RESUMO
The assembly of clathrin-coated vesicles is important for numerous cellular processes, including nutrient uptake and membrane organization. Important contributors to clathrin assembly are four tetrameric assembly proteins, also called adaptor proteins (APs), each of which contains a ß subunit. We identified a single ß subunit, named ß1/2, that contributes to both the AP1 and AP2 complexes of Dictyostelium. Disruption of the gene encoding ß1/2 resulted in severe defects in growth, cytokinesis and development. Additionally, cells lacking ß1/2 displayed profound osmoregulatory defects including the absence of contractile vacuoles and mislocalization of contractile vacuole markers. The phenotypes of ß1/2 null cells were most similar to previously described phenotypes of clathrin and AP1 mutants, supporting a particularly important contribution of AP1 to clathrin pathways in Dictyostelium cells. The absence of ß1/2 in cells led to significant reductions in the protein amounts of the medium-sized subunits of the AP1 and AP2 complexes, establishing a role for the ß subunit in the stability of the medium subunits. Dictyostelium ß1/2 could resemble a common ancestor of the more specialized ß1 and ß2 subunits of the vertebrate AP complexes. Our results support the essential contribution of a single ß subunit to the stability and function of AP1 and AP2 in a simple eukaryote.
Assuntos
Complexo 1 de Proteínas Adaptadoras/metabolismo , Complexo 2 de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/metabolismo , Clatrina/metabolismo , Dictyostelium/fisiologia , Complexo 1 de Proteínas Adaptadoras/genética , Complexo 2 de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/genética , Subunidades alfa do Complexo de Proteínas Adaptadoras/metabolismo , Subunidades beta do Complexo de Proteínas Adaptadoras/genética , Subunidades mu do Complexo de Proteínas Adaptadoras/genética , Subunidades mu do Complexo de Proteínas Adaptadoras/metabolismo , Subunidades sigma do Complexo de Proteínas Adaptadoras/genética , Sequência de Aminoácidos/fisiologia , Agregação Celular/fisiologia , Membrana Celular/metabolismo , Proliferação de Células , Quimiotaxia/fisiologia , Clatrina/genética , Cadeias Pesadas de Clatrina/genética , Cadeias Pesadas de Clatrina/metabolismo , Clonagem Molecular , Citocinese/fisiologia , Dictyostelium/citologia , Carpóforos/citologia , Carpóforos/fisiologia , Técnicas de Inativação de Genes , Complexo de Golgi/metabolismo , Anotação de Sequência Molecular , Dados de Sequência Molecular , Ligação Proteica/fisiologia , Domínios e Motivos de Interação entre Proteínas/fisiologia , Vacúolos/fisiologia , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
The PLEX-ID system is a novel technology that couples PCR amplification and electrospray ionization-mass spectrometry to identify pathogens directly in clinical specimens. The analytical performance of the PLEX-ID Broad Fungal assay was compared with that of traditional culture identification by using 91 characterized fungal culture isolates (64 manufacturer-claimed and 27 nonclaimed organisms) and directly by using 395 respiratory specimens. Discordant results were resolved by D2 large-subunit ribosomal DNA fungal sequencing. Environmental studies were performed to monitor for potential contamination. The PLEX-ID Broad Fungal assay correctly identified 95.6% (87/91) and 81.3% (74/91) of the culture isolates to the genus and species levels, respectively. Of the manufacturer-claimed organisms, 100% (64/64) and 92.2% (59/64) were correctly identified to the genus and species levels, respectively. Direct analysis of respiratory specimens resulted in 67.6% (267/395) and 66.6% (263/395) agreement with culture results to the genus and species levels, respectively, with 16.2% (64/395) of the results discordant with culture and 16.2% (64/395) not detected by the system. The majority (>95%) of the isolates not detected directly by the PLEX-ID system ultimately grew in low quantities in culture (≤ 20 colonies). In 20.3% (35/172) of the respiratory specimens where no growth was observed in culture, the PLEX-ID system identified a fungus, suggesting a potential increase in sensitivity over culture in some instances. The PLEX-ID system provides a rapid method for the detection of a broad array of fungi directly in respiratory specimens and has the potential of impacting turnaround times and patient care by reducing the need to wait for the growth of an organism in culture.
Assuntos
Fungos/química , Fungos/isolamento & purificação , Técnicas Microbiológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Micologia/métodos , Reação em Cadeia da Polimerase/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , Fungos/classificação , Humanos , Micoses/diagnóstico , Infecções Respiratórias/microbiologia , Sensibilidade e EspecificidadeRESUMO
This study examined the relationship between reading women's beauty and fashion magazines and the use of pathogenic dieting methods (laxatives, appetite suppressants/diet pills, skipping two meals a day, intentional vomiting, and restricting calories to 1,200 or less each day) among 502 high school females. Weak to moderate positive associations were found between reading frequency and each of these unhealthful practices except the use of laxatives. When controlling for anxiety about weight and frequency of regular exercise, however, the original bivariate relations between reading frequency and skipping two meals a day, and reading and intentional vomiting, disappeared. Replication and weak specification effects were found when examining the relationships between reading and taking appetite suppressant/weight control pills, and reading and restricting calories, under the control conditions. These findings suggest that two of the most common adolescent dieting methods--restricting calories and taking diet pills--appear to be influenced by the reading of women's beauty and fashion magazines.