RESUMO
OBJECTIVE: To test the hypothesis that the association between levels of mould in floor dust and prevalence of potentially building-related symptoms may depend on the type of ventilation. METHODS: This stratified cross-sectional study is part of the Danish moulds in buildings program (DAMIB), including 503 adolescent schoolboys aged 13-17 years at 15 schools. Besides assessing symptom prevalences in questionnaires, we measured numerous potential risk factors in the school buildings. RESULTS: Stratifying on type of ventilation (natural, exhaust only, or full mechanical ventilation system), the negative effect of high levels of mould in floor dust was more pronounced in rooms with mechanical ventilation system. With a variable combining high level of moulds in floor dust with type of ventilation in the classroom, a significantly increased risk was found for all symptoms in the mechanically ventilated classrooms with high mould concentrations. In multiple logistic regression models, significant odds ratios (OR) ranged from 3.9 [95% confidence interval (95% CI) 1.5-10.1] (nasal congestion) to 17.0 (95% CI 2.1-138) (dizziness). CONCLUSIONS: The combined effect of moulds in dust and ventilation form might be a result of the higher air flow keeping the dust in the breathing zone for a longer time, thereby increasing the exposure for the occupants of the classrooms. It is important in future indoor air research also to focus on the combination effects of risk factors, including the type of ventilation.
Assuntos
Microbiologia do Ar , Poluição do Ar em Ambientes Fechados/efeitos adversos , Poeira , Pisos e Cobertura de Pisos , Fungos , Adolescente , Estudos Transversais , Dinamarca , Humanos , Masculino , Instituições Acadêmicas , Estudantes , VentilaçãoRESUMO
Given the complexity of the mammalian proteome, high-resolution separation technologies are required to achieve comprehensive proteome coverage and to enhance the detection of low-abundance proteins. Among several technologies, Multidimensional Protein Identification Technology (MudPIT) enables the on-line separation of highly complex peptide mixtures directly coupled with mass spectrometry-based identification. Here, we present a variation of the traditional MudPIT protocol, combining highly sensitive chromatography using a nanoflow liquid chromatography system (nano-LC) with a two-dimensional precolumn in a vented column setup. When compared to the traditional MudPIT approach, this nanoflow variation demonstrated better first-phase separation leading to more proteins being characterized while using rather simple instrumentation and a protocol that requires less time and very little technical expertise to perform.
Assuntos
Cromatografia Líquida/métodos , Miócitos Cardíacos/metabolismo , Proteoma/metabolismo , Espectrometria de Massas em Tandem/métodos , Animais , Células Cultivadas , Cromatografia Líquida/instrumentação , Camundongos , NanotecnologiaRESUMO
In this study we show by Northern blot hybridization that the novel human (h) connexin (Cx) genes hCx25, hCx30.2, hCx31.9, hCx40.1, hCx59, and hCx62 are transcribed in different adult tissues. The hCx25 RNA is slightly expressed in placenta, and hCx59 and hCx62 RNA are both transcribed in skeletal muscle, although the latter is also slightly expressed in heart. Expression profiles of three orthologous human (h) and mouse (m) connexin gene pairs, i.e., hCx30.2 versus mCx29, hCx40.1 versus mCx39, and hCx62 versus mCx57, differ strongly, in contrast to other orthologous connexins with higher sequence identities. Thus, several of the new human connexin genes appear to have evolved to different expression patterns and presumably to different functions compared to their orthologues in the mouse genome. (121)
Assuntos
Conexinas/biossíntese , Animais , Sequência de Bases , Northern Blotting , Clonagem Molecular , Conexinas/química , Éxons , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Músculo Esquelético/metabolismo , Miocárdio/metabolismo , Placenta/metabolismo , Reação em Cadeia da Polimerase , RNA/química , RNA Mensageiro/metabolismo , Homologia de Sequência do Ácido Nucleico , Distribuição Tecidual , Transcrição GênicaRESUMO
Connexin alpha1Cx43 has previously been shown to bind to the PDZ domain-containing protein ZO-1. The similarity of the carboxyl termini of this connexin and the lens fiber connexins alpha3Cx46 and alpha8Cx50 suggested that these connexins may also interact with ZO-1. ZO-1 was shown to be highly expressed in mouse lenses. Colocalization of ZO-1 with alpha3Cx46 and alpha8Cx50 connexins in fiber cells was demonstrated by immunofluorescence and by fracture-labeling electron microscopy but showed regional variations throughout the lens. ZO-1 was found to coimmunoprecipitate with alpha3Cx46 and alpha8Cx50, and pull-down experiments showed that the second PDZ domain of ZO-1 was involved in this interaction. Transiently expressed alpha3Cx46 and alpha8Cx50 connexins lacking the COOH-terminal residues did not bind to the second PDZ domain but still formed structures resembling gap junctions by immunofluorescence. These results indicate that ZO-1 interacts with lens fiber connexins alpha3Cx46 and alpha8Cx50 in a manner similar to that previously described for alpha1Cx43. The spatial variation in the interaction of ZO-1 with lens gap junctions is intriguing and is suggestive of multiple dynamic roles for this association.
Assuntos
Conexinas/metabolismo , Cristalino/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Animais , Membrana Celular/metabolismo , Proteínas de Membrana/genética , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Testes de Precipitina , Estrutura Terciária de Proteína , Proteína da Zônula de Oclusão-1RESUMO
A novel gap junction forming protein, mouse connexin-30.2 (Cx30.2) contains 278 amino acid residues, and is 79% identical to human Cx31.9 (GJA11). Northern analysis showed that Cx30.2 is ubiquitously expressed, most prominently in testis. Polyclonal antibodies against Cx30.2 detected a 30 kDa protein in cells overexpressing Cx30.2, and in mouse testis. Immunofluorescence showed that Cx30.2 was expressed in vascular smooth muscle, but also in cell types where Cx31.9 was not detected. These data demonstrate that Cx30.2 is a bona fide gene, and suggest that it is the orthologue of Cx31.9, but that it may have additional roles compared with Cx31.9.
Assuntos
Conexinas/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Northern Blotting , Linhagem Celular , Clonagem Molecular , Conexina 30 , Conexinas/química , DNA , Humanos , Camundongos , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
A novel human connexin gene (GJA11) was cloned from a genomic library. The open reading frame encoded a hypothetical protein of 294 amino acid residues with a predicted molecular mass of 31,933, hence referred to as connexin-31.9 (Cx31.9) or alpha 11 connexin. A clone in GenBank containing the Cx31.9 gene localized to chromosome 17q21.2. Northern analysis of Cx31.9 showed a major 4.4-kilobase transcript, which was expressed at varying levels in all tissues analyzed. Two monoclonal antibodies generated against different domains of Cx31.9 recognized a 30-33-kDa protein from cells overexpressing Cx31.9. Immunofluorescence of overexpressing cells indicated the presence of Cx31.9 between adjacent cells, consistent with its localization to gap junctions. Double voltage clamp analyses of Cx31.9-overexpressing cells, and of paired Xenopus oocytes injected with Cx31.9 cRNA, demonstrated junctional currents indicative of gap junction channel formation. In contrast to previously characterized connexins, Cx31.9 showed no voltage-dependent gating within a physiologically relevant range. Cx31.9 was detected in human tissues by immunoblot analysis, and immunofluorescence localized Cx31.9 expression to vascular smooth muscle cells. Furthermore, it was demonstrated that Cx31.9 interacted with ZO-1. Thus, Cx31.9 represents a novel connexin gene that in vivo generates a protein with unique voltage gating properties.
Assuntos
Conexinas/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/metabolismo , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Conexinas/classificação , Conexinas/genética , Junções Comunicantes/química , Junções Comunicantes/metabolismo , Humanos , Proteínas de Membrana/metabolismo , Dados de Sequência Molecular , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/metabolismo , Oócitos/fisiologia , Fases de Leitura Aberta , Técnicas de Patch-Clamp , Fosfoproteínas/metabolismo , Filogenia , Ligação Proteica , Isoformas de Proteínas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Alinhamento de Sequência , Distribuição Tecidual , Proteínas de Xenopus , Xenopus laevis , Proteína da Zônula de Oclusão-1RESUMO
The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.