RESUMO
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.
Assuntos
Marcadores Genéticos , Testes Genéticos/métodos , Glomerulosclerose Segmentar e Focal/diagnóstico , Doadores Vivos , Programas de Rastreamento , Rim Policístico Autossômico Dominante/diagnóstico , Insuficiência Renal Crônica/diagnóstico , Adulto , Feminino , Glomerulosclerose Segmentar e Focal/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Transplante de Rim , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Rim Policístico Autossômico Dominante/genética , Insuficiência Renal Crônica/genética , Adulto JovemRESUMO
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.
Assuntos
Inibidores da Captação de Neurotransmissores/uso terapêutico , Transtornos de Estresse Pós-Traumáticos/tratamento farmacológico , Tiofenos/uso terapêutico , Distúrbios de Guerra/tratamento farmacológico , Distúrbios de Guerra/epidemiologia , Comorbidade , Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Maior/epidemiologia , Sonhos/efeitos dos fármacos , Cloridrato de Duloxetina , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores da Captação de Neurotransmissores/administração & dosagem , Inibidores da Captação de Neurotransmissores/efeitos adversos , Escalas de Graduação Psiquiátrica , Transtornos de Estresse Pós-Traumáticos/epidemiologia , Tiofenos/administração & dosagem , Tiofenos/efeitos adversos , Fatores de Tempo , Resultado do Tratamento , Veteranos , GuerraRESUMO
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.
Assuntos
Lesão Pulmonar Aguda/etiologia , Anafilaxia/etiologia , Febre/etiologia , Antígenos HLA/imunologia , Teste de Histocompatibilidade/métodos , Isoanticorpos/sangue , Reação Transfusional , Urticária/etiologia , Doença Aguda , Lesão Pulmonar Aguda/imunologia , Adulto , Idoso , Anafilaxia/imunologia , Especificidade de Anticorpos , Reações Antígeno-Anticorpo , Criança , Testes Imunológicos de Citotoxicidade , Ensaio de Imunoadsorção Enzimática , Feminino , Febre/imunologia , Fluorometria , Seguimentos , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Neoplasias/imunologia , Neoplasias/terapia , Urticária/imunologiaRESUMO
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.
Assuntos
Proteínas Sanguíneas/genética , Fator H do Complemento/genética , Variação Genética , Glomerulonefrite Membranoproliferativa/genética , Biópsia , Proteínas do Sistema Complemento , Primers do DNA , Deleção de Genes , Frequência do Gene , Glomerulonefrite Membranoproliferativa/classificação , Glomerulonefrite Membranoproliferativa/patologia , Humanos , Mutação , Reação em Cadeia da Polimerase , Polimorfismo de Nucleotídeo Único , Valores de ReferênciaRESUMO
The Chinese hamster lung (V79) cell was intrinsically 10-times more resistant to peplomycin, a bleomycin-related antitumor antibiotic, than the Chinese hamster ovary (CHO) cell. This may be associated with the 3-times higher levels of recovery of bleomycin hydrolase activity of the V79 cell. The degradation of bleomycin hydrolase molecules in both V79 and CHO cells was examined using a monoclonal antibody specific for the enzyme. Labelling experiments showed that the bleomycin hydrolase in CHO cells was less stable than the comparable enzyme in V79 cells, and that 48 kDa subunits comprising bleomycin hydrolase (a homohexameric enzyme) molecules were degraded into 31 kDa forms in both cell lines. The 105,000 X g pellet (microsomes) fraction obtained after subcellular fractionation of CHO cells contained both 48 kDa subunit and 31 kDa forms of bleomycin hydrolase, while the 105,000 X g supernatant cytosol fraction yielded only 48 kDa subunit forms of the enzyme. Moreover, bleomycin hydrolase activity of both V79 and CHO cells was almost entirely recovered from the cytosol fraction. These results suggest that degradation of the 48 kDa subunit form of bleomycin hydrolase in these two lines of cultured cells into the 31 kDa form occurs on the plasma membrane or the endoplasmic reticulum, with which the resulting large number of bleomycin hydrolase molecules or degraded forms of the enzyme that have lost enzymatic activity are associated.
Assuntos
Bleomicina/farmacologia , Cisteína Endopeptidases , Glicosídeo Hidrolases/metabolismo , Animais , Linhagem Celular , Cricetinae , Resistência a Medicamentos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Imunofluorescência , Histocitoquímica , Técnicas de Imunoadsorção , Pulmão/enzimologia , Substâncias Macromoleculares , Peso Molecular , Ovário/enzimologia , Peplomicina , Frações Subcelulares/enzimologiaRESUMO
Large quantities of recombinant human aldose reductase were produced using Spodoptera frugiperda cells and properties of the enzyme were characterized. Direct purification of the recombinant aldose reductase by affinity column chromatography using Matrex gel orange A yielded a single 36 kDa band, similar in size to the purified human muscle aldose reductase, on a sodium dodecyl sulfate-polyacrylamide gel after silver staining. The isoelectric point of the recombinant enzyme was 5.85 which is identical to the human muscle aldose reductase. Following the treatment with an acylamino-acid releasing enzyme, the blocked NH2-terminal amino acid was identified to be acetylalanine. The successive NH2-terminal sequence and that of the COOH-terminal peptide concurred with the expected translated sequence. Kinetic analyses of the recombinant enzyme activity for various substrates and the cofactor, NADPH, demonstrated a good agreement with the previously reported kinetic data on the purified human aldose reductase. A high concentration of (NH4)2SO4 elicited a significant increase in both Km and Kcat for DL-glyceraldehyde as well as D-glucose. Although IC50 values for most of the aldose reductase inhibitors with recombinant enzyme were found to fall within the comparable range of those obtained with nonhuman mammalian enzymes, the IC50 value for epalrestat was more than 10-fold higher in the recombinant enzyme. These results indicate that the recombinant human aldose reductase expressed in the baculovirus system possesses structurally and enzymatically similar properties as those reported for the native human enzyme and should serve as a superior enzyme preparation to nonhuman mammalian enzymes for the screening of the efficacy and potency of newly developed aldose reductase inhibitors.
Assuntos
Aldeído Redutase/metabolismo , Baculoviridae/genética , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/genética , Aldeído Redutase/isolamento & purificação , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Glicosilação , Humanos , Ponto Isoelétrico , Cinética , Dados de Sequência Molecular , Mariposas , Músculos/enzimologia , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Especificidade por SubstratoRESUMO
Partial assignments for the 1H-NMR resonances of the aromatic residues in human interleukin 6 (IL-6) are reported. The homonuclear Hartmann-Hahn spectrum clearly shows all connectivities for the histidine, tyrosine and tryptophan residues that exist in IL-6. Using a deuterium exchange method, the imidazole proton resonances of His-16 and His-165 have been assigned. Iodination of the tyrosine residues led to the assignment of Tyr-32. Photo-chemically induced dynamic nuclear polarization data have shown that His-16, Tyr-32 and Trp-158 are exposed to solvent, whereas His-165, Tyr-98 and Tyr-101 are buried. Iodination of Tyr-32 gave no significant effect on IL-6 activity, suggesting that Tyr-32 is not responsible for IL-6 activity.
Assuntos
Interleucina-6/química , Ligação Competitiva , Cromatografia Líquida de Alta Pressão , Dicroísmo Circular , Humanos , Concentração de Íons de Hidrogênio , Interleucina-6/metabolismo , Espectroscopia de Ressonância MagnéticaRESUMO
Although the enhanced activity of the polyol pathway has been detected in diabetic glomeruli, the intraglomerular localization of this pathway has not yet been well defined. In this study, we attempted to identify aldose reductase, a key enzyme of the polyol pathway, in cultured rat mesangial cells and to characterize the properties of this enzyme using enzymological and immunological methods. When the aldose reductase (DL-glyceraldehyde-reducing) activity was analyzed in mesangial cell extract, the Lineweaver-Burk plot showed concave downward curvature, and the Michaelis constant was 0.83 mM DL-glyceraldehyde, and this activity was noncompetitively inhibited by an aldose reductase inhibitor, ICI-128,436. The enzyme activity was enhanced by the addition of sulfate ion and partially suppressed by barbital. The enzyme cross-reacted with the antisera against rat lens and testis aldose reductases on Ouchterlony plate, and migrated to the region of molecular weight of about 36,500 Da on Western blotting. The presence of aldose reductase mRNA was also confirmed by Northern analysis using cDNA for rat aldose reductase, 10Q. From these results, it was concluded that the aldose reductase may exist in rat glomerular mesangial cells and may play a role in the development of diabetic glomerulopathy, though the coexistence of aldehyde reductase(s) may not be fully ruled out.
Assuntos
Aldeído Redutase/análise , Mesângio Glomerular/citologia , Mesângio Glomerular/enzimologia , Aldeído Redutase/antagonistas & inibidores , Aldeído Redutase/genética , Animais , Northern Blotting , Western Blotting , Células Cultivadas , DNA/análise , DNA/genética , Sondas de DNA , Expressão Gênica , Imuno-Histoquímica , Cristalino/enzimologia , Masculino , Ftalazinas/farmacologia , RNA Mensageiro/análise , RNA Mensageiro/genética , Ratos , Ratos Endogâmicos , Testículo/enzimologiaRESUMO
We studied the functional consequences of an enhanced polyol pathway activity, elicited with galactose feeding, on the peripheral nerve of transgenic mice expressing human aldose reductase. Nontransgenic littermate mice were used as controls. With a quantitative immunoassay, the expression level of human aldose reductase in the sciatic nerve was 791 +/- 44 ng/mg protein (mean +/- SE), about 25% of that in human sural nerve. When the transgenic mice were fed food containing 30% galactose, significant levels of galactitol accumulated in the sciatic nerve. Galactose feeding of nontransgenic littermate mice led to a 10-fold lower accumulation of galactitol. Galactose feeding for 16 weeks caused a significant and progressive decrease in motor nerve conduction velocity in transgenic mice to 80% of the level of galactose-fed littermate mice, which was not significantly different from that of galactose-free littermate mice. A morphometric analysis of sciatic nerve detected > 10% reduction of mean myelinated fiber size but no alterations of myelinated fiber density in galactose-fed transgenic mice compared with other groups. The functional and structural changes that develop in galactose-fed transgenic mice are similar to those previously reported in diabetic animals. The results of these studies suggest that transgenic mice expressing human aldose reductase may be a useful model not only for defining the role of the polyol pathway in diabetic neuropathy but also for identifying and characterizing effective inhibitors specific for human aldose reductase.
Assuntos
Aldeído Redutase/metabolismo , Galactosemias/metabolismo , Doenças do Sistema Nervoso Periférico/metabolismo , Aldeído Redutase/genética , Animais , Sequência de Bases , Primers do DNA/química , Feminino , Galactitol/biossíntese , Galactose/administração & dosagem , Galactosemias/enzimologia , Regulação Enzimológica da Expressão Gênica , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Condução Nervosa , Nervo Isquiático/enzimologia , Nervo Isquiático/metabolismoRESUMO
Fluorescence resonance energy transfer (FRET) is one of the few methods available to measure the rate at which a folding protein collapses. Using staphylococcal nuclease in which a cysteine residue was engineered in place of Lys64, permitted FRET measurements of the distance between the donor tryptophan 140 and 5-[[2-[(iodoacetyl)-amino]ethyl]amino]naphthalene-1-sulfonic acid-labeled Cys64. These measurements were undertaken on both equilibrium partially folded intermediates at low pH (A states), as well as transient intermediates during stopped-flow refolding. The results indicate that there is an initial collapse of the protein in the deadtime of the stopped-flow instrument, corresponding to a regain of approximately 60% of the native signal, followed by three slower transients. This is in contrast to circular dichroism measurements which show only 20-25% regain of the native secondary structure in the burst phase. Thus hydrophobic collapse precedes the formation of substantial secondary structure. The first two detected transient intermediate species have FRET properties essentially identical with those of the previously characterized equilibrium A state intermediates, suggesting similar structures between the equilibrium and transient intermediates. The effects of anions on the folding of acid-unfolded staphylococcal nuclease, and urea on the unfolding of the resulting A states, indicates that in folding the protein becomes compact prior to formation of major secondary structure, whereas in unfolding the protein expands prior to major loss of secondary structure. Comparison of the kinetics of refolding of staphylococcal nuclease, monitored by FRET, and for a proline-free variant, indicate that folding occurs via two partially folded intermediates leading to a native-like species with one (or more) proline residues in a non-native conformation. For the A states an excellent correlation between compactness measured by FRET, and compactness determined from small-angle X-ray scattering, was observed. Further, a linear relationship between compactness and free energy of unfolding was noted. Formation of soluble aggregates of the A states led to dramatic enhancement of the FRET, consistent with intermolecular fluorescence energy transfer.
Assuntos
Nuclease do Micrococo/química , Nuclease do Micrococo/metabolismo , Dobramento de Proteína , Substituição de Aminoácidos/genética , Naftalenossulfonato de Anilina/metabolismo , Ânions/metabolismo , Ânions/farmacologia , Dicroísmo Circular , Cisteína/genética , Cisteína/metabolismo , Transferência de Energia , Fluorescência , Guanidina/farmacologia , Concentração de Íons de Hidrogênio , Cinética , Nuclease do Micrococo/genética , Modelos Moleculares , Maleabilidade , Prolina/genética , Prolina/metabolismo , Ligação Proteica , Desnaturação Proteica/efeitos dos fármacos , Renaturação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína/efeitos dos fármacos , Termodinâmica , Titulometria , Triptofano/metabolismo , Ureia/farmacologiaRESUMO
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.
Assuntos
Fenda Labial/genética , Fissura Palatina/genética , Análise Mutacional de DNA/métodos , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos/genética , Animais , Ásia , Estudos de Casos e Controles , Bovinos , Galinhas/genética , DNA/genética , Europa (Continente) , Variação Genética/genética , Genética Populacional/métodos , Proteínas de Homeodomínio/química , Proteínas de Homeodomínio/genética , Humanos , Desequilíbrio de Ligação/genética , Fator de Transcrição MSX1 , Camundongos , Dados de Sequência Molecular , Mutação/genética , Polimorfismo Genético/genética , Ratos , Alinhamento de Sequência/métodos , América do Sul , Síndrome , Fatores de Transcrição/química , Fatores de Transcrição/genética , Regiões não Traduzidas/genética , Proteínas de Xenopus/genéticaRESUMO
OBJECTIVE: To clarify the influence of interindividual difference in the level of aldose reductase on the polyol pathway-related metabolism in diabetic patients. RESEARCH DESIGN AND METHODS: The enzyme protein content was determined by a two-site enzyme-linked immunosorbent assay using monoclonal and polyclonal antibodies to recombinant human aldose reductase in erythrocytes from 35 diabetic patients and 11 healthy volunteers. Patients were stratified into two groups by the median of aldose reductase content, and the erythrocyte sorbitol level, the fructose level, and the lactate-to-pyruvate ratio were compared between the two groups. We also examined the correlation of the enzyme content with these metabolic parameters. RESULTS: The group of patients whose enzyme content was above the median showed a significant increase in the levels of sorbitol (34.7 +/- 4.9 vs. 20.4 +/- 2.0 nmol/g Hb, P < 0.05) and fructose (99.8 +/- 17.2 vs. 45.9 +/- 4.6 nmol/g Hb, P < 0.05), along with an elevated lactate-to-pyruvate ratio (28.6 +/- 6.1 vs. 11.7 +/- 1.2, P < 0.05), compared with patients with low enzyme levels. The aldose reductase content in erythrocytes was well correlated with its activity, and there was a significant correlation between the enzyme content and the erythrocyte sorbitol (r = 0.58, P < 0.001) or fructose (r = 0.57, P < 0.001) levels as well as between the enzyme level and the lactate-to-pyruvate ratio (r = 0.38, P < 0.05). CONCLUSIONS: These results suggest that the interindividual variability of aldose reductase content may contribute tangibly to the polyol-pathway flux and cytoplasmic redox alteration in diabetic patients.
Assuntos
Aldeído Redutase/sangue , Diabetes Mellitus Tipo 2/sangue , Eritrócitos/metabolismo , Frutose/sangue , Sorbitol/sangue , Aldeído Redutase/imunologia , Anticorpos , Glicemia/análise , Diabetes Mellitus Tipo 2/enzimologia , Ensaio de Imunoadsorção Enzimática/métodos , Feminino , Hemoglobinas Glicadas/análise , Humanos , Lactatos/sangue , Masculino , Pessoa de Meia-Idade , Oxirredução , Piruvatos/sangue , Proteínas Recombinantes/imunologia , Valores de Referência , Análise de RegressãoRESUMO
Amino acid substitutions of human interleukin-6 (IL-6) were performed. Single substitution Met162----Ala and double substitutions Leu159.166----Val resulted in a significant decrease of IL-6 activity in the production of immunoglobulin (Ig) from B-cells. Single substitution Leu166----Val or Leu159----Val gave a slight or no significant decrease in the Ig-induction activity, respectively. The receptor-binding activity of each IL-6 mutant was also examined. It was observed that the decrease of the receptor-binding activity was generally in parallel with that of the Ig-induction activity. We therefore suggest that hydrophobic side-chains existing in Met162, Leu159, and Leu166 are significantly involved in the receptor-binding of IL-6.
Assuntos
Interleucina-6/genética , Mutagênese Sítio-Dirigida , Animais , Linfócitos B/efeitos dos fármacos , Linfócitos B/imunologia , Sequência de Bases , Ligação Competitiva , Linhagem Celular , Humanos , Interleucina-6/metabolismo , Interleucina-6/farmacologia , Cinética , Dados de Sequência Molecular , Sondas de Oligonucleotídeos , Plasmídeos , Receptores Imunológicos/metabolismo , Receptores de Interleucina-6 , Mapeamento por Restrição , TransfecçãoRESUMO
The nucleotide sequence of a cDNA that encodes a new subunit, named RC7-I, of the 20 S proteasome of rat hepatoma cells has been determined. The polypeptide predicted from the open reading frame consists of 201 amino acid residues with a calculated molecular weight of 22,912 and isoelectric point of 7.25. Approximately 80% of the partial amino acid sequences of several fragments of RC7-I, determined by protein chemical analyses, were found to be in excellent accordance with those deduced from the cDNA sequence. Computer analysis showed that RC7-I belongs to the beta-type subgroup of proteasomes with similarity to the beta-subunit of the archaebacterial proteasome, differing clearing from alpha-type subunits of the proteasome gene family. The overall structure of RC7-I was found to be homologous to that of yeast PRE1, which is necessary for chymotryptic activity.
Assuntos
Quimotripsina/metabolismo , Cisteína Endopeptidases/genética , Complexos Multienzimáticos/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Cisteína Endopeptidases/metabolismo , DNA Complementar , Proteínas Fúngicas/genética , Dados de Sequência Molecular , Complexos Multienzimáticos/metabolismo , Complexo de Endopeptidases do Proteassoma , Ratos , Homologia de Sequência de Aminoácidos , Thermoplasma/genética , Células Tumorais CultivadasRESUMO
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.
Assuntos
Aldeído Redutase/genética , Aldeído Redutase/metabolismo , Receptores ErbB/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Aldeído Redutase/antagonistas & inibidores , Animais , Linhagem Celular , Sobrevivência Celular , Complicações do Diabetes , Diabetes Mellitus/metabolismo , Flavonoides/farmacologia , Sequestradores de Radicais Livres/farmacologia , Humanos , Peróxido de Hidrogênio/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Estresse Oxidativo , Ftalazinas/farmacologia , Proteínas Quinases/metabolismo , Quinazolinas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Transdução de Sinais , Tirfostinas/farmacologia , Regulação para Cima/efeitos dos fármacosRESUMO
The nucleotide sequence of a cDNA that encodes a new subunit, named RC10-II, of the 20S proteasome of rat embryonic brain has been determined. The polypeptide predicted from the open reading frame consists of 205 amino acid residues with a calculated molecular weight of 22,965 and isoelectric point of 6.15. Computer analysis showed that RC10-II belongs to the beta-type subgroup of proteasomes, differing clearly from alpha-type subunits of the proteasome gene family. The primary structure of RC10-II was found to contain the partial amino acid sequences of several fragments of subunit 13, which has a cysteinyl residue critical for the trypsin-like catalytic activity, as reported by L. R. Dick et al. [Biochemistry 31, 7347-7355, 1992], suggesting that RC10-II is a proteasomal subunit necessary for the expression of tryptic activity.
Assuntos
Cisteína Endopeptidases/metabolismo , Complexos Multienzimáticos/metabolismo , Tripsina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Encéfalo/enzimologia , Clonagem Molecular , Cisteína Endopeptidases/biossíntese , DNA Complementar/metabolismo , Biblioteca Gênica , Cinética , Fígado/enzimologia , Substâncias Macromoleculares , Dados de Sequência Molecular , Peso Molecular , Complexos Multienzimáticos/biossíntese , Complexo de Endopeptidases do Proteassoma , Ratos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Saccharomyces cerevisiae/enzimologia , Homologia de Sequência de AminoácidosRESUMO
Site-directed mutagenesis of two sets of three periodic leucine residues which appear at every seventh position in the C-terminal region of human interleukin-6 (IL-6) was performed. Both receptor-binding and immunoglobulin (Ig)-induction activities of a triple mutant Leu168,175,182-->Val were only 1% compared with those of wild-type IL-6. However, the mutant Leu152,159,166-->Val had 13% receptor-binding and 2% Ig-induction activities of those of wild-type IL-6. In order to obtain more direct information on the receptor-binding region, we prepared two synthetic peptides. A significant binding activity was observed for the peptide Leu168-Met185, but not for the peptide Leu152-Arg169. These results indicate that leucine residues in the C-terminal region, especially Leu168, Leu175, and Leu182, play an important role in the receptor-binding and Ig-induction activities.
Assuntos
Interleucina-6/genética , Interleucina-6/farmacologia , Leucina , Mutagênese Sítio-Dirigida , Sequência de Aminoácidos , Formação de Anticorpos/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Sequência de Bases , Ligação Competitiva , Linhagem Celular Transformada , Clonagem Molecular , Escherichia coli/genética , Herpesvirus Humano 4 , Humanos , Interleucina-6/metabolismo , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Fragmentos de Peptídeos/síntese química , Receptores Imunológicos/metabolismo , Receptores de Interleucina-6 , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacologia , Relação Estrutura-AtividadeRESUMO
Aldose reductase (EC 1.1.1.21) has been implicated in a variety of diabetic complications. Here we present the first primary sequence data for the rat lens enzyme, obtained by amino acid and cDNA analysis. We have found structural similarities with another NADPH-dependent oxidoreductase: human liver aldehyde reductase (EC 1.1.1.2). The identity between these two enzymes is 50%. Both enzymes share approx. 40-50% homology with p-crystallin, a major lens protein present only in the frog, Rana pipiens. We propose that aldose reductase, aldehyde reductase and p-crystallin are members of a superfamily of related proteins.
Assuntos
Álcool Desidrogenase/análise , Aldeído Redutase/análise , Cristalinas/análise , Desidrogenase do Álcool de Açúcar/análise , Sequência de Aminoácidos , Animais , DNA/análise , Humanos , Cristalino/enzimologia , Cristalino/metabolismo , Fígado/enzimologia , Rana pipiens , RatosRESUMO
Accelerated formation and accumulation of advanced glycation end products, as well as increased flux of glucose through polyol pathway, have been implicated in the pathogenesis of diabetic vascular complications. We investigated effects of advanced glycation end products on the levels of aldose reductase mRNA, protein, and activity in human microvascular endothelial cells. When endothelial cells were cultured with highly glycated bovine serum albumin, aldose reductase mRNA in endothelial cells demonstrated concentration-dependent elevation. The increase in aldose reductase mRNA was accompanied by elevated protein expression and enzyme activity. Significant increase in the enzyme expression was also observed when endothelial cells were cultured with serum obtained from diabetic patients with end-stage renal disease. Pretreatment of the endothelial cells with probucol or vitamin E prevented the advanced glycation end products-induced increases in aldose reductase mRNA and protein. Electrophoretic mobility shift assays using the nuclear extracts of the endothelial cells treated with advanced glycation end products showed enhancement of specific DNA binding activity for AP-1 consensus sequence. These results indicate that accelerated formation of advanced glycation end products in vivo may elicit activation of the polyol pathway, possibly via augmented oxidative stress, and amplify endothelial cell damage leading to diabetic microvascular dysfunction.
Assuntos
Aldeído Redutase/genética , Endotélio Vascular/enzimologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Albumina Sérica/farmacologia , Transcrição Gênica/efeitos dos fármacos , Aldeído Redutase/biossíntese , Animais , Bovinos , Células Cultivadas , Diabetes Mellitus/sangue , Nefropatias Diabéticas/sangue , Endotélio Vascular/efeitos dos fármacos , Indução Enzimática , Produtos Finais de Glicação Avançada/farmacologia , Humanos , Falência Renal Crônica/sangue , Microcirculação , RNA Mensageiro/genética , Soroalbumina Bovina , Albumina Sérica GlicadaRESUMO
An antibody-sandwich enzyme-linked immunosorbent assay (ELISA) for evaluating tissue levels of aldose reductase was developed using a polyclonal antibody prepared against the recombinant enzyme expressed in a baculovirus system. The specificity of this antibody to aldose reductase was verified by immunoprecipitation, immunoblotting and ELISA. The polyclonal antibody did not crossreact with human aldehyde reductase, an enzyme in the same aldo-keto reductase family structurally and functionally related to aldose reductase. The sensitivity and specificity of this assay method enabled direct determination of aldose reductase level in various human tissues including the erythrocyte. The highest level of aldose reductase was detected in the kidney medulla among tissues investigated. More than a 2-fold variability in the erythrocyte aldose reductase was demonstrated among healthy individuals, indicating the heterogeneity of this enzyme expression in a human population. This assay system may be useful for direct measurement of the level of tissue aldose reductase in conjunction with the evaluation of the efficacy of aldose reductase inhibitors prescribed for the treatment of diabetic complications.