Biochemical limitations to high-level expression of humanized monoclonal antibodies in transgenic maize seed endosperm.

Transgenic plants are potentially valuable systems for the large scale manufacture of therapeutic proteins. To improve this technology, determining the importance of transgene transcript levels on protein accumulation in sink tissues during their development is crucial. In transgenic maize (Zea mays L.) plants expressing humanized monoclonal antibodies (mAbs) in their seed endosperm, steady-state kappa light chain (LC) and gamma heavy chain (HC) mRNA levels were quantified during development and compared to the levels of fully-assembled mAb protein present at seed maturity. RNA blots and non-reducing SDS-PAGE western immunoblots revealed that steady-state LC and HC mRNA and protein levels were undetectable at 10 days after pollination (DAP) but increased quickly thereafter in three transgenic events expressing different mAb molecules. Similar to gamma-zein mRNA, LC and HC messages were highly abundant between 15 and 25 DAP. Quantitative RNA blots and western immunoblots showed that steady-state LC transcript levels during development correlated extremely closely with protein levels in mature seed (r(2)=0.99). For HC, this correlation was not as strong (r(2)=0.85). Consistent with this finding, concomitantly increasing the zygosity levels of the LC and HC transgenes enhanced mAb concentration in mature seed, in contrast to increasing the copy number of the transgene insert, which did not correlate with high seed mAb levels. The results indicate that high-level expression of fully-assembled mAb protein in maize endosperm was favored by high LC and HC mRNA levels and was largely limited by HC protein concentration.

Legitimizing fluvial ecosystems as users of water: an overview.

We suggest that fluvial ecosystems are legitimate users of water and that there are basic ecological principles guiding the maintenance of long-term ecological vitality. This article articulates some fundamental relationships between physical and ecological processes, presents basic principles for maintaining the vitality of fluvial ecosystems, identifies several major scientific challenges and opportunities for effective implementation of the basic ecological principles, and acts as an introduction to three specific articles to follow on biodiversity, biogeochemistry, and riparian communities. All the objectives, by necessity, link climate, land, and fresh water. The basic principles proposed are: (1) the natural flow regime shapes the evolution of aquatic biota and ecological processes, (2) every river has a characteristic flow regime and an associated biotic community, and (3) aquatic ecosystems are topographically unique in occupying the lowest position in the landscape, thereby integrating catchment-scale processes. Scientific challenges for the immediate future relate to quantifying cumulative effects, linking multidisciplinary knowledge and models, and formulating effective monitoring and assessment procedures. Additionally, forecasting the ecological consequences of changing water regimes is a fundamental challenge for science, especially as environmental issues related to fresh waters escalate in the next two to three decades.