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PubMed1990

cis elements and trans-acting factors involved in dimer formation of murine leukemia virus RNA.

The genetic material of all retroviruses examined so far consists of two identical RNA molecules joined at their 5' ends by the dimer linkage structure (DLS). Since the precise location of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analyzed the dimerization process of Moloney murine leukemia virus (MoMuLV) genomic RNA. For this purpose we derived an in vitro model for RNA dimerization. By using this model, murine leukemia virus RNA was shown to form dimeric molecules. Deletion mutagenesis in the 620-nucleotide leader of MoMuLV RNA showed that the dimer promoting sequences are located within the encapsidation element Psi between positions 215 and 420. Furthermore, hybridization assays in which DNA oligomers were used to probe monomer and dimer forms of MoMuLV RNA indicated that the DLS probably maps between positions 280 and 330 from the RNA 5' end. Also, retroviral nucleocapsid protein was shown to catalyze dimerization of MoMuLV RNA and to be tightly bound to genomic dimer RNA in virions. These results suggest that MoMuLV RNA dimerization and encapsidation are probably controlled by the same cis element, Psi, and trans-acting factor, nucleocapsid protein, and thus might be linked during virion formation.

Avian Sarcoma Viruses↗
PubMed1994

Characterization of human immunodeficiency virus type 1 dimeric RNA from wild-type and protease-defective virions.

We have characterized the dimeric genomic RNA in particles of both wild-type and protease (PR)-deficient human immunodeficiency virus type 1 (HIV-1). We found that the dimeric RNA isolated from PR- mutant virions has a lower mobility in nondenaturing gel electrophoresis than that from wild-type virions. It also dissociates into monomers at a lower temperature than the wild-type dimer. Thus, the dimer in PR- particles is in a conformation different from that in wild-type particles. These results are quite similar to recent findings on Moloney murine leukemia virus and suggest that a postassembly, PR-dependent maturation event is a common feature in genomic RNAs of retroviruses. We also measured the thermal stability of the wild-type and PR- dimeric RNAs under different ionic conditions. Both forms of the dimer were stabilized by increasing Na+ concentrations. However, the melting temperatures of the two forms were not significantly affected by the identity of the monovalent cation present in the incubation buffer. This observation is in contrast with recent reports on dimers formed in vitro from short segments of HIV-1 sequence: the latter dimers are specifically stabilized by K+ ions. K+ stabilization of dimers formed in vitro has been taken as evidence for the presence of guanine quartet structures. The results suggest that guanine quartets are not involved in the structure linking full-length, authentic genomic RNA of HIV-1 into a dimeric structure.

Biopolymers↗
PubMed2006

Bronchoalveolar lavage fluid D dimer levels are higher and more prevalent in black patients with pulmonary sarcoidosis.

BACKGROUND: Abnormalities of lung coagulation and fibrinolysis in sarcoidosis are thought to play a role in the pathogenesis of this disease. OBJECTIVE: We previously showed that bronchoalveolar lavage fluid (BALF) D dimer directly correlated with various measures of severity in sarcoidosis. Here, we analyze our observation that BALF D dimer was more frequently found at higher levels in African-American patients with pulmonary sarcoidosis. METHODS: BALF D dimer was measured in 55 subjects with pulmonary sarcoidosis and 31 healthy volunteers by enzyme immunoassay. The healthy group established a normal range of BALF D dimer with 71 ng/ml as the highest measured level. This was the cut point for comparisons among the patients with sarcoidosis. RESULTS: High BALF D dimer levels (>71 ng/ml) were found in younger patients with sarcoidosis and were associated with a significantly lower percent predicted forced expiratory volume in 1 s and greater numbers of BAL lymphocytes. Black patients with sarcoidosis had higher BALF D dimer levels (median 131, range 0-2,040 ng/ml) than white patients (median 18, range 0-605 ng/ml; p = 0.011). Higher than normal BALF D dimer levels were found in 61% of the black subjects with sarcoidosis, but in only 20% of the white individuals (chi(2) = 5.539, p = 0.019). BALF D dimer was the only disease measure that discriminated black from white individuals with sarcoidosis. CONCLUSION: BALF D dimer is an indicator of lung fibrin formation and degradation in sarcoidosis. The relationship of high D dimer levels with greater BAL lymphocytosis and worse lung function may be a marker of active sarcoidosis, especially in African-Americans who tend to suffer a more serious form of the disease.

Adult↗
PubMed2004

Rapid D-dimer test combined a clinical model for deep vein thrombosis. Validation with ultrasonography and clinical follow-up in 383 patients.

An optimal approach to the diagnosis of deep vein thrombosis (DVT) in lower limbs in the emergency department is still unknown. In this prospective cohort study, we aimed to evaluate the accuracy of the widely available plasma D-dimer test (VIDAS) and establish the usefulness of combining D-dimer testing with a clinical model to reduce the need for serial ultra-sonographies and improve the diagnostic strategy of DVT. We performed a cohort study in 383 consecutive outpatients referred to the emergency department of Hospital La Princesa, with clinical suspicion of DVT. The patients were stratified into three pre-test probability categories using an explicit clinical model (Wells score), and underwent a quantitative automated ELISA D-dimer assay (VIDAS D-Dimer bioMérieux). Patients were managed according to the diagnostic strategy based on clinical probability and compression ultrasonography (CU). Patients for whom DVT was considered a high pre-test probability with negative ultrasonographic findings in the initial CU, returned the following week for repeat ultrasonography. All patients with DVT excluded did not receive anticoagulant therapy, and were followed up for three months to monitor the development of venous thromboembolic complications. DVT was confirmed in 102 patients (26.6%): 95 in the initial test, four in the second test, and three who developed venous thromboembolic complications in the three-month follow-up period. The calculated D-dimer cut-off level was 1 micro g/ml. One hundred patients (98%) with DVT had positive D-dimer. D-dimer had a sensitivity of 98% and a negative predictive value of 98.6%. Among the high-probability patients with positive D-dimer tests and initial negative CU, 9.75% had DVT on repeat CU at one week. The study results suggest that the addition of VIDAS D-dimer to this diagnostic algorithm could improve the management of patients with suspected DVT in daily practice. A diagnostic approach of DVT based on D-dimer (cut-off > or =1 microg/ml) as the first diagnostic tool for the exclusion of DVT, and the clinical probability model as the tool that identifies those patients requiring a second ultrasonography is useful and suitable for daily medical practice.

Aged↗
PubMed2005

D-dimer levels in combination with residual venous obstruction and the risk of recurrence after anticoagulation withdrawal for a first idiopathic deep vein thrombosis.

We assessed the predictive value of D-dimer levels in combination with residual venous obstruction (RVO) for recurrent venous thromboembolism (VTE) in a prospective cohort of outpatients after oral anticoagulant therapy (OAT) suspension for a first episode of idiopathic proximal deep vein thrombosis of the lower limbs during a 2-year follow-up. Patients (n=400) were enrolled on the day of OAT suspension when RVO was determined by compression ultrasonography (present in 48.6% of patients). D-dimer (cut-off value: 500 ng/mL) was measured 30+/-10 days afterwards (abnormal in 56.4% of patients). The overall recurrence rate was 16.7% (67/400; 95% confidence intervals - CI -: 13-21 %). The multivariate hazard ratio (HR) for recurrence was 3.32 (95% CI: 1.78-6.75; p<0.0001) for abnormal D-dimer compared to normal D-dimer and 1.2 (95% CI:0.72-2.07; p>0.05) for RVO compared to absent RVO. The recurrence rate was 5.7% (95% CI:2-13%) and 10.4% (95% CI:6-18%), respectively, for normal D-dimer either without or with RVO, 22.9% (95% CI: 14-33%) and 25.9% (95% CI: 18-35%), respectively, for abnormal D-dimer, either without or with RVO. When compared with normal D-dimer without RVO, the multivariate HR for recurrence was similar for abnormal D-dimer either with RVO (4.76 - 95% CI:1.78-12.8) or without RVO (4.3-95%:1.56-11.88). Abnormal D-dimer at one month after OAT withdrawal is an independent risk factor for recurrent VTE, while RVO at the time of OAT withdrawal, either with normal or abnormal D-dimer after one month, does not influence the risk of recurrence.

Administration, Oral↗
PubMed2001

C-reactive protein, fibrin D-dimer, and incident ischemic heart disease in the Speedwell study: are inflammation and fibrin turnover linked in pathogenesis?

Plasma levels of C-reactive protein (CRP, a marker of the reactant plasma protein component of the inflammatory response) and of fibrin D-dimer (a marker of cross-linked fibrin turnover) have each been associated in recent studies with the risk of future ischemic heart disease (IHD). Previous experimental studies have shown that fibrin degradation products, including D-dimer, have effects on inflammatory processes and acute-phase protein responses. In the Speedwell Prospective Study, we therefore measured CRP and D-dimer levels in stored plasma samples from 1690 men aged 49 to 67 years who were followed-up for incident IHD for an average of 75+/-4 months (mean+/-SD) and studied their associations with each other, with baseline and incident IHD, and with IHD risk factors. CRP and D-dimer levels were each associated with age, plasma fibrinogen, smoking habit, and baseline evidence of IHD. CRP was associated with D-dimer (r=0.21, P<0.00001). On univariate analyses, both CRP and D-dimer were associated with incident IHD. The incidence of IHD increased with CRP independently of the level of D-dimer (P=0.0002) and also increased with D-dimer independently of the level of CRP (P=0.048). In multivariate analyses, inclusion of D-dimer and conventional risk factors reduced the strength of the association between CRP and incident IHD; likewise, inclusion of CRP and conventional risk factors reduced the strength of the association between D-dimer and incident IHD. We conclude that although these respective markers of inflammation and fibrin turnover show modest association with each other in middle-aged men, they may have additive associations with risk of incident IHD. Further larger studies are required to test this hypothesis.

Acute-Phase Proteins↗
PubMed2000

Plasma D-dimer levels in operable breast cancer patients correlate with clinical stage and axillary lymph node status.

PURPOSE: To investigate the relationship between preoperative plasma D-dimer levels and extent of tumor involvement in operable breast cancer patients. PATIENTS AND METHODS: A total of 140 preoperative plasma specimens were obtained from women scheduled to undergo diagnostic breast biopsies. Ninety-five patients in the initial group went on to undergo axillary lymph node dissection. Of the 140 patients from whom plasma samples were obtained, 102 were subsequently diagnosed with invasive breast carcinoma, nine were subsequently diagnosed with ductal carcinoma-in-situ, and 20 were subsequently diagnosed with benign breast disease. Plasma D-dimer levels were quantitated using a commercially available immunoassay kit (DIMERTEST; American Diagnostica, Greenwich, CT). The relationships between plasma D-dimer and other prognostic variables (tumor size, estrogen receptor, progesterone receptor, nuclear grade, histologic grade, lymphovascular invasion, and clinical stage grouping) were then examined using univariate and multivariate linear and logistic regression analyses. RESULTS: Median plasma D-dimer levels were significantly higher in patients with invasive carcinoma than those patients with either benign breast disease or carcinoma-in-situ (P =.0001). A significant relationship existed between the presence of elevated D-dimer (> 100 ng/mL) and involved axillary lymph nodes (chi(2) test; P =.001). Elevated D-dimer levels predicted positive lymph node involvement in both univariate regression (P =.0035) and multivariate linear regression (P =.012) models. In addition, elevated D-dimer levels predicted the presence of lymphovascular invasion in univariate logistic regression (P =. 0025) and multivariate logistic regression analysis (P =.0053). Quantitative D-dimer levels were highly correlated with clinical stage grouping (analysis of variance test; P =.002). CONCLUSION: Plasma D-dimer levels were markers of lymphovascular invasion, clinical stage, and lymph node involvement in operable breast cancer. This correlation suggests that detectable fibrin degradation, as measured by plasma D-dimer, is a clinically important marker for lymphovascular invasion and early tumor metastasis in operable breast cancer.

Adult↗
PubMed2005

Latex D-dimer signal in in situ femoral vein thrombus in swine and effect of minidose exogenous tissue plasminogen activator bolus.

STUDY OBJECTIVES: We created in situ femoral vein thrombi in swine to investigate the response of the latex d-dimer signal to acute in situ venous thrombosis, and to determine the minimum dose of exogenous bolus tissue plasminogen activator (t-PA) required to significantly elevate the d-dimer signal. STUDY DESIGN: We studied seven swine (20 to 22 kg) under pentobarbital anesthesia. A 6-cm segment of the proximal femoral vein was surgically exposed and briefly ligated. Thrombin, 250 U, was then injected into the isolated femoral vein segment to create an in situ clot. After clot formation was documented to be complete between the ligatures, they were then released. D-dimer levels were then measured every 15 min for 1 h before and 1 h after clot formation with ligatures released. Time-response curves to establish timing of peak t-PA effect were performed, and then escalating dose-response curves of d-dimer level to minidose t-PA were plotted. RESULTS: After formation of the clot, the release of ligatures resulted in no change in d-dimer levels over 1 h (p = 0.62) in all swine. When a time-response curve to exogenous t-PA bolus in the presence of femoral clot was plotted, there was a maximal increase in d-dimer signal at 30 min after bolus t-PA administration. The subsequent dose-response curves for escalating fivefold boluses of minidose t-PA showed an increase in d-dimer signal at doses of 0.8 mg (p = 0.03) and 4 mg (p = 0.003). CONCLUSION: We conclude the following: (1) in situ femoral vein clot formation does not elevate d-dimer signal for 1 h after ligature release; (2) minidose t-PA boluses of 0.8 mg and 4 mg significantly elevated the latex d-dimer signal above baseline; and (3) there is a potential role of minidose t-PA in enhancing the d-dimer signal in in situ deep venous thrombosis.

Animals↗
PubMed2004

Role of a quantitative D-dimer assay in determining the need for CT angiography of acute pulmonary embolism.

OBJECTIVE: Our goal was to use the results of a quantitative D-dimer assay to determine the need for pulmonary CT angiography in patients suspected of having acute pulmonary embolism. MATERIALS AND METHODS: From July 2001 to December 2002, 755 patients underwent pulmonary CT angiography for the evaluation of acute pulmonary embolism. A rapid, fully automated quantitative D-dimer assay was obtained in more than half the patients. The electronic medical records of the patients were subsequently reviewed to analyze the negative predictive value of the D-dimer assay in the diagnostic workup of acute pulmonary embolism and to determine the outcome of the patients who had negative findings on both D-dimer assay and pulmonary CT angiography at 3-month follow-up. RESULTS: Of the 755 patients who underwent pulmonary CT angiography, 666 (88.2%) had negative findings, 73 (9.7%) had positive findings, and 16 (2.1%) were indeterminate. A total of 426 patients underwent both pulmonary CT angiography and D-dimer level evaluation, and 84 of these had negative findings (< 0.4 microg/mL) on D-dimer assay. Eighty-two of the 84 patients with negative findings on D-dimer assay had negative findings on pulmonary CT angiography; two were indeterminate and both subsequently had low-probability ventilation-perfusion studies. Among patients with positive D-dimer assays, no one with a level between 0.4 and 1.0 microg/mL had pulmonary CT angiography with findings positive for pulmonary embolism. CONCLUSION: A quantitative D-dimer assay was effective in excluding the need for pulmonary CT angiography and had high negative predictive value when the D-dimer level was less than 1.0 microg/mL.

Acute Disease↗
PubMed1999

[Plasma D-dimer levels in patients with deep vein thrombosis].

Hemostatic abnormalities were examined in 40 patients with deep vein thrombosis (DVT), 40 patients treated with warfarin, and 30 healthy volunteers. Plasma D-dimer levels were measured by an enzyme linked fluorescent assay (ELFA; Vidas-D-dimer) and an enzyme immunosorbent assay (ELISA; D-dimer-ELISA). Plasma levels of D-dimer, thrombin-antithrombin complex (TAT), soluble fibrin monomer (SFM) were significantly higher in patients with DVT than in the other groups. Both the sensitivity and specificity of D-dimer for diagnosis of DVT were the highest among hemostatic molecular markers examined. The most adequate cut off value of Vidas-D-dimer was 0.6 microgram/ml. Plasma levels of Vidas-D-dimer were well correlated with D-dimer ELISA, SFM and TAT. As the time of measurement for Vidas-D-dimer is less than one hour, the measurement of D-dimer using a EFLA might be useful for the diagnosis of DVT in bed side.

Enzyme-Linked Immunosorbent Assay↗
PubMedDate not supplied

[Influence of oral anticoagulant treatment on D-dimers levels].

The usefulness of D-dimers determination for the exclusion of deep vein thrombosis (DVT) has been extensively studied. The persistence of high levels of D-dimers has also been suggested as a marker of hypercoagulability in rare studies and might be used to identify patients at risk for recurrent DVT. We have studied the influence of oral anticoagulant treatment in 149 patients, 17 to 84 year-old, with a history of venous thromboembolism; 81 received oral anticoagulant treatment, 68 did not. Patients with known reasons for high level of D-dimers such as cancer were excluded. Thrombophilia was found in 84 patients. D-dimers measurements were performed by ELFA technique using Vidas (bioMérieux, France) analyzer. A significantly lower level of D-dimers was observed in patients under oral anticoagulant compared to patients without this treatment, 197 +/- 134 mug/L versus 399 +/- 239 mug/L, respectively (p < 0.001). A level upper the normal value (500 mug/L) was found in only 3 patients out of 81 receiving an oral anticoagulant treatment as compared with 21 of the 68 patients without treatment. This decrease of D-dimers in patients receiving oral anticoagulants was the same in the different age populations. There was no correlation between INR and D-dimers levels in this study. The clinician should be informed of the decrease of D-dimers in patients treated with anticoagulants. The decrease of D-dimers plasma level during oral anticoagulant treatment suggest that D-dimers concentration in plasma is an indirect marker of reduced clotting activity in vivo.

Administration, Oral↗
PubMed1991

[Clinical usefulness of the measurement of plasma D-dimer levels].

To evaluate the clinical usefulness of D-dimer, various effects on the measurement of D-dimer were examined. Although both fibrinolytic and fibrinogenolytic products were detected by the measurement of FDP, only fibrinolytic products were detected by the measurement of D-dimer. In patients with DIC and other thrombo-embolic diseases, plasma D-dimer levels were significantly higher than in normal persons. A significant positive correlation between plasma D-dimer and serum FDP was found in DIC patients. In patients with DIC associated with acute promyelocytic leukemia, which is thought to be an increased fibrinogenolysis state, serum FDP was higher than the plasma D-dimer which suggests that increased fibrinogenolysis affects the result of serum FDP measurement. Plasma D-dimer significantly increased 5 minutes after endoscopic embolization with thrombin in the patients with esophageal varices. However serum FDP increased 30 minutes after the treatment, which suggests that the D-dimer is more useful for rapid detection of coagulo-fibrinolytic change than serum FDP. Plasma D-dimer was significantly higher in patients with cerebral infarction and increased with age. These finding suggest the usefulness of plasma D-dimer measurement for the specific and rapid evaluation of coagulo-fibrinolytic activation and thrombo-embolic state.

Adult↗
PubMed1990

[Measurement of FDP-D-dimer in DIC and pre-DIC].

We measured FDP-D-dimer value in disseminated intravascular coagulation (DIC), pre-DIC (within 7 days before onset of DIC) and suspected DIC (not completely satisfying the DIC criteria). The level of FDP in many patients with pre-DIC was normal, but the level of FDP-D-dimer in most patients with pre-DIC was increased. FDP was markedly increased one day before onset of DIC but FDP-D-dimer was increased 7 days before the onset. FDP was significantly higher in DIC than in pre-DIC, but it was not higher in pre-DIC than in suspected DIC. In patients with hematological malignancies, FDP-D-dimer was statistically higher in DIC than in pre-DIC and in pre-DIC than in suspected DIC, but in non-hematological malignancies, FDP-D-dimer was not significantly different among the 3 groups. The peak increase of FDP-D-dimer was noted at a DIC score of 7 or 8. The correlation of FDP-D-dimer with FDP was better in pre-DIC than in DIC, and the ratio of FDP-D-dimer to FDP was higher in pre-DIC. FDP-D-dimer was not correlated with fibrinopeptide A or B beta 15-42 in pre-DIC. It is speculated that pre-DIC is a hypercoagulable state and FDP-D-dimer may be useful to the diagnosis of pre-DIC.

Disseminated Intravascular Coagulation↗
PubMed1993

Location of CD4 dimerization site explains critical role of CDR3-like region in HIV-1 infection and T-cell activation and implies a model for complex of coreceptor-MHC.

CD4 cross-linking by antibodies or its natural ligands triggers a tyrosine kinase activity that is one of the necessary steps in the mechanism of human immunodeficiency virus type 1 (HIV-1)-induced syncytium formation and full Th-cell activation. In this study we mapped a part of the dimerization site of human CD4 to amino acids 87-98 using a bivalent CD4 immunoadhesin and a series of overlapping 12-mer peptides of the D1 domain. The dimerization site we found is part of the complementary determining region (CDR) 3-like region of CD4. Using the three-dimensional structure of other immunoglobulin dimers as a basis, a molecular modeling study was performed to dimerize the D1 domains of CD4. Both the peptide binding studies and molecular modeling studies independently led to the conclusion that the CDR3-like region is part of the CD4 dimerization site. The suggested dimerization of CD4 through its CDR3-like region explains the important role that has been ascribed to this region in Th-cell activation and HIV-1-mediated fusion. Based on this model of the CD4 dimer and published results of different mutational analysis studies, a model was proposed for the complex of the CD4 dimer with two MHC-II molecules. The CD4 dimer allows tight binding to a large surface of MHC-II and the complex of CD4 and MHC-II reconciles mutational analysis studies that were previously incompatible. Moreover, the complex suggests how CD4 can dimerize through ligand binding.

Amino Acid Sequence↗
PubMed1993

Quantitative plasma D-dimer levels among patients undergoing pulmonary angiography for suspected pulmonary embolism.

OBJECTIVE: To test the hypothesis that a low D-dimer level has a high negative predictive value for acute pulmonary embolism (PE) among patients undergoing diagnostic pulmonary angiography. DESIGN: Blinded comparison of quantitative plasma D-dimer levels, measured using a monoclonal antibody assay, with pulmonary angiographic results from 173 patients with suspected acute PE. SETTING: Tertiary care setting at fur participating institutions. PATIENTS: Plasma samples were analyzed in 173 patients who underwent diagnostic pulmonary arteriography for suspected acute PE. MAIN OUTCOME MEASURES: Sensitivity, specificity, and predictive values of quantitative plasma D-dimer levels for the diagnosis of PE, using pulmonary angiographic data as the criterion standard test. RESULTS: Of 35 patients with D-dimer values less than 500 ng/mL, only three had abnormal pulmonary angiograms. The negative predictive value of a plasma D-dimer level less than 500 ng/mL for acute PE was 91.4% (95% confidence interval [CI], 76.9% to 98.2%). D-dimer levels were greater than 500 ng/mL in 42 of 45 patients with PE and in 96 of 128 patients without PE (P = .016). Sensitivity, specificity, and positive predictive value of a plasma D-dimer level greater than 500 ng/mL for acute PE were 93.3% (95% CI, 81.7% to 98.6%), 25.0% (95% CI, 17.5% to 32.5%), and 30.4% (95% CI, 22.8% to 38.1%), respectively. CONCLUSIONS: The results of our study indicate that quantitative plasma D-dimer levels can be useful in screening patients with suspected PE who require pulmonary angiography. Plasma D-dimer values less than 500 ng/mL may obviate the need for pulmonary angiography, particularly among medical patients for whom the clinical suspicion of PE is low. The plasma D-dimer value, assayed using a commercially available enzyme-linked immunosorbent assay kit, is a sensitive but nonspecific test for the presence of acute PE.

Adult↗
PubMed1994

Disulfide bond formation between dimeric immunoglobulin A and the polymeric immunoglobulin receptor during hepatic transcytosis.

The polymeric immunoglobulin receptor on rat hepatocytes binds dimeric IgA on the sinusoidal surface and mediates its transport to the canaliculus, where the complex of dimeric IgA and secretory component, the cleaved extracellular domain of polymeric immunoglobulin receptor, is secreted into bile. This process is unique in that disulfide bonds are formed between dimeric IgA and polymeric immunoglobulin receptor during transcytosis, permanently preventing their dissociation. Here we present three lines of evidence that disulfide bonding between dimeric IgA and polymeric immunoglobulin receptor occurs predominantly in a late transcytotic compartment and that hepatic transcytosis can proceed in the absence of disulfide bond formation. First, throughout the course of transcytosis the percentage of intracellular dimeric IgA disulfide bonded to polymeric immunoglobulin receptor is less than half that in bile, suggesting that disulfide bond formation is a late event in transcytosis. Second, dimeric IgA that recycles from early endocytotic compartments into the circulation is mostly noncovalently bound to secretory component. Finally, the rate of transcytosis of dimeric IgA and its appearance in bile are not affected when disulfide bond formation with polymeric immunoglobulin receptor is inhibited by blocking of free thiol groups on dimeric IgA with iodoacetamide. These results are consistent with other findings in the literature and indicate that the main physiological role of disulfide bond formation between dimeric IgA and polymeric immunoglobulin receptor is not to facilitate transcytosis but, rather, to stabilize the dimeric IgA-secretory component complex after its release into external secretions such as bile and intestinal secretions.

Animals↗
PubMed1994

Evidence that Na+/H+ exchanger isoforms NHE1 and NHE3 exist as stable dimers in membranes with a high degree of specificity for homodimers.

In this study, we have investigated whether members of the Na+/H+ exchanger (NHE) family are oligomers and whether such oligomeric structure is required for function. Fibroblasts overexpressing NHE1 were treated briefly at 0 degrees C with the cross-linker disuccinimidyl suberate, then membranes were prepared and proteins analyzed by SDS-polyacrylamide gel electrophoresis. Disuccinimidyl suberate treatment converted quantitatively the immunoreactive monomeric form of NHE1 (110 kDa) to a putative dimeric form (210 kDa). Utilization of NHE1 mutant deleted of the cytoplasmic domain (delta 515TH) demonstrates that the transmembrane domain of the antiporter is sufficient for dimerization. Moreover, coimmunoprecipitation of NHE1 and delta 515TH, coexpressed in the same cell, formally proved the existence of dimers. This dimerization was also shown to take place with the epithelial and apically expressed NHE3 isoform, suggesting that oligomerization is a common feature of these transporters. However, coexpression of NHE1 and NHE3 in the same cells did not lead to the formation of heterodimers demonstrating an isoform specificity for the subunit interaction. The domain(s) involved in the isoform-specific dimerization is (are) likely to be confined within the transmembrane segments, as deletion of the 300 amino acids of the cytoplasmic domain did not disrupt dimerization. Exploiting the dimeric properties of the receptor tyrosine kinases and the fact that dimerization triggers kinase activity, we constructed a NHE1/insulin receptor chimera to probe NHE1 dimerization in vivo. When transfected into hamster fibroblasts, this chimera containing the N-terminal transmembrane domain of NHE1 and the cytoplasmic beta-subunit domain of the insulin receptor generates a functional transporter that is autophosphorylated on tyrosine and that presents properties of a constitutively active insulin receptor. These findings support the notion that NHE1 exists in an oligomeric state in intact cells. Finally, to test whether individual subunits of NHE1 are the minimum functional unit for Na+/H+ exchange, we coexpressed a truncated form of NHE1 (delta 515) together with an inactive mutant of NHE1 (E262I). In spite of good expression of the inactive transporter and its capacity to dimerize with active NHE1, no dominant negative effect was observed on amiloride-sensitive 22Na+ flux. This observation would suggest that individual subunits of NHE1 function independently within the oligomeric state.

Animals↗
PubMed1998

Tetramer-dimer equilibrium of oxyhemoglobin mutants determined from auto-oxidation rates.

One of the main difficulties with blood substitutes based on hemoglobin (Hb) solutions is the auto-oxidation of the hemes, a problem aggravated by the dimerization of Hb tetramers. We have employed a method to study the oxyHb tetramer-dimer equilibrium based on the rate of auto-oxidation as a function of protein concentration. The 16-fold difference in dimer and tetramer auto-oxidation rates (in 20 mM phosphate buffer at pH 7.0, 37 degrees C) was exploited to determine the fraction dimer. The results show a transition of the auto-oxidation rate from low to high protein concentrations, allowing the determination of the tetramer-dimer dissociation coefficient K4,2 = [Dimer] 2/[Tetramer]. A 14-fold increase in K4,2 was observed for addition of 10 mM of the allosteric effector inositol hexaphosphate (IHP). Recombinant hemoglobins (rHb) were genetically engineered to obtain Hb with a lower oxygen affinity than native Hb (Hb A). The rHb alpha2beta2 [(C7) F41Y/(G4) N102Y] shows a fivefold increase in K4,2 at pH 7.0, 37 degrees C. An atmosphere of pure oxygen is necessary in this case to insure fully oxygenated Hb. When this condition is satisfied, this method provides an efficient technique to characterize both the tetramer-dimer equilibrium and the auto-oxidation rates of various oxyHb. For low oxygen affinity Hb equilibrated under air, the presence of deoxy subunits accelerates the auto-oxidation. Although a full analysis is complicated, the auto-oxidation studies for air equilibrated samples are more relevant to the development of a blood substitute based on Hb solutions. The double mutants, rHb alpha2beta2 [(C7) F41Y/(G4) N102A] and rHb alpha2beta2 [(C7) F41Y/(E10) K66T], show a lower oxygen affinity and a higher rate of oxidation than Hb A. Simulations of the auto-oxidation rate versus Hb concentration indicate that very high protein concentrations are required to observe the tetramer auto-oxidation rate. Because the dimers oxidize much more rapidly, even a small fraction dimer will influence the observed oxidation rate.

Adult↗