Helminthic cysteine proteases are well known to play critical roles in tissue invasion, nutrient uptake, and immune evasion of the parasites. In the same manner, the sparganum, the plerocercoid of Spirometra mansoni, is also known to secrete a large amount of cysteine proteases. However, cysteine protease inhibitors regulating the proteolytic activities of the cysteine protease are poorly illustrated. In this regard, we partially purified an endogenous cysteine protease inhibitor from spargana and characterized its biochemical properties. The cysteine protease inhibitor was purified by sequential chromatographies using Resource Q anion exchanger and Superdex 200 HR gel filtration from crude extracts of spargana. The molecular weight of the purified protein was estimated to be about 11 kD on SDS-PAGE. It was able to inhibit papain and 27 kDa cysteine protease of spargana with the ratio of 25.7% and 49.1%, respectively, while did not inhibit chymotrypsin. This finding suggests that the cysteine protease inhibitor of spargana may be involved in regulation of endogenous cysteine proteases of the parasite, rather than interact with cysteine proteases from their hosts.
Animals ; Cystatins/pharmacology ; Cysteine Endopeptidases/metabolism ; Cysteine Proteinase Inhibitors/chemistry/*metabolism/*pharmacology ; Helminth Proteins/*metabolism/*pharmacology ; Spirometra/*metabolism

Cystatin, which widely distributed in both tissues and body fluids of animal and plant, was a superfamily of cysteine proteinase inhibitors. It could form activity-inhibitor complexes with cysteine proteinases to inhibit the hydrolytic activity of proteinases. Cystatin played important roles not only in the inhibition of the proteolytic degradation of fish muscle, but also in biological defense systems against invaders. To explore the functions of fish cystatin and the potential values in fish disease prevention and cure, as well as seafood processing, the recombinant yeast strains which could express Chinese sturgeon cystatin were constructed. First, the cystatin cDNA of Chinese sturgeon, which had been PCR modified, was subcloned into yeast integrated vector pPICZaA. After extracted and purified, the recombinant plasmids were linearized by Sac I. The yeast Pichia pastoris GS115 strain was transformed by use of the Lithium Chloride transformation method, and the recombinant cystatin yeast strains got. After 0.5% methanol induction, SDS-PAGE analysis of the culture supernatant indicated that the yield of recombinant cystatin was about 215mg x L(-1) with the percentage about 73.6%. The recombinant cystatin was purified through Q-Sepharose anion-exchange chromatography, and the purity reached about 94.2%. The inhibitory activity of recombinant cystatin was measured by inhibiting the proteinase activity of papain. The results showed that about 1 microg recombinant cystatin could inhibit the activity of 15 microg papain. Heat stability assay results showed that there was a decrease in inhibitory activity of cystatin with the increasing of temperature. When solution of recombinant cystatin was kept at 70 degrees C for 5min, the inhibitory activity reduced fast. While the recombinant cystatin was heated to 90 degrees C for 5min, the inhibitory activity of recombinant cystatin was undetected. The inhibitory activity for recombinant Chinese sturgeon cystatin was higher than that of CPI (cysteine proteinase inhibitor) from seeds of corn, that about 1 microg purified CIP could inhibited the activity of 0.278 microg papain. But the heat stability of recombinant cystatin is lower than that of the corn CPI. The expression level and the activity of recombinant cystatin from yeast Pichia pastoris were higher than those from E. coli. Moreover, recombinant cystatin from Pichia pastoris was easier to separate and purify. This paper reported that recombinant fish cystatin was produced in a highly efficient expression system based on the methylotrophic yeast, further work will focus on the function of recombinant Chinese sturgeon cystatin to resist fish disease and explore the value of cystatin as a food additive to inhibit cysteine proteinases during surimi processing.
Animals ; Cystatins ; genetics ; metabolism ; pharmacology ; Cysteine Proteinase Inhibitors ; genetics ; metabolism ; pharmacology ; Enzyme Activation ; drug effects ; Fish Proteins ; genetics ; metabolism ; Pichia ; genetics ; metabolism ; Polymerase Chain Reaction ; Protein Stability ; Temperature

Intravascular administration of magnesium (Mg) causes vasodilation and increases renal blood flow. The aim of this study was to investigate the renal effect of Mg following unclamping of the supraceliac aorta. Mongrels were divided into two groups, control (group C, n=7) and Mg group (group Mg, n=7). In group Mg, 30 mg/kg MgSO4 was injected as a bolus immediately prior to unclamping the supraceliac aorta and thereafter as an infusion (10 mg/kg/hr). The group C received an equivalent volume of saline solution. Systemic hemodynamics, renal artery blood flow, renal cortical blood flow (RCBF), renal vascular resistance, and renal function were compared. Following the aortic unclamping, cardiac output and RCBF were less attenuated, and the systemic and renal vascular resistance was elevated to a lesser degree in the group Mg compared to the group C. There was no significant difference in the plasma renin activity, serum creatinine and Cystatin-C between the two groups. The present study shows that Mg infusion improves systemic hemodynamics and RCBF after aortic unclamping. However, we did not observe any improvement in renal function when Mg was administered after supraceliac aortic unclamping.
Animals ; Aorta, Abdominal/physiology/*surgery ; Blood Pressure/drug effects ; Calcium/blood ; Cardiac Output/drug effects ; Comparative Study ; Creatinine/blood ; Cystatins/blood ; Dogs ; Female ; Heart Rate/drug effects ; Magnesium/blood ; Magnesium Sulfate/*pharmacology ; Male ; Renal Circulation/*drug effects ; Renin/blood ; Research Support, Non-U.S. Gov't