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1.
EMBO J ; 37(13)2018 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-29880601

RESUMO

Cytoplasmic mRNA degradation controls gene expression to help eliminate pathogens during infection. However, it has remained unclear whether such regulation also extends to nuclear RNA decay. Here, we show that 145 unstable nuclear RNAs, including enhancer RNAs (eRNAs) and long noncoding RNAs (lncRNAs) such as NEAT1v2, are stabilized upon Salmonella infection in HeLa cells. In uninfected cells, the RNA exosome, aided by the Nuclear EXosome Targeting (NEXT) complex, degrades these labile transcripts. Upon infection, the levels of the exosome/NEXT components, RRP6 and MTR4, dramatically decrease, resulting in transcript stabilization. Depletion of lncRNAs, NEAT1v2, or eRNA07573 in HeLa cells triggers increased susceptibility to Salmonella infection concomitant with the deregulated expression of a distinct class of immunity-related genes, indicating that the accumulation of unstable nuclear RNAs contributes to antibacterial defense. Our results highlight a fundamental role for regulated degradation of nuclear RNA in the response to pathogenic infection.


Assuntos
RNA Nuclear , RNA não Traduzido , Infecções por Salmonella/genética , Sobrevivência Celular , Células HeLa , Humanos , Salmonella enterica/genética , Regulação para Cima
2.
Biochem Biophys Res Commun ; 518(4): 685-690, 2019 10 22.
Artigo em Inglês | MEDLINE | ID: mdl-31472963

RESUMO

Peroxiredoxins (Prxs) detoxify hydrogen peroxide (H2O2), peroxynitrite, and various organic hydroperoxides. However, the differential oxidative status of Prxs reacted with each peroxide remains unclear. In the present study, we focused on the oxidative alteration of Prxs and demonstrated that, in human red blood cells (RBCs), peroxiredoxin 2 (Prx2) is readily reactive with H2O2, forming disulfide dimers, but was not easily hyperoxidized. In contrast, Prx2 was highly sensitive to the relatively hydrophobic oxidants, such as tert-butyl hydroperoxide (t-BHP) and cumene hydroperoxide. These peroxides hyperoxidized Prx2 into oxidatively damaged forms in RBCs. The t-BHP treatment formed hyperoxidized Prx2 in a dose-dependent manner. When organic hydroperoxide-treated RBC lysates were subjected to reverse-phase high performance liquid chromatography, two peaks derived from hyperoxidized Prx2 appeared along with the decrease of that corresponding to native Prx2. Liquid chromatography-tandem mass spectrometry analysis clearly showed that hyperoxidation to sulfonic acid (-SO3H) at Cys-51 residue was more advanced in a newfound hyperoxidized Prx2 compared to another hydrophobic hyperoxidized form previously identified. These results indicate that irreversible hyperoxidation of the Prx2 monomer in RBCs was easily caused by organic hydroperoxide but not H2O2. Thus, it is important to detect the hyperoxidation of Prx2 into sulfinic or sulfonic acid derivates of Cys-51 because hyperoxidized Prx2 is a potential marker of oxidative injury caused by organic hydroperoxides in human RBCs.


Assuntos
Eritrócitos/metabolismo , Peróxido de Hidrogênio/metabolismo , Peróxidos/metabolismo , Peroxirredoxinas/metabolismo , Adulto , Cromatografia de Fase Reversa , Cisteína/química , Cisteína/metabolismo , Dissulfetos/química , Dissulfetos/metabolismo , Humanos , Peróxido de Hidrogênio/química , Pessoa de Meia-Idade , Oxidantes/química , Oxidantes/metabolismo , Oxirredução , Peróxidos/química , Peroxirredoxinas/química , Ácidos Sulfínicos/química , Ácidos Sulfínicos/metabolismo , Ácidos Sulfônicos/química , Ácidos Sulfônicos/metabolismo , Adulto Jovem , terc-Butil Hidroperóxido/química , terc-Butil Hidroperóxido/metabolismo
3.
Biochem Biophys Res Commun ; 495(1): 116-123, 2018 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-29107693

RESUMO

WD repeat-containing protein 74 (WDR74), a nucleolar-localized protein, is the mammalian ortholog of Nsa1, a 60S ribosome assembly factor in yeast. We previously showed that WDR74 associates with MTR4, the nuclear exosome-assisting RNA helicase, whose dissociation is prohibited by an ATPase-deficient mutant of the AAA-type chaperone NVL2. However, the functions and regulation of WDR74 during ribosome biogenesis in cooperation with NVL2 remains unknown. Here, we demonstrated that knockdown of WDR74 leads to significant defects in the pre-rRNA cleavage within the internal transcribed spacer 1 (ITS1), occurring in an early stage of the processing pathway. Interestingly, when the dissociation of WDR74 from the MTR4-containing exonuclease complex was impaired upon expression of the mutant NVL2, the same processing defect, with partial migration of WDR74 from the nucleolus towards the nucleoplasm, was observed. In the nucleoplasm, an increased interaction between WDR74 and MTR4 was detected by in situ proximity ligation assay. Therefore, the dissociation of WDR74 from MTR4 in a late stage of rRNA synthesis is thought to be required for appropriate maturation of the pre-60S particles. These results suggest that the spatiotemporal regulation of ribosome biogenesis in the nucleolus is mediated by the ATPase activity of NVL2.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas de Transporte/metabolismo , Precursores de RNA/metabolismo , ATPases Associadas a Diversas Atividades Celulares/genética , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/genética , Nucléolo Celular/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Transporte Proteico , RNA Helicases/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA , Ribossomos/metabolismo
4.
Biochem Biophys Res Commun ; 467(3): 534-40, 2015 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-26456651

RESUMO

Nuclear VCP-like 2 (NVL2) is a chaperone-like nucleolar ATPase of the AAA (ATPase associated with diverse cellular activities) family, which exhibits a high level of amino acid sequence similarity with the cytosolic AAA-ATPase VCP/p97. These proteins generally act on macromolecular complexes to stimulate energy-dependent release of their constituents. We previously showed that NVL2 interacts with RNA processing/degradation machinery containing an RNA helicase MTR4/DOB1 and an exonuclease complex, nuclear exosome, and involved in the biogenesis of 60S ribosomal subunits. These observations implicate NVL2 as a remodeling factor for the MTR4-exosome complex during the maturation of pre-ribosomal particles. Here, we used a proteomic screen and identified a WD repeat-containing protein 74 (WDR74) as a factor that specifically dissociates from this complex depending on the ATPase activity of NVL2. WDR74 shows weak amino acid sequence similarity with the yeast ribosome biogenesis protein Nsa1 and is co-localized with NVL2 in the nucleolus. Knockdown of WDR74 decreases 60S ribosome levels. Taken together, our results suggest that WDR74 is a novel regulatory protein of the MTR4-exsosome complex whose interaction is regulated by NVL2 and is involved in ribosome biogenesis.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Nucléolo Celular/metabolismo , Exossomos/metabolismo , RNA Helicases/metabolismo , ATPases Associadas a Diversas Atividades Celulares , Linhagem Celular , Humanos , Proteínas de Ligação a RNA , Ribossomos/metabolismo
5.
Biochem Biophys Res Commun ; 464(3): 780-6, 2015 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-26166824

RESUMO

Nuclear VCP-like 2 (NVL2) is a member of the chaperone-like AAA-ATPase family and is involved in the biosynthesis of 60S ribosomal subunits in mammalian cells. We previously showed the interaction of NVL2 with a DExD/H-box RNA helicase MTR4/DOB1, which is a known cofactor for an exoribonuclease complex, the exosome. This finding implicated NVL2 in RNA metabolic processes during ribosome biogenesis. In the present study, we found that a series of mutations within the ATPase domain of NVL2 causes a defect in pre-rRNA processing into mature 28S and 5.8S rRNAs. Co-immunoprecipitation analysis showed that NVL2 was associated with the nuclear exosome complex, which includes RRP6 as a nucleus-specific catalytic subunit. This interaction was prevented by depleting either MTR4 or RRP6, indicating their essential role in mediating this interaction with NVL2. Additionally, knockdown of MPP6, another cofactor for the nuclear exosome, also prevented the interaction by causing MTR4 to dissociate from the nuclear exosome. These results suggest that NVL2 is involved in pre-rRNA processing by associating with the nuclear exosome complex and that MPP6 is required for maintaining the integrity of this rRNA processing complex.


Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas Nucleares/metabolismo , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , ATPases Associadas a Diversas Atividades Celulares , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Substituição de Aminoácidos , Núcleo Celular/metabolismo , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Modelos Biológicos , Mutagênese Sítio-Dirigida , Mutação , Estrutura Terciária de Proteína , RNA Helicases/metabolismo , Interferência de RNA , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo
6.
Leuk Res ; 138: 107454, 2024 03.
Artigo em Inglês | MEDLINE | ID: mdl-38452534

RESUMO

Adult T-cell leukemia/lymphoma (ATL), caused by human T-cell leukemia virus type-1 (HTLV-1) infection, is a malignant hematologic cancer that remains difficult to cure. We herein established a biomarker identification strategy based on the total cell proteomics of cultured ATL cells to search for novel ATL biomarkers. Four protocols with a combination of selected conditions based on lysis buffers and addition agents for total cell proteomics were used for a differential analysis between the ATL cell group (consisting of 11 cell lines), HTLV-1-infected cell group (consisting of 6 cell lines), and HTLV-1-negative cell group (consisting of 6 cell lines). In the analysis, we identified 24 and 27 proteins that were significantly increased (ratio ≥2.0, p < 0.05) and decreased (ratio ≤ 0.5, p < 0.05), respectively, in the ATL group. Previously reported CCL3 and CD30/TNFRSF8 were confirmed to be among significantly increased proteins. Furthermore, correlation analysis between identified proteins and Tax suggested that RASSF2 and GORASP2 were candidates of novel Tax-regulated factors. The biomarker identification strategy established herein is expected to contribute to the identification of biomarkers for ATL and other diseases.


Assuntos
Vírus Linfotrópico T Tipo 1 Humano , Leucemia-Linfoma de Células T do Adulto , Linfoma , Adulto , Humanos , Proteômica , Vírus Linfotrópico T Tipo 1 Humano/metabolismo , Biomarcadores , Digestão , Produtos do Gene tax/metabolismo , Proteínas da Matriz do Complexo de Golgi
7.
Antioxidants (Basel) ; 11(12)2022 Dec 17.
Artigo em Inglês | MEDLINE | ID: mdl-36552694

RESUMO

Peroxiredoxin (Prx) 2 in red blood cells (RBCs) reacts with various reactive oxygen species and changes to hyperoxidized Prx2 (Prx2-SO2/3). Therefore, Prx2 may serve as an indicator of oxidative stress in vivo. This study aimed to analyze Prx2-SO2/3 levels in clinical samples to examine whether the oxidation state of Prx2 in human RBCs reflects the pathological condition of oxidative stress diseases. We first focused on obstructive sleep apnea (OSA), a hypoxic stress-induced disease of the respiratory system, and investigated the levels of Prx2-SO2/3 accumulated in the RBCs of OSA patients. In measurements on a small number of OSA patients and healthy subjects, levels of Prx2-SO2/3 accumulation in patients with OSA were clearly increased compared to those in healthy subjects. Hence, we proceeded to validate these findings with more samples collected from patients with OSA. The results revealed significantly higher levels of erythrocytic Prx2-SO2/3 in patients with OSA than in healthy subjects, as well as a positive correlation between the severity of OSA and Prx2-SO2/3 levels in the RBCs. Moreover, we performed a chromatographic study to show the structural changes of Prx2 due to hyperoxidation. Our findings demonstrated that the Prx2-SO2/3 molecules in RBCs from patients with OSA were considerably more hydrophilic than the reduced form of Prx2. These results implicate Prx2-SO2/3 as a promising candidate biomarker for OSA.

8.
Int J Biochem Cell Biol ; 132: 105919, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33422691

RESUMO

The AAA-ATPase NVL2 associates with an RNA helicase MTR4 and the nuclear RNA exosome in the course of ribosome biogenesis. In our proteomic screen, we had identified a ribosome biogenesis factor WDR74 as a MTR4-interacting partner, whose dissociation is stimulated by the ATP hydrolysis of NVL2. In this study, we report the identification of splicing factor 30 (SPF30), another MTR4-interacting protein with a similar regulatory mechanism. SPF30 is a pre-mRNA splicing factor harboring a Tudor domain in its central region, which regulates various cellular events by binding to dimethylarginine-modified proteins. The interaction between SPF30 and the exosome core is mediated by MTR4 and RRP6, a catalytic component of the nuclear exosome. The N- and C-terminal regions, but not the Tudor domain, of SPF30 are involved in the association with MTR4 and the exosome. The knockdown of SPF30 caused subtle delay in the 12S pre-rRNA processing to mature 5.8S rRNA, even though no obvious effect was observed on the ribosome subunit profile in the cells. Shotgun proteomic analysis to search for SPF30-interacting proteins indicated its role in ribosome biogenesis, pre-mRNA splicing, and box C/D snoRNA biogenesis. These results suggest that SPF30 collaborates with the MTR4-exosome machinery to play a functional role in multiple RNA metabolic pathways, some of which may be regulated by the ATP hydrolysis of NVL2.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Exossomos/genética , RNA Helicases/metabolismo , Fatores de Processamento de RNA/química , Fatores de Processamento de RNA/metabolismo , Estabilidade de RNA , Proteínas do Complexo SMN/química , Proteínas do Complexo SMN/metabolismo , Humanos , Ligação Proteica , Domínios Proteicos
9.
Antioxidants (Basel) ; 10(4)2021 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-33917901

RESUMO

Recent studies have shown that carbonyl stress is a causative factor of schizophrenia, categorized as carbonyl stress-related schizophrenia (CS-SCZ). However, the correlation between carbonyl stress and the pathogenesis of this disease is not well established. In this study, glyoxalase 1(Glo1)-knockout and vitamin B6-deficient mice (KO/VB6 (-) mice), which are susceptible to methylglyoxal (MGO)-induced oxidative damages, were used as a CS-SCZ model to analyze MGO-modified protein and the carbonyl stress status in the brain. A comparison between Wild/VB6(+) mice and KO/VB6(-) mice for accumulated carbonyl proteins levels, with several advanced glycation end products (AGEs) in the brain, revealed that carbonyl protein levels with the Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl) ornithine (MG-H1) moiety were significantly increased in the hippocampus, prefrontal cortex, striatum, cerebral cortex, and brainstem regions of the brain in KO/VB6(-) mice. Moreover, two-dimensional electrophoresis and Liquid chromatography-tandem mass spectrometry analysis showed MG-H1-modified arginine residues in mitochondrial creatine kinase, beta-adrenergic receptor kinase 1, and T-complex protein in the hippocampus region of KO/VB6(-) mice, but not in Wild/VB6(+) mice. In particular, MG-H1 modification of mitochondrial creatine kinase was quite notable. These results suggest that further studies focusing on MG-H1-modified and accumulated proteins in the hippocampus may reveal the onset mechanism of CS-SCZ induced by MGO-induced oxidative damages.

10.
Med Mycol J ; 59(3): E47-E52, 2018.
Artigo em Japonês | MEDLINE | ID: mdl-30175812

RESUMO

Interactions between virulence factors of pathogens and host responses play an important role in the establishment of infection by microbes. We focused on interactions between Cryptococcus neoformans proteins and heparin, which is abundant on host epithelial cells. Surface proteins were extracted and analyzed. Fractions from anion-exchange column chromatography interacted with heparin in surface plasmon resonance analyses. Heparin-binding proteins were purified and then separated by gel electrophoresis; and were identified as transaldolase, glutathione-disulfide reductase, and glyoxal oxidase. These results imply that multifunctional molecules on C. neoformans cells, such as those involved in heparin binding, may play roles in adhesion that trigger responses in the host.


Assuntos
Oxirredutases do Álcool/isolamento & purificação , Oxirredutases do Álcool/metabolismo , Proteínas da Membrana Bacteriana Externa/isolamento & purificação , Proteínas da Membrana Bacteriana Externa/metabolismo , Cryptococcus neoformans/citologia , Cryptococcus neoformans/metabolismo , Glutationa Redutase/isolamento & purificação , Glutationa Redutase/metabolismo , Heparina/metabolismo , Transaldolase/isolamento & purificação , Transaldolase/metabolismo , Fatores de Virulência/isolamento & purificação , Fatores de Virulência/metabolismo , Cryptococcus neoformans/patogenicidade , Ligação Proteica
11.
Hum Cell ; 30(4): 279-289, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28434171

RESUMO

Peroxiredoxin 2 (Prx2) is a redox enzyme that is abundantly expressed in red blood cells (RBCs) and has been the focus of clinical attention for monitoring the oxidative status. We previously developed a method to quantify the reduced and hyperoxidized forms of Prx2 in human RBCs using reverse-phase high-performance liquid chromatography (HPLC). In the present study, we investigated the hyperoxidative status of Prx2 at the molecular level in a post-translational modification analysis using a liquid chromatography-tandem mass spectrometry (LC-MS/MS) system. The LC-MS/MS analysis of the trypsin digests of Prx2 fractionated by reverse-phase HPLC demonstrated that the cysteine-51 residue (Cys-51) of the protein was modified with the hyperoxidative functional groups, sulfinic acid (-SO2H) and sulfonic acid (-SO3H), in RBCs treated with tert-butyl hydroperoxide (t-BHP). Furthermore, a selected ion monitoring (SIM) analysis quantitatively showed that sulfinic acid- and sulfonic acid-induced modifications in Prx2 Cys-51 were increased by the treatment with the oxidant. It was demonstrated that the peroxidatic cysteine of Prx2 separated using our HPLC system for oxidative monitoring was hyperoxidized into sulfinic acid and sulfonic acid in RBCs under an oxidative stress condition.


Assuntos
Cisteína/metabolismo , Eritrócitos/metabolismo , Estresse Oxidativo , Peroxirredoxinas/metabolismo , Ácidos Sulfínicos/metabolismo , Ácidos Sulfônicos/metabolismo , terc-Butil Hidroperóxido/farmacologia , Adulto , Células Cultivadas , Cromatografia Líquida de Alta Pressão , Humanos , Espectrometria de Massas , Oxirredução , Adulto Jovem
12.
FEBS Lett ; 590(17): 2963-72, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27434818

RESUMO

In yeast, the Trf4/5-Air1/2-Mtr4 polyadenylation (TRAMP) complex acts as a cofactor for the nuclear exosome to promote degradation of various RNAs. However, the corresponding machinery in mammals is less characterized. We analyzed the interactions of the human TRAMP-like proteins, PAPD5, ZCCHC7, and MTR4, with the nuclear exosome. PAPD5 and ZCCHC7 exhibited mutual interactions in presence of the exosome catalytic subunit RRP6, whereas MTR4 was dispensable for their assembly. Furthermore, the human TRAMP-like proteins were involved in the RRP6-catalyzed turnover of pre-rRNA 5'ETS fragments. These results suggest the significant role for RRP6 in the assembly of TRAMP-like proteins during nucleolar RNA surveillance.


Assuntos
RNA Helicases/metabolismo , RNA Nucleotidiltransferases/metabolismo , Estabilidade de RNA/genética , Fatores de Transcrição/metabolismo , Exorribonucleases/genética , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Humanos , RNA Helicases/genética , RNA Nucleotidiltransferases/genética , Precursores de RNA/genética , Processamento Pós-Transcricional do RNA , Saccharomyces cerevisiae/genética , Fatores de Transcrição/genética
13.
World J Hepatol ; 6(12): 870-9, 2014 Dec 27.
Artigo em Inglês | MEDLINE | ID: mdl-25544874

RESUMO

Hepatitis C virus (HCV) is a major cause of viral hepatitis and currently infects approximately 170 million people worldwide. An infection by HCV causes high rates of chronic hepatitis (> 75%) and progresses to liver cirrhosis and hepatocellular carcinoma ultimately. HCV can be eliminated by a combination of pegylated α-interferon and the broad-spectrum antiviral drug ribavirin; however, this treatment is still associated with poor efficacy and tolerability and is often accompanied by serious side-effects. While some novel direct-acting antivirals against HCV have been developed recently, high medical costs limit the access to the therapy in cost-sensitive countries. To search for new natural anti-HCV agents, we screened local agricultural products for their suppressive activities against HCV replication using the HCV replicon cell system in vitro. We found a potent inhibitor of HCV RNA expression in the extracts of blueberry leaves and then identified oligomeric proanthocyanidin as the active ingredient. Further investigations into the action mechanism of oligomeric proanthocyanidin suggested that it is an inhibitor of heterogeneous nuclear ribonucleoproteins (hnRNPs) such as hnRNP A2/B1. In this review, we presented an overview of functional foods and ingredients efficient for HCV infection, the chemical structural characteristics of oligomeric proanthocyanidin, and its action mechanism.

14.
Hum Cell ; 27(2): 68-77, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24323765

RESUMO

Adult T-cell leukemia/lymphoma (ATL) is a fatal malignancy caused by infection with human T-lymphotropic virus type-1 and there is no accepted curative therapy for ATL. We searched for biological active substances for the prevention and treatment of ATL from several species of herbs. The ATL cell growth-inhibitory activity and apoptosis assay showed that carnosol, which is an ingredient contained in rosemary (Rosmarinus officinalis), induced apoptosis in ATL cells. Next, to investigate the apoptosis-inducing mechanism of carnosol, we applied proteomic analysis using fluorescent two-dimensional differential gel electrophoresis and mass spectrometry. The proteomic analysis showed that the expression of reductases, enzymes in glycolytic pathway, and enzymes in pentose phosphate pathway was increased in carnosol-treated cells, compared with untreated cells. These results suggested that carnosol affected the redox status in the cells. Further, the quantitative analysis of glutathione, which plays the central role for the maintenance of intracellular redox status, indicated that carnosol caused the decrease of glutathione in the cells. Further, N-acetyl-L-cystein, which is precursor of glutathione, canceled the efficiency of carnosol. From these results, it was suggested that the apoptosis-inducing activity of carnosol in ATL cells was caused by the depletion of glutathione.


Assuntos
Abietanos/farmacologia , Eletroforese em Gel Bidimensional/métodos , Glutationa/metabolismo , Glutationa/fisiologia , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/patologia , Proteômica/métodos , Rosmarinus/química , Abietanos/antagonistas & inibidores , Abietanos/uso terapêutico , Acetilcisteína/farmacologia , Células Cultivadas , Glutationa/deficiência , Glicólise/efeitos dos fármacos , Humanos , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Espectrometria de Massas , Terapia de Alvo Molecular , Oxirredução/efeitos dos fármacos , Via de Pentose Fosfato/efeitos dos fármacos , Fitoterapia
15.
PLoS One ; 6(12): e29074, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-22205997

RESUMO

To find a new biomarker of Tay-Sachs disease and Sandhoff disease. The lyso-GM2 ganglioside (lyso-GM2) levels in the brain and plasma in Sandhoff mice were measured by means of high performance liquid chromatography and the effect of a modified hexosaminidase (Hex) B exhibiting Hex A-like activity was examined. Then, the lyso-GM2 concentrations in human plasma samples were determined. The lyso-GM2 levels in the brain and plasma in Sandhoff mice were apparently increased compared with those in wild-type mice, and they decreased on intracerebroventricular administration of the modified Hex B. The lyso-GM2 levels in plasma of patients with Tay-Sachs disease and Sandhoff disease were increased, and the increase in lyso-GM2 was associated with a decrease in Hex A activity. Lyso-GM2 is expected to be a potential biomarker of Tay-Sachs disease and Sandhoff disease.


Assuntos
Gangliosídeo G(M2)/análogos & derivados , Doença de Sandhoff/metabolismo , Doença de Tay-Sachs/metabolismo , Adulto , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Encéfalo/metabolismo , Proteína Ativadora de G(M2)/deficiência , Gangliosídeo G(M2)/sangue , Gangliosídeo G(M2)/metabolismo , Hexosaminidases/sangue , Humanos , Lactente , Camundongos , Doença de Sandhoff/sangue , Doença de Sandhoff/enzimologia , Doença de Tay-Sachs/sangue , Doença de Tay-Sachs/enzimologia
16.
Hepatol Res ; 40(4): 438-45, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-20236361

RESUMO

AIM: Oxidative stress is involved in the progression of non-alcoholic steatohepatitis (NASH). However, there are few biomarkers that are easily measured and accurately reflect the disease states. The aim of this study was to identify novel oxidative stress markers using the 2-nitrobenzenesulfenyl (NBS) stable isotope labeling method and to examine the clinical utility of these diagnostic markers for NASH. METHODS: Proteins extracted from phosphate buffered saline- and hydrogen peroxide-loaded human primary hepatocyte were labeled with the [(12)C]- and [(13)C]-NBS reagents, respectively. Pairs of peaks with 6-Da differences in which the [(13)C]-NBS labeling was more intense than the [(12)C]-NBS labeling were detected by MALDI-TOF/MS and identified by MS/MS ion searching. RESULTS: Four pairs of peaks, m/z 1705-1711, m/z 1783-1789, m/z 1902-1908 and m/z 2790-2796, were identified as cytochrome c oxidase VIb (COX6B), liver carboxylesterase 1 (CES1), carbamoyl-phosphate synthase 1 (CPS1) and superoxide dismutase (MnSOD), respectively. Furthermore, serum MnSOD protein levels were significantly higher in NASH patients than in simple steatosis (SS) patients. The serum MnSOD levels tended to increase in parallel with the stage of fibrosis. CONCLUSION: The NBS labeling technique was useful to identify biomarkers. Serum MnSOD may be a useful biomarker that can distinguish between SS and NASH.

17.
J Biol Chem ; 284(32): 21165-76, 2009 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-19531480

RESUMO

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma. While searching for new natural anti-HCV agents in agricultural products, we found a potent inhibitor of HCV RNA expression in extracts of blueberry leaves when examined in an HCV subgenomic replicon cell culture system. This activity was observed in a methanol extract fraction of blueberry leaves and was purified by repeated fractionations in reversed-phase high-performance liquid chromatography. The final purified fraction showed a 63-fold increase in specific activity compared with the initial methanol extracts and was composed only of carbon, hydrogen, and oxygen. Liquid chromatography/mass-ion trap-time of flight analysis and butanol-HCl hydrolysis analysis of the purified fraction revealed that the blueberry leaf-derived inhibitor was proanthocyanidin. Furthermore, structural analysis using acid thiolysis indicated that the mean degree of polymerization of the purified proanthocyanidin was 7.7, consisting predominantly of epicatechin. Proanthocyanidin with a polymerization degree of 8 to 9 showed the greatest potency at inhibiting the expression of subgenomic HCV RNA. Purified proanthocyanidin showed dose-dependent inhibition of expression of the neomycin-resistant gene and the NS-3 protein gene in the HCV subgenome in replicon cells. While characterizing the mechanism by which proanthocyanidin inhibited HCV subgenome expression, we found that heterogeneous nuclear ribonucleoprotein A2/B1 showed affinity to blueberry leaf-derived proanthocyanidin and was indispensable for HCV subgenome expression in replicon cells. These data suggest that proanthocyanidin isolated from blueberry leaves may have potential usefulness as an anti-HCV compound by inhibiting viral replication.


Assuntos
Farmacorresistência Viral , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Hepacivirus/genética , Extratos Vegetais/metabolismo , Folhas de Planta/metabolismo , Proantocianidinas/metabolismo , RNA Viral , Replicação Viral/efeitos dos fármacos , Mirtilos Azuis (Planta) , Cromatografia Líquida de Alta Pressão , Cromatografia Líquida/métodos , Concentração Inibidora 50 , Espectrometria de Massas/métodos , Neomicina/farmacologia , Polímeros/química
18.
Cancer Lett ; 271(1): 167-77, 2008 Nov 18.
Artigo em Inglês | MEDLINE | ID: mdl-18691810

RESUMO

Adult T-cell leukemia (ATL) is a fatal malignancy caused by infection with human T lymphotropic virus type-1 (HTLV-1). To search for a new biomarker of ATL, we analyzed sera from ATL patients using ProteinChip arrays. The spectral comparison of ATL patients with HTLV-1 carriers and healthy volunteers showed that the intensities of five peaks (1779, 1866, 2022, 4467, and 8930 m/z) were significantly increased in ATL, while those of four peaks (4067, 4151, 8130, and 8597 m/z) were decreased. From these differentially expressed peaks, we chose peaks of 1779, 1866, and 2022 m/z as biomarker candidates of ATL. MS/MS ion search using tandem mass spectrometry and immunoprecipitation assay using anti-C3 antibody showed that factors derived from these candidate peaks were identified as C3f, which is a component of the complement system and a fragment of complement C3. These results indicate that the complement system was activated in ATL. Further analysis of markers specific to the activation pathways (classical, alternative, and lectin pathways) in the complement system showed that the serum concentration of the marker of the lectin pathway was significantly higher in ATL patients. These results suggest that activation of the complement system in ATL occurs mainly through the lectin pathway.


Assuntos
Ativação do Complemento , Lectinas/metabolismo , Leucemia de Células T/metabolismo , Proteômica , Espectrometria de Massas em Tandem/métodos , Adulto , Estudos de Casos e Controles , Perfilação da Expressão Gênica , Humanos , Imunoprecipitação , Leucemia de Células T/sangue , Proteínas de Neoplasias/sangue , Proteínas de Neoplasias/genética
19.
Intervirology ; 45(3): 125-35, 2002.
Artigo em Inglês | MEDLINE | ID: mdl-12403916

RESUMO

While investigating myoblast fusion using enveloped viruses, we unexpectedly found that the production of hemagglutinating virus of Japan (HVJ; Sendai virus) was suppressed temperature dependently in quail myoblasts transformed with a temperature-sensitive Rous sarcoma virus, which proliferate at 35.5 degrees but differentiate at 41 degrees; viral production was normal at 35.5 degrees but suppressed at 41 degrees irrespective of the species of host cells. The production of some viruses, i.e. measles virus, influenza virus, herpes simplex virus type 1 and poliovirus, was also markedly suppressed at 41 degrees, suggesting that a temperature of 41 degrees affects viral infection generally. To clarify the mechanism of the suppression, the infectious pattern of HVJ was examined both at 37 degrees and at 41 degrees in LLC-MK2 cells. The synthesis of HVJ-specific proteins was inhibited at the transcriptional level at 41 degrees, although viral penetration by envelope fusion was not affected. The transcriptional inhibition was also seen in quail fibroblasts, which do not express a 70-kD heat shock protein (HSP70), suggesting that HSP70 is dispensable for the inhibition of viral gene transcription at 41 degrees. Further, when the infected cells were incubated at 41 degrees after the viral proteins had been synthesized at 37 degrees, viral production was also inhibited. Immunofluorescent staining of the cells exposed to 41 degrees showed that HVJ envelope proteins formed large aggregates on the cell surface, into which both M and NP proteins were assembled. Under the electron microscope, HVJ virions appeared normal even at 41 degrees, but were detected in clusters on the cell surface, unlike at 37 degrees. These observations suggested that the release of HVJ virions from the cell surface was inhibited for some reason at 41 degrees. Consequently, it was indicated that two steps, viral gene transcription and the release of virions, were inhibited at 41 degrees.


Assuntos
Mioblastos/virologia , Infecções por Respirovirus/virologia , Vírus Sendai/fisiologia , Vírus Sendai/patogenicidade , Temperatura , Animais , Linhagem Celular , Linhagem Celular Transformada , Proteínas de Choque Térmico HSP70/metabolismo , Microscopia Eletrônica , Codorniz , Transcrição Gênica , Proteínas Virais/metabolismo , Vírion/metabolismo , Montagem de Vírus
20.
Virology ; 306(2): 244-53, 2003 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-12642098

RESUMO

The influenza virus copies its genomic RNA in the nuclei of host cells, but the viral particles are formed at the plasma membrane. Thus, the export of new genome from the nucleus into the cytoplasm is essential for viral production. Several viral proteins, such as nucleoprotein (NP) and RNA polymerases, synthesized in the cytoplasm, are imported into the nucleus, and form viral ribonucleoprotein (vRNP) with new genomic RNA. vRNP is then exported into the cytoplasm from the nucleus to produce new viral particles. M1, a viral matrix protein, is suggested to participate in the nuclear export of vRNP. It was found unexpectedly that the production of influenza virus was suppressed in MDCK cells at 41 degrees C, although viral proteins were synthesized and the cytopathic effect was observed in host cells. Indirect immunofluorescent staining with anti-NP or M1 monoclonal antibody showed that NP and M1 remained in the nuclei of infected cells at 41 degrees C, suggesting that a suppression of viral production was caused by inhibition of the nuclear export of these proteins. The cellular machinery for nuclear export depending on CRM1, which mediates the nuclear export of influenza viral RNP, functioned normally at 41 degrees C. Glycerol-density gradient centrifugation demonstrated that vRNP also formed normally at 41 degrees C. However, an examination of the interaction between vRNP and M1 by immunoprecipitation indicated that M1 did not associate with vRNP at 41 degrees C, suggesting that the association is essential for the nuclear export of vRNP. Furthermore, when infected cells incubated at 41 degrees C were cultured at 37 degrees C, the interaction between vRNP and M1 was no longer detected even at 37 degrees C. The results suggest that M1 synthesized at 41 degrees C is unable to interact with vRNP and the dissociation of M1 from vRNP is one of the reasons that the transfer of vRNP into the cytoplasm from the nucleus is prevented at 41 degrees C.


Assuntos
Vírus da Influenza A/metabolismo , Nucleoproteínas/metabolismo , Proteínas de Ligação a RNA , Ribonucleoproteínas/metabolismo , Proteínas do Core Viral/metabolismo , Proteínas da Matriz Viral/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Núcleo Celular/virologia , Citoplasma/virologia , Cães , Vírus da Influenza A/genética , Vírus da Influenza A/fisiologia , Substâncias Macromoleculares , Proteínas do Nucleocapsídeo , Temperatura , Replicação Viral
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