RESUMEN
A new reagent for quantification of CgS in plant extracts using a generalized methodology suitable for recognition of homocysteine (Hcy) with luminescence ON response.
Asunto(s)
Benzoxazinas/farmacología , Liasas de Carbono-Oxígeno/análisis , Colorantes Fluorescentes/farmacología , Extractos Vegetales/análisis , Animales , Benzoxazinas/síntesis química , Benzoxazinas/química , Cisteína/análisis , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/química , Células HeLa , Homocisteína/análisis , Humanos , Cinética , Límite de Detección , Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Nicotiana/química , Pez CebraRESUMEN
A total of 221 Salmonella enterica from raw pork (n=64), raw chicken (n=80), and humans (n=77) were characterized for antimicrobial resistance phenotypes and genotypes and virulence plasmid-associated genes. Most Salmonella isolates (95.9%) were multidrug resistant and exhibited high resistance to sulfamethoxazole (96.4%), streptomycin (93.2%), spectinomycin (76.5%), tetracycline (73.3%), ampicillin (70.1%), and trimethoprim (60.2%). Forty-one percent of all isolates were intI1-positive, of which 60% carried class 1 integrons with variable region ranging in size from 0.2 to 2.0 kb. Six integron profiles (IP-I to IP-VI) were defined. The dfrA12-aadA2 cassette was most prevalent (66.7%). Class 1 integrons with the dfrA12-aadA2 cassette in five pork isolates could be horizontally transferred. Three pork isolates carried Salmonella genomic island 1 (SGI1), of which a serovar Anatum harbored SGI1 gene cluster located between thdF and int2. Two single-point mutations (i.e., G-259-T and C-248-T) in gyrA leading to Asp-87-Tyr and Ser-83-Phe substitutions in GyrA, respectively, were detected. Of all plasmid-mediated quinolone resistance genes tested, only qnrS (4.1%) and qnrB (1.8%) were found. The virulence plasmid-associated genes including spvC, pefA, and rck were identified in 8.1%, 1.8%, and 1.4% of all Salmonella isolates, respectively.
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Antiinfecciosos/farmacología , Farmacorresistencia Bacteriana Múltiple/genética , Microbiología de Alimentos , Carne/microbiología , Salmonella enterica/efectos de los fármacos , Animales , Liasas de Carbono-Oxígeno/análisis , Pollos , ARN Helicasas DEAD-box/análisis , Proteínas Fimbrias/análisis , Genotipo , Humanos , Fenotipo , Plásmidos/análisis , Plásmidos/genética , Aves de Corral/microbiología , Proteínas Proto-Oncogénicas/análisis , Salmonella enterica/genética , Salmonella enterica/inmunología , Salmonella enterica/aislamiento & purificación , Porcinos , Tailandia , Factores de Virulencia/genéticaRESUMEN
Phosphorus is an abundant element in living organisms. It is traceable by its X-ray absorption spectrum which shows a strong white line at its K-edge, comparable with that observed for the L(III) edges of rare earth ions. With purple membrane, the variation of the imaginary part of the anomalous dispersion of phosphorus is found to be close to 20 anomalous electron units. Anomalous diffraction experiments at wavelengths near the K-absorption edge of phosphorus confirm this result. The spatial distribution of lipids derived from anomalous diffraction agrees with earlier results from neutron diffraction. Test experiments on single crystals of the carrier protein using 5.76 A photons gave a first low-resolution diffraction pattern. Various techniques of crystal mounting were attempted. In addition, fluorescence measurements on a solution of threonine synthase appear to hint at a change of the phosphate environment of the cofactor upon activator binding.
Asunto(s)
Translocasas Mitocondriales de ADP y ATP/química , Fósforo/análisis , Membrana Púrpura/química , Espectrometría por Rayos X/instrumentación , Espectrometría por Rayos X/métodos , Difracción de Rayos X/instrumentación , Difracción de Rayos X/métodos , Liasas de Carbono-Oxígeno/análisis , Liasas de Carbono-Oxígeno/química , Diseño de Equipo , Análisis de Falla de Equipo , Halobacterium/química , Translocasas Mitocondriales de ADP y ATP/análisis , Conformación Molecular , Fósforo/química , Conformación ProteicaRESUMEN
OBJECTIVE: The mechanism of spinal cord injury has been thought to be related to the vulnerability of spinal motor neuron cells against ischemia. However, the mechanisms of such vulnerability are not fully understood. We previously reported that spinal motor neurons may be lost by programmed cell death and thus now investigate a possible mechanism of neuronal death with immunohistochemical analysis for 8-hydroxy-2'-deoxyguanosine (8-OHdG) and redox factor-1 (Ref-1). METHODS: We used a rabbit spinal cord ischemia model with a balloon catheter. The spinal cord was removed at 8 hours, 1, 2, or 7 days after 15 minutes of transient ischemia, and histologic changes were studied with hematoxylin-eosin staining. Western blot analysis for Ref-1, temporal profiles of 8-OHdG and Ref-1 immunoreactivity, and double-label fluorescence immunocytochemical studies were performed. RESULTS: Most motor neurons were preserved until 2 days but were selectively lost at 7 days of reperfusion. Western blot analysis of a sample from sham control spinal cord showed a characteristic 37-kDa band that was reduced after ischemia. Immunohistochemistry showed the nuclear expression of Ref-1 in motor neurons of control spinal cords, and immunoreactivity was decreased 1 day after ischemia. On the other hand, no nuclear expression was seen of 8-OHdG in motor neurons of control spinal cords, and immunoreactivity was increased 1 day after ischemia. Double-label fluorescence immunocytochemical study revealed that both 8-OHdG and Ref-1 were positive at 8 hours of reperfusion in the same motor neurons, which eventually die. CONCLUSION: These results suggest that Ref-1 decreased in motor neurons after transient spinal cord ischemia and that this reduction preceded oxidative DNA damage. The reduction of Ref-1 protein at the moderately late stage of reperfusion may be one of the factors responsible for the delay in neuronal death after spinal cord ischemia.
Asunto(s)
Apoptosis/inmunología , Liasas de Carbono-Oxígeno/análisis , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxiguanosina/análogos & derivados , Desoxiguanosina/análisis , Ataque Isquémico Transitorio/etiología , Ataque Isquémico Transitorio/inmunología , Enfermedades del Sistema Nervioso/etiología , Enfermedades del Sistema Nervioso/inmunología , Estrés Oxidativo/inmunología , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/inmunología , 8-Hidroxi-2'-Desoxicoguanosina , Animales , Modelos Animales de Enfermedad , Ataque Isquémico Transitorio/patología , Enfermedades del Sistema Nervioso/patología , Neuronas/inmunología , Neuronas/patología , Oxidación-Reducción , Conejos , Índice de Severidad de la Enfermedad , Traumatismos de la Médula Espinal/patología , Factores de TiempoRESUMEN
Apurinic/apyrimidinic endonuclease APE/Ref-1 is a multifunctional protein provided with DNA repair, transcription-factor regulation and anti-apoptotic activities. We have previously reported that, in thyroid cells, TSH regulates both the synthesis and nuclear translocation of APE/Ref-1. We have also shown that nuclear levels of this protein are reduced both in thyroid carcinoma tissues and cell lines. In the present study, APE/Ref-1 expression and cellular localization were analysed by Western blot in hyperfunctioning thyroid nodules from patients with toxic adenoma and/or toxic multinodular goiter. The total content of APE/Ref-1 protein was increased in the majority of the hyperfunctioning tissues with respect to normal adjacent tissue. There was also an increase in the nuclear levels of APE/Ref-1, suggesting enhanced cytoplasm-to-nucleus translocation of the protein in addition to its increased rate of synthesis. These results demonstrate that the phenomenon of nuclear translocation of APE/Ref-1 hypothesized on the basis of cell culture experiments does actually occur in vivo. Together with previous observations in thyroid carcinomas and tumoral cell lines, our findings suggest a two-stage model of APE/Ref-1 behaviour during malignant thyrocyte transformation: an early stage characterized by simple hyperplasia and upregulation of APE/Ref-1 in the nuclear compartment of the cell and a later stage in which nuclear levels of the protein drop to below-normal levels as the cell becomes progressively undifferentiated.
Asunto(s)
Liasas de Carbono-Oxígeno/biosíntesis , Nódulo Tiroideo/patología , Transporte Activo de Núcleo Celular , Adenoma/patología , Anciano , Liasas de Carbono-Oxígeno/análisis , Liasas de Carbono-Oxígeno/metabolismo , Núcleo Celular/enzimología , Transformación Celular Neoplásica/química , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Femenino , Bocio Nodular/patología , Humanos , Masculino , Persona de Mediana Edad , Transporte de Proteínas , Nódulo Tiroideo/enzimología , Regulación hacia ArribaRESUMEN
DNA N-glycosylase/AP (apurinic/apyrimidinic) lyase enzymes of the endonuclease III family (nth in Escherichia coli and Nth1 in mammalian organisms) initiate DNA base excision repair of oxidized ring saturated pyrimidine residues. We generated a null mouse (mNth1(-/-)) by gene targeting. After almost 2 years, such mice exhibited no overt abnormalities. Tissues of mNth1(-/-) mice contained an enzymatic activity which cleaved DNA at sites of oxidized thymine residues (thymine glycol [Tg]). The activity was greater when Tg was paired with G than with A. This is in contrast to Nth1, which is more active against Tg:A pairs than Tg:G pairs. We suggest that there is a back-up mammalian repair activity which attacks Tg:G pairs with much greater efficiency than Tg:A pairs. The significance of this activity may relate to repair of oxidized 5-methyl cytosine residues (5meCyt). It was shown previously (S. Zuo, R. J. Boorstein, and G. W. Teebor, Nucleic Acids Res. 23:3239-3243, 1995) that both ionizing radiation and chemical oxidation yielded Tg from 5meCyt residues in DNA. Thus, this previously undescribed, and hence novel, back-up enzyme activity may function to repair oxidized 5meCyt residues in DNA while also being sufficient to compensate for the loss of Nth1 in the mutant mice, thereby explaining the noninformative phenotype.
Asunto(s)
Liasas de Carbono-Oxígeno/fisiología , Reparación del ADN , Desoxirribonucleasa (Dímero de Pirimidina) , Endodesoxirribonucleasas/fisiología , Proteínas de Escherichia coli , Animales , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Ácido Apurínico/metabolismo , Liasas de Carbono-Oxígeno/análisis , Cruzamientos Genéticos , Metilación de ADN , Reparación del ADN/genética , Reparación del ADN/fisiología , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Endodesoxirribonucleasas/deficiencia , Endodesoxirribonucleasas/genética , Femenino , Marcación de Gen , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Especificidad de Órganos , Fenotipo , Especificidad por Sustrato , Linfocitos T/citología , Linfocitos T/efectos de los fármacos , Linfocitos T/efectos de la radiaciónRESUMEN
APE/Ref-1 is a multifunctional protein possessing both redox and DNA repair functions. Through its redox activity, APE/Ref-1 controls the DNA-binding function of several transcriptional regulators (AP1, NF-kappaB, p53, Pax proteins). We have previously shown that APE/Ref-1 upregulates the transcriptional activity of the thyroid-specific transcription factor Pax8. In thyroid cells, APE/Ref-1 can be detected both in the nuclear and cytoplasmatic compartments. In this study regulatory mechanisms acting on APE/Ref-1 were revealed using the FRTL-5 cell line. TSH induces both cytoplasm-to-nucleus translocation and neosynthesis of APE/Ref-1 protein. Interestingly, only neosynthesis is dependent on cAMP signalling. In contrast, the cytoplasm-to-nucleus translocation is dependent on redox-mediated mechanisms. Based upon the data shown in this study and in others, a bimodal control of APE/Ref-1 by TSH can be delineated.
Asunto(s)
Liasas de Carbono-Oxígeno/análisis , Liasas de Carbono-Oxígeno/metabolismo , AMP Cíclico/farmacología , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Glándula Tiroides/metabolismo , Tirotropina/farmacología , Animales , Transporte Biológico/efectos de los fármacos , Calcio/metabolismo , Liasas de Carbono-Oxígeno/biosíntesis , Línea Celular , Núcleo Celular/química , Núcleo Celular/metabolismo , AMP Cíclico/metabolismo , Citoplasma/química , Citoplasma/metabolismo , Oxidación-Reducción , Ratas , Transducción de Señal , Glándula Tiroides/química , Glándula Tiroides/ultraestructuraRESUMEN
PURPOSE: We determined whether base and nucleotide excision repair is activated in bladder urothelium by chronic persistent low doses of ionizing radiation in male patients with benign prostate hyperplasia and females with chronic cystitis living more than 15 years in Cs contaminated areas after the Chernobyl accident in Ukraine. MATERIALS AND METHODS: Bladder urothelial biopsies from 204 patients were subjected to histological examination and biopsies from 35 were subjected to immunohistochemical study of 8-hydroxy-2'deoxyguanosine, 8-oxoguanine-DNA-glycosylase, apurinic/apyrimidinic endonuclease and xeroderma pigmentosum A endonuclease. RESULTS: Chronic proliferative atypical cystitis with multiple foci of dysplasia and carcinoma in situ were observed in 139 (89%) and in 91 (58%) of 156 group 1 patients from radio contaminated areas, respectively, as well as 10 small transitional cell carcinomas. Chronic cystitis with areas of dysplasia was detected in 9 of 48 patients (19%) in control group 2 from clean (without radio contamination) areas of Ukraine. Greatly elevated levels of 8-hydroxy-2'deoxyguanosine, 8-oxoguanine-DNA-glycosylase, apurinic/apyrimidinic endonuclease and xeroderma pigmentosum A were evident in the urothelium in group 1, accompanied by increased Cs in the urine. CONCLUSIONS: These findings support the hypothesis that significant activation of DNA damage repair (base and nucleotide excision repair) is induced by the oxidative stress generated by long-term low doses of ionizing radiation. The levels of DNA oxidative adducts pointing to mutagenic and carcinogenic potential were in line with the histopathologically diagnosed urothelial lesions.
Asunto(s)
Daño del ADN/efectos de la radiación , Reparación del ADN , Desoxiguanosina/análogos & derivados , Liberación de Radiactividad Peligrosa , Vejiga Urinaria/efectos de la radiación , 8-Hidroxi-2'-Desoxicoguanosina , Adulto , Anciano , Anciano de 80 o más Años , Liasas de Carbono-Oxígeno/análisis , Enfermedad Crónica , Cistitis/patología , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN-Formamidopirimidina Glicosilasa , Desoxiguanosina/análisis , Endonucleasas/análisis , Femenino , Humanos , Inmunohistoquímica , Masculino , Persona de Mediana Edad , N-Glicosil Hidrolasas/análisis , Neoplasias Inducidas por Radiación/genética , Estrés Oxidativo , Hiperplasia Prostática/patología , Ucrania , Vejiga Urinaria/química , Neoplasias de la Vejiga Urinaria/etiología , Neoplasias de la Vejiga Urinaria/genética , Urotelio/química , Urotelio/efectos de la radiaciónRESUMEN
OBJECTIVE: To explore the protective mechanism of fructose-1, 6-diphosphate (FDP) on ischemic brain injury. METHODS: A model of permanent focal cerebral ischemia was performed in rats by intraluminal filament occlusion of middle cerebral artery. TTC staining, immunohistochemistry, Western blotting, and TUNEL staining were used to evaluate the effect of FDP on infarct area, apurinic/apyrimidinic endonuclease (APE/Ref-1) expression, and apoptosis in ischemic brain tissue. RESULT: The infarct areas of FDP intervening group and ischemia for 24 h group were 31.0 +/- 2.9 mm(2) and 47.3 +/- 6.0 mm(2) respectively. The numbers of TUNEL positive cells in ischemic penumbra were 69.3 +/- 2.4/mm(2) and 42.8 +/- 1.7/mm(2) in FDP group and ischemia for 24 h group respectively. FDP upregulated the expression of APE/Ref-1 protein in ischemic penumbra. The numbers of APE/Ref-1 immuno-positive cells in the ischemia for 24 h group and FDP group were 47 +/- 3.4/mm(2) and 26.3 +/- 2.9/mm(2) respectively. The values of optical density by Western blotting in these two groups were 5.3 +/- 3.2 and 13.8 +/- 5.4 respectively. The differences between these two groups were statistically significant. CONCLUSION: Through upregulating the expression of APE/Ref-1 protein, FDP improves the repair ability of brain tissue in the course of ischemic injury and mitigated the quantity of apoptosis in penumbra, thus preventing the extension of cerebral infarction.
Asunto(s)
Isquemia Encefálica/tratamiento farmacológico , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Fructosadifosfatos/farmacología , Fármacos Neuroprotectores/farmacología , Animales , Western Blotting , Infarto Encefálico/tratamiento farmacológico , Isquemia Encefálica/metabolismo , Liasas de Carbono-Oxígeno/análisis , Daño del ADN , Etiquetado Corte-Fin in Situ , Masculino , Ratas , Ratas WistarRESUMEN
PURPOSE: Thioredoxin (TRX), a low molecular weight protein, exerts reduction-oxidation control over a number of transcription factors involved in cell activation and proliferation. High TRX mRNA levels have been found in lung carcinomas, a trait associated with a growth and survival advantage. EXPERIMENTAL DESIGN: In this study, we examined the immunohistochemical expression of human TRX in normal lung and in 102 primary non-small cell lung carcinomas. RESULTS: In normal lung, the staining for TRX was cytoplasmic in the respiratory bronchial epithelium, alveolar epithelium, and alveolar macrophages. Bronchial glandular cells demonstrated a mixed nuclear and cytoplasmic staining. In lung carcinomas, the pattern of expression for TRX was predominantly cytoplasmic and only occasionally nuclear. A strong association between absence of TRX expression and regional lymph node negativity was observed (P = 0.004). High proliferation index, as detected with Ki-67 antibody, was associated with high TRX expression (P = 0.02). A significant correlation between high cytoplasmic p53 reactivity and low TRX expression was observed (P = 0.04). No association with grade, tumor stage, histology, or bcl-2 was noted. A significant coexpression of TRX with human activator protein endonuclease 1 was recorded (P = 0.04). Absence of TRX expression was associated with a better outcome (P < 0.05). CONCLUSIONS: We conclude that overexpression of TRX in non-small cell lung carcinomas is indicative of a more aggressive tumor phenotype and is associated with bad prognostic features and possibly with a poorer outcome.
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Carcinoma de Pulmón de Células no Pequeñas/patología , Neoplasias Pulmonares/patología , Ganglios Linfáticos/patología , Tiorredoxinas/biosíntesis , Anciano , Liasas de Carbono-Oxígeno/análisis , Carcinoma de Pulmón de Células no Pequeñas/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/cirugía , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Femenino , Humanos , Inmunohistoquímica , Antígeno Ki-67/análisis , Pulmón/química , Pulmón/patología , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/cirugía , Ganglios Linfáticos/química , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas Proto-Oncogénicas c-bcl-2/análisis , Análisis de Supervivencia , Proteína p53 Supresora de Tumor/análisisRESUMEN
The DNA repair enzyme, apurinic/apyrimidinic endonuclease (or redox effector factor-1, APE/Ref-1), is involved in base excision repair of apurinic/apyrimidinic sites after oxidative DNA damage. We investigated the expression of APE/Ref-1 and its relationship to oxidative stress after severe traumatic brain injury produced by controlled cortical impact in normal mice, and in mice over- or underexpressing copper-zinc superoxide dismutase (SOD1TG and SOD1KO, respectively). Oxygen free radical-mediated cellular injury was visualized with 8-hydroxyguanine immunoreactivity as a marker for DNA oxidation, and in situ hydroethidine oxidation as a marker for superoxide production. After trauma there was a reduced expression of APE/Ref-1 in the ipsilateral cortex and hippocampus that correlated with the gene dosage levels of cytosolic superoxide dismutase. The decrease in APE/Ref-1 expression preceded DNA fragmentation. There was also a close correlation between APE/Ref-1 protein levels 4 h after trauma and the volume of the lesion 1 week after injury. Our data have demonstrated that reduction of APE/Ref-1 protein levels correlates closely with the level of oxidative stress after traumatic brain injury. We suggest that APE/Ref-1 immunoreactivity is a sensitive marker for oxidative cellular injury.
Asunto(s)
Lesiones Encefálicas/metabolismo , Liasas de Carbono-Oxígeno/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Estrés Oxidativo/fisiología , Animales , Western Blotting , Lesiones Encefálicas/patología , Liasas de Carbono-Oxígeno/análisis , ADN/metabolismo , Fragmentación del ADN , Reparación del ADN , Inmunohistoquímica , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Noqueados , Neuronas/metabolismo , Neuronas/patología , Oxidación-Reducción , Superóxido Dismutasa/genética , Superóxido Dismutasa/metabolismo , Superóxidos/análisis , Superóxidos/metabolismoRESUMEN
For its DNA repair, transcription factor regulation and anti-apoptotic activity, the apurinic/apirimidinic ApeI/Ref-I endonuclease is thought to play a relevant role in human tumorigenesis. In human thyroid tumors, we demonstrated an altered nuclear/cytoplasmic ratio in all the carcinomas examined but not in follicular adenomas. In this study, Ref-I expression and cellular localization were analyzed in a series of human thyroid carcinoma cell lines. We found a reduced nuclear/cytoplasmic ratio in BCPAP, TPC I and ARO cells and not in WRO cells. Such a pattern of expression corresponds to that observed in thyroid tumoral tissues except for the WRO cells which behave as the follicular adenomas rather than carcinomas. Thus, these cell lines represent an excellent in vitro model to analyze the molecular mechanisms involved in Ref-I regulation and activity and clarify its role in thyroid tumorigenesis.
Asunto(s)
Liasas de Carbono-Oxígeno/análisis , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Neoplasias de la Tiroides/enzimología , Adenoma/enzimología , Adenoma/ultraestructura , Western Blotting , Carcinoma/enzimología , Carcinoma/ultraestructura , Núcleo Celular/enzimología , Citoplasma/enzimología , Reparación del ADN , Humanos , Neoplasias de la Tiroides/patología , Neoplasias de la Tiroides/ultraestructura , Células Tumorales CultivadasRESUMEN
To identify new markers of minimal residual disease (MRD) in B-lineage acute lymphoblastic leukemia (ALL), gene expression of leukemic cells obtained from 4 patients with newly diagnosed ALL was compared with that of normal CD19(+)CD10(+) B-cell progenitors obtained from 2 healthy donors. By cDNA array analysis, 334 of 4132 genes studied were expressed 1.5- to 5.8-fold higher in leukemic cells relative to both normal samples; 238 of these genes were also overexpressed in the leukemic cell line RS4;11. Nine genes were selected among the 274 overexpressed in at least 2 leukemic samples, and expression of the encoded proteins was measured by flow cytometry. Two proteins (caldesmon and myeloid nuclear differentiation antigen) were only weakly expressed in leukemic cells despite strong hybridization signals in the array. By contrast, 7 proteins (CD58, creatine kinase B, ninjurin1, Ref1, calpastatin, HDJ-2, and annexin VI) were expressed in B-lineage ALL cells at higher levels than in normal CD19(+)CD10(+) B-cell progenitors (P <.05 in all comparisons). CD58 was chosen for further analysis because of its abundant and prevalent overexpression. An anti-CD58 antibody identified residual leukemic cells (0.01% to 1.13%; median, 0.03%) in 9 of 104 bone marrow samples from children with ALL in clinical remission. MRD estimates by CD58 staining correlated well with those of polymerase chain reaction amplification of immunoglobulin genes. These results indicate that studies of gene expression with cDNA arrays can aid the discovery of leukemia markers. (Blood. 2001;97:2115-2120)
Asunto(s)
Biomarcadores de Tumor/análisis , Médula Ósea/química , Antígenos CD58/análisis , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Perfilación de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Proteínas de Neoplasias/análisis , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Adolescente , Adulto , Anexina A6/análisis , Anexina A6/genética , Biomarcadores de Tumor/genética , Médula Ósea/patología , Antígenos CD58/genética , Proteínas de Unión al Calcio/análisis , Proteínas de Unión al Calcio/genética , Liasas de Carbono-Oxígeno/análisis , Liasas de Carbono-Oxígeno/genética , Proteínas Portadoras/análisis , Proteínas Portadoras/genética , Moléculas de Adhesión Celular Neuronal/análisis , Moléculas de Adhesión Celular Neuronal/genética , Linaje de la Célula , Niño , Preescolar , Creatina Quinasa/análisis , Creatina Quinasa/genética , Forma BB de la Creatina-Quinasa , ADN Complementario/genética , Femenino , Citometría de Flujo , Estudios de Seguimiento , Perfilación de la Expresión Génica/instrumentación , Proteínas del Choque Térmico HSP40 , Proteínas de Choque Térmico/análisis , Proteínas de Choque Térmico/genética , Humanos , Lactante , Isoenzimas/análisis , Isoenzimas/genética , Masculino , Proteínas de Neoplasias/genética , Neoplasia Residual , Factores de Crecimiento Nervioso/análisis , Factores de Crecimiento Nervioso/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangre , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Inducción de Remisión , Sensibilidad y EspecificidadRESUMEN
Mutations of mitochondrial DNA (mtDNA) are associated with different human diseases, including cancer and aging. Reactive oxygen species produced during oxidative phosphorylation are a major source of mtDNA damage. It is not clear, however, whether DNA repair mechanisms, able to abolish effects due to oxidative damage, are present in mitochondria. APE/Ref-1 is a nuclear protein possessing both redox activity (by which activates, "in vitro", the DNA-binding functions of several transcription factors) and DNA repair activity over apurinic/apyrimidinic sites. Immunohistochemical evidences indicate that in follicular thyroid cells, APE/Ref-1 is located in both nucleus and cytoplasm. Electronmicroscopy immunocytochemistry performed in the rat thyroid FRTL-5 cell line, indicates that part of the cytoplasmatic APE/Ref-1 is located in mitochondria. The presence of APE/Ref-1 inside mitochondria is further demonstrated by western blot analysis after cell fractionation. In the Kimol cell line (which is derived from FRTL-5, transformed by the Ki-ras oncogene) the amount of mitochondrial APE/Ref-1 is reduced by three to fourfold with respect to the normal FRTL-5 cells. These results suggest that: (i) a machinery capable of repairing DNA damaged by oxidative stress is present in mitochondria and (ii) mtDNA repair mechanisms may be impaired during cell transformation.
Asunto(s)
Liasas de Carbono-Oxígeno/metabolismo , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Mitocondrias/metabolismo , Glándula Tiroides/metabolismo , Animales , Liasas de Carbono-Oxígeno/análisis , Línea Celular , Reparación del ADN , ADN Mitocondrial/metabolismo , Inmunohistoquímica , Microscopía Electrónica , Microscopía Fluorescente , Mitocondrias/química , Mitocondrias/ultraestructura , Estrés Oxidativo , Ratas , Especies Reactivas de Oxígeno/metabolismo , Fracciones Subcelulares/metabolismo , Glándula Tiroides/citología , Glándula Tiroides/ultraestructura , Proteínas ras/metabolismoRESUMEN
To determine whether redox factor-1 (Ref-1) participates in the pathogenesis of inclusion-body myositis (IBM), we immunolocalized Ref-1 in muscle biopsies of IBM patients by light- and electron-microscopy. Approximately 70-80% of the IBM vacuolated muscle fibers had focal inclusions strongly immunoreactive for Ref-1. By immunoelectronmicroscopy, Ref-1 was localized to paired-helical filaments, 6-10 nm amyloid-like fibrils and amorphous material. Virtually all regenerating and necrotic muscle fibers in various muscle biopsies had diffusely strong Ref-1 immunoreactivity. At all neuromuscular junctions, postsynaptically there was strong Ref-1 immunoreactivity. Our study suggests that Ref-1 plays a role in IBM pathogenesis, and in other pathologic and normal processes of human muscle.
Asunto(s)
Liasas de Carbono-Oxígeno/análisis , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Músculo Esquelético/patología , Miositis por Cuerpos de Inclusión/patología , Biopsia , Humanos , Microscopía Inmunoelectrónica , Músculo Esquelético/ultraestructuraRESUMEN
The DNA base excision repair pathway is responsible for the repair of alkylation and oxidative DNA damage. A crucial step in the base excision repair pathway involves the cleavage of an apurinic/apyrimidinic (AP) site in DNA by an AP endonuclease (APE). The major AP endonuclease in mammalian cells is APE/ref-1, a multifunctional enzyme that acts not only as an AP endonuclease but as a redox-modifying factor for a variety of transcription factors. The purpose of this study was to determine the expression of APE/redox factor-1 (ref-1) in ovarian tissues, particularly ovarian cancers. Formalin-fixed, paraffin-embedded specimens of ovarian tissues (normal, various benign conditions, and epithelial cancers) were studied using both polyclonal and monoclonal antibodies to APE/ref-1. The relationship between APE/ref-1 protein levels and DNA repair activity was studied in ovarian Hey and Hey-C2 cell lines using Western blot and a specific AP-site oligonucleotide cleavage assay. Hey and Hey-C2 cells were fractionated, and the nuclear and cytoplasmic extracts were quantitated for protein levels and assessed for APE/ref-1 with Western blot. Normal ovarian tissues consistently demonstrated strong nuclear staining of the surface epithelium, epithelial inclusions, corpora lutea and albicantia, and stroma. Cytoplasmic staining was absent. A similar pattern was seen for benign conditions including endometriosis. Low malignant potential ovarian cancers stained in a pattern similar to normal ovarian and nonneoplastic tissues; however, two specimens also had areas of cytoplasmic staining. Epithelial ovarian cancers were remarkably different from all other ovarian tissues studied. Both nuclear and cytoplasmic staining of the malignant epithelium were seen and ranged from strong to weak, often with considerable staining heterogeneity within the same tumor. The AP-site oligonucleotide cleavage assay indicated that APE/ref-1 protein levels correlate well with DNA repair activity. The increased levels of APE/ref-1 in the Hey-C2 cells was mainly attributable to increased cytoplasmic enzyme. APE/ref-1 immunoreactivity is altered in malignant ovarian tumors. Further studies will determine whether the altered expression and subcellular location reflect changes in redox regulatory functions.
Asunto(s)
Liasas de Carbono-Oxígeno/análisis , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Enfermedades del Ovario/enzimología , Neoplasias Ováricas/enzimología , Ovario/enzimología , Adenocarcinoma/enzimología , Adenocarcinoma/patología , Adenofibroma/enzimología , Adenofibroma/patología , Cistoadenoma/enzimología , Cistoadenoma/patología , Endometriosis/enzimología , Endometriosis/patología , Femenino , Humanos , Inmunohistoquímica , Enfermedades del Ovario/patología , Neoplasias Ováricas/patología , Ovario/citología , Ovario/patología , Células Tumorales CultivadasRESUMEN
BACKGROUND: The enzyme apurinic/apyriminidic endonuclease/redox factor 1 (Ape1/ref1), a key protein in the base excision repair pathway, displays repair and redox activity. We examined the role of Ape1/ref1 and a protein it regulates, hypoxia inducible factor 1 alpha (HIF-1 alpha) in pediatric yolk sac tumors. PATIENTS AND METHODS: Using an immunohistochemical evaluation, 16 pediatric yolk sac tumors were evaluated for Ape1/ref1 and HIF-1 alpha expression. Samples were obtained from archival tissue. RESULTS: We demonstrated high levels of expression of Ape1/ref1 in 14/16 of the tumors. This expression was limited to the nucleus of the viable portion of each tumor. High levels of HIF-1 alpha expression was noted in half of the same tumors and localized to both the nucleus and cytoplasm of the viable tumor. CONCLUSIONS: The high levels of expression of Ape1/ref1 in this group of chemosensitive tumors may be related to the subcellular location or redox regulatory activity of one of the other factors controlled by Ape1/ref1.
Asunto(s)
Liasas de Carbono-Oxígeno/análisis , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Tumor del Seno Endodérmico/enzimología , Proteínas de Neoplasias/análisis , Preescolar , Proteínas de Unión al ADN/análisis , Femenino , Humanos , Factor 1 Inducible por Hipoxia , Subunidad alfa del Factor 1 Inducible por Hipoxia , Lactante , Recién Nacido , Masculino , Proteínas Nucleares/análisis , Factores de Transcripción/análisisRESUMEN
Apurinic/apyrimidinic endonuclease, a multifunctional protein in the DNA base excision repair pathway, plays a central role in repairing DNA damage caused by reactive oxygen species. We examined protein expression of apurinic/apyrimidinic endonuclease before and after cold injury-induced brain trauma in mice, where we have previously shown reactive oxygen species to participate. Immunohistochemistry showed the nuclear expression of apurinic/apyrimidinic endonuclease in the entire region of control brains. One hour after cold injury-induced brain trauma, nuclear immunoreactivity was predominantly decreased in the inner boundary of the lesion, whereas there was a slight increase in the outer boundary area. Four hours after cold injury-induced brain trauma, nuclear immunoreactivity was almost absent in the entire lesion, and remained so until 24 h. At this time, a marked increase in apurinic/apyrimidinic endonuclease immunoreactivity was seen in the outer boundary zone. Western blot analysis of the sample from the non-ischemic area showed a characteristic 37,000 mol. wt band, which decreased markedly 24 h after cold injury-induced brain trauma. A time-dependent increase in DNA fragmentation was also observed after cold injury-induced brain trauma. Our data provide the first evidence that apurinic/apyrimidinic endonuclease decreased rapidly in the lesion after cold injury-induced brain trauma, whereas it was significantly increased at the outer boundary zone. Although further examination is necessary to elucidate the direct relationship between apurinic/apyrimidinic endonuclease alteration and the pathogenesis of cold injury-induced brain trauma, our results suggest the possibility that an early decrease in apurinic/apyrimidinic endonuclease and failure of the DNA repair mechanism may contribute to DNA-damaged neuronal cell death after cold injury-induced brain trauma.
Asunto(s)
Apoptosis/fisiología , Lesiones Encefálicas/enzimología , Liasas de Carbono-Oxígeno/metabolismo , Frío/efectos adversos , Fragmentación del ADN , Animales , Western Blotting , Edema Encefálico/enzimología , Liasas de Carbono-Oxígeno/análisis , Liasas de Carbono-Oxígeno/biosíntesis , Infarto Cerebral/enzimología , Circulación Cerebrovascular , Reparación del ADN , ADN-(Sitio Apurínico o Apirimidínico) Liasa , Desoxirribonucleasa IV (Fago T4-Inducido) , Electroforesis , Etiquetado Corte-Fin in Situ , Masculino , Ratones , Ratones Endogámicos , Proteínas del Tejido Nervioso/análisis , Neuronas/química , Neuronas/citología , Neuronas/enzimologíaAsunto(s)
Daño del ADN/genética , Reparación del ADN/genética , ADN Mitocondrial/genética , Animales , Liasas de Carbono-Oxígeno/análisis , ADN Ligasas/análisis , ADN Ligasas/fisiología , ADN Polimerasa gamma , ADN Mitocondrial/efectos de los fármacos , ADN Mitocondrial/efectos de la radiación , ADN-(Sitio Apurínico o Apirimidínico) Liasa , ADN Polimerasa Dirigida por ADN/análisis , ADN Polimerasa Dirigida por ADN/fisiología , Desoxirribonucleasa IV (Fago T4-Inducido) , Humanos , VertebradosRESUMEN
Oxidative damage to mitochondrial DNA has been implicated in human degenerative diseases and aging. Although removal of oxidative lesions from mitochondrial DNA occurs, the responsible DNA repair enzymes are poorly understood. By expressing the epitope-tagged proteins in COS-7 cells, we examined subcellular localizations of gene products of human DNA glycosylases: hOGG1, hMYH and hNTH1. A gene encoding for hOGG1 which excises 7,8-dihydro-8-oxoguanine (8-oxoG) from DNA generates four isoforms by alternative splicing (types 1a, 1b, 1c and 2). Three tagged isoforms (types 1b, 1c and 2) were localized in the mitochondria. Type 1a protein, which exclusively contains a putative nuclear localization signal, was sorted to the nucleus and lesser amount to the mitochondria. hMYH, a human homolog gene product of Escherichia coli mutY was mainly transported into the mitochondria. hNTH1 protein excising several pyrimidine lesions was transported into both the nucleus and mitochondria. In contrast to the three DNA glycosylases, translocation of the human major AP endonuclease (hAPE) into the mitochondria was hardly observed in COS-7 cells. These results suggest that the previously observed removal of oxidative base lesions in mitochondrial DNA is initiated by the above DNA glycosylases.