Characterization of monofunctional catalase KatA from radioresistant bacterium Deinococcus radiodurans

Catalase plays a key role in protecting cells against toxic reactive oxygen species. Here we report on the cloning, purification and characterization of a catalase (KatA, DR1998) from the extremely radioresistant bacterium Deinococcus radiodurans. The size of purified D. radiodurans KatA monomer was 65 kDa while gel filtration revealed that the size of the enzyme was 240 kDa, suggesting that KatA formed a homotetramer in solution. Purified KatA displayed a final specific activity of 68,800 U/mg of protein. The catalase activity of KatA was inhibited by sodium azide, sodium cyanide and 3-amino-1,2,4-triazole. The absorption spectrum of KatA exhibited a Soret band at 408 nm. The position of the spectral peak remained unchanged following reduction of KatA with dithionite. No peroxidase activity was found for KatA. These results demonstrate that D. radiodurans KatA is a typical monofunctional heme-containing catalase. The stability of KatA with respect to H2O2 stress was superior to that of commercially available Aspergillus niger and bovine liver catalases. The relative abundance of KatA in cells in addition to the H2O2 resistance property may play a role in the survival strategy of D. radiodurans against oxidative damage.     

Pharmaceutical production of tableting granules in an ultra-small-scale high-shear granulator as a pre-formulation study

Objective: In some of drug developments, the amount of bulk drug powder to use in early stages is limited and it is not easy to supply a sufficient drug amount for conventional preparation methods. Therefore, an ultra-small-scale high-shear granulator (less than 5?g) (USG) was developed and applied to small-scale granulation as a pre-formulation. Method: The sample powder consisted of 66.5% lactose, 28.5% microcrystalline cellulose and 5.0% hydroxypropylcellulose. The granules were obtained to agitate 5?g of the sample powder with 1.0?mL of water at 300?rpm for 5?min after pre-powder mixing for 3?min by the USG and the manual hand (HM) methods. Results: The granules were evaluated by the 10% and 90% accumulated particle size and the recoveries of the granules and the powder solid. Median particle size for the USG and the HM methods was 159.2?±?2.3 and 270.9?±?14.9 μm, respectively. The USG method had a narrower particle size distribution than those by the HM method. The recovery of the granules by USG was significantly larger than that by the HM method. Conclusion: Characteristics of all of the granules indicated that the USG method could produce higher quality granules within a shorter time than the HM methods.     

Nickel-free stainless steel avoids neointima formation following coronary stent implantation

SUS316L stainless steel and cobalt–chromium and platinum–chromium alloys are widely used platforms for coronary stents. These alloys also contain nickel (Ni), which reportedly induces allergic reactions in some subjects and is known to have various cellular effects. The effects of Ni on neointima formation after stent implantation remain unknown, however. We developed coronary stents made of Ni-free high-nitrogen austenitic stainless steel prepared using a N₂-gas pressurized electroslag remelting (P-ESR) process. Neointima formation and inflammatory responses following stent implantation in porcine coronary arteries were then compared between the Ni-free and SUS316L stainless steel stents. We found significantly less neointima formation and inflammation in arteries implanted with Ni-free stents, as compared to SUS316L stents. Notably, Ni²⁺ was eluted into the medium from SUS316L but not from Ni-free stainless steel. Mechanistically, Ni²⁺ increased levels of hypoxia inducible factor protein-1α (HIF-1α) and its target genes in cultured smooth muscle cells. HIF-1α and their target gene levels were also increased in the vascular wall at SUS316L stent sites but not at Ni-free stent sites. The Ni-free stainless steel coronary stent reduces neointima formation, in part by avoiding activation of inflammatory processes via the Ni-HIF pathway. The Ni-free-stainless steel stent is a promising new coronary stent platform.     

Test results after refurbish of cryogenic system for smiles

Superconducting Sub-millimeter-wave Limb-Emission Sounder (SMILES) is to be operated aboard the Japanese Experiment Module (JEM) of the International Space Station (ISS) in 2009. SMILES uses two superconductor-insulator-superconductor (SIS) mixers for sub-millimeter-wave atmospheric observation and they are cooled to 4 K levels by a cryogenic system with a two-stage Stirling cooler, a Joule-Thomson (JT) cycle cooler and a cryostat composed of three stages. Two-stage Stirling cooler precools the JT circuit and also cools radiation shields in the cryostat. JT circuit has three tube-in-tube type heat exchangers and an orifice for JT expansion in the cryostat. The cryogenic system is built, tested and delivered.     

Porcine hemoglobin promotes lipid excretion to feces more strongly than globin protein in rats

In an effort to clarify whether the lipid-lowering effect of hemoglobin is due to globin protein or heme, this study investigated the effects of dietary porcine hemoglobin (PH) and porcine globin (PG) on lipids contents of serum, liver, and feces in rats. Five-week-old male Wistar rats were divided into 3 dietary groups of 7 rats each, with one group receiving a control diet and the other groups receiving diets containing 1.25% (w/w) PH or 1.18% (w/w) PG for 4 weeks. The PH diet decreased triacylglycerol content in serum and cholesterol in serum and liver, whereas the PG diet reduced triacylglycerol content in serum and cholesterol in liver. Fecal lipid excretion in the PH group was significantly higher than in the PG group. Therefore, PH affected lipid excretion to feces due to globin protein as well as heme.     

Direct synthesis of H2O2 acid solutions on carbon cathode prepared from activated carbon and vapor-growing-carbon-fiber by a H2/O2 fuel cell

Direct synthesis of H₂O₂ acid solutions was studied using a gas-diffusion cathode prepared from activated carbon (AC), vapor-growing-carbon-fiber (VGCF) and poly-tetra-fluoro-ethylene (PTFE) powders, with a new H₂/O₂ fuel cell reactor. O₂ reduction to H₂O₂ was remarkably enhanced at the three-phase boundary (O₂(g)-electrode(s)-acid(l)) at the [AC + VGCF] cathode. Fast diffusion processes of O₂ to the active surface and of H₂O₂ to the bulk acid solutions were essential for H₂O₂ accumulation. Synergy of AC and VGCF was observed for the H₂O₂ formation. RRDE and cyclic voltammetry studies indicated that the surface of AC functioned as the active phase for O₂ reduction to HO₂, and VGCF functioned as an electron conductor and a promoter to convert HO₂ to H₂O₂. A maximum H₂O₂ concentration of 353 mM (1.2 wt%) was accomplished under short-circuit conditions (current density 12.7 mA cm⁻², current efficiency 40.1%, geometric area of cathode 1.3 cm², reaction time 6 h).     

On-line sample preconcentration and separation technique based on transient trapping in microchip micellar electrokinetic chromatography

This paper describes a novel on-line sample preconcentration and separation technique named transient trapping (tr-trapping), which improves the efficiencies of separation and concentration by using a partially injected short micellar plug in microchip electrophoresis. Although a longer separation length often provides a better resolution of complexed or closely migrating analytes, our proposed theoretical model indicated that a trap-and-release mechanism enables a short micellar zone, which was partially injected into the separation channel, to work as an effective concentration and separation field. Application of the tr-trapping technique to microchip micellar electrokinetic chromatography (MCMEKC) was performed on a newly fabricated 5-way-cross microchip by using sodium dodecyl sulfate and rhodamine dyes as test micelle and analytes, respectively. When the injection times of micelle (t(inj),M) and sample solution (t(inj),S) were 1.0 and 2.0 s, respectively, both the preconcentration and separation of the dyes were completely finished within only 3.0 s. At t(inj),S of 8.0 s, a 393-fold improvement of the detectability was achieved in comparison with conventional MCMEKC. The resolution obtained with tr-trapping-MCMEKC was also better than that with conventional MCMEKC in spite of the 160-fold shorter length of the injected micellar zone at t(inj),M of 1.0 s. These results clearly demonstrated that the tr-trapping technique in MCMEKC provides a rapid, high-resolution and detectability analysis even in the short separation channel on the microchips.     

Involvement of the adrenal glands and testis in gap junction formation via testosterone within the male rat anterior pituitary gland

We investigated the influence of testicular and adrenal androgens on the presence of gap junctions between folliculo‐stellate cells in the anterior pituitary glands of 60‐day‐old Wistar‐Imamichi strain male rats. The animals were separated into six groups: Group A served as the controls and had free access to a normal diet and water, Group B was given a normal diet and 0.9% NaCl for their drinking water as the controls of adrenalectomized groups, Group C was castrated, Group D was adrenalectomized, Group E was both castrated and adrenalectomized, and Group F was also both castrated and adrenalectomized. In addition, the animals of Group F were administered a dose of testosterone that is known to produce high physiological levels of the hormones in plasma. Five rats from each group were sacrificed 1, 2, 3, 4, 5, 6, and 7 days after their respective operation, and the anterior pituitary glands were removed and prepared for observation by transmission electron microscopy. We quantified the number of follicles and gap junctions and calculated the rate of occurrence as the ratio of the number of gap junctions existing between folliculo‐stellate cells per intersected follicle profile. Simultaneous removal of adrenal glands with castration resulted in a significantly decrease in the number of gap junctions, whereas the administration of testosterone to these rats compensated for this change. These observations indicate that the preservation of gap junctions between folliculo‐stellate cells is mainly dependent on androgens from both the testes and adrenal glands in adult male rats. Microsc. Res. Tech., 2012. © 2012 Wiley Periodicals, Inc.     

Comprehensive evaluation of leukocyte lineage derived from human hematopoietic cells in humanized mice

Recently, humanized animals whereby a part of the animal is biologically engineered using human genes or cells have been utilized to overcome interspecific differences. Herein, we analyzed the detail of the differentiation states of various human leukocyte subpopulations in humanized mouse and evaluated comprehensively the similarity of the leukocyte lineage between humanized mice and humans. Humanized mice were established by transplanting human CD34(+) cord blood cells into irradiated severely immunodeficient NOD/Shi-scid/IL2Rγ(null) (NOG) mice, and the phenotypes of human cells contained in bone marrow, thymus, spleen and peripheral blood from the mice were analyzed at monthly intervals until 4 months after cell transplantation. The analysis revealed that transplanted human hematopoietic stem cells via the caudal vein homed and engrafted themselves successfully at the mouse bone marrow. Subsequently, the differentiated leukocytes migrated to the various tissues. Almost all of the leukocytes within the thymus were human cells. Furthermore, analysis of the differentiation states of human leukocytes in various tissues and organs indicated that it is highly likely that the human-like leukocyte lineage can be developed in mice.     

Hydrogen production from thermo-catalytic decomposition of methane using carbon black catalysts in an indirectly-irradiated tubular packed-bed solar reactor

The solar thermo-catalytic decomposition of methane using carbon black catalysts for CO₂-free hydrogen production is studied in a packed-bed reactor. The indirectly-irradiated reactor is based on a cavity receiver and a tube-type absorber in which a given load of particle catalyst is injected during on-sun operation, while enabling multiple refilling for catalyst replacement. Concentrated solar power is used as an external radiative source for supplying the high temperature process heat and for driving the endothermic reaction. The indirect irradiation via the intermediate opaque tubular absorber results in a more uniform heating of the whole reacting bed volume and thus an easier reaction temperature control and determination. Carbon particles are used for enhancing the rate of the heterogeneous decomposition reaction and the coupling of the reactor with a particle injection system is implemented to operate in semi-continuous mode with possibility of catalyst load renewal after deactivation.