Purification and separation of tomato isolectins by chromatofocusing

Tomato lectin can be rapidly separated from a crude extract of tomato fruit proteins by chromatofocusing. The lectin is recovered from a PBE94 column in two peaks, each with a specific activity comparable to that of lectin purified by affinity chromatography on ovomucoid-Sepharose. Both isolectins consist of a single polypeptide chain (Mr 68,000) and have similar properties.     

Isolation and partial characterization of 3 nontoxic d‐galactose–specific isolectins from seeds of Momordica balsamina

Three isolectins denoted hereforth MBaL‐30, MBaL‐60, and MBaL‐80 were isolated from seeds extract of Momordica balsamina by 30%, 60%, and 80% ammonium sulfate saturations, respectively. The native molecular weights of these lectins, as judged by gel filtration, were 108, 56, and 160 kDa, respectively. On SDS‐PAGE, under reduced condition, 27 kDa band was obtained for all isolectins. The lectins hemagglutinating activities were variably inhibited by d ‐galactose (minimum inhibitory concentrations = 12.5mM, 50mM, and 0.391mM, respectively). MBaL‐30 and ‐60 could agglutinate all human blood types with slight preference for the A and O blood groups, whereas MBaL‐80 did not agglutinate B and AB blood types. The 3 isolectins were purified from crude seeds extract, collectively, in a single step on the affinity matrix Lactamyl‐Seralose 4B; this purified lectin fraction, which contains all isolectins, is termed MBaL. The N‐terminal of MBaL till the 25th amino acid was NLSLSELDFSADTYKSFIKNLRKQL, which shares 88% sequence identity with Momordica charantia lectin type‐2 ribosomal inactivating protein from Momordica charantia and 50% with momordin II from Momordica balsamina . MBaL retained 100% activity at up to 50°C for 30 minutes. MBaL‐30 and MBaL‐60 exhibited maximum activities in the pH range between 4 and 8, while MBaL‐80 was showing maximum activity in the pH range between 3 and 5. Treatment of MBaL‐30 and MBaL‐60 with EDTA completely abolished their hemagglutinating activities. Addition of Zn and Fe ions to the ethylenediaminetetraacetic acid–treated MBaL‐30 and MBaL‐60 lectins did not only regained the loss of activity but also resulted in 200% to 300% increase in activity, respectively. MBaL‐30 and ‐60 agglutinated gram positive Listeria monocytogenes and Staphylococcus aureus, whereas MBaL‐30 could merely agglutinate Escherichia coli . None of these lectins could arrest bacterial growth. Addition of MBaL to cancer cell lines (Gastric cancer cell line (AGS) and Gastric cencer cell line (MKN45), Glioblastoma (ECV‐304), and Human urinary bladder cancer cell line (U87‐MG)) at varying concentrations did not cause statistically significant changes on cell growth and viability.     

Pea (Pisum sativum L.) seed isolectins 1 and 2 and pea root lectin result from carboxypeptidase-like processing of a single gene product

The complete amino acid sequences of the -subunits of pea (Pisum sativum L.) seed and root lectin, the C-terminal amino acids of the -subunits of pea seed lectin, and most of the sequence of the -subunit of pea root lectin were determined. In contrast to earlier reports it was shown that the -subunits of both seed isolectins end at Asn-181. The 1 subunits end at Gln-241 (major fraction) or Lys-240 (minor fraction), whereas the 2 subunits end at Ser-239, Ser-238, Ser-237 or Thr-236. psl cDNA clones from seed are identical to psl cDNA clones from root, and root PSL is identical to seed PSL2, ending at Ser-239, Ser-238 or Ser-237. It seems that the presence of Lys-240 is the sole determinant of the charge difference between pea isolectins. PSL1 can be converted into PSL2 by carboxypeptidase P from Penicillium janthinellum. These results confirm that PSL from roots is encoded by the same gene as PSL from seeds. Thus, it seems that, next to an Asn-X specific protease responsible for the processing at positions 181/182 and 187/188, a carboxypeptidase is responsible for the conversion of PSL1 into PSL2, which is probably the final processing product.     

Purification and characterization of four isolectins of mushroom (Agaricus bisporus)

Four lectins were purified from a mushroom (Agaricus bisporus) by ammonium sulfate fractionation, anion-exchange chromatography, affinity chromatography on bovine submaxillary mucin-Sepharose 4B and preparative isoelectric focusing. They were designated as ABA-I (pI 6.70), II (pI 5.98), III (pI 5.69) and IV (pI 5.53). Polyacrylamide gel electrophoresis of each lectin in the presence of sodium dodecyl sulfate gave a single band with an apparent molecular mass of 16 000 Da. Sedimentation equilibrium analysis suggested that each lectin is a tetramer of subunits. The four lectins were found to have quite similar carbohydrate-binding specificities. The hemagglutination activities of the lectins were effectively inhibited by bovine and porcine submaxillary mucins (BSM and PSM), and NH2-terminal glyco-octapeptides obtained by cyanogen bromide cleavage of human erythrocyte glycophorin A. In addition, desialylated PSM-glycopeptides were more potent inhibitors than untreated PSM-glycopeptides. Among monosaccharides and their glycosides, only methyl N-acetyl-alpha-galactosaminide inhibited lectin binding at a high concentration, but a synthetic oligosaccharide, O-beta-galactopyranosyl-(1----3)-O-(2-acetamido-2-deoxy-alpha-D- galactopyranosyl)-N-tosyl-L-serine, was a strong inhibitor.     

Crystallization and preliminary X-ray studies of two isolectins from the seeds of Lathyrus ochrus

Two isolectins from the seeds of Lathyrus ochrus, LOL I and LOL II, which specifically bind N-acetyllactosamine, have been crystallized using the hanging-drop method and the interface diffusion method, respectively. In the case of LOL I, 2-methylpentane-2,4-diol, polyethylene-glycol 400 or ammonium sulphate have been used as precipitating agents. The best crystals of LOL I were grown at room temperature from a solution of 40% (v/v) methylpentane diol, 50 mM-Hepes at pH 7.5. LOL II crystals have been grown at room temperature from a solution of 32% (v/v) methylpentane diol, 50 mM-2-(N-morpholino)-ethanesulphonic acid at pH 5.5. X-ray examination of the LOL I and LOL II crystals shows that both are monoclinic, space group P2(1). Their cell dimensions are: LOL I, a = 56.4 A, b = 138.8 A, c = 62.9 A, beta = 91 degrees; and LOL II, a = 54.8 A, b = 71.4 A, c = 105.5 A, beta = 105 degrees. Density measurements of the crystals of LOL I indicate that there are two molecules per asymetric unit (Vm = 2.07 A3/dalton). LOL I crystals diffract strongly up to at least 1.8 resolution. Putative crystals of complexes of LOL I with various glycosides were obtained through co-crystallization under the conditions used for the native protein.     

Separation and partial characterization of isolectins with different subunit compositions from Datura stramonium seeds

The lectin from Datura stramonium seeds was separated into three individual isolectins by hydrophobic-interaction chromatography on phenyl-Sepharose. Two of these isolectins are homodimers made up of two A- or two B-subunits, whereas the third is a heterodimer composed of one A- and one B-subunit. Analysis of the homodimeric AA- and BB-isolectins revealed that the A- and B-subunits have similar but not identical M_r values (32 000 and 28 000, respectively), amino acid and carbohydrate compositions. The A-subunit has a higher affinity for N-acetyl-D-glucosamine oligomers than the B-subunit, whereas the latter is more specific for the carbohydrate determinants of some animal glycoproteins such as fetuin, asialofetuin and ovomucoid.     

Characterization of Urtica dioica agglutinin isolectins and the encoding gene family

Urtica dioica agglutinin (UDA) has previously been found in roots and rhizomes of stinging nettles as a mixture of UDA- isolectins. Protein and cDNA sequencing have shown that mature UDA is composed of two hevein domains and is processed from a precursor protein. The precursor contains a signal peptide, two in-tandem hevein domains, a hinge region and a carboxyl-terminal chitinase domain. Genomic fragments encoding precursors for UDA-isolectins have been amplified by five independent polymerase chain reactions on genomic DNA from stinging nettle ecotype Weerselo. One amplified gene was completely sequenced. As compared to the published cDNA sequence, the genomic sequence contains, besides two basepair substitutions, two introns located at the same positions as in other plant chitinases. By partial sequence analysis of 40 amplified genes, 16 different genes were identified which encode seven putative UDA- isolectins. The deduced amino acid sequences share 78.9–98.9% identity. In extracts of roots and rhizomes of stinging nettle ecotype Weerselo six out of these seven isolectins were detected by mass spectrometry. One of them is an acidic form, which has not been identified before. Our results demonstrate that UDA is encoded by a large gene family.     

围栏条件下捕食风险对岩松鼠贮藏核桃种子行为的影响

面对捕食风险,贮藏植物种子的啮齿动物如何通过调整其贮藏行为,权衡捕食风险和贮食效益,需要深入研究。2006 年9 月,在北京东灵山地区,以黄鼬作为捕食风险源,在半自然围栏内比较了有无捕食风险源时,岩松鼠贮藏的核桃种子的状态,以及埋藏种子在实验围栏内的分布、微生境和搬运距离,以探讨捕食风险对岩松鼠贮藏种子行为的影响。结果表明,捕食风险源存在时,搬运和埋藏种子的比例都显著减少,弃置地表种子的比例无显著差异;埋藏于高风险区的种子比例减少,中风险区和低风险区的埋藏种子比例增加,但差异都不显著;位于裸地和草下的埋藏种子比例减少,位于墙基的埋藏种子比例增加,但差异都不显著;此外,埋藏种子的搬运距离也没有显著差异。可见,捕食风险源存在时,岩松鼠显著减少了搬运和埋藏种子的比例,但对埋藏种子时的埋藏区域和微生境选择,以及搬运距离无显著影响。差异不显著可能与围栏效应有关。     

Purification and characterization of complex carbohydrate specific isolectins from wild legume seeds: Acacia constricta is (vinorama) highly homologous to Phaseolus vulgaris lectins

Vinorama isolectins (VL2-VL4) were purified from seeds of Acacia constricta (vinorama) using affinity chromatography on a fetuin-fractogel column followed by cationic-exchange chromatography. Each isolectin fraction presented a characteristic isoelectric point range from 5.5 to 8.4. Under native conditions, VL containing fractions migrated as tetramers of 133 kDa, while in SDS-PAGE, in presence of 2-mercaptoethanol, a single subunit band with M(r) of 34 kDa was observed. VL was found to be a glycoprotein with a 7.5% neutral sugar content. Antibodies to Phaseolus vulgaris lectins PHA and other wild legume lectins as Olneya tesota (palo fierro) PF2 and PF3, and Parkinsonia aculeate (palo verde) PV reacted with VL, but not with anti Glycine max agglutinin SBA or anti Lotus tetragonolobus agglutinin LTA. Furthermore, direct analysis of VL peptides showed sequences homologous to those reported in different lectins of the Phaseolus genus. VL2-VL4 did not have ABO serological or simple sugar specificity, but were inhibited by complex carbohydrates from fetuin and thyroglobulin. Asialofetuin carbohydrates strongly interacted with VL4 and VL3. Vinorama isolectins could be classified as "complex lectins".     

Characterization of two novel defense peptides from pea (Pisum sativum) seeds

A fraction that possesses antifungal activity against Aspergillus niger has been isolated from seeds of the pea (Pisum sativum) by ammonium sulfate fractionation followed by gel filtration on Sephadex G-75. On further purification by reverse-phase high performance liquid chromatography, two small cysteine-rich polypeptides were obtained (Psd1 and Psd2). They are localized primarily in vascular bundles and epidermis tissues of pea pods and exhibit high antifungal activity toward several fungi, displaying IC(50) values ranging from 0.04 to 22 microg/ml. This inhibitory activity decreases when A. niger growth medium is supplemented with cations such as Ca(2+), Mg(2+), Na(+), and K(+). Although the primary sequence of both Psd1 and Psd2 shows homology with other plant defensins, they cannot easily be assigned to any established group.     

Isolation of a trypsin inhibitor from Echinodorus paniculatus seeds by affinity chromatography on immobilized Cratylia mollis isolectins

A highly purified trypsin inhibitor was obtained from Echinodorus paniculatus when an extract prepared from E. paniculatus seed flour (25 gl(-1), with 0.1 M ammonium acetate buffer, pH 8.3, under agitation for 6 min at 28 degrees C) was chromatographed on Sephadex G-25 (12 mlh(-1)), followed by affinity chromatography on immobilized Cratylia mollis isolectins (Cra Iso 1,2,3-Sepharose). The column chromatography was performed at 24 degrees C; the matrix was washed (30 mlh(-1)) with 0.1 M sodium phosphate buffer, pH 7.4 or with the same buffer containing 0.2 M glucose, followed by application of inhibitor sample and elution with 0.015 M sodium borate buffer, pH 7.4, or 1.0 M NaCl. A purified fraction of inhibitor was obtained by gel filtration chromatography (GF-450/HPLC column). Trypsin inhibitory activity was eliminated when the inhibitor was treated with metaperiodate showing that the carbohydrate moiety was important for trypsin inhibition. Binding of inhibitor was also evaluated on immobilized concanavalin A (Con A-Sepharose) using previously described chromatographic conditions with results similar to Cra Iso 1,2,3-Sepharose chromatography.     

Characterization of diphosphonucleotide phosphatase/phosphodiesterase from yellow lupin (Lupinus luteus) seeds

A phosphatase cleaving the pyrophosphate bond in diphosphonucleotides and phosphodiester bond in various phosphodiesters (pH optimum at 6.25) was purified from yellow lupin (Lupinus luteus L.) seeds. The enzyme is 75 kDa monomeric glycoprotein (pI=6.4) with 4.4% of carbohydrate (mannose, N-acetylglucosamine, fucose and xylose). Analysis of its partial amino acid sequence (8 peptides, 101 amino acid residues) together with no divalent cation requirements for catalysis points out that the purified enzyme is different from known plant pyrophosphate cleaving enzymes (apyrases and inorganic pyrophosphatases). Its physiological role could be related to a regulation of diphosphonucleotides level in plant metabolism.     

Characterization of a calcium-dependent protein kinase from Arachis hypogea (groundnut) seeds.

A calcium-dependent protein serine/threonine kinase (GnCDPK) has been detected in groundnut (Arachis hypogea) seeds that specifically phosphorylates a peptide (MLCpep) representing the phosphate-accepting domain of smooth muscle myosin light chains. GnCDPK has been purified to near homogeneity from the soluble fraction of groundnut seeds by ammonium sulfate precipitation, Q Sepharose, Blue Sepharose, and Sephacryl 300 chromatography. The molecular weight of GnCDPK is estimated to be 53,000. Enzyme activity is stimulated about 100-fold in the presence of free Ca2+ (concentration required for half-maximal activation = 0.5 microM). GnCDPK is capable of binding 45Ca2+ ions directly in an electroblot, indicating it to be a calcium-binding protein. Phosphorylation of MLCpep is found to be optimal at an alkaline pH range (pH 9-10). Unlike all other calcium-dependent protein kinases reported from higher plants, GnCDPK does not accept casein or histones as substrate. Sequences related to MLCpep (> 60% homologous) that are present in myosin light chains from skeletal muscles of chicken and rabbit also fail to act as a substrate for GnCDPK. In contrast to the Ca2+/calmodulin dependence of myosin light chain kinases, GnCDPK activity is not affected by the presence of exogenous calmodulin (1-10 microM). However, enzyme activity is considerably inhibited in the presence of calmodulin antagonists like N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide (concentration required for 50% inhibition [IC50] = 30 microM) and calmidazolium (IC50 = 10 microM), indicating an endogenous calmodulin structure to be present in GnCDPK. The probability of GnCDPK being a bona fide plant myosin light chain kinase is discussed.     

Lectins from seeds of jack fruit (Artocarpus integrifolia L.): isolation and purification of two isolectins from the albumin fraction

Two isolectins were isolated from the albumin fraction ofArtocarpus integrifolia L. seeds, by precipitation with ammonium sulfate, and DEAE-cellulose and SP-Sephadex C-50 chromatography. The isolectins, when passed through Sephadex G-100 at pH 7.4, had a molecular mass of 43 000 and when subjected to SDS electrophoresis in the presence of β-mercaptoethanol consisted of two subunits with molecular mass of 11 250 and 15 000. When they were tested with human erythrocytes of all the groups of the ABO system there was no blood specificity. The isolectins formed interference arcs by Ouchterlony double diffusion in agarose gel. Part 1.     

核桃枯枝病新病原及生物学特性

【目的】鉴定四川省广元市剑阁县核桃枯枝病的病原,为该病害的防治奠定基础。【方法】随机采集不同发病阶段的枝干样品30份,采用常规组织分离法分离纯化核桃枯枝病病原菌,用柯赫氏法则对菌株进行致病性验证,结合形态学特征和分子生物学方法对病原菌进行鉴定,采用菌落生长法进行生物学特性研究。【结果】共分离得到180个菌株。致病性测定结果表明核桃枯枝病的病原菌为拟茎点霉Phompsis capsici,这是首次关于Phompsis capsici导致核桃枯枝病在我国发生和研究的报道。在PDA平板上该病原菌菌丝为白色,能产生椭圆形和线形两种分生孢子。病菌菌丝生长以PDA培养基为最适。菌丝能有效利用多种碳、氮源,在供试的9种碳、氮源中,适宜生长的碳源为蔗糖和果糖,氮源为钼氨酸和苯丙氨酸。最适生长温度为25°C,最适pH为6.0-7.0。黑暗条件下菌丝生长最快。【结论】Phompsis capsici最易从伤口侵入致病。     

Secondary removal of seeds dispersed by chimpanzees in a Nigerian montane forest

The effectiveness of chimpanzees as seed dispersers may be influenced by the secondary removal and/or dispersal of seeds by other taxa. This study documents species involvement and their influences on seed treatments (fresh seed, dry seed and seeds rubbed in fresh chimpanzee faeces). Field experiments conducted on ten large‐seed species consumed by chimpanzees in a Nigerian montane forest showed that secondary seed removal after 24 h varied between species. After 96‐h, seed removal still varied between species, but no previous significant differences were observed among treatments, which suggested treatment becomes insignificant with time. Dispersal by chimpanzees may be more important for some large‐seeded species than others. The taxa removing seeds varied across seed species but were mainly restricted to rodents.