Formation and Some Properties of the Acid Phosphatase in Aspergillus terreus

Substrate specificity of purified preparations of phytase from Asp, terreus was examined. The enzyme showed broad specificity. It was found that Asp, terreus produced only one kind of acid phosphatase and it had phytase activity.

Effective materials for the enzyme formation were examined. The formation of the enzyme occurred only during times that mycelia was in contact with inositol.

By differential centrifugation and electron-microscopic autoradiography, it was determined that inositol was incorporated into the mycelia and that it was located at almost the same point as where the active enzyme was located.     

Chemical and Physicochemical Properties of Phytase from Aspergillus terreus

Some chemical and physicochemical properties of the purified phytase preparation produced by Asp. terreus were investigated. From the results of the examination of amino acid analysis, it was suggested that there existed some components other than amino acids in the purified enzyme. Examination of the neutral sugar analysis, therefore, was made by gaschromatography, and it was found that the purified enzyme preparation contained mannose, galactose and a small amount of inositol.

The molecular weight of the enzyme was found to be 214,000 by the Archibald method, and 2.2~2.3×10⁵ by gel-filtration on a Sephadex G–200 column. It was found that by guanidine hydrochloride or by urea, the purified enzyme preparation was dissociated into only one kind of subunit. The native enzyme was supposed to be a homohexamer of the subunits whose molecular weight is 37,000.     

Isolation, characterization and expression of a cDNA encoding an elongation factor-1α from Porphyra yezoensis (Bangiales, Rhodophyta)

We report the isolation, characterization and expression of a cDNA encoding a polypeptide elongation factor‐1α (EF‐1α) from the marine red alga, Porphyra yezoensis. A cDNA clone was isolated from a leafy gametophyte cDNA library and the sequence was analyzed. The clone contained an open reading frame for a protein of 449 amino acids which exhibits sequence similarity to the known EF‐1α. Comparison of the deduced amino acid sequence showed higher similarity to the Porphyra pur‐purea EF‐1αtef‐c (97%) than to the P. purpurea EF‐1αtef‐s (61%). The mRNA was detected both in the leafy gametophyte and the filamentous sporophyte.     

Establishment of a transport system for mouse epididymal sperm at refrigerated temperatures

The exchange of genetically engineered mouse strains between research facilities requires transporting fresh mouse sperm under refrigerated temperatures. Although sperm generally maintains fertility for 48 h at cold temperatures, in vitro fertilization rates of C57BL/6 mouse sperm are low after 48-h cold storage. Furthermore, 48 h is often not sufficient for the specimens to reach their destinations. To increase the availability of this technology, we aimed to extend the cold storage period while maintaining sperm fertility. In this study, we determined the optimal medium for sperm preservation and evaluated the effect of reduced glutathione in the fertilization medium on sperm fertility after cold storage. We found that higher fertility levels were maintained after 72-h cold storage in the preservation medium Lifor compared with storage in paraffin oil, M2 medium, or CPS-1 medium. In addition, 1.0 mM glutathione enhanced sperm fertility. After transporting sperm from Asahikawa Medical University to our laboratory, embryos were efficiently produced from the cold-stored sperm. After transfer, these embryos developed normally into live pups. Finally, we tested the transport system using genetically engineered mouse strains and obtained similar high fertilization rates with all specimens. In summary, we demonstrated that cold storage of sperm in Lifor maintains fertility, and glutathione supplementation increased the in vitro fertilization rates of sperm after up to 96 h of cold storage. This improved protocol provides a simple alternative to transporting live animals or cryopreserved samples for the exchange of genetically engineered mouse strains among research facilities.     

Quantitative microarray-based DNA-DNA hybridization assay for measuring genetic distances among bacterial species and its application to the identification of family Enterobacteriaceae

Quantitative DNA-DNA hybridization to measure the genetic distances among bacterial species is indispensable for taxonomical determination. In the current studies, we developed a method to determine bacterial DNA relatedness on a glass microarray. Reference DNAs representing a total 93 species of Enterobacteriaceae were arrayed on a glass microplate, and signal intensities were measured after 2 hr of hybridization with Cy3-labeled bacterial DNAs. All immobilized DNAs from members of the family Enterobacteriaceae were identified by this method except for DNAs from Yersinia pseudotuberculosis and Y. pestis. These results suggest that quantitative microarray hybridization could be an alternative to conventional DNA-DNA hybridization for measuring chromosome relatedness among bacterial species.     

The effect of a novel leukotriene C4/D4 antagonist, BAY-x-7195, on experimental allergic reaction

The effects of a novel leukotriene (LT) C₄/D₄ antagonist, BAY-x-7195 on experimental allergic reactions in airway and skin were compared to that of ONO-1078. BAY-x-7195 showed an antagonistic action to LTD₄-induced bronchoconstriction in vitro and in vivo. In in vitro experiments, BAY-x-7195 inhibited LTD₄-induced contraction of isolated guinea pig tracheal muscle (pA2=8.03). BAY-x-7195 at doses of 3 – 30 mg/kg clearly inhibited LTD₄-induced increases in respiratory resistance (Rrs) in guinea pigs. In contrast, BAY-x-7195 inhibited significantly U-46619-induced increases in Rrs at a dose of 30 mg/kg in guinea pigs. BAY-x-7195 at doses of 3 — 30 mg/kg inhibited the aerosolized antigen-induced biphasic increase in Rrs in guinea pigs. Moreover BAY-x-7195 inhibited repeated aeroantigen-induced airway hyperreactivity in guinea pigs. In mice, aeroantigen-induced airway inflammation were clearly inhibited by BAY-x-7195. These results show the efficacy of BAY-x-7195 against the antigen-induced increase in airway resistance and antigen-induced airway hyperreactivity in guinea pigs and mice, probably due to anti-LTD₄ antagonistic action and the inhibition of antigen-induced airway inflammation.     

Investigations of motility and fertilization potential in thawed cryopreserved mouse sperm from cold-stored epididymides

Cold transport of epididymides from genetically modified mice is an efficient alternative to the shipment of live animals between research facilities. Mouse sperm from epididymides cold-stored for short periods can maintain viability. We previously reported that cold storage of mouse epididymides in Lifor® perfusion medium prolonged sperm motility and fertilization potential and that the sperm efficiently fertilized oocytes when reduced glutathione was added to the fertilization medium. Cryopreservation usually results in decreased sperm viability; an optimized protocol for cold storage of epididymides plus sperm cryopreservation has yet to be established. Here, we examined the motility and fertilization potential of cryopreserved, thawed (frozen-thawed) sperm from previously cold-stored mouse epididymides. We also examined the protective effect of sphingosine-1-phosphate (S1P) on sperm viability when S1P was added to the preservation medium during cold storage. We assessed viability of frozen-thawed sperm from mouse epididymides that had been cold-transported domestically or internationally and investigated whether embryos fertilized in vitro with these sperm developed normally when implanted in pseudo-pregnant mice. Our results indicate that frozen-thawed sperm from epididymides cold-stored for up to 48 h maintained high fertilization potential. Fertilization potential was reduced after cold storage for 72 h, but not if S1P was included in the cold storage medium. Live pups were born normally to recipients after in vitro fertilization using frozen-thawed sperm from cold-transported epididymides. In summary, we demonstrate an improved protocol for cold-storage of epididymides that can facilitate transport of genetically engineered-mice and preserve sperm viability after cryopreservation.     

Phylogeographic sympatry and isolation of the Eurasian badgers (Meles, Mustelidae, Carnivora): Implications for an alternative analysis using maternally as well as paternally inherited genes

In the present study, to further understand the phylogenetic relationships among the Eurasian badgers (Meles, Mustelidae, Carnivora), which are distributed widely in the Palearctic, partial sequences of the mitochondrial DNA (mtDNA) control region (539-545 base-pairs) as a maternal genetic marker, and the sex-determining region on the Y-chromosome gene (SRY: 1052-1058 base-pairs), as a paternal genetic marker, were examined. The present study revealed ten SRY haplotypes from 47 males of 112 individuals of the Eurasian Continent and Japan. In addition, 39 mtDNA haplotypes were identified from those animals. From the phylogeography of both the uniparentally inherited genes, four lineages were recognized as Japanese, eastern Eurasian, Caucasian, and western Eurasian. The distribution patterns of the mtDNA lineages showed the existence of a sympatric zone between the eastern and western Eurasian lineages around the Volga River in western Russia. Furthermore, the present study suggested that in the Japanese badgers, the larger genetic differentiation of the Shikoku population was attributable to geographic history in the Japanese islands.     

Genetic population structure of the masked palm civet Paguma larvata, (Carnivora: Viverridae) in Japan, revealed from analysis of newly identified compound microsatellites

The masked palm civet Paguma larvata (Carnivora: Viverridae) in Japan has been phylogeographically considered an introduced species from Taiwan. To reveal the population structures and relationships among the P. larvata populations in Japan, seven compound microsatellite loci were isolated from the genome and genotyped for 287 individuals collected from the field. STRUCTURE analysis and factorial correspondence analysis of genotyping data revealed that animals from Japan were divided into four genetic clusters. Geographic distribution of the genetic clusters partly referred to sampling areas, indicating multiple introductions into distinct areas of Japan or independent founding events leading to the generation of different genetic clusters within introduced populations in Japan. The large genetic differentiation of populations in the Shikoku District from those in other areas within Japan suggests that there were at least two introduction routes into Japan, and a possibility that some founders from areas other than Taiwan were also involved in the introduction into Japan. The genetic variation within Japanese populations were not markedly reduced compared with that of Taiwan. The results indicated that the Japanese populations of P. larvata could have retained moderate genetic diversity during founding events, because of multiple introductions, or a large number or high genetic diversity of founders. Although some individuals in Japan showed a sign of admixture between different clusters, there is no evidence that such an admixture markedly increased the genetic diversity within Japanese populations.