Distinct disulfide isomers of μ-Conotoxins KIIIA and KIIIB block voltage-gated sodium channels

In the preparation of synthetic conotoxins containing multiple disulfide bonds, oxidative folding can produce numerous permutations of disulfide bond connectivities. Establishing the native disulfide connectivities thus presents a significant challenge when the venom-derived peptide is not available, as is increasingly the case when conotoxins are identified from cDNA sequences. Here, we investigate the disulfide connectivity of μ-conotoxin KIIIA, which was predicted originally to have a [C1–C9,C2–C15,C4–C16] disulfide pattern based on homology with closely related μ-conotoxins. The two major isomers of synthetic μ-KIIIA formed during oxidative folding were purified and their disulfide connectivities mapped by direct mass spectrometric collision-induced dissociation fragmentation of the disulfide-bonded polypeptides. Our results show that the major oxidative folding product adopts a [C1–C15,C2–C9,C4–C16] disulfide connectivity, while the minor product adopts a [C1–C16,C2–C9,C4–C15] connectivity. Both of these peptides were potent blockers of NaV1.2 (Kd values of 5 and 230 nM, respectively). The solution structure for μ-KIIIA based on nuclear magnetic resonance data was recalculated with the [C1–C15,C2–C9,C4–C16] disulfide pattern; its structure was very similar to the μ-KIIIA structure calculated with the incorrect [C1–C9,C2–C15,C4–C16] disulfide pattern, with an α-helix spanning residues 7–12. In addition, the major folding isomers of μ-KIIIB, an N-terminally extended isoform of μ-KIIIA identified from its cDNA sequence, were isolated. These folding products had the same disulfide connectivities as μ-KIIIA, and both blocked NaV1.2 (Kd values of 470 and 26 nM, respectively). Our results establish that the preferred disulfide pattern of synthetic μ-KIIIA and μ-KIIIB folded in vitro is 1–5/2–4/3–6 but that other disulfide isomers are also potent sodium channel blockers. These findings raise questions about the disulfide pattern(s) of μ-KIIIA in the venom of Conus kinoshitai; indeed, the presence of multiple disulfide isomers in the venom could provide a means of further expanding the snail’s repertoire of active peptides.

Structure of the analgesic μ-Conotoxin KIIIA and effects on the structure and function of disulfide deletion

μ-Conotoxin μ-KIIIA, from Conus kinoshitai, blocks mammalian neuronal voltage-gated sodium channels (VGSCs) and is a potent analgesic following systemic administration in mice. We have determined its solution structure using NMR spectroscopy. Key residues identified previously as being important for activity against VGSCs (Lys7, Trp8, Arg10, Asp11, His12, and Arg14) all reside on an α-helix with the exception of Arg14. To further probe structure−activity relationships of this toxin against VGSC subtypes, we have characterized the analogue μ-KIIIA[C1A,C9A], in which the Cys residues involved in one of the three disulfides in μ-KIIIA were replaced with Ala. Its structure is quite similar to that of μ-KIIIA, indicating that the Cys1−Cys9 disulfide bond could be removed without any significant distortion of the α-helix bearing the key residues. Consistent with this, μ-KIIIA[C1A,C9A] retained activity against VGSCs, with its rank order of potency being essentially the same as that of μ-KIIIA, namely, NaV1.2 > NaV1.4 > NaV1.7 ≥ NaV1.1 > NaV1.3 > NaV1.5. Kinetics of block were obtained for NaV1.2, NaV1.4, and NaV1.7, and in each case, both kon and koff values of μ-KIIIA[C1A,C9A] were larger than those of μ-KIIIA. Our results show that the key residues for VGSC binding lie mostly on an α-helix and that the first disulfide bond can be removed without significantly affecting the structure of this helix, although the modification accelerates the on and off rates of the peptide against all tested VGSC subtypes. These findings lay the groundwork for the design of minimized peptides and helical mimetics as novel analgesics.

Enhancing permanganate chemiluminescence detection for the determination of glutathione and glutathione disulfide in biological matrices

Acidic potassium permanganate chemiluminescence enables direct post-column detection of glutathione, but its application to assess the redox state of a wider range of biological fluids and tissues is limited by its sensitivity. Herein we show that the simple on-line addition of an aqueous formaldehyde solution not only enhances the sensitivity of the procedure by two orders of magnitude, but also provides a remarkable improvement in the selectivity of the reagent towards thiols such as glutathione (compared to phenols and amino acids that do not possess a thiol group). This enhanced mode of detection was applied to the determination of glutathione and its corresponding disulfide species in homogenised striatum samples taken from both wild type mice and the R6/1 transgenic mouse model of Huntington's disease, at both 8 and 12 weeks of age. No significant difference was observed between the GSH/GSSG ratios of wild type mice and R6/1 mice at either age group, suggesting that the early disease progression had not significantly altered the intracellular redox environment.

Effects of combined silicon and molybdenum alloying on the size and evolution of microalloy precipitates in HSLA steels containing niobium and titanium

The effects of combined silicon and molybdenum alloying additions on microalloy precipitate formation in austenite after single- and double-step deformations below the austenite no-recrystallization temperature were examined in high-strength low-alloy (HSLA) steels microalloyed with titanium and niobium. The precipitation sequence in austenite was evaluated following an interrupted thermomechanical processing simulation using transmission electron microscopy. Large (~ 105 nm), cuboidal titanium-rich nitride precipitates showed no evolution in size during reheating and simulated thermomechanical processing. The average size and size distribution of these precipitates were also not affected by the combined silicon and molybdenum additions or by deformation. Relatively fine (< 20 nm), irregular-shaped niobium-rich carbonitride precipitates formed in austenite during isothermal holding at 1173 K. Based upon analysis that incorporated precipitate growth and coarsening models, the combined silicon and molybdenum additions were considered to increase the diffusivity of niobium in austenite by over 30% and result in coarser precipitates at 1173 K compared to the lower alloyed steel. Deformation decreased the size of the niobium-rich carbonitride precipitates that formed in austenite.

Subnanometer molybdenum sulfide on carbon nanotubes as a highly active and stable electrocatalyst for hydrogen evolution reaction

Electrochemically splitting water for hydrogen evolution reaction (HER) has been viewed as a promising approach to produce renewable and clean hydrogen energy. However, searching for cheap and efficient HER electrocatalysts to replace the currently used Pt-based catalysts remains an urgent task. Herein, we develop a one-step carbon nanotube (CNT) assisted synthesis strategy with CNTs' strong adsorbability to mediate the growth of subnanometer-sized MoS(x) on CNTs. The subnanometer MoS(x)-CNT hybrids achieve a low overpotential of 106 mV at 10 mA cm(-2), a small Tafel slope of 37 mV per decade, and an unprecedentedly high turnover frequency value of 18.84 s(-1) at η = 200 mV among all reported non-Pt catalysts in acidic conditions. The superior performance of the hybrid catalysts benefits from the presence of a higher number of active sites and the abundant exposure of unsaturated S atoms rooted in the subnanometer structure, demonstrating a new class of subnanometer-scale catalysts.

Effect of composition and austenite deformation on the transformation characteristics of low-carbon and ultralow-carbon microalloyed steels

Deformation dilatometry has been used to simulate controlled hot rolling followed by controlled cooling of a group of low- and ultralow-carbon microalloyed steels containing additions of boron and/or molybdenum to enhance hardenability. Each alloy was subjected to simulated recrystallization and nonrecrystallization rolling schedules, followed by controlled cooling at rates from 0.1 °C/s to about 100 °C/s, and the corresponding continuous-cooling-transformation (CCT) diagrams were constructed. The resultant microstructures ranged from polygonal ferrite (PF) for combinations of slow cooling rates and low alloying element contents, through to bainitic ferrite accompanied by martensite for fast cooling rates and high concentrations of alloying elements. Combined additions of boron and molybdenum were found to be most effective in increasing steel hardenability, while boron was significantly more effective than molybdenum as a single addition, especially at the ultralow carbon content. Severe plastic deformation of the parent austenite (>0.45) markedly enhanced PF formation in those steels in which this microstructural constituent was formed, indicating a significant effective decrease in their hardenability. In contrast, in those steels in which only nonequilibrium ferrite microstructures were formed, the decreases in hardenability were relatively small, reflecting the lack of sensitivity to strain in the austenite of those microstructural constituents forming in the absence of PF.

Modification of wool fiber using steam explosion

Wool fiber was modified by steam explosion in this study. SEM results show that some scales on the fiber surface were cleaved and tiny grooves generated during the explosion. FTIR results suggest no evident changes in the chemical composition of the fiber after the explosion treatment. However, the crystallinity of the fiber decreased slightly as the steam pressure increased based on the X-ray results. In the thermal analysis, DSC results show that the temperature corresponding to vaporization of absorbed water and cleavage of disulfide bonds respectively decreased as the steam pressure increased. The reduction in thermal decomposition energy of the treated fiber indicates that steam explosion might have destroyed some crystals and crosslinks of macromolecular chains in the fiber. The treatment also led to some alterations of the fiber properties, including reduction in strength, moisture regain and solubility in caustic solution.

Processing and mechanical properties of porous Ti-Mo alloy for biomedical applications

Titanium and some of its alloys are well accepted as load-bearing implant materials due to their excellent mechanical properties, superior corrosion resistance, and outstanding biocompatibility. However, solid implant materials may suffer from the problems of adverse tissue reaction, biomechanical mismatch and lack of new bone tissue ingrowth ability. In the present study, porous titanium-molybdenum (Ti-Mo) alloy was fabricated by the space-holding sintering method. The pore size, pore shape and porosity can be controlled through choosing appropriate space-holding particle materials. Electron scanning microscopy (SEM) was used for the characterisation of the porous Ti-Mo alloy. The mechanical properties of the porous Ti-Mo alloy samples were investigated by compressive tests. Results indicated that the porous Ti-Mo alloy provides promising potential for new implant materials with new bone tissue ingrowth ability and mechanical properties mimicking those of natural bone.

Structural and biochemical characterisation of the oxidoreductase NmDsbA3 from Neisseria meningitidis

DsbA is an enzyme found in the periplasm of Gram-negative bacteria that catalyses the formation of disulfide bonds in a diverse array of protein substrates, many of which are involved in bacterial pathogenesis. Whilst most bacteria possess only a single essential DsbA, Neisseria meningitidis is unusual in that it possesses three DsbAs, although the reason for this additional redundancy is unclear. Two of these N. meningitidis enzymes (NmDsbA1 and NmDsbA2) play an important role in meningococcal attachment to human epithelial cells, whilst NmDsbA3 is considered to have a narrow substrate repertoire. To begin to address the role of DsbAs in the pathogenesis of N. meningitidis, we have determined the structure of NmDsbA3 to 2.3 Å resolution. Although the sequence identity between NmDsbA3 and other DsbAs is low, the NmDsbA3 structure adopted a DsbA-like fold. Consistent with this finding, we demonstrated that NmDsbA3 acts as a thiol-disulfide oxidoreductase in vitro and is reoxidised by Escherichia coli DsbB (EcDsbB). However, pronounced differences in the structures between DsbA3 and EcDsbA, which are clustered around the active site of the enzyme, suggested a structural basis for the unusual substrate specificity that is observed for NmDsbA3.

Cytotoxicity of titanium and titanium alloying elements

It is commonly accepted that titanium and the titanium alloying elements of tantalum, niobium, zirconium, molybdenum, tin, and silicon are biocompatible. However, our research in the development of new titanium alloys for biomedical applications indicated that some titanium alloys containing molybdenum, niobium, and silicon produced by powder metallurgy show a certain degree of cytotoxicity. We hypothesized that the cytotoxicity is linked to the ion release from the metals. To prove this hypothesis, we assessed the cytotoxicity of titanium and titanium alloying elements in both forms of powder and bulk, using osteoblast-like SaOS₂ cells. Results indicated that the metal powders of titanium, niobium, molybdenum, and silicon are cytotoxic, and the bulk metals of silicon and molybdenum also showed cytotoxicity. Meanwhile, we established that the safe ion concentrations (below which the ion concentration is non-toxic) are 8.5, 15.5, 172.0, and 37,000.0 μg/L for molybdenum, titanium, niobium, and silicon, respectively.

High torsional energy disulfides : relationship between cross-strand disulfides and right-handed staples

Redox-active disulfides are capable of being oxidized and reduced under physiological conditions. The enzymatic role of redox-active disulfides in thiol-disulfide reductases is well-known, but redox-active disulfides are also present in non-enzymatic protein structures where they may act as switches of protein function. Here, we examine disulfides linking adjacent β-strands (cross-strand disulfides), which have been reported to be redox-active. Our previous work has established that these cross-strand disulfides have high torsional energies, a quantity likely to be related to the ease with which the disulfide is reduced. We examine the relationship between conformations of disulfides and their location in protein secondary structures. By identifying the overlap between cross-strand disulfides and various conformations, we wish to address whether the high torsional energy of a cross-strand disulfide is sufficient to confer redox activity or whether other factors, such as the presence of the cross-strand disulfide in a strained β-sheet, are required.