The kinetics of baculovirus adsorption to insect cells in suspension culture

The influence of various culture parameters on the attachment of a recombinant baculovirus to suspended insect cells was examined under normal culture conditions. These parameters included cell density, multiplicity of infection, and composition of the cell growth medium. It was found that the fractional rate of virus attachment was independent of the multiplicity of infection but dependent on the cell density. A first order mathematical model was used to simulate the adsorption kinetics and predict the efficiency of virus attachment under the various culture conditions. This calculated efficiency of virus attachment was observed to decrease at high cell densities, which was attributed to cell clumping. It was also observed that virus attachment was more efficient in Sf900II serum free medium than it was in IPL-41 serum-supplemented medium. This effect was attributed to the protein in serum which may coat the cells and so inhibit adsorption. A general discussion relating the observations made in-these experiments to the kinetics of recombinant baculovirus adsorption to suspended insect cells is presented.

Processing of mutated human proinsulin to mature insulin in the non-endocrine cell line, CHO

Heterologous genes encoding proproteins, including proinsulin, generally produce mature protein when expressed in endocrine cells while unprocessed or partially processed protein is produced in non-endocrine cells. Proproteins, which are normally processed in the regulated pathway restricted to endocrine cells, do not always contain the recognition sequence for cleavage by furin, the endoprotease specific to the constitutive pathway, the principal protein processing pathway in non-endocrine cells. Human proinsulin consists of B-Chain-C-peptide-A-Chain and cleavage at the B/C and C/A junctions is required for processing. The B/C, but not the C/A junction, is recognised and cleaved in the constitutive pathway. We expressed a human proinsulin and a mutated proinsulin gene with an engineered furin recognition sequence at the C/A junction and compared the processing efficiency of the mutant and native proinsulin in Chinese Hamster Ovary cells. The processing efficiency of the mutant proinsulin was 56% relative to 0.7% for native proinsulin. However, despite similar levels of mRNA being expressed in both cell lines, the absolute levels of immunoreactive insulin, normalized against mRNA levels, were 18-fold lower in the mutant proinsulin-expressing cells. As a result, there was only a marginal increase in absolute levels of insulin produced by these cells. This unexpected finding may result from preferential degradation of insulin in non-endocrine cells which lack the protection offered by the secretory granules found in endocrine cells.

Plant transformation: Problems and strategies for practical application

Plant transformation is now a core research tool in plant biology and a practical tool for cultivar improvement. There are verified methods for stable introduction of novel genes into the nuclear genomes of over 120 diverse plant species. This review examines the criteria to verify plant transformation; the biological and practical requirements for transformation systems; the integration of tissue culture, gene transfer, selection, and transgene expression strategies to achieve transformation in recalcitrant species; and other constraints to plant transformation including regulatory environment, public perceptions, intellectual property, and economics. Because the costs of screening populations showing diverse genetic changes can far exceed the costs of transformation, it is important to distinguish absolute and useful transformation efficiencies. The major technical challenge facing plant transformation biology is the development of methods and constructs to produce a high proportion of plants showing predictable transgene expression without collateral genetic damage. This will require answers to a series of biological and technical questions, some of which are defined.

Expression of recombinant human follicle stimulating hormone mRNA in Chinese hamster ovary cells under different promoters

Levels of recombinant human follicle stimulating hormone (r-hFSH) mRNA expressed under butyrate and zinc treatment were compared in two CHO-K1 derived cell lines. In King cells under the metallothionein promoter, butyrate induced the increase in both r-hFSH productivity (q(FSH)) and mRNA levels proportionally. In the presence of 1 mM butyrate and 40 mu M zinc, a 4-fold increase in q(FSH) and mRNA levels was achieved as compared to zinc (40) alone; this wasa approximately 6 times higher than in serum free medium. In Darren cells under the beta-actin promotor butyrate induced an increase in q(SFH) but not in mRNA levels.

Effects of process modification and heat pump drying on physical and sensory properties of Chinese style shredded beef

Chinese-style dried, shredded meat is traditionally prepared by sequential cooking, shredding, pre-drying, and final drying (roasting) of lean meat. In this study, shredded dried beef (a(w)<0.6) was prepared by omitting roasting but prolonging pre-drying. Sensory scores of the modified product were lower than those for the traditional product. When heat pump drying replaced traditional oven drying, drying time was shortened without significant difference in quality attributes. Desorption curves were established for shredded beef at several drying temperatures.

Chinese wool auctions: Failed agribusiness reform or future marketing channel?

The spectacular though short-lived operation of China's wool auctions graphically illustrates problems in reforming China's agribusiness sector. A selective coverage of product type and participants, a highly contrived process of price discovery, and an urgent need for parallel reform in related areas all led to the demise of the auctions and are a barrier to their re-emergence as a viable wool marketing channel. Despite the challenges facing wool auctions, this paper argues that they still have much to offer the marketing of wool in China.