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Diabetes Mellitus , Saúde Global , Medicina de Precisão , Humanos , Diabetes Mellitus/terapiaRESUMO
INTRODUCTION: The difference in outcome between right (RCD) and left colonic diverticulitis (LCD) is not well established. The aim of this study was to analyse the presentation and surgical outcome of RCD versus left-sided disease following emergency surgery. METHOD: We conducted a retrospective review of patients presenting with acute diverticulitis over a 10-year period from 2004 to 2014 to a tertiary unit. Patient demographics, Hinchey classification, need for emergency surgery, perioperative outcome and recurrence were evaluated. RESULTS: In total 360 patients presented with acute diverticulitis, 218 (61%) were right-sided and 142 (39%) were left-sided. The mean age (57 yrs vs 68 yrs) and median length of stay (4 days vs 5 days) were significantly less in RCD (p < 0.001). The need for emergency surgery was similar between RCD and LCD (30.7% vs 23.2%, p = 0.12). Sixty-seven (31%) patients with RCD required emergency surgery, 42 (62.7%) of these were based on a presumptive diagnosis of appendicitis and underwent laparoscopic appendicectomy only. Operative morbidity (10.4% vs 51.5%, p < 0.001) and mortality were significantly higher in LCD (1.5% v 15.2%, p = 0.007). Subgroup analysis of non-appendicectomy, RCD patients, showed LCD were more likely to require surgery (11.5% vs 23.2%, p = 0.003). There was no difference in recurrence (p = 0.6). CONCLUSION: Right colonic diverticulitis patients are younger and disease course is more benign compared to LCD. Presentation can be confused with appendicitis without proper imaging. In the rare cases where emergency surgery is required, RCD is associated with a lower operative morbidity and mortality compared to left-sided disease.
Assuntos
Apendicite , Doença Diverticular do Colo , Diverticulite , Doença Diverticular do Colo/cirurgia , Humanos , Pessoa de Meia-Idade , Estudos Retrospectivos , Resultado do TratamentoRESUMO
BACKGROUND: Ligation of intersphincteric tract (LIFT) is a sphincter-saving technique used to treat anal fistulas. Incorporation of a bioprosthesis in LIFT (BioLIFT) aims to improve healing. The use of cross-linked porcine dermal collagen mesh Permacol™ in BioLIFT has never been investigated. The aim of this study was to compare the healing rates and outcome of LIFT and BioLIFT for complex anal fistulas using the Permacol™ biological mesh. METHODS: A retrospective analysis of all patients having LIFT or BioLIFT for complex fistulas from January 2010 to November 2019 was performed in a tertiary referral centre. Patient data from a prospectively collected database of all patients having LIFT or BioLIFT were analyzed. RESULTS: LIFT and BioLIFT were performed in 48 (82.8%) and 10 (17.2%) patients, respectively. All BioLIFT patients had previous interventions for their fistulas compared to 30 (62.5%) of patients who had LIFT, p = 0.023. The primary healing rate for LIFT was 87.5% (42/48) compared to 80% (8/10) in BioLIFT, (p = 0.42). Eight (13.8%) patients developed complications, 6 (12.5%) in the LIFT group vs 2 (20%) in the BioLIFT group (p = 0.62). On univariate analysis, the number of previous operations was predictive of complications (p = 0.03). BioLIFT was not associated with complication (OR = 1.75, 95% CI: 0.30-10.3, p = 0.54) or primary healing (OR = 0.57, 95% CI: 0.97-3.36, p = 0.54). There was no significant difference in recurrence (LIFT 12.5% vs BioLIFT 0%, p = 0.58). Kaplan-Meier analysis found no difference in time to recurrence between the two groups (p = 0.65). CONCLUSION: Permacol™ mesh in BioLIFT is feasible and achieves a high primary healing rate of 80%. Prospective evidence is needed to establish the benefits of BioLIFT and determine whether Permacol™ is superior to the non-cross-linked porcine submucosal mesh.
Assuntos
Bioprótese , Fístula Retal , Canal Anal , Animais , Colágeno , Humanos , Ligadura , Estudos Prospectivos , Recidiva , Estudos Retrospectivos , Telas Cirúrgicas , Suínos , Resultado do TratamentoRESUMO
Optimal interval between neoadjuvant chemoradiotherapy (CRT) and surgery is not elucidated for esophageal squamous carcinoma. The aim of this study is to evaluate the impact of this time interval on patient outcome. Patients treated with neoadjuvant CRT followed by surgery between 2002 and 2009 were analyzed. Patients were divided into two groups based on the median interval to surgery (64 days): A = 64 days (n = 54) and B > 64 days (n = 53). A second analysis was performed by re-classifying patients into three interval groups: A* ≤ 40 days (n = 16); B* 41-80 days (n = 60); C* > 80 days (n = 31). Operative outcome, pathological data, and long-term survival were analyzed. One hundred and seven (n = 107) patients were analyzed. Five patients (9.4%) in group B had an anastomotic leak compared with no leakage from group A (P < 0.021). The complete pathological response was comparable in groups A and B (35% vs. 24.5%, p = 0.23). R0 was significantly lower in group A* (A*: 56.3%, B*: 90%, C*: 74.2%, P = 0.006). In patients with R0 resection, 5-year survival was significantly better in group A than B (71.7% vs. 51%, P = 0.032) and in group A* (A* 100% vs. B* 60.2% & C* 48.3%; A* vs. B*, P = 0.036; A* vs. C*, P = 0.019). Complete pathological response was an independent predictor of survival. Early surgery with R0 resection following neoadjuvant CRT may lead to a better outcome. Further prospective studies are still necessary to provide better insight into the issue. At present, timing of surgery should be individualized and performed at the earliest opportunity.
Assuntos
Carcinoma de Células Escamosas/terapia , Neoplasias Esofágicas/terapia , Esofagectomia , Adulto , Idoso , Idoso de 80 Anos ou mais , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Quimiorradioterapia Adjuvante , Cisplatino/administração & dosagem , Esofagectomia/efeitos adversos , Feminino , Fluoruracila/administração & dosagem , Humanos , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Neoplasia Residual , Estudos Retrospectivos , Taxa de Sobrevida , Fatores de Tempo , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: Advanced hepatocellular carcinoma (HCC) with underlying cirrhosis poses a major operative challenge. Patients have a dismal prognosis without curative resection. The role of hepatic trisectionectomy in these patients is not established. The aim of this study was to analyze and compare the perioperative outcome and prognosis of patients undergoing trisectionectomy with hepatic resection of a lesser extent. METHODS: From 2000 to 2014, 48 patients underwent hepatic trisectionectomy for HCC with background cirrhosis or chronic hepatitis (Group A). Another (Group B) 520 patients underwent liver resection of a lesser extent. Patient demographics, clinicopathological data, perioperative outcome and long-term survival were compared between the 2 groups. RESULTS: Intraoperative bloodloss, operating time and total hospital stay were significantly higher in trisectionectomy patients. Tumors were larger and more advanced in group A. The morbidity rate was 43.8% in group A compared to 27.5% in group B, p = 0.027. In-hospital mortality was 6.3% for group A. Group A had a significantly shorter time to recurrence (4.5months vs 6.2months, p = 0.036), as well as a poorer disease-free survival (DFS) than group B (6.3 months vs 15.7 months, p = 0.02). Overall survival was comparable. Tumor number, size, albumin, INR, microvascular invasions and positive resection margins were predictors of disease-free survival. CONCLUSION: Hepatic trisectionectomy may be associated with a higher morbidity and lower DFS. However, these patients would not be suitable candidates for ablative therapy or liver transplantation. With careful patient selection and meticulous surgical technique, trisectionectomy is feasible and gives these patients the only hope of cure.
Assuntos
Carcinoma Hepatocelular/cirurgia , Hepatectomia/métodos , Neoplasias Hepáticas/cirurgia , Adulto , Idoso , Idoso de 80 Anos ou mais , Ascite/complicações , Perda Sanguínea Cirúrgica/estatística & dados numéricos , Carcinoma Hepatocelular/complicações , Carcinoma Hepatocelular/patologia , Doença Crônica , Intervalo Livre de Doença , Feminino , Hepatectomia/efeitos adversos , Hepatite/complicações , Humanos , Estimativa de Kaplan-Meier , Tempo de Internação/estatística & dados numéricos , Cirrose Hepática/complicações , Neoplasias Hepáticas/complicações , Neoplasias Hepáticas/patologia , Masculino , Margens de Excisão , Pessoa de Meia-Idade , Duração da Cirurgia , Tratamentos com Preservação do Órgão/métodos , Cuidados Pós-Operatórios , Complicações Pós-Operatórias/etiologia , Estudos Prospectivos , Cirurgia Assistida por Computador/métodos , Resultado do Tratamento , Carga Tumoral , Ultrassonografia de Intervenção/métodos , Adulto JovemRESUMO
AIM: In rectal cancer, not all tumours display a response to neoadjuvant treatment. An accurate predictor of response does not exist to guide patient-specific treatment. DNA methylation is a distinctive molecular pathway in colorectal carcinogenesis. Whether DNA methylation is altered by neoadjuvant treatment and a potential response predictor is unknown. We aimed to determine whether DNA methylation is altered by neoadjuvant chemoradiotherapy (CRT) and to determine its role in predicting response to treatment. PATIENTS AND METHODS: Fifty-three (n = 53) patients with locally advanced rectal cancers treated with neoadjuvant CRT followed by surgery were identified from the pathology databases of 2 tertiary referral centres over a 4-year period. Immunohistochemical staining of treatment specimens was carried out using the 5-Methylcytidine (Eurogentec, Seraing, Belgium) antibody. Quantitative analysis of staining was performed using an automated image analysis platform. The modified tumour regression grading system was used to assess tumour response to neoadjuvant therapy. RESULTS: Seven (13%) patients showed complete pathological response while 46 (87%) patients were partial responders to neoadjuvant treatment. In 38 (72%) patients, significant reduction in methylation was observed in post-treatment resection specimens compared to pre-treatment specimens (171.5 vs 152.7, p = 0.01); in 15 (28%) patients, methylation was increased. Pre-treatment methylation correlated significantly with tumour regression (p < 0.001), T-stage (p = 0.005), and was able to predict complete and partial pathological responders (p = 0.01). CONCLUSION: Neoadjuvant CRT appears to alter the rectal cancer epigenome. The significant correlation between pre-treatment DNA methylation with tumour response suggests a potential role for methylation as a biomarker of response.
Assuntos
Adenocarcinoma/terapia , Metilação de DNA/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Neoplasias Retais/terapia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Quimiorradioterapia Adjuvante , Estudos de Coortes , Metilação de DNA/genética , Metilação de DNA/efeitos da radiação , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Masculino , Pessoa de Meia-Idade , Terapia Neoadjuvante , Projetos Piloto , Prognóstico , Neoplasias Retais/genética , Neoplasias Retais/patologia , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Esophagectomy remains the mainstay of treatment for esophageal cancer. The stomach is the commonest organ used to restore intestinal continuity after esophagectomy. Metachronous gastric cancer in the gastric conduit after esophagectomy is rare; the etiology remains unclear. Possible risk factors include Helicobacter pylori infection, biliary or pancreatic reflux and prior radiotherapy. Prognosis of these patients remains poor. Treatment of this particular entity poses unique challenges to the surgeon and oncologist. Early diagnosis by endoscopy may allow endoscopic excision such as endoscopic mucosal resection or endoscopic submucosal dissection. In more advanced cancers, surgery is difficult, reconstruction is complicated, and further radiation may not be feasible because of previous neoadjuvant therapy. In this report, four patients who developed gastric conduit cancers are presented. They were treated with either surgery alone or combined with chemoradiotherapy. All four patients were still alive after at least 21 months, with three patients currently still alive (21-48 months). The literature is also reviewed, in particular addressing the incidence, possible underlying causes, prognosis and options of treatment for this specific clinical scenario.
Assuntos
Adenocarcinoma/cirurgia , Carcinoma de Células em Anel de Sinete/cirurgia , Carcinoma de Células Escamosas/cirurgia , Neoplasias Esofágicas/cirurgia , Segunda Neoplasia Primária/cirurgia , Neoplasias Gástricas/cirurgia , Idoso , Esofagectomia , Feminino , Gastrectomia , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVES: There is lack of consensus regarding concurrent vs. staged approaches, and the prioritisation of staged procedures in cases presenting with colorectal carcinoma (CRC) and abdominal aortic aneurysm (AAA) synchronously. We aim to present our experience, review the literature on this therapeutic dilemma and examine the role of endovascular aortic repair (EVAR). DESIGN, MATERIALS AND METHODS: An observational study of the experience of two centres and a systematic review of the published literature. RESULTS: Twenty-four patients were identified from the prospective databases of two tertiary referral centres between 2001 and 2006. Intervention for both malignancy and aneurysm was performed in 13 patients. In 10 patients, cancer resection was performed initially and was followed by open aneurysm repair (n=3) or EVAR (n=7). Two patients (AAA diameters: 7.0 and 8.0cm) underwent EVAR prior to colonic resection. One patient was selected for synchronous surgery. There were no interval AAA ruptures, graft infection or postoperative mortalities. Literature review identified 269 such cases; of these 101 were treated by combined surgery. In staged surgery, there were nine interval aneurysmal ruptures and one aortic graft infection. CONCLUSIONS: In our experience, staged management can be undertaken, without interval aneurysmal rupture. EVAR has an evolving role in preventing delay in CRC management, in high-risk patients, and during combined intervention.
Assuntos
Aneurisma da Aorta Abdominal/complicações , Neoplasias Colorretais/complicações , Complicações Intraoperatórias/epidemiologia , Complicações Pós-Operatórias/epidemiologia , Idoso , Idoso de 80 Anos ou mais , Aneurisma da Aorta Abdominal/diagnóstico , Aneurisma da Aorta Abdominal/cirurgia , Ruptura Aórtica/epidemiologia , Colectomia/métodos , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/cirurgia , Feminino , Seguimentos , Humanos , Tempo de Internação , Masculino , Pessoa de Meia-Idade , Morbidade/tendências , Estadiamento de Neoplasias/métodos , Prognóstico , Estudos Prospectivos , Taxa de Sobrevida/tendências , Tomografia Computadorizada por Raios X , Reino Unido/epidemiologia , Procedimentos Cirúrgicos Vasculares/métodosRESUMO
BACKGROUND: Shortened trainingtimes duetothe European Working Time Directive (EWTD) and increased public scrutiny of surgical competency have led to a move away from the traditional apprenticeship model of training. Virtual reality (VR) simulation is a fascinating innovation allowing surgeons to develop without the need to practice on real patients and it may be a solution to achieve competency within a shortened training period. METHOD: A Medline search was performed to identify studies and commentaries on the use of VR simulators in endovascular training. FINDINGS: Three studies on carotid stenting and four on peripheral vascular angioplasty demonstrate that simulator training is a valid, feasible and acceptable training tool. One randomised study reports that these skills learned on simulators are transferable to the operating room. CONCLUSION: VR simulators have a role in competency based, structured training of vascular interventionalists and should improve patient safety.
Assuntos
Simulação por Computador , Educação Médica Continuada/métodos , Endoscopia/educação , Interface Usuário-Computador , Doenças Vasculares/cirurgia , Procedimentos Cirúrgicos Vasculares/educação , Competência Clínica , HumanosRESUMO
Amino and carboxyl terminal deletion derivatives of dehalogenase IVa (DehIVa) of Burkholderia cepacia MBA4 were constructed and analyzed for enzyme activity and for protein integrity. The results suggested that the majority of the protein is indispensable. Point mutations on 29 conserved charged and/or polar residues were generated and characterized. Derivatives D11E, D11N, D11S and D181N were totally inactive while mutant N178D was defective in catalysis. Mutations of other conserved residues displayed varying effects. Mutation that enhances DehIVa activity has been shown to be inhibitory in other dehalogenase and essential conserved residues in DehIVa have been shown to be dispensable in others. This suggests there is no general rule for the importance of these conserved residues.
Assuntos
Burkholderia cepacia/enzimologia , Deleção de Genes , Hidrolases/química , Hidrolases/genética , Mutação Puntual , Western Blotting , Burkholderia cepacia/genética , Burkholderia cepacia/crescimento & desenvolvimento , Genes Bacterianos , Hidrolases/metabolismoRESUMO
The most important product of the sequencing of a genome is a complete, accurate catalogue of genes and their products, primarily messenger RNA transcripts and their cognate proteins. Such a catalogue cannot be constructed by computational annotation alone; it requires experimental validation on a genome scale. Using 'exon' and 'tiling' arrays fabricated by ink-jet oligonucleotide synthesis, we devised an experimental approach to validate and refine computational gene predictions and define full-length transcripts on the basis of co-regulated expression of their exons. These methods can provide more accurate gene numbers and allow the detection of mRNA splice variants and identification of the tissue- and disease-specific conditions under which genes are expressed. We apply our technique to chromosome 22q under 69 experimental condition pairs, and to the entire human genome under two experimental conditions. We discuss implications for more comprehensive, consistent and reliable genome annotation, more efficient, full-length complementary DNA cloning strategies and application to complex diseases.
Assuntos
Cromossomos Humanos Par 22 , Biologia Computacional , Genoma Humano , Análise de Sequência com Séries de Oligonucleotídeos , Algoritmos , Processamento Alternativo , Linhagem Celular , DNA Complementar , Éxons , Projeto Genoma Humano , Humanos , Sondas de OligonucleotídeosRESUMO
Haloacid dehalogenases are enzymes that catalyze the hydrolytic removal of halogens from haloalkanoic acids. Dehalogenase IVa (DehIVa) from Burkholderia cepacia MBA4 and dehalogenase CI (DehCI) from Pseudomonas sp. strain CBS3 exhibit 68% identity. Despite their similarity DehIVa is a dimeric enzyme while DehCI is a monomer. In this work, we describe the identification of the domain that confers the dimerization function of DehIVa. Recombinant DNA molecules were constructed by fusion of the respective dehalogenase genes hdlIVa and dehCI. When amino acids 73 to 89 of DehCI were replaced by amino acids 74 to 90 of DehIVa, the recombinant molecule migrated like that of DehIVa in a nondenaturing activity-stained gel. Similarly, when residues 73 to 89 of DehIVa were replaced by the corresponding residues of DehCI, the chimera migrated as a monomer. These 17 amino acid changes were able to determine the aggregation states of the molecules. The retention of the catalytic function in these chimeras indicated that the overall folding of these proteins was not affected. Site-directed mutagenesis on hdlIVa however indicated that amino acids Phe58, Thr65, Leu78, and Phe92 of DehIVa are also important for the aggregation state of the protein. This indicates that the 17 residues are not sufficient for the dimerization of the protein.
Assuntos
Burkholderia cepacia/enzimologia , Hidrolases/química , Hidrolases/metabolismo , Western Blotting , Burkholderia cepacia/química , Burkholderia cepacia/genética , Dimerização , Eletroforese em Gel de Poliacrilamida , Hidrolases/genética , Mutagênese Sítio-Dirigida , Plasmídeos , Reação em Cadeia da Polimerase , Estrutura Terciária de Proteína , Pseudomonas/química , Pseudomonas/enzimologia , Pseudomonas/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Análise de Sequência de DNARESUMO
Flow injection titration was used for the determination of anionic, cationic, nonionic and zwitterionic surfactants. The procedure was based on the micellar-enhanced fluorescence of 1,8-anilino-naphthalene sulfonate (ANS). Samples were injected into a carrier stream of phosphate buffer and 1.0 mol l(-1) NaCl. The sample then passed through a mixing chamber which generated the exponential peak shape needed for the titration as well as diluted the sample in the carrier stream to control the pH and ionic strength of the sample. The peak width was linearly related to the logarithm of the surfactant concentration. The minimum detectable concentration was governed by the critical micelle concentration for anionic, zwitterionic and nonionic surfactants, but below the critical micelle concentration for cationic surfactants. The linear range extended for approximately 1.5 orders of magnitude. Reproducibility ranged from 12% at the lower end of the calibration range to 1.1% at higher concentrations. For SDS recoveries of 82-108% were achieved in matrices as concentrated as 1 mol l(-1) in NaCl or Na(2)SO(4).
RESUMO
Burkholderia cepacia MBA4 has been shown to produce a single dehalogenase batch culture. Moreover, other cryptic dehalogenases were also detected when the cells were grown in continuous culture. In this paper, we report the cloning and characterization of one of the cryptic dehalogenases in MBA4. This cryptic haloacid dehalogenase, designated Chd1, was expressed constitutively in Escherichia coli. This recombinant Chd1 had a relative molecular weight of 58,000 and existed predominantly as a dimer. The subunits had a relative molecular weight of 27,000. Chd1 exhibited isomer specificity, being active towards the L-isomer of 2-monochloropropionic acid only. The structural gene, chd1, was isolated on a 1.7-kb PstI fragment. This fragment contains a functional promoter, because expression of chd1 in E. coli is orientation independent. The nucleotide sequence of this fragment was determined and characterized. An open reading frame of 840 bp encoding a putative peptide of 280 amino acids was identified. This corresponds closely with the size of the subunit. The nucleotide sequence of chd1 did not show any homology with those of other dehalogenase genes. Comparison of the predicted amino acid sequence, however, shows significant homology, ranging from 42 to 50%, with the amino acid sequences of many other dehalogenases. Chd1 is unusual in having a long leader sequence, a property of periplasmic enzymes.
Assuntos
Burkholderia cepacia/genética , Hidrolases/genética , Sequência de Aminoácidos , Sequência de Bases , Burkholderia cepacia/enzimologia , Clonagem Molecular , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Biblioteca Genômica , Halogênios/metabolismo , Hidrolases/biossíntese , Dados de Sequência Molecular , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Recombinantes/biossíntese , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
CPF1 is an abundant basic-helix-loop-helix-ZIP protein that binds to the CDEI motif in Saccharomyces cerevisiae centromeres and in the promoters of numerous genes, including those encoding enzymes of the methionine biosynthetic pathway. Strains lacking CPF1 are methionine auxotrophs, and it has been proposed that CPF1 might positively influence transcription at the MET25 and MET16 genes by modulating promoter chromatin structure. We test this hypothesis and show that the regions surrounding the CDEI motifs in the MET25 and MET16 promoters are maintained in a nucleosome-free state and that this requires the entire CPF1 protein. However, the chromatin structure around the CDEI motifs does not change on derepression of transcription and does not correlate with the methionine phenotype of the cell. An intact CDEI motif but not CPF1 is required for transcriptional activation from a region of the MET25 upstream activation sequence. Our results suggest that CPF1 functions to modulate chromatin structure around the CDEI motif but that these changes at the MET25 and MET16 promoters do not explain how CPF1 functions to maintain methionine-independent growth. The presence of CPF1-dependent chromatin structures at these promoters leads to a weak repression of transcription.
Assuntos
Centrômero/ultraestrutura , Cromatina/ultraestrutura , Proteínas de Ligação a DNA/fisiologia , Proteínas Fúngicas/fisiologia , Regiões Promotoras Genéticas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/ultraestrutura , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Sequências Hélice-Alça-Hélice , Zíper de Leucina , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , RNA Mensageiro/genética , Saccharomyces cerevisiae/genética , Transcrição GênicaRESUMO
CPF1 is a basic helix-loop-helix (bHLH) protein required for optimal centromere function and for maintaining methionine independent growth in yeast. In this work, we show that the region carboxy-terminal to the bHLH domain of CPF1 is essential for CPF1 function in the cell and for dimerisation of CPF1 in solution. The C-terminus of CPF1 contains a potential long amphipathic helix with a hydrophobic face which could provide a suitable protein:protein interface. Point mutations in residues forming this hydrophobic face are sufficient to weaken the interaction between the protein and DNA. By fusing the DNA binding domain or the transcriptional activation domain of GAL4 to the C-terminal 87 amino acids of CPF1, we show that this region is sufficient for mediating protein:protein interactions in vivo. The C-terminal domain of CPF1 can be replaced by the leucine repeat region of the bHLH-ZIP protein USF and the hybrid CPF1-USF protein functions in vivo to provide normal centromere function and methionine independent growth. However, the CPF1-USF hybrid protein is unable to interact with CPF1 suggesting that a dimer of CPF1 is sufficient for maintaining methionine independent growth and normal centromere function.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Zíper de Leucina/genética , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Sequência de Aminoácidos , Sequência de Bases , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Substâncias Macromoleculares , Dados de Sequência Molecular , Plasmídeos/genéticaRESUMO
The structural gene (hdl IVa) for the Pseudomonas cepacia MBA4 2-haloacid halidohydrolase IVa (Hdl IVa) was isolated on a 1.6 kb fragment of Ps. cepacia MBA4 chromosomal DNA. The recombinant halidohydrolase was expressed in Escherichia coli and Pseudomonas putida and the structural gene was subcloned on to the tac expression vector pBTac1. High-level expression from the tac promoter was seen to be temperature-dependent, a consequence of the nucleotide sequence adjacent to the fragment encoding the halidohydrolase. The nucleotide sequence of the fragment encoding the Hdl IVa was determined and analysed. Three ATG codons were identified in one of the open reading frames and the one corresponding to the start of the hdl IVa structural gene was determined by comparison of the predicted amino acid sequences with the experimentally determined N-terminal sequences of halidohydrolase IVa. The hdl IVa gene encoded a 231-amino acid-residue protein of M(r) 25,900. The sequence and predicted structural data are discussed and comparison is made with sequence data for other halidohydrolases.
Assuntos
Burkholderia cepacia/enzimologia , Genes Bacterianos/genética , Hidrolases/genética , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Burkholderia cepacia/genética , Escherichia coli/enzimologia , Escherichia coli/genética , Expressão Gênica , Dados de Sequência Molecular , Pseudomonas putida/enzimologia , Pseudomonas putida/genéticaRESUMO
Yeast repressor activator protein 1 (RAP1) binds in vitro to specific DNA sequences that are found in diverse genetic elements. Expression of the yeast phosphoglycerate kinase gene (PGK) requires the binding of RAP1 to the activator core sequence within the upstream activating sequence (UAS) of PGK. A DNA fragment Z+ which contains the activator core sequence of the PGK(UAS) has been shown to bind RAP1. Here we report that phosphatase treatment of RAP1 affected its binding to the PGK(UAS) but that this depended on the nature of the sequence flanking the 5' end of the activator core sequence. When the sequence flanking the 5' end of the activator core sequence was different from the PGK RAP1-binding site, phosphatase treatment of RAP1 decreased its binding to the DNA. When the 5' end of the binding site was a match to the PGK RAP1-binding site dephosphorylation of RAP1 increased RAP1 binding to the DNA. These observations were reproduced when the minimal functional DNA-binding domain of the RAP1 protein was used, implicating a phosphorylation-dependent binding of RAP1. This is the first evidence for phosphorylation-dependent binding of RAP1.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica , Fosfoglicerato Quinase/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , DNA Fúngico/genética , DNA Fúngico/metabolismo , Genes Fúngicos , Dados de Sequência Molecular , Fosfoglicerato Quinase/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , Fosforilação , Sequências Reguladoras de Ácido Nucleico , Saccharomyces cerevisiae/enzimologiaRESUMO
The UAS of the yeast gene encoding the glycolytic enzyme phosphoglycerate kinase (PGK) contains several different sequence elements involved in transcriptional activation. These elements include the activator core sequence, which is bound by the RAP1 protein, and three copies of the pentamer sequence 5' CTTCC 3'. Upstream of the activator core sequence is a region (Yfp), identified as the site of a strong DNA-protein interaction. The Yfp region contains the consensus binding site for the factor ABF1. We have purified the Y protein, which binds to the Yfp region, to homogeneity. The Y protein migrates as a doublet on SDS-polyacrylamide gel electrophoresis with an apparent molecular weight of 125 KDa. These properties are similar to those of ABF1. ABF1 synthesised in vitro bound strongly to the Yfp region and formed a gel retardation complex of identical mobility to the complex formed by the Y protein. UAS1 of the pyruvate kinase gene (PYK1) promoter contains a RAP1 binding site and single copy of the CTTCC sequence. We have now identified an ABF1 binding site close to the RAP1 binding site and CTTCC sequence in the PYK1 promoter. This site is strongly bound by ABF1 in vitro. The organisation of the PGK and PYK1 UASs is thus similar to each other and to the transcriptional silencer HMR(E) which also contains these sequences.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Regulação Fúngica da Expressão Gênica , Fosfoglicerato Quinase/genética , Piruvato Quinase/genética , Saccharomyces cerevisiae/genética , Fatores de Transcrição/metabolismo , Sequência de Bases , Sítios de Ligação , Ligação Competitiva , Proteínas de Transporte/isolamento & purificação , Proteínas de Transporte/metabolismo , DNA/genética , DNA/metabolismo , Genes Fúngicos , Dados de Sequência Molecular , Fosfoglicerato Quinase/metabolismo , Regiões Promotoras Genéticas , Piruvato Quinase/metabolismo , Sequências Reguladoras de Ácido Nucleico , Proteínas rap de Ligação ao GTPRESUMO
The 827 amino acid yeast RAP1 protein interacts with DNA to regulate gene expression at numerous unrelated loci in the yeast genome. By a combination of amino, carboxy and internal deletions, we have defined an internal 235 amino acid fragment of the yeast RAP1 protein that can bind efficiently to the RAP1 binding site of the PGK Upstream Activation Sequence (UAS). This domain spans residues 361 to 596 of the full length protein and lacks any homology to the DNA binding 'zinc finger' or 'helix-turn-helix' structural motifs. All the RAP1 binding sites we have tested bind domain 361-596, arguing that RAP1 binds all its chromosomal sites via this domain. The domain could not be further reduced in size suggesting that it represents the minimal functional DNA binding domain. The relevance of potential regions of secondary structure within the minimal binding domain is discussed.