RESUMEN
The premotor theory of attention suggests that target processing and generation of a saccade to the target are interdependent. Temporally precise transcranial magnetic stimulation (TMS) was delivered over the human frontal eye fields, the area most frequently associated with the premotor theory in association with eye movements, while subjects performed a visually instructed pro-/antisaccade task. Visual analysis and saccade preparation were clearly separated in time, as indicated by 2 distinct time points of TMS delivery that resulted in elevated saccade latencies. These results show that visual analysis and saccade preparation, although frequently enacted together, are dissociable processes.
Asunto(s)
Lóbulo Frontal/fisiología , Movimientos Sacádicos/fisiología , Estimulación Magnética Transcraneal , Percepción Visual/fisiología , Adulto , Atención/fisiología , Humanos , Masculino , Estimulación Luminosa , Tiempo de Reacción/fisiología , Adulto JovenRESUMEN
P pili are important virulence factors in uropathogenic Escherichia coli. The Cpx two-component signal transduction system controls a stress response and is activated by misfolded proteins in the periplasm. We have discovered new functions for the Cpx pathway, indicating that it may play a critical role in pathogenesis. P pili are assembled via the chaperone/usher pathway. Subunits that go 'OFF-pathway' during pilus biogenesis generate a signal. This signal is derived from the misfolding and aggregation of subunits that failed to come into contact with the chaperone in the periplasm. In response, Cpx not only controls the stress response, but also controls genes necessary for pilus biogenesis, and is involved in regulating the phase variation of pap expression and, potentially, the expression of a panoply of other virulence factors. This study demonstrates how the prototypic chaperone/usher pathway is intricately linked and dependent upon a signal transduction system.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Fimbrias Bacterianas/fisiología , Regulación Bacteriana de la Expresión Génica , Proteínas Periplasmáticas , Transducción de Señal , Factores de Transcripción/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Escherichia coli/genética , Escherichia coli/fisiología , Proteínas Fimbrias , Fimbrias Bacterianas/ultraestructura , Genes Bacterianos , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Proteínas de Choque Térmico/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Operón , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Serina Endopeptidasas/fisiologíaRESUMEN
PapD is an immunoglobulin-like chaperone that mediates the assembly of P pili in uropathogenic strains of Escherichia coli. It binds and caps interactive surfaces on pilus subunits to prevent their premature associations in the periplasm. We elucidated the structural basis of a mechanism whereby PapD also interacts with itself, capping its own subunit binding surface. Crystal structures of dimeric forms of PapD revealed that this self-capping mechanism involves a rearrangement and ordering of the C2-D2 and F1-G1 loops upon dimerization which might ensure that a stable dimer is not formed in solution in spite of a relatively large dimer interface. An analysis of site directed mutations revealed that chaperone dimerization requires the same surface that is otherwise used to bind subunits.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Escherichia coli , Escherichia coli/fisiología , Fimbrias Bacterianas/fisiología , Chaperonas Moleculares/química , Proteínas Periplasmáticas , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Gráficos por Computador , Cristalografía por Rayos X , Dimerización , Fimbrias Bacterianas/genética , Cinética , Sustancias Macromoleculares , Modelos Moleculares , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismoRESUMEN
PapD is the periplasmic chaperone required for the assembly of P pili in pyelonephritic strains of Escherichia coli. It consists of two immunoglobulin-like domains bisected by a subunit binding cleft. PapD is the prototype member of a super family of immunoglobulin-like chaperones that work in concert with their respective ushers to assemble a plethora of adhesive organelles including pilus- and non-pilus-associated adhesins. Three highly conserved residue clusters have been shown to play critical roles in the structure and function of PapD, as determined by site-directed mutagenesis. The in vivo stability of the chaperone depended on the formation of a buried salt bridge within the cleft. Residues along the G1 beta strand were required for efficient binding of subunits consistent with the crystal structure of PapD-peptide complexes. Finally, Thr-53, a residue that is part of a conserved band of residues located on the amino-terminal domain surface opposite the subunit binding cleft, was also found to be critical for pilus assembly, but mutations at Thr-53 did not interfere with chaperone-subunit complex formation.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/fisiología , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas Fimbrias , Chaperonas Moleculares/química , Chaperonas Moleculares/fisiología , Proteínas Periplasmáticas , Adhesinas de Escherichia coli/química , Cromatografía en Agarosa , Cristalografía por Rayos X , Fimbrias Bacterianas/química , Fimbrias Bacterianas/fisiología , Modelos Moleculares , Mutagénesis Sitio-Dirigida , Pruebas de Precipitina , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Factores de TiempoRESUMEN
The molecular basis of how pathogenic bacteria cause disease has been studied by blending a well-developed genetic system with X-ray crystallography, protein chemistry, high resolution electron microscopy, and cell biology. Microbial attachment to host tissues is one of the key events in the early stages of most bacterial infections. Attachment is typically mediated by adhesins that are assembled into hair-like fibers called pili on bacterial surfaces. This article focuses on the structure-function correlates of P pili, which are produced by most pyelonephritic strains of Escherichia coli. P pili are assembled via a chaperone/usher pathway. Similar pathways are responsible for the assembly of over 30 adhesive organelles in various Gram-negative pathogens. P pilus biogenesis has been used as a model system to elucidate common themes in bacterial pathogenesis, namely, the protein folding, secretion, and assembly of virulence factors. The structural basis for pilus biogenesis is discussed as well as the function and consequences of microbial attachment.
Asunto(s)
Proteínas Bacterianas/química , Proteínas de Escherichia coli , Escherichia coli/fisiología , Fimbrias Bacterianas/fisiología , Fimbrias Bacterianas/ultraestructura , Bacterias Gramnegativas/fisiología , Chaperonas Moleculares/química , Chaperonas Moleculares/fisiología , Proteínas Periplasmáticas , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/fisiología , Escherichia coli/genética , Escherichia coli/patogenicidad , Escherichia coli/ultraestructura , Bacterias Gramnegativas/patogenicidad , Bacterias Gramnegativas/ultraestructura , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Conejos , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transducción de Señal , VirulenciaRESUMEN
The initial encounter of a microbial pathogen with the host often involves the recognition of host receptors by different kinds of bacterial adhesive organelles called pili, fimbriae, fibrillae or afimbrial adhesins. The development of over 26 of these architecturally diverse adhesive organelles in various Gram-negative pathogens depends on periplasmic chaperones that are comprised of two immunoglobulin-like domains. All of the chaperones possess a highly conserved sheet in domain 1 and a conserved interdomain hydrogen-bonding network. Chaperone-subunit complex formation depends on the anchoring of the carboxylate group of the subunit into the conserved crevice of the chaperone cleft and the subsequent positioning of the COOH terminus of subunits along the exposed edge of the conserved sheet of the chaperone. We discovered that the chaperones can be divided into two distinct subfamilies based upon conserved structural differences that occur in the conserved sheet. Interestingly, a subdivision of the chaperones based upon whether they assemble rod-like pili or non-pilus organelles that have an atypical morphology defines the same two subgroups. The molecular dissection of the two chaperone subfamilies and the adhesive fibers that they assemble has advanced our understanding of the development of virulence-associated organelles in pathogenic bacteria.
Asunto(s)
Chaperonas Moleculares/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/clasificación , Secuencia Conservada , Cristalografía por Rayos X , Ensayo de Inmunoadsorción Enzimática , Modelos Moleculares , Chaperonas Moleculares/clasificación , Datos de Secuencia Molecular , Conformación ProteicaRESUMEN
In recent years, research on the relationship between brain organization and language processing has benefited tremendously from cross-linguistic comparisons of language disorders among different types of aphasic patients. Results from these cross-linguistic studies have shown that the same aphasic syndromes often look very different from one language to another, suggesting that language-specific knowledge is largely preserved in Broca's and Wernicke's aphasics. In this paper, Chinese aphasic patients were examined with respect to their (in)ability to use classifiers in a noun phrase. The Chinese language, in addition to its lack of verb conjugation and an absence of noun declension, is exceptional in yet another respect: articles, numerals, and other such modifiers cannot directly precede their associated nouns, there has to be an intervening morpheme called a classifier. The appropriate usage of nominal classifiers is considered to be one of the most difficult aspects of Chinese grammar. Our examination of Chinese aphasic patients revealed two essential points. First, Chinese aphasic patients experience difficulty in the production of nominal classifiers, committing a significant number of errors of omission and/or substitution. Second, two different kinds of substitution errors are observed in Broca's and Wernicke's patients, and the detailed analysis of the difference demands a rethinking of the distinction between agrammatism and paragrammatism. The result adds to a growing body of evidence suggesting that grammar is impaired in fluent as well as nonfluent aphasia.