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.     

High yield and nutritional quality of rice for animal feed achieved by continuous irrigation with treated municipal wastewater

Paddy and Water Environment - Two consecutive pilot-scale experiments were conducted to investigate the potential reuse of treated municipal wastewater (TWW) as an irrigation source for cultivating...     

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.     

Identification of a 4-Deoxy-l-erythro-5-hexoseulose Uronic Acid Reductase,FlRed, in an Alginolytic Bacterium Flavobacterium sp. Strain UMI-01

In alginate-assimilating bacteria, alginate is depolymerized to unsaturated monosaccharide by the actions of endolytic and exolytic alginate lyases (EC 4.2.2.3 and EC 4.2.2.11). The monosaccharide is non-enzymatically converted to 4-deoxy-l-erythro-5-hexoseulose uronic acid (DEH), then reduced to 2-keto-3-deoxy-d-gluconate (KDG) by a specific reductase, and metabolized through the Entner–Doudoroff pathway. Recently, the NADPH-dependent reductase A1-R that belongs to short-chain dehydrogenases/reductases (SDR) superfamily was identified as the DEH-reductase in Sphingomonas sp. A1. We have subsequently noticed that an SDR-like enzyme gene, flred, occurred in the genome of an alginolytic bacterium Flavobacterium sp. strain UMI-01. In the present study, we report on the deduced amino-acid sequence of flred and DEH-reducing activity of recombinant FlRed. The deduced amino-acid sequence of flred comprised 254 residues and showed 34% amino-acid identities to that of A1-R from Sphingomonas sp. A1 and 80%–88% to those of SDR-like enzymes from several alginolytic bacteria. Common sequence motifs of SDR-superfamily enzymes, e.g., the catalytic tetrad Asn-Lys-Tyr-Ser and the cofactor-binding sequence Thr-Gly-x-x-x-Gly-x-Gly in Rossmann fold, were completely conserved in FlRed. On the other hand, an Arg residue that determined the NADPH-specificity of Sphingomonas A1-R was replaced by Glu in FlRed. Thus, we investigated cofactor-preference of FlRed using a recombinant enzyme. As a result, the recombinant FlRed (recFlRed) was found to show high specificity to NADH. recFlRed exhibited practically no activity toward variety of aldehyde, ketone, keto ester, keto acid and aldose substrates except for DEH. On the basis of these results, we conclude that FlRed is the NADH-dependent DEH-specific SDR of Flavobacterium sp. strain UMI-01.     

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.     

Structure and properties of the cellulose microfibril

The current structural models of the cellulose microfibril as well as its mechanical and thermal properties are reviewed. The cellulose microfibril can be considered as a single thin and long crystalline entity with highly anisotropic physical properties. The contribution and limit of different methods employed such as electron microscopy, infrared spectroscopy, X-ray scattering and diffraction, solid state nuclear magnetic resonance spectroscopy, and molecular modeling are also discussed.