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1.
The latex agglutination test has been applied as a detection system for lectins, the method being especially useful in locations where the dependence on blood for hemagglutination tests could be minimised. The binding of various glycoproteins and sugars individually to the latex particles facilitated the agglutination with lectins having varying sugar specificities. The glycoproteins used were ovalbumin, horseradish peroxidase, porcine mucin and fetuin, while N-acetylglucosamine, N-acetylgalactosamine comprised the sugars used for binding to latex. The sensitivity of the latex agglutination tests was comparable with that of hemagglutination tests. Sugar binding specificity of the lectins could also be determined by inhibition of the agglutination in the presence of corresponding free sugars. The method proved to be useful in screening crude seed extracts for the presence of lectins. 相似文献
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Summary Fluorescein or rhodamine conjugates of seventeen different lectins were tested for their ability to label the plasma membrane of live plant protoplasts. During the investigation, a strong effect of calcium was observed on the binding of several lectins to protoplasts derived from suspension cultured rose cells (Rosa sp. Paul's Scarlet). The binding of these lectins was increased by elevating the calcium concentration from 1 to 10 mM in the buffer. Other divalent cations had variable, but similar, effects on lectin binding. The mechanism of this effect appeared to involve the protoplast surface rather than the lectins. Although the cell wall-degrading enzymes used to isolate protoplasts had generally no effect on lectin binding, one clear exception was observed. Binding ofArachis hypogaea agglutinin was markedly reduced on protoplasts isolated with Driselase as compared to protoplasts isolated with a combination of Cellulysin and Pectolyase Y-23. Although most of the lectins that labeled protoplasts derived from cultured rose cells or from corn root cortex (Zea mays L. WF9 × Mo17) had specificities for galactose or N-acetylgalactosamine, some differences in protoplast labeling between lectins of the same saccharide specificity were observed. Two different analyses of the interaction betweenRicinus communis agglutinin and rose protoplasts showed that binding was cooperative with an apparent association constant of 7.2 × 105M–1 or 9.8 × 105M–1 with a maximum of approximately 108 lectin molecules bound per protoplast. Treatment of protoplasts with glycosidases which hydrolyze either N- or O-glycosidic linkages of glycoproteins slightly enhanced labeling of protoplasts byRicinus communis agglutinin. Interpretation of these results are discussed.Abbreviations MPR
medium, minimal organic medium (Nothnagel andLyon 1986)
- APA
Abrus precatorius agglutinin
- CSA
Cytisus sessilifolius agglutinin
- ECA
Erythrina cristagalli agglutinin
- GS-I
Griffonia simplicifolia agglutinin
- LcH
Lens culinarus agglutinin
- PNA
Arachis hypogaea agglutinin
- SBA
Glycine max agglutinin
- VAA
Viscum album agglutinin
- VFA
Vicia faba agglutinin
- WGA
Triticum vulgaris agglutinin
- Con A
Canavalia ensiformis agglutinin
- HPA
Helix pomatia agglutinin
- TPA
Tetragonolobus purpureas agglutinin
- RCA
Ricinus communis agglutinin
- DBA
Dolichos biflorus agglutinin
- SJA
Sophora japonica agglutinin
- BPA
Bauhinia purpurea agglutinin
- FITC
fluorescein isothiocyanate
- Ga1NAc
N-acetylgalactosamine
- FDA
fluorescein diacetate
- 2-O-Me-D-Fuc
2-O-methyl-D-fucose
Parts of the work presented here are also submitted in partial fulfillment of requirements for the Ph.D. degree. 相似文献
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M Tunis 《Journal of immunology (Baltimore, Md. : 1950)》1965,95(5):876-879
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Plant protoplast technology: Current status 总被引:2,自引:0,他引:2
Robust and reproducible protoplast-to-plant systems are crucial for underpinning genetic manipulation technology involving
somatic hybridisation and transformation. Novel and effective approaches for maximising the efficiency of such protoplast
cultures include supplementation of media with surfactants and artificial gas carriers, such as perfluorochemicals and haemoglobin.
Physical parameters, particularly electrostimulation, also enhance the development of protoplasts and protoplast-derived cells
in culture. DNA uptake into protoplasts is now a routine and universally accepted procedure in plant biotechnology for introducing
and evaluating both short-term (transient) and long-term (stable) expression of genes in cells and regenerated plants. Importantly,
protoplast fusion overcomes pre- and post-zygotic sexual incompatibility barriers and generates novel germplasm through new
nuclear-cytoplasmic combinations. In this respect, considerable progress has been made in generating somatic hybrid plants,
particularly in citrus, brassicas and potato. Isolated protoplasts are also a unique single cell system for evaluating aspects
of ultrastructure, genetics and physiology, with potential for the biosynthesis of novel secondary products, including commercially-important
recombinant proteins (e.g. antibodies), and as systems in toxicity screening. Recent advances in protoplast technology have benefited from advances
in animal and microbial cell culture, with interesting parallels existing between these systems. Further innovations will
necessitate the strengthening of interdisciplinary links in these research fields and the requirement for continued dialogue
and co-operation between workers with diverse but complementary skills. 相似文献
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A method for the isolation of plant protoplasts is presented that uses inexpensive sources of enzymes. It is suitable for student use in Biotechnology. 相似文献
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M. C. Dornelas F. C. A. Tavares J. C. de Oliviera M. L. C. Vieira 《Plant cell reports》1995,15(1-2):106-110
Summary Protoplasts were isolated from leaf explants ofPassiflora edulis var.flavicarpa (the yellow passion fruit) and from cell suspensions of fivePassiflora species. Chemical fusion was performed using polyethylene glycol and the microcolonies obtained were transferred to growth medium to produce calli. Electrophoresis of soluble proteins and analysis of isoenzymes from calli produced from the fusion experiments were performed to select somatic hybrids. Specific polypeptide bands allowed the identification of somatic hybrids betweenP. edulis var.flavicarpa (+)P. alata, P. edulis var.flavicarpa (+)P. amethystina, P. edulis var.flavicarpa (+)P. cincinnata, P. edulis var.flavicarpa (+)P. giberti andP. edulis var.flavicarpa (+)P. coccinea. An average of 3 to 5% hybrid calli were obtained. With the exception of theP. edulis var.flavicarpa (+)P. coccinea, whole plants were recovered from all hybrids. These somatic hybrids showed 4n=36 chromosomes, which represents a further evidence of their hybridity. 相似文献
10.
Morphologically normal plants have been regenerated from petal protoplasts of petunia (Petunia hybrida) flower. Maximum protoplast yields from petal tissues were obtained within 2 days after anthesis. Protoplasts were cultured on modified Murashige and Skoog's medium in which NH4NO3 and Fe·EDTA concentrations were reduced to 1/3 (7mM) and 1/10 (10 M), respectively. After plating, protoplasts gradually reduced pigment density, and plastids developed near the nucleus. In premitotic petunia petal cells, the nucleus moved from the periphery to the central region of the cell. The first cell divisions were detected after 6–10 days of culture initiation, and the average division frequency was 15% in the best culture condition. The results indicated that the time of the first cell division and cell division frequency were closely related to flower age after anthesis. More than a hundred plants with morphologically normal shoots and roots have been obtained. Those plants grew vigorously in soil.Abbreviations BA
benzylaminopurine
- DAPI
4, 6-diamidino-2-phenylindole
- 2, 4-d
dichlorophenoxyacetic acid
- Fe·EDTA
Fe·ethylenediaminetetraacetate
- IAA
indole-3-acetic acid
- MtSB
microtubule stabilizing buffer
- NAA
-naphthaleneacetic acid
- PIPES
piperazine-N,N-bis(2-ethanesulfonic acid)
- EGTA
ethylene glycol-bis(-aminoethyl ether) N,N,N,N-tetraacetic acid 相似文献
11.
J. J. Rybczyński 《Plant cell reports》1989,8(7):383-386
Explants from hypcotyls and cotyledons of Browalia speciosa were shown to regenerate plantlets.Protoplasts were isolated from etiolated cotyledon material, and, although callus was readily obtained, plantlet regeneration was not observed using numerous hormone regimes.Abbreviations M
Mannitol
- 2,4-D
Dichlorophenoxyacetic acid
- NAA
Naphthalene-acetic acid
- BAP
Benzylaminopurine
- MS medium
Murashige and Skoog (1962) medium
- UM medium
Uchimiya and Murashige (1974) medium
- COT
cotyledon
- SH
shoot
- R
root 相似文献
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A number of commercially available enzymes were used to modify the cell surface of human erythrocytes to varying degrees. In protease-treated erythrocytes the decrease in surface charge (determined by cell electrophoresis or analysis of sialic acid content) correlates with an increase in agglutinability with concanavalin A (ConA) and wheat germ agglutinin (WGA). On the other hand, treatment with neuraminidase leads to very large decrease in surface charge with only an intermediate increase in agglutinability with both lectins. Subsequent protease treatment of these cells enhances their agglutinability appreciably without further altering their surface charge. It is concluded that the increased agglutinability following protease treatment is due both to a decrease in the net negative charge and a removal of peptides and glycopeptides from the cell surface that may sterically hinder the agglutination reaction. 相似文献
14.
Plant lectins as defense proteins against phytophagous insects 总被引:2,自引:0,他引:2
One of the most important direct defense responses in plants against the attack by phytophagous insects is the production of insecticidal peptides or proteins. One particular class of entomotoxic proteins present in many plant species is the group of carbohydrate-binding proteins or lectins. During the last decade a lot of progress was made in the study of a few lectins that are expressed in response to herbivory by phytophagous insects and the insecticidal properties of plant lectins in general. This review gives an overview of lectins with high potential for the use in pest control strategies based on their activity towards pest insects. In addition, potential target sites for lectins inside the insect and the mode of action are discussed. In addition, the effect of plant lectins on non-target organisms such as beneficial insects as well as on human/animal consumers is discussed. It can be concluded that some insecticidal lectins are useful tools that can contribute to the development of integrated pest management strategies with minimal effect(s) on non-target organisms. 相似文献
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Plant protoplast fusion and growth of intergeneric hybrid cells 总被引:5,自引:0,他引:5
16.
The microtubule-disruptive drugs colchicine and vinblastine alter ligand-induced redistribution of cell surface immunoglobulins and lectin receptors. These effects can be duplicated by treatment of cells with the divalent cation ionophores A23187 and X537A. Ionophore activity was dependent upon the presence of Ca2+ (1.8·10?3?4·10?4 M) in the culture medium. The K+-selective ionophore valinomycin had no effect on ligand-induced redistribution of surface receptors. It is suggested that A23187 and X537A impair membrane-associated microtubules involved in transmembrane control of receptor mobility and topography. In contrast to the action of colchicine and vinblastine that bind directly to microtubules, it is proposed that ionophores indirectly affect microtubules by raising the concentration of Ca2+ in the cytoplasm to levels that favor microtubule depolymerization and inhibit microtubule assembly. 相似文献
17.
Plant seed lectins disrupt growth of germinating fungal spores 总被引:2,自引:0,他引:2
Plant seed lectins are well-characterized proteins and glycoproteins whose natural function remains unknown. We found that eleven purified seed lectins (representing five groups of lectin sugar specificities) bound to the germ tubes of asexual spores of Neurospora crassa, Aspergillus amstelodami , and Botryodiplodia theobromae. The lectins caused several types of quantifiable growth disruption during germination of these seed- or soil-borne fungal spores, including sensitivity to osmotic lysis, adventitious branching of the spore germ tubes, and inhibition of germ tube elongation. These anti-fungal effects of purified lectins, which were reversible with the sugar hapten specific for each lectin, were partially duplicated by lectin-like factors in the homologous crude seed extracts. The seed lectins may disrupt fungal growth by interfering with normal cell wall deposition and assembly. 相似文献
18.
Zhang DL Liu SG Yan LF Li LJ Huang GS Fang FD Xia GT He XY Gao BX Bai XH Wang W Ding PG 《Cell biology international》2001,25(10):997-1002
The carcinogenic or tumourigenic testing of seven animal kidney cell lines (F-81, CRFK, MDCK, Vero, Vero-2 cell line, MA-104 and BHK-21) established in China, were carried out in more than 700 nude mice for colony formation in soft agar and for agglutination under different density of plant lectins. Tests showed that there were correlation between cell line chromosome number variations and anchorage independence in soft agar, agglutinability under lectins and tumour-forming ability in nude mice. Since testing in vitro was more economical, simpler and faster and thus thought to be more reliable, we recommend measuring agglutinability, followed by anchorage independence or analysis of karyotype as the initial means for monitoring tumourigenicity of animal cell lines in nude mice. 相似文献
19.
Treatment of untransformed mouse and hamster cells with the tertiary amine local anesthetics dibucaine, tetracaine and procaine increases their susceptibility to agglutination by low doses of the plant lectin concanavalin A. Agglutination of anesthetic-treated untransformed cells by low doses of concanavalin A is accompanied by redistribution of concanavalin A receptors on the cell surface to form patches, similar to that occurring in spontaneous agglutination of virus-transformed cells by concanavalin A. Immunofluorescence and freeze-fracture electronmicroscopic observations indicate that local anesthetics per se do not induce this redistribution of concanavalin A receptors but modify the plasma membrane so that receptor redistribution is facilitated on binding of concanavalin A to the cell surface. Fluorescence polarization measurements on the rotational freedom of the membrane-associated probe, diphenylhexatriene, indicate that local anesthetics produce a small increase in the fluidity of membrane lipids. Spontaneous agglutination of transformed cells by low doses of concanavalin A is inhibited by colchicine and vinblastine but these alkaloids have no effect on concanavalin A agglutination of anesthetic-treated cells. Evidence is presented which suggests that local anesthetics may impair membrane peripheral proteins sensitive to colchicine (microtubules) and cytochalasin-B (microfilaments). Combined treatment of untransformed 3T3 cells with colchicine and cytochalasin B mimics the effect of local anesthetics in enhancing susceptibility to agglutination by low doses of concanavalin A. A hypothesis is presented on the respective roles of colchicine-sensitive and cytochalasin B-sensitive peripheral membrane proteins in controlling the topographical distribution of lectin receptors on the cell surface. 相似文献
20.
Plant lectins: occurrence,biochemistry, functions and applications 总被引:17,自引:0,他引:17
Growing insights into the many roles of glycoconjugates in biorecognition as ligands for lectins indicates a need to compare plant and animal lectins. Furthermore, the popularity of plant lectins as laboratory tools for glycan detection and characterization is an incentive to start this review with a brief introduction to landmarks in the history of lectinology. Based on carbohydrate recognition by lectins, initially described for concanavalin A in 1936, the chemical nature of the ABH-blood group system was unraveled, which was a key factor in introducing the term lectin in 1954. How these versatile probes are produced in plants and how they are swiftly and efficiently purified are outlined, and insights into the diversity of plant lectin structures are also given. The current status of understanding their functions calls for dividing them into external activities, such as harmful effects on aggressors, and internal roles, for example in the transport and assembly of appropriate ligands, or in the targeting of enzymatic activities. As stated above, attention is given to intriguing parallels in structural/functional aspects of plant and animal lectins as well as to explaining caveats and concerns regarding their application in crop protection or in tumor therapy by immunomodulation. Integrating the research from these two lectin superfamilies, the concepts are discussed on the role of information-bearing glycan epitopes and functional consequences of lectin binding as translation of the sugar code (functional glycomics). 相似文献