Effect of short- and long-term administration of baclofen on spatial learning and memory in rats.

Baclofen is the only clinically available metabotropic GABA(B) receptor agonist. In our experiment, we tested the hypothesis that long-term baclofen administration can impair learning and memory in rats. The experiment consisted of three parts. In the first part of the study the drug was administered simultaneously with the beginning of the behavioral tests. In the second and third part of the experiment baclofen was administered daily for 14 days and for one month before the tests. In each part of the experiment, adult rats were randomly divided into four treatment groups. Three groups were given an injection of baclofen at doses of 1 mg/kg, 5 mg/kg, 10 mg/kg, while the fourth group was injected with saline. The injections were given after each session. Spatial learning and memory were tested using the Morris water maze, involving three types of tests: Acquisition, Probe, and Re-acquisition. This work reveals that baclofen did not affect spatial learning at any of the tested doses and regardless of the length of administration. Memory was observed to be affected, but only at the highest dose of baclofen and only temporarily. This conclusion is in line with previously published clinical cases.

Baclofen reversed thermal place preference in rats with chronic constriction injury.

Chronic constriction injury to the sciatic nerve was used as an animal model of neuropathic pain. Instead of frequently used reflex-based tests we used an operant thermal place preference test to evaluate signs of neuropathic pain and the effect of baclofen administration in rats with neuropathy. Chronic constriction injury was induced by four loose ligations of the sciatic nerve. Thermal place preference (45 °C vs. 22 °C and 45 °C vs. 11 °C) was measured after the ligation and after the administration of baclofen in sham and experimental rats. Rats with the chronic constriction injury spent significantly less time on the colder plate compared to sham operated animals at the combination 45 °C vs. 11 °C. After administration of baclofen (10 mg/kg s.c.), the aversion to the colder plate in rats with chronic constriction injury disappeared. At the combination 45 °C vs. 22 °C, no difference in time spent on colder and/or warmer plate was found between sham and experimental animals. These findings show the importance of cold allodynia evaluation in rats with chronic constriction injury and the effectiveness of baclofen in this neuropathic pain model.

CARM1 modulators affect epigenome of stem cells and change morphology of nucleoli.

CARM1 interacts with numerous transcription factors to mediate cellular processes, especially gene expression. This is important for the maintenance of ESC pluripotency or intervention to tumorigenesis. Here, we studied epigenomic effects of two potential CARM1 modulators: an activator (EML159) and an inhibitor (ellagic acid dihydrate, EA). We examined nuclear morphology in human and mouse embryonic stem cells (hESCs, mESCs), as well as in iPS cells. The CARM1 modulators did not function similarly in all cell types. EA decreased the levels of the pluripotency markers, OCT4 and NANOG, particularly in iPSCs, whereas the levels of these proteins increased after EML159 treatment. EML159 treatment of mouse ESCs led to decreased levels of OCT4 and NANOG, which was accompanied by an increased level of Endo-A. The same trend was observed for NANOG and Endo-A in hESCs affected by EML159. Interestingly, EA mainly changed epigenetic features of nucleoli because a high level of arginine asymmetric di-methylation in the nucleoli of hESCs was reduced after EA treatment. ChIP-PCR of ribosomal genes confirmed significantly reduced levels of H3R17me2a, in both the promoter region of ribosomal genes and rDNA encoding 28S rRNA, after EA addition. Moreover, EA treatment changed the nuclear pattern of AgNORs (silver-stained nucleolus organizer regions) in all cell types studied. In EA-treated ESCs, AgNOR pattern was similar to the pattern of AgNORs after inhibition of RNA pol I by actinomycin D. Together, inhibitory effect of EA on arginine methylation and effect on related morphological parameters was especially observed in compartment of nucleoli.

Development of antibodies for determination of alkylresorcinol metabolites in human urine and elucidation of ELISA cross-reactivity.

Alkylresorcinols (ARs) are amphiphilic phenolic lipids and their two main metabolites, 3-(3,5-dihydroxyphenyl)-propanoic acid (DHPPA) and 3,5-dihydroxybenzoic acid (DHBA), can be used as biomarkers of whole grain wheat and rye intake. The aim of this study was to develop antibodies against DHBA and DHPPA for use in ELISA analysis. Good calibration curves were obtained for ELISA using alkaline phosphatase (AP) conjugates. The highest sensitivity for DHPPA was found using a reagent combination of anti-DHPPA-BSA and DHPAA-AP in a direct ELISA (IC50=1.5µmol/L), and for DHBA using a reagent combination of anti-DHBA-OV and DHBA-AP (IC50=1.3µmol/L). Calibration was conducted in the linear range (0.3-27.4µmol/L), with limit of detection (LOD) 0.1µmol/L. Intra and inter CVs was in the range of 0.7-7.2% and 5.1-11.5%, respectively, for DHPPA and 1.3-9.4% and 3.5-20%, respectively, for DHBA. Mean recovery was 104% for DHPPA and 102% for DHBA. The ELISA method developed was then used for analysis of 120 urine samples from free-living men and women that had previously been analysed by gas chromatography-mass spectrometry (GC-MS). ELISA produced several-fold higher values than GC-MS. Application of high-resolution Orbitrap mass spectrometry (HR Orbitrap MS) allowed several compounds, including novel putative AR metabolites, to be identified, synthesised and confirmed as compounds with high ELISA cross-reactivity.

Pain perception in neurodevelopmental animal models of schizophrenia.

Animal models are important for the investigation of mechanisms and therapeutic approaches in various human diseases, including schizophrenia. Recently, two neurodevelopmental rat models of this psychosis were developed based upon the use of subunit selective N-methyl-D-aspartate receptor agonists--quinolinic acid (QUIN) and N-acetyl-aspartyl-glutamate (NAAG). The aim of this study was to evaluate pain perception in these models. QUIN or NAAG was infused into lateral cerebral ventricles neonatally. In the adulthood, the pain perception was examined. The rats with neonatal brain lesions did not show any significant differences in acute mechanical nociception and in formalin test compared to controls. However, the neonatally lesioned rats exhibited significantly higher pain thresholds in thermal nociception. Increased levels of mechanical hyperalgesia, accompanying the sciatic nerve constriction (neuropathic pain), were also observed in lesioned rats. Although hyperalgesia was more pronounced in QUIN-treated animals, the number of c-Fos-immunoreactive neurons of the lumbar spinal cord was similar in experimental and control rats. We conclude that neonatal brain lesions attenuated the thermal perception in both nociceptive and neuropathic pain whereas mechanical pain was increased in the model of neuropathic pain only. Thus, nociceptive and neuropathic pain belongs--in addition to behavioral changes--among the parameters which are affected in described animal models of schizophrenia.

In-house validation of liquid chromatography tandem mass spectrometry for determination of semicarbazide in eggs and stability of analyte in matrix.

An improved LC-MS/MS method for the determination of semicarbazide in whole egg is described. Waters OASIS-MCX cation exchange purification cartridges increased the sensitivity for analysis by LC-MS/MS. The validation study was carried out according to criteria and requirements of Commission Decision 2002/657/EC for confirmatory analysis and provided the data as follows: The correlation coefficient for the matrix calibration curve, in the range of 0-5 microg kg(-1), was r=0.9968. The detection capability and decision limit, measured according to ISO11843-2, were CCalpha=0.20 microg kg(-1) and CCbeta=0.25 microg kg(-1). Repeatability (CVSr) and within-laboratory reproducibility (CVSwr) determined for the concentration levels of 0.2, 0.5 and 1.0 microg kg(-1) SEM ranged from 11.9 to 5.7% and 11.8 to 6.3%, respectively. The validated method was applied to investigate SEM stability in incurred materials (egg homogenates) during long-term storage at -20 degrees C and 4 degrees C. The study proved by a two-sampling test that SEM at levels of 17. 7, 1.2, 10.6 and 0.47 microg kg(-1) was stable for up to 12 months.

Prenatal and perinatal factors influencing nociception, addiction and behavior during ontogenetic development.

This review, which summarizes our findings concerning the long-term effects of pre-, peri- and postnatal factors affecting development, nociception and sensorimotor functions, focuses on three areas: 1) perinatal factors influencing nociception in adult rats were examined in rats with hippocampal lesions, after the administration of stress influencing and psychostimulant drugs (dexamethasone, indomethacine and methamphetamine); 2) the effect of pre- and early postnatal methamphetamine administration was shown to impair the development of sensorimotor functions tested in rat pups throughout the preweaning period; 3) the effect of extensive dorsal rhizotomy of the brachial plexus during the early postnatal period was studied with respect to neuropathic pain development and sensorimotor functions. The present study indicates that prenatal or neonatal stress, as well as various drugs, may disturb the development of the nociceptive system and cause long-term behavioral changes persisting to adulthood and that some types of neuropathic pain cannot be induced during the first two postnatal weeks at all. A mature nervous system is required for the development of the described pathological behaviors.

ELISA for semicarbazide and its application for screening in food contamination.

The development of a direct competitive enzyme-linked immunosorbent assay (ELISA) based on polyclonal antibodies specific for semicarbazide (SEM) is described. Molecular modelling of the hapten mimics and other key components of the assay system was conducted to explain antibody properties in relation to hapten design. The small aliphatic molecule SEM was coupled to 3-carboxybenzaldehyde to produce carboxyphenyl-SEM (CPSEM), for the generation of specific antibodies. Five rabbits produced antibodies against NPSEM (used in direct and indirect ELISA formats) exhibiting a 50% binding inhibition level (IC(50) values) of 0.06-2.28 microgL(-1) in assay buffer for SEM. The most sensitive indirect assay based on the antibody MVK39 showed a high dynamic range providing a linear readout in the range of 0.01-0.2 microgL(-1). Antibody MVK31 (IgG) allowed specific SEM detection at an IC(50)=0.14 microgL(-1) in direct ELISA and was evaluated using solvent extracted SEM-spiked porcine and baby food samples. Recovery levels determined from fortified samples (0.5, 1.0, 1.5, 5, 10 and 20 microgkg(-1)) of porcine and baby food ranged from 82.9 to 105.3%, respectively, with a coefficient of variation less than 15.5%. Respective detection capability and threshold of the assay for porcine muscle, set on the basis of acceptance of no false negative results, was 0.3 and 0.11 microgkg(-1).

Validation of an enzyme-linked immunosorbent assay (ELISA) for the determination of 4-nonylphenol and octylphenol in surface water samples by LC-ESI-MS.

4-Nonylphenol (NP) and octylphenol (OP) were measured by direct ELISA in both laboratory-fortified and surface water samples collected monthly from 10 rivers. In this procedure, samples were concentrated by solid phase extraction (SPE) using Lichrolut RP-18 sorbent with good recoveries obtained for both LC-MS and ELISA, giving a low level of detection (LOD) at the range of low mugL(-1) and good reproducibility. Analysis of 40 surface water samples demonstrated that the ELISA may be a useful screening tool for the determination of the alkylphenols in surface water matrices. The concentration of NP and OP in surface waters ranged from 0.11 to 6.58mugL(-1). A good correlation of results obtained by ELISA and LC-MS within the concentration range of 0.08-6.86mugL(-1) was found in the river samples [R(2)=0.924, n=39]. The influence of various factors on assay determination was also discussed.

GABA(B) receptor agonist baclofen has non-specific antinociceptive effect in the model of peripheral neuropathy in the rat.

Baclofen, which is a specific agonist of the metabotropic GABA(B) receptor, is used in clinical practice for the treatment of spasticity of skeletal muscles. It also exerts an analgesic effect, but this effect is still not clear and especially controversial in neuropathic pain. In this work, we studied the antinociceptive effects of baclofen in a model of chronic peripheral neuropathic pain - loose ligation of the sciatic nerve (chronic constriction injury, CCI). As controls we used sham-operated animals. The changes of thermal pain threshold were measured using the plantar test 15-25 days after the operation. The obtained results suggest that baclofen increases pain threshold in both groups. The antinociceptive effect of baclofen was dose-dependent and the maximum response without motor deficits was observed at a dose of 15 mg/kg s.c. In the rats with CCI, significant differences between affected (ipsilateral) and contralateral hind paw were present. This difference was dose-dependent, the highest value (6.2+/-1.37 s) was found at the dose of 20 mg/kg. Based on our results and previous findings it could be summarized that baclofen has antinociceptive action, which is attenuated in the model of chronic neuropathic pain probably due to the degeneration of GABA interneurons after chronic constriction injury.

[History and the present of metabotropic GABAB receptor]. / Metabotropní GABAB receptor: historie a soucasnost.

GABAB receptor is the main inhibitory metabotropic receptor in mammalian central nervous system. This receptor is a member of the Family 3 G-protein coupled receptors (GPCR). It is related to the metabotropic glutamate receptors, the calcium-sensing receptor and some vomeronasal receptors. The receptor is a heterodimer consisting of two subunits designated BR1 and BR2. Presynaptically. GABAB suppresses transmitter release via inhibition of Ca2+ channels, postsynaptically it increases K+ conductance resulting in hyperpolarization. The receptor is coupled to Gi/Go proteins and its activation can inhibit adenylyl cyclase activity. GABAs is widely distributed in CNS and peripheral tissues and plays an important role in many physiological and pathological processes. Recently, a novel GPCR with close relation to GABAB was cloned. It was termed GABABL, its role in GABAB activation has not been discovered yet.

A highly sensitive flow-through amperometric immunosensor based on the Peroxidase chip and enzyme-channeling principle.

A concept based on the Peroxidase-chip (P-chip), antibody co-immobilization, competitive and enzyme-channeling principle was exploited to develop an integrated flow-through amperometric biosensor for detection of environmental pollutants such as s-triazine herbicides. In this concept, recombinant peroxidase is immobilized on the gold electrode (P-chip) in such a way that direct electron transfer is achieved. The recognition and quantitation the target analyte is realized through the competition between the simazine-glucose oxidase (GOD) conjugate and free simazine for the binding sites of the monoclonal antibody co-immobilized with peroxidase on the gold electrode. The arrangement allows to generate a specific signal in the presence of glucose through the channeling of H2O2 produced by GOD conjugate bound to the antibody. The immunosensor exhibited 50% signal decrease (IC50 value) at approximately 0.02 microg l(-1). A concentration of 0.1 ng l(-1) gave a signal clearly distinguishable from the blank whereas the ELISA using the same antibody had a typical detection limit of about 1 microg l(-1), which is four orders of magnitude higher compared to the presented biosensor system. The results demonstrated that gene engineering biomolecules, in this case recombinant peroxidase, might be attractive reagents for the development of electrochemical immunosensors.

Antibody-based methods for surfactant screening.

This brief overview summarises the immunoassay-based results obtained in the course of two years of the European INCO-Copernicus project BIOTOOLS. The project is aimed at simplifying the procedures for detection of surface active compounds (SAC) using, among others, antibody-based methods, i.e., microtiter plate-based enzyme-linked immunosorbent assays (ELISA), polarisation fluoro immunoassays (PFIA), and enzyme flow injection immunoassays (FIIA). Thirty-three rabbits were immunised with five different sulphophenyl moieties and three p-hydroxyphenyl moieties conjugated to protein immunogens to produce analytical antibodies against linear alkylbenzene sulphonates (LAS) and nonylphenol (NP). Although most of the antibodies exhibited binding reaction in indirect ELISA, only a few showed the required assay sensitivity. The best antibodies for LAS exhibited a 50% binding inhibition at IC50 19.8 microg L(-1) in indirect ELISA. Similar inhibition was observed for direct ELISA using peroxidase tracers. Antibodies against NP allowed the establishment of an indirect assay operating in the mg L(-1) range. A rapid and simple protocol for the screening of NP and LAS using homogeneous PFIA is described. The assay time for 10 samples was 7 minutes, thus allowing fast detection of the selected SAC at the mg L(-1) level. A generic competitive FIIA system, using a protein G column for separation of free and antibody-bound beta-galactosidase (beta-Gal) tracer, was developed for the screening of LAS, NP, and nonylphenol decaethoxylate (NPEO10). The FIIA had a sample throughput (STP) of 5-10 samples per hour, with limits of detection (LOD) for LAS, NP, and NPEO10 of 19.5, 52, and 2.4 microg L(-1), respectively. The developed FIIAs were applied to spiked rain and surface water.

Mapping the agonist-binding site of GABAB type 1 subunit sheds light on the activation process of GABAB receptors.

The gamma-amino-n-butyric acid type B (GABA(B)) receptor is composed of two subunits, GABA(B)1 and GABA(B)2, belonging to the family 3 heptahelix receptors. These proteins possess two domains, a seven transmembrane core and an extracellular domain containing the agonist binding site. This binding domain is likely to fold like bacterial periplasmic binding proteins that are constituted of two lobes that close upon ligand binding. Here, using molecular modeling and site-directed mutagenesis, we have identified residues in the GABA(B)1 subunit that are critical for agonist binding and activation of the heteromeric receptor. Our data suggest that two residues (Ser(246) and Asp(471)) located within lobe I form H bonds and a salt bridge with carboxylic and amino groups of GABA, respectively, demonstrating the pivotal role of lobe I in agonist binding. Interestingly, our data also suggest that a residue within lobe II (Tyr(366)) interacts with the agonists in a closed form model of the binding domain, and its mutation into Ala converts the agonist baclofen into an antagonist. These data demonstrate the pivotal role played by the GABA(B)1 subunit in the activation of the heteromeric GABA(B) receptor and are consistent with the idea that a closed state of the binding domain of family 3 receptors is required for their activation.

Dorsal rhizotomy changes the spontaneous neuronal activity of nuclei in the medial thalamus.

The aim of this study was to examine the influence of unilateral dorsal root section at the cervicothoracic level of the spinal cord on the spontaneous neuronal activity of medial thalamic nuclei in the rat. Single unit extracellular recordings from thalamic nuclei, nc. parafascicularis and nc. centralis lateralis, were obtained with glass micropipettes. The abnormal bursting activity of these nuclei following deafferentation was registered, although a correlation between the occurrence of this activity and the degree of autotomy behavior was not found. Such bursts were never observed in the studied thalamic nuclei of control rats.

The heteromeric GABA-B receptor recognizes G-protein alpha subunit C-termini.

The recently cloned GABA-B receptors are related to the metabotropic glutamate receptors (mGlu receptors), the Ca2+-sensing receptor and one group of vomeronasal receptors. The GABA-B receptors likely function in a heterodimeric form, constituted of GABA-BR1 and GABA-BR2. This novel feature in the G-protein coupled receptors (GPCRs) structure raises questions as to the mechanism of recognition of G-proteins by such receptors. In the present study we show that the GABA-BR1 and BR2 subunits form a functional receptor that recognizes the extreme C-termini of the G alpha i and G alpha o proteins when expressed in HEK293 cells. Indeed, heteromeric GABA-BR1/BR2 receptors do not activate PLC when co-expressed with G alpha q, but do so when co-expressed with the chimeric G alpha qi5 or G alpha qo5 subunits, the G alpha q subunit in which the 5 C-terminal residues are those of G alpha i or G alpha o, respectively. Interestingly, the heteromeric GABA-B receptor did not activate the chimeric G alpha qz5 subunit that contains the 5 C-terminal residues of G alpha z. Among the three residues that are distinct between G alpha qo5 and G alpha qz5 (at position -5, -4 and -1), the amino acid residue at position -4 of G alpha o proteins is critical for specifying the coupling selectivity with the receptor and residue -5 influences the coupling efficacy. Interestingly, these findings correspond to data obtained with the mGluR2 receptor, a distant relative of GABA-B proteins. This shows that the same molecular determinants of the G-protein alpha-subunits are involved in the specific recognition of both the heteromeric GABA-B receptors and the other GPCRs.

[The problem of selecting the structure of a labelled antigen in developing a fluorescence polarization immunoassay for atrazine]. / Problema vybora struktury mechenogo antigena pri razrabotke poliarizatsionnogo fluoroimmunoanaliza atrazina.

Influence of labelled antigens structure on the sensitivity of the polarization fluoroimmunoassay of atrazine was studied. It is shown that the highest sensitivity is provided by the use of the heterologous labelled reagent with the shortest chemical bridge between the antigen and fluorescent label.

Characterization of monoclonal antibodies to 2,4-dichlorophenoxyacetic acid using a piezoelectric quartz crystal microbalance in solution.

Piezoelectric quartz crystals (resonance frequency 10 MHz) were used for an investigation of the immunochemical reaction between 2,4-dichlorophenoxyacetic acid (2,4-D) herbicide and several monoclonal antibodies prepared against 2,4-D. The herbicide was immobilized on the gold electrodes of the crystals silanized with 3-aminopropyltriethoxysilane. The activated carboxylic group of 2,4-D was linked either directly to the silanized surface or through hexamethylenediamine or albumin as a macromolecule. The interaction of the immobilized antigen with monoclonal antibody in solution was followed as a change in the resonant frequency of the crystals. The best results were achieved using 2,4-D attached to albumin. The affinity binding of five monoclonal antibodies was characterized by association (ka) and dissociation (kd) kinetic rate constants and by equilibrium association constants (KA) which were obtained from the experimental frequency vs. time curves.

Sensorimotor synchronization: motor responses to pseudoregular auditory patterns.

Musically trained and untrained subjects (N = 30) were asked to synchronize their finger tapping with stimuli in auditory patterns. Each pattern comprised six successive tonal stimuli of the same duration, the first of which was accented by a different frequency. The duration of inter-stimulus onset intervals (ISIs) gradually increased or decreased in constant steps toward the end of the patterns. Four values of such steps were used in different trials: 20, 30, 45, and 60 msec. Various time-control mechanisms are hypothesized as being simultaneously responsible for subjects' incorrect reproduction of the internal temporal ratios of the stimulus patterns. The mechanism of assimilation (of a central tendency) led subjects to enforce a regular (isochronous) structure on the patterns. The influence of other time-control mechanisms (distinction, subjective expression of an accent, sequential transfer) was expressed mainly in differences between intertap onset intervals (ITIs) and the corresponding ISIs at the beginning of the patterns. The duration of the first two ITIs was in the majority of the trials in an inverse ratio to the ratio of the respective ISIs. The distortions resulting from the timing mechanisms concerned were more pronounced in the performance of nonmusicians than in that of musicians.

Finger tapping in musicians and nonmusicians.

Timing plays an important role in perceiving and performing music. Finger tapping has been successfully used for analyzing timing processes (Fraisse, 1966, Franek et al., 1987, 1988). The aim of this study is to determine differences between musically trained and untrained subjects in their ability to follow repetitive rhythmic tonal patterns by finger tapping. It has been found previously (Povel, 1981; Smith, 1983) that time estimation differs among musicians and nonmusicians under certain conditions. The results presented here show that motor timing revealed by tapping is more accurate in musicians than in nonmusicians.