• Tuberculosis and Respiratory Diseases
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• v.58 no.1
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• pp.31-42
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• 2005

Background : Peroxiredoxins (Prxs) are a relatively newly recognized, novel family of peroxidases that reduce $H_2O_2$ and alkylhydroperoxide into water and alcohol, respectively. There are 6 known isoforms of Prxs present in human cells. Normally, Prxs exist in a head-to-tail homodimeric state in a reduced form. However, in the presence of excess $H_2O_2$, it can be oxidized on its catalytically active cysteine site into inactive oxidized forms. This study surveyed the types of the Prx isoforms present in the pulmonary epithelial, macrophage, endothelial, and other cell lines and observed their response to oxidative stress. Methods : This study examined the effect of exogenous, excess $H_2O_2$ on the Prxs of established cell lines originating from the pulmonary epithelium, macrophages, and other cell lines, which are known to be exposed to high oxygen partial pressures or are believed to be subject to frequent oxidative stress, using non-reducing SDS polyacrylamide electrophoresis (PAGE) and 2 dimensional electrophoresis. Result : The addition of excess $H_2O_2$ to the culture media of the various cell-lines caused the immediate inactivation of Prxs, as evidenced by their inability to form dimers by a disulfide cross linkage. This was detected as a subsequent shift to its monomeric forms on the non-reducing SDS PAGE. These findings were further confirmed by 2 dimensional electrophoresis and immunoblot analysis by a shift toward a more acidic isoelectric point (pI). However, the subsequent reappearance of the dimeric Prxs with a comparable, corresponding decrease in the monomeric bands was noted on the non-reducing SDS PAGE as early as 30 minutes after the $H_2O_2$ treatment suggesting regeneration after oxidation. The regenerated dimers can again be converted to the inactivated form by a repeated $H_2O_2$ treatment, indicating that the protein is still catalytically active. The recovery of Prxs to the original dimeric state was not inhibited by a pre-treatment with cycloheximide, nor by a pretreatment with inhibitors of protein synthesis, which suggests that the reappearance of dimers occurs via a regeneration process rather than via the de novo synthesis of the active protein. Conclusion : The cells, in general, appeared to be equipped with an established system for regenerating inactivated Prxs, and this system may function as a molecular "on-off switch" in various oxidative signal transduction processes. The same mechanisms might applicable other proteins associated with signal transduction where the active catalytic site cysteines exist.

• Journal of Sericultural and Entomological Science
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• v.16 no.1
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• pp.21-48
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• 1974

The studies were carried out to disclose the physical and chemical properties of sericin fraction obtained from silk cocoon shells and its characteristics of swelling and solubility. The following results were obtained. 1. The physical and chemical properties of sericin fraction. 1) In contrast to the easy water soluble sericin, the hard soluble sericin contains fewer amino acids include of polar side radical while the hard soluble amino acid sach as alanine and leucine were detected. 2) The easy soluble amino acids were found mainly on the outer part of the fibroin, but the hard soluble amino acids were located in the near parts to the fibroin. 3) The swelling and solubility of the sericin could be hardly assayed by the analysis of the amino acid composition, and could be considered to tee closely related to the compound of the sericin crystal and secondary structure. 4) The X-ray patterns of the cocoon filament were ring shape, but they disappeared by the degumming treatment. 5) The sericin of tussah silkworm (A. pernyi), showed stronger circular patterns in the meridian than the regular silkworm (Bombyx mori). 6) There was no pattern difference between Fraction A and B. 7) X-ray diffraction patterns of the Sericin 1, ll and 111 were similar except interference of 8.85A (side chain spacing). 8) The amino acids above 150 in molecular weight such as Cys. Tyr. Phe. His. and Arg. were not found quantitatively by the 60 minutes-hydrolysis (6N-HCI). 9) The X-ray Pattern of 4.6A had a tendency to disappear with hot-water, ether, and alcohol treatment. 10) The partial hydrolysis of sericin showed a cirucular interference (2A) on the meridian. 11) The sericin pellet after hydrolysis was considered to be peptides composed with specific amino acids. 12) The decomposing temperature of Sericin 111 was higher than that of Sericin I and II. 13) Thermogram of the inner portioned sericin of the cocoon shell had double endothermic peaks at 165$^{\circ}C$, and 245$^{\circ}C$, and its decomposing temperature was higher than that of other portioned sericin. 14) The infrared spectroscopic properties among sericin I, II, III and sericin extracted from each layer portion of the cocoon shell were similar. II. The characteristics of seriein swelling and solubility related with silk processing. 1) Fifteen minutes was required to dehydrate the free moisture of cocoon shells with centrifugal force controlled at 13${\times}$10$^4$ dyne/g at 3,000 R.P.M. B) It took 30 minutes for the sericin to show positive reaction with the Folin-Ciocaltue reagent at room temperature. 3) The measurable wave length of the visible radiation was 500-750m${\mu}$, and the highest absorbance was observed at the wave length of 650m${\mu}$. 4) The colorimetric analysis should be conducted at 650mu for low concentration (10$\mu\textrm{g}$/$m\ell$), and at 500m${\mu}$ for the higher concentration to obtain an exact analysis. 5) The absorbing curves of sericin and egg albumin at different wave lengths were similar, but the absorbance of the former was slightly higher than that of the latter. 6) The quantity of the sericin measured by the colorimetric analysis, turned out to be less than by the Kjeldahl method. 7) Both temperature and duration in the cocoon cooking process has much effect on the swelling and solubility of the cocoon shells, but the temperature was more influential than the duration of the treatment. 8) The factorial relation between the temperature and the duration of treatment of the cocoon cooking to check for siricin swelling and solubility showed that the treatment duration should be gradually increased to reach optimum swelling and solubility of sericin with low temperature(70$^{\circ}C$) . High temperature, however, showed more sharp increase. 9) The more increased temperature in the drying of fresh cocoons, the less the sericin swelling and solubility were obtained. 10) In a specific cooking duration, the heavier the cocoon shell is, the less the swelling and solubility were obtained. 11) It was considered that there are differences in swelling or solubility between the filaments of each cocoon layer. 12) Sericin swelling or solubility in the cocoon filament was decreased by the wax extraction.. 13) The ionic surface active agent accelerated the swelling and solubility of the sericin at the range of pH 6-7. 14) In the same conditions as above, the cation agent was absorbed into the sericin. 15) In case of the increase of Ca ang Mg in the reeling water, its pH value drifted toward the acidity. 16) A buffering action was observed between the sericin and the water hardness constituents in the reeling water. 17) The effect of calcium on the swelling and solubility of the sericin was more moderate than that of magnecium. 18) The solute of the water hardness constituents increased the electric conductivity in the reeling water.