Nrf2 activation supports cell survival during hypoxia and hypoxia/reoxygenation in cardiomyoblasts; the roles of reactive oxygen and nitrogen species

Adaptive mechanisms involving upregulation of cytoprotective genes under the control of transcription factors such as Nrf2 exist to protect cells from permanent damage and dysfunction under stress conditions. Here we explore of the hypothesis that Nrf2 activation by reactive oxygen and nitrogen species modulates cytotoxicity during hypoxia (H) with and without reoxygenation (H/R) in H9C2 cardiomyoblasts. Using MnTBap as a cell permeable superoxide dismutase (SOD) mimetic and peroxynitrite scavenger and L-NAME as an inhibitor of nitric oxide synthase (NOS), we have shown that MnTBap inhibited the cytotoxic effects of hypoxic stress with and without reoxygenation. However, L-NAME only afforded protection during H. Under reoxygenation, conditions, cytotoxicity was increased by the presence of L-NAME. Nrf2 activation was inhibited independently by MnTBap and L-NAME under H and H/R. The increased cytotoxicity and inhibition of Nrf2 activation by the presence of L-NAME during reoxygenation suggests that NOS activity plays an important role in cell survival at least in part via Nrf2-independent pathways. In contrast, O2 -• scavenging by MnTBap prevented both toxicity and Nrf2 activation during H and H/R implying that toxicity is largely dependent on O2 -.To confirm the importance of Nrf2 for myoblast metabolism, Nrf2 knockdown with siRNA reduced cell survival by 50% during 4h hypoxia with and without 2h of reoxygenation and although cellular glutathione (GSH) was depleted during H and H/R, GSH loss was not exacerbated by Nrf2 knockdown. These data support distinctive roles for ROS and RNS during H and H/R for Nrf2 induction which are important for survival independently of GSH salvage. © 2013 The Authors.

Structure-activity relationships of the N-terminus of calcitonin gene-related peptide:key roles of alanine-5 and threonine-6 in receptor activation

Background and purpose - The N-terminus of calcitonin gene-related peptide (CGRP) is important for receptor activation, especially the disulphide-bonded ring (residues 1-7). However, the roles of individual amino acids within this region have not been examined and so the molecular determinants of agonism are unknown. This study has examined the role of residues 1, 3-6 and 8-9, excluding Cys-2 and Cys-7. Experimental approach - CGRP derivatives were substituted with either cysteine or alanine; further residues were introduced at position 6. Their affinity was measured by radioligand binding and their efficacy by measuring cAMP production in SK-N-MC cells and ß-arrestin 2 translocation in CHO-K1 cells at the CGRP receptor. Key results - Substitution of Ala-5 by cysteine reduced affinity 270-fold and reduced efficacy for production of cAMP in SK-N-MCs. Potency at ß-arrestin translocation was reduced by 9-fold. Substitution of Thr-6 by cysteine destroyed all measurable efficacy of both cAMP and ß-arrestin responses; substitution with either alanine or serine impaired potency. Substitutions at positions 1, 4, 8 and 9 resulted in approximately 10-fold reductions in potency at both responses. Similar observations were made at a second CGRP-activated receptor, the AMY1(a) receptor. Conclusions and implications - Ala-5 and Thr-6 are key determinants of agonist activity for CGRP. Ala-5 is also very important for receptor binding. Residues outside of the 1-7 ring also contribute to agonist activity.

Effect of cooling rate on intercritically reheated microstructure and toughness in high strength low alloy steel

High strength low alloy steels have been shown to be adversely affected by the existence of regions of poor impact toughness within the heat affected zone (HAZ) produced during multipass welding. One of these regions is the intercritically reheated coarse grained HAZ or intercritical zone. Since this region is generally narrow and discontinuous, of the order of 0.5 mm in width, weld simulators are often employed to produce a larger volume of uniform microstructure suitable for toughness assessment. The steel usedfor this study was a commercial quenched and tempered steel of 450 MN m -2 yield strength. Specimen blanks were subjected to a simulated welding cycle to produce a coarse grained structure of upper bainite during the first thermal cycle, followed by a second thermal cycle where the peak temperature T p2 was controlled. Charpy tests carried out for T p2 values in the range 650-850°C showed low toughness for T p2 values between 760 and 790°C, in the intercritical regime. Microstructural investigation of the development of grain boundary martensite-retained austenite (MA) phase has been coupled with image analysis to measure the volume fraction of MAformed. Most of the MA constituent appears at the prior austenite grain boundaries during intercritical heating, resulting in a 'necklace' appearance. For values of T p2 greater than 790°C the necklace appearance is lost and the second phase areas are observed throughout the structure. Concurrent with this is the development of the fine grained, predominantly ferritic structure that is associated with the improvement in toughness. At this stage the microstructure is transforming from the intercritical regime structure to the supercritically reheated coarse grained HAZ structure. The toughness improvement occurs even though the MA phase is still present, suggesting that the embrittlement is associated with the presence of a connected grain boundary network of the MA phase. The nature of the second phase particles can be controlled by the cooling rate during the second cycle and variesfrom MA phase at high cooling rates to a pearlitic structure at low cooling rates. The lowest toughness of the intercritical zone is observed only when MA phase is present. The reason suggested for this is that only the MA particles debond readily, a number of debonded particles in close proximity providing sufficient stress concentration to initiate local cleavage. © 1993 The Institute of Materials.

Empirical expression of tool wear when face milling 416 SS

During a machining process, cutting parameters must be taken into account, since depending on them the cutting edge starts to wear out to the point that tool can fail and needs to be change, which increases the cost and time of production. Since wear is a negative phenomenon on the cutting tool, due to the fact that tool life is reduced, it is important to optimize the cutting variables to be used during the machining process, in order to increase tool life. This research is focused on the influence of cutting parameters such as cutting speed, feed per tooth and axial depth of cut on tool wear during a face milling operation. The Taguchi method is applied in this study, since it uses a special design of orthogonal array to study the entire parameters space, with only few numbers of experiments. Also a relationship between tool wear and the cutting parameters is presented. For the studies, a martensitic 416 stainless steel was selected, due to the importance of this material in the machining of valve parts and pump shafts. Copyright © 2009 by ASME.