PubMed · 4907697
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J R Eshleman. 1970. Pontic selection and design.. https://pubmed.ncbi.nlm.nih.gov/4907697/
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The potential for the feedback inhibition of indole alkaloid synthesis was investigated by spiking suspension cultures of Catharanthus roseus with 0, 9, or 18 mg/L ajmalicine on day 0. The production of ajmalicine, catharanthine, and serpentine were inhibited in a dose-dependent manner. The inhibition was transient as the exogenous ajmalicine was ultimately either metabolized in the medium or within the cell. The addition of neutral resin has previously been shown to enhance ajmalicine production. To minimize product inhibition and product metabolism, Amberlite XAD-7 resin was added to immobilized cultures of C. roseus starting on either day 0, 5, or 15, and fresh resin was exchanged for spent resin every 5 days. The addition of resin did not decrease the viability of the culture. Growth was reduced only in cultures with resin added on day 0. Alkaloid production was enhanced to different extents by the timing of resin addition, suggesting that feedback inhibition or product metabolism was present throughout the culture period. Ajmalicine recovery was nearly 100% when the resin was added initially either on day 0 or day 5. Ajmalicine recovery was reduced to 55% when the resin was added later in the culture period starting on day 15, presumably because of resin saturation or the inaccessibility of alkaloids trapped in the vacuole. Delaying the addition of XAD-7 resin until 5 days after the start of the culture resulted in the highest improvement in ajmalicine production, i.e approximately 70% and also resulted in the complete recovery of ajmalicine from the cell.
Poly(N-isopropylacrylamide), or PNIPAAm, is considered a "smart" polymer because it sharply precipitates when heated above a critical temperature, about 32 degrees C in water, and redissolves when cooled. Conjugates made of PNIPAAm and IgG antibodies also exhibit the same critical temperature behavior. Interestingly, antigens that are complexed with these conjugates can also be phase-separated along with the conjugates. In this work, we conjugated PNIPAAm for the first time to the immunoglobulin Fv fragment, the smallest fragment of an antibody that still retains the antigenic affinity of the whole antibody. For our studies, we used an Fv fragment that strongly binds hen egg white lysozyme (HEL). The purified Fv fragment-polymer conjugate precipitated at the same temperature as did the pure polymer. After addition of the conjugate to a mixture containing HEL and after thermal separation of the conjugate at 37 degrees C, the amount of HEL in solution was reduced by as much as 80%. We were able to demonstrate the reversibility of the separation through three cycles of precipitation and dissolution. It was also possible to recover free HEL by thermal separation of the conjugate in the presence of an eluant, 50 mM diethylamine. The conjugate can then be recycled for second use. In conclusion, immunoseparations can be performed using smart polymer conjugates made with just the variable domains of an antibody. Unlike whole antibodies, fragments of antibodies can be produced in Escherichia coli, allowing easier genetic engineering of the antibody and tailoring of the conjugate.
A novel approach is presented for the controlled intramolecular collapse of linear polymer chains to give well-defined single-molecule nanoparticles whose structure is directly related to the original linear polymer. By employing a combination of living free radical polymerization and benzocyclobutene (BCB) chemistry, nanoparticles can be routinely prepared in multigram quantities with the size being accurately controlled by either the initial degree of polymerization of the linear chain or the level of incorporation of the BCB coupling groups. The latter also allows the cross-link density of the final nanoparticles to be manipulated. In analogy with dendritic macromolecules, a significant reduction of up to 75% in the hydrodynamic volume is observed on going from the starting random coil linear chains to the corresponding nanoparticles. The facile nature of the living free radical process also permits wide variation in monomer selection and functional group incorporation and allows novel macromolecular architectures to be prepared. Furthermore, the use of block copolymers functionalized with benzocyclobutene groups in only one of the blocks gives, after intramolecular collapse, a hybrid architecture in which a single linear polymer chain is attached to the globular nanoparticle.