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.

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.

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.

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.

Recruitment of antigenic gamma-tubulin during mitosis in animal cells: presence of gamma-tubulin in the mitotic spindle.

It has been claimed repeatedly that gamma-tubulin is exclusively localized at the spindle poles in mitotic animal cells, where it plays a role in microtubule nucleation. In addition to this localization, we have observed a gamma-tubulin-specific staining of the mitotic spindle in several animal cells (human, kangaroo rat, mouse, Chinese hamster, Xenopus and Drosophila) using five polyclonal antibodies raised against unique gamma-tubulin sequences and four different fixation protocols. In HeLa and PtK2 cells, gamma-tubulin was detected in the mitotic spindle from late prometaphase to telophase. In contrast, in other cell types, it was detected in metaphase only. In all cases we failed to detect gamma-tubulin in the short aster microtubules at the spindle poles. Electron microscopic observation revealed that at least part of the gamma-tubulin localized on the surface of spindle microtubules with a preferential distribution along kinetochore microtubules. In HeLa cells, the amount of antigenic gamma-tubulin was fairly constant in the spindle poles during mitosis from prometaphase to telophase. In contrast, gamma-tubulin appeared in the mitotic spindles in prometaphase. The amount of gamma-tubulin decreased in telophase, where it relocalized in the interzone. In metaphase cells about 15-25% of the total fluorescence was localized at the spindle poles, while 75-85% of the fluorescence was distributed over the rest of the spindle. These results suggest that the localization and timing of gamma-tubulin during the cell cycle is highly regulated and that is physiological role could be more complex and diverse than initially assumed.

SK-N-BE: a human neuroblastoma cell line containing two subtypes of delta-opioid receptors.

A human neuroblastoma cell line, SK-N-BE, was shown to express a substantial amount of opioid receptors (200-300 fmol/mg of protein). A ligand binding profile of these receptors revealed that they could belong to two distinct subtypes of delta-opioid receptors. Results from sucrose-gradient sedimentation experiments were compared with similar data obtained with the mu-opioid receptor of the rabbit cerebellum and the delta-opioid receptor of the hybrid NG108-15 cell line and have shown that the opioid receptor of the SK-N-BE cell line behaved hydrodynamically as an intermediate between mu- and delta-opioid receptors. Taken together, pharmacological and hydrodynamic studies suggest that the opioid receptors present in the SK-N-BE cell membranes could belong to two delta-opioid receptor subtypes interacting allosterically. Functional experiments suggest that at least one of these subtypes of delta-opioid receptor is negatively coupled to the adenylate cyclase via a Gi protein and that the opiate receptors of the SK-N-BE neuroblastoma cell line undergo a rapid down-regulation when preincubated in the presence of the high-affinity opioid, etorphine.

gamma-Tubulin participates in the formation of the midbody during cytokinesis in mammalian cells.

Animal cells undergoing cytokinesis form an inter-cellular bridge containing two bundles of microtubules interdigitated at their plus ends, which constitute the midbody. Polyclonal antibodies raised against three specific amino acid sequences of gamma-tubulin (EEFATEGGDRKDV, NIIQGEADPTDVHKSL and EYHAATRPDYISWGTQEQ) specifically stained the centrosome in interphase, the spindle poles in all stages of mitosis, and the extremities of the midbody in mammalian cells (Potorous, human, Chinese hamster, mouse). This staining was prevented by the corresponding peptides, by Xenopus gamma-tubulin, but was not modified by purified alpha beta-tubulin heterodimer. An identical staining was obtained with affinity-purified antibodies against the carboxyl-terminal amino acid sequence of human gamma-tubulin. No gamma-tubulin could be detected in the interzone during anaphase and early telophase. Material containing gamma-tubulin first appeared in the two daughter cells on each side of the division plane in late telophase, and accumulated transiently at the minus ends of the two microtubule bundles constituting the midbody for one hour after metaphase. Micro-injection of gamma-tubulin antibodies into anaphase cells prevented the subsequent formation of the microtubule bundles between the two daughter cells. In contrast with previous views, these observations suggest that the microtubules constituting the midbody may be nucleated on special microtubule organizing centres, active during late telophase only, and assembled on each side of the dividing plane between the daughter cells.

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.

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.

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.

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.

5'-Guanylylimidodiphosphate decreases affinity for agonists and apparent molecular size of a frog brain opioid receptor in digitonin solution.

When assayed for specific opiate binding in the presence of 120 mM NaCl, digitonin extracts from frog (Rana ridibunda) brain membranes were found to contain about the same quantity (0.5 pmol/mg of protein) of high (Kdh = 0.4 nM) and of lower (Kdl = 15-20 nM) affinity sites for the opiate agonist [3H]etorphine. The two classes of [3H]etorphine binding sites displayed equally high (Kd = 0.3 nM) affinity for the opiate antagonist [3H]diprenorphine. 5'-Guanylylimidodiphosphate (GppNHp) selectively and potently (IC50 = 0.1 microM) inhibited high affinity binding of the tritiated agonist, and this inhibition resulted from the GppNHp-induced conversion of the high into the lower affinity sites for [3H]etorphine. Following centrifugation of the digitonin extract in sucrose gradients, opioid binding activity was found to be associated with two clearly separated macromolecular components of apparent sedimentation coefficients 11.5 and 9.7 S, respectively. The two components bound [3H]diprenorphine equally well, whereas the fast sedimented component bound [3H]etorphine better than did the slower sedimented one. In addition, labeling of the component of bigger apparent size with [3H]etorphine was considerably reduced in the presence of 50 microM GppNHp. Finally, in soluble extracts which had been (i) preincubated with and (ii) centrifuged in the presence of GppNHp, the fast sedimented component was no longer observed while there was about twice as much of the component of smaller apparent size as in control (no GppNHp) extracts. Together, these results demonstrated the existence of an opioid receptor-G protein complex which, in digitonin solution, was still amenable to regulation (dissociation) by guanine nucleotides.

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.

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.

Differential regulation of two molecular forms of a mu-opioid receptor type by sodium ions, manganese ions and by guanyl-5'-yl imidodiphosphate.

The rabbit cerebellum contains a very high proportion (up to 80%) of mu-opioid receptor sites (Meunier, J.C., Kouakou, Y., Puget, A. and Moisand, C., Mol. Pharmacol. 24, 23-29, 1983). A membrane fraction derived therefrom is labeled either with the opioid agonist, 3H-etorphine or with the opioid 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. Pre-incubation of membranes and radioligand in the presence of 120 mM NaCl results in considerably decreased recovery of the 3H-agonist in 12S form while recovery of the 3H-antagonist in 10S form is substantially increased. The opposite situation is observed when the membranes have been prelabeled with radioligand in the presence of 1 mM MnCl2. Guanyl-5'-yl imidodiphosphate, a metabolically stable structural analog of GTP is found to selectively reduce recovery of labeled 12S receptors while it does not affect that of labeled 10S receptors. These data indicate that the mu-opioid receptor from rabbit cerebellum is capable of existing in two forms which differ in apparent molecular size: an "antagonist" (10S) form of apparent Mr approximately 230,000 which is stabilized in the presence of sodium ions and an "agonist" (12S) form of apparent Mr approximately 300,000 which, unlike the antagonist one, is sensitive to guanyl-5'-yl imidodiphosphate. It is thought that the form of larger apparent size represents the mu-opioid receptor associated with a guanine nucleotide regulatory protein.

Characterization, solubilization and purification of an opioid receptor from the brain of the frog Rana ridibunda.

The brain of the frog R. ridibunda contains a major opioid binding site which in vitro pharmacological profile is different from those of mammalian mu, delta- and kappa-opioid sites. In digitonin extracts, this major opioid site exists as two molecular forms -10S and 12S- which are clearly resolved by sedimentation in sucrose gradients and which are thought to represent the opioid receptor alone (10S) or associated (12S) with a guanine nucleotide regulatory protein. Purification of the digitonin extracts by affinity chromatography on immobilized dynorphin, results in a single major protein component of apparent Mr approximately 64,000.

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.

Multiple opiate binding sites in the central nervous system of the rabbit. Large predominance of a mu subtype in the cerebellum and characterization of a kappa subtype in the thalamus.

We have compared the binding characteristics of [3H]etorphine, a nonselective mu-, delta-, and kappa-opiate agonist, with those of [3H]Tyr-D-Ala-Gly-MePhe-NH(CH2)2OH ([3H]DAGO), a selective mu-agonist, in rabbit cerebellar and thalamic membranes. We have also examined the ability of various unlabeled opioid ligands to compete with the binding of [3H]etorphine in the two preparations. In cerebellar membranes, [3H]DAGO(Kd = 0.7 nM) labels slightly fewer sites than does [3H]etorphine (Kd = 0.06 nM): 0.18 versus 0.24 pmole/mg of protein. In addition, competition studies indicate that up to 75% of the [3H]etorphine binding sites in this preparation display (a) high apparent affinity for unlabeled DAGO and (b) higher apparent affinity for morphine, the prototypical mu-agonist, than for Tyr-D-Ala-Gly-Phe-D-Leu (DADL), a delta-agonist. Together, these results suggest that the rabbit cerebellum contains a very high proportion (0.7-0.8) of mu-opiate binding sites. In thalamic membranes, [3H]DAGO (Kd = 1.1 nM) labels considerably fewer sites than does [3H]etorphine (Kd = 0.08 nM): 0.09 versus 0.27 pmole/mg of protein. In this preparation, the competition curves of DAGO and of DADL resolve binding of [3H]etorphine into two components. The first component accounts for 40-50% of total binding and reflects the interaction of [3H]etorphine with mu-opiate binding sites. The second component (up to 50% of total binding) is unaffected in the presence of DADL at concentrations (1-10 microM) that rule out binding of [3H]etorphine to mu- and delta-opiate binding sites. It disappears readily in the presence of very low concentrations (Ki less than 1 nM) of benzomorphan opiates (bremazocine, cyclazocine, and ethylketocyclazocine) yet it is relatively insensitive to inhibition by mu- and delta-agonists. This second component may therefore reflect [3H]etorphine's interaction with a kappa-opiate binding site. The kappa-opiate binding site is assayed for as that site which binds [3H]etorphine (0.5 nM) in the presence of either DADL (2 microM) or 10 microM of another enkephalin: Tyr-D-Ser-Gly-Phe-Leu-Thr. We find that, in the rabbit central nervous system, the thalamus, followed by frontal cortex and caudate nucleus, shows the highest content of kappa-opiate binding sites.