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The operationalisation of Responsible Research and Innovation is increasingly associated with ethical toolkits. However, scholars remain critical of those toolkits, often referring to them as theoretically problematic, toothless, or too instrumental. Moreover, toolkits imply ideological commitments that are not necessarily made explicit. In this scoping review, we analyse 127 tools designed for technology ethics as part of the RRI Project. We find that (1) these tools tend to frame responsibility as general training or aimed at the development phase of technologies, while monitoring is underrepresented. (2) These toolkits often lack substantive conceptualisations of ethics ignoring contested paradigms. (3) Emerging digital and biotechnologies are over-represented in relation to other socio-technical infrastructures, and (4) there is a risk of a PDF-ization of ethics, as most toolkits are materially constructed as reading material and checklists. We conclude by presenting prompt questions to reflectively reconsider how we design ethical toolkits for technology.

Several perennial snow and ice bodies have been recently studied and monitored in three mountain massifs across the Balkan Peninsula: Prokletije (Northern Albania), Durmitor (Montenegro) and Pirin (Bulgaria), the two glacierets in the Pirin Mountains until soon considered as southernmost of their kind in Europe. In September 2022 a relatively large snow field (about 1.5 ha) was discovered and described for a first time in the glacial cirque Gryka e Kazanit in the Nëmerçka Mountains of Southern Albania. The snow accumulation lies on 1550–1650 m a.s.l. below a vertical limestone cliff with a height of almost 1000 m. The site was visited again in November 2023, when the snow field diminished its size to less than 1 ha. At the same time, multi-annual firn layers were exposed under the last year snow, with depth at least several metres, and indications were observed of ice, buried in the debris cover below. Newest findings indicate that the studied snow-firn body is a glacieret similar to those found in the Pirin Mountains. On a longer-term, nine perennial snow/firm bodies on the Balkans resemble small glaciers rather than snow and ice patches at least under current climatic conditions. Situated on 40°08′ Northern latitude, the newly discovered Nemërçka Glacieret is nominated to be the southernmost glacier in Europe.

This research seeks to develop and test a rapid mapping approach using unmanned aerial vehicles (UAVs) and terrestrial laser scanning to provide precise, high-resolution spatial data for urban areas right after disasters. This mapping aims to support efforts to protect the population and infrastructure while analyzing the situation in affected areas. It focuses on flood-prone regions lacking modern hydrological data and where regular monitoring is absent. This study was conducted in resort villages and adjacent catchments in Bulgaria’s southern Black Sea coast with leading maritime tourism features, after a flash flood on 5 September 2023 caused human casualties and severe material damage. The resulting field data with a spatial resolution of 3 to 5 cm/px were used to trace the effects of the flood on topographic surface changes and structural disturbances. Flood simulation using UAV data and a digital elevation model was performed. The appropriateness of contemporary land use forms and infrastructure location in catchments is discussed. The role of spatial data in the analysis of genetic factors in risk assessment is commented on. The results confirm the applicability of rapid mapping in informing the activities of responders in a period of increased vulnerability following a flood. The results were used by Bulgaria’s Ministry of Environment and Water to analyze the situation shortly after the disaster.

Drought stress affects many aspects of plant biochemistry, with photosynthesis being one the most significantly impaired physiological processes. Melatonin is a natural antioxidant with growth-regulating properties in plants. Its diverse physiological functions have been extensively studied in recent decades. Changes in leaf gas exchange and chlorophyll a fluorescence parameters were investigated in young wheat plants (Triticum aestivum L.) cv. Fermer and cv. Gines which were characterized to differ in their responses to drought, with cv. Gines being more tolerant than cv. Fermer. The plants were subjected to drought for five days by withholding their water supply. Melatonin was applied as a root supplement to the irrigation water before or after the drought period. Analyses were performed before and at the end of the stress period, as well as during the recovery phase. Changes in leaf pigment content, photosynthetic rate, stomatal conductance, and transpiration, as well as some chlorophyll a fluorescence parameters, were recorded. Melatonin alone did not cause considerable changes in the measured traits. We found a significant decrease in leaf gas exchange parameters, Fv/Fm and Fv/F0 values, and leaf pigments due to drought, especially in cv. Fermer. The data show that the application of melatonin favorably influenced the efficiency of the photosynthetic apparatus under water deprivation and during plant recovery. The pre-treatment with melatonin maintained the photosynthesis-related parameters closer to the control levels during the stress period. Both melatonin treatments supported the recovery of photosynthesis when the water supply was restored and the post-drought treatment showed a similar but weaker effect than pre-drought treatment.

The urban heat island effect (UHI) is among the major challenges of urban climate, which is continuously intensifying its impact on urban life and functioning. Against the backdrop of increasingly prolonged heatwaves observed in recent years, practical questions about adaptation measures in cities are growing—questions that traditional meteorological monitoring can hardly answer adequately. On the other hand, UHI has long been the focus of research interest, but due to the technological complexity of providing accurate spatially referenced data at high spatial resolution and the requirement to survey at strictly defined parts of the day, information provision is becoming a major challenge. This is one of the main reasons why UHI research results are less often used directly in urban spatial planning. However, advances in geospatial technologies, including unmanned aerial systems (UASs), are providing more and more reliable tools that can be applied to achieve better and higher-quality information resources that adequately characterize the UHI phenomenon. This paper presents a developed and tested methodology for the rapid and efficient assessment and mapping of the effects of surface urban heat island (SUHI). It is entirely based on the integrated use of data from unmanned aerial systems (UAS)-based remote sensing methods, including thermal photogrammetry and GIS-based analysis methods. The study follows the understanding that correct SUHI research depends on a proper understanding of the urban geosystem, its spatial and structural heterogeneity, and its functional systems, which in turn can only be achieved by supporting the research process with accurate and reliable information resources. In this regard, the possibilities offered by the proposed methodological scheme for efficient geospatial registration of SUHI variations at the microscale, including the calculation of intra-urban SUHI intensity, are discussed in detail. The methodology builds on classical approaches for using local climate zoning (LCZ), adding capabilities for precise delineation of individual zone types and for geostatistical characterization of the urban surface heat island (SUHI). Finally, the proposed scheme is based on state-of-the-art technological tools that provide flexible and automated capabilities to investigate the phenomenon at microscales, including by enabling flexible observation of its dynamics in terms of heat wave manifestation and evolution. Results are presented from a series of sequential tests conducted on the largest residential area in Bulgaria’s capital city, Sofia, in terms of area and population, over a relatively long period from 2021 to 2024.

This study aims to scientifically justify the identification of suitable urban properties for urban green infrastructure (UGI) interventions to optimize its natural regulating functions for long-term pollution mitigation and secondary dust reduction. This study adheres to the perception that planning urban transformations to improve ambient air quality (AQ) requires a thorough understanding of urban structural heterogeneity and its interrelationship with the local microclimate. We apply an approach in which UGI and its potential multifunctionality are explored as a structural–functional element of urban local climatic zones. The same (100 × 100 m) spatial framework is used to develop place-based adapted solutions for intervention in UGI. A complex geospatial analysis of Burgas City, the second largest city (by area) in Bulgaria, was conducted by integrating 12 indicators to reveal the spatial disbalance of AQ regulation’ demand and UGI’s potential to supply ecosystem services. A total of 174 municipally owned properties have been identified, of which 79 are of priority importance, including for transport landscaping, inner-quarter spaces, and social infrastructure. Indicators of population density and location of social facilities were applied with the highest weight in the process of prioritizing sites. The study relies on public data and information from the integrated city platform of Burgas, in cooperation with the city’s government. The results have been discussed with stakeholders and implemented by the Municipality of Burgas in immediate greening measures in support of an ongoing program for Burgas Municipality AQ improvement.

The Nagoya Protocol is an international agreement adopted in 2010 (and entered into force in 2014) which governs access to genetic resources and the fair and equitable sharing of benefits from their utilisation. The agreement aims to prevent misappropriation of genetic resources and, through benefit sharing, create incentives for the conservation and sustainable use of biological diversity. While the equitable sharing of the benefits arising from the utilisation of genetic resources is a widely accepted concept, the way in which the provisions of the Nagoya Protocol are currently being implemented through national access and benefit-sharing legislation places significant logistical challenges on the control of transboundary livestock diseases such as foot-and-mouth disease (FMD). Delays to access FMD virus isolates from the field disrupt the production of new FMD vaccines and other tailored tools for research, surveillance and outbreak control. These concerns were raised within the FMD Reference Laboratory Network and were explored at a recent multistakeholder meeting hosted by the European Commission for the Control of FMD. The aim of this paper is to promote wider awareness of the Nagoya Protocol, and to highlight its impacts on the regular exchange and utilisation of biological materials collected from clinical cases which underpin FMD research activities, and work to develop new epidemiologically relevant vaccines and other diagnostic tools to control the disease.

AbstractAssessment and monitoring of health status in deteriorated forest stands in Gornata Koria and Chuprene Reserves in Western Balkan Range (Bulgaria) were conducted in 2017. An integrated approach (based on remote sensing technologies and terrestrial validation) was applied. A series of subsequent terrain observations were carried out in both protected areas mainly in dead or in poor health status stands. As a result, sixteen insect species were identified in Gornata Koria and nine pests in Chuprene Reserves. The predominant number of pests included bark beetles and weevils (Curculionidae), longhorn beetles (Cerambycidae), etc. Attacks caused by the European bark beetle (Ips typographus) formed the main disturbance in the Norway spruce forests in both reserves. Ten parasitic and ten saprophytic fungi were identified in the stands of the Norway spruce (Picea abies), silver fir (Abies alba) and European beech (Fagus sylvatica). Among the parasitic fungi, six species were identified as destructive (Armillaria sp., Heterobasidion annosum, Fomitopsis pinicola, Fomes fomentarius, Ramaria flava and Pholiota squarrosa).

The worldwide obesity and type 2 diabetes epidemics have led to an increase in nonalcoholic fatty liver disease (NAFLD). NAFLD covers a spectrum of hepatic pathologies ranging from simple steatosis to nonalcoholic steatohepatitis, characterized by fibrosis and hepatic inflammation. Nonalcoholic steatohepatitis predisposes to the onset of hepatocellular carcinoma (HCC). Here, we characterized the effect of a pharmacological activator of the intracellular energy sensor adenosine monophosphate–activated protein kinase (AMPK) on NAFLD progression in a mouse model. The compound stimulated fat oxidation by activating AMPK in both liver and skeletal muscle, as revealed by indirect calorimetry. This translated into an ameliorated hepatic steatosis and reduced fibrosis progression in mice fed a diet high in fat, cholesterol, and fructose for 20 weeks. Feeding mice this diet for 80 weeks caused the onset of HCC. The administration of the AMPK activator for 12 weeks significantly reduced tumor incidence and size. Conclusion: Pharmacological activation of AMPK reduces NAFLD progression to HCC in preclinical models.

This article presents the results of the thermal survey of the capital of Bulgaria (Sofia) carried out in August 2019, with the application of an unmanned aerial system (UAS). The study is based on the concept of local climate zones (LCZs), taking into account the influence of the features of land use/land cover and urban morphology on the urban climate. The basic spatial units used in the study are presented in the form of a regular grid consisting of 3299 cells with sides of 250 × 250 m. A total of 13 types of LCZs were identified, of which LCZs 6, 5, 8, 4, D, and A form the largest share. In the thermal imaging of the surface, a stratified sampling scheme was applied, which allowed us to select 74 cells, which are interpreted as representative of all cells belonging to the corresponding LCZ in the urban space. The performed statistical analysis of the thermal data allowed us to identify both the most thermally loaded zones (LCZs 9, 4, and 5) and the cells forming Urban Cool Islands (mainly in LCZs D and C). The average surface temperature in Sofia during the study period (in the time interval between 8:00 p.m. and 10:00 p.m.) was estimated at 20.9 °C, and between the different zones it varied in the range 17.2–25.1 °C. The highest maximum values of LST (27.9–30.6 °C) were registered in LCZ 4 and LCZ 5. The relation between the spatial structure of the urban thermal patterns and urban surface characteristics was also analyzed. Regression analysis confirmed the hypothesis that as the proportion of green areas increases, surface temperatures decrease, and, vice versa, as the proportion of built-up and impermeable areas increases, surface temperatures increase. A heat load map (via applying a z-transformation to standardize the temperature values), a map of the average surface temperature, and a map of the average intensity of the heat island on the surface were generated in the GIS environment. The results of the study adequately reflect the complex spatial model of the studied phenomenon, which gives grounds to conclude that the research approach used is applicable to similar studies in other cities.

Monitoring the structure parameters and damage to trees plays an important role in forest management. Remote-sensing data collected by an unmanned aerial vehicle (UAV) provides valuable resources to improve the efficiency of decision making. In this work, we propose an approach to enhance algorithms for species classification and assessment of the vital status of forest stands by using automated individual tree crowns delineation (ITCD). The approach can be potentially used for inventory and identifying the health status of trees in regional-scale forest areas. The proposed ITCD algorithm goes through three stages: preprocessing (contrast enhancement), crown segmentation based on wavelet transformation and morphological operations, and boundaries detection. The performance of the ITCD algorithm was demonstrated for different test plots containing homogeneous and complex structured forest stands. For typical scenes, the crown contouring accuracy is about 95%. The pixel-by-pixel classification is based on the ensemble supervised classification method error correcting output codes with the Gaussian kernel support vector machine chosen as a binary learner. We demonstrated that pixel-by-pixel species classification of multi-spectral images can be performed with a total error of about 1%, which is significantly less than by processing RGB images. The advantage of the proposed approach lies in the combined processing of multispectral and RGB photo images.

(1) Background: Persistent Helicobacter pylori infection is the most important risk factor for gastric cancer. The urokinase receptor (uPAR) is upregulated in lesions harboring cancer invasion and inflammation. Circumstantial evidence tends to correlate H. pylori colonization with increased uPAR expression in the human gastric epithelium, but a direct causative link has not yet been established in vivo; (2) Methods: In a mouse model of H. pylori-induced gastritis, we investigated the temporal emergence of uPAR protein expression in the gastric mucosa in response to H. pylori (SS1 strain) infection; (3) Results: We observed intense uPAR immunoreactivity in foveolar epithelial cells of the gastric corpus due to de novo synthesis, compared to non-infected animals. This uPAR induction represents a very early response, but it increases progressively over time as do infiltrating immune cells. Eradication of H. pylori infection by antimicrobial therapy causes a regression of uPAR expression to its physiological baseline levels. Suppression of the inflammatory response by prostaglandin E2 treatment attenuates uPAR expression. Notwithstanding this relationship, H. pylori does induce uPAR expression in vitro in co-cultures with gastric cancer cell lines; (4) Conclusions: We showed that persistent H. pylori colonization is a necessary event for the emergence of a relatively high uPAR protein expression in murine gastric epithelial cells.

Carcinoma-associated fibroblasts are key contributors of the tumor microenvironment that regulates carcinoma progression. They consist of a heterogeneous cell population with diverse origins, phenotypes, and functions. In the present report, we have explored the contribution of bone marrow (BM)-derived cells to generate different fibroblast subsets that putatively produce the matrix metalloproteinase 13 (MMP13) and affect cancer cell invasion. A murine model of skin carcinoma was applied to mice, irradiated, and engrafted with BM isolated from green fluorescent protein (GFP) transgenic mice. We provide evidence that one third of BM-derived GFP+ cells infiltrating the tumor expressed the chondroitin sulfate proteoglycan NG2 (pericytic marker) or α-smooth muscle actin (α-SMA, myofibroblast marker), whereas almost 90% of Thy1+ fibroblasts were originating from resident GFP-negative cells. MMP13producing cells were exclusively α-SMA+ cells and derived from GFP+ BM cells. To investigate their impact on tumor invasion, we isolated mesenchymal stem cells (MSCs) from the BM of wild-type and MMP13-deficient mice. Wild-type MSC promoted cancer cell invasion in a spheroid assay, whereas MSCs obtained from MMP13-deficient mice failed to. Our data support the concept of fibroblast subset specialization with BM-derived α-SMA+ cells being the main source of MMP13, a stromal mediator of cancer cell invasion.

In prostate cancers (PCa), the formation of malignant stroma may substantially influence tumor phenotype and aggressiveness. Thus, the impact of the orthotopic and subcutaneous implantations of hormone-sensitive (H), hormone-insensitive (HI), anaplastic (AT1) Dunning PCa in rats on growth, microcirculation, metabolism was investigated. For this purpose, dynamic contrast-enhanced magnetic resonance imaging and 1H magnetic resonance spectroscopy ([1H]MRS) were applied in combination with histology. Consistent observations revealed that orthotopic H tumors grew significantly slower compared to subcutaneous ones, whereas the growth of HI and AT1 tumors was comparable at both locations. Histologic analysis indicated that glandular differentiation and a close interaction of tumor cells and smooth muscle cells (SMC) were associated with slow tumor growth. Furthermore, there was a significantly lower SMC density in subcutaneous H tumors than in orthotopic H tumors. Perfusion was observed to be significantly lower in orthotopic H tumors than in subcutaneous H tumors. Regional blood volume and permeability-surface area product showed no significant differences between tumor models and their implantation sites. Differences in growth between subcutaneous and orthotopic H tumors can be attributed to tumor-stroma interaction and perfusion. Here, SMC, may stabilize glandular structures and contribute to the maintenance of differentiated phenotype.

The objective of the present study was to confirm the expression and localisation pattern of the urokinase-type plasminogen activator receptor (uPAR) focusing on its possible clinical relevance in patients with urothelial neoplasia of the bladder. uPAR is a central molecule in tissue remodelling during cancer invasion and metastasis and is an established prognostic marker in various cancer diseases other than bladder cancer. Formalin-fixed and paraffin-embedded tumour-tissue blocks from 186 patients treated with radical cystectomy were analysed. uPAR expression was scored as either negative or positive as well as by the actual score. Separate scores were obtained for cancer cells, macrophages and myofibroblasts at the invasive front and in tumour core. We were able to confirm, in an independent patient cohort, the tissue expression and localisation pattern of uPAR as investigated by Immunohistochemistry as well as a significant association between uPAR positivity and increasing tumour stage and tumour grade. This demonstrates the robustness of our previous and current findings. In addition the association between uPAR positive myofibroblasts and poor survival was reproduced. The highest hazard ratios for survival were seen for uPAR positive myofibroblasts both at the invasive front and in tumour core. Evaluating uPAR expression by the actual score showed a significant association between uPAR positive myofibroblasts in tumour core and an increased risk of cancer specific mortality. Our investigations have generated new and valuable biological information about the cell types being involved in tumour invasion and progression through the plasminogen activation system.

Constrained geometric simulations have been performed for the recently published closed-channel state of the nicotinic acetylcholine receptor. These simulations support the theory that correlated motion in the flexible β-sheet structure of the extracellular domain helps to communicate a "conformational wave", spreading from the acetylcholine binding pocket. Furthermore, we have identified key residues that act at the interface between subunits and between domains that could potentially facilitate rapid communication between the binding site and the transmembrane gate., (Copyright © 2014. Published by Elsevier Inc.)

Background and Purpose: It has been proposed that arginine residues lining the intracellular portals of the homomeric 5-HT3 A receptor cause electrostatic repulsion of cation flow, accounting for a single-channel conductance substantially lower than that of the 5-HT3 AB heteromer. However, comparison of receptor homology models for wild-type pentamers suggests that salt bridges in the intracellular domain of the homomer may impart structural rigidity, and we hypothesized that this rigidity could account for the low conductance., Experimental Approach: Mutations were introduced into the portal region of the human 5-HT3 A homopentamer, such that putative salt bridges were broken by neutralizing anionic partners. Single-channel and whole cell currents were measured in transfected tsA201 cells and in Xenopus oocytes respectively. Computational simulations of protein flexibility facilitated comparison of wild-type and mutant receptors., Key Results: Single-channel conductance was increased substantially, often to wild-type heteromeric receptor values, in most 5-HT3 A mutants. Conversely, introduction of arginine residues to the portal region of the heteromer, conjecturally creating salt bridges, decreased conductance. Gating kinetics varied significantly between different mutant receptors. EC50 values for whole-cell responses to 5-HT remained largely unchanged, but Hill coefficients for responses to 5-HT were usually significantly smaller in mutants. Computational simulations suggested increased flexibility throughout the protein structure as a consequence of mutations in the intracellular domain., Conclusions and Implications: These data support a role for intracellular salt bridges in maintaining the quaternary structure of the 5-HT3 receptor and suggest a role for the intracellular domain in allosteric modulation of cooperativity and agonist efficacy., (© 2013 The Authors. British Journal of Pharmacology published by John Wiley &. Sons Ltd on behalf of The British Pharmacological Society.)

The 5-HT₃ receptor is a cation selective member of the pentameric Cys-loop ligand-gated ion channels. While five subunits are known to exist, only two receptor subtypes have been significantly characterized: the homomeric receptor consisting of five A subunits and the heteromeric receptor containing both A and B subunits. The agonist recognition and activation of these receptors is orchestrated by six recognition loops three, A-C, on the principal subunit, and three, D-F, on the complementary subunit. In this study we have focused on the B loop of the principal subunit and loop D of the complementary subunit where aligned amino acids differ between the two subunits. A mutational analysis has been carried out using both 5-HT and m-chlorophenylbiguanide (mCPBG) to characterize receptor activation in the mutant receptors using two-electrode voltage clamp in Xenopus oocytes. The results show that the B loop W178I mutation of the 5-HT3A subunit markedly reduces the efficacy of mCPBG in both the homomeric and heteromeric receptors, while activation by 5-HT remains intact. Replacement of the D loop amino acid triplet RQY of the 5-HT3A subunit, with the aligned residues from the 5-HT3B subunit, QEV, converts 5-HT to a weak partial agonist in both the homomer and heteromer, but does not compromise activation by mCPBG. Exchange of the RQY triplet for the 5-HT3B subunit homologue, QEV, increases the Hill coefficient and decreases the EC₅₀ of this mutant when expressed with the wild type 5-HT3A subunit., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Each subunit of the cation-selective members of the Cys-loop family of ligand-gated ion channels contains a conserved proline residue in the extracellular loop between the second and third transmembrane domains. In the mouse homomeric 5-hydroxytryptamine type 3A (5-HT(3)A) receptor, the effects of substitution of this proline by unnatural amino acids led to the suggestion that trans-cis isomerization of the protein backbone at this position is integral to agonist-induced channel opening [Nature (2005) vol. 438, pp. 248-252]. We explored the generality of this conclusion using natural amino acid mutagenesis of the homologous human 5-HT(3)A receptor. The conserved proline (P303) was substituted by either a histidine or tryprophan and the mutant receptors were expressed in Xenopus oocytes. These mutations did not significantly affect the magnitude of agonist-mediated currents, compromise channel gating by 5-HT or inhibition of 5-HT-induced currents by either picrotoxin or d-tubocurarine. The mutations did, however, result in altered dependence on extracellular Ca(2+) concentration and a 10-fold increase in the rate of receptor desensitization. These results demonstrate an important role for P303 in 5-HT(3)A receptor function but indicate that trans-cis isomerization at this proline is unlikely to be a general mechanism underlying the gating process.

The GABA(A) receptor is a chloride-selective ligand-gated ion channel of the Cys-loop superfamily. The receptor consists of five subunits arranged pseudosymmetrically around a central pore. The predominant form of the receptor in the brain contains alpha(1)-, beta(2)-, and gamma(2)-subunits in the arrangement alphabetaalphagammabeta, counter-clockwise around the pore. GABA(A) receptors containing delta-instead of gamma-subunits, although a minor component of the total receptor population, have interesting properties, such as an extrasynaptic location, high sensitivity to GABA, and potential association with conditions such as epilepsy. They are therefore attractive targets for drug development. Here we addressed the subunit arrangement within the alpha(4)beta(3)delta form of the receptor. Different epitope tags were engineered onto the three subunits, and complexes between receptors and anti-epitope antibodies were imaged by atomic force microscopy. Determination of the numbers of receptors doubly decorated by each of the three antibodies revealed a subunit stoichiometry of 2alpha:2beta:1delta. The distributions of angles between pairs of antibodies against the alpha- and beta-subunits both had peaks at around 144 degrees , indicating that these pairs of subunits were nonadjacent. Decoration of the receptor with ligands that bind to the extracellular domain (i.e., the lectin concanavalin A and an antibody that recognizes the beta-subunit N-terminal sequence) showed that the receptor preferentially binds to the mica extracellular face down. Given this orientation, the geometry of complexes of receptors with both an antibody against the delta-subunit and Fab fragments against the alpha-subunits indicates a predominant subunit arrangement of alphabetaalphadeltabeta, counter-clockwise around the pore when viewed from the extracellular space.

We present two comparative models of the GABA(A) receptor. Model 1 is based on the 4-A resolution structure of the nicotinic acetylcholine receptor from Torpedo marmorata and represents the unliganded receptor. Two agonists, GABA and muscimol, two benzodiazepines, flunitrazepam and alprazolam, together with the general anaesthetic halothane, have been docked to this model. The ion flow is also explored in model 1 by evaluating the interaction energy of a chloride ion as it traverses the extracellular, transmembrane and intracellular domains of the protein. Model 2 differs from model 1 only in the extracellular domain and represents the liganded receptor. Comparison between the two models not only allows us to explore commonalities and differences with comparative models of the nicotinic acetylcholine receptor, but also suggests possible protein sub-domain interactions with the GABA(A) receptor not previously addressed.

The 5-HT3 receptor is a cation-selective ligand-gated ion channel of the Cys-loop superfamily. The receptor is an important therapeutic target, with receptor antagonists being widely used as antiemetics in cancer therapy. The two known receptor subunits, A and B, form homomeric 5-HT 3A receptors and heteromeric 5-HT 3A/B receptors. The heteromeric receptor has the higher single-channel conductance and more closely mimics the properties of the native receptor. We have used atomic force microscopy to study the architecture of 5-HT 3A and 5-HT 3A/B receptors. We engineered different epitope tags onto the A- and B-subunits and imaged receptors that were doubly liganded by anti-epitope antibodies. We found that, for the 5-HT 3A/B receptor, the distribution of angles between antibodies against the A-subunit had a single peak at approximately 144 degrees , whereas the distribution for antibodies against the B-subunit had two peaks at approximately 72 degrees and 144 degrees . Our results indicate that the subunit stoichiometry is 2A:3B and that the subunit arrangement around the receptor rosette is B-B-A-B-A. This arrangement may account for the difference between the agonist Hill coefficients and the single-channel conductances for the two types of receptor.

In women, the late luteal phase or "premenstrual" period is commonly associated with psychological disturbances, which include mood changes and increased aggression. The underlying cause is unknown but one possibility is that fluctuations in levels of neuroactive steroids precipitate changes in expression of GABA(A) receptor subunits that result in functional changes in inhibitory control systems. The present study investigated the levels of expression of alpha4, beta1 and delta GABA(A) receptor subunits in the periaqueductal gray matter (PAG) in rats and whether plasticity occurs during the oestrous cycle in females. In male rats alpha4, beta1 and delta subunit immunoreactive neurones were present throughout the PAG in similar numbers. In female rats in proestrus, oestrus and early dioestrus, the density of alpha4, beta1 and delta subunit immunoreactive cells was similar to males. However, in late dioestrus, the numbers increased significantly, especially in the dorsolateral PAG, a region which is particularly rich in GABAergic interneurones. These parallel changes may reflect an increase in expression of the alpha4beta1delta GABA(A) receptor subtype. Recombinant alpha4beta1delta receptors, expressed in Xenopus oocytes, exhibited and EC(50) for GABA an order of magnitude lower (2.02+/-0.33 microM; mean+/-S.E.M.) than that found for the most ubiquitous alpha1beta2gamma2 GABA(A) receptor (32.8+/-2.5 microM). Increased expression of alpha4beta1delta GABA(A) receptors in the interneurones of the PAG could render the panic circuitry abnormally excitable by disinhibiting the ongoing GABAergic inhibition. Similar changes in neuronal excitability within the PAG in women consequent to falling steroid levels in the late luteal phase of the menstrual cycle could contribute to the development of pre-menstrual dysphoria.

1. Streptavidin is a 60-kDa tetramer which binds four molecules of biotin with extremely high affinity (K(A) approximately 10(14) M(-1)). We have used atomic force microscopy (AFM) to visualize this ligand-protein interaction directly. 2. Biotin was tagged with a short (152-basepair; 50-nm) DNA rod and incubated with streptavidin. The resulting complexes were then imaged by AFM. The molecular volume of streptavidin calculated from the dimensions of the protein particles (105+/-3 nm(3)) was in close agreement with the value calculated from its molecular mass (114 nm(3)). Biotinylation increased the apparent size of streptavidin (to 133+/-2 nm(3)), concomitant with an increase in the thermal stability of the tetramer. 3. Images of streptavidin with one to four molecules of DNA-biotin bound were obtained. When two ligands were bound, the angle between the DNA rods was either acute or obtuse, as expected from the relative orientations of the biotin binding sites. The ratio of acute : obtuse angles (1 : 3) was lower than the expected value (1 : 2), indicating a degree of steric hindrance in the binding of the DNA-biotin. The slight under-representation of higher occupancy states supported this idea. 4. Streptavidin with a single molecule of DNA-biotin bound was used to tag biotinylated beta-galactosidase, a model multimeric enzyme. 5. The ability to image directly the binding of a ligand to its protein target by AFM provides useful information about the nature of the interaction, and about the effect of complex formation on the structure of the protein. Furthermore, the use of DNA-biotin/streptavidin tags could potentially shed light on the architecture of multi-subunit proteins.

1. Phenelzine (PLZ) is an antidepressant with anxiolytic properties. Acute and chronic PLZ administration increase brain GABA levels, an effect due, at least in part, to an inhibition of the activity of the GABA metabolizing enzyme, GABA transaminase (GABA-T). 2. Previous preliminary reports have indicated that acute PLZ treatment also elevates brain alanine levels. As with GABA, the metabolism of alanine involves a pyridoxal phosphate-dependent transaminase. 3. In the study reported here, the effects of acute PLZ treatment on the levels of various amino acids, some of which are also metabolized by pyridoxal phosphate-dependent transaminases were compared in rat whole brain. Of the 6 amino acids investigated, only GABA and alanine levels were elevated (in a time- and dose-dependent manner). 4. The elevation in brain alanine levels could be explained, at least in part, by a time- and dose-dependent inhibitory effect of PLZ on alanine transaminase (ALA-T), although as with GABA the increases are higher than expected from the degree of enzyme inhibition produced. In addition, we also showed that the elevation in alanine levels and the inhibition of alanine transaminase in the brain are retained after 14 days of PLZ treatment, and that PLZ produces a marked increase in extracellular levels of alanine. 5. These results are discussed in terms of their relevance to synaptic function and to the pharmacological profile of PLZ.

Diazepam is widely prescribed as an anxiolytic but its therapeutic application is limited because with daily use tolerance develops to certain aspects of its pharmacological profile. We compared the effects of two dosing paradigms on GABA(A) receptor gene expression and benzodiazepine binding characteristics. Equivalent daily doses of 15 mg/kg/day diazepam were delivered either via constant infusion or daily subcutaneous injection for 14 days. The two distinct treatment regimes produced significantly different changes in GABA(A) receptor alpha4-, beta2-, beta3- and gamma1-subunit mRNA steady-state levels. Similar changes in the GABA enhancement of flunitrazepam binding and the BZ3/BZ2 subtype ratio determined ex vivo were produced, however, significant differences were found in [(3)H]-Ro 15-4513 binding between cortical tissue from diazepam injected animals compared with diazepam infused animals. Our data suggest that it is the diurnal fluctuations in receptor occupancy that are responsible for the different effects produced by these two dosing regimes., (Copyright 2001 Wiley-Liss, Inc.)

Purpose: To present a case of respiratory failure following suboccipital craniectomy for Chiari type I malformation., Clinical Features: A 22-yr-old man presented with a two year history of symptoms and signs suggestive of brainstem compression at the level of the foramen magnum. This was confirmed with magnetic resonance imaging. The procedure of suboccipital craniectomy, upper cervical laminectomy and fourth ventricle exploration was performed. Three hours postoperatively the patient experienced episodes of apnea and subsequently became drowsy. Blood gas analysis revealed hypercapnic respiratory failure. Chest X-ray revealed evidence of pulmonary aspiration. The trachea was re-intubated and the lungs ventilated in intensive care for 72 hr. He was discharged home two weeks postoperatively., Conclusion: Chiari type I malformation is associated with a number of associated anomalies. These patients are at considerable risk of respiratory depression and bulbar dysfunction in the perioperative period. The anesthetic issues are reviewed and discussed.

Zopiclone is a cyclopyrrolone that is used clinically as a hypnotic. Although this drug is known to interact with neuronal gamma-aminobutyric acid type A receptors, its binding site(s) within the receptor oligomer has been reported to be distinct from that of the classical benzodiazepines. After photoaffinity labeling with flunitrazepam, receptors in rat cerebellar membranes showed differentially reduced affinity for flunitrazepam and zopiclone by 50- and 3-fold, respectively. Because histidine 101 of the alpha-subunit is a major site of photolabeling, we have made specific substitutions of this residue and studied the consequences on the binding properties of zopiclone and diazepam using recombinant alpha1beta2gamma2-receptors transiently expressed in tsA201 cells. Both compounds showed similar binding profiles with receptors containing mutated alpha-subunits, suggesting a similar interaction with the residue at position 101. At alpha1beta2gamma3-receptors, flunitrazepam affinity was dramatically decreased by approximately 36-fold, whereas the affinity for zopiclone was decreased only 3-fold, suggesting a differential contribution of the gamma-subunit to the binding pocket. Additionally, we used electrophysiological techniques to examine the contribution of the gamma-subunit isoform in the receptor oligomer to ligand recognition using recombinant receptors expressed in Xenopus oocytes. Both compounds are agonists at alpha1beta2gamma2- and alpha1beta2gamma3-receptors, with flunitrazepam being more potent but less efficacious. In summary, these data suggest that histidine 101 of the alpha1-subunit plays a similar role in ligand recognition for zopiclone, diazepam, and flunitrazepam.

Purpose: To present a case of Moyamoya disease with intracranial hemorrhage complicating pregnancy., Clinical Features: A 36-yr-old parturient at 34 wk gestation presented with left hemiparesis, headache, nausea and vomiting. Subsequent deterioration in level of consciousness and the development of a dilated right pupil necessitated immediate intubation. Urgent non-contrast CT scan revealed a large right intracerebral hematoma with transtentorial herniation. The patient underwent simultaneous emergency Cesarean section and craniotomy. A postoperative angiogram revealed findings consistent with Moyamoya disease. The neonate survived but the patient developed severe cerebral edema and died eleven days postoperatively., Conclusion: Adult patients with Moyamoya disease often present with intracranial hemorrhage which poses unique anesthetic challenges. We report a case of intracerebral hemorrhage during pregnancy, which is known to be associated with high morbidity and mortality. The anesthetic techniques are reviewed and discussed.

The 5-hydroxytryptamine (5-HT)(3) receptor is a member of the ligand-gated ion channel receptor family with significant homology to the nicotinic acetylcholine, gamma-aminobutyric acid(A), and glycine receptors. In this receptor class, the agonist binding site is formed by parts of the extracellular amino-terminal region. This study examines the effects of altering phenylalanine 107 (F107) of the 5-HT(3AL) subunit, obtained from NG108-15 cells, using site-directed mutagenesis. The wild-type (WT) and mutant receptors were expressed in HEK 293 cells and characterized using both whole-cell patch-clamp and radioligand binding. The tyrosine mutant F107Y exhibits a significantly lower affinity for the agonist 5-HT (K(i) = 203 versus 15.6 nM) and an increase of similar magnitude in the EC(50) value (10.6 versus 1.2 microM) compared with WT. The activation kinetics of the maximal currents generated by 5-HT with this mutant were markedly slower than those of the WT receptor, but application of supramaximal concentrations of the agonist markedly decreased the time to half-peak. The asparagine mutant F107N displayed a significantly higher affinity for 5-HT than the WT receptor (1.62 versus 15.6 nM), which was mirrored in direction and magnitude by changes in the EC(50) value for this agonist (0.2 versus 1.2 microM). In contrast to the WT receptor, the mutant F107N was activated by acetylcholine (EC(50) = 260 microM). The response to acetylcholine was blocked by the 5-HT(3) receptor antagonist renzapride with a similar IC(50) value as that determined against currents generated by 5-HT in the WT receptor. These data suggest that F107 is an important determinant of agonist recognition at the 5-HT(3) receptor.

Rationale: Chronic treatment with benzodiazepines results in tolerance to their sedative and anxiolytic effects and there is considerable evidence that different mechanisms underlie the development of tolerance to different behavioural effects., Objective: The purpose of the present experiment was to compare the behavioural effects of chronic treatment with diazepam (15 mg/kg per day) given as daily subcutaneous injections or by osmotic minipump. Both regimens resulted in continual receptor occupancy, but the daily injections also provided a period of higher brain concentrations., Methods: Rats were tested in the holeboard, which provides measures of exploration and locomotor activity, and in the elevated plus-maze and social interaction tests of anxiety. For those in the subcutaneous injection group the tests were 2 h after injection, when brain concentrations were highest., Results: Despite a higher brain concentration in the injected group, both groups showed tolerance to diazepam's sedative effects, after 7 days of treatment. In contrast, in the elevated plus-maze, there was tolerance to the anxiolytic effects in the pump group after 14 days, but a persisting anxiolytic effect in the injected group at 14 and 28 days. Whilst higher brain concentrations could explain this result in the plus-maze, they cannot account for the pattern observed in the social interaction test, where the injection group showed a significant anxiogenic effect at 28 days., Conclusions: Whereas the mechanism underlying tolerance to the sedative effects of diazepam was insensitive to the different treatment regimens, the results suggest that different adaptive mechanisms were triggered in the two tests of anxiety with a differential sensitivity to the treatment regimen. The adaptive mechanism predominating in the social interaction test was favoured by the injection regimen which produced intermittent peak concentrations. This mechanism seems to be an oppositional one, leading to an anxiogenic response, which was manifest despite high brain concentrations of diazepam at the time of testing.

The GABA and benzodiazepine binding sites on GABA(A) receptors are allosterically coupled. The in vitro binding of 2 nM [3H]flunitrazepam to cortical and cerebellar membranes prepared from drug-naive rats was potentiated approximately 1.6-fold by 100 microM GABA. Potentiation in both regions was significantly reduced 4 or 12 but not 24 h after a single dose of 15 mg/kg diazepam. At 24 h after the last of 14 daily doses of diazepam, no differences in GABA potentiation were observed. Diazepam-induced changes in GABA(A) receptor gamma2-subunit gene transcription and alpha1-, beta2-, and gamma2-subunit steady-state mRNA levels did not appear to be temporally related to allosteric uncoupling.

Reported here is a rapid, sensitive and relatively inexpensive procedure using gas chromatography with nitrogen-phosphorus detection (GC-NPD) to quantify buspirone levels in brains of rats. The analyte was directly extracted from brain homogenate with toluene after basification and then subjected to GC-NPD analysis using a capillary column. The calibration curves were linear over the range of 10 to 320 ng per 2 ml of brain homogenate, with typical r2 values >0.99. The assay was highly reproducible and gave peaks with excellent chromatographic properties.

The rate of transcription of the GABA(A) receptor gamma2-subunit gene in rat cortex has been measured using the nuclear run-off transcriptional assay. Exposure of rats to diazepam (15 mg/kg/day for 14 days) caused a significant reduction in the level of nascent GABA(A) receptor gamma2-subunit transcripts. Therefore, a component of the cellular response to chronic benzodiazepine exposure includes events which take place at the level of transcription of a GABA(A) receptor gene.

The effect of chronic zolpidem treatment on the steady-state levels of gamma-aminobutyric acidA alpha1-6, beta1-3 and gamma1-3 subunit mRNAs in rat cortex has been investigated. Male Sprague-Dawley rats were injected once daily, for 7 or 14 days, with 15 mg/kg of zolpidem in sesame oil vehicle. The levels of the alpha4 and beta1 subunit mRNAs were significantly increased after 7 days of treatment and the level of alpha1 subunit mRNA was significantly decreased after 14 days of treatment, as determined by solution hybridization. These results are compared to the previously determined effects of an equivalent schedule of treatment with diazepam.

The 5-HT3 receptor is a ligand-gated ion channel with significant structural similarity to the nicotinic acetylcholine receptor. Several regions that form the ligand binding site in the nicotinic acetylcholine receptor are partially conserved in the 5-HT3 receptor, presumably reflecting the conserved signal transduction mechanism. Specific amino acid differences in these regions may account for their distinct ligand recognition properties. Using site-directed mutagenesis, we have replaced one of these residues, glutamate 106 (E106), with aspartate (D), asparagine (N), alanine (A) or glutamine (Q) and characterized the ligand-binding and electrophysiological properties of the mutant receptors after transient expression in HEK-293 cells. The affinity for the selective 5-HT3 receptor antagonist [3H]GR65630 was decreased 14-fold in the mutant E106D (Kd = 3.69 +/- 0.32 nM) when compared to wildtype (WT, E106) 5-HT3 receptor (0.27 +/- 0.03 nM), while the affinity for E106N was unchanged (0.42 +/- 0.07 nM, means +/- SEM, n = 3-10). Decreased affinities for both E106D and E106N were observed for the antagonists granisetron, ondansetron and renzapride and for the agonists 5-HT (130- and 30-fold) and 2-methyl-5-HT (250- and 20-fold), respectively. Both mutants still formed 5-HT-activatable ion channels, but the high Hill coefficient of the concentration effect curves in wildtype (2.0) was decreased to unity in both cases. The EC50 of 5-HT was increased seven-fold in E106N (8.7 microM) when compared to wildtype (1.2 microM), but unchanged in E106D, and the potency of the antagonist ondansetron for both mutants was decreased. E106A and E106Q expressed poorly preventing a detailed characterization. These data suggest that E106 contributes to the ligand-binding site of the 5-HT3 receptor and may form an ionic or hydrogen bond interaction with the primary ammonium group of 5-HT.

The functional integrity of Alzet osmotic minipumps was assessed using two organic solvents (50% (v/v) dimethyl sulfoxide (DMSO)/50% (v/v) propylene glycol (PG) and 100% tetraglycol) which dissolve diazepam, an aqueous insoluble benzodiazepine. Both solvents showed a significant decrease in output rate over time: the decline with tetraglycol was, however, more marked and variable. Further, the DMSO/PG vehicle demonstrated a comparable decline in rate (1.45%) to that of the control vehicles saline and water (1.12%). DMSO/PG is therefore a suitable solvent for the chronic delivery of diazepam from osmotic minipumps.

The alpha subunit of the gamma-aminobutyric acid type A (GABA(A)) receptor is known to be photoaffinity labeled by the classical benzodiazepine agonist, [3H]flunitrazepam. To identify the specific site for [3H]flunitrazepam photoincorporation in the receptor subunit, we have subjected photoaffinity labeled GABA(A) receptors from bovine cerebral cortex to specific cleavage with cyanogen bromide and purified the resulting photolabeled peptides by immunoprecipitation with an anti-flunitrazepam polyclonal serum. A major photolabeled peptide component from reversed-phase high performance liquid chromatography of the immunopurified peptides was resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The radioactivity profile indicated that the [3H]flunitrazepam photoaffinity label is covalently associated with a 5.4-kDa peptide. This peptide is glycosylated because treatment with the enzyme, peptide-N4-(N-acetyl-beta-glucosaminyl)asparagine amidase, reduced the molecular mass of the peptide to 3.2 kDa. Direct sequencing of the photolabeled peptide by automated Edman degradation showed that the radioactivity is released in the twelfth cycle. Based on the molecular mass of the peptides that can be generated by cyanogen bromide cleavage of the GABA(A) receptor alpha subunit and the potential sites for asparagine-linked glycosylation, the pattern of release of radioactivity during Edman degradation of the photolabeled peptide was mapped to the known amino acid sequence of the receptor subunit. The major site of photoincorporation by [3H]flunitrazepam on the GABA(A) receptor is shown to be alpha subunit residue His102 (numbering based on bovine alpha 1 sequence).

Diazepam and abecarnil produce their overt effects by interaction with the GABAA receptor. Chronic treatment with abecarnil, however, does not induce diazepam-like tolerance. This study investigates the effects of chronic diazepam and abecarnil treatment on expression of GABAA receptor alpha 1-6 beta 1-3 and gamma 1-3 subunit isoform mRNAs in rat cortex. Male Sprague-Dawley rats were injected subcutaneously once daily for 7 or 14 days with 15 mg/kg diazepam or 6 mg/kg abecarnil in sesame-oil vehicle, and steady-state levels of GABAA receptor subunit mRNAs were quantified by solution hybridization. The levels of alpha 4- and alpha-, beta 1- and gamma 3-subunit mRNAs were significantly increased after 7 days of diazepam treatment, and this effect was maintained at 14 days. A significant increase in alpha 3-subunit mRNA was apparent only after 14 days of diazepam treatment and a significant decrease in beta 2-subunit mRNA was seen only after 14 days of abecarnil treatment. Gamma 2-Subunit mRNA was significantly decreased after 14 days of either diazepam or abecarnil exposure. A degree of association between a particular drug treatment and changes in the levels of mRNAs arising from a given gene cluster was noted. Our results are consistent with a model of diazepam dependence based on GABAA receptor subunit isoform switching.

We have used [3H]flunitrazepam and [3H]Ro15-4513 as photoaffinity labelling agents in combination with a chemical cleavage technique to localize the benzodiazepine recognition sites of specific human recombinant alpha 1 beta 1 gamma 2, alpha 1 beta 3 gamma 2 and alpha 6 beta 3 gamma 2 GABAA receptor subtypes. The chemical agent utilized was hydroxylamine, whose substrate is a relatively rare asparagine-glycine amide bond that occurs only in the alpha subunits of the receptors examined in this study. Cleavage products were resolved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of these experiments show that, in the alpha 1 subunit-containing receptors, incorporation of [3H]flunitrazepam occurs within residues 1-103 of the alpha 1 subunit, while incorporation of [3H]Ro15-4513 occurs within the region of the alpha 1 subunit that lies between residue 104 and the C-terminus. Photolabelling of membranes prepared from the alpha 6 beta 3 gamma 2-expressing cell line with [3H]Ro15-4513 resulted in the incorporation of radiolabel into two major protein species of M(r) 56,000 and M(r) 48,000, indicating incorporation into the alpha 6 subunit and possibly also the gamma 2 subunit. Hydroxylamine cleavage of alpha 6-containing receptors labelled with [3H]Ro15-4513 produced a gel profile consistent with the incorporation of the label occurring between residue 125 and the C-terminal. Thus, we have shown that the recognition sites for the agonist [3H]flunitrazepam and the inverse agonist [3H]Ro15-4513 occur within distinct domains of the human GABAA receptor.

We have determined the ultrastructure of 5-hydroxytryptamine3 (5-HT3) serotonin receptors purified from NG108-15 mouse neuroblastoma x rat glioma cells by electron microscopic examination of receptor particles embedded in uranyl acetate stain and metal replicas of rapidly frozen receptors. The 5-HT3 receptor can be modelled as a cylinder 11 nm in length and 8 nm in diameter with a closed end and a central cavity 3 nm in diameter. Analysis of the rotational symmetry of single receptor particles indicates that the 5-HT3 receptor is composed of five subunits arranged symmetrically around a central cavity. Together with evidence obtained for related proteins in other studies using ultrastructural, biochemical, or electrophysiological methods, our observations suggest that all members of the ligand-gated ion channel superfamily may possess a pentameric quaternary structure.

In the transmitter-gated ion channel class of receptors, the members of which are all believed to be heterooligomers, the number and arrangement of the subunits are only known with any certainty for the nicotinic acetylcholine receptor from Torpedo electric fish. That receptor has been shown to possess a pentameric rosette structure, with five homologous subunits (alpha 2, beta gamma delta) arranged to enclose the central ion channel. The data were obtained by electron image analysis of two-dimensional receptor arrays, which form as a consequence of that receptor's exceptionally high abundance in the Torpedo membranes and are therefore not attainable for other receptors. We have applied another direct approach to determine the quaternary structure of native ionotropic GABA receptors. We have purified those receptors from porcine brain cortex and analysed the rotational symmetry of isolated receptors visualized by electron microscopy. The results show the receptor to have a pentameric structure with a central water-filled pore, which can now be said to be characteristic of the entire superfamily.

5-Hydroxytryptamine3 (5-HT3) receptor-type binding sites were solubilised from NG108-15 mouse neuroblastoma x rat glioma hybrid cells using five different detergents [n-octyl-beta-D-glucoside, Triton X-100, 3-[3-(cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS), sodium cholate, and deoxycholate] and the solubilisation efficiencies compared. The equilibrium binding, kinetic properties, and pharmacological profile of solubilised binding sites were similar to those of 5-HT3 receptor-type binding sites (5-HT3R) in membrane preparations determined using [3H]GR65630. The solubilised binding sites were purified using an affinity column constructed by coupling the high-affinity antagonist GR119566X to an Affi-Gel 15 resin. The affinity of purified 5-HT3R for [3H]-GR65630 was reduced threefold compared to the crude soluble preparation, but the pharmacological profile was similar. The sedimentation coefficient of the purified protein (11S, detergent: CHAPS) was determined by sucrose density gradient centrifugation. The apparent molecular mass of the detergent/binding site complex (370 kDa) was determined by size exclusion chromatography in the presence of n-dodecyl-beta-D-maltoside. Gel electrophoresis of the purified protein revealed bands at apparent molecular masses of 36, 40, 50, and 76 kDa. Electron microscopy of the negatively stained purified protein showed the presence of round particles of 8-9 nm diameter with a 2-nm stained pit in the centre, closely resembling the doughnut shapes described for nicotinic acetylcholine receptors.