Acromelic frontonasal dysostosis and ZSWIM6 mutation: phenotypic spectrum and mosaicism.

Acromelic frontonasal dysostosis (AFND) is a distinctive and rare frontonasal malformation that presents in combination with brain and limb abnormalities. A single recurrent heterozygous missense substitution in ZSWIM6, encoding a protein of unknown function, was previously shown to underlie this disorder in four unrelated cases. Here we describe four additional individuals from three families, comprising two sporadic subjects (one of whom had no limb malformation) and a mildly affected female with a severely affected son. In the latter family we demonstrate parental mosaicism through deep sequencing of DNA isolated from a variety of tissues, which each contain different levels of mutation. This has important implications for genetic counselling.

Adolescent Intermittent Alcohol Exposure: Dysregulation of Thrombospondins and Synapse Formation are Associated with Decreased Neuronal Density in the Adult Hippocampus.

BACKGROUND: Adolescent intermittent alcohol exposure (AIE) has profound effects on neuronal function. We have previously shown that AIE causes aberrant hippocampal structure and function that persists into adulthood. However, the possible contributions of astrocytes and their signaling factors remain largely unexplored. We investigated the acute and enduring effects of AIE on astrocytic reactivity and signaling on synaptic expression in the hippocampus, including the impact of the thrombospondin (TSP) family of astrocyte-secreted synaptogenic factors and their neuronal receptor, alpha2delta-1 (α2δ-1). Our hypothesis is that some of the influences of AIE on neuronal function may be secondary to direct effects on astrocytes. METHODS: We conducted Western blot analysis on TSPs 1 to 4 and α2δ-1 from whole hippocampal lysates 24 hours after the 4th and 10th doses of AIE, then 24 days after the last dose (in adulthood). We used immunohistochemistry to assess astrocyte reactivity (i.e., morphology) and synaptogenesis (i.e., colocalization of pre- and postsynaptic puncta). RESULTS: Adolescent AIE reduced α2δ-1 expression, and colocalized pre- and postsynaptic puncta after the fourth ethanol (EtOH) dose. By the 10th dose, increased TSP2 levels were accompanied by an increase in colocalized pre- and postsynaptic puncta, while α2δ-1 returned to control levels. Twenty-four days after the last EtOH dose (i.e., adulthood), TSP2, TSP4, and α2δ-1 expression were all elevated. Astrocyte reactivity, indicated by increased astrocytic volume and area, was also observed at that time. CONCLUSIONS: Repeated EtOH exposure during adolescence results in long-term changes in specific astrocyte signaling proteins and their neuronal synaptogenic receptor. Continued signaling by these traditionally developmental factors in adulthood may represent a compensatory mechanism whereby astrocytes reopen the synaptogenic window and repair lost connectivity, and consequently contribute to the enduring maladaptive structural and functional abnormalities previously observed in the hippocampus after AIE.

Adolescent intermittent alcohol exposure: persistence of structural and functional hippocampal abnormalities into adulthood.

BACKGROUND: Human adolescence is a crucial stage of neurological development during which ethanol (EtOH) consumption is often at its highest. Alcohol abuse during adolescence may render individuals at heightened risk for subsequent alcohol abuse disorders, cognitive dysfunction, or other neurological impairments by irreversibly altering long-term brain function. To test this possibility, we modeled adolescent alcohol abuse (i.e., intermittent EtOH exposure during adolescence [AIE]) in rats to determine whether adolescent exposure to alcohol leads to long-term structural and functional changes that are manifested in adult neuronal circuitry. METHODS: We specifically focused on hippocampal area CA1, a brain region associated with learning and memory. Using electrophysiological, immunohistochemical, and neuroanatomical approaches, we measured post-AIE changes in synaptic plasticity, dendritic spine morphology, and synaptic structure in adulthood. RESULTS: We found that AIE-pretreated adult rats manifest robust long-term potentiation, induced at stimulus intensities lower than those required in controls, suggesting a state of enhanced synaptic plasticity. Moreover, AIE resulted in an increased number of dendritic spines with characteristics typical of immaturity. Immunohistochemistry-based analysis of synaptic structures indicated a significant decrease in the number of co-localized pre- and postsynaptic puncta. This decrease is driven by an overall decrease in 2 postsynaptic density proteins, PSD-95 and SAP102. CONCLUSIONS: Taken together, these findings reveal that repeated alcohol exposure during adolescence results in enduring structural and functional abnormalities in the hippocampus. These synaptic changes in the hippocampal circuits may help to explain learning-related behavioral changes in adult animals preexposed to AIE.

The application of the Yo-Yo intermittent endurance level 2 test to elite female soccer populations.

The aim of this study was to evaluate the application of the Yo-Yo intermittent endurance test level 2 (Yo-Yo IE2) to elite female soccer populations. Elite senior (n = 92), youth (n = 42), domestic (n = 46) and sub-elite female soccer players (n = 19) carried out the Yo-Yo IE2 test on numerous occasions across the season. Test-retest coefficient of variation (CV) in Yo-Yo IE2 test performance in domestic female players was 4.5%. Elite senior female players' Yo-Yo IE2 test performances were better (P < 0.01) than elite youth, domestic and sub-elite players (mean ± standard deviation; 1774 ± 532 vs 1490 ± 447, 1261 ± 449, and 994 ± 373 m). For elite senior female players, wide midfielders (2057 ± 550 m) had a higher Yo-Yo IE2 test performance (P < 0.05) than central defenders (1588 ± 534 m) and attackers (1516 ± 401 m), but not central midfielders (1764 ± 473 m) or full-backs (1964 ± 522 m). Large correlations were observed between Yo-Yo IE2 test performance and the total and high-intensity distance covered (r = 0.55; P < 0.05) during elite senior soccer matches (r = 0.70; P < 0.01). A large correlation was also obtained between Yo-Yo IE2 test performance and (r = 0.68; P < 0.01). Performances in the Yo-Yo IE2 test were greater (P < 0.05) in the middle and the end of the season compared with the preparation period for elite youth female players (1767 ± 539 and 1742 ± 503 vs 1564 ± 504 m) and in elite senior female players, Yo-Yo IE2 test performance increased by 14% (P < 0.01) after completing 4 weeks of intense training prior to the FIFA Women's World Cup Finals (2049 ± 283 vs 1803 ± 342 m). The data demonstrate that the Yo-Yo IE2 test is reproducible and is an indicator of the match-specific physical capacity of female soccer players. Furthermore, the Yo-Yo IE2 test illustrates sensitivity by differentiating intermittent exercise performance of female players in various competitive levels, stages of the season and playing positions.

Binge-pattern ethanol exposure during adolescence, but not adulthood, causes persistent changes in GABAA receptor-mediated tonic inhibition in dentate granule cells.

BACKGROUND: In recent years, it has become clear that acute ethanol (EtOH) affects various neurobiological and behavioral functions differently in adolescent animals than in adults. However, less is known about the long-term neural consequences of chronic EtOH exposure during adolescence, and most importantly whether adolescence represents a developmental period of enhanced vulnerability to such effects. METHODS: We made whole-cell recordings of GABAA receptor-mediated tonic inhibitory currents from dentate gyrus granule cells (DGGCs) in hippocampal slices from adult rats that had been treated with chronic intermittent ethanol (CIE) or saline during adolescence, young adulthood, or adulthood. RESULTS: CIE reduced baseline tonic current amplitude in DGGCs from animals pretreated with EtOH during adolescence, but not in GCs from those pretreated with EtOH during young adulthood or adulthood. Similarly, the enhancement of tonic currents by acute EtOH exposure ex vivo was increased in GCs from animals pretreated with EtOH during adolescence, but not in those from animals pretreated during either of the other 2 developmental periods. CONCLUSIONS: These findings underscore our recent report that CIE during adolescence results in enduring alterations in tonic current and its acute EtOH sensitivity and establish that adolescence is a developmental period during which the hippocampal formation is distinctively vulnerable to long-term alteration by chronic EtOH exposure.

Long-term modulation of A-type K(+) conductances in hippocampal CA1 interneurons in rats after chronic intermittent ethanol exposure during adolescence or adulthood.

BACKGROUND: Chronic alcohol use, especially exposure to alcohol during adolescence or young adulthood, is closely associated with cognitive deficits that may persist into adulthood. Therefore, it is essential to identify possible neuronal mechanisms underlying the observed deficits in learning and memory. Hippocampal interneurons play a pivotal role in regulating hippocampus-dependent learning and memory by exerting strong inhibition on excitatory pyramidal cells. The function of these interneurons is regulated not only by synaptic inputs from other types of neurons but is also precisely governed by their own intrinsic membrane ionic conductances. The voltage-gated A-type potassium current (IA ) regulates the intrinsic membrane properties of neurons, and disruption of IA is responsible for many neuropathological processes including learning and memory deficits. Thus, it represents a previously unexplored cellular mechanism whereby chronic ethanol (EtOH) may alter hippocampal memory-related functioning. METHODS: Using whole-cell electrophysiological recording methods, we investigated the enduring effects of chronic intermittent ethanol (CIE) exposure during adolescence or adulthood on IA in rat CA1 interneurons. RESULTS: We found that the mean peak amplitude of IA was significantly reduced after CIE in either adolescence or adulthood, but IA density was attenuated after CIE in adolescence but not after CIE in adulthood. In addition, the voltage-dependent steady-state activation and inactivation of IA were altered in interneurons after CIE. CONCLUSIONS: These findings suggest that CIE can cause long-term changes in IA channels in interneurons and thus may alter their inhibitory influences on memory-related local hippocampal circuits, which could be, in turn, responsible for learning and memory impairments observed after chronic EtOH exposure.

Functional haploinsufficiency of the human homeobox gene MSX2 causes defects in skull ossification.

The genetic analysis of congenital skull malformations provides insight into normal mechanisms of calvarial osteogenesis. Enlarged parietal foramina (PFM) are oval defects of the parietal bones caused by deficient ossification around the parietal notch, which is normally obliterated during the fifth fetal month. PFM are usually asymptomatic, but may be associated with headache, scalp defects and structural or vascular malformations of the brain. Inheritance is frequently autosomal dominant, but no causative mutations have been identified in non-syndromic cases. We describe here heterozygous mutations of the homeobox gene MSX2 (located on 5q34-q35) in three unrelated families with PFM. One is a deletion of approximately 206 kb including the entire gene and the others are intragenic mutations of the DNA-binding homeodomain (RK159-160del and R172H) that predict disruption of critical intramolecular and DNA contacts. Mouse Msx2 protein with either of the homeodomain mutations exhibited more than 85% reduction in binding to an optimal Msx2 DNA-binding site. Our findings contrast with the only described MSX2 homeodomain mutation (P148H), associated with craniosynostosis, that binds with enhanced affinity to the same target. This demonstrates that MSX2 dosage is critical for human skull development and suggests that PFM and craniosynostosis result, respectively, from loss and gain of activity in an MSX2-mediated pathway of calvarial osteogenic differentiation.

The detection of subtelomeric chromosomal rearrangements in idiopathic mental retardation.

A major challenge for human genetics is to identify new causes of mental retardation, which, although present in about 3% of individuals, is unexplained in more than half of all cases. We have developed a strategy to screen for the abnormal inheritance of subtelomeric DNA polymorphisms in individuals with mental retardation and have detected three abnormalities in 99 patients with normal routine karyotypes. Pulsed-field gel electrophoresis and reverse chromosome painting showed that one case arose from an interstitial or terminal deletion and two from the de novo inheritance of derivative translocation chromosomes. At least 6% of unexplained mental retardation is accounted for by these relatively small chromosomal abnormalities, which will be an important resource in the characterization of the genetic basis of neurodevelopment.

Exclusive paternal origin of new mutations in Apert syndrome.

Apert syndrome results from one or other of two specific nucleotide substitutions, both C-->G transversions, in the fibroblast growth factor receptor 2 (FGFR2) gene. The frequency of new mutations, estimated as 1 per 65,000 live births, implies germline transversion rates at these two positions are currently the highest known in the human genome. Using a novel application of the amplification refractory mutation system (ARMS), we have determined the parental origin of the new mutation in 57 Apert families: in every case, the mutation arose from the father. This identifies the biological basis of the paternal age effect for new mutations previously suggested for this disorder.

Apert syndrome results from localized mutations of FGFR2 and is allelic with Crouzon syndrome.

Apert syndrome is a distinctive human malformation comprising craniosynostosis and severe syndactyly of the hands and feet. We have identified specific missense substitutions involving adjacent amino acids (Ser252Trp and Pro253Arg) in the linker between the second and third extracellular immunoglobulin (Ig) domains of fibroblast growth factor receptor 2 (FGFR2) in all 40 unrelated cases of Apert syndrome studied. Crouzon syndrome, characterized by craniosynostosis but normal limbs, was previously shown to result from allelic mutations of the third Ig domain of FGFR2. The contrasting effects of these mutations provide a genetic resource for dissecting the complex effects of signal transduction through FGFRs in cranial and limb morphogenesis.

Dominant mutations in ROR2, encoding an orphan receptor tyrosine kinase, cause brachydactyly type B.

Inherited limb malformations provide a valuable resource for the identification of genes involved in limb development. Brachydactyly type B (BDB), an autosomal dominant disorder, is the most severe of the brachydactylies and characterized by terminal deficiency of the fingers and toes. In the typical form of BDB, the thumbs and big toes are spared, sometimes with broadening or partial duplication. The BDB1 locus was previously mapped to chromosome 9q22 within an interval of 7.5 cM (refs 9,10). Here we describe mutations in ROR2, which encodes the orphan receptor tyrosine kinase ROR2 (ref. 11), in three unrelated families with BDB1. We identified distinct heterozygous mutations (2 nonsense, 1 frameshift) within a 7-amino-acid segment of the 943-amino-acid protein, all of which predict truncation of the intracellular portion of the protein immediately after the tyrosine kinase domain. The localized nature of these mutations suggests that they confer a specific gain of function. We obtained further evidence for this by demonstrating that two patients heterozygous for 9q22 deletions including ROR2 do not exhibit BDB. Expression of the mouse mouse orthologue, Ror2, early in limb development indicates that BDB arises as a primary defect of skeletal patterning.

Haploinsufficiency of the human homeobox gene ALX4 causes skull ossification defects.

Inherited defects of skull ossification often manifest as symmetric parietal foramina (PFM; MIM 168500). We previously identified mutations of MSX2 in non-syndromic PFM and demonstrated genetic heterogeneity. Deletions of 11p11-p12 (proximal 11p deletion syndrome, P11pDS; MIM 601224) are characterized by multiple exostoses, attributable to haploinsufficiency of EXT2 and PFM. Here we identify ALX4, which encodes a paired-related homeodomain transcription factor, as the PFM disease gene in P11pDS.

Recessive Robinow syndrome, allelic to dominant brachydactyly type B, is caused by mutation of ROR2.

The autosomal recessive form of Robinow syndrome (RRS; MIM 268310) is a severe skeletal dysplasia with generalized limb bone shortening, segmental defects of the spine, brachydactyly and a dysmorphic facial appearance. We previously mapped the gene mutated in RRS to chromosome 9q22 (ref. 4), a region that overlaps the locus for autosomal dominant brachydactyly type B (refs 5,6). The recent identification of ROR2, encoding an orphan receptor tyrosine kinase, as the gene mutated in brachydactyly type B (BDB1; ref. 7) and the mesomelic dwarfing in mice homozygous for a lacZ and/or a neo insertion into Ror2 (refs 8,9) made this gene a candidate for RRS. Here we report homozygous missense mutations in both intracellular and extracellular domains of ROR2 in affected individuals from 3 unrelated consanguineous families, and a nonsense mutation that removes the tyrosine kinase domain and all subsequent 3' regions of the gene in 14 patients from 7 families from Oman. The nature of these mutations suggests that RRS is caused by loss of ROR2 activity. The identification of mutations in three distinct domains (containing Frizzled-like, kringle and tyrosine kinase motifs) indicates that these are all essential for ROR2 function.

In the rat, chronic intermittent ethanol exposure during adolescence alters the ethanol sensitivity of tonic inhibition in adulthood.

BACKGROUND: Alcohol drinking by adolescents is a major public health concern. Adolescents tend to drink in a chronic, intermittent, that is, "binge," pattern, and such patterns of ethanol exposure are associated with increased risk of neurotoxicity and the development of alcohol use disorders (Crews et al., 2000; Hunt, 1993). Both adolescent humans and rats are more sensitive to acute ethanol-induced memory impairment than adults (Acheson et al., 1998; Markwiese et al., 1998). Furthermore, in rats, chronic intermittent ethanol (CIE) exposure during adolescence produces a long-lasting, perhaps permanent, maintenance of the adolescent high sensitivity to ethanol's amnestic effects (White et al., 2000a). We have previously shown that acute ethanol increases tonic inhibitory current mediated by extrasynaptic GABA(A) receptors more efficaciously in dentate granule cells (DGCs) from adolescent than adult rats (Fleming et al., 2007). In this study, we determined if CIE during adolescence produced long-lasting changes in this tonic current. METHODS: Adolescent rats were subjected to a CIE exposure regimen and allowed to mature to full adulthood. Whole-cell voltage-clamp measurements of tonic inhibitory current and mean phasic current were made in vitro in hippocampal brain slices. RESULTS: CIE exposure during adolescence increased the ethanol sensitivity of tonic inhibitory current mediated by extrasynaptic GABA(A) receptors and decreased the ethanol sensitivity of phasic, synaptic GABA(A) receptor-mediated current in adult DGCs. CONCLUSIONS: CIE exposure during adolescence produces long-lasting changes in the function and ethanol sensitivity of extrasynaptic GABA(A) receptors in DGCs. These changes appear to "lock-in" and maintain the high adolescent sensitivity to ethanol in these cells. Furthermore, greater ethanol enhancement of tonic inhibition in the hippocampal formation after CIE is consistent with the greater sensitivity to ethanol-induced memory impairment after adolescent CIE. This finding represents the first demonstration of a long-term, memory-related cellular effect of CIE during adolescence, and the "lock-in" of adolescent ethanol sensitivity that these results suggest could represent a conceptual step forward in understanding the vulnerability of the adolescent brain to alcohol.

Dopamine attenuates evoked inhibitory synaptic currents in central amygdala neurons.

The central nucleus of the amygdala (CeA) plays a critical role in regulating the behavioral, autonomic and endocrine response to stress. Dopamine (DA) participates in mediating the stress response and DA release is enhanced in the CeA during stressful events. However, the electrophysiological effects of DA on CeA neurons have not yet been characterized. Therefore, the purpose of this study was to identify and characterize the effect of DA application on electrophysiological responses of CeA neurons in coronal brain sections of male Sprague-Dawley rats. We used whole-cell patch-clamp electrophysiological techniques to record evoked synaptic responses and to determine basic membrane properties of CeA neurons both before and after DA superfusion. DA (20-250 µM) did not significantly alter membrane conductance over the voltage range tested. However, DA significantly reduced the peak amplitude of evoked inhibitory synaptic currents in CeA neurons. Pretreatment with the D(2) receptor antagonist eticlopride failed to significantly block the inhibitory effects of DA. In contrast, pretreatment with the D(1) receptor antagonist SCH-23390 significantly reduced the effects of DA on evoked inhibitory neurotransmission in these neurons. Moreover, bath superfusion of the specific D(1) receptor agonist SKF-39393, but not the D(2) receptor agonist quinpirole, significantly reduced peak amplitude of evoked inhibitory synaptic events. DA reduced the frequency of miniature IPSCs without altering the amplitude, while having no effect on the amplitude of IPSCs elicited by pressure application of GABA. These results suggest that DA may modulate inhibitory synaptic transmission in CeA through D(1) receptor activation primarily by a presynaptic mechanism.

Differential sensitivity of hippocampal interneurons to ethanol in adolescent and adult rats.

Ethanol (EtOH) promotes GABAergic synaptic transmission in the central nervous system. We have shown that EtOH enhances the frequency of GABA(A) receptor-mediated spontaneous inhibitory postsynaptic currents less powerfully in hippocampal CA1 pyramidal neurons from adolescent animals compared with those from adults. However, we have also shown that EtOH promotes the firing of hippocampal interneurons, located in stratum lacunosum moleculare (SLM), from adolescent animals more potently than in those from adults. Thus the latter finding would seem to be inconsistent with the former. To understand this apparent inconsistency, we have now assessed the effects of EtOH on a different subpopulation of hippocampal interneurons, those with somata located in the stratum oriens (SO). We found that EtOH-induced enhancement of the frequency of spontaneous action potentials (sAPs) was less in interneurons from adolescent rats compared with those from adults. In addition, EtOH-induced reduction of the afterhyperpolarization decay time constant (τ(slow)) was less pronounced in interneurons from adolescent rats, as was the EtOH-induced increase in the amplitude of the hyperpolarization-activated cation current, I(h). The effect of EtOH on sAP firing frequency was blocked by application of the I(h) antagonist 4-ethylphenylamino-1,2-dimethyl-6-methylaminopyrimidinium chloride (ZD7288). These results indicate that although EtOH promotes the firing of hippocampal interneurons, through promotion of I(h), the developmental expression of this effect differs between interneurons with somata located in the SO and SLM.

Role of cannabinoid receptor type 1 desensitization in greater tetrahydrocannabinol impairment of memory in adolescent rats.

Adolescence is a well defined developmental period during which marijuana use is common. However, little is known about the response to marijuana in adolescents compared with adults. We have shown previously that adolescent rats are more impaired than adults by Δ(9)-tetrahydrocannabinol (THC), the main psychoactive compound in marijuana, in a spatial learning task, but the mechanism responsible for this differential impairment is not understood. We determined the role of THC tolerance and cannabinoid receptor type 1 (CB1) regulation in THC-induced spatial learning impairment in adolescent and adult rats. We measured the development of tolerance to THC-induced learning impairment in adolescent (postnatal days 30-35) and adult (postnatal days 70-75) rats. We pretreated them for 5 days with 10 mg/kg THC, and then evaluated the effects of vehicle or THC treatment on learning during training in the Morris water maze. We also determined CB1 number and functional coupling in the hippocampus of adolescents and adults. Finally, we measured the time course of hippocampal CB1 desensitization in adolescents and adults during treatment with 10 mg/kg THC or vehicle. Our results indicate that adults, but not adolescents, become tolerant to the effects of THC during water maze training after 5 days of pretreatment. CB1s in adolescent hippocampus are less functionally coupled to G proteins and desensitize more slowly in response to THC treatment than those of adults. THC may impair learning in adolescents more than in adults because of delayed activation of cellular homeostatic adaptive mechanisms underlying cannabinoid tolerance in the hippocampus.

Genetic screening of 202 individuals with congenital limb malformations and requiring reconstructive surgery.

BACKGROUND: Congenital limb malformations (CLMs) are common and present to a variety of specialties, notably plastic and orthopaedic surgeons, and clinical geneticists. The authors aimed to characterise causative mutations in an unselected cohort of patients with CLMs requiring reconstructive surgery. METHODS: 202 patients presenting with CLM were recruited. The authors obtained G-banded karyotypes and screened EN1, GLI3, HAND2, HOXD13, ROR2, SALL1, SALL4, ZRS of SHH, SPRY4, TBX5, TWIST1 and WNT7A for point mutations using denaturing high performance liquid chromatography (DHPLC) and direct sequencing. Multiplex ligation dependent probe amplification (MLPA) kits were developed and used to measure copy number in GLI3, HOXD13, ROR2, SALL1, SALL4, TBX5 and the ZRS of SHH. RESULTS: Within the cohort, causative genetic alterations were identified in 23 patients (11%): mutations in GLI3 (n = 5), HOXD13 (n = 5), the ZRS of SHH (n = 4), and chromosome abnormalities (n = 4) were the most common lesions found. Clinical features that predicted the discovery of a genetic cause included a bilateral malformation, positive family history, and having increasing numbers of limbs affected (all p<0.01). Additionally, specific patterns of malformation predicted mutations in specific genes. CONCLUSIONS: Based on higher mutation prevalence the authors propose that GLI3, HOXD13 and the ZRS of SHH should be prioritised for introduction into molecular genetic testing programmes for CLM. The authors have developed simple criteria that can refine the selection of patients by surgeons for referral to clinical geneticists. The cohort also represents an excellent resource to test for mutations in novel candidate genes.

MRNIP is a replication fork protection factor.

The remodeling of stalled replication forks to form four-way DNA junctions is an important component of the replication stress response. Nascent DNA at the regressed arms of these reversed forks is protected by RAD51 and the tumor suppressors BRCA1/2, and when this function is compromised, stalled forks undergo pathological MRE11-dependent degradation, leading to chromosomal instability. However, the mechanisms regulating MRE11 functions at reversed forks are currently unclear. Here, we identify the MRE11-binding protein MRNIP as a novel fork protection factor that directly binds to MRE11 and specifically represses its exonuclease activity. The loss of MRNIP results in impaired replication fork progression, MRE11 exonuclease-dependent degradation of reversed forks, persistence of underreplicated genomic regions, chemosensitivity, and chromosome instability. Our findings identify MRNIP as a novel regulator of MRE11 at reversed forks and provide evidence that regulation of specific MRE11 nuclease activities ensures protection of nascent DNA and thereby genome integrity.

The novel micro-opioid receptor antagonist, [N-allyl-Dmt(1)]endomorphin-2, attenuates the enhancement of GABAergic neurotransmission by ethanol.

AIMS: We investigated the effects of [N-allyl-Dmt(1)]endomorphin-2 (TL-319), a novel and highly potent micro-opioid receptor antagonist, on ethanol (EtOH)-induced enhancement of GABA(A) receptor-mediated synaptic activity in the hippocampus. METHODS: Evoked and spontaneous inhibitory postsynaptic currents (eIPSCs and sIPSCs) were isolated from CA1 pyramidal cells from brain slices of male rats using whole-cell patch-clamp techniques. RESULTS: TL-319 had no effect on the baseline amplitude of eIPSCs or the frequency of sIPSCs. However, it induced a dose-dependent suppression of an ethanol-induced increase of sIPSC frequency with full reversal at concentrations of 500 nM and higher. The non-specific competitive opioid receptor antagonist naltrexone also suppressed EtOH-induced increases in sIPSC frequency but only at a concentration of 60 microM. CONCLUSION: These data indicate that blockade of micro-opioid receptors by low concentrations of [N-allyl-Dmt(1)]endomorphin-2 can reverse ethanol-induced increases in GABAergic neurotransmission and possibly alter its anxiolytic or sedative effects. This suggests the possibility that high potency opioid antagonists may emerge as possible candidate compounds for the treatment of ethanol addiction.