Effects of threo-β-hydroxyaspartate derivatives on excitatory amino acid transporters (EAAT4 and EAAT5)

D,L-threo-beta-Benzyloxyaspartate (D,L-TBOA), an analog of threo-beta-hydroxyaspartate (THA) possessing a bulky substituent, is a potent non-transportable blocker for the excitatory amino acid transporters, EAAT1, 2 and 3, while L-threo-beta-methoxyaspartate (L-TMOA) is a blocker for EAAT2, but a substrate for EAAT1 and EAAT3. To characterize the actions of these THA analogs and the function of EAAT4 and EAAT5, we performed electrophysiological analyses in EAAT4 or EAAT5 expressed on Xenopus oocytes. In EAAT4-expressing oocytes, D,L-TBOA acted as a non-transportable blocker, while L-TMOA like D,L-THA was a competitive substrate. In contrast, D,L-THA, D,L-TBOA and L-TMOA all strongly attenuated the glutamate-induced currents generated by EAAT5. Among them, L-TMOA showed the most potent inhibitory action. Moreover, D,L-THA, D,L-TBOA and L-TMOA themselves elicited outward currents at negative potentials and remained inward at positive potentials suggesting that D,L-TBOA and L-TMOA, as well as D,L-THA, not only act as non-transportable blockers, but also block the EAAT5 leak currents. These results indicate that EAATs 4 and 5 show different sensitivities to THA analogs although they share properties of a glutamate-gated chloride channel.     

Enhanced germicidal effects of pulsed UV‐LED irradiation on biofilms

Aims: The major objective of the study was to evaluate the enhanced germicidal effects of low‐frequency pulsed ultraviolet A (UVA)‐light‐emitting diode (LED) on biofilms. Methods and Results: The germicidal effects of UVA‐LED irradiation (365 nm, 0·28 mW cm^?2, in pulsed or continuous mode) on Candida albicans or Escherichia coli biofilms were evaluated by determining colony‐forming units. The morphological change of microbial cells in biofilms was observed using scanning electron microscopy. After 5‐min irradiation, over 90% of viable micro‐organisms in biofilms had been killed, and pulsed irradiation (1–1000 Hz) had significantly greater germicidal ability than continuous irradiation. Pulsed irradiation (100 Hz, 60 min) almost completely killed micro‐organisms in biofilm (>99·9%), and 20‐min irradiation greatly damaged both microbial species. Interestingly, few hyphae were found in irradiated Candida biofilms. Moreover, mannitol treatment, a scavenger of hydroxyl radicals (OH^?), significantly protected viable micro‐organisms in biofilms from UVA‐LED irradiation. Conclusions: The study demonstrated that pulsed UVA‐LED irradiation has a strong germicidal effect (maximum at 100 Hz, over 5‐min irradiation) and causes the disappearance of hyphal forms of Candida. Significance and Impact of the Study: This study can assist in developing a low‐frequency pulsed UVA‐LED system to be applied to pathogenic biofilms for disinfection.     

Development of a novel antimicrobial peptide, AG-30, with angiogenic properties

The utility of various synthetic peptides has been investigated in clinical trials of the treatment of cancers, infectious diseases and endocrine diseases. In the process of functional gene screening with in silico analysis for molecules with angiogenic properties, we generated a small peptide, angiogenic peptide (AG)-30, that possesses both antimicrobial and pro-inflammatory activities. AG-30 has an α-helix structure with a number of hydrophobic or net positively charged amino acids and a propensity to fold into amphipathic structures. Indeed, AG-30 exhibited antimicrobial activity against various bacteria, induced vascular endothelial cell growth and tube formation in a dose-dependent manner and increased neovascularization in a Matrigel plug assay. As a result, AG-30 up-regulated expression of angiogenesis-related cytokines and growth factors for up to 72 hrs in human aortic endothelial cells. To further evaluate the angiogenic effect of AG-30 in vivo , we developed a slow-release AG-30 system utilizing biodegradable gelatin microspheres. In the ischaemic mouse hind limb, slow-release AG-30 treatment results in an increase in angiogenic score, an increase in blood flow (as demonstrated by laser Doppler imaging) and an increase in capillary density (as demonstrated by immunostaining with anti-CD31 antibody). These data suggest that the novel peptide, AG-30, may have therapeutic potential for ischaemic diseases.     

Pre‐dispersal seed predation of bayberry Myrica rubra by Thiotricha pancratiastis (Lepidoptera: Gelechiidae) on Yakushima Island,Japan

We investigated pre‐dispersal seed predation by insects in a bayberry Myrica rubra Sieb. et Zucc. (Myricaceae) on Yakushima Island, Japan. To clarify the patterns of seed fate and predation, all fruit that fell into seed traps were collected to allow any insect larvae within the fruit to emerge, and the fruit were finally dissected to determine whether or not they had been attacked by insect predators. Two lepidopteran species, Thiotricha pancratiastis (Meyrick) (Gelechiidae) and Neoblastobasis spiniharpella Kuznetzov & Sinev (Blastobasidae), emerged from the fruits. Thiotricha pancratiastis is the major seed predator of M. rubra, attacking the fruits intensively during the primary stage of fruit development. Thiotricha pancratiastis had been known as a foliage feeder (leaf miner) of M. rubra, but we revealed that the insect is also an important seed predator of the bayberry.     

Selective detection and transport of fully matched DNA by DNA-loaded microtubule and kinesin motor protein

DNA-loaded microtubules (MTs) moving on a kinesin motor protein-coated substrate can selectively hybridize with a target fully matched DNA over single-base mismatched DNA and transport it. This technique is capable of collecting target biomolecules toward one point site to design new methodology of DNA analysis.     

Comparative genomic analysis revealed a gene for monoglucosyldiacylglycerol synthase, an enzyme for photosynthetic membrane lipid synthesis in cyanobacteria

Cyanobacteria have a thylakoid lipid composition very similar to that of plant chloroplasts, yet cyanobacteria are proposed to synthesize monogalactosyldiacylglycerol (MGDG), a major membrane polar lipid in photosynthetic membranes, by a different pathway. In addition, plant MGDG synthase has been cloned, but no ortholog has been reported in cyanobacterial genomes. We report here identification of the gene for monoglucosyldiacylglycerol (MGlcDG) synthase, which catalyzes the first step of galactolipid synthesis in cyanobacteria. Using comparative genomic analysis, candidates for the gene were selected based on the criteria that the enzyme activity is conserved between two species of cyanobacteria (unicellular [Synechocystis sp. PCC 6803] and filamentous [Anabaena sp. PCC 7120]), and we assumed three characteristics of the enzyme; namely, it harbors a glycosyltransferase motif, falls into a category of genes with unknown function, and shares significant similarity in amino acid sequence between these two cyanobacteria. By a motif search of all genes of Synechocystis, BLAST searches, and similarity searches between these two cyanobacteria, we identified four candidates for the enzyme that have all the characteristics we predicted. When expressed in Escherichia coli, one of the Synechocystis candidate proteins showed MGlcDG synthase activity in a UDP-glucose-dependent manner. The ortholog in Anabaena also showed the same activity. The enzyme was predicted to require a divalent cation for its activity, and this was confirmed by biochemical analysis. The MGlcDG synthase and the plant MGDG synthase shared low similarity, supporting the presumption that cyanobacteria and plants utilize different pathways to synthesize MGDG.