Design of hammerhead ribozymes that cleave murine Sry mRNA in vitro and in vivo

As the first step in investigating the possiblity of applying ribozyme technology to artificial control of the sex ratios at birth in farm animals, where the demand for females exceeds that for males, we designed a hammerhead ribozyme (HHRz) and 2 tRNA(val)-hammerhead ribozyme complexes (tRNARz3 and tRNARz4), and examined their effects upon murine Sry mRNA in vitro and in cells. We demonstrated that HHRz and tRNARz3 could effectively cleave the target Sry mRNA in vitro. For the purpose of experiments in vivo, HHRz was cloned into the highly efficient pUC-CAGGS mammalian expression vector (pCAG/HHRz), and the tRNA ribozyme complexes were cloned into the pol III promoter-driven pPUR-KE vector (pPUR/tRNARz3 and pPUR/tRNARz4); the ribozyme vectors were co-transfected with the target vector (pCAG/Sry). A suppressive action (up to approx. 60%) was confirmed for pCAG/HHRz and pPUR/tRNARz3 upon the transiently expressed exogenously introduced Sry in M15 cultured cells.     

Pattern and Composition of Secondary Succession Beneath Caribbean Pine Plantations of Southwest Sri Lanka

This study examined the vegetation growing beneath Caribbean pine (Pinus caribaea) plantations established within previously abandoned tea lands adjacent to intact rain forest in southwest Sri Lanka. To examine the pattern and composition of secondary succession and factors affecting it, vegetation was sampled in different relative locations (interior and edge conditions) within plantations. Results demonstrated that more vegetation was found near the plantation edges than in the interior, and this pattern was prominent for both wind- and bird- dispersed species. The vegetation represented a mixture of species belonging to a range of successional guilds representing early (17 species, 42.4% in stem count) and late-successional species (52 species, 40.6%), although native long-lived canopy tree species were mostly absent. Bird-dispersed species dominated the flora (80 species, 86.7% in stem count). Abundance of an exotic shrub Clidemia hirta (Melastomataceae) showed a negative correlation with that of other species, indicating its impact on native flora. Underplanting of native canopy species may be effective in assisting secondary succession and control C. hirta in the plantations.     

Ozone treatment of spent medium from Auricularia polytricha cultivation for enzymatic saccharification and subsequent ethanol production

We examined the enzymatic saccharification and ethanol fermentation of the spent media (SMs) from Auricularia polytricha cultivation using wood meals of Falcataria moluccana, Shorea sp., and Tectona grandis. Although the hydrolysis weight decrease and reducing sugar yield were higher in SM of F. moluccana, the ethanol yield was higher in SM of Shorea sp. Ozone treatment of SM further increased the hydrolysis weight decrease, reducing sugar, and ethanol yields in Shorea sp. These results indicate that SM of A. polytricha is a suitable biomass material to produce fermentable sugars for ethanol production, and that ozone treatment is a suitable method for increasing the ethanol yield.     

New sampler for dry deposition in forest canopy

A new type of sampler for dry deposition in a forest canopy was designed and tested to confirm its usefulness. The new sampler is made of Teflon chips packed in a nylon net, and tied on a rod like a branch in the canopy. The validity and characteristics of the new sampler were checked by comparing it with the conventional bucket sampler and the foliar extraction method using hinoki (Chamaecyparis obtusa) trees. The comparison between the deposited amounts on the dummy branch and those on the bucket indicates that the dummy could trap gaseous matter more effective than the bucket. From a comparison with the foliar extracting method, it is concluded that the trapping of particulate matter by the new dummy branch is comparable to that of natural needle leaves. However, its efficiency for gaseous matter is relatively small. This disadvantage should be improved by applying other materials to the dummy. Moreover, it is found that the uptake of gas especially SO₂ through the stomata is an important point in the discussion about dry deposition. To evaluate the amounts of dry deposition in the forest canopy more precisely, combination with the additional method should be useful. This study was supported in part by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan.     

Effects of omega-7 palmitoleic acids on skeletal muscle differentiation in a hyperglycemic condition

Maternal obesity and diabetes are known to be involved in fetal myogenesis, but the later stages of myogenesis are not well understood. In this study, we investigated the influence of a hyperglycemic environment on L6 skeletal myoblast differentiation and the function of omega-7 palmitoleic acids. Exposure to a high concentration of glucose (25 mM) in high-glucose culture medium (HG) increased the expression of myogenic genes (MyoD, Myogenin, MRF4, Myhc2x, and Myhc2a) and the synthesis of myosin. HG also activated the PI3K/AKT pathway revealed muscle cell differentiation. Furthermore, the levels of reactive oxygen species (ROS) and an inflammatory cytokine (Tnfaip3; tumor necrosis factor alpha-induced protein 3), which are crucial for the growth and differentiation of skeletal muscle, were increased by HG. Palmitoleic acids suppressed the expression levels of myogenic regulatory genes and increased the expression level of a cell proliferation-related gene (Pax3). Trans-palmitoleic acid and eicosapentaenoic acid (TPA and EPA) increased the phosphorylation level of MAPK/ERK1/2 and downregulated ROS generation and Tnfaip3 expression. In contrast, cis-palmitoleic acid inactivated MAPK/ERK1/2, leading to increased ROS generation. In conclusion, a hyperglycemic environment mediated by HG induced excessive muscle differentiation. Palmitoleic acids inhibited myoblast differentiation by downregulating muscle-specific genes. Moreover, trans-palmitoleic acids may have beneficial antioxidant and/or anti-inflammatory effects in cells.     

Determining the origin of Al in the outflow from a forest watershed of a suburban forest in Japan by analysis of size distribution

This study clarified the origin of Al in spring water and stream water by analyzing the size distribution of Al of ground water, spring water, stream water, and soil extracts obtained by batch and column experiments using samples from a suburban secondary forest on granite bedrock in Aichi Prefecture. The high concentration of dissolved Al (Al_0.45; fraction through 0.45-μm filter) in water samples obtained from this study site was divided into completely dissolved and small colloidal forms (<0.20 μm; Al_0.20) and large colloidal forms (0.20–0.45 μm). More than 50% of dissolved Al (Al_0.45) in spring water existed in large colloidal form. By comparing the size distribution and concentration of various elements including Al in the samples from soil batch and column experiments, we suggest that the high concentration of Al in spring water is caused by the release of Al from surface soils when ground water emerges as spring water.     

Relationship between <Superscript>137</Superscript>Cs concentration and potassium content in stem wood of Japanese cedar (<Emphasis Type="Italic">Cryptomeria japonica</Emphasis>)

To utilize forest resources in areas affected by fallout from the Fukushima Daiichi Nuclear Power Plant accident, it is important to understand the mechanisms of ¹³⁷Cs movement through the stem wood of contaminated trees. Understanding the mechanism of absorption and migration of ¹³⁷Cs to stem wood is necessary for clues to the future prediction of the transition of ¹³⁷Cs to xylem. In the present study, radial variations in ¹³⁷Cs concentration were investigated in Japanese cedar (Cryptomeria japonica D. Don) trees collected 1 year and 10 months after the accident. Additionally, the relationship between ¹³⁷Cs concentration and potassium (K) content was established. Trees with a higher moisture content and lower lightness value in heartwood tended to have a higher ¹³⁷Cs concentration in the heartwood. In these trees, ¹³⁷Cs concentration peaked at the heartwood–sapwood boundary and gradually decreased toward the pith. By contrast, K content within the heartwood remained nearly constant along the radial direction. The heartwood-to-sapwood ratio of ¹³⁷Cs concentration was significantly positively correlated with that of K content. Based on these results, we suggest that ¹³⁷Cs movement from sapwood to heartwood might be related to the K content ratio of heartwood and sapwood.     

Wood density and carbon and nitrogen concentrations in deadwood of Chamaecyparis obtusa and Cryptomeria japonica

Estimating carbon (C) and nitrogen (N) stocks in deadwood in forests nationwide is required for understanding large-scale C and N cycling. To do so requires estimated values of wood density and C and N concentrations. Additionally, parameters that show variation should be examined. In this study, we clarified the estimated values and the variation in three parameters in each decay class of each of two tree species and examined whether dead log diameter and region contribute to variation in the parameters. Data were collected from 73 Chamaecyparis obtusa (Sieb. et Zucc.) Endl. plantations and 66 Cryptomeria japonica D. Don plantations throughout Japan. Wood densities decreased from 386 to 188?kg?m^?3 for C. obtusa and from 334 to 188?kg?m^?3 for C. japonica in decay classes 1–4. The variation in wood density increased with decay class, and the coefficient of variance increased from 13.9% to 46.4% for C. obtusa and from 15.2% to 48.1% for C. japonica. The N concentrations increased from 1.04 to 4.40?g?kg^?1 for C. obtusa and from 1.11 to 2.97?g?kg^?1 for C. japonica in decay classes 1–4. The variation in N concentration increased with decay class, and the coefficient of variance increased from 51.9% to 76.7% for C. obtusa and from 50.3% to 70.4% for C. japonica. Log diameter and region contributed to variations in wood density and N concentration in decay classes 1 and 2 for C. obtusa and C. japonica. However, no relationship was observed between regional climates and the two parameters. In contrast, C concentrations ranged from 507 to 535?g?kg^?1 and were stable with much lower coefficients of variance throughout the decay classes for both tree species. Thus, we recommend that the same C concentration can be adapted for all decay classes of both tree species.     

Responses of antioxidant enzymes in the needles of hinoki cypress (Chamaecyparis obtusa) seedlings to nutrient solutions containing various calcium/aluminum ratios

An attempt was made to examine the possible connection between the various ratios of calcium/aluminum (Ca/Al) in the nutrient solution of plant cultures and the active oxygen scavenging system of hinoki cypress (Chamaecyparis obtusa) seedlings. The hinoki cypress seedlings were transferred to nutrient solutions containing 5 mM AlCl₃ together with various concentrations of Ca(NO₃)₂ in pots containing glass beads and Teflon tips. The activities of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX) and glutathione reductase (GR) in the needles were estimated at several stages during the 7-day treatment. The samples treated with the lower Ca/Al solutions show the highest SOD activities. The activities of APX and GR, both of them involved in the ascorbate-glutathione cycle, showed the same tendency with decrease to higher Ca/Al ratio. These results indicate that rhizospheric Ca might compete with Al and ameliorate Al toxicity on and in the roots, the Al stress is not transformed to the needles after a few days, and the ascorbate-glutathione cycle in the hinoki cypress needles might fluctuate and be suppressed by the rhizospheric Al stress during the 7 days. This work was supported in part by funding from the Japan Science & Technology Corporation, the CREST program 1996–2001, and the Center for Forest Decline Studies.