When RBL-1 cells were incubated with L-cysteine (7.5 mM) and the ionophore A23187, the slow reacting substances SRS-GSH and SRS-Cys-Gly were formed. When Lcysteine was omitted in the incubation, SRS-GSH and SRS-Cys were isolated but only a trace amount of SRS-Cys-Gly was detectable. Each of the characterized SRSs was accompanied by an as yet uncharacterized structural isomer showing UV absorption at 278 nm. L-Cysteine and other thiols inhibited an aminopeptidase that transforms the highly bioactive SRS of anaphylaxis (SRS-Cys-Gly) into the less bioactive SRS-Cys. SRS-GSH, SRS-Cys-Gly, and SRS-Cys may be readily distinguished from each other by means of their bioactivities, antagonism by FPL 55712, and relative susceptibilities to the actions of soybean lipoxygenase, microsome-bound leucine aminopeptidase, and y-glutamyl transpeptidase. Slow reacting substance (SRS) is generated and released during acute allergic reactions in lung and other tissues (1). Because SRS has long been suspected to be an important mediator in bronchial asthma (2), the isolation and structural characterization of SRSs from various natural sources have been the object of intense investigation. Recently, the isolation of SRS from murine mastocytoma cells was reported (3-5). This SRS was named leukotriene C and was reported to possess the structure 5-hydroxy-6-y-glutamylcysteinylglycyl-7,9, 11,- 14-icosatetraenoic acid (SRS-GSH).It is well documented that rat basophilic leukemia cells (RBL-1) produce SRS in response to the ionophore A23187 (6, 7). RBL SRS has been characterized as a family of thiolipids (8). Recently, two groups described the identification of 5-hydroxy-6-S-cysteinylglycyl-7,9,11,14-icosatetraenoic acid from RBL-1 cells (9, 10).In this paper, we report the identification of several additional SRSs from RBL-1 cells, and we report that cysteine alters the levels of SRSs indirectly by inhibiting an aminopeptidase, an enzyme involved in the inactivation of SRS-Cys-Gly.MATERIALS AND METHODS Materials. Type I soybean lipoxygenase (155,300 units/mg), -y-glutamyltranspeptidase (bovine, 4.0 units/mg), type IV-S leucine aminopeptidase (11 units/mg), prostaglandin B2, arachidonic acid (99%), and L-cysteine were products of Sigma. The SRS antagonist FPL 55712 was supplied by Fisons Limited, Longborough, England. Ionophore A23187 was kindly furnished by R. Hamill of Eli Lilly. Eagle's minimal essential medium, fetal calf serum, newborn calf serum, penicillin, amphotericin B, and streptomycin were products of GIBCO. D-Penicillamine and 3-mercaptopropionic acid were products of Aldrich. The chromatographic adsorbents consisted of Amberlite XAD-7 (Mallinckrodt) and silicic acid (100 mesh, Cells were harvested by centrifugation (400 X g, 10 min), washed once in buffer (150 mM NaCl/3.7 mM KCI/3.0 mM Na2HPO4/3.5 mM KH2PO4/0.9 mM CaCl2, 5.6 mM glucose, adjusted to pH 7.2 with 1 M NaOH), and suspended in this buffer to 1-1.5 X 107 cells per ml. After addition of L-cysteine (7.5 mM), the cell suspension was incubated for 2 min at 38°C. A...
Perfusion of cat paws with compound 48/80 released two slow reacting substances (SRSs) which were isolated and characterized as 5-hydroxy-S-cysteinylglycyl-7,9,-11,14-icosatetraenoic acid (SRS I) and -hydroxy-6S-cysteinyl-7,9,11,14-icosatetraenoic acid (SRS II) on the basis of chemical degradations, amino acid analyses, spectroscopic and enzymic experiments, and comparison with synthetic samples. The smooth muscle-contractile activities of synthetic 5-hydroxy-6 y-glutamylcysteinylglycyl-7,9,11,14-icosatetraenoic acid, synthetic5-hydroxy--S-cysteinyl-7,9,11,14-icosatetraenoic acid, and SRS II were not inactivated by arylsulfatase. On the other hand, the spasmogenic activities produced by synthetic 5-hydroxy-6S-cysteinylglycyl-7,9,11,14-icosatetraenoic acid and SRS I were destroyed at the same rate by the arylsulfatase. This mode of inactivation was attributed to an aminopeptidase activity in the arylsulfatase preparation because 5-ydroxyl-6-Scysteinyl-7,9,11,14-icosatetraenoic acid was isolated and identified as the reaction end product. Because the properties of SRS from cat paws closely resemble those of SRS generated by immunological stimulation of human tissues (SRS-A) and because all known SRS-A are inactivated by arylsulfatases, we contend that 5-hydroxy-6S-cysteinylglycyl-7,9,11,14-icosatetraenoic acid (SRS I) corresponds to SRS-A.In 1938, Feldberg and Kellaway (1) first described a substance that caused the guinea pig ileum to contract more slowly and with more sustained action than did histamine; they named the compound "slow reacting substance (SRS)." Two years later, Kellaway and Trethewie (2) reported that slow reacting substance of anaphylaxis (SRS-A) was released from sensitized tissue by a specific antigen. Subsequently, it was shown (3) that SRS-A was one of several pharmacologic mediators that may be important in human asthma. SRS has since been found to be released from many types of tissues and cell suspensions including guinea pig, rabbit, monkey, bovine, and human lungs (4-6), cat paws (7,8), isolated rat mast cells (9), human leukocytes (10) and nasal polyps (11), human and rat leukemic basophils (12,13), and rat peritoneal fluid (14, 15).Here we present our studies on the purification and identification of two SRSs from cat paws after perfusion with Compound 48/80 (16) (,uC18) preparative column (0.94 X 50 cm) of Partisil-10 M9 ODS (Whatman) was used for SRS purification. The radial compression separation system consisted of a Waters radial compression module (RCM-100) with a radial-Pak C18 cartridge (0.8 X 10 cm).SRS Assay. SRS bioactivity was assayed on guinea pig ileum segments by a modification of the procedure reported by Chakravarty (17). Histamine was assayed in the presence of atropine (1 MM) and SRS was assayed in the presence of atropine (1 ,uM) and mepyramine (1 ,M). One unit of SRS was defined as that amount of SRS which caused a contraction with a peak height equal to that induced by 5 ng of histamine base. After the log-dose-response curve for histamine was determin...
Aims: To determination the prevalence of VTEC in pork products and the surrounding environment of the pork plant (slaughterhouse and cutting plant), and characterization of the VTEC strains isolated (virulence genes and serotype). Methods and Results: Among the 2146 carcass and pork samples and 876 environmental samples (swabs of surfaces or materials), 328 (15%) and 170 (19%) were PCR-positive for stx genes respectively. VTEC strains were recovered from positive samples by colony hybridization or immunoconcentration, serotyped and genetically characterized. Strains of E. coli O157:H7 were not isolated from 3 uidA-positive samples detected by PCR. The VTEC isolates did not harbour eae, ehx and uidA genes. Conclusions: Pigs and pork meat may contain VTEC strains but characterization of the strains based on virulence factors showed that the potential danger of pork meat appears to be low since although all strains harboured a stx gene, they did not have other virulence genes. Significance and Impact of the Study: General hygiene measures appear to be sufficient and specific hygiene measures for VTEC are not necessary at this time. The porcine VTEC strains isolated in our study probably do not present a hazard.
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