Preparation and characterization of monoclonal antibodies against the human thrombin-antithrombin III complex.

Monoclonal antibodies were raised against human thrombin-antithrombin III complex by a hybridoma technique. Among them, five monoclonal antibodies, designated as JITAT-4, -14, -16, -17 and -19, were found to react with thrombin-antithrombin III, but not with its nascent components, alpha-thrombin or antithrombin III. Their respective immunoglobulin classes are IgG1 for JITAT-16 and -19, and IgG2a for JITAT-4, -14 and -17. Besides the thrombin-antithrombin III complex, they all bound to the Factor Xa-antithrombin III complex and the active-site-cleaved two-chain antithrombin III as well. Moreover, the reactivity of these two antibodies to the neoantigens was not affected by heparin, suggesting that their epitopes are independent of heparin-induced conformational changes of antithrombin III. Two of them, JITAT-16 and -17, were categorized as high-affinity antibodies to thrombin-antithrombin III complex, the dissociation constants being 6.7 nM and 4.8 nM, respectively. However, they do not share antigenic determinants. These monoclonal antibodies may allow us to explore more precisely the reaction between antithrombin III and thrombin or its related enzymes.

Calcium ion-dependent monoclonal antibody against human fibrinogen: preparation, characterization, and application to fibrinogen purification.

We have produced a high-affinity monoclonal antibody classified as IgG1 with kappa-type light chains that recognizes the calcium ion(Ca2+)-dependent conformation of the D-domain of human fibrinogen. Binding of fibrinogen in solution to the insolubilized antibody increased in the presence of increasing concentrations of up to 2 mM Ca2+, the half-maximal binding being reached at 130 microM Ca2+. The dissociation constant was estimated to be 1.6 x 10(-8) M at 2 mM Ca2+. The antibody was found also to be dependent on other divalent metal ions including Zn2+, Mn2+, Co2+ and Cu2+, but not Ba2+, Mg2+ or Sr2+. The synthetic Gly-Pro-Arg-Pro-amide peptide, which has recently been shown to bind to close proximity to the calcium binding site in the D-domain, was unable to elicit the conformation for the antigen to be recognized by this antibody. This antibody was found to be a suitable ligand for the immunoaffinity chromatography of normal and abnormal fibrinogens directly from citrated plasma depleted of the vitamin K-dependent proteins or heparinized plasma by eliminating the precipitation procedure widely adopted in conventional techniques of fibrinogen purification. Indeed, fibrinogen Marburg I with the A alpha chains depleted of the carboxy-terminal A alpha(461-610) residue segment has been purified by this technique, although this dysfibrinogen was difficult to purify by conventional precipitation techniques.

An alternative elastase-mediated degradation of fibrinogen and fibrin observed in a patient with herpes simplex encephalitis and pneumonia.

A 74-year-old female developed pneumonia following herpes simplex encephalitis. Her white blood cell counts reached 28,400/microliters, about 90% of which consisted of granulocytes. The polymorphonuclear (PMN) elastase/alpha 1-antitrypsin complex levels increased and reached the maximum of 5,019 ng/ml, indicating the release of a large amount of elastase derived from the granulocytes. The mechanism of PMN elastase release was most likely to be granulocyte destruction associated with phagocytosis. The cleavage of fibrinogen and fibrin by PMN elastase, independent of plasmin, was indicated by the presence of the fragments in immunoprecipitated plasma from the patient corresponding to elastase-induced FDP D and DD fragments and the absence of fragments corresponding to plasmin-induced FDP D and DD fragments on SDS-PAGE. These findings suggested that the large amount of PMN elastase released from the excessive numbers of granulocytes in this patient with herpes simplex encephalitis and pneumonia, induced the cleavage of fibrinogen and fibrin without the participation of plasmin.

Purification and properties of polynucleotide phosphorylase from photosynthetic bacterium Rhodospirillum rubrum.

1. Polynucleotide phosphorylase [polyribonucleotide: orthophosphate nucleotidyltransferase, EC 2.7.7.8] was purified to near homogeneity from the photosynthetic bacterium, Rhodospirillum rubrum. The purified enzyme had a molecular weight of approximately 160,000, and consisted of two equivalent subunits of approximately 76,000 daltons. It catalyzed the three reactions described below. 2. In the exchange reaction of the beta-phosphate of nucleoside diphosphates with Pi by the purified enzyme in the presence of 3.3 mM Pi, 6.7 mMCl2, and 0.33 mM or 1.0 mM nucleotide at pH 8.0 and 20 degrees C, ADP, GDP, and CDP, and CDP were better substrates than UDP, while IDP and deoxyribonucleoside diphosphates hardly served as substrates. The ADP-Pi exchange activity was significantly inhibited by high concentrations of either ADP or Pi. 3. In the polymerization reaction of ribonucleoside diphosphates by the purified enzyme in the presence of 6.7 mM nucleotide and 6.7 mM MgCl2 at pH 8.0 and 20 degrees C, ADP was the best substrate; the activities relative to that with ADP were 55% with UD, 51% with CDP, and 48% with IDP, while GDP hardly served as a substrate, 4. In the phosphoryolysis reaction of polynucleoside diphosphates by the purified enzyme in the presence of 1.0 mM polynucleotide, 6.7 mM Pi, and 6.7 mM MgCl2 at pH 8.0 and 20 degrees C, poly[U] was the best substrate; the activities relative to that with poly[U] were 32% with poly[A], 28% with poly[I], 21% with poly[C], and 2% with yeast RNA, while poly[G] and yeast DNA hardly served as substrates. 5. The three kinds of activities of the purified enzyme described above were stimulated by divalent cations such as Mg2+, Mn2+, Cd2+, and Co2+.

Reversible conversion from Ca(2)+-ATPase activity to Mg(2)+- and Mn(2)+-ATPase activities of coupling factor purified from acetone powder of Rhodospirillum rubrum chromatophores.

It is known that the coupling factor purified from the acetone powder of chromatophores from Rhodospirillum rubrum shows ATPase activity in the presence of Ca(2)+, but not in the presence of Mg(2)+ or Mn(2)+. The present study deals with conditions, under which the Ca(2)+-ATPase activity is reversibly converted into Mg(2)+- and Mn(2)+-ATPase activites with the purified coupling factor. 1. Of the pH indicators tested, 6 kinds coverted the Ca(2)+-ATPase activity into Mg(2)+- and Mn(2)+-ATPase activities in the order, ethyl orange greater than tropaeolin 000 greater than or equal to metanil yellow greater than tropaeolin 00 greater than ethyl red greater than or equal to bromthymol blue. 2. Of the detergents tested, those other than Triton X-100 and Brij 58 caused the conversion described above; dodecylsulfonate was most effective, whereas dodecylpyridinium chloride was moderately effective. 3. 2,4-Dinitrophenol stimulated approximately two-fold the Ca(2)+-ATPase activity, but not the Mg(2)+- or Mn(2)+-ATPase activity at all. However, in the presence of dodecylpyridinium chloride, the pH indicator remarkably stimulated the Mg(2)+- and Mn(2)+-ATPase activities, accompanied with a partial inhibition of the Ca(2)+-ATPase activity. Methyl red and ethyl red showed similar effects. 4. All the nucleoside triphosphates tested can serve as the substrate. ATP was most effective for the Ca(2)+-ATPase activity, whereas dATP was most effective for the Mg(2)+- and Mn(2)+-ATPase activities induced by ethyl orange. 5. In the presence of ethyl orange, the ATPase activity was induced by various divalent cations in the following order of effectiveness, Mg(2)+ greater than Zn(2)+ greater than CO(2)+ greater than Mn(2)+ greater than Ni(2)+. 6. The mechanism of the reversible conversion from the Ca(2)+-ATPase activity to the Mg(2)+- and Mn(2)+-ATPase activities by pH indicators and detergents is discussed.

Effects of pH indicators on various activities of chromatophroes of Rhodospirillum rubrum.

1. The effects of pH indicators on activities for ATP hydrolysis in the dark and ATP-Pi exchange in the dark were examined with chromatophores from Rhodospirillum rubrum. Of thirty-one pH indicators tested, eleven (metanil yellow, 2, 4-dinitrophenol, ethyl orange, bromocresol green, resazurin, neutral red, bromthymol blue, alpha-naphtholphthalein, o-cresolphthalein, phenolphthalein, and alizarin yellow G) almost completely inhibited the activities for ATP formation and ATP-Pi exchange at concentrations of 1 mM, and were studied in detail. 2. Of the eleven pH indicators, those other than alpha-naptholphthalein, o-cresolphthalein and phenolphthalein, when assayed at appropriate concentrations, inhibited ATP-Pi exchange, but not ATP hydrolysis. In ATP-Pi exchange, these eight pH indicators at the concentrations described above were competitive against Pi, and non-competitive against ATP. The remaining three kinds of pH indicators were non-competitive against either Pi or ATP, when assayed at concentrations of the dyes that inhibited both activities. 3. The amounts of pH indicators bound with chromatophores were measured. No correlation was found between the amounts of the bound dyes and the extents of their inhibition of either ATP formation or ATP-Pi exchange. 4. Ethyl orange (pKa=4.1) and 2, 4-dinitrophenol (pKa=3.9) stimulated ATP hydrolysis to the greatest extent. The latter dye was hardly bound with chromatophores. 5. The stimulatory effects of pH indicators on ATP hydrolysis were hardly affected by extraction of quinones from chromatophores. 6. Most of the pH indicators stimulated both succinate-cytochrome c2 and NADH-cytochrome c2 reductions in the dark. 7. The mechanism of uncoupling of the electron transfer system and the phosphorylation system by pH indicators and the mechanism of the coupling are discussed.

A new homogeneous enzyme immunoassay. Its application to measurement of alpha-fetoprotein.

A rapid and sensitive homogeneous enzyme immunoassay (homogeneous EIA) was developed for determination of serum proteins such as alpha-fetoprotein (AFP). There are two assay systems, one is a competitive system including horseradish peroxidase (HRP)-labeled antigen, antibody and substrate, and the other is a non-competitive system including HRP-labeled antibody and substrate. When the aggregate was formed through the binding of HRP-labeled AFP and anti-AFP antibody or through the binding of HRP-labeled anti-AFP antibody and AFP, HRP of the aggregates, as compared with HRP of free conjugates, exhibited marked activity in the presence of 35 mM H2O2. The extent of stimulation of HRP activity depended on the amount of AFP. This new assay method is very simple and sensitive, and can be used for the determination of any kind of protein, hormone, or drug.

Light-induced pH changes and changes in absorbance of pH indicators in Rhodospirillum rubrum chromatophores.

1. The light-induced pH change of chromatophore suspensions from Rhodospirillum rubrum was stimulated significantly and similarly by KCl, NaCl, LiCl, RbCl, CsCl, MgCl2, MnCl2, and CaCl2. In the dark, the pH of chromatophore suspensions decreased immediately and markedly on adding these salts. 2. The light-induced pH change stimulated by KCl plus valinomycin was inhibited by LiCl and NaCl, but not by RbCl. 3. The optimum pH values for light-induced pH change and photosynthetic ATP formation were around 5 and 8, respectively. The amount of chromatophore-bound ubiquinone-10 reduced in the light was independent of pH from 5 to 9. At pH 8, the number of protons incorporated into chromatophores in the light was one-half of the number of ubiquinone-10 molecules reduced in the light. 4. Among several pH indicators tested, bromothymol blue (BTB) and neutral red (NR) showed absorbance changes on illumination of chromatophores. Although the pH change indicated by the absorbance change was opposite to the light-induced pH change of the medium, the effect of KCl on the absorbance changes of BTB and NR, and the effect of valinomycin on that of NR, but not on that of BTB, were similar to those on the light-induced pH change. 5. The light-induced absorbance change of BTB was significantly inhibited by NR, whereas that of NR was hardly influenced by BTB. 6. Oligomycin stimulated the light-induced absorbance change of BTB under either non-phosphorylating or phosphorylating conditions. On the other hand, that of NR under phosphorylating conditions was 50% of that under non-phosphorylating conditions, and was increased by oligomycin.

Disintegration of Rhodospirillum rubrum chromatophore membrane into photoreaction units, reaction centers, and ubiquinone-10 protein with mixture of cholate and deoxycholate.

1. The membrane of Rhodospirillum rubrum chromatophores was disintegrated with mild detergents (cholate and deoxycholate) in order to study the spatial arrangement of the functional proteins in the photochemical apparatus and the electron transport system in the membrane. 2. The components solubilized from the membrane by a mixture of cholate and deoxycholate (C-DOC) were separated into four fractions by molecular-sieve chromatography in the presence of C-DOC; they were designated as F1, F2, F3, and F4 in the order of elution. The fractions were further purified by repeated molecular-sieve chromatography in the presence of C-DOC until each fraction was chromatographically homogeneous. 3. F1 appeared to be conjugated forms of F2. 4. The purified F2 was composed of a rigid complex having a weight of 7 X 10(5) daltons, containing approximately 10 different kinds of protein species with molecular weights of 3.8 X 10(4), 3.6 X 10(4), 3.5 X 10(4), 2.8 X 10(4), 2.7 X 10(4), 2.6 X 10(4), 1.3 X 10(4), 1.2 X 10(4), 1.1 X 10(4), and 1.0 X 10(4). The complex contained 33 bacteriochlorophylls, 4 iron atoms, and 90 phosphates, but no cytochrome, ubiquinone, or phospholipid. It showed the same reaction center activity as chromatophores, indicating that the complex was a unit of the photochemical apparatus (photoreaction unit). Each chromatophore of average size was estimated to possess about 24 photoreaction units. 5. The purified F3 showed an absorbance spectrum characteristic of reaction centers, and contained 3.4 bacteriochlorophylls, 2.0 bacteriopheophytins, and 1.9 acid-labile iron atoms, but no cytochrome or ubiquinone (C-DOC reaction center). It had a weight of 1.2 X 10(5) daltons, and the main components were 4 protein species with molecular weights of 2.8 X 10(4), 2.7 X 10(4), 2.6 X 10(4), and 1.0 X 10(4). 6. The purified F4 showed a molecular weight of about 11,000, and contained one mole of ubiquinone-10 per mole (ubiquinone-10 protein). 7. The reaction center activity of C-DOC reaction centers was stimulated by ubiquinone-10 protein. In addition, the reaction center oxidized reduced cytochrome c2 in the light, provided that ubiquinone-10 protein was present (photo-oxidase activity).

A rapid latex immunoassay for the detection of plasmin-alpha 2-plasmin inhibitor complex. Utilization of two monoclonal antibodies differentially recognizing respective components of the complex.

Among six monoclonal antibodies raised against the human plasmin-alpha 2-plasmin inhibitor complex (PPI), three antibodies were found to recognize the plasmin part (group 1) and another three the alpha 2-plasmin inhibitor (alpha 2-PI) part (group 2) of the complex. One of the group-1 monoclonal antibodies, designated JIPPI-3, specifically reacted with a segment of plasmin containing kringles 2 and 3. Although all three group 2 antibodies reacted with both alpha 2-PI and PPI on immunoblotting and ELISA, one of them, JIPPI-50, was unable to react with alpha 2-PI, when the antibody had been covalently conjugated to Sepharose 4B gels and tested for reactivity against the antigens in solution. The results indicated that the epitope for this antibody had been buried in nascent alpha 2-PI, but had been exposed by complex formation with plasmin or by possible conformational changes induced in the alpha 2-PI molecule on insolubilization to nitrocellulose membranes or immunoplates. By utilizing a set of JIPPI-3 and JIPPI-50, individually coated onto latex beads, PPI could be measured in plasma in the range of 0.8-100 micrograms/ml without interference by coexisting plasminogen (120-200 micrograms/ml) or alpha 2-PI (70 micrograms/ml). This measurable range seems to cover the level of PPI clinically observed under hyperfibrinolytic states.

Monoclonal antibody specific for tissue factor pathway inhibitor-factor Xa complex: its characterization and application to plasmas from patients with disseminated intravascular coagulation and pre-disseminated intravascular coagulation.

Tissue factor pathway inhibitor (TFPI), a Kunitz-type protease inhibitor with three tandem inhibitory domains (K1, K2 and K3), inhibits the initial reactions of the extrinsic blood coagulation pathway through its K1 and K2 domains. We prepared and characterized a monoclonal antibody (Mab8-1) against TFPI-factor Xa (TFPI-Xa) complex. The reactivities of Mab8-1 toward TFPI-Xa complex, TFPI without C-terminal (TFPI-C)-Xa complex, K1K2-Xa complex and K2K3-Xa complex were examined using a surface plasmon resonance analysis (Biacore). The Biacore system allowed a quantitative analysis of antibody-antigen interaction, in real time, from which the association and dissociation rate constants could readily be obtained. The bindings of Mab8-1 to TFPI-Xa complex, TFPI-C-Xa complex and K2K3-Xa complex were each concentration-dependent. However, no binding of Mab8-1 to the K1K2-Xa complex was observed. The binding of Mab8-1 to TFPI or Xa was also not observed. These results suggested that the epitope for Mab8-1 was exposed in the K3 domain of TFPI, which was generated by the conformational change after the formation of TFPI-Xa complex. We then developed an enzyme-linked immunosorbent assay method specific for TFPI-Xa complex using Mab8-1, and we used this assay to measure plasma levels of TFPI-Xa. The normal range assessed from analyses of plasma from 30 normal healthy volunteers was 17.7-66.7 with a mean of 35.5 +/- 11.7 pmol/l. In order to asses the clinical implication of TFPI-Xa complex in the plasma of patients with thrombotic disorders, plasma concentrations were measured in 37 patients with disseminated intravascular coagulation (DIC) caused by a variety of underlying diseases. The TFPI-Xa antigen levels were significantly higher in the patients with DIC (51.9 +/- 21.6 pmol/l) and the 36 patients with pre-DIC (55.1 +/- 20.2 pmol/l) than in the 137 non-DIC patients (37.9 +/- 13.1 pmol/l). In the patients with DIC or pre-DIC, there was no significant correlation between TFPI-Xa complex and the elevated levels of thrombin-antithrombin complex, plasmin-alpha2 plasmin inhibitor complex, D-dimer, soluble fibrin monomer, soluble thrombomodulin or tissue factor. These data indicate that the plasma level of TFPI-Xa seems to be a novel independent molecular marker of DIC and pre-DIC.

Treatment of typhoid fever and other systemic salmonelloses with cefotaxime, ceftriaxone, cefoperazone, and other newer cephalosporins.

Third-generation cephalosporins have been considered for the treatment of systemic salmonelloses because of emerging resistance among Salmonella species to ampicillin, chloramphenicol, and trimethoprim-sulfamethoxazole. Twelve patients with typhoid/paratyphoid fever, nine with nontyphoid salmonella bacteremia, and two with Salmonella meningitis were treated with cefotaxime; one leukemic patient with Salmonella dublin bacteremia received ceftizoxime. All infections were cured except for one in a patient with sickle cell anemia; this patient's illness recurred but was cured with a second course of cefotaxime followed by ceftriaxone. A review of the literature documented cures with cefotaxime in 50 of 61 patients with typhoid/paratyphoid fever, all of four with salmonella osteomyelitis, 12 of 14 with salmonella meningitis, and 44 of 49 with non-typhoid salmonella bacteremia. Ceftriaxone and cefoperazone cured, respectively, 23 of 25 and 32 of 33 patients with typhoid/paratyphoid fever. The relapse rates of typhoid fever treated with cefotaxime, ceftriaxone, and cefoperazone were 6%, 4%, and 0%, respectively. Cefotaxime, ceftriaxone, and cefoperazone are acceptable alternative antibiotics for the treatment of salmonelloses caused by multiresistant organisms.

Assessment of membrane fusion efficiency and its use for distinguishing epitopes on the fusion (F) protein of Sendai virus (HVJ).

Upon incubation of radioiodinated Sendai virus with Ehrlich ascites tumor cells to induce viral envelope-cell membrane fusion, TCA-soluble radioactivity was found to be rapidly released from the virus into the medium. The amount of the TCA-soluble 125I species released into the medium was correlated with the extent of envelope-membrane fusion. Using a new method based on this phenomenon, we evaluated the fusion-inhibiting activity of monoclonal anti-F protein antibodies and could distinguish epitopes within partially overlapping regions of the F protein by their effects on fusion activity. We concluded this method is a reliable and sensitive one for measuring envelope-membrane fusion.

Production and characterization of agglutinating monoclonal antibodies against predominant antigenic factors for Candida albicans.

Two clones, CA4-2 and CA5-4, which produced agglutinating monoclonal immunoglobulin M (IgM) antibodies (MAbs) against mannan antigens of Candida albicans serotype A, were established. The specificity of each MAb was determined by slide agglutination tests for cross-reactivity patterns against the homologous and six other strains of Candida and a strain of Torulopsis: C. albicans serotype B, C. tropicalis, C. guilliermondii, C. krusei, C. parapsilosis, C. pseudotropicalis, and Torulopsis glabrata. The MAb produced by CA4-2 reacted with the homologous, C. tropicalis, and T. glabrata strains, whereas the MAb produced by CA5-4 reacted with the homologous, C. albicans serotype B, and C. tropicalis strains. These results are consistent with results obtained by comparative experiments with several strains of each serotype or species. Specificity of these two MAbs by agglutination was also consistent with the cross-reactivity patterns demonstrated by indirect immunofluorescence staining. The competitive binding experiments by immunofluorescence staining with two MAbs and polyclonal factor sera (PAb factors) 5 and 6 suggested that the MAb from clone CA4-2 did not completely correspond to PAb factor 6 and that the MAb from CA5-4 was distinct from PAb factor 5 in its manner of binding to determinants (the latter was designated 5b), Cross-reactivity patterns, however, furnished evidence that these two MAbs could replace the known PAb factors 6 and 5, respectively, as reagents for aid in the identification of the strains of C. albicans and their serotypes.

Characterization of alternate and truncated forms of murine c-myb proteins.

The expression of c-myb proteins in transformed and normal murine cells was investigated. Two c-myb proteins, p75c-myb and p90c-myb, were detected in normal thymocytes and cell lines with intact c-myb genes. These most likely differ by the inclusion of additional amino acids encoded by an alternatively spliced c-myb mRNA. The use of this alternative exon is therefore not a feature exclusively of those cells with viral integrations in their c-myb gene. Smaller c-myb proteins in myeloid leukemic cell lines with rearranged c-myb genes were also characterized. Viral integration into the 5' region of the c-myb gene in the W265 and W274 cell lines leads to the synthesis in each case of two amino-terminally truncated c-myb proteins. By contrast, in NFS60 cells, viral integration into a more 3' region of the c-myb locus (but upstream of an alternate exon) leads to the production of a single p50c-myb protein.