Molecular cloning and biochemical characterization of a novel erythrose reductase from Candida magnoliae JH110

<p>Abstract</p> <p>Background</p> <p>Erythrose reductase (ER) catalyzes the final step of erythritol production, which is reducing erythrose to erythritol using NAD(P)H as a cofactor. ER has gained interest because of its importance in the production of erythritol, which has extremely low digestibility and approved safety for diabetics. Although ERs were purified and characterized from microbial sources, the entire primary structure and the corresponding DNA for ER still remain unknown in most of erythritol-producing yeasts. <it>Candida magnoliae </it>JH110 isolated from honeycombs produces a significant amount of erythritol, suggesting the presence of erythrose metabolizing enzymes. Here we provide the genetic sequence and functional characteristics of a novel NADPH-dependent ER from <it>C. magnoliae </it>JH110.</p> <p>Results</p> <p>The gene encoding a novel ER was isolated from an osmophilic yeast <it>C. magnoliae </it>JH110. The ER gene composed of 849 nucleotides encodes a polypeptide with a calculated molecular mass of 31.4 kDa. The deduced amino acid sequence of ER showed a high degree of similarity to other members of the aldo-keto reductase superfamily including three ER isozymes from <it>Trichosporonoides megachiliensis </it>SNG-42. The intact coding region of ER from <it>C. magnoliae </it>JH110 was cloned, functionally expressed in <it>Escherichia coli </it>using a combined approach of gene fusion and molecular chaperone co-expression, and subsequently purified to homogeneity. The enzyme displayed a temperature and pH optimum at 42°C and 5.5, respectively. Among various aldoses, the <it>C. magnoliae </it>JH110 ER showed high specific activity for reduction of erythrose to the corresponding alcohol, erythritol. To explore the molecular basis of the catalysis of erythrose reduction with NADPH, homology structural modeling was performed. The result suggested that NADPH binding partners are completely conserved in the <it>C. magnoliae </it>JH110 ER. Furthermore, NADPH interacts with the side chains Lys252, Thr255, and Arg258, which could account for the enzyme's absolute requirement of NADPH over NADH.</p> <p>Conclusions</p> <p>A novel ER enzyme and its corresponding gene were isolated from <it>C. magnoliae </it>JH110. The <it>C. magnoliae </it>JH110 ER with high activity and catalytic efficiency would be very useful for <it>in vitro </it>erythritol production and could be applied for the production of erythritol in other microorganisms, which do not produce erythritol.</p

THE EFFECT OF SHOULDER MOBILITY ON AGONIST AND SYNERGIST DURING SHOULDER PRESS

The purpose of this study was to investigate the effect of shoulder mobility score on agonist and synergist muscle activation during shoulder press and to provide an underpinning fundamental to optimize the training effect while reducing the risk of injuries when instructing training in the field. The participants were divided to two different groups according to individual shoulder mobility score which is part of the Functional Movement Screen (FMS). There were five participants in the score of 3 group (upper group) and six included in the group with the score of less than 3 (lower group). The results of this study revealed that the shoulder mobility score showed a negative correlation with the ratio of the left and right latissimus dorsi/anterior deltoid muscle activation in the concentric contraction phase (

Fabrication of Modified MMT/Glass/Vinylester Multiscale Composites and Their Mechanical Properties

Montmorillonite (MMT) may become a preferred filler material for fiber-reinforced polymer (FRP) composites due to its high aspect ratio, large surface area, and low charge density. In the present paper, MMT/glass/vinylester multiscale composites are prepared with untreated and surface-treated MMT clay particles with an MMT content of 1.0 wt%. Effects of surface treatment on mechanical properties of MMT/glass/vinylester multiscale composites are investigated through tensile and bending tests, which revealed enhanced mechanical properties in the case of surface-treated MMT. Thermal properties are studied through thermogravimetric analysis (TGA) and dynamic mechanical analysis (DMA). X-Ray diffraction is performed to investigate the interaction between MMT and the matrix. Fourier Transform Infrared (FTIR) is also performed for both untreated and surface-treated MMT. Furthermore, Field Emission-Scanning Electron Microscope (FE-SEM) is conducted to investigate the path of fracture propagation within the composite surface, showing that the surface-treated MMT based multiscale composite has better interactions with the host matrix than the untreated MMT multiscale composites. These composites with enhanced mechanical strength can be used for various mechanical applications

Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake

Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glucose uptake have yet to be elucidated.Here, we found that AMPK-induced phospholipase D1 (PLD1) activation is required for (14)C-glucose uptake in muscle cells under glucose deprivation conditions. PLD1 activity rather than PLD2 activity is significantly enhanced by glucose deprivation. AMPK-wild type (WT) stimulates PLD activity, while AMPK-dominant negative (DN) inhibits it. AMPK regulates PLD1 activity through phosphorylation of the Ser-505 and this phosphorylation is increased by the presence of AMP. Furthermore, PLD1-S505Q, a phosphorylation-deficient mutant, shows no changes in activity in response to glucose deprivation and does not show a significant increase in (14)C-glucose uptake when compared to PLD1-WT. Taken together, these results suggest that phosphorylation of PLD1 is important for the regulation of (14)C-glucose uptake. In addition, extracellular signal-regulated kinase (ERK) is stimulated by AMPK-induced PLD1 activation through the formation of phosphatidic acid (PA), which is a product of PLD. An ERK pharmacological inhibitor, PD98059, and the PLD inhibitor, 1-BtOH, both attenuate (14)C-glucose uptake in muscle cells. Finally, the extracellular stresses caused by glucose deprivation or aminoimidazole carboxamide ribonucleotide (AICAR; AMPK activator) regulate (14)C-glucose uptake and cell surface glucose transport (GLUT) 4 through ERK stimulation by AMPK-mediated PLD1 activation.These results suggest that AMPK-mediated PLD1 activation is required for (14)C-glucose uptake through ERK stimulation. We propose that the AMPK-mediated PLD1 pathway may provide crucial clues to understanding the mechanisms involved in glucose uptake

SUCCESSFUL FACTORS OF 540° DWIHURYEOCHAGI IN TAEKWONDO

The purpose of our study was to provide fundamental information about success factors of 540° Dwihuryeochagi in Taekwondo. Twenty Taekwondo athletes who participated in the 2012 Taekwondo Kyukpa Wang (breaking king) championship: ten successful athletes (S, age: 23.1±1.6 yrs, height: 171.0±3.5 cm, body mass: 66.4±7.1 kg) and ten failed athletes (F, age: 22.3±1.8 yrs, height: 172.1±5.4 cm, body mass: 64.4±4.2 kg) were selected. Three-dimensional motion analysis using a system of 3 video cameras with a sampling of 60 fields/s was performed during the competition of 540 ° Dwihuryeochagi. Based on the findings, it is concluded that success factors of 540° Dwihuryeochagi were horizontal velocity of COM during P1, vertical velocity of COM during P2, and the time, kick distance, velocity and angle of lower extremities of P3-P4

Mechanical Stimulation of the HT7 Acupuncture Point to Reduce Ethanol Self-Administration in Rats

Background. Alcoholism, which is a disabling addiction disorder, is a major public health problem worldwide. The present study was designed to determine whether the application of acupuncture at the Shenmen (HT7) point suppresses voluntary alcohol consumption in addicted rats and whether this suppressive effect is potentiated by the administration of naltrexone. Methods. Rats were initially trained to self-administer a sucrose solution by operating a lever. A mechanical acupuncture instrument (MAI) for objective mechanical stimulation was used on rats whose baseline response had been determined. In addition, the effect of HT7 acupuncture on beta-endorphin concentration and ethanol intake via naltrexone were investigated in different groups. Results. We found that ethanol intake and beta-endorphin level in rats being treated with the MAI at the HT7 point reduced significantly. The treatment of naltrexone at high doses reduced the ethanol intake and low-dose injection of naltrexone in conjunction with the MAI also suppressed ethanol intake. Conclusions. The results of the current study indicate that using the MAI at the HT7 point effectively reduces ethanol consumption in rats. Furthermore, the coadministration of the MAI and a low dose of naltrexone can produce some more potent reducing effect of ethanol intake than can acupuncture alone