bluuebunny/crossref_metadata_2025 huggingface dataset:
The medical visit context of treatment decision‐making and the therapeutic relationship
Authors: Roter Debra
Published: March 2003

The ascendance of the autonomy paradigm in treatment decision‐making has evolved over the past several decades to the point where few bioethicists would question that it is the guiding value driving health‐care provider behaviour. In achieving quasi‐legal status, decision‐making has come to be regarded as a formality largely removed from the broader context of medical communication and the therapeutic relationship within which care is delivered. Moreover, disregard for individual patient preference, resistance, reluctance, or incompetence has at times produced pro forma and useless autonomy rituals. Failures of this kind, have been largely attributed to the psychological dynamics of the patients, physicians, illnesses, and contexts that characterize the medical decision. There has been little attempt to provide a framework for accommodating or understanding the larger social context and social influences that contribute to this variation. Applying Paulo Freire’s participatory social orientation model to the context of the medical visit suggests a framework for viewing the impact of physicians’ communication behaviours on patients’ capacity for treatment decision‐making. Physicians’ use of communication strategies can act to reinforce an experience of patient dependence or self‐reliance in regard to the patient‐physician relationship generally and treatment decision‐making, in particular. Certain communications enhance patient participation in the medical visit’s dialogue, contribute to patient engagement in problem posing and problem‐solving, and finally, facilitate patient confidence and competence to undertake autonomous action. The purpose of this essay is to place treatment decision‐making within the broader context of the therapeutic relationship, and to describe ways in which routine medical visit communication can accommodate individual patient preferences and help develop and further patient capacity for autonomous decision‐making.
Eliciting patients’ preferences for adjuvant chemotherapy in breast cancer: development and validation of a bedside decision‐making instrument in a French Regional Cancer Centre
Authors: Carrère Marie‐Odile , Moumjid‐Ferdjaoui Nora , Charavel Marie , Brémond Alain
Published: March 2003

Introduction

In developed countries, the physician‐patient relationship is moving from a paternalistic model to new decision‐making models that take patient preferences into account.

Objectives

Our aim was to develop a Decision Board (DB) and to test its acceptability in a French Regional Cancer Centre regarding the decision on whether or not to use chemotherapy after surgery in postmenopausal women with breast cancer. This paper presents the development process for this instrument and reports the pretesting phase, as well as the corresponding results.

Methods

A working group was created with oncologists, psychologists and economists. Following the first phase, i.e. the development process, a first version of the instrument was presented to health professionals. Their feedback led to important modifications of the instrument. The DB was then presented to experienced patients, which resulted in slight changes. The second phase consisted of pretesting the comprehension, internal and across‐time consistency of the DB on healthy volunteers.

Results

The DB was pretested in a group of 40 healthy volunteers. Eighteen respondents chose chemotherapy and 22 chose not to have chemotherapy. Comprehension rates were very high (≥87.5%). Internal consistency was assessed considering option attitudes based on outcomes and option attitudes based on process. Women shifted their choices in a predictable way. Across‐time consistency was appraised using the test‐retest method with Visual Analog Scales. The Intraclass Correlation Coefficient was 0.97.

Discussion‐conclusion

Due to cultural differences, the DB developed in our French Cancer Centre is quite different from the DBs previously developed elsewhere. Our instrument showed good comprehension and consistency properties, which are corroborated by the DB literature. Whether our DB is acceptable for patients with breast cancer must still be tested. Patients’ reactions will tell us which type of decision‐making model is at work. Further research is needed in order to explore the shared decision‐making process and clarify the concept.
Eliciting patients’ values by use of ‘willingness to pay’: letting the theory drive the method
Authors: Donaldson Cam
Published: March 2003

Objective To describe the three different ways in which ‘willingness to pay’ (WTP) has been used to elicit patients’ values of alternative interventions.

Design For each of the three approaches a survey of patients or the public was undertaken.

Setting, participants and interventions studied For two surveys, the setting was Aberdeen Maternity Hospital, where pregnant women were asked about their WTP for different methods of prenatal screening for cystic fibrosis. In the third survey, parents of primary and secondary schoolchildren were asked about their WTP for different ways of providing child health services.

Main outcome measures Ability of WTP to discriminate between options (i.e. to say whether one option is ‘better’ than another) and the consistency of WTP with stated preferences.

Results Experience with some methods shows that, despite the apparent logic of the technique, it is difficult to elicit consistent responses whereby WTP values derived match the rankings of interventions compared. The most promising technique, the ‘marginal approach’, happens to conform more with economic theory than other approaches. Potential limitations of WTP, such as its association with ability to pay, are discussed, as are approaches to dealing with such problems. Finally, if patients prefer an intervention that is more costly than the status quo, logic dictates that those extra resources will have to be obtained from another health‐care programme. In such contexts, to aid decision‐making, values derived from members of the community for different programmes may be more relevant than values derived from patients. Initial studies in the use of WTP in this broader context of eliciting community values are also outlined.

Conclusions WTP has potential, but its application, and interpretation, are not straightforward. More testing of the ‘marginal approach’ is required and greater use of qualitative research, to assess the validity of the approach, should be made in this area.
Increasing informed uptake and non‐uptake of screening: evidence from a systematic review
Authors: Jepson Ruth G. , Forbes Carol A. , Sowden Amanda J. , Lewis Ruth A.
Published: March 2003

Objective To report data relating to the informed uptake of screening tests.

Search strategy Electronic databases, bibliographies and experts were used to identify relevant published and unpublished studies up until August 2000.

Inclusion criteria RCTs, quasi‐RCTs and controlled trials of interventions aimed at increasing the informed uptake of screening. All participants were eligible as defined by the entry criteria of individual programmes. Studies had to report actual uptake and meet three out of four criteria used to define informed uptake.

Data extraction and synthesis Relevant studies were identified, data extracted and their validity assessed by two reviewers independently. Outcome data included screening uptake, knowledge, informed decision‐making and attitudes to screening. A random‐effects model was used to calculate individual relative risks and 95% confidence intervals.

Main results Six controlled trials (five RCTs and one quasi‐RCT), focusing on antenatal and prostate specific antigen screening, were included. All reported risks/benefits of screening and assessed knowledge. Two also assessed decision‐making. Two reported risks/benefits to all randomized groups and evaluated different ways of presenting information. Neither found that interventions such as videos, information leaflets with decision trees, or touch screen computers conveyed any additional benefits over well‐prepared leaflets.

Conclusions There is some evidence to suggest that changing the format of informed choice interventions in screening does not alter knowledge, satisfaction or decisions about screening. It is not clear whether informed choice in screening affects uptake. More well‐designed RCTs are required and further research should also be directed towards the development of a valid instrument for measuring all components of informed choice in screening.
Consequences of surveying folate awareness
Authors: Watson Lyndsey F. , Watson Maxwell J. , Halliday Jane L. , Bell Robin J.
Published: March 2003

Objectives To assess women's concerns when interviewed about the association between folate and neural tube defects (NTDs) and to determine how this is affected by time, being folate aware, having seen folate promotional material or being pregnant.

Design As part of a community randomized trial outcomes evaluation, independent cross‐sectional follow‐up surveys were carried out in 1997 and 2000.

Setting and participants Six local government areas in the state of Victoria, Australia; 2431 women aged between 15 and 44 years.

Main variables studied Whether or not women knew of the association between folate and NTDs (i.e. were folate aware), whether or not women had been concerned by seeing folate/NTD information and if an interview about folate and NTDs had raised any concerns for them.

Results In the 1997 survey, 36% of women said that the interview had raised concerns and this decreased to 26% in 2000. Women who were folate aware were significantly less likely to have raised concerns than women who were not folate aware (ORadj= 0.38, 95% CI 0.24–0.60). In general, women who had seen promotional material were less likely to feel concern about the interview than those who had not, although this varied with whether or not the promotional material had raised concerns. These effects were greater in women who were pregnant.

Conclusions Women had increased concerns having seen folate promotional material and after being interviewed about it. These results are consistent with the proposition that an initial emotional response to sensitive health information is part of an adaptive response appropriate to the process of health‐related behaviour change.
Deciding how NHS money is spent: a survey of general public and medical views
Authors: Lees Ann , Scott Nicholas , Scott Sheila N. , MacDonald Sara , Campbell Christine
Published: March 2003

Objectives To examine the validity of the Prioritization Scoring Index (PSI) methodology by obtaining the views of our local population and clinicians regarding the criteria and weightings that should be used in deciding how NHS money is spent.

Background We have used a PSI in Argyll and Clyde to allocate new money since 1996 and to determine priorities for our 1999/2000–2003/2004 Health Improvement Programme (HIP). Since the criteria and weightings for this methodology were developed subjectively, we sought to validate these by consulting local people and to change our methodology to take account of wider population views.

Methods A postal questionnaire was sent to 1969 members of the general public, all 314 general practitioners and all 189 hospital consultants in Argyll and Clyde in March 1999. A reminder was sent after 4 weeks. Questions were asked about general funding and prioritization in the NHS and about specific issues relating to potential criteria for prioritization, including those used in our PSI methodology. Responses were analysed quantitatively in the Statistical Package for the Social Sciences (SPSS) and qualitatively through examination of the responses to open questions.

Results The response rate was 51% for the general public and 71% for GPs and consultants. Respondents from the general public were broadly representative of the Argyll and Clyde population. The main findings were that: greater importance should be given to care that improves health, quality of life or prevents ill health rather than to cost, or to government and local health board priorities; half of the general public and most clinicians thought there should be a limit on NHS funding; extra money for the NHS should come from the national lottery (general public) or higher taxes on cigarettes and alcohol (clinicians); doctors should have the greatest influence in deciding how NHS money is spent; a higher priority should not be given to the health‐care needs of younger people rather than older people. Our public and clinicians would allocate approximately 50% of the prioritization weighting to direct patient benefits, 25% to the cost of health‐care and 25% to strategic health issues.

Conclusions Consideration of public and clinician views suggests that a revised PSI should place greater weight on benefits to patients and lower weight on the cost of health‐care.
User involvement in clinical governance
Authors: Pickard Susan , Marshall Martin , Rogers Anne , Sheaff Rod , Sibbald Bonnie , Campbell Stephen , Halliwell Shirley , Roland Martin
Published: March 2003

Objectives To investigate the involvement of users in clinical governance activities within Primary Care Groups (PCGs) and Trusts (PCTs). Drawing on policy and guidance published since 1997, the paper sets out a framework for how users are involved in this agenda, evaluates practice against this standard and suggests why current practice for user involvement in clinical governance is flawed and why this reflects a flaw in the policy design as much as its implementation.

Design Qualitative data comprising semi‐structured interviews, reviews of documentary evidence and relevant literature.

Setting Twelve PCGs/PCTs in England purposively selected to provide variation in size, rurality and group or trust status.

Participants Key stakeholders including Lay Board members (n=12), Chief Executives (CEs) (n= 12), Clinical Governance Leads (CG leads) (n= 14), Mental Health Leads (MH leads) (n= 9), Board Chairs (n=2) and one Executive Committee Lead.

Results Despite an acknowledgement of an organizational commitment to lay involvement, in practice very little has occurred. The role of lay Board members in setting priorities and implementing and monitoring clinical governance remains low. Beyond Board level, involvement of users, patients of GP practices and the general public is patchy and superficial. The PCGs/PCTs continue to rely heavily on Community Health Councils (CHCs) as a conduit or substitute for user involvement; although their abolition is planned, their role to be fulfilled by new organizations called Voices, which will have an expanded remit in addition to replacing CHCs.

Conclusions Clarity is required about the role of lay members in the committees and subcommittees of PCGs and PCTs. Involvement of the wider public should spring naturally from the questions under consideration, rather than be regarded as an end in itself.
Resource allocation, social values and the QALY: a review of the debate and empirical evidence
Authors: Schwappach David L. B.
Published: March 2003

Most health economists agree that public preferences should play a major role in setting criteria for distributing scarce resources. The quality‐adjusted life year (QALY) is used as a preference‐based measure for the outcome of health‐care activities in health economic evaluative studies. Traditionally, health economists proposed maximizing the additional health gain in terms of QALYs so as to maximize social welfare. Evidence has grown however, that neither potential health gain as a single relevant determinant of value, nor the rule of maximizing this health gain are sufficient. Concerns about fairness and equity are also important to the public in distributional decisions. This paper reviews the debate on the role and limitations of the QALY in health‐care priority setting and the empirical evidence surrounding it. A framework is used to systematically explore the available data on factors considered to be important to the public in health‐care resource allocation, and to investigate how these fit with the implicit value judgements inherent in the original QALY formulation. Potential sources of social value are classified into (1) factors that relate to the characteristics of patients and (2) factors related to the characteristics of the intervention's effect on patients' health. As well as these main categories, the article considers preferences for distributional rules. Recent approaches that aim to capture public preferences more comprehensively and to better reflect the value attributed to different health‐care programmes in economic evaluation methods are outlined briefly.
Medical tests: women's reported and preferred decision‐making roles and preferences for information on benefits, side‐effects and false results
Authors: Davey Heather M. , Barratt Alexandra L. , Davey Elizabeth , Butow Phyllis N. , Redman Sally , Houssami Nehmat , Salkeld Glenn P.
Published: March 2003

Objective  To determine women's preferences for and reported experience with medical test decision‐making.

Design  Computer‐assisted telephone survey.

Setting and participants  Six hundred and fifty‐two women resident in households randomly selected from the New South Wales electronic white pages.

Main outcome measures  Reported and preferred test and treatment (for comparison) decision‐making, satisfaction with and anxiety about information on false results and side‐effects; and effect of anxiety on desire for such information.

Results  Overall most women preferred to share test (94.6%) and treatment (91.2%) decision‐making equally with their doctor, or to take a more active role, with only 5.4–8.9% reporting they wanted the doctor to make these decisions on their behalf. This pattern was consistent across all age groups. In general, women reported experiencing a decision‐making role that was consistent with their preference. Women who had a usual doctor were more likely to report experiencing an active role in decision‐making. More women reported receiving as much information as they wanted about the benefits of tests and treatment than about the side‐effects of tests and treatment. Most women wanted information about the possibility of false test results (91.5%) and test side‐effects (95.6%), but many reported the doctor never provided this information (false results = 40.0% and side‐effects = 31.3%). A substantial proportion said this information would make them anxious (false results = 56.6% and side‐effects = 43.1%), but reported they wanted the information anyway (false results = 77.6% and side‐effects = 88.1%).

Conclusions  Women prefer an active role in test and treatment decision‐making. Many women reported receiving inadequate information. If so, this may jeopardize informed decision‐making.
Acceptability of willingness to pay techniques to consumers
Authors: Taylor Susan J. , Armour Carol L.
Published: March 2003

Objective The purpose of this study was to assess and compare the proportion of usable responses and protest votes obtained with two willingness to pay (WTP) techniques, contingent valuation (CV) and discrete choice experiment (DCE) and to assess the acceptability of the techniques to respondents.

Setting and participants Pregnant women attending the public antenatal clinics of a Sydney teaching hospital were surveyed.

Main variables studied Preference for either Treatment A (artificial rupture of the membranes followed by intravenous oxytocin) or Treatment B (prostaglandin E2 gel followed by oxytocin if necessary) was assessed. Then WTP for the preferred treatments was assessed using CV and WTP for specific attributes of the treatments in the DCE. In addition, the acceptability of the two techniques was compared in terms of responses deemed to be valid according to defined criteria, protest votes and comments recorded by consumers.

Results With the CV, 74% of respondents chose gel and their maximum WTP was Aus$178 compared with $133 for the alternative. A total of 68% of responses were deemed to be valid including 5% who may have been expressing a protest vote. With the DCE, respondents were WTP $55 for every 1 h reduction in the length of time from induction to delivery. A total of 72% of responses were deemed valid and only two of these 258 women were considered to have expressed a protest vote.

Conclusions Only a small number of women expressed objections to the use of WTP questions in health‐care and the majority of women completed both questions successfully.
Stabilization of a (βα)8‐barrel protein by an engineered disulfide bridge
Authors: Ivens Andreas , Mayans Olga , Szadkowski Halina , Jürgens Catharina , Wilmanns Matthias , Kirschner Kasper
Published: March 2003

The aim of this study was to increase the stability of the thermolabile (βα)8‐barrel enzyme indoleglycerol phosphate synthase from Escherichia coli by the introduction of disulfide bridges. For the design of such variants, we selected two out of 12 candidates, in which newly introduced cysteines potentially form optimal disulfide bonds. These variants avoid short‐range connections, substitutions near catalytic residues, and crosslinks between the new and the three parental cysteines. The variant linking residues 3 and 189 fastens the N‐terminus to the (βα)8‐barrel. The rate of thermal inactivation at 50 °C of this variant with a closed disulfide bridge is 65‐fold slower than that of the reference dithiol form, but only 13‐fold slower than that of the parental protein. The near‐ultraviolet CD spectrum, the reactivity of parental buried cysteines with Ellman's reagent as well as the decreased turnover number indicate that the protein structure is rigidified. To confirm these data, we have solved the X‐ray structure to 2.1‐Å resolution. The second variant was designed to crosslink the terminal modules βα1 and βα8. However, not even the dithiol form acquired the native fold, possibly because one of the targeted residues is solvent‐inaccessible in the parental protein.
Repression of FasL expression by retinoic acid involves a novel mechanism of inhibition of transactivation function of the nuclear factors of activated T‐cells
Authors: Lee Mi‐Ock , Kang Hyo‐Jin , Kim Young Mi , Oum Ji‐Hyun , Park Jungchan
Published: March 2003

Retinoids are potent immune modulators that inhibit Fas ligand (FasL) expression and thereby repress the activation‐induced apoptosis of immature thymocytes and T‐cell hybridomas. In this study, we demonstrate that all‐trans‐retinoic acid (all‐trans‐RA) directly represses the transcriptional activity of the nuclear factors of activated T‐cells (NFAT), which is an important transactivator of the FasL promoter. The analysis of reporter constructs containing the FasL promoter and wild‐type or mutant NFAT binding‐sites indicated that all‐trans‐RA repression was mediated via an NFAT binding element located in the promoter. A reporter construct comprising the NFAT binding sequence linked to a heterologous SV‐40 promoter showed that NFAT transcriptional activity was significantly inhibited by all‐trans‐RA. Furthermore, all‐trans‐RA inhibited activation of the distal NFAT binding motif present in the interleukin (IL)‐2 promoter, suggesting that the inhibition of NFAT function by all‐trans‐RA was not specific to the FasL promoter. Gel shift assays corroborated the results of the gene reporter studies by showing that all‐trans‐RA decreased the NFAT binding to DNA. All‐trans‐RA blocked translocation of NFATp from the cytosol into the nucleus, which was induced by PMA/ionomycin treatment in HeLa cells transfected with a Flag‐tagged NFATp. Taken together, our results indicate that FasL inhibition by all‐trans‐RA involves a novel mechanism whereby the transcriptional function of NFAT is blocked.
Secretion of a peripheral membrane protein, MFG‐E8, as a complex with membrane vesicles
Authors: Oshima Kenji , Aoki Naohito , Kato Takeo , Kitajima Ken , Matsuda Tsukasa
Published: March 2003

MFG‐E8 (milk fat globule‐EGF factor 8) is a peripheral membrane glycoprotein, which is expressed abundantly in lactating mammary glands and is secreted in association with fat globules. This protein consists of two‐repeated EGF‐like domains, a mucin‐like domain and two‐repeated discoidin‐like domains (C‐domains), and contains an integrin‐binding motif (RGD sequence) in the EGF‐like domain. To clarify the role of each domain on the peripheral association with the cell membrane, several domain‐deletion mutants of MFG‐E8 were expressed in COS‐7 cells. The immunofluorescent staining of intracellular and cell‐surface proteins and biochemical analyses of cell‐surface‐biotinylated and secreted proteins demonstrated that both of the two C‐domains were required for the membrane association. During the course of these studies for domain functions, MFG‐E8, but not C‐domain deletion mutants, was shown to be secreted as membrane vesicle complexes. By size‐exclusion chromatography and ultracentrifugation analyses, the complexes were characterized to have a high‐molecular mass, low density and higher sedimentation velocity and to be detergent‐sensitive. Not only such a exogenously expressed MFG‐E8 but also that endogenously expressed in a mammary epithelial cell line, COMMA‐1D, was secreted as the membrane vesicle‐like complex. Scanning electron microscopic analyses revealed that MFG‐E8 was secreted into the culture medium in association with small membrane vesicles with a size from 100 to 200 nm in diameter. Furthermore, the expression of MFG‐E8 increased the number of these membrane vesicle secreted into the culture medium. These results suggest a possible role of MFG‐E8 in the membrane vesicle secretion, such as budding or shedding of plasma membrane (microvesicles) and exocytosis of endocytic multivesicular bodies (exosomes).
Solution structure of a hydrophobic analogue of the winter flounder antifreeze protein
Authors: Liepinsh Edvards , Otting Gottfried , Harding Margaret M. , Ward Leanne G. , Mackay Joel P. , Haymet A. D. J.
Published: March 2003

The solution structure of a synthetic mutant type I antifreeze protein (AFP I) was determined in aqueous solution at pH 7.0 using nuclear magnetic resonance (NMR) spectroscopy. The mutations comprised the replacement of the four Thr residues by Val and the introduction of two additional Lys‐Glu salt bridges. The antifreeze activity of this mutant peptide, VVVV2KE, has been previously shown to be similar to that of the wild type protein, HPLC6 (defined here as TTTT). The solution structure reveals an α helix bent in the same direction as the more bent conformer of the published crystal structure of TTTT, while the side chain χ1 rotamers of VVVV2KE are similar to those of the straighter conformer in the crystal of TTTT. The Val side chains of VVVV2KE assume the same orientations as the Thr side chains of TTTT, confirming the conservative nature of this mutation. The combined data suggest that AFP I undergoes an equilibrium between straight and bent helices in solution, combined with independent equilibria between different side chain rotamers for some of the amino acid residues. The present study presents the first complete sequence‐specific resonance assignments and the first complete solution structure determination by NMR of any AFP I protein.
Study of substrate specificity of human aromatase by site directed mutagenesis
Authors: Auvray P. , Nativelle C. , Bureau R. , Dallemagne P. , Séralini G.‐E. , Sourdaine P.
Published: March 2003

Human aromatase is responsible for estrogen biosynthesis and is implicated, in particular, in reproduction and estrogen‐dependent tumor proliferation. The molecular structure model is largely derived from the X‐ray structure of bacterial cytochromes sharing only 15–20% identities with hP‐450arom. In the present study, site directed mutagenesis experiments were performed to examine the role of K119, C124, I125, K130, E302, F320, D309, H475, D476, S470, I471 and I474 of aromatase in catalysis and for substrate binding. The catalytic properties of mutants, transfected in 293 cells, were evaluated using androstenedione, testosterone or nor‐testosterone as substrates. In addition, inhibition profiles for these mutants with indane or indolizinone derivatives were obtained. Our results, together with computer modeling, show that catalytic properties of mutants vary in accordance with the substrate used, suggesting possible differences in substrates positioning within the active site. In this respect, importance of residues H475, D476 and K130 was discussed. These results allow us to hypothesize that E302 could be involved in the aromatization mechanism with nor‐androgens, whereas D309 remains involved in androgen aromatization. This study highlights the flexibility of the substrate–enzyme complex conformation, and thus sheds new light on residues that may be responsible for substrate specificity between species or aromatase isoforms.
ORF6 from the clavulanic acid gene cluster of Streptomyces clavuligerus has ornithine acetyltransferase activity
Authors: Kershaw Nadia J. , McNaughton Heather J. , Hewitson Kirsty S. , Hernández Helena , Griffin John , Hughes Claire , Greaves Philip , Barton Barry , Robinson Carol V. , Schofield Christopher J.
Published: March 2003

The clinically used beta‐lactamase inhibitor clavulanic acid is produced by fermentation of Streptomyces clavuligerus. The orf6 gene of the clavulanic acid biosynthetic gene cluster in S. clavuligerus encodes a protein that shows sequence homology to ornithine acetyltransferase (OAT), the fifth enzyme of the arginine biosynthetic pathway. Orf6 was overexpressed in Escherichia coli (at ≈ 15% of total soluble protein by SDS/PAGE analysis) indicating it was not toxic to the host cells. The recombinant protein was purified (to > 95% purity) by a one‐step technique. Like other OATs it was synthesized as a precursor protein which underwent autocatalytic internal cleavage in E. coli to generate α and β subunits. Cleavage was shown to occur between the alanine and threonine residues in a KGXGMXXPX‐‐(M/L)AT (M/L)L motif conserved within all identified OAT sequences. Gel filtration and native electrophoresis analyses implied that the ORF6 protein was an α2β2 heterotetramer and direct evidence for this came from mass spectrometric analyses. Although anomalous migration of the β subunit was observed by standard SDS/PAGE analysis, which indicated the presence of two bands (as previously observed for other OATs), mass spectrometric analyses did not reveal any evidence for post‐translational modification of the β subunit. Extended denaturation with SDS before PAGE resulted in observation of a single major β subunit band. Purified ORF6 was able to catalyse the reversible transfer of an acetyl group from N‐acetylornithine to glutamate, but not the formation of N‐acetylglutamate from glutamate and acetyl‐coenzyme A, nor (detectably) the hydrolysis of N‐acetylornithine. Mass spectrometry also revealed the reaction proceeds via acetylation of the β subunit.
The phosphotransferase system of Streptomyces coelicolor
Authors: Kamionka Annette , Parche Stephan , Nothaft Harald , Siepelmeyer Jörg , Jahreis Knut , Titgemeyer Fritz
Published: March 2003

We have investigated the crr gene of Streptomyces coelicolor that encodes a homologue of enzyme IIAGlucose of Escherichia coli, which, as a component of the phosphoenolpyruvate‐dependent sugar phosphotransferase system (PTS) plays a key role in carbon regulation by triggering glucose transport, carbon catabolite repression, and inducer exclusion. As in E. coli, the crr gene of S. coelicolor is genetically associated with the ptsI gene that encodes the general phosphotransferase enzyme I. The gene product IIACrr was overproduced, purified, and polyclonal antibodies were obtained. Western blot analysis revealed that IIACrr is expressed in vivo. The functionality of IIACrr was demonstrated by phosphoenolpyruvate‐dependent phosphorylation via enzyme I and the histidine‐containing phosphoryl carrier protein HPr. Phosphorylation was abolished when His72, which corresponds to the catalytic histidine of E. coli IIAGlucose, was mutated. The capacity of IIACrr to operate in sugar transport was shown by complementation of the E. coli glucose‐PTS. The striking functional resemblance between IIACrr and IIAGlucose was further demonstrated by its ability to confer inducer exclusion of maltose to E. coli. A specific interaction of IIACrr with the maltose permease subunit MalK from Salmonella typhimurium was uncovered by surface plasmon resonance. These data suggest that this IIAGlucose‐like protein may be involved in carbon metabolism in S. coelicolor.
Chromatin assembly
Authors: Tyler Jessica K.
Published: March 2003

Chromatin is a highly dynamic structure that plays an essential role in regulating all nuclear processes that utilize the DNA template including DNA repair, replication, transcription and recombination. Thus, the mechanisms by which chromatin structures are assembled and modified are questions of broad interest. This minireview will focus on two groups of proteins: (a) histone chaperones and (b) ATP‐dependent chromatin remodeling machines, that co‐operate to assemble DNA and histone proteins into chromatin. The current understanding of how histone chaperones and ATP‐dependent remodeling machines coordinately assemble chromatin in vitro will be discussed, together with the growing body of genetic evidence that supports the role of histone chaperones in the cell.
Effect of coenzymes and thyroid hormones on the dual activities of Xenopus cytosolic thyroid‐hormone‐binding protein (xCTBP) with aldehyde dehydrogenase activity
Authors: Yamauchi Kiyoshi , Nakajima Jun–ichiro
Published: March 2003

A cytosolic thyroid‐hormone‐binding protein (xCTBP), predominantly responsible for the major binding activity of T3 in the cytosol of Xenopus liver, has been shown to be identical to aldehyde dehydrogenase class 1 (ALDH1) [Yamauchi, K., Nakajima, J., Hayashi, H., Horiuchi, R. & Tata, J.R. (1999) J. Biol. Chem. 274, 8460–8469]. Within this paper we surveyed which signaling, and other, compounds affect the thyroid hormone binding activity and aldehyde dehydrogenase activity of recombinant Xenopus ALDH1 (xCTBP/xALDH1) while examining the relationship between these two activities. NAD+ and NADH (each 200 µm), and two steroids (20 µm), inhibit significantly the T3‐binding activity, while NADH and NADPH (each 200 µm), and iodothyronines (1 µm), inhibit the ALDH activity. Scatchard analysis and kinetic studies of xCTBP/xALDH1 indicate that NAD+ and T3 are noncompetitive inhibitors of thyroid‐hormone‐binding and ALDH activities, respectively. These results indicate the formation of a ternary complex consisting of the protein, NAD+ and thyroid hormone. Although the in vitro studies indicate that NAD+ and NADH markedly decrease T3‐binding to xCTBP/xALDH1 at ≈ 10−4 m, a concentration equal to the NAD content in various Xenopus tissues, photoaffinity‐labeling of [125I]T3 using cultured Xenopus cells demonstrates xCTBP/xALDH1 bound T3 within living cells. These results raise the possibility that an unknown factor(s) besides NAD+ and NADH may modulate the thyroid‐hormone‐binding activity of xCTBP/xALDH1. In comparison, thyroid hormone, at its physiological concentration, would poorly modulate the enzyme activity of xCTBP/xALDH1.
Molecular and biochemical characteristics of a gene encoding an alcohol acyl‐transferase involved in the generation of aroma volatile esters during melon ripening
Authors: Yahyaoui Fikri E. L. , Wongs‐Aree Chalermchai , Latché Alain , Hackett Rachel , Grierson Don , Pech Jean‐Claude
Published: March 2003

Two genes (CM‐AAT1 and CM‐AAT2) with strong sequence homology (87% identity at the protein level) putatively involved in the formation of aroma volatile esters have been isolated from Charentais melon fruit. They belong to a large and highly divergent family of multifunctional plant acyl‐transferases and show at most 21% identity to the only other fruit acyl‐transferase characterized so far in strawberry. RT‐PCR studies indicated that both genes were specifically expressed in fruit at increasing rates in the early and mid phases of ripening. Expression was severely reduced in ethylene‐suppressed antisense ACC oxidase (AS) fruit and in wild‐type (WT) fruit treated with the ethylene antagonist 1‐MCP. Cloning of the two genes in yeast revealed that the CM‐AAT1 protein exhibited alcohol acyl‐transferase activity while no such activity could be detected for CM‐AAT2 despite the strong homology between the two sequences. CM‐AAT1 was capable of producing esters from a wide range of combinations of alcohols and acyl‐CoAs. The higher the carbon chain of aliphatic alcohols, the higher the activity. Branched alcohols were esterified at differential rates depending on the position of the methyl group and the nature of the acyl donor. Phenyl and benzoyl alcohols were also good substrates, but activity varied with the position and size of the aromatic residue. The cis/trans configuration influenced activity either positively (2‐hexenol) or negatively (3‐hexenol). Because ripening melons evolve the whole range of esters generated by the recombinant CM‐AAT1 protein, we conclude that CM‐AAT1 plays a major role in aroma volatiles formation in the melon.
Chiral alcohol production by NADH‐dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH
Authors: Itoh Nobuya , Matsuda Michiko , Mabuchi Mariko , Dairi Tohru , Wang Jincun
Published: March 2003

Phenylacetaldehyde reductase (PAR) produced by styrene‐assimilating Corynebacterium strain ST‐10 was used to synthesize chiral alcohols. This enzyme with a broad substrate range reduced various prochiral aromatic ketones and β‐ketoesters to yield optically active secondary alcohols with an enantiomeric purity of more than 98% enantiomeric excess (e.e.). The Escherichia coli recombinant cells which expressed the par gene could efficiently produce important pharmaceutical intermediates; (R)‐2‐chloro‐1‐(3‐chlorophenyl)ethanol (28 mg·mL−1) from m‐chlorophenacyl chloride, ethyl (R)‐4‐chloro‐3‐hydroxy butanoate) (28 mg·mL−1) from ethyl 4‐chloro‐3‐oxobutanoate and (S)‐N‐tert‐butoxycarbonyl(Boc)‐3‐pyrrolidinol from N‐Boc‐3‐pyrrolidinone (51 mg·mL−1), with more than 86% yields. The high yields were due to the fact that PAR could concomitantly reproduce NADH in the presence of 3–7% (v/v) 2‐propanol in the reaction mixture. This biocatalytic process provided one of the best asymmetric reductions ever reported.
Histidine mutagenesis of Arabidopsis thaliana pyruvate dehydrogenase kinase
Authors: Tovar‐Mendez Alejandro , Miernyk Jan A. , Randall Douglas D.
Published: March 2003

Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Analysis of the primary amino‐acid sequences of PDK from various sources reveals that these enzymes include the five domains characteristic of prokaryotic two‐component His‐kinases, despite the fact that PDK exclusively phosphorylates Ser residues in the E1α subunit of the PDC. This seeming contradiction might be resolved if the PDK‐catalyzed reaction employed a phospho‐His intermediate. The results from pH‐stability studies of autophosphorylated Arabidopsis thaliana PDK did not provide any support for a phospho‐His intermediate. Furthermore, site‐directed mutagenesis of the two most likely phosphotransfer His residues (H121 and H168) did not abolish either PDK autophosphorylation or the ability to transphosphorylate E1α. Thus, PDK is a unique type of protein kinase having a His‐kinase‐like sequence but Ser‐kinase activity.
Azidothymidine causes functional and structural destruction of mitochondria, glutathione deficiency and HIV‐1 promoter sensitization
Authors: Yamaguchi Tokio , Katoh Iyoko , Kurata Shun‐ichi
Published: March 2003

Mitochondrial functional and structural impairment and generation of oxidative stress have been implicated in aging, various diseases and chemotherapies. This study analyzed azidothymidine (AZT)‐caused failures in mitochondrial functions, in redox regulation and activation of the HIV‐1 gene expression. We monitored intracellular concentrations of ATP and glutathione (GSH) as the indicators of energy production and redox conditions, respectively, during the time‐course experiments with U937 and MOLT4 human lymphoid cells in the presence of AZT (0.05 mg·mL−1) or H2O2 (0.01 mm) for 15–25 days. Mitochondrial DNA integrity and NF‐κB‐driven HIV‐1 promoter activity were also assessed. ATP concentration began to decrease within several days after exposure to AZT or H2O2, and the decrease continued to reach 30–40% of the normal level. However, decline of GSH was detectable after a retention period for at least 5–6 days, and progressed likewise. PCR analyses found that mitochondrial DNA destruction occurred when the ATP and GSH depletion had progressed, detecting a difference in the deletion pattern between AZT and H2O2‐treated cells. The GSH decrease coincided with HIV‐1 promoter sensitization detected by enhanced DNA binding ability of NF‐κB and induction of the gene expression upon H2O2‐rechallenge. Our results suggest that, in the process of AIDS myopathy development, AZT or oxidative agents directly impair the energy‐producing system of mitochondria, causing dysfunction of cellular redox control, which eventually leads to loss of the mitochondrial DNA integrity. The mechanism of cellular redox condition‐mediated NF‐κB activation is discussed.
Mycobacterium tuberculosis FprA, a novel bacterial NADPH‐ferredoxin reductase
Authors: Fischer Federico , Raimondi Debora , Aliverti Alessandro , Zanetti Giuliana
Published: March 2003

The gene fprA of Mycobacterium tuberculosis, encoding a putative protein with 40% identity to mammalian adrenodoxin reductase, was expressed in Escherichia coli and the protein purified to homogeneity. The 50‐kDa protein monomer contained one tightly bound FAD, whose fluorescence was fully quenched. FprA showed a low ferric reductase activity, whereas it was very active as a NAD(P)H diaphorase with dyes. Kinetic parameters were determined and the specificity constant (kcat/Km) for NADPH was two orders of magnitude larger than that of NADH. Enzyme full reduction, under anaerobiosis, could be achieved with a stoichiometric amount of either dithionite or NADH, but not with even large excess of NADPH. In enzyme titration with substoichiometric amounts of NADPH, only charge transfer species (FAD‐NADPH and FADH2‐NADP+) were formed. At NADPH/FAD ratios higher than one, the neutral FAD semiquinone accumulated, implying that the semiquinone was stabilized by NADPH binding. Stabilization of the one‐electron reduced form of the enzyme may be instrumental for the physiological role of this mycobacterial flavoprotein. By several approaches, FprA was shown to be able to interact productively with [2Fe−2S] iron‐sulfur proteins, either adrenodoxin or plant ferredoxin. More interestingly, kinetic parameters of the cytochrome c reductase reaction catalyzed by FprA in the presence of a 7Fe ferredoxin purified from M. smegmatis were determined. A Km value of 30 nm and a specificity constant of 110 µm−1·s−1 (10 times greater than that for the 2Fe ferredoxin) were determined for this ferredoxin. The systematic name for FprA is therefore NADPH‐ferredoxin oxidoreductase.
Genomic organization of MUC4 mucin gene
Authors: Escande Fabienne , Lemaitre Laurent , Moniaux Nicolas , Batra Surinder K. , Aubert Jean‐Pierre , Buisine Marie‐Pierre
Published: March 2003

The human MUC4 gene encodes a large membrane‐associated mucin, characterized by a mucin tandem repeat domain and a growth factor‐like transmembrane domain. In addition to the originally published sequence (sv0‐MUC4), several MUC4 cDNA sequences (called sv1‐MUC4 to sv21‐MUC4, MUC4/X, MUC4/Y) from various tissues and cell lines have been recently described. They differ from sv0‐MUC4 by deletions and/or insertions located in the 3′ region or, for two of them, by deletion of the central repetitive domain. To establish the nature of the mechanisms responsible for the diversity of MUC4 transcripts, the genomic structure of the 3′ region of the human MUC4 gene was determined. Our results show that it spans approximately 30.8 kb of genomic DNA and is composed of 24 exons, including one alternative exon which was exclusively reported for sv1‐MUC4. Moreover, we have shown that the different MUC4 transcripts are generated by several mechanisms, including the alternative use of cassette exons, exon skipping or use of cryptic splice donor/acceptor sites.
A chloroplast RNA binding protein from stromal thylakoid membranes specifically binds to the 5′ untranslated region of the psbA mRNA
Authors: Ossenbühl Friedrich , Hartmann Kristina , Nickelsen Jörg
Published: March 2003

The intrachloroplastic localization of post‐transcriptional gene expression steps represents one key determinant for the regulation of chloroplast development. We have characterized an RNA binding protein of 63 kDa (RBP63) from Chlamydomonas reinhardtii chloroplasts, which cofractionates with stromal thylakoid memranes. Solubility properties suggest that RBP63 is a peripheral membrane protein. Among RNA probes from different 5′ untranslated regions of chloroplast transcripts, RBP63 preferentially binds to the psbA leader. This binding is dependent on a region comprising seven consecutive A residues, which is required for D1 protein synthesis. A possible role for this newly discovered RNA binding protein in membrane targeting of psbA gene expression is discussed.
Suppression of urokinase receptor expression by bikunin is associated with inhibition of upstream targets of extracellular signal‐regulated kinase‐dependent cascade
Authors: Kobayashi Hiroshi , Suzuki Mika , Kanayama Naohiro , Nishida Takashi , Takigawa Masaharu , Terao Toshihiko
Published: March 2003

Our laboratory showed that bikunin, a Kunitz‐type protease inhibitor, suppresses 4β‐phorbol 12‐myristate 13‐acetate (PMA)‐ or tumor necrosis factor‐alpha (TNFα)‐induced urokinase‐type plasminogen activator (uPA) expression in different cell types. In addition to its effects on protease inhibition, bikunin could be modulating other cellular events associated with the metastatic cascade. To test this hypothesis, we examined whether bikunin was able to suppress the expression of uPA receptor (uPAR) mRNA and protein in a human chondrosarcoma cell line, HCS‐2/8, and two human ovarian cancer cell lines, HOC‐I and HRA. The present study showed that (a) bikunin suppresses the expression of constitutive and PMA‐induced uPAR mRNA and protein in a variety of cell types; (b) an extracellular signal‐regulated kinase (ERK) activation system is necessary for the PMA‐induced increase in uPAR expression, as PD098059 and U0126, which prevent the activation of MEK1, reduce the uPAR expression; (c) bikunin markedly suppresses PMA‐induced phosphorylation of ERK1/2 at the concentration that prevents uPAR expression, but does not reduce total ERK1/2 antigen level; (d) bikunin has no ability to inhibit overexpression of uPAR in cells treated with sodium vanadate; and (e) we further studied the inhibition of uPAR expression by stable transfection of HRA cells with bikunin gene, demonstrating that bikunin secretion is necessary for inhibition of uPAR expression. We conclude that bikunin downregulates constitutive and PMA‐stimulated uPAR mRNA and protein possibly through suppression of upstream targets of the ERK‐dependent cascade, independent of whether cells were treated with exogenous bikunin or transfected with bikunin gene.