RESUMEN

The phosphoinositide 3-kinase (PI3K) pathway is aberrantly activated in many disease states, including tumor cells, either by growth factor receptor tyrosine kinases or by the genetic mutation and amplification of key pathway components. A variety of PI3K isoforms play differential roles in cancers. As such, the development of PI3K inhibitors from novel compound classes should lead to differential pharmacological and pharmacokinetic profiles and allow exploration in various indications, combinations, and dosing regimens. A screening effort aimed at the identification of PI3Kγ inhibitors for the treatment of inflammatory diseases led to the discovery of the novel 2,3-dihydroimidazo[1,2-c]quinazoline class of PI3K inhibitors. A subsequent lead optimization program targeting cancer therapy focused on inhibition of PI3Kα and PI3Kß. Herein, initial structure-activity relationship findings for this class and the optimization that led to the identification of copanlisib (BAY 80-6946) as a clinical candidate for the treatment of solid and hematological tumors are described.

Asunto(s)

Imidazoles/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Pirimidinas/farmacología , Quinazolinas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Antineoplásicos/farmacología , Sitios de Unión , Línea Celular Tumoral , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase Ib/química , Descubrimiento de Drogas , Humanos , Enlace de Hidrógeno , Imidazoles/síntesis química , Imidazoles/química , Simulación del Acoplamiento Molecular , Inhibidores de las Quinasa Fosfoinosítidos-3 , Inhibidores de Proteínas Quinasas/síntesis química , Inhibidores de Proteínas Quinasas/química , Pirimidinas/síntesis química , Pirimidinas/química , Quinazolinas/síntesis química , Quinazolinas/química , Relación Estructura-Actividad

RESUMEN

A new method of estimating the lower flammability limit (LFL) of general organic compounds is presented. The LFL is predicted at 298 K for gases and the lower temperature limit for solids and liquids from structural contributions and the ideal gas heat of formation of the fuel. The average absolute deviation from more than 500 experimental data points is 10.7%. In a previous study, the widely used modified Burgess-Wheeler law was shown to underestimate the effect of temperature on the lower flammability limit when determined in a large-diameter vessel. An improved version of the modified Burgess-Wheeler law is presented that represents the temperature dependence of LFL data determined in large-diameter vessels more accurately. When the LFL is estimated at increased temperatures using a combination of this model and the proposed structural-contribution method, an average absolute deviation of 3.3% is returned when compared with 65 data points for 17 organic compounds determined in an ASHRAE-style apparatus.

Asunto(s)

Incendios , Compuestos Orgánicos/química , Temperatura

RESUMEN

Gene expression profiles permit analysis of host immune response at the transcriptome level. We used the Pax gene Blood RNA (PAX) System and Affymetrix microarrays (HG-U133A&B) to survey profiles in basic military trainees and to classify them as healthy, febrile respiratory illness (FRI) without adenovirus, FRI with adenovirus, and convalescent from FRI with adenovirus. We assessed quality metrics of RNA processing for microarrays. Class prediction analysis discovered nested sets of transcripts that could categorize the phenotypes with optimized accuracy of 99% (nonfebrile vs febrile, P<0.0005), 87% (healthy vs convalescent, P=0.001), and 91% (febrile without vs with adenovirus, P<0.0005). The discovered set for classification of nonfebrile vs febrile patients consisted of 40 transcripts with functions related to interferon induced genes, complement cascades, and TNF and IL1 signaling. The set of seven transcripts for distinguishing healthy vs convalescent individuals included those associated with ribosomal structure, humoral immunity, and cell adhesion. The set of 10 transcripts for distinguishing FRI without vs with adenovirus had functions related to interferon induced genes, IL1 receptor accessory protein, and cell interactions. These results are the first in vivo demonstration of classification of infectious diseases via host signature transcripts and move us towards using the transcriptome in bio-surveillance.

Asunto(s)

Infecciones por Adenovirus Humanos/clasificación , Perfilación de la Expresión Génica , Personal Militar , Infecciones del Sistema Respiratorio/clasificación , Infecciones por Adenovirus Humanos/diagnóstico , Adenovirus Humanos , Formación de Anticuerpos/genética , Adhesión Celular/genética , Convalecencia , Regulación de la Expresión Génica , Humanos , Masculino , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Infecciones del Sistema Respiratorio/diagnóstico , Infecciones del Sistema Respiratorio/virología , Transcripción Genética

RESUMEN

AIM: To assess the usefulness of cord and serum methadone concentrations at 2 days of age in predicting the severity of neonatal abstinence syndrome (NAS) in infants whose mothers received methadone during pregnancy. METHODS: After informed consent, infants were enrolled if they were delivered at 35 weeks gestation or greater. Relevant information was collected from maternal notes. A sample of cord blood was taken at delivery, with a follow up sample at 48 hours of age. The samples were analysed in batches, and the results were unavailable to the attending clinical staff. Infants were treated for NAS on clinical grounds according to a standardised scoring system. RESULTS: Twenty five of 36 eligible infants over the 21 month period of the study were enrolled. Of these, 12 required treatment for NAS. Maternal methadone dose did not predict the need for treatment. However, infants who required treatment had significantly lower methadone concentrations in cord blood than the group who did not receive treatment (31 v 88 ng/ml respectively; p = 0.029). Paired blood samples for methadone concentrations were available for 17 infants. All but one of the 12 infants who required treatment had undetectable concentrations of methadone in the postnatal sample, whereas the median postnatal methadone concentration in untreated infants was 23 ng/ml (p = 0.002). CONCLUSIONS: Methadone concentrations taken from cord blood may identify infants at greater risk of neonatal withdrawal and therefore requiring treatment.

Asunto(s)

Metadona/sangre , Narcóticos/sangre , Síndrome de Abstinencia Neonatal/sangre , Adulto , Esquema de Medicación , Femenino , Sangre Fetal/metabolismo , Humanos , Recién Nacido , Masculino , Intercambio Materno-Fetal , Metadona/administración & dosificación , Metadona/efectos adversos , Persona de Mediana Edad , Narcóticos/administración & dosificación , Narcóticos/efectos adversos , Embarazo , Pronóstico , Factores de Riesgo

RESUMEN

Significant evidence has accumulated suggesting that the inducible form of cyclooxygenase (COX-2), a central enzyme in the prostaglandin biosynthetic pathway, plays an important role in tumor development. To better understand the role of COX-2 in tumorigenesis, we generated transgenic mice that overexpress COX-2 under control of the human keratin 14 promoter, which allows for expression in the epidermis and some other epithelia. Transgenic mice, referred to as K14.COX2 mice, were readily distinguished from their nontransgenic littermates by the appearance of significant alopecia. Administration of a specific COX-2 inhibitor restored hair growth, indicating that the alopecia was attributable to elevated COX-2 enzymatic activity. Unexpectedly, COX-2 overexpression was found to protect, rather than sensitize, K14.COX2 mice to skin tumor development induced by an initiation/promotion protocol. K14.COX2 transgenics developed tumors at a much lower frequency than did their littermate controls (3.3% versus 93%, respectively, on a FVB background and approximately 25% versus 100%, respectively, on an ICR background) and presented with significantly reduced tumor burdens (average, 0.03 versus 12.7 tumors/mouse, respectively, on a FVB background and 0.5 versus 7.1 tumors/mouse, respectively, on an ICR background). Mice fed a COX-2 inhibitor in utero and as weanlings up to the time of promotion also showed a significant resistance to tumor development. These results clearly raise questions regarding the role of COX-2 and elevated prostaglandin levels in skin tumor development.

Asunto(s)

Isoenzimas/biosíntesis , Prostaglandina-Endoperóxido Sintasas/biosíntesis , Neoplasias Cutáneas/enzimología , Neoplasias Cutáneas/prevención & control , Piel/enzimología , 9,10-Dimetil-1,2-benzantraceno , Alopecia/tratamiento farmacológico , Alopecia/enzimología , Animales , Carcinógenos , Celecoxib , Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa 2 , Inhibidores de la Ciclooxigenasa/farmacología , Dinoprostona/metabolismo , Femenino , Folículo Piloso/efectos de los fármacos , Humanos , Isoenzimas/antagonistas & inhibidores , Isoenzimas/genética , Masculino , Proteínas de la Membrana , Ratones , Ratones Endogámicos ICR , Ratones Transgénicos , Fenotipo , Prostaglandina-Endoperóxido Sintasas/genética , Pirazoles , Piel/efectos de los fármacos , Piel/metabolismo , Neoplasias Cutáneas/inducido químicamente , Sulfonamidas/farmacología , Acetato de Tetradecanoilforbol

RESUMEN

Using periodic boundary conditions and a constant applied field, we have simulated current flow through an 8.125-A internal diameter, rigid, atomistic channel with polar walls in a rigid membrane using explicit ions and extended simple point charge water. Channel and bath currents were computed from 10 10-ns trajectories for each of 10 different conditions of concentration and applied voltage. An electric field was applied uniformly throughout the system to all mobile atoms. On average, the resultant net electric field falls primarily across the membrane channel, as expected for two conductive baths separated by a membrane capacitance. The channel is rarely occupied by more than one ion. Current-voltage relations are concentration dependent and superlinear at high concentrations.

Asunto(s)

Iones , Cloruro de Sodio/química , Cloruro de Sodio/metabolismo , Agua/química , Simulación por Computador , Electrólitos/química , Electrofisiología , Modelos Moleculares , Electricidad Estática

RESUMEN

A 44-yr-old male pilot was diagnosed with non-ischemic cardiomyopathy, possibly as a complication of hereditary hemochromatosis, 8 yr after an acquired left bundle branch block was discovered on a routine ECG. Biochemical testing returned high levels of iron and percentage transferrin saturation, and genetic testing for hemochromatosis was remarkable for a heterozygous H63D mutation in the HFE gene on chromosome 6. Hereditary hemochromatosis should be considered in the differential diagnosis when a patient presents with cardiomyopathy and genetic testing for HFE gene variants influencing iron overload is now available as a clinical adjunct for diagnosis and patient management issues. Cardiomyopathy and symptomatic hemochromatosis are aeromedically disqualifying conditions in the U.S. Air Force; however, early identification of hereditary hemochromatosis susceptibility with biochemical or genetic diagnostic tests, followed by education in primary and secondary prevention, will prevent a significant proportion of the possible sequelae.

Asunto(s)

Cardiomiopatías/etiología , Predisposición Genética a la Enfermedad , Hemocromatosis/genética , Proteínas de la Membrana , Personal Militar , Adulto , Bloqueo de Rama/diagnóstico , Antígenos HLA/genética , Hemocromatosis/complicaciones , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Masculino , Mutación Missense , Estados Unidos

RESUMEN

The conductance of sodium ions through a simplified channel-membrane system immersed in a reservoir of 1M NaCl in SPC/E water is examined by molecular dynamics simulation. An applied external potential of 1.1 V drives the ions and water through a channel of length 25 A producing a current of 19.6 pA, in reasonable agreement with experimental findings. The stream of ions and water molecules flows continuously because of the constant applied field and periodic boundary conditions. We also examine the potential profile across the simulation cell, the average density distributions of the various species in the reservoir and radially in the channel, and the ion velocity in the channel.

Asunto(s)

Canales Iónicos/metabolismo , Modelos Biológicos , Fenómenos Biofísicos , Biofisica , Cloruros/metabolismo , Canales Iónicos/química , Modelos Moleculares , Sodio/metabolismo , Termodinámica

RESUMEN

OBJECTIVE: National Women's Hospital is one of two hospitals to report a destructive brain lesion, namely encephaloclastic porencephaly (ECPE), in extremely preterm infants. It has been associated with non-cephalic presentation, early hypotension and the number of chest physiotherapy treatments in the first month. The aim of the present study was to determine the temporal relationship between ECPE and chest physiotherapy use in very low-birth weight (VLBW) infants in our unit. METHODOLOGY: Cerebral ultrasound scan reports, post-mortem reports, clinical and physiotherapy records and, if indicated, original ultrasound films were reviewed for all VLBW babies admitted between 1985 and 1998. RESULTS: Over the 14 year period in question, 2219 babies with a birth weight < or = 1500 g were admitted. Encephaloclastic porencephaly was found in only the 13 previously reported babies born between 1992 and 1994. Encephaloclastic porencephaly was excluded in 1564 (70%) babies. In 621 (28%) babies who did not have late ultrasound scans, ECPE was thought to be unlikely either because the babies never had any chest physiotherapy (n=479) or because they had chest physiotherapy but were known to be neurodevelopmentally normal on follow up (n=142). Data were incomplete for 21 babies (0.9%). The number of chest physiotherapy treatments per baby decreased from a median of 95 prior to 1989 to 38 and the age of starting treatment increased from 5 to 8 days after 1990. The use of chest physiotherapy ceased in 1995. CONCLUSIONS: Encephaloclastic porencephaly emerged as a problem at a time when the use of chest physiotherapy had decreased. The cluster of cases seen between 1992 and 1994, although associated with the number of chest physiotherapy treatments given, began to appear because of some other factor.

Asunto(s)

Lesiones Encefálicas/epidemiología , Recien Nacido Prematuro , Recién Nacido de muy Bajo Peso , Síndrome de Dificultad Respiratoria del Recién Nacido/rehabilitación , Terapia Respiratoria/efectos adversos , Análisis de Varianza , Lesiones Encefálicas/etiología , Distribución de Chi-Cuadrado , Femenino , Humanos , Recién Nacido , Masculino , Nueva Zelanda/epidemiología , Síndrome de Dificultad Respiratoria del Recién Nacido/diagnóstico , Terapia Respiratoria/estadística & datos numéricos , Estudios Retrospectivos , Medición de Riesgo , Factores de Riesgo , Índice de Severidad de la Enfermedad , Análisis de Supervivencia

RESUMEN

Cells exposed to inhibitors of DNA synthesis or suffering DNA damage are arrested or delayed in interphase through the action of checkpoint controls. If the arrested cell is exposed to caffeine, relatively normal cell cycle progression is resumed and, as observed in checkpoint control mutants, loss of checkpoint control activity is associated with a reduction in cell viability. To address the mechanism of caffeine's action on cell progression, fission yeast mutants that take up caffeine but are not sensitized to hydroxyurea (HU) by caffeine were selected. Mutants 788 and 1176 are point mutants of rhp6, the fission yeast homolog of the budding yeast RAD6 gene. Mutant rhp6-788 is slightly HU sensitive, radiosensitive, and exhibits normal checkpoint responses to HU, radiation, or inactivation of DNA ligase. However, the addition of caffeine does not override the associated cell cycle blocks. Both point and deletion mutations show synthetic lethality at room temperature with temperature-sensitive mutations in cyclin B (cdc13-117) or the phosphatase cdc25 (cdc25-22). These observations suggest that the rhp6 gene product, a ubiquitin-conjugating enzyme required for DNA damage repair, promotes entry to mitosis in response to caffeine treatment.

Asunto(s)

Cafeína/farmacología , Ciclo Celular , Ligasas/fisiología , Inhibidores de Fosfodiesterasa/farmacología , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces/fisiología , Enzimas Ubiquitina-Conjugadoras , Proteínas de Ciclo Celular/metabolismo , Supervivencia Celular , Clonación Molecular , Ciclina B/metabolismo , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Fase G2/fisiología , Hidroxiurea/farmacología , Mutagénesis , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Fosfoproteínas Fosfatasas/metabolismo , Temperatura , Factores de Tiempo , Fosfatasas cdc25

RESUMEN

PURPOSE: To review observations of the effects of ionizing radiation on DNA synthesis in eukaryotes. CONTENT: Available information broadly falls into two categories: descriptions of the phenomenon, including dose response data and analysis; and, more recently, investigations utilizing genetic approaches. The down-regulation of DNA replication in the presence of radiation-induced DNA damage appears to be an active cellular response, termed the S-phase damage-sensing (SDS) checkpoint control (Larner et al. 1997). Observations on a variety of eukaryotes, including man, suggest that the regulatory controls involved are highly conserved and may additionally function in G1 and G2 checkpoint controls. Budding yeast, fission yeast and human homologues are identified. CONCLUSIONS: The SDS checkpoint control appears to be comprised of a complex of checkpoint proteins that respond to the stalled replication complex. The replication complex is thought to signal down-regulation of the mitotic kinase, ensuring that the cell does not enter mitosis while S phase is delayed. Concomitantly, the checkpoint complex is believed to transmit a signal via two key checkpoint proteins (Rad3 and Cds1 in the fission yeast), in order to arrest further DNA synthesis initiation.

Asunto(s)

ADN/biosíntesis , ADN/efectos de la radiación , Células Eucariotas/metabolismo , Células Eucariotas/efectos de la radiación , Animales , Replicación del ADN/efectos de la radiación , Humanos

RESUMEN

Two newborn girls had malrotation, small bowel in a subdiaphragmatic location on the right and leftward displacement of the liver. On antenatal scans, each had been diagnosed as having a large intra-abdominal cyst, but this had disappeared in both by the time of delivery. Both infants were asymptomatic at birth. One baby had a wrinkled abdominal wall, which is typically a component of prune belly syndrome. Both babies underwent Ladd procedure for their malrotation. In one, plate-like calcification over the hepatic capsule was the only residue of the previous cyst. In the other, mesenchymal hamartoma of the liver was diagnosed from histology of a collapsed adherent cyst.

Asunto(s)

Quistes/complicaciones , Hamartoma/diagnóstico , Intestino Delgado/anomalías , Hepatopatías/diagnóstico , Calcinosis/diagnóstico , Quistes/diagnóstico , Femenino , Hamartoma/cirugía , Humanos , Recién Nacido , Intestino Delgado/diagnóstico por imagen , Intestino Delgado/cirugía , Hepatopatías/cirugía , Diagnóstico Prenatal , Radiografía , Ultrasonografía

RESUMEN

The protein tyrosine phosphatase CD45 is a critical component of the T cell antigen receptor (TCR) signaling pathway, acting as a positive regulator of Src family protein tyrosine kinases (PTKs) such as Lck. Most CD45-deficient human and murine T cell lines are unable to signal through their TCRs. However, there is a CD45-deficient cell line that can signal through its TCR. We have studied this cell line to identify a TCR signaling pathway that is independent of CD45 regulation. In the course of these experiments, we found that the Syk PTK, but not the ZAP-70 PTK, is able to mediate TCR signaling independently of CD45 and of Lck. For this function, Syk requires functional kinase and SH2 domains, as well as intact phosphorylation sites in the regulatory loop of its kinase domain. Thus, differential expression of Syk is likely to explain the paradoxical phenotypes of different CD45-deficient T cells. Finally, these results suggest differences in activation requirements between two closely related PTK family members, Syk and ZAP-70. The differential activities of these two kinases suggest that they may play distinct, rather than completely redundant, roles in lymphocyte signaling.

Asunto(s)

Precursores Enzimáticos/metabolismo , Proteínas Nucleares , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos T/metabolismo , Familia-src Quinasas/metabolismo , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/metabolismo , Western Blotting , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Citometría de Flujo , Regulación de la Expresión Génica/genética , Humanos , Péptidos y Proteínas de Señalización Intracelular , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Factores de Transcripción NFATC , Fosfoproteínas/análisis , Fosfoproteínas/metabolismo , Fosforilación , Transducción de Señal/fisiología , Quinasa Syk , Factores de Transcripción/metabolismo , Proteína Tirosina Quinasa ZAP-70 , Dominios Homologos src/genética

RESUMEN

Platelets express a single low affinity receptor for immunoglobulin, FcgammaRII, that triggers multiple cellular responses upon interaction with multivalent immune complexes. In this study we show that immobilized IgG is also a potent stimulant of platelet activation triggering adhesion, aggregation, massive dense granule secretion, and thromboxane production. Platelet adhesion to IgG was blocked by the FcgammaRII receptor-specific monoclonal antibody, IV. 3. Pretreatment of the platelets with cytochalasin D to inhibit actin polymerization similarly prevented cell binding to IgG having no effect on platelet binding to fibrinogen. Platelet adhesion to IgG also led to the induction of tyrosine phosphorylation of multiple proteins including pp125(FAK) and p72(SYK). These proteins were also tyrosine-phosphorylated in alphaIIbbeta3-deficient IgG-adherent platelets from patients with Glanzmann's thrombasthenia. These data demonstrate that FcgammaRII mediates pp125(FAK) phosphorylation and platelet adhesion to IgG independent of the integrin alphaIIbbeta3. Treatment of the platelets with bisindolylmaleimide to inhibit protein kinase C prevented phosphorylation of pp125(FAK) as well as several other proteins, but not p72(SYK) phosphorylation. This study establishes that the FcgammaRII receptor mediates pp125(FAK) phosphorylation via protein kinase C.

Asunto(s)

Plaquetas/fisiología , Moléculas de Adhesión Celular/sangre , Inmunoglobulina G/fisiología , Proteínas Tirosina Quinasas/sangre , Receptores de IgG/fisiología , Anticuerpos Monoclonales/farmacología , Plaquetas/inmunología , Quelantes/farmacología , Gránulos Citoplasmáticos/metabolismo , Ácido Egtácico/análogos & derivados , Ácido Egtácico/farmacología , Inhibidores Enzimáticos/farmacología , Precursores Enzimáticos/sangre , Quinasa 1 de Adhesión Focal , Proteína-Tirosina Quinasas de Adhesión Focal , Humanos , Imipramina/farmacología , Técnicas In Vitro , Indoles/farmacología , Péptidos y Proteínas de Señalización Intracelular , Maleimidas/farmacología , Fosforilación , Fosfotirosina , Adhesividad Plaquetaria , Agregación Plaquetaria , Receptores de IgG/efectos de los fármacos , Receptores de IgG/inmunología , Serotonina/sangre , Quinasa Syk , Tromboxano B2/sangre

RESUMEN

Exposure to ionizing radiation temporarily blocks eukaryotic cell cycle progression at the G2/M boundary (G2 delay). The delay probably provides time for repair of DNA damage before chromosome segregation and is thus an active response, indicative of a checkpoint control function. Transition from G2 into mitosis is normally controlled by the activity of a cyclin-dependent kinase, cdc2 in the fission yeast (Schizosaccharomyces pombe). Genetic and cell kinetic evidence suggest that irradiation may impose mitotic delay by inactivation of the cdc2 product, p34cdc2. The activity of p34cdc2 in G2 is regulated by phosphorylation and association with a B-type cyclin, the product of the cdc13 gene, p56cdc13. Previous work does not support a major role for changes in phosphorylation of p34cdc2 in the induction of mitotic delay. Alternatively the kinase may be regulated by changes in the activity/availability of p56cdc13. We have therefore tested the effect of high level, episomal expression of the cdc13 gene on the induction of mitotic delay. No influence of this procedure on the duration of delay was detected, either in a wild-type cell cycle background, or the mutants wee1-50 and cdc2-3w, which show abnormal phosphorylation of p34cdc2.

Asunto(s)

Ciclinas/fisiología , Mitosis/efectos de la radiación , Schizosaccharomyces/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Ciclinas/genética , Fase G2/efectos de la radiación , ARN Mensajero/biosíntesis

RESUMEN

Four pigeons were trained to discriminate between two line orientations in a two-alternative forced-choice procedure. The distribution of reinforcers for the two types of correct response was varied across conditions. Performance on each trial was recorded separately, including the time taken to make a choice response. Discriminability and response-bias measures were calculated for overall performance, and, following a median split of the data from each condition, for faster and slower choice responses in each condition. Discriminability between the stimuli did not vary systematically as a function of choice latency. Variations of the reinforcer distributions produced larger response biases for the faster responses than for the slower responses. Responses on trials following reinforcers were faster and showed a greater effect of the reinforcer distribution than did other responses. Behavioral models of signal detection should consider the speed of the choice response as a factor modulating the effects of reinforcer distributions.

RESUMEN

Exposure of human B-cell precursors (BCP) to ionizing radiation results in cell cycle arrest at the G2-M checkpoint as a result of inhibitory tyrosine phosphorylation of p34cdc2 . Here, we show that ionizing radiation promotes physical interactions between p34cdc2 and the Src family protein-tyrosine kinase Lyn in the cytoplasm of human BCP leading to tyrosine phosphorylation of p34cdc2. Lyn kinase immunoprecipitated from lysates of irradiated BCP as well as a full-length glutathione S-transferase (GST)-Lyn fusion protein-phosphorylated recombinant human p34cdc2 on tyrosine 15. Furthermore, Lyn kinase physically associated with and tyrosine-phosphorylated p34cdc2 kinase in vivo when co-expressed in COS-7 cells. Binding experiments with truncated GST-Lyn fusion proteins suggested a functional role for the SH3 rather than the SH2 domain of Lyn in Lyn-p34cdc2 interactions in BCP. The first 27 residues of the unique amino-terminal domain of Lyn were also essential for the ability of GST-Lyn fusion proteins to bind to p34cdc2 from BCP lysates. Ionizing radiation failed to cause tyrosine phosphorylation of p34cdc2 or G2 arrest in Lyn kinase-deficient BCP, supporting an important role of Lyn kinase in radiation-induced G2 phase-specific cell cycle arrest. Our findings implicate Lyn as an important cytoplasmic suppressor of p34cdc2 function.

Asunto(s)

Linfocitos B/enzimología , Proteína Quinasa CDC2/metabolismo , Familia-src Quinasas/metabolismo , Secuencia de Aminoácidos , Animales , Linfocitos B/citología , Linfocitos B/efectos de la radiación , Sitios de Unión/genética , Proteína Quinasa CDC2/química , Proteína Quinasa CDC2/genética , Reparación del ADN , Fase G2/efectos de la radiación , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/efectos de la radiación , Humanos , Técnicas In Vitro , Datos de Secuencia Molecular , Fosforilación , Leucemia-Linfoma Linfoblástico de Células Precursoras B/enzimología , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Tirosina/química , Tirosina/efectos de la radiación , Familia-src Quinasas/química , Familia-src Quinasas/genética

RESUMEN

To elucidate interactions occurring between B cell protein tyrosine kinases and the signaling components of the B cell antigen receptor, we have co-transfected into COS cells individual tyrosine kinases together with chimeric cell surface receptors containing the cytoplasmic domains of Ig alpha or Ig beta. Of the tyrosine kinases transfected (Lyn, Blk, Hck, Syk, Fyn), only Blk was able to phosphorylate and subsequently associate with cotransfected Ig alpha and Ig beta chimeras in vivo. Association between Blk and the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains was shown by mutational analyses to be the result of an SH2-phosphotyrosine interaction. We identified the tyrosine residues of the Ig alpha and Ig beta cytoplasmic domains phosphorylated by Blk. The enzymatic activity and membrane association of Blk were required for the observed phosphorylation of the Ig alpha and Ig beta chimeras. Sequences within the amino-terminal unique domain of Blk are responsible for recognition and subsequent phosphorylation of the Ig alpha chimera since transfer of the unique region of Blk to Fyn results in the chimeric kinase's ability to phosphorylate the cytoplasmic domain of Ig alpha. These findings indicate that the unique domain of Src family kinases may direct recognition of certain substrates leading to their phosphorylation.

Asunto(s)

Señales de Clasificación de Proteína/metabolismo , Receptores de Antígenos de Linfocitos B/metabolismo , Animales , Sitios de Unión , Línea Celular , Precursores Enzimáticos/genética , Precursores Enzimáticos/metabolismo , Expresión Génica , Humanos , Péptidos y Proteínas de Señalización Intracelular , Mapeo Peptídico , Fosforilación , Señales de Clasificación de Proteína/genética , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Receptores de Antígenos de Linfocitos B/genética , Receptores de IgE/genética , Receptores de IgE/metabolismo , Receptores del Factor de Crecimiento Derivado de Plaquetas/genética , Receptores del Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal , Quinasa Syk , Transfección , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismo

RESUMEN

One of the primary responses observed following antigen-induced cross-linking in mast cells is an increase in the phosphorylation of certain cellular proteins on tyrosine residues. Stimulation of protein-tyrosine kinase activity appears to be necessary for induction of downstream responses such as degranulation. The role of nonreceptor protein-tyrosine kinases in the signal transduction pathway initiated by Fc epsilon RI engagement in an interleukin-3-dependent mast cell line has been examined. The results presented here show that the enzymatic activity of Lyn is increased within seconds of receptor engagement. Syk activity also undergoes a rapid and transient increase, reaching a peak at approximately 30 s. Similarly, the activity of Fer, representing a third class of nontransmembrane protein-tyrosine kinase increases as well, with its activity peak reached at 1 min poststimulation. The enzymatic activities of Syk and Fer were found to correspond to anti-phosphotyrosine antibody reactivity. Phosphorylation of tyrosine residues of the beta and gamma chains of Fc epsilon RI increased concomitant with increased protein-tyrosine kinase activity. These results indicate that at least three classes of nontransmembrane protein-tyrosine kinases are involved in mast cell FceRI signaling and that the activation of these classes of enzymes is temporally regulated.

Asunto(s)

Mastocitos/enzimología , Mastocitos/inmunología , Proteínas Tirosina Quinasas/metabolismo , Receptores de IgE/metabolismo , Animales , Degranulación de la Célula , Línea Celular , Activación Enzimática , Precursores Enzimáticos/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Cinética , Mastocitos/fisiología , Ratones , Fosforilación , Proteínas Tirosina Quinasas/clasificación , Proteínas Proto-Oncogénicas/metabolismo , Receptores de IgE/química , Transducción de Señal , Quinasa Syk , Familia-src Quinasas/metabolismo

RESUMEN

Northern blot analysis of polyadenylated RNA prepared from RBL-2H3 cells revealed the presence of three distinct mRNAs encoding p72Syk, a protein-tyrosine kinase previously shown to be associated with the high affinity IgE receptor present on the surface of these cells (Hutchcroft, J. E., Geahlen, R. L., Deanin, G. G., and Oliver, J. M. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 9107-9111). Here we report the full-length nucleotide sequence of two of these messages, as well as the complete predicted amino acid sequence of the rodent p72Syk protein-tyrosine kinase. In addition, we report evidence indicating alternative splicing of p72Syk mRNAs within RBL-2H3 cells. This splicing event results in the expression of two distinct protein isoforms that differ with respect to the presence of a 23-amino acid insert located within the region of the protein that separates the two SH2 domains from the catalytic domain. Both mRNAs arising from this splicing event appear to encode functional protein-tyrosine kinases, as expression of the corresponding cDNAs in COS cells results in the production of proteins of the expected sizes that possess intrinsic tyrosine specific kinase activity.

Asunto(s)

Empalme Alternativo , Precursores Enzimáticos/genética , Proteínas Tirosina Quinasas/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Northern Blotting , Clonación Molecular , ADN Complementario/genética , Exones/genética , Genoma , Péptidos y Proteínas de Señalización Intracelular , Intrones/genética , Datos de Secuencia Molecular , Ratas , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad de la Especie , Quinasa Syk , Células Tumorales Cultivadas , Proteína Tirosina Quinasa ZAP-70