bluuebunny/crossref_metadata_2025 huggingface dataset:
DAP-kinase induces apoptosis by suppressing integrin activity and disrupting matrix survival signals
Authors: Wang Won-Jing , Kuo Jean-Cheng , Yao Chung-Chen , Chen Ruey-Hwa
Published: October 2002

Death-associated protein kinase (DAP-kinase) is a calcium/calmodulin-dependent serine/threonine kinase, and participates in various apoptosis systems. However, its apoptosis-promoting mechanism is poorly understood. Here, we demonstrate that DAP-kinase suppresses integrin-mediated cell adhesion and signal transduction, whereas dominant-negative interference of this kinase promotes adhesion. This effect of DAP-kinase is neither a consequence of apoptosis nor a result of decreased expression of integrins. Rather, DAP-kinase downregulates integrin activity through an inside-out mechanism. We present evidence indicating that this adhesion-inhibitory effect accounts for a major mechanism of the apoptosis induced by DAP-kinase. First, in growth-arrested fibroblasts, DAP-kinase triggers apoptosis in cells plated on fibronectin, but does not affect the death of cells on poly-l-lysine. Second, in epithelial cells, DAP-kinase induces apoptosis in the anoikis-sensitive MCF10A cells, but not in the anoikis-resistant BT474 cells. Most importantly, the apoptosis-promoting effect of DAP-kinase is completely abolished by enforced activation of integrin-mediated signaling pathways from either integrin itself or its downstream effector, FAK. Finally, we show that integrin or FAK activation blocks the ability of DAP-kinase to upregulate p53. Our results indicate that DAP-kinase exerts apoptotic effects by suppressing integrin functions and integrin-mediated survival signals, thereby activating a p53-dependent apoptotic pathway.
A current transformer modeling
Authors: Le Floch Yann , Guérin Christophe , Boudaud Dominique , Meunier Gérard , Brunotte Xavier
Published: October 2002

This paper presents the modeling of a current transformer by various methods with the FLUX3D software. The technique used is based on the finite element method coupled with electric circuits. A magnetic scalar potential reduced versus T0 formulation (T0ϕ−ϕ) taking into account the electric circuits with an air‐gap is used for this purpose. The air‐gap is described either by a thin volume region or by a surface region.
Integration of control loops in coupled field circuit model to study magnetic devices supplied by power electronic converter and their control
Authors: Manot G. , Lefèvre Y. , Piquet H. , Richardeau F.
Published: October 2002

In this paper, we present a method to take into account the feedback control in software developed from coupled field circuit models. The implementation of control loops is done in a software which is able to simulate electromagnetic devices associated with power electronic circuits having a time dependent topology. The control strategy is implemented in a discrete‐time version and can manage all controllable electronic switches by means of different electric or magnetic quantities calculated during the simulation (flux, currents, voltages, …). Electrical systems involving magnetic devices, power electronic converters and their control devices can be simulated in presence of phenomena such as eddy currents or saturation of the magnetic materials.
Calculation of winding losses using matrix modeling of high frequency transformer
Authors: Cheng K.W.E. , Kwok K.F. , Ho S.L. , Ho Y.L.
Published: October 2002

Calculation of the winding losses of high frequency transformer based on one‐dimensional field analysis is normally inapplicable for complex winding configurations. This paper presents a matrix modeling method which can produce a generalized mechanism to solve the AC winding losses. The transformer is modeled in a matrix connected filament. This is an alternative method to the finite element and is a filament approach. Experimental results and computation results using the proposed method are found to have good agreement.
Linking Web‐based segmentation to pricing tactics
Authors: Iyer Gopalkrishnan R. , Miyazaki Anthony D. , Grewal Dhruv , Giordano Maria
Published: October 2002

While numerous firms have successfully segmented customers using various segment‐based pricing models and tactics, the advent of the Internet has introduced a new element of opportunity with respect to such pricing. This paper identifies the various areas of opportunity for segmenting World Wide Web consumers. Several known pricing tactics are assessed for their application to the World Wide Web consumer markets.
Katanin‐mediated microtubule severing can be regulated by multiple mechanisms
Authors: McNally Karen Perry , Buster Dan , McNally Francis J.
Published: October 2002

Microtubules are essential for a wide range of cellular processes that vary between cell types. Katanin is a microtubule‐severing protein that carries out an essential role in meiotic spindles in Caenorhabditis elegans and a non‐essential role in mitotic spindles of vertebrates. In contrast to these M‐phase associated roles, katanin is also essential for post‐mitotic differentiation events in vertebrate neurons and in Arabidopsis. This diversity of function suggests that katanin's activity might be regulated by multiple mechanisms. Because katanin is active in M‐phase Xenopus extracts but not in interphase extracts, we assayed for regulators of katanin's activity in these extracts. The microtubule‐severing activity of purified katanin was inhibited by interphase Xenopus extracts. Fractionation revealed that this inhibition was due to at least 4 separable components, one of which contains the MAP4 homolog, XMAP230. Inhibition of katanin‐mediated microtubule‐disassembly activity by the XMAP230‐containing fraction was reversible by cyclinB/cdk1, suggesting one possible mechanism for the increased severing activity observed in M‐phase Xenopus extracts. In a previous study, spindle pole association by katanin was essential for its activity during mitosis suggesting that katanin's activity might also be regulated by co‐localization with an activator. The polo‐like kinase, Plx1, co‐localized with katanin at spindle poles in vivo and purified Plx1 increased the microtubule‐severing activity of katanin in vitro. These in vitro experiments illustrate the potential complexity of the regulation of katanin's activity in vivo and may explain how katanin can carry out widely different functions in different cell types. Cell Motil. Cytoskeleton 53:337–349, 2002. © 2002 Wiley‐Liss, Inc.
Mind the gap! Comparingex anteandex postassessments of the costs of complying with environmental regulation
Authors: Bailey Peter D. , Haq Gary , Gouldson Andy
Published: October 2002

This paper considers the question of howex antepredictions of the costs of complying with environmental regulations compare withex postevaluations of actual compliance costs. This is an important issue given the emergence of requirements for regulatory impact assessment (RIA) in many policy arenas (see OECD, 1997) and the conclusion of previous research that the predicted costs of compliance with environmental regulations often exceed actual costs (see Haqet al., 2001). Based upon a review of the different stages of the regulatory decision‐making process, this paper suggests that the reasons for these differences relate to strategic behaviour by affected parties in the policy formulation stage, problems in anticipating the influence of the implementation process, difficulties in forecasting the availability of new technological solutions and incentives for firms to reduce the costs of compliance once environmental regulations have been adopted. Copyright © 2002 John Wiley & Sons, Ltd. and ERP Environment.
Tetanus: Continuing Problem in the Developing World
Authors: Oladiran Idowu , Meier Donald E. , Ojelade A.A. , OlaOlorun David A. , Adeniran Adenike , Tarpley John L.
Published: October 2002

Despite diligent efforts by the World Health Organization and the governments of developing world countries,
tetanus persists as a global health problem. This retrospective study was undertaken to assess the outcome for victims of tetanus presenting to the Baptist Medical Centre in Ogbomoso, Nigeria and to develop better management techniques for future patients. Sixty patients (46
males, 14 females) with nonneonatal tetanus were seen over a 5‐year period (1995–1999). The mean age was 26 years, and 74% were 30 years or younger. All patients were treated with antitetanus serum,
antibiotics, wound débridement (when a wound was identified), and antispasmodics. No patients underwent tracheostomy or gastrostomy. The mortality rate was 44%. Factors significant for predicting mortality were age greater than 14 years, occupation as a farmer, short incubation period, short symptom duration, high degree of severity on presentation, and high temperature during hospital care. The best hope for improvement in the treatment of tetanus in our institution is more conscientious titration of antispasmodics to control spasms without causing significant respiratory depression. The only real hope for reducing the global mortality for tetanus, however, lies in renewed immunization efforts by all health care providers in developing world countries, not just community health workers.
Current National Health Insurance Policies for Thyroid Cancer Prophylactic Surgery in the United States
Authors: Dackiw Alan P.B. , Kuerer Henry M. , Clark Orlo H.
Published: October 2002

The efficacy of prophylactic thyroidectomy in patients with positive RET mutational analysis,
familial thyroid cancer, or both has been reported. As cost has become critical to medical decision‐making, this study was designed to evaluate currently existing coverage policies for prophylactic thyroidectomy. A confidential detailed cross‐sectional nationwide survey of 481 medical directors from the American Association of Health Plans, Medicare, and Medicaid was conducted. Of the 150 respondents, 65% (n = 97) had 100,000 or more enrolled members, and 35% (n = 53) had fewer than 100,000 enrolled members. Only 9% of private plans have specific policies for coverage of prophylactic thyroidectomy for patients with a strong family history of thyroid cancer, 19% provided no coverage, and 72% had no policy.
Only 9% of private plans have specific policies for patients with a known thyroid cancer genetic mutation, 12% provided no coverage, and 79% had no policy. Governmental carriers were less likely to provide coverage for prophylactic surgery: 4% for a strong family history and 6% for a genetic mutation. Altogether, 52% of government carriers provided no coverage for patients with a strong family history, and 50% provided no coverage in patients with a known genetic mutation; 44% of governmental carriers had no policy for either clinical scenario. Limited health insurance coverage for prophylactic thyroidectomy is offered in both private and governmental plans, with variations in coverage. As genetic testing becomes more widespread and with the potential identification of a gene predisposing to familial nonmedullary thyroid cancer, more uniform policies should be established to enable appropriate high risk candidates broader, equal coverage and access to these procedures.
Carotenoids are degraded by free radicals but do not affect lipid peroxidation in unilamellar liposomes under different oxygen tensions
Authors: Chen Gang , Djuric Zora
Published: October 2002

It has been questioned whether carotenoids can act as antioxidants in biological membranes. Biological membranes can be modeled for studies of lipid peroxidation using unilamellar liposomes. Both carotenoid depletion and lipid peroxidation were increased with increasing oxygen tension in unilamellar liposomes. Carotenoids in such liposomes were found to be very sensitive to degradation by free radicals generated from iron and 2,2′‐azobis(2‐amidinopropane) dihydrochloride, but they were not protective against lipid peroxidation. Lycopene and β‐carotene were more sensitive to free radical attack than lutein, zeaxanthin, and β‐cryptoxanthin.
The nuclear localization signal of zebrafish terra is located within the DM domain
Authors: Zhang Lixiang , Hua Zhengchun , Ren Jianguo , Meng Anming
Published: October 2002

Zebrafish Terra is a member of the DM domain‐containing transcription factor family and is involved in somitogenesis. The other known members of this family play a role in sex differentiation across species from Caenorhabditis elegans to human. Using the green fluorescence protein–Terra fusion constructs, we have identified the nuclear localization signal (NLS) of terra by transfecting human HeLa cells. The terra NLS is located between the two intertwined zinc‐binding sites of the DNA‐binding domain. However, the nuclear translocation of terra is independent of the structure required for DNA binding. Mutational analysis demonstrates that basic residues K77 and R78 within the DM domain are absolutely required for the translocation of Terra into the nuclei. Sequence comparison discloses that the NLS of Terra is also present in the other known members of the DM family, indicating the conservative nature of the NLS of this family during evolution.
Sequence‐specific binding property of Arabidopsis thaliana telomeric DNA binding protein 1 (AtTBP1)
Authors: Hwang Moo Gak , Chung In Kwon , Kang Bin Goo , Cho Myeon Haeng
Published: October 2002

We have identified an Arabidopsis thaliana cDNA, designated as AtTBP1, encoding a protein with a predicted size of 70.6 kDa that specifically binds to the plant telomeric repeat sequence TTTAGGG. AtTBP1 is present as a single‐copy gene in Arabidopsis genome and is expressed ubiquitously in various organs. AtTBP1 has a single Myb telomeric DNA binding domain at the C‐terminus and an extensive homology with other known telomere‐binding proteins. The isolated C‐terminus of AtTBP1 is capable of sequence‐specific DNA binding to plant duplex telomeric DNA. These results suggest that AtTBP1 may play important roles in plant telomere function in vivo.
An interferon regulatory factor‐like binding element restricts Xmyf‐5 expression in the posterior somites during Xenopus myogenesis
Authors: Mei Wenyan , Yang Jing , Tao Qinghua , Geng Xin , Rupp Ralph A.W. , Ding Xiaoyan
Published: October 2002

The expression of myf‐5, a key component of myogenic regulatory genes, declines progressively in mature somitic cells during vertebrate myogenesis. Little is known about how this down‐regulation takes place. Here we provide evidence that an interferon regulatory factor binding element (IRF element) within the Xenopus myf‐5 promoter is responsible for the elimination of myf‐5 transcription in mature somitic mesoderm of Xenopus embryos. We show that this IRF element mediates the down‐regulation of Xmyf‐5 transcription in gastrula embryos, and can specifically interact with nuclear proteins of early neurula. Moreover, deletion of this IRF element results in the anterior expansion of reporter gene transcripts within somitic mesoderm in transgenic embryos. Our results, therefore, provide insight into how the negative control of Xmyf‐5 expression takes place.
Imaging domains in model membranes with atomic force microscopy
Authors: Rinia Hilde A. , de Kruijff Ben
Published: October 2002

Lateral segregation in biomembranes can lead to the formation of biologically functional domains. This paper reviews atomic force microscopy studies on domain formation in model membranes, with special emphasis on transbilayer asymmetry, and on lateral domains induced by lipid–lipid interactions or by peptide–lipid interactions.
Identification of novel cellular proteins that bind to the LC8 dynein light chain using a pepscan technique
Authors: Rodrı́guez-Crespo Ignacio , Yélamos Belén , Roncal Fernando , Albar Juan Pablo , Ortiz de Montellano Paul R. , Gavilanes Francisco
Published: October 2002

Dynein is a minus end‐directed microtubule motor that serves multiple cellular functions. We have performed a fine mapping of the 8 kDa dynein light chain (LC8) binding sites throughout the development of a library of consecutive synthetic dodecapeptides covering the amino acid sequences of the various proteins known to interact with this dynein member according to the yeast two hybrid system. Two different consensus sequences were identified: GIQVD present in nNOS, in DNA cytosine methyl transferase and also in GKAP, where it is present twice in the protein sequence. The other LC8 binding motif is KSTQT, present in Bim, dynein heavy chain, Kid‐1, protein 4 and also in swallow. Interestingly, this KSTQT motif is also present in several viruses known to associate with microtubules during retrograde transport from the plasma membrane to the nucleus during viral infection.
Changes in mRNA expression profile underlie phenotypic adaptations in creatine kinase‐deficient muscles
Authors: de Groof A.J.C , Smeets B , Groot Koerkamp M.J.A , Mul A.N , Janssen E.E.W , Tabak H.F , Wieringa B
Published: October 2002

We have studied the mechanisms that regulate the remodeling of the glycolytic, mitochondrial and structural network of muscles of creatine kinase M (M‐CK)/sarcomeric mitochondrial creatine kinase (ScCKmit) knockout mice by comparison of wild‐type and mutant mRNA profiles on cDNA arrays. The magnitudes of changes in mRNA levels were most prominent in M‐CK/ScCKmit (CK−/−) double mutants but did never exceed those of previously observed changes in protein level for any protein examined. In gastrocnemius of CK−/− mice we measured a 2.5‐fold increase in mRNA level for mitochondrial encoded cytochrome c oxidase (COX)‐III which corresponds to the increase in protein content. The level of the nuclear encoded mRNAs for COX‐IV, H+‐ATP synthase‐C, adenine nucleotide translocator‐1 and insulin‐regulatable glucose transporter‐4 showed a 1.5‐fold increase, also in agreement with protein data. In contrast, no concomitant up‐regulation in mRNA and protein content was detected for the mitochondrial inorganic phosphate‐carrier, voltage‐dependent anion channel and certain glycolytic enzymes. Our results reveal that regulation of transcript level plays an important role, but it is not the only principle involved in the remodeling of mitochondrial and cytosolic design of CK−/− muscles.
Differential subcellular localization of hZip1 in adherent and non‐adherent cells
Authors: Milon Béatrice , Dhermy Didier , Pountney David , Bourgeois Monique , Beaumont Carole
Published: October 2002

Two human divalent cation transporters of the ZIP family, hZip1 and hZip2, homologous to Irt1 (Arabidopsis thaliana), the first identified member, have been described. They were shown by transfection into K562 cells to be localized at the plasma membrane and to mediate zinc uptake. Here we report a differential subcellular localization of hZip1 according to cell type. By transient expressions of EGFP‐hZip1, FLAG‐tagged or native hZip1, we observed that hZip1 has a vesicular localization in COS‐7 cells or in several epithelial cell lines, corresponding partially to the endoplasmic reticulum. Using anti‐hZip1 antibodies, we confirmed the intracellular localization of the endogenous protein in PC‐3, a prostate cancer cell line.
Human homolog of mouse tescalcin associates with Na+/H+ exchanger type‐1
Authors: Mailänder Jens , Müller-Esterl Werner , Dedio Jürgen
Published: October 2002

A novel regulatory protein, tescalcin (TSC), recently isolated from mouse embryonic testes, has been implicated in gonadal differentiation. Employing the yeast two‐hybrid system with the Na+/H+ exchanger type‐1 (NHE1) carboxyterminal domain as a bait we have identified a novel NHE1‐associated protein of 214 amino acid residues representing the human homolog of mouse TSC (96.7% identity). Co‐precipitation experiments demonstrated the interaction of human TSC with NHE1 in vitro and in vivo, and 45Ca2+ overlay assay revealed that TSC binds Ca2+. Immunofluorescence studies indicated that TSC is prominent in cellular lamellipodia where it colocalizes with NHE1. Abundant expression of TSC mRNA in the heart suggests that TSC may play important role(s) in concert with NHE1 in cardiac tissues.
MMP–TIMP interaction depends on residue 2 in TIMP‐4
Authors: Stratmann Bernd , Farr Martin , Tschesche Harald
Published: October 2002

Extracellular matrix remodeling and degradation are of great importance in both physiological and pathological situations. Matrix metalloproteinases (MMPs) and their natural occurring inhibitors – tissue inhibitors of metalloproteinases (TIMPs) – are involved in matrix turnover. Among the TIMPs there is only little specificity for inhibiting individual MMPs. In this report we describe the mutational analysis of the interaction of human TIMP‐4 with several MMPs. The effects of different substitutions of residue 2 (Ser2) in the inhibitory domain of TIMP‐4 were determined by kinetic measurements. Size, charge and polarity of residue 2 in the TIMP structure are key factors in MMP inhibition.
Recombinant holophytochrome in Escherichia coli
Authors: Landgraf F.T. , Forreiter C. , Hurtado Picó A. , Lamparter T. , Hughes J.
Published: October 2002

We have successfully co‐expressed two genes from the bilin biosynthetic pathway of Synechocystis together with cyanobacterial phytochrome 1 (Cph1) from the same organism to produce holophytochrome in Escherichia coli. Heme oxygenase was used to convert host heme to biliverdin IXα which was then reduced to phycocyanobilin via phycocyanobilin:ferredoxin oxidoreductase, presumably with the aid of host ferredoxin. In this host environment Cph1 apophytochrome was able to autoassemble with the phycocyanobilin in vivo to form fully photoreversible holophytochrome. The system can be used as a tool for further genetic studies of phytochrome function and signal transduction as well as providing an excellent source of holophytochrome for physicochemical studies.
Plant members of the α1→3/4‐fucosyltransferase gene family encode an α1→4‐fucosyltransferase, potentially involved in Lewisa biosynthesis, and two core α1→3‐fucosyltransferases1
Authors: Bakker Hans , Schijlen Elio , de Vries Theodora , Schiphorst Wietske E.C.M. , Jordi Wilco , Lommen Arjen , Bosch Dirk , van Die Irma
Published: October 2002

Three putative α1→3/4‐fucosyltransferase (α1→3/4‐FucT) genes have been detected in the Arabidopsis thaliana genome. The products of two of these genes have been identified in vivo as core α1→3‐FucTs involved in N‐glycosylation. An orthologue of the third gene was isolated from a Beta vulgaris cDNA library. The encoded enzyme efficiently fucosylates Galβ1→3GlcNAcβ1→3Galβ1→4Glc. Analysis of the product by 400 MHz 1H‐nuclear magnetic resonance spectroscopy showed that the product is α1→4‐fucosylated at the N‐acetylglucosamine residue. In vitro, the recombinant B. vulgaris α1→4‐FucT acts efficiently only on neutral type 1 chain‐based glycan structures. In plants the enzyme is expected to be involved in Lewisa formation on N‐linked glycans.
Gene silencing by S‐adenosylmethionine in muscle differentiation
Authors: Fuso Andrea , Cavallaro Rosaria A. , Orrù Luigi , Buttarelli Francesca R. , Scarpa Sigfrido
Published: October 2002

A well‐characterised experimental system, the myogenin gene in C2C12 muscle cell culture, was chosen to better understand the methylation mechanism underlying the regulation of gene expression. We already demonstrated that demethylation dynamics of a specific CpG site in the 5′‐flanking region of myogenin well correlates with gene expression and terminal differentiation. Here we demonstrate that S‐adenosylmethionine‐sulphate‐p‐toluenesulphonate (SAM) inhibits myogenin expression and myoblast differentiation by delaying the demethylation of specific CpG in differentiating myoblasts. These results suggest new perspectives in methylation mechanisms and the use of SAM in the partial silencing of gene expression, as it could be required in disease treatment.
Neutral fluorescence probe with strong ratiometric response to surface charge of phospholipid membranes
Authors: Duportail Guy , Klymchenko Andrey , Mely Yves , Demchenko Alexander
Published: October 2002

We report on dramatic differences in fluorescence spectra of 4′‐dimethylamino‐3‐hydroxyflavone (probe F) studied in phospholipid membranes of different charge (phosphatidyl glycerol, phosphatidylcholine (PC), their mixture and the mixture of PC with a cationic lipid). The effect consists in variations of relative intensities at two well‐separated band maxima at 520 and 570 nm belonging to normal (N*) and tautomer (T*) excited states of flavone chromophore. Based on these studies we propose a new approach to measure electrostatic potential at the surface layer of phospholipid membranes, which is based on potential‐dependent changes of bilayer hydration and involves very sensitive and convenient ratiometric measurements in fluorescence emission.
The serine protease inhibitor antithrombin III inhibits LPS‐mediated NF‐κB activation by TLR‐4
Authors: Mansell Ashley , Reinicke Anna , Worrall D.Margaret , O'Neill Luke A.J
Published: October 2002

In Drosophila, the Toll family of proteins mediates the innate immune response. Toll is activated by Spaetzle, which is generated in response to pathogens via a serine protease cascade. We wished to investigate if lipopolysaccharides (LPS) might activate Toll‐like receptor (TLR) 4 via a serine protease in humans. The serpin antithrombin III (ATIII) and the thrombin inhibitor hirudin both inhibited nuclear factor (NF)‐κB activation by LPS and Lipid A. ATIII and hirudin were also able to inhibit LPS‐induced NF‐κB activation in cells stably transfected with TLR4. These results suggest that LPS may activate a mammalian serine protease, which generates a product required for TLR4 signalling.
Gelsolin overexpression enhances neurite outgrowth in PC12 cells
Authors: Furnish Elizabeth J. , Zhou Wei , Cunningham Casey C. , Kas Josef A. , Schmidt Christine E.
Published: October 2002

The rational design of therapies for treating nerve injuries requires an understanding of the mechanisms underlying neurite extension. Neurite motility is driven by actin polymerization; however, the mechanisms are not clearly understood. One actin accessory protein, gelsolin, is involved with remodeling the cytoskeleton, although its role in cell motility is unclear. We report a two‐fold upregulation of gelsolin upon differentiation with nerve growth factor. Cells that were genetically modified to overexpress gelsolin have longer neurites and a greater neurite motility rate compared to controls. These data suggest that gelsolin plays an important role in neurite outgrowth.
Thr90 is a key residue of the bacteriorhodopsin proton pumping mechanism
Authors: Perálvarez Alex , Barnadas Ramon , Sabés Manuel , Querol Enric , Padrós Esteve
Published: October 2002

Mutation of Thr90 to Ala has a profound effect on bacteriorhodopsin properties. T90A shows about 20% of the proton pumping efficiency of wild type, once reconstituted into liposomes. Mutation of Thr90 influences greatly the Schiff base/Asp85 environment, as demonstrated by altered λ
max of 555 nm and pK
a of Asp85 (about 1.3 pH units higher than wild type). Hydroxylamine accessibility is increased in both dark and light and differential scanning calorimetry and visible spectrophotometry show decreased thermal stability. These results suggest that Thr90 has an important structural role in both the unphotolysed bacteriorhodopsin and in the proton pumping mechanism.
CLK‐1/Coq7p is a DMQ mono‐oxygenase and a new member of the di‐iron carboxylate protein family
Authors: Rea Shane
Published: October 2002

Strains of Caenorhabditis elegans mutant for clk‐1 exhibit a 20–40% increase in mean lifespan. clk‐1 encodes a mitochondrial protein thought to be either an enzyme or regulatory molecule acting within the ubiquinone biosynthesis pathway. Here CLK‐1 is shown to be related to the ubiquinol oxidase, alternative oxidase, and belong to the functionally diverse di‐iron‐carboxylate protein family which includes bacterioferritin and methane mono‐oxygenase. Construction and analysis of a homology model indicates CLK‐1 is a 2‐polyprenyl‐3‐methyl‐6‐methoxy‐1,4‐benzoquinone mono‐oxygenase as originally predicted. Analysis of known CLK‐1/Coq7p mutations also supports this notion. These findings raise the possibility of developing CLK‐1‐specific inhibitors to test for lifespan extension in higher organisms.
Reversible inhibition of hypoxia‐inducible factor 1 activation by exposure of hypoxic cells to the volatile anesthetic halothane
Authors: Itoh Tatsuya , Namba Tsunehisa , Fukuda Kazuhiko , Semenza Gregg L. , Hirota Kiichi
Published: October 2002

Volatile anesthetics modulate a variety of physiological and pathophysiological responses including hypoxic responses. Hypoxia‐inducible factor 1 (HIF‐1) is a transcription factor that mediates cellular and systemic homeostatic responses to reduced O2 availability in mammals, including erythropoiesis, angiogenesis, and glycolysis. We demonstrate for the first time that the volatile anesthetic halothane blocks HIF‐1 activity and downstream target gene expressions induced by hypoxia in the human hepatoma‐derived cell line, Hep3B. Halothane reversibly blocks hypoxia‐induced HIF‐1α protein accumulation and transcriptional activity at clinically relevant doses.
Tubulin and microtubule are potential targets for brain hexokinase binding
Authors: Wágner Gábor , Kovács János , Löw Péter , Orosz Ferenc , Ovádi Judit
Published: October 2002

The metabolite‐modulated association of a fraction of hexokinase to mitochondria in brain is well documented, however, the involvement of other non‐mitochondrial components in the binding of the hexokinase is controversial. Now we present evidence that the hexokinase binds both tubulin and microtubules in brain in vitro systems. The interaction of tubulin with purified bovine brain hexokinase was characterized by displacement enzyme‐linked immunosorbent assay using specific anti‐brain hexokinase serum (IC50=4.0±1.4 μM). This value virtually was not affected by specific ligands such as ATP or glucose 6‐phosphate. Microtubule‐bound hexokinase obtained in reconstituted systems using microtubule and purified hexokinase or brain extract was visualized by transmission and immunoelectron microscopy on the surface of tubules. The association of purified bovine brain hexokinase with either tubulin or microtubules caused about 30% increase in the activity of the enzyme. This activation was also observed in brain, but not in muscle cell‐free extract. The possible physiological relevance of the multiple heteroassociation of brain hexokinase is discussed.
Both N‐terminal catalytic and C‐terminal RNA binding domain contribute to substrate specificity and cleavage site selection of RNase III
Authors: Conrad Christian , Evguenieva-Hackenberg Elena , Klug Gabriele
Published: October 2002

The double‐stranded RNA‐specific endoribonuclease III (RNase III) of bacteria consists of an N‐terminal nuclease domain and a double‐stranded RNA binding domain (dsRBD) at the C‐terminus. Analysis of two hybrid proteins consisting of the N‐terminal half of Escherichia coli RNase III fused to the dsRBD of the Rhodobacter capsulatus enzyme and vice versa reveals that both domains in combination with the particular substrate determine substrate specificity and cleavage site selection. Extension of the spacer between the two domains of the E. coli enzyme from nine to 20 amino acids did not affect cleavage site selection.
Tailoring the substrate specificity of the β‐glycosidase from the thermophilic archaeon Sulfolobus solfataricus
Authors: Corbett Kevin , Fordham-Skelton Anthony P. , Gatehouse John A. , Davis Benjamin G.
Published: October 2002

The substrate specificity of the thermophilic β‐glycosidase (lacS) from the archaeon Sulfolobus solfataricus (SSβG), a member of the glycohydrolase family 1, has been analysed at a molecular level using predictions from known protein sequences and structures and through site‐directed mutagenesis. Three critical residues were identified and mutated to create catalysts with altered and broadened specificities for use in glycoside synthesis. The wild‐type (WT) and mutated sequences were expressed as recombinant fusion proteins in Escherichia coli, with an added His6‐tag to allow one‐step chromatographic purification. Consistent with side‐chain orientation towards OH‐6, the single Met439→Cys mutation enhances D‐xylosidase specificity 4.7‐fold and decreases D‐fucosidase activity 2‐fold without greatly altering its activity towards other D‐glycoside substrates. Glu432→Cys and Trp433→Cys mutations directed towards OH‐4 and ‐3, respectively, more dramatically impair glucose (Glc), galactose (Gal), fucose specificity than for other glycosides, resulting in two glycosidases with greatly broadened substrate specificities. These include the first examples of stereospecificity tailoring in glycosidases (e.g. WT→W433C, k
cat/K
M (Gal):k
cat/K
M (mannose (Man))=29.4:1→1.2:1). The robustness and high utility of these broad specificity SSβG mutants in parallel synthesis were demonstrated by the formation of libraries of β‐glycosides of Glc, Gal, xylose, Man in one‐pot preparations at 50°C in the presence of organic solvents, that could not be performed by SSβG‐WT.
Abrupt pubertal elevation of DNase I gene expression in human pituitary glands of both sexes
Authors: Yasuda Toshihiro , Takeshita Haruo , Nakajima Tamiko , Mogi Kouichi , Kaneko Yasushi , Ueki Misuzu , Iida Reiko , Kishi Koichiro
Published: October 2002

Deoxyribonuclease I (DNase I) was confirmed to be expressed in the human pituitary gland, particularly the anterior lobe, at levels comparable to those in the pancreas. The DNase I activity and the amount of gene transcript present in the pituitary glands of individuals aged from 1 month to 89 years was significantly age‐dependent, with an abrupt elevation after the neonatal and prepubertal periods irrespective of gender, followed by a gradual age‐dependent decline in males and a marked reduction in females in their postreproductive period. This DNase I age dependence in the pituitary gland was not present in the pancreas and serum. These observations suggest that tissue‐specific up‐regulation of DNase I gene expression in the pituitary gland occur, possibly at the onset of puberty.
The Eps15 homology (EH) domain
Authors: Confalonieri Stefano , Di Fiore Pier Paolo
Published: October 2002

The Eps15 homology (EH) domain was originally identified as a motif present in three copies at the NH2‐termini of Eps15 and of the related molecule Eps15R. Both of these molecules are substrates for the tyrosine kinase activity of the epidermal growth factor receptor and hence the name ‘Eps15 homology’ or EH domain [Wong et al. (1994) Oncogene 9, 1591–1597; Wong et al. (1995) Proc. Natl. Acad. Sci. USA 92, 9530–9534; Fazioli et al. (1993) Mol. Cell. Biol. 13, 5814–5828] was derived. The motif was subsequently found in several proteins from yeast to nematode, thus establishing its evolutionary conservation. Initial studies with filter‐binding assays and phage‐displayed libraries demonstrated its protein:protein interaction abilities and identified specific ligands. Subsequently, structural analyses established the molecular bases of recognition between EH domains and cognate peptides. To date, several EH‐containing and EH‐binding proteins have been identified, which establish in the cell a network of protein:protein interactions, defined as the EH network. This network coordinates cellular functions connected with endocytosis, actin remodeling and intracellular transduction of signals.
The paxillin LD motifs
Authors: Tumbarello David A , Brown Michael C , Turner Christopher E
Published: October 2002

Adapter/scaffold proteins, through their multidomain structure, perform a fundamental role in facilitating signal transduction within cells. Paxillin is a focal adhesion adapter protein implicated in growth factor‐ as well as integrin‐mediated signaling pathways. The amino‐terminus of paxillin contains five leucine‐rich sequences termed LD motifs. These paxillin LD motifs are highly conserved between species as well as within the paxillin superfamily. They mediate interactions with several structural and regulatory proteins important for coordinating changes in the actin cytoskeleton associated with cell motility and cell adhesion as well as in the regulation of gene expression.
Mechanism of oligomerization of Escherichia coli carbamoyl phosphate synthetase and modulation by the allosteric effectors. A site‐directed mutagenesis study
Authors: Mora Paz , Rubio Vicente , Cervera Javier
Published: October 2002

We use site‐directed mutagenesis to clarify the role of effector‐mediated oligomerization changes on the modulation of the activity of Escherichia coli carbamoyl phosphate synthetase (CPS) by its allosteric activator ornithine and its inhibitor UMP. The regulatory domain mutations H975L, L990A and N992A abolished, and N987V decreased CPS oligomerization. The oligomerization domain mutation L421E prevented tetramer but not dimer formation. None of the mutations had drastic effects on enzyme activity or changed the sensitivity or apparent affinity of CPS for ornithine and UMP. Our findings exclude the involvement of oligomerization changes in the control of CPS activity, and show that CPS dimers are formed by the interactions across regulatory domains, and tetramers by the interactions of two dimers across the oligomerization domains. A mechanism for effector‐mediated changes of the oligomerization state is proposed.
Peptide–protein interactions studied by surface plasmon and nuclear magnetic resonances
Authors: Spiga Ottavia , Bernini Andrea , Scarselli Maria , Ciutti Arianna , Bracci Luisa , Lozzi Luisa , Lelli Barbara , Di Maro Daniela , Calamandrei Duccio , Niccolai Neri
Published: October 2002

The structural features of the complexes that α‐bungarotoxin forms with three different synthetic peptides, mimotopes of the nicotinic acetylcholine receptor binding site, have been compared to the corresponding nuclear magnetic resonance (NMR) and surface plasmon resonance (SPR) data. For the considered peptides, the observed different affinities towards the toxin could not be accounted simply by static structural considerations. A combined analysis of the SPR‐ and NMR‐derived dynamic parameters shows new correlations between complex formation and dissociation and the overall pattern of intramolecular and intermolecular nuclear Overhauser effects. These features could be crucial for a rational design of protein ligands.
VHS domain – a longshoreman of vesicle lines
Authors: Lohi Olli , Poussu Anssi , Mao Yuxin , Quiocho Florante , Lehto Veli-Pekka
Published: October 2002

The VHS (ps‐27, rs and TAM) domain is a 140 residue long domain present in the very NH2‐terminus of at least 60 proteins. Based on their functional characteristics and on recent data on the involvement of VHS in cargo recognition in trans‐Golgi, VHS domains are considered to have a general membrane targeting/cargo recognition role in vesicular trafficking. Structurally, VHS is a right‐handed superhelix of eight helices with charged surface patches probably serving as sites of protein–protein recognition and docking.
Exploring the active site of plant glutaredoxin by site‐directed mutagenesis
Authors: Rouhier Nicolas , Gelhaye Eric , Jacquot Jean-Pierre
Published: October 2002

Six mutants (Y26A, C27S, Y29F, Y29P, C30S and Y26W/Y29P) have been engineered in order to explore the active site of poplar glutaredoxin (Grx) (Y26CPYC30). The cysteinic mutants indicate that Cys 27 is the primary nucleophile. Phe is a good substitute for Tyr 29, but the Y29P mutant was inactive. The Y26A mutation caused a moderate loss of activity. The YCPPC and WCPPC mutations did not improve the reactivity of Grx with the chloroplastic NADP‐malate dehydrogenase, a well known target of thioredoxins (Trxs). The results are discussed in relation with the known biochemical properties of Grx and Trx.
DNA intercalation in neutral multilamellar membranes
Authors: Pott Tanja , Roux Didier
Published: October 2002

We report a small angle X‐ray scattering study of the DNA/neutral lipid/water system showing that it is possible to confine DNA into a neutral multilamellar phase at high lipid‐to‐DNA weight ratio, despite the lack of electrostatic interactions in this system. This phase is characterized by a 2D ordering of the DNA molecules intercalated between the neutral bilayers of a 3D smectic phase as shown from the presence of a DNA–DNA correlation peak and the 1D electron density profile of the multilamellar phase. We further demonstrate that it is possible to disperse this phase as small multilamellar vesicles encapsulating high amounts of DNA.
Human monocyte‐derived dendritic cells are deficient in prostaglandin E2 production
Authors: Zelle-Rieser Claudia , Ramoner Reinhold , Artner-Dworzak Erika , Casari Andrea , Bartsch Georg , Thurnher Martin
Published: October 2002

Monocyte‐derived dendritic cells (moDCs) are increasingly used in clinical settings to stimulate tumor immunity. Prostaglandin E2 (PGE2), which is a member of the eicosanoid family of oxygenated arachidonic acid derivatives generated through the action of cyclooxygenases (COXs), is frequently used to enhance the tumor necrosis factor‐α‐induced terminal maturation of moDCs. We show here that one effect of interleukin (IL)‐4, which is used together with GM‐CSF to generate moDCs, is the suppression of endogenous PGE2 production in moDCs. IL‐4 inhibits the cytoplasmic form of phospholipase A2, the enzyme that specifically liberates arachidonic acid from membrane phospholipids. Although moDCs failed to mobilize endogenous arachidonic acid, they converted exogenous arachidonic acid into PGE2 in a COX‐1‐ and COX‐2‐dependent fashion. IL‐4‐mediated suppression of PGE2 biosynthesis in human moDCs explains the previously reported maturation‐enhancing effect of exogenous PGE2. The general suppression of eicosanoid biosynthesis may, however, limit the immunological efficacy of moDCs generated with IL‐4.
Peroxisome proliferator‐activated receptor (PPAR) agonists decrease lipoprotein lipase secretion and glycated LDL uptake by human macrophages
Authors: Gbaguidi F.G , Chinetti G , Milosavljevic D , Teissier E , Chapman J , Olivecrona G , Fruchart J.C , Griglio S , Fruchart-Najib J , Staels B
Published: October 2002

Lipoprotein lipase (LPL) acts independently of its function as triglyceride hydrolase by stimulating macrophage binding and uptake of native, oxidized and glycated LDL. Peroxisome proliferator‐activated receptors (PPARs) are nuclear receptors expressed in monocyte/macrophages, where they control cholesterol homeostasis. Here we study the role of PPARs in the regulation of LPL expression and activity in human monocytes and macrophages. Incubation of human monocytes or macrophages with PPARα or PPARγ ligands increases LPL mRNA and intracellular protein levels. By contrast, PPAR activators decrease secreted LPL mass and enzyme activity in differentiated macrophages. These actions of PPAR activators are associated with a reduced uptake of glycated LDL and could influence atherosclerosis development associated with diabetes.
Induction of mammalian cell death by a plant Bax inhibitor
Authors: Yu Li-Hua , Kawai-Yamada Maki , Naito Mikihiko , Watanabe Kaori , Reed John C , Uchimiya Hirofumi
Published: October 2002

Arabidopsis thaliana AtBI‐1 is an orthologue of mammalian Bax inhibitor‐1 capable of suppressing Bax‐induced cell death in yeast as well as mammalian cells. Here we investigated whether or not AtBI‐1 suppresses Bax‐induced cell death using human fibrosarcoma HT1080 cells. Surprisingly, AtBI‐1 did not block Bax‐induced cell death, but it triggered apoptotic cell death in mammalian cells. The proapoptotic effect of AtBI‐1 was blocked by the X‐linked caspase inhibitor XIAP, suggesting that the cell death caused by AtBI‐1 is similar to that caused by Bax.
Fractionation of cytochromes of phototrophically grown Chloroflexus aurantiacus. Is there a cytochrome bc complex among them?
Authors: Yanyushin Mikhail F
Published: October 2002

The cytochrome‐containing membrane complexes of the phototrophically grown green non‐sulfur bacterium Chloroflexus aurantiacus were fractionated by anion exchange chromatography. Three cytochrome b and four cytochrome c peaks were observed. None of the separated complexes met the features of the cytochrome bc complex. Two main cytochrome b‐containing complexes were further purified: a dimer of identical subunits with unknown function and a succinate:quinone oxidoreductase containing three subunit species. Two novel multisubunit complexes, similar to each other, with two heme c‐bearing subunits were also purified.
Property comparison of recombinant amphibian and mammalian allantoicases
Authors: Vigetti Davide , Pollegioni Loredano , Monetti Claudio , Prati Mariangela , Bernardini Giovanni , Gornati Rosalba
Published: October 2002

Allantoicase is an enzyme involved in uric acid degradation. Although it is commonly accepted that allantoicase is lost in mammals, birds and reptiles, we have recently identified its transcripts in mice and humans. The mouse mRNA seems capable of encoding a functional allantoicase, therefore we expressed the Xenopus and mouse allantoicases (MAlc and XAlc, respectively) in Escherichia coli and characterized the recombinant enzymes. The two recombinant allantoicases show a similar temperature and pH stability but, although XAlc and MAlc share a 54% amino acid identity, they differ in sensitivity to bivalent cations, in substrate affinity and in the level of expression in tissues (as revealed by means of Western blot analysis). We propose that the loss of allantoicase activity in mouse is due to a low substrate affinity and to a reduced expression level of the enzyme.
Shigella apyrase – a novel variant of bacterial acid phosphatases?
Authors: Babu M.Madan , Kamalakkannan S , Subrahmanyam Yerramalli V.B.K , Sankaran Krishnan
Published: October 2002

A virulence‐associated ATP diphosphohydrolase activity in the periplasm of Shigella, identified as apyrase, was found to be markedly similar to bacterial non‐specific acid phosphatases in primary structure. When the Shigella apyrase sequence was threaded in to the recently published 3D structure of the highly similar (73%) Escherichia blattae acid phosphatase it was found to have a highly overlapping 3D structure. Our analysis, which included assays for phosphatase, haloperoxidase and catalase activities, led us to hypothesize that Shigella apyrase might belong to a new class of pyrophosphatase originating as one more variant in the family of bacterial non‐specific acid phosphatases. It revealed interesting structure–function relationships and probable roles relevant to pathogenesis.
Elongation factor‐2 kinase and its newly discovered relatives
Authors: Ryazanov Alexey G.
Published: October 2002

Phosphorylation of elongation factor‐2 (eEF‐2) by the highly specific eEF‐2 kinase results in eEF‐2 inactivation and, therefore, may regulate the global rate of protein synthesis in animal cells. Cloning and sequencing of eEF‐2 kinase led to the discovery of a new family of protein kinases, named α‐kinases, whose catalytic domains display no sequence homology to conventional eukaryotic protein kinases. Several mammalian α‐kinases have recently been cloned. Two of these α‐kinases, named channel‐kinases 1 and 2 (ChaK1 and ChaK2) represent a new type of signaling molecules that are protein kinases fused to ion channels.
Elongation factor G with effector loop from elongation factor Tu is inactive in translocation
Authors: Kolesnikov Alexander , Gudkov Anatoly
Published: October 2002

Elongation factors Tu and G (EF‐Tu and EF‐G) alternately interact with the ribosome during the elongation phase of protein biosynthesis. The function of both factors depends on GTP binding, and the factors are ascribed to a superfamily of G‐proteins . All G‐proteins contain the effector loop, a structural element that is important for the protein's interaction with its target molecule. In this study the effector loop of EF‐G was replaced by the loop taken from EF‐Tu. The EF‐G with EF‐Tu loop has markedly decreased GTPase activity and did not catalyze translocation. We conclude that these loops are not functionally interchangeable since the factors interact with different states of the ribosome.