RESUMO
Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens causing severe gastroenteritis, which may lead to hemolytic uremic syndrome. The Locus of Enterocyte Effacement (LEE), a Pathogenicity Island (PAI), is a major determinant of intestinal epithelium attachment of a group of STEC strains; however, the virulence repertoire of STEC strains lacking LEE, has not been fully characterized. The incidence of LEE-negative STEC strains has increased in several countries, highlighting the relevance of their study. In order to gain insights into the basis for the emergence of LEE-negative STEC strains, we performed a large-scale genomic analysis of 367 strains isolated worldwide from humans, animals, food and the environment. We identified uncharacterized genomic islands, including two PAIs and one Integrative Conjugative Element. Additionally, the Locus of Adhesion and Autoaggregation (LAA) was the most prevalent PAI among LEE-negative strains and we found that it contributes to colonization of the mice intestine. Our comprehensive and rigorous comparative genomic and phylogenetic analyses suggest that the accumulative acquisition of PAIs has played an important, but currently unappreciated role, in the evolution of virulence in these strains. This study provides new knowledge on the pathogenicity of LEE-negative STEC strains and identifies molecular markers for their epidemiological surveillance.
Assuntos
Evolução Molecular , Ilhas Genômicas , Fosfoproteínas/deficiência , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/patogenicidade , Fatores de Virulência/genética , Animais , Modelos Animais de Doenças , Microbiologia Ambiental , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Proteínas de Escherichia coli , Microbiologia de Alimentos , Genótipo , Incidência , Sequências Repetitivas Dispersas , Intestinos/microbiologia , Camundongos , Filogenia , Escherichia coli Shiga Toxigênica/genética , VirulênciaRESUMO
OBJECTIVES: To determine the genetic basis of disordered steroidogenesis in Kuwaiti siblings. STUDY DESIGN: Two siblings (46,XX and 46,XY) had normal female external genitalia and severe glucocorticoid and mineralocorticoid deficiency presenting in the first month of life. Abdominal ultrasonography showed normal size adrenal glands, suggesting cholesterol side chain cleavage enzyme (P450scc) deficiency. The CYP11A1 gene encoding P450scc and the STAR gene encoding the steroidogenic acute regulatory protein (StAR) were directly sequenced from leukocyte DNA. RESULTS: All exons and intron/exon boundaries of the CYP11A1 gene were normal; the STAR gene was homozygous for a novel 14-base deletion/frameshift in exon 4 (g.4643_4656del), so that no functional protein could be produced. Both parents and an unaffected sibling were heterozygous; zygosity was confirmed with a BsmF1 restriction fragment length polymorphism. CONCLUSIONS: Unlike most patients with StAR deficiency, our patients did not have the massive adrenal hyperplasia typical of congenital lipoid adrenal hyperplasia. The distinction between StAR and P450scc deficiency may require gene sequencing.
Assuntos
Insuficiência Adrenal/genética , Enzima de Clivagem da Cadeia Lateral do Colesterol/deficiência , Fosfoproteínas/deficiência , Insuficiência Adrenal/diagnóstico , Enzima de Clivagem da Cadeia Lateral do Colesterol/genética , DNA/metabolismo , Éxons , Saúde da Família , Feminino , Glucocorticoides/deficiência , Homozigoto , Humanos , Recém-Nascido , Íntrons , Kuweit , Leucócitos/metabolismo , Mineralocorticoides/deficiência , Modelos Genéticos , Mutação , Linhagem , Polimorfismo de Fragmento de RestriçãoRESUMO
Treacher Collins syndrome (TCS) is an autosomal dominant disorder of craniofacial development, and mutations in the TCOF1 gene are responsible for over 90% of TCS cases. The knowledge about the molecular mechanisms responsible for this syndrome is relatively scant, probably due to the difficulty of reproducing the pathology in experimental animals. Zebrafish is an emerging model for human disease studies, and we therefore assessed it as a model for studying TCS. We identified in silico the putative zebrafish TCOF1 ortholog and cloned the corresponding cDNA. The derived polypeptide shares the main structural domains found in mammals and amphibians. Tcof1 expression is restricted to the anterior-most regions of zebrafish developing embryos, similar to what happens in mouse embryos. Tcof1 loss-of-function resulted in fish showing phenotypes similar to those observed in TCS patients, and enabled a further characterization of the mechanisms underlying craniofacial malformation. Besides, we initiated the identification of potential molecular targets of treacle in zebrafish. We found that Tcof1 loss-of-function led to a decrease in the expression of cellular proliferation and craniofacial development. Together, results presented here strongly suggest that it is possible to achieve fish with TCS-like phenotype by knocking down the expression of the TCOF1 ortholog in zebrafish. This experimental condition may facilitate the study of the disease etiology during embryonic development.
Assuntos
Modelos Animais de Doenças , Disostose Mandibulofacial/genética , Disostose Mandibulofacial/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra , Sequência de Aminoácidos , Animais , Movimento Celular , Tamanho Celular , Biologia Computacional , Face/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Disostose Mandibulofacial/patologia , Camundongos , Dados de Sequência Molecular , Crista Neural/metabolismo , Crista Neural/patologia , Fenótipo , Fosfoproteínas/química , Fosfoproteínas/deficiência , Homologia de Sequência de Aminoácidos , Crânio/embriologia , Crânio/metabolismo , Fatores de Tempo , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/química , Proteínas de Peixe-Zebra/deficiênciaRESUMO
The long polar fimbriae (Lpf) is one of few adhesive factors of Shiga toxin-producing Escherichia coli (STEC) and it is associated with colonization of the intestine. Studies have demonstrated the presence of lpf genes in several pathogenic E. coli strains, and classification of variants based on polymorphisms in the lpfA1 and lpfA2 genes has been adopted. Using a collection of Argentinean locus of enterocyte effacement (LEE)-negative STEC strains, we determined that the different lpfA types were present in a wide variety of serotypes with no apparent association between the types of lpfA1 or lpfA2 genes and the severity of human disease. The lpfA2-1 was the most prevalent variant identified, which was present in 95.8% of the isolates, and lpfA1-3 and lpfA2-2, proposed as specific biomarkers of E. coli O157:H7, were not found in any of the serotypes studied. The prevalence of lpf genes in a large number of strains is useful to understand the genetic diversity of LEE-negative STEC and to define the association of some of these isolates carrying specific lpf-variants with disease.
Assuntos
Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Fímbrias Bacterianas/genética , Fosfoproteínas/deficiência , Escherichia coli Shiga Toxigênica/genética , Animais , Argentina , Bovinos , Proteínas de Escherichia coli , Variação Genética , Humanos , Sorotipagem , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/isolamento & purificaçãoAssuntos
Diabetes Mellitus Tipo 2/genética , Proteínas de Homeodomínio , Ilhotas Pancreáticas/fisiopatologia , Proteínas Nucleares , Adolescente , Adulto , Idade de Início , Animais , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Criança , Pré-Escolar , Proteínas de Ligação a DNA/genética , Diabetes Mellitus Tipo 2/classificação , Diabetes Mellitus Tipo 2/epidemiologia , Europa (Continente)/epidemiologia , Feminino , Predisposição Genética para Doença , Glucoquinase/genética , Fator 1 Nuclear de Hepatócito , Fator 1-alfa Nuclear de Hepatócito , Fator 1-beta Nuclear de Hepatócito , Fator 4 Nuclear de Hepatócito , Humanos , Lactente , Insulina/metabolismo , Secreção de Insulina , Japão/epidemiologia , Masculino , México/epidemiologia , Camundongos , Camundongos Knockout , Pessoa de Meia-Idade , Mutação , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosfoproteínas/fisiologia , Transativadores/deficiência , Transativadores/genética , Fatores de Transcrição/deficiência , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologiaRESUMO
Using DDRT-PCR, we compared the mRNA content of untreated and TNF-treated mouse embryonic fibroblasts (MEFs). Among differentially represented fragments, we identified and cloned a novel TNF-stimulated gene named Tsg-5. This gene, mapped to mouse chromosome 14, has three exons that can be alternatively spliced giving rise to two mRNA species, one spanning three exons and another that skips the second exon. Analysis of full-length Tsg-5 cDNA revealed a potential start codon within exon 2 encoding an ORF of 40 amino-acids. No homology with known mouse or human sequences, neither at the nucleotide nor at the amino-acid level could be found in public databases. In MEFs, Tsg-5 is induced by tumor necrosis factor-alpha (TNF) and IL-1 beta, albeit with distinct kinetics. TNF-induced Tsg-5 expression is NF-kappa B-dependent as it was inhibited by MG132, lactacystin, Bay 11-7083, and Bay 11-7085. Analysis of Tsg-5 expression in vivo revealed that the gene and its encoded polypeptide are constitutively expressed in the thymus and ovary, whereas, in LPS-treated mice, Tsg-5 mRNA can be detected in the spleen, lung, and brain. Our data suggest that Tsg-5 encodes a new, rare transcript, with a very tight regulation of expression and differential splicing.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/isolamento & purificação , Fator de Necrose Tumoral alfa/fisiologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Clonagem Molecular , DNA Complementar/isolamento & purificação , Proteínas de Ligação a DNA/antagonistas & inibidores , Proteínas de Ligação a DNA/biossíntese , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/fisiologia , Feminino , Regulação da Expressão Gênica/imunologia , Fator Regulador 1 de Interferon , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Dados de Sequência Molecular , NF-kappa B/metabolismo , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Fosfoproteínas/biossíntese , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Reação em Cadeia da PolimeraseRESUMO
In addition to neuroendocrine abnormalities, women with polycystic ovary syndrome have insulin resistance and beta-cell dysfunction associated with a high frequency of metabolic syndrome components, such as glucose intolerance, type 2 diabetes mellitus (DM-2), dyslipidemia and a higher risk for endothelial dysfunction, haemostatic abnormalities, hypertension and cardiovascular disease. Obesity, a common finding in this disorder, plays an important role in the development of metabolic and cardiovascular disorders. Early identification of patients and prompt initiation of insulin sensitizing therapy by pharmacological agents or changes in life style such diet and exercise might improve the metabolic and endocrine abnormalities and reduce the risk of DM-2 and cardiovascular disease in these patients.
Assuntos
Síndrome do Ovário Policístico/complicações , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/prevenção & controle , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/prevenção & controle , Diabetes Gestacional/etiologia , Feminino , Humanos , Hiperandrogenismo/etiologia , Hiperandrogenismo/fisiopatologia , Hiperlipidemias/etiologia , Hipoglicemiantes/uso terapêutico , Proteínas Substratos do Receptor de Insulina , Resistência à Insulina/fisiologia , Síndrome Metabólica/etiologia , Síndrome Metabólica/prevenção & controle , Obesidade/etiologia , Obesidade/prevenção & controle , Fosfoproteínas/deficiência , Síndrome do Ovário Policístico/fisiopatologia , Síndrome do Ovário Policístico/terapia , Gravidez , Receptor de Insulina/fisiologia , Transdução de SinaisRESUMO
In addition to neuroendocrine abnormalities, women with polycystic ovary syndrome have insulin resistance and beta-cell dysfunction associated with a high frequency of metabolic syndrome components, such as glucose intolerance, type 2 diabetes mellitus (DM-2), dyslipidemia and a higher risk for endothelial dysfunction, haemostatic abnormalities, hypertension and cardiovascular disease. Obesity, a common finding in this disorder, plays an important role in the development of metabolic and cardiovascular disorders. Early identification of patients and prompt initiation of insulin sensitizing therapy by pharmacological agents or changes in life style such diet and exercise might improve the metabolic and endocrine abnormalities and reduce the risk of DM-2 and cardiovascular disease in these patients.
Assuntos
Feminino , Humanos , Gravidez , Síndrome do Ovário Policístico/complicações , Diabetes Mellitus Tipo 2/etiologia , Diabetes Mellitus Tipo 2/prevenção & controle , Diabetes Gestacional , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/prevenção & controle , Fosfoproteínas/deficiência , Hipoglicemiantes/uso terapêutico , Hiperandrogenismo , Hiperlipidemias , Obesidade , Receptor de Insulina , Resistência à Insulina/fisiologia , Síndrome do Ovário Policístico/fisiopatologia , Síndrome do Ovário Policístico/terapia , Síndrome Metabólica/etiologia , Síndrome Metabólica/prevenção & controle , Transdução de SinaisRESUMO
BACKGROUND: The cytosolic protein p47-phox (phagocyte oxidase) is one of the essential components of the superoxide generating system in phagocytes and its defect causes approximately 30% of the chronic granulomatous disease (CGD) cases. AIM: Two patients were studied, belonging to the same family, without a consanguinous background, in which deficiency or absence of superoxide generation was found together with recurrent and severe infections in one case and benign infections in the second. METHODS: The presence of gp91-, p67- and p47-phox in patients and controls was determined by Western Blot analysis of granulocytes. Sequencing of PCR amplified DNA was performed by an enzymatic method. RESULTS: Western Blot analysis showed normal expression of gp91 and p67 and absence of p47-phox. The molecular genetic study demonstrated a homocygotic dinucleotide GT (GT) deletion at the beginning of exon 2 of the p47-phox gene. The same mutation has been found in European, American and Japanese patients. CONCLUSIONS: The molecular characterization of this pathology done for the first time in Chile is important for diagnostic classification, patient prognosis, and adequate genetic advice and a possible future therapy.
Assuntos
Doença Granulomatosa Crônica/genética , Fosfoproteínas/deficiência , Adolescente , Adulto , Western Blotting , Estudos de Casos e Controles , Análise Mutacional de DNA , Ensaio de Imunoadsorção Enzimática , Granulócitos/enzimologia , Doença Granulomatosa Crônica/enzimologia , Humanos , Masculino , NADPH Oxidases , Linhagem , Reação em Cadeia da Polimerase , Deleção de SequênciaRESUMO
In order to identify new interferon-stimulated genes that could help in the better understanding of the mechanism of action of interferons (IFNs), we decided to compare, by differential display RT-PCR (DDRT-PCR), the pattern of gene expression between IFN-alpha treated and untreated mouse embryonic fibroblasts (MEFs). Here we describe the initial characterization of a new cDNA fragment, named FRAG-6, that is expressed only upon IFN stimulation. The IFN-induced expression of this new gene can be observed in both wild-type and IRF-1-deficient MEF. FRAG-6 cDNA hybridizes with an mRNA of 6-9 kb that is induced by IFNs in a time-dependent manner. Analysis of the cloned nucleotide sequence revealed a 174 amino acid (aa) open reading frame (ORF) contained within the 576 bp. No significant homology with known nucleotide or protein sequences was observed. FRAG-6 is induced in vitro upon treatment of wild type or IRF-1-null cells with IFN-alpha or -gamma, but not with TNF or IL-1. Treatment of mice with imiquimod, a potent inducer of IFN, led to induced expression of FRAG-6 mRNA in various organs from wild type or IRF-1-deficient mice, but not from STAT-1 or type I IFN receptor deficient animals. Our results demonstrate that FRAG-6 mRNA induction by interferons is IRF-1-independent and it is likely to be activated by the JAK/STAT pathway. Further characterization of FRAG-6 will help us in the understanding of the mechanism of action of IFNs.