RESUMO
STUDY QUESTION: How is the localisation of ovarian follicles affected by ageing and chronic diseases? SUMMARY ANSWER: Ovarian follicles shift deeper towards the medulla, due to thickening of the tunica albuginea (TA), with ageing and some major common chronic diseases. WHAT IS KNOWN ALREADY: The ovary undergoes morphological and functional changes with ageing. The follicular pool follows these changes with alterations in the amount and distribution of residual follicles. Diseases causing a chronic inflammatory process are associated with morphological changes and impaired ovarian function. STUDY DESIGN, SIZE, DURATION: We conducted a cross-sectional study, examining 90 ovaries from 90 female monkeys. The samples were collected from April 2018 to March 2019 at Tsukuba Primate Research Center in National Institutes of Biomedical Innovation, Health and Nutrition, Japan. PARTICIPANTS/MATERIALS, SETTING, METHODS: Ovarian samples were obtained from cynomolgus monkeys that died from natural causes or were euthanised. Ovarian sections were stained with haematoxylin and eosin (H&E) for histological analyses. In ovarian sections from 64 female macaques aged 0-25 years, a total of 13 743 follicles at different developmental stages (primordial, intermediary, primary, early secondary and late secondary) were assessed to determine the depth of each follicle from the outer surface of the ovarian cortex to the far end of the follicle, by using a digital imaging software. TA thickness was measured as sum of basal membrane and tunica collagen layer for each ovary under H&E staining. To explore the possibility of age-related trends in ovarian morphometric characteristics, samples were divided into four different age groups (0-3 years (pre-menarche), 4-9 years, 10-14 years and 15-20 years). To evaluate the effect of common chronic diseases on ovarian morphometric characteristics, macaques with diabetes mellitus (DM) (n = 10), endometriosis (n = 8) or inflammatory bowel disease (IBD) (n = 8) were compared to age-matched controls without chronic diseases. MAIN RESULTS AND THE ROLE OF CHANCE: Ovarian morphometric analysis revealed that the relative location of follicles became deeper in all age groups according to development of follicles (P < 0.05). Total follicle distance from the ovarian surface was increased with ageing (P < 0.05). In a sub-analysis according to developmental stage, only primordial and intermediary follicles were localised deeper with increasing age (P < 0.05). TA thickness was also increased with ageing (P < 0.05). The localisation of the total number of follicles became deeper in ovaries from monkeys with DM, endometriosis or IBD as compared to the control group (P < 0.05). With DM, analysis of follicles distance at almost each developmental stage was significantly deeper compared to controls (P < 0.05) with the exception of early secondary follicles. With endometriosis, follicles at primary and early and late secondary stages were significantly deeper compared to controls (P < 0.05). Also with IBD, follicles at primary and early and late secondary follicles were significantly deeper compared to controls (P < 0.001). The TA was thicker with DM and endometriosis compared to controls (P < 0.05), but not with IBD (P = 0.16). LARGE SCALE DATA: NA. LIMITATIONS, REASONS FOR CAUTION: Two-dimensional histology was used to assess follicle localisation. The possibility of minimal variations between the measured distance to the actual distance in a spherical structure cannot be excluded. Additionally, the severity of disease was not assessed. WIDER IMPLICATIONS OF THE FINDINGS: This study is the first step towards enhancing our understanding of how ageing and chronic diseases affect the relative localisation of dormant and developing follicles. These observations, combined with possible future human studies, may have managerial implications in the field of fertility preservation and other conditions involving ovarian tissue cryopreservation. STUDY FUNDING/COMPETING INTEREST(S): The present work was supported by the Grant-in-Aid for Scientific Research B (19H03801) (to K.K.), Challenging Exploratory Research (18K19624), Japan Agency for Medical Research and Development, Mochida Memorial Foundation for Medical and Pharmaceutical Research, Takeda Science Foundation and Naito Foundation (to K.K.). All authors have no conflicts of interest directly relevant to the content of this article.
Assuntos
Preservação da Fertilidade , Folículo Ovariano , Animais , Doença Crônica , Estudos Transversais , Feminino , Macaca fascicularisRESUMO
Severe acute respiratory syndrome (SARS) spread during the winter of 2003, and attempts have been made to develop vaccines against SARS corona virus (SARS-CoV). The present study provides a strategy to rapidly identify SARS-CoV-derived antigenic peptides recognized by HLA-A2-restricted cytotoxic T lymphocytes (CTLs). Forty-three candidate peptides having HLA-A2-binding motifs were selected in silico and HLA-A2/Db chimeric MHC class I-transgenic mice were immunized with these peptides and a new derivative of muramyl dipeptide that can induce upregulation of HLA-DR, CD80, CD86, and CD40 in human CD14+ antigen presenting cells, was administered as an adjuvant. Six HLAA2-restricted mouse CTL epitopes were identified, including two new epitopes which have never been reported before. One of the novel peptides was naturally processed and successfully induced HLAA2-restricted specific CTLs in both HLA transgenic mice and healthy donors. The method was useful, convenient and efficient for rapid identification of CTL epitopes derived from SARS-CoV proteins and will be possibly applicable for other pathogens to develop a peptide-based vaccine.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/análogos & derivados , Adjuvantes Imunológicos/farmacologia , Antígeno HLA-A2/imunologia , Glicoproteínas de Membrana/imunologia , Linfócitos T Citotóxicos/imunologia , Proteínas do Envelope Viral/imunologia , Animais , Apresentação de Antígeno , Antígenos Virais/imunologia , Epitopos de Linfócito T , Humanos , Imunização , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Glicoproteína da Espícula de Coronavírus , Vacinas Virais/imunologiaRESUMO
In order to study the regulation of the growth differentiation factor-9 (gdf9) gene in a primitive teleost with semelparous life history, we cloned a cDNA encoding shortfinned eel Gdf9, expressed a partial peptide in Escherichia coli, and raised an antiserum to evaluate changes in Gdf9 expression during its pituitary homogenate-induced reproductive cycle. The effects of in vivo and in vitro exposure to the androgen 11-ketotestosterone (11-KT), known to affect previtellogenic (PV) oocyte growth, were also determined. Furthermore, we investigated whether Gdf9 expression was metabolically gated by treating PV fish with recombinant GH in vivo. Immunoreactive proteins of ca. 52 and 55 kDa were identified by western blot analysis. Gdf9 message and protein were most abundant in PV oocytes, and peaked slightly earlier for mRNA than for protein. Captivity resulted in reduced gdf9 mRNA levels, which were restored following pituitary homogenate treatment. As oocytes progressed through induced oogenesis, Gdf9 expression decreased. Neither 11-KT nor GH treatment affected gdf9 mRNA levels in PV fish, although GH could partially restore handling- or captivity-induced decreases in gdf9 mRNA levels. Semelparous eels thus show an expression pattern of Gdf9 during oogenesis that is similar to that seen in other vertebrates, that appears responsive to handling or captivity stress, and whose control remains to be elucidated.
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Androgênios/farmacologia , Anguilla/fisiologia , Fator 9 de Diferenciação de Crescimento/metabolismo , Hormônio do Crescimento/farmacologia , Ovário/metabolismo , Reprodução , Testosterona/análogos & derivados , Anguilla/crescimento & desenvolvimento , Anguilla/metabolismo , Animais , Feminino , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Fator 9 de Diferenciação de Crescimento/química , Fator 9 de Diferenciação de Crescimento/genética , Fator 9 de Diferenciação de Crescimento/isolamento & purificação , Hormônio do Crescimento/análogos & derivados , Imuno-Histoquímica , Hibridização In Situ , Fator de Crescimento Insulin-Like I/metabolismo , Técnicas de Amplificação de Ácido Nucleico , Oogênese , Especificidade de Órgãos , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/crescimento & desenvolvimento , Filogenia , RNA Mensageiro/metabolismo , Análise de Sequência de RNA , Estresse Fisiológico , Testosterona/farmacologiaRESUMO
AIMS: Diffuse large B-cell lymphoma (DLBCL) usually proliferates effacing lymph follicles. In occasional cases, tumour cells show an interfollicular pattern of proliferation preserving lymph follicles. The aim was to analyse clinicopathological findings in DLBCL showing an interfollicular pattern of proliferation to determine whether this type of lymphoma is a distinct entity of DLBCL. METHODS AND RESULTS: Clinicopathological findings in 12 cases of DLBCL showing an interfollicular pattern of proliferation [interfollicular group (IF)] were examined and compared with those in 30 cases of DLBCL with ordinary morphology [control group (CG)]. IF showed a significantly lower lactate dehydrogenase level and International Prognostic Index scores than CG (P = 0.023 and P < 0.01, respectively). The frequency of localized disease, clinical stage 1 and 2, in IF was higher than that in CG (P = 0.016). A morphologically polymorphous pattern of proliferation was found in seven of 12 cases (58.3%) in IF, which was higher than that in CG, five (16.7%) of 30 cases (P < 0.01). Clonality analysis with the polymerase chain reaction method revealed that all 11 IF cases examined showed a monoclonal pattern. Immunohistochemically, the majority (11 of 12) of IF cases showed a non-germinal centre B-cell phenotype and the frequency was higher than that in CG (P = 0.021). CONCLUSION: Diffuse large B-cell lymphoma with an interfollicular pattern of proliferation shows distinct clinical and pathological findings from ordinary DLBCL.
Assuntos
Linfoma Difuso de Grandes Células B/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Proliferação de Células , Feminino , Centro Germinativo/citologia , Humanos , Imuno-Histoquímica , Japão , L-Lactato Desidrogenase/metabolismo , Linfoma Difuso de Grandes Células B/diagnóstico , Masculino , Pessoa de Meia-Idade , Fenótipo , PrognósticoRESUMO
We compared the effects of polypyrimidine tract-binding protein (PTB) on hepatitis C virus (HCV genotype IIa), encephalomyocarditis virus (EMCV) and poliovirus internal ribosome entry site (IRES) activities in vitro. It bound strongly to EMCV IRES, but weakly to PV and HCV RNAs. PV IRES showed the strongest dependency to PTB and it showed less than one-tenth of IRES activity after the immuno-depletion of PTB from HeLa S10 lysate with pre-coated anti-PTB IgG beads, comparing to the normal IgG beads-treated S10 lysate. EMCV IRES activity was approximately 40% of that of normal control after PTB depletion. Especially, HCV IRES activity was approximately 95%, and most weekly affected by the depletion of PTB. Repletion of PTB to depleted S10 lysate restored activities of PV and EMCV IRESs. The data suggest that PTB plays an important role in picornaviral IRESs, but not in HCV IRES.
Assuntos
Vírus da Encefalomiocardite/genética , Regulação Viral da Expressão Gênica/fisiologia , Hepacivirus/genética , Poliovirus/genética , Proteína de Ligação a Regiões Ricas em Polipirimidinas/fisiologia , Sítio de Iniciação de Transcrição/fisiologia , Animais , Anticorpos Antivirais/biossíntese , Cobaias , Células HeLa , Humanos , Proteína de Ligação a Regiões Ricas em Polipirimidinas/metabolismo , Biossíntese de Proteínas , RNA Viral/metabolismo , CoelhosRESUMO
RNA interference (RNAi) can be used to inhibit viral replication in mammalian cells and therefore could be a powerful new antiviral therapy. Small interfering RNA (siRNA) may be effective for RNAi, but there are some technical problems that must be solved in each case, for example, predicting the effective siRNA target site and targeting heterogeneous sequences in a virus population. We show here that diced siRNA generated from long double-stranded RNA (dsRNA) is highly effective for inducing RNAi in HuH-7 cells harboring hepatitis C virus (HCV) replicons and can overcome variations in the HCV genotype. However, in mammalian cells, long dsRNA induced an interferon response and caused cell death. Here we describe an improvement of this method, U6 promoter-driven expression of long hairpin-RNA with multiple point mutations in the sense strand. This can efficiently silence HCV RNA replication and HCV protein expression without triggering the interferon response or cell death normally caused by dsRNA. In conclusion, intracellular-diced dsRNA efficiently induces RNAi, and, despite the high rate of mutation in HCV, it should be a feasible therapeutic strategy for silencing HCV RNA.
Assuntos
Genes Virais , Terapia Genética/métodos , Hepacivirus/genética , Hepatite C/terapia , RNA de Cadeia Dupla/administração & dosagem , RNA Interferente Pequeno/genética , Sequência de Bases , Células Cultivadas , Inativação Gênica , Engenharia Genética , Variação Genética , Vetores Genéticos/genética , Genótipo , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA , Ribonuclease III , Transfecção/métodosRESUMO
Hepatitis C virus (HCV) causes persistent infection in most patients. To clarify the mechanisms underlying establishment of this persistent infection, nucleotide sequences of the E1/E2 region were characterized in 5 patients with acute and chronic HCV infection. We used direct DNA sequencing methods to identify the major sequence of HCV in each patient. Each HCV genome displayed a high frequency of nucleotide sequence variation in the hypervariable region (HVR) of E2. However, patient-specific conserved nucleotide sequences were identified in the E1/E2 region during the course of infection and conserved the higher-order protein structure. In the acute phase HCV infection, amino acid substitution in HVR-1 as the monthly rate of amino acids substitution per site (%) between each point exceeded 10.2%. In the chronic phase HCV infection, a significantly lower rate of amino acid substitution was observed in patients. The host immune responses to HVR-1 of each HCV isolates from all clinical courses were characterized using synthetic peptides and ELISA. One chronic patient serum (genotype 1b) did not react at all to its own HVR-1 peptides, however another patient (genotype 2b) reacted to all clinical course. These results indicated that HVR-1 might not always exhibit neutralizing epitopes of HCV infection. The sequence variation in HVR-1 may instead indicate the existence of various clones in acute phase infection and the adaption of these clones is thought to have caused persistent and chronic infection in each patient.
Assuntos
Hepacivirus/classificação , Hepacivirus/patogenicidade , Análise de Sequência de DNA , Proteínas do Envelope Viral/química , Proteínas Virais/química , Doença Aguda , Adulto , Idoso , Sequência de Aminoácidos , Substituição de Aminoácidos , Sequência de Bases , Doença Crônica , Feminino , Variação Genética , Genótipo , Hepacivirus/genética , Humanos , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Proteínas do Envelope Viral/genética , Proteínas Virais/genéticaRESUMO
A new immuno-radiometric assay (IRMA) to detect hepatitis C virus (HCV) core antigen (HCVcAg) has been developed. The aim of the present study was to investigate the sensitivity and specificity of this IRMA to measure HCV antigenemia, based on the detection of HCV RNA as the gold standard, and to assess the utility of the IRMA in a community-based population. Anti-HCV positive residents in a hyperendemic area of HCV infection in Japan were studied. Serum levels of HCVcAg were measured using IRMA, and the presence of HCV RNA was determined by a qualitative reverse transcription-polymerase chain reaction (RT-PCR) assay. The sensitivity and the specificity of the IRMA were 96.4 and 100%, respectively. The sensitivity of the IRMA was similar between serological HCV group I (HCV genotypes 1a and 1b) (97.6%) and group II (HCV genotypes 2a and 2b) (94.0%). There was a strong correlation between serum HCVcAg level and HCV-RNA measured by a quantitative RT-PCR (r = 0.832, P < 0.0001). There also was a very strong correlation of HCVcAg level between IRMA measurements performed on serum and those performed on plasma (r = 0.984, P < 0.0001). In conclusion, this new IRMA is useful for the detection of HCV core antigen in a community-based population.
Assuntos
Antígenos da Hepatite C/sangue , Ensaio Imunorradiométrico/métodos , Proteínas do Core Viral/análise , Feminino , Humanos , Masculino , RNA Viral/análise , Sensibilidade e EspecificidadeRESUMO
Several lines of evidence suggest that interferon (IFN)-alpha is effective in suppression of liver cirrhosis (LC) as well as hepatitis C virus (HCV) infection, which is a major cause of LC in Japan. However, IFN-alpha often causes systemic toxicity such as flu-like symptoms, which precludes the IFN-alpha dose escalation required for clinical efficacy. Since IFN-alpha is rapidly degraded in the blood circulation, only a small amount of subcutaneously injected IFN-alpha protein can reach the target organ, the liver. It is expected that on-site IFN-alpha production in the liver overcomes the limitation of the conventional parenteral IFN-alpha administration. An adenovirus vector expressing the rat IFN-alpha gene (AxCA-rIFN) was injected intravenously into rats with dimethylnitrosamine-induced LC. While the subcutaneous IFN-alpha protein injection led to a transient elevation of the cytokine both in the liver and serum, the vector-mediated IFN-alpha gene transduction induced a significant amount of IFN-alpha detected in the liver but not in the serum. The injection of AxCA-rIFN prevented the progression of the rat LC, and improved the survival rate of the treated rats. Although no significant toxicity was noted in the animals, we showed that IFN-alpha gene expression in the liver can be efficiently downregulated by the Cre/loxP-mediated shut-off system, in case the IFN-alpha overdose becomes a problem. The study suggested for the first time the advantage and feasibility of IFN-alpha gene therapy for LC.
Assuntos
Adenoviridae/genética , Terapia Genética/métodos , Vetores Genéticos/administração & dosagem , Imunoterapia/métodos , Interferon gama/genética , Cirrose Hepática Experimental/terapia , Animais , Dimetilnitrosamina , Fibrose , Injeções Subcutâneas , Interferon gama/administração & dosagem , Interferon gama/análise , Fígado/imunologia , Fígado/patologia , Cirrose Hepática Experimental/imunologia , Cirrose Hepática Experimental/patologia , Masculino , Ratos , Ratos Sprague-Dawley , Transdução Genética/métodosRESUMO
Hepatitis C virus (HCV) is an important cause of liver disease throughout the world. However, the natural history and pathogenesis of this infection is still not completely understood. The aim of this study was to characterize the evolution of incident, asymptomatic HCV infection in a community-based population in Japan. The Miyazaki Cohort Study is a prospective study of adult residents in two villages, one of which has a very high prevalence of HCV. Nine hundred and seventy-three people from this village were enrolled in the cohort between 1984 and 1995, with antibodies to HCV (anti-HCV) found in 23%. During subsequent visits to annual health screens, new HCV seroconverters were identified among susceptible individuals, and their sequential samples were tested for anti-HCV, HCV-RNA, and HCV core antigen. Fourteen participants (six males, eight females) acquired anti-HCV during the first 11 years of study follow-up, at an incidence rate of 362 per 100 000 person-years. Detectable HCV-RNA and high anti-HCV titres (> 1:2048) were observed for more than 5 years following seroconversion in 80% (8/10) of seroconverters with sufficient information, indicating the development of persistent infection in these subjects. Three (37.5%) of the eight sero converters with persistent infection had fairly consistent, albeit mild, alanine aminotransferase elevations (30-130 IU/L) during the study. Anti-HCV seroconversions occurred at a very high rate in this community-based population in Japan, in which this infection is endemic. Persistence also developed at a high frequency among the cases of newly acquired infection, although the associated liver enzyme abnormalities were mild.
Assuntos
Hepatite C/epidemiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Alanina Transaminase/sangue , Estudos de Coortes , Serviços de Saúde Comunitária , Feminino , Hepacivirus/genética , Hepacivirus/imunologia , Hepatite C/imunologia , Hepatite C/transmissão , Hepatite C/virologia , Anticorpos Anti-Hepatite C/sangue , Antígenos da Hepatite C/sangue , Humanos , Incidência , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Vigilância da População , RNA Viral/sangue , Proteínas do Core Viral/sangueRESUMO
All nuclear-encoded mRNAs contain a 5' cap structure (m7GpppN, where N is any nucleotide), which is recognized by the eukaryotic translation initiation factor 4E (eIF4E) subunit of the eIF4F complex. The eIF4E-binding proteins constitute a family of three polypeptides that reversibly repress cap-dependent translation by binding to eIF4E, thus preventing the formation of the eIF4F complex. We investigated the biological function of 4E-BP1 by disrupting its gene (Eif4ebp1) in the mouse. Eif4ebp1-/- mice manifest markedly smaller white fat pads than wild-type animals, and knockout males display an increase in metabolic rate. The males' white adipose tissue contains cells that exhibit the distinctive multilocular appearance of brown adipocytes, and expresses the uncoupling protein 1 (UCP1), a specific marker of brown fat. Consistent with these observations, translation of the peroxisome proliferator-activated receptor-gamma co-activator 1 (PGC1), a transcriptional co-activator implicated in mitochondrial biogenesis and adaptive thermogenesis, is increased in white adipose tissue of Eif4ebp1-/- mice. These findings demonstrate that 4E-BP1 is a novel regulator of adipogenesis and metabolism in mammals.
Assuntos
Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Proteínas de Membrana/genética , Fatores de Iniciação de Peptídeos/fisiologia , Fosfoproteínas/fisiologia , Biossíntese de Proteínas , Proteínas Repressoras/fisiologia , Fatores de Transcrição/genética , Proteínas Adaptadoras de Transdução de Sinal , Tecido Adiposo/metabolismo , Animais , Metabolismo Basal , Proteínas de Ciclo Celular , Fator de Iniciação 4E em Eucariotos , Fatores de Iniciação em Eucariotos , Regulação da Expressão Gênica , Marcação de Genes , Hipoglicemia/metabolismo , Canais Iônicos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Proteínas Mitocondriais , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Fosfoproteínas/genética , Fosforilação , RNA Mensageiro , Proteínas Repressoras/genética , Proteína Desacopladora 1RESUMO
Latent-class analysis was used to evaluate the usefulness of markers of hepatitis C virus (HCV) infection in characterizing the true, underlying infection in a community-based Japanese population. Antibodies to HCV were detected in 24%, HCV RNA in 22%, and HCV core protein in 19% of stored serum samples from 372 adults. A 2-class model suggested that positive results for any 2 virus markers defined the current HCV infection class, with an estimated prevalence of 22% (95% confidence interval, 18%-26%). The sensitivity for detection of current HCV infection was highest for anti-HCV (97%) and was more moderate for HCV RNA (91%) and HCV core protein (85%). The specificity for each marker was > or =96%. In general, the association between demographic factors and current HCV infection status was strengthened by use of latent-class analysis that combined data for markers of HCV infection, when compared with results of logistic regression analysis for each marker separately.
Assuntos
Hepacivirus , Hepatite C/sangue , RNA Viral/sangue , Proteínas do Core Viral/sangue , Testes de Aglutinação , Biomarcadores/sangue , Feminino , Hepacivirus/isolamento & purificação , Hepatite C/epidemiologia , Hepatite C/virologia , Anticorpos Anti-Hepatite C/sangue , Humanos , Immunoblotting , Japão/epidemiologia , Masculino , Pessoa de Meia-Idade , Prevalência , Sensibilidade e EspecificidadeRESUMO
There are several pathways leading to apoptosis. It is not clear whether cells choose one of them or use multiple processes when they commit to apoptosis. MOLT-4 cells undergo apoptosis after X-irradiation through the p53-dependent pathway and/or ceramide signal. To evaluate the relative contribution of these pathways, we studied effects of the expression of various levels of transfected murine mutant p53 cDNA (TGC-->CGC of codon 173, corresponding to codonl76 in human p53) on the induction of apoptosis in X-irradiated or heated MOLT-4 cells. When survival was determined by the dye-exclusion test at 24 h after irradiation, the percentage of X-ray- or heat-induced dead cells was markedly decreased, depending on the expression level of mutant p53 protein in transfected clones. The appearance of apoptotic cells as determined by morphological changes was also decreased. These inhibitions were almost complete at 24 h after irradiation with X-rays in the case of the highest-expressing clone. p21 WAF1 protein was increased in MOLT-4 cells after X-irradiation, but not in the transfectant. These results suggest that murine mutant p53 protein has a dominant-negative effect against normal p53 in MOLT-4, and that the X-ray-induced apoptosis in MOLT-4 is fully p53-dependent.
Assuntos
Apoptose/genética , Apoptose/efeitos da radiação , Genes p53 , Leucemia de Células T/genética , Leucemia de Células T/patologia , Mutação , Expressão Gênica , Temperatura Alta , Humanos , Transfecção , Células Tumorais CultivadasRESUMO
Hepatitis C virus (HCV) replicates in human and chimpanzee hepatocytes. To characterize the nature of HCV and evaluate antiviral agents, the development of an HCV replication system in a cell culture is essential. We developed a cell line derived from human hepatocytes by fusing them with a hepatoblastoma cell line, HepG2, and obtained several clones. When we tested the clones for their ability to support HCV replication by nested RT-PCR, we found 1 clone (IMY-N9) that was more susceptible to HCV replication than HepG2. The negative-strand HCV RNA was detected in IMY-N9 by strand-specific RT-PCR, and viral RNA was identified in culture supernatant during the culture. Then we monitored HCV RNA titers in IMY-N9 and HepG2, respectively, by real-time detection PCR throughout the culture. A significant increase in the HCV RNA titer was observed only in IMY-N9. Serial passages of HCV culture supernatant were shown in the culture system. Furthermore, we tested several infectious materials for viral infectivity by monitoring HCV RNA titers and/or 50% tissue culture infectious dose (TCID50) of HCV on IMY-N9. In each material, HCV showed various growth patterns and a different TCID50 even though the PCR titer in each material was identical. The results showed that HCV in each material served various growth patterns and different TCID50 even though PCR titer in each material was identical. This cell line is useful for estimating viral activity and for studying cellular factors that may be necessary to HCV replication in human hepatocytes.
Assuntos
Hepacivirus/fisiologia , Hepatite C/diagnóstico , Hepatócitos/fisiologia , Replicação Viral , Fusão Celular , Hepacivirus/genética , Hepacivirus/patogenicidade , Hepatite C/metabolismo , Humanos , RNA Viral/análise , Células Tumorais CultivadasRESUMO
Hepatitis C Virus (HCV) are 55-65 nm spherical particles, but the internal structure of the virion remains to be clarified. To clarify the morphology of HCV core particles, we performed an immune electron microscopy (IEM) using plasma samples from two blood donors with high HCV RNA titers and a detergent-treated anti-HCV core antibody-free plasma sample with high HCV RNA titer (1.5x10(8) copies/ml). Spherical particles, with 33-40 nm in diameter (an average diameter of 37 nm) were found in 1.22-1.25 g/ml fractions after sucrose density gradient centrifugation by conventional electron microscopy (EM). IEM using rabbit polyclonal antibody (RR8) specific to the putative HCV core protein and goat anti-rabbit IgG colloidal gold particles revealed that these spherical particles specifically reacted with RR8. This finding indicates that the spherical particles are naked HCV core particles. Some of the HCV core particles had an icosahedron-like structure. Optical rotation technique showed that the HCV core particle exhibits six-fold symmetry and that the length of regular hexagon side is approximately 20 nm. These findings showed that HCV core particles are spherical particles of 33-40 nm in diameter and that HCV core particles may possess an icosahedron-like structure and a buoyant density of 1.22-1.25 g/ml.
RESUMO
The hepatitis C virus (HCV) nonstructural protein 4B (NS4B) is a relatively hydrophobic 27-kDa protein of unknown function. A tetracycline-regulated gene expression system, a novel monoclonal antibody, and in vitro transcription-translation were employed to investigate the subcellular localization and to characterize the membrane association of this viral protein. When expressed individually or in the context of the entire HCV polyprotein, NS4B was localized in the endoplasmic reticulum (ER), as shown by subcellular fractionation, immunofluorescence analyses, and double-label confocal laser scanning microscopy. In this compartment NS4B colocalized with the other HCV nonstructural proteins. Association of NS4B with the ER membrane occurred cotranslationally, presumably via engagement of the signal recognition particle by an internal signal sequence. In membrane extraction and proteinase protection assays NS4B displayed properties of a cytoplasmically oriented integral membrane protein. Taken together, our findings suggest that NS4B is a component of a membrane-associated cytoplasmic HCV replication complex. An efficient replication system will be essential to further define the role of NS4B in the viral life cycle.
Assuntos
Retículo Endoplasmático/metabolismo , Hepacivirus/patogenicidade , Proteínas de Membrana/metabolismo , Proteínas não Estruturais Virais/metabolismo , Proteínas Virais/metabolismo , Animais , Anticorpos Monoclonais , Retículo Endoplasmático/virologia , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Regulação Viral da Expressão Gênica , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Confocal , Biossíntese de Proteínas , Tetraciclina/farmacologia , Transcrição GênicaRESUMO
Alpha-feto protein (AFP) mRNA levels increase in hepatocellular carcinoma (HCC) cells as compared with non-neoplastic tissue. Therefore, detection of AFP mRNA in blood nuclear cells is useful for the evaluation of treatment efficacy and prognosis of HCC. In this study, simple and reproducible methods were developed to quantify AFP mRNA using the real-time RT-PCR assay (Taq Man assay). By using in vitro synthesized AFP and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA, the sensitivity and dynamic range of the RT-PCR assay were established. AFP mRNA in both HCC and non-neoplastic tissue, as well as in cell lines, were measured using this assay system. The expression of the AFP mRNA level was normalized using the GAPDH house keeping gene product as an endogenous reference. AFP and GAPDH mRNA can be quantified in the range of 10-10(8) copies when using this quantitative assay. Among HCC cell lines, Huh 7 and HepG2 cells, respectively, represented 1.5x10(6) and 6.0x10(5) AFP mRNA/10(6) GAPDH mRNA, in contrast to 6, 23 and 230 AFP mRNA/10(6) GAPDH mRNA for HLE, HLF and PLC/PRF/5 cells, respectively. Other cell lines derived from stomach, pancreas, and colon cancers have 10 AFP mRNA copies/10(6) GAPDH mRNA. In liver tissue from patients with chronic hepatitis, and the non-neoplastic portion of the liver from HCC patients, AFP mRNA distributes from 2.5x10(3) to 5.8x10(4)/10(6) GAPDH transcripts. In contrast, AFP mRNA in tumor cells were more than 100-fold higher than that found in corresponding non-neoplastic portions in two patients who had a high level of AFP in serum. The establishment of the TaqMan quantifying system for AFP mRNA may have important clinical implications.
RESUMO
Persistent hepatitis C virus (HCV) infection often progresses to chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Numerous viruses have been reported to escape from apoptotic mechanism to maintain persistent infection. In the present study, we characterized the effect of HCV proteins on the Fas signal using HCV transgenic mice, which expressed core, E1, E2, and NS2 proteins, regulated by the Cre/loxP switching system. The transgene expression of HCV transgenic mice caused resistance to Fas antibody stimulated lethality. Apoptotic cell death in the liver of HCV protein expressing mice was significantly reduced compared with nonexpressing mice. Histopathological analysis and DNA fragmentation analysis revealed that the HCV proteins suppressed Fas-mediated apoptotic cell death. To identify the target pathway of HCV proteins, we characterized caspase activity. The activation of caspase-9 and -3/7 but not caspase-8 was inhibited by HCV proteins. Cytochrome c release from mitochondria was inhibited in HCV protein expressing mice. These results indicated that the expression of HCV proteins may directly or indirectly inhibit Fas-mediated apoptosis and death in mice by repressing the release of cytochrome c from mitochondria, thereby suppressing caspase-9 and -3/7 activation. These results suggest that HCV may cause persistent infection, as a result of suppression of Fas-mediated cell death.
Assuntos
Grupo dos Citocromos c/metabolismo , Hepacivirus/metabolismo , Transdução de Sinais/fisiologia , Proteínas Virais/metabolismo , Receptor fas/fisiologia , Animais , Apoptose/fisiologia , Caspases/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos TransgênicosRESUMO
The nucleotide sequence of the entire genome of a filamentous cyanobacterium, Anabaena sp. strain PCC 7120, was determined. The genome of Anabaena consisted of a single chromosome (6,413,771 bp) and six plasmids, designated pCC7120alpha (408,101 bp), pCC7120beta (186,614 bp), pCC7120gamma (101,965 bp), pCC7120delta (55,414 bp), pCC7120epsilon (40,340 bp), and pCC7120zeta (5,584 bp). The chromosome bears 5368 potential protein-encoding genes, four sets of rRNA genes, 48 tRNA genes representing 42 tRNA species, and 4 genes for small structural RNAs. The predicted products of 45% of the potential protein-encoding genes showed sequence similarity to known and predicted proteins of known function, and 27% to translated products of hypothetical genes. The remaining 28% lacked significant similarity to genes for known and predicted proteins in the public DNA databases. More than 60 genes involved in various processes of heterocyst formation and nitrogen fixation were assigned to the chromosome based on their similarity to the reported genes. One hundred and ninety-five genes coding for components of two-component signal transduction systems, nearly 2.5 times as many as those in Synechocystis sp. PCC 6803, were identified on the chromosome. Only 37% of the Anabaena genes showed significant sequence similarity to those of Synechocystis, indicating a high degree of divergence of the gene information between the two cyanobacterial strains.
Assuntos
Anabaena/genética , Genoma Bacteriano , Genes Bacterianos , Dados de Sequência Molecular , Fixação de Nitrogênio/genética , Plasmídeos/genética , Análise de Sequência de DNARESUMO
PURPOSE: We reviewed 115 eyes of 65 patients regarding the intraocular pressure(IOP) following laser in situ keratomileusis(LASIK). PATIENTS AND METHODS: The ages averaged 31.2 +/- 10.5(mean +/- standard deviation) years. The preoperative spherical equivalent averaged -6.85 +/- 2.54 (mean +/- standard deviation) D. A noncontact pneumatic tonometer and Goldmann applanation tonometer were used in measuring the IOP. RESULTS: The IOP difference before and after surgery was statistically significant(Mann-Whitney U test). The IOP was significantly corrected with the corrected diopter value, corneal thickness, and corneal curvature(Spearman coefficient by rank). CONCLUSION: The findings show that postoperative IOP may be underestimated due to decreased corneal thickness and curvature. Due attention has to be paid to this feature in evaluating IOP after LASIK.