Serum proteins may often serve as indicators of disease and is a rich source for biomarker discovery. However, the large dynamic range of proteins in serum makes the analysis very challenging because high-abundant proteins tend to mask those of lower abundance. A prefractionation step, such as depletion of a few high-abundant proteins before protein profiling, can assist in the discovery and detection of less abundant proteins that may prove to be informative biomarkers. In the present study, five different depletion columns were investigated considering efficiency, specificity, and reproducibility. Our research included quantitative determination of total protein, albumin, and immunoglobulin G (IgG) concentrations, one- and two-dimensional gels and mass spectrometric analysis of the serum samples before and after the depletion step. Our results showed that all five depletion columns tested removed albumin and IgG with high efficiency. We found that based on reproducibility and binding specificity, the Multiple Affinity Removal Column that removed a total of six high-abundant proteins (albumin, IgG, antitrypsin, IgA, transferring, and haptoglobin) offered the most promising depletion approach. Among the disposable (single-use) products, the ProteoExtract Albumin/IgG Removal kit displayed the best results. Depleted serum from the Multiple Affinity Removal column was further evaluated by 2-D gel electrophoresis (2-DE) analysis, and the results indicated increased resolution and improved intensity of low-abundant proteins in a reproducible fashion. Our study provides a comprehensive investigation of commercially available depletion columns and will be of high importance for future proteomic studies on serum samples.

The crystal structure of Rubisco (ribulose 1,5-bisphosphate carboxylase/oxygenase) from the unicellular green alga Chlamydomonas reinhardtii has been determined to 1.4 Å resolution. Overall, the structure shows high similarity to the previously determined structures of L8S8 Rubisco enzymes. The largest difference is found in the loop between ␤ strands A and B of the small subunit (␤A-␤B loop), which is longer by six amino acid residues than the corresponding region in Rubisco from Spinacia. Mutations of residues in the ␤A-␤B loop have been shown to affect holoenzyme stability and catalytic properties. The information contained in the Chlamydomonas structure enables a more reliable analysis of the effect of these mutations. No electron density was observed for the last 13 residues of the small subunit, which are assumed to be disordered in the crystal. Because of the high resolution of the data, some posttranslational modifications are unambiguously apparent in the structure. These include cysteine and N-terminal methylations and proline 4-hydroxylations.Ribulose 1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39; Rubisco) 1 catalyzes the addition of CO 2 to RuBP (reviewed in Refs. 1-3). This reaction initiates photosynthetic carbon assimilation via the Calvin cycle and results in the net gain of carbon from atmospheric CO 2 into the biosphere. The carboxylation reaction of Rubisco is the major source of carbon needed for life. A competing reaction in which O 2 is added to RuBP results ultimately in a net loss of CO 2 , a process known as photorespiration. Because the reaction catalyzed by Rubisco is unique to photosynthetic CO 2 fixation and nearly all carbohydrate production is dependent on it, the oxygenase reaction qualifies as the most important limiting factor of photosynthetic yield.All Rubisco enzymes have oxygenase activity. This is true also for strictly anaerobic microbes, including some anaerobic archaebacteria (reviewed in Ref. 1). Thus, whereas the ratio of carboxylation to oxygenation at any specified concentrations of CO 2 and O 2 depends on the catalytic efficiency (k cat /K m ) of carboxylation relative to that of oxygenation, referred to as the CO 2 /O 2 specificity factor, net CO 2 fixation is determined by the difference between the rates of carboxylation and oxygenation (4, 5). The kinetic constants that determine carboxylation and oxygenation vary considerably among Rubisco enzymes from different species (6).Based on their quaternary structures, different forms of Rubisco enzymes can be distinguished (reviewed in Refs. 1 and 2). In cyanobacteria and plants with chloroplasts, such as red and brown algae and green plants, a hexadecameric (L8S8) form is found, consisting of eight 55-kDa L subunits and eight 15-kDa S subunits. A dimeric (L2) enzyme, similar in structure to the L subunits of the hexadecameric enzymes, is restricted to some bacteria and alveolates. More recently, a third form was discovered in the thermophilic archaebacterium Thermococcus kodakaraensis that comprises 10 L sub...

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BackgroundIn a dog with joint pain, it is important to determine whether it has suppurative joint disease, characterized by exudation of neutrophils in the synovial fluid, or not, as this affects choice of diagnostic tests and treatments. The aim of this study was to evaluate whether measurement of serum C-reactive protein (CRP) concentration could be used to discriminate between dogs with suppurative arthritis and osteoarthritis (OA). Furthermore, the concentrations of serum and synovial fluid interleukin (IL) 6 concentrations were measured in dogs with joint disease and in healthy dogs, and were correlated to serum CRP concentrations.MethodsDogs with joint pain were enrolled prospectively and were classified to have suppurative arthritis or OA based on synovial fluid analysis and radiographic/arthroscopic findings. Healthy Beagles were enrolled as a comparative group. CRP and IL-6 concentrations were measured with canine-specific immunoassays. The performance of CRP concentration in discriminating between dogs with suppurative arthritis and OA was evaluated using a previously established clinical decision limit for CRP (20 mg/l), and by receiver operator characteristic (ROC) curve and logistic regression analysis. Comparisons of CRP and IL-6 concentrations between groups were performed using t-tests, and correlations by Spearman rank correlation coefficients.ResultsSamples were obtained from 31 dogs with suppurative arthritis, 34 dogs with OA, and 17 healthy dogs. Sixty-two out of 65 dogs with joint disease were correctly classified using the clinical decision limit for CRP. Evaluation of ROC curve and regression analysis indicated that serum CRP concentrations could discriminate between suppurative arthritis and OA. Dogs with suppurative arthritis had higher serum CRP and serum and synovial fluid IL-6 concentrations compared to dogs with OA (p < 0.001). Dogs with OA had higher synovial fluid IL-6 concentrations (p < 0.001), but not higher serum CRP (p = 0.29) or serum IL-6 (p = 0.07) concentrations, compared to healthy dogs. There was a positive correlation between synovial fluid IL-6 and serum CRP concentrations (rs = 0.733, p < 0.001), and between serum IL-6 and serum CRP concentrations (rs = 0.729, p < 0.001).ConclusionCRP concentration was found to discriminate well between dogs with suppurative arthritis and OA.

PI3Kδ is a lipid kinase that is believed to be important in the migration and activation of cells of the immune system. Inhibition is hypothesized to provide a powerful yet selective immunomodulatory effect that may be beneficial for the treatment of conditions such as asthma or rheumatoid arthritis. In this work, we describe the identification of inhibitors based on a thiazolopyridone core structure and their subsequent optimization for inhalation. The initially identified compound (13) had good potency and isoform selectivity but was not suitable for inhalation. Addition of basic substituents to a region of the molecule pointing to solvent was tolerated (enzyme inhibition pIC > 9), and by careful manipulation of the pK and lipophilicity, we were able to discover compounds (20b, 20f) with good lung retention and cell potency that could be taken forward to in vivo studies where significant target engagement could be demonstrated.