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PubMed2002

The conformation of the mature dimeric human immunodeficiency virus type 1 RNA genome requires packaging of pol protein.

The packaging of a mature dimeric RNA genome is an essential step in human immunodeficiency virus type 1 (HIV-1) replication. We have previously shown that overexpression of a protease (PR)-inactive HIV-1 Gag-Pro-Pol precursor protein generates noninfectious virions that contain mainly monomeric RNA (M. Shehu-Xhilaga, S. M. Crowe, and J. Mak, J. Virol. 75:1834-1841, 2001). To further define the contribution of HIV-1 Gag and Gag-Pro-Pol to RNA maturation, we analyzed virion RNA dimers derived from Gag particles in the absence of Gag-Pro-Pol. Compared to wild-type (WT) dimeric RNAs, these RNA dimers have altered mobility and low stability under electrophoresis conditions, suggesting that the HIV-1 Gag precursor protein alone is not sufficient to stabilize the dimeric virion RNA structure. The inclusion of an active viral PR, without reverse transcriptase (RT) and integrase (IN), rescued the stability of the virion RNA dimers in the Gag particles but did not restore the mobility of the RNAs, suggesting that RT and IN are also required for virion RNA dimer maturation. Thin-section electron microscopy showed that viral particles deficient in RT and IN contain empty cone-shaped cores. The abnormal core structure indicates a requirement for Gag-Pro-Pol packaging during core maturation. Supplementing viral particles with either RT or IN via Vpr-RT or Vpr-IN alone did not correct the conformation of the dimer RNAs, whereas expression of both RT and IN in trans as a Vpr-RT-IN fusion restored RNA dimer conformation to that of the WT virus and also restored the electron-dense, cone-shaped virion core characteristic of WT virus. Our data suggest a role for RT-IN in RNA dimer conformation and the formation of the electron-dense viral core.

Cell Line↗
PubMed2003

Sequences downstream of the 5' splice donor site are required for both packaging and dimerization of human immunodeficiency virus type 1 RNA.

Two copies of human immunodeficiency virus type 1 RNA are incorporated into each virus particle and are further converted to a stable dimer as the virus particle matures. Several RNA segments that flank the 5' splice donor site at nucleotide (nt) 289 have been shown to act as packaging signals. Among these, RNA stem-loop 1 (SL1) (nt 243 to 277) can trigger RNA dimerization through a "kissing-loop" mechanism and thus is termed the dimerization initiation site. However, it is unknown whether other packaging signals are also needed for dimerization. To pursue this subject, we mutated stem-loop 3 (SL3) (nt 312 to 325), a GA-rich region (nt 325 to 336), and two G-rich repeats (nt 363 to 367 and nt 405 to 409) in proviral DNA and assessed the effects on RNA dimerization by performing native Northern blot analyses. Our results show that the structure but not the specific RNA sequence of SL3 is needed not only for efficient viral RNA packaging but also for dimerization. Mutations of the GA-rich sequence severely diminished viral RNA dimerization as well as packaging; the combination of mutations in both SL3 and the GA-rich region led to further decreases, implying independent roles for each of these two RNA motifs. Compensation studies further demonstrated that the RNA-packaging and dimerization activity of the GA-rich sequence may not depend on a putative interaction between this region and a CU repeat sequence at nt 227 to 233. In contrast, substitutions in the two G-rich sequences did not cause any diminution of viral RNA packaging or dimerization. We conclude that both the SL3 motif and GA-rich RNA sequences, located downstream of the 5' splice donor site, are required for efficient RNA packaging and dimerization.

Animals↗
PubMed2004

Nucleic acid binding-induced Gag dimerization in the assembly of Rous sarcoma virus particles in vitro.

As also found for other retroviruses, the Rous sarcoma virus structural protein Gag is necessary and sufficient for formation of virus-like particles (VLPs). Purified polypeptide fragments comprising most of Gag spontaneously assemble in vitro at pH 6.5 into VLPs lacking a membrane, a process that requires nucleic acid. We showed previously that the minimum length of a DNA oligonucleotide that can support efficient assembly is 16 nucleotides (nt), twice the protein's binding site size. This observation suggests that the essential role of nucleic acid in assembly is to promote the formation of Gag dimers. In order to gain further insight into the role of dimerization, we have studied the assembly properties of two proteins, a nearly full-length Gag (deltaMBDdeltaPR) capable of proper in vitro assembly and a smaller Gag fragment (CTD-NC) capable of forming only irregular aggregates but with the same pH and oligonucleotide length requirements as for assembly with the larger protein. In analyses by sedimentation velocity and by cross-linking, both proteins remained monomeric in the absence of oligonucleotides or in the presence of an oligonucleotide of length 8 nt (GT8). At pH 8, which does not support assembly, binding to GT16 induced the formation of dimers of deltaMBDdeltaPR but not of CTD-NC, implying that dimerization requires the N-terminal domain of the capsid moiety of Gag. Assembly of VLPs was induced by shifting the pH of dimeric complexes of deltaMBDdeltaPR and GT16 from 8 to 6.5. An analogue of GT16 with a ribonucleotide linkage in the middle also supported dimer formation at pH 8. Even after quantitative cleavage of the oligonucleotide by treatment of the complex with RNase, these dimers could be triggered to undergo assembly by pH change. This result implies that protein-protein interactions stabilize the dimer. We propose that binding of two adjacent Gag molecules on a stretch of nucleic acid leads to protein-protein interactions that create a Gag dimer and that this species has an exposed surface not present in monomers which allows polymerization of the dimers into a spherical shell.

Avian Sarcoma Viruses↗
PubMed2002

Promoter specificity and biological activity of tethered AP-1 dimers.

Activator protein 1 (AP-1) is a group of dimeric transcription factors composed of Jun, Fos, and ATF family proteins. Both gain- and loss-of-function studies have revealed specific roles for individual AP-1 components in cell proliferation, differentiation, apoptosis, and other biological processes. However, little is known about the functions of specific AP-1 dimers. To test the importance of AP-1 composition in transcriptional activation, AP-1 monomers were joined via a flexible polypeptide tether to force specific pairing. The resultant single-chain AP-1 molecules showed DNA binding specificity and transcriptional activation potentials similar to those of untethered dimers, even in the presence of dominant-negative AP-1 monomers. c-Jun-containing dimers showed distinct promoter specificity in transient-transfection experiments, depending on the Fos, Fra, or ATF partner. When stably expressed in NIH 3T3 cells, c-Jun tethered dimer Fra2, but not c-Jun tethered dimer Fra1 and c-Jun tethered dimer cFos (the tilde indicates a tethered dimer), inhibited G(0) arrest at confluency and under low-serum conditions and specifically activated cyclin A expression. These data suggest that the choice of dimerization partner defines the role of c-Jun in gene activation and cell cycle regulation and that single-chain AP-1 molecules provide a powerful tool for assessing the role of specific AP-1 dimers.

3T3 Cells↗
PubMed2003

Adhesive and lateral E-cadherin dimers are mediated by the same interface.

E-cadherin is a transmembrane protein that mediates Ca(2+)-dependent cell-cell adhesion. To study cadherin-cadherin interactions that may underlie the adhesive process, a recombinant E-cadherin lacking free sulfhydryl groups and its mutants with novel cysteines were expressed in epithelial A-431 cells. These cysteine mutants, designed according to various structural models of cadherin dimers, were constructed to reveal cadherin dimerization by the bifunctional sulfhydryl-specific cross-linker BM[PE0]3. Cross-linking experiments with the mutants containing a cysteine at strand B of their EC1 domains did show cadherin dimerization. By their properties these dimers correspond to those which have been characterized by co-immunoprecipitation assay. Under standard culture conditions the adhesive dimer is a dominant form. Calcium depletion dissociates adhesive dimers and promotes the formation of lateral dimers. Our data show that both dimers are mediated by the amino-terminal cadherin domain. Furthermore, the interfaces involved in both adhesive and lateral dimerization appear to be the same. The coexistence of the structurally identical adhesive and lateral dimers suggests some flexibility of the extracellular cadherin region.

Binding Sites↗
PubMed2002

Human T cells resistant to complement lysis by bivalent antibody can be efficiently lysed by dimers of monovalent antibody.

We have previously shown that bivalent human gamma1 CD3 monoclonal antibody (mAb) is ineffective at mediating lysis of human T cells with human complement. In this paper we have used genetic engineering and sulfur chemistry to prepare 2 types of human gamma1 CD3 mAb dimer, with the aim of improving complement lysis activity. The IgG molecules forming the dimers were linked together at their C-termini by stable bismaleimide thioether bridges. The first dimer was composed of 2 bivalent mAb molecules. This dimer proved incapable of lysing human T-cell blasts with human, rabbit, or guinea pig complement. The second dimer consisted of 2 molecules of a monovalent derivative (possessing a single Fab domain) of the bivalent mAb. This dimer was highly lytic with human complement, with a lytic titer 64-fold greater than that of the nondimerized monovalent mAb. The maximum level of lysis of human T-cell blasts achieved with this monovalent mAb dimer was equal to that obtained with the therapeutic antilymphocyte mAb alemtuzumab, but its lytic titer was 4-fold greater. The monovalent mAb dimer was also found to be lytic in the presence of rabbit and guinea pig complement. Dimerization of monovalent antibodies may provide a general strategy for improving the cytolytic activity of other mAbs that are normally unable to induce lysis with complement. The monovalent CD3 mAb dimer may have potential for development as an agent for immunotherapy of T-cell leukemia.

Antibodies, Monoclonal↗
PubMed1997

Activities of tRNA-embedded dimeric minizymes.

Ribozymes have been shown to be potent inhibitors of gene expression and viral function. Effects of ribozyme-mediated repression to target gene in living cells are correlated with the amounts of expression and stabilities of ribozyme molecules. In our previous study, it was demonstrated that a minimized hammerhead ribozyme, minizyme, with high activity forms a dimeric structure with a common stem II. We constructed dimeric minizymes that could cleave the BCR-ABL chimeric (b2a2) mRNA which had been difficult target for conventional hammerhead ribozymes without damaging the normal ABL mRNA. In order to achieve high expression of these dimeric minizymes in vivo for the treatment of CML, we embedded the dimeric minizyme portion downstream of a tRNA(Val) promoter sequence which could be recognized by RNA polymerase III. We determined cleavage activities of tRNA-embedded dimeric minizymes and compared the activities between tRNA-embedded hammerhead ribozyme and tRNA-embedded dimeric minizymes. All tRNA-embedded dimeric minizymes tested were capable of cleavage the target substrate. The activity of the tRNA-embedded dimeric minizyme targeted at BCR-ABL mRNA was almost the same as that of the naked dimeric minizymes. Interestingly, the cleavage activity of tRNA-embedded dimeric minizymes was higher than that of tRNA-embedded conventional hammerhead ribozyme.

Base Sequence↗
PubMed1996

Glycinamide ribonucleotide transformylase undergoes pH-dependent dimerization.

Glycinamide ribonucleotide transformylase (GART) exhibits closely packed dimers in all crystal forms (pH 6.75), but was demonstrated to be monomeric in solution under conditions of optimal catalytic efficiency (pH 7.5). We undertook a study of the pH-dependent behavior of GART in solution to determine whether side-chain ionization is responsible for the observed difference in association state. In the pH range 6.8 to 7.5, dimeric GART reversibly dissociates into a monomeric form as demonstrated by dynamic light scattering. The data give a best fit to a cooperative three-proton transfer mechanism: [formula: see text] A comparison of normalized data obtained from difference UV-absorption spectroscopy with the dynamic light scattering data indicates that two or more tyrosine residues per monomer undergo a local conformational change concomitant with dimerization. Fluorescence studies show that the environment of one or both of the tryptophan residues distal to the dimer interface are also perturbed by dimerization. Fitting of the normalized titration curves yields an apparent pKa = 7.16(+/-0.02) and a subnanomolar KD for the transition. Examination of the dimer interface in the crystal structure indicates that there are two histidine residues, H54 and H73, that are likely responsible for the pH-dependent dimerization. There are also two tyrosine residues, Y67 and Y78, which are adjacent to the interface and which may be exposed during dimerization. Our study indicates that under physiological pH conditions, GART exists as a mixture of monomer and dimer in solution. Taken together, the fact that the monomer-dimer transition displays a sharp pH dependence, and the fact that the enzyme activity is maximal under conditions where it is fully monomeric, suggest that enzyme activity may be modulated by subtle pH changes in the cell.

Acyltransferases↗
PubMed1985

Kinetics and importance of the dimerization step in the folding pathway of the beta 2 subunit of Escherichia coli tryptophan synthase.

During its folding, the polypeptide chain of the beta 2 subunit of Escherichia coli tryptophan synthase (L-serine hydrolyase (adding indole) EC 4.2.1.20) undergoes dimerization. To decide whether this dimerization precedes or follows the formation of the native, functional, tertiary structure of the polypeptide chain, the kinetics of renaturation of beta 2 are studied by monitoring both the regain of native conformation and the dimerization. Dimer formation is followed by the increase of the fluorescence polarization, or by energy transfer between a fluorescence donor and a fluorescence acceptor, which occur upon association of adequately labelled beta chains. Renaturation is followed by the regain of functional properties of beta 2, i.e. its ability to bind pyridoxal-5'-phosphate or to form a fluorescent ternary complex with this coenzyme and L-serine. It is shown that for beta 2 the dimerization obeys first-order kinetics, presumably because it occurs rapidly after a rate-limiting isomerization of the monomer. The dimerization is followed by another isomerization, taking place within the dimer, which leads to the formation of the pyridoxal-5'-phosphate binding site. Still another, slow, isomerization reaction involving the F1 (N-terminal) domain completes the renaturation. With a modified form of beta 2 (trypsin-nicked beta 2) where this isomerization of F1 can be made to occur before the dimerization, the dimer is also shown to appear before the functional properties. It is concluded that a non-native dimer indeed exists as an obligatory intermediate on the folding pathway of nicked beta 2 and of beta 2, and that interdomain interactions are needed to force the polypeptide chains into their native conformations.

Energy Transfer↗
PubMed1989

Monocyte procoagulant activity and membrane-associated D dimer after knee replacement surgery.

We have recently shown that monocyte membrane-associated cross-linked fibrin derivatives (D dimer) can be evidenced by immunogold staining. Using this method, the procoagulant activity (PCA) expressed in vitro by endotoxin-stimulated monocytes has been found to correlate significantly with the number of D dimer-positive monocytes. The incidence of postoperative thrombosis in patients undergoing total knee replacement has been reported by Stulberg et al to be 57%. Since monocytes can play a role, via increased PCA, in the activation of intravascular coagulation, we sought to determine the level of monocyte PCA ex vivo after knee replacement surgery and its possible correlation with the number of D dimer-positive monocytes. Finally, we examined the possible link between these modifications and the occurrence of postoperative deep vein thrombosis (DVT). The PCA expressed by monocytes with or without suboptimal stimulation, the number of D dimer-positive monocytes and the plasma level of D dimer were measured pre- and post-operatively in 11 patients undergoing total knee replacement. Phlebography was performed on day 10 after surgery. A significant increase in the PCA of stimulated monocytes was observed on day 10 after surgery. Moreover, both the number of D dimer-positive monocytes and the plasma level of D dimer increased significantly post-operatively. The number of D dimer-positive monocytes correlated with both monocyte PCA and the plasma D dimer level. The relation between these parameters is discussed. However, neither monocyte PCA nor the number of D dimer-positive monocytes was found to correlate with the occurrence of deep vein thrombosis.

Adult↗
PubMed2006

D-dimer level is associated with the extent of pulmonary embolism.

OBJECTIVES: Our aim was to study the association between the level of D-dimer and the severity of pulmonary embolism (PE) as determined by various biochemical and radiological prognostic markers in order to investigate the potential value of D-dimer as a prognostic marker for the severity of PE. PATIENTS AND METHODS: PE was diagnosed in 100 consecutive out-patients by multi-detector computerized tomography. One patient was excluded and the final cohort consisted of 99 patients. Pulmonary Artery Obstruction Index (PAOI) and Right Ventricular/Left Ventricular (RV/LV) ratio were assessed. RESULTS: The median value for D-dimer was 5.0 mg/L (inter-quartile range: 1.8, 12.2). There was a significant association between log D-dimer, and between log RV/LV (r=0.45), log PAOI (r=0.5), and PaO(2) (r=0.40). The multivariate analysis showed an increased association between log D-dimer and between log RV/LV ratio (r=0.54) and log PAOI (r=0.52) after adjusting for age, gender and for the duration of symptoms. Significant association was found between the level of D-dimer and the most proximal level of PE (p<0.0005). There was a significant dose-response relationship between the level D-dimer and between Troponin-T and the frequency of thrombolysis (p<0.0005). In the subgroup of patients with D-Dimer over the upper quartile (>12.2), 12 (67%) patients had elevated Troponin-T and 8 (32%) patients received thrombolysis, compared to 1 (5%) patient with elevated Troponin-T and none treated with thrombolysis in the subgroup of patients with D-dimer<lower quartile. CONCLUSIONS: We have shown that the level of D-dimer is related to the severity of PE assessed by various radiological, biochemical and clinical markers and might have a potential value as prognostic marker for the severity of PE.

Biomarkers↗
PubMed1989

The channel properties of possible gramicidin dimers.

Extending previous work (Sung & Jordan, 1987 a, Biophys. J. 51, 661-672; 1988, Biophys. J.54, 519-526), we describe channel properties of five possible gramicidin dimers by studying dimerization energies and axial electrical potentials. Unlike the head-to-head dimer (the predominant channel former), both tail-to-tail and head-to-tail dimers with the same beta-helical monomer structure as the head-to-head dimer only form four intermonomer hydrogen bonds and are much less stable. Were channels formed from these dimers to be observed, their electrical potential profiles suggest that they should be cation selective, probably conduct less than the head-to-head dimer, have a central cation binding site, bind cations preferentially if crystallizable, and in the case of the head-to-tail dimer, rectify. Like the antiparallel double stranded helical dimer (a possible minor conducting pathway) the parallel double stranded helical dimer has 28 interstrand hydrogen bonds, but its hydrogen bond network is quite distorted and it is much less stable. If it formed, its electrical potential profile suggests that it would be cation selective, bind anions preferentially if crystallizable, rectify, and at high enough voltages, might exhibit a conductance greater than that of the antiparallel form.

Electrochemistry↗
PubMed2003

Normal D-dimer levels do not exclude thrombotic complications in trauma patients.

BACKGROUND: Deep venous thrombosis (DVT) and pulmonary embolism (PE) are common complications in trauma patients. These diagnoses can be difficult and expensive to make. Recent studies report that a negative D-dimer test excludes thrombotic complications. We questioned the predictive value of a D-dimer test to exclude DVT and PE. METHODS: Adult trauma patients admitted March 1999 to March 2001, with an Injury Severity Score > or =9 and expected length of stay >3 days, were approached for enrollment. Bilateral lower extremity duplex ultrasounds and d-dimer levels were performed within 36 hours of admission, day 3-4, day 7, and weekly until discharge. RESULTS: Twenty-three patients were diagnosed with DVTs, with 18 DVTs detected within the first week of admission. Five DVT patients had normal D-dimer levels. One of three PE patients tested had a normal D-dimer level. The false negative rate for DVT by d-dimer assay was 24%, and the sensitivity was 76%. The negative predictive value for D-dimers was 92%. All false negative d-dimer tests occurred in patients diagnosed with DVT or PE within the 4 days after admission. CONCLUSION: In the early postinjury phase, a negative d-dimer test does not exclude DVT or PE. However, the negative predictive value of a D-dimer test after the first 4 days from admission rose to 100%. Patients with clinical signs and symptoms of DVT or PE in the immediate postinjury phase should undergo further screening to exclude thromboembolic complications.

False Negative Reactions↗
PubMed1999

Contrasting post-traumatic serial changes for D-dimer and PAI-1 in critically injured patients.

We measured D-dimer and plasminogen activator inhibitor-1 (PAI-1) activity in 45 trauma patients to assess their efficacy in predicting the post-traumatic hemostatic perturbations. We found the correlation between D-dimer measured by Simpli Red test and ELISA to be highly significant (p=0.0001). The D-dimer ELISA indicated that the serial changes of D-dimer after trauma were variable. However, the increases of D-dimer were associated with clinical conditions of the patient, such as trauma surgery, infections, or thrombotic complications. A significant correlation was found for D-dimer levels measured by ELISA versus the injury severity score (ISS) in all the trauma patients on day 1 (p=0.0153) and on day 2 (p=0.0495). The PAI-1 activity was increased at admission and showed a progessive decline from day 2 onward, and the correlation for the daily decline of PAI-1 was highly significant (p=0.0001). The PAI-1 activity and plasminogen activator activity showed a significant negative correlation on days 1, 2, and 3. PAI-1 activity correlated moderately with D-dimer level only on day 1 (p=0.0569). Three out of forty-five patients developed thrombotic complications: one patient who died from pulmonary embolism and two patients who developed adult respiratory distress syndrome (ARDS). In summary: 1) PAI-1 activity and D-dimer exhibited contrasting serial changes after trauma. 2) There was also a negative correlation between PAI-1 activity and PA activity. 3) A significant correlation of D-dimer with ISS confirms, as might be anticipated, that there is increased activation of the coagulation mechanism in severe injury, and suggests that D-dimer levels may prove useful to screen for patients at strong risks of thrombotic complications.

Adult↗
PubMed1985

Unexpected similarities in the crystal structures of the Mcg light-chain dimer and its hybrid with the Weir protein.

The covalently linked hybrid of two human lambda-type light chains (Mcg and Weir) crystallizes as trigonal bipyramids in ammonium sulfate [Ely et al., Molec. Immun. 22, 85-92 (1985)]. While markedly different in appearance from the barrel-shaped crystals of the parental Mcg dimer, the bipyramids of the hybrid have the same space group: trigonal P3(1)21. Moreover, the unit cell dimensions are practically identical: a = 72.3 A in both proteins; c = 188.1 A in the hybrid and 185.9 A in the Mcg dimer. These observations imply that the crystal packing and the main features of the three-dimensional structures are closely similar in the Mcg X Weir hybrid and the Mcg dimer. The "constant" domains of the Mcg and Weir proteins belong to the same genetic subclass and were expected to interact in comparable ways in hybrids and parental dimers. However, the overall similarities in the "variable" domain pairs in the hybrid and Mcg dimer were completely unpredicted, since the amino acid sequences of the heterologous variable domains differ by 36 residues. By difference Fourier analysis the Weir light chain has been tentatively identified as monomer 1 (heavy-chain analogue) and the Mcg protein as monomer 2 (light-chain analogue) in the hybrid dimer. Substitutions in key positions in the hypervariable loops explain the differences in binding activity of the Mcg and Weir dimers. In the Mcg dimer bis(dinitrophenyl)lysine spans two relatively spacious subsites (A and B), with primary contacts involving tyrosines 34 and 38 of monomer 2. The Weir dimer, which does not bind dinitrophenyl ligands, has serine and phenylalanine in homologous positions. Moreover, the bilateral replacement of valine 48 and serine 91 in Mcg by leucine and methionine in the Weir dimer should effectively block access to subsite B. In the hybrid binding activity for bis(dinitrophenyl)lysine is restored because the Mcg light chain is present as the monomer 2 subunit.

Amino Acid Sequence↗
PubMed2002

Normal D-dimer levels in emergency department patients suspected of acute pulmonary embolism.

OBJECTIVES: We sought to determine:1) whether normal D-dimer enzyme-linked immunosorbent assay (ELISA) assays predicted the absence of pulmonary embolism (PE) in the high-volume emergency department (ED) of the Brigham and Women's Hospital, and 2) whether ED physicians accepted normal D-dimer levels as confirmation of no PE without further diagnostic testing such as lung scanning, chest computed tomography (CT) scanning, or pulmonary angiography. BACKGROUND: Although the plasma D-dimer ELISA is a sensitive screening test for excluding acute PE, this laboratory marker has not been widely integrated into clinical algorithms such as creatine kinase-MB fraction or troponin testing for acute myocardial infarction. METHODS: We mandated that ED physicians order D-dimer ELISA tests on all patients suspected of acute PE. We reviewed the clinical record of each ED patient initially evaluated for suspected PE during the year 2000. We determined whether additional imaging tests for PE were obtained and whether the final diagnosis was PE. RESULTS: Of 1,106 D-dimer assays, 559 were elevated and 547 were normal. Only 2 of 547 had PE despite a normal D-dimer. The sensitivity of the D-dimer ELISA for acute PE was 96.4% (95% confidence interval [CI]: 87.5% to 99.6%), and the negative predictive value was 99.6% (95% CI: 98.7% to >99.9%). Nevertheless, 24% of patients with normal D-dimers had additional imaging tests for PE. CONCLUSIONS: The D-dimer ELISA has a high negative predictive value for excluding PE. By paying more attention to normal D-dimer results, fewer chest CT scans and lung scans will be required, and improvements may be realized in diagnostic efficiency and cost reduction.

Acute Disease↗
PubMed1996

Thrombogenicity of radiofrequency lesions: results with serial D-dimer determinations.

OBJECTIVES: By measuring plasma levels of D-dimer, a product of fibrin degradation, we sought to investigate whether the application of radiofrequency (RF) energy might activate endogenous thrombotic mechanisms. BACKGROUND: Quantitative determination of D-dimer, a bio-chemical marker of thrombus formation and reactive fibrinolysis, helps to diagnose activation of the coagulation system. It remains controversial whether endocardial lesions produced during RF catheter ablation of arrhythmogenic foci have a thrombogenic effect, and the issue of the need for antithrombotic therapy after RF ablation is still unresolved. METHODS: We made serial determinations of plasma D-dimer levels by enzyme immunoassay before insertion of catheters, after completion of electrophysiologic study (EPS) but before RF ablation, immediately after RF ablation and before discharge (at 48 h) from the hospital in 37 patients undergoing RF ablation (22 men, 15 women; mean [+/-SD] age 37 +/- 18 years, range 12 to 74; 16 +/- 16 lesions produced) of accessory (n = 17) or slow (n = 12) pathways, atrial (n = 4) or ventricular foci (n = 3) or the atrioventricular node (n = 1). D-dimer levels were also measured in 26 age-matched control subjects undergoing EPS only. RESULTS: In the RF ablation group, the mean D-dimer levels increased from a baseline value of 29 +/- 28 to 62 +/- 56 micrograms/liter after EPS (p < 0.0001). However, after RF ablation, D-dimer levels increased to much higher levels (188 +/- 138 micrograms/liter, p < 0.0001). There was no correlation of D-dimer levels with the number of RF lesions produced or the duration of the procedure. At 48 h after the procedure, D-dimer levels decreased (75 +/- 67 micrograms/liter) but still remained significantly elevated compared with baseline values (p = 0.0001). Three were no significant differences in baseline (25 +/- 21 micrograms/liter) and post-EPS (51 +/- 50 micrograms/liter) measurements between control subjects and patients. During RF ablation, intravenous heparin was given to nine patients who still demonstrated high plasma D-dimer levels after RF ablation. CONCLUSIONS: As reflected by elevated plasma D-dimer levels, RF ablation has a thrombogenic effect that persists through 48 h after the procedure. This effect needs to be taken into account when considering antithrombotic therapy in patients undergoing RF ablation.

Adolescent↗
PubMed1996

Cooperativity and anti-cooperativity between ligand binding and the dimerization of ristocetin A: asymmetry of a homodimer complex and implications for signal transduction.

BACKGROUND: Recent work has indicated that dimerization is important in the mode of action of the vancomycin group of glycopeptide antibiotics. NMR studies have shown that one member of this group, ristocetin A, forms an asymmetric dimer with two physically different binding sites for cell wall peptides. Ligand binding by ristocetin A and dimerization are slightly anti-cooperative. In contrast, for the other glycopeptide antibiotics of the vancomycin group that have been examined so far, binding of cell wall peptides and dimerization are cooperative. RESULTS: Here we show that the two halves of the asymmetric homodimer formed by ristocetin A have different affinities for ligand binding. One of these sites is preferentially filled before the other, and binding to this site is cooperative with dimerization. Ligand binding to the other, less favored half of the dimer, is anti-cooperative with dimerization. CONCLUSIONS: In dimer complexes, anti-cooperativity of dimerization upon ligand binding can be a result of asymmetry, in which two binding sites have different affinities for ligands. Such a system, in which one binding site is filled preferentially, may be a mechanism by which the cooperativity between ligand binding and dimerization is fine tuned and may thus have relevance to the control of signal transduction in biological systems.

Binding Sites↗