Artificial immunoglobulin G-binding protein mimetic to staphylococcal protein A. Its production and application to affinity purification of immunoglobulin G.

Staphylococcal protein A consists of a single polypeptide with five immunoglobulin G (IgG)-binding domains, which are linked as E-D-A-B-C in this order from the amino terminal. The DNA coding domains A-B were polymerized one to six times linearly, taking advantage of the non-palindromic nucleotide sequence of the AccI recognition site and the resultant DNAs were inserted in pTRP vector carrying trp promoter. The artificial IgG-binding proteins [pA(AB)1-6], which had been expressed in Escherichia coli JM109, were purified by methods involving IgG-Sepharose affinity chromatography. Among pA(AB)1-6 immobilized on cyanogen bromide-Sepharose, pA(AB)4-Sepharose was the highest in IgG-binding capacity at the same level of mg protein per ml gel, about 30% higher than protein A-Sepharose. At 8 mg protein per ml gel, it bound and eluted about 24 mg of IgG from rabbit serum. Its IgG-binding capacities were the highest with porcine, rabbit, human and guinea pig sera, intermediate with bovine, horse and sheep sera and the lowest with mouse, goat, rat and chicken sera.     

Variations in content and structure of glycosaminoglycans of the vitreous gel from different mammalian species

The vitreous of all species is composed of essentially the same type of extracellular matrix macromolecules organized to a transparent gel. In this study, the composition and fi ne chemical structure of the glycosaminoglycans (GAGs) in the vitreous gel from sheep and goat were determined and compared with those of human and pig vitreous gels. The results showed that, in all examined species; hyaluronan (HA) was the predominant GAG, whereas chondroitin sulphate (CS) was the minor one. In the vitreous gel of the most relative species, i.e. sheep and goat, higher amounts of both of HA and CS were estimated as compared with pig and human tissues. The distribution of hydrodynamic sizes of HA and CS was significantly differed among different species. All HA preparations consisted of molecules with great variability in hydrodynamic sizes. The relative proportions of the large HA molecules (size >1.8 x 10(6) kDa) were significantly higher in sheep and goat as compared with human and pig vitreous gel. The length of CS chains was also of larger size in sheep and goat (50 and 58 kDa, respectively) than the respective chains in human and pig vitreous gel (38 and 28 kDa, respectively). The sulphation patterns of CS preparations were determined following enzymic treatments, HPLC and capillary electrophoretic analyses. The human vitreous-derived CS chains showed quite different sulphation profile than that of CS isolated from other species, since 4-sulphated disaccharides were identified as the dominant moiety. In conclusion, significant compositional and structural variations between the vitreous matrixes of different species at the GAG level were identified. The functional significance of these species-dependent variations is discussed.     

Development of a multiplex system to assess DNA persistence in taphonomic studies

In this study, we have developed a PCR multiplex that can be used to assess DNA degradation and at the same time monitor for inhibition: primers have been designed to amplify human, pig, and rabbit DNA, allowing pig and rabbit to be used as experimental models for taphonomic research, but also enabling studies on human DNA persistence in forensic evidence. Internal amplified controls have been added to monitor for inhibition, allowing the effects of degradation and inhibition to be differentiated. Sequence data for single‐copy nuclear recombination activation gene (RAG‐1) from human, pig, and rabbit were aligned to identify conserved regions and primers were designed that targeted amplicons of 70, 194, 305, and 384 bp. Robust amplification in all three species was possible using as little as 0.3 ng of template DNA. These have been combined with primers that will amplify a bacterial DNA template within the PCR. The multiplex has been evaluated in a series of experiments to gain more knowledge of DNA persistence in soft tissues, which can be important when assessing what material to collect following events such as mass disasters or conflict, when muscle or bone material can be used to aid with the identification of human remains. The experiments used pigs as a model species. When whole pig bodies were exposed to the environment in Northwest England, DNA in muscle tissue persisted for over 24 days in the summer and over 77 days in the winter, with full profiles generated from these samples. In addition to time, accumulated degree days (ADD) were also used as a measure that combines both time and temperature—24 days was in summer equivalent to 295 ADD whereas 77 days in winter was equivalent to 494 ADD.     

Biosensor discrimination of meat juice from various animals using a lectin panel and ellipsometry

In this work, simple microcontact printed gold-wafers were used to make a lectin panel for investigation and discrimination of different meat juices from fresh meat of cattle, chicken, pig, cod, turkey and lamb. Seven different lectins were thus attached to gold surfaces using the streptavidin-biotin method. Lectins recognize and bind specifically to carbohydrate structures present on different proteins. The biorecognition was evaluated with null ellipsometry and the data obtained was related to an internal standard of lactoferrin. The data was evaluated with multivariate data analysis techniques to identify possible discrimination or grouping of data. Scanning ellipsometry was used for visualization of the binding pattern of the lectins and the meat juice proteins. The two-dimensional images obtained could be used to visualize the protein distribution, furthermore, to exclude anomalies. The results showed that the different meat juices from the six different species: cattle, chicken, pig, cod, turkey and lamb could be discriminated from each other. The results showed to be more repetitive for the mammalian meat juices. Using a simple model based on an artificial neuronal net, it was also possible to classify meat juices from the mammals investigated.     

Romancing the "hidden proteome", Anno Domini two zero zero seven

The mechanism of action and properties of a solid-phase ligand library made of hexapeptides, for capturing the "hidden proteome", i.e. the low- and very low-abundance proteins constituting the vast majority of species in any proteome, be it a cell or tissue lysate or a biological fluid, are here reviewed. Mechanisms of adsorption are evaluated, as well as different protocols for en bloc or sequential elution of the captured polypeptides. Examples are given of capture of proteins from serum, human platelet extracts, bacterial extract and egg white. The increment in detection of low-abundance species appears to be of at least four-fold as compared with untreated samples. One particular aspect of this capture is the adsorption of a high proportion of small peptides (in the Mr 600-8000 Da range) that are normally lost upon electrophoretic two-dimensional mapping. Such a peptide population, in human sera, may be of particular importance since it may contain protein cleavage products of diagnostic value.     

Two-dimensional gel electrophoresis as analytical tool for identifying Candida albicans immunogenic proteins

This paper reports the usefulness of two-dimensional gel electrophoresis followed by Western blotting with sera from patients with systemic candidiasis in the identification of the major Candida albicans antigens. In order to have different patterns of protein expression and subcellular localization, three types of protein preparations were obtained: cytoplasmic extracts, protoplast lysates and proteins secreted by protoplasts regenerating their cell wall. These proteins were separated by high-resolution two-dimensional electrophoresis using an immobilized pH gradient. Western blotting with sera from patients with systemic candidiasis allowed the detection of more than 18 immunoreactive proteins. Some of these proteins had different isoforms. All sera reacted with at least three C. albicans proteins and the most reactive serum detected up to eleven proteins. Some of these antigens, e.g., enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), have been identified on the 2-D map. The most reactive proteins were enolase and a 34 kDa protein in the acidic part of the gel (pI 4-4.4) that was only detected in regenerating protoplast-secreted proteins. The identification of all these antigens would be useful for the development of diagnostic strategies.     

Mutation scanning-coupled analysis of haplotypic variability in mitochondrial DNA regions reveals low gene flow between human and porcine Ascaris in endemic regions of China

Haplotypic variation within and among the Ascaris populations representing six provinces in China was investigated. Mitochondrial DNA regions in the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase subunit 1 (nad1) genes were amplified by PCR from total genomic DNA samples (n > 720) from Ascaris individuals from humans and pigs, and subjected to mutation scanning and subsequent selective sequencing. For the cox1, ten different electrophoretic profiles were recorded for human Ascaris, and the same number for pig Ascaris, one of them being common to both host species. For the nad1, 11 different profiles were detected for human Ascaris, and 15 for pig Ascaris. Having defined all haplotypes (20 for pcox1 and 26 for pnad1) by sequencing, their frequencies were estimated in each of the two host species and each of the six provinces. For each mitochondrial region, the frequency of the different haplotypes varied considerably, depending on host species and geographical origin. Analysis of the sequence data (representing all haplotypes for each mitochondrial locus) by F-statistics indicated restricted gene flow between human Ascaris and pig Ascaris, and supported the conclusions from previous molecular epidemiological investigations that pigs are not a significant source of Ascaris infection in humans in endemic regions.     

Gold nanoparticle sensor for homocysteine thiolactone-induced protein modification

Homocysteine thiolactone-induced protein modification (HTPM) is a unique post-translational protein modification that is recognized as an emergent biomarker for cardiovascular disease. HTPM involves the site-specific acylation of proteins at lysine residues by homocysteine thiolactone (HTL) to produce protein homocystamide, which has been found at elevated levels in patients with coronary heart disease. Herein, we report the development of a novel gold nanoparticle (GNP) biochemical sensor for detection of protein homocystamide in an in vitro serum protein-based model system. Human serum albumin (HSA) and human sera were subjected to HTPM in vitro to produce HSA-homocystamide or serum protein homocystamide, respectively, which was subsequently treated with citrate-capped GNPs. This GNP sensor typically provided instantaneous visual confirmation of HTPM in the protein model systems. Transmission electron microscopy images of the GNPs in the presence of HSA-homocystamide suggest that modification-directed nanoparticle assembly is the mechanism by which the biochemical sensor produces a colorimetric signal. The resultant nanoparticle-protein assembly exhibited excellent thermal and dilutional stability, which is expected for a system stabilized by chemisorption and intermolecular disulfide bonding. The sensor typically provided a linear response for modified human sera concentrations greater than approximately 5 mg/mL. The calculated limit of detection and calibration sensitivity for the method in human sera were 5.2 mg/mL and 13.6 AU . (microg/mL)-1, respectively.     

2-D difference gel electrophoresis of the lung squamous cell carcinoma versus normal sera demonstrates consistent alterations in the levels of ten specific proteins

Most lung cancers are diagnosed too late for curative treatment to be possible, therefore early detection is crucial. Serum proteins are a rich source of biomarkers and have the potential to be used as diagnostic and prognostic indicators for lung cancer. In order to examine differences in serum levels of specific proteins associated with human lung squamous carcinoma, immunodepletion of albumin and five other high-abundant serum proteins followed by 2-D difference gel electrophoresis (DIGE) analysis and subsequent MS was used to generate a panel of proteins found to be differentially expressed between the cancer and normal samples. Proteins found to have increased abundance levels in squamous cell carcinoma sera compared to normal sera included apolipoprotein A-IV precursor, chain F; human complement component C3c, haptoglobin, serum amyloid A protein precursor and Ras-related protein Rab-7b. Proteins found to have lower abundance levels in squamous cell carcinoma sera compared to normal sera included alpha-2-HS glycoprotein, hemopexin precursor, proapolipoprotein, antithrombin III and SP40; 40. The data presented here demonstrate that high-abundant protein removal combined with 2-D DIGE is a powerful strategy for the discovery of potential biomarkers. The identification of lung cancer-specific biomarkers is crucial to early detection, which in turn could lead to a dramatic increase in survival rates.     

Methods for research on immune complement activation on modified sensor surfaces

We have developed a methodological system consisting of a new surface sensitive quartz crystal microbalance with dissipation monitoring (QCM-D) sensor surfaces together with different surface modification methods for the investigation of surface associated complement activation in human sera. The QCM-D surface, 10 mm in diameter, was modified by spin-coating of poly(urethane urea) (PUUR) and polystyrene (PS). Some sensor surfaces were also sputtered with titanium (Ti) or modified by hydrophobic self-assembled monolayer (SAM) of an 18-carbon alkane thiol with a ---CH₃ end group. The amount of surface deposited complement protein was investigated by incubation of the modified sensor surfaces in human sera, followed by incubation with antibodies directed against complement factor 3c (C3c). The amounts of bound anti-C3c were then used as an arbitrary measure of surface induced complement activation. The order of complement activation of the different surfaces, as judged by three separate measurements per surface modification, was PUUR>PS=SAM>Ti. The Ti surface had a similar low degree of anti-C3c binding as the negative controls (heat inactivated sera). The novel QCM-D methodology was found to be very simple, accurate, sensitive and well suited as a screening method for complement activation and protein adsorption on different materials. We also compared the sensitivity of QCM-D method with surface plasmon resonance (SPR) for the quantification of protein adsorption and complement activation on gold sensor surfaces. The QCM-D method was equally sensitive as the SPR for the detection of protein adsorption from a solution independently if low flow rate (5 μl/min) was used. A slight increase in sensitivity was found at higher flow rate (30 μl/min). However, we found it difficult to use the SPR method on the Ti, PS and PUUR surfaces due to decreased light penetration of the modified SPR sensor chip.     

Protein profiles in sera of patients with malignant cutaneous melanoma

Seventeen samples of sera from patients with malignant cutaneous melanoma at various stages and 14 samples from healthy subjects were analysed by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. Results highlighted the presence of several protein species at molecular weights lower than 30000 Da, presumably originating from proteolysis, in the sera of the patients with melanoma. These species were completely absent in healthy subjects. In particular, the presence and abundance of species with molecular weights in the range 2500-3500 Da exhibit significant variations related to the different clinical stages of the disease.     

Nickel species analysis of human colonic tissue using liquid chromatography,gel electrophoresis and mass spectrometry

Studies to specify metal-binding species, such as metalloproteins that are present in trace amounts in colonic cell cytosol, using chromatographic separation methods in combination with inductively coupled plasma mass spectrometry (ICP-MS) as element-specific detection require an optimised sample preparation regarding the solubilisation of the proteins. Focus should be taken to avoid metal contamination, enzymatic digestion by different proteases and oxidation. In this article different sample preparation methods are studied to find a suitable method for the isolation and characterisation of Ni species previously found in cytosols from normal and malignant tissues of the human colon. The total Ni concentrations of the cytosols were determined as well as the total protein content. Thus, a Ni-containing protein could be isolated from cytosols of malignant human colonic tissues using size-exclusion chromatography with ICP-MS for element-specific detection. Ni-containing species in the molecular mass range from 10,000 to 20,000 Da were found and pre-concentrated. The determination of the molecular mass of the species was performed through online coupling of reversed-phase chromatography with electrospray ionisation quadrupole time-of-flight MS. Using identical chromatographic conditions and ICP-MS the detected protein was shown to contain Ni.     

Protein binding of quinolonecarboxylic acids. I. Cinoxacin, nalidixic acid and pipemidic acid

Binding of cinoxain (CINX), nalidixic acid (NA) and pipemidic acid (PPA) to serum proteins was investigated by equilibrium dialysis, ultrafiltration and circular dichroism (CD) spectroscopy. CINX and NA were found to bind mainly to albumin in human serum, the latter interacting with the protein about ten times as strongly as CINX at pH 7.4 and 37 degrees C. PPA showed little or no significant binding to human serum albumin (HSA), alpha 1-acid glycoprotein, and globulins, but showed 20-30% binding to protein in human serum. The CD results were suggestive of some weak interaction of PPA with human apotransferrin. Binding of the three drugs to HSA was found to depend on the lipophilicity of their substituents at the 7-position. The degree of protein binding for human, dog and rat sera at 37 degrees C was in the order of NA (92-97%) greater than CINX (68-90%) greater than PPA (20-30%) at drug concentrations of 10-30 micrograms/ml. CINX showed relatively large species dependence in serum protein binding, which seemed to be due to different affinities of this drug to the respective albumins. CINX was found to bind to rat serum albumin as strongly as NA.     

Raman spectroscopy based analysis of milk using random forest classification

The development of a classification system based on the Raman spectra of milk samples is proposed in present study. Such development could be useful for nutritionists in suggesting healthy food to infants for their proper growth. Previously, molecular structures in milk samples have been exploited by Raman spectroscopy. In the current study, Raman spectral data of milk samples of different species is utilized for multi-class classification using a dimensionality reduction technique in combination with random forest (RF) classifier. Quantitative and experimental analysis is based on locally collected milk samples of different species including cow, buffalo, goat and human. This classification is based on the variations (different concentrations of the components present in milk such as proteins, milk fats, lactose etc.) in the intensities of Raman peaks of milk samples. Principal component analysis (PCA) is used as a dimensionality reduction technique in combination with RF to highlight the variations which can differentiate the Raman spectra of milk samples from different species. The proposed technique has demonstrated sufficient potential to be used for differentiation between milk samples of different species as the average accuracy of about 93.7%, precision of about 94%, specificity of about 97% and sensitivity of about 93% has been achieved.     

THE CRYSTAL STRUCTURE OF SILVER CARP INSULIN AT MEDIUM RESOLUTION

It is an important way of surveying the structure-functionrelationship of insulin tostudy insulins from different species. Based on the structure model of an orthorhombic crys-tal obtained by the molecular replacement method, the crystallographic refinement of a hex-amer of silver carp insulin in an asymmetric unit has been carried out with 2.8A resolutiondata using the restrained least- squares method. The comparisons of insulin structures haveshown that the six silver carp insulin molecules have very similar but not identical three- di-mensional structures which are similar to the known 2Zn pig insulin structure but remarka-bly different in some local conformations.     

The crystal structure of silver carp insulin at medium resolution

It is an important way of surveying the structure-function relationship of insulin to study insulins from different species. Based on the structure model of an orthorhombic crystal obtained by the molecular replacement method, the crystallographic refinement of a hexamer of silver carp insulin in an asymmetric unit has been carried out with 2.8 A resolution data using the restrained least-squares method. The comparisons of insulin structures have shown that the six silver carp insulin molecules have very similar but not identical three-dimensional structures which are similar to the known 2 Zn pig insulin structure but remarkably different in some local conformations.     

A novel method for the preparation of T3 free plasma/serum for routine RIA

A novel method to eliminate T₃ from gross sera/plasma based on prior treatment with 8-anilino-1-naphthalene sulphonic acid has been standardized. This method is rapid with a minimum loss of protein and uses only small quantities of charcoal. The treated sera have been tested for acceptance in the RIA system for routine assays.     

Serological proteome analysis of dogs with breast cancer unveils common serum biomarkers with human counterparts

Canine mammary tumor is being touted as a model for investigating the human breast cancer. Breast cancer of the both species has similar biological behavior, histopathologic characteristics, and metastatic pattern. In this study, we used the serological proteome analysis to detect autoantigens that elicit a humoral response in dogs with mammary tumor in order to identify serum biomarkers with potential usefulness as diagnostic markers and to better understand molecular mechanisms underlying canine breast cancer development. Protein extract from a cell line was subject to 2DE followed by Western blotting using sera from 15 dogs with mammary tumor and sera from 15 healthy control dogs. Immunoreactive autoantigens were subsequently identified by the MALDI‐TOF MS. Four autoantigens, including manganese‐superoxide dismutase, triose phosphate isomerase, alpha‐enolase, and phosphoglycerate mutase1, with significantly higher immunoreactivity in the tumor samples than in the normal samples were identified as biomarker candidates. Immunohistochemistry and Western blotting revealed higher expression of these biomarkers in the malignant tumors than in the normal or benign tumors. The autoantigens found in this study have been reported to elicit autoantibody response in the human breast cancer, indicating the similarity of breast cancer proteome profile in dogs with that in human beings.     

A multiplex assay to identify 18 European mammal species from mixtures using the mitochondrial cytochrome b gene

A novel species-specific multiplex to identify 18 common European mammalian species (badger, cat, cow, dog, donkey, fox, goat, guinea pig, harvest mouse, hedgehog, horse, house mouse, human, pig, rabbit, rat, red deer and sheep), many of which are often associated with forensic investigations, has been developed. The assay is based on the mitochondrial cytochrome b gene, which is commonly used in species identification and phylogeny studies. Areas of homology and variation were identified and were used to create universal and species-specific primers. The species-specific primers were designed such that they will only react with the species for which they were designed. Two primer sets were designed for each species making the test self-confirmatory. All primer sets produced the expected results. The multiplex was balanced at template concentration of 40 000 copies (approximately 1.36 pg). Validation was accomplished by analysing the same sample ten times to determine run variation and several samples for each species to determine between-sample variation. Twenty-eight additional mammalian species were reacted with the multiplex. The multiplex provides, for the first time, a definitive method for identification of species in a forensic context.     

茶（叶）碱的流动注射免疫试验法研究

本研究建立了一种多相抗哮喘麻醉药茶碱的酶免疫电化学检测法。此法使用含有Ａ蛋白免疫反应体的流动注射分析装置来分离与抗体结合和与抗体不结合的茶碱磷酸酯酶。抗体、标示剂和试样相继附着在Ａ蛋白免疫反应体上。当ｐＨ值约为中性时，抗体Ａ蛋白和抗体Ａ蛋白标示剂或试样发生反应，合成物则在ｐＨ为酸性情况下洗提。曾用ｐ－磷酸胺基苯（ＰＡＰＰ）和ｐ－胺基酚（ＰＡＰ）作为检测剂。在诸如不同的流动速率和不同的反应物浓度条件下进行了研究，并已用该法测定了血清茶碱，较好地得到了ｒ、ｓ、ｄ值和萃取率，其结果符合要求。