Treatment of Fresh Meat, Fish and Products Thereof with Cold Atmospheric Plasma to Inactivate Microbial Pathogens and Extend Shelf Life.

Assuring the safety of muscle foods and seafood is based on prerequisites and specific measures targeted against defined hazards. This concept is augmented by 'interventions', which are chemical or physical treatments, not genuinely part of the production process, but rather implemented in the framework of a safety assurance system. The present paper focuses on 'Cold Atmospheric pressure Plasma' (CAP) as an emerging non-thermal intervention for microbial decontamination. Over the past decade, a vast number of studies have explored the antimicrobial potential of different CAP systems against a plethora of different foodborne microorganisms. This contribution aims at providing a comprehensive reference and appraisal of the latest literature in the area, with a specific focus on the use of CAP for the treatment of fresh meat, fish and associated products to inactivate microbial pathogens and extend shelf life. Aspects such as changes to organoleptic and nutritional value alongside other matrix effects are considered, so as to provide the reader with a clear insight into the advantages and disadvantages of CAP-based decontamination strategies.

A need for null models in understanding disease transmission: the example of Mycobacterium ulcerans (Buruli ulcer disease).

Understanding the interactions of ecosystems, humans and pathogens is important for disease risk estimation. This is particularly true for neglected and newly emerging diseases where modes and efficiencies of transmission leading to epidemics are not well understood. Using a model for other emerging diseases, the neglected tropical skin disease Buruli ulcer (BU), we systematically review the literature on transmission of the etiologic agent, Mycobacterium ulcerans (MU), within a One Health/EcoHealth framework and against Hill's nine criteria and Koch's postulates for making strong inference in disease systems. Using this strong inference approach, we advocate a null hypothesis for MU transmission and other understudied disease systems. The null should be tested against alternative vector or host roles in pathogen transmission to better inform disease management. We propose a re-evaluation of what is necessary to identify and confirm hosts, reservoirs and vectors associated with environmental pathogen replication, dispersal and transmission; critically review alternative environmental sources of MU that may be important for transmission, including invertebrate and vertebrate species, plants and biofilms on aquatic substrates; and conclude with placing BU within the context of other neglected and emerging infectious diseases with intricate ecological relationships that lead to disease in humans, wildlife and domestic animals.

Signs of Inequitable Access: Users of Private Physiotherapy Services Do Not Reflect the Urban Population in Winnipeg, Manitoba.

Purpose: Both private and public funding cover outpatient physiotherapy (PT) in Canada. Knowledge is lacking in who does and does not access PT services, which limits the ability to identify health/access inequities created by current financing structures. This study characterizes the individuals accessing private PT in Winnipeg to better understand whether inequities exist, given the very limited publicly financed PT. Methods: Patients attending PT in 32 private businesses, sampled for geographic variation, completed a survey online or on paper. We compared the sample's demographic characteristics with Winnipeg population data using chi-square goodness-of-fit tests. Results: In total, 665 adults accessing PT participated. Respondents were older and had higher levels of income and education compared to Winnipeg census data (p < 0.001). Our sample included higher proportions of female and White individuals, and lower proportions of Indigenous persons, newcomers, and people from visible minorities (p < 0.001). Conclusions: There are signs that inequities exist in access to PT in Winnipeg; the cohort who access private PT services does not reflect the wider population, which suggests that some segments of the population are not receiving care.

Objectif : un financement tant public que privé couvre les services ambulatoires de physiothérapie au Canada. On ne sait pas qui y a accès ou non, ce qui limite la possibilité de déterminer les iniquités en matière de santé et d'accès attribuables aux structures actuelles de financement. La présente étude caractérise les personnes qui accèdent à des services de physiothérapie privés à Winnipeg, afin de mieux comprendre s'il existe des iniquités, compte tenu des services de physiothérapie très limités qui sont financés par le secteur public. Méthodologie : des patients recevant des services de physiothérapie dans 32 entreprises privées, échantillonnées d'après leur variation géographique, ont rempli un sondage en ligne ou sur papier. Les chercheurs ont comparé les caractéristiques démographiques de l'échantillon aux données populationnelles de Winnipeg au moyen de tests du chi carré pour la qualité de l'ajustement. Résultats : au total, 665 adultes qui avaient accès à des services de physiothérapie ont participé. Ils étaient plus âgés et avaient un revenu et une scolarisation supérieurs aux données du recensement de Winnipeg (p < 0,001). L'échantillon contenait une plus forte proportion de femmes et de personnes blanches, et une plus faible proportion de personnes autochtones, de nouveaux arrivants et de membres des minorités visibles (p < 0,001). Conclusions : il y a des signes d'iniquité d'accès aux services de physiothérapie à Winnipeg. La cohorte qui a accès aux services de physiothérapie privés ne reflète pas l'ensemble de la population, ce qui indique que certains segments de la population ne reçoivent pas de soins.

Impact of diet moisture on the development of the forensically important blow fly Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae).

The secondary screwworm, Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae), is a carrion-breeding species of veterinary, medical, and forensic importance. It is very abundant in the Nearctic and Neotropical regions and is one of the most common colonizers of vertebrate remains in the southern United States. Therefore, it is of great evidential value in estimating the minimum time of colonization (TOC) of remains related to forensic investigations. So far, studies have investigated the effects of several biotic and abiotic factors on C. macellaria. However, no data on the specific impact of food source moisture on the larval development of this species are known to have been published. In this study, the effects of diet moisture on larval development time, larval length, and weight over time, as well as adult emergence and weight were investigated. C. macellaria was reared on diets prepared from freeze-dried bovine liver with varying moisture content (0.0, 33.0, 50.0, or 70.8%) at 25.6 °C, 77% RH, and 14:10 L:D. Frozen-thawed liver was used as a control. Water content was found to significantly impact immature development time and corresponding life-history traits, both within and among developmental stages of C. macellaria. This result indicates moisture content of the carrion source should be considered when estimating time of colonization and identification of immature stage of flies in forensic investigations. Furthermore, as diet moisture content significantly affected the dry mass of emerging adults, the mass of adults collected at a crime scene could provide useful information as it could be an indicator of the condition the remains were in during time of colonization, including the moisture content of the tissue.

Effects of Photoperiod on the Development of Forensically Important Blow Fly Chrysomya rufifacies (Diptera: Calliphoridae).

Estimation of the time of colonization (TOC) is often based on laboratory studies that document arthropod development. Precise data for forensically important species, such as blow flies (Diptera: Calliphoridae), are essential for accuracy in the estimate of TOC. Calliphorid development is a quantitative trait and thus depends on a host of variables. In calliphorids, studies showed photoperiod can play a role in development. However, there has been little research to date on the effects of photoperiod, and available data indicate the impact is species-specific. In this study, the effects of photoperiod on the development of Chrysomya rufifacies (Macquart), were examined. Chrysomya rufifacies is a fly of great medical and legal importance and is often encountered on vertebrate remains in temperate and tropic regions throughout the world, including North and Central America, Asia, and Australia. Larvae were reared under light regimes of 12, 16, and 24 h of light at 28.5 ± 0.0°C, 86.2 ± 0.3 RH. Minimum development time for each stage did not differ significantly for the applied photoperiods, nor were there significant differences in total minimum postembryonic development time. Photoperiod did not significantly affect larval size or growth rate. The data suggest that light durations investigated in this study do not influence the development of C. rufifacies. This indicates that photoperiod may not be a concern for forensic entomologists in Texas, United States, or other areas with similar conditions when estimating the TOC for this species. Validation efforts are encouraged to verify this conclusion.

Assessment of tenderness of aged bovine gluteus medius muscles using Raman spectroscopy.

A portable 671 nm Raman system was evaluated as a rapid and non-destructive device for the assessment of beef tenderness using 175 gluteus medius muscles (99 for calibration, 76 for validation) aged at -1 °C and 7 °C for fourteen days. Raman and shear force (SF) measurements were performed with the aged beef. The samples stored at -1 °C showed on average only slightly increased SF values. The correlation of Raman spectra with SF using partial least squares regression yielded cross-validated predictions of SF for both storage temperatures with coefficients of determination R(2)cv=0.33-0.79. Validation with independent samples resulted in predictions with R(2)val=0.33. Using thresholds between 30 and 49N, tough and tender samples could be discriminated with partial least squares discriminant analysis with 70-88% and 59-80% accuracy during cross-validation and validation, respectively. These results demonstrate the principle feasibility to predict the SF and thus toughness of raw, aged gluteus beef cuts with a portable Raman device showing potential for grading beef cuts.

Measuring satisfaction with health care in young persons with inflammatory bowel disease--an instrument development and validation study.

BACKGROUND: Patient satisfaction is a relevant prognostic factor in young persons with chronic disease and may be both age and disease specific. To assess health care quality from the patient's view in young persons with inflammatory bowel disease, an easy to use, valid, reliable and informative specific instrument was needed. METHODS: All parts of the study were directed at persons with inflammatory bowel disease aged 15 to 24 ("youth"). A qualitative internet patient survey was used to generate items, complemented by a physician survey and literature search. A 2nd internet survey served to reduce items based on perceived importance and representativeness. Following pilot testing to assess ease of use and face validity, 150 respondents to a postal survey in patients from a paediatric clinical registry were included for validation analyses. Construct validity was assessed by relating summary scores to results from global questions on satisfaction with care using ANOVA. To assess test-retest reliability using intraclass correlation coefficients (ICC), a subset of patients were assessed twice within 3 months. RESULTS: 302 persons with IBD and 55 physicians participated in the item generating internet survey, resulting in 3,954 statements. After discarding redundancies 256 statements were presented in the 2nd internet survey. Of these, 32 items were retained. The resulting instrument assesses both the perceived relevance (importance) of an item as well as the performance of the care giver for each item for calculation of a summary satisfaction score (range 0 to 1). Sensibility testing showed good acceptance for most items. Construct validity was good, with mean scores of 0.63 (0.50 to 0.76), 0.71 (0.69 to 0.74) and 0.81 (0.79 to 0.83) for no, some and good global satisfaction (ANOVA, p < 0.001). Test-retest reliability was satisfactory (ICC 0.6 to 0.7). CONCLUSIONS: We developed an easy to use, patient oriented, valid instrument to assess satisfaction with care in young persons with IBD for use in survey research.

Detecting the effect of Alzheimer's disease on everyday motion behavior.

BACKGROUND: Early detection of behavioral changes in Alzheimer's disease (AD) would help the design and implementation of specific interventions. OBJECTIVE: The target of our investigation was to establish a correlation between diagnosis and unconstrained motion behavior in subjects without major clinical behavior impairments. METHOD: We studied everyday motion behavior in 23 dyads with one partner suffering from AD dementia and one cognitively healthy partner in the subjects' home, employing ankle-mounted three-axes accelerometric sensors. We determined frequency features obtained from the signal envelopes computed by an envelope detector for the carrier band 0.5 Hz to 5 Hz. Based on these features, we employed quadratic discriminant analysis for building models discriminating between AD patients and healthy controls. RESULTS: After leave-one-out cross-validation, the classification accuracy of motion features reached 91% and was superior to the classification accuracy based on the Cohen-Mansfield Agitation Inventory (CMAI). Motion features were significantly correlated with MMSE and CMAI scores. CONCLUSION: Our findings suggest that changes of everyday behavior are detectable in accelerometric behavior protocols even in the absence of major clinical behavioral impairments in AD.

Development of apical membrane organization and V-ATPase regulation in blowfly salivary glands.

Secretory cells in blowfly salivary gland are specialized via morphological and physiological attributes in order to serve their main function, i.e. the transport of solutes at a high rate in response to a hormonal stimulus, namely serotonin (5-HT). This study examines the way that 5-HT-insensitive precursor cells differentiate into morphologically complex 5-HT-responsive secretory cells. By means of immunofluorescence microscopy, immunoblotting and measurements of the transepithelial potential changes, we show the following. (1) The apical membrane of the secretory cells becomes organized into an elaborate system of canaliculi and is folded into pleats during the last pupal day and the first day of adulthood. (2) The structural reorganization of the apical membrane is accompanied by an enrichment of actin filaments and phosphorylated ERM protein (phospho-moesin) at this membrane domain and by the deployment of the membrane-integral part of vacuolar-type H(+)-ATPase (V-ATPase). These findings suggest a role for phospho-moesin, a linker between actin filaments and membrane components, in apical membrane morphogenesis. (3) The assembly and activation of V-ATPase can be induced immediately after eclosion by way of 8-CPT-cAMP, a membrane-permeant cAMP analogue. (4) 5-HT, however, produces the assembly and activation of V-ATPase only in flies aged for at least 2 h after eclosion, indicating that, at eclosion, the 5-HT receptor/adenylyl cyclase/cAMP signalling pathway is inoperative upstream of cAMP. (5) 5-HT activates both the Ca(2+) signalling pathway and the cAMP signalling cascade in fully differentiated secretory cells. However, the functionality of these signalling cascades does not seem to be established in a tightly coordinated manner during cell differentation.

Structural and functional cortical disconnection in Alzheimer's disease: a combined study using diffusion tensor imaging and transcranial magnetic stimulation.

We investigated the functional consequences of compromised white matter integrity in Alzheimer's disease by combining Diffusion Tensor Imaging (DTI) and Transcranial Magnetic Stimulation (TMS) in 19 patients with AD (Alzheimer's disease) and 19 healthy controls. We used a region of interest approach and correlated the ipsilateral silent period (iSP) and the resting motor threshold (RMT) from TMS with fractional anisotropy (FA) and mean diffusivity (MD) values of the corpus callosum and corticospinal tract. AD patients showed significant reductions of FA in intracortical projecting fibre tracts compared to controls and widespread increases in MD. TMS data showed increased latency of iSP in AD patients and a decreased RMT, indicating decreased motor cortical inhibition. Although both TMS and DTI metrics were prominently altered in AD patients, impaired white matter integrity was not associated with increased iSP latency or reduced RMT, as correlation of TMS parameters with FA and MD values in the a priori defined regions showed no significant effects. Therefore, we argue that beside the direct degeneration of the underlying fibre tracts, other pathophysiological mechanisms may account for the observation of decreased transcallosal inhibition and increased motor excitability in AD.

Intra- and inter-cortical motor excitability in Alzheimer's disease.

Transcranial magnetic stimulation (TMS) provides evidence for facilitatory and inhibitory motor dysfunctions in Alzheimer's disease (AD). The corpus callosum (CC) is affected in AD already at early stages consistent with the hypothesis that AD patients exhibit alterations in transcallosally mediated motor inhibition (ipsilateral silent period, iSP). Therefore, here we aimed at investigating the integrity not only of intra-, but also of inter-hemispheric mechanisms of cortical motor excitability in AD. We determined the iSP, the resting motor threshold (RMT), and the amplitude of motor evoked potentials (MEP) in 19 AD patients and 19 healthy controls using single-pulse TMS. Furthermore, we used paired-pulse TMS to study the intra-cortical inhibition (ICI) and intra-cortical facilitation (ICF). All subjects underwent comprehensive neuropsychologic, clinical, and laboratory testing, and neuroimaging to exclude significant co-morbidity. In AD patients, the RMT was significantly reduced (Oneway-ANOVA). An analysis of covariance (ANCOVA) revealed a strong group specific interaction of the inhibitory interstimulus intervals (p = 0.005) with a reduced ICI in AD. Furthermore, we found a significantly prolonged iSP-latency (p = 0.003) in AD compared to controls, whereas the iSP-duration was not different. The iSP-latency correlated significantly with the ICI (ANCOVA) (p = 0.02). The ICF did not differ significantly between groups. Our data suggest comprehensive but still subclinical dysfunctions of motor cortical inhibition in mild to moderate clinical stages of AD with strong interactions of intra- and inter-hemispheric inhibitory phenomena. Future studies are needed to show the potential prognostic relevance of these findings for the further course of the disease.

Microbiological and sensory effects of the combined application of hot-cold organic acid sprays and steam condensation at subatmospheric pressure for decontamination of inoculated pig tissue surfaces.

We studied microbiological and sensory effects of treating pig tissue for 15 s with 55 and 10°C sprays of acetic acid (AA; 0.15 to 0.3 M) and lactic acid (LA; 0.1 to 0.2 M) solutions prior to the tissue being subjected to steam condensation (18 s at 65°C or 10 s at 75°C). LA or AA spraying and then steam treatment resulted in 3- to 4-log average reductions of Pseudomonas fragi and Yersinia enterocolitica inocula (6 to 7 log CFU/cm(2)), regardless of acid temperature or concentration. Buffered LA or 1:1 mixtures of AA:LA and then steam treatment yielded similar reductions. Most of the acid-steam-treated samples had microbial counts below the limit of detection (2 log CFU/cm(2)); thus, the results likely underestimate the potential of this procedure. When the period between inoculation and acid-steam treatment was extended from 0.5 to 24 h, up to a 1-log-higher microbial reduction was observed, due to a 1- to 2-log-greater initial contamination. Increasing the LA contact time to 6 min increased the microbial reduction by 0.8 log. Acid-steam treatment effected lower L* values (darker color) on pigskin, but higher L* values on muscle and fat tissue (paler color). Many muscle samples exhibited lower a* values and off-color brown hues. Off-odors were observed immediately after treatment, but with the exception of fat tissue and AA-treated samples, they largely disappeared during further storage. Off-flavors were only detected in AA-treated muscle samples.

Automated detection of amyloid-ß-related cortical and subcortical signal changes in a transgenic model of Alzheimer's disease using high-field MRI.

In vivo imaging of amyloid-ß (Aß) load as a biomarker of Alzheimer's disease (AD) would be of considerable clinical relevance for the early diagnosis and monitoring of treatment effects. Here, we investigated automated quantification of in vivo T2 relaxation time as a surrogate measure of plaque load in the brains of ten AßPP/PS1 transgenic mice (age 20 weeks) using in vivo MRI acquisitions on a 7T Bruker ClinScan magnet. AßPP/PS1 mice present with rapid-onset cerebral ß-amyloidosis, and were compared with eight age-matched, wild-type control mice (C57Bl/6J) that do not develop Aß-deposition in brain. Data were analyzed with a novel automated voxel-based analysis that allowed mapping the entire brain for significant signal changes. In AßPP/PS1 mice, we found a significant decrease in T2 relaxation times in the deeper neocortical layers, caudate-putamen, thalamus, hippocampus, and cerebellum compared to wildtype controls. These changes were in line with the histological distribution of cerebral Aß plaques and activated microglia. Grey matter density did not differ between wild-type mice and AßPP/PS1 mice, consistent with a lack of neuronal loss in histological investigations. High-field MRI with automated mapping of T2 time changes may be a useful tool for the detection of plaque load in living transgenic animals, which may become relevant for the evaluation of amyloid lowering intervention effects in future studies.

Determinants of female sexual function in inflammatory bowel disease: a survey based cross-sectional analysis.

BACKGROUND: Sexual function is impaired in women with inflammatory bowel disease (IBD) as compared to normal controls. We examined disease specific determinants of different aspects of low sexual function. METHODS: Women with IBD aged 18 to 65 presenting to the university departments of internal medicine and surgery were included. In addition, a random sample from the national patients organization was used (separate analyses). Sexual function was assessed by the Brief Index of Sexual Function in Women, comprising seven different domains of sexuality. Function was considered impaired if subscores were <-1 on a z-normalized scale. Results are presented as age adjusted odds ratios with 95% CI based on multiple logistic regression. RESULTS: 336 questionnaires were included (219 Crohn's disease, 117 ulcerative colitis). Most women reported low sexual activity (63%; 17% none at all, 20% moderate or high activity). Partnership satisfaction was high in spite of low sexual interest in this group. Depressed mood was the strongest predictor of low sexual function scores in all domains. Urban residency and higher socioecomic status had a protective effect. Disease activity was moderately associated with low desire (OR 1.8, 95% CI 1.0 to 3.2). Severity of the disease course impacted most on intercourse frequency (OR 2.3, 95% CI 1.4 to 4.7). Lubrication problems were more common in smokers (OR 2.5, 95% CI 1.3 to 5.1). CONCLUSION: Mood disturbances and social environment impacted more on sexual function in women with IBD than disease specific factors. Smoking is associated with lubrication problems.

The incidence of inflammatory bowel disease in a rural region of Southern Germany: a prospective population-based study.

OBJECTIVE: Although important advances in understanding the aetiology and pathogenesis of inflammatory bowel disease (IBD) have been made, many questions remain unanswered. As the most recent data available on the incidence of IBD in Germany were collected about 15 years ago, we set up a new population-based cohort to determine current incidence data for a defined region in Germany and to establish a basic cohort for prospective follow-up. METHODS: All patients living in the region of Oberpfalz newly diagnosed with IBD between 1 January 2004 and 31 December 2006 were included in this study by setting up a network of reporting clinicians and general practitioners in hospitals as well as in private practices. Demographic and clinical characteristics such as age at first diagnosis, localization of the disease, extraintestinal manifestations or family history on IBD were documented. Age-adjusted incidence rates are presented with 95% Poisson confidence intervals (CIs), based on the European standard population. RESULTS: In total, 286 newly diagnosed patients with IBD were reported in this region, 168 patients suffering from Crohn's disease (CD), 105 patients with ulcerative colitis. Age-standardized incidence rates were 11.0/10(5) (95% CI: 9.1-11.6) for IBD, 6.6/10(5) (95% CI: 5.6-7.7) for CD and 3.9/10(5) (95% CI: 3.2-4.7) for ulcerative colitis. Peak incidences were found in the age interval of 16-24 years for both diseases, predominantly for CD. Age at first diagnosis was lower, extraintestinal manifestations and a positive family history on IBD were more common in patients with CD. CONCLUSION: The incidence rate in IBD seems to be stable in Germany as compared with previously reported data, as is the remarkable predominance of CD. Prospective follow-up studies will be based on this incidence cohort.

Determinants of male sexual function in inflammatory bowel disease: a survey-based cross-sectional analysis in 280 men.

BACKGROUND: Problems with intimacy and sexual performance are among the major concerns of patients with inflammatory bowel disease (IBD). This study was performed to identify disease-related factors associated with low sexual function in men. METHODS: Consecutive patients were surveyed using a standardized questionnaire. A random sample from the national patients' organization was also included. Low sexual function was defined as a score < -1 on a z-normalized scale of the International Index of Erectile Function. Results are presented as adjusted odds ratios (ORs) with 95% confidence interval (CI) based on multiple logistic regression. RESULTS: 280 questionnaires were available for analysis. Scores were similar between the groups and compared with general population means, with the exception of sexual desire. Of the clinical group, 44% felt severely compromised sexually due to their IBD. Erectile function was particularly sensitive to somatic problems (disease activity, OR 2.5, 95% CI: 1.3-4.9; diabetes, OR 7.0, 95% CI: 1.4-35.0). The influence of depressive mood was restricted to aspects of satisfaction (sexual satisfaction, OR 2.3, 95% CI 1.1-4.9; overall satisfaction OR 3.7, 95% CI: 1.7-8.3). CONCLUSIONS: Sexual function was relatively better with longer disease duration and was not affected by the long-term severity of the disease.

Sexual function in persons with inflammatory bowel disease: a survey with matched controls.

BACKGROUND & AIMS: Sexual problems as a result of inflammatory bowel diseases (IBDs) play an important role in patients' worries and concerns. We aimed to evaluate sexual function in men and women with IBD relative to healthy controls. METHODS: A random sample of the national patients organization was surveyed (n = 1000). Age- and sex-matched friends were used as controls; in addition, controls were selected from a large health insurance cohort. Sexual function was evaluated using the Erectile Index of Erectile Function, and the Brief Index of Sexual Function in Women; impaired function was defined as a score less than -1 on a z-normalized scale. The results are reported as age-adjusted odds ratios with 95% confidence intervals based on conditional logistic regression. RESULTS: The response rate was 41% in cases. Overall, 153 male and 181 female matched pairs were available for analysis. The proportion of patients who were married, had a partner, and were sexually active were similar between cases and controls. Depression was the most important determinant of impaired sexual function. Men with IBD in remission or mild activity had similar Erectile Index of Erectile Function scores as compared with controls. Comorbidity and antihypertensive therapy impacted on single subscores. Women with IBD showed impaired function irrespective of disease activity as compared with healthy controls. Results in women varied by type of control. High socioeconomic status was a protective factor for several subscores in women. CONCLUSIONS: Depression is the most important determinant of low sexual function.

The role of helix 8 and of the cytosolic C-termini in the internalization and signal transduction of B(1) and B(2) bradykinin receptors.

Determinants for desensitization and sequestration of G protein-coupled receptors often contain serine or threonine residues located in their C-termini. The sequence context, however, in which these residues have to appear, and the receptor specificity of these motifs are largely unknown. Mutagenesis studies with the B(2) bradykinin receptor (B(2)wt), stably expressed in HEK 293 cells, identified a sequence distal to N338 (NSMGTLRTSI, including I347 but not the basally phosphorylated S348) and in particular the TSI sequence therein, as a major determinant for rapid agonist-inducible internalization and the prevention of receptor hypersensitivity. Chimeras of the noninternalizing B(1) bradykinin receptor (B(1)wt) containing these B(2)wt sequences sequestered poorly, however, suggesting that additional motifs more proximal to N338 are required. In fact, further substitution of the B(1)wt C-terminus with corresponding B(2)wt regions either at C330(7.71) following putative helix 8 (B(1)CB(2)) or at the preceding Y312(7.53) in the NPXXY sequence (B(1)YB(2)) resulted in chimeras displaying rapid internalization. Intriguingly, however, exchange performed at K322(7.63) within putative helix 8 generated a slowly internalizing chimera (B(1)KB(2)). Detailed mutagenesis analysis generating additional chimeras identified the change of V323 in B(1)wt to serine (as in B(2)wt) as being responsible for this effect. The slowly internalizing chimera as well as a B(1)wt point-mutant V323S displayed significantly reduced inositol phosphate accumulation as compared to B(1)wt or the other chimeras. The slow internalization of B(1)KB(2) was also accompanied by a lack of agonist-induced phosphorylation, that in contrast was observed for B(1)YB(2) and B(1)CB(2), suggesting that putative helix 8 is either directly or indirectly (e.g. via G protein activation) involved in the interaction between the receptor and receptor kinases.

Expression levels strongly affect ligand-induced sequestration of B2 bradykinin receptors in transfected cells.

Transfection of cells with expression vectors is one of the most important tools used to assess the effects of receptor mutations on ligand-induced receptor sequestration. Most transfection methods give rise to transiently or stably transfected clones with a wide range of receptor expression levels that may also depend on the mutations made. It is, therefore, important to determine how the regulation of the receptors depends on their numbers per cell. In Chinese hamster ovary (CHO) and human embryonic kidney (HEK)-293 cells expressing high levels of B(2) kinin receptors, we observed poor sequestration indicated by <20% reduction in cell surface receptor number after 10 min of stimulation with 1 microM bradykinin (BK) compared with >70% in low-expressing cells. Whereas the rate of [(3)H]BK internalization (internalized [(3)H]BK in percentage of total bound [(3)H]BK) in low-expressing cells was independent of the ligand-concentration used, in high-expressing cells a strong rate decrease was observed with higher (>1 nM) concentrations. Lower ligand concentrations, however, led to internalization rates identical to those obtained in low-expressing cells. Transiently transfected HEK and COS-7 cells showed results similar to those of stably high-expressing cells. Our results demonstrate the difficulty in determining the internalization pattern of (mutated) B(2) kinin receptors, and possibly of G protein-coupled receptors in general, using a sequestration assay in high-expressing cells or transiently transfected cells with high numbers of receptors per transfected cell. However, the receptor (mutant)-specific internalization rate can be measured, provided that the ligand concentrations used are below a threshold at which the internalization rate is still independent of the ligand concentration.