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In malaria, mosquito saliva and salivary glands play central roles in the multi-faceted interactions that occur among the parasite, its vector, and its host. Analyzing the processes involved in the survival and maintenance of the Plasmodium parasite in mosquito organs, and in its transmission into vertebrate hosts, may lead to the identification of new molecular targets for parasite control. We used comparative two-dimensional gel polyacrylamide electrophoresis (2D-PAGE), surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS), and high-performance liquid chromatography (HPLC), followed by Edman sequencing, to study saliva and salivary gland samples from Anopheles gambiae mosquitoes infected or not with Plasmodium berghei. Quantitative 2D-PAGE profile analysis showed that the intensities of seven spots were affected by the presence of the parasite in the salivary glands. Most of the proteins identified possessed a signal peptide. SELDI-TOF-MS revealed 32 proteins/peptides whose peak intensities differed between the Plasmodium-infected and non-infected control groups. Quantitative comparison of HPLC profiles of low-molecular-weight components from salivary gland extracts revealed several peptides and proteins with levels that were modulated by parasite infection. The results of these complementary approaches suggest that the infection of female A. gambiae mosquitoes by P. berghei alters the production levels of several salivary gland proteins and peptides, some of which (e.g., protein cE5, B3VDI9_ANOGA, and AGAP008216-PA) are known or predicted to be secreted in saliva and involved in blood feeding.

Several compositions of Th2-x/2AnIVx/2(PO4)2(HPO4).H2O (An=U, Np, Pu) were prepared through hydrothermal precipitation from a mixture of nitric solutions containing cations and concentrated phosphoric acid. All the samples were fully characterized by X-ray diffraction, UV-vis, and infrared spectroscopies to check for the existence of thorium-actinide(IV) phosphate hydrogenphosphate hydrates solid solutions. Such compounds were obtained as single phases, up to x=4 for uranium, x=2 for neptunium, and x<4 for plutonium, the cations being fully maintained in the tetravalent oxidation state. In a second step, the samples obtained after heating crystallized precursors at high temperature (1100 degrees C) were characterized. Single-phase thorium-actinide(IV) phosphate-diphosphate solid solutions were obtained up to x=0.8 for Np(IV) and x=1.6 for Pu(IV). For higher substitution rates, polyphase systems composed by beta-TAnPD, An2O(PO4)2, and/or alpha-AnP2O7 were formed. Finally, this hydrothermal route of preparation was applied successfully to the synthesis of an original phosphate-based compound incorporating simultaneously tetravalent uranium, neptunium and plutonium.

Purpose of the Study: In a previous anatomic study of healthy knees, we observed that femoral valgus cannot be attributed to hypoplasia of the lateral femoral condyle. In the present study, in an attempt to determine the site of the femoral deformation, we examined femoral shaft bowing in the frontal plan., Material and Methods: This cadaver study included 41 lower limbs of healthy Caucasian subjects aged over 65 years. The following anatomic landmarks were identified: center of the femoral head (H), center of the intercondylar notch (K), center of the talar dome (A), center of the femoral shaft half way between the apex of the greater trochanter and the middle of the intercondylar notch (S), the tangent line of the femoral condyles (I). Three angles were analyzed: HKA, HKI and SKI. There were 23 normal knees (HKA = 179.1 +/- 1.6 degrees) and 18 valgus knees (HKA = 182.7 +/- 0.8 degrees). Skeletal analysis (the skeleton of an object being defined as the median points of the object) was used to describe the morphology of the femoral shaft then to shape it with a second degree function (y = fx2 + bx + c). The protocol was repeated seven times to achieve accurate measurement. Accuracy was 1 degrees for the HKA angle and 0.45 degrees for the HKI and SKI angles. This accuracy was comparable to that reported in the literature. The Mann and Whitney U test was used to compare means. Spearman's t test was used to search for correlations. The first order risk was set at 0.05., Results: The HKI angle of valgus knees (95.5 +/- 1.1 degrees) was greater than for the normally aligned knees (93.6 +/- 2.4 degrees), confirming the femoral origin of the valgus. The form parameter f for the normal knees (1.33 10(-5) +/- 1.41 10(-5)) was greater than for the valgus knees (5.71 10(-6) +/- 5.27 10(-6)). There was a correlation between the form parameter f and the HKI angle for valgus knees, reflecting a relationship between frontal bowing of the femoral shaft and femoral valgus in this group., Discussion: The difference observed between the two groups of knees regarding the form parameter f and the correlation between f and the HKI angle in the valgus knees led us to consider that a considerable part of constitutional valgus knees can be attributed to the femoral shaft. Thus for equivalent anatomic valgus (SKI), minimal bowing (f) of the femoral shaft in valgus knees leads to greater mechanical valgus (HKI). These results confirm those obtained in our earlier study where we concluded that hypoplasia of the lateral femoral condyle does not contribute to constitutional valgus knees. We hypothesize that the same could be true for degenerative disease of constitutional valgus knees. For surgical cure, the origin of the misalignment in valgus knees dictates the rotation position of the femoral component of total knee arthroplasty and the lengthening technique for the lateral structures.

Mitogenic activity of bone marrow-derived mouse mast cells and mast cell lines P815 and MC/9 on B and T lymphocytes is present in their culture supernatants. To identify this activity, mast cells were incubated in serum-free medium and the supernatant was subjected to differential centrifugation, which resulted in two fractions, the hypodense and dense fraction (pellet). When analyzed for their mitogenic activity on spleen cells, all activity was found to be associated with the dense fraction. Electron microscopy studies revealed the presence in this fraction of small vesicles called exosomes with a heterogeneous size from 60 to 100 nm of diameter. When cocultured with spleen cells, purified exosomes induced blast formation, proliferation, as well as IL-2 and IFN-gamma production, but no detectable IL-4. Similar data were obtained by injecting exosomes into naive mice. In contrast to mast cell lines, a pretreatment with IL-4 is required for bone marrow-derived mast cells to secrete active exosomes. Structurally, exosomes were found to harbor immunologically relevant molecules such as MHC class II, CD86, LFA-1, and ICAM-1. These findings indicate that mast cells can represent a critical component of the immunoregulatory network through secreted exosomes that display mitogenic activity on B and T lymphocytes both in vitro and in vivo.

Bone marrow-derived mouse mast cells (BMMC) and mast cell lines P815 and MC9 have recently been shown to induce antigen-independent B and T lymphocyte activation. It has been demonstrated that a physical contact between mast cells and B and T lymphocytes is not necessary since mast cell supernatants contain full activity. Electron microscopy studies revealed the presence in mast cell supernatants of small vesicles called exosomes with a heterogeneous size from 60 to 100 nm of diameter. When cocultured with spleen cells, purified exosomes induce B and T cell blast formation, proliferation as well as IL-2 and IFN-gamma production. In contrast to P815 and MC9 mast cell lines, a pretreatment with IL-4 is required for BMMC to produce active exosomes. Structurally, these exosomes were found to harbor immunologically relevant molecules such as MHC class II, CD86, LFA-1 and ICAM-1. Here we provide for the first time the evidence that mast cells use exosomes as sophisticated messengers to communicate with cells of the immune system., (Copyright 2001 S. Karger AG, Basel)

5-Hydroxytryptamine-moduline is an endogenous cerebral tetrapeptide that regulates the activity of 5-hydroxytryptamine1B receptors. Direct binding of 5-[3H]hydroxytryptamine-moduline on rat brain homogenate evidenced the existence of two interacting sites for the peptide, very likely corresponding to different conformations of the 5-hydroxytryptamine1B receptor: The peptide first binds to a low-affinity state of the receptor (pIC50 = 7.68+/-0.14) and then induces (or stabilizes) a high-affinity complex (pIC50 = 11.62+/-0.18). This work focuses on the ability of 5-hydroxytryptamine-moduline analogues to recognize the high- and low-affinity sites for 5-hydroxytryptamine-moduline. The results obtained show that the two conformers of the 5-hydroxytryptamine1B receptor have similar but not identical binding pockets for 5-hydroxytryptamine-moduline. These two sites proved to be stereoselective and selective for tetrapeptides and favored the binding of peptides with hydrophobic amino acids in positions 1 and 4, serine in position 2, and a short amino acid in position 3. However, the serine in position 2 seems to be more important for the interaction of the peptide with the low-affinity site than the high-affinity one, which only needs a short hydrophobic amino acid in position 2.

Lithium ion is widely used to treat depressive patients, often as an initial helper for antidepressant drugs or as a mood stabilizer; however, the toxicity of the drug raises serious problems, because the toxic doses of lithium are quite close to the therapeutic ones. Thus, precise characterization of the target(s) involved in the therapeutic activity of lithium is of importance. The present work, carried out at molecular, cellular, and in vivo levels, demonstrates that 5-HT1B receptor constitutes a molecular target for lithium. Several reasons suggest that this interaction is more likely related to the therapeutic properties of lithium than to its undesirable effects. First, the observed biochemical and functional interaction occurs at concentrations that precisely correspond to effective therapeutic doses of lithium. Second, 5-HT1B receptors are well characterized as controlling the activity of the serotonergic system, which is known to be involved in affective disorders and the mechanism of action of various antidepressants. These findings represent progress in our knowledge of the mechanism of action of lithium that may facilitate clinical use of the ion and also open new directions in the research of antidepressant therapies.

This work was intended to determine which enzymatic activities from crude synaptosomal mammalian brain membranes could qualify for the status of 5-hydroxytryptamine-moduline (5-HT-moduline, LSAL, Leu-Ser-Ala-Leu) inactivating enzymes. An enzymatic assay for 5-HT-moduline metabolism was developed using [3H]5-HT-moduline measurement and high performance liquid chromatography (HPLC) technique to identify and quantify 5-HT-moduline metabolites. 5-HT-moduline metabolism displayed all characteristics of metalloprotease activity: sensitivity to divalent ion chelators, reactivation by Zn2+ ions and a pH optimum in the 7-8 range. Bestatin, an aminopeptidase inhibitor, allowed the identification of two enzymatic activities responsible for this metabolism: a bestatin-sensitive aminopeptidase and an endoprotease cleaving 5-HT-moduline into LS (Leu-Ser) and AL (Ala-Leu) dipeptides. This latter enzyme was shown to have a Km of 37.1 +/- 3.6 microM and a Vmax of 5.5 micromol min(-1) l(-1) per mg of protein. Moreover, this enzyme was insensitive to peptidyl dipeptidase A (angiotensin converting enzyme, EC 3.4.15.1), endothelin converting enzyme and neutral endopeptidase (neprylisin, EC 3.4.24.11) inhibitors and displayed some specificity among 5-HT-moduline-analogues and in particular recognized only tetrapeptides. These results, together with the isolation of the LS and AL metabolites [Rousselle, J.C., Massot, O., Delepierre, M., Zifa, E., Rousseau, B., Fillion, G., 1996. Isolation and characterization of an endogenous peptide from rat brain interacting specifically with the serotonergic 1B receptor subtypes. J. Biol. Chem. 271, 726-735] during the purification process of 5-HT-moduline are strong arguments for the physiological implication of this endoprotease in 5-HT-moduline metabolism.

The serotonergic transmission is considered as a neuromodulatory system in the Central Nervous System. 5-HT1B receptors play an important role in this modulatory activity. We have purified from mammalian brain an endogenous peptide, LSAL, we called 5-HT-moduline, interacting specifically with 5-HT1B receptors. This interaction is characterized by a high affinity (Ki = 10(-10) M) and a non-competitive mechanism. Direct [3H]5-HT-moduline binding revealed a single population of sites having an apparent affinity constant close to 10(-10) M. Autoradiographic studies showed a brain distribution of [3H]5-HT-moduline binding sites closely related to the 5-HT1B receptors. In functional studies, the peptide is able to reverse the activity of a 5-HT1B agonist in the nanomolar range. Furthermore, this antagonist effect is also observed in vivo on mice behavior. Immunocytochemistry revealed an heterogeneous distribution of 5-HT-moduline in mouse brain. The labeled structures correspond to cellular profiles with axon-like prolongations. Moreover, in vitro, LSAL is released in a Ca++, K(+)-dependent manner. Therefore, 5-HT-moduline behaves as a neurotransmitter. The fact that 5-HT-moduline induces the desensitization of 5-HT1B receptors reflects the existence of a novel and efficient mechanism able to rapidly modulate the serotonergic activity.

5-Hydroxytryptamine-moduline (5-HT-moduline) is an endogenous tetrapeptide (Leu-Ser-Ala-Leu) recently isolated and characterized from mammalian brain. This compound interacts with 5-HT1B receptors as a non-competitive, high-affinity antagonist and has the properties of an allosteric modulator. 5-HT-moduline could play an important role in the regulation of serotonergic transmission and also, through heteroreceptors, dopaminergic transmission. The aim of this work was to examine the potential ability of 5-HT-moduline to modify the basal extracellular concentration of dopamine and its metabolites (3-methoxytyramine, dihydroxyphenylacetic acid and homovanillic acid), in the rat striatum and to determine its potential interaction with the stimulating activity of a specific 5-HT1B receptor agonist, 3-(1,2,5,6-tetrahydropyrid-4-yl) pyrrolo [3,2-b] pyrid-5-one (CP-93,129), on the release of dopamine. The technique is based on in vivo microdialysis using probes implanted in the striatum of the conscious rat. Results showed that the perfusion of 5-HT-moduline directly into this structure (1.25 mM) increased the striatal level of dopamine by two-fold (104% of the absolute basal release values, P = 0.0015) and that of 3-methoxytyramine by 3-fold (293%, P = 0.0001) without any change in the terminal metabolite concentrations. The intrastriatal administration of CP-93,129 induced a statistically significant, dose-dependent increase of dopamine levels (P < 0.0001). Coperfusion of 5-HT-moduline did not significantly alter the effect of CP-93,129 at 0.1 and 0.5 mM, but appeared to have an additive effect on the lowest dose (P = 0.0406). The results obtained show that 5-HT-moduline directly administered into the striatum increases the release of dopamine in this area. Presumably, this effect results from the desensitization of 5-HT1B receptors located on dopamine terminals. However, the fact that a 5-HT1B receptor agonist (CP-93,129) also increased the release of dopamine in the striatum and that 5-HT-moduline exhibited a slight additive effect with that of a low concentration of CP-93,129 suggests that the two substances interact with different mechanisms.

Early Lyme borreliosis sera with significant titers of anti-outer surface protein C (OspC) borreliacidal antibodies were identified. Human anti-OspC borreliacidal antibodies could be either IgM or IgG. Significant concentrations of borreliacidal activity were detected after vaccination of mice with OspC. Detection of anti-OspC borreliacidal activity was dependent on surface expression of OspC by Borrelia burgdorferi isolate 50772. The ability of OspC to induce borreliacidal antibodies in vivo and after vaccination offers another possible explanation for the ability of vaccination with OspC to protect against infection with B. burgdorferi. Furthermore, detection of anti-OspC borreliacidal antibodies, especially IgM antibodies, in early Lyme borreliosis sera provides additional evidence that borreliacidal antibody detection may be useful for the serodiagnosis of early Lyme borreliosis.

5-HT1B receptors are the predominant auto- and heteroreceptors located on serotonergic and non-serotonergic terminals where they regulate the neuronal release of neurotransmitters. 5-HT-moduline (Leu-Ser-Ala-Leu) has been shown to specifically interact with a very high apparent affinity and in a non-competitive manner with 5-HT1B receptors (Massot et al. 1996; Rousselle et al. 1996). Using transfected cells expressing either 5-HT1B or 5-HT1D receptors, it was shown that 5-HT-moduline prevents the binding of [3H]5-HT to 5-HT1B as well as to 5-HT1D receptors with similar biochemical characteristics: the IC50 of the peptide was 1.2x10(-12) M for 5-HT1B and 9x10(-13) M for 5-HT1D receptors. The observed effect corresponds to a marked decrease of the maximal binding for [3H]5-HT on 5-HT1B (-51.2 +/- 1%) as well as 5-HT1D binding (-47.2 +/- 7.7% of the control binding) whereas the affinity of 5-HT is increased by a factor close to 3. No effect is observed using the "scrambled" peptide (Ala-Leu-Leu-Ser). Parallel assays using transfected cells expressing 5-HT1A or 5-ht6 receptors did not show any significant change induced by the peptide under similar assay conditions. The interaction of the peptide was also studied on the functional activity related to the stimulation of the receptors as measured by the increase in [35S]GTPgammaS binding reflecting the coupling of the receptor to the G-protein. 5-HT-moduline yields an antagonistic effect on the 5-HT induced coupling with a corresponding IC50 = 1.2 +/- 0.7x10(-12) M for 5-HT1B and 9.8 +/- 4.0x10(-12) M for 5-HT1D receptors, respectively. The present results demonstrate that 5-HT-moduline interacts with 5-HT1D as well as 5-HT1B receptors and possesses a non-competitive antagonistic activity, likely corresponding to its role of endogenous allosteric modulator, specific for both 5-HT1B and 5-HT1D receptors.

5-HT-moduline is a cerebral tetrapeptide [Leu-Ser-Ala-Leu] that was recently isolated from bovine brain tissue and shown to interact specifically with 5-HT1B receptors, particularly in rodents. The pharmacological properties of 5-HT1B receptors in rodents are different from those in other species. In order to better understand the role of this peptide in non-rodent species, we determined the distribution of 5-HT-moduline binding sites in guinea-pig brain using both the film autoradiography and digital autoradiography with a newly developed high resolution beta-imaging techniques. We found that 5-HT-moduline binding sites were expressed in various brain regions. Quantitative analysis showed that densities of binding sites were similar to those observed previously in rat brain. Regions with the highest labelling included cortex, septum, hippocampus and some regions of basal ganglia. Our results extend previous data and show that 5-HT-moduline interacts with the two forms of 5-HT1B receptors that are distinct pharmacologically. By this interaction, 5-HT-moduline may play an important role in regulating the functional activity of 5-HT1B receptors, thereby contributes to the pathophysiology of serotonergic transmission., (Copyright 1998 Elsevier Science B.V. All rights reserved.)

5-HT-moduline is an endogenous tetrapeptide [Leu-Ser-Ala-Leu (LSAL)] that was first isolated from bovine brain tissue. To understand the physiological role of this tetrapeptide, we studied the localization of 5-HT-moduline binding sites in rat and mouse brains. Quantitative data obtained with a gaseous detector of beta-particles (beta-imager) indicated that [3H]-5-HT-moduline bound specifically to rat brain sections with high affinity (Kd = 0.77 nM and Bmax = 0. 26 dpm/mm2). Using film autoradiography in parallel, we found that 5-HT-moduline binding sites were expressed in a variety of rat and mouse brain structures. In 5-HT1B receptor knock-out mice, the specific binding of [3H]-5-HT-moduline was not different from background labeling, indicating that 5-HT-moduline targets are exclusively located on the 5-HT1B receptors. Although the distribution of 5-HT-moduline binding sites was similar to that of 5-HT1B receptors, they did not overlap totally. Differences in distribution patterns were found in regions containing either high levels of 5-HT1B receptors such as globus pallidus and subiculum that were poorly labeled or in other regions such as dentate gyrus of hippocampus and cortex where the relative density of 5-HT-moduline binding sites was higher than that of 5-HT1B receptors. In conclusion, our data, based on autoradiographic localization, indicate that 5-HT-moduline targets are located on 5-HT1B receptors present both on 5-HT afferents and postsynaptic neurons. By interacting specifically with 5-HT1B receptors, this tetrapeptide may play a pivotal role in pathological states such as stress that involves the dysfunction of 5-HT neurotransmission.

The localization of 5-HT-moduline, an endogenous cerebral tetrapeptide (LSAL) which specifically interacts with 5-HT1B receptors (Rousselle et al., 1996; Massot et al., 1996) was examined in mouse brain using an immunocytochemistry technique with a polyclonal anti-peptide antibody highly specific for this tetrapeptide. Highest levels of 5-HT-moduline immunoreactivity were observed in the cerebral cortex including cingulate, retrosplenial, parietal and pyriform cortical areas and in the basal ganglia. Intense immunoreactivity also occurred in the hippocampus, subiculum, various hypothalamic and thalamic nuclei and in some midbrain regions such as the substantia nigra and the superior colliculi. Immunoreactive neurons generally showed intense and extensive labelling of the perikarya and dendritic arborizations with moderate to heavy characteristic deposits of reaction product along plasma membranes and within cytoplasm while the nuclei were devoid of reaction product. The results obtained indicated that 5-HT-moduline immunoreactivity was heterogenously distributed in neuronal structures of mouse brain. The distribution of 5-HT-moduline immunoreactivity closely correlated with that of 5-HT-moduline specific binding sites as visualized by autoradiography (Massot et al., 1996). Moreover, it seems to overlap with the distribution of serotonergic innervation and also with that of 5-HT1B receptors in mouse brain (Boschert et al., 1994; Bruinvels et al., 1994; Chopin et al., 1994; Langlois et al., 1995). These data provide evidence that 5-HT-moduline immunoreactivity is located in cells with the morphological appearance of neurones. Its localization in brain areas which also contain 5-HT1B receptors, is in good agreement with previous demonstrations that this peptide specifically interacts with 5-HT1B receptors to regulate their functional activity and accordingly controls the modulatory activity of the serotoninergic system on various CNS functions.

The serotonergic system controls the activity of neurotransmissions involved in numerous physiological functions. It is also thought to be crucially implicated in various pathologies, including psychiatric disorders such as depression, anxiety, and aggressiveness. The properties of 5-hydroxytryptamine (5-HT)-moduline, a novel endogenous peptide, have been tested in vitro and in vivo. Binding studies have shown that the peptide specifically interacts with 5-HT1B/1D receptors via a noncompetitive mechanism corresponding to a high apparent affinity (EC50 = 10(10) M). The interaction was shown in rat and guinea pig brain tissues and in cells transfected with either 5-HT1B or 5-HT1D beta receptor gene. [3H]5-HT-moduline binds to a single population of sites in mammalian brain (Kd = 0.4 nM in rat, Kd = 0.8 nM in guinea pig) as well as in transfected cells expressing the 5-HT1B or the 5-HT1D beta receptors (Kd = 0.2 and 0.6 nM, respectively). Furthermore, the binding is clearly specific of the LSAL sequence. Autoradiographic studies showed an heterogeneous brain distribution of this site. The interaction of 5-HT-moduline with the 5-HT1B/1D receptor corresponds to a decrease in the functional activity of the receptor (i.e., a decrease in the inhibitory effect of a 5-HT1B agonist on the evoked release of [3H]5-HT from synaptosomal preparation). It was also shown that 5-HT-moduline possess an in vivo effect in the social interaction test in mouse. Finally, it was demonstrated that 5-HT-moduline was released from brain synaptosomal preparation by a K+/Ca(2+)-dependent mechanism. In conclusion, 5-HT-moduline is a novel endogenous peptide regulating the serotonergic activity via a direct action at presynaptic 5-HT receptor. It may play an important role in the physiological mechanisms involving the serotonergic system, particularly in mechanisms corresponding to the elaboration of an appropriate response of the central nervous system to a given stimulus.

The existence of endogenous compounds interacting with the serotonergic system was previously postulated. In the present work, rat brain tissues were extracted by acidic and organic procedures. The resulting extract was tested for its capacity to interact with the binding of [3H]5-hydroxytryptamine ([3H]5-HT) to 5-HT1 receptors. Compounds responsible for the observed inhibitory activities were isolated and purified by high pressure liquid chromatography. A tetrapeptide corresponding to a novel amino acid sequence Leu-Ser-Ala-Leu (LSAL) was identified. It reduces the binding of [3H]5-HT to 5-HT1 receptors at low concentration (IC50 = 10(-10) M). This effect corresponds to a specific interaction at 5-HT1B receptors since LSAL does not significantly affect other neurotransmitter bindings. LSAL appears heterogeneously distributed throughout the brain (hippocampus > cerebellum > striatum > brain stem) and in peripheral tissues (kidney > lung > stomach > blood > liver > spleen). Two other peptides, Leu-Ser (LS) and Ala-Leu (AL), were also purified. They hardly affected [3H]5-HT binding compared with LSAL. They presumably represent degradation products of the functional peptide LSAL. The fact that LSAL interacts specifically with 5-HT1B receptors that inhibit the release of neurotransmitters and particularly that of 5-HT itself suggests that this peptide may be involved in mechanisms controlling 5-HT neurotransmission and, accordingly, may play an important role in pathophysiological functions related to 5-HT activity.

5-HT-Moduline (Leu-Ser-Ala-Leu) is a new endogenous peptide purified from rat brain which interacts specifically with 5-HT1B/1D receptors. The binding interaction of 5-HT-Moduline with 5-HT1B/1D receptors appeared to be a non-competitive process, since the Bmax value of [125I] cyanopindolol binding on rat brain cortical membranes was decreased without modification of the Kd. This interaction was conserved on NIH 3T3 cells expressing the 5-HT1B receptor (IC50 = 10(-11)M) suggesting that the binding site for 5-HT-Moduline is localized on the 5-HT1B receptor protein. The observed interaction may lead to functional alterations of 5-HT1B/1D receptors known to play an important role in regulating the release of 5-HT from serotonergic nerve terminals (autoreceptors) as well as the release of other neurotransmitters (heteroreceptors).

The effects of combined physical and psychological stress on cardiovascular and respiratory responses were examined. Thirty-six undergraduate men performed a mental arithmetic task and moderate aerobic exercise, separately and in combination, while physiological measures were recorded continuously using electrocardiography, impedance cardiography, and respiratory gas analysis techniques. Cardiovascular responses during the combination of exercise and mental arithmetic stress were greater than those during either stressor alone, indicating that exercise did not mask the increases in cardiovascular performance evoked by psychological stress. In contrast, respiratory responses to the combined stressor were greater than those during isolated mental arithmetic, but less than those during isolated exercise. Thus the results indicate that physical and psychological stress exert a synergistic impact on cardiac performance, but not necessarily on respiratory performance. The results are consistent with the notion that the cardiovascular response to acute psychological stress exceeds concurrent metabolic demands.

Aluminium industry discharges fluoride into the atmosphere and several studies have shown a slight but significant contribution to the intake of fluoride by children living around aluminium smelters. A monitoring system was set up in 1991, just before a new aluminium smelter came into operation in Loon-Plage, on the North Sea coast, to study the evolution of the urinary fluoride excretion in children around the plant. Every year, 250 children under 14 are sampled in infant clinics, nursery schools and a secondary school. Urinary fluoride excretion was assessed by a potentiometric method on a spot morning urine sample and information on exposure factors was obtained by questionnaire. Urinary fluoride levels decreased with age (r = 0.31) and were higher in children drinking a local bottled water rich in fluoride (geometric mean in mg per gram of creatinine: 0.69 vs 0.52) or taking fluoride tablets (0.82 vs 0.52). The mean urinary fluoride excretion in children did not vary significantly between 1991 (geometric mean: 0.70 mg per gram of creatinine, 95% CI: [0.64-0.77]), 1992 (0.68 [0.62-0.75]) and 1993 (0.68 [0.61-0.76]), even after adjustment for potential confounding factors and despite a moderate increase in atmospheric fluoride levels.

Glial cell membrane fractions were prepared using glial cells preparations isolated from horse brain striatum. [3H]5-HT binding was measured by the filtration technique and the adenylate cyclase activity determined by measuring the cAMP production using a radioimmunoassay. Serotonin binds to glial membrane fractions with an affinity corresponding to a dissociation constant Kd = nM. The corresponding site is serotoninergic specific: [3H]5-HT binding is inhibited by 5-HT agonists (5 OH NM-DMT, 5-MeOHT, 5-MeOH-DMT, NN-DMT) or antagonists (cinanserine, cyproheptadine, methysergide, LSD) and not (or poorly) inhibited by non-serotoninergic related drugs. The population of sites binding 5-HT, present in neuronal membrane preparations and determined in parallel assays is distinct from that observed in glial preparations. The glial membrane fractions contains an adenylate cyclase activated by 5-HT with an apparent affinity constant close to 1 microM. It is serotonin-specific and clearly distinct from the DA-stimulated adenylate cyclase present in the same preparation. The sites binding 5-HT and activating the adenylate cyclase with low affinities might be directly related. This system, clearly distinct from the postsynaptosomal serotoninergic receptor, represents presumably a glial serotoninergic receptor; however, it cannot be totally excluded that these sites may refer to presynaptic membranes.

The solubilization of the serotonergic 5HT1 and 5HT3 sites was performed with digitonin and sodium cholate at 1% (final concentration). Two binding sites for [3H]5HT were observed on rat or horse brain synaptosomal membranes solubilized with these detergents. The corresponding dissociation constants (KD) were 1-3 nM and 13-30 nM respectively. These values were closely similar to those corresponding to 5HT1 and 5HT3 sites located in intact membranes. The solubilized sites specifically bound 5HT. The effect of GTP decreasing the binding to 5HT1 sites was lost on solubilized 5HT1 sites; it was recovered, however, by addition of phospholipids (asolectin 0,2%). The apparent molecular weights of these sites were determined using the gel filtration method (438 and 235 K daltons). The photoactivation of [3H]5HT by U.V. light was used to label 5HT1 and 5HT3 sites irreversively in membranes. The binding of [3H]5HT following U.V. irradiation was not dissociated after dilution; it was saturable and prevented by serotonergic drugs and not by adrenergic or dopaminergic antagonists. Moreover, GTP added prior to the irradiation reduced it markedly thus showing that 5HT1 sites were labelled. Electrophoretic and fluorographic analyses of the labelled material evidenced a 60 K dalton-band specifically labelled with [3H]5HT (5 or 20 nM). These results tend to indicate that the 60 K dalton-proteic band might represent a proteic subunit constituting part of 5HT1 and 5HT3 sites.

Crude membranal fractions isolated from mammalian brain tissue contain two classes of recognition sites capable of binding [3H]5-HT with high affinity constants. These classes of sits are characteristics of the postsynaptosomal membrane fraction for the higher affinity and of a glial cell membrane fraction for the lowest. They are observed with similar properties in cultured neuronal and glial cell respectively. Two 5-HT stimulated adenylate cyclases are present in crude membrane fraction; they are also separable as neuronal and glial components. These observations correspond likely to the existence of two classes of receptors for 5-HT. Their mechanisms of regulation involve presumably structural conformation changes of the recognition site coupled to various states of the activity of the receptor.

Indirect calorimetric studies were performed during a 100 g oral glucose tolerance test in diabetic patients with varying degrees of endocrine pancreatic dysfunction and in a control group of normal subjects. In 3 obese diabetics the study was repeated after a 3 day protein sparing modiefied fast. In diabetic patients the results show alterations of oxidation and storage of carbohydrates, related to insulin secretion deficiency on the one hand, and to overweight on the other. Endocrine pancreatic insufficiency may account directly for alterations observed in individuals with decreased or absent insulin response to glucose load, wheras metabolic factors such as adipose mass, hepatic steatosis, and peripheral insulin resistance appear to be responsible for alterations in carbohydrate oxidation and storage in subjects with relative endocrine pancreatic insufficiency, particularly obese diabetics.

A serotonine-sensitive adenylate cyclase system has been observed in horse brain membrane preparations. In crude mitochondrial fraction two types of activation sites were characterized, the Ka being = 2 nM and 1 micrometer. In purified synaptosomal membranes, a single adenylate cyclase activation was observed corresponding to the highest apparent affinity (KD = 2nM). This activation site might be related to the high affinity binding site (KD = 2NM) were previously described in the same membrane preparations.

Purified glial membrane preparations have been isolated from horse brain striatum. Tritiated 5-HT bound to these membranes with a high affinity (KD = 10 nM); the corresponding binding is reversible and appears specific of the serotoninergic structure. In parallel, 5-HT activates an adenylate cyclase with a low affinity (KD = 1 microM). The sites involved in this binding and in this adenylate cyclase activation appear different from the serotoninergic sites reported in the neuronal membrane preparations.

5 hydroxytryptamine binds to crude brain membrane preparations with two different affinities (KD = 1 to 2 X 10(-9) M for the highest, 1 to 2 X 10(-8) M for the lowest). LSD also binds with two affinities (KD = 3 to 4 X 10(-9) M and KD = 2 to 3 X 10(-8) M). Subcellular distribution of these sites shows that binding involves the two binding affinities in microsomal membranes but solely the high affinity binding sites are present in purified synaptosomal membranes. High affinity sites for 5 HT and for LSD are different as no direct competitive inhibition is observed in that case. On microsomal membranes, direct relationship occurs between low affinity binding for 5 HT and high affinity binding for LSD.

5-HT binding sites of the 5-HT1 type are heterogeneous and appear to comprise several subtypes (5-HT1A, 5-HT1B and 5-HT1C); their physiological role is as yet unclear. The stimulation of adenylate cyclase induced by 5-HT has been investigated in membrane fractions prepared from rat brain cortex. Enzymatic activity was determined by measuring cAMP production with an HPLC technique. It was shown that 5-HT stimulates adenylate cyclase activity with 2 activation constants (Kact): one shows a high apparent affinity (Kact = 0.8 nM) and the other a lower apparent affinity (Kact = 0.30 microM). The latter activity, induced by micromolar concentrations of 5-HT, was inhibited by spiperone at concentrations that block 5-HT1A binding. 5-Methoxytryptamine, bufotenin, and LSD also had a stimulatory biphasic effect on adenylate cyclase activity, whereas trifluoromethylphenylpiperazine, 5-carboxyamidotryptamine, 8-hydroxy-(2-di-n-propylamino)tetralin, RU 24969 had a monophasic effect. Enzyme activation by drugs acting in the micromolar range was inhibited by spiperone (1 microM), suggesting a link between this activation and 5-HT1A sites. On the other hand, the high-affinity activation of the enzyme induced by 5-HT, 5-methoxytryptamine, bufotenin, LSD, and the activation induced by TFMPP were not inhibited by spiperone (1 microM), by propranolol (3 microM), or by mesulergine (0.1 microM), which selectively block 5-HT1A, 5-HT1B, and 5-HT1C sites. Inhibition was produced by dihydroergotamine, methysergide, cinanserin, and mianserin, but not by naloxone, phenoxybenzamine, and phentolamine. Therefore, these activations seem related to 5-HT1 receptors but not to 5-HT1A, 5-HT1B, or 5-HT1C sites. Accordingly, binding of [3H]5-HT to 5-HT1-like sites was examined in the presence of spiperone (1 microM) and propranolol (3 microM); in these conditions, a high-affinity site (KD = 3.4 nM) was indeed revealed. The relative potencies of a series of drugs that stimulate or inhibit the activation of the adenylate cyclase with a high affinity and their ability to inhibit this binding of [3H]5-HT showed a positive correlation, strongly suggesting a direct relation between this recognition site for 5-HT and the production of a second messenger (cAMP). Moreover, this potential receptor is shown to be heterogeneously distributed within the brain, and was localized postsynaptically at serotonergic synapses.

A particular history of a man aged 51 with a right fronto-temporal tumor is analyzed. The main complaint is a paroxysmal need to drink water. Surface electrode electroencephalography and video-telemetry recordings sometimes demonstrated evidence of temporal epileptic abnormalities and therefore seem to have localizing significance. Waterdrinking may thus be added to the widening range of behavioral manifestations associated with epileptic discharges arising in man's temporal lobes.

Ornithine-alpha-ketoglutarate (OAK), a drug commonly used in various catabolic states, was studied for its acute effects on endocrine pancreas. A 30-min infusion of OAK (20 g/m2) induced significant increases in insulin levels (from 15 through 60 min) and in glucagon levels (from 15 through 90 min). However, OAK-induced insulin and glucagon responses were lower than after a 0.5 g/kg arginine infusion. The fluctuations of blood glucose levels were much less marked during OAK infusion than during arginine and especially the late fall was less evident.

Previously, a 5-hydroxytryptamine receptor has been described in neuronal membrane preparations. This receptor corresponds to a recognition site for 5-HT, and is seemingly related to an effector which consists of an adenylate cyclase activated by 5-HT and having a high apparent affinity constant (KD close to 1 nM). Moreover, this neuronal receptor is distinct of the glial serotoninergic receptor (Fillion et al., 1980). The method used to measure the binding of 3H-5-HT has been previously reported (FILLION et al., 1978). Adenylate cyclase activity was determined by measurement of cAMP using a radioimmunoassay (FILLION et al., 1979 a, b). Membranes were pretreated as follows: (1) membranes were preexposed to 5-HT (i.e.: incubated in the presence of 5-HT, generally 10 nM) at 37 degrees C, washed by centrifugation and resuspended in a medium free of 5-HT (or diluted in the same medium) or (2) membranes were incubated with a single concentration of 5-HT for a prolonged duration. Results were as follows: 1. The binding of 3H-5-HT, incubated for 10 min at 37 degrees C, corresponds to a KD close to 20 nM whereas a prolongation of the incubation duration to 15-25 induces the binding of 5-HT with a KD close to 2 nM. 2. Preexposure of the membranes to 5-HT leads to the same increase in affinity with, apparently, a reduced number of sites. 3. N-ethylmaleimide pretreatment of the membranes prior to their preexposure to 5-HT inhibits the change in affinity. 4. GTP, but not GDP, reverses the affinity change. 5. The adenylate cyclase activated by 5-HT is desensitized by preexposure of the membranes to 5-HT. 6. GTP reverses the process of desensitization. These results support the hypothesis of the existence of a regulatory mechanism for the 5-HT neuronal receptor. The regulatory mechanism would involve structural conformation changes of the recognition site corresponding to different affinities of the adenylate cyclase. A nucleotide binding protein is presumably involved in the coupling of these two subunits of the receptor.

Cerebrovascular complications of thrombocytosis may be the presenting symptoms of the disease (thrombocythemia or secondary thrombocytosis). In the light of 3 recently studied cases, the clinical, pathogenic and therapeutic aspects are emphasized. Early diagnosis is fundamental if the occurrence of permanent neurologic dysfunction is to be avoided.

Two different classes of binding sites probably related to serotonergic receptors have already been reported: 5-HT1 binding sites recognize [3H]5-hydroxytryptamine with a high affinity (Kd = 3 nM) and S2 binding sites recognize [3H]spiroperidol and [3H]ketanserine. An additional population of sites has been observed in crude membrane preparations or fractions enriched with synaptosomal membranes obtained from rat brain cortex. This population was observed as a single class of sites in a synaptosomal fraction (L fraction--according to Laduron (1977)). It corresponded to a dissociation constant Kd = 13-15 nM, and Bmax = 0.80 +/- 0.15 pmol/mg protein. Displacement experiments showed that it recognized preferentially the 5-HT structure (bufotenin, 5-MeO-tryptamine). Tryptamine was a weak displacer and 5,7-dihydroxytryptamine totally inefficient. Neither 8-OH-DPAT, nor quipazine had any effect. Methiothepin, cinanserin and cyproheptadine displaced 5-HT from these sites whereas ergot derivatives did not. Contrary to 5-HT1 binding, this recently observed binding was not altered by GTP; alpha-MSH reduced the corresponding Bmax whereas Leu-enkephalin did not. The degenerative lesion of the serotonergic fibers led to a slight increase in the Bmax of the binding without altering the Kd which means that corresponding sites are not located on serotonergic fibers and might be postsynaptically located.