RESUMEN
Related living kidney donors (LKDs) are at higher risk of end-stage renal disease (ESRD) compared with unrelated LKDs. A genetic panel was developed to screen 115 genes associated with renal diseases. We used this panel to screen six negative controls, four transplant candidates with presumed genetic renal disease and six related LKDs. After removing common variants, pathogenicity was predicted using six algorithms to score genetic variants based on conservation and function. All variants were evaluated in the context of patient phenotype and clinical data. We identified causal variants in three of the four transplant candidates. Two patients with a family history of autosomal dominant polycystic kidney disease segregated variants in PKD1. These findings excluded genetic risk in three of four relatives accepted as potential LKDs. A third patient with an atypical history for Alport syndrome had a splice site mutation in COL4A5. This pathogenic variant was excluded in a sibling accepted as an LKD. In another patient with a strong family history of ESRD, a negative genetic screen combined with negative comparative genomic hybridization in the recipient facilitated counseling of the related donor. This genetic renal disease panel will allow rapid, efficient and cost-effective evaluation of related LKDs.
Asunto(s)
Marcadores Genéticos , Pruebas Genéticas/métodos , Glomeruloesclerosis Focal y Segmentaria/diagnóstico , Donadores Vivos , Tamizaje Masivo , Riñón Poliquístico Autosómico Dominante/diagnóstico , Insuficiencia Renal Crónica/diagnóstico , Adulto , Femenino , Glomeruloesclerosis Focal y Segmentaria/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Trasplante de Riñón , Masculino , Persona de Mediana Edad , Mutación , Linaje , Riñón Poliquístico Autosómico Dominante/genética , Insuficiencia Renal Crónica/genética , Adulto JovenRESUMEN
We conducted an in vivo study to evaluate the anticancer effect and toxicity of fine-powder cisplatin suspended in lipiodol (fCDDP/LPD suspension) after a single administration of three different doses to rats via the intrahepatic artery after transplantation of rat ascites hepatoma cells. The toxicity of the fCDDP/LPD suspension was also assessed in the same protocol in noncancer-bearing rats and the observed toxicologic changes were compared among groups administered saline (Sal), an aqueous solution of fCDDP (fCDDP/Sal solution), and LPD alone. In parallel with the toxicity test, plasma CDDP concentrations were compared between the fCDDP/LPD suspension and fCDDP/Sal solution. The mean weight of the tumors in the fCDDP/LPD suspension groups was significantly less than in the LPD-alone group. The pathologic changes in the liver observed in the fCDDP/LPD suspension group increased with dose, were more marked compared with those in the fCDDP/Sal solution and LPD-alone groups, and were reversible. No other toxicologic effects were observed. The concentration of CDDP in the plasma in the fCDDP/LPD suspension group was slightly lower than that in the fCDDP/Sal solution group. In conclusion, the results indicate that the fCDDP/LPD suspension has sufficient anticancer efficacy and tolerability for use in the clinical treatment of hepatocellular carcinoma.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/administración & dosificación , Protocolos de Quimioterapia Combinada Antineoplásica/toxicidad , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Cisplatino/administración & dosificación , Relación Dosis-Respuesta a Droga , Aceite Etiodizado/administración & dosificación , Arteria Hepática , Neoplasias Hepáticas/patología , Masculino , Trasplante de Neoplasias , Tamaño de la Partícula , Polvos , Ratas , Pruebas de Toxicidad , Resultado del TratamientoRESUMEN
INTRODUCTION: Although there is evidence that selective serotonin reuptake inhibitors provide some benefit in the treatment of post-traumatic stress disorder (PTSD), most meta-analytical reviews have concluded that effect sizes are small and, moreover, that there may be relatively little benefit for some populations (e. g., combat veterans with co-morbid major depression, MDD). This study aimed to evaluate the effectiveness and tolerability of the dual reuptake inhibitor duloxetine in the treatment of PTSD and co-morbid MDD. METHODS: Twenty-one treatment refractory, male, combat-related patients with PTSD and co-morbid MDD were enrolled in a naturalistic study and twenty completed the trial. Duloxetine was given between 60 and 120 mg daily over 8 weeks. RESULTS: Duloxetine led to a significant improvement of PTSD-characteristic symptoms as well as co-morbid MDD. Duloxetine effectively reduced nightmares, which is important because decreasing nightmares has been associated with improved sleep in PTSD. DISCUSSION: The results of this naturalistic study suggest that duloxetine is an effective and well-tolerated treatment for patients with PTSD and co-morbid MDD. These initial results need to be extended to the study of women with PTSD.
Asunto(s)
Inhibidores de la Captación de Neurotransmisores/uso terapéutico , Trastornos por Estrés Postraumático/tratamiento farmacológico , Tiofenos/uso terapéutico , Trastornos de Combate/tratamiento farmacológico , Trastornos de Combate/epidemiología , Comorbilidad , Trastorno Depresivo Mayor/tratamiento farmacológico , Trastorno Depresivo Mayor/epidemiología , Sueños/efectos de los fármacos , Clorhidrato de Duloxetina , Humanos , Masculino , Persona de Mediana Edad , Inhibidores de la Captación de Neurotransmisores/administración & dosificación , Inhibidores de la Captación de Neurotransmisores/efectos adversos , Escalas de Valoración Psiquiátrica , Trastornos por Estrés Postraumático/epidemiología , Tiofenos/administración & dosificación , Tiofenos/efectos adversos , Factores de Tiempo , Resultado del Tratamiento , Veteranos , GuerraRESUMEN
To evaluate the specific reactivity of HLA Class I antibodies (HLA-I Abs) in acute non-hemolytic transfusion reactions (ANHTRs) using solid phase assays (SPAs) and conventional complement-dependent lymphocyte cytotoxicity test (LCT). ANHTRs are major issues in transfusion medicine. Anti-leukocyte antibodies have been implicated as one of the causative agents of transfusion-related acute lung injury (TRALI) and febrile reaction. Antibodies to HLA Class I and/or Class II (HLA Abs) have been intensively studied using SPAs for TRALI, but not for febrile reaction. About 107 patients and 186 donors associated with ANHTRs were screened for HLA Abs by SPAs such as enzyme-linked immunosorbent assay (ELISA) and the Luminex method. When HLA-I Ab was detected, its specific reactivity was evaluated by comparing its specificity identified by the Luminex method using recombinant HLA molecules and cognate HLA antigens (Ags), as well as LCT with or without anti-human globulin (AHG). The incidences of HLA Abs were as high as 32.7% of patients' serum samples and 16% of donors' serum samples. The incidence of HLA-I Abs did not differ significantly between cases of febrile and allergic reactions. However, HLA-I Abs associated with febrile reaction showed a significantly higher rate of possessing specific reactivity to cognate HLA Ags than those associated with allergic reactions. In addition, the Luminex method enabled the detection of HLA-I Abs much earlier than AHG-LCT in serum samples from a patient with febrile reaction and platelet transfusion refractoriness (PTR). SPAs seem more useful than AHG-LCT for evaluating reactivity of antibodies in ANHTR cases.
Asunto(s)
Lesión Pulmonar Aguda/etiología , Anafilaxia/etiología , Fiebre/etiología , Antígenos HLA/inmunología , Prueba de Histocompatibilidad/métodos , Isoanticuerpos/sangre , Reacción a la Transfusión , Urticaria/etiología , Enfermedad Aguda , Lesión Pulmonar Aguda/inmunología , Adulto , Anciano , Anafilaxia/inmunología , Especificidad de Anticuerpos , Reacciones Antígeno-Anticuerpo , Niño , Pruebas Inmunológicas de Citotoxicidad , Ensayo de Inmunoadsorción Enzimática , Femenino , Fiebre/inmunología , Fluorometría , Estudios de Seguimiento , Humanos , Japón , Masculino , Persona de Mediana Edad , Neoplasias/inmunología , Neoplasias/terapia , Urticaria/inmunologíaRESUMEN
An audioprofile displays phenotypic data from several audiograms on a single graph that share a common genotype. In this report, we describe the application of audioprofiling to a large family in which a genome-wide screen failed to identify a deafness locus. Analysis of audiograms by audioprofiling suggested that two persons with hearing impairment had a different deafness genotype. On this basis, we reassigned affectation status and identified a p.Cys1837Arg autosomal dominant mutation in alpha-tectorin segregating in all family members except two persons, who segregated autosomal recessive deafness caused by p.Val37Ile and p.Leu90Pro mutations in Connexin 26. One nuclear family in the extended pedigree segregates both dominant and recessive non-syndromic hearing loss.
Asunto(s)
Conexinas/genética , Proteínas de la Matriz Extracelular/genética , Pérdida Auditiva/genética , Glicoproteínas de Membrana/genética , Conexina 26 , Análisis Mutacional de ADN , Familia , Proteínas Ligadas a GPI , Genotipo , Pérdida Auditiva/diagnóstico , Humanos , LinajeRESUMEN
The detection of intracerebral lesions has improved greatly with advancements in MR imaging, especially the greater sensitivity of the 1.5 Tesla unit versus the older 1.0 Tesla unit. We aimed to determine whether improvements in MR imaging have actually improved diagnostic capabilities and treatment outcomes in gamma knife radiosurgery (GKRS) for brain metastases (METs). Ours was a retrospective study of a consecutive series of 1179 patients (441 females, 738 males, mean age: 63 years, range: 19-92 years) with brain METs who underwent GKRS from 1998 to 2004. Our treatment policy was to irradiate all lesions visible on MR images during a single GKRS session. Mean and median tumor numbers were seven and three (range; 1-74). The 1179 patients were divided into two groups: a 1.0 T-group of 660 patients examined using a 1.0 Tesla MR unit before August,2002, and a 1.5 T-group of 519 examined using a 1.5 Tesla MR unit after September 2002. In the 1.5 T-group, lesion volumes as small as 0.004 cc were detected with a 5 mm slice thickness. The corresponding lesion size was 0.013 cc in the 1.0 T-group. One or more lesions invisible on a 5 mm slice study were additionally detected on a 2 mm slice study in 47.8% of patients in the 1.0 T-group and 25.2% in the 1.5 T-group (p<.0001). The median survival time (MST) in the 1.5 T-group was significantly longer than that in the 1.0 T-group (8.4 vs. 6.3 months, p=.0004). Due to biases in patient numbers between the two groups, we analyzed subgroups with KPS of 80% or better, no neurological deficits, stable primary tumors, lung cancer, tumor numbers of four or less and tumor volumes of 10.0 cc or smaller. In every subgroup analysis, the MSTs of the 1.5-Tesla group were significantly longer than those of the 1.0-Tesla group. The prognosis of a cancer patient is undoubtedly influenced by multiple factors. Nevertheless, we conclude that application of the 1.5 Tesla MR unit has had a favorable impact on diagnosis and GKRS treatment results in patients with brain METs.
RESUMEN
INTRODUCTION: Membranoproliferative glomerulonephritis type II or dense deposit disease (MPGN II/DDD) causes chronic renal dysfunction that progresses to end stage renal disease in about half of patients within 10 years of diagnosis. Deficiency of and mutations in the complement factor H (CFH) gene are associated with the development of MPGN II/DDD, suggesting that dysregulation of the alternative pathway of the complement cascade is important in disease pathophysiology. SUBJECTS: Patients with MPGN II/DDD were studied to determine whether specific allele variants of CFH and CFHR5 segregate preferentially with the MPGN II/DDD disease phenotype. The control group was compromised of 131 people in whom age related macular degeneration had been excluded. RESULTS: Allele frequencies of four single nucleotide polymorphisms in CFH and three in CFHR5 were significantly different between MPGN II/DDD patients and controls. CONCLUSION: We have identified specific allele variants of CFH and CFHR5 associated with the MPGN II/DDD disease phenotype. While our data can be interpreted to further implicate complement in the pathogenesis of MPGN II/DDD, these associations could also be unrelated to disease pathophysiology. Functional studies are required to resolve this question.
Asunto(s)
Proteínas Sanguíneas/genética , Factor H de Complemento/genética , Variación Genética , Glomerulonefritis Membranoproliferativa/genética , Biopsia , Proteínas del Sistema Complemento , Cartilla de ADN , Eliminación de Gen , Frecuencia de los Genes , Glomerulonefritis Membranoproliferativa/clasificación , Glomerulonefritis Membranoproliferativa/patología , Humanos , Mutación , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Valores de ReferenciaRESUMEN
Over-expression of aldose reductase (AR) has been observed in many cancer cells. To clarify the role of AR in tumor cells, we investigated the pathways mediating expression of the AR gene induced by 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter. In A549 human lung adenocarcinoma cells, TPA elicited a dose- and time-dependent increase in AR mRNA level with an elevated enzyme activity. The TPA-induced increase in mRNA level and promoter activity of the AR gene was significantly attenuated in the presence of an inhibitor of protein kinase C, tyrosine kinase, or nuclear factor kappaB (NF-kappaB). TPA augmented the NF-kappaB-dependent gene transcription, indicating the involvement of NF-kappaB in this regulation. Accumulation of TPA-treated cells in S phase was almost completely abolished in the presence of ethyl 1-benzyl-3-hydroxy-2(5H)-oxopyrrole-4-carboxylate, an AR inhibitor. Taken together, TPA augmented the promoter activity of the AR gene via the activation of protein kinase and NF-kappaB. The inhibition of AR may assist in the chemotherapy of malignant tumors by suppressing the rapid growth of cancer cells.
Asunto(s)
Aldehído Reductasa/genética , Ciclo Celular/efectos de los fármacos , Acetato de Tetradecanoilforbol/farmacología , Aldehído Reductasa/antagonistas & inhibidores , Aldehído Reductasa/fisiología , Ciclo Celular/fisiología , Línea Celular Tumoral , Cicloheximida/farmacología , Dactinomicina/farmacología , Inhibidores Enzimáticos/farmacología , Expresión Génica/efectos de los fármacos , Humanos , FN-kappa B/metabolismo , Regiones Promotoras Genéticas/genética , Proteína Quinasa C/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Ácido Pirrolidona Carboxílico/análogos & derivados , Ácido Pirrolidona Carboxílico/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
MSX1 has been proposed as a gene in which mutations may contribute to non-syndromic forms of cleft lip and/or cleft palate. Support for this comes from human linkage and linkage disequilibrium studies, chromosomal deletions resulting in haploinsufficiency, a large family with a stop codon mutation that includes clefting as a phenotype, and the Msx1 phenotype in a knockout mouse. This report describes a population based scan for mutations encompassing the sense and antisense transcribed sequence of MSX1 (two exons, one intron). We compare the completed genomic sequence of MSX1 to the mouse Msx1 sequence to identify non-coding homology regions, and sequence highly conserved elements. The samples studied were drawn from a panethnic collection including people of European, Asian, and native South American ancestry. The gene was sequenced in 917 people and potentially aetiological mutations were identified in 16. These included missense mutations in conserved amino acids and point mutations in conserved regions not identified in any of 500 controls sequenced. Five different missense mutations in seven unrelated subjects with clefting are described. Evolutionary sequence comparisons of all known Msx1 orthologues placed the amino acid substitutions in context. Four rare mutations were found in non-coding regions that are highly conserved and disrupt probable regulatory regions. In addition, a panel of 18 population specific polymorphic variants were identified that will be useful in future haplotype analyses of MSX1. MSX1 mutations are found in 2% of cases of clefting and should be considered for genetic counselling implications, particularly in those families in which autosomal dominant inheritance patterns or dental anomalies appear to be cosegregating with the clefting phenotype.
Asunto(s)
Labio Leporino/genética , Fisura del Paladar/genética , Análisis Mutacional de ADN/métodos , Proteínas de Homeodominio/fisiología , Factores de Transcripción/fisiología , Secuencia de Aminoácidos/genética , Animales , Asia , Estudios de Casos y Controles , Bovinos , Pollos/genética , ADN/genética , Europa (Continente) , Variación Genética/genética , Genética de Población/métodos , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Humanos , Desequilibrio de Ligamiento/genética , Factor de Transcripción MSX1 , Ratones , Datos de Secuencia Molecular , Mutación/genética , Polimorfismo Genético/genética , Ratas , Alineación de Secuencia/métodos , América del Sur , Síndrome , Factores de Transcripción/química , Factores de Transcripción/genética , Regiones no Traducidas/genética , Proteínas de Xenopus/genéticaRESUMEN
BACKGROUND: The purpose of this study was to examine whether each exercise and an entire karate training session can achieve: 1). accepted training intensity thresholds for effective aerobic capacity training; 2). energy expenditure (EE) thresholds for total body mass and fat weight loss; and, 3). elevation in excess postexercise oxygen consumption (EPOC). METHODS: We investigated physiological responses during 5 types of karate training in female karate practitioners: basic techniques without (S-Basics) and with (M-Basics) movements, sparring techniques without (TECH I) and with (TECH II) an opponent, and kata. RESULTS: The mean percent of maximum oxygen uptake reserve (%VO2R), percent of maximum heart rate (%HRmax), and maximum heart rate reserve (%HRR) for S-Basics were below the accepted threshold and for M-Basics were marginal or above the threshold for increasing VO2max. For TECH I, TECH II, kata, and the entire 70-min practice, the mean %HRmax and %HRR were well above the threshold, however, %VO2R was below the threshold. Although the mean EPOC measured for 5 min immediately following the entire 70-min karate training did not differ from resting VO2. The blood lactate responses to the 5 types of karate exercises ranged from 1.2+/-0.3 to 2.2+/-0.8 mmol L(-1). The mean EE for each karate exercise ranged from 157+/-10 kJ to 314+/-16 kJ. The mean EE for the entire 70 min practice and EPOC were 1120+/-64 kJ and 28+/-2 kJ, respectively. CONCLUSIONS: The training intensities of karate exercises studied in women were light to moderate, effects of karate training on EPOC was minimal, and the mean EE was marginal to the accepted threshold for total body mass and fat weight loss.
Asunto(s)
Umbral Anaerobio/fisiología , Metabolismo Energético/fisiología , Artes Marciales/fisiología , Consumo de Oxígeno/fisiología , Adulto , Índice de Masa Corporal , Prueba de Esfuerzo , Tolerancia al Ejercicio/fisiología , Femenino , Humanos , Ácido Láctico/sangreRESUMEN
Erectile dysfunction (ED) is a common complication of diabetes mellitus. Erythrocyte aldose reductase (AR) has been implicated in a variety of diabetic complications. The subjects were 62 diabetic patients, of whom 25 were treated with hemodialysis (chronic renal failure CRF group) and the remaining 37 did not have chronic renal failure (DM group). The controls were 20 healthy volunteers age-matched to the patients. The level of AR was measured by the quantitative determination kit for AR in all patients and controls. In this study, ED was diagnosed by 5-item version of the International Index of Erectile Function (IIEF-5). The average level of AR in the CRF group was significantly higher than that in the DM group and controls (P<0.001). The average level of AR in the DM group without ED was significantly lower than that in the DM group with ED and controls (P<0.005). These results suggest that the level of AR may be a useful modality for prediction of ED in diabetic patients.
Asunto(s)
Aldehído Reductasa/metabolismo , Nefropatías Diabéticas/complicaciones , Disfunción Eréctil/diagnóstico , Disfunción Eréctil/enzimología , Eritrocitos/enzimología , Anciano , Biomarcadores , Disfunción Eréctil/complicaciones , Humanos , Masculino , Persona de Mediana Edad , Óxido Nítrico/metabolismo , Valor Predictivo de las PruebasRESUMEN
Aldose reductase (AR) and sorbitol dehydrogenase (SDH) are the enzymes constituting the polyol pathway, an alternate route of glucose metabolism. A wealth of experimental data has indicated the involvement of the polyol pathway in the pathogenesis of diabetic complications. However, there has been surprisingly little research on the relative abundance of SDH to AR in the tissues affected in diabetes. We therefore developed a competitive RT-PCR system to simultaneously determine the mRNA levels of these two enzymes in small amounts of samples, and studied their expression in Schwann cells isolated from adult rat sciatic nerves. Although both AR and SDH mRNA were expressed in the Schwann cells, the levels of SDH cDNA were much lower than those of AR cDNA. The induction of AR mRNA expression in the Schwann cells under hyperosmotic conditions was similarly detected by Northern blot analysis and our competitive RT-PCR method. The RT-PCR system developed in this study may be a useful tool in ascertaining the relative contributions of AR and SDH to the metabolic derangements resulting from the acceleration of polyol pathway activity in the target organ of diabetic complications.
Asunto(s)
Aldehído Reductasa/biosíntesis , Aldehído Reductasa/genética , ADN/biosíntesis , ADN/genética , Regulación Enzimológica de la Expresión Génica/genética , L-Iditol 2-Deshidrogenasa/biosíntesis , L-Iditol 2-Deshidrogenasa/genética , Células de Schwann/enzimología , Animales , Northern Blotting , Células Cultivadas , Cartilla de ADN , Electroforesis en Gel de Agar , Glicerolfosfato Deshidrogenasa/biosíntesis , Glicerolfosfato Deshidrogenasa/genética , Procesamiento de Imagen Asistido por Computador , Indicadores y Reactivos , Masculino , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Nervio Ciático/citología , Nervio Ciático/enzimologíaRESUMEN
The present study was designed to examine the effect of aldose reductase (AR) overexpression on the development of diabetic neuropathy by using mice transgenic for human AR. At 8 weeks of age, transgenic mice (Tg) and non-transgenic littermates (Lm) were made diabetic with streptozotocin. After 8 weeks of untreated diabetes, plasma glucose levels and the reduction in body weight were similar between the groups of diabetic animals. Despite the comparable levels of hyperglycaemia, levels of sorbitol and fructose were significantly greater in the peripheral nerve of diabetic Tg than in diabetic Lm (both P < 0.01). Ouabain sensitive Na(+),K(+)-ATPase activity was similarly decreased in both diabetic Tg and Lm. Protein kinase C activity in the sciatic nerve membrane fraction was unaffected by diabetes in Lm, but was reduced by nearly 40% in the diabetic Tg. Although both groups of diabetic animals exhibited a significant decrease in tibial nerve motor nerve conduction velocity (MNCV), this decrease was significantly more severe (P < 0.01) in diabetic Tg than in diabetic Lm. Consistent with these findings, nerve fibre atrophy was significantly more severe in diabetic Tg than in diabetic Lm (P < 0.01). These findings implicate increased polyol pathway activity in the pathogenesis of diabetic neuropathy. In support of this hypothesis, treating diabetic Tg with an aldose reductase inhibitor (WAY121-509, 4 mg/kg/day) for 8 weeks significantly prevented the accumulation of sorbitol, the decrease in MNCV and the increased myelinated fibre atrophy in diabetic Tg.
Asunto(s)
Aldehído Reductasa/antagonistas & inhibidores , Aldehído Reductasa/genética , Aldehído Reductasa/farmacología , Diabetes Mellitus Experimental/metabolismo , Neuropatías Diabéticas/metabolismo , Inhibidores Enzimáticos/farmacología , Animales , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Neuropatías Diabéticas/genética , Neuropatías Diabéticas/patología , Ensayo de Inmunoadsorción Enzimática , Femenino , Glucosa/metabolismo , Humanos , Hiperglucemia/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Neuronas Motoras/enzimología , Conducción Nerviosa , Proteína Quinasa C/metabolismo , Nervio Ciático/enzimología , Nervio Ciático/patología , ATPasa Intercambiadora de Sodio-Potasio/metabolismoRESUMEN
Kinetic and equilibrium studies of apomyoglobin folding pathways and intermediates have provided important insights into the mechanism of protein folding. To investigate the role of intrinsic helical propensities in the apomyoglobin folding process, a mutant has been prepared in which Asn132 and Glu136 have been substituted with glycine to destabilize the H helix. The structure and dynamics of the equilibrium molten globule state formed at pH 4.1 have been examined using NMR spectroscopy. Deviations of backbone (13)C(alpha) and (13)CO chemical shifts from random coil values reveal high populations of helical structure in the A and G helix regions and in part of the B helix. However, the H helix is significantly destabilized compared to the wild-type molten globule. Heteronuclear [(1)H]-(15)N NOEs show that, although the polypeptide backbone in the H helix region is more flexible than in the wild-type protein, its motions are restricted by transient hydrophobic interactions with the molten globule core. Quench flow hydrogen exchange measurements reveal stable helical structure in the A and G helices and part of the B helix in the burst phase kinetic intermediate and confirm that the H helix is largely unstructured. Stabilization of structure in the H helix occurs during the slow folding phases, in synchrony with the C and E helices and the CD region. The kinetic and equilibrium molten globule intermediates formed by N132G/E136G are similar in structure. Although both the wild-type apomyoglobin and the mutant fold via compact helical intermediates, the structures of the intermediates and consequently the detailed folding pathways differ. Apomyoglobin is therefore capable of compensating for mutations by using alternative folding pathways within a common basic framework. Tertiary hydrophobic interactions appear to play an important role in the formation and stabilization of secondary structure in the H helix of the N132G/E136G mutant. These studies provide important insights into the interplay between secondary and tertiary structure formation in protein folding.
Asunto(s)
Apoproteínas/química , Mutación , Mioglobina/química , Conformación Proteica , Pliegue de Proteína , Apoproteínas/genética , Asparagina/química , Dicroismo Circular , Fluorescencia , Ácido Glutámico/química , Glicina/química , Hidrógeno/química , Enlace de Hidrógeno , Cinética , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Mioglobina/genética , Fragmentos de Péptidos , Estructura Secundaria de ProteínaRESUMEN
Acceleration of the polyol pathway and enhanced oxidative stress are implicated in the pathogenesis of diabetic complications. We and others recently reported that aldose reductase (AR), the rate-limiting enzyme in the polyol pathway, was upregulated by reactive oxygen and nitrogen species in vascular smooth muscle cells. To clarify the molecular mechanisms underlying these findings, we investigated the signal transduction pathways mediating AR expression using the rat vascular smooth muscle cell line A7r5. A selective epidermal growth factor (EGF) receptor kinase inhibitor, tyrphostin AG1478, significantly suppressed the hydrogen peroxide (H2O2)-induced increase in AR mRNA and enzyme activity. Activation of extracellular signal-regulated protein kinase (ERK) by H2O2 was blunted by AG1478. PD98059, a specific inhibitor of ERK kinase (MEK1), reduced H2O2-induced AR expression. EGF alone elicited activation of ERK and induction of AR expression. Increased level of AR transcript was demonstrated in cells treated with oxidized low-density lipoprotein, and this increase was also suppressed by AG1478. Inhibition of p38 MAP kinase by SB203580 also partially suppressed the H2O2-initiated AR induction. The presence of ponalrestat, an AR inhibitor, significantly accelerated H2O2-induced cell death. These results suggested that AR may act as a survival factor in these cells and that the EGF receptor-ERK pathway is the major signaling pathway involved in the upregulation of AR expression under oxidative stress.
Asunto(s)
Aldehído Reductasa/genética , Aldehído Reductasa/metabolismo , Receptores ErbB/metabolismo , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Aldehído Reductasa/antagonistas & inhibidores , Animales , Línea Celular , Supervivencia Celular , Complicaciones de la Diabetes , Diabetes Mellitus/metabolismo , Flavonoides/farmacología , Depuradores de Radicales Libres/farmacología , Humanos , Peróxido de Hidrógeno/farmacología , Músculo Liso Vascular/efectos de los fármacos , Músculo Liso Vascular/metabolismo , Estrés Oxidativo , Ftalazinas/farmacología , Proteínas Quinasas/metabolismo , Quinazolinas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Transducción de Señal , Tirfostinos/farmacología , Regulación hacia Arriba/efectos de los fármacosRESUMEN
OBJECTIVE: To study the causative conception of malignant gestational trophoblastic neoplasms (GTNs), we analyzed malignant GTNs by microsatellite PCR markers. METHOD: DNAs extracted from 12 malignant GTNs were subjected to PCR for five different chromosomal locations. RESULT: Of the 7 cases after a complete mole (CM), 5 were derived from androgenesis, but the remaining 2 were from normal fertilization. Of the 5 cases after nonmolar pregnancies, 2 placental site trophoblastic tumors had alleles from both parents. Of the other 3 choriocarcinomas, 1 was from normal fertilization after spontaneous abortion but 2 originated from androgenesis, suggesting that 1 was from a CM prior to the antecedent abortion, transforming after a long interval. CONCLUSION: By combining the previous cases with these, our analysis of 39 cases demonstrated that trophoblastic neoplasms can arise from at least three different modes of origin (androgenesis, normal fertilization, and parthenogenesis), and antecedent pregnancy is not always identical to the causative conception. Placental site trophoblastic tumors might have different machinery for carcinogenesis because of the predominance of paternal and maternal contributions. In addition, a long dormancy of trophoblasts before malignant transformation, especially for those originating from normal fertilization, was also suggested.
Asunto(s)
Repeticiones de Microsatélite/genética , Polimorfismo Genético , Lugares Marcados de Secuencia , Neoplasias Trofoblásticas/genética , Neoplasias Uterinas/genética , Adulto , Femenino , Humanos , Mola Hidatiforme/genética , Reacción en Cadena de la Polimerasa , EmbarazoRESUMEN
The stability and folding kinetics of wild-type and a mutant staphylococcal nuclease (SNase) at neutral pH are significantly perturbed by the presence of moderate to high concentrations of salts. Very substantial increases in stability toward thermal and urea denaturation were observed; for example, 0.4 M sodium sulfate increased the free energy of wild-type SNase by more than 2 kcal/mol. For the NCA SNase mutant, the presence of the salts abolished the cold denaturation observed at neutral pH with this variant, and substantially increased its stability. Significant effects of salts on the kinetics of refolding were also observed. For NCA SNase, the presence of the salts markedly increased the folding rates (up to 5-fold). On the other hand, chloride, in particular, substantially decreased the rate of folding of the wild-type protein. Since the rates of the slow phases due to proline isomerization were increased by salt, these steps must be coupled to conformational processes. Fluorescence energy transfer between the lone tryptophan (Trp140) and an engineered fluorescent acceptor at residue 64 revealed that the addition of a high concentration of KCl led to the formation of a transient folding intermediate not observed at lower salt concentrations, and in which residues 140 and 64 were much closer than in the native state. The salt-induced effects on the kinetics of folding are attributed to the enhanced stability of the transient folding intermediates. It is likely that the combination of the high net charge, due to the high isoelectric point, and the relatively low intrinsic hydrophobicity, leads to staphylococcal nuclease having only marginal stability at neutral pH. The salt-induced effects on the structure, stability, and kinetics of staphylococcal nuclease are attributed to the binding of counterions, namely, anions, resulting in minimization of intramolecular electrostatic repulsion. This leads to increased stability, more structure, and greater compactness, as observed. Consequently, localized electrostatic repulsion is present at neutral pH in SNase, probably contributing to its marginal stability. The results suggest that, in general, marginally stable globular proteins will be significantly stabilized by salts under conditions where they have a substantial net charge.
Asunto(s)
Nucleasa Microcócica/química , Nucleasa Microcócica/metabolismo , Pliegue de Proteína , Sales (Química)/farmacología , Sustitución de Aminoácidos/genética , Cloruros/farmacología , Dicroismo Circular , Transferencia de Energía , Estabilidad de Enzimas/efectos de los fármacos , Estabilidad de Enzimas/genética , Concentración de Iones de Hidrógeno , Cinética , Nucleasa Microcócica/genética , Espectrometría de Fluorescencia , Sulfatos/farmacología , Ácido Trifluoroacético/farmacología , Triptófano/metabolismoRESUMEN
To investigate the polyol pathway activity in Schwann cells, we determined the mRNA levels of aldose reductase (AR) and sorbitol dehydrogenase (SDH) in cultured cells under hyperglycemic or hyperosmotic conditions using competitive RT-PCR technique. The expressions of AR and SDH mRNAs in Schwann cells were unaltered by high (30 mM) glucose content in the medium. On the other hand, osmotic stress elicited significant increases in AR mRNA without any effect on SDH mRNA expression. The levels of AR mRNA determined by this RT-PCR system were significantly correlated with AR activity, as well as the levels of sorbitol accumulated in Schwann cells cultured under hyperosmotic conditions. These findings suggest that in contrast to the induction of AR expression by osmotic stress, high glucose per se does not up-regulate expression of the enzymes constituting the polyol pathway in Schwann cells. The RT-PCR system developed in this study may be a useful tool in ascertaining the relative contributions of AR and SDH to the metabolic derangements leading to diabetic complications.
Asunto(s)
Aldehído Reductasa/genética , Glucosa/farmacología , L-Iditol 2-Deshidrogenasa/genética , Células de Schwann/enzimología , Animales , Células Cultivadas , Neuropatías Diabéticas/metabolismo , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Masculino , Presión Osmótica , Polímeros/metabolismo , ARN Mensajero/análisis , Rafinosa/farmacología , Ratas , Ratas Sprague-Dawley , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células de Schwann/citología , Células de Schwann/efectos de los fármacosRESUMEN
This study was designed to investigate the levels of IL-2 and its soluble receptor (sIL-2R) in bronchoalveolar lavage fluid (BALF) from patients with systemic sclerosis (SSc). We studied 18 patients with SSc and 10 healthy volunteers. Based on high-resolution computed tomography lung scans the patients were divided into two groups, those with (SSc-ILD group, n= 10) and those without (SSc group, n = 8) evidence of interstitial lung disease (ILD). Both groups showed significantly higher total cell and neutrophil counts in the BALF than controls. The SSc group also showed significantly higher levels of lymphocytes than controls. IL-2 was not detectable in BALF. The patients showed significantly higher levels of sIL-2R than controls (77.8% vs 20%, P=0.005). The median sIL-2R levels detected did not differ between the two patient groups (SSc-ILD 270 pg/ml, SSc 232 pg/ml). This study suggests that SSc patients with or without ILD have elevated levels of sIL-2R in BALF and that in some of these patients this finding could be explained by subclinical pulmonary inflammation.