Nanomechanics combined with HDX reveals allosteric drug binding sites of CFTR NBD1.

Cystic fibrosis (CF) is a frequent genetic disease in Caucasians that is caused by the deletion of F508 (DF508) in the nucleotide binding domain 1 (NBD1) of the CF transmembrane conductance regulator (CFTR). The DF508 compromises the folding energetics of the NBD1, as well as the folding of three other CFTR domains. Combination of FDA approved corrector molecules can efficiently but incompletely rescue the DF508-CFTR folding and stability defect. Thus, new pharmacophores that would reinstate the wild-type-like conformational stability of the DF508-NBD1 would be highly beneficial. The most prominent molecule, 5-bromoindole-3-acetic acid (BIA) that can thermally stabilize the NBD1 has low potency and efficacy. To gain insights into the NBD1 (un)folding dynamics and BIA binding site localization, we combined molecular dynamics (MD) simulations, atomic force spectroscopy (AFM) and hydrogen- deuterium exchange (HDX) experiments. We found that the NBD1 a-subdomain with three adjacent strands from the b-subdomain plays an important role in early folding steps, when crucial non-native interactions are formed via residue F508. Our AFM and HDX experiments showed that BIA associates with this a-core region and increases the resistance of the DF508-NBD1 against mechanical unfolding, a phenomenon that could be exploited in future developments of folding correctors

Reconstruction of the eruptive history of Usu volcano, Hokkaido, Japan, inferred from petrological correlation between tephras and dome lavas

Usu volcano has erupted nine times since 1663. Most eruptive events started with an explosive eruption, which was followed by the formation of lava domes. However, the ages of several summit lava domes and craters remain uncertain. The petrological features of tephra deposits erupted from 1663 to 1853 are known to change systematically. In this study, we correlated lavas with tephras under the assumption that lava and tephra samples from the same event would have similar petrological features. Although the initial explosive eruption in 1663 was not accompanied by lava effusion, lava dome or cryptodome formation was associated with subsequent explosive eruptions. We inferred the location of the vent associated with each event from the location of the associated lava dome and the pyroclastic flow deposit distribution and found that the position of the active vent within the summit caldera differed for each eruption from the late 17th through the 19th century. Moreover, we identified a previously unrecognized lava dome produced by a late 17th century eruption; this dome was largely destroyed by an explosive eruption in 1822 and was replaced by a new lava dome during a later stage of the 1822 event at nearly the same place as the destroyed dome. This new interpretation of the sequence of events is consistent with historical sketches and documents. Our results show that petrological correlation, together with geological evidence, is useful not only for reconstructing volcanic eruption sequences but also for gaining insight into future potential disasters

The Effects of Voluntary, Involuntary, and Forced Exercises on Brain-Derived Neurotrophic Factor and Motor Function Recovery: A Rat Brain Ischemia Model

Author name used in this publication: Xiao-Xiang ZhengAuthor name used in this publication: Kai-Yu Tong2010-2011 > Academic research: refereed > Publication in refereed journalpublished_fina

Stepwise Catalytic Mechanism via Short-Lived Intermediate Inferred from Combined QM/MM MERP and PES Calculations on Retaining Glycosyltransferase ppGalNAcT2

The glycosylation of cell surface proteins plays a crucial role in a multitude of biological processes, such as cell adhesion and recognition. To understand the process of protein glycosylation, the reaction mechanisms of the participating enzymes need to be known. However, the reaction mechanism of retaining glycosyltransferases has not yet been sufficiently explained. Here we investigated the catalytic mechanism of human isoform 2 of the retaining glycosyltransferase polypeptide UDP-GalNAc transferase by coupling two different QM/MM-based approaches, namely a potential energy surface scan in two distance difference dimensions and a minimum energy reaction path optimisation using the Nudged Elastic Band method. Potential energy scan studies often suffer from inadequate sampling of reactive processes due to a predefined scan coordinate system. At the same time, path optimisation methods enable the sampling of a virtually unlimited number of dimensions, but their results cannot be unambiguously interpreted without knowledge of the potential energy surface. By combining these methods, we have been able to eliminate the most significant sources of potential errors inherent to each of these approaches. The structural model is based on the crystal structure of human isoform 2. In the QM/MM method, the QM region consists of 275 atoms, the remaining 5776 atoms were in the MM region. We found that ppGalNAcT2 catalyzes a same-face nucleophilic substitution with internal return (SNi). The optimized transition state for the reaction is 13.8 kcal/mol higher in energy than the reactant while the energy of the product complex is 6.7 kcal/mol lower. During the process of nucleophilic attack, a proton is synchronously transferred to the leaving phosphate. The presence of a short-lived metastable oxocarbenium intermediate is likely, as indicated by the reaction energy profiles obtained using high-level density functionals

CHARACTERISTICS OF VARIOUS CELL TYPES IN PRIMARY INSECT CELL CULTURE FROM LARVAL OVARIES OF SILKWORM (BOMBYX MORI L.)

###EgeUn###As insect cell lines can be used in baculovirus expression systems for producing recombinant proteins, there is a need to establish new cell lines. Insect cell lines are thought to be useful in production of humanized recombinant proteins. Hence, we aimed to establish a culture from larval ovaries of Bombyx mori to investigate the cell characteristics and growth properties. Two sets of primary cell cultures were established from ovaries of fifth instar larvae. Characterization of cell line was carried out by comparing the PCR products of cell line and silkworms from COI (cytochrome c oxidase subunit I) specific primers. In the first set of cultures, the culture population was found to be heterogeneous and cell sizes were variable. Fibroblastic, epithelial-like, spindle-like, and round cells were observed after 7th day. Interestingly, contractions were observed in muscle-like cells at the end of first month, and stayed stable until the fourth month. At the beginning of the fourth month, because cell death increased significantly, cell concentrations decreased. In the second set of cultures, it was determined that doubling time was about 72 h with high viability and confluency of cells were higher in early passages. PCR which was conducted for characterization of cells resulted in a 710 bp product in each cell line and silkworms. In conclusion, growth rate detected in this study was observed to be compatible with literature. Also, it was thought that if primary culture is supported with growth stimulators, growth rate can be more accelerated and this primary culture can be used more efficiently in recombinant protein production studies

Primary insect cell culture from total embryo and embryonic brain tissue of Periplaneta americana: A preliminary study

The aim of this preliminary study was to establish a primary insect cell culture from total embryos and embryonic brain tissues of Periplaneta americana, collected from Izmir, Turkey. Cells were cultured at 29°C in Grace's insect medium for one month. In the embryonic brain tissue culture, single cells and cell clumps containing spherical and ovoid as well as dividing cells were observed. Single bipolar neurons were detected after 4 days in culture. Network systems comprised of bipolar neurons were observed on the 5th day of incubation. In addition, presumably glia cells were observed in the embryonic brain culture. In the total embryo culture, the cell population exhibited variable morphologies, including spherical, spindle-like, polygonal and giant cells after nearly 20 days; the culture covered almost half of the Petri dish area within 30 days. This preliminary study associated with Periplaneta americana primary cell culture is the first of its kind in Turkey. These results should contribute to the development of new insect cell lines that are indigenous to Turkey