Biomedical subjects
J Rafferty
Publications and source records attributed to J Rafferty.
Plant glycerol-3-phosphate-1-acyltransferase (GPAT): structure selectivity studies.
Squash glycerol-3-phosphate-1-acyltransferase has been crystallized and the structure of the enzyme determined, at 1.9-A resolution, using multiple isomorphous replacement of the wild type and a series of individual cysteine mutants. Competitive in vitro substrate selectivity assays have been established that differentiate between selective and non-selective forms of the enzyme. Particular care was taken to use near-physiological concentrations of both substrates. Clear substrate selectivity can be demonstrated with the natural substrate acyl-acyl carrier protein but not with the substrate analogue acyl-CoA. The use of site-directed mutagenesis, coupled to three-dimensional structural determinations, should provide a rational basis for elucidating structural components important in determining the substrate selectivity of this enzyme.
Mutagenesis of squash (Cucurbita moschata) glycerol-3-phosphate acyltransferase (GPAT) to produce an enzyme with altered substrate selectivity.
In an attempt to rationalize the relationship between structure and substrate selectivity of glycerol-3-phosphate acyltransferase (GPAT, 1AT, EC 2.3.1.15) we have cloned a number of cDNAs into the pET overexpression system using a PCR-based approach. Following assay of the recombinant enzyme we noted that the substrate selectivity of the squash (Cucurbita moschata) enzyme had altered dramatically. This form of GPAT has now been crystallized and its full three-dimensional structure elucidated. Since we now have two forms of the enzyme that display different substrate selectivities this should provide a powerful tool to determine the basis of the selectivity changes. Kinetic and structural analyses are currently being performed to rationalize the changes which have taken place.
Protection of committed murine haemopoietic progenitors against BCNU toxicity does not predict protection of primitive, multipotent spleen colony-forming cells - implications for chemoprotective gene therapy.
The effect of expression of an O6-benzylguanine (O6-beG)-resistant mutant (hATPA/GA) of human O6-alkylguanine-DNA alkyltransferase (ATase) on the in vivo toxicity and clastogenicity of the anti-tumour agent N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) to murine bone marrow has been investigated. When compared with control animals, the bipotent granulocyte-macrophage colony-forming (GM-CFC) progenitor population of the hATPA/GA transduced mice were somewhat more resistant to BCNU (1.4-fold, P = 0.047) and this effect was more significant in the presence of the ATase inactivator O6-beG (3. 5-fold, P = 0.001). The polychromatic erythrocytes were also significantly protected against BCNU-induced clastogenicity both in the presence (P < 0.001) and absence of O6-beG (P < 0.05). The primitive, multipotent spleen colony-forming cells (CFU-S) in these animals also showed moderate (1.6-fold, P = 0.034) protection in the absence of O6-beG but in the presence of the inactivator they remained as sensitive to BCNU toxicity as those in the control animals (P = 0.133). This result contrasts with previous findings demonstrating significant hATPA/GA-mediated, O6-beG-resistant protection against the toxicity and clastogenicity of a number of O6-alkylating agents, including temozolomide, fotemustine and chlorozotocin. The possibility that our strategy for protective gene therapy may be highly agent and cell-type specific is unexpected and has possible implications for clinical trials of this approach using BCNU or related agents.
Detrimental effect of nitric oxide synthase inhibition during endotoxemia may be caused by high levels of tumor necrosis factor and interleukin-6.
BACKGROUND: Nitric oxide (NO) production increases during sepsis and endotoxemia. Inhibition of NO synthase has been suggested as a therapeutic modality in sepsis and endotoxemia, but in recent reports NO synthase inhibition increased mortality rate. The mechanism of this phenomenon is not known. Other studies have shown that high levels of tumor necrosis factor (TNF) and interleukin-6 (IL-6) contribute to death during sepsis and endotoxemia. We tested the effect of NO synthase inhibition on survival in endotoxemic rats and hypothesized that inhibition of NO synthase during endotoxemia increases circulating levels of TNF and IL-6. METHODS: Rats were treated with subcutaneous injection of saline solution or 100 mg/kg of the NO synthase inhibitor N-nitro-L-arginine 1 hour before intravenous injection of endotoxin (15 mg/kg) or saline solution. Survival was followed for 24 hours. Plasma nitrite-nitrate (NO2/NO3), TNF, and IL-6 levels were determined at intervals. RESULTS: Endotoxin caused a significant increase in levels of plasma NO2-/NO3-, TNF, and IL-6 and a 33% mortality rate. Pretreatment with N-nitro-L-arginine increased mortality rate to 74%, decreased NO2/NO3, and substantially increased TNF and IL-6 levels. CONCLUSIONS: Inhibition of NO synthase increases mortality rate during endotoxemia. The detrimental effect of NO synthase inhibition during endotoxemia may be caused by excessive production of TNF and IL-6.
Burnout and career-choice regret among family practice physicians in early practice.
OBJECTIVE: Burnout and career-choice regret among physicians in early practice may contribute to physician impairment and attrition as well as patient dissatisfaction. METHOD: Fifty residency-trained family physicians in early practice completed the Maslach Burnout Inventory and a questionnaire on career-choice, demographics, and practice characteristics. RESULTS: The sample showed moderate burnout related to emotional exhaustion and depersonalization of patients but low burnout related to lack of personal accomplishment. Although only 52% reported that they would definitely choose a medical career again, 70% reported that they would definitely choose the specialty of family practice again. At the 0.01 level of significance, those who worked more hours were more emotionally exhausted, and those with children and those with more children under the age of 5 reported less depersonalization. CONCLUSIONS: This sample of physicians in early practice reported slightly less burnout and less specialty-choice regret than a comparable sample of family practice residents, suggesting possible attenuation of burnout with the transition from training to practice. Nonetheless, the importance of overwhelming time demands as a precipitant of exhaustion and potential physician impairment is noted.
Absence of an effect of high vitamin C dosage on the systemic availability of ethinyl estradiol in women using a combination oral contraceptive.
Previous studies in small numbers of women have suggested that the administration of gram quantities of ascorbic acid interferes with the conversion of ethinyl estradiol (EE2) to its sulfates, leading to higher blood levels of EE2. The possibility of such potentiation has been investigated in 37 women using a combination monophasic oral contraceptive (30 micrograms EE2 and 150 micrograms levonorgestrel) for two consecutive cycles. Concomitant daily administration of 1 g ascorbic acid taken 1/2 hour before OC intake, was randomly assigned to the first or second cycle of OC use. On the first and 15th day of OC intake, blood samples were drawn 11 times over a 12-hour interval and Cmax and AUC(0-12 h) calculated. On pill days 10 and 21, only 6-hour post-intake samples were obtained. Samples were analyzed for levels of ascorbic acid, free and sulfated ethinyl estradiol (and a number of other parameters). Cmax and AUC values for EE2 and EE2-sulfate in cycles with and without ascorbic acid were evaluated statistically by the Grizzle model for days 1 and 15 and the ratios of day 15/day 1 for each of the substances. No effect of ascorbic acid was observed (alpha = 0.05, 1-beta = 0.9). Only on day 15 was there a significantly lower AUC for EE2-sulfate in the presence of ascorbic acid intake. Thus, the competition between ascorbic acid and EE2 for sulfation does not lead to an increased systemic availability of EE2 and is, therefore, unlikely to be of any clinical importance. Ascorbic acid can, therefore, be removed from the list of drugs interfering with the pharmacokinetics of ethinyl estradiol.
Effects of habitual caffeine use and acute ingestion: testing a biobehavioral model.
A recently proposed model of the biobehavioral effects of caffeine suggests that acute ingestion impacts physiology and behavior differentially depending on the level of habitual usage of the drug. Acute ingestion and habitual usage are particularly expected to affect arousal and attentional processes. Subjects in the present study were preselected for high and low habitual caffeine use, given caffeine or a placebo, exposed to white noise or no white noise, and asked to perform on several tasks. Included were an arousal/habituation task (pure tones), reaction time, paired associates, anagrams, and vigilance. Electrodermal activity and performance were recorded. As predicted, virtually all effects were on the arousal/habituation and attentional (vigilance) tasks. Both acute ingestion and habitual use increased tonic EDA, and chronic use also reduced phasic responding, especially in the presence of a strong habituating stimulus. Both acute and habitual use also liberalized the vigilance response criterion, in that subjects risked more false alarms in order to attain more hits. In addition, habitual use increased sensitivity and reduced accuracy, and acute ingestion increased vigilance response time in the presence of white noise. Overall, the model was partially supported by these early results, though considerable further research is needed.
Expression of a human O6-alkylguanine-DNA-alkyltransferase cDNA in human cells and transgenic mice.
A truncated human O6-alkylguanine-DNA-alkyltransferase (ATase) cDNA was ligated into an expression vector under the control of the mouse metallothionein-1 gene promotor and upstream of part of the human growth hormone gene to provide splice and polyadenylation signals. Transfection of this construct into human cells resulted in very high levels of ATase expression (more than 300 fmoles/mg protein versus less than 2 fm/mg protein in parent vector transfected control cells). Microinjection of a 4.2 kb fragment of this vector into B6D2F2 mouse embryos and implantation of survivors into pseudopregnant females has so far generated 35 offspring. Southern analysis of tail tip DNA has shown that 11 of the offspring are transgenic for the human ATase gene, between 1 and at least 30 copies of the gene being detected. Human ATase transcripts were detected in total RNA extracted from liver obtained from two male transgenic mice by partial hepatectomy. Cell free extracts of liver samples from five transgenic mice showed up to 4 times higher ATase levels than control livers.
The functional status of elderly people admitted to a local authority residential home.
One hundred and forty-one consecutive admissions to a large local authority residential home were studied to assess their functional level. Both panel (71) and emergency (70) admissions were assessed. Forty-seven per cent of the panel admissions came from hospital and 39% from home compared with 4% and 83% of the emergency admissions. Severe mental impairment was found in 22% panel admissions and 34% of emergencies. Dementia was the commonest underlying condition. Inability to cope at home was the most common reason for referral in both groups. The activities of daily living (ADL) were assessed where possible on admission and after two to four weeks. No statistically significant change was found in any aspect of ADL between the two assessments although 9% of the panel and 19% of the emergency group were assessed as being unacceptable for residential care in one function. Residents initially showed a high level of negative attitude to the concept of residential care but this lessened over the four weeks. This study has shown that although new residents are suitable on admission to residential care, they are marginally so and are likely to become more dependent. This has future implications for staff levels and training in residential homes.
A study of zinc status of elderly institutionalized patients.
In a study of zinc status in the elderly, the mean serum and leucocyte zinc concentrations were found to be significantly lower in continuing-care patients compared with elderly people living in the community. The mean daily dietary intakes of zinc, protein and fibre did not differ between the two groups. Serum zinc levels were found to correlate with serum albumin levels. No significant relationship was found between the mean serum or leucocyte zinc concentrations of patients and either their mental status or the presence of skin pressure damage, leg ulcers or unhealed wounds.
Medical assessment of elderly persons prior to a move to residential care: a review of seven years' experience in Edinburgh.
Assessment prior to a move to residential care has been carried out by the Departments of Geriatric Medicine in the Lothian Region for many years. Results have been reviewed over 7 years in one Department; 688 people were assessed; 7% were placed in alternative accommodation as a result of the assessment and a further 15% were offered specialist advice. The workload was 50 assessments per 10,000 population over 65 years per year. Of these, 58% were already known to the geriatrician and 23% were seen as part of the normal work of the department. It was concluded that the benefits of assessment make the extra work involved well worth while.
Quality of care review: recent experience in Arizona.
The Arizona Health Care Cost Containment System (AHCCCS) is an HMO-oriented Medicaid demonstration project serving approximately 170,000 persons throughout the State of Arizona. To assure that its members are receiving high quality care and because of the potentially adverse incentives of capitation, AHCCCS places particular emphasis on quality-of-care review. A key component of that review is the development of annual, statewide medical audits. In this article, we describe the nature and organization of medical care delivery under AHCCCS, outline the evolution of the annual medical audits, describe the most recent of these audits, and present and discuss the results.
The economics of hospital reimbursement.
Explore the source record for details and available documents.
Behavior of the multiproduct firm. A model of the nonprofit hospital system.
This article presents a hospital model that recognizes the multiproduct nature of hospital output and incorporates trade-offs among various competing goals. A utility function for the hospital is defined over quantity, quality and the net revenues associated with the treatments produced. Utility is constrained by epidemiological factors and by availability of beds. The model is used to draw comparative statics implications, from which econometric hypotheses are developed and tested. This is accomplished by a set of equations that predict variations in patient-specific lengths of stay, admissions, expenses per admission, and occupancy rates. Empirical results are generally consistent with predictions. The model has a variety of policy applications, and these are illustrated.
Development of an item bank.
Explore the source record for details and available documents.
Variations in hospital product: a comparative analysis of proprietary and voluntary hospitals.
Explore the source record for details and available documents.