Structural characterization of proanthocyanidins from adzuki seed coat.

Adzuki bean seed coats contain a variety of pigments with potential bioactivity, including polyphenols, such as proanthocyanidin. To clarify the structure of the pigments from adzuki bean seed coats, the MeOH extract of the seed coats and the extraction residue were subjected to various instrumental analyses and degradation by thiolysis. The MeOH extract was found to contain two types of polymeric red pigments. One of these pigments was assumed to be a simple proanthocyanidin, whereas the other was a complicated polyphenol produced by several modifications and conversions. Direct thiolysis of adzuki seed coat yielded three products as extension subunits. From their chemical structures, a glyoxylic acid-bridged catechin skeleton was suggested as a partial structure of adzuki pigments.

Inhibition of transcription at a coding sequence by alkylating polyamide.

Transcription from DNA sequence-specifically alkylated by a hairpin polyamide (ImPyPy-gamma-ImPyLDu86, 1) was investigated. High-resolution polyacrylamide gel electrophoresis demonstrated that conjugate 1 alkylated a 993-bp DNA fragment, in accordance with the Py-Im recognition rule, predominantly at the one match site on the GFP-encoding strand and at four sites (I'-IV') on the noncoding strand. Alkylation of DNA inhibited the formation of full-length mRNA and caused the transcription of truncated mRNA. Polyacrylamide gel electrophoresis demonstrated that the length of the truncated mRNA was consistent with the alkylated site on the coding strand. Complete inhibition of full-length mRNA synthesis was observed in the presence of 50 nM 1. In clear contrast, the hydrolyzed derivative of 1, designated 2, produced no truncated mRNA, nor did it significantly retard transcription: >80% transcription of full-length mRNA was observed at 50 nM. These results clearly indicate that inhibition of transcription can be achieved with alkylating Py-Im polyamide even in the coding regions of genes.

Regulation of gene expression by sequence-specific alkylating polyamide.

In order to investigate the inhibition of gene expression by a new type of hairpin polyamide-CPI conjugate 1, its ability to inhibit transcription in cell free system was investigated. Sequence-selective alkylation of double-stranded DNA by 1 was investigated by denaturing gel electrophoresis using 1000 bp DNA fragment which codes for green fluorescence protein (GFP) under the control of T7 promoter. Analysis of DNA sequence indicated that 1 alkylated predominantly at the site of 5'-AGTCA-3' in coding region of GFP. The transcript by T7 RNA polymerase using the alkylated DNA as a template was analyzed by PAGE. The results clearly indicate that 1 inhibits transcription by alkylation of coding region at a nanomolar concentration.