Matching SOX: partner proteins and co-factors of the SOX family of transcriptional regulators

SOX transcription factors perform a remarkable variety of important roles in vertebrate development, either activating or repressing specific target genes through interaction with different partner proteins. Surprisingly, these interactions are often mediated by the conserved, DNA-binding HMG domain, raising questions as to how each factor's specificity is generated. We propose a model whereby non-HMG domains may influence partner protein selection and/or binding stability.

Prediction of Golgi Type II membrane proteins based on their transmembrane domains

Motivation: A major issue in cell biology today is how distinct intracellular regions of the cell, like the Golgi Apparatus, maintain their unique composition of proteins and lipids. The cell differentially separates Golgi resident proteins from proteins that move through the organelle to other subcellular destinations. We set out to determine if we could distinguish these two types of transmembrane proteins using computational approaches. Results: A new method has been developed to predict Golgi membrane proteins based on their transmembrane domains. To establish the prediction procedure, we took the hydrophobicity values and frequencies of different residues within the transmembrane domains into consideration. A simple linear discriminant function was developed with a small number of parameters derived from a dataset of Type II transmembrane proteins of known localization. This can discriminate between proteins destined for Golgi apparatus or other locations (post-Golgi) with a success rate of 89.3% or 85.2%, respectively on our redundancy-reduced data sets.

Secondary structure of the third extracellular loop responsible for ligand selectivity of a mammalian gonadotropin-releasing hormone receptor

The extracellular loop 3 (ECL3) of the mammalian gonadotropin-releasing hormone receptor (GnRH-R) contains an acidic amino acid (Glu(301) in the mouse GnRH-R,) that confers agonist selectivity for Are in mammalian GnRH. It is proposed that a specific conformation of ECL3 is necessary to orientate the carboxyl side chain of the acidic residue for interaction with Arg(8) of GnRH, which is supported by decreased affinity for Arg(8) GnRH but not Gln(8) GnRH when an adjacent Pro is mutated to Ala. To probe the structural contribution of the loop domain to the proposed presentation of the carboxyl side chain, we synthesized a model peptide (CGPEMLNRVSEPGC) representing residues 293-302 of mouse ECL3, where Cys and Gly residues are added symmetrically at the N and C termini, respectively, allowing the introduction of a disulfide bridge to simulate the distances at which the ECL3 is tethered to the transmembrane domains 6 and 7 of the receptor. The ability of the ECL3 peptide to bind GnRH with low affinity was demonstrated by its inhibition of GnRH stimulation of inositol phosphate production in cells expressing the GnRH-R. The CD bands of the ECL3 peptides exhibited a superposition of predominantly unordered structure and partial contributions from beta-sheet structure. Likewise, the analysis of the amide I and amide III bands from micro-Raman and FT Raman experiments revealed mainly unordered conformations of the cyclic and of the linear peptide. NMR data demonstrated the presence of a beta-hairpin among an ensemble of largely disordered structures in the cyclic peptide. The location of the turn linking the two strands of the hairpin was assigned to the three central residues L-296, N-297, and R-298. A small population of structured species among an ensemble of predominantly random coil conformation suggests that the unliganded receptor represents a variety of structural conformers, some of which have the potential to make contacts with the ligand. We propose a mechanism of receptor activation whereby binding of the agonist to the inactive receptor state induces and stabilizes a particular structural state of the loop domain, leading to further conformational rearrangements across the transmembrane domain and signal propagating interaction with G proteins. Interaction of the Glu(301) of the receptor with Arg(8) of GnRH induces a folded configuration of the ligand. Our proposal thus suggests that conformational changes of both ligand and receptor result from this interaction.

Reverse transcriptase activity and untranslated region sharing of a new RTE-like, non-long terminal repeat retrotransposon from the human blood fluke, Schistosoma japonicum

A new RTE-like, non-long terminal repeat retrotransposon, termed SjR2, from the human blood fluke, Schistosoma japonicum, is described. SjR2 is similar to3.9 kb in length and is constituted of a single open reading frame encoding a polyprotein with apurinic/apyrimidinic endonuclease and reverse transcriptase domains. The open reading frame is bounded by 5'- and 3'-terininal untranslated regions and, at its 3-terminus, SjR2 bears a short (TGAC)(3) repeat. Phylogenetic analyses based on conserved domains of reverse transcriptase or endonuclease revealed that SjR2 belonged to the RTE clade of non-long terminal repeat retrotransposons. Further, SjR2 was homologous, but probably not orthologous, to SR2 front the African blood fluke, Schistosoma mansoni; this RTE-like family of non-long terminal repeat retrotransposons appears to have arisen before the divergence of the extant schistosome species. Hybridisation analyses indicated that similar to 10,000 copies of SjR2 were dispersed throughout the S. japonicum chromosomes, accounting for up to 14% of the nuclear genome. Messenger RNAs encoding the reverse transcriptase and endonuclease domains of SjR2 were detected in several developmental stages of the schistosome, indicating that the retrotransposon was actively replicating within the genome of the parasite. Exploration of the coding and non-coding regions of SjR2 revealed two notable characteristics. First, the recombinant reverse transcriptase domain of SjR2 expressed in insect cells primed reverse transcription of SjR2 mRNA in vitro. By contrast, recombinant SjR2-endonuclease did not appear to cleave schistosome or plasmid DNA. Second, the 5'-untranslated region of SjR2 was >80% identical to the 3-untranslated region of a schistosome heat shock protein-70 gene (hsp-70) in the antisense orientation, indicating that SjR2-like elements were probably inserted into the non-coding regions of ancestral S. japonicum HSP-70, probably after the species diverged from S. mansoni. (C) 2002 Australian Society for Parasitology Inc. Published by Elsevier Science Ltd. All rights reserved.

Functional significance of a highly conserved glutamate residue of the human noradrenaline transportere

The aim of the study was to investigate the role of glutamate residue 113 in transmembrane domain 2 of the human noradrenaline transporter in determining cell surface expression and functional activity. This residue is absolutely conserved in all members of the Na+- and Cl--dependent transporter family. Mutations to alanine (hE113A), aspartate (hE113D) and glutamine (hE113Q) were achieved by site-directed mutagenesis and the mutants were expressed in transfected COS-7 or HEK-293 cells. Cell surface expression of IIE113A and hE113D, but not hE113Q, was markedly reduced compared with wild type, and functional noradrenaline uptake was detected only for the hE113Q mutant. The pharmacological properties of the hE113Q mutant showed very little change compared with wild type, except for a decrease in V-max values for noradrenaline and dopamine uptake of 2-3-fold. However, the hE113D mutant showed very marked changes in its properties, compared with wild type, with 82-260-fold decreases in the affinities of the substrates, noradrenaline, dopamine and MPP+, and increased Na+ affinity for stimulation of nisoxetine binding. The results of the study show that the size and not the charge of the 113 glutamate residue of the noradrenaline transporter seems to be the most critical factor for maintenance of transporter function and surface expression.

Direct visualization of Ras proteins in spatially distinct cell surface microdomains

Localization of signaling complexes to specific micro-domains coordinates signal transduction at the plasma membrane. Using immunogold electron microscopy of plasma membrane sheets coupled with spatial point pattern analysis, we have visualized morphologically featureless microdomains including lipid rafts, in situ and at high resolution. We find that an inner-plasma membrane lipid raft marker displays cholesterol-dependent clustering in microdomains with a mean diameter of 44 nm that occupy 35% of the cell surface. Cross-linking an outer-leaflet raft protein results in the redistribution of inner leaflet rafts, but they retain their modular structure. Analysis of Ras microlocalization shows that inactive H-ras is distributed between lipid rafts and a cholesterol-independent micro-domain. Conversely, activated H-ras and K-ras reside predominantly in nonoverlapping, cholesterol-independent microdomains. Galectin-1 stabilizes the association of activated H-ras with these nonraft microdomains, whereas K-ras clustering is supported by farnesylation, but not geranylgeranylation. These results illustrate that the inner plasma membrane comprises a complex mosaic of discrete microdomains. Differential spatial localization within this framework can likely account for the distinct signal outputs from the highly homologous Ras proteins.

Identification of residues and domains of Raf important for function in vivo and in vitro

Random mutagenesis and genetic screens for impaired Raf function in Caenorhabditis elegans were used to identify six loss-of-function alleles of lin-45 raf that result in a substitution of a single amino acid. The mutations were classified as weak, intermediate, and strong based on phenotypic severity. We engineered these mutations into the homologous residues of vertebrate Raf-1 and analyzed the mutant proteins for their underlying biochemical defects. Surprisingly, phenotype strength did not correlate with the catalytic activity of the mutant proteins. Amino acid substitutions Val-589 and Ser-619 severely compromised Raf kinase activity, yet these mutants displayed weak phenotypes in the genetic screen. Interestingly, this is because these mutant Raf proteins efficiently activate the MAPK (mitogen-activated protein kinase) cascade in living cells, a result that may inform the analysis of knockout mice. Equally intriguing was the observation that mutant proteins with non-functional Ras-binding domains, and thereby deficient in Ras-mediated membrane recruitment, displayed only intermediate strength phenotypes. This confirms that secondary mechanisms exist to couple Ras to Raf in vivo. The strongest phenotype in the genetic screens was displayed by a S508N mutation that again did not correlate with a significant loss of kinase activity or membrane recruitment by oncogenic Ras in biochemical assays. Ser-508 lies within the Raf-1 activation loop, and mutation of this residue in Raf-1 and the equivalent Ser-615 in B-Raf revealed that this residue regulates Raf binding to MEK. Further characterization revealed that in response to activation by epidermal growth factor, the Raf-S508N mutant protein displayed both reduced catalytic activity and aberrant activation kinetics: characteristics that may explain the C. elegans phenotype.

Initiation of biological agents in patients with ankylosing spondylitis: results of a Delphi study by the ASAS Group

Background: There is ample evidence of important symptomatic efficacy of tumour necrosis factor alpha (TNFalpha) inhibition in ankylosing spondylitis (AS). Moreover, studies suggest that anti-TNF could be considered as the first disease controlling antirheumatic treatment (DC-ART) for AS. Objective: To determine precisely which patients with AS are most likely to benefit from anti-TNFalpha treatment because of the cost and possible long term side effects of such treatment. Methods: Assessment in Ankylosing Spondylitis (ASAS) members were asked to use a Delphi technique to name the characteristics of patients with AS for whom they would start DC-ART, in three different clinical presentations (isolated axial involvement, peripheral arthritis, enthesitis). Results: Among the 62 invited ASAS members, more than 50% actively participated in the four phases of definition according to the Delphi technique. For each of the three clinical presentations, a combination of five to six domains was proposed, with an evaluation instrument and a cut off point defining a minimum level of activity for each domain. Conclusion: This study provides a profile for a patient with AS for considering initiation of biological agents that reflects the opinion of the ASAS members, using a Delphi exercise. Further studies are required to assess their relevance and their consistency with clinical practice.

Core outcome domains for chronic pain clinical trials: IMMPACT recommendations

Objective. To provide recommendations for the core outcome domains that should be considered by investigators conducting clinical trials of the efficacy and effectiveness of treatments for chronic pain. Development of a core set of outcome domains would facilitate comparison and pooling of data, encourage more complete reporting of outcomes, simplify the preparation and review of research proposals and manuscripts, and allow clinicians to make informed decisions regarding the risks and benefits of treatment. Methods. Under the auspices of the Initiative on Methods, Measurement, and Pain Assessment in Clinical Trials (IMMPACT), 27 specialists from academia. governmental agencies, and the pharmaceutical industry participated in a consensus meeting and identified core outcome domains that should be considered in clinical trials of treatments for chronic pain. Conclusions. There was a consensus that chronic pain clinical trials should assess outcomes representing six core domains: (1) pain, (2) physical functioning, (3) emotional functioning, (4) participant ratings of improvement and satisfaction with treatment, (5) symptoms and adverse events, (6) participant disposition (e.g. adherence to the treatment regimen and reasons for premature withdrawal from the trial). Although consideration should be given to the assessment of each of these domains, there may be exceptions to the general recommendation to include all of these domains in chronic pain trials. When this occurs, the rationale for not including domains should be provided. It is not the intention of these recommendations that assessment of the core domains should be considered a requirement for approval of product applications by regulatory agencies or that a treatment must demonstrate statistically significant effects for all of the relevant core domains to establish evidence of its efficacy. (C) 2003 International Association for the Study of Pain.

A high definition, finite difference time domain method

A high definition, finite difference time domain (HD-FDTD) method is presented in this paper. This new method allows the FDTD method to be efficiently applied over a very large frequency range including low frequencies, which are problematic for conventional FDTD methods. In the method, no alterations to the properties of either the source or the transmission media are required. The method is essentially frequency independent and has been verified against analytical solutions within the frequency range 50 Hz-1 GHz. As an example of the lower frequency range, the method has been applied to the problem of induced eddy currents in the human body resulting from the pulsed magnetic field gradients of an MRI system. The new method only requires approximately 0.3% of the source period to obtain an accurate solution. (C) 2003 Elsevier Science Inc. All rights reserved.

Oxide ionic conductivity and microstructures of Sm- or La-doped CeO2-based systems

The concept of crystallographic index termed the effective index is suggested and applied to the design of ceria (CeO2)-based electrolytes to maximize oxide ionic conductivity. The suggested index considers the fluorite structure, and combines the expected oxygen vacancy level with the ionic radius mismatch between host and dopant cations. Using this approach, oxide ionic conductivity of Sm- or La-doped CeO2-based system has been optimized and tested under operating conditions of a solid oxide fuel cell. In the observation of microstructure in atomic scale, both Sm-doped CeO2 and La-doped CeO2 electrolytes had large micro-domains over 10 nm in the lattice. On the other hand, Sm or La and alkaline earth co-doped CeO2-based electrolytes with high effective index had small micro-domains around 1-3 nm in the microstructure. The large micro-domain would prevent oxide ion from passing through the lattice. Therefore, it is concluded that the improvement of ionic conductivity is reflected in changes of microstructure in atomic scale. (C) 2002 Elsevier Science B.V. All rights reserved.

Genomic screen for genes involved in mammalian craniofacial development

Using a subtractive hybridisation approach, we enriched for genes likely to play a role in embryonic development of the mammalian face and other structures. This was achieved by subtracting cDNA derived from adult mouse liver from that derived from 10.5 dpc mouse embryonic branchial arches 1 and 2. Random sequencing of clones from the resultant library revealed that a high percentage correspond to genes with a previously established role in embryonic development and disease, while 15% represent novel or uncharacterised genes. Whole mount in situ hybridisation analysis of novel genes revealed that approximately 50% have restricted expression during embryonic development. In addition to expression in branchial arches, these genes showed a range of expression domains commonly including neural tube and somites. Notably, all genes analysed were found to be expressed not only in the branchial arches but also in the developing limb buds, providing support for the hypothesis that development of the limbs and face is likely to involve analogous molecular processes. (C) 2003 Wiley-Liss, Inc.

CRIM1 Regulates the Rate of Processing and Delivery of Bone Morphogenetic Proteins to the Cell Surface

The Crim1 gene is predicted to encode a transmembrane protein containing six von Willebrand-like cysteine-rich repeats (CRRs) similar to those in the BMP-binding antagonist Chordin (Chrd). In this study, we verify that CRIM1 is a glycosylated, Type I transmembrane protein and demonstrate that the extracellular CRR-containing domain can also be secreted, presumably via processing at the membrane. We have previously demonstrated Crim1 expression at sites consistent with an interaction with bone morphogenetic proteins (BMPs). Here we show that CRIM1 can interact with both BMP4 and BMP7 via the CRR-containing portion of the protein and in so doing acts as an antagonist in three ways. CRIM1 binding of BMP4 and -7 occurs when these proteins are co-expressed within the Golgi compartment of the cell and leads to (i) a reduction in the production and processing of preprotein to mature BMP, (ii) tethering of pre-BMP to the cell surface, and (iii) an effective reduction in the secretion of mature BMP. Functional antagonism was verified by examining the effect of coexpression of CRIM1 and BMP4 on metanephric explant culture. The presence of CRIM1 reduced the effective BMP4 concentration of the media, thereby acting as a BMP4 antagonist. Hence, CRIM1 modulates BMP activity by affecting its processing and delivery to the cell surface