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1.
J Exp Med ; 204(4): 853-63, 2007 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-17420270

RESUMO

Mouse cytomegalovirus (MCMV) susceptibility often results from defects of natural killer (NK) cell function. Here we describe Jinx, an N-ethyl-N-nitrosourea-induced MCMV susceptibility mutation that permits unchecked proliferation of the virus, causing death. In Jinx homozygotes, activated NK cells and cytotoxic T lymphocytes (CTLs) fail to degranulate, although they retain the ability to produce cytokines, and cytokine levels are markedly elevated in the blood of infected mutant mice. Jinx was mapped to mouse chromosome 11 on a total of 246 meioses and confined to a 4.60-million basepair critical region encompassing 122 annotated genes. The phenotype was ascribed to the creation of a novel donor splice site in Unc13d, the mouse orthologue of human MUNC13-4, in which mutations cause type 3 familial hemophagocytic lymphohistiocytosis (FHL3), a fatal disease marked by massive hepatosplenomegaly, anemia, and thrombocytopenia. Jinx mice do not spontaneously develop clinical features of hemophagocytic lymphohistiocytosis (HLH), but do so when infected with lymphocytic choriomeningitis virus, exhibiting hyperactivation of CTLs and antigen-presenting cells, and inadequate restriction of viral proliferation. In contrast, neither Listeria monocytogenes nor MCMV induces the syndrome. In mice, the HLH phenotype is conditional, which suggests the existence of a specific infectious trigger of FHL3 in humans.


Assuntos
Modelos Animais de Doenças , Predisposição Genética para Doença , Infecções por Herpesviridae/metabolismo , Linfo-Histiocitose Hemofagocítica/metabolismo , Linfo-Histiocitose Hemofagocítica/patologia , Proteínas de Membrana/metabolismo , Muromegalovirus/fisiologia , Animais , Células Apresentadoras de Antígenos/metabolismo , Apoptose , Clonagem Molecular , Infecções por Herpesviridae/genética , Infecções por Herpesviridae/patologia , Interferon gama/biossíntese , Linfo-Histiocitose Hemofagocítica/classificação , Linfo-Histiocitose Hemofagocítica/genética , Proteínas de Membrana/genética , Camundongos , Mutação/genética , Fenótipo
2.
Proc Natl Acad Sci U S A ; 106(7): 2097-103, 2009 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-19196968

RESUMO

A mouse neurological mutant, lister, was identified through a genome-wide N-ethyl-N-nitrosourea (ENU) mutagenesis screen. Homozygous lister mice exhibit profound early-onset and progressive neurological and motor dysfunction. lister encodes a RING finger protein, LISTERIN, which functions as an E3 ubiquitin ligase in vitro. Although lister is widely expressed in all tissues, motor and sensory neurons and neuronal processes in the brainstem and spinal cord are primarily affected in the mutant. Pathological signs include gliosis, dystrophic neurites, vacuolated mitochondria, and accumulation of soluble hyperphosphorylated tau. Analysis with a different lister allele generated through targeted gene trap insertion reveals LISTERIN is required for embryonic development and confirms that direct perturbation of a LISTERIN-regulated process causes neurodegeneration. The lister mouse uncovers a pathway involved in neurodegeneration and may serves as a model for understanding the molecular mechanisms underlying human neurodegenerative disorders.


Assuntos
Mutação , Doenças Neurodegenerativas/genética , Ubiquitina-Proteína Ligases/metabolismo , Alelos , Animais , Axônios , Genótipo , Homozigoto , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Mutagênese , Fenótipo , Distribuição Tecidual , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/fisiologia
3.
PLoS Genet ; 3(1): e8, 2007 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-17206865

RESUMO

Premature truncation alleles in the ALMS1 gene are a frequent cause of human Alström syndrome. Alström syndrome is a rare disorder characterized by early obesity and sensory impairment, symptoms shared with other genetic diseases affecting proteins of the primary cilium. ALMS1 localizes to centrosomes and ciliary basal bodies, but truncation mutations in Alms1/ALMS1 do not preclude formation of cilia. Here, we show that in vitro knockdown of Alms1 in mice causes stunted cilia on kidney epithelial cells and prevents these cells from increasing calcium influx in response to mechanical stimuli. The stunted-cilium phenotype can be rescued with a 5' fragment of the Alms1 cDNA, which resembles disease-associated alleles. In a mouse model of Alström syndrome, Alms1 protein can be stably expressed from the mutant allele and is required for cilia formation in primary cells. Aged mice developed specific loss of cilia from the kidney proximal tubules, which is associated with foci of apoptosis or proliferation. As renal failure is a common cause of mortality in Alström syndrome patients, we conclude that this disease should be considered as a further example of the class of renal ciliopathies: wild-type or mutant alleles of the Alström syndrome gene can support normal kidney ciliogenesis in vitro and in vivo, but mutant alleles are associated with age-dependent loss of kidney primary cilia.


Assuntos
Cílios/metabolismo , Proteínas de Ligação a DNA/metabolismo , Rim/citologia , Rim/metabolismo , Anormalidades Múltiplas/patologia , Envelhecimento/metabolismo , Animais , Proteínas de Ciclo Celular , Cílios/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Homeostase , Humanos , Rim/anormalidades , Rim/patologia , Mecanotransdução Celular , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Fragmentos de Peptídeos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Síndrome , Transcrição Gênica
4.
PLoS Med ; 5(3): e54, 2008 Mar 04.
Artigo em Inglês | MEDLINE | ID: mdl-18318598

RESUMO

BACKGROUND: MUC2 mucin produced by intestinal goblet cells is the major component of the intestinal mucus barrier. The inflammatory bowel disease ulcerative colitis is characterized by depleted goblet cells and a reduced mucus layer, but the aetiology remains obscure. In this study we used random mutagenesis to produce two murine models of inflammatory bowel disease, characterised the basis and nature of the inflammation in these mice, and compared the pathology with human ulcerative colitis. METHODS AND FINDINGS: By murine N-ethyl-N-nitrosourea mutagenesis we identified two distinct noncomplementing missense mutations in Muc2 causing an ulcerative colitis-like phenotype. 100% of mice of both strains developed mild spontaneous distal intestinal inflammation by 6 wk (histological colitis scores versus wild-type mice, p < 0.01) and chronic diarrhoea. Monitoring over 300 mice of each strain demonstrated that 25% and 40% of each strain, respectively, developed severe clinical signs of colitis by age 1 y. Mutant mice showed aberrant Muc2 biosynthesis, less stored mucin in goblet cells, a diminished mucus barrier, and increased susceptibility to colitis induced by a luminal toxin. Enhanced local production of IL-1beta, TNF-alpha, and IFN-gamma was seen in the distal colon, and intestinal permeability increased 2-fold. The number of leukocytes within mesenteric lymph nodes increased 5-fold and leukocytes cultured in vitro produced more Th1 and Th2 cytokines (IFN-gamma, TNF-alpha, and IL-13). This pathology was accompanied by accumulation of the Muc2 precursor and ultrastructural and biochemical evidence of endoplasmic reticulum (ER) stress in goblet cells, activation of the unfolded protein response, and altered intestinal expression of genes involved in ER stress, inflammation, apoptosis, and wound repair. Expression of mutated Muc2 oligomerisation domains in vitro demonstrated that aberrant Muc2 oligomerisation underlies the ER stress. In human ulcerative colitis we demonstrate similar accumulation of nonglycosylated MUC2 precursor in goblet cells together with ultrastructural and biochemical evidence of ER stress even in noninflamed intestinal tissue. Although our study demonstrates that mucin misfolding and ER stress initiate colitis in mice, it does not ascertain the genetic or environmental drivers of ER stress in human colitis. CONCLUSIONS: Characterisation of the mouse models we created and comparison with human disease suggest that ER stress-related mucin depletion could be a fundamental component of the pathogenesis of human colitis and that clinical studies combining genetics, ER stress-related pathology and relevant environmental epidemiology are warranted.


Assuntos
Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Retículo Endoplasmático/patologia , Mucinas/metabolismo , Adulto , Animais , Apoptose , Proliferação de Células , Colite Ulcerativa/genética , Colite Ulcerativa/imunologia , Retículo Endoplasmático/ultraestrutura , Regulação da Expressão Gênica , Predisposição Genética para Doença , Células Caliciformes/patologia , Humanos , Inflamação , Mucosa Intestinal/patologia , Mucosa Intestinal/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Mucina-2 , Mucinas/química , Mucinas/genética , Mutação/genética , Dobramento de Proteína , Precursores de Proteínas/metabolismo , Estrutura Quaternária de Proteína
5.
Fly (Austin) ; 1(3): 164-71, 2007.
Artigo em Inglês | MEDLINE | ID: mdl-18820470

RESUMO

The availability of complete genome sequence information for diverse organisms including model genetic organisms has ushered in a new era of protein sequence comparisons making it possible to search for commonalities among entire proteomes using the Basic Local Alignment Search Tool (BLAST). Although the identification and analysis of proteins shared by humans and model organisms has proven an invaluable tool to understanding gene function, the sets of proteins unique to a given model organism's proteome have remained largely unexplored. We have constructed a searchable database that allows biologists to identify proteins unique to a given proteome. The Negative Proteome Database (NPD) is populated with pair-wise protein sequence comparisons between each of the following proteomes: Homo sapiens, Mus musculus, Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, Dictyostelium discoideum, Chlamydomonus reinhardti, Escherichia coli K12, Arabidopsis thaliana and Methanoscarcina acetivorans. Our analysis of negative proteome datasets using the NPD has thus far revealed 107 proteins in humans that may be involved in motile cilia function, 1628 potential pesticide target proteins in flies, 659 proteins shared by flies and humans that are not represented in the less neurologically complex worm proteome, and 180 nuclear encoded human disease associated proteins that are absent from the fly proteome. The NPD is the only online resource where users can quickly perform complex negative and positive comparisons of model organism proteomes. We anticipate that the NPD and the adaptable algorithm which can readily be used to duplicate this analysis on custom sets of proteomes will be an invaluable tool in the investigation of organism specific protein sets.


Assuntos
Bases de Dados de Proteínas , Proteínas de Drosophila/genética , Drosophila/genética , Proteoma , Animais , Genes de Insetos , Humanos , Proteômica/estatística & dados numéricos , Alinhamento de Sequência/estatística & dados numéricos
6.
Proc Natl Acad Sci U S A ; 104(30): 12445-50, 2007 Jul 24.
Artigo em Inglês | MEDLINE | ID: mdl-17640884

RESUMO

Condensins are ubiquitously expressed multiprotein complexes that are important for chromosome condensation and epigenetic regulation of gene transcription, but whose specific roles in vertebrates are poorly understood. We describe a mouse strain, nessy, isolated during an ethylnitrosourea screen for recessive immunological mutations. The nessy mouse has a defect in T lymphocyte development that decreases circulating T cell numbers, increases their expression of the activation/memory marker CD44, and dramatically decreases the numbers of CD4(+)CD8(+) thymocytes and their immediate DN4 precursors. A missense mutation in an unusual alternatively spliced first exon of the kleisin beta gene, a member of the condensin II complex, was shown to be responsible and act in a T cell-autonomous manner. Despite the ubiquitous expression and role of condensins, kleisin beta(nes/nes) mice were viable, fertile, and showed no defects even in the parallel pathway of B cell lymphocyte differentiation. These data define a unique lineage-specific requirement for kleisin beta in mammalian T cell differentiation.


Assuntos
Adenosina Trifosfatases/metabolismo , Diferenciação Celular , Cromossomos de Mamíferos/genética , Proteínas de Ligação a DNA/metabolismo , Complexos Multiproteicos/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Animais , Subpopulações de Linfócitos B/imunologia , Sequência de Bases , Linhagem da Célula , Células Cultivadas , Sequência Conservada , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Vetores Genéticos/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Dados de Sequência Molecular , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Mutação/genética , Fenótipo , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Retroviridae/genética , Alinhamento de Sequência , Baço/metabolismo
7.
Immunity ; 18(6): 751-62, 2003 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-12818157

RESUMO

In a genome-wide ENU mouse mutagenesis screen a recessive mouse mutation, unmodulated, was isolated with profound defects in humoral immune responses, selective deficits in B cell activation by antigen receptors and T cell costimulation by CD28, and gradual development of atopic dermatitis with hyper-IgE. Mutant B cells are specifically defective in forming connections between antigen receptors and two key signaling pathways for immunogenic responses, NF-kappaB and JNK, but signal normally to calcium, NFAT, and ERK. The mutation alters a conserved leucine in the coiled-coil domain of CARMA-1/CARD11, a member of the MAGUK protein family implicated in organizing multimolecular signaling complexes. These results define Carma-1 as a key regulator of the plasticity in antigen receptor signaling that underpins opposing mechanisms of immunity and tolerance.


Assuntos
Formação de Anticorpos/imunologia , Núcleosídeo-Fosfato Quinase/metabolismo , Envelhecimento/imunologia , Sequência de Aminoácidos , Animais , Linfócitos B/imunologia , Dermatite/imunologia , Guanilato Quinases , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Dados de Sequência Molecular , Mutagênese , NF-kappa B/metabolismo , Núcleosídeo-Fosfato Quinase/genética , Núcleosídeo-Fosfato Quinase/imunologia , Linfócitos T/imunologia
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