Adeno-associated virus serotype 2 mediated transduction and coexpression of the human apoAI and SR-BI gene in HepG2 cells

Cholesterol efflux is the first step in the reverse cholesterol transport (RCT) pathway, removing excess cholesterol from tissues, including the arterial wall, thus preventing the development of atherosclerosis. Adeno-associated virus (rAAV) has demonstrated significant promise as a DNA-delivery vector to treat serious human diseases. In this study, we constructed recombinant adeno-associated viruses coexpressing apoAI and SR-BI successfully, the double gene mRNA and protein were both strongly expressed in transduced HepG2 cells. A novel safe and efficient method of promoting the reverse cholesterol transport (RCT) may be established. These results may provide a new method for gene therapy of Arteriosclerosis.     

A novel RGD-toxin protein,Lj-RGD3, from the buccal gland secretion of Lampetra japonica impacts diverse biological activities

RGD (Arg-Gly-Asp) motif toxin proteins from snake venoms, saliva glands secretion of leech or tick have typical characteristics of inhibiting platelet aggregation, angiogenesis, and tumor growth. Here we report cloning and characterization of a novel RGD-toxin protein from the buccal gland of Lampetra japonica. In an attempt to study the activities of anticoagulant in the buccal gland secretion of L. japonica, we established buccal gland cDNA library and identified a gene encoding a predicted protein of 118 amino acids with 3 RGD motifs. The predicted protein was named Lj-RGD3. We generated the cDNA of Lj-RGD3 and obtained the recombinant protein rLj-RGD3. The polyclonal antibodies against rLj-RGD3 recognized the native Lj-RGD3 protein in buccal gland secretion in Western blot analyses. The biological function studies reveal that rLj-RGD3 inhibited human platelet aggregation in a dose-dependent manner with IC₅₀ value at 5.277 μM. In addition, rLj-RGD3 repressed bFGF-induced angiogenesis in the chick chorioallantoic membrane model. rLj-RGD3 also inhibited the adhesion of ECV304 cells to vitronectin. Furthermore, rLj-RGD3 induced apoptosis and significantly inhibited proliferation, migration, and invasion evoked by bFGF in ECV304 cells. Taken together, these results suggested that rLj-RGD3 is a novel RGD-toxin protein possessing typical functions of the RGD-toxin protein.     

中枢神经细胞的高尔基复合体ACP活性分布的电镜观察

本文采用电镜金属盐法—酸性磷酸酶(ACP)细胞化学技术,用30mmol/L pipes缓冲液配制低浓度戊二醛进行固定。对成年大鼠的大脑大锥体细胞,小脑浦肯野氏细胞,脊髓前角运动细胞的高尔基复合体的ACP活性进行了实验研究和探讨。结果发现ACP活性分布在高尔基复合体的部份转移泡、浓缩泡及GERL部位。高尔基复合体呈ACP阳性反应,并显示出多种形态。     

RETRACTED ARTICLE: Circ-NUP214 Promotes Papillary Thyroid Carcinoma Tumorigenesis by Regulating HK2 Expression Through miR-15a-5p

Biochemical Genetics -     

猪流感的诊断技术研究进展

猪流感是由猪流感病毒引起的一种猪的急性、高度接触性呼吸道传染病,不仅给畜禽养殖造成重大经济损失,而且还对人类健康造成威胁,因此快速、敏感、准确诊断猪流感,具有重要的经济价值和公共卫生意义。从传统的实验室诊断技术和分子生物学诊断技术两方面综合阐述了猪流感的诊断研究进展。     

力生长因子在大肠杆菌中的表达及活性分析

MGF(Mechano-growth factor)是一种IGF-1变体形式, 研究发现该因子具有应力敏感性, 并且具有促进肌肉肥大、再生以及神经损伤修复的功能。通过RT-PCR从拉伸刺激的人成骨细胞中克隆MGF cDNA序列, 并去除5'端9 bp的序列, 使N端缺少对肠激酶(Enterokinase, EK)具有抑制作用的脯氨酸, 将截短型MGF (des(1-3) MGF) cDNA序列克隆入pET32a(+)质粒, 构建重组表达质粒。重组质粒转化E. coli BL21(DE3), 在30oC培养下以可溶形式表达融合蛋白Trx/ des(1-3)MGF, 采用离子交换层析和Ni2+金属亲和层析, 获得纯度95%以上的融合蛋白。再对融合蛋白EK酶切, rpHPLC分离获得纯度达98%的des(1-3)MGF, SDS-PAGE电泳及质谱分析蛋白分子量与理论值相符。生物活性实验显示, 所制备的des(1-3)MGF比des(1-3)IGF-1更显著的促进MC3T3-E1细胞的增值和迁移。     

Dexamethasone interferes with osteoblasts formation during osteogenesis through altering IGF-1-mediated angiogenesis

Dexamethasone (Dex), a synthetic glucocorticoid (GC) with long-lasting treatment effects, has been proved to exert a modulatory effect on osteoblast proliferation and differentiation during embryonic osteogenesis. However, it is still controversial if Dex exposure influences endochondral ossification and the underlying mechanism. In this study, chick embryos in vivo and preosteoblast cell cultures in vitro were utilized to investigate the effects of Dex on osteoblast formation and differentiation during the skeletal development. We first demonstrated that Dex exposure could shorten the long bones of 17-day chick embryos in vivo, and also downregulated the expressions of osteogenesis-related genes. Next, we established that Dex exposure inhibited the proliferation and viability of preosteoblasts-MC3TC-E1 cells, and the addition of insulin-like growth factor 1 (IGF-1) could dramatically rescue these negative effects. On the basis of remarkable changes in the rescue experiments, we next verified the important role of angiogenesis in osteogenesis by culturing isolated embryonic phalanges in Dulbecco's modified Eagle's medium culture or on the chick chorioallantoic membrane (CAM). Then, we transplanted MC3T3-E1 cell masses onto the CAM. The data showed that Dex exposure reduced the vessel density within the developed cell mass, concomitantly with the downregulation of IGF-1 pathway. We verified that the inhibition of blood vessel formation caused by Dex could be rescued by IGF-1 treatment using the CAM angiogenesis model. Eventually, we demonstrated that the shortened length of the phalanges in the presence of Dex could be reversed by IGF-1 addition. In summary, these findings suggested that the inhibition of Igf-1 signal caused by Dex exposure exerts a detrimental impact on the formation of osteoblasts and angiogenesis, which consequently shortens long bones during osteogenesis.     

The açaí flavonoid velutin is a potent anti-inflammatory agent: blockade of LPS-mediated TNF-α and IL-6 production through inhibiting NF-κB activation and MAPK pathway

Recent studies have shown that some flavonoids are modulators of proinflammatory cytokine production. In this study, velutin, a unique flavone isolated from the pulp of açaí fruit (Euterpe oleracea Mart.), was examined for its effects in reducing lipopolysaccharide-induced proinflammatory cytokine tumor necrosis factor (TNF)-α and interleukin (IL)-6 production in RAW 264.7 peripheral macrophages and mice peritoneal macrophages. Three other structurally similar and well-studied flavones, luteolin, apigenin and chrysoeriol, were included as controls and for comparative purposes. Velutin exhibited the greatest potency among all flavones in reducing TNF-α and IL-6 production. Velutin also showed the strongest inhibitory effect in nuclear factor (NF)-κB activation (as assessed by secreted alkaline phosphatase reporter assay) and exhibited the greatest effects in blocking the degradation of inhibitor of NF-κB as well as in inhibiting mitogen-activated protein kinase p38 and JNK phosphorylation; all of these are important signaling pathways involved in production of TNF-α and IL-6. The present study led to the discovery of a strong anti-inflammatory flavone, velutin. This compound effectively inhibited the expression of proinflammatory cytokines TNF-α and IL-6 in low micromole levels by inhibiting NF-κB activation and p38 and JNK phosphorylation.     

Fluorescence resonance energy transfer reports properties of syntaxin1a interaction with Munc18-1 in vivo

Syntaxin1A, a neural-specific N-ethylmaleimide-sensitive factor attachment protein receptor protein essential to neurotransmitter release, in isolation forms a closed conformation with an N-terminal alpha-helix bundle folded upon the SNARE motif (H3 domain), thereby limiting interaction of the H3 domain with cognate SNAREs. Munc18-1, a neural-specific member of the Sec1/Munc18 protein family, binds to syntaxin1A, stabilizing this closed conformation. We used fluorescence resonance energy transfer (FRET) to characterize the Munc18-1/syntaxin1A interaction in intact cells. Enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A, or mutants of these proteins, were expressed as donor and acceptor pairs in human embryonic kidney HEK293-S3 and adrenal chromaffin cells. Apparent FRET efficiency was measured using two independent approaches with complementary results that unambiguously verified FRET and provided a spatial map of FRET efficiency. In addition, enhanced cyan fluorescent protein-Munc18-1 and a citrine variant of enhanced yellow fluorescent protein-syntaxin1A colocalized with a Golgi marker and exhibited FRET at early expression times, whereas a strong plasma membrane colocalization, with similar FRET values, was apparent at later times. Trafficking of syntaxin1A to the plasma membrane was dependent on the presence of Munc18-1. Both syntaxin1A(L165A/E166A), a constitutively open conformation mutant, and syntaxin1A(I233A), an H3 domain point mutant, demonstrated apparent FRET efficiency that was reduced approximately 70% from control. In contrast, the H3 domain mutant syntaxin1A(I209A) had no effect. By using phosphomimetic mutants of Munc18-1, we also established that Ser-313, a Munc18-1 protein kinase C phosphorylation site, and Thr-574, a cyclin-dependent kinase 5 phosphorylation site, regulate Munc18-1/syntaxin1A interaction in HEK293-S3 and chromaffin cells. We conclude that FRET imaging in living cells may allow correlated regulation of Munc18-1/syntaxin1A interactions to Ca(2+)-regulated secretory events.