RESUMEN
BACKGROUND: High concentrations of methylglyoxal (MGO) cause cytotoxiticy via formation of advanced glycation endproducts (AGEs) and inflammation. MGO is detoxificated enzymatically by glyoxalase-I (Glo-I). The aim of this study was to analyze the role of Glo-I during the development of cirrhosis. METHODS: In primary hepatocytes, hepatic stellate cells (pHSC) and sinusoidal endothelial cells (pLSEC) from rats with early (CCl4 8wk) and advanced cirrhosis (CCl4 12wk) expression and activity of Glo-I were determined and compared to control. LPS stimulation (24h; 100ng/ml) of HSC was conducted in absence or presence of the partial Glo-I inhibitor ethyl pyruvate (EP) and the specific Glo-I inhibitor BrBzGSHCp2. MGO, inflammatory and fibrotic markers were measured by ELISA and Western blot. Additional rats were treated with CCl4 ± EP 40mg/kg b.w. i.p. from wk 8-12 and analyzed with sirius red staining and Western blot. RESULTS: Expression of Glo-I was significantly reduced in cirrhosis in whole liver and primary liver cells accompanied by elevated levels of MGO. Activity of Glo-I was reduced in cirrhotic pHSC and pLSEC. LPS induced increases of TNF-α, Nrf2, collagen-I, α-SMA, NF-kB and pERK of HSC were blunted by EP and BrBzGSHCp2. Treatment with EP during development of cirrhosis significantly decreased the amount of fibrosis (12wk CCl4: 33.3±7.3%; EP wk 8-12: 20.7±6.2%; p<0.001) as well as levels of α-SMA, TGF-ß and NF-κB in vivo. CONCLUSIONS: Our results show the importance of Glo-I as major detoxifying enzyme for MGO in cirrhosis. The reduced expression of Glo-I in cirrhosis demonstrates a possible explanation for increased inflammatory injury and suggests a "vicious circle" in liver disease. Blunting of the Glo-I activity decrease the amount of fibrosis in established cirrhosis and constitutes a novel target for antifibrotic therapy.
Asunto(s)
Inflamación/patología , Lactoilglutatión Liasa/análisis , Cirrosis Hepática/patología , Hígado/patología , Animales , Células Cultivadas , Citocinas/inmunología , Glutatión/análogos & derivados , Glutatión/farmacología , Inflamación/complicaciones , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Lactoilglutatión Liasa/antagonistas & inhibidores , Lactoilglutatión Liasa/inmunología , Hígado/efectos de los fármacos , Hígado/inmunología , Cirrosis Hepática/complicaciones , Cirrosis Hepática/tratamiento farmacológico , Cirrosis Hepática/inmunología , Masculino , FN-kappa B/inmunología , Piruvaldehído/inmunología , Ratas , Ratas Wistar , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Glyoxalase I (GLO1) is a key enzyme that plays a role in the detoxification of methylglyoxal (MG), a toxic cellular metabolite produced during glycolysis. The present study reports on the preparation and properties of a monoclonal antibody (MAb) directed against mouse GLO1. The antibody was produced by hybridization of mouse myeloma cells with lymph node cells from an immunized rat. The MAb 6F10 specifically recognized GLO1, as evidenced by immunoblotting using a variety of extracts from cultured cells. In immunostaining using MAb 6F10, a diffuse cytoplasmic and nuclear staining pattern was observed. The MAb 6F10 promises to be useful in immunoblotting and immunostaining experiments in various cells and tissues to determine the expression levels of GLO1, as well as to further analyze the biological function of this protein.
Asunto(s)
Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Hibridomas/inmunología , Lactoilglutatión Liasa/inmunología , Animales , Células COS , Chlorocebus aethiops , Ensayo de Inmunoadsorción Enzimática , Técnica del Anticuerpo Fluorescente Indirecta , Humanos , Immunoblotting , Ratones , RatasRESUMEN
Glyoxalase I (GLOI) is the first enzyme of the glyoxalase system that catalyzes the metabolism of reactive dicarbonyls, such as methylglyoxal (MGO). During aging and cataract development, human lens proteins are chemically modified by MGO, which is likely due to inadequate metabolism of MGO by the glyoxalase system. In this study, we have determined the effect of aging on GLOI activity and the immunoreactivity and morphological distribution of GLOI in the human lens. A monoclonal antibody was developed against human GLOI. GLOI immunoreactivity was strongest in the anterior epithelial cells and weaker in rest of the lens. Cultured human lens epithelial cells showed immunostaining throughout the cytoplasm. In the human lens, GLOI activity and immunoreactivity both decreased with age. We believe that this would lead to promotion of MGO-modification in aging lens proteins.
Asunto(s)
Envejecimiento/metabolismo , Senescencia Celular , Células Epiteliales/enzimología , Inmunohistoquímica , Lactoilglutatión Liasa/metabolismo , Cristalino/enzimología , Adolescente , Adulto , Factores de Edad , Anciano , Anciano de 80 o más Años , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos , Células Cultivadas , Niño , Clonación Molecular , Regulación hacia Abajo , Ensayo de Inmunoadsorción Enzimática , Humanos , Lactoilglutatión Liasa/genética , Lactoilglutatión Liasa/inmunología , Cristalino/citología , Persona de Mediana Edad , Adulto JovenRESUMEN
Cereal proteins are known to cause allergic reactions such as Baker's asthma and severe atopic dermatitis to certain populations. In rice allergy, proteins with molecular masses of 14-16, 26, 33, and 56 kDa have been demonstrated to be potentially allergenic. In this study, to identify and characterize the 33-kDa allergen, designated Glb33, this protein was first purified to homogeneity, and its cDNA clone was isolated. When expressed in Escherichia coli, the recombinant Glb33 was shown to be as reactive as the native Glb33 with mouse IgG and patients' IgE antibodies to Glb33. The Glb33 cDNA coded for a protein of 291 amino acids with two 120-amino acid residue repeats, and the amino acid sequence showed similarity to glyoxalase I from various organisms, including human, plant, yeast, and bacterium. As expected, both native Glb33 purified from rice seeds and the recombinant protein had glyoxalase I activity that catalyzes condensation of methylglyoxal and glutathione into S-lactoylglutathione. However, Glb33 had a higher sequence identity to the bacterial glyoxalase I rather than to known plant and yeast enzymes. Both the Glb33 transcript and the protein were detected not only in maturing seeds of rice but also in its stem and leaf. Taken all together, the rice allergen, Glb33, was identified to be a novel type of plant glyoxalase I that is expressed in various plant tissues, including maturing seeds.
Asunto(s)
Alérgenos/genética , Alérgenos/inmunología , Lactoilglutatión Liasa/genética , Lactoilglutatión Liasa/inmunología , Oryza , Proteínas de Plantas/genética , Proteínas de Plantas/inmunología , Alérgenos/análisis , Alérgenos/aislamiento & purificación , Secuencia de Aminoácidos , Clonación Molecular , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Escherichia coli , Lactoilglutatión Liasa/análisis , Lactoilglutatión Liasa/aislamiento & purificación , Datos de Secuencia Molecular , Proteínas de Plantas/análisis , Proteínas de Plantas/aislamiento & purificación , Proteínas Recombinantes/análisis , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Alineación de SecuenciaRESUMEN
A homogenous preparation of glyoxalase I (S-lactoylglutathione-lyase, EC 4.4.1.5) was obtained from Brassica juncea seedlings. The enzyme is a heterodimer with 27,000 and 29,000 M(r) subunits and native M(r) of 56,000. The circular dichroic spectra of the protein showed characteristics of a distinctly helical protein, and magnesium affected the secondary structure. It is a zinc metalloenzyme. Amino acid modification studies suggested the involvement of histidine residues in catalysis. Apo-glyoxalase I was reactivated by divalent cations Mn2+ (0.5 Mm) > Mg2+ (5 Mm) > Zn2+ (0.05 Mm) and Ca2+ (0.01 Mm). Monospecific, polyclonal anti-glyoxalase I antibodies were raised, which showed its presence in seeds, roots, hypocotyl, cotyledon and different flower parts. They showed varied degree of cross reactivity with the extracts from various plants, yeast, bacteria and animal system.
Asunto(s)
Brassica/enzimología , Lactoilglutatión Liasa/metabolismo , Western Blotting , Catálisis , Dicroismo Circular , Reacciones Cruzadas , Ensayo de Inmunoadsorción Enzimática , Lactoilglutatión Liasa/química , Lactoilglutatión Liasa/inmunología , Extractos Vegetales/inmunología , Estructura Secundaria de ProteínaRESUMEN
Glyoxalase-I (Gly-I) is part of the glyoxalase system which converts methylglyoxal to D-lactic acid via an S-D-lactoylglutathione intermediate. This glutathione (GSH)-binding protein was purified from human colon tumors and corresponding normal tissue. The GSH-affinity purified fraction from normal human colon tissue showed enzyme activity of 30.6 +/- 11.5 mumol/min per mg protein, with methylglyoxal as substrate. Corresponding fractions from carcinomas showed significantly elevated Gly-I activity of 54.5 +/- 15 mumol/min per mg protein. Polyclonal antibodies made against human Gly-I cross-reacted weakly with mouse liver Gly-I but not with yeast Gly-I. Isoelectric points of Gly-I from human, mouse and yeast were determined to be 4.6, 4.9 and 7.0, respectively, by horizontal IEF. Immunohistochemical analysis confirmed the increase of Gly-I in human colon carcinoma in 16 out of 21 samples when compared to corresponding normal tissue. The elevated levels of Gly-I in colon tumors may be an indicator of the enhanced proliferative status of the neoplastic condition.
Asunto(s)
Neoplasias del Colon/enzimología , Lactoilglutatión Liasa/análisis , Animales , Anticuerpos/inmunología , Biomarcadores de Tumor/análisis , Neoplasias del Colon/patología , Humanos , Inmunohistoquímica , Focalización Isoeléctrica , Lactoilglutatión Liasa/inmunología , Lactoilglutatión Liasa/aislamiento & purificación , Ratones , Saccharomyces cerevisiaeRESUMEN
Glutathione is the most abundant low molecular mass thiol in human cells. It is involved in the inactivation of genotoxic electrophilic compounds, and a variety of glutathione-linked enzymes catalyze such detoxication reactions. Within this group, the enzymes occurring in highest intracellular concentrations are the glutathione transferases, which catalyze the detoxication of a broad spectrum of alkylating and oxidizing compounds such as epoxides, reactive alkenes and organic hydroperoxides. Multiple forms of glutathione transferase with distinct substrate specificities exist, and their differential expression in cells contributes to differences in detoxication capacities in tissues. Glyoxalase I catalyzes the inactivation of 2-oxoaldehydes and may also be considered as part of the cellular detoxication system. Characterization of the different enzymes and their differential expression in normal and tumor cells will help to clarify their cellular functions and their significance to human cancer. Clear differences in the occurrence of the various enzyme forms in normal and tumor cells have been demonstrated and variations between different tumors appear to be linked to their degree of resistance to alkylating cytostatic drugs. Modulation of catalytic activities in vitro by administration of enzyme inhibitors may help to overcome this resistance.
Asunto(s)
Antineoplásicos/inmunología , Resistencia a Medicamentos/fisiología , Glutatión Transferasa/inmunología , Lactoilglutatión Liasa/inmunología , Neoplasias/enzimología , Animales , Secuencia de Bases , Exones/genética , Glutatión Transferasa/genética , Humanos , Datos de Secuencia Molecular , Neoplasias/inmunología , Polimorfismo Genético , RatasRESUMEN
To evaluate a possible role of major-histocompatibility-complex (MHC)-related immune-response genes for the selective production of drug-induced antibodies, HLA class I (ABC), class II (DR, DQ), class III (BF, C2, C4A, C4B) as well as glyoxalase-1 allotypes were investigated in 26 patients with nomifensine-induced immune hemolytic anemia. No statistically significant deviations of MHC antigen frequencies were noted. The possible implications of these findings are discussed.
Asunto(s)
Anemia Hemolítica/inmunología , Antígenos de Histocompatibilidad/análisis , Nomifensina/efectos adversos , Adulto , Anciano , Anemia Hemolítica/inducido químicamente , Proteínas del Sistema Complemento/análisis , Femenino , Antígenos HLA/análisis , Antígenos HLA-D/análisis , Humanos , Lactoilglutatión Liasa/inmunología , Masculino , Persona de Mediana Edad , FenotipoRESUMEN
Antibodies to glyoxalase I from yeast, rat liver, porcine erythrocytes and human erythrocytes were raised in rabbits. Gel precipitation and immunotitration experiments demonstrated that the mammalian enzymes were immunologically related, but distinct from the yeast enzyme. Fab fragments of the antibodies to human glyoxalase I did not inhibit the catalytic activity, indicating that the antigen binding sites were not directed towards the active site of the enzyme. A radioimmunoassay for glyoxalase I was developed. Quantitative analysis of human adult as well as fetal organs demonstrated that glyoxalase I was present in a concentration of approximately 0.2 micrograms/mg protein in most human tissues.
Asunto(s)
Eritrocitos/enzimología , Lactoilglutatión Liasa/análisis , Hígado/enzimología , Liasas/análisis , Saccharomyces cerevisiae/enzimología , Adulto , Animales , Anticuerpos , Complejo Antígeno-Anticuerpo , Femenino , Feto , Humanos , Inmunodifusión , Inmunoelectroforesis , Inmunoelectroforesis Bidimensional , Fragmentos Fab de Inmunoglobulinas , Lactoilglutatión Liasa/sangre , Lactoilglutatión Liasa/inmunología , Embarazo , Ratas , Especificidad de la Especie , Fracciones Subcelulares/enzimología , Porcinos , Distribución TisularRESUMEN
We determined the gene frequencies for the alleles of the HLA-linked complement markers C2, properdin factor B (BF), C4A (Rodgers) and C4B (Chido), and the red cell enzyme glyoxalase-I in 38 unrelated patients with senile dementia of the Alzheimer type, 42 patients with idiopathic Parkinson's disease, and 59 unaffected, aged-matched control blood donors. In senile dementia of the Alzheimer type and in Parkinson's disease, no significant difference was found in the gene frequencies of alleles at either the BF, C2, or GLO-I locus compared with those of age-matched controls. In senile dementia of the Alzheimer type, a striking increase in the frequency of the rare C4B locus allele, C4*B2, was apparent, resulting in the high relative risk of RR = 8.8 (p less than 0.0001) for this disorder.