RESUMO
CD44 is a complex cell adhesion molecule that mediates communication and adhesion between adjacent cells as well as between cells and the extracellular matrix. CD44 pre-mRNA produces various mRNA isoforms through alternative splicing of 20 exons, among which exons 1-5 (C1-C5) and 16-20 (C6-C10) are constant exons, whereas exons 6-15 (V1-V10) are variant exons. CD44 V10 exon has important roles in breast tumor progression and Hodgkin lymphoma. Here we show that increased expression of hnRNP L inhibits V10 exon splicing of CD44 pre-mRNA, whereas reduced expression of hnRNP L promotes V10 exon splicing. In addition, hnRNP L also promotes V10 splicing of endogenous CD44 pre-mRNA. Through mutation analysis, we demonstrate that the effects of hnRNP L on V10 splicing are abolished when the CA-rich sequence on the upstream intron of V10 exon is disrupted. However, hnRNP L effects are stronger if more CA-repeats are provided. Furthermore, we show that hnRNP L directly contacts the CA-rich sequence. Importantly, we provide evidences that hnRNP L inhibits U2AF65 binding on the upstream Py tract of V10 exon. Our results reveal that hnRNP L is a new regulator for CD44 V10 exon splicing.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas Grupo L/biossíntese , Receptores de Hialuronatos/genética , Íntrons/genética , Splicing de RNA/genética , Adesão Celular/genética , Éxons/genética , Regulação da Expressão Gênica , Células HeLa , Ribonucleoproteínas Nucleares Heterogêneas Grupo L/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Proteínas Nucleares/metabolismo , Ribonucleoproteínas/metabolismo , Fator de Processamento U2AFRESUMO
MDM2, a critical negative regulator of p53, is often overexpressed in leukemia, but few p53 mutations are found, suggesting that p53-independent MDM2 expression occurs due to alterations in MDM2 upstream regulators. In this study, a high MDM2 transcription level was observed (41.17%) regardless of p53 expression in patient with acute myeloid leukemia (AML). Therefore, we performed genome-scale functional screening of the human genes modulating MDM2 expression in a p53-independent manner. We searched co-expression profiles of genes showing a positive or negative pattern with MDM2 expression in a DNA microarray database, selected1089 links, and composed a screening library of 368 genes. Using MDM2 P1 and P2 promoter-reporter systems, we screened clones regulating MDM2 transcriptions in a p53-independent manner by overexpression. Nine clones from the screening library showed enhanced MDM2 promoter activity and MDM2 expression in p53-deficient HCT116 cells. Among them, six clones, including NTRK2, GNA15, SFRS2, EIF5A, ELAVL1, and YWHAB mediated MAPK signaling for expressing MDM2. These results indicate that p53-independent upregulation of MDM2 by increasing selected clones may lead to oncogenesis in AML and that MDM2-modulating genes are novel potential targets for AML treatment.
Assuntos
Regulação Neoplásica da Expressão Gênica , Genoma Humano , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Sequência de Bases , Sobrevivência Celular/genética , Bases de Dados Genéticas , Células HCT116 , Humanos , Leucemia Mieloide Aguda/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Regiões Promotoras Genéticas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Transcrição GênicaRESUMO
OBJECTIVE: To produce an alcoholic beverage containing anthocyanins that can act as antioxidants and have anticarcinogenic activities and antihypertensive effects. RESULTS: High starch-assimilating sake yeast strain of Saccharomyces cerevisiae co-expressing the glucoamylase and α-amylase genes from Debaryomyces occidentalis using the double rDNA-integration system was developed. The new strain grew substantially using 5 % (w/v) purple sweet potato flour as the sole carbon source. Its cell yield reached 14.5 mg ml(-1) after 3 days. This value was 2.4-fold higher than that of the parental wild-type strain. It produced 12 % (v/v) ethanol from 20 % (w/v) purple sweet potato flour and consumed 98 % of the starch content in purple sweet potato flour after 5 days of fermentation. CONCLUSION: We have produced a health-promoting alcoholic beverage abundant in anthocyanins from purple sweet potato.
Assuntos
Bebidas Alcoólicas/análise , Antocianinas/metabolismo , Ipomoea batatas/metabolismo , Saccharomyces cerevisiae/metabolismo , Antocianinas/análise , Debaryomyces/enzimologia , Debaryomyces/genética , Fermentação , Glucana 1,4-alfa-Glucosidase/genética , Glucana 1,4-alfa-Glucosidase/metabolismo , Saccharomyces cerevisiae/genética , Amido/metabolismo , alfa-Amilases/genética , alfa-Amilases/metabolismoRESUMO
The mammalian Y chromosome has unique characteristics compared with the autosomes or X chromosomes. Here we report the finished sequence of the chimpanzee Y chromosome (PTRY), including 271 kb of the Y-specific pseudoautosomal region 1 and 12.7 Mb of the male-specific region of the Y chromosome. Greater sequence divergence between the human Y chromosome (HSAY) and PTRY (1.78%) than between their respective whole genomes (1.23%) confirmed the accelerated evolutionary rate of the Y chromosome. Each of the 19 PTRY protein-coding genes analyzed had at least one nonsynonymous substitution, and 11 genes had higher nonsynonymous substitution rates than synonymous ones, suggesting relaxation of selective constraint, positive selection or both. We also identified lineage-specific changes, including deletion of a 200-kb fragment from the pericentromeric region of HSAY, expansion of young Alu families in HSAY and accumulation of young L1 elements and long terminal repeat retrotransposons in PTRY. Reconstruction of the common ancestral Y chromosome reflects the dynamic changes in our genomes in the 5-6 million years since speciation.
Assuntos
Cromossomos Humanos Y/genética , Evolução Molecular , Pan troglodytes/genética , Cromossomo Y/genética , Animais , Humanos , Masculino , Dados de Sequência Molecular , Sequências Repetitivas de Ácido Nucleico/genética , Alinhamento de Sequência , Análise de Sequência de DNA , Sintenia/genéticaRESUMO
Most Saccharomyces spp. cannot degrade or ferment dextrin, which is the second most abundant carbohydrate in wort for commercial beer production. Dextrin-degrading brewer's bottom and top yeasts expressing the glucoamylase gene (GAM1) from Debaryomyces occidentalis were developed to produce low-carbohydrate (calorie) beers. GAM1 was constitutively expressed in brewer's yeasts using a rDNA-integration system that contained yeast CUP1 gene coding for copper resistance as a selective marker. The recombinants secreted active glucoamylase, displaying both α-1,4- and α-1,6-debranching activities, that degraded dextrin and isomaltose and consequently grew using them as sole carbon source. One of the recombinant strains expressing GAM1 hydrolyzed 96 % of 2 % (w/v) dextrin and 98 % of 2 % (w/v) isomaltose within 5 days of growth. Growth, substrate assimilation, and enzyme activity of these strains were characterized.
Assuntos
Cerveja/microbiologia , Dextrinas/metabolismo , Isomaltose/metabolismo , Saccharomyces cerevisiae/metabolismo , Cromatografia Líquida de Alta Pressão , Glucana 1,4-alfa-Glucosidase/genética , Hidrólise , Plasmídeos/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Fatores de Tempo , Transformação GenéticaRESUMO
Although retinoic acid (RA) is well known to inhibit the differentiation of 3T3-L1 cells into adipocytes both in vivo and in vitro, its molecular mechanism is not fully understood. In this report, we investigate the inhibitory mechanism of adipocyte differentiation by RA in 3T3-L1 cells. Because both RA and Wnt are known to inhibit adipogenesis at a common step involving the inhibition of PPAR-γ expression, we focused on the crosstalk between these two signaling pathways. We found that RA treatment resulted in a dramatic inhibition of adipogenesis, especially at an early phase of differentiation, and led to increased ß-catenin protein expression. Moreover, RA enhances the transcriptional activity of ß-catenin as well as Wnt gene expression during adipogenesis. Taken together, the present study demonstrated that Wnt/ß-catenin signaling may be associated with the RA-induced suppression of adipogenesis and may cooperatively inhibit adipocyte differentiation.
Assuntos
Adipócitos/efeitos dos fármacos , Adipogenia/efeitos dos fármacos , Transdução de Sinais , Tretinoína/farmacologia , Proteínas Wnt/metabolismo , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/metabolismo , Animais , Sequência de Bases , Diferenciação Celular , Cromanos/farmacologia , Primers do DNA , Camundongos , PPAR gama/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Tiazolidinedionas/farmacologia , Troglitazona , beta Catenina/metabolismoRESUMO
A decreased production of interferon gamma (IFNG) has been observed in acute schizophrenia. In order to explore the possible relationship between IFNG and schizophrenia, we attempted to analyze the differentially expressed proteins in the brains of interferon-gamma knockout (Ifng-KO) mice. Five upregulated and five downregulated proteins were identified with 2D gels and MALDI-TOF/TOF MS analyses in Ifng-KO mouse brain. Of the identified proteins, we focused on creatine kinase brain (CKB) and triose phosphate isomerase 1 (TPI1). Consistent with the proteomic data, reverse transcriptase-mediated PCR, immunoblotting, and immunohistochemistry analyses confirmed that the levels of gene expressions of Ckb and Tpi1 were downregulated and upregulated, respectively. When we analyzed the genetic polymorphisms of the single nucleotide polymorphisms (SNPs) of their human orthologous genes in a Korean population, the promoter SNPs of CKB and TPI1 were weakly associated with schizophrenia. In addition, IFNG polymorphisms were associated with schizophrenia. These results suggest that IFNG and proteins affected by IFNG may play a role in the pathogenesis of schizophrenia.
Assuntos
Creatina Quinase Forma BB/metabolismo , Interferon gama/metabolismo , Esquizofrenia/genética , Esquizofrenia/metabolismo , Triose-Fosfato Isomerase/metabolismo , Animais , Encéfalo/metabolismo , Estudos de Casos e Controles , Creatina Quinase Forma BB/genética , Regulação para Baixo , Feminino , Humanos , Interferon gama/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Proteoma/análise , Proteômica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , República da Coreia , Genética Reversa , Triose-Fosfato Isomerase/genética , Regulação para CimaRESUMO
Ethanol production using hemicelluloses has recently become a focus of many researchers. In order to promote D: -xylose fermentation, we cloned the bacterial xylA gene encoding for xylose isomerase with 434 amino acid residues from Agrobacterium tumefaciens, and successfully expressed it in Saccharomyces cerevisiae, a non-xylose assimilating yeast. The recombinant strain S. cerevisiae W303-1A/pAGROXI successfully colonized a minimal medium containing D: -xylose as a sole carbon source and was capable of growth in minimal medium containing 2% xylose via aerobic shake cultivation. Although the recombinant strain assimilates D: -xylose, its ethanol productivity is quite low during fermentation with D: -xylose alone. In order to ascertain the key enzyme in ethanol production from D: -xylose, we checked the expression levels of the gene clusters involved in the xylose assimilating pathway. Among the genes classified into four groups by their expression patterns, the mRNA level of pyruvate decarboxylase (PDC1) was reduced dramatically in xylose media. This reduced expression of PDC1, an enzyme which converts pyruvate to acetaldehyde, may cause low ethanol productivity in xylose medium. Thus, the enhancement of PDC1 gene expression may provide us with a useful tool for the fermentation of ethanol from hemicellulose.
Assuntos
Aldose-Cetose Isomerases/metabolismo , Etanol/metabolismo , Piruvato Descarboxilase/biossíntese , Proteínas de Saccharomyces cerevisiae/biossíntese , Saccharomyces cerevisiae/enzimologia , Xilose/metabolismo , Agrobacterium/enzimologia , Agrobacterium/genética , Aldose-Cetose Isomerases/genética , Clonagem Molecular , Etanol/isolamento & purificação , Piruvato Descarboxilase/isolamento & purificação , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , TransfecçãoRESUMO
To construct Candida tropicalis strains that produce a high yield of xylitol with no requirement for co-substrates, we engineered the yeast with an attenuated xylitol dehydrogenase (XDH) and then assessed the efficiency of xylitol production The mutants, strains XDH-5 (with only one copy of the XDH gene), and ARSdR-16 (with a mutated XDH gene) showed 70 and 40% of wild type (WT) XDH activity, respectively. Conversions of xylose to xylitol by WT, XDH-5, and ARSdR-16 were 62, 64, and 75%, respectively, with productivities of 0.52, 0.54, and 0.62 g l(-1) h(-1), respectively. The ARSdR-16 mutant strain produced xylitol with high yield and high productivity in a simple process that required no co-substrates, such as glycerol. This strain represents a promising alternative for efficient and cost-effective xylitol production.
Assuntos
Candida tropicalis/genética , Candida tropicalis/metabolismo , D-Xilulose Redutase/genética , D-Xilulose Redutase/metabolismo , Xilitol/biossíntese , Sequência de Bases , Biotecnologia , DNA Fúngico/genética , Fermentação , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Genes Fúngicos , Cinética , Mutagênese Sítio-Dirigida , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Mutação , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Xilose/metabolismoRESUMO
Industrial strains of a polyploid, distiller's Saccharomyces cerevisiae that produces glucoamylase and α-amylase was used for the direct fermentation of raw starch to ethanol. Strains contained either Aspergillus awamori glucoamylase gene (GA1), Debaryomyces occidentalis glucoamylase gene (GAM1) or D. occidentalis α-amylase gene (AMY), singly or in combination, integrated into their chromosomes. The strain expressing both GA1 and AMY generated 10.3% (v/v) ethanol (80.9 g l(-1)) from 20% (w/v) raw corn starch after 6 days of fermentation, and decreased the raw starch content to 21% of the initial concentration.
Assuntos
Etanol/metabolismo , Glucana 1,4-alfa-Glucosidase/metabolismo , Saccharomyces cerevisiae/metabolismo , Amido/metabolismo , alfa-Amilases/metabolismo , Clonagem Molecular , Debaryomyces/enzimologia , Debaryomyces/genética , Etanol/análise , Fermentação , Proteínas Fúngicas/metabolismo , Glucana 1,4-alfa-Glucosidase/biossíntese , Glucana 1,4-alfa-Glucosidase/genética , Microbiologia Industrial/métodos , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Amido/análise , alfa-Amilases/biossíntese , alfa-Amilases/genéticaRESUMO
Interferon regulatory factor-1 (IRF-1) is an interferon-induced transcriptional activator that suppresses tumors by impeding cell proliferation. Recently, we demonstrated that the level of SUMOylated IRF-1 is elevated in tumor cells, and that SUMOylation of IRF-1 attenuates its tumor-suppressive function. Here we report that SUMOylated IRF-1 mimics IRF-2, an antagonistic repressor, and shows oncogenic potential. To demonstrate the role of SUMOylated IRF-1 in tumorigenesis, we used SUMO-IRF-1 recombinant protein. Stable expression of SUMO-IRF-1 in NIH3T3 cells resulted in focus formation and anchorage-independent growth in soft agar. Inoculation of SUMO-IRF-1-transfected cells into athymic nude mice resulted in tumor formation and infiltration of adipose tissues. Finally, we demonstrated that SUMO-IRF-1 transforms NIH3T3 cells in a dose-dependent manner suggesting that SUMOylated IRF-1 may act as an oncogenic protein in tumor cells.
Assuntos
Transformação Celular Neoplásica/metabolismo , Fator Regulador 1 de Interferon/metabolismo , Fator Regulador 2 de Interferon/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Animais , Linhagem Celular , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Humanos , Fator Regulador 1 de Interferon/genética , Fator Regulador 2 de Interferon/genética , Camundongos , Células NIH 3T3 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
To develop a strain of Saccharomyces cerevisiae that produces ethanol directly from starch, two integrative vectors were constructed to allow the simultaneous multiple integration of the Aspergillus awamori glucoamylase gene (GA1) and the Debaryomyces occidentalis alpha-amylase gene (AMY) and glucoamylase with debranching activity gene (GAM1) into the chromosomes of an industrial strain of S. cerevisiae. The GA1 and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae using the double delta-integration system. The GAM1 gene was constitutively expressed under the corresponding promoter using the double 18S rDNA-integration system. The recombinant industrial strain secreting biologically active alpha-amylase, glucoamylase and debranching enzyme was able to ferment starch to ethanol in a single step. The new strain produced 8% (v/v) ethanol (62.8 g l(-1)) from 20% (w/v) soluble starch after 2 days, fermentation.
Assuntos
Etanol/metabolismo , Engenharia Genética , Glucana 1,4-alfa-Glucosidase/metabolismo , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Saccharomyces cerevisiae/metabolismo , Amido/metabolismo , alfa-Amilases/metabolismo , Aspergillus/enzimologia , Aspergillus/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expressão Gênica , Vetores Genéticos , Glucana 1,4-alfa-Glucosidase/genética , Sistema da Enzima Desramificadora do Glicogênio/genética , Recombinação Genética , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Saccharomycetales/enzimologia , Saccharomycetales/genética , alfa-Amilases/genéticaRESUMO
OBJECTIVE: Obesity is recognized as the cause of multiple metabolic diseases and is rapidly increasing worldwide. As obesity is due to an imbalance in energy homeostasis, the promotion of energy consumption through browning of white adipose tissue (WAT) has emerged as a promising therapeutic strategy to counter the obesity epidemic. However, the molecular mechanisms of the browning process are not well understood. In this study, we investigated the effects of the GATA family of transcription factors on the browning process. METHODS: We used qPCR to analyze the expression of GATA family members during WAT browning. In order to investigate the function of GATA3 in the browning process, we used the lentivirus system for the ectopic expression and knockdown of GATA3. Western blot and real-time qPCR analyses revealed the regulation of thermogenic genes upon ectopic expression and knockdown of GATA3. Luciferase reporter assays, co-immunoprecipitation, and chromatin immunoprecipitation were performed to demonstrate that GATA3 interacts with proliferator-activated receptor-γ co-activator-1α (PGC-1α) to regulate the promoter activity of uncoupling protein-1 (UCP-1). Enhanced energy expenditure by GATA3 was confirmed using oxygen consumption assays, and the mitochondrial content was assessed using MitoTracker. Furthermore, we examined the in vivo effects of lentiviral GATA3 overexpression and knockdown in inguinal adipose tissue of mice. RESULTS: Gata3 expression levels were significantly elevated in the inguinal adipose tissue of mice exposed to cold conditions. Ectopic expression of GATA3 enhanced the expression of UCP-1 and thermogenic genes upon treatment with norepinephrine whereas GATA3 knockdown had the opposite effect. Luciferase reporter assays using the UCP-1 promoter region showed that UCP-1 expression was increased in a dose-dependent manner by GATA3 regardless of norepinephrine treatment. GATA3 was found to directly bind to the promoter region of UCP-1. Furthermore, our results indicated that GATA3 interacts with the transcriptional coactivator PGC-1α to increase the expression of UCP-1. Taken together, we demonstrate that GATA3 has an important role in enhancing energy expenditure by increasing the expression of thermogenic genes both in vitro and in vivo. CONCLUSION: GATA3 may represent a promising target for the prevention and treatment of obesity by regulating thermogenic capacity.
Assuntos
Tecido Adiposo Marrom/metabolismo , Tecido Adiposo Branco/metabolismo , Fator de Transcrição GATA3/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Proteína Desacopladora 1/metabolismo , Animais , Temperatura Baixa , Metabolismo Energético , Fator de Transcrição GATA3/genética , Humanos , Camundongos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/fisiologia , Regiões Promotoras Genéticas , Termogênese/genética , Proteína Desacopladora 1/genética , Regulação para CimaRESUMO
Clonorchis sinensis, the Chinese liver fluke, is the causative agent of clonorchiasis as well as liver and biliary diseases. The excretory-secretory products (ESPs) of the parasites play important roles in host-parasite interactions. In this study, we have investigated the proteome of ESPs obtained from C. sinensis adult worms. Although the full genome database of C. sinensis is not yet available, we have successfully identified 62 protein spots using 2-DE-based mass analysis and EST database of C. sinensis. The proteins identified include detoxification enzymes, such as glutathione S-transferase and thioredoxin peroxidase, myoglobin and a number of cysteine proteases that are expressed abundantly. In order to identify potential targets for the diagnosis and therapy of clonorchiasis, we conducted immunoblot analysis of the ESPs proteome using the sera obtained from clonorchiasis patients and identified legumains and cysteine proteases as antigens present in the ESPs. Although the cysteine proteases were previously reported to elicit antigenicity, the legumains are found herein for the first time as a serological antigen of C. sinensis. To confirm these findings, we expressed recombinant legumain in Escherichia coli and verified that recombinant legumain also functions as a potent antigen against the sera of clonorchiasis patients. Our results illustrate the validity of immuno-proteomic approaches in the identification of serodiagnostic antigens in the parasites.
Assuntos
Antígenos de Helmintos/metabolismo , Clonorchis sinensis/enzimologia , Cisteína Endopeptidases/metabolismo , Proteínas de Helminto/metabolismo , Proteoma/análise , Testes Sorológicos/métodos , Sequência de Aminoácidos , Animais , Antígenos de Helmintos/genética , Sequência de Bases , Clonorquíase/diagnóstico , Clonorquíase/parasitologia , Clonorchis sinensis/genética , Clonorchis sinensis/imunologia , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/imunologia , Eletroforese em Gel Bidimensional/métodos , Escherichia coli/genética , Proteínas de Helminto/genética , Proteínas de Helminto/imunologia , Immunoblotting , Dados de Sequência Molecular , Proteômica/métodos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reprodutibilidade dos Testes , Alinhamento de SequênciaRESUMO
Clonorchis sinensis, the parasite that causes clonorchiasis, is endemic in many Asian countries, and infection with the organism drives changes in the liver tissues of the host. However, information regarding the molecular events in clonorchiasis remains limited, and little is currently known about host-pathogen interactions in clonorchiasis. In this study, we assessed the gene expression profiles in mice livers via DNA microarray analysis 1, 2, 4, and 6 weeks after induced metacercariae infection. Functional clustering of the gene expression profile showed that the immunity-involved genes were induced in the livers of the mice at the early stage of metacercariae infection, whereas immune responses were reduced in the 6-week liver tissues after infection in which the metacercariae became adult flukes. Many genes involved in fatty acid metabolism, including Peci, Cyp4a10, Acat1, Ehhadh, Gcdh, and Cyp2 family were downregulated in the infected livers. On the other hand, the liver tissues infected with the parasite expressed Wnt signaling molecules such as Wnt7b, Fzd6, and Pdgfrb and cell cycle-regulating genes including cyclin-D1, Cdca3, and Bcl3. These investigations constitute an excellent starting point for increased understanding of the molecular mechanisms underlying host-pathogen interaction during the development of C. sinensis in the host liver.
Assuntos
Clonorquíase/veterinária , Clonorchis sinensis/crescimento & desenvolvimento , Perfilação da Expressão Gênica , Fígado/patologia , Fígado/parasitologia , Animais , Clonorquíase/patologia , Modelos Animais de Doenças , Interações Hospedeiro-Patógeno , Masculino , Camundongos , Análise de Sequência com Séries de OligonucleotídeosRESUMO
In a previously published case study of acute myeloid leukemia, we tracked the dynamics of somatic mutations over 9 years. Interestingly, we observed a group of mutations that expanded during remission, which we named the "remission clone." To determine the nature of the remission clones, we performed flow cytometry-based cell sorting followed by ultradeep sequencing. The remission clone repeatedly expanded after chemotherapeutic cycles and was suppressed during relapse in the myeloid lineage (multipotent hematopoietic stem, progenitor, and myeloid cells). On the other hand, the remission clone was consistently observed in lymphoid lineages (B and T cells) regardless of the disease state. When transfected into the HEK-293 cell line, the NR2C2(A93V) mutant exhibited a growth advantage (all p values < 0.05). The results indicate that the remission clone seems to be another form of clonal hematopoiesis, but without a clear association with leukemia. As the remission clone is present in both myeloid and lymphoid lineages, it likely originates from ancestral hematopoietic cell lineages. More importantly, the remission clone is distinct from the leukemic clone; therefore, mutations expanded during remission require special interpretation when performing next-generation sequencing-based measurable residual disease assessment.
Assuntos
Células-Tronco Hematopoéticas/metabolismo , Leucemia Mieloide Aguda/metabolismo , Células-Tronco Neoplásicas/metabolismo , Adulto , Substituição de Aminoácidos , Linfócitos B/metabolismo , Linfócitos B/patologia , Células HEK293 , Células-Tronco Hematopoéticas/patologia , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Masculino , Mutação de Sentido Incorreto , Células-Tronco Neoplásicas/patologia , Receptores de Esteroides/genética , Receptores de Esteroides/metabolismo , Receptores dos Hormônios Tireóideos/genética , Receptores dos Hormônios Tireóideos/metabolismo , Indução de Remissão , Linfócitos T/metabolismo , Linfócitos T/patologiaRESUMO
Nuclear Factor-kappa B (NF-kappaB) is a transcription factor essential to the control of cell proliferation, survival, differentiation, immune response, and inflammation. Constitutive NF-kappaB activation has been observed in a broad variety of solid tumors and hematological malignancies, which suggests that NF-kappaB signaling may perform a critical role in the development of human cancers. Interferon regulatory factor-2 (IRF-2), an antagonistic transcriptional repressor of IRF-1, evidences oncogenic potential, but little is currently known regarding the mechanism underlying the oncogenic activities of IRF-2. In this study, we report that IRF-2 recruits RelA/p65 transcription factors into the nucleus via physical interaction. While the nuclear recruitment of RelA by IRF-2 augments TNFalpha-induced NF-kappaB dependent transcription, the N-terminal truncated mutant form of IRF-2 inhibits the nuclear localization of RelA, and thus interferes with NF-kappaB activation. Furthermore, the knockdown of IRF-2 by IRF-2 siRNA attenuates TNFalpha-induced NF-kappaB dependent transcription by inhibiting the nuclear localization of RelA. Thus, these results show that IRF-2 regulates NF-kappaB activity via the modulation of NF-kappaB subcellular localization.
Assuntos
Núcleo Celular/metabolismo , Fator Regulador 2 de Interferon/metabolismo , Fator de Transcrição RelA/metabolismo , Transcrição Gênica , Linhagem Celular , Núcleo Celular/química , Humanos , Fator Regulador 2 de Interferon/antagonistas & inibidores , Fator Regulador 2 de Interferon/genética , RNA Interferente Pequeno/genética , Deleção de Sequência , Fator de Transcrição RelA/análise , Transcrição Gênica/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
Phytase liberates inorganic phosphate from phytic acid (myo-inositol hexakisphosphate) which is the major phosphate reserve in plant-derived foods and feeds. An industrial strain of Saccharomyces cerevisiae expressing the Debaryomyces castellii phytase gene (phytDc) and D. occidentalis alpha-amylase gene (AMY) was developed. The phytDc and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae by using the delta-integration system, which contains DNA derived exclusively from yeast. The recombinant industrial strain secreted both phytase and alpha-amylase for the efficient degradation of phytic acid and starch as main components of plant seeds. This new strain hydrolyzed 90% of 0.5% (w/v) sodium phytate within 5 days of growth and utilized 100% of 2% (w/v) starch within 48 h simultaneously.
Assuntos
6-Fitase/metabolismo , Ácido Fítico/metabolismo , Saccharomyces cerevisiae/enzimologia , Amido/metabolismo , alfa-Amilases/metabolismo , 6-Fitase/genética , Ração Animal , Animais , Genes Fúngicos , Humanos , Hidrólise , Reação em Cadeia da Polimerase , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Sementes , Transformação Genética , alfa-Amilases/genéticaRESUMO
Decreased serum high-density lipoprotein (HDL-C) cholesterol is a major risk factor for atherosclerosis and vascular disease. In this study, we assessed the association of 10 uncoupling protein (UCP) 2 and UCP3 polymorphisms with serum HDL cholesterol levels and atherogenic indexes among 658 Korean women. Among the 10 single nucleotide polymorphisms (SNPs) in the UCP2 and UCP3 genes, 2 SNPs in UCP2, -866G>A and +4787C>T (A55V) that were tightly linked (r(2) = 0.97), were significantly associated with decreased HDL cholesterol levels after Bonferroni correction (P = .003 in the recessive model). +4589C>T (Y210Y) in UCP3, a silent variation of Tyr210Tyr in exon 5, was also significantly associated with HDL cholesterol after multiple comparison correction. These 3 SNPs also exhibited some association with increases in the atherogenic index. Source-of-variation analysis revealed that -866G>A SNP accounted for 8.09% of the variation in serum HDL cholesterol levels independent of body mass index. We believe that our results may provide clues to the association of UCP genes with the risk of atherosclerosis through their effects on HDL cholesterol.
Assuntos
HDL-Colesterol/sangue , Canais Iônicos/genética , Proteínas Mitocondriais/genética , Polimorfismo de Nucleotídeo Único , Adulto , Índice de Massa Corporal , Feminino , Humanos , Coreia (Geográfico) , Espécies Reativas de Oxigênio/metabolismo , Proteína Desacopladora 2 , Proteína Desacopladora 3RESUMO
Prohibitin (PHB) is a multifunctional protein conserved in eukaryotic systems and shows various expression levels in tumor cells. However, regulation of PHB is not clearly understood. Here, we focused on the regulation of PHB expression by Wnt signaling, one of dominant regulatory signals in various leukemic cells. High mRNA levels of PHB were found in half of clinical leukemia samples. PHB expression was increased by inhibition of the MAPK pathway and decreased by activation of EGF signal. Although cell proliferating signals downregulated the transcription of PHB, treatment with lithium chloride, an analog of the Wnt signal, induced PHB level in various cell types. We identified the TCF-4/LEF-1 binding motif, CATCTG, in the promoter region of PHB by site-directed mutagenesis and ChIP assay. This ß-catenin-mediated activation of PHB expression was independent of cMYC activation, a product of Wnt signaling. These data indicate that PHB is a direct target of ß-catenin and the increased level of PHB in leukemia can be regulated by Wnt signaling.