A new microbial method for more efficient production of Nalpha-benzyloxycarbonyl-L-aminoadipate delta-semialdehyde and Nalpha-benzyloxycarbonyl-D-aminoadipate delta-semialdehyde

A new biochemical method for more efficient production of Nalpha-benzyloxycarbonyl-L-aminoadipate delta-semialdehyde (Nalpha-Z-L-AASA) and Nalpha-Z-D-AASA was developed with cells of Rhodococcus sp. AIU Z-35-1. Using the cells harvested after 1 d of cultivation, more than 95 mM Nalpha-Z-L-AASA was produced from 100 mM Nalpha-Z-L-lysine by incubating at pH 5.0 for 1 d at 30 degrees C or by incubating at pH 7.0 for 2 d at 10 degrees C. A similar conversion yield of Nalpha-Z-D-AASA was also obtained under the same conditions. These reaction times required were 1/4 and 1/2 of the respective ones by the method with amine oxidase, and the yields of Nalpha-Z-L-AASA and Nalpha-Z-D-AASA were 2 times higher than the respective ones by the method with amine oxidase. In addition, this method had the advantages of not requiring purification of enzyme and addition of catalase. Thus, the microbial method proposed here was superior to the chemical and other biochemical methods in simplicity, reaction rate, and yield.     

Inhibitory Action of Antidepressants on Mouse Betaine/GABA Transporter (BGT1) Heterologously Expressed in Cell Cultures

Betaine/γ-aminobutyric acid (GABA) transporter (BGT1, SLC6A12) is a member of the Na(+)- and Cl(-)-dependent neurotransmitter transporter gene family with a homology to the GABA transporters (GATs), GAT1 (SLC6A1), GAT2 (SLC6A13) and GAT3 (SLC6A11) (HUGO nomenclature). Since antidepressants have been reported to inhibit GABA uptake, we examined those effects on mouse BGT1 (mBGT1) in comparison with other mouse GAT (mGAT) subtypes in the heterologously expressed cell cultures. All antidepressants tested here inhibited the [(3)H]GABA uptake through mBGT1 and mGATs in a rank order of potency with mBGT1 > mGAT1-3. Kinetic analyses for maprotilline, mianserine and trimipramine revealed that they inhibited mBGT1 and mGAT1 noncompetitively, except that mianserine competitively inhibited mBGT1. These results provided a clue to investigate the structure-function relationship of mBGT1 using antidepressants as a tool, leading to the identification of potential candidates for selective and specific inhibitors of mBGT1.     

Sorption kinetics of heavy oil into porous carbons

Sorption kinetics of heavy oil into porous carbons was evaluated by a concept of liquid sorption coefficient obtained from the weight increase of heavy oil with sorption time, which was measured by a wicking test. Exfoliated graphite, carbonized fir fibers and carbon fiber felts were used as porous materials. It was found that the liquid sorption coefficient of fibrous carbons was twice larger than that of exfoliated graphite. Such a difference in the liquid sorption coefficient between the exfoliated graphite and two fibrous carbons was caused by a difference in effective sorption porosity and tortuosity between them. For the exfoliated graphite and carbonized fir fibers, the liquid sorption coefficient and the effective sorption porosity were strongly dependent on their density. The maximum values of both liquid sorption coefficient and effective sorption porosity of the exfoliated graphite were shown at the bulk density around 16 kg/m³. The liquid sorption coefficient of the carbonized fir fibers increased with increasing the density in the range from 6 to 30 kg/m³. When the carbonized fir fibers were densified above 30 kg/m³, the sorption rate was saturated. On the other hand, the sorption kinetics into the carbon fiber felt was almost independent of the bulk density, because the density of the carbon fiber felt is not effective for the pore structure. The effect of bulk density on the sorption kinetics could be supported from an analysis of pore structure of the porous carbons with different densities, which was measured by mercury porosimeter.     

In vitro heat generation by ferrimagnetic maghemite microspheres for hyperthermic treatment of cancer under an alternating magnetic field

Ferrimagnetic materials can be expected to be useful as thermo seeds for hyperthermic treatment of cancer, especially where the cancer is located in deep parts of body, as they can generate heat by magnetic hysteretic loss when they are placed in an alternating magnetic field. Recently, it was reported that ferrimagnetic maghemite (γ-Fe₂O₃) microspheres 20–30 μm in diameter prepared in aqueous solution can show excellent heat generating ability. However, these microspheres have many cracks on their surfaces. In this study, the preparation conditions for the microspheres was further optimized in order to obtain crack-free ferrimagnetic microspheres, and the in vitro heat generation of the obtained microspheres was measured in an agar phantom under an alternating magnetic field. Crack-free γ-Fe₂O₃ microspheres 20–30 μm in diameter were obtained successfully. Their saturation magnetization and coercive force were 68 emu g⁻¹ and 198 Oe, respectively. Their heat generation under an alternating magnetic field of 300 Oe at 100 kHz was estimated to be 42 W g⁻¹. The microspheres showed in vitro heat generation when they were dispersed in an agar phantom and placed under an alternating magnetic field. It is believed that these microspheres may be useful for the in situ hyperthermic treatment of cancer.     

Cytochrome c-552 from gram-negative alkaliphilic Pseudomonas alcaliphila AL15-21T alters the redox properties at high pH

A soluble class I cytochrome c of an alkaliphile was purified and characterized, and its primary structure was determined. This is the first example of a soluble class I cytochrome c in alkaliphiles. Cells the alkaliphilic gram-negative bacterium Pseudomonas alcaliphila AL15-21(T) grown at pH 10 had a soluble cytochrome c content that was more than twofold that of strain AL15-21(T) cells grown at pH 7 under air-limited conditions. Cytochrome c-552, a soluble cytochrome c with a low molecular weight, was purified from strain AL15-21(T) cells grown at pH 10 under air-limited conditions. Cytochrome c-552 had a molecular mass of 7.5 kDa and exhibited an almost fully reduced state in the resting form, which exhibited absorption maxima at wavelengths of 552, 523 and 417 nm. In the oxidized state, it exhibited an absorption maximum at 412 nm when it was oxidized by ferricyanide, its isoelectric point (pI) was 4.3 and it contained one heme c as a prosthetic group. Cytochrome c-552 was autoreduced at pH 10, and the autoreduction was reproducible. On the other hand, the autoreduction of cytochrome c-552 was not observed at pH 7.0. When pH was increased from 7.0 to 8.3, its midpoint redox potentials (E(m) values) increased from +228 mV to +276 mV as determined by redox titrations, and from +217 mV to +275 mV as determined by cyclic voltammetric measurements. The amino acid sequence deduced by cytochrome c-552 gene analysis revealed that the sequence consists of 96 residues, including 19 residues as an amino-terminal signal peptide. A phylogenetic tree based on amino acid sequence indicated that the protein belongs to group 4, cytochrome c(5) in class I cytochrome c.     

Dynamics of the functional gene copy number and overall bacterial community during microcystin-LR degradation by a biological treatment facility in a drinking water treatment plant

Information is limited on the potential for microcystins (MCs) degradation by carrier-attached biofilms obtained in winter that were not exposed to detectable levels of MCs in the preceding months. Under controlled laboratory conditions, we confirmed that microcystin-LR (MCLR) was effectively biodegraded within 5.5 days in cultures of the biofilm sampled in winter. Quantitative polymerase chain reaction (qPCR) assays revealed that seasonal variations in the MCLR-degradation potential of the biofilm were closely related to the initial MCLR-degrader population in the biofilm. Indigenous MCLR-degraders in the biofilm could accumulate by exposure to natural MCLR in the water column, accelerating MCLR-degradation. The qPCR assay suggested that MCLR may be a primary substrate for the degraders in the presence of another labile organic carbon associated with the biofilm under the present study conditions. qPCR and PCR-denaturing gradient gel electrophoresis (DGGE) for 16S rDNA demonstrated that the overall bacterial population from the winter biofilm rapidly increased with the MCLR-degrader population and remained stable after day 3.5, while the overall bacterial community structure shifted throughout the entire biodegradation period. This study is important to the in-depth understanding of microbial degradation of MCs and could facilitate the bioremediation of MCs in polluted habitats.     

Directed evolution by accumulating tailored mutations: thermostabilization of lactate oxidase with less trade-off with catalytic activity

We assumed that adverse effects posed by introducing multiple mutations could be decomposed into those of each of the component mutations and that the risk could be reduced by the accumulation of mutations that were finely tuned for directed improvement of a specific property. We propose here a directed evolution strategy for improving a specific property with less effect on other ones. This strategy is composed of fine-tuning of mutations and their accumulation by our original mutation-assembling method. In this study, we selected lactate oxidase (LOX) as a model enzyme, because its directed evolution had showed a trade-off between thermostability and catalytic activity. Mutation profiling at each of the sites found by error-prone PCR revealed a strong inverse relationship between the two properties. Thermostable mutations with less effect on catalytic activity were selected at each site and accumulated with ideal combinations by our method. The resultant multiple mutants exhibited 5- to 10-fold superior catalytic activity and comparable thermostability with those created by accumulating thermostable mutations, which were not tuned for catalytic activity. This result demonstrates that the accumulation of fine-tuned mutations is an advantageous approach to reduce the risk of adverse effects posed by accumulating multiple mutations.     

Production of catalase by fungi growing at low pH and high temperature

More than 100 fungi were isolated from cultures at pH 1.0 or 2.5 and 42-45 degrees C, and the production of catalases was investigated. Four strains produced a catalase with high stability at pH 2.0, and three of them produced two extracellular catalases and four intracellular catalases. In these four intracellular catalases, two catalases were similar to extracellular catalases in enzymatic properties and pI. These strains belonged to Aspergillus niger.     

Robot simulator in TIPS/Geometric Simulator

With the growth of factory automation, the need for off-line robot programming is increasing rapidly. Off-line programming requires a robot simulator. This is the reason for the development of a TIPS/GS (Geometric Simulator), accompanied by a robot simulator. TIPS/GS has been developed as a project in the TIPS Research Association. The goal of this project is to extend the functions and applications of the solid modeler TIPS-1. Four simulators (i.e. the assembly simulator, engineering, NC simulator and robot simulator) have been developed for these extended applications.

The robot simulator described in this paper has the following special features:

• • When a robot motion is prescribed by the VAL-G language, the result can be seen on a CRT display in several patterns.

• • High-speed dynamic display which can almost keep up with real-time movements.

• • A shaded as well as wire-frame picture is used for the high-speed display entioned above.

• • Supported by the solid modeler, any robot and environment can be used with this system.

• • The preparation of a precise interference checker based on an analytical methods.

This paper is a report on the development of the robot simulator.     

Analysis of flow around impermeable groynes on one side of symmetrical compound channel:An experimental study

This paper presents the results of an experimental study on the influences of floodplain impermeable groynes on flow structure,velocity,and water depth around the groyne(s).A wooden symmetrical compound channel was used.Groyne models with three different groyne relative lengths,0.5,0.75,and 1.0,were used on one floodplain with single and series arrangements.Analysis of the experimental results using the measured flow velocity and water depth values showed that flow structure,velocity,and water depth mainly depend on groyne relative length and the relative distance between series groynes.The flow velocity at the main channel eenterline increased by about 40%,60%,and 85%,and in other parts on the horizontal plane at the floodplain mid-water depth by about 75%,125%,and 175% of its original value in cases of one-side floodplain groyne(s)with relative lengths of 0.5,0.75,and 1.0,respectively.The effective distance between two groynes in series arrangement ranges from 3 to 4 times the groyne length.Using an impermeable groyne with a large relative length in river floodplains increases the generation of eddy and roller zones downstream of the groyne,leading to more scouring and deposition.To avoid that,the groyne relative length must be kept below half the floodplain width.