pMAA-Red: a new pPZP-derived vector for fast visual screening of transgenic Arabidopsis plants at the seed stage

ABSTRACT: BACKGROUND: The production of transgenic plants, either for the overproduction of the protein of interest, for promoter::reporter lines, or for the downregulation of genes is an important prerequisite in modern plant research but is also very time-consuming. RESULTS: We have produced additions to the pPZP family of vectors. Vector pPZP500 (derived from pPZP200) is devoid of NotI sites and vector pPZP600 (derived from pPZP500) contains a bacterial kanamycin resistance gene. Vector pMAA-Red contains a Pdf2.1::DsRed marker and a CaMV::GUS cassette within the T-DNA and is useful for the production of promoter::GUS lines and overexpression lines. The Pdf2.1 promoter is expressed in seeds and syncytia induced by the beet cyst nematode Heterodera schachti in Arabidopsis roots. Transgenic seeds show red fluorescence which can be used for selection and the fluorescence level is indicative of the expression level of the transgene. The advantage is that plants can be grown on soil and that expression of the marker can be directly screened at the seed stage which saves time and resources. Due to the expression of the Pdf2.1::DsRed marker in syncytia, the vector is especially useful for the expression of a gene of interest in syncytia. CONCLUSIONS: The vector pMAA-Red allows for fast and easy production of transgenic Arabidopsis plants with a strong expression level of the gene of interest.     

Mixed-chain phosphatidylcholine bilayers: structure and properties

Calorimetric and X-ray diffraction data are reported for two series of saturated mixed-chain phosphatidylcholines (PCs), 18:0/n:0-PC and n:0/18:0-PC, where the sn-1 and sn-2 fatty acyl chains on the glycerol backbone are systematically varied by two methylene groups from 18:0 to 10:0 (n = 18, 16, 14, 12, or 10). Fully hydrated PCs were annealed at -4 degrees C and their multilamellar dispersions characterized by differential scanning calorimetry and X-ray diffraction. All mixed-chain PCs form low-temperature "crystalline" bilayer phases following low-temperature incubation, except 18:0/10:0-PC. The subtransition temperature (Ts) shifts toward the main (chain melting) transition temperature (Tm) as the sn-1 or sn-2 fatty acyl chain is reduced in length; for the shorter chain PCs (18:0/12:0-PC, 12:0/18:0-PC, and 10:0/18:0-PC), Ts is 1-2 degrees C greater than Tm, and the subtransition enthalpy (delta Hs) is much greater than for the longer acyl chain PCs. Tm decreases with acyl chain length for both series of PCs except 18:0/10:0-PC, while for the positional isomers, n:0/18:0-PC and 18:0/n:0-PC, Tm is higher for the isomer with the longer acyl chain in the sn-2 position of the glycerol backbone. The conversion from the crystalline bilayer Lc phase to the liquid-crystalline L alpha phase with melted hydrocarbon chains occurs through a series of phase changes which are chain length dependent. For example, 18:0/18:0-PC undergoes the phase changes Lc----L beta'----P beta'----L alpha, while the shorter chain PC, 10:0/18:0-PC, is directly transformed from the Lc phase to the L alpha phase. However, normalized enthalpy and entropy data suggest that the overall thermodynamic change, Lc----L alpha, is essentially chain length independent. On cooling, the conversion to the Lc phases occurs via bilayer gel phases, L beta', for the longer chain PCs or through triple-chain interdigitated bilayer gel phases, L beta, for the shorter chain PC 18:0/12:0-PC and possibly 10:0/18:0-PC. Molecular models indicate that the bilayer gel phases for the more asymmetric PC series, 18:0/n:0-PC, must undergo progressive interdigitation with chain length reduction to maintain maximum chain-chain interaction. The L beta phase of 18:0/10:0-PC is the most stable structure for this PC below Tm. The formation and stability of the triple-chain structures can be rationalized from molecular models.     

Adsorption of monovalent and divalent cations by phospholipid membranes. The monomer-dimer problem.

A generalization of the Stern theory is derived to treat the simultaneous adsorption of monovalent cations and divalent cations by single-component phospholipid membranes, where the ion:phospholipid binding stoichiometries are 1:1 for the monovalent cations and 1:1 and/or 1:2 for the divalent cations. This study treats both the situation in which the monovalent and divalent cations compete for membrane binding sites and that in which they do not compete. The general formalism of the screening/binding problem is reviewed, and it is shown how the adsorption problem can be isolated from the electrostatics. The statistical mechanics of mixed 1:1- and 1:2-stoichiometric adsorption (the monomer-dimer problem) is treated, and the problem of simultaneous 1:1 and 1:2 binding is solved. A simple expression for this solution, given in the Bethe approximation, is combined with the electrostatics to yield an adsorption isotherm encompassing both 1:1 monovalent-cation, and 1:1 and 1:2 divalent-cation, binding to charged membranes. A comparison with the simplified treatment of previous authors is made and the significance of their assumptions clarified in light of the present result. The present and previous treatments are plotted for a representative case of Na+ and Ca++ binding to a phosphatidylserine membrane. Criteria are established to permit unambiguous experimental testing of the present vs. previous treatments.     

The probability of obtaining complex kinetic curves for enzyme mechanisms with cubic terms in the pseudo-steady state rate equations

A number of details required for the classification of 3 : 3 double reciprocal plots are provided. It is shown that the ν(S) plot for a 3 : 3 function can have at most four inflexions and at most two inflexions adjacent to a turning point. Using this information, a classification of 3 : 3 ν(S) plots into ten main varieties with several subclasses is reported. The problem of defining the probability with which a given mechanism can give rise to specific curve shape features is considered. Applying this technique, the probability with which four simple enzyme mechanisms can give rise to 3 : 3 curve shapes is computed. It is shown that a 3 : 3 saturation function can have no turning points, at most two inflexions and at most one inflexion in double reciprocal space. The probability with which the available 3 : 3 shapes can arise is also computed. It is concluded that, with realistic values for rate constants, chemically reasonable enzyme mechanisms leading to rate equations of degree n : n can generate most of the kinetic profiles available to a rational function of degree n : n with positive coefficients. The probability of obtaining specific curve shapes is not so characteristic of the particular mechanism for 3:3 rate equations as it is for 2:2 rate equations. The probability of obtaining highly complex curves with several turning points or inflexions is rather lower for the enzyme mechanisms than with general 3 : 3 rational functions. There is a high probability that 3 : 3 mechanisms will generate kinetic curves that are geometrically similar to those possible for degree 2 : 2 but this is not so for binding isotherms. Hence differentiating 3 : 3 from 2 : 2 rate equations from experimental kinetic data is more likely to be successful by non-linear regression to the whole data set than by demonstrating a specific 3 : 3 feature. Binding curves, on the other hand, for three or more sites should give Scatchard plots with inflexions, features not possible with second degree equations which are conic sections in this space.     

Characterization of heparin oligosaccharides binding specifically to antithrombin III using mass spectrometry

Heparan sulfate (HS) is a sulfated glycosaminoglycan attached to a core protein on the cell surface. Protein binding to cell surface HS is a key regulatory event for many cellular processes such as blood coagulation, cell proliferation, and migration. The concept whereby protein binding to HS is not random but requires a limited number of sulfation patterns is becoming clear. Here we describe a hydrophobic trapping assay for screening a library of heparin hexasaccharides for binders to antithrombin III (ATIII). The hexasaccharide compositions are defined with their building block content in the following format: (DeltaHexA:HexA:GlcN:SO 3:Ac). Of five initial compositions present in the library, (1:2:3:6:1), (1:2:3:7:1), (1:2:3:7:0), (1:2:3:8:0), and (1:2:3:9:0), only two are shown to bind ATIII, namely, (1:2:3:8:0) and (1:2:3:9:0). The use of amide hydrophilic interaction (HILIC) liquid chromatography-mass spectrometry permitted reproducible quantitative analysis of the composition of the initial library as well as that of the binding fraction. The specificity of the hexasaccharides binding ATIII was confirmed by assaying their ability to enhance ATIII-mediated inhibition of Factor Xa in vitro.     

Eimeria tenella: specific reversal of t-butylaminoethanol toxicity for parasite and host by choline and dimethylaminoethanol.

t-Butylaminoethanol is an anticoccidial compound that is related structurally to the metabolically active substances, dimethylaminoethanol, and choline. Toxic effects of t-butylaminoethanol for chickens and Eimeria tenella are specifically overcome by feeding sufficient amounts of dimethylaminoethanol or choline. Dietary concentrations of the two above metabolites required to totally overcome toxic effiects of t-butylaminoethanol were determined and are expressed as the reversal ratio, inhibitor (t-butylamino-ethanol): metabolite. The inhibitor:choline ratio for total reversal of toxic effects of the inhibitor in chickens is approximately 1:10 over a concentration range of inhibitor from 0.019 to 0.05%. The inhibitor:choline ratio for reversal of antiparasitic effects is approximately 1:200 with a concentration of 0.01% inhibitor. The inhibitor:Dimethylaminoethanol ratio for reversal of toxic effects of the inhibitor in the chicken is approximately 1:7 with a concentration of 0.015% inhibitor. The inhibitor:dimethylaminoethanol ratio for reversal of antiparasitic effects is approxmately 1:20 wth a concentration of 0.01% inhibitor.     

Effects of phospholipids on the function of (Ca2(+)-Mg2+)-ATPase.

The ATPase activity for the (Ca2(+)-Mg2+)-ATPase purified from rabbit skeletal muscle sarcoplasmic reticulum is lower when reconstituted into bilayers of dimyristoleoylphosphatidylcholine [(C14:1)PC] than when it is reconstituted into dioleoylphosphatidylcholine [(C18:1)PC]. The rate of formation of phosphoenzyme on addition of ATP is slower for (C14:1)PC-ATPase than for the native ATPase or (C18:1)PC-ATPase. The reduction in rate of phosphoenzyme formation is attributed to a reduction in the rate of a conformational change on the ATPase following binding of ATP but before phosphorylation. The level of phosphoenzyme formed from Pi is also less for (C14:1)PC-ATPase than for (C18:1)PC-ATPase. At steady state at pH 6.0 in the presence of ATP Ca2+ is released from (C18:1)PC-ATPase into the medium, but not from (C14:1)PC-ATPase. These effects of (C14:1)PC on the ATPase are reversed by addition of androstenol to a 1:1 molar ratio with (C14:1)PC. The results are interpreted in terms of a kinetic model for the ATPase.     

Thin-layer separation and quantification of bile acids

A method has been developed for quantification of total free and conjugated bile acids separated on silica gel HR coated thin-layer chromatography plates. Aliquots of bile acid solutions are applied to channeled plates which are developed with either ethyl acetate: isooctane: glacial acetic acid 10:10:2 v/v for free bile acid separation, or chloroform:methanol:glacial acetic acid:water 130:50:4:8 v/v for conjugated bile acid separation. Bile acids are determined directly in serial areas of silica gel by treating gel areas suspended in tris buffer with resazurin reagent. The method is quantitative and as little as 0.1 μg of bile acid is readily determined. Application of the method to determinations of bile acids in crude fecal extracts is described.     

Accurate guanine:cytosine discrimination in T4 DNA ligase-based single nucleotide polymorphism analysis using an oxanine-containing ligation fragment

T4 DNA ligase-based mismatch detection methods have been proposed as useful strategies for single nucleotide polymorphism (SNP) analyses. However, there is a critical problem for cytosine/thymine (C/T) SNP analyses: guanine:thymine (G:T) mismatch is not distinguished from guanine:cytosine (G:C). Here we employed chemically modified nucleobases, such as oxanine and hypoxanthine, at the end of a ligation fragment and analyzed their influence on the ligation efficiency between G:C and G:T. Successful ligation for G:C and no ligation for G:T were observed when oxanine was employed adjacent to guanine in the ligation junction. This ligation method using an oxanine-containing fragment has strong potentials for the accurate analysis of C/T SNPs.     

arrayQCplot: software for checking the quality of microarray data

SUMMARY: arrayQCplot is a software for the exploratory analysis of microarray data. This software focuses on quality control and generates newly developed plots for quality and reproducibility checks. It is developed using R and provides a user-friendly graphical interface for graphics and statistical analysis. Therefore, novice users will find arrayQCplot as an easy-to-use software for checking the quality of their data by a simple mouse click. AVAILABILITY: arrayQCplot software is available from Bioconductor at http://www.bioconductor.org. A more detailed manual is available at http://bibs.snu.ac.kr/software/arrayQCplot CONTACT: tspark@stats.snu.ac.kr.     

Inheritance of differences in organ trichome formation in Arabidopsis thaliana (L.) Heynh. plants

It was determined that, according to the character "hairiness of rosette leaves", gl1-1 allele in homozygote state suppresses action of dis2-1 allele (dis2-1 < gl1-1), therefore F2 segregation is identified in a typical for recessive epistasis ratio of 9:3:4. Inheritance of differences in hairiness of stem leaves corresponds to the Mendel scheme of segregation (9:3:3:1), which is typical for dihybrids. Hairiness is absent on stems of triple recessive for dis2-1, er-1, gl1-1 plants, and segregation in F2 is observed in the ratio of 9:3:4.     

Substrate preference in phosphatidylserine biosynthesis for docosahexaenoic acid containing species

Neuronal membranes contain high levels of phosphatidylserine (PS) and docosahexaenoic acid (22:6n-3, DHA). In this study, substrate preference in PS synthesis was determined to gain insight on the biochemical basis for concentrating PS in neuronal membranes where 22:6n-3 is highly enriched. We first established an in vitro assay method using unilamellar vesicles (LUV) of deuterium-labeled substrates and reversed-phase HPLC/electrospray ionization (ESI) mass spectrometry. The PS production by the incubation of deuterium-labeled substrate and microsomal fractions was monitored. We found that tissue-specific substrate preference exists in PS synthesis. Microsomes from the cerebral cortex synthesized PS from 18:0,22:6-PC most favorably among the PC substrates tested, followed by 18:0,22:5-PC, resulting in the PC substrate preference in the order of 18:0,22:6 > 18:0,22:5 > 18:0,20:4 = 18:0,18:1. Liver microsomes also preferred 18:0,22:6-PC as the substrate in PS synthesis but did not use 18:0,22:5-PC favorably. The 18:0,22:5-PC species was converted to PS at the similar extent as 18:0,20:4- or 18:0,18:1-PC species in the liver. Both brain and liver microsomes showed a preference for 18:0 over 16:0 as the sn-1 fatty acid. From these data it was deduced that preferential conversion of 18:0,22:6-PC to the corresponding PS species is at least partly responsible for concentrating PS in neuronal tissues where 22:6n-3 is particularly abundant. The distinctive preference for 18:0,22:5-PS observed with brain microsomes may help to maintain PS at a high level in the brain when 22:6n-3 is replaced by 22:5n-3 as in the case of n-3 fatty acid deficiency.     

A fluorescence study of the interaction of protein synthesis initiation factors 4A, 4E, and 4F with mRNA and oligonucleotide analogs

The initial interaction of mRNA with the protein synthesis machinery presumably involves recognition of the 5'-cap (m7GpppN), although it is not clear at the present time whether this recognition is by eIF-4E or eIF-4F. This process has been studied by direct fluorescence titration experiments. The equilibrium constants for the formation of the binary protein: m7GpppG, protein:mRNA, and protein:protein complexes as well as the ternary mRNA:eIF-4E:eIF-4A complexes were measured. These studies show, for the first time, direct evidence for an eIF-4A:eIF-4E interaction. In contrast to earlier studies, we show that the affinity of eIF-4E and eIF-4F for globin mRNA is similar. Furthermore, the relative affinities of mRNA analogs (capped oligonucleotides) for these initiation factors indicate that the cap is the predominant feature recognized for binding, but other features also contribute to the eIF-4E:mRNA interaction.     

DnaSP version 3: an integrated program for molecular population genetics and molecular evolution analysis

SUMMARY: DnaSP is a Windows integrated software package for the analysis of the DNA polymorphism from nucleotide sequence data. DnaSP version 3 incorporates several methods for estimating the amount and pattern of DNA polymorphism and divergence, and for conducting neutrality tests. AVAILABILITY: For academic uses, DnaSP is available free of charge from: http://www.bio.ub.es/julio/DnaSP.html CONTACT: julio@porthos.bio.ub.es     

乳酸菌素治疗消化性溃疡的临床分析

目的：观察乳酸菌素治疗消化性溃疡的疗效。方法：共28例患者,治疗组12例,对照组16例。治疗组口服乳酸菌素、思密达,对照组口服西米替丁、阿莫西林、硫糖铝片。二组均用药4w。结果：腹疼症状缓解率治疗组为100％,对照组为95％。呕吐症状缓解率均为100％。溃疡愈合率治疗组为100％,对照组为75％。结论：乳酸菌素在抑制幽门螺杆菌感染,保持消化道微生态的平衡和调节机体免疫方面效果明显。     

复方五倍子有效成分的分离鉴定及抑菌活性研究

对渔用抗菌剂复方五倍子有效成分进行薄层色谱与柱层析分离检测,以鉴定出复方中的各种有效成分;选用几种水产动物常见致病菌进行体外抑菌和联合用药试验,以确定各有效成分抑菌活性强弱和各成分间的药物关系,为分析复方五倍子抗菌剂的药物作用机理奠定理论基础,并为其质量控制提供参考依据。用薄层析硅胶GF254制板,以苯:乙醇:甲酸(7:3:1)为展开剂,用氨熏-三氯化铁显色,日光下观察层析结果;通过干法装硅柱,以苯:乙醇:甲酸(7:3:1)作为洗脱液进行洗脱,分离得到各单体,参照理化性质分析及薄层分析,鉴定出复方五倍子抗菌剂总有效成分。结果表明;复方五倍子抗菌剂有效成分为鞣质、没食子酸、槲皮素、黄芩苷、芦荟大黄素、大黄素和1,8-二羟基蒽醌,其中没食子酸含量最大,紫外分光光度法测定为10.47%。用复方的各有效成分别对温和气单胞菌、豚鼠气单胞菌、迟缓爱德华菌和柱状黄杆菌进行抑菌试验,以温和气单胞菌为菌种将复方主要有效成分两两配对进行体外联合抑菌试验,得出复方五倍子各有效成分抑菌活性顺序为:没食子酸>蒽醌(含1,8-二羟基蒽醌、大黄素、芦荟大黄素)>黄芩苷>槲皮素>鞣质。没食子酸和蒽醌具有协同作用,没食子酸和黄芩苷、没食子酸和槲皮素、蒽醌和黄芩苷三组同属于累加作用。       

The sweet spot for biologics: recent advances in characterization of biotherapeutic glycoproteins

Introduction: Glycosylation is recognized as a Critical Quality Attribute for therapeutic glycoproteins such as monoclonal antibodies, fusion proteins and therapeutic replacement enzymes. Hence, efficient and quantitative glycan analysis techniques have been increasingly important for their discovery, development and quality control. The aim of this review is to highlight relevant and recent advances in analytical technologies for characterization of biotherapeutic glycoproteins.

Areas covered: The review gives an overview of the glycosylation trends of biotherapeutics approved in 2016 and 2017 by FDA. It describes current and novel analytical technologies for characterization of therapeutic glycoproteins and is explored in the context of released glycan, glycopeptide or intact glycoprotein analysis. Ultra performance liquid chromatography, mass spectrometry and capillary electrophoresis technologies are explored in this context.

Expert commentary: There is a need for the biopharmaceutical industry to incorporate novel state of the art analytical technologies into existing and new therapeutic glycoprotein workflows for safer and more efficient biotherapeutics and for the improvement of future biotherapeutic design. Additionally, at present, there is no ‘gold-standard’ approach to address all the regulatory requirements and as such this will involve the use of orthogonal glycoanalytical technologies with a view to gain diagnostic information about the therapeutic glycoprotein.     

Determination of the elongate spermatid-Sertoli cell ratio in various mammals

Criteria were devised for determining the elongate spermatid-Sertoli cell ratio in various mammalian species at the electron microscope level. When data from particular species were pooled, the values were: rabbit, 12.17:1, hamster, 10.75:1; gerbil, 10.64:1; rat, 10.32:1; guinea-pig, 10.10:1; vole, 9.75:1; and monkey, 5.94:1. The elongate spermatid-Sertoli cell ratio is a measure of the workload of the Sertoli cell and is a prime factor determining their efficiency. The higher the ratio, the higher the sperm output is likely to be per given weight of seminiferous tubule parenchyma for a particular species.     

A general method for the quantitative analysis of functional chimeras: applications from site-directed mutagenesis and macromolecular association

Two new parameters, I: and C:, are introduced for the quantitative evaluation of functional chimeras: I: (impact) and C: (context dependence) are the free energy difference and sum, respectively, of the effects on a given property measured in forward and retro chimeras. The forward chimera is made by substitution of a part "a" from ensemble A into the analogous position of homologous ensemble B (S:(B --> A)). The C: value is a measure of the interaction of the interrogated position with its surroundings, whereas I: is an expression of the quantitative importance of the probed position. Both I: and C: vary with the evaluated property, for example, kinetics, binding, thermostability, and so forth. The retro chimera is the reverse substitution of the analogous part "b" from B into A, S:(A --> B). The I: and C: values derived from original data for forward and retro mutations in aspartate and tyrosine aminotransferase, from literature data for quasi domain exchange in oncomodulin and for the interaction of Tat with bovine and human TAR are evaluated. The most salient derived conclusions are, first, that Thr 109 (AATase) or Ser 109 (TATase) is an important discriminator for dicarboxylic acid selectivity by these two enzymes (I: < -2.9 kcal/mol). The T109S mutation in AATase produces a nearly equal and opposite effect to S109T in TATase (C: < 0.4 kcal/mol). Second, an I: value of 5.5 kcal/mol describes the effects of mirror mutations D94S (site 1) and S55D (site 2) in the Ca(2+) binding sites of oncomodulin on Ca(2+) affinity. The second mirror set, G98D (site 1) and D59G (site 2), yields a smaller impact (I: = -3.4 kcal/mol) on Ca(2+) binding; however, the effect is significantly more nearly context independent (C: = -0.6 versus C: = -2.7 kcal/mol). Third, the stem and loop regions of HIV and BIV TAR are predominantly responsible for the species specific interaction with BIV Tat(65-81) (I: = -1.5 to -1.6 kcal/mol), whereas I: = 0.1 kcal/mol for bulge TAR chimeras. The C: values are from -0.3 to -1.2 kcal/mol. The analysis described should have important applications to protein design.