Antimicrobial activity and inhibition of aflatoxin B1 formation by olive plant tissue constituents

Ethanolic extracts of olive callus tissues, added at 0.5 or 1.0% to media on which Aspergillus flavus was grown, inhibited aflatoxin production by 90% without inhibiting the fungal growth. The extract was found to contain mainly caffeic acid and, to a lesser extent, catechin and coumarins. The fungicidal and bactericidal activity of caffeic acid, catechin, coumarin and p-, o- or m-coumaric acid were tested and only caffeic acid and o-coumaric acid inhibited aflatoxin production. The inhibitory effect had no correlation with the growth of the fungus. Only coumarin at 10 mmol/1 totally inhibited fungal growth. Of the phenolic constituents of callus tissues tested, catechin and caffeic acid (10 mmol/l) showed bactericidal activity towards Pseudomonas aeruginosa and Staphylococcus aureus.     

Destruction of phagocytosis-suppressing activity of aflatoxin B1 by ozone

The impact of ozone on the immunity-impairing activity of aflatoxin B₁ (AFB₁) was studied. Phagocytosis by rat peritoneal macrophages, which was found to be suppressed in the presence of AFB₁, remained unimpaired when the applied AFB₁ was pretreated with ozone (1.2 mg 1^-1) for 6 min at a flow rate of 40 ml min^-1. Hence, application of ozone on AFB₁-contaminated foodcrops seems to be a promising preventive measure against any adverse immunological disorder in consumers.     

B1 and B2 Bradykinin Receptors Encoded by Distinct mRNAs

Abstract: Bradykinin receptors have been subdivided into at least two major pharmacological subtypes, B₁ and B₂. The cDNAs encoding functional B₂ receptors have recently been cloned, but no molecular information exists at present on the B₁ receptor. In this article, we describe experiments examining the possible relationship between the mRNAs encoding the B₁ and B₂ types of receptor. We showed previously that the Human fibroblast cell line W138 expresses both B₁ and B₂ receptors. In this report, we describe oocyte expression experiments showing that the B₁ receptor in W138 human fibroblast cells is encoded by a distinct mRNA ∼2 kb shorter than that encoding the B₂ receptor. We have used an antisense approach in conjunction with the oocyte expression system to demonstrate that the two messages differ in sequence at several locations throughout the length of the B₂ sequence. Taken together with the mixed pharmacology exhibited in some expression systems by the cloned mouse receptor, the data indicate that B₁-type pharmacology may arise from two independent molecular mechanisms.     

Key role of teichoic acids on aflatoxin B1 binding by probiotic bacteria

Aims:  To assess the ability of five probiotic bacteria to bind aflatoxin B₁ and to determine the key role of teichoic acids in the binding mechanism.
Methods and Results:  The strains were incubated in aqueous solutions containing aflatoxin B₁ (AFB₁). The amount of free toxin was quantified by HPLC. Stability of the bacteria–aflatoxin complex was evaluated by repeated washes with buffer. In order to understand the binding process, protoplasts, spheroplasts and cell wall components of two strains were analysed to assess their capacity to bind AFB₁. Additionally, the role of teichoic acids in the AFB₁ binding process was assessed. Lactobacillus reuteri strain NRRL14171 and Lactobacillus casei strain Shirota were the most efficient strains for binding AFB₁. The stability of the AFB₁–bacteria complex appears to be related to the binding ability of a particular strain; AFB₁ binding was also pH-dependent. Our results suggest that teichoic acids could be responsible for this ability.
Conclusions:  Our results provide information concerning AFB₁ binding by previously untested strains, leading to enhanced understanding of the mechanism by which probiotic bacteria bind AFB₁.
Significance and Impact of the Study:  Our results support the suggestion that some probiotic bacteria could prevent absorption of aflatoxin from the gastrointestinal tract.     

Functional impairment of rat Kupffer cells induced by aflatoxin B1 and its metabolites

Abstract Contamination of food with mycotoxins is a major health problem. Impairment of several immune functions has been repeatedly reported in animals fed with contaminated fodder. Since the liver is a major target of toxicity by aflatoxins, the effects of aflatoxins B1, and its hepatic metabolites Q₁ and M₁ on Kupffer cell function was investigated in vitro. Aflatoxin B₁ induced significant ( P < 0.05) inhibition of phagocytosis, intracellular killing of Candida albicans , and intrinsic anti-Herpes virus activity at concentrations as low as 0.01 pg ml⁻¹. Aflatoxin Q₁ and M₁ had similar effects on phagocytosis and microbicidal activity, but were two- to ten-fold less potent than aflatoxin B₁.     

A monoclonal antibody to aflatoxin B1: detection of the mycotoxin by enzyme immunoassay

A monoclonal antibody (McAb) was produced after fusion of mouse (X63.Ag8.6.5.3) myeloma cells with spleen cells isolated from female Balb-c/NZB F1 hybrid mice immunized with aflatoxin B₁ (oxine)-keyhole limpet haemocyanin conjugate. The hybridoma cell line producing antibody specific for aflatoxin B₁ (AFB₁) was grown in tissue culture and as an ascites tumour. The ascitic fluid gave suitably high dilution titres (1:800 000) by enzyme immunoassay and was conjugated to horseradish peroxidase by a two-step procedure with glutaraldehyde. The conjugate was used to develop a direct competitive enzyme-linked immunosorbent (ELISA) assay for AFB₁. The sensitivity of the ELISA was 0–2 ng/ml with a working range up to 10 ng/ml for AFB₁. The specificity of the McAb was determined and it was shown not to cross-react significantly with any of the metabolites tested. This McAb and the direct competitive ELISA described may prove of use in the detection of AFB₁ in foods and feeds.     

Effect of water activity and temperature on growth and fumonisin B1 and B2 production by Fusarium proliferatum and F. moniliforme on maize grain

S. MARIN, V. SANCHIS, I. VINAS, R. CANELA AND N. MAGAN. 1995. The effect of different water activities ( a _w, 0.968, 0.956, 0.944, 0.925) and temperature (25°C and 30°C) on colonization and production of fumonisin B₁ (FB₁) and B₂ (FB₂) on sterile layers of maize by Fusarium proliferatum and F. moniliforme isolates was determined over periods of 6 weeks. Generally, both F. moniliforme and F. proliferatum grew faster with increasing a _w and best at 30°C. All three isolates produced more FB 1 than FB₂ regardless of a _w or temperature. Very little FB₁ and FB₂ were produced at 0.925 a _w, with maximum produced at 0.956 and 0.968 a _w at both temperatures tested. Most FB₁ and FB₂ were produced by F. moniliforme (25N), followed by F. proliferatum isolates (73N and 131N). At all a _w levels and both temperatures there was an increase in FB₁ and FB₂ concentration with time. Statistical analyses of a _w, temperature, time, two- and three-way interactions showed some significant differences between isolates and FB₁ and FB₂ production.     

Cell wall component and mycotoxin moieties involved in the binding of fumonisin B1 and B2 by lactic acid bacteria

Aims:  The ability of lactic acid bacteria (LAB) to bind fumonisins B₁ and B₂ (FB₁, FB₂) in fermented foods and feeds and in the gastrointestinal tract could contribute to decrease their bioavailability and toxic effects on farm animals and humans. The aim of this work was to identify the bacterial cell wall component(s) and the functional group(s) of FB involved in the LAB–FB interaction.
Methods and Results:  The effect of physicochemical, enzymatic and genetic treatments of bacteria and the removal/inactivation of the functional groups of FB on toxin binding were evaluated. Treatments affecting the bacterial wall polysaccharides, lipids and proteins increased binding, while those degrading peptidoglycan (PG) partially decreased it. In addition, purified PG from Gram-positive bacteria bound FB in a manner analogue to that of intact LAB. For FB, tricarballylic acid (TCA) chains play a significant role in binding as hydrolysed FB had less affinity for LAB.
Conclusions:  Peptidoglycan and TCA are important components of LAB and FB, respectively, involved in the binding interaction.
Significance and Impact of the Study:  Lactic acid bacteria binding efficiency seems related to the peptide moiety structure of the PG. This information can be used to select probiotics with increased FB binding efficiency.     

Vitamin B12 Production by the Gastro-intestinal Microflora of Baboons Fed either a Vitamin B12 Deficient Diet or a Diet Supplemented with Vitamin B12

The vitamin B₁₂-producing capacity of micro-organisms isolated from baboon faeces and gastric contents was measured using Lactobacillus leichmannii . The animals were fed either a diet deficient in or supplemented with vitamin B₁₂ (controls). Samples of gastric and small intestinal contents, obtained at laparotomy from two young vitamin B₁₂-deprived baboons, contained varying quantities of vitamin B₁₂. Many of the organisms isolated from these aspirates produced vitamin B₁₂ in vitro . The highest levels of vitamin B₁₂ were produced by anaerobic organisms. Gastric juice samples from vitamin B₁₂ deprived and control baboons contained similar types of organisms with like vitamin B₁₂-producing capacity. The vitamin B₁₂ content, pH and total bacterial counts of gastric juice samples aspirated after a 6 h fast from vitamin B₁₂ deprived baboons were not significantly different from those of the control animals. The pH values of gastric juice samples aspirated 18 h after feeding, however, were significantly lower than those of 6 h fasting samples in both groups. The mean vitamin B₁₂ levels in the total volumes of gastric juice aspirated after each fasting period were similar. The possible involvement of the gastrointestinal flora in the vitamin B₁₂ status of the baboon is discussed.     

Microbial load in poultry feed and detection of aflatoxin B1 using monoclonal antibody-based enzyme linked immunosorbent assay

Feed samples collected from different poultry farms and feed mills situated in Andaman and Nicobar islands in India were assessed for microflora and aflatoxin B₁ contamination. The bacterial counts ranged from 1.0 times 10⁷ to 8.8 times 10⁷ cfu/g of the feeds, while counts of fungi ranged from 1.0 times 10³ to 8.7 times 10³ cfu/g. The mycoflora comprised mainly of Aspergillus spp., A. flavus being most dominant. Aflatoxin B₁ was detected by monoclonal antibody-based enzyme linked immunosorbent assay technique and the content in different feed samples ranged from 5.5 to 90 ng/g.     

Biosynthesis of vitamin B2 in plants

The biosynthesis of one riboflavin (vitamin B₂) molecule requires one molecule of GTP and two molecules of ribulose 5-phosphate. The imidazole ring of GTP is hydrolytically opened, yielding a 2,5-diaminopyrimidine that is converted to 5-amino-6-ribitylamino-2,4(1 H ,3 H )-pyrimidinedione by a sequence of deamination, side chain reduction and dephosphorylation. Condensation of 5-amino-6-ribitylamino-2,4(1 H ,3 H )-pyrimidinedione with 3,4-dihydroxy-2-butanone 4-phosphate obtained from ribulose 5-phosphate yields 6,7-dimethyl-8-ribityllumazine. Dismutation of the lumazine derivative yields riboflavin and 5-amino-6-ribitylamino-2,4(1 H ,3 H )-pyrimidinedione, which is recycled in the biosynthetic pathway. Characteristic architectural features of most enzymes involved in the plant riboflavin pathway resemble those of eubacteria, whereas the similarities between plants and yeasts are less pronounced. Moreover, riboflavin biosynthesis in plants proceeds by the same reaction steps as in eubacteria, whereas fungi use a somewhat different pathway.     

Marine Brown Algae Requiring Vitamin B12

The brown algae Lithosiphon pusillus, Ectocarpus fasciculatus and Pylaiella litoralis were cultivated in bacteria-free cultures in artificial sea water, ASP 6F. The growth was tested with different additions of vitamins and other metabolites. Lithosiphon pusillus and Ectocarpus fasciculatus were found to require vitamin B₁₂ for optimal growth. The zoospores of Pylaiella litoralis, when cultured in medium Asp 6F with kinetin added, had an absolute requirement for vitamin B₁₂.     

Effect of early aflatoxin B1 exposure on in vivo and in vitro antibody responses in rainbow trout, Salmo gairdneri

Exposure of rainbow trout, Salmo gairdneri , to the carcinogenic mycotoxin, aflatoxin B₁, resulted in a loss of B cell memory, but produced no change in the primary B cell antibody response. This effect was demonstrated both in vivo , in the generation of serum antibodies, and in vitro , in the generation of antibody-producing cells.     

Factors Mediating the Vitamin B12 Requirement of Euglena

SYNOPSIS. Deprived of vitamin B₁₂, Euglena gracilis strain Z ceases to divide which we believe to be a function of the light regime: division inhibition occurs more quickly in continuous light than in alternating (6L : 6D) light and not at all in total darkness. This phenomenon is dependent on the carbon source; cells grown in glutamate-malate medium do not divide regardless of the culture conditions while dl -lactate as carbon source permits growth in darkness in the absence of B₁₂. Conditions which lead to an increased O₂ or decreased CO₂ tension in the medium, such as agitation in darkness or incubation in red or white light, result in inhibition of division. This inhibition can be reversed by re-transferring the cells to still culture in the dark or, in the case of light-induced blockage, by the addition of DCMU.     

K+ATP channel opening prevents succinate-dependent H2O2 generation by plant mitochondria

The role of a recently identified K⁺_ATP channel in preventing H₂O₂ formation was examined in isolated pea stem mitochondria. The succinate-dependent H₂O₂ formation was progressively inhibited, when mitochondria were resuspended in media containing increasing concentration of KCl (from 0.05 to 0.15  M ). This inhibition was linked to a partial dissipation of the transmembrane electrical potential (ΔΨ) induced by KCl. Conversely, the malate plus glutamate-dependent H₂O₂ formation was not influenced. The succinate-sustained H₂O₂ generation was also unaffected by nigericin (a H⁺/K⁺ exchanger), but completely prevented by valinomycin (a K⁺ ionophore). In addition, cyclosporin A (a K⁺_ATP channel opener) inhibited this H₂O₂ formation, while ATP (an inhibitor of the channel opening) slightly increased it. The inhibitory effect of ATP was strongly stimulated in the presence of atractylate (an inhibitor of the adenine nucleotide translocase), thus suggesting that the receptor for ATP on the K⁺ channel faces the intermembrane space. Finally, the succinate-dependent H₂O₂ formation was partially prevented by phenylarsine oxide (a thiol oxidant).     

SUBCELLULAR DISTRIBUTION OF B6 VITAMERS IN CEREBRAL CORTEX

Abstract— The distribution of B₆ vitamers in subcellular fractions of cerebral cortex was examined. No pyridoxine and only traces of pyridoxamine could be detected in cerebral cortex. Significant quantities of pyridoxal were found in the cytoplasmic fraction. No vitamin B₆ was detected in the subcellular fractions carrying microsomes, myelin, vesicles, and small membranes settling above the 1-0 M-sucrose gradient. Pyridoxamine phosphate was the predominant form of vitamin B₆ in cerebral cortex of mature rats and guinea pigs, being present in almost twice the concentration of pyridoxal phosphate. More of the latter compound was present in the cytoplasm than in the mitochondria. In contrast, pyridoxamine phosphate was compartmentalized in extraterminal and intraterminal mitochondria. Of especial interest was the finding that there was a significant amount of pyridoxamine phosphate attached to the presynaptic membrane and a small but detectable amount in the fraction containing postsynaptic membranes.     

Contamination of Indian maize with fumonisin B1 and its effects on chicken macrophage

The natural occurrence of fumonisin B₁ (FB₁) in Indian maize and its impact on the viability and phagocytosis of chicken peritoneal macrophages in vitro were investigated. FB₁ was found to be present at the levels of 300–366 ppm in Fusarium moniliforme -infected maize grains. The infected kernels in a population of 100 ears showed normal (Gaussian) frequency distribution. FB₁, extracted from the contaminated kernels, reduced the viability and phagocytic activity of macrophages. These findings imply that FB₁ exposure may become a potential problem in India and may lead to decreased immune responses with consequent susceptibility of a host to infection.     

Effects of Perinatal Vitamin B6 Deficiency on Dopaminergic Neurochemistry

Long-Evans dams were fed either a vitamin B6-deficient or a control diet from day 13-14 of gestation and throughout lactation. A control pair-fed group was also included because of differences in food intake between vitamin B6-deficient and control ad libitum dams. The progeny of vitamin B6-deficient dams had all the classic symptoms of B6 deficiency. These included weight loss, ataxia, tremor, and epileptic seizures. Concentrations of the neurotransmitter dopamine (DA), and its metabolites 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), as well as D-2 dopamine receptor binding, 3,4-dihydroxyphenylalanine (DOPA) decarboxylase activity, and vitamin B6 levels were measured in the corpus striatum of progeny at 7, 14, and 18 days after birth. Striatal DA and HVA levels were significantly decreased in B6-deficient animals when compared to ad libitum or pair-fed controls. Daily injections of vitamin B6 to deprived animals from the 14th to 18th day after birth improved the abnormal movement and normalized the concentration of DA but not of HVA in corpus striatum. Striatal D-2 dopamine receptor binding using [3H]spiperone as ligand was significantly reduced in 18-day-old animals as compared to ad libitum and pair-fed controls. No significant differences were found at 14 days. The administration of vitamin B6 to deprived animals did not raise the level of D-2 receptor binding during the period of observation. Scatchard plots indicated that the differences in binding were due to changes in receptor number and not in KD. Corpus striatum DOPA decarboxylase activity with and without the addition of exogenous pyridoxal phosphate was significantly reduced in 14- and 18-day-old animals when compared to pair-fed controls.(ABSTRACT TRUNCATED AT 250 WORDS)