Association of a 76 kDa polypeptide with soluble starch synthase I activity in maize (cv B73) endosperm

Soluble starch synthase (SSS) I was purified 361-fold from hand-dissected endosperm tissue of inbred maize (Zea mays, cv. B73) to specific activities ranging between 5 and 9 µmol min⁻¹ mg⁻¹. A key to this purification protocol was the introduction of a size-exclusion chromatography step, a size-based fractionation which provided abundant levels of desalted SSS forms I and II. The native molecular masses calculated for SSS forms I and II were 75.5 kDa and 180 kDa, respectively. SSSI was then further purified by hydrophobic interaction chromatography on Phenyl-Superose and by FPLC on Mono Q. Analysis of column peaks by SDS—PAGE and scanning densitometry revealed that a 76 kDa polypeptide is strongly correlated with SSSI activity. Antibodies were then generated against a 76 kDa polypeptide extracted from starch granules. These antibodies, which were monospecific for the soluble 76 kDa polypeptide, neutralized greater than 90% of SSSI activity, and precipitated the 76 kDa protein. These results establish the 76 kDa protein as an SSSI in the B73 line of inbred maize. An immunologically similar 76 kDa protein also appears to be tightly associated with the starch granule.     

Engineering a therapeutic IgG molecule to address cysteinylation,aggregation and enhance thermal stability and expression

Antibodies can undergo a variety of covalent and non-covalent degradation reactions that have adverse effects on efficacy, safety, manufacture and storage. We had identified an antibody to Angiopoietin 2 (Ang2 mAb) that neutralizes Ang2 binding to its receptor in vitro and inhibits tumor growth in vivo. Despite favorable pharmacological activity, the Ang2 mAb preparations were heterogeneous, aggregated rapidly and were poorly expressed. Here, we report the engineering of the antibody variable and constant domains to generate an antibody with reduced propensity to aggregate, enhanced homogeneity, 11°C elevated T_m, 26-fold improved level of expression and retained activity. The engineered molecule, MEDI-3617, is now compatible with the large scale material supply required for clinical trials and is currently being evaluated in Phase 1 in cancer patients. This is the first report to describe the stability engineering of a therapeutic antibody addressing non canonical cysteine residues and the design strategy reported here is generally applicable to other therapeutic antibodies and proteins.     

Spatial distribution of filament elasticity determines the migratory behaviors of a cell

Any cellular response leading to morphological changes is highly tuned to balance the force generated from structural reorganization, provided by actin cytoskeleton. Actin filaments serve as the backbone of intracellular force, and transduce external mechanical signal via focal adhesion complex into the cell. During migration, cells not only undergo molecular changes but also rapid mechanical modulation. Here we focus on determining, the role of spatial distribution of mechanical changes of actin filaments in epithelial, mesenchymal, fibrotic and cancer cells with non-migration, directional migration, and non-directional migration behaviors using the atomic force microscopy. We found 1) non-migratory cells only generated one type of filament elasticity, 2) cells generating spatially distributed two types of filament elasticity showed directional migration, and 3) pathologic cells that autonomously generated two types of filament elasticity without spatial distribution were actively migrating non-directionally. The demonstration of spatial regulation of filament elasticity of different cell types at the nano-scale highlights the coupling of cytoskeletal function with physical characters at the sub-cellular level, and provides new research directions for migration related disease.     

The irradiated cercariae vaccine model: looking on the bright side of radiation

Schistosomes infect between 200 and 300 million people at any one time. A major strategy to reduce the impact of schistosomiasis on human health is the development of a defined antigen vaccine. Protective immunity induced in mice by irradiated cercariae may serve as a model for the development of a vaccine. In such vaccinated mice, worm burdens resulting from challenge infection can be reduced by more than 90% compared to non-vaccinated mice. During the past three decades, the irradiated-carcariae vaccine model has been dissected in the detail in order to determine factors that may be relevant to vaccination, such as the participating immune compartments, the site and kinetics of the immune response, and the antigens recognized. In this review, Dania Richter, Donald A. Harn and Franz-Rainer Matuschka highlight the research on the vaccine model, focusing on the murine model using gamma-irradiated cercariae of Schistosoma mansoni.     

Induction of Th2 responses and IgE is largely due to carbohydrates functioning as adjuvants on Schistosoma mansoni egg antigens

Infection with the helminth parasite Schistosoma mansoni induces a pronounced Th2-type response that is associated with significant IgE production. To better understand how the parasite drives these responses, we investigated the relative roles of proteins and carbohydrates in driving Th2-type and/or IgE responses using a murine model of intranasal sensitization with soluble egg Ags (SEA) of Schistosoma mansoni. We found that repeated intranasal sensitization with soluble egg Ags led to the induction of both total and specific IgE production and nasal eosinophilia. By comparing the responses of mice sensitized with SEA or metaperiodate-treated SEA we were able to demonstrate that carbohydrates on SEA are the major inducers of IgE production and nasal recruitment of eosinophils. Mice sensitized with periodate-treated SEA displayed a significant decrease in both total and specific IgE levels in comparison to mice sensitized with native SEA. Furthermore, sensitization of mice with periodate-treated SEA significantly reduced levels of Ag-specific IgG1, but had no effect on IgG2a production. Nasal lymphocytes from mice sensitized with native SEA, but not with periodate-treated SEA, produced IL-4, IL-5, and IL-10 when restimulated with native SEA in vitro. On the other hand, lymphocytes from mice sensitized with periodate-treated SEA did not produce any of these same cytokines following in vitro restimulation, suggesting that carbohydrates were required for in vivo induction of Th2 response and for that of associated cytokine responses in this model. Lastly, competitive inhibition ELISA showed that although carbohydrates are required for SEA-specific IgE induction, they are not targets of the induced IgE response.     

Immune biasing by helminth glycans

The ability of helminth parasites to drive polarized Th2 responses has been known for some time. Interestingly, many recent studies have shown that helminth-expressed glycan activation of host immune cells accounts for much of the anti-inflammatory and Th2-biasing observed. This microreview attempts to cover the biology of expression of immunomodulatory glycans in various helminth parasites, the immune cells they interact with including the production of cytokines, chemokines and antibodies. We also discuss the potential cell surface receptors which are capable of binding certain glycans and the known mech-anisms which ultimately lead to production of anti-inflammatory mediators as well as polarizing CD4+ T-cell responses to Th2-type in the host. Lastly, we discuss a novel mechanism for activation of antigen-presenting cells by a specific helminth glycan that leads to maturation of Type 2 dendritic cells.     

Successful genetic transformation of Chinese cabbage using phosphomannose isomerase as a selection marker

A mannose selection system was adapted for use in the Agrobacterium-mediated transformation of Chinese cabbage. This system makes use of the pmi gene that encodes phosphomannose isomerase, which converts mannose-6-phosphate to fructose-6-phosphate. Hypocotyl explants from 4–5-day-old seedlings of Chinese cabbage inbred lines were pre-cultured for 2–3 days and then infected with Agrobacterium. Two genes (l-guluno-γ-lactone oxidase, GLOase, and jasmonic methyl transferase, JMT) were transformed into Chinese cabbage using the transformation procedure developed in this study. We found that supplementing the media with 7 g l⁻¹ mannose and 2% sucrose provides the necessary conditions for the selection of transformed plants from nontransformed plants. The transformation rates were 1.4% for GLOase and 3.0% for JMT, respectively. The Southern blot analysis revealed that several independent transformants (T ₀) were obtained from each transgene. Three different inbred lines were transformed, and most of the T ₁ plants had normal phenotypes. The transformation method presented here for Chinese cabbage using mannose selection is efficient and reproducible, and it can be useful to introduce a desirable gene(s) into commercially useful inbred lines of Chinese cabbage.