A sensitive bacterial luminescence probe for O2 in biochemical systems

1.

1. An oxygen assay method applicable at very low oxygen concentrations has been developed using bioluminescence as an oxygen indicator. The method can be applied with accuracy to the oxygen concentration range between 10⁻⁶ and 10⁻⁸ M.     

Age determination and estimation of larval period in field caught abalone (Haliotis discus hannai Ino 1953) larvae and newly metamorphosed post-larvae by counts of radular teeth rows

We developed an age determination method for larval and newly metamorphosed post-larval abalone Haliotis discus hannai in a laboratory experiment and determined the age of field caught individuals. Laboratory experiments showed that competent veliger larvae (4 days after fertilization) had a radula and regularly added rows of radular teeth with age in the absence of metamorphosis. Under environmentally relevant temperatures (17-22 °C), the number of rows of radular teeth increased linearly with age, but slopes of the regression lines were different among temperatures. Rows of radular teeth were added more slowly at lower temperatures. The effect of temperature on the development rate of the radula was quantified by the regression and the temperature coefficient, Q₁₀. The radular development of newly metamorphosed post-larvae, which had not acquired a peristomal shell (adult shell), was comparable with that of veliger larvae, although older post-larvae had a larger number of rows of radula than those of the same age of veliger larvae. From these results, an age determination method of veliger larvae and newly metamorphosed post-larvae was established, using the number of rows of radular teeth. The age of veliger larvae and newly metamorphosed post-larvae was determined by the age determination method for samples collected in August to October of 2003 and 2004 for which the thermal history of the coastal water of Miyagi Prefecture Japan was available. Only 9.1% of veliger larvae (n = 8) captured in the field had formed a radula and these were estimated to be 4-6 days old. The remaining 90.9% of larvae (n = 80) that had not formed a radula were classified as younger than 4 days old. All newly metamorphosed post-larvae (n = 24) that had metamorphosed on substrata were estimated to be 4-6 days old. Results of the field study indicate that these abalone metamorphosed within a few days after the acquisition of competence (4 days after fertilization) at this site, which has suitable crustose algal habitat.     

Establishment and characterization of monoclonal antibodies against Chuzan virus K-47

We established sixteen mouse monoclonal antibodies reactive to Chuzan virus K-47 strain using P3-X63-Ag8-U1 cells as fusion partner cells. Among them, CG53/2/4 recognized a 100K structural protein of the virus. The 100K antigen lost it's antigenicity for CG53/2/4 after mild periodate oxidation treatment, suggesting that the 100K viral antigen is a glycoprotein. In addition, CG53/2/4 neutralized the viral infectivity. This indicates that the 100K glycoprotein is essential for the infection of the virus. The other monoclonal antibodies reacted with a 41K antigen of the virus. Especially CG1/1 showed the highest reactivity to the virus. Forward step sandwich assay using CG1/1 and biotinylated CG53/2/4 could detect the virus at 10TCID₅₀/ml. Therefore, these monoclonal antibodies can evantually predict the virus infection to the animals before their sideration.     

Oxidation-reduction ratio studies of mitochondria in freeze-trapped samples. NADH and flavoprotein fluorescence signals.

The recording of oxidation-reduction-related fluorescence signals of oxidized flavoprotein (Fp) and reduced pyridine nucleotide (PN) from isolated mitochondria at temperatures below -80 degrees C can be accompanished with a high degree of accuracy and a wide dynamic range. The specific low temperature enhancement of the fluorescence signals due to increased quantum yield and to multiple scattering affords increased accuracy and less interference due to screening pigments such as hemoglobin and myoglobin. Since the metabolic processes are arrested and the recording speed can be greatly diminished, the technique can operate with a much smaller concentration of mitochondria than is needed at room temperature, and the method is suitable for localized oxidation-reduction measurements. The Fp and PN signals originate from the mitochondrial matrix space in which they represent the major fluorochromes. Since Fp and PN are near oxidation-reduction equilibrium, the ratio of the two fluorescence intensities, suitably normalized, approximates the oxidation-reduction ratio of oxidized flavoprotein/reduced pyridine nucleotide. Thus, this technique affords a foundation for the resolution of oxidation-reduction states in two and three dimensions.     

Galectin-8 and galectin-9 are novel substrates for thrombin

Galectin-8 and galectin-9, which each consist of two carbohydrate recognition domains (CRDs) joined by a linker peptide, belong to the tandem-repeat-type subclass of the galectin family. Alternative splicing leads to the formation of at least two and three distinct splice variants (isoforms) of galectin-8 and galectin-9, respectively, with tandem-repeat-type structures. The isoforms share identical CRDs and differ only in the linker region. In a search for differences in biological activity among the isoforms, we found that their isoforms with the longest linker peptide, that is, galectin-8L and galectin-9L (G8L and G9L), are highly susceptible to thrombin cleavage, whereas the predominant isoforms, galectin-8M and galectin-9M (G8M and G9M), and other members of human galectin family so far examined were resistant to thrombin. Amino acid sequence analysis of proteolytic fragments and site-directed mutagenesis showed that the thrombin cleavage sites (-IAPRT- and -PRPRG- for G8L and G9L, respectively) resided within the linker peptides. Although intact G8L stimulated neutrophil adhesion to substrate more efficiently than G8M, the activity of G8L but not that of G8M decreased on thrombin digestion. Similarly, thrombin treatment almost completely abolished eosinophil chemoattractant (ECA) activity of G9L. These observations suggest that G8L and G9L play unique roles in relation to coagulation and inflammation.