Ni(II)-based immobilized metal ion affinity chromatography of recombinant human prolactin from periplasmic Escherichia coli extracts

A novel, two-step preparative technique is described for the purification of authentic recombinant human prolactin (rhPRL) secreted into the periplasm of transformed Escherichia coli cells. The first step is based on immobilized metal ion affinity chromatography of periplasmic extract, using Ni(II) as a relatively specific ligand for hPRL in this system. It gives superior resolution and yield than established ion-exchange chromatography. Size-exclusion chromatography is used for further purification to >99.5% purity. The methodology is reproducible, leading to 77% recovery. Identity and purity of the rhPRL were demonstrated using sodium dodecylsulphate-polyacrylamide electrophoresis, isoelectric focusing, mass spectrometry (matrix-assisted laser desorption ionization time-of-flight), radioimmunoassay, RP-HPLC and high-performance size-exclusion chromatography. In the Nb2 bioassay, the hormone showed a bioactivity of 40.9 IU/mg.     

Laboratory Production of Human Prolactin from CHO Cells Adapted to Serum-Free Suspension Culture

Human prolactin (hPRL) is a polypeptide with 199 amino acids and a molecular mass of 23?kDa. Previously, a eukaryotic hPRL expression vector was used to transfect Chinese hamster ovary (CHO) cells: this work describes a fast and practical laboratory adaptation of these transfected cells, in ~40?days, to grow in suspension in serum-free medium. High cell densities of up to 4.0?×?10⁶ cell/ml were obtained from spinner flask cultures and a stable and continuous production process was developed for at least 30?days. Two harvesting strategies were set up, 50 or 100?% of the total conditioned medium being collected daily and replaced by fresh culture medium. The volumetric productivity was 5?C7???g hPRL/ml, as determined directly in the collected medium via reversed-phase HPLC (RP-HPLC). A two-step process based on a cationic exchanger followed by size exclusion chromatography was applied to obtain purified hPRL from conditioned medium. Two hPRL isoforms, non-glycosylated and glycosylated, could also be separated by high-performance size-exclusion chromatography (HPSEC) and, when analyzed by RP-HPLC, HPSEC, Western blotting, and bioassay, were found to be comparable to the World Health Organization International Reference Reagent of hPRL. These results are useful for the practical scale-up to the pilot and industrial scale of a bioprocess based on CHO cell culture.     

Analysis and Testing of Automotive MR Fluid Shock Absorber Based on Poiseuille Flow Mode

Analysis methodology and design theory of magnetorheological fluid (MR fluid) shock absorber based on of Poiseuille flow mode have been parented using Newton fluid model nonlinear Bingham plastic model. In the light of the technician requirements for Changan mini-automobile, a MR fluid shock absorber has been designed and fabricated using commercial MR fluid. We have validated experimentally the nonlinear MR fluid shock absorber model. The experimental results reveal that the analysis methodology and design theory is reasonable and the MR fluid shock absorber for a specific application can be designed according to the design methodology developed.     

Sequence comparison and environmental adaptation of a bacterial endonuclease

The periplasmic/extracellular bacterial enzyme endonuclease I was chosen as a model system to identify features that might be responsible for temperature- and salt adaptation. A statistical study of amino acid sequence properties belonging to endonuclease I enzymes from three mesophilic habitats (non-marine, brackish water and marine), and three marine temperature groups (psychrophile, intermediate and mesophile) has been conducted. Ten new endonuclease I genes have been sequenced in order to increase the sample size. A bioinformatical method of property dependent statistical analysis of alignments has been applied. To our knowledge this is the first time these methods have been used in order to investigate environmental adaptation of enzymes. Adaptation to low temperature seems to involve increased surface isoelectric point and hydrophobicity in contrast to salt adaptation in which the isoelectric point and hydrophobicity at the surface decreases. Redistribution of charge and hydrophobicity might be the most important signature for cold adaptation and salt adaptation of this enzyme class. The results indicate that general trends of adaptation are possible to elucidate from the amino acid sequences. Also in this paper a new scale of stratified B-factors, derived from the Protein Data Bank, is presented.     

Application of reversed phase high performance liquid chromatography to the analysis of sulphidopeptide leukotrienes in pig bile

Reversed phase HPLC methodology has been developed for separation of peptide leukotrienes and indomethacin in porcine bile. Reproducible recoveries were obtained using radioactive leukotrienes ([3H]LTC4, 57.1 +/- 2.5%; [3H]LTE4, 62.7 +/- 1.9%; [3H]LTD4, 54.3 +/- 2.7%). Radioimmunoassay of column eluant demonstrated that as little as 300 pg of exogenous leukotrienes could be measured in bile fluids, with similar recoveries. Analysis of bile sampled 60-90 min after initiation of experimental endotoxic shock in indomethacin treated pigs revealed a leukotriene concentration of 5.24 +/- 1.16 ng/mL(LTD4). This was significantly greater (p less than 0.05, n = 3) than that observed in samples collected prior to endotoxin (0.42 +/- 0.23 ng/mL), or from untreated animals (0.85 +/- 0.51 ng/mL). This method is thus applicable to investigation of the role of 5-lipoxygenase products in porcine models of human disease, including shock conditions such as endotoxaemia, during cyclooxygenase inhibition by indomethacin.     

Bacterial adhesion inhibition of the quaternary ammonium functionalized silica nanoparticles

Quaternary ammonium compounds have been considered as excellent antibacterial agents due to their effective biocidal activity, long term durability and environmentally friendly performance. In this work, 3-(trimethoxysilyl)-propyldimethyloctadecylammonium chloride as a quaternary ammonium silane was applied for the surface modification of silica nanoparticles. The quaternary ammonium silane provided silica surface with hydrophobicity and antibacterial properties. In addition, the glass surface which was coated with the surface modified silica nanoparticles presented bacterial growth inhibition activity. For comparison of bacterial growth resistance, hydrophobic silane (alkyl functionalized silane) modified silica nanoparticles and pristine silica nanoparticles were prepared. As a result of bacterial adhesion test, the quaternary ammonium functionalized silica nanoparticles exhibited the enhanced inhibition performance against growth of Gram-negative Escherichia coli (96.6%), Gram-positive Staphylococcus aureus (98.5%) and Deinococcus geothermalis (99.6%) compared to pristine silica nanoparticles. These bacteria resistances also were stronger than that of hydrophobically modified silica nanoparticles. It could be explained that the improved bacteria inhibition performance originated from the synergistic effect of hydrophobicity and antibacterial property of quaternary ammonium silane. Additionally, the antimicrobial efficacy of the fabricated nanoparticles increased with decreasing size of the nanoparticles.     

Integrated selective enrichment target--a microtechnology platform for matrix-assisted laser desorption/ionization-mass spectrometry applied on protein biomarkers in prostate diseases

The performance of a miniaturized sample processing platform for matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS), manufactured by silicon microfabrication, called integrated selective enrichment target (ISET) technology was evaluated in a biological context. The ISET serves as both sample treatment device and MALDI-MS target, and contains an array of 96 perforated nanovials, which each can be filled with 40 nL of reversed-phase beads. This methodology minimizes the number of sample transfers and the total surface area available for undesired adsorption of the analytes in order to provide high-sensitivity analysis. ISET technology was successfully applied for characterization of proteins coisolated by affinity chromatography of prostate-specific antigen (PSA) from human seminal fluid. The application of ISET sample preparation enabled multiple analyses to be performed on a limited sample volume, which resulted in the discovery that prolactin inducible protein (PIP) was coisolated from the samples.     

The release of heparinase from the periplasmic space of flavobacterium heparinum by three-step osmotic shock

Heparinase was released from the periplasmic space of Flavobacterium heparinum by three-step osmotic shock procedure. The procedure involves resuspending exponentially growing cells consecutively into (1) 40% sucrose, (2) 10 mM sodium phosphate, 2 mM magnesium chloride, pH 7, and (3) 10 mM sodium phosphate, 300 mM sodium chloride, 2 mM magnesium chloride, pH 7. Typically, 50-75% of the total heparinase activity is recovered by this procedure with an observed 7-15-fold increase in purity. The majority of heparinase activity is released in the final step of the procedure allowing for resolution from cytoplasmic and nonspecific periplasmic material. F. heparinum cells can be stored in 40% sucrose at 4 degrees C for up to one week without significant losses in recovery yields.     

仿生树叶模型的制作及在琼脂糖微流控芯片中的应用

树叶的脉序可以更好模拟人体血管微环境, 但以自然环境中树叶为模板进行实验存在一定的季节局限性; 且不能保证每次实验所用树叶脉序形状相同, 不易控制变量, 且会对环境造成一定破坏. 本文建立了一种简易的仿生树叶模型制作方法, 并通过仿生模型构建了经济、 易操作的琼脂糖微流控芯片. 分别测定了芯片的一、 二、 三级脉序数目及尺寸, 最大脉序尺寸值可达1038.02 μm, 最小脉序尺寸为36.32 μm, 宽度不一的通道构建为细菌趋化性实验提供了稳定可靠的梯度空间, 对探索药物筛选、 微生物利害等研究具有重要意义.     

Determination of ascorbic acid in pharmaceutical preparations by near infrared reflectance spectroscopy

We developed a method for determination of ascorbic acid in pharmaceutical preparations containing various excipients by using near infrared diffuse reflectance spectroscopy and two different calibration methods, viz. stepwise multiple linear regression (SMLR) and partial least-squares (PLS) regression, which provided comparable results and resulted in prediction errors of 1-2%. However, the PLS method provided somewhat better results with the more complex samples.     

Topology of an outer-membrane enzyme: Measuring oxygen and water contacts in solution NMR studies of PagP

The topology of the bacterial outer-membrane enzyme, PagP, in dodecylphosphocholine micelles was studied by solution NMR using oxygen and water contacts as probes of hydrophobicity and topology. The effects of oxygen on amide protons were measured at an oxygen partial pressure of 20 atm through the paramagnetic contribution to the relaxation rates associated with the decay of two-spin order. A significant gradation of paramagnetic rates was observed for backbone amides belonging to the transmembrane residues. These rates were observed to depend on immersion depth, local hydrophobicity, and steric effects. Variations in the paramagnetic relaxation rates due to local hydrophobicity or steric effects could be, to some extent, averaged out by considering an azimuthally averaged quantity. This averaged paramagnetic rate was found to have a distinct maximum exactly in the middle of the transmembrane domain of PagP, assuming the immersion depth axis is tilted by 25 degrees with respect to the barrel axis. Contact between the protein surface and water was assessed by measuring the amide decay rates during water saturation. The comparison of local contrast effects from both water and oxygen allows one to distinguish among steric effects, local hydrophobicity, and immersion depth. For example, the absence of contrast effects from either water or oxygen at the periplasmic end of beta-strands B and C was consistent with protection effects arising from the association with the N-terminal alpha-helix. A parameter defined by the natural logarithm of the ratio of the normalized paramagnetic relaxation rate to the normalized amide decay rate under water saturation was found to correlate with immersion depth of the corresponding backbone amide nuclei. The results suggest that the oxygen/water contrast experiments give direct information regarding membrane protein topology and surface hydrophobicities, thereby complementing existing NMR structure studies and ESR spin-labeling studies.     

A disposable electrochemical immunosensor for prolactin involving affinity reaction on streptavidin-functionalized magnetic particles

A novel electrochemical immunosensor was developed for the determination of the hormone prolactin. The design involved the use of screen-printed carbon electrodes and streptavidin-functionalized magnetic particles. Biotinylated anti-prolactin antibodies were immobilized onto the functionalized magnetic particles and a sandwich-type immunoassay involving prolactin and anti-prolactin antibody labelled with alkaline phosphatase was employed. The resulting bio-conjugate was trapped on the surface of the screen-printed electrode with a small magnet and prolactin quantification was accomplished by differential pulse voltammetry of 1-naphtol formed in the enzyme reaction using 1-naphtyl phosphate as alkaline phosphatase substrate. All variables involved in the preparation of the immunosensor and in the electrochemical detection step were optimized. The calibration plot for prolactin exhibited a linear range between 10 and 2000 ng mL(-1) with a slope value of 7.0 nA mL ng(-1). The limit of detection was 3.74 ng mL(-1). Furthermore, the modified magnetic beads-antiprolactin conjugates showed an excellent stability. The immunosensor exhibited also a high selectivity with respect to other hormones. The analytical usefulness of the immnunosensor was demonstrated by analyzing human sera spiked with prolactin at three different concentration levels.     

Bacterial adhesion to glass and metal-oxide surfaces

Metal oxides can increase the adhesion of negatively-charged bacteria to surfaces primarily due to their positive charge. However, the hydrophobicity of a metal-oxide surface can also increase adhesion of bacteria. In order to understand the relative contribution of charge and hydrophobicity to bacterial adhesion, we measured the adhesion of 8 strains of bacteria, under conditions of low and high-ionic strength (1 and 100 mM, respectively) to 11 different surfaces and examined adhesion as a function of charge, hydrophobicity (water contact angle) and surface energy. Inorganic surfaces included three uncoated glass surfaces and eight metal-oxide thin films prepared on the upper (non-tin-exposed) side of float glass by chemical vapor deposition. The Gram-negative bacteria differed in lengths of lipopolysaccharides on their outer surface (three Escherichia coli strains), the amounts of exopolysaccharides (two Pseudomonas aeruginosa strains), and their known relative adhesion to sand grains (two Burkholderia cepacia strains). One Gram positive bacterium was also used that had a lower adhesion to glass than these other bacteria (Bacillus subtilis). For all eight bacteria, there was a consistent increase in adhesion between with the type of inorganic surface in the order: float glass exposed to tin (coded here as Si-Sn), glass microscope slide (Si-m), uncoated air-side float glass surface (Si-a), followed by thin films of (Co(1-y-z)Fe(y)Cr(z))3O4, Ti/Fe/O, TiO2, SnO2, SnO2:F, SnO2:Sb, A1(2)O3, and Fe2O3 (the colon indicates metal doping, a slash indicates that the metal is a major component, while the dash is used to distinguish surfaces). Increasing the ionic strength from 1 to 100 mM increased adhesion by a factor of 2.0 +/- 0.6 (73% of the sample results were within the 95% CI) showing electrostatic charge was important in adhesion. However, adhesion was not significantly correlated with bacterial charge and contact angle. Adhesion (A) of the eight strains was significantly (P < 10(-25)) correlated with total adhesion free energy (U) between the bacteria and surface (A = 2162e(-1.8U)).Although the correlation was significant, agreement between the model and data was poor for the low energy surfaces (R2 = 0.68), indicating that better models or additional methods to characterize bacteria and surfaces are still needed to more accurately describe initial bacterial adhesion to inorganic surfaces.     

Fast speciation analysis of iodophenol compounds in river waters by capillary electrophoresis-inductively coupled plasma-mass spectrometry with off-line solid-phase microextraction

An analytical methodology for the fast separation and determination of iodophenol species in natural water samples was developed using capillary electrophoresis (CE) coupled to inductively coupled plasma-mass spectrometry (ICP-MS). Based on the element-specific and highly sensitive detection provided by ICP-MS, the methodology has been applied to the analysis of 2-iodophenol, 4-iodophenol, and 2,4,6-triiodophenol. The use of solid-phase microextraction (SPME), after proper optimization, improved the signal by a factor of 100 leading to detection limits in the sub microg.L(-1). Different desorption conditions of iodophenol compounds from the SPME microfiber were studied to achieve the optimum preconcentration factor and best analytical performance. Different CE conditions were studied to achieve complete baseline separation of iodophenols in short migration times. Three different CE buffer systems were evaluated using ICP-MS detection. A buffer solution containing 20 mmol.L(-1) 3-(cyclohexylamino)-1-propanesulfonic acid (CAPS) and an applied potential of +22 kV were finally selected leading to a maximum separation time of 6.6 min. A relative standard deviation (%RSD) of about 5.0% for ten consecutive determinations was obtained. Finally, the speciation methodology developed was utilized for the determination of iodophenol compounds in natural water samples.     

DSC analysis of human synovial fluid samples in the diagnostics of non-septic and septic arthritis

The synovial fluid analysis is an important method in diagnosing and managing septic arthritis. To reach a quick diagnosis, preferably faster than the microbiological cultures, could be a great advantage in the therapy. The differential scanning calorimetry (DSC) has recently been found useful in the differential diagnosis of septic and non-septic periprosthetic conditions. The aim of this study was to evaluate whether there is a specific pattern in the different grades of arthritis, and the effect of three different bacterial strains inoculated in synovial fluid. The sensitivity of our method was also determined by using synovial fluid samples with the different bacterial concentrations. Authors developed a standardized, experimental model to assess the denaturation characteristics of non-septic and inoculated synovial fluid, infected by different bacterial strains. The thermal characteristics [maximal denaturation temperature (T _m) and calorimetric enthalpy change (ΔH _cal)] were monitored by SETARAM Micro DSC-II calorimeter. The denaturation scans clearly demonstrated specific, representative curves in the case of different grades of arthritis, as well as with each individual bacterial strain. Therefore, thermoanalyses of human synovial fluid samples by DSC could be a useful tool in the staging of osteoarthritis and the diagnostics of septic arthritis.     

Tuning the hemolytic and antibacterial activities of amphiphilic polynorbornene derivatives

Amphiphilic cationic polynorbornene derivatives, soluble in water, were prepared from modular norbornene monomers, with a wide range of molecular weights (M(n) = 1600-137 500 g/mol) and narrow polydispersities (PDI = 1.1-1.3). The antibacterial activity determined by growth inhibition assays and the hemolytic activity against human red blood cells were measured and compared to determine the selectivity of the polymers for bacterial over mammalian cells. The effects of monomer repeat unit hydrophobicity and polymer molecular weight on antibacterial and hemolytic activities were determined. The hydrophobicity of the repeat unit was observed to have dramatic effects on antibacterial and hemolytic activities. Lipid membrane disruption activities of the polymers was confirmed by measuring polymer-induced dye leakage from large unilamellar vesicles. By tuning the overall hydrophobicity of the polymer through random copolymerizations of modular norbornene derivatives, highly selective, nonhemolytic antibacterial activities were obtained. For appropriate monomer composition, selectivity against bacteria versus human red blood cells was determined to be over 100.     

Hydrophobic interaction chromatography for the purification of a mycobacterial heat shock protein of relative molecular mass 60,000.

A recombinant mycobacterial heat shock protein of relative molecular mass 60,000 was purified by hydrophobic interaction chromatography. Chromatographic media with ligands of medium hydrophobicity, such as phenyl-Sepharose, bound too strongly to be used for the purification of this heat shock protein. Butyl-Sepharose, with weak hydrophobicity, allowed binding and elution with decreasing concentrations of ammonium sulphate, but only alkyl-Superose allowed the separation of two similar proteins from the Escherichia coli clone expressing the recombinant heat shock protein (relative molecular mass 60,000) of Mycobacterium bovis BCG. The binding parameters of recombinant human heat shock proteins of relative molecular mass 60,000 and 70,000 indicate that phenyl-Sepharose also binds too strongly for the separation of these two heat shock proteins.     

Magnetic solid phase extraction based on phenyl silica adsorbent for the determination of tetracyclines in milk samples by capillary electrophoresis

A magnetic solid phase extraction method coupled to capillary electrophoresis is proposed for the determination of tetracycline, oxytetracycline, chlortetracycline and doxycycline in milk samples. Five different magnetic phenyl silica adsorbents covered with magnetite were synthesized by varying the molar ratio of phenyltrimethylsilane and tetramethylorthosilicate; these adsorbents were evaluated in terms of their pH and degree of hydrophobicity for tetracycline retention. The optimal, selected combination of conditions was a pH of 10.0 and a magnetic sorbent ratio of 4:1; under these conditions, the retention capacity ranged from 99.7% to 101.2% for the four tetracyclines analyzed. The elution conditions and initial sample volume of the proposed extraction method were also optimized, and the best results were obtained with 1×10(-3) M acetic acid in methanol as eluent and a 200 ml of sample volume. Under optimal conditions, average recoveries ranged from 94.2% to 99.8% and the limits of detection ranged from 2 to 9 μg l(-1) for the four tetracyclines. After the proposed method was optimized and validated, 25 milk samples of different brands were analyzed, oxytetracycline residues were detected in five samples, in concentrations ranging from 98 to 213 μg l(-1). Subsequent analysis of positive samples by SPE-CE and magnetic solid phase extraction-HPLC revealed than no significant differences were found from results obtained by the proposed methodology. Thus, the developed magnetic extraction is a robust pre-concentration technique that can be coupled to other analytical methods for the quantitative determination of tetracyclines.     

Determination of secnidazole in human plasma by high-performance liquid chromatography with UV detection and its application to the bioequivalence studies

A simple, accurate, precise and sensitive HPLC-UV method was developed for the determination of secnidazole in human plasma. Secnidazole and tinidazole (IS) were extracted from 0.2 mL of human plasma by ethyl acetate. Secnidazole was then separated by HPLC on a Diamond C(18) column and quantified by ultraviolet detection at 319 nm. The mobile phase consisted of acetonitrile-aqueous 5 mm sodium acetate (30:70, v/v) containing of 0.1% acetic acid adjusted to pH 4.0, and the flow rate was 1.0 mL/min. The low limit of quantification was 0.1 microg/mL. The method was linear over the concentration range 0.1-25.0 microg/mL (R(2) = 1.000). The recovery of secnidazole from human plasma ranged from 76.5 to 89.1%. Inter- and intra-assay precision ranged from 3.3 to 10.7%. Secnidazole in plasma was stable when stored at ambient temperature for 8 h, at -20 degrees C for 2 weeks and at -20 degrees C for three freeze-thaw cycles. The developed method was successfully applied to the pharmacokinetic and bioequivalence studies between test and reference secnidazole tablets following a single 500 mg oral dosage to 20 healthy volunteers of both genders. Pharmacokinetics parameters T(max), C(max), AUC(0-)t, AUC(0-infinity), T(1/2) were determined of both preparations. The analysis of variance (ANOVA) did not show any significant difference between the two preparations and 90% confidence intervals fell within the acceptable range for bioequivalence. It was concluded that the two secnidazole preparations are bioequivalence and may be used interchangeably.     

Isoelectric focusing nonporous silica reversed-phase high-performance liquid chromatography/electrospray ionization time-of-flight mass spectrometry: a three-dimensional liquid-phase protein separation method as applied to the human erythroleukemia cell-line

A liquid-phase three-dimensional protein separation method has been developed that is used to separate the cytosolic fraction of a HEL cell lysate via isoelectric focusing (IEF), nonporous silica (NPS) reversed-phase high-performance liquid chromatography (RP-HPLC) and electrospray ionization time-of-flight mass spectrometry (ESI-TOFMS), respectively. Several hundred unique protein molecular weights were observed in a pI range from 4.8 to 8.5 and a mass range from 5 to 85 kDa. Proteins were positively identified by analysis of the pI (+/-0.5 pI units), an intact protein molecular weight (+/-150 ppm), and peptide mass mapping results. Using the molecular weight (MW) and peptide mapping results of identified proteins it was possible to characterize their posttranslational (PTMs) and/or sequence modifications. PTMs were detected on both forms of cytosolic actin, heat shock 90 beta, HINT and alpha-enolase. Sequence modifications or conflicts were observed for beta-and gamma-actin, ATP beta-synthase and heat shock 90 beta. IEF-NPS-RP-HPLC/ESI-TOFMS was used to determine experimental pI, MW and relative hydrophobicity values for each protein detected. This data was used to generate a 2-D pI-MS protein map, where proteins are displayed according to their pI and molecular weight. Protein molecular weight peaks are represented as bands in the 2-D pI-MS image where the gray scale of each band is proportional to the intensity of the protein molecular weight peak. In addition, a third hydrophobicity dimension (%B) was added as the % acetonitrile elution to generate a 3-D pI-MS-%B plot where each protein can be tagged according to three parameters.