A novel hypothesis for the binding mode of HERG channel blockers

We present a new docking model for HERG channel blockade. Our new model suggests three key interactions such that (1) a protonated nitrogen of the channel blocker forms a hydrogen bond with the carbonyl oxygen of HERG residue T623; (2) an aromatic moiety of the channel blocker makes a pi-pi interaction with the aromatic ring of HERG residue Y652; and (3) a hydrophobic group of the channel blocker forms a hydrophobic interaction with the benzene ring of HERG residue F656. The previous model assumes two interactions such that (1) a protonated nitrogen of the channel blocker forms a cation-pi interaction with the aromatic ring of HERG residue Y652; and (2) a hydrophobic group of the channel blocker forms a hydrophobic interaction with the benzene ring of HERG residue F656. To test these models, we classified 69 known HERG channel blockers into eight binding types based on their plausible binding modes, and further categorized them into two groups based on the number of interactions our model would predict with the HERG channel (two or three). We then compared the pIC(50) value distributions between these two groups. If the old hypothesis is correct, the distributions should not differ between the two groups (i.e., both groups show only two binding interactions). If our novel hypothesis is correct, the distributions should differ between Groups 1 and 2. Consistent with our hypothesis, the two groups differed with regard to pIC(50), and the group having more predicted interactions with the HERG channel had a higher mean pIC(50) value. Although additional work will be required to further validate our hypothesis, this improved understanding of the HERG channel blocker binding mode may help promote the development of in silico predictions methods for identifying potential HERG channel blockers.     

Crystal structure of Thermus caldophilus phosphoglycerate kinase in the open conformation

Phosphoglycerate kinase (PGK) is a key glycolytic enzyme that catalyzes the reversible transfer of a phosphate from 1,3-bisphosphoglycerate to ADP to form 3-phosphoglycerate and ATP in the presence of magnesium. During catalysis, a conformational change occurs that brings the N- and C-domains of PGK closer together. Here we present the 1.8A crystal structure of unliganded PGK from Thermus caldophilus (Tca). Comparison of the structure of TcaPGK (open conformation) with that of Thermotoga maritima (Tma) PGK (closed conformation) revealed that the conformational change reflects a change in the interaction between the domains. We identified Arg148 as a key residue involved in open-to-closed transition. The open conformation of TcaPGK is stabilized by an interdomain salt bridge between Arg148 and Glu375. The binding of 3-PG (or maybe 1,3-BPG) disrupts this salt bridge and, in ternary complex, the formation of new salt bridge between Arg60 and Asp197 stabilizes the closed conformation.     

Dominance of Lysobacter sp. in the rhizosphere of two coastal sand dune plant species, Calystegia soldanella and Elymus mollis

Bacterial diversity in the rhizosphere of beach morning glory (Calystegia soldanella) and wild rye (Elymus mollis), two of the major plant species inhabiting the coastal sane dune in Tae-An, Korea, was studied by the analysis of community 16S rRNA gene clones. The amplified rDNA restriction analysis (ARDRA) of the clones using HaeIII exhibited significant differences in the community composition between the two plant species as well as regional differences, but also identified a specific ARDRA pattern that was most common among the clones regardless of plant species. Subsequent sequence analysis indicated that the pattern was that of Lysobacter spp., which is a member of the family Xanthomonadaceae, class Gamma proteobacteria. The Lysobacter clones comprised 50.6% of the clones derived from C. soldanella and 62.5% of those from E. mollis. Other minor patterns included those of Pseudomonas spp., species of Rhizobium, Chryseobacterium spp. and Pantoea spp. among C. soldanella clones, and Pseudomonas sp. and Aeromonas hydrophila among E. mollis clones. It is not yet clear what kind of roles Lysobacter plays in association with sand dune plants, but its universal presence in the rhizosphere, together with the potential of this taxon for antagonistic activity against plant pathogens, suggests that Lysobacter might form a symbiotic relationship with its host plants.     

Evaluation of formulations of Bacillus licheniformis for the biological control of tomato gray mold caused by Botrytis cinerea

Bacillus licheniformis N1, which has previously exhibited potential as a biological control agent, was investigated to develop a biofungicide to control the gray mold of tomato caused by Botrytis cinerea. Various formulations of B. licheniformis N1 were developed using fermentation cultures of the bacteria in Biji medium, and their ability to control gray mold on tomato plants was evaluated. The results of pot experiments led to the selection of the wettable powder formulation N1E, based on corn starch and olive oil, for evaluation of the disease control activity of this bacterium after both artificial infection of the pathogen and natural disease occurrence under production conditions. In plastic-house artificial infection experiments, a 100-fold diluted N1E treatment was found to be the optimum biofungicide spray formulation. This treatment resulted in the significant reduction of symptom development when N1E was applied before Bo. cinerea infection, but not after the infection. Both artificial infection experiments in a plastic house and natural infection experiments under production conditions revealed that the N1E significantly reduced disease severity on tomato plants and flowers. The disease control value of N1E on tomato plants was 90.5% under production conditions, as compared to the 77% conferred by a chemical fungicide, the mixture of carbendazim and diethofencarb (1:1). The prevention of flower infection by N1E resulted in increased numbers of tomato fruits on each plant. N1E treatment also had growth promotion activity, which showed the increased number of tomato fruits compared to fungicide treatment and non-treated control and the increased fruit size compared the non-treated control under production conditions. This study suggests that the corn starch-based formulation of B. licheniformis developed using liquid fermentation will be an effective tool in the biological control of tomato gray mold.     

Glycogen synthase kinase-3 is an in vivo regulator of hematopoietic stem cell repopulation

The in vivo regulation of hematopoietic stem cell (HSC) function is poorly understood. Here, we show that hematopoietic repopulation can be augmented by administration of a glycogen synthase kinase-3 (GSK-3) inhibitor to recipient mice transplanted with mouse or human HSCs. GSK-3 inhibitor treatment improved neutrophil and megakaryocyte recovery, recipient survival and resulted in enhanced sustained long-term repopulation. The output of primitive Lin(-)c-Kit(+)Sca-1(+) cells and progenitors from HSCs increased upon GSK-3 inhibitor treatment without altering secondary repopulating ability, suggesting that the HSC pool is maintained while overall hematopoietic reconstitution is increased. GSK-3 inhibitors were found to modulate gene targets of Wnt, Hedgehog and Notch pathways in cells comprising the primitive hematopoietic compartment without affecting mature cells. Our study establishes GSK-3 as a specific in vivo modulator of HSC activity, and suggests that administration of GSK-3 inhibitors may provide a clinical means to directly enhance the repopulating capacity of transplanted HSCs.     

Crystal structure of a beta-catenin/BCL9/Tcf4 complex

The canonical Wnt pathway plays critical roles in embryonic development, stem cell growth, and tumorigenesis. Stimulation of the Wnt pathway leads to the association of beta-catenin with Tcf and BCL9 in the nucleus, resulting in the transactivation of Wnt target genes. We have determined the crystal structure of a beta-catenin/BCL9/Tcf-4 triple complex at 2.6 A resolution. Our studies reveal that the beta-catenin binding site of BCL9 is distinct from that of most other beta-catenin partners and forms a good target for developing drugs that block canonical Wnt/beta-catenin signaling. The BCL9 beta-catenin binding domain (CBD) forms an alpha helix that binds to the first armadillo repeat of beta-catenin, which can be mutated to prevent beta-catenin binding to BCL9 without affecting cadherin or alpha-catenin binding. We also demonstrate that beta-catenin Y142 phosphorylation, which has been proposed to regulate BCL9-2 binding, does not directly affect the interaction of beta-catenin with either BCL9 or BCL9-2.     

Extreme regression

We develop a new method for describing patient characteristics associated with extreme good or poor outcome. We address the problem with a regression model composed of extrema (maximum and minimum) functions of the predictor variables. This class of models allows for simple regression function inversion and results in level sets of the regression function which can be expressed as interpretable Boolean combinations of decisions based on individual predictors. We develop an estimation algorithm and present clinical applications to symptoms data for patients with Hodgkin's disease and survival data for patients with multiple myeloma.     

The phox homology domain of phospholipase D activates dynamin GTPase activity and accelerates EGFR endocytosis

Dynamin is a large GTP-binding protein that mediates endocytosis by hydrolyzing GTP. Previously, we reported that phospholipase D2 (PLD2) interacts with dynamin in a GTP-dependent manner. This implies that PLD may regulate the GTPase cycle of dynamin. Here, we show that PLD functions as a GTPase activating protein (GAP) through its phox homology domain (PX), which directly activates the GTPase domain of dynamin, and that the arginine residues in the PLD-PX are vital for this GAP function. Moreover, wild-type PLD-PX, but not mutated PLD-PXs defective for GAP function in vitro, increased epidermal growth factor receptor (EGFR) endocytosis at physiological EGF concentrations. In addition, the silencing of PLDs was shown to retard EGFR endocytosis and the addition of wild-type PLDs or lipase-inactive PLDs, but not PLD1 mutants with defective GAP activity for dynamin in vitro, resulted in the recovery of EGFR endocytosis. These findings suggest that PLD, functioning as an intermolecular GAP for dynamin, accelerates EGFR endocytosis. Moreover, we determined that the phox homology domain itself had GAP activity - a novel function in addition to its role as a binding motif for proteins or lipids.     

Classification methods for the development of genomic signatures from high-dimensional data

Personalized medicine is defined by the use of genomic signatures of patients to assign effective therapies. We present Classification by Ensembles from Random Partitions (CERP) for class prediction and apply CERP to genomic data on leukemia patients and to genomic data with several clinical variables on breast cancer patients. CERP performs consistently well compared to the other classification algorithms. The predictive accuracy can be improved by adding some relevant clinical/histopathological measurements to the genomic data.