Notes on some plant-inhabiting fungi collected from the Nansei Islands (2)

In this paper, four fungi collected in Nansei Islands are reported. One is Strasseria garciniae, which must be transferred to the genus Phyllosticta. This fungus was collected in Okinawa Main Island and Iriomote Island. The other three fungi are newly added to the Japanese fungal flora. Phyllosticta ghaesembillae on Codiaeum variegatum and Cercospora asplenii on Asplenium antiquum were collected in Yoron Island. The last one, Coniella australiensis on Eucalyptus robusta was collected in Okinawa Main Island. Their morphology and symptoms on the host plant are described, with some mycological notes.     

Fungal proteinase expression in the interaction of the plant pathogen Magnaporthe poae with its host.

Infection by pathogenic fungi involves breaching the outer layer of the host by either mechanical or enzymatic means. Subtilisin-like proteinases are considered to be important in the infection process of entomopathogenic, nematophagous, and mycoparasitic fungi. Little is known regarding the expression of such proteinases by plant pathogenic fungi. Magnaporthe poae, a fungal pathogen of Kentucky bluegrass, expressed a subtilisin-like proteinase, proteinase Mp1, in the infected roots. Antibody was produced against the purified enzyme. From immunoblot analysis, expression of the proteinase in infected roots correlated with increasing severity of disease symptoms. Sequence analysis of a genomic clone indicated proteinase Mp1 was homologous to other fungal subtilisin-like proteinases. DNA gel blot analysis indicated proteinase Mp1 was encoded by a small gene family.     

Production, crystallization, and preliminary X-ray analysis of rabbit skeletal muscle troponin complex consisting of troponin C and fragment (1-47) of troponin I.

Troponin is a ternary protein complex consisting of subunits TnC. TnI, and TnT, and plays a key role in calcium regulation of the skeletal and cardiac muscle contraction. In the present study, a partial complex (CI47) was prepared from Escherichia coli-expressed rabbit skeletal muscle TnC and fragment 1-47 of TnI, which is obtained by chemical cleavage of an E. coli-expressed mutant of rabbit skeletal muscle TnI. Within the ternary troponin complex, CI47 is thought to form a core that is resistant to proteolytic digestion, and the interaction within CI47 likely maintains the integrity of the troponin complex. Complex CI47 was crystallized in the presence of sodium citrate. The addition of trehalose improved the diffraction pattern of the crystals substantially. The crystal lattice belongs to the space group P3(1)(2)21, with unit cell dimensions a = b = 48.2 A, c = 162 A. The asymmetric unit presumably contains one CI47 complex. Soaking with p-chloromercuribenzenesulfonate (PCMBS) resulted in loss of isomorphism, but enhanced the quality of the crystals. The crystals diffracted up to 2.3 A resolution, with completeness of 91% and R(merge) = 6.4%. The crystals of PCMBS-derivative should be suitable for X-ray studies using the multiple-wavelength anomalous diffraction technique. This is the first step for elucidating the structure of the full troponin complex.     

Production of keto-disaccharides from aldo-disaccharides in subcritical aqueous ethanol

Isomerization of disaccharides (maltose, isomaltose, cellobiose, lactose, melibiose, palatinose, sucrose, and trehalose) was investigated in subcritical aqueous ethanol. A marked increase in the isomerization of aldo-disaccharides to keto-disaccharides was noted and their hydrolytic reactions were suppressed with increasing ethanol concentration. Under any study condition, the maximum yield of keto-disaccharides produced from aldo-disaccharides linked by β-glycosidic bond was higher than that produced from aldo-disaccharides linked by α-glycosidic bond. Palatinose, a keto-disaccharide, mainly underwent decomposition rather than isomerization in subcritical water and subcritical aqueous ethanol. No isomerization was noted for the non-reducing disaccharides trehalose and sucrose. The rate constant of maltose to maltulose isomerization almost doubled by changing solvent from subcritical water to 80 wt% aqueous ethanol at 220 °C. Increased maltose monohydrate concentration in feed decreased the conversion of maltose and the maximum yield of maltulose, but increased the productivity of maltulose. The maximum productivity of maltulose was ca. 41 g/(h kg-solution).     

On the mechanism of the peroxidase-catalyzed oxygen-transfer reaction

We reported evidence that horseradish peroxidase (HRP) and chloroperoxidase (CPO) catalyze oxygen transfer from H2O2 to thioanisoles [Kobayashi, S., Nakano, M., Goto, T., Kimura, T., & Schaap, A. P. (1986) Biochem. Biophys. Res. Commun. 135, 166-171]. In the present paper, the reaction mechanism of this oxygen transfer is discussed. The oxidation of para-substituted thioanisoles by HRP compound II showed a large negative rho value of -1.46 vs. the sigma + parameter in a Hammett plot. These results are in accord with the formation of a cation radical intermediate in the rate-determining step. Hammett treatments for HRP- and CPO-dependent S-oxygenations did not provide unequivocal proofs to judge the reaction mechanism, because of the poor correlations for sigma + and sigma p parameters. Different behavior was found in kinetics and stereoselectivity between the two enzymes. Results in the present study and recent studies strongly suggested the formation of a cation radical intermediate. The oxygen atom would transfer by reaction of compound II and the cation radical intermediate. Although involvement of the cation radical was not confirmed in the CPO system, a similar mechanism was proposed for CPO.