bluuebunny/crossref_metadata_2025 huggingface dataset:
Perspectives on the American quality movement
Authors: Dory John , Schier Lewis
Published: July 2002

Uses a rich academic and professional literature to demonstrate that the rise and plateau of the US quality movement was the outcome of a complex senior management decision process that extended well beyond the problem of closing a quality gap between US and Japanese competitors. Firms in many industries adopted quality programs to solve simultaneously several problems more serious than quality. The managers of these same firms later revised their strategic agendas by replacing quality with new programs that addressed emerging problems that demanded their attention and resources. The conclusions of this research will help senior managers in many countries to decide whether to commit resources to support new managerial movements, and if so, to determine when to commit these resources.
Business process management: a boundaryless approach to modern competitiveness
Authors: Zairi Mohamed
Published: July 2002

Discusses the concept of business process management as an approach which relies on cross‐functional contributions to organizational performance. Presents arguments on the importance of “hard” and “soft” elements which are all required for determining effective competitiveness. A review of the literature suggests that although the term “process management” is increasingly being used in everyday business language, the practical experience so far is very limited. Presents the concept of business process management through its various key elements, supported by practical examples. Discusses the methodological approaches used in various contexts for creating a culture based on process management. Concludes with some useful guidelines to support efforts in establishing a business process management culture.
A comparison of international quality standards: divergence and agreement
Authors: Guerrero‐Cusumano José‐Luis , Selen Willem J.
Published: July 2002

Summarizes the main points of divergence between the French EAQF, German VDA 6, and USA QS 9000 standards in the automotive sector and evaluates their possible impact on eligibility for certification under the quality standard of comparison. While particular differences are marked, a converging trend based on QS 9000 requirements is emerging. This indicates that, while at first these developments point at a proliferation of quality standards, international quality standards in the automotive sector are in fact converging.
The value of creativity in business process re‐engineering
Authors: Paper David
Published: July 2002

Explores the drivers behind successful business process re‐engineering (BPR). Examines Caterpillar Inc, located in Peoria, Illinois, USA, using data collected over a four‐year period, beginning in 1992. Reveals positive outcomes which can be directly tied to the deployment of the business process simplification and improvement (BSP/I) methodology. BPS/I provides a systematic methodology for analysis, design and implementation of re‐engineering principles. In addition, it provides the environment to help its people generate creative solutions to critical business problems. Examines the link between the steps of BPS/I and the theoretical phases of the problem‐solving process. Also explores the link between BPS/I and Couger’s variant model of creative problem solving. Notes creative opportunities available to Caterpillar as a result of the identification of this link.
Business process management: a review and evaluation
Authors: Lee R.G. , Dale B.G.
Published: July 2002

The area of business process management (BPM) is a relatively new field and there is little academic literature on the subject. This paper critically examines the literature to pinpoint BPM definitions, approaches and principles. A study is reported of a business unit of a “world class” organisation to examine its application of BPM. It was found that the principles of BPM espoused by the corporate organisation and its approach to BPM application is along the lines of best practice as outlined in the literature. However, in the business unit examined, there is an inadequate understanding of BPM and the application of BPM principles is haphazard. For example, it was discovered that employees have too little time to spend on process simplification and improvement and there are too many in‐process measures for process owners to make effective use of the information. It is also pointed out that if BPM is to be truly effective it requires cross‐functional and process orientated management, which is not easy to attain.
The BPR SCOPE concept in leveraging improved supply chain performance
Authors: Berry D. , Evans G.N. , Mason‐Jones R. , Towill D.R.
Published: July 2002

There are three possible outcomes following the undertaking of BPR projects. First, a BPR programme may fail in the short or long term either because it is improperly implemented or because there is no effective follow‐up. Second, a BPR programme may be deemed satisfactory in outcome yet still does not appear to significantly improve bottom‐line performance. Finally, there is the BPR programme which is highly successful and significantly improves the bottom‐line performance. It is identifying the last category which is the subject of this paper. In our view, to maximise the probability of making a major impact on the supply chain bottom line, BPR programmes should be planned to have adequate SCOPE. This is a three‐dimensional model of the change process with breadth, depth and width axes. Our approach to evaluating BPR impact is illustrated by application to the product delivery process of an electronics products supply chain. The prediction of improved performance is demonstrated via a dynamic simulation model of a real‐world supply chain as a function of the particular implementation phase of the BPR programme. The eventual improvement in performance obtained after a number of years progress is very significant. Indeed, in material flow terms, it is now a structurally much improved, robust and internationally competitive supply chain.
ERP adoption: a technological evolution approach
Authors: Hwa Chung Sock , Snyder Charles A.
Published: July 2002

In a competitive environment, firms continually need to establish new business objectives to fulfill their corporate visions. A whole organization should be structured to achieve these objectives. In order to support the objectives, information technology (IT) needs to be aligned to the organization’s mission. IT professionals have provided systems for the organization and, in most cases, these systems consist of individual function, which should be used in an integrated manner. Enterprise Resource Planning (ERP) software has emerged to offer an integrated IT solution. It is suggested that ERP could facilitate achieving compatibility between task characteristics and technology characteristics, a long overdue IT solution. ERP has become one of the major IT investments for many organizations. Unfortunately, little research has been done to provide managers guidance on ERP potential for their organizations. Some useful information about ERP can be found by examining the technological evolution of ERP from MRP.
Extended and virtual enterprises – similarities and differences
Authors: Browne Jim , Zhang Jiangang
Published: July 2002

In today’s global economy, manufacturing enterprises must be viewed in the context of their contribution to the total value chain. Extended or virtual enterprises, consisting of tele‐computing mediated chains of suppliers, manufacturers, assemblers, distributors and customers, compete to supply quasi‐customised products to discerning markets. This paper reviews the thinking behind the extended enterprise and virtual enterprise models of manufacturing systems, identifies the characteristics of each and the similarities and differences between them.
Network design in supply chain management
Authors: Abdinnour‐Helm Sue
Published: July 2002

The distribution function in a supply chain is an important internal service function for any firm, and has been increasingly recognized as playing a strategic role in achieving competitive advantage. This paper proposes improving the distribution function of the supply chain by employing hub‐and‐spoke network designs. Such designs have proven to be effective with third party logistics carriers such as Federal Express, UPS, Norfolk Southern, and Yellow Freight. Several models and heuristic solution techniques have been introduced in the literature in the past ten years. However, the performance of such heuristics, under different transportation environments, has not been examined. This paper acts as a first step in this direction. The performance of two heuristics to solve a hub‐and‐spoke network is compared against the performance of an optimal technique, for various configurations of data. With the results of this study, business managers can, by analyzing the structure of their data, assess the “risk” associated with applying one of the two heuristics. Heuristic developers can also exploit the results of this study to give them insight into areas where heuristics can be developed or strengthened in order to give rise to more robust heuristics.
Assembly process visualization in feature‐based design for assembly
Authors: Chuang Wen‐Chieh , O’Grady Peter
Published: July 2002

Propoes a methodology that enables a dynamic and interactive 3‐Dvisualization model of the assembly process to be developed from the CAD files of each part in the assembly. Makes three main contributions to the research in this area. First, a taxonomy of features for assembly is presented. Second, a process model for generating interactive 3D models of the assembly process is developed and, third, a new algorithm for creating a dynamic assembly model is derived. This algorithm, called the 3D assembly visualization (3AV) algorithm, involves instantiating the motion attributes that specify the motion of each part in the assembly. This dynamic assembly model is then converted to a 3‐D renderable format for visualization. An example is presented that uses virtual reality modeling language (VRML) as the 3‐D representation language. The result is a dynamic and interactive visual representation of the assembly operation. Such visualization can be of considerable use in DFA.
National site licensing and consortia purchasing
Authors: Turner Rollo
Published: July 2002

National site licences appear to bring many advantages to a complex field. However, the way the scheme is set up and administered is vital to the long‐term success of this concept. As currently conceived in the UK (NESLI) the National Site Licence could have some unintended side‐effects to the detriment of libraries, publishers and agents. The problem stems from combining the three functions of the consortium (negotiation, access and purchasing) in a single body, the Managing Agent. This can create a monopolistic situation for the future, which may be undesirable from a number of standpoints. Some solutions are suggested on how to achieve the benefits of National Site Licences without these drawbacks.
Anaesthetists’ records of pre‐operative assessment
Authors: Simmonds Mark , Petterson Jane
Published: July 2002

The pre‐operative anaesthetic records of 195 patients were analysed for the presence of 12 agreed core items of pre‐operative assessment. This study showed that anaesthetists recorded 26.8 per cent of this information. In up to one‐third of patients the following were recorded: smoking history, family history, gastro‐oesophageal reflux, airway assessment, dental assessment, chest examination, heart‐sounds and blood pressure. Previous anaesthesia, drug history and allergies were recorded in one to two‐thirds of patients. Past medical history was recorded in over two‐thirds of patients. With a view to improving the level of record‐keeping, a formatted, pre‐printed pre‐operative assessment record was introduced into practice and two months later the audit was repeated. A small but non‐significant improvement in record keeping was observed. An argument is made for the introduction of an interdisciplinary, unified anaesthetic pre‐operative record.
The impact of alternative presses on scientific communication
Authors: Weintraub Irwin
Published: July 2002

Most grey literature tends to support the disciplines it serves and does not usually raise issues regarding social, political, or ethical considerations. The “other” grey literature, publications issued by alternative presses, attempts to inform readers about aspects of a particular scientific endeavor that may have social ramifications. In the electronic age, the World Wide Web has played a major role in making scientific information accessible to a wide audience more rapidly and efficiently. This democratic approach to information dissemination in science is changing the way science is perceived and implemented in our daily lives. Alternative presses in both electronic and print formats will continue to play a role in shaping the science research and public policy agendas well into the future. This article explores the role of alternative presses and its impact on scientific communication.
Trends and evolution in the development of grey literature: a review
Authors: Luzi Daniela
Published: July 2002

This paper outlines the principal stages in the development of grey literature (GL), from its first appearance in the post‐war period to its evolution into electronic GL. To this end, the study analyses some of the most important studies and conferences organised up to the 1990s. It also examines the first bibliographies and databases, which transformed the way in which GL was collected and distributed. The second part of the paper briefly outlines the main subjects addressed at international GL conferences, identifying both elements of continuity with earlier work and new developments.
Toward sustainable health‐care services: principles, challenges, and a process
Authors: Jameton Andrew , McGuire Catherine
Published: July 2002

Sustainable health care combines three key factors: quality patient care, fiscally responsible budgeting and minimizing environmental impact. Although pollution is well understood as a health problem, US health planners have not fully recognized the need to reduce health‐care pollution. Minimizing health‐care pollution, moreover, requires reducing the throughput of energy and materials. Ultimately, sustaining healthy ecosystems requires that health‐care material and energy utilization be limited. However, traditional conceptions of health‐care ethics maintain a philosophy of rescue that makes limiting life‐saving resources, except at a patient’s request, morally worrisome. Moreover, the media image of health care as technologically intensive, together with the common medical view that nature is the enemy, render suspect philosophical perspectives respectful of Earth’s limits. Nevertheless, academic medical centers have advantages as sites for pursuing sustainability: students often uphold environmental ideals, a public health perspective, and an interest in providing services universally; basic biomedical research on campus permits innovative research combining health and environmental considerations; opportunities exist for including environmental concerns in health professional education; some academic medical centers have already stated environmental criteria for purchasing contracts; and health‐care professionals and institutions are increasingly addressing such environmental concerns as mercury use, latex allergies, dioxin pollution, and waste volume. To address these challenges, a visioning process is proposed, designed to formulate a practical plan by means of public, local, and professional participation in the process of articulating creative and morally sound proposals for change.
Green campuses: the road from little victories to systemic transformation
Authors: Sharp Leith
Published: July 2002

This article contributes to an emerging dialogue about how we can accelerate progress towards institutionalizing a commitment to campus environmental sustainability throughout the university sector. It seeks to utilize progress made to date, in the field of “greening” universities, looking deeply into these experiences, to learn from them and to start revealing how they may inform us to move into the realm of widespread institutional transformation. It presents a range of insights, lessons learned and preferred approaches emerging from seven years of implementing campus environmental programs in universities both in Australia and the USA. Many of these ideas have been further informed during a Churchill Fellowship, spent investigating campus environment initiatives in over 30 universities in Europe and the USA. To assist in giving further weight to the material presented, it draws upon the work of a various authors of organizational change, leadership and management publications. The subject‐matter is wide‐ranging as it is intended as a starting‐point for the reader to pick and choose ideas that may warrant further investigation in their own university context. Even though many of the ideas presented need further exploration and development, in their current state they may prove of some value to the reader as a catalyst for a different level of institutional analysis.
Promoting a subject gateway: a case study from EEVL (Edinburgh Engineering Virtual Library)
Authors: MacLeod Roddy
Published: July 2002

Describes the development of the Edinburgh Engineering Virtual Library (EEVL) (http://eevl.ac.uk) and outlines the services offered. The potential market for EEVL is discussed, and a case study of promotional activities is presented.
Multivariate statistics applied to the evaluation of environmental and chemical data sources
Authors: Voigt Kristina , Welzl Gerhard , Rediske Gerda
Published: July 2002

Constantly expanding chemical and environmental information sources increase the need for descriptive statistical analysis. This paper gives a comparative evaluation of data sources, i.e. online databases, databases on CD‐ROM and Internet resources in the field of environmental chemicals. The evaluation is based on information in three metadatabases for environmental chemicals: DADB‐Metadatabase of Online Databases, DACD‐Metadatabase of CD‐ROMs, DAIN‐Metadatabase of Internet Resources. A data matrix of 50 environmental and chemical descriptors found in DADB, DACD and DAIN is analysed and a technique is applied to transform the data set into a data matrix of a more homogeneous structure. This method is based on algorithms for solving the so‐called travelling salesman problem. Two different ways of analysing the data set are applied and the results are compared. Also, media combination patterns are identified and discussed. For most descriptors the information depth is higher in commercial online databases and databases on CD‐ROM than in free Internet resources. Exceptions, e.g. some health‐related parameters which have a higher percentage in Internet resources, are identified and explained.
Recent relevance research: implications for information professionals
Authors: Greisdorf Howard , Spink Amanda
Published: July 2002

We discuss results from recent relevance research with implications for information professionals. Our studies show that beyond the usual concern with high relevance and non‐relevance judgements, that partially relevant judgements by users are important. We call for the adoption of a more complex view of human relevance judgements in the education and practice of information professionals.
Effective techniques for automatic extraction of Web publications
Authors: Fong A.C.M. , Hui S.C. , Vu H.L.
Published: July 2002

Research organisations and individual researchers increasingly choose to share their research findings by providing lists of their published works on the World Wide Web. To facilitate the exchange of ideas, the lists often include links to published papers in portable document format (PDF) or Postscript (PS) format. Generally, these publication Web sites are updated regularly to include new works. While manual monitoring of relevant Web sites is tedious, commercial search engines and information monitoring systems are ineffective in finding and tracking scholarly publications. Analyses the characteristics of publication index pages and describes effective automatic extraction techniques that the authors have developed. The authors’ techniques combine lexical and syntactic analyses with heuristics. The proposed techniques have been implemented and tested for more than 14,000 Web pages and achieved consistently high success rates of around 90 percent.
From static content to dynamic communities: the evolution of networked educational resources
Authors: Jacobs Neil , Huxley Lesly
Published: July 2002

Five years ago the problems being addressed by major educational resources were how to locate and structure a burgeoning universe of Web sites in order to serve their subject and professional communities with useful and relevant content. Today, new challenges, demands and opportunities are emerging: the linking of content with community, static information with dynamic news. Describes the evolution of three educational resources in the social sciences (SOSIG, Biz/Ed and Regard) and their use of personal profiling, distributed contributions and RSS news channels to serve and gather information. Examines the issues that arise from changing user bases and technologies, sustainability and the need for collaboration, data protection and privacy concerns. Analyses the tensions these and other services face as they move toward a model that links the static with the dynamic, content with community.
A case study of the usability testing of the University of South Florida’s virtual library interface design
Authors: Allen Maryellen
Published: July 2002

Usability testing of Web interfaces for virtual libraries is a crucial factor in the continuing development and improvement of the user interface. In 1999, the University of South Florida Libraries decided to embark on a usability study to coincide with the rollout of a new interface design. Because this type of study had not been conducted with the initial interface, its implementation and completion were paramount in the development of the new design. Details the preliminary activities, testing methodologies and results of usability testing by the USF virtual library project’s usability study group.
Intellectual capital: current issues and policy implications
Authors: Brennan Niamh , Connell Brenda
Published: July 2002

Substantial differences between company book values and market values indicate the presence of assets not recognised and measured in company balance‐sheets. Intellectual capital assets account for a substantial proportion of this discrepancy. At present, companies are not required to report on intellectual capital assets, which leaves the traditional accounting system ineffective for measuring the true impact of such intangibles. Regulations currently in place are analysed in this article. Prior research concerning intellectual capital is presented. Frameworks for intellectual capital are compared. Indicators used for the measurement of intellectual capital are examined. The research methodologies employed for collecting information about the use of intellectual capital accounts in companies are reviewed. Guidelines available to companies for reporting on intellectual capital are considered and also the efforts made towards developing an accounting standard for intellectual capital. Finally, current issues and policy implications of accounting for intellectual capital in the future are examined.
Intellectual capital: Australian annual reporting practices
Authors: Guthrie James , Petty Richard
Published: July 2002

This study reports the results of an empirical examination of Australian annual reporting of intellectual capital. The findings suggest that the development of a model for reporting intangibles is piecemeal and not widely spread. The outcomes of our exploratory investigation are threefold. First, the key components of intellectual capital are poorly understood, inadequately identified, inefficiently managed, and not reported within a consistent framework when reported at all. Second, the main areas of intellectual capital reporting focus on human resources; technology and intellectual property rights; and organisational and workplace structure. Third, even in an Australian enterprise thought of as “best practice” in this regard, a comprehensive management framework for intellectual capital is yet to be developed, especially for collecting and reporting intellectual capital formation. In conclusion, Australian companies do not compare favourably with several European firms in their ability to measure and report their intellectual capital in the annual report.
Measuring intangible corporate assets
Authors: Joia Luiz Antonio
Published: July 2002

Since the beginning of this decade research has been conducted in order to define a feasible and reliable path to measure the intangible assets of a company, also called its intellectual capital. Several models have been defined, though problems still remain to be solved. In this article a heuristic frame addressing the link between intellectual capital and business strategy is developed, according to the author’s proposed intangible corporate asset taxonomy. This model is then applied to a company within the magnesium industry. The “time‐lag trap” issue is presented showing the misconceptions arising from the static rather than the dynamic intangible asset valuing approach. Future trends such as the creation of the IC elasticity concept and some conclusions in this realm are also presented.
Organizational practices for generating human resources in non‐corporate research and technology organizations
Authors: Mrinalini N. , Nath Pradosh
Published: July 2002

In the present context an organization competes in terms of its knowledge intensity. In this article we are focusing on human resource development, sustenance and enhancement as a process in non‐corporate research and technology organizations (RTOs), based on an international study on “Benchmarking the best practices for research and technology organizations”, coordinated by WAITRO. The basic function of these RTOs is to generate knowledge to effectively enhance their client’s competitive strength. A best RTO can be the one that provides a structure, which encourages people to take initiatives to generate new knowledge and effectively translate clients’ technological requirements into research results. For this, the RTO has to be a learning organization. To develop and sustain capabilities is essential for any RTO’s survival and growth. How best an organization performs depends upon its capability to build and enhance this knowledge base, i.e. the human capital. This in turn depends upon the way various activities are organized to generate human resource from the manpower they have.
Mapping intellectual capital in a small manufacturing enterprise
Authors: Heng Michael S.H.
Published: July 2002

The US$1.2 million company faces critical issues of management succession, business strategy, market expansion and innovative product improvements. These would prevent a steady flow of future potential earnings. The core knowledge resides in the management team who has worked in the factory from young. They have not patented the equipment designs or the proprietary processes. The first step in managing its intellectual capital uses the framework of the ISO 9000 standard to provide a mapping structure for capturing its core knowledge in products, process, management and customers. The focus on quality sets the context for shaping and organising the work of capturing its core knowledge. The ISO 9000 standard provides convenient categories for knowledge mapping, and presents a common language for consultant‐client interaction during the mapping process. A limitation in using the ISO 9000 standard for knowledge mapping is its inability to map knowledge concerning the customer base and product opportunities.
Intellectual capital
Authors: Valentine St Leon Mark
Published: July 2002

States that if organisations are to more effectively identify, measure and manage their intellectual capital, the responsibilities for initiating and executing these tasks will fall upon the managers of organisations. Addresses the question “To what extent does organisational type and managerial role define managers’ perception of intellectual capital?”. Two hypotheses were tested using a cross‐sectional design. Specifically, the research was directed at five types of senior managers (general manager, finance, human resources, information technology and marketing) in three broad types of Australian organisations: business, government and non‐profit organisations. Demonstrates tentatively that the perceptions that different types of Australian managers hold of intellectual capital lack uniformity and that different types of Australian organisations have yet to develop suitable policies and strategies that would facilitate its management.
Human capital knowledge value added
Authors: Kannan Gopika , Akhilesh K.B.
Published: July 2002

Knowledge value added is a new cost accounting technique making waves in intangibles accounting. It helps managers conduct a business process audit. However, the managers need a behavioral tool to understand the factors that influence human capital knowledge value add, in order to increase the organizational value add. This tool helps understand the knowledge professional’s perceptions of the organization’s culture towards intellectual enterprise, knowledge management support systems and processes, as well as individual value add, the perceived performance, innovation and consequences of quitting. The tool thus helps formulation of strategies towards effective management of human capital and creating higher value add. A leading Indian infotech organization was studied as a case in point and the results are being used to design more effective knowledge management strategies.
The boardroom agenda: developing the innovative organisation
Authors: Knox Simon
Published: July 2002

Explores the distinctive behaviours and organizational capabilities which enable firms to innovate successfully. Identifies four aspects of innovation which sustain the firm’s abilities to deliver superior customer value. These are: culture and climate; the management of assets and capabilities; structure and controls; and new product and process development. Argues that the over‐reliance on the traditional approach to innovation – the development of new products and services – is too limited a view and may even be preventing business leaders from adopting this broader, organisational approach where the contribution from each area of the business can be multiplicative when kept in balance. Finally, issues the role of business leaders in removing barriers to innovation and nurturing a multi‐faceted approach across the firm and draws conclusions.
Mapping the surface of Escherichia coli peptide deformylase by NMR with organic solvents
Authors: Byerly Douglas W. , McElroy Craig A. , Foster Mark P.
Published: July 2002

Identifying potential ligand binding sites on a protein surface is an important first step for targeted structure‐based drug discovery. While performing control experiments with Escherichia coli peptide deformylase (PDF), we noted that the organic solvents used to solubilize some ligands perturbed many of the same resonances in PDF as the small molecule inhibitors. To further explore this observation, we recorded 15N HSQC spectra of E. coli peptide deformylase (PDF) in the presence of trace quantities of several simple organic solvents (acetone, DMSO, ethanol, isopropanol) and identified their sites of interaction from local perturbation of amide chemical shifts. Analysis of the protein surface structure revealed that the ligand‐induced shift perturbations map to the active site and one additional surface pocket. The correlation between sites of solvent and inhibitor binding highlights the utility of organic solvents to rapidly and effectively validate and characterize binding sites on proteins prior to designing a drug discovery screen. Further, the solvent‐induced perturbations have implications for the use of organic solvents to dissolve candidate ligands in NMR‐based screens.
Properties of Bacillus cereus hemolysin II: A heptameric transmembrane pore
Authors: Miles George , Bayley Hagan , Cheley Stephen
Published: July 2002

The gene encoding hemolysin II (HlyII) was amplified from Bacillus cereus genomic DNA and a truncated mutant, HlyII(ΔCT), was constructed lacking the 94 amino acid extension at the C terminus. The proteins were produced in an E. coli cell‐free in vitro transcription and translation system, and were shown to assemble into SDS‐stable oligomers on rabbit erythrocyte membranes and liposomes. The hemolytic activity of HlyII was measured with rabbit erythrocytes yielding an HC50 value of 1.64 ng mL−1, which is over 15 times more potent than staphylococcal α‐hemolysin. HlyII(ΔCT) was about eight times less potent than HlyII in this assay. Limited proteolysis of the oligomers formed by HlyII and HlyII(ΔCT) on red cell membranes showed that the C‐terminal extension is sensitive to digestion, while HlyII(ΔCT) is protease resistant and migrates with an electrophoretic mobility similar to that of digested HlyII. HlyII forms moderately anion selective, rectifying pores (I+80/I−80 = 0.57, 1 M KCl, pH 7.4) in planar lipid bilayers of diphytanoylphosphatidylcholine with a unitary conductance of 637 pS (1 M KCl, 5 mM HEPES, pH 7.4) and exhibits no gating over a wide range of applied potentials (−160 to +160 mV). In addition, it was demonstrated that HlyII forms a homoheptameric pore by using gel shift electrophoresis aided by a genetically encoded oligoaspartate tag. Although they share limited primary sequence identity (30%), these data confirm that HlyII is a structural and functional homolog of staphylococcal α‐hemolysin.
High‐throughput screening of soluble recombinant proteins
Authors: Shih Yan‐Ping , Kung Wen‐Mei , Chen Jui‐Chuan , Yeh Chia‐Hui , Wang Andrew H.‐J. , Wang Ting‐Fang
Published: July 2002

The aims of high‐throughput (HTP) protein production systems are to obtain well‐expressed and highly soluble proteins, which are preferred candidates for use in structure–function studies. Here, we describe the development of an efficient and inexpensive method for parallel cloning, induction, and cell lysis to produce multiple fusion proteins in Escherichia coli using a 96‐well format. Molecular cloning procedures, used in this HTP system, require no restriction digestion of the PCR products. All target genes can be directionally cloned into eight different fusion protein expression vectors using two universal restriction sites and with high efficiency (>95%). To screen for well‐expressed soluble fusion protein, total cell lysates of bacteria culture (∼1.5 mL) were subjected to high‐speed centrifugation in a 96‐tube format and analyzed by multiwell denaturing SDS‐PAGE. Our results thus far show that 80% of the genes screened show high levels of expression of soluble products in at least one of the eight fusion protein constructs. The method is well suited for automation and is applicable for the production of large numbers of proteins for genome‐wide analysis.
Structural features underlying selective inhibition of protein kinase CK2 by ATP site‐directed tetrabromo‐2‐benzotriazole
Authors: Battistutta Roberto , De Moliner Erika , Sarno Stefania , Zanotti Giuseppe , Pinna Lorenzo A.
Published: July 2002

Two novel crystal structures of Zea mays protein kinase CK2α catalytic subunit, one in complex with the specific inhibitor 4,5,6,7‐tetrabromobenzotriazole (TBB) and another in the apo‐form, were solved at 2.2 Å resolution. These structures were compared with those of the enzyme in presence of ATP and GTP (the natural cosubstrates) and the inhibitor emodin. Interaction of TBB with the active site of CK2α is mainly due to van der Waals contacts, with the ligand fitting almost perfectly the cavity. One nitrogen of the five‐membered ring interacts with two charged residues, Glu 81 and Lys 68, in the depth of the cavity, through two water molecules. These are buried in the active site and are also generally found in the structures of CK2α enzyme analyzed so far, with the exception of the complex with emodin. In the N‐terminal lobe, the position of helix αC is particularly well preserved in all the structures examined; the Gly‐rich loop is displaced from the intermediate position it has in the apo‐form and in the presence of the natural cosubstrates (ATP/GTP) to either an upper (with TBB) or a lower position (with emodin). The selectivity of TBB for CK2 appears to be mainly dictated by the reduced size of the active site which in most other protein kinases is too large for making stable interactions with this inhibitor.
Transmembrane signal transduction of the αIIbβ3 integrin
Authors: Gottschalk Kay E. , Adams Paul D. , Brunger Axel T. , Kessler Horst
Published: July 2002

Integrins are composed of noncovalently bound dimers of an α‐ and a β‐subunit. They play an important role in cell‐matrix adhesion and signal transduction through the cell membrane. Signal transduction can be initiated by the binding of intracellular proteins to the integrin. Binding leads to a major conformational change. The change is passed on to the extracellular domain through the membrane. The affinity of the extracellular domain to certain ligands increases; thus at least two states exist, a low‐affinity and a high‐affinity state. The conformations and conformational changes of the transmembrane (TM) domain are the focus of our interest. We show by a global search of helix–helix interactions that the TM section of the family of integrins are capable of adopting a structure similar to the structure of the homodimeric TM protein Glycophorin A. For the αIIbβ3 integrin, this structural motif represents the high‐affinity state. A second conformation of the TM domain of αIIbβ3 is identified as the low‐affinity state by known mutational and nuclear magnetic resonance (NMR) studies. A transition between these two states was determined by molecular dynamics (MD) calculations. On the basis of these calculations, we propose a three‐state mechanism.
Factors contributing to decreased protein stability when aspartic acid residues are in β‐sheet regions
Authors: Pokkuluri P.R. , Gu M. , Cai X. , Raffen R. , Stevens F.J. , Schiffer M.
Published: July 2002

Asp residues are significantly under represented in β‐sheet regions of proteins, especially in the middle of β‐strands, as found by a number of studies using statistical, modeling, or experimental methods. To further understand the reasons for this under representation of Asp, we prepared and analyzed mutants of a β‐domain. Two Gln residues of the immunoglobulin light‐chain variable domain (VL) of protein Len were replaced with Asp, and then the effects of these changes on protein stability and protein structure were studied. The replacement of Q38D, located at the end of a β‐strand, and that of Q89D, located in the middle of a β‐strand, reduced the stability of the parent immunoglobulin VL domain by 2.0 kcal/mol and 5.3 kcal/mol, respectively. Because the Q89D mutant of the wild‐type VL‐Len domain was too unstable to be expressed as a soluble protein, we prepared the Q89D mutant in a triple mutant background, VL‐Len M4L/Y27dD/T94H, which was 4.2 kcal/mol more stable than the wild‐type VL‐Len domain. The structures of mutants VL‐Len Q38D and VL‐Len Q89D/M4L/Y27dD/T94H were determined by X‐ray diffraction at 1.6 Å resolution. We found no major perturbances in the structures of these Q→D mutant proteins relative to structures of the parent proteins. The observed stability changes have to be accounted for by cumulative effects of the following several factors: (1) by changes in main‐chain dihedral angles and in side‐chain rotomers, (2) by close contacts between some atoms, and, most significantly, (3) by the unfavorable electrostatic interactions between the Asp side chain and the carbonyls of the main chain. We show that the Asn side chain, which is of similar size but neutral, is less destabilizing. The detrimental effect of Asp within a β‐sheet of an immunoglobulin‐type domain can have very serious consequences. A somatic mutation of a β‐strand residue to Asp could prevent the expression of the domain both in vitro and in vivo, or it could contribute to the pathogenic potential of the protein in vivo.
Description of the topographical changes associated to the different stages of the DsbA catalytic cycle
Authors: Vinci Floriana , Couprie Joël , Pucci Piero , Quéméneur Eric , Moutiez Mireille
Published: July 2002

This paper provides a description of the surface topography of DsbA, the bacterial disulfide‐bond forming enzyme, in the different phases of its catalytic cycle. Three representative states, that is, oxidized and reduced protein and a covalent complex mimicking the DsbA‐substrate disulfide intermediate, have been investigated by a combination of limited proteolysis experiments and mass spectrometry methodologies. Protease‐accessible sites are largely distributed in the oxidized form with a small predominance inside the thioredoxin domain. Proteolysis occurs even in secondary structure elements, revealing a significant mobility of the protein. Many cleavage sites disappear in the reduced form and most of the remaining ones appear with strongly reduced kinetics. The protein within the complex shows an intermediate behavior. This variation of flexibility in DsbA is probably the determining factor for the course of its catalytic cycle. In particular, the great mobility of the oxidized protein might facilitate the accommodation of its various substrates, whereas the increasing rigidity from the complexed to the reduced form could help the release of oxidized products. The formation of the complex between PID peptide and DsbA does not significantly protect the enzyme against proteolysis, reinforcing the results previously obtained by calorimetry concerning the weakness of their interaction. The few cleavage sites observed, however, are in favor of the presence of the peptide in the binding site postulated from crystallographic studies. As for the peptide itself, the proteolytic pattern and the protection effect exerted by DsbA could be explained by a preferential orientation within the binding site.
Equilibrium denaturation studies of the Escherichia coli factor for inversion stimulation: Implications for in vivo function
Authors: Hobart Sarah A. , Ilin Sergey , Moriarty Daniel F. , Osuna Robert , Colón Wilfredo
Published: July 2002

The Factor for Inversion Stimulation (FIS) is a dimeric DNA binding protein found in enteric bacteria that is involved in various cellular processes, including stimulation of certain specialized DNA recombination events and transcription regulation of a large number of genes. The intracellular FIS concentration, when cells are grown in rich media, varies dramatically during the early logarithmic growth phase. Its broad range of concentrations could potentially affect the nature of its quaternary structure, which in turn, could affect its ability to function in vivo. Thus, we examined the stability of FIS homodimers under a wide range of concentrations relevant to in vivo expression levels. Its urea‐induced equilibrium denaturation was monitored by far‐ and near‐UV circular dichroism (CD), tyrosine fluorescence, and tyrosine fluorescence anisotropy. The denaturation transitions obtained were concentration‐dependent and showed similar midpoints (Cm) and m values, suggesting a two‐state denaturation process involving the native dimer and unfolded monomers (N2 ↔ 2U). The ΔG for the unfolding of FIS determined from global and individual curve fitting was 14.2 kcal/mole. At concentrations <9 μM, the FIS dimer began to dissociate, as noted by the change in CD signal and size‐exclusion high‐pressure liquid chromatography retention times and peak width. The estimated dimer dissociation constant based on the CD and size‐exclusion chromatography data is in the micromolar range, resulting in a ΔG of at least 5 kcal/mole less than that calculated from the urea denaturation data. This discrepancy suggests a deviation from a two‐state denaturation model, perhaps due to a marginally stable monomeric intermediate. These observations have implications for the stability and function of FIS in vivo.
On the Transition from Two‐Dimensional to Three‐Dimensional Water Waves
Authors: Dias F. , Hărăguş–Courcelle M.
Published: July 2002

There are essentially two types of three‐dimensional water waves: waves that bifurcate from the state of rest (these waves are commonly called short‐crested waves or forced waves), and waves that bifurcate from a two‐dimensional wave of finite amplitude (these waves are sometimes called spontaneous waves). This paper deals with spontaneously generated three‐dimensional waves. To understand this phenomenon better from a mathematical point of view, it is helpful to work on model equations rather than on the full equations. Such an attempt was made formally by Martin in 1982 on the nonlinear Schrödinger equation, but it is shown here that it is hard to justify his results mathematically because of the hyperbolicity of the nonlinear Schrödinger equation for gravity waves. On the other hand, in some parameter regimes, the nonlinear Schrödinger equation becomes elliptic. In that case, the appearance of spontaneous three‐dimensional waves can be shown rigorously by using a dynamical systems approach. The results are extended to the Benney–Roskes–Davey–Stewartson equations when they are both elliptic. Various types of three‐dimensional waves bifurcating from a two‐dimensional periodic wave are obtained.
Subcritical Rossby Waves in Zonal Shear Flows with Nonlinear Critical Layers
Authors: Maslowe S. A. , Clarke S. R.
Published: July 2002

It was shown by Benney and Bergeron [1] that singular neutral modes with nonlinear critical layers are mathematically possible in a variety of shear flows. These are usually subcritical modes; i.e., they occur at values of the flow parameters where their linear, viscous counterparts would be damped. One question raised then is how such modes might be generated.

This article treats the problem of Rossby waves propagating in a mixing layer with velocity profile ū = tanh y. The beta parameter, which is a measure of the stabilizing Coriolis force, is taken to be large enough so that linear instability cannot occur. First, computed dispersion curves are presented for singular modes with nonlinear critical layers. Then, full numerical simulations are employed to illustrate how these modes can be generated by resonant interaction with conventional nonsingular Rossby waves, even when the singular mode is absent initially.