Applying the behaviour change wheel to develop a smartphone application 'stay-active' to increase physical activity in women with gestational diabetes.

BACKGROUND: Physical activity (PA) interventions are an important but underutilised component in the management of gestational diabetes mellitus (GDM). The challenge remains how to deliver cost effective PA interventions that have impact on individual behaviour. Digital technologies can support and promote PA remotely at scale. We describe the development of a behaviourally informed smartphone application (Stay-Active) for women attending an NHS GDM clinic. Stay-Active will support an existing motivational interviewing intervention to increase and maintain PA in this population. METHODS: The behaviour change wheel (BCW) eight step theoretical approach was used to design the application. It provided a systematic approach to understanding the target behaviour, identifying relevant intervention functions, and specifying intervention content. The target behaviour was to increase and maintain PA. To obtain a behavioural diagnosis, qualitative evidence was combined with focus groups on the barriers and facilitators to PA in women with GDM. The findings were mapped onto the Capability Opportunity Motivation-Behaviour (COM-B) model and Theoretical Domains Framework to identify what needs to change for the target behaviour and linked to appropriate intervention functions. Finally, behaviour changes techniques (BCT) and modes of delivery that are most likely to serve the intervention functions were selected. Current evidence, patient focus groups and input from key stakeholders informed Stay-Active's development. RESULTS: We found that psychological capability, reflective and automatic motivation, social and physical opportunity needed to change to increase PA in women with GDM. The four key intervention functions identified were Enablement, Education, Persuasion and Training. Stay-Active incorporates these four intervention functions delivering ten BCTs including: goal setting, credible source, self-monitoring, action planning, prompts and cues. The final design of Stay-Active delivers these BCTs via an educational resource centre, with goal setting and action planning features, personalised performance feedback and individualised promotional messages. CONCLUSION: The BCW has enabled the systematic and comprehensive development of Stay-Active to promote PA in women with GDM within an NHS Maternity service. The next phase is to conduct a trial to assess the feasibility and acceptability of a multi-component intervention that combines Stay-Active with PA Motivational Interviewing.

How point-of-care HbA1c testing changes the behaviour of people with diabetes and clinicians - a qualitative study.

AIM: To explore adults with diabetes and clinician views of point-of-care HbA1c testing. METHODS: Adults with diabetes and HbA1c ≥ 58 mmol/mol (7.5%) receiving HbA1c point-of-care testing in primary care were invited to individual interviews. Participants were interviewed twice, once prior to point-of-care testing and once after 6 months follow-up. Clinicians were interviewed once. A thematic framework based on an a priori framework was used to analyse the data. RESULTS: Fifteen participants (eight women, age range 30-70 years, two Asians, 13 white Europeans) were interviewed. They liked point-of-care testing and found the single appointment more convenient than usual care. Receiving the test result at the appointment helped some people understand how some lifestyle behaviours affected their control of diabetes and motivated them to change behaviours. Receiving an immediate test result reduced the anxiety some people experience when waiting for a result. People thought there was little value in using point-of-care testing for their annual review. Clinicians liked the point-of-care testing but expressed concerns about costs. CONCLUSIONS: This work suggests that several features of point-of-care testing may encourage behavioural change. It helped some people to link their HbA1c result to recent lifestyle behaviours, thereby motivating behavioural change and reinforcing healthy lifestyle choices.

Timings for HbA1c testing in people with diabetes are associated with incentive payments: an analysis of UK primary care data.

AIMS: Guidelines recommend testing HbA1c every 3-6 months in people with diabetes. In the United Kingdom (UK), primary care clinics are financially incentivized to monitor HbA1c at least annually and report proportions of patients meeting targets on 31 March. We explored the hypothesis that this reporting deadline may be associated with over-frequent or delayed HbA1c testing. METHODS: This analysis used HbA1c results from 100 000 people with diabetes during 2005-2014 in the Clinical Practice Research Datalink UK primary care database. Logistic regression was used to explore whether the four months prior to the deadline for quality reporting (December to March) or individual's previous HbA1c were aligned with retesting HbA1c within 60 days or > 1 year from the previous test, and identify other factors associated with the timing of HbA1c testing. RESULTS: Retesting HbA1c within 60 days or > 1 year was more common in December to March compared with other months of the year (odds ratio 1.06, 95% confidence interval 1.04-1.08 for retesting within 60 days). Those with higher HbA1c were more likely to have a repeat test within 60 days and less likely to have a repeat test > 1 year from the previous test. CONCLUSIONS: We have found that retesting HbA1c within 60 days and > 1 year from the previous test was more common in December to March compared with the other months of the year. This work suggests that both practice-centred administrative factors and patient-centred considerations may be influencing diabetes care in the UK.

Women's and healthcare providers' perceptions of long-term complications associated with hypertension and diabetes in pregnancy: a qualitative study.

OBJECTIVES: A diagnosis of hypertensive disorders during pregnancy (HDPs) or gestational diabetes mellitus (GDM) is highly predictive of women at increased risk of developing chronic hypertension, Type 2 diabetes, and cardiovascular disease. This study investigates perceptions of women and healthcare providers in rural India regarding these long-term risks. DESIGN: Qualitative study using modified grounded theory. SETTING: Two states in rural India: Haryana and Andhra Pradesh. POPULATION: Pregnant and postpartum women, community health workers (CHWs), primary care physicians, obstetricians, laboratory technicians, and healthcare officials. METHODS: In-depth interviews and focus group discussions explored: (1) priorities for high-risk pregnant women; (2) detection and management of HDPs and GDM; (3) postpartum management, and (4) knowledge of long-term sequelae of high-risk conditions. A thematic analysis was undertaken. RESULTS: Seven focus group discussions and 11 in-depth interviews (n = 71 participants) were performed. The key priority area for high-risk pregnant women was anaemia. Blood pressure measurement was routinely embedded in antenatal care; however, postpartum follow up and knowledge of the long-term complications were limited. GDM was not considered a common problem, although significant variations and challenges to GDM screening were identified. Knowledge of the long-term sequelae of GDM with regard to an increased risk of Type 2 diabetes and cardiovascular disease among doctors was minimal. CONCLUSIONS: There is a need for improved education, standardisation of testing and postpartum follow up of HDPs and GDM in rural Indian settings. FUNDING: SN is supported by an MRC Clinical Research Training Fellowship (MR/R017182/1). The George Institute for Global Health Global Women's Health programme provided financial support for the research assistant and fieldwork costs in India. TWEETABLE ABSTRACT: Improved education and postpartum care of women with hypertension and diabetes in pregnancy in rural India are needed to prevent long-term risks.

The antepartum stillbirth syndrome: risk factors and pregnancy conditions identified from the INTERGROWTH-21st Project.

OBJECTIVES: To identify risk factors for antepartum stillbirth, including fetal growth restriction, among women with well-dated pregnancies and access to antenatal care. DESIGN: Population-based, prospective, observational study. SETTING: Eight international urban populations. POPULATION: Pregnant women and their babies enrolled in the Newborn Cross-Sectional Study of the INTERGROWTH-21st Project. METHODS: Cox proportional hazard models were used to compare risks among antepartum stillborn and liveborn babies. MAIN OUTCOME MEASURES: Antepartum stillbirth was defined as any fetal death after 16 weeks' gestation before the onset of labour. RESULTS: Of 60 121 babies, 553 were stillborn (9.2 per 1000 births), of which 445 were antepartum deaths (7.4 per 1000 births). After adjustment for site, risk factors were low socio-economic status, hazard ratio (HR): 1.6 (95% CI, 1.2-2.1); single marital status, HR 2.0 (95% CI, 1.4-2.8); age ≥40 years, HR 2.2 (95% CI, 1.4-3.7); essential hypertension, HR 4.0 (95% CI, 2.7-5.9); HIV/AIDS, HR 4.3 (95% CI, 2.0-9.1); pre-eclampsia, HR 1.6 (95% CI, 1.1-3.8); multiple pregnancy, HR 3.3 (95% CI, 2.0-5.6); and antepartum haemorrhage, HR 3.3 (95% CI, 2.5-4.5). Birth weight <3rd centile was associated with antepartum stillbirth [HR, 4.6 (95% CI, 3.4-6.2)]. The greatest risk was seen in babies not suspected to have been growth restricted antenatally, with an HR of 5.0 (95% CI, 3.6-7.0). The population-attributable risk of antepartum death associated with small-for-gestational-age neonates diagnosed at birth was 11%. CONCLUSIONS: Antepartum stillbirth is a complex syndrome associated with several risk factors. Although small babies are at higher risk, current growth restriction detection strategies only modestly reduced the rate of stillbirth. TWEETABLE ABSTRACT: International stillbirth study finds individual risks poor predictors of death but combinations promising.

Short- and medium-term effects of light to moderate alcohol intake on glycaemic control in diabetes mellitus: a systematic review and meta-analysis of randomized trials.

BACKGROUND: People with diabetes are told that drinking alcohol may increase their risk of hypoglycaemia. AIMS: To report the effects of alcohol consumption on glycaemic control in people with diabetes mellitus. METHODS: Medline, EMBASE and the Cochrane Library databases were searched in 2015 to identify randomized trials that compared alcohol consumption with no alcohol use, reporting glycaemic control in people with diabetes. Data on blood glucose, HbA1c and numbers of hypoglycaemic episodes were pooled using random effects meta-analysis. RESULTS: Pooled data from nine short-term studies showed no difference in blood glucose concentrations between those who drank alcohol in doses of 16-80 g (median 20 g, 2.5 units) compared with those who did not drink alcohol at 0.5, 2, 4 and 24 h after alcohol consumption. Pooled data from five medium-term studies showed that there was no difference in blood glucose or HbA1c concentrations at the end of the study between those who drank 11-18 g alcohol/day (median 13 g/day, 1.5 units/day) for 4-104 weeks and those who did not. We found no evidence of a difference in number of hypoglycaemic episodes or in withdrawal rates between randomized groups. CONCLUSIONS: Studies to date have not provided evidence that drinking light to moderate amounts of alcohol, with or without a meal, affects any measure of glycaemic control in people with Type 2 diabetes. These results suggest that current advice that people with diabetes do not need to refrain from drinking moderate quantities of alcohol does not need to be changed; risks to those with Type 1 diabetes remain uncertain.

Estimating the effect of sulfonylurea on HbA1c in diabetes: a systematic review and meta-analysis.

AIMS/HYPOTHESIS: Sulfonylureas are widely prescribed glucose-lowering medications for diabetes, but the extent to which they improve glycaemia is poorly documented. This systematic review evaluates how sulfonylurea treatment affects glycaemic control. METHODS: Medline, EMBASE, the Cochrane Library and clinical trials registries were searched to identify double-blinded randomised controlled trials of fixed-dose sulfonylurea monotherapy or sulfonylurea added on to other glucose-lowering treatments. The primary outcome assessed was change in HbA1c, and secondary outcomes were adverse events, insulin dose and change in body weight. RESULTS: Thirty-one trials with a median duration of 16 weeks were included in the meta-analysis. Sulfonylurea monotherapy (nine trials) lowered HbA1c by 1.51% (17 mmol/mol) more than placebo (95% CI, 1.25, 1.78). Sulfonylureas added to oral diabetes treatment (four trials) lowered HbA1c by 1.62% (18 mmol/mol; 95% CI 1.0, 2.24) compared with the other treatment, and sulfonylurea added to insulin (17 trials) lowered HbA1c by 0.46% (6 mmol/mol; 95% CI 0.24, 0.69) and lowered insulin dose. Higher sulfonylurea doses did not reduce HbA1c more than lower doses. Sulfonylurea treatment resulted in more hypoglycaemic events (RR 2.41, 95% CI 1.41, 4.10) but did not significantly affect the number of other adverse events. Trial length, sulfonylurea type and duration of diabetes contributed to heterogeneity. CONCLUSIONS/INTERPRETATION: Sulfonylurea monotherapy lowered HbA1c level more than previously reported, and we found no evidence that increasing sulfonylurea doses resulted in lower HbA1c. HbA1c is a surrogate endpoint, and we were unable to examine long-term endpoints in these predominately short-term trials, but sulfonylureas appear to be associated with an increased risk of hypoglycaemic events.

Translating research into practice: the introduction of the INTERGROWTH-21st package of clinical standards, tools and guidelines into policies, programmes and services.

The INTERGROWTH-21(st) Project has generated a package of international clinical standards, tools and guidelines. It is now necessary to plan for the next phase of the project: the translation of the research findings into practice through its global dissemination. The plan is to pre-empt barriers to implementation by drawing from the published literature; gathering views and perspectives from policy makers, programmers and practitioners; incorporating input from local 'champions', and collecting and analysing data generated by a monitoring and evaluation system. Working at the global, regional, national and local levels will enable wide dissemination of the package, as well as increase the scope for adaptation and integration in diverse clinical contexts. We seek maximum uptake of the package in policies, guidelines and clinical practice to improve the quality of care offered to mothers and newborns. The strategy will also enhance our understanding of the effectiveness of different approaches to the translation of evidence into practice.

Cancer outcomes and all-cause mortality in adults allocated to metformin: systematic review and collaborative meta-analysis of randomised clinical trials.

AIMS/HYPOTHESIS: Observational studies suggest that metformin may reduce cancer risk by approximately one-third. We examined cancer outcomes and all-cause mortality in published randomised controlled trials (RCTs). METHODS: RCTs comparing metformin with active glucose-lowering therapy or placebo/usual care, with minimum 500 participants and 1-year follow-up, were identified by systematic review. Data on cancer incidence and all-cause mortality were obtained from publications or by contacting investigators. For two trials, cancer incidence data were not available; cancer mortality was used as a surrogate. Summary RRs, 95% CIs and I (2)statistics for heterogeneity were calculated by fixed effects meta-analysis. RESULTS: Of 4,039 abstracts identified, 94 publications described 14 eligible studies. RRs for cancer were available from 11 RCTs with 398 cancers during 51,681 person-years. RRs for all-cause mortality were available from 13 RCTs with 552 deaths during 66,447 person-years. Summary RRs for cancer outcomes in people randomised to metformin compared with any comparator were 1.02 (95% CI 0.82, 1.26) across all trials, 0.98 (95% CI 0.77, 1.23) in a subgroup analysis of active-comparator trials and 1.36 (95% CI 0.74, 2.49) in a subgroup analysis of placebo/usual care comparator trials. The summary RR for all-cause mortality was 0.94 (95% CI 0.79, 1.12) across all trials. CONCLUSIONS/INTERPRETATION: Meta-analysis of currently available RCT data does not support the hypothesis that metformin lowers cancer risk by one-third. Eligible trials also showed no significant effect of metformin on all-cause mortality. However, limitations include heterogeneous comparator types, absent cancer data from two trials, and short follow-up, especially for mortality.

Progesterone receptor isoform expression in response to in utero growth restriction in the fetal guinea pig brain.

Intra-uterine growth restriction (IUGR) is a significant in utero complication that can have profound effects on brain development including reduced myelination and deficits that can continue into adulthood. Progesterone increases oligodendrocyte proliferation and myelin expression, an action that may depend on the expression of progesterone receptor (PR) isoforms A (PRA) and B (PRB). The objective of this study was to determine the effect of IUGR on PR isoform expression in the brain of male and female fetuses and whether effects were associated with a reduction in myelination. We used a guinea pig model that involves selective reduction in maternal perfusion to the placenta at midgestation (35 days, term 70 days). This resulted in a significant reduction in body weight with marked sparing of brain weight. PRA, PRB and myelin basic protein (MBP) expression were measured in the brains of male and female growth-restricted and control fetuses at late gestation. MBP, as a measure of myelination, was found to decrease in association with IUGR in the CA1 hippocampal region with no change observed in the cortical white matter. There was a marked increase in PRA, PRB and total PR expression in the IUGR fetal brain. Control female fetuses demonstrated significantly higher PRA:PRB ratios than males; however, this sex difference was abolished with IUGR. These data suggest the central nervous system effects of clinical use of progesterone augmentation therapy in late pregnancy should be carefully evaluated. The overall upregulation of PR isoforms in association with IUGR suggests increased progesterone action and a possible neuroprotective mechanism.

Evidence for a dose effect of renin-angiotensin system inhibition on progression of microalbuminuria in Type 2 diabetes: a meta-analysis.

AIMS: Renin-angiotensin inhibitors in Type 2 diabetes and microalbuminuria reduce renal and cardiovascular risk, but evidence supporting use of maximal tolerated dose is unclear. We aimed to determine the extent of renin-angiotensin inhibitor dose-dependent effects from randomized trials carried out in a clinical setting. METHODS: In a meta-analysis of randomized clinical trials, alternate doses of angiotensin receptor blockers or angiotensin converting enzyme inhibitors in patients with Type 2 diabetes and microalbuminuria were compared. MEDLINE, EMBASE and the Cochrane Register of Controlled Trials were searched from January 2006 to August 2010. Trials prior to January 2006 were identified from a prior systematic review. Identified outcomes were albumin excretion rate, progression and regression of albuminuria and adverse events. RESULTS: Four trials including 1051 patients compared doses of angiotensin receptor blockers. No trials compared doses of angiotensin converting enzyme inhibitor. The percentage decline in albumin excretion rate from baseline was greater with higher doses (18% higher, 95% CI 8-28%), the regression to normoalbuminuria was greater (OR 1.66, 95% CI 1.22-2.27), with less progression to macroalbuminuria (OR 0.62, CI 0.38-1.02). Adverse events were fewer with lower-dose angiotensin receptor blockers (OR 1.32, 95% CI 0.90-1.92). CONCLUSIONS: Higher-dose compared with lower-dose angiotensin receptor blockers in Type 2 diabetes with microalbuminuria are associated with significantly reduced albumin excretion rate and increased regression to normoalbuminuria. Adverse events are more frequent, but not significantly so. There is potential for trials to determine clinical cardiovascular and renal outcomes at differing doses. Our findings support current recommendations to titrate renin-angiotensin inhibitors to maximum dose whilst considering risk of adverse side effects with higher doses.

Citizens advice in primary care: a qualitative study of the views and experiences of service users and staff.

OBJECTIVES: To examine the views and experiences of staff and users of Citizens Advice Bureau (CAB) services located in general practice, and to identify key factors perceived as contributing to the intervention's effectiveness. STUDY DESIGN: A qualitative study in an urban and rural primary care setting in the UK. METHODS: Semi-structured, face-to-face interviews (n = 22) with primary care and practice staff, CAB advisors and 12 service users. RESULTS: Key positive service features reported by all groups were: the confidential, non-stigmatizing and familiar environment of a general practitioner's (GP) surgery; the ability to make appointments and experienced advisor availability and continuity. Outcomes for service users were described as financial gain, managed debt, and beneficial social and mental health impacts. Perceived staff benefits were appropriate referral and better use of GP consultation time. CONCLUSION: Welfare advice in primary care has financial benefits and was perceived by participants to offer health and other benefits to patients and staff. However, while perceptions of gain from the intervention were evident, demonstration of measurable health improvement and well-being presents challenges. Further empirical work is needed in order to explore these complex cause-effect links and the cost-effectiveness of the intervention.

A new mouse immunoglobulin: IgG3.

A new subclass of mouse IgG for which we propose the name IgG3 has been shown to have a mol wt of 150,000 consistent with an L(2)H(2) structure, and is present in normal mouse serum at a concentration of 0.1-0.2 mg/ml. Its molecular weight, low carbohydrate content, glycopeptide analysis, and C-terminal analysis are all typical of the IgG class. The intact protein had a strong tendency to form noncovalent aggregates with itself which were dissociable in acid. Upon papain digestion an Fab fragment of 47,000 mole wt was generated along with an Fc fragment which was insoluble at neutral pH. As for its biology, the protein did not fix complement, was not cytophilic for gammaG2 receptor sites on macrophages, and did not show passive cutaneous anaphylaxis. It was very efficiently transported across the placenta so that its concentration in the newborn was twice that in the serum of the mother, compared to the concentration of IgG1 and IgG2 proteins which were only present at one-third the concentration of that found in the serum of the mother. The Fc fragment of this protein reacted with and was solubilized by the staphylococcal A protein which also precipitated the intact immunoglobulin. In addition, the myeloma protein which was the prototype for this gammaG subclass exhibited binding activity for levan which was localized to the Fab fragment.

A family of proteins with gamma-adaptin and VHS domains that facilitate trafficking between the trans-Golgi network and the vacuole/lysosome.

We have cloned and characterized members of a novel family of proteins, the GGAs. These proteins contain an NH(2)-terminal VHS domain, one or two coiled-coil domains, and a COOH-terminal domain homologous to the COOH-terminal "ear" domain of gamma-adaptin. However, unlike gamma-adaptin, the GGAs are not associated with clathrin-coated vesicles or with any of the components of the AP-1 complex. GGA1 and GGA2 are also not associated with each other, although they colocalize on perinuclear membranes. Immunogold EM shows that these membranes correspond to trans elements of the Golgi stack and the TGN. GST pulldown experiments indicate that the GGA COOH-terminal domains bind to a subset of the proteins that bind to the gamma-adaptin COOH-terminal domain. In yeast there are two GGA genes. Deleting both of these genes results in missorting of the vacuolar enzyme carboxypeptidase Y, and the cells also have a defective vacuolar morphology phenotype. These results indicate that the function of the GGAs is to facilitate the trafficking of proteins between the TGN and the vacuole, or its mammalian equivalent, the lysosome.

Impact of the Conformational Variability of Oligopeptides on the Computational Prediction of Their CD Spectra.

Although successful in the structural determination of ordered biomolecules, the spectroscopic investigation of oligopeptides in solution is hindered by their complex and rapidly changing conformational ensemble. The measured circular dichroism (CD) spectrum of an oligopeptide is an ensemble average over all microstates, severely limiting its interpretation, in contrast to ordered biomolecules. Spectral deconvolution methods to estimate the secondary structure contributions in the ensemble are still mostly based on databases of larger ordered proteins. Here, we establish how the interpretation of CD spectra of oligopeptides can be enhanced by the ability to compute the same observable from a set of atomic coordinates. Focusing on two representative oligopeptides featuring a known propensity toward an α-helical and ß-hairpin motif, respectively, we compare and cross-validate the structural information coming from deconvolution of the experimental CD spectra, sequence-based de novo structure prediction, and molecular dynamics simulations based on enhanced sampling methods. We find that small conformational variations can give rise to significant changes in the CD signals. While for the simpler conformational landscape of the α-helical peptide de novo structure prediction can already give reasonable agreement with the experiment, an extended ensemble of conformers needs to be considered for the ß-hairpin sequence.

Inhibition of neurosteroid synthesis increases asphyxia-induced brain injury in the late gestation fetal sheep.

Allopregnanolone (AP) is a potent GABAergic agonist that suppresses CNS activity, seizure threshold, and excitotoxicity in the adult brain. AP is present in the fetal sheep brain and increases rapidly after asphyxial insult due to increased 5alpha-reductase type-2 (5alphaR-2) expression. The aim of this study was to use finasteride to suppress fetal neurosteroid synthesis, and then determine the effect on brain injury, particularly in the hippocampus, of asphyxia induced in utero by brief occlusion of the umbilical cord. Catheters and an inflatable umbilical cord cuff were implanted in fetal sheep at approximately 125 days gestation. Five days later the fetuses received either finasteride (20 mg/kg/h) or vehicle (40% hydroxypropyl-beta-cyclodextrin) for 2 h. The umbilical cord was occluded (UCO) for 5 min at 30 min after starting the infusion. The fetal brain was obtained 24 h later for examination of activated caspase-3 expression as an index of apoptosis, and to measure AP content. Finasteride treatment alone significantly reduced AP content and increased the number of caspase-3 positive cells in the hippocampus, cerebellum, and the subcallosal bundle, indicating that AP modulates the normal rate of apoptosis in the developing brain. UCO in vehicle and finasteride-treated fetuses produced a similar, marked decrease in O2 saturation (5.8+/-0.6%), but after finasteride treatment UCO caused a significantly greater increase in the number of caspase-3 positive cells in the hippocampal cornu ammonis 3 (CA3) (57.3+/-1.6%) compared with the vehicle-treated fetuses. Thus, 5alpha-reduced steroids such as AP may be protective in reducing cell death following acute fetal asphyxia. Perturbation of normal fetal neurosteroid levels in late gestation (e.g. due to preterm birth, or maternal synthetic steroid treatment to induce fetal lung maturation) could adversely affect brain development and increase its vulnerability to injury.

The kinetics of mannose 6-phosphate receptor trafficking in the endocytic pathway in HEp-2 cells: the receptor enters and rapidly leaves multivesicular endosomes without accumulating in a prelysosomal compartment.

We have previously shown that in HEp-2 cells, multivesicular bodies (MVBs) processing internalized epidermal growth factor-epidermal growth factor receptor complexes mature and fuse directly with lysosomes in which the complexes are degraded. The MVBs do not fuse with a prelysosomal compartment enriched in mannose 6-phosphate receptor (M6PR) as has been described in other cell types. Here we show that the cation-independent M6PR does not become enriched in the endocytic pathway en route to the lysosome, but if a pulse of M6PR or an M6PR ligand, cathepsin D, is followed, a significant fraction of these proteins are routed from the trans-Golgi to MVBs. Accumulation of M6PR does not occur because when the ligand dissociates, the receptor rapidly leaves the MVB. At steady state, most M6PR are distributed within the trans-Golgi and trans-Golgi network and in vacuolar structures distributed in the peripheral cytoplasm. We suggest that these M6PR-rich vacuoles are on the return route from MVBs to the trans-Golgi network and that a separate stable M6PR-rich compartment equivalent to the late endosome/prelysosome stage does not exist on the endosome-lysosome pathway in these cells.

GGAs: roles of the different domains and comparison with AP-1 and clathrin.

We have previously identified a novel family of proteins called the GGAs (Golgi-localized, gamma-ear-containing, ADP-ribosylation factor-binding proteins). These proteins consist of an NH(2)-terminal VHS domain, followed by a GAT domain, a variable domain, and a gamma-adaptin ear homology domain. Studies from our own laboratory and others, making use of both yeast and mammals cells, indicate that the GGAs facilitate trafficking from the trans-Golgi network to endosomes. Here we have further investigated the function of the GGAs. We find that GGA-deficient yeast are not only defective in vacuolar protein sorting but they are also impaired in their ability to process alpha-factor. Using deletion mutants and chimeras, we show that the VHS domain is required for GGA function and that the VHS domain from Vps27p will not substitute for the GGA VHS domain. In contrast, the gamma-adaptin ear homology domain contributes to GGA function but is not absolutely required, and full function can be restored by replacing the GGA ear domain with the gamma-adaptin ear domain. Deleting the gamma-adaptin gene together with the two GGA genes exacerbates the phenotype in yeast, suggesting that they function on parallel pathways. In mammalian cells, the association of GGAs with the membrane is extremely unstable, which may account for their absence from purified clathrin-coated vesicles. Double- and triple-labeling immunofluorescence experiments indicate that the GGAs and AP-1 are associated with distinct populations of clathrin-coated vesicles budding from the trans-Golgi network. Together with results from other studies, our findings suggest that the GGAs act as monomeric adaptors, with the four domains involved in cargo selection, membrane localization, clathrin binding, and accessory protein recruitment.

Characterization of a fourth adaptor-related protein complex.

Adaptor protein complexes (APs) function as vesicle coat components in different membrane traffic pathways; however, there are a number of pathways for which there is still no candidate coat. To find novel coat components related to AP complexes, we have searched the expressed sequence tag database and have identified, cloned, and sequenced a new member of each of the four AP subunit families. We have shown by a combination of coimmunoprecipitation and yeast two-hybrid analysis that these four proteins (epsilon, beta4, mu4, and sigma4) are components of a novel adaptor-like heterotetrameric complex, which we are calling AP-4. Immunofluorescence reveals that AP-4 is localized to approximately 10-20 discrete dots in the perinuclear region of the cell. This pattern is disrupted by treating the cells with brefeldin A, indicating that, like other coat proteins, the association of AP-4 with membranes is regulated by the small GTPase ARF. Immunogold electron microscopy indicates that AP-4 is associated with nonclathrin-coated vesicles in the region of the trans-Golgi network. The mu4 subunit of the complex specifically interacts with a tyrosine-based sorting signal, indicating that, like the other three AP complexes, AP-4 is involved in the recognition and sorting of cargo proteins with tyrosine-based motifs. AP-4 is of relatively low abundance, but it is expressed ubiquitously, suggesting that it participates in a specialized trafficking pathway but one that is required in all cell types.