Biomedical subjects

I Trakht

Publications and source records attributed to I Trakht.

8 recordsLinked to original sources

PubMed1998

Antipeptide monoclonal antibodies to defined fibrinogen Aalpha chain regions: anti-Aalpha 487-498, a structural probe for fibrinogenolysis.

The fibrinogen alphaC domain (Aalpha 220-610) is one of the earliest targets attacked by plasmin following fibrinolytic system activation. Monoclonal antibodies (MoAbs) to defined sequences within the alphaC domain provide the opportunity to explore the structure-function relationships involved in plasmin's interaction with its Aalpha chain substrate at greater resolution and can serve as reagents with potential clinical use for detecting fibrinogenolysis in vivo. The MoAb F-104 was raised against a multiple antigenic peptide derivative modelled after the hydrophilic 12-residue sequence corresponding to Aalpha 487-498 within the alphaC domain. A sensitive solution phase competitive enzyme-linked immunosorbent assay (ELISA) was developed for MoAb F-104 that can be applied for the direct measurement of intact fibrinogen (purified or plasma; ED50% approximately 5 pmol Aalpha chain equivalents/mL), with negligible cross-reactive interference from peptide cleavage products released by plasmin from the COOH-terminal end of the Aalpha chain (<3%). Immunoblotting and ELISA studies to characterize the fate of the F-104 epitope during fibrinogenolysis in vitro indicated a rapid loss of fibrinogen-associated immunoreactivity that reflected the heterogeneity of plasmin cleavage sites within the alphaC domain; cleavage at the 493-494 arg-his bond destroyed the F-104 epitope, while cleavage at other sites released it in an altered, inaccessible, conformation within the structure of 35- to 40-kD and 17.5- to 18-kD Aalpha chain degradation products. Application of the F-104 ELISA to monitor the course of Aalpha chain proteolysis in a small study population of patients undergoing thrombolytic therapy for myocardial infarction (n = 14) showed that the loss of fibrinogen-associated F-104 immunoreactivity was a very early marker (within 15 to 30 minutes) of in vivo fibrinogenolysis. Additional data obtained suggest that MoAb F-104 may have promise as a reagent for evaluating the creation of an effective lytic state early during therapy, information that could help determine the need for further clinical intervention. Thus, these studies illustrate a rational, targeted, approach towards the development of a novel antifibrinogen MoAb whose application as a structural probe for the region Aalpha 487-498 in vitro and in vivo can provide new insights into the various molecular forms of fibrinogen that circulate under physiologic conditions and in disease.

Amino Acid Sequence↗

PubMed1997

Filamentous phage displaying the extracellular domain of the hLH/CG receptor bind hCG specifically.

We have expressed the extracellular binding domain of the human LH/CG receptor as a fusion with the cpIII filamentous phage coat protein. These fusion phage are able to specifically bind ELISA plates coated with hCG. Preincubation of the phage with hCG before exposure to the coated plates inhibited binding of phage, whereas human FSH had no effect. Furthermore, addition of anti-hLH/CG receptor antisera inhibited binding of phage to the plates. We have also tested the effect of monoclonal antibodies to hCG on phage binding. Monoclonal antibodies that can bind hCG when it is bound to native receptor do not inhibit phage binding. These include B105 and A109 Alternatively, B107 and B109 have an inhibitory effect on phage binding. They cannot bind hCG when it is bound to receptor and are likely directed against epitopes at or near the receptor binding interface. Therefore, these fusion phage exhibit the same binding specificity as the wild-type receptor and likely display the extracellular domain of the hLH/CG receptor on their surface in the native conformation. Phage display is a potentially useful tool for studying the hLH/CG receptor structure and in screening for synthetic compounds that compete with hormone for receptor binding.

Amino Acid Sequence↗

PubMed1996

Three-dimensional structures of gonadotropins.

Most secreted proteins are modified post-translationally with the addition of carbohydrate. It has been difficult to use crystallography to solve the structures of these proteins due to the inherent heterogeneity of the carbohydrate. The structure of the chemically deglycosylated form (hydrogen fluoride treated) of human chorionic gonadotropin (hCG) has been solved through crystallographic techniques. Unfortunately this form of hCG is not biologically active, and exhibits immunochemical differences from native hormone. In addition, subunit interactions appear altered after chemical deglycosylation as indicated by the increased thermal stability of the HF-treated hormone. The Asn 52 glycan on the alpha-subunit of hCG has been identified as being required for biological activity, it is, therefore, of physiological importance to determine the structure of the hormone with its carbohydrate intact. Also, it has not been possible to obtain crystals of the individual glycosylated subunits of hCG. Therefore an alternative method to solve the structure of the biologically active form of the hormone in solution as well as its separated subunits is necessary. Structural information utilizing NMR techniques can be obtained from native hCG subunits in solution if they can be uniformly labeled with 13C and 15N isotopes. We have developed a universal nonradioactive isotope, labeling medium enriched in 13C and 15N which can be used to express uniformly labeled hCG from Chinese hamster ovary cells suitable for solving the structure of the individual subunits and ultimately that of the native, biologically active hormone. The isotopically labeled recombinant hCG and its purified subunits are essentially identical to urinary hCG on comparison by biochemical, immunochemical, biological activity and the ability of the isolated subunits to recombine to form a biologically active dimer. Mass spectrometric analysis and preliminary structural NMR data indicate that the labeling is uniform and there is greater than 90% incorporation, sufficient for complete structural determination studies. This labeled growth medium represents a technological advance which will enable the rapid solution of the structures of the other glycoprotein hormones, as well as other glycoproteins which have proven unsuitable for crystallographic study.

Animals↗

PubMed1996

Comparative structural and functional features of the human fibrinogen alpha C domain and the isolated alpha C fragment. Characterization using monoclonal antibodies to defined COOH-terminal A alpha chain regions.

The alpha C domain of fibrinogen (A alpha-(220-610)) plays a central role in maintaining hemostasis by serving as a substrate for factor XIIIa and plasmin. Monoclonal antibodies that recognize eight distinct epitopes within the COOH-terminal two-thirds of the A alpha chain were employed as structural probes to: 1) isolate the human alpha C domain, 2) compare the topography of the eight epitopes within the alpha C domain of intact fibrinogen and in purified alpha C fragments, and 3) explore the degree to which the alpha C domain's role as a factor XIIIa substrate in intact fibrinogen is preserved within the structure of isolated alpha C fragments. Five antibodies were raised against small, synthetic peptide immunogens (A alpha-(220-230), A alpha-(425-442), A alpha-(487-498), and A alpha-(603-610)), and three were generated against larger cyanogen bromide (A) alpha chain derivatives with each epitope subsequently localized to discrete A alpha chain sequences (A alpha-(259-276), A alpha-(529-539), and A alpha-(563-578)). Human alpha C preparations were isolated from mild plasmin digests of fibrinogen by successive chromatography on concanavalin A-Sepharose, anti-A alpha-(425-442)-Sepharose, and Superdex-75 fast protein liquid chromatography. Immunochemical characterization indicated that the NH2-terminal residue of alpha C fragments was either A alpha-220 or A alpha-231 and that, although the extreme COOH-terminal region, A alpha-(603-610), was absent, all molecules were intact at least through A alpha-(563-578). Solution phase competitive assays indicated that the release of the alpha C domain from intact fibrinogen was associated with several conformational changes, e.g. in the vicinity of A alpha-(220-230), A alpha-(259-276), A alpha-(487-498), and A alpha-(529-539), but that the relative accessibility of other localized structures remained unchanged, e.g. A alpha-(425-442) and A alpha-(563-578). Immunoblotting analysis of alpha C cross-linking in vitro revealed that isolated alpha C fragments could serve as a substrate for factor XIIIa. Immunoblotting studies of the A alpha chain proteolysis that occurs during thrombolytic therapy indicated that alpha C fragments, similar in size and epitope content to those isolated from purified fibrinogen, were released in vivo early during fibrinolytic system activation. The collective findings provide new information about the fine structure of the fibrinogen alpha C domain and its functional implications and also draw attention to the as yet unexplored role of alpha C fragments in the pathophysiology of thrombosis and hemostasis.

Amino Acid Sequence↗

PubMed1995

Alpha-Chain cross-linking in fibrin(ogen) Marburg.

The fibrinogen structural variant, Marburg (A alpha 1-460B beta gamma)2, is comprised of normal B beta and gamma chains but contains severely truncated A alpha chains that are missing approximately one half of their factor XIIIa cross-linking domain. Immunochemical studies of fibrin(ogen) Marburg were conducted to characterize the degree to which deletion of a defined A alpha-chain segment, A alpha 461-610, can affect the process of fibrin stabilization, ie, the factor XIIIa-mediated covalent interaction that occurs between alpha chains of neighboring fibrin molecules and between alpha chains and alpha 2 antiplasmin (alpha 2PI). The ability of Marburg (and control) alpha chains to serve as a substrate for factor XIIIa and undergo cross-linking was examined in an in vitro plasma clotting system. The capacity for alpha-chain cross-linking was evaluated both as the covalent incorporation of the small synthetic peptide, NQEQVSPLTLLK (which represents the first 12 amino acids of alpha 2PI and includes the factor XIIIa-sensitive glutamine residue responsible for the cross-linking of alpha 2PI to fibrin), and as the appearance of native (ie, natural), high-molecular-weight, cross-linked alpha-chain species. Antibodies specific for the (A)alpha and gamma/gamma-gamma chains of fibrin(ogen) and for the peptide and its parent protein, alpha 2PI (68 kD), were used as immunoblotting probes to visualize the various cross-linked products formed during in vitro clotting. Recalcification of Marburg plasma in the presence of increasing concentrations of peptide resulted in the formation of peptide-decorated Marburg alpha-chain monomers. Their size at the highest peptide concentration examined indicated the incorporation of a maximum of 3 to 4 mol of peptide per mole of alpha-chain. In the absence of alpha 2PI 1-12 peptide, the alpha chains of Marburg fibrin cross-linked to form oligomers and polymers, as well as heterodimers that included alpha 2PI. Both the peptide-decorated monomers and the native cross-linked alpha-chain species of Marburg fibrin were smaller than their control plasma counterparts, consistent with the truncated structure of the parent Marburg A alpha chain. Collectively, the findings indicate that, although deletion of the A alpha chain region no. 461-610 in fibrinogen Marburg prevents formation of an extensive alpha polymer network (presumably due to the absence of critical COOH-terminal lysine residues), it does not interfere with initial events in the fibrin stabilization process, namely, factor XIII binding and the ability of alpha chains to undergo limited cross-linking to one another and to alpha 2PI.

Amino Acid Sequence↗

PubMed1995

Monoclonal antibody-based immunoassay for evaluation of lipoprotein oxidation.

Numerous reports indicate that the oxidation of low-density lipoprotein (LDL) can significantly change its metabolic and physiological properties. Most methods for evaluation of LDL oxidation require isolation of lipoprotein, making the procedure laborious and increasing the probability of artifactual modification of LDL. In this paper we describe an immunochemical approach which can be used to measure the oxidation of isolated LDL and apoprotein B in unfractionated serum and to evaluate the effects of antioxidants on these processes. The procedure is based on differential recognition by monoclonal antibodies of native and oxidized lipoproteins. The results obtained with our assay indicate a strong correlation between the changes of apo B epitope expression during oxidation and the formation of conjugated dienes, changes in lipoprotein electrophoretic mobility, and interaction with fibroblast and macrophage receptors. The sensitivity of apo B to oxidation varies greatly among serum samples obtained from individual donors. These differences do not correlate with the differences in sensitivity to oxidation of LDL isolated from the blood samples of the same donors. It is also shown that apo B oxidation in serum can be progressively inhibited in the presence of increasing amounts of various antioxidants.

Animals↗

PubMed1990

Flow sorting of hybrid hybridomas using the DNA stain Hoechst 33342.

A two-step sorting procedure with the fluorescence-activated cell sorter (FACS) is described for the selection of hybrid hybridomas producing bispecific monoclonal antibodies. Parental hybridoma cells were first labelled before fusion with fluorescein isothiocyanate (FITC) and tetramethyl rhodamine isothiocyanate (TRITC); heterofluorescent cells were recovered after fusion. After a period of growth in culture, the cells were then stained with the DNA-specific dye bis-benzimidazole Hoechst 33342 and sorted on the basis of their DNA content. The staining conditions (10 micrograms/ml of Hoechst 33342, 90 min incubation time at 37 degrees C) were found to be optimal for obtaining a well resolved DNA histogram with minimal effect on the growth properties of cells from different mouse hybridoma lines. Employing this method we have isolated hybrid hybridomas synthesizing bispecific monoclonal antibodies reacting with human low density lipoprotein and alkaline phosphatase from calf intestine.

Animals↗

PubMed1989

Monoclonal antibodies to angiotensin-converting enzyme: a powerful tool for lung and vessel studies.

We obtained a series (12 clones) of hybridomas, which produce monoclonal antibodies (Mab) to 5 different epitopes of the angiotensin-converting enzyme (ACE) molecule. These antibodies may be used to (1) map antigenic structure of ACE, including the study of immunologic heterogeneity of ACE from different organs and tissues; (2) study the immunohistochemical distribution of ACE in human tissues, including the diagnosis of sarcoidosis; (3) develop an ACE immunoassay, and (4) prepare an immunosorbent for large-scale ACE isolation and for ACE-apheresis. One of the antibodies, 9B9, when injected into the circulation of rat and monkey, accumulated with high specificity in the lungs as compared with either normal mouse IgG or other organs and blood. The highly specific and nontoxic accumulation of Mab 9B9 suggests that it also may be used for gamma scintigraphy visualization of the pulmonary vascular bed, detection of lung injury and as a vector for targeted drug delivery to the lung.

Animals↗