The Impact of Endogenous Breast Cancer Resistance Protein on Human P-Glycoprotein-Mediated Transport Assays Using LLC-PK1 Cells Transfected With Human P-Glycoprotein

Lilly Laboratories cell porcine kidney 1 (LLC-PK1) cells transfected with human P-glycoprotein (LLC-PK1-P-gp) are widely used in transport assays to identify drug candidates that function as substrates of this efflux transporter. Endogenous transporters expressed in LLC-PK1 cells may complicate the interpretation of findings from P-gp-mediated transport assays. We investigated the impact of porcine breast cancer resistance protein (Bcrp) in P-gp-mediated transport assays in LLC-PK1 cells. Porcine Bcrp mRNA was detected in both LLC-PK1 wildtype (WT) and LLC-PK1-P-gp cells by quantitative RT-PCR. To investigate the activity and impact of porcine Bcrp, we conducted transport assays using 6 typical BCRP substrates in LLC-PK1 cells. Efflux ratios (ER) of the 6 BCRP substrates in LLC-PK1 WT cells were >2, and were reduced in the presence of the BCRP inhibitor Ko143. The efflux activities of the 6 BCRP substrates were confirmed using MDCKII cells transfected with human BCRP. Net ERs of prazosin and fluvastatin, dual substrates of P-gp and BCRP, determined by dividing ERs in LLC-PK1-P-gp cells by those in LLC-PK1 WT cells, were <2, but increased to >2 in the presence of Ko143. These results indicated that endogenous Bcrp in LLC-PK1 cells was involved in the transport of BCRP substrates and may interfere with the identification of P-gp substrates.     

The role of inflammation in regulating platelet production and function: Toll-like receptors in platelets and megakaryocytes

Platelets have been extensively studied as hemostatic regulators, stopping uncontrolled flow of blood from an injured vessel and allowing for repair. However, multiple studies have shown that platelets can interact with bacterial proteins, particularly seen during sepsis and inflammation. Immune cells recognize pathogens through Toll-like Receptors (TLRs). These same receptors allow platelets to recognize bacterial proteins and regulate platelet immunity and function. This review examines the TLRs expressed on platelets and megakaryocytes and how these receptors affect the function of these cells. Through TLRs, platelets go beyond hemostatic regulation and play a pivotal role in inflammation and infection.     

新型酪氨酸激酶抑制剂HHGV678对Bcr-Abl野生型细胞株和伊马替尼耐药细胞株抑制作用的体外研究

本研究比较新型的酪氨酸激酶抑制剂HHGV678与伊马替尼(imatinib,IM)在体外对Bcr-Abl野生型和IM耐药细胞株的抑制作用,探索HHGV678替代IM治疗CML及IM耐药CML患者的可能性。以Bcr-Abl野生型细胞株(K562和32Dp210)及16种IM耐药细胞株(K562R和15种Bcr-Abl点突变细胞株)为研究对象,用MTT法检测HHGV678和IM对上述细胞的生长抑制作用;以DNA梯形条带法和Annexin-V/PI双染色流式细胞术检测细胞凋亡;应用Western blot检测HHGV678对上述细胞BCR-ABL融合蛋白及酪氨酸激酶磷酸化表达的影响。结果表明:HHGV678呈剂量依赖性显著抑制Bcr-Abl野生型细胞株和除T315I点突变细胞株以外的IM耐药细胞株生长。比较IC50发现,HHGV678在低剂量下(0.01-0.3μmol/L)抑制K562和32Dp210细胞生长的作用比IM分别强15.5和28倍;而对除T315I点突变细胞株以外的15种IM耐药细胞株细胞的生长抑制作用比IM强1.4-124.3倍。HHGV678抑制上述细胞酪氨酸激酶磷酸化的能力均强于IM。更重要的是HHGV678在10.0μmol/L剂量下诱导IM强耐药细胞株K562R和T315I点突变细胞株的凋亡率分别达到40.06%和33.32%,显著高于IM的19.77%和10.68%。结论:新型酪氨酸激酶抑制剂HHGV678对Bcr-Abl野生型细胞株和IM耐药细胞株,尤其是对IM强耐药细胞株的生长抑制作用明显强于IM,但HHGV678能否成为治疗CML和IM耐药CML患者新的靶向药物,仍有待进一步的研究。     

Beneficial effects of cannabinoids (CB) in a murine model of allergen-induced airway inflammation: role of CB1/CB2 receptors

The endocannabinoid system (ECS) consists of two cannabinoid (CB) receptors, namely CB₁ and CB₂ receptor, and their endogenous (endocannabinoids) and exogenous (cannabinoids, e.g. delta-9-tetrahydrocannabinol (THC)) ligands which bind to these receptors. Based on studies suggesting a role of THC and the ECS in inflammation, the objective of this study was to examine their involvement in type I hypersensitivity using a murine model of allergic airway inflammation. THC treatment of C57BL/6 wildtype mice dramatically reduced airway inflammation as determined by significantly reduced total cell counts in bronchoalveolar lavage (BAL). These effects were greatest when mice were treated during both, the sensitization and the challenge phase. Furthermore, systemic immune responses were significantly suppressed in mice which received THC during sensitization phase. To investigate a role of CB_1/2 receptors in this setting, we used pharmacological blockade of CB₁ and/or CB₂ receptors by the selective antagonists and moreover CB₁/CB₂ receptor double-knockout mice (CB₁^−/−/CB₂^−/−) and found neither significant changes in the cell patterns in BAL nor in immunoglobulin levels as compared to wildtype mice. Our results indicate that the activation of the ECS by applying the agonist THC is involved in the development of type I allergies. However, CB₁/CB₂ receptor-independent signalling seems likely in the observed results.     

Peptide vaccination is superior to genetic vaccination using a recombineered bacteriophage λ subunit vaccine

Genetic immunization holds promise as a vaccination method, but has so far proven ineffective in large primate and human trials. Herein, we examined the relative merits of genetic immunization and peptide immunization using bacteriophage λ. Bacteriophage λ has proven effective in immune challenge models using both immunization methods, but there has never been a direct comparison of efficacy and of the quality of immune response. In the current study, this vector was produced using a combination of cis and trans phage display. When antibody titers were measured from immunized animals together with IL-2, IL-4 and IFNγ production from splenocytes in vitro, we found that proteins displayed on λ were superior at eliciting an immune response in comparison to genetic immunization with λ. We also found that the antibodies produced in response to immunization with λ displayed proteins bound more epitopes than those produced in response to genetic immunization. Finally, the general immune response to λ inoculation, whether peptide or genetic, was dominated by a Th1 response, as determined by IFNγ and IL-4 concentration, or by a higher concentration of IgG2a antibodies.     

On the energy-dependence of Hoechst 33342 transport by the ABC transporter LmrA

LmrA is an ATP-binding cassette (ABC) multidrug transporter from Lactococcus lactis, and is a structural homologue of the human multidrug resistance P-glycoprotein (ABCB1), the overexpression of which is associated with multidrug resistance in tumours. We recently observed that a truncated version of LmrA lacking the nucleotide-binding domain mediates a proton motive force-dependent ethidium transport reaction by catalyzing proton-ethidium symport. This finding raised the question whether proton motive force-dependent transport can also be observed for other drugs, and whether this reaction is also relevant for full-length LmrA. Furthermore, the observations on LmrA-MD raised the question whether ATP-dependent transport by LmrA in intact cells could be due to the activity of independent ABC transporters that might become upregulated in the lactococcal cells due to the overexpression of LmrA; the recently identified ABC multidrug transporter LmrCD was put forward as a possible candidate. Here, we investigated the energy coupling to the transport of the amphiphilic dye Hoechst 33342 in proteoliposomes containing purified LmrA. For this purpose, LmrA was obtained from lactococcal cells lacking the genomic lmrA and lmrCD genes, in which LmrA was expressed from a plasmid. To separate ATP-dependence from proton motive force-dependence, we also used mutant LmrA proteins, which were affected in their ability to hydrolyse ATP. Our studies in proteoliposomes demonstrate that LmrA can catalyze Hoechst 33342 transport independent of auxiliary proteins, in an ATP-dependent fashion and a transmembrane chemical proton gradient (interior acidic)-dependent fashion.     

野生型及突变型survivin基因重组逆转录病毒的构建及表达研究

目的构建含绿色荧光蛋白和野生型及第34位氨基酸突变型survivin基因的双顺反子逆转录病毒载体,并进行真核细胞表达.方法用PCR的方法由含survivin基因的真核表达载体扩增所需目的片段,然后将该片段插入逆转录病毒载体pMIG.酶切鉴定重组子,脂质体介导下转染包装细胞Phoenix E,病毒上清感染NIH3T3细胞,RT-PCR检测外源基因mRNA的表达、流式细胞仪检测GFP的表达从而确认外源基因的转录.结果经琼脂糖凝胶电泳鉴定,目的片段成功的连接到pMIG载体上.载体转染NIH3T3细胞,RT-PCR显示目的片段mRNA能够表达,流式细胞仪检测GFP获良好的表达.结论成功构建了人survivin基因野生型及突变型逆转录病毒,并在NIH3T3细胞进行了表达,为下一步的研究工作提供了有效的分子工具,并且为将来能在体内应用抑制survivin基因表达的肿瘤治疗奠定基础.     

Oxytocin: The great facilitator of life

Oxytocin (Oxt) is a nonapeptide hormone best known for its role in lactation and parturition. Since 1906 when its uterine-contracting properties were described until 50 years later when its sequence was elucidated, research has focused on its peripheral roles in reproduction. Only over the past several decades have researchers focused on what functions Oxt might have in the brain, the subject of this review.