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Biomedical subjects

G R Matsueda

Publications and source records attributed to G R Matsueda.

At least 55 records · Page 3Linked to original sources
PubMed1988

A sequence-dependent monoclonal antibody specific for single-chain urokinase.

To mimic the sequence spanning the primary site (the Lys158-Ile159 bond) cleaved by plasmin in its conversion of single-chain urokinase plasminogen activator (scuPA) to urokinase, we synthesized the peptide Cys(Acm)-Leu-Arg-Pro-Arg-Phe-Lys-Ile-Ile-Gly-Gly-Glu-Phe-Cys [Cys(Acm)scuPA(153-164)Cys]. Immunization of A/J mice with the Cys(Acm)scuPA(153-164)Cys peptide linked to hemocyanin, followed by somatic cell fusion with a myeloma cell line (SP2/0), yielded a monoclonal antibody (SCOOP1) that bound to single-chain urokinase but not to urokinase or plasmin-treated single-chain urokinase. SCOOP1 could discriminate between single-chain urokinase and urokinase by greater than three orders of magnitude. In a radioimmunoassay, Cys(Acm)scuPA(153-164)Cys completely inhibited SCOOP1 binding to single-chain urokinase, whereas an equimolar mixture of two heptapeptides comprising the amino terminal [Cys-scuPA(153-158)] and carboxy terminal [scuPA(159-164)Cys)] halves of the cleavage site peptide did not. Thus the epitope recognized by SCOOP1 includes the Lys158-Ile159 peptide bond.

Amino Acid Sequence↗
PubMed1988

Conjugation to an antifibrin monoclonal antibody enhances the fibrinolytic potency of tissue plasminogen activator in vitro.

Tissue plasminogen activator (tPA) was covalently linked by disulfide bonds to a monoclonal antibody specific for the amino terminus of the beta chain of fibrin (antibody 59D8). The activity of the tPA-59D8 conjugate was compared with that of tPA, urokinase (UK), and a UK-59D8 conjugate. For lysis of fibrin monomer, tPA was 10 times as potent as UK, whereas both UK-59D8 and tPA-59D8 conjugates were 100 times as potent as UK and 10 times as potent as tPA. Conjugation of tPA or UK to antibody 59D8 produced a 3.2-4.5-fold enhancement in clot lysis in human plasma over that of the respective unconjugated plasminogen activator. However, the UK-59D8 conjugate was only as potent as tPA alone. Antibody-conjugated tPA or UK consumed less fibrinogen, alpha 2-antiplasmin, and plasminogen than did the unconjugated activators, at equipotent fibrinolytic concentrations. Antibody targeting thus appears to increase the concentration of tPA in the vicinity of a fibrin deposit, which thereby leads to enhanced fibrinolysis.

Antibodies, Monoclonal↗
PubMed1988

T cell recognition of fibrinogen. A determinant on the A alpha-chain does not require processing.

Murine T cell hybridomas were used to examine the requirements for processing and presentation of human fibrinogen. In contrast to most protein Ag, fibrinogen (Mr 340,000) did not need to be processed by an APC, inasmuch as intact fibrinogen was presented by both pre-fixed and chloroquine-treated macrophages. Through the use of a variety of protease inhibitors, no involvement of proteases either on the macrophage surface or in the culture medium in the presentation of fibrinogen was observed. The epitope recognized by two T cell hybridomas was localized to the peptide, A alpha (551-578), which was located on the carboxy portion of the A alpha-chain. This portion of the A alpha-chain has no defined secondary structure and must possess the conformational flexibility which allows it to directly associate with an I-Ek molecule. Thus conformational flexibility may be a major factor in determining the processing requirements of a protein Ag.

Animals↗
PubMed1988

Cellular processing of pro-atrial natriuretic factor (pro-ANF): studies using an antiserum that selectively binds ANF-(99-126) after its cleavage from pro-ANF.

Atrial natriuretic factor (ANF) is stored in atrial myocytes as a 15-17K prohormone, but circulates in plasma as a 3K, carboxy (C)-terminal fragment of the prohormone. The tissue location at which the cleavage of pro-ANF to its hormonal form occurs is unknown. In the present study, an immunological approach was taken to address this question. A polyclonal antiserum was generated which recognizes the hormonal form of ANF [ANF-(99-126)] only after its cleavage from the prohormone. This was accomplished by immunizing rabbits with a synthetic peptide corresponding to the seven amino (N)-terminal residues of ANF-(99-126) coupled to carrier protein via a C-terminal cysteine. This antiserum, anti-ANF-(99-105), demonstrated high affinity for ANF-(99-126) (IC50 = 170 pM), but displayed 100-fold less affinity for recombinant pro-ANF [ANF-(2-126)]. The N-terminal specificity of anti-ANF-(99-105) was evident by its failure to bind ANF-(103-126) at concentrations up to 100 nM. The specificity of anti-ANF-(99-105) for the hormonal form of ANF was examined by using thrombin to cleave pro-ANF and testing for the generation of anti-ANF-(99-105) immunoreactivity. Cleavage of atrial pro-ANF or 35S biosynthetically-labeled pro-ANF resulted in the production of immunoreactive material from the prohormone, whereas pro-ANF itself demonstrated no cross-reactivity with anti-ANF-(99-105). Anti-ANF-(99-105) could also recognize ANF released from the isolated perfused rat heart. When anti-ANF-(99-105) was used in immunohistochemical studies of rat atrial myocardium, no staining was observed in unfixed frozen sections. This suggests that proteolytic processing of pro-ANF is not an intracardiocytic event.

Amino Acid Sequence↗
PubMed1987

Characterization of an antibody-urokinase conjugate. A plasminogen activator targeted to fibrin.

The plasminogen activator urokinase was linked covalently to a monoclonal antibody specific for the amino terminus of the beta chain of human fibrin by means of the unidirectional cross-linking reagent N-succinimidyl-3-(2-pyridyldithio)propionate. N-Succinimidyl-3-(2-pyridyldithio)propionate allowed the amino groups on urokinase to be coupled to the sulfhydryl groups on iminothiolane (which had been introduced into the antibody before the coupling reaction). The inter-heavy chain sulfhydryl of the Fab' of this antibody was also linked to N-succinimidyl-3-(2-pyridyldithio)propionate-substituted urokinase. The antibody- or Fab'-urokinase complexes were purified by two affinity chromatography steps. In the first, benzamidine was used as ligand for urokinase, in the second, a heptapeptide consisting of the 7 amino-terminal residues of the beta chain of fibrin (beta peptide) was used as ligand for the antibody. The activity of the purified conjugates was compared with that of urokinase alone in an assay measuring lysis of 125I-fibrin monomer covalently linked to Sepharose CL-4B. For any concentration of either urokinase alone or urokinase-antifibrin antibody conjugate, an equivalent amount of lysis (release of labeled peptide from fibrin monomer-Sepharose) was obtained with 1/250 the concentration (with respect to urokinase content) of antifibrin antibody-urokinase conjugate. The antifibrin Fab'-urokinase conjugate exhibited a similar enhancement of activity in comparison with urokinase. Enhanced fibrinolysis was fully inhibited by beta peptide. These results suggest that antibody targeting enhances the concentration of urokinase in the vicinity of immobilized fibrin monomer, thereby also increasing the local conversion of plasminogen to plasmin, which in turn degrades its substrate, fibrin. Univalent antigen-antibody binding is sufficient for optimal efficiency.

Antibodies, Monoclonal↗
PubMed1987

Evaluation of monoclonal antifibrin antibodies by their binding to human blood clots.

The primary goal of this study was to develop a method for evaluating fibrin-specific antibodies as thrombus detecting agents. The apparatus and assay conditions were chosen by testing antibody 64C5, which binds to the amino terminus of the fibrin beta chain, for its ability to bind to human blood clots. Using 125I-labeled antibody 64C5, the effects of antibody concentration, clot shape, clot mass, temperature, and flow rate were tested. Increased antibody binding was observed when antibody concentration, clot mass and temperature were increased. Under one set of conditions, six 125I-labeled monoclonal antifibrin antibodies (four specific for the beta chain, two specific for the alpha chain) were tested for their binding to retracted clots of human blood. Two radioiodinated antidigoxin antibodies were used as a control. Beta chain-specific antibody 59D8, which provided the highest level of binding to clot, bound 14-fold better than the control antidigoxin antibody. Neither alpha chain-specific antibody bound to clotted blood. To examine the in vivo relevance of the in vitro binding to clots, the uptake of antibody 64C5 was assessed for its binding to human fibrin clotted within the jugular vein of a rabbit. The correlation coefficient between in vitro and in vivo uptake as a function of clot weight was calculated to be 0.91. Thus, in vitro binding of monoclonal antifibrin antibodies to human blood clots was judged to be a realistic method for the comparison and selection of an ideal antifibrin antibody for detailed in vivo testing.

Animals↗
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

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↗
PubMed1986

The hemodynamic response of conscious normotensive rats to atriopeptin III: Lack of a role of the parasympathetic nervous system.

The influence of the parasympathetic nervous system on the cardiovascular response to a synthetic atrial peptide (atriopeptin III) was examined in conscious normotensive rats by utilizing radiolabelled microspheres. Atriopeptin III was infused intravenously for 30 min at a rate of 1 microgram/min per rat in animals pretreated with a bolus intravenous injection of atropine, (150 micrograms/rat, n = 8) or of its vehicle (n = 8). Additional animals (n = 9) received the vehicle of both atropine and atriopeptin III. The atrial peptide decreased mean blood pressure to a similar extent in rats pretreated with atropine (from 124 +/- 4.5 to 108 +/- 5.3 mm Hg, mean +/- S.E.M., P less than 0.05) and in the controls (from 123 +/- 3.8 to 105 +/- 3.8 mm Hg, P less than 0.05). Heart rate rose significantly after administration of atropine. After the 30 min infusion, the cardiac index was significantly lower (P less than 0.05) in both groups infused with atriopeptin III (23.7 +/- 2.3 after atropine pretreatment and 25.1 +/- 2.2 ml X min-1 X 100 g-1 without atropine) than in the group of rats given only the vehicle of both atropine and atriopeptin III (32.4 +/- 2.8 ml X min-1 X 100 g-1). Calculated systemic vascular resistance tended to be higher in the former two groups than in the latter. There was no significant difference in regional blood flow distribution within the three groups of rats. These data therefore indicate that in conscious rats atriopeptin III reduces blood pressure and cardiac output without concomitantly diminishing total peripheral vascular resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Preparation of peptide-protein immunogens using N-succinimidyl bromoacetate as a heterobifunctional crosslinking reagent.

Synthetic peptides derived from human fibrin were unidirectionally conjugated to three carrier proteins (bovine serum albumin, bovine alpha-lactalbumin, and keyhole limpet hemocyanin) by a method that employs N-succinimidyl bromoacetate. This heterobifunctional crosslinking reagent was prepared with a 79% yield in gram quantities from inexpensive starting materials. With this reagent, carrier proteins were first bromoacetylated, then reacted with the thiol groups of cysteine-containing peptides. The extent of peptide conjugation was assessed by amino acid analysis after acid hydrolysis, which liberated 1 mol of S-carboxymethylcysteine for each mole of thioether linkage between peptide and protein. The results of several conjugation experiments indicated that the efficiency of peptide incorporation ranged between 22 and 37% based on the recovery of S-carboxymethylcysteine relative to lysine. When the conjugates were used as immunogens, the S-carboxymethyl linkage was not antigenic in comparison with the S-maleimidobenzoyl linkage, even though their antipeptide immunoreactivities were similar.

Animals↗
PubMed1986

Structural basis for the species selectivity of a fibrin-specific monoclonal antibody.

The structural determinant underlying the species specificity of a monoclonal anti-fibrin antibody (59D8) is the leucyl residue at position 5 in beta-chains of human fibrin. Anti-fibrin antibody 59D8 which had been elicited by immunization with human beta(1-7) peptide, Gly-His-Arg-Pro-Leu-Asp-Lys, binds to human and canine fibrins but not to bovine, ovine, or porcine fibrins. A comparison of the available amino acid sequence data suggested that the ability of anti-fibrin antibody 59D8 to discriminate among various fibrin beta-chains might be due to the amino acid at position 5. This was confirmed by competitive inhibition studies using synthetic fibrin-like peptides and determination of the amino acid sequences of the N-termini of ovine and porcine fibrin beta-chains. Edman degradation employing o-phthalaldehyde blocking permitted use of fibrin monomer rather than its separated constituent polypeptide chains. The same sequencing strategy was used to obtain partial sequence data for the alpha-chains of bovine, ovine, and porcine fibrin.

Amino Acid Sequence↗
PubMed1986

Atrial natriuretic factor: assessment of its structure in atria and regulation of its biosynthesis with volume depletion.

Atrial natriuretic factor (ANF) is a polypeptide that has been isolated from mammalian atrial tissue with potent natriuretic, diuretic and spasmolytic properties. Details of its biosynthesis, cleavage, storage and release are not yet fully defined. Using sequence-specific anti-peptide antisera in immunocytochemical and radioimmunoassay studies, we demonstrated the presence in atrial granules of sequences corresponding to both the large molecular weight ("prohormone") form of ANF and the carboxyterminal peptide believed to be responsible for its biological activity. However, a peptide sequence spanning the proposed site of cleavage of the "signal peptide" from preproANF was undetected. In volume-depleted rats, a 28% decrease in the relative concentrations of atrial ANF mRNA was observed as compared to that of controls (P less than 0.01). There was, however, no significant difference observed between the two groups of animals with respect to atrial levels of ANF, as measured by radioimmunoassay. We conclude from these studies that proANF, but not preproANF, is stored in atrial granules. The concentration of stored ANF remains constant in volume-depleted rats despite a 28% reduction in ambient levels of ANF mRNA. The physiologic significance of these findings is discussed.

Animals↗
PubMed1986

Monoclonal antibodies of predetermined specificity for fibrin: a rational approach to monoclonal antibody production.

Three monoclonal antibodies (MAbs) (55D10, 59D8, and 64C5) have been produced by an immunization strategy that uses a synthetic heptapeptide from the amino terminus of the beta-chain in human fibrin. These MAbs bind to human fibrin in the presence of 4 mg/ml human fibrinogen in solid-phase radioimmunoassay. Ligand specificity studies demonstrate that antifibrin immunoreactivity is completely inhibited by a series of synthetic fibrin-like beta-peptides that bear the position-2 histidyl residue. Although the presence of this residue on the epitope is necessary for its recognition by a fibrin-specific antibody, the presence of the histidyl residue alone is not sufficient for antibody recognition.

Animals↗
PubMed1986

Preparation of Boc-[S-(3-nitro-2-pyridinesulfenyl)]-cysteine and its use for unsymmetrical disulfide bond formation.

The 3-nitro-2-pyridinesulfenyl (Npys) derivative of cysteine was prepared and used to facilitate the formation of an unsymmetrical disulfide bond. Since this derivative is stable in trifluoroacetic acid:CH2 Cl2 (1:1) and anhydrous hydrogen fluoride, Boc-Cys(Npys) could be used directly in solid phase synthesis of the 14-peptide acetyl-Cys(Npys)-Gly-Glu-Gln-Gln-His-His-Pro-Gly-Gly-Gly-Ala-Lys-G ln-Ala-amide. Reaction of this peptide with the free thiol of another peptide, acetyl-Gly-Glu-Gln-His-His-Pro-Gly-Gly-Gly-Ala-Lys-Gln-Cys-amide, gave a single product containing an unsymmetrical disulfide bond. The amino acid composition of this product and HPLC analysis of its dithiothreitol reduction products were consistent with the desired heterodimer. As evidenced by HPLC, the mixed disulfide forms rapidly at alkaline pH and usefully over a wide pH range in aqueous buffers.

Amino Acid Sequence↗