Exchange High Pressure Liquid Chromatography Research Articles

A 6-O-endosulfatase activity assay based on synthetic heparan sulfate oligomers.

Sulf-2 is an extracellular heparan 6-O-endosulfatase involved in the postsynthetic editing of heparan sulfate (HS), which regulates many important biological processes. The activity of the Sulf-2 and its substrate specificity remain insufficiently characterized in spite of more than two decades of studies of this enzyme. This is due, in part, to the difficulties in the production and isolation of this highly modified protein and due to the lack of well-characterized synthetic substrates for the probing of its catalytic activity. We introduce synthetic HS oligosaccharides to fill this gap, and we use our recombinant Sulf-2 protein to show that a paranitrophenol (pNP)-labeled synthetic oligosaccharide allows a reliable quantification of its enzymatic activity. The substrate and products of the desulfation reaction are separated by ion exchange high-pressure liquid chromatography and quantified by UV absorbance. This simple assay allows the detection of the Sulf-2 activity at high sensitivity (nanograms of the enzyme) and specificity. The method also allowed us to measure the heparan 6-O-endosulfatase activity in biological samples as complex as the secretome of cancer cell lines. Our in vitro measurements show that the N-glycosylation of the Sulf-2 enzyme affects the activity of the enzyme and that phosphate ions substantially decrease the Sulf-2 enzymatic activity. This assay offers an efficient, sensitive, and specific measurement of the heparan 6-O-endosulfatase activity that could open avenues to in vivo activity measurements and improve our understanding of the enzymatic editing of the sulfation of heparan.

Quantitation of Inositol Phosphates by HPLC-ESI-MS.

The coupling of anion exchange high-pressure liquid chromatography (HPLC) with electrospray ionization mass spectrometry (ESI-MS) allows for the simultaneous detection of the six forms of inositol phosphate (InsP). Here we describe a rapid quantitative analysis of InsPs by HPLC-ESI-MS, which can be applied to a wide array of sample types. With this method, InsPs could be separated and detected within 20min of sample injection. The detection limit was as low as 25pmol (i.e., ca. 2nmol/g sample) for each type of InsP, which is particularly important for analytes that are often present at low abundance in nature.

Measurement of HbA1c in Gingival Crevicular Blood Using a High-Pressure Liquid Chromatography Procedure.

To validate an ion exchange high-pressure liquid chromatography (HPLC) method for measuring glycated hemoglobin (HbA1c) in gingival crevicular blood (GCB) spotted on filter paper, for use in screening dental patients for diabetes. We collected the GCB specimens for this study from the oral cavities of patients during dental visits, using rigorous strategies to obtain GCB that was as free of debris as possible. The analytical performance of the HPLC method was determined by measuring the precision, linearity, carryover, stability of HbA1c in GCB, and correlation of HbA1c results in GCB specimens with finger-stick blood (FSB) specimens spotted on filter paper. The coefficients of variation (CVs) for the inter- and intrarun precision of the method were less than 2.0%. Linearity ranged between 4.2% and 12.4%; carryover was less than 2.0%, and the stability of the specimen was 6 days at 4°C and as many as 14 days at -70°C. Linear regression analysis comparing the HbA1c results in GCB with FSB yielded a correlation coefficient of 0.993, a slope of 0.981, and an intercept of 0.13. The Bland-Altman plot showed no difference in the HbA1c results from the GCB and FSB specimens at normal, prediabetes, and diabetes HbA1c levels. We validated an HPLC method for measuring HbA1c in GCB; this method can be used to screen dental patients for diabetes.

Hb H Interference on Measurement Of HbA1c With Ion-Exchange HPLC

In this article, an interference caused by hemoglobin H (Hb H), during the measurement of hemoglobin A1c (HbA1c) with ion exchange high pressure liquid chromatography (HPLC) method, was presented in blood sample of a 20-year-old male patient. HbA1c measurement was performed with Agilent 1200 HPLC system using a commercial Recipe HbA1c ion-exchange column. Hemoglobin electrophoresis was performed with Interlab G26 agarose electrophoresis automated compact system. HbA1c level was 18.2% and HbA0 level was 81.5% with ion-exchange HPLC method. Patient’s fasting serum glucose was assessed before HbA1c measurement and the result was 165 mg/dL (9.16 mmol/L). On the other hand, the result of HbA0 was 87.9%, Hb H was 10.8% and Hb A2 was 1.3% with electrophoresis. Whole blood test values were within reference ranges except MCV. MCV value was 79.6 fL. It is important to keep in mind that HbA1c level might be considered falsely high with ion-exchange HPLC method because of Hb H containing sample.

Importance des caractéristiques de la séparation chromatographique en CLHP pour l'interprétation du dosage de l'HbA1c en présence d'un variant d'hémoglobine

The methods of HbA1c assay using ion exchange high pressure liquid chromatography (HPLC) allow the detection of the most common hemoglobin variants. This observation highlights the different behaviour of two HPLC analyzers in the presence of Tatras hemoglobin. By one of the analyzer (Variant II, Bio-Rad) this variant is detected, but not by the other (G8, Tosoh Biosciences). As HbA1c result is crucial for the therapeutic decision, it is important that biologists know the characteristics of the method they use, in order to detect the possible occurence of an hemoglobinopathy and to ensure the best interpretation of the result.

High-resolution preparative separation of glycosaminoglycan oligosaccharides by polyacrylamide gel electrophoresis

Separation of milligram amounts of heparin oligosaccharides ranging in degree of polymerization from 4 to 32 is achieved within 6h using continuous elution polyacrylamide gel electrophoresis (CE–PAGE) on commercially available equipment. The purity and structural integrity of CE–PAGE-separated oligosaccharides are confirmed by strong anion exchange high-pressure liquid chromatography, electrospray ionization Fourier transform mass spectrometry, and two-dimensional nuclear magnetic resonance spectroscopy. The described method is straightforward and time-efficient, affording size-homogeneous oligosaccharides that can be used in sequencing, protein binding, and other structure–function relationship studies.

In vivo measurement of synthesis rate of multiple plasma proteins in humans

Advances in quantitative proteomics have facilitated the measurement of large-scale protein quantification, which represents net changes in protein synthesis and breakdown. However, measuring the rate of protein synthesis is the only way to determine the translational rate of gene transcripts. Here, we report a technique to measure the rate of incorporation of amino acids from ingested protein labeled with stable isotope into individual plasma proteins. This approach involves three steps: 1) production of stable isotope-labeled milk whey protein, oral administration of this intrinsically labeled protein, and subsequent collection of blood samples; 2) fractionation of the plasma and separation of the individual plasma proteins by a combination of anion exchange high-pressure liquid chromatography and gel electrophoresis; and 3) identification of individual plasma proteins by tandem mass spectrometry and measurement of stable isotopic enrichment of these proteins by gas chromatography-mass spectrometry. This method allowed the measurement of the fractional synthesis rate (FSR) of 29 different plasma proteins by using the same precursor pool. We noted a 30-fold difference in FSR of different plasma proteins with a wide range of physiological functions. This approach offers a tremendous opportunity to study the regulation of plasma proteins in humans in many physiological and pathological states.

Proteomic Analysis of Human Neutrophil Granules

Stimulated exocytosis of intracellular granules plays a critical role in conversion of inactive, circulating neutrophils to fully activated cells capable of chemotaxis, phagocytosis, and bacterial killing. The functional changes induced by exocytosis of each of the granule subsets, gelatinase (tertiary) granules, specific (secondary) granules, and azurophil (primary) granules, are poorly defined. To improve the understanding of the role of exocytosis of these granule subsets, a proteomic analysis of the azurophil, specific, and gelatinase granules from human neutrophils was performed. Two different methods for granule protein identification were applied. First, two-dimensional (2D) gel electrophoresis followed by MALDI-TOF MS analysis of peptides obtained by in-gel trypsin digestion of proteins was performed. Second, peptides from tryptic digests of granule membrane proteins were separated by two-dimensional microcapillary chromatography using strong cation exchange and reverse phase microcapillary high pressure liquid chromatography and analyzed with electrospray ionization tandem mass spectrometry (2D HLPC ESI-MS/MS). Our analysis identified 286 proteins on the three granule subsets, 87 of which were identified by MALDI MS and 247 were identified by 2D HPLC ESI-MS/MS. The increased sensitivity of 2D HPLC ESI-MS/MS, however, resulted in identification of over 500 proteins from subcellular organelles contaminating isolated granules. Defining the proteome of neutrophil granule subsets provides a basis for understanding the role of exocytosis in neutrophil biology. Additionally, the described methods may be applied to mobilizable compartments of other secretory cells.

Molecular and Hematological Characterization of Hemoglobin Hope/Hemoglobin E and Hemoglobin Hope/a-Thalassemia 2 in Thai Patients

We report the hematological and molecular characterization of compound heterozygosity for hemoglobin (Hb) Hope/Hb E and double heterozygosity for Hb Hope/ alpha-thalassemia 2 found in 2 unrelated Thai individuals. The first proband presented with slight anemia and mild hypochromic microcytosis. Routine cellulose acetate Hb electrophoresis at pH 8.6 revealed in addition to Hb E another variant migrating slightly more anodic to Hb A. On cation exchange high-pressure liquid chromatography, the variant was eluted in the amount of 60.9% after Hb E The same abnormal Hb was found in a second family in which the proband and her younger sister were both double heterozygotes for this Hb variant and deletional alpha-thalassemia 2, whereas an older sister was a pure carrier of the variant. The amounts of this variant were found to be 34.9%, 35.4%, and 38.3% in the proband, her younger sister, and her older sister, respectively. Direct DNA sequencing of the amplified beta-globin genes of both probands identified the GGT (Gly)-GAT (Asp) mutation at codon 136 corresponding to Hb Hope. beta-Globin gene haplotype analysis demonstrated that all the Thai betaHope genes were associated with the same haplotype, (+ - - - - + +), indicating a single origin of this variant in Thailand. A simple method based on allele-specific polymerase chain reaction for accurate diagnosis of the Hb Hope is described.

Effect of HbE and HbH on HbA1C level by ionic exchange HPLC comparing to immunoturbidimetry

Background: The hemoglobin (Hb)A1C level is widely used to monitor diabetes mellitus patients. The N-terminal amino acid valine of its β chain is glycated. The assay of HbA1C is based on differences in the charge, chemical and structural properties of the protein. Methods: There are fully automated instruments available in clinical chemistry laboratory to assay HbA1C level. The effect of hemoglobinopathies was studied between an ionic exchange high-pressure liquid chromatography (HPLC) (Bio-Rad Laboratories, USA) and immunoturbidimetry (BM/Hitachi 912 with Roche HBA1CII, Germany–Japan) assay. The influence of high level HbF relative to the HbA1C level by ionic exchange HPLC is known. The effect of HbE and HbH to the HbA1C level by ionic exchange HPLC comparing to immunoturbimetry was examined. The evaluation was performed on 34 normal controls (A2A), 17 β thalassemia traits (A2↑A), 36 HbE heterozygotes (EA), 37 HbE homozygotes (EE), 36 β thalassemia/HbE (EF/EFA), 11 EABart's diseases (EABart's), 34 Hb H diseases (A2/CSAH) and 13 cord blood samples (FA). Conclusions: Hemoglobinopathies can impact on the assay of HbA1C level such as HbE and HbH to ionic exchange HPLC. Although not studied as yet, this effect may influence the other methods such as affinity chromatography.

Inhibition of host transcription by vesicular stomatitis virus involves a novel mechanism that is independent of phosphorylation of TATA-binding protein (TBP) or association of TBP with TBP-associated factor subunits.

The matrix (M) protein of vesicular stomatitis virus (VSV) is a potent inhibitor in vivo of transcription by all three host RNA polymerases (RNAP). In the case of host RNA polymerase II (RNAPII), the inhibition is due to lack of activity of the TATA-binding protein (TBP), which is a subunit of the basal transcription factor TFIID. Despite the potency of M protein-induced inhibition in vivo, experiments presented here show that M protein cannot directly inactivate TFIID in vitro. Addition of M protein to nuclear extracts from uninfected cells did not inhibit transcription activity, indicating that the inhibition is indirect and is mediated through host factors. The host factors that are known to regulate TBP activity include phosphorylation by host kinases and association with different TBP-associated factor (TAF) subunits. However, TBP in VSV-infected cells was found to be assembled normally with its TAF subunits, as shown by ion exchange high-pressure liquid chromatography and sedimentation velocity analysis. A normal pattern of phosphorylation of TBP in VSV-infected cells was also observed by pH gradient gel electrophoresis. Collectively, these data indicate that M protein inactivates TBP activity in RNAPII-dependent transcription by a novel mechanism, since the known mechanisms for regulating TBP activity cannot account for the inhibition.

Resistance to glyphosate inLolium rigidum.II. Uptake, translocation, and metabolism

Experiments were conducted to determine potential mechanisms of glyphosate resistance inLolium rigidumfrom Australia.14C-Glyphosate uptake, translocation, and metabolism were compared between resistant (R) and sensitive (S) biotypes. The R seed (48118a) represented the F1progenies of plants having survived a 1.73-kg ae ha−1(4.8 L ha−1) application of a Roundup®formulation. The S seed was a sensitive biotype ofL. rigidumfrom Australia. Plants (one to four tillers, 2 to 4 wk old) were presprayed with a high (1.26 kg ae ha−1) or a low (0.28 kg ae ha−1) dose of formulated glyphosate. The first leaf of the first tiller, which was shielded from the spray, was immediately treated with a14C-glyphosate solution via manual application to the adaxial surface. Harvest was made 6 days after treatment (DAT), and glyphosate residues in the leaf wash, treated leaf, roots, and shoots were quantified based on radioactivity as percentage of applied dose. The overall radioactivity recoveries were very good (90.2 to 97.3% of applied dose). R and S plants showed comparable uptake at the high (79.2 vs. 78.0%) or the low (64.0 vs. 64.7%) doses of glyphosate. About one-half of the absorbed glyphosate in both R and S (32.9 to 38.3% appl.) was translocated into the plant and distributed almost equally into roots (13.6 to 16.0% appl.) and shoots (18.1 to 22.6% appl.). Autoradiography studies demonstrated no difference in tissue localization of glyphosate between the R and S plants. For metabolism studies, tissues from individual plants were homogenized in water, and extracts were analyzed by anion exchange high-pressure liquid chromatography (HPLC) with radioactivity detection. There was little to no metabolism of glyphosate in extracts from various tissues of either R or S plants. Based on these results, we conclude that neither uptake, translocation, nor metabolism play a major role in glyphosate resistance inL. rigidum.

N-acetylaspartic acid (NAA) and N-acetylaspartylglutamic acid (NAAG) in human ventricular, subarachnoid, and lumbar cerebrospinal fluid.

N-Acetylaspartic and N-acetylaspartylglutamic acid concentrations in human ventricular, subarachnoid and lumbar cerebrospinal fluid were measured by combined gas chromatography-mass spectrometry using selected ion monitoring with deuterated internal standards. N-Acetylaspartate concentrations were in the range 55, 9, and 1 microM, respectively; N-acetylaspartylglutamate concentrations in the same fluids were in the range 8, 3 and 4 microM, respectively. There did not appear to be any difference in lumbar fluid concentrations of either compound between control subjects, schizophrenic patients, Alzheimer's disease patients and a pooled group of patients with neurological degeneration. Ventricular concentrations of both compounds were greatly increased in deceased patients suggesting that maintenance of their intracellular concentrations is probably energy dependent. The concentrations of these compounds in lumbar cerebrospinal fluid from living, and ventricular cerebrospinal fluid from deceased subjects were weakly correlated with one another. In lumbar fluid neither compound appeared to be correlated with age. Analysis of serially collected lumbar samples from two subjects showed a weak concentration gradient for both compounds. Neither antipsychotic medication nor the acid transport inhibitor probenecid had any effect on lumbar concentrations of either compound. Attempts to use anion exchange high pressure liquid chromatography with UV detection for measurement of the low concentrations of N-acetylaspartate found in cerebrospinal fluid from living subjects were unsuccessful.

Analytical capillary isotachophoresis of total plasma lipoproteins: a new tool to identify atherogenic low density lipoproteins

Plasma low density lipoproteins from 20 patients were separated by capillary isotachophoresis (ITP). In each patient the apparent diameter of the predominant LDL peak on whole plasma was also determined by nondenaturing gradient gel electrophoresis. Furthermore the concentration of the more electronegatively charged in vivo oxidized LDL- was accomplished using anion exchange high pressure liquid chromatography. By analytical capillary ITP of whole plasma lipoproteins, prestained with a lipophilic dye, LDL were separated into four subfractions. Usually, the predominant subfraction was the slow migrating LDL4, followed by LDL3, and then by the faster LDL2 and LDL1. Slow migrating LDL4 correlated negatively with plasma triglycerides and LDL- and positively with plasma high density lipoprotein (HDL) cholesterol and with the LDL diameter, while the faster LDL1 showed an inverse behavior. The LDL1 + LDL2 to LDL3 + LDL4 ratio showed a strong positive correlation with LDL- concentration (r = 0.87; P < 0.001) and a highly significant inverse correlation with the LDL particle diameter (r = -0.74; P < 0.001). At least three highly atherogenic LDL that could be found in human plasma, namely oxidized, glycated and small-dense, are characterized by a greater electric charge. The LDL profile from capillary ITP and the relative prevalence of faster or slower migrating LDL fractions could indicate the presence of more atherogenic LDL.

Stage-specific Proteophosphoglycan from Leishmania mexicana Amastigotes: STRUCTURAL CHARACTERIZATION OF NOVEL MONO-, DI-, AND TRIPHOSPHORYLATED PHOSPHODIESTER-LINKED OLIGOSACCHARIDES

Intracellular amastigotes of the protozoan parasite Leishmania mexicana secrete a macromolecular proteophosphoglycan (aPPG) into the phagolysosome of their host cell, the mammalian macrophage. The structures of aPPG glycans were analyzed by a combination of high pH anion exchange high pressure liquid chromatography, gas chromatography-mass spectrometry, enzymatic digestions, electrospray-mass spectrometry as well as 1H and 31P NMR spectroscopy. Some glycans are identical to oligosaccharides known from Leishmania mexicana promastigote lipophosphoglycan and secreted acid phosphatase. However, the majority of the aPPG glycans represent amastigote stage-specific and novel structures. These include neutral glycans ([Glcbeta1-3]1-2Galbeta1-4Man, Galbeta1-3Galbeta1-4Man, Galbeta1-3Glcbeta1-3Galbeta1-4Man), several monophosphorylated glycans containing the conserved phosphodisaccharide backbone (R-3-[PO4-6-Gal]beta1-4Man) but carrying stage-specific modifications (R = Galbeta1-, [Glcbeta1-3]1-2Glcbeta1-), and monophosphorylated aPPG tri- and tetrasaccharides that are uniquely phosphorylated on the terminal hexose (PO4-6-Glcbeta1-3Galbeta1-4Man, PO4-6-Glcbeta1-3Glcbeta1-3Galbeta1-4Man, PO4-6-Galbeta1-3Glcbeta1-3Galbeta1-4Man). In addition aPPG contains highly unusual di- and triphosphorylated glycans whose major species are PO4-6-Glcbeta1-3Glcbeta1-3[PO4-6-Gal]beta1-4Man, PO4-6-Galbeta1-3Glcbeta1-3[PO4-6-Gal]beta1-4Man, PO4-6-Galbeta1-3Glcbeta1-3Glcbeta1-3[PO4-6-Gal]beta1-+ ++4Man, PO4-6-Glcbeta1-3[PO4-6-Glc]beta1-3[PO4-6-Gal]beta1-4Man, PO4-6-Galbeta1-3[PO4-6-Glc]beta1-3Glcbeta1-3[PO4-6-Gal]beta1 -4Man, and PO4-6-Glcbeta1-3[PO4-6-Glc]beta1-3Glcbeta1-3[PO4-6-Gal]beta1 -4Man. These glycans are linked together by the conserved phosphodiester R-Manalpha1-PO4-6-Gal-R or the novel phosphodiester R-Manalpha1-PO4-6-Glc-R and are connected to Ser(P) of the protein backbone most likely via the linkage R-Manalpha1-PO4-Ser. The variety of stage-specific glycan structures in Leishmania mexicana aPPG suggests the presence of developmentally regulated amastigote glycosyltransferases which may be potential anti-parasite drug targets.

An inhibitor of glycinamide ribonucleotide formyltransferase is selectively cytotoxic to cells that lack a functional G1 checkpoint.

We studied the effects of purine depletion on the cell cycle using a specific inhibitor of de novo purine biosynthesis, AG2034, an inhibitor of glycinamide ribonucleotide formyltransferase (GARFT). Cytotoxicity was determined by clonogenic assays, and cell cycle perturbations by flow cytometry. Ribonucleotide pools were measured by anion exchange high-pressure liquid chromatography, and DNA strand-breaks were determined by alkaline elution and by the TUNEL assay. When cells were maintained in standard tissue culture medium, which contained 2.2 microM folic acid, AG2034 was cytostatic in all the cell lines tested. Under low-folate conditions (50 nM folic acid), AG2034 caused up to 50% cell death in cell lines that possessed a functional G1 checkpoint (A549, MCF-7), but was only cytostatic to the remaining cells, even at very high concentrations (100 microM). In contrast, AG2034 at 10 nM or 100 nM killed all the cells in cultures of HeLa/S3 or SW480 cells, which lack a functional G1 checkpoint. Flow cytometry studies indicated that in G1 checkpoint-competent cells, AG2034 caused a G1 arrest. Those cells (up to 50%) that were already in S phase died, but the cells that were in G1 arrest maintained viability, based upon clonogenic assays, for many days. In G1 checkpoint-deficient cells, no G1 arrest was seen after AG2034 treatment, all cells progressed into S phase, and all cells died. Measurement of DNA strand-breaks, either by alkaline elution or by the dUTP end-labelling technique, indicated no DNA strand-breaks 24 h after AG2034 treatment, indicating that purine nucleotide depletion can trigger the G1 checkpoint in the absence of DNA damage. Purine depletion causes slow cell death in cells that have passed the G1 checkpoint, but cytostasis in cells that are arrested at the G1 checkpoint. The GARFT inhibitor, at physiological folate concentrations, thus causes selective cytotoxicity to cells lacking a functional G1 checkpoint.

Caged Nicotinic Acid Adenine Dinucleotide Phosphate: SYNTHESIS AND USE

Nicotinic acid adenine dinucleotide phosphate (NAADP) is a metabolite of NADP with Ca2+ mobilizing activity. The Ca2+ release mechanism activated by NAADP as well as the Ca2+ stores that it acts on are different from those activated by either cyclic ADP-ribose or inositol 1,4,5-trisphosphate (IP3) (Lee, H. C., and Aarhus, R. (1995) J. Biol. Chem. 270, 2152-2157). In order to demonstrate unambiguously that NAADP can mobilize Ca2+ stores in live cells, a caged analog was synthesized by reacting NAADP with 1-(2-nitrophenyl)diazoethane. Anion exchange high pressure liquid chromatography (HPLC) was used to purify one particular caged form from the mixture of products. Phosphate analyses following specific enzymatic cleavage indicate that the caging group is on the 2'-phosphate. This is confirmed by 31P NMR spectroscopy, showing that the 2'-phosphate of the caged compound exhibits an altered chemical shift of -2.6 ppm as compared with 2.3 ppm determined for the 2'-phosphate of NAADP. Caged NAADP had no Ca2+ releasing activity at a concentration as high as 1 micro;M when tested on sea urchin egg microsomes. After photolysis, it released Ca2+, was effective in nanomolar range, and was indistinguishable from authentic NAADP. The regeneration of NAADP after photolysis was also confirmed by HPLC analyses. The analog is particularly susceptible to UV and can be efficiently photolyzed using a spectrofluorimeter. To demonstrate its utility in live cells, caged NAADP was microinjected into sea urchin eggs. Photolysis effectively regenerated NAADP and activated Ca2+ oscillations in the eggs. Removal of external Ca2+ did not prevent the Ca2+ oscillations but only delayed the second Ca2+ peak by about 45 s, indicating that the oscillations are due to release from internal stores and not caused by Ca2+ influx. A mechanism based on sensitization of the Ca2+ release by Ca2+ loading is proposed to account for the Ca2+ oscillation observed.

Structures of Five Sulfated Hexasaccharides Prepared from Porcine Intestinal Heparin Using Bacterial Heparinase: STRUCTURAL VARIANTS WITH APPARENT BIOSYNTHETIC PRECURSOR-PRODUCT RELATIONSHIPS FOR THE ANTITHROMBIN III-BINDING SITE

Porcine intestinal heparin was extensively digested with Flavobacterium heparinase and size-fractionated by gel chromatography. Subfractionation of the hexasaccharide fraction by anion exchange high pressure liquid chromatography yielded 10 fractions. Six contained oligosaccharides derived from the repeating disaccharide region, whereas four contained glycoserines from the glycosaminoglycan-protein linkage region. The latter structures were reported recently (Sugahara, K., Tsuda, H., Yoshida, K., Yamada, S., de Beer, T., and Vliegenthart, J.F.G. (1995) J. Biol. Chem. 270, 22914-22923). In this study, the structures of one tetra- and five hexasaccharides from the repeat region were determined by chemical and enzymatic analyses as well as 500-MHz 1H NMR spectroscopy. The tetrasaccharide has the hexasulfated structure typical of heparin. The five hexa- or heptasulfated hexasaccharides share the common core pentasulfated structure delta HexA(2S) alpha 1-4GlcN-(NS, 6S) alpha 1-4IdoA alpha/GlcA beta 1-4GlcN(6S) alpha 1-4GlcA beta 1-4GlcN (NS) with one or two additional sulfate groups (delta HexA, GlcN, IdoA, and GlcA represent 4-deoxy-alpha-L-threo-hex-4-enepyranosyluronic acid, D-glucosamine, L-iduronic acid, and D-glucuronic acid, whereas 2S, 6S and NS stand for 2-O-, 6-O-, and 2-N-sulfate, respectively). Three components have the following hitherto unreported structures: delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S) alpha 1-4GlcA beta 1-4GlcN(NS,6S), delta HexA(2S) alpha 1-4GlcN(NS, 6S) alpha 1-4IdoA alpha 1-4GlcNAc(6S)-alpha 1-4GlcA beta 1-4GlcN(NS, 3S), and delta HexA(2S) alpha 1-4GlcN-(NS,6S) alpha 1-4IdoA (2S) alpha 1-4GlcNAc(6S) alpha 1-4GlcA beta 1-4GlcN(NS, 6S). Two of the five hexasaccharides are structural variants derived from the antithrombin III-binding sites containing 3-O-sulfated GlcN at the reducing termini with or without a 6-O-sulfate group on the reducing N,3-disulfated GlcN residue. Another contains the structure identical to that of the above heptasulfated antithrombin III-binding site fragment but lacks the 3-O-sulfate group and therefore is a pro-form for the binding site. Another has an extra sulfate group on the internal IdoA residue of this pro-form and therefore can be considered to have diverged from the binding site in the biosynthetic pathway. Thus, the isolated hexasacharides in this study include the three overlapping pairs of structural variants with an apparent biosynthetic precursor-product relationship, which may reflect biosynthetic regulatory mechanisms of the binding site.

Caged Cyclic ADP-Ribose: SYNTHESIS AND USE

Cyclic ADP-ribose (cADPR) is a recently discovered cyclic nucleotide with Ca2+ mobilizing activity. Caged cADPR was synthesized by reacting cADPR with 2-nitrophenethyldiazoethane. Elemental analyses, 1H NMR, and extinction coefficient measurements indicate that the product contains only one caging group. Anion exchange high pressure liquid chromatography separated caged cADPR into two forms, which most likely represent isomers. Both forms could be uncaged with equal efficiency by UV exposure to regenerate cADPR. Photolysis of caged cADPR was accomplished effectively with a spectrofluorimeter. The efficiency of uncaging depended on wavelength with UV light shorter than about 320 nm being the most effective. Caged cADPR was biologically inactive and could induce Ca2+ release from sea urchin egg homogenates only after photolysis. Specificity of the Ca2+ release was shown by inhibition by 8-amino-cADPR, a specific antagonist of cADPR. To demonstrate its utility in live cells, caged cADPR was microinjected into sea urchin eggs. Photolysis using a mercury light source effectively regenerated cADPR and resulted in Ca2+ mobilization and activation of cortical exocytosis in the eggs.

Differential Proliferative and Interleukin-10 Responses to Fractionated Filarial Antigens: Preferential Recognition by Patients with Chronic Lymphatic Dysfunction

To characterize filarial antigens that may be associated with the development of chronic lymphatic dysfunction in persons with lymphatic filariasis, T cell responsiveness to Brugia pahangi adult worm extracts and SDS-PAGE antigen fractions were examined among Haitians from an area in which Wuchereria bancrofti is endemic. Greater T cell proliferation and interleukin-10 (IL-10) production were observed in amicrofilaremic patients with hydrocele or elephantiasis than in amicrofilaremic or microfilaremic asymptomatic persons. Antigen fractions that stimulated the highest proliferative responses (in the 25-49 kDa range) and IL-10 production were not identical. Further separation of an immunodominant 30- to 38-kDa fraction by ion exchange high-pressure liquid chromatography identified several subfractions, including a 32-kDa protein band, that elicited T cell responses from patients with elephantiasis or hydrocele. By immunoblot, these patients also had markedly greater humoral reactivity to parasite antigens of approximately 52, 43, 32, and 30 kDa.