Perspective of Pain Clinicians in Three Global Cities on Local Barriers to Providing Care for Chronic Noncancer Pain Patients.

An increasing proportion of the global chronic pain population is managed through services delivered by specialized pain clinics in global cities. This paper describes the results of a survey of pain clinic leaders in three global cities on barriers influencing chronic noncancer pain (CNCP) management provided by those clinics. It demonstrates a pragmatic qualitative approach for characterizing how the global city location of the clinic influences those results. A cross-sectional prospective survey design was used, and data were analyzed using quantitative and qualitative content analysis. Key informants were pain clinicians (n = 4 women and 8 men) responsible for outputs of specialized pain clinics in academic hospital settings in three global cities: Toronto, Kuwait, and Karachi. Krippendorff's thematic clustering technique was used to identify the repetitive themes in the data. All but one of the key informants had their primary pain training from Europe or North America. In Kuwait and Karachi, pain specialists were anesthesiologists and provided CNCP management services independently. In Toronto, pain clinic leaders were part of some form of the multidisciplinary team. Using the results of a question that asked informants to list their top three barriers, ten themes were identified. These themes were artificially organized in three thematic domains: infrastructure, clinical services, and education. In parallel, 31 predefined barriers identified from the literature were scored. The results showed variation in perception of barriers that not only depended on the clinic location but also demonstrated shared experiences across thematic domains. This study demonstrates a simple methodology for informing global and local efforts to improve access to and implementation of CNCP services globally.

Development and evaluation of a continuing pharmacy education (CPE) program in thrombosis management.

BACKGROUND AND PURPOSE: A continuing education (CE) course in thrombosis management for pharmacists was developed through the Office of Continuing Professional Development (CPD) at the University of Toronto to address pharmacists' needs for the knowledge and skills to provide care to patients receiving anticoagulants. This article describes the development of the course as well as the evaluation designed to assess its impact on pharmacists' knowledge, attitudes, and changes in practice. EDUCATIONAL ACTIVITY AND SETTING: A three-day course was developed. Outcomes were evaluated using a feedback questionnaire, pre- and post-session quizzes and semi-structured interviews conducted six months after course completion. Participant satisfaction, knowledge acquisition and perceived change in knowledge, skills and practice were evaluated. FINDINGS: Thirty-seven pharmacists enrolled in the program, 21 of whom participated in a semi-structured interview. More than 90% reported that the program exceeded their expectations. Pharmacists' knowledge in thrombosis care improved significantly after each day of the course. Participants felt the greatest benefits of the program were increases in knowledge and confidence and the opportunity to network. The case-based discussions and practical tips gained from experts and peers were highly ranked. Participants strongly agreed that they were applying what they learned in the course to clinical practice, and they provided numerous examples of how their practice changed because of the program. DISCUSSION AND SUMMARY: The development of this CE course demonstrates application of best practices in continuing education. The evaluation of the program suggests that a CE course in thrombosis improves pharmacist knowledge, confidence and ability to incorporate what was learned into practice. This course design and evaluation can serve as a model for other CE courses for pharmacists as this field continues to grow and encourages thoughtful use of theoretical principles and well-designed evaluation for continual improvement of CE.

Differences in physical activity, eating habits and risk of obesity among Kuwaiti adolescent boys and girls: a population-based study.

The study aimed to assess gender differences among Kuwaiti adolescents in healthy living choices that impact the risk of obesity. A cross-sectional multistage cluster design was employed with a representative sample of 2672 students aged 13-15 years who completed a self-administered Global School-based Student Health (GSHS) survey. The study found that around 48.0% of adolescents were overweight and obese. More boys than girls were obese (28.2% vs. 22.3%, p < 0.0001). However, boys were more likely than girls to report healthy food choices regarding fruit (38.1% vs. 33.2%), and vegetables (21.8% vs. 16.7%). Only 20.7% of adolescents reported physical activity for more than 60 min/day, predominately by boys rather than girls (30.8% vs. 10.5%, respectively, p < 0.0001). Multivariate analysis revealed that male gender, skipping breakfast and physical inactivity were significantly correlated with the risk of overweight and obesity among adolescents. These results suggest that lifestyle education for promoting healthy body masses targeting adolescents should take gender into account.

Chronic Non-Cancer Pain Management Capacity in Karachi.

Chronic non-cancer pain (CNCP) affects people everywhere in the world, but people in developing countries have far less access to therapies that provide relief. There are often missed opportunities to implement these therapies. Karachi shares many characteristics with megacities of the global south and represents Pakistan in the global city league. This review informs readers about the availability of health management and pain services for CNCP in Karachi, and their comparability to those found in other global cities. The literature about CNCP and its management in Karachi and Pakistan is scarce. Nevertheless, some conclusions can be made. In order to inform readers based in other global cities, a brief review of the current health system and pain services in Karachi and Pakistan are discussed together with barriers that impede pain service outputs. The present review employs vignettes to illustrate typical experiences of CNCP patients seeking pain management services in three sectors: public, charitable, and private institutions.

Role of renin-angiotensin system in liver diseases: an outline on the potential therapeutic points of intervention.

INTRODUCTION: The current review aimed to outline the functions of the renin angiotensin system (RAS) in the context of the oxidative stress-associated liver disease. Areas covered: Angiotensin II (Ang II) as the major effector peptide of the RAS is a pro-oxidant and fibrogenic cytokine. Mechanistically, NADPH oxidase (NOX) is a multicomponent enzyme complex that is able to generate reactive oxygen species (ROS) as a downstream signaling pathway of Ang II which is expressed in liver. Ang II has a detrimental role in the pathogenesis of chronic liver disease through possessing pro-oxidant, fibrogenic, and pro-inflammatory impact in the liver. The alternative axis (ACE2/Ang(1-7)/mas) of the RAS serves as an anti-inflammatory, antioxidant and anti-fibrotic component of the RAS. Expert commentary: In summary, the use of alternative axis inhibitors accompanying with ACE2/ Ang(1-7)/mas axis activation is a promising new strategy serving as a novel therapeutic option to prevent and treat chronic liver diseases.

Health Services for Management of Chronic Non-Cancer Pain in Kuwait: A Case Study Review.

The experience of chronic pain is universal, yet pain management services delivered by health professionals vary substantially, depending on the context and patient. This review is a part of a series that has examined the issue of chronic non-cancer pain services and management in different global cities. The review is structured as a case study of the availability of management services for people living with chronic non-cancer pain within the context of the Kuwaiti health systems, and the cases are built from evidence in the published literature identified through a comprehensive review process. The evolution of the organizational structure of the public and private health systems in Kuwait is described. These are discussed in terms of their impact on the delivery of comprehensive chronic pain management service by health professionals in Kuwait. This review also includes a description of chronic pain patient personas to highlight expected barriers as well as compliance issues with services likely to be encountered in Kuwait. The case study analysis and persona descriptions illustrate a need to move beyond pain symptom management towards considering the entire person and his/her individual experience of pain such that health care success is judged by enhancement of patient well-being rather than access to services. A road map for improving integrative chronic pain management in Kuwait is discussed.

Canada's neglected tropical disease research network: who's in the core-who's on the periphery?

BACKGROUND: This study designed and applied accessible yet systematic methods to generate baseline information about the patterns and structure of Canada's neglected tropical disease (NTD) research network; a network that, until recently, was formed and functioned on the periphery of strategic Canadian research funding. METHODOLOGY: MULTIPLE METHODS WERE USED TO CONDUCT THIS STUDY, INCLUDING: (1) a systematic bibliometric procedure to capture archival NTD publications and co-authorship data; (2) a country-level "core-periphery" network analysis to measure and map the structure of Canada's NTD co-authorship network including its size, density, cliques, and centralization; and (3) a statistical analysis to test the correlation between the position of countries in Canada's NTD network ("k-core measure") and the quantity and quality of research produced. PRINCIPAL FINDINGS: Over the past sixty years (1950-2010), Canadian researchers have contributed to 1,079 NTD publications, specializing in Leishmania, African sleeping sickness, and leprosy. Of this work, 70% of all first authors and co-authors (n = 4,145) have been Canadian. Since the 1990s, however, a network of international co-authorship activity has been emerging, with representation of researchers from 62 different countries; largely researchers from OECD countries (e.g. United States and United Kingdom) and some non-OECD countries (e.g. Brazil and Iran). Canada has a core-periphery NTD international research structure, with a densely connected group of OECD countries and some African nations, such as Uganda and Kenya. Sitting predominantly on the periphery of this research network is a cluster of 16 non-OECD nations that fall within the lowest GDP percentile of the network. CONCLUSION/SIGNIFICANCE: The publication specialties, composition, and position of NTD researchers within Canada's NTD country network provide evidence that while Canadian researchers currently remain the overall gatekeepers of the NTD research they generate; there is opportunity to leverage existing research collaborations and help advance regions and NTD areas that are currently under-developed.

Pill characterization data streams for reducing exposure to inadequately identified anti-malarial medication in developing countries.

BACKGROUND: A large fraction of anti-malaria medicines (and indeed many other medicines classes) used in developing countries are inadequately identified. Framing this problem as one of misidentification rather than the more common framing of criminal misrepresentation leads to new solutions sets not currently being considered. METHOD: That reframing led to consideration and analysis of 4 new problems that informed design of a digital platform technology for delivering a distributed medicine characterization system: 1) problematic interests associated with a focus on preventing counterfeiting, 2) the complexity of the many ways that medicines can deviate from expected identities, 3) the challenge of choosing amongst a diversity of attribute characterization technologies, and 4) the need for a flexible and distributed data aggregation mechanism. RESULTS: Analysis of those new problems confirmed an initial insight that a previously described digital technology for tracking malaria tests results in infrastructure limited regions could be adapted for characterizing pill attributes. Feasibility is illustrated by describing how the platform design can be implemented using open-source software and commodity computational and communication technology readily available and supportable in developing countries. DISCUSSION: A system of this type would allow users to answer several questions. Is this medicine what it is supposed to be? Can it be used to treat locally encountered malaria? What has been the experience of others who have used pills having the same identity? Ubiquitous access to global digital telecommunication infrastructure allows the system to generate data streams from these distributed medicine characterization transactions that can be used to map global patterns of use of specifically identified medicines. This can provide feedback necessary to guide efforts to reduce the burden of malaria.

An informatics model for guiding assembly of telemicrobiology workstations for malaria collaborative diagnostics using commodity products and open-source software.

BACKGROUND: Deficits in clinical microbiology infrastructure exacerbate global infectious disease burdens. This paper examines how commodity computation, communication, and measurement products combined with open-source analysis and communication applications can be incorporated into laboratory medicine microbiology protocols. Those commodity components are all now sourceable globally. An informatics model is presented for guiding the use of low-cost commodity components and free software in the assembly of clinically useful and usable telemicrobiology workstations. METHODS: The model incorporates two general principles: 1) collaborative diagnostics, where free and open communication and networking applications are used to link distributed collaborators for reciprocal assistance in organizing and interpreting digital diagnostic data; and 2) commodity engineering, which leverages globally available consumer electronics and open-source informatics applications, to build generic open systems that measure needed information in ways substantially equivalent to more complex proprietary systems. Routine microscopic examination of Giemsa and fluorescently stained blood smears for diagnosing malaria is used as an example to validate the model. RESULTS: The model is used as a constraint-based guide for the design, assembly, and testing of a functioning, open, and commoditized telemicroscopy system that supports distributed acquisition, exploration, analysis, interpretation, and reporting of digital microscopy images of stained malarial blood smears while also supporting remote diagnostic tracking, quality assessment and diagnostic process development. CONCLUSION: The open telemicroscopy workstation design and use-process described here can address clinical microbiology infrastructure deficits in an economically sound and sustainable manner. It can boost capacity to deal with comprehensive measurement of disease and care outcomes in individuals and groups in a distributed and collaborative fashion. The workstation enables local control over the creation and use of diagnostic data, while allowing for remote collaborative support of diagnostic data interpretation and tracking. It can enable global pooling of malaria disease information and the development of open, participatory, and adaptable laboratory medicine practices. The informatic model highlights how the larger issue of access to generic commoditized measurement, information processing, and communication technology in both high- and low-income countries can enable diagnostic services that are much less expensive, but substantially equivalent to those currently in use in high-income countries.

An interfaculty pain curriculum: lessons learned from six years experience.

Minimal pain content has been documented in pre-licensure curricula and students lack important pain knowledge at graduation. To address this problem, we have implemented and evaluated a mandatory Interfaculty Pain Curriculum (IPC) yearly since 2002 for students (N=817 in 2007) from six Health Science Faculties/Departments. The 20-h pain curriculum continues to involve students from Dentistry, Medicine, Nursing, Pharmacy, Physical Therapy, and Occupational Therapy as part of their 2nd or 3rd year program. Evaluation methods based on Kirkpatrick's model now include evaluation of a Comprehensive Pain Management Plan along with the previously used Pain Knowledge and Beliefs Questionnaire (PKPQ) and Daily Content and Process Questionnaires (DCPQ). Important lessons have been learned and subsequent changes made in this iterative curriculum design based on extensive evaluation over the 6-year period. Modifications have included case development more relevant to the diverse student groups, learning contexts that are uni-, inter-, and multi-professional, and facilitator development in working with interprofessional student groups. PKBQ scores have improved in all years with a statistically significant average change on correct responses from 14% to 17%. The DCPQ responses have also indicated consistently that most students (85-95%) rated highly the patient panel, expert-lead clinically focused sessions, and small interprofessional groups. Relevancy and organization of the information presented have been generally rated highly from 80.3% to 91.2%. This curriculum continues to be a unique and valuable learning opportunity as we utilize lessons learned from extensive evaluation to move the pain agenda forward with pre-licensure health science students.

Vulnerability of glial cells to hydrogen peroxide in cultured hippocampal slices.

Massive production of free radicals (FR) has been associated with a variety of pathological conditions in the central nervous system (CNS). We have used the FR generating compound hydrogen peroxide (H2O2) in organotypic hippocampal slice cultures to model oxidative injury in the brain. Necrotic cell death was monitored for up to 48 h using propidium iodide (PI) and confocal microscopy. A 1 h exposure to H2O2 (0.5-2.5 mM) caused a dose-dependent, and region specific cell death in hippocampal slice cultures. Glial cells demonstrated a high degree of vulnerability to H2O2. During the initial 3 h post-injury period, regions of the slice where glial cell bodies predominated showed massive cell death. The majority of neurons in the pyramidal layers were spared, though at later time points they appeared damaged as well. Carboxy-dichlorofluorescein imaging revealed a corresponding early increase in ROS generation in glial cells compared to pyramidal neurons. Immunohistochemistry of PI labeled slices identified astrocytes as the cells most sensitive to H2O2 toxicity. In dissociated cell cultures of hippocampal astrocytes and neurons, astrocytes also exhibited a significantly higher sensitivity to H2O2 than neurons. Hydrogen peroxide-induced cytotoxicity in all regions of the hippocampal slice culture was significantly attenuated by pre-treatment with antioxidants (alpha-tocopherol and glutathione), and was not prevented by blockade of Ca2+ influx, or NMDA channel activation. Cyclosporin A, an inhibitor of mitochondrial permeability transition, reduced cytotoxicity in glial areas by more than 50%, while in the CA2-CA3 pyramidal layers a much smaller, but still significant, attenuation of cytotoxicity was observed. Our results suggest that mitochondria are primary targets of H2O2 toxicity, particularly in astrocytes.

The use of fluorescence enhancement to improve the microscopic diagnosis of falciparum malaria.

BACKGROUND: Giemsa staining of thick blood smears remains the "gold standard" for detecting malaria. However, this method is not very good for diagnosing low-level infections. A method for the simultaneous staining of Plasmodium-parasitized culture and blood smears for both bright field and fluorescence was developed and its ability to improve detection efficiency tested. METHODS: A total of 22 nucleic acid-specific fluorescent dyes were tested for their ability to provide easily observable staining of Plasmodium falciparum-parasitized red blood cells following Giemsa staining. RESULTS: Of the 14 dyes that demonstrated intense fluorescence staining, only SYBR Green 1, YOYO-1 and ethidum homodimer-2 could be detected using fluorescent microscopy, when cells were first stained with Giemsa. Giemsa staining was not effective when applied after the fluorescent dyes. SYBR Green 1 provided the best staining in the presence of Giemsa, as a very high percentage of the parasitized cells were simultaneously stained. When blood films were screened using fluorescence microscopy the parasites were more readily detectable due to the sharp contrast between the dark background and the specific, bright fluorescence produced by the parasites. CONCLUSION: The dual staining method reported here allows fluorescence staining, which enhances the reader's ability to detect parasites under low parasitaemia conditions, coupled with the ability to examine the same cell under bright field conditions to detect the characteristic morphology of Plasmodium species that is observed with Giemsa staining.

Calcium chelation improves spatial learning and synaptic plasticity in aged rats.

Impaired regulation of intracellular calcium is thought to adversely affect synaptic plasticity and cognition in the aged brain. Comparing young (2-3 months) and aged (23-26 months) Fisher 344 rats, stratum radiatum-evoked CA1 field EPSPs were smaller and long-term potentiation (LTP) was diminished in aged hippocampal slices. Resting calcium, in presynaptic axonal terminals in the CA1 stratum radiatum area, was elevated in aged slices. Loading the slice with the calcium chelator, BAPTA-AM, depressed LTP in young slices, but enhanced this plasticity in old slices. Forty-five minutes following LTP-inducing high frequency stimulation, resting calcium levels were significantly increased in both young and old presynaptic terminals, and significantly reduced by pretreatment with BAPTA-AM. In vivo, intraperitoneal administration of BAPTA-AM prior to training in the reference memory version of the Morris water maze test, significantly improved the acquisition of spatial learning in aged animals, without a significant effect in young rats. These results support the hypothesis that increasing intracellular neuronal buffering power for calcium in aged rats ameliorates age-related impaired synaptic plasticity and learning.

Peroxidase catalysed formation of cytotoxic prooxidant phenothiazine free radicals at physiological pH.

The antipsychotic phenothiazines may have other therapeutic applications because of their ability to kill bacteria, plasmids and tumor cells. They are also known to undergo a peroxidase-catalysed oxidation to form cation radicals that are stable at acid pH, but are not detected at a neutral pH. The objective of this project was to determine whether phenothiazine cation radical metabolites could cause oxidative stress at a neutral pH resulting in cytotoxicity. At a neutral pH, catalytic amounts of phenothiazines were found to be oxidised by a peroxidase/H2O2 system and also caused ascorbate, GSH and NADH cooxidation. NADH and GSH co-oxidation was accompanied by oxygen uptake and was increased by the addition of catalytic amounts of superoxide dismutase, indicating that the superoxide radical was formed. The phenothazines were different from other peroxidase substrates in that the NADH, ascorbate or GSH cooxidation was faster at pH 6.0 than pH 7.4, thereby partly reflecting the cation radical stability. The order of catalytic effectiveness found was promazine > chlorpromazine > trifluoperazine. Peroxidase/H2O2 also markedly increased phenothiazine cytotoxicity towards isolated rat hepatocytes at nontoxic phenothiazine concentrations. At both pH 6.0 and 7.4, the same order of phenothiazine catalytic effectiveness was observed as seen in the co-oxidation experiments. Cytotoxicity to hepatocytes could be attributed to oxidative stress as most hepatocyte glutathione oxidation and lipid peroxidation preceded phenothiazine induced cytotoxicity and that cytotoxicity was prevented by the antioxidant butylated hydroxyanisole. This hepatocyte/peroxidase/H2O2 system could be a useful model for studying drug induced idiosyncratic hepatic injury enhanced by inflammation.

Engineering lipobeads: properties of the hydrogel core and the lipid bilayer shell.

We previously reported on a novel system termed Lipobead that consists of hydrogel beads encased within an anchored lipid bilayer. The hydrogel particles are formed by inverse suspension polymerization of dimethylacrylamide with N,N'-ethylenebis(acrylamide). During the polymerization stage, the water in oil emulsion is interfacially stabilized by small molecule surfactants as well as a small percentage of lipid functionalized with a vinyl group. The functionalized lipid becomes tethered to the bead surface and promotes the assembly of a lipid bilayer on the surface of the hydrogel beads. The presence of the functionalized lipid during polymerization dramatically alters the yield, average size, and size distribution of beads produced. This paper examines the effect of various chemical and physical processing parameters on the average size and size distribution of beads produced when lipid is a component of the surfactant mixture. Relationships between the processing parameters, average bead size, and size distribution were established. Macroscopic properties of the lipid bilayers of Lipobeads were also evaluated including phase transition temperature as well as permeability to the small polar molecule, adenosine triphosphate. It was established that the presence of functionalized lipid improves the organization of the bilayer on the Lipobead surface.

H2S cytotoxicity mechanism involves reactive oxygen species formation and mitochondrial depolarisation.

A number of scavengers of reactive oxygen species (ROS) were found to be protective against cell death induced by hydrogen sulfide (H2S) in isolated hepatocytes. The H2O2 scavengers alpha-ketoglutarate and pyruvate, which also act as energy substrate metabolites, were more protective against H2S toxicity than lactate which is only an energy substrate metabolite. All of these results suggest that H2S toxicity is dependent on ROS production. We measured ROS formation directly in hepatocytes using the fluorogenic dichlorofluorescin method. H2S-induced ROS formation was dose dependent and pyruvate inhibited this ROS production. Non-toxic concentrations of H2S enhanced the cytotoxicity of H2O2 generated by glucose/glucose oxidase, which was inhibited by CYP450 inibitors. Furthermore, hepatocyte ROS formation induced by H2S was decreased by CYP450 inhibitors cimetidine and benzylimidazole. These results suggest that CYP450-dependant metabolism of H2S is responsible for inducing ROS production. H2S-induced cytotoxicity was preceded by mitochondrial depolarization as measured by rhodamine 123 fluorescence. Mitochondrial depolarization induced by H2S was prevented by zinc, methionine and pyruvate all of which decreased H2S-induced cell death. Treatment of H2S poisoning may benefit from interventions aimed at minimizing ROS-induced damage and reducing mitochondrial damage.

An integrated undergraduate pain curriculum, based on IASP curricula, for six health science faculties.

Pain education, especially for undergraduates, has been identified as important to changing problematic pain practices, yet, no published data were found describing an integrated, interprofessional pain curriculum for undergraduate students. Therefore, this project aimed to develop, implement, and evaluate an integrated pain curriculum, based on the International Association for the Study of Pain curricula [http://www.iasp-pain.org/curropen.html], for 540 students from six Health Science Faculties/Departments. Over an 18-month period, the University of Toronto Centre for the Study of Pain's Interfaculty Pain Education Committee developed a 20-h undergraduate pain curriculum to be delivered during a 1-week period. Students from Dentistry, Medicine, Nursing, Pharmacy, Physical Therapy, and Occupational Therapy participated as part of their 2nd or 3rd year program. Teaching strategies included large and small groups, Standardized Patients, and 63 facilitators. Evaluation methods included: (a) pre- and post-tests of the Pain Knowledge and Beliefs Questionnaire (PKBQ) and (b) Daily Content and Process Questionnaire (DCPQ) to obtain feedback about process, content, and format across the curriculum's 5 days. A significant improvement in pain knowledge and beliefs was demonstrated (t = 181.28, P < 0.001), although non-responders were problematic at the post-test. DCPQ overall ratings of 'exceeding or meeting expectations' ranged from 74 to 92%. Ratings were highest for the patient-related content and panel, and the small-group discussions with Standardized Patients. Overall evaluations were positive, and statistically significant changes were demonstrated in students' pain knowledge and beliefs. This unique and valuable learning opportunity will be repeated with some modifications next year.

Properties of a self-assembled phospholipid membrane supported on lipobeads.

The overall objective of our work was to make a hydrogel-supported phospholipid bilayer that models a cytoskeleton-supported cell membrane and provides a platform for studying membrane biology. Previously, we demonstrated that a pre-Lipobead, consisting of phospholipids covalently attached to the surface of a hydrogel, could give rise to a Lipobead when incubated with liposomes because the attached phospholipids promote self-assembly of a phospholipid membrane on the pre-Lipobead. We now report the properties of that Lipobead membrane. The lateral diffusion coefficient of fluorescently labeled phosphatidylcholine analogs in the membrane was measured by fluorescence recovery after photobleaching and was found to decrease as the surface anchor density and hydrogel crosslinking density increased. Results from the quenching of phosphatidylcholine analogs suggest that the phospholipid membrane of the Lipobead was composed mostly of a semipermeable lipid bilayer. However, the diffusional barrier properties of the Lipobead membrane were demonstrated by the entrapment of 1.5-3.0 K dextran molecules in the hydrogel core after liposome fusion. This hydrogel-supported bilayer membrane preparation shows promise as a new platform for studying membrane biology and for high throughput drug screening.

Shift in the localization of sites of hydrogen peroxide production in brain mitochondria by mitochondrial stress.

We have determined the underlying sites of H(2)O(2) generation by isolated rat brain mitochondria and how these can shift depending on the presence of respiratory substrates, electron transport chain modulators and exposure to stressors. H(2)O(2) production was determined using the fluorogenic Amplex red and peroxidase system. H(2)O(2) production was higher when succinate was used as a respiratory substrate than with another FAD-dependent substrate, alpha-glycerophosphate, or with the NAD-dependent substrates, glutamate/malate. Depolarization by the uncoupler p-trifluoromethoxyphenylhydrazone decreased H(2)O(2) production stimulated by all respiratory substrates. H(2)O(2) production supported by succinate during reverse transfer of electrons was decreased by inhibitors of complex I (rotenone and diphenyleneiodonium) whereas in glutamate/malate-oxidizing mitochondria diphenyleneiodonium decreased while rotenone increased H(2)O(2) generation. The complex III inhibitors antimycin and myxothiazol decreased succinate-induced H(2)O(2) production but stimulated H(2)O(2) production in glutamate/malate-oxidizing mitochondria. Antimycin and myxothiazol also increased H(2)O(2) production in mitochondria using alpha-glycerophosphate as a respiratory substrate. In substrate/inhibitor experiments maximal stimulation of H(2)O(2) production by complex I was observed with the alpha-glycerophosphate/antimycin combination. In addition, three forms of in vitro mitochondrial stress were studied: Ca(2+) overload, cold storage for more than 24 h and cytochrome c depletion. In each case we observed (i) a decrease in succinate-supported H(2)O(2) production by complex I and an increase in succinate-supported H(2)O(2) production by complex III, (ii) increased glutamate/malate-induced H(2)O(2) generation by complex I and (iii) increased alpha-glycerophosphate-supported H(2)O(2) generation by complex III. Our results suggest that all three forms of mitochondrial stress resulted in similar shifts in the localization of sites of H(2)O(2) generation and that, in both normal and stressed states, the level and location of H(2)O(2) production depend on the predominant energetic substrate.

Characterization of radioligand binding to a transmembrane receptor reconstituted into Lipobeads.

Lipobeads are hydrogel beads surrounded by a lipid bilayer membrane and have been developed to act as a cell analogue. The FLAG-tagged M(2) muscarinic receptor was incorporated onto the surface of the Lipobead by incubating pre-Lipobeads with proteoliposomes containing the receptor. Receptors reconstituted onto the surface of the Lipobeads were functional in that they bound the antagonists quinuclidinylbenzilate and scopolamine with characteristic muscarinic affinities. This demonstrates the feasibility of using Lipobeads to study the binding properties of the M(2) muscarinic receptor and offers a promising approach to the study of transmembrane protein biology in general.