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.     

A cascade of morphogenic signaling initiated by the meninges controls corpus callosum formation

The corpus callosum is the most prominent commissural connection between the cortical hemispheres, and numerous neurodevelopmental disorders are associated with callosal agenesis. By using mice either with meningeal overgrowth or selective loss of meninges, we have identified a cascade of morphogenic signals initiated by the meninges that regulates corpus callosum development. The meninges produce BMP7, an inhibitor of callosal axon outgrowth. This activity is overcome by the induction of expression of Wnt3 by the callosal pathfinding neurons, which antagonize the inhibitory effects of BMP7. Wnt3 expression in the cingulate callosal pathfinding axons is developmentally regulated by another BMP family member, GDF5, which is produced by the adjacent Cajal-Retzius neurons and turns on before outgrowth of the callosal axons. The effects of GDF5 are in turn under the control of a soluble GDF5 inhibitor, Dan, made by the meninges. Thus, the meninges and medial neocortex use a cascade of signals to regulate corpus callosum development.     

Development of a target component extraction method from GC-MS data with an in-house program for metabolite profiling

After gas chromatography-mass spectrometry (GC-MS) analysis, data processing, including retention time correction, spectral deconvolution, peak alignment, and normalization prior to statistical analysis, is an important step in metabolomics. Several commercial or free software packages have been introduced for data processing, but most of them are vendor dependent. To design a simple method for Agilent GC/MS data processing, we developed an in-house program, "CompExtractor", using Microsoft Visual Basic. We tailored the macro modules of an Agilent Chemstation and implanted them in the program. To verify the performance of CompExtractor processing, 30 samples from the three species of the genus Papaver were analyzed with Agilent 5973 MSD GC-MS. The results of CompExtractor processing were compared with those of AMDIS-SpectConnect processing by hierarchical cluster analysis (HCA) and principal component analysis (PCA). The two methods showed good classification according to their species in HCA. The PC1+PC2 scores were 54.32-63.62% for AMDIS-SpectConnect and 56.65-85.92% for CompExtractor in PCA. Although the CompExtractor processing method is an Agilent GC-MS-specific application and the target compounds must be selected first, it can extract the target compounds more precisely in the raw data file with batch mode and simultaneously assemble the matrix text file.     

Selective Gαi subunits as novel direct activators of transient receptor potential canonical (TRPC)4 and TRPC5 channels

The ubiquitous transient receptor potential canonical (TRPC) channels function as non-selective, Ca(2+)-permeable channels and mediate numerous cellular functions. It is commonly assumed that TRPC channels are activated by stimulation of Gα(q)-PLC-coupled receptors. However, whether the Gα(q)-PLC pathway is the main regulator of TRPC4/5 channels and how other Gα proteins may regulate these channels are poorly understood. We previously reported that TRPC4/TRPC5 can be activated by Gα(i). In the current work, we found that Gα(i) subunits, rather than Gα(q), are the primary and direct activators of TRPC4 and TRPC5. We report a novel molecular mechanism in which TRPC4 is activated by several Gα(i) subunits, most prominently by Gα(i2), and TRPC5 is activated primarily by Gα(i3). Activation of Gα(i) by the muscarinic M2 receptors or expression of the constitutively active Gα(i) mutants equally and fully activates the channels. Moreover, both TRPC4 and TRPC5 are activated by direct interaction of their conserved C-terminal SESTD (SEC14-like and spectrin-type domains) with the Gα(i) subunits. Two amino acids (lysine 715 and arginine 716) of the TRPC4 C terminus were identified by structural modeling as mediating the interaction with Gα(i2). These findings indicate an essential role of Gα(i) proteins as novel activators for TRPC4/5 and reveal the molecular mechanism by which G-proteins activate the channels.     

Functional changes in myeloid-derived suppressor cells (MDSCs) during tumor growth: FKBP51 contributes to the regulation of the immunosuppressive function of MDSCs

Myeloid-derived suppressor cells (MDSCs) are increased by tumor-derived factors and suppress anti-tumor immunity. MDSCs obtained at a late time point after tumor injection had stronger suppressive activity than MDSCs obtained at an early time point, as measured by T cell proliferation assays. To find factors in MDSCs that change during tumor growth, we analyzed gene expression profiles from MDSCs at different time points after tumor injection. We found that immune response-related genes were downregulated but protumor function-related genes were upregulated in both monocytic MDSCs (Mo-MDSCs) and polymorphonuclear granulocytic MDSCs (PMN-MDSCs) at the late time point. Among differentially expressed genes, FK506 binding protein 51 (FKBP51), which is a member of the immunophilin protein family and plays a role in immunoregulation, was increased in the Mo-MDSCs and PMN-MDSCs isolated from the late time points. Experiments using small interfering RNA and a chemical inhibitor of FKBP51 revealed that FKBP51 contributes to the regulation of the suppressive function of MDSCs by increasing inducible NO synthase, arginase-1, and reactive oxygen species levels and enhancing NF-κB activity. Collectively, our data suggest that FKBP51 is a novel molecule that can be targeted to regulate the immunosuppressive function of MDSCs.     

Enhanced Recognition and Neutralization of HIV-1 by Antibody-Derived CCR5-Mimetic Peptide Variants

A tyrosine-sulfated CCR5-mimetic peptide, CCR5mim1, inhibits HIV-1 infection more efficiently than sulfopeptides based on the CCR5 amino terminus. Here we characterized sulfopeptide chimeras of CCR5mim1 and the heavy-chain CDR3 of the antibody PG16. Two chimeras bound a range of envelope glycoproteins and neutralized HIV-1 more efficiently than CCR5mim1. An immunoadhesin form of one of these, CCR5mim2-Ig, synergized with CD4-Ig to neutralize HIV-1. These sulfopeptides are among the broadest and most potent CCR5-mimetic peptides described to date.     

Determination of endotoxin through an aptamer-based impedance biosensor

Lipopolysaccharide (LPS) often referred to endotoxin is an undesirable impurity frequently entrained with various recombinant protein therapeutics and plasmid DNA (pDNA) vaccines of bacterial origin. The inherent toxicities (e.g. fever, hypotension, shock and death) of LPS render its early and sensitive detection essential for several biological assays and/or parenteral administrations of biotherapeutics. In this study, an electrochemical biosensor using an LPS specific single stranded DNA (ssDNA) aptamer as a probe was developed. Amine-terminated aptamer exhibiting high affinity (K(d)=11.9 nM) to LPS was immobilized on a gold electrode using 3-mercaptopropionic acid (MPA) as a linker. Each step of the modification process was characterized by cyclic voltammetry (CV) and electrochemical impendence spectroscopy (EIS). A good linear relationship of the changes in the charge-transfer resistance (ΔR(et)) and the logarithmic value of LPS concentration was demonstrated in a broad dynamic detection range of 0.001-1 ng/ml. Furthermore, the aptasensor showed a high selectivity to LPS despite the presence of pDNA, RNA and bovine serum albumin (BSA) and could be regenerated in low pH condition, offering a promising option for detecting LPS often present in a complex milieu.     

Immobilized cell microchannel bioreactor for evaluating fermentation characteristics of mixed substrate consumption and product formation

An immobilized cell microchannel bioreactor was designed to test continuous fermentation. The fermentation set-up included a bottom hydrophilic quartz channel to immobilize cells using 0.4 wt% polyethyleneimine and a top channel designed to continuously remove metabolically generated carbon dioxide using hydrophobic polypropylene. To evaluate fermentation characteristics of immobilized cells, ethanol fermentation was carried out using Saccharomyces cerevisiae and Pichia stipitis. The immobilized cell microchannel bioreactor was used to identify long-term activity of immobilized S. cerevisiae cells. The continuous flow microchannel bioreactor was operated stably over a period of 1 month. The immobilized cell microchannel bioreactor was used to examine the characteristics cells that consumed mixed substrates. The concentration ratio of glucose to xylose for simultaneous utilization of hemicellulosic sugars was evaluated using the microchannel bioreactor and the results were compared with those obtained by using conventional batch fermentation with P. stipitis.