High-throughput assay for detection of soybean lipoxygenase-1

A high-throughput assay was developed to detect soybean lipoxygenase 1 (LOX-1) using a multilabel plate reader. The assay was also adapted to a single cell fluorometer. Fluorescein is degraded by linoleic hydroperoxide produced from soybean lipoxygenase and linoleic acid. The decrease in fluorescence is measured over time, and the area-under-the-curve (AUC) is used to quantify the LOX-1 content of soybean extract. A dose-dependent response is seen with varied dilutions of pure LOX enzyme or soybean extracts. Percent recovery was between 97% and 108%, and relative standard deviation was 4.3%. Advantages of the assay include the reduced preparation time of samples and reduced use of reagents in the high-throughput assay. Multiple samples can be measured in a single run with a multilabel plate reader.     

Antioxidant activity of dodecyl gallate

Dodecyl (C12) gallate exhibits both potent chain-breaking and preventive antioxidant activity. The pyrogallol moiety is responsible for both activities. Dodecyl (lauryl) gallate prevents generation of superoxide radicals by xanthine oxidase, and this activity comes from its ability to inhibit the enzyme. The inhibition kinetics analyzed by Lineweaver-Burk plots found that dodecylgallate is a noncompetitive inhibitor for the generation of superoxide anion. Dodecyl gallate also inhibits formation of uric acid. The inhibition kinetics analyzed by Lineweaver-Burk plots found that dodecyl gallate is a competitive inhibitor for this oxidation. Mitochondrial lipid peroxidation induced by Fe(III)-adenosine 5'-diphosphate/reduced nicotinamide adenine dinucleotide was inhibited by dodecyl gallate while its parent compound, gallic acid, did not show this inhibitory activity. Dodecyl gallate protected mitochondrial functions and human red blood cells against oxidative stresses, but gallic acid showed little effect. The hydrophobic dodecyl group is largely associated with the preventive antioxidative activity.     

Slow-binding inhibition of mushroom (Agaricus bisporus) tyrosinase isoforms by tropolone.

A kinetic study of the inhibition of mushroom tyrosinase by tropolone has been made. Three tyrosinase isoforms were used: two commercial tyrosinases from Fluka and Sigma (isoelectric points of 4. 3 and 4.1, respectively) and one purified isoform from mushroom strain U1 (isoelectric point of 4.5). Tropolone is a slow-binding inhibitor of these mushroom tyrosinase isoforms. Increasing tropolone concentrations provoked a progressive decrease in both the initial velocity and the final (inhibited) steady-state rate in the progress curves of product accumulation. A rapid formation of an enzyme-inhibitor complex, which further undergoes a slow reversible reaction, could take place since the inhibition of the different isoforms was partially reversed by the addition of CuSO(4). The kinetic parameters that described the inhibition by tropolone were evaluated by nonlinear regression fits. Incubation experiments of the different isoforms with tropolone demonstrated that this inhibitor only could bind to the "oxy" form of tyrosinase which justifies a mechanism previously proposed to explain the inhibition of tyrosinase by slow-binding inhibitors.     

Growth inhibition of prostate cancer cells by epigallocatechin gallate in the presence of Cu2+

Green tea is an effective chemopreventive agent to human prostate cancer adenoma (PCA). Epigallocatechin gallate (EGCG) inhibited the growth of PCA cells and induced apoptosis. Cu(2+) is a trace element necessary to our health. Many studies proved that bioactivity of EGCG is altered in the presence of Cu(2+). We investigated the effects of EGCG on PCA cells in the presence of Cu(2+). Also, we explored potential mechanisms via measurement of the relative chemiluminescence of growth medium for PCA cells. Chemiluminscence can be an indication of free radicals. Our test results showed that the addition of EGCG and Cu(2+) to the growth medium decreased the relative viability of androgen-sensitive and androgen-insensitive human prostate cancer cells. However, the effects of EGCG on PCA cells depended on (1) the relative concentrations of added EGCG and Cu(2+) and (2) their order of addition. Our results indicated that few free radicals may be generated in vitro. If so, free radicals generated intracellularly may be a major factor behind apoptosis and growth inhibition observed in the PCA cells. Thus, EGCG might exert its effects intracellularly.     

Variable inhibition of iron uptake by oat phytosiderophore in five soybean cultivars

Abstract

Greenhouse experiment was conducted to evaluate the effect of arbuscular mycorrhizal fungi (AMF) on plant growth, and nutrient uptake in saline soils with different salt and phosphorus (P) levels. The following treatments were included in this experiment: (i) Soil A, with salt level of 16.6 dS m^?1 and P level of 8.4 mg kg^?1; (ii) Soil B, with salt level of 6.2 dS m^?1 and P level of 17.5 mg kg^?1; and (iii) Soil C, with salt level of 2.4 dS m^?1 and P level of 6.5 mg kg^?1. Soils received no (control) or 25 mg P kg^?1 soil as triple super phosphate and were either not inoculated (control) or inoculated with a mixture of AM (AM1) and/or with Glomus intraradices (AM2). All pots were amended with 125 mg N kg^?1 soil as ammonium sulfate. Barley (Hordeum vulgar L., cv. “ACSAD 6”) was grown for five weeks. Plants grown on highly saline soils were severely affected where the dry weight was significantly lower than plants growing on moderately and low saline soils. The tiller number and the plant height were also lower under highly saline condition. The reduced plant growth under highly saline soils is mainly attributed to the negative effect of the high osmotic potential of the soil solution of the highly saline soils which tend to reduce the nutrient and water uptake as well as reduce the plant root growth. Both the application of P fertilizers and the soil inoculation with either inoculum mixture or G. intraradices increased the dry weight and the height of the plants but not the tiller number. The positive effect of P application on plant growth was similar to the effect of AM inoculation. Phosphorus concentration in the plants was higher in the mycorrhizal plant compared to the non mycorrhizal ones when P was not added. On the other hand, the addition of P increased the P concentration in the plants of the non mycorrhizal plants to as high as that of the mycorrhizal plants. Iron (Fe) and zinc (Zn) uptake increased with AM inoculation. The addition of P had a positive effect on micronutrient uptake in soil with low level of soil P, but had a negative effect in soil with high level of soil P. Micronutrient uptake decreases with increasing soil salinity level. Inoculation with AMF decreases sodium (Na) concentration in plants grown in soil of the highest salinity level but had no effect when plants were grown in soil with moderate or low salinity level. The potassium (K) concentration was not affected by any treatment while the K/Na ratio was increased by AM inoculation only when plant were grown in soil of the highest salinity level.     

Effect of bicarbonate on iron reduction by soybean roots 1

Release of reducing compounds by soybean (Glycine max (L.) Merr.] roots has been identified as an adaptive response mechanism to iron‐deficiency conditions which result in chlorosis. These compounds facilitate the conversion of Fe⁺³ to the metabolically active Fe⁺² form, allowing for increased uptake by roots in solution culture experiments. Degree of chlorosis is closely associated with HCO₃ concentration; however, the relationship between that ion and root reduction potential apparently has not been studied. We examined the effect of HCO₃‐ on root reduction potential of ten commercially‐grown soybean cultivars known to differ in chlorosis expression in the field. Root reduction potential was measured spectrophotometrically at 594 nm on samples of nutrient solution containing reduced Fe⁺² . Plants were grown with 5 mM NaHCO₃ or in HCO₃ ^‐‐free solutions. Averaged over cultivars, 0.205 umoles Fe⁺³ were reduced in the HCO₃ ^‐‐free solutions while only 0.009 umoles Fe⁺³ were reduced in the solutions containing HCO₃ ^‐. No significant differences were observed among cultivars for root reduction potential within either HCO₃ ^‐ treatment. Results from this study suggest that HCO₃ ^‐ may inhibit iron absorption by limiting the ability of roots to release reducing compounds which make available Fe⁺2 in the soil solution. This may partially explain the role of HCO₃ ^‐ in reducing chlorosis.     

In vitro inhibition of the activation of Pro-matrix Metalloproteinase 1 (Pro-MMP-1) and Pro-matrix metalloproteinase 9 (Pro-MMP-9) by rice and soybean Bowman-Birk inhibitors

The in vitro inhibitory activity of the rice Bowman-Birk inhibitor (rBBI) or soybean Bowman-Birk inhibitor (sBBI) against trypsin-catalyzed activation of pro-matrix metalloproteinase 1 or 9 (pro-MMP-1 or pro-MMP-9), respectively, was investigated using electrophoresis with silver staining, heparin-enhanced zymography, biotinylated gelatin, Biotrak assay, and fluorescence quenched substrate hydrolysis. rBBI at concentrations of 0.08-0.352 mg/mL dose-dependently inhibited the in vitro activation of 45 microg/mL pro-MMP-1 by trypsin. Heparin-enhanced zymography analysis of pro-MMP-1, trypsin-activated MMP-1, and a mixture of pro-MMP-1-trypsin-rBBI showed clear zones associated with trypsin-activated MMP-1 and the absence of clear zones in lanes containing pro-MMP-1 or a mixture of pro-MMP-1, trypsin, and rBBI. The results of the Biotrak assay also indicated that rBBI dose-dependently suppressed the activation of pro-MMP-1 by trypsin. sBBI dose-dependently inhibited the activation of 100 microg/mL of pro-MMP-9 by trypsin. Biotinylated gelatin assays demonstrated that pro-MMP-9 or pro-MMP-9 in the presence of trypsin and BBI did not hydrolyze gelatin, whereas p-aminophenylmercury acetate (APMA)-activated MMP-9 and trypsin-activated MMP-9 caused significant hydrolysis of gelatin. Quenched fluorescence substrate hydrolysis for total MMP activity showed that pro-MMP-1 or pro-MMP-9 did not hydrolyze the substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2; active MMP-1 or MMP-9 hydrolyzed the substrate, but lower substrate hydrolysis was obtained when pro-MMP-1 or pro-MMP-9 was incubated with trypsin in the presence of increasing concentrations of rBBI. The results are discussed in light of the role of MMP-1 and MMP-9 in the process of angiogenesis and the potential of rBBI or sBBI as a functional food ingredient.     

Noncovalent cross-linking of casein by epigallocatechin gallate characterized by single molecule force microscopy

Interaction of the tea polyphenol epigallocatechin gallate (EGCG) with beta-casein in milk affects the taste of tea and also affects the stability of the tea and the antioxidant ability of the EGCG. In addition, interaction of polyphenols with the chemically similar salivary proline-rich proteins is largely responsible for the astringency of tea and red wine. With the use of single molecule force microscopy, we demonstrate that the interaction of EGCG with a single casein molecule is multivalent and leads to reduction in the persistence length of casein as calculated using the wormlike chain model and a reduction in its radius of gyration. The extra force required to stretch casein in the presence of EGCG is largely entropic, suggesting that multivalent hydrophobic interactions cause a compaction of the casein micelle.     

Lipoxygenase inhibitory activity of octyl gallate

Octyl gallate inhibited soybean lipoxygenase-1 (EC 1.13.11.12, type I) with an IC(50) of 1.3 microM. The inhibition of the enzyme by octyl gallate is a slow and reversible reaction without residual activity. The inhibition kinetics analyzed by Lineweaver-Burk plots indicates that octyl gallate is a competitive inhibitor, and the inhibition constant, K(I), was obtained as 0.54 microM. One molecule of octyl gallate scavenged six molecules of 1,1-diphenyl-2-picrylhydrazyl and inhibited autoxidative lipid peroxidation. In addition, octyl gallate was effective in preventing lipid peroxidation.     

Effect of soybean lipoxygenase on volatile generation and inhibition of Aspergillus flavus mycelial growth

Volatiles generated from lipoxygenase (LOX) normal and LOX deficient soybean (Glycine max) varieties with and without added lipase inhibited Aspergillus flavus mycelial growth and aflatoxin production. Soybean volatiles were analyzed using a solid phase microextraction (SPME) method combined with gas chromatography-mass spectrometry (GC-MS). Twenty-one compounds, including 11 aldehydes, three alcohols, four ketones, one furan, one alkane, and one alkene were detected in the LOX normal soybean line. However, only nine volatile compounds were observed in the LOX deficient soybean variety. The antifungal aldehydes hexanal and (E)-2-hexenal were observed in both LOX normal and LOX deficient lines and were detected at significantly higher amounts in soybean homogenate with added lipase. These aldehydes may be formed through alternate pathways, other than the LOX pathway, and may account for the inhibition of A. flavus growth observed. Other volatiles detected, particularly the ketones and alcohols, may contribute to the antifungal activity observed in both LOX normal and LOX deficient soybean lines. These results suggest that other factors, other than LOX activity, may better explain why soybeans are generally not as severely affected by A. flavus and aflatoxin contamination as other oilseed crops.     

Foliar diagnosis of soybean by dris

Abstract

The Diagnosis and Recommendation Integrated System (DRIS) has proven useful in the interpretation of tissue elemental analyses for many crops, and research was undertaken to apply the same method for foliar diagnosis of soybean (Glycine max [L.] Merr.). Using a data bank in excess of 3500 tissue samples, reference values for evaluating the status of soybean with respect to N, P, K, Ca, Mg, Mn, Fe, Cu, Zn, Mo, B and Al were derived. DRIS diagnoses generally agreed with those obtained by the sufficiency range method. In addition, DRIS assessed the nutrient balance in plant tissue, and identified not only the most‐limiting element, but the order in which other elements would likely become limiting. Further, DRIS was able to diagnose plant nutrient needs earlier in the life of the crop than the sufficiency range method (5 weeks compared to 10 weeks), which would allow remedial steps to be taken earlier. Treatments indicated by DRIS to be needed gave greater yield increases than those indicated by the sufficiency range approach. Geographic differences in DRIS norms were identified, and indicate that regional derivation of diagnostic values may be necessary.     

Antioxidant activity and inhibition of lipid peroxidation in germinating seeds of transgenic soybean expressing OsHGGT

Tocochromanols are potent lipid-soluble antioxidants and essential nutrients for human health. Genetic engineering techniques were used to develop soybeans with enhanced vitamin E levels, including tocotrienols, which are not found in soybean. The gene encoding rice homogentisate geranylgeranyl transferase (HGGT) was overexpressed in soybeans using seed-specific and constitutive promoters. The association between abundance of vitamin E isomers and antioxidant activity was investigated during seed germination. With the exception of β-tocotrienol, all vitamin E isomers were detected in germinating seeds expressing OsHGGT. The antioxidant properties of germinating seed extracts were determined using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) radicals and lipid peroxidation (TBARS). Compared with intact wild-type seeds, transgenic seeds showed increases in radical scavenging of 5.4-17 and 23.2-35.3% in the DPPH and ABTS assays, respectively. Furthermore, the lipid peroxidation levels were 2.0-4.5-fold lower in germinating seeds from transgenic lines than in wild-type seeds. Therefore, it appears that the antioxidant potential of transgenic oil-producing plants such as soybean, sunflower, and corn may be enhanced by overexpressing OsHGGT during seed germination.     

Hydrogen and aluminum inhibition of soybean root extension from limed soil into acid subsurface solutions

Soybean [Glycine max (L.) Merr. cv. ‘Ransom'] root elongation under varying concentrations of solution hydrogen (H) and aluminum (Al) was investigated in a vertical split‐root system. Roots extending from a limed and fertilized soil compartment grew for 12 days into a subsurface compartment with solutions adjusted to either different pH values from 3.7 to 5.5 or a factorial combination of pH (4.0,4.6, and 5.2) and Al (0,7.5, 15, and 30 μM) levels. Ionic forms of Al were estimated with GEOCHEM and solution Al was determined with ferron. Boron (B) (18.5 μM) and zinc (Zn) (0.5 μM) were supplied to all solution treatments, in addition to 2000 μM Ca, after preliminary studies at pH 5.2 without Al indicated that their omission inhibited length of tap roots and their laterals in the subsurface compartment. Both H⁺ and Al inhibited the length of lateral roots more than tap roots. Lateral roots failed to develop on tap roots at pH<4.3 or in treatments with 30 μM Al. Relative tap root length (RRL) among treatments receiving Al correlated with Al as measured by reaction with ferron for 30s. Ferron‐reactive Al was correlated to GEOCHEM‐predicted Al³⁺ activity (r=0.99). A 50% reduction in RRL occurred with either 2.1 μM Al³⁺ activity or 4.9 uM ferron‐reactive Al. The absence of shoot and soil‐root biomass differences among solution treatments in the split‐root system indicated that differences in root growth in the subsurface compartment were not directly confounded with differences in top growth.     

Differential activation of a latent polyphenol oxidase mediated by sodium dodecyl sulfate

A kinetic study of the activity of soluble and membrane-bound latent polyphenol oxidase (PPO) extracted from beet root (Beta vulgaris) was carried out. For the first time, two types of behavior (hyperbolic and sigmoid) are reported in the same enzyme for PPO activation by the surfactant sodium dodecyl sulfate (SDS), depending on substrate nature. A kinetic model based on cooperative systems is developed to describe the activation effect of SDS, enabling the determination of the number of surfactant molecules binding to the enzyme in the activation process. The results indicate that the active site of the enzyme is not affected by SDS and that a stepwise conformational change favors the access of hydrophobic substrates compared to hydrophilic ones. Differential activation of PPO mediated by SDS may be of relevance in the control of PPO activity since the enzyme is able to express activity toward a specific substrate while remaining latent to others.     

Sodium dodecyl sulfate capillary electrophoresis of wheat proteins. 1. Uncoated capillaries.

Four different polymer/buffer systems (a commercial polymer from Bio-Rad, dextran, poly(ethylene oxide) (PEO), and non-crosslinked poly(acrylamide)) were evaluated for use in sodium dodecyl sulfate capillary electrophoresis (SDS-CE) separations of wheat proteins. These polymers were chosen on the basis of published reports of their use in uncoated or dynamically coated capillaries. Each polymer was optimized (where possible) by manipulating the polymer concentration and buffer concentration, and through the use of organic modifiers such as methanol and ethylene glycol. The addition of ethylene glycol to the separation buffer was found to improve the resolution of the separations, despite dilution of the sieving polymers. When PEO was used as the sieving polymer, however, no improvement was seen when ethylene glycol was added. Despite producing similar separations of molecular mass markers, the polymers did not all produce similar wheat protein separations. The commercial reagent and dextran produced similar separations, while the poly(acrylamide) produced faster separations than either. The poly(acrylamide) displayed much lower resolution in the 40-60 kDa range than the other polymers, though this polymer was able to separate the high molecular mass glutenin subunits (HMM-GS) without the use of added organic solvent. PEO produced much different wheat protein separations than the other polymers, despite similar separations of the molecular weight markers. This may have been due to interaction between the wheat proteins and PEO. Each polymer system also predicted different molecular masses of the various wheat protein fractions separated, with the PEO and poly(acrylamide) grossly overestimating the masses for all protein classes. This could have been due to protein-polymer interactions. Further work was done with the Bio-Rad buffer modified by the addition of ethylene glycol. Several different wheat protein fractions as well as proteins extracted from several different cultivars were separated with this buffer and compared. SDS-CE separations were also compared to SDS-poly(acrylamide) gel electrophoresis (PAGE) and several differences in the migration pattern of HMM-GS were noted.     

Oxidation of resveratrol catalyzed by soybean lipoxygenase

In this work the oxidative degradation of resveratrol catalyzed by lipoxygenase-1 (LOX-1) has been studied. The process has been characterized by spectroscopic and polarographic measurements. The oxidation of resveratrol was dependent on the concentration of resveratrol and the enzyme. When resveratrol was incubated in the presence of lipoxygenase at pH 9.0, the reaction displayed a k(M) value of 18.6 x 10(-)(6) M and a catalytic efficiency (k(cat)/k(M)) of 4.3 x 10(4) s(-)(1) M(-)(1). These values are close to those shown by the enzyme when linoleic acid is used as the substrate. The effect of lipoxygenase inhibitors on the lipoxygenase-catalyzed resveratrol oxidation was also evaluated. The rate of resveratrol oxidation was markedly decreased by the presence of NDGA in the incubation mixture. From HPLC measurements, it can be deduced that resveratrol is oxidatively decomposed to a complex mixture of products similar to those obtained when the molecule is oxidized by hydrogen peroxide.     

Kinetics of reduction of ferrylmyoglobin by (-)-epigallocatechin gallate and green tea extract

The hypervalent heme pigment ferrylmyoglobin, a potential prooxidant in muscle tissue and meat, is efficiently reduced by epigallocatechin gallate (EGCG) from green tea and by green tea polyphenol extract (GTP) in neutral or moderately acidic aqueous solution (0.16 M NaCl) to yield metmyoglobin in two parallel processes. The second-order rate constant for direct reduction at pH 7.4 and 25 degrees C was found to have the value 1170 +/- 83 M(-1).s(-1) and activation parameters DeltaH(#) = 70.6 +/- 7.2 kJ.mol(-1) and DeltaS(#) = 50.7 +/- 24.1 J.mol(-1).K(-1) for EGCG and the value 2300 +/- 77 M(-1).s(-1) and parameters DeltaH(#) = 60.6 +/- 2.6 kJ.mol(-1) and DeltaS(#) = 23 +/- 9 J.mol(-1).K(-1) for GTP (based on EGCG concentration). For decreasing pH, the rate increased moderately due to a parallel reduction of protonated ferrylmyoglobin. At physiological pH, EGCG is more efficient in deactivating ferrylmyoglobin than other plant phenols investigated, and the relatively high enthalpy and positive entropy of activation suggest an outer-sphere electron transfer mechanism. The interaction between EGCG and other tea catechins in GTP could be responsible for the even stronger ability for GTP to deactivate ferrylmyoglobin.     

Characterization of kafirin and zein oligomers by preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Quantitative and qualitative analysis of uncooked zein and kafirin fractions were performed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic profiles. Kafirins and zeins present the same oligomer and monomer compositions with the exception of a 66 kDa oligomer that is only present in kafirins. The quantitative analysis showed differences between zein and kafirin. The composition of each oligomer was established via preparative SDS-PAGE. Part of the cooked oligomers resists reduction; the presence of those oligomers could be related to the decrease on protein digestibility with the cooking process.     

Enhanced soybean (Glycine max L.) plant growth and nodulation by Bradyrhizobium japonicum-SB1 in presence of Bacillus thuringiensis-KR1

Abstract

Nodulation and subsequent nitrogen fixation are important factors that determine the productivity of soybean (Glycine max L.). The beneficial effects of nodulation can be enhanced when rhizobial inoculation is combined with plant-growth-promoting bacteria (PGPB). The PGPB strain Bacillus thuringiensis-KR1, originally isolated from the nodules of Kudzu vine (Pueraria thunbergiana), was found to promote growth of soybean plants (variety VL Soya 2) under Jensen's tube and growth pouch conditions, when co-inoculated with Bradyrhizobium japonicum-SB1. Co-inoculation with Bacillus thuringiensis-KR1 (at a cell density of 10 cfu) provided the highest and most consistent increase in nodule number, shoot weight, root weight, root volume, and total biomass, over rhizobial inoculation and control, under both conditions. The results demonstrate the potential benefits of using nonrhizobial nodule occupants of wild legumes for the co-inoculation of soybean, with Bradyrhizobium japonicum-SB1, in order to achieve plant-growth promotion and increased nodulation.