Differentiation of isomeric oligosaccharide structures by ESI tandem MS and GC-MS

A mixture of arabinoxylan oligosaccharides from wheat seedling was permethylated and analyzed by electrospray ion trap MS and GC-MS. The presence of isomeric structures differing in degree of branching and position of the branched residue along the xylose backbone was demonstrated for oligosaccharides with four and five monosaccharide residues. No isomeric structures were found for oligosaccharides with three monosaccharide residues. Linkage analysis by GC-MS reveals that xylose residues were substituted with single arabinoxyl residues at C-3.     

Effect of the cluster size on the micro phase separation in mixtures of beta-lactoglobulin clusters and kappa-carrageenan

The phase separation of globular protein clusters formed by heat-denatured beta-lactoglobulin (beta-lg) in mixtures with the polysaccharide kappa-carrageenan (kappa-car) has been studied at pH 7 and 20 degrees C. The effect of the protein cluster size on the phase separation was investigated by preparing clusters with radii between 20 nm and 1 mum. The formation of protein rich microdomains led to an increase of the turbidity starting at a minimum kappa-car concentration that decreased with increasing cluster size, but was only weakly dependent on the protein concentration. The size and number of microdomains do not depend much on the cluster size, but their density decreases with increasing cluster size leading to a lower turbidity.     

The urokinase receptor: Focused cell surface proteolysis, cell adhesion and signaling

Plasma membrane urokinase-type plasminogen activator (uPA)-receptor (uPAR) is a GPI-anchored protein that binds with high-affinity and activates the serine protease uPA, thus regulating proteolytic activity at the cell surface. In addition, uPAR is a signaling receptor that often does not require its protease ligand or its proteolytic function.uPAR is highly expressed during tissue reorganization, inflammation, and in virtually all human cancers. Since its discovery, in vitro and in vivo models, as well as retrospective clinical studies have shown that over-expression of components of the uPA/uPAR-system correlates with increased proliferation, migration, and invasion affecting the malignant phenotype of cancer. uPAR regulates the cells-extracellular matrix interactions promoting its degradation and turnover through the plasminogen activation cascade.     

Flow-enhanced adhesion regulated by a selectin interdomain hinge

L-selectin requires a threshold shear to enable leukocytes to tether to and roll on vascular surfaces. Transport mechanisms govern flow-enhanced tethering, whereas force governs flow-enhanced rolling by prolonging the lifetimes of L-selectin-ligand complexes (catch bonds). Using selectin crystal structures, molecular dynamics simulations, site-directed mutagenesis, single-molecule force and kinetics experiments, Monte Carlo modeling, and flow chamber adhesion studies, we show that eliminating a hydrogen bond to increase the flexibility of an interdomain hinge in L-selectin reduced the shear threshold for adhesion via two mechanisms. One affects the on-rate by increasing tethering through greater rotational diffusion. The other affects the off-rate by strengthening rolling through augmented catch bonds with longer lifetimes at smaller forces. By forcing open the hinge angle, ligand may slide across its interface with L-selectin to promote rebinding, thereby providing a mechanism for catch bonds. Thus, allosteric changes remote from the ligand-binding interface regulate both bond formation and dissociation.     

High-affinity ligand probes of CD22 overcome the threshold set by cis ligands to allow for binding, endocytosis, and killing of B cells

CD22 (Siglec-2) is a key regulator of B cell signaling whose function is modulated by interaction with extracellular glycan ligands mediated through its N-terminal Ig domain. Its preferred ligand is the sequence Sia alpha2-6Gal that is abundantly expressed on N-linked glycans of B cell glycoproteins, and by binding to CD22 in cis causes CD22 to appear "masked" from binding to synthetic sialoside probes. Yet, despite the presence of cis ligands, CD22 redistributes to sites of cell contact by binding to trans ligands on neighboring cells. In this study, we demonstrate the dynamic equilibrium that exists between CD22 and its cis and trans ligands, using a high-affinity multivalent sialoside probe that competes with cis ligands and binds to CD22 on native human and murine B cells. Consistent with the constitutive endocytosis reported for CD22, the probes are internalized once bound, demonstrating that CD22 is an endocytic receptor that can carry ligand-decorated "cargo" to intracellular compartments. Conjugation of the sialoside probes to the toxin saporin resulted in toxin uptake and toxin-mediated killing of B lymphoma cell lines, suggesting an alternative approach for targeting CD22 for treatment of B cell lymphomas.     

Secreted modular calcium-binding protein-1 localization during mouse embryogenesis

BM-40 is an extracellular matrix-associated protein and is characterized by an extracellular calcium-binding domain as well as a follistatin-like domain. Secreted modular calcium-binding protein-1 (SMOC-1) is a new member of the BM-40 family. It consists of two thyroglobulin-like domains, a follistatin-like domain and a new domain without known homologues and is expressed ubiquitously in many adult murine tissues. Immunofluorescence studies, as well as immunogold electron microscopy, have confirmed the localization of SMOC-1 in or around basement membranes of adult murine skin, blood vessels, brain, kidney, skeletal muscle, and the zona pellucida surrounding the oocyte. In the present work, light microscopic immunohistochemistry has revealed that SMOC-1 is localized in the early mouse embryo day 7 throughout the entire endodermal basement membrane zone of the embryo proper. SMOC-1 mRNA is synthesized, even in early stages of mouse development, by mesenchymal as well as epithelial cells deriving from all three germ layers. In embryonic stage day 12, and fetal stages day 14, 16, and 18, the protein is present in the basement membrane zones of brain, blood vessels, skin, skeletal muscle, lung, heart, liver, pancreas, intestine, and kidney. This broad and organ-specific distribution suggests multifunctional roles of SMOC-1 during mouse embryogenesis.     

Detection and avoidance of an entomopathogenic fungus by a generalist insect predator

Abstract.  1. Increasing evidence suggests that insects can assess their environment based on cues related to mortality risks to themselves or their offspring. Limited knowledge is available on such abilities in relation to entomopathogenic fungi, which can cause significant mortality in insect populations. In laboratory bioassays, the ability of the generalist predator Anthocoris nemorum L. (Heteroptera: Anthocoridae) to detect the presence of its natural enemy, the fungal pathogen Beauveria bassiana (Balsamo) Vuillemin (Ascomycota: Hypocreales) was investigated.
2. Behavioural observations were conducted on adults of A. nemorum foraging in choice and non-choice arenas treated with conidia suspensions of B. bassiana or just the carrier (control). The arenas consisted either of nettle leaves or soil. Additionally, behaviours in response to sporulating nettle aphid cadavers compared with uninfected aphids or paper balls were evaluated on nettle leaves. An oviposition experiment was also conducted in choice arenas on conidia-treated and untreated nettle leaves.
3. Males and females detected and avoided contact with leaf surfaces inoculated with B. bassiana . Females that were forced to enter fungus-treated leaf surfaces were very reluctant to do so. When females encountered cadavers sporulating with B. bassiana they rapidly withdrew compared with harmless paper ball dummies. Soil inoculated with B. bassiana did not affect A. nemorum behaviour or residence time compared with control soil. Females inserted significantly more eggs in control leaf areas compared with areas treated with B. bassiana conidia.
4. All results suggest that A. nemorum detects and avoids the pathogen B. bassiana when it forages on host plants with which it is adapted but not on soil surfaces. The adaptive significance of detection of entomopathogenic fungi is discussed.