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991.
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The 2.0 A structure of a cross-linked complex between snowdrop lectin and a branched mannopentaose: evidence for two unique binding modes 总被引:1,自引:0,他引:1
BACKGROUND: Galanthus nivalis agglutinin (GNA), a mannose-specific lectin from snowdrop bulbs, is a tetrameric member of the family of Amaryllidaceae lectins that exhibit antiviral activity towards HIV. Its subunits are composed of three pseudo-symmetrically related beta sheet domains, each with a conserved mannose-binding site. Crystal structures of monosaccharide and disaccharide complexes of GNA have revealed that all 12 binding sites of the tetramer are functional, and that the degree of occupancy is dependent on the availability of subsidiary interactions from neighboring subunits. The complex of GNA with a branched mannopentaose ((Manalpha1,6-(alpha1, 3-Man)Man-alpha1,6-(alpha1,3-Man)Man) described here simulates a more biologically relevant complex. RESULTS: Two unique mannopentaose binding modes co-exist in the tetragonal structure (1 subunit/asymmetric unit) of the complex. In one, the conserved monosaccharide-binding pocket in domain 1 (CRD 1) is utilized for cross-linkage of twofold related GNA dimers by the outer 3,6 tri-Man arm, which alternates between two orientations consistent with crystal symmetry. Inter-linked dimers assemble helically along the 41 crystal axis forming a pore-like structure. In the second binding mode, the complete 3,6 tri-Man arm binds to an extended binding region in domain 3 (CRD 3) with subsites for each terminal Man and the internal Man positioned in the conserved monosaccharide pocket. The two remaining mannose residues are not visible in either binding mode. CONCLUSIONS: This structure provides insights into possible mechanisms of the cross-linkage that is known to occur when lectins interact with specific multivalent cell surface receptors during events such as agglutination and mitogenic stimulation. By virtue of the large number of sites available for mannose binding, GNA has multiple possibilities of forming unique lattice structures. The two distinctly different binding modes observed in this study confirm that high affinity mannose binding occurs only at the two domain sites located near dimer interfaces. 相似文献
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Lewis rats undergo a relapsing paralytic disease upon challenge with spinal cord emulsified in complete Freund's adjuvant (CFA). Treatment with two intracardiac injections of liposomes composed of whole myelin significantly reduced the severity of disease. Protection was disease-specific since treatment with myelin liposomes did not protect Lewis rats against adjuvant arthritis (AA), a CNS-unrelated T-cell-mediated autoimmune disease. Myelin-liposome-treated, spinal cord/CFA-immunized rats displayed borderline reduction of delayed-type hypersensitivity (DTH) (ear swelling) reactions to myelin and myelin basic protein (MBP), but significantly reduced in vitro lymphnode cell proliferation in response to these antigens. Responses to purified protein derivative of Mycobacterium tuberculosis (PPD) were not reduced, emphasizing the antigen-specific nature of the myelin-liposome-mediated suppression. Spleen cell proliferative responses were inconsistent and often poor. However, when cultured in the presence of NG-monomethyl-L-arginine (MMA), antigen-specific proliferation of spleen cells from both treated and control rats was greatly enhanced, indicating that reactive nitrogen intermediates contributed to the decrease in spleen cell proliferation. Purified splenic T cells from treated rats displayed a pattern of proliferation similar to that of unseparated lymphnode cells. Treatment of rats with a single injection of myelin liposomes after recovery from the first clinical episode significantly reduced the severity of the relapses. 相似文献
997.
F Broly D Marez N Sabbagh M Legrand S Millecamps JM Lo Guidice P Boone UA Meyer 《Canadian Metallurgical Quarterly》1995,5(6):373-384
To detect mutations in the cytochrome P450 CYP2D6 gene (CYP2D6), we developed a strategy based on single-strand conformation polymorphism (SSCP) analysis of the gene amplified by polymerase chain reaction (PCR). The efficiency of the method was evaluated by analysing DNA samples from extensive metabolizers (EM) and poor metabolizers (PM) of debrisoquine. Haplotypes, alleles and mutations of CYP2D6 had previously been characterized in each individual using PCR assays, Xba I restriction fragment length polymorphism (RFLP) and sequencing. PCR-SSCP results were in complete agreement with those obtained using established methods. All previously characterized mutations were associated with particular shifts in the electrophoretic mobility of DNA fragments allowing their identification. We further tested the efficiency of PCR-SSCP for detecting new CYP2D6 mutations. DNA from a PM subject presumed to carry an unknown non-functional mutant allele of CYP2D6 was amplified and bands with aberrant migration patterns were observed on SSCP gels. Sequence analysis of the corresponding DNA fragments revealed the causative mutations. In this way, a novel non-functional allele of the gene, carrying three previously reported mutations and a new mutation in the third exon which results in a premature termination codon, was characterized. Finally, CYP2D6 SSCP analysis was performed on DNA amplified with fluorescent primers and an automated DNA sequencer was used for SSCP analysis of products. We conclude that the PCR-SSCP approach is a powerful method of identifying simultaneously known and new mutations of the CYP2D6 gene. 相似文献
998.
CM Schmidt RG Ehlenfeldt MC Athanasiou LA Duvick H Heinrichs CS David HT Orr 《Canadian Metallurgical Quarterly》1993,151(5):2633-2645
Trophoblast, the only fetal tissue in direct contact with maternal cells, fails to express the polymorphic HLA class I molecules HLA-A and -B, but does express the nonpolymorphic class I molecule HLA-G. It is thought that HLA-G may provide some of the functions of a class I molecule without stimulating maternal immune rejection of the fetal semiallograft. As a first step in identifying the cis-acting DNA regulatory elements involved in the control of class I expression by extraembryonic tissue, several types of transgenic mice were produced. Two HLA-G genomic fragments were used, 5.7 and 6.0 kb in length. These included the entire HLA-G coding region, 1 kb of 3' flanking sequence, and 1.2 or 1.4 kb of 5' flanking sequence, respectively. A hybrid transgene, HLA-A2/G, was produced by replacing the 5' flanking sequence, first exon, and early first intron of HLA-G with the corresponding elements of HLA-A. Comparison of transgene mRNA expression patterns seen in HLA-A2/G and HLA-G transgenic mice suggests that 5' flanking sequences are largely responsible for the differing patterns of expression typical of the classical class I and HLA-G genes. Studies comparing the extraembryonic HLA-G expression levels of founder embryos transgenic for either the 5.7- or 6.0-kb HLA-G transgene showed that the 6.0-kb transgene directed HLA-G expression far more efficiently than did the 5.7-kb HLA-G transgene, producing extraembryonic HLA-G mRNA levels similar to those seen in human extraembryonic tissues. The results of these studies suggest that the 250-bp fragment present at the extreme 5' end of the 6.0-kb HLA-G transgene and absent from the 5.7-kb HLA-G transgene contains an important positive regulatory element. This 250-bp fragment lies further upstream than any of the previously documented class I regulatory regions and may function as a locus control region. 相似文献
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G Blanpied M Blecher A Caracappa C Djalali MA Duval G Giordano K Hicks S Hoblit M Khandaker OC Kistner G Matone L Miceli WK Mize BM Preedom D Rebreyend AM Sandorfi C Schaerf RM Sealock CE Thorn ST Thornton K Vaziri CS Whisnant X Zhao P Wilhelm H Arenh?vel 《Canadian Metallurgical Quarterly》1995,52(2):R455-R459