Kinetic studies of Heck coupling reactions using palladacycle catalysts: experimental and kinetic modeling of the role of dimer species.

Experimental kinetic studies of the coupling of p-bromobenzaldehyde (1) with butyl acrylate (2) using the dimeric palladacycles complex (4) with chelating nitrogen ligands were carried out together with kinetic modeling using a reaction rate expression based on the mechanism shown in Scheme 2. The oxidative addition product of 1 was found to be the resting state within the catalytic cycle. The formation of dimeric Pd species external to the catalytic cycle helped to rationalize a non-first-order rate dependence on catalyst concentration. Theoretical modeling showed how the relative concentrations of the different intermediate species within the catalystic cycle can influence the observed rate dependence on Pd concentration. It was shown how conventional kinetic studies may give reaction orders in substrates which differ from those which would be observed under practical synthetic conditions. Comparison between phosphine- and nonphosphine-based palladacycles suggests that they follow the same reaction mechanism. The role of water in accelerating the initial formation of the active catalyst species is noted.

Evaluation of a fluorodensitometric method for analysis of ergosterol as a fungal marker in compound feeds.

Ergosterol is the principal sterol of fungi and plays an essential role as a component of the cell membrane and other cell constituents. This molecule is considered a good marker of fungal contamination in foods and feeds. This paper reports a rapid and sensitive method to test ergosterol content in compound feeds based on fluorodensitometry after thin-layer chromatography (TLC) separation. This method involves a thermal treatment of TLC plates that leads to the formation of a highly fluorescent ergosterol derivative. Such a dosage allows ergosterol testing in any naturally contaminated samples (limit of detection: 1 ppm of ergosterol) and gives results in close agreement with high-pressure liquid chromatography determination. Moreover, values obtained on mixed feeds for animals at different steps of fungal contamination are linked to quantitative development of storage fungi, evaluated by mycological technique, reinforcing the interest of a rapid method for measuring this fungal marker.

Correlation of desensitisation of platelet activating factor (PAF) receptors with intensity of inflammation and intestinal PAF content during experimental ileitis in guinea pig.

AIM: To determine the kinetics of platelet activating factor (PAF) and prostaglandin E2 (PGE2) receptor desensitisation during intestinal inflammation induced by trinitrobenzenesulphonic acid (TNB) instillation and to study the relation between receptor regulation, inflammatory lesions, and PAF content of the gut wall. METHODS: Receptor desensitisation was assessed on isolated smooth muscle cells from the circular layer. PAF content of the intestinal wall was determined by thin layer chromatography and radioimmunoassay. RESULTS: After an acute inflammatory phase on day 1, subacute changes appeared in TNB instilled ileum, with a maximal intensity on day 6. In control animals, PAF 10 nM and PGE2 10 nM provoked a maximal contraction in the range of 24% of cell shortening. On days 1 and 3 after intestinal instillation of TNB, PAF induced contraction was not altered whereas the effect of PGE2 was progressively desensitised (2 logM rightward shift of its concentration-response curve: Cmax = 1 microM; p < 0.01). Between days 4 and 6, the concentration-response curve of PGE2 shifted by only 1 logM (p < 0.05) whereas the curve of PAF induced contraction shifted by 2 logM (Cmax = 1 microM; p < 0.01). The PAF content of the ileal wall was maximal between days 3 and 5 (300 ng/mg tissue). On days 10 and 15, PAF and PGE2 induced contractions were similar to those observed on day 1, and PAF content returned to basal. CONCLUSION: Inflammation induced by TNB instillation triggers PAF and PGE2 receptor desensitisation; this is dependent on the duration of inflammation and correlates with PAF content in the ileum. This receptor desensitisation may play a protective role by preventing overstimulation of intestinal smooth muscle cells.

Recent acute and subacute mycotoxicoses recognized in France.

Successful investigation and prevention of mycotoxic problems requires close collaboration between scientists from several disciplines ranging from agronomists and technologists required during production of food and feeds, to toxicologists and pathologists examining the effects of mycotoxins on animals and man. Zootoxic metabolites following fungal infection result from four general mechanisms: (i) secondary fungal metabolism (mycotoxins, eg, aflatoxins); (ii) bioconversion of vegetal compounds (eg, dicoumarol); (iii) plant reactions (phytoalexins, eg, coumestrol); and (iv) plant-fungus associations (endophytes, eg, Acremonium/Festuca). In reported pathologic field cases, close cooperation through a selected veterinary network has allowed diagnosis of acute and subacute mycotoxicoses in France. Natural stachybotryotoxicosis may not be limited only to cold climates, but may also occur in mild and warm ones (eg, south west of France, Morocco). A considerable variation was observed in symptoms and lesions depending on toxin levels, ranging from a poor performance in a horse race to a general haemorrhagic syndrome. Several cases of acute equine leucoencephalomalacia, characterized by pathognomonic lesions and recently supported by fumonisin analysis, have been diagnosed in the southern part of France and other countries (eg, New Caledonia and the Ivory Coast). Facial eczema in sheep is endemic in the Basque country, as a result of specific bioclimatic and zootechnic conditions. Reproductive disorders in sheep, cattle, goats and rabbits have been associated with high levels of coumestrol in alfalfa, clover and their derivatives. A few cases of fescue foot disease, associated with the endophyte Acremonium, have been diagnosed recently. In addition, several nervous disorders may be due to unknown mycotoxins. These acute or subacute mycotoxicoses suggest a potentially widespread occurrence of low level toxins and insidious asymptomatic mycotoxicoses, and justify interdisciplinary research in order to improve diagnosis and preventative measures.

Characterization of monascidin A from Monascus as citrinin.

Following our investigations on red pigments and monascidin co-production by Monascus species, the antibiotic called monascidin A was characterized as citrinin. Evidence was given by qualitative methods, mass spectra and NMR. Citrinin, a nephrotoxic agent was produced both by Monascus purpureus and Monascus ruber, either in submerged culture of concentrations of 270 and 340 mg/l, respectively, or in solid state culture of concentration of 100 and 300 mg/kg dried matter, respectively. Since citrinin is a toxic product, it is essential that the production of red pigments as food additives from Monascus spp. avoid the occurrence of citrinin.

Thermostability of Ochratoxin A in wheat under two moisture conditions.

The decomposition of ochratoxin A (OTA) was examined, under different temperature and moisture conditions. The calculated half-lives, corresponding to 50% values, were 707, 201, 12, and 6 min, respectively, at 100, 150, 200, and 250 degrees C for dry wheat and 145, 60, and 19 min, respectively, at 100, 150, and 200 degrees C for wheat heated under wet conditions. The presence of water (50%) increased the decomposition of OTA at 100 and 150 degrees C; the opposite was observed at 200 degrees C. Complete destruction of OTA within the limits of this study (100 to 250 degrees C) was not obtained.

Time of Aspergillus flavus infection and aflatoxin formation in ripening of figs.

Figs in an orchard were inoculated with an aflatoxigenic Aspergillus flavus strain in two ways by spore injection or by dusting at three maturation stages: firm ripe, shrivelled, and dried. Fruits were individually examined for fungal development and analyzed for aflatoxin B1 (AF B1) after 2, 4, 6, 8 and 10 days. Fruit injected at the first stage showed fungal development and AF B1 contamination within two days. The toxin level increased sharply to 1 ppm after 10 days. The mean level of AF B1 (284.75 ng/g) was significantly higher than those observed in other conditions. Figs dusted at the first stage showed only a tiny fungal growth even after 10 days. AF B1 appeared after 6 days with a low frequency (35%), mean level (7.6 ng/g) and a great variation among figs (0.22-15 ng/g). Among fruits inoculated during the shrivelled fig and dried fruit stages, no fungal growth was observed and AF B1 was detected with a lower incidence in association with low mean levels (less than 1.25 ng/g). Methods of prevention of aflatoxin contamination at the critical step, the firm ripe stage, are discussed.

Thermostability of Fumonisin B(1), a Mycotoxin from Fusarium moniliforme, in Corn.

Fumonisin B(1) (FB(1)) is a mycotoxin from Fusarium moniliforme that is frequently associated with corn. Thermal treatments are used in many processes concerning this cereal and its derivatives. The thermostability of this toxin in dry contaminated corn, resulting from F. moniliforme culture, was studied in different time-temperature combinations. FB(1) was quantified by instrumentalized thin-layer chromatography after a two-step sequential development and postchromatographic derivatization by p-anisaldehyde. The identity of FB(1) in extracts, before and after heat treatments, was confirmed by high-pressure liquid chromatography. For each temperature, the natural logarithm of the ratio of resulting FB(1) on initial content (In C/C(0)) is linearly correlated to exposure time. The calculated half-lives (L(50)), corresponding to the 50% value, were 10 min, 38 min, 175 min, and 8 h at 150, 125, 100, and 75 degrees C, respectively. There is a linear relationship between calculated L(50)s on a logarithmic scale and temperature. Therefore FB(1) is not significantly destroyed by the main drying processes of corn or thermal treatments used for its derivatives. Other associated means are required for detoxification.

In vitro effect of diacetoxyscirpenol and deoxynivalenol on microbicidal activity of murine peritoneal macrophages.

Diacetoxyscirpenol and deoxynivalenol, two trichothecene mycotoxins shown previously to exert immunosuppressive effects on the immune system were examined for their in vitro effects on some functions of murine peritoneal macrophages. The cells were pre-incubated for 4 hr with the mycotoxin concentrations of 0.1 ng/ml-1 micrograms/ml. At concentrations that did not affect the cell viability (Specific Lactate Dehydrogenase test), diacetoxyscirpenol and deoxynivalenol suppress microbicidal activity of phagocytic cells. The diacetoxyscirpenol concentrations, which reduce phagocytosis (2 ng/ml), microbicidal activity (1 ng/ml), superoxide anion production (1 ng/ml) and phagosome-lysosome fusion (0.1 ng/ml), indicate that the inhibition of killing mechanism arise from both oxidative and non-oxidative pathways. Phagocytosis, microbicidal activity and superoxide anion production are inhibited by deoxynivalenol at 1 ng/ml whereas phagosome-lysosome fusion is reduce above 100 ng/ml. These results suggest that microbicidal activity inhibition by deoxynivalenol did not depend on non-oxidative pathway (phagosome-lysosome fusion) impairment.

Detection and occurrence of cyclopiazonic acid in cheeses.

Techniques for detection of cyclopiazonic acid in P. camemberti fermented cheeses are described. They include extraction with CHCl3-MeOH, purification, analysis and quantitation by thin-layer chromatography, and confirmation procedures. Recovery from spiked samples was 75-85%, and the lowest detectable level was 0.02 ppm. This toxic metabolite of P. camemberti was found in the crust of 11 of 20 cheeses of different brands, but not in the inner part. The highest levels were found in three samples: 0.4, 1, and 1.5 ppm. Cyclopiazonic acid doses eventually ingested by a consumer appear to be very low: 3 or 4 micrograms in a portion of the most contaminated sample.

Ecotoxinogenesis of Pithomyces chartarum.

Facial eczema is a hepatogenous photosensitivity mycotoxicosis resulting from sporidesmin ingestion. The morphological characters of toxigenic strains of P. chartarum are reported and the effect of temperature on growth and mycotoxin production are studied. The temperature range for which there is an actual risk of toxin accumulation (20-25 degrees C) is much narrower than for an appreciable growth (5-30 degrees C).

Production of aflatoxin B1 by Aspergillus ruber THOM and CHURCH.

Production of aflatoxins by Aspergillus ruber THOM and CHURCH was first reported by KULIK and HOLADAY (1967), although these results have lacked confirmation. In this paper we provide evidence that this fungal strain produces aflatoxins. This finding has implications for food hygiene, especially in countries where such moulds are used in the preparation of foodstuffs.

Gonadotrophin release in ovariectomized ewes fed different amounts of coumestrol.

Three experiments were conducted to study changes in pulsatile secretion of LH and FSH during the breeding season or anoestrus in ovariectomized Ile-de-France ewes fed different amounts of the phyto-oestrogen coumestrol. In Exp. 1, conducted during the breeding season, ewes (3-4 per group) were fed lucerne supplying 4, 18 or 30 mg coumestrol per ewe per day for 15 days. Experiments 2 and 3 were conducted during seasonal anoestrus. In Exp. 2, ewes (4 per group) were fed lucerne supplying coumestrol concentrations ranging from 4 to 38 mg/ewe/day for 15 days. In Exp. 3, ewes (10 per group) were fed lucerne supplying 14 or 125 mg coumestrol/ewe/day for 15 days. During the breeding season, an increased concentration of coumestrol in the diet significantly decreased the amplitude of LH pulses. There were no effects on LH pulse frequency or on FSH concentrations. During seasonal anoestrus, there were no significant effects on LH pulse frequency, or amplitude and no significant effect on FSH concentration. These results show that high concentrations of coumestrol in lucerne diets would not explain seasonal variation in LH pulse frequency in ovariectomized ewes. However, lucerne diets with increased coumestrol concentrations can influence LH release during the breeding season.

Comparative incidence of oral ochratoxicosis and aflatoxicosis on the activity of drug-metabolizing enzymes in rat liver.

Mycotoxicosis has been produced in the rat by daily oral administrations of ochratoxin A (1.5 mg/kg/day) or aflatoxin B1 (1 mg/kg/day). Hepatic microsomal cytochrome P-450 and b5 contents and many phase I and II biotransformation systems have been measured in the course of ochratoxicosis (4 to 15 dosings) and aflatoxicosis (1 to 8 dosings). In case of ochratoxicosis, decreases in cytochrome P-450 level, aminopyrine demethylase and aniline hydroxylase activities were observed in rats receiving 15 administrations of the toxin. Aflatoxicosis induced more severe decreases in cytochrome P-450, aminopyrine demethylase and ethoxycoumarin deethylase following 8 daily gavages. In the two studies, there was no significant change in activities of liver phase II biotransformation enzymes.

[Enhancement factors of penicillic acid production by Penicillium verrucosum var. cyclopium in foodstuffs (author's transl)]. / Facteurs favorisant la production d'acide pénicillique par Penicillium verrucosum var. cyclopium dans les denrées alimentaires.

Main parameters of penicillic acid (P.A.) production by P. verrucosum var. cyclopium were investigated: substrate nature, water content (H: 22-70%), temperature (5-30% degrees C), confined air and incubation delay. 6100, 2820, 1832, 39 and 0.8 mg/kg were the maximal yields obtained in corn, barley, straw, colza and soya. Toxinogenesis rate was greatly enhanced in crushed corn. Maximal yields in corn increased exponentially with H; production and disappearance rates of the free toxin were an increasing function of H and temperature; so, low temperatures may lead to a high accumulation of P.A. within several months. In a confined environment, P.A. concentration was limited for H greater than or equal to 40%; but accession of air induced an accelerated P.A. biogenesis. Maximal yield of P.A. (8000 mg/kg) was obtained in corn with 60% water content, within one week at 25 degrees C.

Cyclopiazonic acid production by Penicillium camemberti Thom and natural occurrence of this mycotoxin in cheese.

Every Penicillium camemberti strain freshly isolated from 20 commercial cheese brands produced cyclopiazonic acid in two culture media at 25, 13, and 4 degrees C; the toxin yield was greatly dependent on the strain and environmental parameters (medium, temperature, and incubation time). The toxigenic ability appeared as a log-normal distribution. This mycotoxin was found in the crust (0.05 to 0.1 microgram/g in three samples, 0.1 to 0.2 microgram/g in five samples, and 0.4, 1, and 1.5 microgram/g in three other samples) but not in the inner part. When its acute toxicity is considered, doses eventually ingested by consumers are very low (lower than 4 microgram). Means for prevention are discussed. A highly toxigenic strength and rate appear to be necessary features leading to natural contamination in cheeses. The distribution of toxigenic ability makes possible without delay a choice of weakly toxic strains.

[Feedstuffs contamination by Penicillium cyclopium Westling. Frequency of penicillic acid producing strains (author's transl)]. / Contamination alimentaire par le Penicillium cyclopium Westling. Fréquence de souches productrices d'acide pénicillique.

For the last six years, 246 samples of feedstuffs, suspected for animal disorder, have been subjected to mycological examination, Penicillium cyclopium was present with more than 10(3) and 10(5) propagules per gram respectively in 42% and 6% of samples. The feeds mainly concerned are, in decreasing order of P. cyclopium frequency and contamination level : barley, maïze mixed feeds (50%), the straw (41%), hays (21%) and milk replacers (10%) (table 1). The penicillic acid bioproduction by fifty strains freshly isolated from these feedstuffs is investigated on wet crushed corn (60% water). After incubation at 25 degrees C for a week, extraction by ethyl acetate and purification, quantification is done by fluorodensitometry on TLC plates after exposure to concentrated ammonia (fig. 1). Among the strains, 50% produces less than 5 ppm, 4% between 10-100 ppm, 20% (100-1 000), 20% (1 000-5 000) and 6% (5 000-10 000 ppm) (table 2). Maximal yield obtained was 8 240 mg/kg. Hypothesis of a log-normal distribution of toxigenic strength is not rejected (fig. 2). On the whole, frequency and toxic yields are higher for strains originating from cereals. One may think that only a quarter of the strains might be considered as actual potential penicillic acid producers in agricultural conditions.

Cyclopiazonic acid bioproduction by Penicillium camemberti Thom: effect of temperature on individual strains.

The effect of temperature on cyclopiazonic acid production varied with the strain used. Accordingly, strains weakly toxigenic at the temperature of cheese ripening and storage can be selected and should be used.

[Lipid metabolism in Aspergillus versicolor (Vuill.) Tiragoschi. Relation to biogenesis of sterigmatocystin]. / Metabolisme lipidique chez l'Aspergillus versicolor (Vuill.) Tiraboschi. Relations avec la biogénèse de la stérigmatocystine

Aspergillus versicolor is cultivated in a synthetic medium for 22 days. Bioproduction of lipids and sterigmatocystin are compared. The fatty acids of the neutral lipid and polar lipids fractions are mainly: C 16:0, C 18:0, C 18:1, C 18:2, C 18:3. Maximal yields of dry weight, neutral lipids and sterigmatocystin occur, respectively, on the 4th, the 7th and the 20th days. These results and their comparison with other works emphasize that a fall of concentration in lipids precedes the phase of highest concentration in secondary metabolites of polyketide type; it appears that fats and particularly palmitic acid are present in biogenesis of these derivatives.