Protocol: Using N-of-1 tests to identify responders to melatonin for sleep disturbance in Parkinson's disease.

BACKGROUND: 40% of Parkinson's Disease (PD) sufferers experience insomnia, impacting health and quality of life for patients and family members, especially carers. There is little evidence that current treatments are effective. OBJECTIVES: To determine the effectiveness of melatonin in reducing insomnia in 44 individuals with PD using N-of-1 trials. To aggregate group data to arrive at population estimates of effectiveness (measured by improvements in PDSS-2) and safety (measured by adverse events) of melatonin in improving insomnia in PD. To assess the feasibility of offering N-of-1 trials for insomnia in PD. METHODOLOGY: Participants will receive either immediate-release melatonin or placebo in random order in 3 paired two-week treatment periods (12 weeks total). Based on their response in a two-week run-in period on 3 mg daily, they will trial either 3 mg or 6 mg. Patients will keep daily sleep diaries and wear a MotionWatch throughout. After the trial patients will discuss their individual report with their doctor, which provides direct feedback about effectiveness and safety of melatonin for them. STATISTICAL METHODS: We will analyse N-of-1 tests 1) individually: effects of melatonin on PDSS-2 and safety will be reported; and 2) aggregated across individual N-of-1 studies, combined using a Bayesian multilevel random effects model, which will account for repeated measures on individuals over time, and will return posterior estimates of overall treatment effect, and effect in each individual. CLINICAL TRIAL REGISTRATION NUMBER: ACTRN12617001103358.

Weighing in on international growth standards: testing the case in Australian preschool children.

Overweight and obesity in preschool-aged children are major health concerns. Accurate and reliable estimates of prevalence are necessary to direct public health and clinical interventions. There are currently three international growth standards used to determine prevalence of overweight and obesity, each using different methodologies: Center for Disease Control (CDC), World Health Organization (WHO) and International Obesity Task Force (IOTF). Adoption and use of each method were examined through a systematic review of Australian population studies (2006-2017). For this period, systematically identified population studies (N = 20) reported prevalence of overweight and obesity ranging between 15 and 38% with most (n = 16) applying the IOTF standards. To demonstrate the differences in prevalence estimates yielded by the IOTF in comparison to the WHO and CDC standards, methods were applied to a sample of N = 1,926 Australian children, aged 3-5 years. As expected, the three standards yielded significantly different estimates when applied to this single population. Prevalence of overweight/obesity was WHO - 9.3%, IOTF - 21.7% and CDC - 33.1%. Judicious selection of growth standards, taking account of their underpinning methodologies and provisions of access to study data sets to allow prevalence comparisons, is recommended.

Sleep-related crash characteristics: Implications for applying a fatigue definition to crash reports.

Sleep-related (SR) crashes are an endemic problem the world over. However, police officers report difficulties in identifying sleepiness as a crash contributing factor. One approach to improving the sensitivity of SR crash identification is by applying a proxy definition post hoc to crash reports. To identify the prominent characteristics of SR crashes and highlight the influence of proxy definitions, ten years of Queensland (Australia) police reports of crashes occurring in ≥100km/h speed zones were analysed. In Queensland, two approaches are routinely taken to identifying SR crashes. First, attending police officers identify crash causal factors; one possible option is 'fatigue/fell asleep'. Second, a proxy definition is applied to all crash reports. Those meeting the definition are considered SR and added to the police-reported SR crashes. Of the 65,204 vehicle operators involved in crashes 3449 were police-reported as SR. Analyses of these data found that male drivers aged 16-24 years within the first two years of unsupervised driving were most likely to have a SR crash. Collision with a stationary object was more likely in SR than in not-SR crashes. Using the proxy definition 9739 (14.9%) crashes were classified as SR. Using the proxy definition removes the findings that SR crashes are more likely to involve males and be of high severity. Additionally, proxy defined SR crashes are no less likely at intersections than not-SR crashes. When interpreting crash data it is important to understand the implications of SR identification because strategies aimed at reducing the road toll are informed by such data. Without the correct interpretation, funding could be misdirected. Improving sleepiness identification should be a priority in terms of both improvement to police and proxy reporting.

Sleep-related vehicle crashes on low speed roads.

BACKGROUND: Very little is known about the characteristics of sleep related (SR) crashes occurring on low speed roads compared with current understanding of the role of sleep in crashes occurring on high speed roads e.g. motorways. To address this gap, analyses were undertaken to identify the differences and similarities between (1) SR crashes occurring on roads with low (≤60km/h) and high (≥100km/h) speed limits, and (2) SR crashes and not-SR crashes occurring on roads with low speed limits. METHOD: Police reports of all crashes occurring on low and high speed roads over a ten year period between 2000 and 2009 were examined for Queensland, Australia. Attending police officers identified all crash attributes, including 'fatigue/fell asleep', which indicates that the police believe the crash to have a causal factor relating to falling asleep, sleepiness due to sleep loss, time of day, or fatigue. Driver or rider involvement in crashes was classified as SR or not-SR. All crash-associated variables were compared using Chi-square tests (Cramer's V=effect size). A series of logistic regression was performed, with driver and crash characteristics as predictors of crash category. A conservative alpha level of 0.001 determined statistical significance. RESULTS: There were 440,855 drivers or riders involved in a crash during this time; 6923 (1.6%) were attributed as SR. SR crashes on low speed roads have similar characteristics to those on high speed roads with young (16-24y) males consistently over represented. SR crashes on low speed roads are noticeably different to not-SR crashes in the same speed zone in that male and young novice drivers are over represented and outcomes are more severe. Of all the SR crashes identified, 41% occurred on low speed roads. CONCLUSION: SR crashes are not confined to high speed roads. Low speed SR crashes warrant specific investigation because they occur in densely populated areas, exposing a greater number of people to risk and have more severe outcomes than not-SR crashes on the same low speed roads.

YGLF motif in the Kaposi sarcoma herpes virus G-protein-coupled receptor adjusts NF-κB activation and paracrine actions.

Kaposi sarcoma (KS) and primary effusion lymphoma (PEL) are two pathologies associated with KS herpes virus (KSHV/HHV-8) infection. KSHV genome contains several oncogenes, among which, the viral G-protein-coupled receptor (vGPCR open reading frame 74) has emerged as a major factor in KS pathogenicity. Indeed, vGPCR is a constitutively active receptor, whose expression is sufficient to drive cell transformation in vitro and tumour development in mice. However, neither the role of vGPCR in KSHV-infected B-lymphocytes nor the molecular basis for its constitutive activation is well understood. Here, we show that vGPCR expression contributes to nuclear factor-κB (NF-κB)-dependent cellular survival in both PEL cells and primary B cells from HIV-negative KS patients. We further identified within vGPCR an AP2 consensus binding motif, Y326GLF, that directs its localization between the plasma membrane and clathrin-coated vesicles. The introduction of a mutation in this site (Y326A) increased NF-κB activity and proinflammatory cytokines production. This correlated with exacerbated morphological rearrangement, migration and proliferation of non-infected monocytes. Collectively, our work raises the possibility that KSHV-infected B-lymphocytes use vGPCR to impact ultimately the immune response and communication within the tumour microenvironment in KSHV-associated pathologies.

Optimizing Clinical Trial Enrollment Methods Through "Goal Programming"

INTRODUCTION: Clinical trials often fail to reach desired goals due to poor recruitment outcomes, including low participant turnout, high recruitment cost, or poor representation of minorities. At present, there is limited literature available to guide recruitment methodology. This study, conducted by researchers at the University of Wisconsin Center for Tobacco Research and Intervention (UW-CTRI), provides an example of how iterative analysis of recruitment data may be used to optimize recruitment outcomes during ongoing recruitment. STUDY METHODOLOGY: UW-CTRI's research team provided a description of methods used to recruit smokers in two randomized trials (n = 196 and n = 175). The trials targeted low socioeconomic status (SES) smokers and involved time-intensive smoking cessation interventions. Primary recruitment goals were to meet required sample size and provide representative diversity while working with limited funds and limited time. Recruitment data was analyzed repeatedly throughout each study to optimize recruitment outcomes. RESULTS: Estimates of recruitment outcomes based on prior studies on smoking cessation suggested that researchers would be able to recruit 240 low SES smokers within 30 months at a cost of $72,000. With employment of methods described herein, researchers were able to recruit 374 low SES smokers over 30 months at a cost of $36,260. DISCUSSION: Each human subjects study presents unique recruitment challenges with time and cost of recruitment dependent on the sample population and study methodology. Nonetheless, researchers may be able to improve recruitment outcomes though iterative analysis of recruitment data and optimization of recruitment methods throughout the recruitment period.

Five population-based interventions for smoking cessation: a MOST trial.

Little is known about the relative, additive, and interactive effects of different population-based treatments for smoking cessation. The goal of this study was to evaluate the main and interactive effects of five different smoking interventions. Using the multiphase optimization strategy (MOST), 1,034 smokers who entered a Web site for smokers (smokefree.gov) were randomly assigned to the "on" and "off" conditions of five smoking cessation interventions: the National Cancer Institute's (NCI) Web site (www.smokefree.gov vs a "lite" Web site), telephone quitline counseling (vs none), a smoking cessation brochure (vs a lite brochure), motivational e-mail messages (vs none), and mini-lozenge nicotine replacement therapy (NRT vs none). Analyses showed that the NCI Web site and NRT both increased abstinence; however, the former increased abstinence significantly only when it was not used with the e-mail messaging intervention (messaging decreased Web site use). The other interventions showed little evidence of effectiveness. There was evidence that mailed nicotine mini-lozenges and the NCI Web site (www.smokefree.gov) provide benefit as population-based smoking interventions.

A stress steroid triggers anxiety via increased expression of α4ßδ GABAA receptors in methamphetamine dependence.

Methamphetamine (METH) is an addictive stimulant drug. In addition to drug craving and lethargy, METH withdrawal is associated with stress-triggered anxiety. However, the cellular basis for this stress-triggered anxiety is not understood. The present results suggest that during METH withdrawal (24h) following chronic exposure (3mg/kg, i.p. for 3-5weeks) of adult, male mice, the effect of one neurosteroid released by stress, 3α,5α-THP (3α-OH-5α-pregnan-20-one), and its 3α,5ß isomer reverse to trigger anxiety assessed by the acoustic startle response (ASR), in contrast to their usual anti-anxiety effects. This novel effect of 3α,5ß-THP was due to increased (three-fold) hippocampal expression of α4ßδ GABAA receptors (GABARs) during METH withdrawal (24h-4weeks) because anxiogenic effects of 3α,5ß-THP were not seen in α4-/- mice. 3α,5ß-THP reduces current at these receptors when it is hyperpolarizing, as observed during METH withdrawal. As a result, 3α,5ß-THP (30nM) increased neuronal excitability, assessed with current clamp and cell-attached recordings in CA1hippocampus, one CNS site which regulates anxiety. α4ßδ GABARs were first increased 1h after METH exposure and recovered 6weeks after METH withdrawal. Similar increases in α4ßδ GABARs and anxiogenic effects of 3α,5ß-THP were noted in rats during METH withdrawal (24h). In contrast, the ASR was increased by chronic METH treatment in the absence of 3α,5ß-THP administration due to its stimulant effect. Although α4ßδ GABARs were increased by chronic METH treatment, the GABAergic current recorded from hippocampal neurons at this time was a depolarizing, shunting inhibition, which was potentiated by 3α,5ß-THP. This steroid reduced neuronal excitability and anxiety during chronic METH treatment, consistent with its typical effect. Flumazenil (10mg/kg, i.p., 3×) reduced α4ßδ expression and prevented the anxiogenic effect of 3α,5ß-THP after METH withdrawal. Our findings suggest a novel mechanism underlying stress-triggered anxiety after METH withdrawal mediated by α4ßδ GABARs.

Conversion of a single-facility pediatric antimicrobial stewardship program to multi-facility application with computerized provider order entry and clinical decision support.

OBJECTIVE: Antimicrobial stewardship programs (ASPs) help meet quality and safety goals with regard to antimicrobial use. Prior to CPOE implementation, the ASP at our pediatric tertiary hospital developed a paper-based order set containing recommendations for optimization of dosing. In adapting our ASP for CPOE, we aimed to preserve consistency in our ASP recommendations and expand ASP expertise to other hospitals in our health system. METHODS: Nine hospitals in our health system adopted pediatric CPOE and share a common domain (Cerner Millenium). ASP clinicians developed sixty individual electronic order sets (vendor reference PowerPlans) to be used independently or as part of larger electronic order sets. Analysis of incidents reported during CPOE implementation and medication variances reports was used to determine the effectiveness of the ASP adaptation. RESULTS: 769 unique PowerPlans were used 15,889 times in the first 30 days after CPOE implementation. Of these, 43 were PowerPlans included in the ASP design and were used a total of 1149 times (7.2% of all orders). During CPOE implementation, 437 incidents were documented, 1.1% of which were associated with ASP content or workflow. Additionally, analysis of medication variance following CPOE implementation showed that ASP errors accounted for 2.9% of total medication variances. DISCUSSION: ASP content and workflow accounted for proportionally fewer incidents than expected as compared to equally complex and frequently used CPOE content. CONCLUSION: Well-defined ASP recommendations and modular design strengthened successful CPOE implementation, as well as the adoption of specialized pediatric ASP expertise with other facilities.

The influence of stress at puberty on mood and learning: role of the α4ßδ GABAA receptor.

It is well-known that the onset of puberty is associated with changes in mood as well as cognition. Stress can have an impact on these outcomes, which in many cases, can be more influential in females, suggesting that gender differences exist. The adolescent period is a vulnerable time for the onset of certain psychopathologies, including anxiety disorders, depression and eating disorders, which are also more prevalent in females. One factor which may contribute to stress-triggered anxiety at puberty is the GABAA receptor (GABAR), which is known to play a pivotal role in anxiety. Expression of α4ßδ GABARs increases on the dendrites of CA1 pyramidal cells at the onset of puberty in the hippocampus, part of the limbic circuitry which governs emotion. This receptor is a sensitive target for the stress steroid 3α-OH-5[α]ß-pregnan-20-one or [allo]pregnanolone, which paradoxically reduces inhibition and increases anxiety during the pubertal period (post-natal day ∼35-44) of female mice in contrast to its usual effect to enhance inhibition and reduce anxiety. Spatial learning and synaptic plasticity are also adversely impacted at puberty, likely a result of increased expression of α4ßδ GABARs on the dendritic spines of CA1 hippocampal pyramidal cells, which are essential for consolidation of memory. This review will focus on the role of these receptors in mediating behavioral changes at puberty. Stress-mediated changes in mood and cognition in early adolescence may have relevance for the expression of psychopathologies in adulthood.

Using pictures to evoke spiritual feelings in breast cancer patients: development of a new paradigm for neuroimaging studies.

This study was designed to develop and validate a method for enhancing spiritual feelings, particularly in women who have received a diagnosis of breast cancer. The protocol specifically was developed to be used in functional magnetic resonance imaging (fMRI) studies. Eighteen breast cancer survivors rated pictures for their ability to enhance feelings of spirituality, happiness, and sadness. Results indicate that presenting carefully selected pictures with spiritual content (e.g., nature scenes, people engaged in contemplative behaviors) can effectively enhance spiritual feelings among breast cancer survivors. Future fMRI studies will explore the use of the protocol developed in this study for investigating neural activity during spiritual feelings and states.

Nanotechnology of emerging targeting systems.

Recent developments in the design and testing of complex nanoscale payload-carrying systems (i.e. systems with payloads that do not exceed 100 nm in size) are the focus of this brief review. Emerging systems include targeted single-walled nanotubes, viral capsids, dendrimers, gold nanoparticles, milled boron carbide nanoparticles, and protein nucleic acid assemblies. Significant advances are emerging with each of these bionanotechnological approaches to cellular targeting.

A preliminary investigation of the effectiveness of a sleep apnea education program.

OBJECTIVES: This study aimed to evaluate a standardised sleep apnea patient education program and develop a study design that may be used to evaluate other such education programs. METHOD: Thirty-four adults diagnosed with obstructive sleep apnea hypopnea syndrome (OSAHS) underwent a standard sleep apnea education program and completed measures of knowledge of and beliefs about sleep apnea before, after, and 3 months following education. Two outcome measures were used: the Apnea Knowledge Test (AKT) and the Apnea Beliefs Scale (ABS). RESULTS: AKT results showed significant knowledge gains posteducation, which were maintained at follow-up. Patients also reported more positive beliefs about their ability to change their behaviour and comply with continuous positive airway pressure (CPAP) treatment recommendations after education. DISCUSSION: Findings from this preliminary investigation suggest that the education program used in this study may improve patients' knowledge of CPAP and promote functional beliefs about OSAHS treatment. This program clearly warrants further research, and ultimately such programs may prove important in improving CPAP compliance.

Psychiatric treatment in prison: a missed opportunity?

National Health Service Executive guidelines require psychiatric services to maintain links with prisoners previously subject to the Care Programme Approach (CPA) and to participate in discharge planning. We are unaware of previous studies assessing the involvement of general psychiatric services with patients in prison or prisoners' perceptions of their needs. Consecutive referrals to a prison psychiatric liaison service over a three-month period were screened for previous psychiatric contact. Half of those interviewed reported previous psychiatric contact. Two-thirds were in contact with services at the time of detention. One-third believed services knew of their imprisonment. Ninety-three per cent believed they would require psychiatric support after release. Few patients received input from general psychiatric services during imprisonment despite a high level of perceived need. Improved liaison would help facilitate both care in prison and discharge planning in the spirit of CPA and the government directive.

Sex differences in anxiety, sensorimotor gating and expression of the alpha4 subunit of the GABAA receptor in the amygdala after progesterone withdrawal.

In a progesterone withdrawal (PWD) model of premenstrual anxiety, we have previously demonstrated that increased hippocampal expression of the alpha4 subunit of the GABAA receptor (GABAA-R) is closely associated with higher anxiety levels in the elevated plus maze. However, several studies indicate that sex differences in regulation of the GABAA-R in specific brain regions may be an important factor in the observed gender differences in mood disorders. Thus, we investigated possible sex differences in GABAA-R subunit expression and anxiety during PWD. To this end, we utilized the acoustic startle response (ASR) to assess anxiety levels in male and female rats undergoing PWD as the ASR is also applicable to the assessment of human anxiety responses. We also investigated GABAA-R alpha4 subunit expression in the amygdala, as the amygdala directly regulates the primary startle circuit. Female rats exhibited a greater ASR during PWD than controls, indicating higher levels of anxiety and arousal. In contrast, male rats undergoing PWD did not demonstrate an increased ASR. The sex differences in the ASR were paralleled by sex differences in the expression of the GABAA-R alpha4 subunit in the amygdala such that alpha4 subunit expression was up-regulated in females during PWD whereas alpha4 levels in males undergoing PWD were not altered relative to controls. These findings might have implications regarding gender differences in human mood disorders and the aetiology of premenstrual anxiety.

Anxiogenic effects of neurosteroid exposure: sex differences and altered GABAA receptor pharmacology in adult rats.

Acute exposure to progesterone or its neurosteroid derivative allopregnanolone (3alpha,5alpha-THP) is anxiolytic, consistent with the GABA modulatory effects of 3alpha,5alpha-THP at the GABA(A) receptor. However, continuous exposure to progesterone increases anxiety in association with increased expression of the benzodiazepine-insensitive GABA(A) receptor alpha4 subunit. Furthermore, negative mood symptoms and altered GABA(A) receptor pharmacology in patients with premenstrual dysphoric disorder occur in the early luteal phase in association with peak circulating levels of progesterone and 3alpha,5alpha-THP. Because sex differences have been reported in steroid-regulated anxiety responses, the present study investigated the role of sex and development in the regulation of anxiety after short-term exposure to 3alpha,5alpha-THP. To this end, we compared the effects of hormone administration in adult male, adult female, and juvenile female rats. Increased anxiety in the elevated plus maze was evident in all groups after 48-h exposure to either 3alpha,5alpha-THP or progesterone. At this time point, alterations in the anxiolytic profile of benzodiazepine agonists and antagonists were also observed in both adult males and females in the elevated plus maze. However, sex differences in the acoustic startle response were observed after short-term hormone treatment such that only female rats displayed an increased response indicative of higher anxiety levels. These results suggest that although neurosteroid exposure may influence both the pharmacological properties of the GABA(A) receptor and the manifestation of anxiety in both sexes, the effects of neurosteroids may be modulated in a sex- and task-specific manner.

Progesterone withdrawal increases the anxiolytic actions of gaboxadol: role of alpha4betadelta GABA(A) receptors.

Hippocampal alpha4betadelta GABA(A) receptors (GABA(A)-R) are increased following progesterone withdrawal (PWD) in a rodent model of premenstrual anxiety. This alpha4betadelta receptor isoform uniquely responds to the GABA agonist gaboxadol (THIP) with a maximum current greater than that gated by GABA, and is potentiated more by pentobarbital than are other GABA(A)-R. We therefore investigated the anxiolytic effects of these drugs using the elevated plus maze. Gaboxadol (1.25 mg/kg) was markedly more anxiolytic in animals undergoing PWD than in controls. Pentobarbital (10 mg/kg) also produced a greater anxiolytic effect during PWD. These results suggest that the pharmacological properties of alpha4betadelta GABA(A)-R following PWD are evident behaviorally. Alterations in the alpha4betadelta GABA(A)-R population may have implications for the etiology and treatment of premenstrual syndrome.

Progesterone withdrawal increases the alpha4 subunit of the GABA(A) receptor in male rats in association with anxiety and altered pharmacology - a comparison with female rats.

Withdrawal from the neurosteroid 3alpha,5alpha-allopregnanolone after chronic administration of progesterone increases anxiety in female rats and up-regulates the alpha4 subunit of the GABA(A) receptor (GABA(A)-R) in the hippocampus. We investigated if these phenomena would also occur in male rats. Progesterone withdrawal (PWD) induced higher alpha4 subunit expression in the hippocampus of both male and female rats, in association with increased anxiety (assessed in the elevated plus maze) comparable to effects previously reported. Because alpha4-containing GABA(A)-R are insensitive to the benzodiazepine (BDZ) lorazepam (LZM), and are positively modulated by flumazenil (FLU, a BDZ antagonist), we therefore tested the effects of these compounds following PWD. Using whole-cell patch clamp techniques, LZM-potentiation of GABA ((EC20))-gated current was markedly reduced in CA1 pyramidal cells of male rats undergoing PWD compared to controls, whereas FLU had no effect on GABA-gated current in control animals but increased it in PWD animals. Behaviorally, both male and female rats were significantly less sensitive to the anxiolytic effects of LZM. In contrast, FLU demonstrated significant anxiolytic effects following PWD. These data suggest that neurosteroid regulation of the alpha4 GABA(A)-R subunit may be a relevant mechanism underlying anxiety disorders, and that this phenomenon is not sex-specific.

CFTR: covalent and noncovalent modification suggests a role for fixed charges in anion conduction.

The goal of the experiments described here was to explore the possible role of fixed charges in determining the conduction properties of CFTR. We focused on transmembrane segment 6 (TM6) which contains four basic residues (R334, K335, R347, and R352) that would be predicted, on the basis of their positions in the primary structure, to span TM6 from near the extracellular (R334, K335) to near the intracellular (R347, R352) end. Cysteines substituted at positions 334 and 335 were readily accessible to thiol reagents, whereas those at positions 347 and 352 were either not accessible or lacked significant functional consequences when modified. The charge at positions 334 and 335 was an important determinant of CFTR channel function. Charge changes at position 334--brought about by covalent modification of engineered cysteine residues, pH titration of cysteine and histidine residues, and amino acid substitution--produced similar effects on macroscopic conductance and the shape of the I-V plot. The effect of charge changes at position 334 on conduction properties could be described by electrodiffusion or rate-theory models in which the charge on this residue lies in an external vestibule of the pore where it functions to increase the concentration of Cl adjacent to the rate-limiting portion of the conduction path. Covalent modification of R334C CFTR increased single-channel conductance determined in detached patches, but did not alter open probability. The results are consistent with the hypothesis that in wild-type CFTR, R334 occupies a position where its charge can influence the distribution of anions near the mouth of the pore.

CFTR: covalent modification of cysteine-substituted channels expressed in Xenopus oocytes shows that activation is due to the opening of channels resident in the plasma membrane.

Some studies of CFTR imply that channel activation can be explained by an increase in open probability (P(o)), whereas others suggest that activation involves an increase in the number of CFTR channels (N) in the plasma membrane. Using two-electrode voltage clamp, we tested for changes in N associated with activation of CFTR in Xenopus oocytes using a cysteine-substituted construct (R334C CFTR) that can be modified by externally applied, impermeant thiol reagents like [2-(trimethylammonium)ethyl] methanethiosulfonate bromide (MTSET+). Covalent modification of R334C CFTR with MTSET+ doubled the conductance and changed the I-V relation from inward rectifying to linear and was completely reversed by 2-mercaptoethanol (2-ME). Thus, labeled and unlabeled channels could be differentiated by noting the percent decrease in conductance brought about by exposure to 2-ME. When oocytes were briefly (20 s) exposed to MTSET+ before CFTR activation, the subsequently activated conductance was characteristic of labeled R334C CFTR, indicating that the entire pool of CFTR channels activated by cAMP was accessible to MTSET+. The addition of unlabeled, newly synthesized channels to the plasma membrane could be monitored on-line during the time when the rate of addition was most rapid after cRNA injection. The addition of new channels could be detected as early as 5 h after cRNA injection, occurred with a half time of approximately 24-48 h, and was disrupted by exposing oocytes to Brefeldin A, whereas activation of R334C CFTR by cAMP occurred with a half time of tens of minutes, and did not appear to involve the addition of new channels to the plasma membrane. These findings demonstrate that in Xenopus oocytes, the major mechanism of CFTR activation by cAMP is by means of an increase in the open probability of CFTR channels.