RESUMO
Junin virus (JUNV), a highly pathogenic New World arenavirus, is the causative agent of Argentine hemorrhagic fever (AHF). The live-attenuated Candid #1 (Can) strain currently serves as a vaccine for at-risk populations. We have previously shown that the Can glycoprotein (GPC) gene is the primary gene responsible for attenuation in a guinea pig model of AHF. However, the mechanisms through which the GPC contributes to the attenuation of the Can strain remain unknown. A more complete understanding of the mechanisms underlying the attenuation and immunogenicity of the Can strain will potentially allow for the rational design of additional safe and novel vaccines. Here, we provide a detailed comparison of both RNA and protein expression profiles between both inter- and intra-segment chimeric JUNV recombinant clones expressing combinations of genes from the Can strain and the pathogenic Romero (Rom) strain. The recombinant viruses that express Can GPC, which were shown to be attenuated in guinea pigs, displayed different RNA levels and GPC processing patterns as determined by Northern and Western blot analyses, respectively. Analysis of recombinant viruses containing amino acid substitutions selected at different mouse brain passages during the generation of Can revealed that altered Can GPC processing was primarily due to the T168A substitution within G1, which eliminates an N-linked glycosylation motif. Incorporation of the T168A substitution in the Rom GPC resulted in a Can-like processing pattern of Rom GPC. In addition, JUNV GPCs containing T168A substitution were retained within the endoplasmic reticulum (ER) and displayed significantly lower cell surface expression than wild-type Rom GPC. Interestingly, the reversion A168T in Can GPC significantly increased GPC expression at the cell surface. Our results demonstrate that recombinant JUNV (rJUNV) expressing Can GPC display markedly different protein expression and elevated genomic RNA expression when compared to viruses expressing Rom GPC. Additionally, our findings indicate that the N-linked glycosylation motif at amino acid positions 166-168 is important for trafficking of JUNV GPC to the cell surface, and the elimination of this motif interferes with the GPC release from the ER.
Assuntos
Motivos de Aminoácidos , Arenavirus do Novo Mundo/imunologia , Glicoproteínas/genética , Glicoproteínas/metabolismo , Febre Hemorrágica Americana , Vacinas Virais , Animais , Arenavirus do Novo Mundo/genética , Linhagem Celular , Células Cultivadas , Cricetinae , Retículo Endoplasmático/metabolismo , Estresse do Retículo Endoplasmático , Expressão Gênica , Regulação Viral da Expressão Gênica , Glicoproteínas/química , Glicoproteínas/imunologia , Glicosilação , Febre Hemorrágica Americana/imunologia , Febre Hemorrágica Americana/metabolismo , Febre Hemorrágica Americana/prevenção & controle , Febre Hemorrágica Americana/virologia , Humanos , Processamento de Proteína Pós-Traducional , Transporte Proteico , Transcrição Gênica , Vacinas Virais/genética , Vacinas Virais/imunologia , VirulênciaRESUMO
OBJECTIVE: To determine the prevalence of JAK2 V617F mutation and its clinical correlation in patients with chronic myeloproliferative disorders (CMD): polycythemia vera (PV), essential thrombocythemia (ET) and idiopathic myelofibrosis (IMF). MATERIALS AND METHODS: Detection of JAK2 V617F mutation by allele specific-PCR. RESULTS: One hundred and three patients with CMD were included in the study. JAK2 V617F distribution was PV 40/45 (89%), ET 30/43 (69%), and IMF 7/15 (47%). In PV and ET patients only, 18 had thrombosis at diagnosis and 12 during follow-up (these were microvascular: 11, venous: 7 and arterial: 12); of these 28/70 (40%) were JAK2pos versus 2/18 (11%) JAK2neg; P=0.02. In a median of 4 years, two patients with PV JAK2pos evolved to myelofibrosis and one patient with PV presented in leukemic transformation (JAK2pos before and after transformation); six patients died: four patients with IMF and two patients with PV. CONCLUSIONS: We found an association between JAK2 V617F and thrombotic events in patients with PV and ET.
Assuntos
Alelos , Janus Quinase 2/genética , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Doença Crônica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos Mieloproliferativos/complicações , Reação em Cadeia da Polimerase , Estudos Prospectivos , Trombose/etiologia , Trombose/genéticaRESUMO
Autosomal recessive nonsyndromic hearing impairment (NSHI) is a heterogeneous condition, for which 53 genetic loci have been reported, and 29 genes have been identified to date. One of these, OTOF, encodes otoferlin, a membrane-anchored calcium-binding protein that plays a role in the exocytosis of synaptic vesicles at the auditory inner hair cell ribbon synapse. We have investigated the prevalence and spectrum of deafness-causing mutations in the OTOF gene. Cohorts of 708 Spanish, 83 Colombian, and 30 Argentinean unrelated subjects with autosomal recessive NSHI were screened for the common p.Gln829X mutation. In compound heterozygotes, the second mutant allele was identified by DNA sequencing. In total, 23 Spanish, two Colombian and two Argentinean subjects were shown to carry two mutant alleles of OTOF. Of these, one Colombian and 13 Spanish subjects presented with auditory neuropathy. In addition, a cohort of 20 unrelated subjects with a diagnosis of auditory neuropathy, from several countries, was screened for mutations in OTOF by DNA sequencing. A total of 11 of these subjects were shown to carry two mutant alleles of OTOF. In total, 18 pathogenic and four neutral novel alleles of the OTOF gene were identified. Haplotype analysis for markers close to OTOF suggests a common founder for the novel c.2905_2923delinsCTCCGAGCGCA mutation, frequently found in Argentina. Our results confirm that mutation of the OTOF gene correlates with a phenotype of prelingual, profound NSHI, and indicate that OTOF mutations are a major cause of inherited auditory neuropathy.
Assuntos
Perda Auditiva Neurossensorial/genética , Proteínas de Membrana/genética , Argentina , Colômbia , Feminino , Genes Recessivos , Humanos , Masculino , Mutação , EspanhaRESUMO
PURPOSE: We describe a program for the promotion of hearing conservation aimed at the adolescent population. The intent of our program is to (a) detect hearing disorders early, as well as to establish their relation to psychosocial and acoustic factors; (b) devise a follow-up procedure to study relevant variables; (c) evaluate the relation between hearing disorders and genetic factors, and (d) raise the social awareness of the effects of noise and its consequences. METHOD: This program, designed to be carried out over a 7-year period, focuses on participants from technical schools in the city of Cordoba, Argentina. Every student will be examined at age 14-15 years and will be reexamined at age 17-18. There will be a yearly follow-up in those cases in which disorders are detected. RESULTS AND CONCLUSIONS: We discuss the organization and planning of this program, together with its launching in the first of the selected schools. We also describe the findings on the following topics: (a) the hearing data on adolescents (age 14-15 years); (b) their recreational habits, personality traits, and attitudes; and (c) the sound immision characteristics these individuals are exposed to during recreational activities.
Assuntos
Promoção da Saúde , Transtornos da Audição/prevenção & controle , Desenvolvimento de Programas , Adolescente , Feminino , Humanos , Percepção Sonora , Masculino , Música , Avaliação de Programas e Projetos de Saúde , RecreaçãoRESUMO
Recent advances in molecular genetics as well as improved strategies for the prevention and control of non-syndromic hearing loss (NSHL) have contributed to the rising importance of their inherited causes. In this study we report 32 families from Argentine with one (sporadic) or more (familial) individuals affected. All the families were initially screened for mutations in three autosomal nuclear genes and one mutation in mitochondrial DNA. These genes have been found in a great number of familial or sporadic cases of congenital deafness in Caucasians. The mutant allele 35 del G of connexin 26 (GJB2, locus DFNB1 on 13q12) was present in three families. We have investigated the gene encoding otoferlin (OTOF, locus DFNB9 on 2p22-p23) and we found the Q829X mutation in heterocigosity in two families. We have also identified in heterocigosity the 342-kb deletion of connexin 30 (GJB6, locus DFNB1 on 13q12) in one family. On the other hand, we have not found any patient with mitochondrial mutation. Since the screening for other mutations is very expensive, our main goal is to investigate the most frequent mutations in each separate gene in the argentine population and to develop simple and specific tests for each frequent mutations.
Assuntos
Perda Auditiva/genética , Mutação , Adulto , Argentina , Criança , Conexina 26 , Conexinas , Surdez/diagnóstico , Surdez/genética , Feminino , Deleção de Genes , Aconselhamento Genético , Perda Auditiva/diagnóstico , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/genética , Humanos , Masculino , LinhagemRESUMO
Los últimos avances en genética molecular como así también el desarrollo de estrategias para la prevención y control de las hipoacusias no sindrómicas (HNS), han contribuido al esclarecimiento de las causas hereditarias de las mismas. En este estudio, se seleccionaron 32 familias argentinas con uno (esporádico) o más (familiar) individuos afectados. El análisis genético consistió en la búsqueda de tres genes autosómicos nucleares y uno en el ADN mitocondrial. Estos genes se localizaron en un gran número de casos familiares o esporádicos de sorderas congénitas en Caucásicos. El alelo mutado 35 del G de la conexina 26 (GJB2, locus DFNB1 en 13g12) se presentó en tres familias. Además se investigó el gen que codifica otoferlina (OTOF locus DFNB9 en 2p22-23) encontrándose en dos familias la mutación Q829X en heterocigocidad. También se identificó en una familia portadora heterocigota la deleción de 342 Kb en la conexina 30 (GJB6, locus DFNB1 en 13g12). Por otro lado, no encontramos ningún paciente con la mutación mitocondrial. Debido a que la búsqueda de otras mutaciones es demasiado costosa, nuestro principal objetivo es investigar aquellas mas frecuentes en la población argentina, a fin de desarrollar test simples y específicos para cada una de ellas. (AU)
Assuntos
Humanos , Masculino , Feminino , Perda Auditiva/genética , Mutação , Perda Auditiva/diagnóstico , Surdez/diagnóstico , Surdez/genética , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/genética , Linhagem , Deleção de Genes , Aconselhamento Genético , ArgentinaRESUMO
Recent advances in molecular genetics as well as improved strategies for the prevention and control of non-syndromic hearing loss (NSHL) have contributed to the rising importance of their inherited causes. In this study we report 32 families from Argentine with one (sporadic) or more (familial) individuals affected. All the families were initially screened for mutations in three autosomal nuclear genes and one mutation in mitochondrial DNA. These genes have been found in a great number of familial or sporadic cases of congenital deafness in Caucasians. The mutant allele 35 del G of connexin 26 (GJB2, locus DFNB1 on 13q12) was present in three families. We have investigated the gene encoding otoferlin (OTOF, locus DFNB9 on 2p22-p23) and we found the Q829X mutation in heterocigosity in two families. We have also identified in heterocigosity the 342-kb deletion of connexin 30 (GJB6, locus DFNB1 on 13q12) in one family. On the other hand, we have not found any patient with mitochondrial mutation. Since the screening for other mutations is very expensive, our main goal is to investigate the most frequent mutations in each separate gene in the argentine population and to develop simple and specific tests for each frequent mutations.
RESUMO
Los últimos avances en genética molecular como así también el desarrollo de estrategias para la prevención y control de las hipoacusias no sindrómicas (HNS), han contribuido al esclarecimiento de las causas hereditarias de las mismas. En este estudio, se seleccionaron 32 familias argentinas con uno (esporádico) o más (familiar) individuos afectados. El análisis genético consistió en la búsqueda de tres genes autosómicos nucleares y uno en el ADN mitocondrial. Estos genes se localizaron en un gran número de casos familiares o esporádicos de sorderas congénitas en Caucásicos. El alelo mutado 35 del G de la conexina 26 (GJB2, locus DFNB1 en 13g12) se presentó en tres familias. Además se investigó el gen que codifica otoferlina (OTOF locus DFNB9 en 2p22-23) encontrándose en dos familias la mutación Q829X en heterocigocidad. También se identificó en una familia portadora heterocigota la deleción de 342 Kb en la conexina 30 (GJB6, locus DFNB1 en 13g12). Por otro lado, no encontramos ningún paciente con la mutación mitocondrial. Debido a que la búsqueda de otras mutaciones es demasiado costosa, nuestro principal objetivo es investigar aquellas mas frecuentes en la población argentina, a fin de desarrollar test simples y específicos para cada una de ellas.
Assuntos
Humanos , Masculino , Feminino , Perda Auditiva/genética , Mutação , Argentina , Surdez/diagnóstico , Surdez/genética , Deleção de Genes , Aconselhamento Genético , Perda Auditiva Neurossensorial/diagnóstico , Perda Auditiva Neurossensorial/genética , Perda Auditiva/diagnóstico , LinhagemRESUMO
Current diagnosis of chronic Chagas disease relies on serologic detection of specific immunoglobulin G against Trypanosoma cruzi. However, the presence of parasites detected by polymerase chain reaction (PCR) in patients without positive conventional serologic testing has been observed. We determined the prevalence and clinical characteristics of persons with seronegative results and T. cruzi DNA detected by PCR in a population at high risk for chronic American trypanosomiasis. We studied a total of 194 persons from two different populations: 110 patients were recruited from an urban cardiology clinic, and 84 persons were citizens from a highly disease-endemic area. Eighty (41%) of persons had negative serologic findings; 12 (15%) had a positive PCR. Three patients with negative serologic findings and positive PCR results had clinical signs and symptoms that suggested Chagas cardiomyopathy. This finding challenges the current recommendations for Chagas disease diagnosis, therapy, and blood transfusion policies.
Assuntos
Doença de Chagas/parasitologia , Trypanosoma cruzi/isolamento & purificação , Adolescente , Adulto , Idoso , Animais , Anticorpos Antiprotozoários/sangue , Argentina/epidemiologia , Doença de Chagas/epidemiologia , Estudos Transversais , DNA de Protozoário/química , DNA de Protozoário/genética , Ensaio de Imunoadsorção Enzimática , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Testes de Inibição da Hemaglutinação , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , População Rural , Estudos Soroepidemiológicos , Trypanosoma cruzi/genética , População UrbanaAssuntos
Humanos , Masculino , Feminino , RESEARCH SUPPORT, NON-U.S. GOVT , Fosfatase Alcalina/diagnóstico , Isoenzimas/diagnóstico , Biomarcadores Tumorais/diagnóstico , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/secundário , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/secundário , Linfoma/diagnóstico , Sensibilidade e Especificidade , Valor Preditivo dos Testes , Teorema de BayesRESUMO
En el plasma humano pueden encontrarse las isoenzimas ósea, hepática e intestinal de fosfatasa alcalina (EC 3.1.3.1). En el plasma de mujeres embarazadas, durante el último trimestre de gestación puede encontrarse otra isoenzima, la fosfatasa alcalina placentaria. Además, en extractos butanólicos de tejido placentario se ha encontrado una isoenzima unida a membrana, la fosfatasa alcalina placentaria de alto peso molecular. En suero de mujeres embarazadas se ha determinado la actividad de fosfatasa alcalina placentaria soluble pero, hasta el momento, no se ha detectado la presencia de la isoenzima de alto peso molecular. En nuestro laboratorio hemos desarrollado un método que permite la detección de fosfatasa alcalina de alto peso molecular en el pellet de plasma centrifugado a 100.000xg. Utilizando el mencionado método hemos determinado la actividad de fosfatasa alcalina placentaria de alto peso molecular en plasma de mujeres embarazadas durante el último trimestre de gestación (AU)
Assuntos
Humanos , Feminino , RESEARCH SUPPORT, NON-U.S. GOVT , Gravidez , Fosfatase Alcalina/sangue , Placenta/enzimologia , Gravidez/sangue , Terceiro Trimestre da Gravidez , Peso Molecular , Fosfatase Alcalina/isolamento & purificação , Fosfatase Alcalina/metabolismoAssuntos
Humanos , Masculino , Feminino , Fosfatase Alcalina , Isoenzimas , Linfoma/diagnóstico , Biomarcadores Tumorais , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/secundário , Neoplasias Ósseas/diagnóstico , Neoplasias Ósseas/secundário , Teorema de Bayes , Valor Preditivo dos Testes , Sensibilidade e EspecificidadeRESUMO
En el plasma humano pueden encontrarse las isoenzimas ósea, hepática e intestinal de fosfatasa alcalina (EC 3.1.3.1). En el plasma de mujeres embarazadas, durante el último trimestre de gestación puede encontrarse otra isoenzima, la fosfatasa alcalina placentaria. Además, en extractos butanólicos de tejido placentario se ha encontrado una isoenzima unida a membrana, la fosfatasa alcalina placentaria de alto peso molecular. En suero de mujeres embarazadas se ha determinado la actividad de fosfatasa alcalina placentaria soluble pero, hasta el momento, no se ha detectado la presencia de la isoenzima de alto peso molecular. En nuestro laboratorio hemos desarrollado un método que permite la detección de fosfatasa alcalina de alto peso molecular en el pellet de plasma centrifugado a 100.000xg. Utilizando el mencionado método hemos determinado la actividad de fosfatasa alcalina placentaria de alto peso molecular en plasma de mujeres embarazadas durante el último trimestre de gestación
Assuntos
Humanos , Feminino , Gravidez , Fosfatase Alcalina/sangue , Placenta/enzimologia , Gravidez/sangue , Fosfatase Alcalina/isolamento & purificação , Fosfatase Alcalina/metabolismo , Peso Molecular , Terceiro Trimestre da GravidezAssuntos
Modalidades de Fisioterapia , Dança , Traumatismos da Medula Espinal , Ajustamento Social , Reabilitação , Paraplegia , Atividades Cotidianas , Autoimagem , Modalidades de Fisioterapia , Dança , Traumatismos da Medula Espinal , Ajustamento Social , Reabilitação , Autoimagem , Paraplegia , Atividades Cotidianas , AutoimagemRESUMO
A partir de la preparación de fosfatasa alcalina (EC 3.1.3.1) parcialmente purificada con etanol, de plasma de personas con grupo 0, se separaron cuatro fracciones con actividad enzimática, mediante cromatografía en columna de DEAE-celulosa, con gradiente discontinuo de NaCI. También se extrajo fosfatasa alcalina de hígado, hueso y mucosa intestinal humanos, a fin de caracterizar las distintas formas moleculares. el uso de inhibidores (L-fenil-alcalina y urea) y electroforesis en gel de poliacrilamida, permitió identificar las isoenzimas presentes en los extractos de tejidos y en los picos de elución cromatográfica de la enzima de plasma. La isoenzima hepática se encuentra en todas las fracciones. La primera eluye con NaCI 40 mM y puede separarse en dos (IA e IB). Ambas contienen las isoenzimas intestinal y ósea, además de formas parcialmente desializadas de la hepática. La segunda fracción (NaCI 60 mM) contiene exclusivamente fosfatasa alcalina hepática. La tercera (NaCI 200 mM), de alto peso molecular, está compuesta por las isoenzimas ósea, intestinal y hepática. El perfil es muy reproducible. La comparación del cromatograma correspondiente a plasma normal con el de un paciente portador de un tumor con metástasis óseas, muestra notables diferencias, indicando la utilidad diagnóstica del método, ya que permite identificar el órgano de origen, en casos de incremento de fosfatasa alcalina en plasma. (AU)
Assuntos
Humanos , Fosfatase Alcalina/sangue , Isoenzimas/isolamento & purificação , Cromatografia por Troca Iônica , DEAE-Celulose/diagnóstico , Etanol/diagnóstico , Cromatografia DEAE-Celulose/instrumentação , Cromatografia DEAE-Celulose/métodos , Neuraminidase/diagnóstico , Fenilalanina/diagnóstico , Ureia/diagnóstico , Cloreto de Sódio/diagnóstico , Fígado/enzimologia , Intestinos/enzimologia , Osso e Ossos/enzimologia , Eletroforese em Gel de Poliacrilamida , Doenças Ósseas/enzimologia , Hepatopatias/enzimologiaRESUMO
A partir de la preparación de fosfatasa alcalina (EC 3.1.3.1) parcialmente purificada con etanol, de plasma de personas con grupo 0, se separaron cuatro fracciones con actividad enzimática, mediante cromatografía en columna de DEAE-celulosa, con gradiente discontinuo de NaCI. También se extrajo fosfatasa alcalina de hígado, hueso y mucosa intestinal humanos, a fin de caracterizar las distintas formas moleculares. el uso de inhibidores (L-fenil-alcalina y urea) y electroforesis en gel de poliacrilamida, permitió identificar las isoenzimas presentes en los extractos de tejidos y en los picos de elución cromatográfica de la enzima de plasma. La isoenzima hepática se encuentra en todas las fracciones. La primera eluye con NaCI 40 mM y puede separarse en dos (IA e IB). Ambas contienen las isoenzimas intestinal y ósea, además de formas parcialmente desializadas de la hepática. La segunda fracción (NaCI 60 mM) contiene exclusivamente fosfatasa alcalina hepática. La tercera (NaCI 200 mM), de alto peso molecular, está compuesta por las isoenzimas ósea, intestinal y hepática. El perfil es muy reproducible. La comparación del cromatograma correspondiente a plasma normal con el de un paciente portador de un tumor con metástasis óseas, muestra notables diferencias, indicando la utilidad diagnóstica del método, ya que permite identificar el órgano de origen, en casos de incremento de fosfatasa alcalina en plasma.