One-step purification of recombinant proteins with the 6xHis tag and Ni-NTA resin.
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Biomedical subjects
Publications and source records attributed to J Crowe.
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The 6xHis/Ni-NTA system allows rapid and efficient affinity purification of recombinant proteins from virtually any expression system. Protocols and tips for purification under both native and denaturing conditions are provided, as well as a rapid spin procedure for protein minipreps.
BACKGROUND: Because sphincterotomy accounts for a major portion of the morbidity and mortality associated with ERCP, we have proposed endoscopic balloon papillary dilation or sphincteroplasty as an alternative. METHODS: We report the outcome in a series of 100 patients in whom balloon sphincteroplasty was attempted for bile duct stones up to 20 mm in diameter, with a median follow-up of 16 months (range 6 to 30). RESULTS: During one ERCP session using sphincteroplasty alone, the bile duct was cleared in 78%, mechanical lithotripsy being required in 10% for stones greater than 12 mm in diameter. Incomplete duct clearance was achieved in a further 4%, all of whom underwent repeat ERCP with successful duct clearance without recourse to sphincterotomy. Failure to clear the bile duct with sphincteroplasty in the remaining 18% was primarily related to large stone size ( > 15 mm). Sphincterotomy was required to clear the duct in 7%. Another 6% comprised elderly high-risk patients with multiple large stones greater than 15 mm who were treated by stent insertion plus ursodeoxycholic acid. No papillary hemorrhage was observed; uncomplicated pancreatitis occurred in 5%. During a median follow-up of 16 months, 2% had recurrent symptomatic bile duct stones considered to have been unrecognized following the initial ERCP: these were removed after repeat sphincteroplasty. No clinical evidence of papillary stenosis was observed during follow-up. CONCLUSIONS: Endoscopic balloon papillary dilation or sphincteroplasty is a safe and effective alternative to sphincterotomy in the management of bile duct stones less than 12 mm; larger stones may require mechanical lithotripsy to facilitate duct clearance.
Replication errors at microsatellite repeats are markers for genomic instability in hereditary nonpolyposis colon carcinoma and in some sporadic cancers. Microsatellite sequences may show alterations in one or both alleles in some tumors, suggesting an error in the DNA replication of dinucleotide repeats. We have investigated microsatellite instability (MSI) in sporadic breast tumors at several loci on the short arm of chromosome 11. Among microsatellites studied we found a high frequency of MSI at one specific locus, D11S988 on chromosome 11p15.5. Most colorectal tumors that exhibit MSI display abnormalities of at least one other locus and usually more. By contrast we have detected only one abnormal microsatellite in all the tumors examined. This marker lies between the TH and HBB genes, a subregion previously suggested to harbor a putative tumor suppressor gene for breast cancer. Loss of heterozygosity for chromosome markers at 11p15 has earlier been correlated with poor prognosis. In an unselected panel of primary breast tumors, we observed that 20 of 69 showed mobility shifts of D11S988 in tumor compared with corresponding normal DNA samples. Tumors with instability at D11S988 were rapidly proliferating compared with tumors without MSI. DNA aneuploidy, estrogen receptor positivity and moderate to poorly differentiated tumor phenotype were also characteristics of tumors with MSI at this locus and the majority also exhibited loss of heterozygosity at one or more of the six 11p loci analyzed. Taken together these data suggest that MSI at the D11S988 locus is a late event in mammary tumorigenesis and may be associated with progression of breast carcinomas.
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The objective of this work was to develop and test a questionnaire to measure health-related quality of life for patients after myocardial infarction (MI). In a cross-sectional survey, 63 patients identified the most frequent and important problems following acute myocardial infarction. The Quality of Life after Myocardial Infarction (QLMI) instrument was developed on the basis of these most frequent and important problems. The QLMI was administered, along with instruments measuring health utilities, social function, and emotional function, in a randomized trial of rehabilitation versus conventional care. The most frequent and important problems fell into areas of symptoms, restriction, confidence, self-esteem, and emotions, each of which is represented in the 26-item QLMI. Effect sizes of the overall QLMI in differentiating between rehabilitation and control groups (0.35), and in detecting improvement over 12 months (1.22) were comparable or larger than any other instrument. The Pearson's correlation coefficient between QLMI administered at 8 and 12 months following AMI varied between 0.75 and 0.87 for the five domains and the overall score. We found substantial correlations of the QLMI with other measures with moderate concordance with predictions about how the instrument should behave if it is a valid measure of health-related quality of life. The QLMI demonstrates a high degree of reliability, and is more responsive than other questionnaires. Relations between the QLMI and other measures provide moderate to strong evidence of its validity in discriminating between patients following AMI according to their health-related quality of life, and in measuring changes in health-related quality of life over time.
Removal of bile duct stones during endoscopic retrograde cholangiopancreatography (ERCP) usually includes papillotomy. Papillotomy is associated with occasional complications and in addition, the longterm sequelae of papillotomy in young patients having laparoscopic cholecystectomy remain unclear. As an alternative to papillotomy, this study prospectively evaluated the efficacy and safety of endoscopic balloon sphincteroplasty to facilitate bile duct clearance. Of 32 patients with bile duct stones (diameter 3-30 mm) at ERCP, sphincteroplasty was considered inappropriate in four patients because of stone size (> 20 mm) necessitating papillotomy for bile duct clearance. Sphincteroplasty was performed in the remaining 28 patients to permit duct clearance by dormier basket, balloon or mechanical lithotripsy. The bile duct was cleared in 22 patients (79%) while additional measures including papillotomy or stent insertion were required in the remaining six patients (21%) because of stone size or technical difficulties. There was no associated papillary haemorrhage. Pancreatitis was seen in one patient (4%) but resolved within 24 hours. Our preliminary experience suggests that sphincteroplasty is a safe and effective sphincter preservation technique that significantly reduces the necessity for papillotomy in the management of bile duct stones.
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Although there are extensive clinical evaluations of cardiac rehabilitation after acute myocardial infarction (AMI), no full economic evaluation is available. Patients with AMI and mild to moderate anxiety or depression, or both, while still in hospital were randomized to either an 8-week rehabilitation intervention (n = 99) or usual care (n = 102). Comprehensive costs and health-related quality of life, measured with the time trade-off preference score, were obtained in a 12-month trial, and together with survival data derived from published meta-analyses, cost-utility and cost-effectiveness of early cardiac rehabilitation were estimated. The best estimate of the incremental net direct 12-month costs for patients randomized to rehabilitation was $480 (United States, 1991)/patient. During 1-year follow-up, rehabilitation patients had fewer "other rehabilitation visits" (p < 0.0001) and gained 0.052 quality-adjusted life-year more than did the group with usual care. The cost-utility ratio was $9,200/quality-adjusted life-year gained with cardiac rehabilitation during the year of follow-up. This economic evaluation of cardiac rehabilitation does not consider the important distinctions between affordability and worth of alternative health-care services. The data provide evidence that brief cardiac rehabilitation initiated soon after AMI for patients with mild to moderate anxiety or depression, or both, is an efficient use of health-care resources and may be economically justified.
Benign papillary stenosis is an uncommon but well recognised cause of recurrent biliary pain usually in post-cholecystectomy patients characterised by biliary dilatation without bile duct calculi or malignancy. Endoscopic or surgical papillotomy is the recommended treatment but may be associated with a higher complication rate than when performed for bile duct stones. We report 2 cases of papillary stenosis treated by endoscopic balloon dilatation or "sphincteroplasty", as a less traumatic intervention which improved biliary drainage and provided symptomatic relief. Our preliminary experience suggests more widespread evaluation of balloon sphincteroplasty should be considered for papillary stenosis.
Gas samples were obtained during colonoscopy for analysis. Patients were prepared with polyethylene glycol (PEG) (N = 23), phosphate enema (N = 34) and mannitol (N = 4). Air insufflation was used in all procedures. High concentrations of hydrogen were detected in 3 out of 38 gas samples in the PEG group, in 2 of 41 samples in the phosphate enema group and in one of the 8 samples in the mannitol group. All patients had a coexisting intracolonic oxygen concentration > 5%. The results suggest that potentially explosive concentrations of hydrogen may occur after conventional bowel preparations, and that insufflation of carbon dioxide during polypectomy should be a routine.
This investigation was designed to determine the impact of a brief period of cardiac rehabilitation, initiated within 6 weeks of acute myocardial infarction (AMI), on both disease-specific and generic health-related quality of life, exercise tolerance and return to work after AMI. With a stratified, parallel group design, 201 low-risk patients with evidence of depression or anxiety, or both, after AMI, were randomized to either an 8-week program of exercise conditioning and behavioral counseling or to conventional care. Although the differences were small, significantly greater improvement was seen in rehabilitation group patients at 8 weeks in the emotions dimension of a new disease-specific, health-related Quality of Life Questionnaire, in their state of anxiety and in exercise tolerance. All measures of health-related quality of life in both groups improved significantly over the 12-month follow-up period. However, the 95% confidence intervals around differences between groups at the 12-month follow-up effectively excluded sustained, clinically important benefits of rehabilitation in disease-specific (limitations, -2.70, 1.40; emotions, -4.86, 1.10, where negative values favor conventional care and positive values favor rehabilitation) and generic health-related quality of life (time trade-off, -0.062, 0.052; quality of well-being, -0.042, 0.035) or in exercise tolerance (-38.5, 52.1 kpm/min); also, return to work was similar in the 2 groups (relative risk, 0.93; confidence interval, 0.71, 1.64).(ABSTRACT TRUNCATED AT 250 WORDS)
The efficacy of ranitidine 150 mg twice daily and Maalox TC three tablets four times daily were compared in patients with endoscopically confirmed duodenal ulcer. Seventy-nine patients were randomly allocated to double-blind, double-dummy treatment, stratified for smokers. Endoscopy was repeated after four weeks. Those unhealed continued treatment for a further two weeks before final endoscopy. Per protocol analysis in 53 patients showed ulcer healing rates at week 4 and at weeks 4 and 6 combined of 78 and 89% on Maalox TC, and of 81 and 91% on ranitidine, respectively. The same analysis gave overall healing rates of 81% in smokers and 100% in nonsmokers, irrespective of treatment. Both treatments provided early ulcer pain relief. Diarrhea was a commoner side effect in patients on Maalox TC. The study showed Maalox TC and ranitidine were equally effective in healing duodenal ulceration.
In order to study the effect of clofazimine, a powerful antimycobacterial and antiinflammatory agent, 49 patients with active Crohn's disease were randomized to either corticosteroids plus clofazimine 100 mg daily (N = 25) or to steroids and matching placebo (N = 24). A total of 28 patients (58%) went into disease remission (clofazimine 16, placebo 12; P = NS) with a fall in disease activity score from 10.5 +/- 4.4 to 3.3 +/- 3.5. Patients were treated for a further eight months with clofazimine or placebo and 18 of 28 maintained their remission and completed the study (clofazimine 12, placebo 6; P = NS). Side effects were minor and consisted of skin rash and increased pigmentation. Clofazimine as a solitary antimycobacterial agent appears ineffective in inducing remission in Crohn's disease but may have a role in either disease maintenance or combination chemotherapy.
In the first year from October 1990 since starting the procedure 65 laparoscopic cholecystectomies were carried out on one surgical service. There were 4 planned open cholecystectomies and 8 laparoscopic procedures converted during the same period: 7 of these were in the first 3 months with only 1 of the last 53 being opened. Surgery was carried out during the same admission in 22 patients presenting as emergencies: acute cholecystitis (9), colic (6), pancreatitis (3), jaundice (4). Two patients had later laparotomies for complications; one patient bled from the umbilical stab and with ongoing peritonism had a pelvic haematoma drained on day 5 and a second was opened following a bile leak caused by a displaced cystic duct clip--both recovered uneventfully. Peroperative cholangiography was performed in 13 patients; 2 were positive (15%) and had ERCP papillotomy 3 days post op without complication. One patient who presented with pancreatitis had ERCP performed post-op without incident. Seven patients had laparoscopic cholecystectomy following papillotomy for common duct stones. The gallbladder was extracted per umbilicus in 45 (3 wound infections) and per right subcostal stab in 20 (no infections). Mean hospital stay was 48 hours (1-4 days) in uncomplicated cases. In conclusion, the learning curve is associated with higher conversion rates. Extraction through pliable hypochondrial muscles is easier and may be safer and less traumatic. Perioperative endoscopic papillotomy is safe and effective for choledochal stones.
We prospectively studied 239 consecutive patients who underwent breast biopsy for 277 nonpalpable lesions characterized by mammographic microcalcifications. Clinical and mammographic characteristics were correlated with histologic findings in an attempt to identify patients more likely to have early breast cancer. The distribution of clinical risk factors was equal between patients with benign or malignant outcomes. The predominant Wolfe pattern on mammography was P2 (38%); however, no relationship was observed between the Wolfe pattern and malignancy. A marked correlation was observed between malignancy and small lesions, more than 15 calcifications, and calcifications in a linear or branching pattern. Twenty-four percent (n = 67) of the biopsy specimens contained either ductal or lobular breast cancer. This study highlights the necessity of an aggressive approach toward suspicious calcifications found by mammography.
An unusual case of a well-documented granulomatous hepatitis in secondary syphilis in a 47-year-old male is presented. This hepatic pathology in secondary syphilis is summarised, and a review of the literature regarding syphilitic granulomatous hepatitis presented.
The efficacy of cisapride, as compared with cimetidine, in the treatment of erosive esophagitis was studied in a double-blind trial. One hundred and twenty-nine patients were assigned to one of four dosage schedules: cisapride 10 mg b.i.d. (20 mg group) or q.i.d. (40 mg group), or cimetidine 400 mg b.i.d. (800 mg group) or q.i.d. (1600 mg group). Treatment lasted 8 to 12 weeks. The degree of esophagitis and the severity of diurnal and nocturnal heartburn and regurgitation were significantly (p less than 0.01) reduced in the four treatment groups. Endoscopy did not show any significant differences among the four groups, although cisapride tended to be more effective in moderate to severe esophagitis, in which cases mucosal healing (i.e. absence of erosions and ulcers) was observed in 69%, 64%, 55% and 55% of the patients treated with cisapride 40 mg, cisapride 20 mg, cimetidine 1600 mg and cimetidine 800 mg. Improvement in reflux symptoms in the two cisapride groups was not significantly different from that in the cimetidine 1600 mg group, but was better (p less than 0.05) than that in the cimetidine 800 mg patients. The severity score for all reflux symptoms had decreased by 79%, 74% (cisapride 40 mg and 20 mg), 69% and 57% (cimetidine 1600 mg and 800 mg) by the end of treatment. These results show that cisapride is at least as effective as acid-suppressing therapy in patients with reflux esophagitis, and is therefore a valuable alternative to it.