Mechanism Underlying Heat Stability of the Rice Endosperm Cytosolic ADP-Glucose Pyrophosphorylase

Rice grains accumulate starch as their major storage reserve whose biosynthesis is sensitive to heat. ADP-glucose pyrophosphorylase (AGPase) is among the starch biosynthetic enzymes severely affected by heat stress during seed maturation. To increase the heat tolerance of the rice enzyme, we engineered two dominant AGPase subunits expressed in developing endosperm, the large (L2) and small (S2b) subunits of the cytosol-specific AGPase. Bacterial expression of the rice S2b with the rice L2, potato tuber LS (pLS), or with the mosaic rice-potato large subunits, L2-pLS and pLS-L2, produced heat-sensitive recombinant enzymes, which retained less than 10% of their enzyme activities after 5 min incubation at 55°C. However, assembly of the rice L2 with the potato tuber SS (pSS) showed significantly increased heat stability comparable to the heat-stable potato pLS/pSS. The S2b assembled with the mosaic L2-pLS subunit showed 3-fold higher sensitivity to 3-PGA than L2/S2b, whereas the counterpart mosaic pLS-L2/S2b showed 225-fold lower sensitivity. Introduction of a QTC motif into S2b created an N-terminal disulfide linkage that was cleaved by dithiothreitol reduction. The QTC enzyme showed moderate heat stability but was not as stable as the potato AGPase. While the QTC AGPase exhibited approximately fourfold increase in 3-PGA sensitivity, its substrate affinities were largely unchanged. Random mutagenesis of S2bQTC produced six mutant lines with elevated production of glycogen in bacteria. All six lines contained a L379F substitution, which conferred enhanced glycogen production in bacteria and increased heat stability. Modeled structure of this mutant enzyme revealed that this highly conserved leucine residue is located in the enzyme’s regulatory pocket that provides interaction sites for activators and inhibitors. Our molecular dynamic simulation analysis suggests that introduction of the QTC motif and the L379F mutation improves enzyme heat stability by stabilizing their backbone structures possibly due to the increased number of H-bonds between the small subunits and increased intermolecular interactions between the two SSs and two LSs at elevated temperature

A proteomic approach to investigating gene cluster expression and secondary metabolite functionality in Aspergillus fumigatus.

A combined proteomics and metabolomics approach was utilised to advance the identification and characterisation of secondary metabolites in Aspergillus fumigatus. Here, implementation of a shotgun proteomic strategy led to the identification of non-redundant mycelial proteins (n = 414) from A. fumigatus including proteins typically under-represented in 2-D proteome maps: proteins with multiple transmembrane regions, hydrophobic proteins and proteins with extremes of molecular mass and pI. Indirect identification of secondary metabolite cluster expression was also achieved, with proteins (n = 18) from LaeA-regulated clusters detected, including GliT encoded within the gliotoxin biosynthetic cluster. Biochemical analysis then revealed that gliotoxin significantly attenuates H2O2-induced oxidative stress in A. fumigatus (p>0.0001), confirming observations from proteomics data. A complementary 2-D/LC-MS/MS approach further elucidated significantly increased abundance (p<0.05) of proliferating cell nuclear antigen (PCNA), NADH-quinone oxidoreductase and the gliotoxin oxidoreductase GliT, along with significantly attenuated abundance (p<0.05) of a heat shock protein, an oxidative stress protein and an autolysis-associated chitinase, when gliotoxin and H2O2 were present, compared to H2O2 alone. Moreover, gliotoxin exposure significantly reduced the abundance of selected proteins (p<0.05) involved in de novo purine biosynthesis. Significantly elevated abundance (p<0.05) of a key enzyme, xanthine-guanine phosphoribosyl transferase Xpt1, utilised in purine salvage, was observed in the presence of H2O2 and gliotoxin. This work provides new insights into the A. fumigatus proteome and experimental strategies, plus mechanistic data pertaining to gliotoxin functionality in the organism

Transferable anticancer innate immunity in spontaneous regression/complete resistance mice

Spontaneous regression/complete resistance (SR/CR) mice resist very high doses of cancer cells that are lethal to WT mice even at low doses. In this study, we show that this resistance is mediated by rapid infiltration of leukocytes, mostly of innate immunity, in both primary and repeated challenges. Formation of rosettes with infiltrating natural killer cells, neutrophils, and macrophages was required for the subsequent destruction of cancer cells through rapid cytolysis. Highly purified natural killer cells, macrophages, and neutrophils from the SR/CR mice independently killed cancer cells in vitro. The independent killing activity by each subset of effector cells is consistent with the observation that the resistance was abolished by depleting total infiltrating leukocytes but not by depleting only one or two subsets of leukocytes. The resistance was completely transferable to WT recipient mice through SR/CR splenocytes, bone marrow cells, or enriched peritoneal macrophages, either for prevention against subsequent cancer challenges or eradication of established malignancy at distant sites

The translational value of non-human primates in preclinical research on infection and immunopathology

The immune system plays a central role in the defense against environmental threats - such as infection with viruses, parasites or bacteria - but can also be a cause of disease, such as in the case of allergic or autoimmune disorders. In the past decades the impressive development of biotechnology has provided scientists with biological tools for the development of highly selective treatments for the different types of disorders. However, despite some clear successes the translation of scientific discoveries into effective treatments has remained challenging. The often-disappointing predictive validity of the preclinical animal models that are used in the selection of the most promising vaccine or drug candidates is the Achilles heel in the therapy development process. This publication summarizes the relevance and usage of non-human primates as pre-clinical model in infectious and autoimmune diseases, in particular for biologicals, which due to their high species-specificity are inactive in lower species. (C) 2015 Elsevier B.V. All rights reserved