Yukihiko Nakashima

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.

A phage-resistant mutant of Lactobacillus casei which permits phage adsorption but not genome injection

A phage-resistant mutant of Lactobacillus casei (strain YIT 9021) was capable of adsorbing both PL-1 and J-1 phages, but did not yield phage-infected cells. The mutant and wild-type strains were identical in morphology and sugar composition of the cell walls. Attempts to induce prophages from strain YIT 9021 were unsuccessful. Electron microscopic examination of negatively stained mixtures of phage PL-1 and YIT 9021 bacteria revealed that the phages were adsorbed to the cells in a tail-first orientation. All the absorbed phages had DNA-filled heads. It was concluded that PL-1 adsorbed normally but was blocked in the injection of the phage genome into the cell.

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 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.

The possible involvement of protein synthesis in the injection of PL-1 phage genome into its host, Lactobacillus casei

The process of genome DNA injection, after adsorption, by phage PL-1 into host cells of Lactobacillus casei was monitored by using the electron microscope. Injection of DNA was inhibited by the protein-synthesis inhibitors chloramphenicol and erythromycin at concentrations where the colony-forming ability of cells not infected by phage was unaffected. The results suggest that protein synthesis may be involved in some way in the process of genome injection.

Differences in TLR9-dependent inhibitory effects of H2O2-induced IL-8 secretion and NF-kappa B/I kappa B-alpha system activation by genomic DNA from five Lactobacillus species

Lactic acid bacteria (LAB) show anti-inflammatory effects, and their genomic DNA was identified as one of the anti-inflammatory components. Despite the differences in anti-inflammatory effects between live LAB dependent not only on genus but also species, this effect has not been compared at the genomic DNA level. We compared the anti-inflammatory effects of the genomic DNA from five Lactobacillus species-Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus gasseri, Lactobacillus plantarum, and Lactobacillus reuteri-using Caco-2 cells. To evaluate anti-inflammatory effects, decreases in H(2)O(2)-induced IL-8 secretion and inhibition of H(2)O(2)-induced NF-κB/IκB-α system activation were examined. All LAB genomic DNAs dose-dependently decreased H(2)O(2)-induced IL-8 secretion and inhibited H(2)O(2)-induced NF-κB/IκB-α system activation. Comparison of these effects between Lactobacillus species showed that the anti-inflammatory effects of L. acidophilus genomic DNA are lower than those of the other species. Furthermore, suppression of Toll-like receptor 9 (TLR9), a specific receptor of bacterial DNA, expression by RNAi abolished the decrease of H(2)O(2)-induced IL-8 secretion and inhibition of H(2)O(2)-induced NF-κB/IκB-α system activation by LAB genomic DNA. Our results demonstrated that the anti-inflammatory effects of genomic DNA differ between Lactobacillus species and TLR9 is one of the major pathways responsible for the anti-inflammatory effect of LAB genomic DNA.

An N-acetylmuramidase induced by PL-1 phage infection of Lactobacillus casei

A lytic enzyme was isolated and purified from PL-1 phage-induced lysates of the host Lactobacillus casei ATCC 27092. The molecular weight of the enzyme was about 30000. Maximum activity on the lysis of the host cell walls occurred at pH 6.0-6.5 and at 45 degrees C. The enzyme activity was inhibited by heavy metal ions, SH- and serine-enzyme inhibitors and o-phenanthroline. The reducing end of the enzymic digest was muramic acid and the enzyme was considered to be an endo-N-acetylmuramidase. However, the enzyme differed from the other known N-acetylmuramidases including hens egg-white lysozyme in several enzymic properties.

Electron microscope studies on the host cell energy requirement for injection of PL-1 phage DNA intoLactobacillus casei

The process of genome DNA injection, after adsorption, by phage PL-1 intoLactobacillus casei ATCC 27092 was monitored by electron microscopy. The DNA injection depended on the incubation temperature, and the apparent activation energy was about 11 kcal. It was inhibited when the cells had been previously starved, where their intracellular ATP contents was lowered less than one-hundredth that of the unstarved cells. There was a good correlation between the ATP contents of cells and the extent of the phage DNA injection. Dicyclohexyl carbodiimide inhibited the process with little effect both on the viability of cells and the infectivity of phages. These results agreed with the view that a high energy level of the host cells would be required for the formation of blender-resistant phage-cell complexes to complete injection of phage DNA into host cells.

Isolation and expression of a gene (CGR1) regulated during the yeast-hyphal transition in Candida albicans.

We used RNA fingerprinting of arbitrarily primed PCR to isolate genes upregulated during the yeast-hyphal transition in Candida albicans. The sequence and expression of one of these genes (CGR1, Candida growth regulation) are presented. Our results suggest that CGR1 expression is associated with a growth cessation of yeast cells, a prerequisite for germination in this organism.

Elevation of dopamine level reduces host-seeking activity in the adult female mosquito Aedes albopictus

BackgroundMosquito-borne viruses are transmitted to human hosts via blood-feeding behavior of female mosquitoes. Female mosquitoes seek a host to take blood meals (host-seeking behavior). In order to prevent virus infections, it is important to understand how they modulate host-seeking behavior. Dopamine (DA) in the central nervous system acts as a neuromediator that regulates a variety of behaviors in insects. In female mosquitoes, host-seeking behavior increases when DA levels in the head decline after emergence. However, it remains unclear whether DA directly modulates host-seeking behavior in female mosquitoes. The aim of this study was to examine whether changes in DA levels in the head affects host-seeking activity in the adult female mosquito Aedes albopictus (Ae. albopictus).FindingsWe compared host-seeking behavior in one group of emerging female adults treated with l-β-3,4-dihydroxyphenylalanine (l-DOPA), the precursor of DA, (l-DOPA group), with that in an untreated control (control group) after confirming elevation of head DA in l-DOPA group by using high-performance liquid chromatography. The content of head DA in l-DOPA group significantly remained higher than that in controls on all days examined. The host-seeking activity in the control group showed a gradual increase over the 6-day experimental period. In contrast, there was no such increase in the host-seeking activity in the l-DOPA group. Therefore, the host-seeking activity of l-DOPA group was significantly lower than that of the controls between day 3 and 6 post-emergence.ConclusionOur results indicate that elevation of DA level reduces host-seeking activity in adult female mosquito Ae. albopictus.