Occurrence of chondroitin sulfate E in glycosaminoglycan isolated from the body wall of sea cucumber Stichopus japonicus

Glycosaminoglycan was isolated from the body wall of sea cucumber Stichopus japonicus by a method consisting of enzymatic digestion, gel filtration, and ion-exchange chromatography. One gram of sea cucumber glycosaminoglycan was composed of 2.50 mmol of sulfate, 0.47 mmol of N-acetylgalactosamine (GalNAc), 0.53 mmol of glucuronic acid (GlcA), 1.73 mmol of fucose, and a small amount of peptide. When mildly hydrolyzed with 0.1 N H2SO4, this glycosaminoglycan released two products, one consisting of fucose plus sulfate and the other of fucose only. Partially hydrolyzed glycosaminoglycan thus obtained was composed of sulfate, GalNAc, GlcA, and fucose at a molar ratio of 3:2:2:1. Partially hydrolyzed glycosaminoglycan was easily digested with chondroitinase AC II. In ion-exchange chromatography, the digest exhibited four sharp peaks whose retention times agreed with those of unsaturated 0-(delta Di-0S), mono-(delta Di-4S and delta Di-6S), and di-(delta Di-SE) sulfated disaccharide, respectively. The disaccharide unit of sea cucumber glycosaminoglycan was composed of 22.4% chondroitin sulfate E, 11.2% chondroitin, 10.4% chondroitin 4-sulfate, and 56.0% chondroitin 6-sulfate.     

An index for microcarrier dispersion inside a horizontally rotating bioreactor for animal cell culture

An index of particle dispersion is used to quantify the degree of particle dispersion inside the horizontally rotating bioreactor. Resin particles were suspended inside a horizontally rotating cylinder. The states of particle dispersion were observed and recorded on video, from which data the index of particle dispersion was calculated. The effect of the operating parameters particle concentration and cylinder rotational speed on the state of particle dispersion were described using the index. By comparing the quantitative and qualitative classifications of particle dispersion it was confirmed that the new index does reflect the degree of particle dispersion. From the comparison it can also be concluded that if the index is greater than 0.96, the particle distribution can be considered uniform.     

Regulation of biological activity and matrix assembly of laminin-5 by COOH-terminal, LG4-5 domain of alpha3 chain

The basement membrane protein laminin-5 (LN5; alpha3beta3gamma2) undergoes specific proteolytic processing of the 190-kDa alpha3 chain to the 160-kDa form after the secretion, releasing its COOH-terminal, LG4-5 domain. To clarify the biological significance of this processing, we tried to express a recombinant precursor LN5 with a 190-kDa alpha3 chain (pre-LN5), in which the cleavage sequence Gln-Asp was changed to Ala-Ala by point mutation. When the wild-type and mutated LN5 heterotrimers were expressed in HEK293 cells, the wild-type alpha3 chain was completely cleaved, whereas the mutated alpha3 chain was partially cleaved at the same cleavage site (Ala-Ala). pre-LN5 was preferentially deposited on the extracellular matrix, but this deposition was effectively blocked by exogenous heparin. This suggests that interaction between the LG4-5 domain and heparan sulfate proteoglycans on the cell surface and/or extracellular matrix is important in the matrix assembly of LN5. Next, we purified both pre-LN5 and the mature LN5 with the processed, 160-kDa alpha3 chain (mat-LN5) from the conditioned medium of the HEK293 cells and compared their biological activities. mat-LN5 showed higher activities to promote cell adhesion, cell scattering, cell migration, and neurite outgrowth than pre-LN5. These results indicate that the proteolytic removal of LG4-5 from the 190-kDa alpha3 chain converts the precursor LN5 from a less active form to a fully active form. Furthermore, the released LG4-5 fragment stimulated the neurite outgrowth in the presence of mat-LN5, suggesting that LG4-5 synergistically enhances integrin signaling as it is released from the precursor LN5.     

N-acetylglucosaminyltransferase III expression is regulated by cell-cell adhesion via the E-cadherin-catenin-actin complex

Recently, our research group investigated the effects of cell-cell interactions on N-linked oligosaccharides (N-glycans). We found that N-acetylglucosaminyltransferase III (GnT-III) activity, and thus, the enzyme product-bisected N-glycans were induced in cells cultured under dense condition in an E-cadherin-dependent manner. To further explore the underlying molecular mechanism, we examined the effects of alpha-catenin, which is a component of the E-cadherin-catenin complex that can bind to actin cytoskeleton, on the regulation of GnT-III expression in the human colon carcinoma DLD-1 cells. GnT-III activity was not substantially increased in cells cultured under dense conditions, compared with those cultured under sparse conditions. However, restoration of alpha-catenin gene to DLD-1 cells resulted in a significant increase in GnT-III activity and in production of the bisected N-glycans, which were detected by E(4)-PHA, suggesting that the E-cadherin-catenin complex is required for the induction. Moreover, treatment with cytochalasin D, an inhibitor of F-actin polymerization, completely blocked the upregulation of GnT-III expression in the dense culture. Taken together, these results strongly suggest that GnT-III expression is tightly regulated by cell-cell adhesion via the E-cadherin-catenin complex and actin cytoskeleton formation.     

Propylthiouracil, a selective inhibitor of NADH-cytochrome b5 reductase

Propylthiouracil inhibited the activity of NADH-cytochrome b5 reductase of rat liver microsomes using potassium ferricyanide as electron acceptor. On the other hand, NADPH-cytochrome P-450 reductase activity was not affected by the compound. NADH-supported reduction of cytochrome b5 was also inhibited by propylthiouracil in the reconstituted system consisting of cytochrome b5 and partially purified NADH-cytochrome b5 reductase.     

Production of double repeated B subunit of Shiga toxin?2e at high levels in transgenic lettuce plants as vaccine material for porcine edema disease

Pig edema disease is a bacterial disease caused by enterohemorrhagic Escherichia coli. E.?coli produces Shiga toxin?2e (Stx2e), which is composed of one A subunit (Stx2eA) and five B subunits (Stx2eB). We previously reported production of Stx2eB in lettuce plants as a potential edible vaccine (Matsui et?al. in Biosci Biotechnol Biochem 73:1628-1634, 2009). However, the accumulation level was very low, and it was necessary to improve expression of Stx2eB for potential use of this plant-based vaccine. Therefore, in this study, we optimized the Stx2eB expression cassette and found that a double repeated Stx2eB (2× Stx2eB) accumulates to higher levels than a single Stx2eB in cultured tobacco cells. Furthermore, a linker peptide between the two Stx2eB moieties played an important role in maximizing the effects of the double repeat. Finally, we generated transgenic lettuce plants expressing 2× Stx2eB with a suitable linker peptide that accumulate as much as 80?mg per 100?g fresh weight, a level that will allow us to use these transgenic lettuce plants practically to generate vaccine material.