毛头鬼伞（Coprinus comatus）中一种碱性蛋白的纯化及其活性

用离子交换层析（CMsepharose FF）和凝胶层析（SuperdexTM75）方法,从新鲜食用菌毛头鬼伞（Coprinus comatus）子实体中分离纯化出一碱性蛋白y3,经SDSPAGE初步确定其分子量约为14.4kD。活性检测结果显示：当其浓度为12.5μg/mL时,对烟草花叶病毒（TMV）在心叶烟枯斑寄主上的侵染抑制率达83.0%;y3对兔血凝集活性滴度为2.5,对人血凝集活性滴度为26,其浓度分别为1.562μg/mL和0.781μg/mL;利用胃癌细胞株MGC803检测y3体外抗肿瘤活性,其IC50为12μg/mL。y3 N端序列为NRDVAACARFIDDFCDTLTP,为一新的蛋白序列。在SWISSPORT上登录号为P83477。     

榆黄蘑中一种抗病毒蛋白的纯化及其抗TMV和HBV的活性

采用阴离子交换层析和凝胶层析方法从新鲜食用菌榆黄蘑 (Plearotuscitrinopileatus)中进行了抗病毒蛋白的纯化 ,结果获得了一个纯化蛋白YP4 6 4 6 ,经SDS PAGE可确定其分子量为 2 7.4kD。以半叶法在枯斑寄主心叶烟上检测该蛋白对烟草花叶病毒 (TMV)的抑制率 ,发现有较好的抗TMV活性 ,其抑制TMV的中浓度为 0 .2 4 μg/mL。同时以HepG2 .2 .2 .15细胞株为模型 ,对所获得的蛋白进行体外抗乙型肝炎病毒效果的评价。结果表明该蛋白对HBsAg的 5 0 %抑制时的浓度为 0 .0 8μg/mL ,但对HBeAg效果不大     

榆黄蘑中一种抗病毒蛋白的纯化及其抗TMV和HBV的活性

采用阴离子交换层析和凝胶层析方法从新鲜食用菌榆黄蘑(Plearotus citrinopileatus)中进行了抗病毒蛋白的纯化,结果获得了一个纯化蛋白YP46-46,经SDS-PAGE可确定其分子量为27.4kD.以半叶法在枯斑寄主心叶烟上检测该蛋白对烟草花叶病毒(TMV)的抑制率,发现有较好的抗TMV活性,其抑制TMV的中浓度为0.24μg/mL.同时以HepG2.2.2.15细胞株为模型,对所获得的蛋白进行体外抗乙型肝炎病毒效果的评价.结果表明该蛋白对HBsAg的50%抑制时的浓度为0.08μg/mL,但对HBeAg效果不大.     

一种食用菌提取物y3对烟草花叶病毒的钝化作用及其机制

测定了从食用菌毛头鬼伞(Coprinus comatus)中提纯的y3蛋白对烟草花叶病毒(Tobacco mosaic virus,TMV)的钝化作用,结果表明y3对TMV有较强的体外钝化作用,在心叶烟枯斑寄主上对TMV的抑制中浓度约为2．0μg／mL;y3在pH9．0时较稳定;TMV与y3混合后刚RNase处理,测得侵染率为61．74％,比未用RNase处理的对照降低了38．26％,说明y3具有一定的体外脱衣壳作用;另外电镜观察发现y3可使部分TMV粒体发生裂解,变短。     

沱茶中抗自由基化学成分的研究

对沱茶(Camellia sinensis var．assamica)的水提取物、50％甲醇提取物、甲醇提取物进行了DPPH自由基清除作用的研究,实验结果表明,水提取物和50％甲醇提取物均具有较强的清除自由基作用,其半数抑制率(IC50)分别为6．07μg／mL和5．01μg／mL,而甲醇提取物清除自由基作用的活性则较弱,其半数抑制率(IC50)为39．63μg/mL。利用层析等手段分别从抗自由基活性较强的水提取物的甲醇可溶部分分离获得了20种化合物,其中,10种化合物(2、5、6、11、12、13、16、17、18、19)为首次从该植物中得到的化合物;DPPH自由基清除活性研究结果表明,其中化合物(1、4、7、9、10、12、14、18)的活性强于阳性对照品抗坏血酸和咖啡酸。比较分析化合物的结构与其活性的关系,发现结构中苯环上有邻位羟基的化合物其清除自由基的活性较强,而且羟基越多活性越强。     

球形孢子丝菌致婴儿固定型孢子丝菌病1例及相关实验研究

报道1例由球形孢子丝菌所致的婴儿固定型孢子丝菌病。患儿女,3个月,因左眼下内侧皮损2个月就诊,皮损脓液标本进行真菌培养,对培养获得菌株进行形态学、生理学和分子生物学鉴定,并进行药物敏感性检测。真菌培养阳性,镜下可见典型的套袖样菌丝。钙调蛋白基因序列分析鉴定为球形孢子丝菌。药敏试验显示特比萘芬和伊曲康唑对该菌株的菌丝相最低抑菌浓度（minimal inhibitorycon centration,MIC）分别为0．5μg／mL和0．5μg／mL;对该菌株的酵母相的MIC值分别为0．25μg／mL和0．5μg／mL。给予患者口服特比萘芬32．5mg／d治疗10周后皮损消退呈瘢痕化修复。依据临床及实验室检查确诊该病例为球形孢子丝菌所致固定型孢子丝菌病,特比萘芬治疗本病例显示较好疗效。     

截短型rhtBIGH3-(RGD)2蛋白通过上调miR-126 表达抑制 人脐静脉内皮细胞的生物活性

目的:探讨miR-126在截短型rhtBIGH3-(RGD)_2蛋白抑制HUVEC细胞生物学活性中的作用。方法:体外培养VEGF孵化的人脐静脉内皮细胞(VEGF-HUVEC),分别加入rhtBIGH3-(RGD)_2蛋白终浓度为0和100μg/mL,作用24、48、72 h条件下,分别检测Caspase-3活性和miR-126表达水平。在rhtBIGH3-(RGD)_2蛋白终浓度为0和100μg/mL时,分别加入miR-126 mimic和miR-126 inhibitor,作用VEGF-HUVEC 48 h后,Real-time PCR检测miR-126表达水平,通过检测Caspase-3活性来检测细胞凋亡情况。结果:在VEGF-HUVEC中,当rhtBIGH3-(RGD)_2蛋白终浓度为100μg/mL条件下,Caspase-3水平升高,miR-126表达水平升高,在48 h下达到最高峰。在VEGF-HUVEC中,当rhtBIGH3-(RGD)_2蛋白终浓度分别为100μg/mL条件下,加入miR-126mimic后,miR-126表达升高,Caspase-3水平升高;加入miR-126 inhibitor后,48 h后检测miR-126表达下降,Caspase-3水平也下降。结论:截短型rhtBIGH3-(RGD)_2蛋白通过上调miR-126表达从而促进细胞凋亡、抑制HUVEC生物活性,从而抑制角膜新生血管的发生     

C12-水解印楝素A的制备、结构鉴定及生物活性

C12-水解印楝素A是将印楝素A C12位上的-COOCH3水解为-COOH而得到的印楝素衍生物。处理后24h和48h,C12-水解印楝素A对斜纹夜蛾3龄幼虫AFC50分别为3．44μg／mL和6．89μg／mL。处理后48h,3μg／mL C12-水解印楝素A对小菜蛾3龄幼虫的拒食率为73．59％,5μg／mL C12-水解印楝素A对棉铃虫3龄幼虫的拒食率为67．76％。3μg／mL C12-水解印楝素A处理后72h,小菜蛾3龄幼虫的校正死亡率为78．16％;5μg／mL C12-水解印楝素A处理后72h,棉铃虫3龄幼虫的校正死亡率为58．69％。     

氧化低密度脂蛋白通过Wnt5a/PKCδ信号通路诱导大鼠肝星状细胞自噬

该文旨在研究氧化低密度脂蛋白(ox-LDL)对大鼠肝星状细胞(HSC-T6)自噬的影响及机制,探讨非酒精性脂肪肝炎的发病机理。体外培养的HSC-T6细胞以不同质量浓度(0、10、20、40、60μg/mL)的ox-LDL分别处理不同时间(0、3、6、12、24 h)后,用Western blot检测LC3 II、Beclin1、p62的含量。不同质量浓度(0、10、20、40、60μg/mL)的ox-LDL处理HSC-T6细胞12 h后,Western blot检测Wnt5a、p-PKCδ、p-STAT3的含量。将HSC-T6细胞分为对照组(Control)、ox-LDL组、ox-LDL+si-NC组和ox-LDL+si-Wnt5a组,经相应处理后用Western blot、qRT-PCR分别检测LC3 II、Beclin1、p62、p-PKCδ、p-STAT3的蛋白和mRNA含量变化;免疫荧光检测LC3 II的变化;油红O染色观察HSC-T6脂滴含量变化;比色法检测各组细胞培养上清中羟脯氨酸(Hyp)含量;ELISA检测细胞培养上清中透明质酸(HA)和层黏连蛋白(LN)含量。PKCδ抑制剂Rottlerin预处理细胞:将HSC-T6细胞分为对照组(Control)、ox-LDL组、ox-LDL+DMSO组和ox-LDL+Rottlerin组,检测方法与敲低Wnt5a一致。经ox-LDL处理后,HSC-T6细胞中LC3 II、Beclin1含量增加(P<0.05),p62含量减少(P<0.01),且在ox-LDL质量浓度为20μg/mL、作用12 h时达到峰值。ox-LDL质量浓度为20μg/mL、作用12 h时,HSC-T6细胞中Wnt5a、p-PKCδ、p-STAT3蛋白表达显著升高(P<0.01)。敲低Wnt5a后,HSC-T6细胞中Wnt5a、LC3 II、Beclin1 mRNA和蛋白表达水平显著降低(P<0.001),p62蛋白表达增多(P<0.01),p-PKCδ、p-STAT3蛋白表达减少(P<0.05),细胞内LC3 II点状聚集减少,脂滴含量减少,细胞培养上清中Hyp、HA、LN含量也减少(P<0.05)。抑制PKCδ后,结果与敲低Wnt5a一致。ox-LDL可通过增强Wnt5a/PKCδ通路诱导HSC-T6细胞自噬。     

蓖麻籽提取物抗雌鼠生育活性成分体外筛选方法的建立

用不同剂量蓖麻籽乙醚提取物处理原代培养大鼠蜕膜细胞和黄体细胞,观察细胞形态变化,同时用MTT法检测细胞活力,探讨蓖麻籽提取物抗雌鼠生育活性成分的作用靶标,从而建立其有效成分的体外筛选方法。结果表明：400μg／mL蓖麻籽提取物处理液对体外培养大鼠蜕膜细胞生长有明显的抑制作用（P〈0．05）;提取物对蜕膜细胞的半抑制浓度（IC50）为568．6±5．3μg／mL,r=0．9790;其抑制作用具有较好的量-效关系。而提取物对黄体细胞活力的抑制作用不显著。结论：用大鼠离体培养蜕膜细胞的活力测定方法,作为蓖麻籽乙醚提取物抗雌鼠有效成分分离、提纯的活性跟踪指标是可行的。     

Purification and characterization of an antibacterial protein in the skin secretion of rockfish Sebastes schlegeli

An antibacterial protein in the skin secretion of rockfish (Sebastes schlegeli) was purified by lectin affinity chromatography on Con A-Sepharose and gel filtration on TSKgel G3000SW. The antibacterial protein featured the high molecular mass and selective action against Gram-negative bacteria. The molecular mass of the protein was estimated to be approximately 150 kDa in gel filtration and approximately 75 kDa by SDS-PAGE, suggesting that it is dimeric. The antibacterial principle was an acidic glycoprotein with pI 4.5, 3.4% reducing sugar and 2.8% amino sugar. Its sugar chains had N-type (high mannose-type) oligosaccharide and sialic acid components. It inhibited strongly the growth of Aeromonas salmonicida, Photobacterium damselae and Shewanella putrefaciens with a minimum inhibitory concentration (MIC) of approximately 3 microg/ml, and moderately the growth of Vibrio parahaemolyticus and A. hydrophila with a MIC of 12.5 microg/ml and 25 microg/ml, respectively. The values of the minimum bactericidal concentration were almost equivalent to those of MIC. The potent sensitivity against virulent pathogens such as A. hydrophila, A. salmonicida and P. damselae may contribute considerably to the innate host defense mechanism to combat microbes on the mucosal surfaces of the rockfish.     

Histone H1: an antimicrobial protein of Atlantic salmon (Salmo salar)

Antimicrobial activity was detected in acid extracts of liver, intestine, and stomach of healthy Atlantic salmon (Salmo salar). An antimicrobial protein was isolated from salmon liver using acid extraction followed by ammonium sulfate precipitation, large-scale gel filtration chromatography, reverse-phase HPLC, and size exclusion HPLC. The salmon antimicrobial (SAM) protein was found to have a molecular mass of 20,734 Da by MALDI TOF mass spectrometry. Peptide mass fingerprinting and partial sequencing by tandem nanoelectrospray mass spectrometry identified the protein as histone H1. The protein had a minimal inhibitory concentration of 31 microg/mL against E. coli D31 in a plate clearing assay. The effect of the SAM protein on bacterial morphology was indistinguishable from that of (Ala-(8,13,18))-magainin II, as shown by scanning electron microscopy, which suggests that the protein disrupts E. coli membranes in a manner similar to that of most antimicrobial peptides. This protein may act as an antimicrobial in vivo through active secretion or by release from cells during infection-related apoptosis.     

Afamin is a novel human vitamin E-binding glycoprotein characterization and in vitro expression

Hydrophobic vitamins are transported in human plasma and extravascular fluids by carrier proteins. No specific protein has been described so far for vitamin E, which plays a crucial role in protecting against oxidative damage and disease. We report here the purification of a 75-kDa glycoprotein with vitamin E-binding properties by stepwise chromatography of lipoprotein-depleted human plasma and monitoring of vitamin E (alpha-tocopherol)-binding activity. Partial sequencing identified this protein as afamin, a previously described member of the albumin gene family with four or five potential N-glycosylation sites. Glycosylation analysis indicated that >90% of the glycans were sialylated biantennary complex structures. The vitamin E-binding properties were confirmed using recombinantly expressed afamin. Qualitative and quantitative analysis of plasma and extravascular fluids revealed an abundant presence of this protein not only in plasma (59.8+/-13.3 microg/mL) but also in extravascular fluids such as follicular (34.4+/-12.7 microg/mL) and cerebrospinal (0.28+/-0.16 microg/mL) fluids, suggesting potential roles for afamin in fertility and neuroprotection. Afamin is partly (13%) bound to plasma lipoproteins. Afamin and vitamin E concentrations significantly correlate in follicular and cerebrospinal fluids but not in plasma. The vitamin E association of afamin in follicular fluid was directly demonstrated by gel filtration chromatography and immunoprecipitation which complements the in vitro findings for purified native and recombinant afamin.     

Inhibition of human P450 enzymes by multiple constituents of the Ginkgo biloba extract

The Ginkgo biloba extract EGb761 was tested for its ability to inhibit the major human cytochrome P450 enzymes (CYPs). The full extract was found to strongly inhibit CYP2C9 (Ki = 14+/- 4 microg/mL), and to a lesser extent, CYP1A2 (Ki = 106 +/- 24 microg/mL), CYP2E1 (Ki = 127 +/- 42 microg/mL), and CYP3A4 (Ki = 155 +/- 43 microg/mL). The terpenoidic and flavonoidic fractions of the extract were tested separately against the same P450s to identify the source of inhibition by EGb761. The terpenoidic fraction inhibited only CYP2C9 (Ki = 15 +/-6 microg/mL) whereas the flavonoidic fraction of EGb761 showed high inhibition of CYP2C9, CYP1A2, CYP2E1, and CYP3A4 (Ki's between 4.9 and 55 microg/mL). The flavonoidic fraction was further fractionated using extraction and chromatography. Inhibition studies indicated that the majority of these fractions inhibited P450s at a significant level (IC50 < 40 microg/mL).     

Synthesis, anti-tuberculosis activity, and 3D-QSAR study of 4-(adamantan-1-yl)-2-substituted quinolines

Structural optimization of the previously identified 4-(adamantan-1-yl)-2-quinolinecarbohydrazide (AQCH, MIC=6.25 microg/mL, 99% inhibition, Mycobacterium tuberculosis H37Rv) has led to two series of 4-(adamantan-1-yl)-2-substituted quinolines (Series 1-2). All new derivatives were evaluated in vitro for antimycobacterial activities against drug-sensitive M. tuberculosis H37Rv strain. Several 4-adamantan-1-yl-quinoline-2-carboxylic acid N'-alkylhydrazides (Series 1) described herein showed promising inhibitory activity. In particular, analogs 7, 9, 20, and 21 displayed MIC of 3.125 microg/mL. Further investigation of AQCH by its reaction with various aliphatic, aromatic, and heteroaromatic aldehydes led to the synthesis of 4-adamantan-1-yl-quinoline-2-carboxylic acid alkylidene hydrazides (Series 2). Analogs 42-44 and 48 have produced promising antimycobacterial activities (99% inhibition) at 3.125 microg/mL against drug-sensitive M. tuberculosis H37Rv strain. The most potent analog 35 of the series produced 99% inhibition at 1.00 microg/mL against drug-sensitive strain, and MIC of 3.125 microg/mL against isoniazid-resistant TB strain. To understand the relationship between structure and activity, a 3D-QSAR analysis has been carried out by three methods-comparative molecular field analysis (CoMFA), CoMFA with inclusion of a hydropathy field (HINT), and comparative molecular similarity indices analysis (CoMSIA). Several statistically significant CoMFA, CoMFA with HINT, and CoMSIA models were generated. Prediction of the activity of a test set of molecules was the best for the CoMFA model generated with database alignment. Based on the CoMFA contours, we have tried to explain the structure-activity relationships of the compounds reported herein.     

Chloropyrimidines as a new class of antimicrobial agents

In the course of our investigations of pyrimidines as antimycotic agents, we have identified a sub-class, with significant in vitro activity against mycobacteria. The salient feature of these pyrimidine derivatives (3a-o and 7a,b) is their appended aryl, heteroaryl and alkylthio substituent at position 6 and also alkylthio substituent at position 2. The rational design, synthesis, and evaluation of the in vitro antibacterial activity against six pathogenic bacteria including virulent and non-virulent strains of Mycobacterium tuberculosis is described. Some of the synthesized compounds (3c, 3h, 3i, 3o) have displayed only potent in vitro antimycobacterial activity with MIC of 0.75 microg/mL except 3i which also demonstrated activity against Escherichia coli at 12.5 microg/mL concentration. Only two compounds, 3a and 3b, demonstrated antibacterial activity against Pseudomonas aeruginosa and E. coli with MIC 12.5 microg/mL. All the synthesized compounds were also evaluated for their antimycotic activity against five pathogenic fungi but only some of them 3j-n and 7a,b were found most potent against Aspergillus fumigatus and Trichophyton mentagrophytes.     

Oligomerization of hydrophobin SC3 in solution: from soluble state to self-assembly

Hydrophobin SC3 is a protein with special self-association properties that differ depending on whether it is in solution, on an air/water interface or on a solid surface. Its self-association on an air/water interface and solid surface have been extensively characterized. The current study focuses on its self-association in water because this is the starting point for the other two association processes. Size-exclusion chromatography was used to fractionate soluble-state SC3. Real-time multiangular light scattering detection of the eluate indicated that SC3 mainly exists as a dimer in buffer, accompanied with a small amount of monomer, tetramer, and larger aggregates. Dimeric SC3 has very likely an elongated shape, as indicated by the hydrodynamic radius determined by using dynamic light scattering (DLS) and fluorescence anisotropy measurements on dansyl-labeled SC3. Size-exclusion chromatography experiments also indicated that the protein oligomerizes very slowly at low temperature (4 degrees C) but rather rapidly at room temperature. Ionic strength plays an important role in the oligomerization; a short-lived monomeric SC3 species could be observed in pure water. Oligomerization was not affected by low pH but was accelerated by high pH. Fluorescence resonance energy transfer showed that dissociation occurred when the protein concentration was lowered; a large population of oligomers, presumably dimers, dissociate when the protein concentration is <4.5 microg/mL. This value is similar to the critical concentration for SC3 self-assembly. Therefore, dimeric SC3 is indicated to be the building block for both aggregation in solution and self-assembly at hydrophobic/hydrophilic interfaces.     

Synthesis and antibacterial activity of C2-fluoro, C6-carbamate ketolides, and their C9-oximes

Novel C6-carbamate ketolides with C2-fluorination and C9-oximation have been synthesized. The best compounds in this series displayed MIC values of 0.03-0.12 microg/mL against streptococci containing erm and mef resistance determinants and 2-4 microg/mL against Haemophilus influenzae. Several compounds also showed measurable activity against erm(B)-containing enterococci with MIC values of 2-8 microg/mL. In vivo activity was adversely affected by fluorination, possibly as a result of increased serum protein binding.     

High-performance liquid chromatography with ultraviolet detection for real-time therapeutic drug monitoring of meropenem in plasma

A simple, rapid, and precise high-performance liquid chromatography (HPLC) method using ultrafiltration to remove plasma protein was developed to determine meropenem concentrations in human plasma in a clinical setting. Plasma was separated by centrifugation at 4 degrees C from blood collected in heparinized vacuum tubes, and meropenem was stabilized by immediately mixing the plasma with 1M 3-morpholinopropanesulfonic acid buffer (pH 7.0) (1:1). Ultrafiltration was used for plasma deproteinization. Meropenem was detected by ultraviolet absorbance at 300 nm with no interfering plasma peak. The calibration curve of meropenem in human plasma was linear from 0.05 to 100 microg/mL. Intraday and interday precision was less than 7.17% (CV), and accuracy was between 97.7% and 106.3% over 0.05 microg/mL. The limit of detection was 0.01 microg/mL. The assay has been clinically applied to a real-time therapeutic drug monitoring in pediatric patients and pharmacokinetic studies.     

Synthesis and biological properties of new 5-nitroindazole derivatives

A series of new 3-alkoxy- or 3-hydroxy-1-[omega-(dialkylamino)alkyl]-5-nitroindazoles have been synthesized and their trichomonacidal, antichagasic and antineoplastic properties studied. Five derivatives (5, 6, 8, 9 and 17) showed remarkable trichomonacidal activity against Trichomonas vaginalis at 10 microg/mL concentration. Three compounds (8, 10, 11) exhibited interesting antichagasic activity and these same compounds moderate antineoplastic activity against TK-10 and HT-29 cell lines. Unspecific cytotoxicity against macrophages has also been evaluated and only compounds 9, 10 and 11 resulted cytotoxic at the higher dose evaluated (100 microg/mL), loosing cytotoxicity at lower doses. QSAR studies have been carried out. X-ray crystallographic study of compound 8 has been performed.