Aminoglycoside uptake increased by tet gene expression

The expression of extrachromosomal tet genes not only confers tetracycline resistance but also increases the susceptibilities of gram-negative bacteria to commonly used aminoglycoside antibiotics. We investigated the possibility that tet expression increases aminoglycoside susceptibility by increasing bacterial uptake of aminoglycoside. Studies of [3H]gentamicin uptake in paired sets of Escherichia coli HB101 and Salmonella typhimurium LT2 expressing and not expressing tet showed that tet expression accelerates energy-dependent [3H]gentamicin uptake. Increased [3H]gentamicin uptake was accompanied by decreased bacterial protein synthesis and bacterial growth. Increased aminoglycoside uptake occurred whether tet expression was constitutive or induced, whether the tet gene was class B or C, and whether the tet gene was plasmid borne or integrated into the bacterial chromosome. tet expression produced no measurable change in membrane potential, suggesting that tet expression increases aminoglycoside uptake either by increasing the availability of specific carriers or by lowering the minimum membrane potential that is necessary for uptake.     

Muscle gene electrotransfer is increased by the antioxidant tempol in mice

Electropermeabilization (EP) is an effective method of gene transfer into different tissues. During EP, reactive oxygen species (ROS) are formed, which could affect transfection efficiency. The role of generated ROS and the role of antioxidants in electrotransfer in myoblasts in vitro and in Musculus tibialis cranialis in mice were, therefore, investigated. We demonstrate in the study that during EP of C2C12 myoblasts, ROS are generated on the surface of the cells, which do not induce long-term genomic DNA damage. Plasmid DNA for transfection (pEGFP-N1), which is present outside the cells during EP, neutralizes the generated ROS. The ROS generation is proportional to the amplitude of the electric pulses and can be scavenged by antioxidants, such as vitamin C or tempol. When antioxidants were used during gene electrotransfer, the transfection efficiency of C2C12 myoblasts was statistically significantly increased 1.6-fold with tempol. Also in vivo, the transfection efficiency of M. tibialis cranialis in mice was statistically significantly increased 1.4-fold by tempol. The study indicates that ROS are generated on cells during EP and can be scavenged by antioxidants. Specifically, tempol can be used to improve gene electrotransfer into the muscle and possibly also to other tissues.     

Cancer gene therapy using a survivin mutant adenovirus

We have constructed a replication-deficient adenovirus encoding a nonphosphorylatable Thr(34)-->Ala mutant of the apoptosis inhibitor survivin (pAd-T34A) to target tumor cell viability in vitro and in vivo. Infection with pAd-T34A caused spontaneous apoptosis in cell lines of breast, cervical, prostate, lung, and colorectal cancer. In contrast, pAd-T34A did not affect cell viability of proliferating normal human cells, including fibroblasts, endothelium, or smooth muscle cells. Infection of tumor cells with pAd-T34A resulted in cytochrome c release from mitochondria, cleavage of approximately 46-kDa upstream caspase-9, processing of caspase-3 to the active subunits of approximately 17 and 19 kDa, and increased caspase-3 catalytic activity. When compared with chemotherapeutic regimens, pAd-T34A was as effective as taxol and considerably more effective than adriamycin in induction of tumor cell apoptosis and enhanced taxol-induced cell death. In three xenograft breast cancer models in immunodeficient mice, pAd-T34A suppressed de novo tumor formation, inhibited by approximately 40% the growth of established tumors, and reduced intraperitoneal tumor dissemination. Tumors injected with pAd-T34A exhibited loss of proliferating cells and massive apoptosis by in situ internucleosomal DNA fragmentation. These data suggest that adenoviral targeting of the survivin pathway may provide a novel approach for selective cancer gene therapy.     

实时荧光定量聚合酶链反应检测大肠癌生存素基因

目的建立实时荧光定量聚合酶链反应(RT-PCR)法检测大肠癌组织生存素(survivin)mRNA的表达。方法用Primer5软件设计RT-PCR引物和TaqMan探针,建立survivin基因RT-PCR法;再用该法检测15例肠道良性疾病组和51例大肠癌及癌旁组织的survivin mRNA表达,以良性疾病组作为正常对照组。结果典型的阳性扩增曲线呈S形,无S形扩增曲线者为阴性,阳性标准质粒标准曲线线性好(r=0.99),正常对照组无survivin表达,大肠癌和癌旁组织的阳性表达率分别为88.24%和33.33%(P0.01),表达量分别是5.24±2.21和3.17±1.02(P0.05)。结论成功构建实时荧光定量RT-PCR法检测survivin基因;大肠癌组织中survivin高表达,表达与性别、肿瘤大小、Dukes分期及淋巴结转移无关(P0.05),正常对照组不表达;RT-PCR法是一简便可行、灵敏的方法,在大肠癌的普查及诊断治疗中有很高的应用价值。     

宫颈癌组织中survivin基因的表达及其临床意义

目的 :探讨宫颈癌组织中survivin基因表达的特征及其临床意义。方法 :应用免疫组织化学方法 (SP法 )检测survivin基因在 41例宫颈癌组织、17例CIN宫颈组织及 10例正常宫颈组织中的表达 ,分析其与临床病理特征的相关性及其与预后的关系。结果 :宫颈癌组织中survivin的阳性表达率较正常组织及CIN均明显升高 (P <0 0 5 ) ;宫颈癌患者中survivin阳性表达的中位生存期明显小于阴性表达者 (P <0 0 5 )。Survivin的表达与宫颈癌的临床分期、病理分级及组织类型无关。结论 :survivin的阳性表达增高与宫颈癌的发生密切相关 ,预示预后不良。     

Transgene expression is increased by photochemically mediated transduction of polycation-complexed adenoviruses

Poor efficiency of adenoviral gene transfer to target cells is a major limitation to adenoviral gene therapy. Inefficient gene transfer occurs in the absence of coxsackie- and adenovirus receptor (CAR) on the cell surface, and can be overcome by enhancing viral entry with cationic molecules. Recombinant adenovirus (Ad) noncovalently complexed with polycations imply a lack of transduction specificity. Therefore, we have investigated the potential of a novel light-specific treatment, named photochemical internalization (PCI), to enhance gene delivery of adenovirus serotype 5 (Ad5) complexed with the cationic agents poly-L-lysine (PLL) and SuperFect trade mark. Cell lines differing in their receptiveness to Ad5 were infected with amounts of virus transducing about 2% of the cells by conventional Ad infection. The combination of polycations and photochemical treatment enabled a substantial increase in reporter gene expression, resulting in up to 75% positive cells. The effect was most prominent in cell lines expressing moderate to low levels of CAR. Furthermore, we show that PCI enables proper gene delivery of fiberless Ad5 at viral concentrations and infection times where transduction of photochemically untreated cells was negligible, both in the absence and presence of PLL. Thus, we conclude that the photochemically induced transduction by adenoviral vectors complexed with polycations present an opportunity to obtain high cell-infectivity levels with low viral doses, also without the fiber-CAR interaction.     

Targeting gene expression to the vascular wall in transgenic mice using the murine preproendothelin-1 promoter.

To develop a system for overexpressing genes in the vascular wall, we created transgenic mice using the reporter gene luciferase and the murine preproendothelin-1 promoter. In vitro analysis suggested that the murine 5'-flanking region contained endothelial-specific elements in a 5.9-kb fragment. Five transgenic mice colonies established from independent founders all exhibited the highest level of luciferase activity in the aorta with up to 8,540 light units per microgram of protein. Immunohistochemistry with anti-luciferase antisera revealed high levels of expression in the endothelial cells of both large and small arteries and lower levels of expression in veins and capillaries. Significant expression was also seen in arterial smooth muscle cells and in select epithelial surfaces which is consistent with the known distribution of endothelin-1 in mammals. The further demonstrate the targeting capability of this system, we overexpressed the lipid-peroxidating enzyme, human 15-lipoxygenase, in the vessel wall of transgenic mice. As with luciferase, expression of active enzyme and immunohistochemical localization in vascular cells were documented in transgenic animals. Hence, this new system can be used to direct expression of molecules to the vascular wall for the purpose of examining the biological significance of either overexpression or inhibition of select proteins.     

靶向干扰Survivin基因对结直肠癌细胞PTTG蛋白的影响

目的探讨重组载体介导的Survivin小干扰RNA(siRNA)对人结肠癌细胞株Lovo中PTTG蛋白的影响。方法运用靶向Survivin siRNA真核表达载体转染结肠癌Lovo细胞,western blot检测PTTG蛋白的表达水平。结果 Survivin基因沉默后24-72h,与正常对照组相比,Survivin和PTTG蛋白表达均明显降低,差异具有统计学意义(P<0.01)。结论 Survivin基因沉默后结直肠癌细胞PTTG蛋白表达显著下调,提示Survivin和PTTG可能相互促进共同参与结直肠癌的发生发展。     

C57BL小鼠survivin基因miRNA表达载体的构建

背景：Survivin在多种肿瘤组织中的高表达,具有调节细胞增殖分裂和强大的抗凋亡功能。目的：利用RNA干扰技术,构建具有特异性阻断C57BL小鼠survivin基因的微小RNA（micro RNA,miRNA）表达载体。设计、时间及地点：单一样本观察,于2008-06/11在重庆医科大学附属第一医院神经内科实验中心完成。材料：环形pcDNATM6.2-GW/EmGFPmiR和BLOCK-iTTM PolII miR RNA干扰Expression Vector Kit with EmGFP为Invitrogen公司产品;DH5a大肠杆菌为实验室保存;xhoI和BamHI酶、壮观霉素均为上海生工生物工程有限公司产品。方法：应用设计软件在C57BL小鼠survivin基因的mRNA上寻找特异性的短核苷酸序列,并设计合成4对寡核苷酸序列,经退火后形成双链DNA片段,采用基因克隆技术,将其克隆到pcDNATM6.2-GW/EmGFPmiR的载体中,转化DH5a大肠杆菌,挑单菌落种于含有壮观霉素LB液体培养基中,提取质粒。主要观察指标：应用测序法和琼脂糖电泳检测对重组体进行鉴定。结果：测序结果显示插入片段与线性载体连接正确,无碱基突变、缺失、插入等异常。双酶切处理pcDNATM6.2-GW/EmGFP-miR重组质粒结果显示片段大小与预期相符。结论：实验结果表明成功构建了C57BL小鼠survivin基因的微小RNA表达载体。     

Honey bee promoter sequences for targeted gene expression

The honey bee, Apis mellifera, displays a rich behavioural repertoire, social organization and caste differentiation, and has an interesting mode of sex determination, but we still know little about its underlying genetic programs. We lack stable transgenic tools in honey bees that would allow genetic control of gene activity in stable transgenic lines. As an initial step towards a transgenic method, we identified promoter sequences in the honey bee that can drive constitutive, tissue‐specific and cold shock‐induced gene expression. We identified the promoter sequences of Am‐actin5c, elp2l, Am‐hsp83 and Am‐hsp70 and showed that, except for the elp2l sequence, the identified sequences were able to drive reporter gene expression in Sf21 cells. We further demonstrated through electroporation experiments that the putative neuron‐specific elp2l promoter sequence can direct gene expression in the honey bee brain. The identification of these promoter sequences is an important initial step in studying the function of genes with transgenic experiments in the honey bee, an organism with a rich set of interesting phenotypes.     

A polymorphism in the resistin gene promoter is related to increased C-reactive protein levels in patients with coronary artery disease.

BACKGROUND: Resistin, a novel adipocyte-derived peptide, has been linked to inflammatory process and coronary artery disease (CAD). The -420C>G polymorphism located in the resistin gene (RETN) promoter has recently been suggested to play a potential role in proinflammatory conditions (e.g., atherogenesis). However, whether this polymorphism has any effect on the inflammatory process in patients with stable CAD is unclear. METHODS: The RETN -420C>G polymorphism was determined by using PCR-restriction fragment length polymorphism. Plasma lipid profiles, glucose and high-sensitivity C-reactive protein (hs-CRP) were measured in fasting state. RESULTS: Patients with variant genotypes (CG+GG) had significantly higher levels of hs-CRP than CC carriers (adjusted p<0.001). In addition, the variant genotypes were observed to be independently associated with higher hs-CRP levels (>3 mg/L, p=0.004). However, no association was found between this polymorphism and plasma lipids or glucose levels. CONCLUSION: Our data suggest that the RETN -420C-to-G variant is associated with increased CRP levels in patients with stable CAD, suggesting that the RETN -420C>G polymorphism may be potentially involved in the inflammatory component of atherogenesis through an enhanced production of CRP.     

生存素基因蛋白和阻抑素在胃癌中的表达及意义

目的探讨生存素基因蛋白(Survivin)和阻抑素(Prohibitin)在胃癌组织中的表达及二者相关的临床意义。方法用免疫组织化学SP法检测胃癌组织及癌旁组织、正常胃黏膜中Prohibitin与Survivin的表达,比较其阳性表达率及Prohibitin与Survivin的关系。结果 Survivin在胃癌中阳性表达率为75.5%(40/53),明显高于癌旁组织和正常胃黏膜(P<0.05);Prohibitin在胃癌中的表达率为84.9%(45/53),明显高于癌旁组织和正常胃黏膜(P<0.01);胃癌组织中Survivin与Prohibitin的表达呈正相关(P<0.01)。结论 Survivin和Prohibitin在胃癌中过度表达,提示二者在胃癌的发生、发展中起重要作用;Survivin和Prohibitin通过抗凋亡机制协同参与胃癌的发生。     

An enhanced autogene-based dual-promoter cytoplasmic expression system yields increased gene expression

The relatively low levels of transfection that can be achieved by current gene-delivery systems have limited the therapeutic utility of gene transfer. This is especially true for nonviral gene-delivery systems, where the levels of gene expression achieved are usually below the levels achieved by viral gene transfer systems. One strategy for increasing gene expression is to design a cytoplasmic expression system that does not require nuclear delivery for gene expression to occur. This can be achieved through the use of an autocatalytic cytoplasmic expression system using phage RNA polymerases. Here we describe cytoplasmic expression systems that yield increased levels of gene expression following in vitro transfection. We demonstrate direct evidence for an exponential, autocatalytic increase in gene expression using autogenes, as well as levels of reporter gene expression that are 20-fold higher than standard CMV-based nuclear expression systems. The development of a high-efficiency plasmid-based expression system could significantly improve the gene expression properties of nonviral gene-delivery systems, thereby increasing their clinical utility.