Neurolathyrism is a unique neurodegeneration disease caused by β-N-oxalyl-L-α, β- diaminopropionic (β-ODAP) present in grass pea seed (Lathyrus stativus L.) and its pathogenetic mechanism is unclear. This issue has become a critical restriction to take full advantage of drought-tolerant grass pea as an elite germplasm resource under climate change. We found that, in a human glioma cell line, β-ODAP treatment decreased mitochondrial membrane potential, leading to outside release and overfall of Ca2+ from mitochondria to cellular matrix. Increased Ca2+ in cellular matrix activated the pathway of ECM, and brought about the overexpression of β1 integrin on cytomembrane surface and the phosphorylation of focal adhesion kinase (FAK). The formation of high concentration of FA units on the cell microfilaments further induced overexpression of paxillin, and then inhibited cytoskeleton polymerization. This phenomenon turned to cause serious cell microfilaments distortion and ultimately cytoskeleton collapse. We also conducted qRT-PCR verification on RNA-sequence data using 8 randomly chosen genes of pathway enrichment, and confirmed that the data was statistically reliable. For the first time, we proposed a relatively complete signal pathway to neurolathyrism. This work would help open a new window to cure neurolathyrism, and fully utilize grass pea germplasm resource under climate change.

三七素是中药三七的止血活性成分,也存在于山黧豆等植物中,称山黧豆神经毒素,其化学成分为β-N-草酰-L-α,p二氨基丙酸,属非蛋白游离氨基酸.本文从传统氨基酸分析、气相、液相色谱及其色谱-质谱联用、毛细管区带电泳、流动注射等方面综述了三七素的分析检测方法,并对相关技术进行了评述,为合理选择三七素检测手段提供参考.

Grass pea (Lathyrus sativus) is a legume with various adverse adaptability and rich nutrition. However, it can lead to the human and animal neurotoxicity after long-term consumption due to its neurotoxin, beta-N-oxalyl-L-alpha, beta-diaminopropionic acid (beta-ODAP), limiting its utilization. This paper summarized the influences of beta-ODAP on osmotic adjustment and growth regulation in grass pea under drought stress, the research progress in analysis methods, toxicological mechanisms and practical utility of beta-ODAP, and the breeding strategies for low- and zero-beta-ODAP. Beta-ODAP synthesis was found to be abundant in grass pea under drought stress and its content was enhanced gradually with the increasing extent of drought stress. beta-ODAP could supply nitrogen for plant growth and seed development, scavenge reactive oxygen species (ROS), involve in osmotic adjustment as a soluble amino acid, transport zinc-ions as a carrier molecule, and impact nodule development. However, increasing the content of sulfur-containing amino acids (methionine and cysteine) could decrease the level of toxicity of grass pea. There were a lot of investigations on collecting genetic resources, cross breeding, tissue culture, and gene manipulation for low- and zero-toxin in grass pea in recent years. Although beta-ODAP could induce excitotoxicity by damaging intracellular Ca2+ homeostasis and as glutamate analogues, it has medicinal value on hemostasis and anti-tumor.

OBJECTIVE To explore the methods for purification of polysaccharides from Calvatia gigantea,and investigate the characteristics of the polysaccharides.METHODS Polysaccharides form Calvatia gigantea were separated and purified by hot water extraction,ethanol precipitation,and fractionation by ionexchange chromatography on DEAE-sepharose fast flow and Sephacryl S-300 HR chromatography.Sephacryl S-300 HR chromatography,agarose gel electrophoresis,SDS-PAGE,UV spectrum were used to identify the homogenicity of the polysaccharides.The apparent average molecular weight was identified by Sephacryl S-300 HR chromatography.The component of the polysaccharides were identified by GC-MS.The content of uronic acid was identified by carbazole-sulphuric acid analysis.The structural characterizations of the polysaccharides were confirmed by NaIO4 oxidation,Smith degradation and IR.RESULTS Three water-soluble polysaccharides CGP-I,CGP-II and CGP-III were obtained from Calvatia gigantea.Three fractions were shown to be homogeneous by Sephacryl S-300 HR chromatography,agarose gel electrophoresis,SDS-PAGE,and UV spectrum.The apparent average molecular weight of CGP-I,CGP-II and CGP-III were estimated to be 9.12×104 Da,18.6×104 Da and 15.6×104 Da,respectively.The component of polysaccharides:mannose,glucose and galactose were found to be in the molar radio of 1.22:11.28:3.92 in CGP-Ⅰ,1.74:26.45:0.36 in CGP-Ⅱ,1.06:9.9:3.29 in CGP-Ⅲ,and an unknown monosaccharide was found.The content of uronic acid in CGP-Ⅱ,CGP-Ⅲ was 17.2 % and 20.5%,respectively,and there was no uronic acid in CGP-Ⅰ.The results of structural characterization showed that they were all branching chain polysaccharide with more 1→4 linking,fewer 1→6 linking.CGP-Ⅰwas neutral polysaccharide which was mainly composed of pyranose and had both β-and α-epimer,CGP-Ⅱ,CGP-Ⅲ were acidic polysaccharides which were composed of pyranose and had β-epimer.CONCLUSION The preliminary property and structural characterization of the polysaccharides purified from calvatia gigantea were investigated.

用PEG6000模拟干旱对山黧豆进行胁迫。结果发现,胁迫初期（0~48 h）,突然的水分亏缺使气孔导度（Sc）和蒸腾速率（Tr）迅速下降,而净光合速率（Pn）和水分有效利用率（WUE）基本维持稳定。胁迫后期（48~108 h）,上述四个光合指标均不同程度地向对照水平恢复;脯氨酸和相对电导率（REC）受胁迫强度影响较小,但与胁迫时间呈明显正相关,分别在48和60 h增幅最大,而且当REC上升至对照的50%左右时,脯氨酸含量已达对照的14倍之多。由此认为,山黧豆可能主要通过迅速减小Sc和Tr以及大量而急剧的积累脯氨酸来减轻干旱所造成的伤害。

The chromatographic behaviors of 20 protein amino acids and a few non-protein amino acids including ODAP(β-N-oxalyl-L-α,β-diaminopropioni acid) in Lathyrus sativus were studied on the silica gel thin layers.Butyl alcohol/ acetic acid /water were used as a developer and ninhydrin as a determiner.The results showed that it was difficult to separated ODAP from several amino acid mixture using one-way or two-way TLC analysis.The resolution of ODAP was,however,easily obtained by pre-separation of ODAP from amino acids using cation exchange column(H+) before one-way TLC analysis.

To screen the bacteria of degrading the neurotoxin ODAP from 30 kinds of bacteria by microbial fermentation,the consumed amounts of ODAP from Lathyrus sativus extraction as culture medium which contained fixed quantify of ODAP was determined phthaladehyde colorimetric method.The results showed that there were two kinds of bacteria which had the property of consuming obviously ODAP.In addition,the rescarch of this paper included the relativity of the growth and the degradation of ODAP,and the enzyme kinetics of reaction velocity and time.Then it was concluded that it was capable of producing ODAP degrading enzyme by microbial fermentation in culture medium of ODAP.

Two strains B_2 and B_(13) degraded from the toxinβ-ODAP were screened and selected.Two suc- cessive solid state fermentations are processed for the two selected strains.After sterilization the seeds were inoculated with B_2.The fermentation was carried out at 37℃for 3 days and was stopped by steam.The sec- ond inoculum B_(13) was added after cooling,and the second fermentation period was processed at 37℃for 3 days and was stopped by steam.The control and processed seed powders were extracted for 1 hour at 0～4℃with 0.2mol/L HClO_4.The contents ofα,β-ODAP were measured by precolumn derivatization with 1-fluo- ro-2,4-dinitrobenzene(FDNB).As a result,the contents ofβ-ODAP andα-ODAP in control seed powders were 1.146mg/g and 0.653 mg/g,respectively.The contents ofβ-ODAP andα-ODAP processed seed pow- ders were 0.203mg/g and 0.0909 mg/g.After fermentation,the detoxification efficiency ofβ-ODAP was 82. 2% and the content ofα-ODAP was decreased by 86%,and the protein level of the processed was significant- ly surpassed that of the control.

Annual plant Lathyrus sativus L. belongs to the Leguminosae family, containingβ-N-oxalyl-α,β-diaminopropionic (β-ODAP), which is the cause of Lathyrism. Preliminary results of the study indicate thatβ-ODAP can form from the acylization of L-α,β-diaminopropionic acid (DAPA), the acylization of DAPA need the participation of oxalyl-Co-A also, and oxalyl-Co-A can form from the combination of oxalic acid and Co-A. That is to say, the oxalic acid is one of the precursors ofβ-ODAP biosynthesis. It is known that glycolate oxidase (GO) exists in the leaf of C3 plant generally. GO not only can catalyse ethyl alcohol acid to oxidize into aldehydic acid, but also can catalyse aldehydic acid to oxidize into oxalic acid. This paper is based on the former work on the relationship between the content change of oxalic acid and the accumulation and biosynthesis ofβ-ODAP in different organs, different development stages of grass pea by exogenous oxalic acid or butyl-2- hydroxybutynoate (BHB, inhibitor of GO synthesis) and under water stress, and the relationship between resistance of grass pea and oxalic acid,β-ODAP, and to offer a new train of thought and new technologies to develop the resistance and low toxicity of grassspea.

Interrelationship among abscisic acid (ABA) content, accumulation of free polyamines and biosynthesis of beta-N-oxalyl-l-alpha,beta-diaminopropionic acid (ODAP) was studied in grass pea (Lathyrus sativus L.) seedlings under drought stress induced by 10% polyethylene glycol (PEG6000). Increase of ABA content occurred prior to that of ODAP and polyamine contents, and was found significantly positive correlation between ABA content and ODAP content. Addition of exogenous ABA increased ODAP content in leaves. On the other hand, pretreatment with alpha-difluoromethylarginine (DFMA), a polyamine biosynthesis inhibitor, significantly suppressed the accumulation of free putrescine (Put), free spermidine (Spd) and free spermine (Spm), which in turn inhibited biosynthesis of ODAP in well-watered leaves. Meanwhile, addition of exogenous Put alleviated DFMA-induced inhibition on the biosynthesis of Put and Spd, but did not affect the biosynthesis of Spm and ODAP in well-watered leaves. Same result was also achieved in drought-stressed leaves. Increasing accumulation of ODAP was significantly correlated with increasing Spm content (R=0.7957**) but not with that of Spd and Put. Therefore, it can be argued that ABA stimulated the biosynthesis of ODAP simultaneously with increasing the level of free Spm under drought stress condition.

In order to assess the influence of nutrient elements on the accumulation of β-N-oxalyl-L-α, β-diaminopropionic acid (β-ODAP; the probable cause of lathyrism) in Lathyrus sativus L. (grass pea), it was first examined under field conditions during the lifespan of a grass pea plant using high-performance liquid chromatography (HPLC). β-ODAP mainly accumulated in young seedlings, in developing and mature seeds and in young leaves, especially in young seedlings. In contrast, all mature leaves, roots, and stems showed a low level of β-ODAP. The β-ODAP accumulation pattern in seedlings grown in a nutrient-deficient solution was highest (3·57 mg/g) in shoots at 7 d growing in the nitrogen-deficient solution and higher compared to the control (2·31 mg/g) in zinc-, calcium-, phosphorus- and molybdenum-deficient shoots (P<0·05). The contents of β-ODAP in seedlings growing in other element-deficient solutions were similar to controls. When the content of β-ODAP in grass pea seedlings fertilized with different forms of organic nitrogen was assayed the results indicated that amino acids such as glutamine and serine, as well as nucleotide nitrogen, all significantly enhanced the accumulation of β-ODAP in young seedlings relative to controls (P<0·05). Taken together, these data suggest that β-ODAP accumulation in grass pea might be related to the level of total free nitrogenous compounds and that nitrogen and phosphate may be the crucial nutrient factors influencing β-ODAP content under field conditions. Thus, the application of appropriate nitrogen and phosphorus fertilizers to the soil could decrease the content of β-ODAP in the seeds and leaves of grass pea.

The effects of exogenous abscissic acid (ABA) application on the antioxidant defenses were investigated in grass pea (Lathyrus sativus L.) seedlings. Four treatment combinations of ABA (1 x 10(-3) nM ABA) and PEG (10% polyethylene glycol, PEG 6000) were designed to evaluate their short-term (48 h) effect: (1) Well-watered group (Control group 1), (2) PEG treatment, (3) ABA treatment, and (4) PEG + ABA treatment. In addition, 2 other treatments were used to evaluate the long-term (15 d) effect of ABA: well-watered group (Control group 2) and ABA treatment. Time-course analyses of ABA content, the production of malondialdehyde (NIDA) and H2O2, and the activities of superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) and peroxidase (POD) in water-stressed leaves showed that a significant increase in ABA content preceded that of MDA and H2O2 in long term experiment, which was followed by a substantial increase in the activities of four antioxidant enzymes. Under the short-term drought stress, ABA application promoted the activities of antioxidant enzymes, and reduced the accumulation of MDA and H2O2 significantly. On the contrary, long term application of exogenous ABA increase the generation of MDA and H2O2 significantly. It could be argued that during a successive application of exogenous ABA, ABA played a dual role by which the beneficial role in the initial stage shifted to a detrimental one under prolonged treatment in up-regulating protective defense strategies in plants.

Different protein extraction methods were comparatively examined with Lathyrus sativus leaves as the experimental material and on the basis of this the sample preparation was studied.The choices of IPG strips and the protocols,parameters and staining methods of the first phase IEF and the second-phase SDS-PAGE were compared and their conditions were optimized.The results showed that when TCA-acet precipitation was adopted to extract protein,Tris-base was added to the lysis solution as the protease inhibitor,when isoelectric focusing electrophoresis was adopted,the low voltage was prolonged(30 V,12 h;500 V,1 h;1 000 V,2 h)to help the salt ion electrophoresis and thus conduct the two-dimension electrophorsis of leaf protein of L.sativus,and when Coomassie brilliant blue and compound silver staining were adopted to stain the gel,the two dimension electrophoretograms with high reproduction and resolution power of leaf protein of L.sativus were obtained,wihich indicated that the optimized two-dimension electrophoresis of leaf protein of L.sativus was characterized with quality sample preparation and high electrophoresis resolution thus being completely suited to further proteomics research.

Lathyrus sativits L., a member of the annual Leguminosae family, is not only resistant to drought and cold, but also to many plant diseases, insect pests, and barrenness. Moreover, it is highly nutritive and hence an ideal forage crop. However, the seeds of Lathyrus sativus contain P-N-oxalyl-alpha,beta-diaminopropionic acid (ODAP), the main element causing lathyrism. Previous studies indicated that ODAP could form from the oxalylation Of L-alpha,beta-diaminopropionic acid (DAPA), while DAPA synthesis needed the participation of oxalyl-Co-A resulting from the combination of oxalic acid and Co-A; i.e., oxalic acid is one of the precursors of ODAP biosynthesis. Glycolate oxidase (GO), generally existing in the leaves of the C, plant, can catalyze not only the transformation of glycolic acid to glyoxalic acid, but also that of glyoxalic acid to oxalic acid. This study investigated the relationship between the change of oxalic acid and the accumulation of ODAP in different organs at different development stages of grass pea treated with exogenous oxalic acid, butyl hydroxybutynoate (BHB, inhibitor of GO synthesis), and under water stress; evaluated the relationship between the adversity and the accumulation of oxalic acid and ODAP; and provided a new strategy for selecting grass pea with environment-tolerance and low toxicity.

OBJECTIVE:To study the polysaccharide of Condonopsis pilosula.METHOD:The polysaccharide, CPP-1, was purified by DE-52 cellulose and Sephadex G-200 gel column chromatography. Purity and molecular weight of the polysaccharide were determined by gel permeation chromatography. Methylation analysis, periodate oxidation and degradation, IR, 1H-NMR and 13C-NMR methods were adopted to elucidate the chemical structure.RESULT:The molecular weight of CPP-1 was estimated to be 7.5 x 10(4), and the structure of CPP-1 was a beta-(2 --> 1) linked beta-D-fructosan.CONCLUSION:CPP-1 was a neutral homosaccharide.

Drought tolerance and toxicity variation of grass pea (Lathyrus Sativus L.) were studied in the presence and absence of earth element Eu3+ under the pot-culture condition. The addition of exogenous Eu3+, increases the survival days (SD) and lowered the stomatal sensitivity of grass pea seedlings under the decreasing soil moisture from - 0. 2 MPa to - 1 MPa, suggesting that Eu3+ can improve the ability of grass pea seedlings to tolerate drought. But in this case, Eu3+ is found to significantly inhibit the increase in contents of plant toxicity beta-N-oxalyl-L-a, P-diaminopropionic acid (ODAP) level in leaves. Therefore, the results demonstrate that Eu3+ application is a potential agronomic approach to improve drought tolerance while lowering the toxicity level in grass pea productivity.

L.sativus is a feed crop with a long cultivated history. However, its cultivation has diminished in many regions because of its toxicity. In order to reuse this fine kind of forage crop, intensive researches for developing varieties with low and zero toxin have been carried out around the world. The toxic biochemistry of L.sativus, including composition, biosynthesis and the effect of environment on biosynthesis of toxin, was reviewed in this paper. In addition, research of removal toxin from L.sativus by breeding, processing, tissue cultures and transgenesis were summarized. Finally, the probable biological significance of toxin was discussed and the research prospects for L.sativus were also presented.

α-D-Glucan (ASP-1) was isolated from the hot water extract of Angelica sinensis roots by ethanol precipitation, anion-exchange and gel-permeation chromatography. Smith degradation, NMR spectroscopy, composition and methylation analyses confirmed the ASP-1 structure comprises a (1→6) linked α-D-glucopyranosyl backbone, coupled with two branches per unit bonded through α(1→3) linkages, where each branch contains Glc-(1→3)-Glc-(1-and Glc-(1→4)-Glc-(1- chains at either end.

A simple method is proposed for the simultaneous analysis of the neurotoxin β-N-oxalyl-α, β-diaminopropionic acid (β-ODAP) and its isomer α-ODAP, as well as other free amino acids, in Lathyrus sativus extract based on reversed-phase (C18 column) high-performance liquid chromatography (HPLC) of their para-nitro-benzyloxycarbonyl chloride (PNZ-Cl) derivatives. Samples were derivatized in 0.5 M NaHCO3 within 2 min at ambient temperature by adding 50 mM PNZ-Cl in acetonitrile. The PNZ-derivatives were quantified by their UV-absorption at 260 nm. Detection limits were approximately 8 ng mL−1 for both α-, β-isomers, 9 ng mL−1 for homoarginine at a signal-to-noise ratio of 3:1. The coefficients of determination were 0.9967–0.9991. The method was validated by the determination of β-, α-ODAP and homoarginine contents in ethanol-water (30:70, v/v) extracts from the seed and foliage of Lathyrus sativus.

A sensitive catalytic method is developed for the spectrophotometric determination of oxalic acid. The procedure is based on the effect of oxalate on the oxidation of Victoria blue B by dichromate in dilute sulfuric acid medium. The reaction is quantitatively estimated by measuring the decrease in absorbance of Victoria blue B at the maximum wavelength of 610 nm after quenching the reaction with tap water. The factors effecting the sensitivity and reproducibility of the reaction were studied. The method is not interfered with by foreign species generally associated with oxalate and oxalic acid. The described method is simple, specific, inexpensive and suitable for oxalic acid concentrations of between 0.06 and 9.0 µg mL−1. It was validated with satisfactory results by determining oxalic acid content in water extracts from plant materials such as spinach and Lathyrus sativus.