bluuebunny/crossref_metadata_2025 huggingface dataset:
Estimating historical changes in land cover:North American croplands from 1850 to 1992
Authors: Ramankutty Navin , Foley Jonathan A.
Published: March 2003

1. We present a simple algorithm for reconstructing spatially explicit historical changes in croplands. We initialize our simulation with a satellite‐derived characterization of present‐day croplands c. 1992. This data set of croplands is then used within a simple model, along with historical cropland inventory data at the national and subnational level, to reconstruct historical crop cover. We present an annual data set of cropland areas in North America between 1850 and 1992, at a spatial resolution of 5 min (≈10 km).

2. The reconstructed changes in North American crop cover are generally consistent with qualitative descriptions of change. Crop cover is initially concentrated in the eastern portions of the continent, and subsequently migrates westward into the Midwestern United States and the Prairie Provinces of Canada. We also see cropland abandonment in the eastern portions of the continent during the 20th century. The simulation, however, fails to characterize adequately the changes in crop cover in Mexico.

3. We also estimate the extent to which the different vegetation types of North America have been cleared for cultivation. We find that savannas/grasslands/steppes and forests/woodlands have undergone the most extensive conversion (1.68 and 1.40 million km2 cleared, respectively, since 1850). We further discuss the wider implications of such large‐scale changes in land cover.
On the role of dynamic atmosphere–vegetation interactions under increasing radiative forcing
Authors: Füssler Jürg S. , Gassmann Fritz
Published: March 2003

A zero‐dimensional model of local atmosphere–vegetation interaction is presented. The model includes essentials of water related two‐way feedbacks, such as the influence of vegetation on evapotranspiration, and the impact of temperature and drought on biomass growth and mortality. The simple model serves as a framework for the preliminary investigation of vegetation related feedbacks under climate change scenarios.
Vegetational history of the Korean Peninsula
Authors: Kong Woo‐Seok
Published: March 2003

1 The vegetational and environmental history of the Korean Peninsula has been reconstructed by the use of both macrofossils and pollen data. These data were analysed within the time‐frame between the Permian and the present day.

2 The continuous appearances of presently occurring conifers and dicotyledons since the Cretaceous indicate both the absence of catastrophic environmental changes in the past, and a degree of long‐term climatic continuity. There were, however, climatic fluctuations occurring within the full time‐frame considered.

3 The presence of a relatively rich flora in both the Oligocene and Miocene suggests the existence of a warm climate. The removal of the Tertiary and Quaternary conifers from the Holocene deposits and the continued survival of cryophilous conifers since the later Pleistocene may be due to climatic deterioration.

4 Overall, an analysis on the vegetational history seems to provide information for the better understanding of the present vegetation as well as providing data on environmental change in the region.
Sensitization to horse hair, symptoms and lung function in grooms
Authors: Tutluoǧlu B. , Atış S. , Anakkaya A. N. , Altuǧ E. , Tosun G. A. , Yaman M.
Published: March 2003

Objective This study aimed to investigate the rate of occupational sensitization to horse hair in grooms and whether occupational exposure to horse hair increases respiratory and allergic symptoms and affects lung function in grooms or not.

Methods This is a cross‐sectional study. Two hundred grooms were randomly selected among 1000 grooms working in Veliefendi Hippodrome of Istanbul. One hundred and twenty‐five subjects agreed to enter the study. Ninety‐two workers who worked in the different parts of this hippodrome enrolled as the control group. A detailed questionnaire including respiratory and allergic symptoms was filled in, physical examination, skin prick tests and pulmonary function tests were performed.

Results Sensitization to horse hair was 12.8% in grooms and 4.3% in controls. The difference was statistically significant (P = 0.0035). Asthma was found in 14.4% of the grooms and 5.4% of the controls, allergic rhinitis in 42.4% of the grooms and 18.4% of the controls, allergic conjunctivitis in 35.2% of the grooms and 15.2% of the controls, and allergic skin diseases in 32.8% of the grooms and 13% of the controls. The differences were statistically significant (P = 0.043, P = 0.0002, P = 0.001 and P = 0.0008, respectively). The means of FEV1, FEV1/FVC and FVC parameters were significantly lower in the groom group (P = 0.006, P = 0.001 and P = 0.003, respectively). In the multivariate analysis, being in the groom group and working years were found to be predictive factors for impairments of lung function (P < 0.001 and P = 0.002, respectively).

Conclusion Occupational exposure to horse increases the sensitization to horse hair, induces asthma and allergic symptoms and also impairs lung functions.
Influence of different dosage schedules of inhaled fluticasone propionate on peripheral blood cytokine concentrations in childhood asthma
Authors: Visser M. J. , Postma D. S. , Brand P. L. P. , Arends L. R. , Duiverman E. J. , Kauffman H. F.
Published: March 2003

Background Asthma is characterized by eosinophilic airways inflammation with elevated levels of IL‐4, IL‐5 and sICAM‐1, and reduced levels of IL‐10 and IFN‐γ. Inhaled corticosteroids powerfully reduce airways inflammation.

Objective To investigate if eosinophil counts, serum eosinophilic cationic protein (ECP) and sICAM‐1 levels, as well as serum and production of cytokines (IL‐4, IL‐5, IL‐10, IFN‐γ) by peripheral blood monocytes (PBMCs) are useful markers to monitor therapy with inhaled fluticasone propionate (FP) in asthmatic children.

Methods In a double‐blind, 1‐year study, 55 asthmatic children (aged 6–10 years) stopped inhaled corticosteroids for a mean period of 24 days and were randomized to receive either FP 200 µg/day (constant dose group), or a starting dose of FP 1000 µg/day with two monthly reductions to 500, 200 and 100 µg/day (stepdown group). Hyper‐responsiveness, symptom scores and blood sampling were performed at 2‐month intervals.

Results Symptoms and hyper‐responsiveness improved significantly in both treatment groups after reintroduction of FP. Eosinophil counts decreased significantly more during the first 2 months of FP in the stepdown group than in the constant dose group (P = 0.03). We found a trend towards a dose‐dependent response in changes of eosinophil counts and serum ECP levels during treatment. Serum IL‐4 and IL‐5 levels were undetectable in the majority of children. No significant effect of the dose of FP on the release of IL‐4, IL‐5, IL‐10 or IFN‐γ by Con A stimulated PBMCs was found. sICAM‐1 levels did not significantly differ at any time point between the two groups.

Conclusion Serum ECP as well as peripheral blood eosinophils, cytokine production by PBMCs and sICAM‐1 levels are insensitive markers in titrating and monitoring therapy with inhaled corticosteroids over a wide dose range in childhood asthma.
Lotic meiofaunal community dynamics: colonisation, resilience and persistence in a spatially and temporally heterogeneous environment
Authors: Robertson Anne L.
Published: March 2003

1. The spatial and temporal dynamics of lotic meiofaunal communities were examined with a focus on colonisation, maintenance of populations in lotic environments and persistence of meiofaunal communities.

2. Lotic meiofaunal colonisation of new habitats may take place via a number of mechanisms and is rapid at both the patch scale (within hours–days) and the drainage basin scale (within 20 years). Successional patterns in lotic meiofaunal communities are evident although data are extremely limited.

3. Lotic meiofaunal communities appear to be resilient to high flow disturbances. Resilience is moderated by the availability of in‐stream refugia and habitat hydrology. Lotic meiofauna may also adopt a ‘refuge as habitat‘ approach to such disturbances.

4. Lotic cyclopoid copepods possess a common suite of life history characteristics that confers resilience to disturbances. Compared to pelagic planktonic species they have short generation times, many descendants per reproductive cycle and long lifespans. Females in ‘source‘ populations are likely to survive disturbances and so could continuously reproduce over a long period of time producing large numbers of offspring which develop rapidly and recolonise ‘sink‘ areas of the stream.

5. Persistence of lotic meiofaunal communities is highly variable. Meiofaunal persistence does not increase with increasing proportions of in‐stream flow refugia.
Biogeographic variation in nest placement: a case study with conservation implications
Authors: Parody Jennifer M. , Parker Timothy H.
Published: March 2003

Abstract. Local habitat characteristics are often used to describe a species’ niche despite the fact that habitat use can vary across the geographical range. We sought to quantify variation in habitat preferences by asking how nesting habit varies within and between populations of Bell’s vireo (Vireo bellii) across its geographical range. Analyses of detailed nest placement data from three localities (Kansas, eastern New Mexico and western New Mexico) showed a general trend towards use of dense vegetation. However, there was substantial variation in nest placement and vegetation at nest sites between localities. Furthermore, a review of nest placement data from the literature shows strong differences in nest heights and species of trees even between populations less than 100 km apart. We evaluate these results in light of conservation and suggest that to be most effective, habitat conservation plans should be based on data collected at the locality where the population of interest occurs.
The use of evidence by health care user organizations
Authors: Glenton Claire , Oxman Andrew D.
Published: March 2003

Objectives To explore the use of research evidence by consumer and patient organizations and the extent to which their goals and activities are consistent with evidence‐based health care and patient‐centred care.

DesignA mailed survey, telephone and face‐to‐face interviews of leaders of organizations representing health care users.

SettingNorway.

Participants Sixty‐nine of 109 questionnaires that were mailed were included in our analysis and approximately 20 interviews were conducted with representatives of general consumer and patient advocacy groups and interest groups that focus on particular diseases or disabilities.

MeasurementsInformation was collected on the goals of the organizations, the nature of their everyday work, the extent to which research information is required in this work and how research information is accessed and appraised.

ResultsAn important focus of many user groups is peer support. They tend to emphasize experience‐based knowledge. A total of 82% of the respondents said that they often or sometimes had use for research results in their work. Research‐based information is most often obtained indirectly through physicians or researchers.

ConclusionsNorwegian health care user organizations do not appear to promote evidence‐based health care. To the extent that they help to disseminate scientific information, they appear to do so uncritically, relying on few sources and traditional authorities.
The relationship between expectations and satisfaction: a qualitative study of patients' experiences of surgery for gynaecological cancer
Authors: Haas Marion
Published: March 2003

It is important that a patient perspective is introduced to the identification and measurement of the outcomes of health care. The aim of this study was to use qualitative methods to examine the presence or absence of expectations prior to the experience of health care and the relationship between expectations, satisfaction and dissatisfaction in a group of women undergoing surgery in a large teaching hospital. Nineteen women with a diagnosis of gynaecological cancer were interviewed on two occasions, before and after surgery. A thematic analysis was undertaken. The results suggest that there is not a clear relationship between expectations and satisfaction. Women had different levels of expectations about different types of care and different aspects of care. Unfulfilled expectations did not lead to less satisfaction. The women were able to express satisfaction either with the care overall or with specific aspects of care, as well as being able to distinguish aspects of care with which they were dissatisfied.
Participation in treatment decision‐making by women with early stage breast cancer
Authors: Kenny Patricia , Quine Susan , Shiell Alan , Cameron Sue
Published: March 2003

Objective This study aimed to assess the way women treated for early stage breast cancer perceived the treatment selection process. The purpose was to understand more fully patients’ experiences of the decision process and their preferences for participation in treatment decisions.

Setting and participants The study informants were 40 women, treated at a teaching hospital in Sydney Australia, who were interviewed face to face 1 year after their first treatment for stage I or stage II breast cancer.

Methods This study used a qualitative approach, based on the analysis of interview transcripts. The main areas covered were how the informants’ treatment decisions were made and their preferences for participation in treatment decisions. Content and thematic analyses were conducted with findings presented using verbatim quotations for illustration.

Results and conclusions Many of the informants who preferred not to participate in decisions also failed to recognize the need for value judgements (as well as medical expertise) in the decision‐making process. Some informants believed they ought to be responsible for the consequences whilst others did not. Difficulties were identified in patient utilization of medical information for treatment decision‐making, and also in establishing preferences for the risks and benefits of treatments where few patients had prior experience of the potential outcomes. The findings indicate that patient participation in treatment decision‐making is a more complex issue than simply giving patients information and choices. Ways of enhancing patients’ involvement in the treatment selection process are discussed.
Patient participation in service improvement: the initial Measures Project experience
Authors: Millett  , Devlin  , Adams  , Gill
Published: March 2003

Background ‘Measures’ is a 2‐year, part‐funded European Community project. It began in March 1998 and has, as its primary foci, the aims of continuing to improve the quality and efficiency of patient care by, (1) re‐examining healthcare processes and (2) determining how best to take advantage of information technology. Central to this has been patient involvement in the project. Initially the project is focused on aspects of rheumatology, diabetes and spinal injury services based across Pinderfields and Pontefract NHS Hospitals Trust.

Approach Patient questionnaires, interactive cross‐specialty and individual specialty focus groups have been employed. The re‐enforcement of relationships with voluntary organizations has featured prominently.

Conclusion Initial perception from patients participating in the process is highly positive and supportive. Specific areas to be considered for change have been addressed.
Doctor‐patient communication patterns in breast cancer adjuvant therapy discussions
Authors: Siminoff Laura A. , Ravdin Peter , Colabianchi Natalie , Sturm Christina M. Saunders
Published: March 2003

Objective To identify variables within the patient‐oncologist communication pattern that impact overall patient comprehension and satisfaction within the breast cancer adjuvant therapy (AT) setting.

Setting and participants Fifty patients were recruited from a number of academic and community‐based oncology practices. Fifteen oncologists participated.

Main variables Three communication variables were identified: percentage of total utterances spoken by the patient, percentage of total physician utterances that were coded as affective (i.e. emotional), and total number of questions asked by the patient during the consultation. Knowledge and satisfaction were assessed by a variety of outcome measures, including knowledge items and satisfaction as measured by VASs, the satisfaction with decision scale and the decisional conflict scale.

Results The level of patient knowledge about breast cancer and satisfaction with the clinical encounter showed a tendency to correlate with the variables measuring aspects of patient‐physician communication style. Patients who spoke more or asked more questions tended to be more knowledgeable whilst patients whose physicians used more affective language tended to know less but to be more satisfied with their clinical encounter.

Conclusions In order to optimize patients’ degree of comprehension and satisfaction with their breast cancer adjuvant therapy, physicians need to increase their affective participation in clinical encounters whilst encouraging patients to ask questions and to actively participate in the decision‐making process.
Enhancing prevention in primary care: are interventions targeted towards consumers’ and providers’ perceived needs?
Authors: Beaulieu Marie‐Dominique , Talbot Yves , Jadad Alejandro R. , Xhignesse Marianne
Published: March 2003

Objective

To explore perceived barriers to the implementation of prevention guidelines, with a particular interest to perceived information needs from the point of view of health professionals and consumers.

Study Design

Focus group.

Setting and Participants

Eight focus groups were held in three Canadian cities: three with consumer, three with family physician, and two with primary care nurses.

Analysis

Inductive analysis based on transcribed interviews. The material was analysed by two of the investigators. Agreement on interpretation was checked independently by three other researchers on 10% of the material.

Results

Lack of motivation, discontinuity of care and lack of adequate remuneration were perceived as the strongest barriers to prevention implementation. Computerized information management systems were not perceived by physicians and nurses as strong facilitating factors. Consumers expressed strongly a need for information on non‐traditional preventive interventions. Physicians and nurses expressed a need for patient education material more than for practice guidelines. Research evidence was not considered as the first criteria to judge the value of preventive information.

Conclusions

Evidence‐based medicine has triggered a massive effort to develop technologies to support the dissemination of evidence‐based information on the assumption that poor access to such information is an important barrier to implementation of effective practices. Our results suggest that such an assumption may not be correct. Providing only evidence‐based information from the realm of traditional medicine will appear restrictive to most users, particularly to consumers, and may not be as valued as anticipated considering the expressed scepticism toward research evidence.
The role of health professionals in informing cancer patients: findings from The Teamwork Project (phase one)
Authors: Smith
Published: March 2003

Background

The Teamwork Project is managed by the National Cancer Alliance (NCA) and funded jointly by the National Lottery Charities Board and the Department of Health. The aim of the Project is to produce a Personal Information File to help people with cancer work in partnership with health professionals. Phase one was carried out between September 1998 and April 2000.

The Teamwork Project arose as a direct result of the NCA report, ‘Patient‐Centred Cancer Services’? – What Patients Say,1 one of a number of studies that found people with cancer want to be involved in decisions about their treatment and care. The study also found that, for this involvement to be successful, health professionals need to support patients in accessing information relevant to their individual needs and help them understand and apply that information. The focus of The Teamwork Project is to help provide a practical solution to meeting this information need.

Approach

The Teamwork Project has used a wide‐range of methods including literature appraisal; patient questionnaires; focus groups; semi‐structured interviews and a consultation exercise. Throughout the Project there has been on‐going involvement from both patients and professionals.

Conclusions

There may be a divergence of views among health professionals in cancer services regarding their role as providers of patient information. Consequently, there may also be a significant variance in how their patients are informed in practice. This finding needs to be validated and the reasons for this understood if the full potential of the forthcoming National Health Service (NHS) Cancer Information Strategy is to be realised.
Involving consumers in a needs‐led research programme: a pilot project
Authors: Oliver Sandy , Milne Ruairidh , Bradburn Jane , Buchanan Phyll , Kerridge Lynn , Walley Tom , Gabbay John
Published: March 2003

Objectives To describe the methods used for involving consumers in a needs‐led health research programme, and to discuss facilitators, barriers and goals.

Design In a short action research pilot study, we involved consumers in all stages of the Health Technology Assessment (HTA) Programme: identifying and prioritizing research topics; commissioning and reporting research; and communicating openly about the programme. We drew on the experience of campaigning, self‐help and patients’ representative groups, national charities, health information services, consumer researchers and journalists for various tasks. We explored consumer literature as a potential source for research questions, and as a route for disseminating research findings. These innovations were complemented by training, one‐to‐one support and discussion. A reflective approach included interviews with consumers, co‐ordinating staff, external observers and other programme contributors, document analysis and multidisciplinary discussion (including consumers) amongst programme contributors.

Results When seeking research topics, face‐to‐face discussion with a consumer group was more productive than scanning consumer research reports or contacting consumer health information services. Consumers were willing and able to play active roles as panel members in refining and prioritizing topics, and in commenting on research plans and reports. Training programmes for consumer involvement in service planning were readily adapted for a research programme. Challenges to be overcome were cultural divides, language barriers and a need for skill development amongst consumers and others. Involving consumers highlighted a need for support and training for all contributors to the programme.

Conclusions Consumers made unique contributions to the HTA Programme. Their involvement exposed processes which needed further thought and development. Consumer involvement benefited from the National Co‐ordinating Centre for Health Technology Assessment (NCCHTA) staff being comfortable with innovation, participative development and team learning. Neither recruitment nor research capacity were insurmountable challenges, but ongoing effort is required if consumer involvement is to be sustained.
Information and involvement
Authors: Thornton H.
Published: March 2003

This article discusses the beliefs that provision of good quality information is the key to (a) successful and satisfying involvement of patients in their own decision‐making and (b) involvement of lay people in the research process, in debate and other involvement in wider health issues. Education of children, health professionals, the public and the media is advocated, enabling critical appraisal skills and good quality health information to lead to improved involvement of citizens in health‐care decisions of all kinds, both individual and societal. Examples of individual, group and specific group involvement through research projects, debates about screening, Citizens’ Juries, etc. are used to illustrate benefits to patients and to health provision in general.
Healthcare in a land called PeoplePower: nothing about me without me
Authors: Delbanco Tom , Berwick Donald M. , Boufford Jo Ivey , Edgman‐Levitan  , Ollenschläger Günter , Plamping Diane , Rockefeller Richard G.
Published: March 2003

In a 5‐day retreat at a Salzburg Seminar attended by 64 individuals from 29 countries, teams of health professionals, patient advocates, artists, reporters and social scientists adopted the guiding principle of ‘nothing about me without me’ and created the country of PeoplePower. Designed to shift health care from ‘biomedicine’ to ‘infomedicine’, patients and health workers throughout PeoplePower join in informed, shared decision‐making and governance. Drawing, where possible, on computer‐based guidance and communication technologies, patients and clinicians contribute actively to the patient record, transcripts of clinical encounters are shared, and patient education occurs primarily in the home, school and community‐based organizations. Patients and clinicians jointly develop individual ‘quality contracts’, serving as building blocks for quality measurement and improvement systems that aggregate data, while reflecting unique attributes of individual patients and clinicians. Patients donate process and outcome data to national data banks that fuel epidemiological research and evidence‐based improvement systems. In PeoplePower hospitals, constant patient and employee feedback informs quality improvement work teams of patients and health professionals. Volunteers work actively in all units, patient rooms are information centres that transform their shape and decor as needs and individual preferences dictate, and arts and humanities programmes nourish the spirit. In the community, from the earliest school days the citizenry works with health professionals to adopt responsible health behaviours. Communities join in selecting and educating health professionals and barter systems improve access to care. Finally, lay individuals partner with professionals on all local, regional and national governmental and private health agencies.
The role of patients' meta‐preferences in the design and evaluation of decision support systems
Authors: Dowie Jack
Published: March 2003

The arrival of new analysis‐based decision technologies will necessitate a profound rethinking both of the nature of the patient–doctor relationship and of the way aids and support systems designed to improve decision‐making within that relationship are designed and evaluated. One‐dimensional typologies of the traditional `paternalist/shared/informed' sort do not provide the complexity called for by the heterogeneity of patient's `meta‐preferences' regarding their relationship with a doctor on the one hand and regarding the analytical level of judgement and decision‐making on the other. A multidimensional matrix embodying this distinction is proposed as a framework of the minimal complexity required for the design and evaluation of the full range of decision aids and decision modes. Essentially aids should be conceived of and evaluatedcell‐specificallyand the search for universally satisfactory decision support systems abandoned. `shared' and `informed' are best interpreted as attributes which may or not be in line with a patient's meta‐preferences. Future research should focus on the higher level goal ofbetterdecision‐making, a goal that will need to respect and reflect these meta‐preferences.
Proteolysis of bovine β‐lactoglobulin during thermal treatment in subdenaturing conditions highlights some structural features of the temperature‐modified protein and yields fragments with low immunoreactivity
Authors: Iametti Stefania , Rasmussen Patrizia , Frøkiær Hanne , Ferranti Pasquale , Addeo Francesco , Bonomi Francesco
Published: March 2003

Bovine β‐lactoglobulin was hydrolyzed with trypsin or chymotrypsin in the course of heat treatment at 55, 60 and 65 °C at neutral pH. At these temperatures β‐lactoglobulin undergoes significant but reversible structural changes. In the conditions used in the present study, β‐lactoglobulin was virtually insensitive to proteolysis by either enzyme at room temperature, but underwent extensive proteolysis when either protease was present during the heat treatment. High‐temperature proteolysis occurs in a progressive manner. Mass spectrometry analysis of some large‐sized breakdown intermediates formed in the early steps of hydrolysis indicated that both enzymes effectively hydrolyzed some regions of β‐lactoglobulin that were transiently exposed during the physical treatments and that were not accessible in the native protein. The immunochemical properties of the products of β‐lactoglobulin hydrolysis were assessed by using various β‐lactoglobulin‐specific antibodies, and most epitopic sites were no longer present after attack of the partially unfolded protein by the two proteases.
Protein methylation as a marker of aspartate damage in glucose‐6‐phosphate dehydrogenase‐deficient erythrocytes
Authors: Ingrosso Diego , Cimmino Amelia , D'Angelo Stefania , Alfinito Fiorella , Zappia Vincenzo , Galletti Patrizia
Published: March 2003

The ‘Mediterranean’ variant of glucose‐6‐phosphate dehydrogenase (G6PD) deficiency is due to the C563CT point mutation, leading to replacement of Ser with Phe at position 188, resulting in acute haemolysis triggered by oxidants. Previous work has shown increased formation of altered aspartate residues in membrane proteins during cell ageing and in response to oxidative stress in normal erythrocytes. These abnormal residues are specifically recognized by the repair enzyme l‐isoaspartate (d‐aspartate) protein O‐methyltransferase (PCMT; EC 2.1.1.77).

The aim of this work was to study the possible involvement of protein aspartate damage in the mechanism linking the G6PD defect and erythrocyte injury, through oxidative stress. Patients affected by G6PD deficiency (Mediterranean variant) were selected. In situ methylation assays were performed by incubating intact erythrocytes in the presence of methyl‐labelled methionine. Altered aspartate residues were detected in membrane proteins by methyl ester quantification.

We present here evidence that, in G6PD‐deficient erythrocytes, damaged residues are significantly increased in membrane proteins, in parallel with the decay of pyruvate kinase activity, used as a cell age marker. Erythrocytes from patients were subjected to oxidative stress in vitro, by treatment with t‐butylhydroperoxide, monitored by a rise in concentration of both methaemoglobin and thiobarbituric acid‐reactive substances. l‐Isoaspartate residues increased dramatically in G6PD‐deficient erythrocytes in response to such treatment, compared with baseline conditions.

The increased susceptibility of G6PD‐deficient erythrocytes to membrane protein aspartate damage in response to oxidative stress suggests the involvement of protein deamidation/isomerization in the mechanisms of cell injury and haemolysis.
Kinetic analysis of hydroxylation of saturated fatty acids by recombinant P450foxy produced by an Escherichia coli expression system
Authors: Kitazume Tatsuya , Tanaka Akinori , Takaya Naoki , Nakamura Akira , Matsuyama Shigeru , Suzuki Takahisa , Shoun Hirofumi
Published: March 2003

Cytochrome P450foxy (P450foxy, CYP505) is a fused protein of cytochrome P450 (P450) and its reductase isolated from the fungus Fusarium oxysporum, which catalyzes the subterminal (ω‐1∼ω‐3) hydroxylation of fatty acids. Here, we produced, purified and characterized a fused recombinant protein (rP450foxy) using the Escherichia coli expression system. Purified rP450foxy was catalytically and spectrally indistinguishable from the native protein, but most of the rP450foxy was recovered in the soluble fraction of E. coli cells unlike the membrane‐bound native protein. The results are consistent with our notion that the native protein is targeted to the membrane by a post‐translational modification mechanism. We also discovered that P450foxy could use shorter saturated fatty acid chains (C9 and C10) as a substrate. The regiospecificity (ω‐1∼ω‐3) of hydroxylation due to the enzymatic reaction for the short substrates (decanoate, C10; undecanoate, C11) was the same as that for longer substrates. Steady state kinetic studies showed that the kcat values for all substrates tested (C9‐C16) were of the same magnitude (1200–1800 min−1), whereas the catalytic efficiency (kcat/Km) was higher for longer fatty acids. Substrate inhibition was observed with fatty acid substrates longer than C13, and the degree of inhibition increased with increasing chain length. This substrate inhibition was not apparent with P450BM3, a bacterial counterpart of P450foxy, which was the first obvious difference in their catalytic properties to be identified. Kinetic data were consistent with the inhibition due to binding of the second substrate. We discuss the inhibition mechanism based on differences between P450foxy and P450BM3 in key amino acid residues for substrate binding.
Structural studies on the core and the O‐polysaccharide repeating unit of Pseudomonas aeruginosa immunotype 1 lipopolysaccharide
Authors: Bystrova Olga V. , Shashkov Aleksander S. , Kocharova Nina A. , Knirel Yuriy A , Lindner Buko , Zähringer Ulrich , Pier Gerald B.
Published: March 2003

The structure of the lipopolysaccharide (LPS) of Pseudomonas aeruginosa immunotype 1 was studied after mild acid and strong alkaline degradations by MS and NMR spectroscopy. Three types of LPS molecules were found, including those with an unsubstituted glycoform 1 core (A) or an isomeric glycoform 2 core substituted with one O‐polysaccharide repeating unit (B) or with a long‐chain O‐polysaccharide. Therefore, of two core glycoforms, only glycoform 2 accepts the O‐polysaccharide.

In the structures A and B, Kdo, Hep, Hep7Cm, GalNAcAN3Ac, GalNFoAN, QuiNAc, GalNAla represent 3‐deoxy‐d‐manno‐octulosonic acid, l‐glycero‐d‐manno‐heptose, 7‐O‐carbamoyl‐l‐glycero‐d‐manno‐heptose, 2‐acetamido‐3‐O‐acetyl‐2‐deoxygalacturonamide, 2‐formamido‐2‐deoxygalacturonamide, 2‐acetamido‐2,6‐dideoxyglucose and 2‐(l‐alanylamino)‐2‐deoxygalactose, respectively; all sugars are in the pyranose form and have the d configuration unless otherwise stated. One or more phosphorylation sites may be occupied by diphosphate groups. In a minority of the LPS molecules, an O‐acetyl group is present in the outer core region at unknown position.

 The site and the configuration of the linkage between the O‐polysaccharide and the core and the structure of the O‐polysaccharide repeating unit were defined in P. aeruginosa immunotype 1. The QuiNAc residue linked to the Rha residue of the core was found to have the β configuration, whereas in the interior repeating units of the O‐polysaccharide this residue is in the α‐configuration. The data obtained are in accordance with the initiation of biosynthesis of the O‐polysaccharide of P. aeruginosa O6, which is closely related to immunotype 1, by transfer of d‐QuiNAc‐1‐P to undecaprenyl phosphate followed by synthesis of the repeating O‐antigen tetrasaccharide.
Expression of recombinant murine pregnancy‐associated plasma protein‐A (PAPP‐A) and a novel variant (PAPP‐Ai) with differential proteolytic activity
Authors: Søe Rikke , Overgaard Michael T. , Thomsen Anni R. , Laursen Lisbeth S. , Olsen Inger M. , Sottrup‐Jensen Lars , Haaning Jesper , Giudice Linda C. , Conover Cheryl A. , Oxvig Claus
Published: March 2003

Murine pregnancy‐associated plasma protein‐A (PAPP‐A) cDNA encoding a 1545 amino‐acid protein has been cloned. We have also identified and cloned cDNA that encodes a novel variant of PAPP‐A, PAPP‐Ai, carrying a 29‐residue highly basic insert. The point of insertion corresponds to a junction between two exons in the human PAPP‐A gene. The human intron flanked by these exons does not encode a homologous corresponding insert, which is unique to the mouse. The overall sequence identity between murine and human PAPP‐A is 91%, and murine PAPP‐A contains sequence motifs previously described in the sequence of human PAPP‐A. Through expression in mammalian cells, we show that murine PAPP‐A and PAPP‐Ai are active metalloproteinases, both capable of cleaving insulin‐like growth factor binding protein (IGFBP)‐4 and ‐5. Cleavage of IGFBP‐4 is dramatically enhanced by the addition of IGF, whereas cleavage of IGFBP‐5 is slightly inhibited by IGF, as previously established with human PAPP‐A. Surprisingly, however, quantitative analyses demonstrate that the murine PAPP‐Ai cleaves IGFBP‐4 very slowly compared to PAPP‐A, even though its ability to cleave IGFBP‐5 is unaffected by the presence of the insert. By RT‐PCR analysis, we find that both variants are expressed in several tissues. The level of mRNA in the murine placenta does not exceed the levels of other tissues analyzed. Furthermore, the IGFBP‐4‐proteolytic activity of murine pregnancy serum is not elevated. This is in striking contrast to the increase seen in human pregnancy serum, and the expression of PAPP‐A in the human placenta, which exceeds other tissues at least 250‐fold. Interestingly, the position of the insert of PAPP‐Ai, within the proteolytic domain, lies in close proximity to the cysteine residue, which in human PAPP‐A forms a disulfide bond with the proform of eosinophil major basic protein (proMBP). ProMBP functions as a proteinase inhibitor in the PAPP‐A–proMBP complex, but whether any mechanistic parallel on regulation of proteolytic activity can be drawn between the insert of PAPP‐Ai and the linkage to proMBP is not known. Importantly, these data support the development of the mouse as a model organism for the study of PAPP‐A, which must take into account the differences between the mouse and the human.
Nuclear receptor‐dependent transcription with chromatin
Authors: Kraus W Lee , Wong Jiemin
Published: March 2003

Nuclear receptors (NRs) are ligand‐regulated, DNA‐binding transcription factors that function in the chromatin environment of the nucleus to alter the expression of subsets of hormone‐responsive genes. It is clear that chromatin, rather than being a passive player, has a profound effect on both transcriptional repression and activation mediated by NRs. NRs act in conjunction with at least three general classes of cofactors to regulate transcription in the context of chromatin: (a) chromatin remodelers; (b) corepressors; and (c) coactivators, many of which have distinct enzymatic activities that remodel nucleosomes or covalently modify histones (e.g. acetylases, deacetylases, methyltransferases, and kinases). In this paper, we will present a brief overview of these enzymes, their activities, and how they assist NRs in the repression or activation of transcription in the context of chromatin.
A functional role of the membrane‐proximal extracellular domains of the signal transducer gp130 in heterodimerization with the leukemia inhibitory factor receptor
Authors: Timmermann Andreas , Küster Andrea , Kurth Ingo , Heinrich Peter C. , Müller‐Newen Gerhard
Published: March 2003

gp130 is the common signal transducing receptor subunit of interleukin (IL)‐6‐type cytokines. gp130 either homodimerizes in response to IL‐6 and IL‐11 or forms heterodimers with the leukemia inhibitory factor (LIF) receptor (LIFR) in response to LIF, oncostatin M (OSM), ciliary neurotrophic factor (CNTF), cardiotrophin‐1 (CT‐1) or cardiotrophin‐like cytokine resulting in the onset of cytoplasmic tyrosine phosphorylation cascades. The extracellular parts of both gp130 and LIFR consist of several Ig‐like and fibronectin type III‐like domains. The role of the membrane‐distal domains of gp130 (D1, D2, D3) and LIFR in ligand binding is well established. In this study we investigated the functional significance of the membrane‐proximal domains of gp130 (D4, D5, D6) in respect to heterodimerization with LIFR. Deletion of each of the membrane‐proximal domains of gp130 (Δ4, Δ5 and Δ6) leads to LIF unresponsiveness. Replacement of the gp130 domains by the corresponding domains of the related GCSF receptor either restores weak LIF responsiveness (D4‐GCSFR), leads to constitutive activation of gp130 (D5‐GCSFR) or results in an inactive receptor (D6‐GCSFR). Mutation of a specific cysteine in D5 of gp130 (C458A) leads to constitutive heterodimerization with the LIFR and increased sensitivity towards LIF stimulation. Based on these findings, a functional model of the gp130–LIFR heterodimer is proposed that includes contacts between D5 of gp130 and the corresponding domain D7 of the LIFR and highlights the requirement for both receptor dimerization and adequate receptor orientation as a prerequisite for signal transduction.
Substrate specificity of human kallikrein 2 (hK2) as determined by phage display technology
Authors: Cloutier Sylvain M. , Chagas Jair Ribeiro , Mach Jean‐Pierre , Gygi Christian M. , Leisinger Hans‐Jurg , Deperthes David
Published: March 2003

Human glandular kallikrein 2 (hK2) is a trypsin‐like serine protease expressed predominantly in the prostate epithelium. Recently, hK2 has proven to be a useful marker that can be used in combination with prostate specific antigen for screening and diagnosis of prostate cancer. The cleavage by hK2 of certain substrates in the proteolytic cascade suggest that the kallikrein may be involved in prostate cancer development; however, there has been very little other progress toward its biochemical characterization or elucidation of its true physiological role. In the present work, we adapt phage substrate technology to study the substrate specificity of hK2. A phage‐displayed random pentapeptide library with exhaustive diversity was generated and then screened with purified hK2. Phages displaying peptides susceptible to hK2 cleavage were amplified in eight rounds of selection and genes encoding substrates were transferred from the phage to a fluorescent system using cyan fluorescent protein (derived from green fluorescent protein) that enables rapid determination of specificity constants. This study shows that hK2 has a strict preference for Arg in the P1 position, which is further enhanced by a Ser in P′1 position. The scissile bonds identified by phage display substrate selection correspond to those of the natural biological substrates of hK2, which include protein C inhibitor, semenogelins, and fibronectin. Moreover, three new putative hK2 protein substrates, shown elsewhere to be involved in the biology of the cancer, have been identified thus reinforcing the importance of hK2 in prostate cancer development.
A complex fruit‐specific type‐2 ribosome‐inactivating protein from elderberry (Sambucus nigra) is correctly processed and assembled in transgenic tobacco plants
Authors: Chen Ying , Vandenbussche Frank , Rougé Pierre , Proost Paul , Peumans Willy J. , Van Damme Els J. M.
Published: March 2003

Fruits of elderberry (Sambucus nigra) express small quantities of a type‐2 ribosome‐inactivating protein with an exclusive specificity towards the NeuAc(α2,6)Gal/GalNAc disaccharide and a unique molecular structure typified by the occurrence of a disulfide bridge between the B‐chains of two adjacent protomers. A cDNA clone encoding this so‐called Sambucus nigra fruit specific agglutinin I (SNA‐If) was isolated and expressed in tobacco (Samsun NN) under the control of the 35S cauliflower mosaic virus promoter. Characterization of the purified protein indicated that the recombinant SNA‐If from tobacco leaves has the same molecular structure and biological activities as native SNA‐If from elderberry fruits, demonstrating that transgenic tobacco plants are fully capable of expressing and correctly processing and assembling a type‐2 ribosome‐inactivating protein with a complex molecular structure. None of the transformants showed a phenotypic effect, indicating that the ectopically expressed SNA‐If does not affect the viability of the tobacco cells. Bioassays further demonstrated that none of the transgenic lines exhibited a decreased sensitivity to infection with tobacco mosaic virus suggesting that the elderberry type‐2 RIP SNA‐If does not act as an antiviral agent in planta.
Regulation of transcription of the Dnmt1 gene by Sp1 and Sp3 zinc finger proteins
Authors: Kishikawa Shotaro , Murata Takehide , Kimura Hiromichi , Shiota Kunio , Yokoyama Kazunari K
Published: March 2003

The Sp family is a family of transcription factors that bind to cis‐elements in the promoter regions of various genes. Regulation of transcription by Sp proteins is based on interactions between a GC‐rich binding site (GGGCGG) in DNA and C‐terminal zinc finger motifs in the proteins. In this study, we characterized the GC‐rich promoter of the gene for the DNA methyltransferase (Dnmt1) that is responsible for methylation of cytosine residues in mammals and plays a role in gene silencing. We found that a cis‐element (nucleotides −161 to −147) was essential for the expression of the mouse gene for Dnmt1. DNA‐binding assays indicated that transcription factors Sp1 and Sp3 bound to the same cis‐element in this region in a dose‐dependent manner. In Drosophila SL2 cells, which lack the Sp family of transcription factors, forced expression of Sp1 or Sp3 enhanced transcription from the Dnmt1 promoter. Stimulation by Sp1 and Sp3 were independent phenomena. Furthermore, cotransfection reporter assays with a p300‐expression plasmid revealed the activation of the promoter of the Dnmt1 gene in the presence of Sp3. The transcriptional coactivator p300 interacted with Sp3 in vivo and in vitro. Our results indicate that expression of the Dnmt1 gene is controled by Sp1 and Sp3 and that p300 is involved in the activation by Sp3.
An active site homology model of phenylalanine ammonia‐lyase from P. crispum
Authors: Röther Dagmar , Poppe László , Morlock Gaby , Viergutz Sandra , Rétey János
Published: March 2003

The plant enzyme phenylalanine ammonia‐lyase (PAL, EC 4.3.1.5) shows homology to histidine ammonia‐lyase (HAL) whose structure has been solved by X‐ray crystallography. Based on amino‐acid sequence alignment of the two enzymes, mutagenesis was performed on amino‐acid residues that were identical or similar to the active site residues in HAL to gain insight into the importance of this residues in PAL for substrate binding or catalysis. We mutated the following amino‐acid residues: S203, R354, Y110, Y351, N260, Q348, F400, Q488 and L138. Determination of the kinetic constants of the overexpressed and purified enzymes revealed that mutagenesis led in each case to diminished activity. Mutants S203A, R354A and Y351F showed a decrease in kcat by factors of 435, 130 and 235, respectively. Mutants F400A, Q488A and L138H showed a 345‐, 615‐ and 14‐fold lower kcat, respectively. The greatest loss of activity occurred in the PAL mutants N260A, Q348A and Y110F, which were 2700, 2370 and 75 000 times less active than wild‐type PAL. To elucidate the possible function of the mutated amino‐acid residues in PAL we built a homology model of PAL based on structural data of HAL and mutagenesis experiments with PAL. The homology model of PAL showed that the active site of PAL resembles the active site of HAL. This allowed us to propose possible roles for the corresponding residues in PAL catalysis.