Opioid receptor polymorphism A118G associated with clinical severity in a drug overdose population.

Genetic variations in the human mu-opioid receptor gene (OPRM1) mediate individual differences in response to pain and opiate addiction. We studied whether the common A118G (rs1799971) mu-opioid receptor single nucleotide polymorphism (SNP) was associated with overdose severity in humans. In addition, we examined an SNP responsible for alternative splicing of OPRM1 (rs2075572). We assessed allele frequencies of the above SNPs and associations with clinical severity in patients presenting to the emergency department (ED) with acute drug overdose. This work was designed as an observational cohort study over a 12-month period at an urban teaching hospital. Participants consisted of consecutive adult ED patients with suspected acute drug overdose for whom discarded blood samples were available for analysis. Specimens were linked with clinical variables (demographics, urine toxicology screens, clinical outcomes) then deidentified prior to genetic SNP analysis. Blinded genotyping was performed after standard DNA purification and whole genome amplification. In-hospital severe outcomes were defined as either respiratory arrest (RA; defined by mechanical ventilation) or cardiac arrest (CA; defined by loss of pulse). We analyzed 179 patients (61% male, median age 32) who overall suffered 15 RAs and four CAs, of whom three died. The 118G allele conferred 5.3-fold increased odds of CA/RA (p<0.05), while the rs2075572 variant allele was not associated with CA/RA. The 118G variant allele in the OPRM1 gene is associated with worse clinical severity in patients with acute drug overdose. These findings mark the first time that the 118G variant allele is linked with clinical drug overdose vulnerability.

Dopamine receptor D1 and postsynaptic density gene variants associate with opiate abuse and striatal expression levels.

Opioid drugs are highly addictive and their abuse has a strong genetic load. Dopamine-glutamate interactions are hypothesized to be important for regulating neural systems central for addiction vulnerability. Balanced dopamine-glutamate interaction is mediated through several functional associations, including a physical link between discs, large homolog 4 (Drosophila) (DLG4, PSD-95) and dopamine receptor 1 (DRD1) within the postsynaptic density to regulate DRD1 trafficking. To address whether genetic associations with heroin abuse exist in relation to dopamine and glutamate and their potential interactions, we evaluated single-nucleotide polymorphisms of key genes within these systems in three populations of opiate abusers and controls, totaling 489 individuals from Europe and the United States. Despite significant differences in racial makeup of the separate samples, polymorphisms of DRD1 and DLG4 were found to be associated with opiate abuse. In addition, a strong gene-gene interaction between homer 1 homolog (Drosophila) (HOMER1) and DRD1 was predicted to occur in Caucasian subjects. This interaction was further analyzed by evaluating DRD1 genotype in relation to HOMER1b/c protein expression in postmortem tissue from a subset of Caucasian subjects. DRD1 rs265973 genotype correlated with HOMER1b/c levels in the striatum, but not cortex or amygdala; the correlation was inversed in opiate abusers as compared with controls. Cumulatively, these results support the hypothesis that there may be significant, genetically influenced interactions between glutamatergic and dopaminergic pathways in opiate abusers.

Dense linkage disequilibrium mapping in the 15q11-q13 maternal expression domain yields evidence for association in autism.

Autism [MIM 209850] is a neurodevelopmental disorder exhibiting a complex genetic etiology with clinical and locus heterogeneity. Chromosome 15q11-q13 has been proposed to harbor a gene for autism susceptibility based on (1) maternal-specific chromosomal duplications seen in autism and (2) positive evidence for linkage disequilibrium (LD) at 15q markers in chromosomally normal autism families. To investigate and localize a potential susceptibility variant, we developed a dense single nucleotide polymorphism (SNP) map of the maternal expression domain in proximal 15q. We analyzed 29 SNPs spanning the two known imprinted, maternally expressed genes in the interval (UBE3A and ATP10C) and putative imprinting control regions. With a marker coverage of 1/10 kb in coding regions and 1/15 kb in large 5' introns, this map was employed to thoroughly dissect LD in autism families. Two SNPs within ATP10C demonstrated evidence for preferential allelic transmission to affected offspring. The signal detected at these SNPs was stronger in singleton families, and an adjacent SNP demonstrated transmission distortion in this subset. All SNPs showing allelic association lie within islands of sequence homology between human and mouse genomes that may be part of an ancestral haplotype containing a functional susceptibility allele. The region was further explored for recombination hot spots and haplotype blocks to evaluate haplotype transmission. Five haplotype blocks were defined within this region. One haplotype within ATP10C displayed suggestive evidence for preferential transmission. Interpretation of these data will require replication across data sets, evaluation of potential functional effects of associated alleles, and a thorough assessment of haplotype transmission within ATP10C and neighboring genes. Nevertheless, these findings are consistent with the presence of an autism susceptibility locus in 15q11-q13.

[Study of tensile bond strength of 3 different adhesive systems associated with composites on dentinal surfaces]. / Estudo de resistência à tração de três sistemas adesivos associados a resina composta em superfícies dentinárias.

The aim of this in vitro study was to compare the tensile bond strength of 3 different bonding systems, associated to composite resins, bonded to dentinal surfaces. Forty-four dentinal surfaces were obtained from recently extracted human molars. A standardized smear layer was obtained and the surfaces were divided in 3 groups: G1) self etch + microhybrid composite; G2) single-component adhesive + phosphoric acid + microhybrid composite and G3) conventional system (acid + primer + bond) + microhybrid composite. Specimens made of composite resin were constructed in the shape of an inverted truncated cone with 3 mm of diameter. Tensile bond strength test was performed at the speed of 0.5 mm/min, and the results were expressed in MPa. The analysis of variance ANOVA (p < 0.05) determined that the type of bonding system used influenced tensile bond strength. Tukey's test, however, showed that the results of the comparison between G2 and G3 were the only statistically significant ones, with G2 showing greater values of tensile bond strength.

Patients who want their family and physician to make resuscitation decisions for them: observations from SUPPORT and HELP. Study to Understand Prognoses and Preferences for Outcomes and Risks of Treatment. Hospitalized Elderly Longitudinal Project.

OBJECTIVE: To determine the extent to which older or seriously ill inpatients would prefer to have their family and physician make resuscitation decisions for them rather than having their own stated preferences followed if they were unable to decide themselves. DESIGN: Analysis of existing data from the Hospitalized Elderly Longitudinal Project (HELP) and the Study to Understand Prognoses and Preferences for Outcomes and Risks of Treatment (SUPPORT). SETTING: Five teaching hospitals in the United States. PARTICIPANTS: 2203 seriously ill adult inpatients (SUPPORT) and 1226 older inpatients (HELP) who expressed preferences about resuscitation and about advance decision-making. MEASURES: We used a logistic regression model to determine which factors predicted preferences for family and physician decision-making. RESULTS: Of the 513 HELP patients in this analysis, 363 (70.8%) would prefer to have their family and physician make resuscitation decisions for them whereas 29.2% would prefer to have their own stated preferences followed if they were to lose decision-making capacity. Of the 646 SUPPORT patients, 504 (78.0%) would prefer to have their family and physician decide and 22.0% would prefer to have their advance preferences followed. Independent predictors of preference for family and physician decision-making included not wanting to be resuscitated and having a surrogate decision-maker. CONCLUSIONS: Most inpatients who are older or have serious illnesses would not want their stated resuscitation preferences followed if they were to lose decision-making capacity. Most patients in both groups would prefer that their family and physician make resuscitation decisions for them. These results underscore the need to understand resuscitation preferences within a broader context of patient values.

Beta-adrenergic regulation of cyclic AMP synthesis in cultured human syncytiotrophoblast.

Isolated elements of the beta-adrenergic/adenyl cyclase signal transduction system have been studied previously using purified membranes. We used cultured syncytiotrophoblast cells to identify components of this signalling system and the interactions which regulate syncytial adenyl cyclase. Generation of cyclic AMP (cAMP) was stimulated in these cells by both forskolin and isoproterenol but not by dopamine, adenosine, carbachol or prostaglandin E1. Synthesis was also stimulated by treatment with cholera toxin, indicating the involvement of the G-protein, Gs. Somatostatin inhibited isoproterenol- or forskolin-stimulated cAMP generation, an effect which could be blocked by pretreatment of the cells with pertussis toxin, demonstrating the mediation of somatostatin action by Gi. Furthermore, secretion of human chorionic gonadotrophin (hCG) was increased significantly by isoproterenol while somatostatin blocked the isoproterenol-stimulated release of hCG. These results clearly demonstrate that adenyl cyclase in syncytiotrophoblast is controlled by a stimulatory pathway operating through Gs and inhibitory pathway acting through Gi.

Newborn penile glans amputation during circumcision and successful reattachment.

Circumcision, the most common operation in male patients in the United States, is performed by a variety of health care professionals. Although not technically difficult, it results in a large number of reported and unreported complications annually. We report the successful reattachment of a distal penile glans, which was amputated when the Sheldon clamp was used for newborn circumcision. The literature is reviewed, and prevention and treatment of this type of circumcision injury are described.

Dual regulation of human syncytial adenylyl cyclase.

The dual (stimulatory and inhibitory) regulation of adenylyl cyclase was studied in syncytiotrophoblast basal membranes prepared from term human placenta. Stimulation of adenylyl cyclase activity with GTP, non-hydrolyzable GTP analogs, isoproterenol and PGE1 was observed, confirming the presence of an intact stimulatory pathway in these membranes. Investigations of the inhibitory pathway revealed tight coupling of the G-protein, Gi alpha, to catalytic adenylyl cyclase, with high doses of GTP producing 80 per cent inhibition of GTP/forskolin-stimulated activity. Confirming Gi alpha involvement, pertussis toxin (PTX) treatment of basal membranes augmented the responses of adenylyl cyclase to both GTP and forskolin. In addition, immunoblotting of basal membrane proteins revealed the presence of the G-protein subunits, Gs alpha, Gi alpha, and G beta/gamma. The response of adenylyl cyclase was measured to a series of agonists known to inhibit adenylyl cyclase in other tissues, however a reproducible inhibitory effect was produced only by somatostatin (approximately 80 per cent). Treatment of basal membranes with PTX caused a degree of reversal of the somatostatin-mediated adenylyl cyclase inhibition. However, the intoxication was insufficient to restore GTP/forskolin-stimulated activity.

Differentiation of type II cells during explant culture of human fetal lung is accelerated by endogenous prostanoids and adenosine 3',5'-monophosphate.

Explant culture of the fetal lung, in the absence of serum or hormones, results in precocious biochemical and morphological differentiation of alveolar epithelial cells. In this study we tested the hypothesis that these maturational events are induced by endogenous cAMP. During culture of human fetal lung the content of tissue cAMP increased 140% from days 3-6. Treatment with isobutylmethylxanthine caused a further doubling of cAMP content, and indomethacin blocked most of the increase in cAMP. Isobutylmethylxanthine accelerated and indomethacin inhibited the increases during culture in surfactant protein-A (SP-A), SP-A mRNA, SP-B mRNA, phosphatidylcholine content, and activity of fatty acid synthetase. There was no effect of these treatments on the content of SP-C mRNA, which did not increase during culture. Increasing concentrations of prostaglandins E1 and E2 in the presence of indomethacin produced a parallel stimulation of cAMP content, SP-A, and fatty acid synthetase activity. The temporal increase in SP-A was also blocked by inhibitors of protein kinase-A. In morphological studies, indomethacin-treated explants appeared less mature, with decreased intralumenal volume and more columnar epithelial cells. We conclude that increased tissue cAMP levels, stimulated by endogenous prostaglandins, accelerate differentiation of alveolar epithelial cells during explant culture.

Control of the sodium-proton antiporter in human placental microvillous membranes by transport substrates.

The microvillous membrane of the human placental syncytiotrophoblast contains an amiloride-inhibitable, electroneutral, Na+/H+ antiporter. The kinetic characteristics of this antiporter have been investigated to determine its response to alterations in intracellular and extracellular H+ and Na+ concentrations. Antiporter activity was measured using a pH-sensitive fluorescent probe entrapped in placental microvillous vesicles. We report here on the kinetic characterization of the antiporter, a transporter which displays simple, saturable kinetics for the external site but complex kinetics at the internal site. Measurement of the external Na+ and H+ dependences demonstrated that Na+ and H+ compete for binding to a single external binding site which displays saturation kinetics. The external Km determined for Na+ was 8.2 +/- 4.0 mM, while the external pK was 7.29 +/- 0.02. The Vmax calculated from these experiments was 0.57 +/- 0.10 nequiv./s per mg membrane protein. By contrast, the internal dependences for both Na+ and H+ showed significant deviations from simple linear kinetics. Decreasing internal pH to 6.0 stimulated Na+/H+ exchange to a greater degree than predicted for a single-site saturable binding model, in a manner which suggested allosteric activation. At the other extreme, Na+/H+ exchange ceased above an internal pH of 7.1, despite the existence of an inwardly-directed Na+ gradient. Increasing intracellular Na+ caused inhibition of Na+/H+ exchange but the intracellular Na+ dependence showed that the effect is due to a mechanism more complex than simple, competitive inhibition between Na+ and H+. These results show that the microvillous Na+/H+ antiporter is insensitive to changes in extracellular Na+ and H+ concentrations in the physiological range. Changes in intracellular Na+ and H+ however are likely to cause marked changes in antiporter activity. These characteristics suggest that cellular Na+ and H+ concentrations are tightly controlled in the placental syncytiotrophoblast and that the Na+/H+ antiporter may play a significant role in their regulation.

Simultaneous preparation of paired, syncytial, microvillous and basal membranes from human placenta.

A method for the simultaneous preparation of microvillous and basal membrane vesicles from human placental syncytiotrophoblast is described. Mg2(+)-aggregated basal membranes are separated from microvillous membranes by low-speed centrifugation after initial homogenization and centrifugation steps. Microvillous membranes (MVM) are obtained from the low speed supernatant while basal membranes (BM) contained in the Mg2(+)-aggregated material are resuspended and further purified on a sucrose step gradient. MVM and BM prepared by this method were enriched 20-fold and 11-fold as determined by the membrane marker enzymes, alkaline phosphatase (MVM) and adenylate cyclase (BM). There was minimal cross-contamination of the two isolated plasma membrane fractions and the yields obtained were 26% (MVM) and 21% (BM) compared to the initial homogenate. The MVM and BM fractions were free from contamination by mitochondrial or lysosomal membranes and showed only minor contamination by microsomal membranes. The two membrane fractions were also tested for the presence of non-syncytial plasma membranes by electrophoretic immunoblotting. Contamination of both MVM and BM by fibroblast, endothelial, macrophage and cytotrophoblast plasma membranes amounted to less than 15% of the total membrane protein as determined by immunoblotting. Vesicle orientation, determined from the latency of specific concanavalin A binding, was 88 +/- 4% right-side out for MVM and 73 +/- 12% right-side out for BM. This simple preparative procedure produces a high yield of both MVM and BM from human placenta. The analytical data demonstrates that 'paired' MVM and BM fractions derived from the same placental tissue have a high purity in terms not only of contamination by intracellular membranes, but also in terms of contamination by non-syncytial plasma membranes.

Beta-adrenergic receptors and cAMP response increase during explant culture of human fetal lung: partial inhibition by dexamethasone.

We studied beta-adrenergic receptors and responses in human fetal lung (15-25 wk gestation) maintained in explant culture with and without added dexamethasone. To determine beta-adrenergic receptor concentration, we performed radioligand binding assays with [125I]-iodocyanopindolol. We also examined the ability of isoproterenol to stimulate cAMP generation as a measure of response to beta-adrenergic receptor occupancy. In control cultures, beta-receptor concentration increased significantly from d 0 to 3 of culture and thereafter remained stable. The kd (approximately 24 pM) of [125I]-iodocyanopindolol did not change with time in culture. The ability of isoproterenol to stimulate cAMP generation over basal levels increased in controls throughout the 5 d in explant culture. Addition of dexamethasone (10 nM) to the culture medium partially blocked the increase in beta-receptor concentration and decreased both cAMP content and generation (basal and stimulated) in a dose-dependent manner (median effective concentration approximately 1 nM). In these same explants, dexamethasone increased the activity of fatty acid synthetase, an enzyme important in surfactant synthesis, more than 2-fold. Our results indicate that beta-adrenergic receptors and isoproterenol stimulation of cAMP generation increase spontaneously in human fetal lung grown in explant culture. Dexamethasone, which accelerates other aspects of human lung development in vitro, decreases beta-adrenergic receptor concentration and inhibits beta-adrenergic responses.

Metabolism of the potent carcinogen 3-methylcholanthrylene by rat liver microsomes.

The products formed in the metabolism of 3-methylcholanthrylene (3MCE), either in the presence or in the absence of an epoxide hydrolase inhibitor, 3,3,3-trichloropropylene 1,2-oxide (TCPO), with an NADPH-regenerating system and liver microsomes from 3-methylcholanthrene (3MC)-treated male Sprague-Dawley rats were separated by reversed-phase and normal-phase HPLC. The metabolites were characterized by UV-visible absorption spectral analysis, and by comparing their retention times on reversed-phase and normal-phase HPLC with authentic 3MC derivatives whenever available. In addition to 3MC trans-1,2-diol, 3MC-1-one, and 3MC-2-one reported earlier by other investigators, 3-hydroxymethylcholanthrylene (3-OHMCE), 3-OHMCE trans-11,12-dihydrodiol, 3MCE trans-11,12-dihydrodiol, 3MCE trans-9, 10-dihydrodiol. 9- and 10-hydroxy-3MCE. 3MC-2-one trans-9,10-dihydrodiol, and a chemically unstable 3MCE 1,2-epoxide were identified as metabolites of 3MCE. 3MC cis-1,2-diol, a previously reported metabolite of 3MCE, was not detectable. In the presence of TCPO, metabolites that have been identified include 3-OHMCE, 3-OHMCE 11,12-epoxide. 3MCE 11,12-epoxide, 3MC-2-one, 3MC-1-one, 9-hydroxy-3MCE, 10-hydroxy-3MCE, and an unstable metabolic intermediate 3MCE 1,2-epoxide. The results suggest that 3MCE 1,2-epoxide, 3MCE 9,10-diol-7,8-epoxide, and 3MC-2-one 9,10-diol-7,8-epoxide may be involved in the metabolic activation of 3MCE to carcinogenic form.

Potassium inhibition of DMH-induced small intestinal tumors in rats.

This is a preliminary report that shows that supplemental potassium partially prevents 1,2-dimethylhydrazine (DMH) induction of tumors of the small intestine in Sprague-Dawley rats. Male rats were injected weekly with 20 mg DMH/kg body wt for 20 weeks. Potassium chloride was provided in the drinking water one week before the first DMH treatment and was continued until sacrifice 14 weeks after the last DMH treatment. There were four groups of rats and they were identified as follows: DMH, DMH + K, K, and untreated control. Based on 24-hour food and water consumption data and food and water compositions, rats provided 0.5% potassium (from KCl in the drinking water) were ingesting 287.5 +/- 9.2 mg potassium per 24 hours (K/Na = 4.18) and the unsupplemented groups were ingesting 180.3 +/- 18.4 mg potassium per 24 hours (K/Na = 2.62). At sacrifice, the incidence of DMH-induced small intestinal tumors was significantly (p less than 0.05) reduced from 46% (6/13) in the DMH group to 6% (1/17) in the DMH + K group (p less than 0.05). The potassium supplement also significantly (p less than 0.05) reduced the cumulative small intestinal tumor incidence from 40% (8/20) in the DMH group to 5% (1/20) in the DMH + K group. The incidence of colon tumors and of zymbal gland tumors appeared to be reduced by the potassium supplement; however, these were not statistically significant observations (p greater than 0.05). Based on the complete blood count and other blood parameters measured, the level of potassium supplement used induced no apparent toxic effects.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention, recognition, and treatment of perinatal asphyxia.

Management of perinatal asphyxia is one of those rare opportunities in clinical medicine when death or life-long disability can be prevented with several minutes of skillful and judicious action. Fetal and neonatal asphyxia is approached most successfully as a joint obstetric, pediatric, and anesthetic effort. This article reflects the team approach to perinatal asphyxia.

Ontogeny of fetal adenylate cyclase; mechanisms for regulation of beta-adrenergic receptors.

Transmembrane second messenger signalling systems regulate differentiation, growth and homeostatic responses during fetal development. The beta-adrenergic adenylate cyclase system is the best studied of these and has been used as a model to investigate the control of developmental processes. In tissues such as lung, heart and parotid, beta-adrenergic responsiveness of adenylate cyclase increases during development. In the developing fetal lung beta-receptor concentration increases during gestation or after glucocorticoid treatment, but cannot fully explain enhanced adrenergic responsiveness. To probe developmental and hormonal effects on beta-receptor function, we asked if advancing gestation or glucocorticoid treatment alters beta-receptor-Gs interactions in fetal rabbit lung membrane particulates. Before 25 days gestation, 1-isoproterenol competes for 3H-dihydroalprenolol (DHA), a radiolabelled beta-antagonist, with a single low affinity, later in gestation, high and low affinities of isoproterenol for the beta-receptor are present which can be shifted to the lower affinity by addition of guanyl nucleotide. High affinity binding is precociously induced in 25 days--fetal lung particulates as early as 3 h after maternal betamethasone treatment, but beta-adrenoreceptor concentration in treated fetuses was increased over controls only after 24 h of treatment. Cholera toxin catalyzed ADP ribosylation of membrane particulates showed cholera toxin substrate (Gs) was not altered by glucocorticoid treatment. Stimulation of adenylate cyclase activity with isoproterenol (100mM) and GTP (100mM) resulted in no incremental increase over that produced by GTP (100mM) alone in glucocorticoid treated or control particulates, either early or late in gestation. These data demonstrate that beta-receptor-Gs interactions are not sufficient to produce full agonist responses. Although both beta-adrenergic receptors and Gs are present in fetal rabbit lung early in gestation, interaction of these two adenylate cyclase components appears subsequently. This developmental event can be rapidly induced by maternal betamethasone treatment.

Respiratory diseases in pregnancy.

Pregnant women with respiratory problems pose a special challenge. Pregnancy alters lung function and the natural history of common pulmonary disorders. Respiratory problems in pregnancy must be approached with an understanding of the unique, inter-dependent physiologic and psychological status of mother and fetus.

Estrogen reduces beta-adrenoceptor-mediated cAMP production and the concentration of the guanyl nucleotide-regulatory protein, Gs, in rabbit myometrium.

The uterine contractile response to adrenergic agonists or sympathetic stimulation is influenced dramatically by the hormonal milieu. Rabbit uterine contraction is mediated by alpha 1-adrenoceptors, whereas relaxation in response to the same stimulus is mediated by beta 2-adrenoceptors. Whether uterine contractility is increased or decreased by adrenergic stimulation is determined by the gonadal steroids estrogen and progesterone: uterine contraction prevails in the estrogen-dominant or the ovariectomized animal, but in the progesterone-dominant rabbit, uterine relaxation is observed. In previous studies, we have demonstrated that changes in the concentration or agonist affinity of these adrenoceptors cannot account for the changes in contractile response. In the present studies, we tested whether sex steroids might alter beta-adrenergic response by acting on events distal to receptor occupancy, and whether this could explain the conversion of contractile response. We found that myometrial cAMP generation is potently stimulated by beta-agonists in progesterone-treated and also in ovariectomized animals, but this stimulation is absent after estrogen treatment. Similar, but smaller, changes were observed for cAMP generation in response to prostaglandin E2 and forskolin. Stimulation of adenylate cyclase in uterine particulates by agents which act on the guanyl nucleotide-sensitive stimulatory transducer, Gs, is unchanged after estrogen treatment. However, specific labeling of Gs catalyzed by cholera toxin is reduced in membrane particulates from estrogen-treated animals. Recombination of extracts of uterine membranes from the differently treated animals also suggested qualitative differences in Gs. We conclude that at least one component of the adenylate cyclase cascade beyond the beta-adrenoceptor, i.e., Gs, is a target for ovarian steroids; estrogen reduces Gs labeling and beta-adrenoceptor-mediated cAMP production. However, uterine Gs labeling and cAMP production are similar in ovariectomized and in progesterone-treated rabbits. Since these uteri exhibit different contractile responses, the observed changes are not sufficient to explain sex steroid-mediated conversion of myometrial contractile response.