Insertional re-activation of a chloramphenicol acetyltransferase misfolding mutant protein

The deletion of nine residues from the C-terminus of the bacterialchloramphenicol acetyltransferase (CAT) results in depositionof the mutant protein in cytoplasmic inclusion bodies and lossof chloramphenicol resistance in Escherichia coli. This foldingdefect is relieved by C-terminal fusion of the polypeptide withas few as two residues. Based on these observations, efficientpositive selection for the cloning of DNA fragments has beendemonstrated. The cloning vector encodes a C-terminally truncatedCAT protein. Restriction sites in front of the stop codon allowthe insertion of target DNA, resulting in the production ofproperly folded CAT fusion proteins and regained chloramphenicolresistance. The positive selection of recombinants is accomplishedby growth of transformants on chloramphenicol-containing agarplates. The method appears particularly convenient for the cloningof DNA fragments amplified by the PCR because minimal informationto restore CAT folding can be included in the primers. The cloningof random sequences shows that the folding defect can be relievedby fusion to a wide variety of peptides, providing great flexibilityto the positive selection system. This vector may also contributeto the determination of the role of the C-terminus in CAT folding.     

Active,passive and transpassive dissolution of a nickel base super alloy in concentrated acid mixture solution

The electrochemical and corrosion behaviour of a nickel base super alloy (C-263) has been investigated in the deaerated binary and ternary solution mixture of concentrated phosphoric acid, acetic acid, sulphuric acid, nitric acid or water using potentiostatic technique at 35°C. The possibilities of electropolishing of this alloy in these solution mixtures have been also explored. The alloy showed distinct active, passive and transpassive behaviour in the experimental solutions. The alloy remained active and turned passive in the negative potential region. Transpassive dissolution of the alloy is observed and electropolishing is achieved in this region. The best electropolishing is obtained in 50% H₃PO₄ + 40% CH₃COOH + 10% H₂SO₄. Higher content of water in the electrolytic solution is not useful for electropolishing of the alloy The experimental results also suggest that a current plateau in the transpassive potential region is not a sufficient condition to achieve electropolishing.     

Characterization of allergoids from ovalbumin in vitro and in vivo

Several in vivo and in vitro methods for monitoring immunological properties of two allergoids obtained by formaldehyde treatment of ovalbumin (OA) were developed. The calculated molecular weight of allergoids was 80 kD (OA-F1) and 165 kD (OA-F2), respectively. The allergenic activity in vitro of allergoids in mast-cell histamine release assay was 1000 times lower than of OA. Both allergoids showed reduced ability to induce passive cutaneous anaphylaxis in the Sprague-Dawley rats or systemic anaphylaxis in Dunkin-Harley guinea-pigs. The ability of OA and allergoids to bind to the OA-specific IgE antibodies was measured in vivo by the inhibition of passive cutaneous anaphylaxis (PCA-inhibition). Allergoid binding to IgE was 51-66% lower than the native allergen. Moreover, the avidity of OA-specific IgG antibodies, measured by ELISA-inhibition, for allergoids and allergen was of the same order. Allergoids induced a different pattern of humoral immune response from that, induced by the native allergen. Thus, after immunization of BALB/c mouse, both allergoids induced a higher production of IgG and a lower production of IgE than OA, only OA-F2 induced a lower production of IgG1. The differences in the IgA response to the immunogens was not significant. Delayed hypersensitivity studies in the BALB/c mouse showed that allergoids were 5- to 12-times less effective in inducing a cell-mediated immune response than OA. The present study provides a battery of immunological methods for preclinical testing of modified allergens.     

Increased concentrations of octachlorodibenzo-p-dioxin in cases with breast cancer--results from a case-control study

Organochlorines are persistent and highly lipophilic environmental contaminants which bioaccumulate in the food chain. Some of these chemicals, 2,2-bis(p-chlorophenyl)-1,1,1-trichloroethane (DDT) and polychlorinated biphenyls (PCBs), have been suggested to be of significance in the aetiology of breast cancer. 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is an anti-oestrogen in animal studies and should be thus lower the risk of breast cancer. The other isomers of polychlorinated dibenzo-p-dioxins (PCDDs) or the chemically related polychlorinated dibenzofurans (PCDFs) have not been tested regarding carcinogenesis of the breast. The purpose of this study was to investigate whether PCDDs or PCDFs influence the risk for breast cancer. Consecutive patients who underwent surgery for a breast disease between 1993 and 1995 were recruited for the study. Cases were 22 patients with infiltrative breast cancer and controls were 19 patients operated for a benign breast disease during the same time period. Approximately 10 g of breast tissue free from tumour was taken from the specimen and frozen until analysis. Fat was extracted, cleaned and analysed with a high-resolution gas chromatograph coupled to a high-resolution mass spectrometer. Median concentrations of octachlorinated dibenzo-p-dioxin (OCDD) were 598 (170-14,880) and 396 (103-1,847) pg/g lipid in the cases and in the controls, respectively. In a multivariate logistic regression analysis controlling for other risk factors for breast cancer increased odds ratio (OR) was obtained for OCDD: 401-1000 pg/g lipid yielded OR 3.8, 95% confidence interval (CI) 0.4-39, > 1000 pg/g lipid gave OR 5.2, CI 0.4-72. When the lipid OCDD variable was examined as a continuous risk factor there was a 1.09 (9%), CI 0.95-1.25, increase in the adjusted OR for breast cancer per 100 unit (pg/g lipid) increase in OCDD. No differences were found between cases and controls for the other six tested PCDDs. Mean concentration of TCDD was in the cases 3.6 (1.0-7.9) and in the controls 3.3 (1.1-6.3) pg/g lipid. For PCDFs no significant differences were found between cases and controls. The results were not changed if oestrogen or progesterone receptor status, S-phase fraction and DNA ploidy were considered. Breast tissue concentration of OCDD was increased in cancer patients, whereas the concentrations of other PCDDs and PCDFs were equal in cases and controls.