Absorption of 3-chloro-4-(dichloromethyl)-5-hydroxy-2-[5H] furanone (MX) through rat small intestine in vitro.

The intestinal absorption of 3-chloro-4-(dichloromethyl)-5-hydroxy-2-[5H] furanone (MX), a highly mutagenic furanone found in chlorinated waters, was studied using an in vitro everted rat gut sac system, using reverse mutation in Salmonella typhimurium to detect mutagens transported from the mucosal to the serosal compartments. Absorption was measurable, but limited, with significant increase in bacterial revertants (serosal compartment) noted at a dose of 50 micrograms/ml MX (mucosal compartment, p < 0.05). Gut sac incubation with MX and glutathione (GSH, 1.0 mM) resulted in no detectable absorption of mutagens. Preincubation with diethylmaleate to deplete mucosal GSH resulted in increased absorption of MX-derived mutagens compared to controls (a significant induction of revertant colonies was noted at a dose of 25 micrograms/ml MX p < 0.05). Gut sac incubation with chlorinated fulvic acids resulted in no detectable absorption of mutagens. In vitro studies to assess the possibility of beta-lyase activation of the postulated MX-GSH conjugate showed no mutagenic activation.

Fate of fluazifop butyl in rat and human skin in vitro.

Enzyme mediated hydrolysis of fluazifop butyl has been measured with rat and human skin post-mitochondrial fractions. Rat skin had a ten times greater capacity to metabolise fluazifop butyl than human skin, but the enzyme affinities were similar. The post-mitochondrial fraction metabolism was compared to that seen during absorption in a flow through diffusion cell with viable skin. Limited hydrolysis of absorbed fluazifop butyl was seen in rat skin, but increased two fold if the stratum corneum was removed. The stratum corneum was found to retain fluazifop butyl. When the skin was pre-incubated with the esterase inhibitor bis (p-nitrophenol) phosphate (BNPP), reduced metabolism was seen. No metabolism of fluazifop butyl was seen in human skin during absorption. Retention of the compound by the stratum corneum is postulated to restrict the accessibility of the compound to the enzyme site, thus influencing the observed metabolism during the absorption process.

Prevention of postoperative renal dysfunction in patients with obstructive jaundice: a comparison of mannitol-induced diuresis and oral sodium taurocholate.

The effect of oral sodium taurocholate was compared with that of intravenous mannitol when used independently to determine their relative effect on protecting postoperative renal function in patients with obstructive jaundice. A total of 20 patients with obstructive jaundice were randomized to receive either mannitol or sodium taurocholate before surgery. Their preoperative and postoperative renal function was monitored using serum creatinine and predicted creatinine clearance over a 4-day period. There was no difference in postoperative renal function between the two groups and no patient from either group suffered significant renal dysfunction.

Investigation of the effects of beta-2 stimulation on free fatty acids in man.

In this study we present evidence that lipolysis in man is under beta-2 adrenergic control and that beta-2 stimulation produces a characteristic profile of individual free fatty acid (FFA) release. Twelve healthy volunteers received infusions of placebo (N Saline), terbutaline (a selective beta-2 agonist) and dilevalol (a new non-selective beta-blocker with beta-2 agonist activity). Plasma FFA concentrations during and after the infusions were measured using gas chromatography. A significant rise in total and individual FFAs was seen after 30 min of terbutaline infusion. This was most marked for oleic acid. Total and individual FFA concentrations also rose after 30 min of dilevalol infusion; this was only significant for oleic acid and was approximately 15% of the rise induced by terbutaline infusion. Placebo infusion did not cause any significant changes in FFA levels.

A comparison of two procedures for labelling the surface of the hydatid disease organism, Echinococcus granulosus, with 125I.

Living, intact protoscoleces of the British horse and sheep strains of Echinococcus granulosus were subjected to surface radioiodination procedures using 125I and Iodogen and 125I-Bolton Hunter reagent. Subsequent combined electron microscopy and autoradiography revealed specific surface membrane labelling with the Iodogen procedure, but significant tegumental labelling with the Bolton-Hunter reagent. The two parasite strains yielded different profiles of electrophoretically separated labelled proteins; the Iodogen method, not surprisingly, resulted in a less complex pattern of labelled polypeptides than the Bolton and Hunter reagent.

Regional specialization of the surface of a parasitic nematode.

A monoclonal antibody (NIM-M7) has been prepared which reacts with a major surface antigen of adult males and females of Trichinella spiralis. This specificity is only demonstrable when the antigen is liberated by detergent solubilization of surface-labelled worms. When reacted with living adults, on the other hand, NIM-M7 reacts well with only the eversible cloaca, or copulatory bell, of the male, binding weakly, if at all, to other surface areas of male or female worms. A similar staining pattern is also given by Concanavalin A. The differential staining given by NIM-M7 must indicate a molecular difference between the organization of the same surface antigen on the cuticular surface of the copulatory bell and other areas of the worm surface. This example of regional specialization demonstrates that the nematode cuticle is not necessarily chemically uniform.

Identification of an antigen of Onchocerca volvulus of possible diagnostic use.

Adult Onchocerca volvulus worms obtained by enzyme digestion from nodules of infected Mexicans were radio-isotope labelled by the chloramine-T or Bolton-Hunter methods. No antigenic determinants were detected in extracts of worms labelled by the chloramine-T method but 3 antigens were detected in extracts of the Bolton-Hunter labelled worms. Two were present in such small amounts that it was impractical to investigate them further, but a major component of mol. wt 20 kDa was purified by gel filtration and used in a serological survey of inhabitants of villages in Southern Mexico. Using the 20 kDa antigen, which is superficially located on both sexes of O. volvulus, sera from both non-endemic and endemic regions were analysed by radio-immunoprecipitation of this antigen. In Southern Mexico, the average sensitivity of the test was 92%, and the specificity 98%. Whilst the 20 kDa antigen did not detect antibodies in the sera of Trinidadians infected with Wuncheria bancrofti or Mansonella ozzardi, this antigen detected high levels of antibodies in Indians exposed to W. bancrofti.

The occurrence of antibodies to hidden and exposed determinants of surface antigens of Trichinella spiralis.

Mice were infected per os with Trichinella spiralis and their lymphocytes were removed and fused with mouse myeloma cell line P3 x 63Ag8653P3 for the selection of monoclonal antibodies to biochemically defined, stage-specific surface antigens of 3 parasite developmental stages: muscle larvae, adults and newborn larvae. Two separate antibodies against a defined single surface antigen of each stage were isolated. In each separate case the pair of monoclonal antibodies precipitated the same component from detergent-solubilized surface antigen preparations, but only one was able to bind to the surface of the living worm. The other must therefore be directed against an antigenic epitope which is obscured in the intact worm surface. The latter type of antibody is unlikely to be involved in the initial phase of parasite rejection and hence is another example of a non-protective host antibody response. The stimulus for its synthesis may be release of surface antigen, which does occur in vitro. One surface antigen of the newborn larvae is only detected by antibody in the first 6 h after birth; thereafter its presence is obscured as other antigens appear. The major surface antigen of the infective larvae contains carbohydrate determinants which are not available at the parasite surface. In addition, it displays great molecular heterogeneity but all variants appear to be derived from a common polypeptide structure.

Stage specific secreted and somatic antigens of Trichinella spiralis.

Infective larvae, adult males and newborn larvae of Trichinella spiralis were cultured with [35S]methionine in vitro. Total secreted and total somatic (sodium deoxycholate-soluble) proteins were analyzed by electrophoresis in the presence of sodium dodecyl sulphate (SDS-PAGE). Secreted proteins were relatively few in number and were different for each stage, whereas somatic proteins gave an unresolved smear in all cases. Immune precipitation with serum from infected mice revealed only one major antigen in secretions of all stages. In a similar investigation of the solubilised somatic antigens, the complexity of antigens ranged from none in infective larvae, through few in the adult, to many in the newborn larvae. The total concanavalin A-binding glycoproteins of each stage exhibited considerable individuality, and hence stage specificity, when resolved by two dimensional gel analysis. These results extend our knowledge of stage specific components of T. spiralis, and allow a rational approach towards the construction of diagnostic procedures.

A major change in surface antigens during the maturation of newborn larvae of Trichinella spiralis.

Newborn larvae of Trichinella spiralis were collected for 30 min from female worms in culture, incubated in vitro for various times up to 18 h, and surface-labelled with iodine. The detergent-solubilised products were examined by SDS-polyacrylamide gel electrophoresis. At time periods up to 6 h these larvae expressed only one Mr 64 000 iodine-labelled surface protein. Some time between 6 h and 18 h a further three components (apparent Mr 58 000, 34 000 and 32 000) became accessible to surface labelling. All four of these components are antigenic in that they can be immunoprecipitated with T. spiralis immune sera. Tryptic peptide analysis revealed that the 32 and 34 kDa antigens were structurally very similar, but the 58 and 64 kDa proteins differed from each other and the 32-34 kDa pair. Thus T. spiralis not only undergoes a total change in surface antigens between moults, but also major changes in surface antigen expression within one stage.

Non-covalent interactions result in aggregation of surface antigens of the parasitic nematode Trichinella spiralis.

Surface antigens of three stages of the nematode worm Trichinella spiralis has been labelled with iodine and examined by sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis under reducing and non-reducing conditions. A variety of interactions were defined: the infective larva surface antigens formed a spectrum of aggregates from 50kDa to greater than 1000kDa from subunits of 47kDa and 90kDa; in the adult worms of 60kDa complex arose fron interaction between two dissimilar molecules of 40kDa and 20kDa; the new-born larvae components formed homologous dimers from a 58kDa molecule. Aggregating molecules were adherent to lentil lectin-Sepharose and are therefore glycoproteins. The interactions observed were completely abolished by boiling in SDS/mercaptoethanol, but only partially destroyed by boiling in SDS/iodoacetamide. Based upon this, the associations can be characterized as non-covalent, but disulphide-bond-dependent. It is suggested, but not proved, that the aggregates arise from strong non-covalent hydrophobic interaction sites which are stabilized by intrachain disulphide bonds in the molecules concerned.

Positive and negative selection of cells by hapten-modified antibodies.

A method for simultaneous positive and negative selection of cells identified by an antibody is described. It is an adaption of the panning technique using anti-benzene arsonate (Ars)-coated Petri dishes to select the Ars-antibody-coated cells. The adherent cell population is recovered in a suitable state by hapten elution with Ars-azo-tyrosine.