Bronchoalveolar lavage fluid protein composition in patients with sarcoidosis and idiopathic pulmonary fibrosis: a two-dimensional electrophoretic study

We used two-dimensional (2-D) electrophoresis to analyze the protein composition of fluid recovered by bronchoalveolar lavage (BALF) from patients with sarcoidosis and idiopathic pulmonary fibrosis, two forms of interstitial lung disease with different cellular composition and cytokine profile in BALF. They are also characterized by different pathogenesis and clinical evolution, idiopathic pulmonary fibrosis being less favorable than sarcoidosis due to rapidly progressive pulmonary fibrosis. Thirty-eight proteins or protein fragments, never previously assigned in BALF samples, were identified by various methods including mass fingerprinting of tryptic digests. Comparison of the BALF protein maps of the two groups of patients showed 32 spots with statistically significant disease-related variations in relative abundance. In sarcoidosis we found an increase in the amount of several plasma proteins, while in idiopathic pulmonary fibrosis we observed a statistically significant increase in low-molecular-weight proteins, many of which are involved in inflammatory processes (such as MIF and calgranulin) or antioxidant response (such as antioxidant peroxysomal enzyme and thioredoxin peroxidase 2). 2-D electrophoresis allowed us to identify new BALF proteins and to characterize protein composition in patients with sarcoidosis and idiophatic pulmonary fibrosis. Comparison of the gels of the two diseases showed that they differ in BALF protein profiles as they do in type of immune response.     

Liquid chromatographic determination of rifampin in human plasma, bronchoalveolar lavage fluid, and alveolar cells

A technique is presented for the measurement of rifampin in humans by reversed-phase column chromatography and postcolumn photo irradiation. The assay employs diazepam as an internal standard and provides specific, rapid, and reliable determinations for drug concentration in plasma, bronchoalveolar lavage (BAL), and alveolar cells (AC). The preparation of plasma and AC samples utilizes a simple deproteinization step, whereas BAL supernatants require a solid-phase extraction. The assay produces sharp peaks with retention times of 6.2 and 15 min for rifampin and diazepam, respectively. The detection limits for rifampin were 0.5 microgram/mL for plasma, 0.015 microgram/mL for BAL supernatants, and 0.03 microgram/mL for AC suspensions. The assay has excellent performance characteristics, making it suitable for pharmacological studies in humans, and it is being used to support a study of the intrapulmonary pharmacokinetics of rifampin.     

Determination of cefquinome in pig plasma and bronchoalveolar lavage fluid by high-performance liquid chromatography combined with electrospray ionization mass spectrometry

The aim of this study was to develop a rapid and sensitive method for the quantification of cefquinome in animal plasma and bronchoalveolar lavage (BAL) fluid using high-performance liquid chromatography combined with electrospray tandem mass spectrometry (LC-ESI-MS/MS). Cefadroxil is used as internal standard. For plasma, the sample preparation includes a simple deproteinization step with a Microcon filter. This allows detecting the unbound cefquinome concentration, which is correlated with the concentration in other body fluids, such as BAL fluid. To be able to detect the total plasma concentration, deproteinization with acetonitrile, followed by a back-extraction of actonitrile with dichloromethane was performed. The BAL fluid is centrifuged to precipitate floating particles. Chromatographic separation is achieved on a PLRP-S column using 0.005% formic acid and methanol as mobile phase. For plasma, good linearity was observed in the range of 5-2500 ng ml(-1) for both the unbound and total concentration. The response in BAL fluid was linear in the range of 4-1000 ng ml(-1). The limit of quantification (LOQ) was set at 5.00 ng ml(-1) for plasma and at 4.00 ng ml(-1) for BAL fluid. The limit of detection (LOD) was 3.12 ng ml(-1) and 0.41 ng ml(-1) for the unbound and total concentration in plasma, respectively, and was 1.43 ng ml(-1) for BAL fluid. The method was shown to be of use in a pharmacokinetic study in pigs, where the correlation between cefquinome concentrations in plasma and BAL fluid of pigs was studied.     

高效液相色谱-蒸发光散射检测器测定大鼠支气管肺泡灌洗液中肺泡表面活性物质

为准确快速地对支气管肺泡灌洗液中的磷脂进行定量分析,采用蒸发光散射检测器建立了该物质的高效液相色谱测定方法.该方法使用硅胶色谱柱,流动相A为V(甲醇)∶V(水)＝11∶2,流动相B为纯氯仿,A与B体积比为1∶39;以V(氯仿)∶V(甲醇)∶V(水)＝10∶10∶3溶解样品;得到的校正曲线回归方程,相关系数R2=0.9971,日间精密度为10.9%～13.4%,加标回收率为98%～115%.本法的重复性和标准曲线的线性能够满足大鼠支气管肺泡灌洗液中肺泡表面活性物质定量分析的要求.     

Measurement of histamine in rat bronchoalveolar lavage fluid by high-performance cation-exchange chromatography coupled with post-column derivatization to o-phthaldialdehyde.

To better define involvement of mast cell derived mediators in the pulmonary response to fibrogenic dusts, a rapid and accurate method was required to analyze samples of bronchoalveolar lavage fluid for histamine. Samples of rat lung lavage were analyzed for histamine via high-performance cation-exchange chromatography coupled with post-column derivatization with o-phthaldialdehyde. The fluorescent derivative could be detected to ca. 1 ng/ml of lavage. Recoveries averaged 94.2% with an average relative standard deviation of +/- 5.3%. There were no correlations between amount or fibrogenicity of inhaled dust and subsequent release of histamine into lavage fluid.     

High-performance liquid chromatographic determination of pyrazinamide in human plasma, bronchoalveolar lavage fluid, and alveolar cells

A technique is presented for the measurement of pyrazinamide in human plasma, bronchoalveolar lavage, and alveolar cells by reversed-phase column chromatography. The assay utilizes acetazolamide as an internal standard, ultraviolet detection at 268 nm, and an acetonitrile-based mobile phase. Preparation of plasma samples requires a simple deproteinization step, resulting in the development of sharp peaks with retention times of 8.4 and 17 minutes for pyrazinamide and acetazolamide, respectively. Bronchoalveolar lavage and alveolar cell suspensions require an acid extraction with ethyl acetate, evaporation to dryness, and reconstitution. This method provides specific, rapid, and reliable determinations of drug concentrations and therefore is suitable for pharmacological studies, particularly those that are designed to quantitate the intrapulmonary concentrations of pyrazinamide.     

A high-pressure liquid chromatographic-tandem mass spectrometric method for the determination of ethambutol in human plasma, bronchoalveolar lavage fluid, and alveolar cells

A technique is presented for the specific and sensitive determination of ethambutol concentrations in plasma, bronchoalveolar lavage (BAL), and alveolar cells (AC) using a high-pressure liquid chromatographic (HPLC)-tandem mass spectrometric (MS-MS) method. The preparation of samples requires a deproteinization step with acetonitrile. The retention times for ethambutol, neostigmine bromide, and propranolol are 2.0, 1.4, and 1.1 min, respectively, with a total run time of 2.8 min. The detection limits for ethambutol are 0.05 microg/mL for plasma and 0.005 microg/mL for the BAL supernatants and AC suspensions. The assay has excellent performance characteristics and has been used to support a study of the intrapulmonary pharmacokinetics of ethambutol in human subjects.     

Dynamics of protein and mixed protein/surfactant adsorption layers at the water/fluid interface

The adsorption behaviour of proteins and systems mixed with surfactants of different nature is described. In the absence of surfactants the proteins mainly adsorb in a diffusion controlled manner. Due to lack of quantitative models the experimental results are discussed partly qualitatively. There are different types of interaction between proteins and surfactant molecules. These interactions lead to protein/surfactant complexes the surface activity and conformation of which are different from those of the pure protein. Complexes formed with ionic surfactants via electrostatic interaction have usually a higher surface activity, which becomes evident from the more than additive surface pressure increase. The presence of only small amounts of ionic surfactants can significantly modify the structure of adsorbed proteins. With increasing amounts of ionic surfactants, however, an opposite effect is reached as due to hydrophobic interaction and the complexes become less surface active and can be displaced from the interface due to competitive adsorption. In the presence of non-ionic surfactants the adsorption layer is mainly formed by competitive adsorption between the compounds and the only interaction is of hydrophobic nature. Such complexes are typically less surface active than the pure protein. From a certain surfactant concentration of the interface is covered almost exclusively by the non-ionic surfactant. Mixed layers of proteins and lipids formed by penetration at the water/air or by competitive adsorption at the water/chloroform interface are formed such that at a certain pressure the components start to separate. Using Brewster angle microscopy in penetration experiments of proteins into lipid monolayers this interfacial separation can be visualised. A brief comparison of the protein adsorption at the water/air and water/n-tetradecane shows that the adsorbed amount at the water/oil interface is much stronger and the change in interfacial tension much larger than at the water/air interface. Also some experimental data on the dilational elasticity of proteins at both interfaces measured by a transient relaxation technique are discussed on the basis of the derived thermodynamic model. As a fast developing field of application the use of surface tensiometry and rheometry of mixed protein/surfactant mixed layers is demonstrated as a new tool in the diagnostics of various diseases and for monitoring the progress of therapies.     

A high-performance liquid chromatographic-tandem mass spectrometric method for the determination of cethromycin (ABT-773) in human plasma, bronchoalveolar lavage fluid, and alveolar cells

A method is developed for the specific and sensitive determination of cethromycin concentrations in plasma, bronchoalveolar lavage (BAL), and alveolar cells (AC), using a high-performance liquid chromatographic-tandem mass spectrometry (MS) method. The mobile phase consists of 50% acetonitrile-0.05% acetic acid-5mM ammonium acetate; the column used is a C(8) reversed-phase stationary phase. The preparation of samples requires a solvent extraction step. The retention times for cethromycin and the internal standard are approximately 2.0 and 2.7 min, respectively, with a total run time of 3.5 min. Detection is carried out using electrospray MS in a multiple reaction monitor mode. The detection limits for cethromycin are 1 ng/mL for plasma and 0.2 ng/mL for BAL supernatants and AC suspensions. The assay has excellent performance characteristics and has been used to support a study of the intrapulmonary pharmacokinetics of cethromycin in human subjects.     

Profiling stage-dependent changes of protein expression in Caenorhabditis elegans by mass spectrometric proteome analysis leads to the identification of stage-specific marker proteins

Proteome maps obtained by synchronization of the wild-type Caenorhabditis elegans development reflected stage-dependent molecular differences and revealed dynamic cytoskeletal processes during ontogenesis. Distinct protein spots that may function as molecular markers for the corresponding developmental stages were mass spectrometrically identified. The amount of the Cu(2+)- Zn(2+) superoxide dismutase (CE23550) and an aspartyl proteinase (CE21681) was highest in the first larval stage (L1) and decreased during the ontogenesis from the first larval stage to the adult. Tropomyosin III (CE29059) was prominently present in the first and second larval stage (L1/L2). Abundances of actin 1 or 4 (CE12358 or CE13148) and tropomyosin I (CE28782) were particularly high in multiple spots in the third larval stage (L3). Interestingly, the amount of DIM-1 protein (CE27706), reflected by two spots, was the lowest in this stage. A particular splicing factor (CE31089) was detected only in the fourth larval stage (L4), whereas a spot with high abundance representing the cuticle collagen (CE02272) was only found highly expressed in adult animals (A). In addition, a Ca(2+)-binding protein (CE12368) and one protein spot which has not yet been identified, both reached their maximal spot intensities in the adult stage (A). Moreover, the ASP-1, CCT-5, GPD-1, GPD-2, HSP-6, HSP-16.2, IFB-2, LEC-2, LIN-53, LMN-1, MDH-1, NUD-1, RPA-0, RSP-12, SOD-1, TBB-1, TBB-2, TMY-1, UNC-60, and VIT-2 proteins for which mutants are available and two still unidentified protein spots which were present in all developmental stages, have been reproducibly localized in proteome maps of distinct ontogenesis states.     

Substrate specificity analysis of protein kinase complex Dbf2-Mob1 by peptide library and proteome array screening

Background  

The mitotic exit network (MEN) is a group of proteins that form a signaling cascade that is essential for cells to exit mitosis in Saccharomyces cerevisiae. The MEN has also been implicated in playing a role in cytokinesis. Two components of this signaling pathway are the protein kinase Dbf2 and its binding partner essential for its kinase activity, Mob1. The components of MEN that act upstream of Dbf2-Mob1 have been characterized, but physiological substrates for Dbf2-Mob1 have yet to be identified.     

A chemical inhibitor reveals the role of Raf kinase inhibitor protein in cell migration

Raf kinase inhibitor protein (RKIP) is a modulator of cell signaling that functions as an endogenous inhibitor of multiple kinases. We demonstrate here a positive role for RKIP in the regulation of cell locomotion. We discovered that RKIP is the relevant cellular target of locostatin, a cell migration inhibitor. Locostatin abrogates RKIP's ability to bind and inhibit Raf-1 kinase, and it acts by disrupting a protein-protein interaction, an uncommon mode of action for a small molecule. Small interfering RNA-mediated silencing of RKIP expression also reduces cell migration rate. Overexpression of RKIP converts epithelial cells to a highly migratory fibroblast-like phenotype, with dramatic reduction in the sensitivity of cells to locostatin. RKIP is therefore the compound's valid target and a key regulator of cell motility.     

Methodological approach to the characterization of diacylphosphatidyl cholines in rabbit lung lavage fluid by fast-atom bombardment mass spectrometry

Strong evidence exists that the distribution of phospholipids in the lung is a function of the degree of adult respiratory distress syndrome. The capabilities of positive-ion fast-atom bombardment (FAB) mass spectrometry for the determination of the relative distribution of intact diacylphosphatidylcholine species in lung lavage fluid were investigated. Two different FAB matrices and two different isolation/purification procedures--extraction/thin layer chromatography--high performance liquid chromatography (TLC-HPLC)--have been evaluated. In addition the relative fatty acid composition of the diacylphosphatidylcholines was determined by negative-ion FAB mass spectrometry using the [RCOO]- fragment ion. These results were compared with those obtained by gas chromatographic determination of the fatty acid methylesters.     

Numerical characterization of ultraviolet ink fluid agglomeration and the surfactant effect in nanoinkjet printing

Ultraviolet (UV) ink is a major ink type used in additive manufacturing via 3D inkjet printing. A major challenge in nanoinkjet printing is ink agglomeration. Among the UV ink components, oligomers have the highest tendency to agglomerate which can agitate the stability and quality of the printing fluid and possibly lead to nanoscale nozzle clogging. In this work, the first numerical study on the UV ink fluid, UV ink is modeled by using dissipative particle dynamics to study mesoscale agglomeration. The constituents of the ink model are composed of polystyrene and polyethylene glycol as photopolymers, BZP as a photoinitiator, and SDS as a surfactant. Styrene is a prevalent and established commercial photopolymer in present 3D inkjet applications, while ethylene glycol is a photopolymer known to improve ink viscosity. The morphological characteristics of the UV ink are studied here, where the results for different models from four cases considered here show how the kind of photopolymers and their constituent ratios affect the agglomeration morphology of the fluidic system. The existence of both oligomers and monomers results in mutual morphological benefits against agglomeration, while the photoinitiator occurs between photopolymers. In addition, we find that the surfactant can reduce the average size of agglomeration and improve the dispersion uniformity by increasing the number of agglomerates. These results highlight the important role additives can play to prevent, reduce, and control various forms of agglomeration to achieve enhanced nanoinkjet printing quality. Copyright © 2017 John Wiley & Sons, Ltd.     

Validation of a new HPLC‐UV method for determination of the antibiotic linezolid in human plasma and in bronchoalveolar lavage

A rapid and selective HPLC‐UV method was developed for the quantification of linezolid (LNZ) in human plasma and bronchoalveolar lavage (BAL) at the concentrations associated with therapy. Plasma samples were extracted by solid‐phase extraction followed by evaporation to dryness and reconstitution in mobile phase solution. The chromatographic separation was carried out on a C₁₈ column with an isocratic mobile phase consisting of dihydrogen phosphate buffer 50 mm (pH 3.5) and acetonitrile (60:40 v/v). The detection was performed using a photodiode array. Under these conditions, a single chromatographic run could be completed within 12 min. The method was validated by estimating the precision and the accuracy for inter‐ and intra‐day analysis in the concentration range of 25–25600 ng/mL. The method was linear over the investigated range with all the correlation coefficients R > 0.999. The intra‐ and inter‐day precision was within 8.90% and the accuracy ranged from ?4.76 to +5.20%. This rapid and sensitive method was fully validated and could be applied to pharmacokinetic study for the determination of LNZ levels in human plasma and BAL samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Recognition of essential purines by the U1A protein

Background  

The RNA recognition motif (RRM) is one of the largest families of RNA binding domains. The RRM is modulated so that individual proteins containing RRMs can specifically recognize RNA targets with diverse sequences and structures. Understanding the principles governing this specificity will be important for the rational modification and design of RRM-RNA complexes.     

Mechanical load induces a 100-fold increase in the rate of collagen proteolysis by MMP-1

Although mechanical stress is known to profoundly influence the composition and structure of the extracellular matrix (ECM), the mechanisms by which this regulation occurs remain poorly understood. We used a single-molecule magnetic tweezers assay to study the effect of force on collagen proteolysis by matrix metalloproteinase-1 (MMP-1). Here we show that the application of ～10 pN in extensional force causes an ～100-fold increase in proteolysis rates. Our results support a mechanistic model in which the collagen triple helix unwinds prior to proteolysis. The data and resulting model predict that biologically relevant forces may increase localized ECM proteolysis, suggesting a possible role for mechanical force in the regulation of ECM remodeling.