Genetic succession and spatial genetic structure in a natural old growth Cryptomeria japonica forest revealed by nuclear and chloroplast microsatellite markers

Spatial genetic structure and diversity of Cryptomeria japonica trees in old growth forest but selectively logged in approximately 300 years ago were analyzed using seven nuclear microsatellite markers and one chloroplast microsatellite marker. The individuals were sampled from a 4-ha natural forest plot (200 m × 200 m), which are consisted of three size classes including newly regenerated individuals such as regenerated individuals (REG class; stem diameter at breast height (DBH), 5–110 cm), remaining individuals (REM class; DBH ≥ 110 cm), and old stumps. Genetic diversity was similar between the three groups. These results suggest that genes from the old growth forest may have been passed on to the next generation (i.e., the current forest). DNA extraction and analysis from old stumps was partly successful and we could compare the genetic diversity between old stumps and living trees using four microsatellite loci. Patterns of spatial genetic structure detected by analyses of the nuclear and chloroplast markers differed substantially; all nuclear markers showed significant positive autocorrelation (P < 0.05) over short distances, but the chloroplast marker showed no significant autocorrelation over any distance. We concluded that Moran's I spatial autocorrelation pattern for the chloroplast microsatellite may have been influenced by the extensive pollen flow characteristic of this species, because chloroplast DNA of C. japonica is paternally inherited through pollen and is a wind-pollinated species that produces abundant pollen every few years. In contrast, Moran's I spatial autocorrelation pattern for nuclear microsatellites may reflect limited seed dispersal in gaps where the forest has regenerated and low levels of seed shadow overlap due to past logging. The average seed dispersal distance was 86.0 m at the study site and about 34.0% of the seeds were from the outside of the site, which revealed by parentage analysis. The individuals remaining after logging, and the extensive pollen flow from outside our study plot, may play important roles in maintaining the forests on this island. Gaps created by the selective logging activities appear to have been important for the regeneration of this species in the past but in the present time, natural disturbances such as typhoon and landslide are also important for gap creation.     

Protective Effect of Chitin Urocanate Nanofibers against Ultraviolet Radiation

Urocanic acid is a major ultraviolet (UV)-absorbing chromophore. Chitins are highly crystalline structures that are found predominantly in crustacean shells. Alpha-chitin consists of microfibers that contain nanofibrils embedded in a protein matrix. Acid hydrolysis is a common method used to prepare chitin nanofibrils (NFs). We typically obtain NFs by hydrolyzing chitin with acetic acid. However, in the present study, we used urocanic acid to prepare urocanic acid chitin NFs (UNFs) and examined its protective effect against UVB radiation. Hos: HR-1 mice coated with UNFs were UVB irradiated (302 nm, 150 mJ/cm²), and these mice showed markedly lower UVB radiation-induced cutaneous erythema than the control. Additionally, sunburn cells were rarely detected in the epidermis of UNFs-coated mice after UVB irradiation. Although the difference was not as significant as UNFs, the number of sunburn cells in mice treated with acetic acid chitin nanofibrils (ANFs) tended to be lower than in control mice. These results demonstrate that ANFs have a protective effect against UVB and suggest that the anti-inflammatory and antioxidant effects of NFs influence the protective effect of ANFs against UVB radiation. The combination of NFs with other substances that possess UV-protective effects, such as urocanic acid, may provide an enhanced protective effect against UVB radiation.     

Improvement of application method of liquid fertilizer with irrigation water for uniform topdressing and change in nitrogen concentration of flooded water after application

(pp. 811–816)
The application method of liquid fertilizer with irrigation water was tested in large scale paddy fields using a newly developed fertilizer supplier which can make a supplying rate of liquid fertilizer constant. Uniformity of fertilization, change in the nitrogen concentration of flooded water and absorption of applied nitrogen by paddy rice were examined. High uniformity of fertilization was achieved with this method as compared with a conventional broadcast application of granular fertilizer, or an inflow fertilization of granule-like fertilizer with irrigation water.
When urea was applied as liquid fertilizer, the fertilizer-N disappeared quickly from ponding water under shallow conditions of ponding water depth. In the case of the same depth of ponding water, the rate of reduction was large under conditions of large permeability of water.
The nitrogen utilization rates of urea and ammonium sulfate labeled with ¹⁵N were not related to the depth of ponding water at the time of fertilization. When ammonium sulfate was supplied the nitrogen utilization rate was around 50% regardless of fertilization conditions however, the urea-N utilization rate fell to 40% or less at a permeability of ponding water of 0.8 cm day⁻¹ or less, while it increased to 50% equivalent to ammonium sulfate, at 0.8 cm day⁻¹ or more permeability.     

Follicular development after ovum pick-up and fertilizability of retrieved oocytes in postpartum dairy cattle

This study aimed to evaluate gonadotropin secretion and the developmental competence of follicular oocytes in dairy cattle during the early postpartum (PP) period. The number of follicles developed after transvaginal ultrasound-guided ovum pick-up (OPU) and fertilizability of retrieved oocytes were compared between cows in which the first dominant follicle (DF) ovulated (ovulated group, n=4) and did not ovulate (non-ovulated group, n=3), and between early PP (early PP group, n=2) and after the resumption of the estrous cycle (cyclic group, n=2). Follicular ablation was performed 2-4 days after the detection of DF in the second follicular wave PP. OPU was repeated 3-5 times at 3 or 4-day intervals from 3-4 days after the follicular ablation. At OPU, the follicles were enumerated and all those > or = 5 mm in diameter were aspirated. Recovered oocytes were subjected to in vitro maturation and fertilization. Both criteria were similar between ovulated and non-ovulated groups, and between early PP and cyclic groups. These results suggest that FSH/LH secretions required for follicle recruitment and subsequent follicular growth during the early PP period are similar to those after resumption of the estrous cycle. They also indicate that follicular oocytes during the early PP period have developmental competence.     

Sperm motility, plasma membrane integrity, and binding capacity to homologous zona pellucida of cryopreserved epididymal spermatozoa in the domestic cat

We examined motility, plasma membrane integrity, and binding capacity to homologous zona pellucidae (ZP) of frozen/thawed epididymal cat sperm as a model species for endangered felines. Epididymal spermatozoa from 20 domestic cats were frozen with freezing egg-yolk extender containing 3.0% glycerol in 0.25-ml straws. Post-thaw motility and plasma membrane integrity of the frozen/thawed spermatozoa were 31.8 +/- 2.4% and 32.2 +/- 4.2%, respectively. The frozen/thawed spermatozoa were co-cultured with frozen/thawed immature homologous oocytes with intact ZP for 3 h to examine their ability to bind to the ZP. Sixteen of the 20 frozen/thawed sperm samples demonstrated the ability to bind to ZP. These results indicated that the freezing system for epididymal sperm used in the present study gives appropriate information for banking the genetic resources of wild felid species.     

Export rate of dissolved organic matter from paddy fields during cultivation-activity events

The aim of this study is to quantify the dissolved organic carbon (DOC) of drainage water from paddy fields in agricultural areas of Tottori prefecture, Japan. In four experimental paddy fields, DOC concentration varied much from 1.1 to 10.1 mg C l⁻¹, and was the highest during heavy runoff that occurred in April when there was a non-agricultural period. However, variation in DOC concentration did not always correspond to rainfall, but depended more on cultivation-activity events such as tilling, planting, draining in summer, and final draining in autumn. The water discharge rate from each experimental field was estimated by using a hydrologic model (the Tank Model and a genetic algorithm). Daily DOC export rate per unit area of three experimental paddy fields was calculated to be 0.0074, 0.0052, and 0.0081 kg a⁻¹ day⁻¹, respectively. The daily DOC export rate showed large seasonal variation with the highest value in May and June. It can be concluded that DOC export from paddy fields can be a substantial source of DOC in receiving waters, and the export rate depends much on cultivation method practice. It might be suggested that DOC export from paddy fields can be controlled by a better water management practice of farmers.     

Bioavailability and tissue distribution of anthocyanins in bilberry (Vaccinium myrtillus L.) extract in rats

To clarify how structural diversity of anthocyanins relates to their in vivo function, bioavailability was precisely studied in rats using bilberry (Vaccinium myrtillus L.) extract (Bilberon 25) as an anthocyanin source that contains 15 different anthocyanins. The bilberry extract was orally or intravenously administered to rats, and the plasma levels of each anthocyanin were determined by high-performance liquid chromatography. As the result, all anthocyanins except peonidin 3-O-alpha-L-arabinoside were detectable in the blood plasma. The plasma concentration of anthocyanins as a whole reached the maximum level of 1.2 microM at 15 min after oral administration of 400 mg/kg bilberry extract (153.2 mg/kg as anthocyanins) and then decreased with time. Uptake and decay profiles of each anthocyanin in the plasma were almost the same for all anthocyanins except a few with their maximum after 30 min. Among the anthocyanins carrying the same aglycone, the plasma level after 15 min of oral administration was as follows: galactoside > glucoside > arabinoside. Plasma clearance of anthocyanins after intravenous administration clearly showed that arabinoside disappeared more rapidly than glucoside and galactoside. On the other hand, when anthocyanins carrying the same sugar moiety were compared, the half disappearance time of plasma anthocyanins was in the following order: delphinidin > cyanidin > petunidin = peonidin > malvidin. The bioavailability of anthocyanins was in the range of 0.61-1.82% and was 0.93% as the anthocyanin mixture. The bioavailability of anthocyanins carrying the same aglycone was in the following order: Galactoside showed the highest followed by glucoside and arabinoside for cyanidin and delphinidin, but arabinoside and galactoside showed a higher bioavailability than glucoside for petunidin and malvidin. Anthocyanins recovered in urine and bile during the first 4 h after intravenous administration were only 30.8 and 13.4%, respectively. Anthocyanin profiles in tissues were quite different from those in blood plasma. The major anthocyanins distributed in liver and kidney were the O-methyl anthocyanins such as peonidin, malvidin, and other O-methyl anthocyanins derived from delphinidin, cyanidin, and petunidin-glycosides.     

Effects of administration of glucocorticoids and feeding status on plasma leptin concentrations in dogs

OBJECTIVE: To investigate effects of short- and long- term administration of glucocorticoids, feeding status, and serum concentrations of insulin and cortisol on plasma leptin concentrations in dogs. ANIMALS: 20 nonobese dogs. PROCEDURE: For experiment 1, plasma leptin concentrations and serum concentrations of insulin and cortisol were monitored for 24 hours in 4 dogs administered dexamethasone (0.1 mg/kg, IV) or saline (0.9% NaCl) solution for fed and nonfed conditions. For experiment 2, 11 dogs were administered prednisolone (1 mg/kg, PO, q 24 h for 56 days [7 dogs] and 2 mg/kg, PO, q 24 h for 28 days [4 dogs]) and 5 dogs served as control dogs. Plasma leptin and serum insulin concentrations were monitored weekly. RESULTS: For experiment 1, dexamethasone injection with the fed condition drastically increased plasma leptin concentrations. Furthermore, injection of saline solution with the fed condition increased plasma leptin concentrations. These increases in plasma leptin concentrations correlated with increases in serum insulin concentrations. Dexamethasone injection with the nonfed condition increased plasma leptin concentrations slightly but continuously. Injection of saline solution with the nonfed condition did not alter plasma leptin concentrations. For experiment 2, prednisolone administration at either dosage and duration did not alter plasma leptin concentrations in any dogs. CONCLUSIONS AND CLINICAL RELEVANCE: Dexamethasone injection and feeding increased plasma leptin concentrations in dogs. In addition, dexamethasone administration enhanced the effect of feeding on increases in plasma leptin concentrations. Daily oral administration of prednisolone (1 or 2 mg/kg) did not affect plasma leptin concentrations in dogs.     

Postprandial changes in leptin concentrations of cerebrospinal fluid in dogs during development of obesity

OBJECTIVE: To evaluate postprandial changes in the leptin concentration of CSF in dogs during development of obesity. ANIMALS: 4 male Beagles. PROCEDURES: Weight gain was induced and assessments were made when the dogs were in thin, optimal, and obese body conditions (BCs). The fat area at the level of the L3 vertebra was measured via computed tomography to assess the degree of obesity. Dogs were evaluated in fed and unfed states. Dogs in the fed state received food at 9 AM. Blood and CSF samples were collected at 8 AM, 4 PM, and 10 PM. RESULTS: Baseline CSF leptin concentrations in the thin, optimal, and obese dogs were 24.3 +/- 2.7 pg/mL, 86.1 +/- 14.7 pg/mL, and 116.2 +/- 47.3 pg/mL, respectively. In the thin BC, CSF leptin concentration transiently increased at 4 PM. In the optimal BC, baseline CSF leptin concentration was maintained until 10 PM. In the obese BC, CSF leptin concentration increased from baseline value at 4 PM and 10 PM. Correlation between CSF leptin concentration and fat area was good at all time points. There was a significant negative correlation between the CSF leptin concentration-to-serum leptin concentration ratio and fat area at 4 PM; this correlation was not significant at 8 AM and 10 PM. CONCLUSIONS AND CLINICAL RELEVANCE: Decreased transport of leptin at the blood-brain barrier may be 1 mechanism of leptin resistance in dogs. However, leptin resistance at the blood-brain barrier may not be important in development of obesity in dogs.