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Biomedical subjects

D J Fitzgerald

Publications and source records attributed to D J Fitzgerald.

At least 37 records · Page 2Linked to original sources
PubMed2001

O-acetylsalicylhydroxamic acid, a novel acetylating inhibitor of prostaglandin H2 synthase: structural and functional characterization of enzyme-inhibitor interactions.

Aspirin is unique among clinically used nonsteroidal antiinflammatory drugs in that it irreversibly inactivates prostaglandin (PG) H2 synthase (PGHS) via acetylation of an active-site serine residue. We report the synthesis and characterization of a novel acetylating agent, O-acetylsalicylhydroxamic acid (AcSHA), which inhibits PGE2 synthesis in vivo and blocks the cyclooxygenase activity of PGHS in vitro. AcSHA requires the presence of the active-site residue Ser-529 to be active against human PGHS-1; the S529A mutant is resistant to inactivation by the inhibitor. Analysis of PGHS inactivation by AcSHA, coupled with the X-ray crystal structure of the complex of ovine PGHS-1 with AcSHA, confirms that the inhibitor elicits its effects via acetylation of Ser-529 in the cyclooxygenase active site. The crystal structure reveals an intact inhibitor molecule bound in the enzyme's cyclooxygenase active-site channel, hydrogen bonding with Arg-119 of the enzyme. The structure-activity profile of AcSHA can be rationalized in terms of the crystal structure of the enzyme-ligand complex. AcSHA may prove useful as a lead compound to facilitate the development of new acetylating inhibitors.

Acetylation↗
PubMed2001

Dual-function vaccine for Pseudomonas aeruginosa: characterization of chimeric exotoxin A-pilin protein.

Pseudomonas aeruginosa is the major infectious agent of concern for cystic fibrosis patients. Strategies to prevent colonization by this bacterium and/or neutralize its virulence factors are clearly needed. Here we characterize a dual-function vaccine designed to generate antibodies to reduce bacterial adherence and to neutralize the cytotoxic activity of exotoxin A. To construct the vaccine, key sequences from type IV pilin were inserted into a vector encoding a nontoxic (active-site deletion) version of exotoxin A. The chimeric protein, termed PE64Delta553pil, was expressed in Escherichia coli, refolded to a near-native conformation, and then characterized by various biochemical and immunological assays. PE64Delta553pil bound specifically to asialo-GM1, and, when injected into rabbits, produced antibodies that reduced bacterial adherence and neutralized the cell-killing activity of exotoxin A. Results support further evaluation of this chimeric protein as a vaccine to prevent Pseudomonas colonization in susceptible individuals.

ADP Ribose Transferases↗
PubMed2001

Selective inhibition of COX-2 in humans is associated with less gastrointestinal injury: a comparison of nimesulide and naproxen.

BACKGROUND: Selective inhibitors of cyclooxygenase (COX)-2 may provoke less gastric damage and platelet inhibition than conventional non-steroidal anti-inflammatory drugs. AIMS: We compared the biochemical and gastrointestinal effects of nimesulide, a potent and selective COX-2 inhibitor, with naproxen which exhibits no selectivity. SUBJECTS: Thirty six healthy volunteers were randomised to nimesulide 100 mg or naproxen 500 mg twice daily for two weeks in a double blind, crossover study with a washout between treatments. METHODS: Gastrointestinal side effects were assessed by endoscopy, and by estimation of small intestinal absorption-permeability and inflammation. Comparisons were made between variables at the end of each treatment phase. RESULTS: Nimesulide caused significantly less gastric injury using the modified Lanza score (p<0.001) as well as reduced duodenum injury (p=0.039). Nimesulide had lower visual analogue scores (VAS) for haemorrhage and erosive lesions in the stomach (p<0.001) and for mucosal injection in the duodenum (p=0.039). Naproxen increased excretion of calprotectin, a marker of intestinal inflammation (5.5 (1.2) to 12.1 (2.1) mg/l) while nimesulide had no effect (treatment difference p=0.03). Naproxen abolished platelet aggregation to arachidonic acid and suppressed serum thromboxane B(2) (TXB(2)) by 98%, indices of COX-1 activity. In contrast, nimesulide had no significant effect on platelet aggregation, although it reduced serum TXB(2) by 29%. Production of prostaglandin E(2) and prostacyclin by gastric biopsies, also COX-1 dependent, was inhibited by naproxen, but not by nimesulide. COX-2 activity, determined as endotoxin induced prostaglandin E(2) formation in plasma, was markedly suppressed by both treatments. INTERPRETATION: Nimesulide has preferential selectivity for COX-2 over COX-1 in vivo at full therapeutic doses and induces less gastrointestinal damage than that seen with naproxen in the short term.

Adolescent↗
PubMed2001

Inhibition of cyclooxygenase-2 aggravates doxorubicin-mediated cardiac injury in vivo.

The clinical use of doxorubicin, an anthracycline chemotherapeutic agent, is limited by cardiotoxicity, particularly when combined with herceptin, an antibody that blocks the HER2 receptor. Doxorubicin induces cyclooxygenase-2 (COX-2) activity in rat neonatal cardiomyocytes. This expression of COX-2 limits doxorubicin-induced cardiac cell injury, raising the possibility that the administration of a prostaglandin may protect the heart during the in vivo administration of doxorubicin. Doxorubicin (15 mg/kg) administered to adult male Sprague Dawley rats induced COX-2 expression and activity in cardiac tissue. Prostacyclin generation measured as the excretion of 2,3-dinor-6-keto-PGF(1alpha) also increased, and this was blocked by a COX-2 inhibitor, SC236. In contrast, administration of a COX-1 inhibitor SC560 at a dose that reduced serum thromboxane B2 by more than 80% did not prevent the doxorubicin-induced increase in prostacyclin generation. Doxorubicin increased cardiac injury, detected as a rise in plasma cardiac troponin T, serum lactate dehydrogenase, and cardiomyocyte apoptosis; this was aggravated by coadministration of SC236 but not SC560. The degree of injury in animals treated with a combination of doxorubicin and SC236 was attenuated by prior administration of the prostacyclin analogue iloprost. These data raise the possibility of protecting the heart during the administration of doxorubicin by prior administration of prostacyclin.

6-Ketoprostaglandin F1 alpha↗
PubMed2001

Vascular biology of thrombosis: the role of platelet-vessel wall adhesion.

Arterial thrombosis is initiated after atherosclerotic plaque rupture. The ruptured plaque triggers a cascade of platelet-mediated events that result in the formation of a platelet-rich thrombus. Thrombus development ultimately leads to vessel occlusion, the precipitating event in most myocardial infarctions and many strokes. Arterial thrombosis involves a process of platelet adhesion, activation, and aggregation. Platelets first form a monolayer by adhering to blood vessel subendothelial matrix proteins (collagen and von Willebrand factor) via interactions with platelet membrane glycoprotein (GP) receptors. This interaction produces an intracellular signal that activates platelets to increase expression of a specific GP receptor, GPIIb/IIIa, with a high affinity for fibrinogen and other adhesion molecules. Binding of fibrinogen to adjacent platelets results in irreversible platelet aggregation and the formation of the platelet aggregate. Understanding of this process has led to considerable research into potential antiplatelet compounds. A number of effective antiplatelet drugs are now available, which affect different stages along the platelet adhesion/activation/aggregation pathway. These include aspirin, dipyridamole, and the adenosine diphosphate antagonists. Much effort is now focused on development of thrombin receptor antagonists. However, this is proving to be difficult. Considerable interest has surrounded a new class of platelet drugs, the GPIIb/IIIa receptor antagonists. These drugs have proved effective in short-term infusion trials. However, long-term studies of oral GPIIb/IIIa receptor antagonists have shown increases in mortality. It is believed that these antagonists may act as partial agonists and may increase the risk for thrombosis.

Cerebral Arteries↗
PubMed2001

Occupancy of the internal and external pools of glycoprotein IIb/IIIa following abciximab bolus and infusion.

The internal pool of GPIIb/IIIa, which is expressed upon platelet activation, may be inaccessible to inhibition by GPIIb/IIIa antagonists. To determine the occupancy of the internal and external pools of GPIIb/IIIa and platelet function following an abciximab bolus and infusion, 15 patients undergoing elective percutaneous transluminal coronary angioplasty were administered abciximab as a bolus and 36-h infusion. GPIIb/IIIa receptor number and occupancy in resting and TRAP-6 (20 microM)-activated samples (to expose the internal pool of GPIIb/IIIa) was quantified using a monoclonal antibody-based assay. Antibody binding was quantified by flow cytometry and platelet inhibition by light transmittance aggregation and by the rapid platelet function analyser (Accumetrics, San Diego, CA). The target of >80% receptor occupancy (range 82--99% occupancy) of the external pool of GPIIb/IIIa was achieved in all patients at 3 min. Receptor occupancy of the combined internal and external pools of GPIIb/IIIa was less, ranging from 75 to 93% and again was maximal at 3 min. Platelet aggregation was markedly inhibited to 20 microM ADP (maximal, 11 +/- 2% of baseline), but less so to 5 microM TRAP-6 (maximal, 36 +/- 25% of baseline). Following discontinuation of the drug, there was a gradual fall in receptor occupancy over 15 days coinciding with the disappearance of abciximab from the platelet surface. Maximum inhibition of platelet function and receptor occupancy of the external pool of GPIIb/IIIa occurs within 3 min of an abciximab bolus and infusion. However, some internal receptors that are expressed by potent agonists are not occupied, which may explain the incomplete inhibition of platelet aggregation.

Abciximab↗
PubMed2000

The platelet integrin alpha IIbbeta 3 has an endogenous thiol isomerase activity.

Integrins are cysteine-rich heterodimeric cell-surface adhesion molecules that alter their affinity for ligands in response to cellular activation. The molecular mechanisms involved in this activation of integrins are not understood. Treatment with the thiol-reducing agent, dithiothreitol, can induce an activation-like state in many integrins suggesting that cysteine-cysteine dithiol bonds are important for the receptor's tertiary structure and may be involved in activation-induced conformational changes. Here we demonstrate that the platelet-specific integrin, alpha(IIb)beta(3), contains an endogenous thiol isomerase activity, predicted from the presence of the tetrapeptide motif, CXXC, in each of the cysteine-rich repeats of the beta(3) polypeptide. This motif comprises the active site in enzymes involved in disulfide exchange reactions, including protein-disulfide isomerase (EC ) and thioredoxin. Intrinsic thiol isomerase activity is also observed in the related integrin, alpha(v)beta(3), which shares a common beta-subunit. Thiol isomerase activity within alpha(IIb)beta(3) is time-dependent and saturable, and is inhibited by the protein-disulfide isomerase inhibitor, bacitracin. Furthermore, this activity is calcium-sensitive and is regulated in the EDTA-stabilized conformation of the integrin. This novel demonstration of an enzymatic activity associated with an integrin subunit suggests that altered thiol bonding within the integrin or its substrates may be locally modified during alpha(IIb)beta(3) activation.

Amino Acid Sequence↗
PubMed2000

Evidence of platelet activation during treatment with a GPIIb/IIIa antagonist in patients presenting with acute coronary syndromes.

OBJECTIVES: The study was done to determine the role of partial agonist activity in the lack of effectiveness of the oral GPIIb/IIIa antagonist orbofiban. BACKGROUND: Orbofiban, an oral GPIIb/IIIa antagonist, was found to increase the mortality of patients with acute coronary syndromes (ACS) in the OPUS-TIMI-16 trial, despite the fact that it is a very potent anti-platelet agent and that IV agents have proven very effective. METHODS: Patients (n = 520) with ACS were randomized to orbofiban 30 mg, 40 mg or 50 mg twice daily or 50 mg once daily or placebo. Platelet activity was assessed in 175 patients by examining GPIIb/IIIa receptor conformation, expression of CD63 antigen, and platelet aggregation. RESULTS: Plasma concentrations of orbofiban at the highest dose (74 +/- 6 ng/ml peak, 61 +/- 5 ng/ml trough) exceeded the IC50 for platelet aggregation to adenosine diphosphate (ADP) (29 +/- 6 ng/ml) and thrombin-activating peptide (61 +/- 18 ng/ml). Orbofiban induced a conformational change in GPIIb/IIIa detected as the displacement of the monoclonal antibody mAb2; such conformational changes have been linked to partial agonist activity. Consistent with this, platelet expression of CD63 ex vivo was significantly increased at five time points during the study. In vitro, orbofiban increased platelet aggregation to a submaximal concentration of epinephrine (67 +/- 19% vs. 27 +/- 9%, n = 5) and increased thromboxane formation when the platelet GPIIb/IIIa were clustered using monoclonal antibodies to the receptor. CONCLUSIONS: Orbofiban is both an antagonist and a partial agonist of platelet GPIIb/IIIa. At low concentrations of the drug, this partial agonist activity may enhance platelet aggregation. Along with suboptimal plasma drug levels, these findings may help explain the lack of efficacy seen with orbofiban in patients with ACS.

Acute Disease↗
PubMed2000

Cyclooxygenase-1 and -2-dependent prostacyclin formation in patients with atherosclerosis.

BACKGROUND: The formation of prostacyclin (PGI(2)), thromboxane (TX) A(2), and isoprostanes is markedly enhanced in atherosclerosis. We examined the relative contribution of cyclooxygenase (COX)-1 and -2 to the generation of these eicosanoids in patients with atherosclerosis. METHODS AND RESULTS: The study population consisted of 42 patients with atherosclerosis who were undergoing surgical revascularization. COX-2 mRNA was detected in areas of atherosclerosis but not in normal blood vessel walls, and there was evidence of COX-1 induction. The use of immunohistochemical studies localized the COX-2 to proliferating vascular smooth muscle cells and macrophages. Twenty-four patients who did not previously receive aspirin were randomized to receive either no treatment or nimesulide at 24 hours before surgery and then for 3 days. Eighteen patients who were receiving aspirin were continued on a protocol of either aspirin alone or a combination of aspirin and nimesulide. Urinary levels of 11-dehydro-TXB(2) and 2,3-dinor-6-keto-PGF(1alpha), metabolites of TXA(2) and PGI(2), respectively, were elevated in patients with atherosclerosis compared with normal subjects (3211+/-533 versus 679+/-63 pg/mg creatinine, P<0.001; 594+/-156 versus 130+/-22 pg/mg creatinine, P<0.05, respectively), as was the level of the isoprostane 8-iso-PGF(2alpha). Nimesulide reduced 2, 3-dinor-6-keto-PGF(1alpha) excretion by 46+/-5% (378.3+/-103 to 167+/-37 pg/mg creatinine, P<0.01) preoperatively and blunted the increase after surgery. Nimesulide had no significant effect on 11-dehydro-TXB(2) before (2678+/-694 to 2110+/-282 pg/mg creatinine) or after surgery. The levels of both products were lower in patients who were taking aspirin, and no further reduction was seen with the addition of nimesulide. None of the treatments influenced urinary 8-iso-PGF(2alpha) excretion. CONCLUSIONS: Both COX-1 and -2 are expressed and contribute to the increase in PGI(2) in patients with atherosclerosis, whereas TXA(2) is generated by COX-1.

6-Ketoprostaglandin F1 alpha↗
PubMed2000

Surface expression of glycoprotein ib alpha is dependent on glycoprotein ib beta: evidence from a novel mutation causing Bernard-Soulier syndrome.

Bernard-Soulier syndrome is a rare bleeding disorder caused by a quantitative or qualitative defect in the platelet glycoprotein (GP) Ib-IX-V complex. The complex, which serves as a platelet receptor for von Willebrand factor, is composed of 4 subunits: GPIb alpha, GPIb beta, GPIX, and GPV. We here describe the molecular basis of a novel form of Bernard-Soulier syndrome in a patient in whom the components of the GPIb-IX-V complex were undetectable on the platelet surface. Although confocal imaging confirmed that GPIb alpha was not present on the platelet surface, GPIb alpha was readily detectable in the patient's platelets. Moreover, immunoprecipitation of plasma with specific monoclonal antibodies identified circulating, soluble GPIb alpha. DNA-sequence analysis revealed normal sequences for GPIb alpha and GPIX. There was a G to A substitution at position 159 of the gene encoding GPIb beta, resulting in a premature termination of translation at amino acid 21. Studies of transient coexpression of this mutant, W21stop-GPIb beta, together with wild-type GPIbalpha and GPIX, demonstrated a failure of GPIX expression on the surface of HEK 293T cells. Similar results were obtained with Chinese hamster ovary alpha IX cells, a stable cell line expressing GPIbalpha that retains the capacity to re-express GPIX. Thus, we found that GPIbbeta affects the surface expression of the GPIb-IX complex by failing to support the insertion of GPIb alpha and GPIX into the platelet membrane. (Blood. 2000;96:532-539)

Animals↗
PubMed2000

Glycoprotein IIb/IIIa antagonists induce apoptosis in rat cardiomyocytes by caspase-3 activation.

The platelet integrin glycoprotein (GP) IIb/IIIa, which mediates platelet aggregation, has been the target for novel antiplatelet agents, the GPIIb/IIIa antagonists. Several GPIIb/IIIa antagonists have been developed based on the peptide RGDS present in adhesion proteins, including the principle ligand fibrinogen. The apoptosis enzyme, procaspase-3, contains an RGD-recognition sequence and is activated by RGDS. We examined the effects of RGDS and several GPIIb/IIIa antagonists on cell death and procaspase-3 activation in rat neonatal cardiomyocytes. These antagonists do not recognize rat integrins, yet RGDS, orbofiban, and xemilofiban induced dose-dependent apoptosis and procaspase-3 activation in cardiomyocytes over 72 h, particularly under hypoxic conditions. Scrambled peptide, the monoclonal antibody 7E3 or integrelin (a peptide containing a KGD sequence), had little or no effect. Immunoprecipitation of procaspase-3 followed by treatment with the compounds showed that procaspase-3 was activated directly by RGDS, orbofiban, xemilofiban, and by monoclonal 7E3 antibody, the latter demonstrating that compounds must enter cells to induce apoptosis through caspase activation. Integrelin had no effect. Binding studies with (3)H-SC52012B, a GPIIb/IIIa antagonist analogue of orbofiban, showed no specific binding to cardiomyocytes, but the radioligand accumulated intracellularly over 72 h. (3)H-SC52012B also bound directly to human recombinant caspase-3 (K(d), 59 +/- 2 nm), and this was prevented by orbofiban, xemilofiban, and the monoclonal 7E3 antibody but not by integrelin. Finally confocal microscopy showed that RGDS co-localized with caspase-3 inside the cell. These data show that RGDS and its mimetics induce cardiomyocyte apoptosis by direct activation of procaspase-3.

Alanine↗
PubMed2000

Streptokinase-induced platelet activation involves antistreptokinase antibodies and cleavage of protease-activated receptor-1.

Streptokinase activates platelets, limiting its effectiveness as a thrombolytic agent. The role of antistreptokinase antibodies and proteases in streptokinase-induced platelet activation was investigated. Streptokinase induced localization of human IgG to the platelet surface, platelet aggregation, and thromboxane A(2) production. These effects were inhibited by a monoclonal antibody to the platelet Fc receptor, IV.3. The platelet response to streptokinase was also blocked by an antibody directed against the cleavage site of the platelet thrombin receptor, protease-activated receptor-1 (PAR-1), but not by hirudin or an active site thrombin inhibitor, Ro46-6240. In plasma depleted of plasminogen, exogenous wild-type plasminogen, but not an inactive mutant protein, S(741)A plasminogen, supported platelet aggregation, suggesting that the protease cleaving PAR-1 was streptokinase-plasminogen. Streptokinase-plasminogen cleaved a synthetic peptide corresponding to PAR-1, resulting in generation of PAR-1 tethered ligand sequence and selectively reduced binding of a cleavage-sensitive PAR-1 antibody in intact cells. A combination of streptokinase, plasminogen, and antistreptokinase antibodies activated human erythroleukemic cells and was inhibited by pretreatment with IV.3 or pretreating the cells with the PAR-1 agonist SFLLRN, suggesting Fc receptor and PAR-1 interactions are necessary for cell activation in this system also. Streptokinase-induced platelet activation is dependent on both antistreptokinase-Fc receptor interactions and cleavage of PAR-1. (Blood. 2000;95:1301-1308)

Adenosine Diphosphate↗
PubMed2000

Polymorphisms in glycoprotein (GP) lb alpha are not associated with adverse outcomes in primigravidae.

The platelet glycoprotein lb-IX complex is critical in platelet adhesion under conditions of high shear stress. Three genetic polymorphisms in the GPlbalpha subunit have been variably associated with thrombotic disorders. Women with serious obstetrical complications have an increased incidence of polymorphisms in other coagulation factors predisposing them alpha to thrombosis. Three GPlb variants were genotyped in 271 women in an Irish primigravid population, 258 with recorded outcomes, and in 22 patients with recurrent thrombosis and 15 patients with recurrent fetal loss. These were: the 13 amino acid variable number of tandem repeats (VNTR A/B/C/D), the Thr145Met antigen (HPA2) and a variant in the 'Kozak' sequence 5 bases upstream of the methionine initiation codon (T-5C). The VNTR B and HPA2b (145Met) alleles were strongly associated with each other, as found in previous analyses of Caucasian populations (estimated haplotype frequency 5%). The Kozak rarer C variant was found at an allele frequency of 9.1%, and appeared completely associated with the commonest haplotype of the other markers (VNTR/C, 145Thr). GPlb alpha genotype did not have any marked influence on pregnancy outcome. The Kozak/C variant was not significantly elevated in the 15 primigravidae patients who miscarried compared with 233 normal outcomes (OR=2.09, CI=0.7-6.4), neither was the 145Met variant (P =0.38). The Kozak/C variant was not elevated in the six primigravidae patients who developed pre-eclampsia (OR=0.84, CI=0.1-7.3). Comparison of the 271 primigravidae with 15 recurrent fetal loss patients showed no association between Kozak/C and recurrent fetal loss (OR=0.65, CI=0.1-3.0), nor with the 145Met variant (OR=0.62, CI=0.1-4.9). Combining miscarriage cases, recurrent thrombosis cases, pre-eclampsia, and intrauterine growth retardation outcome groups together, there is still no significant genotypic difference compared with normal outcome (OR=0.90, CI=0.4-1.9). Genetic variants of GPlbalpha are unlikely to contribute to adverse events associated with thrombosis in a primigravidae population.

Journal Article↗
PubMed2000

Prospective evaluation of the risk conferred by factor V Leiden and thermolabile methylenetetrahydrofolate reductase polymorphisms in pregnancy.

Factor V (FV) Leiden and thermolabile methylenetetrahydrofolate reductase (MTHFR) are 2 common polymorphisms that have been implicated in vascular thrombosis. We determined whether these mutations predicted an adverse outcome in pregnancy. Second, we looked for an interaction between these 2 mutations in patients with recurrent fetal loss or thrombosis in pregnancy. Primigravid subjects at their booking visit to the National Maternity Hospital (Holles Street, Dublin, Ireland) were screened for the polymorphisms. Thermolabile MTHFR and FV Leiden genotypes were detected by either restriction fragment length polymorphism or heteroduplex capillary chromatography. The carrier frequency of FV Leiden in the screened primigravid population was 2.7% (allele frequency 1.36%), all being heterozygous for the mutation. This value was lower than expected from previous studies in European populations. Forty-nine percent of the screened population (289 of 584) were heterozygous for thermolabile MTHFR, and 10.6% were homozygous (62 of 584). The frequency of the 2 polymorphisms was no higher in those who subsequently developed preeclampsia (n=12) or intrauterine growth retardation (n=9), and none of the screened population developed thrombosis. However, the frequency of FV Leiden was higher in patients who subsequently miscarried after the first trimester of pregnancy (allele frequency of 5.5%, P=0.0356). Among those positive for FV Leiden, 3 of 27 miscarried, compared with 24 of 572 of FV Leiden-negative patients (11% versus 4.2%). No interaction was found between the 2 mutations in the control or patient populations. In patients with a prior history of venous thrombosis, the carrier rate of FV Leiden was increased (4 of 33, allele frequency of 7.6%, P=0. 0115). In contrast, the carrier frequency for thermolabile MTHFR was no higher, and there was no interaction between the 2 mutations. Neither mutation occurred at a significantly higher frequency in patients with a prior history of recurrent fetal loss. In conclusion, FV Leiden is a risk factor for thrombosis in pregnancy and possibly for second-trimester miscarriage independent of thermolabile MTHFR. However, prospective analysis suggests that the risk conferred by FV Leiden is low in a primigravid population. The thermolabile MTHFR genotype was not implicated in any adverse outcome.

Abortion, Habitual↗
PubMed2000

An automated heteroduplex assay for the Pi(A) polymorphism of glycoprotein IIb/IIIa, multiplexed with two prothrombotic genetic markers.

Screening for the PIA polymorphism has been made faster and simpler with the advent of heteroduplex technology. Simultaneous screening for three common prothrombotic polymorphisms pl(A), factor V Leiden, and MTHFR(C677T) has been achieved with multiplex heteroduplex analysis. We describe a quick and simple method for PlA heteroduplex probe production. The probe was multiplexed with heteroduplex probes for MTHFR(C677T) and factor V Leiden polymorphisms in a one tube assay, allowing rapid automated genotyping of all three. This automated multiplex assay was applied to a cohort of 165 patients and showed excellent correlation with gel-based assays, both PAGE and RFLP. This approach will facilitate the analysis of multiple polymorphisms in complex disease in large populations.

Cohort Studies↗
PubMed2000

Glycoprotein IIb/IIIa receptor number and occupancy during chronic administration of an oral antagonist.

Long-term treatment with oral glycoprotein (GP)IIb/IIIa antagonists has failed to produce significant clinical benefit. We have examined the pharmacology of xemilofiban in the evaluation of oral xemilofiban in controlling thrombotic events (EXCITE) trial. The EXCITE trial was a multicenter study of xemilofiban in 7232 patients undergoing percutaneous coronary intervention. Thirty-two patients randomized to xemilofiban (10 or 20 mg three times daily) or placebo were followed for up to 6 months. GPIIb/IIIa receptor number and occupancy were quantified using two monoclonal antibodies mAb1 and mAb2. mAb1 was used to quantify receptor number. mAb2 recognizes an epitope that is lost due to a ligand-induced conformational change in GPIIb/IIIa and is a marker of receptor occupancy. Platelet aggregation was performed by light transmission. In vitro, the active metabolite of xemilofiban (SC-54701) inhibited mAb2 binding (IC(50) of 0.5 +/- 0.1 x 10(-8) M) but not mAb1. In vivo, long-term therapy with xemilofiban did not alter GPIIb/IIIa receptor number. mAb2 binding was inhibited throughout the treatment period and recovered slowly after drug withdrawal. Maximum inhibition of ADP-induced aggregation occurred at 4 to 7 h after the first dose of study medication. However, inhibition of platelet aggregation was low (between 24 and 45%) before dosing on days 60 and 180. There was no significant rebound increase in platelet aggregation after drug withdrawal. Long-term xemilofiban therapy does not alter platelet GPIIb/IIIa receptor number. Inhibition of platelet aggregation was poor at the end of each dosing interval and this may explain the failure of xemilofiban to alter clinical events.

Adenosine Diphosphate↗
PubMed1999

DNA distortion as a factor in nucleosome positioning.

In a previous report we constructed a synthetic DNA sequence that directed the deposition of histone octamers to a single site, and it was proposed that DNA distortion was involved in the positioning effect. In the present study we utilized the chemical probe potassium permanganate to identify sites of DNA distortion in the synthetic positioning sequence. A permanganate hypersite was identified 15 bp from the nucleosome pseudo-dyad at a site known to display DNA distortion in the mature nucleosome. The sequence of the site contained a TA step flanked by an oligo-pyrimidine tract. A series of substitutions were made in the region of the permanganate hypersite and the resulting constructs tested for affinity for histone octamers and translational positioning in in vitro studies. The results revealed that either a single base substitution at the TA step or in the adjacent homopolymeric tract dramatically affected affinity and positioning activity. The rotational orientation of the permanganate-sensitive sequence was shown to be important for functions, since altering the orientation of the site in a positioning fragment reduced positioning activity and octamer affinity, while altering the rotational orientation of the sequence in a non-positioning fragment had the opposite effects. A reconstituted 5 S rDNA positioning sequence from Lytechinus variegatus was also shown to display a permanganate hypersite 16 bp from its pseudo-dyad.

Animals↗