Production and characterization of antibodies to the β-(1→6)-galactotetraosyl group and their interaction with arabinogalactan-proteins

Rabbit antisera were raised against -(16)-galactotetraose coupled to bovine serum albumin (Gal₄-BSA). The antisera reacted with arabinogalactan-proteins (AGPs) isolated from seeds, roots, or leaves of radish (Raphanus sativus L.) as revealed by immunodiffusion analysis. Extensive removal of -l-arabinofuranosyl residues from these AGPs enhanced the formation of precipitin with the antisera. The antisera did not react with such other polysaccharides as soybean arabinan-4-galactan, -(14)-galactan, and -(13)-galactan, indicating their high specificity toward the consecutive -(16)-galactosyl side chains of AGPs. The antibodies were purified by affinity chromatography on a column of immobilized -(16)-galactotetraose as ligand. The specificity of the antibodies toward consecutive (16)-linked -galactosyl residues was confirmed by enzyme-linked immunosorbent assay for hapten inhibition against Gal₄-BSA as antigen, which revealed that -(16)-galactotriose and-tetraose were potent inhibitors, while -(13)-or -(14)-galactobioses and -trioses were essentially unreactive. Electron-microscopic observation of immunogold-stained tissues demonstrated that AGPs were localized in the middle lamella as well as at the plasma membrane of primary roots of radish. Agglutination of protoplasts prepared from cotyledons occurred with the antibodies, supporting the evidence for localization of AGPs in the plasma membrane. The antibody-mediated agglutination was inhibited by addition of AGPs or -(16)-galactotetraose.Abbreviations AGP arabinogalactan-protein - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - Gal₃-BSA -(16)-galactotriose coupled to BSA - Gal₄-BSA -(16)-galactotetraose coupled to BSA - Ig immunoglobulin - 4-Me-GlcpA 4-O-methyl-d-glucopyranosyluronic acid - M_r relative molecular mass The authors wish to thank Dr. J. Ohnishi of Department of Biochemistry, Saitama University, for his help in preparing protoplasts.     

Immunolocalization of H+-ATPases in the plasma membrane of pollen grains and pollen tubes ofLilium longiflorum

Summary A heterogeneous distribution of H⁺-ATPase was visualized in germinated pollen ofLilium longiflorum using monoclonal antibodies raised against plasma membrane H⁺-ATPase. Immunolocalization studies of protoplasts and subprotoplasts derived from pollen tubes and sectioned pollen grains and pollen tubes show that H⁺-ATPases are abundant in the plasma membrane of pollen grains but are absent or sparsely distributed in the plasma membrane of pollen tubes. This polar distribution of H⁺-ATPases is probably the basis of the endogenous current pattern measured in growing lily pollen and involved in pollen tube tip growth.Abbreviations BSA bovine serum albumine - Hepes N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - Mes 2-(N-morpholino)-ethane sulphonic acid - PBS phosphate buffered saline - Pipes piperazine-N,N-bis(2-ethanesulfonic acid) - Tris 2-amino-2-hydroxymethyl-1,3-propandiol     

In vivo secretion and association of clusterin (SGP-2) in luminal fluid with spermatozoa in the rat testis and epididymis.

Clusterin (sulfated glycoprotein-2) is a heterodimeric glycoprotein synthesized and secreted by rat Sertoli cells. An antigenically similar form is synthesized and secreted by the epididymis. The goal of this study was to define the epididymal regions in which clusterin is present and the regions in which clusterin is secreted and interacts with developing spermatozoa. Seminiferous tubule (STF), caput, corpus, and cauda fluids were collected by micropuncture and/or microperfusion and two-dimensional Western blot analysis was performed with a polyclonal antibody directed against Sertoli cell clusterin. Clusterin was found in both STF and epididymal fluid. STF contained predominantly the clusterin heavy chain (45 kd); however, a 70 Kd heterodimer was present under nonreducing conditions. Two subunits of clusterin with lower molecular weights (41 kd, heavy chain; 32 kd, light chain) and higher isoelectric points were present in the luminal fluid of all epididymal regions. The intraluminal levels of the heavy and light chains decreased from caput to cauda. Analysis by two-dimensional gel electrophoresis of proteins secreted directly into the epididymal luminal fluid revealed that clusterin was secreted by caput epithelium and not by the corpus and cauda epithelium. Western blots of membrane extracts from testicular, caput, and cauda spermatozoa revealed that testicular clusterin was associated with testicular sperm and epididymal clusterin with predominantly caput sperm. Our findings suggest that clusterin is secreted into the caput epididymal lumen, where it binds to sperm and then dissociates from sperm to be endocytosed by cells of the distal epididymal epithelium.     

Characterization of a major cell wall antigen and potential adhesin in three strains of Candida albicans

Selected strains of Candida albicans were examined to reveal the surface antigenicity and biochemical nature of major cell wall proteins that also were shown to serve as cellular adhesins on human buccal epithelial cells. Confirmation of the adhesive properties of these cells was made by scanning electron microscopy and immunofluorescence microscopy. Particular attention was directed at the clinical isolate KM-302. By means of indirect immunofluorescence staining, the KM-302 blastoconidia absorbed rabbit anti-C. albicans ATCC-32354 serum, revealing specific localization of surface antigens on germ tubes and pseudohyphae. Extracellular polymeric material and the cell wall extract of C. albicans KM-302 blastoconidia were found to contain a major surface antigen of 49 kDa that exhibited 42% adhesion inhibition in vitro. Of considerable significance is that immunogold localization by electron microscopy showed the antigen to be almost exclusively cell wall bound. This major antigen, identified in affinity and gel filtration chromatography fractions, was composed of 4% carbohydrate and 95.7% protein and had an isoelectric point of 6.1. The major antigen also showed a high level of similarity with that of C. albicans strain SC-5314 inasmuch as the major antigen of that strain had carbohydrate and protein compositions of 4 and 95.5%, respectively. Both of these strains also possessed the same percent of adhesion inhibition of human buccal epithelial cells.Abbreviations BECs buccal epithelial cells - CWE cell wall extract - EPP extracellular polymers and proteins - FITC fluorescein isothiocyanate - mAg major antigen - OD ₆₀₀ optical density at 600 nm - PBS phosphate buffered saline - TEM transmission electron microscopy - YNB yeast nitrogen base     

达氏鲟生长激素基因cDNA克隆、表达及免疫荧光定位研究

为研究达氏鲟(Acipenser dabryanus)生长激素(Growth Hormone, GH)基因的功能, 合成了达氏鲟垂体SMART cDNA, 克隆得到GH全长cDNA序列。达氏鲟GH全长cDNA序列为1008 bp, 由52 bp的5'端非编码区(Untranslated region, UTR)、编码214个氨基酸的645 bp开放阅读框(Open reading frame, ORF)和311 bp的3'UTR构成。运用GH氨基酸序列构建进化树分析发现, 达氏鲟与两栖类、爬行类和哺乳类的一致性要高于真骨鱼类。实时荧光定量PCR结果表明, 达氏鲟GH mRNA主要在垂体和下丘脑中表达, 且垂体中GH的表达量约为下丘脑的110倍; Western-blot研究结果与qRT-PCR一致, 仅在垂体和下丘脑中检测到生长激素蛋白, 且垂体中GH的表达量远高于下丘脑。免疫荧光定位结果显示, GH主要定位于垂体中部, 下丘脑中也有少量荧光信号; 苏木精-伊红组织切片染色研究表明, GH主要是由嗜酸性的生长激素分泌细胞分泌。研究为深入研究脊椎动物生长激素基因的进化和人工养殖达氏鲟的生长调控提供了基础。       

Tissue specific expression of potent insecticidal, Allium sativum leaf agglutinin (ASAL) in important pulse crop, chickpea (Cicer arietinum L.) to resist the phloem feeding Aphis craccivora

The phloem sap-sucking hemipteran insect, Aphis craccivora, commonly known as cowpea aphid, cause major yield loss of important food legume crop chickpea. Among different plant lectins Allium sativum leaf agglutinin (ASAL), a mannose binding lectin was found to be potent antifeedant for sap sucking insect A. craccivora. Present study describes expression of ASAL in chickpea through Agrobacterium-mediated transformation of “single cotyledon with half embryo” explant. ASAL was expressed under the control of CaMV35S promoter for constitutive expression and phloem specific rolC promoter for specifically targeting the toxin at feeding site, using pCAMBIA2301 vector containing plant selection marker nptII. Southern blot analysis demonstrated the integration and copy number of chimeric ASAL gene in chickpea and its inheritance in T₁ and T₂ progeny plants. Expression of ASAL in T₀ and T₁ plants was confirmed through northern and western blot analysis. The segregation pattern of ASAL transgene was observed in T₁ progenies, which followed the 3:1 Mendelian ratio. Enzyme linked immunosorbant assay (ELISA) determined the level of ASAL expression in different transgenic lines in the range of 0.08–0.38% of total soluble protein. The phloem tissue specific expression of ASAL gene driven by rolC promoter has been monitored by immunolocalization analysis of mature stem sections. Survival and fecundity of A. craccivora decreased to 11–26% and 22–42%, respectively when in planta bioassay conducted on T₁ plants compared to untransformed control plant which showed 85% survival. Thus, through unique approach of phloem specific expression of novel insecticidal lectin (ASAL), aphid resistance has been successfully achieved in chickpea. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Ethylene in induced conifer defense: cDNA cloning, protein expression, and cellular and subcellular localization of 1-aminocyclopropane-1-carboxylate oxidase in resin duct and phenolic parenchyma cells

Members of the Pinaceae family have complex chemical defense strategies. Conifer defenses associated with specialized cell types of the bark involve constitutive and inducible accumulation of phenolic compounds in polyphenolic phloem parenchyma cells and oleoresin terpenoids in resin ducts. These defenses can protect trees against insect herbivory and fungal colonization. The phytohormone ethylene has been shown to induce the same anatomical and cellular defense responses that occur following insect feeding, mechanical wounding, or fungal inoculation in Douglas fir (Pseudotsuga menziesii) stems (Hudgins and Franceschi in Plant Physiol 135:2134–2149, 2004). However, very little is known about the genes involved in ethylene formation in conifer defense or about the temporal and spatial patterns of their protein expression. The enzyme 1-aminocyclopropane-1-carboxylate oxidase (ACO) catalyzes the final step in ethylene biosynthesis. We cloned full-length and near full-length ACO cDNAs from three conifer species, Sitka spruce (Picea sitchensis), white spruce (P. glauca), and Douglas fir, each with high similarity to Arabidopsis thaliana ACO proteins. Using an Arabidopsis anti-ACO antibody we determined that ACO is constitutively expressed in Douglas fir stem tissues and is up-regulated by mechanical wounding, consistent with the wound-induced increase of ethylene levels. Immunolocalization showed cytosolic ACO is predominantly present in specialized cell types of the wound-induced bark, specifically in epithelial cells of terpenoid-producing cortical resin ducts, in polyphenolic phloem parenchyma cells, and in ray parenchyma cells.J.W. Hudgins and Steven G. Ralph contributed equally to this work.     

Immunogold localization of the cell-wall-matrix polysaccharides rhamnogalacturonan I and xyloglucan during cell expansion and cytokinesis inTrifolium pratense L.; implication for secretory pathways

We have localized two cell-wall-matrix polysaccharides, the main pectic polysaccharide, rhamnogalacturonan I (RG-I), and the hemicellulose, xyloglucan (XG), in root-tip and leaf tissues of red clover (Trifolium pratense L.) using immunoelectron microscopy. Our micrographs show that in both leaf and root tissues RG-I is restricted to the middle lamella, with 80–90% of the label associated with the expanded regions of the middle lamella at the corner junctions between cells. Xyloglucan, however, is nearly exclusively located in the cellulose-microfibril-containing region of the cell wall. Thus, these cell-wall-matrix polysaccharides are present in distinct and complementary regions of the cell wall. Our results further show that during cell expansion both RG-I and XG are present within Golgi cisternae and vesicles, thus confirming that the Golgi apparatus is the main site of synthesis of the non-cellulosic cell-wall polysaccharides. No label is seen over the endoplasmic reticulum, indicating that synthesis of these complex polysaccharides is restricted to the Golgi. The distribution of RG-I and XG in root-tip cells undergoing cell division was also examined, and it was found that while XG is present in the Golgi stacks and cell plate during cytokinesis, RG-I is virtually absent from the forming cell plate.Abbreviations ER endoplasmic reticulum - RG-I rhamnogalacturonan I - XG xyloglucan