The identification of antigens incorporated into immune complexes (IC-antigens) is important for studying the pathophysiology of immunological diseases. Immune complexome analysis identifies ...IC-antigens by analyzing ICs collected from biological fluids by IC-capturing beads. In this study, we optimized the method to improve its comprehensiveness while maintaining selectivity for IC-antigens by comparing the number of identified peptides (model IC experiment) or proteins (human pooled serum) eluted from Protein G beads using different pH solutions (pH 2.0 – 11.0).
•Critical step is sample extraction and preparation in analysis of biomolecules.•Materials and techniques are improving for extracting nucleotides and nucleosides.•How to analyze and extract the ...proteins in an exact lesion of diseased tissue.
This review describes the recent developments in sample extraction and preparation techniques for mass spectrometric analysis of nucleotides, nucleosides, and proteins. Unique materials and techniques have been developed for highly selective extraction of nucleotides and nucleosides by solid-phase extraction strategies using various affinities. However, for proteins, the analysis of small-scale sections of diseased tissues (formalin-fixed, paraffin-embedded tissues) and the direct analysis of an exact lesion on the surface of diseased tissues (liquid extraction surface analysis) have become important advances in this field. In this review, we focus on the latest developments of these techniques and strategies.
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•Benzofuran-2-boronic acid can be used as a fluorescent sensor for Pd2+.•Fluorescence enhancement reaction proceeds rapidly even at room temperature.•The proposed fluorescent sensor ...exhibits excellent selectivity for Pd2+.
Benzofuran-2-boronic acid could be used as a fluorescent sensor for the detection of Pd2+ because it was rapidly converted to highly fluorescent derivative after mixing with Pd2+ under basic condition at room temperature. We found that dimerization of benzofuran was occurred to form fluorescent derivative by the catalytic activity of palladium. The fluorescence intensity at 360nm increased with increasing the concentration of Pd2+. The excellent selectivity for Pd2+ was demonstrated among other metal ions. Based on this findings, we successfully applied benzofuran-2-boronic acid to develop a microplate-based assay for high-throughput measurement of Pd2+. The detection limit (blank+3SD) for Pd2+ of the proposed assay was 9.8nM.
Comprehensive identification and profiling of antigens in immune complexes (IC-antigens) is useful to provide insights into pathophysiology and could form the basis for novel diagnostic and treatment ...strategies for many immune-related diseases. Immune complexome analysis is the method for comprehensively identifying and profiling IC-antigens in biological fluids (such as serum and cerebrospinal fluid). Here, we describe an IC-antigen detection method; specifically, ICs in biological fluids are captured by using protein G- or protein A-coated beads, are subjected to papain-digestion, elution, and tryptic digestion, and are analyzed by nano-liquid chromatography-tandem mass spectrometry (nano-LC-MS/MS).
Antioxidants have gained marked attention owing to their ability to prevent the oxidation of biological components and to protect the body from reactive oxygen species, thereby maintaining human ...health. Thus, antioxidant-rich dietary supplements and natural foods can be effective against oxidative stress and can even act as chemopreventive agents. Therefore, a simple and rapid assay for evaluation of antioxidant capacity and assessment of their distribution profile in natural sources is vital. Herein, we report a rapid, innovative chemiluminescence (CL) platform for evaluation and visualization of antioxidant capacity. We found that intense and long-lasting CL was formed upon the redox reaction of quinones, e.g., menadione, with antioxidants, e.g., l-ascorbic acid, in the presence of luminol. The produced CL intensities were proportional to the antioxidants' concentrations with a detection limit of 0.18 μM for the model antioxidant, l-ascorbic acid. As the formed CL was long-lasting, it could be easily captured and detected with a charge-coupled device (CCD) camera. To evaluate the quantification ability of the CCD camera, we developed a smart and fast microplate-based assay based on photographing the generated CL with a cooled CCD camera. The photographed CL intensities were linearly proportional with the antioxidant concentrations, and then the method was applied for photographing multiple food sample extracts. Ultimately, we utilized our method for the distribution profiling of antioxidant capacity in food cut sections. Samples were dipped in luminol and then in quinone, followed by CCD camera photography, without the need for any pulverization/extraction procedure, giving precise antioxidant distribution information.
Quinones are frequently used as derivatization reagents in HPLC analysis to improve detection sensitivity. In the present study, a simple, sensitive, and selective chemiluminescence (CL) ...derivatization strategy for biogenic amines, prior to their HPLC-CL analysis, was developed. The novel CL derivatization strategy was established based on using anthraquinone-2-carbonyl chloride as derivatizing agent for amines and then using the unique property of the quinones' moiety to generate reactive oxygen species (ROS) in response to UV irradiation. Typical amines such as tryptamine and phenethylamine were derivatized with anthraquinone-2-carbonyl chloride and then injected into an HPLC system equipped with an online photoreactor. The anthraquinone-tagged amines are separated and then UV-irradiated when they pass through a photoreactor to generate ROS from the quinone moiety of the derivative. Tryptamine and phenethylamine can be determined by measuring the chemiluminescence intensity produced by the reaction of the generated ROS with luminol. The chemiluminescence disappears when the photoreactor is turned off, suggesting that ROS are no longer generated from the quinone moiety in the absence of UV irradiation. This result indicates that the generation of ROS could be controlled by turning the photoreactor on and off. Under the optimized conditions, the limits of detection for tryptamine and phenethylamine were 124 and 84 nM, respectively. The developed method is successfully applied to determine the concentrations of tryptamine and phenethylamine in wine samples.
Oxidative stress refers to elevated reactive oxygen species (ROS) levels, and NADPH oxidases (NOXs), which are one of the most important sources of ROS. Oxidative stress plays important roles in the ...etiologies, pathological mechanisms, and treatment strategies of vascular diseases. Additionally, oxidative stress affects mechanisms of carcinogenesis, tumor growth, and prognosis in malignancies. Nearly all solid tumors show stimulation of neo-vascularity, termed angiogenesis, which is closely associated with malignant aggressiveness. Thus, cancers can be seen as a type of vascular disease. Oxidative stress-induced functions are regulated by complex endogenous mechanisms and exogenous factors, such as medication and diet. Although understanding these regulatory mechanisms is important for improving the prognosis of urothelial cancer, it is not sufficient, because there are controversial and conflicting opinions. Therefore, we believe that this knowledge is essential to discuss observations and treatment strategies in urothelial cancer. In this review, we describe the relationships between members of the NOX family and tumorigenesis, tumor growth, and pathological mechanisms in urological cancers including prostate cancer, renal cell carcinoma, and urothelial cancer. In addition, we introduce natural compounds and chemical agents that are associated with ROS-induced angiogenesis or apoptosis.
Osteoclasts are multinucleated bone-resorbing cells that are formed by the fusion of macrophages. Recently, we identified Rab44, a large Rab GTPase, as an upregulated gene during osteoclast ...differentiation that negatively regulates osteoclast differentiation. However, the molecular mechanisms by which Rab44 negatively regulates osteoclast differentiation remain unknown. Here, we found that the GDP form of Rab44 interacted with the actin-binding protein, Coronin1C, in murine macrophages. Immunoprecipitation experiments revealed that the interaction of Rab44 and Coronin1C occurred in wild-type and a dominant-negative (DN) mutant of Rab44, but not in a constitutively active (CA) mutant of Rab44. Consistent with these findings, the expression of the CA mutant inhibited osteoclast differentiation, whereas that of the DN mutant enhanced this differentiation. Using a phase-contrast microscope, Coronin1C-knockdown osteoclasts apparently impaired multinuclear formation. Moreover, Coronin1C knockdown impaired the migration and chemotaxis of RAW-D macrophages. An in vivo experimental system demonstrated that Coronin1C knockdown suppresses osteoclastogenesis. Therefore, the decreased cell formation and fusion of Coronin1C-depleted osteoclasts might be due to the decreased migration of Coronin1C-knockdown macrophages. These results indicate that Coronin1C is a GDP-specific Rab44 effector that controls osteoclast formation by regulating cell motility in macrophages.
We have developed an HPLC-UV method for the determination of pyrroloquinoline quinone (PQQ), which utilizes a redox-based colorimetric reaction. In the proposed colorimetric reaction, the redox ...reaction between PQQ and dithiothreitol generates superoxide anion radicals that can convert 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride (INT) to formazan dye. After PQQ separation on an octadecyl silica column, it was mixed online with dithiothreitol and INT, and the formed formazan dye was monitored by absorbance at 490 nm. The detection limit (S/N = 3) of the proposed method was 7.6 nM (152 fmol/injection). The proposed method could selectively detect PQQ in food products without any clean-up procedures.
It is important to evaluate the amount of daptomycin (DAP) distributed to skeletal muscles to elucidate the mechanisms related to penetration and side effects, such as myopathies. However, no attempt ...has been made to measure DAP concentrations in skeletal muscles. The study’s aim to investigate the feasibility of trypsin digestion, as a muscle sample preparation technique for the determination of DAP in murine skeletal muscle, was evaluated in conjunction with a conventional HPLC-UV analysis. Compared with trypsin digestion, DAP was less recovered from spiked skeletal muscle by the conventional extraction, including homogenization, centrifugation, and filtration, because of its incorporation into the muscle protein. On the other hand, a sample preparation technique involving enzymatic digestion employing trypsin fully recovered DAP from the spiked skeletal muscle. Based on the spike recovery assay results, we proposed an efficient muscle sample preparation method involving trypsin digestion. HPLC analysis in conjunction with the sample preparation method has successfully determined DAP concentrations of skeletal muscles collected from mice administrated subcutaneously with DAP. The proposed method is suitable for application to investigations that include animal experiments on drug migration into muscle and mechanism underlying skeletal muscle injury as a side reaction, such as myopathies, of DAP therapy.
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