Regulation of Eukaryotic Initiation Factor 4E and Its Isoform： Implications for Antiviral Strategy in Plants

In recent years, biotechnology has permitted regulation of the expression of endogenous plant genes to improve agronomlcally important traits. Genetic modification of crops has benefited from emerging knowledge of new genes, especially genes that exhibit novel functions, one of which is eukaryotlc initiation factor 4E （eIF4E）. eIF4E Is one of the most important translation initiation factors Involved in eukaryotic initiation. Recent research has demonstrated that virus resistance mediated by eIF4E and Its isoform elf （Iso）4E occurs in several plant-virus interactions, thus indicating a potential new role for eIF4E/elF（Iso）4E In resistance strategies against plant viruses. In this review, we briefly describe eIF4E activity In plant translation, its potential role, and functions of the eIF4E subfamily In plant-virus interactions. Other initiation factors such as elF4G could also play a role In plant resistance against viruses. Finally, the potential for developing eIF4E-mediated resistance to plant viruses in the future Is discussed. Future research should focus on elucidation of the resistance mechanism and spectrum mediated by eIF4E. Knowledge of a particu- lar plant-virus interaction will help to deepen our understanding of eIF4E and other eukaryotic Initiation factors, and their involvement in virus disease control.     

Triple light chain antibodies: Factors that influence its formation in cell culture

THIOMABs are recombinant antibodies engineered with reactive cysteines, which can be covalently conjugated to drugs of interest to generate targeted therapeutics. During the analysis of THIOMABs secreted by stably transfected Chinese Hamster Ovary (CHO) cells, we discovered the existence of a new species—Triple Light Chain Antibody (3LC). This 3LC species is the product of a disulfide bond formed between an extra light chain and one of the engineered cysteines on the THIOMAB. We characterized the 3LC by size exclusion chromatography, mass spectrometry, and microchip electrophoresis. We also investigated the potential causes of 3LC formation during cell culture, focusing on the effects of free light chain (LC) polypeptide concentration, THIOMAB amino acid sequence, and glutathione (GSH) production. In studies covering 12 THIOMABs produced by 66 stable cell lines, increased free LC polypeptide expression—evaluated as the ratio of mRNA encoding for LC to the mRNA encoding for heavy chain (HC)—correlated with increased 3LC levels. The amino acid sequence of the THIOMAB molecule also impacted its susceptibility to 3LC formation: hydrophilic LC polypeptides showed elevated 3LC levels. Finally, increased GSH production—evaluated as the ratio of the cell‐specific production rate of GSH (q_GSH) to the cell‐specific production rate of THIOMAB (q_p)—corresponded to decreased 3LC levels. In time‐lapse studies, changes in extracellular 3LC levels during cell culture corresponded to changes in mRNA LC/HC ratio and q_GSH/q_p ratio. In summary, we found that cell lines with low mRNA LC/HC ratio and high q_GSH/q_p ratio yielded the lowest levels of 3LC. These findings provide us with factors to consider in selecting a cell line to produce THIOMABs with minimal levels of the 3LC impurity. Biotechnol. Bioeng. 2010. 105: 748–760. © 2009 Wiley Periodicals, Inc.     

Tyrosine phosphatase SHP-2 regulates IL-1 signaling in fibroblasts through focal adhesions

Interleukin-1beta (IL-1beta) mediates destruction of matrix collagens in diverse inflammatory diseases including arthritis, periodontitis, and pulmonary fibrosis by activating fibroblasts, cells that interact with matrix proteins through integrin-based adhesions. In vitro, IL-1beta signaling is modulated by focal adhesions, supramolecular protein complexes that are enriched with tyrosine kinases and phosphatases. We assessed the importance of tyrosine phosphatases in regulating cell-matrix interactions and IL-1beta signaling. In human gingival fibroblasts plated on fibronectin, IL-1beta enhanced the maturation of focal adhesions as defined by morphology and enrichment with paxillin and alpha-actinin. IL-1beta also induced activation of ERK and recruitment of phospho-ERK to focal complexes/adhesions. Treatment with the potent tyrosine phosphatase inhibitor pervanadate, in the absence of IL-1beta, recapitulated many of these responses indicating the importance of tyrosine phosphatases. Immunoblotting of collagen bead-associated complexes revealed that the tyrosine phosphatase, SHP-2, was also enriched in focal complexes/adhesions. Depletion of SHP-2 by siRNA or by homologous recombination markedly altered IL-1beta-induced ERK activation and maturation of focal adhesions. IL-1beta-induced tyrosine phosphorylation of SHP-2 on residue Y542 promoted focal adhesion maturation. Association of Gab1 with SHP-2 in focal adhesions correlated temporally with activation of ERK and was abrogated in cells expressing mutant (Y542F) SHP-2. We conclude that IL-1beta mediated maturation of focal adhesions is dependent on tyrosine phosphorylation of SHP-2 at Y542, leading to recruitment of Gab1, a process that may influence the downstream activation of ERK.     

Overexpression of inducible heat shock protein 70 and its mutants in astrocytes is associated with maintenance of mitochondrial physiology during glucose deprivation stress

Wild-type inducible Hsp70 (WT) and 2 folding deficient mutants protect the brain against focal cerebral ischemia in vivo and brain cells from oxygen-glucose deprivation (OGD) in vitro, but the protective mechanisms remain unclear. Mitochondria are central to both normal physiological function and the regulation of cell death. We tested the effect of overexpressing Hsp70 and 2 mutants, Hsp70-K71 E, an adenosine triphosphatase (ATPase)-deficient point mutant, and Hsp70-381-640, a deletion mutant lacking the ATPase domain on mitochondrial physiology under glucose deprivation (GD) stress in primary cultured astrocytes. Mitochondrial membrane potential was assessed using a potentiometric fluorescent dye tetramethylrhodamine ethyl ester (TMRE). By 5 hours of GD, the mitochondria in the LXSN control transfected astrocytes had markedly reduced membrane potential. However, in the Hsp70-WT, -K71E, and -381-640 groups, there was no apparent change in TMRE signal during 5 hours of GD. Oxygen consumption was measured to assess oxidative respiration. Overexpression of Hsp70-K71 E and -381-640 prevented the decrease in state III respiration observed at 5 hours, and all 3 prevented the increase in state IV respiration found in LXSN controls after 5 hours of GD. Reactive oxygen species (ROS) production was assessed with hydroethidine. Hsp70 and its mutants all significantly reduced the increases in ROS accumulation during 5 hours of GD. The results demonstrate that the protective effect of the carboxyl-terminal half of Hsp70 and of the full-length molecule is associated with better maintained mitochondrial membrane potential, better maintained state IV respiration, and reduced ROS generation during GD.     

Designing a conservation plan for protecting the habitat for giant pandas in the Qionglai mountain range, China

Population viability of the giant panda ( Ailuropoda melanoleuca ) is threatened by small population sizes in scattered isolated habitat areas. Designing a conservation plan for protecting and connecting the fragmented habitat will improve the chances for survival of this endangered species. For such a plan, this study assessed the overall habitat suitability for the species in the Qionglai mountain range (Sichuan, China) using Landsat TM imagery acquired in 2001, geographical data, field surveys, and information acquired in previous researches. Results show that the habitat is separated by roads and rivers, as well as by human settlements and cropland areas, into four main habitat blocks. Overlapping these four habitat blocks with the current nature reserve network reveals that only 36% of the total habitat is protected within nature reserves. Thus, the current nature reserve network is failing to preserve essential habitat for dispersal and genetic exchange. In this study, five key areas and four linkage areas were identified and suggested as nature reserves and/or corridors. These areas, together with the six currently established nature reserves in the mountain range, will form a conservation unit for facilitating the exchange of giant panda individuals among previously isolated habitat blocks. Policies recently implemented by the Chinese government, including the Natural Forest Conservation Program (NFCP) and the Grain-to-Green Program (GTGP), could aid in the formation of such a conservation unit.     

Significance of organic nitrogen acquisition for dominant plant species in an alpine meadow on the Tibet plateau, China

Though the potential of plants to take up organic N (e.g., amino acids) is well established, the true significance of organic N acquisition to plant N nutrition has not yet been quantified under field conditions. Here we demonstrate that organic N contributes significantly to the annual N uptake of three dominant plant species (Kobresia humilis, Saussurea superba and Stipa aliena) of alpine meadows on the Tibet Plateau, China. This was achieved by using double-labelled (¹⁴C and ¹⁵N) algae as a source for slow and continuous release of amino acids, and tracing both labels in the above- and below-ground plant biomass. Four months after addition of algae, between 0.5% and 2.6% of ¹⁴C and 5% and 14% of ¹⁵N from added algae were recovered in the plants, which translate into an uptake of organic N between 0.3 mg N m⁻² and 1.5 mg N m⁻². The calculated contribution of organic N to total N uptake was estimated to range between 21% and 35% for K. humilis, and between 13% and 21% for S. aliena and S. superba, respectively, implying that organic N uptake by grassland plants is quantitatively significant under field conditions in the studied alpine meadows. This finding has important ecological implications with regard to competition for organic N between microorganisms and plant roots.