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J.l. Guéant - One of the best experts on this subject based on the ideXlab platform.
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Methamphetamine-induced turning behavior in rats transfected with several plasmids.
2013Co-Authors: Carlos Enrique Orozco-barrios, Shyue-fang Battaglia-hsu, Martha Ligia Arango-rodriguez, Jose Ayala-davila, Celine Chery, Jean-marc Alberto, Henry Schroeder, Jean-luc Daval, Daniel Martinez-fong, J.l. GuéantAbstract:The plasmids were pCMV-TCII-OLEO coding for Transcobalamin-oleosin (TO), pCMV-OLEO-TCII coding for oleosin-Transcobalamin (OT), pCMV-TCII coding for Transcobalamin II (T), pCMV-OLEO coding for oleosin (O), and pCDNA3 (P). The values are the mean±SEM of 3 animals per group. ** = Significantly different from control groups. P
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Analysis of apoptosis in N1E-115 cells stably transfected with different plasmids.
2013Co-Authors: Carlos Enrique Orozco-barrios, Shyue-fang Battaglia-hsu, Martha Ligia Arango-rodriguez, Jose Ayala-davila, Celine Chery, Jean-marc Alberto, Henry Schroeder, Jean-luc Daval, Daniel Martinez-fong, J.l. GuéantAbstract:The plasmids were pCMV-TCII-OLEO coding for Transcobalamin-oleosin (TCII-OLEO), pCMV-OLEO-TCII coding for oleosin-Transcobalamin (OLEO-TCII), pCMV-TCII coding for Transcobalamin II (TCII), pCMV-OLEO coding for oleosin (OLEO), and pCDNA3. The immunofluorescence was done with a rabbit polyclonal antibody to cleaved Caspase-3 and a donkey antirabbit IgG fluorescein labeled. Before fixation, cells were incubated with 4 µM propidium iodide for 10 min. Cell nuclei were counterstained with Hoechst 33258. Calibration bars = 100 µm.
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Expression of Transcobalamin/oleosin chimeric proteins in transfected N1E-115 cells.
2013Co-Authors: Carlos Enrique Orozco-barrios, Shyue-fang Battaglia-hsu, Martha Ligia Arango-rodriguez, Jose Ayala-davila, Celine Chery, Jean-marc Alberto, Henry Schroeder, Jean-luc Daval, Daniel Martinez-fong, J.l. GuéantAbstract:A: Transgenic expression in N1E-115 cells at 48 h after transfection using lipofectamine. The cells were transfected with one of the following plasmids: pCMV-TCII-OLEO coding for Transcobalamin-oleosin (lane 1), pCMV-OLEO-TCII coding for oleosin-Transcobalamin (lane 2), pCMV-TCII coding for Transcobalamin II (lane 3), pCMV-OLEO coding for oleosin (lane 4), pCDNA3 (lane 5), and pCMV-GFP-TCII-OLEO coding for GFP-TCII-OLEO (lane 6). The housekeeping gene was β-actin. The size in bp of the amplified products was 1347 for Transcobalamin-oleosin (TCII-OLEO), 1240 for oleosin-Transcobalamin (OLEO-TCII), 551 for Transcobalamin II (TCII), 275 for oleosin (OLEO), and 349 for β-actin. B: Western blotting of homogenate of N1E-115 cells transfected with the various plasmids. From lane 1 to 6: homogenates from cells transfected with pCMV-TCII-OLEO, pCMV-OLEO-TCII, pCMV-TCII, pCMV-OLEO, empty plasmid, pCMV-GFP-TCII-OLEO, respectively. C: Vitamin B12 binding capacity in transfected cells. 57Co-labeled Cobalamin (Cbl, ∼300 µCi per µg) was incorporated into culture medium (30,000 dpm/mL) for three days. The total amount of radioactivity taken by each cell lines was measured in pellets and supernatants. Mean and S.E.M. are indicated. D: Indirect immunofluorescence of TCII in N1E-115 cells transiently transfected with lipofectamine. The four constructs and the empty plasmid were tranfected in N1E-115 cells (1–5). The immunofluorescence was done with a goat polyclonal antibody to TCII and a donkey antigoat IgG fluorescein labeled. Cell nuclei were counterstained with Hoechst 33258. Calibration bars = 10 µm. E: Confocal analysis showing co-localization of the protein GFP-TCII-OLEO with endoplasmic reticulum in transfected N1E-115 cells. The cells were transfected with the plasmid pCMV-GFP-TCII-OLEO coding for GFP-Transcobalamin-oleosin (GFP-TCII-OLEO), using lipofectamine. Cell nuclei were counterstained with Hoechst 33258 (1, 5). Co-localization was evidenced with fluorescence from GFP (2, 6), immuno-fluorescence with a mouse monoclonal antibody to the human golgin-97 (3) or a rabbit polyclonal antibody to calreticulin (7) and merge fluorescence (4,8). The secondary antibodies include a donkey IgG anti-mouse TRITC labeled or a donkey IgG anti-rabbit TRITC labeled. Calibration bars = 20 µm.
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Crohn's disease and vitamin B_12 metabolism
Digestive Diseases and Sciences, 1996Co-Authors: Daniel Lambert, F. Felden, Samira Benhayoun, C. Adjalla, Marie-andree Gélot, Pascal Renkes, Philippe Gérard, Francine Belleville, Pierre Gaucher, J.l. GuéantAbstract:The concentrations of vitamin B_12, its analogs, and the haptocorrin and Transcobalamin carriers in 21 patients suffering from Crohn's disease and a group of controls (20 adults) were measured. There were no significant differences in the mean values for vitamin B_12, total corrinoids (vitamin B_12 + analogs), or vitamin B_12 or total corrinoids bound to haptocorrin or Transcobalamin of the Crohn's and control patients. There was a significant increase in the binding capacity of Transcobalamin in the Crohn's patients compared to the controls ( P
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Receptor binding of Transcobalamin II-cobalamin in human colon adenocarcinoma HT 29 cell line
The Journal of Nutritional Biochemistry, 1996Co-Authors: Rhizlane Bougrine, E. Nexø, Jean Pierre Nicolas, C. Masson, Renée Hatier, J.l. GuéantAbstract:Abstract Transcobalamin II is the blood cobalamin binding protein that delivers cobalamin to target cells via a receptor-mediated endocytosis. We have studied the receptor binding of human Transcobalamin II in differentiated HT 29 cells. The on-rate constant and the off-rate constant were estimated at 5.8 nM −1 min −1 and 0.015 min −1 , respectively. Scatchard analysis of the Transcobalamin II-[ 57 Co] cobalamin binding to HT 29 cells showed a Ka at 0.14 pM −1 and 31,000 receptor sites per 15 days-aged cell. The binding was inhibited by EDTA and nearly abolished at pH 5.0. The Transcobalamin II-[ 57 Co] cobalamin bound to plasma membrane was eluted as a high molecular cross-linked complex in Superose 6 gel filtration. Electron microscope radioautography showed the endocytosis of iodinated rabbit Transcobalamin II in HT 29 cells. In conclusion, Transcobalamin II receptor is present in HT 29 cells and may be involved in a receptor-mediated endocytosis of Transcobalamin-cobalamin complex.
Ebba Nexo - One of the best experts on this subject based on the ideXlab platform.
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Size exclusion chromatography of B12 in infant formula no. 3 (NAN1).
2016Co-Authors: Eva Greibe, Ebba NexoAbstract:The x-axis indicates fraction number. Elution volume for void volume (V0), Transcobalamin (TC), free B12, and total volume (Vt) are indicated. B12 was on a free form, and no B12 eluted together with the elution profile of Transcobalamin or other B12-binding proteins. The graphics were created in Graph Pad Prism version 5.
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Three family members with elevated plasma cobalamin, Transcobalamin and soluble Transcobalamin receptor (sCD320).
Clinical chemistry and laboratory medicine, 2013Co-Authors: Elke Hoffmann-lücke, Johan F. B. Arendt, Peter H. Nissen, Gustav Mikkelsen, Jan Aasly, Ebba NexoAbstract:BACKGROUND Plasma cobalamin is requested in order to diagnose cobalamin deficiency and low levels confirm a deficient state. Here, we present three family members with unexpected high levels of cobalamin. METHODS We included a patient referred for cobalamin measurement due to neurological symptoms, her son and her daughter. Mother and son both suffered from myotonic dystrophy type II, while the daughter tested negative for this disease. Blood samples were analyzed for cobalamin, haptocorrin, Transcobalamin, holoTC, and sCD320. We employed gel filtration and antibody precipitation for further characterization. The protein coding region of the TCN2 gene, encoding Transcobalamin, was sequenced. RESULTS The patient, her {son} and [daughter] all had cobalamin levels above the measurement range of the routine method employed (>1476 pmol/L). Total Transcobalamin and (holoTC) were 5980 (1500), {5260 (2410)} and [5630 (1340)] pmol/L, which is well above the upper reference limits of 1500 (160) pmol/L. The sCD320 concentration was also well above the upper reference limit of 97 arb.u.: 1340, {1510} and [1090] arb.u. Haptocorrin levels were within the reference range and no signs of cobalamin deficiency were found. DNA sequencing of the TCN2 gene revealed several known polymorphisms not associated with highly elevated Transcobalamin levels. Upon gel filtration, sCD320 eluted as a larger molecule than previously reported. By incubation with anti-Transcobalamin antibodies, we precipitated both Transcobalamin and part of sCD320. CONCLUSIONS The high cobalamin levels were mainly explained by high levels of holoTC, possibly caused by complex formation with its soluble receptor, sCD320. The family occurrence points to a genetic explanation.
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Cobalamin Related Parameters and Disease Patterns in Patients with Increased Serum Cobalamin Levels
2012Co-Authors: Johan F. B. Arendt, Ebba NexoAbstract:BackgroundMeasurement of serum cobalamin levels is routinely used to diagnose cobalamin deficiency. Surprisingly, approximately 15% of patients have high cobalamin levels and no consensus exists regarding the clinical implications. MethodsHospital-treated patients above 18 years of age referred for serum cobalamin measurement were included in groups of patients [percentage cobalamin supplemented] with low (1000, n = 199 [53%]) cobalamin levels. Total and cobalamin-saturated (holo) Transcobalamin, total haptocorrin, soluble TC receptor, sCD320, and methylmalonic acid were analyzed. Data on diagnoses and medical prescriptions was obtained through medical files and the Aarhus University Prescription Database. ResultsAmong patients not cobalamin supplemented median total haptocorrin and holo Transcobalamin levels were markedly higher in the groups with high/very high cobalamin levels compared to groups with low/normal cobalamin levels. Median total Transcobalamin and sCD320 levels were similar across the groups. A number of diagnoses were significantly associated to very high Cbl levels (odds ratio (95% confidence interval)): alcoholism (5.74 (2.76–11.96)), liver disease (8.53 (3.59–20.23)), and cancer (5.48 (2.85–10.55)). Elevated haptocorrin levels were seen in patients with alcoholism, cancer, liver-, renal-, autoimmune-, and bronchopulmonary disease. No clinical associations to sCD320 and total and holo Transcobalamin levels were found. ConclusionIn non-supplemented patients, high cobalamin levels were associated to high haptocorrin levels, and several diagnoses, including alcoholism, liver disease and cancer. Our study emphasizes that clinicians should take high serum cobalamin levels into consideration in the diagnostic process.
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Cobalamin Related Parameters and Disease Patterns in Patients with Increased Serum Cobalamin Levels
2012Co-Authors: Johan F. B. Arendt, Ebba NexoAbstract:Background: Measurement of serum cobalamin levels is routinely used to diagnose cobalamin deficiency. Surprisingly, approximately 15 % of patients have high cobalamin levels and no consensus exists regarding the clinical implications. Methods: Hospital-treated patients above 18 years of age referred for serum cobalamin measurement were included in groups of patients [percentage cobalamin supplemented] with low (,200 pmol/L, n = 200 [6%]), normal (200–600, n = 202 [6%]) high (601–1000, n = 217 [27%]) and very high (.1000, n = 199 [53%]) cobalamin levels. Total and cobalamin-saturated (holo) Transcobalamin, total haptocorrin, soluble TC receptor, sCD320, and methylmalonic acid were analyzed. Data on diagnoses and medical prescriptions was obtained through medical files and the Aarhus University Prescription Database. Results: Among patients not cobalamin supplemented median total haptocorrin and holo Transcobalamin levels were markedly higher in the groups with high/very high cobalamin levels compared to groups with low/normal cobalamin levels. Median total Transcobalamin and sCD320 levels were similar across the groups. A number of diagnoses were significantly associated to very high Cbl levels (odds ratio (95 % confidence interval)): alcoholism (5.74 (2.76–11.96)), liver disease (8.53 (3.59–20.23)), and cancer (5.48 (2.85–10.55)). Elevated haptocorrin levels were seen in patients with alcoholism, cancer, liver-, renal-, autoimmune-, and bronchopulmonary disease. No clinical associations to sCD320 and total and holo Transcobalamin levels were found
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Cobalamin related parameters in diagnoses.
2012Co-Authors: Johan F. B. Arendt, Ebba NexoAbstract:Median (interquartile ranges) [reference ranges] levels of Cbl related parameters divided according to diagnoses in patients referred for measurement of serum Cbl levels and not in Cbl supplementation therapy (n = 632). Abbreviations: Cbl: cobalamin, vitamin B12; TC: Transcobalamin; holoTC: Cbl-saturated Transcobalamin; HC: haptocorrin; sCD320: soluble Transcobalamin receptor CD320.
Edward V. Quadros - One of the best experts on this subject based on the ideXlab platform.
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Cellular uptake of cobalamin: Transcobalamin and the TCblR/CD320 receptor.
Biochimie, 2013Co-Authors: Edward V. Quadros, Jeffrey M. SequeiraAbstract:Cellular uptake of cobalamin is facilitated by a receptor-mediated endocytosis process involving Transcobalamin, a plasma protein that binds cobalamin and a cell surface receptor that specifically binds Transcobalamin saturated with cobalamin. Intracellular Cbl concentration is maintained by modulating the expression of the receptor, which is cell cycle associated with highest expression in actively proliferating cells and an efflux system that shunts the excess cobalamin out of the cells for mobilization to other tissues where it is most needed. This review describes the process, proteins involved and genes encoding these proteins.
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Characterization of a monoclonal antibody with specificity for holo-Transcobalamin
Nutrition & Metabolism, 2006Co-Authors: Lars Örning, Sergey N. Fedosov, Anne Rian, Andrew Campbell, Jeff Brady, Birgit Bramlage, Keith Thompson, Edward V. QuadrosAbstract:Background HoloTranscobalamin, cobalamin-saturated Transcobalamin, is the minor fraction of circulating cobalamin (vitamin B12), which is available for cellular uptake and hence is physiologically relevant. Currently, no method allows simple, direct quantification of holoTranscobalamin. We now report on the identification and characterization of a monoclonal antibody with a unique specificity for holoTranscobalamin. Methods The specificity and affinity of the monoclonal antibodies were determined using surface plasmon resonance and recombinant Transcobalamin as well as by immobilizing the antibodies on magnetic microspheres and using native Transcobalamin in serum. The epitope of the holoTranscobalamin specific antibody was identified using phage display and comparison to a de novo generated three-dimensional model of Transcobalamin using the program Rosetta. A direct assay for holotrnscobalamin in the ELISA format was developed using the specific antibody and compared to the commercial assay HoloTC RIA. Results An antibody exhibiting >100-fold specificity for holoTranscobalamin over apoTranscobalamin was identified. The affinity but not the specificity varied inversely with ionic strength and pH, indicating importance of electrostatic interactions. The epitope was discontinuous and epitope mapping of the antibody by phage display identified two similar motifs with no direct sequence similarity to Transcobalamin. A comparison of the motifs with a de novo generated three-dimensional model of Transcobalamin identified two structures in the N-terminal part of Transcobalamin that resembled the motif. Using this antibody an ELISA based prototype assay was developed and compared to the only available commercial assay for measuring holoTranscobalamin, HoloTC RIA. Conclusion The identified antibody possesses a unique specificity for holoTranscobalamin and can be used to develop a direct assay for the quantification of holoTranscobalamin.
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direct assay for cobalamin bound to Transcobalamin holo Transcobalamin in serum
Clinical Chemistry, 2002Co-Authors: Marius Andreas Ulleland, Sheldon P. Rothenberg, Edward V. Quadros, Sergey N. Fedosov, Ingar Eilertsen, Sundrehagen Erling, Lars ÖrningAbstract:Background: Only cobalamin carried by Transcobalamin (holo-Transcobalamin) is available for cellular uptake and hence is physiologically relevant. However, no reliable or accurate methods for quantifying holo-Transcobalamin are available. We report a novel holo-Transcobalamin assay based on solid-phase capture of Transcobalamin. Methods: A monoclonal antibody specific for human Transcobalamin with an affinity constant >1010 L/mol was immobilized on magnetic microspheres to capture and concentrate Transcobalamin. The cobalamin bound to Transcobalamin was then released and assayed by a competitive binding radioassay. The quantification of holo-Transcobalamin was accomplished using calibrators composed of recombinant, human holo-Transcobalamin. Results: The assay was specific for holo-Transcobalamin and had a detection limit of 5 pmol/L. Within-run and total imprecision (CV) was 5% and 8–9%, respectively. The working range (CV <20%) was 5–370 pmol/L. Dilutions of serum were linear in the assay range. The recovery of recombinant, human holo-Transcobalamin added to serum was 93–108%. A 95% reference interval of 24–157 pmol/L was established for holo-Transcobalamin in 105 healthy volunteers 20–80 years of age. For 72 of these sera, holo-haptocorrin and total cobalamin were also determined. Whereas holo-haptocorrin correlated well ( r 2 = 0.87) with total cobalamin, holo-Transcobalamin correlated poorly ( r 2 = 0.23) with total cobalamin or holo-haptocorrin. Conclusions: The solid-phase capture assay provides a simple, reliable method for quantitative determination of holo-Transcobalamin in serum.
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Direct assay for cobalamin bound to Transcobalamin (holo-Transcobalamin) in serum.
Clinical chemistry, 2002Co-Authors: Marius Andreas Ulleland, Sheldon P. Rothenberg, Edward V. Quadros, Sergey N. Fedosov, Ingar Eilertsen, Sundrehagen Erling, Lars ÖrningAbstract:Background: Only cobalamin carried by Transcobalamin (holo-Transcobalamin) is available for cellular uptake and hence is physiologically relevant. However, no reliable or accurate methods for quantifying holo-Transcobalamin are available. We report a novel holo-Transcobalamin assay based on solid-phase capture of Transcobalamin. Methods: A monoclonal antibody specific for human Transcobalamin with an affinity constant >1010 L/mol was immobilized on magnetic microspheres to capture and concentrate Transcobalamin. The cobalamin bound to Transcobalamin was then released and assayed by a competitive binding radioassay. The quantification of holo-Transcobalamin was accomplished using calibrators composed of recombinant, human holo-Transcobalamin. Results: The assay was specific for holo-Transcobalamin and had a detection limit of 5 pmol/L. Within-run and total imprecision (CV) was 5% and 8–9%, respectively. The working range (CV
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4 Transcobalamin II and the membrane receptor for the Transcobalamin II-cobalamin complex
Baillière's Clinical Haematology, 1995Co-Authors: Sheldon P. Rothenberg, Edward V. QuadrosAbstract:Summary Transcobalamin II is a plasma protein that binds vitamin B 12 (cobalamin) as it is absorbed in the terminal ileum and distributes it to tissues. The circulating Transcobalamin II-cobalamin complex binds to receptors on the plasma membrane of tissue cells and is then internalized by receptor-mediated endocytosis. A number of genetic abnormalities are characterized either by a failure to express Transcobalamin II or by synthesis of an abnormal protein. These disorders result in cellular cobalamin deficiency and megaloblastic anaemia. In this chapter we review the structural and functional properties of Transcobalamin II, the receptor for the Transcobalamin-cobalamin complex and the clinical disorders that are associated with perturbation of circulating Transcobalamin II. In addition, we provide emerging data about the molecular genetics of Transcobalamin II which has emanated from our own and other laboratories.
Lars Örning - One of the best experts on this subject based on the ideXlab platform.
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Characterization of a monoclonal antibody with specificity for holo-Transcobalamin
Nutrition & Metabolism, 2006Co-Authors: Lars Örning, Sergey N. Fedosov, Anne Rian, Andrew Campbell, Jeff Brady, Birgit Bramlage, Keith Thompson, Edward V. QuadrosAbstract:Background HoloTranscobalamin, cobalamin-saturated Transcobalamin, is the minor fraction of circulating cobalamin (vitamin B12), which is available for cellular uptake and hence is physiologically relevant. Currently, no method allows simple, direct quantification of holoTranscobalamin. We now report on the identification and characterization of a monoclonal antibody with a unique specificity for holoTranscobalamin. Methods The specificity and affinity of the monoclonal antibodies were determined using surface plasmon resonance and recombinant Transcobalamin as well as by immobilizing the antibodies on magnetic microspheres and using native Transcobalamin in serum. The epitope of the holoTranscobalamin specific antibody was identified using phage display and comparison to a de novo generated three-dimensional model of Transcobalamin using the program Rosetta. A direct assay for holotrnscobalamin in the ELISA format was developed using the specific antibody and compared to the commercial assay HoloTC RIA. Results An antibody exhibiting >100-fold specificity for holoTranscobalamin over apoTranscobalamin was identified. The affinity but not the specificity varied inversely with ionic strength and pH, indicating importance of electrostatic interactions. The epitope was discontinuous and epitope mapping of the antibody by phage display identified two similar motifs with no direct sequence similarity to Transcobalamin. A comparison of the motifs with a de novo generated three-dimensional model of Transcobalamin identified two structures in the N-terminal part of Transcobalamin that resembled the motif. Using this antibody an ELISA based prototype assay was developed and compared to the only available commercial assay for measuring holoTranscobalamin, HoloTC RIA. Conclusion The identified antibody possesses a unique specificity for holoTranscobalamin and can be used to develop a direct assay for the quantification of holoTranscobalamin.
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Direct assay for cobalamin bound to Transcobalamin (holo-Transcobalamin) in serum.
Clinical chemistry, 2002Co-Authors: Marius Andreas Ulleland, Sheldon P. Rothenberg, Edward V. Quadros, Sergey N. Fedosov, Ingar Eilertsen, Sundrehagen Erling, Lars ÖrningAbstract:Background: Only cobalamin carried by Transcobalamin (holo-Transcobalamin) is available for cellular uptake and hence is physiologically relevant. However, no reliable or accurate methods for quantifying holo-Transcobalamin are available. We report a novel holo-Transcobalamin assay based on solid-phase capture of Transcobalamin. Methods: A monoclonal antibody specific for human Transcobalamin with an affinity constant >1010 L/mol was immobilized on magnetic microspheres to capture and concentrate Transcobalamin. The cobalamin bound to Transcobalamin was then released and assayed by a competitive binding radioassay. The quantification of holo-Transcobalamin was accomplished using calibrators composed of recombinant, human holo-Transcobalamin. Results: The assay was specific for holo-Transcobalamin and had a detection limit of 5 pmol/L. Within-run and total imprecision (CV) was 5% and 8–9%, respectively. The working range (CV
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direct assay for cobalamin bound to Transcobalamin holo Transcobalamin in serum
Clinical Chemistry, 2002Co-Authors: Marius Andreas Ulleland, Sheldon P. Rothenberg, Edward V. Quadros, Sergey N. Fedosov, Ingar Eilertsen, Sundrehagen Erling, Lars ÖrningAbstract:Background: Only cobalamin carried by Transcobalamin (holo-Transcobalamin) is available for cellular uptake and hence is physiologically relevant. However, no reliable or accurate methods for quantifying holo-Transcobalamin are available. We report a novel holo-Transcobalamin assay based on solid-phase capture of Transcobalamin. Methods: A monoclonal antibody specific for human Transcobalamin with an affinity constant >1010 L/mol was immobilized on magnetic microspheres to capture and concentrate Transcobalamin. The cobalamin bound to Transcobalamin was then released and assayed by a competitive binding radioassay. The quantification of holo-Transcobalamin was accomplished using calibrators composed of recombinant, human holo-Transcobalamin. Results: The assay was specific for holo-Transcobalamin and had a detection limit of 5 pmol/L. Within-run and total imprecision (CV) was 5% and 8–9%, respectively. The working range (CV <20%) was 5–370 pmol/L. Dilutions of serum were linear in the assay range. The recovery of recombinant, human holo-Transcobalamin added to serum was 93–108%. A 95% reference interval of 24–157 pmol/L was established for holo-Transcobalamin in 105 healthy volunteers 20–80 years of age. For 72 of these sera, holo-haptocorrin and total cobalamin were also determined. Whereas holo-haptocorrin correlated well ( r 2 = 0.87) with total cobalamin, holo-Transcobalamin correlated poorly ( r 2 = 0.23) with total cobalamin or holo-haptocorrin. Conclusions: The solid-phase capture assay provides a simple, reliable method for quantitative determination of holo-Transcobalamin in serum.
E. Nexø - One of the best experts on this subject based on the ideXlab platform.
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Characterization of the cobalamins attached to Transcobalamin I and Transcobalamin II in human plasma.
Scandinavian Journal of Haematology, 2009Co-Authors: E. NexøAbstract:Insolubilized antibody to Transcobalamin I was used to separate Transcobalamin I and Transcobalamin II. By bioautography of the extracted cobalamins it was shown that Transcobalamin II bound more deoxyadenosylcobalamin than did Transcobalamin I, and that methylcobalamin accounts for most of the cobalamins attached to Transcobalamin I. This finding may indicate that Transcobalamin I has a function in the metabolism of methylcobalamin in man.
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Receptor binding of Transcobalamin II-cobalamin in human colon adenocarcinoma HT 29 cell line
The Journal of Nutritional Biochemistry, 1996Co-Authors: Rhizlane Bougrine, E. Nexø, Jean Pierre Nicolas, C. Masson, Renée Hatier, J.l. GuéantAbstract:Abstract Transcobalamin II is the blood cobalamin binding protein that delivers cobalamin to target cells via a receptor-mediated endocytosis. We have studied the receptor binding of human Transcobalamin II in differentiated HT 29 cells. The on-rate constant and the off-rate constant were estimated at 5.8 nM −1 min −1 and 0.015 min −1 , respectively. Scatchard analysis of the Transcobalamin II-[ 57 Co] cobalamin binding to HT 29 cells showed a Ka at 0.14 pM −1 and 31,000 receptor sites per 15 days-aged cell. The binding was inhibited by EDTA and nearly abolished at pH 5.0. The Transcobalamin II-[ 57 Co] cobalamin bound to plasma membrane was eluted as a high molecular cross-linked complex in Superose 6 gel filtration. Electron microscope radioautography showed the endocytosis of iodinated rabbit Transcobalamin II in HT 29 cells. In conclusion, Transcobalamin II receptor is present in HT 29 cells and may be involved in a receptor-mediated endocytosis of Transcobalamin-cobalamin complex.
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Circadian variation of plasma cobalamin, Transcobalamin-bound cobalamin and unsaturated binding capacity of Transcobalamin and haptocorrin in healthy elderly.
Journal of affective disorders, 1995Co-Authors: K S Björkstén, L H Thorell, E. NexøAbstract:Blood samples were drawn every second hour during a 24-h period from healthy elderly subjects. Plasma levels of cobalamin, albumin, Transcobalamin-bound cobalamin and unsaturated binding capacity of Transcobalamin and haptocorrin were determined. During night, all components decreased in parallel with plasma albumin. The nocturnal decreases were therefore ascribed to plasma volume changes due to bed-rest. Reports on the recently proposed role of cobalamin for the biological clock were reviewed, but the proposed role could not be explained by this study.
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Transcobalamin II - cobalamin binding sites are present on rabbit germ cells
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 1992Co-Authors: E. Boukhzer, E. Nexø, A. Ennya, F. Felden, A. Gerard, Jean Pierre Nicolas, H. Gérard, J.l. GuéantAbstract:Abstract Specific binding sites for rabbit Transcobalamin II have been found on isolated adult rabbit germ cells. Scatchard analysis revealed a single class of binding sites for [57Co]cyanocobalamin-Transcobalamin II with an association constant ( K a ) of 1.3 · 1010 M−1 and 700 sites per cell. Binding was reversible, saturable and calcium dependent. Electron microscope radioautography following incubation with iodinated Transcobalamin II at 4°C led to a detectable labeling mainly restricted to the plasma membrane.
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Synthesis and secretion of a cobalamin-binding protein by HT 29 cell line
Biochemical Journal, 1991Co-Authors: H. Schohn, E. Nexø, J.l. Guéant, M. Girr, L Baricault, Alain Zweibaum, Jean Pierre NicolasAbstract:An HT 29 cell line derived from human colonic carcinoma was shown to synthesize and release a cobalamin-binding protein. The cobalamin-binding protein was classified as Transcobalamin (TC). By gel filtration on Sephacryl S200 HR, we observed that the secreted protein bound to cobalamin had the same size as plasma Transcobalamin. Like Transcobalamin, the cobalamin-binding protein bound cobalamin but not cobinamide. Purification of the cobalamin-binding protein was performed by heparin-Sepharose affinity chromatography and by Sephacryl S200 gel filtration. The molecular mass of the purified protein was estimated at 44 kDa by SDS/PAGE. The isoelectric point was determined to be 6.4. The purified cobalamin-binding protein reacted with an antiserum produced against human Transcobalamin. A 44 kDa band was also identified by SDS/PAGE of an immunoprecipitated homogenate from HT 29 cells labelled with [35S]methionine and in a Western blot of cell homogenates. The secretion of the cobalamin-binding protein was maximal between 10 and 12 days of cell culture and was inhibited by cycloheximide.
