bluuebunny/crossref_metadata_2025 huggingface dataset:
Market entry decisions in China
Authors: Reid David McHardy , Walsh John
Published: April 2004

Since the inception of the open‐door policy, in 1978, China has acted as a magnet for firms wishing to
locate there to gain access to the billion plus consumers. However, the scramble to establish a presence in the
country has led firms to make a number of hasty decisions, to revise their plans, and in some cases, to withdraw
from the market. Research in the PRC, primarily in Shanghai, among leading international consumer goods firms
has revealed a variety of shortcomings in our practical understanding of the internationalization process and
the theoretical underpinning of the market entry decisions that emerge. It has also yielded insights into how
adaptation to the advancing PRC business environment may be successfully effected. © 2003 Wiley
Periodicals, Inc.
Kenya
Authors: Toroitich Oliver K. , Debrah Yaw A.
Published: April 2004

Arguably, Kenya is the “darling” of tourists who are brave enough to “explore” Africa
and to find out for themselves the myths and reality of life on a continent that always receives negative media
report overseas. Other tourists are attracted to Kenya because Hollywood movies based on Africa and set in Kenya
(e.g., Out of Africa) have depicted the romanticized life of Europeans/White
settlers/expatriates in the “deep” and “remote” African frontier. Thus, those with a
stereotypical picture of Africa acquired from Western television screens expect to find an extremely undeveloped
country. But arrival at Jomo Kenyatta International airport in Nairobi reveals a relatively well‐developed
city that is very much integrated into the world capitalist system/economy. With a relatively strong economy
Kenya has been successful in attracting both tourists and foreign investors. This article attempts to provide a
clearer picture of the business climate in the country. Among other issues, it highlights the fact that the
country has enjoyed peace and political stability while many of its neighbors remain deeply entrenched in wars
and ethnic conflicts. This has resulted in some degree of economic prosperity that has eluded many countries in
sub‐Saharan Africa. While highlighting the numerous investment opportunities especially in the
telecommunications sector, the article also sheds light on some of the challenges that confront foreign investors
and how to tackle them. © 2003 Wiley Periodicals, Inc.
Motives for foreign direct investment in the United States
Authors: Zitta Steven J. , Powers Thomas L.
Published: April 2004

As foreign direct investment becomes an important element of the global marketplace it is important to
understand why firms use FDI as an investment option. This article reports research that identifies motivations
that influence factor‐ and market‐seeking inward FDI in the United States. Based on a study of 127
foreign companies investing in the United States, the research identifies motivators that differentiate and
explain these two types of investment behaviors. © 2003 Wiley Periodicals, Inc.
The gas pedal and the brake … toward a global balance of diverging cultural determinants in managerial mindsets
Authors: Beer Lawrence A.
Published: April 2004

Is the American culturally driven inclination for short‐term results and constant change coupled with a
focus on the individual as opposed to the group influencing the way transnational corporate business is
conducted? Such an issue is raised via an introduction to cultural determinants as theorized by the
underlying research of Hofstede, Tromprenaars, and Hampden‐Turrner regarding time orientation as well as
the contrasting of American and foreign value systems in respect to change acceptance as driven by the culturally
limiting national mind set of the company manager. Using global, publicly traded companies and the pressure on
them for quarterly performance attainment as fueled by the Street, financial analysts, the effect of the American
culturally driven need to continually show financial progress at abbreviated intervals or provide for some
pronounced judicious changes in the interim may have begun to spiral out to managerial decision making around the
world. Such phenomenon may begin to upset the historic balance that the polar culturally based applications of
time, change, and decision making provided the historic global commercial environment. Such proposed effect could
also influence traditional worldwide economic and social development. This concept is raised but not empirically
answered, and is offered to stimulate further evaluation and research into the potential conflict in international
decision making when opposite mindsets collide. © 2003 Wiley Periodicals, Inc.
Economic malaise in Japan: Changing economic order and the future of the U.S.–Japan economic relationship
Authors: Messerschmidt Dan C.
Published: April 2004

B. Gao. 2001. Japan's Economic Dilemma: The Institutional Origins of Prosperity and
Stagnation.
Cambridge: Cambridge University Press. 300 pages; ISBN 0‐521‐79025‐5.

F. Bergsten, T. Ito, & M. Noland. 2001. No More Bashing: Building a New Japan‐United States
Economic Relationship.
Washington, DC: Institute for International Economics. 328 pages; ISBN 0‐88132‐286‐5.
Consequences of growth variation in northern Baltic herring for assessment and management
Authors: Rahikainen Mika , Stephenson Robert L.
Published: April 2004

Growth rates of herring in the northern Baltic Sea differ among areas, and have changed substantially over time in some areas in response to environmental change. Weight-at-age of adult herring in some areas of the Finnish herring fishery fluctuated by as much as 60% over the past three decades. Elsewhere, there have been similar but more subtle changes. Growth variation has implications for stock assessment and management: differences suggest a need for considering a smaller spatial structure, at least at the scale of the ICES subdivision, in the case of northern Baltic herring. Changes in growth have an impact on the calculation and use of common biological reference points, and erode the capability of yield projections beyond the short term. Investigation of the impact of growth variation on common reference points of northern Baltic herring revealed that F0.1 was a robust reference despite the marked variability in growth, whereas Fx%SPR (e.g. F35%SPR) was less robust, depending on the definition of maximum spawning-per-recruit. Herring in different areas of the northern Baltic Sea probably require different reference points and possibly different management strategies, as a consequence of differences and variability in growth characteristics.
Infections and some other conditions affecting the skin and subcutis of the naso-ocular region of cats—Clinical experience 1987–2003
Authors: Malik Richard , Vogelnest Linda , O'Brien Carolyn R. , White Joanna , Hawke Christine , Wigney Denise I. , Martin Patricia , Norris Jacqueline M.
Published: April 2004

Infections of the skin or subcutis of the naso-ocular region develop through two mechanisms. Cases with lesions but without concomitant signs of nasal disease probably result from cat scratch injuries. Under certain circumstances, such lacerations result in the introduction of saprophytic microorganisms in such large numbers that host defence mechanisms are overwhelmed. This results in localised, variably invasive, disease in an otherwise immunocompetent host. An unpredictable range of organisms can give rise to such infections including a variety of fungal and bacterial genera. Causal organisms will likely vary from one geography to another as a result of differences in soil type and related environmental factors. Accordingly, procurement of appropriate tissue specimens for culture and susceptibility testing is essential to guide therapy, as these cases require medical and sometimes surgical intervention in order to effect a favourable outcome. In contrast, patients with naso-ocular lesions and concurrent signs of nasal disease have a different pathogenesis. Primary infection of the sinonasal region likely results from the inhalation of infectious propagules, with the infection subsequently penetrating overlying bones to invade the subcutaneous space. These lesions are typically the result of cryptococcosis or aspergillosis and must be distinguished from invasive nasal malignancies. An approach to the investigation and treatment of these patients is presented together with photographs of representative cases.
Further developments in closure experiments for surface diffuse irradiance under cloud‐free skies at a continental site
Authors: Halthore Rangasayi N. , Miller Mark A. , Ogren John A. , Sheridan Patrick J. , Slater Donald W. , Stoffel Thomas
Published: April 2004

Direct and diffuse surface irradiances measured with the shaded and ventilated Black and White Eppley pyranometer under cloud‐free skies at a continental site are compared with radiative transfer model calculated values. For the measurements in 2002, calculations of irradiance utilizing surface‐measured aerosol single scattering albedo (SSA) agree with that measured to within the mutual uncertainties. For the days in 2001 and 2003, the modeled irradiance values agree with that measured only if highly absorbing aerosols of SSA ∼0.7, well below surface‐measured SSA, are used. Modeled diffuse/direct irradiance ratios continue to exceed those measured in the visible and near‐IR using silicon detector‐based radiometers, suggesting problems with measurements and/or model estimates of atmospheric, most likely, aerosol, absorption. A detailed study is therefore required to establish the role of aerosol absorption in shortwave radiative transfer.
Noise in two‐color electronic distance meter measurements revisited
Authors: Langbein John
Published: April 2004

Frequent, high‐precision geodetic data have temporally correlated errors. Temporal correlations directly affect both the estimate of rate and its standard error; the rate of deformation is a key product from geodetic measurements made in tectonically active areas. Various models of temporally correlated errors are developed and these provide relations between the power spectral density and the data covariance matrix. These relations are applied to two‐color electronic distance meter (EDM) measurements made frequently in California over the past 15–20 years. Previous analysis indicated that these data have significant random walk error. Analysis using the noise models developed here indicates that the random walk model is valid for about 30% of the data. A second 30% of the data can be better modeled with power law noise with a spectral index between 1 and 2, while another 30% of the data can be modeled with a combination of band‐pass‐filtered plus random walk noise. The remaining 10% of the data can be best modeled as a combination of band‐pass‐filtered plus power law noise. This band‐pass‐filtered noise is a product of an annual cycle that leaks into adjacent frequency bands. For time spans of more than 1 year these more complex noise models indicate that the precision in rate estimates is better than that inferred by just the simpler, random walk model of noise.
A numerical study on global propagations and amplitude growths of large‐scale gravity wave packets
Authors: Zhang Shaodong , Yi Fan
Published: April 2004

By using a full‐implicit‐continuous‐Eulerian (FICE) scheme and taking a set of basic atmospheric motion equations in spherical coordinates as governing equations, a fully nonlinear numerical model for the dynamics of the middle and upper atmosphere is established to numerically study the nonlinear global propagations and amplitude growths of large‐scale gravity wave packets. The simulation results show that the newly established model can successfully exhibit the essential characteristics of the global propagations of gravity waves: wave energies propagate upward along the ray paths derived from the linear gravity wave theory, and wave disturbance amplitudes increase with the increasing heights. During the upward propagation of gravity wave packets, there occurs evident enhancement of background wind along the propagation direction. Moreover, our simulation demonstrates that Earth rotation has little influences on the wave energy propagations and amplitude growths. However, the primary nonlinear curvature terms ( tan and − tanθ) play an important role in the growths of gravity wave amplitudes, which causes a evident latitudinal dependence of wave amplitude growth. In the Northern Hemisphere, with the same spatial scales and initial zonal disturbance amplitudes, the waves propagating in lower‐latitude regions have larger disturbance amplitudes.
Solar influence on the O(1D) dayglow emission rate: Global‐scale measurements by WINDII on UARS
Authors: Zhang S. P. , Shepherd G. G.
Published: April 2004

More than 130,000 emission rate profiles of the O(1D) dayglow (630 nm) were observed by WINDII during 1991–1995, which provide an unprecedented resource for studying the O(1D) layer, and related physics and chemistry in the thermosphere and ionosphere. This article describes the general properties of the O(1D) emission layer and an empirical model derived from WINDII measurements for the O(1D) emission rate as a function of the solar zenith angle and solar irradiance using the F10.7 cm flux as a proxy.
Location of the Carlsberg Fault zone from seismic controlled‐source fan recordings
Authors: Nielsen Lars , Thybo Hans
Published: April 2004

We locate the concealed Carlsberg Fault zone in the city of Copenhagen from seismic fan recordings. The fault is part of a fault system close to the border between the Danish Basin and the Baltic Shield. Recent earthquakes indicate that this area is tectonically active. The fault zone is a seismic low‐velocity zone. Fan shots were recorded on three receiver arrays (1.5–2.4 km long arcs) across the fault. Sources were placed inside and up to ∼500 m away from the ∼400–700 m wide fault zone at offsets of up to ∼7 km. Shots inside the fault zone show: 1) weak, delayed first arrivals inside the fault zone; 2) stronger first arrivals outside the fault zone; 3) guided waves inside the fault zone. The fault is a shadow zone for shots detonated outside the fault zone. Our approach facilitates fault mapping in densely urbanized areas where seismic profiling is not feasible.
Cell signalling in vascular cells exposed to cyclic strain: the emerging role of protein phosphatases
Authors: Lee Taeseung , Sumpio Bauer E.
Published: April 2004

Phosphorylation of tyrosine or serine/threonine residues of intracellular proteins is a reversible and dynamic process which is essential in controlling cellular growth, migration and survival. The phosphorylation states of numerous intermediary signalling proteins are governed by the opposing activities of protein kinases and phosphatases. Abnormal phosphorylation states have been linked with many human diseases, including cancer, diabetes, hypertension and cardiac hypertrophy. Recently, several reports have described the role of phosphatases in regulating critical cellular functions and signalling pathways in vascular cells. This Review will focus on the significance of several of these phosphatases and present information on the role of protein phosphatase type 2a in endothelial cells exposed to haemodynamic forces.
Shear‐stress preconditioning and tissue‐engineering‐based paradigms for generating arterial substitutes
Authors: Baguneid Mohamed , Murray David , Salacinski Henryk J. , Fuller Barry , Hamilton George , Walker Michael , Seifalian Alexander M.
Published: April 2004

In situ tissue engineering using shear‐stress preconditioning and adhesive biomolecules is a new approach to autologous tissue engineering. In the present study, novel tissue‐engineering grafts (TEGs) were preconditioned within an in vitro pulsatile flow circuit, with and without the addition of fibronectin (FN), to establish whether low‐shear‐stress conditions promoted endothelial cell (EC) retention and differentiation. TEGs (n=24) were generated by the contraction and compaction of collagen(I) by porcine aortic smooth‐muscle cells (SMCs) on to a compliant polyester graft scaffold. ECs were radiolabelled with [111In]indium tropolonate and seeded on to the luminal surface of the TEGs. Following organ culture in a bioreactor (7 days), TEGs were split into four groups (n=six TEGs per group): Group A acted as controls with TEGs unmodified and seeded with radiolabelled ECs; Group B underwent luminal pre‐coating with FN (75 μg/ml) prior to EC seeding; Group C underwent preconditioning within a pulsatile flow circuit at 10–20 μN (1–2 dyn)/cm2 for 7 days prior to EC seeding, and Group D TEGs were preconditioned for 7 days at 1–2 dyn/cm2, followed by luminal pre‐coating with FN prior to EC seeding. The resistance to physiological shear stress of the seeded ECs was assessed using a γ‐radiation counter within a physiological flow circuit producing an arterial waveform with a mean shear stress of 93.2 μN (9.32 dyn)/cm2. Environmental scanning electron microscopy (ESEM) was used to determine the distribution and degree of differentiation of the attached Ecs, and tissue‐type‐plasminogen‐activator (tPA) assays provided a measure of function and viability. EC resistance to shear stress at 93.2 μN/cm2 was significantly enhanced by a period of preconditioning (Group C) at 10–20 μN/cm2, surface modification with FN (Group B), or both (Group D) when compared with control grafts (Group A). However, TEGs coated with FN whether preconditioned (Group D) or not (Group B) demonstrated the best results for EC retention. ESEM demonstrated near‐confluent differentiated flattened ECs in both these cases. EC function was demonstrated by a steady increase in tPA production. Low‐shear‐stress preconditioning of TEGs enhances EC retention in vitro with an additional advantage demonstrated by pre‐treatment with FN prior to endothelialization. These findings may be exploited in the development of tissue‐engineered constructs to maintain a confluent endothelial lining.
Grafts of microencapsulated pancreatic islet cells for the therapy of diabetes mellitus in non‐immunosuppressed animals
Authors: Calafiore Riccardo , Basta Giuseppe , Luca Giovanni , Calvitti Mario , Calabrese Giuseppe , Racanicchi Leda , Macchiarulo Giacomo , Mancuso Francesca , Guido Lucia , Brunetti Paolo
Published: April 2004

Pancreatic‐islet‐cell transplantation may reverse hyperglycaemia in diabetic recipients that undertake general pharmacological immunosuppression. A major challenge that remains is the need to avoid immunosuppression associated with the use of allogeneic or heterologous islet cells. In the present study we demonstrate the use of microencapsulation of cells using artificial biocompatible and permselective membranes prepared with alginic acid derivatives and polyamino acids. While characterization of the microcapsule constituent polymers continues to progress, other technical issues such as definition of the immunobarrier capacity, biocompatibility, size, shape and graft site have come into sharper focus. Assessment of microcapsules properties, in order to establish possible guidelines for fabrication of reproducible membranes, and results from both in vitro functional testing, and in vivo encapsulated‐islet‐transplant outcome in several animal models of diabetes are reported.
Mutations in and near the second calcium-binding domain of integrin alphaIIb affect the structure and function of integrin alphaIIbbeta3
Authors: GIDWITZ Susan , TEMPLE Brenda , WHITE Gilbert C.
Published: April 2004

Calcium-binding domains in the α-subunit of integrins contain a central loop structure. To examine the importance of the loop structure, a series of αIIb mutants containing changes to the calcium-liganding amino acids have been constructed. Significantly, none of the mutant αIIbβ3 complexes was detected on the surface of transfected cells, but mutant pro-αIIb was detected in cell lysates in complex with β3. To study the importance of the regions flanking the second calcium-binding domain for ligand-binding and ligand-binding specificity, three αIIb/α5 chimaeras containing α5 sequences flanking or flanking and including the second calcium-binding domain were constructed. The chimaera containing both α5-flanking regions was not expressed on the cell surface, but FR1 and FR2, substituting either the first or second flanking region, were expressed. FR1β3-transfected cells lost the ability to adhere to fibrinogen and to support aggregation and had minimal fibrinogen-binding ability. The heterodimer complex was less stable than the wild-type. FR2β3-transfected cells adhered to fibrinogen and bound soluble fibrinogen with higher affinity when compared with wild-type. In addition, the heterodimer complex was more stable than wild-type. These results indicate that the conformation of the second calcium-binding domain is critical for maturation of the αIIbβ3 complex and expression on the cell surface and that the surrounding sequences are critical for αIIbβ3 function.
Proline residues in transmembrane segment IV are critical for activity, expression and targeting of the Na+/H+ exchanger isoform 1
Authors: SLEPKOV Emily R. , CHOW Signy , LEMIEUX M. Joanne , FLIEGEL Larry
Published: April 2004

NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167→Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE.
Studies on regulation of IGF (insulin-like growth factor)-binding protein (IGFBP) 4 proteolysis by pregnancy-associated plasma protein-A (PAPP-A) in cells treated with phorbol ester
Authors: SIVANANDAM Arun S. , MOHAN Subburaman , KITA Hirohito , KAPUR Sanjay , CHEN Shin-Tai , LINKHART Thomas A. , BAGI Gyorgy , BAYLINK David J. , QIN Xuezhong
Published: April 2004

PAPP-A (pregnancy-associated plasma protein-A) is produced by hSFs (human skin fibroblasts) and hOBs (human osteoblasts) and enhances the mitogenic activity of IGFs (insulin-like growth factors) by degradation of IGFBP-4 (insulin-like growth factor-binding protein 4). PKC (protein kinase C) activation in these cells led to reduction in IGFBP-4 proteolysis. This study was undertaken to determine the mechanism by which activation of PKC suppresses IGFBP-4 proteolysis. Treatment of hSFs/hOBs with TPA (PMA; 100 nM) reduced IGFBP-4 proteolysis without significantly decreasing the PAPP-A level in the CM (conditioned medium). Immunodepletion of the proform of eosinophil major basic protein (proMBP), a known PAPP-A inhibitor, from CM of TPA-treated cells (TPA CM) failed to increase IGFBP-4 proteolytic activity. Transduction of hSFs with proMBP retrovirus increased the concentration of proMBP up to 30 ng/ml and led to a moderate reduction in IGFBP-4 proteolysis. In contrast, TPA treatment blocked IGFBP-4 proteolysis but failed to induce a detectable amount of proMBP in the CM. While proMBP overexpression led to the formation of a covalent proMBP–PAPP-A complex and reduced the migration of PAPP-A on SDS/PAGE, TPA treatment dose- and time-dependently increased the conversion of a ≈470 kDa PAPP-A form (PAPP-A470) to a ≈400 kDa PAPP-A form (PAPP-A400). Since unreduced PAPP-A400 co-migrated with the 400 kDa recombinant PAPP-A homodimer and since PAPP-A monomers from reduced PAPP-A470 and PAPP-A400 co-migrated on SDS/PAGE, conversion of PAPP-A470 to PAPP-A400 is unlikely to be caused by proteolytic cleavage of PAPP-A. Consistent with the data showing that the increase in the ratio of PAPP-A400/PAPP-A470 is correlated with the extent of reduction in IGFBP-4 proteolysis, partially purified PAPP-A400 exhibited a 4-fold reduction in IGFBP-4 proteolytic activity compared with PAPP-A470. These data suggest that a novel mechanism, namely conversion of PAPP-A470 to the less-active PAPP-A400, could account for the TPA-induced suppression of PAPP-A activity.
Relocalized redox-active lysosomal iron is an important mediator of oxidative-stress-induced DNA damage
Authors: KURZ Tino , LEAKE Alan , von ZGLINICKI Thomas , BRUNK Ulf T.
Published: April 2004

Oxidative damage to nuclear DNA is known to involve site-specific Fenton-type chemistry catalysed by redox-active iron or copper in the immediate vicinity of DNA. However, the presence of transition metals in the nucleus has not been shown convincingly. Recently, it was proposed that a major part of the cellular pool of loose iron is confined within the acidic vacuolar compartment [Yu, Persson, Eaton and Brunk (2003) Free Radical Biol. Med. 34, 1243–1252; Persson, Yu, Tirosh, Eaton and Brunk (2003) Free Radical Biol. Med. 34, 1295–1305]. Consequently, rupture of secondary lysosomes, as well as subsequent relocation of labile iron to the nucleus, could be an important intermediary step in the generation of oxidative damage to DNA. To test this concept we employed the potent iron chelator DFO (desferrioxamine) conjugated with starch to form an HMM-DFO (high-molecular-mass DFO complex). The HMM-DFO complex will enter cells only via fluid-phase endocytosis and remain within the acidic vacuolar compartment, thereby chelating redox-active iron exclusively inside the endosomal/lysosomal compartment. Both free DFO and HMM-DFO equally protected lysosomal-membrane integrity against H2O2-induced oxidative disruption. More importantly, both forms of DFO prevented H2O2-induced strand breaks in nuclear DNA, including telomeres. To exclude the possibility that lysosomal hydrolases, rather than iron, caused the observed DNA damage, limited lysosomal rupture was induced using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride; subsequently, hardly any DNA damage was found. These observations suggest that rapid oxidative damage to cellular DNA is minimal in the absence of redox-active iron and that oxidant-mediated DNA damage, observed in normal cells, is mainly derived from intralysosomal iron translocated to the nucleus after lysosomal rupture.
Heparan sulphate proteoglycans modulate fibroblast growth factor-2 binding through a lipid raft-mediated mechanism
Authors: CHU Chia Lin , BUCZEK-THOMAS J. Ann , NUGENT Matthew A.
Published: April 2004

We investigated how lipid raft association of HSPG (heparan sulphate proteoglycans) modulates FGF-2 (fibroblast growth factor-2/basic fibroblast growth factor) interactions with vascular smooth-muscle cells. When lipid rafts were disrupted with sterol-binding agents, methyl-β-cyclodextrin and filipin, FGF-2 binding to HSPG was reduced 2–5-fold, yet the amount and turnover of cell-surface HSPG were unaffected. Approx. 50–65% of bound FGF-2 was in lipid raft-associated fractions based on insolubility in cold Triton X-100 and flotation in OptiPrep density gradients, and this level was increased with higher FGF-2 concentrations. Less FGF-2 (50–90%) was associated in raft fractions when cholesterol was depleted or HSPG were degraded with heparinase III. To investigate how lipid raft–HSPG interactions altered binding, we compared the rates of FGF-2 dissociation with native, MβCD (methyl-β-cyclodextrin)- and filipin-treated cells. We found that FGF-2 dissociation rates were increased when lipid rafts were disrupted. These results suggest that localization of HSPG within lipid rafts creates high local concentrations of binding sites such that dissociation of FGF-2 is hindered. The localization of FGF-2 and HSPG to lipid rafts also correlated with the activation of protein kinase Cα. Thus raft association of HSPG might create growth factor traps resulting in increased binding and signal transduction to enhance cell sensitivity.
NapGH components of the periplasmic nitrate reductase of Escherichia coli K-12: location, topology and physiological roles in quinol oxidation and redox balancing
Authors: BRONDIJK T. Harma C. , NILAVONGSE Arjaree , FILENKO Nina , RICHARDSON David J. , COLE Jeffrey A.
Published: April 2004

Nap (periplasmic nitrate reductase) operons of many bacteria include four common, essential components, napD, napA, napB and napC (or a homologue of napC). In Escherichia coli there are three additional genes, napF, napG and napH, none of which are essential for Nap activity. We now show that deletion of either napG or napH almost abolished Nap-dependent nitrate reduction by strains defective in naphthoquinone synthesis. The residual rate of nitrate reduction (approx. 1% of that of napG+H+ strains) is sufficient to replace fumarate reduction in a redox-balancing role during growth by glucose fermentation. Western blotting combined with β-galactosidase and alkaline phosphatase fusion experiments established that NapH is an integral membrane protein with four transmembrane helices. Both the N- and C-termini as well as the two non-haem iron–sulphur centres are located in the cytoplasm. An N-terminal twin arginine motif was shown to be essential for NapG function, consistent with the expectation that NapG is secreted into the periplasm by the twin arginine translocation pathway. A bacterial two-hybrid system was used to show that NapH interacts, presumably on the cytoplasmic side of, or within, the membrane, with NapC. As expected for a periplasmic protein, no NapG interactions with NapC or NapH were detected in the cytoplasm. An in vitro quinol dehydrogenase assay was developed to show that both NapG and NapH are essential for rapid electron transfer from menadiol to the terminal NapAB complex. These new in vivo and in vitro results establish that NapG and NapH form a quinol dehydrogenase that couples electron transfer from the high midpoint redox potential ubiquinone–ubiquinol couple via NapC and NapB to NapA.
Inhibition of hydroxyapatite formation by osteopontin phosphopeptides
Authors: PAMPENA David A. , ROBERTSON Karen A. , LITVINOVA Olga , LAJOIE Gilles , GOLDBERG Harvey A. , HUNTER Graeme K.
Published: April 2004

Osteopontin (OPN) is an acidic phosphoglycoprotein that is believed to function in the prevention of soft tissue calcification. In vitro studies have shown that OPN can inhibit the formation of hydroxyapatite (HA) and other biologically relevant crystal phases, and that this inhibitory activity requires phosphorylation of the protein; however, it is not known which phosphorylated residues are involved. We have synthesized peptides corresponding to four phosphoserine-containing sequences in rat OPN: OPN7–17, containing phosphoserines 10 and 11; OPN41–52, containing phosphoserines 46 and 47; OPN248–264, containing phosphoserines 250, 257 and 262; and OPN290–301, containing phosphoserines 295–297. The abilities of these peptides to inhibit de novo HA formation were determined using a constant-composition autotitration assay. All four OPN phosphopeptides caused a dose-dependent increase in nucleation lag time, but did not significantly affect subsequent formation of the crystals. However, OPN41–52 (inhibitory constant 73.5 min/µM) and OPN290–301 (72.2 min/µM) were approx. 4 times more potent inhibitors than OPN7–17 (19.7 min/µM) and OPN247–264 (16.3 min/µM). ‘Scrambling’ the amino acid sequence of OPN290–301 resulted in decreased potency (45.6 min/µM), whereas omission of the phosphate groups from this peptide caused a greater decrease (5.20 min/µM). These findings have identified phosphorylated sequences that are important for the ability of rat bone OPN to inhibit HA crystal formation, and suggest that negative-charge density is an important factor in this activity.
HNF1alpha is involved in tissue-specific regulation of CFTR gene expression
Authors: MOUCHEL Nathalie , HENSTRA Sytse A. , McCARTHY Victoria A. , WILLIAMS Sarah H. , PHYLACTIDES Marios , HARRIS Ann
Published: April 2004

The CFTR (cystic fibrosis transmembrane conductance regulator) gene shows a complex pattern of expression with tissue-specific and temporal regulation. However, the genetic elements and transcription factors that control CFTR expression are largely unidentified. The CFTR promoter does not confer tissue specificity on gene expression, suggesting that there are regulatory elements outside the upstream region. Analysis of potential regulatory elements defined as DNase 1-hypersensitive sites within introns of the gene revealed multiple predicted binding sites for the HNF1α (hepatocyte nuclear factor 1α) transcription factor. HNF1α, which is expressed in many of the same epithelial cell types as CFTR and shows similar differentiation-dependent changes in gene expression, bound to these sites in vitro. Overexpression of heterologous HNF1α augmented CFTR transcription in vivo. In contrast, antisense inhibition of HNF1α transcription decreased the CFTR mRNA levels. Hnf1α knockout mice showed lower levels of CFTR mRNA in their small intestine in comparison with wild-type mice. This is the first report of a transcription factor, which confers tissue specificity on the expression of this important disease-associated gene.
Phosphorylation regulates nucleophosmin targeting to the centrosome during mitosis as detected by cross-reactive phosphorylation-specific MKK1/MKK2 antibodies
Authors: CHA Hyukjin , HANCOCK Chad , DANGI Surabhi , MAIGUEL Dony , CARRIER France , SHAPIRO Paul
Published: April 2004

Phosphorylation-specific antibodies provide a powerful tool for analysing the regulation and activity of proteins in the MAP (mitogen-activated protein) kinase and other signalling pathways. Using synchronized cells, it was observed that phosphorylation-specific antibodies developed against the active form of MKK1/MKK2 (MAP kinase kinase-1 and -2) reacted with a protein that was approx. 35 kDa during G2/M-phase of the cell cycle. Failure of the 35 kDa protein to react with phosphorylation-independent MKK1/MKK2 antibodies suggested that this protein was not related to MKK1 or MKK2. Thus the 35 kDa protein was isolated by immunoprecipitation with the phospho-MKK1/MKK2 antibody and identified by MS. Peptide sequence analysis revealed matches with NPM (nucleophosmin/B23), a phosphoprotein involved in nucleolar assembly, centrosome duplication and ribosome assembly and transport. Biochemical and immunocytochemistry analyses verified that the phospho-MKK1/MKK2 antibodies cross-reacted with NPM that was phosphorylated at Thr234 and Thr237 during G2/M-phase, which are the same sites that are targeted by Cdc2 (cell division cycle protein-2) during mitosis. Using phosphorylation site mutants, we show that phosphorylation of Thr234 and Thr237 is required for NPM immunoreactivity with the phospho-MKK1/MKK2 antibody. Moreover, phosphorylation of Thr234 and Thr237 was demonstrated to regulate NPM localization to the centrosome after nuclear envelope breakdown in mitotic cells. These findings reveal a new insight into the role of phosphorylation in regulating NPM targeting during mitosis. However, caution should be used when using commercially available phospho-MKK1/MKK2 antibodies to examine the regulation of MKK1/MKK2 during mitotic transitions, owing to their cross-reactivity with phosphorylated NPM at this time of the cell cycle.
Copper stimulates trafficking of a distinct pool of the Menkes copper ATPase (ATP7A) to the plasma membrane and diverts it into a rapid recycling pool
Authors: PASE Luke , VOSKOBOINIK Ilia , GREENOUGH Mark , CAMAKARIS James
Published: April 2004

MNK (Menkes copper-translocating P-type ATPase, or the Menkes protein; ATP7A) plays a key role in regulating copper homoeostasis in humans. MNK has been shown to have a dual role in the cell: it delivers copper to cuproenzymes in the Golgi compartment and effluxes excess copper from the cell. These roles can be achieved through copper-regulated trafficking of MNK. It has previously been shown to undergo trafficking from the trans-Golgi network to the plasma membrane in response to elevated copper concentrations, and to be endocytosed from the plasma membrane to the trans-Golgi network upon the removal of elevated copper. However, the fundamental question as to whether copper influences trafficking of MNK to or from the plasma membrane remained unanswered. In this study we utilized various methods of cell-surface biotinylation to attempt to resolve this issue. These studies suggest that copper induces trafficking of MNK to the plasma membrane but does not affect its rate of internalization from the plasma membrane. We also found that only a specific pool of MNK can traffic to the plasma membrane in response to elevated copper. Significantly, copper appeared to divert MNK into a fast-recycling pool and prevented it from recycling to the Golgi compartment, thus maintaining a high level of MNK in the proximity of the plasma membrane. These findings shed new light on the cell biology of MNK and the mechanism of copper homoeostasis in general.
A proteomic approach to identify early molecular targets of oxidative stress in human epithelial lens cells
Authors: PARON Igor , D'ELIA Angela , D'AMBROSIO Chiara , SCALONI Andrea , D'AURIZIO Federica , PRESCOTT Alan , DAMANTE Giuseppe , TELL Gianluca
Published: April 2004

Oxidative stress is one of the most relevant contributors of cataractogenesis. To identify early protein targets of oxidative stress in lens cells, we used a differential proteomics approach to CD5A human epithelial lens cells treated with 500 µM H2O2 for 30 min. This dose of H2O2 was assayed to induce efficiently a block of cellular proliferation and to activate the oxidative stress-early inducible transcription factor EGR-1 (early growth response gene product 1), previously reported as stimulated factor in a model of cataractogenesis [Nakajima, Nakajima, Fukiage, Azuma and Shearer (2002) Exp. Eye Res. 74, 231–236]. We identified nine proteins, which sensitively reacted to H2O2 treatment by using two-dimensional gel electrophoresis and matrix-assisted laserdesorption ionization–time-of-flight-MS. In addition to cytoskeletal proteins (tubulin 1α and vimentin) and enzymes (phosphoglycerate kinase 1, ATP synthase β, enolase α, nucleophosmin and heat-shock cognate 54 kDa protein), which presented quantitative differences in expression profiles, peroxiredoxin and glyceraldehyde 3-phosphate dehydrogenase showed changes in pI as a result of overoxidation. Mass-mapping experiments demonstrated the specific modification of peroxiredoxin I active-site cysteine into cysteic acid, thus providing an explanation for the increase in negative charge measured for this protein. With respect to other global differential approaches based on gene expression analysis, our results allowed us to identify novel molecular targets of oxidative stress in lens cells. These results indicate that a combination of different approaches is required for a complete functional understanding of the biological events triggered by oxidative stress.
Purification and biochemical characterization of the D6 chemokine receptor
Authors: BLACKBURN Paul E. , SIMPSON Clare V. , NIBBS Robert J. B. , O'HARA Maureen , BOOTH Rhona , POULOS Jemma , ISAACS Neil W. , GRAHAM Gerard J.
Published: April 2004

There is much interest in chemokine receptors as therapeutic targets in diseases such as AIDS, autoimmune and inflammatory disorders, and cancer. Hampering such studies is the lack of accurate three-dimensional structural models of these molecules. The CC-chemokine receptor D6 is expressed at exceptionally high levels in heterologous transfectants. Here we report the purification and biochemical characterization of milligram quantities of D6 protein from relatively small cultures of transfected mammalian cells. Importantly, purified D6 retains full functional activity, shown by displaceable binding of 125I-labelled MIP-1β (macrophage inflammatory protein-1β) and by complete binding of the receptor to a MIP-1α affinity column. In addition, we show that D6 is decorated on the N-terminus by N-linked glycosylation. Mutational analysis reveals that this glycosylation is dispensable for ligand binding and high expression in transfected cells. Metabolic labelling has revealed the receptor to also be sulphated and phosphorylated. Phosphorylation is ligand independent and is not enhanced by ligand binding and internalization, suggesting similarities with the viral chemokine receptor homologue US28. Like US28, an analysis of the full cellular complement of D6 in transfected cells indicates that >80% is found associated with intracellular vesicular structures. This may account for the high quantities of D6 that can be synthesized in these cells. These unusual properties of D6, and the biochemical characterization described here, leads the way towards work aimed at generating the three-dimensional structure of this seven-transmembrane-spanning receptor.
Pyridoxine biosynthesis in yeast: participation of ribose 5-phosphate ketol-isomerase
Authors: KONDO Hiroki , NAKAMURA Yoriko , DONG Yi-Xin , NIKAWA Jun-ichi , SUEDA Shinji
Published: April 2004

To identify the genes involved in pyridoxine synthesis in yeast, auxotrophic mutants were prepared. After transformation with a yeast genomic library, a transformant (A22t1) was obtained from one of the auxotrophs, A22, which lost the pyridoxine auxotrophy. From an analysis of the plasmid harboured in A22t1, the RKI1 gene coding for ribose 5-phosphate ketol-isomerase and residing on chromosome no. 15 was identified as the responsible gene. This notion was confirmed by gene disruption and tetrad analysis on a diploid prepared from the wild-type and the auxotroph. The site of mutation on the RKI1 gene was identified as position 566 with a transition from guanine to adenine, resulting in amino acid substitution of Arg-189 with lysine. The enzymic activity of the Arg189→Lys (R189K) mutant of ribose 5-phosphate ketolisomerase was 0.6% when compared with the wild-type enzyme. Loss of the structural integrity of the protein seems to be responsible for the greatly diminished activity, which eventually leads to a shortage of either ribose 5-phosphate or ribulose 5-phosphate as the starting or intermediary material for pyridoxine synthesis.
Over-expression of sterol-regulatory-element-binding protein-1c (SREBP1c) in rat pancreatic islets induces lipogenesis and decreases glucose-stimulated insulin release: modulation by 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR)
Authors: DIRAISON Frédérique , PARTON Laura , FERRÉ Pascal , FOUFELLE Fabienne , BRISCOE Celia P. , LECLERC Isabelle , RUTTER Guy A.
Published: April 2004

Accumulation of intracellular lipid by pancreatic islet β-cells has been proposed to inhibit normal glucose-regulated insulin secretion (‘glucolipotoxicity’). In the present study, we determine whether over-expression in rat islets of the lipogenic transcription factor SREBP1c (sterol-regulatory-element-binding protein-1c) affects insulin release, and whether changes in islet lipid content may be reversed by activation of AMPK (AMP-activated protein kinase). Infection with an adenovirus encoding the constitutively active nuclear fragment of SREBP1c resulted in expression of the protein in approx. 20% of islet cell nuclei, with a preference for β-cells at the islet periphery. Real-time PCR (TaqMan®) analysis showed that SREBP1c up-regulated the expression of FAS (fatty acid synthase; 6-fold), acetyl-CoA carboxylase-1 (2-fold), as well as peroxisomal-proliferator-activated receptor-γ (7-fold), uncoupling protein-2 (1.4-fold) and Bcl2 (B-cell lymphocytic-leukaemia proto-oncogene 2; 1.3-fold). By contrast, levels of pre-proinsulin, pancreatic duodenal homeobox-1, glucokinase and GLUT2 (glucose transporter isoform-2) mRNAs were unaltered. SREBP1c-transduced islets displayed a 3-fold increase in triacylglycerol content, decreased glucose oxidation and ATP levels, and a profound inhibition of glucose-, but not depolarisation-, induced insulin secretion. Culture of islets with the AMPK activator 5-amino-4-imidazolecarboxamide riboside decreased the expression of the endogenous SREBP1c and FAS genes, and reversed the effect of over-expressing active SREBP1c on FAS mRNA levels and cellular triacylglycerol content. We conclude that SREBP1c over-expression, even when confined to a subset of β-cells, leads to defective insulin secretion from islets and may contribute to some forms of Type II diabetes.
Ligand control of interaction in vivo between ecdysteroid receptor and ultraspiracle ligand-binding domain
Authors: BERGMAN Thomas , HENRICH Vincent C. , SCHLATTNER Uwe , LEZZI Markus
Published: April 2004

Ecdysteroids (Ecs) enhance the formation of Ec receptor–ultraspiracle protein (EcR–USP) heterodimers which regulate gene transcription. To study EcR–USP heterodimerization, fusion proteins were constructed from the LBDs (ligand-binding domains) of Drosophila EcR or USP and the activation or DNA-binding region of GAL4 respectively. Reporter gene (lacZ) activation was fully dependent on the co-expression of the two fusion proteins and thus constitutes a reliable measure for the interaction in vivo between the two LBDs in the yeast cell. To identify structures involved in heterodimerization, a total of 27 point mutations were generated in the EcR and USP LBDs at selected sites. Heterodimerization and its inducibility by ligand were mainly affected by mutations in the dimerization interface and in the ligand-binding pocket of EcR respectively. However, also mutations not located in these structures or even in the LBD of USP influenced ligand-dependent heterodimerization. Together with previously reported ligand-binding studies, the existence of such local, intra- and inter-molecular mutation effects suggest that ligand-induced dimerization results from a synergistic interaction between ligand-binding and heterodimerization functions in EcR LBD, and that it depends on global features of the LBDs of EcR and USP and on their mutual surface compatibility.