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Cysteine is the simplest thiolated, chiral amino acid and is often used as the anchor for studies of self-assembled monolayers (SAMs) of complex biomolecules such as peptides. Understanding the interaction of SAMs of cysteine with low-energy secondary electrons (SEs) produced by X-rays can further our understanding of radiation damage in biomolecules. In particular, if the electrons are polarized, chiral-selective chemistry could have bearing on the origin of homochirality in nature. In the present paper, we use synchrotron radiation-based X-ray photoelectron spectroscopy to determine the changes that occur in the bonding of self-assembled layers of cysteine on gold as a result of soft X-ray irradiation. To investigate the possibility of chiral selectivity resulting from the interaction of low-energy, spin-polarized SEs (SPSEs), measurements were conducted on cysteine adsorbed on a 3 nm-thick gold layer deposited on a CoPt thin-film multilayer with perpendicular magnetic anisotropy. Time-dependent measurements of the C 1s, N 1s, O 1s, S 2p, and Au 4f core levels are used to follow the changes in surface chemistry and determine reaction cross-sections as a function of SE exposure. Analysis of the data results in cross-sections in the range of 5–7 Mb and suggests possible reaction pathways. Changing the magnetization direction of the CoPt multilayer produces SPSEs with opposite polarity. Some evidence of spin-dependent reactions is indicated but is inconclusive. Possible reasons for the discrepancy are posited.

Spin transitions of cobalt ions in LaCoO3single crystals have been studied by the method of X-ray magnetic circular dichroism (XMCD) at the K- and L2,3-edges of Co3+ions. The orbital momentum of cobalt ions obtained for the K-edge at the 3dlevel in the region of the spin transition in the temperature range from 25 to 120 K increases by a factor of approximately 1.6, whereas the slope of the magnetization curve value in the same temperature range and magnetic field increases by a factor of more than 10. XMCD experiments at the cobalt L2,3-edges demonstrate gradual growth of the ratio of the orbital momentum to the spin one L/Sfrom 0.48 to 0.53 in the temperature range from 60 K to 120 K.

Le tonomètre Icare®Home est un nouveau tonomètre à rebond, développé pour l’automesure de la pression intraoculaire (PIO). L’objectif principal de notre étude était d’évaluer la fiabilité et la reproductibilité des mesures prises avec le tonomètre Icare®Home, chez des patients glaucomateux, en comparaison avec le tonomètre à aplanation de Goldmann. Un objectif secondaire était de rechercher les facteurs pouvant influencer la reproductibilité de ces mesures.

Évaluer l’efficacité à 1 an de la trabéculoplastie sélective au laser (SLT) sur la pression intraoculaire (PIO) et identifier des différences selon le nombre de traitements topiques antiglaucomateux pré-SLT dans le glaucome primitif à angle ouvert (GPAO) et l’hypertonie oculaire (HTO).

Background:Obesity is strongly associated with prevalence of obstructive sleep apnea (OSA), and weight loss has been shown to reduce disease severity.Objective:To investigate whether liraglutide 3.0 mg reduces OSA severity compared with placebo using the primary end point of change in apnea–hypopnea index (AHI) after 32 weeks. Liraglutide’s weight loss efficacy was also examined.Subjects/Methods:In this randomized, double-blind trial, non-diabetic participants with obesity who had moderate (AHI 15–29.9 events h−1) or severe (AHI ⩾30 events h−1) OSA and were unwilling/unable to use continuous positive airway pressure therapy were randomized for 32 weeks to liraglutide 3.0 mg (n=180) or placebo (n=179), both as adjunct to diet (500 kcal day−1deficit) and exercise. Baseline characteristics were similar between groups (mean age 48.5 years, males 71.9%, AHI 49.2 events h−1, severe OSA 67.1%, body weight 117.6 kg, body mass index 39.1 kg m−2, prediabetes 63.2%, HbA1c5.7%).Results:After 32 weeks, the mean reduction in AHI was greater with liraglutide than with placebo (−12.2 vs −6.1 events h−1, estimated treatment difference: −6.1 events h−1(95% confidence interval (CI), −11.0 to −1.2), P=0.0150). Liraglutide produced greater mean percentage weight loss compared with placebo (−5.7% vs −1.6%, estimated treatment difference: −4.2% (95% CI, −5.2 to −3.1%), P<0.0001). A statistically significant association between the degree of weight loss and improvement in OSA end points (P<0.01, all) was demonstrated post hoc. Greater reductions in glycated hemoglobin (HbA1c) and systolic blood pressure (SBP) were seen with liraglutide versus placebo (both P<0.001). The safety profile of liraglutide 3.0 mg was similar to that seen with doses ⩽1.8 mg.Conclusions:As an adjunct to diet and exercise, liraglutide 3.0 mg was generally well tolerated and produced significantly greater reductions than placebo in AHI, body weight, SBP and HbA1cin participants with obesity and moderate/severe OSA. The results confirm that weight loss improves OSA-related parameters.

Évaluer la reproductibilité intra- et inter-examinateur de la mesure automatique de l’épaisseur du complexe cellulaire ganglionnaire (ganglion cell complex, GCC) maculaire par OCT SD Canon HS-100 (Tokyo, Japon) chez des yeux normaux, hypertones et glaucomateux.

La détermination clinique des limites externes du disque optique (DO) ne correspond pas toujours aux véritables limites anatomiques de la tête du nerf optique définies par l’extrémité de la membrane de Bruch (bruch membrane opening-BMO). Un nouvel indice d’analyse du DO en tomographie par cohérence optique (OCT), le minimal rim width(BMO-MRW) évalue la plus petite épaisseur de l’anneau neuro-rétinien comprise entre le BMO et la membrane limitante interne. Le but de cette étude est d’évaluer un nouveau logiciel permettant une mesure automatique du BMO-MRW.

Recycling of organic wastes via their incorporation in cultivated lands is known to alter soil structural stability. Aggregate stability tests are commonly used to express quantitatively the susceptibility of soil structural stability to deformation. The objective of this study was to investigate the effects of biosolids addition, namely composted manure (MC) and activated sludge (AS), and spiking of the soils with orthophosphate (OP), phytic acid (PA) or humic acid (HA), on soil aggregate stability of semi-arid loamy sand, loam and clay soils before and after subjecting the soils to six rain storms (each 30mm rain with a break of 3–4 days). Aggregate stability was determined from water-retention curves at high matric potential. The effects of the applied amendments on pre- and post-rain aggregate stability were inconsistent and soil-dependent. For the pre-rain state, all of the tested amendments improved aggregate stability relative to the control. For the post-rain condition, aggregate stability was lower in the MC, OP and PA treatments and higher in the AS and HA treatments than in the control. The coarse-textured loam and loamy sand soils were more affected by the soil amendments than the clay soil. For the pre-rain state, addition of organic matter significantly improved macro-porosity and hence the stability of apparent macro-aggregate (>250μm). Our results indicate a possible advantage for separation of aggregates into macro- and micro-aggregates for more precise evaluation and understanding of the effects organic amendments might have on aggregate stability.

Le solriamfetol est un inhibiteur de la recapture de la dopamine et de la noradrénaline approuvé dans l’UE et aux États-Unis pour traiter la somnolence diurne excessive (SDE) du syndrome d’apnées-hypopnées obstructives du sommeil (SAHOS; 37,5–150mg/jour). Cette analyse a permis d’évaluer si le traitement de la SDE par solriamfetol affectait l’utilisation du traitement primaire (TPSAHOS).

We have performed temporally resolved X-ray excited optical luminescence measurements on Si nanowires. By performing Si K edge X-ray excitation measurements we have determined that the short-lifetime components have primarily Si character, while the long-lifetime component stems from the SiO 2shell and SiO 2−Si interface. The lifetime of the short-lifetime component decreases with increasing luminescence energy, i.e., as the size of the nanosilicon species decreases.

In this paper, we investigate the release of two drugs, nicotine and caffeine, from poly ϵ-caprolactone (PCL) matrices, as a model for the delivery of highly hydrophilic drugs. Since PCL does not degrade over the period of our experiments (<30 days), drug diffusion through the matrix is expected to be the dominant mechanism of release. Contrary to expectations, we find that the drug diffusion coefficient increases with increasing drug loading, weakly for caffeine and strongly for nicotine. The water content in the PCL matrices (after all of the drug was released) was found to be orders of magnitude higher than the expected value, increasing with increasing drug loading. We suggest that these phenomena arise from the semicrystalline nature of PCL under our experimental conditions, which inhibits matrix collapse when the drug is released, thereby creating voids into which water can diffuse. We apply a quantitative model for these systems that considers counter-diffusion of water intothe matrix with drug diffusion out of the matrix. The high solubility of both drugs in aqueous solutions leads to drug partitioning into the polymer-encapsulated water, thereby increasing the effective rate of drug diffusion and release. The model is shown to fit the experimental data of both drugs using only one fit parameter.

We report the synthesis and characterization of 1D Si−CdSe heteronanostructures with different morphologies such as coaxial, biaxial, sandwiched, pattern wrapping, coiling, structures etc., via a one-step metal catalyzed thermal evaporation method. Both Si and CdSe exhibit single crystalline characteristics in the heterostructures, as revealed by scanning transmission electron microscopy. The Si nanowires formed directly from the Si substrate via the solid−liquid−solid process acts as the absorption site for CdSe deposition as well as the template for the formation of 1D Si−CdSe heterostructures. Time-resolved X-ray excited optical luminescence from the 1D Si−CdSe heteronanostructures reveals two main emission features at 530 and 637 nm with slow and fast decay lifetime, respectively. The 530 and 637 nm emission is associated with the Si and CdSe component of the heterostructures, respectively.

Zusammenfassung: Lokoregionale Rezidive stellen ein relevantes Problem in der chirurgischen Onkologie dar. Intraluminale Lokalrezidive sind in vielen Fällen Ausdruck einer unzureichenden Primärresektion, die mit nicht ausreichenden Sicherheitsabständen durchgeführt wurde. Die Nachresektion mit ausreichenden Sicherheitsabständen stellt, wann immer möglich, das Therapieverfahren der Wahl dar. Das Auftreten eines Rezidivs im Tumorbett ist nach optimaler primärer Resektion Ausdruck einer bereits primär lokal weit fortgeschrittenen Erkrankung. Die Indikation zur Nachresektion ist hier immer sehr kritisch abzuwägen. Aussicht auf längerfristige Tumorkontrolle besteht nur, wenn die Zweitresektion residualtumorfrei durchgeführt werden kann und wenn sie sinnvoll in ein multimodales Behandlungskonzept eingebettet ist. Für Rezidive im Bereich der Lymphabstrombahn, der sog. 4. Dimension, besteht in der Regel keine zweite kurative Behandlungsoption.

Environmentally sound management of the use of composts in agriculture relies on matching the rate of release of available N from compost‐amended soils to the crop demand. To develop such management it is necessary to (i) characterize the properties of composts that control their rates of decomposition and release of N and (ii) determine the optimal amount of composts that should be applied annually to wheat (Triticum aestivumL.). Carbon and N mineralization were measured under controlled conditions to determine compost decomposition rate parameters, and the NCSOIL model was used to derive the organic wastes parameters that control the rates of N and C transformations in the soil. We also characterized the effect of a drying period to estimate the effects of the dry season on C and N dynamics in the soil. The optimized compost parameters were then used to predict mineral N concentration dynamics in a soil–wheat system after successive annual applications of compost. Sewage sludge compost (SSC) and cattle manure compost (CMC) mineralization characteristics showed similar partitioning into two components of differing ease of decomposition. The labile component accounted for 16 to 20% of total C and 11 to 14% of total N, and it decomposed at a rate of 2.4 × 10−2d−1, whereas the resistant pool had a decomposition rate constant of 1.2 to 1.4 × 10−4d−1The main differences between the two composts resulted from their total C and N and inorganic N contents, which were determined analytically. The long‐term effect of a drying period on C and N mineralization was negligible. Use of these optimization results in a simulation of compost mineralization under a wheat crop, with a modified plant‐effect version of the NCSOIL model, enabled us to evaluate the effects of the following factors on the C and N dynamics in soil: (i) soil temperature, (ii) mineral N uptake by plants, and (iii) release of very labile organic C in root exudates. This labile organic C enhanced N immobilization following application, and so decreased the N available for uptake by plants.

The overall objective of the present study was to determine the loading limits of composts that should be applied annually to irrigated wheat. We conducted a container experiment in a greenhouse during four years. It included eight treatments: sewage sludge compost (SSC) and cattle manure compost (CMC), each applied annually to a sandy soil, at rates equivalent to 3, 6, and 12 kg m−2, and two controls, one fertilized and one unfertilized. Total dry matter (DM), grain production, and the amount of N, P, and K taken up by plants increased with increasing compost rate. Nitrogen uptake by the plants of the fertilized control was much higher than by the plants of the highest compost rate. Phosphorus and K uptake by the plants amended with the highest compost rate was much higher than by the fertilized control plants. Inorganic N quantity in the soil increased with increasing compost rate and with successive applications. The net N mineralization during the first year of wheat growth was very low, less than 3.5% of the applied organic N under all compost application rates. The contribution of the organic N mineralization increased during the second and third years. Most of the N increase in the compost treatment was found in the upper layer of 0 to 15 cm, whereas in the fertilized treatment N accumulated from the surface to the bottom of the container, 0 to 55 cm. The successive application of high rates of composts resulted in P and K accumulation in the soil profile.

The association between hemostatic activation, stroke mechanism, and outcome is poorly defined. The Hemostatic System Activation Study (HAS) investigators measured serial levels of prothrombin fragment F1.2, a marker of thrombin generation, in patients enrolled in the Warfarin Aspirin Recurrent Stroke Study (WARSS).

The detection of disseminated tumor cells in peripheral blood is limited by the presence of very few tumor cells within a large number of blood cells. Therefore, tumor cell detection calls for enrichment systems with effective depletion of blood cells and high tumor cell recovery. We compared the new density gradient centrifugation method OncoQuick with the standard method of Ficoll. The enriched cell fractions were quantified. Tumor cell spiking experiments examined the recovery of tumor cells as detected by immunocytochemistry and cytokeratin-20 reverse transcriptase–polymerase chain reaction (RT-PCR). Clinical application of OncoQuick was evaluated in 37 peripheral blood samples of patients with gastrointestinal carcinomas. The depletion of mononuclear cells (MNCs) in the enriched cell fraction after OncoQuick centrifugation was 632-fold, with an average cell number of 9.5 × 10⁴, compared with Ficoll, with a depletion factor of 3.8 and a mean number of 1.6 × 10⁷ MNCs. The mean tumor cell recovery rates were 87% for OncoQuick and 84% for Ficoll. The increased depletion of MNCs with OncoQuick centrifugation further simplified immunocytochemical evaluation by reducing the number of cytospins and increasing the tumor cell density. Due to the reduced number of co-enriched MNCs by OncoQuick, the blood volume, which could be analyzed in one RT-PCR reaction, was increased up to 30 ml. Examination of peripheral blood samples from 37 patients with gastrointestinal tumors showed a cytokeratin-20 detection rate of 30% and a significant correlation with the presence of distant metastases (P &lt; 0.02). OncoQuick significantly reduced the co-enriched number of MNCs, with a high tumor cell recovery rate. Processing blood from tumor patients with OncoQuick increased the chance of detecting circulating tumor cells. Cytometry 49:150–158, 2002. © 2002 Wiley-Liss, Inc.

Patients with end-stage renal failure suffer from severe plasma trace metal deficiency that is not corrected by dialysis. Trace metals, including Zn2+, are critical for cell differentiation and replication. Zn2+also plays important role in cell apoptosis. Both processes are known to be impaired in uremia. The present study was undertaken to evaluate the effect of Zn2+supplementation on apoptosis of cultured peripheral blood mononuclear cells (PBMC) from patients on chronic hemodialysis versus those from healthy control subjects, concomitantly with assessment of mitogen-induced cell proliferation. The results showed that (1) basal total cell-associated Zn2+was elevated in uremic PBMC, compared to normal controls (23.9 [plusmn] 5.64 v10.5 [plusmn] 2.64 [mu ]mol/L/mg protein). The gap persisted following incubation in Zn2+-enriched medium (63.3 [plusmn] 26.12 v81.6 [plusmn] 13.4 [mu ]mol/L/mg protein, P[lt ] .005). (2) Basal proliferative response to phytohemagglutinin (PHA) was significantly decreased in uremic PBMC compared to normal controls (12,000 [plusmn] 1,560 cpm v16,600 [plusmn] 1,460 cpm, P[lt ] .01). Incubation of uremic PBMC in Zn2+-enriched medium improved their proliferative response to PHA, yielding counts per minute significantly higher compared to their normal counterparts (37,000 [plusmn] 7,500 cpm v22,000 [plusmn] 3,000 cpm, P[lt ] .001). (3) Basal apoptosis rate in uremic PBMC was significantly elevated compared to normal control cells (7.6% v2.6%, P[lt ] .05). Following incubation in Zn2+-enriched medium, apoptosis was increased both in normal and uremic PBMC. Percent apoptosis of uremic PBMC remained significantly elevated compared to control cells (11.7% v5.7%). We conclude that uremic PBMC are more responsive to exogenous Zn2+in culture than their normal counterparts. This, among other abnormalities, might reflect an abnormal regulation of Zn2+transport by uremic mononuclear cell menbranes. The resultant increase in total cell-associated Zn2+content improves poor proliferative responsiveness of uremic PBMC. On the other hand, increased total cell-associated Zn2+stimulates enhanced apoptosis in uremic PBMC, which, probably by eliminating defective cells, contributes to the functional capability of the population as a whole. The net effect of the 2 processes is still augmentation of cell proliferation.

To understand how 2-O-sulfation of uronic acid residues influences the biosynthesis of anticoagulant heparan sulfate, the cDNA encoding glucosaminyl 3-O-sulfotransferase-1 (3-OST-1) was introduced into wild-type Chinese hamster ovary cells and mutant pgsF-17 cells, which are defective in 2-O-sulfation. 3-OST-1-transduced cells gained the ability to bind to antithrombin. Structural analysis of the heparan sulfate chains showed that 3-OST-1 generates sequences containing GlcUA-GlcN(SO(3))3(SO(3)) and GlcUA-GlcN(SO(3))3(SO(3))6(SO(3)) in both wild-type and mutant cells. In addition, IdoUA-GlcN(SO(3))3(SO(3)) and IdoUA-GlcN(SO(3))3(SO(3))6(SO(3)) accumulate in the mutant chain. These disaccharides were also observed by tagging [6-(3)H]GlcN-labeled pgsF-17 heparan sulfate in vitro with [(35)S]PAPs and purified 3-OST-1. Heparan sulfate derived from the transduced mutant also had approximately 2-fold higher affinity for antithrombin than heparan sulfate derived from the transduced wild-type cells, and it inactivated factor Xa more efficiently. This study demonstrates for the first time that (i) 3-O-sulfation by 3-OST-1 can occur independently of the 2-O-sulfation of uronic acids, (ii) 2-O-sulfation usually occurs before 3-O-sulfation, (iii) 2-O-sulfation blocks the action of 3-OST-1 at glucosamine residues located to the reducing side of IdoUA units, and (iv) that alternative antithrombin-binding structures can be made in the absence of 2-O-sulfation.

A cDNA encoding a new cytochrome P450 was isolated from a mouse brain library. Sequence analysis reveals that this 1,958-base pair cDNA encodes a 57-58-kDa 502-amino acid polypeptide that is 70-91% identical to CYP2J subfamily P450s and is designated CYP2J9. Recombinant CYP2J9 was co-expressed with NADPH-cytochrome P450 oxidoreductase (CYPOR) in Sf9 cells using a baculovirus system. Microsomes of CYP2J9/CYPOR-transfected cells metabolize arachidonic acid to 19-hydroxyeicosatetraenoic acid (HETE) thus CYP2J9 is enzymologically distinct from other P450s. Northern analysis reveals that CYP2J9 transcripts are present at high levels in mouse brain. Mouse brain microsomes biosynthesize 19-HETE. RNA polymerase chain reaction analysis demonstrates that CYP2J9 mRNAs are widely distributed in brain and most abundant in the cerebellum. Immunoblotting using an antibody raised against human CYP2J2 that cross-reacts with CYP2J9 detects a 56-kDa protein band that is expressed in cerebellum and other brain segments and is regulated during postnatal development. In situ hybridization of mouse brain sections with a CYP2J9-specific riboprobe and immunohistochemical staining with the anti-human CYP2J2 IgG reveals abundant CYP2J9 mRNA and protein in cerebellar Purkinje cells. Importantly, 19-HETE inhibits the activity of recombinant P/Q-type Ca(2+) channels that are known to be expressed preferentially in cerebellar Purkinje cells and are involved in triggering neurotransmitter release. Based on these data, we conclude that CYP2J9 is a developmentally regulated P450 that is abundant in brain, localized to cerebellar Purkinje cells, and active in the biosynthesis of 19-HETE, an eicosanoid that inhibits activity of P/Q-type Ca(2+) channels. We postulate that CYP2J9 arachidonic acid products play important functional roles in the brain.

The response of granulosa cells to luteinizing hormone (LH) and follicle-stimulating hormone (FSH) is mediated mainly by cAMP/protein kinase A (PKA) signaling. Notably, the activity of the extracellular signal-regulated kinase (ERK) signaling cascade is elevated in response to these stimuli as well. We studied the involvement of the ERK cascade in LH- and FSH-induced steroidogenesis in two granulosa-derived cell lines, rLHR-4 and rFSHR-17, respectively. We found that stimulation of these cells with the appropriate gonadotropin induced ERK activation as well as progesterone production downstream of PKA. Inhibition of ERK activity enhanced gonadotropin-stimulated progesterone production, which was correlated with increased expression of the steroidogenic acute regulatory protein (StAR), a key regulator of progesterone synthesis. Therefore, it is likely that gonadotropin-stimulated progesterone formation is regulated by a pathway that includes PKA and StAR, and this process is down-regulated by ERK, due to attenuation of StAR expression. Our results suggest that activation of PKA signaling by gonadotropins not only induces steroidogenesis but also activates down-regulation machinery involving the ERK cascade. The activation of ERK by gonadotropins as well as by other agents may be a key mechanism for the modulation of gonadotropin-induced steroidogenesis.

To study the in vivo expression of the murine Tie2 gene, we have targeted the hypoxanthine phosphoribosyltransferase (Hprt) gene locus to generate two single-copy transgenic mice: T1, containing the 2,100-bp Tie2 promoter upstream from the beta-galactosidase (LacZ) gene, and T5, which also included an enhancing element originating from the first intron of the Tie2 gene. Comparing T1 and T5 embryos at day E10.5 revealed differential endothelial cell-specific expression of LacZ, whereas colocalization analyses showed that the expression was confined to endothelial cells. Moderate reporter gene activity was observed in the brain and kidney of T1 adults, whereas extensive LacZ gene expression was seen in the vasculature of most organs of the T5 adults. This study demonstrates the feasibility of targeting the Hprt locus with endothelial cell-specific sequences to analyze the spatial-temporal expression of transgenes. Of particular importance is the observation that the analysis of a single transgene copy in a defined locus allows for an accurate and rapid comparison of transcriptional activity among regulatory DNA sequences.

Epoxyeicosatrienoic acids (EETs), products of the cytochrome P-450 monooxygenase metabolism of arachidonic acid, can regulate the activity of ion channels. We examined the effects of EETs on cardiac L-type Ca2+ channels that play important roles in regulating cardiac contractility, controlling heart rate, and mediating slow conduction in normal nodal cells and ischemic myocardium. Our experimental approach was to reconstitute porcine L-type Ca2+ channels into planar lipid bilayers where we could control the aqueous and lipid environments of the channels and the regulatory pathways that change channel properties. We found that 20 to 125 nM EETs inhibited the open probability of reconstituted L-type Ca2+ channels, accelerated the inactivation of the channels, and reduced the unitary current amplitude of open channels. There was no selectivity among different EET regioisomers or stereoisomers. When 11,12-EET was esterified to the sn-2 position of phosphatidylcholine, restricting it to the hydrophobic phase of the planar lipid bilayer, the reconstituted channels were similarly inhibited, suggesting that the EET interacts directly with Ca2+ channels through the lipid phase. The inhibitory effects of EET persisted in the presence of microcystin, an inhibitor of protein phosphatases 1 and 2A, suggesting that dephosphorylation was not the mechanism through which these eicosanoids down-regulate channel activity. This inhibition may be an important protective mechanism in the setting of cardiac ischemia where arachidonic acid levels are dramatically increased and EETs have been shown to manifest preconditioning-like effects.

To understand the mechanisms that control anticoagulant heparan sulfate (HSact) biosynthesis, we previously showed that HSact production in the F9 system is determined by the abundance of 3-O-sulfotransferase-1 as well as the size of the HSact precursor pool. In this study, HSact precursor structures have been studied by characterizing [6-3H]GlcN metabolically labeled F9 HS tagged with 3-O-sulfates in vitro by 3'-phosphoadenosine 5'-phospho-35S and purified 3-O-sulfotransferase-1. This later in vitro labeling allows the regions of HS destined to become the antithrombin (AT)-binding sites to be tagged for subsequent structural studies. It was shown that six 3-O-sulfation sites exist per HSact precursor chain. At least five out of six 3-O-sulfate-tagged oligosaccharides in HSact precursors bind AT, whereas none of 3-O-sulfate-tagged oligosaccharides from HSinact precursors bind AT. When treated with low pH nitrous or heparitinase, 3-O-sulfate-tagged HSact and HSinact precursors exhibit clearly different structural features. 3-O-Sulfate-tagged HSact hexasaccharides were AT affinity purified and sequenced by chemical and enzymatic degradations. The 3-O-sulfate-tagged HSact hexasaccharides exhibited the following structures, DeltaUA-[6-3H]GlcNAc6S-GlcUA-[6-3H]GlcNS3(35)S+/-6S-++ +IdceA2S-[6-3H]Glc NS6S. The underlined 6- and 3-O-sulfates constitute the most critical groups for AT binding in view of the fact that the precursor hexasaccharides possess all the elements for AT binding except for the 3-O-sulfate moiety. The presence of five potential AT-binding precursor hexasaccharides in all HSact precursor chains demonstrates for the first time the processive assembly of specific sequence in HS. The difference in structures around potential 3-O-sulfate acceptor sites in HSact and HSinact precursors suggests that these precursors might be generated by different concerted assembly mechanisms in the same cell. This study permits us to understand better the nature of the HS biosynthetic pathway that leads to the generation of specific saccharide sequences.

3-O-Sulfated glucosaminyl residues are rare constituents of heparan sulfate and are essential for the activity of anticoagulant heparan sulfate. Cellular production of the critical active structure is controlled by the rate-limiting enzyme, heparan sulfate D-glucosaminyl 3-O-sulfotransferase-1 (3-OST-1) (EC 2.8.2.23). We have probed the expressed sequence tag data base with the carboxyl-terminal sulfotransferase domain of 3-OST-1 to reveal three novel, incomplete human cDNAs. These were utilized in library screens to isolate full-length cDNAs. Clones corresponding to predominant transcripts were obtained for the 367-, 406-, and 390-amino acid enzymes 3-OST-2, 3-OST-3A, and 3-OST-3B, respectively. These type II integral membrane proteins are comprised of a divergent amino-terminal region and a very homologous carboxyl-terminal sulfotransferase domain of approximately 260 residues. Also recovered were partial length clones for 3-OST-4. Expression of the full-length enzymes confirms the 3-O-sulfation of specific glucosaminyl residues within heparan sulfate (Liu, J., Shworak, N. W., Sinaÿ, P., Schwartz, J. J. Zhang, L., Fritze, L. M. S., and Rosenberg, R. D. (1999) J. Biol. Chem. 274, 5185-5192). Southern analyses suggest the human 3OST1, 3OST2, and 3OST4 genes, and the corresponding mouse isologs, are single copy. However, 3OST3A and 3OST3B genes are each duplicated in humans and show at least one copy each in mice. Intriguingly, the entire sulfotransferase domain sequence of the 3-OST-3B cDNA (774 base pairs) was 99.2% identical to the same region of 3-OST-3A. Together, these data argue that the structure of this functionally important region is actively maintained by gene conversion between 3OST3A and 3OST3B loci. Interspecific mouse back-cross analysis identified the loci for mouse 3Ost genes and syntenic assignments of corresponding human isologs were confirmed by the identification of mapped sequence-tagged site markers. Northern blot analyses indicate brain exclusive and brain predominant expression of 3-OST-4 and 3-OST-2 transcripts, respectively; whereas, 3-OST-3A and 3-OST-3B isoforms show widespread expression of multiple transcripts. The reiteration and conservation of the 3-OST sulfotransferase domain suggest that this structure is a self-contained functional unit. Moreover, the extensive number of 3OST genes with diverse expression patterns of multiple transcripts suggests that the novel 3-OST enzymes, like 3-OST-1, regulate important biologic properties of heparan sulfate proteoglycans.

The 3-O-sulfation of glucosamine residues is an important modification during the biosynthesis of heparan sulfate (HS). Our previous studies have led us to purify and molecularly clone the heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST-1), which is the key enzyme converting nonanticoagulant heparan sulfate (HSinact) to anticoagulant heparan sulfate (HSact). In this study, we expressed and characterized the full-length cDNAs of 3-OST-1 homologous genes, designated as 3-OST-2, 3-OST-3A, and 3-OST-3B as described in the accompanying paper (Shworak, N. W., Liu, J., Petros, L. M., Zhang, L., Kobayashi, M., Copeland, N. G., Jenkins, N. A., and Rosenberg, R. D. (1999) J. Biol. Chem. 274, 5170-5184). All these cDNAs were successfully expressed in COS-7 cells, and heparan sulfate sulfotransferase activities were found in the cell extracts. We demonstrated that 3-OST-2, 3-OST-3A, and 3-OST-3B are heparan sulfate D-glucosaminyl 3-O-sulfotransferases because the enzymes transfer sulfate from adenosine 3'-phosphophate 5'-phospho-[35S]sulfate ([35S]PAPS) to the 3-OH position of glucosamine. 3-OST-3A and 3-OST-3B sulfate an identical disaccharide. HSact conversion activity in the cell extract transfected by 3-OST-1 was shown to be 300-fold greater than that in the cell extracts transfected by 3-OST-2 and 3-OST-3A, suggesting that 3-OST-2 and 3-OST-3A do not make HSact. The results of the disaccharide analysis of the nitrous acid-degraded [35S]HS suggested that 3-OST-2 transfers sulfate to GlcA2S-GlcNS and IdoA2S-GlcNS; 3-OST-3A transfers sulfate to IdoA2S-GlcNS. Our results demonstrate that the 3-O-sulfation of glucosamine is generated by different isoforms depending on the saccharide structures around the modified glucosamine residue. This discovery has provided evidence for a new cellular mechanism for generating a defined saccharide sequence in structurally complex HS polysaccharide.

3-O-Sulfation of glucosamine by heparan sulfate D-glucosaminyl 3-O-sulfotransferase (3-OST-1) is the key modification in anticoagulant heparan sulfate synthesis. However, the heparan sulfates modified by 3-OST-2 and 3-OST-3A, isoforms of 3-OST-1, do not have anticoagulant activity, although these isoforms transfer sulfate to the 3-OH position of glucosamine residues. In this study, we characterize the substrate specificity of purified 3-OST-3A at the tetrasaccharide level. The 3-OST-3A enzyme was purified from Sf9 cells infected with recombinant baculovirus containing 3-OST-3A cDNA. Two 3-OST-3A-modified tetrasaccharides were purified from the 3-O-(35)S-sulfated heparan sulfate that was digested by heparin lyases. These tetrasaccharides were analyzed using nitrous acid and enzymatic degradation combined with matrix-assisted laser desorption/ionization-mass spectrometry. Two novel tetrasaccharides were discovered with proposed structures of DeltaUA2S-GlcNS-IdoUA2S-[(35)S]GlcNH(2)3S and DeltaUA2S-GlcNS-IdoUA2S-[3-(35)S]GlcNH(2)3S6S . The results demonstrate that 3-OST-3A sulfates N-unsubstituted glucosamine residues, and the 3-OST-3A modification sites are probably located in defined oligosaccharide sequences. Our study suggests that oligosaccharides with N-unsubstituted glucosamine are precursors for sulfation by 3-OST-3A. The intriguing linkage between N-unsubstituted glucosamine and the 3-O-sulfation by 3-OST-3A may provide a clue to the potential biological functions of 3-OST-3A-modified heparan sulfate.