Surface characteristics of necrotrophic secondary hyphae produced by the bean anthracnose fungus, Colletotrichum lindemuthianum

During infection of bean (Phaseolus vulgaris), the hemibiotrophic anthracnose pathogen, Colletotrichum lindemuthianum, initially produces biotrophic primary hyphae that are large-diameter and entirely intracellular, followed by necrotrophic secondary hyphae that are narrower and either intercellular or intracellular. In the present study, transmission electron microscopy of infected tissues prepared by high-pressure freezing and freeze-substitution showed that secondary hyphae have much thinner cell walls (25–40 nm) than primary hyphae (100–130 nm) and are not surrounded by an extracellular matrix. Immunofluorescence labelling with a panel of monoclonal antibodies showed that glycoproteins which are present on conidia, germ-tubes, appressoria, primary hyphae and mycelium grown in vitro are absent from the surface of secondary hyphae. Chitin, detected with the lectin wheat germ agglutinin, was the only surface component shared by secondary hyphae and the other fungal cell types. The results suggest that the fungal cell surface becomes modified during necrotrophic growth, with none of the glycoproteins associated with earlier stages of the infection process being produced.     

Patterns of protein glycosylation in bovine placentomes as a function of gestational age and in retained versus non‐retained placenta

The formation of placenta at the beginning of pregnancy and its separation at parturition require not only deep remodelling of extracellular matrix, which mainly consists of proteins conjugated with sugar moieties, but also the cooperation with cells from both maternal and foetal parts of placenta. The aim of the study was to compare the patterns of selected conjugated proteins with sugar moieties between pregnant and term placenta as well as between released and retained placenta in cows. Placental samples from healthy pregnant cows (3–5 months of pregnancy) were collected at a slaughterhouse (n = 6), and parturient samples were collected during caesarean section at term and retrospectively divided into retained (n = 6) and released (n = 6). The pattern of selected sugar moieties conjugated with proteins was detected by use of lectin blotting with Phaseolus Vulgaris leucoagglutinin, Maackia Amurensis and Sambucus Nigra (Elderberry). The comparison and analysis of obtained band patterns showed differences between their number, molecular weight and abundance related to the intensity of staining. Samples from 3 to 4 months showed similarities, while at the 5th month, clear differences were visible in all 3 lectins, which were used in this study. Samples from retained/released placenta expressed significant differences in PHA‐L and SNA pattern in the foetal part. Obtained results indicate that the development of placenta related to extracellular matrix and accompanying cells from both sides of placenta shows dynamic changes during pregnancy. Moreover, in the case of animals with the retention of foetal membranes the patterns of proteins conjugated with sugar moieties are altered, suggesting that the changes in extracellular matrix metabolism can be involved in the attachment and detachment of the placenta in cows.     

Characterization of the complex carbohydrates in the zona pellucida of mammalian oocytes using lectin histochemistry

The objective of this study was to characterize the glycoconjugates present in the zona pellucida of the follicular oocytes in sheep, goats and pigs. The zona pellucida was stained with periodic acid-Schiff, low iron diamine, high iron diamine, and nine different lectin horseradish conjugates: Con-A, SBA, DBA, PNA, RCA-I, GSA-II, WGA, LTA and UEA-I. Staining with DBA, PNA, SBA and RCA-I was performed with and without saponification with KOH and sialidase digestion. The results showed the presence of neutral and acidic glycoconjugates with different terminal sugars and also sialic acid radicals in the zona pellucida of all the animals studied. In particular, the positive staining with WGA, SBA, PNA and RCA-I suggests the presence of oligosaccharides with N-acetyl-d-glucosamine and sialic acid linked to the penultimate -N-acetyl-d-galactosamine and to the disaccharide galactosyl-(1-3)-N-acetyl-d-galactosamine. The terminal trisaccharide sialic acid galactosyl-(1-4)-N-acetyl-d-glucosamine was identified only in the zona pellucida of ovine and procine oocytes. Thus, the zona pellucida exhibited species-specific variations in the content and distribution of lectin-binding patterns that may reflect the species specificity of gamete interaction.Abbreviations HID high iron diamine - HRP horseradish peroxidase - LID low iron diamine - PAS periodic acid-Schiff - PBS phosphate-buffered saline; see also Tables I and II     

Exploration for anti-insect properties of lectin from seeds of soybean (Glycine max) usingBactrocera cucurbitae as a model

Lectin fromGlycine max L. was extracted and purified by affinity chromatography using asialofetuin-linked porous amino-activated silica beads. The concentration-dependent effect of lectin was studied on freshly laid eggs (0–8 h old) of the melon flyBactrocera cucurbitae (Coquillett); lectin failed to influence egg hatching. However, treating second instar larvae (64–72 h old) with increasing concentrations of lectin significantly reduced the development period, number of pupae and number of emergingB. cucurbitae, and was negatively correlated with the increase in the lectin concentration. The LC₅₀ value, 54μg ml⁻¹, was calculated on the basis of adult emergence. Treatment of the larvae (64–72 h old) with the LC₅₀ concentration resulted in a decrease in pupal weight. The activity of three hydrolase enzymes (esterases, acid and alkaline phosphatases), one oxidoreductase (catalase) and one group transfer enzyme (glutathione S-transferase) was assayed in second instar larvae at the LC₅₀ concentration of lectin after exposure for 24, 48 and 72 h. The activity of esterases increased significantly (P<0.01) at the three exposure intervals, whereas the activities of the three other hydrolyses and the transferases were significantly suppressed (P<0.01). http://www.phytoparasitica.org posting Sept. 13, 2006.     

高效的水稻基因枪转化和可育转基因植株再生

从水稻品种台北309成熟胚诱导生长3~4周的愈伤组织,经过1~3次继代培养后,可以形成大量颗粒状胚性愈伤组织,适用于基因枪转化实验。将分别含有雪花莲凝集素(GNA)基因、h pt基因的质粒pIP860和p35H混合包裹在金粉上轰击转化上述胚性愈伤组织,在含30~50mg/L潮霉素的培养基上进行筛选、预再生、再生及长根培养。使用干燥处理     

Identification of a mannose‐binding lectin‐like protein recognized by the anti‐CD25 antibody in haemocytes from Cherax quadricarinatus

The crustacean haemolymph contains three main cell populations; however, it is not clear which mechanisms participate in the regulation of cells related to innate immunity. This work aimed to identify potential interleukin‐like receptors that could regulate cellular responses in Cherax quadricarinatus. By histochemical analysis with murine anti‐CD25 staining (targeting the α‐chain of the IL‐2 receptor), we identified that this antibody recognizes cytoplasmic granules in semigranular and granular haemocytes. In haemocytes stimulated with phorbol myristate acetate (PMA), increased fluorescence was observed in these cytoplasmic granules, whereas staining with a human IL‐2 antibody after stimulation with 1–10 ng/ml PMA revealed no overexpression of the receptor or oxidative burst in haemocytes. Two‐dimensional Western blot analysis of haemocyte lysates showed that anti‐CD25 identified a 27.4‐kDa protein with an isoelectric point (pI) of 7.7 and a 46‐kDa protein with a pI of 6.9. De novo sequencing of these proteins identified that they had 32% homology with a mannose‐binding lectin (MBL) from Pacifastacus leniusculus. Our results indicate that a mannose‐binding lectin‐like protein could exert a protective effect that prevents damage from other activated immune responses.     

凡纳滨对虾C型凝集素（LvLc1）的特征和活性分析

C型凝集素是一种依赖于Ca~(2+)而发挥功能的糖蛋白,在一线的固有免疫防御过程中发挥着重要作用。围绕对虾C型凝集素开展深入研究,不仅可以丰富无脊椎动物固有免疫学内容,还有望将其开发为具有免疫增强效果的活性饵料,应用于对虾的健康养殖。本实验根据实验室前期转录组信息提示克隆获得了凡纳滨对虾一种新的C型凝集素基因(LvLc1,Gen Bank注册号:KY937940)。生物信息学分析显示LvLc1基因的开放阅读框全长891 bp,编码296个氨基酸,该基因编码的蛋白质含有一个保守的糖识别结构域(carbohydrate recognition domain,CRD),该结构域中具有潜在的半乳糖结合位点(QPD motif),进化发生分析显示LvLc1与来自节肢动物的甘露糖结合凝集素家族成员聚类在一起。对LvLc1基因的CRD结构域进行了原核重组表达与蛋白活性分析研究,结果显示:重组目的蛋白(rLvLc1)在Ca~(2+)存在的条件下,对多种病原菌(G~+、G~–和真菌)具有凝集作用,其凝集活性可被半乳糖、甘露糖、脂多糖等多种病原相关分子模式所抑制。研究表明,LvLc1作为C-型凝集素家族一个新成员,可能通过重要的模式识别受体作用,参与机体应答病原微生物侵染的防御过程。     

日本沼虾血清凝集素特性研究

取日本沼虾血淋巴液,利用血凝实验和糖抑制实验测定其血清凝集素的凝血活性和糖专一性,对血清凝集素热稳定性、pH值、盐度、重金属离子、EDTA-Na2、有机磷农药、黄酮和多糖类物质对其活性的影响进行了探讨。结果显示:日本沼虾血清凝集素可以对兔、鸡、小鼠、蟾蜍和鲢鱼5种动物血红细胞产生凝集反应,以对小鼠和鸡血红细胞的凝集效价较高。能够专一性结合D-葡萄糖,D-果糖而产生抑制效果。在50~60℃范围内凝集活性最强,80℃及以上凝集素发生变性,无凝集活性。该凝集素pH值作用范围广泛,在pH值2～12都可以使鸡红细胞发生凝集,pH值3～7范围内凝集效果较好。盐度为13～15时,凝集活性最强。凝集素活性能被浓度高于16mmol/L(含16mmol/L)的EDTA-Na2完全抑制,对重金属离子Cu2+和Zn2+敏感性强,其活性被完全抑制。乙酰甲胺磷和敌百虫对日本沼虾血清凝集素活性有一定的影响,黄酮和多糖类物质可显著增强虾血清凝集素的凝集作用。     

Cloning, expression, and characterization of a novel anti-HIV lectin from the cultured cyanobacterium, Oscillatoria agardhii

OAA, the potent anti-HIV protein from Oscillatoria agardhii NIES-204 belongs to a new lectin family, shows strict binding specificity for high-mannose N-glycans, and has an extremely high association constant in the picomolar range for recombinant gp120, an envelope protein of HIV. In this study we have cloned the gene encoding OAA from the genomic DNA of the cyanobacterium, and efficiently expressed the recombinant lectin (rOAA) in Escherichia coli. The rOAA expressed as a His-tagged fusion protein was recovered in a soluble form and purified in high yield (48 mg/1 l-culture) by metal chelate chromatography. The fusion protein was cleaved with factor Xa, and the resulting rOAA was isolated in a final yield of 14.8 mg/1 l-culture by reversed-phase HPLC. Both the N-terminal sequence and the molecular mass of rOAA were found to be identical with those of OAA. The rOAA was fully functional with the same properties as OAA, as evidenced by hemagglutination activity, hapten-inhibition test, and binding specificity for high-mannose-type N-glycans. This rOAA should be applicable as a specific probe for high-mannose N-glycans and should contribute to elucidation of the molecular basis of its strict carbohydrate-binding specificity and potent anti-HIV activity.