Cd, Pb and Zn oral bioaccessibility of urban soils contaminated in the past by atmospheric emissions from two lead and zinc smelters.

Ingestion of dust or soil particles could pose a potential health risk due to long-term metal trace element (MTE) exposure. Twenty-seven urban topsoil samples (kitchen garden and lawn) were collected and analyzed for Cd, Pb and Zn using the unified Bioaccessibility Research Group of Europe (BARGE) method (UBM) test to estimate the human bioaccessibility of these elements. The quantities of Cd, Pb and Zn extracted from soils indicated, on average, 68, 62 and 47% bioaccessibility, respectively, in the gastric phase and 31, 32 and 23% bioaccessibility, respectively, in the gastro-intestinal phase. Significant positive correlations were observed between concentrations extracted with UBM and total MTE contents. Stepwise multiple regression analysis showed that human bioaccessibility was also affected by some physico-chemical soil parameters (i.e. total nitrogen, carbonates, clay contents and pH). The unified test presents some valuable data for risk assessment. Indeed, the incorporation of oral bioaccessible concentrations into risk estimations could give more realistic information for health risk assessment.

[Potassium channels, genetic and acquired diseases]. / Canaux potassiques, maladies héréditaires et acquises.

INTRODUCTION: K(+) channels allow the passive and selective transport of K(+) ions through the membranes. They control K(+) homeostasis, neuronal and muscular excitabilities, and neurotransmitter and hormone release. EXEGESIS: K(+) channels are composed of pore-forming subunits associated with regulatory subunits. Many different K(+) channels have been identified. CONCLUSION: This diversity is stressed by the growing number of genetic and acquired diseases associated with these channels.

Long term treatment with the somatostatin analog SMS 201-995 in a patient with a thyrotropin- and growth hormone-secreting pituitary adenoma.

A patient with a mixed pituitary tumor secreting TSH and GH was treated, starting 3 months after partial adenomectomy, with the somatostatin analog SMS 201-995 for 8 months. Somatostatin itself inhibited TSH, GH, and alpha-subunit release by the tumor both in vivo and in vitro. Long term treatment with twice daily sc injections of SMS 201-995 resulted in decreased TSH secretion and lower serum thyroid hormone levels. However, euthyroidism was achieved only when the patient was treated with three daily 200-micrograms injections of SMS 201-995. After 30 weeks of SMS 201-995 therapy, TSH secretion increased, while GH secretion remained suppressed. After withdrawal for 6 months, SMS 201-995 (100 micrograms, sc, twice daily) again completely inhibited TSH secretion. SMS 201-995 did not alter the volume of the residual adenomatous tissue. We conclude that SMS 201-995 may be a valuable therapeutic agent for the management of patients with a thyrotroph adenoma. However, desensitization may occur during long term treatment.

Fine structural studies of growth-hormone-releasing-factor (GRF)-immunoreactive neurons and their synaptic connections in the guinea pig arcuate nucleus.

The fine structure of neurons containing human growth-hormone-releasing factor (hGRF) immunoreactivity located in the arcuate nucleus of the guinea pig was studied by means of the preembedding immunohistochemical technique. The perikaryon of labeled neurons was fusiform or ovoid; the nucleus was regular in shape and contained a prominent nucleolus. The main ultrastructural features of the hGRF-immunoreactive neurons were the presence of numerous labeled secretory granules (100-120 nm in diameter) and the abundance and the enlargement of the organelles involved in the synthesis of the peptides: a well-developed rough endoplasmic reticulum and a conspicuous Golgi apparatus. Synaptic inputs were observed on immunoreactive perikarya but, above all, on the labeled dendrites. The unstained presynaptic nerve endings most often contained only small clear vesicles and formed symmetrical contacts. In rare cases, the presynaptic terminals exhibited both small clear and large dense vesicles and constituted asymmetrical contacts. Immunoreactive nerve endings were also observed in this area: the synaptic boutons contained large, stained vesicles and small, unlabeled, clear vesicles. These axon terminals made synaptic contacts with unstained dendritic processes; the contacts were symmetrical. The results indicate that hGRF-immunoreactive neurons of the guinea pig arcuate nucleus present morphological features of neuroendocrine cells. Moreover, the presence of hGRF-labeled nerve endings in the arcuate nucleus itself suggests that a substance related to hGRF might be a neuromodulator, at least in this area.

GABA and enkephalin in the lateral septum of the guinea pig: light and electron microscopic evidence for interrelations.

Tract tracing techniques combined with immunohistochemistry in rats and guinea pigs have demonstrated the existence of a hypothalamo-lateral septum enkephalinergic pathway. Numerous enkephalinergic nerve endings encompass cell bodies located in the lateral septum. The present immunocytochemical study, at light and electron microscopic levels, was undertaken in the guinea pig brain to determine whether the contacted perikarya contain gamma-aminobutyric acid (GABA). The antisera against GABA revealed the presence of immunoreactive cell bodies throughout the lateral septum. At the light microscopic level, most GABA neurons appeared round while others were oval with one or two emerging dendrites. Ultrastructurally, cell bodies displayed a moderate number of organelles and a pale nucleus with frequent indentations of the nuclear envelope. The precise relationship between GABA neurons and enkephalinergic terminals was examined by means of a double-immunostaining method showing that 60% of cell bodies receiving synaptic inputs from enkephalinergic afferents contained GABA. These results show that the hypothalamo-septal enkephalinergic pathway prominently innervates GABA-containing neurons and also provide anatomical basis suggesting a disinhibitory role for this enkephalinergic tract.

Affinity of bronchial secretion glycoproteins and cells of human bronchial mucosa for Ricinus communis lectins.

The coupling of Ricinus communis lectins to Sephadex G 25 was used in order to study mucins and other glycoproteins from human bronchial secretion. The major part of human bronchial mucins and other glycoproteins such as immunoglobulins A, bronchotransferrin and alpha1-antichymotrypsin were isolated by this procedure. A parallel study of human bronchial mucosa was achieved with peroxidase labeled Ricinus communis lectins; this study characterized goblet cells and mucous cells which contain mucins, and serous cells which are involved in the synthesis or the secretion of the other glycoproteins.

Effects of inhibition of GABA catabolism by aminooxyacetic acid on enkephalinergic neurons--immunocytochemical and ultrastructural study in the enkephalinergic hypothalamoseptal tract of the guinea-pig.

The immunocytochemical and ultrastructural features within [Met]enkephalin neurons of the guinea-pig hypothalamoseptal tract were investigated under chronic inhibition of GABAergic catabolism. This was achieved by raising the brain GABA concentration with aminooxyacetic acid which inhibits GABA-transaminase, the enzyme responsible for the catabolism of GABA. Guinea-pigs were injected intraperitoneally with 10 or 20 mg/kg per day aminooxyacetic acid for two, four or eight days and killed 16 h post-dose. Repeated injections of aminooxyacetic acid produced a great increase in immunoreactivity for GABA in nerve endings surrounding enkephalinergic perikarya in the magnocellular dorsal nucleus of the guinea-pig. Extensive immunocytochemical studies stressed the increase and redistribution of the immunoreaction for [Met]enkephalin in the perikarya of the magnocellular dorsal nucleus under such GABAergic activation. Quantitative and statistical analyses showed that administration of aminooxyacetic acid for eight days significantly increased the intensity of labelling within stimulated perikarya (P less than 0.001). A concomitant accumulation of immunopositive large granules in the enkephalinergic boutons of the lateral septum was observed. In the same way, ultrastructural changes in enkephalinergic cell bodies were analysed and reflected disturbances in the biosynthetic and digestive activities of enkephalinergic perikarya. We postulate that chronic inhibition of the GABAergic catabolism leads to modification in the metabolism of enkephalinergic neurons and to an inhibitory action of GABA on the [Met]enkephalin release from nerve endings. This study give morphological support to the complex functional interactions between GABA and opioid peptide transmitter system.

Localization of mu opioid receptors on the membranes of nerve endings and tanycytes in the guinea-pig median eminence by electron microscopic radioautography.

The high density of opioid-containing nerve endings in the median eminence together with the absence of direct effects of opioids upon pituitary suggest a local action of opioids in the median eminence. The aim of this work was to address the occurrence of mu-opioid binding sites in the median eminence at the electron microscopic level, using the highly selective radioligand [125I]FK 33-824. mu-Opioid receptors were labeled in vitro on slightly prefixed slices of mediobasal hypothalamus. The labeling was essentially detected in the external part of the median eminence. Most of the silver grains overlaid membrane appositions. Two overall types of appositions were concerned: nerve terminal-nerve terminal or nerve terminal-tanycyte. Detailed analysis of the silver grain distribution indicated that mu receptors were observed on membranes of different types of nerve endings but also of tanycytes. All the binding sites were localized out of synaptic junctions since the median eminence is totally devoid of these structures. Our results suggest that in the median eminence, opioid peptides have a paracrine and/or autocrine action occurring at least via mu receptors located on nerve terminals but also on tanycytes.

Combination of immunocytochemistry and in situ hybridization in the same semi-thin sections: detection of Met-enkephalin and pro-enkephalin mRNA in the hypothalamic magnocellular dorsal nucleus of the guinea pig.

We describe a procedure for combining pre-embedding peroxidase immunocytochemistry with pre-embedding autoradiographic in situ hybridization in the same vibratome sections of paraformaldehyde-fixed brain tissue. The simultaneous detection of Met-enkephalin (Met-enk)-immunoreactive product and pro-enkephalin (PE) mRNA in neurons of the magnocellular dorsal nucleus (MDN) in the guinea pig hypothalamus was carried out as a model for this procedure. Vibratome slices were processed for Met-enk immunodetection followed by the incubation with a 45-base synthetic oligonucleotide complementary to PE mRNA labeled with 35S. Tissues were embedded in araldite, cut into semi-thin sections, and processed for autoradiography. Many neurons double labeled for Met-enk and PE mRNA were viewed in the MDN. The histological quality and the spatial resolution of both signals were optimized, since precise intracellular localization of hybridization sites was possible. This method allows simultaneous study of peptide immunoreactivity and mRNA expression levels in neurons within the same semi-thin sections. It may be useful for a variety of quantitative analyses, and might also be extended to ultrastructural analysis.

Use of lectins for detection of glycoconjugates in the glandular cells of the human bronchial mucosa.

Paraffin-embedded human bronchial biopsies, obtained in macroscopically healthy areas, were examined using nine peroxidase-bound lectins. These were either isolated or purified by affinity column chromatography (Ulex europeus, Triticum vulgare, Glycine max, and Arachis hypogatea lectins) or commercial preparations (Lotus tetragonolobus, Canavalia ensiformis, Helix pomatia, Phaseolus vulgaris, and Lens culinaris lectins) and conjugated to peroxidase (except for concanavalin A). These labeled lectins were used as specific molecular probes to localize differences in carbohydrate-containing components present in the different types of glandular cells of human bronchial mucosa. The choice of fixative was crucial and tests used for this study have shown that Carnoy's solution seems to be the most appropriate solution to preserve mucous glycoproteins in situ. Comparison of the affinity of several lectins for human bronchial glycoproteins and for bronchial mucosa demonstrates the predominance of serum-type glycoproteins in serous cells of the submucosal glands and mucin-type glycoproteins in mucous cells of the submucosal glands and in goblet cells of the bronchial epithelium. Furthermore the data obtained with some lectins, such as Helix pomatia agglutinin, suggest that there are some differences in the mucins synthesized by goblet cells and by mucous cells.

Limulus polyphemus lectin sites in human bronchial mucosa.

The lectin from Limulus polyphemus (limulin) has an affinity for glycoproteins containing N-glycolyl or N-acetylneuraminic residues. By using a peroxidase labeled limulin on human bronchial mucosa sections, it has been possible to demonstrate selectively a diffuse staining within the apical part of the epithelial goblet cells. Whereas, in the submucosal glands serous cells, the labeling appears as small granules localized either at the apical pole or in the whole cytoplasm. In our experimental conditions the labeled limulin has no affinity for mucous cells that are known to contain sialic acid residues.

Catecholamine innervation of enkephalinergic neurons in guinea pig hypothalamus: demonstration by an in vitro autoradiographic technique combined with a post-embedding immunogold method.

To study the relationship between the catecholamine (CA) nerve endings and the enkephalinergic cell bodies in the magnocellular dorsal nucleus (MDN) of guinea pig hypothalamus, double-labeling experiments were performed on the same tissue section at the electron microscopic level. An in vitro autoradiographic (ARG) method for [3H]-norepinephrine (NE) or [3H]-dopamine (DA) was combined with a post-embedding immunogold cytochemical technique for Met-enkephalin (Met-enk) in colchicine-treated animals. Hypothalamic slices (450 micrograms) were perfused with [3H]-NE or [3H]-DA at the fluid-gas interface, then fixed by immersion with glutaraldehyde and osmic acid. Semi-thin sections processed from the thickness of the slices showed adequate penetration of the tracers to all parts of the tissue. Frontal sections permitted visualization of some CA-uptake structures distributed around the cells. At the ultrastructural level, preservation appeared good on about 60% of the thickness of slices, and [3H]-CA structures were easily distinguished. Ultra-thin sections were successively incubated with Met-enk and colloidal gold-labeled antisera, followed by ARG processing. At the electron microscopic level, the good integrity of the tissue made possible visualization of [3H]-CA nerve terminals making synaptic contacts with enkephalinergic perikarya. These results provide morphological evidence for direct catecholaminergic control of enkephalinergic neurons of the MDN.

Use of an antiserum against deglycosylated human mucins for cellular localization of their peptide precursors: antigenic similarities between bronchial and intestinal mucins.

Highly glycosylated regions of mucins, or glycopeptides, were obtained by proteolysis of human bronchial mucins. They were deglycosylated by treatment with a trifluoromethane sulfonic acid/anisole mixture and subsequent solvolysis with anhydrous liquid hydrogen fluoride. The resulting peptides were then used to raise an immune serum in rabbit. This immune serum was used to localize the peptide precursors of human respiratory mucins within bronchial cells, using an immunohistochemical method. Two main patterns of labeling were observed in the goblet cells: the entire cytoplasm of some goblet cells was immunoreactive, whereas in other cells the labeling was concentrated around the nucleus. In the respiratory mucous glands, the labeling was localized around or below the nucleus. The serous cells were not stained. Similar labeling was observed in human colon goblet cells. This immune serum seems to be specific for mucin-secreting cells and has a strong affinity for the perinuclear region of these cells.

Plasticity in expression of immunoreactivity for neuropeptide Y, enkephalins and neurotensin in the hypothalamic tubero-infundibular dopaminergic system during lactation in mice.

In lactating nursing vs lactating pup-deprived mice, single or multiple immunolabeling was performed to compare immunoreactivities (ir) for neuropeptide Y (NPY), enkephalins (ENK) and neurotensin (NT) in the tyrosine hydroxylase (TH)-ir (ENK) and neurotensin (NT) in the tyrosine hydroxylase (TH)-ir hypothalamic tubero-infundibular dopaminergic (TIDA) system. NPY-, ENK- and NT-irs were intensely expressed and coexisted in virtually all TH-ir endings in the median eminence (ME) of nursing mice. Removal of the pups induced a marked depletion of the peptide-irs from the ME TH-ir endings. In the arcuate nucleus (ARC) of colchicine-treated nursing mice which received peripheral injections of Fluoro-Gold (FG) to retrogradely label neuroendocrine cells, virtually all dorsal A12 TH-ir perikarya simultaneously expressed, with individual variations, NPY-, ENK- and NT-irs, and all contained FG. These results suggest that the synthesis of NPY, ENK and NT is enhanced in TIDA neurons during lactation and that these neuromessengers may be co-released together with DA from the ME to regulate the suckling-induced prolactin secretion at the hypothalamic and/or pituitary levels.

Early transient expression of somatostatin (SRIF) immunoreactivity in dorsal root ganglia during ontogenesis in the rat.

Immunofluorescence was used in the rat to study the early ontogenetic expression of somatostatin (SRIF) in the dorsal root ganglia (DRGs) from gestational day 10.5 to day 15.5. SRIF-immunoreactivity (IR) was not detectable in day-10.5 embryos, was first observed in DRGs at day 11.5, reached a peak in intensity and distribution at around day 13.5 and thereafter decreased to become undetectable by day 15.5 in the DRGs of the trunk region. The dynamic expression of SRIF-IR in DRG perikarya could be correlated with its expression in nerve fibers located in the limbs and the abdominal mesenchyme. Thus, SRIF-IR is expressed at a time when sensory fibers could have established connections with their embryonic targets and when DRG neurons could have undergone their final mitotic phase. These data showing the earliest and transient expression of a neuropeptide in developing DRGs confirm and extend the notion that SRIF plays an important role in developmental processes.

GABA axon terminals in synaptic contact with enkephalin neurons in the hypothalamus of the guinea pig. Demonstration by double immunocytochemistry.

By using a method combining pre-embedding immunoperoxidase staining for enkephalin and postembedding immunocolloidal gold labeling for gamma-aminobutyric acid (GABA) it has been demonstrate that many GABAergic boutons made synapses on enkephalin-reacting soma in the magnocellular dorsal nucleus of the guinea pig hypothalamus. The gold particles revealing the presence of GABA were essentially located over the small clear vesicles and mitochondria present in these GABAergic nerve endings. All the synapses observed were symmetrical. Taking into account the great number of these nerve endings, we conclude for a strong regulatory role of GABA on enkephalin-containing cells of the magnocellular dorsal nucleus.

Dystrophic peptidergic neurites in senile plaques of Alzheimer's disease hippocampus precede formation of paired helical filaments.

The relationship between peptidergic dystrophic neurites and paired helical filament (PHF)-positive neurites in Alzheimer's disease (AD) senile plaques (SPs) was studied using combined fluorescence and bright-field optics. Cryostat sections of AD hippocampi were first stained with thioflavine-S and immunolabelled with antisera raised against different neuropeptides: somatostatin-28(1-12), somatostatin-14, neuropeptide Y, cholecystokinin (CCK) and substance P. Secondly, using the elution-restaining procedure, sections were immunolabelled with anti-tau/PHF. In immature SPs, clusters of abnormal, swollen neurites were found. The dystrophic, strongly peptidic-positive neurites contained fewer PHFs than the poorly positive ones. Cell bodies, exhibiting a peptidic content, could be found within SPs without any alteration. These results suggest the following sequence of events: an extracellular poisoning mechanism, perhaps the amyloid substance, first changes the structure of presynaptic endings and causes the formation of ballooning dystrophic neurites filled with their normal peptidic content. Subsequently, intracellular degradation occurs with formation of the PHFs. Then the other structures such as dendrites and perikarya are damaged by the same mechanism. Therefore, this phenomenon seems to precede any formation of PHFs in SPs.

The fetal expression of proenkephalin mRNAs and Met-enkephalin immunoreactivity in the hypothalamoseptal tract and adjacent hypothalamic areas of the guinea pig brain.

The development of the enkephalinergic hypothalamoseptal tract in the guinea pig brain was studied from embryonic day 30 until birth. Proenkephalin (PE) mRNAs were detected in the hypothalamic magnocellular dorsal nucleus (MDN) by in situ hybridization with a synthetic 35S-labeled oligonucleotide. The Met-enkephalin-like immunoreactivity (Met-enk-LI) in the MDN and the lateral septum (LS) was detected with antibodies against Met-enkephalin, on adjacent cryostat sections. At the same time, an immunohistochemical study of the arrangement of enkephalinergic axon terminals in the LS at birth was performed at the electron microscopic level. PE mRNAs were first found to be expressed in the MDN at embryonic day 32 (E32) and increased to reach a maximal level at E48. Met-enk-LI was consistently detectable from E38 in numerous perikarya of the MDN as well as in nerve terminals of the LS. The number of Met-enk-LI cells of the MDN decreased after this stage until birth, whereas positive nerve endings in the LS increased. At the electron microscopic level, numerous cell bodies of the LS at birth were consistently surrounded by Met-enk immunoreactive nerve terminals. Cells expressing the PE gene and Met-enk-LI were also observed from E38 to E44 in the periventricular area. Some of these cells were found double-labeled with Met-enkephalin and Somatostatin antisera. The enkephalinergic system of the hypothalamoseptal tract appears at early embryonic stages and may be essential in regulating septal neuronal functions early in gestation. Differing ontogenic onsets of the enkephalinergic hypothalamoseptal and periventricular-median eminence tracts suggest possible developmental and functional differences.

Immunocytochemical and ultrastructural identification of luteinizing hormone-releasing hormone (LH-RH)-containing neurons in the vascular organ of the lamina terminalis (OVLT) of the squirrel monkey.

By comparison of immunocytochemical data and classical electron microscopic results, luteinizing hormone-releasing hormone (LH-RH)-immunoreactive neurons are described in the vascular organ of the lamina terminalis (OVLT) of the squirrel monkey. These cells contain neurosecretory granules (NSG) essentially located at their periphery. The rough endoplasmic reticulum and the Golgi apparatus are well developed. Some NSG are also associated with the Golgi complex. Nerve endings containing NSG are observed at the surface of the ependymocytes.

GABAergic afferents modulate proenkephalin mRNA expression in the guinea pig hypothalamic magnocellular dorsal nucleus.

The proenkephalin (PE) gene expression within Met-enkephalin (ME) neurons of the guinea pig magnocellular dorsal nucleus (MDN) was measured following activation of GABAergic transmission by aminooxyacetic acid treatment (AOAA). A combination of preembedding ME immunocytochemistry and PE in situ hybridization was used to investigate PE mRNA and enkephalin immunoreactivity levels. In perikarya of the MDN, chronic AOAA-treatment resulted in an important decline in PE mRNA signal (-128%). In double-labeled neurons, the PE mRNA decrease was associated to an increase in ME immunoreactivity. These results indicate that the increase in ME immunoreactivity in the MDN following AOAA-treatment is not due to a rise in PE gene transcription. This study provides additional morphological evidence supporting a role for GABA in modulating the enkephalinergic hypothalamoseptal tract of the guinea pig.