RESUMEN
⦠BACKGROUND: The use of pH-neutral peritoneal dialysis (PD) fluids low in glucose degradation products (GDP) may better preserve the peritoneal membrane and have fewer systemic effects. The effects of conversion from conventional to neutral-pH, low-GDP PD fluids in prevalent patients are unclear. Few studies on the role of neutral-pH, low-GDP PD have studied residual renal function, ultrafiltration, peritonitis incidence and technique failure, transport characteristics, and local and systemic markers of inflammation in prevalent PD patients. ⦠METHODS: In a multi-center open-label randomized clinical trial (RCT), we randomly assigned 40 of 78 stable continuous ambulatory PD (CAPD) and automated PD (APD) patients to treatment with bicarbonate/lactate, neutral-pH, low-GDP PD fluid (Physioneal; Baxter Healthcare Corporation, Deerfield, IL, USA) and compared them with 38 patients continuing their current standard lactate-buffered PD fluid (PDF) (Dianeal; Baxter Healthcare Corporation, Deerfield, IL, USA) during 2 years. Primary outcome was residual renal function (RRF) and ultrafiltration (UF) during peritoneal equilibration test (PET); peritonitis incidence was a secondary outcome. Furthermore, clinical parameters as well as several biomarkers in effluents and serum were measured. ⦠RESULTS: During follow-up, RRF did not differ between the groups. In the Physioneal group ultrafiltration (UF) during PET remained more or less stable (-20 mL [confidence interval (CI): -163.5 - 123.5 mL]; p = 0.7 over 24 months), whereas it declined in the Dianeal group (-243 mL [CI: -376.6 to -109.4 mL]; p < 0.0001 over 24 months), resulting in a difference of 233.7 mL [95% CI 41.0 - 425.5 mL]; p = 0.017 between the groups at 24 months. The peritonitis rate was lower in the Physioneal group: adjusted odds ratio (OR) 0.38 (0.15 - 0.97) p = 0.043. No differences were observed between the 2 groups in peritoneal adequacy or transport characteristics nor effluent markers of local inflammation (cancer antigen [CA]125, hyaluronan [HA], vascular endothelial growth factor [VEGF], macrophage chemo-attractant protein [MCP]-1, HA and transforming growth factor [TGF]ß-1). ⦠CONCLUSION: In prevalent PD patients, our study did not find a difference in RRF after conversion from conventional to neutral-pH, low-GDP PD fluids, although there is a possibility that the study was underpowered to detect a difference. Decline in UF during standardized PET was lower after 2 years in the Physioneal group.
Asunto(s)
Soluciones para Diálisis/química , Guanosina Difosfato/análisis , Fallo Renal Crónico/terapia , Diálisis Peritoneal Ambulatoria Continua/métodos , Tampones (Química) , Femenino , Estudios de Seguimiento , Humanos , Concentración de Iones de Hidrógeno , Incidencia , Masculino , Persona de Mediana Edad , Países Bajos/epidemiología , Compuestos Orgánicos/farmacología , Peritoneo , Peritonitis/epidemiología , Estudios Prospectivos , Factores de TiempoRESUMEN
Directional translocation of the ribosome through the mRNA open reading frame is a critical determinant of translational fidelity. This process entails a complex interplay of large-scale conformational changes within the actively translating particle, which together coordinate the movement of tRNA and mRNA substrates with respect to the large and small ribosomal subunits. Using pre-steady state, single-molecule fluorescence resonance energy transfer imaging, we tracked the nature and timing of these conformational events within the Escherichia coli ribosome from five structural perspectives. Our investigations revealed direct evidence of structurally and kinetically distinct late intermediates during substrate movement, whose resolution determines the rate of translocation. These steps involve intramolecular events within the EF-G-GDP-bound ribosome, including exaggerated, reversible fluctuations of the small-subunit head domain, which ultimately facilitate peptidyl-tRNA's movement into its final post-translocation position.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Factor G de Elongación Peptídica/metabolismo , ARN Bacteriano/metabolismo , Aminoacil-ARN de Transferencia/metabolismo , Ribosomas/metabolismo , Escherichia coli/química , Proteínas de Escherichia coli/análisis , Transferencia Resonante de Energía de Fluorescencia , Guanosina Difosfato/análisis , Guanosina Difosfato/metabolismo , Modelos Moleculares , Factor G de Elongación Peptídica/análisis , Transporte de Proteínas , ARN Bacteriano/análisis , Aminoacil-ARN de Transferencia/análisis , Ribosomas/químicaRESUMEN
A novel amphiphilic Tb(3+) complex (TbL(3+)(I)) consisting of a +3 charged head and a hydrophobic alkyl chain has been developed. It spontaneously self-assembles in water and forms stable vesicles at neutral pH. TbL(3+)(I) has no aromatic groups (functioning as an antenna), and its intrinsic luminescence is thus minimized. These features lead to the self-assembling TbL(3+)(I) receptor molecules demonstrating an increased luminescence intensity upon binding of nucleotides. Upon addition of guanosine triphosphate (GTP), the luminescence from Tb(3+) was notably promoted (127-fold), as the light energy absorbed by the guanine group of GTP was efficiently transferred to the Tb(3+) center. In the case of guanosine diphosphate (GDP) and guanosine monophosphate (GMP), respectively, 78-fold and 43-fold increases in luminescence intensity were observed. This enhancement was less significant than that observed for GTP, due to fewer negative charges on GDP and GMP. No other nucleotides or the tested nonphosphorylated nucleosides affected the luminescence intensity to any notable extent. In marked contrast, all tested nucleotides, including guanine nucleotides, barely promoted the luminescence of molecularly dispersed receptors, TbL(3+)(II), indicating that the confinement and organization of molecules in a nanointerface play vital roles in improving the performance of a sensing system. This Tb(3+) complex nanointerface is successfully used for monitoring the GTP-to-GDP conversion.
Asunto(s)
Técnicas Biosensibles , Guanosina Trifosfato/análisis , Luminiscencia , Nanopartículas/química , Guanosina Difosfato/análisis , Tamaño de la Partícula , Terbio/química , Agua/químicaRESUMEN
An off-the-shelf supramolecular sensing system was designed to discriminate biologically relevant phosphates in neutral water using multivariate data analysis. The system is based on an indicator displacement assay comprising only two unmodified commercially available components: a dendritic poly-electrolyte and a common fluorescent dye. Effective discrimination of nucleotide diphosphates and inorganic diphosphate was achieved through principal component analysis (PCA).
Asunto(s)
Adenosina Difosfato/análisis , Citidina Difosfato/análisis , Colorantes Fluorescentes , Guanosina Difosfato/análisis , Fosfatos/análisis , Agua/química , Concentración de Iones de Hidrógeno , Polímeros/química , Análisis de Componente PrincipalRESUMEN
The development of novel fluorescence methods for the detection of key biomolecules is of great interest, both in basic research and in drug discovery. Particularly relevant and widespread molecules in cells are ADP and GDP, which are the products of a large number of cellular reactions, including reactions catalysed by nucleoside triphosphatases and kinases. Previously, biosensors for ADP were developed in this laboratory, based on fluorophore adducts with the bacterial actin homologue ParM. It is shown in the present study that one of these biosensors, tetramethylrhodamine-ParM, can also monitor GDP. The biosensor can be used to measure micromolar concentrations of GDP on the background of millimolar concentrations of GTP. The fluorescence response of the biosensor is fast, the response time being <0.2 s. Thus the biosensor allows real-time measurements of GTPase and GTP-dependent kinase reactions. Applications of the GDP biosensor are exemplified with two different GTPases, measuring the rates of GTP hydrolysis and nucleotide exchange.
Asunto(s)
Técnicas Biosensibles/métodos , Guanosina Difosfato/análisis , Rodaminas/metabolismo , Actinas/metabolismo , Proteínas de Escherichia coli/metabolismo , Colorantes Fluorescentes , GTP Fosfohidrolasas/análisis , Proteínas ras/metabolismoRESUMEN
A new, homogeneous, high-throughput-compatible assay method is described for the fluorescence-based quantitation of nanomolar concentrations of ribonucleoside diphosphates (rNDPs). The principle of the method is the conversion of the rNDPs to RNA by the enzyme polynucleotide phosphorylase (EC 2.7.7.8) and detection of the RNA by the increased fluorescence of a commercial nucleic acid detection dye. A commercial RNA homopolymer complementary to the RNA product is included to increase the sensitivity for ADP and UDP. Standard curves for nanomolar concentrations of ADP, UDP, GDP, and CDP are shown. The assay detected 75 nM ADP produced by the pyruvate kinase-catalyzed phosphorylation of pyruvate with a signal-to-baseline ratio of 2.8. The assay may be used in either a continuous or a discontinuous mode.
Asunto(s)
Adenosina Difosfato/análisis , Espectrometría de Fluorescencia/métodos , Citidina Difosfato/análisis , Colorantes Fluorescentes/química , Guanosina Difosfato/análisis , Ensayos Analíticos de Alto Rendimiento/métodos , Cinética , Fosforilación , Piruvato Quinasa/metabolismo , Piruvatos/metabolismo , ARN/química , Uridina Difosfato/análisisRESUMEN
Since the adequate supply of guanine nucleotides is vital for cellular activities, limitation of their syntheses would certainly result in modulation of cellular fate toward differentiation and apoptosis. The aim of this study was to set a correlation between the intracellular level of GTP and the induction of relevant signaling pathways involved in the cell's fate toward life or death. In that regard, we measured the GTP level among human leukemia K562 cells exposed to mycophenolic acid (MPA) or 3-hydrogenkwadaphnin (3-HK) as two potent inosine monophosphate dehydrogenase inhibitors. Our results supported the maturation of the cells when the intracellular GTP level was reduced by almost 30-40%. Under these conditions, 3-HK and/or MPA caused up-regulation of PKCα and PI3K/AKT pathways. Furthermore, co-treatment of cells with hypoxanthine plus 3-HK or MPA, which caused a reduction of about 60% in the intracellular GTP levels, led to apoptosis and activation of mitochondrial pathways through inverse regulation of Bcl-2/Bax expression and activation of caspase-3. Moreover, our results demonstrated that attenuation of GTP by almost 60% augmented the intracellular ROS and nuclear localization of p21 and subsequently led to cell death. These results suggest that two different threshold levels of GTP are needed for induction of differentiation and/or ROS-associated apoptosis.
Asunto(s)
Apoptosis , Diferenciación Celular , Guanosina Trifosfato/análisis , Adenosina Trifosfato/análisis , Diterpenos/farmacología , Guanosina Difosfato/análisis , Humanos , Hipoxantina/farmacología , IMP Deshidrogenasa/antagonistas & inhibidores , Células K562 , Ácido Micofenólico/farmacología , Fosfatidilinositol 3-Quinasas/fisiología , Proteína Quinasa C-alfa/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Especies Reactivas de Oxígeno/metabolismoRESUMEN
The 21-kD protein Ras of the low-molecular-weight GTP-binding (LMWG) family plays an important role in transduction of extracellular signals. Ras functions as a 'molecular switch' in transduction of signals from the membrane receptors of many growth factors, cytokines, and other second messengers to the cell nucleus. Numerous studies have shown that in multiple malignant tumors and hematopoietic malignancies, faulty signal transduction via the Ras pathway plays a key role in tumorigenesis. In this work, a non-radioactive assay was used to quantify Ras activity in hematologic malignancies. Ras activation was measured in six different cell lines and 24 patient samples, and sequence analysis of N- and K-ras was performed. The 24 patient samples comprised of seven acute myelogenous leukemia (AML) samples, five acute lymphocytic leukemia (ALL) samples, four myeloproliferative disease (MPD) samples, four lymphoma samples, four juvenile myelomonocytic leukemia (JMML) samples, and WBC from a healthy donor. The purpose of this study was to compare Ras activity determined by percentage of Ras-GTP with the mutational status of the Ras gene in the hematopoietic cells of the patients. Mutation analysis revealed ras mutations in two of the seven AML samples, one in codon 12 and one in codon 61; ras mutations were also found in two of the four JMML samples, and in one of the four lymphoma samples (codon 12). We found a mean Ras activation of 23.1% in cell lines with known constitutively activating ras mutations, which was significantly different from cell lines with ras wildtype sequence (Ras activation of 4.8%). Two of the five activating ras mutations in the patient samples correlated with increased Ras activation. In the other three samples, Ras was probably activated through "upstream" or "downstream" mechanisms.
Asunto(s)
Neoplasias Hematológicas/química , Neoplasias Hematológicas/genética , Proteínas ras/análisis , Proteínas ras/genética , Análisis Mutacional de ADN , Guanosina Difosfato/análisis , Guanosina Trifosfato/análisis , Neoplasias Hematológicas/etiología , Humanos , Leucemia Mieloide Aguda , Leucemia Mielomonocítica Juvenil , Linfoma , Mutación , Trastornos Mieloproliferativos , Oncogenes , Leucemia-Linfoma Linfoblástico de Células Precursoras , Transducción de Señal/genética , Células Tumorales CultivadasAsunto(s)
Metabolismo , Saccharomyces cerevisiae/metabolismo , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Acetilcoenzima A/análisis , Adenosina Difosfato/análisis , Adenosina Trifosfato/análisis , Técnicas Citológicas , Guanosina Difosfato/análisis , Guanosina Trifosfato/análisis , Saccharomyces cerevisiae/citologíaRESUMEN
Enzymatic cleavage of the P-chiral diastereoisomers of the 5' mRNA cap analogue bearing phosphorothioate moiety in alfa position of 5',5'-triphosphate bridge (m(7)Gppp(S)G D1 and D2) was performed by human Decapping Scavenger (DcpS) enzyme. Analysis of the degradation products allowed to estimate the absolute configuration at the asymmetric phosphorus atoms in examined compounds via correlation with the R(P) and S(P) diastereoisomers of guanosine 5'-O-(1-thiodiphosphate) (GDPalphaS).
Asunto(s)
Endorribonucleasas/química , Conformación de Ácido Nucleico , Análogos de Caperuza de ARN/química , Caperuzas de ARN/química , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/análisis , Humanos , Hidrólisis , Tionucleótidos/análisisRESUMEN
In the heart beta1-adrenergic (beta1R) and adenosine A1 (A1R) and A2A (A2AR) receptors modulate contractile and metabolic function. The interaction between these receptors was investigated at the level of G-protein cycling by determining the effect of receptor agonists on the binding of GTP to G-proteins and displacement of G alpha-subunit-bound GDP by GTP. Crude membranes from rat heart or brain were stimulated by agonists for beta1R (isoproterenol; ISO), A1R (chlorocyclopentyladenosine, CCPA) and A2AR (CGS-21680; CGS). GTP binding to membranes was increased by ISO (17%), CCPA (6%) and CGS (12%). Binding values observed with incubation using ISO and CCPA together were significantly less than values obtained by the incubation of individual agents alone. With ISO, GTP binding to G alpha(s) subunits as determined by immunoprecipitation was increased 79% in heart and 87% in brain. These increases were attenuated by CCPA, an effect that was inhibited by CGS. GDP release by membranes was increased 6.9% and 4.6% by ISO and CCPA, respectively. After co-incubation of these agonists, release was increased less than determined by the addition of the individual agent responses. CGS inhibited the reduced release caused by of CCPA. Adenylyl cyclase activity stimulated by ISO was attenuated 33% by CCPA, an effect inhibited by CGS. Together, these results indicate that A1R exert an antiadrenergic action at the level of beta1R stimulated G(s)-protein cycling and that A2AR reduce this action.
Asunto(s)
Agonistas Adrenérgicos beta/farmacología , Proteínas de Unión al GTP/metabolismo , Ventrículos Cardíacos/metabolismo , Receptor de Adenosina A1/fisiología , Receptores de Adenosina A2/fisiología , Adenosina/análogos & derivados , Adenosina/farmacología , Adenilil Ciclasas/análisis , Adenilil Ciclasas/metabolismo , Animales , Guanosina Difosfato/análisis , Guanosina Difosfato/metabolismo , Isoproterenol/farmacología , Proteínas de la Membrana/metabolismo , Membranas/efectos de los fármacos , Membranas/metabolismo , Fenetilaminas/farmacología , Pruebas de Precipitina , Ratas , Ratas Sprague-DawleyRESUMEN
We describe a capillary electrophoresis (CE) assay to detect G protein-coupled receptor (GPCR)-stimulated G protein GTPase activity in cell membranes expressing alpha2A adrenoreceptor-Galphao1 wild-type (wt) or C351I mutant fusion proteins using a fluorescent, hydrolyzable GTP analogue. As no change in total fluorescence is observed by conversion of substrate to product, CE is used to separate the fluorescent substrate (*GTP) from the fluorescent product (*GDP). Using the assay, the alpha2a adrenoceptor agonist UK14,304 was shown to simulate specific production of *GDP in membranes from HEK293T cells expressing receptor-G protein fusion to 525% of basal levels with an EC50 of 0.48 +/- 0.20 microM. The EC50 increased to 9.4 +/- 5 muM with addition of the antagonist yohimbine. Nucleotide hydrolysis was increased further over agonist-stimulated levels with addition of the in vivo modulator protein RGS (regulator of G protein signaling). It is envisioned that this technique could be used for screening for novel GPCR ligands or other G protein signaling modifiers.
Asunto(s)
Electroforesis Capilar/métodos , GTP Fosfohidrolasas/análisis , Receptores Acoplados a Proteínas G , Línea Celular , Membrana Celular/enzimología , Evaluación Preclínica de Medicamentos/métodos , Colorantes Fluorescentes/análisis , GTP Fosfohidrolasas/metabolismo , Guanosina Difosfato/análisis , Guanosina Difosfato/biosíntesis , Guanosina Trifosfato/análisis , Guanosina Trifosfato/metabolismo , Humanos , Ligandos , Receptores Adrenérgicos alfa 2 , TransfecciónRESUMEN
Ras guanine nucleotide binding protein (GTPase) activation is a widely assessed readout in cell biological studies. We describe an improved approach for the quantitative analysis of total GDP and GTP bound to Ras. The present method involves HPLC separation and online detection/quantitation of Ras-bound [(32)P]-labelled GDP/GTP. As compared to standard approaches that are time consuming and/or provide only semi-quantitative data, this technique allows the rapid processing of large numbers of samples for the quantitative determination of Ras-bound GDP and GTP.
Asunto(s)
Biofisica/métodos , Cromatografía Líquida de Alta Presión/métodos , Guanosina Difosfato/análisis , Guanosina Trifosfato/análisis , Proteínas ras/metabolismo , Animales , Células COS , Cromatografía en Capa Delgada , Genes ras/genética , Pruebas de Precipitina , Factores de TiempoRESUMEN
This paper describes for the first time the direct measurement of boric acid (B(OH)(3)) and borate (B(OH)(4) (-)) adduction to NAD(+) and NADH by electrospray ionization mass spectrometry (ESI-MS) and (11)B NMR spectroscopy. The analysis demonstrates that borate binds to both cis-2,3-ribose diols on NAD(+) forming borate monoesters (1 : 1 addition), borate diesters (1 : 2 addition) and diborate esters (2 : 1 addition), whereas, only borate monoesters were formed with NADH. MS in the negative ion mode showed borate was bound to a cis-2,3-ribose diol and not to the hydroxyl groups on the phosphate backbone of NAD(+), and MS/MS showed that the 1 : 1 addition monoester contained borate bound to the adenosine ribose. Boron shifts of borate monoesters and diesters with NAD(+) were observed at 7.80 and 12.56 ppm at pH 7.0 to 9.0. The esterifications of borate with NAD(+) and NADH were pH dependent with maximum formation occurring under alkaline conditions with significant formation occurring at pH 7.0. Using ESI-MS, the limit of detection was 50 micro M for NAD(+) and boric acid (1 : 1) to detect NAD(+)-borate monoester at pH 7.0. These results suggest esterification of borate with nicotinamide nucleotides could be of biological significance.
Asunto(s)
Boratos/química , NAD/química , Esterificación , Guanosina Difosfato/análisis , Guanosina Difosfato/química , Concentración de Iones de Hidrógeno , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Espectrometría de Masa por Ionización de ElectrosprayRESUMEN
To understand the polymerization dynamics of FtsZ, a bacterial cell division protein similar to tubulin, insight is required into the nature of the nucleotide bound to the polymerized protein. In a previous study, we showed that the FtsZ polymers contain mostly GDP. A recent study challenged this result, suggesting that the polymerized FtsZ is in a GTP-bound state. Here, we show that, when radiolabelled [gamma-32P]-GTP is used to polymerize FtsZ, GTP is hydrolysed instantaneously. The FtsZ polymer contains both GDP and the radiolabelled inorganic phosphate.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biopolímeros/química , Proteínas del Citoesqueleto , Guanosina Trifosfato/metabolismo , Cromatografía en Capa Delgada , Dimerización , Guanosina Difosfato/análisis , Hidrólisis , Fosfatos/análisisRESUMEN
The CDC25 gene product is a guanine nucleotide exchange factor for Ras proteins in yeast. Recently it has been suggested that the intracellular levels of guanine nucleotides may influence the exchange reaction. To test this hypothesis we measured the levels of nucleotides in yeast cells under different growth conditions and the relative amount of Ras2-GTP. The intracellular GTP/GDP ratio was found to be very sensitive to growth conditions: the ratio is high, close to that of ATP/ADP during exponential growth, but it decreases rapidly before the beginning of stationary phase, and it drops further under starvation conditions. The addition of glucose to glucose-starved cells causes a fast increase of the GTP/GDP ratio. The relative amount of Ras2-GTP changes in a parallel way suggesting that there is a correlation with the cytosolic GTP/GDP ratio. In addition 'in vitro' mixed-nucleotide exchange experiments done on purified Ras2 protein demonstrated that the GTP and GDP concentrations influence the extent of Ras2-GTP loading giving further support to their possible regulatory role.
Asunto(s)
Proteínas Fúngicas/metabolismo , Nucleótidos de Guanina/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Proteínas ras/metabolismo , Citosol/metabolismo , Glucosa/farmacología , Guanosina Difosfato/análisis , Guanosina Difosfato/química , Guanosina Trifosfato/análisis , Guanosina Trifosfato/química , Saccharomyces cerevisiae/crecimiento & desarrollo , Transducción de Señal/efectos de los fármacos , Regulación hacia Arriba , Proteínas ras/químicaRESUMEN
Dexras1 is a novel GTP-binding protein that shares structural similarity with the Ras family of small molecular weight GTPases and is strongly and rapidly induced during treatment with dexamethasone. The function of Dexras1 and its contribution to glucocorticoid-dependent signaling in the corticotroph cell are unknown. The present study was undertaken to examine the potential role of Dexras1 in the regulation of peptide hormone secretion in the AtT-20 corticotroph cell line. To determine the effects of Dexras1 expressed independently of glucocorticoid treatment, expression plasmids for wild-type and constitutively active mutant Dexras1 proteins were cotransfected with human GH (hGH), which provides an ectopic marker for the stimulus-coupled secretory pathway. GTP binding properties and the GTP to GDP ratio of wild-type and mutant Dexras1 proteins were examined in transiently transfected AtT-20 and COS-7 cells. Stimulated and constitutive components of secretion were assessed after 2-h incubations with 5 mM 8-Br-cAMP or control. cAMP treatment led to a 2-fold increase in hGH secretion relative to control. Cotransfection of wild-type Dexras1 had no effect on cAMP-stimulated hGH secretion, but a constitutively active mutant, Dexras[A178V], attenuated stimulated secretion by 86% (P < 0.01). A double-mutant containing a deletion of the carboxyl terminus isoprenylation site, Dexras[A178V/C277term], did not inhibit cAMP-stimulated hGH secretion, indicating that the effect is prenylation dependent. These findings suggest that activation of Dexras1 has important functional consequences leading to inhibition of stimulus-secretion coupling in corticotroph cells. Because Dexras1 messenger RNA is strongly and rapidly induced during glucocorticoid treatment, these results raise the possibility that Dexras1 may participate in the signal transduction pathways that govern the rapid regulatory effects of glucocorticoids on peptide hormone secretion in corticotroph cells.
Asunto(s)
Hormona Adrenocorticotrópica/metabolismo , AMP Cíclico/farmacología , Hormona de Crecimiento Humana/metabolismo , Proteínas de Unión al GTP Monoméricas/fisiología , Adenohipófisis/metabolismo , Proteínas ras , 8-Bromo Monofosfato de Adenosina Cíclica/farmacología , Secuencia de Aminoácidos , Animales , Western Blotting , Células COS , Línea Celular , Dexametasona/farmacología , Proteínas de Unión al GTP/química , Expresión Génica , Glucocorticoides/farmacología , Guanosina Difosfato/análisis , Guanosina Trifosfato/análisis , Guanosina Trifosfato/metabolismo , Hormona de Crecimiento Humana/genética , Humanos , Ratones , Datos de Secuencia Molecular , Proteínas de Unión al GTP Monoméricas/química , Proteínas de Unión al GTP Monoméricas/genética , Fosfatos/metabolismo , Radioisótopos de Fósforo , Adenohipófisis/efectos de los fármacos , ARN Mensajero/biosíntesis , Proteínas Recombinantes , Alineación de Secuencia , TransfecciónRESUMEN
The neurotoxic effects of low-level lead (Pb) during senescence are increasing interests of importance. We investigated the effects of low-level Pb on the brain in a normal condition and a pathophysiological condition of energy shortage that is commonly found in age-related neurological diseases. Middle-aged rats (15 months old) were exposed to 200 mg/l Pb acetate in drinking water for 2 months and thereafter received bilateral intracerebroventricular injections of streptozotocin (STZ). After 1 month's additional exposure to the same level of Pb solution as before the rats were sacrificed. Blood and brain Pb levels were measured by graphite furnace atomic absorption spectrophotometry. Energy-rich phosphate levels in the brain were determined by high-performance liquid chromatography equipped with a UV detector. Astroglial activation and glucose-regulated protein (GRP)94 expression were examined immunohistochemically. Exposure to Pb increased the blood Pb level to 10.8 microg/dl and the brain Pb level to 0.052 microg/g. But a significant additional increase in the brain Pb level, to 0.101 microg/g, became obvious in rats treated with Pb + STZ. Both Pb and STZ induced perturbation in brain energy metabolism, but no further alteration in energy metabolite levels was found in rats treated with Pb + STZ. Astroglial activation and GRP94-positive astrocytes and neurons were found only in the brains of Pb + STZ-treated rats. These results suggest that exposure to low-level Pb can perturb brain energy metabolism and the brain becomes more vulnerable to Pb when it is under energy stress.
Asunto(s)
Envejecimiento/efectos de los fármacos , Química Encefálica/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Metabolismo Energético/efectos de los fármacos , Plomo/toxicidad , Adenosina Difosfato/análisis , Adenosina Trifosfato/análisis , Envejecimiento/metabolismo , Animales , Astrocitos/citología , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Encéfalo/patología , Creatina/análisis , Guanosina Difosfato/análisis , Guanosina Trifosfato/análisis , Proteínas HSP70 de Choque Térmico/biosíntesis , Inyecciones Intraventriculares , Plomo/análisis , Plomo/sangre , Masculino , Proteínas de la Membrana/biosíntesis , Fosfocreatina/análisis , Ratas , Ratas Wistar , Estreptozocina/administración & dosificaciónRESUMEN
Ras functions as an essential upstream regulator of growth-factor-receptor-coupled signal transduction pathways. Ras is converted from an inactive GDP-bound state to an active GTP-bound state in response to receptor activation. Thus, the ratio of GTP/GDP bound to Ras is a measure of its state of activation. Mutations that stabilize the GTP-bound form of Ras result in constitutive activation and cellular transformation. The most widely used method for measuring Ras activation utilizes [32P]PO4 to label cellular nucleotide pools and is therefore limited to use with cultured cells. We have modified and adapted an enzyme-based method for rapid, precise measurement of Ras-bound GTP and GDP in normal and psoriatic human skin. This method does not require radiolabeling of cellular nucleotides. In cultured fibroblasts, the enzymatic and [32P]PO4 incorporation methods yielded similar results. Application of the enzymatic method to human skin revealed that 6% of Ras was in the active GTP-bound state in normal skin, compared to 15.4% of Ras in psoriatic lesions. The total amount of Ras normalized to protein content was similar in normal and psoriatic skin. Enhanced activation of Ras is likely a critical mediator of the increased cell growth characteristic of psoriatic lesions.
Asunto(s)
Fibroblastos/fisiología , Psoriasis/metabolismo , Receptores de Factores de Crecimiento/fisiología , Piel/crecimiento & desarrollo , Proteínas ras/metabolismo , Animales , Técnicas de Cultivo de Célula , Cricetinae , Embrión de Mamíferos/citología , Femenino , Guanosina Difosfato/análisis , Guanosina Trifosfato/análisis , Humanos , Fosfotransferasas/fisiología , Pruebas de Precipitina , Embarazo , Transducción de SeñalRESUMEN
The detection of non-covalent complexes in the mass range 19,000-34,000 Da, using electrospray ionization mass spectrometry (ESI-MS), is reviewed. The examples discussed include (1) a protein-ligand interaction (ras-GDP), (2) an inhibitor-protein-ligand interaction (SCH 54292/SCH 54341-ras-GDP), (3) a protein-protein interaction (gamma-IFN homodimer) and (4) a protein-metal complex [HCV (1-181)-Zn]. In each case, the ESI-MS method is capable of releasing the intact non-covalent complex from its native solution state into the gas phase in the form of multiply-charge ions. The molecular masses of these complexes were determined with a mass accuracy of better than 0.01%, which is far superior to the traditional methods of sodium dodecyl sulfate polyacrylamide gel electrophoresis and gel permeation chromatography. The method provides the researcher with a quick, reliable and reproducible method for probing difficult biological problems. The key to success in the study of non-covalent complexes depends on careful understanding and manipulation of ESI source parameters and sample solution conditions; special care must be taken with the source orifice potential and the solution pH and organic co-solvents must be avoided. This paper also illustrates the usefulness of ESI-MS for addressing biological problems leading to the discovery of new therapeutics; the approach involves the rapid screening of potential drug candidates, such as weakly bound inhibitors.