Growth dynamics of domains in ternary fluid vesicles

We have studied the growth dynamics of domains on ternary fluid vesicles composed of saturated (dipalmitoylphosphatidylcholine), unsaturated (dioleoylphosphatidylcholine) phosphatidylcholine lipids, and cholesterol using a fluorescence microscopy. The domain coarsening processes are classified into two types: normal coarsening and trapped coarsening. For the normal coarsening, the domains having flat circular shape grow in a diffusion-and-coalescence manner and phenomenologically the mean size grows as a power law of approximately t(2/3). The observed growth law is not described by a two-dimensional diffusion-and-coalescence growth mechanism following the Saffman and Delbrück theory, which may originate from the two-body hydrodynamic interactions between domains. For trapped coarsening, on the other hand, the domain coarsening is suppressed at a certain domain size because the repulsive interdomain interactions obstruct the coalescence of domains. The two-color imaging of the trapped domains reveals that the repulsive interactions are induced by the budding of domains. The model free energy consisting of the bending energy of domains, the bending energy of matrix, the line energy of domain boundary, and the translation energy of domains can describe the observed trapped coarsening. The trapping of domains is caused by the coupling between the phase separation and the membrane elasticity under the incompressibility constraint.     

Development of a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys (<Emphasis Type="Italic">Macaca fascicularis</Emphasis>)

To develop a microsatellite marker set applicable to genome-wide screening of cynomolgus monkeys (Macaca fascicularis), 148 microsatellite markers were selected from the human genome database. The polymorphisms and inheritance of PCR products were determined by screening twenty unrelated monkeys and by analysis of three families, respectively. As a result, 106 primers (72%) gave PCR products of the size expected for humans and rhesus monkeys. Among these products, polymorphism and single-gene inheritance in cynomolgus monkeys was observed for 66 markers (62%). The average number of alleles at the 66 polymorphic loci was 5.86 (range 2–10), and average heterozygosity was 0.63 (range 0.10–0.88). This is the first report of microsatellite markers for cynomolgus monkeys. Chromosomal mapping of these markers is now in progress.     

Carbenoxolone inhibits TRPV4 channel‐initiated oxidative urothelial injury and ameliorates cyclophosphamide‐induced bladder dysfunction

Carbenoxolone (CBX) is a clinically prescribed drug for the treatment of digestive ulcer and inflammation. It is also a widely used pharmacological inhibitor of several channels in basic research. Given that the overactivity of several channels, including those inhibitable by CBX, underlies bladder dysfunction, we tested the potential therapeutic application and mechanism of CBX in the treatment of voiding dysfunction. In a mouse model of cystitis induced by cyclophosphamide (CYP), CBX administration prevented the CYP‐elicited increase in bladder weight, oedema, haemorrhage, and urothelial injury. CBX also greatly improved micturition pattern, as manifested by the apparently decreased micturition frequency and increased micturition volume. Western blot results showed that CBX suppressed CYP‐induced increase in protein carbonyls, COX‐2, and iNOS. Further analysis using cultured urothelial cells revealed that acrolein, the major metabolite of CYP, caused protein oxidation, p38 activation, and urothelial injury. These effects of acrolein were reproduced by TRPV4 agonists and significantly prevented by antioxidant NAC, p38 inhibitor SB203580, TRPV4 antagonist RN‐1734, and CBX. Further studies showed that CBX potently suppressed TRPV4 agonist‐initiated calcium influx and subsequent cell injury. CBX attenuated CYP‐induced cystitis in vivo and reduced acrolein‐induced cell injury in vitro, through mechanisms involving inhibition of TRPV4 channels and attenuation of the channel‐mediated oxidative stress. CBX might be a promising agent for the treatment of bladder dysfunction.     

Vertebrate WRNIP1 and BLM are required for efficient maintenance of genome stability

Bloom syndrome (BS) is rare autosomal recessive disorder associated with chromosomal instability. The gene responsible for BS, BLM, encodes a protein belonging to the RecQ helicase family. Disruptions of the SGS1 gene of Saccharomyces cerevisiae, which encodes the RecQ helicase homologue in the budding yeast, causes accelerated aging, and this phenotype is enhanced by the disruption of MGS1, the budding yeast homologue for WRNIP1. To examine the functional relationship between RecQ and WRNIP1 in vertebrate cells, we generated and characterized wrnip1/blm cells derived from the chicken B-lymphocyte line DT40. wrnip1/blm cells showed an additive elevation of sister chromatid exchange (SCE), suggesting that both genes independently contribute to the suppression of excess SCE formation. The double mutants were more sensitive to DNA damage from camptothecin (CPT), but not to damage from methyl methanesulfonate, than either single mutant. This result suggests that WRNIP1 and BLM function independently to repair DNA or induce tolerance to the lesions induced by CPT.     

Structure-activity relationship studies of imidazo[1,2-c]pyrimidine derivatives as potent and orally effective Syk family kinases inhibitors

Spleen tyrosine kinase (Syk) and zeta-associated protein kinase of 70k Da (ZAP-70) are members of the Syk family and non-receptor-type protein tyrosine kinases, which play crucial roles in B- and T-cell activation. Therefore, a Syk family tyrosine kinases inhibitor would be a useful therapeutic agent for the treatment of various allergic disorders and autoimmune diseases. Previously, we reported that 1,2,4-triazolo[4,3-c]pyrimidine derivative 1 and 1,2,4-triazolo[1,5-c]pyrimidine derivative 2 showed strong inhibitory activities against Syk family kinases. These compounds also exhibited high-level suppression of IL-2 in cellular assays. However, their oral efficacies were poor in a mouse model of IL-2 production. To improve oral effectiveness, we investigated a new series of Syk family kinases inhibitors. We found that imidazo[1,2-c]pyrimidine derivatives potently inhibited the Syk family kinases. Among these agents, compound 9f not only showed strong inhibitory activities against Syk and ZAP-70 kinases in vitro, but its oral administration resulted in the in vivo suppression of both the passive cutaneous anaphylaxis reaction and Concanavalin A-induced IL-2 production in a mouse model.     

Identification and biochemical characterization of the SLC9A7 interactome

Organellar and cytosolic pH homeostasis is central to most cellular processes, including vesicular trafficking, post-translational modification/processing of proteins, and receptor-ligand interactions. SLC9A7 (NHE7) was identified as a unique (Na+, K+)/H+ exchanger that dynamically cycles between the trans-Golgi network (TGN), endosomes and the plasma membrane. Here we have used mass spectrometry to explore the affinity-captured interactome of NHE7, leading to the identification of cytoskeletal proteins, cell adhesion molecules, membrane transporters, and signaling molecules. Among these binding proteins, calcium-calmodulin, but not apo-calmodulin, binds to NHE7 and regulates the organellar transporter activity. Vimentin was co-immunoprecipitated with endogenous NHE7 protein in human breast cancer MDA-MB-231 cells. A sizable population of NHE7 relocalized to focal complexes in migrating cells and showed colocalization with vimentin and actin in focal complexes. Among the NHE7-binding proteins identified, CD44, a cell surface glycoprotein receptor for hyaluronate and other ligands, showed regulated interaction with NHE7. Pretreatment of the cells with phorbol ester facilitated the NHE7-CD44 interaction and the lipid raft association of CD44. When lipid rafts were chemically disrupted, the NHE7-CD44 interaction was markedly reduced. These results suggest potential dual roles of NHE7 in intracellular compartments and subdomains of cell-surface membranes.