Expression systems tissue

The distribution of endosulfan and endosulfan sulfate was evaluated in the brains of cats given a single intravenous injection of 3 mg/kg endosulfan (Khanna et al. 1979). Peak concentrations of endosulfan in the brain were found at the earliest time point examined (15 minutes after administration) and then decreased. When tissue levels were expressed per gram of tissue, little differential was observed in distribution among the brain areas studied. However, if endosulfan levels were expressed per gram of tissue lipid, higher initial levels were observed in the cerebral cortex and cerebellum than in the spinal cord and brainstem. Loss of endosulfan was most rapid from those areas low in Upid. Endosulfan sulfate levels peaked in the brain at 1 hour postadministration. In contrast, endosulfan sulfate levels in liver peaked within 15 minutes postadministration. The time course of neurotoxic effects observed in the animals in this study corresponded most closely with endosulfan levels in the central nervous system tissues examined. 

Simpson JE, Newcombe J, Cuzner ML, Woodroofe MN (1998) Expression of monocyte chemoattractant protein-1 and other beta-chemokines by resident glia and inflammatory cells in multiple sclerosis lesions. J Neuroimmunol 84 238-249 Simpson J, Rezaie P, Newcombe J, Cuzner ML, Male D, Woodroofe MN (2000) Expression of the beta-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosis central nervous system tissue. J Neuroimmunol 108 192-200... 

Simpson J, Rezaie P, Newcombe J, et al. Expression of the beta-chemokine receptors CCR2, CCR3 and CCR5 in multiple sclerosis central nervous system tissue. J Neuroimmunol 2000 108 192-200. 

In this review, we focus on the use of plant tissue culture to produce foreign proteins that have direct commercial or medical applications. The development of large-scale plant tissue culture systems for the production of biopharmaceutical proteins requires efficient, high-level expression of stable, biologically active products. To minimize the cost of protein recovery and purification, it is preferable that the expression system releases the product in a form that can be harvested from the culture medium. In addition, the relevant bioprocessing issues associated with bioreactor culture of plant cells and tissues must be addressed. 

Compared with whole plants, there has been limited development of foreign protein expression systems specifically for use in tissue culture. Some modifications of expression constructs have resulted in improved protein accumulation or have allowed simplified protein recovery. However, in general, modified expression systems have been tested only in a restricted number of cases and have not resulted in the large increases in product yield required for plant cultures to compete with other foreign protein production vehicles. Transient expression techniques, for example using viral vectors, that have been developed for use in whole plants have not yet been applied in plant tissue culture. 

A vector that facilitates high-level protein expression in plant tissue culture, particularly a transient expression system that could be applied to existing wild-type cultures, would be advantageous for in vitro foreign protein production. However, such a system has not yet been developed. The success of this approach depends in part on whether appropriate levels of viral infection, replication and transmission can be established within tissue culture systems. 

The presence of foreign protein in the medium of plant cultures does not necessarily mean that all or even most of the product can be recovered from the medium. In many expression systems where an appropriate signal sequence has been used, considerable amounts of foreign protein remain within the plant cells and/or tissues. For example, in a comparison of IgG antibody production in tobacco cell suspension and hairy root cultures, a maximum of 72% of the total antibody was found in the medium of the suspension cultures whereas only 26% was found in the medium of the hairy root cultures [17]. This result could indicate that secretion and/or transport across the cell wall was slower in the hairy roots alternatively, it could indicate poorer stability of the secreted protein in the hairy root medium. If foreign proteins are to be purified from the medium, improved secretion and extracellular product stability are desirable. 

By using specific catalytic activities, the differences in V ax due to any factor are automatically compensated for in the calculation. Because this method establishes relative contribution, there is no a priori need to use the same units for both components of the RAF calculation as long as the same units are used within the cDNA-expressed enzyme data set and the human tissue data set. Moreover, knowledge of the P450 content in either the cDNA expression system or in human liver microsomes is not necessary in order to make an interpretation. 

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