Antioxidant activity of 3-methyl-1-phenyl-2-pyrazolin-5-one

Summary

The antioxidant activity of an anti-ischemic agent, 3-methyl-1-phenyl-2-pyrazolin-5-one (MCI-186), was examined. The pKa value of MCI-186 is 7.0 and the rate of oxidation of MCI-186 initiated with an azo compound increased with increasing pH, suggesting that the anionic form of MCI-186 is much more reactive than the non-ionic form. The major products were 3-methyl-1-phenyl-2-pyrazolin-4,5-dione (4,5-dione) and 2-oxo-3-(phenylhydrazono)-butanoic acid (OPB). Hydrolysis of 4,5-dione gave OPB. The minor intermediate product was 4-hydroxy-4-(3-methyl-1-phenyl-1H-pyrazolin-5-on-4-yl)-3-methyl-1-phenyl-1H-pyrazolin-5-one (BPOH). The nucleophilic attack of the anionic form of MCI-186 to 4,5-dione is likely to give BPOH. MCI-186 (50 μM) inhibited the aerobic oxidation at 37°C of 5.2 mM unilamellar soybean phosphatidylcholine (PC) liposomal membranes, initiated with a water-soluble initiator, as efficientlyas did ascorbate (100 μM). MCI-186 (50 μM) also inhibited the oxidation of the same PC liposomal membranes, this time initiated with a lipid-soluble initiator, almost as efficiently as did α-tocopherol (2 μM). Furthermore, the combination of MCI-186 with ascorbate or α-tocopherol showed almost complete inhibition of PC oxidation induced by both initiators. These data suggest that MCI-186 may work as a good antioxidant in cellular systems as well as in cell-free systems.     

Novel bioresources for studies of Brassica oleracea: identification of a kale MYB transcription factor responsible for glucosinolate production

Plants belonging to the Brassicaceae family exhibit species‐specific profiles of glucosinolates (GSLs), a class of defence compounds against pathogens and insects. GSLs also exhibit various human health–promoting properties. Among them, glucoraphanin (aliphatic 4‐methylsulphinylbutyl GSL) has attracted the most attention because it hydrolyses to form a potent anticancer compound. Increased interest in developing commercial varieties of Brassicaceae crops with desirable GSL profiles has led to attempts to identify genes that are potentially valuable for controlling GSL biosynthesis. However, little attention has been focused on genes of kale (Brassica oleracea var. acephala). In this study, we established full‐length kale cDNA libraries containing 59 904 clones, which were used to generate an expressed sequence tag (EST) data set with 119 204 entries. The EST data set clarified genes related to the GSL biosynthesis pathway in kale. We specifically focused on BoMYB29, a homolog of Arabidopsis MYB29/PMG2/HAG3, not only to characterize its function but also to demonstrate its usability as a biological resource. BoMYB29 overexpression in wild‐type Arabidopsis enhanced the expression of aliphatic GSL biosynthetic genes and the accumulation of aliphatic GSLs. When expressed in the myb28myb29 mutant, which exhibited no detectable aliphatic GSLs, BoMYB29 restored the expression of biosynthetic genes and aliphatic GSL accumulation. Interestingly, the ratio of methylsulphinyl GSL content, including glucoraphanin, to that of methylthio GSLs was greatly increased, indicating the suitability of BoMYB29 as a regulator for increasing methylsulphinyl GSL content. Our results indicate that these biological resources can facilitate further identification of genes useful for modifications of GSL profiles and accumulation in kale.     

Sequence and phylogenetic analyses of the water‐soluble glucan synthesizing‐glucosyltransferase genes of Streptococcus dentirousetti

Two tandemly aligned glucosyltransferase (GTF) genes whose gene products are responsible for water‐soluble glucan synthesis were isolated from Streptococcus dentirousetti NUM1303 and sequenced. One of the GTF genes of S. dentirousetti consisted of a 4110 bp open reading frame (ORF) that encoded for a 1369 amino acid protein and was revealed to be a S. sobrinus gtfS homolog. The percent similarity of amino acid sequences of the GTF‐S from S. dentirousetti compared to those from S. sobrinus was 99%. In addition, a putative gtfT was found in tandem in the downstream region of the S. dentirousetti gtfS. The gtfT of S. dentirousetti consisted of a 4527 bp ORF encoding for 1508 amino acids. The similarity of amino acid sequences of the GTF‐T from S. dentirousetti and S. sobrinus was 94%. Phylogenetic analysis based on amino acid sequences from other related streptococcal GTFs suggested that both GTF‐S and GTF‐T of S. dentirousetti are closely related to S. sobrinus.     

Accurate prediction of hot spot residues through physicochemical characteristics of amino acid sequences

Hot spot residues of proteins are fundamental interface residues that help proteins perform their functions. Detecting hot spots by experimental methods is costly and time‐consuming. Sequential and structural information has been widely used in the computational prediction of hot spots. However, structural information is not always available. In this article, we investigated the problem of identifying hot spots using only physicochemical characteristics extracted from amino acid sequences. We first extracted 132 relatively independent physicochemical features from a set of the 544 properties in AAindex1, an amino acid index database. Each feature was utilized to train a classification model with a novel encoding schema for hot spot prediction by the IBk algorithm, an extension of the K‐nearest neighbor algorithm. The combinations of the individual classifiers were explored and the classifiers that appeared frequently in the top performing combinations were selected. The hot spot predictor was built based on an ensemble of these classifiers and to work in a voting manner. Experimental results demonstrated that our method effectively exploited the feature space and allowed flexible weights of features for different queries. On the commonly used hot spot benchmark sets, our method significantly outperformed other machine learning algorithms and state‐of‐the‐art hot spot predictors. The program is available at http://sfb.kaust.edu.sa/pages/software.aspx . Proteins 2013; 81:1351–1362 © 2013 Wiley Periodicals, Inc.     

Facile synthesis of the cyclohexane fragment of enacloxins,a series of antibiotics isolated from Frateuria sp. W-315

An efficient and good yield synthesis of the cyclohexane moiety of enacyloxins, a series of antibiotics isolated from Frateuria sp. W-315, was achieved from d-quinic acid using a successive Barton–McCombie deoxygenation.     

The Photochemical Reaction of Pentachlorophenol

A herbicide, sodium pentachlorophenoxide (Na-PCP), used in Japan, is easily decomposed with sunlight after its application in the rice field. The photochemical reaction of Na-PCP in an aqueous solution on exposure to sunlight afforded numerous products which were mainly accompanied with chloranilic acid and a yellow compound (I). The chemical structure of the yellow compound I was established as being 3, 4, 5-trichloro-6-(2′-hydroxy-3′, 4′, 5′, 6′-tetrachlorophenoxy)-o-benzoquinone by chemical and spectroscopic evidences.

Minor decomposition products of sodium pentachlorophenoxide (Na-PCP) in an aqueous solution by sunlight have been isolated. Chemical structures of them are described, and infrared and ultraviolet spectra are presented in support of these stractures. These illustrate a new type of oxidative reaction of phenols.     

2′,4′-BNA/LNA aptamers: CE-SELEX using a DNA-based library of full-length 2′-O,4′-C-methylene-bridged/linked bicyclic ribonucleotides

DNA-based aptamers that contain 2′-O,4′-C-methylene-bridged/linked bicyclic ribonucleotides (B/L nucleotides) over the entire length were successfully obtained using a capillary electrophoresis systematic evolution of ligands by exponential enrichment (CE-SELEX) method. A modified DNA library was prepared with an enzyme mix of KOD Dash and KOD mutant DNA polymerases. Forty 2′-O,4′-C-methylene bridged/locked nucleic acid (2′,4′-BNA/LNA) aptamers were isolated from an enriched pool and classified into six groups according to their sequence. 2′,4′-BNA/LNA aptamers of groups V and VI bound human thrombin with K_d values in the range of several 10 nanomolar levels.     

Direct Immunochemiluminescent Assay for proBNP and Total BNP in Human Plasma proBNP and Total BNP Levels in Normal and Heart Failure

Background

Recent studies have shown that in addition to brain (or B-type) natriuretic peptide (BNP) and the N-terminal proBNP fragment, levels of intact proBNP are also increased in heart failure. Moreover, present BNP immunoassays also measure proBNP, as the anti-BNP antibody cross-reacts with proBNP. It is important to know the exact levels of proBNP in heart failure, because elevation of the low-activity proBNP may be associated with the development of heart failure.

Methodology/Principal Findings

We therefore established a two-step immunochemiluminescent assay for total BNP (BNP+proBNP) and proBNP using monoclonal antibodies and glycosylated proBNP as a standard. The assay enables measurement of plasma total BNP and proBNP within only 7 h, without prior extraction of the plasma. The detection limit was 0.4 pmol/L for a 50-µl plasma sample. Within-run CVs ranged from 5.2%–8.0% in proBNP assay and from 7.0%–8.4% in total BNP assay, and between-run CVs ranged from 5.3–7.4% in proBNP assay and from 2.9%–9.5% in total BNP assay, respectively. The dilution curves for plasma samples showed good linearity (correlation coefficients = 0.998–1.00), and analytical recovery was 90–101%. The mean total BNP and proBNP in plasma from 116 healthy subjects were 1.4±1.2 pM and 1.0±0.7 pM, respectively, and were 80±129 pM and 42±70 pM in 32 heart failure patients. Plasma proBNP levels significantly correlate with age in normal subjects.

Conclusions/Significance

Our immunochemiluminescent assay is sufficiently rapid and precise for routine determination of total BNP and proBNP in human plasma.     

Feeding responses of an oligophagous bean aphid, Megoura crassicauda, to primary and secondary substances in Vicia angustifolia

The feeding behaviour of the aphid Megoura crassicauda Mordivilko (Homoptera: Aphididae), which feeds selectively on plants in the genus Vicia (Fabaceae), was studied. The aphids deposited proteinaceous stylet sheaths intercellularly towards the phloem tissues of host plants. Similar stylet sheaths were formed on a Parafilm membrane when host‐specific acylated flavonoid glycosides [two 2″‐O‐(E)‐p‐coumaroyl esters of quercetin 3‐O‐diglycosides] present in the extracts of the narrow vetch, Vicia angustifolia L., were supplied in the solution covered by the membrane. In contrast, their corresponding deacyl analogues, present more abundantly in the host plant tissues, were not stimulatory, which suggested specificity in the structural requirements of the probing stimulants. While the aphids imbibed an artificial diet composed of primary nutrients (e.g., sucrose and amino acids) and produced a large quantity of honeydew, acylated flavonoids alone and non‐acylated flavonoids supplied with the nutrients more or less suppressed honeydew production. These findings implied that the acylated flavonoids serve as a cue to navigate the stylet sheath towards the phloem prior to sap‐sucking, whereas non‐acylated flavonoids may serve as a negative stimulus to refrain from sucking during tissue penetration before tapping the phloem, although the distribution of these compounds in the plant tissues remains unknown. Thus, the feeding behaviour of M. crassicauda appears to be controlled by multiple chemical stimuli in the process of the settling on its host plant.