Aims To establish a proper protoplast-preparation route from enriched motile flagellates and nonmotile resting cells of Haematococcus pluvialis . Methods and Results Through cultivations in two mixotrophic media, enriched Haematococcus flagellates and resting cells were respectively produced and applied in enzymatic protoplast preparations. Great differences of enzymatic sensitivity and osmotic-lability were identified between them. Flagellates showed the same osmotic-lability as protoplasts and the extracellular matrix-removing rate was applied for an evaluation of protoplast-releasing. During the treatment of flagellates, an addition of more than 0·2 mmol l −1 Ca 2+ was found to be essential for maintenance of high cellular viability. More than 80% cellular viability and a 90% protoplast-releasing rate were obtained simultaneously after 2–3 h treatment of 0·06% proteinase K in 0·05 mol l −1 Tris-HCl (pH7·8) buffer with 0·2 mmol l −1 CaCl 2 and 0·2 mol l −1 sorbitol/mannitol. For resting cells, a treatment of both 0·12% proteinase K and a combination of 2% cellulase + 1% snailase could function similarly in order to degrade the cellulosic cell wall, while the protoplast yield was limited to about 40%, due to the existence of an undegradable secondary wall in the mature resting cell. Conclusion Proteinase K was efficient for protoplast-releasing from either flagellates or resting cells. Due to the great difference of enzymatic sensitivity and osmotic-lability between flagellates and resting cells, it was necessary to select a different enzymatic treating process based upon the main cell type in the culture. A better protoplast-preparing efficiency was obtained from Haematococcus cells when flagellates were in the majority. Significance and Impact of the Study The protoplast preparation of H. pluvialis was firstly established based on two main cell types of H. pluvialis , motile flagellates and nonmotile resting cells. Increase of flagellate stability and viable protoplast-preparing efficiency through addition of Ca 2+ in enzymatic solution was firstly reported.
Mingxin Ye,Hui Li,Hongjun Luo,Yongyin Zhou,Wenhong Luo,Zhexuan Lin
Homocysteine is an amino acid containing a free sulfhydryl group, making it probably contribute to the antioxidative capacity in the body. We recently found that plasma total homocysteine (total-Hcy) concentration increased with time when whole blood samples were kept at room temperature. The present study was to elucidate how increased plasma total-Hcy is produced and explore the potential physiological role of homocysteine. Erythrocytes and leukocytes were separated and incubated in vitro; the amount of total-Hcy released by these two kinds of cells was then determined by HPLC-MS. The effects of homocysteine and methionine on reactive oxygen species (ROS) production, osmotic fragility, and methemoglobin formation in erythrocytes under oxidative stress were studied. The reducing activities of homocysteine and methionine were tested by ferryl hemoglobin (Hb) decay assay. As a result, it was discovered that erythrocytes metabolized methionine to homocysteine, which was then oxidized within the cells and released to the plasma. Homocysteine and its precursor methionine could significantly decrease Rosup-induced ROS production in erythrocytes and inhibit Rosup-induced erythrocyte’s osmotic fragility increase and methemoglobin formation. Homocysteine (but not methionine) was demonstrated to enhance ferryl Hb reduction. In conclusion, erythrocytes metabolize methionine to homocysteine, which contributes to the antioxidative capability under oxidative stress and might be a supplementary protective factor for erythrocytes against ROS damage.Keywords:homocysteine;methionine;oxidative stress;antioxidant
Aluminum (Al) is the most abundant metal in the earth’s crust. Al accumulates in erythrocyte and causes toxicity on erythrocyte membrane. The dysfunction of erythrocyte membrane is a potential risk to hypertension. The high Al content in plasma was associated with hypertension. To investigate the effect of AlCl 3 on blood pressure and the function of erythrocyte membrane, the rats were intragastrically exposed to 0, 64(1/20 LD 50 ), 128(1/10 LD 50 ), and 256(1/5 LD 50 ) mg/kg body weight AlCl 3 in double distilled water for 120 days, respectively. Then, we determined the systolic and mean arterial blood pressures of rats, the osmotic fragility, the percentage of membrane proteins, the activities of Na + /K + -ATPase, Mg 2+ -ATPase, Ca 2+ -ATPase, catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-pX), and malondialdehyde (MDA) content of the erythrocyte membrane in this experiment. The results showed that AlCl 3 elevated the systolic and mean arterial blood pressure of rats, increased the osmotic fragility, decreased the percentage of membrane protein, inhibited the activities of Na + /K + -ATPase, Mg 2+ -ATPase, Ca 2+ -ATPase, CAT, SOD and GSH-pX, and increased the MDA content of erythrocyte membrane. These results indicate that AlCl 3 may induce hypertension by disturbing the function of erythrocyte membrane.
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