Morihiko Sakaguchi

Inhibition of amino acid decarboxylase activity of Enterobacter aerogenes by active components in spices

The water and ethanol extracts of several commercially available spices were examined for their inhibitory action on the decarboxylase activity of a crude extract of Enterobacter aerogenes . The water extracts had a negligible effect on histidine decarboxylase activity, except for water extract of cloves which reduced the activity by about 40%. However, the ethanol extracts had a rather higher inhibitory action upon histidine, lysine, and ornithine decarboxylases. Of the spices used, cloves, cinnamon, sage, nutmeg, and allspice were very effective in inhibiting the decarboxylases. Among the components of those spices, cinnamaldehyde and eugenol were found to be effective inhibitors.

Antioxidant activities of phycocyanobilin prepared from Spirulina platensis

The antioxidative activity of phycocyanobilin fromSpirulina platensis was evaluated againstoxidation of methyl linoleate in a hydrophobic systemor with phosphatidylcholine liposomes. Phycocyanobilin as well as phytochemicals includingα-tocopherol, caffeic acid and zeaxanthin,effectively inhibited the peroxidation of methyllinoleate and produced a prolonged induction period.Oxidation of phosphatidylcholine liposomes was alsocontrolled markedly by adding phycocyanobilin orα-tocopherol. Phycocyanobilin was distributedoutside in the liposomes to scavenge radicals fromAAPH and to prevent initiation of radical chainreactions. When the concentrations of phycocyanin andphycocyanobilin in the reaction mixture were adjustedequally on a phycocyanobilin basis, the activity ofphycocyanobilin was almost the same as that ofphycocyanin in the AAPH-containing reaction mixture.The antioxidizing action of phycocyanin prepared fromspray-dried Spirulina almost agreed with thatfrom fresh Spirulina in the AAPH-containingreaction mixture. These results suggest thatphycocyanobilin is responsible for the majority of theantioxidative activity of phycocyanin and may act asan effective antioxidant in a living human body.

Hemocyte components in crustaceans convert hemocyanin into a phenoloxidase-like enzyme

The functional conversion of hemocyanin (Hc), an oxygen transporter, into an enzyme was investigated in crustaceans. Hc is converted into a phenoloxidase-like enzyme by hemocyte components, which is triggered by beta-1,3-glucan. This activation is severely hampered with leupeptin and E-64 treatment, indicating that the serine/cysteine proteases in the hemocytes are involved in the activation. In a SDS-PAGE-analysis, no change was observed between normal and activated Hc under reduced conditions. However, under non-reduced condition of normal Hc, several minor bands were observed at oligomeric position of Hc subunit, which disappeared upon activation. These results indicate that a split of the reductive bond, such as the disulfide bond between subunits, is essential for Hc activation. This is the first report to show the enzymatic conversion of Hc and the presence of the covalent bond in the Hc subunit of crustaceans.

Combined effect of sodium chloride and clove on growth and biogenic amine formation of Enterobacter aerogenes in mackerel muscle extract.

The inhibitory effects of clove (0.5%) and sodium chloride (1-5%) on the growth and biogenic amine (histamine and cadaverine) production of Enterobacter aerogenes in mackerel muscle broth at 30°C were investigated. At 1% level, sodium chloride was favorable for growth, whereas higher levels slightly reduced the growth in comparison to the control. The maximum population numbers obtained in the presence of sodium chloride were essentially the same as that of the control. Amine production was remarkably enhanced by the presence of 1 % NaCl alone. Only a little increase was observed for higher levels, and sodium chloride at more than 3% had no stimulatory effect on the amine formation. Addition of clove at a level of 0.5% to the broth resulted in a delay in the growth and the amine formation. The presence of NaCl as low as 2% in combination with clove (0.5%) completely inhibited the growth and amine production of E. aerogenes in mackerel broth. Synergistic effects of clove essential oils and sodium chloride could be considered as the probable reason for the inactivation of the bacterium.

A hyperosmotic stress-induced mRNA of carp cell encodes Na+- and Cl−-dependent high affinity taurine transporter

A cDNA clone encoding a Na(+)- and Cl(-)-dependent high affinity taurine transporter was isolated from a common carp cell line, Epithelioma papulosum cyprini (EPC), as a hyperosmotic stress-inducible gene by RNA arbitrarily primed PCR. The clone contained a 2.5-kb cDNA fragment including an open reading frame of 1878 bp encoding a protein of 625 amino acids. The deduced amino acid sequence of carp taurine transporter shows 78-80% identity to those of cloned mammalian taurine transporters. The functional characteristics of the cloned transporter were analyzed by expression in COS-7 cells. Transfection with the cDNA induced Na(+)- and Cl(-)-dependent taurine transport activity with an apparent K(m) of 56 microM. The Na(+)/Cl(-)hepatopancreas. Taurine transporter mRNA level increased up to 7.5-fold on raising the ambient osmolality from 300 to 450 mosmol/kgH(2)O. These data suggest the significant role of taurine as an osmolyte in carp cells.

A Critical Look at Whether ‘Freshness’ Can Be Determined

Abstract The measurement of ‘freshness’ has proved to be elusive and there are still no widely agreed objective methods for its determination. We point out that the reasons for this are several: (1) conceptual, (2) measurement of time elapsed since death at a set temperature is insufficient, and (3) no test so far proposed has the correct characteristics. We put forward a system in which ‘freshness’ can be carefully defined and various criteria and methods can be specified and agreed on by all interested parties. This system overcomes the terminological and conceptual difficulties that exist when the term ‘freshness’ is used. Furthermore, we outline the characteristics that a test should possess and discuss the reasons why many proposed measures fail to be used in practice.

Isolation and characterization of a Japanese flounder clonal line, reversed, which exhibits reversal of metamorphic left-right asymmetry.

We have isolated a clonal line reversed (rev) of homozygous Japanese flounder through gynogenesis. The homozygous offspring gynogenetically produced from rev exhibited reversal of organization of the metamorphic L/R asymmetry such as the direction of eye-migration at a high frequency (20-30%). The molecular analysis using a left-specific marker pitx2 revealed that the embryonic L/R axis was ambiguously established: in more than half of rev embryos, pitx2 was expressed bilaterally in the lateral plate mesoderm (LPM). Previous studies in other animals demonstrated that ectopic pitx2 expression in the LPM could cause laterality defects of the visceral organs. Likewise, our results using rev imply that bilateral pitx2 expression could lead to randomization of the visceral organs. Coincidence of ectopic pitx2 expression and reversal of the direction of eye-migration in the population of rev offspring suggests that the rev locus is critical in specification of both the metamorphic and the visceral L/R asymmetries. However, reversal of the sidedness of the orientation of the visceral organs was not always accompanied by reversal of the direction of metamorphic eye-migration, suggesting that different mechanisms should be involved downstream of the rev locus in directing these two phases of asymmetric morphogenesis in the Japanese flounder.

Sequence and expression of a cDNA encoding the red seabream androgen receptor

The cDNA of the androgen receptor (AR) has been isolated from the ovary of red seabream, Pagrus major, and sequenced. The amino acid sequence of red seabream AR (rsAR) shows about 45% identity with those of Xenopus, rat, mouse, and human ARS. It is shown that rsAR has the ability to trans-activate the responsive gene depending on the presence of androgen.

A stable line of transgenic medaka (Oryzias latipes) carrying the CAT gene

Abstract A stable homozygous line of transgenic medaka (Oryzias latipes) was produced by injecting plasmid DNA containing rainbow trout metallothionein A promoter region followed by bacterial acetyltransferase gene (rtMT-A-CAT), into the cytoplasm of 111 fertilized eggs. The line transmitted active CAT gene to all of the offsprings until sixth generation in mendelian fashion. The Southern blot analysis and the crossing experiments indicated that the DNA was integrated into the chromosome. These results reveal that the medaka is a good model for basic studies of the production of transgenic fish.

Transgenic Medaka Overexpressing a Melanin-Concentrating Hormone Exhibit Lightened Body Color but No Remarkable Abnormality

Abstract: Melanin-concentrating hormone (MCH) is a cyclic heptadecapeptide that concentrates melanin granules in the melanophores and lightens the body color of a fish. To investigate the utility of MCH as a reporter gene, a transgenic medaka strain overexpressing the MCH gene was established and its phenotypic features were examined. The salmon MCH gene driven by cytomegalovirus promoter was injected into 100 fertilized eggs of the HNI-1 medaka strain, which exhibits black body color. One F0 female transmitted the transgene and a lightened body color phenotype to the F1 generation. A homozygous transgenic strain was established by crossing F2 fish homozygous for the transgene. Expression of the transgene was detected in several organs by Northern blotting. The melanin granules of transgenics were highly shrunk. Bioassay using scales confirmed the secretion of MCH into blood, and the MCH concentration was estimated between 0.5 and 5 μM. Development, growth, feeding behavior, and reproduction of transgenics did not differ significantly among transgenic and nontransgenic siblings. The result whereby enhanced MCH expression induced a change in body color, but no remarkable abnormality, suggests the usefulness of MCH as a novel reporter gene with unique features.