bluuebunny/crossref_metadata_2025 huggingface dataset:
Unravelling the competitiveness debate
Authors: Williams Evan , Macdonald Kenneth , Kind Vanessa
Published: October 2002

As the global marketplace has developed, businesses have become increasingly concerned about their ability to compete. The question of whether environmental regulation may be an influential factor in this respect has been the subject of considerable debate, much of which has focused on the costs associated with regulatory compliance. In a global sense many of the key points in this debate may be academic since it might be argued that companies operating under widely different market conditions should be expected to experience different levels of competitiveness. However, within the setting of the EU, where it is intended that industry should operate on a level playing field, the issue becomes much more important and the debate takes on greater significance. This paper considers the opposing sides of the argument in an attempt to gain a clearer indication of whether differences in regulation might lead to differences in competitiveness. In so doing, the paper examines the Porter hypothesis (Porter and van der Linde, 1995), which suggests that strict environmental regulation may be a spur to increased industrial competitiveness. After exploring the logic underlying the hypothesis, the paper considers various efforts to corroborate or refute the argument. On the surface, the hypothesis should be relatively simple to test with the question ‘does new environmental regulation result in a net increase in costs of production relative to those of a firm's competitors?’. Unfortunately this question has proved virtually insoluble, and in this paper we attempt to explore why this has proved to be such an intractable problem. Copyright © Crown Copyright 2002. Recorded with the permission of the Controller of Her Majesty's Stationery Office. Published by John Wiley & Sons, Ltd.
Characterisation of CuInS2/ Zn(Se,O)/ZnO solar cells as a function of Zn(Se,O) buffer deposition kinetics in a chemical bath
Authors: Chaparro A. M. , Gutiérrez M. T. , Herrero J. , Klaer J. , Romero M. J. , Al‐Jassim M. M.
Published: October 2002

Thin‐film solar cells of CuInS2/Zn(Se,O)/ZnO configuration have been studied from the point of view of their dependence on the Zn(Se,O) chemical bath deposition (CBD) conditions. The kinetics of deposition of the Zn(Se,O) buffer is followed during cell processing with a quartz crystal microbalance (QCM). Two different CBD growth mechanisms yield buffer layers with different properties. Under a predominant electroless deposition reaction, the resulting buffer layer has mixed ZnSe–ZnO composition. The solar cells with this buffer type show higher fill factor (FF) and lower open‐circuit voltage (Voc). Under a chemical growth regime, the buffer layer has higher ZnSe proportion, giving rise to cells with higher VOC, but lower FF and stability. The parameters of this second type of cell also show major dependence on illumination effects (light‐soaking effects). Electron‐beam‐induced current (EBIC) and cathodoluminescence (CL) measurements are carried out to characterise the CuInS2/Zn(Se,O) junctions formed under the two buffer growth regimes. Cross‐sectional EBIC shows a wider space charge region (SCR) than expected for p‐CuInS2 in contact with Zn(Se,O), and the p–n junction is driven within the CuInS2 phase. These results reflect a chemical modification of CuInS2, most probably caused by the ammonia of the bath solution. CL shows more defective interfaces when Zn(Se,O) is deposited under the chemical mechanism (slower deposition rate, hence longer contact time of the CuInS2 with the bath solution) than under the electroless kinetics (faster deposition rate). Copyright © 2002 John Wiley & Sons, Ltd.
Design code development for fibre‐reinforced polymer structures and repairs
Authors: Machida Atsuhiko , Maruyama Kyuichi
Published: October 2002

This paper presents the issues and solutions in developing design codes and standards for the use of fibre‐reinforced polymer (FRP)‐reinforced concrete structures. First, the codes for new construction, referring mainly to the Japan Society of Civil Engineers (JSCE) Recommendations for Design and Construction of Concrete Structures using CFRM (Continuous Fibre Reinforcing Materials), the Canadian Highway Bridge Design Code (CHBDC), and the American Concrete Institute (ACI) Committee 440 recommendations are outlined. Methods discussed are: structural analysis; determination of design values; examination of flexural and shear capacity; precautions to ensure ductility or deformability; and calculations of deformation and development length. Similarly, design codes developed for strengthening of concrete structures with FRP, namely the recommendations by the JSCE, ACI Committee 440, and the fib code are compared and discussed with respect to the ways in which FRP may contribute to improvement of the performance of structures in flexure, shear and ductility. Test methods for different material properties of FRP and the bond of FRP to concrete are also presented, with reference to the JSCE recommendations.
Hemodynamic Effect of Transdermal Glyceryl Trinitrate on Newly Constructed Arteriovenous Fistula
Authors: Akin Emin Baris , Topçu Ömer , Özcan Hasan , Ersöz Sadik , Aytaç Suat , Anadol Erdal
Published: October 2002

Transdermal glyceryl trinitrate (GTN)
administration may have a beneficial effect in the creation of an arteriovenous fistula (AVF) by increasing blood flow through the access and by inhibiting platelet aggregation. We evaluated the hemodynamic effects of transdermal GTN administration on newly constructed arteriovenous fistula. Radiocephalic fistula at the wrist (Brescia fistula) was constructed as the initial vascular access in 31 uremia patients (study = 16, control = 15). The patient demographics and the etiology of chronic renal disease were similar in the two groups. None of the patients had severe vasculitis. The mean duration of chronic renal disease was 8 months (1–24 months). The diameter,
blood flow rate, and blood output at the drainage vein and the subclavian vein were measured by duplex ultrasonography 24 hours after the procedure. The measurements were performed again with transdermal GTN (10 mg/24 hours adhesive patch) administration in the study group and, without any medication, in the control group 4 hours after the initial measurements were taken. In the study group, all of the hemodynamic parameters were significantly increased over the initial measurements (p <0.05) whereas in the control group all hemodynamic parameters were unchanged, except the diameter of the subclavian vein (p <0.05). The actual change in hemodynamic parameters was significant in the study group when compared to the control group (p <0.05). Our data showed that transdermal GTN administration at the forearm increases flow through the Brescia fistula.
Familial Nonmedullary Thyroid Carcinoma Characterized by Multifocality and a High Recurrence Rate in a Large Study Population
Authors: Uchino Shinya , Noguchi Shiro , Kawamoto Hitoshi , Yamashita Hiroyuki , Watanabe Shin , Yamashita Hiroto , Shuto Shigeru
Published: October 2002

First‐degree relatives of persons with thyroid cancer are known to be at relatively high risk for the disease.
To better understand the clinicopathologic characteristics of familial nonmedullary thyroid carcinoma (FNMTC), we carried out a retrospective study in which we identified individuals treated at our institution who had at least one first‐degree relative with the disease. We used data obtained from our patient records to compare the features of 258 cases of the disease with the features of sporadic papillary or follicular thyroid carcinoma in another group of patients. The 258 patients represented 154 families and were selected from among 6458 patients with papillary or follicular thyroid carcinoma who underwent thyroidectomy between 1946 and 2000. Compared to the patients with sporadic disease, the FNMTC patients were more likely to have intraglandular dissemination (28.5% vs. 40.7%; p <
0.0001) and multiple benign nodules (29.8% vs. 41.5%;
p <0.0001). There were no significant differences between the two types of patients in terms of gender, age, tumor diameter, adhesion to or invasion of the surrounding tissues,
macroscopic metastasis observed at surgery, histology, presence of single benign nodules, presence of chronic thyroiditis, microscopic metastasis, or rate of lymph node metastasis. Recurrence was statistically frequent in the FNMTC patients compared with that in the sporadic disease patients (16.3% vs. 9.6%; p =
0.0005), and the disease‐free survival rate was significantly poorer in the FNMTC patients (p = 0.0041 by the Wilcoxon test and p <0.0001 by the log‐rank test). No significant difference in the overall survival rate was found between the two groups. Multivariate analysis by Cox’s proportional hazards method showed FNMTC to be an independent predictor of shorter disease‐free survival (risk ratio 1.88; confidence interval 1.35–2.54;
p = 0.0003). Locoregional recurrence in the ipsilateral or contralateral lymph nodes and contralateral thyroid lobe was significantly more frequent in the FNMTC patients than in the sporadic disease patients, whereas no difference was found regarding distant metastases. We conclude that FNMTC is a clinically distinct entity with an aggressive nature. Because of the frequent presence of benign nodules, multifocality, and high rate of locoregional recurrence, total or near‐total thyroidectomy with modified radical neck dissection in FNMTC patients is recommended.
Videolaparoscopic Resection of Insulinomas: Experience in Two Institutions
Authors: Gramática Luis , Herrera Miguel F. , Mercado‐Luna Andrés , Sierra Mauricio , Verasay Guillermo , Brunner Noemí
Published: October 2002

Laparoscopic resection of islet cell tumors has been performed in some selected cases. The aim of the study was to analyze the experience of two institutions in the laparoscopic management of insulinomas. In a 4‐year period, videolaparoscopic resection of sporadic insulinomas was performed in 9 patients. All patients had hypoglycemia/hyperinsulinism and a solitary tumor demonstrated by image studies. Demographics, surgical findings,
results, and complications were analyzed. Mean age of the patients was
43 years. One patient was male and eight were females. One tumor was located in the head of the pancreas, 4 in the body, and 4 in the tail.
Laparoscopic resection was completed in all patients. Procedures included 4 enucleations and 5 distal pancreatectomies. Pancreatic resection with splenic preservation was achieved in 4 cases.
Intraoperative ultrasound was used in 7 patients. Mean size of the tumors was 1.6 cm. All patients became normoglycemic after surgery.
Complications included one pancreatic fistula, one pleural effusion,
and one peripancreatic fluid collection. All resolved spontaneously. In a follow‐up period between 3 and 48 months no evidence of recurrence has been observed. This series supports laparoscopic resection of preoperatively localized benign solitary insulinomas. The operation provides the advantages of minimally invasive surgery and can be safely performed in most cases.
Does functional photosytem II complex have an oxygen channel?
Authors: Anderson Jan M
Published: October 2002

Photosystem II complex (PSII) of thylakoid membranes uses light energy to oxidise extremely stable water and produce oxygen (2H2O→O2+4H++4e−). PSII is compared with cytochrome c oxidase that catalyses the opposite reaction coupled to proton translocation. Cytochrome c oxidase has proton and water channels, and a tentative oxygen channel. I propose that functional PSII complexes also need a specific oxygen channel to direct O2 from the water molecules bound to specific Mn atoms of the Mn cluster within PSII out to the membrane surface. The function of this channel will be to prevent oxygen being accessible to the radical pair P680+Pheo−, thereby preventing singlet oxygen generation from the triplet P680 state in functional PSII. The important role of singlet oxygen in structurally perturbed non‐functional photosystem II is also discussed.
Slow vacuolar channels from barley mesophyll cells are regulated by 14‐3‐3 proteins
Authors: van den Wijngaard Paul W.J. , Bunney Tom D. , Roobeek Ilja , Schönknecht Gerald , de Boer Albertus H.
Published: October 2002

The conductance of the vacuolar membrane at elevated cytosolic Ca2+ levels is dominated by the slow activating cation selective (SV) channel. At physiological, submicromolar Ca2+ concentrations the SV currents are very small. Only recently has the role of 14‐3‐3 proteins in the regulation of voltage‐gated and Ca2+‐activated plasma membrane ion channels been investigated in Drosophila, Xenopus and plants. Here we report the first evidence that plant 14‐3‐3 proteins are involved in the down‐regulation of ion channels in the vacuolar membrane as well. Using the patch‐clamp technique we have demonstrated that 14‐3‐3 protein drastically reduces the current carried by SV channels. The current decline amounted to 80% and half‐maximal reduction was reached within 5 s after 14‐3‐3‐addition to the bath. The voltage sensitivity of the channel was not affected by 14‐3‐3. A coordinating role for 14‐3‐3 proteins in the regulation of plasma membrane and tonoplast ion transporters is discussed.
Caspase activation in response to cytotoxic Rana catesbeiana ribonuclease in MCF‐7 cells
Authors: Hu Chih-Chi Andrew , Tang Chih-Hang Anthony , Wang Jaang-Jiun
Published: October 2002

Rana catesbeiana ribonuclease (RC‐RNase) and onconase were proven to own anti‐tumor activity. While molecular determinants of onconase‐induced cell death have become more explicit, the RC‐RNase‐induced death pathway remains presently unknown. Here we demonstrated that RC‐RNase‐induced molecular cascades in caspase‐3‐deficient MCF‐7 cells did not include activation of initiation caspase‐8 and ‐9. Cleavage timing suggested that procaspase‐2 and ‐6 might be processed by active caspase‐7 in MCF‐7 cells. Caspase‐7 was also responsible for cleavage of the poly(ADP‐ribose) polymerase. Furthermore, we reported that overexpression of Bcl‐XL could raise the survival rates of MCF‐7 cells treated with RC‐RNase and onconase.
Neuronal nicotinic receptors: from protein structure to function
Authors: Itier Valérie , Bertrand Daniel
Published: October 2002

Neuronal nicotinic acetylcholine receptors are a prototype of ligand‐gated channels that mediate transmission in the central and peripheral nervous system. Structure–function studies performed at the amino acid level are now unraveling the determinant residues either for the properties of the ligand‐binding domain or the ionic pore. In this work we review, in the light of the latest finding, the structure–function relationship of these receptors and their implication in neurological diseases.
Semliki Forest virus vectors: efficient vehicles for in vitro and in vivo gene delivery
Authors: Lundstrom Kenneth , Schweitzer Christophe , Rotmann Daniel , Hermann Danielle , Schneider Edith M. , Ehrengruber Markus U.
Published: October 2002

Rapidly generated high‐titer Semliki Forest virus (SFV) vectors can infect numerous mammalian cell lines and primary cell cultures, and result in high levels of transgene expression. SFV‐based expression of transmembrane receptors has been characterized by specific ligand‐binding activity and functional responses. Adaptation of the SFV technology for mammalian suspension cultures has allowed the production of hundreds of milligrams of recombinant receptor for purification and structural studies. The same SFV stock solutions used for the infection of mammalian cells in culture have also been successfully applied for efficient transgene expression in organotypic hippocampal slices, as well as in vivo in rodent brain.
Enhanced expression of human ABC‐transporter tap is associated with cellular resistance to mitoxantrone
Authors: Lage Hermann , Perlitz Christin , Abele Rupert , Tampé Robert , Dietel Manfred , Schadendorf Dirk , Sinha Pranav
Published: October 2002

Multidrug resistance (MDR) phenotypes have been associated with the overexpression of various members of the superfamily of ATP binding cassette (ABC) transporters. Here we demonstrate that a member of the ABC‐transporter family, the heterodimer ‘transporter associated with antigen processing’ (TAP), physiologically involved in major histocompatibility complex class I‐restricted antigen presentation, is significantly overexpressed in the human gastric carcinoma cell line EPG85‐257RNOV exhibiting a mitoxantrone‐resistant phenotype. This tumor cell line shows an atypical MDR phenotype in the absence of ‘P‐glycoprotein’ or ‘MDR‐associated protein’ overexpression but with an enforced ‘breast cancer resistance protein’ expression level. Transfection of both TAP subunits encoding cDNA molecules, TAP1 and TAP2, into the drug‐sensitive parental gastric carcinoma cell line EPG85‐257P conferred a 3.3‐fold resistance to mitoxantrone but not to alternative anti‐neoplastic agents. Furthermore, cell clones transfected with both, but not singularly expressed TAP1 or TAP2, reduced cellular mitoxantrone accumulation. Taken together, the data suggest that the heterodimeric TAP complex possesses characteristics of a xenobiotic transporter and that the TAP dimer contributes to the atypical MDR phenotype of human cancer cells.
Internalization‐defective mutants of somatostatin receptor subtype 2 exert normal signaling functions in hematopoietic cells
Authors: Oomen Sigrid P.M.A. , Hofland Leo J. , Lamberts Steven W.J. , Löwenberg Bob , Touw Ivo P.
Published: October 2002

The regulatory peptide somatostatin (SST) acts via a family of G‐protein‐coupled receptors comprising five subtypes (SSTR1–5). G‐protein‐coupled receptors activate multiple signaling mechanisms, which variably depend on internalization and intracellular routing of activated receptors. We have recently demonstrated that hematopoietic precursors express SSTR2 and that SST is a chemoattractant for these cells. Herein, we characterize critical regions in SSTR2 involved in endocytosis and describe how ligand‐induced internalization impacts on two major signaling functions of SSTR2 in hematopoietic cells, the activation of the Erk pathway and the induction of promigratory responses.
Two‐dimensional crystallization of membrane proteins: the lipid layer strategy
Authors: Levy Daniel , Chami Mohamed , Rigaud Jean-Louis
Published: October 2002

Due to the difficulty to crystallize membrane proteins, there is a considerable interest to intensify research topics aimed at developing new methods of crystallization. In this context, the lipid layer crystallization at the air/water interface, used so far for soluble proteins, has been recently adapted successfully to produce two‐dimensional (2D) crystals of membrane proteins, amenable to structural analysis by electron crystallography. Besides to represent a new alternative strategy, this approach gains the advantage to decrease significantly the amount of material needed in incubation trials, thus opening the field of crystallization to those membrane proteins difficult to surexpress and/or purify. The systematic studies that have been performed on different classes of membrane proteins are reviewed and the physico‐chemical processes that lead to the production of 2D crystals are addressed. The different drawbacks, advantages and perspectives of this new strategy for providing structural information on membrane proteins are discussed.
The protease inhibitor chagasin of Trypanosoma cruzi adopts an immunoglobulin‐type fold and may have arisen by horizontal gene transfer
Authors: Rigden Daniel J , Monteiro Ana C.S , Grossi de Sá M.Fatima
Published: October 2002

Chagasin, a protein from Trypanosoma cruzi, is the first member of a new family of cysteine protease inhibitors. Despite its lack of significant sequence identity with known proteins, convincing structural models, using variable light chain templates, could be constructed on the basis of threading results. Experimental support for the final structure came from inhibition data for overlapping oligopeptides spanning the chagasin sequence. Chagasin therefore exemplifies a new protease inhibitor structural class and a new natural use for an immunoglobulin‐like domain. Limited sequence resemblance suggests that chagasin may represent the result of a rare horizontal gene transfer from host to parasite.
A new FACIT of the collagen family: COL21A1
Authors: Fitzgerald Jamie , Bateman John F.
Published: October 2002

Interrogation of the Human Genome data for sequences related to the von Willebrand factor A‐domain module identified a previously unreported 4.1 kb full‐length cDNA that is predicted to encode a new member of the collagen superfamily of extracellular matrix proteins, collagen XXI. The domain organization of collagen XXI comprised an N‐terminal signal sequence, followed by single von Willebrand factor A‐domain and thrombospondin domains, and an interrupted collagen triple helix. The organization of these motifs predict that collagen XXI is a new member of the FACIT collagen sub‐family. Expression analysis indicated that COL21A1 mRNA is present in many tissues including heart, stomach, kidney, skeletal muscle and placenta, and radiation hybrid mapping localized the COL21A1 gene to 6p11‐12.
Antisense inhibition of Chk2/hCds1 expression attenuates DNA damage‐induced S and G2 checkpoints and enhances apoptotic activity in HEK‐293 cells
Authors: Yu Qiang , Rose Ji Hyun La , Zhang Hongliang , Pommier Yves
Published: October 2002

The cellular response to DNA damage involves checkpoint controls that delay cell cycle progression in order to provide time for repair of damaged DNA. Chk2/hCds1 is a recently identified homolog of the yeast Cds1 kinase that is involved in cell cycle checkpoint response to DNA damage. To investigate the functions of Chk2/hCds1 in response to DNA damage in mammalian cells, we established a stable human kidney embryonic cell line (HEK‐293) that expresses antisense Chk2/hCds1 (Chk2AS) under the control of an inducible promoter. Cells that express Chk2AS display defective S‐phase delay in response to DNA replication‐mediated DNA damage induced by the topoisomerase I inhibitor camptothecin. The defective G2 checkpoint was also observed in Chk2AS cells exposed to the DNA damaging agent VP‐16 or γ‐radiation. Enhanced apoptosis was observed in Chk2AS cells after exposure to γ‐radiation or camptothecin. No p53 activation was observed after DNA damage in HEK‐293 or Chk2AS cells. Our results indicate that perturbation of Chk2/hCds1 expression adversely affects the S‐ and G2‐phase checkpoints following DNA damage or DNA replication block, and suggest that reduced expression of Chk2/hCds1 might promote a p53‐independent apoptotic response.
Lectin‐like oxidized low density lipoprotein receptor‐1 (LOX‐1) supports cell adhesion to fibronectin
Authors: Shimaoka Takeshi , Kume Noriaki , Minami Manabu , Hayashida Kazutaka , Sawamura Tatsuya , Kita Toru , Yonehara Shin
Published: October 2002

Lectin‐like oxidized lipoprotein receptor‐1 (LOX‐1) is a specific receptor for atherogenic oxidized low density lipoprotein (OxLDL) which belongs to the scavenger receptor family. In the present report, we show that LOX‐1 can also support cell adhesion to fibronectin (FN) in a divalent cation‐independent fashion. CHO‐K1 cells stably expressing bovine LOX‐1 (BLOX‐1‐CHO), but not untransfected CHO‐K1 cells, can adhere to FN‐coated plates, but not to collagen‐coated plates, in the presence of EDTA. BLOX‐1‐CHO adhesion to FN‐coated plates can also be suppressed by scavenger receptor ligands, such as OxLDL, polyinosinic acid (poly I), and dextran sulfate, but not by native LDL, acetylated LDL, polycytidylic acid (poly C), or chondroitin sulfate. Cultured bovine aortic endothelial cells can similarly adhere to FN‐coated plates, which was inhibited by OxLDL, poly I, and dextran sulfate in the presence of EDTA. LOX‐1 may play an important role in cell adhesion to FN in an integrin‐independent manner.
Is Ffh required for export of secretory proteins?
Authors: Kim Jinoh , Rusch Sharyn , Luirink Joen , Kendall Debra A.
Published: October 2002

In Escherichia coli, protein export from the cytoplasm may occur via the signal recognition particle (SRP)‐dependent pathway or the Sec‐dependent pathway. Membrane proteins utilize the SRP‐dependent route, whereas many secretory proteins use the cytoplasmic Sec machinery. To examine the possibility that signal peptide hydrophobicity governs which targeting route is utilized, we used a series of PhoA signal sequence mutants which vary only by incremental hydrophobicity changes. We show that depletion of SRP, but not trigger factor, affects all the mutants examined. These results suggest secretory proteins with a variety of signal sequences, as well as membrane proteins, require SRP for export.
Xenopus kidney hyaluronidase‐1 (XKH1), a novel type of membrane‐bound hyaluronidase solely degrades hyaluronan at neutral pH1
Authors: Reitinger Stephan , Müllegger Johannes , Lepperdinger Günter
Published: October 2002

In search for Xenopus laevis hyaluronidase genes, a cDNA encoding a putative PH‐20‐like enzyme was isolated. In the adult frog, this mRNA was only found to be expressed in the kidney and therefore named XKH1. When expressed by means of cRNA injection into frog oocytes, XKH1 solely exhibited at physiologic ionic strength hyaluronidase activity at neutral pH and in weakly acidic solutions. The enzyme was inactive below pH 5.4. In addition to hyaluronic acid hydrolysis, chondroitin sulfate also was degraded at low yield as assessed by fluorophore‐assisted carbohydrate electrophoresis analysis of the degradation products. The enzyme is sorted to the outer surface of the cell membrane of XKH1 expressing oocytes. From there, it could not be removed by phospholipase C nor was secreted hyaluronidase activity detectable. We conclude that XKH1 represents a membrane‐bound hyaluronan‐degrading enzyme exclusively expressed in cells of the adult frog kidney where it either may be involved in the reorganization of the extracellular architecture or in supporting physiological demands for proper renal functions.
Accelerated telomere shortening in Fanconi anemia fibroblasts – a longitudinal study
Authors: Adelfalk Caroline , Lorenz Mario , Serra Violeta , von Zglinicki Thomas , Hirsch-Kauffmann Monica , Schweiger Manfred
Published: October 2002

Fanconi anemia (FA) is a fatal inherited disease displaying chromosomal instability, disturbances in oxygen metabolism and a high burden of intracellular radical oxygen species. Oxygen radicals can damage DNA including telomeric regions. Insufficient repair results in single strand breaks that can induce accelerated telomere shortening. In a longitudinal study we demonstrate that telomeric DNA is continuously lost at a higher rate in FA fibroblasts compared to healthy controls. Furthermore, we show that this loss is caused rather by an increased shortening per cell division in regularly replicating cells than by apoptosis.
RXRβ isoforms in neuroblastoma cells and evidence for a novel 3′‐end transcript
Authors: Rana Birju , Pearson Andrew D.J. , Redfern Christopher P.F.
Published: October 2002

RXRβ is predominantly involved in retinoid responses in neuroblastoma cells, in particular the N‐type SH SY 5Y cells and the S‐type SH S EP cells, both derivatives of a mixed phenotype neuroblastoma cell line. The aim of this study was to identify RXRβ isoforms expressed in neuroblastoma cells and to characterise a putative novel RXRβ transcript. RXRβ1 and RXRβ2 were expressed in these neuroblastoma cells. An isoform with an insertion into the ligand binding domain, RXRβSLSR (referred to in previous studies as RXRβ3), was expressed at a similar level to RXRβ. A novel RXRβ transcript was identified by RNase protection assays and was at least as abundant as the expected RXRβ transcript and expressed in other cell types. Evidence suggests that this novel transcript was transcribed from an internal promoter between exons 5 and 6, contained a retained intron (intron 6) and was alternatively spliced with and without the SLSR insertion. These data show that the pattern of RXRβ expression is complex. The relative abundance of the novel RXRβ transcript suggests that it may be an important aspect of RXRβ function or regulation in a range of cell types.
Disruption of Thermus thermophilus genes by homologous recombination using a thermostable kanamycin‐resistant marker
Authors: Hashimoto Yasuko , Yano Takato , Kuramitsu Seiki , Kagamiyama Hiroyuki
Published: October 2002

Genes of an extremely thermophilic bacterium, Thermus thermophilus, were disrupted by homologous recombination using a recently developed, thermostable kanamycin‐resistant marker. First, the trpE gene was disrupted with various constructions of DNA. The transformation efficiency was exponentially increased as the length of the homologous regions flanking the marker gene increased above the minimum length (200–300 bp). We then disrupted five genes of the nucleotide excision repair system and examined their phenotypes. The convenience and high reliability of this method should prompt its application to the high‐throughput systematic disruption of the genes of this thermophilic bacterium.
Cardiolipin binds nonyl acridine orange by aggregating the dye at exposed hydrophobic domains on bilayer surfaces
Authors: Mileykovskaya Eugenia , Dowhan William , Birke Ronald L. , Zheng Donghong , Lutterodt Lydia , Haines Thomas H.
Published: October 2002

10‐N‐Nonyl acridine orange (NAO) has been used at low concentrations as a fluorescent indicator for cardiolipin (CL) in membranes and bilayers. The mechanism of its selective fluorescence in the presence of CL, and not any other phospholipids, is not understood. The dye might recognize CL by its high pK (pK
2>8.5). To investigate that, we established that NAO does not exhibit a pK in a pH range between 2.3 and 10.0. A second explanation is that the dye aggregates at hydrophobic domains on bilayers exposed by the CL. We found that a similar spectral shift occurs in the absence of CL in a concentrated solution of the dye in methanol and in the solid state. A model is proposed in which the nonyl group inserts in the bilayer at the hydrophobic surface generated by the presence of four chains on the phospholipid.
Increased resistin expression in the adipose tissue of male prolactin transgenic mice and in male mice with elevated androgen levels
Authors: Ling Charlotte , Kindblom Jon , Wennbo Håkan , Billig Håkan
Published: October 2002

The aim of this study was to investigate the regulation of resistin, a recently identified adipocyte‐secreted peptide, in the adipose tissue of prolactin (PRL)‐transgenic (tg) mice using ribonuclease protection assay. The level of resistin mRNA increased 3.5‐fold in the adipose tissue of untreated male PRL‐tg mice compared to controls. However, there was no difference in resistin expression in the adipose tissue of female PRL‐tg mice compared to control mice. PRL‐tg male mice have elevated serum testosterone levels and we therefore analyzed the effects of testosterone alone on resistin mRNA expression. Furthermore, the effects of elevated androgen levels on PRL receptor (PRLR) mRNA expression in the adipose tissue were investigated. Resistin mRNA increased 2.6‐fold in the adipose tissue of control male mice with elevated serum androgen levels. In addition, PRLR mRNA expression was increased in the adipose tissue of male mice with elevated testosterone. These results suggest testosterone to be a regulator of resistin and PRLR mRNA expression in the adipose tissue of male mice.
Structural mimicry of DH domains by Arfaptin suggests a model for the recognition of Rac–GDP by its guanine nucleotide exchange factors
Authors: Cherfils Jacqueline
Published: October 2002

Small G proteins cycle between an inactive form bound to GDP, and an active form bound to GTP. The two forms have different conformations and interact specifically with different partners, hence, the ability of G proteins to function as molecular switches. This view has been challenged by recent structural and biochemical studies of the Arfaptin/Por protein, which interacts equally well with the GDP‐ and GTP‐bound forms of the G protein Rac. Here it is shown that the dimeric helical domain of Arfaptin superimposes with a monomeric helical domain from the Dbl homology domain of Tiam, a guanine nucleotide exchange factor (GEF) for Rac, in their respective complexes with Rac. This unexpected structural mimicry suggests that the Rac–GDP–Arfaptin complex resembles the low‐affinity Rac–GDP–GEF complex that initiates the exchange reaction. This provides a model for the exchange mechanism where DH domains first dock onto Rac–GDP at the switch 2 before they undergo domain closure to catalyze GDP dissociation.
Characterisation of Rac activation in thrombin‐ and collagen‐stimulated human blood platelets
Authors: Soulet Carine , Gendreau Sandra , Missy Karine , Benard Valérie , Plantavid Monique , Payrastre Bernard
Published: October 2002

In this study, we characterised the mechanisms of Rac GTPase activation in human platelets stimulated by two physiological agonists, either thrombin, acting through membrane receptors coupled to heterotrimeric G‐proteins, or collagen which is known to mobilise a tyrosine kinase‐dependent pathway. Both agonists induced a rapid activation of Rac that was not significantly affected by the inhibition of integrin αIIbβ3 engagement. Using pharmacological inhibitors, we found that phospholipase C activation and calcium mobilisation were essential for platelet Rac activation by either thrombin or collagen whereas protein kinase C inhibition was without effect. In contrast to Rac, Cdc42 activation was independent of phospholipase C activation, indicating that the two GTPases are differently regulated. We also found that phosphoinositide 3‐kinase was not required for Rac activation in response to thrombin but was involved in its activation by collagen.
Identification and tissue distribution of two differentially spliced variants of the rat carnitine transporter OCTN2
Authors: Brooks Hilary , Krähenbühl Stephan
Published: October 2002

In this paper we show that the only known Na+ dependent transporter of carnitine in mammals, organic cation transporter number 2 (OCTN2), is subject to differential splicing. Cloning of OCTN2 in different rat tissues identified two splicing variants. We have developed a real time quantitative polymerase chain reaction method for quantification of these splice variants. Both splice variants could be detected in all tissues examined with a relative abundance of 0.1–1% of the full length transcript. We also draw attention to the previously described mutations in clinical examples of primary carnitine deficiency in humans where the described mutations appear to be those of a splicing or mis‐splicing event.
Dioxin induces a novel nuclear factor, DIF‐3, that is implicated in spermatogenesis
Authors: Ohbayashi Tetsuya , Oikawa Kosuke , Iwata Ryoko , Kameta Akemi , Evine Kazumi , Isobe Taku , Matsuda Yoichi , Mimura Junsei , Fujii-Kuriyama Yoshiaki , Kuroda Masahiko , Mukai Kiyoshi
Published: October 2002

2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin (TCDD; dioxin), a member of a class of environmental pollutants represented by polychlorinated dibenzo‐p‐dioxins and dibenzofurans, is one of the most toxic artificial compounds ever developed. In this study, we identified a novel TCDD target gene, DIF‐3 (dioxin inducible factor‐3), by cDNA representational difference analysis. DIF‐3 protein is a nuclear factor and possesses a zinc‐finger motif at its N‐terminus. High DIF‐3 mRNA expression in the testes was demonstrated by Northern blot analysis and abundant DIF‐3 protein was detected during spermatogenesis. Thus, these results suggest that DIF‐3 may be a target gene mediating the reproductive toxicity induced by TCDD.
Anti‐apoptotic protein survivin plays a significant role in tubular morphogenesis of human coronary arteriolar endothelial cells by hypoxic preconditioning
Authors: Zhu Li , Fukuda Shoji , Cordis Gerald , Das Dipak K , Maulik Nilanjana
Published: October 2002

Brief exposure of endothelial cells to oxidative stress induced by hypoxia followed by reoxygenation enhances tube formation. Our study provides evidence that hypoxic preconditioning accelerates tubular morphogenesis along with the activation of reactive oxygen species‐inducible nuclear transcription factor‐κB (NF‐κB), phosphatidylinositol 3‐kinase (PI3‐kinase) and broad‐spectrum anti‐apoptotic protein survivin in human coronary arteriolar endothelial cells (HCAEC). The formation of tubular morphogenesis was inhibited by using the PI3‐kinase and NF‐κB antagonists LY294002 and SN50 respectively. The activation of survivin by hypoxic preconditioning was also inhibited by LY294002 and SN50 along with increased apoptosis in HCAEC. These data demonstrate a crucial role of PI3‐kinase/Akt/NF‐κB/survivin signaling in tubular morphogenesis of HCAEC triggered by hypoxic preconditioning.
NF‐κB is required for TNF‐α‐directed smooth muscle cell migration
Authors: Wang Zhongbiao , Castresana Manuel R. , Newman Walter H.
Published: October 2002

Migration of vascular smooth muscle cells (VSMC) is a crucial event in the formation of vascular stenotic lesions. Tumor necrosis factor‐α (TNF‐α) is elaborated by VSMC in atherosclerosis and following angioplasty. We investigated the role of nuclear factor‐κB (NF‐κB) in human VSMC migration induced by TNF‐α. Adenoviral expression of a mutant form of the inhibitor of NF‐κB, IκB‐αM, suppressed TNF‐α‐triggered degradation of cellular IκB‐α, inhibited activation of NF‐κB, and attenuated TNF‐α‐induced migration. Further, IκB‐αM suppressed TNF‐α‐stimulated release of interleukin‐6 and ‐8 (IL‐6 and IL‐8). Neutralization of IL‐6 and IL‐8 with appropriate antibodies reduced TNF‐α‐induced VSMC migration. Addition of recombinant IL‐6 and IL‐8 stimulated migration. Collectively, our data provide initial evidence that TNF‐α‐mediated VSMC migration requires NF‐κB activation and is associated with induction of IL‐6 and IL‐8 which act in an autocrine manner.
Ursolic acid enhances nitric oxide and tumor necrosis factor‐α production via nuclear factor‐κB activation in the resting macrophages
Authors: You Ho Jin , Choi Chul Yung , Kim Ji Young , Park Sung Jun , Hahm Kyung-Soo , Jeong Hye Gwang
Published: October 2002

Ursolic acid (UA), a pentacyclic triterpene acid, is reported to have anti‐tumor activities; however, the mechanism underlying its anti‐tumorigenic effects is poorly understood. To further determine the mechanism of UA, we investigated the effects of UA on the release of nitric oxide (NO) and tumor necrosis factor‐α (TNF‐α), and on the level of inducible nitric oxide synthase (iNOS) and TNF‐α gene expression in mouse resting macrophages. We found that UA elicited a dose‐dependent increase in NO and TNF‐α production, and the level of iNOS and TNF‐α mRNA. Transient expression and electrophoretic mobility shift assays with nuclear factor‐κB (NF‐κB) binding sites revealed that the increased level of iNOS mRNA and TNF‐α mRNA induced by UA were mediated by the NF‐κB transcription factor complex. These results demonstrate that UA stimulates NO and TNF‐α release and is able to upregulate iNOS and TNF‐α expression through NF‐κB transactivation in the resting macrophages.
Relevance of fatty acid oxidation in regulation of the outer mitochondrial membrane permeability for ADP
Authors: Toleikis Adolfas , Liobikas Julius , Trumbeckaite Sonata , Majiene Daiva
Published: October 2002

The present study on saponin‐treated rat heart muscle fibers has revealed a new function of the fatty acid oxidation system in the regulation of the outer mitochondrial membrane (OMM) permeability for ADP. It is found that oxidation of palmitoyl‐CoA+carnitine, palmitoyl‐L‐carnitine and octanoyl‐L‐carnitine (alone or in combination with pyruvate+malate) dramatically decreased a very high value of apparent K
m of oxidative phosphorylation for ADP. Octanoyl‐D‐carnitine, as well as palmitate, palmitoyl‐CoA, and palmitoyl‐L‐carnitine were not effective in this respect, when their oxidation was prevented by the absence of necessary cofactors or blocked with rotenone. Our data suggest that oxidation, but not transport of fatty acids into mitochondria, induces an increase in the OMM permeability for ADP.
A critical appraisal of the mitochondrial coenzyme Q pool
Authors: Lenaz Giorgio
Published: October 2002

The function of the coenzyme Q (CoQ) pool in the inner mitochondrial membrane is reviewed in view of recent findings suggesting a supramolecular organization of the mitochondrial respiratory complexes. In spite of the structural evidence for preferential aggregations of the inner membrane components, most kinetic evidence is in favor of a dispersed organization based on random collisions of the small connecting redox components, in particular CoQ, with the individual complexes. The shape of the CoQ molecule in the pool, suggested to be a folded one, is in agreement with its very rapid lateral diffusion mobility in the membrane midplane. Since the structural evidence in favor of specific supercomplexes is rather strong, it cannot be excluded that electron transfer may follow either pool behavior or preferential channeling depending on the physiological conditions. Another function ascribed to the CoQ pool is the antioxidant action of the reduced CoQ molecules; although it cannot be excluded that protein‐bound ubisemiquinones may be a source of oxygen radicals, particularly at the level of complex III, the available evidence suggests that the mitochondrial pool only behaves as an antioxidant under physiological conditions.
Q/R RNA editing of the AMPA receptor subunit 2 (GRIA2) transcript evolves no later than the appearance of cartilaginous fishes
Authors: Kung Shieh-Shiuh , Chen Yu-Chia , Lin Wei-Hsiang , Chen Chun-Chen , Chow Wei-Yuan
Published: October 2002

The amino acid, either a glutamine (Q) or an arginine (R), at the Q/R site of the pore‐lining segment (M2) of a vertebrate AMPA receptor subunit critically influences the properties of the receptor. The R codon of the mammalian AMPA receptor subunit 2 (GRIA2) transcript is not coded by the chromosomal sequence, but is created by posttranscriptional RNA editing activities. On the other hand, the R codons of some teleost GRIA2 homologs are coded by chromosomal sequences. To elucidate the evolution of the utilization of Q/R RNA editing in modifying vertebrate GRIA2 transcripts, the GRIA2 genes of five fish species and an amphibian were studied. The putative hagfish GRIA2 homolog (hfGRIA2) encodes an R codon, whereas shark and bullfrog GRIA2 genes specify a Q codon at the genomic Q/R site. All gnathostoma GRIA2 genes possess an intron splitting the coding regions of M2 and the third hydrophobic region (M3). The intronic components required for Q/R RNA editing are preserved in all the Q‐coding vertebrate GRIA2 genes but are absent from the R‐coding GRIA2 genes. Interestingly, the hfGRIA2 is intronless, suggesting that hfGRIA2 is unlikely evolved from a Q/R editing‐competent gene. Results of this study suggest that modification of GRIA2 transcripts by Q/R editing is most likely acquired after the separation of the Agnatha and Gnathostome.
A pool of β‐tubulin is hyperphosphorylated at serine residues in Alzheimer disease brain
Authors: Vijayan Shrijay , El-Akkad Ezzat , Grundke-Iqbal Inge , Iqbal Khalid
Published: October 2002

In Alzheimer disease (AD) brain, activities of protein phosphatase (PP)‐2A/PP‐1 which are known to be associated with microtubules are compromised and are probably a cause of neurofibrillary degeneration through hyperphosphorylation of microtubule proteins. In the present study, an increase of ∼11 pmol phosphate/μg protein in 100 000×g pellet from AD compared with age‐matched control brains was found. Tau protein, which is hyperphosphorylated in AD can only account for ∼4 pmol phosphate/μg protein, suggesting the presence of non‐tau hyperphosphorylated proteins in the diseased brain. Western blot analysis with phosphoserine antibodies revealed a ∼54 kDa non‐tau protein to be significantly hyperphosphorylated in AD compared with age‐matched control cases in the particulate fraction. The ∼54 kDa protein was purified by preparative sodium dodecyl sulfate–polyacrylamide gel electrophoresis and identified as β‐tubulin by immunolabeling with specific antibodies, mass spectrometry analysis and by N‐terminal amino acid sequencing. The purified protein was hyperphosphorylated at serine residues in AD.
Staurosporine treatment and serum starvation promote the cleavage of emerin in cultured mouse myoblasts: involvement of a caspase‐dependent mechanism
Authors: Columbaro Marta , Mattioli Elisabetta , Lattanzi Giovanna , Rutigliano Cristina , Ognibene Andrea , Maraldi Nadir Mario , Squarzoni Stefano
Published: October 2002

Emerin is a nuclear membrane‐anchored protein which is absent or mutated in patients affected by Emery–Dreifuss muscular dystrophy. In this study, we induced apoptosis in cultured mouse myoblasts to evaluate emerin fate during the nuclear destabilization involved in programmed cell death. Emerin proteolysis was observed in myocytes during the apoptotic process. Myoblast apoptosis and emerin degradation were associated with chromatin compaction and detachment from the nuclear lamina, as detected by electron microscopy. In vivo specific inhibition of caspase 3 or caspase 6 activity completely abolished emerin proteolysis. These results show that the process of programmed cell death in muscle cells leads to emerin proteolysis, which appears to be related to caspase 6 activation and to cleavage of other nuclear envelope proteins, that share sequence homologies or functional features with emerin.
The homeotic protein dlk is expressed during peripheral nerve development
Authors: Costaglioli Patricia , Côme Christophe , Knoll-Gellida Anja , Salles Jérôme , Cassagne Claude , Garbay Bertrand
Published: October 2002

To investigate the molecular events controlling myelination of the peripheral nervous system, we compared gene expression of normal mouse sciatic nerves to that of the trembler mouse, whose Schwann cells are blocked in a pre‐myelinating phenotype. Using cDNA array, we assessed expression levels of 1176 genes, and we found that delta‐like protein (dlk), an epidermal growth factor‐like homeotic protein, was expressed in the normal developing nerves, but at a low level in the dysmyelinating mutant trembler. Moreover, dlk expression was down‐regulated when myelin protein expression was up‐regulated, and no expression was observed in the developing brain. These results suggest that dlk expression is required for Schwann cell acquisition of the myelinating phenotype.
Reactive oxygen species generated from the mitochondrial electron transport chain induce cytochrome c dissociation from beef‐heart submitochondrial particles via cardiolipin peroxidation. Possible role in the apoptosis
Authors: Petrosillo Giuseppe , Ruggiero Francesca Maria , Pistolese Marilva , Paradies Giuseppe
Published: October 2002

Cytochrome c release from mitochondria is a critical event in the apoptosis induction. Dissociation of cytochrome c from the mitochondrial inner membrane (IMM) is a necessary first step for cytochrome c release. In the present study, the effect of reactive oxygen species (ROS) on the dissociation of cytochrome c from beef‐heart submitochondrial particles (SMP) and on the cardiolipin content was investigated. Exposure of SMP to mitochondrial‐mediated ROS generation resulted in a large dissociation of cytochrome c from SMP and in a parallel loss of cardiolipin. Both these effects were directly and significantly correlated and also abolished by superoxide dismutase+catalase. These results demonstrate that ROS generation induces the dissociation of cytochrome c from IMM via cardiolipin peroxidation. The data may prove useful in clarifying the molecular mechanism underlying the release of cytochrome c from the mitochondria to the cytosol.