Immunologic studies on nucleic acids and their components II. Reversible inhibition of anti-nucleoside antibodies in aqueous pyridine and its application in antibody purification

The effect of aqueous pyridine on a hapten—antihapten system was investigated by the quantitative precipitin reaction and by the membrane filtration method. It was found that dilute solutions of pyridine inhibited the reaction between isopentenyladenosine and its antiserum. Other solvents examined were less effective. The effect of pyridine was reversible at concentrations where complete inhibition occurred, thus indicating its use for the dissociation of antigen—antibody complexes. The inhibitory effect of pyridine was exploited in a single-step purification method for anti—isopentenyladenosine and antideoxy-adenylate antibodies. In addition, generally applicable methods for linking nucleosides and nucleotides to aminoethyl-Sepharose are described.

Inhibition of the biosynthesis of isoprenoid compounds by phenolic acids in the rat brain

The incorporation of [2-14C]mevalonate into nonsaponifiable lipids by rat brain homogenates is inhibited by phenolic acids derived from tyrosine. The phenyl acids derived from phenylalanine are inhibitory only at very high concentrations compared with phenolic acids. The brain is more sensitive to inhibition by the phenolic acids than the liver. These studies indicate a possible role for phenolic acids in the impairment of cerebral sterol metabolism in phenylketonuria.

Tax compliance and psychic costs: Behavioral experimental evidence using a physiological marker

Although paying taxes is a key element of a well-functioning society, there is still limited understanding as to why people actually pay their taxes. Models emphasizing that taxpayers make strategic, financially motivated compliance decisions seemingly assume an overly restrictive view of human nature. Law abidance may be more accurately explained by social norms, a concept that has gained growing importance as research attempts to understand the tax compliance puzzle. This study analyzes the influence of psychic stress generated by the possibility of breaking social norms in the tax compliance context. We measure psychic stress using heart rate variability (HRV), which captures the psychobiological or neural equivalents of psychic stress that may arise from the contemplation of real or imagined actions, producing immediate physiologic discomfort. The results of our laboratory experiments provide empirical evidence of a positive correlation between psychic stress and tax compliance, thus underscoring the importance of moral sentiments for tax compliance. We also identify three distinct types of individuals who differ in their levels of psychic stress, tax morale, and tax compliance.

Inhibition of Human Two-Pore Domain K+ Channel TREK1 by Local Anesthetic Lidocaine: Negative Cooperativity and Half-of-Sites Saturation Kinetics

TWIK-related K+ channel TREK1, a background leak K+ channel, has been strongly implicated as the target of several general and local anesthetics. Here, using the whole-cell and single-channel patch-clamp technique, we investigated the effect of lidocaine, a local anesthetic, on the human (h) TREK1 channel heterologously expressed in human embryonic kidney 293 cells by an adenoviral-mediated expression system. Lidocaine, at clinical concentrations, produced reversible, concentration-dependent inhibition of hTREK1 current, with IC50 value of 180 mu M, by reducing the single-channel open probability and stabilizing the closed state. We have identified a strategically placed unique aromatic couplet (Tyr352 and Phe355) in the vicinity of the protein kinase A phosphorylation site, Ser348, in the C-terminal domain (CTD) of hTREK1, that is critical for the action of lidocaine. Furthermore, the phosphorylation state of Ser348 was found to have a regulatory role in lidocaine-mediated inhibition of hTREK1. It is interesting that we observed strong intersubunit negative cooperativity (Hill coefficient = 0.49) and half-of-sites saturation binding stoichiometry (half-reaction order) for the binding of lidocaine to hTREK1. Studies with the heterodimer of wild-type (wt)-hTREK1 and Delta 119 C-terminal deletion mutant (hTREK1(wt)-Delta 119) revealed that single CTD of hTREK1 was capable of mediating partial inhibition by lidocaine, but complete inhibition necessitates the cooperative interaction between both the CTDs upon binding of lidocaine. Based on our observations, we propose a model that explains the unique kinetics and provides a plausible paradigm for the inhibitory action of lidocaine on hTREK1.

Molecular mechanism for the specific inhibition of reverse transcriptase of rous sarcoma virus by the copper complexes of isonicotinic acid hydrazide

Isonicotinic acid hydrazide (isoniazid), one of the most potent antitubercular drugs, was recently shown, in our laboratory, to form two different complexes with copper, depending upon the oxidation state of the metal ion. Both the complexes have been shown to possess antiviral activity against Rous sarcoma virus, an RNA tumor virus. The antiviral activity of the complexes has been attributed to their ability to inhibit the endogenous reverse transcriptase activity of RSV. More recent studies in our laboratory indicate that both these complexes inhibit both endogenous and exogenous reactions. As low a final concentration as 50 μM of the cupric and the cuprous complexes inhibits the endogenous reaction to the extent of 93 and 75 per cent respectively. Inhibition of the exogenous reaction varies with the templates. The inhibition can be reversed by either β-mercaptoethanol or ethylene-diamine-tetra-acetic acid. The specificity of this inhibition has been ascertained by using a synthetic primer-template, −(dG)not, vert, similar15−(rCm)n, which is highly specific for reverse transcriptases. The inhibition is found to be template specific. The studies carried out, using various synthetic primer-templates, show the inhibition of both the steps of reverse transcription by the copper complexes of isoniazid.

Heparin-binding growth-associated molecule (HB-GAM) in activity-dependent neuronal plasticity in hippocampus

Cell adhesion and extracellular matrix (ECM) molecules play a significant role in neuronal plasticity both during development and in the adult. Plastic changes in which ECM components are implicated may underlie important nervous system functions, such as memory formation and learning. Heparin-binding growthassociated molecule (HB-GAM, also known as pleiotrophin), is an ECM protein involved in neurite outgrowth, axonal guidance and synaptogenesis during perinatal period. In the adult brain HB-GAM expression is restricted to the regions which display pronounced synaptic plasticity (e.g., hippocampal CA3-CA1 areas, cerebral cortex laminae II-IV, olfactory bulb). Expression of HB-GAM is regulated in an activity-dependent manner and is also induced in response to neuronal injury. In this work mutant mice were used to study the in vivo function of HB-GAM and its receptor syndecan-3 in hippocampal synaptic plasticity and in hippocampus-dependent behavioral tasks. Phenotypic analysis of HBGAM null mutants and mice overexpressing HB-GAM revealed that opposite genetic manipulations result in reverse changes in synaptic plasticity as well as behavior in the mutants. Electrophysiological recordings showed that mice lacking HB-GAM have an increased level of long-term potentiation (LTP) in the area CA1 of hippocampus and impaired spatial learning, whereas animals with enhanced level of HB-GAM expression have attenuated LTP, but outperformed their wild-type controls in spatial learning. It was also found that GABA(A) receptor-mediated synaptic transmission is altered in the transgenic mice overexpressing HB-GAM. The results suggest that these animals have accentuated hippocampal GABAergic inhibition, which may contribute to the altered glutamatergic synaptic plasticity. Structural studies of HB-GAM demonstrated that this protein belongs to the thrombospondin type I repeat (TSR) superfamily and contains two β-sheet domains connected by a flexible linker. It was found that didomain structure is necessary for biological activity of HB-GAM and electrophysiological phenotype displayed by the HB-GAM mutants. The individual domains displayed weaker binding to heparan sulfate and failed to promote neurite outgrowth as well as affect hippocampal LTP. Effects of HB-GAM on hippocampal synaptic plasticity are believed to be mediated by one of its (co-)receptor molecules, namely syndecan-3. In support of that, HB-GAM did not attenuate LTP in mice deficient in syndecan-3 as it did in wild-type controls. In addition, syndecan-3 knockout mice displayed electrophysiological and behavioral phenotype similar to that of HB-GAM knockouts (i.e. enhanced LTP and impaired learning in Morris water-maze). Thus HB-GAM and syndecan-3 are important modulators of synaptic plasticity in hippocampus and play a role in regulation of learning-related behavior.

On the expressive power of behavioral profiles

Behavioral profiles have been proposed as a behavioral abstraction of dynamic systems, specifically in the context of business process modeling. A behavioral profile can be seen as a complete graph over a set of task labels, where each edge is annotated with one relation from a given set of binary behavioral relations. Since their introduction, behavioral profiles were argued to provide a convenient way for comparing pairs of process models with respect to their behavior or computing behavioral similarity between process models. Still, as of today, there is little understanding of the expressive power of behavioral profiles. Via counter-examples, several authors have shown that behavioral profiles over various sets of behavioral relations cannot distinguish certain systems up to trace equivalence, even for restricted classes of systems represented as safe workflow nets. This paper studies the expressive power of behavioral profiles from two angles. Firstly, the paper investigates the expressive power of behavioral profiles and systems captured as acyclic workflow nets. It is shown that for unlabeled acyclic workflow net systems, behavioral profiles over a simple set of behavioral relations are expressive up to configuration equivalence. When systems are labeled, this result does not hold for any of several previously proposed sets of behavioral relations. Secondly, the paper compares the expressive power of behavioral profiles and regular languages. It is shown that for any set of behavioral relations, behavioral profiles are strictly less expressive than regular languages, entailing that behavioral profiles cannot be used to decide trace equivalence of finite automata and thus Petri nets.

GABAa Receptor Mediated Signalling in the Brain: Inhibition, Shunting and Excitation

Within central nervous system, the simple division of chemical synaptic transmission to depolarizing excitation mediated by glutamate and hyperpolarizing inhibition mediated by γ-amino butyric acid (GABA), is evidently an oversimplification. The GABAa receptor (GABAaR) mediated responses can be of opposite sign within a single resting cell, due to the compartmentalized distribution of cation chloride cotransporters (CCCs). The K+/Cl- cotransporter 2 (KCC2), member of the CCC family, promotes K+ fuelled Cl- extrusion and sets the reversal potential of GABA evoked anion currents typically slightly below the resting membrane potential. The interesting ionic plasticity property of GABAergic signalling emerges from the short-term and long-term alterations in the intraneuronal concentrations of GABAaR permeable anions (Cl- and HCO3-). The short-term effects arise rapidly (in the time scale of hundreds of milliseconds) and are due to the GABAaR activation dependent shifts in anion gradients, whereas the changes in expression, distribution and kinetic regulation of CCCs are underlying the long-term effects, which may take minutes or even hours to develop. In this Thesis, the differences in the reversal potential of GABAaR mediated responses between dopaminergic and GABAergic cell types, located in the substantia nigra, were shown to be attributable to the differences in the chloride extrusion mechanisms. The stronger inhibitory effect of GABA on GABAergic neurons was due to the cell type specific expression of KCC2 whereas the KCC2 was absent from dopaminergic neurons, leading to a less prominent inhibition brought by GABAaR activation. The levels of KCC2 protein exhibited activity dependent alterations in hippocampal pyramidal neurons. Intense neuronal activity, leading to a massive release of brain derived neurotrophic factor (BDNF) in vivo, or applications of tyrosine receptor kinase B (TrkB) agonists BDNF or neurotrophin-4 in vitro, were shown to down-regulate KCC2 protein levels which led to a reduction in the efficacy of Cl- extrusion. The GABAergic transmission is interestingly involved in an increase of extracellular K+ concentration. A substantial increase in interstitial K+ tends to depolarize the cell membrane. The effects that varying ion gradients had on the generation of biphasic GABAaR mediated responses were addressed, with particular emphasis on the novel idea that the K+/Cl- extrusion via KCC2 is accelerated in response to a rapid accumulation of intracellular Cl-. The KCC2 inhibitor furosemide produced a large reduction in the GABAaR dependent extracellular K+ transients. Thus, paradoxically, both the inefficient KCC2 activity (via increased intracellular Cl-) and efficient KCC2 activity (via increased extracellular K+) may promote excitation.

Feedback-type inhibition of activity of 3-hydroxy-3-methylglutaryl coenzyme a reductase by ubiquinone

Accompanying the decrease in serum cholesterol and increase in concentration of ubiquinone in liver and its microsomes, the activity, but not the protein, of HMG-CoA reductase decreased in ubiquinone-supplemented rats. A soluble 58-kDa preparation of HMG-CoA reductase was partially inhibited on addition of ubiquinone indicating a possible feedback type of action.

Inhibition of replication of rinderpest virus by 5-fluorouracil

5-Fluorouracil (5FU), an analogue of uracil, was found to inhibit the production of infectious particles of rinderpest virus (RPV) in Vero cells (African green monkey kidney cells) by 99%, at a concentration of 1 μg/ml. The levels of individual mRNA specific for five of the virus genes were also reduced drastically, while the level of mRNA for a cellular housekeeping gene—glyceraldehyde-3-phosphate dehydrogenase (GAPDH)—was unaltered by fluorouracil treatment of infected cells. Both virus RNA and protein synthesis showed inhibition in a dose-dependent manner. The virions which budded out of 5-fluorouracil-treated cells also contained reduced amounts of virus proteins compared with virus particles from untreated cells.

Generation and Characterization of the Cation-Chloride Cotransporter KCC2 Hypomorphic Mouse

The cation-Cl- cotransporter (CCC) family comprises of Na+-Cl- cotransporter (NCC), Na+-K+-2Cl- cotransporters (NKCC1-2), and four K+-Cl- cotransporters (KCC1-4). These proteins are involved in several physiological activities, such as cell volume regulation. In neuronal tissues, NKCC1 and KCC2 are important in determining the intracellular Cl- levels and hence the neuronal responses to inhibitory neurotransmitters GABA and glycine. One aim of the work was to elucidate the roles for CCC isoforms in the control of nervous system development. KCC2 mRNA was shown to be developmentally up-regulated and follow neuronal maturation, whereas NKCC1 and KCC4 transcripts were highly expressed in the proliferative zones of subcortical regions. KCC1 and KCC3 mRNA displayed low expression throughout the embryogenesis. These expression profiles suggest a role for CCC isoforms in maturation of synaptic responses and in the regulation of neuronal proliferation during embryogenesis. The major aim of this work was to study the biological consequences of KCC2-deficiency in the adult CNS, by generating transgenic mice retaining 15-20% of normal KCC2 levels. In addition, by using these mice as a tool for in vivo pharmacological analysis, we investigated the requirements for KCC2 in tonic versus phasic GABAA receptor-mediated inhibition. KCC2-deficient mice displayed normal reproduction and life span, but showed several behavioral abnormalities, including increased anxiety-like behavior, impaired performance in water maze, alterations in nociceptive processing, and increased seizure susceptibility. In contrast, the mice displayed apparently normal spontaneous locomotor activity and motor coordination. Pharmacological analysis of KCC2-deficient mice revealed reduced sensititivity to diazepam, but normal gaboxadol-induced sedation, neurosteroid hypnosis and alcohol-induced motor impairment. Electrophysiological recordings from CA1-CA3 subregions of the hippocampus showed that KCC2 deficiency affected the reversal potentials of both the phasic and tonic GABA currents, and that the tonic conductance was not affected. The results suggest that requirement for KCC2 in GABAergic neurotransmission may differ among several functional systems in the CNS, which is possibly due to the more critical role of KCC2 activity in phasic compared to tonic GABAergic inhibition.

Characterization of inhibition of platelet function by paracetamol and its interaction with diclofenac and parecoxib

Aim: To characterize the inhibition of platelet function by paracetamol in vivo and in vitro, and to evaluate the possible interaction of paracetamol and diclofenac or valdecoxib in vivo. To assess the analgesic effect of the drugs in an experimental pain model. Methods: Healthy volunteers received increasing doses of intravenous paracetamol (15, 22.5 and 30 mg/kg), or the combination of paracetamol 1 g and diclofenac 1.1 mg/kg or valdecoxib 40 mg (as the pro-drug parecoxib). Inhibition of platelet function was assessed with photometric aggregometry, the platelet function analyzer (PFA-100), and release of thromboxane B2. Analgesia was assessed with the cold pressor test. The inhibition coefficient of platelet aggregation by paracetamol was determined as well as the nature of interaction between paracetamol and diclofenac by an isobolographic analysis in vitro. Results: Paracetamol inhibited platelet aggregation and TxB2-release dose-dependently in volunteers and concentration-dependently in vitro. The inhibition coefficient was 15.2 mg/L (95% CI 11.8 - 18.6). Paracetamol augmented the platelet inhibition by diclofenac in vivo, and the isobole showed that this interaction is synergistic. Paracetamol showed no interaction with valdecoxib. PFA-100 appeared insensitive in detecting platelet dysfunction by paracetamol, and the cold-pressor test showed no analgesia. Conclusions: Paracetamol inhibits platelet function in vivo and shows synergism when combined with diclofenac. This effect may increase the risk of bleeding in surgical patients with an impaired haemostatic system. The combination of paracetamol and valdecoxib may be useful in patients with low risk for thromboembolism. The PFA-100 seems unsuitable for detection of platelet dysfunction and the cold-pressor test seems unsuitable for detection of analgesia by paracetamol.