Unpredictable cycloisomerization of 1,11-dien-6-ynes by a common cobalt catalyst.

1,11-Dien-6-ynes undergo cycloisomerization in the presence of the cobalt catalytic system CoBr2/phosphine ligand/Zn/ZnI2 giving cyclohexene, diene or cyclopropane structures depending on the type of the phosphine ligand. This unpredictable behaviour suggests that, although the availability of the cobalt catalytic system is appealing, the development of well-defined catalysts is desirable for further progress.

CO2 Electroreduction on Silver Foams Modified by Ionic Liquids with Different Cation Side Chain Length.

Ionic liquids (ILs) are capable of tuning the kinetics of electroreduction processes by modifying a catalyst interface. In this work, a group of hydrophobic imidazolium-based ILs were immobilized on Ag foams by using a procedure known as "solid catalyst with ionic liquid layer" (SCILL). The derived electrocatalysts demonstrated altered selectivity and CO production rates for the electrochemical reduction of CO2 compared to the unmodified Ag foam. The activity change caused by the IL was dependent on the length of the N-alkyl substituent. The rate of CO production is optimized at moderate chain length and IL loadings. The observed trends are attributed to a local enrichment of CO2-based species in the proximity of the catalyst and a modification of the environment of its active sites. On the contrary, high loadings or long IL chains render the surface inaccessible and favor the hydrogen evolution reaction.

Loss of retrograde endocannabinoid signaling and reduced adult neurogenesis in diacylglycerol lipase knock-out mice.

Endocannabinoids (eCBs) function as retrograde signaling molecules at synapses throughout the brain, regulate axonal growth and guidance during development, and drive adult neurogenesis. There remains a lack of genetic evidence as to the identity of the enzyme(s) responsible for the synthesis of eCBs in the brain. Diacylglycerol lipase-alpha (DAGLalpha) and -beta (DAGLbeta) synthesize 2-arachidonoyl-glycerol (2-AG), the most abundant eCB in the brain. However, their respective contribution to this and to eCB signaling has not been tested. In the present study, we show approximately 80% reductions in 2-AG levels in the brain and spinal cord in DAGLalpha(-/-) mice and a 50% reduction in the brain in DAGLbeta(-/-) mice. In contrast, DAGLbeta plays a more important role than DAGLalpha in regulating 2-AG levels in the liver, with a 90% reduction seen in DAGLbeta(-/-) mice. Levels of arachidonic acid decrease in parallel with 2-AG, suggesting that DAGL activity controls the steady-state levels of both lipids. In the hippocampus, the postsynaptic release of an eCB results in the transient suppression of GABA-mediated transmission at inhibitory synapses; we now show that this form of synaptic plasticity is completely lost in DAGLalpha(-/-) animals and relatively unaffected in DAGLbeta(-/-) animals. Finally, we show that the control of adult neurogenesis in the hippocampus and subventricular zone is compromised in the DAGLalpha(-/-) and/or DAGLbeta(-/-) mice. These findings provide the first evidence that DAGLalpha is the major biosynthetic enzyme for 2-AG in the nervous system and reveal an essential role for this enzyme in regulating retrograde synaptic plasticity and adult neurogenesis.

Electroreductive 5-Hydroxymethylfurfural Dimerization on Carbon Electrodes.

The electrochemical conversion of biomass-based compounds to fuels and fuel precursors can aid the defossilization of the transportation sector. Herein, the electrohydrodimerization of 5-hydroxymethylfurfural (HMF) to the fuel precursor 5,5'-bis(hydroxymethyl)hydrofuroin (BHH) was investigated on different carbon electrodes. Compared to boron-doped diamond (BDD) electrodes, on glassy carbon (GC) electrodes a less negative HMF reduction onset potential and a switch in product selectivity from BHH to the electrocatalytic hydrogenation product 2,5-di(hydroxymethyl)furan (DHMF) with increasing overpotential was found. On BDD, the electrohydrodimerization was the dominant process independent of the applied potential. An increase in the initial HMF concentration led to suppression of the competing hydrogen evolution reaction and DHMF formation, resulting in higher BHH faradaic efficiencies. In contrast, BHH selectivity decreased with higher initial HMF concentration, which was attributed to increased electrochemically induced HMF degradation. Finally, it was demonstrated that even a simple graphite foil can function as an active HMF electroreduction catalyst.

Genetic and functional analysis of human P2X5 reveals a distinct pattern of exon 10 polymorphism with predominant expression of the nonfunctional receptor isoform.

P2X5 is a member of the P2X family of ATP-gated nonselective cation channels, which exist as trimeric assemblies. P2X5 is believed to trimerize with another member of this family, P2X1. We investigated the single-nucleotide polymorphism (SNP) at the 3' splice site of exon 10 of the human P2X5 gene. As reported previously, presence of a T at the SNP location results in inclusion of exon 10 in the mature transcript, whereas exon 10 is excluded when a G is present at this location. Our genotyping of human DNA samples reveals predominance of the G-bearing allele, which was exclusively present in DNA samples from white American, Middle Eastern, and Chinese donors. Samples from African American donors were polymorphic, with the G allele more frequent. Reverse transcription-polymerase chain reaction analysis of lymphocytes demonstrated a 100% positive correlation between genotype and P2X5 transcript. Immunostaining of P2X1/P2X5 stably coexpressing cell lines showed full-length P2X5 to be expressed at the cell surface and the exon 10-deleted isoform to be cytoplasmic. Fluorometric imaging-based pharmacological characterization indicated a ligand-dependent increase in intracellular calcium in 1321N1 astrocytoma cells transiently expressing full-length P2X5 but not the exon 10-deleted isoform. Likewise, electrophysiological analysis showed robust ATP-evoked currents when full-length but not the exon 10-deleted isoform of P2X5 was expressed. Taken together, our findings indicate that most humans express only a nonfunctional isoform of P2X5, which is in stark contrast to what is seen in other vertebrate species in which P2X5 has been studied, from which only the full-length isoform is known.

A General and Facile Approach for the Electrochemical Reduction of Carbon Dioxide Inspired by Deep Eutectic Solvents.

Deep eutectic solvents (DESs) were applied to the electrochemical CO2 reduction reaction (CO2 RR). Choline-based DESs represent a non-toxic and inexpensive alternative to room-temperature ionic liquids (RTILs) as additives to the system or as electrolyte. Following the study on choline-based DESs this approach was generalized and simple and organic-soluble systems were devised based on the combination of organic chloride salts with ethylene glycol (EG), allowing the chlorides to be readily used as cocatalysts in the CO2 RR. This approach negates the need for anion exchange and, because the chloride salt is usually the least expensive one, substantially reduces the cost of the electrolyte and opens the way for high-throughput experimentation.

Novel pharmacological activity of loperamide and CP-339,818 on human HCN channels characterized with an automated electrophysiology assay.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels underlie the pacemaker currents in neurons (I(h)) and cardiac (I(f)) cells. As such, the identification and characterization of novel blockers of HCN channels is important to enable the dissection of their function in vivo. Using a new IonWorks HT electrophysiology assay with human HCN1 and HCN4 expressed stably in cell lines, four HCN channel blockers are characterized. Two blockers known for their activity at opioid/Ca(2+) channels and K(+) channels, loperamide and CP-339,818 (respectively), are described to block HCN1 more potently than HCN4. The known HCN blocker ZD7288 was also found to be more selective for HCN1 over HCN4, while the HCN blocker DK-AH269 was equipotent on HCN4 and HCN1. Partial replacement of the intracellular Cl(-) with gluconate reduced the potency on both channels, but to varying degrees. For both HCN1 and HCN4, ZD7288 was most sensitive in lower Cl(-) solutions, while the potency of loperamide was not affected by the differing solutions. The block of HCN1 for all compounds was voltage-dependent, being relieved at more negative potentials. The voltage-dependent, Cl(-) dependent, HCN1 preferring compounds described here elaborate on the current known pharmacology of HCN channels and may help provide novel tools and chemical starting points for the investigation of HCN channel function in natively expressing systems.

Postnatal development of the hyperpolarization-activated excitatory current Ih in mouse hippocampal pyramidal neurons.

The hyperpolarization-activated excitatory current I(h) shapes rhythmic firing and other components of excitability in differentiating neurons, and may thus influence activity-dependent CNS development. We therefore studied developmental changes in I(h) and underlying hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits in pyramidal neurons of neonatal mouse hippocampus using electrophysiological and immunofluorescence approaches. I(h) conductance (at -80 mV) tripled in CA3 neurons and quintupled in CA1 neurons between postnatal day 1 (P1) and P20; parallel changes in membrane area resulted in current density maxima at P5 in CA3 and P10 in CA1. Concurrently, I(h) activation times fell sevenfold in CA3 and 10-fold in CA1. A computational model indicates that a decrease in I(h) activation time will increase the rhythmic firing rate. Two mechanisms contributed to more rapid I(h) activation at P20 in CA3 and CA1 neurons: a fall in the intrinsic time constants of two kinetic components, tau(fast) and tau(slow), to 35-40% (at -90 mV) of their P1 values, and a preferential increase in fast component amplitude and contribution to I(h) (from approximately 35% to approximately 74% of total). HCN1, HCN2, and HCN4 immunoreactivities showed independent temporal and spatial developmental patterns. HCN1 immunoreactivity was low at P1 and P5 and increased by P20. HCN2 immunoreactivity was detected at P1 and increased steadily up to P20. HCN4 immunoreactivity was initially low and showed a small increase by P20. We suggest that developmental increases in I(h) amplitude and activation rate reflect changes in the number and underlying structure of I(h) channels, and that I(h) maturation may shape rhythmic activity important for hippocampal circuit maturation.

Development of a novel automated ion channel recording method using "inside-out" whole-cell membranes.

Efforts to develop novel methods for recording from ion channels have been receiving increased attention in recent years. In this study, the authors report a unique "inside-out" whole-cell configuration of patch-clamp recording that has been developed. This method entails adding cells into a standard patch pipette and, with positive pressure, obtaining a gigaseal recording from a cell at the inside tip of the electrode. In this configuration, the cell may be moved through the air, first rupturing part of the cellular membrane and enabling bath access to the intracellular side of the membrane, and then into a series of wells containing differing solutions, enabling robotic control of all the steps in an experiment. The robotic system developed here fully automates the electrophysiological experiments, including gigaseal formation, obtaining whole-cell configuration, data acquisition, and drug application. Proof-of-principle experiments consisting of application of intracellularly acting potassium channel blockers to K+ channel cell lines resulted in a very rapid block, as well as block reversal, of the current. This technique allows compound application directly to the intracellular side of ion channels and enables the dissociation of compound in activities due to cellular barrier limitations. This technique should allow for parallel implementation of recording pipettes and the future development of larger array-based screening methods.

A novel high-throughput screening assay for HCN channel blocker using membrane potential-sensitive dye and FLIPR.

Hyperpolarization-activated cation nonselective (HCN) channels represent an interesting group of targets for drug development. In this study, the authors report the development of a novel membrane potential-sensitive dye (MPSD) assay for HCN channel modulators that has been miniaturized into 384-well fluorescent imaging plate reader (FLIPR) high-throughput screening (HTS) format. When optimized (by cell plating density, plate type, cell recovery from cryopreservation), the well-to-well signal variability was low, with a Z' = 0.73 and coefficient of variation = 6.4%, whereas the MPSD fluorescence signal amplitude was -23,700 +/- 1500 FLIPR(3) relative fluorescence units (a linear relationship was found between HCN1 MPSD fluorescence signal and the cell plating density) and was completely blocked by 30 microM ZD7288. The assay tolerated up to 1% DMSO, inclusion of which did not significantly change the signal kinetics or amplitude. A single-concentration screening of an ion channel-focused library composed of 4855 compounds resulted in 89 HCN1 blocker hits, 51 of which were subsequently analyzed with an 8-point concentration-response analysis on the IonWorks HT electrophysiology platform. The correlation between MPSD and the electrophysiology assay was moderate, as shown by the linear regression analysis (r(2) = 0.56) between the respective IC(50)s obtained using these 2 assays. The reported HTS-compatible HCN channel blocker assay can serve as a tool in drug discovery in the pursuit of HCN channel isoform-selective small molecules that could be used in the development of clinically relevant compounds.

Direct inhibition of Ih by analgesic loperamide in rat DRG neurons.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are responsible for the functional hyperpolarization-activated current (I(h)) in dorsal root ganglion (DRG) neurons, playing an important role in pain processing. We found that the known analgesic loperamide inhibited I(h) channels in rat DRG neurons. Loperamide blocked I(h) in a concentration-dependent manner, with an IC(50) = 4.9 +/- 0.6 and 11.0 +/- 0.5 microM for large- and small-diameter neurons, respectively. Loperamide-induced I(h) inhibition was unrelated to the activation of opioid receptors and was reversible, voltage-dependent, use-independent, and was associated with a negative shift of V(1/2) for I(h) steady-state activation. Loperamide block of I(h) was voltage-dependent, gradually decreasing at more hyperpolarized membrane voltages from 89% at -60 mV to 4% at -120 mV in the presence of 3.7 microM loperamide. The voltage sensitivity of block can be explained by a loperamide-induced shift in the steady-state activation of I(h). Inclusion of 10 microM loperamide into the recording pipette did not affect I(h) voltage for half-maximal activation, activation kinetics, and the peak current amplitude, whereas concurrent application of equimolar external loperamide produced a rapid, reversible I(h) inhibition. The observed loperamide-induced I(h) inhibition was not caused by the activation of peripheral opioid receptors because the broad-spectrum opioid receptor antagonist naloxone did not reverse I(h) inhibition. Therefore we suggest that loperamide inhibits I(h) by direct binding to the extracellular region of the channel. Because I(h) channels are involved in pain processing, loperamide-induced inhibition of I(h) channels could provide an additional molecular mechanism for its analgesic action.

Regulation of an inactivating potassium current (IA) by the extracellular matrix protein vitronectin in embryonic mouse hippocampal neurones.

Integrins are a class of intrinsic membrane receptors for extracellular matrix ligands. In the central nervous system, integrins and their ligands influence neuronal growth and synaptic function, but relatively little is known about their potential to regulate intrinsic excitability. To explore this area, we examined the effects of matrix components on potassium currents in developing mouse hippocampal neurones, using electrophysiological and immunochemical approaches. We tested the effects of three integrin ligands present in the hippocampus, fibronectin, laminin and vitronectin, on electrogenesis in late embryonic hippocampal pyramidal neurones. Explants cultured in serum-free medium were exposed to ligands (fibronectin at 3 microg ml-1, laminin at 5 microg ml-1, vitronectin at 10 microg ml-1) for 3-4 days, and voltage-gated potassium currents were recorded from presumptive CA3 pyramidal neurones. Of the three matrix components, only vitronectin affected potassium currents, selectively increasing the amplitude of the inactivating potassium current (IA, or A-current) by about 75 % over control levels, and its density (current per unit area) by about 40 % (measured after 3 day exposures from embryonic day 15.5). Other potassium currents were spared, except to the extent that membrane area was increased. The actions of vitronectin were sensitive to RGD (Arg-Gly-Asp)-sequence-containing peptide, indicating the involvement of integrins as vitronectin receptors. The kinetic properties of IA, including the voltage-dependence of activation and inactivation, inactivation rate and the rate of recovery from inactivation, were minimally affected by vitronectin and were consistent with enhanced functional expression of Kv4-family subunits. Analyses of Kv4.2 and Kv1.4 immunoreactivity also suggested a preferential increase in Kv4.2 levels, with lesser effects on Kv1.4 levels. These results indicate that vitronectin can selectively regulate IA, and together with other observations suggest that modulation of neuronal excitability by integrins and their ligands occurs commonly.

Regulation of the hyperpolarization-activated cationic current Ih in mouse hippocampal pyramidal neurones by vitronectin, a component of extracellular matrix.

Because the hyperpolarization-activated cation-selective current I(h) makes important contributions to neural excitability, we examined its long-term regulation by vitronectin, an extracellular matrix component commonly elevated at injury sites and detected immunochemically in activated microglia. Focusing on mouse hippocampal pyramidal neurones in organotypic slice cultures established at postnatal day 0 or 1 and examined after 3-4 days in vitro, we observed differences in the amplitude and activation rate of I(h) between neurones in naive and vitronectin-exposed slices (10 microg ml(-1) added to serum-free medium), and between neurones in slices derived from wild-type and vitronectin-deficient mice. The potassium inward rectifier I(K(ir)), activated at similar voltages to I(h), was not affected by vitronectin. In CA1, differences in I(h) amplitude primarily reflected changes in maximum conductance (G(max)): a 23.3% increase to 3.18 +/- 0.64 nS from 2.58 +/- 0.96 nS (P < 0.05) in vitronectin-exposed neurones, and a 17.9% decrease to 2.24 +/- 0.26 nS from 2.73 +/- 0.64 nS (P < 0.05) in neurones from vitronectin-deficient slices. The voltage of one-half maximum activation (V(1/2)) was not significantly affected by vitronectin exposure (-78.1 +/- 2.3 mV versus -80.0 +/- 4.9 mV in naive neurones; P > 0.05) or vitronectin deficiency (-83.8 +/- 3.1 mV versus -82.0 +/- 2.9 mV in wild-type neurones; P > 0.05). In CA3 neurones, changes in I(h) reflected differences in both G(max) and V(1/2): in vitronectin-exposed neurones there was a 35.4% increase in G(max) to 1.30 +/- 0.49 nS from 0.96 +/- 0.26 nS (P < 0.01), and a +3.0 mV shift in V(1/2) to -89.8 mV from -92.8 mV (P < 0.05). The time course of I(h) activation could be fitted by the sum of two exponential functions, fast and slow. In both CA1 and CA3 neurones the fast component amplitude was preferentially sensitive to vitronectin, with its relatively larger contribution to total current in vitronectin-exposed cells contributing to the acceleration of I(h) activation. Further, HCN1 immunoreactivity appeared elevated in vitronectin-exposed slices, while HCN2 levels appeared unaltered. We suggest that vitronectin-stimulated increases in I(h) may potentially affect excitability under pathological conditions.