Screening for cold tolerance genes in C. elegans, whose expressions are affected by anticancer drugs camptothecin and leptomycin B.

Temperature is a vital environmental factor affecting organisms' survival as they determine the mechanisms to tolerate rapid temperature changes. We demonstrate an experimental system for screening chemicals that affect cold tolerance in Caenorhabditis elegans. The anticancer drugs leptomycin B and camptothecin were among the 4000 chemicals that were screened as those affecting cold tolerance. Genes whose expression was affected by leptomycin B or camptothecin under cold stimuli were investigated by transcriptome analysis. Abnormal cold tolerance was detected in several mutants possessing genes that were rendered defective and whose expression altered after exposure to either leptomycin B or camptothecin. The genetic epistasis analysis revealed that leptomycin B or camptothecin may increase cold tolerance by affecting a pathway upstream of the insulin receptor DAF-2 that regulates cold tolerance in the intestine. Our experimental system combining drug and cold tolerance could be used for a comprehensive screening of genes that control cold tolerance at a low cost and in a short time period.

Lipid Bilayer Reformation Using the Wiping Blade for Improved Ion Channel Analysis.

The measurement of ion permeation activity across planar lipid bilayers is a useful technique for the functional analysis and drug evaluation of ion channels at the single-molecule level. To enhance the data throughput, parallelization of lipid bilayers is desirable. However, existing parallelized approaches face challenges in simultaneously and efficiently measuring ion channel activities under various conditions on one chip. In this study, we propose an approach to overcome these limitations by developing a device capable of repeated measurements of ion channels incorporated into individually arrayed lipid bilayers. Our device forms an array of a lipid bilayer at a micropore on a separator by merging two lipid monolayers assembled on the surface of aqueous droplets. We introduce a vertically moving, blade-shaped module─referred to as a "wiping blade"─which enables controlled disruption and reformation of the bilayer at the micropore. By optimizing the surface properties and clearance of the wiping blade, we successfully achieved repeated bilayer formation. The arrayed lipid bilayer device with the integrated wiping blade module demonstrates a 5-fold improvement in data throughput during ion channel activity measurements. Finally, we validate the practical utility of our device by evaluating the effects of an ion channel inhibitor. The developed device opens new avenues for high-throughput analysis and screening of ion channels, leading to significant advancements in drug discovery and functional studies of membrane proteins. It offers a powerful tool for researchers in the field and holds promise for accelerating drug development by targeting ion channels.

Discovery of Novel HCN4 Blockers with Unique Blocking Kinetics and Binding Properties.

The hyperpolarization-activated cyclic nucleotide-gated 4 (HCN4) channel underlies the pacemaker currents, called "If," in sinoatrial nodes (SANs), which regulate heart rhythm. Some HCN4 blockers such as ivabradine have been extensively studied for treating various heart diseases. Studies have shown that these blockers have diverse state dependencies and binding sites, suggesting the existence of potential chemical and functional diversity among HCN4 blockers. Here we report approaches for the identification of novel HCN4 blockers through a random screening campaign among 16,000 small-molecule compounds using an automated patch-clamp system. These molecules exhibited various blockade profiles, and their blocking kinetics and associating amino acids were determined by electrophysiological studies and site-directed mutagenesis analysis, respectively. The profiles of these blockers were distinct from those of the previously reported HCN channel blockers ivabradine and ZD7288. Notably, the mutagenesis analysis showed that blockers with potencies that were increased when the channel was open involved a C478 residue, located at the pore cavity region near the cellular surface of the plasma membrane, while those with potencies that were decreased when the channel was open involved residues Y506 and I510, located at the intracellular region of the pore gate. Thus, this study reported for the first time the discovery of novel HCN4 blockers by screening, and their profiling analysis using an automated patch-clamp system provided chemical tools that will be useful to obtain unique molecular insights into the drug-binding modes of HCN4 and may contribute to the expansion of therapeutic options in the future.

Electrophysiological measurement of ion channels on plasma/organelle membranes using an on-chip lipid bilayer system.

Ion channels are located in plasma membranes as well as on mitochondrial, lysosomal, and endoplasmic reticulum membranes. They play a critical role in physiology and drug targeting. It is particularly challenging to measure the current mediated by ion channels in the lysosomal and the endoplasmic reticulum membranes using the conventional patch clamp method. In this study, we show that our proposed device is applicable for an electrophysiological measurement of various types of ion channel in plasma and organelle membranes. We designed an on-chip device that can form multiple electrical contacts with a measurement system when placed on a mount system. Using crude cell membranes containing ion channels extracted from cultured cells without detergents, we detected open/close signals of the hERG, TRPV1, and NMDA channels on plasma membranes, those of the TRPML1 channels on lysosomal membranes, and open/close signals of the RyR channels on SR membranes. This method will provide a highly versatile drug screening system for ion channels expressed by various cell membranes, including plasma, SR, mitochondrial, Golgi, and lysosomal membranes.

SOx Tolerant Pt/TiO2 Catalysts for CO Oxidation and the Effect of TiO2 Supports on Catalytic Activity.

We developed a new technique for mitigating catalyst deactivation caused by SO2 in exhaust gases. A series of 0.1 wt %-Pt/TiO2 catalysts with different surface, crystal, and pore structures were prepared and tested for CO oxidation activity in the presence of SO2 and H2O. The order of the CO oxidation activity under the influence of SO2 was much different from that in the absence of SO2. Catalysts with a high ratio of larger pores exhibited higher catalytic activity under the influence of SO2 and H2O in the temperature range of 250-300 °C, whereas other parameters, such as BET surface area and crystal structure of the TiO2 support, had minor effects on the CO oxidation activity. The oxidation state of Pt differed significantly depending on the kind of TiO2 support. Some catalysts were less active without H2 reduction pretreatment due to the presence of oxidized Pt species.

A novel Na(+) -Independent alanine-serine-cysteine transporter 1 inhibitor inhibits both influx and efflux of D-Serine.

NMDA receptor dysfunctions are hypothesized to underlie the pathophysiology of schizophrenia, and treatment with D-serine (D-Ser), an NMDA receptor coagonist, may improve the clinical symptoms of schizophrenia. Thus, upregulating the synaptic D-Ser level is a novel strategy for schizophrenia treatment. Na(+) -independent alanine-serine-cysteine transporter 1 (asc-1) is a transporter responsible for regulating the extracellular D-Ser levels in the brain. In this study, we discovered a novel asc-1 inhibitor, (+)-amino(1-(3,5-dichlorophenyl)-3,5-dimethyl-1H-pyrazol-4-yl)acetic acid (ACPP), and assessed its pharmacological profile. ACPP inhibited the D-[(3) H]Ser uptake in human asc-1-expressing CHO cells and rat primary neurons with IC50 values of 0.72 ± 0.13 and 0.89 ± 0.30 µM, respectively. In accordance with the lower asc-1 expression levels in astrocytes, ACPP did not inhibit D-Ser uptake in rat primary astrocytes. In a microdialysis study, ACPP dose dependently decreased the extracellular D-Ser levels in the rat hippocampus under the same conditions in which the asc-1 inhibitor S-methyl-L-cysteine (SMLC) increased it. To obtain insights into this difference, we conducted a D-[(3) H]Ser efflux assay using asc-1-expressing CHO cells. ACPP inhibited D-[(3) H]Ser efflux, whereas SMLC increased it. These results suggest that ACPP is a novel inhibitor of asc-1. © 2016 Wiley Periodicals, Inc.

Transient receptor potential canonical 3 (TRPC3) mediates thrombin-induced astrocyte activation and upregulates its own expression in cortical astrocytes.

Reactive astrogliosis, defined by abnormal morphology and excessive cell proliferation, is a characteristic response of astrocytes to CNS injuries, including intracerebral hemorrhage. Thrombin, a major blood-derived serine protease, leaks into the brain parenchyma upon blood-brain barrier disruption and can induce brain injury and astrogliosis. Transient receptor potential canonical (TRPC) channels, Ca(2+)-permeable, nonselective cation channels, are expressed in astrocytes and involved in Ca(2+) influx after receptor stimulation; however, their pathophysiological functions in reactive astrocytes remain unknown. We investigated the pathophysiological roles of TRPC in thrombin-activated cortical astrocytes. Application of thrombin (1 U/ml, 20 h) upregulated TRPC3 protein, which was associated with increased Ca(2+) influx after thapsigargin treatment. Pharmacological manipulations revealed that the TRPC3 upregulation was mediated by protease-activated receptor 1 (PAR-1), extracellular signal-regulated protein kinase, c-Jun NH(2)-terminal kinase, and nuclear factor-κB signaling and required de novo protein synthesis. The Ca(2+) signaling blockers BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate and a selective TRPC3 inhibitor, pyrazole-3, attenuated TRPC3 upregulation, suggesting that Ca(2+) signaling through TRPC3 contributes to its increased expression. Thrombin-induced morphological changes at 3 h upregulated S100B, a marker of reactive astrocytes, at 20 h and increased astrocytic proliferation by 72 h, all of which were inhibited by Ca(2+)-signaling blockers and specific knockdown of TRPC3 using small interfering RNA. Intracortical injection of SFLLR-NH(2), a PAR-1 agonist peptide, induced proliferation of astrocytes, most of which were TRPC3 immunopositive. These results suggest that thrombin dynamically upregulates TRPC3 and that TRPC3 contributes to the pathological activation of astrocytes in part through a feedforward upregulation of its own expression.

High-vacuum vapor deposition and in situ monitoring of N-carboxy anhydride benzyl glutamate polymerization.

A high-vacuum physical vapor deposition (PVD) method was used to grow polypeptide chains from gold surfaces coupled with in situ monitoring using surface plasmon resonance spectroscopy (SPR). Polymerization of N-carboxy anhydride of benzyl-L-glutamate (BLG-NCA) was demonstrated, forming grafted polybenzyl-L-glutamate (PBLG) films. 2-Aminoethanethiol (AET) was used as the initiating site either deposited as a self-assembled monolayer (SAM), as a PVD film, or codeposited on the gold substrates. PBLG films up to 30 nm thick in which the formation of different secondary structures was monitored by in situ SPR and ex situ Fourier transform infrared spectroscopy (FT-IR) methods based on the parameters for deposition were prepared. Furthermore, surface topology and film morphology properties were determined on the basis of AFM measurements and were found to depend largely on the deposition process of the AET. Besides investigation of the AET surface density effects, the orientation of the PBLG chains was also characterized using ex situ FT-IR.

Ca2+ mobilization mediated by transient receptor potential canonical 3 is associated with thrombin-induced morphological changes in 1321N1 human astrocytoma cells.

Activated astrocytes show various patterns of Ca(2+) mobilization under pathological conditions. In the present study we revealed a novel function of astrocytic Ca(2+) dynamics through investigation of thrombin-induced unique Ca(2+) entry. Using 1321N1 human astrocytoma cells, which have been shown to be a good model for detecting morphological dynamics, we observed rapid retraction of bipolar protrusions that were reversibly evoked by 0.03-3 U/mL thrombin. Morphological changes were predominantly dependent on a specific thrombin receptor subtype, proteinase-activated receptor 1 (PAR-1). In parallel, Fura-2 imaging of intracellular Ca(2+) concentration ([Ca(2+)](i)) showed that thrombin induced heterogeneous Ca(2+) responses with asynchronous repetitive peaks. These oscillations were found to be a result of repetitive Ca(2+) release from intracellular stores, followed by refilling of Ca(2+) from the extracellular region without a direct [Ca(2+)](i) increase. Pharmacological manipulation with BAPTA-AM, cyclopiazonic acid, and 2-aminoethoxydiphenyl borate indicated that Ca(2+) mobilization was involved in thrombin-induced morphological changes. We further addressed the role of Ca(2+) entry using small interfering RNA (siRNA) for transient receptor potential canonical 3 (TRPC3). As a result, both thrombin-induced morphological changes and oscillatory Ca(2+) responses were significantly attenuated in siRNA-transfected cells. Inhibition of TRPC3 with pyrazole-3 also provided support for the contribution of Ca(2+) influx. Moreover, TRPC3-mediated Ca(2+) dynamics regulated thrombin-induced phosphorylation of myosin light chain 2. These results suggest a novel function of astrocytic Ca(2+) dynamics, including Ca(2+) entry, in the pathophysiological effects of PAR-1-mediated astrocytic activation. TRPC3 forms a functional Ca(2+) channel and might modulate astrocytic activation in response to brain hemorrhaging.

The promoting effect of energetic activation of a methane molecular-beam in the direct catalytic partial oxidation of methane.

Energetic activation of a methane molecular-beam promoted remarkably the direct catalytic partial oxidation on Pt and Rh foils, in particular, hydrogen formation was dramatically enhanced.

IR chemiluminescence probe of the vibrational energy distribution of CO2 formed during steady-state CO oxidation on Pt(111) and Pt(110) surfaces.

The infrared (IR) chemiluminescence spectra of CO2 were measured during the steady-state CO + O2 reaction over Pt(110) and Pt(111) surfaces. Analysis of the IR emission spectra indicates that the bending vibrational temperature (TVB), as well as the antisymmetric vibrational temperature (TVAS), was higher on Pt(110) than on Pt(111). On the Pt(110) surface, the highly excited bending vibrational mode compared to the antisymmetric vibrational mode was observed under reaction conditions at low CO coverage (theta(CO) < 0.2) or at high surface temperatures (TS > or = 700 K). This can be related to the activated complex of CO2 formation in a more bent form on the inclined (111) terraces of the Pt(110)(1 x 2) structure. On the other hand, at high CO coverage (theta(CO) > 0.2) or at low surface temperatures (TS < 650 K), TVAS was higher than TVB, which can be caused by the reconstruction of the Pt(110)(1 x 2) surface to the (1 x 1) form with high CO coverage.

Scallop DMT functions as a Ca2+ transporter.

We identified a DMT (divalent metal transporter) homologous protein that functions as a Ca(2+) transporter. Scallop DMT cDNA encodes a 539-amino-acid protein with 12 putative membrane-spanning domains and has a consensus transport motif in the fourth extracellular loop. Since its mRNA is significantly expressed in the gill and intestine, it is assumed that scallop DMT transports Ca(2+) from seawater by the gill and from food by the intestine. Scallop DMT lacks the iron-responsive element commonly found in iron-regulatory proteins, suggesting that it is free of the post-transcriptional regulation from intracellular Fe(2+) concentration. Scallop DMT distinctly functions as a Ca(2+) transporter unlike other DMTs, however, it also transports Fe(2+) and Cd(2+) similar to them.

Structure of activated complex of CO2 formation in a CO + O2 reaction on Pd(110) and Pd(111).

Examinations of CO2 formed during steady-state CO oxidation reactions were performed using infrared (IR) chemiluminescence. The CO2 was formed using a molecular-beam method over Pd(110) and Pd(111). The CO2 formation rate is temperature dependent under various partial pressure conditions. The temperature of the maximum formation rate is denoted as TSmax. Analyses of IR emission spectra at surface temperatures higher than TSmax showed that the average vibrational temperature (TVAV) is higher for Pd(111) than for Pd(110). The antisymmetric vibrational temperature (TVAS) is almost equal on both surfaces. These results suggest that the activated CO2 complex is more bent on Pd(111) and straighter on Pd(110). Furthermore, the difference in the TVAV value was small for surface temperatures less than TSmax. The TVAS value was much higher than TVAV on both surfaces. These phenomena were observed only when the surface temperature was lower than TSmax: they became more pronounced at lower temperatures, suggesting that the activated complex of CO2 formation is much straighter on both Pd surfaces than that observed at higher surface temperatures. Combined with kinetic results, the higher CO coverage at the lower surface temperatures is inferred to be related to the linear activated complex of CO2 formation.

Infrared chemiluminescence study of CO2 formation in CO + NO reaction on Pd(110) and Pd(111) surfaces.

Infrared (IR) chemiluminescence studies of CO2 formed during steady-state CO + NO reaction over Pd(110) and Pd(111) surfaces were carried out. Kinetics of the CO + NO reaction were studied over Pd(110) using a molecular-beam reaction system in the pressure range of 10-2-10-1 Torr. The activity of the CO + NO reaction on Pd(110) was much higher than that of Pd(111), which was quite different from the result of other experiments under a higher pressure range. On the basis of the experimental data on the dependence of the reaction rate on CO and NO pressures and the reaction rate constants obtained by using a reaction model, the coverage of NO, CO, N, and O was calculated under various flux conditions. From the analysis of IR emission spectra in the CO + O2 reaction on Pd(110) and Pd(111), the antisymmetric vibrational temperature (TVAS) was seen to be higher than the bending vibrational temperature (TVB) on Pd(110). In contrast, TVB was higher than TVAS on Pd(111). These behaviors suggest that the activated complex for CO2 formation is more bent on Pd(111) than that on Pd(110), which is reflected by the surface structure. Both TVB and TVAS for the CO + O2 reaction on Pd(110) and Pd(111) increased gradually with increasing surface temperature (TS). On the other hand, in the case of the CO + NO reaction on Pd(110) and Pd(111), TVAS decreased and TVB increased significantly with increasing TS. TVB was lower than TVAS at lower TS, while TVB was higher than TVAS at higher TS. Comparison of the data obtained for the two reactions indicates that TVB in the CO + NO reaction on Pd(110) at TS = 800 and 850 K is much higher than that in the CO + O2 reaction on Pd(110).