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Biomedical subjects

E Haber

Publications and source records attributed to E Haber.

At least 127 records · Page 7Linked to original sources
PubMed1987

Thrombolysis by a fibrin-specific antibody Fab'-urokinase conjugate.

The unidirectional crosslinking agent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) was used to covalently couple fibrin-specific antibody Fab' to low-molecular-weight urokinase by means of the sulfhydryl groups of the inter-heavy-chain disulfide bonds. At enzymatic activities equal with respect to the substrate pyroglutamyl-glycyl-arginyl-p-nitroanilide-hydrochloride, the Fab'-urokinase conjugate lysed fibrin monomer linked to sepharose with a potency 95 times that of urokinase (which is essentially identical to the potency previously reported for an intact antibody-urokinase conjugate). The Fab'-urokinase conjugate also lysed human thrombi in fresh citrated plasma with a maximal potency 4.4 times that of uncoupled urokinase. These findings indicate that univalent antibody binding is sufficient to enhance fibrinolysis and that the increased potency of antifibrin-urokinase conjugates can also be demonstrated when cross-linked thrombi are used as substrate in the presence of plasma.

Antibodies↗
PubMed1987

Analysis of structure-activity relationships in renin substrate analogue inhibitory peptides.

On the basis of the minimal octapeptide sequence of the renin substrate, a series of peptides was synthesized containing (3S,4S)-4-amino-3-hydroxy-6-methylheptanoic acid (statine) or (3S,4S)-4-amino-3-hydroxy-5-phenylpentanoic acid (AHPPA) at the P1P1' position. Some of these peptides also contained Nin-formyltryptophan at the P5, P3, or P3' position. Renin-inhibitory potency varied over a wide range (from inactive to IC50 = 3 nM). Potency was reduced by at least 10-fold when the peptide was shortened by two residues at either the amino or carboxy terminus. The AHPPA-containing inhibitors were several-fold less potent than the statine-containing inhibitors. Analysis of models for the three-dimensional structure of inhibitors at the active site of human renin suggests that the diminished potency of the AHPPA peptides in comparison with the statine-containing peptides was caused by a shift in the peptide backbone due to a steric conflict between the phenyl ring of the AHPPA residue and the S1 subsite. The importance of the side chain and the 3(S)-hydroxyl group of the statine residue was demonstrated by substituting 5-aminovaleric acid for a dipeptide unit at the P1P1' position, which resulted in a peptide devoid of renin-inhibitory activity. Substitutions of other basic amino acids for histidine at the P2 position caused a great loss in potency, possibly due to disruption of a hydrogen bond as suggested by molecular modeling. Studies on the plasma renins of four nonhuman species suggest that the isoleucine-histidine segment at the P2'P3' position is important to defining the human specificity of the substrate. This work suggests a number of properties important to the design of potent renin inhibitors, and demonstrates the usefulness of three-dimensional models in the interpretation of structure-activity data.

Amino Acids↗
PubMed1987

Construction and expression of a recombinant antibody-targeted plasminogen activator.

Covalent linkage of tissue-type plasminogen activator (t-PA) to a monoclonal antibody specific for the fibrin beta chain (anti-fibrin 59D8) results in a thrombolytic agent that is more specific and more potent than t-PA alone. To provide a ready source of this hybrid molecule and to allow tailoring of the active moieties for optimal activity, we have engineered a recombinant version of the 59D8-t-PA conjugate. The rearranged 59D8 heavy chain gene was cloned and combined in the expression vector pSV2gpt with sequence coding for a portion of the gamma 2b constant region and the catalytic beta chain of t-PA. This construct was transfected into heavy chain loss variant cells derived from the 59D8 hybridoma. Recombinant protein was purified by affinity chromatography and analyzed with electrophoretic transfer blots. These revealed a 65-kDa heavy chain-t-PA fusion protein that is secreted in association with the 59D8 light chain in the form of a 170-kDa disulfide-linked dimer. Chromogenic substrate assays showed the fusion protein to have 70% of the peptidolytic activity of native t-PA and to activate plasminogen as efficiently as t-PA. In a competitive binding assay, reconstituted antibody was shown to have a binding profile similar to that of native 59D8. Thus, by recombinant techniques, we have produced a hybrid protein capable of high-affinity fibrin binding and plasminogen activation.

Antibodies, Monoclonal↗
PubMed1987

Antibody-enhanced thrombolysis: targeting of tissue plasminogen activator in vivo.

Tissue plasminogen activator (tPA) was modified by the unidirectional crosslinking reagent N-succinimidyl 3-(2-pyridyldithio)propionate and coupled to iminothiolane-modified anti-fibrin antibody 59D8 by the formation of disulfide bonds at neutral pH. Purification by two affinity-chromatography steps yielded tPA-anti-fibrin antibody conjugate (tPA-59D8) possessing both tPA and anti-fibrin antibody activities. In a quantitative rabbit thrombolysis model, the activity of the purified conjugate was compared with that of tPA alone and that of a conjugate between tPA and a digoxin-specific monoclonal antibody. After correction for spontaneous lysis (10.9 +/- 2.5%), tPA-59D8 was shown to be 2.8-9.6 times more potent than tPA alone. Unconjugated tPA and tPA-digoxin were equipotent. At equivalent thrombolytic concentrations, tPA-59D8 degraded less fibrinogen and consumed less alpha 2-antiplasmin than did tPA alone. This suggests that tPA can be efficiently directed to the site of a thrombus by conjugation to an anti-fibrin monoclonal antibody, resulting in both more potent and more selective thrombolysis.

Animals↗
PubMed1987

Monoclonal antibody modification with chelate-linked high-molecular-weight polymers: major increases in polyvalent cation binding without loss of antigen binding.

Polyethyleneimine or polylysines of differing molecular sizes were substituted with either EDTA or DTPA and then with succinic acid groups. These polymers were then reacted with the amino groups on myosin-specific monoclonal antibody or its Fab using a water soluble carbodiimide. The polymer-antibody complexes were capable of binding up to 150 di- or trivalent ions per mole (Mn++, Gd , or 111In ) without attendant loss of antigen binding. The polylysine derivatives of the intact antibody were rapidly cleared and sequestered in the liver, whereas the polylysine 14-kilodalton (kd) derivative of Fab was cleared from the circulation with minimal hepatic and kidney sequestration. This differed from the biodistribution of intact antimyosin or its Fab labeled with 111In via direct attachment of DTPA to the epsilon amino group of the lysyl residues. Applications in magnetic resonance and nuclear imaging are envisioned.

Antibodies, Monoclonal↗
PubMed1987

Models for the three-dimensional structure of renin inhibitors bound in the active site of human renin: an analysis of the properties that produce tight binding.

Three-dimensional models of the octapeptide segment of renin substrate and of several inhibitors characterized by a modified scissile bond were constructed at the active site of human renin. The substrate and inhibitor models were based on the structure of pepstatin solved by x-ray crystallography to a resolution of 1.8 A. The renin structure, previously reported by us, was based on the structures of homologous aspartyl proteases solved by x-ray diffraction techniques to a resolution of 2.1 A or higher. An energy minimization program, CHARMM, was used to refine these structures with respect to the optimal interaction of the enzyme and its inhibitor, and to calculate the energy of each inhibitor-enzyme interaction. The results indicate that the most significant difference between the binding of inhibitors and of the natural substrate is caused by a reduction in electrostatic rather than conformational strain.

Binding Sites↗
PubMed1987

Indium 111-monoclonal antimyosin antibody imaging in the diagnosis of acute myocarditis.

A definitive diagnosis of myocarditis requires right ventricular biopsy. Despite its specificity, however, right ventricular biopsy may lack sensitivity due to the focal nature of the disease. Because indium 111-monoclonal antimyosin antibody imaging can be used to detect myocardial necrosis, this procedure was performed on 28 patients clinically suspected of having myocarditis, 25 of whom had left ventricular ejection fractions of less than 45%, and the results were compared with those of right ventricular biopsy performed within 48 hr of the scan. Antimyosin scans were positive in nine patients who had evidence of myocarditis on right ventricular biopsy, and negative in 11 who had no evidence of myocarditis by biopsy. The remaining eight had positive antimyosin scans but showed no evidence of myocarditis on right ventricular biopsy. On the basis of a right ventricular biopsy standard, the sensitivity of this method was 100%, the specificity 58%. We conclude that antimyosin antibody imaging is a reliable screening method for the evaluation of patients suspected of having myocarditis, and that a positive antimyosin scan indicates the need for right ventricular biopsy to establish the histologic diagnosis.

Acute Disease↗
PubMed1987

The search for a hypothalamic Na+,K+-ATPase inhibitor.

Accumulating experimental evidence suggests that natriuresis in response to intravascular volume expansion is promoted by an endogenous regulator of Na+,K+-adenosine triphosphatase (ATPase). Efforts to purify this substance by a number of laboratories have as yet been unsuccessful. The properties of partially purified inhibitors from plasma, urine, and tissue often fail to possess the characteristics thought to be consistent with those of a physiological regulator. These include potency (Ki of approximately 1 nM), reversibility of inhibition, specificity for Na+,K+-ATPase, and responsiveness to relevant physiological stimuli. Two rather different candidate substances, extracted from urine and hypothalamus, have been purified to a high degree. Neither is a peptide, and both are of low molecular weight and resistant to acid hydrolysis. The substance from urine is rather nonpolar and interacts with digoxin-specific antibodies, while that from hypothalamus is polar and does not appear to share epitopes with the cardiac glycosides. On the serosal surface of the toad urinary bladder, the hypothalamic substance causes a reversible inhibition of Na+ transport, inhibits rubidium uptake in red blood cells by acting on the membrane's exterior surface, inhibits binding of ouabain to purified Na+,K+-ATPase, and reversibly inhibits hydrolysis of adenosine 5'-triphosphate by the enzyme with a Ki of 1.4 nM. The hypothalamic inhibitor may be differentiated from ouabain by their respective ionic requirements for optimal inhibition of enzymatic activity, and although both ouabain and the hypothalamic inhibitor fix Na+,K+-ATPase in its E2 conformation, the hypothalamic inhibitor does not promote phosphorylation of the enzyme by inorganic phosphate in the presence of Mg2+. Ionic requirements for inhibition also differentiate the hypothalamic inhibitor from vanadate ion, as does the inhibitor's activity in the presence of norepinephrine. Further enzymological and physiological studies will be facilitated by structural characterizations of the inhibitory substances and by the availability of a method to measure their concentrations in physiological fluids.

Animals↗
PubMed1987

Detection of early necrosis in a canine model of low flow myocardial ischemia using 125I-antimyosin (Fab')2.

Low flow myocardial ischemia in the distribution of the left anterior descending coronary artery was established in 32 anesthetized dogs. The early development of irreversible myocardial injury was identified using 125I-antimyosin (Fab')2--a specific immunological marker of myocardial necrosis. The simultaneous injection of 131I-nonspecific (Fab')2 controlled for variables unrelated to specific antimyosin activity. It was also possible to calculate the relative uptake of 125I-antimyosin (Fab')2 in the ischemic area. In order to prevent recirculation of the (Fab')2, and to limit the time of exposure to the myocardium, the fragments were injected directly into the coronary circulation during diversion of the coronary sinus effluent. The antibody fragments were given either two, three or five hours after the onset of low flow in different groups of dogs and the animals were sacrificed 60 minutes later. Selective accumulation of 125I-antimyosin (Fab')2 in the ischemic area occurred between two and three hours after the onset of low flow ischemia indicating that necrosis was already established at this time. Macroautoradiographs of cross sections of the left ventricle and autoradiographs of microscopic sections of the ischemic myocardium independently confirmed selective accumulation of 125I-antimyosin (Fab')2 and hence necrosis, at three hours after the onset of low flow. In this study the use of 125I-antimyosin (Fab')2, in contrast to the use of histology, as a marker of necrosis avoided error due to sampling and also permitted the detection of cell death early during the ischemic process. The demonstration, within a narrow time frame, of early necrosis in this low flow preparation provides a potential model with which to evaluate agents for myocardial preservation in obstructive coronary disease.

Animals↗
PubMed1987

Production and characterization of human renin antibodies prepared with synthetic immunogens.

Renin is an aspartyl protease that plays a key role in regulating blood pressure. The aim of this study was to use synthetic peptides to produce antibodies able to recognize the entire human renin molecule and to inhibit its enzymatic activity. Two three-dimensional renin models were used to predict 17 putative epitopes. Then 18 peptides related to 13 potential antigenic determinants were synthesized by solid- or liquid-phase technique, and 11 were shown to be antigenic when tested by their binding to several polyclonal human renin antibodies. The peptides were injected into rabbits, and antisera tested by radio-immunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA) showed that nine peptides were immunogenic. The peptide related to the flap region which lies across the renin catalytic cleft showed potentially useful characteristics. Immunoglobulin G, purified from antibodies raised against this epitope, bound labelled renin and inhibited its enzymatic activity in a dose-dependent manner. This approach constitutes the basis for the development of a synthetic antirenin vaccine able to inhibit the renin-angiotensin system specifically.

Amino Acid Sequence↗
PubMed1987

Renin inhibitors: a search for principles of design.

Principles for the design of renin inhibitors were examined by utilizing a three-dimensional model of the enzyme with inhibitory peptides in its catalytic site. Optimal conformations were subjected to energy minimization by the computer program CHARMM. The results were then compared with the inhibitory potencies of these peptides as assessed by an in vitro renin assay. The results of the modeling study and enzymatic assay were generally congruent, showing that residues at the P5, P4 and P3 positions contributed importantly to binding, that an acetyl group can replace Pro-His at the P5, P6 position, that the lesser potency of AHPPA-containing as compared with statine-containing peptides may be related to the better fit into the S1 subsite of an isobutyl as opposed to a benzyl side chain, that histidine in the P2 position forms an essential hydrogen bond with residue 230 (which cannot be formed by another basic residue), and that there is a general correlation between inhibitory potency and calculated energy of interaction as deduced from the model. Major determinants of energy of binding are conformational strain and electrostatic interaction.

Amino Acid Sequence↗
PubMed1987

Myocardial damage delineated by indium-111 antimyosin Fab and technetium-99m pyrophosphate.

Technetium-99m-labeled pyrophosphate and radiolabeled antimyosin antibodies are two infarct localizing agents with apparently different kinetics of localization. To determine whether these agents localize in a similar fashion in early acute myocardial necrosis, we studied the simultaneous distribution of 111In-labeled antimyosin and 99mTc-labeled pyrophosphate in dogs after intracoronary (i.c.) (n = 9) or intravenous (i.v.) (n = 9) administration of a mixture of these two agents in a reperfused infarct model. The mean infarct size (+/- s.d.) delineated by pyrophosphate [20.2 +/- 14.1 (i.v.), 29.8 +/- 12.3 (i.c.)] was larger than that by antimyosin [14.2 +/- 11.3 (i.v.) (p = 0.05), 20.0 +/- 11.8 (i.c.) (p = 0.05)] which was larger than that by triphenyl tetrazolium chloride [13.9 +/- 8.0 (i.v.) (p = 0.05), 15.3 +/- 6.5 (i.c.) (p = 0.05)]. This overestimation persisted whether the radiopharmaceuticals were administered by intracoronary or intravenous injections, although the latter with antimyosin was only slightly larger (TTC:AM = 13.9:14.2) (p = n.s.). There was a good correlation, however, between antimyosin and pyrophosphate delineated infarct sizes in dogs with intracoronary injection (y = 0.82x + 13.33, r = 0.79) or i.v. injection (y = 1.208x + 3.01, r = 0.97) of the mixture of the two agents. Since the images of the 111In and 99mTc activities were obtained consecutively by identical methods, the overestimation of infarct size by pyrophosphate cannot be due to differences in spatial resolution of the techniques used. The differences in the areas of myocardial damage delineated by pyrophosphate and antimyosin in our study most probably denote the area of viable but compromised myocardium.

Animals↗
PubMed1987

Acute myocardial infarct imaging with indium-111-labeled monoclonal antimyosin Fab.

Indium-111 monoclonal antimyosin Fab scintigraphy was used to detect myocardial necrosis in 52 of 54 patients (96.3%) with acute myocardial infarction. Infarcts were visualized when coronary arteries were persistently occluded (n = 10), became patent after thrombolysis (n = 33), or became patent after spontaneous reperfusion (n = 7). Posteroinferolateral visualizations were obtained in two patients with clinical and enzymatic evidence of infarction but normal electrocardiograms. Of the two patients in whom no infarcts were visualized, one had an anterior myocardial infarct. This patient underwent successful thrombolytic therapy, with attendant minimization of creatine kinase release. The other patient had a small, nonreperfused inferior myocardial infarct. Five patients with a history of remote infarction and acute necrosis showed antimyosin uptake only in regions concordant with the acute episodes of infarction, and radiolabeled antimyosin Fab localized in neither old infarcts nor normal, noninfarcted myocardium. Antimyosin Fab scintigraphy, thus, appears to be a highly specific means of delineating necrotic myocardium, at least in this limited and selected group of patients.

Antibodies, Monoclonal↗
PubMed1986

Static accessibility model of protein antigenicity: the case of scorpion neurotoxin.

Scorpion neurotoxins are a family of homologous, 64 to 65 residue containing proteins with four invariant disulfide bridges. Previous experimental work established four antigenic epitopes in the Androctonus australis neurotoxin and localized them in the amino acid sequence. Using crystallographic coordinates of the Centruroides sculpturatus neurotoxin and computing its large sphere (radius 1 nm or 10 A) accessibility profiles, we identified six antigenic sites clustered into four surface regions. Three of four computed sites coincided with the epitopes identified and localized experimentally in the A. australis neurotoxin, while two of the computed sites partially overlapped the fourth epitope. To investigate the relationship between antigenicity and segmental flexibility, 8-ps molecular dynamics simulations were performed on the C. sculpturatus structure, average backbone temperature factors computed from the simulation, and results compared with the X-ray-derived B values. Most of the neurotoxin structure and, in particular, three of the four antigenic sites were found inflexible, as judged by the computed and/or crystallographic temperature factors. The remaining epitope was associated with only marginal above-average maxima of backbone B values, corresponding to root mean square atomic displacements of 0.5 A (50 pm). We conclude that neurotoxin antigenicity is determined by an exceptional surface exposure of relatively short loop segments and that segmental flexibility is not an essential component of antigenicity.

Animals↗
PubMed1986

Location of antigenic epitopes on antibody molecules.

Using X-ray crystallographic co-ordinates of immunoglobulins, surface regions accessible to a large spherical probe, comparable in size to an antibody domain, were computed. Locations of these exposed regions were compared with those of experimentally determined antigenic sites, i.e. idiotypic, allotypic and isotypic serological markers. In all cases, an excellent agreement was found. The most prominent computed epitopes correspond to convex parts of antibody surface made by reverse turn segments of the polypeptide chain. The computed epitopes occur in homologous positions in all the immunoglobulin domains, and most of the beta-sheet surfaces on the domains are poorly antigenic. The CH2 domain (Fc fragment) has many more antigenic sites than the Fab fragments (antigen-binding fragments). Variable domain epitopes (idiotypes) involve both hypervariable and framework residues, and only about 25% of the hypervariable residues are strongly antigenic. The results indicate that, in a vertebrate body, each antibody molecule may be recognized, and its concentration regulated, by at least 40 complementary anti-immunoglobulin antibodies; therefore, a possibility of an "immune network" with much higher connectivity than is generally assumed should be seriously contemplated.

Animals↗
PubMed1986

Immunofluorescence studies of florid rheumatic Aschoff lesions.

We studied cardiac tissues of a patient who died of severe rheumatic myocarditis. Multiple Aschoff lesions were present throughout both ventricles and auricles. Immunofluorescence studies showed large monocytoid cells staining with OKM1 and anti-Leu M-3 as well as anti-Ia. Scattered T cells in areas of focal myocarditis stained with OKT3. Parallel staining for cardiac myosin-heavy chain antigens showed patchy dissolution of cardiac muscle fibers and traces of cardiac myosin within large monocytoid Aschoff cells.

Adult↗