Screening of crude drug extracts for prolyl endopeptidase inhibitory activity.

Prolyl endopeptidase (PEP, EC 3.4.21.26) is an enzyme to play a role in metabolism of proline-containing neuropeptides, such as vasopressin, substance P and thyrotropin-releasing hormone (TRH), which were suggested to be involved with learning and memory processes. Then, specific inhibitor of PEP is expected to have antiamnesic effects, and thus we screened forty-six water- and methanol-extracts from crude drugs selected on the basis of traditional Chinese medicine theory, for Flavobacterium prolyl endopeptidase inhibition. Among them, the water-extracts of Rhodiola sacra (IC50, 0.77 microgram/ml) and the methanol-extracts of Lycopodium clavatum (IC50, 1.3 micrograms/ml), Paeonia lactiflora var. trichocarpa (IC50, 5.7 micrograms/ml), Paeonia veitchii (IC50, 2.4 micrograms/ml) and Rhodiola sacra (IC50, 0.67 microgram/ml) showed strong inhibitory activity. In addition, we also examined the PEP inhibitory activity of eleven compounds from Salvia deserta, and found that in addition to a catechol group alpha-hydroxy-para-quinone group may be related to the PEP inhibition.     

Submergence tolerance in immature stages of the stalk-eyed fly Sphyracephala detrahens (Diptera: Diopsidae)

Sphyracephala detrahens (Walker, 1860) (Diptera: Diopsidae) inhabits the riparian zones of streams and rivers. Because of the limited dispersal ability of S. detrahens during egg, larval, and pupal stages, immature individuals are at risk of being submerged by floodwater after heavy rain. In this study, I evaluated the submergence tolerances of immatures of S. detrahens by comparing them to immatures of Drosophila melanogaster, which also feed on rotten fruits but are not restricted to the riparian zone. The results showed that S. detrahens eggs were susceptible to desiccation, but more than 80% of eggs hatched after full submergence. Later instar larvae were more resistant to full submergence than earlier instar larvae. The duration of submergence causing 50% pupation (PD₅₀) in the first, second, and third-instar larvae of S. detrahens were 15.88, 58.46, and 91.74 h, respectively. The PD₅₀ of the third-instar larvae of D. melanogaster was 20.01 h. Third-instar S. detrahens larvae continued to develop in water for a longer duration than D. melanogaster larvae of the same instar. In the pupal stages, late pupae tended to remain afloat longer than early pupae. The duration of submergence causing 50% emergence (ED₅₀) of adults from early and late pupae were 40.70 and 104.74 h, respectively. In the larval and pupal stages, individuals in the later developmental phases tended to be more tolerant to full submergence. The submergence tolerance of the immature stages of S. detrahens may reflect adaptation to an environment with fluctuating water levels.     

Fgf4 is required for left-right patterning of visceral organs in zebrafish

Fgf signaling plays essential roles in many developmental events. To investigate the roles of Fgf4 signaling in zebrafish development, we generated Fgf4 knockdown embryos by injection with Fgf4 antisense morpholino oligonucleotides. Randomized LR patterning of visceral organs including the liver, pancreas, and heart was observed in the knockdown embryos. Prominent expression of Fgf4 was observed in the posterior notochord and Kupffer's vesicle region in the early stages of segmentation. Lefty1, lefty2, southpaw, and pitx2 are known to play crucial roles in LR patterning of visceral organs. Fgf4 was essential for the expression of lefty1, which is necessary for the asymmetric expression of southpaw and pitx2 in the lateral plate mesoderm, in the posterior notochord, and the expression of lefty2 and lefty1 in the left cardiac field. Fgf8 is also known to be crucial for the formation of Kupffer's vesicle, which is needed for the LR patterning of visceral organs. In contrast, Fgf4 was required for the formation of cilia in Kupffer's vesicle, indicating that the role of Fgf4 in the LR patterning is quite distinct from that of Fgf8. The present findings indicate that Fgf4 plays a unique role in the LR patterning of visceral organs in zebrafish.     

Correction: Lack of discrimination of sex and maturity of conspecifics in the copulation attempts of the male stalk-eyed fly,Sphyracephala detrahens (Diptera: Diopsidae)

Journal of Ethology -     

Identification of hematoxylin-stainable protein in epidermal keratohyalin granules as phosphorylated cystatin alpha by protein kinase C.

The hematoxylin-stainable protein (HSP) in keratohyalin granules of the newborn rat epidermis was found to have the same amino acid composition and the same inhibitory and immunological properties as cystatin alpha. However, only its pI value (4.7) differed from that of cystatin alpha (5.3). Alkaline phosphatase treatment of HSP changed its pI value from 4.7 to 5.3. This pI change was inhibited by EDTA, an inhibitor of alkaline phosphatase. Furthermore, 32P from [gamma-32P]ATP was incorporated into recombinant cystatin alpha by a protein kinase C (PKC) preparation in the presence of phosphatidyl serine and Ca2+ ions as co-factors. The incorporation increased dose-dependently with the added cystatin alpha and was inhibited significantly by H-7, a specific inhibitor of PKC. SDS-PAGE autoradiography of the 32P-labeled proteins showed that 32P was incorporated into the cystatin alpha. This incorporation was not observed by the action of cAMP-dependent protein kinase. Therefore, it is highly possible that the HSP is a phosphorylated cystatin alpha and that the phosphorylation is catalyzed specifically by PKC.