RESUMO
INTRODUCTION: A comfortable treatment position in radiotherapy may promote patient stability and improve outcomes such as accuracy. The aim of this study was to identify, prioritise and determine the feasibility of delivery of intervention components as part of a radiotherapy comfort intervention package. METHODS: Prior research, consisting of a systematic review and qualitative interviews with patient and therapeutic radiographers, was triangulated and 15 intervention components developed. An online nominal group technique consensus meeting with 7 patients who received radiotherapy exceeding 10 min for one of three anatomical cancer sites and 3 therapeutic radiographers (TRs) participated. Four activities were undertaken: 1) discussion of comfort intervention components; 2) initial vote; 3) prioritisation of intervention components; and 4) discussion of feasibility in radiotherapy and were analysed using established quantitative and qualitative methods. RESULTS: One intervention component was added from initial discussions to the 15 pre-determined components being discussed. 11 components were recommended as 'accepted' (n = 5) or 'accepted with caution' (n = 6) to proceed to development. The highest scoring intervention components were 'Compassionate & empathetic communication training for TRs' and 'Tailored information, e.g., TRs provide the required information only as part of preparation for treatment'. Anther that followed closely was 'Adjustments & supports provided for arms or legs during treatment by TRs'. Those 'accepted with caution' included 'Soft pads/mattress under the body to alleviate body discomfort managed by TRs'. Qualitative analysis highlighted concerns over the radiation environment and emphasised the importance of resources such as equipment, training, and time. CONCLUSION: The recommended comfort interventions have potential to improve patient comfort during radiotherapy and should be considered to incorporate into positioning and immobilisation guidelines. However, specific intervention strategies to address these components will need to be developed and robustly evaluated. IMPLICATIONS FOR PRACTICE: Comfort interventions might help patients relax and stay still during treatment, which could improve treatment accuracy and efficacy. Introducing these comfort interventions in practice have potential to lead to a more positive patient experience and improved overall quality of care during radiotherapy.
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Conforto do Paciente , Radioterapia , Humanos , ConsensoRESUMO
With an increasingly complex healthcare environment, ethics is becoming a more critical part of medical education. We aimed to explore European paediatric trainees' experiences of facing ethical dilemmas and their medical ethics education whilst assessing their perceptions of ethical dilemmas in current and future practice. The Young Sections of the European Academy of Paediatrics and European Society of Paediatric and Neonatal Intensive Care developed an explorative online survey covering demographics, ethical dilemmas faced and ethics training. The survey was made available in nine languages from November 2019 to January 2020 via newsletters and social media. Participants (n = 253) from 22 countries, predominantly female (82%) and residents (70%), with a median age of 29-years, completed the survey. The majority (58%) faced ethical dilemmas monthly or more frequently. Most ethics training was received by ethics lectures in medical school (81%) and on the job (60%). A disagreement between the healthcare team and patient/family was the most frequently faced moral dilemma (45%); the second was withholding/withdrawing life-prolonging measures (33%). The latter was considered the most challenging dilemma to resolve (50%). Respondents reported that ethical issues are not sufficiently addressed during their training and wished for more case-based teaching. Many have been personally affected by moral dilemmas, especially regarding withholding/withdrawing life-prolonging measures, and often felt inadequately supported.Conclusion: Paediatric trainees face many moral issues in daily practice and consider that training about managing current and future ethical dilemmas should be improved, such as by the provision of a core European paediatric ethics curriculum. What is Known: ⢠Paediatric services are becoming more complex with an increase in ethical dilemmas asking for rigorous training in ethics. ⢠Ethics training is often lacking or covered poorly in both pre- and postgraduate medical education curricula. ⢠Existing ethics training for European paediatric trainees is haphazard and lacks standardisation. What is New: ⢠The PaEdiatric Residents and Fellows Ethics (PERFEct) survey provides insight into the European paediatric trainees' views regarding ethical dilemmas in their current and future practice. ⢠European paediatric trainees report a lack of ethics training during paediatric residency and fellowship. ⢠This study provides content suggestions for standardised medical ethics training for paediatric trainees in Europe.
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Educação Médica , Pediatria , Adulto , Criança , Currículo , Ética Médica , Feminino , Humanos , Recém-Nascido , Princípios MoraisRESUMO
OBJECTIVES: Describe independently living older peoples' views of oral-health, and their experiences of accessing dental care-services within community settings. METHODS: Dentistry and Oral-Sciences Source, CINAHL, MEDLINE and AMED databases were searched to 15 January 2020. Assessment of methodological quality was undertaken using the JBI Critical Appraisal Checklist. Extracted data underwent meta-aggregative synthesis; findings were assigned levels of credibility and categorised according to similarity. These categories were subsequently synthesised into themes. RESULTS: Five studies were identified and included within analysis. Forty-six findings were aggregated into 18 descriptive categories, which were synthesised into five themes. These themes were Aggregated oral health experience; Taking responsibility for individual oral health; Practical issues related to accessing dental services; Negotiation of cost associated with care and Role of the dental professional. CONCLUSIONS: Previous dental healthcare experiences influence older peoples' health perceptions and health-seeking behaviours. Dental service provision and the perceptions of dental professionals require adaptation to accommodate the needs of adults as they age. This includes provision of domiciliary services and nationally subsidised dental care.
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Atenção à Saúde , Saúde Bucal , Adulto , Assistência Odontológica , Humanos , Pesquisa QualitativaRESUMO
OBJECTIVES: The aim of this review was to search existing literature to identify comfort interventions that can be used to assist an adult patient to undergo complex radiotherapy requiring positional stability for periods greater than 10 min. The objectives of this review were to; 1) identify comfort interventions used for clinical procedures that involve sustained inactivity similar to radiotherapy; 2) define characteristics of comfort interventions for future practice; and 3) determine the effectiveness of identified comfort interventions. The Preferred Reporting Items for Systematic Reviews and meta-analyses statement and the Template-for-Intervention-Description-and Replication guide were used. KEY FINDINGS: The literature search was performed using PICO criteria with five databases (AMED, CINAHL EMBASE, MEDLINE, PsycINFO) identifying 5269 titles. After screening, 46 randomised controlled trials met the inclusion criteria. Thirteen interventions were reported and were grouped into four categories: Audio-visual, Psychological, Physical, and Other interventions (education/information and aromatherapy). The majority of aromatherapy, one audio-visual and one educational intervention were judged to be clinically significant for improving patient comfort based on anxiety outcome measures (effect size ≥ 0.4, mean change is greater than the Minimal-Important-Difference and low-risk-of-bias). Medium to large effect sizes were reported in many interventions where differences did not exceed the Minimal-Important-Difference for the measure. These interventions were deemed worthy of further investigation. CONCLUSION: Several interventions were identified that may improve comfort during radiotherapy assisting patients to sustain and endure the same position over time. This is crucial for the continual growth of complex radiotherapy requiring a need for comfort to ensure stability for targeted treatment. IMPLICATIONS FOR PRACTICE: Further investigation of comfort interventions is warranted, including tailoring interventions to patient choice and determining if multiple interventions can be used concurrently to improve effectiveness.
Assuntos
Ansiedade , Conforto do Paciente , Adulto , Ansiedade/prevenção & controle , Humanos , Cooperação do PacienteAssuntos
Enfermagem de Cuidados Críticos , Pesquisa em Enfermagem , Enfermagem Pediátrica , Publicações Periódicas como Assunto , Editoração/estatística & dados numéricos , Austrália , Criança , Pré-Escolar , Humanos , Lactente , Recém-Nascido , Unidades de Terapia Intensiva Neonatal , Unidades de Terapia Intensiva Pediátrica , Nova ZelândiaRESUMO
UNLABELLED: Theme: Leadership, management, nursing education. INTRODUCTION: Family-centered care (FCC) in NICUs is related to staff culture and the organization of the unit. AIM: To describe the organizational characteristics and services for families in Italian NICUs. METHODS: This survey involved 105 NICUs in Italy. The Italian version of the 'FCC in the NICUs: A Self-Assessment Inventory' developed by the Institute for FCC was sent to the nurse managers in January 2015. RESULTS: Forty-seven NICUs answered (49%). The means of the NICU characteristics are number of beds: 20; newborns discharged/year: 331, of which very low birth weight infant: 68; unit's rooms: 3.7). The total mean score of the 10 areas explored by questionnaire was 2.6 (on 5 points Likert scale) for the 'status' and of 2.3 (on 3 points scale) for priority for change. CONCLUSION: The results show an organizational lack, but also the consciousness of the need of change. Sharing new organizational strategies could be an important issue for the future.
Assuntos
Terapia Intensiva Neonatal/métodos , Assistência Centrada no Paciente/estatística & dados numéricos , Qualidade da Assistência à Saúde/normas , Família , Feminino , Humanos , Recém-Nascido , Terapia Intensiva Neonatal/organização & administração , Terapia Intensiva Neonatal/estatística & dados numéricos , Itália , Liderança , Masculino , Enfermeiros Administradores/normas , Inovação Organizacional , Assistência Centrada no Paciente/normas , Qualidade da Assistência à Saúde/estatística & dados numéricos , Inquéritos e QuestionáriosRESUMO
Convergent evidence has emerged over the past decade to highlight the role of manganese as a key player in the defenses that many organisms are building to fight oxidative stress. For redox processes replacing iron by manganese requires adaptation at different levels. The aim of this perspective is to summarize recent important observations and to analyze the implications of the present knowledge for resolving future issues.
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Manganês , Estresse Oxidativo/efeitos dos fármacos , Homeostase , Humanos , FerroRESUMO
The heterodinuclear complexes [Fe(III)Mn(II)(L-Bn)(µ-OAc)(2)](ClO(4))(2) (1) and [Fe(II)Mn(II)(L-Bn)(µ-OAc)(2)](ClO(4)) (2) with the unsymmetrical dinucleating ligand HL-Bn {[2-bis[(2-pyridylmethyl)aminomethyl]]-6-[benzyl-2-(pyridylmethyl)aminomethyl]-4-methylphenol} were synthesized and characterized as biologically relevant models of the new Fe/Mn class of nonheme enzymes. Crystallographic studies have been completed on compound 1 and reveal an Fe(III)Mn(II)µ-phenoxobis(µ-carboxylato) core. A single location of the Fe(III) ion in 1 and of the Fe(II) ion in 2 was demonstrated by Mössbauer and (1)H NMR spectroscopies, respectively. An investigation of the temperature dependence of the magnetic susceptibility of 1 revealed a moderate antiferromagnetic interaction (J = 20 cm(-1)) between the high-spin Fe(III) and Mn(II) ions in 1, which was confirmed by Mössbauer and electron paramagnetic resonance (EPR) studies. The electrochemical properties of complex 1 are described. A quasireversible electron transfer at -40 mV versus Ag/AgCl corresponding to the Fe(III)Mn(II)/Fe(II)Mn(II) couple appears in the cyclic voltammogram. Thorough investigations of the Mössbauer and EPR signatures of complex 2 were performed. The analysis allowed evidencing of a weak antiferromagnetic interaction (J = 5.72 cm(-1)) within the Fe(II)Mn(II) pair consistent with that deduced from magnetic susceptibility measurements (J = 6.8 cm(-1)). Owing to the similar value of the Fe(II) zero-field splitting (D(Fe) = 3.55 cm(-1)), the usual treatment within the strong exchange limit was precluded and a full analysis of the electronic structure of the ground state of complex 2 was developed. This situation is reminiscent of that found in many diiron and iron-manganese enzyme active sites.
Assuntos
Complexos de Coordenação/química , Compostos Férricos/química , Compostos Ferrosos/química , Manganês/química , Cresóis/química , Cristalografia por Raios X , Ferro/química , Ligantes , Modelos Moleculares , Piridinas/químicaRESUMO
BACKGROUND: Transition from paediatric to adult healthcare has received little attention in the Netherlands. This study aimed to: (i) map experiences with the transfer to adult care of young adults with chronic conditions; and (ii) identify recommendations for transitional care of young adults, their parents and healthcare providers. METHODS: Semi-structured interviews with 24 young adults after transfer (aged 15-22 years; diagnosed with haemophilia, diabetes mellitus, spina bifida, congenital heart disorders, cystic fibrosis, juvenile rheumatoid arthritis or sickle cell disease), 24 parents and 17 healthcare providers. Thematic analysis was performed. RESULTS: Only the haemophilia department offered a structured transition programme, most patients had not been prepared for transition. Experiences and views of patients, parents and professionals mainly overlapped and were condensed into four core themes. Two are related to moving to adult care: (1) 'leaving paediatric care is a logical step'. Leaving familiar surroundings was harder for parents than for young adults who displayed a positive 'wait-and-see' attitude; and (2) 'transition is complicated by cultural gaps between paediatric and adult services'. Young adults and parents felt lost after transfer and recommended their peers 'to be alert and involved'. Providers also recognized the cultural chasm between both services and worried about non-compliance, lost to follow-up and lack of independence. Two other themes indicated priorities for improvement: (3) 'better patient and parent preparation' for differences between healthcare settings and for new roles and responsibilities with respect to self-management; and (4) 'more collaboration and personal links' between paediatric and adult care providers. CONCLUSIONS: Action is required to cross the chasm between paediatric and adult-oriented care. Preparation for transition should start early and focus on strengthening adolescents' independency without undermining parental involvement. Building bridges between services, gaining trust and investing in new personal relations is a challenge for all parties involved: transition is about responding and bonding.
Assuntos
Atenção à Saúde/métodos , Relações Pais-Filho , Poder Familiar , Qualidade da Assistência à Saúde/normas , Transição para Assistência do Adulto/organização & administração , Adolescente , Fatores Etários , Doença Crônica , Feminino , Acessibilidade aos Serviços de Saúde , Necessidades e Demandas de Serviços de Saúde , Humanos , Masculino , Pediatria/organização & administração , Desenvolvimento de Programas , Avaliação de Programas e Projetos de Saúde , Pesquisa Qualitativa , Qualidade da Assistência à Saúde/estatística & dados numéricos , Fatores de Tempo , Adulto JovemRESUMO
BACKGROUND: Since 1999 a multidisciplinary follow-up programme for parents and children with major anatomical congenital anomalies is in place in our hospital, run by a dedicated team. The aim of the present study was to evaluate the services of this team from a parental perspective. METHODS: Parents completed a questionnaire including open and closed questions about satisfaction with the various professional disciplines involved in the follow-up, statements on usefulness of the follow-up services and suggestions for improvement. RESULTS: Four hundred and sixty-nine surveys were sent out, of which 71% were returned. Non-responding parents included significantly more parents of non-Dutch origin (P= 0.038) and parents who never responded to invitations for follow-up examinations (P < 0.001). Parental satisfaction differed for the various disciplines. Eighty per cent of the parents were (very) satisfied with the social worker, compared with 92% with nurses. More than half of the parents agreed that the follow-up services give peace of mind. Almost a quarter of parents, however, considered the follow-up services as redundant. The children of these parents had significantly shorter intensive care unit stay (P= 0.02), were older at the time of the questionnaire (P= 0.04), of higher socio-economic status (P= 0.001) and less likely to be of non-Dutch origin (P= 0.008). Sixty-one per cent of the parents had contacted the 24-h helpline. Ninety per cent of the parents were satisfied with the intensive care unit, almost 80% with the general ward. CONCLUSION: Overall, parents were satisfied with the services of the follow-up team. Some parents, however, saw room for improvement related to better communication, recognizability of the team and better planning and organization.
Assuntos
Assistência ao Convalescente/normas , Serviços de Saúde da Criança/normas , Anormalidades Congênitas/reabilitação , Comportamento do Consumidor , Pais/psicologia , Criança , Pesquisas sobre Atenção à Saúde , Humanos , Países Baixos , Inquéritos e QuestionáriosRESUMO
In Bacillus subtilis, the transcription factor PerR is an iron dependant sensor of H(2)O(2). The sensing mechanism relies on a selective metal catalysed oxidation of two histidine residues of the regulatory site. Here we present the first crystal structure of the active PerR protein in complex with a Mn(2+) ion. In addition, X-ray absorption spectroscopy experiments were performed to characterize the corresponding iron form of the protein. Both studies reveal a penta-coordinate arrangement of the regulatory site that involves three histidines and two aspartates. One of the histidine ligand belongs to the N-terminal domain. Binding of this residue to the regulatory metal allows the protein to adopt a caliper-like conformation suited to DNA binding. Since this histidine is conserved in all PerR and a vast majority of Fur proteins, it is likely that the allosteric switch induced by the regulatory metal is general for this family of metalloregulators.
Assuntos
Bacillus subtilis/metabolismo , Proteínas de Bactérias/metabolismo , Peróxido de Hidrogênio/metabolismo , Proteínas Repressoras/metabolismo , Bacillus subtilis/genética , Proteínas de Bactérias/genética , Sítios de Ligação , Regulação Bacteriana da Expressão Gênica , Magnésio/metabolismo , Modelos Moleculares , Estrutura Quaternária de Proteína , Proteínas Repressoras/genética , Análise Espectral , Raios XRESUMO
BACKGROUND: Although recent resuscitation guidelines are supportive of family presence during cardiopulmonary resuscitation literature from the last decade suggests that it is often discouraged, and the subject remains a controversial issue. OBJECTIVES: To determine the experiences and attitudes of European paediatric critical care nurses about parental presence during the resuscitation of a child. DESIGN: A survey design was employed. PARTICIPANTS: A convenience sample of European paediatric critical care nurses was used. METHODS: A structured questionnaire was used, which incorporated a series of attitude statements that were rated using a 5-point Likert scale. Differences in attitudes were explored in three areas: decision-making, processes and outcomes of resuscitation. RESULTS: The results from this survey suggest that European paediatric nurses are very supportive of parental presence during cardiopulmonary resuscitation. Only a few nurses reported that their unit had a policy that covered parental presence during cardiopulmonary resuscitation and most nurses did not support the use of a dedicated nurse to look after the parents during resuscitation. CONCLUSIONS: Compared with previous studies relating to adult cardiopulmonary resuscitation, paediatric nurses experience family member presence more frequently than adult critical care nurses and appear to be more supportive of relatives' presence. It is recommended that paediatric intensive care units establish local policies that cover parental presence during cardiopulmonary resuscitation.
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Atitude do Pessoal de Saúde , Reanimação Cardiopulmonar/enfermagem , Cuidados Críticos/psicologia , Recursos Humanos de Enfermagem Hospitalar/psicologia , Pais/psicologia , Visitas a Pacientes/psicologia , Adulto , Análise de Variância , Reanimação Cardiopulmonar/psicologia , Criança , Cuidados Críticos/organização & administração , Tomada de Decisões , Europa (Continente) , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Masculino , Pesquisa Metodológica em Enfermagem , Política Organizacional , Avaliação de Processos e Resultados em Cuidados de Saúde , Pais/educação , Quartos de Pacientes/organização & administração , Enfermagem Pediátrica/organização & administração , Relações Profissional-Família , Apoio Social , Inquéritos e Questionários , Visitas a Pacientes/educaçãoRESUMO
This paper presents the results of a survey into the experiences and attitudes of 124 European critical care nurses to the presence of family members during cardiopulmonary resuscitation (CPR). Nurses from mainland Europe were less experienced and less sure about the consequences of relatives witnessing resuscitation than United Kingdom (UK) nurses. Generally, nurses supported the presence of family members, although UK nurses held significantly more positive attitudes than their non-UK counterparts in the areas of decision-making, processes and outcomes of resuscitation. Differences in attitudes are explored in the discussion. On the basis of results from this study, it is recommended that further policy guidance is required.
Assuntos
Atitude do Pessoal de Saúde , Reanimação Cardiopulmonar/psicologia , Cuidados Críticos/psicologia , Família , Recursos Humanos de Enfermagem Hospitalar/psicologia , Visitas a Pacientes , Adulto , Viés , Reanimação Cardiopulmonar/enfermagem , Cuidados Críticos/organização & administração , Comparação Transcultural , Tomada de Decisões , Europa (Continente) , Família/psicologia , Feminino , Conhecimentos, Atitudes e Prática em Saúde , Humanos , Unidades de Terapia Intensiva , Masculino , Papel do Profissional de Enfermagem , Pesquisa Metodológica em Enfermagem , Recursos Humanos de Enfermagem Hospitalar/educação , Política Organizacional , Avaliação de Processos e Resultados em Cuidados de Saúde , Relações Profissional-Família , Reino Unido , Visitas a Pacientes/psicologiaRESUMO
New dissymmetric tertiary amines (N(3)SR) with varying N/S donor sets have been synthesized to provide mono- and dinuclear complexes. Acetate ions are used to complete the octahedral coordination sphere around nickel(II) atom(s). The facile conversion of mononuclear to dinuclear systems can be controlled to produce either mono- or dinuclear complexes from the same ligand. The dinuclear complex a(BPh(4))(2) ([Ni(2)(N(3)SSN(3))(OAc)(2)](BPh(4))(2)) has been characterized in the solid state by X-ray diffraction techniques as solvate: a(BPh(4))(2).(1/2)[5(CH(3)OH).(CH(3)CN).(CH(3)CH(2)OH)]. The two Ni atoms are six-coordinated and bridged by a disulfide group and two bidentate acetates. Magnetic susceptibility reveals a weak ferromagnetic exchange interaction between the two Ni atoms with J = 2.5(7) cm(-1). UV-vis studies suggest that the six-coordinated structure persists in solution. The (1)H NMR spectrum of a(BPh(4))(2) exhibits sharp significantly hyperfine shifted ligand signals. A complete assignment of resonances is accomplished by a combination of methods: 2D-COSY experiments, selective chemical substitution, and analysis of proton relaxation data. Proton isotropic hyperfine shifts are shown to originate mainly from contact interactions and to intrinsically contain a small J-magnetic coupling and/or zero-field splitting contribution. A temperature dependence study of longitudinal relaxation times indicates that a very unusual paramagnetic Curie dipolar mechanism is the dominant relaxation pathway in these weakly ferromagnetically spin-coupled dinickel(II) centers. The mononuclear nickel(II) analogue exhibits extremely broader (1)H NMR signals and only partial analysis could be performed. These data are consistent with a shortening of electronic relaxation times in homodinuclear compounds with respect to the corresponding mononuclear species.
Assuntos
Aminas/síntese química , Níquel/química , Compostos Organometálicos/química , Compostos Organometálicos/síntese química , Algoritmos , Aminas/química , Catálise , Fenômenos Químicos , Físico-Química , Cristalografia por Raios X , Análise de Fourier , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estrutura Molecular , Piridinas/química , Espectrofotometria Ultravioleta , Relação Estrutura-Atividade , Difração de Raios XRESUMO
Fur (ferric uptake regulation protein) is a bacterial global regulator that uses iron as a cofactor to bind to specific DNA sequences. It has been suggested that metal binding induces a conformational change in the protein, which is subsequently able to recognize DNA. This mechanism of activation has been investigated here using selective chemical modification monitored by mass spectrometry. The reactivity of each lysine residue of the Fur protein was studied, first in the apo form of the protein, then after metal activation and finally after DNA binding. Of particular interest is Lys76, which was shown to be highly protected from modification in the presence of target DNA. Hydrogen-deuterium exchange experiments were performed to map with higher resolution the conformational changes induced by metal binding. On the basis of these results, together with a secondary structure prediction, the presence in Fur of a non-classical helix-turn-helix motif is proposed. Experimental results show that activation upon metal binding induces conformational modification of this specific motif. The recognition helix, interacting directly with the major groove of the DNA, would include the domain [Y55-F61]. This helix would be followed by a small "wing" formed between two beta strands, containing Lys76, which might interact directly with DNA. These results suggest that Fur and DtxR (diphtheria toxin repressor), another bacterial repressor, share not only the function of being iron concentration regulators, and the structure of their DNA-binding domain.
Assuntos
Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/química , DNA/metabolismo , Ferro/metabolismo , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Sequência de Aminoácidos , Apoproteínas/química , Apoproteínas/metabolismo , DNA/genética , Proteínas de Ligação a DNA/metabolismo , Deutério/metabolismo , Sequências Hélice-Volta-Hélice , Imidoésteres/metabolismo , Lisina/química , Lisina/metabolismo , Manganês/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Alinhamento de Sequência , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
The dinucleating ligand 2,6-bis[(bis(2-pyridylmethyl)amino)methyl]-4-methylphenol (H-BPMP) has been used to synthesize the three dinuclear Cu(II) complexes [Cu2(BPMP)(OH)][ClO4](2).0.5C4H8O (1), [Cu2(BPMP)(H2O)2](ClO4)(3).4H2O (2), and [Cu2(H-BPMP)][(ClO4)4].2CH3CN (3). X-ray diffraction studies reveal that 1 is a mu-hydroxo, mu-phenoxo complex, 2 a diaqua, mu-phenoxo complex, and 3 a binuclear complex with Cu-Cu distances of 2.96, 4.32, and 6.92 A, respectively. Magnetization measurements reveal that 1 is moderately antiferromagnetically coupled while 2 and 3 are essentially uncoupled. The electronic spectra in acetonitrile or in water solutions give results in accordance with the solid-state structures. 1 is EPR-silent, in agreement with the antiferromagnetic coupling between the two copper atoms. The X-band spectrum of powdered 2 is consistent with a tetragonally elongated square pyramid geometry around the Cu(II) ions, in accordance with the solid-state structure, while the spectrum in frozen solution suggests a change in the coordination geometry. The EPR spectra of 3 corroborate the solid-state and UV-visible studies. The 1H NMR spectra also lead to observations in accordance with the conclusions from other spectroscopies. The electrochemical behavior of 1 and 2 in acetonitrile or in water solutions shows that the first reduction (Cu(II)Cu(II)-Cu(II)Cu(I) redox couple) is reversible and the second (formation of Cu(I)Cu(I) irreversible. In water, 1 and 2 are reversibly interconverted upon acid/base titration (pK 4.95). In basic medium a new species, 4, is reversibly formed (pK 12.0), identified as the bishydroxo complex. Only 1 exhibits catecholase activity (oxidation of 3,5-di-tert-butylcatechol to the corresponding quinone, vmax = 1.1 x 10(-6) M-1 s-1 and KM = 1.49 mM). The results indicate that the pH dependence of the catalytic abilities of the complexes is related to changes in the coordination sphere of the metal centers.
Assuntos
Catecóis/metabolismo , Cobre/química , Catálise , Cristalografia por Raios X , Eletroquímica , Espectroscopia de Ressonância de Spin Eletrônica , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância Magnética , Conformação Molecular , Espectrofotometria Ultravioleta , Água/químicaRESUMO
Selective chemical modification of thiol groups combined with mass spectrometry analysis was used to characterize cysteine ligands in the zinc-binding site of the Fur protein. Fur is a metalloregulatory protein involved in the regulation of almost all bacterial genes related to iron uptake in Gram-negative bacteria such as Escherichia coli. In addition to the iron site, Fur also possesses a tight-binding zinc site that likely comprises two cysteines. Using a new procedure, we confirm the involvement of two cysteines in zinc binding and identify them within the two pairs of cysteines present in the protein. The protein was treated under nondenaturing conditions with iodoacetamide, and the progressive alkylation of the thiol groups monitored by quenching the reaction at different times and measuring the extent of alkylation by mass spectrometry. Complementary experiments were carried out in the absence or presence of EDTA, a strong zinc chelator, to determine which of the cysteines were protected from alkylation by the zinc atom. Enzymatic digestion of the modified protein and analysis of the peptide mixture by mass spectrometry enabled fast identification of reactive and protected thiol groups. Two cysteines, Cys92 and Cys95, were thus assigned as zinc ligands. Examination of the sequence comprising the zinc site indicates that it may belong to a new type of structural zinc site. Furthermore, Cys132 was shown to be the fastest reacting cysteine, implying it is a surface-exposed residue.
Assuntos
Proteínas de Bactérias/química , Cisteína/química , Escherichia coli/química , Compostos Férricos/química , Metaloproteínas/química , Proteínas Repressoras/química , Zinco/química , Alquilação , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cisteína/isolamento & purificação , Cisteína/metabolismo , Ácido Edético/química , Compostos Férricos/metabolismo , Hidrólise , Iodoacetamida/química , Espectrometria de Massas , Metaloproteínas/metabolismo , Dados de Sequência Molecular , Proteínas Repressoras/metabolismo , Serina Endopeptidases/química , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Fatores de Tempo , Zinco/metabolismoRESUMO
The Fur apoprotein has been purified and reconstituted with Co2+ and Mn2+ ions. These samples have been analyzed by UV-visible, EPR, and 1H NMR spectroscopies, by XAS, and by magnetization measurements. The apo-Fur protein is able to bind one metal dication (Co2+ or Mn2+) per monomer. A saturation magnetization study confirms the presence of a high-spin metal dication [Mn(II) S = 5/2 and Co(II) S = 3/2]. The two metal ions per Fur dimer are not in magnetic interaction (|J| < 0.1 cm-1 ). The UV-visible spectrum of the cobalt-substituted form (Co-Fur) presents two main bands at 660 nm and 540(br) nm with epsilon540 nm = 65 M-1 cm-1. The EPR spectrum gives the following g values: gx = 5.0(5), gy = 4.0(2), and gz = 2. 3(1), which are in accordance with a nearly axial (E/D < 0.11) site. The value of 55 cm-1 for the splitting (Delta) between the ground and the first excited state has been derived from an EPR saturation study and is in agreement with magnetization data. The EXAFS data of Co-Fur indicate a metal environment comprising five nitrogen/oxygen atoms at 2.11 A, the absence of sulfur, and the presence of histidines as ligands. 1H NMR of Co-Fur in H2O and D2O shows at least two exchangeable signals coming from histidine NH protons and shows the signature of carboxylate group(s). The combined spectroscopic data allow us to propose that the main metal site of Fur in Co-Fur contains at least two histidines, at least one aspartate or glutamate, and no cysteine as ligands and is in an axially distorted octahedral environment.
Assuntos
Proteínas de Bactérias/química , Escherichia coli/química , Proteínas Repressoras/química , Apoproteínas/química , Apoproteínas/isolamento & purificação , Proteínas de Bactérias/isolamento & purificação , Sítios de Ligação , Cobalto/química , Espectroscopia de Ressonância de Spin Eletrônica , Ligantes , Magnetismo , Manganês/química , Proteínas Repressoras/isolamento & purificação , Espectrofotometria Ultravioleta , EstereoisomerismoRESUMO
The zinc K-edge X-ray absorption spectra of the Fur (ferric uptake regulation) protein isolated from Escherichia coli have been analyzed in frozen solution to determine details of the zinc coordination. The spectra of apoFur and of the cobalt-substituted protein have been analyzed and compared in order to see the influence of the cobalt incorporation on the geometry of the zinc site. EXAFS analysis gave for both samples (apoFur and CoFur) a tetrahedral environment for the zinc atom with two sulfur donor ligands at a distance of 2.3 A from the zinc and two N/O donor ligands at 2.0 A. The two sulfur donor ligands are probably two of the four cysteines present in each Fur monomer and could be Cys92 and Cys95, which are known from mutagenesis studies to be essential for Fur activity [Coy, M., Doyle, C., Besser, J., and Neilands, J. B. (1994) BioMetals 7, 292-298]. The distances obtained from our fits were always too short to be compatible with penta or hexa coordination. The typical pattern observed for the Fourier transform of the EXAFS oscillations suggests the presence of at least one imidazole ligand. The XANES of these two forms of the protein are similar but significantly different. This suggests a change of the conformation of the zinc site upon cobalt incorporation. The present study provides the first unambiguous evidence for the presence of a structural zinc site in the Fur protein from Escherichia coli.