PACAP-PAC1R modulates fear extinction via the ventromedial hypothalamus.

Exposure to traumatic stress can lead to fear dysregulation, which has been associated with posttraumatic stress disorder (PTSD). Previous work showed that a polymorphism in the PACAP-PAC1R (pituitary adenylate cyclase-activating polypeptide) system is associated with PTSD risk in women, and PACAP (ADCYAP1)-PAC1R (ADCYAP1R1) are highly expressed in the hypothalamus. Here, we show that female mice subjected to acute stress immobilization (IMO) have fear extinction impairments related to Adcyap1 and Adcyap1r1 mRNA upregulation in the hypothalamus, PACAP-c-Fos downregulation in the Medial Amygdala (MeA), and PACAP-FosB/ΔFosB upregulation in the Ventromedial Hypothalamus dorsomedial part (VMHdm). DREADD-mediated inhibition of MeA neurons projecting to the VMHdm during IMO rescues both PACAP upregulation in VMHdm and the fear extinction impairment. We also found that women with the risk genotype of ADCYAP1R1 rs2267735 polymorphism have impaired fear extinction.

Medical cannabis vs. synthetic cannabinoids: What does the future hold?

The medical use of cannabis has an intricate therapeutic history that finds its roots in ancient China (∼2700 BC). The main psychoactive component of cannabis, Δ(9) -tetrahydrocannabinol (Δ(9) -THC), was discovered in 1964. This was a significant breakthrough, as it allowed the generation of synthetic analogs of Δ(9) -THC, the discovery of cannabinoid receptors, and the generation of synthetic small molecules. Despite this, today there is still a paucity of drugs that target the cannabinoid system.

Multiple casualties in the Bastion UK Role 3 hospital: personal reflections on the positive evolution of deployed military hospital care.

In this article a snapshot of casualty presentations to the UK Role 3 hospital in Camp Bastion, Afghanistan, will be briefly described. The observations allow reflection on the advances and strength of clinical provision at the time of the incident, written from a medical command perspective.

The atypical cannabinoid O-1602 stimulates food intake and adiposity in rats.

AIMS: Cannabinoids are known to control energy homeostasis. Atypical cannabinoids produce pharmacological effects via unidentified targets. We sought to investigate whether the atypical cannabinoid O-1602 controls food intake and body weight. METHODS: The rats were injected acutely or subchronically with O-1602, and the expression of several factors involved in adipocyte metabolism was assessed by real-time polymerase chain reaction. In vivo findings were corroborated with in vitro studies incubating 3T3-L1 adipocytes with O-1602, and measuring intracellular calcium and lipid accumulation. Finally, as some reports suggest that O-1602 is an agonist of the putative cannabinoid receptor GPR55, we tested it in mice lacking GPR55. RESULTS: Central and peripheral administration of O-1602 acutely stimulates food intake, and chronically increases adiposity. The hyperphagic action of O-1602 is mediated by the downregulation of mRNA and protein levels of the anorexigenic neuropeptide cocaine- and amphetamine-regulated transcript. The effects on fat mass are independent of food intake, and involve a decrease in the expression of lipolytic enzymes such as hormone sensitive lipase and adipose triglyceride lipase in white adipose tissue. Consistently, in vitro data showed that O-1602 increased the levels of intracellular calcium and lipid accumulation in adipocytes. Finally, we injected O-1602 in GPR55 -/- mice and found that O-1602 was able to induce feeding behaviour in GPR55-deficient mice. CONCLUSIONS: These findings show that O-1602 modulates food intake and adiposity independently of GPR55 receptor. Thus atypical cannabinoids may represent a novel class of molecules involved in energy balance.

Cannabinoids and bone: endocannabinoids modulate human osteoclast function in vitro.

BACKGROUND AND PURPOSE: Both CB(1) and CB(2) cannabinoid receptors have been shown to play a role in bone metabolism. Crucially, previous studies have focussed on the effects of cannabinoid ligands in murine bone cells. This study aimed to investigate the effects of cannabinoids on human bone cells in vitro. EXPERIMENTAL APPROACH: Quantitative RT-PCR was used to determine expression of cannabinoid receptors and liquid chromatography-electrospray ionization tandem mass spectrometry was used to determine the presence of endocannabinoids in human bone cells. The effect of cannabinoids on human osteoclast formation, polarization and resorption was determined by assessing the number of cells expressing α(v) ß(3) or with F-actin rings, or measurement of resorption area. KEY RESULTS: Human osteoclasts express both CB(1) and CB(2) receptors. CB(2) expression was significantly higher in human monocytes compared to differentiated osteoclasts. Furthermore, the differentiation of human osteoclasts from monocytes was associated with a reduction in 2-AG levels and an increase in anandamide (AEA) levels. Treatment of osteoclasts with LPS significantly increased levels of AEA. Nanomolar concentrations of AEA and the synthetic agonists CP 55 940 and JWH015 stimulated human osteoclast polarization and resorption; these effects were attenuated in the presence of CB(1) and/or CB(2) antagonists. CONCLUSIONS: AND IMPLICATIONS Low concentrations of cannabinoids activate human osteoclasts in vitro. There is a dynamic regulation of the expression of the CB(2) receptor and the production of the endocannabinoids during the differentiation of human bone cells. These data suggest that small molecules modulating the endocannabinoid system could be important therapeutics in human bone disease. LINKED ARTICLES: This article is part of a themed section on Cannabinoids in Biology and Medicine. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2012.165.issue-8. To view Part I of Cannabinoids in Biology and Medicine visit http://dx.doi.org/10.1111/bph.2011.163.issue-7.

International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB1 and CB2.

There are at least two types of cannabinoid receptors (CB(1) and CB(2)). Ligands activating these G protein-coupled receptors (GPCRs) include the phytocannabinoid Δ(9)-tetrahydrocannabinol, numerous synthetic compounds, and endogenous compounds known as endocannabinoids. Cannabinoid receptor antagonists have also been developed. Some of these ligands activate or block one type of cannabinoid receptor more potently than the other type. This review summarizes current data indicating the extent to which cannabinoid receptor ligands undergo orthosteric or allosteric interactions with non-CB(1), non-CB(2) established GPCRs, deorphanized receptors such as GPR55, ligand-gated ion channels, transient receptor potential (TRP) channels, and other ion channels or peroxisome proliferator-activated nuclear receptors. From these data, it is clear that some ligands that interact similarly with CB(1) and/or CB(2) receptors are likely to display significantly different pharmacological profiles. The review also lists some criteria that any novel "CB(3)" cannabinoid receptor or channel should fulfil and concludes that these criteria are not currently met by any non-CB(1), non-CB(2) pharmacological receptor or channel. However, it does identify certain pharmacological targets that should be investigated further as potential CB(3) receptors or channels. These include TRP vanilloid 1, which possibly functions as an ionotropic cannabinoid receptor under physiological and/or pathological conditions, and some deorphanized GPCRs. Also discussed are 1) the ability of CB(1) receptors to form heteromeric complexes with certain other GPCRs, 2) phylogenetic relationships that exist between CB(1)/CB(2) receptors and other GPCRs, 3) evidence for the existence of several as-yet-uncharacterized non-CB(1), non-CB(2) cannabinoid receptors; and 4) current cannabinoid receptor nomenclature.

Biochanin A, a naturally occurring inhibitor of fatty acid amide hydrolase.

BACKGROUND AND PURPOSE: Inhibitors of fatty acid amide hydrolase (FAAH), the enzyme responsible for the metabolism of the endogenous cannabinoid (CB) receptor ligand anandamide (AEA), are effective in a number of animal models of pain. Here, we investigated a series of isoflavones with respect to their abilities to inhibit FAAH. EXPERIMENTAL APPROACH: In vitro assays of FAAH activity and affinity for CB receptors were used to characterize key compounds. In vivo assays used were biochemical responses to formalin in anaesthetized mice and the 'tetrad' test for central CB receptor activation. KEY RESULTS: Of the compounds tested, biochanin A was adjudged to be the most promising. Biochanin A inhibited the hydrolysis of 0.5 microM AEA by mouse, rat and human FAAH with IC(50) values of 1.8, 1.4 and 2.4 microM respectively. The compound did not interact to any major extent with CB(1) or CB(2) receptors, nor with FAAH-2. In anaesthetized mice, URB597 (30 microg i.pl.) and biochanin A (100 microg i.pl.) both inhibited the spinal phosphorylation of extracellular signal-regulated kinase produced by the intraplantar injection of formalin. The effects of both compounds were significantly reduced by the CB(1) receptor antagonist/inverse agonist AM251 (30 microg i.pl.). Biochanin A (15 mg.kg(-1) i.v.) did not increase brain AEA concentrations, but produced a modest potentiation of the effects of 10 mg.kg(-1) i.v. AEA in the tetrad test. CONCLUSIONS AND IMPLICATIONS: It is concluded that biochanin A, in addition to its other biochemical properties, inhibits FAAH both in vitro and peripherally in vivo.

Differential effects of hypothalamic long-chain fatty acid infusions on suppression of hepatic glucose production.

Our objective was to investigate whether the direct bilateral infusion of the monounsaturated fatty acid (MUFA) oleic acid (OA) within the mediobasal hypothalamus (MBH) is sufficient to reproduce the effect of administration of OA (30 nmol) in the third cerebral ventricle, which inhibits glucose production (GP) in rats. We used the pancreatic basal insulin clamp technique (plasma insulin ∼20 mU/ml) in combination with tracer dilution methodology to compare the effect of MBH OA on GP to that of a saturated fatty acid (SFA), palmitic acid (PA), and a polyunsaturated fatty acid (PUFA), linoleic acid (LA). The MBH infusion of 200 but not 40 pmol of OA was sufficient to markedly inhibit GP (by 61% from 12.6 ± 0.6 to 5.1 ± 1.6 mg·kg(-1)·min(-1)) such that exogenous glucose had to be infused at the rate of 6.0 ± 1.2 mg·kg(-1)·min(-1) to prevent hypoglycemia. MBH infusion of PA also caused a significant decrease in GP, but only at a total dose of 4 nmol (GP 5.8 ± 1.6 mg·kg(-1)·min(-1)). Finally, MBH LA at a total dose of 0.2 and 4 nmol failed to modify GP compared with rats receiving MBH vehicle. Increased availability of OA within the MBH is sufficient to markedly inhibit GP. LA does not share the effect of OA, whereas PA can reproduce the potent effect of OA on GP, but only at a higher dose. It remains to be determined whether SFAs need to be converted to MUFAs to exert this effect or whether they activate a separate signaling pathway to inhibit GP.

Fluorescent ligand binding reveals heterogeneous distribution of adrenoceptors and 'cannabinoid-like' receptors in small arteries.

BACKGROUND AND PURPOSE: Pharmacological analysis of synergism or functional antagonism between different receptors commonly assumes that interacting receptors are located in the same cells. We have now investigated the distribution of alpha-adrenoceptors, beta-adrenoceptors and cannabinoid-like (GPR55) receptors in the mouse arteries. EXPERIMENTAL APPROACH: Fluorescence intensity from vascular tissue incubated with fluorescent ligands (alpha(1)-adrenoceptor ligand, BODIPY-FL-prazosin, QAPB; beta-adrenoceptor ligand, TMR-CGP12177; fluorescent angiotensin II; a novel diarylpyrazole cannabinoid ligand (Tocrifluor 1117, T1117) was measured with confocal microscopy. Small mesenteric and tail arteries of wild-type and alpha(1B/D)-adrenoceptor-KO mice were used. KEY RESULTS: T1117, a fluorescent form of the cannabinoid CB(1) receptor antagonist AM251, was a ligand for GPR55, with low affinity for CB(1) receptors. In mesenteric arterial smooth muscle cells, alpha(1A)-adrenoceptors were predominantly located in different cells from those with beta-adrenoceptors, angiotensin receptors or cannabinoid-like (GPR55) receptors. Cells with beta-adrenoceptors predominated at arterial branches. Endothelial cells expressed beta-adrenoceptors, alpha-adrenoceptors and cannabinoid-like receptors. Only endothelial alpha-adrenoceptors appeared in clusters. Adventitia was a rich source of G protein-coupled receptors (GPCRs), particularly fibroblasts and nerve tracts, where Schwann cells bound alpha-adrenoceptor, beta-adrenoceptor and CB-receptor ligands, with a mix of separate receptor locations and co-localization. CONCLUSIONS AND IMPLICATIONS: Within each cell type, each GPCR had a distinctive heterogeneous distribution with limited co-localization, providing a guide to the possibilities for functional synergism, and suggesting a new paradigm for synergism in which interactions may be either between cells or involve converging intracellular signalling processes.

Evidence that the plant cannabinoid cannabigerol is a highly potent alpha2-adrenoceptor agonist and moderately potent 5HT1A receptor antagonist.

BACKGROUND AND PURPOSE: Cannabis is the source of at least seventy phytocannabinoids. The pharmacology of most of these has been little investigated, three notable exceptions being Delta(9)-tetrahydrocannabinol, cannabidiol and Delta(9)-tetrahydrocannabivarin. This investigation addressed the question of whether the little-studied phytocannabinoid, cannabigerol, can activate or block any G protein-coupled receptor. EXPERIMENTAL APPROACH: The [(35)S]GTPgammaS binding assay, performed with mouse brain membranes, was used to test the ability of cannabigerol to produce G protein-coupled receptor activation or blockade. Its ability to displace [(3)H]CP55940 from mouse CB(1) and human CB(2) cannabinoid receptors and to inhibit electrically evoked contractions of the mouse isolated vas deferens was also investigated. KEY RESULTS: In the brain membrane experiments, cannabigerol behaved as a potent alpha(2)-adrenoceptor agonist (EC(50)= 0.2 nM) and antagonized the 5-HT(1A) receptor agonist, R-(+)-8-hydroxy-2-(di-n-propylamino)tetralin (apparent K(B)= 51.9 nM). At 10 microM, it also behaved as a CB(1) receptor competitive antagonist. Additionally, cannabigerol inhibited evoked contractions of the vas deferens in a manner that appeared to be alpha(2)-adrenoceptor-mediated (EC(50)= 72.8 nM) and displayed significant affinity for mouse CB(1) and human CB(2) receptors. CONCLUSIONS AND IMPLICATIONS: This investigation has provided the first evidence that cannabigerol can activate alpha(2)-adrenoceptors, bind to cannabinoid CB(1) and CB(2) receptors and block CB(1) and 5-HT(1A) receptors. It will now be important to investigate why cannabigerol produced signs of agonism more potently in the [(35)S]GTPgammaS binding assay than in the vas deferens and also whether it can inhibit noradrenaline uptake in this isolated tissue and in the brain.

Modulation of sensory neuron potassium conductances by anandamide indicates roles for metabolites.

BACKGROUND AND PURPOSE: The endogenous cannabinoid anandamide (AEA) acts at cannabinoid (CB(1)) and vanilloid (TRPV(1)) receptors. AEA also shows antinociceptive properties; although the underlying mechanism for this is not fully understood, both CB(1) and TRPV(1) may be involved. Voltage-activated Ca(2+) channels in rat-cultured dorsal root ganglion (DRG) neurons are modulated by AEA. However, AEA in different populations of neurons enhanced or attenuated KCl-evoked Ca(2+) influx; these effects were linked with soma size. The aim of this study was to determine how AEA or its metabolites might produce these variable responses. EXPERIMENTAL APPROACH: The whole cell patch-clamp technique and fura-2 Ca(2+) imaging were used to characterize the actions of AEA on action potential firing and voltage-activated K(+) currents and to determine whether AEA metabolism plays any role in its effects on cultured DRG neurons. KEY RESULTS: AEA attenuated multiple action potential firing evoked by 300 ms depolarizing current commands in a subpopulation of DRG neurons. Application of 1 microM AEA attenuated voltage-activated K(+) currents and the recovery of KCl-evoked Ca(2+) transients. The insensitivity of these responses to the CB(1) receptor antagonist rimonabant (100 nM) and preincubation of DRG neurons with pertussis toxin suggested that these actions are not CB(1) receptor-mediated. Preincubating DRG neurons with the fatty acid amide hydrolase (FAAH) inhibitor phenylmethylsulphonyl fluoride (PMSF) attenuated the inhibitory actions of AEA on K(+) currents and Ca(2+) influx. CONCLUSION AND IMPLICATIONS: These data suggest that the products of AEA metabolism by FAAH contribute to the attenuation of K(+) conductances and altered excitability of cultured sensory neurons.

Chronic exposure of sensory neurones to increased levels of nerve growth factor modulates CB1/TRPV1 receptor crosstalk.

BACKGROUND: Anandamide (AEA) activates both cannabinoid CB(1) and TRPV1 receptors, which are expressed on cultured dorsal root ganglion neurones. Increased levels of nerve growth factor (NGF) are associated with chronic pain states. EXPERIMENTAL APPROACH: The aim of this study was to compare of the effects of AEA on CB(1) receptor signalling and TRPV1-CB(1) crosstalk in low and high concentrations of NGF, using voltage-clamp electrophysiology and Fura-2 calcium imaging. KEY RESULTS: Chronic exposure to high NGF (200 ng ml(-1)) as compared to low NGF (20 ng ml(-1)) increases the proportion of neurones that exhibit an inward current in response to AEA (1 microM), from 7 to 29%. In contrast, inhibition of voltage-gated calcium currents by AEA is not significantly different in low NGF (33+/-9%, compared to high NGF 28+/-6%). Crosstalk between CB and TRPV1 receptors is modulated by exposure to high NGF. In low NGF, exposure to the CB(1) receptor antagonist, SR141716A, (100 nM) increases the percentage of neurones in which AEA elicits an increase in [Ca(2+)](i), from 10 to 23%. In high NGF, the antagonist does not alter the percentage of responders (33 to 30%). In low NGF, exposure to the CB receptor agonist, WIN55 (1 microM) reduces capsaicin-mediated increases in [Ca(2+)](i) to 28+/-8% of control as compared to an enhancement to 172+/-26% of control observed in high NGF. CONCLUSIONS AND IMPLICATIONS: We conclude that cannabinoid-mediated modulation of TRPV1 receptor activation is altered after exposure to high NGF.

Tuning the endocannabinoid system: allosteric modulators of the CB1 receptor.

Cannabinoid CB1 receptor antagonists are novel therapeutics with potential for the treatment of a number of conditions including obesity, nicotine addition and metabolic syndrome. In 2005, Price et al. demonstrated that the cannabinoid CB1 receptor contains an allosteric-binding site which binds synthetic small molecules. In this issue of the British Journal of Pharmacology, Horswill et al. have extended these observations. They demonstrate that a structurally similar small molecule allosterically modulates the cannabinoid CB1 receptor and reduces body weight and food intake in an acute feeding model. Allosteric modulation now contends as a new strategy in the therapeutic exploitation of cannabinoid receptors that may offer certain advantages over the more familiar small molecules targeting the orthosteric site.

Cannabidiol displays unexpectedly high potency as an antagonist of CB1 and CB2 receptor agonists in vitro.

BACKGROUND AND PURPOSE: A nonpsychoactive constituent of the cannabis plant, cannabidiol has been demonstrated to have low affinity for both cannabinoid CB1 and CB2 receptors. We have shown previously that cannabidiol can enhance electrically evoked contractions of the mouse vas deferens, suggestive of inverse agonism. We have also shown that cannabidiol can antagonize cannabinoid receptor agonists in this tissue with a greater potency than we would expect from its poor affinity for cannabinoid receptors. This study aimed to investigate whether these properties of cannabidiol extend to CB1 receptors expressed in mouse brain and to human CB2 receptors that have been transfected into CHO cells. EXPERIMENTAL APPROACH: The [35S]GTPS binding assay was used to determine both the efficacy of cannabidiol and the ability of cannabidiol to antagonize cannabinoid receptor agonists (CP55940 and R-(+)-WIN55212) at the mouse CB1 and the human CB2 receptor. KEY RESULTS: This paper reports firstly that cannabidiol displays inverse agonism at the human CB2 receptor. Secondly, we demonstrate that cannabidiol is a high potency antagonist of cannabinoid receptor agonists in mouse brain and in membranes from CHO cells transfected with human CB2 receptors. CONCLUSIONS AND IMPLICATIONS: This study has provided the first evidence that cannabidiol can display CB2 receptor inverse agonism, an action that appears to be responsible for its antagonism of CP55940 at the human CB2 receptor. The ability of cannabidiol to behave as a CB2 receptor inverse agonist may contribute to its documented anti-inflammatory properties.

The psychoactive plant cannabinoid, Delta9-tetrahydrocannabinol, is antagonized by Delta8- and Delta9-tetrahydrocannabivarin in mice in vivo.

BACKGROUND AND PURPOSE: To follow up in vitro evidence that Delta(9)-tetrahydrocannabivarin extracted from cannabis (eDelta(9)-THCV) is a CB(1) receptor antagonist by establishing whether synthetic Delta(9)-tetrahydrocannabivarin (O-4394) and Delta(8)-tetrahydrocannabivarin (O-4395) behave as CB(1) antagonists in vivo. EXPERIMENTAL APPROACH: O-4394 and O-4395 were compared with eDelta(9)-THCV as displacers of [(3)H]-CP55940 from specific CB(1) binding sites on mouse brain membranes and as antagonists of CP55940 in [(35)S]GTPgammaS binding assays performed with mouse brain membranes and of R-(+)-WIN55212 in mouse isolated vasa deferentia. Their ability to antagonize in vivo effects of 3 or 10 mg kg(-1) (i.v.) Delta(9)-tetrahydrocannabinol in mice was then investigated. KEY RESULTS: O-4394 and O-4395 exhibited similar potencies to eDelta(9)-THCV as displacers of [(3)H]-CP55940 (K (i)=46.6 and 64.4 nM, respectively) and as antagonists of CP55940 in the [(35)S]GTPgammaS binding assay (apparent K (B)=82.1 and 125.9 nM, respectively) and R-(+)-WIN55212 in the vas deferens (apparent K (B)=4.8 and 3.9 nM respectively). At i.v. doses of 0.1, 0.3, 1.0 and/or 3 mg kg(-1) O-4394 and O-4395 attenuated Delta(9)-tetrahydrocannabinol-induced anti-nociception (tail-flick test) and hypothermia (rectal temperature). O-4395 but not O-4394 also antagonized Delta(9)-tetrahydrocannabinol-induced ring immobility. By themselves, O-4395 and O-4394 induced ring immobility at 3 or 10 mg kg(-1) (i.v.) and antinociception at doses above 10 mg kg(-1) (i.v.). O-4395 also induced hypothermia at 3 mg kg(-1) (i.v.) and above. CONCLUSIONS AND IMPLICATIONS: O-4394 and O-4395 exhibit similar in vitro potencies to eDelta(9)-THCV as CB(1) receptor ligands and as antagonists of cannabinoid receptor agonists and can antagonize Delta(9)-tetrahydrocannabinol in vivo.

Effects of poor glucose handling on arterial stiffness and left ventricular mass in normal children.

AIM: Cardiovascular risk factors can be present in children and young adults. We previously found abnormal microvascular function in children who had glucose intolerance and insulin resistance. The aim of the present study was to investigate whether they also have abnormalities in left ventricular mass (LVM) and arterial stiffness. METHODS: We measured heart dimensions and LVM using echocardiography, and arterial stiffness using pulse wave analysis in 23 children with good glucose handling (postfeeding glucose: 3.9 to 5 mmol/L) and 21 with poor glucose handling (7.7 to 11.4 mmol/L). RESULTS: The time to pulse reflection was slightly shorter in the poorer glucose handlers (mean+/-SD: 143+/-10 vs 153+/-20 ms, P=0.04), suggestive of increased arterial stiffness. Also in this group, there were significant relationships between intraventricular septal thickness, blood pressure and body mass index, but not in the normal glucose handlers. CONCLUSIONS: We have found that normal children who are in the lowest quintile of glucose tolerance in comparison with their peers are exhibiting the first signs of arterial stiffening. In addition, we have seen the beginnings of a relationship between blood pressure, body mass index and left ventricular enlargement in this group. While these changes may not yet be clinically significant, their emergence might be further evidence of early predisposition to cardiovascular disease.

Loss of one HuD allele on chromosome #1p selects for amplification of the N-myc proto-oncogene in human neuroblastoma cells.

In human neuroblastoma tumors, amplification of the N-myc proto-oncogene and loss of all or part of the short arm of chromosome #1 are both associated with a poor prognosis. Accruing evidence indicates that it is the absence of one allele of the HuD (ELAVL4) gene, encoding the neuronal-specific RNA-binding protein HuD and localized to 1p34, that is linked to amplification. In 12 human neuroblastoma cell lines, N-myc amplification correlates with loss of one HuD allele and decreased HuD expression. Transfection experiments demonstrate that modulating HuD expression affects N-myc gene copy number as well as expression. Introduction of a sense HuD construct into two N-myc amplified cell lines considerably increases N-myc expression whereas gene copy number decreases. Conversely, expression of antisense HuD in N-myc nonamplified SH-SY5Y cells reduces HuD and N-myc mRNA levels even as cells show amplification of the N-myc gene. Thus, N-myc gene copy number is modulated by alteration of HuD expression. We propose that haploinsufficiency of HuD due to chromosome #1p deletion in neuroblastoma selects for cells that amplify N-myc genes. Application of these findings could lead to more effective therapies in the treatment of those patients with the worst prognosis.

Medical support to littoral manoeuvre.

My personal view, after some 16 years away from 3 Commando Brigade, is that there has been a vast improvement in the medical infrastructure, logistics and communications at Unit level. The defined treatment timelines provide medical staff with much needed leverage in securing scarce SH assets and the casualty evacuation plan from Role 1 to Role 2 is cogent and workable. The Role 2 capability is much more flexible and robust and can now be projected forward to where it is needed. And, of course, we are now supported by a superb Role 3 facility afloat. All in all medical support to Lit M is in good shape, even if we are all still "dripping" about our tents!

Cutting-balloon-associated vascular rupture after failed standard balloon angioplasty.

The following case reports illustrate a possible complication of vascular rupture when cutting balloon dilatation is performed immediately after failed standard balloon angioplasty to the same diameter. Deferral of the cutting balloon dilatation should be considered in such circumstances.