Molecular analysis and physicochemical properties of electrophoretic variants of wild soybean Glycine soja storage proteins

Cultivated soybeans (Glycine max) are derived from wild soybeans (Glycine soja) and can be crossed with them to produce fertile offspring. The latter exhibit greater genetic variation than the former, suggesting a possibility that wild soybeans contain storage proteins with properties different from and better than those of cultivated soybeans. To identify a wild soybean suitable for breeding a new soybean cultivar, we analyzed seed proteins from 390 lines of wild soybeans by electrophoresis. We found some lines containing electrophoretic variants of glycinin and beta-conglycinin subunits: one line containing a small alpha' subunit of beta-conglycinin and two and five lines containing small A3 and large A4 polypeptides of glycinin, respectively. Beta-Conglycinin and glycinin containing such variant subunits exhibited solubility and emulsifying ability similar to those of the predominant types of wild and cultivated soybeans. Glycinins containing small A3 and large A4 gave a shoulder derived from the start of denaturation at a temperature 4 degrees C lower than that of glycinin from the predominant types of wild and cultivated soybeans, although their thermal denaturation midpoint temperatures were very similar to each other. Cloning and sequencing of the predominant and variant subunit cDNAs revealed that the small alpha' and the small A3 lacked 24 amino acid residues in the extension region and four amino acid residues in the hypervariable region, respectively, and that the large A4 did not have an insert corresponding to the difference in the electrophoretic mobility but Arg279 and Gln305 were replaced by glutamine and histidine, respectively, in the hypervariable region. These suggest that small differences even in the hypervariable region can affect the thermal stability, as well as the electrophoretic mobilities, of the proteins.     

Changes in some enzyme activities and the nicotinamide nucleotide coenzymes in barley seedlings during vernalization

Respiratory metabolism was investigated by determining the following enzyme activities and nicotinamide nucleotide coenzyme contents in barley shoots (Hordeum vulgare L., var. Akashinriki) during vernalization : glucose-6-phosphate dehydrogenase, phosphoglucoisomerase, enolase, isocitrate dehydrogenase, malate dehydrogenase, and glutamic dehydrogenase. The activities of glucose-6-phosphate de hydrogenase and phosphoglucoisomerase in vernalized shoots were slightly lower than those in non-vernalized samples. Malate dehydrogenase activity increased gradually in vernalized shoots and was always higher than in non-vernalized shoots. Activation was found in enolase, isocitrate dehydrogenase and glutamic dehydrogenase after a long vernalization treatment.

The contents of total nicotinamide nucleotide coenzymes increased during the early germination stage in both treatments, then decreased gradually in the succeeding stage.     

Estimation of mycorrhizal colonization of the roots of oak seedlings inoculated with an ectomycorrhizal fungus,Laccaria amethystea

Six-month-old seedlings of Quercus serrata and Quercus glauca in a nursery were inoculated with the ectomycorrhizal fungus Laccaria amethystea encapsulated in alginate gel and grown in the nursery. The seedlings were collected at 1, 3, and 5 months after the inoculation and examined for colonization of the root system with ectomycorrhizal fungi. The roots within 5 months after the inoculation showed rudimentary ectomycorrhizal colonization. The level of colonization of the root system was estimated based on the intensity of hyphal covering on the root tips by staining with a fluorescent dye and expressed as an index of mycorrhizal colonization (IMC). IMC increased with the time after inoculation and reached values of 4 and 12% in Q. serrata and Q. glauca, respectively at 5 months after the inoculation. The determination of IMC may enable to assess the development of mycorrhizal colonization of the root system that shows rudimentary ectomycorrhizas after the inoculation.     

Partial purification and characterization of polyphenol oxidase from banana (Musa sapientum L.) peel

Polyphenol oxidase (EC 1.10.3.1, o-diphenol: oxygen oxidoreductase, PPO) of banana (Musa sapientum L.) peel was partially purified about 460-fold with a recovery of 2.2% using dopamine as substrate. The enzyme showed a single peak on Toyopearl HW55-S chromatography. However, two bands were detected by staining with Coomassie brilliant blue on PAGE: one was very clear, and the other was faint. Molecular weight for purified PPO was estimated to be about 41 000 by gel filtration. The enzyme quickly oxidized dopamine, and its Km value (Michaelis constant) for dopamine was 3.9 mM. Optimum pH was 6.5 and the PPO activity was quite stable in the range of pH 5-11 for 48 h. The enzyme had an optimum temperature at 30 degrees C and was stable up to 60 degrees C after heat treatment for 30 min. The enzyme activity was strongly inhibited by sodium diethyldithiocarbamate, potassium cyanide, L-ascorbic acid, and cysteine at 1 mM. Under a low buffer capacity, the enzyme was also strongly inhibited by citric acid and acetic acid at 10 mM.     

Structure-activity relationships of tea compounds against human cancer cells

The content of the biologically active amino acid theanine in 15 commercial black, green, specialty, and herbal tea leaves was determined as the 2,4-dinitrophenyltheanine derivative (DNP-theanine) by a validated HPLC method. To define relative anticarcinogenic potencies of tea compounds and teas, nine green tea catechins, three black tea theaflavins, and theanine as well as aqueous and 80% ethanol/water extracts of the same tea leaves were evaluated for their ability to induce cell death in human cancer and normal cells using a tetrazolium microculture (MTT) assay. Compared to untreated controls, most catechins, theaflavins, theanine, and all tea extracts reduced the numbers of the following human cancer cell lines: breast (MCF-7), colon (HT-29), hepatoma (liver) (HepG2), and prostate (PC-3) as well as normal human liver cells (Chang). The growth of normal human lung (HEL299) cells was not inhibited. The destruction of cancer cells was also observed visually by reverse phase microscopy. Statistical analysis of the data showed that (a) the anticarcinogenic effects of tea compounds and of tea leaf extracts varied widely and were concentration dependent over the ranges from 50 to 400 microg/mL of tea compound and from 50 to 400 microg/g of tea solids; (b) the different cancer cells varied in their susceptibilities to destruction; (c) 80% ethanol/water extracts with higher levels of flavonoids determined by HPLC were in most cases more active than the corresponding water extracts; and (d) flavonoid levels of the teas did not directly correlate with anticarcinogenic activities. The findings extend related observations on the anticarcinogenic potential of tea ingredients and suggest that consumers may benefit more by drinking both green and black teas.     

Kinetics of light-induced cis-trans isomerization of four piperines and their levels in ground black peppers as determined by HPLC and LC/MS

The pungent compounds piperine and isomers thereof, secondary metabolites present in black and white pepper fruit, undergo light-induced isomerizations. To facilitate studies in this area, an HPLC method has been developed for analysis and isolation of the following four possible piperine-derived photoinduced isomers: piperine, isopiperine, chavicine, and isochavicine. The limits of detection (LOD) estimated from calibration plots were approximately 15-30 ng for each isomer. Reproducibilities of the analyses were excellent, and recoveries of spiked samples were as follows (average +/- SD; n = 3): chavicine, 98.4 +/- 2.1%; isopiperine, 96.2 +/- 3.2%; piperine, 104 +/- 3.8%; isochavicine, 98.9 +/- 3.0%. To determine the kinetics of these isomerizations, fluorescent light, sunlight, and UV radiation at 254 nm was used to induce cis-trans geometric isomerization as a function of light intensities and time of exposure determined with the aid of high-performance liquid chromatography (HPLC) and liquid chromatography with diode array UV detection-mass spectrometry (LC-DAD/MS). HPLC was also used to determine the distribution of the isomers in four commercial ground black pepper products used as spices in culinary practice. Isomerization increased with light intensities and time of exposure and leveled off at the so-called photostationary phases. The piperine levels of the four products were quite similar, ranging (in wt %) from 10.17 to 11.68. The amounts of the other three isomers ranged from 0.01 to 0.07 of the total for chavicine; from 0.15 to 0.23 for isopiperine; and from 0.37 to 0.42 for isochavicine. The results establish the utility of the HPLC method for simultaneous analysis of the four isomers both in pure form and in black pepper extracts. The dietary significance of the results is discussed.     

New piezoelectric moduli of wood: <Emphasis Type="Italic">d</Emphasis><Subscript>31</Subscript> and <Emphasis Type="Italic">d</Emphasis><Subscript>32</Subscript>

This article reports the piezoelectric moduli of wood d ₃₁, d ₃₂, and d ₃₆. The piezoelectric moduli of wood d ₃₁ and d ₃₂ have not been previously reported, although there has been much research on the d ₁₄ and d ₂₅ moduli of wood. The moduli d ₃₁, d ₃₂, and d ₃₆ were measured carefully because their absolute values were considerably smaller than those of d ₁₄ and d ₂₅. For Softwoods, d ₃₆ values were mostly negative, whereas the values for hardwoods had either positive or negative values. The other moduli, d ₃₁ and d ₃₂, were a mixture of positive and negative values in softwoods and hardwoods. The existence of d ₃₁ and d ₃₂ suggests the presence of an electrical polarity of the cellulose crystal in the fiber direction of the wood. The polarities of d ₃₁ and d ₃₂ became clear from wood in the outer part of the trunk, where the crystallinity of cellulose is large and the alignment of the crystals becomes parallel to the fiber direction.     

Changes of crystallinity in wood cellulose by heat treatment under dried and moist conditions

The different effects of heat treatment on wood, especially on the cellulose crystallites of wood under ovendried and highly moist conditions, were investigated by X-ray diffractometer. Heat was found to increase significantly the crystallinity of wood cellulose; moreover, almost twice as much crystallization was observed after heat treatment of spruce and buna under a highly moist condition than under the oven-dried condition. In pure cellulose almost the same crystallization was observed under both the conditions, whereas more crystallization occurred in wood cellulose than in pure cellulose. Absolute crystallization was observed for the wood species and pure cellulose under both conditions, considering the thermal decomposition of the amorphous region in addition to crystallization. Our results suggested that other components accomparying wood cellulose were involved in the increase of crystallinity by heat treatment, and that wood cellulose contained more quasicrystalline regions than pure cellulose. Moreover, calculated apparent activation energies revealed that crystallization and decrystallization in pure and wood cellulose under heat treatment of highly moist condition were some-what easier than those under the oven-dried condition. The behavior of the piezoelectric modulusd ₂₅ almost paralleled that of crystallinity.     

Lateral bud outgrowth on decapitated shoots of low-pruned mulberry (Morus alba L.)

After decapitation, lateral shoot growth of mulberry coppice (Morus alba L. cv. Shin-ichinose) from 10-year-old stumps had an extremely acrotonic form compared with that of intact one-year-old stems of trees during the spring. When one dominant shoot from each stump was decapitated, only a few upper lateral buds grew out and elongated. Defoliation of the decapitated shoots resulted in an increase in the number of shoots sprouting and a decrease in the rate of elongation of the laterals. Further enhancement of laterals on decapitated dominant shoots resulted from the removal of competing intact coppice shoots from the stumps. 1-Naphthaleneacetic acid (NAA) applied as a spray reduced the number of lateral buds sprouting from both defoliated, decapitated erect shoots and intact horizontally trained shoots. The results suggest that management of mulberry coppice could increase the supply of leaves for commercial silk production.     

Astaxanthin Exerts Immunomodulatory Effect by Regulating SDH-HIF-1α Axis and Reprogramming Mitochondrial Metabolism in LPS-Stimulated RAW264.7 Cells

Astaxanthin (AX) is a carotenoid that exerts potent antioxidant activity and acts in cell membranes and mitochondria, which consist of the bilayer molecules. Targeting mitochondria to ameliorate inflammatory diseases by regulating mitochondrial metabolism has become possible and topical. Although AX has been shown to have anti-inflammatory effects in various cells, the mechanisms are quite different. In particular, the role of AX on mitochondrial metabolism in macrophages is still unknown. In this study, we investigated the effect of AX on mitochondria-mediated inflammation and its mechanisms in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. AX attenuated the mitochondrial O₂⁻ production and maintained the mitochondrial membrane potential, implying that AX preserved mitochondrial homeostasis to avoid LPS stimulation-induced mitochondrial dysfunction. Additionally, AX prevented the decrease in mitochondrial complexes I, II, and III, which were caused by LPS stimulation. Especially, AX inhibited the reduction in mitochondrial succinate dehydrogenase (SDH; complex II) activity and upregulated the protein and mRNA level of SDH complex, subunit B. Furthermore, AX blocked the IL-1β expression by regulating the SDH-HIF-1α axis and suppressed the energy shift from an OXPHOS phenotype to a glycolysis phenotype. These findings revealed important effects of AX on mitochondrial enzymes as well as on mitochondrial energy metabolism in the immune response. In addition, these raised the possibility that AX plays an important role in other diseases caused by SDH mutation and metabolic disorders.