Caspases ; genetics ; Genetic Predisposition to Disease ; Humans ; Neoplasms ; genetics ; Polymorphism, Genetic

We postulated that apolysis was processed in accordance with apoptotic changes occurring in a cestode, Spirometra erinacei (Pseudophyllidea). We cloned the novel putative apoptosis-associated gene from S. erinacei via screening of a S. erinacei cDNA library with a ced-3 gene (activator of apoptosis) probe from Caenorhabditis elegans. We identified a 261-bp cDNA sequence, which encodes for an 86-amino acid protein. The cloned gene expression was observed in the neck and gravid proglottids via Northern blotting, using cloned cDNA inserts as probes, but the clone was not expressed in any of other tissues. We suggest that this gene may be involved in the apolysis of S. erinacei during normal tissue development and differentiation in cestode parasites.
Spirometra/*genetics/growth & development/physiology ; Molecular Sequence Data ; Gene Library ; Cloning, Molecular ; Caspases/genetics ; Caenorhabditis elegans Proteins/genetics ; Base Sequence ; Apoptosis/*genetics ; Animals ; Amino Acid Sequence

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

Primary neuronal culture is a powerful tool to study neuronal development, aging, and degeneration. However, cultured neurons show signs of cell death after 2 or 3 weeks. Although the mechanism underlying this phenomenon has not been elucidated, several preventive methods have been identified. Here we show that the neuronal loss in primary cortical culture involves calpain activation and subsequent neuronal cell death. Neuronal loss during cultivation showed destruction of neurites and synapses, and a decrease in neuron numbers. micro-Calpain and micro-calpain were initially activated and accumulated by increased RNA expression. This neuronal death exhibited neurodegenerative features, such as conversion of p35 to p25, which is important in the developmental process and in the pathogenesis of Alzheimer's disease. But, postnatal and aged rat cortex did not show calpain activation and prolonged processing of p35 to p25, in contrast to the long-term culture of cortical neurons. In addition, the inhibition of calpains by ALLM or ALLN blocked the conversion of p35 to p25, indicating that the calpain activity is essential for the neurodegenerative features of cell death. Taken together, this study shows that the neuronal loss in primary cortical cultures involves neurodegeneration-like cell death through the activation of calpains and the subsequent processing of p35 to p25, but not developmental apoptosis or aging. Our results suggest that the long term primary culture of cortical neurons represent a valuable model of neurodegeneration, such as Alzheimer's disease.
Transcription, Genetic/genetics ; Time Factors ; Rats ; Phosphotransferases/*metabolism ; Neurons/*cytology/*metabolism ; Cells, Cultured ; Cell Shape ; Caspases/antagonists & inhibitors/metabolism ; Calpain/antagonists & inhibitors/genetics/*metabolism ; *Apoptosis ; Animals

OBJECTIVETo evaluate antisense technology for human telomerase inhibition in the treatment of endometrial cancer.

METHODSAn antisense oligodeoxynucleotides (AODN) directed against the human telomerase transcriptase (hTERT), designed and synthesized to serve as a telomerase inhibitor, was transfected into endometrial carcinoma cell line HEC-1A by lipofectin. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot were used to test the expression of hTERT mRNA and hTERT protein before and after transfection. Telomerase activity was tested by telomeric repeat amplification protocol. The proliferation and growth of HEC-1A were also studied by methyl thiazolyl tetrazolium and cell growth curve before and after transfection.

RESULTSAODN could down-regulate the expression of hTERT mRNA and protein, inhibiting telomerase activity and proliferation of endometrial cancer cell line in a dose- and period-dependent manner.

CONCLUSIONAntisense oligodeoxynucleotides of human telomerase transcriptase definitely inhibits the proliferation of endometrial cancer cell line. Telomerase inhibitor may thus become a new gene therapeutic agent for endometrial carcinoma.

Caspases ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Endometrial Neoplasms ; pathology ; therapy ; Female ; Genetic Therapy ; Humans ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; analysis ; Telomerase ; antagonists & inhibitors ; genetics

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

OBJECTIVETo investigate the single nucleotide polymorphisms (SNPs) and the distribution of their haplotypes in caspase-3 gene in Zhejiang Han nationality in China.

METHODSDenaturing high-performance liquid chromatography (DHPLC) and DNA sequencing were used to detect the SNPs in the regulatory region and the exons 2-7 and their flanking sequences in caspase-3 gene. Expectation maximization (EM) algorithm was used for haplotype frequencies analysis and pairwise linkage disequilibrium test.

RESULTS(1) Three SNPs were identified in caspase-3 gene; the three sites C829A, A17532C and C20541T were located in 5' regulatory region, intron 4 and 3' regulatory region, respectively. (2) Strong linkage disequilibrium was found among these SNPs; site A17532C and C20541T were in complete linkage disequilibrium. (3) C-829/A-17532/C-20541 (54.3%) was the main haplotype of Zhejiang Han nationality.

CONCLUSIONThe above findings indicated there is strong linkage disequilibrium among the three SNPs in caspase-3 gene in Han nationality of Zhejiang province and the main haplotype of Han nationality is obviously different from that of North American.

Asian Continental Ancestry Group ; genetics ; Caspase 3 ; Caspases ; genetics ; China ; ethnology ; DNA Mutational Analysis ; Gene Frequency ; Genotype ; Haplotypes ; Humans ; Introns ; Linkage Disequilibrium ; Polymorphism, Single Nucleotide

OBJECTIVETo search for the susceptibility variant (s) of type 2 diabetes in the susceptible regions on chr.1 (1p36.23-36.33, 1q24.3-25.1, and 1q42.12-42.13) by genotyping SNP markers in case-control DNA samples and identifying the haplotype associated with type 2 diabetes.

METHODSTotally 124 SNPs in 33 candidate genes in the mapped regions were chosen from public SNP data or identified by sequencing the samples that were used to search for SNP locus. Sequencing method was used to genotype the loci for 236 sporadic type 2 diabetes patients and 152 normal subjects in Northern Han Chinese population. The haplotypes with significant difference were further analyzed.

RESULTSOf 124 SNPs successfully typed, 4 SNPs that showed association with diabetes status were found: rs203849 (P=0.005, OR=1.60) and rs203826 (P=0.016, OR=1.60) located in sAC gene, rs7535528 (P=0.028, OR=1.45) located in PANK4, rs884363 (P=0.043, OR=1.37) located in CASP9 gene. In addition, the frequencies of two combination types from these 4 SNP genotypes were significantly different between case and control groups (P < 0.001). Furthermore, four haplotypes associated with diabetes were found in haplotype analysis of sAC gene.

CONCLUSIONsAC, PANK4, and CA SP9 may be associated with type 2 diabetes in Han population in north China, and it seems that the synergetic effect of these genes is responsible for the development of type 2 diabetes.

Adult ; Apoptosis ; genetics ; Caspase 9 ; Caspases ; genetics ; China ; Chromosomes, Human, Pair 1 ; genetics ; Diabetes Mellitus, Type 2 ; genetics ; Female ; Genetic Predisposition to Disease ; Genetic Testing ; Genetic Variation ; Genotype ; Haplotypes ; genetics ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide

OBJECTIVETo investigate polymorphisms in the coding exons and their splicing areas of caspase10 gene (CASP10) in Chinese of Han nationality.

METHODSPolymerase chain reaction (PCR), denaturing high-performance liquid chromatography (DHPLC), direct sequencing and clonal sequencing were used to analyze the exon 9 and its flanking sequences.

RESULTSIn 70 blood samples of Chinese subjects researched the known single nucleotide polymorphisms (SNPs) in the exon 9 of CASP10 gene published in dbSNP of NCBI were not identified. We found a short sequence with more than ten continuous and repeated T nucleotides within the intron 8 near the exon 9 and the length of repeated sequence nucleotides T was different in samples. Clonal sequencing analysis indicated that it was a microsatellite of single nucleotide-repeated sequence. The recurrence and specificity of same result was further confirmed by PCR with high fidelity polymerase and sequencing. Furthermore all of 70 blood samples detected by DHPLC showed heterozygous profiles. We named it as IVS8-13(T)n temperately.

CONCLUSIONOur research suggested that the site be a microsatellite of single nucleotide-repeated sequence. Meanwhile, our data further showed that it was quite different from that by querying SNP database of non-Chinese population in NCBI, in the same region a SNP of type of insertion deletion existed. It could be inferred that the difference between the populations might be associated with the genetic characteristics of ethnics. The significance of the microsatellite in CASP10 gene in Chinese of Han nationality remains to be elucidated.

Asian Continental Ancestry Group ; genetics ; Base Sequence ; Caspase 10 ; Caspases ; genetics ; China ; ethnology ; Ethnic Groups ; Genetics, Population ; Humans ; Introns ; genetics ; Microsatellite Repeats ; genetics ; Molecular Sequence Data ; Polymorphism, Single Nucleotide ; Sequence Analysis, DNA

OBJECTIVETo investigate the mechanism of U87 cell apoptosis induced by inhibiting miR-21 expression.

METHODSAntisense oligonucleotides of miR-21 were chemically synthesized and transfected into U87 cells. The apoptosis, proliferation, and invasion of the cells were evaluated. The relationship between miR-21 and PTEN or caspase was identified by bioinformatics and Western blot.

RESULTSInhibiting miR-21 expression led to U87 cell growth suppression, apoptosis induction, invasion reduction, caspase-3 activity elevation and caspase-9 activation, but did not affect PTEN and caspase-8 expression.

CONCLUSIONmiR-21 may function as an antiapoptotic miRNA in U87 cells. Inhibiting miR-21 expression could induce U87 cell apoptosis via caspase-9 and 3 activation, but not PTEN activation.

Animals ; Apoptosis ; drug effects ; genetics ; Caspases ; metabolism ; Cell Line, Tumor ; Cell Proliferation ; Enzyme Activation ; genetics ; Gene Expression Regulation, Neoplastic ; Glioma ; genetics ; pathology ; Humans ; MicroRNAs ; antagonists & inhibitors ; genetics ; Oligoribonucleotides, Antisense ; genetics ; pharmacology ; PTEN Phosphohydrolase ; metabolism ; Transfection