Dieckol,a natural polyphenolic drug,inhibits the proliferation and migration of colon cancer cells by inhibiting PI3K,AKT, and mTOR phosphorylation

This study investigated that dieckol (DKL), a natural drug, inhibits colon cancer cell proliferation and migration by inhibiting phosphoinositide-3-kinase (PI3K), protein kinase B (AKT), and mammalian target of rapamycin (mTOR) phosphorylation in HCT-116 cells. The cells were treated with DKL in various concentrations (32 and 50 μM) for 24 h and then analyzed for various experiments. MTT (tetrazolium bromide) and crystal violet assay investigated DKL-mediated cytotoxicity. Dichlorodihydrofluorescein diacetate staining was used to assess the reactive oxygen species (ROS) measurement, and apoptotic changes were studied by dual acridine orange and ethidium bromide staining. Protein expression of cell survival, cell cycle, proliferation, and apoptosis protein was evaluated by western blot analysis. Results indicated that DKL produces significant cytotoxicity in HCT-116, and the half-maximal inhibitory concentration was found to be 32 μM for 24-h incubation. Moreover, effective production of ROS and enhanced apoptotic signs were observed upon DKL treatment in HCT-116. DKL induces the expression of phosphorylated PI3K, AKT, and mToR-associated enhanced expression of cyclin-D1, proliferating cell nuclear antigen, cyclin-dependent kinase (CDK)-4, CDK-6, and Bcl-2 in HCT-116. In addition, proapoptotic proteins such as Bax, caspase-9, and caspase-3 were significantly enhanced by DKL treatment in HCT-116. Hence, DKL has been considered a chemotherapeutic drug by impeding the expression of PI3K-, AKT-, and mTOR-mediated inhibition of proliferation and cell cycle-regulating proteins.     

热稳定性伯克霍尔德菌脂肪酶A突变体的筛选

摘要:【目的】为更好地提高伯克霍尔德菌ZYB002脂肪酶LipA在TMP纸浆造纸工艺中的应用,有必要利用蛋白质工程技术,提高其热稳定性。【方法】基于B-factor值筛选LipA多肽链中潜在的突变位点,利用迭代饱和诱变技术,构建突变文库,筛选热稳定性提高的突变体。【结果】利用上述方法,从4个突变文库中分别筛选到在55℃下,半衰期较野生型脂肪酶LipA分别提高了1.8倍、3倍、2.2倍和1.7倍的脂肪酶突变体。【结论】基于B-factor值选择突变位点,利用迭代饱和突变技术,快速筛选到热稳定性有显著提高的突变体。     

The P2 purinoceptors in prostate cancer

P2 purinoceptors are composed of ligand-gated ion channel type (P2X receptor) and G protein-coupled metabolite type (P2Y receptor). Both these receptors have played important roles in the prostate cancer microenvironment in recent years. P2X and P2Y receptors can contribute to prostate cancer’s growth and invasiveness. However, the comprehensive mechanisms have yet to be identified. By summarizing the relevant studies, we believe that P2X and P2Y receptors play a dual role in cancer cell growth depending on the prostate cancer microenvironment and different downstream signalling pathways. We also summarized how different signalling pathways contribute to tumor invasiveness and metastasis through P2X and P2Y receptors, focusing on understanding the specific mechanisms led by P2X4, P2X7, and P2Y2. Statins may reduce and prevent tumor progression through P2X7 so that P2X purinergic receptors may have clinical implications in the management of prostate cancer. Furthermore, P2X7 receptors can aid in the early detection of prostate cancer. We hope that this review will provide new insights for future mechanistic and clinical investigations into the role of P2 purinergic receptors in prostate cancer.     

来源于铜绿假单胞杆菌(Pseudomonas aeruginosa)所产蛋白酶PT121克隆表达及其在小肽合成上的应用

【目的】基于前期筛选到的P.aeruginosa PT121所产有机溶剂耐受性蛋白酶,本研究对该蛋白酶进行克隆表达,研究了重组蛋白的酶学性质及小肽合成上的应用。【方法】参考文献中报道的相似蛋白酶pseudolysin设计引物从菌株PT121基因组中克隆到耐有机溶剂蛋白酶PT121基因las B。构建诱导表达重组质粒p ET22b-las B,于大肠杆菌中进行表达。考察蛋白酶PT121酶学性质及小肽合成上的应用。【结果】序列分析表明las B基因编码信号肽、前肽及成熟肽3个部分,成熟肽部分含有301个氨基酸,分子量约33 k Da,属于金属蛋白酶M4家族。通过破碎条件优化,一步法制备得到较为纯净的重组蛋白酶PT121,其比活力达7700 U/mg,该酶呈现了较高的热稳定性,p H稳定性及溶剂耐受性,与野生菌P.aeruginosa PT121所产蛋白酶性质一致。在50%DMSO体系中高效催化合成了多种小肽,其中阿斯巴甜前体(Cbz-Asp-Phe-NH2)产率高达91%。【结论】蛋白酶PT121基因在大肠杆菌中克隆表达为进一步研究相关催化机理及分子改造奠定了基础。     

Serum- and Glucocorticoid-Inducible Kinase-1 (SGK-1) Plays a Role in Membrane Trafficking in Caenorhabditis elegans

The mammalian serum- and glucocorticoid-inducible kinase SGK1 regulates the endocytosis of ion channels. Here we report that in C. elegans sgk-1 null mutants, GFP-tagged MIG-14/Wntless, the sorting receptor of Wnt, failed to localize to the basolateral membrane of intestinal cells; instead, it was mis-sorted to lysosomes. This effect can be explained in part by altered sphingolipid levels, because reducing glucosylceramide biosynthesis restored the localization of MIG-14::GFP. Membrane traffic was not perturbed in general, as no obvious morphological defects were detected for early endosomes, the Golgi apparatus, and the endoplasmic reticulum (ER) in sgk-1 null animals. The recycling of MIG-14/Wntless through the Golgi might be partially responsible for the observed phenotype because the subcellular distribution of two plasma membrane cargoes that do not recycle through the trans-Golgi network (TGN) was affected to a lesser degree. Consistently, knockdown of the ArfGEF gbf-1 altered the distribution of SGK-1 at the basolateral membrane of intestinal cells. In addition, we found that sgk-1(RNAi) induced unfolded protein response in the ER, suggesting at least an indirect role of SGK-1 early in the secretory pathway. We propose that SGK-1 function is required for lipid homeostasis and that it acts at different intracellular trafficking steps.     

Molecular Mechanism of Nicotine Degradation by a Newly Isolated Strain,Ochrobactrum sp. Strain SJY1

A newly isolated strain, SJY1, identified as Ochrobactrum sp., utilizes nicotine as a sole source of carbon, nitrogen, and energy. Strain SJY1 could efficiently degrade nicotine via a variant of the pyridine and pyrrolidine pathways (the VPP pathway), which highlights bacterial metabolic diversity in relation to nicotine degradation. A 97-kbp DNA fragment containing six nicotine degradation-related genes was obtained by gap closing from the genome sequence of strain SJY1. Three genes, designated vppB, vppD, and vppE, in the VPP pathway were cloned and heterologously expressed, and the related proteins were characterized. The vppB gene encodes a flavin-containing amine oxidase converting 6-hydroxynicotine to 6-hydroxy-N-methylmyosmine. Although VppB specifically catalyzes the dehydrogenation of 6-hydroxynicotine rather than nicotine, it shares higher amino acid sequence identity with nicotine oxidase (38%) from the pyrrolidine pathway than with its isoenzyme (6-hydroxy-l-nicotine oxidase, 24%) from the pyridine pathway. The vppD gene encodes an NADH-dependent flavin-containing monooxygenase, which catalyzes the hydroxylation of 6-hydroxy-3-succinoylpyridine to 2,5-dihydroxypyridine. VppD shows 62% amino acid sequence identity with the hydroxylase (HspB) from Pseudomonas putida strain S16, whereas the specific activity of VppD is ∼10-fold higher than that of HspB. VppE is responsible for the transformation of 2,5-dihydroxypyridine. Sequence alignment and phylogenetic analysis suggested that the VPP pathway, which evolved independently from nicotinic acid degradation, might have a closer relationship with the pyrrolidine pathway. The proteins and functional pathway identified here provide a sound basis for future studies aimed at a better understanding of molecular principles of nicotine degradation.