Profiling of membrane protein variants in a baculovirus system by coupling cell-surface detection with small-scale parallel expression

Production of structure-grade mammalian membrane proteins in substantial quantities has been hindered by a lack of methods for effectively profiling multiple constructs expression in higher eukaryotic systems such as insect or mammalian cells. To address this problem, a specialized small-scale eukaryotic expression platform by Thomson Instrument Company (Vertiga-IM) was developed and used in tandem with a Guava EasyCyte microcapillary 96-well cytometer to monitor cell density and health and evaluate membrane protein expression. Two proof of concept experiments were conducted using the human beta(2)-adrenergic receptor (beta(2)AR) and the gap junction protein connexin26 (Cx26) in a baculovirus expression system. First, cell surface expression was used to assess the expression levels of 14 beta(2)AR truncation variants expressed using the Vertiga-IM shaker. Three of these variants were then compared to wild-type beta(2)AR using three metrics: cell surface expression, saturation ligand binding and protein immunoblot analysis of dodecylmaltoside extracted material. Second, a series of systematic Cx26 truncation variants were evaluated for expression by protein immunoblot analysis. The cumulative results for these two systems show that the Vertiga-IM instrument can be used effectively in the parallel insect cell microexpression of membrane protein variants, and that the expression of cell surface molecules as monitored with the Guava EasyCyte instrument can be used to rapidly assess the production of properly folded proteins in the baculovirus expression system. This approach expedites the in vitro evaluation of a large number of mammalian membrane protein variants.     

Infection of bovine oviduct cell cultures with Chlamydophila abortus

Bovine infertility is a major cause of loss in the livestock industry. In the present study bovine oviduct cell cultures were infected with a Chlamydophila abortus strain. A direct evaluation of infection was performed by means of May Grünwald-Giemsa and immunocytochemistry for chlamydial LPS, which revealed inclusion bodies and vacuolisation. SEM and TEM analysis of infected cells showed various degrees of cell damage and conglutination of microvilli. This finding suggests that cattle infertility may result from an alteration of oviduct environment caused by multiplication of C. abortus. This microorganism, among other infectious agents, could be considered a potential causative agent of bovine infertility.     

Prediction of protein secondary structure content for the twilight zone sequences

Secondary protein structure carries information about local structural arrangements, which include three major conformations: alpha-helices, beta-strands, and coils. Significant majority of successful methods for prediction of the secondary structure is based on multiple sequence alignment. However, multiple alignment fails to provide accurate results when a sequence comes from the twilight zone, that is, it is characterized by low (<30%) homology. To this end, we propose a novel method for prediction of secondary structure content through comprehensive sequence representation, called PSSC-core. The method uses a multiple linear regression model and introduces a comprehensive feature-based sequence representation to predict amount of helices and strands for sequences from the twilight zone. The PSSC-core method was tested and compared with two other state-of-the-art prediction methods on a set of 2187 twilight zone sequences. The results indicate that our method provides better predictions for both helix and strand content. The PSSC-core is shown to provide statistically significantly better results when compared with the competing methods, reducing the prediction error by 5-7% for helix and 7-9% for strand content predictions. The proposed feature-based sequence representation uses a comprehensive set of physicochemical properties that are custom-designed for each of the helix and strand content predictions. It includes composition and composition moment vectors, frequency of tetra-peptides associated with helical and strand conformations, various property-based groups like exchange groups, chemical groups of the side chains and hydrophobic group, auto-correlations based on hydrophobicity, side-chain masses, hydropathy, and conformational patterns for beta-sheets. The PSSC-core method provides an alternative for predicting the secondary structure content that can be used to validate and constrain results of other structure prediction methods. At the same time, it also provides useful insight into design of successful protein sequence representations that can be used in developing new methods related to prediction of different aspects of the secondary protein structure.     

Induction of growth inhibition and differentiation of human leukemia HL-60 cells by a Tunisian gerboui olive leaf extract

Cancer protection associated with the consumption of olive products is well established, but not for leukemia. The protective effects of olive (Olea europaea L.) leaves were investigated by incubating human promyelocytic leukemia HL-60 cells with olive leaf extracts (OLEs) from seven principal Tunisian olive varieties, namely, Chemchali, Chemlali, Chétoui, Gerboui, Sayali, Zalmati and Zarrazi. The results showed significant growth inhibition of HL-60 cells incubated for 48 h with a 100-fold dilution of each OLE which had been obtained by incubating 10 g of dried leaves in 100 ml of 70% ethanol for one week with subsequent ultrafiltration. DNA fragmentation was observed in the cells incubated for 19 h with a 100-fold dilution of the Chemchali, Chemlali and Zalmati extracts. The results of a nitroblue tetrazolium (NBT) assay revealed NBT reduction, a differentiation marker, by the OLE-treated cells after an overnight incubation. The Gerboui extract showed the highest NBT reduction ability at more than 90%. An HPLC analysis revealed the presence of apigenin 7-glucoside in the extract, which was found in subsequent experiments to be responsible for the Gerboui extract-mediated cell differentiation.     

Growth rate and carrageenan analyses in four strains of <Emphasis Type="Italic">Kappaphycus alvarezii</Emphasis> (Rhodophyta,Gigartinales) farmed in the subtropical waters of São Paulo State,Brazil

Growth rate, semi-refined and refined carrageenan yields, 3,6-anhydrogalactose and sulphate contents, and gel strength were investigated in four strains of Kappaphycus alvarezii (brown, green and red tetrasporophytic strains, and one strain derived from tetraspores progeny, called G11) farmed in subtropical waters of São Paulo State, Brazil. All studied strains showed higher growth rates from February to May, decreasing from July to December. The G11 strain exhibited lower growth rates, but had semi-refined and refined carrageenan yields significantly higher than the others and similar to a commercial sample. A negative correlation between growth rate and semi-refined carrageenan yield was verified for all strains. The brown strain had the lowest content of 3,6-anhydrogalactose, while G11 and green strains showed higher values. No clear pattern of variation and no relationship were observed for sulphate and gel strength. However, all strains presented gel strength values near the one obtained from commercial sample. These results show that the carrageenan extracted from brown, green, red, and G11 strains of K. alvarezii cultured in subtropical waters of São Paulo State, Brazil with commercial potentials.     

A manually curated database of tetrapod mitochondrially encoded tRNA sequences and secondary structures

Background  

Mitochondrial tRNAs have been the subject of study for structural biologists interested in their secondary structure characteristics, evolutionary biologists have researched patterns of compensatory and structural evolution and medical studies have been directed towards understanding the basis of human disease. However, an up to date, manually curated database of mitochondrially encoded tRNAs from higher animals is currently not available.     

Recognition of functional genetic polymorphism using ESE motif definition: a conservative evolutionary approach to CYP2D6/CYP2C19 gene variants

Genetica - Although predicting the effects of variants near intron-exon boundaries is relatively straightforward, predicting the functional Exon Splicing Enhancers (ESEs) and the possible effects...     

印度藏茴香植物精油对温室棉蚜的熏蒸毒性及其成分分析（英文）

【目的】棉蚜Aphis gossypii是世界各地室内和室外果树、蔬菜和观赏植物上最具危害性的害虫之一。这一害虫取食植物汁液,产生蜜露,传播植物病毒,对植物从质和量上产生破坏。为了控制温室中的这一害虫,植物精油可用作化学农药的替代药物。本实验研究了印度藏茴香Carum copticum植物精油对棉蚜成虫的熏蒸毒性。【方法】将研磨的印度藏茴香干种子用改良的挥发油提取器(Clevenger-type apparatus)进行水蒸馏。所有生物测定均在27±2℃,相对湿度65%±5%和光周期16L∶8D条件下进行。研究采用完全随机设计,6个处理(5个不同浓度的精油加对照)。每一浓度3次重复,每一重复20头成虫。【结果】结果表明,棉蚜成蚜接触印度藏茴香精油24 h后出现明显的死亡。该精油对棉蚜成蚜的致死中浓度(LC50)为1.21μL/L空气。棉蚜成蚜的死亡百分率对精油的施用表现出较高的敏感性。精油浓度为1.21μL/L空气时,估计的棉蚜的致死中时(LT50)为11.79 h。这一精油的熏蒸毒性与浓度和接触时间之间具有一定的相关性。GC/MS组分分析结果表明,该精油由18种化合物组成,最重要的是一些化合物引起了熏蒸毒性,如麝香草酚(占50.07%)、γ-萜品烯(占23.99%)和对异丙基苯甲烷(占22.90%)。【结论】本研究结果表明印度藏茴香植物精油对棉蚜具有较好的杀虫效果。印度藏茴香精油对棉蚜产生较大的影响,由于它具有较高的熏蒸毒性潜力,因此可在温室中用于这一害虫的综合治理。