Therapy of chronic relapsing experimental allergic encephalomyelitis and the role of the blood-brain barrier: elucidation by the action of Brequinar sodium.

The immunosuppressive effect of the novel 4-quinoline carboxylic acid derivative Brequinar sodium on the chronic relapsing experimental allergic encephalomyelitis CREAE model in the Biozzi AB/H mouse was investigated. Although Brequinar sodium actively inhibited peripheral immune responses, it showed a limited potential to control an ongoing disease of the central nervous system (CNS). Doses of 25 mg/kg inhibited in vivo induced proliferative response and prevented EAE when treated from day 9 post-inoculation (p.i.). However, when administered from day 12 p.i. or during the post-acute remission phase-limited effects on the course of disease were observed. By comparison, treatment with a single high dose of cyclophosphamide (200 mg/kg) at these time points was significantly effective in controlling disease. As a possible explanation of the observed results it is suggested that for a compound to be effective in treating an ongoing immune response in the CNS, it must be capable of crossing the blood-brain barrier and act on the disease-inducing cells activated within the CNS. This hypothesis is supported by the finding that intracerebral injections of Brequinar sodium on day 12 p.i. significantly inhibited disease progression. This suggests that strategies aimed at controlling immune-mediated disease of the CNS require therapeutic doses of the compounds to be delivered into the CNS.

Inhibition of aflatoxin biosynthesis by tolnaftate.

Tolnaftate [2-napthyl-N-methyl-N-(m-tolyl)thionocarbamate], an antifungal drug, is widely used to control superficial fungal infections in humans and other animals. In this study the effect of tolnaftate on aflatoxin biosynthesis by Aspergillus parasiticus NRRL 3240 was investigated. Tolnaftate changed the morphology of A. parasiticus to yeastlike forms and inhibited aflatoxin formation. The formation of aflatoxin G was blocked considerably, indicating a metabolic block in the conversion of aflatoxin B to aflatoxin G. The incorporation of [1-14C]acetate into aflatoxin was significantly inhibited at a concentration of 1 mM tolnaftate. The presence of zinc in the resuspension buffer resulted in reversal of the tolnaftate-induced inhibition of aflatoxin G1 biosynthesis.

Induction of mixed function oxidases on oral administration of dieldrin.

The administration of dieldrin (30 mg/kg body weight) caused an increase in the liver weight of rats. The metabolism of aflatoxins B1 and G1 by the microsomes obtained from the liver of dieldrin-treated animals was enhanced significantly as compared to the controls showing that dieldrin increased the activity of mixed function hydroxylases. Dieldrin caused an increase in the activity of liver microsomal NADPH oxidase and a decrease in the lipid peroxidation. Dieldrin brought about an increase in the phosphatidylcholine content of rat liver.

Effect of Zinc on tricarboxylic acid cycle intermediates and enzymes in relation to aflatoxin biosynthesis.

The stimulatory action of zinc on aflatoxin production by Aspergillus parasiticus NRRL3240 has been investigated by studying the levels of tricarboxylic acid (TCA) cycle intermediates and related enzymes in the fungal mycelium. During the stationary phase of growth, the levels of alpha-keto acids declined in zinc-sufficient cultures compared with those in zinc-dificient cultures. TCA cycle enzymes did not show any significant changes due to zinc availability. In zinc-deficient cultures, enzymes of the TCA cycle had maximum activity on the fourth day, after which their activity declined. In zinc-sufficient cultures, some enzymes showed maximum activity on the fourth day, others on the second day.

Regulation of aflatoxin biosynthesis. III. Comparative study of adenine nucleotide pool in aflatoxigenic and non-aflatoxigenic strains.

The adenine nucleotide pools were studied in toxigenic (NRRL 3240) and non-toxigenic (NRRL 3537) strains of Aspergillus flavus group in relation to aflatoxin biosynthesis. The levels of ADP and AMP were comparatively low in the non-toxigenic strain throughout the growth period. In the toxigenic strain the levels of ADP and AMP increased significantly during the stationary phase. The ATP content of the toxigencic strain had higher ATP levels during the stationary phase compared to the toxigenic strain. The inorganic phosphate levels of the non-aflatoxigenic strain were higher when compared with the aflatoxigenic strain. High levels of inorganic phosphate have been reported to be inhibitory for secondary biosynthesis including aflatoxins. The energy charge values of the non-toxigenic strain, compared to the toxigenic strain, were lower during exponential phase but higher during stationary phase. The observed changes have been correlated with aflatoxin biosynthesis.

Regulation of aflatoxin biosynthesis. 2 Comparative study of tricarboxylic acid cycle in aflatoxigenic and non-aflatoxigenic strains of Aspergillus flavus.

The presence of a functional TCA cycle and its intermediates has been shown in Aspergillus parasiticus and Aspergillus flavus. The role of the TCA cycle in aflatoxin biosynthesis has been studied. In A. parasiticus the TCA cycle was activated on the second day of growth (exponential growth phase) resulting in the onset of aflatoxin biosynthesis. Pyruvate accumulation to toxic levels in A. parasiticus is suggested to shift the growth phase from exponential to stationary with the onset of aflatoxin biosynthesis. In A. flavus, a non-aflatoxigenic strain, no pyruvate accumulation was observed. Moreover, the onset of aflatoxin production in A. parasiticus was followed by a decline in the levels of TCA cycle intermediates indicating their possible utilization in secondary metabolite synthesis. Accumulation of TCA cycle intermediates in A. flavus is suggested to be due to non-diversion to aflatoxin biosynthesis.

Regulation of aflatoxin biosynthesis. 1 Comparative study of mycelial composition and glycolysis in aflatoxigen and nonaflatoxigenic strains.

A comparative biochemical study of an aflatoxigenic strain Aspergillus parasiticus NRRL 3240 and a nonaflatoxigenic strain A. flavus NRRL 3237 was carried out in order to have a better idea of regulation of aflatoxin biosynthesis. The results obtained revealed continuous primary metabolic activity (protein synthesis) in the nonaflatoxigenic strain while the aflatoxigenic stain showed inhibition of protein and nucleic acid synthesis. The aflatoxigenic strain showed higher levels of oxygen uptake, RNA, NAD, FMN and activities of glycolytic enzymes. Furthermore, it had lower of lipids and reduced activity of glucose-6-phosphate dehydrogenase, which is a source for NADPH. The differences observed have been discussed in relation to aflatoxin biosynthesis and its regulation.

Effect of zinc on adenine nucleotide pools in relation to aflatoxin biosynthesis in Aspergillus parasiticus.

The adenylic acid systems of Aspergillus parasiticus were studied in zinc-replete and zinc-deficient media. The adenosine 5'-triphosphate levels of the fungus were high during exponential phase and low during stationary phase in zinc-replete cultures. On the other hand, the levels of adenosine 5'-diphosphate and adenosine 5'-monophosphate were low during exponential phase of growth and high during stationary phase. The adenosine 5'-triphosphate levels during exponential phase may indicate higher primary metabolic activity of the fungus. On the other hand, high adenosine 5'-monophosphate levels during stationary phase may inhibit lipid formation and may enhance aflatoxin levels. The inorganic phosphorus content was low in a zinc-replete medium throughout the growth period, thereby favoring aflatoxin biosynthesis. The energy charge during the exponential phase was high but low during the stationary phase. In general the energy charge values were lower because of high adenosine 5'-monophosphate content.

Inhibition of aflatoxin formation by 2-mercaptoethanol.

2-Mercaptoethanol inhibits growth of Aspergillus parasiticus NRRL 3240 and aflatoxin formation by the fungus. When added to the resuspended medium, 2-mercaptoethanol inhibited [1-14C]acetate incorporation into both aflatoxins and neutral lipids, thereby showing that it acts at an early stage of aflatoxin biosynthesis. The inhibition is probably due to its chelating action on zinc, which is essential for aflatoxin production. It is proposed that any chelating agent that selectively binds to zinc will inhibit aflatoxin formation.