Molecular cloning and expression of human chondroitin N-acetylgalactosaminyltransferase: the key enzyme for chain initiation and elongation of chondroitin/dermatan sulfate on the protein linkage region tetrasaccharide shared by heparin/heparan sulfate.

Based on sequence homology with the recently cloned human chondroitin synthase, we identified a novel beta1,4-N-acetylgalactosaminyltransferase, which consisted of 532 amino acids with a type II transmembrane protein topology. The amino acid sequence displayed 27% identity to that of human chondroitin synthase. The expression of a soluble form of the protein in COS-1 cells produced an active enzyme, which transferred beta1,4-N-acetylgalactosamine (GalNAc) from UDP-[(3)H]GalNAc not only to a polymer chondroitin representing growing chondroitin chains (beta-GalNAc transferase II activity) but also to GlcUAbeta1--3Galbeta1-O-C(2)H(4)NH-benzyloxycarbonyl, a synthetic substrate for beta-GalNAc transferase I that transfers the first GalNAc to the core tetrasaccharide in the protein linkage region of chondroitin sulfate. Hence, the enzyme is involved in the biosynthetic initiation and elongation of chondroitin sulfate and is the key enzyme responsible for the selective chain assembly of chondroitin/dermatan sulfate on the linkage region tetrasaccharide common to various proteoglycans containing chondroitin/dermatan sulfate or heparin/heparan sulfate chains. The coding region of this enzyme was divided into seven discrete exons and localized to chromosome 8. Northern blot analysis revealed that the chondroitin GalNAc transferase gene exhibited a ubiquitous but markedly differential expression in human tissues and that the expression pattern was similar to that of chondroitin synthase. Thus, more than two distinct enzymes forming the novel gene family are required for chain initiation and elongation in chondroitin/dermatan sulfate as in the biosynthesis of heparin/heparan sulfate.     

Negative effects of acorns on the wood mouse <Emphasis Type="Italic">Apodemus speciosus</Emphasis>

 Antinutritional effects of acorns and tannic acid on the Japanese wood mouse Apodemus speciosus were examined in the laboratory. The first feeding experiment was conducted for 15 days using three types of diet: control diet (laboratory chow for mice), acorns of Quercus serrata (QS), and acorns of Q. mongolica var. grosseserrata (QM), which differ in tannin content (control, tannin free; QS, 2.7% tannic acid equivalent; QM, 8.5%). Six and one of eight mice died in the QM and QS groups, respectively, whereas all mice survived in good health in the control group. Body weight in the QM and QS groups decreased as much as 23.6% and 16.8% in the first 5 days, respectively, whereas that in the control group did not change significantly. Dry matter intake in the QM group was 50.0% and 38.7% less than that in the control and QS, respectively. Apparent dry matter digestibility was not different among the diets, but apparent nitrogen digestibility did differ between the two acorn groups (QM, −17.5%; QS, 12.0%). The logistic regression analyses revealed that the survival of mice was synergistically influenced by both dry matter intake and apparent nitrogen digestibility. In the second experiment, wood mice fed the tannin-free formula diet, which is nutritionally matched to QS and QM acorns except for the tannin, did not suffer antinutritional effects, whereas mice fed the tannin-supplemented formula diets suffered body weight loss and negative nitrogen digestibility. These results indicate that the tannins in acorns could cause serious damage to the wood mouse, which may rely on acorns as a usual diet. Plausible hypotheses explaining how the wood mice could overcome the deleterious effects of the acorns are discussed. Received: May 9, 2002 / Accepted: December 6, 2002     

Microsatellite loci in Japanese quail and cross-species amplification in chicken and guinea fowl

In line with the Gifu University''s initiative to map the Japanese quail genome, a total of 100 Japanese quail microsatellite markers isolated in our laboratory were evaluated in a population of 20 unrelated quails randomly sampled from a colony of wild quail origin. Ninety-eight markers were polymorphic with an average of 3.7 alleles per locus and a mean heterozygosity of 0.423. To determine the utility of these markers for comparative genome mapping in Phasianidae, cross-species amplification of all the markers was tested with chicken and guinea fowl DNA. Amplification products similar in size to the orthologous loci in quail were observed in 42 loci in chicken and 20 loci in guinea fowl. Of the cross-reactive markers, 57.1% in chicken and 55.0% in guinea fowl were polymorphic when tested in 20 birds from their respective populations. Five of 15 markers that could cross-amplify Japanese quail, chicken, and guinea fowl DNA were polymorphic in all three species. Amplification of orthologous loci was confirmed by sequencing 10 loci each from chicken and guinea fowl and comparing with them the corresponding quail sequence. The microsatellite markers reported would serve as a useful resource base for genetic mapping in quail and comparative mapping in Phasianidae.     

Complex formation between Tap and p15 affects binding to FG-repeat nucleoporins and nucleocytoplasmic shuttling.

Mammalian Tap-p15 and yeast Mex67p-Mtr2p are conserved and essential mRNA export factor complexes that transport mRNPs through the nuclear pore. Here, we report that the small subunit p15 affects the binding of the large subunit Tap to repeat nucleoporins. BIAcore measurements revealed that recombinant Tap binds with high affinity (K(d) in the nm range) to repeat nucleoporins and dissociates from them very slowly. In contrast, when recombinant Tap was bound to p15, the derived heterodimeric complex exhibited a significant lower affinity to FG-repeat nucleoporins (K(d) in the microm range). Furthermore, when recombinant Tap lacking the N-terminal nuclear localization sequences (TapDeltaNLS) was microinjected in mammalian cells, it did not shuttle; however, TapDeltaNLS with bound p15 efficiently shuttles between nucleus and cytoplasm. We conclude that heterodimerization of Tap and p15 is required for shuttling of the functional Tap-p15 mRNA exporter complex.     

Kinetic model for synthesis of fructosyl-stevioside using suspended β-fructofuranosidase

The synthesis experiments of fructosyl-stevioside were conducted under the various conditions of the initial concentrations of the substrates and the enzyme. The transfructosylation of stevioside with sucrose and the hydrolyses of sucrose and fructosyl-stevioside simultaneously occurred. The fructosyl-stevioside synthesis was inhibited by the side products, glucose and fructose. A kinetic model was constructed by considering the Ping-Pong Bi Bi mechanism for the transfructosylation, the apparent Ordered Uni Bi mechanism for the hydrolysis and the competitive inhibition by the side products. The model constants were estimated by fitting the model equations with the experimental results for the sucrose hydrolysis and the fructosyl-stevioside synthesis. The model can predict not only the appropriate conditions to efficiently synthesize the fructosyl-stevioside, but also the reaction time giving the maximum conversion.     

Templates of food–habitat resources for the organization of soil animals in temperate and tropical forests

The functioning and structure of terrestrial ecosystems are shaped and maintained by plant–decomposer interactions. The food and habitat of animal populations are biogenic and are mainly of plant origin (plant litter) in terrestrial ecosystems. Primary resources of the food-habitat template for the organization of soil animals are provided by the primary production of plants, and are then modified through decomposition processes by microbial populations. In the microbial decomposition system, the efficiency of carbon utilization by microbial decomposers characterizes the decomposition processes between tropical and temperate forest ecosystems. Tropical forests show poor development of soil reservoir systems because of the high efficiency of lignin decomposition by microbial populations. The decomposition processes of leaf litter are described briefly for the understanding of organization of soil animal communities in tropical and temperate forests. A comparison of decomposition processes shows qualitative differences in decomposition between temperate and tropical forests. The composition of functional groups of soil animals is well explained by the decomposition processes in both forests.     

Isolation and characterization of anthocyanin 5-O-glucosyltransferase from flowers of Dahlia variabilis

In the cyanic flowers ofDahlia variabilis (Asteraceae), an enzyme was demonstrated which catalyzes a glucosyl group transfer from UDP-glucose to the 5 position of anthocyanidin 3-O-glucoside and 3-O-malonylglucoside. The anthocyanin 5-O-glucosyltransferase (5GT) was purified 88-fold at 8 percnt; yield by (NH₄)₂SO₄ precipitation followed by successive chromatography on DEAE-cellulose, Sephacryl S-200 and Mono P. 5GT exhibited a pH optimum at 8.0 and a pI of 4. 2. Its apparent molecular weight calculated from Sephacryl S-200 was 53 kDa. Its activity was stimulated by 2-ME and DTE but strongly inhibited by PCMB and NEM. It was slightly activated by Mg²⁺ and Ca²⁺ but strongly inhibited by Hg²⁺, Zn²⁺, Cu²⁺, Mn²⁺, Fe³⁺ and Al³⁺. No effect of EDTA was observed. The apparent Km values for cyanidin 3-O-glucoside, cyanidin 3-O-(6′′-O-malonyl)glucoside and UDP-glucose were 120 μmol/L, 75 μmol/L and 250 μmol/L, respectively. Pelargonidin 3-O-glucoside and malonylglucoside were also considerable substrates, but low relative activity was observed for delphinidin 3-O-glucoside which has yet not been found inDahlia flowers.Dahlia 5GT showed substrate specificities different from those reported forSilene, Petunia, Matthiola andPerilla. Neither ADP-glucose nor UDP-galactose could serve as glycosyl donor.     

Distribution of lysozyme and protease, and amino acid concentration in the guts of a wood-feeding termite, Reticulitermes speratus (Kolbe): possible digestion of symbiont bacteria transferred by trophallaxis

Distribution of lysozyme and protease, and amino acid concentration in the guts of a wood‐feeding termite, Reticulitermes speratus (Kolbe) (Isoptera, Rhinotermitidae) were studied to examine the possibility that termites digest symbiont bacteria transferred by trophallaxis. Total lysozyme activity was found predominantly in the salivary gland and to a minor extent in the digestive tracts. However, specific lysozyme activity was high in the foregut as well as in the salivary gland. The similarity of the lysozyme pH profile of the salivary gland and of the foregut suggested that the foregut lysozyme came from the salivary gland. Major protease activity having the optimum pH of 7.5 was found in the midgut. Total free amino acid amount and concentration in the midgut was higher than elsewhere in the digestive tract. The possibility that lysozyme secreted from the salivary gland into the foregut digests hindgut bacteria transferred by trophallaxis was discussed.     

Preparation of High Yields of Algal Protoplasts Using Buccal Juice of Sea Hare and Commercial Cellulase

Buccal juice of the sea hare Aplysia juliana was found to degrade algal polysaccharides. The optimal enzyme composition for protoplast preparation from Undaria pinnatifida was protein at 48 μg/ml buccal juice from sea hare, 10 mg/ml cellulase Onozuka-RS, 0.4 M NaCl, 0.8 M sorbitol, 2 mg/ml dextran sulfate sodium salt, and 1 μl/ml 2-mercaptoethanol in 10 mM MES buffer (pH 6.0). Protoplasts of Eisenia bicyclis, Endarachne binghamiae (Phaeophyta), and Ulva pertusa (Chlorophyta) could also be prepared in a similar manner. Yields of these protoplasts were about 10⁷ cells per gram of fresh weight alga. Received January 26, 1998; accepted September 17, 1998.