Application of an enzyme-linked immunosorbent assay for screening of T-2 toxin-producing Fusarium spp.

Culture filtrates of Fusarium species were subjected without clean-up procedures to an indirect competitive enzyme-linked immunosorbent assay with anti-T-2 toxin monoclonal antibody. Fusarium sporotrichioides, F. poae, F. tricinctum, and F. culmorum strains were positive for T-2 toxin, with a minimum detection limit of 5 pg per assay (100 pg/ml of culture filtrate), and the assay data correlated well with the gas-liquid chromatographic data.     

A male-specific renal cytochrome P-450 isozyme(s) responsible for mutagenic activation of 3-methoxy-4-aminoazobenzene in mice

Renal microsomes from male mice (BALB/c, DBA/2 and BALB/c x DBA/2 F1) showed about 10-fold greater activity for mediating mutagenic activation of 3-methoxy-4-aminoazobenzene (3-MeO-AAB) toward Salmonella typhimurium TA98 than did the corresponding hepatic microsomes, as compared on the basis of nmol of microsomal cytochrome P-450. On the other hand, female renal microsomes and other extrahepatic microsomes (lung, small intestine and colon) in both sexes of mice showed little or no activity for converting 3-MeO-AAB to mutagen(s). The mutagenic activation of 3-MeO-AAB with the male renal enzyme(s) was definitely inhibited by cytochrome P-450 inhibitors, 7,8-benzoflavone and SKF 525A. All these findings suggest that in mice, there is a male-specific renal 3-MeO-AAB activation enzyme(s), a cytochrome P-450 isozyme(s), which is different, at least in proportion and/or in nature, from hepatic cytochrome P-450 isozymes.     

Mycological survey of Korean cereals and production of mycotoxins by Fusarium isolates.

The fungal species isolated from Korean cereals (barley, polished barley, wheat, rye, and malt) were Alternaria spp., Aspergillus spp., Chaetomium spp., Drechslera spp., Epicoccum sp., Fusarium spp., and Penicillium spp., etc. The number of Fusarium strains isolated was 36, and their ability to produce Fusarium mycotoxins on rice was tested. Nivalenol (NIV) was produced by Fusarium graminearum (7 of 9 isolates), Fusarium oxysporum (3 of 10 isolates), and Fusarium spp. (7 of 15 isolates). Of 15 isolates of Fusarium spp., 6 formed deoxynivalenol (DON). Fusarenon-X and 3-acetyl-DON were produced by most NIV- and DON-forming isolates, respectively. Zearalenone was produced by 3 isolates of F. graminearum, 1 isolate of Fusarium equiseti, and 11 isolates of Fusarium spp. T-2 toxin was not produced by any Fusarium isolates. The highest concentrations of mycotoxins produced by Fusarium isolates were 77.4 (NIV), 5.3 (DON), 138.3 (fusarenon-X), 40.6 (3-acetyl-DON), and 23.2 (zearalenone) micrograms/g.     

Isolation, and catalytic and immunochemical properties of cathepsin D-like acid proteinase from rat erythrocytes

An erythrocyte membrane-associated cathepsin D-like acid proteinase, termed "EMAP," was purified to homogeneity from freshly collected rat blood in a yield of 60-65%. The molecular weight of the enzyme was determined to be 80,000-82,000 by Sephadex G-100 gel filtration. The enzyme was inhibited strongly by pepstatin and partially by HgCl2, Pb(NO3)2, and iodoacetic acid. The preferred substrate for the enzyme was hemoglobin. The enzyme also hydrolyzed serum albumin and casein, but to lesser extents, with an optimum pH of 3.5-4.0. However, it could not hydrolyze leucyl-2-naphthylamide, benzyloxycarbonyl-Phe-Arg-4-methyl-7-coumarylamide or other synthetic substrates at pH values ranging from 3.5 to 9.5. The enzyme was very similar to human EMAP in a number of enzymatic properties, whereas it differed from rat cathepsin D in several respects, such as pH stability, molecular weight, isoelectric point, and chromatographic properties. Immunologically, the enzyme cross-reacted with the rabbit antibody prepared against human EMAP. The patterns of immunoelectrophoresis, immunoblotting, and immunoprecipitation of the enzyme were remarkably similar, if not identical, to those of human EMAP. In contrast, rat EMAP showed no reaction with the rabbit antibody raised to rat spleen cathepsin D. These results indicate that EMAP is a unique cathepsin D-like acid proteinase different from ordinary cathepsin D.     

Selectivity and contribution of lecithin: cholesterol acyltransferase to plasma cholesterol ester formation

Selectivity factors (Vm/Km) for human and rat lecithin: cholesterol acyltransferases (LCAT) for the transfer of various acyl groups from the 2-position of phosphatidylcholine were determined. By multiplying these values by the proportions of acyl groups at the 2-position of phosphatidylcholine, one can predict the proportions of molecular species of cholesterol ester which will be synthesized by LCAT. In human subjects fasted overnight, the molecular composition of plasma cholesterol ester was found to reflect the LCAT selectivity relatively accurately. This result supports the concepts that hepatic acyl-CoA:cholesterol acyltransferase (ACAT) does not contribute significantly to the synthesis of plasma cholesterol ester and that removal of cholesterol ester from plasma is not selective with respect to molecular species under these conditions. In contrast to the results with humans, the molecular composition of plasma cholesterol ester formed in spontaneously hypertensive rats fed a high-cholesterol diet and then fasted overnight differs from that which is predicted from LCAT selectivity and the proportion of various fatty acids at the 2-position of phosphatidylcholine: these results suggest that cholesterol ester is formed mainly via the ACAT reaction.     

Release of endogenous prostaglandins by mild hyperosmotic saline inhibits tetragastrin-stimulated gastric acid secretion in rats

Filling of the gastric lumen of rats with 1.0 M NaCl solution (5 ml) for 10 min under urethane anesthesia caused an increase in the gastric fluid concentrations of prostaglandin (PG) E₂, 13, 14-dihydro-15-keto-PGE₂ and 6-keto-PGF_1α as determined by radioimmunoassay. PGE₂ was the major PG generated. The levels of PGE₂ in the gastric fluid were increased dose-dependently after filling the lumen with 0.3, 0.5, 0.7 or 1.0 M NaCl solutions. The pH of the gastric fluid increased similarly after 0.5 to 1.0 M NaCl solutions. Indomethacin (10 mg/kg, i.p.) suppressed the PGE₂ increase caused by 1.0 M NaCl solution, but did not prevent the increase of the pH of the gastric fluid induced by intragastric 1.0 M NaCl. Infusion of tetragastrin (62.5 μg/kg/hr, i.v., for 10 min) caused a marked increase of acid secretion without modifying intragastic concentration of PGE₂. The acid secretion due to tetragastrin was completely inhibited after intragastric administration of 1.0 M NaCl solution, while indomethacin restored the tetragastrin-induced acid secretion, with prevention of a rise of intragastric PGE₂ levels. These observations suggest that 1.0 M NaCl solutions suppress basal intragastric acid through a mechanism which is independent of prostaglandins. In contrast, the suppression of tetragastrin-induced acid secretion by intragastric 1.0 M NaCl solution appears to be mediated through a release of prostaglandins     

Differential effects of recombinant human interferon-gamma and interleukin 2 on natural killer cell activity of peripheral blood in early human development

Ontogenic development and the lymphokine responsiveness of human NK cell activity against K562 target cells in peripheral blood lymphocytes were evaluated in fetuses, premature infants, and term neonates by using a 4-hr 51Cr-release assay. Basal NK activity and NK boosting by lymphokines were comparatively assayed after an 18-hr incubation with medium alone, recombinant human IFN-gamma (1000 U/ml), and recombinant human IL 2 (25 U/ml), respectively. Lymphocytes from 20-wk-old fetuses lacked NK cell activity even after the pretreatment with IFN-gamma. Low, but significant levels of NK activity and NK boosting by IFN-gamma were observed in premature infants after 27 wk of gestation, with a progressive intrauterine maturation of these activities. Both basal NK activity and NK boosting by IFN-gamma in term neonates were still lower than those of adult controls. The grade of NK boosting by IFN-gamma appeared to depend on the development of basal NK activity. Contrary to IFN-gamma, IL 2 could induce marked NK activity even in 20-wk-old fetuses who lacked both basal and IFN-gamma inducible NK activities. NK boosting by IL 2 was much more efficient than that by IFN-gamma at any period of human life. The facts that IL 2-induced NK boosting could occur without any appreciable production of IFN-gamma in neonatal lymphocytes, and that ample neutralizing doses of anti-IFN-gamma antibody hardly suppressed IL 2-mediated NK boosting even in adult lymphocytes, indicated that the effect of IL 2 on NK boosting might be independent of IFN-gamma production. On the basis of the ontogenic differences in the development of the lymphokine responsiveness of NK cell activity and on the different NK boosting mechanisms of these lymphokines it was suggested that so-called human "pre-NK cells" might be divided into IFN-gamma sensitive and IL 2-sensitive cells. Whether these cell populations belong to different cell lineages or different maturation stages of the same cell line, however, remains unsettled.     

Circadian rhythms of urinary excretions of water and electrolytes in patients receiving total parenteral nutrition (TPN)

In order to determine whether the usual feeding pattern actually modifies the circadian rhythms of urinary excretion of water and electrolytes, we compared the circadian rhythm characteristics in patients receiving total parenteral nutrition (TPN group) with those in patients on an ordinary hospital diet (control group). Statistically significant circadian rhythms were detected in all of the urinary variables investigated herein by using the population mean-cosinor method in both groups. In addition, there were no statistically significant differences of the mesor, the %-amplitude and the acrophase between the two groups. These results suggest that the usual feeding pattern is not a main determinant in forming the circadian rhythm characteristics of human urinary variables.     

Involvement of C-terminal 14 residues of alkali light chain in binding to the heavy chain of myosin

The alkali light chain of rabbit skeletal muscle myosin, A1, was cyanylated with 2-nitro-5-thiocyanobenzoic acid, and the peptide bond at Cys 177 was subsequently cleaved in the presence of 0.05 M CaCl2. Two peptide fragments, from the N-terminal to the residue 176 (CF1) and from the residue 177 to the C-terminal (CF2), were obtained. The CD spectrum and the difference UV absorption spectrum induced by CaCl2 suggested that CF1 largely retained the higher order structure of A1. The CF1 fragment, however, could neither incorporate subfragment-1 (S-1) by an exchange reaction, nor bind with the renatured 20K fragment of S-1 heavy chain. On the other hand, the C-terminal fragment of 14 residues, CF2, could bind with the 20K fragment of S-1 heavy chain. These results indicate that the binding site of the alkali light chain for the heavy chain of myosin is located within the C-terminal 14 residues.