Urochordate Histoincompatible Interactions Activate Vertebrate-Like Coagulation System Components

The colonial ascidian Botryllus schlosseri expresses a unique allorecognition system. When two histoincompatible Botryllus colonies come into direct contact, they develop an inflammatory-like rejection response. A surprising high number of vertebrates' coagulation genes and coagulation-related domains were disclosed in a cDNA library of differentially expressed sequence tags (ESTs), prepared for this allorejection process. Serine proteases, especially from the trypsin family, were highly represented among Botryllus library ortholgues and its “molecular function” gene ontology analysis. These, together with the built-up clot-like lesions in the interaction area, led us to further test whether a vertebrate-like clotting system participates in Botryllus innate immunity. Three morphologically distinct clot types (points of rejection; POR) were followed. We demonstrated the specific expression of nine coagulation orthologue transcripts in Botryllus rejection processes and effects of the anti-coagulant heparin on POR formation and heartbeats. In situ hybridization of fibrinogen and von Willebrand factor orthologues elucidated enhanced expression patterns specific to histoincompatible reactions as well as common expressions not augmented by innate immunity. Immunohistochemistry for fibrinogen revealed, in naïve and immune challenged colonies alike, specific antibody binding to a small population of Botryllus compartment cells. Altogether, molecular, physiological and morphological outcomes suggest the involvement of vertebrates-like coagulation elements in urochordate immunity, not assigned with vasculature injury

2-Mercapto-Quinazolinones as Inhibitors of Type II NADH Dehydrogenase and Mycobacterium tuberculosis:Structure-Activity Relationships, Mechanism of Action and Absorption, Distribution, Metabolism, and Excretion Characterization

<i>Mycobacterium tuberculosis</i> (<i>MTb</i>) possesses two nonproton pumping type II NADH dehydrogenase (NDH-2) enzymes which are predicted to be jointly essential for respiratory metabolism. Furthermore, the structure of a closely related bacterial NDH-2 has been reported recently, allowing for the structure-based design of small-molecule inhibitors. Herein, we disclose <i>MTb</i> whole-cell structure–activity relationships (SARs) for a series of 2-mercapto-quinazolinones which target the <i>ndh</i> encoded NDH-2 with nanomolar potencies. The compounds were inactivated by glutathione-dependent adduct formation as well as quinazolinone oxidation in microsomes. Pharmacokinetic studies demonstrated modest bioavailability and compound exposures. Resistance to the compounds in <i>MTb</i> was conferred by promoter mutations in the alternative nonessential NDH-2 encoded by <i>ndhA</i> in <i>MTb</i>. Bioenergetic analyses revealed a decrease in oxygen consumption rates in response to inhibitor in cells in which membrane potential was uncoupled from ATP production, while inverted membrane vesicles showed mercapto-quinazolinone-dependent inhibition of ATP production when NADH was the electron donor to the respiratory chain. Enzyme kinetic studies further demonstrated noncompetitive inhibition, suggesting binding of this scaffold to an allosteric site. In summary, while the initial <i>MTb</i> SAR showed limited improvement in potency, these results, combined with structural information on the bacterial protein, will aid in the future discovery of new and improved NDH-2 inhibitors

Alternative Splicing Events Are a Late Feature of Pathology in a Mouse Model of Spinal Muscular Atrophy

Spinal muscular atrophy is a severe motor neuron disease caused by inactivating mutations in the SMN1 gene leading to reduced levels of full-length functional SMN protein. SMN is a critical mediator of spliceosomal protein assembly, and complete loss or drastic reduction in protein leads to loss of cell viability. However, the reason for selective motor neuron degeneration when SMN is reduced to levels which are tolerated by all other cell types is not currently understood. Widespread splicing abnormalities have recently been reported at end-stage in a mouse model of SMA, leading to the proposition that disruption of efficient splicing is the primary mechanism of motor neuron death. However, it remains unclear whether splicing abnormalities are present during early stages of the disease, which would be a requirement for a direct role in disease pathogenesis. We performed exon-array analysis of RNA from SMN deficient mouse spinal cord at 3 time points, pre-symptomatic (P1), early symptomatic (P7), and late-symptomatic (P13). Compared to littermate control mice, SMA mice showed a time-dependent increase in the number of exons showing differential expression, with minimal differences between genotypes at P1 and P7, but substantial variation in late-symptomatic (P13) mice. Gene ontology analysis revealed differences in pathways associated with neuronal development as well as cellular injury. Validation of selected targets by RT–PCR confirmed the array findings and was in keeping with a shift between physiologically occurring mRNA isoforms. We conclude that the majority of splicing changes occur late in SMA and may represent a secondary effect of cell injury, though we cannot rule out significant early changes in a small number of transcripts crucial to motor neuron survival

Reconstruction of the 869 Jogan Earthquake, the predecessor of the 2011 Tohoku earthquake, by geological evidence combined with tsunami simulation

International Symposium on Backwards Problem in Geotechnical Engineering and Monitoring of Geo-Construction, Green Hall, Kensetsu-Koryu-kan, 2011/07/14-15We reconstructed the tsunami inundation area of the 869 Jogan earthquake by geological evidence such as tsunami deposit, and inferred fault source model by tsunami simulation. From analysis of nearly 400 geological core samples obtained from the Sendai and Ishinomaki Plains, it is inferred that the 869 Jogan tsunami inundated to 3-4 km further inland from shoreline and was generated from subduction megathrust fault which has 200 km long and 100 km width off Miyagi and Fukushima. Magnitude and source of the Jogan tsunami is very similar to the 2011 Tohoku earthquake

High-performance liquid chromatography accelerator mass spectrometry: Correcting for losses during analysis by internal standardization

A method was developed to account for analytical losses of 14C-analyte when determining the concentration in biological samples using chromatographic separation and analysis by accelerator mass spectrometry. From the equations of J. Vogel and A.H. Love (in: A.L. Burlingame (Ed.), Methods in Enzymology, Academic Press, New York, 2005), new equations were derived to describe the isotopic dilution of a chromatographically isolated 14C-analyte. The analytical recovery for each sample was determined by the use of the UV response for nonlabeled analyte, as an internal standard against a standard curve. The slope of the curve was substituted into the equations to provide a method of accurately determining the analyte concentration. © 2008 Elsevier Inc. All rights reserved

Constraining sediment provenance for tsunami deposits using distributions of grain size and foraminifera from the Kujukuri coastline and shelf, Japan

Tsunami deposits preserved in the geological record provide a more comprehensive understanding of their patterns of frequency and intensity over longer timescales; but recognizing tsunami deposits can prove challenging due to post-depositional changes, lack of contrast between the deposits and surrounding sedimentary layers, and differentiating between tsunami and storm deposition. Modern baseline studies address these challenges by providing insight into modern spatial distributions that can be compared with palaeotsunami deposits. This study documents the spatial fingerprint of grain size and foraminifera from Hasunuma Beach and the Kujukuri shelf to provide a basis from which tsunami deposits can be interpreted. At Hasunuma Beach, approximately 50 km east of Tokyo, the spatial distribution of three common proxies (foraminiferal taxonomy, foraminiferal taphonomy and sediment grain size) for tsunami identification were mapped and clustered using Partitioning Around Medoids cluster analysis. Partitioning Around Medoids cluster analysis objectively discriminated two coastal zones corresponding to onshore and offshore sample locations. Results show that onshore samples are characterized by coarser grain sizes (medium to coarse sand) and higher abundances of Pararotalia nipponica (27 to 63%) than offshore samples, which are characterized by finer grain sizes (fine to medium sand), lower abundances of Pararotalia nipponica (2 to 19%) and Ammonia parkinsoniana (0 to 10%), higher abundances of planktonics (15 to 58%) and species with fragile tests including Uvigerinella glabra. When compared to grain-size and foraminiferal taxonomy, foraminiferal taphonomy; i.e. surface condition of foraminifera, a proxy not commonly used to identify tsunami deposits, was most effective in discriminating modern coastal zones (identified supratidal, intertidal and offshore environments) and determining sediment provenance for tsunami deposits at Kujukuri. This modern baseline study assists the interpretation of tsunami deposits in the geological record because it provides a basis for sediment provenance to be determined.NRF (Natl Research Foundation, S’pore)MOE (Min. of Education, S’pore)Published versio