Enantioselective hydrolysis of racemic styrene oxide by epoxide hydrolase of<Emphasis Type="Italic">Rhodosporidium kratochvilovae</Emphasis> SYU-08

Enantioselective hydrolysis for the production of chiral styrene oxide was investigated using the epoxide hydrolase activity of a newly isolatedRhodosporidium kratochvilovae SYU-08. The effects of reaction prameters—buffer type, pH, temperature, initial substrate concentrations, phenyl-1,2-ethanediol concentrations on hydrolysis rate, and enantioselectivity—were analyzed. Optically active (S)-styrene oxide with an enantiomeric excess higher than 99 % was obtained from its racemate with a yield of 38 % (theoretically 50% maximum yield) from an initial concentration of 80 mM.     

Isolation and Antifungal and Antioomycete Activities of Aerugine Produced by Pseudomonas fluorescens Strain MM-B16

The bacterial strain MM-B16, which showed strong antifungal and antioomycete activity against some plant pathogens, was isolated from a mountain forest soil in Korea. Based on the physiological and biochemical characteristics and 16S ribosomal DNA sequence analysis, the bacterial strain MM-B16 was identical to Pseudomonas fluorescens. An antibiotic active against Colletotrichum orbiculare and Phytophthora capsici in vitro and in vivo was isolated from the culture filtrates of P. fluorescens strain MM-B16 using various chromatographic procedures. The molecular formula of the antibiotic was deduced to be C₁₀H₁₁NO₂S (M⁺, m/z 209.0513) by analysis of electron impact mass spectral data. Based on the nuclear magnetic resonance and infrared spectral data, the antibiotic was confirmed to have the structure of a thiazoline derivative, aerugine [4-hydroxymethyl-2-(2-hydroxyphenyl)-2-thiazoline]. C. orbiculare, P. capsici, and Pythium ultimum were most sensitive to aerugine (MICs for these organisms were approximately 10 μg ml⁻¹). However, no antimicrobial activity was found against yeasts and bacteria even at concentrations of more than 100 μg ml⁻¹. Treatment with aerugine exhibited a significantly high protective activity against development of phytophthora disease on pepper and anthracnose on cucumber. However, the control efficacy of aerugine against the diseases was in general somewhat less than that of the commercial fungicides metalaxyl and chlorothalonil. This is the first study to isolate aerugine from P. fluorescens and demonstrate its in vitro and in vivo antifungal and antioomycete activities against C. orbiculare and P. capsici.     

Comparative Molecular Modeling Study of Arabidopsis NADPH-Dependent Thioredoxin Reductase and Its Hybrid Protein

2-Cys peroxiredoxins (Prxs) play important roles in the protection of chloroplast proteins from oxidative damage. Arabidopsis NADPH-dependent thioredoxin reductase isotype C (AtNTRC) was identified as efficient electron donor for chloroplastic 2-Cys Prx-A. There are three isotypes (A, B, and C) of thioredoxin reductase (TrxR) in Arabidopsis. AtNTRA contains only TrxR domain, but AtNTRC consists of N-terminal TrxR and C-terminal thioredoxin (Trx) domains. AtNTRC has various oligomer structures, and Trx domain is important for chaperone activity. Our previous experimental study has reported that the hybrid protein (AtNTRA-(Trx-D)), which was a fusion of AtNTRA and Trx domain from AtNTRC, has formed variety of structures and shown strong chaperone activity. But, electron transfer mechanism was not detected at all. To find out the reason of this problem with structural basis, we performed two different molecular dynamics (MD) simulations on AtNTRC and AtNTRA-(Trx-D) proteins with same cofactors such as NADPH and flavin adenine dinucleotide (FAD) for 50 ns. Structural difference has found from superimposition of two structures that were taken relatively close to average structure. The main reason that AtNTRA-(Trx-D) cannot transfer the electron from TrxR domain to Trx domain is due to the difference of key catalytic residues in active site. The long distance between TrxR C153 and disulfide bond of Trx C387-C390 has been observed in AtNTRA-(Trx-D) because of following reasons: i) unstable and unfavorable interaction of the linker region, ii) shifted Trx domain, and iii) different or weak interface interaction of Trx domains. This study is one of the good examples for understanding the relationship between structure formation and reaction activity in hybrid protein. In addition, this study would be helpful for further study on the mechanism of electron transfer reaction in NADPH-dependent thioredoxin reductase proteins.     

Eclalbasaponin II Ameliorates the Cognitive Impairment Induced by Cholinergic Blockade in Mice

Eclalbasaponin II derived from Eclipta prostrata L. (Asteraceae) has been reported to have anti-fibrotic, anti-bacterial and autophagic activities, but its effect on cognitive function has not been investigated. We studied the effect of eclalbasaponin II on cholinergic blockade-induced memory impairment in mice using the passive avoidance, Y-maze, and Morris water maze tasks. Eclalbasaponin II (10 or 20 mg/kg, p.o.) significantly ameliorated the cognitive dysfunction induced by scopolamine in the passive avoidance, Y-maze, and the Morris water maze tasks. To identify the mechanism of the memory-ameliorating effect of eclalbasaponin II, acetylcholinesterase (AChE) activity assay, Western blot analysis and electrophysiology were conducted. Eclalbasaponin II inhibited the AChE activity in ex vivo study, and the administration of eclalbasaponin II and its metabolite, echinocystic acid, increased the phosphorylation levels of memory-related signaling molecules, including protein kinase B (Akt) and glycogen synthase kinase-3β (GSK-3β), in the hippocampus. Although eclalbasaponin II did not affect hippocampal long term potentiation (LTP), echinocystic acid significantly enhanced hippocampal LTP formation (30 μM). These results suggest that eclalbasaponin II ameliorates cholinergic blockade-induced cognitive impairment via AChE inhibition, LTP formation and the activation of Akt-GSK-3β signaling, and that eclalbasaponin II may be a useful to treat cognitive impairment derived from cholinergic dysfunction.     

Cell separation from high cell density broths of Alcaligenes eutrophus by using a coagulant

Summary Alcaligenes eutrophus was successfully recovered from high cell density broths by pre-treatment with polyaluminium hydroxide chloride silicate as a coagulant at 36–90 mg Al/l. The optimum pH range for cell coagulation was 10–12. Subsequent centrifugation (45×g) and filtration (pore size 0.5 mm) gave a cell recovery of higher than 90%. The energy demand for cell recovery with the coagulant was only 3–11% of that without it.     

Centromere-Linkage Analysis and Consolidation of the Zebrafish Genetic Map

The ease of isolating mutations in zebrafish will contribute to an understanding of a variety of processes common to all vertebrates. To facilitate genetic analysis of such mutations, we have identified DNA polymorphisms closely linked to each of the 25 centromeres of zebrafish, placed centromeres on the linkage map, increased the number of mapped PCR-based markers to 652, and consolidated the number of linkage groups to the number of chromosomes. This work makes possible centromere-linkage analysis, a novel, rapid method to assign mutations to a specific linkage group using half-tetrads.     

Synthesis of methyl beta-D-fructoside catalyzed by levansucrase from Rahnella aquatilis

Methyl beta-D-fructoside(MF) was formed from sucrose and methanol by a transfructosylation reaction using recombinant levansucrase from Rahnella aquatilis. The increase in the yield of MF formation was achieved by increasing methanol concentration. The enzyme stability at higher concentrations of methanol was maintained by lowering the reaction temperature. The optimum temperature and sucrose concentration for MF formation was 10 degrees C and 50 gL(-1) respectively and the yield of MF was 70%.     

Targeted and passive environmental DNA approaches outperform established methods for detection of quagga mussels,Dreissena rostriformis bugensis in flowing water

1. The early detection of invasive non‐native species (INNS) is important for informing management actions. Established monitoring methods require the collection or observation of specimens, which is unlikely at the beginning of an invasion when densities are likely to be low. Environmental DNA (eDNA) analysis is a highly promising technique for the detection of INNS—particularly during the early stages of an invasion.
2. Here, we compared the use of traditional kick‐net sampling with two eDNA approaches (targeted detection using both conventional and quantitative PCR and passive detection via metabarcoding with conserved primers) for detection of quagga mussel, Dreissena rostriformis bugensis, a high priority INNS, along a density gradient on the River Wraysbury, UK.
3. All three molecular tools outperformed traditional sampling in terms of detection. Conventional PCR and qPCR both had 100% detection rate in all samples and outperformed metabarcoding when the target species was at low densities. Additionally, quagga mussel DNA copy number (qPCR) and relative read count (metabarcoding) were significantly influenced by both mussel density and distance from source population, with distance being the most significant predictor.
4. Synthesis and application. All three molecular approaches were more sensitive than traditional kick‐net sampling for the detection of the quagga mussel in flowing water, and both qPCR and metabarcoding enabled estimates of relative abundance. Targeted approaches were more sensitive than metabarcoding, but metabarcoding has the advantage of providing information on the wider community and consequently the impacts of INNS.

     

An immunohistochemical study of the pancreatic endocrine cells of the ddN mouse

The regional distribution and frequency of the pancreatic endocrine cells in the ddN mouse were studied using specific antisera against insulin, glucagon, somatostatin and human pancreatic polypeptide (hPP). In the pancreatic islets, most of insulin-immunoreactive (IR) cells were located in the central region, and glucagon-, somatostatin and hPP-IR cells were located in the peripheral region regardless of the lobe. In the splenic part, glucagon-IR cells were also located in the central regions, and more numerous somatostatin-IR cells were detected in the central regions as compared with the duo-denal part. hPP-IR cells were restricted to the peripheral regions in both lobes but more numerous cells were detected in the duodenal portion. In the exocrine parenchyma of the splenic lobe, only insulin- and glucagon-IR cells were detected but all four kinds of IR cells were observed in the duodenal portion. In addition, insulin and hPP-IR cells were also demonstrated in the pancreatic duct regions. In conclusion, some strain-dependent characteristic distributional patterns of pancreatic endocrine cells were found in the ddN mouse with somewhat different distributional patterns between the two pancreatic lobes.