RESUMO
Mice with the recessive motheaten (me) or the allelic viable motheaten (mev) mutations express a severe autoimmune and immunodeficiency syndrome. We have shown that the basic defect in these mice involves lesions in the gene which encodes haematopoietic cell phosphatase (HCP). These mice thus provide excellent models for investigating the roles of phosphatases in haematopoiesis and the nature of the genetic and cellular events linking impaired haematopoiesis to severe immunodeficiency and expression of systemic autoimmunity.
Assuntos
Doenças Autoimunes/genética , Células-Tronco Hematopoéticas/enzimologia , Síndromes de Imunodeficiência/genética , Camundongos Mutantes/genética , Mutação , Proteínas Tirosina Fosfatases/genética , Alopecia/genética , Alopecia/imunologia , Alopecia/patologia , Animais , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Sequência de Bases , Medula Óssea/enzimologia , Medula Óssea/metabolismo , Medula Óssea/patologia , Mapeamento Cromossômico , Sequência Consenso , Análise Mutacional de DNA , Genes Recessivos , Células-Tronco Hematopoéticas/patologia , Síndromes de Imunodeficiência/imunologia , Síndromes de Imunodeficiência/patologia , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Endogâmicos C57BL/genética , Camundongos Mutantes/imunologia , Dados de Sequência Molecular , Fenótipo , Fosforilação , Reação em Cadeia da Polimerase , Processamento de Proteína Pós-Traducional , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/deficiência , Splicing de RNA , RNA Mensageiro/genética , Alinhamento de Sequência , Deleção de SequênciaRESUMO
Hematopoietic cell phosphatase (HCP), encoded by the hcph gene, (also called PTP1C, SHP, SH-PTP1, and PTPN6) is deficient in motheaten (me/me), and the allelic viable motheaten (me(v)/me(v)) mice. Since HCP is expressed in many cell types and protein phosphorylation is a major mechanism of regulating protein function, it is not surprising that the motheaten phenotype is pleiotropic. It is commonly thought that immune system involvement causes this disease. If so, the motheaten disease ought to be alleviated when the recombination activation gene-1 (RAG-1) is disrupted because there will be no V(D)J rearrangement and thus impaired development of B and T cells. We bred homozygous, double-mutant me(v)/me(v) x RAG 1 -/- mice and found that, in fact, inflamed paws, and splenomegaly with elevated myelopoiesis. Thus, except for autoantibodies, the motheaten phenotype does not depend on the presence of B and T cells. This observation cautions the use of motheaten mice as a model of autoimmune disease.
Assuntos
Doenças Autoimunes/genética , Proteínas de Homeodomínio , Linfócitos , Mutação , Proteínas Tirosina Fosfatases/genética , Proteínas/genética , Animais , Linfócitos B , Dermatite/patologia , Modelos Animais de Doenças , Genótipo , Peptídeos e Proteínas de Sinalização Intracelular , Pulmão/patologia , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Baço/patologia , Análise de Sobrevida , Linfócitos TRESUMO
OBJECTIVES: Studying post-infliximab gene expression changes could provide insights into the pathogenesis of ankylosing spondylitis (AS). METHODS: Gene expression changes were screened by microarray on peripheral blood RNA of 16 AS patients at baseline and 2 weeks post-infliximab, and selected results were confirmed by quantitative real-time (qRT)-PCR. Corresponding serum-soluble LIGHT (sLIGHT) was estimated by ELISA and the fold change in sLIGHT was correlated to the fold change in erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and the Bath AS disease activity index. RESULTS: Post-infliximab, 69% of the patients (11/16) achieved an ASAS20 response. Six candidate genes were differentially expressed by microarray; four of which were validated by qRT-PCR. sLIGHT showed the most significant difference. There was good correlation of baseline sLIGHT with CRP (R = 0.60; p = 0.01) and ESR (R = 0.51; p = 0.04). The fold change in sLIGHT correlated with change in both CRP (R = 0.71, p = 0.002) and ESR (R = 0.77, p<0.001). CONCLUSION: LIGHT is significantly downregulated by infliximab. sLIGHT correlated well with changes in inflammatory markers.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Antirreumáticos/uso terapêutico , Proteínas Sanguíneas/metabolismo , Espondilite Anquilosante/tratamento farmacológico , Adulto , Biomarcadores/sangue , Sedimentação Sanguínea , Proteína C-Reativa/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Humanos , Infliximab , Masculino , Pessoa de Meia-Idade , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Índice de Gravidade de Doença , Espondilite Anquilosante/sangue , Membro 14 da Superfamília de Ligantes de Fatores de Necrose Tumoral/sangue , Adulto JovemRESUMO
Motheaten mice have a mutation that causes abnormal splicing of the SHP-1 gene producing transcripts that are out of frame. Thus, motheaten mice cannot produce normal SHP-1 protein. However, we have found that the SHP-1 locus in normal mice is expressed as multiple in-frame splice variant transcripts. We report here that the motheaten SHP-1 gene is likewise expressed in multiple spliced forms, two of which are in frame. One of these two variants, which is also present in normal mice, lacks the exon containing the motheaten mutation and is therefore expected to encode an active phosphatase with only one of the two SH2 domains of SHP-1. We showed that both of these variants produce phosphatases with a higher specific activity than SHP-1, suggesting that motheatein mice are not SHP-1 null. The possibility that motheaten mice produce disregulated phosphatases offered a simple explanation for the puzzling observation that substrates of SHP-1 are hypo-phosphorylated in motheaten mice. We tested this by measuring for SHP-1 protein and activity in motheaten macrophages. However, we did not detect specific activity, and found that one of these variant proteins was unstable. These findings likewise suggest that little or no SHP-1 variant proteins exist in normal cells.
Assuntos
Variação Genética , Proteínas Tirosina Fosfatases/genética , Processamento Alternativo , Animais , Sequência de Bases , Células COS , Primers do DNA/genética , Estabilidade Enzimática , Peptídeos e Proteínas de Sinalização Intracelular , Isoenzimas/genética , Isoenzimas/metabolismo , Macrófagos/enzimologia , Camundongos , Camundongos Mutantes , Mutação , Fases de Leitura Aberta , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/metabolismo , Baço/enzimologia , TransfecçãoRESUMO
A recombinant phage clone containing the 5' end of the gene HRS encoding human histidyl-tRNA synthetase (HRS) has been isolated. Primer extension analyses indicated that there are two types of HRS transcripts. The longer transcripts were initiated from a single transcription start point (tsp) located approximately 455 bp upstream and the shorter transcripts were initiated from multiple tsp located approximately 38 to 82 bp upstream from the HRS ATG start codon. Functionally, we have identified two regions (+1 to -122; -185 to -502), each of which when placed 5' of a promoterless cat construct can initiate transcription in both orientations after transfection into HeLa cells. A pair of imperfect inverted repeats (IIR) was located within the region +1 to -122. Using mobility shift assays, we have identified a nuclear factor that binds specifically to each half of the IIR. However, this pair of IIR (-73 to -110) was not sufficient for bidirectional transcription activity. At least one copy of a 27-bp oligodeoxyribonucleotide (oligo), which spans -94 to -120, was required in order to facilitate bidirectional transcription activity. From mobility shift assays using HeLa cell nuclear extracts and this 27-bp oligo, we have identified two DNA-protein complexes, both of which are presumably required to initiate bidirectional transcription.
Assuntos
Histidina-tRNA Ligase/genética , Sequências Reguladoras de Ácido Nucleico , Transcrição Gênica , Sequência de Aminoácidos , Animais , Sequência de Bases , Cricetinae , DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Humanos , Dados de Sequência Molecular , Regiões Promotoras Genéticas , RNA Mensageiro/metabolismo , TransfecçãoRESUMO
MBD is a rare disorder strongly associated with alcoholism. It is characterized pathologically by demyelination and necrosis of the corpus callosum. MBD presents with severe neurologic deficits and significant sequelae developing in most survivors. We report a patient with total clinical recovery. Serial MR imaging demonstrated typical lesions with restricted diffusion in the acute stage and total resolution without atrophy or cystic change.
Assuntos
Alcoolismo/patologia , Imageamento por Ressonância Magnética , Doença de Marchiafava-Bignami/patologia , Recuperação de Função Fisiológica , Cerebelo/patologia , Corpo Caloso/patologia , Humanos , Cápsula Interna/patologia , Masculino , Pessoa de Meia-Idade , Remissão EspontâneaRESUMO
This report is about a rare case of a lumbar spinal dural arteriovenous fistula (DAVF) with distant brainstem symptoms of vertigo and ataxia. Brain magnetic resonance imaging (MRI) demonstrated a high signal abnormality in the lower pons, medulla, and cervico-medullary junction, presumably related to venous hypertension. Spinal MRI revealed markedly dilated veins along the dorsal aspect of the cord, while spinal angiography confirmed the presence of a lumbar spinal DAVF. Because lumbar DAVF may cause remote brainstem symptoms, spinal DAVF should be considered in the differential diagnosis of high signal MRI abnormalities localized in the brainstem. Properly extending the scanning range is essential in the diagnosis.
Assuntos
Fístula Arteriovenosa/diagnóstico , Imageamento por Ressonância Magnética , Idoso , Angiografia , Fístula Arteriovenosa/complicações , Ataxia/etiologia , Diagnóstico Diferencial , Dura-Máter/irrigação sanguínea , Evolução Fatal , Humanos , Região Lombossacral/irrigação sanguínea , Masculino , Vertigem/etiologiaRESUMO
Mice homozygous for the autosomal recessive motheaten (me) or the allelic viable motheaten (mev) mutations manifest a unique immunological disease associated with severe immunodeficiency and autoimmunity. Over the past few years, our group has used the motheaten mouse as a model system for elucidating the genetic and cellular events that contribute to expression of normal hematopoietic and immune cell function. To this end, we have sought to identify the gene responsible for the motheaten phenotype. In our initial studies, our general approach involved the use of subtractive hybridization to identify genes that were differentially expressed in the mutant versus control mice and which might thus provide clues as to the primary gene defect. Using this approach, we showed that genes encoding stefin A cysteine proteinase inhibitors are markedly overexpressed in bone marrow cells of me and mev mice compared to bone marrow cells of normal congenic animals. However, the motheaten mutation has been mapped to mouse choromosome 6 while the stefin A gene cluster was localized to mouse chromosome 16. Stefin gene therefore does not represent the primary gene defect. Our second strategy aimed at identifying the primary gene defect underlying the motheaten phenotype was prompted by the recent localization of a protein tyrosine phosphatase gene to human chromosome 12p12-p13, a region containing a large segment of homology with the region on mouse chromosome 6 where the motheaten locus has been mapped. We have shown that abnormal Hcph transcripts are expressed in me and mev bone marrow cells and that the generation of these altered transcripts is due to RNA splicing defects caused by single basepair changes in the Hcph genes of the mutant mice. These mutant mice thus provide a valuable model system for elucidating the biological roles of HCP in vivo and defining the mechanism whereby defective function of a hematopoietic cell phosphatase leads to expression of the motheaten phenotype of severe immunodeficiency and systemic autoimmunity.
Assuntos
Doenças Autoimunes/genética , Proteínas Tirosina Fosfatases/genética , Imunodeficiência Combinada Severa/genética , Sequência de Aminoácidos , Animais , Doenças Autoimunes/metabolismo , Sequência de Bases , Medula Óssea/metabolismo , Camundongos , Camundongos Mutantes , Dados de Sequência Molecular , Fenótipo , Mutação Puntual , Imunodeficiência Combinada Severa/metabolismoRESUMO
The Ah receptor, which regulates induction of aryl hydrocarbon hydroxylase (cytochrome P1-450), can be assayed using [3H] 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) as the radioligand. Sucrose density gradient (SDG) centrifugation is the most reliable method for detecting and quantitating Ah receptor; however, previous gradient assays in swinging bucket rotors (SBR) have required overnight centrifugation to achieve adequate separation of specific receptor peaks from nonspecific binding to other cytosolic components. These lengthy runs limit the number of assays which can be performed and could permit excessive dissociation of radioligand from the receptor. Thus, we tested brief (2-h) SDG centrifugation assays in a vertical tube rotor (VTR) as a possible alternative to prolonged SBR runs. Two-hour VTR runs yield gradient profiles very similar to those obtained with 16-h runs in an SBR and the calculated sedimentation coefficient (in low ionic strength buffer) is about 9.6 S for the Ah receptor assayed in either rotor. The concentration of Ah receptor in cytosols from liver, lung, kidney, and testis of C57BL/6J mice is the same by VTR analysis as when measured by SBR analysis. Thus, extensive dissociation of [3H]TCDD from the Ah receptor does not appear to occur in cytosols from a variety of tissues even with run times up to 16 h. Assays performed by rapid VTR centrifugation appear equally valid to those obtained by overnight SBR centrifugation.
Assuntos
Hidrocarboneto de Aril Hidroxilases/genética , Dioxinas/metabolismo , Genes Reguladores , Dibenzodioxinas Policloradas/metabolismo , Animais , Sítios de Ligação , Centrifugação com Gradiente de Concentração/métodos , Citosol/metabolismo , Fígado/metabolismo , Camundongos , Camundongos Endogâmicos C57BLRESUMO
SHP-1 is a protein-tyrosine phosphatase with two Src homology 2 (SH2) domains. These SH2 domains determine which proteins SHP-1 associates with, but they also autoregulate the activity of the catalytic domain. In this report, we find that the murine SHP-1 transcript is processed to yield a series of alternatively spliced in-frame transcripts, the majority of which exclude exons encoding one or the other SH2 domain. We have examined the corresponding protein isoforms in several ways. First, our measurements of V(max) and K(m) under different conditions indicate that the SH2 variants have elevated activity because of lessened autoregulation. Second, to ascertain whether regulation by the SH2 domains reflects intra- or intermolecular effects, we analyzed the state of SHP-1 by high performance liquid chromatography and sucrose density gradient centrifugation. Our results showed that SHP-1 is a monomer and, thus, is regulated in an intramolecular manner. Third, our analyses detected shape differences between SHP-1 and the active splice variant protein deleted of the amino-terminal SH2 domain; i.e. SHP-1 was globular and resistant to proteolytic digestion, while the splice variant protein was "rod-shaped" and more susceptible to proteolytic digestion.
Assuntos
Processamento Alternativo , Variação Genética , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Domínio Catalítico , Linhagem Celular , Cromatografia em Gel , Éxons , Homeostase , Peptídeos e Proteínas de Sinalização Intracelular , Íntrons , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Cinética , Camundongos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/química , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Mapeamento por Restrição , Proteínas Tirosina Fosfatases Contendo o Domínio SH2 , Domínios de Homologia de srcRESUMO
Immunoreactive N-acetylserotonin and melatonin in rat serum were extracted by ethyl acetate at less than or equal to pH 3 and chloroform at greater than or equal to pH 10, respectively, following protein precipitation and were quantified by radioimmunoassays. There were diurnal rhythms of N-acetylserotonin and melatonin in the serum of rats (housed in 121/12d; lights on from 06.00-18.00 h for two weeks). Serum melatonin levels 20.00-04.00 h are higher than those at 08.00-16.00 h. Serum N-acetylserotonin levels were, however, higher at 24.00-08.00 h and lower at 12.00-20.00 h. The concentration of N-acetylserotonin in individual samples ranged from a minimum of 0.45 ng/ml at 12.00 h to a maximum of 2.78 ng/ml at 08.00 h. The diurnal study on N-acetylserotonin levels in the rat serum was repeated and a similar result was obtained. The findings of the presence of N-acetylserotonin in the rat serum and a diurnal rhythm of serum N-acetylserotonin are in keeping with the notions that N-acetylserotonin may be secreted by the pineal gland and that N-acetylserotonin may be another indole with hormonal activities.
Assuntos
Ritmo Circadiano , Melatonina/sangue , Serotonina/análogos & derivados , Animais , Masculino , Radioimunoensaio , Ratos , Serotonina/sangueRESUMO
Stefins or Type 1 cystatins belong to a large, evolutionarily conserved protein superfamily, the members of which inhibit the papain-like cysteine proteinases. We report here on the molecular cloning and chromosomal localization of three newly identified members of the murine stefin gene family. These genes, designated herein as mouse stefins 1, 2, and 3, were isolated on the basis of their relatively increased expression in moth-eaten viable compared to normal congenic mouse bone marrow cells. The open reading frames of the stefin cDNAs encode proteins of approximately 11.5 kDa that show between 50 and 92% identity to sequences of stefins isolated from various other species. Data from Southern analysis suggest that the murine stefin gene family encompasses at least 6 and possibly 10-20 members, all of which appear to be clustered in the genome. Analysis of interspecific backcross mice indicates that the genes encoding the three mouse stefins all map to mouse chromosome 16, a localization that is consistent with the recent assignment of the human stefin A gene to a region of conserved homology between human chromosome 3q and the proximal region of mouse chromosome 16.