RESUMO
The methylotrophic yeast Pichia pastoris has been used extensively for expressing recombinant proteins because it combines the ease of genetic manipulation, the ability to provide complex posttranslational modifications and the capacity for efficient protein secretion. The most successful and commonly used secretion signal leader in Pichia pastoris has been the alpha mating factor (MATα) prepro secretion signal. However, limitations exist as some proteins cannot be secreted efficiently, leading to strategies to enhance secretion efficiency by modifying the secretion signal leader. Based on a Jpred secondary structure prediction and knob-socket modeling of tertiary structure, numerous deletions and duplications of the MATα prepro leader were engineered to evaluate the correlation between predicted secondary structure and the secretion level of the reporters horseradish peroxidase (HRP) and Candida antarctica lipase B. In addition, circular dichroism analyses were completed for the wild type and several mutant pro-peptides to evaluate actual differences in secondary structure. The results lead to a new model of MATα pro-peptide signal leader, which suggests that the N and C-termini of MATα pro-peptide need to be presented in a specific orientation for proper interaction with the cellular secretion machinery and for efficient protein secretion.
Assuntos
Proteínas Fúngicas/genética , Fator de Acasalamento/genética , Peptídeos/genética , Pichia/genética , Proteínas Recombinantes de Fusão/genética , Sequência de Aminoácidos , Dicroísmo Circular , Eletroforese em Gel de Poliacrilamida , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Peroxidase do Rábano Silvestre/genética , Peroxidase do Rábano Silvestre/metabolismo , Lipase/genética , Lipase/metabolismo , Fator de Acasalamento/química , Fator de Acasalamento/metabolismo , Modelos Moleculares , Mutação , Peptídeos/química , Peptídeos/metabolismo , Pichia/metabolismo , Precursores de Proteínas/química , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Sinais Direcionadores de Proteínas/genética , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/metabolismo , Deleção de SequênciaRESUMO
The Ku70/Ku80 heterodimer plays a crucial role in non-homologous end-joining during DNA repair, and is also involved in multiple cellular processes such as telomere maintenance, transcription, and apoptosis. In this study, we investigate the regulation of AtKu genes in higher plants. Promoters of the AtKu70 and AtKu80 were isolated from Arabidopsis and their activities characterised using GUS reporter constructs. AtKu promoter activities were relatively higher in hypocotyls and cotyledons upon germination and in stigma and siliques as well at their early developing stages. Furthermore, AtKu promoter activities could be enhanced by gibberellic acid, auxins, and jasmonic acid, but repressed by abscisic acid, salicylic acid, heat, drought and cold, respectively. Deletion analysis demonstrates minimal lengths of ~400 bp and 600 bp upstream of transcription start site for functional promoters of AtKu70 and AtKu80, respectively. Taken together, expressions of Ku genes are regulated both by developmental programs as well as by plant hormones and environmental stresses.
RESUMO
BACKGROUND: Pain is often inadequately treated in patients with cancer. Previous studies have demonstrated the effectiveness of the World Health Organization (WHO) analgesic ladder in cancer pain management. METHODS: A total of 131 consecutive patients with advanced cancer referred to a hospice home care program were enrolled over one year period from Jan. 1 to Dec. 31, 2000. We assessed the adequacy of prescribed analgesic drugs using guidelines developed by the WHO. Age, gender, Eastern Cooperative Oncology Group performance status, pain mechanism at referral, pain and symptom intensity, and doses and days of drug administration during the course of treatment were recorded at regular intervals. RESULTS: Eighty-two percent of the patients (107 of 131) had pain symtoms at referral. Forty-seven patients were excluded from this study due to inadequate follow-up times or inability to express the pain intensity. Sixty patients who had measurable pain intensity requiring analgesic therapy were followed up until death for a mean duration of 65 days. At referral, 46% of the patients (28 of 60) received inadequate treatment. In the last week of life, 2%, 26% and 70% of patients were taking non-opioid drugs, moderate opioids and strong opioids, respectively. A significant improvement in pain and symptom intensity was achieved after referral. A minority of the patients (10%) had inadequate pain control in the last week of life. CONCLUSIONS: This study demonstrates that a managed hospice home care system enables patients to receive adequate pain treatment, according to WHO guidelines.