The tumor antigen capable of inducing tumor resistance (tumor rejection antigen; TRA) was obtained in a solubilized form by sodium dodecyl sulfate (SDS) extraction of plasma membrane fraction from Rous sarcoma virus (RSV)‐induced CSA1M fibrosarcoma cells (BALB/c origin). Analyses by Sephacryl S‐300 gel filtration and SDS‐polyacrylamide gel electrophoresis revealed that TRA activity was recovered in the fraction with a molecular weight of approximately 60 kD. Unfractionated crude SDS‐solubilized preparation contained gp70 as detected by rabbit anti‐gp70 antiserum, whereas such reactivity was lost in the fraction exhibiting the molecular weight of about 60 kD. Since this fraction retained pp60 src activity, the relation of TRA to pp60 src was further investigated. pp60 v‐ src was also obtained from the lysate of v‐src‐expressing yeast transformant. Immunization of BALB/c mice with such pp60 v‐ src ‐containing lysate failed to induce any significant tumor protection. The above 60 kD fraction of CSA1M solubilized antigens was allowed to bind to Sepharose beads coupled with anti‐pp60 src monoclonal antibody and separated into the bead‐bound and bead‐unbound fractions. The bead‐bound fraction that was recovered from pp60 src ‐binding beads (pp60 src ‐positive fraction) did not exhibit the TRA activity. In contrast, immunization with the fraction depleted of pp60 src activity (bead‐unbound fraction) resulted in potent tumor protection. These results indicate that the solubilized membranous component(s) of CSA1M with a molecular weight of approximately 60 kD, which is distinct from functional pp60 src , functions as the TRA against RSV‐induced CSA1M tumor cells.
6‐Aminohexanoic acid cyclic dimer hydrolase produced by Achromobacter guttatus cells grown on 6‐aminohexanoic acid cyclic dimer (1,8‐diazacyclotetradecane‐2,9‐dione) was purified until it was homogeneous according to polyacrylamide disc gel electrophoresis, sodium dodecylsulfate polyacrylamide gel electrophoresis, Sephadex G‐200 column chromatography, and analytical centrifugation. The following characteristics of the purified enzyme were observed. (1) The molecular weight of the enzyme, estimated to be 110 000 by column chromatography on Sephadex G‐200 and 100 000 by sedimentative equilibrium, together with the minimum molecular weight of 55 000 obtained by sodium dodecylsulfate polyacrylamide gel electrophoresis, indicated it is a dimeric enzyme. The sedimentation constant ( s 20 ) was estimated to be 5.7 S from the sedimentation velocity. (2) The enzyme had an optimum pH of 7.3 when 6‐aminohexanoic acid cyclic dimer was used as substrate. It was stable in a pH range between 5.5 to 8.5, and 50% and 100% of activity was lost after 10 min of heating at 45°C and 50°C respectively. The optimum temperature for activity was 33°C. The Michaelis‐Menten constant ( K m ) was 6 mM toward the dimer, and the turnover number was 8 s −1 assuming a molecular weight of 100 000. (3) This enzyme was strongly inhibited by 1 μM diisoprpylphosphofluoridate and 10 μM p ‐chloromercuribenzoate but scarcely inhibited by 100 mM ethylenediaminetetraacetic acid. The enzyme inhibited by p ‐chloromercuribenzoate could be reactivated by 2‐mercaptoethanol. (4) This enzyme was only active on 6‐aminohexanoic acid cyclic dimer to form 6‐aminohexanoyl‐6‐aminohexanoic acid. It was inactive on 6‐aminohexanoic acid oligomers (degree of polymerization 2 to 6), various cyclic amides, cyclic diamides, amides, oligopeptides, and casein. (5) The absorption coefficient ( A 1%280 nm) of the enzyme was 19.4. The enzyme was composed of 500 amino acid residues including 3 cysteines; from this the molecular weight was calculated to be 103 200 as a dimer. (6) 6‐Aminohexanoic acid cyclic dimer hydrolase was classified as a new cyclic amide hydrolase.
Abstract Polyclonal differentiation of unprimed B cells into IgM‐producing cells induced by lipopolysaccharide (LPS) or T cell‐derived lymphokine B151‐TRF2 has been shown to contain a process of I‐A‐restricted B‐B cell interaction, so that the B cell responses are inhibited by monoclonal antibodies (mAb) specific for I‐A molecules. On the other hand, the B cell responses are also inhibited by anti‐I‐E mAb, although I‐E molecules are not involved in such B‐B cell interaction. In this study, we examined the mechanism underlying the anti‐I‐E‐mediated inhibition of the B cell responses. The B cell responses induced by LPS or B151‐TRF2 were inhibited by either anti‐I‐A or anti‐I‐E mAb added on day 0 over a 5‐day culture period, whereas when added on day 3 the responses were inhibited only by anti‐I‐E mAb and not by anti‐I‐A mAb. To gain insight into the mechanism underlying the anti‐I‐E‐mediated inhibition, we prepared monovalent Fab and divalent F(ab') 2 fragments of anti‐I‐A and anti‐I‐E mAb and examined their effects on the B cell responses. We found that the B cell responses were inhibited by the F(ab') 2 but not Fab fragment of anti‐I‐E mAb, whereas the Fab fragment of anti‐I‐A mAb still gave effective inhibition. The F(ab') 2 but not Fab fragment of anti‐I‐E mAb induced increases in cyclic AMP (cAMP) levels in B cells, whereas the undigested anti‐I‐A mAb did not induce such increases. Furthermore, adenylate cyclase inhibitors, which inhibit cellular cAMP accumulation, circumvented the B cell responses inhibited by anti‐I‐E but not anti‐I‐A mAb. Thus, these results indicate that the anti‐I‐E‐mediated inhibition of the B cell responses requires increases in intracellular cAMP levels induced by cross‐linking of I‐E molecules. In contrast, anti‐I‐A mAb inhibits the B cell responses without cross‐linking of I‐A molecules and cAMP accumulation. These results reinforce a unique function of I‐A molecules as restriction elements in the Ia‐restricted B‐B cell interaction.
To examine the role of airway inflammation in airway hyperresponsiveness (AHR), we developed an animal model of AHR in guinea pigs and examined the histopathologic changes of these airways. Guinea pigs were actively sensitized with dinitrophenylated Ascaris suum extract and challenged with inhalation of the same extract. Six and 24 h after antigen challenge, airway responsiveness to inhaled acetylcholine (ACh), bronchoalveolar lavage fluid (BALF) and lung histology were studied. Airway responsiveness to inhaled ACh increased 6 h after antigen challenge (p < 0.05), but an increase in airway responsiveness was not observed 24 h after antigen challenge as determined by PC300 (the minimum concentration of ACh at which the respiratory resistance exceeded 300% of baseline value). The number of eosinophils and neutrophils in BALF increased 6 and 24 h after antigen challenge compared to sensitized, nonchallenged guinea pigs, peaking at 24 h after antigen challenge. On the other hand, the numbers of infiltrating eosinophils in bronchial and bronchiolar tissues increased 6 and 24 h after antigen challenge compared to sensitized, nonchallenged guinea pigs, peaking at 6 h after antigen challenge. We therefore conclude that AHR after allergen exposure in sensitized guinea pigs is associated with an increase in infiltrating eosinophils in lung tissue but not with BAL eosinophilia or BAL neutrophilia.
The antiallergic drug repirinast (30 mg/kg i.p.) significantly inhibited antigen-induced early and late pulmonary responses in guinea pigs. The same dose of repirinast significantly inhibited bronchoalveolar lavage eosinophilia and neutrophilia 5 h after antigen challenge (at the peak of the late response). Histologic examination revealed that repirinast markedly inhibited leukocyte (predominantly eosinophils) infiltration into bronchial tissue. Repirinast also blocked antigen-induced airway hyperresponsiveness to inhaled acetylcholine. The inhibitory effect of repirinast on the infiltration of eosinophils and/or neutrophils into the airway may reflect the inhibition of late pulmonary response and airway hyperresponsiveness.
A method for the synthesis of NAD + ‐ N 6 ‐[ N ‐( N ‐acryloyl‐1‐methoxycarbonyl‐5‐aminopentyl)‐propioamide] (monomeric NAD + derivative), a new NAD + derivative carrying a vinyl group, was described. By copolymerization with acrylamide 90% of the NAD + derivative was converted to a water‐soluble macromolecular NAD + derivative (polymeric NAD + derivative). Both NAD + derivatives were reduced completely with yeast alcohol dehydrogenase. High cofactor activities relative to free NAD + and NADH were obtained for the monomeric derivatives of NAD + (71–86%) and NADH (66–87%) with yeast alcohol, horse liver alcohol, lactate, and malate dehydrogenases. Lower but substantial relative cofactor activities were obtained for the polymeric derivatives of NAD + (18–33%) and NADH (28–60%) with yeast alcohol, horse liver alcohol, and malate dehydrogenases. But lactate dehydrogenase had negligible activity both for the polymeric NAD + and NADH derivatives. Kinetic studies were carried out with yeast alcohol dehydrogenase and lactate dehydrogenase, and the following kinetic constants were determined: the maximum velocity ( V ), the limiting Michaelis constants for coenzyme ( K a ) and for substrate ( K b ), and the dissociation constant of the enzyme‐coenzyme complex ( K ia ). In the reaction system with the monomeric NAD + derivative and yeast alcohol dehydrogenase, the K ia value was similar to that for NAD + and the values of V, K a , and K b were 38, 30, 21% of those for NAD + . In the reaction system with the polymeric NAD + derivative and yeast alcohol dehydrogenase, the values of V, K a , K b , and K ia were 0.5, 4, 3, 3 times larger than those for the monomer. In the reaction system with the monomeric NAD + derivative and lactate dehydrogenase, the values of V, K a , K b and K ia were 39, 16, 36, 57% of those for NAD + . From these results the following conclusions were obtained: (a) the modification of NAD + by alkylating at position 6 in the adenine moiety of NAD + caused a decrease in the V value, possibly due to configurational alterations of the binary or ternary coenzyme complexes of the enzymes resulting in decreased cofactor activity of NAD + , but the decrease in the activity was somewhat compensated by the decrease in the values of K a , K b , and K ia also caused by the modification; (b) the incorporation of the NAD + derivative into the side chain of polyacrylamide caused decrease in the affinity for the enzyme and further decrease in the V value, and thus decreased the cofactor activity to below that of the monomeric NAD + derivative.
Abstract The culture supernatant (SN) from a cloned line of thymic stroma-derived cells in fibroblastic form (TSCF) contained a factor capable of supporting the growth of the interleukln (IL) 2-dependent, antigen-specific helper T cell (Th) clone 9-16 without requiring IL2 and antigen. This active substance, designated as thymic stroma-derived T-cell growth factor (TSTGF), was partially purified through DEAE-Sephacel chromatography and PBE 94 chromatofocusing. The original SN did not contain IL1, IL2, IL3, IL4, or interferon activities; but an appreciable magnitude of colony-stimulating factor (CSF) activity in addition to TSTGF was present, whereas the partially purified preparation of TSTGF was depleted of any type of CSF activity. The elution profile of TSTGF activity on the chromatofocusing has revealed that TSTGF has an isoelectric point (pI) of about 6.0. When a purified TSTGF sample was applied to Sephacryl S-200 column chromatography and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, TSTGF activity was eluted in a single peak around an apparent molecular weight of about 25,000. The activity of TSTGF also was shown to be relatively stable with heat treatment and in the wide range of pH, but it was abolished by treatment with either trypsin or dithiothreitol. These results indicate that TSTGF, a novel T-cell growth factor, is the protein that has an apparent molecular weight of about 25,000 and a pI of 6.0, and in the intact molecule, it contains the disulfide bond(s) required to maintain and/or express its biologic activity.
Purified alpha-fetoprotein (AFP), fucosylated AFP mixtures, and 40 sera from patients with AFP-producing hepatocellular carcinoma were analysed by monoclonal enzyme immunoassay (EIA) to distinguish fucosylated and nonfucosylated AFP molecular variants. FUC-AFP-25 was discriminated from FUC-AFP-75 by the EIA using monoclonal antibody 18H4 in the range of total AFP concentrations from 100 to 800 ng/mL. In addition, sera from 40 patients with hepatocellular carcinoma, with AFP concentrations from 100 to 1270 ng/mL and with fucosylated AFP from 0 to 100% by conventional cross immuno-affinoelectrophoresis, were also analysed by the present EIA. A statistically significant correlation was obtained between the data from the present EIA and from the conventional crossed immuno-affinoelectrophoresis in the range of fucosylated AFP more than 20% and the serum concentration of AFP more than 100 ng/mL. These results indicate that the present EIA is useful for clinical detection of hepatocellular carcinoma during the follow-up of patients with chronic liver diseases.
