Investigating the interaction between osteoprotegerin and receptor activator of NF-kappaB or tumor necrosis factor-related apoptosis-inducing ligand: evidence for a pivotal role for osteoprotegerin in regulating two distinct pathways

Osteoprotegerin (OPG) binds the ligand for receptor activator of nuclear factor kappaB (RANKL) to prevent association with its receptor RANK and inhibit osteoclast-mediated bone resorption. OPG has been reported, recently, to inhibit tumor necrosis factor-related apoptosis-induced ligand (TRAIL)-induced tumor cell apoptosis. This raises the possibility that OPG may play a unique role in regulating these two signaling pathways. However, there are little data on the interactions between OPG, RANKL, and TRAIL, and the relative affinity of OPG for these two ligands is unknown. In the present study we examined the ability of OPG to bind native human TRAIL and RANKL under physiological conditions. Native TRAIL was expressed in Escherichia coli, purified to homogeneity, and shown to induce human myeloma cell apoptosis. OPG inhibited native TRAIL from binding the TRAILR1 at 37 degrees C in vitro. Similarly, OPG prevented RANKL from binding to RANK. TRAIL also prevented OPG-mediated inhibition of RANKL from binding RANK. The affinity of OPG for native TRAIL and RANKL at 37 degrees C was determined by plasmon surface resonance analysis. OPG had a binding affinity for TRAIL of 45 nM, whereas the affinity of OPG for RANKL was 23 nM. These data suggest that OPG can bind both RANKL and TRAIL and that the affinity of OPG for these two ligands is of a similar order of magnitude. Furthermore, OPG prevented TRAIL-mediated reductions in cell viability, whereas TRAIL inhibited OPG-mediated inhibition of osteoclastogenesis in vitro. This highlights the pivotal role of OPG in regulating the biology of both RANKL and TRAIL.     

Effects of lipopolysaccharide on glial phenotype and activity of glutamate transporters: Evidence for delayed up-regulation and redistribution of GLT-1

Excitatory amino acid transporters (EAATs) are responsible for homeostasis of extracellular L-glutamate, and the glial transporters are functionally dominant. EAAT expression or function is altered in acute and chronic neurological conditions, but little is known about the regulation of EAATs in reactive astroglia found in such neuropathologies. These studies examined the effects of the bacterial endotoxin lipopolysaccharide (LPS) on glial EAATs in vitro. The effects of LPS (1 microg/ml, 24-72 h) on EAAT activity and expression were examined in primary cultures of mouse astrocytes. [(3)H]D-aspartate uptake increased to 129% of control by 72 h treatment with LPS. Saturation analysis revealed that apparent K(m) was unchanged whilst V(max) was significantly increased to 172% of control by 72 h LPS treatment. Biotinylation and Western blotting indicated that cell-surface expression of GLT-1 was significantly elevated (146% control) by LPS treatment whereas GLAST expression was unchanged. Confocal analyses revealed that LPS treatment resulted in cytoskeletal changes and stellation of astrocytes, with rearrangement of F-actin (as shown by phalloidin labelling). Immunocytochemistry revealed clustering of GLAST, and increased expression and redistribution of GLT-1 to the cell-surface following treatment with LPS. Similar experiments were conducted in microglia, where LPS (50 ng/ml) was found to up-regulate expression of GLT-1 at 24 and 72 h in concert with cytoskeletal changes accompanying activation. These findings suggest an association of cytoskeletal changes in glia with EAAT activity, with the predominant adaptation involving up-regulation and redistribution of GLT-1.     

The plant glycosyltransferase clone collection for functional genomics

The glycosyltransferases (GTs) are an important and functionally diverse family of enzymes involved in glycan and glycoside biosynthesis. Plants have evolved large families of GTs which undertake the array of glycosylation reactions that occur during plant development and growth. Based on the Carbohydrate‐Active enZymes (CAZy) database, the genome of the reference plant Arabidopsis thaliana codes for over 450 GTs, while the rice genome (Oryza sativa) contains over 600 members. Collectively, GTs from these reference plants can be classified into over 40 distinct GT families. Although these enzymes are involved in many important plant specific processes such as cell‐wall and secondary metabolite biosynthesis, few have been functionally characterized. We have sought to develop a plant GTs clone resource that will enable functional genomic approaches to be undertaken by the plant research community. In total, 403 (88%) of CAZy defined Arabidopsis GTs have been cloned, while 96 (15%) of the GTs coded by rice have been cloned. The collection resulted in the update of a number of Arabidopsis GT gene models. The clones represent full‐length coding sequences without termination codons and are Gateway^® compatible. To demonstrate the utility of this JBEI GT Collection, a set of efficient particle bombardment plasmids (pBullet) was also constructed with markers for the endomembrane. The utility of the pBullet collection was demonstrated by localizing all members of the Arabidopsis GT14 family to the Golgi apparatus or the endoplasmic reticulum (ER). Updates to these resources are available at the JBEI GT Collection website http://www.addgene.org/ .     

Structural and sequence motifs in dermatan sulfate for promoting fibroblast growth factor-2 (FGF-2) and FGF-7 activity

Glycosaminoglycans have been implicated in the binding and activation of a variety of growth factors, cytokines, and chemokines. In this way, glycosaminoglycans are thought to participate in events such as development and wound repair. In particular, heparin and heparan sulfate have been well studied, and specific aspects of their structure dictate their participation in a variety of activities. In contrast, although dermatan sulfate participates in many of the same biological processes as heparin and heparan sulfate, the interactions of dermatan sulfate have been less well studied. Dermatan sulfate is abundant in the wound environment and binds and activates growth factors such as fibroblast growth factor-2 (FGF-2) and FGF-7, which are present during the wound repair process. To determine the minimum size and sulfation content of active dermatan sulfate oligosaccharides, dermatan sulfate was first digested and then separated by size exclusion high pressure liquid chromatography, and the activity to facilitate FGF-2 and FGF-7 was assayed by the cellular proliferation of cell lines expressing FGFR1 or FGFR2 IIIb. The minimum size required for the activation of FGF-2 was an octasaccharide and for FGF-7 a decasaccharide. Active fractions were rich in monosulfated, primarily 4-O-sulfated, disaccharides and iduronic acid. Increasing the sulfation to primarily 2/4-O-sulfated and 2/6-O-sulfated disaccharides did not increase activity. Cell proliferation decreased or was abolished with higher sulfated dermatan sulfate preparations. This indicated a preference for specific dermatan sulfate oligosaccharides capable of promoting FGF-2- and FGF-7-dependent cell proliferation. These data identify critical oligosaccharides that promote specific members of the FGF family that are important for wound repair and angiogenesis.     

Behavioral response of manatees to variations in environmental sound levels

Florida manatees (Trichechus manatus latirostris) inhabit coastal regions because they feed on the aquatic vegetation that grows in shallow waters, which are the same areas where human activities are greatest. Noise produced from anthropogenic and natural sources has the potential to affect these animals by eliciting responses ranging from mild behavioral changes to extreme aversion. Sound levels were calculated from recordings made throughout behavioral observation periods. An information theoretic approach was used to investigate the relationship between behavior patterns and sound level. Results indicated that elevated sound levels affect manatee activity and are a function of behavioral state. The proportion of time manatees spent feeding and milling changed in response to sound level. When ambient sound levels were highest, more time was spent in the directed, goal‐oriented behavior of feeding, whereas less time was spent engaged in undirected behavior such as milling. This work illustrates how shifts in activity of individual manatees may be useful parameters for identifying impacts of noise on manatees and might inform population level effects.     

Bioturbation in matgrounds at Lake Bogoria in the Kenya Rift Valley: implications for interpreting the heterogeneous early Cambrian seafloor

Modern burrowing organisms feed on microbial organic matter in matgrounds near hot springs on the margins of Lake Bogoria, a saline alkaline lake in the Kenya Rift Valley. The burrowers produce a low-diversity trace assemblage similar to those produced by undermat miners during the Ediacaran–Cambrian transition. Despite obvious differences in body plans and phylogenetic affinities, these modern animals feed on microbes in similar ways to those inferred for primitive bilaterians. With increasing distance from hot-spring vents, outflow channels and adjacent matgrounds, the diversity and depth of the traces increase and mixgrounds become dominant. This modern extreme environment gives clues for interpreting the heterogeneous early Cambrian seafloor, with: (1) the restriction of ‘pre-agronomic revolution’ matground substrates; and (2) expansion of adjacent ‘post-agronomic revolution’ mixground areas.