Reactive oxygen species impair Slo1 BK channel function by altering cysteine-mediated calcium sensing

Vascular dysfunction is a hallmark of many diseases, including coronary heart disease, stroke and diabetes. The underlying mechanisms of these disorders, which are intimately associated with inflammation and oxidative stress caused by excess reactive oxygen species (ROS), have remained elusive. Here we report that ROS are powerful inhibitors of vascular smooth muscle calcium-dependent Slo1 BK or Maxi-K potassium channels, an important physiological determinant of vascular tone. By targeting a cysteine residue near the Ca(2+) bowl of the BK alpha subunit, H(2)O(2) virtually eliminates physiological activation of the channel, with an inhibitory potency comparable to a knockout of the auxiliary subunit BK beta 1. These results reveal a molecular structural basis for the vascular dysfunction involving oxidative stress and provide a solid rationale for a potential use of BK openers in the prevention and treatment of cardiovascular disorders.     

Development of flight performance in the brown booby

How do birds acquire flight skills after fledging? This issue is important, as it is closely related to variation in the duration of offspring care, the causes of which remain unknown. In this study, we raised hatchling brown boobies, Sula leucogaster, and attached an acceleration data logger to each bird at fledging to record its movements. This allowed us to quantify precisely the time spent flapping, gliding and resting. The duration of foraging trips and proportion of time spent gliding during flight increased with the number of days since fledging, whereas the proportion of time spent in flight decreased. This indicates that brown boobies gradually acquire efficient flight skills during the post-fledging period, which might be the proximate cause of the long postfledging care period in this species. To the authors' knowledge, this is the first study to record precisely the ontogeny of flight behaviour in birds.     

Guanylyl cyclase and cGMP-specific phosphodiesterase participate in the acrosome reaction of starfish sperm

In the starfish, Asterias amurensis, the cooperation of three components of the egg jelly, i.e. ARIS (acrosome reaction-inducing substance), Co-ARIS and asterosap, is responsible for inducing the acrosome reaction. Experimentally, ARIS and asterosap are sufficient for the induction. However, when sperm are treated only with asterosap, they become unresponsive to the egg jelly to undergo the reaction. In this study, we analysed the mechanism of the acrosome reaction, using sperm inactivation by asterosap as a clue. Asterosap causes a rapid and transient increase in intracellular cGMP through the activation of the asterosap receptor, a guanylyl cyclase, and causes an increase in intracellular Ca(2+). When sperm were pretreated with asterosap, the guanylyl cyclase seemed to be inactivated irreversibly by dephosphorylation. They were still responsive to ARIS but no longer to asterosap. However, in the presence of IBMX or zaprinast, inhibitors against phosphodiesterases (PDEs), they retained their capacity to undergo the acrosome reaction in response to the egg jelly or ARIS alone. IBMX and zaprinast suppressed the intracellular catabolism of cGMP, but not of cAMP. These results suggest that guanylyl cyclase and cGMP-specific, IBMX- and zaprinast-susceptible PDEs are involved in the regulation of the acrosome reaction.     

Evaluation of systemic blood NO dynamics by EPR spectroscopy: HbNO as an endogenous index of NO

The measurement of hemoglobin-nitric oxide (NO) adduct (HbNO) in whole blood by the electron paramagnetic resonance (EPR) method seems relevant for the assessment of systemic NO levels. However, ceruloplasmin and unknown radical species overlap the same magnetic field as that of HbNO. To reveal the EPR spectrum of HbNO, we then introduced the EPR signal subtraction method, which is based on the computer-assisted subtraction of the digitized EPR spectrum of HbNO-depleted blood from that of sample blood using the software. Rats were treated with N(omega)-nitro-L-arginine methyl ester (L-NAME; 120 mg. kg-1. day-1) for 1 wk to obtain HbNO-depleted blood. When this method was applied to the analysis of untreated fresh whole blood, the five-coordinate state of HbNO was observed. HbNO concentration in pentobarbital-anesthetized rats was augmented (change in [HbNO] = 1.6-5.5 microM) by infusion of L-arginine (0.2-0.6 g/kg) but not D-arginine. Using this method, we attempted to evaluate the effects of temocapril on HbNO dynamics in an L-NAME-induced rat endothelial dysfunction model. The oral administration of L-NAME for 2 wk induced a serious hypertension, and the HbNO concentration was reduced (change in [HbNO] = 5.7 microM). Coadministration of temocapril dose dependently improved both changes in blood pressure and the systemic HbNO concentration. In this study, we succeeded in measuring the blood HbNO level as an index of NO by the EPR HbNO signal subtraction method. We also demonstrated that temocapril improves abnormalities of NO dynamics in L-NAME-induced endothelial dysfunction rats using the EPR HbNO signal subtraction method.     

Cystatin 10, a novel chondrocyte-specific protein,may promote the last steps of the chondrocyte differentiation pathway

This study attempts to characterize cystatin 10 (Cst10), which we recently identified as a novel protein implicated in endochondral ossification. Expression of Cst10 was specific to cartilage, localized in the cytosol of prehypertrophic and hypertrophic chondrocytes of the mouse growth plate. In the mouse chondrogenic cell line ATDC5, Cst10 expression preceded type X collagen expression and increased in synchrony with maturation. When we compared ATDC5 cells transfected with Cst10 cDNA with cells transfected with a mock vector, hypertrophic maturation and mineralization of chondrocytes were promoted by Cst10 gene overexpression in that type X collagen expression was observed earlier, and alizarin red staining was stronger. On the other hand, type II collagen expression and Alcian blue staining, both of which are markers of the early stage of chondrocyte differentiation, were similar in both cells. Overexpression of the Cst10 gene also caused fragmentation of nuclei, the appearance of annexin V, a change in the mitochondrial membrane potential, and activation of caspases. These results strongly suggest that Cst10 may play an important role in the last steps of the chondrocyte differentiation pathway as an inducer of maturation, followed by apoptosis of chondrocytes.     

Phosphoenolpyruvate carboxylase: three-dimensional structure and molecular mechanisms

Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) catalyzes the irreversible carboxylation of phosphoenolpyruvate (PEP) to form oxaloacetate and Pi using Mg2+ or Mn2+ as a cofactor. PEPC plays a key role in photosynthesis by C4 and Crassulacean acid metabolism plants, in addition to its many anaplerotic functions. Recently, three-dimensional structures of PEPC from Escherichia coli and the C4 plant maize (Zea mays) were elucidated by X-ray crystallographic analysis. These structures reveal an overall square arrangement of the four identical subunits, making up a "dimer-of-dimers" and an eight-stranded beta barrel structure. At the C-terminal region of the beta barrel, the Mn2+ and a PEP analog interact with catalytically essential residues, confirmed by site-directed mutagenesis studies. At about 20A from the beta barrel, an allosteric inhibitor (aspartate) was found to be tightly bound to down-regulate the activity of the E. coli enzyme. In the case of maize C4-PEPC, the putative binding site for an allosteric activator (glucose 6-phosphate) was also revealed. Detailed comparison of the various structures of E. coli PEPC in its inactive state with maize PEPC in its active state shows that the relative orientations of the two subunits in the basal "dimer" are different, implicating an allosteric transition. Dynamic movements were observed for several loops due to the binding of either an allosteric inhibitor, a metal cofactor, a PEP analog, or a sulfate anion, indicating the functional significance of these mobile loops in catalysis and regulation. Information derived from these three-dimensional structures, combined with related biochemical studies, has established models for the reaction mechanism and allosteric regulation of this important C-fixing enzyme.     

A consensus linkage map for sugi (Cryptomeria japonica) from two pedigrees, based on microsatellites and expressed sequence tags

A consensus map for sugi (Cryptomeria japonica) was constructed by integrating linkage data from two unrelated third-generation pedigrees, one derived from a full-sib cross and the other by self-pollination of F1 individuals. The progeny segregation data of the first pedigree were derived from cleaved amplified polymorphic sequences, microsatellites, restriction fragment length polymorphisms, and single nucleotide polymorphisms. The data of the second pedigree were derived from cleaved amplified polymorphic sequences, isozyme markers, morphological traits, random amplified polymorphic DNA markers, and restriction fragment length polymorphisms. Linkage analyses were done for the first pedigree with JoinMap 3.0, using its parameter set for progeny derived by cross-pollination, and for the second pedigree with the parameter set for progeny derived from selfing of F1 individuals. The 11 chromosomes of C. japonica are represented in the consensus map. A total of 438 markers were assigned to 11 large linkage groups, 1 small linkage group, and 1 nonintegrated linkage group from the second pedigree; their total length was 1372.2 cM. On average, the consensus map showed 1 marker every 3.0 cM. PCR-based codominant DNA markers such as cleaved amplified polymorphic sequences and microsatellite markers were distributed in all linkage groups and occupied about half of mapped loci. These markers are very useful for integration of different linkage maps, QTL mapping, and comparative mapping for evolutional study, especially for species with a large genome size such as conifers.     

CrkL directs ASAP1 to peripheral focal adhesions

Searching for proteins in platelets that can interact with the N-terminal SH3 domain of CrkL (using a combination of a pull-down assay followed by mass spectrometry), we have found that human platelets express an ADP-ribosylation factor (Arf)-specific GTPase-activating protein (GAP), ASAP1, as a CrkL-binding protein. In spreading platelets, most endogenous ASAP1 is localized at peripheral focal adhesions. To determine the physiologic significance of the CrkL-ASAP1 association, we overexpressed CrkL, ASAP1, or both in combination in COS7 cells. Unlike endogenous ASAP1 in platelets, overexpressed ASAP1 showed diffuse cytoplasmic distribution. However, when co-expressed with wild-type CrkL, both endogenous and expressed ASAP1 accumulated at CrkL-induced focal adhesions. An SH2-mutated CrkL, which cannot localize at focal adhesions, failed to recruit ASAP1 into focal adhesions. Thus, CrkL appears to be a lynchpin between ASAP1 and peripheral focal adhesions.