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PURPOSE: The aim of this study was to examine the effect of vibration on motor unit (MU) firing behavior and physical performance of antagonist muscles in healthy young adult males. METHODS: Fourteen males (age = 24.3 ± 3.6 years) were included in this study. There were two conditions, one in which participants received 80 Hz vibration in the distal tendon of the hamstring for 30 s and the control condition (no vibration). High-density surface electromyography (HD-SEMG) signals and maximal voluntary contraction (MVC) of knee extensor muscles were evaluated before and after the respective conditions and recorded from the vastus lateralis muscle during submaximal ramp-up and sustained contractions at 30% MVC. Convolution blind source separation was used to decompose the HD-SEMG signals into individual MU firing behaviors. RESULTS: In total, 739 MUs were detected (control; 360 MUs and vibration; 379 MUs), and a total of 312 matched MUs were identified across both submaximal contraction conditions (control: 150 MUs; vibration: 162 MUs). Vibration significantly increased the discharge rate (p = 0.047) and decreased the recruitment threshold before and after intervention (p = 0.001) but not in the control condition. Furthermore, the recruitment threshold is a factor that influences discharge rate. Significant correlations were observed between the recruitment threshold and both the ∆ discharge rate and the ∆ recruitment threshold under the vibration condition (p < 0.001). CONCLUSION: Vibration increased in the discharge rate and decreased the recruitment threshold of the antagonist muscle. These findings suggested that vibration contributes to immediate changes in the neural control of antagonist muscles.
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The purpose of this study was to examine the moral concerns and problem-solving behavior for outpatient nurses in palliative cancer care. The target of this study was 284 outpatient nurses(22.9%)out of 1,241 respondents. As a result, it was concluded that outpatient nurses providing palliative cancer care have higher ethical concerns than nurses working in acute care hospitals. In addition, the more moral concerns there were, the more nurses manage their care according to patient's individual circumstances. In the future, it is necessary to provide education on the moral concerns of outpatient nurses and the problem-solving behavior for nurses so that patients in the final stages of life and their families can spend a better time.
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Principios Morales , Cuidados Paliativos , Solución de Problemas , Femenino , Humanos , Persona de Mediana Edad , Adulto , Masculino , Pacientes Ambulatorios , Encuestas y Cuestionarios , Atención AmbulatoriaRESUMEN
Fibroblast growth factor 23 (FGF-23), a hormone mainly secreted by osteocytes and osteoblasts, regulates phosphate and vitamin D levels. However, the in vivo significance of FGF-23 is not fully elucidated. This case report describes a 12-year-old girl with systemic lupus erythematosus (SLE), lupus nephritis, and an elevated serum FGF-23 level. The patient was treated with active vitamin D and oral sodium phosphate medications to manage low serum phosphate levels (2.2 mg/dL). Magnetic resonance imaging (MRI) revealed a high-intensity area in the left femur, but somatostatin receptor scintigraphy images did not indicate tumor-induced osteomalacia. SLE treatment using mycophenolate mofetil (1500 mg/day) was initiated, and serum complements levels increased as FGF-23 level increased. Serum FGF-23 level gradually decreased as urinary protein levels decreased after treatment with steroids; however, there was no change in the high-intensity area on MRI. Recent studies have reported that serum FGF-23 level is associated with iron deficiency and inflammation; yet, the mechanism related to these associations is not fully elucidated. The findings from this case suggest that elevated serum FGF-23 levels noted in our patient were related to silent lupus nephritis and lupus nephritis activity.
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Lupus Eritematoso Sistémico , Nefritis Lúpica , Niño , Femenino , Factor-23 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos , Humanos , Lupus Eritematoso Sistémico/complicaciones , Nefritis Lúpica/complicaciones , Nefritis Lúpica/diagnóstico , Fosfatos , Vitamina DRESUMEN
SUMMARY: Ammonium acid urate (AAU) crystals are rare in industrialized countries. Furthermore, the number of children with diabetic ketoacidosis (DKA) who develop severe acute kidney injury (AKI) after hospitalization is small. We encountered two patients with AKI caused by AAU crystals during the recovery phase of DKA upon admission. They were diagnosed with severe DKA and hyperuricemia. Their urine volume decreased and AKI developed several days after hospitalization; however, acidosis improved in both patients. Urine sediment analysis revealed AAU crystals. They were treated with urine alkalization and diuretics. Excretion of ammonia in the urine and urine pH levels increased after treatment of DKA, which resulted in the formation of AAU crystals. In patients with severe DKA, the urine and urine sediment should be carefully examined as AAU can form in the recovery phase of DKA. LEARNING POINTS: Ammonium acid urate crystals could be formed in the recovery phase of diabetic ketoacidosis. Diabetic ketoacidosis patients may develop acute kidney injury caused by ammonium acid urate crystals. Urine and urine sediment should be carefully checked in patients with severe DKA who present with hyperuricemia and volume depletion.
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We retrospectively analyzed endocrine late effects in 81 childhood cancer survivor (CCS) patients who had been referred to our endocrinology department in Chiba Children's Hospital between January 1, 2008 and December 31, 2016. Among 69 eligible patients (33 male, 36 female), endocrine late effects were identified in 56 patients (81.1%). The median age at the last visit to our endocrinology department was 17.4 years (range: 7.1-35.3 years). The most common primary cancer was acute lymphoblastic leukemia (22 patients, 31.8%). Forty-four patients (64%) were treated using radiation therapy. A primary brain tumor and high doses (≥6 g/m2) of cyclophosphamide were significantly associated with growth hormone deficiency (GHD). Our present study suggests that high doses of cyclophosphamide is a risk factor for GHD. Adult heights and pubertal growth spurts of patients treated with radiation therapy were significantly lower than patients not treated with radiation therapy. Our retrospective study reconfirmed that hematopoietic stem cell transplantation and chronic graft versus host disease (GVHD) were associated with elevated risks of primary hypothyroidism. However, it is unclear whether GVHD induces thyroid dysfunction. Gonadal radiation and busulfan were associated with primary hypogonadism as reported in previous studies. We found high doses of cyclophosphamide to be involved in pituitary disorders. We suggest that pediatric endocrinologists should discuss the potential effects of radiation therapy on adult height and pubertal growth spurt in CCS patients. Moreover, patients who have been treated with high doses of cyclophosphamide or have chronic GVHD require long-term follow-up for endocrine late effects.
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Supervivientes de Cáncer , Trastornos del Crecimiento/epidemiología , Hormona de Crecimiento Humana/deficiencia , Hipertiroidismo/epidemiología , Hipogonadismo/epidemiología , Hipotiroidismo/epidemiología , Neoplasias/terapia , Pubertad Precoz/epidemiología , Adolescente , Adulto , Antineoplásicos/uso terapéutico , Neoplasias Encefálicas/terapia , Busulfano/uso terapéutico , Niño , Enfermedad Crónica , Ciclofosfamida/uso terapéutico , Femenino , Enfermedad Injerto contra Huésped/epidemiología , Neoplasias Hematológicas/terapia , Trasplante de Células Madre Hematopoyéticas/métodos , Histiocitosis de Células de Langerhans/terapia , Humanos , Japón/epidemiología , Efectos Adversos a Largo Plazo/epidemiología , Masculino , Neuroblastoma/terapia , Radioterapia/métodos , Estudios Retrospectivos , Rabdomiosarcoma/terapia , Factores de Riesgo , Adulto JovenRESUMEN
Maintenance hemodialysis outpatients must limit salt and water intake to maintain electrolyte balance and blood pressure. In Kawashima Hospital, nationally registered dietitians provide hemodialysis patients with monthly nutritional counseling. We investigated whether nutritional counseling affects interdialytic weight gain (IDWG) and blood pressure. We investigated 48 hemodialysis patients whose monthly average IDWG ratio to dry weight exceeded 5.1% and who had not had a long-term hospital admittance of > 1 month. After the 48-month nutritional counseling period, the IDWG ratio had improved in 37 of the patients (77.1%), significantly decreasing from 6.0±0.7 to 5.3±0.9%. Estimated salt and water intake decreased significantly from 13.3±2.7 to 11.8±2.4 g/day and 2528±455 to 2332±410 ml/day, respectively. During the intervention period, normalized protein catabolic rate and body mass index did not change substantially. Pre-hemodialysis systolic and diastolic blood pressures had significantly decreased from 149±19 to 134±18 mmHg, and 82±13 to 75±10 mmHg for 48 months after study initiation, respectively. The dosage of antihypertensive drugs had significantly decreased in the group that experienced improvement in the IDWG ratio. Long-term nutritional counseling by nationally registered dietitians may improve the IDWG ratio and blood pressure of hemodialysis patients by decreasing their salt and water intake. J. Med. Invest. 64: 129-135, February, 2017.
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Consejo , Estado Nutricional , Diálisis Renal , Adulto , Atención Ambulatoria , Antihipertensivos/administración & dosificación , Presión Sanguínea , Femenino , Humanos , Hipertensión/etiología , Hipertensión/prevención & control , Masculino , Persona de Mediana Edad , Terapia Nutricional , Diálisis Renal/efectos adversos , Aumento de PesoRESUMEN
Ribavirin (RBV) has been widely used as an antiviral reagent, specifically for patients with chronic hepatitis C. We previously demonstrated that adenosine kinase, which monophosphorylates RBV into the metabolically active form, is a key determinant for RBV sensitivity against hepatitis C virus RNA replication. However, the precise mechanism of RBV action and whether RBV affects cellular metabolism remain unclear. Analysis of liver gene expression profiles obtained from patients with advanced chronic hepatitis C treated with the combination of pegylated interferon and RBV showed that the adenosine kinase expression level tends to be lower in patients who are overweight and significantly decreases with progression to advanced fibrosis stages. In our effort to investigate whether RBV affects cellular metabolism, we found that RBV treatment under clinically achievable concentrations suppressed lipogenesis in hepatic cells. In this process, guanosine triphosphate depletion through inosine monophosphate dehydrogenase inhibition by RBV and adenosine monophosphate-activated protein kinase-related kinases, especially microtubule affinity regulating kinase 4, were required. In addition, RBV treatment led to the down-regulation of retinoid X receptor α (RXRα), a key nuclear receptor in various metabolic processes, including lipogenesis. Moreover, we found that guanosine triphosphate depletion in cells induced the down-regulation of RXRα, which was mediated by microtubule affinity regulating kinase 4. Overexpression of RXRα attenuated the RBV action for suppression of lipogenic genes and intracellular neutral lipids, suggesting that down-regulation of RXRα was required for the suppression of lipogenesis in RBV action. Conclusion: We provide novel insights about RBV action in lipogenesis and its mechanisms involving inosine monophosphate dehydrogenase inhibition, adenosine monophosphate-activated protein kinase-related kinases, and down-regulation of RXRα. RBV may be a potential reagent for anticancer therapy against the active lipogenesis involved in hepatocarcinogenesis. (Hepatology Communications 2017;1:550-563).
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BACKGROUND: Ribavirin (RBV) is a potential partner of interferon-based therapy and recently approved therapy using direct acting antivirals for patients with chronic hepatitis C. However, the precise mechanisms underlying RBV action against hepatitis C virus (HCV) replication are not yet understood. To clarify this point, we attempted to develop RBV-resistant cells from RBV-sensitive HCV RNA-replicating cells. METHODOLOGY/PRINCIPAL FINDINGS: By repetitive RBV (100 µM) treatment (10 weeks) of 3.5-year-cultured OL8 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates, dozens of colonies that survived RBV treatment were obtained. These colonies were mixed together and further treated with high doses of RBV (up to 200 µM). By such RBV treatment, we successfully established 12 RBV-survived genome-length HCV RNA-replicating cell lines. Among them, three representative cell lines were characterized. HCV RNA replication in these cells resisted RBV significantly more than that in the parental OL8 cells. Genetic analysis of HCV found several common and conserved amino acid substitutions in HCV proteins among the three RBV-resistant cell species. Furthermore, using cDNA microarray and quantitative RT-PCR analyses, we identified 5 host genes whose expression levels were commonly altered by more than four-fold among these RBV-resistant cells compared with the parental cells. Moreover, to determine whether viral or host factor contributes to RBV resistance, we developed newly HCV RNA-replicating cells by introducing total RNAs isolated from RBV-sensitive parental cells or RBV-resistant cells into the HCV RNA-cured-parental or -RBV-resistant cells using an electroporation method, and evaluated the degrees of RBV resistance of these developed cells. Consequently, we found that RBV-resistant phenotype was conferred mainly by host factor and partially by viral factor. CONCLUSIONS/SIGNIFICANCE: These newly established HCV RNA-replicating cell lines should become useful tools for further understanding the anti-HCV mechanisms of RBV.
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Antivirales/farmacología , Farmacorresistencia Viral , Hepacivirus/metabolismo , Hepatocitos/virología , Fenotipo , Ribavirina/farmacología , Replicación Viral , Técnicas de Cultivo de Célula/métodos , Línea Celular Tumoral , Genes Virales , Hepacivirus/efectos de los fármacos , Hepacivirus/fisiología , HumanosRESUMEN
Upon reacting 3',4'-unsaturated cytosine (8 and 9) and adenine nucleosides (13 and 14) with XeF(2)/BF3 · OEt(2), the respective novel 3',4'-difluoro-3'-deoxyribofuranosyl nucleosides (10-12 and 15-18) could be obtained. Formation of anti-adducts (11, 16 and 18) revealed that the fluorination involved oxonium ions as incipient intermediates. TBDMS-protected 3',4'-unsaturated adenosine provided the ß-face adducts as sole stereoisomers whereas α-face-selectivity was observed with the TBDPS-protected adenosine 14. The evaluation of the novel 3'-deoxy-3',4'-difluororibofuranosylcytosine-(19-21) and adenine nucleosides (22-25) against antitumor and antiviral activities revealed that 3',4'-difluorocordycepin (24) was found to possess anti-HCV activity. The SI of 24 was comparable to that of the anti-HCV drug ribavirin. However, sofosbuvir, FDA-approved novel anti-HCV drug, showed better SI value. Our finding revealed that the introduction of the fluoro-substituent into the 4'-position of cordycepin derivatives decreased the cytotoxicity to the host cell with retention of the antiviral activity.
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Antivirales/química , Antivirales/farmacología , Desoxirribonucleósidos/química , Desoxirribonucleósidos/farmacología , Hepacivirus/efectos de los fármacos , Antivirales/síntesis química , Línea Celular , Desoxiadenosinas/síntesis química , Desoxiadenosinas/química , Desoxiadenosinas/farmacología , Desoxirribonucleósidos/síntesis química , Halogenación , Hepatitis C/tratamiento farmacológico , Humanos , Relación Estructura-ActividadRESUMEN
Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although the sustained virologic response rate in the treatment of genotype 1 using new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has been improved by more than 70%, several severe side effects such as skin rash/ageusia and advanced anemia have become a problem. Under these circumstances, a new type of anti-HCV oral drug with few side effects is needed. Our recently developed HCV drug assay systems, including the HuH-7 cell line-derived OR6 and AH1R, and the Li23 cell line-derived ORL8 and ORL11, allow genome-length HCV RNAs (several strains of genotype 1b) encoding renilla luciferase to replicate efficiently. Using these systems as anti-HCV candidates, we have identified numerous existing medicines that can be used against HCV with few side effects, such as statins and teprenon. To obtain additional anti-HCV candidates, we evaluated a number of oral health supplements, and found that the capsule but not the liquid form of Cordyceps militaris (CM) (Ascomycotinanorth, North Chinese caterpillar fungus), which is used as a Chinese herbal medicine, exhibited moderate anti-HCV activity. In combination with interferon-α or ribavirin, CM exhibited an additive inhibitory effect. Among the main components of CM, cordycepin, but not ergosterol, contributed to the anti-HCV activity of CM. In consideration of all these results, we suggest that CM would be useful as an oral anti-HCV agent in combination with interferon-α and/or ribavirin.
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Antivirales/farmacología , Cordyceps/química , Desoxiadenosinas/farmacología , Medicamentos Herbarios Chinos/farmacología , Cápsulas Fúngicas , Hepacivirus/efectos de los fármacos , Línea Celular Tumoral , Suplementos Dietéticos , Ergosterol/farmacología , Humanos , Interferón-alfa/farmacología , Salud Bucal , ARN Viral/biosíntesis , Ribavirina/farmacología , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: The most distinguishing genetic feature of hepatitis C virus (HCV) is its remarkable diversity and variation. To understand this feature, we previously performed genetic analysis of HCV in the long-term culture of human hepatoma HuH-7-derived HCV RNA-replicating cell lines. On the other hand, we newly established HCV RNA-replicating cell lines using human hepatoma Li23 cells, which were distinct from HuH-7 cells. METHODOLOGY/PRINCIPAL FINDINGS: Li23-derived HCV RNA-replicating cells were cultured for 4 years. We performed genetic analysis of HCVs recovered from these cells at 0, 2, and 4 years in culture. Most analysis was performed in two separate parts: one part covered from the 5'-terminus to NS2, which is mostly nonessential for RNA replication, and the other part covered from NS3 to NS5B, which is essential for RNA replication. Genetic mutations in both regions accumulated in a time-dependent manner, and the mutation rates in the 5'-terminus-NS2 and NS3-NS5B regions were 4.0-9.0×10(-3) and 2.7-4.0×10(-3) base substitutions/site/year, respectively. These results suggest that the variation in the NS3-NS5B regions is affected by the pressure of RNA replication. Several in-frame deletions (3-105 nucleotides) were detected in the structural regions of HCV RNAs obtained from 2-year or 4-year cultured cells. Phylogenetic tree analyses clearly showed that the genetic diversity of HCV was expanded in a time-dependent manner. The GC content of HCV RNA was significantly increased in a time-dependent manner, as previously observed in HuH-7-derived cell systems. This phenomenon was partially due to the alterations in codon usages for codon optimization in human cells. Furthermore, we demonstrated that these long-term cultured cells were useful as a source for the selection of HCV clones showing resistance to anti-HCV agents. CONCLUSIONS/SIGNIFICANCE: Long-term cultured HCV RNA-replicating cells are useful for the analysis of evolutionary dynamics and variations of HCV and for drug-resistance analysis.
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Carcinoma Hepatocelular/metabolismo , Hepacivirus/genética , ARN Viral/biosíntesis , Antivirales/química , Secuencia de Bases , Carcinoma Hepatocelular/virología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Codón , Evolución Molecular , Eliminación de Gen , Genes Virales , Variación Genética , Genoma Viral , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virología , Datos de Secuencia Molecular , Mutación , Sistemas de Lectura Abierta , Fenotipo , FilogeniaRESUMEN
[This corrects the article on p. e72519 in vol. 8.].
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BACKGROUND: Persistent hepatitis C virus (HCV) infection causes chronic liver diseases and is a global health problem. Although new triple therapy (pegylated-interferon, ribavirin, and telaprevir/boceprevir) has recently been started and is expected to achieve a sustained virologic response of more than 70% in HCV genotype 1 patients, there are several problems to be resolved, including skin rash/ageusia and advanced anemia. Thus a new type of anti-HCV drug is still needed. METHODOLOGY/PRINCIPAL FINDINGS: Recently developed HCV drug assay systems using HCV-RNA-replicating cells (e.g., HuH-7-derived OR6 and Li23-derived ORL8) were used to evaluate the anti-HCV activity of drug candidates. During the course of the evaluation of anti-HCV candidates, we unexpectedly found that two preclinical antimalarial drugs (N-89 and its derivative N-251) showed potent anti-HCV activities at tens of nanomolar concentrations irrespective of the cell lines and HCV strains of genotype 1b. We confirmed that replication of authentic HCV-RNA was inhibited by these drugs. Interestingly, however, this anti-HCV activity did not work for JFH-1 strain of genotype 2a. We demonstrated that HCV-RNA-replicating cells were cured by treatment with only N-89. A comparative time course assay using N-89 and interferon-α demonstrated that N-89-treated ORL8 cells had more rapid anti-HCV kinetics than did interferon-α-treated cells. This anti-HCV activity was largely canceled by vitamin E. In combination with interferon-α and/or ribavirin, N-89 or N-251 exhibited a synergistic inhibitory effect. CONCLUSIONS/SIGNIFICANCE: We found that the preclinical antimalarial drugs N-89 and N-251 exhibited very fast and potent anti-HCV activities using cell-based HCV-RNA-replication assay systems. N-89 and N-251 may be useful as a new type of anti-HCV reagents when used singly or in combination with interferon and/or ribavirin.
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Antimaláricos/análisis , Antimaláricos/farmacología , Hepacivirus/genética , Hepacivirus/fisiología , ARN Viral/metabolismo , Replicación Viral/efectos de los fármacos , Línea Celular Tumoral , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Genoma Viral/genética , Genotipo , Hepacivirus/efectos de los fármacos , Humanos , Interferón-alfa/farmacología , Ribavirina/farmacología , Factores de Tiempo , Vitamina E/farmacologíaRESUMEN
This study assessed the radiographic appearance of bone graft domes longitudinally after osteotome sinus floor elevation using cone beam computed tomography (CBCT). This study presents the radiological findings of a 6-month follow-up CBCT study in maxillary osteotome sinus floor elevation. We examined 52 patients with a crestal bone height of less than 8 mm in the posterior maxilla who required sinus augmentation. Implants (n = 91) were subsequently placed in regenerated bone following osteotome sinus floor elevation; autogenous bone was used as the augmentation material. In all cases, the grafted augmentation material tended to be absorbed, but at least 1 mm of grafted augmentation material was recognized around the implant fixtures on CBCT at the second implant operation. The border between the grafted augmentation material and the existing bone was indistinct. The grafted area apical to the implants undergoes shrinkage and remodeling. It was suggested that sufficient grafted autogenous bone changes into bone to support an implant.
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BACKGROUND & AIM: FKBP8/FKBP38 is a unique FK506-binding protein with a C-terminal membrane anchor and localizes at the outer membranes of mitochondria and the endoplasmic reticulum. Similar to some immunophilins, such as FKBP51, FKBP52 and Cyclophilin 40, FKBP8/FKBP38 contain a putative Calmodulin-binding domain and a tetratricopeptide-repeat (TPR) domain for the binding of Hsp90. Both Hsp90 and the non-structural protein 5A (NS5A) of the hepatitis C virus (HCV) interact specifically with FKBP8/FKBP38 through its TPR domain, and the ternary complex formation plays a critical role in HCV RNA replication. The goal of this study is to evaluate that the host factor inhibits the ternary complex formation and the replication of HCV in vitro and in vivo. METHODS: S100 proteins, FKBP38, FKBP8, HCV NS5A, Hsp90, and calmodulin were expressed in E.coli and purified. In vitro binding studies were performed by GST pull-down, S-tag pull-down and surface plasmon resonance analyses. The effect of S100 proteins on HCV replication was analysed by Western blotting using an HCV NS3 antibody following transfection of S100 proteins into the HCV replicon harbouring cell line (sO cells). RESULTS: In vitro binding studies showed that S100A1, S100A2, S100A6, S100B and S100P directly interacted with FKBP8/FKBP38 in a Ca(2+) -dependent manner and inhibited the FKBP8/FKBP38-Hsp90 and FKBP8/FKBP38-NS5A interactions. Furthermore, overexpression of S100A1, S100A2 and S100A6 in sO cells resulted in the efficient inhibition of HCV replication. CONCLUSION: The association of the S100 proteins with FKBP8/FKBP38 provides a novel Ca(2+) -dependent regulatory role in HCV replication through the NS5A-host protein interaction.
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Calcio/metabolismo , Proteínas HSP90 de Choque Térmico/metabolismo , Hepacivirus/fisiología , ARN Viral/biosíntesis , Proteínas S100/metabolismo , Proteínas no Estructurales Virales/metabolismo , Escherichia coli , Hepacivirus/genética , Humanos , Plásmidos/genética , Proteínas Recombinantes/metabolismo , Resonancia por Plasmón de Superficie , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
UNLABELLED: Ribavirin (RBV) is often used in conjunction with interferon-based therapy for patients with chronic hepatitis C. There is a drastic difference in the anti-hepatitis C virus (HCV) activity of RBV between the HuH-7-derived assay system, OR6, possessing the RBV-resistant phenotype (50% effective concentration [EC50 ]: >100 µM) and the recently discovered Li23-derived assay system, ORL8, possessing the RBV-sensitive phenotype (EC50 : 8 µM; clinically achievable concentration). This is because the anti-HCV activity of RBV was mediated by the inhibition of inosine monophosphate dehydrogenase in RBV-sensitive ORL8 cells harboring HCV RNA. By means of comparative analyses using RBV-resistant OR6 cells and RBV-sensitive ORL8 cells, we tried to identify host factor(s) determining the anti-HCV activity of RBV. We found that the expression of adenosine kinase (ADK) in ORL8 cells was significantly higher than that in RBV-resistant OR6 cells harboring HCV RNA. Ectopic ADK expression in OR6 cells converted them from an RBV-resistant to an RBV-sensitive phenotype, and inhibition of ADK abolished the activity of RBV. We showed that the differential ADK expression between ORL8 and OR6 cells was not the result of genetic polymorphisms in the ADK gene promoter region and was not mediated by a microRNA control mechanism. We found that the 5' untranslated region (UTR) of ADK messenger RNA in ORL8 cells was longer than that in OR6 cells, and that only a long 5' UTR possessed internal ribosome entry site (IRES) activity. Finally, we demonstrated that the long 5' UTR functioned as an IRES in primary human hepatocytes. CONCLUSION: These results indicate that ADK acts as a determinant for the activity of RBV and provide new insight into the molecular mechanism underlying differential drug sensitivity.
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Adenosina Quinasa/fisiología , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepatitis C/patología , Hepatocitos/efectos de los fármacos , Ribavirina/farmacología , Antivirales/uso terapéutico , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Línea Celular Tumoral , Farmacorresistencia Viral , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Hepatitis C/metabolismo , Hepatocitos/patología , Hepatocitos/virología , Humanos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Fenotipo , ARN Viral/metabolismo , Ribavirina/uso terapéutico , Resultado del TratamientoRESUMEN
Persistent hepatitis C virus (HCV) infection frequently causes hepatocellular carcinoma. However, the mechanisms of HCV-associated hepatocarcinogenesis and disease progression are unclear. Although the human hepatoma cell line, HuH-7, has been widely used as the only cell culture system for robust HCV replication, we recently developed new human hepatoma Li23 cell line-derived OL, OL8, OL11, and OL14 cells, in which genome-length HCV RNA (O strain of genotype 1b) efficiently replicates. OL, OL8, OL11, and OL14 cells were cultured for more than 2 years. We prepared cured cells from OL8 and OL11 cells by interferon-γ treatment. The cured cells were also cultured for more than 2 years. cDNA microarray and RT-PCR analyses were performed using total RNAs prepared from these cells. We first selected several hundred highly or moderately expressed probes, the expression levels of which were upregulated or downregulated at ratios of more than 2 or less than 0.5 in each set of compared cells (e.g., parent OL8 cells versus OL8 cells cultured for 2 years). From among these probes, we next selected those whose expression levels commonly changed during a 2-year culture of genome-length HCV RNA-replicating cells, but which did not change during a 2-year culture period in cured cells. We further examined the expression levels of the selected candidate genes by RT-PCR analysis using additional specimens from the cells cultured for 3.5 years. Reproducibility of the RT-PCR analysis using specimens from recultured cells was also confirmed. Finally, we identified 5 upregulated genes and 4 downregulated genes, the expression levels of which were irreversibly altered during 3.5-year replication of HCV RNA. These genes may play roles in the optimization of the environment in HCV RNA replication, or may play key roles in the progression of HCV-associated hepatic diseases.
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Perfilación de la Expresión Génica , Hepacivirus/fisiología , Hepatocitos/virología , Neoplasias Hepáticas/genética , ARN Viral/genética , Replicación Viral , Línea Celular Tumoral , Regulación de la Expresión Génica , Hepacivirus/genética , Hepatocitos/metabolismo , Humanos , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/virología , Masculino , Persona de Mediana Edad , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Viral/metabolismo , Células Tumorales CultivadasRESUMEN
We developed a new cell culture drug assay system (AH1R), in which genome-length hepatitis C virus (HCV) RNA (AH1 strain of genotype 1b derived from a patient with acute hepatitis C) efficiently replicates. By comparing the AH1R system with the OR6 assay system that we developed previously (O strain of genotype 1b derived from an HCV-positive blood donor), we demonstrated that the anti-HCV profiles of reagents including interferon-γ and cyclosporine A significantly differed between these assay systems. Furthermore, we found unexpectedly that rolipram, an anti-inflammatory drug, showed anti-HCV activity in the AH1R assay but not in the OR6 assay, suggesting that the anti-HCV activity of rolipram differs depending on the HCV strain. Taken together, these results suggest that the AH1R assay system is useful for the objective evaluation of anti-HCV reagents and for the discovery of different classes of anti-HCV reagents.
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Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Hepatitis C/virología , Pruebas de Sensibilidad Microbiana/métodos , Replicación Viral/efectos de los fármacos , Línea Celular , Hepacivirus/aislamiento & purificación , HumanosRESUMEN
Postmenopausal women with chronic hepatitis C exhibited a poor response to interferon (IFN) therapy compared to premenopausal women. Osteoporosis is the typical complication that occurs in postmenopausal women. Recently, it was reported that an osteoporotic reagent, vitamin D3, exhibited anti-hepatitis C virus (HCV) activity. Therefore, we investigated whether or not another osteoporotic reagent, raloxifene, would exhibit anti-HCV activity in cell culture systems. Here, we demonstrated that raloxifene inhibited HCV RNA replication in genotype 1b and infection in genotype 2a. Raloxifene enhanced the anti-HCV activity of IFN-α. These results suggest a link between the molecular biology of osteoporosis and the HCV life cycle.
RESUMEN
The budding ascidian Polyandrocarpa misakiensis proliferates asexually by budding. The atrial epithelium is a multipotent but differentiated tissue, which transdifferentiates into various tissues and organs after the bud separates from the parental body. We isolated cDNA clones homologous to the myc proto-oncogene from P. misakiensis. The cDNA, named Pm-myc, encoded a polypeptide of 639 amino acid residues, containing Myc-specific functional motifs, Myc box I and Myc box II, and the basic helix-loop-helix domain. Expression of Pm-myc was observed in the atrial epithelium in the organ-forming region of the developing bud, where the epithelial cells dedifferentiate and re-enter the cell cycle. The expression was also observed in fibroblast-like cells, which are known to participate in the organogenesis together with the epithelial cells. Unexpectedly, the atrial epithelium expressed Pm-myc more than one day before the dedifferentiation. The organogenesis was disturbed by Pm-myc-specific double-stranded RNA. In situ hybridization revealed that Pm-myc-positive fibroblast-like cells disappeared around the organ primordium of the dsRNA-treated bud. The results suggest that the mesenchymal-epithelial transition of fibroblast-like cells is important for the organogenesis in this budding ascidian species.