Comparative study of embryonic thermoresistance of two inbred strains of the medaka (Oryzias latipes)

Summary By using inbred strains (HO4C and HB32C) of the medaka,Oryzias latipes, the involvement of genetic factor(s) in the determination of thermoresistance of fish was investigated. The thermoresistance of embryos of the medaka was quantitated by the fraction of the embryos surviving 1 day after heat treatment. At early stages of development (st. 13 and st. 20–21), the HO4C strain was more resistant than the HB32C strain. At st. 20–21, the HO4C strain was more resistant than the HB32C strain at all temperatures used (42, 43, and 44°C). At later stages of development (st. 27 and st. 32), however, the HB32C strain was more resistant than the HO4C strain.The results of genetic cross experiments raised the following possibilities; the thermoresistance of embryos at early developmental stages can be lowered by some factor(s) inherited in the HO4C strain and/or increased by those in the HB32C strain. By contrast, the sensitivity of embryos at later stages of development was not affected by factor(s) of their parents, but by their own genetic constitution.     

Demonstration of endothelin (ET) receptors on cultured rabbit chondrocytes and stimulation of DNA synthesis and calcium influx by ET-1 via its receptors

Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with ¹²⁵ I-ET-1, two major bands of 43 kDa and 46 kDa were separated by SDS-PAGE. Scatchard analysis demonstrated two classes of ET receptors with K_d values of 1 × 10^?10 M and 5 × 10^?9 M. The numbers of high- and low- affinity receptors were 1 × 10⁴ and 2 × 10⁵ per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-β1, IL-1β, RA and EGF. ET-1 stimulated DNA synthesis in cultured rabbit chondrocytes. ET-1 also stimulated calcium incorporation through the cell membrane of chondrocytes. These findings indicate that ET-1 has a physiological effect on chondrocytes via its receptors on the cells.     

Preparation of a novel (1→4)-β-D-glycan by Acetobacter xylinum — a proposed mechanism for incorporation of a N-acetylglucosamine residue into bacterial cellulose

A novel polysaccharide having a N-acetylglucosamine (GlcNAc) residue as one of the constituents was synthesized by incubation of Acetobacter xylinum in a modified Schramm-Hestrin medium containing lysozyme-susceptible phosphoryl chitin (P-chitin) andd-glucose. HPLC of the culture medium snowed that the P-chitin added was depolymerized to monomeric and oligomeric P-chitins during the incubation, and the P-chitins with permeable sizes were utilized as a carbon source by the bacteria.¹³C NMR analysis revealed that the P-chitin consists mainly of GlcNAc 6-P residues. Furthermore, monomeric GlcNAc 6-phosphate was also found to enhance the incorporation of GlcNAc residues into the polysaccharide. However, no incorporation of the GlcNAc residues was observed when A. xylinum was incubated in a medium containing either highly phosphorylated chitin (DS = 1.90) or its oligomers produced by acid hydrolysis.     

Thy-1-positive cells in the subodontoblastic layer possess high potential to differentiate into hard tissue-forming cells

The cells of the subodontoblastic cell-rich layer in dental pulp are speculated to contain odontoblast progenitor cells because of their positional relationship with odontoblasts as well as their high alkaline phosphatase (ALP) activity. However, it has yet to be determined whether these cells have the ability to differentiate into odontoblastic cells. In the present study, we firstly found that the majority of cells in the subodontoblastic layer expressed Thy-1, a cell-surface marker of stem and progenitor cells. Then, we evaluated the capacity of Thy-1 high- and low-expressing (Thy-1(high) and Thy-1(low)) cells separated from rat dental pulp cells by use of a fluorescence-activated cell sorter to differentiate into hard tissue-forming cells in vitro and in vivo. Following stimulation with bone morphogenetic protein-2, Thy-1(high) cells in vitro showed accelerated induction of ALP activity and formation of alizarin red-positive mineralized matrix compared with Thy-1(low) cells. Furthermore, subcutaneous implantation of Thy-1(high) cells efficiently induced the formation of bone-like matrix. These results collectively suggest that Thy-1-positive dental pulp cells localized in the subodontoblastic layer had the ability to differentiate into hard tissue-forming cells, and thus these cells may serve as a source of odontoblastic cells.     

Neuron-specific recombination by Cre recombinase inserted into the murine tau locus

To determine the neuronal function of genes in vivo, the neuron-specific deletion of a target gene in animals is required. Tau, a microtubule-associated protein, is expressed abundantly in neurons but scarcely in glias and other tissues. Therefore, to generate mice that express Cre recombinase in neurons, we inserted Cre recombinase into the tau locus. By crossing these tau-Cre mice with ROSA26 lacZ reporter mice, we observed Cre recombinase activity in the neurons from most of the central nervous system, but not in glias nor in non-neuronal tissues. This neuronal-specific activity appeared during embryogenesis. We further crossed tau-Cre mice with rab8 ‘floxed’ mice, and showed that the recombination was nearly complete in the brain, but incomplete or non-detectable in other tissues. Thus, tau-Cre knockin mouse is a useful tool for studying the neuronal function of a gene in vivo.     

Mutual conversion of substrate specificities of Thermoactinomyces vulgaris R-47 alpha-amylases TVAI and TVAII by site-directed mutagenesis

Thermoactinomyces vulgaris R-47 produces two alpha-amylases, TVAI and TVAII, differing in substrate specificity from each other. TVAI favors high-molecular-weight substrates like starch, and scarcely hydrolyzes cyclomaltooligosaccharides (cyclodextrins) with a small cavity. TVAII favors low-molecular-weight substrates like oligosaccharides, and can efficiently hydrolyze cyclodextrins with various sized cavities. To understand the relationship between the structure and substrate specificity of these enzymes, we precisely examined the roles of key residues for substrate recognition by X-ray structural and kinetic parameter analyses of mutant enzymes and successfully obtained mutants in which the substrate specificity of each enzyme is partially converted into that of another.     

Components Contributing to the Improvement of Meat Taste during Storage

The changes in both taste and taste components of beef, pork, and chicken during storage were examined.

The brothy taste intensity of pork and chicken was significantly stronger after conditioning than before. On the other hand, for beef, there was no significant difference in the brothy taste intensity before or after conditioning. The analysis of major taste components showed that the levels of free amino acids in all meats were higher after conditioning than before. The differences in the levels of free amino acids before versus after conditioning were large in pork and chicken and very small in beef. Oligopeptide levels were lower in beef after conditioning than before, but they were higher in pork and chicken after conditioning than before. These results corresponded to results of the sensory evaluation studies described above, indicating that free amino acids and oligopeptides contributed to the improvement of meat taste during storage.