Development of a novel quadruple auxotrophic host transformation system by argB gene disruption using adeA gene and exploiting adenine auxotrophy in Aspergillus oryzae

We previously designed a triple auxotrophic host-vector system in Aspergillus oryzae by isolating red-colored adenine auxotrophic mutants upon UV mutagenesis of a double auxotrophic host (niaD-sC-). In the present study an effort to exploit this system and construct a novel quadruple auxotrophic host was made by disrupting the argB gene involved in arginine biosynthesis. The argB gene-disruption cassette was generated by fusion PCR, which required only two steps of PCR to insert the selectable marker, adeA, into the target argB gene. The chimeric DNA fragment was transformed into the triple auxotrophic strain (niaD-sC-adeA-) and the argB disruptants were obtained with a high rate of efficiency (approximately 40%). The argB disruptants were characterized by normal colony color and reversal of arginine auxotrophy by introduction of the wild-type argB gene. Quadruple auxotrophic strains (niaD-sC-DeltaargB adeA- or niaD-sC-DeltaargB adeB-) were subsequently isolated upon UV mutagenesis of the triple auxotrophic strain (niaD-sC-DeltaargB) followed by screening of red-colored colonies for adenine auxotrophy. The results obtained showed that the adeA gene served as an efficient selection marker in developing a novel host-vector system with quadruple auxotrophy in A. oryzae, thus providing a powerful tool to breed multiple auxotrophic mutants from a deuteromycete wherein sexual crossing is impossible.     

Large-scale single-nucleotide polymorphism (SNP) and haplotype analyses, using dense SNP Maps, of 199 drug-related genes in 752 subjects: the analysis of the association between uncommon SNPs within haplotype blocks and the haplotypes constructed with haplotype-tagging SNPs

To optimize the strategies for population-based pharmacogenetic studies, we extensively analyzed single-nucleotide polymorphisms (SNPs) and haplotypes in 199 drug-related genes, through use of 4,190 SNPs in 752 control subjects. Drug-related genes, like other genes, have a haplotype-block structure, and a few haplotype-tagging SNPs (htSNPs) could represent most of the major haplotypes constructed with common SNPs in a block. Because our data included 860 uncommon (frequency <0.1) SNPs with frequencies that were accurately estimated, we analyzed the relationship between haplotypes and uncommon SNPs within the blocks (549 SNPs). We inferred haplotype frequencies through use of the data from all htSNPs and one of the uncommon SNPs within a block and calculated four joint probabilities for the haplotypes. We show that, irrespective of the minor-allele frequency of an uncommon SNP, the majority (mean +/- SD frequency 0.943+/-0.117) of the minor alleles were assigned to a single haplotype tagged by htSNPs if the uncommon SNP was within the block. These results support the hypothesis that recombinations occur only infrequently within blocks. The proportion of a single haplotype tagged by htSNPs to which the minor alleles of an uncommon SNP were assigned was positively correlated with the minor-allele frequency when the frequency was <0.03 (P<.000001; n=233 [Spearman's rank correlation coefficient]). The results of simulation studies suggested that haplotype analysis using htSNPs may be useful in the detection of uncommon SNPs associated with phenotypes if the frequencies of the SNPs are higher in affected than in control populations, the SNPs are within the blocks, and the frequencies of the SNPs are >0.03.     

Assay method for Escherichia coli photolyase activity using single-strand cis-syn cyclobutane pyrimidine dimer DNA as substrate

A high-performance liquid chromatography method for the assay of Escherichia coli photolyase activity was developed. When cis-syn cyclobutane pyrimidine dimer was used as substrate, the Michaelis constant (K(m)) value for the photolyase activity was 100 nM. The linear range of the calibration curve of the photolyase activity was 0.026-6.64 microU/assay tube. The correlation coefficient for this linearity was 0.998. The limit of detection (S/N = 3) was 26 nU/assay tube. The photolyase activity was increased 1.6-fold in the presence of 5,10-methenyltetrahydrofolic acid in the enzyme reaction mixture.     

Dibasic amino acid residues at the carboxy-terminal end of kinase homology domain participate in the plasma membrane localization and function of phosphatidylinositol 5-kinase gamma

Type I phosphatidylinositol 4-phosphate (PI(4)P) 5-kinases (PIP5Ks) catalyze the synthesis of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)), an essential lipid molecule involved in various cellular processes such as regulation of actin cytoskeleton and membrane traffic. The protein localizes to the plasma membrane where its activity has been shown to be regulated by small GTPase ARFs and/or phosphatidic acid. Deletion analysis of amino- or carboxy-terminal sequences of PIP5Kgamma fused with EGFP demonstrated that the presence of central kinase homology domain (KHD), a 380 amino acid-long region highly conserved among PIP5K family, was necessary and sufficient for the plasma membrane localization of PIP5Kgamma. Particularly, the dibasic Arg-Lys sequence located at the carboxy-terminal end of KHD was shown to be crucial for the plasma membrane targeting of PIP5Kgamma, since the deletion or charge-reversal mutation of this dibasic sequence resulted in the mislocalization of the protein to the cytoplasm. Mislocalized mutants also failed to complement the temperature-sensitive growth of Saccharomyces cerevisiae mss4-1 mutant defective in PIP5K function. The presence of dibasic residues at the C-terminal end of KHD was conserved among mammalian as well as invertebrate PIP5K family members, but not in the type II PIPKs that are not targeted to the plasma membrane, suggesting that the conserved dibasic motif provides a mechanism essential for the proper localization and cellular function of PIP5Ks.     

Patterns of intracellular calcium oscillations in horse oocytes fertilized by intracytoplasmic sperm injection: possible explanations for the low success of this assisted reproduction technique in the horse

In all species studied, fertilization induces intracellular Ca2+ ([Ca2+]i) oscillations required for oocyte activation and embryonic development. This species-specific pattern has not been studied in the equine, partly due to the difficulties linked to in vitro fertilization in this species. Therefore, the objective of this study was to use intracytoplasmic sperm injection (ICSI) to investigate fertilization-induced [Ca2+]i signaling and, possibly, ascertain problems linked to the success of this technology in the horse. In vivo- and in vitro-matured mare oocytes were injected with a single motile stallion sperm. Few oocytes displayed [Ca2+]i responses regardless of oocyte source and we hypothesized that this may result from insufficient release of the sperm-borne active molecule (sperm factor) into the oocyte. However, permeabilization of sperm membranes with Triton-X or by sonication did not alleviate the deficient [Ca2+]i responses in mare oocytes. Thus, we hypothesized that a step downstream of release, possibly required for sperm factor function, is not appropriately accomplished in horse oocytes. To test this, ICSI-fertilized horse oocytes were fused to unfertilized mouse oocytes, which are known to respond with [Ca2+]i oscillations to injection of stallion sperm, and [Ca2+]i monitoring was performed. Such pairs consistently displayed [Ca2+]i responses demonstrating that the sperm factor is appropriately released into the ooplasm of horse oocytes, but that these are unable to activate and/or provide the appropriate substrate that is required for the sperm factor delivered by ICSI to initiate oscillations. These findings may have implications to improve the success of ICSI in the equine and other livestock species.     

Dehydrotrametenonic acid and dehydroeburiconic acid from Poria cocos and their inhibitory effects on eukaryotic DNA polymerase alpha and beta

A new lanostane-type triterpene acid, (20xi)-3-oxolanosta-7,9(11),24-trien-21-oic acid (1; dehydrotrametenonic acid), along with a known triterpene acid, dehydroeburiconic acid (2), were isolated from the epidermis of the sclerotia of Poria cocos. The structure of 1 was analyzed on the basis of spectroscopic methods. Compounds 1 and 2 inhibited calf DNA polymerase alpha and rat DNA polymerase beta, with the 50% inhibition values of 45.5 microM (1) and 40.8 microM (2), and 86.5 microM (1) and 30.0 microM (2), respectively.     

Supplemental effects and metabolic fate of crystalline arginine in juvenile shrimp Marsupenaeus japonicus

Feeding experiments with juvenile kuruma shrimp (Marsupenaeus japonicus) were conducted to understand the effects of supplemental levels of crystalline arginine hydrochloride on the growth and assimilation of arginine. In experiment 1 the juvenile shrimp were maintained on diets with and without arginine supplements. The addition of 3.0% arginine to a casein-based diet was slightly effective in improving the growth of the juveniles. In experiment 2, tracer experiments using [14C] arginine were conducted to clarify the ingestion and assimilation of arginine, 9 and 24 h after feeding, at different levels of supplemental arginine. Tracer experiments showed that the leaching rate of arginine 1 h after feeding ranged from 16 to 26% in the diets with different levels (0, 0.1, 1.5, 3.0 and 6.0%) of supplemental arginine; that is, 74-84% of the given arginine was actually ingested by the shrimp fed the diets. However, with increasing levels of supplemental arginine the incorporation rate of arginine into the whole body decreased and the utilization of absorbed arginine for body protein synthesis was reduced, whereas the excretion of absorbed arginine was increased. Thus, the absorbed arginine was not effectively utilized for body protein synthesis when large amounts of arginine were supplemented to the diets.     

Interleukin 15 induces the signals of epidermal proliferation through ERK and PI 3-kinase in a human epidermal keratinocyte cell line,HaCaT

Interleukin 15 (IL-15) is a potent stimulator of proliferation and an inhibitor of apoptosis in lymphocytes. We attempted to elucidate the mechanism of IL-15 function in HaCaT keratinocytes. We found that 5-bromo-2(')-deoxyuridine incorporation increased in a dose-dependent manner with IL-15. This was blocked by MEK inhibitor U0126 or PI 3-K inhibitor LY294002. ERK1/2 and Akt phosphorylation by IL-15 were detected in a dose- and time-dependent manner. U0126 and LY294002 abolished ERK1/2 and Akt phosphorylation, respectively. DNA fragmentation and Annexin V binding accompanied by UVB-induced apoptosis were reduced by 30-50% with IL-15. Taken together, IL-15 induced cellular proliferation and had an anti-apoptotic effect on keratinocytes, in which ERK1/2 and Akt phosphorylation played crucial roles. The signal transduction pathways of IL-15 in keratinocytes were partially elucidated; they share a substantial part with growth signals induced by EGF. These results suggest a therapeutic approach to inflammatory skin diseases by controlling these signals.