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

Sulfur is an essential element for the entire biological kingdom because of its incorporation into amino acids, proteins and other biomolecules. Sulfur atoms are also important in the iron-containing flavoenzymes. Unlike humans, plants can use inorganic sulfur to synthesize sulfur-containing amino acids. Therefore, plants are an important source of sulfur for humans. Sulfur-containing compounds are found in all body cells and are indispensable for life. Some of sulfur-containing antioxidant compounds are, cysteine, methionine, taurine, glutathione, lipoic acid, mercaptopropionylglycine, N-acetylcysteine, and the three major organosulfur compounds of garlic oil, diallylsulfide, diallyldisulfide and diallyltrisulfide. In a comparison of the structure-function relationship among these sulfur-containing antioxidant compounds, dihydrolipoic acid (the reduced form of LA) is the most effective antioxidant. Dihydrolipoic acid contains two sulfhydryl groups and can undergo further oxidation reaction to form lipoic acid. The antioxidative activities of sulfur-containing compounds follow a general trend, the more highly reduced forms are stronger antioxidants and the number of sulfur atoms determine, at least in part, their modulatory activites on the glutathione related antioxidant enzymes. In this article, the antioxidant effects and the antioxidative activities, of sulfur-containing amino acids, are reviewed. In addition, the general antioxidant effects and the structure-function relationship of some sulfur-containing compounds are also reviewed.
Acetylcysteine/pharmacology ; Amino Acids, Sulfur/*pharmacology ; Antioxidants/*pharmacology ; Cysteine/pharmacology ; Glutathione/pharmacology ; Methionine/pharmacology ; Structure-Activity Relationship ; Taurine/pharmacology ; Thioctic Acid/pharmacology ; Thiopronine/pharmacology

The purpose of the present study is to investigate the effect of L-cysteine on colonic motility and the underlying mechanism. Immunohistochemical staining and Western blot were used to detect the localization of the HS-generating enzymes cystathionine-β-synthase (CBS) and cystathionine-γ-lyase (CSE). Organ bath system was used to observe the muscle contractile activities. Whole-cell patch-clamp technique was applied to record ionic channels currents in colonic smooth muscle cells. The results showed that both CBS and CSE were localized in mucosa, longitudinal and circular muscle and enteric neurons. L-cysteine had a dual effect on colonic contraction, and the excitatory effect was blocked by pretreatment with CBS inhibitor aminooxyacetate acid (AOAA) and CSE inhibitor propargylglycine (PAG); L-cysteine concentration-dependently inhibited L-type calcium channel current (I) without changing the characteristic of L-type calcium channel (P < 0.01); In contrast, the exogenous HS donor NaHS increased I at concentration of 100 μmol/L, but inhibited I and modified the channel characteristics at concentration of 300 μmol/L (P < 0.05); Furthermore, L-cysteine had no effect on large conductance calcium channel current (I), but NaHS significantly inhibited I (P < 0.05). These results suggest that L-cysteine has a potential dual effect on colonic smooth muscle and the inhibitory effect might be directly mediated by L-type calcium channel while the excitatory effect might be mediated by endogenous HS.
Cystathionine beta-Synthase ; Cystathionine gamma-Lyase ; Cysteine ; pharmacology ; Hydrogen Sulfide ; Muscle, Smooth

A 29 kDa cysteine protease of Taenia solium metacestodes was purified by Mono Q anion-exchanger and Superose 6 HR gel filtration chromatography. The enzyme was effectively inhibited by cysteine protease inhibitors, such as iodoacetic acid (IAA) and trans-epoxy-succinyl-L-leucyl-amido (4-guanidino) butane (E-64) while inhibitors acting on serine- or metallo-proteases did not affect the enzyme activity. The purified enzyme degraded human immunoglobulin G (IgG), collagen and bovine serum albumin (BSA), but human IgG was more susceptible for proteolysis by the enzyme. To define the precise biological roles of the enzyme, more detailed biochemical and functional studies would be required.
Taenia solium/*enzymology ; Serum Albumin, Bovine/metabolism ; Leucine/analogs & derivatives/pharmacology ; Iodoacetic Acid/pharmacology ; Immunoglobulin G/metabolism ; Humans ; Cysteine Proteinase Inhibitors/pharmacology ; Cysteine Endopeptidases/chemistry/*isolation & purification/metabolism ; Collagen/metabolism ; Chromatography, Ion Exchange ; Chromatography, Gel ; Animals

OBJECTIVEApoptosis of the cells of liver cancer cell line HepG2 could be induced by TNF alpha and actinomycin D (Act D). In the current study, the molecular mechanism of the apoptosis protection of stronger neo-minophagen C (SNMC) to HepG2 cells was investigated.

METHODSSNMC was added to the HepG2 cell culture medium when the cell concentration reached 0, 2, 20, 100, 200, 800 microg/ml 30 min before their apoptosis were inducted with TNF alpha and Act D. A flow cytometry assay was performed to detect the cell apoptosis rate; electromicroscopy was employed to visualize the subcellular structure after apoptosis. DNA ladder formation was checked with genomic DNA agarose electrophoresis. The expression pattern of apoptosis related protein Caspase-3, Bcl-2 and Bax was detected by Western blot.

RESULTSAfter pretreatment with various concentrations of SNMC and 12 hours after treatment with TNF alpha and Act D, the HepG2 cell apoptosis rate and DNA ladder formation decreased dramatically when the SNMC concentration was higher in the media; the intracellular inactive form of Caspase-3 increased while the 17*10(3) active Caspase-3 decreased gradually. In addition, the expression of Bcl-2 increased and the expression of Bax decreased. Under the electromicroscope, the typical nucleolus condensation of HepG2 induced by TNF alpha and Act D was not seen among the 100 microg/ml SNMC treated cells.

CONCLUSIONSNMC inhibits TNF alpha and Act D induced HepG2 cell apoptosis. This protective action may be regulated by intracellular apoptosis related factors.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; pathology ; Cell Line, Tumor ; Cysteine ; pharmacology ; Drug Combinations ; Glycine ; pharmacology ; Glycyrrhiza ; Humans ; Liver Neoplasms ; pathology ; Oleanolic Acid ; analogs & derivatives ; pharmacology

OBJECTIVETo observe the protective effect of the proteasome inhibitor MG-132 in rats with severe acute pancreatitis (SAP) and the associated lung injury.

METHODSIn rat models of the SAP established with injection of 5% sodium taurocholate into the biliary-pancreatic duct, the changes of the serum amylase and myeloperoxidase (MPO) activity in the pancreatic and lung tissues were evaluated. The pathological changes of the pancreatic and lung tissues were also observed.

RESULTSMG-132 significantly decreased serum amylase, pancreatic weight/body weight ratio, and pancreatic and pulmonary myeloperoxidase activity (P<0.05). Histopathological examinations revealed milder edema, cellular damage, and inflammation in the pancreatic and lung tissues of rats pretreated with the peptide (P<0.05).

CONCLUSIONMG-132 ameliorates SAP and the associated lung injury in rats.

Acute Disease ; Amylases ; blood ; Animals ; Cysteine Proteinase Inhibitors ; pharmacology ; Leupeptins ; pharmacology ; Lung ; pathology ; Lung Injury ; drug therapy ; Pancreas ; pathology ; Pancreatitis ; drug therapy ; Peroxidase ; blood ; Rats ; Rats, Sprague-Dawley

This study was aimed to investigate the effect of proteasome inhibitor MG-132 at different doses on cultured K562 cell apoptosis. MTT assay was used to observe the activity of K562 cell proliferation inhibition rate after treating for 48 hours at different doses (0, 2, 4, 8, 16, 32 micromol/L). Immunocytochemistry was used to detect the NF-kappaB activity and glucocorticoid receptor (GR) expression. Flow cytometry was used to determine the K562 cell apoptosis. The results indicated that proliferation inhibition rate of K562 cells after treated for 48 hours showed dose-dependent, the inhibitory rates of cell proliferation in test groups were significant higher than that in control group, and the effect in 32 micromol/L test group was the most obvious (45.24 +/- 4.12)% (p < 0.05). The NF-kappaB activity and GR expression after treating for 48 hours showed dose-dependent. Compared with control group, the NF-kappaB activities in test groups were lower (p < 0.05), and the NF-kappaB activity in 32 micromol/L test group was the lowest (63.60 +/- 2.95); the GR expression in test groups was higher (p < 0.05), and the GR expression in 16 micromol/L test group was the highest (75.62 +/- 2.70). The K562 cell apoptosis rate after treating for 48 hours also showed dose-dependent. Compared with control group, the K562 cell apoptosis rates in test groups were higher (p < 0.05), the K562 cell apoptosis rate in 32 micromol/L test group was the highest (21.37 +/- 2.02)%. It is concluded that the MG-132 may induce K562 cell apoptosis and proliferation inhibition through up-regulation of NF-kappaB activity and down-regulation of GR expression both in dose-dependent manner.
Apoptosis ; drug effects ; Cell Proliferation ; drug effects ; Cysteine Proteinase Inhibitors ; pharmacology ; Humans ; K562 Cells ; Leupeptins ; pharmacology ; NF-kappa B ; metabolism ; Receptors, Glucocorticoid ; metabolism

The present study aimed to investigate the protective effect of S-propargyl-cysteine (SPRC) on atherosclerosis progression in mice. A mouse model of vulnerable atherosclerotic plaque was created in ApoE^-/- mice by carotid artery tandem stenosis (TS) combined with a Western diet. Macrophotography, lipid profiles, and inflammatory markers were measured to evaluate the antiatherosclerotic effects of SPRC compared to atorvastatin as a control. Histopathological analysis was performed to assess the plaque stability. To explore the protective mechanism of SPRC, human umbilical vein endothelial cells (HUVECs) were cultured in vitro and challenged with oxidized low-density lipoprotein (ox-LDL). Cell viability was determined with a Cell Counting Kit-8 (CCK-8). Endothelial nitric oxide synthase (eNOS) phosphorylation and mRNA expression were detected by Western blot and RT-qPCR respectively. The results showed that the lesion area quantified by en face photographs of the aortic arch and carotid artery was significantly less, plasma total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) were reduced, plaque collagen content was increased and matrix metalloproteinase-9 (MMP-9) was decreased in 80 mg/kg per day SPRC-treated mice compared with model mice. These findings support the role of SPRC in plaque stabilization. In vitro studies revealed that 100 μmol/L SPRC increased the cell viability and the phosphorylation level of eNOS after ox-LDL challenge. These results suggest that SPRC delays the progression of atherosclerosis and enhances plaque stability. The protective effect may be at least partially related to the increased phosphorylation of eNOS in endothelial cells.
Animals ; Humans ; Mice ; Atherosclerosis ; Cholesterol/metabolism* ; Cysteine/pharmacology* ; Human Umbilical Vein Endothelial Cells/metabolism* ; Lipoproteins, LDL/pharmacology* ; Nitric Oxide Synthase Type III/metabolism* ; Phosphorylation ; Plaque, Atherosclerotic/pathology*

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

OBJECTIVETo investigate the inhibition mechanisms of BAY11-7082 (IkappaB-alpha phosphorylation inhibitor) and Lactacystein (proteosome inhibitor) in CD154-induced NF-kappaB activation.

METHODSWe used recombinant CD154 to stimulate EBV/LMP1 negative Ramos B cell and observed the effects of BAY11-7082 and Lactacystein in CD154-induced NF-kappaB luciferase activation, phosphorylation and degradation of IkappaB-alpha, phosphorylation of p65, and nuclear translocation of NF-kappaB subunits upon CD154 stimulation.

RESULTSBoth BAY11-7082 and Lactacystein abrogated CD154-induced NF-kappaB luciferase activation in Ramos cells. While CD154-induced phosphorylation of p65, phosphorylation and degradation of IkappaB-alpha, and nuclear translocation of p50, p65, and c-Rel were all blocked by BAY11-7082; Lactacystein only inhibited degradation of IkappaB-alpha and p65 nuclear translocation.

CONCLUSIONBAY11-7082 and Lactacystein inhibit CD154-induced NF-kappaB activation through different mechanisms.

Acetylcysteine ; analogs & derivatives ; pharmacology ; Apoptosis ; drug effects ; Burkitt Lymphoma ; pathology ; CD40 Ligand ; pharmacology ; Cysteine Proteinase Inhibitors ; pharmacology ; Enzyme Activation ; drug effects ; Humans ; NF-kappa B ; antagonists & inhibitors ; metabolism ; Nitriles ; pharmacology ; Sulfones ; pharmacology ; Tumor Cells, Cultured