Vascular Amputees: A Study in Disappointment.

Despite optimistic reports about the results of amputation for advanced vascular disease, the patient's assessment of advantages and disadvantages is seldom acknowledged. A detailed social study of 67 amputees has revealed considerable disparity between the patient's views and those of the medical staff. About a third of the patients are forced to retire from active work by the amputation; about three-quarters report a serious decline in their social activities; only about half are really independent with prostheses in the long term; a quarter report severe and intractable symptoms related to their amputation stumps; only about a quarter feel that the amputation was definitely beneficial; and only about one in five feel that the medical staff have provided adequate support during their hospital stay. Amputees face physical and financial disability, isolation, and discomfort. Every effort must be made to explain the implications of amputation honestly and realistically and to ensure continuing patient assessment and support.

Electron Cooling in a Magnetically Expanding Plasma.

Electron cooling in a magnetically expanding plasma, which is a fundamental process for plasma flow and detachment in magnetic nozzles, is experimentally investigated using a radio frequency plasma source and magnetic nozzle (MN). Probe measurements of the plasma density, potential, and electron temperature along the center line of the MN indicate that the expansion follows a polytropic law with exponent γ_{e}=1.15±0.03. This value contradicts isothermal electron expansion, γ_{e}=1, which is commonly assumed in MN models. The axial variation of the measured quantities can be described by a simple quasi-1D fluid model with classical electron thermal conduction, for which it has been previously shown that a value of γ_{e}≈1.19 is expected in the weakly collisional limit. A new criterion, derived from the model, ensures efficient ion acceleration when a critical value for the ratio of convected to conducted power is exceeded.

Estimating ON and OFF contributions to the photopic hill: normative data and clinical applications.

BACKGROUND: With progressively brighter stimuli, the amplitude of the b-wave of the human photopic electroretinogram (ERG) first increases to a maximal value (Vmax) and then decreases to finally reach a plateau, a phenomenon known as the photopic hill (PH). A mathematical model combining a Gaussian (G) and a logistic (L) growth function was previously proposed to fit this unusual luminance-response curve, where the G and L functions were suggested to represent, respectively, the OFF and ON retinal pathway contributions to the building of the PH. METHOD: The PHs of patients presenting stationary diseases affecting specifically the ON (3 CSNB-1) or OFF (4 CPCPA) retinal pathways as well as patients affected with retinitis pigmentosa (14 RP) of different stages or etiology were analyzed using this mathematical model and compared to the PHs of a group of 28 normal subjects. RESULTS: The PH of the CSNB-1 patients had a much larger contribution from the G function compared to normal subjects, whereas the opposite was observed for the CPCPA patients. On the other hand, analysis of data from RP patients revealed variable G-L contributions to the building of their PH. CONCLUSION: In this study, we confirm the previous claim that the luminance-response function of the photopic ERG b-wave can be decomposed into a Gaussian function and a logistic growth function representing, respectively, the OFF and ON retinal pathways. Furthermore, our findings suggest that this mathematical decomposition could be useful to further segregate and potentially follow the progression of retinopathies such as RP.

An empirical study of tissue banking in Australia: navigating regulatory and ethical challenges.

Collections of tumour samples can be an invaluable resource for medical research. There are, however, numerous ethical and legal challenges associated with tumour banking. While there has been extensive discussion of these issues in the legal and ethical literature, there are few available empirical data in relation to the activities of tumour banks in Australia, their practices around ethically charged issues, and their success in implementing complex regulatory guidelines. The aim of this study was to gain more information about the activities of tumour banks in New South Wales, Australia, with a particular focus on their management of, and attitudes towards, ethical and regulatory issues. A survey of 27 tumour collection and research facilities was conducted using a 55-item questionnaire. There is significant heterogeneity of research methodologies as well as of methods for gaining consent and ensuring donor privacy, and there is general concern among the research community about ethical and regulatory issues related to tumour banking. Heterogeneity of practice and uncertainty about ethical and regulatory requirements is problematic in its potential to hinder research and its potential to generate the space for unethical practice, whether intentional or unintentional. There is a pressing need to address these issues so that tumour banks can be used in the most ethical and efficient way possible.

The GKT diploma in dental sedation--a judgement.

AIMS AND OBJECTIVES: The use of sedation in the management of pain and anxiety for the provision of dental care is as vital to the dental profession as are windscreen wipers to a motor vehicle. Not for use on every patient or every occasion, but in times of need to wipe away the tears, and essential for effective work. Training in sedation techniques should be a part of the undergraduate curriculum, and postgraduate opportunities need to be developed to support this important aspect of care. This paper examines a particular training course provided within the Department of Sedation and Special Care Dentistry at GKT Dental Institute, King's College London, leading to the Diploma in Conscious Sedation for Dentistry (Dip.D.Sed). The aim of this study was to investigate what impact the course has had on the practice of sedation. Three objectives were defined: 1) Students' evaluation of the course; 2) Students' practice in sedation prior to and on completion of the course; 3) Students' involvement in sedation training of dentists or dental nurses following completion of the course. METHOD: Information was obtained by postal questionnaire from students who had attended the course since its inception in 1997 to 2000. RESULTS: 30 students completed and returned the questionnaire which represented a 100% response. There was an overall expression of satisfaction from students on the course content and the experience they had obtained. The range of experience was 70-100 treatment episodes over 40 clinical sessions. An increase in both the practice of sedation and the involvement in training (dental nurses and dentists) was also shown. The greatest clinical change was the increase in use of intravenous sedation by the students from the community dental service. CONCLUSION AND RECOMMENDATION: This study concluded that the objectives of the course had been achieved. The importance of providing training that enables the safe and effective provision of sedation within primary care as an operator sedationist was strengthened by this study. The value of an intermediate level between the two day section 63 course and the six month diploma course was suggested by students in this study. The development of a clinical attachment based on The Standard Course in Conscious Sedation was proposed as a possible option to fill the gap. The provision of postgraduate training in sedation is limited particularly in some areas of the UK. This problem should be addressed by increasing the opportunity for postgraduate training in sedation by dental schools and postgraduate deaneries. Work towards increasing the funding and opportunities for training in this important area of care needs to be undertaken.

Human UDP-glucuronosyltransferase 2B7.

UDP-Glucuronosyltransferases (UGTs) are glycoproteins, localized in endoplasmic reticulum (ER) and nuclear membranes, which catalyze the confugation of a broad variety of lipophilic aglycon substrates with glucuronic acid using UDP-glucuronic acid (UDP-GlcUA) as the sugar donor. The major function of glucuronidation is to change hydrophobic compounds into hydrophilic derivatives, a process which facilitates their detoxification and excretion. However, it is also widely recognized that glucuronidation can result in compounds which are biologically active or demonstrate increased toxicity. UGTs, like other drug-metabolizing enzymes, have been postulated to be involved in controlling the steady state concentrations of nuclear receptor ligands for interactions with nuclear receptors [1,2]. One of the isoforms from the UGT2B subfamily, UGT2B7, has been found to be a major human UGT2B isoform, involved in the glucuronidation of a variety of endogenous compounds and xenobiotics. In this review, we included all available information from our studies and those of other investigators on a) the history of the identification and expression of UGT2B7 in human tissues, b) the substrate specificity of UGT2B7, c) the extrahepatic localization of UGT2B7 d) the nuclear localization of UGT2B7 and e) characterization of the UGT2B7 gene and promoter.

Healthcare rationing: constraints and equity.

It is no longer possible or ethically acceptable to deny that rationing occurs in medical practice. We ration already by using "contraindications to treatment". There are no rationing criteria that are universally ethically acceptable. We need ways to establish community preferences if we are to develop responsible methods of rationing healthcare services.

Ethical thinking and stakeholders.

Modern medical practice increasingly requires us to think beyond the confines of the doctor-patient relationship to other stakeholders. Problems and issues that can be identified as being of interest to a number of people are defined as "focal entities". Stakeholders are those with an interest in the outcome of the problem or issue. The salience of stakeholders is defined by their power, legitimacy and urgency. The process of thinking about stakeholders can help to clarify our ethical thinking about matters of importance to people beyond the doctor-patient relationship.

Glucuronidation of linoleic acid diols by human microsomal and recombinant UDP-glucuronosyltransferases: identification of UGT2B7 as the major isoform involved.

Recent reports suggest that linoleic acid (LA) epoxides and diols are associated with important physiological, pharmacological, and pathological events in vivo. We have shown recently that LA-diols are excellent substrates for human liver microsomal UDP-glucuronosyltransferases (UGTs); however, it is not known if other human tissues glucuronidate LA-diols or which UGT isozyme(s) is involved. The present studies with human intestinal microsomes indicate that glucuronidation of LA-diols occurs throughout the gastrointestinal tract, with the highest activity in the small intestine. LA-diols yielded exclusively hydroxyl-linked glucuronides, whereas LA yielded the carboxyl-linked glucuronide. Studies with human recombinant UGTs demonstrated that only UGT2B7 glucuronidated LA and LA-diols. Kinetic analysis with UGT2B7 yielded apparent K(m) values in the range of 40-70 microM and V(max) values from 4.5 to 5.4 nmol/mg x min. These studies indicate that LA and LA-diols are excellent substrates for intestinal UGTs and provide the first evidence for UGT2B7 being the major isoform involved.

Confidentiality.

Issues of confidentiality are complicated by the relationships we have to patients and others who have valid interests in the confidential information. There are no straightforward answers to problems which involve complex relationships and sensitive information. The best we can do is to think thoroughly and carefully about the issues in each case, and use our knowledge of the people involved to reach a decision. Doctors faced with difficult decisions of this kind should be assured that everyone finds them difficult. Sharing the burden with experienced colleagues can be helpful.

13-hydroxy- and 13-oxooctadecadienoic acids: novel substrates for human UDP-glucuronosyltransferases.

Although there are numerous studies of glucuronidation of endogenous compounds, information on the glucuronidation of fatty acids is lacking. In the present studies, both linoleic acid (LA) and its biologically active oxidized derivatives, 13-hydroxyoctadecadienoic acid (13-HODE) and 13-oxooctadecadienoic acid (13-OXO), have been shown to be effective substrates for human liver UDP-glucuronosyltransferases (UGT) and recombinant UGT2B7. LA (carboxyl glucuronide) and 13-OXO (carboxyl glucuronide, unproven) were actively glucuronidated by human liver microsomes (HLM) and human recombinant UGT2B7 with similar activities, in the range of 2 nmol/mg. min. The hydroxyl derivative of LA, 13-HODE, was glucuronidated at both the hydroxyl and carboxyl functions with carboxyl glucuronidation predominating (ratio of COOH/OH, 2:1). For all substrates, the K(m) for formation of the carboxyl-linked glucuronide was in the range of 100 to 200 microM while that for the hydroxyl-linked glucuronide was somewhat lower (>100 microM). This is the first demonstration of glucuronidation of LA and its oxidized derivatives, 13-HODE and 13-OXO, by HLM and recombinant UGT2B7.

Application of photoaffinity labeling with [(3)H] all trans- and 9-cis-retinoic acids for characterization of cellular retinoic acid--binding proteins I and II.

Cellular retinoic acid-binding proteins (CRABPs) are carrier proteins thought to play a crucial role in the transport and metabolism of all-trans-retinoic acid (atRA) and its derivatives within the cell. This report describes a novel photoaffinity-based binding assay involving competition between potential ligands of CRABP and [(3)H]atRA or [(3)H]-9-cis-RA for binding to the atRA-binding sites of CRABP I and II. Photoaffinity labeling of purified CRABPs with [(3)H]atRA was light- and concentration-dependent, saturable, and protected by several retinoids in a concentration-dependent manner, indicating that binding occurred in the CRABP atRA-binding site. Structure-function relationship studies demonstrated that oxidative changes to the atRA beta-ionone ring did not affect ligand potency. However, derivatives lacking a terminal carboxyl group and some cis isomers did not bind to CRABPs. These studies also identified two novel ligands for CRABPs: 5,6-epoxy-RA and retinoyl-beta-D-glucuronide (RAG). The labeling of both CRABPs with 9-cis-RA occurred with much lower affinity. Experimental evidence excluded nonspecific binding of RAG to CRABPs and UDP-glucuronosyltransferases, the enzymes responsible for RAG synthesis. These results established that RAG is an effective ligand of CRABPs. Therefore, photoaffinity labeling with [(3)H]atRA can be used to identify new ligands for CRABP and retinoid nuclear receptors and also provide information concerning the identity of amino acid(s) localized in the atRA-binding site of these proteins.

Glucuronidation of estrogens and retinoic acid and expression of UDP-glucuronosyltransferase 2B7 in human intestinal mucosa.

We have recently shown that, in human intestine, glucuronidation of androsterone and testosterone was on the nanomolar level and increased from proximal to distal intestine. In the present study, we have characterized estrogen UDP-glucuronosyltransferase activity in microsomes from intestine of seven human subjects. Intestinal microsomes from all segments of intestine from both males and females (except for one male) glucuronidated estrone (0.2-2.6 nmol/mg x min) and estradiol (0.5-3.1 nmol/mg x min) at levels 2 to 15 times higher than found with human liver microsomes (0.04-0.1 and 0.16-0.25 nmol/mg x min, for estrone and estradiol, respectively). Only with estriol were there significant hepatic glucuronidation (2. 2-4.5 nmol/mg x min) and intestinal glucuronidation activities (0.2-2.2 nmol/mg x min) that were lower than those in liver. All-trans-retinoic acid was glucuronidated by all segments of intestine from both sexes at levels 50 to 80% of those found with human liver but quite low compared with estrogen glucuronidation. In the two subjects for whom stomach was available, there was no measurable activity in stomach microsomes toward any of the substrates. UGT2B RNA expression was examined in mucosa from stomach to colon from two subjects. There was significant expression of UGT2B7, but not of UGT2B4 or UGT2B15, in all segments of intestine. To our knowledge, this is the first direct demonstration of glucuronidation of estrogens by human intestinal microsomes. Thus, in humans, the intestine may be considered as part of the overall mechanism of detoxification via glucuronidation.

Linoleic acid diols are novel substrates for human UDP-glucuronosyltransferases.

Linoleic acid diol glucuronides have been isolated previously from urine of patients suffering from generalized peroxisomal disorders. Glucuronidation of linoleic acid and linoleic acid diols by human liver microsomes was studied to investigate the role of glucuronide conjugation in the metabolism of linoleic acid diols. Glucuronide products were isolated and analyzed by TLC and HPLC-MS. HPLC-MS showed ions with (m/z) corresponding to singly glucuronidated linoleic acid diols while TLC revealed that the glucuronidation was at a hydroxyl position. Kinetic analysis gave apparent K(m) values in the range of 50-200 microM and V(max) rates from 5 to 12 nmol/mg x min. These rates are substantially higher than activities seen for most endogenous hydroxylated substrates. Assays using each of the four individually purified linoleic acid diol enantiomers suggest that glucuronidation occurs at only one of the two hydroxyl groups of each enantiomer. These results show for the first time that hydroxylated fatty acids are actively glucuronidated by human liver microsomes and suggest that glucuronidation may play a significant role in the biotransformation of linoleic acid diols in humans.

Direct interaction of all-trans-retinoic acid with protein kinase C (PKC). Implications for PKC signaling and cancer therapy.

Protein kinase C (PKC) regulates fundamental cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. All-trans-retinoic acid (atRA) modulates PKC activity, but the mechanism of this regulation is unknown. Amino acid alignments and crystal structure analysis of retinoic acid (RA)-binding proteins revealed a putative atRA-binding motif in PKC, suggesting existence of an atRA binding site on the PKC molecule. This was supported by photolabeling studies showing concentration- and UV-dependent photoincorporation of [(3)H]atRA into PKCalpha, which was effectively protected by 4-OH-atRA, 9-cis-RA, and atRA glucuronide, but not by retinol. Photoaffinity labeling demonstrated strong competition between atRA and phosphatidylserine (PS) for binding to PKCalpha, a slight competition with phorbol-12-myristate-13-acetate, and none with diacylglycerol, fatty acids, or Ca(2+). At pharmacological concentrations (10 micrometer), atRA decreased PKCalpha activity through the competition with PS but not phorbol-12-myristate-13-acetate, diacylglycerol, or Ca(2+). These results let us hypothesize that in vivo, pharmacological concentrations of atRA may hamper binding of PS to PKCalpha and prevent PKCalpha activation. Thus, this study provides the first evidence for direct binding of atRA to PKC isozymes and suggests the existence of a general mechanism for regulation of PKC activity during exposure to retinoids, as in retinoid-based cancer therapy.

Variability of human hepatic UDP-glucuronosyltransferase activity.

The availability of a unique series of liver samples from human subjects, both control patients (9) and those with liver disease (6; biliary atresia (2), retransplant, chronic tyrosinemia type I, tyrosinemia, Wilson's disease) allowed us to characterize human hepatic UDP-glucuronosyltransferases using photoaffinity labeling, immunoblotting and enzymatic assays. There was wide inter-individual variation in photoincorporation of the photoaffinity analogs, [32P]5-azido-UDP-glucuronic acid and [32P]5-azido-UDP-glucose and enzymatic glucuronidation of substrates specific to the two subfamilies of UDP-glucuronosyltransferases. However, the largest differences were between subjects with liver disease. Glucuronidation activities toward one substrate from each of the UDP-glucuronosyltransferases subfamilies, 1A and 2B, for control and liver disease, respectively, were 1.7-4.5 vs 0.4-4.7 nmol/mg x min for hyodeoxycholic acid (2B substrate) and 9.2-27.9 vs 8.1-75 nmol/mg x min for pchloro-m-xylenol (1A substrate). Microsomes from a patient with chronic tyrosinemia (HL32) photoincorporated [32P]5-azido-UDP-glucuronic acid at a level 1.5 times higher than the other samples, was intensely photolabeled by [32P]5-azido-UDP-glucose and had significantly higher enzymatic activity toward p-chloro-m-xylenol. Immunoblot analysis using anti-UDP-glucuronosyltransferase antibodies demonstrated wide inter-individual variations in UDP-glucuronosyltransferase protein with increased UDP-glucuronosyltransferase protein in HL32 microsomes, corresponding to one of the bands photolabeled by both probes. Detailed investigation of substrate specificity, using substrates representative of both the 1A (bilirubin, 4-nitrophenol) and 2B (androsterone, testosterone) families was carried out with HL32, HL38 (age and sex matched control) and HL18 (older control). Strikingly increased (5-8-fold) glucuronidation activity was seen in comparison to HL18 only with the phenolic substrates. The results indicate that one or more phenol-specific UDP-glucuronosyltransferase 1A isoforms are expressed at above normal levels in this tyrosinemic subject.

Structural and functional studies of UDP-glucuronosyltransferases.

UDP-Glucuronosyltransferases (UGTs) are glycoproteins localized in the endoplasmic reticulum (ER) which catalyze the conjugation of a broad variety of lipophilic aglycon substrates with glucuronic acid using UDP-glucuronic acid (UDP-GIcUA) as the sugar donor. Glucuronidation is a major factor in the elimination of lipophilic compounds from the body. In this review, current information on the substrate specificities of UGT1A and 2B family isoforms is discussed. Recent findings with regard to UGT structure and topology are presented, including a dynamic topological model of UGTs in the ER. Evidence from experiments on UGT interactions with inhibitors directed at specific amino acids, photoaffinity labeling, and analysis of amino acid alignments suggest that UDP-GIcUA interacts with residues in both the N- and C-terminal domains, whereas aglycon binding sites are localized in the N-terminal domain. The amino acids identified so far as crucial for substrate binding and catalysis are arginine, lysine, histidine, proline, and residues containing carboxylic acid. Site-directed mutagenesis experiments are critical for unambiguous identification of the active-site architecture.

Vacuolation in renal tubular epithelium of Cd-1 mice: an incidental finding.

Prominent cytoplasmic vacuoles were observed in renal tubular epithelial cells of the outer medulla in several kidneys from test article-dosed mice (Crl:CD-1 (ICR)BR VAF/PLUS) during routine light microscopic (LM) examination. Because the vacuolar change was detected infrequently and was not found in any control mice from that study, it was not clear whether the vacuolation represented a drug-induced change. To address this question, kidney sections from mice from multiple unrelated studies were examined by LM for similar vacuolar changes. Vacuolation was seen by LM in 2.3% of the control and 2.8% of the test article-dosed mice. Transmission electron microscopy (TEM) was also performed on kidneys with prominent light microscopic vacuoles in 5 control mice and 2 test article-dosed mice to further characterize the vacuoles. Ultrastructurally, the vacuoles contained fibrillar and finely stipled granular material or membranous whorls. Kidneys from control mice lacking light microscopic evidence of vacuolation had smaller vacuoles containing similar material when examined by TEM. Because vacuoles were present in both control mice and test article-dosed mice, it was concluded that the vacuoles were incidental and unrelated to compound administration. These studies also demonstrated that vacuoles can be expected to be observed by LM examination in 2-3% of Crl:CD-1 (ICR)BR VAF/PLUS, mice.