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1.
Virus Res ; 304: 198528, 2021 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-34339771

RESUMEN

Enterovirus D68 is an emerging respiratory disease pathogen causing multiple outbreaks worldwide. Enterovirus D68 strain US/KY/14-18953 was adapted to propagate in Vero cells resulting alteration of seven amino acids. The Vero cell adapted virus was inactivated with Formalin and immunized in mice. Formalin inactivated vaccine elicited high virus specific IgG antibody titer and neutralization titer. Avidity of the IgG antibodies elicited by two different doses of formalin inactivated vaccine is moderately high which got augmented by alum adjuvanted formulations. Formalin inactivated unadjuvanted vaccine elicited a balanced IgG1 type and IgG2a type antibody indicating a more balanced Th2/Th1 type immune response while alum formulated formalin inactivated antigen elicited significantly high IgG1 antibody in immunized sera and Th2 cytokines in mice splenocytes denoting Th2 type T cell immune response. Additionally, the formalin inactivated vaccine formulations has displayed excellent serum mediated invivo protective efficacy. These data suggested that formalin inactivated Enterovirus D68 is a promising vaccine candidate.


Asunto(s)
Enterovirus Humano D , Vacunas Virales , Adyuvantes Inmunológicos , Animales , Anticuerpos Neutralizantes , Anticuerpos Antivirales , Chlorocebus aethiops , Formaldehído , Inmunoglobulina G , Ratones , Ratones Endogámicos BALB C , Vacunas de Productos Inactivados , Células Vero
2.
J Biol Chem ; 287(29): 24387-96, 2012 Jul 13.
Artículo en Inglés | MEDLINE | ID: mdl-22532564

RESUMEN

Because human prostate-distributed UDP-glucuronosyltransferase (UGT) 2B15 metabolizes 5α-dihydrotestosterone (DHT) and 3α-androstane-5α,17ß-diol metabolite, we sought to determine whether 2B15 requires regulated phosphorylation similar to UGTs already analyzed. Reversible down-regulation of 2B15-transfected COS-1 cells following curcumin treatment and irreversible inhibition by calphostin C, bisindolylmaleimide, or röttlerin treatment versus activation by phorbol 12-myristate 13-acetate indicated that 2B15 undergoes PKC phosphorylation. Mutation of three predicted PKC and two tyrosine kinase sites in 2B15 caused 70-100 and 80-90% inactivation, respectively. Anti-UGT-1168 antibody trapped 2B15-His-containing co-immunoprecipitates of PKCα in 130-140- and >150-kDa complexes by gradient SDS-PAGE analysis. Complexes bound to WT 2B15-His remained intact during electrophoresis, whereas 2B15-His mutants at phosphorylation sites differentially dissociated. PKCα siRNA treatment inactivated >50% of COS-1 cell-expressed 2B15. In contrast, treatment of 2B15-transfected COS-1 cells with the Src-specific activator 1,25-dihydroxyvitamin D(3) enhanced activity; treatment with the Src-specific PP2 inhibitor or Src siRNA inhibited >50% of the activity. Solubilized 2B15-His-transfected Src-free fibroblasts subjected to in vitro [γ-(33)P]ATP-dependent phosphorylation by PKCα and/or Src, affinity purification, and SDS gel analysis revealed 2-fold more radiolabeling of 55-58-kDa 2B15-His by PKCα than by Src; labeling was additive for combined kinases. Collectively, the evidence indicates that 2B15 requires regulated phosphorylation by both PKCα and Src, which is consistent with the complexity of synthesis and metabolism of its major substrate, DHT. Whether basal cells import or synthesize testosterone for transport to luminal cells for reduction to DHT by 5α-steroid reductase 2, comparatively low-activity luminal cell 2B15 undergoes a complex pattern of regulated phosphorylation necessary to maintain homeostatic DHT levels to support occupation of the androgen receptor for prostate-specific functions.


Asunto(s)
Dihidrotestosterona/metabolismo , Glucuronosiltransferasa/metabolismo , Proteína Quinasa C-alfa/metabolismo , Familia-src Quinasas/metabolismo , Animales , Células COS , Inhibidores Enzimáticos/farmacología , Glucuronosiltransferasa/genética , Humanos , Inmunoprecipitación , Masculino , Ratones , Mutación , Fosforilación/genética , Fosforilación/fisiología , Unión Proteica , Proteína Quinasa C-alfa/antagonistas & inhibidores , Familia-src Quinasas/genética
3.
J Virol ; 85(24): 12881-9, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-21976647

RESUMEN

Hepatitis C virus (HCV) often leads to persistent infection. Interferon (IFN) and IFN-stimulated genes (ISGs) are amplified during HCV infection but fail to eliminate virus from the liver in a large number of infected patients. We have observed previously that HCV infection induces IFN-ß production in immortalized human hepatocytes (IHH) as early as 24 h after infection, although virus replication is not inhibited. To gain insights on possible countermeasures of virus for the suppression of host antiviral response, the cellular transcriptional profiles of ISGs were examined after various treatments of IHH. The majority of ISGs were upregulated in IFN-treated IHH from the level for mock-treated cells. However, the comparison of ISG expression in IFN-treated IHH and IFN-pretreated, HCV genotype 2a-infected IHH indicated that virus infection suppresses the upregulation of a subset of effector molecules, including ISG56 and IFITM1. Similar results were observed for HCV-infected Huh7 cells. Subsequent study suggested that the exogenous expression of ISG56 or IFITM1 inhibits HCV replication in IHH or Huh7 cells, and the knockdown of these genes enhanced HCV replication. Further characterization revealed that the overexpression of these ISGs does not block HCV pseudotype entry into Huh7 cells. Taken together, our results demonstrated that ISG56 and IFITM1 serve as important molecules to restrict HCV infection, and they may have implications in the development of therapeutic modalities.


Asunto(s)
Antígenos de Diferenciación/metabolismo , Hepacivirus/inmunología , Hepacivirus/patogenicidad , Interacciones Huésped-Patógeno , Factores de Transcripción/metabolismo , Replicación Viral , Proteínas Adaptadoras Transductoras de Señales , Línea Celular , Perfilación de la Expresión Génica , Hepacivirus/crecimiento & desarrollo , Hepatocitos/inmunología , Hepatocitos/virología , Humanos , Evasión Inmune , Proteínas de Unión al ARN
4.
Hepatology ; 53(2): 406-14, 2011 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-21274862

RESUMEN

UNLABELLED: The role of autophagy in disease pathogenesis following viral infection is beginning to be elucidated. We have previously reported that hepatitis C virus (HCV) infection in hepatocytes induces autophagy. However, the biological significance of HCV-induced autophagy has not been clarified. Autophagy has recently been identified as a novel component of the innate immune system against viral infection. In this study, we found that knockdown of autophagy-related protein beclin 1 (BCN1) or autophagy-related protein 7 (ATG7) in immortalized human hepatocytes (IHHs) inhibited HCV growth. BCN1- or ATG7-knockdown IHHs, when they were infected with HCV, exhibited increased expression of interferon-ß, 2',5'-oligoadenylate synthetase 1, interferon-α, and interferon-α-inducible protein 27 messenger RNAs of the interferon signaling pathways in comparison with infected control IHHs. A subsequent study demonstrated that HCV infection in autophagy-impaired IHHs displayed caspase activation, poly(adenosine diphosphate ribose) polymerase cleavage, and apoptotic cell death. CONCLUSION: The disruption of autophagy machinery in HCV-infected hepatocytes activates the interferon signaling pathway and induces apoptosis. Together, these results suggest that HCV-induced autophagy impairs the innate immune response.


Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Autofagia/genética , Hepacivirus/inmunología , Hepatocitos/virología , Inmunidad Innata/inmunología , Proteínas de la Membrana/genética , Enzimas Activadoras de Ubiquitina/genética , Apoptosis/genética , Apoptosis/fisiología , Autofagia/fisiología , Proteína 7 Relacionada con la Autofagia , Beclina-1 , Caspasas/metabolismo , Línea Celular , Línea Celular Tumoral , Hepacivirus/crecimiento & desarrollo , Hepatitis C Crónica/inmunología , Hepatitis C Crónica/metabolismo , Hepatitis C Crónica/patología , Hepatocitos/metabolismo , Hepatocitos/patología , Humanos , Interferones/metabolismo , ARN Interferente Pequeño/genética , Transducción de Señal/fisiología
5.
J Virol ; 84(21): 10991-8, 2010 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-20810735

RESUMEN

Hepatitis C virus (HCV) establishes chronic infection in a significant number of infected humans, although the mechanisms for chronicity remain largely unknown. We have previously shown that HCV infection in immortalized human hepatocytes (IHH) induces beta interferon (IFN-ß) expression (T. Kanda, R. Steele, R. Ray, and R. B. Ray, J. Virol. 81:12375-12381, 2007). However, the regulation of the downstream signaling pathway for IFN-α production by HCV is not clearly understood. In this study, the regulation of the IFN signaling pathway following HCV genotype 1a (clone H77) or genotype 2a (clone JFH1) infection of IHH was examined. HCV infection upregulated expression of total STAT1 but failed to induce phosphorylation and efficient nuclear translocation. Subsequent study revealed that HCV infection induces IFN-stimulated response element activation, as evidenced by upregulation of 2',5'-oligoadenylate synthetase 1. However, nuclear translocation of IRF-7 was impaired following HCV infection. In HCV-infected IHH, IFN-α expression initially increased (up to 24 h) and then decreased at later time points, and IFN-α-inducible protein 27 was not induced. Interestingly, HCV infection blocked IRF-7 nuclear translocation upon poly(I-C) or IFN-α treatment of IHH. Together, our data suggest that HCV infection enhances STAT1 expression but impairs nuclear translocation of IRF-7 and its downstream molecules. These impairments in the IFN-α signaling pathway may, in part, be responsible for establishment of chronic HCV infection.


Asunto(s)
Transporte Activo de Núcleo Celular , Hepatocitos/virología , Factor 7 Regulador del Interferón/metabolismo , Interferón-alfa/biosíntesis , Factor de Transcripción STAT1/genética , Línea Celular , Hepatocitos/metabolismo , Humanos , Fosforilación , Transducción de Señal , Regulación hacia Arriba/genética
6.
Cancer Res ; 70(5): 1925-31, 2010 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-20179194

RESUMEN

Breast cancer is one of the most common cancers among women in the United States. Although there are effective drugs for treating advanced stages of breast cancers, women eventually develop resistance. One of the approaches to control breast cancer is prevention through diet, which inhibits one or more neoplastic events and reduces cancer risk. In this study, we have used human breast cancer cells, MCF-7 and MDA-MB-231, and primary human mammary epithelial cells as an in vitro model to assess the efficacy of bitter melon (Momordica charantia) extract (BME) as an anticancer agent. BME treatment of breast cancer cells resulted in a significant decrease in cell proliferation and induced apoptotic cell death. Apoptosis of breast cancer cells was accompanied by increased poly(ADP-ribose) polymerase cleavage and caspase activation. Subsequent studies showed that BME treatment of breast cancer cells inhibited survivin and claspin expression. Fluorescence-activated cell sorting analysis suggested that MCF-7 cells treated with BME accumulated during the G2-M phase of the cell cycle. Further studies revealed that BME treatment enhanced p53, p21, and pChk1/2 and inhibited cyclin B1 and cyclin D1 expression, suggesting an additional mechanism involving cell cycle regulation. Together, these results show that BME modulates signal transduction pathways for inhibition of breast cancer cell growth and can be used as a dietary supplement for prevention of breast cancer.


Asunto(s)
Apoptosis/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Genes cdc/efectos de los fármacos , Momordica charantia/química , Extractos Vegetales/farmacología , Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/biosíntesis , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Caspasas/metabolismo , Proteínas de Ciclo Celular/antagonistas & inhibidores , Proteínas de Ciclo Celular/genética , División Celular/efectos de los fármacos , División Celular/genética , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Activación Enzimática/efectos de los fármacos , Femenino , Fase G2/efectos de los fármacos , Fase G2/genética , Humanos , Proteínas Inhibidoras de la Apoptosis , Proteínas Asociadas a Microtúbulos/antagonistas & inhibidores , Proteínas Asociadas a Microtúbulos/biosíntesis , Survivin
7.
FEMS Microbiol Lett ; 303(2): 107-15, 2010 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-20030720

RESUMEN

Chronological analysis of 125 Vibrio cholerae O139 strains isolated during 1993-2005 in Kolkata revealed the prevalence of two new genotypes of cholera toxin (CT) and novel combinations of ctxB and rstR alleles resulting in variant CTX prophages. One of the new genotypes of ctxB, which first appeared in 1996 with the re-emerged V. cholerae O139 strains that had CTX Calcutta phage, was designated as genotype 4. In 1998, another new genotype, designated as genotype 5, was detected that prevailed mostly in CTX phages with El Tor rstR. The prototype El Tor CTX phage with genotype 3 gradually disappeared in O139, and since 2002 the predominant CTX prophages in O139 are Calcutta phages with genotype 4 and El Tor phages with genotype 5. Results showed that V. cholerae O139 strains of Kolkata, isolated over a decade, harboured CTX prophages in the large chromosome having no RS1 downstream of CTX prophage. During the course of its intermittent incidence over a decade, five types of O139 strains were detected based on CT genotypes. Such abrupt genetic changes in O139 strains might not favour its continued prevalence in human cases in Kolkata, India.


Asunto(s)
Proteínas Bacterianas/genética , Toxina del Cólera/genética , Profagos/genética , Proteínas Represoras/genética , Vibrio cholerae O139/genética , Vibrio cholerae O139/virología , Alelos , Cólera/microbiología , Evolución Molecular , Genotipo , Humanos , India , Epidemiología Molecular , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , Vibrio cholerae O139/aislamiento & purificación
8.
Cancer Res ; 69(24): 9354-9, 2009 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-19934312

RESUMEN

Breast cancer is the leading cause of cancer death among women. We have shown previously an antiproliferative effect of MBP-1 on several human cancer cells. In this study, we have examined the potential of MBP-1 as a gene therapeutic candidate in regression of breast cancer growth and metastasis in an immunocompetent mouse model. For this, we have used a mouse breast cancer cell line (EO771) and syngeneic C57BL/6 mice. EO771 cells were implanted into the mammary fat pad of C57BL/6 mice. Replication-deficient recombinant adenovirus expressing MBP-1 was administered intratumorally to determine gene therapeutic potential. The results showed a significant regression of primary and distant (lung) tumor growth. Animals exhibited prolonged survival on treatment with MBP-1 compared with the control group (dl312). Subsequent studies suggested that MBP-1 inhibits matrix metalloproteinase expression in human breast cancer cells. Cells transduced with MBP-1 displayed inhibition of migration in a wound-healing assay. The conditioned medium from MBP-1-transduced cells blocked in vitro tube formation assay and inhibited expression of several angiogenic molecules. Taken together, our study shows that MBP-1 acts as a double-edged sword by inhibiting primary and metastatic tumor growth and modulating matrix metalloproteinase expression with a therapeutic potential against breast cancer progression.


Asunto(s)
Proteínas de Unión al ADN/biosíntesis , Neoplasias Mamarias Experimentales/terapia , Adenoviridae/genética , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Procesos de Crecimiento Celular/genética , Línea Celular Tumoral , Movimiento Celular/fisiología , Proteínas de Unión al ADN/genética , Células Endoteliales/patología , Terapia Genética/métodos , Humanos , Inmunocompetencia , Neoplasias Mamarias Experimentales/irrigación sanguínea , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/patología , Metaloproteinasa 2 de la Matriz/biosíntesis , Metaloproteinasa 2 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/metabolismo , Neovascularización Patológica/patología
9.
J Med Microbiol ; 58(Pt 2): 239-247, 2009 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-19141743

RESUMEN

Retrospective analysis led to the detection of two Vibrio cholerae variant O1 strains (VC51 and VC53), which were isolated in 1992 in Kolkata from clinical cases, with identical traits to 2004 Mozambique variant O1 strains. The Mozambique O1 strains that caused a huge outbreak in 2004 have been shown to have phenotypic traits of both classical and El Tor biotypes, and thereby have been reported as variant. Our study demonstrated that two O1 strains isolated in Kolkata during 1992 were of the El Tor background as evidenced by polymyxin B (50 U ml(-1)) resistance, positivity in Voges-Proskauer reactions and sensitivity to biotype-specific vibrio phages. With the features of classical CTX prophage, localization in the small chromosome, and an absence of RS1 and pTLC, both Mozambique and Kolkata strains appeared to be identical. Furthermore, two Kolkata strains exhibited an identical ribotype to that of the Mozambique variant, displaying ribotype pattern RI that had been assigned to Kolkata V. cholerae O1 strains isolated on or before 1992. NotI pulsotype analysis indicated that these 1992 Kolkata strains along with the Mozambique variant O1 belonged to very closely related clones. Considering the chronological events, and the typical identity at the phenotypic and the genotypic level between the two O1 strains isolated during 1992 from Kolkata and during 2004 from Mozambique, we propose that some of the 1992 Kolkata O1 strains might have acted as progenitors for Mozambique variant O1 strains.


Asunto(s)
Cólera/microbiología , Vibrio cholerae O1/clasificación , Vibrio cholerae O1/aislamiento & purificación , Antibacterianos/farmacología , Técnicas de Tipificación Bacteriana , Bacteriófagos/crecimiento & desarrollo , Cromosomas Bacterianos , Dermatoglifia del ADN , ADN Bacteriano/genética , Electroforesis en Gel de Campo Pulsado , Genotipo , Humanos , India , Epidemiología Molecular , Plásmidos , Polimixina B/farmacología , Profagos/genética , Estudios Retrospectivos , Ribotipificación , Vibrio cholerae O1/genética , Vibrio cholerae O1/fisiología
11.
Indian J Med Res ; 128(6): 695-8, 2008 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-19246791

RESUMEN

Considering the recent emergence of "hybrid biotype" and "El Tor variant", we propose to redefine the biotyping scheme for Vibrio cholerae O1 serogroup. The existing biotyping scheme has limitations and causes confusion as many of the hybrid biotype and El Tor variant strains have phenotypic and genetic changes. A revised biotyping scheme will play a significant role to understand the ecology, epidemiology and nature of infection of V. cholerae O1 strains in future.


Asunto(s)
Técnicas de Tipificación Bacteriana/métodos , Vibrio cholerae/clasificación , Toxina del Cólera/clasificación , Genotipo , Vibrio cholerae/genética
12.
J Infect ; 55(5): 431-8, 2007 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-17854903

RESUMEN

OBJECTIVES: To study the phenotypic and genotypic characterization of newly emerged V. cholerae O1, Inaba strains isolated from patients with diarrhoea. METHODS: Bacterial characterization was made using polymerase chain reaction, ribotyping, PFGE and RFLP. RESULTS: After its first appearance in July 2004, O1 Inaba became the dominant serotype by March 2005 and totally replaced the former dominant serotype, Ogawa from May 2005. Most of the Inaba isolates belong to a new ribotype RIV. Ogawa and also some Inaba strains isolated during the same period were identified as RIII. Similarly, the majority of the Inaba isolates belong to 'H1' pulsotype and one isolate is type 'H', while the Ogawa isolates were mostly 'H' pulsotype. Presence of CTX prophage was detected in a single site of the chromosome with at least two RS elements. CONCLUSIONS: There has been a switch of dominant serotype from Ogawa to Inaba in the Kolkata region. This is not necessarily due to emergence of a new clone but does serve as an epidemiological marker. Further analysis at the molecular level will be required to define this trend and to monitor future spread to other regions.


Asunto(s)
Cólera/microbiología , Epidemiología Molecular , Vibrio cholerae O1/clasificación , Vibrio cholerae O1/genética , Cólera/epidemiología , Toxina del Cólera/genética , Cromosomas Bacterianos/genética , Dermatoglifia del ADN , ADN Bacteriano/genética , Electroforesis en Gel de Campo Pulsado , Hospitalización , Humanos , India/epidemiología , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Profagos/genética , Ribotipificación , Vibrio cholerae O1/aislamiento & purificación
13.
J Med Microbiol ; 56(Pt 6): 824-832, 2007 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-17510270

RESUMEN

During 2003, Vibrio cholerae O1 Ogawa was the predominant serotype among diarrhoeal patients admitted to different hospitals in India. With the exception of 3 strains from Kolkata, none of 172 strains examined exhibited resistance to tetracycline, but 45.7 % showed reduced susceptibility to ciprofloxacin. Extensive molecular characterization using randomly amplified polymorphic DNA analysis, ribotyping and PFGE revealed that almost all the strains within a serogroup were clonally related. Along with the H pulsotype, a newly described L pulsotype of recently emerged O1 Inaba strains was detected among the O1 Ogawa strains from 2003. The striking similarity in their molecular properties and antibiograms indicated that at least certain clones of recently emerged Inaba strains from 2004 may have evolved from O1 Ogawa strains. This view was further supported by the detection of a nearly identical wbeT region among the O1 Ogawa and recently emerged Inaba strains, the latter differing only by a single point mutation. Since 2003, a hiatus in the isolation of serogroup O139 was observed and these strains share the same PFGE profiles as those isolated during 2000. Organization of tandemly arranged CTX(El), CTX(Cal) and truncated CTX(Cal) (devoid of ctxAB) prophages was unique among the majority of these O139 strains.


Asunto(s)
Cólera/epidemiología , Cólera/microbiología , Vibrio cholerae O139/genética , Vibrio cholerae O139/fisiología , Vibrio cholerae O1/genética , Vibrio cholerae O1/fisiología , Antibacterianos/farmacología , Cólera/virología , Ciprofloxacina/farmacología , Análisis por Conglomerados , Dermatoglifia del ADN , ADN Bacteriano/genética , ADN Viral/genética , Farmacorresistencia Bacteriana , Electroforesis en Gel de Campo Pulsado , Evolución Molecular , Genotipo , Humanos , India/epidemiología , Epidemiología Molecular , Fenotipo , Profagos/genética , Técnica del ADN Polimorfo Amplificado Aleatorio , Ribotipificación , Tetraciclina/farmacología , Vibrio cholerae O1/aislamiento & purificación , Vibrio cholerae O139/aislamiento & purificación , Factores de Virulencia/genética
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