RESUMEN
Two residues have been shown to be critical for the kinase activity of the receptor for epidermal growth factor (EGF): lysine-721, which functions in the binding of ATP by correctly positioning the gamma-phosphate for phosphoryl transfer, and aspartate-813, which functions as the catalytic base of the kinase. Mutation of either of these two residues has been shown to disrupt kinase activity of the receptor. However, studies performed in different laboratories had suggested that while EGF receptors mutated at lysine-721 are unable to stimulate significant increases of [(3)H]thymidine incorporation into DNA in response to EGF treatment, cells expressing EGF receptors mutated at aspartate-813 do stimulate significant incorporation of [(3)H]thymidine into DNA in response to EGF. In the present study, EGF receptors mutated at lysine-721 or aspartate-813 (K721R and D813A, respectively), as well as wild-type EGF receptors, were expressed in the same cellular background, Chinese hamster ovary cells, and side-by-side experiments were performed to investigate possible signaling-related differences. Our results indicate that while there are measurable differences in the abilities of the two mutant receptors to stimulate [(3)H]thymidine incorporation between 20 and 24 h after addition of EGF, these differences cannot be correlated with significant differences in EGF-stimulated tyrosine phosphorylation of mutant EGF receptor and endogenous ErbB2, the extent of receptor internalization, EGF-stimulated ion uptake, stimulation of SHC activity, or receptor association with Grb2. Flow cytometric data suggest that populations of cells expressing either kinase-impaired mutant EGF receptor progress similarly into S phase in response to addition of EGF. These observations suggest that D813A and K721R retain similar ability to stimulate mitogenic signaling events through transactivation of ErbB2 with only subtle temporal differences, and they emphasize the importance of expressing mutant receptors in an identical cellular context to make valid comparisons of functions.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Factor de Crecimiento Epidérmico/farmacología , Receptores ErbB/genética , Receptores ErbB/metabolismo , Mitógenos/farmacología , Mutación , Animales , Células CHO , Dominio Catalítico/genética , Cricetinae , Dimerización , Proteína Adaptadora GRB2 , Transferasas Intramoleculares/metabolismo , Mitosis , Fosforilación , Transporte de Proteínas , Proteínas/metabolismo , Receptor ErbB-2/biosíntesis , Receptor ErbB-2/genética , Rubidio/metabolismo , Transducción de Señal , Activación TranscripcionalRESUMEN
To investigate the physiological function of syntaxin 4 in the regulation of GLUT4 vesicle trafficking, we used homologous recombination to generate syntaxin 4-knockout mice. Homozygotic disruption of the syntaxin 4 gene results in early embryonic lethality, whereas heterozygous knockout mice, Syn4(+/-), had normal viability with no significant impairment in growth, development, or reproduction. However, the Syn4(+/-) mice manifested impaired glucose tolerance with a 50% reduction in whole-body glucose uptake. This defect was attributed to a 50% reduction in skeletal muscle glucose transport determined by 2-deoxyglucose uptake during hyperinsulinemic-euglycemic clamp procedures. In parallel, insulin-stimulated GLUT4 translocation in skeletal muscle was also significantly reduced in these mice. In contrast, Syn4(+/-) mice displayed normal insulin-stimulated glucose uptake and metabolism in adipose tissue and liver. Together, these data demonstrate that syntaxin 4 plays a critical physiological role in insulin-stimulated glucose uptake in skeletal muscle. Furthermore, reduction in syntaxin 4 protein levels in this tissue can account for the impairment in whole-body insulin-stimulated glucose metabolism in this animal model.
Asunto(s)
Glucosa/metabolismo , Resistencia a la Insulina/genética , Proteínas de la Membrana/genética , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Músculo Esquelético/fisiología , Adipocitos/fisiología , Tejido Adiposo Pardo , Animales , Transporte Biológico , Técnica de Clampeo de la Glucosa , Prueba de Tolerancia a la Glucosa , Transportador de Glucosa de Tipo 4 , Glucógeno/metabolismo , Glucólisis , Heterocigoto , Hígado/metabolismo , Ratones , Ratones Noqueados , Proteínas Qa-SNARERESUMEN
To investigate the physiological function of the VAMP3 vesicle SNARE (v-SNARE) isoform in the regulation of GLUT4 vesicle trafficking, we generated homozygotic VAMP3 null mice by targeted gene disruption. The VAMP3 null mice had typical growth rate and weight gain, with normal maintenance of fasting serum glucose and insulin levels. Analysis of glucose disposal and insulin sensitivity demonstrated normal insulin and glucose tolerance, with no evidence for insulin resistance. Insulin stimulation of glucose uptake in isolated primary adipocytes was essentially the same for the wild-type and VAMP3 null mice. Similarly, insulin-, hypoxia-, and exercise-stimulated glucose uptake in isolated skeletal muscle did not differ significantly. In addition, other general membrane trafficking events including phagocytosis, pinocytosis, and transferrin receptor recycling were also found to be unaffected in the VAMP3 null mice. Taken together, these data demonstrate that VAMP3 function is not necessary for either regulated GLUT4 translocation or general constitutive membrane recycling.
Asunto(s)
Insulina/metabolismo , Proteínas de la Membrana/genética , Proteínas Musculares , Condicionamiento Físico Animal , Proteínas de Transporte Vesicular , Adipocitos/metabolismo , Animales , Glucemia/metabolismo , Western Blotting , Peso Corporal/genética , Bovinos , Células Cultivadas , ADN Complementario/metabolismo , Embrión de Mamíferos/metabolismo , Femenino , Fibroblastos/metabolismo , Eliminación de Gen , Glucosa/farmacocinética , Transportador de Glucosa de Tipo 4 , Homocigoto , Hipoxia , Insulina/sangre , Masculino , Proteínas de la Membrana/química , Ratones , Modelos Genéticos , Proteínas de Transporte de Monosacáridos/metabolismo , Músculo Esquelético/metabolismo , Mutagénesis Sitio-Dirigida , Fagocitosis , Pinocitosis , Isoformas de Proteínas , Receptores de Transferrina/metabolismo , Proteínas SNARE , Factores Sexuales , Factores de Tiempo , Distribución Tisular , Transferrina/metabolismo , Proteína 3 de Membrana Asociada a VesículasRESUMEN
The State of Texas had the most (cumulative) tuberculous cattle herds of any state in the United States during the decade ending in 1997. Of the cumulative 18 infected herds in Texas, 12 herds were concentrated in El Paso County (designated the 'El Paso milkshed'). To identify whether non-bovine reservoirs were a source of Mycobacterium bovis infection of cattle in this region, an investigation was conducted on the premises of 14 dairy herds (12 tuberculous and 2 non-affected herds) between May 1995 and June 1997. None of the 670 mammalian, avian and environmental (soil, water and air) samples collected and cultured from the premises of these herds was positive for the presence of M. bovis. None of the 119 human urine samples obtained from employees of these dairies was culture positive for M. bovis. Of 124 dairy-farm workers with tuberculin skin-test results, 48 showed positive test results. There was, however, no difference in percentages of positive skin-test results between farms without, and farms having, bovine tuberculosis within the last two years or longer. The percentage of positive reactions did not increase with length of time employed at a dairy with a history of confirmed tuberculosis. These findings suggest that non-bovine reservoirs appear not to be a factor responsible for tuberculosis of cattle in the El Paso milkshed.
Asunto(s)
Reservorios de Enfermedades/veterinaria , Mycobacterium bovis/aislamiento & purificación , Enfermedades Profesionales/epidemiología , Tuberculosis Bovina/epidemiología , Tuberculosis Bovina/prevención & control , Adulto , Animales , Aves , Bovinos , Industria Lechera , Microbiología Ambiental , Femenino , Humanos , Masculino , Prevalencia , Roedores , Texas/epidemiologíaRESUMEN
Insulin-stimulated glucose transport and GLUT4 translocation require regulated interactions between the v-SNARE, VAMP2, and the t-SNARE, syntaxin 4. We have isolated a novel syntaxin 4-binding protein, Synip, which specifically interacts with syntaxin 4. Insulin induces a dissociation of the Synip:syntaxin 4 complex due to an apparent decrease in the binding affinity of Synip for syntaxin 4. In contrast, the carboxyterminal domain of Synip does not dissociate from syntaxin 4 in response to insulin stimulation but inhibits glucose transport and GLUT4 translocation. These data implicate Synip as an insulin-regulated syntaxin 4-binding protein directly involved in the control of glucose transport and GLUT4 vesicle translocation.
Asunto(s)
Adipocitos/metabolismo , Proteínas Portadoras/metabolismo , Insulina/farmacología , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas de Transporte Vesicular , Adipocitos/efectos de los fármacos , Secuencia de Aminoácidos , Animales , Unión Competitiva , Transporte Biológico/efectos de los fármacos , Línea Celular , Clonación Molecular , Cricetinae , Genes Dominantes , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4 , Humanos , Ratones , Datos de Secuencia Molecular , Mutación , Orgánulos/metabolismo , Unión Proteica/efectos de los fármacos , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , ARN Mensajero/análisis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Levaduras/genéticaRESUMEN
There is growing attention to the health benefits of mind/body interventions, particularly relaxation and meditation. Biomedical research has provided undeniable evidence of the interconnectedness of the mind and body. The field of psychoneuroimmunology has defined the role of stress in reducing effectiveness of the immune system in combating infection and growth of malignant tumors. This article explains the development of meditation practice and explores the indications that the practice of meditation is effective reducing the harmful effects of stress. In addition, there are encouraging reports of studies citing the influence of melatonin on breast and prostate tumors. A preliminary study finds an association between meditation practice and levels of melatonin produced by the pineal gland.
Asunto(s)
Meditación , Relaciones Metafisicas Mente-Cuerpo , Neoplasias de la Próstata/prevención & control , Neoplasias de la Próstata/psicología , Estrés Psicológico/complicaciones , Estrés Psicológico/prevención & control , Antioxidantes/farmacología , Neoplasias de la Mama/inmunología , Neoplasias de la Mama/prevención & control , Ejercicios Respiratorios , Femenino , Humanos , Acontecimientos que Cambian la Vida , Masculino , Meditación/métodos , Melatonina/farmacología , Melatonina/fisiología , Neoplasias de la Próstata/etiología , Neoplasias de la Próstata/inmunología , Psiconeuroinmunología , YogaAsunto(s)
Insulina/fisiología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas de Transporte Vesicular , Animales , Transporte Biológico , Compartimento Celular , Endocitosis , Glucosa/metabolismo , Transportador de Glucosa de Tipo 4 , Humanos , Proteínas de la Membrana/metabolismo , Modelos Biológicos , Proteínas SNARE , Vesículas Sinápticas/metabolismoRESUMEN
Insulin stimulates glucose transporter (GLUT) 4 vesicle translocation from intracellular storage sites to the plasma membrane in 3T3L1 adipocytes through a VAMP2- and syntaxin 4-dependent mechanism. We have observed that Munc18c, a mammalian homolog of the yeast syntaxin-binding protein n-Sec1p, competed for the binding of VAMP2 to syntaxin 4. Consistent with an inhibitory function for Munc18c, expression of Munc18c, but not the related Munc18b isoform, prevented the insulin stimulation of GLUT4 and IRAP/vp165 translocation to the plasma membrane without any significant effect on GLUT1 trafficking. As expected, overexpressed Munc18c was found to co-immunoprecipitate with syntaxin 4 in the basal state. However, these complexes were found to dissociate upon insulin stimulation. Furthermore, endogenous Munc18c was predominantly localized to the plasma membrane and its distribution was not altered by insulin stimulation. Although expression of enhanced green fluorescent protein-Munc18c primarily resulted in a dispersed cytosolic distribution, co-expression with syntaxin 4 resulted in increased localization to the plasma membrane. Together, these data suggest that Munc18c inhibits the docking/fusion of GLUT4-containing vesicles by blocking the binding of VAMP2 to syntaxin 4. Insulin relieves this inhibition by inducing the dissociation of Munc18c from syntaxin 4 and by sequestering Munc18c to an alternative plasma membrane binding site.
Asunto(s)
Adipocitos/efectos de los fármacos , Insulina/farmacología , Proteínas de Transporte de Monosacáridos/metabolismo , Proteínas Musculares , Proteínas del Tejido Nervioso , Proteínas/metabolismo , Proteínas de Transporte Vesicular , Células 3T3 , Adipocitos/metabolismo , Animales , Unión Competitiva , Transporte Biológico , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Transportador de Glucosa de Tipo 1 , Transportador de Glucosa de Tipo 4 , Antagonistas de Insulina/farmacología , Proteína Antagonista del Receptor de Interleucina 1 , Proteínas de la Membrana/metabolismo , Ratones , Proteínas Munc18 , Proteínas Qa-SNARE , Proteínas Qb-SNARE , Proteínas Qc-SNARE , Proteínas R-SNARE , Proteínas SNARE , Sialoglicoproteínas/metabolismo , Fracciones Subcelulares/metabolismoRESUMEN
Dormant Artemia salina cysts contain desiccated gastrulae that are metabolically inactive, and physiologically arrested. Following rehydration, embryos resume development via alterations in protein expression, in the complete absence of cell division. In mammals, activation of p70 ribosomal S6 kinase (p70S6k) has been implicated in translational control, in particular the selective up-regulation of translation of mRNAs with polypyrimidine tracts at their 5' start sites. We therefore investigated ribosomal S6 kinase activity in preemergence development. We demonstrate that an S6 kinase activity is rapidly stimulated (within < 15 min) following rehydration and coincides with the onset of ribosomal S6 subunit phosphorylation. This S6 kinase activity displays chromatographic and biochemical characteristics that are similar to those of mammalian p70S6k. Partially purified Artemia S6 kinase was inactivated by treatment with protein phosphatase 2A. Activation of S6 kinase activity was shown to be due to an enzymatic step(s), and not simply rehydration of stored, active enzyme. The temporal profile of activation of S6 kinase activity is compatible with a regulatory function for p70S6k in early preemergence development of encysted Artemia. These studies identify activated Artemia cysts as a system for biochemical studies of p70S6k regulation.
Asunto(s)
Artemia/embriología , Proteínas Quinasas S6 Ribosómicas/metabolismo , Secuencia de Aminoácidos , Animales , Western Blotting , Cromatografía Liquida/métodos , Citosol/enzimología , Deshidratación , Embrión no Mamífero/enzimología , Embrión no Mamífero/fisiología , Activación Enzimática/efectos de los fármacos , Datos de Secuencia Molecular , Oligopéptidos/metabolismo , Fosfoproteínas Fosfatasas/farmacología , Proteína Fosfatasa 2 , Proteínas Quinasas S6 Ribosómicas/efectos de los fármacos , Especificidad por SustratoRESUMEN
Young male and female Sprague-Dawley rats (30 days old) were assigned randomly to three treatment groups: (1) alcohol treatment--received beer with 5% ethanol added, food, and water ad libitum; (2) pair-fed treatment--received nonalcoholic beer plus sucrose and food to match intake by the alcohol-treated animals; and (3) control treatment--received food and water ad libitum. Animals were tested for alcohol preference for 24 hr and then received their assigned treatments for a period of 30 days, followed by a period of abstinence before alcohol preference testing again at 74 days of age. Males given free access to beer and water did not drink large quantities of beer. Females given free access to beer and water drank a lot of beer on the first day, but decreased intake until approximately 52 days of age. A developmental change in young female rats at approximately 52 days of age resulted in increased voluntary ethanol intake, possibly caused by hormonal changes associated with the establishment of estrous cycles. When the animals were tested for alcohol preference at 74 days of age after a period of abstinence, males and females in the pair-fed group had greater alcohol preference than animals in the other groups. Females in the pair-fed group had greater alcohol intake based on body weight than males in the pair-fed group and males and females in all other groups. These results provide insight into sex differences in the development of voluntary drinking behavior and responses of drinking behavior to the early stress of pair-feeding.
Asunto(s)
Consumo de Bebidas Alcohólicas/fisiopatología , Caracteres Sexuales , Maduración Sexual/fisiología , Factores de Edad , Animales , Cerveza , Estro/fisiología , Femenino , Hormonas Esteroides Gonadales/fisiología , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
The epidermal growth factor (EGF) receptor, like other protein tyrosine kinases, shows a preference for substrates having acidic residues in the vicinity of the tyrosyl residue that undergoes phosphorylation. We have developed a peptide substrate for the EGF receptor, termed tyrsub, which is based upon the highly acidic amino terminal sequence of human erythrocyte Band 3. Tyrsub possesses the lowest apparent Km(Km(app) = 32 microM) for phosphorylation by the EGF receptor of any peptide substrate reported to date. Using tyrsub, as well as analogs containing either Ser (sersub) or Phe (phesub) in place of Tyr, we investigated the relative importance of characteristics of the hydroxyaminoacyl residue in substrate recognition. Sersub was unable either to act as a substrate or serve as an effective inhibitor of tyrsub phosphorylation by the EGF receptor. Phesub was also unable to inhibit EGF-stimulable tyrsub phosphorylation, suggesting that the phenolic hydroxyl of the tyrosyl residue, rather than the aromatic ring, predominates in substrate recognition. These results indicate that for peptide substrates, at least, binding consists of two steps, recognition, in which the tyrosyl side chain plays the central role, and docking, in which residues surrounding the tyrosyl residue contribute to stabilizing binding interactions.
Asunto(s)
Receptores ErbB/metabolismo , Oligopéptidos/metabolismo , Secuencia de Aminoácidos , Receptores ErbB/antagonistas & inhibidores , Gastrinas/farmacología , Cinética , Datos de Secuencia Molecular , Oligopéptidos/síntesis química , Oligopéptidos/farmacología , Fosforilación , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
The residue proposed to serve as the catalytic base for phosphoryl transfer, Asp-813, of the human epidermal growth factor receptor (EGFR) was mutated to Ala, and the mutant receptor (D813A) was expressed in Chinese hamster ovary (CHO) cells. Partially purified D813A exhibited no detectable kinase activity in the absence or presence of EGF. A low level of EGF-stimulable phosphorylation of D813A was detectable in intact cells, apparently due to the activity of an associated Tyr kinase(s). As previously observed for kinase-inactive Lys-721 mutants, EGF binding to D813A stimulates mitogen-activated protein kinase activity. Surprisingly, and unlike results reported for Lys-721 mutants, D813A is capable of stimulating both 86Rb+ uptake and DNA synthesis in response to EGF. These data suggest not only that Asp-813 is critical to the catalytic activity of the EGFR but also that differences may exist in the signaling properties of kinase-negative Lys-721 and kinase-negative Asp-813 EGFR mutants.