Isolation and1H-NMR spectroscopic identification of the glucose-containing lipid-linked precursor oligosaccharide ofN-linked carbohydrate chains

The lipid-linked precursor ofN-type glycoprotein oligosaccharides was isolated from porcine thyroid microsomes after in cubation with UDP[³H] Glucose. The carbohydrate was released from dolichol pyrophosphate by mild acid hydrolysis, purified by gel filtration and characterized by 500-MHz¹H-NMR spectroscopy in combination with enzymatic degradation. The parent oligosaccharide was found to be Glc₃Man₉Glc-NAc₂. The three glucose residues are present in the linear sequence Glcα1-2Glα1-3 Glc, the latter being α(1-3)-linked to one of the mannose residues. In order to establish the branch location of the triglucosyl unit, the parent compound was digested with jack-bean α-mannosidase. The oligosaccharide product was purified by gel filtration, and identified by¹H-NMR as Glc₃Man₅GlcNAc₂ lacking the mannose residues A, D₂, B and D₃. Therefore, the structure of the precursor oligosaccharide is as follows: $$\begin{gathered} c b a D_1 C 4 \hfill \\ Glc\alpha 1 - 2Glc\alpha 1 - 3Glc\alpha 1 - 3Man\alpha 1 - 2Man\alpha 1 - 2Man\alpha 1 \hfill \\ 3 \swarrow 3 2 1 \hfill \\ Man\alpha 1 - 2Man\alpha 1 Man\beta 1 - 4GlcNAc\beta 1 - 4GlcNAc \hfill \\ D_{2 } A 3 6 \hfill \\ Man\alpha 1 \hfill \\ 6 \hfill \\ Man\alpha 1 - 2Man\alpha 1 \nwarrow 4 \hfill \\ D_3 B \hfill \\ \end{gathered} $$      

Preliminary X-ray crystallographic analysis of a modified basic fibroblast growth factor

Human basic fibroblast growth factor (hbFGF) has been modified, with Ala3 and Ser5 substituted by glutamic acid, and the purified recombinant protein has been crystallized. The crystals are triclinic (space group P1) with unit cell parameters a = 31.0 A, b = 33.6 A, c = 34.7 A, alpha = 88 degrees, beta = 85 degrees, gamma = 76 degrees, and they diffract to at least 2 A.     

Deformability Assessment of Waterborne Protozoa Using a Microfluidic-Enabled Force Microscopy Probe

Many modern filtration technologies are incapable of the complete removal of Cryptosporidium oocysts from drinking-water. Consequently, Cryptosporidium-contaminated drinking-water supplies can severely implicate both water utilities and consumers. Existing methods for the detection of Cryptosporidium in drinking-water do not discern between non-pathogenic and pathogenic species, nor between viable and non-viable oocysts. Using FluidFM, a novel force spectroscopy method employing microchannelled cantilevers for single-cell level manipulation, we assessed the size and deformability properties of two species of Cryptosporidium that pose varying levels of risk to human health. A comparison of such characteristics demonstrated the ability of FluidFM to discern between Cryptosporidium muris and Cryptosporidium parvum with 86% efficiency, whilst using a measurement throughput which exceeded 50 discrete oocysts per hour. In addition, we measured the deformability properties for untreated and temperature-inactivated oocysts of the highly infective, human pathogenic C. parvum to assess whether deformability may be a marker of viability. Our results indicate that untreated and temperature-inactivated C. parvum oocysts had overlapping but significantly different deformability distributions.     

Investigation of two new putative pheromone components of the invasive Australian redback spider,Latrodectus hasseltii,with potential applications for control

The Australian redback spider, Latrodectus hasseltii preys on at least 10 endemic species in New Zealand, highlighting a need for control. Male redbacks are attracted to virgin females by an airborne pheromone. The aim of this study was to analyse the response of male redback spiders to two volatile chemicals found on the silk of virgin but not mated females, to determine whether these compounds constitute components of the airborne pheromone. Mature male redback spiders were placed in an olfactometer where they had a choice of two stimuli. We compared their response to paired combinations of a control, virgin silk, butyric acid and isovaleric acid. Male redbacks responded equally strongly to butyric acid and virgin silk, in terms of time spent near the stimulus. The identification of butyric acid as a component of the airborne sex pheromone of L. hasseltii provides the groundwork for developing a pheromone-based control.     

A temporal analysis shows major histocompatibility complex loci in the Scandinavian wolf population are consistent with neutral evolution

The major histocompatibility complex (MHC) has an integral role in the immune system, and hence diversity at its genes may be of particular importance for the health of populations. In large populations, balancing selection maintains diversity in MHC genes, but theoretical expectations indicate that this form of selection is absent or inefficient in small populations. We examine the level of diversity at three MHC class II loci in the wolf population of Scandinavia, a population naturally recolonized with a genetic contribution from as few as three founders, and in four neighbouring wolf populations. In the Scandinavian wolf population, two alleles were found for each locus and the distribution of alleles is compatible with their linkage into two haplotypes. Changes in the level of heterozygosity over time since recolonization demonstrate the effects of the proposed arrival of an immigrant wolf. The maintenance of diversity is shown to be compatible with a neutral, random allocation of alleles, in conjunction with crossing between packs. A total of 15 DRB1, seven DQA and 10 DQB1 alleles are found in four neighbouring wolf populations, with substantial sharing across populations. Even in these larger populations, bottlenecks and fragmentation with consequent genetic drift are likely to have resulted in few indicators for balancing selection and significant differentiation of populations.     

CD1d-restricted NKT cells express a chemokine receptor profile indicative of Th1-type inflammatory homing cells

CD1d-restricted T cells (NKT cells) are innate memory cells activated by lipid Ags and play important roles in the initiation and regulation of the immune response. However, little is known about the trafficking patterns of these cells or the tissue compartment in which they exert their regulatory activity. In this study, we determined the chemokine receptor profile expressed by CD1d-restricted T cells found in the peripheral blood of healthy volunteers as well as CD1d-restricted T cell clones. CD1d-restricted T cells were identified by Abs recognizing the invariant Valpha24 TCR rearrangement or by binding to CD1d-Fc fusion tetramers loaded with alpha-GalCer. CD1d-restricted T cells in the peripheral blood and CD1d-restricted T cell clones expressed high levels of CXCR3, CCR5, and CCR6; intermediate levels of CXCR4 and CXCR6; and low levels of CXCR1, CCR1, CCR2, and CX(3)CR1, a receptor pattern often associated with tissue-infiltrating effector Th1 cells and CD8+ T cells. Very few of these cells expressed the lymphoid-homing receptors CCR7 or CXCR5. CCR4 was expressed predominantly on CD4+, but not on double-negative CD1d-restricted T cells, which may indicate differential trafficking patterns for these two functionally distinct subsets. CD1d-restricted T cell clones responded to chemokine ligands for CXCR1/2, CXCR3, CXCR4, CXCR6, CCR4, and CCR5 in calcium flux and/or chemotaxis assays. These data indicate that CD1d-restricted T cells express a chemokine receptor profile most similar to Th1 inflammatory homing cells and suggest that these cells perform their function in peripheral tissue sites rather than in secondary lymphoid organs.