RESUMO
OBJECTIVES: Self-expanding metal stents (SEMS) are increasingly being used to treat malignant colorectal obstruction. However, complications have been reported in up to 50% of patients. There is limited information on long-term outcomes of these patients. The aim of this study was to retrospectively assess the long-term clinical success of SEMS in patients with malignant colorectal obstruction in a single tertiary center and to identify possible predictive factors of developing complications. METHODS: A total of 47 attempts to insert colorectal SEMS were made in 47 patients during a 5-year period. Stents of 9-cm length were placed under endoscopic and radiologic monitoring. After 24 h, all patients underwent abdominal X-ray to verify correct positioning of the stent. Patients were followed at the outpatient clinic. RESULTS: Insertion success was achieved in 44 (94%) patients. Acceptable initial colonic decompression was observed in 44 out of 47 (94%) attempts and in all (100%) successfully inserted stents. The stents were placed in the rectum (n=7, 15%), sigmoid (n=33, 70%), left colon (n=4, 9%), or anastomosis (n=3, 6%). The majority of patients had stage IV disease (n=40, 85%). SEMS served as a bridge to scheduled surgery in 9 (20%) patients and as a palliative definitive treatment in 38 (80%) cases. Three patients were lost to follow-up, so the outcome was evaluated in 41 patients. Long-term clinical failure occurred in 21 (51%) patients and was due to complications such as: migration (n=9, 22%), obstruction (n=7, 17%), perforation (n=3, 7%), and tenesmus (n=2, 5%). Perforations occurred 3, 4, and 34 days after insertion, and all patients died. In the bridge-to-surgery group, primary anastomosis was possible in only four of nine patients (44%). Clinical failure was not associated with any tumor-related factor. However, eight of nine patients with stent migration and two of three patients with perforation had been previously treated with chemotherapy. CONCLUSIONS: Placement of SEMS does not seem to be as effective as suggested because of late complications. For patients with potentially curable lesions, the use of colonic stents for malignant obstruction should only be considered when surgery is scheduled shortly after the stent insertion. Moreover, in patients with incurable obstructing colorectal cancer eligible for chemotherapy and a long life expectancy, palliative treatments other than SEMS should be considered.
Assuntos
Neoplasias Colorretais/complicações , Obstrução Intestinal/cirurgia , Cuidados Paliativos/métodos , Falha de Prótese , Stents/efeitos adversos , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Coortes , Colonoscopia/efeitos adversos , Colonoscopia/métodos , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Feminino , Seguimentos , Humanos , Obstrução Intestinal/etiologia , Obstrução Intestinal/mortalidade , Obstrução Intestinal/patologia , Estimativa de Kaplan-Meier , Masculino , Metais , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Complicações Pós-Operatórias/diagnóstico , Complicações Pós-Operatórias/mortalidade , Valor Preditivo dos Testes , Probabilidade , Desenho de Prótese , Estudos Retrospectivos , Medição de Risco , Estatísticas não Paramétricas , Fatores de Tempo , Resultado do TratamentoRESUMO
BACKGROUND AND AIM: The diagnosis of gastrointestinal stromal tumors (GISTs) has important prognostic and therapeutic implications. The specific diagnosis of GIST has to be based on immunocytochemistry. This study aimed to prospectively compare in a crossover manner the accuracy of endoscopic ultrasound (EUS)-guided fine-needle aspiration (EUS-FNA) and EUS-guided trucut biopsy (EUS-TCB) in the specific diagnosis of gastric GISTs. We hypothesized that EUS-TCB is superior to EUS-FNA in this respect. PATIENTS AND METHODS: Forty patients with gastric subepithelial tumors suspected on the basis of EUS of being a GIST underwent both EUS-FNA and EUS-TCB. The sequence in which the techniques were employed was randomly assigned to avoid bias. RESULTS: Forty tumors were sampled (mean number of passes: 2.1 +/- 0.9 with EUS-TNB and 1.9 +/- 0.8 with EUS-FNA; P = not significant, NS). Final diagnoses were: GIST (n = 27), carcinoma (n = 2), leiomyoma (n = 1), schwannoma (n = 1), and no diagnosis possible (n = 9). Device failure occurred in 6 patients with EUS-TCB. A cytohistological diagnosis of mesenchymal tumor (n = 29) and carcinoma (n = 2) was made in 70 % of cases by EUS-FNA and in 60 % of cases by EUS-TCB ( P = NS). Among the samples that were adequate, immunohistochemistry could be performed in 74 % of EUS-FNA samples and in 91 % of EUS-TCB samples ( P = 0.025). When inadequate samples were included, the overall diagnostic accuracy of EUS-FNA was 52 % and that of EUS-TCB was 55 % ( P = NS). There were no complications. CONCLUSIONS: EUS-TCB is not superior to EUS-FNA in GISTs because of the high rate of technical failure of trucut. However, when an adequate sample is obtained with EUS-TCB, immunohistochemical phenotyping is almost always possible. EUS-TCB can be safely performed in this set of patients.
Assuntos
Biópsia por Agulha , Endossonografia , Tumores do Estroma Gastrointestinal/diagnóstico , Neoplasias Gástricas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha Fina , Estudos Cross-Over , Feminino , Tumores do Estroma Gastrointestinal/patologia , Gastroscopia , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Neoplasias Gástricas/patologiaRESUMO
Synchrotron radiation techniques have enabled us to record meridional x-ray diffraction patterns from frog sartorius muscle at resolutions ranging from approximately 2,800 to 38 nm (i.e., overlapping with the optical microscope and the region normally accessible with low angle diffraction cameras). These diffraction patterns represent the transform of the low resolution structure of muscle projected on the sarcomere axis and sampled by its repeat. Altering the sarcomere length results in the sampling of different parts of this transform, which induces changes in the positions and the integrated intensities of the diffraction maxima. This effect has been used to determine the transform of the mass projection on the muscle axis in a quasicontinuous fashion. The results reveal the existence of maxima arising from long-range periodicities in the structure. Determination of the zeroes in the transforms has been used to obtain phase information from which electron density maps have been calculated. The x-ray diffraction diagrams and the resulting electron density maps show the existence of a series of mass bands, disposed transversely to the sarcomere axis and distributed at regular intervals. A set of these transverse structures is associated with thin filaments, and their 102.0-nm repeat suggests a close structural relationship with their known molecular components. A second set, spaced by approximately 230.0 nm, is also present; from diffraction theory one has to conclude that this repeat simultaneously exists in thick and thin filament regions.
Assuntos
Músculos/ultraestrutura , Miofibrilas/ultraestrutura , Sarcômeros/ultraestrutura , Animais , Rana esculenta , Difração de Raios X/métodosRESUMO
Wireless capsule endoscopy is a new technique that allows complete exploration of the small bowel without external wires. Its role has been analyzed in many small bowel diseases such as obscure gastrointestinal bleeding, Crohn's disease and gastrointestinal polyposis syndromes with promising results. Studies on other pathologies (i.e. small bowel tumour, celiac disease) are under evaluation to define the role of this technique.
Assuntos
Endoscopia por Cápsula/métodos , Hemorragia Gastrointestinal/terapia , Doenças Inflamatórias Intestinais/terapia , Doença Celíaca/metabolismo , Ensaios Clínicos como Assunto , Endoscopia , Trato Gastrointestinal/patologia , Hemorragia , Humanos , Sensibilidade e EspecificidadeRESUMO
OBJECTIVES: To evaluate the preconditioning effect of sildenafil administered preoperatively in kidneys subjected to a period of warm ischemia (WI), hypothermic perfusion (HP) or cold storage (CS) and finally, autotransplant (AT). MATERIAL AND METHOD: We studied 6 groups of autotransplanted kidneys: no-WI-inmediate AT (Group A); 45 min of WI + immediate AT (Group B); 45 min of WI + 60 min of HP + autotransplant (Group C); 45 min of WI + 60 min of CS + autotransplant (Group D); 100 mg of oral sildenafil preoperatively + 45 min of WI + autotransplant (Group E); 100 mg of oral sildenafil preoperatively + 45 min of WI+60 min of HP + autotransplant (Group F). Belzer solution was used for HP; UW-Viaspan for CS. Inmediately after the autotransplant (reperfusion period), we recorded in real time for 60 min the values of Renal vascular Flow (RVF) and Renal Vascular Resistance (RVR). Nitric Oxide levels in the cava and renal graft vein were recorded every 15 min during the 60 min of the reperfusion-study period. Conventional & Electronic microscopy were completed after the process. RESULTS: We obtained significant higher values of RVF and lower values of RVR in sildenafil groups (E and F) in comparison to the other groups (A-D) (Table 1). NO levels were also significantly higher in groups E and F (Fig. 1). Groups A, B, E and F showed integrity of tubule and endothelium in comparison to groups C and D in the microscopic study. CONCLUSIONS: We showed a beneficious effect of sildenafil in inmediate post-transplant reperfusion hemodynamic and biochemical parameters of kidneys subjected to a critical period of warm-ischemia.
Assuntos
Precondicionamento Isquêmico/métodos , Transplante de Rim , Piperazinas/uso terapêutico , Sulfonas/uso terapêutico , Isquemia Quente , Animais , Purinas/uso terapêutico , Citrato de Sildenafila , SuínosRESUMO
INTRODUCTION: Delayed graft function alter living donor transplantation is a subject of debate. Delayed graft function can be partially explained by renal ischemia-reperfusion injury, when severe is associated with decreased graft survival. In this experimental living donor model study, we analyze the hemodynamic, histological and biochemical effects of laparoscopic nephrectomy. We also, analyze the effect of a pulsatile machine perfusion for kidney preservation during cold ischemia time. MATERIAL AND METHODS: Twenty large-white pigs (average weight 40-45 kgrs) were divided in 4 experimental groups: Group A: Laparoscopic nephrectomy+ immediate graft perfusion in pulsatile vacuum pump+autotransplant Group B: Laparoscopic nephrectomy+ immediate graft perfusion by gravity+autotransplant Group C: Open nephrectomy+immediate graft perfusion in pulsatile vacuum pump+autotransplant Group D: Open nephrectomy+ immediate graft perfusion by gravity+autotransplant Both laparoscopic and open nephrectomy were completed transperitoneally according to standardized technique. Hypothermic perfusion was done in a system designed in our lab. RESULTS: We observed a decreased renal artery flow in kidneys procured laparoscopically compared to open nephrectomy. We found an artery flow recovery during the first 60 minutes after revascularization. Renal machine perfusion during cold ischemia time seems to have no beneficial effect, but shows a deleterious effect on hemodynamic event for renal transplantation. Lower plasma nitric oxide level is observed in kidneys obtained by laparoscopy compared with open surgical technique. And finally, we also found higher histological damage in proximal tubular and endothelial cell, in kidneys obtained by laparoscopy compared with open surgery. CONCLUSIONS: In our experience: Laparoscopic nephrectomy versus open nephrectomy produces, in a model of living donor transplant, a lower value or renal blood flow and a higher value of renal vascular resistanse. These hemodynamic findings tend to normalize by 60 min after the reperfusion. A lower blood concentration of nitric oxide after the transplant was detected in laparoscopic group Vs open surgery group.
Assuntos
Transplante de Rim , Laparoscopia , Nefrectomia/efeitos adversos , Nefrectomia/métodos , Preservação de Órgãos/métodos , Traumatismo por Reperfusão/etiologia , Animais , Doadores Vivos , SuínosRESUMO
OBJECTIVE: The primary aim of this systematic review was to determine if inertial flywheel resistance training is superior to gravity-dependent resistance training in improving muscle strength. The secondary aim was to determine whether inertial flywheel resistance training is superior to gravity-dependent resistance training in improving other muscular adaptations. DESIGN: A systematic review with meta-analyses of randomised and non-randomised controlled trials. METHODS: We searched MEDLINE, Scopus, SPORTDiscus, Web of Science and Cochrane Central Register of Controlled Trials with no publication date restrictions until November 2016. We performed meta-analyses on randomised and non-randomised controlled trials to determine the standardized mean difference between the effects of inertial flywheel and gravity-dependent resistance training on muscle strength. A total of 76 and 71 participants were included in the primary and secondary analyses, respectively. RESULTS: After systematic review, we included three randomised and four non-randomised controlled trials. In the primary analysis for the primary outcome muscle strength, the pooled results from randomised controlled trials showed no difference (SMD=-0.05; 95%CI -0.51 to 0.40; p=0.82; I2=0%). In the secondary analyses of the primary outcome, the pooled results from non-randomised controlled trials showed no difference (SMD=0.02; 95%CI -0.45 to 0.49; p=0.93; I2=0%; and SMD=0.03; 95%CI -0.43 to 0.50; p=0.88; I2=0%). Meta-analysis on secondary outcomes could not be performed. CONCLUSION: Based on the available data, inertial flywheel resistance training was not superior to gravity-dependent resistance training in enhancing muscle strength. Data for other strength variables and other muscular adaptations was insufficient to draw firm conclusions from.
Assuntos
Força Muscular , Treinamento Resistido/instrumentação , Treinamento Resistido/métodos , Humanos , Ensaios Clínicos Controlados Aleatórios como AssuntoRESUMO
BACKGROUND AND STUDY AIM: Acute pancreatitis as a complication of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) of pancreatic lesions is rarely observed. However, there is little information on the incidence of hyperamylasemia after EUS-FNA of the pancreas and its clinical significance. This study aimed to supply this lack of information. PATIENTS AND METHODS: Patients who underwent EUS-FNA of a pancreatic lesion between October 2004 and October 2005 were studied prospectively. Exclusion criteria were: (i) platelet count under 50,000/mm (3) and/or prothrombin time < 50 %; (ii) performance of surgery, endoscopic retrograde cholangiopancreatography (ERCP), a percutaneous biopsy attempt, or another invasive procedure within 7 days before EUS-FNA; (iii) lack of informed consent. Serum amylase levels were determined before and 8 and 24 h after the procedure. Hyperamylasemia was defined by amylase levels above 104 UI/L (and higher than baseline levels) 8 h after the procedure. Acute pancreatitis was defined by upper abdominal pain (with or without nausea and/or vomiting) accompanied by elevation of serum amylase or lipase to at least twice baseline levels. RESULTS: A total of 100 patients underwent EUS-FNA of a pancreatic lesion (58 men, 42 women; mean age 60 +/- 13 years). Eleven patients (11 %) showed hyperamylasemia 8 h after the puncture (298 +/- 293 UI/L, range 105 - 1044 UI/L), but only two of them developed acute mild pancreatitis after EUS-FNA. Hyperamylasemia was not related either to the type of lesion (cystic or solid) or to its location, the duration of the procedure, or the number of passes performed. CONCLUSIONS: Pancreatitis after pancreatic EUS-FNA occurs in 2 % of patients, with some more cases of silent hyperamylasemia. This complication may have to be included in the information given to patients for their informed consent.
Assuntos
Biópsia por Agulha Fina/efeitos adversos , Endossonografia/efeitos adversos , Hiperamilassemia/etiologia , Pancreatite/epidemiologia , Pancreatite/etiologia , Doença Aguda , Distribuição por Idade , Idoso , Análise de Variância , Biópsia por Agulha Fina/métodos , Estudos de Casos e Controles , Endossonografia/métodos , Feminino , Seguimentos , Humanos , Hiperamilassemia/epidemiologia , Hiperamilassemia/fisiopatologia , Incidência , Masculino , Pessoa de Meia-Idade , Pancreatopatias/diagnóstico por imagem , Testes de Função Pancreática , Pancreatite/sangue , Estudos Prospectivos , Valores de Referência , Medição de Risco , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Distribuição por SexoRESUMO
BACKGROUND/AIMS: There is little information concerning the potential role of fine-needle aspiration guided by endoscopic ultrasonography in the pathologic diagnosis of intraductal papillary mucinous tumors of the pancreas. METHODOLOGY: Patients with an intraductal papillary mucinous tumor of the pancreas suggested by endoscopic ultrasonography underwent fine-needle aspiration guided by endoscopic ultrasonography in order to investigate the presence of mucin and/or cytologic changes consistent with this diagnosis. A group of 111 patients with other pancreatic lesions explored during the same period of time was used as a control group. RESULTS: Fine-needle aspiration guided by endoscopic ultrasonography was safely performed in 19 patients and supported the diagnosis in 17 of them. Nine out of the 17 patients with suspicion of intraductal papillary mucinous tumors of the pancreas went to surgery and this diagnosis was confirmed in the resected specimen in all of them. Sensitivity, specificity, positive predictive value, negative predictive value and accuracy of EUS FNA in the diagnosis of IPMT were 82%, 100%, 100%, 92% and 94% respectively. CONCLUSIONS: Fine-needle aspiration guided by endoscopic ultrasonography is a good technique to support the diagnosis of intraductal papillary mucinous tumors of the pancreas and should be considered in this group of patients if pathologic confirmation is judged to be necessary.
Assuntos
Carcinoma Ductal Pancreático/diagnóstico por imagem , Carcinoma Ductal Pancreático/patologia , Carcinoma Papilar/diagnóstico por imagem , Carcinoma Papilar/patologia , Endossonografia , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Biópsia por Agulha Fina/métodos , Dilatação Patológica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Ductos Pancreáticos/patologia , Estudos Retrospectivos , Sensibilidade e EspecificidadeRESUMO
BACKGROUND/AIMS: Despite the existence of published recommendations, various studies of antibiotic prophylaxis have reached conflicting conclusions, and controversy exists regarding the role of antibiotic prophylaxis in ERCP. The aim of this study was to analyze the efficacy of the intramuscular administration of clindamicine and gentamicine before ERCP. METHODOLOGY: Sixty-one consecutive patients referred for ERCP were prospectively randomized to receive either clindamicine 600mg and gentamicine 80mg, both intramuscularly one hour before the ERCP (group I; 31 patients) or not (group II; 30 patients). Two blood samples were obtained from every patient (just before endoscopy and within 5 minutes of withdrawal of the endoscope) and were incubated for 7 days and examined daily for growth of bacteria. Patients were closely monitored for 7 days after endoscopy to detect the development of infectious complications. RESULTS: Only 7 cultures from 7 patients were positive. Four were obtained post-ERCP (two patients in group I and two in group II) and the remaining three before endoscopy. The post-ERCP isolated bacteria were: Streptococcus mitis, Peptoestreptococcus anaerobious, Moraxella spp and Escherichia coli. Two patients, one from each group, developed post-ERCP cholangitis that were solved with medical treatment. CONCLUSIONS: Our findings indicate that ERCP induce bacteremia in a small group of patients and suggest that prophylactic administration of clindamicine plus gentamicine does not reduce the incidence of bacteremia and cholangitis, and do not support the routine use of prophylactic antibiotics prior to ERCP.
Assuntos
Antibacterianos/uso terapêutico , Antibioticoprofilaxia , Colangiopancreatografia Retrógrada Endoscópica/efeitos adversos , Clindamicina/uso terapêutico , Gentamicinas/uso terapêutico , Idoso , Antibacterianos/administração & dosagem , Bacteriemia/epidemiologia , Bacteriemia/etiologia , Bacteriemia/prevenção & controle , Colangite/epidemiologia , Colangite/etiologia , Colangite/prevenção & controle , Clindamicina/administração & dosagem , Feminino , Gentamicinas/administração & dosagem , Humanos , Incidência , Masculino , Pessoa de Meia-Idade , Resultado do TratamentoRESUMO
PURPOSE: We performed a clinical trial to determine whether postoperative adjuvant chemotherapy with two drugs versus one drug could prolong survival. PATIENTS AND METHODS: From 1985 to 1996, 85 patients with completely resected locally advanced gastric cancer were enrolled. The subjects were randomized into two treatment groups, as follows: mitomycin (MMC) 10 to 20 mg/m2 intravenously (i.v.) on day 1 every 6 weeks plus ftorafur (FT) 500 mg/m2/d for 36 consecutive days; or MMC alone, 10 to 20 mg/m2 i.v. every 6 weeks. All courses were repeated four times. RESULTS: After a median follow-up duration of 62 months, the overall 5-year survival rates were 67% for the MMC-FT group versus 44% for the MMC group (P = .04). Subgroup analysis to compare survival curves using the method of Mantel-Cox showed survival rates significantly in favor of the MMC-FT group in the subsets of patients with node-negative disease (P = .01) and those whose disease was stage IB or II (P = .008). CONCLUSION: Significantly better survival results were observed for MMC-FT versus MMC alone. Subset analysis suggest a strong benefit in patients with node-negative and early-stage resected gastric cancer.
Assuntos
Adenocarcinoma/tratamento farmacológico , Antibióticos Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Mitomicinas/uso terapêutico , Neoplasias Gástricas/tratamento farmacológico , Adenocarcinoma/cirurgia , Adulto , Idoso , Quimioterapia Adjuvante , Feminino , Gastrectomia , Humanos , Excisão de Linfonodo , Masculino , Pessoa de Meia-Idade , Mitomicinas/administração & dosagem , Esplenectomia , Neoplasias Gástricas/cirurgia , Análise de Sobrevida , Tegafur/administração & dosagemRESUMO
X-ray patterns have been obtained from solutions of microtubules and tubulin rings using synchrotron radiation. They are interpreted on the basis of the oriented fiber X-ray diagram of microtubules, and by comparing them with the calculated scattering traces of model structures. The data are consistent with models in which rings consist of coiled protofilaments. This is in agreement with some published ring models while it excludes others.
Assuntos
Tubulina (Proteína) , Microscopia Eletrônica , Microtúbulos/ultraestrutura , Modelos Moleculares , Modelos Estruturais , Conformação Proteica , Espalhamento de Radiação , Raios XRESUMO
GDP liganded tubulin, which is inactive in microtubule assembly, polymerizes into rings more readily than the active GTP liganded protein. The structure of double rings made of highly purified GDP-tubulin has been characterized to 3 nm resolution with synchrotron X-ray solution scattering. The scattering profile has characteristic maxima due to closely packed double rings of 38 nm mean diameter, with a 5.5 nm mean spacing between the rings, and a 4.2 nm centre-to-centre spacing between non-globular tubulin monomers within both rings. There are probably 24 and 32 monomers in the inner and outer ring, respectively, and the double ring population is more than 75% homogeneous in size. Comparison of this double ring structure to the lattice of tubulin molecules in microtubules indicates that the tubulin rings are equivalent to pairs of protofilament segments curved tangentially to the microtubule surface, with bending angles of 30 degrees and 22.5 degrees per tubulin alpha beta dimer. When the rings are modelled employing the same non-globular tubulin monomer as in microtubules, the best computer fitted scattering profiles correspond to monomer orientations equivalent to two microtubule protofilaments coiled sideways, with same or opposite polarity. Rings constitute the equilibrium assembly state of GDP-tubulin, which is tensioned inside microtubules after GTP hydrolysis, causing their functional instability. In analogy with other nucleotide binding proteins, the inactive/active structural switch of tubulin is induced by the binding of the gamma phosphate and a coordinated Mg ion. It should involve domain rearrangements which cancel the bending of the tubulin dimer in the ring structure.
Assuntos
Guanosina Difosfato/química , Microtúbulos/química , Tubulina (Proteína)/química , Microtúbulos/ultraestrutura , Modelos Moleculares , Conformação Proteica , Espalhamento de Radiação , Soluções , Tubulina (Proteína)/ultraestruturaRESUMO
In vitro DNA packaging activity in a defined system derived from bacteriophage phi 29 depends upon the chemical integrity of the connector protein p10. Proteolytic cleavage of p10 rendered the proheads inactive for DNA packaging. A similar treatment on isolated connectors abolished the DNA-binding activity of the native p10, but the general shape and size of the connector was not changed as revealed by electron microscopy. Analytical ultracentrifugation showed that the proteolyzed connectors had a smaller sedimentation coefficient, while amino acid analysis after dialysis of the proteolyzed p10 confirmed the loss of 16 and 19 amino acids from the amino and carboxy termini, respectively. Low angle X-ray scattering revealed that proteolysis was followed by a small decrease in the radius of gyration and a reorganization of the distal domain of the cylindrical inner part of the connector. Characterization of the cleavage sites in the primary sequence allowed us to propose the location of the DNA-binding domain in the connector model.
Assuntos
Bacteriófagos/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas Virais/metabolismo , Sequência de Aminoácidos , Aminoácidos/análise , Proteínas de Ligação a DNA/análise , Microscopia Eletrônica , Dados de Sequência Molecular , Conformação Proteica , Espalhamento de Radiação , Tripsina , Ultracentrifugação , Proteínas Virais/análiseRESUMO
The acrosomal process of Limulus sperm contains a bundle of filaments composed of actin and a 102 kDa protein in a 1:1 molar ratio. The structure of the bundle in true discharge was investigated by electron cryomicroscopy, X-ray scattering and crystallographic image analysis. A bundle can be characterized as a quasi-crystal with continuously varying views along the bundle axis. Each segment of the bundle is found to obey the symmetry of space group P1, with a = b = 147 A, c = 762 A, alpha = 90 degrees, beta = 90.6 degrees, gamma = 120 degrees. A unit cell contains a helical repeat of the filament with a selection rule following that of an actin filament. A 24 A projection map based on the h0l view was reconstructed after averaging 5300 unit cells from six electron images. Filaments in this projection are well separated and clearly display a 21 screw symmetry. This screw symmetry results from the helical parameters of the bundle filament and is found to be a non-crystallographic symmetry element present in the unit cell. Our structural analysis has led to the proposal that the assembly of a stable bundle with a defined maximum diameter can be controlled by the crystallographic packing of the twisted filaments.
Assuntos
Acrossomo/química , Actinas/química , Caranguejos Ferradura/química , Difração de Raios X , Acrossomo/ultraestrutura , Actinas/isolamento & purificação , Actinas/ultraestrutura , Animais , Técnica de Fratura por Congelamento , Caranguejos Ferradura/ultraestrutura , Processamento de Imagem Assistida por Computador , Masculino , Peso Molecular , Peptídeos/química , Peptídeos/isolamento & purificaçãoRESUMO
Addition of the antimitotic drug vinblastine to solutions of purified tubulin induces the formation of helical polymers whose structure and type of aggregation is determined by the concentration of magnesium. While paracrystalline arrangements of single coils are observed at low concentrations of the ion, for concentrations higher than 6 mM free double-coiled spirals are obtained, which are indistinguishable from those obtained in the presence of microtubule-associated proteins (MAPs). This result is consistent with a similar effect of magnesium and MAPs in neutralizing negative charges on the tubulin molecule and so allowing for lateral contacts between protofilaments. The effects that temperature has on the structure of both types of polymers, free spirals or paracrystals, have been monitored using time-resolved X-ray solution scattering. This study shows that a temperature increase: (1) affects the length and lateral aggregation of the spirals in the paracrystalline sample; (2) induces a reversible increase of the helical pitch in both types of polymers that closely follows the temperature change; (3) produces an irreversible aggregation of some of the protein in both types of polymers; and (4) can induce a reversible transformation from one type of structure to the other when the concentration of Mg2+ is in the boundary between the two ranges. We suggest that the changes in pitch are due to a temperature-induced conformational change of the tubulin molecule. This effect may be related to the structural modifications that result in the temperature-induced assembly of microtubules in vitro under normal conditions of assembly.
Assuntos
Tubulina (Proteína)/química , Vimblastina/farmacologia , Animais , Química Encefálica , Magnésio/farmacologia , Modelos Moleculares , Polímeros , Conformação Proteica , Espalhamento de Radiação , Soluções , Suínos , Temperatura , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/isolamento & purificação , Tubulina (Proteína)/ultraestrutura , Difração de Raios X , Raios XRESUMO
During normal contractions of vertebrate striated muscle, it is believed that the cross-bridges which produce the sliding force undergo asynchronous cyclical changes in their structure. Thus, an X-ray diffraction diagram from a muscle under these conditions will give structural information averaged over the whole range of cross-bridge states. Such diagrams show characteristic and informative differences from those given by relaxed muscle, but can give little information about changes in the configuration of the cross-bridges at different stages of their working stroke. However, it is possible to effect a partial synchronization of these changes by applying very rapid changes in length, completed in less than one millisecond to an otherwise isometrically contracting muscle. If the amplitude of these length changes is comparable to the length of the cross-bridge stroke (say 100 A per half-sarcomere), then it should bring about a transient but significant redistribution of cross-bridge states, which would show up in the X-ray diagram. We have made use of synchrotron radiation as a high intensity X-ray source in order to record such patterns with the necessary time resolution (1 ms or less) and have found major changes in the intensity of the 143 A meridional reflection accompanying the rapid length changes of the muscle. These changes appear to arise from specific configurational changes in the cross-bridges during the working stroke. A model is suggested in which the 143 A meridional intensity in a contracting muscle arises mainly from attached cross-bridges and is generated by the part of the myosin head near the S1-S2 junction. During normal contraction, cross-bridges go through their structural cycle asynchronously with each other, since they start at different times, but if the S2 changes in length rather little, then the configurational changes in the myosin heads are synchronized with the actin filament movement in such a way that the S1-S2 junction remains relatively fixed in its axial position. In a quick release, it is suggested that bringing many S1 heads simultaneously to the end of their working strokes on actin disrupts the 143 A axial repeat of their distal ends near S2, and brings about the large decrease of the 143 A meridional reflection. This model therefore involves a large change in the position of part of the myosin head structure relative to actin during the working stroke of the cross-bridge.
Assuntos
Contração Muscular , Músculos/ultraestrutura , Animais , Contração Isométrica , Substâncias Macromoleculares , Modelos Estruturais , Miosinas , Aceleradores de Partículas , Rana esculenta , Fatores de Tempo , Difração de Raios XRESUMO
The structure of microtubules has been characterized to 3 nm resolution employing time-resolved X-ray scattering. This has revealed detailed structural features of microtubules not observed before in solution. The polymerization of highly purified tubulin, induced by the antitumour drug taxol, has been employed as a microtubule model system. This assembly reaction requires Mg2+, is optimal at a 1:1 taxol to tubulin heterodimer molar ratio, proceeds with GTP or GDP and is intrinsically reversible. The X-ray scattering profiles are consistent with identical non-globular alpha and beta-tubulin monomers ordered within the known helical surface lattice of microtubules. Purified tubulin-taxol microtubules have a smaller mean diameter (approx. 22 nm) than those induced by microtubule associated proteins or glycerol (approx. 24 nm), but nearly identical wall substructure to the resolution of the measurements. This is because the majority of the former consist of only 12 protofilaments instead of the typical 13 protofilaments, as confirmed by electron microscopy of thin-sectioned, negatively stained and ice-embedded taxol microtubules. It may be concluded that taxol induces a slight reduction of the lateral contact curvature between tubulin monomers. The main fringe pattern observed in cryo-electron micrographs is consistent with a simple 12 protofilament 3-start skewed lattice model. Cylindrical closure of this lattice can be achieved by tilting the lattice 0.8 degrees with respect to the microtubule axis. The closure implies a discontinuity in the type of lateral contacts between the tubulin monomers (regardless of whether these are of the -alpha-beta- or the -alpha-alpha-/-beta-beta- type), which indicates that lateral contacts and the subunit specificity of taxol binding are, to a large degree, equivalent.
Assuntos
Alcaloides/farmacologia , Microtúbulos/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Glicerol , Nucleotídeos de Guanina/metabolismo , Magnésio/metabolismo , Microscopia Eletrônica , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/ultraestrutura , Paclitaxel , Espalhamento de Radiação , Raios XRESUMO
BACKGROUND: The demand for gastrointestinal endoscopy is increasing in most developed countries, resulting in an important rise in overall costs and waiting lists for endoscopic procedures. Therefore, adherence to appropriate indications for these procedures is essential for the rational use of finite resources in an open-access system. AIM: To assess indications and appropriateness of colonoscopy according to the European Panel on the Appropriateness of Gastrointestinal Endoscopy (EPAGE) criteria. METHODS: From May to June 2004, all consecutive patients referred to our Unit for open-access colonoscopy were considered for inclusion in this prospective study. Appropriateness of each colonoscopy was established according to the EPAGE criteria. In order to evaluate whether appropriateness of use correlated with the diagnostic yield of colonoscopy, relevant endoscopic findings were also recorded. RESULTS: A total of 350 consecutive patients were included in the study. In 38 of them, the colonoscopy indication was not listed in the EPAGE guidelines and, consequently, they were not evaluated. In the remaining 312 patients, the indication for the procedure was considered inappropriate in 73 (23%) patients. Both referring doctor characteristics (specialty and health care setting) and patient data (age) correlated with appropriateness of endoscopy. The diagnostic yield was significantly higher for appropriate colonoscopies (42%) than in those judged inappropriate (21%) (P = 0.001). CONCLUSIONS: A noteworthy proportion of patients referred for colonoscopy to an open-access endoscopy unit are considered inappropriate because of their indication, with significant differences among specialties. These results suggest that implementation of validated guidelines for its appropriate use could improve this situation and, considering the correlation between appropriateness and diagnostic yield, even contribute to improve the prognosis of patients with colorectal diseases.