Dual function of the propeptide of prouroguanylin in the folding of the mature peptide: disulfide-coupled folding and dimerization.

Guanylyl cyclase activating peptide II (GCAP-II), an endogenous ligand of guanylyl cyclase C, is produced via the processing of the precursor protein (prepro-GCAP-II). We have previously shown that the propeptide in pro-GCAP-II functions as an intramolecular chaperone in the proper folding of the mature peptide, GCAP-II (Hidaka, Y., Ohno, M., Hemmasi, B., Hill, O., Forssmann, W.-G., and Shimonishi, Y. (1998) Biochemistry 37, 8498-8507). Here, we report an essential region in pro-GCAP-II for the correct disulfide pairing of the mature peptide, GCAP-II. Five mutant proteins, in which amino acid residues were sequentially deleted from the N terminus, and three mutant proteins of pro-GCAP-II, in which N-terminal 6, 11, or 17 amino acid residues were deleted, were overproduced using Escherichia coli or human kidney 293T cells, respectively. Detailed analysis of in vivo or in vitro folding of these mutant proteins revealed that one or two amino acid residues at the N terminus of pro-GCAP-II are critical, not only for the chaperone function in the folding but also for the net stabilization of pro-GCAP-II. In addition, size exclusion chromatography revealed that pro-GCAP-II exists as a dimer in solution. These data indicate that the propeptide has two roles in proper folding: the disulfide-coupled folding of the mature region and the dimerization of pro-GCAP-II.

[Effect of coix seed on the changes in peripheral lymphocyte subsets].

Coix seed has been used in patients with verruca vulgaris and verruca planae juveniles, which have been considered to be induced by viral infection. Moreover, coixenolide, component in the seeds of coix, was reported to show anti-tumor activity. Possibly coix seed may have some influence on the cytotoxic activity of peripheral lymphocytes but there has been no data on this. Then we investigated the changes in number of cytotoxic lymphoid cells in seven volunteers before, during (four weeks) and after taking six coix seed tablets. Lymphocyte subsets were analyzed with monoclonal antibodies using a flow cytometer. The level of CD3+CD56+ (MHC-non restricted cytotoxic T cells) markedly increased at four weeks (before 1.9 +/- 0.5% vs four weeks 4.2 +/- 0.7%, p less than 0.01). The level of CD16+CD57- (the mature, most active natural killer cells) increased at three weeks (before 4.5 +/- 0.8% vs three weeks 5.2 +/- 0.8%, p less than 0.05). The level of CD3-CD56+ (natural killer cells) and the level of CD16+CD57+ (the variable active natural killer cells) decreased at one week and returned to normal level thereafter (before 13.7 +/- 2.1% vs one week 11.2 +/- 1.5%, p less than 0.05; before 8.8 +/- 1.5% vs one week 6.9 +/- 1.3%, p less than 0.05, respectively). These results indicate that coix seed modulate the peripheral blood lymphocyte subsets and may be effective to virus disease through the enhancement of cytotoxic activity.

Chinese medicine, Coix seeds increase peripheral cytotoxic T and NK cells.

Coix seeds, a Chinese medicine have been used in Japan and reported to be effective in patients with verruca vulgaris and verrucae planae juveniles. We investigated the in vivo effects on lymphocyte subsets in seven healthy volunteers who took six tablets of Coix seeds three times a day (a typical dose) for four weeks. Leukocyte counts and the percentage of total lymphocytes did not change but the percentages of CD3+CD56+ cells and CD16+CD57- cells increased significantly. These results indicate that Coix seeds increase peripheral cytotoxic lymphocytes and may be effective to viral infection through the enhancement of cytotoxic activity.