Synthesis and insecticidal activity of benzoheterocyclic analogues of N'-benzoyl-N-(tert-butyl)benzohydrazide: Part 2. Introduction of substituents on the benzene rings of the benzoheterocycle moiety

A series of N'-benzoheterocyclecarbonyl-N-tert-butyl-3,5-dimethylbenzohydrazide analogues possessing a variety of substituents on the benzene rings of the benzoheterocyle moieties were synthesized and tested for their insecticidal activity. The introduction of a methyl group at the R1 position of the benzoheterocycle moiety strongly increased the insecticidal activity. Among the analogues synthesized, N'-tert-butyl-N'-(3,5-dimethylbenzoyl)-5-methyl-6-chromanecarbohydrazide showed the highest insecticidal activity (LC50 = 0.89 mg litre(-1)).     

Microsatellite polymorphism in Japanese mongrel dogs

The genetic variability of 182 unrelated mongrel dogs living in various areas of Japan (from Hokkaido to Okinawa) was studied by collecting their blood. Ten microsatellite loci were chosen from different autosomal chromosomes. After combining a few rare adjoining alleles to allelic classes, it was confirmed that the Hardy-Weinberg equilibrium was attained in each locus. The polymorphic information contents (PICs) of the loci, Ren37A11, Ren48E01, AHTk253, ZuBeCa30, Ren277K09, Ren42N13, AHT130, PEZ03, PEZ12, and AHT121, were 0.58, 0.63, 0.67, 0.67, 0.68, 0.71, 0.79, 0.80, 0.80, and 0.80, and the power of discriminations (PDs) were 0.80, 0.85, 0.87, 0.88, 0.88, 0.89, 0.94, 0.94, 0.94, and 0.94, respectively. The combined mean exclusion chance (MEC) was 0.9995, indicating that these microsatellite loci are useful for kinship testing of Japanese dogs.     

The TIR–NBS but not LRR domains of two novel N-like proteins are functionally competent to induce the elicitor p50-dependent hypersensitive response

The tobacco N protein recognizes the helicase domain (p50) of the Tobacco mosaic virus (TMV) replicase as an elicitor and mediates hypersensitive response (HR). We obtained two cDNA clones encoding novel N-like (NL) proteins NL-C26 and NL-B69 from Nicotiana tabacum cv. Samsun NN. NL-C26 and NL-B69 had a Toll-interleukin-1 receptor/nucleotide-binding site/leucine-rich repeat (TIR–NBS–LRR) structure and showed 78% and 73% identities to N, respectively. The NL-C26 and NL-B69 genes were also expressed in N. tabacum cv. Samsun nn, which lacks the N gene. Unlike N, NL-C26 and NL-B69, when coexpressed with p50, failed to induce HR on the sites of agroinfiltration in Samsun nn leaves. However, the elicitor-dependent HR in Samsun nn was induced efficiently by chimeric N proteins with the continuous TIR–NBS domains of NL-C26 and NL-B69. On the other hand, the efficiency of HR induction varied significantly among chimeric N proteins with either of the TIR and NBS domains of the NL proteins. In contrast, chimeras carrying the LRR domains of the NL proteins did not induce HR. Thus, the TIR–NBS domains of NL-C26 and NL-B69 could functionally adapt to the LRR domain of N, which may determine the specificity for the elicitor. We speculate that the NL genes are potential HR-inducing resistance genes for undetermined pathogens other than TMV.     

Glutathione supplementation to semen extender improves the quality of frozen-thawed canine spermatozoa for transcervical insemination

The present study was conducted to evaluate whether supplementation of semen extender with glutathione (GSH) can maintain the quality of frozen-thawed canine spermatozoa. Eighteen ejaculates were obtained from 5 dogs and placed in extender (20% egg yolk, Tris, citric acid, lactose, raffinose, antibiotics and 6.5% glycerol) containing 0 (control), 2.5, 5, 7.5 or 10 mM GSH. The samples were cooled to 4 C and then frozen in liquid nitrogen vapor. Motility parameters of the sperm were evaluated at 0, 1, 2, 3, 4, 12 and 24 h after thawing. Sperm motility was higher in the 5 mM GSH group than in the control or 2.5 and 10 mM GSH groups; this effect was observed at 1 to 24 h after thawing (P < 0.05). The 5 mM GSH group had a higher sperm viability index at 12 and 24 h after thawing compared with the other groups (P < 0.05). Acrosome integrity, evaluated at 4 h after thawing, was greater in two of the GSH-treated groups (5 and 10 mM) compared with the control. Lipid peroxidation (LP) levels immediately after thawing were lower in the 5 and 10 mM GSH groups compared with the control, while those at 12 h after thawing did not differ significantly. Frozen-thawed semen in the 5 mM GSH group was used for transcervical insemination of 4 bitches, resulting in delivery of 5 puppies from 2 bitches. These results indicate that supplementation of semen extender with 5 mM GSH was effective in improving motility, longevity and acrosomal integrity and inhibiting LP levels in post-thaw canine spermatozoa, without any adverse impacts on full-term development after transcervical insemination.     

Resveratrol production potential of grape flowers and green berries to screen genotypes for gray mold and powdery mildew resistance

The ability of grape cultivars to produce resveratrol in response to UV radiation is associated with their resistance to fungal pathogens. In this study, we evaluated the UV-induced resveratrol production potential of grape flowers and green berries of 72 grape genotypes. Their ability to produce resveratrol in response to UV radiation was used to establish a selection criterion for screening genotypes for resistance to gray mold and powdery mildew. We quantified resveratrol in grape flower extracts from pre-bloom and early bloom flowers after exposure to UV radiation. There was a strong negative correlation between UV-induced resveratrol production and susceptibility to Botrytis infection. The grape genotype was considered to be susceptible to gray mold when the resveratrol content of whole flower clusters was less than 10 μg g⁻¹ FW after the UV treatment. We determined resveratrol production in response to UV radiation in whole grape berries from 0 to 30 days after full bloom. The ability to produce resveratrol in response to UV increased by 8- to 20-times during this period, depending on the genotype. At 30 days after full bloom, Vitis vinifera genotypes generally had low levels of resveratrol (<50 μg g⁻¹ FW), while interspecific hybrids, especially the North American cultivars, had high levels (>50 μg g⁻¹ FW). There were clear varietal differences in resistance to Erysiphe (powdery mildew) infection. Susceptibility to Erysiphe infection was strongly negatively correlated with UV-induced resveratrol production.     

Effect of halomethyl-1,3,5-triazines on nitrification of ammonia

Nitrification inhibitory activity of halomethyl-1,3,5-triazines was determined by measuring the inhibitory activities on ammonia-oxidation to nitrate (NO₃⁻-N) in an upland soil and on ammonia-oxidation to nitrite (NO₂⁻-N) by Nitrosomonas europaea ATCC 25978 (ATCC) and Nitrosomonas sp TK 794 (TK). Within the chlorinated trimethyl-1,3,5-triazines, those bearing at least one trichloromethyl group inhibited nitrification more strongly, both in soil and in cell suspension of ATCC, than other mono- or dichlorinated methyl-1,3,5-triazines. Introduction of an amino group to 2,4,6-tris(trichloromethyl)-1,3,5-triazine gave 10- and 100-fold increases of nitrification inhibitory activity in soil and ATCC cell culture, respectively. Within the trihalomethyl-1,3,5-triazines, those having tribromomethyl group(s) exhibited rather weaker nitrification inhibition in soil than the corresponding trichloromethyl-1,3,5-triazines, although they showed a strong inhibition in cell suspension. Ammonium oxidation in ATCC was inhibited more strongly than that in TK. In QSAR studies, the optimum log P values were calculated as c 4.30. By using this value it will become possible to design highly active trichloromethyl-1,3,5-triazine nitrification inhibitors.     

Overcoming hybrid lethality in a cross between Japanese pear and apple using gamma irradiation and confirmation of hybrid status using flow cytometry and SSR markers

Although there have been some attempts to obtain intergeneric hybrids between Japanese pear (Pyrus pyrifolia Nakai) and apple (Malus × domestica Borkh.) mature hybrids have not been generated due to hybrid lethality. Shoots from immature hybrid embryos were gamma irradiated and cultured at normal temperature conditions to obtain viable intergeneric plants. Gamma irradiation doses of 50 and 100 Gy cultured at 25 °C killed all shoots but there was one survivor at 150 Gy and 25 °C. Hybrid status of the shoot was confirmed by using morphology, flow cytometry and SSR markers. The intergeneric plant had similar leaf serrations to apple and similar petioles to Japanese pear. Flow cytometric analysis revealed that the intergeneric shoot had a DNA content intermediate between the parents. SSR analysis showed one allele from each parent for all 16 SSR loci investigated. These data strongly suggest that the obtained shoot is a true hybrid between Japanese pear and apple.     

Purification and characterization of two novel cysteine protease inhibitors, Eel-CPI-2 and Eel-CPI-3, in the skin mucus of the Japanese eel Anguilla japonica

We have discovered multiple acidic cysteine protease inhibitors, in addition to the known Eel-CPI-1, in the skin mucus extract of the Japanese eel Anguilla japonica by using the two-dimensional gel system of gelatin reverse zymography. Two of the acidic inhibitors, which we have named Eel-CPI-2 and Eel-CPI-3, were purified to homogeneity by anion exchange chromatography on a column of DEAE-Sepharose CL-6B, followed by fast protein liquid chromatography on Superdex 75 10/300 GL and HiTrap Q HP columns. The amino acid compositions of Eel-CPI-2 and Eel-CPI-3 were found to be almost identical and closely similar to that of the eel galectin AJL1. The molecular masses of Eel-CPI-2 and Eel-CPI-3 were elucidated to be 16,089.080 and 16,089.137 Da, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The apparent dissociation constant of Eel-CPI-2 and Eel-CPI-3 for cysteine protease papain was determined to be 1.79 × 10⁻⁷ and 1.05 × 10⁻⁷ M, respectively, by a quartz crystal microbalance technique.