A Disulfide Stabilized beta-Sandwich Defines the Structure of a New Cysteine Framework M-Superfamily Conotoxin

The structure of a new cysteine framework (-C-CC-C-C-C) ``M''-superfamily conotoxin, Mo3964, shows it to have a beta-sandwich structure that is stabilized by inter-sheet cross disulfide bonds. Mo3964 decreases outward K+ currents in rat dorsal root ganglion neurons and increases the reversal potential of the Na(V)1.2 channels. The structure of Mo3964 (PDB ID: 2MW7) is constructed from the disulfide connectivity pattern, i.e., 1-3, 2-5, and 4-6, that is hitherto undescribed for the ``M''-superfamily conotoxins. The tertiary structural fold has not been described for any of the known conus peptides. NOE (549), dihedral angle (84), and hydrogen bond (28) restraints, obtained by measurement of (h3)J(NC') scalar couplings, were used as input for structure calculation. The ensemble of structures showed a backbone root mean square deviation of 0.68 +/- 0.18 angstrom, with 87% and 13% of the backbone dihedral (phi, psi) angles lying in the most favored and additional allowed regions of the Ramachandran map. The conotoxin Mo3964 represents a new bioactive peptide fold that is stabilized by disulfide bonds and adds to the existing repertoire of scaffolds that can be used to design stable bioactive peptide molecules.

Numerical exploration of new features on three-dimensional transition of a cylinder wake

The three-dimensional transition of the wake flow behind a circular cylinder is studied in detail by direct numerical simulations using 3D incompressible N-S equations for Reynolds number ranging from 200 to 300. New features and vortex dynamics of the 3D transition of the wake are found and investigated. At Re = 200, the flow pattern is characterized by mode A instability. However, the spanwise characteristic length of the cylinder determines the transition features. Particularly for the specific spanwise characteristic length linear stable mode may dominate the wake in place of mode A and determine the spanwise phase difference of the primary vortices shedding. At Re = 250 and 300 it is found that the streamwise vortices evolve into a new type of mode - "dual vortex pair mode" downstream. The streamwise vortex structures switch among mode A, mode B and dual vortex pair mode from near wake to downstream wake. At Re = 250, an independent low frequency f(m) in addition to the vortex shedding frequency f(s) is identified. Frequency coupling between f(m) and f(s) occurs. These result in the irregularity of the temporal signals and become a key feature in the transition of the wake. Based on the formation analysis of the streamwise vorticity in the vicinity of cylinder, it is suggested that mode A is caused by the emergence of the spanwise velocity due to three dimensionality of the incoming flow past the cylinder. Energy distribution on various wave numbers and the frequency variation in the wake are also described.

Exploration of new drug delivery pathways: I. Mechanism of folate uptake in cultured human cells. II. Role of nuclear localization signal peptides in the delivery of oligonucleotide into reconstituted nuclei

The roles of the folate receptor and an anion carrier in the uptake of 5- methyltetrahydrofolate (5-MeH_4folate) were studied in cultured human (KB) cells using radioactive 5-MeH_4folate. Binding of the 5-MeH_4folate was inhibited by folic acid, but not by probenecid, an anion carrier inhibitor. The internalization of 5-MeH_4folate was inhibited by low temperature, folic acid, probenecid and methotrexate. Prolonged incubation of cells in the presence of high concentrations of probenecid appeared to inhibit endocytosis of folatereceptors as well as the anion carrier. The V_(max) and K_M values for the carrier were 8.65 ± 0.55 pmol/min/mg cell protein and 3.74 ± 0.54µM, respectively. The transport of 5-MeH4folate was competitively inhibited by folic acid, probenecid and methotrexate. The carrier dissociation constants for folic acid, probenecid and methotreate were 641 µM, 2.23 mM and 13.8 µM, respectively. Kinetic analysis suggests that 5-MeH_4folate at physiological concentration is transported through an anion carrier with the characteristics of the reduced-folate carrier after 5-MeH_4folate is endocytosed by folate receptors in KB cells. Our data with KB cells suggest that folate receptors and probenecid-sensitive carriers work in tandem to transport 5-MeH_4folate to the cytoplasm of cells, based upon the assumption that 1 mM probenecid does not interfere with the acidification of the vesicle where the folate receptors are endocytosed.

Oligodeoxynucleotides designed to hybridize to specific mRNA sequences (antisense oligonucleotides) or double stranded DNA sequences have been used to inhibit the synthesis of a number of cellular and viral proteins (Crooke, S. T. (1993) FASEB J. 7, 533-539; Carter, G. and Lemoine, N. R. (1993) Br. J. Cacer 67, 869-876; Stein, C. A. and cohen, J. S. (1988) Cancer Res. 48, 2659-2668). However, the distribution of the delivered oligonucleotides in the cell, i.e., in the cytoplasm or in the nucleus has not been clearly defined. We studied the kinetics of oligonucleotide transport into the cell nucleus using reconstituted cell nuclei as a model system. We present evidences here that oligonucleotides can freely diffuse into reconstituted nuclei. Our results are consistent with the reports by Leonetti et al. (Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 2702-2706, April 1991), which were published while we were carrying this research independently. We also investigated whether a synthetic nuclear localization signal (NLS) peptide of SV40 T antigen could be used for the nuclear targeting of oligonucleotides. We synthesized a nuclear localization signal peptide-conjugated oligonucleotide to see if a nuclear localization signal peptide can enhance the uptake of oligonucleotides into reconstituted nuclei of Xenopus. Uptake of the NLS peptide-conjugated oligonucleotide was comparable to the control oligonucleotide at similar concentrations, suggesting that the NLS signal peptide does not significantly enhance the nuclear accumulation of oligonucleotides. This result is probably due to the small size of the oligonucleotide.

Development of a new modelling framework to estimate the C footprint from Basque dairy farms

19 p.

New climate scenario framework implementation in the GCAM integrated assessment model

48 p.

The new exploration for proton-implanted silicon: the conversion of a surface-region-purification-induced p-n junction into a p-i-n electrical structure approaching silicon on insulator

A surface-region-purification-induced p-n junction, a puzzle discovered at Brookhaven National Laboratory, in a silicon-on-defect-layer (SODL) material has been explored by carrying out various annealing conditions and subsequent measurements on electrical properties. The origin of the pn junction has been experimentally investigated. Furthermore, the p-n junction has been transformed into a p-i-n electrical structure by adding a high temperature annealing process to the previously used SODL procedure, making the SODL material approach silicon on insulator (SOI). The control of the initial oxygen amount in the silicon material is suggested to be critical for the experimental results.