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Mat J A P Daemen - One of the best experts on this subject based on the ideXlab platform.
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molecular mri of early thrombus formation using a bimodal α2 antiplasmin based contrast agent
Jacc-cardiovascular Imaging, 2009Co-Authors: Robbertjan J H M Miserus, Johan W. M. Heemskerk, Veronica Herias, Lenneke Prinzen, Marc B I Lobbes, Robertjan Van Suylen, Anouk Dirksen, Tilman Mathias Hackeng, Jos M A Van Engelshoven, Mat J A P DaemenAbstract:Objectives We aimed to investigate whether early thrombus formation can be visualized with in vivo magnetic resonance imaging (MRI) by the use of a novel bimodal α2-antiplasmin–based contrast agent (CA). Background Thrombus formation plays a central role in several vascular diseases. During the early phases of thrombus formation, activated factor XIII (FXIIIa) covalently cross-links α2-antiplasmin to fibrin, indicating the potential of α2-antiplasmin–based CAs in the detection of early thrombus formation. Methods A bimodal CA was synthesized by coupling gadolinium-diethylene triamine pentaacetic acid and rhodamine to an α2-antiplasmin–based peptide. For the control CA, a glutamine residue essential for cross-linking was replaced by alanine. In vitro-generated thrombi were exposed to both CAs and imaged by MRI and 2-photon laser-scanning microscopy. Immunohistochemistry was performed on human pulmonary thromboemboli sections to determine the presence of α2-antiplasmin and FXIII in different thrombus remodeling phases. In vivo feasibility of the CA in detecting early thrombus formation specifically was investigated with MRI. Results In vitro–generated thrombi exposed to the α2-antiplasmin–based CA showed hyperintense magnetic resonance signal intensities at the thrombus edge. No hyperintense signal was observed when we used the α2-antiplasmin–based CA in the presence of FXIII inhibitor Dansylcadaverine nor when we used the control CA. Two-photon laser-scanning microscopy demonstrated that the α2-antiplasmin–based CA bound to fibrin. Immunohistochemistry demonstrated substantial α2-antiplasmin staining in fresh compared with lytic and organized thrombi. The administration of CA in vivo within seconds after inducing thrombus formation increased contrast-to-noise ratios (CNRs 2.28 ± 0.39, n=6) at the site of thrombus formation compared with the control CA (CNRs −0.14 ± 0.55, p = 0.003, n = 6) and α2-antiplasmin–based CA administration 24 to 48 h after thrombus formation (CNRs 0.11 ± 0.23, p = 0.006, n = 6). Conclusions A bimodal CA was developed, characterized, and validated. Our results showed that this bimodal CA enabled noninvasive in vivo magnetic resonance visualization of early thrombus formation.
Robert J Smith - One of the best experts on this subject based on the ideXlab platform.
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insulin like growth factor i receptor internalization regulates signaling via the shc mitogen activated protein kinase pathway but not the insulin receptor substrate 1 pathway
Journal of Biological Chemistry, 1998Co-Authors: Jesse C Chow, Gerolama Condorelli, Robert J SmithAbstract:Insulin-like growth factor-I (IGF-I) receptors activate divergent signaling pathways by phosphorylating multiple cellular proteins, including insulin receptor substrate-1 (IRS-1) and the Shc proteins. Following hormone binding, IGF-I receptors cluster into clathrin-coated pits and are internalized via an endocytotic mechanism. This study investigates the relationship between IGF-I receptor internalization and signaling via IRS-1 and Shc. A mutation in the C terminus of the IGF-I receptor decreased both the rate of receptor internalization and IGF-I-stimulated Shc phosphorylation by more than 50%, but did not affect IRS-1 phosphorylation. Low temperature (15 degrees C) decreased IGF-I receptor internalization and completely inhibited Shc phosphorylation. Although receptor and IRS-1 phosphorylation were decreased in accordance with delayed binding kinetics at 15 degrees C, the ratio of IRS-1 to receptor phosphorylation was increased more than 2-fold. Dansylcadaverine decreased receptor internalization and Shc phosphorylation, but did not change receptor or IRS-1 phosphorylation. Consistent with these findings, Dansylcadaverine inhibited IGF-I-stimulated Shc-Grb2 association, mitogen-activated protein kinase phosphorylation, and p90 ribosomal S6 kinase activation, but did not affect the association of phosphatidylinositide 3-kinase with IRS-1 or activation of p70 S6 kinase. These data support the concept that Shc/mitogen-activated protein kinase pathway activation requires IGF-I receptor internalization, whereas the IRS-1 pathway is activated by both cell surface and endosomal receptors.
J W C M Jansen - One of the best experts on this subject based on the ideXlab platform.
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cross linking of α2 antiplasmin to fibrin is a key factor in regulating blood clot lysis species differences
Blood Coagulation & Fibrinolysis, 1993Co-Authors: J J J Van Giezen, J Minkema, Bonno N. Bouma, J W C M JansenAbstract:: The basal lysis rates of ex-vivo prepared blood clots from rats, mice, hamsters, dogs, and humans and levels of alpha 2-antiplasmin (alpha 2-AP) cross-linked to fibrin have been studied in the presence and absence of factor XIII (FXIII) inhibitors using a blood clot lysis assay and an alpha 2-AP binding assay. Clots prepared from rat or human blood lysed spontaneously within 3-5 h. Clots from hamster blood lysed completely within 30 min but clots prepared from murine or canine blood lysed only after addition of 1.0 IU human t-PA. To study the effect of activated FXIII (FXIIIa) inhibition on blood clot lysis the FXIII inhibitors L722151 and mono-Dansylcadaverine (dansyl) were used. In the presence of the FXIII inhibitors human, murine, and canine blood clots showed increased lysis rates. The lysis rate of rat blood clots was not affected. Effects on hamster blood clots could not be detected because of the high basal lysis rate. In clots prepared from human, murine, or canine plasma about 20% of the plasma alpha 2-AP concentration was cross-linked to fibrin. FXIII inhibitors inhibited cross-linking by more than 80%. No significant cross-linking of alpha 2-AP could be detected in rat and hamster plasma clots. When 0.1 volume of human plasma was added to 0.9 volume of rat plasma the level of alpha 2-AP cross-linking was equal to that in human plasma indicating that rat alpha 2-AP can be cross-linked to human fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)
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cross linking of α2 antiplasmin to fibrin is a key factor in regulating blood clot lysis species differences
Blood Coagulation & Fibrinolysis, 1993Co-Authors: J J J Van Giezen, J Minkema, Bonno N. Bouma, J W C M JansenAbstract:: The basal lysis rates of ex-vivo prepared blood clots from rats, mice, hamsters, dogs, and humans and levels of alpha 2-antiplasmin (alpha 2-AP) cross-linked to fibrin have been studied in the presence and absence of factor XIII (FXIII) inhibitors using a blood clot lysis assay and an alpha 2-AP binding assay. Clots prepared from rat or human blood lysed spontaneously within 3-5 h. Clots from hamster blood lysed completely within 30 min but clots prepared from murine or canine blood lysed only after addition of 1.0 IU human t-PA. To study the effect of activated FXIII (FXIIIa) inhibition on blood clot lysis the FXIII inhibitors L722151 and mono-Dansylcadaverine (dansyl) were used. In the presence of the FXIII inhibitors human, murine, and canine blood clots showed increased lysis rates. The lysis rate of rat blood clots was not affected. Effects on hamster blood clots could not be detected because of the high basal lysis rate. In clots prepared from human, murine, or canine plasma about 20% of the plasma alpha 2-AP concentration was cross-linked to fibrin. FXIII inhibitors inhibited cross-linking by more than 80%. No significant cross-linking of alpha 2-AP could be detected in rat and hamster plasma clots. When 0.1 volume of human plasma was added to 0.9 volume of rat plasma the level of alpha 2-AP cross-linking was equal to that in human plasma indicating that rat alpha 2-AP can be cross-linked to human fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)
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cross linking of alpha 2 antiplasmin to fibrin is a key factor in regulating blood clot lysis species differences
Blood Coagulation & Fibrinolysis, 1993Co-Authors: J J J Van Giezen, J Minkema, Bonno N. Bouma, J W C M JansenAbstract:The basal lysis rates of ex-vivo prepared blood clots from rats, mice, hamsters, dogs, and humans and levels of alpha 2-antiplasmin (alpha 2-AP) cross-linked to fibrin have been studied in the presence and absence of factor XIII (FXIII) inhibitors using a blood clot lysis assay and an alpha 2-AP binding assay. Clots prepared from rat or human blood lysed spontaneously within 3-5 h. Clots from hamster blood lysed completely within 30 min but clots prepared from murine or canine blood lysed only after addition of 1.0 IU human t-PA. To study the effect of activated FXIII (FXIIIa) inhibition on blood clot lysis the FXIII inhibitors L722151 and mono-Dansylcadaverine (dansyl) were used. In the presence of the FXIII inhibitors human, murine, and canine blood clots showed increased lysis rates. The lysis rate of rat blood clots was not affected. Effects on hamster blood clots could not be detected because of the high basal lysis rate. In clots prepared from human, murine, or canine plasma about 20% of the plasma alpha 2-AP concentration was cross-linked to fibrin. FXIII inhibitors inhibited cross-linking by more than 80%. No significant cross-linking of alpha 2-AP could be detected in rat and hamster plasma clots. When 0.1 volume of human plasma was added to 0.9 volume of rat plasma the level of alpha 2-AP cross-linking was equal to that in human plasma indicating that rat alpha 2-AP can be cross-linked to human fibrin.(ABSTRACT TRUNCATED AT 250 WORDS)
Robbertjan J H M Miserus - One of the best experts on this subject based on the ideXlab platform.
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molecular mri of early thrombus formation using a bimodal α2 antiplasmin based contrast agent
Jacc-cardiovascular Imaging, 2009Co-Authors: Robbertjan J H M Miserus, Johan W. M. Heemskerk, Veronica Herias, Lenneke Prinzen, Marc B I Lobbes, Robertjan Van Suylen, Anouk Dirksen, Tilman Mathias Hackeng, Jos M A Van Engelshoven, Mat J A P DaemenAbstract:Objectives We aimed to investigate whether early thrombus formation can be visualized with in vivo magnetic resonance imaging (MRI) by the use of a novel bimodal α2-antiplasmin–based contrast agent (CA). Background Thrombus formation plays a central role in several vascular diseases. During the early phases of thrombus formation, activated factor XIII (FXIIIa) covalently cross-links α2-antiplasmin to fibrin, indicating the potential of α2-antiplasmin–based CAs in the detection of early thrombus formation. Methods A bimodal CA was synthesized by coupling gadolinium-diethylene triamine pentaacetic acid and rhodamine to an α2-antiplasmin–based peptide. For the control CA, a glutamine residue essential for cross-linking was replaced by alanine. In vitro-generated thrombi were exposed to both CAs and imaged by MRI and 2-photon laser-scanning microscopy. Immunohistochemistry was performed on human pulmonary thromboemboli sections to determine the presence of α2-antiplasmin and FXIII in different thrombus remodeling phases. In vivo feasibility of the CA in detecting early thrombus formation specifically was investigated with MRI. Results In vitro–generated thrombi exposed to the α2-antiplasmin–based CA showed hyperintense magnetic resonance signal intensities at the thrombus edge. No hyperintense signal was observed when we used the α2-antiplasmin–based CA in the presence of FXIII inhibitor Dansylcadaverine nor when we used the control CA. Two-photon laser-scanning microscopy demonstrated that the α2-antiplasmin–based CA bound to fibrin. Immunohistochemistry demonstrated substantial α2-antiplasmin staining in fresh compared with lytic and organized thrombi. The administration of CA in vivo within seconds after inducing thrombus formation increased contrast-to-noise ratios (CNRs 2.28 ± 0.39, n=6) at the site of thrombus formation compared with the control CA (CNRs −0.14 ± 0.55, p = 0.003, n = 6) and α2-antiplasmin–based CA administration 24 to 48 h after thrombus formation (CNRs 0.11 ± 0.23, p = 0.006, n = 6). Conclusions A bimodal CA was developed, characterized, and validated. Our results showed that this bimodal CA enabled noninvasive in vivo magnetic resonance visualization of early thrombus formation.
Laszlo Lorand - One of the best experts on this subject based on the ideXlab platform.
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the intermediate filament protein vimentin in the lens is a target for cross linking by transglutaminase
Journal of Biological Chemistry, 1998Co-Authors: Sophie Clement, Pauline T Velasco, James H. Wilson, Robert D. Goldman, S Prasanna N Murthy, Thomas J Lukas, Laszlo LorandAbstract:Mere addition of Ca2+ to a lens cortical homogenate (bovine) generates a series of products composed of a variety of high molecular weight vimentin species. The Ca2+-induced cross-linking of this cytoskeletal element seems to be mediated by the intrinsic transglutaminase of lens, because the reaction could be blocked at the monomeric state of vimentin by the inclusion of small synthetic substrates of the enzyme Dansylcadaverine or dansyl-e-aminocaproyl-Gln-Gln-Ile-Val. These compounds are known to compete against the Gln or Lys functionalities of proteins that would participate in forming the N e(γ-glutamyl)lysine protein-to-protein cross-links. The cytosolic transglutaminase-catalyzed reactions could be reproduced with purified bovine lens vimentin and also with recombinant human vimentin preparations. Employing the latter system, we have titrated the transglutaminase-reactive sites of vimentin and, by sequencing the dansyl-tracer-labeled segments of the protein, we have shown that residues Gln453 and Gln460 served as acceptor functionalities and Lys97, Lys104, Lys294, and Lys439 as electron donor functionalities in vimentin. The transglutaminase-dependent reaction of this intermediate filament protein might influence the shape and plasticity of the fiber cells, and the enzyme-catalyzed cross-linking of vimentin, in conjunction with other lens constituents, may contribute to the process of cataract formation.
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the pancornulins a group of small proline rich related cornified envelope precursors with bifunctional capabilities in isopeptide bond formation
Journal of Investigative Dermatology, 1995Co-Authors: Mary Ann Greco, K N Parameswaran, Laszlo Lorand, Howard P Baden, William S Lane, Joseph C KvedarAbstract:In this report, the pancornulins are identified as members of the spr (small, proline-rich) multigene family by amino acid sequence and mass spectrometry analyses. One of the pancornulins (14.9 kDa) is identical to the protein predicted by spr-1 clone 128. The other pancornulins (16.9 kDa and 22 kDa) are novel members of the spr family. Immunoelectron microscopy of purified cornified envelopes with a pancornulin-specific antibody established these proteins more definitively as cornified envelope precursors. In addition, two-dimensional electrophoretic analyses of keratinocyte extracts labeled enzymatically with Dansylcadaverine (to identify amine acceptors) or dansylPGGQQIV (to Identify amine donors) showed that both glutamine and lysine residues within the pancornulins participate in the isopeptide linkage characteristic of cornified envelope formation. These results contrasted with those obtained using involucrin, a prominent cornified envelope protein shown capable of acting only as an amine acceptor in this systems Novel partial cDNAs obtained after reverse transcription and polymerase chain reaction amplification of total messenger RNA with pancornulin-specific primers suggest that the spr multigene family may be even larger than previously described, The bifunctional reactivity of the pancornulins in cross-linking and the large number of family members identified to date suggest that the pancornulins and other spr-1-related proteins may be more important in cornified envelope formation than previously considered, perhaps functioning as "bridge" molecules during the early phases of cornified envelope assembly.
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amide bond cleavage monitored continuously through detection of a Dansylcadaverine leaving group
Biochemical and Biophysical Research Communications, 1992Co-Authors: Laszlo Lorand, K N Parameswaran, Pauline T Velasco, S N P Murthy, James H. WilsonAbstract:The transglutaminase-catalyzed incorporation of the fluorescent amine, Dansylcadaverine, into casein derivatives, such as N,N-dimethylcasein, is accompanied by a large increase in intensity of emission (Lorand et al., Anal. Biochem. 44, 221-231, 1971). We have sought to make use of this sensitive detection device for the continuous, on-line monitoring of an amide-splitting reaction in which Dansylcadaverine served as the leaving group. The transglutaminase-coupled test system comprised gamma-glutamylDansylcadaverine as the first substrate and gamma-glutamylamine cyclotransferase as the enzyme responsible for releasing Dansylcadaverine from the gamma-amide. At close to saturating levels of transglutaminase, the measured rate of increase of fluorescence, i.e. the steady-state rate of Dansylcadaverine incorporation into N,N-dimethylcasein, showed a near-linear relationship with the concentration of gamma-glutamylamine cyclotransferase present in the assay mixture. The general approach developed may be applicable to the assay of other amide cleaving enzymes.
