Makoto Ohkubo

Development of non-competitive enzyme-linked immunosorbent assays for mummichog Fundulus heteroclitus gonadotropins - examining seasonal variations in plasma FSH and LH levels in both sexes.

The mummichog Fundulus heteroclitus is an excellent experimental fish for reproductive physiology because of its adequate size, easiness for rearing, and controllable reproduction under laboratory conditions. Furthermore, it is the only species that the native GtHs and their subunits have been purified among small experimental fishes. In this study, homologous non-competitive enzyme-linked immunosorbent assays (ELISAs) for the mummichog FSH and LH were developed by raising monoclonal and polyclonal antibodies against the purified GtHs or their subunits, and the plasma hormone levels in various seasons were examined. The cross-reactivity of LH in the FSH ELISA and the cross-reactivity of FSH in the LH ELISA were low, 2.3% and 0.2% respectively, indicating high specificities of both GtH assays. The practical detection limits were 10 pg/well (0.125 ng/ml plasma) for the FSH ELISA and 8 pg/well (0.1 ng/ml plasma) for the LH ELISA. Plasma FSH levels in females indicated distinct correlations with ovarian stages: they were almost undetectable (<0.125 ng/ml) during the post-spawning immature phase (September), low values (0.3 ng/ml) during the cortical alveoli accumulation phase (December), considerably high (1.8 ng/ml) in the vitellogenic phase (February), and very high values (12 ng/ml) during the spawning season (June). The male FSH levels showed similar pattern of changes to that of females, also indicating distinct correlations with testicular activities. Plasma LH levels were considerably high during the spawning period in both sexes (3.3 ng/ml in females and 4.5 ng/ml in males). They were low or undetectable values in non-spawning seasons, and clear correlation with the gonadal stages was not observed. These results indicate the importance of FSH for various reproductive events in multiple spawning fishes, and are consistent with the general understanding that the LH is responsible for final gametes maturation in both sexes. Nonetheless, they further suggest that the role of LH for various reproductive events other than the final maturation may be limited.

Myofibrillar proteolysis by myofibril-bound serine protease from white croaker Argyrosomus argentatus

The modori phenomenon is defined as heat-induced myofibrillar degradation caused by endogenous serine protease(s) of fish muscle during Kamaboko fish meat gel production. This study was undertaken to analyze myofibrillar proteolysis of white croaker Argyrosomus argentatus muscle, which is an ingredient of high quality Kamaboko, by myofibril-bound serine protease (MBSP) under conditions corresponding to the modori phenomenon. White croaker MBSP was stable between pH 2–11 and below 65°C, and about 60% of its initial activity remained after incubation for 2 h under the conditions at 65°C and pH 7.5. About 60% of the enzyme activity was suppressed by 0.5 M NaCl. White croaker MBSP degraded various myofibrillar proteins between 40 and 70°C and pH 6.0–9.0, and preferentially degraded myosin heavy chain rather than other myofibrillar proteins. The enzyme degraded the myosin heavy chain most strongly at 55°C and pH 7.0, and a major part of the bands of myosin heavy chain and its degradation products disappeared for a period of 2 h. These degradation characteristics are very similar to those observed during the modori phenomenon, indicating that MBSP could be a modori-inducing protease involved in the modori phenomenon of white croaker Kamaboko production.

Multiple spawning of amphidromous type ayu Plecoglossus altivelis in a large river, Mogami River System

Female ayu Plecoglossus altivelis were sampled from the Mogami River System, Yamagata Prefecture to investigate the occurrence of multiple spawning that has previously been reported in small rivers. Spawning frequencies of the females were estimated by histological observations of the ovary mainly from the degree of degeneration of postovulatory follicles. In the middle and late part of the spawning period, most females showed characteristics of having spawned two or three times, indicating that multiple spawning is a common phenomenon also in a large river. Mean relative batch fecundity (RBF) and condition factor (without gonads) decreased with the number of spawning batches. Size dependency in the occurrence of the multiple spawning and the RBF values was not observed. These results are similar to those reported in previous studies of ayu from a small river near the Mogami River System, and indicate that the occurrence and properties of multiple spawning are almost the same regardless of the river scale. However, it is possible that the ratios of multiply spawned females may differ due to variations in survival rates during the spawning season.

Haplotype of the mitochondrial DNA control region of a neonatal finless porpoise stranded around the Yokohama Port

62008 33 86 Received 11.4.2007 ; a cepted 8.11.2007 Copyright

A functional motif discovery algorithm for invertebrate EST sequence data

We have developed a new algorithm for invertebrate expressed sequence tag (EST) analysis, termed as the fmEST algorithm, which consists of a systematic homology search, functional motif scanning, and clustering alignment. This study was undertaken to evaluate the validity of our fmEST algorithm in functional motif discovery for invertebrate EST sequence data. Out of 200 unidentified invertebrate ESTs, including 100 arthropod ESTs and 100 mollusk ESTs, 18 arthropod ESTs and 21 mollusk ESTs were identified as fmESTs that contained functional motifs. The nucleotide lengths of arthropod fmEST and mollusk fmEST sequences were distributed from 388 to 954 bp and from 222 to 742 bp, respectively. This result allowed us to annotate these invertebrate fmESTs as various functional genes, while they showed no significant homology to the gene information recorded in the international DNA databases using the conventional BLAST homology search program. In addition, another 1 arthropod EST and 23 mollusk ESTs were assembled into contigs with any identified fmESTs by clustering alignment. Based on these findings, we have concluded that our fmEST algorithm, involving the functional motif discovery procedure, is a valuable approach, enabling us to break new ground in undeveloped invertebrate EST analysis.