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PubMed1995

Photo-CIDNP study of pyrimidine dimer splitting. I: Reactions involving pyrimidine radical cation intermediates.

The light-induced splitting of pyrimidine dimers was studied using the electron acceptor anthraquinone-2-sulfonate (AQS) as a photosensitizer. To this end, photochemically induced dynamic nuclear polarization (photo-CIDNP) experiments were performed on a series of pyrimidine monomers and dimers. The CIDNP spectra demonstrate the existence of both the dimer radical cation, which is formed by electron transfer from the dimer to the photoexcited sensitizer AQS*, and its dissociation product, the monomer radical cation. In spectra of 1,1'-trimethylene bridged cis, syn pyrimidine dimers, polarization is observed that originates from a spin-sorting process in the dimer radical pair. This points to a relatively long lifetime of the dimer radical cation involved, which is presumably due to stabilization by the trimethylene bridge. Polarization originating from a dimer radical pair is detected in the spectrum of trans,anti (1,3-dimethyluracil) dimer as well. The spectra of the bridged pyrimidines also demonstrate the reversibility of the dissociation of dimer radical cation into monomer radical cation, which is concluded from the observation of polarization in the dimer as a result of spin sorting in the monomer radical pair.

Cations↗
PubMed2001

Role of stem B, loop B, and nucleotides next to the primer binding site and the kissing-loop domain in human immunodeficiency virus type 1 replication and genomic-RNA dimerization.

Stem-loop B is a 12-nucleotide [nt]-long completely conserved sequence postulated to form a 4-bp stem and a 4-nt internal loop under the kissing-loop hairpin (klh) (nt 248 to 270) of human immunodeficiency virus type 1 (HIV-1) genomic RNA. We investigated its role in viral replication, genomic RNA dimerization, and dimerization of partial HIV-1 RNA transcripts. The putative CUCG246-CGAG277 duplex was replaced by nine alternative complementary sequences, five likely to base pair only in short RNAs and four likely to base pair in long (approximately 500-nt) RNAs, as assessed by the algorithm mfold. Among the five former sequences, none preserved genome dimerization and all reduced viral replication by 98 to 99.9%. Among the four latter sequences, three (MB6, -9, and -10) preserved genome dimerization, one (MB7) did not significantly inhibit it, and two (MB9 and -10) preserved viral replication. We conclude that duplex formation by stem B nucleotides is necessary for viral infectivity and complete genome dimerization. Deleting the 5' or 3' side of loop B or of stem B had little impact on dimerization of partial RNA transcript and no impact on klh folding (and, for loop B mutations, on stem B folding), but each deletion inhibited genome dimerization almost as much as klh destruction. This suggests that loop B is required for complete genome dimerization and that loop B and stem B stimulate dimerization only in very long RNAs and/or in the presence of unidentified viral and cellular factors. Finally, we asked if nine deletions or nucleotide substitutions within nt 200 to 242 and/or nt 282 to 335 could influence genome dimerization. These mutations had intermediate inhibitory impacts consistent with their predicted influence on stem B, loop B, and klh formation. Two exceptions were Delta200-226 and Delta236-242 genomic RNAs, which dimerized relatively poorly despite having neutral or positive influences on stem B, loop B, and klh folding.

Animals↗
PubMed2002

Bipartite signal for genomic RNA dimerization in Moloney murine leukemia virus.

Retroviral virions each contain two identical genomic RNA strands that are stably but noncovalently joined in parallel near their 5' ends. For certain viruses, this dimerization has been shown to depend on a unique RNA stem-loop locus, called the dimer initiation site (DIS), that efficiently homodimerizes through a palindromic base sequence in its loop. Previous studies with Moloney murine leukemia virus (Mo-MuLV) identified two alternative DIS loci that can each independently support RNA dimerization in vitro but whose relative contributions are unknown. We now report that both of these loci contribute to the assembly of the Mo-MuLV dimer. Using targeted deletions, point mutagenesis, and antisense oligonucleotides, we found that each of the two stem-loops forms as predicted and contributes independently to dimerization in vitro through a mechanism involving autocomplementary interactions of its loop. Disruption of either DIS locus individually reduced both the yield and the thermal stability of the in vitro dimers, whereas disruption of both eliminated dimerization altogether. Similarly, the thermal stability of virion-derived dimers was impaired by deletion of both DIS elements, and point mutations in either element produced defects in viral replication that correlated with their effects on in vitro RNA dimerization. These findings support the view that in some retroviruses, dimer initiation and stability involve two or more closely linked DIS loci which together align the nascent dimer strands in parallel and in register.

Animals↗
PubMed2001

Dimerization of receptor protein-tyrosine phosphatase alpha in living cells.

BACKGROUND: Dimerization is an important regulatory mechanism of single membrane-spanning receptors. For instance, activation of receptor protein-tyrosine kinases (RPTKs) involves dimerization. Structural, functional and biochemical studies suggested that the enzymatic counterparts of RPTKs, the receptor protein-tyrosine phosphatases (RPTPs), are inhibited by dimerization, but whether RPTPs actually dimerize in living cells remained to be determined. RESULTS: In order to assess RPTP dimerization, we have assayed Fluorescence Resonance Energy Transfer (FRET) between chimeric proteins of cyan- and yellow-emitting derivatives of green fluorescent protein, fused to RPTPalpha, using three different techniques: dual wavelength excitation, spectral imaging and fluorescence lifetime imaging. All three techniques suggested that FRET occurred between RPTPalpha -CFP and -YFP fusion proteins, and thus that RPTPalpha dimerized in living cells. RPTPalpha dimerization was constitutive, extensive and specific. RPTPalpha dimerization was consistent with cross-linking experiments, using a non-cell-permeable chemical cross-linker. Using a panel of deletion mutants, we found that the transmembrane domain was required and sufficient for dimerization. CONCLUSIONS: We demonstrate here that RPTPalpha dimerized constitutively in living cells, which may be mediated by the transmembrane domain, providing strong support for the model that dimerization is involved in regulation of RPTPs.

Cross-Linking Reagents↗
PubMed2002

The clinical usefulness of D-dimer testing in cancer patients with suspected deep venous thrombosis.

BACKGROUND: Little is known about the diagnostic value of a D-dimer test in cancer patients with clinically suspected deep venous thrombosis (DVT). OBJECTIVE: To evaluate the clinical utility of a whole blood rapid D-dimer test (SimpliRED) in cancer patients compared with noncancer patients. METHODS: In consecutive patients with suspected lower limb DVT, a D-dimer test and ultrasonogram were performed. Cancer status was recorded at presentation. If the D-dimer test and ultrasonogram results were normal, DVT was considered absent. If the D-dimer result was abnormal, ultrasonography was performed again 1 week later. Anticoagulant therapy was only instituted in those patients with an abnormal ultrasonography result. All patients were followed up for 3 months to record subsequent thromboembolic events. The accuracy of the D-dimer test was assessed, and the efficiency and safety of withholding additional ultrasonography in cancer patients with normal results on both D-dimer and ultrasonography was compared with noncancer patients. RESULTS: A total of 1739 consecutive patients were studied, 217 (12%) of whom had cancer. The negative predictive value of the D-dimer test was 97% in both cancer and noncancer patients. In 63 (29%) of all 217 cancer patients, the D-dimer and ultrasonography results were normal at referral; therefore, the diagnosis of DVT was refuted and anticoagulant treatment was withheld. In these 63 patients, one thromboembolic event occurred during follow-up (1.6%; 95% confidence interval, 0.04%-8.53%). CONCLUSIONS: The negative predictive value of a whole blood D-dimer test in cancer patients seems as high as in noncancer patients. In a substantial proportion of cancer patients, the diagnosis can likely be refuted at referral, based on normal D-dimer test and ultrasonogram results. Furthermore, it seems safe to withhold anticoagulant therapy in these patients.

Adult↗
PubMed1988

Mechanism of fluorescence concentration quenching of carboxyfluorescein in liposomes: energy transfer to nonfluorescent dimers.

When 5(6)-carboxyfluorescein (6CF) is encapsulated in liposomes at 0.2 M, 97-98% of the fluorescence is quenched. We have studied the mechanism of this effect. The dye-liposome system is a special case of concentration quenching of dyes, a phenomenon recognized for 100 years. Absorption spectra of encapsulated dye show that 6CF dimerizes, and the dimer is nonfluorescent. The dimerization constant was estimated, and it was concluded that dimerization can account for only part of the quenching. In 6CF solutions, the fluorescence lifetime decreased drastically as concentration was changed over the narrow range 0.02-0.05 M, a finding which was attributed to energy transfer to dimers. Inhibition of dimerization by propylene glycol also inhibited the shortening of lifetime. Förster critical transfer distances were calculated to be 51 and 57 A for monomer-monomer and monomer-dimer transfer, respectively. Monomer-monomer transfer was demonstrated directly by steady-state or time-resolved anisotropy experiments, while transfer to dimer was modeled by using sulforhodamine B, which has a critical transfer distance like that for the dimer and also quenches 6CF emission. No direct evidence for collisional self-quenching of 6CF could be found, although a model compound, salicylate, did quench weakly. For xanthene dyes, the rate of energy transfer is much faster than that for quenching collisions, implying that collisional quenching in the usual 6CF-liposome system is insignificant. The reason why 6CF is not 100% quenched in liposomes is attributed to dye interaction with lipid as evidenced by (i) multiexponential decay of 6CF in liposomes with a long component of 3-4 ns, (ii) inhibition of dimerization in liposomes, (iii) partial protection of dye from quenching by KI, (iv) differing amounts of dimerization in liposomes made from different kinds of phospholipid, and (v) enhancement of fluorescence lifetime in the presence of Triton X-100.

Chemical Phenomena↗
PubMed1990

The use of haemoregulatory peptides (pEEDCK monomer and dimer) for reduction of cytostatic drug induced haemopoietic damage.

We have previously shown that the stem cell inhibitory peptide pGlu-Glu-Asp-Cys-Lys (pEEDCK monomer) leads to a good tolerance of otherwise lethal multiple ara-C doses and an increased survival of ara-C + peptide treated mice. This effect was due to the prevention of drug-induced CFU-S proliferation, thus keeping stem cells in a quiescent state insensitive to ara-C. Here we show that the pEEDCK monomer also inhibits stem cell proliferation after clinically relevant (non-lethal) ara-C doses. This leads to a sustained (100%) stem cell number in the femoral bone marrow, which was greatly reduced without protective peptide treatment (27%). We have measured the kinetics of influx of CFU-S into the empty S-phase (after two consecutive ara-C injections). This influx reached peak levels of 60-70%; pEEDCK treatment reduced it to 25-30%. Due to its cysteine content the pEEDCK monomer is easily oxidized and forms a symmetric disulfide-bonded dimer (pEEDCK)2. This dimer is a potent stimulator of haemopoiesis. Various modes of protective peptide treatment (monomer and dimer) were investigated in conjunction with a standardized protocol of 2 x 300 mg/kg ara-C given 12 h apart. (a) ara-monomer-ara: The administration of pEEDCK-monomer 2 h before the second ara-C injection retarded the onset of neutropenia, shortened its duration and improved recovery after depression. The degree of short-term neutropenia was not changed. (b) ara-ara-HN2-dimer: Post chemotherapy infusion of the stimulatory (pEEDCK)2 dimer led to considerable increases of progenitor levels (6.8 CFU-GM/1000 bone marrow cells vs. 1.2 CFU-GM/1000 in normal mice) 2 days after cytostatic treatment when CFU-GM were not detectable in unprotected mice. This increase was followed by greatly elevated granulocyte counts (8000 PMN/mm3 vs. 750 PMN/mm3 in normal mice). In the dimer-treated mice, up to 75% of the peripheral leukocytes were mature PMN (normal, 10%). (c) ara-monomer-ara-dimer: ara-C and monomer treatment as above (a) followed by dimer infusion led to complete protection of haemopoiesis. Mice treated with the protective pEEDCK monomer plus stimulatory dimer did not develop the leukocyte depression seen in unprotected animals. Our results show that the haemoregulatory peptide monomer and dimer can be used to improve the haematological status of mice treated with clinically relevant doses of cytostatic drugs (anti-metabolite and alkylating, alone and in combination). The pEEDCK monomer and dimer are equally active also on human haemopoietic cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1997

Discrepancy between latex and ELISA D-dimer values in sepsis may be caused by human neutrophil elastase.

We have recently shown that D-dimers are degraded by human neutrophil elastase (HNE) in vitro, causing rapid decrease in the D-dimer levels measured by a Latex test, but not with an ELISA test employing the same monoclonal antibody against D-dimer. To see if such discrepant D-dimer concentrations occurred in patients with high HNE concentration, we examined 80 plasma samples from 8 patients with sepsis with a Latex and an ELISA test and calculated the ratio between the D-dimer values obtained with the two tests. Twenty healthy pregnant and twenty pre-eclamptic patients, who are known to have raised D-dimer but low HNE concentrations, were chosen as controls. HNE levels were estimated by determining the HNE-alpha 1-proteinase inhibitor complex (HNE-A1PI) concentration. HNE-A1PI concentration was increased in sepsis patients compared with pre-eclamptic patients (p < 0.0005) and healthy pregnant women (p < 0.0005). In sepsis patients, the D-dimer results were skewed towards lower ratios between Latex and ELISA values compared to pre-eclamptic patients (p = 0.008) and healthy pregnant women (p = 0.0001). In plasma samples from patients with the largest discrepancy between Latex and ELISA D-dimer values, Western blotting with immunostaining indicated degradation of D-dimers to D-like fragments similar to those observed following degradation of cross-linked fibrin by HNE in vitro. We conclude that in sepsis patients there is a marked discrepancy between Latex and ELISA D-dimer values that may be caused by HNE. In such patients Latex D-dimer assays may cause severe underestimation of fibrinolysis.

Adult↗
PubMed1996

Significance of D-dimer concentrations during and after cardiopulmonary bypass.

OBJECTIVE: To determine whether D-dimer fragments predictably increase during cardiopulmonary bypass (CPB), and if so, whether increases correlate with postoperative blood loss or predict postoperative coagulopathy. DESIGN: Prospective observational study of 65 consecutive patients undergoing first-time coronary artery bypass graft (CABG) or first-time valve replacement. SETTING: Single center University teaching hospital. PARTICIPANTS: Male and female patients between the ages of 30 and 90 years undergoing first-time CABG or valve replacement surgery using CPB. Patients were excluded from study for prolonged preoperative bleeding time, preoperative warfarin therapy, perioperative intra-aortic balloon pump support, thrombolytic therapy in the week preceding operation, reoperation, and emergency operation. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Blood sampling for platelet count, prothrombin time, partial thromboplastin time, thrombin time, fibrinogen, activated coagulation time (ACT) and D-dimer concentrations was obtained at four times during each case; (1) preoperatively, after insertion of the internal jugular introducer, before insertion of pulmonary artery catheter; (2) during CPB at 28 degrees C, immediately before rewarming; (3) after heparin neutralization (20 minutes after initial protamine dose); (4) 12 to 24 hours postoperatively. Blood loss in the intensive care unit was calculated by measuring total mediastinal drainage output at 1 and 4 hours after arrival from the operating room. An initial decrease in fibrinogen was noted during bypass, but no increase in D-dimer was identified. A few patients developed a modest increase in D-dimer after heparin neutralization, but none greater than 2.0 ug/mL. Postoperatively, fibrinogen concentration increased toward baseline levels. However, this is when six patients developed significant (> 2.0 ug/mL) D-dimer formation. Results suggest appropriate physiologic response-normalization of fibrinogen with new synthesis and remodeling of clot in the operative site causing D-dimer formation. Patients with highest D-dimer levels at 12 to 24 hours postoperatively had the highest blood loss at 4 hours postoperatively, suggesting that early postoperative excess bleeding predisposed to increased clot formation and subsequent clot remodeling causing elevated D-dimer concentrations. CONCLUSIONS: D-dimer concentration is not usually elevated in patients undergoing CPB when adequately anticoagulated as monitored using the ACT. When mild elevation of D-dimer occurs, it is most often after heparin neutralization and/or in the postoperative period and is not predictive of increased postoperative blood loss. Elevations of D-dimer concentrations in the postoperative period without corresponding decreases in fibrinogen concentrations may occur and do not signify coagulopathy.

Adult↗
PubMed1992

Apple four in human blood coagulation factor XI mediates dimer formation.

Human blood coagulation factor XI is a dimer composed of two identical subunits. Each subunit contains four apple domains as tandem repeats followed by a serine protease region. A disulfide bridge between Cys321 of each fourth apple domain links the subunits together. The role of Cys321 in the dimerization of factor XI was examined by mutagenesis followed by expression of its cDNA in baby hamster kidney cells. The recombinant proteins were then purified from the tissue culture medium and shown to have full biological activity. Normal recombinant factor XI was secreted as a dimer as determined by SDS-PAGE, while recombinant factor XI-Cys321 Ser migrated as a monomer under these conditions. Gel filtration studies, however, revealed that each protein existed as a dimer under native conditions, indicating that the disulfide bond between Cys321 of each factor XI monomer was not necessary for dimer formation. The fourth apple domain (apple4) of factor XI was then introduced into tissue plasminogen activator (tPA) to investigate its role in the dimerization of other polypeptide chains. The fusion protein, containing apple4 (apple4-tPA), formed dimers as detected by SDS-PAGE and gel filtration. Furthermore, dimerization was specific to apple4, while apple3 had no effect on dimerization. These data further indicated that the apple4 domain of factor XI mediates dimerization of the two subunits and the interchain disulfide bond involving Cys321 was not essential for dimer formation.

Amino Acid Sequence↗
PubMed1992

Construction of a stable dimer of Bacillus stearothermophilus lactate dehydrogenase.

A molecular graphics analysis of the features which prevent cytosolic malate dehydrogenase dimers from forming tetramers was evaluated by its success in predicting the synthesis of a version of the LDH framework which is a stable dimer. Surface residues responsible for malate dehydrogenases being dimers were revealed by superimposing the structures of two dimers of pig cytosolic malate dehydrogenase on one homologous tetramer of L-lactate dehydrogenase from Bacillus stearothermophilus. Four regions were identified as composing the P-axis dimer-dimer interface. Two regions of the dimer were surface loops that collided when built as a tetramer: a large loop (residues 203-207, KNOBI) and a small loop (residues 264-269, KNOBII), and these were candidates to explain the dimeric character of malate dehydrogenase. The analysis was tested by constructing a synthetic B. stearothermophilus lactate dehydrogenase (KNOBI) containing the large malate dehydrogenase loop (residues 203-207 being AYIKLQAKE, and extra four amino acids). The new construct was thermotolerant (90 degrees C) and enzymically active with kcat and KM (pyruvate) values similar to those of the wild-type enzyme. However, whereas the allosteric activator fructose 1,6-bisphosphate decreased KM 100 times for wild type, it had no influence on KNOBI. The molecular volumes of 1-120 microM concentrations of the construct were measured by time-resolved decay of tryptophan fluorescence anisotropy and by gel filtration. Both methods showed the molecular weight of wild type increased from dimer to tetramer with Kd about 20 microM dimer. KNOBI remained a dimer under these conditions.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Slowly interchanging conformers of bovine neurophysin-I in the unliganded dimeric state.

The effect of neurophysin dimerization on Tyr-49, a residue adjacent to the hormone-binding site, was investigated by proton NMR in order to analyze the basis of the dimerization-induced increase in neurophysin hormone affinity. Dimerization-induced changes in Tyr-49 resonances, in two unliganded bovine neurophysins, suggested that Tyr-49 perturbation is an intrinsic consequence of dimerization, although Tyr-49 is distant from the monomer-monomer interface in the crystalline liganded state. To determine whether this perturbation reflects a conformational difference between liganded and unliganded states that places Tyr-49 at the interface in the unliganded state, or a dimerization-induced change in secondary (2 degrees) or tertiary (3 degrees) structure, the more general structural consequences of dimerization were further analyzed. No change in 2 degrees structure upon dimerization was demonstrable by CD. On the other hand, a general similarity of regions involved in dimerization in unliganded and liganded states was indicated by NMR evidence of participation of His-80 and Phe-35 in dimerization in the unliganded state; both residues are at the interface in the crystal structure and distant from Tyr-49. Consistent with a lack of direct participation of Tyr-49 at the monomer-monomer interface, dimerization induced at least two distinct slowly exchanging environmental states for the 3.5 ring protons of Tyr-49 without significantly increased dipolar broadening relative to the monomer. Two environments were also found in the dimer of des-1-8 neurophysin-I for the methyl protons of Thr-9, another residue distant from the monomer-monomer interface and close to the binding site in the liganded state.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2002

Factors affecting metal-metal bonding in the face-shared d(3)d(3) bioctahedral dimer systems, MM'Cl(9)(5-) (M, M' = V, Nb, Ta).

Density functional theory (DFT) calculations have been used to investigate the d(3)d(3) bioctahedral complexes, MM'Cl(9)(5-), of the vanadium triad. Broken-symmetry calculations upon these species indicate that the V-containing complexes have optimized metal-metal separations of 3.4-3.5 A, corresponding to essentially localized magnetic electrons. The metal-metal separations in these weakly coupled dimers are elongated as a consequence of Coulombic repulsion, which profoundly influences (and destabilizes) the gas-phase structures for such dimers; nevertheless, the intermetallic interactions in the V-containing dimers involve significantly greater metal-metal bonding character than in the analogous Cr-containing dimers. These observations all show good agreement with existing experimental (solid state) results for the chloride-bridged, face-shared dimers V(2)Cl(9)(5-) and V(2)Cl(3)(thf)(6)(+). In contrast to the V-containing dimers, complexes featuring only Nb and Ta have much shorter intermetallic distances (approximately 2.4 A) consistent with d-electron delocalization and formal metal-metal triple bond formation; again, good agreement is found with available experimental data. Calculations on the complexes V(2)(mu-Cl)(3)(dme)(6)(+), Nb(2)(mu-dms)(3)Cl(6)(2-), and Ta(2)(mu-dms)(3)Cl(6)(2-), which are closely related to compounds for which crystallographic structural data exist, have been pursued and provide an insight into the intermetallic interactions in the experimentally characterized complexes. Analysis of the contributions from d-orbital overlap (E(ovlp)) stabilization, as well as spin polarization (exchange) stabilization of localized d electrons (E(spe)), has also been attempted for the MM'Cl(9)(5-) dimers. While E(ovlp) clearly dominates over E(spe) as a stabilizing factor in those dimers containing only Nb and Ta metal atoms, detailed assessment of the competition between E(ovlp) and E(spe) for V-containing dimers is obstructed by the instability of triply bonded V-containing dimers against Coulombic explosion. On the basis of the periodic trends in E(ovlp) versus E(spe), the V-triad dimers have a greater propensity for metal-metal bonding than do their Cr-triad or Mn-triad counterparts.

Journal Article↗
PubMed1999

D-dimers in relation to the severity of arteriosclerosis in patients with stable angina pectoris after myocardial infarction.

BACKGROUND: Plasma concentrations of D-dimers show the extent of intravascular fibrinolysis of cross-linked fibrin. Higher concentrations of D-dimers are found in the plasma of arteriosclerosis patients with increased fibrin metabolism. The present study was performed in order to investigate whether there is a relationship between the severity of arteriosclerosis and fibrinolytic activity indicated by plasma levels of D-dimer. METHODS: The study populations consisted of 1112 men and 299 women with stable angina pectoris, on average 36+/-5.6 days after a myocardial infarction, as well as 326 men and 138 women with no clinical signs of cardiovascular disease. In addition to cardiological and angiological examinations, the lipid status and levels of fibrinogen, plasma viscosity, F 1+2, plasminogen, plasminogen activator inhibitor-1, D-dimer, and C-reactive protein of the participants were determined. RESULTS: The plasma concentration of D-dimers increases with age, both in the group with coronary artery disease and in the control group, with the female gender showing consistently higher concentrations in both groups. D-dimers correlate with other parameters of the lipid and coagulation systems, which explains 32.0% and 39.2% of the variance in D-dimer values in men and women, respectively. A significant increase in the level of D-dimers can be found in participants with generalized arteriosclerosis, with a left ventricular ejection fraction </=40% as well as those with left-ventricular aneurysm. CONCLUSION: This study indicates that there is increased fibrinolytic activity in patients with severe arteriosclerosis. This finding gives further support to the hypothesis that D-dimer concentration is dependent on the amount of fibrin associated with arteriosclerotic thrombi. However, because of the low specificity and wide overlap of D-dimer values between patients and controls, enhanced D-dimer values are of limited relevance above and beyond other lipid metabolism risk indicators for coronary artery disease or coronary artery disease and peripheral arterial occlusive disease.

Adult↗
PubMed1994

Identification of the primary site of the human immunodeficiency virus type 1 RNA dimerization in vitro.

The diploid genome of all retroviruses is made of two homologous copies of RNA intimately associated near their 5' end, in a region called the dimer linkage structure. Dimerization of genomic RNA is thought to be important for crucial functions of the retroviral life cycle (reverse transcription, translation, encapsidation). Previous in vitro studies mapped the dimer linkage structure of human immunodeficiency virus type 1 (HIV-1) in a region downstream of the splice donor site, containing conserved purine tracts that were postulated to mediate dimerization, through purine quartets. However, we recently showed that dimerization of HIV-1 RNA also involves sequences upstream of the splice donor site. Here, we used chemical modification interference to identify nucleotides that are required in unmodified form for dimerization of a RNA fragment containing nucleotides 1-707 of HIV-1 RNA. These nucleotides map exclusively in a restricted area upstream of the splice donor site and downstream of the primer binding site. They are centered around a palindromic sequence (GUGCAC279) located in a hairpin loop. Our results support a model in which dimer formation is initiated by the annealing of the palindromic sequences, possibly by a loop-loop interaction between the two monomers. Further experiments show that the deletion of the stem-loop or base substitutions in the loop abolish dimerization, despite the presence of the previously postulated dimer linkage structure. On the other hand, deletions of the purine tracts downstream of the splice donor site do not prevent dimerization. Therefore, we conclude that the palindromic region represents the dimerization initiation site of genomic RNA.

Base Sequence↗
PubMed1995

The G protein gamma subunit. Requirements for dimerization with beta subunits.

Guanine nucleotide-binding protein beta and gamma subunits form a tightly bound complex that can only be separated by denaturation. Assembly of beta and gamma subunits is a complicated process. The beta 1 and gamma 2 subunits can be synthesized in vitro in rabbit reticulocyte lysate and then assembled into dimers, but beta 1 cannot form beta gamma dimers when synthesized in a wheat germ extract. In contrast, gamma 2 translated in either system can dimerize with beta 1, suggesting that dimerization-competent gamma 2 can be synthesized without the aid of specific chaperonins or other cofactors. Dimerization-competent gamma 2 in solution forms an asymmetric particle with a Stokes radius of about 21 +/- 0.4 A (n = 4), s20,w of 0.9 S (range 0.8-1.0 S, n = 2), and frictional ratio of 1.57 (assuming no hydration). To define the part of gamma 2 that is needed for native beta gamma dimer formation, a series of N- and C-terminal truncations were generated, synthesized in vitro, and incubated with beta 1. Dimerization was assessed by stabilization of beta 1 to tryptic proteolysis. Truncation of up to 13 amino acids at the C terminus did not affect dimerization with beta 1, whereas removal of 27 amino acids prevented it. Therefore, a region between residues 45 and 59 of gamma 2 is important for dimerization. Truncation of 15 amino acids from the N terminus greatly diminished the formation of beta gamma dimers, while removal of 25 amino acids entirely blocked it. Thus, another region important for forming native beta gamma is near the N terminus. Extension of the N terminus by 12 amino acids that include the influenza virus hemagglutinin epitope did not prevent beta gamma dimerization. Furthermore, in intact 35S-labeled COS cells, epitope-tagged gamma 2 coimmunoprecipitates with beta and alpha subunits. The N-terminal epitope tag must lie at the surface of the heterotrimer since it prevents neither heterotrimer formation nor access of the antibody.

Amino Acid Sequence↗
PubMed1994

Dimerization of human immunodeficiency virus type 1 RNA involves sequences located upstream of the splice donor site.

The retroviral genome consists of two homologous RNA molecules associated close to their 5' ends. We studied the spontaneous dimerization of four HIV-1 RNA fragments (RNAs 1-707, 1-615, 311-612, and 311-415) containing the previously defined dimerization domain, and a RNA fragment (RNA 1-311) corresponding to the upstream sequences. Significant dimerization of all RNAs is observed on agarose gels when magnesium is included in the electrophoresis buffer. In contrast to dimerization of RNAs 311-612 and 311-415, dimerization of RNAs 1-707, 1-615 and 1-311 strongly depends on the size of the monovalent cation present in the incubation buffer. Also, dimerization of RNAs 1-707, 1-615, and 1-311 is 10 times faster than that of RNAs 311-612 and 311-415. The dimers formed by the latter RNAs are substantially more stable than that of RNA 1-615, while RNA 1-311 dimer is 5-7 degrees C less stable than RNA 1-615 dimer. These results indicate that dimerization of HIV-1 genomic RNA involves elements located upstream of the splice donor site (position 305), i.e. outside of the previously defined dimerization domain.

Base Sequence↗
PubMed2001

Practical utility of the D-dimer assay for excluding thromboembolism in severely injured trauma patients.

BACKGROUND: We have advocated the use of a D-dimer assay to exclude the diagnosis of pulmonary embolism (PE) and deep venous thrombosis (DVT) in surgical and trauma patients suspected of having these diagnoses. Injury is known to increase D-dimer levels independent of thromboembolism. The purpose of this study was to assess the period after injury over which the D-dimer assay remains positive because of injury exclusive of thromboembolism. METHODS: We prospectively sampled the plasma of severely injured patients for D-dimer using an enzyme-linked immunosorbent assay method at admission; at hours 8, 16, 24, and 48; and at days 3, 4, 5, and 6. Patients were then screened for DVT with a routine duplex Doppler at day 7. Patients were followed for PE, adult respiratory distress syndrome, and disseminated intravascular coagulation. RESULTS: One hundred fifty-four patients (mean Injury Severity Score of 23) underwent a total of 1,230 D-dimer assays. Twenty-six (17%) had thromboembolism. Nine (6%) patients developed DVT, 2 (1%) developed PE, 13 (8%) developed disseminated intravascular coagulation, and 11 (7%) developed severe adult respiratory distress syndrome. None of the trauma patients with thromboembolism had a (false) negative D-dimer at or after the time of their thromboembolic complication. True-negative D-dimer results as a function of time from injury are: 0 hours, 18%; 8 hours, 16%; 16 hours, 17%; 24 hours, 22%; 48 hours, 37%; day 3, 34%; day 4, 32%; day 5, 30%; and day 6, 30%. The negative predictive value of the assay was 100%. D-dimer levels were significantly higher in those who developed a thromboembolic complication than in those who did not (independent of Injury Severity Score). CONCLUSION: These data serve to validate D-dimer as a means of excluding thromboembolism, specifically in patients with severe injury (100% negative predictive value). Before 48 hours after injury, however, the vast majority of these patients without thromboembolism had positive D-dimer assays. Because of the high false-positive rate early after severe injury, the D-dimer assay may be of little value before postinjury hour 48.

Adult↗