基于HaCaT细胞建立化妆品刺激性分级评价细胞模型.选取化妆品成品及原料,将样品作用HaCaT细胞一定时间,以生理盐水作对照,用MTT法测定相对细胞活力,根据相对细胞活力结果对样品进行刺激性分级评价.选取了 9 种化妆品成品建立了刺激性评价细胞模型,并基于该模型评价了 8 种化妆品成品、2 种化妆品原料的刺激性.结论显示基于HaCaT细胞建立的化妆品刺激性分级评价细胞模型,可实现对化妆品刺激性快速、高效的评价.

Organic anion transporting polypeptide 1B1 (OATP1B1) is specifically expressed at the basolateral membrane of human hepatocytes and plays important roles in the uptake of various endogenous and exogenous compounds including many drugs. The proper functioning of OATP1B1, hence, is essential for the bioavailability of various therapeutic agents and needs to be tightly regulated. Dileucine-based signals are involved in lysosomal targeting, internalization, and trans-Golgi network to endosome transporting of membrane proteins. In the current study, we analyzed the 3 intracellular and 13 transmembrane dileucine motifs (DLMs) within the sequence of OATP1B1. It was found that the simultaneous replacement of I332 and L333 with alanine resulted in a significantly reduced level of the mature form of OATP1B1. The cell surface expression of I332A/L333A could be partially rescued by MG132, as well as agents that prevent clathrin-dependent protein internalization, suggesting that this dileucine motif may be involved in the endocytosis of OATP1B1. On the other hand, I376/L377 and I642/L643, which are localized at transmembrane helices (TM) 8 and 12, respectively, are involved in the interaction of the transporter with its substrates. I642A/L643A exhibited a significantly decreased protein level compared to that of the wild-type, implying that the motif is important for maintaining the stability of OATP1B1 as well.

Abstract The authors have requested that this preprint be removed from Research Square.

目的 研究乳源五肽脯氨酸-甘氨酸-脯氨酸-异亮氨酸-脯氨酸(PGPIP)缓解小鼠慢性酒精性肝损伤及其相关的分子机制.方法 40只C57BL/6小鼠,随机均分为对照组、模型组、谷胱甘肽(GSH)组、PGPIP组.采用10 d Lieber-Decarli酒精饲料喂养加1次急性酒精灌胃的方法建立小鼠慢性酒精性脂肪肝模型,造模的同时给予药物干预.根据已有的基因表达综合(GE O)数据库中RN A测序数据,采用聚类热图对肝脏内质网应激相关基因差异表达分析.肝脏苏木素-伊红(HE)染色分析各处理组对小鼠酒精性肝损伤的病理学影响,油红O染色分析各处理组对小鼠肝脏脂滴积累的影响.透射电子显微镜分析酒精代谢引起的脂滴积累对肝细胞显微结构的影响并验证PGPIP的作用效果.蛋白质免疫印迹法(Western blot)检测内质网应激相关信号转导蛋白表达变化.结果 PGPIP组病理学检查类似正常对照组,小鼠肝脏损伤明显减轻.模型组小鼠肝脏中有明显的脂滴积累,表现为大小不同程度的混合型脂滴,PGPIP处理后明显减少酒精诱导的肝脏脂滴积累.PGPIP对PERK-eIF2α-ATF4通路有较大影响,且对转录激活因子6(ATF6)和Cleaved Caspase 3蛋白的表达有显著性的效果.结论 五肽PGPIP能缓解小鼠慢性酒精性脂肪肝和肝损伤,其机制可能是通过减少肝细胞中的脂滴积累,并且减少内质网应激和肝细胞凋亡从而发挥作用.

目的 研究乳源六肽脯氨酸-甘氨酸-脯氨酸-异亮氨酸-脯氨酸-天冬酰胺(PGPIPN)和其截短五肽脯氨酸-甘氨酸-脯氨酸-异亮氨酸-脯氨酸(PGPIP)缓解小鼠慢性酒精性肝损伤及其相关的分子机制.方法 60只昆明小鼠,随机均分为对照组、模型组、谷胱甘肽(GSH)组、PG-PIPN组、截短五肽PGPIP组.采用梯度酒精灌胃的方法建立小鼠慢性酒精性肝损伤模型,造模的同时给予药物干预,共12周.肝脏HE染色分析各处理组对小鼠酒精性肝损伤的病理学影响.体外分离培养小鼠原代肝细胞和人正常肝细胞系L-02,水溶性四氮唑-1(WST-1)细胞增殖及细胞毒性检测确定各种细胞合适的PGPIPN诱导浓度.持续诱导L-02细胞不同时间,Western blot检测人叉头框蛋白O3(FoxO3a)和磷酸化FoxO3a蛋白质的表达,确定合适的诱导时间.免疫荧光染色检测FoxO3a在L-02细胞中的亚细胞定位.实时荧光定量PCR(qRT-PCR)检测不同处理组小鼠原代肝细胞和L-02细胞FoxO3a和锰超氧化物歧化酶(Mn-SOD)基因mRNA的变化.结果 PGPIPN组和PGPIP组病理学检查类似GSH组,小鼠肝脏损伤明显减轻.分别选择中浓度和高浓度PGPIPN诱导小鼠原代肝细胞和L-02细胞.在16 h,L-02细胞FoxO3a蛋白表达显著增加,并且FoxO3a蛋白主要表达于细胞核内.此外,在相应剂量PGPIPN诱导后,两种类型细胞中mRNA水平的显著增加.结论 PG-PIPN和截短五肽PGPIP能减少小鼠慢性酒精性肝损伤,其机制可能是通过FoxO3a-MnSOD信号通路,减少酒精诱导的氧化应激从而发挥作用.

本文以多糖含量丰富的铁皮石斛、细茎石斛和鼓槌石斛为对象,探讨了石斛多糖对秀丽隐杆线虫寿命、产卵量、运动行为能力、热应激能力等的影响.结果表明:3种石斛多糖在不影响秀丽隐杆线虫正常生殖能力的条件下显著延长了线虫的平均寿命,80 mg/L的细茎石斛多糖处理的线虫平均寿命较对照组增加了33.3％;不同浓度的石斛多糖能明显提升线虫的运动能力,其头部摆动频率、身体弯曲频率、热应激能力均随着多糖浓度的升高而增加.该研究结果为阐述石斛多糖延缓衰老的作用机理和更好地开发利用石斛提供了参考.

N-glycosylation plays critical roles in protein secretion, sorting, stability, activity modulation, and interactions to other molecules in the eukaryotic organisms. Fungal β-1,4-mannanases have been widely used in the agri-food industry and contribute to the pathogenesis on plants. However, the information on N-glycosylation of a specific fungal carbohydrate-active enzyme (CAZyme) is currently limited. Herein, a cDNA was cloned from Aspergillus aculeatus QH1, displaying a full length of 1302 bp with an open reading frame of 1134 bp encoding for a GH5 subfamily 7 β-1, 4-mannanase, namely AacMan5_7A. The enzyme was purified and exhibited an optimal activity at pH 4.6 and 60 °C, hydrolyzing glucomannan and galactomannan, but not yeast mannan. AacMan5_7A is an N-glycosylated protein decorated with a high-mannose type glycan. Further through UPLC-ESI-MS/MS analysis, one of the four predicted N-glycosylation sites at N255 position was experimentally verified. The present study expands the information of N-glycosylation in fungal CAZymes, providing scientific bases for enhancing the production of fungal enzymes and their applications in food, feed, and plant biomass conversions.

Organic anion transporting polypeptides (OATPs, gene symbol SLCO) mediate sodium-independent transport of endogenous compounds such as bile salts, hormones and their conjugates as well as toxins and drugs. OATP1B1 is the major OATP specifically expressed at the basolateral membrane of human hepatocytes and many clinically important drugs have been shown to be substrates of the transporter. According to the computer-based hydropathy analysis, a large intracellular loop 3 (IL3) is situated between transmembrane domain 6 and 7 of OATPs, in which a conserved NPxY motif is found. In the current study, HEK293 cells expressing the HA-tagged OATP1B1 was utilized to investigate the role of the NPxY motif for the function and expression of the transporter. Alanine replacement of N335 or P336 retained substantial uptake function; while simultaneous mutation of these residues resulted in a double mutant that lost almost all the transport activity. On the other hand, Y338A showed >80% reduction for estrone-3-sulfate uptake. Plasma membrane protein analysis revealed that N335/P336A completely lost its cell surface protein expression; while that of Y338A is dramatically reduced. Further investigation with pharmacological inhibitors and immunocytochemistry demonstrated that N335/336A is detained in the Golgi apparatus and Y338A exhibited accelerated protein degradation rate compared to that of the wild-type. Conservative replacement of Y338 with phenylalanine fully recovered uptake and expression of the transporter. In summary, a new role was observed for the NPxY motif located in the IL3 of OATP1B1, which may affect processing and stability of the transporter.

Organic anion transporting polypeptides (OATPs, gene symbol SLCO) are important membrane transporter proteins that mediate the uptake of wide ranges of endogenous and exogenous compounds. OATP2B1 has been found in multiple organs and tissues, including the liver, small intestine, kidney, brain, placenta, heart, skin, as well as skeletal muscle, and is proposed to be involved in the uptake of orally administered drugs. Quite a few reports have demonstrated that transmembrane domains (TMs) are crucial for proper functions of OATP family members. Comparative modeling proposed that TM1, along with TM2, 4, and 5 of the N-terminal half of OATP2B1, may be localized within the substrate interaction pocket and are important for uptake function of the transporter. Alanine scanning of the putative transmembrane domain 1 of OATP2B1 revealed that substitution of L58 with alanine dramatically altered the Km value, and mutation of V52, H55, Q59, and L69 resulted in significantly reduced substrate turnover number, whereas A61V, Q62A, and S66A exhibited significant change in both Km and Vmax values. In addition, phenylalanine at position 51 seems to play an important role in maintaining proper folding of OATP2B1 because alanine replacement of F51 caused accelerated degradation of the transporter protein. Although proteasome and lysosome inhibitors could partially recover protein level, the mutant transporter remained nonfunctional. Taken together, the identification of nine essential amino acid residues within TM1 of OATP2B1 suggested that the transmembrane domain is important for maintaining proper function of the transporter.

酶工程是现代生物技术四大核心工程之一,作为生物技术专业的主干课程,在学生的专业知识构建中具有重要的地位.本文介绍了华南农业大学在酶工程课程教学内容的优化,教学方法和教学手段的改进,考核评价体系的完善等方面进行的探索与实践,旨在进一步提高酶工程教学质量,满足社会对生物技术专业人才培养的要求.

Organic anion-transporting polypeptides play important roles in the uptake of various endogenous and exogenous compounds. It has been proposed that OATP family members, as membrane proteins, may form oligomers. However, oligomerization status of OATPs is still largely unclear. In the present study, HEK293 cells stably expressing OATP1B1 were generated to investigate the oligomerization status of the transporter. Chemical cross-linking and coimmunoprecipitation experiments revealed that OATP1B1 may form homo-oligomers, possibly through disulfide bonds. When wild-type OATP1B1 was coexpressed with a loss-of-function mutant W258A, cells showed reduced uptake of prototypic substrate estrone-3-sulfate (ES). Interestingly, such a coexpression did not affect OATP1B1 transport activity of high concentrations ES, implicating that oligomerization status may affect only the high affinity component of ES. OATP1B1 possesses three GXXXG motifs that have been associated with protein dimerization in other membrane proteins. When glycine residues were replaced with alanine, G219A and G393A showed drastically reduced uptake function. Further studies revealed that G219A has a similar association capability to that of the wild-type, while mutation at Gly393 may affect oligomerization status of the transporter. Kinetic analysis showed that both G219A and G393A have a dramatically reduced Vmax for ES uptake. Km of G219A was increased while that of G393A exhibited a decreased value for high affinity component of ES binding. Our studies demonstrated that OATP1B1 may function as oligomers in the high affinity site of ES while acting as monomers for the low affinity binding component of the substrate.

本科教学是普通高等学校办学的生命线，课程考核在本科教学过程中具有考核评定、质量区分、教学预测和反馈、激励导向等多种功能。本文以华南农业大学2013年开始实施的本科课程考核管理新规定为对象，对该规定实施的积极意义、遭遇的困境进行分析，并提出相应的建议，以期为高等学校本科课程改革提供参考。

Organic anion transporting polypeptides (OATPs, gene symbol SLCO) are membrane proteins that mediate the sodium-independent transport of a wide range of endogenous and exogenous compounds. Due to their broad substrate specificity, wide tissue distribution, and involvement in drug-drug interactions, OATPs have been considered as key players in drug absorption, distribution, and excretion. Transmembrane domains (TMs) are crucial structural features involved in proper functions of many transporters. According to computer-based modeling and previous studies of our laboratory and others, TM11 of OATP1B1 may face the substrate interaction pocket and thus play an important role in the transport function of the protein. Alanine-scanning of the transmembrane domain identified seven critical amino acid residues within the region. Further analysis revealed that alanine substitution of these residues resulted in reduced protein stability, which led to significantly decreased protein expression on the plasma membrane. In addition, all mutants exhibited an altered Km for ES uptake (either high affinity or low affinity component, or both), though Km for taurocholate transport only changed in R580A, G584A, and F591A. These results suggested that critical residues in TM11 not only affect protein stability of the transporter, but its interaction with substrates as well. The identification of seven essential residues out of 21 TM amino acids highlighted the importance of this transmembrane domain in the proper function of OATP1B1.

Organic anion-transporting polypeptides are members of the solute carrier (SLC) family and key determinants for the transmembrane transport of a wide variety of compounds. OATP1B1 is predominantly expressed at the basolateral membrane of human hepatocytes and play an important role in drug clearance from the body. It has been demonstrated to be responsible for the hepatic uptake of various drugs. Computer-based hydropathy analysis predicted several putative phosphorylation sites at the amino and carboxyl termini and at intracellular loop 3 of OATP family members. Therefore, their transport functions may be regulated by phosphorylation. Previous studies have demonstrated that uptake function of OATP2B1 and OATP1A2 is regulated by protein kinase C (PKC). In the present study, we treated HEK293 cells stably expressing OATP1B1 with different PKC modulators and measured their transport activity for prototypic substrate estrone-3-sulfate. It was found that OATP1B1 uptake function was reduced upon PKC activation. Further studies indicated that PKC may affect OATP1B1 activity through regulation of the cell surface protein level. Moreover, we found out that PKC activator phorbol 12-myristate 13-acetate (PMA) not only affects the internalization of OATP1B1 but its recycling as well. Immunocytochemistry analysis revealed that internalized OATP1B1 co-localized with early and recycling endosomal markers and the co-localization of OATP1B1 with recycling endosome is dependent on PKC activation. Taken together, our present study demonstrated that PKC regulates the function of OATP1B1 by affecting internalization and recycling of the transporter protein.

文章通过分析生物类实验教学的特点，探讨了导生制教学模式在生物类实验教学中的应用，并对导生的选拔、培训、课堂实施以及实施导生制对实验教学的积极意义进行了综述。

The development of the Experimental Teaching Center on Modern Biotechnology of South China Agricultural University is closely concentrated on the talent demand and the regional planning for biotechnology on Pearl River Delta. Som certain measures have been taken to reform from the experimental teaching system and contents,teaching model and methods,the allocation of experimental teaching resources,the open sharing mechanism and so on,which has been quitely contributed to build an excellent experimental teaching platform,furtherly to improve and perfect the experimental teaching mode and talent training mechanism. The development experience of the Experimental Teaching Center on Modern Biotechnology would have a helpful demonstration effect on experiment teaching of modern biotechnology.

Objective: Organic anion transporting polypeptides (OATPs) are a family of important uptake transporters. It was demonstrated previously that OATP uptake function is regulated by protein kinase C (PKC). However, the phosphorylation site(s) modified involved in PKC regulation remains unknown. Methods: HEK293 cells stably expressing organic anion transporting polypeptides 1B1 (OATP1B1) were treated by PKC modulators to investigate its regulatory effect on OATP1B1. In addition, site‐directed mutagenesis was used to identify the possible PKC regulation sites within OATP1B1. Results: We found out that OATP1B1 uptake function is regulated by PKC modulators. Further studies indicated that PKC may affect OATP1B1 activity through its regulation of the protein expression on the cell surface. Moreover, we found out that PKC activator PMA not only affect the internalization of OATP1B1 but its recycling as well. Mutagenesis studies revealed that several amino acids located at intracellular loops and the carboxyl terminus of OATP1B1 are involved in the phosphorylation regulation process. Conclusions: OATP1B1 transport function is regulated by PKC, the action of which leads to accelerated internalization and reduced recycling of the transporter protein.Grant Funding Source: Supported by National Natural Science Foundation of China Grant #81373473 and #U1332124

Because of their wide distribution and capability of transporting a large variety of compounds, organic anion-transporting polypeptides (OATPs) have been extensively recognized as crucial players in absorption, distribution, and excretion of various drugs. OATP1A2 was the first cloned human OATP and has been found to transport wide range of endogenous and exogenous compounds. Bovine Oatp1a2 (bOatp1a2) shares high homology with human OATP1A2 and is considered the functional ortholog of the latter. Previous study in our laboratory demonstrated that bOatp1a2 transport of estrone-3-sulfate (ES) exhibited biphasic saturation kinetics. In the present study, we investigated the transport function of bOatp1a2 for four different quinolone antibacterial agents (enrofloxacin, levofloxacin, norfloxacin, and ciprofloxacin) and found that all the tested fluoroquinolones can be transported by bOatp1a2. Further studies showed that different binding sites are responsible for the transport of different fluoroquinolones. Both ciprofloxacin and norfloxacin exhibited biphasic saturation kinetics. The K(m)s of the high-and low-affinity components for ciprofloxacin were 3.80 +/- 0.85 mu M and 182 +/- 31 mu M, respectively, while those for norfloxacin were 24.7 +/- 0.1 mu M and 393 +/- 79 mu M, respectively. Enrofloxacin and levofloxacin showed an inhibitory effect on the uptake of only the high concentration of ES and thus may be transported by the low-affinity site for ES. Interestingly, enrofloxacin and levofloxacin demonstrated an activation effect on ES uptake at the high-affinity binding site. These results suggested that multiple binding sites within the structure of bOatp1a2 may be responsible for the uptake of different quinolone antimicrobial agents.