Synthesis of the acceptor analog αFuc(1→2)αGal-O(CH2)7 CH3: A probe for the kinetic mechanism of recombinant human blood group B glycosyltransferase

We report the chemical synthesis of Fuc(12)Gal-O(CH₂)₇CH₃ (1) an analog of the natural blood group (O)H disaccharide Fuc(12)Gal-OR. Compound 1 was a good substrate for recombinant blood group B glycosyltransferase (GTB) and was used as a precursor for the enzymatic synthesis of the blood group B analog Gal(3)[Fuc(12)]Gal-O(CH₂)₇CH₃ (2). To probe the mechanism of the GTB reaction, kinetic evaluations were carried out employing compound 1 or the natural acceptor disaccharide Fuc(12)Gal-O(CH₂)₇CH₃ (3) with UDP-Gal and UDP-GalNAc donors. Comparisons of the kinetic constants for alternative donor and acceptor pairs suggest that the GTB mechanism is Theorell-Chance where donor binding precedes acceptor binding. GTB operates with retention of configuration at the anomeric center of the donor. Retaining reactions are thought to occur via a double-displacement mechanism with formation of a glycosyl-enzyme intermediate consistent with the proposed Theorell-Chance mechanism.     

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%.     

Cell Specific,Cross-Species Expression of Myrosinases in Brassica Napus,Arabidopsis Thaliana and Nicotiana Tabacum

A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific expression to idioblast myrosin cells of immature and mature seeds and myrosin cells of phloem of B. napus. In A. thaliana the B. napus promoter directs expression to guard cells similar to the expression pattern of TGG1. The Myr1.Bn1 signal peptide targets the gene product to the reticular myrosin grains of myrosin cells. Our results indicate that myrosinase gene promoters from Brassicaceae direct cell, organ and developmental specific expression in B. napus, A. thaliana and N. tabacum.     

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.