The factor IXa heparin-binding exosite is a cofactor interactive site: mechanism for antithrombin-independent inhibition of intrinsic tenase by heparin

Therapeutic heparin concentrations selectively inhibit the intrinsic tenase complex in an antithrombin-independent manner. To define the molecular target and mechanism for this inhibition, recombinant human factor IXa with alanine substituted for solvent-exposed basic residues (H92, R170, R233, K241) in the protease domain was characterized with regard to enzymatic activity, heparin affinity, and inhibition by low molecular weight heparin (LMWH). These mutations only had modest effects on chromogenic substrate hydrolysis and the kinetics of factor X activation by factor IXa. Likewise, factor IXa H92A and K241A showed factor IXa-factor VIIIa affinity similar to factor IXa wild type (WT). In contrast, factor IXa R170A demonstrated a 4-fold increase in apparent factor IXa-factor VIIIa affinity and dramatically increased coagulant activity relative to factor IXa WT. Factor IXa R233A demonstrated a 2.5-fold decrease in cofactor affinity and reduced ability to stabilize cofactor half-life relative to wild type, suggesting that interaction with the factor VIIIa A2 domain was disrupted. Markedly (R233A) or moderately (H92A, R170A, K241A) reduced binding to immobilized LMWH was observed for the mutant proteases. Solution competition demonstrated that the EC(50) for LMWH was increased less than 2-fold for factor IXa H92A and K241A but over 3.5-fold for factor IXa R170A, indicating that relative heparin affinity was WT > H92A/K241A > R170A > R233A. Kinetic analysis of intrinsic tenase inhibition demonstrated that relative affinity for LMWH was WT > K241A > H92A > R170A > R233A, correlating with heparin affinity. Thus, LMWH inhibits intrinsic tenase by interacting with the heparin-binding exosite in the factor IXa protease domain, which disrupts interaction with the factor VIIIa A2 domain.     

A new endemic species of Helichrysum Gaertn. (Asteraceae-Inuleae) from south Anatolia

A new species of Helichrysum Gaertn., H. orbicularifolium Sümbül, R. S. Göktürk & O. D. Düşen sp. nov. , is described and illustrated. The species is restricted to the south Anatolia Province, Antalya, Turkey, growing in humid calcareous rock crevices and on limestone cliffs. The species is compared with the closely related H. chasmolycicum . © 2003 The Linnean Society of London, Botanical Journal of the Linnean Society , 2003, 141 , 251–254.     

Oligomerized Ced-4 kills budding yeast through a caspase-independent mechanism.

In Caenorhabdtis elegans, Ced-3, Ced-4, and Ced-9 are components of a cell suicide program. Ced-4 facilitates the proteolytic activation of the caspase, Ced-3, while Ced-9 opposes Ced-3/Ced-4 killing. To examine the interactions among these proteins they were expressed in Saccharomyces cerevisiae. Ced-3 and Ced-4 were lethal when expressed alone, revealing an intrinsic Ced-4 killing activity. Coexpression of Ced-9 blocked Ced-3- and Ced-4-induced killing, showing Ced-9 can independently antagonize the action of both proteins. Ced-3- but not Ced-4-toxicity was attenuated by coexpression of the caspase inhibitors, CrmA and p35. Thus, besides its Ced-3- and Ced-9-dependent action in C. elegans, Ced-4 has an additional Ced-9-dependent, Ced-3-independent killing mechanism in yeast. Two-hybrid analysis confirmed that Ced-4 formed heteromers with Ced-9. In addition, Ced-4 formed homomers and mutation of its nucleoside triphosphate binding motif eliminated both homomerization and cell killing. We suggest the caspase-independent lethality of Ced-4 in yeast is mediated by a Ced-4 homomer.     

A circadian rhythm of locomotor activity in newly emerged Ceratophyllus sciurorum

Abstract Circadian rhythm in newly emerged individuals of the Red Squirrel ( Scuirus vulgaris ) flea C.s. sciurorum was studied in a constant environment, using an insect activity monitor. Trials were run over 7 days using two start times (08.00 and 17.00 hours). The results show that, regardless of start time, the fleas display a 24 h activity rhythm. The presence of a rhythm under constant conditions gives a strong indication that C. s.sciurorum has a self-sustaining clock which is started by disturbance and is most likely to be linked to host activity patterns.     

Spatial distribution and prediction of seed production by Eucalyptus microcarpa in a fragmented landscape

Woodlands worldwide have been greatly modified by clearing for agriculture, and their conservation and restoration requires understanding of tree recruitment processes. Seed production is one possible point of recruitment failure, and one that the spatial arrangement of trees may affect. We sampled 118 Eucalyptus microcarpa (Myrtaceae) trees to compare and analyse the determinants of seed production in this dominant tree of modified, fragmented temperate grassy woodlands, which extend over much of southeastern Australia. Fecundity was estimated as the seed crop measured on leaf mass and whole tree bases and was compared between categories of tree configuration. We also modelled fecundity using boosted regression trees, a new and flexible tool. Fecundity on a leaf mass basis was predominantly influenced by environmental factors (topographic ‘wetness’, slope, soil type), rather than by local tree density and configuration. Fewer seed per unit leaf mass were produced on flat and topographically wet sites, reflecting poor tolerance of waterlogging by E. microcarpa. By contrast, whole tree fecundity was little influenced by environmental factors. Local tree density and configuration did influence whole tree fecundity, which was high in solitary and woodland‐spaced trees and reduced under high local density. We found little evidence for reduced fecundity of E. microcarpa in solitary trees. This points to the importance of scattered trees as sources of seed for tree recruitment and for natural regeneration of landscape level tree cover. Considerable uncertainty remains in modelled seed supply, and may be reduced with sampling across multiple years and greater environmental and spatial domains.