Arthur A. Grey

Effect of sodium tanshinone IIA sulfonate in the rabbit myocardium and on human cardiomyocytes and vascular endothelial cells

Sodium tanshinone IIA sulfonate (STS) is a derivative of tanshinone IIA. The latter is a pharmacologically active component isolated from the rhizome of the Chinese herb Salvia miltiorrhiza. Liquid chromatographically pure STS was found to reduce myocardial infarct size by 53.14 +/- 22.79% relative to that in the saline control in a rabbit 1 hr-ischemia and 3 hr-reperfusion model. This effect was comparable to that of Trolox (a better characterized antioxidant serving as a reference cytoprotector), which salvaged the myocardium in the same infarct model by 62.13 +/- 18.91%. Also, like Trolox, STS did not inhibit oxygen uptake by xanthine oxidase (XO), a key enzyme in free radical generation. However, in contrast to Trolox, STS significantly prolonged the survival of cultured human saphenous vein endothelial cells but not human ventricular myocytes in vitro when these cells were separately exposed to XO-generated oxyradicals. Note that the endothelium is recognized to be a key site of oxidant generation and attack. Our findings in vitro and in vivo support the interpretation that STS is a cardioprotective substance, and that it may exert a beneficial effect on the clinically important vascular endothelium.

Molecular properties and myocardial salvage effects of morin hydrate

Morin hydrate is a bioactive pigment found in yellow Brazil wood. Recently, we reported that morin hydrate prolongs the survival of three types of cells from the human circulatory system against oxyradicals generated in vitro. The protection excels that given by equimolar concentrations of ascorbate, mannitol, and Trolox. Here, we demonstrate that, in vivo, morin hydrate at 5 mumol/kg actually reduced by > 50% the tissue necrosis in post-ischemic and reperfused rabbit hearts. Mechanistically, morin hydrate not only scavenges oxyradicals, but also moderately inhibits xanthine oxidase, a free-radical generating enzyme from the ischemic endothelium. Among other possibilities, morin hydrate appears to chelate some metal ions (e.g. Fe2+) in oxyradical formation, although this needs to be examined further. Nuclear magnetic resonance (at 500 mHz) and electron-impact mass spectrometry also supported a molecular formula of C15H10O7 for morin hydrate. Only by X-ray crystallography was it clearly revealed that there are two water molecules attached by intermolecular hydrogen bonds to a morin molecule. Also, the three rings of morin hydrate approach coplanarity. This conformation favours a delocalization of electrons after oxyradical reduction, making morin an effective antioxidant. Thus, we have documented some of the molecular properties and myocardial salvage effects of morin hydrate.

Control of glycoprotein synthesis. Characterization of (1 → 4)-N-acetyl-β-d-glucosaminyltransferases acting on the α-d-(1 → 3)- and α-d-(1 → 6)-linked arms of N-linked oligosaccharides

Abstract Hen oviduct membranes contain at least three N -acetyl-β- d -glucosaminyltransferases (GlcNAc-T) that attach a βGlcNAc residue in (1-4)-linkage to a d -Man p residue of the N -linked oligosaccharide core, i.e., (1 → 4)-β- d -GlcNAc-T III which adds a “bisecting” GlcNAc group to form the β- d -Glc p NAc-(1 → 4)-β- d -Man p -(1 → 4)- d -GlcNAc moiety; (1 → 2)-β- d -GlcNAc-T IV which adds a GlcNAc group to the (1 → 3)-α- d -Man arm to form the β- d -Glc p NAc-(1 → 4)-[β- d Glc p NAc-(1 → 2)]-α- d -Man p -(1 → 3)-β- d -Man p -(1 → 4)- d -Glc p NAc component; and (1 → 4)-β- d -GlcNAc-T VI which adds a GlcNAc group to the α- d -Man p residue of β- d -Glc p NAc-(1 → 6)-[β- d -Glc p NAc-(1 → 2)]-α- d -Man p -R to form β- d -Glc p NAc-(1 → 6)-[β- d -Glc p NAc-(1 → 4)]-[β- d -Glc p NAc-(1 → 2)]-α- d -Man p -R. We now report a novel (1 → 4)-(β- d -GlcNAc-T activity (GlcNAc-T VI′) in hen oviduct membranes that transfers GlcNAc to β- d -Glc p NAc-(1 → 2)-α- d -Man p -(1 → 6)-β- d -Man p -R to form β- d -Glc p NAc-(1 → 4)-[β- d -Glc p NAc-(1 → 2)]-α- d -Man p -(1 → 6)-β- d -Man p -R. The structure of the enzyme product was confirmed by 1 H NMR spectroscopy, FAB-mass spectrometry and methylation analysis. Previous work with GlcNAc-T IV was carried out with biantennary substrates; we now show that hen oviduct membrane GlcNAc-T IV can also transfer GlcNAc to monoantennary β- d -Glc p NAc-(1 → 2)-α- d -Man p -(1 → 3)-β- d -Man p -R to form β- d -Glc p NAc-(1 → 4)-[β- d -Glc p NAc-(1 → 2)]-α- d -Man p -(1 → 3)-β- d -Man p -R. The findings that GlcNAc-T VI′ and IV have similar kinetic characteristics and that hen oviduct membranes can convert methyl β- d -Glc p NAc-(1 → 2)-α- d -Man p to methyl β- d -Glc p NAc-(1 → 4)-[β- d -Glc p NAc-(1 → 2)]-α- d -Man p suggest that these two activities may be due to the same enzyme. The R-group of the β- d -Glc p NAc-(1 → 2)-α- d -Man p -(1 → 6)-β- d -Man p (or Glc p )-R substrate has an important influence on GlcNAc-T VI′ enzyme activity. When R is GlcNAc or βGlc-allyl, the activity is drastically reduced. This may be due to conformational factors and may explain why hen oviduct membranes add a GlcNAc residue in (1 → 4)-β-linkage mainly to the (1 → 3)-α- d -Man arm of the bi-antennary substrate β- d -Glc p NAc-(1 → 2)-α- d -Man p -(1 → 6)-[β- d -Glc p NAc-(1 → 2)-α- d -Man p -(1 → 3)]-β- d -Man p -R to form β- d -Glc p NAc-(1 → 2)-α- d -Man p -(1 → 6)-{β- d -Glc p NAc-(1 → 2)-[β- d -Glc p NAc-(1 → 4)]-α- d -Man p -(1 → 3)}-β- d -Man p -R.

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)2(GlcNAc)2 (Fuc)1 to (GlcNAc)6(Man)3(GlcNAc)2 were obtained from human α1-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.

Synthesis of pentasaccharide analogues of the N-glycan substrates of N-acetylglucosaminyltransferases III, IV and V using tetrasaccharide precursors and recombinant β-(1 → 2)-N-acetylglucosaminyltransferase II

Recombinant human UDP-GlcNAc: alpha-Man-(1-->6)R beta-(1-->2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143, GlcNAc-T II) was produced in the Sf9 insect cell/baculovirus expression system as a fusion protein with a (His)6 tag and partially purified by affinity chromatography on a metal chelating column. The partially purified enzyme was used to catalyze the transfer of GlcNAc from UDP-GlcNAc to R-alpha-Man(1-->6)(beta-GlcNAc(1-->2)alpha-Man(1-->3))beta-Man-O-octyl to form beta-GlcNAc(1-->2)R-alpha-Man(1-->6)(beta-GlcNAc(1-->2)alpha- Man(1-->3))beta-Man-O-octyl where there is either no modification of the alpha-Man(1-->6) residue (7), or where R is 3-deoxy (8), 4-deoxy (9) or 6-deoxy (10). The yields ranged from 64-80%. Products were characterized by 1H and 13C nuclear magnetic resonance spectroscopy and fast atom bombardment mass spectrometry. Compounds 7-10 are pentasaccharide analogues of the biantennary N-glycan substrates of N-acetylglucosaminyltransferases III, IV and V.

Purification, characterization, and structural elucidation of the active moiety of the previously called "suppressor activating factor (SAF)".

Upon extensive purification of the serum-free supernatant produced by a mutant T cell line (6T-CEM), an immunosuppressive activity was found to reside in an oxidized product of spermine, spermine dialdehyde (SDA). The activity was purified to homogeneity from a serum-free supernatant by using gel filtration chromatography and reverse-phase C18 HPLC. Fast Atom Bombardment (FAB) mass spectral analysis revealed its MW to be 202 and Electron Impact (EI) analysis of the acetylated material identified the purified molecule to be spermine. In the presence of human or rodent plasma, spermine exhibited no immunosuppressive activity up to 2 mg/ml. However, when assayed in the presence of FCS, which contains polyamine oxidase (PAO), spermine is oxidized to its corresponding dialdehyde which is active at 0.1 microM/ml. We have previously described a high molecular weight suppressor activating factor (SAF) found in the serum-containing supernatant of the 6T-CEM cell line. Our preliminary biological data suggest that SDA is probably responsible for the immunosuppressive activities previously observed for the SAF. The strong affinity of SDA for proteins and thiocompounds may account for the apparent high MW previously reported for SAF.

Synthesis of Trideuteriomethyl 5-Deuterium-β-D-Mannopyranoside

Abstract The title compound was prepared by first converting trideuteriomethyl 2,3,4-tri-O-benzyl-β-D-mannopyranoside to a 6-bromo-6-deoxy derivative which on elimination by using DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) or DBN (1,5-diazabi-cyclo[4.3.0]non-5-ene) gave a hex-5-enopyranoside derivative. The deuteroboration of the hex-5-enopyranoside followed by oxidation and subsequent deblocking produced trideuteriomethyl 5-deuterium-β-D-mannopyranoside.