A 5000-YEAR FOSSIL RECORD OF LARVAL SHELL MORPHOLOGY OF SUBMARINE CAVE MICROSHELLS

A 5000-year fossil series of minute submarine cave bivalves was studied using morphometric and evolutionary analyses. The obtained results indicate that the shapes of larval shells of studied species were labile, whereas the size of the larval shell was stable in each species studied. This result is different than that previously reported in most other studies in which size change is more common than shape change. This unique evolutionary pattern of these bivalves might be attributed to their refugial lifestyle.     

The involvement of immunoproteasomes in induction of MHC class I-restricted immunity targeting Toxoplasma SAG1

The ubiquitin-proteasome system (UPS) plays an indispensable role in inducing MHC class I-restricted CD8+ T cells and was exploited in the development of a DNA vaccine against the intracellular protozoan Toxoplasma gondii by constructing a chimeric DNA encoding a fusion protein between murine ubiquitin and the toxoplasma antigen SAG1. The SAG1 peptide was promptly degraded in antigen-presenting cells (APCs) transfected with the chimeric DNA. Degradation, however, was hampered by incubating the APCs with the proteasome inhibitor epoxomicin. Mice vaccinated with the DNA acquired potent protective immunity mediated by MHC class I-restricted CD8+ T cells against infection by the highly virulent Toxoplasma. The accelerated degradation and induction of immunity were dependent on the UPS since mice lacking an immuno-subunit of 20S proteasome, LMP7, lost these functions, although they were independent of the proteasome regulator PA28alpha/beta complex.     

Aquaporin-2 trafficking is regulated by PDZ-domain containing protein SPA-1

Targeted positioning of water channel aquaporin-2 (AQP2) strictly regulates body water homeostasis. Trafficking of AQP2 to the apical membrane is critical to the reabsorption of water in renal collecting ducts. Controlled apical positioning of AQP2 suggests the existence of proteins that interact with AQP2. A biochemical search for AQP2-interacting proteins led to the identification of PDZ-domain containing protein, signal-induced proliferation-associated gene-1 (SPA-1) which is a GTPase-activating protein (GAP) for Rap1. The distribution of SPA-1 coincided with that of AQP2 in renal collecting ducts. The site of colocalization was concomitantly relocated by hydration status. AQP2 trafficking to the apical membrane was inhibited by the SPA-1 mutant lacking Rap1GAP activity and by the constitutively active mutant of Rap1. AQP2 trafficking was impaired in SPA-1-deficient mice. Our results show that SPA-1 directly binds to AQP2 and regulates at least in part AQP2 trafficking.     

Interaction of myosin.ADP.fluorometal complexes with fluorescent probes and direct observation using quick-freeze deep-etch electron microscopy

Myosin forms stable ternary complexes with ADP and phosphate analogues of fluorometals that mimic different ATPase reaction intermediates corresponding to each step of the cross-bridge cycle. In the present study, we monitored the formation of ternary complexes of myosin.ADP.fluorometal using the fluorescence probe prodan. It has been reported that the fluorescence changes of the probe reflect the formation of intermediates in the ATPase reaction [Hiratsuka (1998) Biochemistry 37, 7167-7176]. Prodan bound to skeletal muscle heavy-mero-myosin (HMM).ADP.fluorometal, with each complex showing different fluorescence spectra. Prodan bound to the HMM.ADP.BeFn complex showed a slightly smaller red-shift than other complexes in the presence of ATP, suggesting a difference in the localized conformation or a difference in the population of BeFn species of global shape. We also examined directly the global structure of the HMM.ADP.fluorometal complexes using quick-freeze deep-etch replica electron microscopy. The HMM heads in the absence of nucleotides were mostly straight and elongated. In contrast, the HMM heads of ternary complexes showed sharply kinked or rounded configurations as seen in the presence of ATP. This is the first report of the direct observation of myosin-ADP-fluorometal ternary complexes, and the results suggest that these complexes indeed mimic the shape of the myosin head during ATP hydrolysis.     

Global methylation screening in the Arabidopsis thaliana and Mus musculus genome: applications of virtual image restriction landmark genomic scanning (Vi-RLGS)

Understanding the role of ‘epigenetic’ changes such as DNA methylation and chromatin remodeling has now become critical in understanding many biological processes. In order to delineate the global methylation pattern in a given genomic DNA, computer software has been developed to create a virtual image of restriction landmark genomic scanning (Vi-RLGS). When using a methylation- sensitive enzyme such as NotI as the restriction landmark, the comparison between real and in silico RLGS profiles of the genome provides a methylation map of genomic NotI sites. A methylation map of the Arabidopsis genome was created that could be confirmed by a methylation-sensitive PCR assay. The method has also been applied to the mouse genome. Although a complete methylation map has not been completed, a region of methylation difference between two tissues has been tested and confirmed by bisulfite sequencing. Vi-RLGS in conjunction with real RLGS will make it possible to develop a more complete map of genomic sites that are methylated or demethylated as a consequence of normal or abnormal development.