Characterization of the retinoid orphan-related receptor-alpha coactivator binding interface: A structural basis for ligand-independent transcription

The retinoid orphan-related receptor-alpha (RORalpha) is a member of the ROR subfamily of orphan receptors and acts as a constitutive activator of transcription in the absence of exogenous ligands. To understand the basis of this activity, we constructed a homology model of Rill using the closely related TRbeta as a template. Molecular modeling suggested that bulky hydrophobic side chains occupy the RORa ligand cavity leaving a small but distinct cavity that may be involved in receptor stabilization. This model was subject to docking simulation with a receptor-interacting peptide from the steroid receptor coactivator, GR-interacting protein-1, which delineated a coactivator binding surface consisting of the signature motif spanning helices 3-5 and helix 12 [activation function 2 (AF2)]. Probing this surface with scanning alanine mutagenesis showed structural and functional equivalence between homologous residues of RORalpha and TRbeta. This was surprising (given that Rill is a ligand-independent activator, whereas TRbeta has an absolute requirement for ligand) and prompted us to use molecular modeling to identify differences between Rill and TRbeta in the way that the All helix interacts with the rest of the receptor. Modeling highlighted a nonconserved amino acid in helix 11 of RORa (Phe491) and a short-length of 3.10 helix at the N terminus of AF2 which we suggest i) ensures that AF2 is locked permanently in the holoconformation described for other liganded receptors and thus 2) enables ligand-independent recruitment of coactivators. Consistent with this, mutation of RORa Phe491 to either methionine or alanine (methionine is the homologous residue in TRbeta), reduced and ablated transcriptional activation and recruitment of coactivators, respectively. Furthermore, we were able to reconstitute transcriptional activity for both a deletion mutant of Ill lacking All and Phe491 Met, by overexpression of a GAL-AF2 fusion protein, demonstrating ligand-independent recruitment of AF2 and a role for Phe491 in recruiting AF2.

Genetic variability in cultivated common bean beyond the two major gene pools

It is generally accepted that two major gene pools exist in cultivated common bean (Phaseolus vulgaris L.), a Middle American and an Andean one. Some evidence, based on unique phaseolin morphotypes and AFLP analysis, suggests that at least one more gene pool exists in cultivated common bean. To investigate this hypothesis, 1072 accessions from a common bean core collection from the primary centres of origin, held at CIAT, were investigated. Various agronomic and morphological attributes (14 categorical and 11 quantitative) were measured. Multivariate analyses, consisting of homogeneity analysis and clustering for categorical data, clustering and ordination techniques for quantitative data and nonlinear principal component analysis for mixed data, were undertaken. The results of most analyses supported the existence of the two major gene pools. However, the analysis of categorical data of protein types showed an additional minor gene pool. The minor gene pool is designated North Andean and includes phaseolin types CH, S and T; lectin types 312, Pr, B and K; and mostly A5, A6 and A4 types alpha-amylase inhibitor. Analysis of the combined categorical data of protein types and some plant categorical data also suggested that some other germplasm with C type phaseolin are distinguished from the major gene pools.

Syntheses of polycationic dendrimers on lipophilic peptide core for complexation and transport of oligonucleotides

Synthesis of novel polycationic lipophilic peptide core(s) was accomplished and these agents successfully transfected human retinal pigment epithelium cells with ODN1 upon complexation with the oligonucleotide. The level of transfection was indirectly measured by the decreased production of the protein hVEGF (human vascular endothelial growth factor) in comparison to the transfection agent cytofectin GSV(TM). (C) 2002 Elsevier Science Ltd. All rights reserved.

Towards synthetic vaccines built on carbohydrate cores

Lipophilic polyfunctional carbohydrate core/templates have been designed and developed for drug/vaccine delivery. Three carbohydrate-based templates containing four protected N-terminal arms were synthesised from glucose and galactose. Methyl alpha-D-glucopyranoside was converted to two derivatives bearing a carboxylic acid handle for attachment to solid supports, spacer arms of differing hydrophilicity, and phthaloyl-protected amino groups suitable for peptide chain extension. beta-D-Galactopyranosyl azide was converted to a template bearing a carboxylic acid handle and four BOC-protected amines. All the templates were found to be suitable for attachment to solid supports and subsequent cleavage from resins, using either BOC- or FMOC-methodologies.

A lipid core peptide construct containing a conserved region determinant of the group a streptococcal M protein elicits heterologous opsonic antibodies

The study reported here investigated the immunogenicity and protective potential of a lipid core peptide (LCP) construct containing a conserved region determinant of M protein, defined as peptide J8. Parenteral immunization of mice with LCP-J8 led to the induction of high-titer serum immunoglobulin G J18-specific antibodies when the construct was coadministered with complete Freund's adjuvant (CFA) or administered alone. LCP-J8 in CFA had significantly enhanced immunogenicity compared with the monomeric peptide J8 given in CFA. Moreover, LCP-J8/CFA and LCP-J8 antisera opsonized four different group A streptococcal (GAS) strains, and the antisera did not cross-react with human heart tissue proteins. These data indicate the potential of an LCP-based M protein conserved region GAS vaccine in the induction of broadly protective immune responses in the absence of a conventional adjuvant.

Starch degradation in 'Kensington' mango fruit following heat treatments

Changes in carbohydrate metabolism of 'Kensington' mango fruit from 2 major production regions in Queensland were measured after conditioning fruit with hot air at 40degreesC for 0, 2, 4, 8 and 16 h or at 22degreesC for 16 h (control) followed by hot-water treatment at either 45degreesC fruit-core temperature for 30 min or 47degreesC fruit-core temperature held for 15 min. Advancing physiological maturity of 'Kensington' mango fruit was correlated with increased starch concentration within the mesocarp. An alpha-amylase inhibitor was present in unripe 'Kensington' mesocarp. alpha-Amylase activity was promoted by conditioning fruit at 40degreesC for 8 h, and this enhanced enzyme activity persisted until the fruit were ripe. Consequently, starch degradation was accelerated and the concentration of total soluble solids was higher in fruit conditioned at 40degreesC for 8 h than in fruit left at the lower temperature of 22degreesC for 16 h or not conditioned. Immediately on removal of fruit from hot-water treatment, activities of alpha-amylase and phosphorylase were inhibited. This inhibition was correlated with higher starch concentration and starch layer and starch spot injuries in these fruit. A positive correlation was also found between increased sucrose concentration and greater starch loss in 40degreesC conditioned 'Kensington' fruit. It is proposed that increased sugar concentration in the mesocarp increased the level of fruit heat tolerance.

Human peripheral blood V alpha 24(+) V beta 11(+) NKT cells expand following administration of alpha-galactosylceramide-pulsed dendritic cells

Background and Objectives We have undertaken the first clinical trial involving the administration of alpha-GalactosylCeramine (alpha-GalCer)-pulsed dendritic cells (DCs) to human subjects, to determine safety, optimal dose, optimal administration route and immunological effects. Materials and Methods Subjects (n = 4) with metastatic malignancy received two infusions of alpha-GalCer-pulsed DCs intravenously, and two infusions intradermally. The percentages of Valpha24 Vbeta11 NKT cells in peripheral blood (PB) were determined by three-colour flow cytometry and the PB NKT cell numbers were calculated using the total number of PB lymphocytes/ml determined by automated full-blood counts. Results No serious treatment related adverse events were observed during the study period. Administration of alpha-GalCer-pulsed DCs in vivo can significantly (P < 0.03) increase PB Valpha24(+) Vbeta11(+) NKT cell numbers above pretreatment baseline levels after the transient fall in the NKT numbers within 48 h. Conclusions Administration of alpha-GalCer-pulsed DCs is well tolerated, modulates PB Valpha24(+) Vbeta11(+) NKT cells and may have a role in the therapy of malignancies sensitive to activities of Valpha24(+) Vbeta11(+) NKT cells, or for autoimmune diseases.

A new level of conotoxin diversity, a non-native disulfide bond connectivity in alpha-conotoxin AuIB reduces structural definition but increases biological activity

alpha-Conotoxin AuIB and a disulfide bond variant of AuIB have been synthesized to determine the role of disulfide bond connectivity on structure and activity. Both of these peptides contain the 15 amino acid sequence GCCSYPPCFATNPDC, with the globular (native) isomer having the disulfide connectivity Cys(2-8 and 3-15) and the ribbon isomer having the disulfide connectivity Cys(2-15 and 3-8). The solution structures of the peptides were determined by NAIR spectroscopy, and their ability to block the nicotinic acetylcholine receptors on dissociated neurons of the rat parasympathetic ganglia was examined. The ribbon disulfide isomer, although having a less well defined structure, is surprisingly found to have approximately 10 times greater potency than the native peptide. To our knowledge this is the first demonstration of a non-native disulfide bond isomer of a conotoxin exhibiting greater biological activity than the native isomer.

Enhancing the immunogenicity and modulating the fine epitope recognition of antisera to a helical group A streptococcal peptide vaccine candidate from the M protein using lipid-core peptide technology

A conserved helical peptide vaccine candidate from the M protein of group A streptococci, p145, has been described. Minimal epitopes within p145 have been defined and an epitope recognized by protective antibodies, but not by autoreactive T cells, has been identified. When administered to mice, p145 has low immunogenicity. Many boosts of peptide are required to achieve a high antibody titre (> 12 800). To attempt to overcome this low immunogenicity, lipid-core peptide technology was employed. Lipid-core peptides (LCP) consist of an oligomeric polylysine core, with multiple copies of the peptide of choice, conjugated to a series of lipoamino acids, which acts as an anchor for the antigen. Seven different LCP constructs based on the p145 peptide sequence were synthesized (LCP1-->LCP7) and the immunogenicity of the compounds examined. The most immunogenic constructs contained the longest alkyl side-chains. The number of lipoamino acids in the constructs affected the immunogenicity and spacing between the alkyl side-chains increased immunogenicity. An increase in immunogenicity (enzyme-linked immunosorbent assay (ELISA) titres) of up to 100-fold was demonstrated using this technology and some constructs without adjuvant were more immunogenic than p145 administered with complete Freund's adjuvant (CFA). The fine specificity of the induced antibody response differed for the different constructs but one construct, LCP4, induced antibodies of identical fine specificity to those found in endemic human serum. Opsonic activity of LCP4 antisera was more than double that of p145 antisera. These data show the potential for LCP technology to both enhance immunogenicity of complex peptides and to focus the immune response towards or away from critical epitopes.

Role of PGF(2 alpha) and oxytocin in parturition in the brushtail possum (Trichosurus vulpecula)

Maturation of the fetal pituitary and adrenal glands allows the secretion of cortisol, which in turn leads to an increase in prostaglandin and mesotocin production. The production of prostaglandin and mesotocin results in an increase in uterine contractions and initiates birth in marsupials. The major metabolite of PGF(2alpha), 13,14-dihydro-15-keto-prostaglandin F-2alpha (PGFM), has been found in the plasma of the possum at the time of birth and administration of PGF(2alpha) to female possums induced the adoption of the birth position. Evidence that mesotocin is an integral hormone of birth in the tammar wallaby indicates that both PGF(2alpha) and mesotocin or oxytocin are required for marsupial birth. The presence of PGF(2alpha) receptors in the uterus and corpus luteum of the possum, and the in vitro uterine responsiveness to PGF(2alpha) or oxytocin, were examined. PGF(2alpha) receptors were not observed in possum uteri and the inability of PGF(2alpha) to cause contractions indicates that PGF(2alpha) is not involved directly in contraction of the uterus at parturition. The presence of oxytocin and mesotocin receptors in the uterus of possoms and the ability of oxytocin to induce uterine contraction in vitro supports the view that mesotocin is required for expulsion of the young from the uterus. Low numbers of PGF(2alpha) receptors were found in the possum corpus luteum at birth, indicating an involvement of PGF(2alpha) in regression of the corpus luteum.

Microstructural evidence for the mechanism of the alpha-beta phase transformation in ytterbium SiAlON ceramics

Detailed microstructural evidence for the mechanism of the alpha-beta phase transformation in ytterbium SiAlON ceramics is presented. Grains, which show partial transformation, have been examined using transmission electron microscopy. We suggest that the transformation proceeds as a discernable reaction front and the accompanying lattice mismatch is accommodated be a series of complex dislocations. The stabilizing cation is ejected from the transformed alpha- phase and diffuse along the dislocation to accumulate as isolated pockets in a way similar to that observed in metal systems and termed pipe diffusion. High-resolution electron microscopy reveals the details of each of these features.

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 structural basis for the selection of dominant alpha beta T cell receptors in antiviral immunity

We have examined the basis for immunodominant or public TCR usage in an antiviral CTL response. Residues encoded by each of the highly selected genetic elements of an immunodominant clonotype recognizing Epstein-Barr virus were critical to the antigen specificity of the receptor. Upon recognizing antigen the immunodominant TCR undergoes extensive conformational changes in the complementarity determining regions (CDRs), including the disruption of the canonical structures of the germline-encoded CDR1alpha and CDR2alpha loops to produce an enhanced fit with the HLA-peptide complex. TCR ligation induces conformational changes in the TCRalpha constant domain thought to form part of the docking site for CD3epsilon. These findings indicate that TCR immunodominance is associated with structural properties conferring receptor specificity and suggest a novel structural link between TCR ligation and intracellular signaling.

Explicit/implicit partitioning and a new explicit form of the generalized alpha method

Most finite element packages use the Newmark algorithm for time integration of structural dynamics. Various algorithms have been proposed to better optimize the high frequency dissipation of this algorithm. Hulbert and Chung proposed both implicit and explicit forms of the generalized alpha method. The algorithms optimize high frequency dissipation effectively, and despite recent work on algorithms that possess momentum conserving/energy dissipative properties in a non-linear context, the generalized alpha method remains an efficient way to solve many problems, especially with adaptive timestep control. However, the implicit and explicit algorithms use incompatible parameter sets and cannot be used together in a spatial partition, whereas this can be done for the Newmark algorithm, as Hughes and Liu demonstrated, and for the HHT-alpha algorithm developed from it. The present paper shows that the explicit generalized alpha method can be rewritten so that it becomes compatible with the implicit form. All four algorithmic parameters can be matched between the explicit and implicit forms. An element interface between implicit and explicit partitions can then be used, analogous to that devised by Hughes and Liu to extend the Newmark method. The stability of the explicit/implicit algorithm is examined in a linear context and found to exceed that of the explicit partition. The element partition is significantly less dissipative of intermediate frequencies than one using the HHT-alpha method. The explicit algorithm can also be rewritten so that the discrete equation of motion evaluates forces from displacements and velocities found at the predicted mid-point of a cycle. Copyright (C) 2003 John Wiley Sons, Ltd.