Nonresponse to hepatitis B vaccines and the kinetics of anti-HBs production

The present paper demonstrates the immunohistochemical distribution of proteoglycan (PG) molecules carrying chondroitin sulfate (CS) chains with 6-sulfated hexosamine residues, or CS and/or dermatan sulfate (DS) chains with 4-sulfated residues in the developing bovine ruminal papillae (RP) using monoclonal antibodies (mAbs) 3B3, 2B6, and MO225. These PGs carrying chondroitin 6-sulfate that were detected by mAb 3B3, and the glucuronic acid 2-sulfate-N-acetyl-galactosamine 6-sulfate unit that was detected by the mAb MO225 were distributed in the mesenchyme and epithelial basement membrane in the rumen, and were thus correlated to the outgrowth of the RP. The PG carrying DS was detected by the mAb 2B6 and was distributed in the lower region of the mesenchyme and intermuscular connective tissue during the development of the RP. These findings suggest that PGs carrying CS chains with 6-sulfation are involved in the outgrowth of the RP, and that PGs carrying DS are involved in organization in the mesenchyme.     

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.     

Morphological and biochemical features affecting the antithrombotic properties of the aorta in adult rabbits and rabbit pups

We hypothesised that there are important physiologic differences in arterial wall structure and function with respect to antithrombotic activity in the very young (pre-puberty) compared to adults. Electron microscopy, gel electrophoresis, and activity assays were used to examine differences in aorta structure and function comparing prepubertal rabbits (pups) to adult rabbits. Differences in endothelial function, extracellular matrix structure, proteoglycan (PG) distribution and glycosaminoglycan (GAG) content and function were shown. In both intima and media, total PG, chondroitin sulfate (CS) PG and heparan sulfate (HS) PG content were significantly increased in pups compared to adult rabbits. These findings corresponded to increased concentrations by mass analyses of CS GAG and DS GAG in aortas from pups. There was also a significant increase in antithrombin activity in pups due to HS GAG. In conclusion, differences in both structure and antithrombin activity of aortas from pups compared to adult rabbits suggest that young arteries may have greater antithrombotic potential that is, at least in part, related to increased HS GAG.     

Identification of regulatory sequences of juvenile hormone-sensitive and -insensitive serum protein-encoding genes

Proteoglycans (PGs) from bovine cornea showed a protective effect on liposome peroxidation induced by Fe2+. Both chondroitin sulfate, dermatan sulfate-containing PG (CS,DS-PG) and keratan sulfate-containing PG (KS-PG) inhibited thiobarbituric acid-reactive substance formation when incubated with liposomes and Fe2+, CS,DS-PG being more effective than KS-PG. The native structure of PGs contributed markedly to antioxidant activity. Papain digestion of core protein reduced the protective effect of CS,DS-PG, whereas it abolished completely that of KS-PG. Apparently only hexuronate-containing glycosaminoglycan (GAG) chains may exert a significant antioxidant activity and this was confirmed using standard GAGs. Quasielastic laser light scattering was used to evaluate the structural consequence of peroxidative damage induced by Fenton reagent on liposomes. After exposure to the free-radical-generating system, a bimodal distribution of liposomes was observed, probably depending on the loss of native structure and fragmentation. Both CS,DS-PG and KS-PG prevented liposome breakdown. Again, free KS chains were ineffective against liposome damage, whereas DS and CS maintained the normal distribution of liposome size. These data support the hypothesis that PGs may represent part of the antioxidant mechanisms of organisms and suggest that modifications of PG content and/or composition might affect tissue sensitivity to oxidative stress.     

Urinary glycosaminoglycan excretion in NIDDM subjects: its relationship to albuminura

Nephropathy is a serious microvascular complication of diabetes mellitus which is preceded by a period of microalbuminura. Increased loss of proteoglycan (PG) from glomerular basement (GBM) has been postulated to alter glomerular charge selectivity which contributes to urinary loss of albumin. In this study we measured the excretion of urinary glycosaminoglycans (GAG), the degradation products of PG, in 82 non-insulin-dependent (NIDDM) (Type 2) diabetic and 34 non-diabetic subjects. We found that diabetic subjects had a significantly higher GAG urinary excretion rate compared to non-diabetic subjects (12.54 +/- 5.67 vs 8.80 +/- 3.99 micrograms glucuronic acid min-1, p = 0.0001). Categorizing for albuminuric status shows that the diabetic normo-, micro- and macroalbuminuric groups have a higher GAG excretion rate than non-diabetic subjects. Heparan sulphate (HS) GAG urinary excretion was measured in 25 samples from diabetic subjects and 18 non-diabetic subjects. Diabetic subjects excreted more HS GAG than controls both as a rate or as a percentage of total GAG (3.70 +/- 1.94 vs 2.38 +/- 1.48 micrograms glucosamine min-1, p = 0.02; 31.6% +/- 12.5 vs 23.1% +/- 10.4, p = 0.02). Categorizing for albuminuric status shows that micro- and macro-albuminuric groups have a significantly higher HS GAG excretion rate than non-diabetic subjects. We conclude that, as in IDDM, excretion of GAG and HS GAG is higher in NIDDM and may precede the development of microalbuminuria.     

Assessment of glycosaminoglycan-protein linkage tetrasaccharides as acceptors for GalNAc- and GlcNAc-transferases from mouse mastocytoma

Two glycosaminoglycan-protein linkage tetrasaccharide-serine compounds, GlcAbeta1-3Galbeta1-3Galbeta1-4Xylbeta1-O-Ser and GlcAbeta1-3Gal(4-O-sulfate)beta1-3Galbeta1-4Xylbeta1-O -Ser, were tested as hexosamine acceptors, using UDP-[3H]GlcNAc and UDP-[3H]GalNAc as sugar donors, and solubilized mouse mastocytoma microsomes as enzyme source. The nonsulfated Ser-tetrasaccharide was found to function as an acceptor for a GalNAc residue, whereas the Ser-tetrasaccharide containing a sulfated galactose unit was inactive. Characterization of the radio-labelled product by digestion with alpha-N-acetylgalactosaminidase and beta-N-acetylhexosaminidase revealed that the [3H]GalNAc unit was alpha-linked, as in the product previously synthesized using serum enzymes, and not beta-linked as found in the chondroitin sulfate polymer. Heparan sulfate/heparin biosynthesis could not be primed by either of the two linkage Ser-tetrasaccharides, since no transfer of [3H]GlcNAc from UDP-[3H]GlcNAc could be detected. By contrast, transfer of a [3H]GlcNAc unit to a [GlcAbeta1-4GlcNAcalpha1-4]2-GlcAbeta1-4-aMan hexasaccharide acceptor used to assay the GlcNAc transferase involved in chain elongation, was readily detected. These results are in agreement with the recent proposal that two different N-acetylglucosaminyl transferases catalyse the biosynthesis of heparan sulfate. Although the mastocytoma system contains both the heparan sulfate/heparin and chondroitin sulfate biosynthetic enzymes the Ser-tetrasaccharides do not seem to fulfil the requirements to serve as acceptors for the first HexNAc transfer reactions involved in the formation of these polysaccharides.     

Boar spermadhesins AQN-1 and AQN-3: oligosaccharide and zona pellucida binding characteristics

AQN-1 and AQN-3 form part of the complement of surface-associated lectins which coat the plasma membrane overlying the acrosomal cap of in vitro capacitated boar spermatozoa. They belong to the spermadhesin protein family and have binding affinity for glycoconjugates of the zona pellucida, the extracellular investment surrounding mammalian eggs. The oligosaccharide and zona pellucida binding characteristics of spermadhesins AQN-1 and AQN-3 were investigated using a solid-phase assay and biotinylated glycoprotein ligands. Both sperm proteins bind glycoproteins containing Gal beta (1-4)-GlcNAc and Gal beta-(1-3)-GalNAc oligosaccharide sequences with dissociation constants (Kd) of 0.08 to 0.8 microM, and to zona pellucida glycoproteins with Kd = 0.15-0.25 microM. However, 5-N-acetyl neuraminic acid alpha (2-3/6)-linked to the galactose residue decreases the affinity of glycosylated ligands to AQN-1 three-fold, although it did not affect oligosaccharide binding to AQN-3. In addition, AQN-3 binds preferentially to glycoproteins with either a linear or tri- and tetraantennary carbohydrates than to those containing diantennary N-acetyllactosamine structures. The similar but distinct oligosaccharide recognition capabilities of spermadhesins AQN-1 and AQN-3 (this work) and AWN-1 (Dostálová, Z, Calvete, J.J., Sanz, L., and T?pfer-Petersen, E. (1995) Eur. J. Biochem. 230, 329-336) suggest that, in the pig, sperm-zona pellucida binding might be mediated by lectins displaying similar although distinct carbohydrate-recognition abilities.     

Biosynthesis of the escherichia coli K4 capsule polysaccharide. A parallel system for studies of glycosyltransferases in chondroitin formation

Escherichia coli K4 bacteria synthesize a capsule polysaccharide (GalNAc-GlcA(fructose))n with the carbohydrate backbone identical to chondroitin. GlcA- and GalNAc-transferase activities from the bacterial membrane were assayed with acceptors derived from the capsule polysaccharide and radiolabeled UDP-[14C]GlcA and UDP-[3H]GalNAc, respectively. It was shown that defructosylated oligosaccharides (chondroitin) could serve as substrates for both the GlcA- and the GalNAc-transferases. The radiolabeled products were completely degraded with chondroitinase AC; the [14C]GlcA unit could be removed by beta-D-glucuronidase, and the [3H]GalNAc could be removed by beta-N-acetylhexosaminidase. A fructosylated oligosaccharide acceptor tested for GlcA-transferase activity was found to be inactive. These results indicate that the chain elongation reaction of the K4 polysaccharide proceeds in the same way as the polymerization of the chondroitin chain, by the addition of the monosaccharide units one by one to the nonreducing end of the polymer. This makes the biosynthesis of the K4 polysaccharide an interesting parallel system for studies of chondroitin sulfate biosynthesis. In the biosynthesis of capsule polysaccharides from E. coli, a similar mechanism has earlier been demonstrated for polysialic acid (NeuNAc)n (Rohr, T. E., and Troy, F. A. (1980) J. Biol. Chem. 255, 2332-2342) and for the K5 polysaccharide (GlcAbeta1-4GlcNAcalpha1-4)n (Lidholt, K., Fjelstad, M., Jann, K., and Lindahl, U. (1994) Carbohydr. Res. 255, 87-101). In contrast, chain elongation of hyaluronan (GlcAbeta1-3GlcNAcbeta1-4)n is claimed to occur at the reducing end (Prehm, P. (1983) Biochem. J. 211, 181-189).     

Administration of a crystalloid fluid preload does not prevent the decrease in arterial blood pressure after induction of anaesthesia with propofol and fentanyl

Five oligosaccharide alpha1-phosphates and one sulfated glycopeptide have been isolated from the hemofiltrate of one patient with end-stage renal disease. Isolation of these compounds has been achieved using reverse osmosis, ion-exchange and size-exclusion chromatography and high performance liquid chromatography. The structures were predominantly elucidated by one- and two-dimensional 1H and 31P NMR spectroscopy. The chemical structures were determined to be: 1 NeuAc alpha2-3Gal alpha1-OPO3H2; 2 NeuAc alpha2-6Galbeta1-4GlcNAc alpha1-OPO3H2; 3 NeuAc alpha2-3Galbeta1-3GalNAc alpha1-OPO3H2; 4 NeuAc alpha2-3Galbeta1-3[NeuAc alpha2-6]GalNAc alpha1-OPO3H2 (proposed structure); 5 Fuc alpha1-2Galbeta1-4[Fuc alpha1-3]GlcNAc alpha1-OPO3H2; 6 HOSO3-4Fuc alpha1-6GlcNAcbeta1-NAsn. While 2 and 3 have been previously characterized as compounds of urine and hemofiltrate, the oligosaccharide alpha1-phosphates 1, 4, and 5 could be isolated--to our knowledge--for the first time from biological material. Compound 6 is the first glycopeptide reported to contain a 4-sulfated fucose residue.     

Neisseria gonorrhoeae that infect men have lipooligosaccharides with terminal N-acetyllactosamine repeats

Infectious Neisseria gonorrhoeae make relatively large lipooligosaccharides (LOS) that structurally resemble human glycosphingolipids. MS11mkC is an LOS variant of N. gonorrhoeae strain MS11 which was isolated from men at the onset of dysuria (Schneider, H., Griffiss, J. M., Boslego, J. W., Hitchcock, P. J., Zahos, K. M., and Apicella, M. A. (1991) J. Exp. Med. 174, 1601-1605). Delayed extraction matrix-assisted laser desorption and ionization and electrospray ionization mass spectrometry of O-deacylated MS11mkC LOS produced ions consistent with known LOS which have lacto-N-neotetraose (Galbeta1-->4GlcNAcbeta1-->3Galbeta1-->4Glc; paraglobosyl; monoclonal antibodies (mAbs) 1B2(+) and 06B4(+)) and GalNAc-->lacto-N-neotetraose (gangliosyl; mAb 1-1-M+) oligosaccharides. Ion peaks for a larger LOS which also bound mAb 1B2 indicated the addition of a hexose (+162 Da) to gangliosyl LOS or the addition of a hexose and a N-acetylhexosamine (+365 Da) to paraglobosyl LOS. Analysis of HF-treated and O-deacylated LOS revealed three major components present in a phosphoethanolamine (PEA)0 and a PEA1 series. Digestion of MS11mkC LOS by beta-N-acetylhexosaminidase and beta-galactosidase, alone and sequentially, combined with mAb binding patterns, confirmed the presence of a nonreducing terminal repeating LacNAc ((Galbeta1-->4GlcNAc)2) on the largest LOS, rather than a parallel oligosaccharide structure.     

Biosynthesis of branched polylactosaminoglycans. Embryonal carcinoma cells express midchain beta1,6-N-acetylglucosaminyltransferase activity that generates branches to preformed linear backbones

Two types of beta1,6-GlcNAc transferases (IGnT6) are involved in in vitro branching of polylactosamines: dIGnT6 (distally acting), transferring to the penultimate galactose residue in acceptors like GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-R, and cIGnT6 (centrally acting), transferring to the midchain galactoses in acceptors of the type (GlcNAcbeta1-3)Galbeta1-4GlcNAcbeta1-3Galbeta1-+ ++4GlcNAcbeta1-R. The roles of the two transferases in the biosynthesis of branched polylactosamine backbones have not been clearly elucidated. We report here that cIGnT6 activity is expressed in human (PA1) and murine (PC13) embryonal carcinoma (EC) cells, both of which contain branched polylactosamines in large amounts. In the presence of exogenous UDP-GlcNAc, lysates from both EC cells catalyzed the formation of the branched pentasaccharide Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-4 GlcNAc from the linear tetrasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc. The PA1 cell lysates were shown to also catalyze the formation of the branched heptasaccharides Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1-3(+ ++GlcNAcbeta1-6)Galbeta1 -4GlcNAc and Galbeta1-4GlcNAcbeta1-3(GlcNAcbeta1-6)Galbeta1-+ ++4GlcNAcbeta1-3Galbeta1 -4GlcNAc from the linear hexasaccharide Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAcbeta1- 3Galbeta1-4GlcNAc in reactions characteristic to cIGnT6. By contrast, dIGnT6 activity was not detected in the lysates of the two EC cells that were incubated with UDP-GlcNAc and the acceptor trisaccharide GlcNAcbeta1-3Galbeta1-4GlcNAc. Hence, it appears likely that cIGnT6, rather than dIGnT6 is responsible for the synthesis of the branched polylactosamine chains in these cells.     

The effects of high-dose methotrexate on the development of cartilage lesions in a lapine model of osteoarthrosis

To determine whether systemic administration of methotrexate (MTX) can prevent joint destruction in experimental osteoarthrosis (OA) in rabbits, the disorder was induced unilaterally in the knee joints of 40 rabbits by partial medial meniscectomy and sectioning of the medial collateral and both cruciate ligaments. A sham operation (arthrotomy only) was performed in another four animals. Effects on the cartilage of the femoral condyles were studied after 6 and 12 weeks. Twelve weeks after induction, femoral and tibial osteophyte formation was demonstrated on radiographs in all cases. Marked cartilage damage was found histologically (median Mankin score 10 vs 1 for non-operated controls; P < 0.05, Wilcoxon test). Cartilage proteoglycan (GAG) content (dye binding assay) was reduced in operated joints [63 +/- 8 (mean +/- SEM) vs 75 +/- 6 micrograms chondroitin sulfate/mg cartilage wet weight], and the leukocyte count in the joints was elevated (226 +/- 14 vs 7 +/- 3 leukocytes per microliter joint aspirate after injection of 0.5 ml saline solution; both P < 0.05, Wilcoxon test). The rate of GAG synthesis was unchanged (ex vivo labelling with 35S-sulfate). Treatment with MTX (30 mg x kg body weight-1 x week-1 i.m., starting 12 h postoperatively) reduced cartilage damage (median Mankin score 8 vs 10 for placebo, P < 0.05, Mann-Whitney U-test), but had no significant effect on the other parameters tested. No significant MTX effects were observed on cartilage from nonoperated joints. Our data indicate that MTX may have a limited therapeutic effect in experimental OA in the rabbit.     

Successful treatment of intraoperative myocardial ischemia with nicorandil

The relationship between sulfation and polymerization in chondroitin sulfate (CS) biosynthesis has been poorly understood. In this study, we investigated the specificity of bovine serum UDP-GalNAc: CS beta-GalNAc transferase responsible for chain elongation using structurally defined acceptor substrates. They consisted of tetra- and hexasaccharide-serines that were chemically synthesized and various regular oligosaccharides with a GlcA residue at the nonreducing terminus, prepared from chondroitin and CS using testicular hyaluronidase. The enzyme preparation was obtained from fetal bovine serum by means of heparin-Sepharose affinity chromatography. The preparation did not contain the alpha-GalNAc transferase recently demonstrated in fetal bovine serum (Kitagawa et al., J. Biol. Chem., 270, 22190-22195, 1995), that utilizes common acceptor substrates. The beta-GalNAc transferase used as acceptors, two hexasaccharide-serines GlcA beta 1-3GalNAc beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser and GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal (4-sulfate) beta 1-3Gal beta 1-4Xyl beta 1-O-Ser, but neither the monosulfated hexasaccharide-serine GlcA beta 1-3GalNAc(4-sulfate) beta 1-4GlcA beta 1-3Gal beta 1-3Gal beta 1-4Xyl beta 1-O-Ser nor tetrasaccharide-serines with or without a sulfate group at C-4 of the third sugar residue Gal-3 from the reducing end. The results indicated that the sulfate group at the Gal-3 C-4 markedly affected the transfer of GalNAc to the terminal GlcA. In addition, a sulfate group at C-4 of the reducing terminal GalNAc of regular tetrasaccharides remarkably enhanced the GalNAc transfer, suggesting that the enzyme recognizes up to the fourth saccharide residue from the nonreducing end. The level of incorporation into a tetra- or hexasaccharide containing a terminal 2-O-sulfated GlcA residue was significant, whereas there was no apparent incorporation into tetra- or hexasaccharides containing a terminal 3-O-sulfated GlcA or penultimate 4,6-O-disulfated GalNAc residue. These results indicated that sulfation reactions play important roles in chain elongation and termination.     

Synthesis of tri- and tetrasaccharide fragments of the Shigella dysenteriae type 1 O-antigen deoxygenated and fluorinated at position 3 of the methyl alpha-D-galactopyranoside terminus

The blockwise synthesis of methyl alpha tri- and tetrasaccharide analogs of the biochemical repeating unit of the Shigella dysenteriae type 1 O-polysaccharide is described. Modifications include deoxygenation and deoxyfluorination at position 3 of the galactopyranoside residue. Methyl 4,6-O-benzylidene-3-deoxy-alpha-D-xylo-hexopyranoside (8) and methyl 4,6-O-benzylidene-3-deoxy-3-fluoro-alpha-D-galactopyranoside (9) were condensed with (2,3,4-tri-O-benzoyl-alpha-L-rhamnopyranosyl)-(1-->3) -2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl chloride to give, after deprotection, the target trisaccharide methyl alpha-L-rhamnopyranosyl-(1-->3)-alpha-L- rhamnopyranosyl-(1-->2)-3-deoxy-alpha-D-xylo-hexopyranoside and the corresponding fluorinated oligosaccharide. For the tetrasaccharide synthesis, the glycosyl acceptors 8 and 9 were condensed with the temporarily protected (2,4-di-O-benzoyl-3-O-chloroacetyl-alpha-L- rhamnopyranosyl)-(1-->3)-2,4-di-O-benzoyl-alpha-L-rhamnopyranosyl chloride. Removal of the chloroacetyl group was followed by condensation of the resulting selectively deblocked trisaccharides with 3,4,6-tri-O-acetyl-2-azido-2-deoxy-alpha-D-glucopyranosyl chloride. Reduction and deprotection then gave the free methyl 2-acetamido-2-deoxy- alpha-D-glucopyranosyl-(1-->3)-alpha-L-rhamnopyranosyl- (1-->3)-alpha-L-rhamnopyranosyl-(1-->2)-3-deoxy-alpha-D-xylo-hexopyra noside and the fluorinated analog.     

Glycosaminoglycan binding properties of annexin IV, V, and VI

We have previously demonstrated that annexin IV, one of the calcium/phospholipid-binding annexin family proteins, binds to glycosaminoglycans (GAGs) in a calcium-dependent manner (Kojima, K., Yamamoto, K., Irimura, T., Osawa, T., Ogawa, H., and Matsumoto, I. (1996) J. Biol. Chem. 271, 7679-7685). In this study, we investigated the GAG binding specificities of annexins IV, V, and VI by affinity chromatography and solid phase assays. Annexin IV was found to bind in a calcium-dependent manner to all the GAG columns tested. Annexin V bound to heparin and heparan sulfate columns but not to chondroitin sulfate columns. Annexin VI was adsorbed to heparin and heparan sulfate columns in a calcium-independent manner, and to chondroitin sulfate columns in a calcium-dependent manner. An N-terminal half fragment (A6NH) and a C-terminal half fragment (A6CH) of annexin VI, each containing four units, were prepared by digestion with V8 protease and examined for GAG binding activities. A6NH bound to heparin in the presence of calcium but not to chondroitin sulfate C, whereas A6CH bound to heparin calcium-independently and to chondroitin sulfate C calcium-dependently. The results showed that annexin IV, V, and VI have different GAG binding properties. Some annexins have been reported to be detected not only in the cytoplasm but also on the cell surface or in extracellular components. The findings suggest that the some annexins function as recognition elements for GAGs in extracellular space.     

Cyclooxygenase-1 and -2 of endothelial cells utilize exogenous or endogenous arachidonic acid for transcellular production of thromboxane

The presence of prostaglandin (PG) H2 in the supernatant of human umbilical vein endothelial cells (HUVEC) stimulated by thrombin restores the capacity of aspirin-treated platelets to generate thromboxane (TX) B2. Induction of cyclooxygenase-2 (Cox-2) by interleukin (IL)-1alpha or a phorbol ester increases this formation. HUVEC treated with aspirin lost their capacity to generate PGs but recovery occurred after 3- or 6-h induction of Cox-2 with phorbol ester or IL-1alpha. Enzyme activity of the newly synthesized Cox-2 in aspirin-treated cells, evaluated after immunoprecipitation, was similar to untreated cells but after 18 h of cell stimulation only 50-60% recovery of Cox-1 was observed. The use of SC58125, a selective Cox-2 inhibitor, confirmed these findings in intact cells. Cyclooxygenase activity was related to the amount of Cox proteins present in the cells, but after induction of Cox-2, contribution of the latter to PG production was 6-8-fold that of Cox-1. Aspirin-treated or untreated cells were incubated in the absence or presence of SC58125 and stimulated by thrombin, the ionophore A23187, or exogenous arachidonic acid. The production of endogenous (6-keto-PGF1alpha, PGE2, PGF2alpha) versus transcellular (TXB2) metabolites was independent of the inducer, the source of arachidonic acid and the Cox isozyme. However, in acetylsalicylic acid-treated cells, after 6-h stimulation with IL-1alpha, newly synthesized Cox-2 produced less TXB2 than 6-keto-PGF1alpha compared to untreated cells. At later times (>18 h), there was no metabolic difference between the cells. These studies suggest that in HUVEC, Cox compartmentalization occurring after short-term activation may selectively affect transcellular metabolism, but not constitutive production, of PGs.     

A neurite outgrowth-inhibitory proteoglycan expressed during development is similar to that isolated from adult brain after isomorphic injury

The expression of proteoglycans (PGs) in the mammalian central nervous system (CNS) appears to be strictly regulated both during development and after damage to the mammalian CNS. Recently, we have isolated from membranes of injured adult brain a neurite outgrowth-inhibitory proteoglycan (IMP), the activity of which could be specifically counteracted by a monoclonal antibody (mAB) against the PG. We described in this report the characterization of perinatal membrane proteoglycan (PMP), a heparan-sulfate/chondroitin-sulfate-containing PG expressed during brain development. Its maximal expression was observed around postnatal day 3, decreasing strongly in normal adult tissue. This PG was purified and characterized using mABs generated against IMP. The comparison of PMP and IMP properties indicates that the two PGs are highly related and share expression patterns, biochemical characteristics, and the ability to inhibit neurite initiation in culture. However, IMP and PMP displayed a distinct effect on neurite elongation, which may be explained by their differences in glycosilation pattern. The data presented in this report support the idea that proteoglycans expressed during CNS development are re-expressed following injury.     

Molecular cloning and characterization of an N-acetylglucosamine-6-O-sulfotransferase

We isolated a cDNA clone encoding mouse N-acetylglucosamine-6-O-sulfotransferase based on sequence homology to the previously cloned mouse chondroitin 6-sulfotransferase. The cDNA clone contained an open reading frame that predicts a type II transmembrane protein composed of 483 amino acid residues. The expressed enzyme transferred sulfate to the 6 position of nonreducing GlcNAc in GlcNAcbeta1-3Galbeta1-4GlcNAc. Galbeta1-4GlcNAcbeta1-3Galbeta1-4GlcNAc and various glycosaminoglycans did not serve as acceptors. Expression of the cDNA in COS-7 cells resulted in production of a cell-surface antigen, the epitope of which was NeuAcalpha2-3Galbeta1-4(SO4-6)GlcNAc; double transfection with fucosyltransferase IV yielded Galbeta1-4(Fucalpha1-3)(SO4-6)GlcNAc antigen. The sulfotransferase mRNA was strongly expressed in the cerebrum, cerebellum, eye, pancreas, and lung of adult mice. In situ hybridization revealed that the mRNA was localized in high endothelial venules of mesenteric lymph nodes. The sulfotransferase was concluded to be involved in biosynthesis of glycoconjugates bearing the 6-sulfo N-acetyllactosamine structure such as 6-sulfo sialyl Lewis X. The products of the sulfotransferase probably include glycoconjugates with intercellular recognition signals; one candidate of such a glycoconjugate is an L-selectin ligand.