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Halogen bonding is a highly directional interaction and a potential tool in functional material design through self-assembly. Herein, we describe two fundamental supramolecular strategies to synthesize molecularly imprinted polymers (MIPs) with halogen bonding-based molecular recognition sites. In the first method, the size of the σ-hole was increased by aromatic fluorine substitution of the template molecule, enhancing the halogen bonding in the supramolecule. The second method involved sandwiching hydrogen atoms of a template molecule between iodo substituents, which suppressed competing hydrogen bonding and enabled multiple recognition patterns, improving the selectivity. The interaction mode between the functional monomer and the templates was elucidated by 1H NMR, 13C NMR, X-ray absorption spectroscopy, and computational simulation. Finally, we succeeded in the effective chromatographic separation of diiodobenzene isomers on the uniformly sized MIPs prepared by multi-step swelling and polymerization. The MIPs selectively recognized halogenated thyroid hormones via halogen bonding and could be applied to screening endocrine disruptors.
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Similar to psychostimulants, the peripheral administration of menthol promotes mouse motor activity, and the neurotransmitter dopamine has been suggested to be involved in this effect. The present study aimed to elucidate the effects of l-menthol on parts of the central nervous system that are involved in motor effects. The subcutaneous administration of l-menthol significantly increased the number of c-Fos-like immunoreactive nuclei in the dorsal striatum of the mice, and motor activity was promoted. It also increased the extracellular dopamine level in the dorsal striatum of the mice. These observations indicated that after subcutaneous administration, l-menthol enhances dopamine-mediated neurotransmission, and activates neuronal activity in the dorsal striatum, thereby promoting motor activity in mice.
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Conducta Animal/efectos de los fármacos , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Mentol/farmacología , Actividad Motora/efectos de los fármacos , Neuronas/metabolismo , Proteínas Proto-Oncogénicas c-fos/metabolismo , Transmisión Sináptica/efectos de los fármacos , Animales , Núcleo Celular/metabolismo , Masculino , Ratones , Ratones Endogámicos ICRRESUMEN
Prohydrojasmon (PDJ) is a synthetic jasmonate derivative that is primarily used as a growth regulator, but its mechanism of action is unclear. In this study, we elucidated the effects of PDJ on phytochemical production in red leaf lettuce. The PDJ treatments promoted the accumulation of phenolic compounds in aerial plant parts. An LC-MS analysis revealed that these accumulated compounds were identified as cyanidin-3-O-glucoside, cyanidin-3-O-(6â³-O-malonyl)-glucoside and cyanidin-3-O-(6â³-O-malonyl)-glucoside methyl ester. The abundance of these compounds in lettuce extracts increased significantly in response to the PDJ treatment. Additionally, the LC-MS analysis also identified the accumulated phenolic compounds in the extracts of PDJ-treated lettuce, including caffeoyltartaric acid, chlorogenic acid, caffeoylmalic acid, chicoric acid, and dicaffeoylquinic acid. Gene expression analyses indicated the PDJ treatments upregulated the expression of PAL, F3H, and ANS genes in lettuce. These results suggest that PDJ treatments enhance the expression of genes involved in the synthesis of anthocyanins and phenolic compounds, resulting in an increase in the quantities of these compounds, which reportedly have various functions affecting human physiology.
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Proteins are typically separated by an immune reaction, such as an enzyme-linked immunosorbent assay, and are detected by selective fluorescent labeling. This has potential for complicated procedures and the denaturation of proteins by labeling, and is cost consuming. In this study, we propose a technique for the selective separation and detection of a target protein using a molecularly imprinted hydrogel (PI gel) with fluorescent monomers. We focused on 8-anilino-1-naphthalenesulfonic acid (ANS), where the fluorescence intensity is easily changed by the interaction with proteins, and successfully synthesized the ANS monomer and a poly(ethylene glycol) (PEG) conjugated ANS monomer. The PI gel with the ANS monomers using bovine serum albumin (BSA) as a template showed the selective adsorption of BSA and the fluorescence intensity increased due to the adsorption of BSA.
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Hidrogeles , Impresión Molecular , Adsorción , Polietilenglicoles , Albúmina Sérica BovinaRESUMEN
A novel acidic morpholine-derivative containing glyceride (M-glyceride) was isolated from the cells of two strains of the thraustochytrid, Aurantiochytrium. The glyceride accounted for approximately 0.1 -0.4% of the lyophilized cells. The glyceride consisted of peaks I (85%) and II (15%). The structures of the intact and acetylated glycerides were elucidated by liquid chromatography-quadrupole time-of-flight chromatograph mass spectrometer (LC-Q/TOF) and NMR spectroscopy. The hydrate type of M-glyceride was detected as a minor component by LC-MS/MS. By 2D-NMR experiments, peaks I of the intact M-glyceride were elucidated as 1,2-didocosapentaenoyl-glyceryl-2'-oxy-3'-oxomorpholino propionic acid, and peak II was estimated 1,2-palmitoyldocosapentaenoyl- and/or 1,2-docosapentaenoylpalmitoyl-glyceryl-2'-oxy-3'-oxomorpholino propionic acid. The double bond position of docosapentaenoic acid was of the ω - 6 type (C22 = 5.ω - 6). The M-glyceride content varied by the cell cycle. The content was 0.4% of lyophilized cells at the mid logarithmic phase, and decreased to 0.1% at the mid stationary phase. When cells were grown in 1.0 µM M-glyceride-containing growth media, cell growth was stimulated to 110% of the control. With 0.1 µM acetyl M-glyceride, stimulation of 113% of the control was observed. Finding morpholine derivatives in biological components is rare, and 2-hydroxy-3-oxomorpholino propionic acid (auranic acid) is a novel morpholine derivative.
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Neurotoxic chemicals that pass through the blood-brain barrier (BBB) can influence brain function. Efficient methods to test the permeability of the BBB to specific chemicals would facilitate identification of potentially neurotoxic agents. We report here a simultaneous blood and brain microdialysis in a free-moving mouse to test BBB permeability of different chemicals. Microdialysis sampling was conducted in mice at 3-5 days after implantation of a brain microdialysis probe and 1 day after implantation of a blood microdialysis probe. Therefore, mice were under almost physiological conditions. Results of an intravenous injection of lucifer yellow or uranine showed that the BBB was functioning in the mice under the experimental conditions. Mice were given phenyl arsenic compounds orally, and concentration-time profiles for phenyl arsenic compounds such as diphenylarsinic acid, phenylarsonic acid, and phenylmethylarsinic acid in the blood and brain dialysate samples were obtained using simultaneous blood and brain microdialysis coupled with liquid chromatography-tandem mass spectrometry. Peak area-time profiles for linalool and 2-phenethyl alcohol (fragrance compounds or plant-derived volatile organic chemicals) were obtained using simultaneous blood and brain microdialysis coupled with gas chromatography-mass spectrometry in mice given lavender or rose essential oils intraperitoneally. BBB function was confirmed using lucifer yellow in these mice, and results indicated that the phenyl arsenic compounds, linalool and 2-phenethyl alcohol, passed through the BBB. The present study demonstrates that simultaneous blood and brain microdialysis in a free-moving mouse makes it possible to test the BBB permeability of chemicals when coupled with appropriate chemical analysis methods.
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An interlaboratory study on the National Institute for Environmental Studies (NIES) certified reference material (CRM) No. 28 Urban Aerosols, collected on the filters of a central ventilating system in a building in Beijing city center, was performed to obtain informative values of Hg isotopic composition and total Hg (THg) mass fraction. The THg mass fraction was determined by four organizations using atomic absorption spectrometry; it resulted in the mean value of 1.19 ± 0.12 mg/kg (2SD, n = 24). The Hg isotopic composition of the CRM was measured and intercompared at two different institutions by cold vapor generation system coupled to multicollector inductively coupled plasma mass spectrometry. Subsequently, a conventional dissolution method that uses a mixture of HNO3/HCl/H2O2 in Hotblock® and two different dissolution methods that use a mixture of HNO3/HCl with a microwave and a digestion bomb were applied. The Hg isotopic compositions were δ202Hg = - 1.26 ± 0.17, Δ199Hg = - 0.23 ± 0.06, Δ200Hg = 0.01 ± 0.07, and Δ201Hg = - 0.22 ± 0.09 (2SD, n = 18) for the conventional method, which agree well with those obtained using microwave and bomb digestion. Our results indicate that, for the quality control of particulate matter analyses, this CRM is appropriate for use in environmental and geochemical studies. Graphical abstract.
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The halogen-π (X-π) interaction is an intermolecular interaction between the electron-poor region of bonded halogen atoms and aromatic rings. We report an experimental evaluation of the halogen-π (X-π) interaction using liquid chromatography with carbon-material coated columns providing strong π interactions in the normal phase mode. A C70-fullerene (C70)-coated column showed higher retentions for halogenated benzenes as the number of halogen substitutions increased as a result of X-π interactions. In addition, the strength of the X-π interaction increased in the order of F < Cl < Br < I. Changes to the UV absorption of C70 and the brominated benzenes suggested that the intermolecular interaction changed from the π-π interaction to X-π interaction as the number of bromo substitutions increased. Computer simulations also showed that the difference in dipole moments among structural isomers affected the strength of the π-π interaction. Furthermore, we concluded from small peak shifts in 1H NMR and from computer simulations that the orbital interaction contributes to the X-π interactions. Finally, we succeeded in the one-pot separation of all isomers of brominated benzenes using the C70-coated column by optimizing the mobile phase conditions.
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We report hydrogen/deuterium (H/D) isotope effects based on weak intermolecular interactions with polar functional groups and aromatic rings in liquid chromatography (LC). Various LC experiments with different aromatic analytes, separation media, and nonpolar mobile phases were conducted under normal phase LC conditions, where the hydrophobic interaction was completely suppressed. The separation media that had polar functional groups, such as silanol groups, allowed for higher separation efficiencies for the pairs of aromatic H/D isotopologues. In comparing the 13C NMR spectra of protiated and deuterated aromatic analytes, the electron density of the deuterated analyte was found to be slightly higher than that of the protiated analytes. In the case of silanol functional groups, aromatic rings of the analyte acted as donors through the OH-π interaction to hydrogen atoms in the silanol groups. Thus, the deuterated analytes were able to be greatly retained by the stronger OH-π interactions. Furthermore, a C70-fullerene bonded monolithic column (C70 column), which effectively provides CH-π interactions, allowed the opposite isotope effect. Briefly, an electrostatic attraction based on the dipole-(induced) dipole interaction dominated in the CH-π interactions, according to a van't Hoff analysis. Hence, the bonding lengths of the C-H or D bonds were sensitively affected, such that we were able to conclude that the CH-π interaction depended on the geometric effect. Applying these opposing H/D isotope effects, we were able to finally demonstrate effective H/D isotopologue separations by utilizing the complementary action of the OH-π and CH-π interactions.
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Bacterial production has been often estimated from DNA synthesis rates by using tritium-labeled thymidine. Some bacteria species cannot incorporate extracellular thymidine into their DNA, suggesting their biomass production might be overlooked when using the conventional method. In the present study, to evaluate appropriateness of deoxyribonucleosides for evaluating bacterial production of natural bacterial communities from the viewpoint of DNA synthesis, incorporation rates of four deoxyribonucleosides (thymidine, deoxyadenosine, deoxyguanosine and deoxycytidine) labeled by nitrogen stable isotope (15N) into bacterial DNA were examined in both ocean (Sagami Bay) and freshwater (Lake Kasumigaura) ecosystems in July 2015 and January 2016. In most stations in Sagami Bay and Lake Kasumigaura, we found that incorporation rates of deoxyguanosine were the highest among those of the four deoxyribonucleosides, and the incorporation rate of deoxyguanosine was approximately 2.5 times higher than that of thymidine. Whereas, incorporation rates of deoxyadenosine and deoxycytidine were 0.9 and 0.2 times higher than that of thymidine. These results clearly suggest that the numbers of bacterial species which can incorporate exogenous deoxyguanosine into their DNA are relatively greater as compared to the other deoxyribonucleosides, and measurement of bacterial production using deoxyguanosine more likely reflects larger numbers of bacterial species productions.
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ADN Bacteriano/biosíntesis , ADN Bacteriano/química , Isótopos de Nitrógeno/metabolismo , Bahías/microbiología , Biomasa , Desoxiadenosinas/metabolismo , Desoxicitidina/metabolismo , Desoxiguanosina/metabolismo , Ecosistema , Japón , Cinética , Lagos/microbiología , Consorcios Microbianos/fisiología , Timidina/metabolismoRESUMEN
Microcystis aeruginosa is a water bloom-forming cyanobacterium found in fresh and brackish water ecosystems worldwide. Previously, we showed that several instances of M. aeruginosa bloom in brackish water can be explained by the proliferation of salt-tolerant M. aeruginosa strains harboring genes for a compatible solute sucrose. However, evolutionary history of sucrose genes in M. aeruginosa remains unclear because salt-tolerant strains have been poorly described. Here, we characterized a novel salt-tolerant strain of M. aeruginosa (NIES-4325) isolated from the brackish water of Lake Abashiri, Japan. A whole-genome analysis of M. aeruginosa NIES-4325 identified genes for sucrose synthesis (sppA, spsA and susA). Quantitative sucrose and gene expression analyses suggested that sucrose is implicated in acclimation to high salt in NIES-4325. Notably, the sucrose genes of M. aeruginosa are monophyletic, yet sucrose genes of NIES-4325 are highly divergent from those of other salt-tolerant M. aeruginosa strains. This suggests an early sucrose gene import into M. aeruginosa from other cyanobacteria, followed by multiple losses during intraspecific diversification. One of a few survivors of salt-tolerant strains is a likely donor of recent horizontal spreads of sucrose genes across M. aeruginosa lineages.
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Evolución Molecular , Agua Dulce/microbiología , Microcystis/genética , Tolerancia a la Sal , Sacarosa/metabolismo , ADN Bacteriano/genética , Genes Bacterianos , Genotipo , Japón , Secuenciación Completa del GenomaRESUMEN
Microcystins (MCs) are a group of cyclic heptapeptide hepatotoxins produced by Microcystis and several other genera of cyanobacteria. Many structural variants have been characterized using various methods such as liquid chromatography-mass spectrometry (LC-MS) analysis, enzyme-linked immunosorbent assay (ELISA) and protein phosphatase 2A (PP2A) inhibition assay. The representative MC, MC-LR, and related cyanobacterial toxins strongly inhibit PP2A activity and can therefore be assayed by measuring the extent of PP2A inhibition. However, these methods require reference toxin standards for the quantification and identification of known MCs. To obtain various MC-producing cyanobacterial strains, we surveyed and collected MC-producing cyanobacteria from environmental sources of water in Okinawa, Japan. Using a dual assay (LC-MS analysis and PP2A inhibition assay), we identified and isolated Microcystis strains producing five MC variants (MC-LR, -RR, -LA, -FR and -WR). Approximately 4 mg of MC-WR and -FR toxins were purified from the laboratory culture of the Microcystis isolate NIES-4344. Pure MC-WR and -FR variants were prepared for future use as toxin standards in LC-MS analysis. Phylogenetic analysis based on ftsZ revealed that the NIES-4344 strain belongs to the identified groups in Microcystis aeruginosa. This is the first report of Microcystis strains producing mainly MC-WR and -FR toxins in Japan.
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Microcistinas/análisis , Microcystis/aislamiento & purificación , Contaminantes del Agua/aislamiento & purificación , Animales , Línea Celular , Monitoreo del Ambiente , Eutrofización , Agua Dulce/microbiología , Insectos , Japón , Microcistinas/genética , Microcistinas/metabolismo , Microcystis/metabolismo , Filogenia , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/genética , Proteína Fosfatasa 2/metabolismoRESUMEN
Multigenerational adverse effects from the environment such as nutrition and chemicals are among important concerns in environmental health issues. Previously, we have found that arsenite exposure of only F0 females during their pregnancy increases hepatic tumors in the F2 males in C3H mice. In the current study, we investigated the association of DNA methylation with the hepatic tumor increase in the F2 males of the arsenite group. Reduced-representation bisulfite sequencing analysis newly identified that DNA methylation levels of regions around the transcriptional start sites of Tmem54 and Cd74 were decreased and the expression of these genes were significantly increased in the hepatic tumors of F2 males of the arsenite group. The associations between DNA methylation in these regions and gene expression changes were confirmed by treatment of murine hepatoma cell lines and hepatic stellate cell line with 5-aza-2'-deoxycytidine. Overexpression of Cd74 in Hepa1c1c7 cells increased Trib3 expression and suppressed the expression of tumor suppressor genes Id3 and Atoh8. Human database analysis using the Cancer Genome Atlas indicated that TMEM54, CD74, and TRIB3 were significantly increased and that ATOH8 was decreased in hepatocellular carcinoma. The data also showed that high expression of TMEM54 and TRIB3 and low expression of ATOH8 were associated with poor survival. These results suggested that an increase in Tmem54 and Cd74 expression via DNA methylation reduction was involved in the tumor increase in the F2 male offspring by gestational arsenite exposure of F0 females. This study also suggested that genes downstream of Cd74 were involved in tumorigenesis.
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Arsenitos/efectos adversos , Carcinoma Hepatocelular/genética , Metilación de ADN/genética , Neoplasias Hepáticas/genética , Animales , Antígenos de Diferenciación de Linfocitos B/genética , Antígenos de Neoplasias/genética , Carcinoma Hepatocelular/inducido químicamente , Línea Celular Tumoral , Femenino , Expresión Génica/genética , Antígenos de Histocompatibilidad Clase II/genética , Neoplasias Hepáticas/inducido químicamente , Masculino , Ratones , Ratones Endogámicos C3H , EmbarazoRESUMEN
Convex-concave π conjugated surfaces in hemispherical bucky bowl such as corannulene (Crn) have shown increasing utility in constructing self-assembled new functional materials owing to its unique π electrons and strong dipole. Here, we investigate these specific molecular recognitions on Crn by developing new silica-monolithic capillary columns modified with Crn and evaluating their performance in the separation of different aromatic compounds by liquid chromatography (LC). We synthesized two Crn derivatives and conjugated them onto the surface of a silica monolith. The first Crn derivative was edge functionalized, which can undergo free inversion of a convex-concave surface. The second Crn derivative was synthesized by modifying the spoke of Crn, which suppresses the convex-concave inversion. Results of LC suggest that each surface showed different shape recognition based on π interaction. Furthermore, the concave surface of Crn showed strong CH-π interaction with a planar molecule, coronene, demonstrated by the shifts of the 1H NMR signals of both Crn and coronene resulting from the multiple interactions between Crn and π electrons in coronene. These results clearly demonstrated the presence of CH-π interactions at multiple points, and the role of shape recognition.
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We report an efficient screening procedure for the selective detection of compounds that are actively bound to estrogen receptor (ER) from environmental water samples using a receptor-mimic adsorbent prepared by a molecularly imprinted polymer (MIP). To mimic the recognition ability of ER, we improved the typical MIP preparation procedure using a hydrophilic matrix with a polyethylene glycol (PEG)-based crosslinker and a hydrophobic monomer to imitate the hydrophobic pocket of ER. An optimized MIP prepared with methacrylic acid as an additional functional monomer and estriol (E3), an analogue of 17ß-estradiol (E2), exhibited highly selective adsorption for ER-active compounds such as E2 and E3, with significant suppression of non-specific hydrophobic adsorption. The prepared MIP was then applied to the screening of ER-active compounds in sewage samples. The fraction concentrated by the MIP was evaluated by in vitro bioassay using the yeast two-hybrid (Y2H) method and liquid chromatography-quadrupole time-of-flight mass spectrometry (LC-Q-TOFMS). Compared to an authentic adsorbent, styrene-divinylbenzene (SDB)-based resin, the fraction concentrated by the MIP had 120% ER activity in the Y2H assay, and only 25% peak volume was detected in LC-Q-TOFMS. Furthermore, a few ER-active compounds were identified only from the fraction concentrated by the MIP, although they could not be determined in the fraction concentrated by the SDB-based resin due to ion suppression along with high levels of hydrophobic compounds. These results indicated that the newly developed MIP effectively captured ER-active compounds and while allowing most non-ER-active compounds to pass through.
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Impresión Molecular/métodos , Imitación Molecular , Polímeros/química , Receptores de Estrógenos/metabolismo , Agua/química , Adsorción , Estradiol/química , Estradiol/aislamiento & purificación , Estriol/química , Estriol/aislamiento & purificación , Interacciones Hidrofóbicas e Hidrofílicas , Polietilenglicoles , Técnicas del Sistema de Dos HíbridosRESUMEN
Microcystis aeruginosa is a bloom-forming cyanobacterium found in eutrophic water bodies worldwide. M. aeruginosa blooms usually occur in freshwater; however, they have also been reported to occur in brackish water. Because M. aeruginosa often produces the cyanotoxin microcystin, they are a major concern to public health and environment. Despite this, the ecology, genomic basis, and evolutionary process underlying the M. aeruginosa bloom invasion from fresh to brackish water have been poorly investigated. Hence, in the present study, we have sequenced and characterized genomes of two newly discovered salt-tolerant M. aeruginosa strains obtained from Japanese brackish water lakes (Lakes Shinji and Tofutsu). Both genomes contain a set of genes for the synthesis of osmolyte sucrose (sppA, spsA, and susA), hitherto identified in only one strain (PCC 7806) of M. aeruginosa. Chemical and gene expression analyses confirmed sucrose accumulation induced by salt. A comprehensive genetic survey of >200 strains indicated that sucrose genes are extremely rare in M. aeruginosa. Most surprisingly, comparative genome analyses of the three strains indicated extremely low genetic diversity in the sucrose genes compared with other core genome genes, suggesting very recent acquisitions via horizontal transfer. Invasion of M. aeruginosa blooms into brackish water may be a recent event triggered by anthropogenic eutrophication of brackish water.
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Particulate organic phosphorus (P) compounds were examined in ultraoligotrophic Lake Saiko, Japan. A cartridge filter was used to collect sufficient amount of suspended particles for analysis by a two-dimensional NMR (1H-31P heteronuclear multiple bond correlation). 2-Aminoethylphosphonic acid (2-AEP), a phosphonate, was detected in suspended particles in Lake Saiko. The identity of the phosphonate was confirmed by comparison with a commercially available compound. Because 2-AEP is bioavailable, microorganisms can store and use this compound under extremely P-limited conditions. This is the first study to detect 2-AEP in an ultra-oligotrophic environment.
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Ácido Aminoetilfosfónico/análisis , Lagos/análisis , Contaminantes Químicos del Agua/análisis , Japón , Imagen por Resonancia Magnética/métodos , Organofosfonatos/análisis , Fósforo/análisisRESUMEN
We report on a molecularly imprinted hydrogel for the selective fluorescent detection of a targeting protein. To achieve the fluorescent detection of proteins, a fluorescent functional monomer was synthesized with fluorescein isothiocyanate and allylamine. The monomer (FITC-AA) provided a gradable alteration of the absorption spectrum, and a decrease of the fluorescent intensity due to an interaction with the model protein, lysozyme. Also, the imprinted hydrogel (PI-gel) was simply prepared with FITC-AA, the anionic functional monomers, poly(ethylene glycol) dimethacrylate, and lysozyme by radical photo polymerization. The PI-gel showed selective adsorption for lysozyme and the imprinting factor reached to 2.7. Additionally, the PI-gel indicated a gradual decrease of the fluorescent intensity corresponding to the lysozyme concertation similar to a competitive enzyme-linked immunosorbent assay detection.
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Ensayo de Inmunoadsorción Enzimática/métodos , Fluoresceína-5-Isotiocianato/química , Colorantes Fluorescentes/química , Hidrogel de Polietilenoglicol-Dimetacrilato/síntesis química , Impresión Molecular , Muramidasa/análisis , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Espectrometría de FluorescenciaRESUMEN
Selective adsorption of carbohydrates and glycoproteins was effectively achieved by molecularly imprinted hydrogels (MIHs) with a poly(ethylene glycol) (PEG)-based crosslinker and 4-vinylphenylboronic acid. In addition, an MIH with a novel boronic acid monomer provided selective adsorption and enabled visible detection of fructose.
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Ácidos Borónicos/química , Carbohidratos/química , Fructosa/análisis , Glicoproteínas/química , Hidrogeles/química , Impresión Molecular , Compuestos de Vinilo/química , Adsorción , Polietilenglicoles/química , Propiedades de SuperficieRESUMEN
An ozone-sensitive mutant was isolated from T-DNA-tagged lines of Arabidopsis thaliana. The T-DNA was inserted at a locus on chromosome 3, where two genes encoding glycolate oxidases, GOX1 and GOX2, peroxisomal enzymes involved in photorespiration, reside contiguously. The amounts of the mutant's foliar transcripts for these genes were reduced, and glycolate oxidase activity was approximately 60% of that of the wild-type plants. No difference in growth and appearance was observed between the mutant and the wild-type plants under normal conditions with ambient air under a light intensity of 100 µmol photons m-2 s-1. However, signs of severe damage, such as chlorosis and ion leakage from the tissue, rapidly appeared in mutant leaves in response to ozone treatment at a concentration of 0.2 µl l-1 under a higher light intensity of 350 µmol photons m-2 s-1 that caused no such symptoms in the wild-type plant. The mutant also exhibited sensitivity to sulfur dioxide and long-term high-intensity light. Arabidopsis mutants with deficiencies in other photorespiratory enzymes such as glutamate:glyoxylate aminotransferase and hydroxypyruvate reductase also exhibited ozone sensitivities. Therefore, photorespiration appears to be involved in protection against photooxidative stress caused by ozone and other abiotic factors under high-intensity light.