Inhibition of L- and P-selectin by a rationally synthesized novel core 2-like branched structure containing GalNAc-Lewisx and Neu5Acalpha2- 3Galbeta1-3GalNAc sequences

The selectins interact in important normal and pathological situations with certain sialylated, fucosylated glycoconjugate ligands containing sialyl Lewisx(Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcN Ac). Much effort has gone into the synthesis of sialylated and sulfated Lewisxanalogs as competitive ligands for the selectins. Since the natural selectin ligands GlyCAM-1 and PSGL-1 carry sialyl Lewisxas part of a branched Core 2 O-linked structure, we recently synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(SE-3Galbeta1++ +-3)GalNAc1alphaOMe and found it to be a moderately superior ligand for L and P-selectin (Koenig et al. , Glycobiology 7, 79-93, 1997). Other studies have shown that sulfate esters can replace sialic acid in some selectin ligands (Yeun et al. , Biochemistry, 31, 9126-9131, 1992; Imai et al. , Nature, 361, 555, 1993). Based upon these observations, we hypothesized that Neu5Acalpha2-3Galbeta1-3GalNAc might have the capability of interacting with L- and P-selectin. To examine this hypothesis, we synthesized Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-6(Neu5Acalpha2++ +-3Galbeta1-3)- GalNAc alpha1-OB, which was found to be 2- to 3-fold better than sialyl Lexfor P and L selectin, respectively. We also report the synthesis of an unusual structure GalNAcbeta1-4(Fucalpha1- 3)GlcNAcbeta1-OMe (GalNAc- Lewisx-O-methyl glycoside), which also proved to be a better inhibitor of L- and P-selectin than sialyl Lewisx-OMe. Combining this with our knowledge of Core 2 branched structures, we have synthesized a molecule that is 5- to 6-fold better at inhibiting L- and P-selectin than sialyl Lewisx-OMe, By contrast to unbranched structures, substitution of a sulfate ester group for a sialic acid residue in such a molecule resulted in a considerable loss of inhibition ability. Thus, the combination of a sialic acid residue on the primary (beta1-3) arm, and a modified Lexunit on the branched (beta1-6) arm on an O-linked Core 2 structure generated a monovalent synthetic oliogosaccharide inhibitor superior to SLexfor both L- and P-selectin.      

Beneficial effect of fluorocarbon emulsion media on the function of neuromuscular preparations in vitro

The effects of liquid fluorocarbons as bathing media were determined by use of in vitro neuromuscular preparations. Rat hemidiaphragms were bathed in either oxygenated fluorocarbon (FC) emulsion or standard oxygenated Krebs solution. Contractile force in response to simple supramaximal nerve stimuli as well as to high frequency stimulation was greater, while twitch:tetanus ratio was smaller in FC emulsion. With such medium, post-tetanic potentiation of contraction was also more consistently observed. Indirectly stimulated diaphragms survived longer in FC emulsion. After cessation of oxygenation, oxygen tension (ρO(2)) of the medium declined more rapidly with Krebs than with FC emulsion; ρO(2) directly correlated with force of contraction. Similarly, in the chick biventer cervicis preparation, FC emulsion enhanced nerve-stimulated force of contraction; returning the preparation to standard Krebs solution reversed this phenomenon. Dose-resonse curves of muscle contraction in response to acetycholine and KCl administration were shifted upward during FC emulsion superfusion. Frequency of miniature endplate potentials was lower in FC emulsion than that observed in Krebs solution, measured from the same cell of the rat diaphragm. Resting membrane potentials were also greater in muscle cells sampled from FC emulsion-bathed preparations. These data suggest that FC emulsion is superior to standard Krebs solution as a bathing medium for in vitro neuromuscular preparations by virtue of the high solubility of oxygen in it.     

Fluorescently-labeled fimbrin decorates a dynamic actin filament network in live plant cells

Recently it has been established, through a detailed biochemical analysis, that recombinant Arabidopsis thaliana fimbrin 1 (AtFim1) is a member of the fimbrin/plastin family of actin filament bundling or cross-linking proteins [D.R. Kovar et al. (2000) Plant J 24:625-636]. To determine whether AtFim1 can function as an F-actin-binding protein in the complex environment of the plant cell cytoplasm, we created a fluorescent protein analog and introduced it by microinjection into live Tradescantia virginiana L. stamen hair cells. AtFim1 derivatized with Oregon Green 488 had biochemical properties similar to unlabeled fimbrin, including the Kd value for binding to plant F-actin and the ability to cross-link filaments into higher-order structures. Fluorescent-fimbrin decorated an array of fine actin filaments in the cortical cytoplasm of stamen hair cells, which were shown with time-course studies to be highly dynamic. These data establish AtFim1 as a bona fide member of the fimbrin/plastin family, and represent the first use of a plant actin-binding protein as a powerful cytological tool for tracking the spatial and temporal redistribution of actin filaments in individual cells.     

Implications in the difference of anti-Mi-2 and -p155/140 autoantibody prevalence in two dermatomyositis cohorts from Mexico City and Guadalajara

Introduction

Autoantibodies and clinical manifestations in polymyositis/dermatomyositis (PM/DM) are affected by both genetic and environmental factors. The high prevalence of DM and anti-Mi-2 in Central America is thought to be associated with the high UV index of the area. The prevalences of autoantibodies and the clinical manifestations of PM/DM were evaluated comparing two cohorts in Mexico.

Methods

Ninety-five Mexican patients with PM/DM (66 DM, 29 PM; 67 Mexico City, 28 Guadalajara) were studied. Autoantibodies were characterized by immunoprecipitation using ³⁵S-methionine labeled K562 cell extract. Clinical information was obtained from medical records.

Results

DM represented 69% of PM/DM and anti-Mi-2 was the most common autoantibody (35%), followed by anti-p155/140 (11%); however, anti-Jo-1 was only 4%. The autoantibody profile in adult-onset DM in Mexico City versus Guadalajara showed striking differences: anti-Mi-2 was 59% versus 12% (P = 0.0012) whereas anti-p155/140 was 9% versus 35% (P = 0.02), respectively. A strong association of anti-Mi-2 with DM was confirmed and when clinical features of anti-Mi-2 (+) DM (n = 30) versus anti-Mi-2 (-) DM (n = 36) were compared, the shawl sign (86% versus 64%, P < 0.05) was more common in the anti-Mi-2 (+) group (P = 0.0001). Levels of creatine phosphokinase (CPK) were higher in those who were anti-Mi-2 (+) but they responded well to therapy.

Conclusions

Anti-Mi-2 has a high prevalence in Mexican DM and is associated with the shawl sign and high CPK. The prevalence of anti-Mi-2 and anti-p155/140 was significantly different in Mexico City versus Guadalajara, which have a similar UV index. This suggests roles of factors other than UV in anti-Mi-2 antibody production.     

Rpp25 is a major target of autoantibodies to the Th/To complex as measured by a novel chemiluminescent assay

Introduction

Autoantibodies to the Th/To antigen have been described in systemic sclerosis (SSc) and several proteins of the macromolecular Th/To complex have been reported to react with anti-Th/To antibodies. However, anti-Th/To has not been clinically utilized due to unavailability of commercial tests. The objective of the present study is to evaluate the newly developed ELISA and chemiluminescent immunoassay (CLIA) to measure autoantibodies to Rpp25 (a component of the Th/To complex) using immunoprecipitation (IP) as the reference method.

Methods

The first cohort consisted of 123 SSc patients including 7 anti-Th/To positive samples confirmed by IP. Additional seven anti-Th/To positive samples from non-SSc patients were also tested. For evaluation of the QUANTA Flash Rpp25 CLIA (research use only), 8 anti-Th/To IP positives, a cohort of 70 unselected SSc patients and sera from various disease controls (n = 357) and random healthy individuals (n = 10) were studied.

Results

Anti-Rpp25 antibodies determined by ELISA were found in 11/14 anti-Th/To IP positive but only in 1/156 (0.6%) negative samples resulting in a positive percent agreement of 78.6% (95% confidence interval [CI] 49.2, 95.3%) and a negative percent agreement of 99.4% (95% CI 96.4, 100.0%). To verify the results using a second method, 53 samples were tested by ELISA and CLIA for anti-Rpp25 reactivity and the results were highly correlated (rho = 0.71, 95% CI 0.56, 0.81; P < 0.0001). To define the cutoff of the CLIA, anti-Th/To IP positive and negative sera were tested using the anti-Rpp25 CLIA. At the cutoff selected by receiver operating characteristic (ROC) analysis 8/8 (100.0%) of the anti-Th/To positive sera but only 2/367 (0.5%) of the controls were positive for anti-Rpp25 antibodies. The positive and negative percent agreements were 100.0% (95% CI 63.1, 100.0%) and 99.5% (95% CI 98.0, 99.9%), respectively. In the disease cohorts 2/70 (2.9%) of the SSc patients were positive for anti-Rpp25 antibodies compared to 2/367 (0.5%) of the controls (P = 0.032). ROC analysis showed discrimination between SSc patients and controls with an area under the curve value of 0.732 (95% CI 0.655, 0.809).

Conclusion

Rpp25 is a major target of autoantibodies to the Th/To autoantigen complex. Further studies are needed to evaluate the clinical utility of the new assays.