Recombinant Polypeptides Derived from the Fibrin Binding Domain of Fibronectin Are Potential Agents for the Imaging of Blood Clots

1997 ◽  
Vol 77 (04) ◽  
pp. 796-803 ◽  
Author(s):  
N Ezov ◽  
A Nimrod ◽  
B Parizada ◽  
M M Werber ◽  
A Goldlust ◽  
...  
Keyword(s):  
Thrombus Formation ◽  
Recombinant Polypeptide ◽  
Covalent Linkage ◽  
Steel Coil ◽  
Amino Terminal ◽  
Imaging Tool ◽  
Recombinant Polypeptides ◽  
Amino Terminal Domain

SummaryThrombus formation in the circulation is accompanied by covalent linkage of fibronectin (FN) through transglutamination of glutamine no. 3 in the fibrin binding amino terminal domain (FBD) of FN. We have exploited this phenomenon for thrombus detection by the employment of radioactively-labelled recombinant polypeptide molecules derived from the 5-finger FBD of human FN. Three recombinant FBD polypeptides, 12 kDa (“2 fingers”), 18.5 kDa (“3 fingers”) and 31 kDa FBD (“5 fingers”), were prepared and compared to native FN-derived 31 kDa-FBD with respect to their ability to attach to fibrin clots in vitro and in vivo. The accessibility of Gln-3 in these molecules was demonstrated by the incorporation of stoichiometric amounts of 14C- putrescine in the presence of plasma transglutaminase. Competitive binding experiments to fibrin have indicated that, although the binding affinities of the FBD molecules are lower than that of FN, substantial covalent linkage was obtained in the presence of transglutaminase, and even in the presence of excess FN or heparin. The biological clearance rates of radioactively labelled FBD molecules in rats and rabbits were much higher than those of FN and fibrinogen, thus indicating their potential advantage for use as a diagnostic imaging tool. Of the three molecules, the 12 kDa FBD exhibited the highest rate of clearance. The potential of the 12 kDa and 31 kDa FBDs as imaging agents was examined in a stainless steel coil-induced thrombus model in rats and in a jugular vein thrombus model in rabbits, using either [125I] or [111ln]-labelled materials. At 24 h, clot-to-blood ratios ranged between 10 and 22 for [125I]-12 kDa FBD and 40 and 60 for [luIn]-12 kDa FBD. In the rat model, heparin did not inhibit the uptake of FBD. Taken together, the results indicate that recombinant 12 kDa FBD is a good candidate for the diagnosis of venous thrombosis.

scholarly journals The amino-terminal domain of pyrrolysyl-tRNA synthetase is dispensable in vitro but required for in vivo activity

FEBS Letters ◽  
2007 ◽  
Vol 581 (17) ◽  
pp. 3197-3203 ◽  
Author(s):  
Stephanie Herring ◽  
Alexandre Ambrogelly ◽  
Sarath Gundllapalli ◽  
Patrick O'Donoghue ◽  
Carla R. Polycarpo ◽  
...  
Keyword(s):  
Trna Synthetase ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain

scholarly journals Formation of a Tap/NXF1 Homotypic Complex Is Mediated through the Amino-Terminal Domain of Tap and Enhances Interaction with Nucleoporins

2008 ◽  
Vol 19 (1) ◽  
pp. 327-338 ◽  
Author(s):  
Leah H. Matzat ◽  
Stephen Berberoglu ◽  
Lyne Lévesque
Keyword(s):  
Nuclear Export ◽  
Rna Binding ◽  
Direct Interaction ◽  
Nuclear Pore ◽  
Amino Terminal ◽  
Multimeric Complex ◽  
Rna Export ◽  
Amino Terminal Domain

Nuclear export of mRNAs is mediated by the Tap/Nxt1 pathway. Tap moves its RNA cargo through the nuclear pore complex by direct interaction with nucleoporin phenylalanine-glycine repeats. This interaction is strengthened by the formation of a Tap/Nxt1 heterodimer. We now present evidence that Tap can form a multimeric complex with itself and with other members of the NXF family. We also show that the homotypic Tap complex can interact with both Nxt1 and nucleoporins in vitro. The region mediating this oligomerization is localized to the first 187 amino acids of Tap, which overlaps with its RNA-binding domain. Removal of this domain greatly reduces the ability of Tap to bind nucleoporins in vitro and in vivo. This is the first report showing that the Tap amino terminus modulates the interaction of Tap with nucleoporins. We speculate that this mechanism has a regulatory role for RNA export independent of RNA binding.

scholarly journals Residues near the Amino Terminus of Rns Are Essential for Positive Autoregulation and DNA Binding

2008 ◽  
Vol 190 (7) ◽  
pp. 2279-2285 ◽  
Author(s):  
Georgeta N. Basturea ◽  
Maria D. Bodero ◽  
Mario E. Moreno ◽  
George P. Munson
Keyword(s):  
Dna Binding ◽  
Amino Terminus ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Carboxy Terminal Domain ◽  
Carboxy Terminal ◽  
Amino Terminal Domain ◽  
Regulated Promoters

ABSTRACT Most members of the AraC/XylS family contain a conserved carboxy-terminal DNA binding domain and a less conserved amino-terminal domain involved in binding small-molecule effectors and dimerization. However, there is no evidence that Rns, a regulator of enterotoxigenic Escherichia coli virulence genes, responds to an effector ligand, and in this study we found that the amino-terminal domain of Rns does not form homodimers in vivo. Exposure of Rns to the chemical cross-linker glutaraldehyde revealed that the full-length protein is also a monomer in vitro. Nevertheless, deletion analysis of Rns demonstrated that the first 60 amino acids of the protein are essential for the activation and repression of Rns-regulated promoters in vivo. Amino-terminal truncation of Rns abolished DNA binding in vitro, and two randomly generated mutations, I14T and N16D, that independently abolished Rns autoregulation were isolated. Further analysis of these mutations revealed that they have disparate effects at other Rns-regulated promoters and suggest that they may be involved in an interaction with the carboxy-terminal domain of Rns. Thus, evolution may have preserved the amino terminus of Rns because it is essential for the regulator's activity even though it apparently lacks the two functions, dimerization and ligand binding, usually associated with the amino-terminal domains of AraC/XylS family members.

scholarly journals BBK07, a Dominant In Vivo Antigen of Borrelia burgdorferi, Is a Potential Marker for Serodiagnosis of Lyme Disease

2009 ◽  
Vol 16 (11) ◽  
pp. 1569-1575 ◽  
Author(s):  
Adam S. Coleman ◽  
Utpal Pal
Keyword(s):  
Lyme Disease ◽  
Borrelia Burgdorferi ◽  
Amino Acid Identity ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Potential Marker ◽  
Mammalian Infection ◽  
Amino Terminal Domain

ABSTRACT One of the recently identified Borrelia burgdorferi immunogens, BBK07, is characterized for its expression in the spirochete infection cycle and evaluated for its potential use as a serodiagnostic marker for Lyme disease. We show that the BBK07 gene is expressed at extremely low levels in vitro and in ticks but is dramatically induced by spirochetes once introduced into the host and is highly expressed throughout mammalian infection. In contrast, the expression of BBK12, a paralog of BBK07 with 87% amino acid identity, although expressed in vitro, remained undetectable in vivo throughout murine infection and in ticks. BBK07 is localized in the outer membrane, and the amino-terminal domain of the antigen is exposed on the microbial surface. A truncated BBK07 protein representing the amino-terminal domain is able to effectively detect antibodies to B. burgdorferi, both in experimentally infected mice and in humans. Further characterization of the immunodominant antigens of B. burgdorferi, such as BBK07, could contribute to the development of novel serodiagnostic markers for detection of Lyme disease.

scholarly journals ADA3, a putative transcriptional adaptor, consists of two separable domains and interacts with ADA2 and GCN5 in a trimeric complex.

1995 ◽  
Vol 15 (3) ◽  
pp. 1203-1209 ◽  
Author(s):  
J Horiuchi ◽  
N Silverman ◽  
G A Marcus ◽  
L Guarente
Keyword(s):  
Activation Domains ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Trimeric Complex ◽  
Nonoverlapping Domains ◽  
Carboxyl Terminal Domain ◽  
Carboxyl Terminal ◽  
Amino Terminal Domain

Mutations in yeast ADA2, ADA3, and GCN5 weaken the activation potential of a subset of acidic activation domains. In this report, we show that their gene products form a heterotrimeric complex in vitro, with ADA2 as the linchpin holding ADA3 and GCN5 together. Further, activation by LexA-ADA3 fusions in vivo are regulated by the levels of ADA2. Combined with a prior observation that LexA-ADA2 fusions are regulated by the levels of ADA3 (N. Silverman, J. Agapite, and L. Guarente, Proc. Natl. Acad. Sci. USA 91:11665-11668, 1994), this finding suggests that these proteins also form a complex in cells. ADA3 can be separated into two nonoverlapping domains, an amino-terminal domain and a carboxyl-terminal domain, which do not separately complement the slow-growth phenotype or transcriptional defect of a delta ada3 strain but together supply full complementation. The carboxyl-terminal domain of ADA3 alone suffices for heterotrimeric complex formation in vitro and activation of LexA-ADA2 in vivo. We present a model depicting the ADA complex as a coactivator in which the ADA3 amino-terminal domain mediates an interaction between activation domains and the ADA complex.

scholarly journals Dual Cyclin-Binding Domains Are Required for p107 To Function as a Kinase Inhibitor

1998 ◽  
Vol 18 (9) ◽  
pp. 5380-5391 ◽  
Author(s):  
Enrique Castaño ◽  
Yelena Kleyner ◽  
Brian David Dynlacht
Keyword(s):  
Mammalian Cells ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Growth Suppression ◽  
Kinase Substrate ◽  
Amino Terminal ◽  
Terminal Domain ◽  
Amino Terminal Domain

ABSTRACT The retinoblastoma (pRB) family of proteins includes three proteins known to suppress growth of mammalian cells. Previously we had found that growth suppression by two of these proteins, p107 and p130, could result from the inhibition of associated cyclin-dependent kinases (cdks). One important unresolved issue, however, is the mechanism through which inhibition occurs. Here we present in vivo and in vitro evidence to suggest that p107 is a bona fide inhibitor of both cyclin A-cdk2 and cyclin E-cdk2 that exhibits an inhibitory constant (Ki ) comparable to that of the cdk inhibitor p21/WAF1. In contrast, pRB is unable to inhibit cdks. Further reminiscent of p21, a second cyclin-binding site was mapped to the amino-terminal portions of p107 and p130. This amino-terminal domain is capable of inhibiting cyclin-cdk2 complexes, although it is not a potent substrate for these kinases. In contrast, a carboxy-terminal fragment of p107 that contains the previously identified cyclin-binding domain serves as an excellent kinase substrate although it is unable to inhibit either kinase. Clustered point mutations suggest that the amino-terminal domain is functionally important for cyclin binding and growth suppression. Moreover, peptides spanning the cyclin-binding region are capable of interfering with p107 binding to cyclin-cdk2 complexes and kinase inhibition. Our ability to distinguish between p107 and p130 as inhibitors rather than simple substrates suggests that these proteins may represent true inhibitors of cdks.

A Comparison of the in Vitro and in Vivo Thrombogenic Activity of Factor IX Concentrates Using Stasis (Wessler) and Non-Stasis Rabbit Models

1980 ◽  
Vol 44 (02) ◽  
pp. 081-086 ◽  
Author(s):  
C V Prowse ◽  
A E Williams
Keyword(s):  
Platelet Rich Plasma ◽  
Thrombus Formation ◽  
Factor Ix ◽  
Coagulation System ◽  
In Vitro Tests ◽  
Clinical Use ◽  
Low Activity

SummaryThe thrombogenic effects of selected factor IX concentrates were evaluated in two rabbit models; the Wessler stasis model and a novel non-stasis model. Concentrates active in either the NAPTT or TGt50 in vitro tests of potential thrombogenicity, or both, caused thrombus formation in the Wessler technique and activation of the coagulation system in the non-stasis model. A concentrate with low activity in both in vitro tests did not have thrombogenic effects in vivo, at the chosen dose. Results in the non-stasis model suggested that the thrombogenic effects of factor IX concentrates may occur by at least two mechanisms. A concentrate prepared from platelet-rich plasma and a pyrogenic concentrate were also tested and found to have no thrombogenic effect in vivo.These studies justify the use of the NAPTT and TGt50 in vitro tests for the screening of factor IX concentrates prior to clinical use.

Neutralization of a Low Molecular Weight Heparin (LHN-1) and Conventional Heparin by Protamine Sulfate in Rats

1986 ◽  
Vol 56 (03) ◽  
pp. 318-322 ◽  
Author(s):  
V Diness ◽  
P B Østergaard
Keyword(s):  
Molecular Weight ◽  
Low Molecular Weight Heparin ◽  
Thrombus Formation ◽  
Experimental Models ◽  
Protamine Sulfate ◽  
Low Molecular Weight ◽  
Antithrombotic Effect ◽  
Higher Doses

SummaryThe neutralization of a low molecular weight heparin (LHN-1) and conventional heparin (CH) by protamine sulfate has been studied in vitro and in vivo. In vitro, the APTT activity of CH was completely neutralized in parallel with the anti-Xa activity. The APTT activity of LHN-1 was almost completely neutralized in a way similar to the APTT activity of CH, whereas the anti-Xa activity of LHN-1 was only partially neutralized.In vivo, CH 3 mg/kg and LHN-1 7.2 mg/kg was given intravenously in rats. The APTT and anti-Xa activities, after neutralization by protamine sulfate in vivo, were similar to the results in vitro. In CH treated rats no haemorrhagic effect in the rat tail bleeding test and no antithrombotic effect in the rat stasis model was found at a protamine sulfate to heparin ratio of about 1, which neutralized APTT and anti-Xa activities. In LHN-1 treated rats the haemorrhagic effect was neutralized when APTT was close to normal whereas higher doses of protamine sulfate were required for neutralization of the antithrombotic effect. This probably reflects the fact that in most experimental models higher doses of heparin are needed to induce bleeding than to prevent thrombus formation. Our results demonstrate that even if complete neutralization of APTT and anti-Xa activities were not seen in LHN-1 treated rats, the in vivo effects of LHN-1 could be neutralized as efficiently as those of conventional heparin. The large fall in blood pressure caused by high doses of protamine sulfate alone was prevented by the prior injection of LHN-1.

Surface Modification by Nanobiomaterials for Vascular Tissue Engineering Applications

2020 ◽  
Vol 27 (10) ◽  
pp. 1634-1646 ◽  
Author(s):  
Huey-Shan Hung ◽  
Shan-hui Hsu
Keyword(s):  
Tissue Engineering ◽  
Vascular Tissue ◽  
Thrombus Formation ◽  
Vascular Grafts ◽  
Vascular Tissue Engineering ◽  
Great Success ◽  
Natural Polymers ◽  
Vascular Biomaterials

Treatment of cardiovascular disease has achieved great success using artificial implants, particularly synthetic-polymer made grafts. However, thrombus formation and restenosis are the current clinical problems need to be conquered. New biomaterials, modifying the surface of synthetic vascular grafts, have been created to improve long-term patency for the better hemocompatibility. The vascular biomaterials can be fabricated from synthetic or natural polymers for vascular tissue engineering. Stem cells can be seeded by different techniques into tissue-engineered vascular grafts in vitro and implanted in vivo to repair the vascular tissues. To overcome the thrombogenesis and promote the endothelialization effect, vascular biomaterials employing nanotopography are more bio-mimic to the native tissue made and have been engineered by various approaches such as prepared as a simple surface coating on the vascular biomaterials. It has now become an important and interesting field to find novel approaches to better endothelization of vascular biomaterials. In this article, we focus to review the techniques with better potential improving endothelization and summarize for vascular biomaterial application. This review article will enable the development of biomaterials with a high degree of originality, innovative research on novel techniques for surface fabrication for vascular biomaterials application.

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