酶的自杀底物及其应用

酶的自杀底物是治疗酶学上一类斩新的课题,对于医疗实践有着重要的意义,然而,酶的自杀底物究竟是怎么回事,它的作用机理如何等问题,现已逐渐为广大生物化学工作者和临床医务人员所关注,本文想就此作一些介绍。 我们从“生物化学”中可以知道,酶在进行酶促反应时,必须先形成酶——底物复合体,然后由复合体分解成最终产物。

甲状腺机能减退症患者激素替代治疗对血脂及心肌酶学的影响

目的:观察原发性甲状腺机能减退症(甲减)患者激素替代治疗前后血脂及心肌酶学的变化。方法:76例甲减患者治疗前和治疗12周后分别测定空腹血清游离三碘甲状腺原氨酸(FT3)、游离甲状腺素(FT4)、促甲状腺激素(sTSH)、总胆固醇(TC)、甘油三酯(TG)、肌酸磷酸激酶(CK)及同工酶(CKMB)。结果:治疗前TC、TG、CK、CKMB较对照组升高(P<0.01或P<0.05);治疗后上述指标均恢复至正常。结论:甲减与脂代谢、心肌酶学异常关系密切。

Effective siRNA targets screening for human telomerase reverse transcriptase

AIM: To study the inhibitory effects of siRNAs targeting different hTERT sequences and to screen the effective siRNA sequence.METHODS: Five double-stranded siRNAs targeting coding and non-coding regions of hTERT gene were designed and synthesized by T7 transcription system in vitro. siRNA4sequence was screened by full length gene targeting technique and the rest of the siRNA sequences were selected randomly. After being purified by ethanol precipitation, the siRNAs were transfected to the human hepatocellular carcinoma cell (HepG2) by Lipofectamine 2000TM. At 48-72 h after siRNAs transfection, MTT assay,RT-PCR and Western-blot were applied to evaluate the effects of siRNAs on cell growth, mRNA and protein expression level of hTERT gene, respectively.RESULTS: Compared to the control cells, the cells treated with the five double-stranded siRNAs exhibited different degrees of inhibition of cell proliferation in a dose-dependent manner. siRNA2 and siRNA4, exhibited obvious effects of inhibiting hTERT mRNA and protein expression in HepG2cells.CONCLUSION: siRNAs targeting different hTERT sequences have significantly various inhibitory effects on hTERT gene expression. The siRNA sequence screened by full length gene targeting technique has comparable inhibitory effect with the rest siRNA sequences screened by random selection, suggesting that siRNAs and antisense oligonucleic acids may have the same effective target sites. Compared with chemical synthesis method,synthesizing double-stranded siRNA by T7 transcription system in vitro is a rapid, simple, and inexpensive method suitable for screening high-effect siRNA targeting site for specific gene.

Inhibition of histone deacetylase for the treatment of biliary tract cancer:A new effective pharmacological approach

AIM： To investigate in vitro and in vivo therapeutic effects of histone deacetylase inhibitors NVP-LAQ824 and NVP-LBH589 on biliary tract cancer. METHODS： Cell growth inhibition by NVP-LAQ824 and NVP-LBH589 was studied in vitro in 7 human biliary tract cancer cell lines by MTT assay. In addition, the antitumoral effect of NVP-LBH589 was studied in a chimeric mouse model. Anti-tumoral drug mechanism was assessed by immunoblotting for acH4 and p21^WAFl/CIP-1, PARP assay, cell cycle analysis, TUNEL assay, and immunhistochemistry for MIB-1. RESULTS： In vitro treatment with both compounds significantly suppressed the growth of all cancer cell lines [mean IC50 （3 d） 0.11 and 0.05 μmol/L, respectively], and was associated with hyperacetylation of nucleosomal histone H4, increased expression of p21^WAF-1/CIP-1, induction of apoptosis （PARP cleavage）, and cell cycle arrest at G2/M checkpoint. After 28 d, NVP- LBH589 significantly reduced tumor mass by 66% （bile duct cancer） and 87% （gallbladder cancer） in vivo in comparison to placebo, and potentiated the efficacy of gemcitabine. Further analysis of the tumor specimens revealed increased apoptosis by TUNEL assay and reduced cell proliferation （MIB-1）. CONCLUSION： Our findings suggest that NVP-LBH589 and NVP-LAQ824 are active against human biliary tract cancer in vitro. In addition, NVP-LBH589 demonstrated significant in vivo activity and potentiated the efficacy of gemcitabine. Therefore, further clinical evaluation of this new drug for the treatment of biliary tract cancer is recommended.

Hepatic preconditioning of doxorubicin in stop-flow chemotherapy:NF-κB/IκB-α pathway and expression of HSP72

AIM:To provide hepatic protection through administration of doxorubicin before stop-flow chemotherapy (SFC) and to investigate the expression of heat shock protein 72 (HSP72) and role of nuclear factor kappa B (NF-κB) in this effect. METHODS: The hepatic preconditioning of doxorubicin was established in a porcine model by injection of doxorubicin (1 mg/kg) before SFC. The experimental animals were randomized into two groups: groups receiving doxorubicin (DOX) and normal saline (NS). Serial serum and tissue samples were taken from both groups to evaluate the protection of doxorubicin. Western blot and immunoprecipitation were applied to detect the expression of HSP72, NF-κB p65 protein, inhibitor κB-α (IκB-α) and phosphorylated IκB-α as well. The expression of tumor necrosis factor α (TNF-α) was estimated by semiquantitative RT-PCR. And the extent of the hepatic injury was estimated with the level of serum aminotransferases. RESULTS: An abundance production of HSP72 in porcine liver was observed after 24 h of intravenous administration of doxorubicin, but without any change in the expression of NF-κB p65 subunit in cytoplasm. NF-κB p65 subunit accumulated in nuclei at the end of SFC and reached its highest level at 30 min after the restoration of the abdominal circulation and decreased gradually during the 6 h after SFC in NS group, while there was little change in DOX group. There was also a slight decrease of IκB-α at 30 min after the restoration of the abdominal circulation in NS group accompanying with the appearance of phosphorylated IκB-α. The expression of TNF-α was significantly higher in NS group than that in DOX group (average 1.40±0.17 vs 0.62±0.22, P<0.01) at serial time points after SFC. Serum ALT and AST levels of NS group were higher after 24 h than those of DOX group (93.2±7.8 IU/L vs 53.3±13.9 IU/L, 217.0±29.4 IU/L vs 155.0±15.6 IU/L for ALT and AST respectively, P<0.05) and after 48 h than those of DOX group (66.6±18.1 IU/L vs 43.3±16.7 IU/L, 174.4±21.3 IU/L vs 125.7±10.5 IU/L for ALT and AST respectively, P<0.05). CONCLUSION: Doxorubicin renders the liver to be tolerant to the hepatic influence in SFC in a porcine model through the NF-κB/IκB-αpathway with the expression of HSP72.

Expression and significance of microRNA-32 in multiple myeloma

Objective: This study was aimed to explore the expression of microRNA-32(miR-32) in multiple myeloma(MM), and study its association with β2-microglobulin and staging of MM by Durie-Salmon classification. Methods: The expression level of miR-32 in bone marrow mono-nuclear cells of MM were examined by real-time polymerase chain reaction(real-time PCR), and the correlations between the expression level of miR-32 and related clinic pathologic features β2-microglobulin, staging of MM by Durie-Salmon classification were further analyzed. Results: The expression of miR-32 in MM patients was obviously higher than that in normal control(P < 0.05). The expression of miR-32 in relapsed/ refractory MM patients was obviously higher than that in newly diagnosed MM patients. The expression of miR-32 in MM patients decreased after chemical therapy than that before treatment, especially in effective group(P < 0.05). There was no statistically significant change in ineffective/progress group after chemical therapy(P > 0.05).The expression of miR-32 was associated to staging of MM and β2-microglobulin level. Conclusion: Expression level of miR-32 in MM patients is significantly higher than that in normal bone marrow, these data indicated that miR-32 may play an important role in the development of MM. High-regulated expression of miR-32 was associated with β2-microglobulin and staging of MM by Durie-Salmon classification.

Effects of combined drug therapy on HIV-1 infection dynamics

Infection of human immunodeficiency virus （HIV） is determined through the decay of healthy CD44- T-cells in a well-mixed compartment, such as a bloodstream. A mathe- matical model is considered to illustrate the effects of combined drug therapy, i.e. reverse transcription plus protease inhibitor, on viral growth and T-cell population dynamics. This model is used to explain the existence and stability of infected and uninfected steady states in HIV growth. An analytical technique, called variational iteration method （VIM）, is used to solve the mathematical model. This method is modified to obtain the rapidly convergent successive approximations of the exact solution. These approximations are obtained without any restrictions or the transformations that may change the physical behavior of the problem. Numerical simulations are computed and exhibited to illustrate the effects of proposed drug therapy on the growth or decay of infection.