The Role of Vacuole and Vacuolar H+-ATPase in Tolerance to Cadmium in Saccharomyces Cerevisiae

รายงานการวิจัย--มหาวิทยาลัยเทคโนโลยีราชมงคลพระนคร, 2553Heavy metal is one of the major environmental pollutants. However, the protective cellular mechanisms in response to heavy metal stress are not well-understood yet. Previously, it has been found that the yeast deletion mutants lacking vacuolar H+-ATPase (V-ATPase) activity, which functions in vacuolar acidification, exhibited growth defects under cadmium stress conditions. In addition, several genes involved in H+-ATPase have been recently found to be required for resistance to aluminum. To investigate the roles of vacuolar H+-ATPase, plasma membrane H+-ATPase, and mitochondrial H+-ATPase in tolerance to cadmium, we examined the growth of yeast deletion mutants lacking different types of H+-ATPase in YPD media containing 80 μM of cadmium. Our results showed that a number of mutants lacking V-ATPase activity were sensitive to cadmium, suggesting the important role of V-ATPase in cadmium detoxification. We next examined the growth of mutants lacking genes responsible for different vacuolar functions in order to investigate the functional activities of vacuoles involved in cadmium detoxification mechanism. We found that the mutants lacking vacuolar protein-sorting genes exhibited high sensitivity to cadmium as well, suggesting the importance of protein transportation during cadmium stress.Rajamangala University of Technology Phra Nakho

Metabolic disturbance and phytochemical changes in Andrographis paniculata and possible action mode of andrographolide

Objective: To explore the effect of gibberellic acid (GA3) and its inhibitor paclobutrazol (PBZ) on chemical composition and their pharmacological effects on Andrographis paniculata (Burm. f.) Wall. ex Nees, and to clarify action mode of andrographolide. Methods: The chemical composition was extracted by sequential extraction with hexane, dichloromethane, ethyl acetate and methanol, respectively. Andrographolide and its derivatives were evaluated by HPLC. Moreover, the metabolic profiling was analyzed by GC-MS. Inhibitory effect of crude extracts was tested against Staphylococcus aureus using agar well diffusion method. Mode of action was tested against mutant yeast by spotting assay. Andrographolide were tested for their mode of action against eukaryotes. Results: Among different solvents, dichloromethane gave the highest yield of crude (3.58% DW), with the highest andrographolide content (8.3 mg/g DW). The effect of plant hormone (10 mg/L GA3 or PBZ) on phytochemical variations and bioactivity of Andrographis paniculata was demonstrated. It was found that PBZ promoted sesquiterpene compounds about 3.5 times over than GA3 treatment. But inhibitory effect of extracts against Staphylococcus aureus was highest in GA3 treated plants; andrographolide and 14-deoxy-11,12-didehydroandrographolide contents were significantly higher than those of water or PBZ. It was found that there were 11 strains involving in ergosterol biosynthesis, V-ATPase activity and homeostasis, and superoxide detoxification process. In this regard, andrographolide might cause the damage on the lipid bilayer of yeast cell and plasma membrane by interfering ergosterol biosynthesis. Conclusions: It is found that GA3 promotes andrographolide and 14-deoxy-11,12-didehydroandrographolide content while PBZ promotes sesquiterpene content. Andrographolide might cause the damage on the lipid bilayer of yeast cell and plasma membrane by interfering ergosterol biosynthesis. It might also affect mitochondria electron transport chain, leading to the occurrence of ROS, which can further harm cell organelles. However, the library screening is the first step to investigate mode of action of andrographolide