RESUMO
BACKGROUND: The European Union faces severe and worsening personnel shortages in healthcare. Coaching has emerged as a human-centred strategy to enhance sustainable employment and retention. While the number of efficacy studies on coaching continues to grow, knowledge about the barriers and facilitators to implementing coaching interventions among healthcare professionals (HCPs) remains scarce. OBJECTIVES: This systematic review aimed to describe common barriers and facilitators to the implementation of coaching interventions for HCPs. METHODS: In April 2023, five databases were searched for eligible articles. Barriers and facilitators were systematically identified and mapped onto the constructs of the Consolidated Framework for Implementation Research (CFIR). Directed content analysis yielded thematic areas and a reporting frequency. RESULTS: A total of thirty (n = 30) studies were included in this review, representing twenty-five (n = 25) distinct coaching programmes. Implementation determinants were clustered under two CFIR domains: the Inner Setting (8 facilitators, 5 barriers) and Implementation Process (6 facilitators, 1 barrier). Barriers included (i) limited organisational capacity, (ii) lack of psychological safety, (iii) competing work demands, and (iv) insufficient leadership buy-in, while facilitators were the (i) allocation of protected time for participants and coaches, (ii) promotion through opinion leaders, (iii) embeddedness in existing Continuous Professional Development programmes, and (iv) programme co-creation. CONCLUSION: The findings of this study provide practical insights to guide the future implementation of coaching interventions at an organisational level. In particular, the identified barriers and facilitators suggest, for optimal efficacy and sustainment, coaching interventions must be implemented within a safe, supportive organisational climate.
Assuntos
Pessoal de Saúde , Tutoria , Humanos , Pessoal de Saúde/educação , Liderança , Tutoria/organização & administração , Desenvolvimento de ProgramasRESUMO
OBJECTIVE: The paediatric knee is prone to pure chondral shear-off lesions due to the developing osteochondral unit. Refixation of the chondral fragment is commonly done using metalwork or absorbable biomaterials. Both fixation methods come with biomaterial-related drawbacks. Earlier work on chondral allografts for cartilage repair in adults has shown successful osteochondral integration when the chondral allograft is treated with multiple incisions and then glued to the subchondral bone using fibrin glue. This is commonly referred to as the "hedgehog technique." This study investigates the feasibility of a modification of the hedgehog technique in autologous cartilage to repair shear-off lesions in children. DESIGN: Three consecutive patients (aged 11, 12, and 14 years) with shear-off chondral fragments of 2, 5, and 8 cm2 were treated using this modified hedgehog technique. The calcified side of the chondral fragments were multiply incised and trimmed obliquely for an interlocking fit in the defect site. Fibrin glue and, if indicated sutures, were applied to fix the fragment to the defect. In 1 patient, an anterior cruciate ligament (ACL) repair was also performed. Patients were evaluated clinically and by magnetic resonance imaging (MRI) up to 12 months postoperatively. RESULTS: Twelve months after surgery, all patients reported no pain and showed complete return to sport and full range of motion. MRI showed no signs of fragment loosening. CONCLUSIONS: The modified hedgehog technique is a feasible treatment option to repair pure chondral shear-off lesions in the paediatric knee. This was the first time this technique was used in autografting.
Assuntos
Doenças das Cartilagens/cirurgia , Cartilagem Articular/cirurgia , Adesivo Tecidual de Fibrina , Articulação do Joelho/patologia , Joelho/cirurgia , Criança , Feminino , Humanos , Traumatismos do Joelho/patologia , Traumatismos do Joelho/cirurgia , Articulação do Joelho/cirurgia , Masculino , Resultado do TratamentoRESUMO
BACKGROUND: Low-dose radiotherapy (LDRT) for pain reduction in osteoarthritis (OA) is a frequently used treatment in Germany and Eastern European countries. The evidence on the effects of LDRT on pain in patients with OA remains unclear. This study evaluated the effect of LDRT on pain in patients with severe OA of the hip or knee joint. METHODS: This prospective study included a total of 16 joints in 12 patients (4 hips and 12 knees). The inclusion criteria were: patients older than 50 years, severe OA (Kellgren-Lawrence grade III-IV) of the hip or knee joint, patients not responding to conservative treatment and patients who are inoperable or not willing to undergo surgery. The joint was irradiated with a total dose of 6.0 Gray. The Numeric Rating Scale for pain (NRS-pain) and patient-reported outcome measures were obtained at pre-, 6, 13, 26, 39 and 52 weeks post-radiation. A decrease of two points on the NRS-pain was defined as clinical relevant. RESULTS: The median age of the included patients was 74 years (range 58-89). In 50% of the joints (n = 8, 3 hip and 5 knee joints), a clinical relevant difference in pain at 6 weeks post-radiation was observed. This clinical relevant difference decreased to 25% at 52 weeks post-radiation. CONCLUSION: LDRT showed a clinical relevant pain relief at 6 weeks after radiotherapy. The long-term effect of LDRT, however, was limited. A randomized placebo-controlled trial is necessary to assess the effect of LDRT on pain in patients with OA of the hip or knee joint.
Assuntos
Osteoartrite do Quadril/radioterapia , Osteoartrite do Joelho/radioterapia , Dosagem Radioterapêutica , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Feminino , Articulação do Quadril/diagnóstico por imagem , Humanos , Articulação do Joelho/diagnóstico por imagem , Masculino , Pessoa de Meia-Idade , Osteoartrite do Quadril/classificação , Osteoartrite do Joelho/classificação , Medição da Dor , Medidas de Resultados Relatados pelo Paciente , Planejamento da Radioterapia Assistida por Computador , Índice de Gravidade de Doença , Tomografia Computadorizada por Raios XRESUMO
PURPOSE: The aim of this multicentre cohort study was to evaluate the midterm outcomes and survival after cementless stemless resurfacing arthroplasty (CSRA) in a series of 33 shoulders in 27 patients with primary osteoarthritis. METHODS: Clinical outcome assessment included: Constant-Murley score (CMS); Simple Shoulder Test (SST); Disability of Arm, Shoulder, Hand (DASH); EuroQol-5D (EQ-5D) utility scores; Numerous Rating Scale (NRS) for pain. Radiographs were assessed by two independent observers for oversizing, radiolucency, glenohumeral subluxation, glenoid erosion and subsidence. Correlations between the clinical and radiological outcomes were calculated. Complications were registered, and revision and survival rates were calculated. RESULTS: Mean age at time of surgery and mean follow-up time were, respectively, 67.7 (range 50.2-85.1) and 7.2 years (range 5.7-9.3 years). Means (SD) for CMS, age- and gender-adjusted CMS, SST, DASH and EQ-5D utility scores were: 56.4 (20.2), 76.5 (25.0), 54.0 (29.8), 37.6 (23.3) and 0.8 (0.1), respectively. NRS for pain was 2.0 and 3.8, respectively, in rest and during activities. Radiographic assessment of the CSRAs showed oversizing in 54.5%; radiolucency in 18.2%; superior glenohumeral subluxation in 33.3%; glenoid erosion in 45.5%; and subsidence in 3.0%. Perioperative complications did not occur. Revision surgery was performed in one patient (3.0%). CONCLUSION: For primary osteoarthritis, the CSRA showed good clinical but poor radiological outcomes at midterm follow-up.
Assuntos
Hemiartroplastia/métodos , Osteoartrite/cirurgia , Articulação do Ombro/cirurgia , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Hemiartroplastia/efeitos adversos , Humanos , Luxações Articulares/etiologia , Estimativa de Kaplan-Meier , Masculino , Pessoa de Meia-Idade , Falha de Prótese , Reoperação , Estudos Retrospectivos , Articulação do Ombro/diagnóstico por imagem , Articulação do Ombro/fisiopatologiaRESUMO
BACKGROUND: In this case report, we describe a left-arm both-bone forearm fracture in a 15-year-old girl who fell off a swing. Conservative treatment with an above-elbow cast failed, resulting in a malunion with functional impairment. The pro- and supination were 90/0/10, respectively. The patient complained of difficulties performing daily activities. For this pediatric case, a corrective osteotomy was proposed using a CT-guided technique aiming for maximum anatomical and functional outcome. It was the first time this technique was used in our hospital. METHODS: A corrective osteotomy of the patient's left arm was performed using 3D printed templates to guide the osteotomy orientation. These templates were produced using specialized software in which CT images of her malunited left forearm were overlaid with the mirrored images of her healthy right forearm. RESULTS: The postoperative CT-scan showed a near-anatomical reduction with close to 1° correction in all three planes, as compared to the preoperative planning. Three months after surgery, the patient had regained full function of her left forearm. CONCLUSION: Although this was the first time this technique was used in our hospital, it resulted in excellent anatomical and functional outcomes making it a safe, reliable and precise treatment option that may be useful for even more complex corrections. LEVEL OF EVIDENCE: Level V.
Assuntos
Fraturas Mal-Unidas/diagnóstico por imagem , Fraturas Mal-Unidas/cirurgia , Fraturas do Rádio/cirurgia , Cirurgia Assistida por Computador/métodos , Fraturas da Ulna/cirurgia , Adolescente , Feminino , Antebraço/fisiopatologia , Fraturas Mal-Unidas/fisiopatologia , Humanos , Osteotomia/métodos , Pronação , Fraturas do Rádio/diagnóstico por imagem , Supinação , Tomografia Computadorizada por Raios X , Fraturas da Ulna/diagnóstico por imagemRESUMO
AIMS: This prospective randomised controlled trial was designed to evaluate the outcome of both the MRI- and CT-based patient-specific matched guides (PSG) from the same manufacturer. PATIENTS AND METHODS: A total of 137 knees in 137 patients (50 men, 87 women) were included, 67 in the MRI- and 70 in the CT-based PSG group. Their mean age was 68.4 years (47.0 to 88.9). Outcome was expressed as the biomechanical limb alignment (centre hip-knee-ankle: HKA-axis) achieved post-operatively, the position of the individual components within 3° of the pre-operatively planned alignment, correct planned implant size and operative data (e.g. operating time and blood loss). RESULTS: The patient demographics (e.g. age, body mass index), correct planned implant size and operative data were not significantly different between the two groups. The proportion of outliers in the coronal and sagittal plane ranged from 0% to 21% in both groups. Only the number of outliers for the posterior slope of the tibial component showed a significant difference (p = 0.004) with more outliers in the CT group (n = 9, 13%) than in the MRI group (0%). CONCLUSION: The post-operative HKA-axis was comparable in the MRI- and CT-based PSGs, but there were significantly more outliers for the posterior slope in the CT-based PSGs. TAKE HOME MESSAGE: Alignment with MRI-based PSG is at least as good as, if not better, than that of the CT-based PSG, and is the preferred imaging modality when performing TKA with use of PSG. Cite this article: Bone Joint J 2016;98-B:786-92.
Assuntos
Artroplastia do Joelho/instrumentação , Desenho Assistido por Computador , Articulação do Joelho/diagnóstico por imagem , Prótese do Joelho , Imageamento por Ressonância Magnética , Ajuste de Prótese/instrumentação , Tomografia Computadorizada por Raios X , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Articulação do Joelho/cirurgia , Masculino , Pessoa de Meia-Idade , Osteoartrite do Joelho/cirurgia , Cuidados Pré-Operatórios/métodos , Estudos ProspectivosRESUMO
Published clinical trials who studied the accuracy of patient-specific guides (PSG) for total knee arthroplasty exclude patients with articular deformity of the knee joint. We prospectively analysed a series of 30 patients with post-traumatic osteoarthritis of the knee joint with use of PSG. At 1 year post-operative, the achieved biomechanical (HKA) axis and varus/valgus of the femur and tibia components were measured on anterior-posterior (AP) long-standing weight-bearing radiographs. Flexion/extension of the femoral and AP slope of the tibia component was measured on standard lateral radiographs. Percentages >3° deviation of the pre-operative planned HKA axis and individual implant components were considered as outliers. Approved and used implant size, median blood loss (ml) and operation time (min) were obtained from the operation records. Pre- and 1-year post-operative patient-reported outcome measures (PROMs) were performed. Eighty-three per cent of the patients had a HKA axis restored <3° of the pre-operative planned alignment. Varus/valgus outliers were 0.0 and 6.7 % for the femoral and tibial components, respectively. Percentages of outliers of flexion/extension were 36.7 % for the femoral component and 10.0 % for the AP slope of the tibial component. Median blood loss was 300 ml (50-700), while operation time was 67 min (44-144). In 20 % of all cases, the approved implant size was changed into one size smaller. One-year post-operative PROMs improved significantly. We conclude that the accuracy of CT-based PSG is not impaired in patients with post-traumatic osteoarthritis and this modality can restore biomechanical limb alignment.
Assuntos
Artroplastia do Joelho/métodos , Traumatismos do Joelho/complicações , Osteoartrite do Joelho/cirurgia , Idoso , Perda Sanguínea Cirúrgica , Feminino , Fixação Intramedular de Fraturas/métodos , Humanos , Prótese do Joelho , Masculino , Pessoa de Meia-Idade , Duração da Cirurgia , Osteoartrite do Joelho/diagnóstico por imagem , Osteoartrite do Joelho/etiologia , Avaliação de Resultados da Assistência ao Paciente , Cuidados Pré-Operatórios/métodos , Estudos Prospectivos , Radiografia Intervencionista/métodos , Sensibilidade e Especificidade , Tomografia Computadorizada por Raios X/normas , Resultado do TratamentoRESUMO
Cartilage has a poor regenerative capacity. Tissue-engineering approaches using porous scaffolds seeded with chondrocytes may improve cartilage repair. The aim of this study was to examine the effect of pore size and pore interconnectivity on cartilage repair in osteochondral defects treated with different scaffolds seeded with allogenic chondrocytes. Scaffolds consisting of 55 wt% poly(ethylene oxide terephthalate) and 45 wt% poly(butylene terephthalate) (PEOT/PBT) with different pore sizes and interconnectivities were made, using a compression moulding (CM) and a three-dimensional fibre (3DF) deposition technique. In these scaffolds, allogenic chondrocytes were seeded, cultured for 3 weeks and implanted in osteochondral defects of skeletally mature rabbits. At 3 weeks no difference in cartilage repair between an empty osteochondral defect, CM or 3DF scaffolds was found. Three months post-implantation, cartilage repair was significantly improved after implantation of a 3DF scaffold compared to a CM scaffold. Although not significant, Mankin scores for osteoarthritis (OA) indicated less OA in the 3DF scaffold group compared to empty defects and CM-treated defects. It is concluded that scaffold pore size and pore interconnectivity influences osteochondral repair and a decreased pore interconnectivity seems to impair osteochondral repair.
Assuntos
Osso e Ossos/patologia , Cartilagem Articular/patologia , Regeneração , Engenharia Tecidual/métodos , Alicerces Teciduais/química , Animais , Condrócitos/citologia , Feminino , Osteoartrite/patologia , Poliésteres/química , Polietilenoglicóis/química , Polietilenotereftalatos , Polímeros/química , Porosidade , Coelhos , CicatrizaçãoRESUMO
Transgenic animals are commonly employed to explore the function of individual proteins. Transgenic animal models include the mouse, the zebrafish, and the South African clawed toad Xenopus laevis. In contrast to mice and zebrafish, with Xenopus transgenesis DNA integration is mostly achieved in the one-cell stage. Moreover, Xenopus (as well as zebrafish) eggs are relatively large, the embryos are transparent, a large offspring is generated, and maintenance of the offspring is easy. In our transgenic studies in Xenopus, we focus on the well-characterized neuroendocrine melanotrope cells of the pituitary pars intermedia that are regulated during the process of adaptation of Xenopus to a changing environment. When the animal is placed on a black background, the melanotrope cells produce and process large amounts of the prohormone proopiomelanocortin (POMC). We apply stable melanotrope-specific transgenesis that is achieved by mixing a Xenopus POMC-promoter/transgene construct with sperm nuclei and injecting this mixture into unfertilized eggs. Since in the melanotrope cells the POMC promoter is much more active in black-adapted animals, the level of transgene expression can be manipulated by placing the animal on either a black or a white background. In this paper we review the possibilities of the Xenopus melanotrope-specific transgenic approach. Following a brief overview of the functioning of Xenopus melanotrope cells, stable melanotrope-specific transgenesis is discussed and our transgenic studies on brain-derived neurotrophic factor and secretory pathway components are described as examples of the transgenic approach in a physiological context and close to the in vivo situation.
Assuntos
Sistemas Neurossecretores/metabolismo , Xenopus laevis/metabolismo , Animais , Animais Geneticamente Modificados , Regulação da Expressão Gênica , Humanos , Modelos Animais , Pró-Opiomelanocortina/metabolismoRESUMO
The prohormone convertases (PCs) 1/3 and 2 accomplish the major proteolytic cleavage events in neuroendocrine tissues; each of these convertases has a small associated binding protein that inhibits convertase action in the secretory pathway. The proSAAS protein binds to PC1/3, whereas the 7B2 protein binds to PC2. However, both convertase-binding proteins are more widely expressed than their cognate enzymes, suggesting that they may perform other functions as well. All known mammalian proSAASs are over 85% conserved; thus, identifying functionally important segments has been impossible. Here, we report the first identification of nonmammalian proSAAS molecules, from Xenopus and zebrafish (Danio rerio). Although these two proteins show an overall amino acid sequence identity of only 29 and 30% with mouse proSAAS, two 14-16 residue hydrophobic segments (predicted to form alpha-helices) and two, nine through 11 residue sequences containing basic convertase cleavage sites are highly conserved; therefore, these sequences may be of functional importance. Confidence that these nonmammalian molecules represent authentic proSAAS is supported by the finding that both inhibit mouse PC1/3 with nanomolar inhibition constants; human furin was not inhibited. In vitro, the two proteins were cleaved by PC2 and furin to three or more peptide products. Both zebrafish and Xenopus proSAAS exhibited neural and endocrine distributions, as assessed by in situ and PCR experiments, respectively. In summary, the identification of proSAAS molecules in lower vertebrates provides clues as to functional regions within this widely expressed neuroendocrine protein.
Assuntos
Neuropeptídeos/química , Neuropeptídeos/genética , Pró-Proteína Convertases/antagonistas & inibidores , Homologia de Sequência de Aminoácidos , Vertebrados/genética , Sequência de Aminoácidos , Animais , Sequência Conservada , Embrião não Mamífero , Inibidores Enzimáticos/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Dados de Sequência Molecular , Células Neuroendócrinas/metabolismo , Neurônios/metabolismo , Neuropeptídeos/isolamento & purificação , Neuropeptídeos/metabolismo , Filogenia , Estrutura Secundária de Proteína/genética , Xenopus laevis/genética , Peixe-Zebra/genéticaRESUMO
OBJECTIVES: An investigation of the hearing status of musicians of professional symphony orchestras. Main questions are: (1) Should musicians be treated as a special group with regard to hearing, noise, and noise related hearing problems (2) Do patterns of hearing damage differ for different instrument types (3) Do OAE have an added value in the diagnosis of noise induced hearing loss (NIHL) in musicians. METHODS: 241 professional musicians, aged between 23-64 participated. A brief medical history and the subjective judgment of their hearing and hearing problems were assessed. Musicians were subjected to an extensive audiological test battery, which contained testing of audiometric thresholds, loudness perception, diplacusis, tinnitus, speech perception in noise, and otoacoustic emissions. RESULTS: Most musicians could be categorized as normal hearing, but their audiograms show notches at 6 kHz, a frequency that is associated with NIHL. Musicians often complained about tinnitus and hyperacusis, while diplacusis was generally not reported as a problem. Tinnitus was most often localized utmost left and this could not be related to the instrument. It was usually perceived in high frequency areas, associated with NIHL. In general, musicians scored very well on the speech-in-noise test. The results of the loudness perception test were within normal limits. Otoacoustic emissions were more intense with better pure-tone thresholds, but due to large individual differences it can still not be used as an objective test for early detection of NIHL. CONCLUSIONS: Musicians show more noise induced hearing loss than could be expected on the basis of age and gender. Other indicators, such as complaints and prevalence of tinnitus, complaints about hyperacusis and prevalence of diplacusis suggest that musicians' ears are at risk. Continuing education about the risks of intensive sound exposure to musicians, with the emphasis on the possible development of tinnitus and hyperacusis and the need for good hearing protection is warranted.
Assuntos
Perda Auditiva Provocada por Ruído/etiologia , Hiperacusia/etiologia , Ruído/efeitos adversos , Doenças Profissionais/etiologia , Zumbido/etiologia , Adulto , Audiometria de Tons Puros/métodos , Limiar Auditivo/classificação , Limiar Auditivo/fisiologia , Feminino , Perda Auditiva Provocada por Ruído/diagnóstico , Perda Auditiva Provocada por Ruído/fisiopatologia , Humanos , Hiperacusia/diagnóstico , Hiperacusia/fisiopatologia , Masculino , Pessoa de Meia-Idade , Música , Doenças Profissionais/diagnóstico , Doenças Profissionais/fisiopatologia , Inquéritos e Questionários , Zumbido/diagnóstico , Zumbido/fisiopatologia , Adulto JovemRESUMO
STUDY DESIGN: The authors monitored a father and his six children, all of whom had idiopathic scoliosis for more than 13 years. OBJECTIVE: To analyze the similarities and differences in curve pattern between the family members. SUMMARY OF BACKGROUND DATA: The etiology and pathogenesis of idiopathic scoliosis are not well understood. Genetic factors play an important role, as is shown by the high concordance in monozygotic twins. METHODS: Radiographs from all family members were analyzed. Location and direction of the curve, Cobb angle, location of the apex, and rib-vertebra angle difference were compared. RESULTS: A father and his six children had idiopathic scoliosis. The curves differed within the family and changed during follow-up. The father had a left convex thoracic scoliosis of 42 degrees. Only three of the children had a curve of similar severity, and only four children had a left convex thoracic scoliosis. In three children the curve pattern changed during follow-up. CONCLUSIONS: This report of idiopathic scoliosis in a father and six children supports the present theories on a genetic origin of the disorder but also stresses the importance of dynamic processes in the development of scoliosis.
Assuntos
Saúde da Família , Escoliose/diagnóstico por imagem , Coluna Vertebral/diagnóstico por imagem , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Seguimentos , Predisposição Genética para Doença , Humanos , Lactente , Cifose/diagnóstico por imagem , Cifose/genética , Lordose/diagnóstico por imagem , Lordose/genética , Masculino , Radiografia , Escoliose/genéticaRESUMO
Most authors state that there is strong evidence for a genetic origin of adolescent idiopathic scoliosis (AIS). This conclusion is mainly based on the fact that the rate of concordance for AIS in monozygotic twins is significantly higher than that in dizygotic twins. However, it is of interest to determine whether all elements of scoliosis formation are genetically predetermined. If this were the case, there would perhaps be less place for closed treatment. We surveyed the literature for monozygotic twin pairs in which both members suffered from idiopathic scoliosis and added 3 pairs from our own patient group. The total group consisted of 32 twin pairs. We found that gender, direction of the convexity, the level of the apex and the kyphotic angle were determined more by genetic factors than the lateral Cobb angle of the scoliotic curve. This suggests that variations in the environment may affect the curve patterns in monozygotic twins.
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Testes Genéticos , Escoliose/diagnóstico por imagem , Escoliose/genética , Gêmeos Monozigóticos/genética , Adolescente , Braquetes , Feminino , Seguimentos , Humanos , Radiografia , Escoliose/reabilitação , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Resultado do TratamentoRESUMO
This study was undertaken to determine the long-term outcome of conservatively treated popliteal cysts in children. It was based on a review of 20 cysts, none of which were initially treated surgically. The records of children with popliteal cysts between 1986 and 1992 were studied, and a group of patients without other diseases or other physical abnormality in the knee region was selected. Twenty-one patients were included and were subjected to a clinical and ultrasound investigation. Three were lost to follow-up, leaving 18 patients with 20 cysts available for analysis. In the follow-up period, which ranged from 5 to 10 years, 14 cysts were treated conservatively with a mean follow-up of 7 years, whereas 6 cysts were operated on after a mean period of 2 years. Of the conservatively treated cysts, eight had disappeared and six had shrunk. Although spontaneous remission is not to be expected in all cases, asymptomatic popliteal cysts in children can be treated conservatively with good results.
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Cisto Popliteal/terapia , Criança , Pré-Escolar , Feminino , Seguimentos , Humanos , Masculino , Fatores de TempoRESUMO
Vacuolar H+-ATPases (V-ATPases) mediate the acidification of multiple intracellular compartments, including secretory granules in which an acidic milieu is necessary for prohormone processing. A search for genes coordinately expressed with the prohormone proopiomelanocortin (POMC) in the melanotrope cells of Xenopus intermediate pituitary led to the isolation of a cDNA encoding the complete amino-acid sequence of the type I transmembrane V-ATPase accessory subunit Ac45 (predicted size 48 kDa). Comparison of Xenopus and mammalian Ac45 sequences revealed conserved regions in the protein that may be of functional importance. Western blot analysis showed that immunoreactive Ac45 represents a approximately 40-kDa product that is expressed predominantly in neuroendocrine tissues; deglycosylation resulted in a approximately 27-kDa immunoreactive Ac45 product which is smaller than predicted for the intact protein. Biosynthetic studies revealed that newly synthesized Xenopus Ac45 is an N-glycosylated protein of approximately 60 kDa; the nonglycosylated, newly synthesized form is approximately 46 kDa which is similar to the predicted size. Immunocytochemical analysis showed that in Xenopus pituitary, Ac45 is highly expressed in the biosynthetically active melanotrope cells. We conclude that the regionally conserved Xenopus Ac45 protein is synthesized as an N-glycosylated approximately 60-kDa precursor that is intracellularly cleaved to an approximately 40-kDa product and speculate that it may assist in the V-ATPase-mediated acidification of neuroendocrine secretory granules.
Assuntos
Hipófise/enzimologia , ATPases Translocadoras de Prótons/biossíntese , ATPases Vacuolares Próton-Translocadoras , Sequência de Aminoácidos , Animais , Sequência de Bases , Evolução Biológica , Western Blotting , Clonagem Molecular , DNA Complementar , Humanos , Hidrólise , Imuno-Histoquímica , Dados de Sequência Molecular , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/metabolismo , Homologia de Sequência de Aminoácidos , Transfecção , Xenopus laevisRESUMO
Faecal samples were collected from healthy volunteers in two regions in Venezuela, the village of Grulla (n = 195) and the city of Mérida (n = 181), and analysed for the prevalence of antibiotic resistant faecal Escherichia coli as well as the antibiotic susceptibility of the strains isolated. The highest prevalences of resistance were observed for amoxicillin, oxytetracycline, sulfamethoxazole and trimethoprim. The percentages found for Grulla were 46, 38, 44 and 30%, respectively; for Mérida 39, 65, 56 and 36%, respectively. In Mérida, a significantly higher prevalence of resistance for oxytetracycline was found (P < 0.05). Significant differences in the distribution of the MIC values between Grulla and Mérida were observed for amoxicillin, chloramphenicol and oxytetracycline (P < 0.05). In Grulla, the most frequent pattern was resistance to amoxicillin only and in Mérida to oxytetracycline only. Amoxicillin resistance was due to production of TEM1 beta-lactamase.
Assuntos
Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/isolamento & purificação , Fezes/microbiologia , Vigilância da População , Adolescente , Adulto , Idoso , Criança , Pré-Escolar , Resistência Microbiana a Medicamentos , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Prevalência , População Rural , População Urbana , Venezuela/epidemiologiaRESUMO
Ac45 is a type I transmembrane protein associated with vacuolar H+-ATPase, a proton pump mediating the acidification of multiple intracellular organelles. In this study, we examined the intracellular routing of Ac45 in transfected CV-1 fibroblasts. Steady state immunolabeling showed that Ac45 is located on the plasma membrane and in a vacuolar compartment in the juxtanuclear region. Antibody internalization experiments revealed that Ac45 is rapidly retrieved from the cell surface and is targeted to the vacuolar structures. The 26-residue cytoplasmic tail of Ac45 was intrinsically capable of mediating endocytosis of the cell surface protein Tac, indicating that the tail contains an autonomous internalization signal. Immunolocalization studies on cells expressing carboxy-terminally truncated Ac45 mutants showed the presence of essential routing information in the membrane-distal region of the cytoplasmic tail. Further mutational analysis of this region, which lacks the recognized tyrosine- or di-leucine-based sorting motifs, suggested that multiple sites rather than a short linear sequence are responsible for the internalization. Collectively, our results indicate that the cytoplasmic tail of Ac45 contains autonomous targeting information distinct from previously described routing determinants.
Assuntos
Membrana Celular/enzimologia , Proteínas de Membrana/metabolismo , ATPases Translocadoras de Prótons/metabolismo , ATPases Vacuolares Próton-Translocadoras , Vacúolos/enzimologia , Proteínas de Xenopus , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Transporte Biológico , Linhagem Celular , Citoplasma/enzimologia , Análise Mutacional de DNA , Endocitose , Membranas Intracelulares/enzimologia , Proteínas de Membrana/química , Proteínas de Membrana/genética , Microscopia de Fluorescência , Dados de Sequência Molecular , Mutação , Sinais Direcionadores de Proteínas , ATPases Translocadoras de Prótons/química , ATPases Translocadoras de Prótons/genética , Receptores de Interleucina-2/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , TransfecçãoRESUMO
An improved method for the quantitative derivatization of amino acids with fluorenylmethyl chloroformate (FMOC-Cl) is described. Amino acids are derivatized in borate buffer at pH 11.4 for 40 min at ambient temperature. All amino acids resulted in stable derivatives. In particular, improved derivatization was obtained with the troublesome amino acids His and Tyr: exclusively monosubstituted His and disubstituted Tyr were formed, eluting as free peaks in the chromatogram. These derivatives show a higher fluorescence response than their disubstituted and monosubstituted counterparts, respectively, resulting from other protocols. Under the new conditions, considerable less of the hydrolysis product of FMOC-Cl is seen in the chromatograms. Baseline noise was substantially reduced at a higher emission wavelength (630 nm instead of 313 or 340 nm). With simple precautions, extensive adsorption of the disubstituted derivatives (Lys, Hyl, and Tyr) on plastic or glass surfaces could be prevented. Calibration curves were linear over a 10 to 300 molar ratio of FMOC-Cl to total amino acid. The detection limits are in the femtomole range and the derivatives are stable for more than 48 h, thus permitting automated analysis of multiple samples.
Assuntos
Aminoácidos/análise , Cromatografia Líquida de Alta Pressão/métodos , Aminoácidos/química , Angiotensina II/análise , Angiotensina II/química , Fluorenos/química , Histidina/análise , Histidina/química , Humanos , Indicadores e Reagentes/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Tirosina/análise , Tirosina/químicaRESUMO
Secretogranin III (SgIII) is an acidic protein of unknown function that is present in the storage vesicles of many neuroendocrine cells. It is coexpressed with the prohormone proopiomelanocortin in the intermediate pituitary of Xenopus laevis. We developed an antiserum to investigate the biosynthesis of SgIII in pulse-chase incubated Xenopus neurointermediate lobes. SgIII was synthesized as a 61- or 63-kDa (N-glycosylated) protein and processed to a 48-kDa form which, in turn, was partially cleaved to fragments of 28 and 20 kDa. The 48-, 28-, and 20-kDa cleavage products, but not their precursors, were secreted. This secretion is regulated and can be blocked in parallel with that of proopiomelanocortin-derived peptides by the hypothalamic factors dopamine, gamma-aminobutyric acid, and neuropeptide Y. Coexpression of Xenopus SgIII with prohormone convertase (PC)1 or PC2 in transfected fibroblasts was sufficient to reconstitute the processing events observed in the neurointermediate lobes. Site-directed mutagenesis revealed that Xenopus SgIII is cleaved at two dibasic sites, namely Lys68-Arg69 and Arg237-Arg238. Pulse-chase incubations of lobes with Na2[35S]SO4 showed that SgIII is sulfated in the trans-Golgi network before it is processed. Finally, SgIII processing was found in several neuroendocrine cell types from various species. We conclude that SgIII is a precursor protein and that the intact molecule can only have an intracellular function, whereas an extracellular role can only be attributed to its cleavage products.
Assuntos
Hipófise/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Ésteres do Ácido Sulfúrico/metabolismo , Adaptação Fisiológica , Animais , Ácido Aspártico Endopeptidases/metabolismo , Sequência de Bases , Compartimento Celular , Linhagem Celular , Cromograninas , Cor , Glândulas Endócrinas/metabolismo , Hormônios Estimuladores de Melanócitos/metabolismo , Camundongos , Dados de Sequência Molecular , Neurônios/metabolismo , Hipófise/citologia , Pró-Proteína Convertase 2 , Pró-Proteína Convertases , Subtilisinas/metabolismo , Xenopus laevisRESUMO
The biosynthetic machinery in the melanotrope cells of the Xenopus intermediate pituitary is primarily dedicated to the generation of proopiomelanocortin (POMC)-derived, melanophore-stimulating peptides. Transfer of the animal to a black background stimulates the production of these peptides and causes a dramatic increase in POMC mRNA levels. To identify genes involved in the biosynthesis and regulated release of peptide hormones, we differentially screened an intermediate pituitary cDNA library of toads adapted to a black background with cDNA probes derived from intermediate pituitary mRNA of black- and white-adapted animals. Here we report the identification of twelve distinct genes whose expression levels in the melanotropes are regulated in coordination with that of POMC. Four of these genes are novel while the others code for translocon-associated proteins, a lumenal cysteine protease of the endoplasmic reticulum, prohormone-processing enzymes, members of the granin family and a transmembrane protein presumably involved in the assembly and/or specific functioning of vacuolar H(+)-ATPase from secretory granules. Our results indicate that a wide variety of both soluble and membrane-associated components of the secretory pathway is recruited in physiologically activated, peptide hormone-producing cells.