RESUMEN
Autophagy is a fundamental catabolic process that uses a unique post-translational modification, the conjugation of ATG8 protein to phosphatidylethanolamine (PE). ATG8 lipidation also occurs during non-canonical autophagy, a parallel pathway involving conjugation of ATG8 to single membranes (CASM) at endolysosomal compartments, with key functions in immunity, vision, and neurobiology. It is widely assumed that CASM involves the same conjugation of ATG8 to PE, but this has not been formally tested. Here, we discover that all ATG8s can also undergo alternative lipidation to phosphatidylserine (PS) during CASM, induced pharmacologically, by LC3-associated phagocytosis or influenza A virus infection, in mammalian cells. Importantly, ATG8-PS and ATG8-PE adducts are differentially delipidated by the ATG4 family and bear different cellular dynamics, indicating significant molecular distinctions. These results provide important insights into autophagy signaling, revealing an alternative form of the hallmark ATG8 lipidation event. Furthermore, ATG8-PS provides a specific "molecular signature" for the non-canonical autophagy pathway.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Autofagosomas/metabolismo , Familia de las Proteínas 8 Relacionadas con la Autofagia/metabolismo , Autofagia , Proteínas Asociadas a Microtúbulos/metabolismo , Fosfatidilserinas/metabolismo , Procesamiento Proteico-Postraduccional , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Autofagosomas/efectos de los fármacos , Autofagosomas/genética , Autofagosomas/patología , Familia de las Proteínas 8 Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/genética , Proteínas Relacionadas con la Autofagia/metabolismo , Cisteína Endopeptidasas/genética , Cisteína Endopeptidasas/metabolismo , Femenino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Virus de la Influenza A/patogenicidad , Macrólidos/farmacología , Masculino , Ratones , Proteínas Asociadas a Microtúbulos/genética , Monensina/farmacología , Fagocitosis , Fosfatidiletanolaminas/metabolismo , Células RAW 264.7 , Transducción de SeñalRESUMEN
Polycomb-repressive complex 1 (PRC1) has a central role in the regulation of heritable gene silencing during differentiation and development. PRC1 recruitment is generally attributed to interaction of the chromodomain of the core protein Polycomb with trimethyl histone H3K27 (H3K27me3), catalyzed by a second complex, PRC2. Unexpectedly we find that RING1B, the catalytic subunit of PRC1, and associated monoubiquitylation of histone H2A are targeted to closely overlapping sites in wild-type and PRC2-deficient mouse embryonic stem cells (mESCs), demonstrating an H3K27me3-independent pathway for recruitment of PRC1 activity. We show that this pathway is mediated by RYBP-PRC1, a complex comprising catalytic subunits of PRC1 and the protein RYBP. RYBP-PRC1 is recruited to target loci in mESCs and is also involved in Xist RNA-mediated silencing, the latter suggesting a wider role in Polycomb silencing. We discuss the implications of these findings for understanding recruitment and function of Polycomb repressors.
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Células Madre Embrionarias/metabolismo , Histonas/metabolismo , Proteínas Represoras/metabolismo , Animales , Línea Celular , Fibroblastos/metabolismo , Ratones , Complejo Represivo Polycomb 1 , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Proteínas Represoras/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
BACKGROUND: Older adults, including those with long-term conditions (LTCs), are vulnerable to social isolation. They are likely to have become more socially isolated during the Coronavirus Disease 2019 (COVID-19) pandemic, often due to advice to "shield" to protect them from infection. This places them at particular risk of depression and loneliness. There is a need for brief scalable psychosocial interventions to mitigate the psychological impacts of social isolation. Behavioural activation (BA) is a credible candidate intervention, but a trial is needed. METHODS AND FINDINGS: We undertook an external pilot parallel randomised trial (ISRCTN94091479) designed to test recruitment, retention and engagement with, and the acceptability and preliminary effects of the intervention. Participants aged ≥65 years with 2 or more LTCs were recruited in primary care and randomised by computer and with concealed allocation between June and October 2020. BA was offered to intervention participants (n = 47), and control participants received usual primary care (n = 49). Assessment of outcome was made blind to treatment allocation. The primary outcome was depression severity (measured using the Patient Health Questionnaire 9 (PHQ-9)). We also measured health-related quality of life (measured by the Short Form (SF)-12v2 mental component scale (MCS) and physical component scale (PCS)), anxiety (measured by the Generalised Anxiety Disorder 7 (GAD-7)), perceived social and emotional loneliness (measured by the De Jong Gierveld Scale: 11-item loneliness scale). Outcome was measured at 1 and 3 months. The mean age of participants was aged 74 years (standard deviation (SD) 5.5) and they were mostly White (n = 92, 95.8%), and approximately two-thirds of the sample were female (n = 59, 61.5%). Remote recruitment was possible, and 45/47 (95.7%) randomised to the intervention completed 1 or more sessions (median 6 sessions) out of 8. A total of 90 (93.8%) completed the 1-month follow-up, and 86 (89.6%) completed the 3-month follow-up, with similar rates for control (1 month: 45/49 and 3 months 44/49) and intervention (1 month: 45/47and 3 months: 42/47) follow-up. Between-group comparisons were made using a confidence interval (CI) approach, and by adjusting for the covariate of interest at baseline. At 1 month (the primary clinical outcome point), the median number of completed sessions for people receiving the BA intervention was 3, and almost all participants were still receiving the BA intervention. The between-group comparison for the primary clinical outcome at 1 month was an adjusted between-group mean difference of -0.50 PHQ-9 points (95% CI -2.01 to 1.01), but only a small number of participants had completed the intervention at this point. At 3 months, the PHQ-9 adjusted mean difference (AMD) was 0.19 (95% CI -1.36 to 1.75). When we examined loneliness, the adjusted between-group difference in the De Jong Gierveld Loneliness Scale at 1 month was 0.28 (95% CI -0.51 to 1.06) and at 3 months -0.87 (95% CI -1.56 to -0.18), suggesting evidence of benefit of the intervention at this time point. For anxiety, the GAD adjusted between-group difference at 1 month was 0.20 (-1.33, 1.73) and at 3 months 0.31 (-1.08, 1.70). For the SF-12 (physical component score), the adjusted between-group difference at 1 month was 0.34 (-4.17, 4.85) and at 3 months 0.11 (-4.46, 4.67). For the SF-12 (mental component score), the adjusted between-group difference at 1 month was 1.91 (-2.64, 5.15) and at 3 months 1.26 (-2.64, 5.15). Participants who withdrew had minimal depressive symptoms at entry. There were no adverse events. The Behavioural Activation in Social Isolation (BASIL) study had 2 main limitations. First, we found that the intervention was still being delivered at the prespecified primary outcome point, and this fed into the design of the main trial where a primary outcome of 3 months is now collected. Second, this was a pilot trial and was not designed to test between-group differences with high levels of statistical power. Type 2 errors are likely to have occurred, and a larger trial is now underway to test for robust effects and replicate signals of effectiveness in important secondary outcomes such as loneliness. CONCLUSIONS: In this study, we observed that BA is a credible intervention to mitigate the psychological impacts of COVID-19 isolation for older adults. We demonstrated that it is feasible to undertake a trial of BA. The intervention can be delivered remotely and at scale, but should be reserved for older adults with evidence of depressive symptoms. The significant reduction in loneliness is unlikely to be a chance finding, and replication will be explored in a fully powered randomised controlled trial (RCT). TRIAL REGISTRATION: ISRCTN94091479.
Asunto(s)
COVID-19/psicología , Depresión/prevención & control , Promoción de la Salud/métodos , Servicios de Salud para Ancianos , Soledad , Pandemias , Aislamiento Social , Anciano , Ejercicio Físico , Femenino , Conductas Relacionadas con la Salud , Humanos , Internet , Masculino , Proyectos Piloto , Evaluación de Programas y Proyectos de Salud , SARS-CoV-2 , Participación Social , Medicina Estatal , Reino UnidoRESUMEN
Epigenetic marks such as posttranslational histone modifications specify the functional states of underlying DNA sequences, though how they are maintained after their disruption during DNA replication remains a critical question. We identify the mammalian SWI/SNF-like protein SMARCAD1 as a key factor required for the re-establishment of repressive chromatin. The ATPase activity of SMARCAD1 is necessary for global deacetylation of histones H3/H4. In this way, SMARCAD1 promotes methylation of H3K9, the establishment of heterochromatin, and faithful chromosome segregation. SMARCAD1 associates with transcriptional repressors including KAP1, histone deacetylases HDAC1/2 and the histone methyltransferase G9a/GLP and modulates the interaction of HDAC1 and KAP1 with heterochromatin. SMARCAD1 directly interacts with PCNA, a central component of the replication machinery, and is recruited to sites of DNA replication. Our findings suggest that chromatin remodeling by SMARCAD1 ensures that silenced loci, such as pericentric heterochromatin, are correctly perpetuated.
Asunto(s)
Cromatina/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , Histonas/metabolismo , Acetilación , Adenosina Trifosfatasas/metabolismo , Animales , Western Blotting , Línea Celular , Células Cultivadas , Cromatina/genética , ADN Helicasas/genética , Regulación de la Expresión Génica , Células HEK293 , Células HeLa , Heterocromatina/genética , Heterocromatina/metabolismo , Histona Desacetilasa 1/metabolismo , Histona Metiltransferasas , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Metilación , Ratones , Células 3T3 NIH , Antígeno Nuclear de Célula en Proliferación/metabolismo , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Fase SRESUMEN
BACKGROUND: Older adults were more likely to be socially isolated during the COVID-19 pandemic, with increased risk of depression and loneliness. We aimed to investigate whether a behavioural activation intervention delivered via telephone could mitigate depression and loneliness in at-risk older people during the COVID-19 pandemic. METHODS: BASIL+ (Behavioural Activation in Social Isolation) was a pragmatic randomised controlled trial conducted among patients recruited from general practices in England and Wales, and was designed to assess the effectiveness of behavioural activation in mitigating depression and loneliness among older people during the COVID-19 pandemic. Eligible participants were aged 65 years and older, socially isolated, with a score of 5 or higher on the Patient Health Questionnaire-9 (PHQ-9), and had multiple long-term conditions. Participants were allocated in a 1:1 ratio to the intervention (behavioural activation) or control groups by use of simple randomisation without stratification. Behavioural activation was delivered by telephone; participants were offered up to eight weekly sessions with trained BASIL+ Support Workers. Behavioural activation was adapted to maintain social connections and encourage socially reinforcing activities. Participants in the control group received usual care with existing COVID-19 wellbeing resources. The primary clinical outcome was self-reported depression severity, assessed by the PHQ-9, at 3 months. Outcomes were assessed masked to allocation and analysis was by treatment allocation. This trial is registered with the ISRCTN registry (ISRCTN63034289). FINDINGS: Between Feb 8, 2021, and Feb 28, 2022, 449 eligible participants were identified and 435 from 26 general practices were recruited and randomly assigned (1:1) to the behavioural activation intervention (n=218) or to the control group (usual care with signposting; n=217). The mean age of participants was 75·7 years (SD 6·7); 270 (62·1%) of 435 participants were female, and 418 (96·1%) were White. Participants in the intervention group attended an average of 5·2 (SD 2·9) of eight remote behavioural activation sessions. The adjusted mean difference in PHQ-9 scores between the control and intervention groups at 3 months was -1·65 (95% CI -2·54 to -0·75, p=0·0003). No adverse events were reported that were attributable to the behavioural activation intervention. INTERPRETATION: Behavioural activation is an effective and potentially scalable intervention that can reduce symptoms of depression and emotional loneliness in at-risk groups in the short term. The findings of this trial add to the range of strategies to improve the mental health of older adults with multiple long-term conditions. These results can be helpful to policy makers beyond the pandemic in reducing the global burden of depression and addressing the health impacts of loneliness, particularly in at-risk groups. FUNDING: UK National Institute for Health and Care Research.
Asunto(s)
COVID-19 , Ocimum basilicum , Humanos , Femenino , Anciano , Masculino , Gales/epidemiología , Pandemias/prevención & control , COVID-19/epidemiología , COVID-19/prevención & control , Inglaterra/epidemiologíaRESUMEN
INTRODUCTION: Depression is a leading mental health problem worldwide. People with long-term conditions are at increased risk of experiencing depression. The COVID-19 pandemic led to strict social restrictions being imposed across the UK population. Social isolation can have negative consequences on the physical and mental wellbeing of older adults. In the Behavioural Activation in Social IsoLation (BASIL+) trial we will test whether a brief psychological intervention (based on Behavioural Activation), delivered remotely, can mitigate depression and loneliness in older adults with long-term conditions during isolation. METHODS: We will conduct a two-arm, parallel-group, randomised controlled trial across several research sites, to evaluate the clinical and cost-effectiveness of the BASIL+ intervention. Participants will be recruited via participating general practices across England and Wales. Participants must be aged ≥65 with two or more long-term conditions, or a condition that may indicate they are within a 'clinically extremely vulnerable' group in relation to COVID-19, and have scored ≥5 on the Patient Health Questionnaire (PHQ9), to be eligible for inclusion. Randomisation will be 1:1, stratified by research site. Intervention participants will receive up to eight intervention sessions delivered remotely by trained BASIL+ Support Workers and supported by a self-help booklet. Control participants will receive usual care, with additional signposting to reputable sources of self-help and information, including advice on keeping mentally and physically well. A qualitative process evaluation will also be undertaken to explore the acceptability of the BASIL+ intervention, as well as barriers and enablers to integrating the intervention into participants' existing health and care support, and the impact of the intervention on participants' mood and general wellbeing in the context of the COVID-19 restrictions. Semi-structured interviews will be conducted with intervention participants, participant's caregivers/supportive others and BASIL+ Support Workers. Outcome data will be collected at one, three, and 12 months post-randomisation. Clinical and cost-effectiveness will be evaluated. The primary outcome is depressive symptoms at the three-month follow up, measured by the PHQ9. Secondary outcomes include loneliness, social isolation, anxiety, quality of life, and a bespoke health services use questionnaire. DISCUSSION: This study is the first large-scale trial evaluating a brief Behavioural Activation intervention in this population, and builds upon the results of a successful external pilot trial. TRIAL REGISTRATION: ClinicalTrials.Gov identifier ISRCTN63034289, registered on 5th February 2021.
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COVID-19 , Ocimum basilicum , Anciano , Análisis Costo-Beneficio , Depresión/prevención & control , Humanos , Soledad , Pandemias , Calidad de Vida , Ensayos Clínicos Controlados Aleatorios como Asunto , Aislamiento SocialRESUMEN
BACKGROUND: Behavioural and cognitive interventions remain credible approaches in addressing loneliness and depression. There was a need to rapidly generate and assimilate trial-based data during COVID-19. OBJECTIVES: We undertook a parallel pilot RCT of behavioural activation (a brief behavioural intervention) for depression and loneliness (Behavioural Activation in Social Isolation, the BASIL-C19 trial ISRCTN94091479). We also assimilate these data in a living systematic review (PROSPERO CRD42021298788) of cognitive and/or behavioural interventions. METHODS: Participants (≥65 years) with long-term conditions were computer randomised to behavioural activation (n=47) versus care as usual (n=49). Primary outcome was PHQ-9. Secondary outcomes included loneliness (De Jong Scale). Data from the BASIL-C19 trial were included in a metanalysis of depression and loneliness. FINDINGS: The 12 months adjusted mean difference for PHQ-9 was -0.70 (95% CI -2.61 to 1.20) and for loneliness was -0.39 (95% CI -1.43 to 0.65).The BASIL-C19 living systematic review (12 trials) found short-term reductions in depression (standardised mean difference (SMD)=-0.31, 95% CI -0.51 to -0.11) and loneliness (SMD=-0.48, 95% CI -0.70 to -0.27). There were few long-term trials, but there was evidence of some benefit (loneliness SMD=-0.20, 95% CI -0.40 to -0.01; depression SMD=-0.20, 95% CI -0.47 to 0.07). DISCUSSION: We delivered a pilot trial of a behavioural intervention targeting loneliness and depression; achieving long-term follow-up. Living meta-analysis provides strong evidence of short-term benefit for loneliness and depression for cognitive and/or behavioural approaches. A fully powered BASIL trial is underway. CLINICAL IMPLICATIONS: Scalable behavioural and cognitive approaches should be considered as population-level strategies for depression and loneliness on the basis of a living systematic review.
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COVID-19 , Ocimum basilicum , Humanos , Depresión , Proyectos Piloto , Salud Pública , Ensayos Clínicos Controlados Aleatorios como Asunto , Aislamiento Social/psicología , Reino UnidoRESUMEN
Deimination is the post-translational conversion of arginine residues to citrulline. It has been implicated as a causative factor in autoimmune diseases such as multiple sclerosis and rheumatoid arthritis and more recently, as a marker of neurodegeneration. We have investigated the effect of the post-translational modification of arginine residues on the structure of recombinant ovine prion protein. Deiminated prion protein exhibited biophysical properties characteristic of the scrapie-associated conformer of prion protein viz. an increased beta-sheet secondary structure, congophilic structures indicative of amyloid and proteinase K resistance which could be templated onto normal unmodified prion protein. In the light of these findings, a potential role of post-translational modifications to prion protein in disease initiation or propagation is discussed.
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Priones/química , Naftalenosulfonatos de Anilina , Animales , Arginina/química , Arginina/metabolismo , Birrefringencia , Dicroismo Circular , Citrulina/química , Citrulina/metabolismo , Electroforesis en Gel de Poliacrilamida , Endopeptidasa K/metabolismo , Colorantes Fluorescentes , Conformación Proteica/efectos de los fármacos , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/química , Ovinos , Espectroscopía Infrarroja por Transformada de FourierRESUMEN
Prion diseases, or transmissible spongiform encephalopathies (TSEs) are typically characterised by CNS accumulation of PrP(Sc), an aberrant conformer of a normal cellular protein PrP(C). It is thought PrP(Sc) is itself infectious and the causative agent of such diseases. To date, no chemical modifications of PrP(Sc), or a sub-population thereof, have been reported. In this study we have investigated whether chemical modification of amino acids within PrP might cause this protein to exhibit aberrant properties and whether these properties can be propagated onto unmodified prion protein. Of particular interest were post-translational modifications resulting from physiological conditions shown to be associated with TSE disease. Here we report that in vitro exposure of recombinant PrP to conditions that imitate the end effects of oxidative/nitrative stress in TSE-infected mouse brains cause the protein to adopt many of the physical characteristics of PrP(Sc). Most interestingly, these properties could be propagated onto unmodified PrP protein when the modified protein was used as a template. These data suggest that post-translational modifications of PrP might contribute to the initiation and/or propagation of prion protein-associated plaques in vivo during prion disease, thereby high-lighting novel biochemical pathways as possible therapeutic targets for these conditions.
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Priones/química , Procesamiento Proteico-Postraduccional , Scrapie/metabolismo , Amiloide/química , Animales , Encéfalo/metabolismo , Encéfalo/patología , Endopeptidasa K/química , Cinética , Ratones , Nitrógeno/química , Estrés Oxidativo , Unión Proteica , Proteínas Recombinantes/química , Ovinos , Tirosina/análogos & derivados , Tirosina/químicaRESUMEN
High molecular weight material recovered from the culture filtrate of cell suspension cultured Pyrus communis was composed of 81% carbohydrate, 13% protein and 5% inorganic material. This material was separated into three fractions (one neutral (Fraction A) and two acidic (Fractions B and C)), by anion-exchange chromatography on DEAE-Sepharose CL-6B using a gradient of imidazole-HCl at pH 7.0. The monosaccharide and linkage composition of each fraction was determined after carboxyl reduction of uronic acid residues. From the combined results of the carbohydrate analyses, we conclude that the high molecular weight extracellular material consists of three major and two minor polysaccharides: a (fucogalacto)xyloglucan (36%) in the unbound neutral Fraction A; a type II arabinogalactan (as an arabinogalactan-protein, 29%) and an acidic (glucurono)arabinoxylan (2%) in Fraction B; and a galacturonan (33%) and a trace of heteromannan in Fraction C. The main amino acids in the proteins were Glx, Thr, Ser, Hyp/Pro and Gly. Further separation of Fraction B by solvent partition, SDS-PAGE and analysis by LC-MS/MS identified the major proteins as two chitanases, two thaumatin-like proteins, a beta-1,3-glucanase, an extracellular dermal glycoprotein and a pathogenesis-related protein.
Asunto(s)
Glicoproteínas/metabolismo , Polisacáridos/metabolismo , Pyrus/metabolismo , Células Cultivadas , Cromatografía Liquida , Electroforesis en Gel de Poliacrilamida , Glicoproteínas/química , Polisacáridos/química , Pyrus/citología , Espectrometría de Masas en TándemRESUMEN
PURPOSE: To test a theoretically and empirically based model linking potential protective resources (hardiness, coworker and supervisor support) to the outcomes of work stress and job satisfaction and replicating the relationship of work stress to job satisfaction while accounting for the potential influence of negative affectivity. DESIGN: A cross-sectional research design using survey data collected from two convenience samples. SETTING: Two worksites: (1) a high-tech company and (2) a government agency. SUBJECTS: High-tech employees (N = 310; response rate, 73.8%) and government agency employees (N = 745; response rate, 49.7%). MEASURES: The Dispositional Resilience Scale measured hardiness and the Positive and Negative Affect Schedule measured negative affectivity. Coworker and supervisor support were measured using the Coworker Support Scale and the Supervisor Support Scale, respectively. The Perceived Work Stress Scale measured work stress, and a single item from the Job Satisfaction Scale assessed overall job satisfaction. RESULTS: A multiple-group path analysis examined the proposed model. Similar patterns of association were found for both samples and suggested a more parsimonious model without the path from negative affectivity to job satisfaction. The model supports the protective nature of hardiness and support at work with regard to work stress and job satisfaction. CONCLUSION: Explanations of relationships depicted in the model, practical implications for reducing work stress and enhancing job satisfaction, limitations and future directions are discussed.
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Satisfacción en el Trabajo , Apoyo Social , Estrés Psicológico/psicología , Lugar de Trabajo/psicología , Adulto , Estudios Transversales , Femenino , Agencias Gubernamentales , Humanos , Masculino , Personalidad , TecnologíaRESUMEN
Matrix-assisted laser desorption/ionization (MALDI)-time-of-flight (TOF)-mass spectrometry (MS) is now routinely used in many laboratories for the rapid and sensitive identification of proteins by peptide mass fingerprinting (PMF). We describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by one- or two-dimensional gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and de-staining, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF MS of the tryptic peptides, and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method.
Asunto(s)
Mapeo Peptídico/métodos , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Bases de Datos de Proteínas , Humanos , Peso Molecular , Péptidos/química , Proteínas/química , Programas InformáticosRESUMEN
PURPOSE: To investigate the reliability and validity of a single-item overall job satisfaction measure. METHODS: Public agency employees (n = 745) were surveyed regarding job satisfaction, work, personality, and health variables. The single-item measure underwent the following analyses: correction for attenuation formula to estimate minimum reliability; correlations with multiple-item job satisfaction, work, personality, and health measures to determine concurrent validity, construct validity, and specific relevance to health promotion; and logistic regression to determine the predictability of turnover intention. RESULTS: For the single-item measure the minimum reliability estimate was high, all correlational tests for validity were significant, logistic regression indicated substantial predictability of turnover intention, and correlations with the health measures were significant. DISCUSSION: These results are consistent with other studies and support the psychometric properties of this single-item overall job satisfaction measure. Limitations of the study and its implications for worksite health promotion are discussed.
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Satisfacción en el Trabajo , Adulto , Estudios Transversales , Femenino , Humanos , Masculino , Persona de Mediana Edad , Estados UnidosRESUMEN
The GIMAPs (GTPases of the immunity-associated proteins) are a family of small GTPases expressed prominently in the immune systems of mammals and other vertebrates. In mammals, studies of mutant or genetically-modified rodents have indicated important roles for the GIMAP GTPases in the development and survival of lymphocytes. No clear picture has yet emerged, however, of the molecular mechanisms by which they perform their function(s). Using biotin tag-affinity purification we identified a major, and highly specific, interaction between the human cytosolic family member GIMAP6 and GABARAPL2, one of the mammalian homologues of the yeast autophagy protein Atg8. Chemical cross-linking studies performed on Jurkat T cells, which express both GIMAP6 and GABARAPL2 endogenously, indicated that the two proteins in these cells readily associate with one another in the cytosol under normal conditions. The GIMAP6-GABARAPL2 interaction was disrupted by deletion of the last 10 amino acids of GIMAP6. The N-terminal region of GIMAP6, however, which includes a putative Atg8-family interacting motif, was not required. Over-expression of GIMAP6 resulted in increased levels of endogenous GABARAPL2 in cells. After culture of cells in starvation medium, GIMAP6 was found to localise in punctate structures with both GABARAPL2 and the autophagosomal marker MAP1LC3B, indicating that GIMAP6 re-locates to autophagosomes on starvation. Consistent with this finding, we have demonstrated that starvation of Jurkat T cells results in the degradation of GIMAP6. Whilst these findings raise the possibility that the GIMAPs play roles in the regulation of autophagy, we have been unable to demonstrate an effect of GIMAP6 over-expression on autophagic flux.
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Proteínas Adaptadoras Transductoras de Señales/metabolismo , GTP Fosfohidrolasas/metabolismo , Sistema Inmunológico/metabolismo , Proteínas de Microfilamentos/metabolismo , Fagosomas/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Anticuerpos Monoclonales/inmunología , Autofagia , Familia de las Proteínas 8 Relacionadas con la Autofagia , Western Blotting , Ensayo de Inmunoadsorción Enzimática , GTP Fosfohidrolasas/antagonistas & inhibidores , GTP Fosfohidrolasas/genética , GTP Fosfohidrolasas/inmunología , Células HEK293 , Células Endoteliales de la Vena Umbilical Humana , Humanos , Técnicas para Inmunoenzimas , Inmunoprecipitación , Células Jurkat , Ratones , Proteínas de Microfilamentos/genética , Mutagénesis Sitio-Dirigida , Mutación/genética , ARN Mensajero/genética , ARN Interferente Pequeño/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
MALDI-TOF mass spectrometers are now commonplace and their relative ease of use means that most non-specialist labs can readily access the technology for the rapid and sensitive analysis of biomolecules. One of the main uses of MALDI-TOF-MS is in the identification of proteins, by peptide mass fingerprinting (PMF). Here we describe a simple protocol that can be performed in a standard biochemistry laboratory, whereby proteins separated by 1D or 2D gel electrophoresis can be identified at femtomole levels. The procedure involves excision of the spot or band from the gel, washing and destaining, reduction and alkylation, in-gel trypsin digestion, MALDI-TOF-MS of the tryptic peptides and database searching of the PMF data. Up to 96 protein samples can easily be manually processed at one time by this method.
Asunto(s)
Proteínas/análisis , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Alquilación , Bases de Datos de Proteínas , Electroforesis en Gel Bidimensional , Laboratorios , Límite de Detección , Fragmentos de Péptidos/análisis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/metabolismo , Mapeo Peptídico , Proteínas/química , Proteínas/aislamiento & purificación , Proteínas/metabolismo , Tinción con Nitrato de Plata , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/instrumentación , Tripsina/metabolismoRESUMEN
Ino80 is an ATP-dependent nucleosome-remodeling enzyme involved in transcription, replication, and the DNA damage response. Here, we characterize the fission yeast Ino80 and find that it is essential for cell viability. We show that the Ino80 complex from fission yeast mediates ATP-dependent nucleosome remodeling in vitro. The purification of the Ino80-associated complex identified a highly conserved complex and the presence of a novel zinc finger protein with similarities to the mammalian transcriptional regulator Yin Yang 1 (YY1) and other members of the GLI-Krüppel family of proteins. Deletion of this Iec1 protein or the Ino80 complex subunit arp8, ies6, or ies2 causes defects in DNA damage repair, the response to replication stress, and nucleotide metabolism. We show that Iec1 is important for the correct expression of genes involved in nucleotide metabolism, including the ribonucleotide reductase subunit cdc22 and phosphate- and adenine-responsive genes. We find that Ino80 is recruited to a large number of promoter regions on phosphate starvation, including those of phosphate- and adenine-responsive genes that depend on Iec1 for correct expression. Iec1 is required for the binding of Ino80 to target genes and subsequent histone loss at the promoter and throughout the body of these genes on phosphate starvation. This suggests that the Iec1-Ino80 complex promotes transcription through nucleosome eviction.
Asunto(s)
Nucleosomas/metabolismo , Nucleótidos/metabolismo , Fosfatos/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Factores de Transcripción/metabolismo , Dedos de Zinc , Adenina/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Regulación Fúngica de la Expresión Génica , Análisis por Micromatrices , Datos de Secuencia Molecular , Proteínas de Schizosaccharomyces pombe/genética , Factores de Transcripción/genéticaRESUMEN
A cellulose/xyloglucan framework is considered to form the basis for the mechanical properties of primary plant cell walls and hence to have a major influence on the biomechanical properties of growing, fleshy plant tissues. In this study, structural variants of xyloglucan have been investigated as components of composites with bacterial cellulose as a simplified model for the cellulose/xyloglucan framework of primary plant cell walls. Evidence for molecular binding to cellulose with perturbation of cellulose crystallinity was found for all xyloglucan types. High molecular mass samples gave homogeneous centimeter-scale composites with extensive cross-linking of cellulose with xyloglucan. Lower molecular mass xyloglucans gave heterogeneous composites having a range of microscopic structures with little, if any, cross-linking. Xyloglucans with reduced levels of galactose substitution had evidence of self-association, competitive with cellulose binding. At comparable molecular mass, fucose substitution resulted in a modest promotion of microscopic features characteristic of primary cell walls. Taken together, the data are evidence that galactose substitution of the xyloglucan core structure is a major determinant of cellulose composite formation and properties, with additional fucose substitution acting as a secondary modulator. These conclusions are consistent with reported structural and mechanical properties of Arabidopsis mutants lacking specific fucose and/or galactose residues.