Study on the polarographic catalytic wave of vitamin P in the presence of persulfate and its application

The polarographic catalytic wave of vitamin P in the presence of persulfate was studied by linear potential scan polarography and cyclic voltammetry. Vitamin P yielded a single reduction wave in acidic aqueous solution, which was ascribed to a 2e(-), 2H(+) reduction of the carbonyl group in the C-4 position. Actually, the carbonyl group C=O first underwent a 1e(-), 1H(+) reduction to form a neutral free radical, and the further 1e(-), 1H(+) reduction of the free radical was simultaneous with its following chemical reactions. When S(2)O(2-)(8) was present, the free radical of vitamin P was oxidized by both S(2)O(2-)(8) and its reduction intermediate, the sulfate radical anion SO(*-)(4), to regenerate the original, which resulted in the production of a polarographic catalytic wave of vitamin P. Based on this catalytic wave, a novel method for the determination of vitamin P was proposed. In 0.02 M tartaric acid-sodium tartrate (pH 3.3) buffer containing 5.0 x 10(-3) M K(2)S(2)O(8), the peak potential of the catalytic wave was -1.42 V (vs SCE) and the peak current was rectilinear to the vitamin P concentration in the range of 8.0 x 10(-9)-1.0 x 10(-6) M (r = 0.9994, n = 13). The catalytic wave of 2.0 x 10(-7) M vitamin P enhanced the polarographic current 70 times compared with the corresponding reduction wave. The detection limit was 2.0 x 10(-9) M, and the relative standard deviation at the 2.0 x 10(-7) M level was 0.7% (n = 15). The proposed method was used for the determination of vitamin P content in the pharmaceutical preparation of tablets and the medicinal plant Sophora japonica L. without previous separation.     

Direct syntheses of S-alkylthio-D-galactono-, D-mannono-1,4-lactones, S-alkylthio-L-galactitols and D-mannitols displaying amphiphilic and mesophasic properties

Alkylthio-L-galactitols and D-mannitols were obtained in good yields (70-81%) by reduction, with NaBH4, of the corresponding 6-S-alkyl-6-thio-D-hexono-1,4-lactones.     

Synthesis of (+)-(2R,3S,4R)-2,3,4-trihydroxycyclohexanone from D-glucose

We have described the synthesis of (+)-(2R,3S,4R)-2,3,4-trihydroxycyclohexanone by the reduction of a keto-conduritol derivative, the latter being prepared in five steps from (-)-(2S,3R,4S,5S)-2,3,4-tribenzyloxy-5-hydroxycyclohexanone, which is in turn readily synthesized from D-glucose.     

Structural analyses of O-glycan sugar chains on IgA1 hinge region using SELDI-TOFMS with various lectins

The aim of the study was to develop a simple and precise method for identifying glycosylation of the IgA hinge region using surface-enhanced laser desorption/ionization (SELDI)-TOFMS with a lectin-coupled ProteinChip array. Serum IgA was isolated using an anti-IgA antibody column. Following reduction, alkylation, and trypsin digestion, the IgA fragments were applied on the ProteinChip coupled with jacalin, peanut agglutinin (PNA), or Vilsa villosa lectin (VVL). The SELDI-TOFMS peaks corresponding to the fragments containing IgA1 hinge glycopeptides trapped by each lectin were compared. The jacalin-, PNA-, and VVL-immobilized ProteinChips detected 13, 4, and 2 peaks, respectively. One major peak was confirmed as a glycopeptide by MS/MS analysis. These results suggest that a lectin-immobilized ProteinChip assay can be used to simplify the procedures for the analyses of the O-glycans in IgA1 hinge. This method potentially makes it possible to identify a disease-specific glycoform by selecting the appropriate ligand-coupled ProteinChip array.     

Activity-guided isolation of antiplasmodial dihydrochalcones and flavanones from Piper hostmannianum var. berbicense

The bioassay-guided purification of an n-hexane extract from the leaves of Piper hostmannianum var. berbicense led to the isolation of four monoterpene or prenyl-substituted dihydrochalcones (1a, 1b, 2, 3) as well as the known compounds 2',6'-dihydroxy-4'-methoxydihydrochalcone (4), linderatone (5), strobopinin (6), adunctin E (7) and (-)-methyllinderatin (8). Their structures were established on the basis of NMR and X-ray analysis. (-)-Methyllinderatin, linderatone and 2',6'-dihydroxy-4'-methoxydihydrochalcone exhibited the most potent antiplasmodial activity with IC50 values of 5.64, 10.33 and 12.69 microM, respectively against both chloroquine-sensitive and resistant strains of Plasmodium falciparum (F32,FcB1). The activity of (-)-methyllinderatin was confirmed in vivo against Plasmodium vinckei petteri in mice (80% of reduction of parasitemia) at a dose of 20 mg/kg/day.     

Synthesis of the sugar moiety of TIME-EA4, a glycopeptide isolated from silkworm diapause eggs

We describe the efficient synthesis of the tetrasaccharide, 2-O-acetyl-3,4,6-tri-O-benzyl-alpha-D-mannopyranosyl-(1-->6)-2,4-di-O-acetyl-3-O-allyl-beta-D-mannopyranosyl-(1-->4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl-(1-->4)-3,6-di-O-benzyl-2-deoxy-2-phthalimido-beta-D-glucopyranosyl azide, which is the protected form of the sugar unit of TIME-EA4 that is isolated from the diapausing eggs of the silkworm, Bombyx mori. The beta-linked D-mannoside of the tetrasaccharide was obtained using the conventional oxidation-reduction method for inversion of the configuration at the C-2 hydroxyl group of beta-D-glucoside. The reduction was effected with NaBH(4) in a methanolic solution in a ratio of 98:2 in favor of the beta-D-mannoside that was obtained in 87% yield.     

Field trial on the control effect of fipronil bait against German cockroaches

A field trial on the control effect of fipronil poison bait against German cockroaches (Blatella germanica) was carried out at different restaurant types in Sinchon, Seoul, Republic of Korea. Monitoring was performed applying food baited traps for 2 days per week. Reduction rates of German cockroaches by applying fipronil baits were 90.9% at Korean restaurants, 96.4% at Chinese restaurants, and 89.4% in beer hall kitchens after 4 weeks of the treatment. Overall average of the reduction rate was 93.9%. As the natural reduction rate at untreated restaurants was 11.5% after 4 weeks, a correction of the average reduction rate by applying the Abbot formula was 93.1%.     

A high-performance liquid chromatography-based assay of glutathione transferase omega 1 supported by glutathione or non-physiological reductants

The unusual glutathione S-transferase GSTO1 reduces, rather than conjugates, endo- and xenobiotics, and its role in diverse cellular processes has been proposed. GSTO1 has been assayed spectrophotometrically by measuring the disappearance of its substrate, S-(4-nitrophenacyl)glutathione (4-NPG), in the presence of 2-mercaptoethanol that regenerates GSTO1 from its mixed disulfide. To assay GSTO1 in rat liver cytosol, we have developed a high-performance liquid chromatography (HPLC)-based procedure with two main advantages: (i) it measures the formation of the 4-NPG reduction product 4-nitroacetophenone, thereby offering improved sensitivity and accuracy, and (ii) it can use glutathione, the physiological reductant of GSTO1, which is impossible to do with the spectrophotometric procedure. Using the new assay, we show that (i) the GSTO1-catalyzed reduction of 4-NPG in rat liver cytosol also yields 1-(4-nitrophenyl)ethanol, whose formation from 4-nitroacetophenone requires NAD(P)H; (ii) the two assays measure comparable activities with 2-mercaptoethanol or tris(2-carboxyethyl)phosphine used as reductant; (iii) the cytosolic reduction of 4-NPG is inhibited by GSTO1 inhibitors (KT53, 5-chloromethylfluorescein diacetate, and zinc), although the inhibitory effect is strikingly influenced by the type of reductant in the assay and by the sequence of reductant and inhibitor addition. Characterization of GSTO1 inhibitors with the improved assay provides better understanding of interaction of these chemicals with the enzyme.     

Adenosyl coenzyme and pH dependence of the [4Fe-4S]2+/1+ transition in lysine 2,3-aminomutase

5'-[N-[(3S)-3-Amino-carboxypropyl]-N-methylamino]-5(')-deoxyadenosine (azaSAM), an analog of S-adenosyl-L-methionine (SAM), was used to study the cofactor-dependent reduction of the [4Fe-4S](2+) center in lysine 2,3-aminomutase to the +1 oxidation state. azaSAM has a tertiary nitrogen in place of the sulfonium center of SAM. The analog binds to lysine 2,3-aminomutase with K(d)s of 1.4+/-0.3 microM at pH 8.0 and 2.2+/-0.6 microM at pH 6.5. Reduction of the [4Fe-4S](2+) center in the presence of this analog gives a 10K [4Fe-4S](1+) electron paramagnetic resonance (EPR) signal similar to that seen with SAM or S-adenosyl-L-homocysteine (SAH). The pH dependence of cofactor-induced reduction was examined to determine whether ionization of the tertiary nitrogen (pK(a)=7.08) might affect reduction of the [4Fe-4S](2+) center. The results show similar behavior in azaSAM and SAH, demonstrating that ionization of the aza group in azaSAM does not account for pH dependence in cofactor-dependent reduction of the [4Fe-4S](2+) center. The signal shape of the low-temperature EPR signal for the [4Fe-4S](1+) center in the SAM-induced reduction displayed a pH dependence that was not observed in the azaSAM- or SAH-induced spectra. Unique features of the signal are at a maximum at the pH activity optimum of pH 8 and are diminished as the pH is lowered or raised. These features are also absent in the spectra at all pHs examined when reduction is induced by azaSAM or SAH.     

Ent-3,4-seco-labdane and ent-labdane diterpenoids from Croton stipuliformis (Euphorbiaceae)

From a methanolic extract of the leaves of Croton stipuliformis, three ent-3,4-seco-labdanes (1-3) and an ent-labdane (4) together with the known compounds 6-hydroxynidorellol (5), maravuic acid, and sitosterol were isolated and identified from their spectroscopic data. The absolute stereochemistry of compound 4 was determined by application of Mosher's method in the NMR tube.     

Protective effects of solvent fractions of Mentha spicata (L.) leaves evaluated on 4-nitroquinoline-1-oxide induced chromosome damage and apoptosis in mouse bone marrow cells

Spearmint leaves (Mentha spicata L.) contain high levels of antioxidants that are known to protect against both exogenous and endogenous DNA damage. In this study, the protective effects of the hexane fraction (HF), chloroform fraction (CF) and ethyl acetate fraction (EAF) in an ethanol extract from M. spicata were evaluated against 4-nitroquinoline-1-oxide (4-NQO) induced chromosome damage and apoptosis in bone marrow cells of Swiss albino mice. Two (EAF; 80 and 160 mg/ kg body weight - bw) or three (HF and CF; 80, 160 and 320 mg/ kg bw) doses of solvent fractions or vehicle control (25% DMSO in water) were administered orally for five consecutive days. Upon the sixth day, 4-NQO was injected intraperitoneally. The animals were killed the following day. Other control groups were comprised of animals treated with either the vehicle control or the various doses of solvent fractions, but with no 4-NQO treatment. 4-NQO induced micro-nucleated polychromatic erythrocytes (MnPCEs) in all the test groups. However, pre-treatment of animals with the solvent fractions significantly reduced the 4-NQO-induced MnPCEs as well as the percentage of apoptotic cells. The reduction of both MnPCE and apoptosis was more evident following the pre-treatment of animals with 160 mg/kg bw EAF.     

Optimizing the connectivity in disulfide-rich peptides: alpha-conotoxin SII as a case study

We describe a strategy for the efficient, unambiguous assignment of disulfide connectivities in alpha-conotoxin SII, of which approximately 30% of its mass is cysteine, as an example of a generalizable technique for investigation of cysteine-rich peptides. alpha-Conotoxin SII was shown to possess 3-8, 2-18, and 4-14 disulfide bond connectivity. Sequential disulfide bond connectivity analysis was performed by partial reduction with Tris(2-carboxyethyl)phosphine and real-time mass monitoring by direct-infusion electrospray mass spectrometry (ESMS). This method achieved high yields of the differentially reduced disulfide bonded intermediates and economic use of reduced peptide. Intermediates were alkylated with either N-phenylmaleimide or 4-vinylpyridine. The resulting alkyl products were assigned by ESMS and their alkyl positions sequentially identified via conventional Edman degradation. The methodology described allows a more efficient, rapid, and reliable assignment of disulfide bond connectivity in synthetic and native cysteine-rich peptides.     

Transformations of 4- and 17alpha-substituted testosterone analogues by Fusarium culmorum

A series of 4- and/or 17alpha-substituted testosterone analogues has been incubated with the hydroxylating fungus Fusarium culmorum AM282. It was found that 19-norandrostenedione, 19-nortestosterone, 4-methoxytestosterone, 4-methyltestosterone, and 4-chloro-17alpha-methyltestosterone were hydroxylated exclusively or mainly at the 6beta-position. The mixtures of 6beta-, 15alpha-, and 12beta- or 11alpha-monohydroxy derivatives were obtained from 17alpha-methyltestosterone and 17alpha-ethyl-19-nortestosterone--the substrates with alkyl group at C-17alpha. 4-Chlorotestosterone was predominantly hydroxylated at 15alpha-position, but the reaction was accompanied by the reduction of 4-en-3-one system, which proceeded in the sequence: reduction of ketone to 3beta-alcohol and then reduction of the double 4,5 bond. The results obtained indicate an influence of stereoelectronic and steric effects of substitutes on regioselectivity of the hydroxylation of 4-en-3-one steroids by F. culmorum.     

Identification and quantitative determination of carbohydrates in ethanolic extracts of two conifers using 13C NMR spectroscopy

We developed a method for the direct identification and quantification of carbohydrates in raw vegetable extracts using (13)C NMR spectroscopy without any preliminary step of precipitation or reduction of the components. This method has been validated (accuracy, precision and response linearity) using pure compounds and artificial mixtures before being applied to authentic ethanolic extracts of pine needles, pine wood and pine cones and fir twigs. We determined that carbohydrates represented from 15% to 35% of the crude extracts in which pinitol was the principal constituent accompanied by arabinitol, mannitol, glucose and fructose.     

Regulation of interleukin-4 signaling by extracellular reduction of intramolecular disulfides

Interleukin-4 (IL-4) contains three structurally important intramolecular disulfides that are required for the bioactivity of the cytokine. We show that the cell surface of HeLa cells and endotoxin-activated monocytes can reduce IL-4 intramolecular disulfides in the extracellular space and inhibit binding of IL-4 to the IL-4Rα receptor. IL-4 disulfides were in vitro reduced by thioredoxin 1 (Trx1) and protein disulfide isomerase (PDI). Reduction of IL-4 disulfides by the cell surface of HeLa cells was inhibited by auranofin, an inhibitor of thioredoxin reductase that is an electron donor to both Trx1 and PDI. Both Trx1 and PDI have been shown to be located at the cell surface and our data suggests that these enzymes are involved in catalyzing reduction of IL-4 disulfides. The pro-drug N-acetylcysteine (NAC) that promotes T-helper type 1 responses was also shown to mediate the reduction of IL-4 disulfides. Our data provides evidence for a novel redox dependent pathway for regulation of cytokine activity by extracellular reduction of intramolecular disulfides at the cell surface by members of the thioredoxin enzyme family.     

Synthesis and biological evaluation of a radiolabeled analog of methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside directed towards influencing cellular glycosaminoglycan biosynthesis.

Two methods are presented for the synthesis of methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside. The first method employs the Barton-McCombie deoxygenation methodology, and the second method utilizes an oxidation-beta-elimination methodology that allows for the incorporation of hydrogen isotopes into the title compound. Hence, methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside (4) and methyl 2-acetamido-2,4-dideoxy-beta-D-xylo-hexopyranoside-6-t (14) were synthesized and evaluated for their ability to inhibit hepatocyte, cell-surface glycosaminoglycan biosynthesis and to incorporate a [(3)H] radiolabel into isolated glycosaminoglycans, respectively. Compound 4, at a concentration of 1.0 mM, demonstrated a reduction of D-[(3)H]glucosamine and [(35)S]sulfate incorporation into isolated glycosaminoglycans by 69 and 59%, of the control cultures, respectively. At 10 and 20 mM, 4 demonstrated a maximum inhibition of incorporation of both radiolabels to approximately 10% of the control cultures. Compound 14 demonstrated a maximum incorporation of a [(3)H] radiolabel into isolated cell-surface glycosaminoglycans at 10 and 20 mM. The mechanism of inhibition of glycosaminoglycan biosynthesis is due, in part, to the incorporation of a 4-deoxy moiety into glycosaminoglycan chains resulting in premature chain termination.     

Threshing efficiency as an incentive for rapid domestication of emmer wheat

Background and Aims

The harvesting method of wild and cultivated cereals has long been recognized as an important factor in the emergence of domesticated non-shattering ear genotypes. This study aimed to quantify the effects of spike brittleness and threshability on threshing time and efficiency in emmer wheat, and to evaluate the implications of post-harvest processes on domestication of cereals in the Near East.

Methods

A diverse collection of tetraploid wheat genotypes, consisting of Triticum turgidum ssp. dicoccoides – the wild progenitor of domesticated wheat – traditional landraces, modern cultivars (T. turgidum ssp. durum) and 150 recombinant (wild × modern) inbred lines, was used in replicated controlled threshing experiments to quantify the effects of spike brittleness and threshability on threshing time and efficiency.

Key Results

The transition from a brittle hulled wild phenotype to non-brittle hulled phenotype (landraces) was associated with an approx. 30 % reduction in threshing time, whereas the transition from the latter to non-brittle free-threshing cultivars was associated with an approx. 85 % reduction in threshing time. Similar trends were obtained with groups of recombinant inbred lines showing extreme phenotypes of brittleness and threshability.

Conclusions

In tetraploid wheat, both non-brittle spike and free-threshing are labour-saving traits that increase the efficiency of post-harvest processing, which could have been an incentive for rapid domestication of the Near Eastern cereals, thus refuting the recently proposed hypothesis regarding extra labour associated with the domesticated phenotype (non-brittle spike) and its presumed role in extending the domestication episode time frame.     

Impact of commonly used agrochemicals on bacterial diversity in cultivated soils

The effects of three selected agrochemicals on bacterial diversity in cultivated soil have been studied. The selected agrochemicals are Cerox (an insecticide), Ceresate and Paraquat (both herbicides). The effect on bacterial population was studied by looking at the total heterotrophic bacteria presence and the effect of the agrochemicals on some selected soil microbes. The soil type used was loamy with pH of 6.0–7.0. The soil was placed in opaque pots and bambara bean (Vigna subterranean) seeds cultivated in them. The agrochemicals were applied two weeks after germination of seeds at concentrations based on manufacturer’s recommendation. Plant growth was assessed by weekly measurement of plant height, foliage appearance and number of nodules formed after one month. The results indicated that the diversity index (Di) among the bacteria populations in untreated soil and that of Cerox-treated soils were high with mean diversity index above 0.95. Mean Di for Ceresate-treated soil was 0.88, and that for Paraquattreated soil was 0.85 indicating low bacterial populations in these treatment-type soils. The study also showed that application of the agrochemicals caused reduction in the number of total heterotrophic bacteria population sizes in the soil. Ceresate caused 82.50% reduction in bacteria number from a mean of 40 × 10⁵ cfu g⁻¹ of soil sample to 70 × 10⁴ cfu g⁻¹. Paraquat-treated soil showed 92.86% reduction, from a mean of 56 × 10⁵ cfu g⁻¹ to 40 × 10⁴ cfu g⁻¹. Application of Cerox to the soil did not have any remarkable reduction in bacterial population number. Total viable cell count studies using Congo red yeast-extract mannitol agar indicated reduction in the number of Rhizobium spp. after application of the agrochemicals. Mean number of Rhizobium population numbers per gram of soil was 180 × 10⁴ for the untreated soil. Cerox-treated soil recorded mean number of 138 × 10⁴ rhizobial cfu g⁻¹ of soil, a 23.33% reduction. Ceresate- and Paraquat-treated soils recorded 20 × 10⁴ and 12 × 10⁴ cfu g⁻¹ of soil, respectively, representing 88.89% and 93.33% reduction in Rhizobium population numbers. Correspondingly, the mean number of nodules per plant was 44 for the growth in untreated soil, 30 for the plant in the Cerox-treated soil, 8 for the plant in Paraquat-treated soil and 3 for the plant in Ceresate-treated soil. The study has confirmed detrimental effect of insecticide on bacterial populations in the soil. Total heterotrophic counts, rhizobial counts as well as the number of nodules of all samples taken from the chemically treated soils were all low as compared to values obtained for the untreated soil. However, the effect of the insecticide was minimal in all cases as compared to the effects of the herbicides on the soil fauna. Indiscriminate use of agrochemicals on farms can therefore affect soil flora and subsequently food production.     

Phytoestrogens as inhibitors of fungal 17beta-hydroxysteroid dehydrogenase

Different phytoestrogens were tested as inhibitors of 17beta-hydroxysteroid dehydrogenase from the fungus Cochliobolus lunatus (17beta-HSDcl), a member of the short-chain dehydrogenase/reductase superfamily. Phytoestrogens inhibited the oxidation of 100microM 17beta-hydroxyestra-4-en-3-one and the reduction of 100microM estra-4-en-3,17-dione, the best substrate pair known. The best inhibitors of oxidation, with IC(50) below 1microM, were flavones hydroxylated at positions 3, 5 and 7: 3-hydroxyflavone, 3,7-dihydroxyflavone, 5,7-dihydroxyflavone (chrysin) and 5-hydroxyflavone, together with 5-methoxyflavone. The best inhibitors of reduction were less potent; 3-hydroxyflavone, 5-methoxyflavone, coumestrol, 3,5,7,4'-tetrahydroxyflavone (kaempferol) and 5-hydroxyflavone, all had IC(50) values between 1 and 5microM. Docking the representative inhibitors chrysin and kaempferol into the active site of 17beta-HSDcl revealed the possible binding mode, in which they are sandwiched between the nicotinamide moiety and Tyr212. The structural features of phytoestrogens, inhibitors of both oxidation and reduction catalyzed by the fungal 17beta-HSD, are similar to the reported structural features of phytoestrogen inhibitors of human 17beta-HSD types 1 and 2.