Recombinant protein expression plasmids optimized for industrial E. coli fermentation and plant systems produce biologically active human insulin-like growth factor-1 in transgenic rice and tobacco plants.

Human insulin-like growth factor-1 (hIGF-1) is a growth factor with clinical significance in medicine. The therapeutic potential of recombinant hIGF-1 (rthIGF-1) stems from the fact that hIGF-1 resembles insulin in many aspects of physiology. The expression of hIGF-1 in transgenic tobacco and rice plants using different expression cassettes is reported here. In the present study, two coding sequences were tested, one with the original human sequence, but partially optimized for expression in E. coli and the other with a plant-codon-optimized sequence that was expected to give a higher level of expression in plant systems. Three different hIGF-1 recombinant expression constructs were generated. All expression constructs utilized the maize ubiquitin 1 promoter with or without a signal sequence. Analyses conducted using a hIGF-1 specific ELISA kit showed all transgenic plants produced hIGF-1 and the accumulated hIGF-1 increased from the E. coli codon bias to higher levels when the hIGF-1 coding sequence was codon-optimized to match that of the maize zeamatin protein--the most transcribed gene in maize endosperm suspension cells. Further analyses that compared the functionality of the bacterial signal peptide Lam B in plants showed that this leader peptide led to lower expression levels when compared to transgenic plants that did not contain this sequence. This indicated that this expression construct was functional without removal of the bacterial signal sequence. The maize ubiquitin 1 promoter was found to be more active in rice plants than tobacco plants indicating that in this case, there was a class preference that was biased towards a monocot host. Biological analyses conducted using protein extracts from transgenic plants showed that the rthIGF-1 was effective in stimulating the in vitro growth and proliferation of human SH-SY5Y neuroblastoma cells. This indicated that the plant-produced rthIGF-1 was stable and biologically active. As some plants have been reported to express an endogenous insulin-like protein, we also looked for any effect of the human growth factor in transgenic plants, but no developmental or morphological differences with wild type tobacco or rice plants were detected. Since insulin and hIGF-1 share some overlapping roles, hIGF-1 may become a substitute therapeutic agent in subjects with certain defects in their insulin receptor signaling. Hence, if the full beneficial potential of rthIGF-1 is achieved, it is expected that in the future the demand will likely increase significantly.

Twenty years later: a reevaluation of the contribution of plasma thyroglobulin to the diagnosis of thyroid dysgenesis in infants with congenital hypothyroidism.

BACKGROUND: The definition of the type of thyroid dysgenesis in congenital hypothyroidism (CH), ectopy, or athyreosis is important for monitoring patients and for genetic investigations. We have recently encountered infants who in spite of undetectable Technetium uptake on scintigraphy had biochemical results making athyreosis unlikely. OBJECTIVE: To reevaluate the utility of plasma thyroglobulin (Tg) in this clinical context using new sensitive Tg assays. SUBJECTS AND METHODS: Plasma Tg was retrospectively determined by two immunoassay systems on specimens obtained at diagnosis in 31 hypothyroid infants with thyroid dysgenesis. RESULTS: Scintigraphy led to the diagnosis of ectopy in 19 infants and of athyreosis in 12. Seven (58%) of the infants classified as athyreotic by scintigraphy had detectable plasma Tg (>0.2 microg/l), indicating that they had functional thyroid tissue. CONCLUSIONS: An undetectable plasma Tg should be documented to validate a scintigraphic diagnosis of athyreosis. Conversely, when plasma Tg is undetectable, scintigraphy could be avoided.