The human necdin gene, NDN, is maternally imprinted and located in the Prader-Willi syndrome chromosomal region.

Prader-Willi syndrome (PWS) is a neurogenetic disorder that results from the absence of a normal paternal contribution to the 15q11-13 region. The clinical manifestations of PWS are a transient severe hypotonia in the newborn period, with mental retardation, hypogonadism and obesity observed later in development. Five transcripts with exclusive expression from the paternal allele have been isolated, but none of these has been shown to be involved in PWS. In this study, we report the isolation and characterization of NDN, a new human imprinted gene. NDN is exclusively expressed from the paternal allele in the tissues analysed and is located in the PWS region. It encodes a putative protein homologous to the mouse brain-specific NECDIN protein, NDN; as in mouse, expression in brain is restricted to post-mitotic neurons. NDN displays several characteristics of an imprinted locus, including allelic DNA methylation and asynchronous DNA replication. A complete lack of NDN expression in PWS brain and fibroblasts indicates that the gene is expressed exclusively from the paternal allele in these tissues and suggests a possible role of this new gene in PWS.

[Role of DNA microarrays in the diagnosis of pleural exudates: a feasibility study]. / Apport des puces à ADN dans le diagnostic étiologique des pleurésies: étude de faisabilité

INTRODUCTION: Establishing the cause of exudative pleural effusions is sometimes difficult, especially in the context of possible malignant pleural mesothelioma (MPM). Therefore, the development of new biological tools is necessary. The aim of this study was to determine the feasibility and the diagnostic contribution of genomic analysis of cells contained in pleural fluid, using DNA microarray techniques. METHODS: Patients with pleural effusion requiring diagnostic thoracocentesis were eligible to participate in the study. Five hundred mls of pleural fluid were then collected. RNA was extracted from pleural fluid cells and its integrity was assessed. Gene expression was studied using pangenomic DNA microarrays. RESULTS: Seventeen patients were included (4 MPM, 8 secondary malignant pleurisies, 5 benign pleurisies). Three patients offered fully exploitable samples. Taking into account the results of control experiments, gene expression study from pleural fluid was reproducible. The comparison of samples showed significant differences in gene expression. Samples from 14 patients were not exploitable because of RNA degradation. CONCLUSIONS: Gene expression study of cells from pleural fluid is feasible but remains difficult, essentially in relationship with RNA weakness.

A Distal-less-like gene is induced in the regenerating central nervous system of the urodele Pleurodeles waltl.

We report the cloning of a Distal-less-like gene (PwDlx-3) and its pattern of expression during embryonic development and adult tail regeneration in the urodele Pleurodeles waltl. Using RT-PCR and in situ hybridization experiments we determined that, during regeneration, PwDlx-3 is expressed in the epidermis, the cells associated with muscle masses and in the ventrolateral parts of the ependymal tube. PwDlx-3 localization in the muscle masses and in cells of the ependymal tube, which give rise during regeneration to the ventral roots and the spinal ganglia, suggests that this gene might be expressed in cells which have some neural crest cell potentialities. PwDlx-3 is the first homeobox gene shown to be expressed in the regenerating spinal cord but not in the adult one and could thus be involved in the regeneration of the nervous system.

The alpha(1A) subunits of rat brain calcium channels are developmentally regulated by alternative RNA splicing.

Calcium influx through voltage-gated calcium channels governs important aspects of CNS development. Multiple alternative splicings of the pore-forming alpha(1) subunits have been evidenced in adult brain but little information about their expression during ontogenesis is presently available. The aim of this study was to focus on the expression of three rat voltage-gated calcium channel alpha(1A) splice variants (alpha(1A-a), alpha(1A-b) and alpha(1A-EFe)) during brain ontogenesis in vivo. Using a reverse transcription-polymerase chain reaction strategy, we found that the three isoforms have different timings of development throughout the brain: alpha(1A-b) is expressed from embryonic to the adult stage, alpha(1A--EFe) is restricted to the embryonic period whereas alpha(1A-a) is expressed only postnatally. In situ hybridization indicated that alpha(1A-a) and alpha(1A-b) isoforms develop with different regional and cellular patterns. In hippocampus and cerebellum, alpha(1A-b) represented the predominant isoform at all developmental stages. Taken together, these data reveal that alternative RNA splicing may modulate the alpha(1A) calcium channel properties during development.

Tissue-specific expression and methylation of the human CYP2E1 gene.

The level and number of CYP2E1 gene transcripts were investigated by northern blot analysis in various human adult tissues including liver, lung, placenta, skin and neurinoma. Three transcripts of 1.8, 2.6 and 4 Kb were expressed in a tissue-specific manner. The origin of the various transcripts was studied and showed that both 4 and 2.6 Kb mRNAs contained sequences from the 3' non-translated region of the gene and that the 4 Kb also contained region localized in the 5' non-translated region. Furthermore, it clearly appeared that a catalytically active CYP2E1 enzyme (as proved by NDMA demethylase activity) was only detected in tissues expressing the 1.8 Kb. The human CYP2E1 was also identified through immunohistochemical techniques. Finally, we observed a relation between the hypomethylation of the human CYP2E1 gene and the hypoexpression of the corresponding protein.

Early alterations at the plasma membrane of breast cancer cell lines in response to estradiol and hydroxytamoxifen.

The time course of the early stage of estradiol-17 beta (E2) and hydroxytamoxifen (OHTAM) action at the plasma membrane of hormone-responsive MCF-7 and non-responsive MDA-MB-231 (MDA) breast cancer cell lines was investigated using scanning electron microscopy (SEM), electron probe X-ray microanalysis and microelectrophysiology analysis. SEM showed a marked increase in the density and the length of microvilli (MV) on MCF-7 cells treated with 1 nM estradiol for 1 min. This membrane response disappeared at 5 min. No early effect was obtained with OHTAM, but both compounds produced a similar surge of heterogeneous MV at 15 min of treatment. The morphological change induced by E2 subsided at 60 min, whereas that of OHTAM persisted. X-ray microanalysis and computer determination of peak/background ratios permitted the demonstration that these morphological alterations were concomitant with a rise in the intracellular level of potassium. Microelectrophysiology analysis showed a sharp transitory decrease in the membrane potential of MCF-7 cells in response to estradiol. In the estrogen-insensitive MDA cells, the hormone did not modify the membrane potential and K levels decreased at 1 and 5 min before rising again to control levels at minute 15 when MV appeared. With OHTAM, potassium decreased significantly at 60 min of treatment. These initial and transitory changes in surface morphology paralleled by alterations in potassium level may be consistent with the occurrence of estrogen membrane receptors on target cells, a new aspect of steroid hormone action.

Increase in mRNAs encoding neonatal II and III sodium channel alpha-isoforms during kainate-induced seizures in adult rat hippocampus.

Subtypes I, II and III of sodium channel alpha-subunit mRNAs were analyzed in adult rat brain areas after kainate-induced seizures. Tissue samples were microdissected from occipital neocortex, CA1 and CA3 hippocampus areas and dentate gyrus. Three reverse transcriptase-polymerase chain reaction (RT-PCR) protocols were undertaken to amplify these mRNAs. Amplification products were then distinguished after digestion by restriction enzymes, electrophoresis separation and densitometric analysis of gel profiles. PCR 1 evidenced the relative percentage of mRNAs I, II and III as well as neonatal II and III subtype isoforms, which resulted from an alternative splicing. PCR 2 and 3 were performed to focus on the neonatal vs. adult ratio in II and III subtypes, respectively. Seizures were shown to induce an increase in both neonatal subtypes, which suggested an alteration at the splicing level. These changes exhibited a peculiar brain regional distribution, the maximal effect being observed in dentate gyrus and hippocampus CA1 area. In situ hybridization experiments, using a digoxigenin-labeled oligonucleotide probe-specific for neonatal II and III mRNAs, confirmed this increase in neonatal mRNA subtypes. These changes were transient, reaching a maximum 6 h after drug injection, then disappearing between 12 and 48 h. They were prevented by a pre-treatment of animals by MK-801, a non-competitive antagonist of NMDA receptors. This work, thus, suggested that KA-induced seizures can be accompanied by transient alteration in the splicing pattern of sodium channel alpha-subunit mRNAs which resulted in an increase in expression of their neonatal isoforms within localized areas of adult rat brain.

Autoimmune alterations in the neurohypophysis of rabbits immunized against vasopressin.

Rabbits immunized against vasopressin developed clinical signs of diabetes insipidus persisting for up to two months after the last boost. Ultrastructural and immunocytochemical studies of the neurohypophysis demonstrated autoimmune alterations: infiltration by immune cells and extracellular deposits of immunoglobulins. No alterations were observed in the hypothalamic nuclei synthesizing vasopressin nor in hormone-target cells of the kidney. Immunization against vasopressin may provide the first example of an autoimmune disease in the hypothalamo-neurohypophysial system.

Distribution of benzodiazepine receptors in the rat superior colliculus: a light and electron microscope quantitative autoradiographic study.

The distribution of benzodiazepine (Bdz) receptors of the central type was analysed in the superficial grey layer of the rat superior colliculus from light and electron microscope autoradiographs, using the highly specific partial reverse agonist [3H]Ro 15-4513, a radioligand which can be crosslinked to its binding sites by ultraviolet rays. Biochemical characteristics of the binding were first defined by liquid scintillation count on unfixed cryostat mesencephalic brain slices. Saturation curves (1.6-20 nM) and Scatchard plot indicated that the radioligand bound with a high affinity (Kd = 11 nM) to a single population of sites (Bmax = 650 fmol/mg dry tissue). A slight primary chemical fixation of the brain did not significantly modify the binding characteristics. The consolidation of the specific binding by ultraviolet light on prefixed brain slices was found to be optimal after a 45-min illumination period. The distribution of Bdz sites on light and electron microscope autoradiographs was then analysed by applying these binding conditions. Prefixed brain slices (50 micron thick, Vibratome) were incubated in the 15 nM radioligand in the absence (total binding) or in the presence (non-specific binding) of the non-radioactive antagonist Ro 15-1788 (10(-5) M). Quantitative light microscopic study of Epon-embedded semithin sections showed that 95% of the silver grains of the specific label were located on the neuropil to the detriment of the neuronal and glial cell compartments. In the electron microscopic study, the distribution of the specific binding sites was statistically analysed over a total of more than 10 identified single or junctional tissue compartments, using the 50% probability circle method (Williams, 1969). Apart from a slight labeling of varicose profiles, the specific labeling was found to be concentrated on two particular tissue compartments: the percentage of grains associated with contacts between varicosities and dendrites was 32%, and that associated with axodendritic synapses was 16% of the total specific labeling measured over all compartments combined. A low proportion (33%) of the labeled axodendritic interfaces was characterized by a synaptic differentiation. These results suggest that both synaptic and non-synaptic Bdz receptors are present in the rat superior colliculus, and may each modulate neuronal cell activity in a different way.

Quantitative analysis of fetal rat brain neurons developing in primary cultures. I. Stereological study of the neuronal differentiation.

An ultrastructural stereological analysis was performed to analyze the morphological differentiation of primary cultures of fetal rat brain neurons, growing for two weeks in a serum-free medium. The number of neurons and of gliofibrillary acidic protein (GFAP)-positive glial cells was estimated by light microscopy counting in the culture wells. These cultures provided a quasi-pure neuronal population, since the number of GFAP-positive glial cells was found to be 1% (day 7) and 2% (day 14) respectively of the total number of cultured cells. Cell counts and the stereological measurements were related to the surface area of the culture well. The neuronal differentiation was characterized by an increase in the plasma membrane surface area (x9) and volume (x8) of neurites, contrasting with the decrease in the perikarya surface area and volume. These primary stereological data were combined with the number of neurons to obtain parameters characterizing an average neuron. The increase in membrane surface area of an average neuron was found to be a linear function of time, 29 micron 2 and 445 micron 2 of new membrane being added per day of culture to perikarya and neurites respectively. The number of chemical synapses was also counted and compared to the changes in the plasma membrane surface area. After 7 days in vitro they increased in number more rapidly than the increase in the plasma membrane surface area of neurons.

Quantitative analysis of rat brain neurons developing in primary cultures. II. Changes in the distribution of N-CAM associated to neuronal cell surfaces.

Cultured rat fetal brain cells underwent morphological differentiation, as quantitatively described in the companion paper. In the same system, biochemical and immunolabeling studies were performed to analyze the developmental changes in neural cell adhesion molecule (N-CAM) distribution and quantity at the cell surface of neurons. The cell surface-associated N-CAM, related to the culture protein content, remained stable during the two-week period under study, as demonstrated by 125I-protein A binding assays. Immunogold labeling experiments, both in transmission and scanning electron microscopy, indicated a dramatic decrease in N-CAM site density in each membrane compartment, perikarya and neurites. This temporal variation of N-CAM distribution was not accompanied by differences in N-CAM site density between these two membrane compartments. On the other hand, individual perikarya, observed in scanning electron microscopy, showed various levels of labeling. In addition, immunoblot experiments demonstrated the absence of chemical modulation of N-CAM during the period under study, since the high molecular weight (embryonic) form remained dominant. Moreover, an increase in the total N-CAM amount was detected, contrasting with the stable quantity of cell surface-associated N-CAM. This suggested the existence of an N-CAM intracellular pool in cultured neurons. Finally, since the neurite membrane surface area increased 9-fold (companion paper) and since only a 5-fold decrease in N-CAM site density was observed in this compartment, N-CAM supply to neurite membranes was postulated.

Ultrastructural localization of voltage-sensitive sodium channels using [125I]alpha scorpion toxin.

The distribution of alpha scorpion toxin (alpha-ScTx) receptors was examined in differentiated mouse neuroblastoma cell cultures (N IE 115 clone) by electron microscope autoradiography using [125I]alpha-ScTx. This neurotoxin binds specifically to voltage-sensitive sodium channels, slowing down the inactivation of the sodium permeability. Quantitative analysis demonstrated that only plasma membranes were labelled. The alpha-ScTx receptors seemed to be randomly dispersed on both cell bodies and cell processes. Microvilli protruding from the cell bodies carried more sodium channels than other parts of the membrane. The specific binding site density for alpha-ScTx varied from 4 (cell body membrane) to 13 (cell process membrane) per square micrometer.

Early appearance of cells bearing Na+ channels in developing mouse brain. A quantitative analysis using light microscopic autoradiography.

125I-alpha-Scorpion toxin (alpha-ScTx) binds to a component of the voltage-sensitive Na+ channel. We have previously shown that receptor capacity on dissociated mouse brain cells increases between days 12 and 19 of fetal life as does the expression of neurotoxin-sensitive 22Na+ influx. In the present study we have investigated the distribution of Na+ channels at the cellular level. Quantitative analysis by light-microscopic autoradiography was carried out on dissociated brain cells labeled with 125I-alpha-ScTx at 13, 15 and 18 fetal days. We have shown that at day 13 a large population of cells (39% of total) is alpha-ScTx-labeled, providing direct confirmation for a wide-spread presence of Na+ channels at an early stage of mouse brain development. The subsequent increase in receptor number with age is due both to an increase in alpha-ScTx-labeled cells (to 53% and 97% at days 15 and 18, respectively) and to an increase in the receptor density on these cells (10.9, 12.7 and 34.5 silver grains/1000 microns2 of cell surface for the 3 stages studied).

Ultrastructural visualization of Na+-channel associated [125I]alpha-scorpion toxin binding sites on fetal mouse nerve cells in culture.

Purified neurotoxin II from the scorpion Androctonus australis Hector (alpha-ScTx) has previously been shown to bind specifically to the voltage-sensitive Na+ channels of excitable cells. Recent studies, using high specific activity 125I-labeled alpha-ScTx, demonstrated specific binding to neuronal cells derived from fetal mouse brains. In the present study, 125I-labeled alpha-ScTx was used to localize the voltage-sensitive Na+ channels in cultured fetal mouse brain cells. By quantitative electron microscope autoradiography we demonstrate that specific alpha-ScTx binding sites are selectively located at the plasma membrane. Estimates of their density revealed that neurites at 13 days in vitro carry at least 6 X more specific alpha-ScTx sites than cell body membrane.

mRNA coding for voltage-gated sodium channel beta2 subunit in rat central nervous system: cellular distribution and changes following kainate-induced seizures.

The cellular distribution of sodium channel beta2 subunit mRNA was examined in the central nervous system from adult Wistar rats using a non-radioactive in situ hybridization method with digoxigenin-labeled cRNA probes. The expression of the subunit was strong in cerebral and cerebellar cortex, in medulla oblongata and in the spinal cord whereas heterogeneous in hippocampus. The distribution was evaluated in hippocampus and cerebral cortex from 1 to 72 h after kainate injection and compared to control rats using densitometric analysis. In these areas, a transient increase was seen 1 h after the drug administration, followed, in the hippocampus, by a significant decrease. These variations differ from those we previously reported for alpha subunits and might play a role in cellular excitability changes occurring in the course of seizures.

Voltage-sensitive Na+ channels in the neurohypophysis of the rat as demonstrated by 125I-labelled scorpion toxin.

Fresh rat neural lobe slices were incubated in the presence of [125I] alpha-scorpion toxin (ScTX), a specific marker of Na+ channels. Quantitative electron microscope autoradiography revealed preferential, irregularly spaced labeling of the axolemma of neurosecretory axons, with a significantly higher crude specific activity than any other neuronal or non-neuronal compartment. The number of specific binding sites at the neural lobe surface was calculated to be about 23 per microns2 of axolemma.

[Severe bronchial stenosis with upstream bronchiectasis in an arc welder: causal relation or epiphenomenon?]. / Sténose bronchique serrée avec bronchectasies d'amont chez un soudeur à l'arc: relation causale ou épiphénomène?

This case concerns an arc welder who presented suppurative bronchiectasis and episodes of purulent left side pleurisy in relation to cystic bronchiectasis of the left lower lobe and a very severe stenosis at the origin of the main left bronchus. The medicolegal problem was to assess the causal relationship between these lesions and occupational exposure. They do not come under the heading of table 44 of the General List and we made this the aim of discretionary award in front of a regional committee of compensation for occupational disease.

[An experimental study on the systemic distribution of calibrated talc after intra-pleural injection]. / Etude expérimentale de la distribution systémique de talc calibré après injection intrapleurale.

INTRODUCTION: Among the agents used to produce pleural symphysis talc is the most effective and least expensive. However, its use is controversial on account of the description of respiratory complications associated with subsequent systemic spread of the talc particles. This hypothesis rests on clinical and experimental observations of talc particles in the viscera. However, all talc preparations are not identical and this extra-pleural spread could be dependent on particle size. This experimental study was undertaken to determine whether there was systemic spread of a calibrated talc preparation used routinely in clinical practice following intra-pleural administration in rats. METHODS: 48 rats received 20 mg (11 rats) and 40 mg (33 rats) of calibrated talc suspended in 1 ml of physiological saline by intra-pleural injection. The animals were randomised for sacrifice at 24 hours (22 rats) and 72 hours (22 rats) after the injection. The lungs, parietal pleura, diaphragm, liver, spleen, pericardium, brain and blood were examined by light microscopy and polarised light to search for bi-refringent particles. RESULTS: No deaths occurred during the procedure. At the time of sacrifice no pleural symphysis was seen. In 5 animals some talc particles were seen in the extra-thoracic organs: in the liver in 3 in the spleen in 1 and one particle in the brain of one animal examined by electron microscopy. No talc particles were found in the blood. CONCLUSIONS: Intra-pleural injection of calibrated talc, (Steritalc-Novatech-Plan de Grasse-France) has a weak systemic spread in > small animals. These results may be related to the diameter of the talc particles used (mean 33.6 microns; median 31.3 microns). The hypothesis that systemic spread is influenced by the diameter of the talc particles needs to be supported by experimental studies using talc particles of smaller diameter in order to compare the systemic distribution of the different preparations.

[Are we sectioning the cochlear efferent system during vestibular neurotomy?]. / Sectionne-t-on le système efférent cochléaire au cours de la neurotomie vestibulaire?

INTRODUCTION: In addition to sensory neurons which transmit information from the inner ear to the brain, there is a system of efferent feedback fibers, called the olivocochlear system, carrying signals from the brain to the ear. Over the past half-century, the efferent system has been extensively studied in animals and results provided theories as to the functional significance of these efferents: to improve signal-to-noise ratio in the auditory periphery, to mediate selective attention, and to protect the inner ear from acoustic overexposure. The results of several studies conducted in man rely on the study of patients who have undergone a vestibular neurectomy. Indeed, anatomical data show that olivocochlear efferents could travel along or inside the vestibular part of the auditory nerve before reaching the organ of Corti. Therefore, these patients may be considered as an experimental model of unilaterally de-efferented subjects. However, to date, none has reported the existence of olivocohlear efferents in the vestibular section following neurectomy. MATERIALS AND RESULTS: In this study, we present the histological results from 18 vestibular sections and show the absence of olivocochlear efferents. CONCLUSION: These results provide a reason to reconsider the results of previous experiments conducted in similar patients and ask for further studies on the olivocochlear efferents pathways.

Immunolabelling and flow cytometry as new tools to explore dysferlinopathies.

Dysferlinopathies are autosomal recessive muscular dystrophies caused by DYSF mutations, which lead to a reduced amount or a complete lack of dysferlin. One step in dysferlinopathies diagnosis consists in Western blot analysis of proteins extracted from muscle biopsy, or blood monocytes. We have taken advantage of dysferlin expression in monocytes to develop a whole blood flow cytometry (WBFC), using antibodies directed against dysferlin. Six patients were submitted to WBFC analysis and immunofluorescence analysis on monocytes. Results obtained are correlated to Western blot from monocytes and muscle biopsies. The possible usefulness of this flow cytometry analysis in routine diagnosis is presented.