cDNA cloning of Japanese oyster stress protein homologous to the mammalian 78-kDa glucose regulated protein and its induction by heatshock

ABSTRACT:    The 78-kDa glucose regulated protein (GRP78), a member of stress proteins, was cloned from a cDNA library of Japanese oyster Crassostrea gigas . The analysis on Japanese oyster GRP78 clone of approximately 2.6 kb revealed that the entire open reading frame was 1983 bp long and encoded 661 amino acid residues. At the DNA sequence level, the coding region of Japanese oyster GRP78 gene was 72, 62, and 62% identical to those of chicken GRP78, Japanese flounder HSP70, and Japanese flounder HSC71 genes, respectively. Deduced amino acid sequence of Japanese oyster GRP78 was 84, 62, and 62% identical to those of chicken GRP78, Japanese flounder HSP70, and Japanese flounder HSC71, respectively. Japanese oyster GRP78 contained an 18-residue sequence at the N-terminus that exhibits characteristics of a cleavable signal sequence. It also contained an ATPase domain, and a peptide-binding domain in addition to a Lys-Asp-Glu-Leu (KDEL) peptide motif that is involved in determining endoplasmic reticulum localization. Northern blot analysis showed that GRP78 mRNA was induced with heatshock treatment in the oyster tissues.     

Solubilization of type I and V collagens in Japanese flounder muscle during chilled storage

ABSTRACT:   The post-mortem changes of type I and V collagens in Japanese flounder muscle during chilled storage were examined. The muscle softened significantly within 12 h under chilled storage. Both type I and V collagens isolated by ion-exchange chromatographies showed no remarkable changes in band patterns even after 24 h of storage, suggesting that degraded collagen molecules may be removed from the collagen fraction by a conventional preparation method. In contrast, type I and V collagen molecules were detected by Western blotting with each specific antibody in a saline extract and gradually increased during chilled storage. These results suggest that both type I and V collagens may participate in the post-mortem softening of fish muscle.     

Characteristics of effluent load from a watershed including irrigation ponds

This paper describes an attempt to estimate the mass budget of irrigation ponds within a watershed and the possibilities to control the effluent load of nutrients from the watershed located in the Kyotanabe area. The paddy field lots were irrigated by the ponds and several mountain streams. In the study watershed, there were two ponds located on the upper and lower sides of a paddy area. Water could be pumped up from the lower pond to the upper pond as necessary. At the ponds, the total amount of nitrogen and phosphorus in the inflow loads including the sediment release was larger than those in the outflow loads. In SS, the effect of load reduction in the ponds was high. It is assumed that organic load reduction was not expected in the ponds. The study result indicates that it is possible to remove the nutrients in the ponds when a larger amount of water is pumped up than when the ponds are only in irrigation use. Moreover, the removal capacities of the nutrients could probably be increased in both ponds by controlling the amount of sediment releases. Electronic Publication     

Sediment-complex-binding cellulose breakdown in wetlands of rivers

We have been assessing the activity level of cellulase in wetland sediments to clarify the significance of cellulase for the turnover of plant cellulose in wetlands. In this study, we investigated the cellulose degrading function of sediment in wetlands to clarify the biochemical breakdown mechanism of cellulose. Specifically, we measured cellulase activities of sediments collected from wetlands of the Chikugo River (Fukuoka Prefecture), Midori River (Kumamoto Prefecture), Hamado River (Kumamoto Prefecture), Yodo River (Osaka Prefecture), and Tanaka River (Mie Prefecture). The results revealed that the activity levels differed significantly among rivers. Additionally, the cellulase activities of the sediment were not completely suppressed in the presence of chloramphenicol. These findings suggested that a portion of the cellulase activities were derived from cellulases binding to the components of sediments. Actually, sediments also showed the ability to bind fungal cellulase. Comparison of the binding ability of clay and plant residues, the main components of sediments, revealed that plant residues had significantly higher abilities to bind cellulase. This finding was supported by the fact that there was a strong correlation between the organic matter content in the sediment and the cellulase-binding ability (R = 0.66). Results of this study show that sediment complexes harboring cellulases might function as a bioreactor to degrade cellulose in wetlands.     

Function of meiobenthos and microorganisms in cellulose breakdown in sediments of wetlands with different origins in Hokkaido

To validate the mechanism of cellulose breakdown in cold climate wetlands, we investigated cellulase activity in sediments collected from 17 wetland sites in Hokkaido, the northern area of Japan. We evaluated cellulase activity by quantitative analysis of glucose released from carboxymethyl cellulose and found that sediments from peat fens demonstrated high activity, followed by sediments from lagoons and estuaries. Sediments from peat fens also contained greater amounts of organic matter, followed by lagoons and estuaries, thereby suggesting a strong positive correlation between organic matter content and cellulase activity. Evaluation of cellulase activity by qualitative cellulose zymographic analysis showed that various cellulases with different molecular sizes were implicated in cellulose breakdown in wetlands. Among them, cellulose breakdown in Meguma Pond (peat fen), Notsuke Gulf (peat fen), and Lake Utonai (lagoon) was potentially due to microorganism cellulase, while that in Lake Chobushi (lagoon) was ascribed to meiobenthos (Oligochaeta species) cellulase. The findings presented herein suggest that the origin and activity level of cellulase vary depending on the type of cold climate wetland.     

Hydraulic conductivity, photosynthesis and leaf water balance in six evergreen woody species from fall to winter

To confirm that freeze-thaw embolism is a primary stress for evergreen woody species in winter, hydraulic conductivity, photosynthesis and leaf water potential were measured during fall and winter in trees growing in a cool temperate zone (Nikko) and in a warm temperate zone (Tokyo). We examined two evergreen conifers that naturally occur in the cool temperate zone (Abies firma Siebold & Zucc. and Abies homolepis Siebold & Zucc.), and four evergreen broad-leaved woody species that are restricted to the warm temperate zone (Camellia japonica L., Cinnamomum camphora (L.) J. Presl, Ilex crenata Thunb. and Quercus myrsinaefolia Blume). In Tokyo, where no freeze-thaw cycles of xylem sap occurred, hydraulic conductivity, photosynthesis and water balance remained constant during the experimental period. In Nikko, where there were 38 daily freeze-thaw cycles by February, neither of the tracheid-bearing evergreen conifers showed xylem embolism or leaf water deficits. Similarly, the broad-leaved evergreen trees with small-diameter vessels did not exhibit severe embolism or water deficits and maintained CO(2) assimilation even in January. In contrast, the two broad-leaved evergreen trees with large-diameter vessels showed significantly reduced hydraulic conductivity and shoot die-back in winter. We conclude that freeze-thaw embolism restricts evergreen woody species with large-diameter vessels to the warm temperate zone, whereas other stresses limit the distribution of broad-leaved trees, that have small-diameter vessels, but which are restricted to the warm temperate zone.     

Termite resistance and wood-penetrability of chemically modified tannin and tannin-copper complexes as wood preservatives

We examined the ability of chemically modified tannin and tannin-copper complexes to penetrate wood and the ability of the treated wood to resist termites. Only the tannin-treated wood retained the agents after treatment. Wood with untreated mimosa tannin (MT) retained the least amount, followed by wood with resorcinolated tannin (RMT) and that with catecholated tannin (CMT). When RMT or CMT was mixed with ammonia-copper, the wood retained twice as much of these solutions as the MT -ammonia-copper solution. The degree of retention of RMT-NH₃-Cu and CMT-NH₃-Cu ranged from 268 to 326kg/m³. The solutions penetrated 2–13 mm from the tangential sections of the logs. We also measured the termite resistance conferred by these solutions. Most of the tannin-NH₃-Cu solutions showed contact lethality for termites in the contact toxicity test. However, the termites were fed cellulose treated with those solutions and most survived the oral toxicity test (14 days). Moreover, these solutions reduced the amount of damage to the wood by termites. However, the mortality rate of the termites during the eating-damage test (>21 days) did not reach 100% for any of the solutions except for RMT. As a result of the field stake test with the same log's used for the penetrability test, the mass loss of wood treated with RMT or CMT alone or with RMT + NH₃ + CuCl₂, was about one-third to one-half that of the controls. Because these solutions have good wood penetrability and good termite resistance, they have potential use as low-toxicity wood preservatives.     

Molecular cloning of canine mucosal addressin cell adhesion molecule-1 cDNA and its expression in normal tissues

A cDNA encoding canine mucosal addressin cell adhesion molecule-1 (MAdCAM-1) was cloned. The entire open reading frame of canine MAdCAM-1 cDNA comprises 1137 bp, corresponding to 378 amino acid residues. The deduced amino acid sequence of canine MAdCAM-1 was 55.2%, 53.7%, and 52.4% identical to rat, mouse, and human MAdCAM-1, respectively. Canine MAdCAM-1 appeared to contain two immunoglobulin-like domains at the N-terminus, followed by a mucin-like domain and a third immunoglobulin-like domain. The structures of the dog, rat, and mouse proteins are likely similar because all of the cysteine residues in the immunoglobulin-like domains were conserved. Canine MAdCAM-1 mRNA was confirmed to express extremely in the mesenteric lymph node by RT-PCR.     

Cloning of canine Toll-like receptor 7 gene and its expression in dog tissues

Toll-like receptor 7 (TLR7) is activated by single strand RNA and imidazoquinoline compounds, and induces interferon production. In this study, canine TLR7 cDNA was cloned and sequenced. The full-length cDNA of canine TLR7 gene was 3419bp, encoding 1032 amino acids. The similarities of canine TLR7 with human and mouse TLR7 were 84 and 80% at the nucleotide sequence level, and 86 and 79% at amino acid sequence level, respectively. Further, the expression of TLR7 mRNA was investigated in canine normal tissues by semiquantitative RT-PCR analysis. The common expression level of TLR7 mRNA in tissues from three dogs examined was in large intestine, lung, pancreas, small intestine and skin, though the expression level in each tissue was varied among these healthy dogs. In other tissues (kidney, liver, lymph node, spleen, adrenal gland, and PBMCs), the level of TLR7 mRNA expression was different in individuals.