Bullfrog larval cathepsin E as digestive enzyme

Bullfrog larval cathepsin E as digestive enzyme

Sl18 P29-4 P29-6 Expressed sequence tags (ESTs) from the sea louse Lepeophtheirus salmonis and study of trypsin cDNAs S.C. Johnson, K.V. Ewart, J.A...

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P29-6

Expressed sequence tags (ESTs) from the sea louse Lepeophtheirus salmonis and study of trypsin cDNAs S.C. Johnson, K.V. Ewart, J.A. Osborne, L. Stratton, N.W. Ross and S. Macintosh NRC Institute for Marine Biosciences 141 I Oxford St., Halifax NS, Canada B3H 321 Expressed sequence tags (ESTs) are individual cDNA clones from a tissue or whole animal that are catalogued and partially sequenced (tagged) for gene identification. An EST collection provides a “shotgun” approach to finding relevant cDNA clones from organisms of interest. This is ideal for use in animals that have not yet been well studied because it provides instant tools for investigating aspects of their biochemistry and physiology. L. salmonis is a parasite of Atlantic salmon. We are currently seeking clones that (1) could be useful tools in better understanding the biology of L. salmonis and its adaptations to host parasitism and (2) would serve as vaccine candidates for salmon. A cDNA library was prepared from L. salmonis and 392 clones were sequenced. Of these, 60% have matches to known genes sequences from other animals. Among them are five cDNAs encoding proteins in the trypsin family. The trypsin family clones may be useful in studying the role of louse-derived typsin-like enzymes found in mucus of infected salmon. The role of trypsin in infection and the potential use of the related clones in studying the enzyme are discussed.

Bullfrog larval cathepsin E as digestive enzyme. Mineta T’,‘., Kobayashi K’., Uemura I’., Inokuchi T’ and Yasugi S’. ‘Life Science Institute, Sophia University, 7-l Kioi-cho, Chiyoda-ku, Tokyo, Japan. ‘Department of Biology, Faculty of Science, Tokyo Metropolitan University, Tokyo, Japan. ‘Department of Biology, Faculty of Education, Utsunomiya University, Utsunomiya, Japan. Cathepsin E is a major aspartic proteinase in larval fore-gut of bullfrog, Rana catesbeiana.The results of immunohistochemical analysis with anti-cathepsin E antiserum showed that cathepsin E exists in the epithelium of both manicotto, which is a stomach-like hypertrophic organ, and the proximal part of duodenum. Particularly, the mucous layer of duodenum was intensively stained, and the luminal side of the epithelial cells was immunopositive. The contents in the lumen of fore-gut was also immunopositive. The immunocytochemical localization of cathepsin E was examined by a post-embedding and protein A-gold method at the ultrastructural level. Cathepsin E localized to mucous granules, called dense granules, in the surface mucous cells of duodenum. These findings indicate that cathepsin E functions as a digestive enzyme secreted into the lumen of fore-gut. On the other hand, cathepsin E was hardly observed in the manicotto gland cells. The gland cells were identified as oxyntic cells from the physiological and ultrastructural observations and from the immunohistochemical observation with anti-porcine H+/K+-ATPase antiserum. These results showed evidently that manicotto is specialized for acid secretion and protein digestion takes place in the extensive area of fore-gut including manicotto. We propose the functional model of anuran larval fore-gut. P29-7

P29-5 Central versus peripheral regulation of thyroidal status in a teleost fish, Sciaenups ocelfatus: evidence for a free-running circadian rhythm of T, secretion.

Leiner KA and MacKenzie DS Department of Biology, Texas A&M University,

College

Station, Texas, USA 77843. There is debate about the extent of central regulation in dctennining thyroidal status in fish. The red drum, S. ocellatus, exhibits a high amplitude diurnal rhythm of circulating T, levels. We hypothesized that there may be a dynamic daily activation of the hypothalamo-pituitary-thyroid (HPT) axis in this fish. Red drum were maintained under a 12L: 12D photoperiod and fed once daily at variable times before the lighting was switched to constant dim illumination for up to four days. In one experiment fish continued to be fed during constant illumination, while in another they were not fed. Complete diurnal profiles of T, and T; were obtained by sampling tanks of fish every 3 hours beginning on the last day of the 12L: 12D photoperiod. The rhythm of circulating T, freeran for 2 days under dim illumination in fish fed throughout the experiment. The rhythm also free-ran for 3 days in constant conditions in feed-restricted fish, although with a diminished amplitude over time. Photoperiod appears to be the main environtnental cue which dctennines the phase of the T, cycle, while the amplitude and duration of these component peaks can be tnodified by nutrient intake. These data provide evidence fol a circadian rhythm of T, secretion from the thyroid gland, presumably via central stimulation, that is entrained by dawn.

Retinoic acid stimulates the development of adult-type chromatophores in the flounder. b&&j

and Yamano K

Inland Station, National Research Institute of Aquaculture, Tamaki, Mie 5 19-04, Japan Different doses of 9-cis retinoic acid (9&A) were administered to flounder (teleost) larvae to study the role of retinoic acid in the development of adult-type (ad-) chromatophores during metamorphosis. The highest dose (25 nM) of 9cRA stimulated the ad-melanophore development not only on the eyed side of the fish but also on the blind side, resulting in ambicolored flounder. When flounder larvae were treated with 9cRA at 4 different metamorphic stages, strong stimulatory effect of 9cRA on the chromatophore development was only observed in the fish treated at premetamorphosis. 9cRA administered at later 2 stages had no effect, while ad-chromatophores actually appear at the end of metamorphosis. These data suggest that 9&A stimulate the determination of the developmental fate of neural crest cells for the ad-chromatophores during premetamorphosis. The present results also suggest the presence of neural crest cells or immature chromatophores on both sides of the larval body and that the ad-chromatophore development is somehow inhibited on the blind side in spontaneous metamorphosis. Alltrans retinoic acid (atRA) similarly stimulated the development of ad-chromatophores and caused deformity of fins, as well as 9cRA. The teratogenic effect of atRA was much stronger than that of 9cRA.