RESUMEN
Although a modulatory role has been reported for α-lipoic acid (LA) on T-type Ca2+ channels in the nervous system, the acute effects of LA in vivo, particularly on nociceptive transmission in the trigeminal system, remain to be determined. The aim of the present study was to investigate whether acute intravenous LA administration to rats attenuates the excitability of wide dynamic range (WDR) spinal trigeminal nucleus caudalis (SpVc) neurons in response to nociceptive and non-nociceptive mechanical stimulation in vivo. Extracellular single unit recordings were made from seventeen SpVc neurons in response to orofacial mechanical stimulation of pentobarbital-anesthetized rats. Responses to both non-noxious and noxious mechanical stimuli were analyzed in the present study. The mean firing frequency of SpVc WDR neurons in response to both non-noxious and noxious mechanical stimuli was significantly and dose-dependently inhibited by LA (1-100â¯mM, i.v.) and maximum inhibition of the discharge frequency of both non-noxious and noxious mechanical stimuli was seen within 5â¯min. These inhibitory effects lasted for approximately 10â¯min. These results suggest that acute intravenous LA administration suppresses trigeminal sensory transmission, including nociception, via possibly blocking T-type Ca2+ channels. LA may be used as a therapeutic agent for the treatment of trigeminal nociceptive pain.
Asunto(s)
Nocicepción/efectos de los fármacos , Nociceptores/efectos de los fármacos , Ácido Tióctico/farmacología , Núcleo Espinal del Trigémino/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Administración Intravenosa , Animales , Electrofisiología , Cara/inervación , Masculino , Dolor Nociceptivo/tratamiento farmacológico , Dolor Nociceptivo/patología , Nociceptores/patología , Nociceptores/fisiología , Estimulación Física , Ratas Wistar , Piel/inervación , Núcleo Espinal del Trigémino/citología , Núcleo Espinal del Trigémino/patologíaRESUMEN
BACKGROUND: Cystinuria has been proposed to be an inherited defect of apical membrane transport systems for cystine and basic amino acids in renal proximal tubules. Although the mutations of the recently identified transporter BAT1/b(0,+)AT have been related to nontype I cystinuria, the function and localization of human BAT1 (hBAT1)/b(0,+)AT have not been well characterized. METHODS: The cDNA encoding hBAT1 was isolated from human kidney. Fluorescence in situ hybridization was performed to map the hBAT1 gene on human chromosomes. Tissue distribution and localization of expression were examined by Northern blot and immunohistochemical analyses. hBAT1 cDNA was transfected to COS-7 cells with rBAT cDNA, and the uptake and efflux of 14C-labeled amino acids were measured to determine the functional properties. The roles of protein kinase-dependent phosphorylation were investigated using inhibitors or activators of protein kinases. RESULTS: The hBAT1 gene was mapped to 19q12-13.1 on the human chromosome, which is the locus of nontype I cystinuria. hBAT1 message was expressed predominantly in kidney. hBAT1 protein was localized in the apical membrane of proximal tubules in human kidney. When expressed in COS-7 cells with a type II membrane glycoprotein rBAT (related to b(0,+)-amino acid transporter), hBAT1 exhibited the transport activity with the properties of amino acid transport system b(0,+), which transported cystine as well as basic and neutral amino acids presumably via a substrate exchange mechanism. BAT1-mediated transport was reduced by the protein kinase A activator and enhanced by the tyrosine kinase inhibitor. CONCLUSIONS: hBAT1 exhibited the properties expected for a transporter subserving the high-affinity cystine transport system in renal proximal tubules. The hBAT1 gene was mapped to the locus of nontype I cystinuria, confirming the involvement of hBAT1 in cystinuria.
Asunto(s)
Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Cistinuria/genética , Cistinuria/metabolismo , Secuencia de Aminoácidos , Aminoácidos/metabolismo , Animales , Secuencia de Bases , Transporte Biológico Activo , Células COS , Mapeo Cromosómico , ARN Helicasas DEAD-box , Cartilla de ADN/genética , ADN Complementario/genética , Femenino , Humanos , Hibridación Fluorescente in Situ , Técnicas In Vitro , Datos de Secuencia Molecular , Fosforilación , Proteínas Quinasas/metabolismo , ARN Helicasas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Homología de Secuencia de Aminoácido , Distribución Tisular , XenopusRESUMEN
We isolated a cDNA for the human homologue of system asc transporter Asc-1 from human brain. The encoded protein designated as hAsc-1 (human Asc-1) exhibited 91 % sequence identity to mouse Asc-1. Consistent with mouse Asc-1, hAsc-1 required 4F2 heavy chain for its functional expression in Xenopus oocytes. hAsc-1 exhibited the properties of amino acid transport system asc which transports small neutral amino acids in a Na(+)-independent manner. hAsc-1 transported D-serine at high affinity with a K(m) value of 22.8 microM. In brain, 2.0 kb mRNA was highly expressed. hAsc-1 gene was mapped to human chromosome 19, region q12-q13.1. Because of the high-affinity transport with the K(m) value close to the physiological concentration of D-serine, together with the high levels of expression in brain, hAsc-1 is proposed to play significant roles in the D-serine mobilization in brain.
Asunto(s)
Sistema de Transporte de Aminoácidos y+ , Química Encefálica , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Cromosomas Humanos Par 19 , Serina/metabolismo , Sistemas de Transporte de Aminoácidos , Animales , Northern Blotting , Mapeo Cromosómico , Clonación Molecular , ADN Complementario , Expresión Génica/fisiología , Humanos , Hibridación Fluorescente in Situ , Ratones , Datos de Secuencia Molecular , Oocitos/fisiología , ARN Mensajero/análisis , Receptores de N-Metil-D-Aspartato/metabolismo , Homología de Secuencia de Aminoácido , XenopusRESUMEN
Although L-3,4-dihydroxyphenylalanine (L-DOPA) is claimed to be a neurotransmitter in the central nervous system (CNS), receptor or transporter molecules for L-DOPA have not been determined. In an attempt to identify a transporter for L-DOPA, we examined whether or not an active and high affinity L-DOPA transport system is expressed in Xenopus laevis oocytes injected with poly A(+) RNA prepared from several tissues. Among the poly A(+) RNAs tested, rabbit intestinal epithelium poly A(+) RNA gave the highest transport activity for L-[(14)C]DOPA in the oocytes. The uptake was approximately five times higher than that of water-injected oocytes, and was partially Na(+)-dependent. L-Tyrosine, L-phenylalanine, L-leucine and L-lysine inhibited this transport activity, whereas D-DOPA, dopamine, glutamate and L-DOPA cyclohexylester, an L-DOPA antagonist did not affect this transport. Coinjection of an antisense cRNA, as well as oligonucleotide complementary to rabbit rBAT (NBAT) cDNA almost completely inhibited the uptake of L-[(14)C]DOPA in the oocytes. On the other hand, an antisense cRNA of rabbit 4F2hc barely affected this L-[(14)C]DOPA uptake activity. rBAT was thus responsible for the L-[(14)C]DOPA uptake activity expressed in X. laevis oocytes injected with poly A(+) RNA from rabbit intestinal epithelium. As rBAT is localized at the target regions of L-DOPA in the CNS, rBAT might be one of the components involved in L-DOPAergic neurotransmission.
Asunto(s)
Sistemas de Transporte de Aminoácidos Básicos , Aminoácidos/metabolismo , Proteínas Portadoras/metabolismo , Levodopa/farmacocinética , Glicoproteínas de Membrana/metabolismo , Neurotransmisores/farmacocinética , ARN Mensajero/metabolismo , Animales , Transporte Biológico , Proteínas Portadoras/genética , Femenino , Expresión Génica , Mucosa Intestinal/metabolismo , Intestino Delgado/metabolismo , Iones , Cinética , Masculino , Glicoproteínas de Membrana/genética , Microinyecciones/métodos , Oocitos/metabolismo , ARN Mensajero/administración & dosificación , Conejos , Ratas , Ratas Wistar , Sodio/metabolismo , Xenopus laevisRESUMEN
A cDNA was isolated from the mouse brain that encodes a novel Na(+)-independent neutral amino acid transporter. The encoded protein, designated as Asc-1 (asc-type amino acid transporter 1), was found to be structurally related to recently identified mammalian amino acid transporters for the transport systems L, y(+)L, x(C)(-), and b(0,+), which are linked, via a disulfide bond, to the type II membrane glycoproteins, 4F2 heavy chain (4F2hc), or rBAT (related to b(0,+) amino acid transporter). Asc-1 required 4F2hc for its functional expression. In Western blot analysis in the nonreducing condition, a 118-kDa band, which seems to correspond to the heterodimeric complex of Asc-1 and 4F2hc, was detected in the mouse brain. The band shifted to 33 kDa in the reducing condition, confirming that Asc-1 and 4F2hc are linked via a disulfide bond. Asc-1-mediated transport was not dependent on the presence of Na(+) or Cl(-). Although Asc-1 showed a high sequence homology (66% identity at the amino acid level) to the Na(+)-independent broad scope neutral amino acid transporter LAT2 (Segawa, H., Fukasawa, Y., Miyamoto, K., Takeda, E., Endou, H., and Kanai, Y. (1999) J. Biol. Chem. 274, 19745-19751), Asc-1 also exhibited distinctive substrate selectivity and transport properties. Asc-1 preferred small neutral amino acids such as Gly, L-Ala, L-Ser, L-Thr, and L-Cys, and alpha-aminoisobutyric acid as substrates. Asc-1 also transported D-isomers of the small neutral amino acids, in particular D-Ser, a putative endogenous modulator of N-methyl-D-aspartate-type glutamate receptors, with high affinity. Asc-1 operated preferentially, although not exclusively, in an exchange mode. Asc-1 mRNA was detected in the brain, lung, small intestine, and placenta. The functional properties of Asc-1 seem to be consistent with those of a transporter subserving the Na(+)-independent small neutral amino acid transport system asc.
Asunto(s)
Aminoácidos/metabolismo , Proteínas Portadoras/metabolismo , Sodio/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos , Animales , Transporte Biológico , Proteínas Portadoras/química , ADN Complementario , Concentración de Iones de Hidrógeno , Ratones , Datos de Secuencia Molecular , Ratas , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , XenopusRESUMEN
In cholinergic neurons, high-affinity choline uptake in presynaptic terminals is the rate-limiting step in acetylcholine synthesis. Using information provided by the Caenorhabditis elegans Genome Project, we cloned a cDNA encoding the high-affinity choline transporter from C. elegans (cho-1). We subsequently used this clone to isolate the corresponding cDNA from rat (CHT1). CHT1 is not homologous to neurotransmitter transporters, but is homologous to members of the Na+-dependent glucose transporter family. Expression of CHT1 mRNA is restricted to cholinergic neurons. The characteristics of CHT1-mediated choline uptake essentially match those of high-affinity choline uptake in rat brain synaptosomes.
Asunto(s)
Proteínas Portadoras/química , Proteínas Portadoras/genética , Proteínas de Transporte de Membrana , Secuencia de Aminoácidos , Animales , Unión Competitiva/efectos de los fármacos , Células CHO , Caenorhabditis elegans , Proteínas Portadoras/metabolismo , Membrana Celular/metabolismo , Colina/metabolismo , Colina/farmacocinética , Clonación Molecular , Cricetinae , ADN Complementario/genética , Hemicolinio 3/farmacología , Datos de Secuencia Molecular , Proteínas de Transporte de Monosacáridos/genética , Neuronas/metabolismo , Inhibidores de la Captación de Neurotransmisores/farmacología , Oocitos/citología , Oocitos/metabolismo , Especificidad de Órganos , Filogenia , ARN Mensajero/biosíntesis , Ratas , Homología de Secuencia de Aminoácido , Sodio/metabolismoRESUMEN
We have isolated a cDNA from rat small intestine that encodes a novel Na+-independent neutral amino acid transporter with distinctive characteristics in substrate selectivity and transport property. The encoded protein, designated L-type amino acid transporter-2 (LAT-2), shows amino acid sequence similarity to the system L Na+-independent neutral amino acid transporter LAT-1 (Kanai, Y., Segawa, H., Miyamoto, K., Uchino, H., Takeda, E., and Endou, H. (1998) J. Biol. Chem. 273, 23629-23632) (50% identity) and the system y+L transporters y+LAT-1 (47%) and KIAA0245/y+LAT-2 (45%) (Torrents, D., Estevez, R., Pineda, M., Fernandez, E., Lloberas, J., Shi, Y.-B., Zorzano, A., and Palacin, M. (1998) J. Biol. Chem. 273, 32437-32445). LAT-2 is a nonglycosylated membrane protein. It requires 4F2 heavy chain, a type II membrane glycoprotein, for its functional expression in Xenopus oocytes. LAT-2-mediated transport is not dependent on Na+ or Cl- and is inhibited by a system L-specific inhibitor, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), indicating that LAT-2 is a second isoform of the system L transporter. Compared with LAT-1, which prefers large neutral amino acids with branched or aromatic side chains, LAT-2 exhibits remarkably broad substrate selectivity. It transports all of the L-isomers of neutral alpha-amino acids. LAT-2 exhibits higher affinity (Km = 30-50 microM) to Tyr, Phe, Trp, Thr, Asn, Ile, Cys, Ser, Leu, Val, and Gln and relatively lower affinity (Km = 180-300 microM) to His, Ala, Met, and Gly. In addition, LAT-2 mediates facilitated diffusion of substrate amino acids, as distinct from LAT-1, which mediates amino acid exchange. LAT-2-mediated transport is increased by lowering the pH level, with peak activity at pH 6.25, because of the decrease in the Km value without changing the Vmax value. Because of these functional properties and a high level of expression of LAT-2 in the small intestine, kidney, placenta, and brain, it is suggested that the heterodimeric complex of LAT-2 and 4F2 heavy chain is involved in the trans-cellular transport of neutral amino acids in epithelia and blood-tissue barriers.
Asunto(s)
Proteínas Portadoras/genética , Intestino Delgado/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos , Aminoácidos/metabolismo , Animales , Antígenos CD/genética , Transporte Biológico , Proteínas Portadoras/química , Clonación Molecular , Proteína-1 Reguladora de Fusión , Concentración de Iones de Hidrógeno , Cinética , Leucina/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/genética , Datos de Secuencia Molecular , Oocitos/metabolismo , ARN Complementario/genética , ARN Mensajero/metabolismo , Ratas , Alineación de Secuencia , Especificidad por Sustrato , XenopusRESUMEN
Experiment was designed to determine whether heat stress suppresses neutrophil function and injections of selenium and vitamin E prior to heat stress prevent suppression of neutrophil function in goats. Twelve female goats were divided into 2 groups of 6 each and were kept at 25 degrees C. Goats in the treatment group were injected intramuscularly with 0.1 mg/kg of selenium and 2.72 IU/kg of vitamin E at 8 and 1 day prior to the initiation of heat stress. The other group was kept as control. All goats were exposed to hot environment at 38 degrees C from day 0 through 8. Decreased tendency in plasma cortisol concentrations and temporary increase in plasma glucose concentrations were shown in both groups. In the control group, plasma selenium concentration gradually increased and alpha-tocopherol concentration decreased during the first 2 days. After the second injection with selenium and vitamin E, plasma selenium and alpha-tocopherol concentration significantly increased and remained higher than those in the control group. Whole blood glutathione peroxidase (GSH-Px) activity in the treatment group tended to be greater than that in the control group, but no significant difference was observed between 2 groups. The nitroblue tetrazolium (NBT) reduction by activated neutrophils significantly decreased on day 6 in the control group but not in the treatment group. The NBT reduction by resting neutrophils significantly decreased in both groups. These data suggest that heat stress depresses neutrophil function, and selenium and vitamin E injection prior to heat stress has no apparent effect on neutrophil function during the stress.
Asunto(s)
Cabras/sangre , Trastornos de Estrés por Calor/veterinaria , Indicadores y Reactivos/metabolismo , Neutrófilos/efectos de los fármacos , Nitroazul de Tetrazolio/metabolismo , Selenio/farmacología , Vitamina E/farmacología , Animales , Glucemia/metabolismo , Temperatura Corporal , Femenino , Glutatión Peroxidasa/sangre , Trastornos de Estrés por Calor/sangre , Hematócrito , Hidrocortisona/sangre , Recuento de Leucocitos , Neutrófilos/metabolismo , Respiración , Selenio/sangre , Vitamina E/sangreRESUMEN
Nucleobindin (Nuc) was originally identified as a 55 kDa protein that enhanced anti-DNA antibody production in cultures of autoimmune MRL/lpr mouse spleen cells. cDNA cloning and the production of recombinant protein (rNuc) have revealed that rNuc binds to DNA and Ca2+ by means of leucine zipper and EF-hand motif structures. In vitro and in vivo effects of rNuc to induce autoantibodies including anti-dsDNA antibody in normal mice have been demonstrated; however, whether Nuc is involved in the state of autoimmunity occurred in MRL/lpr, C3H/gld and male BXSB mice has not been elucidated. Here we report that the expression of Nuc mRNA is upregulated with age in the lymphatic organs and is positively correlated to the serum levels of Nuc in these lupus-prone strains of mice. Upon immunization with KLH in Freund's complete adjuvant, normal BALB/c mice also showed an elevated level of Nuc in their serum and elevated Nuc mRNA in their lymphatic organs. In addition, in vitro stimulation of BALB/c splenocytes with Con A or LPS remarkably enhanced Nuc mRNA expression. These results suggest that upregulation of Nuc mRNA in lymphatic organs and serum Nuc of lupus-prone mice is related to spontaneous activation of immunocompetent cells; the present data are also consistent with our previous hypothesis on the role of Nuc in the induction or enhancement of autoimmunity in lupus models of mice.
Asunto(s)
Anticuerpos Antinucleares/biosíntesis , Proteínas de Unión al ADN/biosíntesis , Sustancias de Crecimiento/biosíntesis , Lupus Eritematoso Sistémico/genética , Linfocitos/inmunología , Bazo/inmunología , Animales , Proteínas de Unión al Calcio , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/fisiología , Femenino , Regulación de la Expresión Génica , Sustancias de Crecimiento/química , Sustancias de Crecimiento/fisiología , Leucina Zippers , Lupus Eritematoso Sistémico/inmunología , Activación de Linfocitos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos , Ratones Mutantes , Proteínas del Tejido Nervioso , Nucleobindinas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Especificidad de la Especie , Transcripción GenéticaAsunto(s)
Sistemas de Transporte de Aminoácidos Básicos , Proteínas Portadoras/metabolismo , Glutamina/metabolismo , Testículo/metabolismo , Animales , Transporte Biológico , Northern Blotting , Proteínas Portadoras/química , Proteínas Portadoras/genética , Clonación Molecular , ADN Complementario/genética , Expresión Génica , Masculino , Proteínas de la Membrana/metabolismo , Ratones , Modelos Moleculares , Oocitos/metabolismo , Filogenia , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , ARN Mensajero/metabolismo , Especificidad por Sustrato , XenopusRESUMEN
A cDNA was isolated from mouse testis which encodes a Na+-dependent neutral amino acid transporter. The encoded protein, designated ASCT2, showed amino acid sequence similarity to the mammalian glutamate transporters (40-44% identity), Na+-dependent neutral amino acid transporter ASCT1 (57% identity; Arriza, J. L., Kavanaugh, M. P., Fairman, W. A., Wu, Y.-N., Murdoch, G. H., North, R. A., and Amara, S. G.(1993) J. Biol. Chem. 268, 15329-15332; Shafqat, S., Tamarappoo, B. K., Kilberg, M. S., Puranam, R. S., McNamara, J. O., Guadano-Ferraz, A., and Fremeau, T., Jr. (1993) J. Biol. Chem. 268, 15351-15355) and a mouse adipocyte differentiation-associated gene product AAAT (94% identity; Liao, K., and Lane, D.(1995) Biochem. Biophys. Res. Commun. 208, 1008-1015). When expressed in Xenopus laevis oocytes, ASCT2 exhibited Na+-dependent uptakes of neutral amino acids such as L-alanine, L-serine, L-threonine, L-cysteine, and L-glutamine at high affinity with Km values around 20 microM. L-Methionine, L-leucine, L-glycine, and L-valine were also transported by ASCT2 but with lower affinity. The substrate selectivity of ASCT2 was typical of amino acid transport system ASC, which prefers neutral amino acids without bulky or branched side chains. ASCT2 also transported L-glutamate at low affinity (Km = 1.6 mM). L-Glutamate transport was enhanced by lowering extracellular pH, suggesting that L-glutamate was transported as protonated form. In contrast to electrogenic transport of glutamate transporters and the other ASC isoform ASCT1, ASCT2-mediated amino acid transport was electroneutral. Na+ dependence of L-alanine uptake fits to the Michaelis-Menten equation, suggesting a single Na+ cotransported with one amino acid, which was distinct from glutamate transporters coupled to two Na+. Northern blot hybridization revealed that ASCT2 was mainly expressed in kidney, large intestine, lung, skeletal muscle, testis, and adipose tissue. Functional characterization of ASCT2 provided fruitful information on the properties of substrate binding sites and the mechanisms of transport of Na+-dependent neutral and acidic amino acid transporter family, which would facilitate the structure-function analyses based on the comparison of the primary structures of ASCT2 and the other members of the family.
Asunto(s)
Aminoácidos/metabolismo , Proteínas Portadoras/metabolismo , Testículo/metabolismo , Secuencia de Aminoácidos , Sistemas de Transporte de Aminoácidos , Animales , Secuencia de Bases , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/química , Clonación Molecular , Cartilla de ADN , ADN Complementario , Femenino , Concentración de Iones de Hidrógeno , Cinética , Masculino , Ratones , Ratones Endogámicos ICR , Ratones Endogámicos , Datos de Secuencia Molecular , Oocitos/fisiología , Especificidad de Órganos , Reacción en Cadena de la Polimerasa , Homología de Secuencia de Aminoácido , Estereoisomerismo , Especificidad por Sustrato , Xenopus laevisRESUMEN
EAAC1 is a neuronal and epithelial high affinity glutamate transporter previously cloned from rabbit intestine. Here we report the isolation of EAAC 1 from rat brain* and its expression in the central nervous system based on in situ hybridization. Strong signals were detected in brain, spinal cord and retina. Expression of EAAC1 was particularly strong in pyramidal cells of the cerebral cortex, pyramidal cells of the hippocampus, mitral cells of the olfactory bulb, various thalamic nuclei and cells of certain retinal layers. EAAC1 was also expressed in non-glutamatergic neurons such as GABAergic cerebellar Purkinje cells and alpha-motor neurons of the spinal cord. We propose that EAAC1 is not only involved in the sequestration of glutamate at glutamatergic synapses and in protecting neurons from glutamate excitotoxicity, but also in the cellular metabolism involving glutamate.
Asunto(s)
Sistema de Transporte de Aminoácidos X-AG , Proteínas Portadoras/metabolismo , Sistema Nervioso Central/metabolismo , Ácido Glutámico/metabolismo , Neuronas/metabolismo , Simportadores , Secuencia de Aminoácidos , Animales , Transporte Biológico , Northern Blotting , Química Encefálica , Proteínas Portadoras/genética , Proteínas Portadoras/aislamiento & purificación , Sistema Nervioso Central/citología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , ADN Complementario/biosíntesis , ADN Complementario/aislamiento & purificación , Transportador 3 de Aminoácidos Excitadores , Proteínas de Transporte de Glutamato en la Membrana Plasmática , Hibridación in Situ , Masculino , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Retina/citología , Retina/metabolismo , Sodio/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
The heterotrimeric G protein, G alpha i2, transduces signals from seven membrane spanning receptors to effectors such as adenylyl cyclase and ion channels. The purpose of this study was to identify these or other cellular proteins that interact with G alpha i2 by use of the yeast two-hybrid system. A human B cell cDNA library was screened by this system using full length G alpha i2. Four positive colonies were obtained. Two of the four were identified as nucleobindin, a calcium binding protein and a putative antigen to which anti-nuclear antibodies are generated in mice with a disorder that resembles systemic lupus erythematosus. Nucleobindin has a leucine zipper, EF hands, and a signal peptide sequence and is thought to localize to the nucleus as well as being secreted. The specificity of intehraction between G alpha i2 and nucleobindin was confirmed by an in vitro binding assay using recombinant proteins. Transfection of G alpha i2 and nucleobindin in COS cells increased G alpha i2 expression relative to cells transfected with G alpha i2 and mock vector. Our results indicate that the yeast two-hybrid system provides a means to identify novel proteins that interact with G alpha proteins. Nucleobindin appears to represent one of those proteins.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Sustancias de Crecimiento/metabolismo , Animales , Anticuerpos Monoclonales , Linfocitos B/fisiología , Secuencia de Bases , Proteínas de Unión al Calcio , Línea Celular , Chlorocebus aethiops , Cartilla de ADN , ADN Complementario , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/química , Proteínas de Unión al GTP/biosíntesis , Biblioteca de Genes , Glutatión Transferasa/biosíntesis , Sustancias de Crecimiento/biosíntesis , Sustancias de Crecimiento/química , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso , Nucleobindinas , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Transfección , beta-Galactosidasa/biosíntesisRESUMEN
Our previous works have shown that nucleobindin (Nuc) or recombinant (r) Nuc not only augments anti-DNA antibody production in vitro but also accelerates autoimmune response in vivo in MRL/+/+ (MRL/n) mice which are the substrain of autoimmune MRL/lpr/lpr (MRL/l) mice. To investigate whether rNuc can induce autoimmune response similarly in naive mice, we carried out intraperitoneal (i.p.) injection of rNuc (5 micrograms) without adjuvant into 8-week-old female BALB/c mice and continued injection twice a week for 12 weeks. About 5 weeks after the first injection, all the mice began to show IgG hypergammaglobulinemia (HG) followed by elevation of a number of autoantibodies of the IgG class such as anti-double-stranded (ds) DNA, anti-U1 ribonuclear protein (RNP), anti-ssB(La) and anti-Fc antibodies (RF), but not by anti-Sm antibodies. However, the IgG anti-dsDNA antibody response and histopathological changes in the kidney of these BALB/c mice were not so noticeable as those in MRL/n mice induced by rNuc in our previous experiment. In contrast, the IgG anti-rNuc antibody response of normal BALB/c mice induced by rNuc was stronger than that of MRL/n mice induced by rNuc. Since the titers of each autoantibody of BALB/c mice induced by rNuc were not always associated with the level of IgG HG, and either IgG HG or IgG autoantibodies could not be induced by control administration of extracts (5 micrograms) of Escherichia coli with or without harboring plasmid alone, polyclonal B cell activation (PBA) appeared not to be the mechanism of this autoimmunity.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Autoanticuerpos/biosíntesis , Enfermedades Autoinmunes/inmunología , Proteínas de Unión al ADN/inmunología , Sustancias de Crecimiento/inmunología , Animales , Anticuerpos Antinucleares/biosíntesis , Anticuerpos Antinucleares/sangre , Autoanticuerpos/sangre , Autoanticuerpos/inmunología , Proteínas de Unión al Calcio , ADN/sangre , ADN/inmunología , Femenino , Inmunidad Innata , Inmunización , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Inmunoglobulina G/inmunología , Trastornos Linfoproliferativos/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Mutantes , Proteínas del Tejido Nervioso , Nucleobindinas , Proteínas Recombinantes/inmunología , Ribonucleoproteína Nuclear Pequeña U1/inmunologíaRESUMEN
Three new nor-cucurbitacin glucosides were isolated from the root of Caputo nigri (Cabeça-de-negro) and their structures established as 29-nor-1,2,3,4,5,10-dehydro-2,3,16 alpha,20R, 22 zeta,25-hexahydroxy-11-oxocucurbita-6,23-diene 2-O-beta-D-glucopyranoside and 2-O-beta-D-glucopyranosyl(1-2)beta-D- glucopyranoside(sophorose), and 1,2,3,4,5,10-dehydro-2,3,16 alpha-trihydroxy-4, 9,14-trimethyl-19-norpregn-6-ene-11,20-dione 2-O-beta-D-glucopyranoside on the basis of detailed spectroscopic analysis.
Asunto(s)
Glucósidos/aislamiento & purificación , Plantas Medicinales/química , Glucósidos/química , Espectroscopía de Resonancia Magnética , Espectrofotometría InfrarrojaRESUMEN
High affinity transport of glutamate across plasma membranes of brain neurons and epithelial is mediated by a Na(+)- and K(+)-coupled electrogenic transporter. Here we report the primary structure and functional characterization of the human high affinity glutamate transporter (HEAAC1). A unique characteristic of HEAAC1-mediated transport is that the affinity for glutamate and the maximal transport rate are strongly dependent on membrane potential. Our data provide new insights into individual steps of high affinity glutamate transport and show that the transport mechanism is distinct from that of the gamma-aminobutyric acid transporter GAT-1 and the Na+/glucose transporter SGLT1. Under voltage clamp condition, HEAAC1 mediated large substrate-evoked inward currents (up to 1 microA). The substrate specificity, stereospecificity, the Km value (30 +/- 3 microM at -60 mV) of the L-glutamate-evoked current, and Northern analysis all agree with previously reported characteristics of high affinity glutamate transport in brain. In contrast to SGLT1 and GAT-1, voltage jump studies of HEAAC1 yielded only minor relaxation currents. Classic inhibitors of brain glutamate uptake such as DL-threo-beta-hydroxyaspartate, L-trans-pyrrolidine 2,4,-dicarboxylic acid (PDC), and dihydrokainate were found to be either transport substrates or to have no significant effect on glutamate transport. We also found that the maximal transport rate for PDC was markedly reduced compared to that for L-glutamate. We propose that PDC most likely reduces the turnover rate of the transporter. A search of the sequence data bases revealed weak homology of HEAAC1 to the H(+)-coupled vesicular monoamine transporter, suggesting an evolutionary link between plasma membrane and vesicular transporters.
Asunto(s)
Glicoproteínas/metabolismo , Neuronas/metabolismo , Adulto , Secuencia de Aminoácidos , Sistema de Transporte de Aminoácidos X-AG , Animales , Transporte Biológico , Clonación Molecular , ADN Complementario , Epitelio/metabolismo , Antagonistas de Aminoácidos Excitadores , Glutamatos/metabolismo , Ácido Glutámico , Glicoproteínas/química , Humanos , Activación del Canal Iónico , Potenciales de la Membrana , Datos de Secuencia Molecular , Homología de Secuencia de Aminoácido , Transmisión Sináptica , XenopusRESUMEN
We previously purified a 55 kDa protein that preferentially expands anti-DNA antibody production both in vitro and in vivo across the H-2 barrier from culture supernatants of KML1-7 cells, cloned from a lupus-prone MRL/lpr mouse. By using the purified protein, termed nucleobindin (Nuc), we cloned cDNA and produced recombinant(r) Nuc in Escherichia coli. To elucidate the function of rNuc in vivo, we initially injected intraperitoneally 5 micrograms of rNuc without adjuvant into female MRL/n mice at 8 weeks of age and continued injection twice a week. As early as 5 weeks after administration, all mice treated showed an increase in IgG anti-double stranded (ds) DNA antibodies accompanied by IgG hypergammaglobulinemia (HG). Of particular interest was that these mice also produced anti-U1RNP antibodies and rheumatoid factor (RF) of IgG class, but not anti-Sm antibodies. Histopathologically, hypercellularity with occasional crescents in the glomeruli was observed, but evidence for lupus nephritis was lacking, indicating that some factors other than Nuc are necessary for the development of a lupus syndrome observed in MRL/lpr mice. Similar administration of lipopolysaccharide into MRL/n mice failed to induce autoantibodies except for a slight increase in serum IgG, suggesting that these autoimmune responses are not due simply to polyclonal B-cell activation. The presence of rNuc will give us a clue for further understanding of autoimmunity.