We have investigated the interaction between GH (growth hormone) and GHR (GH receptor). We previously demonstrated that a truncated GHR that possesses a transmembrane domain but no cytoplasmic domain blocks receptor signalling. Based on this observation we investigated the impact of tethering the receptor's extracellular domain to the cell surface using a native lipid GPI (glycosylphosphatidylinositol) anchor. We also investigated the effect of tethering GH, the ligand itself, to the cell surface and demonstrated that tethering either the ecGHR (extracellular domain of GHR) or the ligand itself to the cell membrane via a GPI anchor greatly attenuates signalling. To elucidate the mechanism for this antagonist activity, we used confocal microscopy to examine the fluorescently modified ligand and receptor. GH–GPI was expressed on the cell surface and formed inactive receptor complexes that failed to internalize and blocked receptor activation. In conclusion, contrary to expectation, tethering an agonist to the cell surface can generate an inactive hormone receptor complex that fails to internalize.
cytokine antagonist; growth hormone; growth hormone receptor; glycosylphosphatidylinositol anchor; signal transduction; receptor trafficking; CMV, cytomegalovirus; GFP, green fluorescent protein; GH, growth hormone; GHR, growth hormone receptor; ecGHR, extracellular domain of GHR; GPI, glycosylphosphatidylinositol; HEK, human embryonic kidney; LHRE, lactogenic hormone response element; LL, long linker; PE, phycoerythrin; RFP, red fluorescent protein; RL, Renilla luciferase; wtGH, wild-type GH
Fibroblast Growth Factor (FGF)-10 promotes the proliferation and survival of murine hepatoblasts during early stages of hepatogenesis through a Wnt-β-catenin dependent pathway. To determine the mechanism by which this occurs, we expanded primary culture of hepatoblasts enriched for progenitor markers CD133 and CD49f from embryonic day (E) 12.5 fetal liver and an established tumor initiating stem cell line from Mat1a−/− livers in media conditioned with recombinant (r) FGF10 or rFGF7. FGF Receptor (R) activation resulted in the downstream activation of MAPK, PI3K-AKT, and β-catenin pathways, as well as cellular proliferation. Additionally, increased levels of nuclear β-catenin phosphorylated at Serine-552 in cultured primary hepatoblasts, Mat1a−/− cells, and also in ex vivo embryonic liver explants indicate AKT-dependent activation of β-catenin downstream of FGFR activation; conversely, the addition of AKT inhibitor Ly294002 completely abrogated β-catenin activation. FGFR activation-induced cell proliferation and survival were also inhibited by the compound ICG-001, a small molecule inhibitor of β-catenin-CREB Binding Protein (CBP) in hepatoblasts, further indicating a CBP-dependent regulatory mechanism of β-catenin activity.
Conclusion: FGF signaling regulates the proliferation and survival of embryonic and transformed progenitor cells in part through AKT-mediated activation of β-catenin and downstream interaction with the transcriptional co-activator CBP.
Sequential multispectral imaging is an acquisition technique that involves collecting images
of a target at different wavelengths, to compile a spectrum for each pixel. In surgical
applications it suffers from low illumination levels and motion artefacts. A three-channel
rigid endoscope system has been developed that allows simultaneous recording of stereoscopic
and multispectral images. Salient features on the tissue surface may be tracked during the
acquisition in the stereo cameras and, using multiple camera triangulation techniques, this
information used to align the multispectral images automatically even though the tissue or
camera is moving. This paper describes a detailed validation of the set-up in a controlled
experiment before presenting the first in vivo use of the device in a porcine
minimally invasive surgical procedure. Multispectral images of the large bowel were acquired
and used to extract the relative concentration of haemoglobin in the tissue despite motion due
to breathing during the acquisition. Using the stereoscopic information it was also possible to
overlay the multispectral information on the reconstructed 3D surface. This experiment
demonstrates the ability of this system for measuring blood perfusion changes in the tissue
during surgery and its potential use as a platform for other sequential imaging modalities.
(170.2150) Endoscopic imaging; (170.3010) Image reconstruction techniques; (170.6510) Spectroscopy, tissue diagnostics
ARHGAP22 is a RhoGAP protein comprising an N-terminal PH domain, a RhoGAP domain and a C-terminal coiled-coil domain. It has recently been identified as an Akt substrate that binds 14-3-3 proteins in response to treatment with growth factors involved in cell migration. We used a range of biophysical techniques to investigate the weak interaction between 14-3-3 and a truncated form of ARHGAP22 lacking the coiled-coil domain. This weak interaction could be stabilized by chemical cross-linking which we used to show that: a monomer of ARHGAP22 binds a dimer of 14-3-3; the ARHGAP22 PH domain is required for the 14-3-3 interaction; the RhoGAP domain is unlikely to participate in the interaction; Ser16 is the more important of two predicted 14-3-3 binding sites; and, phosphorylation of Ser16 may not be necessary for 14-3-3 interaction under the conditions we used. Small angle X-ray scattering and cross-link information were used to generate solution structures of the isolated proteins and of the cross-linked ARHGAP22:14-3-3 complex, showing that no major rearrangement occurs in either protein upon binding, and supporting a role for the PH domain and N-terminal peptide of ARHGAP22 in the 14-3-3 interaction. Small-angle X-ray scattering measurements of mixtures of ARHGAP22 and 14-3-3 were used to establish that the affinity of the interaction is ∼30 µM.
Insulin exerts many of its metabolic actions via the canonical phosphatidylinositide 3 kinase (PI3K)/Akt pathway, leading to phosphorylation and 14-3-3 binding of key metabolic targets. We previously identified a GTPase-activating protein (GAP) for Rac1 called RhoGAP22 as an insulin-responsive 14-3-3 binding protein. Insulin increased 14-3-3 binding to RhoGAP22 fourfold, and this effect was PI3K dependent. We identified two insulin-responsive 14-3-3 binding sites (pSer16 and pSer395) within RhoGAP22, and mutagenesis studies revealed a complex interplay between the phosphorylation at these two sites. Mutating Ser16 to alanine blocked 14-3-3 binding to RhoGAP22 in vivo, and phosphorylation at Ser16 was mediated by the kinase Akt. Overexpression of a mutant RhoGAP22 that was unable to bind 14-3-3 reduced cell motility in NIH-3T3 fibroblasts, and this effect was dependent on a functional GAP domain. Mutation of the catalytic arginine of the GAP domain of RhoGAP22 potentiated growth factor-stimulated Rac1 GTP loading. We propose that insulin and possibly growth factors such as platelet-derived growth factor may play a novel role in regulating cell migration and motility via the Akt-dependent phosphorylation of RhoGAP22, leading to modulation of Rac1 activity.
We report a case where a patient slipped from the operating table during positioning for a laparoscopic colectomy. This occurred due to failure of the Velcro holding the mattress to the operating table. On further inspection, although the amount of Velcro was satisfactory to hold the patient, it had failed due to a combination of its poor positioning, table design and the build up of ‘fluff’ on the Velcro from the use of incorrect cleaning products. The episode was reported as a critical incident and review lead to the implementation of changes across theatres to prevent such an episode happening again.
The use of multiple robots for performing complex tasks is becoming a common practice for many robot applications. When different operators are involved, effective cooperation with anticipated manoeuvres is important for seamless, synergistic control of all the end-effectors. In this paper, the concept of Collaborative Gaze Channelling (CGC) is presented for improved control of surgical robots for a shared task. Through eye tracking, the fixations of each operator are monitored and presented in a shared surgical workspace. CGC permits remote or physically separated collaborators to share their intention by visualising the eye gaze of their counterparts, and thus recovers, to a certain extent, the information of mutual intent that we rely upon in a vis-à-vis working setting. In this study, the efficiency of surgical manipulation with and without CGC for controlling a pair of bimanual surgical robots is evaluated by analysing the level of coordination of two independent operators. Fitts’ law is used to compare the quality of movement with or without CGC. A total of 40 subjects have been recruited for this study and the results show that the proposed CGC framework exhibits significant improvement (p < 0.05) on all the motion indices used for quality assessment. This study demonstrates that visual guidance is an implicit yet effective way of communication during collaborative tasks for robotic surgery. Detailed experimental validation results demonstrate the potential clinical value of the proposed CGC framework.
Robotic surgery; Human–robot interface; Eye tracking; Perceptual docking; Collaborative surgical task
Background: Akt plays a major role in insulin regulation of metabolism.
Results: Akt operates at 5–22% of its dynamic range. This lacks concordance with Akt substrate phosphorylation, GLUT4 translocation, and protein synthesis.
Conclusion: Akt is a demultiplexer that splits the insulin signal into discrete outputs.
Significance: This study provides better understanding of the Akt pathway and has implications for the role of Akt in diseases.
Akt plays a major role in insulin regulation of metabolism in muscle, fat, and liver. Here, we show that in 3T3-L1 adipocytes, Akt operates optimally over a limited dynamic range. This indicates that Akt is a highly sensitive amplification step in the pathway. With robust insulin stimulation, substantial changes in Akt phosphorylation using either pharmacologic or genetic manipulations had relatively little effect on Akt activity. By integrating these data we observed that half-maximal Akt activity was achieved at a threshold level of Akt phosphorylation corresponding to 5–22% of its full dynamic range. This behavior was also associated with lack of concordance or demultiplexing in the behavior of downstream components. Most notably, FoxO1 phosphorylation was more sensitive to insulin and did not exhibit a change in its rate of phosphorylation between 1 and 100 nm insulin compared with other substrates (AS160, TSC2, GSK3). Similar differences were observed between various insulin-regulated pathways such as GLUT4 translocation and protein synthesis. These data indicate that Akt itself is a major amplification switch in the insulin signaling pathway and that features of the pathway enable the insulin signal to be split or demultiplexed into discrete outputs. This has important implications for the role of this pathway in disease.
Adipocyte; Akt PKB; Insulin Resistance; Protein Synthesis; Signal Transduction; IRS1; PDGF; mTOR; Rapamycin
Visceral adipose tissue (VAT) is more closely linked to insulin resistance than subcutaneous adipose tissue (SAT). We conducted a quantitative analysis of the secretomes of VAT and SAT to identify differences in adipokine secretion that account for the adverse metabolic consequences of VAT.
RESEARCH DESIGN AND METHODS
We used lectin affinity chromatography followed by comparison of isotope-labeled amino acid incorporation rates to quantitate relative differences in the secretomes of VAT and SAT explants. Because adipose tissue is composed of multiple cell types, which may contribute to depot-specific differences in secretion, we isolated preadipocytes and microvascular endothelial cells (MVECs) and compared their secretomes to those from whole adipose tissue.
Although there were no discrete depot-specific differences in the secretomes from whole adipose tissue, preadipocytes, or MVECS, VAT exhibited an overall higher level of protein secretion than SAT. More proteins were secreted in twofold greater abundance from VAT explants compared with SAT explants (59% versus 21%), preadipocytes (68% versus 0%), and MVECs (62% versus 15%). The number of proteins in the whole adipose tissue secretome was greater than the sum of its cellular constituents. Finally, almost 50% of the adipose tissue secretome was composed of factors with a role in angiogenesis.
VAT has a higher secretory capacity than SAT, and this difference is an intrinsic feature of its cellular components. In view of the number of angiogenic factors in the adipose tissue secretome, we propose that VAT represents a more readily expandable tissue depot.
Adaptive phenotypic plasticity allows organisms to cope with environmental variability, and yet, despite its adaptive significance, phenotypic plasticity is neither ubiquitous nor infinite. In this review, we merge developmental and population genetic perspectives to explore costs and limits on the evolution of plasticity. Specifically, we focus on the role of modularity in developmental genetic networks as a mechanism underlying phenotypic plasticity, and apply to it lessons learned from population genetic theory on the interplay between relaxed selection and mutation accumulation. We argue that the environmental specificity of gene expression and the associated reduction in pleiotropic constraints drive a fundamental tradeoff between the range of plasticity that can be accommodated and mutation accumulation in alternative developmental networks. This tradeoff has broad implications for understanding the origin and maintenance of plasticity and may contribute to a better understanding of the role of plasticity in the origin, diversification, and loss of phenotypic diversity.
costs and limits; development; modularity; mutation accumulation; phenotypic plasticity; pleiotropy; relaxed selection
Introduction. Appropriate prevention of infection is a key area of research in natural orifice translumenal endoscopic surgery (NOTES), as identified by the Natural Orifice Surgery Consortium for Assessment and Research (NOSCAR). Methods. A review of the literature was conducted evaluating the evidence base for access orifice preparation/treatment in NOTES procedures in the context of infectious
complications. Recommendations based on the Oxford Centre for Evidence-Based Medicine guidelines were made.
Results. The most robust evidence includes several experimental randomised controlled trials assessing infectious complications in the transgastric approach to NOTES. Transvaginal procedures are long established for accessing the peritoneal cavity following disinfection with antiseptic. Only experimental case series for transcolonic and transvesical approaches are described. Conclusion. Grade C recommendation requiring no preoperative preparation can be made for the transgastric approach. Antiseptic irrigation is recommended for transvaginal (grade C) NOTES access, as is current practice. Further human trials need to be conducted to corroborate the current evidence base for transgastric closure. It is important that future trials are conducted in a methodologically robust fashion, with emphasis on clinical outcomes and standardisation of enterotomy closure and postoperative therapy.
The tendency for some individuals to partake in high-risk behaviors (eg, substance abuse, gambling, risky sexual activities) is a matter of great public health concern, yet the characteristics and neural bases of this vulnerability are largely unknown. Recent work shows that this susceptibility can be partially predicted by laboratory measures of reward seeking under risk, including the Balloon Analog Risk Task. Rats were trained to respond on two levers: one of which (the ‘add lever') increased the size of a potential food reward and a second (the ‘cash-out lever') that led to delivery of accrued reward. Crucially, each add-lever response was also associated with a risk that the trial would fail and no reward would be delivered. The relative probabilities that each add-lever press would lead to an addition food pellet or to trial failure (risk) were orthogonally varied. Rats exhibited a pattern of responding characteristic of incentive motivation and risk aversion, with a subset of rats showing traits of high-risk taking and/or suboptimal responding. Neural inactivation studies suggest that the orbitofrontal cortex supports greater reward seeking in the presence or absence of risk, whereas the medial prefrontal cortex is required for optimization of patterns of responding. These findings provide new information about the neural circuitry of decision making under risk and reveal new insights into the biological determinants of risk-taking behaviors that may be useful in developing biomarkers of vulnerability.
decision; frontal; addiction; reward; risk; cognition; Animal models; Cognition; Addiction & Substance Abuse; Behavioral Science; Depression, Unipolar/Bipolar; decision; frontal; reward; addiction; risk
Objectives. To describe conservative and surgical foot care in patients with RA in England and explore factors that predict the type of foot care received.
Methods. Use of podiatry and type of foot surgery were outcomes recorded in an inception cohort involving nine rheumatology centres that recruited patients with RA between 1986 and 1998 across England. Associations between patient-specific factors and service use were identified using univariate logistic regression analyses. The independence of these associations was then verified through multiple binary logistic regression modelling.
Results. Data were collected on 1237 patients with RA [66.9% females, mean (s.d.) age at disease onset = 54.36 (14.18) years, median DAS = 4.09 (1st quartile = 3.04, 3rd quartile = 5.26), median HAQ = 1 (0.50, 1.63)]. Interventions involving the feet in the cohort were low with only 364 (30%) out of 1218 receiving podiatry and 47 (4%) out of 1237 patients having surgery. At baseline, female gender, increasing age at onset, being RF positive and higher DAS scores were each independently associated with increased odds of seeing a podiatrist. Gender, age of onset and baseline DAS were independently associated with the odds of having foot surgery.
Conclusions. Despite the known high prevalence of foot pathologies in RA, only one-third of this cohort accessed podiatry. While older females were more likely to access podiatry care and younger patients surgery, the majority of the RA population did not access any foot care.
Rheumatoid arthritis; Foot; Podiatry; Surgery; Orthopaedic surgery; Foot care; Access to service
TOC summary: Introduction of exotic hosts can support unexpected emergence of unknown parasites.
Cercarial dermatitis (swimmer’s itch) is caused by the penetration of human skin by cercariae of schistosome parasites that develop in and are released from snail hosts. Cercarial dermatitis is frequently acquired in freshwater habitats, and less commonly in marine or estuarine waters. To investigate reports of a dermatitis outbreak in San Francisco Bay, California, we surveyed local snails for schistosome infections during 2005–2008. We found schistosomes only in Haminoea japonica, an Asian snail first reported in San Francisco Bay in 1999. Genetic markers place this schistosome within a large clade of avian schistosomes, but do not match any species for which there are genetic data. It is the second known schistosome species to cause dermatitis in western North American coastal waters; these species are transmitted by exotic snails. Introduction of exotic hosts can support unexpected emergence of an unknown parasite with serious medical or veterinary implications.
Schistosome; cercarial dermatitis; swimmer’s itch; marine snail; nonnative; Haminoea; San Francisco Bay; California; research
The UK Clinical Aptitude Test (UKCAT) was introduced in 2006 as an additional tool for the selection of medical students. It tests mental ability in four distinct domains (Quantitative Reasoning, Verbal Reasoning, Abstract Reasoning, and Decision Analysis), and the results are available to students and admissions panels in advance of the selection process. As yet the predictive validity of the test against course performance is largely unknown.
The study objective was to determine whether UKCAT scores predict performance during the first two years of the 5-year undergraduate medical course at Nottingham.
We studied a single cohort of students, who entered Nottingham Medical School in October 2007 and had taken the UKCAT. We used linear regression analysis to identify independent predictors of marks for different parts of the 2-year preclinical course.
Data were available for 204/260 (78%) of the entry cohort. The UKCAT total score had little predictive value. Quantitative Reasoning was a significant independent predictor of course marks in Theme A ('The Cell'), (p = 0.005), and Verbal Reasoning predicted Theme C ('The Community') (p < 0.001), but otherwise the effects were slight or non-existent.
This limited study from a single entry cohort at one medical school suggests that the predictive value of the UKCAT, particularly the total score, is low. Section scores may predict success in specific types of course assessment.
The ultimate test of validity will not be available for some years, when current cohorts of students graduate. However, if this test of mental ability does not predict preclinical performance, it is arguably less likely to predict the outcome in the clinical years. Further research from medical schools with different types of curriculum and assessment is needed, with longitudinal studies throughout the course.
Insulin triggers glucose uptake into muscle and adipose tissue by stimulating the translocation of the glucose transporter glut4 from intracellular vesicles to the plasma membrane (pm). insulin leads to a rapid increase in glut4 at the pm from ∼5% to 40–50%. this effect is time and dose-dependent, reaching a new steady state after 30 min of insulin stimulation. previous kinetic analyses in adipocytes has revealed that this is regulated by two mechanisms—increasing the amount of glut4 in the endosomal recycling system and increasing the exocytosis rate constant. fazakerley et al.1 focuses on GLUT4 kinetics in the L6 skeletal muscle cell line. Despite displaying a similar redistribution of GLUT4 to the cell surface with insulin to that seen in adipocytes, the mechanism for this effect in L6 cells was completely different. Insulin had a modest effect to increase the amount of GLUT4 in the recycling system with the dominant effect being on reduction of the endocytosis rate constant. Similar findings were observed with AMPK agonists. These studies indicate that different cell types are capable of achieving the same cell biological endpoint but using completely distinct mechanisms.
trafficking; kinetics; GLUT4; adipocytes; L6 muscle cell line; AMPK; endocytosis; insulin action; recycling
The pharmacological activity of different nuclear receptor (NR) ligands is reflected by their impact on receptor structure. Thus, we asked whether differential presentation of protein-protein interaction surfaces on the androgen receptor (AR), a surrogate assay of receptor conformation, could be used in a prospective manner to define the pharmacological activity of bound ligands. To this end, we identified over 150 proteins/polypeptides whose ability to interact with AR is influenced in a differential manner by ligand binding. The most discriminatory of these protein-AR interactions were used to develop a robust compound-profiling tool that enabled the separation of ligands into functionally distinguishable classes. Importantly, the ligands within each class exhibited similar pharmacological activities, a result that highlights the relationship between receptor structure and activity and provides direction for the discovery of novel AR modulators.
Objectives To determine whether the UK Clinical Aptitude Test (UKCAT) adds value to the selection process for school leaver applicants to medical and dental school, and in particular whether UKCAT can reduce the socioeconomic bias known to affect A levels.
Design Cohort study
Setting Applicants to 23 UK medical and dental schools in 2006.
Participants 9884 applicants who took the UKCAT in the UK and who achieved at least three passes at A level in their school leaving examinations (53% of all applicants).
Main outcome measures Independent predictors of obtaining at least AAB at A level and
UKCAT scores at or above the 30th centile for the cohort, for the subsections and the entire test.
Results Independent predictors of obtaining at least AAB at A level were white ethnicity (odds ratio 1.58, 95% confidence interval 1.41 to 1.77), professional or managerial background (1.39, 1.22 to 1.59), and independent or grammar schooling (2.26, 2.02 to 2.52) (all P<0.001). Independent predictors of achieving UKCAT scores at or above the 30th centile for the whole test were male sex (odd ratio 1.48, 1.32 to 1.66), white ethnicity (2.17, 1.94 to 2.43), professional or managerial background (1.34, 1.17 to 1.54), and independent or grammar schooling (1.91, 1.70 to 2.14) (all P<0.001). One major limitation of the study was that socioeconomic status was not volunteered by approximately 30% of the applicants. Those who withheld socioeconomic status data were significantly different from those who provided that information, which may have caused bias in the analysis.
Conclusions UKCAT was introduced with a high expectation of increasing the diversity and fairness in selection for UK medical and dental schools. This study of a major subgroup of applicants in the first year of operation suggests that it has an inherent favourable bias to men and students from a higher socioeconomic class or independent or grammar schools. However, it does provide a reasonable proxy for A levels in the selection process.
The insulin-stimulated trafficking of GLUT4 to the plasma membrane in muscle and fat tissue constitutes a central process in blood glucose homeostasis. The tethering, docking, and fusion of GLUT4 vesicles with the plasma membrane (PM) represent the most distal steps in this pathway and have been recently shown to be key targets of insulin action. However, it remains unclear how insulin influences these processes to promote the insertion of the glucose transporter into the PM. In this study we have identified a previously uncharacterized role for cortical actin in the distal trafficking of GLUT4. Using high-frequency total internal reflection fluorescence microscopy (TIRFM) imaging, we show that insulin increases actin polymerization near the PM and that disruption of this process inhibited GLUT4 exocytosis. Using TIRFM in combination with probes that could distinguish between vesicle transport and fusion, we found that defective actin remodeling was accompanied by normal insulin-regulated accumulation of GLUT4 vesicles close to the PM, but the final exocytotic fusion step was impaired. These data clearly resolve multiple steps of the final stages of GLUT4 trafficking, demonstrating a crucial role for actin in the final stage of this process.
At Nottingham University more than 95% of entrants to the traditional 5-year medical course are school leavers. Since 2003 we have admitted graduate entrants (GEM) to a shortened (4-year) course to 'widen access to students from more disadvantaged backgrounds'. We have recently shown that the GEM course widens academic and socio-demographic diversity of the medical student population. This study explored whether GEM students also bring psychological diversity and whether this could be beneficial.
We studied: a) 217 and 96 applicants to the Nottingham 5- and 4-year courses respectively, applying in the 2002-3 UCAS cycle, and, b) 246 school leavers starting the 5-year course and 39 graduate entrants to the 4-year course in October 2003. The psychological profiles of the two groups of applicants and two groups of entrants were compared using their performance in the Goldberg 'Big 5' Personality test, the Personal Qualities Assessment (PQA; measuring interpersonal traits and interpersonal values), and the Lovibond and Lovibond measure of depression, anxiety and stress. For the comparison of the Entrants we excluded the 33 school leavers and seven graduates who took the tests as Applicants.
Statistical analyses were undertaken using SPSS software (version 16.0).
Graduate applicants compared to school leaver applicants were significantly more conscientious, more confident, more self controlled, more communitarian in moral orientation and less anxious. Only one of these differences was preserved in the entrants with graduates being less anxious. However, the graduate entrants were significantly less empathetic and conscientious than the school leavers.
This study has shown that school leaver and graduate entrants to medical school differ in some psychological characteristics. However, if confirmed in other studies and if they were manifest in the extreme, not all the traits brought by graduates would be desirable for someone aiming for a medical career.
In L6 myotubes, redistribution of a hemagglutinin (HA) epitope-tagged GLUT4 (HA-GLUT4) to the cell surface occurs rapidly in response to insulin stimulation and AMP-activated protein kinase (AMPK) activation. We have examined whether these separate signaling pathways have a convergent mechanism that leads to GLUT4 mobilization and to changes in GLUT4 recycling. HA antibody uptake on GLUT4 in the basal steady state reached a final equilibrium level that was only 81% of the insulin-stimulated level. AMPK activators (5-aminoimidazole-4-carboxyamide ribonucleoside (AICAR) and A-769662) led to a similar level of antibody uptake to that found in insulin-stimulated cells. However, the combined responses to insulin stimulation and AMPK activation led to an antibody uptake level of ∼20% above the insulin level. Increases in antibody uptake due to insulin, but not AICAR or A-769662, treatment were reduced by both wortmannin and Akt inhibitor. The GLUT4 internalization rate constant in the basal steady state was very rapid (0.43 min−1) and was decreased during the steady-state responses to insulin (0.18 min−1), AICAR (0.16 min−1), and A-769662 (0.24 min−1). This study has revealed a nonconvergent mobilization of GLUT4 in response to activation of Akt and AMPK signaling. Furthermore, GLUT4 trafficking in L6 muscle cells is very reliant on regulated endocytosis for control of cell surface GLUT4 levels.
Cell/Endocytosis; Cell/Exocytosis; Diseases/Diabetes; Hormones/Insulin; Membrane/Recycling; Membrane/Trafficking; Phosphorylation/Kinases/Serine-Threonine; Transport/Glucose
Cocrystallization with a peptide, free-interface diffusion crystal chips and crystal dehydration were important in the production of diffraction-quality crystals of the Munc18c protein that helps to regulate membrane fusion.
The production of diffraction-quality crystals of Munc18c, a protein involved in regulating vesicular exocytosis in mammals, is reported. The diffraction resolution of Munc18c crystals was optimized by (i) cocrystallizing with a peptide fragment of the Munc18c functional binding partner syntaxin4, (ii) using nanolitre free-interface diffusion crystallization-screening chips and microlitre hanging-drop vapour diffusion and (iii) applying a post-crystallization dehydration treatment. Crystals belonging to the cubic space group P213, with unit-cell parameters a = b = c = 170.8 Å, α = β = γ = 90°, were generated that diffract to 3.7 Å resolution on a laboratory X-ray source.
Munc18c; syntaxin4; free-interface diffusion; dehydration