毛细管电泳四色荧光检测法分析茶树SSR标记

将毛细管电泳四色荧光栓测技术应用于茶树SSR标记分析.该方法采用三引物PCR扩增SSR位点,三引物即在5'端加有M13尾巴序列(5'-CACGACGTTGTAAAACGAC-3')的特异正向引物、特异反向引物及带有荧光标记的通用型M13引物:为了运用四色荧光检测系统使通过一次毛细管电泳能同时检测3个以上的SSR位点,采用蓝、绿、黑3种不同颜色的荧光染料分别对3个M13引物进行标记. 应用该方法对42个茶树品种(系)的16个SSR位点进行遗传分析的结果表明:此法具有简便、可靠、低成本及高通量的优点;且随着所分析SSR位点数的增加,降低成本的效果更加显著.采用建立的方法,还筛选获得了11个多态性丰富的可应用于茶树遗传研究的SSR标记.     

Maltodextrins as Chiral Selectors in CE: Molecular Structure Effect of Basic Chiral Compounds on the Enantioseparation

Prediction of chiral separation for a compound using a chiral selector is an interesting and debatable work. For this purpose, in this study 23 chiral basic drugs with different chemical structures were selected as model solutes and the influence of their chemical structures on the enantioseparation in the presence of maltodextrin (MD) as chiral selector was investigated. For chiral separation, a 100‐mM phosphate buffer solution (pH 3.0) containing 10% (w/v) MD with dextrose equivalent (DE) of 4‐7 as chiral selector at the temperature of 25°C and voltage of 20 kV was used. Under this condition, baseline separation was achieved for nine chiral compounds and partial separation was obtained for another six chiral compounds while no enantioseparation was obtained for the remaining eight compounds. The results showed that the existence of at least two aromatic rings or cycloalkanes and an oxygen or nitrogen atom or –CN group directly bonded to the chiral center are necessary for baseline separation. With the obtained results in this study, chiral separation of a chiral compound can be estimated with MD‐modified capillary electrophoresis before analysis. This prediction will minimize the number of preliminary experiments required to resolve enantiomers and will save time and cost. Chirality 26:620–628, 2014. © 2014 Wiley Periodicals, Inc.     

Theoretical basis of single breath gas absorption tests

Absorption of gas from alveoli is examined in a simplified model of the respiratory system during a stylized single breath consisting of constant inspiratory flow, constant expiratory flow, and breathholding. The equations describing gas behavior are general since they are based upon conservation of mass. The equations simplify considerably when gases that are not soluble in pulmonary tissue and/or blood are utilized. In a three-compartment model, diffusing capacity of the lung for carbon monoxide (D _CO ) will be underestimated except when both uneven distribution of lung volume andD _CO are present; under most circumstances, the standard clinical 10-s method [9] is at least as accurate as any other. When pulmonary capillary blood flow is calculated by the one point method [2] in a one-compartment lung, it is underestimated; in the three-compartment model, it is underestimated except when both uneven distribution of . and lung volume are present. The multiple single breath method [2] accurately measuresD _CO and . Measurement of pulmonary tissue volume is improved by correcting the value of the intercept of acetylene absorption to the time when carbon monoxide apparently began rather than utilizing the beginning of inspiration.Nomenclature D _CO diffusing capacity of the lung for CO (ml CO, STPD/min/mm Hg) - pulmonary capillary blood flow rate (L/min) - V _t pulmonary tissue volume (L) - V _A alveolar compartment volume (L) - V _Ao alveolar compartment volume at conclusion of inspiratory flow (L) - inspiratory flow rate (L/sec) - expiratory flow rate (L/sec) - Bunsen coefficient of pulmonary tissue for test gas (ml test gas/ml tissue/atm) - Bunsen coefficient of pulmonary tissue for test gas (ml test gas/ml blood/atm) - F _A fractional pressure of test gas in the alveolar compartment (atm)     

Roles of interstitial fraction and load conditions on the dynamic binding capacity of proteins on capillary‐channeled polymer fiber columns

Capillary‐channeled polymer (C‐CP) fibers are used as a stationary phase for ion‐exchange chromatography of proteins. Collinear packing of the fibers permits operation at high linear velocities (U_o > 100 mm s^?1) and low backpressure (<2,000 psi) on analytical‐scale columns. Rapid solvent transport is matched with very efficient solute mass transfer as fibers are virtually non‐porous with respect to the size of the target protein molecules. Lack of porosity of course limits the equilibrium binding capacity of stationary phases. Breakthrough curves and frontal analysis are used to better understand trade‐offs between the kinetic and thermodynamic properties as C‐CP fibers are applied in preparative situations. Fiber columns packed to different interstitial fraction values affect both the total fiber surface area (e.g., equilibrium binding capacity [EBC]) and the permittivity to flow and mass transport characteristics (e.g., dynamic binding capacity [DBC]). The EBC of the nylon 6 C‐CP fibers was found to be 1.30 mg g^?1, with isotherms that were best matched by a Moreau model, showing linearity up to solute concentrations of ～0.4 mg mL^?1. Isotherms generated under flow conditions were equally well approximated using Langmuir, Freundlich, and Moreau isotherm models. Fairly linear responses were seen up to the maximum load concentration of 1.2 mg mL^?1. Counterintuitively, dynamic studies revealed that conditions of high column porosity yielded a DBC that is ～70% higher than the EBC. These findings point to potential advantages in terms downstream processing applications, where protein throughput and yield are critical metrics. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:97–109, 2015     

Methods of nitric oxide detection in plants: A commentary

Over the last decade nitric oxide (NO) has been shown to influence a range of processes in plants. However, when, where and even if NO production occurs is controversial in several physiological scenarios in plants. This arises from a series of causes: (a) doubts have arisen over the specificity of widely used 4,5-diaminofluorescein diacetate (DAF-2DA)/4-amino-5-methylamino-2,7-difluorofluorescein (DAF-FM) dyes for NO, (b) no plant nitric oxide synthase (NOS) has been cloned, so that the validity of using mammalian NOS inhibitors to demonstrate that NO is being measured is debatable, (c) the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (cPTIO) needs to be used with caution, and (d) some discrepancies between assays for in planta measurements and another based on sampling NO from the gas phase have been reported. This review will outline some commonly used methods to determine NO, attempt to reconcile differing results obtained by different laboratories and suggest appropriate approaches to unequivocally demonstrate the production of NO.     

A scale‐down mimic for mapping the process performance of centrifugation,depth and sterile filtration

In the production of biopharmaceuticals disk‐stack centrifugation is widely used as a harvest step for the removal of cells and cellular debris. Depth filters followed by sterile filters are often then employed to remove residual solids remaining in the centrate. Process development of centrifugation is usually conducted at pilot‐scale so as to mimic the commercial scale equipment but this method requires large quantities of cell culture and significant levels of effort for successful characterization. A scale‐down approach based upon the use of a shear device and a bench‐top centrifuge has been extended in this work towards a preparative methodology that successfully predicts the performance of the continuous centrifuge and polishing filters. The use of this methodology allows the effects of cell culture conditions and large‐scale centrifugal process parameters on subsequent filtration performance to be assessed at an early stage of process development where material availability is limited. Biotechnol. Bioeng. 2016;113: 1934–1941. © 2016 The Authors. Biotechnology and Bioengineering Published by Wiley Periodicals, Inc.     

Chiral Separation of Uncharged Pomalidomide Enantiomers Using Carboxymethyl‐β‐Cyclodextrin: A Validated Capillary Electrophoretic Method

The racemic mixture of pomalidomide (POM), a second‐generation immunomodulatory uncharged drug, was separated into enantiomers by capillary zone electrophoresis for the first time. Seven different chargeable cyclodextrin (CD) derivatives were screened as complexing agents and chiral selectors, investigating the stability of the POM‐CD inclusion complexes and their enantiodiscriminating capacities. Based on preliminary experiments, carboxymethyl‐β‐CD (CM‐β‐CD) was found to be the most effective chiral selector. Factors influencing enantioseparation were systematically optimized, using an orthogonal experimental design. Optimal parameters (background electrolyte [BGE]: 50 mM Tris‐acetate buffer, pH 6.5, containing 15 mM CM‐β‐CD; capillary temperature: 20°C; voltage applied +15 kV) allowed baseline separation of POM enantiomers with a resolution as high as 4.87. The developed method was validated, in terms of sensitivity (limit of detection and limit of quantification), linearity, accuracy, repeatability, and intermediate precision. Chirality 28:199–203, 2016. © 2015 Wiley Periodicals, Inc.     

Isolation and characterization of porcine somatotropin containing a succinimide residue in place of aspartate129.

Aspartate129 in porcine somatotropin was converted into a cyclic imide residue (succinimide) under acidic solution conditions. Reversed-phase high performance liquid chromatography was utilized to isolate and quantitate this altered species, which accounted for approximately 30% of the total protein. The molecular mass of this modified species was determined by electrospray mass spectrometry to be 18 Da less than normal porcine somatotropin, indicative of a loss of 1 H2O molecule. Tryptic peptide mapping demonstrated that the peptide composed of residues 126-133 was altered in this modified protein. Amino acid analysis, amino acid sequencing, mass spectrometry, and capillary zone electrophoresis were used to demonstrate that aspartate129 in this peptide had been converted into a succinimide residue. Further confirmation that this peptide contained a succinimide was obtained by hydrolyzing the modified peptide at pH 9.0, which yielded both the aspartate and isoaspartate peptides.     

Dopamine transporter is essential for the maintenance of spontaneous activity of auditory nerve neurones and their responsiveness to sound stimulation

Dopamine, a neurotransmitter released by the lateral olivocochlear efferents, has been shown tonically to inhibit the spontaneous and sound-evoked activity of auditory nerve fibres. This permanent inhibition probably requires the presence of an efficient transporter to remove dopamine from the synaptic cleft. Here, we report that the dopamine transporter is located in the lateral efferent fibres both below the inner hair cells and in the inner spiral bundle. Perilymphatic perfusion of the dopamine transporter inhibitors nomifensine and N-[1-(2-benzo[b]thiophenyl)cyclohexyl]piperidine into the cochlea reduced the spontaneous neural noise and the sound-evoked compound action potential of the auditory nerve in a dose-dependent manner, leading to both neural responses being completely abolished. We observed no significant change in cochlear responses generated by sensory hair cells (cochlear microphonic, summating potential, distortion products otoacoustic emissions) or in the endocochlear potential reflecting the functional state of the stria vascularis. This is consistent with a selective action of dopamine transporter inhibitors on auditory nerve activity. Capillary electrophoresis with laser-induced fluorescence (EC-LIF) measurements showed that nomifensine-induced inhibition of auditory nerve responses was due to increased extracellular dopamine levels in the cochlea. Altogether, these results show that the dopamine transporter is essential for maintaining the spontaneous activity of auditory nerve neurones and their responsiveness to sound stimulation.     

毛细管电泳及其在蛋白质折叠研究中的应用

简要介绍了毛细管电泳的常用分离模式及其原理,并对毛细管电泳在蛋白质化学领域中的新应用——研究蛋白质折叠和发展前景作了评述。