Increased cytochrome P-450 activity in Aspergillus fumigatus after xenobiotic exposure.

Microsomal fractions were isolated from Aspergillus fumigatus cultures that had been treated with dimethylsulphoxide (DMSO), ethanol, benzopyrene, phenobarbital or naphthalene. All these xenobiotics increased microsomal cytochrome P-450 content. Cytochrome P-450 reductase activity remained unaffected and no alteration in the microsomal protein profile was detectable by SDS PAGE. Benzopyrene, dimethylaniline, hexobarbitol and p-nitroanisole were metabolised by A. fumigatus microsomes, while aniline, ethoxyresorufin and pentoxyresorufin were not. No xenobiotic elicited a more specific oxidation of any of the substrates. A fumigatus microsomes also catalyzed the cell-free biosynthesis of ergosterol and quantitative analysis of assay metabolites showed that exposure to ethanol, benzopyrene or phenobarbitone in vivo resulted in enhanced ergosterol biosynthesis in vitro.

Improved synthesis and characterisation of a hydrazide derivative of diethylenetriaminepentaacetic acid for site-specific labelling of monoclonal antibodies with 111In.

The monohydrazide derivative of diethylenetriaminepentaacetic acid, (HOOCCH2)2NCH2CH2N(CH2CO-OH)CH2CH2N(CH2COOH++ +)(CH2CO.NHNH2), is a bifunctional chelator designed for attaching the radiometal 111In selectively to the carbohydrate side chains of pre-oxidised monoclonal antibodies. A simple synthesis of this chelator (from diethylenetriaminepentaacetic acid cyclic anhydride and hydrazine), and its purification and chemical characterisation, are described. Rabbit IgG was oxidised with periodate, and the aldehyde groups thus generated were reacted with the linker forming a conjugate that was readily labelled in high yield and purity with 111In.

Glial fibrillary acidic protein in the cytoskeletal and soluble protein fractions of the developing rat brain.

The distribution of glial fibrillary acidic protein (GFAP) into cytoskeletal and soluble protein fractions during development of the rat brain has been studied by quantitative immunoblotting and enzyme-linked immunosorbent assay (ELISA). These assays indicate that cytoskeletal GFAP accounts for nearly all the total GFAP in the adult rat brain, and that the developmental increase in the GFAP content of the rat brain is due to accumulation of GFAP into the cytoskeleton. A small and constant amount of the total GFAP was detected in the soluble protein fraction. This GFAP had an apparent molecular mass (Mr) similar to that of the highest Mr form of GFAP detected in the cytoskeletal fraction. In contrast to the assays for cytoskeletal GFAP, no significant increase in the GFAP concentration of the soluble protein fraction could be measured during development. Sensitive, calibrated immunoblotting of cytoskeletal and soluble protein with [125I]protein A confirmed these findings, and showed that both cytoskeletal and soluble GFAP are first detected during the same period of foetal rat brain development. A finite and reproducible amount of lower Mr forms of GFAP were observed in the cytoskeletal fraction even when prepared in the presence of stringent proteolytic inhibitors. These presumed proteolytic degradation products of GFAP increased in abundance during development, parallel to the increase in cytoskeletal GFAP content of the rat brain. However, the abundant proteolytic degradation products of GFAP found in the cytoskeletal fraction were not detected in the soluble protein fraction at any age studied.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental changes in the rate of production of an unusual testosterone metabolite, 4-androstene-3 alpha, 17 beta-diol, by chick liver microsomes.

Testosterone is produced by the chick embryo testis from the 13th day of incubation. We have investigated the ability of microsomes prepared from the fetal and neonatal liver to metabolize testosterone and have found that the principal metabolite generated by microsomes in the presence of NADPH is 4-androstene-3 alpha, 17 beta-diol. The rate of production of this metabolite declined sharply over the time of hatching. Conversely, 16 alpha-hydroxytestosterone production increased transiently just after hatching. Our findings indicate that chick liver microsomes contain a 3 alpha-hydroxysteroid dehydrogenase (3 alpha-hydroxysteroid: NAD(P) oxidoreductase, EC 1.1.1.50) whose activity changes during development.

A study into the effects of 2-acetylaminofluorene on hepatic monooxygenase activities in the chick embryo.

2AAF is a potent inducer of cytochrome P-450 in the chick embryo liver. The induction has been characterized with respect to a range of monooxygenase activities and the regiospecificity of 2AAF hydroxylation. Similarities to the response elicited by both PB and 3MC were noted. 2AAF was rapidly deacetylated by hepatic microsomes prepared from control animals to 2AF, an inhibitor of monooxygenase activity. Metabolites generated in vivo and carried over in vitro might have therefore interfered with the subsequent kinetic analysis. In general terms induction of a unique cytochrome P-450 subform(s) could not be attributed to 2AAF in the chick embryo. The data is discussed with respect to the reported resistance of avian species to the hepatocarcinogenic effects of 2AAF. Two possibilities are highlighted, a diversion of 2AAF to ring hydroxylated metabolites and/or deacetylation of 2AAF. Both effects could reduce carcinogenicity by decreasing the concentration of proximate carcinogen and/or promoter(s).

The induction of cytochrome P-450 and monooxygenase activities in the chick embryo by 2-acetylaminofluorene.

Administration of 2-acetylaminofluorene to chick embryos increases the cytochrome P-450 level 3.4 fold but causes no increase in total epoxide hydrase activity or other microsomal electron transport enzymes. The induction response shows some similarity to that elicited by phenabarbitone both in terms of the monooxygenase activities induced and their inhibition characteristics. Induction of a specific cytochrome P-450 subform by this agent may increase its detoxification and in part account for the resistance of avian species to its hepatocarcinogenic effect.

In vitro studies of the characteristics of the release of prostaglandins from viscous solutions.

The diffusion rates of prostaglandin E2 in different viscous solutions used in clinical practice for inducing abortion and term labour have been studied in the laboratory. The results indicate that of those currently in use, solutions of Dextran might be more appropriate for the purposes. By increasing the viscosity of solutions in use at present, clinical results may be further improved by aiding retention in the vagina or uterus while producing a slow sustained linear release of prostaglandin.

Diurnal fluctuation in the rate of synthesis of a specific protein fraction in the rat brain.

A protein fraction has been identified in microsomes prepared from the rat hypothalamus whose rate of synthesis fluctuates diurnally in ovariectomized animals.

Plasma concentrations of prolactin and thyrotrophin during suckling in urethane-anaesthetized rats.

Plasma levels of prolactin and TSH were determined by radioimmunoassay in urethaneanaesthetized lactating rats during suckling. Oxytocin release was monitored by recording intramammary pressure. Application of ten pups, 3 h after administration of urethane (1-1 g/kg, i.p.), evoked a parallel rise in prolactin and TSH concentrations which reached a maximum during the 3rd hour of suckling and then declined. Peak hormone concentrations represented a 25-fold increase in prolactin and a ten-fold increase in TSH. Suckling also elicited a pulsatile (every 5-10 min) release of 0-5--1-0 mu. oxytocin. The gradual rise in prolactin and TSH occurred between the 1st and 20th oxytocin pulses. Intravenous injection of thyrotrophin-releasing hormone (TRH) into unsuckled, anaesthetized lactating rats resulted in a 7- to 30-fold increase in TSH concentration, whereas prolactin levels showed no substantial change. These results indicate that suckling releases TSH as well as prolactin in the urethaneanaesthetized rat. However, the absence of prolactin release after injections of TRH makes it unlikely that both endocrine responses are regulated solely by the actions of this one releasing hormone.

Restoration by oestradiol benzoate of a neural and hormonal rhythm in the ovariectomized rat.

The rate of [35S]methionine incorporation into protein in discrete cerebral areas was measured before and after the administration of oestradiol benzoate (OB) to chronically ovariectomized rats. The circadian rhythm of incorporation which is normally seen in the intact cyclic female rat was deleted by ovariectomy. A daily rhythm of incorporation reappeared, however, in all the brain areas studied 30 h after a single injection of OB (20 mug), and was still present 12 days later. The release of luteinizing hormone (LH) after administration of 20 mug OB was measured in chronically ovariectomized animals and was found to be biphasic. High levels of LH after ovariectomy were initially reduced by negative feedback, but this phase was followed 52 h later by a facilitation of LH release between 15.00 and 18.00 h. The facilitation of LH release at this time of day was still detectable 12 days after the initial injection. The evidence for a functional link between the rhythm of neural activity which is reflected by [35S]methionine incorporation, and the ability to 'time' the facilitation of LH release is discussed.