The transgenic animal platform for biopharmaceutical production

The recombinant production of therapeutic proteins for human diseases is currently the largest source of innovation in the pharmaceutical industry. The market growth has been the driving force on efforts for the development of new therapeutic proteins, in which transgenesis emerges as key component. The use of the transgenic animal platform offers attractive possibilities, residing on the low production costs allied to high productivity and quality of the recombinant proteins. Although many strategies have evolved over the past decades for the generation of transgenic founders, transgenesis in livestock animals generally faces some challenges, mainly due to random transgene integration and control over transgene copy number. But new developments in gene editing with CRISPR/Cas system promises to revolutionize the field for its simplicity and high efficiency. In addition, for the final approval of any given recombinant protein for animal or human use, the production and characterization of bioreactor founders and expression patterns and functionality of the proteins are technical part of the process, which also requires regulatory and administrative decisions, with a large emphasis on biosafety. The approval of two mammary gland-derived recombinant proteins for commercial and clinical use has boosted the interest for more efficient, safer and economic ways to generate transgenic founders to meet the increasing demand for biomedical proteins worldwide.     

Expression and regulation of the Escherichia coli glutamate dehydrogenase gene (gdh) in Rhizobium japonicum

The glutamate dehydrogenase (gdh) gene of Escherichia coli was transferred into an ammonium assimilation deficient mutant (Asm^-) of Rhizobium japonicum (CJ9) using plasmid pRP301, a broad host range derivative of RP₄. Exconjugants capable of growth on ammonia as sole N-source occurred at a frequency of 6.8×10^-6. Assimilatory GDH (NADP⁺) activity was detected in the strain carrying the E. coli gdh gene and the pattern of ammonia assimilation via GDH was similar to that of the Asm⁺ wild type strain. However, GDH mediated ammonia assimilation was not subject to regulation by l-glutamate. Nitrogenase activity was expressed ex planta in R. japonicum CJ9 harbouring the gdh gene, however, the presence of the gdh gene did not restore symbiotic effectiveness to the CJ9 Asm^- strain in nodules. The gdh plasmid was maintained in approximately 90% of the isolates recovered from soybean nodules.Abbreviations gdh glutamate dehydrogenase - Asm^- mutant ammonia assimilation deficient mutant     

Progestogens and cardiovascular reactions associated with oral contraceptives and a comparison of the safety of 50- and 30-microgram oestrogen preparations.

Progestogens probably have metabolic effects that may contribute to the increased risk of cardiovascular reactions associated with combined oestrogen-progestogen oral contraceptives. This possibility was investigated by a study of nearly 2000 reports to the Committee on Safety of Medicines from 1964 to 1977. The reports concerned preparations in which norethisterone acetate in doses of 1.0, 2.5, 3.0, or 4.0 mg was combined with 50 microgram of ethinyloestradiol and those in which levonorgestrel in doses of 150 or 250 microgram was combined with 30 microgram of ethinyloestradiol. Observed and expected numbers of reports were compared, using retail pharmacy purchase figures as a measure of the use of different preparations. There was a significant positive association between the dose of norethisterone acetate and deaths from stroke and ischaemic heart disease (IHD); this association was also found for all cases of these two conditions, fatal plus non-fatal. There were no associations of dose of norethisterone acetate with hypertension or venous thrombosis. The higher dose of levonorgestrel was associated with a possible excess of deaths, non-venous plus venous, and an excess of strokes. There was no association between dose of levonorgestrel and hypertension or venous thrombosis. The reports were also used to assess the relative safety of 30-microgram and 50-microgram oestrogen preparations. Those with 30 microgram of oestrogen were associated with significantly fewer reports of death and IHD (both fatal, and fatal plus non-fatal) than those with 50 microgram of oestrogen. In view of the large-scale move towards preparations with progressively lower oestrogen doses, there are no grounds for major changes in oral contraceptive practice. Within the range of preparations currently in use, however, there is a case for minimising the dose of progestogen to reduce the chances of thromboembolism.     

Antigen-presenting cells that induce anti-H-2K T-cell responses: Differences in stimulator-cell requirements for induction of proliferation and cell-mediated lympholysis

Splenic T or B cells, which have been depleted of adherent cells by passage through Sephadex G10 columns, fail to stimulate allogeneic lymph-node cells (LN) in primary mixed lymphocyte reactions (MLR) both when the stimulating antigens are H-2 plus Ia and H-2K only. This failure cannot be ascribed to lack of viability of G10-passed cells, since by dye exclusion they are 95 percent viable and can be induced to proliferate in vitro by exposure to LPS or allogeneic cells. Stimulation of MLR activity could be restored by addition of small numbers of plastic-adherent spleen cells (SAC) which had to be syngeneic with the G10-passed stimulator cells. Further, SAC alone without G10-passed cells induced MLR activity which was, on a cell-for-cell basis, 40 times more effective than that induced by unfractionated spleen cells. If the SAC were first depleted of Ia⁺ cells, no stimulation was obtained. This result was observed both in cases where responder and stimulator strains differed across the entireH-2-gene complex and in a mutant-wild type combination (CBA and H-2^km1) in which the difference between the two strains has been mapped to theK region only. These results indicate that Ia⁺ SAC contain a subset(s) of cells which are responsible for stimulation in MLR, regardless of whether the alloantigenic differences involve either Ia or H-2K. In contrast to the inability of G10-passed splenic cells to stimulate MLR activity, these cells were able to stimulate CTL from cytotoxic T lymphocyte precursors (CTL.P) in combinations where the antigenic differences between responder and stimulator were at the entireH-2 complex or atH-2K only. However, SAC were more potent stimulators of cell-mediated lympholysis (CML) activity on a cell-for-cell basis. Thus, either CTL.P can be stimulated by nonadherent spleen cells or they are specifically sensitive to a small subpopulation of contaminating cells that cannot readily be removed by G10 passage.     

Ammonia-assimilating enzymes in bryophytes

Ammonia can be incorporated into amino acids by reductive amination of oxoglutarate, or by the glutamate synthase cycle via glutamine. The majority of plants possess the enzymes necessary for the operation of both these pathways although nitrogen is thought to be assimilated via the glutamate synthase cycle in most cases. Measurements of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase activities are presented from a selection of bryophytes. The genus Sphagnum was found to be unique in lacking measurable glutamate dehydrogenase activity. The relevance of this in the nitrogen-poor acid bog is briefly considered.     

Isolation of an Asm− mutant of Rhizobium japonicum defective in symbiotic N2-fixation

Abstract A mutant strain of Rhizobium japonicum (CJ9) unable to assimilate ammonium (Asm⁻) was isolated following mutagenesis with N -methyl N -nitro-nitrosoguanidine (NTG). Glutamate synthase activity was not detectable in cell-free extracts of the mutant strain in contrast to the wild type and revertant strains. Although mutant CJ9 induced nitrogenase activity in an 'in vitro' assay system under microaerobic conditions, it failed to fix nitrogen (acetylene reduction) in soybean root nodules. These properties of mutant CJ9 constitute a new Asm⁻ mutant class in Rhizobium spp.