Effect of spice essential oils on growth and aflatoxin B1 production by Aspergillus flavus

Aflatoxin B¹ production by Aspergillus flavus was studied in yeast extract sucrose broth in the presence of cinnamon, clove, almond and cardamom oils. Growth and aflatoxin B₁ production was inhibited by 0.5 μl cinnamon oil ml^-1 medium and by 1 μl clove oil ml_-1. Almond and cardamom oils only affected growth when their concentration exceeded 1.25 μl ml^-1 medium. Aflatoxin B₁ production was stimulated by 0.75 and 1 μl almond oil ml^-1 medium or by 0.25 and 0.5 μl cardamom oil ml^-1.     

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.     

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.     

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.     

M1 Muscarinic Receptors Stimulate Rapid and Prolonged Phases of Neuronal Nitric Oxide Synthase Activity: Involvement of Different Calcium Pools

Abstract: This study shows that activation of M₁ muscarinic receptors, when coexpressed in Chinese hamster ovary (CHO)-K1 cells with neuronal nitric oxide (NO) synthase (nNOS), produces early and late phases of elevation of both intracellular Ca²⁺ concentration and nNOS activity. We examined the relationship between receptor-mediated increases in intracellular Ca²⁺ concentration and activation of nNOS over both short and long intervals using guanosine 3',5'-cyclic monophosphate (cGMP) formation as a measure of nNOS activity. The rapid phase of nNOS activation was dependent on release of Ca²⁺ from intracellular stores in both the CHO M₁/nNOS transfected cells and in neuroblastoma (N1E-115) cells, in which muscarinic receptors and nNOS are endogenously expressed. Two single point mutations in the M₁ muscarinic receptor that have previously been shown to uncouple differentially the receptor from phosphoinositide hydrolysis produced parallel attenuation of the rapid phase of nNOS activation. Characterization of the prolonged phase of nNOS activation was done using the conversion of l -[³H]arginine to l -[³H]citrulline as well as cGMP formation following stimulation of M₁ muscarinic receptors for 60 min. Both responses were dependent on influx of extracellular Ca²⁺ and were accompanied by prolonged formation of NO at functionally effective levels as late as 60 min following receptor activation. Therefore, this study demonstrates for the first time the existence of two mechanistically distinct phases of nNOS activation that are dependent on different sources of Ca²⁺.     

Enhancement of vitamin B6 levels in seeds through metabolic engineering

As a versatile cofactor for many enzymes catalyzing important biochemical reactions, vitamin B₆ is required for all cellular organisms. In contrast to bacteria, fungi and plants, which have the ability to synthesize vitamin B₆ de novo , animals have to take up the vitamin from their diet. Plants are the major source of vitamin B₆ for animals. The recent identification of vitamin B₆ biosynthetic enzymes PDX1 and PDX2 in plants makes it possible to regulate the biosynthesis of this important vitamin. In this study, we generated Arabidopsis plants overexpressing the PDX1 and/or PDX2 gene and used a liquid chromatography/mass spectrometry/mass spectrometry method to determine the levels of different forms of vitamin B₆ in these transgenic plants. It was found that expression of the PDX genes under control of the CaMV 35S promoter caused only a limited increase in pyridoxine contents in dry seeds but not in shoots or roots. When using the Arabidopsis seed-specific 12S promoter to drive the expression of the PDX genes, the levels of vitamin B₆ increased more than twofold in transgenic plants. Our work demonstrates that it is feasible to enhance vitamin B₆ content in seeds by metabolic engineering.     

Fumonisin B1 production by Fusarium proliferatum strains isolated from Allium fistulosum plants and seeds in Japan

Aims:  To test the fumonisin B₁ - producing ability of Fusarium proliferatum strains isolated from Welsh onion ( Allium fistulosum ) plants and seeds of commercial cultivars in Japan and to examine the applicability of PCR-based assays to discriminate between fumonisin B₁-producing and nonproducing isolates.
Methods and Results:  Fumonisin B₁ levels in 20 Fusarium isolates obtained from Welsh onion plants and seeds of seven commercial cultivars were determined by HPLC. Thirteen of the 20 isolates produced fumonisin B₁. PCR assay with FUM1 gene-specific primers amplified a DNA fragment (700 bp) only from fumonisin-producing isolates.
Conclusions:  Fusarium proliferatum isolates that can produce fumonisin B₁ were often associated with wilted Welsh onion plants and seeds of some commercial cultivars. The PCR assay with FUM1 gene-specific primers has the potential to discriminate between fumonisin B₁-producing and nonproducing isolates.
Significance and Impact of the Study:  This study revealed that F. proliferatum producing fumonisin B₁ is associated with Welsh onion plants and that commercial cultivar seeds may be contaminated with the fungus. PCR amplification of FUM1 gene can be a useful tool for the rapid identification of fumonisin B₁-producing F. proliferatum isolates.     

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.     

VITAMIN B6 TRANSPORT IN THE CENTRAL NERVOUS SYSTEM: IN VIVO STUDIES

Abstract— The total concentrations of vitamin B₆ (B₆) in plasma, choroid plexus, CSF and brain of adult New Zealand white rabbits, measured fluorometrically, were 0.30, 15.10, 0.39 and 8.90 μ mol/l or kg respectively. The mechanisms by which B₆ enters and leaves brain, choroid plexus and CSF were investigated by injecting [³H]pyridoxine (PIN) intravenously, intraventricularly and intraarterially. [³H]PIN, with or without unlabelled PIN, was infused intravenously at a constant rate into conscious rabbits. At 150 min, [³H]B₆ readily entered CSF, choroid plexus and brain. The addition of 0.5 mmol/kg carrier PIN to the infusion solution depressed the relative entry of [³H]B₆ into CSF, choroid plexus and brain by about 80%. After intraventricular injection, [³H]PIN readily entered brain from CSF. The intraventricular injection of carrier PIN with [³H]PIN decreased the amount of [³H]B₆ in brain and also decreased the percentage of [³H]B₆ in CSF and brain that was phosphorylated. During one pass through the cerebral circulation, [³H]PIN (1 μ m ) was cleared from the circulation no more rapidly than mannitol. These results were interpreted as showing that the entry of B₆ from blood into CSF and presumably the extracellular space of brain and thence into brain cells involves one or more saturable transport and/or metabolic steps.     

SOME EFFECTS OF DIETARY VITAMIN B6 DEFICIENCY AND 4-DEOXYPYRIDOXINE ON γ-AMINOBUTYRIC ACID METABOLISM IN RAT BRAIN

Abstract— Investigations of the respective effects of dietary vitamin B₆ deficiency and 4-deoxypyridoxine (a vitamin B₆ antagonist) on GABA metabolism in rat brain have been carried out. No convulsions were observed in rats subjected to either treatment. GABA levels were lowered by both treatments, the greatest diminutions being found with the dietary deficiency. Glutamic acid decarboxylase activity was reduced under both conditions, but the loss of activity in the B₆ deficiency experiments could be attributed to cofactor depletion, whereas in the deoxypyridoxine experiments the loss of activity appears to be due to lower levels of available apoenzyme. The activity of GABA-transaminase was not affected by deoxypyridoxine treatment and only moderately reduced in the B.5 deficient animals.     

Response of phenolic metabolism to the application of carbendazim plus boron in tobacco

In view of the essential role of phenolic compounds in the development of pathogen resistance in plants, and given the influence that fungicides and boron (B) exert over phenolic metabolism, the aim of the present study was to determine the individual effect of the application of a fungicide, as well as to determine the joint effect of the fungicide and B on the metabolism of phenolic compounds in tobacco plants ( Nicotiana tabacum L. cv. Tennessee 86). The fungicide applied was carbendazim (carb), a preventative fungicide, with a purity of 100% at a concentration of 2.6 m M . Boron was applied in the form of H₃BO₃ at: 1.6 m M (B₁), 4 m M (B₂), 8 m M (B₃), 16 m M (B₄), 32 m M (B₅), or 64 m M (B₆). In all, there were eight treatments: one without carb and without B (control), one with only carb, and six combinations of carb with each concentration of B. The results indicated that the foliar application with carb alone led to increases in phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) activity and a foliar accumulation of phenols. This effect of the carb alone could signify an additional tolerance mechanism to pathogenic infection, given the participation of phenolic compounds in the lignification of plant cell walls. The joint application of carb and B increased both the biosynthesis and the oxidation of the phenolic compounds, especially in carb plus B₃, while the application of carb plus B₅ or carb plus B₆ reduced these processes as well as the foliar biomass.     

Muscarinic Modulation of Striatal Dopamine, Glutamate, and GABA Release, as Measured with In Vivo Microdialysis

Abstract: Intrastriatal microdialysis was used to administer muscarinic drugs in freely moving rats for 40 min at a flow rate of 2 µl/min. Administration of the nonselective agonist pilocarpine at 10 m M increased striatal dopamine release and decreased extracellular GABA and glutamate overflow. Perfusion with the muscarinic M₂ antagonist methoctramine at 75 µ M increased extracellular dopamine and glutamate concentrations but exerted no changes on extracellular GABA levels. Intrastriatal administration of the M₁ antagonist pirenzepine at 0.05 µ M decreased extracellular dopamine overflow. Application of pirenzepine (0.05 and 5 µ M ) exerted no effects on the measured GABA or glutamate levels. There are thus important differences in applied doses of muscarinic drugs needed to obtain modulatory effects. High doses of agonists are probably needed to superimpose on the background of tonic influences of striatal acetylcholine, whereas antagonists can block the receptors in small doses. We further suggest that M₁ receptors might tonically facilitate striatal dopamine release, that M₂ receptors might tonically inhibit striatal glutamate efflux, and that acetylcholine does not exert tonic effects on striatal GABA release. The link with the pilocarpine animal model for temporal lobe epilepsy will be discussed.     

Respiratory Q10 of marigold (Tagetes patula) in response to long-term temperature differences and its relationship to growth and maintenance respiration

Acclimation of respiration to temperature is not well understood. To determine whether whole plant respiration responses to long-term temperature treatments can be described using the Q₁₀ concept, the CO₂ exchange rate of marigolds ( Tagetes patula L. 'Queen Sophia'), grown at 20°C or 30°C, was measured for 62 days. When plants of the same age were compared, plants grown at 20°C consistently had a higher specific respiration (R_spc) than plants grown at 30°C (long-term Q₁₀= 0.71–0.97). This was due to a combination of greater dry mass at 30°C and a decrease in R_spc with increasing mass. When plants of the same dry mass were compared, the long-term Q₁₀ was 1.35–1.55; i.e. R_spc was higher at 30°C than at 20°C. Whole plant respiration could be accurately described by dividing respiration into growth and maintenance components. The maintenance respiration coefficient was higher at 30°C than at 20°C, while the growth respiration coefficient was lower at 30°C, partly because of temperature-dependent changes in plant composition. These results suggest difficulties with interpreting temperature effects on whole plant respiration, because conclusions depend greatly on whether plants of the same age or mass are compared. These difficulties can be minimized by describing whole plant respiration on the basis of growth and maintenance components.     

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.     

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.     

Effects of alkaloids on feeding by Phormia reginaconfirm the critical role of sensory inhibition

Abstract. Inhibition of peripheral chemoreceptors by micromolar concentrations of alkaloids may account for feeding deterrence. Earlier work showed a reduction in both the proboscis extension response and sensory activity recorded from sugar-sensitive cells in tarsal D and labellar 'largest' hairs when the stimulating solution was sucrose mixed with alkaloids. In the present work a similar behavioural effect was also seen when alkaloids were mixed with pyranose and furanose sugars as well as with water and deuterium oxide. Behavioural deterrence continued after the stimulus was removed. Complete recovery occurred after 120 s. To check against the possibility of a central inhibitory state (CIS) having been established two sets of experiments were run in which quinine was applied to a single tarsus followed by a sucrose post-test to the contralateral tarsus. In one half of the tests sugar was applied first (S₁Q₂), and in the other half quinine first (Q₁S₂). Comparable tests were run with contralateral labellar hairs. No CIS was set up by Q₁; however, a central excitatory state (CES) was set up by S₁. In order to test (a) whether quinine had different effects on other known or unknown receptors and (b) post-ingestive effects, volumes imbibed in single drinks of sucrose with and without quinine were compared. Volumes of the mixture imbibed were not significantly different from what was predicted based on the known reduction in sensory acitivty.     

Occurrence of aflatoxin in some liver curative herbal medicines

Fifty herbal medicine samples of seven different taxa known to cure liver disorders were analysed for aflatoxin contamination. Twenty-three samples, out of 50, were contaminated with various levels of aflatoxins. Amongst the 23 contaminated samples the maximum level of aflatoxin B¹ recorded was 2.23 μg g^-1 in Asparagus racemosus and the minimum 0.28 μg g^-1 in Emblica officinalis . Aflatoxin G¹ was only found in one species, Terminalia belarica . Aflatoxin production of the isolates of Aspergillus flavus was also examined and the highest levels were produced by isolates from A. racemosus (1.07-2.47 μg ml^-1). Aflatoxin contamination of herbal drugs may be a risk for patients because the level of aflatoxins is much higher than the tolerance level fixed by the WHO for foods.     

THE STABILITY OF VITAMIN B6 ACCUMULATION AND PYRIDOXAL KINASE ACTIVITY IN RABBIT BRAIN AND CHOROID PLEXUS

The effects of changes in the concentrations of pyridoxal phosphate and blogenic amines in brain on: (I) pyridoxal kinase (EC 2.7.1.35) activity in brain and choroid plexus; and (2) vitamin B₆ accumulation by brain slices and isolated, intact choroid plexuses were studied. New Zealand white rabbits were treated parenterally with 200 mg/kg pyridoxine-HCl for 3 days or 120 mg/kg 4-deoxypyridoxine HCI or 5 mg/kg reserpine I day before death. After these treatments the mean concentration of pyridoxal phosphate in brain was elevated by 39% by pyridoxine and decreased by 57% by 4-deoxypyridoxine. Reserpine had no effect. However, the ability of brain slices and isolated, intact choroid plexuses from the treated rabbits to accumulate [³H] vitamin B₆ (with [³H]pyridoxine in the medium) was not different from untreated controls. Also, the specific activity of pyridoxal kinase in brain and choroid plexus of treated rabbits was not different from controls. These results show that vitamin B₆ accumulation and pyridoxal kinase activity in brain and choroid plexus are independent of both pyridoxal phosphate and reserpine-sensitive biogenic amine concentrations in brain. In vitro studies with pyridoxal kinase showed that. in both choroid plexus and brain. pyridoxal kinase was a single enzyme with a molecular weight of 43.000 and a K_m , for pyridoxine of 2.0 μM Crude and partially-purified pyridoxal kinase from brain was not inhibited by biogenic amines (1 mM) or pyridoxal phosphate (5 μM). These in vitro data are consistent with the lack of effect of changes in pyridoxal phosphate and biogenic amine concentrations (in brain) on pyridoxal kinase activity in brain in vivo.     

Up-Regulation of the Neuronal Form of Nitric Oxide Synthase in Response to Prolonged Muscarinic M1 Receptor Stimulation

Abstract: It is generally believed that the neuronal form of nitric oxide synthase (nNOS) is constitutively expressed and that regulation of this enzyme's activity is mediated solely by changes in cytosolic calcium concentration. Serendipitously, however, we observed that pretreatment of Chinese hamster ovary (CHO) cells, which coexpress muscarinic M₁ receptors and nNOS, with 3.3 µ M or 1 m M carbachol (CCh) for 48 h resulted in marked enhancement of maximal muscarinic receptor-stimulated nNOS activity as determined by l -[³H]citrulline and cyclic [³H]GMP production. This was accompanied by a decrease in the potency of CCh. Muscarinic receptor density was reduced in the agonist-pretreated cells, as determined by specific [ N-methyl -³H]scopolamine methyl chloride binding, whereas competition binding studies revealed no changes in agonist affinity. Both receptor-stimulated inositol phosphate formation and elevation of intracellular calcium concentrations were found to be desensitized in agonist-pretreated cells in a manner dependent on CCh pretreatment concentration. It is interesting that ionomycin-stimulated nNOS activity was greater in CCh-pretreated cells. Also, western analysis revealed increased nNOS immunoreactivity in pretreated cells. A similar increase in nNOS immunoreactivity following agonist treatment was demonstrated in N1E-115 neuroblastoma cells, which endogenously express nNOS and muscarinic M₁ receptors. Thus, the enhancement of maximal receptor-stimulated nNOS activity following agonist pretreatment can be attributed to up-regulation of nNOS. It is interesting that this augmentation of the response takes place in spite of receptor down-regulation and desensitization of multiple steps involved in nNOS activation.