OBJECTIVETo investigate the role of caspase-3 and its inhibitor Ac-DEVD-CHO in rat lens epithelial cell apoptosis induced by hydrogen peroxide (H(2)O(2)) in vitro.

METHODSRat lenses were incubated in modified Eagle's medium containing 2 mmol/L H(2)O(2) to induce apoptosis in vitro. Apoptosis in lens epithelial cells was assessed by transmission electron microscopy and annexin V-propidium iodide (PI) double staining flow cytometry after 12, 24 and 48 h of incubation. The activity of caspase-3 was analyzed by western blotting.

RESULTSObservations under transmission electron microscopy revealed that 2 mmol/L H(2)O(2) could effectively induce lens epithelial cell apoptosis in vitro. Caspase-3 activity increased during cell apoptosis and the peak measurement occurred at 24 h after treatment with H(2)O(2). Cell apoptosis was blocked by caspase-3 inhibitor Ac-DEVD-CHO.

CONCLUSIONSThe activation of caspase-3 plays an important role in executing apoptosis in H(2)O(2)-treated lens epithelial cells and in the formation of cataract. The caspase-3 inhibitor Ac-DEVD-CHO may effectively prevent lens epithelial cell apoptosis caused by oxidative injury.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; Caspase Inhibitors ; Caspases ; metabolism ; physiology ; Cysteine Proteinase Inhibitors ; pharmacology ; Enzyme Activation ; Epithelial Cells ; cytology ; Female ; Hydrogen Peroxide ; pharmacology ; Lens, Crystalline ; cytology ; Oligopeptides ; pharmacology ; Organ Culture Techniques ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate whether proteasome inhibitor MG-132 induces apoptosis of human erythroleukemia cell line K562 and possible mechanisms.

METHODSK562 cells were incubated with RPMI 1640 and exposed to 0, 1, 5, 10, 15 micromol/L of MG-132 for 24 hrs, respectively. The apoptosis of cells were detected by fluorescence microscope, DNA fragments and flow cytometry. The NF-kappaB mRNA expression was quantified by reverse transcription-polymerase chain reaction (RT-PCR). Expression of NF-kappaB and caspase-3 was semiquantitatively analyzed with SABC techniques. Caspase-3 activities were measured with a colorimetric method.

RESULTSThe growth of K562 cells was inhibited and the apoptosis of the cells increased after MG-132 treatment in a dose-dependent manner. After 24 hrs of 15 micromol/L MG-132 treatment, the percentage of apoptotic cells (26.5+/-0.6%) increased significantly when compared with the untreated controls (1.2+/-0.1%) (P<0.01). MG-132 treatment decreased the mRNA and protein expression of NF-kappaB, and increased the protein expression of caspase-3.

CONCLUSIONSMG-132 can induce apoptosis of human erythroleukemia cell line K562 through the down-regulation of NF-kappaB expression and up-regulation of caspase-3 expression.

Antineoplastic Agents ; pharmacology ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Cysteine Proteinase Inhibitors ; pharmacology ; Dose-Response Relationship, Drug ; Humans ; K562 Cells ; Leupeptins ; pharmacology ; NF-kappa B ; Proteasome Inhibitors ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the effect of interleukin-6 (IL-6) on the apoptosis of annulus fibrosus (AF) cell induced by interleukin-1beta (IL-1beta).

METHODSCultured AF cells were divided into 6 groups and treated with no drug, 10 ng/mL IL-6, 10 ng/mL IL-1beta, 10 ng/mL IL-1beta and Z-VAD-FMK (a caspase-9 inhibitor), 10 ng/mL IL-1beta and 10 ng/mL IL-6, 10 ng/mL IL-1beta and 100 ng/mL IL-6, respectively. After three days of culture, the apoptosis rate, the positive rates of caspase-3, -8, and -9 of AF cells were detected with flow cytometry.

RESULTSThe apoptosis rates of cells in group 1 to 6 were 2.67% +/- 1.08%, 2.71% +/- 0.53%, 20.37% +/- 1.57%, 11.34% +/- 0.67%, 18.17% +/- 0.74%, and 9.42% +/- 1.08%, respectively. There was no significant difference between group 1 and 2, while the apoptosis rates of group 4, 5, and 6 were significantly lower than group 3 (P = 0.001, P = 0.172, and P = 0.001, respectively). Positive rates of caspase-3 in group 5 (12.35% +/- 0.64%) and 6 (9.26% +/- 0.36%) were significantly lower than group 3 (17.14% +/- 0.72%; P = 0.001 and P < 0.001, respectively). And positive rates of caspase-9 in group 5 (15.13% +/- 1.45%) and 6 (10.17% +/- 2.50%) were significantly lower than group 3 (19.4% +/- 0.98% ; P = 0.014 and P = 0.004, respectively). But there was not obvious change of caspase-8 activity after IL-6 was added.

CONCLUSIONIL-6 is capable of protecting AF cells from IL-1beta induced apoptosis in vitro. Mechanism of the protection is related with the inhibition of caspase-3 and -9 activities.

Amino Acid Chloromethyl Ketones ; pharmacology ; Animals ; Apoptosis ; drug effects ; Caspase Inhibitors ; Cells, Cultured ; Cysteine Proteinase Inhibitors ; pharmacology ; Interleukin-1beta ; pharmacology ; Interleukin-6 ; pharmacology ; Intervertebral Disc ; cytology ; drug effects ; enzymology ; Rabbits

OBJECTIVETo investigate the effect of z-DEVD-fmk, a caspase-3 inhibitor on the neuronal apoptosis in ischemia-reperfusion region (IRR) of rat cerebral cortex.

METHODSRats prepared by middle cerebral artery occlusion and reperfusion were used as the research model. The animals were divided into A group (untreated), B group (DMSO control) and C group (treated with z-DEVD-fmk). Before reperfusion, z-DEVD-fmk (7 microg/kg) was injected into the ischemic side of ventriculus cerebri of C group rats. The expression and activation of caspase-3, expression and cleavage of poly (ADP-ribose) polymerase (PARP), and apoptotic neurons in the temporal-parietal cortex IRRs (SPAB method) of all the rats were studied using Western blotting, in situ apoptotic detection (TUNEL method) and immunohistochemistry.

RESULTSIn the cerebral IRRs of A, B, C groups reperfused for 1 h and 24 h, the quantities of caspase-3 precursor were 16.7 +/- 3.0, 11.5 +/- 3.0 and 47.5 +/- 3.5, and 76.1 +/- 3.5, 71.3 +/- 6.4 and 88.2 +/- 5.5, respectively; the caspase-3 fragments (12,000) 8.2 +/- 2.3, 9.4 +/- 1.2 and 4.3 +/- 1.6, and 59.0 +/- 6.3, 60.5 +/- 7.2 and 17.3 +/- 2.8, respectively; the PARP 12.6 +/- 3.0, 13.9 +/- 2.0 and 53.7 +/- 4.1, and 67.5 +/- 8.6, 61.1 +/- 6.6 and 93.6 +/- 4.1, respectively; the PARP fragments (24,000) 6.0 +/- 0.7, 6.6 +/- 1.2, 3.6 +/- 1.1, and 27.4 +/- 2.6, 25.8 +/- 3.2, 12.1 +/- 2.8 (relative quantity, x+/- s); the densities of apoptotic neurons 83.3 +/- 7.5, 84.3 +/- 5.7 and 45.7 +/- 4.0, and 197.4 +/- 11.8, 185.2 +/- 11.2 and 99.1 +/- 5.8 (cell number/0.1 mm(2), x+/- s). These results showed that in the cerebral IRRs of both A and B groups, all caspase-3 expression and activation, PARP expression and cleavage, and neuronal apoptosis were increased relevantly along with prolongation of the reperfusion time (P < 0.05 - 0.001). At each time point of the reperfusion, caspase-3 activation, PARP cleavage and neuronal apoptosis in the cerebral IRR of C group were significantly less than those of the former two groups (P < 0.05 - 0.001). The variations of the 5 parameters of A, B and C groups correlated positively with one another (r = 0.630 - 0.942, P < 0.01). The cells expressing PARP were mainly neurons in the cerebral IRRs of all the animals, but the difference of their number was not distinct among the 3 groups.

CONCLUSIONSIt is an important mechanism resulting in apoptosis of the injured neurons in the cerebral IRR that caspase-3 expression and activation abnormally increased by the reperfusion have more PARP rapidly inactivated by over-cleavage. z-DEVD-fmk may decrease PARP cleavage by inhibiting activity and auto-activation of caspase-3, and prevent the injured neurons from apoptosis.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase Inhibitors ; Cerebral Cortex ; drug effects ; metabolism ; pathology ; Cysteine Proteinase Inhibitors ; pharmacology ; Infarction, Middle Cerebral Artery ; complications ; Male ; Neurons ; drug effects ; Oligopeptides ; pharmacology ; Poly(ADP-ribose) Polymerases ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Reperfusion Injury ; etiology ; metabolism ; physiopathology

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

BACKGROUNDAtrial fibrillation (AF) is accompanied by atrial structural remodeling. Calpain activity is induced during AF. To test a causal relationship between calpain activation and atrial structural changes, N-acetyl-Leu-Leu-Met (ALLM), a calpain inhibitor, was utilized in a canine AF model.

METHODSFifteen dogs were randomly divided into 3 groups: sham-operated group, control group and calpain inhibitor group; each with 5 dogs. Sustained AF was induced by rapid right atrium pacing at 600 beats per minute for 3 weeks. ALLM was administered at a dosage of 1.0 mg x kg(-1) x d(-1) in the calpain inhibitor group. Three weeks later, the proteolysis, protein expression of TnT and myosin, calpain I localization and expression and structural changes were examined in left atrial free walls, right atrial free walls and the interatrial septum respectively. Atrial size and contractile function were also measured by echocardiography.

RESULTSLong-term rapid atrial pacing induced marked structural changes such as enlarged atrial volume, myolysis, degradation of TnT and myosin, accumulation of glycogen and changes in mitochondrial shape and size, which were paralleled by an increase in calpain activity. The positive correlation between calpain activity and the degree of myolysis (r(s) = 0.90 961, P < 0.0001) was demonstrated. In addition to structural abnormalities, pacing-induced atrial contractile dysfunction was observed in this study. The pacing-induced atrial structural alterations and loss of contractility were partially prevented by the calpain inhibitor ALLM.

CONCLUSIONSActivation of calpain represents key features in the progression towards overt structural remodeling. Calpain inhibitor, ALLM, suppressed the increased calpain activity and reversed structural remodeling caused by sustained atrial fibrillation in the present model. Calpain inhibition may therefore provide a possibility for therapeutic intervention in AF.

Animals ; Atrial Fibrillation ; pathology ; Calpain ; antagonists & inhibitors ; Cysteine Proteinase Inhibitors ; pharmacology ; Disease Models, Animal ; Dogs ; Heart Atria ; pathology ; ultrastructure ; Myosins ; analysis ; Troponin T ; analysis

According to the amino acids sequence of OC-IdeltaD86 gene and Escherichia coli codon usage, we synthesized this gene by overlap extension PCR method with 7 oligonucleotides DNA fragments. The PCR fragment was inserted into pGEM-T-easy vector and the recombined plasmid was named pGEM-T-OC-IdeltaD86. Two oligonucleotides into which the BamH I and Xho I sites were introduced were designed and synthesized based on pGEM-T-OC-IdeltaD86 and pet21b, and the PCR fragment into which the BamH I and Xho I sites were introduced was obtained. After digesting it with BamH I and Xho I, OC-IdeltaD86 gene was cloned into the corresponding sites of pet21b and obtained prokaryotic expression vector pet21b-OC-IdeltaD86. OC-IdeltaD86 gene was expressed in E. coli (BL21(DE3)plysS) after IPTG(Isopropyl beta-D-1-thiogalactopyranoside) inducement for 5 hours. The fusion protein of OC-IdeltaD86:6His gene accounted for 11.4% of total protein and 16.4% of soluble protein, which had been successfully purified by Ni-NTA and concentrated by PEG20000. This protein can effectively inhibit papain activity in vitro and may be used in anti-nematode research in vivo.
Cloning, Molecular ; Cystatins ; biosynthesis ; genetics ; Cysteine Endopeptidases ; metabolism ; Cysteine Proteinase Inhibitors ; genetics ; Escherichia coli ; genetics ; metabolism ; Genes, Plant ; genetics ; Mutation ; Oligonucleotides ; chemical synthesis ; genetics ; Oryza ; genetics ; Papain ; antagonists & inhibitors ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; metabolism

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

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