RESUMO
The primary purpose of the study was to evaluate whether a pericardiectomy (PERI) alters training- or myocardial infarction (MI)-induced left ventricular hypertrophy (LVH), chamber geometry, gene expression and/or running performance. Mice were randomized into 6 groups: naïve control (CONT)-sedentary (Sed), CONT-trained (Tr), PERI-Sed, PERI-Tr, MI-Sed and MI-Tr. MI mice also received a pericardiectomy as part of the MI surgical procedure. 10 weeks of treadmill running resulted in enhanced running performance-to-exhaustion in all 3 trained groups (CONT-Tr, PERI-Tr, MI-Tr) compared to sedentary cohorts (P<0.001). Training also resulted in similar increases in normalized LVH (LV/BW) in CONT-Tr and PERI-Tr mice. 2D-echocardiographic evaluation of LV internal chamber dimensions revealed that stroke diameter (SD) was larger in PERI compared to MI (P<0.01) but not CONT mice. Ventricular B-type natriuretic peptide mRNA (BNP) was elevated only in the 2 MI groups. Left ventricle ß1-adrenergic receptor (ß1-AR) and melusin transcripts both demonstrated an overall increase in trained compared to sedentary mice (both P<0.05). Additionally long-term pericardiectomy did not further enhance running performance or increase LV/BW in either sedentary or trained mice.
Assuntos
Hipertrofia Ventricular Esquerda/fisiopatologia , Infarto do Miocárdio/fisiopatologia , Pericardiectomia , Condicionamento Físico Animal , Animais , Proteínas do Citoesqueleto/fisiologia , Ecocardiografia , Feminino , Expressão Gênica , Ventrículos do Coração/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Musculares/fisiologia , Peptídeo Natriurético Encefálico/fisiologia , Distribuição Aleatória , Receptores Adrenérgicos beta/fisiologiaRESUMO
The clinical and pathologic features of 13 patients with biopsy-proven reticulum cell sarcoma of the orbit are reviewed. All of the patients were adults, in contrast to many previous reports that have emphasized how common this tumor is in children. Eight patients presented with proptosis and without prior diagnosis of lymphoma, and 5 patients developed orbital deposits in the course of a known systemic lymphoma. Of the 8 patients whose orbital biopsies established the diagnosis, 4 were found concomitantly to have other sites of disease, and 4 patients developed other evidence of lymphoma from 2 to 15 months after orbital biopsy. None of the cases in this series was interpreted as a primary orbital lymphoma. The histopathologic findings, the problem of histogenesis, and the need for systemic staging of cytologically malignant orbital lymphoma are discussed.
Assuntos
Linfoma não Hodgkin , Neoplasias Orbitárias , Adulto , Idoso , Diagnóstico Diferencial , Feminino , Humanos , Linfoma não Hodgkin/diagnóstico , Linfoma não Hodgkin/etiologia , Linfoma não Hodgkin/patologia , Masculino , Pessoa de Meia-Idade , Órbita/patologia , Neoplasias Orbitárias/diagnóstico , Neoplasias Orbitárias/etiologia , Neoplasias Orbitárias/patologia , PrognósticoRESUMO
The cases of twenty-four patients who had twenty-six osteotomies of the tibial tubercle in conjunction with total knee replacement were analyzed with regard to complications and technical considerations. The patients were followed for a minimum of two years (average, three years and six months). Major complications related to the surgical technique occurred in 23 per cent of the knees and complications not related to the technique, in an additional 8 per cent. Rheumatoid arthritis and a history of at least one previous operation about the knee were predisposing factors for these complications.
Assuntos
Prótese do Joelho , Osteotomia/métodos , Tíbia/cirurgia , Adulto , Idoso , Artrite Reumatoide/complicações , Artrite Reumatoide/cirurgia , Feminino , Humanos , Articulação do Joelho/fisiopatologia , Ligamentos Articulares , Masculino , Pessoa de Meia-Idade , Movimento , Osteotomia/efeitos adversos , Falha de Prótese , Ruptura Espontânea , Tíbia/fisiopatologiaRESUMO
Effective laundry protease performance against susceptible stains depends upon both the enzyme itself and the environment in which it must work. In order to technically design superior laundry proteases, a model for protease's mechanism of action in detergents was developed which has been substantiated through-the-wash. While evaluation of this model and/or a given protease's effectiveness could be judged by a variety of methods, the utility of using visual wash performance comparisons, analytical, and stain characterization studies is described. Finally, data comparing the performance of wild type Subtilisin proteases with mutants designed via the projected model are given, demonstrating possible utility of the system.
Assuntos
Detergentes , Lavanderia/métodos , Engenharia de Proteínas , Subtilisinas , Catálise , Corantes , Hidrólise , Cinética , Modelos Químicos , Mutação , Subtilisinas/genéticaRESUMO
The successful recruitment of medical staff to country areas is a difficult process. This paper outlines strategies designed to increase the probability of a successful recruitment program. Strategies include determining if the position is truly required, designing an advertising campaign that reaches the target audience and addressing the significant regional and medical factors influencing the attractiveness of positions. Other areas discussed include the role of local hospitals, factors unique to individual medical practitioners, contracts and two possible long-term solutions--familiarising medical students with rural practice and recruiting overseas doctors.
Assuntos
Área Carente de Assistência Médica , Seleção de Pessoal/métodos , Médicos/provisão & distribuição , Área de Atuação Profissional , Serviços de Saúde Rural , Publicidade , Diretores de Hospitais , Serviços Contratados , Hospitais Rurais/organização & administração , Renda , Corpo Clínico Hospitalar/provisão & distribuição , Diretores Médicos , Médicos/normas , Cônjuges , Vitória , Recursos HumanosRESUMO
This paper reviews the advantages and disadvantages of medical quality assurance methods commonly used in hospitals. A computer-based literature search up to April 1993 was done using the terms quality assurance, quality control, audit, utilization review, accreditation, occurrence screening and total quality management. In addition, a manual search was undertaken of references of papers obtained by the computer search and of the contents of the following journals: Quality Review Bulletin, Australian Clinical Review and the British Medical Journal. An assessment of the advantages and disadvantages of 15 quality assurance methods used in hospitals was made. Many quality assurance methods used in hospitals have significant disadvantages. A systematic method that provides meaningful and useful information to clinicians and improves patient care is urgently required. New methods such as occurrence screening and total quality management are slowly emerging and have much potential.
Assuntos
Administração Hospitalar/normas , Garantia da Qualidade dos Cuidados de Saúde/organização & administração , Austrália , Autopsia/normas , Médicos Legistas/normas , Credenciamento/normas , Administração Hospitalar/métodos , Mortalidade Hospitalar , Auditoria Médica/normas , Privilégios do Corpo Clínico/normas , Satisfação do Paciente , Guias de Prática Clínica como Assunto , Gestão de Riscos , Gestão da Qualidade Total/normas , Resultado do Tratamento , Revisão da Utilização de Recursos de Saúde/normasRESUMO
OBJECTIVE: To determine whether adverse occurrence screening of inpatient discharge summaries by means of a limited number of criteria can provide an effective medical quality control system without requiring substantial additional resources. DESIGN: The medical records department used 14 criteria to screen all inpatient discharge summaries shortly after discharge. Summaries that met particular criteria were sent for medical review. If an adverse patient event was found, appropriate action was determined by a committee of four doctors. SETTING: 200 bed base hospital in country Victoria. PATIENTS: All inpatients discharged over a two year period. MAIN OUTCOME MEASURES: The incidence of negative patient outcomes and the cost of resources required to conduct the program. RESULTS: A reduction in the incidence of some negative patient outcomes was achieved and the nature of postgraduate education meetings was changed. Minimal additional resources were required to run the program. CONCLUSION: Limited adverse occurrence screening has provided a simple, inexpensive and effective medical quality control system in a medium-sized hospital.
Assuntos
Administração Hospitalar/normas , Avaliação de Processos e Resultados em Cuidados de Saúde/organização & administração , Gestão de Riscos/métodos , Sistemas de Notificação de Reações Adversas a Medicamentos , Protocolos Clínicos , Hospitais com 100 a 299 Leitos , Humanos , Tempo de Internação , Prontuários Médicos , Alta do Paciente , Garantia da Qualidade dos Cuidados de Saúde , Controle de Qualidade , VitóriaRESUMO
The objective of the present study was to determine the rate of adverse patient occurrences in a medium sized hospital and to assess the effectiveness and efficiency of limited adverse occurrence screening as a method of medical quality control. The medical records of inpatients discharged from a base hospital in Horsham, Victoria, were screened by the medical records department using eight general outcome criteria. Histories found to meet a criterion were sent to a medical reviewer to determine if an adverse patient occurrence had taken place. A random sample of histories not meeting any criteria was also reviewed. The main outcome measures were the rate of adverse patient occurrences and the proportion of these events detected by limited occurrence screening, the accuracy of the screening process, the time taken and the cost of finding adverse events. The total adverse patient occurrence rate was estimated to be 2.75% (95% CI 1.36-4.14%). Limited adverse occurrence screening using eight screening criteria detected 49.1% (95% CI 32.6-99.3%) of all adverse patient occurrences and 64.4% (95% CI 37.8-100%) of all adverse occurrences of major severity. This was achieved by reviewing the records of 9.72% of all patients discharged. Screening was quick and accurate (false positive rate 2.0%, false negative rate 0.4%). Medical review took on average 5 min (s.d. +/- 3.03). The method required 500 h of staff time over one year and cost $22000 (0.1% of total hospital budget). The proportion of adverse patient occurrences found by limited screening was much higher than that found by traditional quality assurance methods. Limited adverse occurrence screening using retrospective review requires a small proportion of total budget that should be available to most hospitals for medical quality assurance activities.
Assuntos
Administração Hospitalar/normas , Doença Iatrogênica , Auditoria Médica/métodos , Estudos de Casos e Controles , Cuidados Críticos , Mortalidade Hospitalar , Humanos , Capacitação em Serviço/economia , Razão de Chances , Readmissão do Paciente , Transferência de Pacientes , Valor Preditivo dos Testes , Estudos Retrospectivos , Gestão de Riscos , Fatores de Tempo , Resultado do Tratamento , VitóriaRESUMO
OBJECTIVES: To determine whether continuous detection of adverse patient occurrences followed by analysis and medical intervention can alter the rate of adverse occurrences. DESIGN AND PARTICIPANTS: 15912 patients discharged from one hospital were reviewed in two stages. Medical records were screened retrospectively by medical records staff for one or more of eight general patient outcome criteria. Those that screened positive for the criteria were reviewed by one of four doctors. If an adverse occurrence was confirmed, further analysis and recommendations for action to prevent its recurrence were made at meetings of the four doctors, and forwarded to a committee of visiting medical officers who decided on the appropriate course of action. SETTING: A rural base hospital in Horsham, Victoria, between July 1991 and June 1994. MAIN OUTCOME MEASURES: The rate and severity of adverse patient occurrences in each year. RESULTS: 1465 records were screened positive for one or more criteria, and an adverse patient occurrence was confirmed in 155. 88 cases were determined to be minor or not preventable and further action (mostly by changes to hospital policies) was recommended for the remaining 67. Over the three years, the number of adverse occurrences fell from 69 (1.35% of all patient discharges in the first year) to 33 (0.58% of all patient discharges in the third year) (P < 0.0001) and there was no significant change in severity. CONCLUSIONS: The rate of adverse patient occurrences can be significantly reduced by their continuous detection using retrospective screening in conjunction with review, analysis and action to prevent recurrences.
Assuntos
Hospitais Rurais , Avaliação de Resultados em Cuidados de Saúde , Revisão por Pares , Garantia da Qualidade dos Cuidados de Saúde , Adolescente , Adulto , Austrália , Criança , Pré-Escolar , Interpretação Estatística de Dados , Feminino , Humanos , Lactente , Recém-Nascido , Pacientes Internados , Masculino , Prontuários Médicos , Pessoa de Meia-Idade , Estudos RetrospectivosRESUMO
Nonunion of proximal tibial osteotomy is a rare occurrence. Treatment goals should emphasize preservation of proximal tibial bone stock in view of possible subsequent total knee arthroplasty (TKA). Previous reports, in mostly smaller series, have emphasized the use of external fixation in the management of this problem. There have been no previous reports regarding the use of internal fixation in the treatment of nonunion occurring after tibial osteotomy performed proximal to the tibial tubercle. In this report, the results of internal fixation in the treatment of six cases of nonunion after proximal tibial osteotomy are analyzed with regard to functional and roentgenographic results. Healing of the nonunion was obtained in all six patients. Realignment of angulatory deformities was uniformly achieved. Achievement of union and correction of axial malalignment, in addition to creating an improved substrate for later TKA, provided other objectives as well. Elimination of pain and instability at the nonunion site allowed these patients to derive some of the intended benefits from the original osteotomy.
Assuntos
Fixação Interna de Fraturas/métodos , Fraturas não Consolidadas/etiologia , Osteotomia/efeitos adversos , Tíbia/cirurgia , Feminino , Fraturas não Consolidadas/diagnóstico por imagem , Fraturas não Consolidadas/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Radiografia , Reoperação , Tíbia/diagnóstico por imagem , Tíbia/fisiopatologia , CicatrizaçãoRESUMO
Healthcare delivery involves complex systems Preventable injuries occur more frequently than in other complex industries. Clinical risk management aims to reduce the probability of adverse patient events occurring. Adverse events can be detected by medical record review, clinical incident reporting and other methods. Events are analysed to determine latent and active errors and ranked in order of risk severity. Action is then planned and implemented to prevent the event from recurring. Effective actions include simplifying systems, standardising procedures, introducing constraints, using reminders and checklists, providing timely information, and small-group interactive education. Organisational factors that may increase the probability of successfully reducing medical errors in hospitals include the availability of adequate resources and education, the presence of clinical, executive and board of management quality champions, and quality improvement and risk management as key objectives in a hospital's strategic plan. Governments can aid the implementation of risk management programs by financially rewarding high quality care.
Assuntos
Erros Médicos/prevenção & controle , Gestão de Riscos/organização & administração , Algoritmos , Procedimentos Clínicos , Guias como Assunto , Humanos , Prontuários Médicos , Avaliação de Resultados em Cuidados de Saúde/métodos , Satisfação do Paciente , Análise e Desempenho de Tarefas , VitóriaRESUMO
OBJECTIVE: To determine if retrospective medical record screening and clinical review followed by appropriate action can effectively and efficiently detect and reduce adverse events in an emergency department. METHOD AND PARTICIPANTS: The medical records of 20 050 patients who attended the emergency department over a two year period were screened for adverse events using five general patient outcome criteria. Records that screened positive were reviewed by the hospital's clinical risk manager. If an adverse event was detected, the record was also reviewed by the director of emergency. For the first three months details of adverse events were recorded to determine a baseline adverse event rate, but no further action was taken. When an adverse event was found in the remaining 21 months, further analysis and recommendations for action to prevent a recurrence were made to relevant hospital staff. SETTING: A rural base hospital in the Wimmera region of Victoria, Australia between October 1997 and September 1999. RESULTS: Of all the patient attendances 573 (2.85%) were screened positive for one or more criteria. An adverse event was confirmed in 250 patient attendances (1.24% of all attendances). Of the adverse occurrences, 81 (32.4%) were determined to be of major severity and 169 (67.6%) of minor severity. Quality improvement activities, mostly changes to hospital policies and work processes, were implemented with the aim of preventing the recurrence of specific adverse patient events. Over two years the number of adverse events fell from 84 (3.26% of all patient attendances) in the pre-intervention quarter to 12 (0.48% of all patient attendances) in the final quarter (relative risk reduction 85.3% (95% CI, 62.7% to 100%)). CONCLUSIONS: Adverse events in emergency departments can be efficiently detected and their rate reduced using retrospective medical record screening together with clinical review, analysis and action to prevent recurrences.
Assuntos
Serviço Hospitalar de Emergência/normas , Erros Médicos/prevenção & controle , Prontuários Médicos , Garantia da Qualidade dos Cuidados de Saúde , Serviço Hospitalar de Emergência/estatística & dados numéricos , Hospitais Rurais/normas , Humanos , Auditoria Médica , Gestão de Riscos , VitóriaRESUMO
Hospital accreditation has been criticized for its emphasis on structure and documentation. Less attention is given to the clinical process and outcomes of care. How can the accreditation process become a meaningful part of day-to-day hospital management? Four approaches are discussed. (1) The development of industrial type quality assurance programmes that detect negative patient outcomes and improve patient care. (2) The appointment of a Quality Assurance/Accreditation Coordinator with appropriate authority. (3) The establishment of an effective Quality Assurance/Accreditation Committee. (4) The continuous review of accreditation standards through normal committee function and department review, and trial surveys. Such strategies will enable hospital accreditation to develop beyond a paper exercise and to provide the foundation for excellence in health care delivery.
Assuntos
Acreditação/métodos , Hospitais/normas , Garantia da Qualidade dos Cuidados de Saúde/organização & administração , Austrália , Guias como Assunto , Avaliação de Resultados em Cuidados de Saúde , Revisão dos Cuidados de Saúde por Pares/métodos , Comitê de Profissionais/organização & administraçãoRESUMO
Transformation of Mucor circinelloides is routinely achieved by using a plasmid containing the wild-type leuA gene to complement the leucine requirement of an auxotrophic host strain. As is the case for other zygomycetes, the transforming DNA is usually not integrated into the genome of M. circinelloides, but is maintained as an autonomously replicating plasmid. However, even under selective conditions, the plasmid is segregationally unstable, resulting in a rather low number of cells carrying the plasmid. We report here on a new transformation vector based on a dominant selection marker conferring resistance to geneticin, which allows for plasmid maintenance in high copy numbers. The vector was also used to transform Mucor rouxii and Rhizomucor pusillus, and should therefore be a valuable tool for gene expression studies in zygomycetes. The functionality and regulatory properties of the promoter of the M. circinelloides gpd1 gene (which codes for glyceraldehyde-3P-dehydrogenase) were demonstrated in R. pusillus using geneticin selection. In this work, we have also determined the molecular basis of the Leu(-) phenotype of the M. circinelloides host strain R7B. The leucine requirement is due to a single point mutation in the leuA gene that results in the replacement of a glutamic acid by a lysine residue.
Assuntos
Fungos/genética , Vetores Genéticos , Mucor/genética , Sequência de Aminoácidos , Sequência de Bases , Farmacorresistência Fúngica/genética , Genes Fúngicos , Gentamicinas/farmacologia , Mutação , Transformação GenéticaRESUMO
Although family-planning clinics are numerous and well-accepted in most Australian capital cities, such clinics have been difficult to establish in some Victorian rural towns. One such clinic was established at a rural base hospital in a Victorian town in 1984. The history of the clinic's development and its mode of operation are discussed. The profiles of 157 consecutive new patients who attended the clinic are presented. Comparisons are made with the patient profiles from a Melbourne family-planning clinic. The clinic also provides a separate weekly educational programme. The effect of the educational service in attracting patients to the clinic also was evaluated. Over all, it was concluded that the successful establishment of this clinic has been a result of the appointment of female medical and nursing staff members, the support of the local base hospital, an active media campaign and the development of a separate and concurrent educational programme that was conducted by clinic staff members. It is recommended that these factors be considered in the future development of such clinics in rural areas.
Assuntos
Serviços de Planejamento Familiar , Ambulatório Hospitalar/organização & administração , Educação de Pacientes como Assunto , Saúde da População Rural , Adolescente , Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Gravidez , VitóriaRESUMO
Because of trauma, metabolic bone disease, congenital deformity, or prior osteotomy, an extraarticular deformity may be present in patients requiring total knee arthroplasty. If the extraarticular deformity is not corrected extraarticularly, it must be corrected by compensatory distal femoral or proximal tibial wedge resection to produce overall limb alignment. Because such a wedge resection between the proximal and distal attachments of the collateral ligaments will produce asymmetrical ligament length, complex instabilities may result. This article, through overlay templates and trigonometric analysis, evaluates all the issues confronting the surgeon deciding whether to pursue intraarticular or extraarticular correction. The conclusions are as follows: (1) the closer a deformity is to the knee, the greater its importance, (2) femoral deformities are more difficult to correct intraarticularly than tibial deformities because femoral compensatory wedge resection produces instability only in extension, and (3) intraarticular correction of varus deformities produces lateral instability that is usually better tolerated than medial instability, and some extraarticular deformities are best treated by extraarticular correct, independent, or total knee arthroplasty.
Assuntos
Articulação do Joelho/cirurgia , Prótese do Joelho , Idoso , Artrite/complicações , Artrite/cirurgia , Artroplastia/métodos , Feminino , Fêmur/anormalidades , Humanos , Instabilidade Articular/etiologia , Masculino , Pessoa de Meia-Idade , Tíbia/anormalidadesRESUMO
The vacuolar aspartyl protease proteinase A (PrA) of Saccharomyces cerevisiae is encoded as a preproenzyme by the PEP4 gene and transported to the vacuole via the secretory route. Upon arrival of the proenzyme proPrA to the vacuole, active mature 42 kDa PrA is generated by specific proteolysis involving the vacuolar endoprotease proteinase B (PrB). Vacuolar activation of proPrA can also take place in mutants lacking PrB activity (prb1). Here an active 43 kDa species termed pseudoPrA is formed, probably by an autocatalytic process. When the PEP4 gene is overexpressed in wild-type cells, mature PrA can be found in the growth medium. We have found that prb1 strains overexpressing PEP4 can form pseudoPrA extracellularly. N-terminal amino acid sequence determination of extracellular, as well as vacuolar pseudoPrA showed that it contains nine amino acids of the propeptide, indicating a cleavage between Phe67 and Ser68 of the preproenzyme. This cleavage site is in accordance with the known substrate preference for PrA, supporting the notion that pseudoPrA is formed by autoactivation. When a multicopy PEP4 transformant of a prb1 mutant was grown in the presence of the aspartyl protease inhibitor pepstatin A, a significant level of proPrA was found in the growth medium. Our analyses show that overexpression of PEP4 leads to the secretion of proPrA to the growth medium where the zymogen is converted to pseudoPrA or mature PrA in a manner similar to the vacuolar processing reactions. Amino acid sequencing of secreted proPrA confirmed the predicted cleavage by signal peptidase between Ala22 and Lys23 of the preproenzyme.
Assuntos
Ácido Aspártico Endopeptidases/metabolismo , Proteínas Fúngicas/metabolismo , Processamento de Proteína Pós-Traducional , Saccharomyces cerevisiae/enzimologia , Vacúolos/metabolismo , Compartimento Celular , Ativação Enzimática , Mutação , Inibidores de Proteases/farmacologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae , Serina EndopeptidasesRESUMO
OBJECTIVES: To determine if an integrated clinical risk management program that detects adverse patient events in a hospital, analyses their risk and takes action can alter the rate of adverse events. DESIGN: Longitudinal survey of adverse patient events over eight years of progressive implementation of the risk management program. PARTICIPANTS AND SETTING: 49,834 inpatients (July 1991 to September 1999) and 20,050 emergency department patients (October 1997 to September 1999) at a rural base hospital in the Wimmera region of Victoria. MAIN OUTCOME MEASURES: Rates of adverse events detected by medical record review and clinical incident and general practitioner reporting. RESULTS: The annual rate of inpatient adverse events decreased between the first and eighth years of the study from 1.35% of all patient discharges (69 events) to 0.74% (49 events) (P<0.001). Absolute risk reduction was 0.61% (95% CI, 0.23%-0.99%), and relative risk reduction was 44.9% (95% CI, 16.9%-72.9%). The quarterly rate of emergency department adverse events decreased between the first and eighth quarters of monitoring from 3.26% of all attendances (84 events) to 0.48% (12 events) (P< 0.001). Absolute risk reduction was 2.78% (95% CI, 2.04%-3.52%), and relative risk reduction was 85.3% (95% CI, 62.7%-100%). CONCLUSIONS: Adverse patient events can be detected, and their frequency reduced, using multiple detection methods and clinical improvement strategies as part of an integrated clinical risk management program.
Assuntos
Competência Clínica/normas , Hospitais Rurais/normas , Doença Iatrogênica/prevenção & controle , Erros Médicos/prevenção & controle , Gestão de Riscos/organização & administração , Serviço Hospitalar de Emergência/normas , Medicina de Família e Comunidade/normas , Pesquisa sobre Serviços de Saúde , Hospitais Rurais/organização & administração , Humanos , Estudos Longitudinais , Erros Médicos/estatística & dados numéricos , Erros Médicos/tendências , Modelos Organizacionais , Avaliação de Resultados em Cuidados de Saúde , Satisfação do Paciente , Avaliação de Programas e Projetos de Saúde , Estudos Retrospectivos , Medição de Risco , Fatores de Risco , Análise de Sistemas , Vitória/epidemiologiaRESUMO
Hexose oxidase (D-hexose:O(2)-oxidoreductase, EC 1.1.3.5, HOX) normally found in the red alga Chondrus crispus was produced heterologously in different host systems. Full-length HOX polypeptide was produced in Escherichia coli, but no HOX activity could be detected. In contrast, active HOX could be produced in the methylotrophic yeast Pichia pastoris. Several growth physiological and genetic approaches for optimization of hexose oxidase production in P. pastoris were investigated. Our results indicate that specific growth conditions are essential in order to produce active HOX with the correct conformation. Furthermore, HOX seems to be activated by proteolytic cleavage of the full-length polypeptide chain into two fragments, which remain physically associated. Attempts to direct HOX to the extracellular compartment using the widely used secretion signals from Saccharomyces cerevisiae invertase or alpha-mating factor failed. However, we show in this study that HOX is transported out of P. pastoris via a hitherto unknown mechanism and that it is possible to enhance this secretion by mutagenesis from below the detection limit to at least 250 mg extracellular enzyme per liter.
Assuntos
Oxirredutases do Álcool/genética , Pichia/enzimologia , Pichia/genética , Proteínas Recombinantes/biossíntese , Rodófitas/enzimologia , Rodófitas/genética , Oxirredutases do Álcool/imunologia , Oxirredutases do Álcool/metabolismo , Oxirredutases do Álcool/efeitos da radiação , Formação de Anticorpos/genética , Especificidade de Anticorpos/genética , Western Blotting , Clonagem Molecular , Cobre/metabolismo , Meios de Cultivo Condicionados/metabolismo , Endopeptidases/metabolismo , Ativação Enzimática/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Flavina-Adenina Dinucleotídeo/metabolismo , Regulação Enzimológica da Expressão Gênica , Vetores Genéticos/síntese química , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hidrólise , Fator de Acasalamento , Mutagênese , Peptídeos/genética , Peptídeos/metabolismo , Pichia/efeitos da radiação , Dobramento de Proteína , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/imunologia , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/efeitos da radiação , Rodófitas/fisiologia , Raios Ultravioleta , beta-FrutofuranosidaseRESUMO
Yeast proteinase A is synthesized as a zymogen which transits through the endoplasmic reticulum, the Golgi complex and the endosome to the vacuole. On arrival in the vacuole, activation takes place. It has previously been found that proteinase A can activate autocatalytically; however, the propeptide of proteinase A shows essentially no similarity to other known aspartic proteinase propeptides. To understand why proteinase A activation occurs rapidly in the vacuole but not at all in earlier compartments, we have purified the zymogen and investigated the conditions that trigger autoactivation and the mechanism of autoactivation. Autoactivation was triggered by acidic pH and its rate increased with increasing ionic strength. Kinetic evidence indicates that autoactivation mainly occurs via a bimolecular product-catalysed mechanism in which an active proteinase A molecule activates a zymogen molecule. Both the pH- and ionic-strength-dependence and the predominance of a product-catalysed mechanism are well adapted to the situation in vivo, since slow activation in the absence of active proteinase A helps to prevent activation in prevacuolar compartments, whereas, on delivery to the vacuole, lower pH, higher ionic strength and the presence of already active proteinases ensure rapid activation. Product-catalysed autoactivation may be a general mechanism by which cells ensure autoactivation of intracellular enzymes to be both rapid and compartmentalized.