Yuzaburo Ishida

Chattonella and Fibrocapsa (Raphidophyceae): First observation of, potentially harmful, red tide organisms in Dutch coastal waters

Species of the potentially toxic and red-tide-forming marine-phytoplankton genera Chattonella and Fibrocapsa (Raphidophyceae) were observed for the first time in 1991 in samples taken in Dutch coastal waters; they were again recorded and enumerated in the following years. Chattonella spp. cell numbers varied with the season, with a maximum in May or June in the Dutch Wadden Sea. Cell numbers of Chattonella and F. japonica Toriumi et Takano were up to 6.0 . 10(3) cells . dm(-3) in the Dutch Wadden Sea, except at one station in June 1993 when over 10(4) cells . dm(-3) Chattonella were counted. In May 1993, a minor bloom (over 2.0 . 10(5) cells . dm(-3)) was observed at a station in the southern central North Sea, 100 km northwest of the island of Terschelling. The potentially neurotoxic species Chattonella marina (Subrahmanyan) Hara et Chihara was identified and discriminated from morphologically related species within the class of Raphidophyceae by immunofluorescence. F. japonica could only be clearly identified in live samples; in fixed samples cell morphology was severely affected. The identification of this species was supported by the presence of mucocysts, structures that can be observed readily by optical and electron microscopy.

An extremely heat‐stable extracellular proteinase (aeropyrolysin) from the hyperthermophilic archaeon Aeropyrum pernix K1

An extracellular metalloproteinase, which we had designated aeropyrolysin, from the aerobic marine hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820), was purified by ammonium sulfate precipitation, anionic exchange chromatography, and gel filtration chromatography. The purified enzyme was composed of a single polypeptide chain with a molecular mass of 52 kDa as determined by SDS‐PAGE. The proteinase had a broad pH optimum (pH 5–9) with a maximal activity at pH 6–8 for azocasein hydrolysis. The optimum temperature for enzyme activity was 100°C in the absence of 1 mM CaCl2 and 110°C in the presence of 1 mM CaCl2. The enzyme activity was completely inhibited by EDTA and EGTA, indicating that it was a metalloproteinase. The enzyme was highly resistant to the denaturing reagents urea, guanidine‐HCl, dithiothreitol, 2‐mercaptoethanol and SDS. The enzyme also showed a high activity with the metalloproteinase specific substrate MOCAc‐Pro‐Leu‐Gly‐Leu‐A2pr(Dnp)‐Ala‐Arg‐NH2. The enzyme was extremely thermostable showing half‐lives of 2.5 h at 120°C and 1.2 h at 125°C in the presence of 1 mM CaCl2. These results indicate that this enzyme is one of the most thermostable extracellular proteinases reported to date.

Detection of the red tide-causing plankton Chattonella marina using a piezoelectric immunosensor

Red tide is a phenomenon caused by small algae or phytoplankton where upon seawater appears red. Both toxic and non-toxic red tides have occurred throughout recorded history but in recent years there has been a global increase in the number of these events due to coastal pollution and to other unclear factors; for example, long-distance transport of species across oceans. Some kinds of red tides cause mass deaths of cultured fish and thus cause serious economic problems for countries where fish is one of the important protein supplies. In Japan, the Chattonella species is one of the dominant representatives among many kinds of flagellates recognized in red tides. In this paper, determination of Chattonella marina was performed using a piezoelectric immunosensor comprising a gold-coated piezoelectric crystal coated with monoclonal antibody MR-21. This antibody shows highly specific reactivity towards the cytoplasmic membrane of C. marina. The resonance frequency of the piezoelectric crystal is associated with a mass change at the surface and measurement of the frequency shift by reaction between the antibody MR-21 immobilized on the piezoelectric crystal and C. marina enables to detect C. marina. This immunosensor system is able to detect 102 to 104 cells ml−1 of C. marina directly in seawater.

Detection of the red tide-causing plankton Alexandrium affine by a piezoelectric immunosensor using a novel method of immobilizing antibodies

In this paper, the determination of Alexandrium affine in sea water was performed using piezoelectric immunosensors. In addition, we report a novel method of immobilizing antibodies on the quartz crystal immunosensors using plasma-polymerized ethylenediamine film. The films formed on the quartz crystals are extremely thin, homogeneous and incorporate amino groups. Sensors produced using this method show higher sensitivity than sensors made using conventional immobilization methods for example polyethylenimine or γ-aminopropyltrimethoxysilane.