Characterization of Pneumocystis carinii Preparations Developed for Lipid Analysis

Pneumocystis carinii organisms were isolated from viral antibody-negative rats that had been infected by intratracheal intubation of organism preparations tested negative for common bacteria and fungi. Infection scores of lungs from infected animals at the time of parasite isolation was > 5 (100-1,000 organisms/oil immersion field). Electron microscopy of heavily infected lungs revealed that the pathogens adhered to Type I pneumocytes and to each other, resulting in obstructions up to several cell layers thick, which extended into the alveolar lumen. Protocols for purifying the organisms were developed to optimize separation from each other and from host cells, and to optimize preparation purity, recovery efficiency, and organism viability. The study tested mucolytic agents, sieving, various centrifugation speeds, lysis of host cells by osmotic shock and filtration through membranes of different pore diameter. Final preparations contained no intact host cells as determined by light microscopy. Only minor amounts (< 5%) of host debris were detected by electron microscopy. Most organisms and their pellicles were ultrastructurally intact but no longer adhered to one another. The final preparation was characterized biochemically by quantitation of the specific lung surfactant marker surfactant protein A, which indicated > 99.5% purity. The total non-P. carinii protein in the final preparation (< 6%, depending on the level of infection) was estimated by the protein content of pelletable material resulting from processing uninfected lungs in an identical manner. Elimination of free cholesterol and phospholipids from host lung tissue was monitored during the purification process. Exogenous stigmasterol, added as an extracellular marker, decreased during the purification process and was undetectable in the final organism preparation. Yields of 10⁸-10⁹ organisms/rat were routinely obtained. Viability, assessed by the calcein acetoxymethyl ester-propidium iodide assay, was 80–95%.     

Design and Characterization of a Membrane Protein Unfolding Platform in Lipid Bilayers

Accurate measurement of membrane protein stability—and particularly how it may vary as a result of disease-phenotypic mutations—ideally requires a denaturant that can unfold a membrane-embedded structure while leaving the solubilizing environment unaffected. The steric trap method fulfills this requirement by using monovalent streptavidin (mSA) molecules to unfold membrane proteins engineered with two spatially close biotin tags. Here we adapted this method to an 87-residue helix-loop-helix (hairpin) construct derived from helices 3 and 4 in the transmembrane domain of the human cystic fibrosis transmembrane conductance regulator (CFTR), wherein helix-helix tertiary interactions are anticipated to confer a portion of construct stability. The wild type CFTR TM3/4 hairpin construct was modified with two accessible biotin tags for mSA-induced unfolding, along with two helix-terminal pyrene labels to monitor loss of inter-helical contacts by pyrene excimer fluorescence. A series of eight constructs with biotin tags at varying distances from the helix-terminal pyrene labels were expressed, purified and labeled appropriately; all constructs exhibited largely helical circular dichroism spectra. We found that addition of mSA to an optimized construct in lipid vesicles led to a complete and reversible loss in pyrene excimer fluorescence and mSA binding, and hence hairpin unfolding—results further supported by SDS-PAGE visualization of mSA bound and unbound species. While some dimeric/oligomeric populations persist that may affect quantitation of the unfolding step, our characterization of the design yields a promising prototype of a future platform for the systematic study of membrane protein folding in a lipid bilayer environment.     

深海嗜热噬菌体GVE2头尾连接蛋白EV8的原核表达和功能分析

为了解头尾连接蛋白在噬菌体感染和装配中起的作用,对高温噬茵体GVE2(virulent bacteriophage of Geobacillus sp.E263)的头尾连接蛋白EV8进行了原核表达和功能鉴定.将EV8编码序列克隆入pGEX4T-2原核表达载体,并转染大肠埃希氏菌BL21.诱导表达后用SDS-PAGE进行鉴定,显示获得相对分子质量约36kD的谷胱甘肽硫转移酶(GST-EV8)融合蛋白.Western blot检测发现EV8在GVE2感染后2h开始表达,说明该蛋白为噬菌体晚期表达蛋白.免疫电镜定位表明头尾连接蛋白位于噬菌体头尾的连接处,为进一步研究高温噬菌体的组装和表达调控奠定了基础.     

Characterization of Engineered Channelrhodopsin Variants with Improved Properties and Kinetics

Channelrhodopsin 2 (ChR2), a light-activated nonselective cationic channel from Chlamydomonas reinhardtii, has become a useful tool to excite neurons into which it is transfected. The other ChR from Chlamydomonas, ChR1, has attracted less attention because of its proton-selective permeability. By making chimeras of the transmembrane domains of ChR1 and ChR2, combined with site-directed mutagenesis, we developed a ChR variant, named ChEF, that exhibits significantly less inactivation during persistent light stimulation. ChEF undergoes only 33% inactivation, compared with 77% for ChR2. Point mutation of Ile¹⁷⁰ of ChEF to Val (yielding “ChIEF”) accelerates the rate of channel closure while retaining reduced inactivation, leading to more consistent responses when stimulated above 25 Hz in both HEK293 cells and cultured hippocampal neurons. In addition, these variants have altered spectral responses, light sensitivity, and channel selectivity. ChEF and ChIEF allow more precise temporal control of depolarization, and can induce action potential trains that more closely resemble natural spiking patterns.     

Electro-Optical Properties Characterization of Fish Type III Antifreeze Protein

Antifreeze proteins (AFPs) are ice-binding proteins that depress the freezing point of water in a non-colligative manner without a significant modification of the melting point. Found in the blood and tissues of some organisms (such as fish, insects, plants, and soil bacteria), AFPs play an important role in subzero temperature survival. Fish Type III AFP is present in members of the subclass Zoarcoidei. AFPIII are small 7-kDa—or 14-kDa tandem—globular proteins. In the present work, we study the behavior of several physical properties, such as the low-frequency dielectric permittivity spectrum, circular dichroism, and electrical conductivity of Fish Type III AFP solutions measured at different concentrations. The combination of the information obtained from these measurements could be explained through the formation of AFP molecular aggregates or, alternatively, by the existence of some other type of interparticle interactions. Thermal stability and electro-optical behavior, when proteins are dissolved in deuterated water, were also investigated.     

Study of Antimicrobial Properties of Preparations Based on Maleic-Acid Copolymers Containing Silver Nanoparticles and Phenolic Residues

Applied Biochemistry and Microbiology - In this work, water-soluble polymer composites containing two antimicrobial components, silver nanoparticles and phenol ligands, are obtained. The...     

An ER Protein Functionally Couples Neutral Lipid Metabolism on Lipid Droplets to Membrane Lipid Synthesis in the ER

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Effect of the Fluorescein to Protein Ratio on Anti-Treponema pallidum Conjugates

The effect of using different labeling techniques and various amounts of fluorescein-isothiocyanate was tested with several pools of antisera to Treponema pallidum. Optimal labeling occurred in all serum pools at a fluorescien to protein ratio of 15. The use of pure dye and the dialysis method of labeling is recommended as the method of choice because of its effectiveness in the reduction of nonspecific staining.     

Physicochemical Properties and Specific Structural Features of Protein–Lipid Composites of Increased Nutritive Value

Fractional and component compositions of protein–lipid composites with increased nutritive value (compared to the protein preparations from which they were produced) were studied based on solubility and electrophoretic behavior. Differences in the fractional compositions of proteins and the amounts of hydrogen, ionic, and hydrophobic bonds were found. It was demonstrated that the water-, salt-, and alkali-soluble fractions of proteins changed during the manufacturing of the composites with soybean and wheat bran flour; the water- and alkali-soluble fractions, with protein concentrate from bran. Heterogeneity of the compositions and specific conformational features of composite proteins resulting from disulfide bonds were found. It was demonstrated that, during the manufacturing of composites, proteins of soybean flour aggregated (with the involvement of disulfide bonds), whereas protein products from wheat bran disaggregated. Breaks of interchain (wheat) or intrachain (concentrate) disulphide bonds accompanied the disaggregation. Overall the properties and specific structural features of the protein–lipid composites studied depended on the nature of the protein (soybean or wheat), type of initial preparations (flour or concentrate), and method of their production (emulsifying or drying).     

The Lipid and Protein Staining Properties of Sudan II, III and IV and Their Components

Saturated solutions of commercial Sudan II, III and IV in 60% ethanol were found to stain heat-coagulated fat-free purified human serum albumin and defatted whole serum. The protein staining components were isolated and identified by means of paper chromatography on mineral oil-impregnated filter paper with 95% ethanol as the developing agent. The brownish and yellowish fractions which migrated rapidly in this system stained proteins well, whereas the red, pink and orange fractions, which migrated slowly, stained lipids only. The solubility and staining power of the slowly migrating lipid coloring fractions remained satisfactory in absence of the rapidly moving protein staining fractions.     

Ligand Extraction Properties of the GM2 Activator Protein and Its Interactions with Lipid Vesicles

The GM2 activator protein (GM2AP) is an accessory protein required for the enzymatic conversion of GM2 to GM3 by hydrolases in the lysosomal compartments of cells. Here, GM2AP interactions with lipid vesicles are investigated by sucrose-loaded vesicle sedimentation and gel filtration assays, and the effects of pH and lipid composition on membrane binding and lipid extraction are characterized. The sedimentation experiments allow for facile quantification of the percentage of protein in solution and on the bilayer surface, with detailed analysis of the protein:lipid complex that remains in solution. Optimum binding and ligand extraction is found for pH 4.8 where <15% of the protein remains surface associated regardless of the lipid composition. In addition to extracting GM2, we find that GM2AP readily extracts dansyl-headgroup-labeled lipids as well as other phospholipids from vesicles. The ability of GM2AP to extract dansyl-DHPE from vesicles is altered by pH and the specific ligand GM2. Although the unique endosomal lipid, bis(monoacylglycero)phosphate, is not required for ligand extraction, it does enhance the extraction efficiency of GM2 when cholesterol is present in the vesicles.     

汉坦病毒核壳蛋白重组抗原的制备和基因分型的研究

根据HFRSV汉滩型（HTN）代表株76－118和汉城型（SEO）代表株R22基因资料,设计了两组引物,用电脑软件分析证明设计符合引物标准。以一组引物克隆全长S基因片段和N端的部分S基因片段,并使它们在T7系统进行融合表达和非融合表达。非融合表达产量虽不及融合表达高,但生物活性好。以非融合表达的两个S基因片段产物作间接ELISA的包被抗原,其工作浓度均达1：100000用另一组引物建立了逆转录-聚合酶链式反应（RT-PCR）,检测我国不同地区由8种主要宿主分离的37个HFRSV毒株,2个阳性标准对照毒株和5个阴性对照标本,并与cELISA法比较,二者阳性检出率分别为100％和84．6％,符合率为84．6％,但前者比后者敏感性高15．4％。对其中20个毒株的PCR扩增产物先后用RsaⅠ和HindⅢ作二级酶切,建立了逆转录-聚合酶链式反应-限制性片段长度多态性分析（RT-PCR－RFLP）分型法。被定为HTN型的9株,SEO型的8株,余3株未能定型。此20个毒株曾用血清学方法分型,仅11株分型成功．与RT-PCR－RFLP法结果符合。分型成功率RT-PCR－RFLP法比血清法高30％。     

汉坦病毒核壳蛋白重组抗原的制备和基因分型的研究

根据HFRSV汉滩型(HTN)代表株76-118和汉城型(SEO)代表株R22基因资料,设计了两组引物,用电脑软件分析证明设计符合引物标准.以一组引物克隆全长S基因片段和N端的部分S基因片段,并使它们在T7系统进行融合表达和非融合表达.非融合表达产量虽不及融合表达高,但生物活性好.以非融合表达的两个S基因片段产物作间接ELISA的包被抗原,其工作浓度均达1:10 000.用另一组引物建立了逆转录-聚合酶链式反应(RT-PCR),检测我国不同地区由8种主要宿主分离的37个HFRSV毒株,2个阳性标准对照毒株和5个阴性对照标本,并与cELISA法比较,二者阳性检出率分别为100%和84.6%,符合率为84.6%,但前者比后者敏感性高15.4%.对其中20个毒株的PCR扩增产物先后用RsaⅠ和HindⅢ作二级酶切,建立了逆转录-聚合酶链式反应-限制性片段长度多态性分析(RT-PCR-RFLP)分型法.被定为HTN型的9株,SEO型的8株,余3株未能定型.此20个毒株曾用血清学方法分型,仅11株分型成功,与RT-PCR-RFLP法结果符合.分型成功率RT-PCR-RFLP法比血清法高30%.     

基于性别与季节差异的赤腹松鼠糖脂代谢指标测定

以2015年1—12月捕自四川省荥经县的171只成体赤腹松鼠Callosciurus erythraeus为研究对象,首次测定了其糖脂代谢8项指标值,并分析了性别、季节及妊娠对指标的影响。结果显示:(1)赤腹松鼠糖脂代谢指标的性别差异无统计学意义。(2)高密度脂蛋白、载脂蛋白B浓度的季节差异无统计学意义,总胆固醇、甘油三酯、低密度脂蛋白、极低密度脂蛋白、葡萄糖浓度为夏、秋季高于春、冬季,载脂蛋白AI浓度为夏、秋季低于春、冬季。(3)妊娠鼠甘油三酯、极低密度脂蛋白浓度高于未妊娠鼠。结果表明,雌、雄赤腹松鼠糖脂代谢指标具有同一性,且大多数指标具有季节差异,这可能与雌、雄鼠的繁殖状态及生活环境的多样性变化有关。     

Physicochemical and Biological Studies on Various Preparations of Tuberculin Purifield Protein Derivative

Tuberculin purified protein derivative (PPD) has been prepared by seven different precipitation methods from culture filtrate of Mycobacterium tuberculosis var. hominis. It was found to contain 48 to 99% tuberculoprotein, depending on the method of precipitation. The remaining percentage is represented by nucleic acid, polysaccharide, and ash. Activation analysis on tuberculin PPD and on tubercle bacilli has revealed the presence of trace elements. The molecular weight of tuberculin PPD has been found to be of the order of 14,800 to 27,800. The biological activity of tuberculin PPD varies from lot to lot and from method to method. A correlation between its molecular weight and its biological activity seems to exist.     

Isolation and Characterization of a Lipid Transfer Protein Gene (BplLTP1) from Betula platyphylla

Although nonspecific lipid transfer proteins (nsLTPs) are widely present in plants, their functions are not fully understood. Here, we isolated and characterized a putative nsLTP gene, BplLTP1, from Betula platyphylla. The full-length cDNA of BplLTP1 is 638 bp long, including a 363-bp open reading frame (GenBank accession no. JQ409562). The putative protein BplLTP1 contains an N-terminal signal sequence and possesses the characteristic features of nsLTPs. An amino acid sequence alignment revealed that BplLTP1 shares a high level of similarity with other known nsLTPs. A 3D model of BplLTP1 was also constructed based on the homology model. Quantitative real time-PCR analysis showed that there were no obvious differences in the expression levels of BplLTP1 among different tissues. BplLTP1 displayed distinctly higher expression levels in young tissues than in older tissues. Moreover, BplLTP1 was upregulated at the mononuclear microspore developmental stages in male inflorescences. Expression analysis was performed using 3-month-old cultured seedlings, and the results revealed that the expression of BplLTP1 was upregulated by exogenous abscisic acid and salicylic acid, downregulated by exogenous methyl jasmonate, and not significantly altered by exogenous gibberellin A. In addition, a prokaryotic expression system was constructed with pET32a-BplLTP1 and Escherichia coli strain BL21 and subjected to abiotic stress resistance analysis. The results indicated that the expression of BplLTP1 improved the resistance of the recombinant strain to salt (NaCl) and drought (polyethylene glycol) stress, but not to alkali (NaHCO₃) stress.