In order to analyze the phylogeny of soybean-nodulating bacteria in alkaline soils in Vietnam, indigenous soybean-nodulating bacteria were isolated from root nodules by cultivating three kinds of &j-soybean cultivam on two alkaline soils in Vietnam. The 120 isolates were classified into two major genera of soybean-nodulating rhizobia, namely Brudyrhizobium and Sinorhizobium genera, based on a growth analysis on medium and PCR-RFLP analyses of 16s rDNA and of the 165-23s rDNA internal transcribed spacer (ITS) region. Most of the isolates of B. juponicum were extra-slow-growing and their ITS types were similar to that of B. juponicum USDA 136. They were not isolated €kom the soybean cultivar CNS used as &jm, genotype. Isolates of Sinorhizobium were divided into two groups, S. fredii and S. sp., based on a PCR-RFLP analysis of 16s rDNA. Furthermore, PCR-RFLP analysis of the 165-23s rDNA ITS region enabled to separate them into Ave types, three ITS types associated with S. fkedii and two with S. sp. Sinorhizobium was frequently isolated from the three soybean cultivars on two soils. From the isolate ratio, it was suggested that B. juponicum strains similar to B. juponicum USDA 136 and S.Pedii predominated in the alkaline soils of Vietnam. Additionally, our findings indicated that the &j-genotypes affected not only the compatibility, but also the preference for nodulation between the host soybean and rhizobia.

The acidic polysaccharide alginate represents a promising marine biomass for the microbial production of biofuels, although the molecular and structural characteristics of alginate transporters remain to be clarified. In Sphingomonas sp. A1, the ATP-binding cassette transporter AlgM1M2SS is responsible for the import of alginate across the cytoplasmic membrane. Here, we present the substrate-transport characteristics and quaternary structure of AlgM1M2SS. The addition of poly- or oligoalginate enhanced the ATPase activity of reconstituted AlgM1M2SS coupled with one of the periplasmic solute-binding proteins, AlgQ1 or AlgQ2. External fluorescence-labeled oligoalginates were specifically imported into AlgM1M2SS-containing proteoliposomes in the presence of AlgQ2, ATP, and Mg(2+). The crystal structure of AlgQ2-bound AlgM1M2SS adopts an inward-facing conformation. The interaction between AlgQ2 and AlgM1M2SS induces the formation of an alginate-binding tunnel-like structure accessible to the solvent. The translocation route inside the transmembrane domains contains charged residues suitable for the import of acidic saccharides.

Central nervous system neurons in adult mammals display limited regeneration after injury, and functional recovery is poor following complete transection (>4 mm gap) of a rat spinal cord. A novel combination scaffold composed of 3D nanofibrous hydrogel PuraMatrix and a honeycomb collagen sponge was used to promote spinal repair and locomotor functional recovery following complete transection of the spinal cord in rats. We transplanted this scaffold into 5 mm spinal cord gaps and assessed spinal repair and functional recovery using the Basso, Beattie, and Bresnahan (BBB) locomotor scale. The BBB score of the scaffold-transplanted group was significantly higher than that of the PBS-injected control group from 24 d to 4 months after the operation (P < 0.001-0.01), reaching 6.0  ±  0.75 (mean ± SEM) in the transplant and 0.70  ±  0.46 in the control groups. Neuronal regeneration and spinal repair were examined histologically using Pan Neuronal Marker, glial fibrillary acidic protein, growth-associated protein 43, and DAPI. The scaffolds were well integrated into the spinal cords, filling the 5 mm gaps with higher numbers of regenerated and migrated neurons, astrocytes, and other cells than in the control group. Mature and immature neurons and astrocytes in the scaffolds became colocalized and aligned longitudinally over >2 mm, suggesting their differentiation, maturation, and function. The spinal cord NF200 content of the transplant group, analyzed by western blot, was more than twice that of the control group, supporting the histological results. Transplantation of this novel scaffold promoted functional recovery, spinal repair, and neuronal regeneration.

The primary culture of neuronal cells plays an important role in neuroscience. There has long been a need for methods enabling the long-term culture of primary neurons at low density, in defined serum-free medium. However, the lower the cell density, the more difficult it is to maintain the cells in culture. Therefore, we aimed to develop a method for long-term culture of neurons at low density, in serum-free medium, without the need for a glial feeder layer. Here, we describe the work leading to our determination of a protocol for long-term (>2 months) primary culture of rat hippocampal neurons in serum-free medium at the low density of 3×104 cells/mL (8.9×103 cells/cm2) without a glial feeder layer. Neurons were cultured on a three-dimensional nanofibrous hydrogel, PuraMatrix, and sandwiched under a coverslip to reproduce the in vivo environment, including the three-dimensional extracellular matrix, low-oxygen conditions, and exposure to concentrated paracrine factors. We examined the effects of varying PuraMatrix concentrations, the timing and presence or absence of a coverslip, the timing of neuronal isolation from embryos, cell density at plating, medium components, and changing the medium or not on parameters such as developmental pattern, cell viability, neuronal ratio, and neurite length. Using our method of combining the sandwich culture technique with PuraMatrix in Neurobasal medium/B27/L-glutamine for primary neuron culture, we achieved longer neurites (≥3,000 µm), greater cell viability (≥30%) for 2 months, and uniform culture across the wells. We also achieved an average neuronal ratio of 97%, showing a nearly pure culture of neurons without astrocytes. Our method is considerably better than techniques for the primary culture of neurons, and eliminates the need for a glial feeder layer. It also exhibits continued support for axonal elongation and synaptic activity for long periods (>6 weeks).

Fruit-bodies of Agaricales are known to show positive phototropism during the early stage of development, but negative gravitropism at the later stage after the onset of basidiospore formation. However, when exposed to light from below, the fruit-bodies of Tephrocybe tesquorum and Coprinus spp. grew downward through all stages of development, even after the onset of basidiospore formation. Primordium formation, fruit-body development and basidiospore formation were not disturbed under such conditions. In these downward-growing fruit-bodies, gills stood straight upward. In T.tesquorum, caps often became swollen and stipes sometimes became twisted anticlockwise, contrary to those in light from above, while such behaviours were not observed in Coprinus spp.