Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin.

We have previously shown that replacing the P1-site residue (Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lys results in the acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively. However, the inhibitory activities thus induced are not strong. In the present study, we introduced additional amino acid replacements around the reactive site to try to make the P1-site mutants more effective inhibitors of chymotrypsin or trypsin. The amino acid replacement Asp-->Tyr at the P2' site of OMCHI3(P1Met) resulted in conversion to a 35000-fold more effective inhibitor of chymotrypsin with an inhibitor constant (K(i)) of 1. 17x10(-11) M. The K(i) value of OMCHI3(P1Met, P2'Ala) indicated that the effect on the interaction with chymotrypsin of removing a negative charge from the P2' site was greater than that of introducing an aromatic ring. Similarly, enhanced inhibition of trypsin was observed when the Asp-->Tyr replacement was introduced into the P2' site of OMCHI3(P1Lys). Two additional replacements, Asp-->Ala at the P4 site and Arg-->Ala at the P3' site, made the mutant a more effective inhibitor of trypsin with a K(i) value of 1. 44x10(-9) M. By contrast, Arg-->Ala replacement at the P3' site of OMCHI3(P1Met, P2'Tyr) resulted in a greatly reduced inhibition of chymotrypsin, and Asp-->Ala replacement at the P4 site produced only a small change when compared with a natural variant of OMCHI3. These results clearly indicate that not only the P1-site residue but also the characteristics, particularly the electrostatic properties, of the amino acid residues around the reactive site of the protease inhibitor determine the strength of its interactions with proteases. Furthermore, amino acids with different characteristics are required around the reactive site for strong inhibition of chymotrypsin and trypsin.     

Construction of a plasmid for high-level expression of goat alpha-lactalbumin and its derivative in E. coli

The high-level expression system of goat alpha-lactalbumin (alpha-LA) in E. coli was established by fusing the alpha-LA cDNA to porcine adenylate kinase cDNA and expressing the fused gene under the control of tac promoter. For high-level expression, elimination of 3'-noncoding region of the alpha-LA cDNA was found to be necessary.     

Effector cells working for rejection of Ad12 tumors in mice

Immune spleen cells (ISC) capable of inhibiting the growth of adenovirus type 12 (Ad12) tumors were raised in C57BL/6 mice by immunization with Ad12, fractionated according to their affinity for plastic and nylon-wool substrates or treated with various antisera plus complement, and subjected to the tumor-neutralization test (Winn) to define the effector cells for the cell species. Full antitumor activity of ISC was recovered in the cell fractions nonadherent to the two substrates; the antitumor activity of ISC was abrogated entirely by anti-Thy-1,2 serum and almost entirely by anti-Lyt-2.2 ascites fluid plus complement. These results clearly indicate that T-lymphocytes, particularly those bearing Lyt-2.2 antigen, are the principal effectors in ISC against Ad12 tumors in animals.     

Use of paired abdominal flaps for release of adduction contractures of the thumb.

A method for correction of an adduction contracture of the thumb is presented. Paired flaps provide good cover to the palmar and dorsal sides of the web space. This method produces better cosmetic and functional results than the traditional methods.     

Characterization of insulin receptors in primary cultures of quail (Coturnix coturnix japonica) oviduct cells. The level of insulin receptor is regulated by steroid and peptide hormones

1. We have characterized the insulin receptor in primary cultured quail oviduct cells and examined the hormonal regulation of its level. 2. We have also shown the recycling pathway of insulin receptors in the cultured cells using specific inhibitors (tunicamycin, chloroquine, monensin, and brefeldin A). 3. Our data suggest that glucocorticoids play important physiological roles in egg-white protein synthesis through increasing the number of insulin receptors and insulin through enhancing the transport of amino acids.     

Ultrastructural preservation of plasma membranes by non-lethal slow freezing to liquid nitrogen temperature

Secondary hyphae of Lyophyllum ulmarium were shown to tolerate slow freezing, which allowed extracellular freezing, to -196 degrees C. A freeze-fracture study showed that under this non-lethal freezing condition, the plasma membrane of the secondary hyphae did not show any ultrastructural changes as compared with the control, except gross cellular shrinkage. Tertiary hyphae of Lyophyllum ulmarium, on the other hand, were completely injured by slow freezing to -196 degrees C, and the plasma membrane showed distinct intramembrane particle aggregation as a result of direct membrane contact caused by severe cellular deformation. It is suggested that the absence of freezing injury in the secondary hyphae was due to ultrastructural preservation of the plasma membrane, which resulted from avoidance of severe cellular deformation, while occurrence of freezing injury in the tertiary hyphae is considered to be due to ultrastructural changes in the plasma membrane caused by severe cellular deformation.     

Screening of marine cyanobacteria for high palmitoleic acid production

Abstract Screening of fatty acid composition in 150 strains of marine microalgae, cyanobacteria and green algae was carried out, and 20 strains showed relatively high contents of palmitoleic acid. Among them, two cyanobacteria, Phormidium sp. NKBG 041105 and Oscillatoria sp. NKBG 091600, showed an unusually high cis -palmitoleic acid content (54.5% and 54.4% of total fatty acid, respectively). Phormidium sp. NKBG 041105 had the highest cis -palmitoleic acid content per biomass (46.3 mg (g dry cell weight)⁻¹), and cis -palrnitoleic acid composition was found to be constant with varying temperature. These results indicate that this cyanobacterium could be considered as a new source for palmitoleic acid.     

A Theoretical Model of Jigsaw-Puzzle Pattern Formation by Plant Leaf Epidermal Cells

Plant leaf epidermal cells exhibit a jigsaw puzzle–like pattern that is generated by interdigitation of the cell wall during leaf development. The contribution of two ROP GTPases, ROP2 and ROP6, to the cytoskeletal dynamics that regulate epidermal cell wall interdigitation has already been examined; however, how interactions between these molecules result in pattern formation remains to be elucidated. Here, we propose a simple interface equation model that incorporates both the cell wall remodeling activity of ROP GTPases and the diffusible signaling molecules by which they are regulated. This model successfully reproduces pattern formation observed in vivo, and explains the counterintuitive experimental results of decreased cellulose production and increased thickness. Our model also reproduces the dynamics of three-way cell wall junctions. Therefore, this model provides a possible mechanism for cell wall interdigitation formation in vivo.