Identification of group specific component/vitamin D-binding protein (GC/DBP) mutants by isoelectric focusing in immobilized pH gradients

The six common genetic types of the group specific component/vitamin D-binding protein (GC/DBP) system are usually classified by isoelectric focusing in carrier ampholytes, followed by visualization of the GC proteins by immunoprinting with monospecific antiserum. In addition, more than 120 mutant GC types have been discovered. For their identification additional methods were necessary, including polyacrylamide gel electrophoresis, isoelectric focusing in the presence of 3 M urea as well as isoelectric focusing in immobilized pH gradients. The application of the last method is described in detail and several examples of GC/DBP mutants identified thereby are presented.     

Subtyping of group specific component (GC) in human semen, blood and vaginal fluid by isoelectric focusing in immobilized pH gradients

The group specific component (GC) is stable and well suited for forensic casework. Isoelectric focusing of common GC variants from semen, seminal fluid, vaginal fluid and semen stains, on Immobiline DryPlates, pH 4.5-5.4, is of practical value in criminal investigations of sexual deliquencies. GC is present in normospermia and azoospermia seminal fluids and found in about 20% of the vaginal secretions. The GC patterns observed were similar and in accordance with the bands of the individual GC type in plasma/serum.     

Interaction of the vitamin D-binding protein (group-specific component) and its ligand 25-hydroxy-vitamin D3: binding differences of the various genetic types disclosed by isoelectric focusing

The three common variants of the vitamin D binding protein, also known as group specific component (Gc), namely types 1S, 1F and 2, as well as some rare variants were studied by thin-layer polyacrylamide gel isoelectric focusing in a pH 4.5-5.4 carrier ampholyte generated pH gradient, additionally containing N-(2-acetamido)-2-aminoethanesulfonic acid (ACES). Prior to isoelectric focusing, whole serum or purified preparations of the vitamin D binding protein were incubated with 25-hydroxycholecalciferol at various ligand/protein ratios. Binding differences were found for the anodal and cathodal isoforms of Gc 1 variants and also for various allelic types. Isoforms with higher isoelectric points generally had a lower affinity for the ligand than the variants with lower isoelectric points.     

Subtyping of group specific component (GC) in micro-bloodstains and semen stains by isoelectric focusing in ultrathin immobilized pH gradient gels followed by enzyme immunodetection

A powerful method for group specific component (GC) subtyping with good resolution of GC bands by isoelectric focusing on ultrathin immobilized pH gradients is described. After separation, GC detection is achieved using a highly sensitive alkaline phosphatase conjugated enzyme-immuno system. The efficiency of the method in forensic case work for subtyping GC from diluted bloodstain extracts, blood micro-stains and semen stains is demonstrated. Furthermore, GC subtyping and selective detection of human GC provides the evidence for human origin of the stain. This is important when analyzing microstains with limited stain consumption.     

Comparative analysis of adaptive neuro-fuzzy inference system (ANFIS) and RSRM models to predict DBP (trihalomethanes) levels in the water treatment plant

Since disinfection by-products are a growing concern, it is important to know their quantities in water treatment plants before they are released to the public. As a result, there is a requirement for constant monitoring of disinfection by-products (DBPs), which can have major consequences for human health and productivity. Consequently, in previous studies, several models for predicting disinfection byproduct formation in drinking water have been developed which were either linear/log-linear, hybrid or neural network (radial basis function). In this study, an adaptive neuro-fuzzy inference system (ANFIS) is proposed for predicting trihalomethane levels in real distribution systems. To train and verify the proposed model, 24 sets of data were used, including THMs levels (TCM, BDCM, DBCM and T-THM levels) and five parameters (pH, temperature, UVA₂₅₄, residual chlorine, and dissolved organic carbon). As compared to response surface modeling (RSRM) coefficient of determination, R² is between 0.727 < R² < 0.886, average absolute deviation, AAD = 4.07–10.99 %), MAE = 0.01 – 0.978, and RMSE = 0.017 – 1.449. Further, ANFIS for THMs (T-THMs, TCM, BDCM, and DBCM) prediction consistently show higher regression coefficients between 0.956 < R² < 0.989, average absolute deviation, AAD = 0.350 – 1.977 %), MAE = 0.002 – 0.133, and RMSE = 0.007 – 0.401, Consequently, based on the statistical indices obtained, ANFIS, on the other hand, proved to be effective for predicting the formation of THMs, and thus allowed improved DBPs monitoring in water treatment systems.     

Adsorbate-geometry specific subsurface relaxation in the CO/Pt(111) system

A dramatic multilayer substrate relaxation is observed for the (square root 19 x square root 19)-13CO adlayer phase on a Pt(111) electrode by surface X-ray scattering. Within the (square root 19 x square root 19) unit cell, a vertical expansion of 0.28 A was determined for the Pt atoms under near-top-site CO molecules, whereas only 0.04 A was found under near-bridge-site CO molecules. The lateral displacements involve small rotations toward more symmetric bonding. Both the expansions and rotations extend into the bulk with a decay length of 1.8 Pt layers. This nonuniform layer expansion, hitherto unseen, appears to be a manifestation of the differential stress induced by CO adsorption at different sites.     

An integrated GC/FT-IR system for the analysis of environmental pollutants

An integrated gas chromatography/Fourier transform infrared spectrometry (GC/FT-IR) system developed for the analysis of environmental pollutants is described. The versatility of the system allows the utilization of many different techniques of sample introduction and manipulation during analysis. The sample can be introduced by direct injection or thermal desorption from an adsorbent cartridge, and can then be separated on one of two capillary columns and detected by FT-IR or an FID. Cold traps and collection cartridges incorporated in the system permit recovery and additional fractionation of samples. Recovered sample and sample fractions can then be re-analyzed by GC/FT-IR or subsequently analyzed by GC/MS or other methods.     

气相色谱-质谱联用法同时测定动物尿液中克伦特罗和沙丁胺醇

建立了尿液中克伦特罗和沙丁胺醇同时测定的GC-MS方法. 尿液在碱性条件下以异丁醇提取后, 用Waters Oasis MCX小柱净化, 吹干后用BSTFA进行硅烷化衍生, 采用GC-EI-MS法, 选择离子方式采集数据. 结果表明, 克伦特罗和沙丁胺醇的线性范围为0.002～1.00 mg/L, 相关系数分别为0.9993和0.9985, 方法检出限可达0.5 μg/L (S/N=10). 尿液中克伦特罗和沙丁胺醇的回收率分别为75.9%～89.2%和73.6%～89.5%, 相对标准偏差(RSD)均不大于10%.     

Alkoxy/siloxy group exchange in the system vinyltrialkoxysilane-iridium(I) siloxide complex

A study of reactions of dimeric siloxide iridium complex, [[(cod)Ir(mu-OSiMe3)]2] (1) with vinyltriethoxysilane and vinyltrimethoxysilane has revealed a new type of the reation--alkoxy group transfer from silicon to iridium with a simultaneous transfer of a siloxy group from iridium to silicon--as a result of which vinyldialkoxytrimethyldisiloxane and dimeric alkoxide iridium complex [[(cod)Ir(mu-OR)]2] (3) are formed. The structure of [[(cod)Ir(mu-OEt)]2] (3a) has been solved by X-ray diffraction.     

Three-way principal component analysis of the volatile fraction by HS-SPME/GC of aceto balsamico tradizionale of modena

The present research is aimed at monitoring the evolution of the volatile organic compounds of different samples of aceto balsamico tradizionale of modena (ABTM) during ageing. The flavouring compounds, headspace fraction, of the vinegars of four batterie were sampled by solid phase microextraction technique (SPME), and successively analysed by gas chromatography. Obtaining a data set characterized by different sources of variability such as, different producers, samples of different age and chromatographic profile. The gas chromatographic signals were processed by a three-way data analysis method (Tucker3), which allows an easy visualisation of the data by furnishing a distinct set of graphs for each source of variability. The obtained results indicate that the samples can be separated according to their age highlighting the chemical constituents, which play a major role for their differentiation. The present study represents an example of how the application of Tucker3 models, on gas chromatographic signals may help to follow the transformation processes of food products.     

蛋白受体结合筛选-气质联用法测定动物组织中的氯霉素残留

建立了动物组织中氯霉素残留的蛋白受体结合筛选和GC MS分析方法。受体结合生物筛选的筛选限为0.1μg kg,假阴性率为零,其它常用抗生素的交叉反应率均<0.1%。筛选阳性样本经液液分配萃取、C18SPE固相萃取净化后衍生进行GC MS的NCI选择离子监测分析。其定量检出限为0 1μg kg,样品添加水平0 1～10μg kg时,回收率为76 2%～103 5%,相对标准偏差为7 5%～10 6%。     

The utilization of TG/GC/MS in the establishment of the mechanism of poly(styrene) degradation

General purpose poly(styrene) is a large volume commodity polymer used in a variety of applications. It is widely used in food packaging, particularly for baked goods. In this application, the presence of styrene monomer, which has a distinctive taste and aroma, cannot be tolerated. Processing of the polymer and forming of the food container at an unacceptably high temperature leads to the formation of styrene monomer and finished articles with unacceptable aroma characteristics. An examination of the thermal degradation of poly(styrene) has revealed the origin of monomer formation. The thermal decomposition of poly(styrene) has been widely studied. However, most studies have been carried out at high temperature (>300°C) where many processes are occurring simultaneously. Degradation at lower temperature, 280°C, occurs in two well-defined steps. The first is thermolysis of a head-to-head bond present in the mainchain as a consequence of polymerization termination by radical coupling. This generates macroradicals which smoothly depolymerize to expel styrene monomer. The nature of the degradation is readily apparent from kinetic analysis of the isothermal thermogravimetry (TG) data and the identity of the single volatile product may be readily established by gas chromatography/mass spectrometry (GC/MS) analysis of the effluent from the TG analysis.     

GC/MS analysis of pesticides in the Ferrara area (Italy) surface water: a chemometric study

The development of a network to monitor surface waters is a critical element in the assessment, restoration and protection of water quality. In this study, concentrations of 42 pesticides--determined by GC-MS on samples from 11 points along the Ferrara area rivers--have been analyzed by chemometric tools. The data were collected over a three-year period (2002-2004). Principal component analysis of the detected pesticides was carried out in order to define the best spatial locations for the sampling points. The results obtained have been interpreted in view of agricultural land use. Time series data regarding pesticide contents in surface waters has been analyzed using the Autocorrelation function. This chemometric tool allows for seasonal trends and makes it possible to optimize sampling frequency in order to detect the effective maximum pesticide content.     

The potent anti-HIV protein cyanovirin-N contains two novel carbohydrate binding sites that selectively bind to Man(8) D1D3 and Man(9) with nanomolar affinity: implications for binding to the HIV envelope protein gp120.

Cyanovirin-N (CVN) is a monomeric 11 kDa cyanobacterial protein that potently inactivates diverse strains of human immunodeficiency virus (HIV) at the level of cell fusion by virtue of high affinity interactions with the surface envelope glycoprotein gp120. Several lines of evidence have suggested that CVN-gp120 interactions are in part mediated by N-linked complex carbohydrates present on gp120, but experimental evidence has been lacking. To this end we screened a comprehensive panel of carbohydrates which represent structurally the N-linked carbohydrates found on gp120 for their ability to inhibit the fusion-blocking activity of CVN in a quantitative HIV-1 envelope-mediated cell fusion assay. Our results show that CVN specifically recognizes with nanomolar affinity Man(9)GlcNAc(2) and the D1D3 isomer of Man(8)GlcNAc(2). Nonlinear least squares best fitting of titration data generated using the cell fusion assay show that CVN binds to gp120 with an equilibrium association constant (K(a)) of 2.4 (+/- 0.1) x 10(7) M(-1) and an apparent stoichiometry of 2 equiv of CVN per gp120, Man(8)GlcNAc(2) D1D3 acts as a divalent ligand (2 CVN:1 Man(8)) with a K(a) of 5.4 (+/- 0.5) x 10(7) M(-1), and Man(9)GlcNAc(2) functions as a trivalent ligand (3 CVN:1 Man(9)) with a K(a) of 1.3 (+/- 0.3) x 10(8) M(-1). Isothermal titration calorimetry experiments of CVN binding to Man(9)GlcNAc(2) at micromolar concentrations confirmed the nanomolar affinity (K(a) = 1.5 (+/- 0.9) x 10(8) M(-1)), and the fitted data indicated a stoichiometry equal to approximately one (1 Man(9):1 CVN). The 1:1 stoichiometry at micromolar concentrations suggested that CVN has not only a high affinity binding site-relevant to the studies at nM concentrations-but a lower affinity site as well that facilitates cross-linking of CVN-oligomannose at micromolar concentrations or higher. The specificity of CVN for Man(8) D1D3 and Man(9) over the D1D2 isomer of Man(8) indicated that the minimum structure required for high affinity binding comprises Manalpha1 --> 2Manalpha. By following the (1)H-(15)N correlation spectrum of (15)N-labeled CVN upon titration with this disaccharide, we unambiguously demonstrate that CVN recognizes and binds to the disaccharide Manalpha1 --> 2Manalpha via two distinct binding sites of differing affinities located on opposite ends of the protein. The high affinity site has a K(a) of 7.2 (+/- 4) x 10(6) M(-1) and the low affinity site a K(a) of 6.8 (+/- 4) x 10(5) M(-1) as determined by isothermal titration calorimetry. Mapped surfaces of the carbohydrate binding sites are presented, and implications for binding to gp120 are discussed.