Single molecular force spectroscopy of modular proteins in the nervous system.
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The derailed (drl) gene encodes a receptor tyrosine kinase (RTK) that governs aspects of axon guidance and muscle-epidermal interactions in the Drosophila embryo. To determine the types of neurons that express drl, we have examined a series of drl promoter fusions to axon-targeted reporters. We have identified enhancers that drive reporter expression in four distinct subtypes of embryonic neurons, all of which project axons in the anterior commissure of the developing nervous system. We also identified enhancers driving expression in the drl-expressing muscles and epidermal attachment cells. These enhancers define the classes of neurons projecting in the anterior commissure and can be used to precisely define axon pathfinding errors in drl and other mutants.
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We have designed a computerised vascular registry (CVR) combining storage of complete patient histories in minute detail, including reoperations and long-term follow-up, with clinical applicability. The basic concept of this registry is the storage of data in a structure of cycles (one cycle per treatment episode), modules (clusters of logistically correlated data) and data-chapters (clusters of clinically correlated data). The registry was designed to minimally interfere with routine clinical practice, for instance by collecting the data step-by-step at the wards and out-patient clinics, quite similar to traditional record keeping. The CVR enables production of inventories of all stored data. More importantly, and in addition to other registries, the structure of our registry adequately enables analyses of data of patients with multiple interventions and patients with long-term follow-up. A microcomputer was used for the input of data, which were stored in a structure enabling effortless transportation of the data to a mainframe computer. Standard software programs were used. Simple inventories and analyses were performed on a microcomputer, and a mainframe computer was used for more complex analyses. The performance and applicability of the newly designed CVR was thoroughly tested in comprehensive retrospective studies. On the basis of these experiences several adjustments were carried out after which the CVR was introduced into clinical practice.
Object perception may involve seeing, recognition, preparation of actions, and emotional responses--functions that human brain imaging and neuropsychology suggest are localized separately. Perhaps because of this specialization, object perception is remarkably rapid and efficient. Representations of componential structure and interpolation from view-dependent images both play a part in object recognition. Unattended objects may be implicitly registered, but recent experiments suggest that attention is required to bind features, to represent three-dimensional structure, and to mediate awareness.
Recent phylogenetic and structural analyses of multidomain phosphoryl transfer proteins of bacteria have revealed that interdomain (but not intradomain) splicing and fusion, as well as domain duplication and deletion, have occurred frequently during evolution. These events have been found to be exceedingly rare in certain other protein families. Domain-shuffling events are illustrated by examples from the superfamilies of phosphoenolpyruvate-dependent sugar phosphotransferase systems, their transcriptional regulatory protein targets of phosphorylation, sensor autokinase/response regulator signal transduction systems, and permeases of the ATP-binding-cassette type.
A range of di- and tri-substituted triazenes have been synthesized from a polymer-supported diazonium salt and various primary and secondary amines. The triazenes obtained were treated with transition metal salts to form polymer-supported metal complexes in firstly a general screen and then in a specific manner.
A range of polymer-supported triazenes and their metal-bound analogues were screened for use in catalysis. Fe, Cu and Zr complexes were, together with the polymer-supported triazene ligand alone, screened in the addition of Et(2)Zn to benzaldehyde. A supported Pd triazene complex was screened for activity in Suzuki and Sonogashira reactions and a supported Ru triazene complex screened for transfer hydrogenation.
Recent research on multisensory perception suggests a number of general principles for crossmodal integration and that the standard model in the field--feedforward convergence of information--must be modified to include a role for feedback projections from multimodal to unimodal brain areas.
The nephrons of the vertebrate kidney originate from mesenchymal tissue that is recruited and incorporated into a branching epithelium. Key features of this unusual manner of specifying functional units within a tubular organ have now been found to be similarly employed during development of the insect renal system.
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Tumour necrosis factor (TNF) receptor family members regulate processes that range from cell proliferation to programmed cell death. The extracellular, ligand-binding domains of these proteins consist of small, cysteine-rich subdomains, first observed in the three-dimensional structures of the type I TNF receptor. A structure-based alignment of TNFR family members indicates that the extracellular domains are constructed primarily of two small polypeptide modules. These modules play distinctive structural roles in the architecture of the domains. Analogues of at least one of these modules can be found in the domains of other receptors and extracellular proteins. Variations in their sequence and order of assembly are expected to account for differences in shape, flexibility and ligand specificity.
Cloning of polyether polyketide synthase (PKS) genes for salinomycin biosynthesis was attempted from Streptomyces albus. Seven beta-ketoacyl synthase (KS) core regions were obtained by PCR amplification using primers designed based on the conserved KS domains of type I PKSs. Using the KS fragment as a probe, screening of an S. albus genomic DNA library was carried out by colony hybridization. From the positive cosmid clone isolated, a 4.5-kbBamHI fragment was subcloned and sequenced. It showed high homology with bacterial type I PKSs and was deduced to code for KS, malonyl transferase, and ketoreductase motifs. By gene disruption with this 4.5-kb BamHI fragment, the cloned gene was shown to be a part of the salinomycin biosynthetic gene cluster of S. albus.
Group II introns are large catalytic RNAs with a remarkable repertoire of reactions. Here we present construct designs and protocols that were used to develop a set of kinetic frameworks for studying the structure and reaction mechanisms of group II introns and ribozymes derived from them. In addition, we discuss application of these systems to structure/function analysis of the ai5gamma group II intron.
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BACKGROUND: Many active peptides are synthesized by nonribosomal peptide synthetases (NRPSs), large multimodular enzymes. Each module incorporates one amino acid, and is composed of two domains: an activation domain that activates the substrate amino acid and a condensation domain for peptide-bond formation. Activation domains sometimes contain additional activities (e.g. N-methylation or epimerization). Novel peptides can be generated by swapping domains. Exchange of domains containing N-methylation activity has not been reported, however. RESULTS: The actinomycin NRPS was used to investigate domain swapping. The first two amino acids of actinomycin are threonine and valine. We replaced the valine activation domain of module 2 with an N-methyl valine (MeVal) activation domain. The recombinant NRPS (AcmTmVe) catalyzes the formation of threonyl-valine. In the presence of S-adenosyl-methionine, valine was converted to MeVal but subsequent dipeptide formation was blocked. When acyl-threonine (the natural intermediate) was present at module 1, formation of acyl-threonine-MeVal occurred. The epimerization domain of AcmTmVe was impaired. CONCLUSIONS: A simple activation domain can be replaced by one with N-methylation activity. The same condensation domain can catalyze peptide-bond formation between N-methyl and nonmethylated amino acids. Modification of the upstream amino acid (i.e. acylation of threonine), however, was required for condensation with MeVal. Steric hindrance reduces chemical reactivity of N-methyl amino acids - perfect substrate positioning may only be achieved with acylated threonine. Loss of the epimerase activity of AcmTmVe suggests N-methyltransferase and epimerase domains, not found together naturally, are incompatible.
FHA domains are conserved sequences of 65-100 amino acid residues found principally within eukaryotic nuclear proteins, but which also exist in certain prokaryotes. The FHA domain is thought to mediate protein-protein interactions, but its mode of action has yet to be elucidated. Here, we show that the two highly divergent FHA domains of Saccharomyces cerevisiae Rad53p, a protein kinase involved in cell cycle checkpoint control, possess phosphopeptide-binding specificity. We also demonstrate that other FHA domains bind peptides in a phospho-dependent manner. These findings indicate that the FHA domain is a phospho-specific protein-protein interaction motif and have important implications for mechanisms of intracellular signaling in both eukaryotes and prokaryotes.