Idiotypic analysis of anti-GAT antibodies. VII. Common idiotype on hybridoma antibodies to poly(Glu60 Ala40)

A single DBA/2 mouse, immunized with L-glutamic acid60-L-alanine40 (GA), was used to produce hybridoma cell lines. Seven hybridoma anti-GA antibodies were obtained for idiotypic analyses. Two hybridoma anti-L-glutamic acid60-L-alanine30-L-tyrosine10 (GAT) antibodies, preferentially reactive to GA, were studied in parallel. Anti-idiotypic antisera to purified anti-GAT and anti-GA serum antibodies and to hybridoma anti-GA antibodies were analyzed by idiotype binding and inhibition of idiotype binding assays. Five of the nine hybridoma antibodies exhibited common GA-1 idiotypic specificities previously demonstrated on the majority of anti-GA antibodies of inbred mouse strains of differing immunoglobulin heavy chain linkage groups; these hybridoma antibodies also possessed private idiotypic determinants. Two GA-1 negative hybridoma anti-GA antibodies appeared identical by immunochemical criteria, arguing that somatic hybridization does not artifactually generate private idiotypic determinants. The results demonstrate that the common GA-1 idiotype system is associated with a family of nonidentical but idiotypically related antibody molecules present in a single DBA/2 mouse, and these antibodies are part of the "GA-1 idiotypic family".     

Planting Sentinel European Trees in Eastern Asia as a Novel Method to Identify Potential Insect Pest Invaders

Quarantine measures to prevent insect invasions tend to focus on well-known pests but a large proportion of the recent invaders were not known to cause significant damage in their native range, or were not even known to science before their introduction. A novel method is proposed to detect new potential pests of woody plants in their region of origin before they are introduced to a new continent. Since Asia is currently considered to be the main supplier of insect invaders to Europe, sentinel trees were planted in China during 2007-2011 as an early warning tool to identify the potential for additional Asian insect species to colonize European trees. Seedlings (1-1.5 m tall) of five broadleaved (Quercus petraea, Q. suber, Q. ilex, Fagus sylvatica, and Carpinus betulus) and two conifer species (Abies alba and Cupressus sempervirens) were planted in blocks of 100 seedlings at two widely separated sites (one in a nursery near Beijing and the other in a forest environment near Fuyang in eastern China), and then regularly surveyed for colonization by insects. A total of 104 insect species, mostly defoliators, were observed on these new hosts, and at least six species were capable of larval development. Although a number of the insects observed were probably incidental feeders, 38 species had more than five colonization events, mostly infesting Q. petraea, and could be considered as being capable of switching to European trees if introduced to Europe. Three years was shown to be an appropriate duration for the experiment, since the rate of colonization then tended to plateau. A majority of the identified species appeared to have switched from agricultural crops and fruit trees rather than from forest trees. Although these results are promising, the method is not appropriate for xylophagous pests and other groups developing on larger trees. Apart from the logistical problems, the identification to species level of the specimens collected was a major difficulty. This situation could be improved by the development of molecular databases.     

Integration of cell line and process development to overcome the challenge of a difficult to express protein

This case study addresses the difficulty in achieving high level expression and production of a small, very positively charged recombinant protein. The novel challenges with this protein include the protein's adherence to the cell surface and its inhibitory effects on Chinese hamster ovary (CHO) cell growth. To overcome these challenges, we utilized a multi‐prong approach. We identified dextran sulfate as a way to simultaneously extract the protein from the cell surface and boost cellular productivity. In addition, host cells were adapted to grow in the presence of this protein to improve growth and production characteristics. To achieve an increase in productivity, new cell lines from three different CHO host lines were created and evaluated in parallel with new process development workflows. Instead of a traditional screen of only four to six cell lines in bioreactors, over 130 cell lines were screened by utilization of 15 mL automated bioreactors (AMBR) in an optimal production process specifically developed for this protein. Using the automation, far less manual intervention is required than in traditional bench‐top bioreactors, and much more control is achieved than typical plate or shake flask based screens. By utilizing an integrated cell line and process development incorporating medium optimized for this protein, we were able to increase titer more than 10‐fold while obtaining desirable product quality. Finally, Monte Carlo simulations were performed to predict the optimal number of cell lines to screen in future cell line development work with the goal of systematically increasing titer through enhanced cell line screening. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1201–1211, 2015     

Engineering trypsin for inhibitor resistance

The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [k_cat/K_M = (1.2 ± 0.3) × 10⁷ M⁻¹ s⁻¹. Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.     

Transferrin receptor 2-alpha supports cell growth both in iron-chelated cultured cells and in vivo

In most cells, transferrin receptor (TfR1)-mediated endocytosis is a major pathway for cellular iron uptake. We recently cloned the human transferrin receptor 2 (TfR2) gene, which encodes a second receptor for transferrin (Kawabata, H., Yang, R., Hirama, T., Vuong, P. T., Kawano, S., Gombart, A. F., and Koeffler, H. P. (1999) J. Biol. Chem. 274, 20826-20832). In the present study, the regulation of TfR2 expression and function was investigated. A select Chinese hamster ovary (CHO)-TRVb cell line that does not express either TfR1 or TfR2 was stably transfected with either TfR1 or TfR2-alpha cDNA. TfR2-alpha-expressing cells had considerably lower affinity for holotransferrin when compared with TfR1-expressing CHO cells. Interestingly, in contrast to TfR1, expression of TfR2 mRNA in K562 cells was not up-regulated by desferrioxamine (DFO), a cell membrane-permeable iron chelator. In MG63 cells, expression of TfR2 mRNA was regulated in the cell cycle with the highest expression in late G(1) phase and no expression in G(0)/G(1). DFO reduced cell proliferation and DNA synthesis of CHO-TRVb control cells, whereas it had little effect on TfR2-alpha-expressing CHO cells when measured by clonogenic and cell cycle analysis. In addition, CHO cells that express TfR2-alpha developed into tumors in nude mice whereas CHO control cells did not. In conclusion, TfR2 expression may be regulated by the cell cycle rather than cellular iron status and may support cell growth both in vitro and in vivo.     

Mobile elements create strain-level variation in the services conferred by an aphid symbiont

Heritable, facultative symbionts are common in arthropods, often functioning in host defence. Despite moderately reduced genomes, facultative symbionts retain evolutionary potential through mobile genetic elements (MGEs). MGEs form the primary basis of strain-level variation in genome content and architecture, and often correlate with variability in symbiont-mediated phenotypes. In pea aphids (Acyrthosiphon pisum), strain-level variation in the type of toxin-encoding bacteriophages (APSEs) carried by the bacterium Hamiltonella defensa correlates with strength of defence against parasitoids. However, co-inheritance creates difficulties for partitioning their relative contributions to aphid defence. Here we identified isolates of H. defensa that were nearly identical except for APSE type. When holding H. defensa genotype constant, protection levels corresponded to APSE virulence module type. Results further indicated that APSEs move repeatedly within some H. defensa clades providing a mechanism for rapid evolution in anti-parasitoid defences. Strain variation in H. defensa also correlates with the presence of a second symbiont Fukatsuia symbiotica. Predictions that nutritional interactions structured this coinfection were not supported by comparative genomics, but bacteriocin-containing plasmids unique to co-infecting strains may contribute to their common pairing. In conclusion, strain diversity, and joint capacities for horizontal transfer of MGEs and symbionts, are emergent players in the rapid evolution of arthropods.