Plant-based Paste Fermented by Lactic Acid Bacteria and Yeast: Functional Analysis and Possibility of Application to Functional Foods.

A plant-based paste fermented by lactic acid bacteria and yeast (fermented paste) was made from various plant materials. The paste was made of fermented food by applying traditional food-preservation techniques, that is, fermentation and sugaring. The fermented paste contained major nutrients (carbohydrates, proteins, and lipids), 18 kinds of amino acids, and vitamins (vitamin A, B1, B2, B6, B12, E, K, niacin, biotin, pantothenic acid, and folic acid). It contained five kinds of organic acids, and a large amount of dietary fiber and plant phytochemicals. Sucrose from brown sugar, used as a material, was completely resolved into glucose and fructose. Some physiological functions of the fermented paste were examined in vitro. It was demonstrated that the paste possessed antioxidant, antihypertensive, antibacterial, anti-inflammatory, anti-allergy and anti-tyrosinase activities in vitro. It was thought that the fermented paste would be a helpful functional food with various nutrients to help prevent lifestyle diseases.

Physical and mechanical properties of highly textured polycrystalline Nb4AlC3 ceramic.

Highly textured polycrystalline Nb4AlC3 ceramic was fabricated by slip casting in a strong magnetic field followed by spark plasma sintering. Its Lotgering orientation factor was determined on the textured top and side surfaces as f(00l) ∼1.0 and f(hk0)=0.36, respectively. This ceramic showed layered microstructure at the scales ranging from nanometers to millimeters. The as-prepared ceramic had excellent anisotropic physical properties. Along the c-axis direction, it showed higher hardness, bending strength, and fracture toughness of 7.0 GPa, 881 MPa and 14.1 MPa m1/2, respectively, whereas higher values of electrical conductivity (0.81×106 Ω-1 m-1), thermal conductivity (21.20 W m-1 K-1) and Young's modulus (365 GPa) were obtained along the a- or b-axis direction.

Aqueous colloidal processing of single-wall carbon nanotubes and their composites with ceramics.

A unique combination of acid treatment, aqueous colloidal processing, and spark-plasma sintering (SPS) has been used to fabricate high-density Al2O3 /single-wall carbon nanotube (SWNT) composites with well-distributed SWNTs and other carbon nanostructures ('nano-onions', diamond) at Al2O3 grain boundaries. This approach could be used to obtain well-controlled microstructures of ceramic/SWNT composites for tailored mechanical, electrical, and thermal properties. In addition, the colloidal approach for dispersing SWNTs presented here could be used for the controlled manipulation of SWNTs.

Selenophosphate synthetase genes from lung adenocarcinoma cells: Sps1 for recycling L-selenocysteine and Sps2 for selenite assimilation.

A labile selenium donor compound monoselenophosphate is synthesized from selenide and ATP by selenophosphate synthetase (SPS). In the present study, Sps1 and Sps2 were cloned from a cDNA library prepared from human lung adenocarcinoma cells (NCIH441). The human lung Sps1 has been cloned as an ORF of 1,179 bp, identical in sequence to that of the recently revised human liver Sps1. The in-frame TGA codon of the lung Sps2 was genetically altered to TGT (Cys) to obtain the Sps2Cys gene. Expression of the recombinant plasmids containing Sps1 or Sps2Cys was highly toxic to Escherichia coli host cells grown aerobically. Accordingly, the human lung Sps homologs were characterized by an in vivo complementation assay using a selD mutant strain. An added selenium source and a low salt concentration (0.1-0.25% NaCl) in the medium were required for reproducible and sensitive in vivo complementation. Sps2Cys effectively complemented the selD mutant, and the resulting formate dehydrogenase H activity was as high as that of WT E. coli MC4100. In contrast, only a weak complementation of the selD mutant by the Sps1 gene was observed when cells were grown in selenite media. Better complementation with added l-selenocysteine suggested involvement of a selenocysteine lyase for mobilization of selenium. Based on this apparent substrate specificity of the Sps1 and Sps2 gene products we suggest that the Sps1-encoded enzyme depends on a selenium salvage system that recycles l-selenocysteine, whereas the Sps2 enzyme can function with a selenite assimilation system.

Contact-damage-resistant ceramic/single-wall carbon nanotubes and ceramic/graphite composites.

There has been growing interest in incorporating single-wall carbon nanotubes (SWNTs) as toughening agents in brittle ceramics. Here we have prepared dense Al(2)O(3)/SWNT composites using the spark-plasma sintering (SPS) method. Vickers (sharp) and Hertzian (blunt) indentation tests reveal that these composites are highly contact-damage resistant, as shown by the lack of crack formation. However, direct toughness measurements, using the single-edge V-notch beam method, show that these composites are as brittle as dense Al(2)O(3) (having a toughness of 3.22 MPa m(0.5)). This type of unusual mechanical behaviour was also observed in SPS-processed, dense Al(2)O(3)/graphite composites. We argue that the highly shear-deformable SWNTs or graphite heterogeneities in the composites help redistribute the stress field under indentation, imparting the composites with contact-damage resistance. These composites may find use in engineering and biomedical applications where contact loading is important.

Assay method for antitumor L-methionine gamma-lyase: comprehensive kinetic analysis of the complex reaction with L-methionine.

L-Methionine gamma-lyase (EC 4.4.1.11) is a pyridoxal 5'-phosphate-dependent multifunctional enzyme. Measuring the initial velocity of alpha-ketobutyrate production by alpha,gamma-elimination of L-methionine catalyzed by L-methionine gamma-lyase is not very feasible, because the enzyme simultaneously catalyzes both gamma-replacement and alpha,gamma-elimination. To develop an accurate enzyme assay, the comprehensive enzyme kinetics needed to be elucidated by progress curve analysis on the basis of a reaction model for conversion of L-methionine to alpha-ketobutyrate, methanethiol, and ammonia with pyridoxal 5'-phosphate as a cofactor. Kinetic parameters were determined by linear transformation using an approximation of a Maclaurin series from the whole velocity of alpha-ketobutyrate production including alpha,gamma-elimination and gamma-replacement. The significance of gamma-replacement was revealed both theoretically and practically by the kinetic analysis. The enzyme activity was standardized and represented as the Vmax value taking into consideration gamma-replacement in the presence of L-methionine at 37 degrees C and pH 8.0. The novel method that we proposed is accurate, sensitive, reproducible, and linear over a wide range for the determination of L-methionine gamma-lyase activity.

Recombinant expression, biochemical characterization and stabilization through proteolysis of an L-glutamate oxidase from Streptomyces sp. X-119-6.

L-glutamate oxidase (LGOX) from Streptomyces sp. X-119-6 is a protein of 150 kDa that has hexamer structure alpha2beta2gamma2. The gene encoding LGOX was cloned and heterologously expressed in Escherichia coli. LGOX isolated from the E. coli transformant had the structure of a one chain polypeptide. Although the recombinant LGOX exhibited catalytic activity, it was inferior to the LGOX isolated from Streptomyces sp. X-119-6 in catalytic efficiency. The recombinant LGOX exhibited low thermostability compared to the LGOX isolated from Streptomyces sp. X-119-6 and was an aggregated form. Proteolysis of the recombinant LGOX with the metalloendopeptidase from Streptomyces griseus (Sgmp) improved its catalytic efficiency at various pH. Furthermore, the Sgmp-treated recombinant LGOX had a subunit structure of alpha2beta2gamma2 and nearly the same enzymological character as the LGOX isolated from Streptomyces sp. X-119-6. A higher molecular species observed for the recombinant LGOX was not detected for the Sgmp-treated recombinant LGOX. These results prove that proteolysis by Sgmp is involved in the stabilization of the recombinant LGOX.

Identification of thermoacidophilic bacteria and a new Alicyclobacillus genomic species isolated from acidic environments in Japan.

Sixty strains of thermoacidophilic bacteria have been isolated from soil and water samples obtained from various acidic environments in Japan. An initial comparative sequence analysis of the hypervariable regions of the 16S rDNA revealed that all strains could be assigned to the Alicyclobacillus acidocaldarius- Alicyclobacillus genomic species 1 group, which could be further subdivided into three clusters (Clusters I-III). On the basis of phenotypic characteristics, chemotaxonomic profiles, and phylogenetic data of six selected strains, five strains were identified as either A. acidocaldarius or Alicyclobacillus genomic species 1; however, one strain (MIH 332) could not be determined to belong to either of these species. 16S rDNA sequence homology values between strain MIH 332 and the reference strains of A. acidocaldarius (ATCC 27009(T)) and Alicyclobacillus genomic species 1 (DSM 11984) were 98.8% and 99.1%, respectively, which were higher than the corresponding similarity between the reference strains (98.4%). On the other hand, DNA-DNA hybridization levels between strain MIH 332 and the reference strains were 39% and 44%, respectively, which were lower than the value between the reference strains (59% or 65%). However, the phenotype of strain MIH 332 was also similar to those of the reference strains, and a typical phenotype could not be found for the strain, thus indicating that the strain may be a new genomic species of A. acidocaldarius, for which the name Alicyclobacillus genomic species 2 is tentatively proposed. The results of this study suggest that A. acidocaldarius and its related species are widely distributed in acidic environments in Japan, with slight regional variations in morphological and genotypic characteristics.

Biochemical and molecular characterization of the NAD(+)-dependent isocitrate dehydrogenase from the chemolithotroph Acidithiobacillus thiooxidans.

An isocitrate dehydrogenase (ICDH) with an unique coenzyme specificity from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The native enzyme was homodimeric with a subunit of M(r) 45000 and showed a 78-fold preference for NAD(+) over NADP(+). The cloned ICDH gene (icd) was expressed in an icd-deficient strain of Escherichia coli EB106; the activity was found in the cell extract. The gene encodes a 429-amino acid polypeptide and is located between open reading frames encoding a putative aconitase gene (upstream of icd) and a putative succinyl-CoA synthase beta-subunit gene (downstream of icd). A. thiooxidans ICDH showed high sequence similarity to bacterial NADP(+)-dependent ICDH rather than eukaryotic NAD(+)-dependent ICDH, but the NAD(+)-preference of the enzyme was suggested due to residues conserved in the coenzyme binding site of the NAD(+)-dependent decarboxylating dehydrogenase.

A convenient synthetic pathway for multivalent assembly of aminoglycoside antibiotics starting from amikacin.

Vinylpolymers carrying a kanamycin cluster at the side chain were prepared via regioselective N-acylation of amikacin with N-succinimidyl p-vinylbenzoate, followed by radical homo- and co-polymerization with acrylamide. Two independent biological assays disclosed that the polyvalent kanamycin models showed neither antibacterial activity nor inhibitory activity against rRNA-based protein synthesis, suggesting that the multivalency-binding approach is not valid for integrating the potential of aminoglyoside anitibiotics.

An in vitro screening method for DNA cytosine-C5-methylase inhibitor.

A specific inhibitor of DNA cytosine C5-methylases would be useful for studying genomic imprinting, X-chromosome inactivation, carcinogenesis, and regulation of tissue-specific gene expression, for these physiological phenomena appears to be regulated through DNA methylation in promoter sequences. This paper reports a novel convenient in vitro assay method for screening DNA cytosine C5-methylase inhibitor. Our method uses a commercially available Hae III methylase (cytosine C5 methylase), its corresponding Hae III endonuclease, and lambda DNA as their substrate.

Phylogenetic characterization of a new thermoacidophilic bacterium isolated from hot springs in Japan.

Three strains of thermophilic-acidophilic bacteria isolated previously from different hot springs in Japan were characterized by molecular genetic methods. The strategy taken involved PCR amplification, sequencing and restriction pattern analysis of 16S rDNA, 16S-23S rDNA spacer polymorphism analysis and genomic DNA-DNA hybridization. A phylogenetic analysis based on 16S rDNA sequences showed that the new thermoacidophilic isolates formed a genetically coherent group at the species level and fell into a major cluster together with members of the genera Alicyclobacillus and Sulfobacillus with A. acidocaldarius and A. acidoterrestris as their closest relatives. The levels of binary sequence similarity between the isolates and the two Alicyclobacillus species were 97.6 to 97.9%, values considered low enough to warrant placement of the isolates in a distinct species of the genus Alicyclobacillus. The 16S rDNA restriction pattern analysis, but not 16S-23S rDNA spacer polymorphism analysis, was useful for differentiating the isolates from the established Alicyclobacillus species. DNA-DNA hybridization assays demonstrated a distinct phylogenetic position of our isolates as a genospecies within the genus Alicyclobacillus. On the basis of these results, the thermoacidophilic isolates should be classified into a new species of Alicyclobacillus. The results of this study suggest that this new genospecies of Alicyclobacillus is widely distributed in hot springs in Japan.