Predictors of state-based changes in wanting and liking.

People report wanting food when they are hungry, and on eating it they typically report liking the experience. After eating, both wanting and liking decline, but wanting declines to a greater extent, which we term the 'affective discrepancy effect'. In this study we examine the predictors - state, sensory and memory-based - of these affective changes. Hungry participants undertook three tasks: (1) written recollections of what certain foods are like to eat; (2) ratings of wanting and expected flavour liking and fillingness when looking at snacks, and ratings of food and flavour liking when eating them; (3) ratings of bodily state. These tasks were then repeated after lunch. State-based changes in food liking were best predicted by changes in flavour liking. For state-based change in wanting, memory-based information about flavour liking and fillingness from tasks (1) and (2) were all significant predictors. For recollections about eating (task 1), mentions of food fillingness significantly increased pre-to post-lunch and this was the best predictor of the affective discrepancy effect. Recollections of food fillingness are state-dependent, and can arise unbidden (i.e., such recollective content was unprompted). This may reflect one way that memory may selectively influence wanting, and hence whether food intake is initiated or not.

The psychological basis of reductions in food desire during satiety.

Satiety-the reduced desire to eat, drink or have sex in their respective aftermath-is particularly important for feeding, where it assists energy balance. During satiety, the anticipated pleasure of eating is far less than the actual pleasure of eating. Here we examine two accounts of this effect: (i) satiety signals inhibit retrieval of pleasant food memories that form desirable images, allowing unpleasant memories into mind; (ii) feelings of fullness reflect what eating would be like now, negating the need for imagery. To test these accounts, participants undertook two tasks pre- and post-lunch: (i) judging desire for palatable foods either with or without imagery impairing manipulations; (ii) explicitly recollecting food memories. Impairing imagery reduced desire equally, when hungry and sated. Food-memory recollections became more negative/less positive when sated, with this correlating with changes in desire. These findings support the first account and suggest imagery is used when hungry and when sated to simulate eating, and that the content of these memory-based simulations changes with state. The nature of this process and its implications for satiety more generally are discussed.

Synthetic Cannabinoid Receptor Agonists in Postmortem Casework in Scotland.

Synthetic cannabinoid receptor agonists (SCRAs) have been a concern to forensic toxicologists since their emergence as drugs of abuse in the mid-late 2000s. The extent of their use in Scotland appears to be low especially when compared to other drug groups such as opioids and benzodiazepines. There is a concern, however, that the use is widespread in prison populations in particular. In this work, samples of blood and urine collected during routine postmortem examination between April 2017 and March 2019 were subjected to analysis of SCRA compounds. Circumstantial and demographic information was collected on positive cases to build up a body of evidence for where SCRAs may be most likely to contribute to the cause of death. Thirteen out of 133 cases (10%) tested were positive for one or more compound in one or more matrix. Overall, the detection of 5F-MDMB-PINACA or its O-desmethyl acid metabolite was most common, followed by the metabolite shared by AB-FUBINACA and MMB-FUBINACA. SCRA-positive cases were predominantly males (92%), and the age range of all decedents was 21-49 years old (median 36 years). The majority of cases were certified as drug-related deaths (DRDs, 38%), natural/medical (31%) or suicide (23%), and two of the DRDs mentioned SCRAs specifically in the cause of death. The concentrations of SCRAs detected did not seem to be as important to the determination of the cause of death as their mere presence, but quantitative results were reported (where possible) in order to build up a body of evidence for SCRA concentrations in different case types.

Recollections about food when hungry and sated.

Memory processes may have several roles in appetite regulation. Here we examine one such role, derived from the animal literature, in which satiety cues lead to the inhibition of rewarding food-related memories. We tested this idea over three studies (n's of 58, 67, 50 respectively), by presenting participants with visual or verbal food cues, and asking them to describe what these foods were like to eat. This recollection task was undertaken hungry and sated. The resulting recollections were then coded and contrasted across state. Irrespective of state, participants took the same time to make their recollections, they were of similar length and included the same amount of sensory detail and affective content. However, in all three studies, sated recollections tended to include more reports about how filling a food would be. This increase in reports of food fillingness across state, was significantly correlated with increases in reports of stomach distension across state. While these results are consistent with the operation of memory inhibition, a further possibility is considered, whereby interoceptive satiety cues are integrated into food-related recollections (but not other recollections) to form a memory-inteorception-combination, thereby drawing attention to the consequences of eating when sated.

Lifestyle risk behaviours among adolescents: a two-year longitudinal study of the impact of the COVID-19 pandemic.

OBJECTIVE: To examine changes in the prevalence of six key chronic disease risk factors (the "Big 6"), from before (2019) to during (2021) the COVID-19 pandemic, among a large and geographically diverse sample of adolescents, and whether differences over time are associated with lockdown status and gender. DESIGN: Prospective cohort study. SETTING: Three Australian states (New South Wales, Queensland and Western Australia) spanning over 3000 km. PARTICIPANTS: 983 adolescents (baseline Mage=12.6, SD=0.5, 54.8% girl) drawn from the control group of the Health4Life Study. PRIMARY OUTCOMES: The prevalence of physical inactivity, poor diet (insufficient fruit and vegetable intake, high sugar-sweetened beverage intake, high discretionary food intake), poor sleep, excessive recreational screen time, alcohol use and tobacco use. RESULTS: The prevalence of excessive recreational screen time (prevalence ratios (PR)=1.06, 95% CI=1.03 to 1.11), insufficient fruit intake (PR=1.50, 95% CI=1.26 to 1.79), and alcohol (PR=4.34, 95% CI=2.82 to 6.67) and tobacco use (PR=4.05 95% CI=1.86 to 8.84) increased over the 2-year period, with alcohol use increasing more among girls (PR=2.34, 95% CI=1.19 to 4.62). The prevalence of insufficient sleep declined across the full sample (PR=0.74, 95% CI=0.68 to 0.81); however, increased among girls (PR=1.24, 95% CI=1.10 to 1.41). The prevalence of high sugar-sweetened beverage (PR=0.61, 95% CI=0.64 to 0.83) and discretionary food consumption (PR=0.73, 95% CI=0.64 to 0.83) reduced among those subjected to stay-at-home orders, compared with those not in lockdown. CONCLUSION: Lifestyle risk behaviours, particularly excessive recreational screen time, poor diet, physical inactivity and poor sleep, are prevalent among adolescents. Young people must be supported to find ways to improve or maintain their health, regardless of the course of the pandemic. Targeted approaches to support groups that may be disproportionately impacted, such as adolescent girls, are needed. TRIAL REGISTRATION NUMBER: Australian New Zealand Clinical Trials Registry (ACTRN12619000431123).

An Insight into Gabapentin and Pregabalin in Scottish Prisoners.

The aim of this study was to evaluate the prevalence and abuse potential of antiepileptic drugs (AEDs) among prison populations in Scotland, UK. Participants consisted of all admitted and released prisoners over a 1 month period who consented to provide samples. Urine samples were collected and analyzed by liquid chromatography coupled with triple quadrupole tandem mass spectrometry using a method validated for the simultaneous quantification of 21 AEDs in urine. A total of 904 samples were collected. The samples were also screened for drugs of abuse by using point-of-care testing kits. A total of 18% of the samples were positive for AEDs. Gabapentin (GBP) was identified in 118 samples (13%) and pregabalin (PRG) in 32 samples (3.5%). Interestingly, 12 samples contained both drugs (1.3%). The concentrations ranged from 0.5 to 1,100 mg/L (median, 15 mg/L) for GBP and from 0.5 to 440 mg/L (median, 7.3 mg/L) for PRG. Four samples were found to have concentrations >400 mg/L, two samples for GBP and two samples for PRG. These concentrations are at least 20 times above the median concentrations. Other AEDs detected were levetiracetam (four samples), vigabatrin (four samples), lamotrigine (three samples), valproic acid (three samples), carbamazepine (two samples) and topiramate (one sample). Illicit or non-prescribed drugs were detected in 81% of urine samples of which 80% were from admitted prisoners and 20% from released prisoners. Benzodiazepines, opiates and cannabis were the most frequently detected drugs. Other drugs found in positive AED samples were methadone (26%), cocaine (18%), buprenorphine (17%), amphetamines (4%), methamphetamines (4%) and barbiturates (4%). This study shows a high prevalence of AEDs within the Scottish prison system, primarily due to GBP and PRG; however, due to the anonymity of the sample collection, it is unknown if these are prescribed or illicit drug ingestions.

Gas Chromatography-Mass Spectrometry Method for the Quantitative Identification of 23 New Psychoactive Substances in Blood and Urine.

New psychoactive substances (NPSs) have become an integral part of the recreational drug market with "new" compounds being reported by the European Monitoring Centre for Drugs and Drug Addiction weekly. Due to the changing nature of NPSs, it is impractical to carry out single analyte or even simple class quantitation. Although several gas chromatography-mass spectrometry (GC-MS) methods have been developed these are typically class specific. We present a validated GC-MS method for the quantitation of 2-DPMP, 3-MeO-PCE, 3-MeO-PCP, 5-APB, 6-APB, benzedrone, butylone, ethylone, flephedrone, methiopropamine, MDPV, mephedrone, methoxetamine, methylone, naphyrone, 25B-NBOME, 25C-NBOME, 25D-NBOMe, 25E-NBOME, 25H-NBOME, 25I-NBOME, Mescaline-NBOME and 25P-NBOME in blood and urine samples. Sample preparation was carried out using solid-phase extraction followed by derivatisation and analysis by GC-MS. Parameters investigated for validation included bias, precision, linear calibration model, carryover, interferences, limit of detection, limit of quantification, and autosampler and freeze/thaw stability. All drugs yielded successful results for each of these parameters as per SWGTOX guidelines. The GC-MS method was used for the reanalysis of 12 blood samples (eight cases) where 25I-NBOMe, 25C-NBOMe, methoxetamine and methylone had previously been detected by NMS laboratories. This GC-MS method was able to quantitatively detect these drugs in 75% of the blood samples, 42% of which contained either 25C-NBOMe or 25I-NBOMe. This method accurately allows for the simultaneous quantification of a wide variety of compounds via GC-MS, in particular NBOMe compounds which are typically analysed by liquid chromatography-tandem mass spectrometry which is not available in all laboratories.

Application of a Urine and Hair Validated LC-MS-MS Method to Determine the Effect of Hair Color on the Incorporation of 25B-NBOMe, 25C-NBOMe and 25I-NBOMe into Hair in the Rat.

NBOMes are a group of new psychoactive substances derived from phenethylamines. Recreational abuse is thought to have begun in 2010 and they are commonly associated with the "club drug" scene. They are administered in liquid form or as blotters due to their high potency. An LC-MS-MS method was validated using Scientific Working Group for Forensic Toxicology parameters for the detection of 25B-, 25C- and 4-iodo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (25I-NBOMe) using 4-bromo-2,5-dimethoxy-N-[(2-methoxyphenyl)methyl]-benzeneethanamine (25B-NBOMe)-D3 as internal standard for urine and hair. Calibration graphs with R2 values >0.99 were observed for urine and hair for concentrations ranging from 0.1 to 100 ng/mL and 0.025 to 2.5 ng/mg, respectively. Urine LODs ranged from 5 to 25 pg/mL and had an LOQ of 50 pg/mL. Hair LOD and LOQs ranged from 3 to 5 pg/mg and 6.25 to 12.5 pg/mg, respectively. Intra- and inter-day precision was <20% and accuracy was within ±20% for both matrices. The method was shown to be selective for both exogenous and endogenous compounds. No matrix effects were observed for either matrix. LLE recovery ranged from 90 to 103% for urine samples and solid phase extraction recovery ranged from 80 to 107% for hair samples. Long-Evans rats (n = 55) were administered 25B-, 25C- or 25I-NBOMe at doses ranging from 30 to 300 µg/kg over a period of 10 days. Rats were shaved prior to their first dose and re-shaved after the 10-day period. Hair was separated by color (black: n = 55 and white: n = 55) and analyzed using the validated LC-MS-MS method to assess the impact hair color has on the incorporation of these drugs. All drugs were successfully detected in black hair. 25B-NBOMe from rats receiving the highest dose and 25C-NBOMe from rats receiving the medium and high doses were quantified in white hair. 25I-NBOMe was detected but fell below the limit of quantification. A dose-dependent concentration increase was observed in the black hair. All pooled urine samples tested positive for their expected NBOMes.

Analysis and clinical findings of cases positive for the novel synthetic cannabinoid receptor agonist MDMB-CHMICA.

CONTEXT: MDMB-CHMICA is a synthetic cannabinoid receptor agonist which has caused concern due to its presence in cases of adverse reaction and death. METHOD: 43 cases of suspected synthetic cannabinoid ingestion were identified from patients presenting at an Emergency Department and from post-mortem casework. These were subjected to liquid-liquid extraction using tertiary-butyl methyl ether and quantitatively analysed by Electrospray Ionisation Liquid Chromatography-tandem Mass Spectrometry. For positive samples, case and clinical details were sought and interrogated. RESULTS: 11 samples were found positive for MDMB-CHMICA. Concentrations found ranged from <1 to 22 ng/mL (mean: 6 ng/mL, median: 3 ng/mL). The age range was 15-44 years (mean: 26 years, median: 21 years), with the majority (82%) of positive results found in males. Clinical presentations included hypothermia, hypoglycaemia, syncope, recurrent vomiting, altered mental state and serotonin toxicity, with corresponding concentrations of MDMB-CHMICA as low as <1 ng/mL. Duration of hospitalisation ranged from 3 to 24 h (mean: 12 h, median: 8 h). DISCUSSION: The concentration range presented in this case series is indicative of MDMB-CHMICA having a high potency, as is known to be the case for other synthetic cannabinoid receptor agonists. The age range and gender representation were consistent with that reported for users of other drugs of this type. The clinical presentations observed were typical of synthetic cannabinoid receptor agonists and show the difficulties in identifying reactions potentially associated with drugs of this type. CONCLUSION: The range of MDMB-CHMICA concentrations in Emergency Department presentations (n = 9) and post-mortem cases (n = 2) was reported. No correlation between the concentration of this drug and clinical presentation or cause of death was reported in this sample. However, the potential for harm associated with low concentrations of MDMB-CHMICA and the symptoms of toxicity being non-specific were highlighted.

Simultaneous analysis of 22 antiepileptic drugs in postmortem blood, serum and plasma using LC-MS-MS with a focus on their role in forensic cases.

In recent years, there has been a growth in reports of antiepileptic drugs (AEDs) being misused on their own or in combination with other drugs of abuse in a variety of toxicological case types such as drug abuse, suicide, overdose and drug facilitated crime. To our knowledge, there are no simultaneous quantification methods for the analysis of the most commonly encountered AEDs in postmortem whole blood and clinical plasma/serum samples at the same time. A simple, accurate and cost-effective liquid chromatography-tandem mass spectrometric (LC-MS-MS) method has been developed and validated for the simultaneous quantification of carbamazepine (CBZ) and its metabolite CBZ-10,11-epoxide, eslicarbazepine acetate, oxcarbazepine and S-licarbazepine as a metabolite, gabapentin, lacosamide, lamotrigine, levetiracetam, pregabalin, phenobarbital, phenytoin and its metabolite 5-(p-hydroxyphenyl)-5-phenylhydantoin, retigabine (ezogabine) and its metabolite N-acetyl retigabine, rufinamide, stiripentol, topiramate, tiagabine, valproic acid, vigabatrin and zonisamide in postmortem whole blood, serum and plasma which would be suitable for routine forensic toxicological analysis and therapeutic drug monitoring. All AEDs were detected and quantified within 17 min without endogenous interferences. The correlation coefficient (R(2)) was >0.995 for all AEDs with accuracy ranging from 90 to 113% and precision <13% for all analytes. The recovery ranged from 70 to 98%. No carryover was observed in a blank control injected after the highest standard and the matrix effect was acceptable and ranged from 90 to 120%. The method has been successfully verified using authentic case samples that had previously been quantified using different methods.

Simultaneous detection and quantification of amphetamines, diazepam and its metabolites, cocaine and its metabolites, and opiates in hair by LC-ESI-MS-MS using a single extraction method.

A liquid chromatography-tandem mass spectrometry method was developed and validated for the simultaneous identification and quantification of amphetamines, diazepam and its metabolites, cocaine and its metabolites, and opiates from hair using a single extraction method. As part of the method development, Gemini C18, Synergi Hydro RP, and Zorbax Stablebond-Phenyl LC columns were tested with three different mobile phases. Analyte recovery and limit of detection were evaluated for two different solid-phase extraction methods that used Bond Elut Certify and Clean Screen cartridges. Phosphate buffer (pH 5.0) was chosen as the optimum hair incubation medium because of the high stability of cocaine and 6-monoacetylmorphine using this method and faster sample preparation. The optimized method was fully validated. Linearity was established over the concentration range 0.2-10 ng/mg hair, and the correlation coefficients were all greater than 0.99. Total extraction recoveries were greater than 76%, detection limits were between 0.02 and 0.09 ng/mg, and the intra- and interday imprecisions were generally less than 20% in spiked hair. The intra- and interbatch imprecision of the method for a pooled authentic hair sample ranged from 1.4 to 23.4% relative standard deviation (RSD) and 8.3 to 25.4% RSD, respectively, for representative analytes from the different drug groups. The percent matrix effect ranged from 63.5 to 135.6%, with most analytes demonstrating ion suppression. Sixteen postmortem samples collected from suspected drug-related deaths were analyzed for the 17 drugs of abuse and metabolites included in the method. The method was sufficiently sensitive and specific for the analysis of drugs and metabolites in postmortem hair samples. There is scope for the inclusion of other target drugs and metabolites in the method.

Active Rab11 and functional recycling endosome are required for E-cadherin trafficking and lumen formation during epithelial morphogenesis.

The correct targeting and trafficking of the adherens junction protein epithelial cadherin (E-cadherin) is a major determinant for the acquisition of epithelial cell polarity and for the maintenance of epithelial integrity. The compartments and trafficking components required to sort and transport E-cadherin to the basolateral cell surface remain to be fully defined. On the basis of previous data, we know that E-cadherin is trafficked via the recycling endosome (RE) in nonpolarized and newly polarized cells. Here we explore the role of the RE throughout epithelial morphogenesis in MDCK monolayers and cysts. Time-lapse microscopy in live cells, altering RE function biochemically, and expressing a dominant-negative form of Rab11 (DN-Rab11), each showed that the RE is always requisite for E-cadherin sorting and trafficking. The RE remained important for E-cadherin trafficking in MDCK cells from a nonpolarized state through to fully formed, polarized epithelial monolayers. During the development of epithelial cysts, DN-Rab11 disrupted E-cadherin targeting and trafficking, the subapical localization of pERM and actin, and cyst lumen formation. This final effect demonstrated an early and critical interdependence of Rab11 and the RE for E-cadherin targeting, apical membrane formation, and cell polarity in cysts.

Detection of benzodiazepines in hair using ELISA and LC-ESI-MS-MS.

This study was designed to validate an enzyme-linked immunosorbent assay (ELISA) and liquid chromatography-tandem mass spectrometry (LC-MS-MS) method for the detection of nine benzodiazepines in hair. Sixteen hair case samples were tested from drug-related deaths where a positive benzodiazepine blood result was obtained. The case samples were decontaminated with 0.1% sodium dodecyl sulfate, distilled water, and dichloromethane. For ELISA analysis, the samples were extracted by incubation in monobasic phosphate buffer for 1 h and then neutralized with dibasic phosphate buffer. They were diluted 1:5 with phosphate buffer saline (PBS) prior to analysis. For LC-MS-MS, the samples were incubated overnight in methanol/25% ammonium hydroxide (20:1). The benzodiazepines were extracted by solid phase. Thirteen samples were confirmed positive by LC-MS-MS. The benzodiazepines detected included diazepam, nordiazepam, temazepam, oxazepam, nitrazepam, and lorazepam. Using a cut-off concentration of 0.1 ng/mg oxazepam, the Immunalysis Benzodiazepine Microplate ELISA demonstrated a sensitivity and specificity of 100% and 81%, respectively, compared with LC-MS-MS results.

Validation of the immunalysis microplate ELISA for the detection of methamphetamine in hair.

The object of this study was to validate the Immunalysis Methamphetamine Microplate ELISA for detecting methamphetamine in hair. Twenty-nine scalp hair samples were obtained as routine cases submitted to the National Institute of Scientific Investigation in Seoul by the police. The hair samples were washed with 0.1% sodium dodecyl sulfate, distilled water, and dichloromethane. The samples were screened using the Immunalysis Methamphetamine Microplate ELISA and confirmed using gas chromatography-mass spectrometry (GC-MS). Twenty-eight hair samples were screened and confirmed as positive for methamphetamine. For ELISA analysis, the samples were extracted by incubation in monobasic phosphate buffer for 1 h at 60 degrees C. For GC-MS, the samples were extracted for 20 h in methanol containing 1% hydrochloric acid. The methanol/acid solution was evaporated to dryness and the resulting residue was derivatized with trifluoroacetic anhydride. Methamphetamine and amphetamine were detected using selective ion monitoring (SIM) mode. The Immunalysis Methamphetamine Microplate ELISA demonstrated a sensitivity and specificity of 97% and 100%, respectively, using a cut-off concentration of 0.5 ng/mg d-methamphetamine. The ELISA kit showed 63% cross-reactivity with d,l-methamphetamine and did not cross-react to any significant extent with the licit l-methamphetamine isomer. The intra- and interassay precisions were 2.5% and 3.7%, respectively.

Polarized trafficking of E-cadherin is regulated by Rac1 and Cdc42 in Madin-Darby canine kidney cells.

E-cadherin is a major cell-cell adhesion protein of epithelia that is trafficked to the basolateral cell surface in a polarized fashion. The exact post-Golgi route and regulation of E-cadherin transport have not been fully described. The Rho GTPases Cdc42 and Rac1 have been implicated in many cell functions, including the exocytic trafficking of other proteins in polarized epithelial cells. These Rho family proteins are also associated with the cadherin-catenin complexes at the cell surface. We have used functional mutants of Rac1 and Cdc42 and inactivating toxins to demonstrate specific roles for both Cdc42 and Rac1 in the post-Golgi transport of E-cadherin. Dominant-negative mutants of Cdc42 and Rac1 accumulate E-cadherin at a distinct post-Golgi step. This accumulation occurs before p120(ctn) interacts with E-cadherin, because p120(ctn) localization was not affected by the Cdc42 or Rac1 mutants. Moreover, the GTPase mutants had no effect on the trafficking of a targeting mutant of E-cadherin, consistent with the selective involvement of Cdc42 and Rac1 in basolateral trafficking. These results provide a new example of Rho GTPase regulation of basolateral trafficking and demonstrate novel roles for Cdc42 and Rac1 in the post-Golgi transport of E-cadherin.

Syntaxin 6 and Vti1b form a novel SNARE complex, which is up-regulated in activated macrophages to facilitate exocytosis of tumor necrosis Factor-alpha.

A key function of activated macrophages is to secrete proinflammatory cytokines such as TNFalpha; however, the intracellular pathway and machinery responsible for cytokine trafficking and secretion is largely undefined. Here we show that individual SNARE proteins involved in vesicle docking and fusion are regulated at both gene and protein expression upon stimulation with the bacterial cell wall component lipopolysaccharide. Focusing on two intracellular SNARE proteins, Vti1b and syntaxin 6 (Stx6), we show that they are up-regulated in conjunction with increasing cytokine secretion in activated macrophages and that their levels are selectively titrated to accommodate the volume and timing of post-Golgi cytokine trafficking. In macrophages, Vti1b and syntaxin 6 are localized on intracellular membranes and are present on isolated Golgi membranes and on Golgi-derived TNFalpha vesicles budded in vitro. By immunoprecipitation, we find that Vti1b and syntaxin 6 interact to form a novel intracellular Q-SNARE complex. Functional studies using overexpression of full-length and truncated proteins show that both Vti1b and syntaxin 6 function and have rate-limiting roles in TNFalpha trafficking and secretion. This study shows how macrophages have uniquely adapted a novel Golgi-associated SNARE complex to accommodate their requirement for increased cytokine secretion.

Regulation of endocytosis, nuclear translocation, and signaling of fibroblast growth factor receptor 1 by E-cadherin.

Fibroblast growth factor (FGF) receptors (FGFRs) signal to modulate diverse cellular functions, including epithelial cell morphogenesis. In epithelial cells, E-cadherin plays a key role in cell-cell adhesion, and its function can be regulated through endocytic trafficking. In this study, we investigated the location, trafficking, and function of FGFR1 and E-cadherin and report a novel mechanism, based on endocytic trafficking, for the coregulation of E-cadherin and signaling from FGFR1. FGF induces the internalization of surface FGFR1 and surface E-cadherin, followed by nuclear translocation of FGFR1. The internalization of both proteins is regulated by common endocytic machinery, resulting in cointernalization of FGFR1 and E-cadherin into early endosomes. By blocking endocytosis, we show that this is a requisite, initial step for the nuclear translocation of FGFR1. Overexpression of E-cadherin blocks both the coendocytosis of E-cadherin and FGFR1, the nuclear translocation of FGFR1 and FGF-induced signaling to the mitogen-activated protein kinase pathway. Furthermore, stabilization of surface adhesive E-cadherin, by overexpressing p120ctn, also blocks internalization and nuclear translocation of FGFR1. These data reveal that conjoint endocytosis and trafficking is a novel mechanism for the coregulation of E-cadherin and FGFR1 during cell signaling and morphogenesis.

N4WBP5A (Ndfip2), a Nedd4-interacting protein, localizes to multivesicular bodies and the Golgi, and has a potential role in protein trafficking.

N4WBP5A (Ndfip2) belongs to an evolutionarily conserved group of Nedd4-interacting proteins with two homologues in mammalian species. We have previously shown that N4WBP5A expression in Xenopus oocytes results in increased cell-surface expression of the epithelial sodium channel. N4WBPs are characterized by one or two amino terminal PPxY motifs and three transmembrane domains. Here we show that both PPxY motifs of N4WBP5A mediate interaction with WW domains of Nedd4 and that N4WBP5A can physically interact with the WW domains of several Nedd4-family proteins. N4WBP5A is ubiquitinated and ubiquitination does not significantly affect the turnover of N4WBP5A protein. Ubiquitination of N4WBP5A is enhanced by Nedd4 and Nedd4-2 expression. N4WBP5A localizes to the Golgi, vesicles associated with the Golgi complex and to multivesicular bodies. We show that the ectopic expression of N4WBP5A inhibits receptor-mediated endocytosis of labelled epidermal growth factor. N4WBP5A overexpression inhibits accumulation of EGF in large endocytic/lysosomal vesicles suggestive of a role for N4WBP5A in protein trafficking. We propose that N4WBP5A acts as an adaptor to recruit Nedd4 family ubiquitin-protein ligases to the protein trafficking machinery.

GAIP participates in budding of membrane carriers at the trans-Golgi network.

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans-Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.

The t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic and cytokine secretion.

Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response. TNFalpha plays a key role in inflammatory disease; yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that, in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.