麻叶荨麻籽抑菌成分和抑菌特性的研究

目的：为了确定麻叶荨麻籽的抑菌成分和其抑菌特性。方法：以麻叶荨麻籽的不同提取物为抑菌剂进行抑菌。结果：麻叶荨麻籽的抑菌活性部位是石油醚和乙酸乙脂提取物,其中石油醚提取物的抑菌作用最强,对大肠杆菌和枯草芽孢杆菌的最低抑菌浓度是1．25mg/mL,金黄色葡萄球菌是2．5mg/mL。麻叶荨麻籽抗菌活性成分是：酚类、鞣质、有机酸等化合物。石油醚提取物对三种细菌脲酶活性均有不同程度的抑制作用。结论：麻叶荨麻籽提取物是较为有效的抑菌物。     

Mature Amaranthus hypochondriacus seeds contain non-processed 11S precursors

Amaranth is a dicotyledonous plant whose major seed storage proteins are globulins and glutelins. An unique feature of amaranth seeds is the presence of a fraction named albumin-2, that is extractable with water only after an exhaustive extraction of globulins and albumin-1. In this work, we tested the hypothesis that albumin-2 fraction could be constituted by a non-processed 11S globulin (proglobulin). To this end, the gene encoding the amaranth 11S subunit was cloned and expressed in Escherichia coli. Subsequently, the recombinant proglobulin and albumin-2 purified from seeds were treated with a sunflower vacuolar processing enzyme (VPE). A 55 kDa component of albumin-2 was specifically cleaved into 38 and 17-15 kDa polypeptides, as a consequence of this endoproteolytic cleavage a change of the oligomeric state from trimeric to hexameric was observed. Amaranth 11S globulin fraction was not modified under these proteolysis conditions. Using VPE-specific antibodies, it was shown that amaranth expresses a 57 kDa VPE, and that both developing and mature amaranth seeds have VPE activity, although the increase of this activity during amaranth seed development is higher than that observed for sunflower seeds. These results confirm the presence of unprocessed 11S precursors in mature amaranth seeds; this phenomenon cannot, however, be attributed to low VPE activity during developing of amaranth seeds.     

Activation of Enzymes during Germination: Amylopectin-1,6-glucosidase in Peas

Development of amylopectin-1, 6-glucosidase (amylopectin 6- glucanohydrolase) activity in germinating peas occurs under conditions in which protein synthesis is inhibited and α- and β-amylase fail to increase in activity. An increase in activity of amylopectin 6-glucanohydrolase can be obtained in cell free homogenates of imbibed pea seeds under autolytic conditions. Activation is shown to occur in a particulate fraction and to be due to the action of a proteolytic enzyme in the homogcnates. A system could be constituted consisting of a particulate fraction and trypsin which lead to a rapid activation of the amylopectin 6-glucanohvdrolase. This is regarded as proof that enzymes in germinating seeds can be formed by liberation or activation of precursor forms. The implications of this are discussed.     

Lipid peroxidation, carbohydrate hydrolysis, and Amadori-Maillard reaction at early stages of dry seed aging

Processes underlying aging of air-dry seeds were investigated. Naturally aged seeds of pea (Pisum sativum L.), cucumber (Cucumis sativus L.), and buckwheat (Fagopyrum esculentum Moench.) were separated into three fractions of different quality according to their room temperature phosphorescence (RTP): fraction I contained strong seeds; fraction II comprised weak seeds; and fraction III was composed of dead seeds. These seed fractions were used to identify the processes in air-dry seeds that account for the initial deterioration in seed quality at early stages of aging and for the possible transient improvement of seed quality at further aging. Experiments with seed powders showed that the increase in thermochemiluminescence (TCL) in the temperature range of 50?C110°C was determined by the presence of lipid peroxidation products. No difference was observed between TCL levels in fraction I and fraction II seeds; hence, lipid peroxidation is not responsible for the transition of strong seeds to the fraction of weak seeds. The TCL intensity monitored upon heating in the range of 50?C110°C increased only in the fraction of dead seeds. In fraction II seeds, a twofold increase in TCL upon heating at 150°C was observed in aged seeds; this indicates that the products of oligosaccharide hydrolysis (glucose) were more abundant in fraction II seeds than in fraction I seeds under similar conditions. This assumption was confirmed by direct determination of glucose content with a glucometer. Hence, the transition of air-dry seeds from fraction I (strong seeds) to fraction II (weak seeds) occurred due to the activation of carbohydrate hydrolysis in aging seeds. Since air-dry seeds contain no free water during aging, the seed moisture content decreases during hydrolysis, presumably, because the bound water participates in the hydrolysis reaction. The decrease in ??improved?? seeds of glucose content and the increase in moisture content suggest the activation of amino-carbonyl reaction. It is proposed that the amino-carbonyl (Amadori-Maillard) reaction is responsible for closing water channels in improved seeds, as well as for the decreased water permeability of cell membranes during seed imbibition.     

Lipid peroxidation associated with accelerated aging of soybean axes

Soybean seeds age rapidly during storage at high temperature and high relative humidity. The axes of such aged seeds contain high levels of malondialdehyde, a product of the peroxidation of unsaturated fatty acids. The levels of linoleic (18:2) and linolenic (18:3) acids in a polar lipid (phospholipid) fraction decrease during aging and more dramatically during postaging deterioration. None of these changes occurred in seeds that have been stored at high temperature but low relative humidity. No superoxide dismutase activity was detected in any nonimbibed seed. In viable seeds, activity was detectable 1.5 hours after the onset of imbibition, but none was found in aged seeds up to 5 hours. It is suggested that aging leads to peroxidative changes to lipids and that these could contribute to loss of viability.     

The influence of poly(A)-binding proteins on translation of poly(A)+ RNA in a cell-free system from embryo axes of dry pea seeds

Ribonucleoproteins of the ribosomal fraction of germinated pea embryo axes, containing translationally active mRNA, differ from analogous ribonucleoproteins of dry pea seeds, which contain stored mRNA, by the presence of a 60 kDa protein fraction showing affinity to poly(A). The above protein fraction largely affects the activity of poly(A)⁺ RNA translation in cell-free system. An activating effect is clearly seen at a weight ratio of poly(A)-binding proteins:poly(A)⁺ RNA of 3:1, whereas with an increase in the concentration of these proteins the translational activity drops. The effect of poly(A)-binding proteins containing the 60 kDa fraction on poly(A)⁺ RNA dependent cell-free translation can be efficiently reduced by simultaneous addition of synthetic poly(adenylic acid). It was also proved that activation of translation does not influence its products. It is concluded that poly(A)-binding proteins from the ribosomal fraction of embryo axes of pea seeds, especially the 60 kDa fraction, are involved in regulation of the translational activity of poly(A)⁺ RNA.     

One-step purification of Kunitz soybean trypsin inhibitor

A fast and simple method for the extraction and purification of Kunitz trypsin inhibitor from soybean seeds is described. The first step consisted in the heat treatment of whole soybean seeds in water at 60 degrees C for 90 min. It was found that 8.4% of total trypsin inhibitory activity of the seeds was secreted during heat treatment. The aqueous medium was loaded onto an affinity chromatography column with immobilized trypsin. The retained fraction, eluted with 0.01 N HCl, contained the purified Kunitz trypsin inhibitor, which was subsequently stabilized by freeze-drying without loss of activity. From 1g soybean seeds, 0.7 mg inhibitor with a specific trypsin inhibitory (TI) activity of 11,430 TIU/mg was obtained. The yield was greater than that obtained with established procedures. Due to the ease of the procedure proposed, the method is readily scalable to pilot plant or industrial preparations.     

Antinociceptive activity and chemical composition of constituents from Caragana microphylla seeds

This investigation was undertaken to ascertain the antinociceptive activity of Caragana microphylla Lam. seeds and isolate and characterize the constituents. Antinociceptive activity was screened using acetic acid-induced abdominal constriction in ICR mice. The 75% ethanol extract and some fractions showed analgesic activity, but the antinociceptive activity of chloroform fraction was the strongest and was more productive than other fractions. Seven compounds were isolated from it and identified as: (1) machaeric acid, (2) beta-sitosterol, (3) stigmasterol, (4) pratol, (5) dehydrocavidine, (6) formononetin and (7) sucrose. Caragana microphylla Lam. seeds showed analgesic activity, with the chloroform fraction showing the strongest analgesic activity among the fractions.     

Transgenic rice established to express corn cystatin exhibits strong inhibitory activity against insect gut proteinases

Corn cystatin (CC), a phytocystatin, shows a wide inhibitory spectrum against various cysteine proteinases. We produced transgenic rice plants by introducing CC cDNA under CaMV 35S promoter as a first step to obtain a rice plant with insecticidal activity. This attempt was based on the observation that many insect pests, especially Coleoptera, have cysteine proteinases, probably digestive enzymes, and also that oryzacystatin, an intrinsic rice cystatin, shows a narrow inhibition spectrum and is present in ordinary rice seeds in insufficient amounts to inhibit the cysteine proteinases of rice insect pests. The transgenic rice plants generated contained high levels of CC mRNA and CC protein in both seeds and leaves, the CC protein content of the seed reaching ca. 2% of the total heat soluble protein. We also recovered CC activity from seeds and found that the CC fraction efficiently inhibited both papain and cathepsin H, whereas the corresponding fraction from non-transformed rice seeds showed much lower or undetectable inhibitory activities against these cysteine proteinases. Furthermore, CC prepared from transgenic rice plants showed potent inhibitory activity against proteinases that occur in the gut of the insect pest, Sitophilus zeamais.     

Benzyl isothiocyanate is the chief or sole anthelmintic in papaya seed extracts

Papaya (Carica papaya) seeds were extracted in an aqueous buffer or in organic solvents, fractionated by chromatography on silica and aliquots tested for anthelmintic activity by viability assays using Caenorhabditis elegans. For all preparations and fractions tested, anthelmintic activity and benzyl isothiocyanate content correlated positively. Aqueous extracts prepared from heat-treated seeds had no anthelmintic activity or benzyl isothiocyanate content although both appeared when these extracts were incubated with a myrosinase-containing fraction prepared from papaya seeds. A 10 h incubation of crude seed extracts at room temperature led to a decrease in anthelmintic activity and fractionated samples showed a lower benzyl isothiocyanate content relative to non-incubated controls. Benzyl thiocyanate, benzyl cyanide, and benzonitrile were not detected in any preparations and cyanogenic glucosides. which were present, could not account for the anthelmintic activity detected. Thus, our results are best explained if benzyl isothiocyanate is the predominant or sole anthelmintic agent in papaya seed extracts regardless of how seeds are extracted.     

Soybean trypsin inhibitor and beta-amylase stimulate macrophages

Mature soybean seeds were found to contain macrophage stimulation activity. The activity existed in a water-soluble fraction of soybean whey. Gel filtration, DEAE-cellulose column chromatography and high-performance liquid chromatography of the water-soluble fraction gave two active proteins. By N-terminal and total amino acid analyses, these were effectively stimulated by each of the two proteins to produce nitrite.     

The auxin conjugate 1-O-indole-3-acetyl-beta-D-glucose is synthesized in immature legume seeds by IAGlc synthase and may be used for modification of some high molecular weight compounds

Immature seeds of some dicotyledonous plants contain IAGlc synthase catalysing the synthesis of 1-O-IAGlc. This enzyme activity is comparable with 1-O-IAGlc synthase activity investigated earlier in liquid endosperm of Zea mays. Polyclonal antibodies against maize 1-O-IAGlc synthase cross-react with partially purified 1-O-IAGlc synthase from immature pea and rape seeds. Single immunoreactive bands were observed at a locus corresponding to 45.7 kDa and 43.7 kDa from pea and rape enzyme preparations, respectively, unlike that from the 50 kDa molecular mass of the maize enzyme. It was also observed that some high molecular weight compounds of pea seeds are labelled in vivo by [(14)C] IAA, and unlabelled 1-O-IAGlc inhibits that labelling. In immature pea seeds 43-49.8% of the IAA-modified high molecular weight compounds, obtained after ultracentrifugation, was found in the soluble fraction and 50.1-57% in the insoluble fraction. Ester-linked IAA accounted for about 6-9% and 38-45.6% in soluble and insoluble material, respectively, estimated after hydrolysis in 1 N NaOH. Enzymatic hydrolysis of IAA-labelled high molecular weight compounds gives free IAA and compound(s) corresponding to IAGlc isomers. These results suggest that 1-O-IAGlc synthesized in legume seeds may be used for the modification of some high molecular weight compounds.     

Tissue and subcellular distribution of the lectin from Datura stramonium (thorn apple).

Plants of Datura stramonium (thorn-apple) were dissected into their component tissues and examined for the presence of the Datura lectin. This lectin was easily detected in seeds and in various parts of the flowers of adult plants. Traces were also found in green (emerged) cotyledons and roots of seedlings. The specific lectin activity in seeds contained within the fruits increased as the seeds matured. Mature seeds were homogenized in sucrose and separated by differential centrifugation into four fractions, three of which were clearly of distinct composition. Most of the lectin activity sedimented with the low-speed (cell-wall/protein-body) pellet, but a similar specific activity was recovered from the other fractions. However, if EDTA was included in the homogenization medium, three or four times more lectin activity was recovered in the soluble fraction. Immunofluorescent staining of formaldehyde-fixed sections showed that the lectin was localized in the cytoplasm, with little associated with cell walls. The possible relevance of these results to the function of the lectin in plant cells is discussed.     

Glycinin A4A5 subunit digesting protease in soybean seeds.

Endopeptidase was partially purified from the globulin fraction of 4-hr-imbibed soybean seeds. The protease fraction obtained had proteolytic activity on the glycinin A4A5 subunit at both pH 4 and 8. A suitable peptidic substrate for the endopeptidase was isolated from the tryptic digest of the carboxymethylated A4A5 subunit. Using the tryptic peptide of glycinin A5 subunit, a simple assay system for the soybean endopeptidase activity has been established. The activity was significantly inhibited by phenylmethylsulfonyl fluoride, indicating the endopeptidase is a serine protease.     

Synergism between gibberellic acid and low Pfr levels inducing germination of Kalanchoë seeds

Sown on water, seeds of Kalanchoëbiossfetdiana Poelln. cv. Feuerblute are absolutely light-requiring and show full red/far-red reversibility. In seeds, sown on 2 ×10^-3 M gibberellic acid, red/far-red reversibility disappears and both short red and far-red irradiations induce germination. Gibberellic acid alone does not induce germination, but it increases the physiological activity of P_fr to the extent, that the low P_fr level obtained by far-red irradiation becomes very effective. The synergism between gibberellic acid and far-red light appears after a two-day incubation; period. The nature of this lag phase was examined by measuring both germination and uptake of labelled gibberellic acid in intact seeds and seeds with a punctured seed coat. The lag phase was shown to be independent of the uptake kinetics of gibberellic acid and allows development to a specific stage, necessary for germination after phytochrome-phototransformation. The kinetics of the uptake of gibberellic acid by intact seeds and embryos of intact seeds are different. In intact seeds most of the gibberellic acid is retained in the seed coat; only a small fraction actually penetrates to the embryo where it can exert its physiological activity.     

DNA topoisomerase I from rice: Enzyme synthesis in germination, and partial purification from cultured cells

DNA topoisomerase I activity was observed in two-day-old seeds of rice when the seeds started germination at 30°. Partially purified enzyme from cultured rice cells showed maximum activity at pH 7.0 with 75 mM NaCl in the absence of ATP, and showed resistance to camptothecin and DNA-intercalating reagents. The M_r was ca 80 000 using gel permeation on a Sephacryl S-200 column. After fractionation of the homogenate from cultured rice cells by centrifugation, the activity was observed mainly in the crude nuclear fraction.     

Involvement of glyoxysomal lipase in the hydrolysis of storage triacylglycerols in the cotyledons of soybean seedlings

The total cotyledon extract of soybean (Glycine max [L.] Merr. var. Coker 136) seedlings underwent lipolysis as measured by the release of fatty acids. The highest lipolytic activity occurred at pH 9. This lipolytic activity was absent in the dry seeds and increased after germination concomitant with the decrease in total lipids. Using spherosomes (lipid bodies) isolated from the cotyledons during the peak stage of lipolysis (5-7 days) as substrates, about 40% of the lipase activity was found in the glyoxysomes after organelle breakage had been accounted for; the remaining activity was distributed among other subcellular fractions but none was found in the spherosomal fraction. The glyoxysomal lipase had maximal activity at pH 9, and catalyzed the hydrolysis of tri-, di-, and monoacylglycerols of linoleic acid, the most abundant fatty acid in soybean. The spherosomes contained a neutral lipase that could hydrolyze monolinolein and N-methylindoxylmyristate, but not trilinolein. This spherosomal lipase activity dropped off rapidly during early seedling growth, preceding lipolysis. Spherosomes isolated from either dry or germinated seeds did not possess lipolytic activity, and spherosomes from germinated seeds but not from dry seeds could serve as substrates for the glyoxysomal lipase. It is concluded that the glyoxysomal lipase is the enzyme catalyzing the initial hydrolysis of storage triacylglycerols.     

Physiological and cellular mechanisms of natural herbicide resource from Aglaia odorata Lour. on bioassay plants

A bioassay-guided fractionation of the allelochemicals of the ethyl acetate fraction extracted from Aglaia odorata led to isolation of a bisamide, odorine. The growth inhibitory effects of odorine and ethyl acetate fraction were studied for comparison on Echinochloa crus-galli. Odorine and ethyl acetate fraction of A. odorata could inhibit the germination and seedling growth of E. crus-galli, with ethyl acetate fraction being more potent. Thus, ethyl acetate fraction was selected for further experiment, E. crus-galli seeds were studied the effects of the ethyl acetate fraction from A. odorata leaves in wettable powder formulation on imbibition and ??-amylase activity. It was found that treated seeds showed lower imbibition and ??-amylase activity. The results of cytogenetic bioassay in Allium cepa roots showed that ethyl acetate fraction inhibited cell mitosis and induced mitotic abnormalities resulting from its action on chromatin organization and mitotic spindle in the exposed roots.     

SEED DORMANCY AND GERMINATION IN MELAMPYRUM LINEARE

Immature seeds of Melampyrum lineare Desr. have very high germination percentages and dormancy is induced in a variable fraction of the seed crop during ripening. Correlated with this is the endogenous gibberellin-like activity which is found in considerable amounts in immature seeds, less in batches of ripe seeds, and is not detectable in batches containing only dormant seeds. For germination dormant seeds require activation followed by cold storage. In the laboratory activation is produced by allowing moist, dormant seeds to respire freely for several weeks at 20 C, or by treatment with exogenous GA₃. Dormancy appears to be most directly related to inability of the embryo to hydrolyze the thickened, mannan-containing endosperm cell walls. Embryos excised from dormant seed can be grown on agar enriched with whole macerated dormant seeds or with the ethanol-extractable materials from these (mostly sucrose and a glycoside). However, dormant seed material does not support growth when extracted to remove benzene- and ethanol-soluble materials.     

Enhancement by gibberellic acid and asymmetric distribution of lysophospholipase in germinating barley

Germination of whole barley seeds for 4 and 6 days followed by measurement of lysophospholipase (lysolecithin acyl hydrolase, LAH) in the embryo-containing and embryo-free halves revealed a gradient of activity between the two halves of the seed. Most of the activity appeared in the embryo-containing half. This gradient decreased slightly in the aleurone and dramatically in the starchy endosperm during the 2 day germination interval. Embryo-containing and embryo-free half seeds of surface sterilized barley were placed separately on sterile agar plates. After 4 and 6 days LAH was observed in both the aleurone and starchy endosperm of the embryo-containing halves. In the embryo-free halves, LAH appeared at low levels in the aleurone and was virtually absent in the starchy endosperm. The scutellum of germinating seeds contains LAH activity. Exposure of embryo-free half seeds to GA₃ for 24 hr showed enhancement of acidic and alkaline LAH activities in the aleurone fraction and in the GA₃-medium in which the half seeds were treated. The LAH activity of the starchy endosperm of these half seeds was little changed by GA₃ treatment. Exposure of isolated aleurones to GA₃ for 24 hr resulted in substantial enhancement of acidic and alkaline LAH activities in the bathing medium and in fractions prepared from the aleurone. The physiological significance of the influence of GA₃ on LAH activity during barley germination is discussed.