bluuebunny/crossref_metadata_2025 huggingface dataset:
Chromatic, Flow and
Reliability Polynomials: The
Complexity of their Coefficients
Authors: OXLEY JAMES , WELSH DOMINIC
Published: September 2002

We study the complexity of computing the coefficients of three classical polynomials, namely
the chromatic, flow and reliability polynomials of a graph. Each of these is a specialization
of the Tutte polynomial Σtijxiyj. It is shown that, unless NP = RP, many of the relevant
coefficients do not even have good randomized approximation schemes. We consider the
quasi-order induced by approximation reducibility and highlight the pivotal position of the
coefficient t10 = t01, otherwise known as the beta invariant.

Our nonapproximability results are obtained by showing that various decision problems
based on the coefficients are NP-hard. A study of such predicates shows a significant
difference between the case of graphs, where, by Robertson–Seymour theory, they are
computable in polynomial time, and the case of matrices over finite fields, where they are
shown to be NP-hard.
Being more certain about Random Assignment in Social Policy Evaluations
Authors: Stafford Bruce
Published: September 2002

Social experiments have been widely utilised in evaluations of social programmes in the US to identify ‘what works’, whilst in the UK their use is more controversial. This paper explores the paradigmatic, technical and practical issues evaluators confront in using randomised experiments to evaluate social policies. Possible remedies to some of these problems are outlined. It is argued that although no evaluation methodology is problem-free, policy makers and researchers should be more confident about the merits of using random assignment, provided it is used in conjunction with other methodologies more suited to understanding why and how interventions work.
Cloning and characterization of the 5′-flanking region of the oxalate decarboxylase gene from Flammulina velutipes
Authors: AZAM Mohammad , KESARWANI Meenu , CHAKRABORTY Subhra , NATARAJAN Krishnamurthy , DATTA Asis
Published: September 2002

The oxalate-degrading enzyme, oxalate decarboxylase (OXDC), was purified and characterized from Flammulina velutipes, a basidiomycetous fungus [Mehta and Datta (1991) J. Biol. Chem. 266, 23548—23553]. The cDNA cloning and analyses revealed that OXDC transcription was induced by oxalic acid. However, in this report, we show that OXDC transcription is induced by low pH, not by oxalate. To understand the regulatory mechanism of OXDC expression, we have cloned and analysed a 580-bp genomic fragment from the 5′-flanking region of the OXDC gene. Sequence analysis showed the presence of several eukaryotic transcription factor binding motifs within the −580bp of the upstream region. Electrophoretic-mobility-shift assays with partially purified cell extracts revealed specific binding of a factor in acid-induced, but not in uninduced, extracts. Furthermore, DNase I protection assays using the partially purified fraction from oxalic acid-induced extract revealed a footprint of a 13-bp sequence 5′GCGGGGTCGCCGA3′, termed low pH responsive element (LPRE), corresponding to the −287 to −275bp region of the OXDC promoter. Our results suggest that in F. velutipes cells, activation of OXDC transcription in response to low pH is mediated by the binding of a novel transcription factor through the LPRE site in the OXDC promoter.
Control of eukaryotic protein synthesis by upstream open reading frames in the 5′-untranslated region of an mRNA
Authors: MEIJER Hedda A. , THOMAS Adri A.M.
Published: September 2002

Control of gene expression is achieved at various levels. Translational control becomes crucial in the absence of transcription, such as occurs in early developmental stages. One of the initiating events in translation is that the 40S subunit of the ribosome binds the mRNA at the 5′-cap structure and scans the 5′-untranslated region (5′-UTR) for AUG initiation codons. AUG codons upstream of the main open reading frame can induce formation of a translation-competent ribosome that may translate and (i) terminate and re-initiate, (ii) terminate and leave the mRNA, resulting in down-regulation of translation of the main open reading frame, or (iii) synthesize an N-terminally extended protein. In the present review we discuss how upstream AUGs can control the expression of the main open reading frame, and a comparison is made with other elements in the 5′-UTR that control mRNA translation, such as hairpins and internal ribosome entry sites. Recent data indicate the flexibility of controlling translation initiation, and how the mode of ribosome entry on the mRNA as well as the elements in the 5′-UTR can accurately regulate the amount of protein synthesized from a specific mRNA.
Non-essential roles of cysteine residues in functional expression and redox regulatory pathways for canine glutamate/aspartate transporter based on mutagenic analysis
Authors: TAMAHARA Satoshi , INABA Mutsumi , SATO Kota , MATSUKI Naoaki , HIKASA Yoshiaki , ONO Ken-ichiro
Published: September 2002

A redox regulatory mechanism and a molecular link between oxidative and excitotoxic neurodegeneration have been postulated for high-affinity Na+-dependent glutamate transporters. In the present study, mutations were introduced at three cysteine residues in canine glutamate/aspartate transporter (GLAST) to investigate the functional significance of thiol groups in response to oxidation. Cys(-) GLAST, in which all cysteines were replaced by other amino acids, as well as other mutants with disruption of one of three cysteine residues, showed insoluble oligomer formation, which was considered to be due to spontaneous and excessive oxidation as observed in wild-type GLAST. The mutant transporters also showed plasma-membrane localization and glutamate-transport kinetics that were very similar to those of wild-type GLAST. Glutamate-transport activities in COS-7 cells transfected with wild-type and Cys(-) GLAST were inhibited to the same degree when cells were exposed to Hg2+ and were recovered by the addition of thiol-specific reductant dithiothreitol. These findings suggest that cysteine residues are not critical in functional expression of GLAST and the redox-sensing pathway via glutamate transporters.
Structural and functional characterization of the USP11 deubiquitinating enzyme, which interacts with the RanGTP-associated protein RanBPM
Authors: IDEGUCHI Haruko , UEDA Atsuhisa , TANAKA Masatsugu , YANG Jun , TSUJI Takashi , OHNO Shigeru , HAGIWARA Eri , AOKI Akiko , ISHIGATSUBO Yoshiaki
Published: September 2002

RanBPM is a RanGTP-binding protein required for correct nucleation of microtubules. To characterize the mechanism, we searched for RanBPM-binding proteins by using a yeast two-hybrid method and isolated a cDNA encoding the ubiquitin-specific protease USP11. The full-length cDNA of USP11 was cloned from a Jurkat cell library. Sequencing revealed that USP11 possesses Cys box, His box, Asp and KRF domains, which are highly conserved in many ubiquitin-specific proteases. By immunoblotting using HeLa cells, we concluded that 921-residue version of USP11 was the predominant form, and USP11 may be a ubiquitous protein in various human tissues. By immunofluorescence assay, USP11 primarily was localized in the nucleus of non-dividing cells, suggesting an association between USP11 and RanBPM in the nucleus. Furthermore, the association between USP11 and RanBPM in vivo was confirmed not only by yeast two-hybrid assay but also by co-immunoprecipitation assays using exogenously expressed USP11 and RanBPM. We next revealed proteasome-dependent degradation of RanBPM by pulse—chase analysis using proteasome inhibitors. In fact, ubiquitinated RanBPM was detected by both in vivo and in vitro ubiquitination assays. Finally, ubiquitin conjugation to RanBPM was inhibited in a dose-dependent manner by the addition of recombinant USP11. We conclude that RanBPM was the enzymic substrate for USP11 and was deubiquitinated specifically.
High expression in involuting reproductive tissues of uterocalin/24p3, a lipocalin and acute phase protein
Authors: RYON Joel , BENDICKSON Lee , NILSEN-HAMILTON Marit
Published: September 2002

During reproduction the mass and number of cells in the uterus and the mammary gland increase rapidly and then diminish more rapidly after their reproductive functions are completed. The diminishment of tissue mass, known as involution, involves an ordered series of events that includes apoptosis of resident cells, neutrophil invasion, the release of degradative enzymes and phagocytosis of cellular debris. Local signals are believed to regulate the progression of involution in each tissue. Here we show that the mammary gland and uterus express high levels of uterocalin, a protein that specifically induces apoptosis in neutrophils and other leucocytes. In the mammary gland, uterocalin expression is induced by weaning. In both tissues, uterocalin is expressed at extremely high levels such that it constitutes an average of 0.2—0.5% of the total extractable protein at its peak. Epithelial cells in the uterus and mammary gland produce uterocalin. In each case, the protein is secreted into the tissue lumen, with mammary-derived uterocalin being found in the milk. The period of highest uterocalin expression invivo is consistent with the hypothesis that one of its physiological roles is to induce apoptosis of infiltrating neutrophils and thereby delay the entry of neutrophils into the tissue. It is proposed that the role of uterocalin during involution is to provide a window of time during which resident cells are protected from the degradative enzymes, free radicals and other secreted products of activated phagocytes to allow these cells to prepare to survive the processes of involution.
Isolation and structural elucidation of biologically active phospholipids from Scytonema julianum (cyanobacteria)
Authors: ANTONOPOULOU Smaragdi , OIKONOMOU Alexandra , KARANTONIS Haralabos C. , FRAGOPOULOU Elizabeth , PANTAZIDOU Adriani
Published: September 2002

The role of platelet-activating factor (PAF) as a mediator appeared in rather primitive organisms like protozoans and was maintained in more evolved organisms. No reports exist for the presence of PAF or PAF analogues—or even compounds that exhibit PAF-like activity—in cyanobacteria, even though they belong to a a group of organisms at a low evolutionary level where the content of alkylacyl forms of ether lipids is expected to be high. In addition, cyanobacteria serve as a rich source of novel bioactive metabolites. In the present study the total lipids of a strain of Scytonema julianum, a filamentous cyanobacterium isolated from a Greek cave, were separated into neutral lipids and phospholipids, the latter being further fractionated by HPLC. Each phospholipid fraction was tested in vitro for its ability to inhibit PAF-, arachidonic acid- and ADP-induced washed-rabbit-platelet aggregation and/or to cause platelet aggregation. Two types of phospholipids causing platelet aggregation were detected and shown to be an acetylsphingomyelin and an acylacetylglycerol phosphoacetylated glycolipid. The existence of the sphingomyelin analogues is very important, since ceramides, cerebrosides and related lipids are intracellular second messengers. The identification of the phosphoglycoglycerolipid demonstrates a new type of lipid in cyanobacteria, namely one that exhibits a biological activity very similar to that of PAF. Its presence reinforces the concept that PAF is a member of a large family of lipid mediators, apparently having different physiological roles in prokaryotic and eukaryotic organisms. In addition, Scytonema julianum contains a phosphatidylcholine (C16:0/18:2), even though bacteria in general seldom contain choline-containing phosphoacylglycerols.
Induction of apoptosis in human leukaemic cells by IPENSpm, a novel polyamine analogue and anti-metabolite
Authors: FRASER Alison V. , WOSTER Patrick M. , WALLACE Heather M.
Published: September 2002

Human promyelogenous leukaemic cells (HL-60) were treated with novel spermine analogue, (S)-N1-(2-methyl-1-butyl)-N11-ethyl-4,8-diazaundecane (IPENSpm), and the effects on growth and intracellular polyamine metabolism were measured. IPENSpm was cytotoxic to these cells at concentrations greater than 2.5μM. It induced apoptosis in a caspase-dependent manner and its toxicity profile was comparable with etoposide, a well-known anti-tumour agent and inducer of apoptosis. IPENSpm decreased intracellular polyamine content as a result of changes in ornithine decarboxylase activity and increases in spermidine/spermine N1-acetyltransferase and polyamine export. Analysis showed spermine and spermidine as the major intracellular polyamines, while putrescine and acetyl-polyamines were the main export compounds. IPENSpm used the polyamine transporter system for uptake and its accumulation in cells was prevented by polyamine transport inhibitors. IPENSpm can be classified as a polyamine anti-metabolite and it may be a promising new lead compound in terms of treatment of some human cancers.
Cathepsin B expression and down-regulation by gene silencing and antisense DNA in human chondrocytes
Authors: ZWICKY Roman , MÜNTENER Kathrin , GOLDRING Mary B. , BAICI Antonio
Published: September 2002

Cathepsin B, a marker of the dedifferentiated chondrocyte phenotype, contributes to cartilage destruction in osteoarthritis and pathological proteolysis in rheumatoid arthritis and cancer. In search of possible means for neutralizing the action of this enzyme, we compared its expression, biosynthesis and distribution in articular chondrocytes and two lines of immortalized human chondrocytes. Native articular chondrocytes in primary culture and the polyclonal T/C-28a2 chondrocyte cell line were similar with respect to the number of endosomes and lysosomes, the distribution of three alternatively spliced cathepsin B mRNA forms, and the cathepsin B activity. In contrast, the clonal C-28/I2 cell line contained four times higher levels of intracellular cathepsin B activity, slightly higher numbers of endosomes and lysosomes, and uniform distribution of all three cathepsin B transcripts and thus resembled subcultured chondrocytes at an early stage of dedifferentiation. Transfection of T/C-28a2 chondrocytes with double-stranded cathepsin B mRNA resulted in inhibition of cathepsin B biosynthesis by up to 70% due to RNA interference, and single-stranded antisense DNAs of various sizes decreased cathepsin B biosynthesis by up to 78%. An antisense oligonucleotide designed to hybridize to the end of cathepsin B's exons 1 and the beginning of exon 3 was successful in specifically inhibiting the mRNA splice variant lacking exon 2. These results indicate that cathepsin B expression and activity may be targeted for gene silencing by RNA interference and antisense DNA in chondrocytes. Furthermore, the differential expression and distribution of cathepsin B and presence of the necessary molecular apparatus for gene silencing in the immortalized human chondrocyte cell lines indicate that they may serve as a useful model for studying the function of relevant enzymes in cartilage pathologies.
Plausible molecular mechanism for activation by fumarate and electron transfer of the dopamine β-mono-oxygenase reaction
Authors: WIMALASENA D. Shyamali , JAYATILLAKE Samantha P. , HAINES Donovan C. , WIMALASENA Kandatege
Published: September 2002

A series of fumarate analogues has been used to explore the molecular mechanism of the activation of dopamine β-mono-oxygenase by fumarate. Mesaconic acid (MA) and trans-glutaconic acid (TGA) both activate the enzyme at low concentrations, similar to fumarate. However, unlike fumarate, TGA and MA interact effectively with the oxidized enzyme to inhibit it at concentrations of 1—5mM. Monoethylfumarate (EFum) does not activate the enzyme, but inhibits it. In contrast with TGA and MA, however, EFum inhibits the enzyme by interacting with the reduced form. The saturated dicarboxylic acid analogues, the geometric isomer and the diamide of fumaric acid do not either activate or inhibit the enzyme. The phenylethylamine—fumarate conjugate, N-(2-phenylethyl)fumaramide (PEA-Fum), is an 600-fold more potent inhibitor than EFum and behaves as a bi-substrate inhibitor for the reduced enzyme. The amide of PEA-Fum behaves similarly, but with an inhibition potency 20-fold less than that of PEA-Fum. The phenylethylamine conjugates of saturated or geometric isomers of fumarate do not inhibit the enzyme. Based on these findings and on steady-state kinetic analysis, an electrostatic model involving an interaction between the amine group of the enzyme-bound substrate and a carboxylate group of fumarate is proposed to account for enzyme activation by fumarate. Furthermore, in light of the recently proposed model for the similar copper enzyme, peptidylglycine α-hydroxylating mono-oxygenase, the above electrostatic model suggests that fumarate may also play a role in efficient electron transfer between the active-site copper centres of dopamine β-mono-oxygenase.
Molecular cloning and biochemical characterization of rabbit factor XI
Authors: SINHA Dipali , MARCINKIEWICZ Mariola , GAILANI David , WALSH Peter N.
Published: September 2002

Human factor XI, a plasma glycoprotein required for normal haemostasis, is a homodimer (160kDa) formed by a single interchain disulphide bond linking the Cys-321 of each Apple 4 domain. Bovine, porcine and murine factor XI are also disulphide-linked homodimers. Rabbit factor XI, however, is an 80kDa polypeptide on non-reducing SDS/PAGE, suggesting that rabbit factor XI exists and functions physiologically either as a monomer, as does prekallikrein, a structural homologue to factor XI, or as a non-covalent homodimer. We have investigated the structure and function of rabbit factor XI to gain insight into the relation between homodimeric structure and factor XI function. Characterization of the cDNA sequence of rabbit factor XI and its amino acid translation revealed that in the rabbit protein a His residue replaces the Cys-321 that forms the interchain disulphide linkage in human factor XI, explaining why rabbit factor XI is a monomer in non-reducing SDS/PAGE. On size-exclusion chromatography, however, purified plasma rabbit factor XI, like the human protein and unlike prekallikrein, eluted as a dimer, demonstrating that rabbit factor XI circulates as a non-covalent dimer. In functional assays rabbit factor XI and human factor XI behaved similarly. Both monomeric and dimeric factor XI were detected in extracts of cells expressing rabbit factor XI. We conclude that the failure of rabbit factor XI to form a covalent homodimer due to the replacement of Cys-321 with His does not impair its functional activity because it exists in plasma as a non-covalent homodimer and homodimerization is an intracellular process.
Genistein inhibits CCAAT/enhancer-binding protein β (C/EBPβ) activity and 3T3-L1 adipogenesis by increasing C/EBP homologous protein expression
Authors: HARMON Anne W. , PATEL Yashomati M. , HARP Joyce B.
Published: September 2002

The tyrosine kinase inhibitor genistein inhibits 3T3-L1 adipogenesis when present during the first 72h of differentiation. In this report, we investigated the underlying mechanisms involved in the anti-adipogenic effects of genistein. We found that genistein blocked the DNA binding and transcriptional activity of CCAAT/enhancer-binding protein β (C/EBPβ) during differentiation by promoting the expression of C/EBP homologous protein, a dominant-negative member of the C/EBP family. Loss of C/EBPβ activity was manifested as a loss of differentiation-induced C/EBPα and peroxisome-proliferator-activated receptor γ protein expression and a dramatic reduction in lipid accumulation. Further, we documented for the first time that C/EBPβ was tyrosine-phosphorylated in vivo during differentiation and in vitro by activated epidermal growth factor receptor. Genistein inhibited both of these events. Collectively, these results indicate that genistein blocks adipogenesis and C/EBPβ activity by increasing the level of C/EBP homologous protein and possibly by inhibiting the tyrosine phosphorylation of C/EBPβ.
Correlations between the functional integrity of the endoplasmic reticulum and polarized Ca2+ signalling in mouse lacrimal acinar cells: a role for inositol 1,3,4,5-tetrakisphosphate
Authors: HARMER Alexander R. , GALLACHER David V. , SMITH Peter M.
Published: September 2002

Ca2+ signalling in exocrine acinar cells has been shown to be both polarized and pulsatile in all cell types tested, except acutely isolated mouse lacrimal acinar cells. Lacrimal cells are also unusual in that they display a very low sensitivity to Ins(1,4,5)P3 (InsP3) that may be enhanced by placing the cells in primary culture for 12—72h or by intracellular infusion of a low concentration of Ins(1,3,4,5)P4 (InsP4). We have proposed previously that this atypical behaviour stemmed from vesiculation of the endoplasmic reticulum (ER) incurred during isolation of the cells and, furthermore, that time in culture or InsP4 increased sensitivity to InsP3 by increasing ER integrity [Smith, Harmer, Letcher and Irvine (2000) Biochem. J. 347, 77—82]. We have measured the half time for fluorescence recovery after photobleaching (FRAP) of a fluorescent marker (Mag-fluo 4) loaded into the ER lumen in order to determine directly the functional integrity of the ER in lacrimal cells. The half-time for FRAP was increased (indicating a reduction in the functional integrity of the ER) following exposure to anti-microtubule agents (taxol and nocodazole) known to perturb ER structure and decreased (indicating an increase in the functional integrity of the ER) by time in culture and exposure to InsP4. The action of InsP4 was particularly pronounced because it occurred under patch-clamp whole-cell conditions that were themselves found to reduce ER functional integrity. These data show that ER remodelling could be a physiological regulator of Ca2+ signalling and indicate a role for InsP4 in control of this process.
Isoprenoid biosynthesis in higher plants and in Escherichia coli: on the branching in the methylerythritol phosphate pathway and the independent biosynthesis of isopentenyl diphosphate and dimethylallyl diphosphate
Authors: HOEFFLER Jean-François , HEMMERLIN Andréa , GROSDEMANGE-BILLIARD Catherine , BACH Thomas J. , ROHMER Michel
Published: September 2002

In the bacterium Escherichia coli, the mevalonic-acid (MVA)-independent 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway is characterized by two branches leading separately to isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). The signature of this branching is the retention of deuterium in DMAPP and the deuterium loss in IPP after incorporation of 1-[4-2H]deoxy-d-xylulose ([4-2H]DX). Feeding tobacco BY-2 cell-suspension cultures with [4-2H]DX resulted in deuterium retention in the isoprene units derived from DMAPP, as well as from IPP in the plastidial isoprenoids, phytoene and plastoquinone, synthesized via the MEP pathway. This labelling pattern represents direct evidence for the presence of the DMAPP branch of the MEP pathway in a higher plant, and shows that IPP can be synthesized from DMAPP in plant plastids, most probably via a plastidial IPP isomerase.
Cloning and characterization of anti-cathepsin L single chain variable fragment whose expression inhibits procathepsin L secretion in human melanoma cells
Authors: GUILLAUME-ROUSSELET Nathalie , JEAN Didier , FRADE Raymond
Published: September 2002

We previously demonstrated that increase of procathepsin L secretion by human melanoma cells strongly increased their tumourigenicity and switched their phenotype from low to highly metastatic. Thus, we herein analysed whether it was possible to inhibit procathepsin L secretion using anti-cathepsin L ScFv. For this purpose, we produced different forms of fusion cathepsin L in prokaryotic or eukaryotic expression systems. An anti-cathepsin L monoclonal antibody (mAb), named 3D8, was isolated from mice immunized with purified procathepsin L-His. This 3D8 mAb interacted with an epitope localized on the 156—197 amino acid sequence of cathepsin L and recognized recombinant or native forms of cathepsin L synthesized by human melanoma cells. An active anti-cathepsin L ScFv was generated and characterized from 3D8 mAb heavy and light variable chains. Then, human melanoma cells were transiently co-transfected with 3D8 ScFv and cathepsin L cDNAs. Data demonstrated that increase of 3D8 ScFv expression in human melanoma cells totally inhibited procathepsin L secretion and induced accumulation of intracellular procathepsin L. Our results constitute the first demonstration that anti-cathepsin L ScFv could be used in human melanoma cells to inhibit procathepsin L secretion. This ScFv represents a new molecular tool to explore cell therapy of human melanomas.
Cytidine diphosphate-diacylglycerol synthesis in Mycobacterium smegmatis
Authors: NIGOU Jérôme , BESRA Gurdyal S.
Published: September 2002

Recent studies have demonstrated that, during infection of macrophages by mycobacteria, phospholipids (PLs) are released from the mycobacterial cell wall within infected macrophages and transported out of this compartment into intracellular vesicles. The release of these PLs may have functions that influence the outcome of mycobacterial infections. Despite their important role, little is known about the biosynthesis of PLs in mycobacteria. In all organisms, PL biosynthesis begins with acylation of sn-glycerol 3-phosphate to form phosphatidic acid (PA), which is then converted to the central liponucleotide intermediate, cytidine diphosphate-diacylglycerol (CDP-DAG) via the CDP-DAG synthase (CDS). The present work examines CDS activity in Mycobacterium smegmatis extracts, with regard to subcellular localization, pH dependence, bivalent and univalent cation requirement, substrate specificity and regulation by nucleotides. We show that CDS activity, which is mainly found within the cytoplasmic membrane, is Mg2+-dependent and activated by K+ ions. Among PAs containing saturated fatty acids, dipalmitoyl-PA is the preferred substrate [Km = 0.23±0.03mM for Triton X-100 (v/v)/PA in the ratio 5:1]. Moreover, CDS activity is inhibited by the reaction products PPi (IC50 = 1.5mM), CDP-DAG (IC50 = 0.3mM) and the nucleotides ATP, UTP and GTP. This study contributes to the delineation of PL biosynthesis in mycobacteria.
Cell-cycle arrest by PD184352 requires inhibition of extracellular signal-regulated kinases (ERK) 1/2 but not ERK5/BMK1
Authors: SQUIRES Matthew S. , NIXON Paula M. , COOK Simon J.
Published: September 2002

Serum and growth factors activate both the canonical extracellular signal-regulated kinase (ERK) 1/2 pathway and the ERK5/big mitogen-activated protein kinase 1 (BMK) 1 pathway. Pharmacological inhibition of the ERK1/2 pathway using PD98059 and U0126 prevents cyclin D1 expression and inhibits cell proliferation, arguing that the ERK1/2 pathway is rate limiting for cell-cycle re-entry. However, both PD98059 and U0126 also inhibit the ERK5/BMK1 pathway, raising the possibility that the anti-proliferative effect of such drugs may be due to inhibition of ERK5 or both pathways. Here we characterize the effect of the novel mitogen-activated protein kinase/ERK kinase (MEK) inhibitor, PD184352, on the ERK1/2 and ERK5 pathways in the Chinese hamster fibroblast cell line CCl39. In quiescent cells, serum-stimulated ERK1 activity was completely inhibited by PD184352 with an IC50 below 1μM, whereas ERK5 activation was unaffected even at 20μM. Serum-stimulated DNA synthesis and cyclin D1 expression was inhibited by low doses of PD184352, which abolished ERK1 activity but had no effect on ERK5. Similarly, in cycling cells PD184352 caused a dose-dependent G1 arrest and inhibition of cyclin D1 expression at low doses, which inhibited ERK1 but were without effect on ERK5. These results indicate that the anti-proliferative effect of PD184352 is due to inhibition of the classical ERK1/2 pathway and does not require inhibition of the ERK5 pathway.
Distinct roles of activating transcription factor 6 (ATF6) and double-stranded RNA-activated protein kinase-like endoplasmic reticulum kinase (PERK) in transcription during the mammalian unfolded protein response
Authors: OKADA Tetsuya , YOSHIDA Hiderou , AKAZAWA Rieko , NEGISHI Manabu , MORI Kazutoshi
Published: September 2002

In response to accumulation of unfolded proteins in the endoplasmic reticulum (ER), a homoeostatic response, termed the unfolded protein response (UPR), is activated in all eukaryotic cells. The UPR involves only transcriptional regulation in yeast, and approx. 6% of all yeast genes, encoding not only proteins to augment the folding capacity in the ER, but also proteins working at various stages of secretion, are induced by ER stress [Travers, Patil, Wodicka, Lockhart, Weissman and Walter (2000) Cell (Cambridge, Mass.) 101, 249–258]. In the present study, we conducted microarray analysis of HeLa cells, although our analysis covered only a small fraction of the human genome. A great majority of human ER stress-inducible genes (approx. 1% of 1800 genes examined) were classified into two groups. One group consisted of genes encoding ER-resident molecular chaperones and folding enzymes, and these genes were directly regulated by the ER-membrane-bound transcription factor activating transcription factor (ATF) 6. The ER-membrane-bound protein kinase double-stranded RNA-activated protein kinase-like ER kinase (PERK)-mediated signalling pathway appeared to be responsible for induction of the remaining genes, which are not involved in secretion, but may be important after cellular recovery from ER stress. In higher eukaryotes, the PERK-mediated translational-attenuation system is known to operate in concert with the transcriptional-induction system. Thus we propose that mammalian cells have evolved a strategy to cope with ER stress different from that of yeast cells.
Rapid insertion of GLUT2 into the rat jejunal brush-border membrane promoted by glucagon-like peptide 2
Authors: AU Anita , GUPTA Alina , SCHEMBRI Paul , CHEESEMAN Chris. I.
Published: September 2002

A possible role for GLUT2 transiently expressed in the rat jejunal brush-border membrane (BBM) under the influence of glucagon-like peptide 2 (GLP-2) was investigated using in vivo perfusion of the intestinal lumen as well as isolation of membrane proteins and immunohistochemistry. A 1h vascular infusion of GLP-2 in vivo doubled the rate of fructose absorption and this increase could be blocked by luminal phloretin. Immunohistochemistry of frozen sections of rat jejunum showed the expression of GLUT2 in both the basolateral and BBMs of mature enterocytes. Perfusion of the intestinal lumen with 50mM d-glucose or vascular infusion of 800pM GLP-2 for 1h increased the expression of GLUT2 in the BBM. Quantification of these changes using Western blotting of biotinylated surface-exposed protein showed a doubling of the expression of GLUT2 in the BBM, but the effects of glucose and GLP-2 were not additive. These results indicate that vascular GLP-2 can promote the insertion of GLUT2 into the rat jejunal BBM providing a low-affinity/high-capacity route of entry for dietary hexoses.
The evidence for two opposite, ATP-generating and ATP-consuming, extracellular pathways on endothelial and lymphoid cells
Authors: YEGUTKIN Gennady G. , HENTTINEN Tiina , SAMBURSKI Sergei S. , SPYCHALA Jozef , JALKANEN Sirpa
Published: September 2002

Extracellular purines are important signalling molecules in the vasculature that are regulated by a network of cell surface ectoenzymes. By using human endothelial cells and normal and leukaemic lymphocytes as enzyme sources, we identified the following purine-converting ectoenzymes: (1) ecto-nucleotidases, NTP diphosphohydrolase/CD39 (EC 3.6.1.5) and ecto-5′-nucleotidase/CD73 (EC 3.1.3.5); (2) ecto-nucleotide kinases, adenylate kinase (EC 2.7.4.3) and nucleoside diphosphate kinase (EC 2.7.4.6); (3) ecto-adenosine deaminase (EC 3.5.4.4). Evidence for this was obtained by using enzyme assays with 3H-labelled nucleotides and adenosine as substrates, direct evaluation of γ-phosphate transfer from [γ-32P]ATP to AMP/NDP, and bioluminescent measurement of extracellular ATP synthesis. In addition, incorporation of radioactivity into an approx. 20kDa surface protein was observed following incubation of Namalwa B cells with [γ-32P]ATP. Thus two opposite, ATP-generating and ATP-consuming, pathways coexist on the cell surface, where basal ATP release, re-synthesis of high-energy phosphoryls, and selective ecto-protein phosphorylation are counteracted by stepwise nucleotide breakdown with subsequent adenosine inactivation. The comparative measurements of enzymic activities indicated the predominance of the nucleotide-inactivating pathway via ecto-nucleotidase reactions on the endothelial cells. The lymphocytes are characterized by counteracting ATP-regenerating/adenosine-eliminating phenotypes, thus allowing them to avoid the lymphotoxic effects of adenosine and maintain surrounding ATP at a steady-state level. These results are in agreement with divergent effects of ATP and adenosine on endothelial function and haemostasis, and provide a novel regulatory mechanism of local agonist availability for nucleotide- or nucleoside-selective receptors within the vasculature.
The molecular mechanism for the spectral shifts between vertebrate ultraviolet- and violet-sensitive cone visual pigments
Authors: COWING Jill A. , POOPALASUNDARAM Subathra , WILKIE Susan E. , ROBINSON Phyllis R. , BOWMAKER James K. , HUNT David M.
Published: September 2002

The short-wave-sensitive (SWS) visual pigments of vertebrate cone photoreceptors are divided into two classes on the basis of molecular identity, SWS1 and SWS2. Only the SWS1 class are present in mammals. The SWS1 pigments can be further subdivided into violet-sensitive (VS), with λmax (the peak of maximal absorbance) values generally between 400 and 430nm, and ultraviolet-sensitive (UVS), with a λmax<380nm. Phylogenetic evidence indicates that the ancestral pigment was UVS and that VS pigments have evolved separately from UVS pigments in the different vertebrate lineages. In this study, we have examined the mechanism of evolution of VS pigments in the mammalian lineage leading to present day ungulates (cow and pig). Amino acid sequence comparisons of the UVS pigments of teleost fish, amphibia, reptiles and rodents show that site 86 is invariably occupied by Phe but is replaced in bovine and porcine VS pigments by Tyr. Using site-directed mutagenesis of goldfish UVS opsin, we have shown that a Phe-86→Tyr substitution is sufficient by itself to shift the λmax of the goldfish pigment from a wild-type value of 360nm to around 420nm, and the reverse substitution of Tyr-86—Phe into bovine VS opsin produces a similar shift in the opposite direction. The substitution of this single amino acid is sufficient to account therefore for the evolution of bovine and porcine VS pigments. The replacement of Phe with polar Tyr at site 86 is consistent with the stabilization of Schiff-base protonation in VS pigments and the absence of protonation in UVS pigments.
Origins of the difference in Ca2+ requirement for activation of μ- and m-calpain
Authors: DUTT Previn , SPRIGGS Cherie N. , DAVIES Peter L. , JIA Zongchao , ELCE John S.
Published: September 2002

The μ- and m-calpains are closely related Ca2+-dependent cysteine proteases having different in vitro Ca2+ requirements (Kd), of approx. 25 and 325μM respectively. The two isoforms are heterodimers of slightly different large (80kDa) subunits and an identical small (28kDa) subunit, so that the difference in Kd values must reside in the large subunits. As assayed here, these Kd values relate to the Ca2+ required for the first phase of calpain activation and do not reflect the lower Ca2+ then required by fully activated calpain. On the basis of sequence comparison and the X-ray structure of m-calpain, many m-type residues in the C-terminal EF-hand-containing domain IV were converted into the corresponding μ-type residues, but these mutations did not produce the expected decrease in Kd. In a series of hybrid (μ/m) large-subunit calpains, the Kd values decreased progressively towards that of μ-calpain as the proportion of μ-type sequence increased from 0 to 90%. Kd values cannot therefore be ascribed to one or a few specific intramolecular interactions, but reflect the global response of the whole molecule to Ca2+ binding. Nonetheless, 25% of the difference in Kd values between μ- and m-calpain can be ascribed to the N-terminal peptide of the large subunit, whereas the C-terminal EF-hand-containing domain IV accounts for 65% of the difference.
Elasticity analysis and design for large metabolic responses produced by changes in enzyme activities
Authors: ORTEGA Fernando , ACERENZA Luis
Published: September 2002

Metabolic control analysis has been extensively used to describe how the sensitivity properties of the component enzymes in a metabolic pathway (represented by the elasticity coefficients) determine the way in which metabolic variables respond (described by the control coefficients). Similarly, metabolic control design addresses the inverse problem of obtaining the sensitivity properties of the component enzymes that are required for the system to show a pre-established pattern of responses. These formalisms, including what is called elasticity analysis and design, were developed for small, strictly speaking infinitesimal, changes. Here we extend them to large metabolic responses. The new approach can be applied to simple two-step pathways or to any arbitrary metabolic system divided into two groups linked by one intermediate. General expressions that relate control and elasticity coefficients for large changes are derived. Concentration and flux connectivity relationships are obtained. The relationships for large changes indicate that the pattern of responses is not necessarily the same as the one obtained with the traditional infinitesimal approach, in some cases the patterns being qualitatively different. The general analysis is used to study the control of ketogenesis in rat liver mitochondria, starting from data available in the literature. The control profile of the pathway subject to large changes shows both quantitative and qualitative differences from the one obtained from an analysis that is performed with infinitesimal coefficients. This exemplifies the type of errors that may be introduced when drawing conclusions about large metabolic responses from results obtained with an infinitesimal treatment.
Site-directed mutagenesis reveals a novel catalytic mechanism of Mycobacterium tuberculosis alkylhydroperoxidase C
Authors: CHAUHAN Radha , MANDE Shekhar C.
Published: September 2002

Mycobacterium tuberculosis alkylhydroperoxidase C (AhpC) belongs to the peroxiredoxin family, but unusually contains three cysteine residues in its active site. It is overexpressed in isoniazid-resistant strains of M. tuberculosis. We demonstrate that AhpC is capable of acting as a general antioxidant by protecting a range of substrates including supercoiled DNA. Active-site Cys to Ala mutants show that all three cysteine residues are important for activity. Cys-61 plays a central role in activity and Cys-174 also appears to be crucial. Interestingly, the C174A mutant is inactive, but double mutant C174/176A shows significant revertant activity. Kinetic parameters indicate that the C176A mutant is active, although much less efficient. We suggest that M. tuberculosis AhpC therefore belongs to a novel peroxiredoxin family and might follow a unique disulphide-relay reaction mechanism.
A novel role for farnesyl pyrophosphate synthase in fibroblast growth factor-mediated signal transduction
Authors: REILLY John F. , MARTINEZ Shawndra D. , MICKEY Gregory , MAHER Pamela A.
Published: September 2002

Farnesyl pyrophosphate synthase (FPPS) catalyses the formation of a key cellular intermediate in isoprenoid metabolic pathways. Here we describe a novel role for this enzyme in fibroblast growth factor (FGF)-mediated signalling. We demonstrate the binding of FPPS to FGF receptors (FGFRs) using the yeast two-hybrid assay, pull-down assays and co-immunoprecipitation. The interaction between FPPS and FGFR is regulated by the cellular metabolic state and by treatment with FGF-2. Overexpression of FPPS inhibits FGF-2-induced cell proliferation, accompanied by a failure of the FGF-2-mediated induction of cyclins D1 and E. Overexpression of FPPS in fibroblasts also promotes increased farnesylation of Ras, and temporally extends FGF-2-stimulated activation of the Ras/ERK (extracellular-signal-regulated kinase) cascade. These data suggest that, in addition to its role in isoprenoid biosynthesis, FPPS may function as a modulator of the cellular response to FGF treatment.
Ance, a Drosophila angiotensin-converting enzyme homologue, is expressed in imaginal cells during metamorphosis and is regulated by the steroid, 20-hydroxyecdysone
Authors: SIVITER Richard J. , TAYLOR Christine A.M. , COTTAM Deborah M. , DENTON Adrian , DANI M. Paulina , MILNER Martin J. , SHIRRAS Alan D. , ISAAC R. Elwyn
Published: September 2002

Ance is a single domain homologue of mammalian angiotensin-converting enzyme (ACE) and is important for normal development and reproduction in Drosophila melanogaster. Mammalian ACE is responsible for the synthesis of angiotensin II and the inactivation of bradykinin and N-acetyl-Ser-Asp-Lys-Pro, but the absence of similar peptide hormones in insects suggests novel functions for Ance. We now provide evidence in support of a role for Ance during Drosophila metamorphosis. The transition of larva to pupa was accompanied by a 3-fold increase in ACE-like activity, which subsequently dropped to larval levels on adult eclosion. This increase was attributed to the induction of Ance expression during the wandering phase of the last larval instar in the imaginal cells (imaginal discs, abdominal histoblasts, gut imaginal cells and imaginal salivary gland). Ance expression was particularly strong in the presumptive adult midgut formed as a result of massive proliferation of the imaginal midgut cells soon after pupariation. No Ance transcripts were detected in the midgut of the fully differentiated adult intestine. Ance protein and mRNA were not detected in imaginal discs from wandering larvae of flies homozygous for the ecd1 allele, a temperature-sensitive ecdysone-less mutant, suggesting that Ance expression is ecdysteroid-dependent. Physiological levels of 20-hydroxyecdysone induced the synthesis of ACE-like activity and Ance protein by a wing disc cell line (Cl.8+), confirming that Ance is an ecdysteroid-responsive gene. We propose that the expression of Ance in imaginal cells is co-ordinated by exposure to ecdysteroid (moulting hormone) during the last larval instar moult to increase levels of ACE-like activity during metamorphosis. The enzyme activity may be required for the processing of a developmental peptide hormone or may function in concert with other peptidases to provide amino acids for the synthesis of adult proteins.
Signal transducers and activators of transcription 3 (STAT3) inhibits transcription of the inducible nitric oxide synthase gene by interacting with nuclear factor κB
Authors: YU Zhiyuan , ZHANG Wenzheng , KONE Bruce C.
Published: September 2002

Prolific generation of NO by inducible nitric oxide synthase (iNOS) can cause unintended injury to host cells during glomerulonephritis and other inflammatory diseases. While much is known about the mechanisms of iNOS induction, few transcriptional repressors have been found. We explored the role of signal transducers and activators of transcription 3 (STAT3) proteins in interleukin (IL)-1β- and lipopolysaccharide (LPS)+interferon (IFN)-γ-mediated iNOS induction in murine mesangial cells. Both stimuli induced rapid phosphorylation of STAT3 and sequence-specific STAT3 DNA-binding activity. Supershift assays with a STAT3 element probe demonstrated that nuclear factor κB (NF-κB) p65 and p50 complexed with STAT3 in the DNA—protein complex. The direct interaction of STAT3 and NF-κB p65 was verified in vivo by co-immunoprecipitation and in vitro by pull-down assays with glutathione S-transferase-NF-κB p65 fusion protein and in vitro-translated STAT3α. Overexpression of STAT3 dramatically inhibited IL-1β- or LPS+IFN-γ-mediated induction of iNOS promoter-luciferase constructs that contained the wild-type iNOS promoter or ones harbouring mutated STAT-binding elements. In tests of indirect inhibitory effects of STAT3, overexpression of STAT3 dramatically inhibited the activity of an NF-κB-dependent promoter devoid of STAT-binding elements without affecting NF-κB DNA-binding activity. Thus STAT3, via direct interactions with NF-κB p65, serves as a dominant-negative inhibitor of NF-κB activity to suppress indirectly cytokine induction of the iNOS promoter in mesangial cells. These results provide a new model for the termination of NO production by activated iNOS following exposure to pro-inflammatory stimuli.
Overexpression and functional characterization of an ABC (ATP-binding cassette) transporter encoded by the genes drrA and drrB of Mycobacterium tuberculosis
Authors: CHOUDHURI Baisakhee Saha , BHAKTA Sanjib , BARIK Rajib , BASU Joyoti , KUNDU Manikuntala , CHAKRABARTI Parul
Published: September 2002

The genes encoding ATP-binding cassette (ABC) transporters occupy 2.5% of the genome of Mycobacterium tuberculosis. However, none of these putative ABC transporters has been characterized so far. We describe the development of expression systems for simultaneous expression of the ATP-binding protein DrrA and the membrane integral protein DrrB which together behave as a functional doxorubicin efflux pump. Doxorubicin uptake in Escherichia coli or Mycobacterium smegmatis expressing DrrAB was inhibited by reserpine, an inhibitor of ABC transporters. The localization of DrrA to the membrane depended on the simultaneous expression of DrrB. ATP binding was positively regulated by doxorubicin and daunorubicin. At the same time, DrrB appeared to be sensitive to proteolysis when expressed alone in the absence of DrrA. Simultaneous expression of the two polypeptides was essential to obtain a functional doxorubicin efflux pump. Expression of DrrAB in E. coli conferred 8-fold increased resistance to ethidium bromide, a cationic compound. 2′,7′-bis-(2-Carboxyethyl)-5(6)-carboxyfluorescein (BCECF), a neutral compound, also behaved as a substrate of the reconstituted efflux pump. When expressed in M. smegmatis, DrrAB conferred resistance to a number of clinically relevant, structurally unrelated antibiotics. The resistant phenotype could be reversed by verapamil and reserpine, two potent inhibitors of ABC transporters.