Immune carrier properties of acid-treated Salmonella minnesota R595 bacteria. The immune response to TNP-bacterial conjugates in rabbits and mice.

Salmonella minnesota R595 bacteria from which the core region of the lipopolysaccharide on the cell wall had previously been removed by mild acid treatment were trinitrophenylated. Differing amounts of these trinitrophenyl naked bacterial conjugates (TNP-NB), covering a range of epitope densities, were used for immunising mice and rabbits via the intraperitoneal or intravenous routes without adjuvants. It was found that such acid-treated, naked bacteria were effective carriers for the covalently linked hapten, TNP, with an optimum epitope density of 15 micrograms TNP/mg NB. Significant immune responses were obtained with dose levels as low as 50 ng TNP. The possible applications of acid-treated, naked bacteria as universal carriers having inherent adjuvant activity are discussed.

Acid-treated, naked bacteria as immune carriers for protein antigens.

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.

Metabolic activation and deactivation of fusarin C, a mutagen produced by Fusarium moniliforme.

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.

Mechanism for the stabilization in vivo of the aziridine precursor --(4-acetoxyphenyl)-2-chloro-N-methyl-ethylammonium chloride by serum proteins.

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.

The influence of fish oil supplementation on plasma lipoproteins and arterial lipids in vervet monkeys with established atherosclerosis.

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 use of peroxidase anti-peroxidase (PAP) complexes in the detection of plant viruses by ELISA.

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 mutagen produced by Fusarium moniliforme.

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.

The chemical and enzymatic interaction of glutathione with the fungal metabolite, fusarin C.

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.

The role of rat liver microsomal enzymes in the metabolism of the fungal metabolite fusarin C.

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.

Comparison of the effect of the amount and degree of unsaturation of dietary fat on plasma low density lipoproteins in vervet monkeys.

The effects of the degree of unsaturation and of the amount of dietary fat on low density lipoprotein (LDL) concentration and composition were determined in vervet monkeys. Diets with fat contents of 41, 31 and 18% energy, each with a low and a high polyunsaturated to saturated fatty acid ratio (P/S; 0.27-0.38 and 1.13-1.47) were fed to six female vervet monkeys for two months. Another six females were given a low fat, high P/S diet for the same period of time, to serve as a reference. The cholesterol contents of the diets were low (21-33 mg per day) and relatively constant. LDL cholesterol concentrations decreased significantly (P < or = 0.01) when the dietary fat content decreased from 31 to 18% of energy. The dietary P/S ratio only affected LDL cholesterol concentrations during moderate (31% of energy) fat intake, where LDL cholesterol increased (P < or = 0.01) with a decrease in dietary P/S. Substantial individual variations were observed in LDL cholesterol concentration responses to dietary fat changes. The changes in LDL cholesterol concentrations were the result of changes in the concentration of LDL particles, as the molecular composition did not differ significantly between dietary periods. The high density lipoprotein cholesterol and the plasma triacylglycerol concentrations were not influenced by the dietary fat changes. During the high P/S diets, the percentage of 18:2 (linoleic acid) increased (P < or = 0.01) and that of 18:1 (oleic acid) decreased (P < or = 0.01) in LDL esterified cholesterol, as compared to the low P/S diets. In adipose tissue triacylglycerol the percentage of 18:2 was three times higher (P < or = 0.01) during the high P/S diets than during the low P/S diets.(ABSTRACT TRUNCATED AT 250 WORDS)

METAMOD: software for steady-state modelling and control analysis of metabolic pathways on the BBC microcomputer.

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.

Tube inserts as aids in preparative ultracentrifugation.

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.

An apparatus for the concentration of large volumes of dilute protein solutions to a predetermined volume.

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.

The chemical composition and stability of bacillomycin S.

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.

Electro-extraction of viruses from infected plant tissue.

An apparatus is described which was used for rapid extraction of viruses from frozen and thawed infected plant tissues. The novel principle is the establishment of a potential gradient of 15 to 20 volts/cm at approximately 90 degrees across the leaves of the infected plant while the leaves are surrounded by buffer of low molarity and of the appropriate hydrogen ion concentration. To keep the leaves in the correct orientation they were placed as single layers between coarse rigid plastic gauze. The method, termed electro-extraction, was used as the initial step in the purification of turnip yellow mosaic, tobacco mosaic and maize streak viruses. An electron micrograph of the purified maize streak virus is presented.

Determination of the number of Fab1 pieces which react with high molecular weight antigens.

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.

A novel method of electrophoresis for the separation of individual human serum glycoproteins.

A method for the isolation of protein components by a combination of polyacrylamide gel electrophoresis and electro-extraction of slices of gel contained in cellophane dialysis tubing, using the H tube electrophoresis apparatus, is described. The method was applied to the purification of individual human serum glycoproteins. Although the glycoproteins have widely differing electrophoretic mobilities, they appeared to be antigenically interrelated judging from their complex "spurring" in Ouchterlony double gel diffusion tests.

Eliciting antibodies in chickens to human dimeric IgA. Removal of factors from human colostrum depressing anti IgA antibody production.

Contrary to expectation chickens did not readily elicit antibodies to IgA dimers when untreated human colostrum was used as antigen. When colostrum was fractionated by means of a column of 8% granulated agarose equilibrated with 10mM phosphate buffer, pH 7.2, a major and a minor fraction were obtained. The major or "1st fraction" consisted of two components with sedimentation coefficients of 10.9 S and 14.1 S, respectively. The minor or "2nd fraction" consisted of components of S values ranging from 2 to 6 and small amounts of 10.9 and 14.1 S material. When chickens were immunized with the "1st fraction" antibodies to dimeric IgA were produced. When the "1st and 2nd fractions" of the column were remixed and used for immunization of chickens, the immune response was as poor as when the chickens were injected with untreated colostrum. An immuno-depressing agent in colostrum was indicated. When rabbits were immunized with clarified human colostrum, antibodies against five antigens were elicited, one of the antigens being dimeric IgA. The immuno-depressing agent is therefore not universal. The purified agent suppressed antibody formation in chickens against the haemocyanin of Jasus lalandii. The "activity" is therefore not specific for IgA and the remaining four antigens in human colostrum. The purified component is a glyco-protein with a hexose content in excess of 10%. The derivatized sugars prepared from it were shown by gas liquid chromatography to be an equimolar mixture of galactose, mannose and fucose. The molecular weight (Mr) of the purified component was found to be 72,000 by sedimentation and diffusion and 80,000 by SDS page using Mr reference standards. The properties of the immuno-suppressor strongly suggest that it is the secretory piece of dimeric IgA.

Eliciting antibodies in chickens to human dimeric IgA removal of factors from human colostrum depressing anti IgA antibody production.

Contrary to expectation chickens did not readily elicit antibodies to IgA dimers when untreated human colostrum was used as antigen. When colostrum was fractionated by means of a column of 8% granulated agarose equilibrated with 10mM phosphate buffer, pH 7.2, a major and a minor fraction were obtained. The major or "1st fraction" consisted of two components with sedimentation coefficients of 10.9 S and 14.1 S, respectively. The minor or "2nd fraction" consisted of components of S values ranging from 2 to 6 and small amounts of 10.9 and 14.1 S material. When chickens were immunized with the "1st fraction" antibodies to dimeric IgA were produced. When the "1st and 2nd fractions" of the column were remixed and used for immunization of chickens, the immune response was poor as when the chickens were injected with untreated colostrum. An immuno-depressing agent in colostrum was indicated. When rabbits were immunized with clarified human colostrum, antibodies against five antigens were elicited, one of the antigens being dimeric IgA. The purified agent suppressed antibody formation in chickens against the haemocyanin of Jasus lalandii. The "activity" is therefore not specific for IgA and the remaining four antigens in human colostrum. The purified component is a glyco-protein with a hexose content in excess of 10%. The derivatized sugars prepared from it were shown by gas liquid chromatography to be an equimolar mixture of galactose, mannose and fucose. The molecular weight (Mr) of the purified component was found to be 72,000 by sedimentation and diffusion and 80,000 by SDS page using Mr reference standards. The properties of the immuno-suppressor strongly suggest that it is the secretory piece of dimeric IgA.

Bead centrifuge tube inserts--a semi-quantitative theory.

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.