Sample prep for proteomics of breast cancer: proteomics and gene ontology reveal dramatic differences in protein solubilization preferences of radioimmunoprecipitation assay and urea lysis buffers.

BACKGROUND: An important step in the proteomics of solid tumors, including breast cancer, consists of efficiently extracting most of proteins in the tumor specimen. For this purpose, Radio-Immunoprecipitation Assay (RIPA) buffer is widely employed. RIPA buffer's rapid and highly efficient cell lysis and good solubilization of a wide range of proteins is further augmented by its compatibility with protease and phosphatase inhibitors, ability to minimize non-specific protein binding leading to a lower background in immunoprecipitation, and its suitability for protein quantitation. RESULTS: In this work, the insoluble matter left after RIPA buffer extraction of proteins from breast tumors are subjected to another extraction step, using a urea-based buffer. It is shown that RIPA and urea lysis buffers fractionate breast tissue proteins primarily on the basis of molecular weights. The average molecular weight of proteins that dissolve exclusively in urea buffer is up to 60% higher than in RIPA.Gene Ontology (GO) and Directed Acyclic Graphs (DAG) are used to map the collective biological and biophysical attributes of the RIPA and urea proteomes. The Cellular Component and Molecular Function annotations reveal protein solubilization preferences of the buffers, especially the compartmentalization and functional distributions.It is shown that nearly all extracellular matrix proteins (ECM) in the breast tumors and matched normal tissues are found, nearly exclusively, in the urea fraction, while they are mostly insoluble in RIPA buffer. Additionally, it is demonstrated that cytoskeletal and extracellular region proteins are more soluble in urea than in RIPA, whereas for nuclear, cytoplasmic and mitochondrial proteins, RIPA buffer is preferred.Extracellular matrix proteins are highly implicated in cancer, including their proteinase-mediated degradation and remodelling, tumor development, progression, adhesion and metastasis. Thus, if they are not efficiently extracted by RIPA buffer, important information may be missed in cancer research. CONCLUSION: For proteomics of solid tumors, a two-step extraction process is recommended. First, proteins in the tumor specimen should be extracted with RIPA buffer. Second, the RIPA-insoluble material should be extracted with the urea-based buffer employed in this work.

Dramatic down-regulation of oxidoreductases in human hepatocellular carcinoma hepG2 cells: proteomics and gene ontology unveiling new frontiers in cancer enzymology.

BACKGROUND: Oxidoreductases are enzymes that catalyze many redox reactions in normal and neoplastic cells. Their actions include catalysis of the transformation of free, neutral oxygen gas into oxygen free radicals, superoxide, hydroperoxide, singlet oxygen and hydrogen peroxide. These activated forms of oxygen contribute to oxidative stress that modifies lipids, proteins, DNA and carbohydrates. On the other hand, oxidoreductases constitute one of the most important free radical scavenger systems typified by catalase, superoxide dismutase and glutathione peroxidase.In this work, proteomics, Gene Ontology mapping and Directed Acyclic Graphs (DAG) are employed to detect and quantify differential oxidoreductase enzyme expressions between HepG2 cells and normal human liver tissues. RESULTS: For the set of bioinformatics calculations whose BLAST searches are performed using the BLAST program BLASTP 2.2.13 [Nov-27-2005], DAG of the Gene Ontology's Molecular Function annotations show that oxidoreductase activity parent node of the liver proteome contains 331 annotated protein sequences, 7 child nodes and an annotation score of 188.9, whereas that of HepG2 cells has 188 annotated protein sequences, 3 child nodes and an annotation score of only 91.9. Overwhelming preponderance of oxidoreductases in the liver is additionally supported by the isomerase DAGs: nearly all the reactions described in the normal liver isomerase DAG are oxidoreductase isomerization reactions, whereas only one of the three child nodes in the HepG2 isomerase DAG is oxidoreductase. Upon normalization of the annotation scores to the parent Molecular Function nodes, oxidoreductases are down-regulated in HepG2 cells by 58%.Similarly, for the set of bioinformatics calculations whose BLAST searches are carried out using BLASTP 2.2.15 [Oct-15-2006], oxidoreductases are down-regulated in HepG2 cells by 56%. CONCLUSION: Proteomics and Gene Ontology reveal, for the first time, differential enzyme activities between HepG2 cells and normal human liver tissues, which may be a promising new prognostic marker of Hepatocellular carcinoma.Two independent sets of bioinformatics calculations that employ two BLAST program versions, and searched different databases, arrived at essentially the same conclusion: oxidoreductases are down-regulated in HepG2 cells by approximately 57%, when compared to normal human liver tissues. Down-regulation of oxidoreductases in hepatoma is additionally supported by Gene Ontology analysis of isomerises.

Combination treatment strategies in early rheumatoid arthritis.

Combinations of disease modifying antirheumatic drugs (DMARDs) are increasingly being used in patients with early rheumatoid arthritis (RA) when long term results with sequential DMARD monotherapy are disappointing. Combination DMARD therapy may be more effective than monotherapy, and has no additional short term adverse events. The evidence for using combination DMARD therapy is still weak, however, and further trials are needed.

Does waiting matter? A randomized controlled trial of new non-urgent rheumatology out-patient referrals.

OBJECTIVE: To examine the effect of waiting times on the health status of patients referred for a non-urgent rheumatology opinion. METHODS: The study was a randomized controlled clinical study evaluating a 'fast track' appointment with a 6-week target waiting time against an 'ordinary' appointment in the main city out-patient clinic of the rheumatology service for the Lothian and Borders region (population approximately 1 million). Health status was measured using the SF12 physical and mental summary component T-scores and pain was measured with a 100 mm visual analogue pain scale. Secondary outcomes were health utility and perceived health both measured with the EuroQol instrument, mental health measured with the Hospital Anxiety and Depression scale, disability with the modified Health Assessment Questionnaire and economic costs measured from a societal perspective. RESULTS: Mean waiting times were 43 days (sigma = +/-16) and 105 days (sigma = +/-51) for 'fast track' and 'ordinary' appointments, respectively. Both groups showed significant improvements in mean [95% confidence interval (CI)] scores for pain: 11 (7, 16)(P < 0.001); physical health status: 4 (2, 5) (P < 0.001); mental health status: 2 (0.1, 4) (P < 0.02); and health utility: 0.11 (0.07, 0.16) (P < 0.001) by the end of the 15-month period of the study, but there was no significant difference between either arm of the study. CONCLUSIONS: Rationing by delay was not detrimental to either mental or physical health and patients in both arms of the study showed significant and similar improvement in health by 15 months. Expenditure of resources on waiting times without regard to clinical outcomes is likely to be wasteful and additional resources should be directed at achieving the greatest clinical benefit. More research into effective methods of controlling demand and better identification of those who would benefit from access to specialist care is needed.

Is day care equivalent to inpatient care for active rheumatoid arthritis? Randomised controlled clinical and economic evaluation.

OBJECTIVE: To test the clinical equivalence and resource consequences of day care with inpatient care for active rheumatoid arthritis. DESIGN: Randomised controlled clinical trial with integrated cost minimisation economic evaluation. SETTING: Rheumatic diseases unit at a teaching hospital between 1994 and 1996. SUBJECTS: 118 consecutive patients with active rheumatoid arthritis randomised to receive either day care or inpatient care. MAIN OUTCOME MEASURES: Clinical assessments recorded on admission, discharge, and follow up at 12 months comprised: the health assessment questionnaire, Ritchie articular index, erythrocyte sedimentation rate, hospital anxiety and depression scale, and Steinbrocker functional class. Resource estimates were of the direct and indirect costs relating to treatment for rheumatoid arthritis. Secondary outcome measures (health utility) were ascertained by time trade off and with the quality of well being scale. RESULTS: Both groups had improvement in scores on the health assessment questionnaire and Ritchie index and erythrocyte sedimentation rate after hospital treatment (P < 0.0001) but clinical outcome did not differ significantly between the groups either at discharge or follow up. The mean hospital cost per patient for day care, 798 Pounds (95% confidence interval 705 Pounds to 888 Pounds), was lower than for inpatient care, 1253 Pounds (1155 Pounds to 1370 Pounds), but this difference was offset by higher community, travel, and readmission costs. The difference in total cost per patient between day care and inpatient care was small (1789 Pounds (1539 Pounds to 2027 Pounds) v 2021 Pounds (1834 Pounds to 2230 Pounds)). Quantile regression analysis showed a cost difference in favour of day care up to the 50th centile (374 Pounds; 639 Pounds to 109 Pounds). CONCLUSIONS: Day care and inpatient care for patients with uncomplicated active rheumatoid arthritis have equivalent clinical outcome with a small difference in overall resource cost in favour of day care. The choice of management strategy may depend increasingly on convenience, satisfaction, or more comprehensive health measures reflecting the preferences of patients, providers, and service commissioners.

An audit of methotrexate and folic acid for rheumatoid arthritis. Experience from a teaching centre.

We describe an audit of 158 patients with RA treated with weekly methotrexate and 5 mg of folic acid 24 h later. Our aim was to assess the safety and efficacy of this regime in our hands compared with published clinical trials of methotrexate in RA, and to examine patient outcomes. Treatment improved ESR, but only 69% of patients continuing therapy for prolonged periods believed their arthritis to be better on treatment. Health Assessment Questionnaire and Hospital Anxiety and Depression questionnaire scores in prospectively studied patients were not significantly altered by treatment. Toxicity occurred frequently (59% in those continuing and 89% in those ceasing therapy) and cessation of therapy solely due to lack of efficacy was rare. The probability of patients continuing with methotrexate and folic acid after 1, 2, 3 and 4 yr was 87, 76, 74 and 74%, respectively, figures that are at the upper end of the reported range for methotrexate alone.

Health economics as an aspect of health outcome: basic principles and application in rheumatoid arthritis.

Health economic evaluation comprises the systematic appraisal of the costs, the benefits and the relative economic efficiency of different medical interventions. The first section of this paper outlines the techniques of economic analysis and how they relate to the efficient use of health resources. This is followed by a review of the health economics of rheumatoid arthritis as a model for the wider application of health economics in the field of rheumatology.

A pilot study of the economic cost and clinical outcome of day patient vs inpatient management of active rheumatoid arthritis.

The aims of this pilot study, which compares day patient with inpatient care for management of active RA were (i) to test the feasibility of a trial protocol design including the method of randomization and the practicality of data collection, and (ii) to obtain preliminary information on economic cost and clinical outcome of these two methods of management. Twenty consecutive patients requiring admission for management of active RA were randomized to receive either day patient or inpatient care. All hospital, transport, community and indirect costs incurred over a 6-month period from recruitment were collected for each patient. Disease activity and clinical outcome were assessed using the Ritchie articular index, ESR, Health Assessment Questionnaire, Functional Independence Measure and Hospital Anxiety and Depression Scale. The trial protocol was found to be feasible and no patient allocated to the day patient group requested or required to be transferred to inpatient care. Day care was significantly cheaper than inpatient care despite higher transport costs; the total cost of treating 10 day patients was UK 10,272 pounds compared with 14,528 pounds for 10 inpatients. Clinical outcome was comparable in both groups for all parameters studied and there was no obvious detrimental effect on patients receiving day care. This pilot study demonstrates that day care is feasible and acceptable to patients with active RA. The preliminary data suggest that day care is substantially cheaper than inpatient care and does not apparently compromise clinical outcome.

Monoarthritis caused by Mycobacterium avium complex in a liver transplant recipient.

A 38-yr-old liver transplant recipient presented with a monoarthritis caused by Mycobacterium avium complex (MAC). Diagnosis was not obtained with repeated microscopy and culture of SF aspirate, and required synovial biopsy.

Multicentric reticulohistiocytosis with arthritis and cardiac infiltration: regression following treatment for underlying malignancy.

The case is reported of a 63 year old man presenting with a rapidly destructive symmetrical polyarthritis and widespread papular nodular skin lesions, confirmed by a biopsy to be due to multicentric reticulohistiocytosis. Biventricular cardiac failure developed secondary to extensive myocardial infiltration with multicentric reticulohistiocytosis, a complication of this disease which has not previously been reported. The joint, skin, and cardiac manifestations of multicentric reticulohistiocytosis substantially regressed following resection of an associated squamous cell carcinoma. This report adds to the small amount of published work which suggests that multicentric reticulohistiocytosis can be a paraneoplastic disease that may respond to treatment directed at the underlying tumour.

Hand and upper limb problems of instrumental musicians.

Instrumental musicians are prone to a variety of occupationally determined upper limb problems that produce significant disability and loss of earnings. As the majority of these affect the musculoskeletal system in one way or another they assume a particular relevance to the practice of rheumatology. Recent advances in our understanding of the aetiology of these conditions are described together their mode of presentation, differential diagnosis, investigation and the therapeutic options available for them.

Lack of correlation between IgG T-lymphocyte flow cytometric crossmatches with primary renal allograft outcome.

The flow cytometric crossmatch (FCXM) has been reported to be more sensitive and capable of detecting very low levels of antibodies than the normally used complement dependent cytotoxicity test. We studied both the two colour IgG T cell FCXM and CDC-XM in 146 renal allograft recipients, 111 primary and 35 regrafts, of which 26% (29/111) of 1st and 20% (7/35) of regrafts had a positive FCXM. There was no overall correlation between the FCXM results and early graft outcome in primary renal allografts. The FCXM did not appear to have any advantage over the CDC-XM in predicting graft outcome in unsensitized first grafts. In the small number of regrafts studied, a positive FCXM was associated with a higher degree of graft failure. FCXM can exhibit false negative results if sera are used solely neat although these prozone phenomena do not influence subsequent graft outcome.

The effect of high-dose short-term ibuprofen on antihypertensive control with hydrochlorothiazide.

The effect of high-dose ibuprofen, a nonsteroidal anti-inflammatory drug (NSAID), on the blood pressure of treated hypertensive patients was evaluated in a randomized, placebo-controlled, double-blind, crossover trial with 24-hour ambulatory blood pressure monitoring. Twelve middle-aged black women with essential hypertension, controlled with 50 mg hydrochlorothiazide per day, randomly received 3200 mg ibuprofen and a placebo for 8 days. Each treatment phase was separated by a 1-week washout period. Ambulatory blood pressure monitoring (ABPM), body weight, and 24-hour urinary excretion of sodium, creatinine, and prostaglandin E2 (PGE2) were determined at the end of each treatment phase. Mean (+/- SEM) 24-hour systolic and diastolic blood pressures were 122/85 (+/- 2.9/1.7) and 125/85 (+/- 3.0/1.1) during the placebo and ibuprofen phases, respectively. Mean ABPM during six consecutive 4-hour periods also revealed no significant differences between placebo and ibuprofen. We conclude that 3200 mg ibuprofen per day for up to 1 week induced little change in blood pressure in hypertensive who are receiving hydrochlorothiazide.

Conjugal mobility of the multicopy plasmids NTP1 and NTP16.

Mobilization of the multicopy plasmid NTP16, like that of ColE1, is promoted by a range of conjugal plasmids. However, the mechanisms employed for NTP16 mobilization differ between groups. Mobilization by the IncI1 plasmid R64 requires trans-acting products from NTP16 plus a cis-acting region of the small plasmid. In contrast, this system is used inefficiently by the F plasmid and instead, a high-frequency conduction process occurs. Analysis of exconjugant cells reveals that F-mediated mobilization of NTP16 frequently involves rearrangements of NTP16 DNA, promoted by the Tn1000 transposon of F and/or by the kanamycin resistance transposon (Tn4352) of NTP16. Possible mechanisms for the high-frequency F-mediated mobilization of NTP16 are discussed. The plasmid NTP1, which is closely related to NTP16, is also mobilized efficiently by R64. It is not however efficiently mobilized by F, demonstrating the requirement for the Tn4352 element, which is not present in this plasmid, for effective F-mediated transfer.

Characterization of the drug resistance plasmid NTP16.

A functional and physical analysis of the multicopy plasmid NTP16 is presented. The plasmid-encoded drug resistance determinants are located, as are regions encoding the origin of replication, incompatibility functions, copy number determinants, and mobility functions. It is demonstrated that NTP16 probably arose from the closely related plasmid NTP1 by the acquisition of a novel kanamycin resistance transposon, Tn4352, followed by deletion of some NTP1 sequences. The incompatibility behavior of NTP16 derivatives indicates a system of control rather more complex than that which operates in ColE1. In addition to the RNA I/primer RNA system, the production of a further trans-acting product is demonstrated and its site of action located. A series of derivative plasmids have been created which may prove useful as vectors for genetic engineering.

The effect of phenylpropanolamine on ambulatory blood pressure.

Phenylpropanolamine (PPA) is a sympathomimetic amine and component of many over-the-counter decongestants and anorectic agents. It has been reported to cause elevated blood pressure and even hypertensive crises. The pressor effects with therapeutic doses are not well established. We monitored the effects of acute and chronic PPA dosing using 24-hour ambulatory blood pressure recording as a sensitive method of monitoring blood pressure variability. Eighteen normotensive male subjects were randomly assigned to receive 75 mg PPA (sustained-release preparation) or placebo in a double-blind crossover design with blood pressure monitored on days 1 (D1) and 6 (D6) of each period. There was no significant difference in blood pressure when compared as either 2-hour intervals or 24-hour global means: (placebo) 116/68 (D1), 117/68 (D6); (PPA) 118/69 (D1), 119/69 (D6). Our results document the absence of pressor effect with PPA in therapeutic doses even with repeated measurements and further confirm the reproducibility of 24-hour blood pressure monitoring.