Alleviation of cadmium-induced photoinhibition and oxidative stress by melatonin in Chlamydomonas reinhardtii.

As one of the most threatening challenges to the natural environment and human health, cadmium (Cd) pollution has seriously impacted natural organisms. Green algae, such as Chlamydomonas reinhardtii (C. reinhardtii), can provide a safer, lower cost, and more effective ecological approach to the treatment of heavy metal ions in wastewater due to their sorption properties. However, heavy metal ions affect C. reinhardtii when adsorbed. Melatonin is able to protect the plant body from damage when the plant is under biotic/abiotic stress. Therefore, we investigated the effects of melatonin on the cell morphology, chlorophyll content, chlorophyll fluorescence parameters, enzymatic activity of the antioxidant system, gene expression, and the ascorbic acid (AsA)-glutathione (GSH) cycle of C. reinhardtii under the stress of Cd (13 mg/L). Our results indicated that Cd significantly induced photoinhibition and overaccumulation of reactive oxygen species (ROS). By application with the concentration of 1.0 µM melatonin, the algal solute of C. reinhardtii under the Cd stress gradually regained its green color, the cell morphology became intact, and the photosynthetic electron transport function was retained. However, in the melatonin-silenced strain, there was a significant decrease in all of the above indicators. In addition, the use of exogenous melatonin or the expression of endogenous melatonin genes could enhance the intracellular enzyme activities of catalase (CAT), peroxidase (POD), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR). It also upregulated the expression of active enzyme genes such as SOD1, CAT1, FSD1, GSH1, GPX5, and GSHR1. These results indicate that the presence of melatonin effectively protects the activity of photosynthetic system II in C. reinhardtii, enhances antioxidant activity, upregulates gene expression in the AsA-GSH cycle, and reduces the level of ROS, thereby alleviating the damage caused by Cd toxicity.

Structural variation and evolution of chloroplast tRNAs in green algae.

As one of the important groups of the core Chlorophyta (Green algae), Chlorophyceae plays an important role in the evolution of plants. As a carrier of amino acids, tRNA plays an indispensable role in life activities. However, the structural variation of chloroplast tRNA and its evolutionary characteristics in Chlorophyta species have not been well studied. In this study, we analyzed the chloroplast genome tRNAs of 14 species in five categories in the green algae. We found that the number of chloroplasts tRNAs of Chlorophyceae is maintained between 28-32, and the length of the gene sequence ranges from 71 nt to 91 nt. There are 23-27 anticodon types of tRNAs, and some tRNAs have missing anticodons that are compensated for by other types of anticodons of that tRNA. In addition, three tRNAs were found to contain introns in the anti-codon loop of the tRNA, but the analysis scored poorly and it is presumed that these introns are not functional. After multiple sequence alignment, the Ψ-loop is the most conserved structural unit in the tRNA secondary structure, containing mostly U-U-C-x-A-x-U conserved sequences. The number of transitions in tRNA is higher than the number of transversions. In the replication loss analysis, it was found that green algal chloroplast tRNAs may have undergone substantial gene loss during the course of evolution. Based on the constructed phylogenetic tree, mutations were found to accompany the evolution of the Green algae chloroplast tRNA. Moreover, chloroplast tRNAs of Chlorophyceae are consistent with those of monocotyledons and gymnosperms in terms of evolutionary patterns, sharing a common multi-phylogenetic pattern and rooted in a rich common ancestor. Sequence alignment and systematic analysis of tRNA in chloroplast genome of Chlorophyceae, clarified the characteristics and rules of tRNA changes, which will promote the evolutionary relationship of tRNA and the origin and evolution of chloroplast.

Conjugation of staphylokinase with the arabinogalactan-PEG conjugate: Study on the immunogenicity, in vitro bioactivity and pharmacokinetics.

Staphylokinase (SAK) is a bacterial protein with profibrinolytic activity. However, SAK suffers from short serum half-life and high immunogenicity. PEGylation with high Mw (20 kDa or 40 kDa) could decrease the immunogenicity and prolong the serum half-life of the proteins. However, the PEGylated protein could induce the anti-PEG antibodies and its bioactivity was significantly decreased. Arabinogalactan (AG) is a health-promoting substance with numerous biological activities. Conjugation of AG is an alternative strategy to solve the above-mentioned problems. However, conjugation with AG significantly decreased the bioactivity of a protein by shielding the bioactive domain. Here, AG conjugation and PEGylation were combined to improve the therapeutic efficacy of SAK. PEG with low Mw (2 kDa or 5 kDa) acted as a linker to conjugate AG from Larix. As compared with SAK-AG (22.3%), the conjugates (SAK-P2K-AG and SAK-P5K-AG) largely maintained the bioactivity of SAK (73.8% and 62.9%). The two conjugates both showed an 8-fold decrease in the SAK-specific IgG titers and a prolonged serum half-life. Moreover, the conjugates did not render any apparent toxicity to the heart, liver and renal functions of mice. Thus, our conjugation strategy is promising for the development of an effective long-acting therapeutic protein.

Conjugation with Eight-Arm PEG Markedly Improves the In Vitro Activity and Prolongs the Blood Circulation of Staphylokinase.

Staphylokinase (SAK) is a profibrinolytic protein and can be used for therapy of acute myocardial infarction and coronary thrombosis. However, SAK suffers from a short serum half-life time (∼6 min) that limits its clinical application. PEGylation prolongs the half-life time of SAK, whereas it significantly decreases the bioactivity of SAK for the steric shielding effect of PEG. To improve the bioactivity and prolong the half-life time of SAK, 8-arm PEG maleimide (8-arm PEG) was used for conjugation of multiple SAK molecules in one entity. C terminus of SAK was engineered with cysteine residue, followed by reaction with the maleimide moieties of 8-arm PEG to obtain the conjugate (SAKp-PEG). Conjugation with 8-arm PEG retained the secondary structure of SAK, slightly perturbed the tertiary structure of SAK, and essentially maintained its in vitro bioactivity by the multivalence of SAK. Conjugation with 8-arm PEG increased the hydrodynamic volume and thus significantly prolonged the half-life time of SAK. SAKp-PEG elicited a 1.4-fold increase in the SAK-specific IgG titers as compared with SAK, and rendered no apparent toxicity to the cardiac, liver and renal functions of mice. Thus, multiple conjugation of a protein with 8-arm PEG was an effective strategy to develop a long-acting protein drug with improved bioactivity and prolonged blood circulation.