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1.
Gac. méd. Méx ; 156(4): 348-351, Jul.-Aug. 2020. graf
Article in English | LILACS | ID: biblio-1249923

ABSTRACT

Abstract Introduction: Reports of dermatological manifestations in patients with COVID-19 suggest a possible cutaneous tropism of SARS-CoV-2; however, the capacity of this virus to infect the skin is unknown. Objective: To determine the susceptibility of the skin to SARS-CoV-2 infection based on the expression of viral entry factors ACE2 and TMPRSS2 in this organ. Method: A comprehensive analysis of human tissue gene expression databases was carried out looking for the presence of the ACE2 and TMPRSS2 genes in the skin. mRNA expression of these genes in skin-derived human cell lines was also assessed. Results: The analyses showed high co-expression of ACE2 and TMPRSS2 in the gastrointestinal tract and kidney, but not in the skin. Only the human immortalized keratinocyte HaCaT cell line expressed detectable levels of ACE2, and no cell line originating in the skin expressed TMPRSS2. Conclusions: Our results suggest that cutaneous manifestations in patients with COVID-19 cannot be directly attributed to the virus. It is possible that cutaneous blood vessels endothelial damage, as well as the effect of circulating inflammatory mediators produced in response to the virus, are the cause of skin involvement.


Resumen Introducción: Reportes de manifestaciones dermatológicas en pacientes con COVID-19 sugieren un posible tropismo cutáneo del virus SARS-CoV-2; sin embargo, se desconoce la capacidad de este virus para infectar la piel. Objetivo: Determinar la susceptibilidad de la piel a la infección por SARS-CoV-2 con base en la expresión de los factores de entrada viral ACE2 y TMPRSS2 en dicho órgano. Método: Se buscaron los genes ACE2 y TMPRSS2 en la piel, para lo cual se realizó un análisis extenso de las bases de datos de expresión genética en tejidos humanos. Asimismo, se evaluó la expresión de dichos genes en líneas celulares humanas derivadas de la piel. Resultados: Los análisis mostraron alta expresión conjunta de ACE2 y TMPRSS2 en el tracto gastrointestinal y en los riñones, pero no en la piel. Solo la línea celular de queratinocitos humanos inmortalizados HaCaT expresó niveles detectables de ACE2 y ninguna línea celular de origen cutáneo expresó TMPRSS2. Conclusiones: Los resultados sugieren que las manifestaciones dermatológicas en pacientes con COVID-19 no pueden ser atribuidas directamente al virus; es posible que sean originadas por el daño endotelial a los vasos sanguíneos cutáneos y el efecto de los mediadores inflamatorios circulantes producidos en respuesta al virus.


Subject(s)
Humans , Pneumonia, Viral/complications , Serine Endopeptidases/genetics , Skin Diseases, Viral/virology , Coronavirus Infections/complications , Peptidyl-Dipeptidase A/genetics , Pneumonia, Viral/genetics , Skin/virology , Cell Line , Gene Expression Regulation , Coronavirus Infections/genetics , Genetic Predisposition to Disease , Virus Internalization , Viral Tropism/physiology , Pandemics , Betacoronavirus/isolation & purification , Angiotensin-Converting Enzyme 2 , SARS-CoV-2 , COVID-19
2.
Gac. méd. Méx ; 156(4): 324-329, Jul.-Aug. 2020. graf
Article in English | LILACS | ID: biblio-1249919

ABSTRACT

Abstract In the efforts to explain COVID-19 pathophysiology, studies are being carried out on the correspondence between the expression of SARS-CoV-2 cell receptors and viral sequences. ACE2, CD147 and TMPRSS2 receptors expression could indicate poorly explored potential infection targets. For the genomic analysis of SARS-CoV-2 receptors, using BioGPS information was decided, which is a portal that centralizes genetic annotation resources, in combination with that of The Human Protein Atlas, the largest portal of human transcriptome and proteome data. We also reviewed the most recent articles on the subject. RNA and viral receptor proteins expression was observed in numerous anatomical sites, which partially coincides with the information reported in the literature. High expression in testicular cells markedly stood out, and it would be therefore important ruling out whether this anatomical site is a SARS-CoV-2 reservoir; otherwise, germ cell damage, as it is observed in infections with other RNA viruses, should be determined.


Resumen En el afán por explicar la fisiopatogenia de COVID-19 se están realizando estudios en torno a la correspondencia entre la expresión de receptores celulares de SARS-CoV-2 y las secuencias virales. La expresión de los receptores ACE2, CD147 y TMPRSS2 podría indicar blancos de infección poco explorados. Para el análisis genómico de los receptores de SARS-CoV-2 se optó por utilizar la información del BioGPS, un portal que centraliza los recursos de anotación genética, en combinación con la de The Human Protein Atlas, el portal más grande de datos del transcriptoma y proteoma humanos. También se revisaron los artículos más recientemente respecto al tema. En numerosos sitios anatómicos se observó la expresión de ARN y proteínas de los receptores del virus, que coinciden parcialmente con la información reportada en la literatura. Resaltó la alta expresión en las células de los testículos, por lo que sería importante descartar si este sitio anatómico es un reservorio de SARS-CoV-2; de no ser así, determinar el daño en las células germinales, tal como sucede en infecciones por otros virus ARN.


Subject(s)
Humans , Pneumonia, Viral/virology , Testis/virology , Coronavirus Infections/virology , Betacoronavirus/isolation & purification , Pneumonia, Viral/physiopathology , Serine Endopeptidases/genetics , Gene Expression Regulation , Virus Latency , Coronavirus Infections/physiopathology , Peptidyl-Dipeptidase A/genetics , Basigin/genetics , Pandemics , Angiotensin-Converting Enzyme 2 , SARS-CoV-2 , COVID-19
3.
Electron. j. biotechnol ; 29: 7-12, sept. 2017. ilus, graf, tab
Article in English | LILACS | ID: biblio-1016095

ABSTRACT

Background: DegP is a serine protease that specifically cleaves and refolds unfolding proteins in the periplasmic space of the cells. To date, there is no information regarding DegP from halophilic bacteria. Chromohalobacter salexigens BKL5 is a moderately halophilic bacterium that has the ability to grow in a media containing more than 15% salt. Therefore, the objectives of this work were to clone and overexpress DegP-encoding gene from C. salexigens BKL5 and characterize its biochemical properties. Results: DegP-encoding gene was overexpressed in Escherichia coli BL21(DE3) CodonPlus in an active form. SDS-PAGE analysis showed that the molecular weight of the recombinant DegP was 45 kDa. Size-exclusion chromatography analysis suggested that recombinant DegP was present in two multimeric states, hexameric and dodecameric, with molecular weights of 297.9 and 579.12 kDa, respectively. Both conformations were enzymatically active when casein was used as substrate for enzymatic assay. Circular dichroism analysis showed that recombinant DegP was composed of 0.21­0.29 helical content, which was comparable to the helical content in the crystal structure of E. coli DegP. The basic/acidic residue ratio of recombinant DegP was 0.56, which was slightly higher than that of DegP from extreme halophiles (average, 0.45) but significantly lower than that of DegP from nonhalophiles (average, 0.94). Conclusions: Recombinant DegP from C. salexigens BKL5 showed proteolytic activity when ß-casein was used as a substrate. In silico analysis indicated that recombinant DegP had characteristics similar to those of halophilic proteins depending on its amino acid composition.


Subject(s)
Serine Endopeptidases/genetics , Periplasmic Proteins/genetics , Chromohalobacter/enzymology , Proteolysis , Heat-Shock Proteins/genetics , Recombinant Proteins , Serine Endopeptidases/metabolism , Caseins , Chromatography, Gel , Circular Dichroism , Cloning, Molecular , Periplasmic Proteins/metabolism , Electrophoresis, Polyacrylamide Gel , Escherichia coli , Salinity , Chromohalobacter/genetics , Heat-Shock Proteins/metabolism , Molecular Weight
4.
Biomédica (Bogotá) ; 37(supl.2): 135-142, jul.-set. 2017. tab, graf
Article in Spanish | LILACS | ID: biblio-888532

ABSTRACT

Resumen Introducción. Aedes aegypti y Ae. albopictus son reconocidos vectores de arbovirus como los del dengue, la fiebre amarilla, el chikungunya y el Zika, en regiones tropicales y subtropicales del mundo. En Colombia, la distribución geográfica de Ae. albopictus ha sufrido un incremento y hoy incluye ciudades como Cali y Medellín. Hasta ahora, sin embargo, no se ha recabado información concluyente sobre su infección viral y su capacidad de transmisión a los humanos. Objetivo. Determinar la infección natural por dengue en ejemplares de Ae. albopictus recolectados en un área urbana de Medellín. Materiales y métodos. Se recolectaron individuos de Ae. albopictus en el campus de la Universidad Nacional de Colombia, sede Medellín. Se confirmó su clasificación taxonómica mediante el análisis del gen citocromo oxidasa I (COI), y se extrajo el ARN total para la identificación del virus del dengue y de los respectivos serotipos. La presencia del genotipo DENV se infirió mediante el análisis del gen NS3. Resultados. El análisis del COI corroboró el estatus taxonómico de Ae. albopictus. Uno de los mosquitos procesados fue positivo para DENV-2 y el análisis del NS3 mostró una gran similitud con el genotipo asiático-americano. Conclusión. Se reporta la infección con DENV-2 en Ae. albopictus en Medellín, Colombia. La presencia del genotipo asiático-americano en una zona urbana sugiere su posible circulación entre humanos y en Ae. albopictus, lo cual alerta sobre su eventual papel en la transmisión del DENV-2, y sobre la necesidad de incluir esta especie en la vigilancia entomológica en Colombia.


Abstract Introduction: Aedes aegypti and Ae. albopictus are recognized vectors of dengue, yellow fever, chikungunya and Zika arboviruses in several countries worldwide. In Colombia, Ae. albopictus geographical distribution has increased to include highly populated cities such as Cali and Medellín. Although this species has been frequently found in urban and semi-urban zones in the country, its role as vector of the dengue fever is poorly known. Objective: To identify the presence of Ae. albopictus specimens naturally infected with dengue virus collected in Medellín. Materials and methods: Insects were collected in the Universidad Nacional de Colombia campus in Medellín. Individuals were classified as Ae. albopictus and confirmed by DNA barcode region analysis. Mosquitoes were processed for dengue virus identification, and a fragment of the NS3 gen was sequenced and compared with DENV-2 genotypes reported in the literature. Results: Sequence analysis of COI indicated Ae. albopictus individuals were similar to those recently reported in Colombia, and genetically close to those from other regions worldwide. Among the pools tested one was positive for DENV-2, and the NS3 analysis indicated it belonged to the Asian-American clade. Conclusion: We report the presence Ae. albopictus naturally infected with the Asian-American genotype of DENV-2 in Colombia. The presence of Ae. albopictus specimens carrying the most common genotype infecting humans in a highly populated city such as Medellín indicates its potential role as dengue vector in Colombia and highlights the relevance of including it in current vector surveillance strategies.


Subject(s)
Animals , Humans , Aedes/virology , Dengue/epidemiology , Dengue Virus/isolation & purification , Mosquito Vectors/virology , Serine Endopeptidases/genetics , Serotyping , Polymerase Chain Reaction , Cities , Viral Nonstructural Proteins/genetics , Colombia/epidemiology , DNA, Complementary/analysis , Electron Transport Complex IV/genetics , Insect Proteins/genetics , Aedes/genetics , RNA Helicases/genetics , Dengue/transmission , Dengue Virus/classification , Dengue Virus/genetics , Genotype
5.
Ann. hepatol ; 16(1): 77-85, Jan.-Feb. 2017. graf
Article in English | LILACS | ID: biblio-838089

ABSTRACT

Abstract: Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease worldwide. We have previously shown that hepatic reticuloendothelial system (RES) iron deposition is associated with an advanced degree of nonalcoholic steatohepatitis (NASH) in humans. In this study, we aimed to determine differentially expressed genes related to iron overload, inflammation and oxidative stress pathways, with the goal of identifying factors associated with NASH progression. Seventy five patients with NAFLD were evaluated for their biochemical parameters and their liver tissue analyzed for NASH histological characteristics. Gene expression analysis of pathways related to iron homeostasis, inflammation and oxidative stress was performed using real-time PCR. Gene expression was compared between subjects based on disease status and presence of hepatic iron staining. We observed increased gene expression of hepcidin (HAMP) (2.3 fold, p = 0.027), transmembrane serine proteinase 6 (TMPRSS6) (8.4 fold, p = 0.003), signal transducer and activator of transcription 3 (STAT3) (5.5 fold, p = 0.004), proinflammatory cytokines; IL-1β (2.7 fold, p = 0.046) and TNF-α (3.8 fold, p = 0.001) in patients with NASH. TMPRSS6, a negative regulator of HAMP, is overexpressed in patients with NASH and HIF1α (hypoxia inducible factor-1) is downregulated. NAFLD patients with hepatic iron deposition exhibited higher hepcidin expression (3.1 fold, p = 0.04) but lower expression of cytokines. In conclusion, we observed elevated hepatic HAMP expression in patients with NASH and in NAFLD patients who had hepatic iron deposition, while proinflammatory cytokines displayed elevated expression only in patients with NASH, suggesting a regulatory role for hepcidin in NAFL to NASH transition and in mitigating inflammatory responses.


Subject(s)
Humans , Male , Female , Middle Aged , Oxidative Stress/genetics , Iron Overload/genetics , Non-alcoholic Fatty Liver Disease/genetics , Inflammation/genetics , Iron/analysis , Liver/chemistry , Serine Endopeptidases/genetics , Gene Expression Regulation , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/blood , Inflammation Mediators/blood , Iron Overload/diagnosis , Iron Overload/blood , STAT3 Transcription Factor/genetics , Interleukin-1beta/genetics , Interleukin-1beta/blood , Real-Time Polymerase Chain Reaction , Hepcidins/genetics , Non-alcoholic Fatty Liver Disease/diagnosis , Non-alcoholic Fatty Liver Disease/blood , Inflammation/diagnosis , Inflammation/blood , Liver/pathology , Membrane Proteins/genetics
6.
Biomédica (Bogotá) ; 37(1): 131-140, ene.-feb. 2017. graf
Article in Spanish | LILACS | ID: biblio-888451

ABSTRACT

Resumen Introducción: El dengue es una enfermedad causada por uno de los cuatro serotipos del virus del dengue (DENV) y es endémica en, aproximadamente, 130 países. Su incidencia ha aumentado notablemente en las últimas décadas, así como la frecuencia y la magnitud de los brotes. A pesar de los esfuerzos, no existen tratamientos profilácticos ni terapéuticos contra la enfermedad y, en ese contexto, el estudio de los procesos que gobiernan el ciclo de infección del DENV es esencial para desarrollar vacunas o terapias antivirales. Una de las moléculas del DENV más prometedoras es la proteína no estructural 3 (NS3), la cual es indispensable para la replicación viral y es uno de los principales blancos inmunológicos durante la infección. Objetivo: Producir anticuerpos policlonales para contribuir a los futuros estudios sobre las interacciones entre la proteína NS3 y otras proteínas celulares. Materiales y métodos: Se expresaron dos proteínas recombinantes del dominio helicasa de NS3 del DENV de serotipo 2, las cuales se emplearon para inmunizar ratas y producir anticuerpos policlonales. Resultados: Los anticuerpos producidos fueron útiles en ensayos de Western blot e inmunofluorescencia y se reportó por primera vez un anticuerpo policlonal anti-NS3 que permitió la inmunoprecipitación de la proteína viral y la detecta con Western blot sin necesidad de inducir sobreexpresión de NS3 o de usar extractos de células marcados metabólicamente con radioisótopos. Conclusión: Las proteínas recombinantes expresadas y los anticuerpos producidos constituyen herramientas valiosas para estudiar procesos infecciosos del DENV que involucren a la proteína NS3 y evaluar pruebas dirigidas a interferir las funciones de esta proteína.


Abstract Introduction: Dengue is a disease caused by one of four serotypes of the dengue virus (DENV) and is endemic in approximately 130 countries. The incidence of dengue has increased dramatically in recent decades, as well as the frequency and magnitude of outbreaks. Despite all efforts, there are no prophylactic or therapeutic treatments for the disease. Accordingly, research on the processes governing the DENV infection cycle is essential to develop vaccines or antiviral therapies. One of the most attractive DENV molecules to investigate is nonstructural protein 3 (NS3), which is essential for viral replication and a major immune target for infection. Objective: To produce antibodies to support future studies on NS3 and its cellular interactions with other proteins. Materials and methods: Two recombinant proteins of the helicase domain of DENV NS3 serotype 2 were expressed, and used to immunize mice and produce polyclonal antibodies. Results: The antibodies produced were useful in Western blot and immunofluorescence tests. We report an NS3 antibody that immunoprecipitates the viral protein and detects it in Western blot with no need to over-express it or use cell extracts with metabolic radiolabeling.


Subject(s)
Animals , Humans , Mice , Virus Replication/physiology , Viral Nonstructural Proteins/genetics , Viral Nonstructural Proteins/metabolism , Dengue/virology , Dengue Virus/immunology , Antibodies, Viral/immunology , Virus Replication/genetics , Virus Replication/immunology , Serine Endopeptidases/genetics , Serine Endopeptidases/metabolism , Serine Endopeptidases/chemistry , Blotting, Western , Viral Nonstructural Proteins/chemistry , RNA Helicases/genetics , RNA Helicases/metabolism , RNA Helicases/chemistry , Antibodies, Viral/metabolism , Antibodies, Viral/chemistry
7.
Biol. Res ; 49: 1-10, 2016. ilus, graf
Article in English | LILACS | ID: biblio-950843

ABSTRACT

Schizophrenia is a severe psychiatric disorder that results in a significant disability for the patient. The disorder is characterized by impairment of the adaptive orchestration of actions, a cognitive function that is mainly dependent on the prefrontal cortex. This behavioral deficit, together with cellular and neurophysiological alterations in the prefrontal cortex, as well as reduced density of GABAergic cells and aberrant oscillatory activity, all indicate structural and functional deficits of the prefrontal cortex in schizophrenia. Among the several risk factors for the development of schizophrenia, stress during the prenatal period has been identified as crucial. Thus, it is proposed that prenatal stress induces neurodevelopmental alterations in the prefrontal cortex that are expressed as cognitive impairment observed in schizophrenia. However, the precise mechanisms that link prenatal stress with the impairment of prefrontal cortex function is largely unknown. Reelin is an extracellular matrix protein involved in the development of cortical neural connectivity at embryonic stages, and in synaptic plasticity at postnatal stages. Interestingly, down-regulation of reelin expression has been associated with epigenetic changes in the reelin gene of the prefrontal cortex of schizophrenic patients. We recently showed that, similar to schizophrenic patients, prenatal stress induces down-expression of reelin associated with the methylation of its promoter in the rodent prefrontal cortex. These alterations were paralleled with altered prefrontal cortex functional connectivity and impairment in prefrontal cortex-dependent behavioral tasks. Therefore, considering molecular, cellular, physiological and behavioral evidence, we propose a unifying framework that links prenatal stress and prefrontal malfunction through epigenetic alterations of the reelin gene.


Subject(s)
Humans , Female , Pregnancy , Prenatal Exposure Delayed Effects/physiopathology , Schizophrenia/etiology , Schizophrenia/physiopathology , Stress, Physiological/physiology , Brain/embryology , Serine Endopeptidases/genetics , Cell Adhesion Molecules, Neuronal/genetics , Extracellular Matrix Proteins/genetics , Epigenesis, Genetic/physiology , Nerve Tissue Proteins/genetics , Social Behavior Disorders/physiopathology , Brain/physiopathology , Gene Expression , Risk Factors , Cognition Disorders/physiopathology , DNA Methylation
8.
Braz. j. microbiol ; 46(3): 879-883, July-Sept. 2015. tab
Article in English | LILACS | ID: lil-755800

ABSTRACT

Studies investigating rickettsial infections in ticks parasitizing wild animals in the Northeast region of Brazil have been confined to the detection of Rickettsia amblyommii in immature stages of Amblyomma longirostre collected from birds in the state of Bahia, and in immatures and females of Amblyomma auriculariumcollected from the striped hog-nosed skunk (Conepatus semistriatus) and armadillos (Euphractus sexcinctus) in the state of Pernambuco. The current study extends the distribution of R. amblyommii (strain Aranha), which was detected in A. longirostre collected from the thin-spined porcupine Chaetomys subspinosus and the hairy dwarf porcupine Coendou insidiosus. In addition, we report the first detection of Rickettsia bellii in adults of A. longirostre collected from C. insidiosus in the state of Bahia.

.


Subject(s)
Animals , Ixodidae/microbiology , Rickettsia Infections/microbiology , Rickettsia/genetics , Rickettsia/isolation & purification , Tick Infestations/microbiology , Animals, Wild , Armadillos , Base Sequence , Birds , Brazil , Bacterial Outer Membrane Proteins/genetics , Bacterial Proteins/genetics , Citrate (si)-Synthase/genetics , DNA, Bacterial , Heat-Shock Proteins/genetics , Mephitidae , Molecular Sequence Data , Molecular Typing , Porcupines , Periplasmic Proteins/genetics , Sequence Analysis, DNA , Serine Endopeptidases/genetics
9.
Trends psychiatry psychother. (Impr.) ; 36(1): 52-58, Jan-Mar/2014. tab
Article in English | LILACS | ID: lil-707279

ABSTRACT

INTRODUCTION: Day hospitals in psychiatry are a major alternative to inpatient care today, acting as key components of community and social psychiatry. Objective: To study trends in the use of psychiatric day hospitals over the last decades of the 20th century and the first decade of the 21st century, focusing on patient age, sex, and diagnostic group, using data from Centro Hospitalar São João, Porto, Portugal. METHODS: Data corresponding to years 1970 to 2009 were collected from patient files. Patients were classified into seven diagnostic groups considering their primary diagnoses only. RESULTS: Mean age upon admission rose from 32.7±12.1 years in the second half of the 1970s to 43.5±12.2 years in 2005-2009 (p for trend < 0.001). Most patients were female (63.2%), however their proportion decreased from nearly 70% in the 1970s to 60% in the first decade of the 21st century. In males, until the late 1980s, neurotic disorders (E) were the most common diagnosis, accounting for more than one third of admissions. In the subsequent years, this proportion decreased, and the number of admissions for schizophrenia (C) exceeded 50% in 2004- 2009. In females, until the late 1980s, affective disorders (D) and neurotic disorders (E), similarly distributed, accounted for most admissions. From the 1990s on, the proportion of neurotic disorders (E) substantially decreased, and affective disorders (D) came to represent more than 50% of all admissions. CONCLUSIONS: Mean age upon admission rose with time, as did the percentage of female admissions, even though the latter tendency weakened in the last 10 years assessed. There was also an increase in the proportion of patients with schizophrenia. .


INTRODUÇÃO: Os hospitais de dia em psiquiatria representam atualmente uma das principais alternativas ao internamento, atuando como componentes chave na psiquiatria comunitária e social. OBJETIVO: Avaliar tendências na utilização de um hospital de dia no período compreendido entre as últimas décadas do século 20 e a primeira década do século 21, com foco em idade, sexo e grupo diagnóstico, usando dados do Centro Hospitalar São João, Porto, Portugal. MÉTODOS: Dados correspondentes aos anos 1970 a 2009 foram coletados dos prontuários clínicos. Os pacientes foram classificados em sete grupos diagnósticos, tendo em conta o diagnóstico principal. Resultados: A idade média na admissão aumentou de 32.7±12.1 anos na segunda metade da década de 1970 para 43.5±12.2 anos em 2005-2009 (p < 0.001). A maioria dos pacientes era do sexo feminino (63.2%), no entanto sua proporção diminuiu de cerca de 70% na década de 1970 para 60% na primeira década do século 21. Nos homens, até o final dos anos 1980, o grupo das perturbações neuróticas (E) era o diagnóstico mais comum, representando mais de um terço das admissões. Durante os anos seguintes, essa proporção diminuiu, e o número de admissões por esquizofrenia (C) alcançou mais de 50% no período de 2004-2009. Nas mulheres, até o final dos anos 1980, as perturbações afetivas (D) e as perturbações neuróticas (E), distribuídas similarmente, respondiam pela maioria das admissões. A partir dos anos 1990, a proporção das perturbações neuróticas (E) diminuiu substancialmente, e as perturbações afetivas (D) passaram a corresponder a mais de 50% do total das admissões. Conclusões: A idade média na admissão ...


Subject(s)
Adolescent , Child , Female , Humans , Male , Young Adult , Adenoma/genetics , Germ-Line Mutation , Neoplastic Syndromes, Hereditary/genetics , Serine Endopeptidases/genetics , Thyroid Neoplasms/genetics , Adenoma/metabolism , Adenoma/pathology , Genetic Predisposition to Disease , Pedigree , Sequence Analysis, DNA , Serine Endopeptidases/metabolism , Thyroid Neoplasms/metabolism , Thyroid Neoplasms/pathology
10.
Braz. j. med. biol. res ; 46(6): 546-554, 02/jul. 2013. tab, graf
Article in English | LILACS | ID: lil-679208

ABSTRACT

Multidrug resistance (MDR) poses a serious impediment to the success of chemotherapy for laryngeal cancer. To identify microRNAs and mRNAs associated with MDR of human laryngeal cancer Hep-2 cells, we developed a multidrug-resistant human laryngeal cancer subline, designated Hep-2/v, by exposing Hep-2 cells to stepwise increasing concentrations of vincristine (0.02-0.96'µM). Microarray assays were performed to compare the microRNA and mRNA expression profiles of Hep-2 and Hep-2/v cells. Compared to Hep-2 cells, Hep-2/v cells were more resistant to chemotherapy drugs (∼45-fold more resistant to vincristine, 5.1-fold more resistant to cisplatin, and 5.6-fold more resistant to 5-fluorouracil) and had a longer doubling time (42.33±1.76 vs 28.75±1.12'h, P<0.05), higher percentage of cells in G0/G1 phase (80.98±0.52 vs 69.14±0.89, P<0.05), increased efflux of rhodamine 123 (95.97±0.56 vs 12.40±0.44%, P<0.01), and up-regulated MDR1 expression. A total of 7 microRNAs and 605 mRNAs were differentially expressed between the two cell types. Of the differentially expressed mRNAs identified, regulator of G-protein signaling 10, high-temperature requirement protein A1, and nuclear protein 1 were found to be the putative targets of the differentially expressed microRNAs identified. These findings may open a new avenue for clarifying the mechanisms responsible for MDR in laryngeal cancer.


Subject(s)
Humans , Drug Resistance, Neoplasm/genetics , Laryngeal Neoplasms/genetics , MicroRNAs/isolation & purification , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , RNA, Messenger/isolation & purification , Antineoplastic Agents/pharmacology , Basic Helix-Loop-Helix Transcription Factors/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cisplatin/pharmacology , Flow Cytometry , Fluorouracil/pharmacology , G1 Phase Cell Cycle Checkpoints , Genes, MDR , Laryngeal Neoplasms/drug therapy , Neoplasm Proteins/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , RGS Proteins/genetics , /pharmacokinetics , Serine Endopeptidases/genetics , Tissue Array Analysis , Vincristine/pharmacology
11.
Article in English | WPRIM | ID: wpr-214940

ABSTRACT

PURPOSE: The purpose of this study was to determine the pharmacogenetic effects of complement factor H (CFH) Y402H, LOC387715 and high-temperature requirement factor A1 (HTRA1) genotypes on the treatment of exudative age-related macular degeneration (AMD) by intravitreal bevacizumab injection in a Korean population. METHODS: Seventy-five patients diagnosed with exudative AMD were treated with intravitreal bevacizumab (2.5 mg) monotherapy. All patients received three initial intravitreal bevacizumab injections every four weeks and were then treated "as needed" based on clinical findings, optical coherence tomography and fluorescein angiography during the 12 month follow-up period after the third injection. RESULTS: The difference in visual acuity improvement among the three genotypes of LOC387715 were statistically significant at six months post-treatment (logarithm of the minimum angle of resolution; TT, 0.346; GT, 0.264; GG, 0.188; p = 0.037). Among the LOC387715 genotypes, the number of additional injections was lower in patients who had the risk T allele (GG, 2.143; GT, 2.000; TT, 1.575; p = 0.064). There was no significant difference between visual acuity and central macular thickness change in the CFH Y402H polymorphism group during the 12 month follow-up period. However, the TC group of CFH Y402H required more additional bevacizumab injections than the TT group (TT, 1.517; TC, 3.363; p = 0.020). CONCLUSIONS: This study demonstrated that different LOC387715/HTRA1 genotypes resulted in different bevacizumab treatment responses on exudative AMD. Patients with the risk allele had an improved treatment response and less need for additional injections. However, patients with the CFH Y402H risk allele needed more additional injections of bevacizumab in order to improve visual acuity. This study illustrates how pharmacogenetic factors may help determine treatment modality and dosing. This could ultimately provide basic data for 'personalized medicine' in AMD.


Subject(s)
Aged , Alleles , Angiogenesis Inhibitors/administration & dosage , Antibodies, Monoclonal, Humanized/administration & dosage , DNA/genetics , Female , Follow-Up Studies , Genotype , Humans , Intravitreal Injections , Macular Degeneration/drug therapy , Male , Pharmacogenetics/methods , Polymorphism, Genetic , Retrospective Studies , Serine Endopeptidases/genetics , Vascular Endothelial Growth Factor A/antagonists & inhibitors , Visual Acuity
12.
Indian J Hum Genet ; 2011 May; 17(2): 65-69
Article in English | IMSEAR | ID: sea-138937

ABSTRACT

The present study was carried out to determine the prevalence of families having mental retardation in Pakistani population. We enrolled seven mentally retarded (MR) families with two or more affected individuals. Family history was taken to minimize the chances of other abnormalities. Pedigrees were drawn using the Cyrillic software (version 2.1). The structure of pedigrees shows that all the marriages are consanguineous and the families have recessive mode of inheritance. All the families were studied by linkage analysis to mental retardation locus (MRT1)/gene PRSS12. Three STR markers (D4S191, D4S2392, and D4S3024) in vicinity of mental retardation (MR) locus (MRT1)/gene PRSS12 were amplified on all the sample of each family by PCR. The PCR products were then genotyped on non denaturing polyacrylamide gel electrophoresis (PAGE). The Haplotype were constructed to determine the pattern of inheritance and also to determine that a family was linked or unlinked to gene PRSS12. One out of the seven families was potentially linked to gene PRSS12, while the other six families remain unlinked.


Subject(s)
Family , Genetic Linkage/genetics , Genetic Predisposition to Disease , Humans , Intellectual Disability/epidemiology , Intellectual Disability/genetics , Molecular Diagnostic Techniques/methods , Pakistan/epidemiology , Serine Endopeptidases/genetics
13.
Article in Korean | WPRIM | ID: wpr-151633

ABSTRACT

Severe congenital neutropenia is a rare hematological disease characterized by a selective decrease in circulating neutrophils, maturation arrest of granulocytic precursors at the promyelocyte stage, and recurrence of infections. A 2-month-old male infant (patient A) and a 14-month-old female child (patient B) were referred to our hospital due to severe neutropenia. Sequencing analysis of ELA2 and HAX1 genes was performed. Two single nucleotide polymorphisms of HAX1 gene were found. They were 5,104T-->G point mutation of exon 1 and 5,474A-->G point mutation of intron 1 in HAX1 gene. The mutation of ELA2 gene was not found. The patient A showed a good response to granulocyte colony-stimulating factor (G-CSF) treatment and the absolute neutrophil count recovered to 1,195/microliter. But the patient B showed a partial response to G-CSF treatment and experienced several episodes of herpetic gingivostomatitis, oral ulcer, acute pharyngotonsillitis and otitis media during follow-up.


Subject(s)
Adaptor Proteins, Signal Transducing/genetics , Bone Marrow/pathology , Female , /adverse effects , Humans , Infant , Male , Neutropenia/congenital , Neutrophils/cytology , Oral Ulcer/etiology , Otitis Media/etiology , Polymorphism, Single Nucleotide , Serine Endopeptidases/genetics , Stomatitis, Herpetic/etiology
14.
Braz. j. med. biol. res ; 41(1): 12-17, Jan. 2008. graf
Article in English | LILACS | ID: lil-469981

ABSTRACT

A serine proteinase with thrombin-like activity was isolated from the venom of the Central American pit viper Bothrops asper. Isolation was performed by a combination of affinity chromatography on aminobenzamidine-Sepharose and ion-exchange chromatography on DEAE-Sepharose. The enzyme accounts for approximately 0.13 percent of the venom dry weight and has a molecular mass of 32 kDa as determined by SDS-PAGE, and of 27 kDa as determined by MALDI-TOF mass spectrometry. Its partial amino acid sequence shows high identity with snake venom serine proteinases and a complete identity with a cDNA clone previously sequenced from this species. The N-terminal sequence of the enzyme is VIGGDECNINEHRSLVVLFXSSGFL CAGTLVQDEWVLTAANCDSKNFQ. The enzyme induces clotting of plasma (minimum coagulant dose = 4.1 µg) and fibrinogen (minimum coagulant dose = 4.2 µg) in vitro, and promotes defibrin(ogen)ation in vivo (minimum defibrin(ogen)ating dose = 1.0 µg). In addition, when injected intravenously in mice at doses of 5 and 10 µg, it induces a series of behavioral changes, i.e., loss of the righting reflex, opisthotonus, and intermittent rotations over the long axis of the body, which closely resemble the `gyroxin-like' effect induced by other thrombin-like enzymes from snake venoms.


Subject(s)
Animals , Mice , Blood Coagulation , Bothrops , Coagulants/isolation & purification , Crotalid Venoms/enzymology , Serine Endopeptidases/isolation & purification , Amino Acid Sequence , Antivenins/therapeutic use , Blood Coagulation/drug effects , Chromatography, Agarose , Chromatography, Ion Exchange , Costa Rica , Coagulants/administration & dosage , Coagulants/pharmacology , Drug Evaluation, Preclinical , Fibrinogen/metabolism , Serine Endopeptidases/chemistry , Serine Endopeptidases/genetics , Serine Endopeptidases/pharmacology , Snake Bites/physiopathology , Thrombin/chemistry
15.
Article in English | IMSEAR | ID: sea-37297

ABSTRACT

Research into actions of resveratrol, abundantly present in red grape skin, has been greatly stimulated by its reported beneficial health influence. Since it was recently proposed as a potential prostate cancer chemopreventive agent, we here performed an in vivo experiment to explore its effect in the Transgenic Rat for Adenocarcinoma of Prostate (TRAP) model, featuring the rat probasin promoter/SV 40 T antigen. Resveratrol suppressed prostate cancer growth and induction of apoptosis through androgen receptor (AR) down-regulation, without any sign of toxicity. Resveratrol not only downregulated androgen receptor (AR) expression but also suppressed the androgen responsive glandular kallikrein 11 (Gk11), known to be an ortholog of the human prostate specific antigen (PSA), at the mRNA level. The data provide a mechanistic basis for resveratrol chemopreventive efficacy against prostate cancer.


Subject(s)
Adenocarcinoma/drug therapy , Angiogenesis Inhibitors/therapeutic use , Animals , Anticarcinogenic Agents/therapeutic use , Apoptosis/drug effects , Blotting, Western , Body Weight/drug effects , COS Cells , Chlorocebus aethiops , Disease Models, Animal , Estradiol/blood , Gene Expression Regulation, Neoplastic/drug effects , Heterozygote , Humans , Male , Mice , Mice, Nude , Prostatic Neoplasms/drug therapy , Protein Biosynthesis , RNA, Messenger , Rats , Rats, Transgenic , Receptors, Androgen/antagonists & inhibitors , Reverse Transcriptase Polymerase Chain Reaction , Serine Endopeptidases/genetics , Stilbenes/therapeutic use , Testosterone/blood , Ubiquitin/metabolism
16.
Rev. chil. cardiol ; 27(1): 43-50, 2008. tab
Article in Spanish | LILACS | ID: lil-499090

ABSTRACT

Introducción: Variaciones comunes en el gen PCSK9 han sido asociadas a elevadas concentraciones de colesterol plasmático. Entre ellas, el polimorfismo 23968A>G ha sido relacionado con hipercolesterolemia poligénica en individuos europeos y japoneses. Objetivos: Determinar la frecuencia de la variante 23968A>G del gen PCSK9 en pacientes con enfermedad arterial coronaria y controles, y evaluar su posible contribución al desarrollo de esta patología en individuos chilenos. Métodos: Se efectuó genotipificación de la variante 23968A>G del gen PCSK9 a 218 individuos no relacionados de la Región de La Araucanía: 110 con enfermedad coronaria confirmada por angiografía (estenosis >70 por ciento) y 108 sujetos controles, mediante la técnica de reacción en cadena de la polimerasa seguida de restricción enzimática (PCR-RFLP). Resultados: La distribución de genotipos para el polimorfismo 23968A>G del gen PCSK9 en los pacientes con enfermedad coronaria (AA: 95.5 por ciento, AG: 4.5 por ciento, GG: 0 por ciento) y controles (AA: 95.4 por ciento, AG: 4.6 por ciento, GG: 0 por ciento) fue similar (p= 0.769). La frecuencia del alelo mutado G fue también semejante entre casos y controles (0.0227 vs. 0.0231, p=0.771). La odds ratio (OR) relacionada al alelo mutado G fue 0.98 (I.C. 95 por ciento: 0.28 - 3.44) confirmando la ausencia de asociación. Conclusiones: El polimorfismo 23968A>G del gen PCSK9 no contribuye para el desarrollo de enfermedad arterial coronaria en la población estudiada.


Background: Common variants of the PCSK9 gene are associated to elevation of plasma cholesterol levels. Among them, the 23968A>G variant has been related to polygenic hipercholesterolemia in European and in Japanese subjects. Aim: to determine the frequency of the 23968A>G variant of the PCSK9 gene in patients with coronary artery disease and controls and to estimate its significance in the development of CAD in Chilean subjects. Methods: Genotypes of the 23968A>G variant of the PCSK9 gene were determined in 218 unrelated subjectsin the Region de La Araucania. One- hundred ten had angiography proven significant coronary artery disease(>70 percent stenosis) and 108 were controls. PCR followed by enzymatic restriction was used. Results: The genotype distribution for 23,968A>G variant in CAD patients (AA: 95.5 percent, AG: 4.5 percent, GG: 0 percent) and controls (AA: 95.4 percent, AG: 4.6 percent, GG: 0 percent) was similar (p=0.769). The relative frequency of mutated 23,968G allele in CAD and controls was not significantly different (0.0227 vs. 0.0231, p=0.771). Moreover, the odds ratio (OR) shown that the G allele was not associated with CAD (OR: 0.98; 95 percent C.I. = 0.28 - 3.44, P = NS). Conclusion: The 23968A>G polymorphism of the PCSK9 gene is not related to the presence of CAD in this population.


Subject(s)
Humans , Male , Female , Adult , Middle Aged , Cardiovascular Diseases/genetics , Hypercholesterolemia/genetics , Polymorphism, Genetic , Serine Endopeptidases/blood , Case-Control Studies , Chile/epidemiology , Cholesterol, HDL/blood , Cholesterol, LDL/blood , DNA , Genetic Predisposition to Disease , Genetic Variation , Mutation , Polymerase Chain Reaction , Serine Endopeptidases/genetics
17.
J Genet ; 2005 Aug; 84(2): 131-6
Article in English | IMSEAR | ID: sea-114266

ABSTRACT

Genetic polymorphisms associated with structural changes of their gene product are important in terms of their potential relation with diseases. Therefore, in this study, splice-site variants of the transmembrane serine protease gene TMPRSS4, nephronophthisis gene NPHP4, and organic-cation transporter gene ORCTL4, were selected from the dbSNP single nucleotide polymorphism database as candidates to identify genetic polymorphisms associated with a structural change in their mRNA transcripts. The allele frequencies of the TMPRSS4 c.4-7A>G, NPHP4 c.2818-2A>T, and ORCTL4 c.517-2A>C polymorphisms in a Japanese population were determined to be 0.42, 0.10, and 0.27, respectively, by PCR-SSCP analysis. Next, the effect of these polymorphisms on the mode of pre-mRNA splicing was investigated by RT-PCR analysis followed by sequencing analysis. The TMPRSS4, NPHP4, and ORCTL4 polymorphisms were associated with the production of the r.4-6_4-1ins transcript, the r.2818_2823del and r.2818_2859del transcripts, and the r.517-94_517-1ins; r.517-2a>c and r.517_620del transcripts, respectively. Since the proteins encoded by all these transcripts are associated with relatively significant structural changes in the form amino acid insertion/deletion and premature termination, their functional ability may be greatly reduced. Our demonstration of structural changes in mRNA transcripts as a result of splice-site polymorphisms implies that they may be of biological significance in certain pathological conditions.


Subject(s)
Alleles , Base Sequence , DNA, Complementary/genetics , Gene Expression , Gene Frequency , Humans , Japan , Membrane Proteins/genetics , Organic Cation Transport Proteins/genetics , Polymorphism, Genetic , Proteins/genetics , RNA Splice Sites/genetics , RNA, Messenger/genetics , Serine Endopeptidases/genetics
18.
Article in Korean | WPRIM | ID: wpr-16719

ABSTRACT

BACKGROUND/AIMS: X-linked inhibitor of apoptosis (XIAP) is the most potent member of the IAP family that exerts antiapoptotic effects. Recently, XIAP-associated factor 1 (XAF1) and two mitochondrial proteins, Smac/DIABLO and HtrA2, have been identified to negatively regulate the caspase-inhibiting activity of XIAP. We explored the candidacy of XAF1, Smac/DIABLO and HtrA2 as a tumor suppressor in colonic carcinogenesis. METHODS: Expression and mutation status of the genes in 10 colorectal carcinoma cell lines and 40 primary tumors were examined by quantitative PCR analysis. RESULTS: XAF1 transcript was not expressed or present at extremely low levels in 60% (6/10) of cancer cell lines whereas Smac/DIABLO and HtrA2 are normally expressed in all cell lines examined. Tumor-specific loss or reduction of XAF1 was also found in 35% (14/40) of matched tissue sets obtained from the same patients. XAF1 transcript was reactivated in all the low expressor cell lines by treatment with the demethylating agent 5-aza-2'-deoxycytidine. Moreover, bisulfite DNA sequencing analysis for 34 CpG sites in the promoter region revealed a strong association between hypermethylation and gene silencing. Restoration of XAF1 expression resulted in enhanced apoptotic response to etoposide and 5-flurouracil, whereas knockdown of XAF1 expression by siRNA transfection significantly inhibited chemotherapeutic drug-induced apoptosis. CONCLUSIONS: XAF1 undergoes epigenetic gene silencing in a considerable proportion of human colon cancers by aberrant promoter hypermethylation, suggesting that XAF1 inactivation might be implicated in colonic tumorigenesis.


Subject(s)
Cell Line, Tumor , Colonic Neoplasms/genetics , DNA Methylation , English Abstract , Gene Expression Regulation, Neoplastic , Gene Silencing , Humans , Intracellular Signaling Peptides and Proteins/genetics , Mitochondrial Proteins/genetics , Neoplasm Proteins/genetics , Promoter Regions, Genetic , Serine Endopeptidases/genetics
19.
Article in English | WPRIM | ID: wpr-56736

ABSTRACT

Leader peptidase is a novel serine protease in Escherichia coli, which catalyzes the cleavage of amino-terminal signal sequences from exported proteins. It is an integral membrane protein containing two transmembrane segments with its carboxy-terminal catalytic domain residing in the periplasmic space. Recently, the x-ray crystal structure of signal peptidase-inhibitor complex showed that Asp 280, a highly conserved consensus sequence of E. coli leader peptidase is the closest charged residue in the vicinity of two catalytic dyad, Ser 90 and Lys 145, and it is likely held in place by a salt bridge to Arg 282. Possible roles of Asp 280 and Arg 282 in the structure-catalytic function relationship were investigated by the site-directed mutagenesis of Asp 280 substituted with alanine, glutamic acid, glycine, or asparagine and of Arg 282 with methionine. All of mutants purified with nickel affinity chromatography were inactive using in vitro assay. It is surprising to find complete lose of activity by an extension of one carbon units in the mutant where Asp 280 is substituted with glutamic acid. These results suggest that Asp 280 and Arg 282 are in a sequence which constitutes catalytic crevice of leader peptidase and are essential for maintaining the conformation of catalytic pocket.


Subject(s)
Aspartic Acid/chemistry , Bacterial Outer Membrane Proteins/metabolism , Blotting, Western , Escherichia coli/enzymology , Escherichia coli/chemistry , Micrococcal Nuclease/metabolism , Mutagenesis, Site-Directed , Oligonucleotides , Protein Precursors/metabolism , Serine Endopeptidases/metabolism , Serine Endopeptidases/genetics , Serine Endopeptidases/chemistry , Structure-Activity Relationship
20.
Article in English | WPRIM | ID: wpr-186197

ABSTRACT

A heterozygous GTG to ATG (Val297Met) mutation was detected in a patient with inherited protein C deficiency and deep vein thrombosis. Cosegregation of the mutation with protein C deficiency was observed through a family pedigree study. Molecular models of the serine protease domains of wild type and mutant protein C were constructed by standard comparative method. Val 297 was found to be located in the hydrophobic core of the protein. Although the substitution of Met for Val does not greatly alter the hydrophobicity of the protein, it introduces a bulkier side chain, which yields steric hindrance between this residue and adjacent residues, such as Met364, Tyr393, Ile321, Ile323, and Val378. It seems that the Met can not fit into the tight packing into which it is trapped, thereby probably inducing misfolding and/or greater instability of the protein. Such misfolding and/or instability thereby eventually disturbs the catalytic triad, in consistent with the observed type I deficiency state.


Subject(s)
Adult , Base Sequence , Female , Humans , Male , Middle Aged , Models, Molecular , Molecular Sequence Data , Pedigree , Point Mutation , Polymerase Chain Reaction/methods , Protein C/genetics , Protein C/chemistry , Protein C Deficiency/genetics , Protein Conformation , Serine Endopeptidases/genetics , Serine Endopeptidases/chemistry , Venous Thrombosis/genetics
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