Kinetic and Structural Analysis of the Mg2+ -binding Site of the Guanine Nucleotide-binding Protein p21 H-ras.

The coordination and binding of the Mg2+ ion in the nucleotide−binding site of p21 have been investigated using site−directed mutagenesis, kinetic methods, and phosphorous NMR. Mg2+ in the p21.nucleotide.Mg2+ complex appears to be in fast equilibrium with the solvent. The dissociation constant between Mg2+ and the p21.GDP complex was determined to be 2.8 microM. It decreases 30− or 16−fold on substituting Ser−17 or Asp−57 with alanine, respectively, whereas the T35A mutation has no effect. All three mutations influence the dissociation constants and the association and dissociation rate constants of the interaction between guanine nucleotides and p21, but to a different degree. We conclude that Thr−35 is only complexed to Mg2+ in the GTP conformation and both Asp−57 and Ser−17 appear to be critical for both GDP and GTP binding. 31P NMR spectra of the GDP and Gpp(NH)p (guanosine−5'−(beta,gamma−imido)triphosphate) complexes of mutated p21 show a remarkable perturbation of the guanine nucleotide− binding site compared to wild−type protein. The mutant proteins show reduced GTPase rates, which are not stimulated by the GTPase−activating protein GAP. p21(S17A) has been reported to function just as p21(S17N) as a dominant negative inhibitor of normal p21. We find that it inhibits oncogenic p21−induced survival of primary neuron

Coupled-channels analysis of the 16^{{\bf 16}}O+208^{{\bf 208}}Pb fusion barrier distribution

Analyses using simplified coupled-channels models have been unable to describe the shape of the previously measured fusion barrier distribution for the doubly magic $^{16}$O+$^{208}$Pb system. This problem was investigated by re-measuring the fission excitation function for $^{16}$O+$^{208}$Pb with improved accuracy and performing more exact coupled-channels calculations, avoiding the constant-coupling and first-order coupling approximations often used in simplified analyses. Couplings to the single- and 2-phonon states of $^{208}$Pb, correctly taking into account the excitation energy and the phonon character of these states, particle transfers, and the effects of varying the diffuseness of the nuclear potential, were all explored. However, in contrast to other recent analyses of precise fusion data, no satisfactory simultaneous description of the shape of the experimental barrier distribution and the fusion cross-sections for $^{16}$O+$^{208}$Pb was obtained.Comment: RevTex, 29 pages, 7 postscript figures, to appear in PR

Parity nonconservation in heavy atoms: The radiative correction enhanced by the strong electric field of the nucleus

Parity nonconservation due to the nuclear weak charge is considered. We demonstrate that the radiative corrections to this effect due to the vacuum fluctuations of the characteristic size larger than the nuclear radius $r_0$ and smaller than the electron Compton wave-length $\lambda_C$ are enhanced because of the strong electric field of the nucleus. The parameter that allows one to classify the corrections is the large logarithm $\ln(\lambda_C/r_0)$. The vacuum polarization contribution is enhanced by the second power of the logarithm. Although the self-energy and the vertex corrections do not vanish, they contain only the first power of the logarithm. The value of the radiative correction is 0.4% for Cs and 0.9% for Tl, Pb, and Bi. We discuss also how the correction affects the interpretation of the experimental data on parity nonconservation in atoms.Comment: 4 pages, 3 figures, RevTe

Mapping the molecular surface of the analgesic NaV1.7-selective peptide Pn3a reveals residues essential for membrane and channel interactions

Compelling human genetic studies have identified the voltage-gated sodium channel NaV1.7 as a promising therapeutic target for the treatment of pain. The analgesic spider venom-derived peptide µtheraphotoxin-Pn3a is an exceptionally potent and selective inhibitor of NaV1.7, however, little is known about the structure-activity relationships or channel interactions that define this activity. We rationally designed seventeen Pn3a analogues and determined their activity at hNaV1.7 using patchclamp electrophysiology. The positively charged amino acids K22 and K24 were identified as crucial for Pn3a activity, with molecular modeling identifying interactions of these residues with the S3-S4 loop of domain II of hNaV1.7. Removal of hydrophobic residues Y4, Y27 and W30 led to a loss of potency (>250-fold), while replacement of negatively charged D1 and D8 residues with a positively charged lysine led to increased potencies (>13-fold), likely through alterations in membrane lipid interactions. Mutating D8 to an asparagine led to the greatest improvement in Pn3a potency at NaV1.7 (20-fold), whilst maintaining >100-fold selectivity over the major off-targets NaV1.4, NaV1.5 and NaV1.6. The Pn3a[D8N] mutant retained analgesic activity in vivo, significantly attenuating mechanical allodynia in a clinically relevant mouse model of post-surgical pain at doses 3-fold lower than wild-type Pn3a, without causing motor adverse effects. Results from this study will facilitate future rational design of potent and selective peptidic NaV1.7 inhibitors for the development of more efficacious and safer analgesics but also to further investigate the involvement of NaV1.7 in pain

Analgesic Effects of GpTx-1, PF-04856264 and CNV1014802 in a Mouse Model of NaV1.7-Mediated Pain

Loss-of-function mutations of NaV1.7 lead to congenital insensitivity to pain, a rare condition resulting in individuals who are otherwise normal except for the inability to sense pain, making pharmacological inhibition of NaV1.7 a promising therapeutic strategy for the treatment of pain. We characterized a novel mouse model of NaV1.7-mediated pain based on intraplantar injection of the scorpion toxin OD1, which is suitable for rapid in vivo profiling of NaV1.7 inhibitors. Intraplantar injection of OD1 caused spontaneous pain behaviors, which were reversed by co-injection with NaV1.7 inhibitors and significantly reduced in NaV1.7−/− mice. To validate the use of the model for profiling NaV1.7 inhibitors, we determined the NaV selectivity and tested the efficacy of the reported NaV1.7 inhibitors GpTx-1, PF-04856264 and CNV1014802 (raxatrigine). GpTx-1 selectively inhibited NaV1.7 and was effective when co-administered with OD1, but lacked efficacy when delivered systemically. PF-04856264 state-dependently and selectively inhibited NaV1.7 and significantly reduced OD1-induced spontaneous pain when delivered locally and systemically. CNV1014802 state-dependently, but non-selectively, inhibited NaV channels and was only effective in the OD1 model when delivered systemically. Our novel model of NaV1.7-mediated pain based on intraplantar injection of OD1 is thus suitable for the rapid in vivo characterization of the analgesic efficacy of NaV1.7 inhibitors

Contamination Control and Assay Results for the Majorana Demonstrator Ultra Clean Components

The MAJORANA DEMONSTRATOR is a neutrinoless double beta decay experiment utilizing enriched Ge-76 detectors in 2 separate modules inside of a common solid shield at the Sanford Underground Research Facility. The DEMONSTRATOR has utilized world leading assay sensitivities to develop clean materials and processes for producing ultra-pure copper and plastic components. This experiment is now operating, and initial data provide new insights into the success of cleaning and processing. Post production copper assays after the completion of Module 1 showed an increase in U and Th contamination in finished parts compared to starting bulk material. A revised cleaning method and additional round of surface contamination studies prior to Module 2 construction have provided evidence that more rigorous process control can reduce surface contamination. This article describes the assay results and discuss further studies to take advantage of assay capabilities for the purpose of maintaining ultra clean fabrication and process design.Comment: Proceedings of Low Radioactivity Techniques (LRT May 2017, Seoul