Cellular delivery and enhanced anticancer activity of berberine complexed with a cationic derivative of γ–cyclodextrin

A cationic derivative of γ-cyclodextrin (GCD) modified with propylenediamine (PDA) was synthesized. It was shown that the derivative (GCD–PDA) is mucoadhesive and resistant to the digestion with?∝-amylase indicating that it may constitute an efficient oral delivery vehicle. GCD-PDA formed an inclusion complex with berberine (BBR), an alkaloid displaying a multitude of beneficial physiological effects. The complexed BBR penetrates a lipid membrane easier than the free one. Both uncomplexed BBR and that complexed with GCD-PDA was delivered to normal (NMuMG) and cancerous (4T1) murine mammary gland cells. In the normal cells both free and complexed BBR was homogeneously dispersed in the cytoplasm and was nontoxic up to 131?μM. In the cancerous cells uncomplexed BBR was also homogeneously dispersed but it was toxic to about 25% of cells at 131?μM, while the GCD-PDA/BBR complex was preferably localized in lysosomes and its toxicity doubled at this concentration compared to that of free BBR. Moreover, free BBR and GCD-PDA/BBR showed even more efficient inhibitory effect against murine melanoma (B16-F10) cells than against 4T1 cells.     

Generation of chimeric HBc proteins with epitopes in E.coli: formation of virus-like particles and a potent inducer of antigen-specific cytotoxic immune response and anti-tumor effect in vivo

The major aim of the project was to develop the virus-like particles (VLPs) displaying single or multi-epitope of hepatocellular carcinomas (HCC) in Escherichia coli and to evaluate the effect on inducing Ag-specific CD8(+) T cell response and antitumor efficacy as candidate vaccines. To this end, hepatitis B virus core (HBc) particles were used as a carrier of HCC epitopes. Four HCC epitopes MAGE-1(278-286aa), MAGE-3(271-279aa), AFP1 (158-166aa) or AFP2 (542-550aa) were fused to the 3' terminus of the truncated HBV core gene, respectively, or conjunctively. Not all recombinant plasmids led to expression of chimeric proteins in expression strain E. coli BL21 (DE3), but chimeric proteins which are expressed in inclusion bodies resulted in the formation of complete "mature" VLPs. E. coli-derived truncated HBc(1-144) chimeric protein self-assembled into VLPs that both morphologically and physically are similar to the wild-type ones and they still remained activity after purification and refolding from 6M urea solution. We also showed that they could be internalized and presented by DCs in vitro. Additionally, DCs pulsed with the chimeric HBc-VLPs could induce stronger CTL activity and greater IFN-gamma secretion by responding T cells compared with peptid-pulsed DCs. In the B16-pIR-HH tumor therapy model, the growth of established tumors was significantly inhibited by immunization using VLP-pulsed DCs, resulting in significantly higher survival rate of immunized animals. Thus, the results of the current study have demonstrated the principal possibility of using VLP on the basis of HBcAg for creation of a new type of HCC-specific immunogen.     

Plasmodium yoelii: the effect of second blood meal and anti-sporozoite antibodies on development and gene expression in the mosquito vector, Anopheles stephensi

The sporogonic development of the malaria parasite takes place in the mosquito and a wide range of factors modulates it. Among those, the contents of the blood meal can influence the parasite development directly or indirectly through the mosquito response to the infection. We have studied the effect of a second blood meal in previously infected mosquitoes and the effect of anti-sporozoite immune serum on parasite development and mosquito response to the infection. The prevalence and intensity of infection and gene expression of both Plasmodium yoelii and Anopheles stephensi was analyzed. We verified that a second blood meal and its immune status interfere with parasite development and with Plasmodium and mosquito gene expression.     

Cellular functions of NSF: not just SNAPs and SNAREs

N-ethylmaleimide sensitive factor (NSF) is an ATPases associated with various cellular activities protein (AAA), broadly required for intracellular membrane fusion. NSF functions as a SNAP receptor (SNARE) chaperone which binds, through soluble NSF attachment proteins (SNAPs), to SNARE complexes and utilizes the energy of ATP hydrolysis to disassemble them thus facilitating SNARE recycling. While this is a major function of NSF, it does seem to interact with other proteins, such as the AMPA receptor subunit, GluR2, and beta2-AR and is thought to affect their trafficking patterns. New data suggest that NSF may be regulated by transient post-translational modifications such as phosphorylation and nitrosylation. These new aspects of NSF function as well as its role in SNARE complex dynamics will be discussed.     

p38MAP Kinase activity is required for human primary adipocyte differentiation

Little is known about the role of p38MAPK in human adipocyte differentiation. Here we showed that p38MAPK activity increases during human preadipocytes differentiation. Pharmacological inhibition of p38MAPK during adipocyte differentiation of primary human preadipocytes markedly reduced triglycerides accumulation and adipocyte markers expression. Cell cycle arrest or proliferation was not affected by p38MAPK inhibition. Although induction of C/EBPbeta was not altered by the p38MAPK inhibitor, its phosphorylation on Threonine(188) was decreased as well as PPARgamma expression. These results indicate that p38MAPK plays a positive role in human adipogenesis through regulation of C/EBPbeta and PPARgamma factors.     

Effect of acyl-CoA oxidase activity on the accumulation of gamma-decalactone by the yeast Yarrowia lipolytica: a factorial approach

beta-Oxidation is a cyclic pathway involved in the degradation of lipids. In yeast, it occurs in peroxisomes and the first step is catalyzed by an acyl-CoA oxidase (Aoxp). The yeast Yarrowia lipolytica possesses several genes (POX) coding for Aoxps. This study is based on the factorial analysis of results obtained with the many POX derivative strains that have been constructed previously. The effect of interactions between Aoxps on the acyl-CoA oxidase (Aox) activity was important even at the second order. We then investigated the effect of Aox activity on growth and lactone production. Aox activity was correlated with acidification of the medium by cells and with cellular growth but not with lactone production, although Aox activity on short chains was inversely correlated with lactone accumulation. Due to the poor correlation between Aox activity and lactone production, the modeling of this parameter gave no satisfactory results but growth depending on Aox activity was modeled.     

人γ－精浆蛋白的亲和纯化及其糖基化分析

目的:从人精液中提纯γ-精浆蛋白并对其生物学特性进行鉴定。 方法:应用饱和硫酸铵法从小鼠腹水中纯化出抗γ-精浆蛋白的单克隆抗体E4B7,将其共价交联到CNBr-Sepharose4B上,制备成免疫亲和层析柱,用该层析柱亲和纯化经饱和硫酸铵粗提的精液标本。纯化产物分别用SDS-PAGE、Western-blot及ELISA进行生物学特性鉴定,最后经PNGase F、Endo-H酶消化后进行糖基化分析。结果:SDS-PAGE电泳表明纯化蛋白的相对分子量约为44KD,Western-blot及ELISA鉴定显示该蛋白可与抗γ-精浆蛋白的单抗E4B7特异性结合。对纯化蛋白无论是单用Endo-H酶消化,还是同时用Endo-H酶和PNGase F酶共同消化,消化产物电泳后相对分子量均降低到34KD左右,而单独用PNGase F酶消化后相对分子量没有改变。Western-blot及ELISA鉴定显示糖苷酶处理后的纯化蛋白仍可与单抗E4B7特异性结合。结论:成功纯化出N-糖基化形式的γ-精浆蛋白,这为下一步筛选人源化抗体奠定了纯的抗原基础。     

硝黄散外敷联合加味五虎汤口服治疗痰热闭肺型肺炎支原体肺炎患儿对肺功能和血清TNF-α、IFN-γ、IL-4水平的影响

目的:观察硝黄散外敷联合加味五虎汤口服治疗痰热闭肺型肺炎支原体肺炎(MPP)患儿对肺功能和血清肿瘤坏死因子-α(TNF-α)、γ-干扰素(IFN-γ)、白介素-4(IL-4)水平的影响。方法:研究对象为我院2019年6月~2021年1月期间收治的MPP患儿80例,采用随机数字表法将患儿分为对照组(n=40,阿奇霉素抗感染治疗)和研究组(n=40,对照组基础上加用硝黄散外敷联合加味五虎汤口服治疗),均治疗7 d。比较两组患儿临床疗效,比较两组治疗前、治疗7 d后的中医证候积分、临床症状改善情况、肺功能和血清TNF-α、IFN-γ、IL-4水平。结果:研究组治疗7 d后的临床总有效率为92.50%(37/40),高于对照组的72.50%(29/40),差异有统计学意义(P<0.05)。与对照组相比,研究组的症状(咳嗽憋喘、发热、肺部干湿啰音)消失时间均更短(P<0.05)。与对照组相比,研究组治疗7 d后用力肺活量(FVC)、最高呼气峰流速(PEF)、第1秒最大呼气容积(FEV1)、FEV1/FVC均更高(P<0.05),研究组治疗7 d后的中医证候积分及血清TNF-α、IFN-γ和IL-4水平均更低(P<0.05)。结论:痰热闭肺型MPP患儿采用硝黄散外敷联合加味五虎汤口服治疗,可有效缩短患儿症状消失时间,显著改善其肺功能、血清炎症因子水平,疗效显著。