OBJECTIVETo design hammerhead ribozymes against mouse caspase-7 and to study their expression and cleavage activity in vitro.

METHODSThe secondary structures of ribozyme and caspase-7 genes were analyzed and simulated by computer. Ribozymes DNA sequences were synthesized by automatic synthetic apparatus. Caspase-7 DNA sequence was acquired by reverse transcription PCR. Ribozymes and caspase-7 DNA sequences were separately cloned into pBSKneo U6 and pGEM-T vectors. Ribozymes and caspase-7 mRNA were obtained by transcription in vitro, and ribozymes cleavage activity was identified by cleavage experiment in vitro.

RESULTSTwo ribozymes named Rz333 and Rz394 targeting 333 and 394 sites in caspase-7 mRNA were designed by computer software, and their DNA sequences were synthesized. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed by in vitro transcription. In vitro cleavage experiments showed that Rz333 cleaved caspase-7 mRNA and produced 243nt and 744nt segments. The cleavage efficiency is 67.98%, while Rz394 cannot cleave caspase-7 mRNA.

CONCLUSIONSRz333 can site-specifically cleave caspase-7 mRNA.

Animals ; Base Sequence ; Caspase 7 ; Caspase Inhibitors ; Caspases ; genetics ; Cloning, Molecular ; Mice ; Mice, Inbred BALB C ; Molecular Sequence Data ; RNA, Catalytic ; biosynthesis ; genetics ; metabolism ; RNA, Messenger ; biosynthesis ; genetics

To investigate the role of mitochondria in neuronal apoptosis, ischemia-reperfusion mediated neuronal cell injury model was established by depriving of glucose, serum and oxygen in media. DNA fragmentation, cell viability, cytochrome C releasing, caspase3 activity and mitochondrial transmembrane potential were observed after N2a cells suffered the insults. The results showed that N2a cells in ischemic territory exhibited survival damage, classical cell apoptosis change, DNA ladder and activation of caspase3. Apoptosis-related alterations in mitochondrial functions, including release of cytochrome C and depression of mitochondrial transmembrane potential (deltapsim) were testified in N2a cells after mimic ischemia-reperfusion. Moreover, activation of caspase3 occurred following the release of cytochrome C. However, the inhibitor of caspase3, Ac-DEVD-CHO, couldn't completely rescue N2a cells from apoptosis. Administration of cyclosporine A, an inhibitor of mitochondria permeability transition pore only partly inhibited caspase3 activity and reduced DNA damage. Interestingly, treatment of Z-IETD-FMK, an inhibitor of caspase8 could completely reverse DNA fragmentation, but can't completely inhibit caspase3 activity. It was concluded that there were caspase3 dependent and independent cellular apoptosis pathways in N2a cells suffering ischemia-reperfusion insults. Mitochondria dysfunction may early trigger apoptosis and amplify apoptosis signal.
Animals ; Apoptosis ; physiology ; Caspase 3 ; Caspases ; biosynthesis ; Cytochromes c ; biosynthesis ; Mice ; Mitochondria ; physiology ; Neuroblastoma ; pathology ; Neurons ; pathology ; Reperfusion Injury ; metabolism ; pathology ; Tumor Cells, Cultured

OBJECTIVETo study the effect and mechanism of berbamine on the apoptosis of multidrug resistant leukemia K562/Adr cells and in reversing the drug resistance.

METHODSIC50 value of K562/Adr cell was determined with MTT method, cell apoptosis rate was analyzed by flow cytometry with Annexin V FITC-PI assay, with the peak and cell cycle detected by PI staining. At the same time, flow cytometry was also used in determining Caspase-3, P-GP protein expression and drug accumulating capacity in cells, and RT-PCR method was used to analyze the gene expression of mdr-1.

RESULTSBerbamine could inhibit human leukemia K562/Adr cell growth in dose-dependent manner, it could also induce cell apoptosis, increase the protein expression of Caspase-3 and the drug excretion capacity of cells, reduce the mRNA and protein expression levels of mdr-1 gene.

CONCLUSIONBerbamine could activate Caspase-3 to induce human leukemia K562/Adr cell apoptosis, and by reducing mdr-1 gene expression to reverse its multidrug resistance.

ATP-Binding Cassette, Sub-Family B, Member 1 ; biosynthesis ; genetics ; Alkaloids ; pharmacology ; Antineoplastic Agents, Phytogenic ; pharmacology ; Apoptosis ; drug effects ; Benzylisoquinolines ; pharmacology ; Caspase 3 ; Caspases ; biosynthesis ; genetics ; Drug Resistance, Neoplasm ; drug effects ; genetics ; Humans ; K562 Cells ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVETo study the expression of hypoxia-inducible factor (HIF)-1alpha and related genes in abdominal aorta aneurysm (AAA) and explore the underlying pathogenesis.

METHODSTwenty-two AAA specimens were collected and 5 normal abdominal aorta tissue were used as control. Northern blot, western blot and immunohistochemistry were used to evaluated the expression of HIF-1alpha mRNA and protein product. Western blot and immunohistochemistry method were also used to determine the expression of vascular endothelial growth factor (VEGF) and caspase-3. Microvessel density (MVD) was studied by immunohistochemistry stain of CD34.

RESULTSThe expression of HIF-1alpha mRNA and protein product were significantly higher in AAA than that in normal abdominal aorta (P < 0.01). The expression of VEGF and caspase-3 were also higher in AAA and both had a significantly positive relationship with HIF-1alpha expression (P < 0.01). Most of the positive cells located in VSMC and adventitia of AAA. The MVD counts were higher in AAA.

CONCLUSIONHIF-1alpha may have an important role the development of AAA, which maybe obtained by regulating the expression of VEGF or caspase-3.

Aged ; Aortic Aneurysm, Abdominal ; etiology ; genetics ; metabolism ; Caspase 3 ; Caspases ; biosynthesis ; Female ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit ; Immunohistochemistry ; Male ; Middle Aged ; Neovascularization, Pathologic ; pathology ; RNA, Messenger ; genetics ; Transcription Factors ; biosynthesis ; genetics ; Vascular Endothelial Growth Factor A ; biosynthesis

OBJECTIVETo explore the mechanism of injurious effect of succinic acid on human fibroblast and it's role in bacteroides fragilis infection.

METHODSIn vitro cultured human fibroblasts were challenged by succinic acid in concentrations of 5, 10, 20 and 30 mmol/L (pH5.5), respectively. The cellular activity, apoptosis rate, the collagen synthesis in the supernatant of the cell culture, and the activity of caspase-3 were determined 24 hours after challenge. Isotonic saline challenged fibroblast were employed as control and the changes in the indices before and after succinic acid challenge were observed.

RESULTSAlong with the increase in the concentration of succinic acid, the fibroblast proliferation rate was decreased and so was the collagen synthesis. But the apoptosis rate and caspase-3 activity were increased. The activity of caspase-3 was markedly higher than that in normal control when the succinic acid concentration was 10-30 mmol/L. The cellular activity and collagen synthesis were significantly lower and the apoptosis rate was obviously higher than those in control group when the succinic acid concentration was 20 or 30 mmol/L (P < 0.05).

CONCLUSIONThe proliferation and collagen synthesis in fibroblast culture could be significantly inhibited and the cellular apoptosis could be promoted by succinic acid. The process of wound healing of the wounds infected by bacteroides fragilis would be delayed due to the production of succinic acid by the bacteria.

Apoptosis ; drug effects ; Caspase 3 ; Caspases ; metabolism ; Cells, Cultured ; Collagen ; biosynthesis ; Dose-Response Relationship, Drug ; Fibroblasts ; drug effects ; metabolism ; physiology ; Humans ; Succinic Acid ; pharmacology

To explore the new gene therapeutic method for pancreatic carcinoma, the recombinant Caspases-3 gene (r-Caspases-3) was constructed by molecular biologic method. The eukaryotic expression plasmid pcDNA 3.1 (+)/r-Caspase-3 was constructed by rearrangement of the large subunit and small subunit of caspases-3, and then it was transfected into pancreatic carcinoma cells strain (PC-II). After being transfected, the expression of r-Caspase-3 mRNA in pancreatic carcinoma cells was detected by RT-PCR and its apoptotic activity was detected by FCM. The sequencing of the recombinant molecules (r-Caspases-3) confirmed that its small subunit preceded its large subunit. After the pancreatic carcinoma cells were transfected with the pcDNA3.1(+)/r-Caspases-3 by liposomes, an 894 bp strap was observed by means of RT-PCR. No strap was found in control groups. A transparent hypodiploid karyotype peak was revealed by FCM. The above data indicate that the gene of r-Caspase-3 has been constructed successfully, r-Caspase-3 has apoptotic activity and can be used as target gene in gene therapy for pancreatic carcinoma.
Apoptosis ; Caspase 3 ; Caspases ; biosynthesis ; genetics ; physiology ; Cloning, Molecular ; Genetic Therapy ; Humans ; Pancreatic Neoplasms ; pathology ; therapy ; Plasmids ; genetics ; Transfection ; Tumor Cells, Cultured

OBJECTIVE: To investigate the expression of caspase-3 during skin wound healing and explore the applicability of caspase-3 to determination of wound age. METHODS: The expression of caspase-3 were performed on 33 mice skin incised wounds at different posttraumatic intervals and 3 mice as control by immunohistochemical technique. RESULTS: Expression of caspase-3 was detectable in polymorphonuclear cells (PMNs) in the wound specimens aged 6 h. In the wound specimens aged from 12 h to 24 h after injury, caspase-3 was identified in a large number of infiltrating PMNs and part of mononuclear cells (MNCs). Afterwards, the MNCs and fibroblastic cells (FBCs) accounted in the most part of the caspase-3 positive cells. Morphometrically, the ratio of the number of the caspase-3 stained PMNs, MNCs and FBCs to total number of the cells in the wounds was evaluated and calculated. The ratio of the caspase-3 positive cells was low in the wound specimens aged from 0 h to 3 h (4.53 +/- 6.53)%, and maximized in the wound specimens aged 3 day (62.66 +/- 4.84)%. Thereafter, the ratio decreased and minimized in the specimens aged 14 day (21.56 +/- 4.54)%. CONCLUSION The results suggest that caspase-3 may play an important role in inducing neutrophil, macrophage and fibroblast apoptosis during skin wound healing. Furthermore, caspase-3 may be used as a marker for the wound age determination.
Animals ; Apoptosis ; Caspase 3 ; Caspases/biosynthesis* ; Female ; Immunohistochemistry ; Male ; Mice ; Skin/metabolism* ; Staining and Labeling ; Time Factors ; Wound Healing ; Wounds and Injuries/physiopathology*

OBJECTIVE: To investigate the expression of caspase-3 in different posttraumatic intervals and severity of brain injury. METHODS: The study examined brain tissue samples of slight (n = 24), severe (n = 24) brain injury and control (n = 6) of rat, using immunohistochemical staining, western-blot and RT-PCR method. RESULTS: Up-regulating of caspase-3 expression was found in tissue from traumatic brain injury compared with controls in early 1 hour after TBI, and lasted for 14 days. The gray degree and threshold area of caspase-3 positive cells is different in different severity of brain injury. CONCLUSION The increasing of caspase-3 expression indicates that TBI exists. The gray degree, threshold area of caspase-3 positive cells and the cleavage degree of pro-caspase-3 have association with the severity of brain injury.
Animals ; Apoptosis ; Blotting, Western ; Brain Injuries/pathology* ; Caspase 3 ; Caspases/genetics* ; Immunohistochemistry ; RNA, Messenger/biosynthesis* ; Rats ; Reverse Transcriptase Polymerase Chain Reaction ; Time Factors

The pathogenesis of chronic cyclosporine A (CsA) nephrotoxicity has not been elucidated, but apoptosis is thought to play an important role in CsA induced tubular atrophy. Recently Fas-Fas ligand system mediated apoptosis has been frequently reported in many epithelial cells as well as in T lymphocytes. We investigated the ability of CsA to induce apoptosis in cultured human proximal tubular epithelial cells and also the effect of -MSH on them. Fas, Fas ligand, and an intracellular adaptor protein, Fas-associating protein with death domain (FADD) expression, and poly-ADP ribose polymerase (PARP) cleavage were also studied. CsA induced apoptosis in cultured tubular epithelial cells demonstrated by increased number of TUNEL positive cells and it was accompanied by a significant increase in Fas mRNA and Fas ligand protein expressions. FADD and the cleavage product of PARP also increased, indicating the activation of caspase. In -MSH co-treated cells, apoptosis markedly decreased with downregulation of Fas, Fas ligand and FADD expressions and also the cleavage product of PARP. In conclusion, these data suggest that tubular cell apoptosis mediated by Fas system may play a role in tubular atrophy in chronic CsA nephrotoxicity and pretreatment of -MSH may have a some inhibitory effect on CsA induced tubular cell apoptosis.
Antigens, CD95/genetics ; Apoptosis/*drug effects ; Carrier Proteins/biosynthesis ; Caspases/physiology ; Cells, Cultured ; Cyclosporine/*toxicity ; Human ; Immunosuppressive Agents/*toxicity ; Kidney Tubules, Proximal/cytology/*drug effects/metabolism ; Membrane Glycoproteins/biosynthesis ; NAD+ ADP-Ribosyltransferase/metabolism ; RNA, Messenger/analysis ; alpha-MSH/*pharmacology

OBJECTIVETo investigate the effect of Astragalus injection (AI) on left ventricular remodeling and the expression of apoptotic gene caspase-3 in rats after myocardial infarction (MI).

METHODSThe MI model was established. The experimental animals were divided into 4 groups, Group I (the sham-operated group), Group II (the sham-operated plus AI group), Group III (the model group), Group IV (the model plus AI group). Animals in the II and IV group were intraperitoneally injected with 2 ml AI once a day after operation, while animals in the I and III group were treated with normal saline of equal volume. After treated for 4 weeks successively, the structural change of left ventricle and the level of oxyproline in myocardium were observed, and expression of caspase-3 was determined by immunohistochemical method.

RESULTSAs compared with Group Ill, the ultrastructure of myocardium and indexes of left ventricular remodeling were improved, the myocardial content of oxyproline decreased (P < 0.05), the caspase-3 positive cells reduced and caspase-3 mRNA expression significantly down-regulated (P < 0.05).

CONCLUSIONAI can improve left ventricular remodeling, inhibit apoptosis by down-regulate the expression of apoptotic gene caspase-3 in rats after MI.

Animals ; Astragalus membranaceus ; Caspase 3 ; Caspases ; biosynthesis ; genetics ; Down-Regulation ; Drugs, Chinese Herbal ; pharmacology ; Injections ; Male ; Myocardial Infarction ; metabolism ; pathology ; physiopathology ; Myocardium ; ultrastructure ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Wistar ; Ventricular Remodeling ; drug effects