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PubMed1989

Isolation of dimeric forms of human pituitary growth hormone.

A procedure is described which for the first time allows the isolation of noncovalently-linked dimeric human pituitary growth hormone. Isomers of this dimeric species were prepared as were also, for the first time, isomers of covalently-linked dimers. Chromatography on DEAE-Sepharose CL-6B revealed the existence of noncovalently-linked dimers composed of monomers of 22K hGH, 20K hGH and 20K1 hGH (the latter is a new form of 20K hGH with a scission in the peptide chain) and covalent dimers containing 22K hGH and 24K hGH (the latter a 22K hGH with a scission). The different dimers all occurred as charge isomers and subsequent HPLC on an anion exchanger followed by zone electrophoresis in agarose suspension made possible the isolation of four noncovalently-linked isomers: one form of (20K-20K)hGH, two forms of (20K-22K)hGH and one form of (22K-22K)hGH; and of three covalently-linked isomers: one form of (22K-22K)hGH and two forms of (22K-24K)hGH.

Chemical Phenomena↗
PubMed2002

A comparison of spiral computed tomography and latex agglutination D-dimer assay in acute pulmonary embolism using pulmonary arteriography as gold standard.

OBJECTIVE: To compare the diagnostic accuracy of contrast medium enhanced spiral computed tomography of the pulmonary arteries (s-CTPA) and a latex agglutination D-dimer assay in patients with suspected acute pulmonary embolism (PE) by using pulmonary arteriography (PA) and clinical follow-up as reference method. DESIGN: Ninety hemodynamically stable patients with symptoms of acute pulmonary embolism were prospectively evaluated with s-CTPA and pulmonary arteriography (PA) within 24 h from admission. Plasma D-dimer levels on admittance were analyzed using a rapid latex agglutination D-dimer assay. The outcome of D-dimer concentrations in plasma below 0.25 and 0.5 mg/l was studied. RESULTS: All PA and s-CTPA investigations were regarded as of acceptable diagnostic quality in a consensus reading. Thirty-three patients had a positive PA (37%). Three patients had false negative and two patients had false positive s-CTPA findings. s-CTPA had 91% sensitivity, 96% specificity, 94% positive predictive value (PPV) and 95% negative predictive value (NPV). The sensitivity and specificity for D-dimer below 0.5 mg/l were 79 and 88%, respectively. The PPV and NPV were 81 and 87%. If a cut-off level of 0.25 mg/l was used the corresponding figures were 91, 65, 63 and 92%. CONCLUSION: s-CTPA has a higher sensitivity and specificity than latex agglutination D-dimer. A cut-off level of 0.25 mg/l can be used as screening method, but s-CTPA must be performed to exclude false positive cases.

Adolescent↗
PubMed1996

Beta 1 integrin subunit dimerization via disulfide bonds.

Integrins of the beta 1 family were isolated from human smooth muscle. SDS-PAGE analysis and subsequent immunoblotting demonstrated that integrin samples contain a protein immunologically related to beta 1 integrin subunit with the previously undescribed apparent molecular mass 205 kD. One-dimensional peptide mapping showed that the 205 kD protein is not a novel beta 1 related integrin subunit, but a beta 1 integrin subunit dimer. After reduction the major part of the beta 1 immunoreactive material migrated from the 205 kD to 130 kD region, indicating that beta 1 integrin subunit dimers were formed via disulfide bonds. When electrophoretically pure beta 1 monomer and dimer forms were analized it was found that during SDS-PAGE about 30% of beta 1 integrin subunit monomers were organized into dimers while approximately 70% of the beta 1 dimer form was partly disrupted into monomers. It was suggested that this steady-state process is a result of a reversible reaction between intra- and intermolecular disulfide bonds. Possible in vivo dimerization of integrins via disulfide bonds is discussed.

Chromatography, Affinity↗
PubMed1995

Intercellular adhesion molecule-1 dimerization and its consequences for adhesion mediated by lymphocyte function associated-1.

Intercellular adhesion molecule-1 (ICAM-1, CD54) is a ligand for the integrins lymphocyte function associated-1 (LFA-1, CD11a/CD18) and complement receptor-3 (Mac-1, CD11b/CD18) making it an important participant in many immune and inflammatory processes. Modified recombinant soluble ICAM-1 formed dimers. This result indicated that the ectodomain of ICAM-1 contains homophilic interaction sites. Soluble ICAM-1 dimers bind to solid-phase purified LFA-1 with high avidity (dissociation constant [Kd] = 8 nM) in contrast to soluble ICAM-1 monomers whose binding was not measurable. Cell surface ICAM-1 was found to be dimeric based on two distinct criteria. First, a monoclonal antibody specific for monomeric soluble ICAM-1, CA7, binds normal ICAM-1 poorly at the cell surface; this antibody, however, binds strongly to two mutant forms of ICAM-1 when expressed at the cell surface, thus identifying elements required for dimer formation. Second, chemical cross-linking of cell surface ICAM-1 on transfected cells and tumor necrosis factor-activated endothelial cells results in conversion of a portion of ICAM-1 to a covalent dimer. Cell surface ICAM-1 dimers are more potent ligands for LFA-1-dependent adhesion than ICAM-1 monomers. While many extracellular matrix-associated ligands of integrins are multimeric, this is the first evidence of specific, functionally important homodimerization of a cell surface integrin ligand.

Animals↗
PubMed1989

DNA damage and repair in normal, xeroderma pigmentosum and XP revertant cells analyzed by gel electrophoresis: excision of cyclobutane dimers from the whole genome is not necessary for cell survival.

Human fibroblasts were embedded in microscopic-sized agarose beads and lysed in situ to produce chromosome-sized DNA trapped inside the beads. Conventional alkaline agarose gel electrophoresis of trapped DNA from cells damaged by X-rays before embedding gave mol. wts similar to those obtained with alkaline sucrose gradients. When cells were irradiated with UV light before embedding in agarose and incubated with UV endonuclease to cleave cyclobutane pyrimidine dimer sites, UV damage was detectable by gel electrophoresis over a range of 2.6-52 J/m2 as a linear function of dose, and repair was detectable within 6 h of irradiation. Two independently derived UV-resistant xeroderma pigmentosum (XP) revertants did not remove cyclobutane dimers up to 48 h after irradiation. Incubation of normal and XP revertant cells with cytosine arabinoside after UV irradiation resulted in similar numbers of single-strand breaks; these breaks represent repair of non-dimer photoproducts. Evidently, excision of cyclobutane pyrimidine dimers from the whole genome is not necessary for survival of human cells after UV irradiation; rather, repair of non-dimer photoproducts such as photoproducts in the genome as a whole or cyclobutane dimers in a small number of genes may be more biologically important.

Cell Line↗
PubMed1983

1H NMR study of an ethidium dimer poly(dA-dT) complex: evidence of a transition between bis and monointercalation.

Comparative 1H NMR and optical studies of the interaction between poly(dA-dT), ethidium bromide (Et) and ethidium dimer (Et2) in 0.7 M NaCl are reported as a function of the temperature. Denaturation of the complexes followed at both polynucleotide and drug levels leads to a biphasic melting process for poly(dA-dT) complexed with ethidium dimer (t1/2 = 75 degrees C; 93 degrees C) but a monophasic one in poly(dA-dT): ethidium bromide complex (t1/2 = 74 degrees C). In both cases drug signals exhibit monophasic thermal dependence (Et = 81 degrees C; Et2 = 95 degrees C). Evidence is presented showing that the ethidium dimer bisintercalates into poly(dA-dT) in high salt, based on the observation that i) dimer and monomer ring protons exhibit similar upfield shifts upon DNA binding, ii) upfield shifts of DNA sugar protons are twice as large with the dimer than with ethidium bromide. Comparison between native DNA fraction and bound drug fraction indicates that ethidium covers, n = 2.5-3 base pairs. The dimer bisintercalates and covers, n = 5.7 base pairs when the helix fraction is high but as the number of available sites decreases the binding mode changes and the drug monointercalates (n = 2.9).

Chemical Phenomena↗
PubMed1996

Bacteriophage T4 regA protein binds RNA as a monomer, overcoming dimer interactions.

The stoichiometry of the complex formed between the T4 translational repressor protein regA and the 16 nt gene 44 recognition element (gene 44RE) RNA has been determined. Under quantitative binding conditions, the association of wild-type regA protein with gene 44RE RNA exhibits saturation at a 1:1 ratio of protein to RNA. It is known that regA protein exists as a dimer in protein crystals. Thus, the stoichiometry may be indicative of a regA dimer bound to two RNAs or a regA monomer bound to one RNA. Gel filtration through Sephadex G-75 revealed that wild-type and R91L regA proteins (14.6 kDa) elute at a mass of 29 kDa, consistent with the mass of a dimer. However, wild-type regA preincubated with gene 44RE (1:1) resulted in a complex that eluted at approximately 20 kDa, consistent with a regA monomer-RNA complex. Covalent crosslinking of surface lysines with glutaraldehyde confirmed that wild-type and R91L proteins exist as dimers and higher oligomers in solution. However, the addition of RNA to wild-type regA protein prior to crosslinking inhibited the formation of crosslinked dimers. Thus, the regA protein-protein interactions observed in solution are disrupted or blocked in the presence of gene 44RE RNA. Together, these studies demonstrate that regA protein binds RNA as a monomer, although free protein exists predominantly as a dimer.

Chromatography, Gel↗
PubMed1976

Histone dimers: a fundamental unit in histone assembly.

Histone interactions which occur, at moderate ionic strengths, when several types of purified, renatured histones are mixed at equimolar ratios have been studied. The four histones H2A,H2B,H3 and H4 complex and form dimers. Histone H1 does not interact with the other four histone types and does not form dimers. Mixing of single histone species with preformed histone pairs as well as mixing of two different types of histone pairs, leads to exchange of histones among the pairs and formation of dimers. No trimers are formed. The dimers are in equilibrium with high-molecular weight histone structures. The results indicate that histone dimers may serve as a stable intermediate in histone assembly. Because each histone type (except H1) can interact with itself as well as with each of the other three histone types we suggest that each histone type should be considered as an interchangeable subunit of a multichain protein in which the dimer species is the most stable structure.

Animals↗
PubMed1996

Biochemical properties of the monomeric mutant of human cathepsin E expressed in Chinese hamster ovary cells: comparison with dimeric forms of the natural and recombinant cathepsin E.

Cathepsin E (CE) is the only known aspartic proteinase that exists as a homodimer consisting of two fully catalytically active monomers, which are covalently bound by a disulfide bond between two cysteine residues at the NH2-terminal region (Cys43 in human pro-CE). To understand the physiological significance of the dimer formation, the monomeric mutant of human CE was constructed by site-directed mutagenesis (Cys43 ->Ser43) and expressed in Chinese hamster ovary (CHO) cells. Immunolocalization of the mutant protein at both the light and electron microscopic levels revealed the monomeric CE to be associated predominantly with the endoplasmic reticulum and the non-lysosomal endocytic organelles. The cellular localization of the monomeric protein was compatible with that of the wild-type (dimeric form) of recombinant human CE expressed in the same cells. The monomeric protein was generated primarily as the 46-kDa pro-CE with a high-mannose-type oligosaccharide chain in the cells. In addition to the maximal activation at around pH 3.5, a substantial proportion of the monomeric pro-CE was converted to the mature form by incubation at pH 7 and 37 degrees C for 5 min. In contrast, the dimeric pro-CE was scarcely activated by treatment at pH7. Although catalytic properties of the in vitro-activated monomeric CE appeared to be indistinguishable from those of the dimeric forms of natural and recombinant CE, the monomeric form was more unstable to pH and temperature changes than these dimeric forms. These results indicate that the dimerization of CE is not necessarily required for proper folding to express activity, correct intracellular localization and carbohydrate modification, but that it may be essential to structurally stabilize the molecule in vivo.

Amino Acid Sequence↗
PubMed1995

Dimeric chemotactic peptides discriminate between chemotaxis and superoxide production of human neutrophils.

A series of dimeric fMLPs were synthesized by cross-linking with hydrophilic linkers, mono-, di-, tri-, and tetraethylene glycols. All of the dimers showed higher activities than monomeric fMLP in chemotaxis assays for human neutrophils. The magnitude of the activity enhancement was dependent on the linker length. On the other hand, production of superoxide anion was not as enhanced as chemotaxis. The experimental results indicate that these dimers, especially the triethylene glycol-bridged fMLP dimer, D-fMLP-G3, are selective agonists for chemotaxis. In order to distinguish between biological activities and intracellular signaling, rapid mobilization of the intracellular calcium was also measured. D-fMLP-G3 strongly enhanced the calcium mobilization. These results suggest that chemotaxis was correlated with the increase of the intracellular calcium concentration, whereas NADPH oxidase was not activated to generate superoxide by this elevation of cytoplasmic calcium caused by the dimeric ligand, D-fMLP-G3. The dimeric fMLPs might act as useful ligands for studying the intracellular signaling mechanisms of human neutrophils.

Amino Acid Sequence↗
PubMed1995

Production of hetero-dimeric dihydrofolate reductase-thymidylate synthase bifunctional enzyme.

As a result of the expression of a single open reading frame composed of the coding sequence for a cysteine-free mutant (Cys85-->Ala, Cys152-->Ser) of Escherichia coli dihydrofolate reductase (DHFR; 18K monomeric protein), that for the E. coli thymidylate synthase (TS; dimeric protein with a 30K promoter), and a spacer sequence (coding 7 amino acids) with a Shine-Dargarno sequence, an active hetero-dimeric bifunctional enzyme with 50K DHFR-TS and 30K TS polypeptides was stably produced in the transformed E. coli cell in addition to an overproduction of the TS dimeric enzyme. The highly purified hetero-dimeric enzyme has similar Vmax and Km values in both DHFR and TS activities to those of the natural counterparts, monomeric DHFR and dimeric TS. Although the hetero-dimeric enzyme did not show an apparent channeling transfer of dihydrofolate (the intermediate substrate) between the spatially discrete DHFR and TS active sites, the coupling efficiency of the TS and DHFR reactions in the artificial enzyme was better than that in the separated enzymes, as shown by a decrease in the intermediate concentration at the steady state in the coupled reaction.

Amino Acid Sequence↗
PubMed1982

Interaction of specific amino acid derivatives with dimeric alpha-chymotrypsin.

Binding and catalytic activities of dimeric alpha-chymotrypsin from specific amino acid derivatives were investigated with special reference to the equilibrium between active and inactive monomeric forms of this enzyme occurring in a low pH range. The low catalytic activity of the dimeric enzyme towards these specific substrates was revealed to be due to the difficulty in binding of the dimer. However the free tryptophanates and N-acetyl-L-tryptophan, which are slightly smaller (in molecular size) than the above substrates and comparable to the nonspecific phenyl acetate substrates (towards which the dimeric alpha-chymotrypsin showed an distinct catalytic activity in our previous study [J. Biochem. 87, 871-880 (1980)]), were bound to the dimer more strongly than to the monomeric enzyme. Hence they enhanced dimer formation when added at low concentrations.

Amino Acids↗
PubMed1982

pH dependence of the formation of dimeric alpha-chymotrypsin and its catalytic activity.

The dimerization of alpha-chymotrypsin (alpha-CT) has been known to involve a specific interaction between the Tyr-146 alpha-carboxyl of one molecule and the His-57 imidazole, which is a member of the catalytic triad in the active site, of the other. This interaction determines the pH dependences of the dimerization constant and of the catalytic activities of the monomeric and dimeric enzymes. We compared the pKa values for catalytic activities with known pKa values for the dimerization constant in order to assign pKa values to residues of the enzyme. In the monomeric enzyme, the catalytic triad has a pKa value of 3.6 at the site between the Asp-102 carboxyl and His-57 N delta 1, and the Tyr-146 alpha-carboxyl has a pKa value of 4.6. In the dimeric enzyme, the site between the Asp-102 carboxyl and His-57 N delta 1 has a pKa value of around 5.5 and the site between His-57 N epsilon 2 and the Tyr-146 alpha-carboxyl has a pKa value around 2.4. Protonation at the site between the Asp-102 carboxyl and His-57 N delta 1 reduced the catalytic activity of the dimeric enzyme for p-nitrophenyl propionate, indicating that the Asp-102 carboxyl plays an important role in the monomeric alpha-CT-catalyzed reactions.

Biopolymers↗
PubMed1990

Dimeric character of a basic phospholipase A2 from cobra venom: experimental and modelling study.

When it is gel filtered on Sephadex in the absence of calcium ions, basic phospholipase A2 from Naja nigricollis venom elutes as a dimer. In order to study the possibility of this dimerization from a structural point of view, three-dimensional models of both monomeric and dimeric N. nigricollis phospholipases A2 have been graphically built on the basis of homologies with the phospholipases A2 from pancreatic bovine and Crotalus atrox venom. The building of a dimeric model is made possible by the deletion of a particular loop of the bovine structure. The predicted models of N. nigricollis phospholipase A2 have been checked using molecular mechanics and molecular dynamics techniques according to a suitable protocol which has been developed starting from refined X-ray structures of phospholipases A2 as the test case. The observed stability of the dimeric model, in the absence of calcium, agrees with the hypothesis of the dimerization of the basic phospholipase A2. Particularly, Arg31, which replaces the hydrophobic residue present in pancreatic bovine and C.atrox venom phospholipases A2, contributes to this stability.

Amino Acid Sequence↗
PubMed1993

A comparative evaluation of ELISAs for D-dimer and related fibrin(ogen) degradation products.

This study was designed to (i) characterize two new monoclonal antibodies that react with neoepitopes on fibrin(ogen) fragments D-dimer and D1 and (ii) compare the specificity of these antibodies with that of two commercially available 'D-dimer' ELISAs [Dimertest EIA (American Diagnostica, Inc.) and Asserachrom D-Di (Diagnostica Stago)] and also with an ELISA for fibrinolytic fragments containing the E domain complexed with a D (or D-dimer) moiety [Fibrinostika FbDP (Organon Teknika)]. All assays were in a capture (sandwich) ELISA format. Fibrin(ogen) degradation products were isolated in high yield by a novel method involving hydrophobic interaction chromatography. The results disclosed considerable differences in sensitivity and specificity for purified fibrinolytic fragments among all five ELISAs. Although the diagnostic utility of monitoring plasma fibrinolytic fragments is not questioned, our results provide a rational basis for understanding why mAb-based ELISAs for these analytes are not well correlated. If, as our data suggest, the D-dimer ELISAs are not actually measuring the same analytes, then interpretation of studies that determine D-dimer concentrations with different methods will be problematic and standardization of D-dimer measurements across assays will be impossible.

Antibodies, Monoclonal↗
PubMed1999

Contribution of a new, rapid, quantitative and automated method for D-dimer measurement to exclude deep vein thrombosis in symptomatic outpatients.

Ninety-nine consecutive outpatients with symptoms suggestive of deep vein thrombosis (DVT) were tested for the presence of D-dimer using a new, rapid method (BC D-Dimer) based on the agglutination principle and performed on a BCT analyzer. Dimertest Gold EIA and VIDAS D-Dimer devices were used as comparative methods. Venography was performed in all patients, with DVT diagnosed in 39 of them (34 proximal). The BC D-Dimer test proved to be rapid, automated and well suited for individual tests with a good reproducibility (coefficient of variation % 1.5-5.3). Its performance was comparable with that of the other methods, as indicated by the areas under the receiver operating characteristic curves (Gold EIA 0.95; VIDAS 0.95; BC D-Dimer 0.91) and the coefficients of correlation (Pearson's coefficients from 0.832 to 0.876). On the basis of the Kappa coefficients, there was quite a good concordance between the three tests. At the cut-off levels that provided the highest sensitivity (97.4%, for all the tests), the negative predictive values were similar for all methods, at over 95%. The corresponding specificity ranged from 62.7 to 81.7%. In conclusion, this study shows that the new method can be included in prospective clinical trials to test the utility of D-dimer measurement in combination with other non-invasive diagnostic procedures in the management of the diagnosis of DVT.

Adult↗
PubMed2004

Prognostic value of a new quantitative D-dimer test in critically ill patients 24 and 48 h following admission to the intensive care unit.

A D-dimer assay may predict mortality in medical critically ill patients, although no consensus on the clinical utility of this diagnostic test has been reached. A prospective single-center study was designed to evaluate whether D-dimer levels, as measured by a new, rapid assay, correlate with poor outcome in critically ill patients. A total of 95 blood samples were collected from medical and surgical adult patients 24 and 48 h following admission to the intensive care unit (ICU). D-dimer was assayed by the Miniquant quantitative test and correlated to the ICU length of stay, the hospital length of stay, the Acute Physiology and Chronic Health Evaluation (APACHE) II score and the Simplified Acute Physiology Score (SAPS) 24 and 48 h following admission to the ICU, organ system failure index and hospital mortality. The 24-h D-dimer level correlated with the 48 h APACHE II and SAPS scores (r = 0.41, P = 0.01; and r = 0.39, P = 0.01, respectively). The 48-h D-dimer level correlated with the APACHE II and SAPS scores at 48 h and with the organ system failure index (number of organ failure) (r = 0.54, P = 0.0008; r = 0.60, P = 0.0001; and r = 0.37, P = 0.02, respectively). Neither the 24-h nor the 48-h D-dimer levels were predictive of in-hospital mortality in a multivariate model. We conclude that this simple and new laboratory test may serve as an additional tool to predict the clinical severity of patients admitted to the ICU.

APACHE↗
PubMed2000

Predictive value of a rapid semiquantitative D-dimer assay in critically ill patients with suspected venous thromboembolic disease.

OBJECTIVE: To evaluate the performance of a new, rapid semi-quantitative assay for the detection of circulating D-dimer in whole blood from critically ill patients with suspected venous thromboembolic disease. DESIGN: Prospective, blinded, single-center study. SETTING: Medical intensive care unit (ICU) of Barnes-Jewish Hospital, St. Louis, MO, a university-affiliated urban teaching hospital. PATIENTS: Two hundred thirty-nine adult patients with clinical suspicion of venous thromboembolic disease admitted to a medical ICU. INTERVENTIONS: Collection of blood samples within 24 hrs of clinical suspicion of venous thromboembolic disease. MEASUREMENTS AND MAIN RESULTS: The main outcome measures evaluated included the occurrence of venous thromboembolic disease (i.e., lower extremity venous thrombosis, pulmonary embolism, catheter-associated venous thrombosis) and hospital mortality. Fifty-seven patients (23.8%) were classified as having venous thromboembolic disease during their ICU stays (pulmonary embolism, 21 patients; lower extremity thrombosis, 44 patients; line-associated venous thrombosis, 3 patients). The semiquantitative whole-blood assay for circulating D-dimer had a 96.4% sensitivity and a negative predictive value of 92.1% for identifying patients with venous thromboembolic disease. The specificity of this assay was 19.7%, and its positive predictive value was 26.9%. There was a strong correlation between the semiquantitative assay for circulating D-dimer and the quantitative measurement of circulating D-dimer using an enzyme immunoassay (Spearman's correlation coefficient, 0.8643; p<.001). We also identified a strong correlation between both the quantitative and semiquantitative measurements of circulating D-dimer with the sepsis classification proposed by the American College of Chest Physicians/Society of Critical Care Medicine (i.e., systemic inflammatory response syndrome, sepsis, severe sepsis, septic shock) for patients without venous thromboembolic disease (n = 182; quantitative measure: Spearman's correlation coefficient, 0.207; p = .002; semiquantitative measure: Spearman's correlation coefficient, 0.3519; p<.001). CONCLUSIONS: These preliminary findings suggest that a rapid whole-blood assay for the semiquantitative detection of circulating D-dimer may be a useful diagnostic tool for the exclusion of venous thromboembolic disease among critically ill patients.

Adolescent↗