A distinctive variable region 14-positive (V14+) a chain (V.14') of the T-cell antigen receptor is predominantly expressed in multiple mouse subspecies. The VJ14 family has two members, Val4.1 and Val4.2, which differ by only three amino acids at positions 50-52. Based on the EcoRI restriction fragment length polymorphism of the gene encoding V.14, mice can be divided into three groups:type I with an 11.2-kilobase (kb) fragment, type II with a 2.0-kb fragment, and type m with the 2.0-kb and 11.2-kb fragments.Usage of V,14-J.281, where J.281 is an a-chain joining segment, with a one-base, N region dominates at the level of 0.02-1.5% of a chains in all laboratory strains, Mus musculus castaneus, and Mus musculus domesticus but not in Mus musculus molossinus, Mus musculus musculus, and Mus spicilegus samples. The preferential Val4Ja281 expression seems to be due to positive selection because the V-Jjunctional region is always glycine, despite the ability of the V.14 gene to associate with J. other than JL281. As Va14-Ja281 expression is independent of known maijor histocompatibility complex antigens, including H-2, TLA, Qa, and HMIT, the selecting ligand must be a monomorphic molecule of the mouse, expressed in a subspecies-specific manner. Additional observations, such as the expression of homogeneous V.14-J.281 in athymic mice, suggest that the positive selection of V.14' T cells occurs extrathymically.
We have developed a sensitive and rapid method for detection and cloning of cDNA amplified by double-step and inverse polymerase chain reaction (PCR) techniques. The blunt-ended double-strand cDNA libraries were circularized with T4 ligase and subsequently amplified by double-step PCR with two sets of primers of outward orientation (at amounts of 1-10 pmol in the first step and 100 pmol in the second step) which hybridize with the known region of the target DNA. This method is useful for analysis of the repertoires of TCR or immunoglobulins, in particular TCR alpha-chains, which are encoded by a single known constant region gene and greater than 100 unknown variable and joining region gene segments. By using this method, we detected at least 10(4) copies of TCR alpha-chain transcripts in the original samples which are equivalent to 10(3) T cells. The use of 1-10 ng of cytoplasmic RNA allowed us to make approximately 10(3) independent TCR alpha-chain libraries and to determine the sequence of unknown TCR alpha-chain cDNA by this method. We also show the frequency of a TCR alpha-chain usage in naive spleen and tumor-infiltrating lymphocytes.
Abstract. We established a new and facile model to investigate allergic mechanism and assess the effect of antiallergic compounds. Male Wistar rats were actively or passively sensitized. Active sensitization was performed by injection of both dinitrophenylated-ovalbumin (DNP-OA) and Bordetella pertussis. Nine days later, DNP-OA was injected into the right hind footpad. This antigen challenge induced a biphasic footpad swelling that consisted of an early-phase (EPR) and a late-phase response (LPR). In rats passively sensitized with rat anti-DNP-OA serum, DNP-OA induced only EPR. The EPR was suppressed by disodium cromoglycate, a mast cell stabilizer, but not by cyclosporin A, an immunosuppressant, while the LPR was suppressed by cyclosporin A. Furthermore, to investigate these two allergic responses determined by the interactions between the hapten and the carrier proteins, two distinct haptenated antigens were created. DNP-Ascaris (DNP-As) induced a marked EPR and LPR in DNP-As-sensitized rats. However, DNP-As induced only EPR in DNP-OA-sensitized rats, indicating that the usage of the same carrier protein in both sensitization and challenge was necessary for induction of LPR. These data suggest that this actively sensitization model in which EPR and LPR are functionally distinguishable should be useful for evaluating the efficacy of antiallergic compounds.
When Citrobacter freundii cephalosporinase was incubated with 6~-[3-(2-chlorophenyl)-5-methyl-4-isoxazolyl]penicillin sulfone (cloxacillin sulfone) in phosphate buffer, the enzyme was suddenly inactivated just after the completion of enzymatic degradation of the cloxacillin sulfone. Such delayed inactivation was due to a secondary inhibitor formed from cloxacillin sulfone during the incubation period. The inactivation was delayed due to the protection of the enzyme by cloxacillin sulfone from the attack of the secondary inhibitor. Phosphate anions were essential for the formation of the secondary inhibitor.However, once the secondary inhibitor was formed, the inactivation occurred in the absence of phosphate anions although the degree of the inactivation depended on the length of the preincubation period with phosphate anions. The main species (more than 80%) of the inactivated enzyme was detected as a single protein band with a slightly lower pI value than that of the native enzyme on isoelectric focusing on a plate.Oxidation of the thiazolidine sulfur of a /3-lactamase-stable penicillin is known to convert the antibiotic into a "mechanism-based" inhibitor of (3-lactamase1). A sulfone may accelerate (9-elimination of the 6a proton and cleavage of the bond between C-5 and the sulfur, resulting in a transiently stable 6-aminoacrylate chromophore, which causes enzyme inactivation1,2). 649-[3-(2-Chlorophenyl)-5-methyl-4-isoxazolyl]penicillin (cloxacillin) is a semisynthetic penicillin that is stable for many types of ~-lactamases, an exception being OXA-type penicillinases3). In our previous study4) , we proved that cloxacillin sulfone (Fig. 1) is, as predicted from the stability of cloxacillin against enzymatic hydrolysis, a strong suicide inhibitor for some P-lactamases.However, cloxacillin sulfone showed no progressive inactivation of Citrobacter freundii cephalosporinase, even though cloxacillin is a very poor substrate for the cephalosporinase.On the other hand, we found an unexpected phenomenon concerning the interaction between cloxacillin sulfone and the cephalosporinase, the cephalosporinase was irreversibly inactivated just after completion of the enzymatic degradation of the cloxacillin sulfone by the enzyme. This "delayed inactivation" was most likely not caused by contamination of the cloxacillin sulfone preparation because the inactivation was seen even after repeated purification of the preparation by HPLC using different columns and elution systems. This study was carried out to elucidate the mechanism of the delayed inactivation.
In a previous study1}, we demonstrated that cloxacillin sulfone (1A) is a powerful mechanismbased inactivator for TEM-type penicillinases, but not for Citrobacter freundii cephalosporinase.On the other hand, we found the interesting phenomenonthat 1A was converted into a specific inhibitor for the cephalosporinase during its incubation with phosphate anions. This finding was originally referred to as a delayed inactivation phenomenon because the inhibitory effect of the specific inhibitor appeared just after completion of 1A degradation^. Wecalled the specific inhibitor the "secondary inhibitor".The phenomenon was assumedto be due to the compound(s) derived from 1A through a phosphate-mediated reaction. The reasons for this assumption were as follows : 1) The inhibitor was not produced in the absence of phosphate anions. 2) When 1A was preincubated with phosphate anions, the secondary inhibitor activity was detected even after the free anions had been removed from the reaction mixture.3) The level of the inhibitory activity increased with increasing phosphate anion concentration and increasing preincubation period. In this study, we tried to isolate the secondary inhibitor after prolonged incubation of 1A with phosphate anions. Cephalosporinase was purified from cells of C. freundii GN346 as previously described3).1A ( Fig. 1) was synthesized from cloxacillin via cloxacillin sulfoxide and then purified by HPLC, as reported previously1}. The 6-epimer of 1A (IB) (Fig. 1) IB was separated from 1A by HPLC. As shown in Fig. 2A, freshly solubilized 1A gave one major HPLCpeak, PI (at 37 minutes)accompanied by several minor peaks, P2 (at 36 minutes) and P3s (ranging from 29 to 33 minutes). The P2 and P3s compounds were identified as IB and hydrolysis products, respectively, on the basis of the HPLCpattern for an authentic sample of IB and alkaline hydrolyzates of 1A (data not shown). When 1A was incubated in 50 mMphosphate buffer (pH 7.0) at 30°C for 15 hours, the decrease in peak PI was followed not only by concomitant increases in peaks P2 and P3s, but also by the appearance of a new peak at 21 minutes (P4) (Fig. 2B). Peak P4 did not appear when 1A was incubated in distilled water (Fig. 2C), whereas the decrease in peak PI followed by increases in peaks P2 and P3s was observed even in distilled water. It should be noted that 1A preincubated in distilled water did not exhibit the secondary inhibitor activity2). It could therefore be presumed that the P4 compound is the secondary inhibitor. It was also confirmed that the amount of the P4 compoundwas largely dependent upon the concentration of phosphate anions and the length of the incubation period (data not shown). The peak PI, P2, P3s and P4 fractions were pooled separately, then they were examined as to the secondary inhibitor activity in the absence of phosphate anions (Fig. 3). Only the P4 fraction showed strong irreversible inactivation
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