Neurotransmitter selection by monoamine oxidase isoforms, dissected in terms of functional groups by mixed double mutant cycles.

Double mutant cycles were constructed using neurotransmitters and synthetic substrates that measure their selective binding to one monoamine oxidase (MAO) enzyme isoform over another as a function of structural change. This work measures a reduction in selectivity for the MAOB isoform of 3 to 9.5 kJ mol-1 upon the addition of hydroxy functional groups to a phenethylamine scaffold. Replacement of hydroxy functional groups on the phenethylamine scaffold by hydrophobic substituents measures an increase in selectivity for MAOB of -1.1 to -6.9 kJ mol-1. The strategies presented here can be applied to the development of competitive reversible inhibitors of MAO enzymes and other targets with structurally related isoforms.

Interethnic differences at the thermometric response to cold test: functional disorders of blood circulation in hand fingers and exposure to hand-arm vibration.

OBJECTIVES: To report some notable aspects regarding thermometric response to cold test in black African subjects compared with Caucasians: both groups comprised persons exposed to hand-arm vibration and controls. METHODS: An overall sample of 48 workers was examined in order to study their blood circulation in hand fingers: a control group of 12 healthy Caucasian workers never exposed before to hand-arm vibration; 12 Caucasian workers exposed for several years to vibrating tools and affected by occupational Raynaud's phenomenon; 12 healthy black African workers exposed to hand-arm vibration for almost 3 years; and 12 healthy black African workers never exposed to hand-arm vibration. Computerized skin thermometry was performed and thermometric curves were analyzed according to thermometric interpretation criteria such as the area-over-curve (AOC), the fifth minute of recovery/baseline temperature ratio (5REC/BT) and the temperature at the tenth minute of recovery (10REC) after cold test. RESULTS: Thermometric parameters in Caucasian subjects confirmed the basis of the existing literature in controls (basal finger temperature higher than 32 degrees C and complete recovery to the initial temperature after the cold test) and also in patients with Raynaud's phenomenon (basal temperature often lower than control subjects and slow recovery of finger temperature after cold test). Statistically significant difference was found between healthy Caucasians and healthy black subjects in all the parameters tested: healthy black subjects showed values of AOC and 10REC suggesting almost constantly lower finger temperatures during the thermometry test. Black people, both exposed and non-exposed to hand-arm vibration showed thermometric parameters suggesting poor blood microcirculation, which seems even poorer than in Caucasian people complaining Raynaud's phenomenon. CONCLUSIONS: Our chronothermometric tests suggest some significant interethnic differences in peripheral microcirculation, which seems rather poor in black African subjects in comparison with Caucasians.

[Evidence Based Occupational Medicine: ten year experience with audiometric examination in a handicraft company]. / Evidence Based Occupational Medicine: 10 anni di esami audiometrici in aziende artigianali.

This work describes the audiometry threshold assessment of 1000 workers employed in different artisan categories during a period of ten-year noise professional exposure. The hearing loss noise-induced rates were determined by analysing audiometric tests at the beginning of our period of study and after 5 and 10 years of noise exposure. Environmental noise exposures were on average 88 dB(A), but near 90 dB(A) in some work categories. Workers widely used hearing protection devices, nearly at 93%, during the period we studied. The Evidence Based Occupational Medicine should find out points of reference proving the efficiency and effectiveness of occupational physicians: in this case, a positive trend in the reduction of hearing loss rate will be expected to confirm the goodness of prevention practice. Our study suggests that the levels of protection so far accepted are not effective enough in order to reduce the incidence of noise-induced hearing loss in the course of the years: in despite of most accredited predicting models for hearing conservation programs, a significant percentage of workers exposed to industrial noise continues to present a high incidence of hearing loss. The Evidence Based Occupational Medicine suggests that the proposed prevention activities carried out in the described factories were not enough effective.

GABA(B1a), GABA(B1b) AND GABA(B2) mRNA variants expression in hippocampus resected from patients with temporal lobe epilepsy.

The aim of this study was to investigate the mRNA expression of the two GABA(B1) receptor isoforms and the GABA(B2) subunit, in human postmortem control hippocampal sections and in sections resected from epilepsy patients using quantitative in situ hybridisation autoradiography. Utilising human control hippocampal sections it was shown that the oligonucleotides employed were specific to the receptor. Hippocampal slices from surgical specimens obtained from patients with hippocampal sclerosis and temporal lobe epilepsy were compared with neurologically normal postmortem control subjects for neuropathology and GABA(B) mRNA expression. Neuronal loss was observed in most of the hippocampal subregions, but in the subiculum no significant difference was detected. The localisation of GABA(B1a) and GABA(B1b) isoform mRNAs in human control hippocampal sections supported and extended earlier studies using the GABA(B1) pan probe, which does not distinguish between the two GABA(B1) isoforms. Moreover, the GABA(B2) mRNA location confirmed the heterodimerisation of the receptor. Thus, although there was an apparent correlation between GABA(B1b) and GABA(B2), GABA(B1a) exhibited no such relationship. GABA(B1b) and GABA(B2) showed a similar intensity of expression whilst GABA(B1a) displayed a lower hybridisation signal. Comparison of the expression of the three mRNAs between control and epileptic subjects showed significant decreases or increases in different hippocampal subregions.GABA(B) isoforms and subunit mRNA expression per remaining neuron was significantly increased in the hilus and dentate gyrus. These results demonstrate that altered GABA(B) receptor mRNA expression occurs in human TLE; possibly the observed changes may also serve to counteract ongoing hyperexcitability.

Biological monitoring of occupational exposure to N,N-dimethylacetamide with identification of a new metabolite.

AIMS: To study the concentration of N,N-dimethylacetamide (DMA) and its metabolite, N-methylacetamide (NMA), in urine of workers occupationally exposed to DMA in a factory producing synthetic acrylic fibres. METHODS: During the first phase, 223 workers exposed to low environmental concentrations of DMA provided urine samples at the end of a work shift. High concentrations of the unmodified solvent and its metabolite were found in a group of workers whose job was to start up machinery. The second and third phases focused on conditions favouring high uptake of DMA. RESULTS: The highest concentrations of unmodified solvent and NMA were found in the urine of workers recently engaged in starting up machinery. NMA in urine was 1.5-173.6 mg/g creatinine (median 20.5). In spite of the low environmental concentration, about 20% of the urine concentration of NMA was higher than 30 mg/g creatinine. Dermal absorption of DMA was high. A shower and a change of clothing at the end of the work shift, and washing away any solvent left on the skin, ensured that dermal absorption of DMA did not continue. This significantly reduced the NMA urinary concentration at values lower than 30 mg/g creatinine. In some urine samples, S-acetamidomethyl-mercapturic acid was identified by NMR analysis; this is probably a metabolite of N,N-dimethylacetamide--it has never before been identified in humans or animals. CONCLUSIONS: Even at low environmental concentrations of DMA, dermal absorption can be considerable. Unmodified DMA and NMA concentrations in urine are good biomarkers for monitoring occupational exposure to the solvent.

[Biological monitoring of occupational exposure to acrylonitrile]. / Monitoraggio biologico delle esposizioni professionali ad acrilonitrile.

Acrylonitrile (ACN) is a solvent widely used in industry especially as raw material in the manufacturing of acrylic fibres, clothes and domestic furniture. It is also used in manufacturing of resins (ACN-butadiene-styrene...) and for production of nitrilic elastomers. Some researchers proposed the biological monitoring of occupational exposure to ACN by measuring the solvent in the urine, but results were widely spread especially in relation to the analytical method used. This article reports the main aspects that can reduce the variability of results. We checked several ACN solutions in water and urine after heating at 90 degrees C for 1, 3, 5, 8 and 16 hours. Water solutions maintained their ACN concentration in all the checked conditions, while urine concentrations of ACN in urine deceased during the conditioning time until 80% of their initial concentration. The analysis of ACN in urine provided by workers potentially exposed to ACN and by control subjects gave median results of 1.9 and 2.0 micrograms/g creat, without any statistical difference. The results split in relation to the smoking habit showed a statistic difference: the median values of ACN were 1.7 and 4.7 micrograms/g creat, respectively among the 175 non-smokers and 57 smokers.

Studies of GABA(B) receptors labelled with [(3)H]-CGP62349 in hippocampus resected from patients with temporal lobe epilepsy.

1 The aim of this study was to investigate the binding of a novel GABA(B) receptor radioligand, [(3)H]-CGP62349, to human post-mortem control and epileptic hippocampal sections using quantitative receptor autoradiography. Utilizing human control hippocampal sections it was shown that [(3)H]-CGP62349 bound with high affinity (K(D) 0.5 nM) to this tissue. 2 Hippocampal slices from surgical specimens obtained from patients with hippocampal sclerosis (HS) and temporal lobe epilepsy (TLE) were compared with neurologically normal post-mortem control subjects for neuropathology and GABA(B) receptor density and affinity. Neuronal loss was observed in most of the hippocampal subregions, but in the subiculum no significant difference was detected. 3 The localization of GABA(B) receptors with the antagonist [(3)H]-CGP62349 in human control hippocampal sections supported and extended earlier studies using the agonist ligand [(3)H]-GABA. 4 The kinetics of binding to the GABA(B) receptor in human hippocampus using this novel compound was comparable to previous data obtained in rat hippocampal membranes. 5 GABA(B) receptor density (B(max)) was significantly reduced in CA3, hilus, and dentate gyrus (DG); the affinity was increased exclusively in DG. The trend is identical in all the hippocampal subregions with the agonist and the antagonist, although significant differences with the antagonist where recorded in CA3 and hilus, whereas with the agonist a significant reduction was reported in all of the hippocampal subfields. 6 GABA(B) receptor expression per remaining neuron appeared significantly increased in CA3 and hilus. These results suggest altered GABA(B) receptor function may occur in human TLE, possibly as a result of synaptic reorganization, and may contribute to epileptogenesis.

Generation of a selective 5-HT3B subunit-recognising polyclonal antibody; identification of immunoreactive cells in rat hippocampus.

The present study generated a polyclonal antibody (AP86/3) that recognises a peptide sequence of the h5-HT(3B) receptor subunit. Western blot analysis of homogenates prepared from cell lines expressing either homomeric (h5-HT(3A)) or heteromeric (h5-HT(3A/3B)) receptors, as well as immunocytochemical studies with the same cell lines, indicated that AP86/3 recognised, selectively, the 5-HT(3B) subunit. Immunohistochemical labelling was also apparent in cells in the rat hippocampus that displayed the distribution and morphology of interneurones.

Distribution of GABA(B(1a)), GABA(B(1b)) and GABA(B2) receptor protein in cerebral cortex and thalamus of adult rats.

The distribution of GABA(B) receptor subunits GABA(B(1a)), GABA(B(1b)) and GABA(B2), has been examined in the cerebral cortex and thalamus of adult rats using an immunocytochemical technique. GABA(B(1a)) and GABA(B(1b)) subunits co-localized with GABA(B2) in the cortex, where afferent thalamic GABAergic axons project to pyramidal neurones. The expression patterns of GABA(B(1a)), GABA(B(1b)) and GABA(B2) were similar throughout the thalamus. The data suggest that the GABA(B(1b)) subunit might be the presynaptic isoform in the thalamo-cortical pathway with the GABA(B(1a)) subunit possibly present at postsynaptic sites on cell bodies. This contrasts with our previous data, obtained in cerebellum and spinal cord which indicate opposite locations. Thus, it seems unlikely that functional role along with cellular location can be assigned in a general manner to specific GABA(B) receptor subunit splice variants.

GABAB receptor protein and mRNA distribution in rat spinal cord and dorsal root ganglia.

The presence of metabotropic receptors for GABA, GABAB, on primary afferent terminals in mammalian spinal cord has been previously reported. In this study we provide further evidence to support this in the rat and show that the GABAB receptor subunits GABAB1 and GABAB2 mRNA and the corresponding subunit proteins are present in the spinal cord and dorsal root ganglion. We also show that the predominant GABAB1 receptor subunit mRNA present in the afferent fibre cell body appears to be the 1a form. In frozen sections of lumbar spinal cord and dorsal root ganglia (DRG) GABAB receptors were labelled with [3H]CGP 62349 or the sections postfixed with paraformaldehyde and subjected to in situ hybridization using oligonucleotides designed to selectively hybridize with the mRNA for GABAB(1a), GABAB(1b) or GABAB2. For immunocytochemistry (ICC), sections were obtained from rats anaesthetized and perfused-fixed with paraformaldehyde. The distribution of binding sites for [3H]CGP 62349 mirrored that previously observed with [3H]GABA at GABAB sites. The density of binding sites was high in the dorsal horn but much lower in the ventral regions. By contrast, the density of mRNA (pan) was more evenly distributed across the laminae of the spinal cord. The density of mRNA detected with the pan probe was high in the DRG and distributed over the neuron cell bodies. This would accord with GABAB receptor protein being formed in the sensory neurons and transported to the primary afferent terminals. Of the GABAB1 mRNA in the DRG, approximately 90% was of the GABAB(1a) form and approximately 10% in the GABAB(1b) form. This would suggest that GABAB(1a) mRNA may be responsible for encoding presynaptic GABAB receptors on primary afferent terminals in a manner similar to that we have previously observed in the cerebellar cortex. GABAB2 mRNA was also evenly distributed across the spinal cord laminae at densities equivalent to those of GABAB1 in the dorsal horn. GABAB2 mRNA was also detected to the same degree within the DRG. Immunocytochemical analysis revealed that GABAB(1a), GABAB(1b) and GABAB2 were all present in the spinal cord. GABAB(1a) labelling appeared to be more dense than GABAB(1b) and within the superficial dorsal horn GABAB(1a) was present in the neuropil whereas GABAB(1b) was associated with cell bodies in this region. Both 1a and 1b immunoreactivity was expressed in motor neurons in lamina IX. GABAB2 immunoreactivity was expressed throughout the spinal cord and was evident within the neuropil of the superficial laminae.

Distribution of GABA(B) receptor protein in somatosensory cortex and thalamus of adult rats and during postnatal development.

In the present study we report the immunolocalisation of gamma-aminobutyric acid (GABA)(B) receptors within the cerebral somatosensory cortex (S1) and thalamus of adult and young (1-22 postnatal days) rats. The antibody used recognises a peptide in the carboxy-terminal domain and therefore did not distinguish between the different isoforms GABA(B)1a or GABA(B)1b. The results showed that GABA(B) receptor protein was widely distributed in the brain of both adult and young rats, with different degrees of labelling in separate cerebral nuclei. Antibody labelling was localised both on cells and the neuropil. In the cerebral cortex of adult animals the highest immunolabelling was evident in layers V and VIb, although immunoreactivity was also present in the superficial layers. The strongest signal was evident in the medial habenula.The thalamus showed labelling in the reticular, ventrobasal and geniculate nuclei. In the first postnatal days GABA(B) expression was evident in the cortical cells of layer V, VIb and in the cortical plate. The pattern of labelling in the cerebral cortex of young rats became indistinguishable from that of adult rats by day 12. In the thalamus, the main difference compared to the adult pattern was observed in the mediodorsal nucleus which, in early development, showed a high immunosignal, however, by postnatal day 22 the immunoreactivity decreased with only some scattered cells labelled in the adult brain.

Layer-specific immunocytochemical localization of GABA(B)R1a and GABA(B)R1b receptors in the rat piriform cortex.

A peculiar, layer-segregated immunoreactive distribution of GABABR1a and GABABR1b receptor antibodies is present in the piriform cortex of adult rats. The GABABR1a antibody selectively marked the neuropile in layer Ia, where afferent olfactory fibres and intrinsic GABAergic (gamma-aminobutyric acid) axons terminate on the distal apical dendrites of pyramidal neurons. The GABABR1b antibody was detected in the soma and the large basal dendrites of layer II and III neurons. The pattern of distribution observed supports the hypothesis that (presynaptic) GABABR1a receptors in the superficial molecular layer modulate neurotransmitter release in a feedforward synaptic circuit, whereas GABABR1b (postsynaptic) receptors mediate feedback inhibitory potentials on principal cells.

Expression of the SMN gene, the spinal muscular atrophy determining gene, in the mammalian central nervous system.

The survival motor neuron (SMN) gene is the putative disease gene for human spinal muscular atrophy (SMA), an autosomal recessive disorder characterized by progressive degeneration of lower motor neurons. Two copies of the gene, centromeric and telomeric, are present in the same 5q13 chromosomal region in humans. However, only the telomeric gene is affected in SMA. The SMN gene(s) encode(s) a novel protein of unknown function. To gain insights into the role of SMN in neurons, we have identified the SMN gene ortholog in the rat, and investigated SMN expression in the CNS of rat, monkey and humans by immunocytochemistry and in situ hybridization experiments. Antibodies against the SMN amino-terminus specifically recognized a single protein identical to the in vitro translation products of human and rat SMN cDNAs. The SMN gene transcript and product were widely but unevenly expressed throughout cerebral and spinal cord areas. The SMN protein was localized mainly in the cytoplasm of specific neuronal systems, and it was particularly expressed in lower motor neurons of newborn and adult animals. Likewise, a strong hybridization signal was detected in lamina IX of the spinal ventral horn. These results support the relevance of SMN for the motor neuron function and the pathogenetic role of the SMN gene in the neuronal degeneration associated with SMA.

Changes in protein kinase C and its presynaptic substrate B-50/GAP-43 after intrauterine exposure to methylazoxy-methanol, a treatment inducing cortical and hippocampal damage and cognitive deficit in rats.

The involvement of protein kinase C (PKC)-dependent processes in adaptive and plastic changes underlying neuronal plasticity was tested in an in vivo animal model characterized by targeted cellular ablation of cortical and hippocampal neurons, cognitive impairment and lack of induction of long-term potentiation. [3H]Phorbol ester binding performed on brain slices revealed a 67.4 and 35.0% increase in membrane-bound protein kinase C in the cortex and hippocampus respectively of rats treated with methylazoxy-methanol acetate compared with saline-treated control rats, and there was no modification in the expression of mRNAs of different protein kinase C isozymes. In situ phosphorylation experiments performed with 32Pi-labelled synaptosomes from the affected areas demonstrated that the phosphorylation of the nervous tissue-specific presynaptic membrane-associated protein kinase C substrate B-50/GAP-43 was increased by 51.4 and 44.8% in cortex and hippocampus respectively. Western blot analysis of protein kinase C in synaptosomal cytosol and membrane fractions prepared from cortex and hippocampus showed an increased proportion of protein kinase C in the membrane compartment in treated animals, but no change in the total synaptosomal protein kinase C activity. Our data are consistent with increased activity of presynaptic protein kinase C and predict a sustained increase in glutamate release in methylazoxy-methanol-treated rats.

A reticuloreticular commissural pathway in the rat thalamus.

To further characterize the communication between the thalami of the two hemispheres, a connection linking the rostral reticular nuclei of the two thalamic sides was investigated in the rat by retrograde and anterograde tracing. The rostral reticular nucleus can be divided into a medial region, with densely packed fusiform neurons, and a lateral region, with less densely packed, polymorphic neurons. After injections of Fluorogold (FG) in the medial region, retrogradely labeled, small fusiform neurons were found in the corresponding contralateral region. The retrograde labeling data were confirmed by the anterograde-tracing experiments. Thin, beaded axons, anterogradely labeled after injection of biocytin or biotinylated dextranamine in the medial region, innervate the corresponding region in the contralateral reticular nucleus. The present data suggest the existence of a commissural pathway specifically devoted to the crosstalk between the rostral reticular nuclei of the two thalamic sides. The commissural gamma aminobutyric acid (GABA)-ergic input on the GABAergic neurons of the rostral reticular nucleus could modulate the generation of sleep spindles. The reticuloreticular pathway may, moreover, synchronize the diffuse modulatory effect of the rostral reticular nucleus on nonprimary cortical areas through the bilateral projections of the nucleus to the ventromedial, intralaminar, and anterior thalamic nuclei.

Effects of postnatal or adult chronic acetylcholinesterase inhibition on muscarinic receptors, phosphoinositide turnover and m1 mRNA expression.

Muscarinic receptor number, receptor-stimulated phosphoinositide hydrolysis and m1 mRNA expression were examined in the cerebral cortex and hippocampus of rats treated during postnatal development or in adult age with the organophosphate diisopropylfluorophosphate. Developing rats were treated from postnatal days 4-9 or from postnatal days 4-20 and killed on days 10 and 21, respectively, 24 h after the last administration of diisopropylfluorophosphate. Adult animals were treated for 14 days. Acetylcholinesterase activity and muscarinic receptor number were significantly reduced in all groups of treatment. Muscarinic receptor-stimulated phosphoinositide turnover, however, was significantly reduced in postnatal days 4-20 and adult treated rats but not in the postnatal days 4-9 group. No differences were observed in ED50 values. Conversely, m1 mRNA expression was significantly reduced both in the cerebral cortex and hippocampus of postnatal days 4-9 treated rats, but not of postnatal days 4-20 and adult treated rats. These results indicate that chronic inhibition of acetylcholinesterase in developing rats results in significant alterations in muscarinic neurotransmission. These alterations may delay the maturation of the cholinergic system and, therefore, may account for some of the long-lasting neurotoxic effects observed after developmental exposure to organophosphate pesticides.