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1.
Sci Rep ; 7(1): 16697, 2017 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-29196670

RESUMO

Tuberous sclerosis complex (TSC) is a rare autosomal dominant disorder causing benign tumors in the brain and other vital organs. The genes implicated in disease development are TSC1 and TSC2. Here, we have performed mutational analysis followed by a genotype-phenotype correlation study based on the clinical characteristics of the affected individuals. Twenty unrelated probands or families from Greece have been analyzed, of whom 13 had definite TSC, whereas another 7 had a possible TSC diagnosis. Using direct sequencing, we have identified pathogenic mutations in 13 patients/families (6 in TSC1 and 7 in TSC2), 5 of which were novel. The mutation identification rate for patients with definite TSC was 85%, but only 29% for the ones with a possible TSC diagnosis. Multiplex ligation-dependent probe amplification (MLPA) did not reveal any genomic rearrangements in TSC1 and TSC2 in the samples with no mutations identified. In general, TSC2 disease was more severe than TSC1, with more subependymal giant cell astrocytomas and angiomyolipomas, higher incidence of pharmacoresistant epileptic seizures, and more severe neuropsychiatric disorders. To our knowledge, this is the first comprehensive TSC1 and TSC2 mutational analysis carried out in TSC patients in Greece.


Assuntos
Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/genética , Esclerose Tuberosa/patologia , Adulto , Criança , Análise Mutacional de DNA , Éxons , Feminino , Deleção de Genes , Estudos de Associação Genética , Grécia , Humanos , Masculino , Mutação de Sentido Incorreto , Linhagem , Estrutura Terciária de Proteína , Esclerose Tuberosa/genética
2.
Apoptosis ; 15(2): 128-38, 2010 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-19882353

RESUMO

A major hallmark of the terminal stages of apoptosis is the internucleosomal DNA fragmentation. The endonuclease responsible for this type of DNA degradation is the DNA fragmentation factor (DFF). DFF is a complex of the endonuclease DFF40 and its chaperone/inhibitor, DFF45. In vitro work has shown that histone H1 and HMGB1/2 recruit/target DFF40 to the internucleosomal linker regions of chromatin and that histone H1 directly interacts with DFF40 conferring DNA binding ability and enhancing its nuclease activity. The histone H1 family is comprised of many subtypes, which recent work has shown may have distinct roles in chromatin function. Thus we studied the binding association of DFF40 with specific H1 subtypes and whether these binding associations are altered after the induction of apoptosis in an in vivo cellular context. The apoptotic agent used in this study is the histone deacetylase inhibitor, trichostatin A (TSA). We separated the insoluble chromatin-enriched fraction from the soluble nuclear fraction of the NB4 leukemic cell line. Using MNase digestion, we provide evidence which strongly suggests that the heterodimer, DFF40-DFF45, is localized to the chromatin fraction under apoptotic as well as non-apoptotic conditions. Moreover, we present results that show that DFF40 interacts with the all H1 subtypes used in this study, but preferentially interacts with specific H1 subtypes after the induction of apoptosis by TSA. These results illustrate for the first time the association of DFF40 with individual H1 subtypes, under a specific apoptotic stimulus in an in vivo cellular context.


Assuntos
Núcleo Celular/enzimologia , Desoxirribonucleases/metabolismo , Histonas/metabolismo , Ácidos Hidroxâmicos/farmacologia , Leucemia/enzimologia , Proteínas/metabolismo , Apoptose/efeitos dos fármacos , Proteínas Reguladoras de Apoptose , Western Blotting , Caspase 3/metabolismo , Caspase 9/metabolismo , Linhagem Celular Tumoral , Núcleo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Fracionamento Químico , Cromatina/metabolismo , Humanos , Imunoprecipitação , Proteínas de Ligação a Poli-ADP-Ribose , Transporte Proteico/efeitos dos fármacos , Especificidade por Substrato/efeitos dos fármacos
3.
Leuk Res ; 34(6): 786-92, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-19782399

RESUMO

Histone deacetylase inhibitors (HDACIs) inhibit deacetylases and the accumulation of high levels of acetylation results in chromatin remodeling events which may lead to cell cycle arrest and apoptosis. This work investigates the sensitivity of four leukemic cell lines to the HDACI, trichostatin A (TSA) as compared to normal lymphocytes with respect to acetylation and apoptotic levels. Specifically, this study analyzes the time kinetics of histone H4 and alpha-tubulin acetylation and associates these findings to the time course of TSA-induced PARP cleavage and DFF45 proteolysis. The results of this study show (1) that a non-responsive leukemic cell line to the apoptotic effects of TSA does not have increased acetylation levels in contrast to the responsive leukemic cell lines that show a hyperacetylated profile. This indicates that acetylation levels may be of special importance in accessing the potential sensitivities of leukemic cells to HDACIs, (2) TSA induced apoptosis in lymphocytes but at lower levels and (3) the lack of PARP cleavage and DFF45 proteolysis found in lymphocytes clearly differentiates the final stages apoptosis of human peripheral blood lymphocytes from those of the TSA-sensitive leukemic cell lines. Of value is that the results of this study show that the evaluation of the acetylation levels of target proteins may possibly have the potential of being used as additional indicators of the responsiveness or sensitivity of different cancer cell types to this continuously growing class of anticancer agents.


Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Inibidores de Histona Desacetilases/farmacologia , Ácidos Hidroxâmicos/farmacologia , Leucemia/patologia , Acetilação/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Fragmentação do DNA/efeitos dos fármacos , Avaliação Pré-Clínica de Medicamentos , Células HL-60 , Histona Acetiltransferases/metabolismo , Histonas/metabolismo , Humanos , Ácidos Hidroxâmicos/uso terapêutico , Células K562 , Leucemia/genética , Leucemia/metabolismo , Leucemia/fisiopatologia , Poli(ADP-Ribose) Polimerases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Tubulina (Proteína)/metabolismo
4.
Biochem Genet ; 41(7-8): 235-43, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-14587666

RESUMO

Nuclei from Bactrocera oleae and Ceratitis capitata larvae contain a major protein that shares most of the characteristics of vertebrate high mobility group (HMG) proteins. Proteins are extracted from nuclei with 0.35 M NaCl, are soluble in 5% perchloric acid, are relatively small (molecular weight in the range of 10-16 kDa), and have both a high basic and a high acidic amino acid content. The amino acid constitution of these proteins is similar to that of the HMGB protein family of vertebrates. The proteins cross-react with antibodies raised against the HMGD chromosomal protein of Drosophila melanogaster. The possible relatedness of these proteins to high mobility group proteins is discussed.


Assuntos
Ceratitis capitata/genética , Proteínas de Grupo de Alta Mobilidade/genética , Animais , Western Blotting , Ceratitis capitata/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Eletroforese em Gel de Poliacrilamida , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/metabolismo , Proteínas Nucleares/metabolismo , Tephritidae/genética , Tephritidae/metabolismo
5.
Biochem Genet ; 41(1-2): 39-46, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-12645872

RESUMO

Nuclei from Plodia interpunctella larvae contain four major proteins, which are extracted by 5% perchloric acid and 0.35 M NaCl. The proteins have been designated PL1, PL2, PL3, and PL4. The amino acid analyses of these proteins show that they have high proportions of acidic and basic amino acid residues, a property characteristic of the high mobility group (HMG) proteins isolated from vertebrate tissues. Immunological characterication of these proteins clearly shows that PL1, PL2, and PL4 are more closely related to HMG1 dipteran proteins, while PL3 is more closely related to HMG1 dipteran proteins. The possible relatedness of these proteins to HMG proteins is discussed.


Assuntos
Proteínas de Grupo de Alta Mobilidade/química , Proteínas de Insetos/química , Mariposas/química , Aminoácidos/análise , Animais , Núcleo Celular/química , Cromatografia Líquida de Alta Pressão , Reações Cruzadas , Proteínas de Grupo de Alta Mobilidade/isolamento & purificação , Proteínas de Grupo de Alta Mobilidade/fisiologia , Proteínas de Insetos/isolamento & purificação , Proteínas de Insetos/fisiologia
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