Effects of facial fanning on local exercise performance and thermoregulatory responses during hyperthermia

To investigate the effects of hyperthermia and facial fanning during hyperthermia on hand-grip exercise performance and thermoregulatory response, we studied eight male subjects, aged 20-53 years. Subjects exercised at 20% of maximal hand-grip strength in the sitting position under three conditions: normothermia (NT), hyperthermia without fanning (HT-nf) or with fanning at 5.5 m X sec-1 wind speed (HT-f). Hyperthermia (0.5 degrees C higher oesophageal temperature than in NT) was induced by leg immersion in water at 42 degrees C. Mean exercise performance was markedly reduced from 716 contractions (NT) to 310 (HT-nf) by hyperthermia (P less than 0.01) and significantly (P less than 0.05) improved to 431 (HT-f) by facial fanning. Hyperthermic exercise was accompanied by significant increases in forearm blood flow (71%) and the local sweat rate on the thigh (136%) at the end of exercise compared with that in NT. Heart rate (HR) and rating of perceived exertion (RPE) increased during exercise and were higher in HT-nf than in NT at any given time of exercise. Oesophageal, tympanic (Tty) and mean skin temperatures were also significantly higher in HT-nf than in NT. Facial fanning caused a marked decrease in forehead skin temperature (1.5-2.0 degrees C) and a slight decrease in Tty, HR and PRE compared with that in HT-nf at any given time of exercise. These results suggested that hyperthermia increased thermoregulatory demands and reduced exercise performance. Facial fanning caused decreases in face skin and brain temperatures, and improved performance.     

SOD1 dimerization monitoring using a novel split NanoLuc,NanoBit

In the present study, we applied a highly sensitive NanoLuc‐based technology to understand the status of superoxide dismutase 1 (SOD1) within mammalian cells. Two fragments of NanoLuc (NanoBit), large N‐terminal and small C‐terminal regions, were fused with wild‐type (wt) and mutant human SOD1 (hSOD1) genes and transfected into cells. Luciferase activity through NanoBit assembly was only detected in NanoBit‐tagged wtSOD1‐expressing cells. Furthermore, the developed NanoLuc system was used to investigate the role of protein‐protein interactions in the pathogenesis of amyotrophic lateral sclerosis (ALS). In addition to SOD1, we also applied this NanoBit system for detecting the dimerization of wild‐type, M337V‐mutated human TAR‐binding protein 43 kDa (hTDP43) and its cleaved C‐terminal fragment (TDP25_M337V) as well as their interactions with SOD1. Luciferase activities of NanoBit‐tagged mutant SOD1, TDP43, or TDP25 were negligible. Finally, we found that a zinc chelator partially reduced the luciferase activity of NanoBit‐wtSOD1. Collectively, these results show that the present assay is sensitive and convenient to appreciate ALS and to develop useful agents for the modulation of SOD1 conformation.     

Effects of human menstrual cycle on thermoregulatory vasodilation during exercise

To investigate the effects of the menstrual cycle and of exercise intensity on the relationship between finger blood flow (FBF) and esophageal temperature (Tes), we studied four women, aged 20-32 years. Subjects exercised at 40% and 70% VO2max in the semi-supine posture at an ambient temperature of 20 degrees C. Resting Tes was higher during the luteal phase than the follicular phase (P less than 0.01). There were no significant differences between the two phases in FBF, oxygen consumption, carbon dioxide production, heart rate or minute ventilation at rest and during exercise, respectively. Each regression line of the FBF-Tes relationship consists of two distinct segments of FBF change to Tes (slope 1 and 2). FBF increased at a threshold Tes for vasodilation ([Tes 0]) and the rate of FBF rise became greater at ([Tes 0]) and the rate of FBF rise became greater at another Tes above this threshold ([Tes 0']). For both levels of exercise, [Tes 0] and [Tes 0'] were shifted upward during the luteal phase, but the slopes of the FBF-Tes relationship were almost the same in the two phases of the menstrual cycle. Increasing exercise intensity induced a significant decrease in slope 1 of the FBF-Tes relationship during the follicular (P less than 0.01) and the luteal phases (P less than 0.02), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Expression of sulfotransferases involved in the biosynthesis of chondroitin sulfate E in the bone marrow derived mast cells

Bone marrow-derived mast cells (BMMCs) contain chondroitin sulfate (CS)-E comprised of GlcA-GalNAc(4SO4) units and GlcA-GalNAc(4,6-SO4) units. GalNAc 4-sulfate 6-O-sulfotransferase (GalNAc4S-6ST) transfers sulfate to position 6 of GalNAc(4SO4) residues of CS. On the basis of the specificity of GalNAc4S-6ST, it is thought that CS-E is synthesized in BMMC through the sequential sulfation by chondroitin 4-sulfotransferase (C4ST)-1 and GalNAc4S-6ST. In this paper, we investigated whether GalNAc4S-6ST and C4ST-1 are actually expressed in BMMCs in which CS-E is actively synthesized. As the bone marrow cells differentiate to BMMCs, level of C4ST-1 and GalNAc4S-6ST messages increased, whereas chondroitin 6-sulfotransferase (C6ST)-1 message decreased. In the extract of BMMCs, activity of GalNAc4S-6ST and C4ST but not C6ST were detected. The recombinant mouse GalNAc4S-6ST transferred sulfate to both nonreducing terminal and internal GalNAc(4SO4) residues; the activity toward nonreducing terminal GalNAc(4SO4) was increased with increasing pH. When CS-E synthesized by BMMCs was metabolically labeled with 35SO4 in the presence of bafilomycin A, chloroquine or NH4Cl, the proportion of the nonreducing terminal GalNAc(4,6-SO4) was increased compared with the control, suggesting that GalNAc4S-6ST in BMMC may elaborate CS-E in the intracellular compartment with relatively low pH where sulfation of the internal GalNAc(4SO4) by GalNAc4S-6ST preferentially occurs.     

Conformational fluctuations of the Ca2+-ATPase in the native membrane environment. Effects of pH, temperature, catalytic substrates, and thapsigargin

Digestion with proteinase K or trypsin yields complementary information on conformational transitions of the Ca(2+)-ATPase (SERCA) in the native membrane environment. Distinct digestion patterns are obtained with proteinase K, revealing interconversion of E1 and E2 or E1 approximately P and E2-P states. The pH dependence of digestion patterns shows that, in the presence of Mg(2+), conversion of E2 to E1 pattern occurs (even when Ca(2+) is absent) as H(+) dissociates from acidic residues. Mutational analysis demonstrates that the Glu(309) and Glu(771) acidic residues (empty Ca(2+)-binding sites I and II) are required for stabilization of E2. Glu(309) ionization is most important to yield E1. However, a further transition produced by Ca(2+) binding to E1 (i.e. E1.2Ca(2+)) is still needed for catalytic activation. Following ATP utilization, H(+)/Ca(2+) exchange is involved in the transition from the E1 approximately P.2Ca(2+) to the E2-P pattern, whereby alkaline pH will limit this conformational transition. Complementary experiments on digestion with trypsin exhibit high temperature dependence, indicating that, in the E1 and E2 ground states, the ATPase conformation undergoes strong fluctuations related to internal protein dynamics. The fluctuations are tightly constrained by ATP binding and phosphoenzyme formation, and this constraint must be overcome by thermal activation and substrate-free energy to allow enzyme turnover. In fact, a substantial portion of ATP free energy is utilized for conformational work related to the E1 approximately P.2Ca(2+) to E2-P transition, thereby disrupting high affinity binding and allowing luminal diffusion of Ca(2+). The E2 state and luminal path closure follow removal of conformational constraint by phosphate.