bluuebunny/crossref_metadata_2025 huggingface dataset:
Efficient Prenylation by a Plant Geranylgeranyltransferase-I Requires a Functional CaaL Box Motif and a Proximal Polybasic Domain
Authors: Caldelari Daniela , Sternberg Hasana , Rodrı́guez-Concepción Manuel , Gruissem Wilhelm , Yalovsky Shaul
Published: October 2002

Geranylgeranyltransferase-I (GGT-I) is a heterodimeric enzyme that shares a common α-subunit with farnesyltransferase (FTase) and has a distinct β-subunit. GGT-I preferentially modifies proteins, which terminate in a CaaL box sequence motif. Cloning of Arabidopsis GGT-I β-subunit (AtGGT-IB) was achieved by a yeast (Saccharomyces cerevisiae) two-hybrid screen, using the tomato (Lycopersicon esculentum) FTase α-subunit (FTA) as bait. Sequence and structure analysis revealed that the core active site of GGT-I and FTase are very similar. AtGGT-IA/FTA and AtGGT-IB were co-expressed in baculovirus-infected insect cells to obtain recombinant protein that was used for biochemical and molecular analysis. The recombinant AtGGT-I prenylated efficiently CaaL box fusion proteins in which the a2 position was occupied by an aliphatic residue, whereas charged or polar residues at the same position greatly reduced the efficiency of prenylation. A polybasic domain proximal to the CaaL box motif induced a 5-fold increase in the maximal reaction rate, and increased the affinity of the enzyme to the protein substrate by an order of magnitude. GGT-I retained high activity in a temperature range between 24°C and 42°C, and showed increased activity rate at relatively basic pH values of 7.9 and 8.5. Reverse transcriptase-polymerase chain reaction, protein immuno-blots, and transient expression assays of green fluorescent protein fusion proteins show that GGT-IB is ubiquitously expressed in a number of tissues, and that expression levels and protein activity were not changed in mutant plants lacking FTase β-subunit.
Chemical Composition of thePrunus laurocerasusLeaf Surface. Dynamic Changes of the Epicuticular Wax Film during Leaf Development
Authors: Jetter Reinhard , Schäffer Stefanie
Published: October 2002

The seasonal development of adaxial Prunus laurocerasus leaf surfaces was studied using newly developed methods for the mechanical removal of epicuticular waxes. During epidermal cell expansion, more than 50 μg leaf−1 of alkyl acetates accumulated within 10 d, forming an epicuticular wax film approximately 30 nm thick. Then, alcohols dominated for 18 d of leaf development, before alkanes accumulated in an epicuticular wax film with steadily increasing thickness (approximately 60 nm after 60 d), accompanied by small amounts of fatty acids, aldehydes, and alkyl esters. In contrast, the intracuticular waxes stayed fairly constant during development, being dominated by triterpenoids that could not be detected in the epicuticular waxes. The accumulation rates of all cuticular components are indicative for spontaneous segregation of intra- and epicuticular fractions during diffusional transport within the cuticle. This is the first report quantifying the loss of individual compound classes (acetates and alcohols) from the epicuticular wax mixture. Experiments with isolated epicuticular films showed that neither chemical conversion within the epicuticular film nor erosion/evaporation of wax constituents could account for this effect. Instead, transport of epicuticular compounds back into the tissue seems likely. Possible ecological and physiological functions of the coordinate changes in the composition of the plant surface layers are discussed.
The Short-Chain Alcohol Dehydrogenase ABA2 Catalyzes the Conversion of Xanthoxin to Abscisic Aldehyde[W]
Authors: González-Guzmán Miguel , Apostolova Nadezda , Bellés José M. , Barrero José M. , Piqueras Pedro , Ponce María R. , Micol José L. , Serrano Ramón , Rodríguez Pedro L.
Published: October 2002

Mutants able to germinate and perform early growth in medium containing a high NaCl concentration were identified during the course of two independent screenings and named salt resistant (sre) and salobreño (sañ). The sre and sañ mutants also were able to germinate in high-osmoticum medium, indicating that they are osmotolerant in a germination assay. Complementation analyses revealed that sre1-1, sre1-2, sañ3-1, and sañ3-2 were alleles of the abscisic acid (ABA) biosynthesis ABA2 gene. A map-based cloning strategy allowed the identification of the ABA2 gene and molecular characterization of four new aba2 alleles. The ABA2 gene product belongs to the family of short-chain dehydrogenases/reductases, which are known to be NAD- or NADP-dependent oxidoreductases. Recombinant ABA2 protein produced in Escherichia coli exhibits a K  m value for xanthoxin of 19 μM and catalyzes in a NAD-dependent manner the conversion of xanthoxin to abscisic aldehyde, as determined by HPLC–mass spectrometry. The ABA2 mRNA is expressed constitutively in all plant organs examined and is not upregulated in response to osmotic stress. The results of this work are discussed in the context of previous genetic and biochemical evidence regarding ABA biosynthesis, confirming the xanthoxin→abscisic aldehyde→ABA transition as the last steps of the major ABA biosynthetic pathway.
Structural characterization of crystals of α-glycine during anomalous electrical behaviour
Authors: Langan Paul , Mason Sax A. , Myles Dean , Schoenborn Benno P.
Published: October 2002

The crystal structure of α-glycine has been investigated in the temperature range 288–427 K using neutron diffraction. The molecular structure does not change significantly and the putative crystallographic phase transition associated with anomalous electrical behaviour in this temperature range is not observed. The unit cell expands anisotropically with increasing temperature, with the unique monoclinic b axis, corresponding to the stacking direction of molecular layers, changing the most. The increasing separation of antiferroelectric molecular layers with increasing temperature is driven by an increase in molecular libration about an axis that lies perpendicular to the b axis. There is also a weakening of the interlayer hydrogen bonds with temperature. These structural and dynamic changes will affect the response of molecular dipoles to an applied electric field and provide a possible mechanism for the anomalous electrical behaviour.
The mapping of electronic energy distributions using experimental electron density
Authors: Tsirelson Vladimir G.
Published: October 2002

It is demonstrated that the approximate kinetic energy density calculated using the second-order gradient expansion with parameters of the multipole model fitted to experimental structure factors reproduces the main features of this quantity in a molecular or crystal position space. The use of the local virial theorem provides an appropriate derivation of approximate potential energy density and electronic energy density from the experimental (model) electron density and its derivatives. Consideration of these functions is not restricted by the critical points in the electron density and provides a comprehensive characterization of bonding in molecules and crystals.
X-ray studies on crystalline complexes involving amino acids and peptides. XXXIX. Crystal structures of malonic acid complexes of DL- and L-histidine. Preservation of aggregation pattern on reversal of chirality
Authors: Saraswathi N.T. , Vijayan M.
Published: October 2002

The malonic acid complexes of DL- and L-histidine are made up of zwitterionic positively charged histidinium ions and semimalonate (hydrogen malonate) ions. They crystallise in space groups P21/n and P21, respectively, with nearly the same unit-cell parameters. The molecules aggregate in the two complexes in a remarkably similar manner. The two sets of crystallographically independent molecules are related by a pseudo-glide plane. This pseudo-symmetry is almost exact except in the case of the α-carboxylate group and, to some extent, the α-C and the α-N atoms. Preservation of the aggregation pattern to such an extent on the reversal of chirality of half the amino-acid molecules is observed for the first time in amino-acid complexes. This is achieved at the cost of considerable conformational strain in one of the two histidinium ions in the L-histidine complex.
Crystal structure of 5-oxatricyclo[5.1.0.01,3]octane-4-one: two polymorphs in one crystal
Authors: Yufit D. S. , Kozhushkov S. I. , Howard J. A. K. , de Meijere A.
Published: October 2002

Two subsets of data, corresponding to different crystalline modifications of the title compound, 5-oxatricyclo[5.1.0.01,3]octane-4-one (C7H8O2), have been obtained from the same experiment. Both structures were successfully solved and refined. The packing of identical layers of molecules is different for monoclinic and orthorhombic forms.
Simple model designed to generate new crystal structures derived from a mother phase; application to molecular compounds
Authors: Gervais Claire , Coquerel Gérard
Published: October 2002

The basic principles of a model predicting new lattices from a known crystal structure are described. The first of the two-step procedure consists of extracting one- or two-dimensional periodic fragments (PF) from the mother structure. In the second step, symmetry operators are added to the PFs in order to generate one or several new three-dimensional lattices consistent with the 230 space groups. Most of the examples are related to polymorphism, but relationships between racemic compounds and enantiomers, twinning and lamellar epitaxy phenomena are also exemplified.
Optimization of the PD coefficient in a flight simulator control via genetic algorithms
Authors: Yurt Sait N. , Özkol I˙brahim , Kaya Metin O. , Haciyev Chingiz
Published: October 2002

In this study the design of motion‐based flight simulators is carried out by specifying the performance required of the motion cueing mechanism, to generate translational and angular motions as a 6–3 Stewart Platform Mechanism (SPM). These motions are intended to approximate the specific forces and angular accelerations encountered by the pilot in the simulated aircraft. Firstly, the dynamics of this 6–3 SPM is given in closed form as in our earlier study. Then, for the control of obtained dynamic model, a leg‐length based PD algorithm is applied. In the optimization of the applied PD algorithm's coefficients, Real Coded Genetic Algorithms are used. So as to have faster and effective system's performance, the fitness function chosen, in Genetic Algorithms, having maximum overshoot value, settling time and steady state error which are obtained from the unit step response. The performance of the system studied is compared to the similar studies in the literature exist.
Transitions into research careers in Switzerland
Authors: Julia Leemann Regula
Published: October 2002

This study examines the importance of gender and social origin for the transition of university graduates into the first phase of a research career, the doctoral programme. It also considers whether women and members of lower social classes have better career opportunities in certain fields than in others. The empirical analysis makes use of a panel database that provides information on the education and career paths of Swiss university graduates in all fields of study as of the beginning of the 1990s. The results show that women and people from less educated families are less commonly recruited for research careers than are men and people from university‐educated backgrounds. However, the degree of inequality of opportunity is not the same in all of the fields.
Employment equity in South Africa: lessons from the global school
Authors: Thomas Adèle
Published: October 2002

South African companies employing 50 or more employees or those with a specified financial turnover, are required to comply with the provisions of the newly introduced Employment Equity Act. Prior to the introduction of the Act, large companies in the country had already introduced strategies of affirmative action to achieve employment equity. This paper highlights some of the strategic and operational concerns surrounding employment equity legislation in South Africa and reviews broad practices of employment equity in Malaysia, India, the USA, Canada, Britain and Zimbabwe. Lessons for the effective implementation of employment equity in South Africa are identified.
Implementation of intelligent decision support systems in health care
Authors: Smith A.E. , Nugent C.D. , McClean S.I.
Published: October 2002

The full implementation of any intelligent system in health care, which is designed for decision support, has several stages, from initial problem identification through development and, finally, cost‐benefit analysis. Central to this is formal objectivist evaluation with its core component of inherent performance of the outputs from these systems. A Medline survey of one type of intelligent system is presented, which demonstrates that this issue is not being addressed adequately. Lack of criteria for dealing with the outputs from these “black box” systems to prescribe adequate levels of inherent performance may be preventing their being accepted by those in the health‐care domain and, thus, their being applied widely in the field.
Barriers to entry in industrial markets
Authors: Karakaya Fahri
Published: October 2002

This study examines the importance of 25 barriers to market entry in industrial markets. A survey of 93 firms indicates that majority of business executives consider cost advantages and capital requirements to enter markets as the two most important barriers to entry followed by incumbents having a superior production process, capital intensity of the market, and customer loyalty. The least important barriers perceived by the executives in the study are government licensing requirements, followed by heavy advertising. In addition, the study investigates the underlying dimensions of barriers to entry in industrial market through a factor analysis. The results indicate that there are four major underlying dimensions of entry barriers in industrial markets.
Selecting and promoting service encounter elements in passenger rail transportation
Authors: Tripp Carolyn , Drea John T.
Published: October 2002

A critical issue in the effective development and promotion of services is the identification of which service components are most closely related to attitude toward the service provider and repeat purchase behavior. The present study disaggregates a service (passenger rail transportation) into pre‐core and core service performances, and uses these elements to create a model of the effects on relevant service attitudes and intentions. Using a sample of 2,529 Amtrak riders, the present research finds that the core service elements of on‐board conditions, cafe car conditions, and on‐time performance were most strongly related to attitude toward the service provider. Advertising implications for developing repeat purchase behavior are discussed.
Effect of thermal radiation from fabrics on human body
Authors: Nakajima Toshinari , Hachino Yuki , Yamano Haruko
Published: October 2002

The effectiveness of the far infrared radiation processing cloth as clothing material is discussed. Temperature rise of the irradiation plane is more rapid than the heating by heat conduction and convection for the radiant heating. Skin temperature change and thermal sensation of the examinee wearing the sweater with a plastic heater at back were examined. “The relation of the inverse proportion of heat intensity to the time needed until the extent of thermal stimulation perceived was examined. Individual examinees tested, by oral contact, the extent of the pleasant sensation produced by the warming condition. They judged the extent of the warming according to whether it felt comfortable. Individually, they differed a great extent in their opinion of what was comfortable. However, for all examinees, the relationship of warming and feeling comfortable was confined within narrow parameters. In this experiment, a unit of the radiant heat stimulation was determined by dividing mW/cm2 by the warming period, since intermittent warming was carried out.” The degree of the skin temperature perceived by the sensation was almost fixed at 33–35, even if the radiant heat strength differed. The rapid thermal stimulation by radiant heating can be perceived even for a slight temperature rise of the skin. It is also necessary to consider the temperature rise speed, except for the temperature rise of the cloth surface, when the effectiveness of the far infrared radiation processing cloth was discussed.
Applying digital manufacturing technology to ship production and the maritime environment
Authors: Kim Hongtae , Lee Jong‐Kap , Park Jin‐Hyoung , Park Beom‐Jin , Jang Dong‐Sik
Published: October 2002

This paper is mainly concerned with the digital manufacturing technologies in the context of the shipbuilding industry. New concepts such as digital shipbuilding, virtual shipyard, and simulation‐based design (SBD) will be explored. After reviewing the digital shipbuilding, a case study will be presented using the virtual assembly simulation system for shipbuilding (VASSS), a simulation based tool, to evaluate block erection sequence taking account of shipyard facilities, operational efficiency and equipment replacement time.
The synergistic leadership theory
Authors: Irby Beverly J. , Brown Genevieve , Duffy Jo Ann , Trautman Diane
Published: October 2002

Modernist theories in leadership were traditionally dominated by masculine incorporation and lacked feminine presence in development and language. The synergistic theory of leadership (SLT) seeks to explicate the need for a post‐modernist leadership theory by providing an alternative to, and not a replacement for, traditional theories. Six aspects particular to the SLT influence the ideas and include issues concerning diversity and the inclusion of the female voice in the theory. Four factors are key to the relational and interactive nature of the theory, which provides a useful framework for building and understanding the interdependent relationships. In a tetrahedron model, the theory uses four factors, including leadership behavior, organizational structure, external forces, and attitudes, beliefs, and values to demonstrate aspects not only of leadership but its effects on various institutions and positions. Developed through a qualitative approach, the theory has been validated qualitatively and quantitatively nationwide and is currently being validated internationally.
A numerical study of cavitation foot‐prints in liquid‐lubricated asymmetrical herringbone grooved journal bearings
Authors: Junmei Wan , Lee T.S. , Shu C. , Jiankang Wu
Published: October 2002

A numerical model is presented in this paper to better describe the cavitated fluid flow phenomena in liquid‐lubricated Asymmetrical Herringbone Grooved Journal Bearings (HGJBs). An effective “follow the groove” grid transformation method is used in the present study to capture all the groove boundaries. A singularity at the groove edges is avoided with this approach. Symmetrical groove patterns as well as asymmetrical groove patterns can be accurately computed with the proposed method. The difficult problem of abrupt changes of oil film thickness in the liquid‐lubricated HGJB physical domain is modeled here through a series expansion approach. Results are comparable with available experimental and known numerical data from other investigators. Cavitation footprints, pressure distributions and their corresponding load characteristics are presented in this study. Effects of the critical transitional flow phenomena on the performance of the asymmetrical HGJBs are also determined through the present study.
Supplier selection problem: a comparison of the total cost of ownership and analytic hierarchy process approaches
Authors: Bhutta Khurrum S. , Huq Faizul
Published: October 2002

Supplier selection and evaluation are arguably one of the most critical functions for the success of an organization. Several approaches exist in the literature to objectively evaluate suppliers, including analytic hierarchy process and total cost of ownership. Analytic hierarchy process provides a framework to cope with multiple criteria situations involving supplier selection, while the total cost of ownership is a methodology and philosophy, which look beyond just the price of a purchase to better understand and manage costs in selecting and maintaining relationships with suppliers. This paper illustrates the two approaches and provides a comparison.
Resource adequacy in new product development: a discriminant analysis
Authors: Huang Xueli , Soutar Geoffrey N. , Brown Alan
Published: October 2002

New product development (NPD) is crucial to the survival and thriving of a business entity and a firm’s sources of advantages are important to the NPD success. This paper explores the marketing and technical resources adequacy of Australian small and medium enterprises (SMEs) in NPD. A survey of 276 Australian SMEs in the chemical and machinery industries was conducted. Analytical procedures include factor analysis, cluster analysis, and discriminant analysis. Findings from these analyses suggest that three distinct groups in terms of their NPD resources exist in Australian SMEs: one group with rich marketing and technical resources and skills, one with rich technical resource only, and one with rich technical skill only. The organisational and managerial characteristics of each group of these firms are described. The findings imply that different resource groups need to adopt different strategies in NPD.
“Sustainability” in higher education
Authors: Wals Arjen E.J. , Jickling Bob
Published: October 2002

It is higher education’s responsibility to continuously challenge and critique value and knowledge claims that have prescriptive tendencies. Part of this responsibility lies in engaging students in socio‐scientific disputes. The ill‐defined nature of sustainability manifests itself in such disputes when conflicting values, norms, interests, and reality constructions meet. This makes sustainability – its need for contextualization and the debate surrounding it – pivotal for higher education. It offers an opportunity for reflection on the mission of our universities and colleges, but also a chance to enhance the quality of the learning process. This paper explores both the overarching goals and process of higher education from an emancipatory view and with regard to sustainability.
The Ludwig report: implications for corporate governance
Authors: Dunne Theresa M. , Helliar Christine V.
Published: October 2002

The Ludwig report concerning currency trading losses at AIB plc was issued in March 2002. This paper reviews the Ludwig report and assesses possible implications of the report for internal control and corporate governance procedures in treasury departments.
Characterization and multiple molecular forms of TorD from Shewanella massilia, the putative chaperone of the molybdoenzyme TorA
Authors: Tranier Samuel , Mortier‐Barrière Isabelle , Ilbert Marianne , Birck Catherine , Iobbi‐Nivol Chantal , Méjean Vincent , Samama Jean‐Pierre
Published: October 2002

Several bacteria use trimethylamine N‐oxyde (TMAO) as an exogenous electron acceptor for anaerobic respiration. This metabolic pathway involves expression of the tor operon that codes for a periplasmic molybdopterin‐containing reductase of the DMSO/TMAO family, a pentahemic c‐type cytochrome, and the TorD cytoplasmic chaperone, possibly required for acquisition of the molybdenum cofactor and translocation of the reductase by the twin‐arginine translocation system. In this report, we show that the TorD chaperone from Shewanella massilia forms multiple and stable oligomeric species. The monomeric, dimeric, and trimeric forms were purified to homogeneity and characterized by analytical ultracentrifugation. Small‐angle X‐ray scattering (SAXS) and preliminary diffraction data indicated that the TorD dimer is made of identical protein modules of similar size to the monomeric species. Interconversion of the native oligomeric forms occurred at acidic pH value. In this condition, ANS fluorescence indicates a non‐native conformation of the polypeptide chain in which, according to the circular dichroism spectra, the α‐helical content is similar to that of the native species. Surface plasmon resonance showed that both the monomeric and dimeric species bind the mature TorA enzyme, but that the dimer binds its target protein more efficiently. The possible biologic significance of these oligomers is discussed in relation to the chaperone activity of TorD, and to the ability of another member of the TorD family to bind the Twin Arginine leader sequences of the precursor of DMSO/TMAO reductases.
The effect of prime‐site occupancy on the hepatitis C virus NS3 protease structure
Authors: Casbarra Annarita , Piaz Fabrizio Dal , Ingallinella Paolo , Orrù Stefania , Pucci Piero , Pessi Antonello , Bianchi Elisabetta
Published: October 2002

We recently reported a new class of inhibitors of the chymotrypsin‐like serine protease NS3 of the hepatitis C virus. These inhibitors exploit the binding potential of the S′ site of the protease, which is not generally used by the natural substrates. The effect of prime‐site occupancy was analyzed by circular dichroism spectroscopy and limited proteolysis‐mass spectrometry. Generally, nonprime inhibitors cause a structural change in NS3. Binding in the S′ site produces additional conformational changes with different binding modes, even in the case of the NS3/4A cofactor complex. Notably, inhibitor binding either in the S or S′ site also has profound effects on the stabilization of the protease. In addition, the stabilization propagates to regions not in direct contact with the inhibitor. In particular, the N‐terminal region, which according to structural studies is endowed with low structural stability and is not stabilized by nonprime inhibitors, was now fully protected from proteolytic degradation. From the perspective of drug design, P‐P′ inhibitors take advantage of binding pockets, which are not exploited by the natural HCV substrates; hence, they are an entry point for a novel class of NS3/4A inhibitors. Here we show that binding of each inhibitor is associated with a specific structural rearrangement. The development of a range of inhibitors belonging to different classes and an understanding of their interactions with the protease are required to address the issue of the most likely outcome of viral protease inhibitor therapy, that is, viral resistance.
Plasticity in structure and interactions is critical for the action of indolicidin, an antibacterial peptide of innate immune origin
Authors: Nagpal Sushma , Kaur Kanwal J. , Jain Deepti , Salunke Dinakar M.
Published: October 2002

The comparative analysis of two cationic antibacterial peptides of the cathelicidin family—indolicidin and tritrypticin—enabled addressing the structural features critical for the mechanism of indolicidin activity. Functional behavior of retro‐indolicidin was found to be identical to that of native indolicidin. It is apparent that the gross conformational propensities associated with retro‐peptides resemble those of the native sequences, suggesting that native and retro‐peptides can have similar structures. Both the native and the retro‐indolicidin show identical affinities while binding to endotoxin, the initial event associated with the antibacterial activity of cationic peptide antibiotics. The indolicidin–endotoxin binding was modeled by docking the indolicidin molecule in the endotoxin structure. The conformational flexibility associated with the indolicidin residues, as well as that of the fatty acid chains of endotoxin combined with the relatively strong structural interactions, such as ionic and hydrophobic, provide the basis for the endotoxin–peptide recognition. Thus, the key feature of the recognition between the cationic antibacterial peptides and endotoxin is the plasticity of molecular interactions, which may have been designed for the purpose of maintaining activity against a broad range of organisms, a hallmark of primitive host defense.
The role of disulfide bond in the amyloidogenic state of β2‐microglobulin studied by heteronuclear NMR
Authors: Katou Hidenori , Kanno Takashi , Hoshino Masaru , Hagihara Yoshihisa , Tanaka Hiroyuki , Kawai Tomoji , Hasegawa Kazuhiro , Naiki Hironobu , Goto Yuji
Published: October 2002

β2‐Microglobulin (β2‐m) is a major component of dialysis‐related amyloid fibrils. Although recombinant β2‐m forms needle‐like fibrils by in vitro extension reaction at pH 2.5, reduced β2‐m, in which the intrachain disulfide bond is reduced, cannot form typical fibrils. Instead, thinner and flexible filaments are formed, as shown by atomic force microscopy images. To clarify the role of the disulfide bond in amyloid fibril formation, we characterized the conformations of the oxidized (intact) and reduced forms of β2‐m in the acid‐denatured state at pH 2.5, as well as the native state at pH 6.5, by heteronuclear NMR. {1H}‐15N NOE at the regions between the two cysteine residues (Cys25–Cys80) revealed a marked difference in the pico‐ and nanosecond time scale dynamics between that the acid‐denatured oxidized and reduced states, with the former showing reduced mobility. Intriguingly, the secondary chemical shifts, ΔCα, ΔCO, and ΔHα, and 3JHNHα coupling constants indicated that both the oxidized and reduced β2‐m at pH 2.5 have marginal α‐helical propensity at regions close to the C‐terminal cysteine, although it is a β‐sheet protein in the native state. The results suggest that the reduced mobility of the denatured state is an important factor for the amylodogenic potential of β2‐m, and that the marginal helical propensity at the C‐terminal regions might play a role in modifying this potential.
Protein stability indicates divergent evolution of PD‐(D/E)XK type II restriction endonucleases
Authors: Fuxreiter Monika , Simon István
Published: October 2002

Type II restriction endonucleases recognize 4–8 base‐pair‐long DNA sequences and catalyze their cleavage with remarkable specificity. Crystal structures of the PD‐(DE)XK superfamily revealed a common α/β core motif and similar active site. In contrast, these enzymes show little sequence similarity and use different strategies to interact with their substrate DNA. The intriguing question is whether this enzyme family could have evolved from a common origin. In our present work, protein structure stability elements were analyzed and compared in three parts of PD‐(DE)XK type II restriction endonucleases: (1) core motif, (2) active‐site residues, and (3) residues playing role in DNA recognition. High correlation was found between the active‐site residues and those stabilization factors that contribute to preventing structural decay. DNA recognition sites were also observed to participate in stabilization centers. It indicates that recognition motifs and active sites in PD‐(DE)XK type II restriction endonucleases should have been evolutionary more conserved than other parts of the structure. Based on this observation it is proposed that PD‐(DE)XK type II restriction endonucleases have developed from a common ancestor with divergent evolution.
Patterns of intracellular and intercellular Ca2+ waves in the longitudinal muscle layer of the murine large intestine In vitro
Authors: Hennig Grant W. , Smith Christian B. , O'Shea Deirdre M. , Smith Terence K.
Published: October 2002

Ca2+ wave activity was monitored in the longitudinal (LM) layer of isolated murine caecum and proximal colon at 35 °C with fluo‐4 AM and an iCCD camera. Both intracellular (within LM cells) and intercellular (also spreading from cell to cell) Ca2+ waves were observed. Intracellular Ca2+ waves were associated with a lack of muscle movement whereas intercellular Ca2+ waves, which were five times more intense than intracellular waves, were often associated with localized contractions. Several intracellular Ca2+ waves were present at the same time in individual LM cells. Waves in adjacent LM cells were not coordinated and were unaffected by TTX (1 μM) but were blocked by IP3 receptor antagonists xestospongin‐C (Xe‐C; 2 μM) or 2‐aminoethyl diphenylborate (2‐APB; 25 μM), and by ryanodine (10 μM). Caffeine (5 mm) restored wave activity following blockade with Xe‐C. NiCl2 (1 mm) blocked intracellular Ca2+ waves, and nicardipine (2 μM) reduced their frequency and intensity, but did not affect their velocity, suggesting the sarcoplasmic reticulum may be fuelled by extracellular Ca2+ entry. Intercellular Ca2+ waves often occurred in bursts and propagated rapidly across sizeable regions of the LM layer and were blocked by heptanol (0.5 mm). Intercellular Ca2+ waves were dependent upon neural activity, external Ca2+ entry through L‐type Ca2+ channels, and amplification via calcium‐induced calcium release (CICR). In conclusion, intracellular Ca2+ waves, which may reduce muscle excitability, are confined to individual LM cells. They depend upon Ca2+ release from internal Ca2+ stores and are likely to be fuelled by extracellular Ca2+ entry. Intercellular Ca2+ waves, which are likely to underlie smooth muscle tone, mixing and propulsion, depend upon neural activity, muscle action potential propagation and amplification by CICR.
Long‐term study of Ca2+ homeostasis and of survival in collagenase‐isolated muscle fibres from normal and mdx mice
Authors: De Backer F. , Vandebrouck C. , Gailly P. , Gillis J. M.
Published: October 2002

Skeletal muscles of the mdx mouse lack dystrophin offering the possibility to study the role of intracellular Ca2+ ions in fibre degeneration. Flexor digitorum brevis muscles of 3‐month‐old mdx and normal mice were dissociated with collagenase; fibres were maintained in culture for 6 days (d0 to d5) and their survival was assessed. Cytosolic [Ca2+], passive Mn2+ influx (indicative of Ca2+ influx) and activity of mechanosensitive/voltage‐independent Ca2+ channels were studied over the same period. Survival of normal fibres declined steadily from d0 to d3, but an acceleration of fibre death occurred in mdx fibres from d1 to d2. This could be greatly reduced but not abolished by lowering external [Ca2+] 10‐fold. In the d0‐d5 period, both mdx and normal fibres showed transient increases of Mn2+ influx and activity of the Ca2+ channels; these peaked at d1 and disappeared by d3–d4. Increases were always significantly larger in mdx fibres. Altogether, over the 6 days, 130 paired measurements of [Ca2+]i and Mn2+ influx were made on 68 fibres from mdx and 62 fibres from normal mice. In 90 % of the fibres, [Ca2+]i remained within the 25–85 nm limits while Mn2+ influx varied more than 10‐fold. The median for Mn2+ influx was 45 % greater in fibres from mdx mice than in fibres from control C57 mice. However, there was no significant difference between [Ca2+]i medians in fibres from normal and mdx mice. Addition of 25–75 nm of a Ca2+ ionophore (4‐bromo‐A23187) to the medium did not affect the level of cytosolic [Ca2+] in both types of fibres, while markedly increasing the rate of Mn2+ influx, as expected. Thus, Ca2+ homeostasis was equally robust in mdx and normal fibres. The remaining 10 % of the fibres showed, at d1, high levels of Mn2+ influx and/or elevated [Ca2+]i above 100 nm. This did not affect survival of normal fibres but was probably responsible of the increased death rate in mdx fibres.
Excitation of smooth muscles isolated from the guinea‐pig gastric antrum in response to depolarization
Authors: Kito Yoshihiko , Fukuta Hiroyasu , Yamamoto Yoshimichi , Suzuki Hikaru
Published: October 2002

In small segments of circular smooth muscle bundle isolated from the guinea‐pig gastric antrum, depolarization of the tissue with intracellular current stimuli evoked regenerative slow potentials after a refractory period of 5–10 s. The refractory period changed inversely with the amplitude and duration of the stimulating depolarization. Thapsigargin (an inhibitor of calcium‐ATPase at internal stores), 2‐aminoethoxydiphenyl borate (2‐APB, an inhibitor of inositol 1,4,5‐trisphosphate (IP3)‐receptor‐mediated Ca2+ release), and carbonyl cyanide m‐chlorophenyl‐hydrazone (a mitochondrial protonophore) reduced the amplitude of slow potentials, with no significant alteration of the refractory period. Bisindolylmaleimide I or chelerythrine (inhibitors of protein kinase C, PKC) increased the refractory period and inhibited the amplitude of slow potentials. These results indicate that the refractory period and amplitude of slow potentials are related to the activation of PKC and the amount of Ca2+ released from the internal stores through activation of IP3 receptors, respectively. Acetylcholine (ACh) reduced the refractory period and increased the amplitude of slow potentials: the former was antagonized by chelerythrine and the latter by 2‐APB. The results suggest that ACh has dual actions; stimulation of the metabolism of inositol phosphate and activation of PKC. Phorbol‐12‐myristate‐13‐acetate, a selective stimulant of PKC, at low concentrations (< 10 nm) mimicked the actions of ACh and at high concentrations reduced the frequency of slow potentials and increased the refractory period. The possible involvement of the concentration‐dependent differences in the actions of phorbol ester on the translocation of PKC was considered.
Nitric oxide‐induced Cl− secretion in isolated rat colon is mediated by the release of thromboxane A2
Authors: Sakai Hideki , Suzuki Tomoyuki , Murota Miki , Takahashi Yuji , Takeguchi Noriaki
Published: October 2002

We have shown previously that thromboxane A2 (TXA2), which may be released by the anti‐tumour drug irinotecan and by platelet‐activating factor (PAF), causes Cl− secretion in the isolated rat colon. In the present study, the involvement of TXA2 in nitric oxide‐induced Cl− secretion in isolated rat colon was investigated. In colonic mucosa set between Ussing chambers, the NO donor sodium nitroprusside (SNP; 100 μM) caused Cl− secretion, an effect that was almost completely inhibited by the NO scavenger carboxy‐PTIO at 200 μM. The SNP‐induced Cl− secretion was inhibited in a concentration‐dependent manner by the TXA2 receptor antagonist ONO‐3708 (IC50= 2 μM) and the TX synthase inhibitor Y‐20811 (IC50= 0.4 μM). SNP significantly increased the release of TXA2 (measured as TXB2 release) from the mucosa. The SNP‐induced increases in Cl− secretion and TXA2 release were blocked by a NO‐sensitive guanylate cyclase inhibitor (ODQ). Dibutyryl cGMP (500 μM) also induced Cl− secretion, which was sensitive to ONO‐3708 (10 μM) and Y‐20811 (1 μM), and increased the release of TXA2 from the mucosa. PAF‐induced (10 μM) Cl− secretion was inhibited by carboxy‐PTIO (200 μM) and ODQ (10 μM), whereas irinotecan‐induced (500 μM) Cl− secretion was not significantly inhibited by these drugs. A stable TXA2 analogue (STA2) but not SNP (100 μM) changed the membrane potential of epithelial cells in isolated colonic crypts under the whole‐cell current‐clamp condition. These results indicate that PAF elicits the NO‐cGMP pathway and then stimulates the release of TXA2, which is a stimulant of colonic Cl− secretion. In contrast, the NO‐cGMP pathway is not involved in the TXA2‐mediated Cl− secretion induced by irinotecan.
Presynaptic and postsynaptic mechanisms underlie paired pulse depression at single GABAergic boutons in rat collicular cultures
Authors: Kirischuk Sergei , Clements John D. , Grantyn Rosemarie
Published: October 2002

Paired pulse depression (PPD) is a common form of short‐term synaptic plasticity. The aim of this study was to characterise PPD at the level of a single inhibitory bouton. Low‐density collicular cultures were loaded with the Ca2+ indicator Oregon Green‐1, active boutons were stained with RH414, and action potentials were blocked with TTX. Evoked IPSCs (eIPSCs) and presynaptic Ca2+ transients were recorded in response to direct presynaptic depolarisation of an individual bouton. The single bouton eIPSCs had a low failure rate (< 0.1), large average quantal content (3‐6) and slow decay (τ1= 15 ms, τ2= 81 ms). The PPD of eIPSCs had two distinct components: PPDfast and PPDslow (τ= 86 ms and 2 s). PPDslow showed no dependence on extracellular Ca2+ concentration, or on the first eIPSC's failure rate or amplitude. Most probably, it reflects a release‐independent inhibition of exocytosis. PPDfast was only observed in normal or elevated Ca2+. It decreased with the failure rate and increased with the amplitude of the first eIPSC. It coincided with paired pulse depression of the presynaptic Ca2+ transients (τ= 120 ms). The decay of the latter was accelerated by EGTA, which also reduced PPDfast. Therefore, a suppressive effect of residual presynaptic Ca2+ on subsequent Ca2+ influx is considered the most likely cause of PPDfast. PPDfast may also have a postsynaptic component, because exposure to a low‐affinity GABAA receptor antagonist (TPMPA; 300 μM) counteracted PPDfast, and asynchronous IPSC amplitudes were depressed for a short interval following an eIPSC. Thus, at these synapses, PPD is produced by at least two release‐independent presynaptic mechanisms and one release‐dependent postsynaptic mechanism.
Interleukin‐6 release from the human brain during prolonged exercise
Authors: Nybo Lars , Nielsen Bodil , Klarlund Pedersen Bente , Møller Kirsten , Secher Niels H.
Published: October 2002

Interleukin (IL)‐6 is a pleiotropic cytokine, which has a variety of physiological roles including functions within the central nervous system. Circulating IL‐6 increases markedly during exercise, partly due to the release of IL‐6 from the contracting skeletal muscles, and exercise‐induced IL‐6 may be linked with central fatigue, which is enhanced by hyperthermia. Exercise‐induced IL‐6 may also stimulate hepatic glycogenolysis, which is important during prolonged and repeated exercise. Thus, in a randomised order and separated by 60 min of rest, eight young male subjects completed two 60 min exercise bouts: one bout with a normal (38 °C) and the other with an elevated (39.5 °C) core temperature. The cerebral IL‐6 response was determined on the basis of internal jugular venous to arterial IL‐6 differences and global cerebral blood flow. There was no net release or uptake of IL‐6 in the brain at rest or after 15 min of exercise, but a small release of IL‐6 was observed after 60 min of exercise in the first bout (0.06 ± 0.03 ng min−1). This release of IL‐6 from the brain was five‐fold greater at the end of the second bout (0.30 ± 0.08 ng min−1; P < 0.05) with no separate influence of hyperthermia. In conclusion, IL‐6 is released from the brain during prolonged exercise in humans and it appears that the duration of the exercise rather than the increase in body temperature dictates the cerebral IL‐6 response.
CFD Calculation of a Mixed Flow Pump Characteristic From Shutoff to Maximum Flow
Authors: Muggli Felix A. , Holbein Peter , Dupont Philippe
Published: October 2002

The behavior of the flow in a vertical semi-axial mixed flow pump has been analyzed by numerical flow simulations of the entire stage, and the results have been compared to test data. As the flow is expected to be unsteady at part load in such a pump, the steady-state simulations were complemented with unsteady flow simulations of the entire machine at one part load operating point. Pressure measurements at different locations in the casing of the pump provided valuable data for the validation of the calculated pressure head. This paper shows that the pump characteristic can be quite accurately predicted from full load to part load by modern numerical tools. Simulations of the unsteady flow, which use much more computer resources, are also feasible in an industrial environment and yield detailed information about the flow patterns and pressure fluctuations in the pump.
Design Synthesis of Path Generating Compliant Mechanisms by Evolutionary Optimization of Topology and Shape
Authors: Tai Kang , Cui Guang Yu , Ray Tapabrata
Published: October 2002

This work demonstrates the successful synthesis of path generating compliant mechanisms by the process of topology and shape design optimization. As geometric topology variation of continuum structures is difficult to treat and analysis of the displacement path or trajectory of such structures is computationally intensive, a highly effective and efficient optimal design procedure is needed. This paper describes the use of a recently developed morphological geometric representation scheme coupled with an evolutionary algorithm to synthesize the mechanism. The scheme uses arrangements of skeleton and “flesh” to define structural geometry, which facilitates transmission of topological/shape characteristics across generations in the evolutionary process and will not render any geometrically invalid designs. The evolutionary algorithm solves the problem as a discrete optimization problem, with a proficient constraint handling capability.
Methods to Estimate the Rock Strength and Tooth Wear While Drilling With Roller-Bits—Part 1: Milled-Tooth Bits
Authors: Karasawa H. , Ohno T. , Kosugi M. , Rowley J. C.
Published: October 2002

This paper proposes new methods to estimate both the rock strength and tooth wear while drilling with roller-bits. Laboratory drilling tests were conducted to obtain the penetration rate, bit weight and torque using milled-tooth bits with different tooth wear (T0, T4, T7). Drilling media used for the tests were soft to medium-hard rocks whose uniaxial compressive strength ranged from 14 to 118 MPa. Based on the test results, a parameter, which presents the rock strength independent of the tooth wear, was first investigated. The investigation revealed that a parameter related to the axial energy and the rotary energy required to drill rock is effective to estimate the rock strength independent of the tooth wear. Second, methods to estimate the tooth wear were studied based on the same parameter that represents the rock strength. From the results of this study, methods to measure the tooth wear are proposed.
H ∞ Synthesis of Nonlinear Feedback Systems in a Volterra Representation
Authors: Glass J. W. , Franchek M. A.
Published: October 2002

Presented in this paper is a procedure for selecting the H∞ performance weights for a class of nonlinear feedback systems modeled by a truncated Volterra series. The performance weight selection addresses the trade-offs between closed-loop performance and closed-loop stability. The class of feedback systems considered are regulating systems subject to step disturbances. The methodology is based on two recent results concerning closed loop stability for this class of nonlinear feedback systems. These stability conditions can be formulated as an H∞ problem where the performance weights are systematically identified. This synthesis procedure is applied to the idle speed control of a Ford 4.6L V-8 fuel injection engine. Through this application, the design trade-off between closed-loop performance and stability is illustrated.
Quad-Layer: Layered Quadrilateral Meshing of Narrow Two-Dimensional Domains by Bubble Packing and Chordal Axis Transformation
Authors: Yamakawa Soji , Shimada Kenji
Published: October 2002

This paper presents a computational method for quadrilateral meshing of a thin, or narrow, two-dimensional domain for finite element analysis. The proposed method creates a well-shaped single-layered, multi-layered, or partially multi-layered quadrilateral mesh. Element sizes can be uniform or graded. A high quality, layered quadrilateral mesh is often required for finite element analysis of a narrow two-dimensional domain with a large deformation such as in the analysis of rubber deformation or sheet metal forming. Fully automated quadrilateral meshing is performed in two stages: (1) extraction of the skeleton of a given domain by discrete chordal axis transformation, and (2) discretization of the chordal axis into a set of line segments and conversion of each of the line segments to a single quadrilateral element or multiple layers of quadrilateral elements. In each step a physically-based computational method called bubble packing is applied to discretize a curve into a set of line segments of specified sizes. Experiments show that the accuracy of a large-deformation FEM analysis can be significantly improved by using a well-shaped quadrilateral mesh created by the proposed method.