Increases in endothelial Ca(2+) activate K(Ca) channels and elicit EDHF-type arteriolar dilation via gap junctions

In skeletal muscle arterioles, the pathway leading to non-nitric oxide (NO), non-prostaglandin-mediated endothelium-derived hyperpolarizing factor (EDHF)-type dilations is not well characterized. To elucidate some of the steps in this process, simultaneous changes in endothelial intracellular Ca(2+) concentration ([Ca(2+)](i)) and the diameter of rat gracilis muscle arterioles (approximately 60 microm) to acetylcholine (ACh) were measured by fura 2 microfluorimetry (in the absence of NO and prostaglandins). ACh elicited rapid increases in endothelial [Ca(2+)](i) (101 +/- 7%), followed by substantial dilations (73 +/- 2%, coupling time: 1.3 +/- 0.2 s) that were prevented by endothelial loading of an intracellular Ca(2+) chelator [1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid]. Arteriolar dilations to ACh were also inhibited by intraluminal administration of the Ca(2+)-activated K(+) (K(Ca)) channel blockers charybdotoxin plus apamin or by palmitoleic acid, an uncoupler of myoendothelial gap junctions without affecting changes in endothelial [Ca(2+)](i). The presence of large conductance K(Ca) channels on arteriolar endothelial cells was demonstrated with immunohistochemisty. We propose that in skeletal muscle arterioles, EDHF-type mediation is evoked by an increase in endothelial [Ca(2+)](i), which by activating endothelial K(Ca) channels elicits hyperpolarization that is conducted via myoendothelial gap junctions to the smooth muscle resulting in decreases in [Ca(2+)](i) and consequently dilation.     

Loop L5 acts as a conformational latch in the mitotic kinesin Eg5

All members of the kinesin superfamily of molecular motors contain an unusual structural motif consisting of an α-helix that is interrupted by a flexible loop, referred to as L5. We have examined the function of L5 in the mitotic kinesin Eg5 by combining site-directed mutagenesis of L5 with transient state kinetics, molecular dynamics simulations, and docking using cryo electron microscopy density. We find that mutation of a proline residue located at a turn within this loop profoundly slows nucleotide-induced structural changes both at the catalytic site as well as at the microtubule binding domain and the neck linker. Molecular dynamics simulations reveal that this mutation affects the dynamics not only of L5 itself but also of the switch I structural elements that sense ATP binding to the catalytic site. Our results lead us to propose that L5 regulates the rate of conformational change in key elements of the nucleotide binding site through its interactions with α3 and in so doing controls the speed of movement and force generation in kinesin motors.     

Satellite-based monitoring of tropical seagrass vegetation: current techniques and future developments

Decline of seagrasses has been documented in many parts of the world. Reduction in water clarity, through increased turbidity and increased nutrient concentrations, is considered to be the primary cause of seagrass loss. Recent studies have indicated the need for new methods that will enable early detection of decline in seagrass extent and productivity, over large areas. In this review of current literature on coastal remote sensing, we examine the ability of remote sensing to serve as an information provider for seagrass monitoring. Remote sensing offers the potential to map the extent of seagrass cover and monitor changes in these with high accuracy for shallow waters. The accuracy of mapping seagrasses in deeper waters is unclear. Recent advances in sensor technology and radiometric transfer modelling have resulted in the ability to map suspended sediment, sea surface temperature and below-surface irradiance. It is therefore potentially possible to monitor the factors that cause the decline in seagrass status. When the latest products in remote sensing are linked to seagrass production models, it may serve as an early-warning system for seagrass decline and ultimately allow a better management of these susceptible ecosystems.     

ABA Increases the Desiccation Tolerance of Photosynthesis in the Afromontane Understorey Moss Atrichum androgynum

The effect of pretreatment with abscisic acid (ABA) on the physiologyof the moss Atrichum androgynum during a desiccation–rehydrationcycle was examined. During rehydration following desiccationfor 16 h, net CO₂fixation recovered much more slowly than photosystemII (PSII) activity, conditions conducive to the formation ofreactive oxygen species (ROS) in the photosynthetic apparatus.Pretreatment with ABA increased the rate of recovery of photosynthesisand PSII activity, and also doubled non-photochemical quenching(NPQ). Increased NPQ activity will reduce ROS formation, andmay explain in part how ABA hardens the moss to desiccation.In ABA-pretreated, but not untreated mosses, desiccation significantlyincreased the concentration of soluble sugars. Sugar accumulationmay promote vitrification of the cytoplasm and protect membranesduring desiccation. Starch concentrations in freshly collectedA. androgynum were only approx. 40 mg g^-1dry mass; they roseslightly during desiccation but were only slightly affectedby ABA pretreatment. ABA did not reduce chlorophyll breakdownduring desiccation. Copyright 2001 Annals of Botany Company Moss, desiccation, abscisic acid, photosynthesis, chlorophyll fluorescence     

NMR-driven secondary and tertiary structure model of Ca2+-loaded calexcitin

Calexcitin (CE) is a Ca2+-binding protein which is expressed in neuronal cells and is a member of the sarcoplasmic Ca2+-binding protein subfamily. The peptide backbone of Ca2+-CE has been assigned by NMR and it shows that CE is composed of nine alpha-helices-forming four EF-hands and an additional helix near the C-terminus. A structural model of CE suggests the presence of a putative recessed hydrophobic pocket that may be involved in Ca2+-mediated protein-ligand interactions. This feature is unique to CE and is absent in other SCPs, such as those from Branchiostoma and Nereis, and from calerythrin.     

Phosphatidylglycerol Depletion Induces an Increase in Myxoxanthophyll Biosynthetic Activity in Synechocystis PCC6803 Cells

Phosphatidylglycerol (PG) depletion suppressed the oxygen-evolvingactivity of Synechocystis PCC6803 pgsA mutant cells. Shortageof PG led to decreased photosynthetic activity, which, similarto the effect of high light exposure, is likely to generatethe production of reactive oxygen species (ROS) or free radicals.Protection of the PG-depleted cells against light-induced damageincreased the echinenone and myxoxanthophyll content of thecells. The increased carotenoid content was localized in a solublefraction of the cells as well as in isolated thylakoid and cytoplasmicmembranes. The soluble carotenoid fraction contained carotenederivatives, which may bind to proteins. These carotene–proteincomplexes are similar to orange carotenoid protein that is involvedin yielding protection against free radicals and ROS. An increasein the content of myxoxanthophyll and echinenone upon PG depletionsuggests that PG depletion regulates the biosynthetic pathwayof specific carotenoids.     

Lipid binding properties of 4E10, 2F5, and WR304 monoclonal antibodies that neutralize HIV-1

Two human mAbs (2F5 and 4E10), originally derived from HIV-1-infected patients, are important, but rare, mAbs that exhibit broad cross-clade neutralizing activities against HIV-1. In addition to peptide sequences on the gp41 envelope protein, both antibodies reportedly also bound specifically to several phospholipid antigens. However, the phospholipid binding property of 2F5 has been disputed and, because of uncertainly regarding phospholipid binding, the modeling of neutralizing mechanisms has been difficult. To explore this issue, we examined the binding of 4E10 and 2F5 to a broad range of lipid antigens by ELISA. 4E10 and 2F5 both bound to a variety of purified phospholipids, and 4E10 bound, but 2F5 did not bind, to cardiolipin. Both mAbs also bound to a sulfated glycolipid, sulfogalactosyl ceramide (sulfatide), and to two neutral glycolipids, galactosyl ceramide and glucosyl ceramide, but not to other galactosyl glycolipids. 4E10, but not 2F5, also bound to cholesterol, although both mAbs bound to squalene. Interestingly, 4E10, but not 2F5, exhibited striking binding to lipid A, the lipid moiety of Gram-negative bacterial lipopolysaccharide. The binding properties of 4E10 to phospholipids, sulfatide, cholesterol, squalene, and lipid A were similar to those of a neutralizing murine mAb (WR304) induced by liposomes containing phosphatidylinositol phosphate and lipid A, although WR304 did not bind to neutral glycolipids. The discovery of a binding specificity of 4E10 for lipid A, a widely used vaccine adjuvant, suggests that innate immunity stimulated by lipid A could have played a role for induction of multispecific antibodies that simultaneously recognize both HIV-1 protein and lipid antigens.