Generation of Monoclonal Antibody for 15-deoxy-Δ12,14-Prostaglandin J2 and Development of Enzyme-Linked Immunosorbent Assay for Its Quantification in Culture Medium of Adipocytes

15-deoxy-??^12,14-Prostaglandin J₂ (15d-PGJ₂) is a biologically active molecule serving as a pro-adipogenic factor or an anti-inflammatory regulator. This compound is one of naturally occurring derivatives formed by the non-enzymatic dehydration of PGD₂. To determine the endogenous synthesis of 15d-PGJ₂, a convenient immunological approach is useful. At first, we established a cloned hybridoma cell line to secrete a monoclonal antibody specific for 15d-PGJ₂. For the development of a solid-phase enzyme-linked immunosorbent assay (ELISA), the immobilized antigen using a protein conjugate of 15d-PGJ₂ was allowed to react competitively with a monoclonal antibody in the presence of free 15d-PGJ₂. Under the optimized conditions, a sensitive calibration curve was generated able to determine the amount of 15d-PGJ₂ from 0.5?pg to 9.7?ng with 71?pg of 50% displacement in one assay. Our monoclonal antibody did not recognize other related prostanoids except PGJ₂ with cross-reaction of 4%. Our ELISA was demonstrated to be reliable for the quantification of 15d-PGJ₂ in the maturation medium of cultured adipocytes by confirming the accuracy and specificity of its determination. The application of our assay revealed that the non-enzymatic formation of 15d-PGJ₂ became more evident after several hours of incubation with authentic PGD₂ at 37?°C. The results indicate the usefulness of our developed solid-phase ELISA with the monoclonal antibody for further studies on the endogenous synthesis of 15d-PGJ₂ and its roles in various cells and tissues.     

High-performance liquid chromatography–tandem mass spectrometry for identification of isoflavones and description of the biotransformation of kudzu root

High-performance liquid chromatography-tandem mass spectrometry has been used to identify isoflavone aglycones and glycosides in kudzu root. Fourteen isoflavones were detected. Among these, six were identified by comparison with authentic standards. Tentative identifications of the other isoflavones are based on UV spectra, mass spectra of protonated and deprotonated molecules, and MS-MS data. Several are reported for the first time in kudzu root. The bioactivity and bioavailability of isoflavone aglycones are usually greater than those of their glycosides. To improve the bioavailability of kudzu root isoflavones, crude beta-glycosidases prepared from microbes were used to hydrolyze the isoflavone glycosides. Several MS modes are combined not only to identify the isoflavones in kudzu root, but also to describe the biotransformation of kudzu root isoflavone glycosides. It is also proved that crude beta-glycosidases have high selectivity toward the O-glycosides of isoflavones.     

Direct affinity screening chromatography–mass spectrometry assay for identification of antibacterial agents from natural product sources

A direct affinity screening – mass spectrometry assay, coupled to liquid chromatography, is presented as a tool for natural product drug discovery. Using the assay, fractionated extracts from a Caribbean gorgonian coral were shown to contain a new chemical entity (NCE) which binds to a mimic of the Gram positive bacterial cell wall (lysine–d-alanine–d-alanine). Conditions for observation of a specific noncovalent complex between the NCE and the target mimic using electrospray ionization-mass spectrometry were validated in a series of positive and negative control experiments, which featured flow injection analysis-based titrations. While the structural identity of the NCE could not be determined due to limited sample quantities, this work provides proof-of-principle for such an approach to potentially accelerate drug discovery from natural product sources.     

Concise synthesis of glyconoamidines as affinity ligands for the purification of β-glucosidase involved in control of some biological events including plant leaf movement

Glycosidases are involved in deactivation or storage of some endogenous bioactive substances through biologically intriguing processes. For example, nyctinastic leaf movement is controlled by a biological clock through the regulation of β-glucosidase activity. Ganem’s glyconoamidine (1) is used as a micromolar inhibitor of glucosidase in biochemical studies and would be useful as an affinity ligand for purification of glycosidase. However, its use for the specific inhibition of glucosidase which is highly specific to a glycoside with voluntary aglycon is seriously restricted because no universal method for the synthesis of N-alkylated glyconoamidine has been reported. Here, we report a concise synthesis of N-alkylated Ganem’s glyconoamidine with voluntary aglycon using a non-protected sugar derivative.     

Rapid screening and identification of α-glucosidase inhibitors from mulberry leaves using enzyme-immobilized magnetic beads coupled with HPLC/MS and NMR

α-Glucosidase plays important roles in the digestion and absorption of carbohydrates in the small intestine. The inhibition of α-glucosidase is regarded as a potential way to treat diabetes. We established an approach to screening α-glucosidase inhibitors from medicinal plants using enzyme-coated magnetic bead. Using 1-(3-dimethyl-aminopropyl)-3-ethylcarbodiimide and N-hydroxysuccinimide as reaction reagents, α-glucosidase was immobilized on the magnetic beads by covalent linkage. The conjugation of α-glucosidase to the magnetic beads was characterized using scanning electron microscope and X-ray diffractometer. The proposed approach was applied in fishing potential α-glucosidase inhibitors from extract of Morus alba, a Chinese medicinal plant. The structures of potential active compounds were identified via liquid chromatography-mass spectrometry and nuclear magnetic resonance. The results demonstrated that two flavonoids (isoquercitrin and astragalin) could bind to α-glucosidase, which was confirmed via conventional α-glucosidase inhibitory assay. Our findings suggested that enzyme-coated magnetic beads may be suitable for discovering active compounds from medicinal plants.     

Monodisperse magnetic poly(glycidyl methacrylate) microspheres for isolation of autoantibodies with affinity for the 46 kDa form of unconventional Myo1C present in autoimmune patients

Monodisperse nonmagnetic macroporous poly(glycidyl methacrylate) (PGMA) microspheres were synthesized by multistep swelling polymerization of glycidyl methacrylate, ethylene dimethacrylate and 2-[(methoxycarbonyl)methoxy]ethyl methacrylate (MCMEMA). This was followed (a) by ammonolysis to modify the microspheres with amino groups, and (b) by incorporation of iron oxide (γ-Fe₂O₃) into the pores to render the particles magnetic. The resulting porous and magnetic microspheres were characterized by scanning and transmission electron microscopy (SEM and TEM), atomic absorption and Fourier transform infrared spectroscopy (AAS and FTIR), elemental analysis, vibrating magnetometry, mercury porosimetry and Brunauer-Emmett-Teller adsorption/desorption isotherms. The microspheres are meso- and macroporous, typically 5 μm in diameter, contain 0.9 mM · g^?1 of amino groups and 14 wt.% of iron according to elemental analysis and AAS, respectively. The particles were conjugated to p46/Myo1C protein, a potential biomarker of autoimmune diseases, to isolate specific autoantibodies in the blood of patients suffering from multiple sclerosis (MS). The p46/Myo1C loaded microspheres are shown to enable the preconcentration of minute quantities of specific immunoglobulins prior to their quantification via SDS-PAGE. The immunoglobulin M (IgM) with affinity to Myo1C was detected in MS patients.

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Graphical abstract Monodisperse magnetic poly(glycidyl methacrylate) microspheres were synthesized, conjugated with 46 kDa form of unconventional Myo1C protein (p46/Myo1C) via carbodiimide (DIC) chemistry, and specific autoantibodies isolated from blood of multiple sclerosis (MS) patients; immunoglobulin M (IgM) level increased in MS patients.

     

Hydrophilic Nb-immobilized magnetic core–shell microsphere – A novel immobilized metal ion affinity chromatography material for highly selective enrichment of phosphopeptides

Rapid and selective enrichment of phosphopeptides from complex biological samples is essential and challenging in phosphorylated proteomics. In this work, for the first time, niobium ions were directly immobilized on the surface of polydopamine-coated magnetic microspheres through a facile and effective synthetic route. The Fe₃O₄@polydopamine-Nb⁵⁺ (denoted as Fe₃O₄@PD-Nb⁵⁺) microspheres possess merits of high hydrophilicity and good biological compatibility, and demonstrated low limit of detection (2 fmol). The selectivity was also basically satisfactory (β-casein:BSA = 1:500) to capture phosphopeptides. They were also successfully applied for enrichment of phosphopeptides from real biological samples such as human serum and nonfat milk. Compared with Fe₃O₄@PD-Ti⁴⁺ microspheres, the Fe₃O₄@PD-Nb⁵⁺ microspheres exhibit superior selectivity to multi-phosphorylated peptides, and thus may be complementary to the conventional IMAC materials.     

Static mass spectrographs of a new type using Euler’s homogeneous electric and magnetic fields. II: Conditions of high-order double focusing for two-cascade schemes

Static mass spectrometers are masked by more modern and more sophisticated mass spectrometric instruments, but their potential nowhere is almost exhausted. One of the principle advantages of static mass spectrometers is that they are the only type of mass spectrometric instruments capable of operating in a spectrographic mode by measuring the whole range of masses per a single run. However, systems with good spectrographic properties significantly differ from the optical point of view from systems with good spectrometric properties, and the development of such devices leads to completely new optical problems and, accordingly, to novel methods of their solution. In this paper, we consider possibilities related to the application of electrostatic and magnetostatic fields, homogeneous in Euler’s terms, to the development of two-cascade mass spectrographs with double focusing.     

Investigation into mercury bound to biothiols: structural identification using ESI–ion-trap MS and introduction of a method for their HPLC separation with simultaneous detection by ICP-MS and ESI-MS

Mercury in plants or animal tissue is supposed to occur in the form of complexes formed with biologically relevant thiols (biothiols), rather than as free cation. We describe a technique for the separation and molecular identification of mercury and methylmercury complexes derived from their reactions with cysteine (Cys) and glutathione (GS): Hg(Cys)₂, Hg(GS)₂, MeHgCys, MeHgGS. Complexes were characterised by electrospray mass spectrometry (MS) equipped with an ion trap and the fragmentation pattern of MeHgCys was explained by using MP2 and B3LYP calculations, showing the importance of mercury–amine interactions in the gas phase. Chromatographic baseline separation was performed within 10 min with formic acid as the mobile phase on a reversed-phase column. Detection was done by online simultaneous coupling of ES-MS and inductively coupled plasma MS. When the mercury complexes were spiked in real samples (plant extracts), no perturbation of the separation and detection conditions was observed, suggesting that this method is capable of detecting mercury biothiol complexes in plants. Figure Separation and structural identification of Hg and MeHg biothiols A part of this work was presented as a poster at the European Winter Conference on Plasma Spectrochemistry, 2007, held in Taormina, Italy.     

Simple and efficient protocol for the synthesis of benzoxazole,benzoimidazole and benzothiazole heterocycles using Fe(Ⅲ)–Schiff base/SBA-15 as a nanocatalyst

Benzimidazoles, benzoxazoles, and benzothiazoles derivatives were synthesized from condensation of aldehydes and 1,2-phenylenediamine or ortho-aminophenol or ortho-aminothiophenol in the presence of catalytic amount of Fe(Ⅲ)–Schiff base/SBA-15 in water medium. Short reaction times, good to excellent yields, easy availability, reusability, and use of an eco-friendly catalyst are some of the significant attributes of the present method.     

A new method of using supercritical carbon dioxide as a green solvent for synthesis and purification of 5,5‴-bis(tridecafluorohexyl)-2,2′:5′,2″:5″,2‴-quaterthiophene,which is one of n-type organic semiconducting materials

We have investigated synthesis as well as purification of 5,5?-bis(tridecafluorohexyl)-2,2′:5′,2″:5″,2?-quaterthiophene (BFH-4?T, n-type organic semiconducting material) using supercritical carbon dioxide (scCO₂) as a green solvent. BFH-4T was obtained in good selectivity and high yield by TDAE/PdCl₂-catalyzed reductive coupling reaction of 5-bromo-5′-(tridecafluorohexyl)-2,2′-bithiophene in scCO₂. We have also successfully established purification of the reaction mixture by passing scCO₂ in the reaction vessel. The product was yellow powder of BFH-4T with purity of more than 99% and Pd catalyst was not contained.     

A novel magnetic biosorbent prepared using an oak shell waste material as an efficient adsorbent for consecutive removal of Pb2+, Ag+, Ba2+, Sr2+, and CrO42− from aqueous solutions

In this research, the performance of a new biosorbent prepared using low-cost oak shell waste materials for consecutive removal of particular cations and chromate anions from aqueous solutions was studied. The adsorbent impregnated with Pb²⁺, Ag⁺, Ba²⁺, and Sr²⁺ was used to remove chromate anions by the formation of an appropriate insoluble precipitate. The biosorbent was characterized by FTIR, XRD, SEM, TG-DTG, and VSM techniques. The adsorption capacities of 133.84, 53.12, 50.12, and 40.39 mg g^?1 were obtained for chromate for the samples containing Pb²⁺, Ag⁺, Ba²⁺, and Sr²⁺ cations, respectively. The chromate uptake was inversely proportional to the k_sp of the chromate precipitate and increased with the cation content of the adsorbent. The adsorption process was kinetically fast and the equilibrium was established within 10 min. The experimental data were analyzed by the Langmuir, Freundlich, Sips, and Redlich-Peterson isotherm models. The data were fitted to the Langmuir isotherm, indicating that chromate was adsorbed homogeneously on the adsorbent surface.     

Synthesis of one-pot pyrazolo[4′,3′:5,6]pyrano[2,3-c]phenazin-15-yl) methanone derivatives via a multi-component using Fe3O4@TiO2-SO3H as a recoverable magnetic catalyst under microwave irradiation

ABSTRACT

A new one-pot method for the synthesis four-component of pyrazolo[4′,3′:5,6]pyrano[2,3-c]phenazin-15-yl)methanone derivatives has been developed in the presence of nano Fe₃O₄@TiO₂-SO₃H catalysts (heterogeneous acid) under microwave conditions and in a solvent-free environment at 180?W. One of the benefits of using this catalyst was its re-use in subsequent stages of its reaction without much loss in its activity, which was carried out by an external magnet and recovered. The catalyst was synthesized and characterized by XRD, EDX, TEM, FESEM, TGA-DTA, BET, VSM and AFM. The productivity of the products obtained from this protocol (MAOS) is significantly high and the shorter reaction time in the synthesis process over the reflux method. These results showed advantages for synthesis, such as mild reaction conditions, no use of toxic catalysts in the laboratory, solvent-free environment, low energy consumption and Economically Affordable.