Production and characterization of antibodies cross-reactive with major aflatoxins

Antibodies cross-reactive with 4 major aflatoxins were demonstrated three weeks after immunization of rabbits with an immunogen which was prepared by conjugating aflatoxin B3 to bovine serum albumin. Aflatoxin B3 was first converted to its hemisuccinate before conjugation to the protein. Tritiated aflatoxin B1 (AFB1) was used as the marker ligand both for antibody titer determination as well as for analysis of antibody specificity. Competitive RIA revealed that the antibodies have good cross-reactivity with aflatoxins B1, B2, G1, and G2 when tritiated AFB1 was used as the marker ligand. The concentrations causing 50% inhibition of binding of 3H-AFB1 to the antibodies by unlabeled aflatoxins B1, B2, G1, G2 and B3 were found to be 0.25, 3.34, 0.32, 4.0 and 0.53 ng/assay, respectively. The antibodies could be used for simultaneous analysis of aflatoxins B1 and G1, two of the most important toxic metabolites produced by Aspergillus flavus and A. parasiticus.     

Production and characterization of antibody against aflatoxin M1

Summary Antibody against aflatoxin M₁ was obtained after immunization of rabbits with bovine serum albumin-afla M₁ oxime conjugate. The antibody has greatest binding efficiency for afla M₁, and was less efficient for afla B₁. Cross-reaction of antibody with aflatoxin Q₁, aflatoxicol, and aflatoxin B_2a was weak. Aflatoxin B₂, G₁, and G₂ and afla B₁-guanine adduct showed almost no cross-reaction with the antibody. The sensitivity of the binding assay for aflatoxin M₁ detection is in the range of 1–10 ng per assay. Detailed methods for the preparation of the conjugate, production of immune serum, and methods for antibody determination are described.Supported by the College of Agricultural and Life Sciences, North Central Regional project NC-129, the University of Wisconsin-Madison, and by Public Health Service research grant number CA 15064 from the National Cancer Institute, NIH.The authors wish to thank Dr R.C. Garner for providing aflatoxin-B-guanine adduct, and Dr Dennis H. Hseih for providing aflatoxicol and aflatoxin Q₁.     

The use of fungal protoplasts in the study of aflatoxin biosynthesis

Summary Protoplasts derived fromAspergillus flavus are shown to be capable of synthesizing aflatoxins when incubated in a chemically defined medium.¹⁴C-Acetate and¹⁴C-Versicolorin A, added to protoplasts from 3-day-old mycelium, are incorporated into aflatoxin B₁.     

Covalent binding of aflatoxin B1 to liver DNA in rats pretreated with ethanol

Summary Male Fischer F-344 rats were given ethanol in the drinking water and/or by single oral administration. Following this, the animals received p.o. 100 ng/kg of the hepatocarcinogen [³H]aflatoxin B₁ (AFB₁). 24 h later, the level of DNA-bound AFB₁ was determined in the liver and was found not to be affected by any type of ethanol pretreatment. A cocarcinogenic effect of ethanol in the liver is therefore unlikely to be due to an effect on the metabolic activation and inactivation processes governing the formation of DNA-binding AFB₁ metabolites.To whom correspondence should be addressed.Acknowledgment. We thank the European Science Foundation for the Toxicology Research Fellowship awarded to M.M.     

Aflatoxin metabolism and absence of cytochrome P-450 in rat colon tissue during vitamin A malnutrition

Summary Homogenized mucosal linings prepared from vitamin A adequate and deficient male rats were used in metabolic studies of aflatoxin B₁ (AFB₁). Cytochrome P-420 was identified in both groups which metabolized AFB₁ to 4 metabolic products in vitro. The implications of this observation are discussed in relation to colon carcinoma.Acknowledgments. This work was supported by USPHS (NIEHS), grant R01 ES 00336 and R01 CA 00270. Hoffman-La Roche Foundation Research Corporation awarded to T. C. C., Research Career Development Awardee of the NIEHS. Present address: Division of Nutritional Sciences, Cornell University, Ithaca, New York, USA.     

Monoclonal antibodies to L-asparaginase

Summary Five hybridomas secreting monoclonal antibody toE. coli L-asparaginase were isolated. These monoclonal antibodies were classified into 3 different subclasses; Ig G₁ (1 clone), Ig G₂ (2 clones) and Ig G₃ (2 clones). One of them possessed anti-L-asparaginase neutralizing activity. Four antibodies examined demonstrated a linear Langmuir binding plot and binding affinities, with equilibrium dissociation constant (K_d) ranging between 2.5×10^–9M and 6.3×10^–10 M. The monoclonal antibodies should be useful probes for investigation of the enzyme activity.     

Chromosomenaberrationen in den Wurzelspitzen vonAllium cepa durch Aflatoxin B1

Summary After treatment of roots ofAllium cepa with aflatoxin B₁ in 0.3% dimethylformamide, chromosome bridges, C-mitose chromosomes and a reduction of the mitotic index were observed. The aberrations occurred especially frequently when the roots had grown in 200 µg/ml toxin for 48 h. In its cytotoxic effect onAllium cepa root tips, aflatoxin B₁ acts similarly to the chemically related coumarin.     

The use of cell free extracts derived from fungal protoplasts in the study of aflatoxin biosynthesis

Summary A supernatant fraction derived from protoplasts ofAspergillus flavus was shown to be capable of converting both sterigmatocystin and versiconal hemiacetal acetate to aflatoxin B₁. Versicolorin A was not converted under the same conditions.     

Reduction of aflatoxin B1 with zinc borohydride: An efficient preparation of aflatoxicol

Summary A simple and mild reduction of aflatoxin B₁, involving treatment of aflatoxin B₁ with ethereal zinc borohydride to give 57–65% yield of diastereomeric aflatoxicols, is described.This work was supported by the College of Agricultural and Life Sciences, the University of Wisconsin, Madison, and by Public Health Service Research, grant No. CA 15064, from the National Cancer Institute, and by funds from contributions of food industries to the Food Research Institute. We are grateful to Professor Dennis Hsieh for a gift of natural aflatoxicol, to Mr William Harder for technical assistance throughout this investigation, and to Mr Gary Girdaukas for taking mass spectra.     

Über den Vitamin-B12-Gehalt von Organen verschieden ernährter Ratten

Summary Adult, male rats bred for over 10 generations on a soy meal-corn-diet had vitamin B₁₂ values of liver and kidney about 10 times lower than the controls. If the deficient ration was supplemented with 5µg/kg of B₁₂, these values were still about 1/5 of the controls. 3 weeks on the deficient diet lowered the B₁₂ levels in the livers and kidneys of previously undepleted rats to about 1/2, and a similar diet containing 0.1% of iodized casein lowered these levels to about 1/3 of the normal values but did not lower the B₁₂-concentration of organs of already deficient rats.Rats bred on the deficient diet and receiving for 1 month a supplement of 30µg/kg of vitamin B₁₂ or the stock diet with a similar B₁₂-content, had normal B₁₂-levels in livers and kidneys.     

Insulin activates Gαil,2 protein in rat hepatoma (HTC) cell membranes

Insulin action is initiated by binding to its cognate receptor, which then triggers multiple cellular responses by activating different signaling pathways. There is evidence that insulin receptor signaling may involve G protein activation in different target cells. We have studied the activation of G proteins in rat hepatoma (HTC) cells. We found that insulin stimulated binding of guanosine 5′-O-(3-thiotriphosphate) (GTP-γ-³⁵S) to plasma membrane proteins of HTC cells, in a dose-dependent manner. This effect was completely blocked by pertussis toxin treatment of the membranes, suggesting the involvement of G proteins of the Gα _i/Gα _o family. The expression of these Gα proteins was checked by Western blotting. Next, we used blocking antibodies to sort out the specific Gα protein activated by insulin stimulation. Anti-Gα _il,2 antibodies completely prevented insulin-stimulated GTP binding, whereas anti-Gα _o,i3 did not modify this effect of insulin on GTP binding. Moreover, we found physical association of the insulin receptor with Gα _i1,2 by copurification studies. These results further support the involvement of a pertussis toxin-sensitive G protein in insulin receptor signaling and provides some evidence of specific association and activation of Gα _i1,2 protein by insulin. These findings suggest that Gα _i1,2 proteins might be involved in insulin action. Received 23 September 1998; received after revision 23 November 1998; accepted 25 November 1998     

Role of lactone ring of aflatoxin B1 in toxicity and mutagenicity

Summary Cleavage of the lactone ring of aflatoxin B₁ results in a nonfluorescent compound that has greatly reduced biological activity. Mutagenicity, as measured by the Ames test, is reduced 450-fold compared to that of B₁, and toxicity, as measured by the chick embryo test, is reduced 18-fold.     

Binding of aflatoxins B1 and G1 to human serum proteins

Zusammenfassung In vitro Experimente mit¹⁴C-markiertem, gereinigtem Aflatoxin zur Untersuchung der Bindung von Aflatoxin B₁ und G₁ an verschiedene Serumproteine ergaben, dass Aflatoxin B₁ hauptsächlich mit-Globulin, G₁ dagegen vorwiegend mit Albumin bindet.

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This work was supported by part by a grant from the China Medical Board of New York, Inc., and was performed during one of us (S.S.L.) received a class C. research award from the National Science Council, Republic of China.     

The cell cycle of an established line ofDrosophila melanogaster cells in vitro

Riassunto La durata media delle fasi del ciclo cellulare (G₁, S e G₂) e del tempo di generazione totale (G _T ) in una linea stabilizzata di cellule diDrosophila melanogaster risulta essere rispettivamente di 1.8, 10.0, 7.2 e 18.8 ore.     

The effect of dietary fat on the anticoagulant activity of aflatoxin B1

Summary A single i.p. dose of aflatoxin B₁ had no significant effect on the thrombotest clotting times of monkeys subsisting on low-fat and high-fat dietary regimens, respectively. There was a significant interaction between aflatoxin and dietary fat level.     

Activation of CD47 receptors causes histamine secretion from mast cells

Mast cells play pivotal roles in allergic and inflammatory processes via distinct activation pathways. Mucosal and serosal mast cells are activated by the IgE/FcɛRI pathway, while only serosal mast cells are activated by basic secretagogues. We show that CD47 receptors are expressed on rat peritoneal mast cells. 4N1K, a peptide agonist of CD47, rapidly caused exocytosis. Such exocytosis required increased intracellular calcium and was inhibited by pertussis toxin and an antibody against the βγ dimer of a G_i protein. Cooperation with integrins and glycosylphosphatidylinositol-anchored proteins was necessary, since anti-integrin antibodies and pretreatment with phosphatidylinositol-phospholipase C reduced exocytosis. Depletion of membrane cholesterol inhibited exocytosis and decreased CD47 in lipid rafts, consistent with a CD47/integrin/G_i protein complex being located in rafts. An anti-CD47 antibody inhibited exocytosis induced by 4N1K and by mastoparan and spermine, suggesting that basic secretagogues might target CD47. We propose that 4N1K-stimulated mast cell exocytosis involves a CD47/integrin/G_i protein complex. Received 8 December 2008; received after revision 12 January 2009; accepted 29 January 2009     

Interphase differential sensitivity to protein synthesis inhibition

Summary The effect of protein synthesis inhibition during interphase ofAllium cepa L. root meristem cells was studied. Anisomycin and cycloheximide were used in intermittent treatments during interphase of a synchronous cell subpopulation labelled as binucleate by caffeine, and the delays in reaching prophase were recorded. High sensitivity to protein synthesis inhibition was detected in G₁ and in the S/G₂ boundary, while protein synthesis in most of the S and G₂ phase was not required for the normal timing of next prophase.     

The structures of minor congeners of detoxin complex,the selective antagonist of blasticidin S1

Summary The structures of the minor congeners of detoxin complex, viz., detoxins E₁, C₁, C₂, C₃, B₁, B₃ and A₁ have been established on the basis of spectral and degradative evidence.Acknowledgment. This work was supported by a Grant-in-Aid for Special Project Research (510208) from the Ministry of Education, Science and Culture, Japan. We are grateful to Kaken Chemical Co. Ltd for the supply of the detoxin complex. This is Part V of studies of Detoxin Complex, the Selective Antagonists of Blasticidin S'. For Part IV, see Ogita et al.⁸.     

Augmentation of natural antiganglioside IgM antibodies in lower motor neuron disease (LMND) and role of CD5+ B cells

IgM antibodies directed against neuronal gangliosides GM₁ , GM₂ , GD_1a , GD_1b and GT_1b occur in normal individuals and their level significantly decreases with age. Patients with lower motor neuron disease (LMND) produce high levels of these autoantibodies. AntiGM₁ IgM is selectively augmented. In these patients, the CD5+ (B1) and CD5− (B2) subsets of B cells are not distinct entities but range from those without detectable CD5 marker to those with high CD5+ expression. B1 B cells were sorted to homogeneity, but B2 B cell cannot be isolated to homogeneity because of the presence of B1 cells with low CD5 expression. In short term cultures both the subsets produced IgM antibodies, but the antibodies reacted better with desialylated GM₁ than with GM₁ . Cycloheximide (Cx) (0.35 mM) largely blocked IgM synthesis of the B1 B cells but inhibition of the B2 B cells was incomplete, possibly due to shedding of cytophilic antibodies as well as to the presence of B1 phenotype with loss of CD5 expression. CD5+ B cells may be involved in the production of antiglycolipid IgM. Received 9 June 1997; received after revision 21 July 1997; accepted 28 July 1997     

Cell cycle determination of phytohemagglutinin-stimulated lymphocytes from the opossum,Didelphis virginiana

Résumé La courbe PLM (pourcentages indiqués de mitoses) pour les lymphocytes d'opossum in vitro a fait preuve d'unT _c (temps générateur) que n'atteignait pas la somme de G₂, S, et M (mitosis). Tandis que les calculations pour les phases de G₂ et S sont représentées avec exactitude sur la courbe, il y a des variations dans le G₁ parmi les population de lymphocytes au fonctionnement divers (au nombre de deux ou peut-être plus parmi cellesci) qui sont responsables de ce désaccord apparent.