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Oral and intraperitoneal administration of 2-(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride (Compound A), an analogue of phenyl aziridine precursors that occur in the shrub Salsola tuberculatiformis Botsch, had a contraceptive effect on female Wistar rats with a concomitant decrease in total body, uterus, and every mass and an increase in abronal mass. Compound A elicited a Type II difference spectrum and inhibited the Type I deoxycorticosterone (DOC) induced difference spectrum of sheep adrenal cytochrome P450c11 in a manner similar to that of S2, a biologically active fraction isolated from S. tuberculatiformis. The effects of Compound A on the spectral properties of P450c11 were diminished with time in PBS. Electrospray mass spectrometry (ES-MS) indicated that the rate of cyclization of Compound A to the corresponding aziridine followed a time course similar to the attenuation of cytochrome P450c11 inhibition. It was concluded that the aziridine precursor. Compound A, rather than aziridine itself, was the inhibiting agent of sheep adrenal P450c11. Addition of sheep and rat plasma prevented the attenuation of the effect of Compound A on the spectral properties of cytochrome P450c11. Subsequent ES-MS analysis indicated that Compound A was stabilized in plasma by sex hormone binding globulin and corticosteroid binding globulin. These results suggest a mechanism whereby natural plant products, which are highly reactive and unstable in vitro, can be stabilized by binding to plasma proteins, and so remain biologically active in vivo.

Cytochrome P450c17 (P450c17), together with cytochrome P450c21 (P450c21), plays an important role in progesterone metabolism in the mammalian adrenal cortex. Low levels of expression and the presence of other steroidogenic enzymes in adrenal cortex endoplasmic reticulum (ER) impedes purification and characterisation of wild type as well as mutant forms of the hemoprotein. Heterologous gene expression systems have previously been used successfully to express active P450c17. Heterologous expression can also be used for the preparation of anti-P450c17-IgG. For antibody production larger amounts of pure P450c17 peptide, rather than the active protein, is, however, desirable. If the expressed protein can be affinity tagged and secreted into the medium, isolation and purification will be facilitated. Saccharomyces cerevisiae, YPH259, was transformed with a modified YCplac111 yeast expression-secretion vector (pPRL2). The gene coding for a truncated human P450c17 (signal anchor sequence 1-18 was removed) was inserted, in reading frame, downstream from the leader sequence MF alpha. A histidine tag was incorporated at the C-terminus. The modified yeast expression vector was expressed in yeast, the secreted P450c17-peptide purified by affinity chromatography and identified by immunoblot analysis.

Ingestion of the Namibian shrub Salsola tuberculatiformis Botsch. by virgin female rats extends the dioestrus period of their oestrous cycles. Methanol extracts of the plant also inhibit adrenal steroidogenesis in the rat. With the aid of a bioassay, in which vaginal smears were used to follow the oestrous cycles of virgin rats, active fractions could be obtained which indicated that the plant contains a number of active compounds. The most active of these are highly unstable compounds which could not be isolated in pure form. However, two stable but less active compounds were identified as 4-hydroxyacetophenone and 4-hydroxy-3-methoxyacetophenone. This study investigated the influence of these acetophenones, their glucosides, and that of ethanol extracts of S. tuberculatiformis on adrenal steroidogenesis. Acetovanillon, a structurally related natural product also known as compound Z, was included in this study. Results show that the shrub contains active substances which interfere with adrenal 11 beta-hydroxylase, the terminal enzyme in glucocorticoid biosynthesis. This interaction with the cytochrome P-450(11)beta-dependent hydroxylase, as well as the inhibition of the conversion of deoxycorticosterone to corticosterone, was used to develop two sensitive and reliable assays for the rapid identification of small amounts of active compounds from S. tuberculatiformis.

There is controversy about whether supplementing diets with marine fish oil can regress, promote or prevent atherosclerosis. Therefore the effects of an Atlantic pilchard oil (FO) supplement and dietary change were measured in a proven atherosclerosis model. Vervet or African Green monkeys were fed an atherogenic diet (AD) for long enough to ensure progression before treatments started. Matched groups were then treated for 20 months, either by adding FO to the AD (AD/FO), or by changing to a therapeutic diet with FO (TD/FO). Control treatments consisted of supplementing with sunflower oil (SO) instead of FO, so that treatments were AD/SO and TD/SO. The same total polyunsaturates were supplied by the FO and SO and the dose of FO was realistic (2.5% of total energy). A reference group (R) received the TD with no oil supplements. Supplementing with FO did not change the concentrations of total, low or high density lipoprotein cholesterol in plasma. After The AD/FO the intimas of aortas contained more total (p < or = 0.001), free (p < or = 0.05) and esterified (p < or = 0.05) cholesterol, total phospholipid (p < or = 0.01) and sphingomyelin (p < or = 0.05) than after the AD/SO. After FO supplementation eicosapentaenoic acid was significantly higher and arachidonic acid significantly lower in the plasma and aorta intima phosphatidylcholine. None of these changes was anti-atherogenic in terms of atherosclerosis measured in the same individuals (1). Nor did FO increase the efficacy of the TD.

The metabolic activation and deactivation of fusarin C, a mutagen produced by Fusarium moniliforme strain MRC 826, was studied by the Salmonella typhimurium mutagenicity assay using tester strain TA 100. A microsomal monooxygenase, preferably induced by phenobarbitone (PB) activities the mutagen to its active mutagenic form. Deactivation of the mutagenic metabolite seems to occur through chemical binding to thiol groups and by enzymatic conjugation mediated by a cytosolic glutathione -S-transferase.

The H tube electrophoresis technique was used for isolating human IgG by the net effect of electrophoresis and electro-endosmosis in agarose gel containing human serum. In a similar experiment human serum albumin was purified by extracting the agarose gel slice containing the albumin following zone electrophoresis of serum in agarose. By taking advantage of the relatively high reverse electro-endosmotic flow compared to the migration of IgG in agarose gel, sheep anti chicken immunoglobulin was fractionated into active and inactive portions. It was estimated that 90% of the nonspecific IgG could be eliminated in this manner. The specific immunoglobulin fraction showed a higher mobility to the positive electrode than the bulk of the non specific IgG. The H tube proved to be ideal for zone electrophoresis in concentration gradients of inert substances. For this purpose sugar gradients were used for electrophoretic separation of proteins and viruses which are not affected by high osmotic pressures but, for separating animal cells it was necessary to use Ficoll gradients. A mixture of yellow Atypical Tomato Mosaic Virus and the haemocyanin of Burnupena cincta were separated into discrete zones and chicken IgY anti haemocyanin was subfractionated. The specific fraction showed a lower migration rate to the positive pole than the bulk of the non specific material.

An apparatus was designed and manufactured that can be used for the concentration of large quantities of dilute protein solutions to a predetermined volume. The method is based on the osmotic transfer of water to a concentrated hydrophilic polymer (poly ethylene glycol, PEG) through a protein-stopping dialysis membrane and is a refinement of a method previously reported by van Oss. The apparatus is made of perspex. Concentration takes place through a commercially available dialysis tube and is aided by a 40% polyethylene glycol solution. Large volumes (5l) of dilute protein solution could be reduced to 50 ml at a rate of 30 ml per hour with no significant loss in biological activity.

Conventional preparative ultracentrifugation was modified by using inserts in the centrifuge tubes, causing a reduction of the time necessary to centrifuge a protein at reduced rotor velocity from suspension.

Salmonella minnesota R595 bacteria were stripped of their natural antigenic determinants to yield acid-treated, naked bacteria. The proteins, human apolipoprotein A1 and carcino-embryonic antigen, were adsorbed to naked bacteria and these complexes were used to immunise rabbits. Although the antibody titres obtained were comparable to those achieved using Freund's adjuvant emulsions, much less antigen was needed for immunisation. This technique could be of great value where the amount of protein available for immunisation is very small.

Bacillomycin S, an antifungal antibiotic from culture filtrates of Bacillus subtilis, was shown to consist of proteinaceous matter (83.6 g/100 g) and lipid material (16 g/100 g). The amino acid and fatty acid components were determined qualitatively and quantitatively. It was shown that bacillomycin S is stable at physiological pH and it is suggested that its antifungal activity resides in the intact molecule.

Granulated agarose gels suitable for gel exclusion chromatography of proteins of any molecular weight may now be prepared. This was made possible by the observation that agarose solutions of 16% polysaccharide may be prepared by displacing 8% agarose from solution with 8% polyethylene glycol Mr 6000. The displaced polysaccharide concentrates in a viscous mass occupying half the volume of the original carbohydrate solution. By diluting the displaced polysaccharide with hot watery solutions of electrolyte and allowing the solutions to congeal, gels of any desired concentration, ranging from low to the maximum of 16%, may be prepared.

Glutathione (GSH) interacts both chemically and enzymatically with fusarin C, a mutagenic metabolite produced by Fusarium moniliforme. The chemical reaction, which is pH-dependent, results in the formation of both fusarin A and a compound that lacks the 2-pyrrolidone moiety thereby suggesting an interaction at the C-13-C-14 epoxide. Enzymatic interaction of fusarin C with GSH also appears to occur at this site as fusarins A and D, which lack the epoxide, do not serve as substrates for GSH-S-transferases. The interaction of GSH with fusarin C appears to be an important deactivation step which could explain the lack of carcinogenicity observed for fusarin C in rats.

A mutagenic compound produced by Fusarium moniliforme on maize was isolated by CHCl3--iso-PrOH extraction, solvent partitioning and liquid chromatography on silica gel and Sephadex LH-20. HPLC studies showed that different mutagenic and non-mutagenic forms can be derived from the mutagen (P3) and that prolonged exposure to longwave u.v. light and to high temperatures causes a total loss of its u.v. absorption and mutagenic characteristics. Spectral data presented for P3 include u.v., i.r., mass spectra as well as 1H NMR and 13C NMR. Mass spectral data indicated a molecular formula of C23H29NO7.

METAMOD, a BBC microcomputer-based software package for steady-state modelling and control analysis of model metabolic pathways, is described, The package consists of two programs. METADEF allows the user to define the pathway in terms of reactions, rate equations and initial concentrations of metabolites. METACAL uses one of two algorithms to calculate the steady-state concentrations and fluxes. One algorithm uses the current ratio of production and consumption rates of variable metabolites to adjust iteratively their concentrations in such a way that they converge towards the steady state. The other algorithm solves the roots of the system equations by means of a quasi-Newtonian procedure. Control analysis allows the calculation of elasticity, control and response coefficients, by means of finite difference approximation. METAMOD is interactive and easy to use, and suitable for teaching and research purposes.

The metabolic activation of fusarin C by a rat liver microsomal monooxygenase resulted in the formation of a water-soluble mutagenic metabolite. However, fusarin C incubated in the presence of a microsomal preparation, but in the absence of an NADPH-generating system, led to the formation of fusarin PM1, a highly water-soluble compound which, like fusarin C, requires metabolic activation to be mutagenic. Enzyme studies using as substrates fusarins A and D, compounds structurally related to fusarin C, together with structural studies of fusarin PM1 indicated that fusarin PM1 was formed by the action of carboxylesterase which hydrolyses the C-20 methyl ester group to a free carboxylic acid.

An ELISA test system for detection of plant viruses in field samples is described, based on the unlabelled antibody method using a peroxidase-antiperoxidase (PAP) complex. Novel features of the system include the use of acid-treated naked bacteria as combined carrier-adjuvants for the production of rabbit antiviral antibodies, and the use of acid-treated chicken antibodies (IgY) for antigen trapping in the ELISA. Systems for detection of potato virus Y (PVY), potato leafroll virus (PLRV), grapevine fanleaf virus (GFV) and grapevine virus A (GVA) were developed and compared with conventional direct double antibody sandwich (DAS) systems in tests with both purified virus and field samples. The PAP systems offer improved sensitivity, no background problems in the outer rows of the microtitre plates and are much easier to visualize with the naked eye if no plate reader is available.

A semi-quantitative theory is proposed for the sedimentation of particles in centrifuge tubes filled with polymer beads acting as sedimentation aids. The theory concerns the attainment of approximate sedimentation-diffusion equilibrium in minimum time of centrifugation. It is shown empirically that the time to reach this state is approximately proportional to the diameter of the polymer beads as predicted by the theory.

The sedimenting-droplet technique was used for the determination of the masses of immune Fab1 pieces which may bind to antigenic sites on three haemocyanins. Similar experiments were conducted on three viruses which belong to the Tobamo group. It was calculated that 384 homologous Fab1 pieces could bind to a molecule of the haemocyanin of Burnupena cincta and 950 homologous Fab1 pieces to a virion of tobacco mosaic virus. By taking special precautions the number of undegraded antibody molecules which can bind to a Tobacco mosaic virus particle may be determined with the same technique.

Moiety-conserved cycles are metabolic structures that interconvert different forms of a chemical moiety (such as ATP-ADP-AMP, the different forms of adenylate), while the sum of these forms remains constant. Their metabolic behaviour is treated within the framework of control analysis [Kacser, H. & Burns, J.A. (1973) Symp. Soc. Exp. Biol 27, 65-104]. To explain the importance of the conserved sum of cycle metabolites as a parameter of the system, the cycle is first regarded as a 'black box'. The interactions of the cycle with the rest of the system are expressed in terms of 'cycle elasticities' and 'cycle control coefficients' by the usual connectivity properties. The conserved sum is seen to be an 'external' parameter in the sense that its effect is described by a combined response expression. All cycle coefficients can be written in terms of elasticities and concentrations of cycle metabolites. The treatment shows how connectivity expressions should be modified when moiety-conserved cycles are present and establishes new summation and connectivity properties. The analysis is applied to a two-member moiety-conserved cycle and its general application is discussed.

A new method for the removal of the stabilizing substrate, deoxycorticosterone, from adrenal cytochrome P-450(11) beta, has been developed. Dextran coated charcoal is used for the adsorption of the steroid and the adsorbed steroid is separated from the cytochrome P-450-preparation by low speed centrifugation. The substrate-free enzyme, obtained in this manner, has all the characteristic spectral properties of low-spin cytochrome P-450(11) beta and may be converted to the high-spin form by the addition of deoxycorticosterone. The dextran coated charcoal method has the following advantages over the previously used method of substrate removal. It does not require the addition of the cofactors for cytochrome P-450-dependant hydroxylation of deoxycorticosterone, small amounts of enzyme may be prepared in a short time and the enzyme preparation is not diluted to any great extent during the process.