Nobuhiro Kashige

Mechanism of the photocatalytic inactivation of Lactobacillus casei phage PL-1 by titania thin film

The mechanism of the inactivation of Lactobacillus casei phage PL-1 suspended in a phosphate buffer by black-light (BL) -catalytic titanium dioxide (TiO2) thin film was studied. Generation of both superoxide anions (O2−) and hydroxyl radicals ( · OH) was confirmed in the aqueous medium in which TiO2 film was settled with BL irradiation under gentle shaking. With BL-irradiation alone without TiO2 film, only O2− was generated to some extent. The genome DNA inside the phage particles was found to be fragmented by the treatment of PL-1 phages with BL-catalytic TiO2 film. The phage inactivation by BL-catalytic TiO2 film was inhibited by the addition of albumin in a concentration-dependent manner. BL-catalytic TiO2 film was considered to cause primarily the damage to the capsid protein through the generation of active oxygen species such as · OH, followed by damage to the genome DNA inside the phage particles.

Purification and characterization of intracellular α-l-rhamnosidase from Pseudomonas paucimobilis FP2001

Abstractα-l-Rhamnosidase was extracted and purified from the cells of Pseudomonas paucimobilis FP2001 with a 19.5% yield. The purified enzyme, which was homogeneous as shown by SDS-PAGE and isoelectric focusing, had a molecular weight of 112,000 and an isoelectric point of 7.1. The enzyme activity was accelerated by Ca2+ and remained stable for several months when stored at –20 °C. The optimum pH was 7.8; the optimum temperature was 45 °C. The Km, Vmax and kcat for p-nitrophenyl α-l-rhamnopyranoside were 1.18 mM, 92.4 μM · min–1 and 117,000 · min–1, respectively. Examination of the substrate specificity using various synthetic and natural l-rhamnosyl glycosides showed that this enzyme had a relatively broader substrate specificity than those reported so far.

Inactivation of Lactobacillus Bacteriophage PL-1 by Microwave Irradiation

The effect of microwave irradiation on the survival of bacteriophage PL‐1, which is specific for Lactobacillus casei, was studied using a commercial 2,450 MHz microwave oven. The phages were inactivated by microwave irradiation according to almost first‐order reaction kinetics. The rate of phage inactivation was not affected by the difference in the continuous or intermittent irradiation, nor by the concentrations of phages used, but was affected by the volume of phage suspensions, which prevented the loss of generated heat. Microwave irradiation of phage suspensions produced a number of ghost phages with empty heads, but fragmentation of the tail was hardly noticed. The breakage of phage genome DNA was primarily caused by the heat generated by microwave irradiation, whereas the phage DNA was not affected by the same temperature achieved by heat from outside. Thus we concluded that the phage‐inactivating effect of microwave irradiation was mainly attributed to a thermal microwave effect, which was much stronger than a simple thermal exposure.

Effect of ionic strength on the inactivation of micro-organisms by microwave irradiation.

Microwave irradiation at 2450 MHz inactivated the cells of Escherichia coli, Staphylococcus aureus and Candida albicans suspended in a phosphate buffer. The rate of cell inactivation was proportional to that of the increase in temperature accompanied by microwave irradiation. The inactivation rates of E. coli and C. albicans were affected by addition of NaCl and KCl, but not by sucrose. The maximal inactivation effect was exerted at concentrations of 0·5–1·0 mol l−1, and the end‐point temperature was the highest at the same salt concentrations. Correlation of both the electroconductivity and di‐electric loss of ionic solutions with the heating by microwave irradiation was discussed.

New compounds derived from dihydropyrazines having DNA strand-breakage activity

Abstract Dihydropyrazine derivatives such as 2,3-dihydro-5,6-dimethylpyrazine (1), 2,3-dihydro-2,5,6-trimethyl-pyrazine (2) and 2,3-dihydro-2,2,5,6-teramethylpyrazine (3) were found to be transformed into (2R∗, 3S∗, 5R∗)-1,2 ethylene-imino-1,7,10-triaza-2,3,6-trimethyl-3-hydroxy-spiro [4,5] decan-6-ene (4), the stereo-isomeric mixtures of 2,4aR∗,7,9aS∗-tetramethylcyclohexano [1,2-e: 4,5-e′]-dipiperazin-6-ene (5) and (4aR∗, 9aS∗)-2,2,4a,7,7,9a-hexamethylcyclohexano [1,2-e: 4,5-e′]-dipiperazin-6-ene (6), respectively. These dimerized compounds (4, 5 and 6), whose structures were determined by X-ray and nmr spectral analyses, showed almost the same DNA strand-breakage activity as their parent dihydropyrazines. The dimerization pathway is discussed on the basis of the PM3 calculation data.

Superoxide dismutase from the silkworm, Bombyx mori: sequence, distribution, and overexpression.

Superoxide dismutase (SOD) is an enzyme facilitating the removal of superoxide anions from living organisms. This study focused on SOD from the silkworm, Bombyx mori (bmSOD). cDNA encoding bmSOD was amplified by reverse transcriptase–polymerase chain reaction. The deduced amino acid sequence of bmSOD indicated that the residues forming the Cu/Zn binding site are conserved and that the sequence is in 60% identity to that of the Drosophila melanogaster. B. mori SOD was also close to the D. melanogaster SOD in a phylogenetic tree. The bmSOD mRNA and the enzyme activity were widely distributed in diverse tissues. bmSOD functionally overexpressed in Escherichia coli in a soluble form was purified, and its stability was examined. bmSOD at 4 °C retained almost all of its original activity after incubation at pH 4–11 for 24 h. Incubation (pH 7) for 30 min at temperatures below 40 °C also affected activity insignificantly.

Trace metal zinc stimulates secretion of antimicrobial peptide LL-37 from Caco-2 cells through ERK and p38 MAP kinase.

Infectious diseases, especially, diarrhoea, are responsible for high mortality rates in developing countries. Zinc supplementation shows beneficial effects against such diseases, but the mechanism of action is poorly understood. Here, we examined whether zinc supplementation can improve mucosal innate immunity through induction of antimicrobial peptide secretion from intestinal epithelial cells. Zinc was found to induce secretion of the antimicrobial peptide LL-37 from Caco-2 cell in a dose (0.63±0.09ng/mL and 0.54±0.06ng/mL at 20μM and 50μM respectively) and time dependent manner. LL-37 secretion increased immediately (1h) after exposure to 20μM Zn (0.29±0.04ng/mL), which continued up to 48h of exposure (0.58±0.05ng/mL). Zinc induces the phosphorylation of ERK and p38 MAP kinase and regulates LL-37 secretion through these MAP kinases. Zinc supplementation may have beneficial effects on mucosal innate immunity via secretion of LL-37.

Cloning, Sequence Analysis, and Expression of the Gene Encoding Sphingomonas paucimobilis FP2001 α- L -Rhamnosidase

The gene (rhaM) encoding the α-L-rhamnosidase of Sphingomonas paucimobilis FP2001 was cloned, sequenced, and expressed in Escherichia coli. The rhaM consisted of 3,354 nucleotides and had a promoter and Shine-Dalgarno sequences typical in bacteria. The rhaM encoding a protein (Rham) deducted from the sequence consisted of 1,117 amino acids and had a putative signal peptide of 25 amino acids. Rham has no similarity to other known rhamnosidases. Rham has a sugar-binding domain of glycoside hydrolase family 2, which has been well conserved in β-glucuronidase, β-mannosidase, and β-galactosidase, in its C-terminal region. Rham is possibly a member of a new bacterial subfamily in glycoside hydrolase family 78 (α-L-rhamnosidase). RT-PCR analysis of rhaM mRNA indicated that the induction of α-L-rhamnosidase by the addition of L-rhamnose occurred on the transcriptional level.

Structure-activity relationships in the induction of single-strand breakage in plasmid pBR322 DNA by amino sugars and derivatives

Structure-activity relationships in the induction of strand breakage in plasmid pBR322 DNA by amino sugars and their derivatives were investigated using agarose gel electrophoresis. The coexistence of a potential free aldehyde group at the C-1 position and a free amino group at the C-2 position in the molecules was indispensable for the display of DNA strand-breaking activity in both mono- and oligo-aminosaccharides. The activity was increased by the introduction of an acidic group, especially a phosphate group, at the C-6 position. The activity was also increased by the addition of Cu2+. The order of activity of the amino monosaccharides tested was D-isoglucosamine > D-mannosamine > D-galactosamine > D-glucosamine, and it is suggested that this order is correlated with the portion of acyclic (aldehydo) form in the solution of each sugar. The possible chemical basis for DNA strand breakage by amino sugars is discussed.

Cloning, sequence analysis, and expression of Lactobacillus casei phage PL-1 lysis genes

Summary. The genes encoding the host cell wall-lytic proteins were searched in the genome DNA of phage PL-1 active against Lactobacillus casei ATCC 27092 by comparing the amino acid sequences with those of others using a computer software of the DDBJ data base. The gene regions found were cloned into E. coli by inserting PCR-amplified DNA fragments into the EcoRI site of pUC19, and the nucleotide sequences were determined. One of the ORFs (hol) consisted of 270 bp encoding 90 amino acids. The hol product (holin) possessed a putative secretion signal, two putative transmembrane helices, and a highly charged C-terminus. Another ORF (lys) consisted of 1050 bp encoding an N-acetylmuramoyl-L-alanine amidase of 350 amino acids. The gene lys was expressed in E. coli using pCALn expression vector, and the purified gene product hydrolysed the amide linkage in the peptidoglycans of L. casei. The amino acid sequence of PL-1 amidase showed a high homology to those of Lactococcus lactis phage rlt and Listeria monocytogenes phage A511. It was suggested that the N-terminal region was involved in enzyme activity and the C-terminal region in binding the enzyme to the cell wall substrate, respectively.