Inhibition of HSP70 Gene Expression by Modified Antisense and Its Effects on Embryonic Sensitivity to Heat Shock

Experiments were performed to evaluate the efficiency of inhibition of HSP70 gene expressionby antisense oligonucleotides complementary to the mRNA of HSP70 and to test the effects ofinhibition of HSP70 gene expression on subsequent embryonic sensitivity to heat shock. Theresults showed that transfection of pre-implantation embryos at 4-cell stage with 5 Mantisense oligo had no effect on in vitro blastocyst development. However, transfection with10 to 40 M antisense oligo had reduced in vitro blastocyst development to 15, 10% and 0; Forthe embryos which exposed to 40 M As arrested at the 16-cell stage, there was no blastocystformation within the heat shock groups. In contrast, transfection had no effect on embryonicsensitivity to heat shock, above 25% of embryos developed to blastocyst stage in controlgroups.

INDUCTION OF C-MYC GENE AMPLIFICATION BY HYDROXYUREA AND ITS INHIBITION BY HOMOHARRINGTONINE

Induction of c-myc gene amplification in L1210 cells by hydroxyurea and its inhibition by homohar-ringtonine were investigated using the DNA-DNA molecular hybridization technique. When the cells were treated with hydroxyurea 1.0 mM for 16 hours, and incubated a further 16 hours in a drug-free medium, the c-myc gene amplified 23.5-fold. If homohar-ringtonine 50 μM was used at the same time as hydroxyurea, gene amplification did not occur. Cycloheximide, an inhibitor of protein biosynthesis, produced a similar effect. Our results indicated that a (or some) protein factor(s) might be involved in gene amplification. Detailed analysis showed that the synthesis of this protein factor(s) started 4 hours before the initiation of the S phase but did not continue in the S phase. It was also found that this protein factor(s) was very labile and began to degrade 2 hours after its appearance.

Down-regulation of Notch-1 gene expression inhibits growth of TJ-905 glioblastoma

Objective To study the inhibitory effect of siRNA on glioblastoma (GBM) Notch-1 gene expression in addition to the growth of TJ-905 glioblastoma. Methods Three small interference RNAs (siRNAs) targeting Notch1 gene,named siRNA1,siRNA2,siRNA3,synthesized chemically in vitro with gene bank BLAST. TJ-905 cells were transfected twice with the siRNA by using Oligofectamine

Gene transfer of huCTLA4-Ig to inhibit the acute rejection of liver allograft in rats

To observe the effect of gene transfer of huCTLA4-Ig to inhibit the acute rejection of liver allograft in rats.Methods With AdEasy vector system,the recombinant adenovirus containing huCTLA4-Ig gene was constructed.Using ex vivo gene transfer technique,exogenous gene was introduced to the liver graft during cold preservation and expressed locally in the graft.The effect of inhibition of acute rejection and inducing liver graft tolerance was observed.Results No recipients in group A (without any treatment,n=5) or group B (treated with Ad-GFP,n=4) died within 3 weeks after transplantation and severe acute rejection (massive periportal infiltration,endothelilitis,damage to biliary epithelium and severe tissue destruction) was confirmed pathologically in the graft.In contrast,all recipients in group C (treated with Ad-huCTLA4-Ig,n=5) achieved long-term liver allograft survival (>150 days).Histological examination of Ad-huCTLA4-Ig transduced allografts demonstrated a mild to moderate periportal inflammation and mild injury to liver graft on day 8 posttransplant.A mild mononuclear infiltration was observed;however,there was complete preservation of the bild ducts and no evidence of vascular injury on day 150 posttransplant.The mean IL-2 concentration in serum was (362.09±45.84) ng/L at day 1 pretransplant.In control animals (groups A and B),serum IL-2 concentration was elevated to a high level within 7 days posttransplant,which was about 1.5 to 2.5 times as much as that before transplant.In contrast,in huCTLA4-Ig-treated animals (groups C),IL-2 concentration in serum was maintained at a relative low level,which was near or less than that before transplant (P<0.01).Conclusion Using ex vivo gene transfer technique,huCTLA4-Ig gene can be introduced to the liver graft during cold preservation.The modified graft can express and excrete immunoregulatory protein locally,which can suppress acute alloimmune response and is responsible for prolongation of graft survival without using routine immunosuppressive drugs.These findings provide some experimental evidence that gene delivery of sequences encoding immunoregulatory proteins can be applied to clinical liver transplantation for inhibiting the acute alloimmune response and achieving graft tolerance.7 refs,2 tabs.

Growth-inhibitory effects of MOB2 on human hepatic carcinoma cell line SMMC-7721

AIM:To investigate the growth-inhibiting and apoptosis-inducing effects of the gene MOB2 on human hepatic carcinoma cell line SMMC-7721.METHODS:The full-length cDNA of the MOB2 gene was amplified from human umbilical vein endothelial cells.The correct full-length MOB2 cDNA was subcloned into the eukaryotic expression vector pEGFP-C1.After lipofection of the MOB2 gene into cancer cells,the levels of MOB2 protein in the cancer cells were detected by immunoblotting.To transfect the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells,the cells were cultured in Dulbecco's Modified Eagle'sMedium with 10% fetal calf serum and glutamine,and then mixed with liposomes,Lipofectamine 2000 and the plasmid vector pEGFP-CI-MOB2.RESULTS:We observed the growth and proliferation of SMMC-7721 cells containing pEGFP-CI-MOB2 and analyzed their apoptosis and growth cycle phases by flow cytometry.We successfully transfected the recombined plasmid vector pEGFP-CI-MOB2 into SMMC-7721 cells and screened for a single clone cell containing MOB2.After transfection,MOB2 enhanced growth suppression,induced apoptosis,increased the ratio of G0/G1,significantly inhibited the advance of cell cycle phase,and arrested cells in G0/G1 phase.CONCLUSION:MOB2 overexpression induces apoptosis and inhibits the growth of human hepatic cancer cells,which may be useful in gene therapy for hepatic carcinoma.

Invertebrate Iridovirus Modulation of Apoptosis

Programmed cell death （apoptosis） is a key host response to virus infection. Viruses that can modulate host apoptotic responses are likely to gain important opportunities for transmission. Here we review recent studies that demonstrate that particles of Invertebrate iridescent virus 6 （IIV-6） （Iridoviridae, genus Iridovirus）, or an IIV-6 virion protein extract, are capable of inducing apoptosis in lepidopteran and coleopteran cells, at concentrations 1000-fold lower than that required to shut-off host macromolecular synthesis. Induction of apoptosis depends on endocytosis of one or more heat-sensitive virion component（s）. Studies with a JNK inhibitor （SP600125） indicated that the JNK signaling pathway is significantly involved in apoptosis in IIV-6 infections of Choristoneurafumiferana cells. The genome of IIV-6 codes for an inhibitor of apoptosis iap gene （193R） that encodes a protein of 208 aa with 15% identity and 28% similarity in its amino acid sequence to IAP-3 from Cydia pomonella ganulovirus （CpGV）. Transcription of IIV-6 iap did not require prior DNA or protein synthesis, indicating that it is an immediate-early class gene. Transient expression and gene knockdown studies have confirmed the functional nature of the IIV-6 lap gene. We present a tentative model for IIV-6 induction and inhibition of apoptosis in insect cells and discuss the potential applications of these findings in insect pest control.