IFN-γ production in response to <Emphasis Type="Italic">in vitro</Emphasis> stimulation with collagen type II in rheumatoid arthritis is associated with HLA-DRB1<Superscript>*</Superscript>0401 and HLA-DQ8

Introduction

Despite much work over past decades, whether antigen-specific immune reactions occur in rheumatoid arthritis (RA) and to what extent such reactions are directed towards joint-specific autoantigens is still questionable. One strong indicator for antigenic involvement in RA is the fact that certain major histocompatibility complex (MHC) class II genotypes [human leucocyte antigen (HLA)-DR4 and HLA-DR1] predispose for the development of the disease [1]. In the present report, collagen type II (CII) was studied as a putative autoantigen on the basis of both clinical and experimental data that show an increased frequency of antibodies to CII in RA patients [2,3,4] and that show that CII can induce experimental arthritis [5].It is evident from the literature that RA peripheral blood mononuclear cells (PBMCs) respond poorly to antigenic stimulation [6,7,8], and in particular evidence for a partial tolerization to CII has been presented [9]. The strategy of the present work has accordingly been to reinvestigate T-cell reactivity to CII in RA patients, to relate it to the response to commonly used recall antigens and to analyze IFN-γ responses as an alternative to proliferative responses.     

Genetic transformation via particle bombardment of Catharanthus roseus plants through adventitious organogenesis of buds

In vitro propagation of Catharanthus roseus was achieved using nodal explants. Bud induction was best on medium containing 1.0 mg benzyl aminopurine l^–1. Hardening of rooted shoots to soil was very successful with 98% survival. Genetically transformed C. roseus plantlets were obtained after bombardment of nodal explants, which were then micropropagated, with DNA coated particles with green fluorescent protein (GFP) or -glucuronidase (GUS) reporter genes. Histological studies showed that the gene insertion method proved effective with many cells and different tissues displaying the reporter gene signals, showing that gene expressions were rather stable.     

Intensity-based analysis of two-colour microarrays enables efficient and flexible hybridization designs

In two-colour microarrays, the ratio of signal intensities of two co-hybridized samples is used as a relative measure of gene expression. Ratio-based analysis becomes complicated and inefficient in multi-class comparisons. We therefore investigated the validity of an intensity-based analysis procedure. To this end, two different cRNA targets were hybridized together, separately, with a common reference and in a self-self fashion on spotted 65mer oligonucleotide microarrays. We found that the signal intensity of the cRNA targets was not influenced by the presence of a target labelled in the opposite colour. This indicates that targets do not compete for binding sites on the array, which is essential for intensity-based analysis. It is demonstrated that, for good-quality arrays, the correlation of signal intensity measurements between the different hybridization designs is high (R > 0.9). Furthermore, ratio calculations from ratio- and intensity-based analyses correlated well (R > 0.8). Based on these results, we advocate the use of separate intensities rather than ratios in the analysis of two-colour long-oligonucleotide microarrays. Intensity-based analysis makes microarray experiments more efficient and more flexible: It allows for direct comparisons between all hybridized samples, while circumventing the need for a reference sample that occupies half of the hybridization capacity.     

Measuring and modelling leaf diffusive conductance in juvenile silver birch, <Emphasis Type="Italic">Betula pendula</Emphasis>

Leaf diffusive conductance for water (g_l) and twig xylem pressure (_xt) was measured in juvenile silver birch, Betula pendula, under field conditions in southern Sweden. Data from one site were used to parameterise two different multiplicative models for g_l (dependent data), and measurements from another site were used to validate these models (independent data). In addition, experiments were performed in controlled environments to validate the g_l response functions used in the models. The driving variables in the D-model were photosynthetic photon flux density, air temperature and water vapour pressure deficit of the air (D_a), while the DH-model also included the accumulated hours after sunrise each day with D_a above a certain threshold (H). Both models satisfactorily predicted the variation in g_l in dependent as well as in independent data, and the g_l response functions used were supported by the experiments in controlled environments. The DH-model was more successful in predicting g_l than the D-model by accounting for the observation that g_l was lower at higher H under similar weather conditions. There was a considerable variation in maximum g_l during the season, as well as between the two sites. On relatively warm and dry days _xt rapidly declined during the morning and then stabilized around a constant value until the late afternoon, with the stomatal regulation effectively preventing _xt from decreasing below this value. We suggest that these models could be used to simulate the g_l in juvenile birch if maximum g_l is locally estimated and if the response functions are not extrapolated beyond the climate range for this study.     

Molecular tools for a molecular medicine: analyzing genes, transcripts and proteins using padlock and proximity probes

Procedures and reagents are needed to specifically detect all the macromolecules that are being identified in the course of genome projects. We discuss how this challenge may be met using a set of ligation-based reagents termed padlock probes and proximity ligation probes. These probes include elements with affinity for specific nucleic acid and protein molecules, respectively, along with unique identifier DNA sequence elements that encode the identity of the recognized target molecules. The information content of DNA strands that form in the detection reactions are recorded after amplification, allowing the recognized target molecules to be identified. The procedures permit highly specific solution-phase or localized analyses of large sets of target molecules as required in future molecular analyses.     

Reappraisal of the regulation of lactococcal L-lactate dehydrogenase

Lactococcal lactate dehydrogenases (LDHs) are coregulated at the substrate level by at least two mechanisms: the fructose-1,6-biphosphate/phosphate ratio and the NADH/NAD ratio. Among the Lactococcus lactis species, there are strains that are predominantly regulated by the first mechanism (e.g., strain 65.1) or by the second mechanism (e.g., strain NCDO 2118). A more complete model of the kinetics of the regulation of lactococcal LDH is discussed.     

Applications and performance of a MALDI-ToF mass spectrometer with quadratic field reflectron technology

A new matrix-assisted laser desorption/ionization time of flight mass spectrometer (MALDI-ToF MS), developed specifically for the identification and characterization of proteins and peptides in proteomic investigations, is described. The mass spectrometer which can be integrated with the 2-D gel electrophoresis workflow is a bench-top instrument, enabling rapid, reliable and unattended protein identification in low-, as well as high-throughput proteomics applications. To obtain precise information on peptide sequences, the instrument utilizes a timed ion gate and a unique quadratic field reflectron (Z2 technology), allowing single-run, post-source decay (PSD) of selected peptides. In this study, the performance of the instrument in reflectron, PSD and linear mode, respectively, was investigated. The results showed that the limit of detection for a single peptide in reflectron mode was 125 amol with a signal to noise ratio exceeding 20. Average mass resolution for peptides larger than 2000 u was around 13,000 full width, half maximum (FWHM). The limit for protein identification during peptide mass fingerprinting (PMF) was 500 amol with a sequence coverage of 18%. Mass error during PMF analysis was less than 15 ppm for 17 out of 25 (68%) identified peptides. In PSD mode, a complete series of y-ions of a CAF-derivatized peptide could be obtained from 3.75 fmol of material. The average mass error of PSD-generated fragments was less than 0.14 u. Finally, in linear mode, intact proteins with molecular masses greater than 300,000 u were detected with mass errors below 0.2%.     

Conserving tropical nature: current challenges for ecologists

Tropical biodiversity continues to erode unabated, which calls for ecologists to address the problem directly, placing less reliance on indirect interventions, such as community-based development schemes. Ecologists must become more assertive in providing scientifically formulated and adaptively managed interventions, involving biodiversity payments, to serve local, regional and global interests in tropical nature. Priorities for tropical ecologists thus include the identification of key thresholds to ecological resilience, and the formulation of clear monitoring protocols and management strategies for implementation by local resource managers. A particular challenge is to demonstrate how nature reserves contribute to the adaptive capacity of regional land-use matrices and, hence, to the provision of sustainable benefits at multiple spatial and temporal scales.     

Joint explorative analysis of neuroreceptor subsystems in the human brain: application to receptor-transporter correlation using PET data

Positron emission tomography (PET) has proved to be a highly successful technique in the qualitative and quantitative exploration of the human brain's neurotransmitter-receptor systems. In recent years, the number of PET radioligands, targeted to different neuroreceptor systems of the human brain, has increased considerably. This development paves the way for a simultaneous analysis of different receptor systems and subsystems in the same individual. The detailed exploration of the versatility of neuroreceptor systems requires novel technical approaches, capable of operating on huge parametric image datasets. An initial step of such explorative data processing and analysis should be the development of novel exploratory data-mining tools to gain insight into the "structure" of complex multi-individual, multi-receptor data sets. For practical reasons, a possible and feasible starting point of multi-receptor research can be the analysis of the pre- and post-synaptic binding sites of the same neurotransmitter. In the present study, we propose an unsupervised, unbiased data-mining tool for this task and demonstrate its usefulness by using quantitative receptor maps, obtained with positron emission tomography, from five healthy subjects on (pre-synaptic) serotonin transporters (5-HTT or SERT) and (post-synaptic) 5-HT(1A) receptors. Major components of the proposed technique include the projection of the input receptor maps to a feature space, the quasi-clustering and classification of projected data (neighbourhood formation), trans-individual analysis of neighbourhood properties (trajectory analysis), and the back-projection of the results of trajectory analysis to normal space (creation of multi-receptor maps). The resulting multi-receptor maps suggest that complex relationships and tendencies in the relationship between pre- and post-synaptic transporter-receptor systems can be revealed and classified by using this method. As an example, we demonstrate the regional correlation of the serotonin transporter-receptor systems. These parameter-specific multi-receptor maps can usefully guide the researchers in their endeavour to formulate models of multi-receptor interactions and changes in the human brain.