A new surfactant series, the N-alkylamino-1-deoxylactitols: application for extraction of 'op' opiate receptors from frog brain.

A new series of surfactants, the N-alkylamino-1-deoxylactitols, was prepared and employed to extract 'op' opiate receptors from frog brain. These surfactants are both cheap and convenient to prepare. Receptors were reproducibly extracted in a good yield using N-nonylamino-1-deoxylactitol. This derivative, which was not denaturing during the extraction process, could thus be used instead of the more costly digitonin, whose rather variable purity affects yield.

Species differences in the localization of neuropeptide FF receptors in rodent and lagomorph brain and spinal cord.

Quantitative in vitro receptor autoradiography of [125I][D-Tyr1,(NMe)Phe3]NPFF was used to study the regional distribution of neuropeptide FF receptors in rodent and lagomorph brain. In rat, mouse, rabbit, and Afghan pika [125I][D-Tyr1,(NMe)Phe3]NPFF binding sites were enriched in the superficial layers of dorsal horn of the spinal cord and in parabrachial nucleus, central gray matter, hypothalamus, and reunions thalamic nucleus. In other neuroanatomical regions, important species differences in NPFF receptor patterns are observed. In marked contrast, the brain and the spinal cord of the Octodon degus are devoid of NPFF receptors. The present study shows that in different species regional variations in brain NPFF receptor binding occur.

Divalent cations: do they stabilize the agonist (12S) form of the mu opioid receptor?

In rabbit cerebellum membranes, millimolar concentrations of Mn++ ions while slightly reducing the Kd of 3H-etorphine counteract, to a large extent, sodium inhibition of equilibrium binding of the tritiated agonist to the mu opioid receptor. In digitonin extracts of membranes which have been incubated either with 3H-etorphine or with 3H-diprenorphine in the presence of Mn++ ions, recovery of radioligand bound to the agonist form of the receptor (sedimenting at position 12S) is substantially increased while, at the same time, recovery of radioligand bound to the antagonist form of the receptor (10S) is markedly reduced. Taken together these results suggest that Mn++ ions stabilize the agonist (12S) form of the mu opioid receptor. Mn++ ions may do so by promoting association of the receptor and of a guanine nucleotide regulatory protein.

Solubilization of two molecular forms of the frog brain opioid receptor.

In equilibrium binding studies, using 3H-etorphine and 3H-diprenorphine, digitonin extracts of frog brain membranes are found to contain two classes of sites, one of which is seen only in the presence of Na+ ions. Centrifugation of the extracts in sucrose gradients separates two macromolecular components (10S and 12S) which display specific opiate binding activity. The 12S component appears to carry the site that binds opiates in the absence of Na+ ions while the 10S component would carry the other site, i.e. the one which is seen only in the presence of Na+ ions in equilibrium binding studies. Preliminary evidence is also given that in extracts of frog brain membranes which have been pre-incubated with 120 mM NaCl, the balance of the two components is shifted in favor of the slower sedimenting (10S) one. These results are discussed in terms of the regulation of the state of equilibrium between an agonist (12S) and an antagonist (10S) form of the opioid receptor.

The natural tolerance of the afghan pika (Ochotona rufescens) to morphine.

A lagomorph, the afghan pika, Ochotona rufescens showed no effect whatever following the subcutaneous injection of morphine in doses up to 50 mg per 100 g of body weight, i.e. 250 times the ED50 for the rat. Higher doses were toxic and induced convulsions. However, the pika is responsive to synthetic opiates such as etorphine, pentazocine and phenoperidine. Interestingly enough, morphine antagonized the opiate response elicited by those narcotics to which the animal is sensitive. Pharmacokinetic analysis demonstrated that morphine enters the pika's brain as readily as it does the rat's. In addition, opiate receptor sites, which are present in normal amounts in pika brain retained their high affinity for 3H-etorphine (KD = 0.3 nM), 3H-naloxone (KD = 1.2 nM) and morphine. Moreover, binding of morphine to pika brain homogenates was inhibited in the presence of sodium ions (agonist response). Therefore, the antagonism of phenoperidine action by morphine appeared not to occur at the opiate receptor site; the mechanism of the pika's natural tolerance to morphine may reside in molecular events that normally preceed (metabolism?) or follow (enzyme activation?) the interaction between the drug and its specific recognition sites.

Apparent precoupling of kappa- but not mu-opioid receptors with a G protein in the absence of agonist.

Rabbit and guinea-pig cerebellum membranes contain a very high (greater than 80%) proportion of mu- and kappa-opioid receptors, respectively. Rabbit (mu) and guinea-pig (kappa) cerebellum membranes were (i) labeled either with the opiate agonist, [3H]etorphine (Kd = 0.1-0.2 nM), or with the opiate antagonist, [3H]diprenorphine (Kd = 0.1 nM), in the absence or presence of Na+ and/or 5'-guanylylimidodiphosphate (GppNHp), (ii) solubilized with digitonin (1%, w:v) and (iii) the radioactivity in the soluble extracts analyzed by ultracentrifugation in sucrose gradients. In the soluble extracts from rabbit cerebellum (mu) membranes, bound [3H]etorphine sedimented faster (S20,w congruent to 12S) than bound [3H]diprenorphine (10S), while in those from guinea-pig cerebellum (kappa) membranes, bound [3H]etorphine and bound [3H]diprenorphine sedimented at the same position (12S). Na+ selectively decreased recovery of the bound tritiated agonist in the two soluble preparations. When they had been generated in the presence of GppNHp but in the absence of Na+, the [3H]etorphine complexes of the mu- and kappa-opioid receptors as well as the [3H]diprenorphine complex of the kappa-opioid receptor were all recovered at position 10S, indicating that GppNHp had induced a decrease of the apparent molecular size of the two types of opioid receptors. These data are interpreted in terms of mu- and kappa-opioid receptors being capable of physically interacting with a G protein (GTP binding regulatory protein) yet, unlike the mu-opioid receptor which does so only in the presence of an agonist, the kappa-opioid receptor appears to be precoupled with a G protein.

Regulation by Na+ ions and GppNHp of the interaction between a G protein and the amphibian type of opioid receptor.

Digitonin treatment of frog brain membranes in 50 mM Tris-HCl yields a soluble extract that contains nearly equal amounts of free and G protein-bound opioid receptor molecules (Mollereau et al., 1988, J. Biol. Chem. 263, 18003). We report here that the balance of the two forms of the opioid receptor in digitonin solution is dependent on the environment of the membrane suspension at the time of solubilization with the detergent. Preincubating the membrane suspension with 50 microM GppNHp or with 120 mM NaCl results, in the two cases, in a digitonin extract that no longer displays the G protein-bound form of the receptor, i.e., the form of the receptor which exhibits high affinity for the opiate agonist etorphine in binding studies, as well as large apparent molecular size in sucrose gradients. Assuming that the situation in soluble extracts faithfully reflects the one in the membrane, these results would exclude the possibility that in a physiological environment the opioid receptor is in part precoupled with a G protein in the absence of an opioid agonist.

Evidence for a new type of opioid binding site in the brain of the frog Rana ridibunda.

The crude membrane fraction from the brain of the frog Rana ridibunda was shown to contain 0.7-0.8 pmol/mg protein for a site with high (KD = 0.1 nM) and about 3.2 pmol/mg protein for a site with lower (KD = 10-15 nM) affinity for the opiate agonist [3H]etorphine and for the opiate antagonist [3H]diprenorphine. In addition to its very high affinity for the two tritiated oripavine derivatives, the high affinity site displayed (i) a considerably reduced ability to bind the agonist but not the antagonist in the presence of Na+ ions and (ii) pronounced stereospecificity. These properties are all typical of an opioid receptor site. The lower affinity site, which was about four times as abundant as the other exhibited none of the aforementioned characteristics and is therefore probably not opioid in nature. Detailed testing of the potency of various unlabelled opioid ligands to inhibit the binding of [3H]etorphine at the high affinity site showed that the latter consists of a mixture of several types of opioid sites, including a major type with an apparent binding profile clearly different from those of mammalian brain mu, delta- and kappa-opioid sites. In particular, this major type of site, which accounted for about 70% of the opioid binding in frog brain membranes, bound mu ([D-Ala2,MePhe4,Glyol5]enkephalin), delta ([D-Thr2,Leu5]enkephalyl-Thr) and kappa (U50,488) selective ligands with much lower affinity than did mu-, delta- and kappa-opioid receptor sites, respectively.

Physical separation of the agonist and antagonist forms of a mu opiate receptor?

The rabbit cerebellum contains up to 80% of mu opiate binding sites (J.C. Meunier, Life Sci. 31, 1327-1330 (1982) ). A crude membrane fraction derived therefrom was labelled either with the opiate agonist: 3H-etorphine or with the opiate antagonist: 3H-diprenorphine, and solubilized with digitonin. Centrifugation of the soluble extracts in linear sucrose gradients reveals that bound 3H-etorphine sediments faster than does bound 3H-diprenorphine: 12S vs 10S. The 3H-etorphine macromolecular complex (12S) is virtually absent in extracts of membranes that have been labelled in the presence of 120 mM NaCl or of 50 microM Gpp(NH)p (guanyl-5'-yl imidodiphosphate). Under the same conditions, recovery of 3H-diprenorphine in bound (10S) form is either not affected (Gpp(NH)p) or substantially increased (NaCl). These data are consistent with the notion that cerebellar mu opiate receptors may exist under physically separable agonist and antagonist states.

Distinct molecular forms of opiate binding in the frog brain.

In frog brain membranes, equilibrium saturation binding of the opiate agonist: 3H-etorphine and of the opiate antagonist: 3H-diprenorphine is biphasic. In the presence of sodium ions (120 mM), "kinetic conversions" (unchanged Bmax) are observed: high affinity binding of the 3H-agonist is decreased while high affinity binding of the antagonist is increased. Frog brain membranes were labelled either with 3H-etorphine or with 3H-diprenorphine and solubilized with digitonin. The soluble extracts were centrifuged in sucrose gradients: two distinct components were labelled which sedimented respectively faster (approximately 12 S) and slower (approximately 10 S) than did catalase, used as marker. Pre-incubation of membrane and of 3H-ligand in the presence of 120 mM NaCl resulted in 1) considerably reduced recovery of the 12 S component in labelled form and 2) substantially enhanced labelling of the 10 S component with the 3H-antagonist. Gpp(NH)p (guanyl-5'-yl imidodiphosphate) exerted similar effects as did sodium ions. Taken together, these data suggest that the two components in question represent physically distinct agonist and antagonist forms of frog brain opiate receptors.

Opioid receptor types in the brain of the Afghan pika (Ochotona rufescens), a species which is naturally tolerant to morphine.

The rabbit is normally sensitive to morphine while another lagomorph, the Afghan pika Ochotona rufescens is naturally tolerant to the analgesic effects elicited by the opium alkaloid. In spite of the different responsiveness of the two species to morphine we find that the pika brain and the rabbit brain both contain a mixture of mu-, delta- and kappa-opioid sites in nearly the same proportions: 46-47% mu, 23% delta and 28-30% kappa. Moreover, apparent binding of morphine in pika and rabbit brain membranes is inhibited in the presence of Na+ ions and/or of 5-guanylylimidodiphosphate indicating that morphine should behave as an opiate agonist (analgesic) not only in rabbits, which it does but also in pikas, which it does not. Taken together these results suggest that the natural tolerance of the Afghan pika to morphine may not reside in modified opioid receptor types and that its origin should be sought elsewhere.

Morphine metabolism in the naturally morphine-tolerant afghan pika: a preliminary study.

The afghan pika (Ochotona rufescens), a lagomorph which is naturally tolerant to the analgesic action of morphine, metabolizes morphine into morphine 3-glucuronide apparently faster than does the rabbit, another lagomorph which is however normally responsive to morphine. In the two species, following morphine administration, another unidentified component appears very soon (5 min) in pika blood plasma and much later (60 min) in rabbit blood plasma. This unknown component which appears not to be morphine derived might be involved in the natural resistance of the Afghan pika to morphine.

Impact of travel on the seroprevalence of hepatitis A in children.

BACKGROUND: Recent data about hepatitis A virus (HAV) seroprevalence in industrialized countries and the impact of travels to endemic areas are sparse or absent, particularly for children. OBJECTIVE: To determine the impact of travel to endemic areas on HAV seroprevalence and estimate the overall HAV seroprevalence in children in France. To identify risk factors for positive HAV serologic results. STUDY DESIGN: This prospective multicentre cross-sectional seroprevalence study took place in eight paediatric emergency units throughout France. Children 1-16 years of age following all inclusion and exclusion criteria were included. Demographic, socioeconomic, and travel data were prospectively collected with a standardized questionnaire before measurement of specific HAV antibodies. HAV seroprevalence was determined and its association with diverse variables assessed by univariate and multivariate analyses. RESULTS: 430 children were included, of whom 116 had travelled to endemic areas. The HAV seroprevalence in the overall population was 5% (95%CI, 3-7) and was higher among the travellers (12% [95%CI, 6-18]) than among the others (2% [95%CI, 0-3]), OR=7.0 [95%CI, 2.6-18.8]. Risk factors identified for positive serologic results for HAV were travel to an endemic area >7 days (adjusted OR [aOR]=4.3 [95%CI, 1.5-12]), age of 14-16 years (aOR=7.7 [95%CI, 1.6-38.3]) and mother's birth in an endemic area (aOR=5.2 [95%CI, 1.8-14.8]). CONCLUSION: Statistical evidence showed that travel to endemic areas and parents' place of birth both play a role in HAV serologic results in children with a significant difference of HAV seroprevalence between traveller and non-traveller children in France.

Early psychological care of the French victims of the Costa Concordia shipwreck.

Most of the French passengers who survived the shipwreck of the cruise ship Costa Concordia were repatriatedfrom Italy to Marseille, one of the stopovers of the cruise. The shipwreck happened during the nightof 13th-14th January 2012 and entailed the forced evacuation of 4195 passengers and crewmembers.Thirty-two persons died and 2 others are still reported missing. The massive and unexpected inflow of402 French citizens in the port of Marseille required the quick setting up of welcome facilities, not only tosolve logistical problems, but also to address psychological and sometimes even medical problems. ThePrehospital Psychological Emergency Service (CUMP) and the Prehospital Emergency Medical Service(SAMU) of Marseille examined 196 persons in total, and were able to avoid a great number of emergencyadmissions deemed necessary because of difficult psychological situations (death, missing or lost persons,acute stress). The objective of this report is to rapidly present the emergency committee as a whole andto describe in more detail the work that the CUMP accomplished during the 36 hours necessary to takecharge of the majority of the French passengers of the Costa Concordia.

Spatial-symbolic Query Engine in Anatomy.

OBJECTIVES: Currently, the primary means for answering anatomical questions such as 'what vital organs would potentially be impacted by a bullet wound to the abdomen?' is to look them up in textbooks or to browse online sources. In this work we describe a semantic web service and spatial query processor that permits a user to graphically pose such questions as joined queries over separately defined spatial and symbolic knowledge sources. METHODS: Spatial relations (e.g. anterior) were defined by two anatomy experts, and based on a 3-D volume of labeled images of the thorax, all the labeled anatomical structures were queried to retrieve the target structures for every query structure and every spatial relation. A web user interface and a web service were designed to relate existing symbolic information from the Foundational Model of Anatomy ontology (FMA) with spatial information provided by the spatial query processor, and to permit users to select anatomical structures and define queries. RESULTS: We evaluated the accuracy of results returned by the queries, and since there is no independent gold standard, we used two anatomy experts' opinions as the gold standard for comparison. We asked the same experts to define the gold standard and to define the spatial relations. The F-measure for the overall evaluation is 0.90 for rater 1 and 0.56 for rater 2. The percentage of observed agreement is 99% and Cohen's kappa coefficient reaches 0.51. The main source of disagreement relates to issues with the labels used in the dataset, and not with the tool itself. CONCLUSIONS: In its current state the system can be used as an end-user application but it is likely to be of most use as a framework for building end-user applications such as displaying the results as a 3-D anatomical scene. The system promises potential practical utility for obtaining and navigating spatial and symbolic data.

[Metabolism and pharmacokinetics of methoxy-9-ellipticine and its principal metabolite, hydroxy-9-ellipticine]. / Métabolisme et pharmacocinétique de la méthoxy-9-ellipticine et son métabolite principal, l'hydroxy-9-ellipticine.

The biliary and urinary excretion of 9-methoxyellipticine in Rats yield the 0-demethylated metabolite 9-hydroxyellipticine, partly present as the glucuronide. Whole blood kinetics of 9-methoxyellipticine in Rabbits following a single i.v. administration is extremely fast, but is deeply modified after repeated dosage.

Comparative study of erythrocyte glycolytic enzymes in man and in two species of Lagomorphae. (Ochotona rufescens rufescens and Oryctolagus cuniculus).

The authors made a comparative study of red cell enzyme glycolysis in man and in two species of Lagomorphae, the pika and the rabbit. The activities of the 12 enzymes of Embden-Meyerhoff pathway and of the two dehydrogenases of pentose shunt (glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase) were determined. Phosphoglycerate kinase and pyruvate kinase showed quite similar activities in pika erythrocytes and in erythrocytes from human umbilical cord. The levels of these enzymes differed significantly in the pika and in the rabbit. No differences were noted between pyruvate kinases from the rabbit and the neonatal man. The other activities gave values either identical to those found in the adult man or intermediate between the adult and the neonatal man. In the rabbit the levels of glycolitic enzymes were generally lower than in the pika except for lactate dehydrogenase and glycose-6-phosphate dehydrogenase. Some characteristics of erythrocyte metabolism specific to the pika may account for the differences observed in this species. The influence of red cell age cannot explain the variations observed for no significant reticulocytosis was observed in the circulating blood. The percentages found in the pika and the rabbit were essentially identical at determination.

[Induction of cutaneous or subcutaneous fibroblastic tumors in the Afghan pika (Ochotona rufescens rufescens) by injection or bovine papilloma virus]. / Induction de tumeurs fibroblastiques cutanées ou sous-cutanées chez l'Ochotone afghan (Ochotona rufescens rufescens) par inoculation du virus du papillome bovin

Newborn Afghan pikas have been inoculated with bovine papilloma virus via the subcutaneous route. Cutaneous or subcutaneous fibromas and fibrosarcomas were observed after a mean incubation period of nine months. The transmission of these tumors by homograft has been obtained. Bovine papilloma virus antibodies have been demonstrated in most of the animals inoculated at birth. They have not been detected in animals bearing transplanted tumors.