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Pascale Simard Hugo Galarneau Sébastien Marois Daniel Rusu Caroline D Hoemann Patrice E Poubelle Hani El-Gabalawy Maria JG Fernandes 《Arthritis research & therapy》2009,11(3):R74-10
Introduction
Osteoarthritis is characterized by the progressive destruction of cartilage in the articular joints. Novel therapies that promote resurfacing of exposed bone in focal areas are of interest in osteoarthritis because they may delay the progression of this disabling disease in patients who develop focal lesions. Recently, the addition of 80% deacetylated chitosan to cartilage microfractures was shown to promote the regeneration of hyaline cartilage. The molecular mechanisms by which chitosan promotes cartilage regeneration remain unknown. Because neutrophils are transiently recruited to the microfracture site, the effect of 80% deacetylated chitosan on the function of neutrophils was investigated. Most studies on neutrophils use preparations of chitosan with an uncertain degree of deacetylation. For therapeutic purposes, it is of interest to determine whether the degree of deacetylation influences the response of neutrophils to chitosan. The effect of 95% deacetylated chitosan on the function of neutrophils was therefore also investigated and compared with that of 80% deacetylated chitosan. 相似文献4.
Levin AM Coroneus JG Cocco MJ Weiss GA 《Protein science : a publication of the Protein Society》2006,15(3):478-486
The techniques of phage-displayed homolog shotgun scanning, oligomer complementation, NMR secondary structure analysis, and computational docking provide a complementary suite of tools for dissecting protein-protein interactions. Focusing these tools on the interaction between the catalytic sub-unit of protein kinase A (PKAcat) and caveolin-1 scaffolding domain (CSD) reveals the first structural model for the interaction. Homolog shotgun scanning varied each CSD residue as either a wild-type or a homologous amino acid. Wild-type to homolog ratios from 116 different homologous CSD variants identified side-chain functional groups responsible for precise contacts with PKAcat. Structural analysis by NMR assigned an alpha-helical conformation to the central residues 84- 97 of CSD. The extensive mutagenesis data and NMR secondary structure information provided constraints for developing a model for the PKAcat-CSD interaction. Addition of synthetic CSD to phage-displayed CSD resulted in oligomer complementation, or enhanced binding to PKAcat. Together with previous experiments examining the interaction between CSD and endothelial nitric oxide synthase (eNOS), the results suggest a general oligomerization-dependent enhancement of binding between signal transducing enzymes and caveolin-1. 相似文献
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Rod and cone photoreceptors project from the outer retinal surface into a
carbohydrate-rich interphotoreceptor matrix (IPM). Unique IPM
glycoconjugates are distributed around rods and cones. Wheat germ
agglutinin (WGA) strongly decorates the rod matrix domains and weakly
decorates the cone matrix domains. This study characterizes the major
WGA-binding glycoprotein in the human IPM, which we refer to as SPACR
(sialoprotein associated with cones and rods). SPACR, which has a molecular
weight of 147 kDa, was isolated and purified from the IPM by lectin
affinity chromatography. A polyclonal antibody to SPACR was prepared that
colocalizes in tissue preparations with WGA-binding domains in the IPM.
Sequential digestion of SPACR with N- and O- glycosidases results in a
systematic increase in electrophorectic mobility, indicating the presence
of both N- and O-linked glycoconjugates. Complete deglycosylation results
in a reduction in the relative molecular mass of SPACR by about 30%.
Analysis of lectin binding allowed us to identify some of the structural
characteristics of SPACR glycoconjugates. Treatment with neuraminidase
exposes Galbeta1- 3GalNAc disaccharide as indicated by positive peanut
agglutinin (PNA) staining, accompanied by the loss of WGA staining. Maackia
amurensis agglutinins (MAA-1 and MAA-2), specific for sialic acid in
alpha2-3 linkage to Gal, bind SPACR, while Sambucus nigra agglutinin (SNA),
specific for alpha2-6 linked sialic acid, does not, indicating that the
dominant glycoconjugate determinant on SPACR is the O-linked carbohydrate,
NeuAcalpha2-3Galbeta1-3GalNAc. The abundance of sialic acid in SPACR
suggests that this glycoprotein may contribute substantially to the
polyanionic nature of the IPM. The carbohydrate chains present on SPACR
could also provide sites for extensive crosslinking and participate in the
formation of the ordered IPM lattice that surrounds the elongate
photoreceptors projecting from the outer retinal surface.
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Purification and characterization of Dolichos lablab lectin 总被引:1,自引:0,他引:1
The mannose/glucose-binding Dolichos lablab lectin (designated DLL) has
been purified from seeds of Dolichos lablab (hyacinth bean) to
electrophoretic homogeneity by affinity chromatography on an ovalbumin-
Sepharose 4B column. The purified lectin gave a single symmetric protein
peak with an apparent molecular mass of 67 kDa on gel filtration
chromatography, and five bands ranging from 10 kDa to 22 kDa upon SDS-PAGE.
N-Terminal sequence analysis of these bands revealed subunit heterogeneity
due to posttranslational proteolytic truncation at different sites mostly
at the carboxyl terminus. The carbohydrate binding properties of the
purified lectin were investigated by three different approaches:
hemagglutination inhibition assay, quantitative precipitation inhibition
assay, and ELISA. On the basis of these studies, it is concluded that the
Dolichos lablab lectin has neither an extended carbohydrate combining site,
nor a hydrophobic binding site adjacent to it. The carbohydrate combining
site of DLL appears to most effectively accommodate a nonreducing terminal
alpha-d-mannosyl unit, and to be complementary to the C-3, C-4, and C-6
equatorial hydroxyl groups of alpha-d-mannopyranosyl and
alpha-d-glucopyranosyl residues. DLL strongly precipitates murine IgM but
not IgG, and the recent finding that this lectin interacts specifically
with NIH 3T3 fibroblasts transfected with the Flt3 tyrosine kinase receptor
and preserves human cord blood stem cells and progenitors in a quiescent
state for prolonged periods in culture, make this lectin a valuable tool in
biomedical research.
相似文献
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We have broadly defined the DNA regions regulating esterase6 activity in
several life stages and tissue types of D. melanogaster using P-
element-mediated transformation of constructs that contain the esterase6
coding region and deletions or substitutions in 5' or 3' flanking DNA.
Hemolymph is a conserved ancestral site of EST6 activity in Drosophila and
the primary sequences regulating its activity lie between -171 and -25 bp
relative to the translation initiation site: deletion of these sequences
decrease activity approximately 20-fold. Hemolymph activity is also
modulated by four other DNA regions, three of which lie 5' and one of which
lies 3' of the coding region. Of these, two have positive and two have
negative effects, each of approximately twofold. Esterase6 activity is
present also in two male reproductive tract tissues; the ejaculatory bulb,
which is another ancestral activity site, and the ejaculatory duct, which
is a recently acquired site within the melanogaster species subgroup.
Activities in these tissues are at least in part independently regulated:
activity in the ejaculatory bulb is conferred by sequences between -273 and
-172 bp (threefold decrease when deleted), while activity in the
ejaculatory duct is conferred by more distal sequences between -844 and
-614 bp (fourfold decrease when deleted). The reproductive tract activity
is further modulated by two additional DNA regions, one in 5' DNA (-613 to
-284 bp; threefold decrease when deleted) and the other in 3' DNA (+1860 to
+2731 bp; threefold decrease when deleted) that probably overlaps the
adjacent esteraseP gene. Collating these data with previous studies
suggests that expression of EST6 in the ancestral sites is mainly regulated
by conserved proximal sequences while more variable distal sequences
regulate expression in the acquired ejaculatory duct site.
相似文献
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Nucleotide variation at the hypervariable esterase 6 isozyme locus of Drosophila simulans 总被引:2,自引:0,他引:2
Esterase 6 (Est-6/EST6) is polymorphic in both Drosophila melanogaster and
D. simulans for two common allozyme forms, as well as for several other
less common variants. Parallel latitudinal clines in the frequencies of the
common EST6-F and EST6-S allozymes in these species have previously been
interpreted in terms of a shared amino acid polymorphism that distinguishes
the two variants and is subject to selection. Here we compare the sequences
of four D. simulans Est-6 isolates and show that overall estimates of
nucleotide heterozygosity in both coding and 5' flanking regions are more
than threefold higher than those obtained previously for this gene in D.
melanogaster. Nevertheless, the ratio of replacement to exon silent-site
polymorphism in D. simulans is less than the ratio of replacement to silent
divergence between D. simulans and D. melanogaster, which could be the
result of increased efficiency of selection against replacement
polymorphisms in D. simulans or to divergent selection between the two
species. We also find that the amino acid polymorphisms separating EST6- F
and EST6-S in D. simulans are not the same as those that separate these
allozymes in D. melanogaster, implying that the shared clines do not
reflect shared molecular targets for selection. All comparisons within and
between the two species reveal a remarkable paucity of variation in a
stretch of nearly 400 bp immediately 5' of the gene, indicative of strong
selective constraint to retain essential aspects of Est-6 promoter
function.
相似文献
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The recent structure determinations of the mammalian effector enzyme adenylyl cyclase reveal the structure of its catalytic core, provide new insights into its catalytic mechanism and suggest how diverse signaling molecules regulate its activity. 相似文献