[Apolipoprotein E and bleomycin hydrolase. Polymorphisms: association with neurodegenerative diseases]. / Maladies neurodégénératives et polymorphisme des gènes de l'apolipoprotéine E et de la bléomycine hydrolase.

Several studies indicate a possible association between different genes and chronic neurodegenerative diseases including Alzheimer's disease (DTA). To further investigate, we have analyzed association between the apolipoprotein E (apo E) and bleomycin hydrolase (BH) polymorphisms and three groups of elderly patients: control subjects (T) (n = 68), late-onset sporadic DTA patients (DTAst) (n = 65) and other non vascular neurodegerative diseases (MNDA) (n = 52). Apo E-epsilon4 and BH-G alleles frequencies (%) are: 8.2 (T), 31.5 (DTAst), 16.4 (MNDA) and 41.4 (T), 35.6 (DTAst). No association has been observed between carrying the G allele and DTA in epsilon4 negative subjects but, our data have confirmed the earlier reports: carrying the epsilon4 allele is a dose-dependent risk factor for the DTAst (OR: 6.0, IC 95 %: 2.6-13.7) and decrease the age of symptom onset (p < 0.005). They have also suggested that apo E genotyping may be of interest to perform differential diagnosis of neurodegenerative diseases in elderly subjects.

Structure and level of genetic diversity in various bean types evidenced with microsatellite markers isolated from a genomic enriched library.

We have constructed a common bean genomic library enriched for microsatellite motifs (ATA), (CA), (CAC) and (GA). After screening, 60% of the clones selected from the library enriched for the (ATA) repeat contained microsatellites versus 21% of the clones from the library enriched for (GA) (CA) and (CAC) repeats. Fifteen primer pairs have been developed allowing for the amplification of SSR loci. We have evaluated the genetic diversity of these loci between 45 different bean lines belonging to nine various quality types. A total of 81 alleles were detected at the 15 microsatellite loci with an average of 5.3 alleles per locus. We have investigated the origin of allelic size polymorphism at the locus PvATA20 in which the number of repeats ranges from 24 to 85. We have related these large differences in repeat number to unequal crossing-over between repeated DNA regions. The diversity analysis revealed contrasted levels of variability according to the bean type. The lower level was evidenced for the very fine French bean, showing the effect of breeders intensive selection.

Variability of cytochrome P450 1A2 activity over time in young and elderly healthy volunteers.

AIMS: To assess the age-associated changes over time of plasma paraxanthine/caffeine (PAX/CAF) ratios used as a probe for CYP1A2 activity. METHODS: Intraindividual and interindividual variabilities in PAX/CAF ratio were compared by phenotyping with caffeine, 16 young and 16 elderly healthy subjects on five occasions. RESULTS: PAX/CAF ratio variability was comparable regardless of age (intraindividual CV: 17.6 +/- 6% and 16.2 +/- 5.9%, interindividual CV: 48.1 +/- 2.9% and 42.7 +/- 3.6% in young and elderly, respectively). The PAX/CAF ratio was lower in elderly than in young subjects (95% CI for the difference: 0.004, 0.32) but the difference was not significant in nonsmokers compared separately. CONCLUSIONS: The variability over time of the PAX/CAF ratio is not influenced by age.

A large multiple endocrine neoplasia type 1 family with clinical expression suggestive of anticipation.

We describe a large multigenerational multiple endocrine neoplasia Type 1 (MEN1) family with clinical expression suggestive of anticipation. In the second and third generations, two deceased obligate gene carriers died at the ages of 85 and 76 without the history of MEN1, whereas two other living gene carriers above the age of 65 have had no clinical evidence of MEN1 to date. In the fourth generation, eight members were affected, with four having severe MEN1-related and atypical malignancies: a case of metastatic endocrine pancreatic tumor, two cases of metastatic thymic carcinoids, and a case of spinal ependymoma. In the fifth generation, all five patients were below the age of 22 when the disease was detected. MEN1 was confirmed in the family by linkage analysis using MEN1-linked microsatellite markers and by identification of a nonsense mutation in the MEN1/menin gene. Alleotyping showed loss of heterozygosity (LOH) involving the wild-type alleles in seven tumors in the family including the ependymoma, which is the first MEN1-related case that shows genetic abnormality in chromosome 11q13, suggesting that MEN1 gene might be involved in the tumorigenesis of a subset of ependymomas. In relation to clinical anticipation, repeated expansion studies were carried out but failed to detect any expansion. We conclude that this is a unique MEN1 family and that an unknown genetic mechanism might be contributing to the anticipation phenomenon. We demonstrate in this family that all gene carriers, including the very young members, will need close and careful follow-up.

Nicotine inhibits slowly inactivating K+ currents in rat cultured striatal neurons.

As shown on cultured striatal neurons recorded in whole-cell configuration, both acetylcholine (in the presence of atropine) and nicotine reduced voltage-dependent outward currents. Although, at early postnatal ages, outward currents in these cells are mainly carried by rapidly and slowly inactivating K+ channels, these inhibitions resulted from a selective and reversible effect on the slowly inactivating K+ conductance (IK+). This action was blocked by the nicotinic antagonist dihydro-beta-erythroïdine and reproduced by nicotinic agonists. When neurons were recorded under current-clamp conditions, nicotine increased reversibly their firing rate generated by step depolarizations. Therefore, in addition to its well documented muscarinic effects, acetylcholine also controls K+ currents in striatal neurons through mechanisms mediated by nicotinic receptors.

Nicotine-induced inhibition of neuronal phospholipase A2.

A protective effect of nicotine against glutamate-induced neurotoxicity has previously been reported in cultured striatal and cortical neurons. The aim of this study was to investigate whether nicotine also inhibits glutamate-evoked arachidonic acid release from cultured striatal neurons. (-)-Nicotine selectively inhibited the release of [3H]-arachidonic acid induced by the joint stimulation of alpha-amino-3-isoxazol-5-propionic acid and metabotropic receptors, whereas the response evoked by the sole activation of N-methyl-D-aspartate receptors remained unchanged. The inhibitory effect of (-)-nicotine was not mediated by nicotinic receptors because it was neither reproduced by acetylcholine (in the presence of atropine) or 1,1-dimethyl-4-phenyl piperazinium, nor reversed by dihydro-beta-erythroidine or hexamethonium, two central nicotinic receptor antagonists. (-)-Nicotine, which induced rapidly desensitizing inward currents in 17% of striatal neurons, did not alter the alpha-amino-3-isoxazol-5-propionic acid-evoked currents. Moreover, (-)-nicotine did not inhibit the accumulation of inositol phosphate derivatives induced by agonists of glutamate metabotropic receptors. In fact, using the fluorogenic phospholipase A2 substrate 1,2-bis-(1-pyrenedecanoyl)-sn-glycero-3-phosphocholine, (-)-nicotine was found to inhibit both particulate and soluble phospholipase A2 activities from striatal neurons. Therefore, (-)-nicotine can modulate a neuronal response (arachidonic acid release) evoked by glutamate but this process is not involved in the neuroprotective effect of the drug on glutamate-induced neurotoxicity.

Calcium-dependent, slowly inactivating potassium currents in cultured neurons of rat neocortex.

Slowly inactivating outward currents were examined in neurons from rat anterior cortex dissociated at postnatal day 1 and recorded after 7-48 days in vitro by the use of whole-cell patch-clamp technique, in the presence of 0.5-0.8 microM tetrodotoxin (TTX). 50 microM carbachol and 1-5 mM CsCl2. Experiments were often carried out in the additional presence of 1-5 mM CsCl2, which blocks the anomalous, inwardly rectifying IQ, the fast Ca(2+)-dependent K+ current (IC), and 50 microM carbachol, which depresses the IM current. These currents were evoked by depolarizing steps to -40 +/- 5 mV from a conditioning hyperpolarization to -110 +/- 10 mV. Their sensitivity to elevation from 2.5 to 12.5 mM in extracellular K+ concentration, together with their sensitivity to 5-15 mM tetraethylammonium, suggests that they are mainly carried by K+ ions. Their activation and inactivation curves show that the threshold for activation is -65 mV, that their inactivation is achieved at -75 mV and that potentials more negative than -120 mV are needed to abolish it. The time-dependence of de-inactivation gives a maximal current amplitude for conditioning hyperpolarizations of 2 s and is best described by a monoexponential function with a time constant of 0.7 s. Slow transient K+ currents were depressed by low doses of 4-aminopyridine (30-100 microM), which indicates the occurrence of an ID-type component in the recorded K+ currents. No slowly declining K+ current was expressed when a recording solution containing 10 mM 1,2-bis (2-aminophenoxy)ethane-N,N,N'-N'-tetraacetic acid (BAPTA), instead of 1-5 mM BAPTA, was used. When recorded without Ca2+ chelator in the pipette, slowly declining K+ currents were blocked by bath-applied 40-50 microM BAPTA-aminoethoxy, revealing a large-amplitude, rapidly inactivating outward current. This residual component is insensitive to 50 microM 4-aminopyridine and may include a current more related to the IA-type. Our data provide evidence that, in cultured cortical neurons from rat, the expression of an ID-like K+ current is highly dependent on internal Ca2+ concentration.

[Does the placebo effect exist in newborn infants?]. / L'effet placebo existe-t-il chez le nouveau-né?

Although comparison with a placebo is necessary to demonstrate the "true" effect of a drug, neonatologists are usually reluctant to use a placebo. The reason given is the lack of placebo effect in neonates. We studied heart and respiration rates and behaviour in normal neonates during heelstick for diagnosis of phenylketonuria. In this open randomized study we compared no treatment with an "analgesic" treatment consisting of water and sucrose. There was no difference in heart and respiration rates and behaviour between the two groups. These results do not demonstrate a "suggested" placebo effect and can in part be explained by the model and tools used to measure pain. The results do not support the non-use of placebo in drug evaluation trials in children.

Reduction of desensitization of a glutamate ionotropic receptor by antagonists.

The glutamate receptor channel subtype that responds to both quisqualate (QA) and alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionate (AMPA) was expressed in Xenopus oocytes injected with rat cerebral cortex mRNA. Voltage-clamp current responses to QA, AMPA, and glutamate (GLU) exhibited a rapid increase followed by a decrease to a desensitized steady state (DS). Perfusion with high agonist concentrations produced smaller DS responses than perfusion with low concentrations. During the DS, the current was increased by lowering of the concentration of agonist or by application of low concentrations of a competitive antagonist, 6,7-dinitroquinoxaline-2,3-dione (DNQX). This paradoxical increase of the agonist-induced currents during the DS was also observed in cultured Purkinje cells with another competitive antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX). Dose-response curves obtained in oocytes were bell shaped, with a negative slope for high concentrations of QA. DNQX shifted these bell-shaped curves to the right. Together, these results indicate that the agonists are able to reversibly inhibit the AMPA receptor. The classical desensitization model of Katz and Thesleff [J. Physiol. (Lond.) 138:63-80 (1957)] cannot account for our observations.

[Pneumopathy due to sulfasalazopyrine. A case with a challenge test]. / Pneumopathie à la sulfasalazopyrine. Une observation avec test de réintroduction.

The complications of treatment with sulfasalazopyrine(SSP) are rare. We report a case of a 68 year old patient who was hospitalised for a hectic fever with weight loss, chest pain and dyspnoea of effort. This patient had been treated for 2 1/2 months for a haemorrhagic proctocolitis with 3 gms. per day of SSP. There was a leucocytosis of 23 700/mm3 of which there were 3,318 eosinophils/mm3. The pulmonary radiograph showed a predominantly interstitial pattern at the apices in the axillary region and in the costo-phrenic angle. The broncho alveolar lavage (LBA) was normal. A thoracic scan confirmed the interstitial syndrome and the existence of pleural thickening in the upper part of the lungs. As drug induced pulmonary disease was suspected SSP was stopped. The patient became apyrexial in 48 hours without any further treatment. Thirty days later the pulmonary radiograph and the laboratory investigations had returned to 'normal and a challenge test using the drug was given whilst the patient was still in hospital. After 24 hours there was a febrile peak of 38.7 degrees C and 36 hours later there was a leucocytosis once more of 19 700/mm3 of which there were 1,100 eosinophils/mm3. The pulmonary radiograph showed diffuse bilateral interstitial opacities. The treatment was stopped and the fever disappeared within 24 hours. After a review of the literature, two points of this case were discussed in particular: the frequency of pulmonary disease occurring as a complication of SSP and the indication for challenge test.

Developmental changes in neuronal sensitivity to excitatory amino acids in area CA1 of the rat hippocampus.

The laminar distribution of decreases in extracellular free calcium and concomitant field potentials induced by repetitive orthodromic stimulation, ionophoretic application of N-methyl-D-aspartate and quisqualate, was studied in the CA1 field of rat hippocampal slices, at two different stages during postnatal development. While stimulation-elicited and quisqualate-induced signals remain maximal in stratum pyramidale during the first postnatal month, the laminar profiles of responses to N-methyl-D-aspartate (NMDA) depend on age: the responses to this agonist are maximal in stratum pyramidale in 5-9-day-old rats and in stratum radiatum in 12-30-day-old rats. Our findings suggest that, during the second postnatal week, the apical dendrites of pyramidal neurons in area CAl become more sensitive to NMDA, which is expressed by big influxes of calcium at this level.

Voltage-dependency of the responses of cerebellar Purkinje cells to excitatory amino acids.

The voltage-dependency of the responses of Purkinje cells to excitatory amino acids was examined in rat cerebellar slices, using intrasomatic recordings with the single electrode voltage-clamp. In standard perfusion medium, the depolarizations evoked in these neurones by ionophoretic pulse applications (less than 300 ms) of L-glutamate, L-aspartate and quisqualate in their dendritic fields had underlying inward currents which did not increase or even decreased, as the holding potential was shifted to values more negative than -65 mV. This 'abnormal' voltage-dependency was still present in Mg2+ -free solution but was abolished in the presence of CsCl2 (10 mM) in the perfusion medium. When TTX (5 microM) and CdCl2 (0.1 mM) were further added to the bath in order to block regenerative conductances, thus broadening the range of the clamp voltages to more positive values than -50 mV, the current-voltage relation between -80 and 0 mV for responses to L-glutamate and L-asparate was almost linear. Our results support the view that low doses of both amino acids act on Purkinje cells essentially via the activation of receptors which are not of the N-methyl-D-aspartate type.

An N-methyl-D-aspartate receptor-independent excitatory action of partial reduction of extracellular [Mg2+] in CA1-region of rat hippocampal slices.

Partial reduction of [Mg2+]o from 2 to 1 mM markedly enhanced neuronal responses evoked by Schaffer collateral-commissural fiber stimulation in the CA1-region of rat hippocampal slices. The amplitude of extracellular population potentials recorded in the CA1-pyramidal cell layer and maximum dV/dt of extracellular population EPSP's recorded in the CA1-pyramidal apical dendritic layer were both increased. However, unlike findings from slices where Mg2+ was completely removed from the bathing medium, there was no spontaneous or evoked epileptiform activity, and the N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphonovalerate (2-APV) did not antagonize the enhancement of evoked responses. These results indicate that, in addition to the participation of NMDA receptors in the epileptiform activity observed when Mg2+ is completely removed from the bathing medium, there is also an NMDA receptor-independent excitatory action of partial reduction of [Mg2+]o in hippocampal slices.

Development of two types of calcium channels in cultured mammalian hippocampal neurons.

Calcium influx through voltage-gated membrane channels plays a crucial role in a variety of neuronal processes, including long-term potentiation and epileptogenesis in the mammalian cortex. Recent studies indicate that calcium channels in some cell types are heterogeneous. This heterogeneity has now been shown for calcium channels in mammalian cortical neurons. When dissociated embryonic hippocampal neurons from rat were grown in culture they first had only low voltage-activated, fully inactivating somatic calcium channels. These channels were metabolically stable and conducted calcium better than barium. Appearing later in conjunction with neurite outgrowth and eventually predominating in the dendrites, were high voltage-activated, slowly inactivating calcium channels. These were metabolically labile and more selective to barium than to calcium. Both types of calcium currents were reduced by classical calcium channel antagonists, but the low voltage-activated channels were more strongly blocked by the anticonvulsant drug phenytoin. These findings demonstrate the development and coexistence of two distinct types of calcium channels in mammalian cortical neurons.

The action of valproate on spontaneous epileptiform activity in the absence of synaptic transmission and on evoked changes in [Ca2+]o and [K+]o in the hippocampal slice.

The effects of valproate (VPA) on neuronal excitability and on changes in extracellular potassium ([K+]o) and calcium ([Ca2+]o) were investigated with ion selective-reference electrode pairs in area CA1 of rat hippocampal slices. Field potential responses to single ortho- and antidromic stimuli were unaltered by VPA (1-5 mM). The afferent volley evoked in the Schaffer-commissural fibers was also unaffected. In contrast, VPA (1 mM) depressed frequency potentiation and paired pulse facilitation markedly. Decreases in [Ca2+]o induced either by repetitive stimulation or by application of the excitatory amino acids N-methyl-D-aspartate and quisqualate were reduced, and the latter results suggest that VPA interferes with postsynaptic Ca2+ entry. When synaptic transmission was blocked by lowering [Ca2+]o (0.2 mM) and elevating [Mg2+]o (7 mM), prolonged afterdischarges elicited by antidromic stimulation were blocked by VPA. VPA also suppressed the spontaneous epileptiform activity seen when [Ca2+]o was lowered to 0.2 mM, without elevating [Mg2+]o. The amplitudes of the rises in [K+]o induced by repetitive orthodromic stimulation were only slightly depressed and those elicited by antidromic stimulation were generally unaltered by VPA, as were laminar profiles of stimulus-evoked [K+]o signals. These results indicate that VPA has membrane actions in addition to known effects on excitatory and inhibitory transmitter pools.

Epileptiform activity in combined slices of the hippocampus, subiculum and entorhinal cortex during perfusion with low magnesium medium.

Reduction of [Mg2+]o induced spontaneous epileptiform activity consisting of 40-100-ms bursts of population spikes in hippocampal slices. This activity disappeared from area CA1 when the connections to area CA3 were cut, but persisted in isolated minislices of area CA3. Spontaneous activity was also observed in the dentate gyrus, provided that the connections to the subiculum and entorhinal cortex (EC) were intact. In the parasubiculum and EC longer lasting epileptiform events were observed which resembled seizure-like behaviour. The epileptiform activity was completely suppressed by 2-aminophosphonovalerate (30 microM) suggesting that N-methyl-D-aspartate receptors for excitatory amino acid transmitters participate in the generation of this activity. These findings show that the EC possesses properties which permit the generation of seizure-like activity in contrast to the hippocampus where the activity resembled recurrent interictal events.

Effects of GABA and bicuculline on N-methyl-D-aspartate- and quisqualate-induced reductions in extracellular free calcium in area CA1 of the hippocampal slice.

Decreases in extracellular free calcium ([Ca2+]o) and concomitant field potentials were recorded from the dendritic and cell body layers of the CA1 field in transverse hippocampal slices. They were elicited by tetanic stimulation of Schaffer collaterals and commissural fibers or by iontophoretic application of the excitatory amino acids N-methyl-D-aspartate (NMDA) and quisqualate (Quis). Under control conditions, decreases in [Ca2+]o were found to be maximal in stratum pyramidale (SP). In stratum radiatum (SR), 100 micron away from SP, decreases in [Ca2+]o were half the size of those observed in SP. Bicuculline methiodide, bath-applied at concentrations of 10-100 microM, enhanced the reductions in [Ca2+]o, increased the field potentials in all layers and also induced "spontaneous" epileptiform activity. In the presence of bicuculline, the decreases in [Ca2+]o were particularly enhanced in SR and were often greater than those recorded in SP. This was the case for changes in [Ca2+]o induced either by repetitive electrical stimulation or by application of NMDA and Quis. When synaptic transmission was blocked by perfusing the slices with a low Ca2+ medium, all NMDA and Quis-induced changes in [Ca2+]o were predictably reduced but there was a relative enhancement of changes in [Ca2+]o in SR with respect to those in SP. We propose that, under normal conditions, an inhibitory control mediated by GABA limits the reductions of [Ca2+]o particularly in SR. In support of this proposal, we found that bath-applied GABA had a depressant action on changes in [Ca2+]o.