Identification of cytochrome P450 and glutathione-S-transferase genes preferentially expressed in chemosensory organs of the swallowtail butterfly, Papilio xuthus L

The swallowtail butterfly, Papilio xuthus L., feeds exclusively on members of the plant family, Rutaceae. Female butterflies lay eggs in response to specific chemicals contained in their host plants. They perceive a variety of polar compounds as oviposition stimulants through the tarsal chemosensilla of the foreleg by drumming upon the leaf surface. We undertook an expressed sequence tag (EST) analysis to identify the chemosensory-related genes that are expressed in chemosensilla on the tarsus of P. xuthus. Several genes that showed similarity with biotransformation enzymes were identified from the ESTs. Among them, a cytochrome P450 and a glutathione-S-transferase (GST) were preferentially expressed in the chemosensory organs. We have determined the structure of both cDNA and genomic sequences encoding these enzymes and designated the P450 as CYP341A2, a novel member of CYP341A subfamily, and the GST as GST-pxcs1, respectively. We observed a localized expression of CYP341A2 at the base of tarsal chemosensilla by in situ hybridization. These results suggest that these degrading enzymes play a role in the chemosensory reception for host plant recognition.     

Development of an orexin-2 receptor selective agonist, [Ala(11), D-Leu(15)]orexin-B

Investigation of L-alanine and D-amino acid replacement of orexin-B revealed that three L-leucine residues at the positions of 11, 14, and 15 in orexin-B were important to show selectivity for the orexin-2 receptor (OX(2)) over the orexin-1 receptor (OX(1)). L-Alanine substitution at position 11 and D-leucine substitution at positions 14 and 15 maintained the potency of orexin-B to mobilize [Ca(2+)](i) in CHO cells expressing the OX(2), while their potency for the OX(1) was significantly reduced. In combined substitutions, we identified that [Ala(11), D-Leu(15)]orexin-B showed a 400-fold selectivity for the OX(2) (EC(50)=0.13nM) over OX(1) (EC(50)=52nM). [Ala(11), D-Leu(15)]orexin-B is a beneficial tool for addressing the functional roles of the OX(2).     

Fixed-point observation of Oncomelania nosophora in Kofu Basin--establishment of monitoring system of schistosomiasis japonica in Japan

There are still many Oncomelania snails that inhabit the Kofu Basin, Yamanashi Prefecture, Japan, which had been declared free of schistosomiasis japonica. Due to the need to monitor the situation, a fixed-point observation system using GIS from GPS is being examined. In addition, in broad present or former endemic areas, survey areas are being managed by remote sensing with satellite images or aerial photographs. A simple and effective monitoring method by mobile GIS using PDAs was developed, risk or hazard maps were prepared and a system that would enable a response in the event of reemergence is being examined.     

Synthesis and evaluation of novel pyrrolidinyl sordaricin derivatives as antifungal agents

N-Benzyl pyrrolidinyl sordaricin derivatives have been synthesized from cis-4-hydroxy-D-proline in a stereocontrolled manner. These compounds maintained moderate antifungal activity against several pathogenic fungal strains. Their MIC values against Candida albicans were in the range of 0.25-2 microg/mL.     

Synthesis and evaluation of N-substituted 1,4-oxazepanyl Sordaricins as selective fungal EF-2 inhibitors

Sordaricin analogues possessing 6-methoxy-7-methyl-1,4-oxazepane moiety instead of the sugar part were synthesized and evaluated. It was found that N-substituents on the oxazepane ring had influence on biological activity. In particular, N-(2-methylpropenyl) derivative 12p exhibited potent in vitro antifungal activity. Furthermore, 12p maintained significant activity (MIC 0.25 microg/mL) against Candida albicans SANK51486 even in the presence of 20% horse serum.     

Genomic structure of the sponge,Halichondria okadai calcyphosine gene

Calcyphosine is an EF-hand Ca(2+)-binding protein, which was first isolated from the canine thyroid. It is phosphorylated in a cyclic AMP (cAMP)-dependent manner; then it is thought to be implicated in the cross-signaling between the cAMP and calcium-phosphatidylinositol cascades. Here, we isolated the DNA complementary to RNA (cDNA) of an EF-hand Ca(2+)-binding protein from the sponge, Halichondria okadai and determined its genomic structure. The deduced sequence of the sponge Ca(2+)-binding protein showed significant similarity (about 40% identity) with those of mammal calcyphosines, and the intron positions were well conserved between the sponge and human calcyphosine genes. We considered that the isolated cDNA was that of sponge calcyphosine, and that sponge and mammalian calcyphosines evolved from a common ancestor gene. Recent cDNA projects have revealed that a calcyphosine cDNA is also expressed by human, mouse, and the ascidia. These cDNAs have more than 60% identity with sponge calcyphosine and each other, and all are composed of 208 amino acid residues. On the constructed phylogenetic trees, calcyphosines are essentially divided into two groups, types-I and -II calcyphosines. Type-I calcyphosine may be specific to mammals, and type-II is widely distributed among metazoan species. This suggests that type-II calcyphosine is a rather ancient gene with some essential function.     

Increased expression of kisspeptin and GnRH forms in the brain of scombroid fish during final ovarian maturation and ovulation

ABSTRACT: BACKGROUND: Kisspeptins (Kiss) are prime players in the control of reproductive function through their regulation of gonadotropin-releasing hormone (GnRH) expression in the brain. The experimental scombroid fish, chub mackerel (Scomber japonicus) expresses two kiss (kiss1 and kiss2) and three gnrh (gnrh1, gnrh2, and gnrh3) forms in the brain. In the present study, we analyzed expression changes of kiss and gnrh mRNAs in the brain and corresponding GnRH peptides in the brain and pituitary during final ovarian maturation (FOM) and ovulation. METHODS: Female fish possessing late vitellogenic oocytes were injected with GnRH analogue to induce FOM and ovulation. Fish were observed for daily spawning activities and sampled one week post-injection at germinal vesicle migration (GVM), oocyte hydration, ovulation, and post-ovulatory time periods. Changes in relative mRNA levels of kiss and gnrh forms in the brain were determined using quantitative real-time PCR. Changes in GnRH peptides in the brain and pituitary were analyzed using time-resolved fluoroimmunoassay. RESULTS: Both kiss1 and kiss2 mRNA levels in the brain were low at late vitellogenic stage and increased significantly during the GVM period. However, kiss1 mRNA levels decreased during oocyte hydration before increasing again at ovulatory and post-ovulatory periods. In contrast, kiss2 mRNA levels decreased at ovulatory and post-ovulatory periods. Levels of gnrh1 mRNA in the brain increased only during post-ovulatory period. However, levels of gnrh2 and gnrh3 mRNAs were elevated during GVM and then, decreased during oocyte hydration before increasing again at ovulatory period. During post-ovulatory period, both gnrh2 and gnrh3 mRNA levels declined. Peptide levels of all three GnRH forms in the brain were elevated during GVM and oocyte hydration; their levels were significantly lower during late vitellogenic, ovulatory, and post-ovulatory periods. In contrast, pituitary GnRH peptide levels did not show any significant fluctuations, with the GnRH1 peptide levels being many-fold higher than the GnRH2 and GnRH3 forms. CONCLUSION: The results indicate increased expression of multiple Kiss and GnRH forms in the brain and suggest their possible involvement in the regulation of FOM and ovulation in captive female chub mackerel.     

Neurofibrillary Tangles and the Deposition of a Beta Amyloid Peptide with a Novel N-Terminal Epitope in the Brains of Wild Tsushima Leopard Cats

Beta amyloid (Aβ) deposits are seen in aged individuals in many of the mammalian species that possess the same Aβ amino acid sequence as humans. Conversely, neurofibrillary tangles (NFT), the other hallmark lesion of Alzheimer’s disease (AD), are extremely rare in these animals. We detected Aβ deposits in the brains of Tsushima leopard cats (Prionailurus bengalensis euptilurus) that live exclusively on Tsushima Island, Japan. Aβ42 was deposited in a granular pattern in the neuropil of the pyramidal cell layer, but did not form argyrophilic senile plaques. These Aβ deposits were not immunolabeled with antibodies to the N-terminal of human Aβ. Sequence analysis of the amyloid precursor protein revealed an amino acid substitution at the 7th residue of the Aβ peptide. In a comparison with other mammalian animals that do develop argyrophilic senile plaques, we concluded that the alternative Aβ amino acid sequence displayed by leopard cats is likely to be related to its distinctive deposition pattern. Interestingly, most of the animals with these Aβ deposits also developed NFTs. The distributions of hyperphosphorylated tau-positive cells and the two major isoforms of aggregated tau proteins were quite similar to those seen in Alzheimer’s disease. In addition, the unphosphorylated form of GSK-3β colocalized with hyperphosphorylated tau within the affected neurons. In conclusion, this animal species develops AD-type NFTs without argyrophilic senile plaques.