Vitamin D 3 hydroxylase (Vdh) isolated from actinomycete Pseudonocardia autotrophica is a cytochrome P450 (CYP) responsible for the biocatalytic conversion of vitamin D 3 (VD 3 ) to 1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 VD 3 ) by P. autotrophica. Although its biological function is unclear, Vdh is capable of catalyzing the two-step hydroxylation of VD 3 , i.e. the conversion of VD 3 to 25-hydroxyvitamin D 3 (25(OH)VD 3 ) and then of 25(OH)VD 3 to 1␣,25(OH) 2 VD 3 , a hormonal form of VD 3 . Here we describe the crystal structures of wild-type Vdh (Vdh-WT) in the substrate-free form and of the highly active quadruple mutant (Vdh-K1) generated by directed evolution in the substrate-free, VD 3 -bound, and 25(OH)VD 3 -bound forms. Vdh-WT exhibits an open conformation with the distal heme pocket exposed to the solvent both in the presence and absence of a substrate, whereas Vdh-K1 exhibits a closed conformation in both the substrate-free and substrate-bound forms. The results suggest that the conformational equilibrium was largely shifted toward the closed conformation by four amino acid substitutions scattered throughout the molecule. The substratebound structure of Vdh-K1 accommodates both VD 3 and 25(OH)VD 3 but in an anti-parallel orientation. The occurrence of the two secosteroid binding modes accounts for the regioselective sequential VD 3 hydroxylation activities. Moreover, these structures determined before and after directed evolution, together with biochemical and spectroscopic data, provide insights into how directed evolution has worked for significant enhancement of both the VD 3 25-hydroxylase and 25(OH)VD 3 1␣-hydroxylase activities.3 is a B-ring opening secosteroid involved in a wide variety of biological functions in mammals (1). In humans, VD 3 is converted into its physiologically active form, 1␣,25-dihydroxyvitamin D 3 (1␣,25(OH) 2 VD 3 ), via hydroxylation reactions that are catalyzed by several cytochrome P450s (CYPs) (1, 2). The first hydroxylation is done at the C25 position of VD 3 by CYP27A1 (2, 3) and CYP2R1 (2, 4) in the liver to produce 25-hydroxyvitamin D 3 (25(OH)VD 3 ). The second proceeds at the C1␣ position of 25(OH)VD 3 by CYP27B1 in the kidney (5) (Fig. 1). The final product, 1␣,25(OH) 2 VD 3 , functions as a hormone with a critical role in maintaining calcium and phosphate homeostasis as well as in controlling the differentiation and proliferation of multiple cell types (1, 2, 6). Indeed, the many symptoms associated with VD 3 deficiency and the VD metabolic disorder, which include psoriasis, osteoporosis, rickets, and hypoparathyroidism, are treated using 1␣,25(OH) 2 VD 3 and its derivatives (1).Although the chemical synthesis of 1␣,25(OH) 2 VD 3 from cholesterol is an established method, it is inefficient, the maximum yield is no more than 1% (7). Alternatively, biocatalytic conversion by the actinomycete Pseudonocardia autotrophica is currently in practical use for the industrial production of 1␣,25(OH) 2 VD 3 (8, 9). We have recently cloned the gene encoding the VD 3 hydroxy...

PURPOSE: Red ginseng (RG) has been reported to improve the blood and organ lipid profile when combined with exercise. However, the effect of RG on energy metabolism during exercise is poorly understood. Therefore, this study was designed to investigate whether RG treatment alters fat utilization during exercise; METHODS: We used seven-week-old ICR mice (n = 42). RG (1 g/kg) was administered orally daily during two weeks of endurance training. All mice were randomized into two groups: training only group (CON group) and training with RG group (RG group). Endurance training consisted of 20~25 m/min on a slope of 8° for one hour five times a week. After a two-week experimental period, we measured substrate utilization during exercise at the same intensity and duration of training using a respiratory calorimetry chamber. Mice were dissected for glycogen measurement of muscles and liver before, immediately after, and one hour after the exercise; RESULT: Fat oxidation during the initial 20 min of the one-hour exercise significantly increased in the RG group compared to the CON group. In addition, the liver glycogen stores significantly decreased immediately after the one-hour exercise compared to at rest in the RG group, but did not differ between immediately after the one-hour exercise and at rest in the RG group. The glycogen concentration in white and red gastrocnemius muscle did not differ between the groups immediately after the one-hour exercise; CONCLUSIONS: These results suggest that RG treatment for two weeks promotes fat oxidation and a glycogen-sparing effect during exercise. This might lead to a delay in peripheral fatigue during endurance exercise performance.

Vitamin D 3 hydroxylase (Vdh) is a novel cytochrome P450 monooxygenase isolated from the actinomycete Pseudonocardia autotrophica and consisting of 403 amino-acid residues. Vdh catalyzes the activation of vitamin D 3 via sequential hydroxylation reactions: these reactions involve the conversion of vitamin D 3 (cholecalciferol or VD3) to 25-hydroxyvitamin D 3 [25(OH)VD3] and the subsequent conversion of 25(OH)VD3 to 1,25-dihydroxyvitamin D 3 [calciferol or 1,25(OH) 2 VD3]. Overexpression of recombinant Vdh was carried out using a Rhodococcus erythropolis expression system and the protein was subsequently purified and crystallized. Two different crystal forms were obtained by the hanging-drop vapour-diffusion method at 293 K using polyethylene glycol as a precipitant. The form I crystal belonged to the trigonal space group P3 1 , with unit-cell parameters a = b = 61.7, c = 98.8 Å . There is one Vdh molecule in the asymmetric unit, with a solvent content of 47.6%. The form II crystal was grown in the presence of 25(OH)VD3 and belonged to the orthorhombic system P2 1 2 1 2 1 , with unit-cell parameters a = 63.4, b = 65.6 c = 102.2 Å . There is one Vdh molecule in the asymmetric unit, with a solvent content of 46.7%. Native data sets were collected to resolutions of 1.75 and 3.05 Å for form I and form II crystals, respectively, using synchrotron radiation. The structure solution was obtained by the molecular-replacement method and model refinement is in progress for the form I crystal.

Many studies in the literature have examined older adults’ past and current lifestyles in either positive or negative association with their life satisfaction levels. Health capabilities naturally decline with aging and can consequently be related to older adults’ life satisfaction levels. Thus, the present study sought to examine the effects of age difference, lifestyles, and health capabilities on older adults’ life satisfaction levels. A total of 290 older adults from three clinical research centers in the United States completed a self-administered questionnaire on their lifestyles and life satisfaction levels, and their health capability assessments were evaluated. There was a significant effect of advancing age on life satisfaction levels among older adults. Additionally, engagement in exercise or physical activity significantly influenced life satisfaction levels. However, there were no statistical effects of vital signs and functional assessments of health capabilities on life satisfaction among older adults. The findings suggest that advancing age itself is the strongest factor in older adults’ life satisfaction. Additionally, engagement in exercise and physical activity can enhance life satisfaction levels as a supplemental factor among older adults. These findings can be beneficial to optimize life satisfaction levels through appropriate programs to encourage positive lifestyles among older adults.

To investigate the effects of long-term lithium treatment and low intensity endurance exercise on brain-derived neurotrophic factor (BDNF) expression and glycogen synthase kinase 3 beta (GSK3β) activity in the hippocampus of obese rats. Fifty 10-week-old male Sprague-Dawley rats were selected. There was a control group of 10 rats (chow control group) while the other forty rats were fed on a high-fat diet for eight weeks to induce obesity. Rats were then assigned into four random groups. The rats were given 10 mg/kg lithium chloride (LiCl) dissolved in 1 mL sterile distilled water once a day, 5 times a week. The rats did 20 min of treadmill walking with an exercise intensity of 40% maximal oxygen uptake (VO2 max) (12 m/min, slope 0%). This was performed for 20 min a day, 3 days a week. Twelve weeks of lithium treatment or endurance exercise significantly reduced body weight and body fat mass in obese rats, without showing additive effects when the treatments were given in parallel or significant toxic responses in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in blood and kidney and liver tissues. BDNF expression in the hippocampus was significantly increased both in exercise and lithium groups with synergistic effects found in the group where both exercise and lithium treatments were given in parallel. On the other hand, the decrease in GSK3β activity was shown only in the lithium treatment group, without showing additive effects when the treatments were given in parallel. Lithium and low-intensity endurance exercise for 12 weeks increased the expression of BDNF, a neuroprotective factor in the hippocampus of obese mice. Lithium treatment alone inhibited the activity of GSK3β. This can be interpreted as a positive indication of applicability of the two factors in the prevention of neurodegenerative diseases.