Presynaptic actions of 4-Aminopyridine and γ-aminobutyric acid on rat sympathetic ganglia in vitro

Responses to bath-applications of 4-aminopyridine (4-AP) and -aminobutyric acid (GABA) were recorded intracellularly from neurones in the rat isolated superior cervical ganglion. 4-aminopyridine (0.1–1.0 mmol/l) usually induced spontaneous action potentials and excitatory postsynaptic potentials (EPSPs), which were blocked by hexamethonium. Membrane potential was unchanged; spike duration was slightly increased. Vagus nerve B-and C-fibre potentials were prolonged. In 4-AP solution (0.1–0.3 mmol/l), GABA (0.1 mmol/l), 3-aminopropanesulphonic acid or muscimol evoked bursts of spikes and EPSPs in addition to a neuronal depolarization. These bursts, which were not elicited by glycine, glutamate, taurine or (±)-baclofen, were completely antagonised by hexamethonium, tetrodotoxin or bicuculline methochloride. It is concluded that: (a) 4-AP has a potent presynaptic action on sympathetic ganglia; (b) presynaptic actions of GABA can be recorded postsynaptically in the presence of 4-AP; and (c) the presynaptic GABA-receptors revealed in this condition are similar to those on the postsynaptic membrane

The development of novel LTA4H modulators to selectively target LTB4 generation

The pro-inflammatory mediator leukotriene B4 (LTB4) is implicated in the pathologies of an array of diseases and thus represents an attractive therapeutic target. The enzyme leukotriene A4 hydrolase (LTA4H) catalyses the distal step in LTB4 synthesis and hence inhibitors of this enzyme have been actively pursued. Despite potent LTA4H inhibitors entering clinical trials all have failed to show efficacy. We recently identified a secondary anti-inflammatory role for LTA4H in degrading the neutrophil chemoattractant Pro-Gly-Pro (PGP) and rationalized that the failure of conventional LTA4H inhibitors may be that they inadvertently prevented PGP degradation. We demonstrate that these inhibitors do indeed fail to discriminate between the dual activities of LTA4H, and enable PGP accumulation in mice. Accordingly, we have developed novel compounds that potently inhibit LTB4 generation whilst leaving PGP degradation unperturbed. These novel compounds could represent a safer and superior class of LTA4H inhibitors for translation into the clinic

PIP5KIβ Selectively Modulates Apical Endocytosis in Polarized Renal Epithelial Cells

Localized synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] at clathrin coated pits (CCPs) is crucial for the recruitment of adaptors and other components of the internalization machinery, as well as for regulating actin dynamics during endocytosis. PtdIns(4,5)P2 is synthesized from phosphatidylinositol 4-phosphate by any of three phosphatidylinositol 5-kinase type I (PIP5KI) isoforms (α, β or γ). PIP5KIβ localizes almost exclusively to the apical surface in polarized mouse cortical collecting duct cells, whereas the other isoforms have a less polarized membrane distribution. We therefore investigated the role of PIP5KI isoforms in endocytosis at the apical and basolateral domains. Endocytosis at the apical surface is known to occur more slowly than at the basolateral surface. Apical endocytosis was selectively stimulated by overexpression of PIP5KIβ whereas the other isoforms had no effect on either apical or basolateral internalization. We found no difference in the affinity for PtdIns(4,5)P2-containing liposomes of the PtdIns(4,5)P2 binding domains of epsin and Dab2, consistent with a generic effect of elevated PtdIns(4,5)P2 on apical endocytosis. Additionally, using apical total internal reflection fluorescence imaging and electron microscopy we found that cells overexpressing PIP5KIβ have fewer apical CCPs but more internalized coated structures than control cells, consistent with enhanced maturation of apical CCPs. Together, our results suggest that synthesis of PtdIns(4,5)P2 mediated by PIP5KIβ is rate limiting for apical but not basolateral endocytosis in polarized kidney cells. PtdIns(4,5)P2 may be required to overcome specific structural constraints that limit the efficiency of apical endocytosis. © 2013 Szalinski et al

A Novel, “Double-Clamp” Binding Mode for Human Heme Oxygenase-1 Inhibition

The development of heme oxygenase (HO) inhibitors is critical in dissecting and understanding the HO system and for potential therapeutic applications. We have established a program to design and optimize HO inhibitors using structure-activity relationships in conjunction with X-ray crystallographic analyses. One of our previous complex crystal structures revealed a putative secondary hydrophobic binding pocket which could be exploited for a new design strategy by introducing a functional group that would fit into this potential site. To test this hypothesis and gain further insights into the structural basis of inhibitor binding, we have synthesized and characterized 1-(1H-imidazol-1-yl)-4,4-diphenyl-2-butanone (QC-308). Using a carbon monoxide (CO) formation assay on rat spleen microsomes, the compound was found to be ∼15 times more potent (IC50 = 0.27±0.07 µM) than its monophenyl analogue, which is already a potent compound in its own right (QC-65; IC50 = 4.0±1.8 µM). The crystal structure of hHO-1 with QC-308 revealed that the second phenyl group in the western region of the compound is indeed accommodated by a definitive secondary proximal hydrophobic pocket. Thus, the two phenyl moieties are each stabilized by distinct hydrophobic pockets. This “double-clamp” binding offers additional inhibitor stabilization and provides a new route for improvement of human heme oxygenase inhibitors

Sensory Experience Differentially Modulates the mRNA Expression of the Polysialyltransferases ST8SiaII and ST8SiaIV in Postnatal Mouse Visual Cortex

Polysialic acid (PSA) is a unique carbohydrate composed of a linear homopolymer of α-2,8 linked sialic acid, and is mainly attached to the fifth immunoglobulin-like domain of the neural cell adhesion molecule (NCAM) in vertebrate neural system. In the brain, PSA is exclusively synthesized by the two polysialyltransferases ST8SiaII (also known as STX) and ST8SiaIV (also known as PST). By modulating adhesive property of NCAM, PSA plays a critical role in several neural development processes such as cell migration, neurite outgrowth, axon pathfinding, synaptogenesis and activity-dependent plasticity. The expression of PSA is temporally and spatially regulated during neural development and a tight regulation of PSA expression is essential to its biological function. In mouse visual cortex, PSA is downregulated following eye opening and its decrease allows the maturation of GABAergic synapses and the opening of the critical period for ocular dominance plasticity. Relatively little is known about how PSA levels are regulated by sensory experience and neuronal activity. Here, we demonstrate that while both ST8SiaII and ST8SiaIV mRNA levels decrease around the time of eye opening in mouse visual cortex, only ST8SiaII mRNA level reduction is regulated by sensory experience. Using an organotypic culture system from mouse visual cortex, we further show that ST8SiaII gene expression is regulated by spiking activity and NMDA-mediated excitation. Further, we show that both ST8SiaII and ST8SiaIV mRNA levels are positively regulated by PKC-mediated signaling. Therefore, sensory experience-dependent ST8SiaII gene expression regulates PSA levels in postnatal visual cortex, thus acting as molecular link between visual activity and PSA expression

Novel, Real-Time Cell Analysis for Measuring Viral Cytopathogenesis and the Efficacy of Neutralizing Antibodies to the 2009 Influenza A (H1N1) Virus

A novel electronic cell sensor array technology, the real-time cell analysis (RTCA) system, was developed to monitor cell events. Unlike the conventional methods labeling the target cells with fluorescence, luminescence, or light absorption, the RTCA system allows for label-free detection of cell processes directly without the incorporation of labels. Here, we used this new format to measure the cytopathic effect (CPE) of the 2009 influenza A (H1N1) virus and the efficacy of neutralizing antibodies in human sera to this virus. The real-time dynamic monitoring of CPE was performed on MDCK cell cultures infected with the H1N1 virus, ranging from 5.50×102 to 5.50×107 copies/mL. The resulting CPE kinetic curves were automatically recorded and were both time and viral load dependent. The CPE kinetics were also distinguishable between different H1N1 stains, as the onset of CPE induced by the A/Shanghai/37T/2009 H1N1 virus was earlier than that of the A/Shanghai/143T/2009 H1N1 virus. Furthermore, inhibition of H1N1 virus-induced CPE in the presence of human specific anti-sera was detected and quantified using the RTCA system. Antibody titers determined using this new neutralization test correlated well with those obtained independently via the standard hemagglutination inhibition test. Taken together, this new CPE assay format provided label-free and high-throughput measurement of viral growth and the effect of neutralizing antibodies, illustrating its potential in influenza vaccine studies

Heterozygosity increases microsatellite mutation rate, linking it to demographic history

<p>Abstract</p> <p>Background</p> <p>Biochemical experiments in yeast suggest a possible mechanism that would cause heterozygous sites to mutate faster than equivalent homozygous sites. If such a process operates, it could undermine a key assumption at the core of population genetic theory, namely that mutation rate and population size are indpendent, because population expansion would increase heterozygosity that in turn would increase mutation rate. Here we test this hypothesis using both direct counting of microsatellite mutations in human pedigrees and an analysis of the relationship between microsatellite length and patterns of demographically-induced variation in heterozygosity.</p> <p>Results</p> <p>We find that microsatellite alleles of any given length are more likely to mutate when their homologue is unusually different in length. Furthermore, microsatellite lengths in human populations do not vary randomly, but instead exhibit highly predictable trends with both distance from Africa, a surrogate measure of genome-wide heterozygosity, and modern population size. This predictability remains even after statistically controlling for non-independence due to shared ancestry among populations.</p> <p>Conclusion</p> <p>Our results reveal patterns that are unexpected under classical population genetic theory, where no mechanism exists capable of linking allele length to extrinsic variables such as geography or population size. However, the predictability of microsatellite length is consistent with heterozygote instability and suggest that this has an important impact on microsatellite evolution. Whether similar processes impact on single nucleotide polymorphisms remains unclear.</p

Endocannabinoids Generated by Ca2+ or by Metabotropic Glutamate Receptors Appear to Arise from Different Pools of Diacylglycerol Lipase

The identity and subcellular sources of endocannabinoids (eCBs) will shape their ability to affect synaptic transmission and, ultimately, behavior. Recent discoveries support the conclusion that 2-arachidonoyl glycerol, 2-AG, is the major signaling eCB, however, some important issues remain open. 2-AG can be synthesized by a mechanism that is strictly Ca2+-dependent, and another that is initiated by G-protein coupled receptors (GPCRs) and facilitated by Ca2+. An important question is whether or not the 2-AG in these cases is synthesized by the same pool of diacylglycerol lipase alpha (DAGLα). Using whole-cell voltage-clamp techniques in CA1 pyramidal cells in acute in vitro rat hippocampal slices, we investigated two mechanistically distinct eCB-mediated responses to address this issue. We now report that pharmacological inhibitors of DGLα have quantitatively different effects on eCB-mediated responses triggered by different stimuli, suggesting that functional, and perhaps physical, distinctions among pools of DAGLα exist

Observation of associated near-side and away-side long-range correlations in √sNN=5.02 TeV proton-lead collisions with the ATLAS detector

Two-particle correlations in relative azimuthal angle (Δϕ) and pseudorapidity (Δη) are measured in √sNN=5.02  TeV p+Pb collisions using the ATLAS detector at the LHC. The measurements are performed using approximately 1  μb-1 of data as a function of transverse momentum (pT) and the transverse energy (ΣETPb) summed over 3.1<η<4.9 in the direction of the Pb beam. The correlation function, constructed from charged particles, exhibits a long-range (2<|Δη|<5) “near-side” (Δϕ∼0) correlation that grows rapidly with increasing ΣETPb. A long-range “away-side” (Δϕ∼π) correlation, obtained by subtracting the expected contributions from recoiling dijets and other sources estimated using events with small ΣETPb, is found to match the near-side correlation in magnitude, shape (in Δη and Δϕ) and ΣETPb dependence. The resultant Δϕ correlation is approximately symmetric about π/2, and is consistent with a dominant cos⁡2Δϕ modulation for all ΣETPb ranges and particle pT