Immunomodulatory activity of a methionine aminopeptidase-2 inhibitor on B cell differentiation.

Methionine aminopeptidase-2 (MetAP-2) inhibitors have potent anti-angiogenesis activity and are being developed for the treatment of solid tumours. The recently observed specific expression of MetAP-2 in germinal centre B cells suggests that it has a role in regulating B cell function. We have demonstrated a potent MetAP-2-dependent inhibitory effect on the antibody secretion from B cell receptor and CD40 co-stimulated primary human B cells in the presence of interleukin-21. The effect of MetAP-2 inhibition on antibody secretion was due to a block in differentiation of B cells into plasma cells. Immunohistochemical analysis of germinal centres from human, mouse and marmoset spleen showed a similar expression pattern of MetAP-2 in the marmoset and man, whereas mouse spleen showed no detectable expression. In a marmoset, T dependent immunization model, the MetAP-2 inhibitor suppressed an antigen-specific antibody response. Furthermore, histological analysis showed loss of B cells in the spleen and disrupted germinal centre formation. These results provide experimental evidence to support a novel role for MetAP-2 in immunomodulation. These effects of MetAP-2 are mediated by disruption of the germinal centre reaction and a block in the differentiation of B cells into plasma cells.

Monomeric and multimeric blockers of selectins: comparison of in vitro and in vivo activity.

The potency of the oligosaccharides SiaLe(x), SiaLe(a), HSO(3)Le(x), and HSO(3)Le(a), their conjugates with polyacrylamide (PAA, 40 kD), and other monomeric and polymeric selectin inhibitors has been compared with that of the polysaccharide fucoidan. The following assay systems were used: 1) a 96-well assay based either on the use of recombinant E-, P-, and L-selectins or an analogous assay with natural P-selectin isolated from human platelets; 2) a platelet-based P-selectin cell assay; and 3) a rat model of peritoneal inflammation. IC(50) values for the neoglycoconjugate SiaLe(a)-PAA were 6, 40, and 85 microM for recombinant E-, P-, and L-selectins, respectively; all monomeric inhibitors were about two orders of magnitude weaker. PAA-conjugates, containing as a ligand tyrosine-O-sulfate (sTyr) in addition to one of the sialylated oligosaccharides, were the most potent synthetic blockers in vitro. Compared with fucoidan, the most potent known P- and L-selectin blocker, the bi-ligand glycoconjugate HSO(3)Le(a)-PAA-sTyr displayed similar inhibitory activity in vitro towards L-selectin and about ten times lower activity towards P-selectin. All of the tested synthetic polymers displayed a similar ability to inhibit neutrophil extravasation in the peritonitis model (in vivo) at 10 mg/kg. The data provide evidence that monomeric SiaLe(x) is considerably more effective as a selectin blocker in vivo than in vitro, whereas the opposite is true for fucoidan and the bi-ligand neoglycoconjugate HSO(3)Le(a)-PAA-sTyr.

Rituxan (anti-CD20 antibody)-induced translocation of CD20 into lipid rafts is crucial for calcium influx and apoptosis.

Rituxan, a chimeric anti-CD20 antibody, is the first antibody approved for immunotherapy in non-Hodgkin's B-cell lymphoma and other B-cell lymphoproliferative disorders. Additionally, efficacy of Rituxan treatment has been reported in nonmalignant autoimmune diseases such as rheumatoid arthritis. Crosslinking of CD20 molecules by Rituxan induces therapeutic B-cell depletion. CD20 is a B-lymphocyte specific integral membrane protein, proposed to function as a store-operated calcium channel, which is activated upon receptor-stimulated calcium depletion of intracellular stores. Crosslinking of CD20 by antibodies has been reported to induce a redistribution of CD20 molecules to specialized microdomains at the plasma membrane known as lipid rafts. Here, we report that in the absence of Rituxan, CD20 exhibits a low affinity to lipid rafts. However, binding of Rituxan significantly increases the affinity of CD20 for lipid rafts resulting in its redistribution to a fraction resistant to Triton X-100 solubilization. Furthermore, we demonstrate that disturbing the raft integrity by cholesterol extraction results in dissociation of CD20 from a Triton X-100 resistant fraction followed by complete inhibition of Rituxan-induced calcium entry and apoptosis. The integrity of lipid rafts seems to play a crucial role for CD20-induced caspase activation. These data show, for the first time, that Rituxan-induced translocation of CD20 to lipid rafts is important for increased intracellular Ca(2+) levels and downstream apoptotic signalling.

Prevalence of narcolepsy symptomatology and diagnosis in the European general population.

OBJECTIVE: To determine the prevalence of narcolepsy in the general population of five European countries (target population 205,890,882 inhabitants). METHODS: Overall, 18,980 randomly selected subjects were interviewed (participation rate 80.4%). These subjects were representative of the general population of the UK, Germany, Italy, Portugal, and Spain. They were interviewed by telephone using the Sleep-EVAL expert system, which provided narcolepsy diagnosis according to the International Classification of Sleep Disorders (ICSD). RESULTS: Excessive daytime sleepiness was reported by 15% of the sample, with a higher prevalence in the UK and Germany. Napping two times or more in the same day was reported by 1.6% of the sample, with a significantly higher rate in Germany. Cataplexy (episodes of loss of muscle function related to a strong emotion), a cardinal symptom of narcolepsy, was found in 1.6% of the sample. An ICSD narcolepsy diagnosis was found in 0.047% of the sample: The narcolepsy was severe for 0.026% of the sample and moderate in 0.021%. CONCLUSION: This is the first epidemiologic study that estimates the prevalence of narcolepsy in the general population of these five European countries. The disorder affects 47 individuals/100,000 inhabitants.

Single-nucleotide polymorphism alleles in the insulin receptor gene are associated with typical migraine.

We have identified a migraine locus on chromosome 19p13.3/2 using linkage and association analysis. We isolated 48 single-nucleotide polymorphisms within the locus, of which we genotyped 24 in a Caucasian population comprising 827 unrelated cases and 765 controls. Five single-nucleotide polymorphisms within the insulin receptor gene showed significant association with migraine. This association was independently replicated in a case-control population collected separately. We used experiments with insulin receptor RNA and protein to investigate functionality for the migraine-associated single-nucleotide polymorphisms. We suggest possible functions for the insulin receptor in migraine pathogenesis.

How age and daytime activities are related to insomnia in the general population: consequences for older people.

OBJECTIVES: To determine the role of activity status and social life satisfaction on the report of insomnia symptoms and sleeping habits. DESIGN: Cross-sectional telephone survey using the Sleep-EVAL knowledge base system. SETTING: Representative samples of three general populations (United Kingdom, Germany, and Italy). PARTICIPANTS: 13,057 subjects age 15 and older: 4,972 in the United Kingdom, 4,115 in Germany, and 3,970 in Italy. These subjects were representative of 160 million inhabitants. MEASUREMENTS: Clinical questionnaire on insomnia and investigation of associated pathologies (psychiatric and neurological disorders). RESULTS: Insomnia symptoms were reported by more than one-third of the population age 65 and older. Multivariate models showed that age was not a predictive factor of insomnia symptoms when controlling for activity status and social life satisfaction. The level of activity and social interactions had no influence on napping, but age was found to have a significant positive effect on napping. CONCLUSIONS: These results indicate that the aging process per se is not responsible for the increase of insomnia often reported in older people. Instead, inactivity, dissatisfaction with social life, and the presence of organic diseases and mental disorders were the best predictors of insomnia, age being insignificant. Healthy older people (i.e., without organic or mental pathologies) have a prevalence of insomnia symptoms similar to that observed in younger people. Moreover, being active and satisfied with social life are protective factors against insomnia at any age.

Missionary positions: Christian, modernist, postmodernist.

In the late 1960s and early 1970s "the missionary position" became widespread as a technical expression for face-to-face man-on-top sexual intercourse. It was accompanied by standard (and undocumented) stories as to the origin of the expression, stories featuring missionaries and either Polynesians, Africans, Chinese, Native Americans, or Melanesians. By the late 1980s and 1990s the expression had become a core symbol in modernist and postmodernist moral discourses. This paper examines accounts of the origin of the expression, provides evidence that it originated in Kinsey's (mis)reading of Malinowski, analyzes the symbolic elements of the missionary-position narrative as synthesizing modernist objections to Christian morality, analyzes the "missionary position" in postmodernist narratives as synthesizing postmodernist objections to modernist morality, and explores some of the functions of this myth within the academy.

Prevalence and patterns of problematic sleep among older adolescents.

OBJECTIVE: Despite many constraints on time schedules among teenagers, epidemiological data on sleep complaints in adolescence remain limited and are nonexistent for sleep disorders. This study provides additional data on sleep habits and DSM-IV sleep disorders in late adolescence. METHOD: A representative sample of 1,125 adolescents aged 15 to 18 years was interviewed by telephone using the Sleep-EVAL system. These adolescents came from 4 European countries: France, Great Britain, Germany, and Italy. Information was collected about sociodemographic characteristics, sleep/wake schedule, sleep habits, and sleep disorders and was compared with information from 2,169 young adults (19-24 years of age). RESULTS: Compared with young adults, adolescents presented with a distinct sleep/wake schedule: they went to sleep earlier, they woke up later, and they slept longer than young adults did. On weekends and days off, they also slept more than young adults did. However, the prevalence rates of sleep symptoms and sleep disorders were comparable in both groups. Approximately 25% reported insomnia symptoms and approximately 4% had a DSM-IV insomnia disorder. Fewer than 0.5% had a circadian rhythm disorder. CONCLUSIONS: Prevalence of insomnia disorders is lower in the adolescent population than in middle-aged or elderly adults. However, a rate of 4% in this young population is important given their young age and the consequences for daytime functioning.

Is sleep-disordered breathing an independent risk factor for hypertension in the general population (13,057 subjects)?

UNLABELLED: OBJECTIVES Sleep-disordered breathing has been hypothesized to have a close relationship with hypertension but previous studies have reported mixed results. This is an important health issue that requires further clarification because of the potential impact on the prevention and control of hypertension. METHODS: The relationship between hypertension and three forms of sleep-disordered breathing (chronic snoring, breathing pauses and obstructive sleep apnea syndrome (OSAS)) was assessed using representative samples of the non-institutionalized population of the UK, Germany and Italy (159 million inhabitants). The samples were comprised of 13,057 individuals aged 15-100 years who were interviewed about their sleeping habits and their sleep symptoms over the telephone using the Sleep-EVAL system. RESULTS: OSAS was found in 1.9% (95% CI: 1.2% to 2.3%) of the UK sample, 1.8% (95% CI: 1.4% to 2.2%) of the German sample and 1.1% (95% CI: 0.8% to 1.4%) of the Italian sample. OSAS was an independent risk factor (odds ratio (OR): 9.7) for hypertension after controlling for possible confounding effects of age, gender, obesity, smoking, alcohol consumption, life stress, and, heart and renal disease. CONCLUSIONS: Results from three of the most populated countries in Western Europe indicate that OSAS is an independent risk factor for hypertension. Snoring and breathing pauses during sleep appeared to be non-significant predictive factors.

The place of confusional arousals in sleep and mental disorders: findings in a general population sample of 13,057 subjects.

Confusional arousals, or sleep drunkenness, occur upon awakening and remain unstudied in the general population. We selected a representative sample from the United Kingdom, Germany, and Italy (N = 13,057) and conducted telephone interviews. Confusional arousals were reported by 2.9% of the sample: 1% (95% confidence interval: .8 to 1.2%) of the sample also presented with memory deficits (53.9%), disorientation in time and/or space (71%), or slow mentation and speech (54.4%), and 1.9% (1.7% to 2.1%) reported confusional arousals without associated features. Younger subjects (< 35 years) and shift or night workers were at higher risk of reporting confusional arousals. These arousals were strongly associated with the presence of a mental disorder with odds ratios ranging from 2.4 to 13.5. Bipolar and anxiety disorders were the most frequently associated mental disorders. Furthermore, subjects with Obstructive Sleep Apnea Syndrome (OSAS), hypnagogic or hypnopompic hallucinations, violent or injurious behaviors, insomnia, and hypersomnia are more likely to suffer from confusional arousals. Confusional arousals appears to occur quite frequently in the general population, affecting mostly younger subjects regardless of their gender. Physicians should be aware of the frequent associations between confusional arousals, mental disorders, and OSAS. Furthermore, the high occurrence of confusional arousals in shift or night workers may increase the likelihood of inappropriate response by employees sleeping at work.

Four-year changes in college athletes' ethical value choices in sports situations.

Positive values for fairness in competition are supposed to undergird the behavior of athletes engaged in sport. Whether athletes' values actually develop over 4 years in a college that emphasizes character development is the focus of this study. Athletes' (N = 631) use of deontological ethics (Hahm, Beller & Stoll, 1989) in 21 sports value dilemmas were evaluated. At entrance, as well as near graduation, intercollegiate athletes' value scores were lower than intramural athletes' scores. Both groups' scores declined while they were in college. Individual-sport athletes had higher scores than team-sport athletes but manifested a greater decline over 4 years. The findings are consistent with other studies that show decreases in "sportsmanship orientation" and an increase in "professional" attitudes associated with participation in sport.

Activation of nitric oxide synthase by beta 2-adrenoceptors in human umbilical vein endothelium in vitro.

1. Some animal studies suggest that beta-adrenoceptor-mediated vasorelaxation is in part mediated through nitric oxide (NO) release. Furthermore, in humans, we have recently shown that forearm blood flow is increased by infusion of beta2-adrenergic agonists into the brachial artery, and the nitric oxide synthase (NOS) inhibitor N(G)-monomethyl-L-arginine (L-NMMA) inhibits this response. 2. The purpose of the present study was to determine whether stimulation of human umbilical vein endothelial beta-adrenoceptors causes vasorelaxation and nitric oxide generation, and whether this might be mediated by cyclic adenosine-3',5'-monophosphate (cyclic AMP). 3. Vasorelaxant responses were determined in umbilical vein rings to the nonselective beta-adrenergic agonist isoprenaline and to the cyclic AMP analogue dibutyryl cyclic AMP, following precontraction with prostaglandin F2alpha. 4. NOS activity was measured in cultured human umbilical vein endothelial cells (HUVEC) by the conversion of [3H]-L-arginine to [3H]-L-citrulline, and adenylyl cyclase activity by the conversion of [alpha-32P]-ATP to [32P]-cyclic AMP. 5. Isoprenaline relaxed umbilical vein rings, and this vasorelaxation was abolished by beta2- (but not beta1-) adrenergic blockage, and by endothelium removal or 1 mM L-NMMA. In addition, vasorelaxant responses to dibutyryl cyclic AMP were inhibited by 1 mM L-NMMA, with a reduction in Emax from 90.0+/-9.3% to 50.5+/-9.9% (P<0.05). 6. Isoprenaline 1 microM increased NOS activity in HUVEC (34.0+/-5.9% above basal, P<0.001). Furthermore, isoprenaline increased adenylyl cyclase activity in a concentration-dependent manner; this response was inhibited by beta2 (but not beta1-) adrenergic blockade. Forskolin 1 microM and dibutyryl cyclic AMP 1 mM each increased NOS activity in HUVEC, to a degree similar to isoprenaline 1 microM. The increase in L-arginine to L-citrulline conversion observed with each agent was abolished by coincubation with NOS inhibitors. 7. These results indicate that endothelial beta2-adrenergic stimulation and cyclic AMP elevation activate the L-arginine/NO system, and give rise to vasorelaxation, in human umbilical vein.

Potentiation of cyclic AMP-mediated vasorelaxation by phenylephrine in pulmonary arteries of the rat.

Alpha1-adrenoceptor agonists may potentiate relaxation to beta-adrenoceptor agonists, although the mechanisms are unclear. We compared relaxations induced by beta-adrenoceptor agonists and cyclic AMP-dependent vasodilators in rat pulmonary arteries constricted with prostaglandin F2alpha (PGF2alpha) or the alpha1-adrenoceptor agonist phenylephrine (PE). In addition, we examined whether differences were related to cyclic AMP- or nitric oxide (NO) and cyclic GMP-dependent pathways. Isoprenaline-induced relaxation was substantially potentiated in arteries constricted with PE compared with PGF2alpha. Methoxamine was similar to PE, whereas there was no difference between PGF2alpha and 30 mM KCl. The potentiation was primarily due to a marked increase in the NO-independent component of relaxation, from 9.1+/-1.7% for PGF2alpha to 55.1+/-4.4% for PE. NO-dependent relaxation was also enhanced, but to a lesser extent (50%). Relaxation to salbutamol was almost entirely NO-dependent in both groups, and was potentiated approximately 50% by PE. Relaxation to forskolin (activator of adenylate cyclase) was also enhanced in PE constricted arteries. Part of this relaxation was NO-dependent, but the major effect of PE was to increase the NO-independent component. Propranolol diminished but did not abolish the potentiation. There was no difference in response to CPT cyclic AMP (membrane permeant analogue) between PE and PGF2alpha, suggesting that mechanisms distal to the production of cyclic AMP were unchanged. Relaxation to sodium nitroprusside (SNP) was the same for PE and PGF2alpha, although relaxation to acetylcholine (ACh) was slightly depressed. This implies that potentiation by PE does not involve the cyclic GMP pathway directly. Mesenteric arteries constricted with PE did not show potentiation of isoprenaline-induced relaxation compared to those constricted with PGF2alpha, suggesting that this effect may be specific to the pulmonary circulation. These results clearly show that PE potentiates both the NO-independent and -dependent components of cyclic AMP-mediated relaxation in pulmonary arteries of the rat, although the effect on the former is more profound. We suggest that potentiation of both components is largely due to direct activation of adenylate cyclase via alpha1-adrenoceptors, within the smooth muscle and endothelial cells respectively.

Night terrors, sleepwalking, and confusional arousals in the general population: their frequency and relationship to other sleep and mental disorders.

BACKGROUND: Arousal parasomnias (night terrors, sleepwalking, and confusional arousals) have seldom been investigated in the adult general population. Clinical studies of parasomnias, however, show that these disorders may be indicators of underlying mental disorders and may have serious consequences. METHOD: A representative sample of the United Kingdom population (N = 4972) was interviewed by telephone with the Sleep-EVAL system. RESULTS: Night terrors were reported by 2.2% (95% CI = 1.8% to 2.6%) of the sample, sleepwalking by 2.0% (1.6% to 2.4%), and confusional arousals by 4.2% (3.6% to 4.8%). The rate of these 3 parasomnias decreased significantly with age, but no gender difference was observed. Multivariate models identified the following independent factors as associated with confusional arousals (odds ratio [OR]): age of 15-24 years (OR = 4.1), shift work (OR = 2.1), hypnagogic hallucinations (OR = 3.3), deep sleep (OR = 1.6), daytime sleepiness (OR = 1.9), sleep talking (OR = 1.7), daily smoking (OR = 1.7), adjustment disorder (OR = 3.1), and bipolar disorder (OR = 13.0). Factors associated with night terrors were subjective sense of choking or blocked breathing at night (OR = 5.1), obstructive sleep apnea syndrome (OR = 4.1), alcohol consumption at bedtime (OR = 3.9), violent or injury-causing behaviors during sleep (OR = 3.2), hypnagogic hallucinations (OR = 2.2), and nightmares at least 1 night per month (OR = 4.0). Factors associated with sleepwalking were age of 15-24 years (OR = 5.2), subjective sense of choking or blocked breathing at night (OR = 5.1), sleep talking (OR = 5.0), and a road accident in the past year (OR = 3.9) after controlling for possible effects of sleep deprivation, life stress, and mental and sleep disorders. CONCLUSION: Arousal parasomnias, especially night terrors and confusional arousals, are often the expression of a mental disorder. Other life or medical conditions, such as shift work or excessive need of sleep for confusional arousals and stressful events for sleepwalking, may also trigger parasomnias. Prevalence rates are based on self-reported data and, consequently, are likely underestimated.

The prevalence of depressive disorders in the United Kingdom.

BACKGROUND: The prevalence of major psychiatric disorders in the general population is difficult to pinpoint owing to widely divergent estimates yielded by studies employing different criteria, methods, and instruments. Depressive disorders, which represent a sizable mental health care expense for the public purse, are no exception to the rule. METHODS: The prevalence of depressive disorders was assessed in a representative sample (n = 4972) of the U.K. general population in 1994. Interviews were performed over the telephone by lay interviewers using an expert system that tailored the questionnaire to each individual based on prior responses. Diagnoses and symptoms lists were based on the DSM-IV. RESULTS: Five percent (95% confidence interval = 4.4-5.6%) of the sample was diagnosed by the system with a depressive disorder at the time of the interview, with the rate slightly higher for women (5.9%) than men (4.2%). Unemployed, separated, divorced, and widowed individuals were found to be at higher risk for depression. Depressive subjects were seen almost exclusively by general practitioners (only 3.4% by psychiatrists). Only 12.5% of them consulted their physician seeking mental health treatment, and 15.9% reported being hospitalized in the past 12 months. CONCLUSIONS: The study indicates that mental health problems in the community are seriously underdetected by general practitioners, and that these professionals are highly reluctant to refer patients with depressive disorders to the appropriate specialist.

Modulation of sialyl Lewis X dependent binding to E-selectin by glycoforms of alpha-1-acid glycoprotein expressed in rheumatoid arthritis.

Alpha-1-acid glycoprotein (AGP) is an extensively glycosylated acute phase protein of imprecisely defined physiological function. Nonetheless it is known that the oligosaccharide component comprising 42% of the 41 kDa molecular weight is critical to the previously described multifarious immunomodulatory functions of AGP in vitro. Complex oligosaccharides were enzymically released from AGP purified from the blood of rheumatoid arthritis sufferers by our oligosaccharide protective method. Oligosaccharide profiling was by means of high pH anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Monosaccharide composition analysis revealed increased fucosylation of inflammatory AGP oligosaccharide chains, suggesting the potential for expression of the tetrasaccharide antigen and E-Selectin ligand, sialyl Lewis X (sLeX). The hypothesis that AGP may function to inhibit blood cell binding to activated endothelium at E-Selectin was tested in a microtitre cell-protein binding assay. In this system we have shown that the oligosaccharide moiety of AGP, as expressed in inflammatory disease, can inhibit the sLeX/E-Selectin interaction. Thus we have identified a correlation between the abnormal glycosylation of AGP in rheumatoid arthritis and suppression of sLeX dependent cell adhesion through inhibition of E-selectin binding which could be the basis of a novel, site specific, anti-inflammatory agent.

Characterization of E-selectin-binding epitopes expressed by skin-homing T cells.

The glycoprotein counter-receptors for E-selectin borne on skin-homing T cells are poorly defined. In this study we have used flow cytometry to investigate the surface expression of potential carbohydrate ligands for E-selectin on HUT78, a skin-homing cutaneous T-cell lymphoma. These cells possessed high surface expression of the KM-93 epitope but not HECA 452 or CSLEX1 epitopes. The KM-93 antibody also blocked the binding of HUT78 cells to E-selectin. All these antibodies are reported to recognize sialyl Lewis X (sLex)-like molecules. Using an E-selectin affinity matrix, the main glycoprotein isolated from HUT78 cells was a molecular species of 90 000 MW. Other minor species of molecular weights 40 000, 60 000, 100 000, 120 000 and 200 000 were also identified as potential counter-receptors for E-selectin. Four of the purified counter-receptors (90 000, 100 000, 120 000 and 200 000 MW) stained positive with the KM-93 antibody. Immunoblot analysis of these purified glycoproteins established the identity of the 90 000 MW glycoprotein as l-selectin. Furthermore, an anti-l-selectin antibody inhibited the binding of HUT78 cells to E-selectin, probably by steric inhibition of the carbohydrate ligand for E-selectin that is borne on the C-type lectin domain of l-selectin. These results suggest that a carbohydrate epitope on l-selectin may act as a ligand for E-selectin on skin-homing T cells.

Membrane potential-dependent and -independent vasodilation in small pulmonary arteries from chronically hypoxic rats.

Chronic hypoxia is associated with altered pulmonary vasoreactivity, and it has been suggested that an increased response to voltage-dependent vasodilators may relate to enhanced Ca++ entry via voltage-dependent channels, secondary to depolarization. Few studies have been performed on small pulmonary arteries, and it is unknown whether they are depolarized after chronic hypoxia. We examined the resting membrane potential, and the actions of voltage-dependent (verapamil, levcromakalim) and -independent (isoproterenol, forskolin, papaverine) vasodilators in small ( approximately 300 microm internal diameter) pulmonary arteries from chronically hypoxic rats. The resting membrane potential was more positive in arteries after chronic hypoxia (control: -60 +/- 0.5 mV; hypoxic: -54.4 +/- 1.1 mV; P < .01), and this was reflected by a shift to the left of the response curves for K+ and 4-aminopyridine. In arteries constricted with prostaglandin F2alpha the response to verapamil and levcromakalim was increased after chronic hypoxia, although maximum prostaglandin F2alpha-induced tension was unchanged, which implies a reduction in voltage-independent constrictor mechanisms. Although vasorelaxation to isoproterenol was depressed in arteries from hypoxic rats, forskolin-induced relaxation was enhanced substantially, and because the response to the phosphodiesterase inhibitor papaverine was unchanged, we suggest that this reflects an up-regulation of adenylate cyclase. In conclusion, chronic hypoxia resulted in a significant depolarization in small pulmonary arteries, but this may explain only partly the increased efficacy of voltage-dependent vasodilators. Whether the reduction in voltage-independent constrictor mechanisms is related to the apparent up-regulation of adenylate cyclase remains to be elucidated.

Scintillation proximity assay for E-, P-, and L-selectin utilizing polyacrylamide-based neoglycoconjugates as ligands.

In this study, a novel scintillation proximity assay (SPA) that uses radiolabeled soluble neoglycoconjugates as synthetic alternatives to the natural E-, P-, and L-selectin counterligands was developed. The neoglycoconjugates contained sialyl LewisX or sialyl LewisA attached via a three-carbon spacer to a poly[N-(hydroxyethyl)acrylamide] backbone, thus presenting the carbohydrates in a multivalent form. Selectin-ZZ fusion proteins were immobilized on anti-rabbit IgG-coated SPA beads via a rabbit IgG bridge. The neoglycoconjugate ligands bound to all three bead-immobilized selectins, with the highest binding levels apparent with E-selectin. Saturation binding studies with E-selectin revealed a complex interaction indicative of two or more binding affinities. The response to carbohydrate inhibitors was comparable in E-selectin assays that used either the neoglycoconjugates or the tritium-labeled HL60 cells as selectin counterligands. The incorporation of tyrosine sulfate groups into the backbone of the neoglycoconjugate resulted in enhanced binding avidity to both P- and L-selectin, indicating that the sulfate-containing neoglycoconjugates are viable synthetic mimics of the natural P- and L-selectin counterligands. The use of these radiolabeled neoglycoconjugates in conjunction with SPA results in a format ideally suited for the high-throughput screening for selectin antagonists. Furthermore, this approach can potentially be used to measure other low-avidity lectin-carbohydrate interactions.