The Tim9p-Tim10p complex binds to the transmembrane domains of the ADP/ATP carrier

The soluble Tim9p-Tim10p (Tim, translocase of inner membrane) complex of the mitochondrial intermembrane space mediates the import of the carrier proteins and is a component of the TIM22 import system. The mechanism by which the Tim9p-Tim10p complex assembles and binds the carriers is not well understood, but previous studies have proposed that the conserved cysteine residues in the 'twin CX3C' motif coordinate zinc and potentially generate a zinc-finger-like structure that binds to the matrix loops of the carrier proteins. Here we have purified the native and recombinant Tim9p-Tim10p complex, and show that both complexes resemble each other and consist of three Tim9p and three Tim10p. Results from inductively coupled plasma--mass spectrometry studies failed to detect zinc in the Tim9p-Tim10p complex. Instead, the cysteine residues seemingly formed disulfide linkages. The Tim9p-Tim10p complex bound specifically to the transmembrane domains of the ADP/ATP carrier, but had no affinity for Tim23p, an inner membrane protein that is inserted via the TIM22 complex. The chaperone-like Tim9p-Tim10p complex thus may prevent aggregation of the unfolded carrier proteins in the aqueous intermembrane space.     

N-terminal tyrosine residues within the potassium channel Kir3 modulate GTPase activity of Galphai

trkB activation results in tyrosine phosphorylation of N-terminal Kir3 residues, decreasing channel activation. To determine the mechanism of this effect, we reconstituted Kir3, trkB, and the mu opioid receptor in Xenopus oocytes. Activation of trkB by BDNF (brain-derived neurotrophic factor) accelerated Kir3 deactivation following termination of mu opioid receptor signaling. Similarly, overexpression of RGS4, a GTPase-activating protein (GAP), accelerated Kir3 deactivation. Blocking GTPase activity with GTPgammaS also prevented Kir3 deactivation, and the GTPgammaS effect was not reversed by BDNF treatment. These results suggest that BDNF treatment did not reduce Kir3 affinity for Gbetagamma but rather acted to accelerate GTPase activity, like RGS4. Tyrosine phosphatase inhibition by peroxyvanadate pretreatment reversibly mimicked the BDNF/trkB effect, indicating that tyrosine phosphorylation of Kir3 may have caused the GTPase acceleration. Tyrosine to phenylalanine substitution in the N-terminal domain of Kir3.4 blocked the BDNF effect, supporting the hypothesis that phosphorylation of these tyrosines was responsible. Like other GAPs, Kir3.4 contains a tyrosine-arginine-glutamine motif that is thought to function by interacting with G protein catalytic domains to facilitate GTP hydrolysis. These data suggest that the N-terminal tyrosine hydroxyls in Kir3 normally mask the GAP activity and that modification by phosphorylation or phenylalanine substitution reveals the GAP domain. Thus, BDNF activation of trkB could inhibit Kir3 by facilitating channel deactivation.     

Peptides containing cyclin/Cdk-nuclear localization signal motifs derived from viral initiator proteins bind to DNA when unphosphorylated

A single phosphorylation event at T-antigen residue Thr124 regulates initiation of simian virus 40 DNA replication. To explore this regulatory process, a series of peptides were synthesized, centered on Thr124. These peptides contain a nuclear localization signal (NLS) and a recognition site for cyclin/Cdk kinases. When unphosphorylated, the "CDK/NLS" peptides inhibit T-antigen assembly and bind non-sequence specifically to DNA. However, these activities are greatly reduced upon phosphorylation of Thr124. Similar results were obtained by using peptides derived from the CDK/NLS region of bovine papillomavirus E1. Related studies indicate that residues in the NLS bind to DNA, whereas those in the CDK motif regulate binding. These findings are discussed in terms of the control of T-antigen double hexamer assembly and initiation of viral replication.     

Action spectrum for cryptochrome-dependent hypocotyl growth inhibition in Arabidopsis

Cryptochrome blue-light photoreceptors are found in both plants and animals and have been implicated in numerous developmental and circadian signaling pathways. Nevertheless, no action spectrum for a physiological response shown to be entirely under the control of cryptochrome has been reported. In this work, an action spectrum was determined in vivo for a cryptochrome-mediated high-irradiance response, the blue-light-dependent inhibition of hypocotyl elongation in Arabidopsis. Comparison of growth of wild-type, cry1cry2 cryptochrome-deficient double mutants, and cryptochrome-overexpressing seedlings demonstrated that responsivity to monochromatic light sources within the range of 390 to 530 nm results from the activity of cryptochrome with no other photoreceptor having a significant primary role at the fluence range tested. In both green- and norflurazon-treated (chlorophyll-deficient) seedlings, cryptochrome activity is fairly uniform throughout its range of maximal response (390-480 nm), with no sharply defined peak at 450 nm; however, activity at longer wavelengths was disproportionately enhanced in CRY1-overexpressing seedlings as compared with wild type. The action spectrum does not correlate well with the absorption spectra either of purified recombinant cryptochrome photoreceptor or to that of a second class of blue-light photoreceptor, phototropin (PHOT1 and PHOT2). Photoreceptor concentration as determined by western-blot analysis showed a greater stability of CRY2 protein under the monochromatic light conditions used in this study as compared with broad band blue light, suggesting a complex mechanism of photoreceptor activation. The possible role of additional photoreceptors (in particular phytochrome A) in cryptochrome responses is discussed.     

Differential regulation of the chick dorsal thoracic dermal progenitors from the medial dermomyotome

The chick dorsal feather-forming dermis originates from the dorsomedial somite and its formation depends primarily on Wnt1 from the dorsal neural tube. We investigate further the origin and specification of dermal progenitors from the medial dermomyotome. This comprises two distinct domains: the dorsomedial lip and a more central region (or intervening zone) that derives from it. We confirm that Wnt1 induces Wnt11 expression in the dorsomedial lip as previously shown, and show using DiI injections that some of these cells, which continue to express Wnt11 migrate under the ectoderm, towards the midline, to form most of the dorsal dermis. Transplantation of left somites to the right side to reverse the mediolateral axis confirms this finding and moreover suggests the presence of an attractive or permissive environment produced by the midline tissues or/and a repellent or inadequate environment by the lateral tissues. By contrast, the dorsolateral dermal cells just delaminate from the surface of the intervening space, which expresses En1. Excision of the axial organs or the ectoderm, and grafting of Wnt1-secreting cells, shows that, although the two populations of dermal progenitors both requires Wnt1 for their survival, the signalling required for their specification differs. Indeed Wnt11 expression relies on dorsal neural tube-derived Wnt1, while En1 expression depends on the presence of the ectoderm. The dorsal feather-forming dermal progenitors thus appear to be differentially regulated by dorsal signals from the neural tube and the ectoderm, and derive directly and indirectly from the dorsomedial lip. As these two dermomyotomal populations are well known to also give rise to epaxial muscles, an isolated domain of the dermomyotome that contains only dermal precursors does not exist and none of the dermomyotomal domains can be considered uniquely as a dermatome.     

Structural basis of tropism of Escherichia coli to the bladder during urinary tract infection

The first step in the colonization of the human urinary tract by pathogenic Escherichia coli is the mannose-sensitive binding of FimH, the adhesin present at the tip of type 1 pili, to the bladder epithelium. We elucidated crystallographically the interactions of FimH with D-mannose. The unique site binding pocket occupied by D-mannose was probed using site-directed mutagenesis. All but one of the mutants examined had greatly diminished mannose-binding activity and had also lost the ability to bind human bladder cells. The binding activity of the mono-saccharide D-mannose was delineated from this of mannotriose (Man(alpha 1-3)[Man(alpha 1-6)]Man) by generating mutants that abolished D-mannose binding but retained mannotriose binding activity. Our structure/function analysis demonstrated that the binding of the monosaccharide alpha-D-mannose is the primary bladder cell receptor for uropathogenic E. coli and that this event requires a highly conserved FimH binding pocket. The residues in the FimH mannose-binding pocket were sequenced and found to be invariant in over 200 uropathogenic strains of E. coli. Only enterohaemorrhagic E. coli (EHEC) possess a sequence variation within the mannose-binding pocket of FimH, suggesting a naturally occurring mechanism of attenuation in EHEC bacteria that would prevent them from being targeted to the urinary tract.     

In planta protein-protein interactions assessed using a nanovirus-based replication and expression system

The multipartite genome of the nanovirus Faba bean necrotic yellows virus, which consists of one gene on each DNA component, was exploited to construct a series of virus-based episomal vectors designed for transient replication and gene expression in plants. This nanovirus based expression system yields high levels of protein which allows isolation of recombinant protein and protein complexes from plant tissues. As examples, we demonstrated in planta interaction between the nanovirus F-box protein Clink and SKP1, a constituent of the ubiquitin-dependent protein turnover pathway. Thus, replicative nanovirus vectors provide a simple and efficient means for in planta characterization of protein-protein interaction.     

A knowledge-driven agent-centred framework for data mining in EMG

In this paper, we present a multi-agent framework for data mining in electromyography. This application, based on a web interface, provides a set of functionalities allowing to manipulate 1000 medical cases and more than 25,000 neurological tests stored in a medical database. The aim is to extract medical information using data mining algorithms and to supply a knowledge base with pertinent information. The multi-agent platform gives the possibility to distribute the data management process between several autonomous entities. This framework provides a parallel and flexible data manipulation.     

Defining a pathway of communication from the C-terminal peptide binding domain to the N-terminal ATPase domain in a AAA protein

AAA proteins remodel other proteins to affect a multitude of biological processes. Their power to remodel substrates must lie in their capacity to couple substrate binding to conformational changes via cycles of nucleotide binding and hydrolysis, but these relationships have not yet been deciphered for any member. We report that when one AAA protein, Hsp104, engages polypeptide at the C-terminal peptide-binding region, the ATPase cycle of the C-terminal nucleotide-binding domain (NBD2) drives a conformational change in the middle region. This, in turn, drives ATP hydrolysis in the N-terminal ATPase domain (NBD1). This interdomain communication pathway can be blocked by mutation in the middle region or bypassed by antibodies that bind there, demonstrating the crucial role this region plays in transducing signals from one end of the molecule to the other.     

Stu1p is physically associated with beta-tubulin and is required for structural integrity of the mitotic spindle

Formation of the bipolar mitotic spindle relies on a balance of forces acting on the spindle poles. The primary outward force is generated by the kinesin-related proteins of the BimC family that cross-link antiparallel interpolar microtubules and slide them past each other. Here, we provide evidence that Stu1p is also required for the production of this outward force in the yeast Saccharomyces cerevisiae. In the temperature-sensitive stu1-5 mutant, spindle pole separation is inhibited, and preanaphase spindles collapse, with their previously separated poles being drawn together. The temperature sensitivity of stu1-5 can be suppressed by doubling the dosage of Cin8p, a yeast BimC kinesin-related protein. Stu1p was observed to be a component of the mitotic spindle localizing to the midregion of anaphase spindles. It also binds to microtubules in vitro, and we have examined the nature of this interaction. We show that Stu1p interacts specifically with beta-tubulin and identify the domains required for this interaction on both Stu1p and beta-tubulin. Taken together, these findings suggest that Stu1p binds to interpolar microtubules of the mitotic spindle and plays an essential role in their ability to provide an outward force on the spindle poles.