Depression of cell-mediated immunity by tumour cell products: induction of resistance by immunotherapeutically active extracts of bovine ocular squamous cell carcinoma

Summary Tumours produce substances that inhibit the expression of cell-mediated immunity, in the form of delayed-type hypersensitivity in mice. Phenol-saline extracts of bovine ocular squamous cell carcinoma (BOSCC) which have immunotherapeutic activity in cattle were able to immunize mice against this depressive effect. Such immunization was effective against products of BOSCC, a spontaneous rat tumour, three of four human tumour cell lines and (in other experiments) mouse tumours. Phenol-saline extracts of mouse tumour cell lines were immunogenic (protective against depression of delayed-type hypersensitivity) in mice. Fractions of BOSCC phenol-saline extracts which were immunotherapeutically active in cattle were generally also protective in mice. The protective activity was lost after treatment with proteinase K, and was present in the supernatant after precipitation with 55% ammonium sulphate. It was not affected by treatment with RNase or DNase or by heating to 50 °C for 2 h. It was present in gel filtration fractions with an apparent molecular weight of 10,000–37,000 daltons. The immunogenic factor in mice and the immunotherapeutic factor in cattle may be related to each other.     

Comparison of different methods of immobilization for lignin peroxidase production by Phanerochaete chrysosporium

Summary Phanerochaete chrysosporium was immobilized in agar, agarose and -carrageenan gel beads, nylon web, and polyurethane foam, and used for the production of lignin peroxidase in shake cultures on a carbon-limited medium. Nylon was found to be the best carrier, with the maximum lignin peroxidase activity (340 U/l) reached on the 7th day. The enzyme production rate was significantly lower with freely suspended mycelial pellets. Both nylon and polyurethane based biocatalysts were active for at least 38 days after the addition of veratryl alcohol. Best results were obtained when a spore inoculum was used instead of day-old pellets. -Carrageenan was found unsuitable as a carrier for lignin peroxidase production.     

Immunological Detection and Quantitation of Tryptophan Decarboxylase in Developing Catharanthus roseus Seedlings

l-Tryptophan decarboxylase (TDC) (EC 4.2.1.27) enzyme activity was induced in cell suspension cultures of Catharanthus roseus after treatment with a Pythium aphanidermatum elicitor preparation. The enzyme was extracted from lyophilized cells containing high levels of TDC and the protein was purified to homogeneity. The pure protein was used to produce highly specific polyclonal antibodies, and an enzyme-linked immunosorbent assay (ELISA) was developed to quantitate the level of TDC antigen during seedling development and in leaves of the mature plant. Western immunoblotting of proteins after SDS-PAGE with anti-TDC antibodies detected several immunoreactive proteins (40, 44, 54.8, 55, and 67 kilodaltons) which appeared at different stages during seedling development and in leaves of the mature plant. The major 54.8 and 55 kilodalton antigenic proteins in immunoblots appeared transiently between days 1 to 5 and 5 to 8 of seedling development, respectively. The 54.8 kilodalton protein was devoid of TDC enzyme activity, whereas the appearance of the 55 kilodalton protein coincided with the appearance of this decarboxylase activity. The minor immunoreactive proteins (40, 44, and 67 kilodaltons) appeared after day 5 of seedling development and in older leaves of the mature plant, and their relationship, if any, to TDC is presently unknown. Results suggest that the synthesis and degradation of TDC protein is highly regulated in Catharanthus roseus and that this regulation follows a preset developmental program.     

The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.     

Synthesis of the penicillin precursor, δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine (ACV), by an immobilized enzyme preparation

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV) synthetase activity has been partially-purified from cell-free extracts of Streptomyces clavuligerus by ammonium sulfate precipitation. The salt precipitated enzyme was immobilized on an anion exchange resin and synthesis of ACV was observed by exposing the immobilized enzyme preparation to a reaction mixture containing l--aminoadipic acid, l-valine and l-cysteine in the presence of appropriate cofactors. Reaction mixtures containing l--aminobutyric acid(aB) in place of l-valine synthesized the ACV analog ACaB. Immobilized ACV synthetase can be reused, and after six cycles of reaction, 28.9% of original activity remains.     

Transformation by Complementation of an Adenine Auxotroph of the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium

Swollen basidiospores of an adenine auxotroph of Phanerochaete chrysosporium were protoplasted with Novozyme 234 and transformed to prototrophy by using a plasmid containing the gene for an adenine biosynthetic enzyme from Schizophyllum commune. Transformation frequencies of 100 transformants per μg of DNA were obtained. Southern blot analysis of DNA extracted from transformants demonstrated that plasmid DNA was integrated into the chromosomal DNA in multiple tandem copies. Analysis of conidia and basidiospores from transformants demonstrated that the transforming character was mitotically and meiotically stable on both selective and nonselective media. Genetic crosses between double mutants transformed for adenine prototrophy and other auxotrophic strains yielded Ade⁻ progeny, which indicated that integration occurred at a site(s) other than the resident adenine biosynthetic gene.     

Spectral evidence for non-calcium interactions of intracellular Indo-1

Indo-1 is widely used to measure intracellular free calcium, [Ca2+]i, by comparing the fluorescence emission at 2 or more wavelengths with the emissions, which are assumed to be known, of Indo-1 when it is fully calcium-bound and when it is fully calcium-free. Accurate quantitation requires that these "reference" values be obtained on intracellular dye, and the full spectra of this study show that the reason is a significant spectral shift of the calcium-free peak, but not the calcium-bound. A mathematical analysis shows that the new peak must be a new state of the Indo-1 molecule, since it cannot be simply due to residual calcium in the cell. When intracellular "reference" spectra were used in the data analysis, [Ca2+]i could be calculated from whole spectra or from the ratio of observations at two wavelengths with good agreement. When extracellular "reference" spectra were used, the value calculated by the ratio method depended on the choice of wavelengths.