Expression analysis of polyphenol oxidase isozymes by active staining method and tissue browning of head lettuce (Lactuca sativa L.).

Browning of plant tissue is generally considered attributable to enzymatic oxidation by polyphenol oxidase (PPO). Electrophoresis followed by activity staining has been used as an effective procedure to visually detect and isolate isozymes; however, it has not been applied for examination of various PPO isozymes in lettuce. Our study demonstrated that different lettuce PPO isozymes could be detected at different pH in active staining, and multiple isozymes were detected only under alkaline conditions. As a result, we concluded that activity staining with approximately pH 8 enabled to detect various PPO isozymes in lettuce. By expression analysis of the PPO isozymes after wounding, PPO isozymes that correlated with time-course of tissue browning were detected. The wound-induced PPO may play a key role in enzymatic browning.

The effect of amino acids on the C14-sphingosine-triggered germination of Nomuraea rileyi, and surface amino acids on Spodoptera litura fabricius.

D-erythro-C14-Sphingosine (C14-Sph) accelerated the germination of Nomuraea rileyi in a solution containing peptone, but activity declined to a large degree in water. This suggests the presence of a co-factor in C14-Sph-triggered germination. Since the main role of peptone is to supply nitrogen constituents, we examined the effects of various nitrogen constituents. It was found that Ala and His were highly effective for C14-Sph-triggered germination.

Potential of D-erythro-C14-Sphingosine as an adjuvant for a fungal pesticide of Nomuraea rileyi.

D-erythro-C(14)-Sphingosin (C(14)-Sph) was isolated as the germination accelerating factor in Nomuraea rileyi in our previous study. This activity was expected to support fungal infection by reduction of the infection time between conidial adhesion and invasion into the insect. In this study, we estimated the effect of C(14)-Sph with regard to the infection time. Conidia activated by C(14)-Sph shortened the time to about half, indicating the potential of C(14)-Sph as an adjuvant for fungal pesticide of N. rileyi.

Isolation of a bioactive substance from the silkworm (Bombyx mori Linnaeus) that accelerates the germination of the entomopathogenic fungus Nomuraea rileyi (Farlow) Samson.

The conidium of the entomopathogenic fungus Nomuraea rileyi has been found to germinate rapidly in the presence of host insect-derived extracts. Thus the extract appears to contain an important factor involved in host recognition by N. rileyi. However, the substance responsible for such unique germination behavior has yet to be identified. Hence we attempted to purify this substance. One thousand g of dried silkworm pupae was subjected to methanol extraction, followed by methanolysis, two different solvent partitions, and three different column chromatographies. A total of 12.4 mg of substance was obtained in the active fraction. The substance obtained exhibited an activity more than 46,000 times higher than that of the methanol extract. The substance was detected as a single peak on Sephadex LH20 column chromatography and as a single band on high-performance thin-layer chromatography. These data indicate that the concentrated fraction contained a high-purity substance.

Characterization of a germination-accelerating factor from the silkworm (Bombyx mori Linnaeus) of entomopathogenic fungus Nomuraea rileyi (Farlow) Samson.

The conidium of the entomopathogenic fungus, Nomuraea rileyi, has been found to germinate rapidly in the presence of a host insect-derived extract. This extract therefore appears to contain an important factor involved in host recognition by N. rileyi, although the substance (germination-accelerating factor, GAF) responsible for such unique germination behavior has yet to be identified. Our previous study was extended to the isolation of GAF from pupae of the silkworm, a host insect of N. rileyi. This present work subjects GAF to a structural analysis. The chemical structure of GAF is characterized as 2S-amino-tetradeca-4-ene-1,3R-diol (D-erythro-C(14)-sphingosine) based on spectroscopic data. An examination of the structure-activity relationship shows that the activity of D-erythro-C(14)-sphingosine was superior to that of sphingosines with shorter and longer carbon chains. It is suggested that the molecular species with a 14-carbon chain of a sphingosine is important for host recognition.