Determination of carbohydrate structures N-linked to soluble CD154 and characterization of the interactions of CD40 with CD154 expressed in Pichia pastoris and Chinese hamster ovary cells.

CD40-CD154 (CD40 ligand) interactions are essential for the development of protective immunity. Previous studies have described the CD40 binding site as a shallow groove formed between two monomers of CD154. However, these studies have not examined the structure or biological function of the carbohydrate on CD154. Human CD154 contains a single N-linked glycosylation site at asparagine 240. We have characterized the interactions between CD40 and soluble (s) CD154 in which sCD154 contains different types of carbohydrates. Detailed carbohydrate analysis revealed high-mannose structures on sCD154 purified from Pichia pastoris, whereas CD154 purified from Chinese hamster ovary E1A contained heterogeneous populations of complex carbohydrates. sCD154 purified from either system was trimeric, it bound to CD40 with similar affinities of 10-30 nM, and it functionally induced CD69 and CD95 expression on primary B cells. Together, these results indicate that the presence of varied types of N-linked glycans on asparagine 240 of CD154 does not play a significant role in the CD40-CD154 interactions.

A comparative study of the asparagine-linked oligosaccharides on siglec-5, siglec-7 and siglec-8, expressed in a CHO cell line, and their contribution to ligand recognition.

The siglecs (sialic acid-binding immunoglobulin-like lectins) mediate sialic acid-dependent cellular interactions and may in some cases signal through SH2-binding domains. In addition to the previously characterized siglecs, sialoadhesin, CD22, CD33 and myelin-associated glycoprotein, several new ones, siglec-5, siglec-7 and siglec-8, have recently been cloned. Although these novel receptors have generated considerable interest as therapeutic targets because of their expression pattern on immune cells, very little is known about how their lectin activity is regulated. Previous studies with sialoadhesin, CD22 and CD33 have shown that siglec glycosylation has significant effects on binding. To determine any differences in the glycan composition of siglec-5, siglec-7 and siglec-8 that may modify their function, we released and characterized the N-linked oligosaccharide distribution in these three glycoproteins. The glycan pools from siglec-5 and siglec-7 contained a larger proportion of sialylated and core-fucosylated biantennary, triantennary and tetra-antennary oligosaccharides, whereas the carbohydrate mixture released from siglec-8 is noticeably less sialylated and is more abundant in 'high-mannose'-type glycans. In addition, we show that, in contrast with CD22 and CD33, mutating the conserved potentially N-linked glycosylation site in the first domain has no effect on binding mediated by siglec-5 or siglec-7.

High resolution of oligosaccharide mixtures by ultrahigh voltage micellar electrokinetic capillary chromatography.

Oligosaccharide mixtures released from ribonuclease B and human IgG have been separated using micellar electrokinetic capillary chromatography operated at 100 kV. The resolution of these closely related analytes at this high voltage was found to be superior to that obtained at 20 kV, a voltage which is ordinarily used in most capillary electrophoresis separations.

Early indication of effects of puromycin aminonucleoside using a fluorimetric assay of 2-aminoacridone-derivatized carbohydrates in urine.

This paper describes a novel noninvasive method to study the changes in free carbohydrates excreted in urine as a result of toxicity in the rat induced by the administration of puromycin aminonucleoside (PAN). Urine samples were collected for 24 h prior to dosing and at 8, 24, and 32 h postdosing. For each sample, free carbohydrates were extracted from the urine using a graphitized carbon column and then labeled with 2-aminoacridone (2-AMAC) prior to analysis by hydrophilic interaction liquid chromatography (HILC). Dramatic changes were seen in the profile of the carbohydrates at the 8- and 24-h time points. These changes in carbohydrate profiles may be useful as early indicators of toxicity.

A probe for the versatile analysis and characterization of N-linked oligosaccharides.

A novel fluorescent probe, 3-(acetylamino)-6-aminoacridine (AA-Ac), has been synthesized and its applicability to the analysis of picomole levels of N-linked glycans investigated. AA-Ac was found to be an excellent derivatization reagent for N-linked glycans, giving at least twice the intensity of fluorescence as its predecessor 2-aminoacridone. AA-Ac-labeled glycans were analyzed by both normal and reversed-phase HPLC. They were also amenable to enzymatic sequencing and analysis by MALDI-TOF mass spectrometry, free zone capillary electrophoresis, and capillary electrophoresis/electrospray ionization mass spectrometry.

Analysis of oligosaccharides by microbore high-performance liquid chromatography.

A 1-mm microbore hydrophilic interaction column has been used for the separation of 2-aminoacridone (2-AMAC)-derivatized glycan mixtures, released from naturally occurring and recombinant proteins. Primary structure identification of the 2-AMAC glycan derivatives was carried out by HPLC using fluorescence and mass spectrometric detection. In some cases, enzymatic digestion of the 2-AMAC glycans was applied to confirm glycan structure. This strategy is considerably more rapid than methods normally used in glycan analysis, which involves manual collection of separated oligosaccharide derivatives and analysis of individual fractions by mass spectrometry.

An integrated proteomic approach to studying glomerular nephrotoxicity.

A single dose of puromycin aminonucleoside (PAN) given parenterally to rats induces ultrastructural glomerular changes and a nephrotic syndrome similar in many respects to human minimal change nephropathy. The exact aetiologies of both the human and the experimental syndromes are unknown, and are probably multifactorial. However, among the observed consequences in humans and rats is increased plasma protein excretion in urine, beginning in the latter typically 3-6 days after PAN administration. In view of this, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) has been used to profile urinary proteins during PAN-induced nephrotoxicity and subsequent recovery in the rat. In addition, urinary high performance liquid chromatography (HPLC) profiles and high resolution proton nuclear magnetic resonance (NMR) spectroscopy has been utilised to simultaneously detect toxin-induced changes in the relative concentrations of a number of metabolites. The proteomic approach, in conjunction with these other techniques, has the potential to provide significantly more mechanistic information than is provided readily by traditional clinical chemistry.

Carbohydrate release from picomole quantities of glycoprotein and characterisation of glycans by high-performance liquid chromatography and mass spectrometry.

Samples of 5 to 20 microg of human IgG were subjected to dithiothreitol treatment to reduce disulphide bridges, followed by tryptic digestion. Glycans released from the tryptic peptide mixture by PNGase F digestion were then derivatised with 2-aminoacridone. Labelled oligosaccharides were separated by normal-phase high-performance liquid chromatography and individual components were collected for matrix-assisted laser desorption ionization time-of-flight and electrospray mass spectrometric analysis.

Non-thiazolidinedione antihyperglycaemic agents. Part 3: The effects of stereochemistry on the potency of alpha-methoxy-beta-phenylpropanoic acids.

Rhizopus delemar lipase catalysed ester hydrolysis of the alpha-methoxy-beta-phenylpropanoate 1 affords the (R)-(+) and (S)-(-) isomers in > 84% enantiomeric excess. Absolute stereochemistry was determined by a single crystal X-ray analysis of a related synthetic analogue. The activity of these two enantiomers on glucose transport in vitro and as anti-diabetic agents in vivo is reported and their unexpected equivalence attributed to an enzyme-mediated stereospecific isomerisation of the (R)-(+) isomer. Binding studies using recombinant human PPARgamma (peroxisomal proliferator activated receptor gamma), now established as a molecular target for this compound class, indicate a 20-fold higher binding affinity for the (S) antipode relative to the (R) antipode.

A chromatographic and mass spectrometric strategy for the analysis of oligosaccharides: determination of the glycan structures in porcine thyroglobulin.

Oligosaccharides released from porcine thyroglobulin were first derivatised with 2-aminoacridone (2-AMAC) and analysed by capillary electrophoresis to determine the complexity of this glycan pool. The same glycan mixture was then subjected to either a sialidase digest or a sialidase and fucosidase digest prior to derivatisation with 2-AMAC and analysis by normal phase high pressure liquid chromatography (HPLC). Comparison of the three chromatographic profiles with known standards allowed an initial identification of the glycan structures. The 2-AMAC derivatised glycans were then collected from HPLC for matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) analysis and the molecular weights of predicted structures were confirmed. This study demonstrates that a two enzyme array and subsequent MALDI-TOF analysis can be used successfully to assign the major glycans present in a complex mixture.

Profiling of 2-aminoacridone derivatised glycans by electrospray ionization mass spectrometry.

Three different types of N-glycans, high-mannose, complex and hybrid, have been derivatised with 2-aminoacridone and analysed by electrospray mass spectrometry and tandem mass spectrometry (MS/MS) using a hybrid quadrupole orthogonal acceleration time-of-flight mass spectrometer, fitted with a nanoflow electrospray ion source. Relatively simple MS/MS fragmentation patterns have been observed for each type of glycan, allowing rapid elucidation of the order in which the monosaccharide residues making up these glycans are linked to one another.

A dual-detection strategy in the chromatographic analysis of 2-aminoacridone-derivatized oligosaccharides.

A protocol has been developed involving the derivatization of glycan mixtures with 2-aminoacridone and co-injection with a dextran ladder derivatized with methyl 4-aminobenzoate (M-4AB). These two derivatizing agents have very different ultraviolet absorbance and fluorescence characteristics. A chromatographic separation using a normal-phase column support followed by in-series UV and fluorescence detection allowed simultaneous analysis of the two mixtures of the separately derivatized carbohydrates without any interference. This new approach uses the M-4AB dextran ladder derivatives as internal standards spanning the whole chromatogram, allowing an accurate and detailed comparison of glycosylation profiles. It also saves much time by avoiding the necessity of "sandwiching" an unknown glycan mixture between two chromatographic runs of a dextran ladder. The use of this technique has been demonstrated in the case of glycans released from ribonuclease B and human IgG.

Two-dimensional chromatography in the analysis of complex glycans from transferrin.

Oligosaccharides released from human transferrin have been derivatized with 2-aminoacridone (2-AMAC) prior to analysis by either reversed- or normal-phase high-performance liquid chromatography. These separation methods are complementary and allow distinction between isomeric mono- and disialylated oligosaccharides, which terminate with Sia alpha 2-6Gal or Sia alpha 2-3Gal at the nonreducing end. Collected fractions of 2-AMAC-derivatized glycans were analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, before and after desialylation.

Direct structural analysis of 2-aminoacridone derivatized oligosaccharides by high-performance liquid chromatography/mass spectrometric detection.

In this preliminary study linear and branched oligosaccharides, derivatized with 2-aminoacridone, have been separated by reverse-phase high-performance liquid chromatography, and detected by positive-ion electrospray mass spectrometry. Tandem Mass spectra of the protonated carbohydrate analytes gave simple fragmentation patterns from which sequence information could be readily obtained.

Randomised double blind controlled study of recurrence of gastric ulcer after treatment for eradication of Helicobacter pylori infection.

OBJECTIVE: To determine whether eradication of Helicobacter pylori infection reduces recurrence of benign gastric ulceration. DESIGN: Randomised, double blind, controlled study. Patients were randomised in a 1:2 ratio to either omeprazole 40 mg once daily for eight weeks or the same treatment plus amoxycillin 750 mg twice daily for weeks 7 and 8. A 12 month untreated follow up ensued. SETTING: Teaching and district general hospitals between 1991 and 1994. SUBJECTS: 107 patients with benign gastric ulcer associated with H pylori. MAIN OUTCOME MEASURES: Endoscopically confirmed relapse with gastric ulcer (analysed with life table methods), H pylori eradication, and healing of gastric ulcers (Mantel-Haenszel test). RESULTS: 172 patients were enrolled. Malignancy was diagnosed in 19; 24 were not infected with H pylori; four withdrew because of adverse events; and 18 failed to attend for start of treatment, leaving 107 patients eligible for analysis (35 omeprazole alone; 72 omeprazole plus amoxycillin). In the omeprazole/amoxycillin group 93% (67/72; 95% confidence interval 84% to 98%) of gastric ulcers healed and 83% (29/35; 66% to 94%) in the omeprazole group (P = 0.103). Eradication of H pylori was 58% (42/72; 46% to 70%) and 6% (2/35; 1% to 19%) (P < 0.001) and relapse after treatment was 22% (16/72) and 49% (17/35) (life table analysis, P < 0.001), in the two groups, respectively. The recurrence rates were 7% (3/44) after successful H pylori eradication and 48% (30/63) in those who continued to be infected (P < 0.001). CONCLUSIONS: Eradication of H pylori reduces relapse with gastric ulcer over one year. Eradication rates achieved with this regimen, however, are too low for it to be recommended for routine use.

Minimizing cationization effects in the analysis of complex mixtures of oligosaccharides.

We outline simple methodology for the rapid and selective analysis of 2-aminoacridone (2-AMAC) derivatised oligosaccharides by matrix-assisted laser desorption/ionization mass spectrometry. This involves the addition of small amounts of lithium chloride to the matrix before evaporation of solvent and crystallization. Signals mainly attributed to proton, sodium and potassium adducts are suppressed to a great extent, and a single signal due to (M + Li)+ is observed. This technique is rapid and is most useful for the direct analysis of complex glycan mixtures, after derivatization with 2-aminoacridone and without separation of the individual components.

Comparison of the effect of two dose schedules of oral omeprazole with oral ranitidine on gastric aspirate pH and volume in patients undergoing elective surgery.

We have compared gastric aspirate pH and volume at induction of anaesthesia in 222 patients who had received either omeprazole or ranitidine before elective operations. Omeprazole was given orally either as 40 mg on the evening before and 40 mg on the morning of surgery or as 80 mg on the morning of surgery. Ranitidine 150 mg was given orally on the evening before surgery and 2 h before anaesthesia. Treatment success was defined as aspirate pH > or = 2.5 and volume < 25 ml at induction of anaesthesia. Treatment was successful in 84% (95% confidence interval (CI) 73-91%) of patients in the omeprazole 40 + 40 mg group, 84% (95% CI 73-91%) in the ranitidine group and 73% (95% CI 61-83%) in the omeprazole 80 mg group. There were no statistically significant differences between the groups. Twelve patients in the omeprazole 80 mg group had gastric pH < 2.5 and four had volume > 25 ml. Only three patients had a gastric pH < 2.5 in the omeprazole 40 + 40 mg group and none had volume > 25 ml, which compared well with the ranitidine group. Omeprazole, given as 40 mg in the evening and 40 mg on the morning of operation, has a potential role for use in patients at risk for aspiration during general anaesthesia.

The effect of phytic acid on the resolution of peptides and proteins in capillary electrophoresis.

Studies are reported on the effect of the sodium salt of phytic acid on the resolution of peptides and proteins. Improved separation in the case of peptides is shown to be due to ion-ion pairing interactions between the positively charged peptides and the phytic acid polyanionic species. The improved peak shapes related to the proteins can be interpreted in terms of the sample preconcentration due to injection of analytes from a water medium to one of high ionic strength.