Radical scavenging by N-aminoazaaromatics

N-Aminoazaaromatics were found to react with nitric oxide in the presence of oxygen to afford deaminated products in high yields. The reaction proceeded almost instantaneously in various solvents including water, and one to two equivalent of NO was consumed depending upon the amount of oxygen coexisted, and 1 equivalent of N2O was released in the reaction. In addition, N-aminoazoles were deaminated by potassium superoxide to give parent azoles in good yields. Two equivalents of superoxide was consumed, and about half equivalents of both nitrite and nitrate ion were released. The results demonstrated that N-aminoazoles have ability to protect the biological system against the oxidation promoted by radicals such as nitrogen oxides and superoxide.     

Effect of 1,25(OH)2D3 on expression of estrogen receptor-alpha mRNA on rat osteosarcoma cell line (ROS 17/2.8)

In order to investigate the regulatory mechanisms of estrogen receptors (ER) in bone cells, changes in ER-alpha mRNA levels of rat osteosarcoma cell line (ROS 17/2.8) before and after exposure to 1,25(OH)2D3 and 17-beta estradiol respectively were measured by quantitative polymerase chain reaction using an internal standard. ER mRNA levels in the ROS 17/2.8 cultured with the medium alone had 5.029 +/- 1.623 mol/g total RNA x 10(-13) and were not statistically different from those cultured in the presence of 1,25(OH)2D3 at concentrations of 10(-12) M and less. ER mRNA levels in the ROS 17/2.8 cell line showed a small but a significant increase as a result of stimulation by 1,25(OH)2D3 at concentrations of 10(-10) and 10(-11) M. However, ER mRNA levels in ROS 17/2.8 cultured in the presence of 1,25(OH)2D3 at concentrations of 10(-9) M were not statistically different from those of the control. On the other hand, the expression of ER in ROS 17/2.8 cells cultured for 3 hours with various doses of 1,25(OH)2D3 showed, by immunoblotting methods, a significant increase at the dose of 10(-10) M in the expression of ER. Although a physiological significance is obscure, these observations suggest that 1,25(OH)2D3 plays a part in the expression of ER in ROS 17/2.8. No significant changes were seen in the expression of ER mRNA and the synthesis of ER as a result of stimulation by the estradiol.     

Treatment of Irradiated Mice with High-Dose Ascorbic Acid Reduced Lethality

Ascorbic acid is an effective antioxidant and free radical scavenger. Therefore, it is expected that ascorbic acid should act as a radioprotectant. We investigated the effects of post-radiation treatment with ascorbic acid on mouse survival. Mice received whole body irradiation (WBI) followed by intraperitoneal administration of ascorbic acid. Administration of 3 g/kg of ascorbic acid immediately after exposure significantly increased mouse survival after WBI at 7 to 8 Gy. However, administration of less than 3 g/kg of ascorbic acid was ineffective, and 4 or more g/kg was harmful to the mice. Post-exposure treatment with 3 g/kg of ascorbic acid reduced radiation-induced apoptosis in bone marrow cells and restored hematopoietic function. Treatment with ascorbic acid (3 g/kg) up to 24 h (1, 6, 12, or 24 h) after WBI at 7.5 Gy effectively improved mouse survival; however, treatments beyond 36 h were ineffective. Two treatments with ascorbic acid (1.5 g/kg × 2, immediately and 24 h after radiation, 3 g/kg in total) also improved mouse survival after WBI at 7.5 Gy, accompanied with suppression of radiation-induced free radical metabolites. In conclusion, administration of high-dose ascorbic acid might reduce radiation lethality in mice even after exposure.     

Endurance Training-Induced Increase in Circulating Irisin Levels Is Associated with Reduction of Abdominal Visceral Fat in Middle-Aged and Older Adults

To elucidate the effects of endurance training on circulating irisin levels in young and middle-aged/older adults, and to determine the association between endurance training-induced alteration of irisin and reduction in body fat. Twenty-five healthy young (age 21 ± 1 years; 16 men, 9 women) and 28 healthy middle-aged/older adults (age 67 ± 8 years; 12 men, 16 women) participated in the study. Each age cohort was divided into two groups: the endurance-training group (14 young, 14 middle-aged/older) and the control group. Subjects in the training groups completed an 8-week endurance-training program (cycling at 60-70% peak oxygen uptake [V˙O_2peak] for 45 min, 3 days/week). Before and after the intervention, we evaluated serum irisin level, V˙O_2peak, and body composition. The increase in V˙O_2peak in the young and middle-aged/older training groups after the intervention period was significantly greater than those in the young and middle-aged/older control groups (P < 0.05). Serum irisin level was significantly increased in the middle-aged/older training group after the intervention period (P < 0.01), but not in the young training group. Furthermore, in the middle-aged/older training group, the endurance training-induced reduction in visceral adipose tissue area was negatively correlated with the change in serum irisin level (r = −0.54, P < 0.05). These results suggest a possible role for secreted irisin in the exercise-induced alteration of abdominal visceral fat in middle-aged and older adults.     

Cumulative Incidence and Predictors of Progression in Corticosteroid-Na?ve Patients with Sarcoidosis

Background

Assessment of the clinical course of sarcoidosis requires long-term observation. However, the appropriate period of follow-up for sarcoidosis remains unclear, especially in patients without indication of corticosteroid therapy at the time of diagnosis.

Objective

This study aimed to clarify the cumulative incidence and identify risk factors for disease progression in corticosteroid-naïve sarcoidosis patients.

Methods

The clinical courses of 150 Japanese patients with sarcoidosis, who were followed for more than 2 years and had no indication for corticosteroid therapy at diagnosis, were retrospectively reviewed. Disease progression was defined as worsening of pulmonary sarcoidosis, development of new organ involvement, or extrapulmonary organ damage. The cumulative incidence of progression was estimated by generating a cumulative incidence curve with the Fine and Gray method.

Results

The median follow-up duration was 7.7 years (interquartile range, 4.7–13.6 years). Thirty-two (21%) patients experienced disease progression. New organ involvement appeared in 16 patients (11%). The 6-month, and 1-, 5-, 10-, and 15-year cumulative incidence of progression was 2%, 5%, 15%, 28%, and 31%, respectively. The number of organs involved at diagnosis was an independent predictor for progression with a multifactorial adjusted hazard ratio of 1.71 (95% confidence interval, 1.11–2.62). The optimal cut-off of the number of organs involved at diagnosis to identify future progression was three.

Conclusions

In corticosteroid-naïve sarcoidosis patients, the risks of disease progression are comparable from 0–5 years and 5–10 years after diagnosis. The number of organs involved at diagnosis is a useful predictor for progression of sarcoidosis.     

TAE226, a Bis-Anilino Pyrimidine Compound,Inhibits the EGFR-Mutant Kinase Including T790M Mutant to Show Anti-Tumor Effect on EGFR-Mutant Non-Small Cell Lung Cancer Cells

TAE226, a bis-anilino pyrimidine compound, has been developed as an inhibitor of focal adhesion kinase (FAK) and insulin-like growth factor-I receptor (IGF-IR). In this study, we investigated the effect of TAE226 on non-small-cell lung cancer (NSCLC), especially focusing on the EGFR mutational status. TAE226 was more effective against cells with mutant EGFR, including the T790M mutant, than against cells with wild-type one. TAE226 preferentially inhibited phospho-EGFR and its downstream signaling mediators in the cells with mutant EGFR than in those with wild-type one. Phosphorylation of FAK and IGF-IR was not inhibited at the concentration at which the proliferation of EGFR-mutant cells was inhibited. Results of the in vitro binding assay indicated significant differences in the affinity for TAE226 between the wild-type and L858R (or delE746_A750) mutant, and the reduced affinity of ATP to the L858R (or delE746_A750) mutant resulted in good responsiveness of the L858R (or delE746_A750) mutant cells to TAE226. Of interest, the L858R/T790M or delE746_A750/T790M mutant enhanced the binding affinity for TAE226 compared with the L858R or delE746_A750 mutant, resulting in the effectiveness of TAE226 against T790M mutant cells despite the T790M mutation restoring the ATP affinity for the mutant EGFR close to that for the wild-type. TAE226 also showed higher affinity of about 15-fold for the L858R/T790M mutant than for the wild-type one by kinetic interaction analysis. The anti-tumor effect against EGFR-mutant tumors including T790M mutation was confirmed in mouse models without any significant toxicity. In summary, we showed that TAE226 inhibited the activation of mutant EGFR and exhibited anti-proliferative activity against NSCLCs carrying EGFR mutations, including T790M mutation.     

Secondary succession of plant communities in a subtropical mountainous region of SW China

Since 1985, originally forested mountainous areas of China have been allowed to return to their natural state after years of exploitation including agriculture, development, and logging. The reforms began earlier in less accessible locations, so that today the successional process is more advanced there. The vegetation in Luquan, Qiongzhusi, and Xishan near Kunming, central Yunnan, exhibits, in a limited area, a range of stages of plant succession that are widely encountered throughout the broader region, and thus affords a special opportunity for a comprehensive study. We analyzed the successional sequence of these various plant communities. They ranged from pioneer coniferous and/or pioneer deciduous broad-leaved stands to pre-mature semi-humid evergreen broad-leaved stands, through mixed coniferous and broad-leaved or mixed deciduous and evergreen broad-leaved stands. The succession proceeded from pioneer coniferous Pinus and Keteleeria, and deciduous Platycarya and Alnus, to late-successional evergreen broad-leaved Cyclobalanopsis and Castanopsis. Two regeneration types of woody species in either the early successional (15–50 years), the mid-successional (40–80 years), or the late-successional (80–180 years) stage were classified. Relatively high species diversity was found in the seral phase at the three study sites. The late-successional stage was commonest where human disturbance was least evident. Poor soil chemical properties under pioneer Pinus were seen as a limitation to plant growth, while the abundance of Alnus at the early stage led to an improved level of organic matter and nitrogen.     

Landscape genetic structure of <Emphasis Type="Italic">Betula maximowicziana</Emphasis> in the Chichibu mountain range,central Japan

We evaluated the genetic structure of 16 Betula maximowicziana populations in the Chichibu mountain range, central Japan, located within a 25-km radius; all but two populations were at altitudes of 1,100–1,400 m. The results indicate the effects of geographic topology on the landscape genetic structure of the populations and should facilitate the development of local-scale strategies to conserve and manage them. Analyses involving 11 nuclear simple sequence repeat loci showed that most populations had similar intrapopulation genetic diversity parameters. Population differentiation (F _ST = 0.021, G′_ST = 0.033) parameters for the populations examined were low but were relatively high compared to those obtained in a previous study covering populations in a much larger area with a radius of approximately 1,000 km (F _ST = 0.062, G′_ST = 0.102). Three populations (Iriyama, Kanayamasawa, and Nishizawa) were differentiated from the other populations by Monmonier’s and spatial analysis of molecular variance algorithms or by STRUCTURE analysis. Since a high mountain ridge (nearly 2,000 m) separates the Kanayamasawa and Nishizawa populations from the other 14 populations and the Kanayamasawa and Nishizawa populations are themselves separated by another mountain ridge, the genetic structure appears to be partly due to mountain ridges acting as genetic barriers and restricting gene flow. However, the Iriyama population is genetically different but not separated by any clear geographic barrier. These results show that the landscape genetic structure is complex in the mountain range and we need to pay attention, within landscape genetic studies and conservation programs, to geographic barriers and local population differentiation.     

Purification and characterization of renin binding protein (RnBP) from porcine kidney

Renin binding protein (RnBP) was purified from porcine kidney using pepstatin affinity column chromatography, DEAE-Sepharose column chromatography, gel filtration on Ultrogel-AcA 34, aminohexyl-Sepharose 4B column chromatography, and high performance liquid chromatography (HPLC) on TSK-gel G-3000 SW. The purified preparation was homogeneous as judged by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, polyacrylamide disc gel electrophoresis and isoelectric focusing on polyacrylamide gel. The isoelectric point was at pH 4.85, and the apparent molecular weight of RnBP was estimated to be 42,000 by SDS-polyacrylamide gel electrophoresis. The preparation did not show any renin activity and was stable for 30 min at 37 degrees C between pH 5.0 and 9.0 or on storage for 4 weeks at 4 degrees C or -80 degrees C. The activity of renin was greatly inhibited by RnBP. From the kinetic analysis of the inhibition we roughly estimated the dissociation constant between renin and RnBP to be about 0.2 nM, assuming that the stoichiometry in the complex, i.e., high molecular weight (HMW) renin, is one to one, and that the complex is inactive. The inhibitory activity of RnBP was lost by acidification at pH 3.0 and the activity of renin was restored. The purified RnBP formed a single precipitin line with the antiserum prepared with the purified HMW renin as antigen, which is RnBP-renin complex (Takahashi, S., et al. (1983) J. Biochem. 93, 265-274), and this line fused with one of the two precipitin lines formed between HMW renin and anti-HMW renin antiserum. The other of the two lines was between renin and anti-HMW renin antiserum. The purified preparation was thus identified as RnBP. The HMW renin was reconstituted with the purified RnBP and renin, and the apparent molecular weight of the reconstituted specimen was estimated to be 60,000 by gel filtration on Ultrogel AcA 44.