The polar neutral and basic taxoids isolated from needles and twigs of Taxus cuspidata and their biological activity

Twelve basic taxoids and 22 neutral taxoids were isolated from basic and polar neutral fractions of the extracts of needles and twigs of Taxus cuspidata. Among them, taxine NA-13, 3,11-cyclotaxinine NN-1, taxinine NN-6, 11(15→1)abeo-taxinine NN-1, taxine NA-8, and taxine NA-4 were isolated first from natural sources by us. The cytotoxic activity of isolated compounds was evaluated against three human cell lines: normal human fibroblast cells (WI-38), malignant tumor cells induced from WI-38 (VA-13), and human liver tumor cells (HepG2). 7-Epitaxol, 7-epicephalomannine, taxinine NN-6, taxine NA-2, taxuspine H, and taxagifine were active toward VA-13 cells and 7-epitaxol, 7-epicephalomannine, taxinine NN-1, 9,10-deacetyltaxinine, and taxagifine were active toward HepG2 cells. The multidrug-resistant (MDR) cancer reversal activity of isolated compounds was evaluated on the basis of the amount of vincristine (VCR) accumulated in MDR human ovarian cancer 2780 AD. Taxine NA-8, taxine NA-2, 5-cinnamoyl-10-acetyltaxicin II, and taxinine NN-1 indicated stronger MDR cancer reversal activity than verapamil. The result of primary screening based on 39 human cancer cell lines suggests that taxinine NN-1 belongs to a new mechanistic class and is a new anticancer agents. 7-Epicephalomannine was found to be an effective anticancer agent with tubulin as its molecular target, which is the same as paclitaxel. Part of this work was presented at the 74th Annual Meeting of the Chemical Society of Japan, Tanabe, March 1998, and the 41st Symposium on the Chemistry of Terpenes, Essential Oils, and Aromatics, Gifu, December 1998. The results of this work were also presented as patent documents at PCT Int: Appl WO 2001007040 A1, February 2001; Appl WO 2000-Jp 5036, 27 July 2000. Priority: JP 1999-214273, 28 July 1999; JP 1999-224652, 6 August 1999; JP 2000-76404, 14 March 2000     

Large-Scale Purification and Characterization of Barley Limit Dextrinase,a Member of the α-Amylase Structural Family

Homogeneous barley limit dextrinase (LD) was isolated on a large scale in a yield of 9 mg/kg of 10-day germinated green malt. This represents a 9,400-fold purification and 29% recovery of the activity in a flour extract in 0.2M NaOAc (pH 5.0) containing 5 mM ascorbic acid. The purification protocol consists of precipitation from the extract at 20–70% saturated ammonium sulfate (AMS), followed by diethylaminoethyl (DEAE) 650S Fractogel anion-exchange chromatography, and affinity chromatography on β-cyclodextrin-Sepharose in the presence of 2M AMS. LD was eluted by 7 mMβ-cyclodextrin and contains a single polypeptide chain of 105 kDa (SDS-PAGE) and pI 4.3. Sequence analysis of tryptic fragments, prepared from 2-vinylpyridinylated LD and purified by RP-HPLC, identified short motifs recognized in β-strand 2, 3, and 5 characteristic of a catalytic (β/α)₈-barrel domain of the α-amylase family of amylolytic enzymes. Barley LD has ≈50 and 85% sequence identity to bacterial pullulanases and rice starch debranching enzyme, respectively. By using ¹H-NMR spectroscopy, LD hydrolyzes specifically α-1,6-glucosidic linkages in pullulan and a branched oligodextrin, 6²-O-α-maltotriosyl-maltotriose, with retention of the α-anomeric configuration. β-Cyclodextrin competitively inhibits the LD activity with K_i of 40 μM, while K_i is 1.9 mM and 2.4 mM for α-cyclodextrin and γ-cyclodextrin, respectively.     

Microwave heating of tea residue yields polysaccharides, polyphenols, and plant biopolyester

Microwave heating was used to produce aqueous-soluble components from green, oolong, and black tea residues. Heating at 200-230 degrees C for 2 min extracted 40-50% of polysaccharides and 60-70% of the polyphenols. Solubilization of arabinose and galactose by autohydrolysis occurred with heating above 170 degrees C, whereas heating above 200 degrees C was necessary to solubilize xylose. Catechins were soluble in water by heating at low temperature (110 degrees C); however, new polyphenols having strong antioxidant activity were produced above 200 degrees C. The amount of solubilized materials and antioxidant activity increased with increased fermentation of harvested tea leaves (green tea < oolong tea < black tea). Cutin, a plant biopolyester, remained in the residue after heating as did cellulose and lignin/tannin. The predominant cutin monomer that was recovered was 9,10-epoxy-18-hydroxyoctadecanoic acid, followed by dihydroxyhexadecanoic acid and 9,10,18-trihydroxyoctadecanoic acid.     

Close linkage of a blast resistance gene,Pias(t), with a bacterial leaf blight resistance gene,Xa1-as(t), in a rice cultivar ‘Asominori’

It has long been known that a bacterial leaf blight-resistant line in rice obtained from a crossing using ‘Asominori’ as a resistant parent also has resistance to blast, but a blast resistance gene in ‘Asominori’ has not been investigated in detail. In the present study, a blast resistance gene in ‘Asominori’, tentatively named Pias(t), was revealed to be located within 162-kb region between DNA markers YX4-3 and NX4-1 on chromosome 4 and to be linked with an ‘Asominori’ allele of the bacterial leaf blight resistance gene Xa1, tentatively named Xa1-as(t). An ‘Asominori’ allele of Pias(t) was found to be dominant and difference of disease severity between lines having the ‘Asominori’ allele of Pias(t) and those without it was 1.2 in disease index from 0 to 10. Pias(t) was also closely linked with the Ph gene controlling phenol reaction, suggesting the possibility of successful selection of blast resistance using the phenol reaction. Since blast-resistant commercial cultivars have been developed using ‘Asominori’ as a parent, Pias(t) is considered to be a useful gene in rice breeding for blast resistance.     

Quantitative trait loci that control body weight and obesity in an F2 intercross between C57BL/6J and DDD.Cg-Ay mice

I have developed a congenic mouse strain for the A(y) allele at the agouti locus in an inbred DDD/Sgn strain, DDD.Cg-A(y). DDD.Cg-A(y) females are extremely obese and significantly heavier than B6.Cg-A(y) females. The objectives of this study were to determine the genetic basis of obesity in DDD.Cg-A(y) mice, and to determine whether or not their high body weight was due to the presence of DDD background-specific modifiers. I performed quantitative trait locus (QTL) analyses for body weight and body mass index in two types of F(2) mice [F2 A(y) (F(2) mice carrying the A(y) allele) and F(2) non-A(y) (F2 mice without the A(y) allele)] produced by crossing C57BL/6J females and DDD.Cg-A(y) males. The results of the QTL analysis of F(2) A(y) mice were very similar to those obtained for F(2) non-A(y) mice. It was unlikely that the high body weight of DDD.Cg-A(y) mice was due to the presence of specific modifiers. When both F(2) datasets were merged and analyzed, four significant body weight QTLs were identified on chromosomes 6, 9, and 17 (2 loci) and four significant obesity QTLs were identified on chromosomes 1, 6, 9, and 17. Although the presence of DDD background-specific modifiers was not confirmed, a multifactorial basis of obesity in DDD.Cg-A(y) females was thus revealed.     

Mathematical Model of Lateral Oxygen Diffusion and Respiration of Roots for Estimating the Diffusion Coefficient and Affinity to Oxygen

We propose herein a mathematical model, for calculating the oxygen diffusion coefficient (D) and K _m value of a cell (M) based on oxygen concentration-respiration rate curve obtained under the condition that oxygen was supplied by lateral diffusion in roots. The model was deductively developed on the assumption that the roots were uniform and cylindrical and that the oxygen concentration and respiration rate were interdependent.

We estimated D and M from the oxygen concentration-respiration curves of rice (cv. Fujisaka 5) and wheat (cv. Chinese Spring) using this model. These values showed a certain range as follows. In rice, 0.003 cm² h^-1 ≤ D < 0.037 cm² h^-1, and 0.55 μmol mL^-1 ≤ M < 0.75 μmol mL^-1, in wheat, 0.016 cm² h^-1 ≤ D < 0.093 cm² h^-1, and 2.1 μmol mL^-1 ≤ M < 2.2 μmol mL^-1. By comparing the values, rice appeared to be more adapted to hypoxia than wheat, because rice had lower D and M values implying the existence of a safeguard mechanism and high affinity to oxygen. Higher oxygen uptake rate under hypoxia in rice was derived from the lower M value, which exceeded the low D.     

Molecular mapping and identification of candidate gene conferring organophosphate-sensitive reaction in sorghum (Sorghum bicolor)

Some sorghum cultivars are sensitive to organophosphate insecticides, which cause red to purple injury spots and, in severe cases, leaf death. Sensitivity to organophosphates is conditioned by a single locus on chromosome 5. We constructed a high-density genetic map around the locus, termed osr, using DNA markers, and delimited osr to a 377-kb region on the short arm of chromosome 5. Sequence analysis of this region predicted 19 gene candidates. Four of the candidates are homologous to a disease resistance NBS-LRR gene. mRNA-Seq analysis of gene expression and SNPs from two resistant cultivars (‘JN43’ and ‘Greenleaf’) and one sensitive cultivar (‘Nakei MS3B’) suggest that one or more members of this NBS-LRR gene family are osr. Our results suggested that the recessive allele of osr results in sensitivity to OP insecticides.     

Physicochemical properties of water-soluble myofibrillar proteins prepared from chicken breast muscle

The solubility of skeletal muscle myofibrillar proteins in water was examined. The solubility of the proteins was found to be sensitive to ionic strength and pH of the solution. At the ionic strength of less than 12 mM and neutral pH, more than 80% of myofibrillar proteins were solubilized. Heating at a temperature of more than 70°C was required for the proteins to retain their solubility. The solubility of freeze‐dried protein powder prepared from water‐soluble myofibrillar proteins was also examined, and it was found that addition of trehalose and heating were essential for re‐solubilization in water. Amino acid composition of water‐soluble myofibrillar proteins was found to be almost the same as that of myofibrillar proteins.     

Apolipoprotein gene polymorphisms as cause of cholesterol QTLs in mice

Quantitative trait locus (QTL) analyses of plasma cholesterol levels were carried out in three sets of F(2) mice that were formed in a 'round-robin' manner from C57BL/6J, KK (-A(y)), and RR strains. Six QTLs were identified on chromosomes 1 (Cq1, Cq2, and Cq6), 3 (Cq3), and 9 (Cq4 and Cq5); of these, Cq2 colocalized with Cq6, and Cq4 colocalized with Cq5. The major candidate gene for Cq2 and Cq6 is Apoa2, and that for Cq4 and Cq5 is Apoa4. The adequacy of polymorphisms in candidate genes as cause of QTLs was investigated in this study. For Apoa2, three different alleles (Apoa2(a), Apoa2(b), and Apoa2(c)) are known. Since there was no significant physiologic difference between Apoa2(a) and Apoa2(c) alleles, previous hypothesis that Apoa2(b) was different from Apoa2(a) and Apoa2(c) in the ability to increase cholesterol levels was further supported. Presumably, G-to-A substitution at nucleotide 84 and/or C-to-T substitution at nucleotide 182 are crucial to make the Apoa2(b) unique. On the other hand, for Apoa4, the most striking polymorphism was the number of Glu-Gln-Ala/Val-Gln repeats in carboxyl end; however, this might not be responsible for QTLs. Instead, a silent mutation, C-to-T substitution at nucleotide 771, was shown to be completely correlated with the occurrence of QTLs in a total of six F(2) intercrosses. Provisionally, but reasonably, these base substitutions are qualified as primary causes that constitute QTL effect. The potential strategy for identifying genes and base substitutions underlying QTLs is discussed.     

Amorphous clay minerals of recent volcanic ash soils in hokkaido (ii)

In the previous study (1), it was shown that the size-fractions finer than 2 μ of the soil samples were abundant in siliceous amorphous materials. These materials are more siliceous in the specimens of the A horizons than in the C and tend to become more siliceous with increase of particle-size. Discrete amorphous silica is present in all the size-fractions finer than 2 μ and its amount is considerably greater in coarser clay fractions. Since the proportions of coarser clay fractions are greater in the whole clay fractions, it is considered that the amorphous silica of the coarser clay fractions play a significant role in determining the properties of all of the amorphous clay minerals contained. Amorphous silica exists not only in the fractions finer than 2 μ, but also in the fractions coarser than 2 μ, even in the fine sand fractions of the A horizons.