Reduction of the infectivity of scrapie agent as a model for BSE in the manufacturing process of Trasylol.

The Trasylol manufacturing process was investigated with respect to its capacity for the inactivation/removal of infectivity causing bovine spongiform encephalopathy (BSE). Four process steps were selected for this investigation and scaled down to laboratory scale. Authentic samples of bovine lungs used in the Trasylol manufacturing plant were taken and spiked in laboratory scale experiments with high infectious titres of the rodent adapted scrapie strain ME 7 which served as model for BSE. After performing the respective process steps the output samples collected were tested in C57BL mice carrying the Sinc gene. An overall reduction of the infectious agent in the order of 18 log10 was observed, indicating a very high capacity of the Trasylol process for the inactivation/removal of the BSE/scrapie agent. The discussed safety strategy for the product leads to the conclusion that Trasylol is BSE safe.

Regions of an Eimeria tenella antigen contain sequences which are conserved in circumsporozoite proteins from Plasmodium spp. and which are related to the thrombospondin gene family.

Coccidiosis, caused by Eimeria spp., is a major disease of economic importance to the poultry industry. The cloning and characterisation of genes coding for antigens of those species infecting chickens is an initial step in the identification of protective antigens suitable for the development of a genetically engineered vaccine. This report describes the molecular characterisation of an antigen of E. tenella produced by the recombinant lambda amp3 bacteriophage EtHL6. Three native polypeptides corresponding to the EtHL6 antigen, with sizes between 110 and 94 kDa, have been identified on both sporozoites and second generation merozoites of E. tenella by mouse antisera raised against the EtHL6 fusion protein. The DNA insert is a 722-bp EcoRI fragment encoding a polypeptide comprising three tandem blocks of amino acids which are highly homologous to each other. Each region, A, B and C, contains a strongly hydrophilic domain and two pairs of cysteine residues. Computer analysis has identified similarities with a group of proteins which include the circumsporozoite antigen and thrombospondin-related anonymous protein (TRAP) of malaria parasites, human thrombospondin, mouse properdin and the terminal components of the complement pathway.

Characterization of coccidial proteins by two-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis.

Two dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (2D SDS-PAGE) has been used to produce 'fingerprint' maps of the proteins from each of the 7 species of Eimeria which infect the chicken. All 7 species could be identified from their array of polypeptides but few differences were detected between strains of the same species. Alterations to the polypeptide array associated with the stage of sporulation of the oocysts were observed. Iodination of sporozoites, 2D SDS-PAGE, autoradiography and immunoblotting techniques were combined to identify polypeptides with a surface moiety and those which were antigenic.

Eimeria tenella: immunological diversity between asexual generations.

In the development of a normal strain, WIS, of Eimeria tenella there are three generations of schizogony whereas in an attenuated line, WIS-F-96, derived from WIS, the second and third are absent. Chickens immunized by infection with WIS-F-96, however, were highly resistant to oral challenge with sporulated oocysts of WIS, and histological studies indicated that the immune response was directed against the sporozoites from that challenge inoculum. When challenge of the WIS-F-96-primed chickens consisted of second generation merozoites of WIS (inoculated intracaecally), immunity was less pronounced and the histological data indicated that the merozoites proceeded to develop normally in these birds. These indications of immunological diversity between the merozoites of the first and second generations of schizogony of E. tenella WIS correlated with the results of preliminary studies of the antigenic composition of these developmental stages.

Eimeria tenella sporozoites: the method of excystation affects the surface membrane proteins.

Eimerian sporozoites can be recovered from intestinal washings after oral administration of oocysts to chickens but suspensions of sporozoites are usually prepared in the laboratory by incubation of sporocysts or fractured oocysts in vitro, at body temperatures, with relatively high concentrations of trypsin and bile salts. Since these agents affect membrane structure, the surface membrane of proteins of Eimeria tenella sporozoites excysted in vivo and in vitro have been compared. Surface radio-iodination followed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that more 125I was incorporated into polypeptides on sporozoites excysted in vivo than on sporozoites excysted in vitro. The 125I-polypeptide profile of sporozoites excysted in vivo was more resistant to subsequent incubation with pure trypsin than that of sporozoites excysted in vitro, but incubation with bile salts resulted in the loss of some iodinated polypeptides from both preparations of iodinated sporozoites. Reaction with combinations of crude trypsin and bile salts led to the lysis of sporozoites. The method of excystation had no effect on the reaction of convalescent chicken serum with Western blots of sporozoites but the results of immunofluorescent staining carried out with mouse monoclonal antibodies indicated that the structure of the cell surface was altered and some antigenic determinants were lost from sporozoites excysted in vitro. In contrast, neither the infectivity of sporozoites determined in vivo, nor their invasion of cultured cells was changed by the method of excystation.

Isolation of lambda amp3 genomic recombinants coding for antigens of Eimeria tenella.

Eimeria are the causative agents of coccidosis, a disease which is of world wide economic importance in the poultry industry. Immunity resulting from infection is species-specific and both antibody and cell-mediated responses have been implicated. As an initial step in the development of a genetically-engineered vaccine against coccidiosis, libraries of EcoRI-digested genomic DNA from E. tenella have been constructed in Escherichia coli using the expression vector lambda amp3. Screening of the libraries with serum from chickens immunized by infection has identified at least 24 different recombinant phage which produce eimeria antigens fused to beta-galactosidase. A significant proportion of the Eimeria DNA inserts cross-hybridise with each other and contain sequences which are highly represented in the genome. The identification of these clones will enable the isolation of intact genes from E. tenella DNA and facilitate detailed analysis of the antigens and immune responses.

Identification of the sporozoite antigens of Eimeria tenella.

The surface membranes of Eimeria tenella sporozoites were labelled with 125I and polypeptides resolved by polyacrylamide gel electrophoresis in sodium dodecyl sulphate (SDS-PAGE). The most heavily labelled polypeptides were 47, 26, 21 and less than or equal to 18 kDa but significant amounts of 125I were incorporated into a number of polypeptides with molecular weights ranging from greater than 200 000 to less than 18 000. Similar 125I-polypeptide profiles were observed after the surface labelling of sporozoites of E. acervulina, E. maxima and E. nieschulzi. Sporozoites of E. tenella were also radiolabelled by incubation in medium containing [35S]methionine and SDS-PAGE resolved more than 35 radiolabelled polypeptides with molecular weights from greater than 200 000 to less than 18 000. 125I and 35S-labelled sporozoites of E. tenella were solubilised in the detergents Triton X-100 or sodium deoxycholate and immunoprecipitated with serum from chickens immunized by infection with this species. Polypeptides of unlabelled E. tenella sporozoites, resolved by SDS-PAGE, were blotted onto nitrocellulose and the antigens, which reacted with the chicken serum, identified by immunoperoxidase staining. There was some variation between different sporozoite preparations in the number and molecular weights of antigens identified by these techniques but, consistently, the major surface polypeptides that were specifically immunoprecipitated were 104, 47 and 43 kDa. Specifically immunoprecipitated 35S-labelled antigens were of 123-94 kDa, 54-42 kDa and 32-25 kDa and antigens detected on Western blots were within the following ranges: 113-96 kDa, 73-67 kDa, 54-42 kDa, 37-32 kDa and 18-14 kDa.

The large-scale preparation of purified sporozoites of Eimeria spp. by metrizamide density-gradient centrifugation.

A method is described for the reproducible purification of greater than 1.0 X 10(8) sporozoites of Eimeria spp. by centrifugation on metrizamide density-gradients. The mean contamination of the purified sporozoite fraction by sporocysts and sporocyst debris was less than 6% and the recovered sporozoites were fully viable, infective and ultrastructurally intact.

Hydrodynamic characterization of the photoaffinity-labeled insulin receptor solubilized in Triton X-100.

The insulin receptor in isolated rat liver plasma membranes was covalently labeled with the photoreactive insulin analogue NB-29-[(4-azido-2-nitrophenyl)acetyl]insulin and solubilized with the nondenaturing detergent Triton X-100. The resulting protein-detergent complex was characterized by gel filtration on Sepharose 6B, sedimentation rate determination in linear sucrose gradients, and equilibrium isopycnic centrifugation in NaBr and CsCl. The labeled insulin receptor was found in two forms. The Strokes radii and s20,w's of the two receptor-detergent complexes (R1 and R2) were (mean +/- SEM) 7.08 +/- 0.04 and 3.62 +/- 0.05 nm and 10.45 +/- 0.04 and 6.54 +/- 0.15 S, respectively. The two forms appeared to have the same buoyant density, 1.285 +/- 0.002 g cm-3. The dissociation of R2 from R1, or its reaggregation, either with itself or with other unlabeled proteins, to give R1 proceeded without chemical modification. Mild reduction of disulfide bonds (1 mM 1,4-dithiothreitol) increased the dissociation of R2 from R1. These results indicate that the solubilized receptor binds significant amounts of detergents, that the insulin binding component of the receptor binds to other receptor components by hydrophobic interactions, and that one or more components of the insulin receptor contain intrachain disulfide bonds.

Insulin action on adipocytes. Evidence that the anti-lipolytic and lipogenic effects of insulin are mediated by the same receptor.

1. The dose-response relationships of insulin stimulation of lipogenesis and inhibition of lipolysis were studied simultaneously by using rat adipocytes to determine whether these different effects of insulin are mediated through the same or different sets of receptors. 2. The sensitivity (defined as the concentration of insulin required to produce a half-maximal effect) of the stimulated lipogenic response to insulin was not significantly different from the sensitivity of the anti-lipolytic response to insulin. The addition of different adrenaline and glucose concentrations did not alter the half-maximal concentration of insulin required to inhibit lipolysis. 3. The specificities of the lipogenic and antilipolytic responses were studied by using insulin analogues. The sensitivities of the lipogenic and anti-lipolytic responses were the same for five chemically modified insulins and hagfish insulin, which have potencies compared with bovine insulin of between 3 and 90%. 4. Starving rats for 48h significantly increased the sensitivities of both the antilipolytic and lipogenic responses to insulin, but the changes in the sensitivities of both lipogenesis and anti-lipolysis returned to that of fed rats. 5. We conclude that insulin stimulates lipogenesis and inhibits lipolysis over the same concentration range. These observations provide powerful evidence that the different effects of insulin are mediated through the same set of receptors.

Preparation of plasma-membrane subfractions from isolated rat hepatocytes.

1. Rat livers were dissociated into their constituent cells by perfusion through the portal vein with a medium containing collagenase, and hepatocytes separated from non-parenchymal cells. 2. It is shown that the procedure described by Wisher & Evans [(1975) Biochem. J. 146, 375-388] for preparation of plasma membranes from liver tissue when applied to isolated hepatocytes also yielded subfractions of similar morphology and marker-enzyme distribution. 3. Thus the distribution of alkaline phosphodiesterase, 5'-nucleotidase and the basal and glucagon-stimulated adenylate cyclase among two 'light' vesicular and one 'heavy' junction-containing plasma-membrane subfractions paralleled that reported for tissue-derived plasma-membrane subfractions. 4. Increased recoveries and specific activities of plasma-membrane marker enzymes were obtained when soya-bean trypsin inhibitor was included in the collagenase-containing perfusion media used to dissociate the liver. 5. Polyacrylamide-gel-electrophoretic analysis of the corresponding plasma-membrane subfractions prepared from liver tissue and isolated hepatocytes were generally similar. 6. The results indicate that the functional polarity of the hepatocyte's plasma membrane is retained after tissue dissociation. The damage occurring to plasma-membrane ectoenzymes by the collagenase-perfusion procedure is discussed.

The lipid composition of plasma membrane subfractions originating from the three major functional domains of the rat hepatocyte cell surface.

1. The neutral and phospholipid compositions of three rat liver plasma membrane subfractions originating predominantly from the three major functional domains of the hepatocyte viz the blood sinusoidal, contiguous and bile canalicular fractions, were determined. 2. The sinusoidal and canalicular plasma membrane subfractions, both of which were vesicular, contained a higher lipid to protein weight ratio than the contiguous plasma membrane subfraction that consisted of membrane strips, junctional complexes and some larger vesicles. The three plasma membrane subfractions contained a similar neutral lipid to phospholipid ratio. The highest unesterified cholesterol content was associated with the canalicular plasma membrane subfraction. 3. The phospholipid profiles of the three subfractions were generally similar. However, the canalicular plasma membrane subfraction contained a higher proportion of sphingomyelin than the other subfractions. 4. Correlations between the neutral and phospholipid composition of the subfractions and membrane integrity and function are discussed, especially with respect to a possible role of lipids in governing the resilience of the canalicular plasma membrane to the action of bile salts.

Functional polarity of the rat hepatocyte surface membrane. Isolation and characterization of plasma-membrane subfractions from the blood-sinusoidal, bile-Canalicular and contiguous surfaces of the hepatocyte.

1. Six rat liver plasma-membrane subfractions of different density and morphological, enzymic and chemical properties were prepared from homogenates by a combination of differential, rate-zonal and density-gradient centrifugation. They consisted of three vesicular 'light' subfractions of density 1.12-1.13 and three 'heavy' subfractions of density 1.16-1.18 containing membrane strips and intercellular junctions. 2. All six subfractions contained a basal adenylate cyclase activity. One of the 'light' subfractions that showed the highest glucagon-stimulated adenylate cyclase activity was identified as deriving form the blood-sinusoidal face of the hepatocyte. This subfraction, unlike the others, was contaminated by Golgi components, as indicated by its morphological properties and the presence of galactosyl- and sialyl-transferase activities. 3. All the six subfractions showed high activities of the following plasma-membrane marker enzymes: 5'-nucleotidase, alkaline phosphodiesterase (nucleotide pyrophosphatase), alkaline phosphatase, leucine naphthylamidase and Mg2+-activated adenosine triphosphatase. A 'light' subfraction that showed the highest specific activities of all the above marker enzymes, but lacked a glucagon-stimulated adenylate cyclase activity, was identified as deriving from the bile-canalicular face of the hepatocyte. 4. The 'heavy' subfractions, which showed generally the lowest activities of the above plasma-membrane enzyme markers, and were characterized by the presence of desmosomes and gap junctions, were taken to originate from the contiguous faces of the hepatocyte. 5. The protein composition of the six subfractions was generally similar, as shown by polyacrylamide-gel electrophoresis. Differences in the amounts of various protein and glycoprotein bands among the subfractions correlated with their morphology, enzymic composition and sialic acid content. 6. Hormonal and histochemical evidence supporting the identification of a bile-canalicular subfraction, a blood-sinusoidal subfraction and contiguous-face subfractions is discussed.