Robert Julian Dickinson

Competitive inhibition at the glycine site of the N-methyl-D-aspartate receptor by the anesthetics xenon and isoflurane: evidence from molecular modeling and electrophysiology.

Background:Inhibition of N-methyl-d-aspartate (NMDA) receptors by anesthetic gases and vapors may play an important role in anesthesia and neuroprotection. However, the site of action of these agents on the NMDA receptor is unknown. The authors show that xenon and isoflurane compete for the binding of the coagonist glycine on the NMDA receptor NR1 subunit. Methods:Using a novel application of grand canonical Monte Carlo simulations, the authors predict the binding site of xenon on NMDA receptors. They test this prediction using electrophysiology on recombinant NMDA receptors. Results:The authors’ modeling predicts that xenon binds at the glycine site of the NMDA receptor. The authors show that inhibition of NMDA receptors by xenon and isoflurane increases as glycine concentration is decreased, consistent with the prediction of competitive inhibition at the glycine site. Lineweaver-Burk analysis shows that isoflurane inhibition seems purely competitive with glycine, but for xenon, there is an additional component of noncompetitive inhibition. The loss of inhibitory effect of xenon and isoflurane in mutant NR1(F639A)/NR2A receptors is explained by increased glycine affinity of the mutant receptors, and inhibition is restored at low glycine concentrations. Conclusions:Xenon and isoflurane inhibit NMDA receptors by binding at the same site as the coagonist glycine. This finding may have important implications for general anesthesia and neuroprotection. Neuroprotectants that act at the glycine site of the NMDA receptor antagonists are well tolerated in patients, being devoid of psychotomimetic side effects, and the mechanism of inhibition may play a role in their clinical profile.

A propofol binding site on mammalian GABAA receptors identified by photolabeling

Propofol is the most important intravenous general anesthetic in current clinical use. It acts by potentiating GABAA receptors, but where it binds to this receptor is not known and has been a matter of some controversy. We have synthesized a novel propofol analogue photolabeling reagent that has a biological activity very similar to that of propofol. We confirmed that this reagent labeled known propofol binding sites in human serum albumin which have been identified using X-ray crystallography. Using a combination of the protiated label and a deuterated version, and mammalian receptors labeled in intact membranes, we have identified a novel binding site for propofol in GABAA receptors consisting of both β3 homopentamers and α1β3 heteropentamers. The binding site is located within the β subunit, at the interface between the transmembrane domains and the extracellular domain, and lies close to known determinants of anesthetic sensitivity in transmembrane segments TM1 and TM2.

Observation by MR imaging of in vivo temperature changes induced by radio frequency hyperthermia.

Volunteers have undergone radio frequency hyperthermia in a magnetic resonance (MR) imaging system to investigate which of several possible MR parameters would be most convenient and sensitive to use to observe in vivo temperature changes. Measurements were made for T1, T2, and perfusion and diffusion variations, although the number of sequences needed at each temperature meant that the number of data points obtainable was limited. However, in the temperature range studied (around 28-42 degrees C), changes in both T1 and the diffusion coefficient were observed that agreed quite well with those predicted theoretically (respectively around 1.3%/degrees C and 2.4/degrees C).

A random phased array device for delivery of high intensity focused ultrasound

Randomized phased arrays can offer electronic steering of a single focus and simultaneous multiple foci concomitant with low levels of secondary maxima and are potentially useful as sources of high intensity focused ultrasound (HIFU). This work describes laboratory testing of a 1 MHz random phased array consisting of 254 elements on a spherical shell of radius of curvature 130 mm and diameter 170 mm. Acoustic output power and efficiency are measured for a range of input electrical powers, and field distributions for various single- and multiple-focus conditions are evaluated by a novel technique using an infrared camera to provide rapid imaging of temperature changes on the surface of an absorbing target. Experimental results show that the array can steer a single focus laterally to at least +/-15 mm off axis and axially to more than +/-15 mm from the centre of curvature of the array and patterns of four and five simultaneous foci +/-10 mm laterally and axially whilst maintaining low intensity levels in secondary maxima away from the targeted area in good agreement with linear theoretical predictions. Experiments in which pork meat was thermally ablated indicate that contiguous lesions several cm(3) in volume can be produced using the patterns of multiple foci.

Stereoselective loss of righting reflex in rats by isoflurane.

BACKGROUND Although it is accepted widely that optically active intravenous general anesthetics produce stereoselective effects in animals, the situation regarding volatile agents is confused. Conventional studies with scarce isoflurane enantiomers have been limited to small numbers of animals and produced conflicting results. By injecting these volatile enantiomers intravenously, however, it is possible to study large numbers of animals and obtain reliable results that can help to identify the molecular targets for isoflurane. METHODS Pure isoflurane enantiomers were administered intravenously to rats after solubilization in a lipid emulsion. The ability of each enantiomer to produce a loss of righting reflex was determined as a function of dose, and quantal dose-response curves were constructed. In addition, sleep times were recorded with each enantiomer. Chiral gas chromatography was used to measure relative enantiomer concentrations in the brains of rats injected with racemic isoflurane. RESULTS The S(+)-enantiomer was 40 +/- 8% more potent than the R(-)-enantiomer at producing a loss of righting reflex. The S(+)-enantiomer induced longer sleep times (by about 50%) than did the R(-)-enantiomer. Rats anesthetized by a dose of racemic isoflurane sufficient to achieve a half-maximal effect had essentially identical brain concentrations of the two enantiomers. CONCLUSIONS The S(+)-enantiomer of the general anesthetic isoflurane is significantly (P < 0.001) more potent than the R(-)-enantiomer at causing a loss of righting reflex in rats. This confirms the view that isoflurane acts by binding to chiral sites. The observed degree of stereoselectivity provides a useful guide for ascertaining from in vitro experiments which molecular targets are most likely to play major roles in the loss of righting reflex caused by isoflurane.

Evaluation of droplet dispersion during non-invasive ventilation, oxygen therapy, nebuliser treatment and chest physiotherapy in clinical practice: implications for management of pandemic influenza and other airborne infections.

BACKGROUND Influenza viruses are thought to be spread by droplets, but the role of aerosol dissemination is unclear and has not been assessed by previous studies. Oxygen therapy, nebulised medication and ventilatory support are treatments used in clinical practice to treat influenzal infection are thought to generate droplets or aerosols. OBJECTIVES Evaluation of the characteristics of droplet/aerosol dispersion around delivery systems during non-invasive ventilation (NIV), oxygen therapy, nebuliser treatment and chest physiotherapy by measuring droplet size, geographical distribution of droplets, decay in droplets over time after the interventions were discontinued. METHODS Three groups were studied: (1) normal controls, (2) subjects with coryzal symptoms and (3) adult patients with chronic lung disease who were admitted to hospital with an infective exacerbation. Each group received oxygen therapy, NIV using a vented mask system and a modified circuit with non-vented mask and exhalation filter, and nebulised saline. The patient group had a period of standardised chest physiotherapy treatment. Droplet counts in mean diameter size ranges from 0.3 to > 10 µm were measured with an counter placed adjacent to the face and at a 1-m distance from the subject/patient, at the height of the nose/mouth of an average health-care worker. RESULTS NIV using a vented mask produced droplets in the large size range (> 10 µm) in patients (p = 0.042) and coryzal subjects (p = 0.044) compared with baseline values, but not in normal controls (p = 0.379), but this increase in large droplets was not seen using the NIV circuit modification. Chest physiotherapy produced droplets predominantly of > 10 µm (p = 0.003), which, as with NIV droplet count in the patients, had fallen significantly by 1 m. Oxygen therapy did not increase droplet count in any size range. Nebulised saline delivered droplets in the small- and medium-size aerosol/droplet range, but did not increase large-size droplet count. CONCLUSIONS NIV and chest physiotherapy are droplet (not aerosol)-generating procedures, producing droplets of > 10 µm in size. Due to their large mass, most fall out on to local surfaces within 1 m. The only device producing an aerosol was the nebuliser and the output profile is consistent with nebuliser characteristics rather than dissemination of large droplets from patients. These findings suggest that health-care workers providing NIV and chest physiotherapy, working within 1 m of an infected patient should have a higher level of respiratory protection, but that infection control measures designed to limit aerosol spread may have less relevance for these procedures. These results may have infection control implications for other airborne infections, such as severe acute respiratory syndrome and tuberculosis, as well as for pandemic influenza infection.

Determinants of the anesthetic sensitivity of two-pore domain acid-sensitive potassium channels: molecular cloning of an anesthetic-activated potassium channel from Lymnaea stagnalis.

Certain two-pore domain K+ channels are plausible targets for volatile general anesthetics, yet little is known at the molecular level about how these simple agents cause channel activation. The first anesthetic-activated K+ current IK(An) that was characterized was discovered in the mollusk Lymnaea stagnalis and is remarkable for both its sensitivity to general anesthetics and its stereoselective responses to anesthetic enantiomers (Franks, N. P., and Lieb, W. R. (1988) Nature 333, 662–664 and Franks, N. P., and Lieb, W. R. (1991) Science 254, 427–430). Here we report the molecular cloning of a two-pore domain K+ channel LyTASK from L. stagnalis and show that, when expressed in HEK-293 cells, it displays the same biophysical characteristics as the anesthetic-activated K+ current IK(An). Sequence analysis and functional properties show it to be a member of the TASK family of channels with ∼47% identity at the amino acid level when compared with human TASK-1 and TASK-3. By using chimeric channel constructs and site-directed mutagenesis we have identified the specific amino acid 159 to be a critical determinant of anesthetic sensitivity, which, when mutated to alanine, essentially eliminates anesthetic activation in the human channels and greatly reduces activation in LyTASK. The L159A mutation in LyTASK disrupts the stereoselective response to isoflurane while having no effect on the pH sensitivity of the channel, suggesting this critical amino acid may form part of an anesthetic binding site.

Liver Resection With a New Multiprobe Bipolar Radiofrequency Device

HYPOTHESIS Liver resection can be associated with marked blood loss. A novel multiprobe bipolar radiofrequency device (Habib 4X; RITA Medical Systems Inc, Fremont, California) has been developed to assist in liver resection and to reduce intraoperative blood loss. DESIGN Prospective study. SETTING Tertiary referral unit. PATIENTS Sixty-two patients requiring liver resection between November 1, 2004, and February 28, 2006, primarily for metastatic cancer. INTERVENTION Liver resection with the radiofrequency device. MAIN OUTCOME MEASURES Intraoperative blood loss, liver parenchyma transection time, and complications. RESULTS There were 51 minor and 11 major hepatectomies. Mean (SD) transection time was 39 (27) seconds per square centimeter. Mean (SD) blood loss was 4.8 (5.6) mL per square centimeter. No patient required hepatic inflow occlusion. One patient required blood transfusion. There were no deaths, and the morbidity rate was 18%. Mean (SD) hospital stay was 8 (3) days. CONCLUSIONS This new bipolar radiofrequency device allows minor and major hepatectomies to be performed with minimal blood loss, low blood transfusion requirement, and reduced mortality and morbidity rates.

Probing the molecular dimensions of general anaesthetic target sites in tadpoles (Xenopus laevis) and model systems using cycloalcohols

1 The series of cycloalcohols C6, C7, C8 and C10 have been used to probe the molecular dimensions of a variety of general anaesthetic target sites. 2 The general anaesthetic EC50 concentrations of the cycloalcohols were determined for tadpoles (Xenopus laevis). All of the cycloalcohols tested were found to be potent general anaesthetics (on average EC50/Csat = 0.03). 3 The effects of the cycloalcohols on highly purified luciferase enzymes from fireflies (Photinus pyralis) and bacteria (Vibrio harveyi) were also investigated. Both enzymes were inhibited competitively, with the cycloalcohols competing with firefly luciferin for binding to the firefly enzyme and with n‐decanal for binding to the bacterial enzyme. 4 The binding site on the firefly enzyme could accommodate two molecules of cycloalcohols C6 and C7 but only a single molecule of the larger cycloalcohols (C8 and C10), implying a volume of the binding site of about 250 cm3 mol−1. In contrast, the binding site on the bacterial luciferase could bind only a single cycloalcohol molecule between C6 and C10. 5 While all of the cycloalcohols were potent inhibitors of the firefly luciferase enzyme (on average EC50/Csat = 0.015), they were very weak inhibitors of the bacterial luciferase enzyme (on average EC50/Csat = 0.12). Since both enzymes bind long‐chain aliphatic n‐alcohols tightly, the differing affinities of the cycloalcohols for the two enzymes is probably a consequence of geometrical factors. 6 The cycloalcohols produced very small effects on lipid bilayers. At EC50 concentrations which produce general anaesthesia, lipid bilayer phase transitions were shifted, on average, by only 0.43°C. 7 We conclude that the general anaesthetic effects of the cycloalcohols can most economically be explained by assuming that the cycloalcohols act at protein binding sites in the central nervous system. These target sites would have binding properties similar to those of the anaesthetic‐binding site on firefly luciferase, but their average volume would be somewhat smaller than 250 cm3 mol−1.

Effects of temperature on the anaesthetic potency of halothane, enflurane and ethanol in Daphnia magna (Cladocera: Crustacea)

1. The effects of temperature on the anesthetic potencies of halothane, enflurane and ethanol have been studied in the water flea Daphnia magna. 2. In the absence of anaesthetics, decreasing temperature resulted in decreased activity by the daphnids. 3. Potencies in the gas phase decreased with increasing temperature for all of the anaesthetics, while aqueous potency decreased for halothane and enflurane but increased for ethanol. 4. Enthalpy calculations suggest that the observed potency changes for the inhalational anaesthetics cannot be accounted for in terms of changing solubility in lipid bilayers but most likely reflect more specific interactions with animal target sites.