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Transfection is used to introduce a gene of interest into a cell. To interpret the downstream results, understanding which effects are the true biological responses to the gene and which, if any, are off-target effects can be difficult. In order to discriminate true biological effects from off-target effects, we transfected a breast cancer cell line, MCF7, with a vector encoding either a reporter gene or the identical vector without the reporter gene insert. Both resulted in similar numbers of differentially expressed transcripts, suggesting that very few of the responses were directly due to the introduction of the reporter gene. We postulate that many differentially expressed transcripts are the result of the introduction of foreign DNA, as the biological processes associated with these genes are primarily associated with an immune response to a viral infection. Interestingly, different transfection reagents resulted in >10-fold difference in the number of differentially expressed transcripts. This suggests the importance of testing multiple reagents and selecting the best transfection reagent along with the appropriate vector within the context of the experimental model system to ensure that the majority of the observed responses are biological effects of the gene of interest and not based on a particular transfection process used.

The Nicotiana tabacum lectin, called Nictaba, is a nucleocytoplasmic plant lectin expressed in tobacco leaves after exogenous application of specific jasmonates and upon insect herbivory. Since the lectin concentrations are rather low, huge amounts of plant material are needed to purify milligram quantities of the protein. In addition, the purified lectin fractions are always contaminated with low molecular weight compounds such as phenols. In an attempt to improve and facilitate the purification of the tobacco lectin in reasonable amounts, an in vitro-coupled transcription/translation system based on an Escherichia coli lysate was used to express the lectin gene. Recombinant expression levels could be enhanced by an adapted codon usage. Recombinant lectin was purified, biochemically characterized and found to be biologically active. The biological activity of the recombinant lectin towards insect epithelial midgut cells was clearly demonstrated in a functional bio-assay and the internal cellular localization was analyzed using immunocytochemical techniques.

A series of substituted allylbenzene derivatives was tested in vitro for ability to inhibit cell growth in the KB cell line. None of the compounds was as active as Δ9-tetrahydrocannabinol. It is suggested that inhibition of cell growth by cannabinoids requires a polycyclic system which may act by an intercalaction mechanism.

A method, utilizing brine shrimp (Artemia salina Leach), is proposed as a simple bioassay for natural product research. The procedure determines LC (50) values in microg/ml of active compounds and extracts in the brine medium. Activities of a broad range of known active compounds are manifested as toxicity to the shrimp. Screening results with seed extracts of 41 species of Euphorbiaceae were compared with 9KB and 9PS cytotoxicities. The method is rapid, reliable, inexpensive, and convenient as an in-house general bioassay tool.

A line of cells (IP-3), persistently infected with defective measles virus, was developed from co-cultures of subacute sclerosing panencephalitis-derived brain cells with monkey kidney cells (BSC-1). The line, carried for more than 45 serial passages, produced neither infectious virus nor hemagglutinin. Cultures consistently displayed a cycling focal cytopathic pattern of infection characterized by formation of syncytia, necrosis, and plaques followed by healing. Fluorescent measles antigen was present in damaged areas only, especially in syncytia. The development of syncytia was suppressed by measles antibody. Syncytia were lysed by antibody plus rabbit complement. A variant noncytopathic subline (IP-3-Ca) was derived from the 16th passage. Infectious virus was not detected. Cells were minimally positive for hemadsorption. Fluorescent measles antigen was present in all cells. Co-cultures of IP-3-Ca cells with normal BSC-1 cells developed massive syncytia and extensive destruction. The IP-3-Ca cells multiplied in the presence of measles antibodies and were destroyed by antibodies plus complement. The possible relevance of these findings to the pathogenesis of subacute sclerosing panencephalitis is discussed.

A NADH-ferricyanide reductase of the external surface of intact mouse ascites tumor cells grown in culture was shown. The oxidation/reduction reaction was due to enzymatic rather than inorganic iron catalysis as demonstrated by the kinetics and specificity of the reaction. Activities of three markers for cytoplasmic contents were lacking with the intact tumor cells. The dehydrogenase activity was inhibited by p-chloromercuribenzoate, bathophenanthroline sulfonate, and the anticancer drug adriamycin. Sodium azide and potassium cyanide inhibited partially. The response to inhibitors resembled that of isolated plasma membranes rather than that of mitochondria. Concurrent with these findings, neither superoxide dismutase nor rotenone affected the redox activity. The findings provide evidence for the operation of a plasma membrane redox system at the surface of intact, living cells.