Probing conformational changes of prestin with thiol-reactive optical switches

Thiol-reactive optical switch probes were used to examine conformational changes of prestin-based membrane motor. Because this motor is based on mechanoelectric coupling similar to piezoelectricity, the motile activity can be monitored by charge movements across the plasma membrane, which appears as nonlinear capacitance. When the plasma membrane is conjugated with the probes, optically induced spiro-merocyanine transition positively shifted nonlinear capacitance of outer hair cells and prestin-transfected cells by ∼10 mV. These shifts were reversible and were eliminated by pretreatment with iodoacetamide. However, they were little affected by pretreatment with biotin maleimide, which cannot reach the cytoplasmic surface. Our results showed that merocyanine states, with a larger dipole moment, interact with the motor's extended conformation stronger than with the compact conformation by 1.6 × 10⁻²¹ J/molecule. The interaction sites are near the cytoplasmic side of the motor protein.     

Tissue stretch decreases soluble TGF-beta1 and type-1 procollagen in mouse subcutaneous connective tissue: evidence from ex vivo and in vivo models

Vascular smooth muscle cells (SMC) may be directly exposed to blood flow after an endothelial-denuding injury. It is not known whether direct exposure of SMC to shear stress reduces SMC turnover and contributes to the low rate of restenosis after most vascular interventions. This study examines if laminar shear stress inhibits SMC proliferation or stimulates apoptosis. Bovine aortic SMC were exposed to arterial magnitudes of laminar shear stress (11 dynes/cm(2)) for up to 24 h and compared to control SMC (0 dynes/cm(2)). SMC density was assessed by cell counting, DNA synthesis by (3)[H]-thymidine incorporation, and apoptosis by TUNEL staining. Akt, caspase, bax, and bcl-2 phosphorylation were assessed by Western blotting; caspase activity was also measured with an in vitro assay. Analysis of variance was used to compare groups. SMC exposed to laminar shear stress had a 38% decrease in cell number (n = 4, P = 0.03), 54% reduction in (3)[H]-thymidine incorporation (n = 3, P = 0.003), and 15-fold increase in TUNEL staining (n = 4, P < 0.0001). Akt phosphorylation was reduced by 67% (n = 3, P < 0.0001), whereas bax/bcl-2 phosphorylation was increased by 1.8-fold (n = 3, P = 0.01). Caspase-3 activity was increased threefold (n = 5, P = 0.03). Pretreatment of cells with ZVAD-fmk or wortmannin resulted in 42% increased cell retention (n = 3, P < 0.01) and a fourfold increase in apoptosis (n = 3, P < 0.04), respectively. Cells transduced with constitutively-active Akt had twofold decreased apoptosis (n = 3, P < 0.002). SMC exposed to laminar shear stress have decreased proliferation and increased apoptosis, mediated by the Akt pathway. These results suggest that augmentation of SMC apoptosis may be an alternative strategy to inhibit restenosis after vascular injury.     

Litter decomposition and nitrogen mineralization of soils in subtropical plantation forests of southern China,with special attention to comparisons between legumes and non-legumes

Litter decomposition and nitrogen mineralization were investigated in subtropical plantation forests in southern China. The CO₂ –C release from incubated litter and the forest floor of Acacia mangium, Acacia auriculaeformis, Eucalyptus citriodora, Pinus elliotii and Schima superba stands were used to estimate relative rates of litter decomposition. Decomposition was not positively correlated with litter nitrogen. E. citridora litter decomposed most rapidly and A. mangium litter most slowly, both with and without the addition of exotic nitrogen. Aerobic incubation and intact soil core incubation at 30 °C over a period of 30 days were used to assess nitrogen mineralization of six forest soils. Although there were differences in results obtained using the two methods, patterns between legume and non-legume species were the same regardless of method. All soils had pH values below 4.5, but this did not prevent nitrification. The dominant form of mineral nitrogen was nitrate for legume species and ammonium for non-legume species. The nitrogen mineralization potential was highest for soils in which legumes were growing.     

Cross-continent comparisons reveal differing environmental drivers of growth of the coral reef fish, <Emphasis Type="Italic">Lutjanus bohar</Emphasis>

Biochronologies provide important insights into the growth responses of fishes to past variability in physical and biological environments and, in so doing, allow modelling of likely responses to climate change in the future. We examined spatial variability in the key drivers of inter-annual growth patterns of a widespread, tropical snapper, Lutjanus bohar, at similar tropical latitudes on the north-western and north-eastern coasts of the continent of Australia. For this study, we developed biochronologies from otoliths that provided proxies of somatic growth and these were analysed using mixed-effects models to examine the historical drivers of growth. Our analyses demonstrated that growth patterns of fish were driven by different climatic and biological factors in each region, including Pacific Ocean climate indices, regional sea level and the size structure of the fish community. Our results showed that the local oceanographic and biological context of reef systems strongly influenced the growth of L. bohar and that a single age-related growth trend cannot be assumed for separate populations of this species that are likely to experience different environmental conditions. Generalised predictions about the growth response of fishes to climate change will thus require adequate characterisation of the spatial variability in growth determinants likely to be found throughout the range of species that have cosmopolitan distributions.     

Synthesis and pharmacokinetic and pharmacodynamic evaluation of the forodesine HCl analog BCX-3040

Forodesine HCl is a potent inhibitor of the enzyme purine nucleoside phosphorylase (PNP) and is currently in clinical trials for the treatment of leukemia and lymphoma. Animal models indicated that forodesine HCl would have low oral bioavailability in humans and it was initially developed as an intravenous formulation. We were interested in identifying analogs of forodesine HCl with improved oral bioavailability. The 2'-deoxy analog (BCX-3040) was synthesized and its pharmacokinetic and pharmacodynamic properties compared with forodesine HCl.     

CORKSCREW1 defines a novel mechanism of domain specification in the maize shoot

In higher plants, determinate leaf primordia arise in regular patterns on the flanks of the indeterminate shoot apical meristem (SAM). The acquisition of leaf form is then a gradual process, involving the specification and growth of distinct domains within the three leaf axes. The recessive corkscrew1 (cks1) mutation of maize (Zea mays) disrupts both leaf initiation patterns in the SAM and domain specification within the mediolateral and proximodistal leaf axes. Specifically, cks1 mutant leaves exhibit multiple midribs and leaf sheath tissue differentiates in the blade domain. Such perturbations are a common feature of maize mutants that ectopically accumulate KNOTTED1-like homeobox (KNOX) proteins in leaf tissue. Consistent with this observation, at least two knox genes are ectopically expressed in cks1 mutant leaves. However, ectopic KNOX proteins cannot be detected. We therefore propose that CKS1 primarily functions within the SAM to establish boundaries between meristematic and leaf zones. Loss of gene function disrupts boundary formation, impacts phyllotactic patterns, and leads to aspects of indeterminate growth within leaf primordia. Because these perturbations arise independently of ectopic KNOX activity, the cks1 mutation defines a novel component of the developmental machinery that facilitates leaf-versus-shoot development in maize.     

Generation and characterization of novel stromal specific antibodies

Rheumatoid synovial fibroblasts were used as an immunogen to produce monoclonal antibodies selected for their reactivity with stromal cell antigens. Mice were immunised with low passage whole cell preparations and the subsequent hybridomas were screened by immunohistochemistry on rheumatoid synovium and tonsil sections. The aim was to identify those antibodies that recognised antigens that were restricted to stromal cells and were not expressed on CD45 positive leucocytes. A significant number of antibodies detected antigen that identified endothelial cells. These antibodies were further characterised to determine whether the vessels identified by these antibodies were vascular or lymphatic. From five fusions clones were identified with predominant reactivity with： 1） fibroblasts and endothelial cells; or 2） broad stromal elements （fibroblast, endothelium, epithelium, follicular dendritic cells）. A fibroblast-specific antibody that did not also identify vessels was not generated. Examples of each reactivity pattern are discussed.