2 phosphorylation but are regulated through independent pathways downstream of Syk.

Asunto(s)

Proteínas Adaptadoras Transductoras de Señales , Plaquetas/fisiología , Proteínas Portadoras/metabolismo , Integrinas/metabolismo , Isoenzimas/metabolismo , Proteínas de la Membrana , Fosfoproteínas/metabolismo , Activación Plaquetaria/fisiología , Fosfolipasas de Tipo C/metabolismo , Tirosina/metabolismo , Animales , Plaquetas/metabolismo , Activación Enzimática , Humanos , Ratones , Fosfolipasa C gamma , Fosforilación , Receptores de Colágeno

RESUMEN

Recent data indicate that several members of the Tec family of protein tyrosine kinases function in antigen receptor signal transduction. Txk, a Tec family protein tyrosine kinase, is expressed in both immature and mature T cells and in mast cells. By overexpressing Txk in T cells throughout development, we found that Txk specifically augments the phospholipase C (PLC)-gamma1-mediated calcium signal transduction pathway upon T cell antigen receptor (TCR) engagement. Although Txk is structurally different from inducible T cell kinase (Itk), another Tec family member expressed in T cells, expression of the Txk transgene could partially rescue defects in positive selection and signaling in itk(-)(/)(-) mice. Conversely, in the itk(+/+) (wild-type) background, overexpression of Txk inhibited positive selection of TCR transgenic thymocytes, presumably due to induction of cell death. These results identify a role for Txk in TCR signal transduction, T cell development, and selection and suggest that the Tec family kinases Itk and Txk perform analogous functions.

Asunto(s)

Activación de Linfocitos , Proteínas Tirosina Quinasas/fisiología , Linfocitos T/inmunología , Animales , Señalización del Calcio , Células Cultivadas , Antígenos Comunes de Leucocito/fisiología , Ratones , Ratones Transgénicos , Receptores de Antígenos de Linfocitos T alfa-beta/fisiología