Designing Boron Nitride Islands in Carbon Materials for Efficient Electrochemical Synthesis of Hydrogen Peroxide.

Heteroatom-doped carbons have drawn increasing research interest as catalysts for various electrochemical reactions due to their unique electronic and surface structures. In particular, co-doping of carbon with boron and nitrogen has been shown to provide significant catalytic activity for oxygen reduction reaction (ORR). However, limited experimental work has been done to systematically study these materials, and much remains to be understood about the nature of the active site(s), particularly with regards to the factors underlying the activity enhancements of these boron-carbon-nitrogen (BCN) materials. Herein, we prepare several BCN materials experimentally with a facile and controlled synthesis method, and systematically study their electrochemical performance. We demonstrate the existence of h-BN domains embedded in the graphitic structures of these materials using X-ray spectroscopy. These synthesized structures yield higher activity and selectivity toward the 2e- ORR to H2O2 than structures with individual B or N doping. We further employ density functional theory calculations to understand the role of a variety of h-BN domains within the carbon lattice for the ORR and find that the interface between h-BN domains and graphene exhibits unique catalytic behavior that can preferentially drive the production of H2O2. To the best of our knowledge, this is the first example of h-BN domains in carbon identified as a novel system for the electrochemical production of H2O2.

MAPK signaling pathway regulates cerebrovascular receptor expression in human cerebral arteries.

BACKGROUND: Cerebral ischemia results in enhanced expression of contractile cerebrovascular receptors, such as endothelin type B (ET(B)), 5-hydroxytryptamine type 1B (5-HT(1B)), angiotensin II type 1 (AT(1)) and thromboxane (TP) receptors in the cerebral arteries within the ischemic area. The receptor upregulation occurs via activation of the mitogen-activated protein kinases (MAPK) pathway. Previous studies have shown that inhibitors of the MAPK pathway diminished the ischemic area and contractile cerebrovascular receptors after experimental cerebral ischemia. The aim of this study was to examine if the upregulation of contractile cerebrovascular receptors after 48 h of organ culture of human cerebral arteries involves MAPK pathways and if it can be prevented by a MEK1/2 inhibitor. Human cerebral arteries were obtained from patients undergoing intracranial tumor surgery. The vessels were divided into ring segments and incubated for 48 h in the presence or absence of the specific MEK1/2 inhibitor U0126. The vessels were then examined by using in vitro pharmacological methods and protein immunohistochemistry. RESULTS: After organ culture of the cerebral arteries the contractile responses to endothelin (ET)-1, angiotensin (Ang) II and thromboxane (TP) were enhanced in comparison with fresh human arteries. However, 5-carboxamidotryptamine (5-CT) induced decreased contractile responses after organ culture as compared to fresh arteries. Incubation with U0126 diminished the maximum contraction elicited by application of ET-1, Ang II and U46619 in human cerebral arteries. In addition, the MEK1/2 inhibitor decreased the contractile response to 5-CT. Immunohistochemistry revealed that organ culture resulted in increased expression of endothelin ET(A), endothelin ET(B) angiotensin AT(2), 5-hydroxytryptamine 5-HT(1B) and thromboxane A2 receptors, and elevated levels of activated pERK1/2, all localized to the smooth muscle cells of the cerebral arteries. Co-incubation with U0126 normalized these proteins. CONCLUSION: The study demonstrated that there is a clear association between human cerebrovascular receptor upregulation via transcription involving activation of the MAPK pathway after organ culture. Inhibition of the MAPK pathways attenuated the vasoconstriction mediated by ET, AT and TP receptors in human cerebral arteries and the enhanced expression of their receptors. The results indicate that MAPK inhibition might be a novel target for treatment of cerebrovascular disorders.

Comparison of responses to vasoactive drugs in human and rat cerebral arteries using myography and pressurized cerebral artery method.

BACKGROUND: Dilatation of cranial vessels has been proposed as a part of the cascade that initiates an episode of migraine. This is based on the observation that intravenous administration of several substances with vasodilator properties can trigger migraine-like symptoms in migraineurs. METHODS: We used in vitro myography of human cerebral arteries and in vitro pressurized arteriography of rat middle cerebral artery (MCA) to evaluate the vasomotor responses of cerebral arteries to increasing concentrations of vasoactive substances used to elicit migraine-like attacks. RESULTS: All substances except carbachol induced a strong vasodilatory response when applied to the abluminal side of a rat MCA but negligible response when applied to the luminal side. Luminal carbachol gave a strong dilatory response but a weak response at the abluminal side. The prostaglandins PGE(2) and epoprostenol constricted the rat MCA while human cerebral arteries relaxed. The pEC(50) of carbachol, histamine, epoprostenol, VIP and sildenafil differed significantly between cerebral arteries from man and rat. The differences in pEC(50) for SNP, αCGRP, PACAP-27 and PACAP-38 were not significant between the species. PGE(2) had no noticeable effect on human arteries in vitro. CONCLUSION: All tested substances with the exception of VIP and carbachol have been found to elicit migraine-like attacks in migraineurs. Since these two agents have vasodilatory effects in humans, it suggests that vasodilatation is not the only reason for eliciting a migraine-like attack in migraineurs. In addition, there are significant species differences that show the importance of performing experiments in human vessels.

Effect of the calcitonin gene-related peptide (CGRP) receptor antagonist telcagepant in human cranial arteries.

INTRODUCTION: Calcitonin gene-related peptide (CGRP) is a neuronal messenger in intracranial sensory nerves and is considered to play a significant role in migraine pathophysiology. MATERIALS AND METHODS: We investigated the effect of the CGRP receptor antagonist, telcagepant, on CGRP-induced cranial vasodilatation in human isolated cerebral and middle meningeal arteries. We also studied the expression of the CGRP receptor components in cranial arteries with immunocytochemistry. Concentration response curves to αCGRP were performed in human isolated cerebral and middle meningeal arteries in the absence or presence of telcagepant. Arterial slices were stained for RAMP1, CLR and actin in a double immunofluorescence staining. RESULTS: In both arteries, we found that: (i) telcagepant was devoid of any contractile or relaxant effects per se; (ii) pretreatment with telcagepant antagonised the αCGRP-induced relaxation in a competitive manner; and (iii) immunohistochemistry revealed expression and co-localisation of CLR and RAMP1 in the smooth muscle cells in the media layer of both arteries. CONCLUSIONS: Our findings provide morphological and functional data on the presence of CGRP receptors in cerebral and meningeal arteries, which illustrates a possible site of action of telcagepant in the treatment of migraine.

Absorbed dose and dose rate using the Varian OBI 1.3 and 1.4 CBCT system.

According to published data, the absorbed dose used for a CBCT image acquisition with Varian OBI v1.3 can be as high as 100 mGy. In 2008 Varian released a new OBI version (v1.4), which promised to reduce the imaging dose. In this study, absorbed doses used for CBCT image acquisitions with the default irradiation techniques of Varian OBI v1.3 and v1.4 are measured. TLDs are used to derive dose distributions at three planes inside an anthropomorphic phantom. In addition, point doses and dose profiles inside a 'stack' of three CTDI body phantoms are measured using a new solid state detector, the CT Dose Profiler. With the CT Dose Profiler, the individual pulses from the X-ray tube are also studied. To verify the absorbed dose measured with the CT Dose Profiler, it is compared to TLD. The image quality is evaluated using a Catphan phantom. For OBI v1.3, doses measured in transverse planes of the Alderson phantom range between 64 mGy and 144 mGy. The average dose is around 100 mGy. For OBI v1.4, doses measured in transverse planes of the Alderson phantom range between 1 mGy and 51 mGy. Mean doses range between 3-35 mGy depending on CBCT mode. CT Dose Profiler data agree with TLD measurements in a CTDI phantom within the uncertainty of the TLD measurements (estimated SD +/- 10%). Instantaneous dose rate at the periphery of the phantom can be higher than 20 mGy/s, which is 10 times the dose rate at the center. The spatial resolution in v1.4 is not as high as in v1.3. In conclusion, measurements show that the imaging doses for default modes in Varian OBI v1.4 CBCT system are significantly lower than in v1.3. The CT Dose Profiler is proven fast and accurate for CBCT applications.

The U1 snRNP-associated factor Luc7p affects 5' splice site selection in yeast and human.

yLuc7p is an essential subunit of the yeast U1 snRNP and contains two putative zinc fingers. Using RNA-protein cross-linking and directed site-specific proteolysis (DSSP), we have established that the N-terminal zinc finger of yLuc7p contacts the pre-mRNA in the 5' exon in a region close to the cap. Modifying the pre-mRNA sequence in the region contacted by yLuc7p affects splicing in a yLuc7p-dependent manner indicating that yLuc7p stabilizes U1 snRNP-pre-mRNA interaction, thus reminding of the mode of action of another U1 snRNP component, Nam8p. Database searches identified three putative human yLuc7p homologs (hLuc7A, hLuc7B1 and hLuc7B2). These proteins have an extended C-terminal tail rich in RS and RE residues, a feature characteristic of splicing factors. Consistent with a role in pre-mRNA splicing, hLuc7A localizes in the nucleus and antibodies raised against hLuc7A specifically co-precipitate U1 snRNA from human cell extracts. Interestingly, hLuc7A overexpression affects splicing of a reporter in vivo. Taken together, our data suggest that the formation of a wide network of protein-RNA interactions around the 5' splice site by U1 snRNP-associated factors contributes to alternative splicing regulation.

Tackling Capacity Fading in Vanadium Redox Flow Batteries with Amphoteric Polybenzimidazole/Nafion Bilayer Membranes.

Vanadium flow batteries are among the most promising technologies for stationary energy storage applications if their cost of storage can be further decreased. Capacity fading resulting from imbalanced vanadium crossover is a key operating cost component. Herein, a new approach is reported to avoid this cost by balancing electrolyte transport with amphoteric bilayer Nafion/meta-polybenzimidazole membranes. Within this system, the anion- and cation-exchange capacity can be tuned in a straightforward manner by changing the thickness of the respective polymer layer to balance electrolyte transport for a given current density. At high current densities, a net migrative flux of vanadium directed towards the positive side is observed owing to the higher average charge of vanadium ions present at the negative side. The coulombic repulsion between the vanadium ions and the positive charges in the membrane counteracts this migrative transport and can reverse the direction of the net vanadium flux. For a technically relevant current density of 120 mA cm-2 , a PBI thickness of 3-4 µm is required to balance the vanadium crossover and to minimize capacity fading.

Arthritis suppression by NADPH activation operates through an interferon-beta pathway.

BACKGROUND: A polymorphism in the activating component of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex, neutrophil cytosolic factor 1 (NCF1), has previously been identified as a regulator of arthritis severity in mice and rats. This discovery resulted in a search for NADPH oxidase-activating substances as a potential new approach to treat autoimmune disorders such as rheumatoid arthritis (RA). We have recently shown that compounds inducing NCF1-dependent oxidative burst, e.g. phytol, have a strong ameliorating effect on arthritis in rats. However, the underlying molecular mechanism is still not clearly understood. The aim of this study was to use gene-expression profiling to understand the protective effect against arthritis of activation of NADPH oxidase in the immune system. RESULTS: Subcutaneous administration of phytol leads to an accumulation of the compound in the inguinal lymph nodes, with peak levels being reached approximately 10 days after administration. Hence, global gene-expression profiling on inguinal lymph nodes was performed 10 days after the induction of pristane-induced arthritis (PIA) and phytol administration. The differentially expressed genes could be divided into two pathways, consisting of genes regulated by different interferons. IFN-gamma regulated the pathway associated with arthritis development, whereas IFN-beta regulated the pathway associated with disease protection through phytol. Importantly, these two molecular pathways were also confirmed to differentiate between the arthritis-susceptible dark agouti (DA) rat, (with an Ncf-1DA allele that allows only low oxidative burst), and the arthritis-protected DA.Ncf-1E3 rat (with an Ncf1E3 allele that allows a stronger oxidative burst). CONCLUSION: Naturally occurring genetic polymorphisms in the Ncf-1 gene modulate the activity of the NADPH oxidase complex, which strongly regulates the severity of arthritis. We now show that the Ncf-1 allele that enhances oxidative burst and protects against arthritis is operating through an IFN-beta-associated pathway, whereas the arthritis-driving allele operates through an IFN-gamma-associated pathway. Treatment of arthritis-susceptible rats with an NADPH oxidase-activating substance, phytol, protects against arthritis. Interestingly, the treatment led to a restoration of the oxidative-burst effect and induction of a strikingly similar IFN-beta-dependent pathway, as seen with the disease-protective Ncf1 polymorphism.

Angiotensin II-induced vasodilatation in cerebral arteries is mediated by endothelium-derived hyperpolarising factor.

The angiotensin II-induced vasodilatation was evaluated in rat middle cerebral artery, especially regarding endothelium-derived hyperpolarising factor (EDHF), by use of a pressurised arteriograph. The angiotensin II dilatation was partly antagonised by inhibitors of nitric oxide synthase and cyclo-oxygenase. The remaining dilatation was inhibited by the potassium channel blockers, charybdotoxin and apamin, providing direct evidence that angiotensin II induces EDHF-mediated dilatation in cerebral arteries. The angiotensin II dilatation was blocked by the angiotensin AT1 and AT2 receptor blockers candesartan and PD 123319. Both angiotensin AT1 and AT2 receptors were detected on the endothelium by immunohistochemistry.

Smoking particles enhance endothelin A and endothelin B receptor-mediated contractions by enhancing translation in rat bronchi.

BACKGROUND: Smoking is known to cause chronic inflammatory changes in the bronchi and to contribute to airway hyper-reactivity, such as in bronchial asthma. To study the effect of smoking on the endothelin system in rat airways, bronchial segments were exposed to DMSO-soluble smoking particles (DSP) from cigarette smoke, to nicotine and to DMSO, respectively. METHODS: Isolated rat bronchial segments were cultured for 24 hours in the presence or absence of DSP, nicotine or DMSO alone. Contractile responses to sarafotoxin 6c (a selective agonist for ETB receptors) and endothelin-1 (an ETA and ETB receptor agonist) were studied by use of a sensitive myograph. Before ET-1 was introduced, the ETB receptors were desensitized by use of S6c. The remaining contractility observed was considered to be the result of selective activation of the ETA receptors. ETA and ETB receptor mRNA expression was analyzed using real-time quantitative PCR. The location and concentration of ETA and ETB receptors were studied by means of immunohistochemistry together with confocal microscopy after overnight incubation with selective antibodies. RESULTS: After being cultured together with DSP for 24 hours the bronchial segments showed an increased contractility mediated by ETA and ETB receptors, whereas culturing them together with nicotine did not affect their contractility. The up-regulation of their contractility was blunted by cycloheximide treatment, a translational inhibitor. No significant change in the expression of ETA and ETB receptor mRNA through exposure to DMSO or to nicotine exposure alone occurred, although immunohistochemistry revealed a clear increase in ETA and ETB receptors in the smooth muscle after incubation in the presence of DSP. Taken as a whole, this is seen as the presence of a translation mechanism. CONCLUSION: The increased contractility of rat bronchi when exposed to DSP appears to be due to a translation mechanism.

GIS supported calculations of (137)Cs deposition in Sweden based on precipitation data.

It is of interest to know the spatial variation and the amount of (137)Cs e.g. in case of an accident with a radioactive discharge. In this study, the spatial distribution of the quarterly (137)Cs deposition over Sweden due to nuclear weapons fallout (NWF) during the period 1962-1966 was determined by relating the measured deposition density at a reference site to the amount of precipitation. Measured quarterly values of (137)Cs deposition density per unit precipitation at three reference sites and quarterly precipitation at 62 weather stations distributed over Sweden were used in the calculations. The reference sites were assumed to represent areas with different quarterly mean precipitation. The extent of these areas was determined from the distribution of the mean measured precipitation between 1961 and 1990 and varied according to seasonal variations in the mean precipitation pattern. Deposition maps were created by interpolation within a geographical information system (GIS). Both integrated (total) and cumulative (decay corrected) deposition densities were calculated. The lowest levels of NWF (137)Cs deposition density were noted in north-eastern and eastern parts of Sweden and the highest levels in the western parts of Sweden. Furthermore the deposition density of (137)Cs, resulting from the Chernobyl accident was determined for an area in western Sweden based on precipitation data. The highest levels of Chernobyl (137)Cs in western Sweden were found in the western parts of the area along the coast and the lowest in the east. The sum of the deposition densities from NWF and Chernobyl in western Sweden was then compared to the total activity measured in soil samples at 27 locations. Comparisons between the predicted values of this study show a good agreement with measured values and other studies.

Picture book support for preparing children ahead of and during day surgery.

Aim To develop and evaluate the use of a specific picture book aiming to prepare children for anaesthesia and surgery. Methods An intervention comparing two different information methods before ear, nose and throat day surgery was performed. The intervention involved using a specific information sheet and a specific picture book. Parents (n=104) of children aged 2-12 years completed open-ended questions that were analysed with qualitative content analysis. They were divided into two groups: one group received routine information and one received routine information and the intervention. Findings The picture sheet and picture book were valuable aids to prepare small children for anaesthesia and surgery by explaining the procedures that would take place. The parents expressed that knowledge of the procedures made them and the child feel secure. Conclusion Peri-operative information through pictures supports children and their parents during day surgery and may be helpful in future healthcare visits.

Molecular architecture of the recombinant human MCM2-7 helicase in complex with nucleotides and DNA.

DNA replication is a key biological process that involves different protein complexes whose assembly is rigorously regulated in a successive order. One of these complexes is a replicative hexameric helicase, the MCM complex, which is essential for the initiation and elongation phases of replication. After the assembly of a double heterohexameric MCM2-7 complex at replication origins in G1, the 2 heterohexamers separate from each other and associate with Cdc45 and GINS proteins in a CMG complex that is capable of unwinding dsDNA during S phase. Here, we have reconstituted and characterized the purified human MCM2-7 (hMCM2-7) hexameric complex by co-expression of its 6 different subunits in insect cells. The conformational variability of the complex has been analyzed by single particle electron microscopy in the presence of different nucleotide analogs and DNA. The interaction with nucleotide stabilizes the complex while DNA introduces conformational changes in the hexamer inducing a cylindrical shape. Our studies suggest that the assembly of GINS and Cdc45 to the hMCM2-7 hexamer would favor conformational changes on the hexamer bound to ssDNA shifting the cylindrical shape of the complex into a right-handed spiral conformation as observed in the CMG complex bound to DNA.

Drug absorption from the isolated perfused rat lung--correlations with drug physicochemical properties and epithelial permeability.

The pulmonary absorption of nine low-molecular-weight (225-430 Da) drugs (atenolol, budesonide, enalaprilat, enalapril, formoterol, losartan, metoprolol, propranolol and terbutaline) and one high-molecular-weight membrane permeability marker compound (FITC-dextran 10000 Da) was investigated using the isolated, perfused and ventilated rat lung (IPL). The relationships between pulmonary transport characteristics, epithelial permeability of Caco-2 cell monolayers and drug physicochemical properties were evaluated using multivariate data analysis. Finally, an in vitro-in vivo correlation was made using in vivo rat lung absorption data. The absorption half-life of the investigated drugs ranged from 2 to 59 min, and the extent of absorption from 21 to 94% in 2 h in the isolated perfused rat lung model. The apparent first-order absorption rate constant in IPL (ka(lung)) was found to correlate to the apparent permeability (P(app)) of Caco-2 cell monolayers (r = 0.87), cLog D(7.4) (r = 0.70), cLog P, and to the molecular polar surface area (%PSA) (r = -0.79) of the drugs. A Partial Least Squares (PLS)-model for prediction of the absorption rate (log ka(lung)) from the descriptors log P(app), %PSA and cLogD(7.4) was found (Q2 = 0.74, R2 = 0.78). Furthermore, a strong in vitro-in vivo correlation (r = 0.98) was found for the in vitro (IPL) drug absorption half-life and the pulmonary absorption half-life obtained in rats in vivo, based on a sub-set of five compounds.

Management of internal root resorption on permanent teeth.

Internal root resorption (IRR) is a particular category of pulp disease characterized by the loss of dentine as a result of the action of clastic cells stimulated by pulpal inflammation. This review article explains the etiology, the prevalence of IRR, and, in addition to the clinical data, the contribution of the three-dimensional imaging (CBCT) to the diagnosis, the clinical decision, and the therapeutic management of IRR. The authors discussed the various therapeutic options including the orthograde or retrograde fillings of the root canal resorption area. Root canal treatment remains the treatment of choice of internal root resorption as it removes the granulation tissue and blood supply of the clastic cells. The authors describe with different clinical cases the modern endodontic techniques including optical aids, ultrasonic improvement of chemical debridement, and the use of alternative materials such as calcium silicate combined with thermoplastic filling (warm gutta-percha). In these conditions, the prognosis of the conservative treatment of internal resorptions, even if root walls are perforated, is good.

Differential vasoactive effects of sildenafil and tadalafil on cerebral arteries.

Phosphodiesterase 5 (PDE5) is associated with migraine pathophysiology, stroke recovery and vasospasm treatment. The potential vascular interplay of PDE5 inhibitors sildenafil, tadalafil and UK-114,542 was studied by intra- versus extra-luminal administration in rat middle cerebral arteries in vitro and on middle meningeal arteries in vivo. By Western blot PDE5 was detected in both cerebral and meningeal arteries, though with minor variations in band intensity between vascular beds. Rat middle cerebral artery diameter was investigated using pressurised arteriography, applying UK-114,542, sildenafil, and tadalafil intra- or extra-luminally. Effects on the dural middle meningeal artery were studied in the in vivo closed cranial window model. At high concentrations, abluminal sildenafil and UK-114,542, but not tadalafil, induced dilatation of the middle cerebral artery. Luminal application elicited a contraction of 4% (sildenafil, P=0.03) and 10% (tadalafil, P=0.02). In vivo, sildenafil, but not tadalafil, dose-dependently dilated middle meningeal artery concomitant to blood pressure reduction (1-3mg/kg);1mg/kg sildenafil inducing 60 ± 14% (P=0.04) and vehicle (DMSO) 13 ± 6% dilatation. In conclusion, PDE5 inhibitors applied luminally had minor contractile effect, whereas abluminal sildenafil induced middle cerebral artery dilatation above therapeutic levels. In vivo, sildenafil dilated middle meningeal artery concomitant with a reduction in blood pressure. Tadalafil had no dilatory effects. PDE5 inhibitors show differential vascular activity in cerebral arteries from healthy animals; arterial dilatation is seen primarily above therapeutic levels. Such findings support clinical studies showing no vasodilator effects of sildenafil on cerebral arteries in healthy subjects.

Interpretation and management of INR results: a case history based survey in 13 countries.

INTRODUCTION: Standardisation of treatment with vitamin K antagonists (VKAs) is still an issue after 60 years of use. The study aimed to explore aspects of VKA monitoring in primary and secondary care. METHODS: Two case histories were distributed to physicians in 13 countries. Case history A focused on a patient with atrial fibrillation on stable anticoagulation (latest INR 2.3). Physicians were asked about frequency of INR measurement, when to change the VKA dose, and the patient's annual risk of ischemic stroke and bleeding. Case history B focused on a patient with an unexpected INR of 4.8, asking for the patient's 48-hour bleeding risk, the immediate dose reduction and time until a repeat INR. RESULTS: Altogether, 3016 physicians responded (response rate 8 - 38%), of which 82% were from primary care and 18% from secondary care. Answers varied substantially within and between countries regardless of level of care and VKA used. Median number of weeks between INR measurements was 4 - 6 weeks. Median threshold INR for increasing or decreasing the VKA dose was 1.9 and 3.1, respectively. Risk of ischemic stroke and bleeding were overestimated 2 - 3 times. In case history B, the median dose reduction the two first days was 75% for GPs and 55% for specialists, irrespective of estimates of bleeding risk; with one week to a repeat INR. CONCLUSION: Variation in VKA monitoring is substantial implying clinical consequences. Guidelines seem either unknown or may be considered impracticable. Further efforts towards standardisation of VKA management are needed.

Crystal structure of the open conformation of the mammalian chaperonin CCT in complex with tubulin.

Protein folding is assisted by molecular chaperones. CCT (chaperonin containing TCP-1, or TRiC) is a 1-MDa oligomer that is built by two rings comprising eight different 60-kDa subunits. This chaperonin regulates the folding of important proteins including actin, α-tubulin and ß-tubulin. We used an electron density map at 5.5 Å resolution to reconstruct CCT, which showed a substrate in the inner cavities of both rings. Here we present the crystal structure of the open conformation of this nanomachine in complex with tubulin, providing information about the mechanism by which it aids tubulin folding. The structure showed that the substrate interacts with loops in the apical and equatorial domains of CCT. The organization of the ATP-binding pockets suggests that the substrate is stretched inside the cavity. Our data provide the basis for understanding the function of this chaperonin.

Relationship between AMPK and the transcriptional balance of clock-related genes in skeletal muscle.

Circadian clocks coordinate physiological, behavioral, and biochemical events with predictable daily environmental changes by a self-sustained transcriptional feedback loop. CLOCK and ARNTL are transcriptional activators that regulate Per and Cry gene expression. PER and CRY inhibit their own transcription, and their turnover allows this cycle to restart. The transcription factors BHLHB2 and BHLHB3 repress Per activation, whereas orphan nuclear receptors of the NR1D and ROR families control Arntl expression. Here we show the AMP-activated protein kinase (AMPK)gamma(3) subunit is involved in the regulation of peripheral circadian clock function. AMPKgamma3 knockout (Prkag3(-/-)) mice or wild-type littermates were injected with saline or an AMPK activator, 5-amino-4-imidazole-carboxamide riboside (AICAR), and white glycolytic gastrocnemius muscle was removed for gene expression analysis. Genes involved in the regulation of circadian rhythms (Cry2, Nr1d1, and Bhlhb2) were differentially regulated in response to AICAR in wild-type mice but remained unaltered in Prkag3(-/-) mice. Basal expression of Per1 was higher in Prkag3(-/-) mice compared with wild-type mice. Distinct diurnal changes in the respiratory exchange ratio (RER) between the light and dark phase of the day were observed in wild-type mice but not Prkag3(-/-) mice. In summary, the expression profile of clock-related genes in skeletal muscle in response to AICAR, as well as the diurnal shift in energy utilization, is impaired in AMPKgamma(3) subunit knockout mice. Our results indicate AMPK heterotrimeric complexes containing the AMPKgamma(3) subunit may play a specific role in linking circadian oscillators and energy metabolism in skeletal muscle.

Contractile responses to ergotamine and dihydroergotamine in the perfused middle cerebral artery of rat.

The vasomotor effects of ergotamine and dihydroergotamine (DHE) on the middle cerebral artery (MCA) of rats were studied using the pressurised arteriography method and in vitro myographs. MCAs from Sprague-Dawley rats were mounted on two glass micropipettes using the arteriograph, pressurised to 85 mmHg and luminally perfused. All vessels used attained spontaneous contractile tone (34.9+/-1.8% of resting tone) and responded to luminal adenosine triphosphate (ATP) with dilatation (24.1+/-4.0%), which showed functioning endothelium. Luminally added ergotamine or DHE induced maximal contractions of 16.8+8% and 22.4+/-0.9%, respectively, compared to the resting diameter, with a pEC(50) of 8.7+/-0.1 for ergotamine and 9.0+/-0.1 for DHE. Abluminal application of ergotamine and DHE also caused concentration-dependent contractions of the perfused MCA by 21.4+/-2.1% and 23.1+/-7.0%, respectively, with pEC(50) values of 7.6+/-0.2 for ergotamine and 8.4+/-0.5 for DHE. The responses were blocked by the 5-HT(2A) receptor antagonist ketanserin (concentration 10(-12) to 10(-5) M) and partially with the 5-HT(1B) receptor antagonist BRL-11557PM-B. The 5-HT(1D) receptor antagonist SB-224289-A had no significant effect. Using a myograph technique, isolated ring segments of the MCA with intact endothelium were mounted on two metal wires. Neither agonist caused relaxation of resting vessels, however, they both responded by weak contractile responses (26+/-3% of submaximal contractile capacity relative to 60 mM potassium). The contractions were typically slow in on and off set (about 30-60 min). The long duration of ergots should be investigated further in an attempt to design drugs with less recurrence.