RESUMEN
Fibroblast to myofibroblast transdifferentiation mediates numerous fibrotic disorders, such as idiopathic pulmonary fibrosis (IPF). We have previously demonstrated that non-muscle myosin II (NMII) is activated in response to fibrotic lung extracellular matrix, thereby mediating myofibroblast transdifferentiation. NMII-A is known to interact with the calcium-binding protein S100A4, but the mechanism by which S100A4 regulates fibrotic disorders is unclear. In this study, we show that fibroblast S100A4 is a calcium-dependent, mechanoeffector protein that is uniquely sensitive to pathophysiologic-range lung stiffness (8-25 kPa) and thereby mediates myofibroblast transdifferentiation. Re-expression of endogenous fibroblast S100A4 rescues the myofibroblastic phenotype in S100A4 KO fibroblasts. Analysis of NMII-A/actin dynamics reveals that S100A4 mediates the unraveling and redistribution of peripheral actomyosin to a central location, resulting in a contractile myofibroblast. Furthermore, S100A4 loss protects against murine in vivo pulmonary fibrosis, and S100A4 expression is dysregulated in IPF. Our data reveal a novel mechanosensor/effector role for endogenous fibroblast S100A4 in inducing cytoskeletal redistribution in fibrotic disorders such as IPF.
Asunto(s)
Fibrosis Pulmonar Idiopática , Mecanotransducción Celular , Miofibroblastos , Proteína de Unión al Calcio S100A4 , Animales , Ratones , Transdiferenciación Celular , Fibrosis , Fibrosis Pulmonar Idiopática/metabolismo , Fibrosis Pulmonar Idiopática/patología , Pulmón/metabolismo , Miofibroblastos/metabolismo , Miofibroblastos/patología , Proteína de Unión al Calcio S100A4/genética , Proteína de Unión al Calcio S100A4/metabolismoRESUMEN
OBJECTIVE: A method for the determination of 41 pesticides including 26 organophosphorus pesticides, 6 kinds of carbamate, 7 pryethoids insecticides, 1 kinds of organochlorine and fungicide in vegetables and fruits, was established based on optimized QuEChERS with on line gel permeation chromatography-gas chromatography-mass spectrometry (GPC-GC-MS/MS). METHODS: The method obtained the optimized conditions by comparing different methods of extraction and purification. The analytes were extracted from the samples using acetonitrile-buffer of acetic acid and sodium acetate. Then the extracts were determined by GC-MS/MS in multireaction monitoring (MRM) mode after being cleaned up with mixed solid-phase dispersion. And the external standard method was applied to quantify. RESULTS: All the 41 pesticides had good linearity in the ranges of 2-200 ng/mL with the correlation coefficients higher than 0.995. The average recoveries of the most of 41 pesticides in celery and apple were in the range of 71.0%-125.4% at the spiked levels of 5, 10 and 50 µg/kg,with relative standard deviations (RSDs) of 0.34%-15.0%. The limits of quantification (LOQs) of the method at the spiked levels of 5 µg/kg were 0.107-10.100 µg/kg. CONCLUSION: The method is simple, rapid, sensitive and specific.
Asunto(s)
Contaminación de Alimentos/análisis , Frutas/química , Cromatografía de Gases y Espectrometría de Masas/métodos , Residuos de Plaguicidas/análisis , Verduras/química , Acetonitrilos , Plaguicidas , Espectrometría de Masas en Tándem/métodosRESUMEN
UNLABELLED: Latently infected cells remain a primary barrier to eradication of HIV-1. Over the past decade, a better understanding of the molecular mechanisms by which latency is established and maintained has led to the discovery of a number of compounds that selectively reactivate latent proviruses without inducing polyclonal T cell activation. Recently, the histone deacetylase (HDAC) inhibitor vorinostat has been demonstrated to induce HIV transcription from latently infected cells when administered to patients. While vorinostat will be given in the context of antiretroviral therapy (ART), infection of new cells by induced virus remains a clinical concern. Here, we demonstrate that vorinostat significantly increases the susceptibility of CD4(+) T cells to infection by HIV in a dose- and time-dependent manner that is independent of receptor and coreceptor usage. Vorinostat does not enhance viral fusion with cells but rather enhances the kinetics and efficiency of postentry viral events, including reverse transcription, nuclear import, and integration, and enhances viral production in a spreading-infection assay. Selective inhibition of the cytoplasmic class IIb HDAC6 with tubacin recapitulated the effect of vorinostat. These findings reveal a previously unknown cytoplasmic effect of HDAC inhibitors promoting productive infection of CD4(+) T cells that is distinct from their well-characterized effects on nuclear histone acetylation and long-terminal-repeat (LTR) transcription. Our results indicate that careful monitoring of patients and ART intensification are warranted during vorinostat treatment and indicate that HDAC inhibitors that selectively target nuclear class I HDACs could reactivate latent HIV without increasing the susceptibility of uninfected cells to HIV. IMPORTANCE: HDAC inhibitors, particularly vorinostat, are currently being investigated clinically as part of a "shock-and-kill" strategy to purge latent reservoirs of HIV. We demonstrate here that vorinostat increases the susceptibility of uninfected CD4(+) T cells to infection with HIV, raising clinical concerns that vorinostat may reseed the viral reservoirs it is meant to purge, particularly under conditions of suboptimal drug exposure. We demonstrate that vorinostat acts following viral fusion and enhances the kinetics and efficiency of reverse transcription, nuclear import, and integration. The effect of vorinostat was recapitulated using the cytoplasmic histone deacetylase 6 (HDAC6) inhibitor tubacin, revealing a novel and previously unknown cytoplasmic mechanism of HDAC inhibitors on HIV replication that is distinct from their well-characterized effects of long-terminal-repeat (LTR)-driven gene expression. Moreover, our results suggest that treatment of patients with class I-specific HDAC inhibitors could induce latent viruses without increasing the susceptibility of uninfected cells to HIV.
Asunto(s)
Linfocitos T CD4-Positivos/virología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Inhibidores de Histona Desacetilasas/farmacología , Ácidos Hidroxámicos/farmacología , Linfocitos T CD4-Positivos/química , Linfocitos T CD4-Positivos/efectos de los fármacos , Regulación Viral de la Expresión Génica/efectos de los fármacos , Duplicado del Terminal Largo de VIH , VIH-1/genética , Humanos , Cinética , Transcripción Reversa/efectos de los fármacos , Integración Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Replicación Viral/efectos de los fármacos , VorinostatRESUMEN
Human parainfluenza virus type 3 (HPIV3), one of the paramyxoviruses, uses its accessory C protein as an antagonist against interferon (IFN)-mediated host innate immunity. We have previously shown that the C protein significantly decreased the IFN-induced phosphorylation of signal transducer and activator of transcription (Stat) 1 and the formation of gamma IFN activation factor (GAF) complex, thus abrogating the antiviral activity of the IFNs against vesicular stomatitis virus (VSV) replication. Here, by mutational analyses we demonstrated that the N-terminal truncation of the C protein (CNdelta25 and CNdelta50) substantially (approximately 50%) recovers the IFN-induced responses, suggesting the critical role of the N-terminal region of the C protein in IFN signaling. Furthermore, our results indicate that the charged amino acid residues within the N-terminal region of the C protein regulate the antagonistic effect of the C protein on IFN signaling.
Asunto(s)
Interferones/metabolismo , Virus de la Parainfluenza 3 Humana/metabolismo , Transducción de Señal , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencia de Aminoácidos , Antivirales/metabolismo , Células HeLa , Humanos , Interferones/farmacología , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Fosforilación/efectos de los fármacos , Estructura Terciaria de Proteína , Factor de Transcripción STAT1/metabolismo , Transducción de Señal/efectos de los fármacos , Relación Estructura-Actividad , TYK2 Quinasa/antagonistas & inhibidores , Transcripción Genética/efectos de los fármacos , Vesiculovirus/efectos de los fármacos , Vesiculovirus/metabolismoRESUMEN
Human parainfluenza virus type 3 (HPIV3) is an important respiratory tract pathogen of infants and children. There are no vaccines or antivirals currently approved for prevention or treatment of HPIV3 infection. Towards developing an antiviral therapy to combat HPIV3 infection, we have established a green fluorescent protein (GFP)-tagged HPIV3 infected-cell assay and used it for screening of a small molecule library obtained from ChemBridge Diver. Two novel small molecules (C5 and C7) which shared structural similarities were identified and their inhibitory effects on HPIV3 were confirmed in CV-1 and human lung epithelium A549 cells by plaque assay, Western blot and Northern blot analyses. C5 and C7 effectively prevented the cytopathic effect in cells infected with HPIV3, achieving IC(50) values of 2.36 microM and 0.08 microM, respectively, for infectious virus production. The inhibition appears to be at the primary transcriptional level of HPIV3 life cycle based on sequential time course test, binding and internalization assays, and finally by a minigenome transcription assay in cells as well as measuring viral transcripts in cells in the presence of anisomycin. Interestingly, vesicular stomatitis virus (VSV), another member of mononegavirales order, was also inhibited by these compounds, whereas poliovirus-a picornavirus was not. Use of these inhibitors has a strong potential to develop novel antiviral agents against this important human pathogen.
Asunto(s)
Antivirales/farmacología , Virus de la Parainfluenza 3 Humana/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/farmacología , Animales , Línea Celular , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , Evaluación Preclínica de Medicamentos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Estructura Molecular , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Transcripción Genética/efectos de los fármacos , Ensayo de Placa Viral , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
It has been suggested that cellular proteins are involved in hepatitis C virus (HCV) RNA replication. By using the yeast two-hybrid system, we isolated seven cDNA clones encoding proteins interacting with HCV RNA polymerase (NS5B) from a human liver cDNA library. For one of these, alpha-actinin, we confirmed the interaction by coimmunoprecipitation, immunofluorescent staining and confocal microscopic analysis. Experiments with deletion mutants showed that domains NS5B(84-95), NS5B(466-478), and alpha-actinin(621-733) are responsible for the interaction. Studies of the HCV subgenomic replicon system with small interference RNA indicate that alpha-actinin is essential for HCV RNA replication. Our results suggest alpha-actinin may be a component of the HCV replication complex.
Asunto(s)
Actinina/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Hepacivirus/metabolismo , Actinina/química , Actinina/genética , Animales , Células COS , Línea Celular Tumoral , Chlorocebus aethiops , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Biblioteca de Genes , Hepacivirus/genética , Hepacivirus/ultraestructura , Humanos , Microscopía Confocal , Pruebas de Precipitina , Estructura Terciaria de Proteína , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/farmacología , ARN Viral/biosíntesis , ARN Viral/genética , ARN Viral/metabolismo , Replicón/genética , Eliminación de Secuencia , Técnicas del Sistema de Dos Híbridos , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiologíaRESUMEN
AIM: To establish an efficient, sensitive, cell-based assay system for NS3 serine protease in an effort to study further the property of hepatitis C virus (HCV) and develop new antiviral agents. METHODS: We constructed pCI-neo-NS3/4A-SEAP chimeric plasmid, in which the secreted alkaline phosphatase (SEAP) was fused in-frame to the downstream of NS4A/4B cleavage site. The protease activity of NS3 was reflected by the activity of SEAP in the culture media of transient or stable expression cells. Stably expressing cell lines were obtained by G418 selection. Pefabloc SC, a potent irreversible serine protease inhibitor, was used to treat the stably expressing cell lines to assess the system for screening NS3 inhibitors. To compare the activity of serine proteases from 1b and 1a, two chimeric clones were constructed and introduced into both transient and stable expression systems. RESULTS: The SEAP activity in the culture media could be detected in both transient and stable expression systems, and was apparently decreased after Pefabloc SC treatment. In both transient and stable systems, NS3/4A-SEAP chimeric gene from HCV genotype 1b produced higher SEAP activity in the culture media than that from 1a. CONCLUSION: The cell-based system is efficient and sensitive enough for detection and comparison of NS3 protease activity, and screening of anti-NS3 inhibitors. The functional difference between NS3/4A from 1a and 1b subtypes revealed by this system provides a clue for further investigations.
Asunto(s)
Hepacivirus/genética , Hepatitis C/virología , Proteínas no Estructurales Virales/genética , Animales , Northern Blotting , Western Blotting , Células COS , Carcinoma Hepatocelular , Línea Celular Tumoral , Regulación Viral de la Expresión Génica , Genotipo , Hepacivirus/enzimología , Humanos , Neoplasias Hepáticas , ARN Mensajero/análisis , Proteínas Recombinantes de Fusión/genética , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Transfección , Proteínas no Estructurales Virales/metabolismoRESUMEN
The C protein of human parainfluenza virus type 3 (HPIV3) is a multifunctional accessory protein that inhibits viral transcription and interferon (IFN) signaling. In the present study, we found that removal of N-terminal 25 or 50 amino acid residues from the C protein (CNDelta25 or CNDelta50) totally abolished viral RNA synthesis in the HPIV3 minigenome system. Further N-terminal or C-terminal deletion impaired the inhibitory ability of CNDelta25 and CNDelta50. Subsequent mutagenesis analysis suggested that the N-terminal-charged amino acid residues (K3, K6, K12, E16, and R24) contribute to the higher inhibition caused by CNDelta25 than the C protein. Consistent with viral RNA synthesis inhibition, the growth of HPIV3 was significantly decreased by 5 logs in HeLa-derived cell line expressing CNDelta25. Interestingly, replication of respiratory syncytial virus (RSV), another important respiratory tract pathogen, was also strongly inhibited in the presence of CNDelta25. These findings provide a promising potential to use CNDelta25 as an antiviral agent against the clinically important respiratory tract diseases caused by HPIV3 and RSV.
Asunto(s)
Antivirales/farmacología , Virus de la Parainfluenza 3 Humana/fisiología , Eliminación de Secuencia , Proteínas Virales/genética , Replicación Viral/efectos de los fármacos , Células HeLa , Humanos , Proteínas Mutantes/genética , Proteínas Mutantes/farmacología , Virus de la Parainfluenza 3 Humana/efectos de los fármacos , ARN Viral/biosíntesis , Virus Sincitiales Respiratorios/fisiología , Proteínas Virales/farmacologíaRESUMEN
A method for monitoring foodborne pathogenic bacteria by multiplex polymerase chain reaction (PCR)--capillary electrophoresis (CE) with a laser induced fluorescence detector was developed. Three sets of primers were designed to amplify the gene segments of uidA gene in E. coli. O157:H7, invA gene in salmonella and ipaH gene in Shigella, individually. The multiple PCR system and the separation conditions of CE were optimized. Using a capillary coated with linear polyacrylamide and sieving buffer of 7.0 g/L methyl cellulose (MC) under 8.3 kV of electric voltage, the proposed method was able to simultaneously detect the PCR products of specific genes existing in the three kinds of pathogenic bacteria within 22 min. The relative standard deviations of migration time for the detected DNA fragments were ranging from 1.47% to 2.07%. In comparison with agarose gel electrophoresis, the proposed method is rapid, sensitive and accurate.
Asunto(s)
Electroforesis Capilar/métodos , Microbiología de Alimentos , Reacción en Cadena de la Polimerasa/métodos , Espectrometría de Fluorescencia/métodos , Antígenos Bacterianos/genética , Antígenos Bacterianos/aislamiento & purificación , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , ADN Bacteriano/genética , Electroforesis en Gel de Agar , Reproducibilidad de los ResultadosRESUMEN
RNase L, a principal mediator of innate immunity to viral infections in higher vertebrates, is required for a complete IFN antiviral response against certain RNA stranded viruses. dsRNA produced during viral infections activates IFN-inducible synthetases that produce 5'-phosphorylated, 2',5'-oligoadenylates (2-5A) from ATP. 2-5A activates RNase L in a wide range of different mammalian cell types, thus blocking viral replication. However, 2-5A has unfavorable pharmacologic properties; it is rapidly degraded, does not transit cell membranes, and leads to apoptosis. To obtain activators of RNase L with improved drug-like properties, high-throughput screening was performed on chemical libraries by using fluorescence resonance energy transfer. Seven compounds were obtained that activated RNase L at micromolar concentrations, and structure-activity relationship studies resulted in identification of an additional four active compounds. Two lead compounds were shown to have a similar mechanistic path toward RNase L activation as the natural activator 2-5A. The compounds bound to the 2-5A-binding domain of RNase L (as determined by surface plasmon resonance and confirmed by computational docking), and the compounds induced RNase L dimerization and activation. Interestingly, the low-molecular-weight activators of RNase L had broad-spectrum antiviral activity against diverse types of RNA viruses, including the human pathogen human parainfluenza virus type 3, yet these compounds by themselves were not cytotoxic at the effective concentrations. Therefore, these RNase L activators are prototypes for a previously uncharacterized class of broad-spectrum antiviral agents.
Asunto(s)
Antivirales/metabolismo , Endorribonucleasas/metabolismo , Activadores de Enzimas/metabolismo , Inmunidad Innata/fisiología , Virus de la Parainfluenza 3 Humana/metabolismo , Nucleótidos de Adenina/metabolismo , Animales , Antivirales/farmacología , Línea Celular , Cromatografía Líquida de Alta Presión , Dimerización , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Activadores de Enzimas/farmacología , Transferencia Resonante de Energía de Fluorescencia , Ratones , Modelos Moleculares , Oligonucleótidos/genética , Oligorribonucleótidos/metabolismo , Virus de la Parainfluenza 3 Humana/efectos de los fármacos , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Resonancia por Plasmón de Superficie , Replicación Viral/efectos de los fármacosRESUMEN
A method for the determination of chlorhexidine acetate in disinfectors using capillary electrophoresis (CE) was developed using the following conditions: detection wavelength, 254 nm; applied voltage, 15 kV; uncoated fused silica capillary column, 50 cm x 75 microm i.d.; buffer, 15 mmol/L phosphate and acetonitrile (60:40, v/v). Under the optimum conditions, chlorhexidine acetate in disinfectors was determinated in 4 min. The effects of different parameters, such as buffer component, buffer concentration, buffer pH value and electrophoretic voltage, on the CE analysis of chlorhexidine acetate were studied in detail. The linear range of the proposed method was 0.01-0.10 g/L. The detection limit was 0.004 mg/L. The relative standard deviation (RSD) of absorbance was 3.97% and the RSD of migration time was 2.99%. The spiked recoveries of samples were 91.4%-116.6%. The method was compared with the high performance liquid chromatographic method and the relative error was less than 4.0%. The proposed method was simple, quick and suitable for disinfector analysis.
Asunto(s)
Clorhexidina/análisis , Desinfectantes/análisis , Electroforesis CapilarRESUMEN
BACKGROUND: To construct the full-length complementary DNA of HCV genome from an HCV infected patient. METHODS: Four HCV gene fragments (1.6, 3.5, 2.4 kb and 2.6 kb) were amplified by RT-PCR from serum of a Chinese patient and fused and connected together to produce a 9.2 kb subgenomic fragment, which was further cloned into a cassette vector with fixed 5-prime and 3-prime termini of HCV to make the full-length cDNA. The cDNA heterogeneity was analyzed by comparing the sequences of 4 isolated HVR1 regions. The prokaryotic expressed Core, NS3 protease, NS3 helicase were detected for their specific reactivities with patient serum by Western blot analysis. And the protease activity of NS3 was evaluated in a cell-based NS3/4A-SEAP expression system. RESULTS: The cDNA covered the near full-length of HCV genome from the patient's serum. The difference among HVR1 regions indicated no selection of HCV variants during RT-PCR and the quasi-species characteristic of the amplified cDNA. The prokaryotic expressed viral proteins could be identified by patient serum. In the NS3/4A-SEAP system, NS3 could cleave the 4A-4B site between NS4A and SEAP proteins and resulted in the secretion of SEAP in culture media. CONCLUSION: These results suggest that the cloned HCV cDNA encodes a complete and functional open reading frame and will be useful for further construction of infectious cDNA clone.