Sensitive detection of the c‐KIT c.1430G>T mutation by mutant‐specific polymerase chain reaction in feline mast cell tumours

Here, we describe the establishment of mutant‐specific polymerase chain reaction (PCR) for detection of a c‐KIT c.1430G>T mutation in feline mast cell tumours. Several mutations in feline c‐KIT have been identified, with the c.1430G>T mutation accounting for a significant portion of feline mast cell tumour mutations. The c.1430G>T mutation in c‐KIT exon 9 was detected in 15.7% (11 of 70) of samples by mutant‐specific PCR but in only 7.1% (5 of 70) by PCR–restriction fragment length polymorphism (RFLP) in the genomic DNA isolated from 70 formalin‐fixed paraffin‐embedded sections or cells collected by fine needle aspiration. Mutant‐specific PCR showed remarkably higher detection rate than did PCR–RFLP. DNA sequence analysis did not always yield identical results to those of mutant‐specific PCR, suggesting heterogeneity of tumour cells. Mutant‐specific PCR is a valid and efficient screening tool for detection of the c‐KIT c.1430G>T point mutation in feline mast cell tumours compared with PCR–RFLP and sequencing analysis.     

Performance of a closed recirculating system with foam separation, nitrification and denitrification units for intensive culture of eel: towards zero emission

The development of a closed recirculating aquaculture system that does not discharge effluents would reduce a large amount of pollutant load on aquatic bodies. In this study, eel were reared in a closed recirculating system, which consisted of a rearing tank, a foam separation unit, a nitrification unit and a denitrification unit. The foam separation unit has an inhalation-type aerator and supplies air bubbles to the rearing water. The growth of eel, which were fed a commercial diet, was satisfactory, with gross weight increases of up three times in 3 months. The survival rate under the congested experimental conditions was 91%. The foam separation unit maintained oxygen saturation in the rearing water at about 80%. Furthermore, fine colloidal substances were absorbed on the stable foam formed from eel mucus and were removed from the rearing water by foam separation. Ammonia oxidation and the removal of suspended solids were accomplished rapidly and simultaneously in the nitrification unit. The ammonia concentration and turbidity were kept at less than 1.2 mg of N per litre and 2.5 units, respectively. When the denitrification process was operated, nitrate that accumulated in the rearing water (151 mg of N per litre) was reduced to 40 mg of N per litre. The sludge was easily recovered from the nitrification and denitrification tanks, and the components were found suitable as compost. Based on these results, the intensive aquaculture of freshwater fish such as eel can be achieved using a closed recirculating system without emission.     

Programmed Cell Death Induced by (?)-8,9-Dehydroneopeltolide in Human Promyelocytic Leukemia HL-60 Cells under Energy Stress Conditions

(+)-Neopeltolide is a marine macrolide natural product that exhibits potent antiproliferative activity against several human cancer cell lines. Previous study has established that this natural product primarily targets the complex III of the mitochondrial electron transport chain. However, the biochemical mode-of-actions of neopeltolide have not been investigated in detail. Here we report that (−)-8,9-dehydroneopeltolide (8,9-DNP), a more accessible synthetic analogue, shows potent cytotoxicity against human promyelocytic leukemia HL-60 cells preferentially under energy stress conditions. Nuclear morphology analysis, as well as DNA ladder assay, indicated that 8,9-DNP induced significant nuclear condensation/fragmentation and DNA fragmentation, and these events could be suppressed by preincubating the cells with a pan-caspase inhibitor, N-benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (zVAD). Immunoblot analysis demonstrated the release of cytochrome c from the mitochondria and the cleavage of full-length caspase-3 and poly(ADP-ribose) polymerase (PARP). These results indicated that 8,9-DNP induced caspase-dependent apoptotic programmed cell death under energy stress conditions. It was also found that 8,9-DNP induced non-apoptotic cell death in the presence/absence of zVAD under energy stress conditions. Immunoblot analysis showed the intracytosolic release of apoptosis-inducing factor (AIF), although it did not further translocate to the nucleus. It appears most likely that, in the presence of zVAD, 8,9-DNP triggered necrotic cell death as a result of severe intracellular ATP depletion.     

Reproductive season and female maturity size of coconut crab <Emphasis Type="Italic">Birgus latro</Emphasis> on Hatoma Island,southern Japan

Information about reproductive season, size at sexual maturity and size-dependent reproductive potentials is important for efficient resource management. Such information is necessary to determine seasonal closures and minimum legal size limits. To conserve resources of coconut crab Birgus latro, which are in danger of extinction due to excessive exploitation, their reproductive season, female size at functional maturity and relationship between female size and reproductive potentials were investigated. The reproductive season started in early June and ended late August, and females with higher reproductive potentials showed a tendency to spawn early in the reproductive season. The prohibition of exploiting the resources during the reproductive season, especially early in the season, would be appropriate for sustainability of high reproductive outputs. Fifty percent of investigated females matured functionally at 24.5-mm thoracic length (TL). However, the size at which all females reached functional maturity was 32.3 mm TL, and there was a wide variation in the size of functional maturity among individuals. We must determine minimum legal size by taking the variation into account to allow all individuals to mate at least once before they are caught, which may prevent serious depression in reproductive rate and genetic diversity.     

Distribution of immunoglobulin-containing cells in calves inoculated orally with ruminal Bacteroides succinogenes and Selenomonas ruminantium

Distribution of immunoglobulin(Ig)-containing cells was investigated in calves inoculated orally with live organisms of both Bacteroides succinogenes and Selenomonas ruminantium. Pathological changes and many Ig-containing cells were observed in calves which inoculated three times at 2, 3 and 26 days of age. Follicular germinal center was increased in number and size of the lymph nodes associated with the forestomach, suggesting activation of lymph apparatus. In the associated lymph nodes, IgG-containing cells were predominant and were located in both cortex and medulla, mainly in the medullary cord, B lymphocyte areas. Only a few IgA- and IgM-containing cells were observed in the lymph nodes. Accordingly, the inoculated bacteria may stimulate IgG-containing B lymphocyte populations. A few IgG-containing cells were detected in the mucosa of the forestomach. Ig-containing cells, predominantly IgG, were observed in the mucosa of the abomasum and intestine, and in the mesenteric lymph nodes. However, number of the cells in the mesenteric lymph nodes was smaller than that of the forestomach associated lymph nodes. The results suggest that the intraorally inoculated bacteria may stimulate the maturation of IgG positive lymphocytes in the lymph nodes associated with the forestomach.     

Seed productivity test of CMS lines of Japanese bunching onion (Allium fistulosum L.) possessing the cytoplasm of a wild species, A. galanthum Kar. et Kir.

In a previous study, we developed male sterile lines of Japanese bunching onion (Allium fistulosum L.) possessing the cytoplasm of a wild species, A. galanthum Kar. et Kir., by backcrossing. To evaluate seed productivity of the male sterile lines in practise, they were crossed with the male fertile line, cultivar 'Kujyo', using honeybees as pollinators under field conditions. The number of florets and seeds per inflorescence, seed set and seed germination of the material were investigated. Although variation was observed among the male sterile lines, there were several lines having seed productivity equal to cultivar 'Kujyo'. Our data demonstrate that the male sterile lines of A. fistulosum possessing the cytoplasm of A. galanthum are useful as seed parents for the commercial F seed production of A. fistulosum.     

Developmental changes of phyllochron in near-isogenic lines of rice (Oryza sativa L.) with different growth durations

The repeated elements called phytomers, which consist of leaf, node, internode and axillary bud, play an important role in the development of modular organization in plants. Rice has the striking feature that the rate of the phytomer production is closely synchronized with the rate of leaf emergence (phyllochron). We examined developmental changes of phyllochron by using 10 near-isogenic lines (NILs) showing diversified growth durations in rice. The NILs were established by backcrosses with a strain practically insensitive to photoperiod, and they consisted of early- or late-flowering NILs whose differences of growth duration were caused by the combinations of alleles at 6 loci. The developmental patterns of phyllochron were evaluated by means of a quartic polynomial, which fitted well in most cases. The results indicated that phyllochron greatly changed during development, especially in late-flowering NILs as well as the recurrent parents, although the fluctuation of phyllochron was not so marked in the early-flowering NILs. Thus, the developmental change of phyllochron was highly dependent upon the genotypes and/or growth duration; however, it was associated with neither floral initiation nor temperature, indicating that the change of phyllochron might reflect internal or physiological changes which occur during the life cycle of rice. This revised version was published online in July 2006 with corrections to the Cover Date.     

Indica-japonica differentiation in Chinese rice landraces

Summary Ninety Chinese rice landraces were examined with special reference to the indica-japonica differentiation in terms of traditional criteria, isozyme analysis and PCR analysis of the chloroplast DNA (cpDNA). Cultivars were separated into indica and japonica defined by a discriminant function (Z) based on key characters, as well as by isozyme genotypes. Most indica landraces had chloroplast DNAs with a deletion at the Pst-12 fragment, while most japonica landraces had cpDNAs without the deletion. Two traditionally recognized varietal groups in China, keng and hsien, corresponded largely to the respective japonica and indica revealed in our study. The results obtained in this study showed good agreement for classification of indica and japonica types by the three methods: discriminant analysis by Z value, isozyme analysis, and PCR analysis for cpDNA.     

Construction and characterization of a BAC library of soybean

We have constructed a soybean (Glycine max (L.) Merrill) bacterial artificial chromosome (BAC) library from green leaf protoplasts of the cultivar, Misuzudaizu. The library contains 53,760 clones with an average insert size of 116 kb. About 2.9% chloroplast DNA origin was revealed by PCR and colony hybridization. Apart from 2.8% clones having no insert, this library represents 5.2 genome equivalents. With this genome coverage, the probability of having any DNA sequence represented in the library is higher than 99.5%. Three-dimensional pools of the BAC library in combination with the use of a high efficiency genome scanning (HEGS) electrophoresis facilitate rapid and efficient PCR-based screening. An average of five positive clones were identified after screening the BAC library with SSR and STS markers. BAC-end walking was performed for three SSR associated BACs. This library will provide a good resource for positional cloning of agronomically and biologically important QTL genes that Misuzudaizu possesses.