bluuebunny/crossref_metadata_2025 huggingface dataset:
Roles of synorganisation, zygomorphy and heterotopy in floral evolution: the gynostemium and labellum of orchids and other lilioid monocots
Authors: RUDALL PAULA J. , BATEMAN RICHARD M.
Published: September 2002

A gynostemium, comprising stamen filaments adnate to a syncarpous style, occurs in only three groups of monocots: the large family Orchidaceae (Asparagales) and two small genera Pauridia (Hypoxidaceae: Asparagales) and Corsia (Corsiaceae, probably in Liliales), all epigynous taxa. Pauridia has actinomorphic (polysymmetric) flowers, whereas those of Corsia and most orchids are strongly zygomorphic (monosymmetric) with a well‐differentiated labellum. In Corsia the labellum is formed from the outer median tepal (sepal), whereas in orchids it is formed from the inner median tepal (petal) and is developmentally adaxial (but positionally abaxial in orchids with resupinate flowers). Furthermore, in orchids zygomorphy is also expressed in the stamen whorls, in contrast to Corsia. In Pauridia a complete stamen whorl is suppressed, but the ‘lost’ outer whorl is fused to the style. The evolution of adnation and zygomorphy are discussed in the context of the existing phylogenetic framework in monocotyledons. An arguably typological classification of floral terata is presented, focusing on three contrasting modes each of peloria and pseudopeloria. Dynamic evolutionary transitions in floral morphology are assigned to recently revised concepts of heterotopy (including homeosis) and heterochrony, seeking patterns that delimit developmental constraints and allow inferences regarding underlying genetic controls. Current evidence suggests that lateral heterotopy is more frequent than acropetal heterotopy, and that full basipetal heterotopy does not occur. Pseudopeloria is more likely to generate a radically altered yet functional perianth, but is also more likely to cause acropetal modification of the gynostemium. These comparisons indicate that there are at least two key genes or sets of genes controlling adnation, adaxial stamen suppression and labellum development in lilioid monocots; at least one is responsible for stamen adnation to the style (i.e. gynostemium formation), and another controls adaxial stamen suppression and adaxial labellum formation in orchids. Stamen adnation to the style may be a product of over‐expression of the genes related to epigyny (i.e. a form of hyper‐epigyny). If, as seems likely, stamen‐style adnation preceded zygomorphy in orchid evolution, then the flowers of Pauridia may closely resemble those of the immediate ancestors of Orchidaceae, although existing molecular phylogenetic data indicate that a sister‐group relationship is unlikely. The initial radiation in Orchidaceae can be attributed to the combination of hyper‐epigyny, zygomorphy and resupination, but later radiations at lower taxonomic levels that generated the remarkable species richness of subfamilies Orchidoideae and Epidendroideae are more likely to reflect more subtle innovations that directly influence pollinator specificity, such as the development of stalked pollinaria and heavily marked and or spur‐bearing labella.
Carbohydrates: a limit on bacterial diversity within the colon
Authors: RABIU BODUN A. , GIBSON GLENN R.
Published: September 2002

The human large intestine is recognised as a physiologically important organ responsible for the conservation of water and salts. Through its resident bacteria, it is also capable of complex, enzyme catalysed, hydrolyticdigestive functions that have a high biological impact on the host. These microorganisms metabolise dietary components, principally complex carbohydrates that are not hydrolysed or absorbed in the upper gastrointestinal tract, and in this way, sequester energy for the host, through fermentation. This process involves a series of anaerobic, energy‐yielding, catabolic reactions which complete digestive processes in the gut, resulting in end products that in turn influence the distribution of microbial species present as well as having some systemic effects. Some of the bacteria are thought to possess important health‐promoting activities, especially with respect to their influence on mucosal and systemic immune responses to disease. These bioactivities can be modulated by substrates that support and influence microbial development, growth and survival. For these reasons, it is necessary to review dietary factors that may delimit bacterial diversity, to be able to predict responses and sensitivities to various environmental pressures and manipulations that occur in this area of human microbiology.
The Influence of Culture and of Institutional Factors in Social Policy: French Social Policy in Martinique
Authors: Révauger Jean-Paul
Published: September 2002

The standards and structure of social policy in Martinique are now very similar to those in France. However, in spite of its funding by France, welfare remains problematic. Although the staff are local, the structure and concepts are French, which technically makes policy implementation difficult, and creates uneasiness. The implementation of French welfare in Martinique runs counter to the local politics of identity and the drive for autonomy. Welfare focuses the chief ambiguity of Martinique, which craves for local control, but would like to maintain the current level of funding from Europe.
Neither Welfare State nor Welfare Society: The Case of Hong Kong
Authors: Wong Chack-kie , Chau Kenneth Kin-lam , Wong Timothy Ka-ying
Published: September 2002

This article points to the simple fact that modern welfare systems are neither welfare states nor are they welfare societies. In reality, they are a combination of both. The current interpretation of the supposed dichotomy of these two institutions has inhibited our understanding of reality. Consequently, this has obscured the complicated ways in which the state and the society have worked together to harmonize the needs for social protection and active citizenship. In the present article, the example of a Chinese welfare system has been used to illustrate that even a welfare system usually not regarded as a welfare state can develop strong universal welfare state programmes. In order to demonstrate the significance of this situation, findings related to public attitudes have been cited to reflect the support of both policy assumptions underlying social protection and active citizenship in the welfare system that is under review.
Themed Section on the Policy Process and Social Policy
Authors: Bochel Hugh M.
Published: September 2002

Despite the frequent comparisons and discussions of continuity between the Conservatives' social policies and those of New Labour, it is certainly true that the mechanisms which each has used to develop and implement social policy have been rhetorically, and often practically, different. Under the Conservatives the emphasis on markets and marketisation, with the centrality of the ‘consumer’, was reflected in social policy including through devices such as privatisation, compulsory competitive tendering, the creation of internal markets, managerialism and the use of ‘Next Steps’ agencies for delivery. New Labour's approach has made familiar terms such as ‘joined-up’ government, ‘evidence-based’ policy, ‘partnership’, ‘modernisation’ and ‘democratic renewal’. And, whatever might be said about their social policies, New Labour have shown a degree of radicalism in their governmental and constitutional reforms, most notably perhaps through devolution to Northern Ireland, Scotland and Wales, and the passing of the Human Rights Act. At the same time, the EU has been developing as a political and decision-making entity with an emerging social dimension, that has, as Duncan argues here, the potential for some impact on UK social policy.
Policy Discourses on ‘Reconciling Work and Life’ in the EU
Authors: Duncan Simon
Published: September 2002

This paper outlines the development EU policy discourse on ‘the reconciliation of work and family life’. This imposes a policy disjuncture on New Labour, for, while the British government may be ideologically more attracted to the liberal US model of ‘flexible’ labour, it is bound by EU law to implement a more corporatist gender equality model. The paper notes how themes of economic competition, democratisation, and protecting gender contracts emerged at the foundation EU gender policy. It traces these themes into an ‘equal opportunities at work’ discourse during the 1970s and 1980s and, with the increasing importance of the ‘demographic time bomb’ discourse and of Scandinavian style gender equality, into discourses stressing the ‘reconciliation of paid work with family life’ and gender mainstreaming. The paper ends by addressing the ‘half-empty or half-full’ assessments of EU gender policy.
Delivery Structures and Policy Development in Post-Devolution Scotland
Authors: Parry Richard
Published: September 2002

The policy process in the devolved Scottish system reconciles the Scottish themes of delivering social policy from the centre, through channels of advice and professional direction, and the New Labour theme of broad social policy strategies aiming at better service delivery and employment outcomes. Beneath the surface issues there is a trend to re-structure some services. The Scottish Executive's strategy Social Justice, set out in annual reports, relates devolved and non-devolved responsibilities in a way that has implications for the structure of Executive departments and the policy-making demands made upon civil servants. The research reported here uses interviews with officials to explore the structures of policy making in the Executive within a context of expectations about ministerial and official roles inherited from the previous administrative devolution.
Health Policy in Wales – Distinctive or Derivative?
Authors: McClelland Siobhan
Published: September 2002

This article examines the policy process in Wales prior to the introduction of a National Assembly for Wales and uses this as a framework for assessing the potential impact of devolution. Health and social services form the largest part of the budget of the National Assembly for Wales and the article focuses on health policy as a key area of interest for Assembly Members. The period 1992 to 1997 demonstrates an emphasis on managing both demand and finances thereby promoting the continued dominance, in terms of both resource allocation and prioritisation, of the acute hospital sector. The article then explores how far this has changed and what potential there is for changing this assumptive reality in a New Labour and devolved NHS.
Who is for Kids and Who is just Kidding? Members of the Israeli Knesset's Activities on Children's Issues between 1990–2001
Authors: Ben-Arieh Asher
Published: September 2002

This article reports the findings of a study which explored the activities of members of four Israeli Knessets (MKs) between 1990 and 2001 (the 12th to the 15th Knessets), and sought to explore possible connections between their activities in relation to child welfare and a number of social and political variables. The study was based on archival and quantitative data pertaining to MKs' activities. A short overview of the Israeli political system and the child welfare policy arena is presented, focusing on the changes they both went through between 1990 and 2001. The variety of MKs' activities on issues concerning children are described and possible connections between social and political variables and their activities are explored.
Putting the ‘Policy’ back into Social Policy
Authors: Newman Janet
Published: September 2002

This paper argues that the study of social policy can be enriched by a greater focus on the dynamics of the policy process itself. Such a focus needs to transcend the usual descriptive accounts of institutions and implementation methods. The paper draws on a range of theoretical approaches that illuminate the shaping and delivery of policy, from classic theories of power and the state to analyses of the micro-dynamics of the policy–action relationship. The paper explores the contribution of recent developments in governance theory, and assesses contemporary debates about the ‘modernisation’ of the policy process and the focus on evaluating ‘what works’ in social policy.
Iron chelation-induced senescence-like growth arrest in hepatocyte cell lines: association of transforming growth factor β1 (TGF-β1)-mediated p27Kip1 expression
Authors: YOON Gyesoon , KIM Hyun-Jung , YOON Young-Sil , CHO Hyeseong , LIM In K. , LEE Jae-Ho
Published: September 2002

Iron is essential for cellular proliferation in all organisms. When deprived of iron, the growth of cells is invariably inhibited. However, the mechanism involved remains largely unclear. In the present study, we have observed that subcytotoxic concentrations of desferroxamine mesylate (DFO), an iron chelator, specifically inhibited the transition from G1 to S-phase of Chang cells, a hepatocyte cell line. This was accompanied by the appearance of senescent biomarkers, such as enlarged and flattened cell morphology, senescence-associated β-galactosidase activity and reduced expression of poly(ADP-ribose) polymerase. Concomitantly, p27Kip1 (where Kip is kinase-inhibitory protein) was induced markedly, whereas other negative cell-cycle regulators, such as p21Cip1 (where Cip is cyclin-dependent kinase-interacting protein), p15INK4B and p16INK4A (where INK is inhibitors of cyclin-dependent kinase 4), were not, implying its association in the G1 arrest. Furthermore, the induction of p27Kip1 was accompanied by an increased level of transforming growth factor β1 (TGF-β1) mRNA. When neutralized with an anti-(TGF-β1) antibody, p27Kip1 induction was completely abolished, indicating that TGF-β1 is the major inducer of p27Kip1. Finally, DFO-induced senescence-like arrest was found to be independent of p53, since cell-cycle arrest was still observed with two p53-negative cell lines, Huh7 and Hep3B cells. In conclusion, DFO induced senescence-like G1 arrest in hepatocyte cell lines and this was associated with the induction of p27Kip1 through TGF-β1, but was independent of p53.
Antizyme induction by polyamine analogues as a factor of cell growth inhibition
Authors: MITCHELL John L.A. , LEYSER Aviva , HOLTORFF Michelle S. , BATES Jill S. , FRYDMAN Benjamin , VALASINAS Aldonia L. , REDDY Venodhar K. , MARTON Laurence J.
Published: September 2002

The polyamines spermidine and spermine and their diamine precursor putrescine are essential for mammalian cell growth and viability, and strategies are sought for reducing polyamine levels in order to inhibit cancer growth. Several structural analogues of the polyamines have been found to decrease natural polyamine levels and inhibit cell growth, probably by stimulating normal feedback mechanisms. In the present study, a large selection of spermine analogues has been tested for their effectiveness in inducing the production of antizyme, a key protein in feedback inhibition of putrescine synthesis and cellular polyamine uptake. Bisethylnorspermine, bisethylhomospermine, 1,19-bis-(ethylamino)-5,10,15-triazanonadecane, longer oligoamine constructs and many conformationally constrained analogues of these compounds were found to stimulate antizyme synthesis to different levels in rat liver HTC cells, with some producing far more antizyme than the natural polyamine spermine. Uptake of the tested compounds was found to be dependent on, and limited by, the polyamine transport system, for which all these have approximately equal affinity. These analogues differed in their ability to inhibit HTC cell growth during 3days of exposure, and this ability correlated with their antizyme-inducing potential. This is the first direct evidence that antizyme is induced by several polyamine analogues. Selection of analogues with this potential may be an effective strategy for maximizing polyamine deprivation and growth inhibition.
Characterization of the human ephrin-A4 promoter
Authors: MUNTHE Else , AASHEIM Hans-Christian
Published: September 2002

Expression of the ephrin-A4 ligand, a family member of ligands binding the Eph receptor tyrosine kinases, is induced after an antigen–receptor stimulation of lymphocytes. To understand the transcription regulation of the ephrin-A4 gene, its promoter was identified and regulating elements were characterized. The ephrin-A4 promoter contains cis elements directing the cell-specific expression. By deletion studies, three specific regions, which were contributing to the transcription activity in lymphoid cells, were localized. In one of these regions, an inverted CCAAT box was identified and shown to bind the transcription activator nuclear factor-Y (NF-Y). The importance of NF-Y binding for the ephrin-A4 promoter activity is shown by a total abrogation of promoter activity after destruction of its binding site. NF-Y binding and activity are also crucially dependent on the integrity of the surrounding sequence. In addition, electrophoretic mobility-shift assay and serial-mutation analysis of the two remaining regulating regions revealed cis regulatory elements contributing to the transcription activity of the ephrin-A4 promoter.
Stereochemical features of the hydrolysis of 9,10-epoxystearic acid catalysed by plant and mammalian epoxide hydrolases
Authors: SUMMERER Stephan , HANANO Abdulsamie , UTSUMI Shigeru , ARAND Michael , SCHUBER Francis , BLÉE Elizabeth
Published: September 2002

cis-9,10-Epoxystearic acid was used as a tool to probe the active sites of epoxide hydrolases (EHs) of mammalian and plant origin. We have compared the stereochemical features of the hydrolysis of this substrate catalysed by soluble and membrane-bound rat liver EHs, by soluble EH (purified to apparent homogeneity) obtained from maize seedlings or celeriac roots, and by recombinant soybean EH expressed in yeast. Plant EHs were found to differ in their enantioselectivity, i.e. their ability to discriminate between the two enantiomers of 9,10-epoxystearic acid. For example, while the maize enzyme hydrated both enantiomers at the same rate, the EH from soybean exhibited very high enantioselectivity in favour of 9R,10S-epoxystearic acid. This latter enzyme also exhibited a strict stereoselectivity, i.e. it hydrolysed the racemic substrate with a very high enantioconvergence, yielding a single chiral diol product, threo-9R,10R-dihydroxystearic acid. Soybean EH shared these distinctive stereochemical features with the membrane-bound rat liver EH. The stereochemical outcome of these enzymes probably results from a stereoselective attack by the nucleophilic residue on the oxirane ring carbon having the (S)-configuration, leading to the presumed (in plant EH) covalent acyl–enzyme intermediate. In sharp contrast, the reactions catalysed by cytosolic rat liver EH exhibited a complete absence of enantioselectivity and enantioconvergence; this latter effect might be ascribed to a regioselective formation of the acyl–enzyme intermediate involving C-10 of 9,10-epoxystearic acid, independent of its configuration. Thus, compared with soybean EH, the active site of rat liver soluble EH displays a very distinct means of anchoring the oxirane ring of the fatty acid epoxides, and therefore appears to be a poor model for mapping the catalytic domain of plant EHs.
The family of hepatoma-derived growth factor proteins: characterization of a new member HRP-4 and classification of its subfamilies
Authors: DIETZ Frank , FRANKEN Sebastian , YOSHIDA Kenya , NAKAMURA Hideji , KAPPLER Joachim , GIESELMANN Volkmar
Published: September 2002

Hepatoma-derived growth factor (HDGF)-related proteins (HRPs) comprise a family of polypeptides named after HDGF, which was identified by its mitogenic activity towards fibroblasts. In the present study, we describe a hitherto unknown HRP, termed HRP-4. The cDNA of bovine HRP-4 (bHRP-4) predicts a polypeptide of 235 amino acids. Northern- and Western-blot analyses of various bovine tissues demonstrated that HRP-4 is only expressed in the testis. Recombinantly produced bHRP-4 and murine HDGF (mHDGF) histidine-tagged polypeptides display growth-factor activity for cultured primary human fibroblasts at an optimum concentration of 1ng/ml in serum-free medium. The growth-factor activity declines with increasing concentrations to reach background levels at 1μg/ml. The expression of the fusion proteins, bHRP-4–green fluorescent protein and mHDGF–green fluorescent protein, in HEK-293 cells demonstrates nuclear localization of the proteins. bHRP-4 and mHDGF bind to the glycosaminoglycans heparin and heparan sulphate, but not to chondroitin sulphate. Affinity constants determined for these interactions are between 6 and 42nM. Comparison of the bHRP-4 amino acid sequence with HRP-1–3 and p52/75/lens epithelium-derived growth factor (LEDGF) shows that these proteins share a conserved N-terminal part of 91 amino acids but have C-termini of different lengths and charge. This demonstrates the modular structure of these proteins and allows its classification into three groups based on charge, size and sequence comparison. HRP-4, HRP-1 and HDGF are small acidic proteins, HRP-3 is a small basic protein, and HRP-2 and p52/75/LEDGF are larger basic proteins.
Identification of two highly sialylated human tear-fluid DMBT1 isoforms: the major high-molecular-mass glycoproteins in human tears
Authors: SCHULZ Benjamin L. , OXLEY David , PACKER Nicolle H. , KARLSSON Niclas G.
Published: September 2002

Human open eye tear fluid was separated by low-percentage SDS/PAGE to detect high-molecular-mass protein components. Two bands were found with apparent molecular masses of 330 and 270kDa respectively. By peptide-mass fingerprinting after tryptic digestion, the proteins were found to be isoforms of the DMBT1 gene product, with over 30% of the predicted protein covered by the tryptic peptides. By using gradient SDS/agarose/polyacrylamide composite gel electrophoresis and staining for glycosylation, it was shown that the two isoforms were the major high-molecular-mass glycoproteins of >200kDa in human tear fluid. Western blotting showed that the proteins expressed sialyl-Lea. After the release of oligosaccharides by reductive β-elimination from protein blotted on to PVDF membrane, it was revealed by liquid chromatography-MS that the O-linked oligosaccharides were comprised mainly of highly sialylated oligosaccharides with up to 16 monosaccharide units. A majority of the oligosaccharides could be described by the formula dHex0→2NeuAc1→xHexxHexNAcx(-ol), x = 1–6, where Hex stands for hexose, dHex for deoxyhexose, HexNAc for N-acetylhexosamine and NeuAc for N-acetylneuraminate. The number of sialic acids in the formula is less than 5. Interpretation of collision-induced fragmentation tandem MS confirmed the presence of sialic acid and suggested the presence of previously undescribed structures carrying the sialyl-Lea epitopes. Small amounts of neutral and sulphated species were also present. This is the first time that O-linked oligosaccharides have been detected and described from protein variant of the DMBT1 gene.
Interaction of heparin with internally quenched fluorogenic peptides derived from heparin-binding consensus sequences, kallistatin and anti-thrombin III
Authors: PIMENTA Daniel C. , NANTES Iseli L. , SOUZA Eduardo S. de , BONNIEC Bernard Le , ITO Amando S. , TERSARIOL Ivarne L.S. , OLIVEIRA Vitor , JULIANO Maria A. , JULIANO Luiz
Published: September 2002

Internally quenched fluorogenic (IQF) peptides bearing the fluorescence donor/acceptor pair o-aminobenzoic acid (Abz)/N-(2,4-dinitrophenyl)ethylenediamine (EDDnp) at N- and C-terminal ends were synthesized containing heparin-binding sites from the human serpins kallistatin and antithrombin, as well as consensus heparin-binding sequences (Cardin clusters). The dissociation constant (Kd), as well as the stoichiometry for the heparin–peptide complexes, was determined directly by measuring the decrease in fluorescence of the peptide solution. Experimental procedures were as sensitive as those used to follow the fluorescence change of tryptophan in heparin-binding proteins. The conformation of the peptides and the heparin–peptide complexes were obtained from measurements of time-resolved fluorescence decay and CD spectra. Kallistatin (Arg300–Pro319)-derived peptide (HC2) and one derived from antithrombin III helix D [(AT3D), corresponding to Ser112–Lys139], which are the heparin-binding sites in these serpins, showed significant affinity for 4500Da heparin, for which Kd values were 17nM and 100nM respectively. The CD spectra of the heparin–HC2 peptide complex did not show any significant α-helix content, different from the situation with peptide AT3D, for which complex-formation with heparin resulted in 24% α-helix content. The end-to-end distance distribution and the time-resolved fluorescence-decay measurements agree with the CD spectra and Kd values. The synthetic α-methyl glycoside pentasaccharide AGA∗IAM (where A represents N,6-O-sulphated α-d-glucosamine; G, β-d-glucuronic acid; A∗, N,3,6-O-sulphated α-d-glucosamine; I, 2-O-sulphated α-l-iduronic acid; and AM, α-methyl glycoside of A) also binds to AT3D and other consensus heparin-binding sequences, although with lower affinity. The interaction of IQF peptides with 4500Da heparin was displaced by protamine. In conclusion, IQF peptides containing Abz/EDDnp as the donor/acceptor fluorescence pair are very promising tools for structure–activity relationship studies on heparin–peptide complexes, as well as for the development of new peptides as heparin reversal-effect compounds.
Soya phytoestrogens, genistein and daidzein, decrease apolipoprotein B secretion from HepG2 cells through multiple mechanisms
Authors: BORRADAILE Nica M. , DREU Linda E. de , WILCOX Lisa J. , EDWARDS Jane Y. , HUFF Murray W
Published: September 2002

Diets containing the soya-derived phytoestrogens, genistein and daidzein, decrease plasma cholesterol in humans and experimental animals. The mechanisms responsible for the hypocholesterolaemic effects of these isoflavones are unknown. The present study was conducted to determine if genistein and daidzein regulate hepatocyte cholesterol metabolism and apolipoprotein (apo) B secretion in cultured human hepatoma (HepG2) cells. ApoB secretion was decreased dose-dependently by up to 63% and 71% by genistein and daidzein (100μM; P<0.0001) respectively. In contrast, no effect on apoAI secretion was observed. Cellular cholesterol synthesis was inhibited 41% by genistein (100μM; P<0.005) and 18% by daidzein (100μM; P<0.05), which was associated with significant increases in 3-hydroxy-3-methylglutaryl-CoA reductase mRNA. Cellular cholesterol esterification was decreased 56% by genistein (100μM; P<0.04) and 29% by daidzein (100μM; P<0.04); however, mRNA levels for acyl-CoA:cholesterol acyltransferase (ACAT) 1 and ACAT2 were unaffected. At 100μM, both isoflavones equally inhibited the activities of both forms of ACAT in cells transfected with either ACAT1 or ACAT2. Genistein (100μM) and daidzein (100μM) significantly decreased the activity of microsomal triacylglycerol transfer protein (MTP) by 30% and 24% respectively, and significantly decreased MTP mRNA levels by 35% and 55%. Both isoflavones increased low-density lipoprotein (LDL)-receptor mRNA levels by 3- to 6-fold (100μM; P<0.03) and significantly increased the binding, uptake and degradation of 125I-labelled LDL, suggesting that enhanced reuptake of newly secreted apoB-containing lipoproteins contributed to the net decrease in apoB secretion. These results indicate that genistein and daidzein inhibit hepatocyte apoB secretion through several mechanisms, including inhibition of cholesterol synthesis and esterification, inhibition of MTP activity and expression and increased expression of the LDL-receptor.
Foxa3 (HNF-3γ) binds to and activates the rat proglucagon gene promoter but is not essential for proglucagon gene expression
Authors: LIU Yuanfang , SHEN Wei , BRUBAKER Patricia L. , KAESTNER Klaus H. , DRUCKER Daniel J.
Published: September 2002

Members of the Forkhead box a (Foxa) transcription factor family are expressed in the liver, pancreatic islets and intestine and both Foxa1 and Foxa2 regulate proglucagon gene transcription. As Foxa proteins exhibit overlapping DNA-binding specificities, we examined the role of Foxa3 [hepatocyte nuclear factor (HNF)-3γ] in control of proglucagon gene expression. Foxa3 was detected by reverse transcriptase PCR in glucagon-producing cell lines and binds to the rat proglucagon gene G2 promoter element in GLUTag enteroendocrine cells. Although Foxa3 increased rat proglucagon promoter activity in BHK fibroblasts, augmentation of Foxa3 expression did not increase proglucagon promoter activity in GLUTag cells. Furthermore, adenoviral Foxa3 expression did not affect endogenous proglucagon gene expression in islet or intestinal endocrine cell lines. Although Foxa3-/- mice exhibit mild hypoglycaemia during a prolonged fast, the levels of proglucagon-derived peptides and proglucagon mRNA transcripts were comparable in tissues from wild-type and Foxa3-/- mice. These findings identify Foxa3 as a member of the proglucagon gene G2 element binding-protein family that, unlike Foxa1, is not essential for control of islet or intestinal proglucagon gene expression in vivo.
Luteolin, an emerging anti-cancer flavonoid, poisons eukaryotic DNA topoisomerase I
Authors: CHOWDHURY Arnab Roy , SHARMA Shalini , MANDAL Suparna , GOSWAMI Anindya , MUKHOPADHYAY Sibabrata , MAJUMDER Hemanta K.
Published: September 2002

Luteolin, a naturally occurring flavonoid, is abundant in our daily dietary intake. It exhibits a wide spectrum of pharmacological properties, but little is known about its biochemical targets other than the fact that it induces topoisomerase II-mediated apoptosis. In the present study, we show that luteolin completely inhibits the catalytic activity of eukaryotic DNA topoisomerase I at a concentration of 40μM, with an IC50 of 5μM. Preincubation of enzyme with luteolin before adding a DNA substrate increases the inhibition of the catalytic activity (IC50 = 0.66μM). Treatment of DNA with luteolin before addition of topoisomerase I reduces this inhibitory effect. Subsequent fluorescence tests show that luteolin not only interacts directly with the enzyme but also with the substrate DNA, and intercalates at a very high concentration (>250μM) without binding to the minor groove. Direct interaction between luteolin and DNA does not affect the assembly of the enzyme–DNA complex, as evident from the electrophoretic mobility-shift assays. Here we show that the inhibition of topoisomerase I by luteolin is due to the stabilization of topoisomerase-I DNA-cleavable complexes. Hence, luteolin is similar to camptothecin, a class I inhibitor, with respect to its ability to form the topoisomerase I-mediated ‘cleavable complex'. But, unlike camptothecin, luteolin interacts with both free enzyme and substrate DNA. The inhibitory effect of luteolin is translated into concanavalin A-stimulated mouse splenocytes, with the compound inducing SDS–K+-precipitable DNA–topoisomerase complexes. This is the first report on luteolin as an inhibitor of the catalytic activity of topoisomerase I, and our results further support its therapeutic potential as a lead anti-cancer compound that poisons topoisomerases.
Oligomeric structure of proclavaminic acid amidino hydrolase: evolution of a hydrolytic enzyme in clavulanic acid biosynthesis
Authors: ELKINS Jonathan M. , CLIFTON Ian. J. , HERNÁNDEZ Helena , DOAN Linh X. , ROBINSON Carol V. , SCHOFIELD Christopher J. , HEWITSON Kirsty S.
Published: September 2002

During biosynthesis of the clinically used β-lactamase inhibitor clavulanic acid, one of the three steps catalysed by clavaminic acid synthase is separated from the other two by a step catalysed by proclavaminic acid amidino hydrolase (PAH), in which the guanidino group of an intermediate is hydrolysed to give proclavaminic acid and urea. PAH shows considerable sequence homology with the primary metabolic arginases, which hydrolyse arginine to ornithine and urea, but does not accept arginine as a substrate. Like other members of the bacterial sub-family of arginases, PAH is hexameric in solution and requires Mn2+ ions for activity. Other metal ions, including Co2+, can substitute for Mn2+. Two new substrates for PAH were identified, N-acetyl-(l)-arginine and (3R)-hydroxy-N-acetyl-(l)-arginine. Crystal structures of PAH from Streptomyces clavuligerus (at 1.75Å and 2.45Å resolution, where 1Å = 0.1nm) imply how it binds β-lactams rather than the amino acid substrate of the arginases from which it evolved. The structures also suggest how PAH selects for a particular alcohol intermediate in the clavam biosynthesis pathway. As observed for the arginases, each PAH monomer consists of a core of β-strands surrounded by α-helices, and its active site contains a di-Mn2+ centre with a bridging water molecule responsible for hydrolytic attack on to the guanidino group of the substrate. Comparison of structures obtained under different conditions reveals different conformations of a flexible loop, which must move to allow substrate binding.
A peptide mimic of an antigenic loop of α-human chorionic gonadotropin hormone: solution structure and interaction with a llama VHH domain
Authors: FERRAT Gilles , RENISIO Jean-Guillaume , MORELLI Xavier , SLOOTSTRA Jerry , MELOEN Rob , CAMBILLAU Christian , DARBON Hervé
Published: September 2002

The X-ray structure of a ternary complex between human chorionic gonadotropin hormone (hCG) and two Fvs recognizing its α and β subunits has been recently determined. The Fvs recognize the elongated hCG molecule by its two ends, one being the Leu-12–Cys-29 loop of the α subunit. We have designed and synthesized a 17-amino-acid peptide (named PepH14) derived from the sequence of this antigenic loop with the purpose of mimicking its three-dimensional structure and its affinity for antibodies. We have determined the solution structure of PepH14 by homonuclear NMR spectroscopy and derived distance restraints. Comparison of this structure with that of the corresponding antigenic loop of α-hCG reveals strong conformational similarities. In particular, the two pairs of residues that establish crucial contacts with the Fv fragment share the same conformation in PepH14 and in the authentic hormone loop. We propose a three-dimensional model of interaction of PepH14 with a llama VHH (VHH-H14) fragment cloned from a single-chain llama immunoglobulin raised against α-hCG. This model has been constrained by the chemical shift variations of the H14 1HN and 15N resonances monitored upon binding with PepH14. Mapping of the backbone chemical shift variations on the VHH structure determined by NMR indicates that PepH14 binds to VHH-H14 and forms a complex using the three complementary determining regions (CDRs). They define a shallow groove encompassing residues Thr-31, Ala-56, Tyr-59 and Trp-104 which have been shown to be in conformational exchange [Renisio, Pérez, Czisch, Guenneugues, Bornet, Frenken, Cambillau and Darbon (2002) Proteins 47, 546–555] and also Phe-37 and Ala-50. This groove is close to the hydrophobic interface area observed between VH and VL domains in Fvs from classical antibodies, which explains the rather lateral binding of PepH14 on the VHH.
The AtNFS2 gene from Arabidopsis thaliana encodes a NifS-like plastidial cysteine desulphurase
Authors: LÉON Sébastien , TOURAINE Brigitte , BRIAT Jean-François , LOBRÉAUX Stéphane
Published: September 2002

NifS-like proteins are cysteine desulphurases required for the mobilization of sulphur from cysteine. They are present in all organisms, where they are involved in iron–sulphur (Fe–S) cluster biosynthesis. In eukaryotes, these enzymes are present in mitochondria, which are the major site for Fe–S cluster assembly. The genome of the model plant Arabidopsis thaliana contains two putative NifS-like proteins. A cDNA corresponding to one of them was cloned by reverse-transcription PCR, and named AtNFS2. The corresponding transcript is expressed in many plant tissues. It encodes a protein highly related (75% similarity) to the slr0077-gene product from Synechocystis PCC 6803, and is predicted to be targeted to plastids. Indeed, a chimaeric AtNFS2–GFP fusion protein, containing one-third of AtNFS2 from its N-terminal end, was addressed to chloroplasts. Overproduction in Escherichia coli and purification of recombinant AtNFS2 protein enabled one to demonstrate that it bears a pyridoxal 5′-phosphate-dependent cysteine desulphurase activity in vitro, thus being the first NifS homologue characterized to date in plants. The putative physiological functions of this gene are discussed, including the attractive hypothesis of a possible role in Fe–S cluster assembly in plastids.
The human homologue of yeast ArgRIII protein is an inositol phosphate multikinase with predominantly nuclear localization
Authors: NALASKOWSKI Marcus M. , DESCHERMEIER Christina , FANICK Werner , MAYR Georg W.
Published: September 2002

The function of the transcription regulator ArgRIII in the expression of several genes involved in the metabolism of arginine in yeast has been well studied. It was previously reported that it is also an inositol phosphate multikinase and an important factor of the mRNA export pathway [reviewed by Shears (2000) Bioessays 22, 786–789]. In the present study we report the cloning of a full-length 1248-bp cDNA encoding a human inositol phosphate multikinase (IPMK). This protein has a calculated molecular mass of 47.219kDa. Functionally important motifs [inositol phosphate-binding site, ATP-binding site, catalytically important SSLL (Ser-Ser-Leu-Leu) domain] are conserved between the human IPMK and yeast ArgRIII. Bacterially expressed protein demonstrated an inositol phosphate multikinase activity similar to that of yeast ArgRIII. Ins(1,4,5)P3 is phosphorylated at positions 3 and 6 up to Ins(1,3,4,5,6)P5. The human IPMK fused with a fluorescent protein tag is localized predominantly in the nucleus when transiently expressed in mammalian cells. A basic cluster in the protein's C-terminus is positively involved in nuclear targeting. These findings are consistent with the concept of a nuclear inositol phosphate signalling and phosphorylation pathway in mammalian cells.
Inducible nitric oxide synthase (NOS2) expressed in septic patients is nitrated on selected tyrosine residues: implications for enzymic activity
Authors: LANONE Sophie , MANIVET Philippe , CALLEBERT Jacques , LAUNAY Jean-Marie , PAYEN Didier , AUBIER Michel , BOCZKOWSKI Jorge , MEBAZAA Alexandre
Published: September 2002

Tyrosine nitration is a post-translational protein modification with potentially significant biological implications. In the present study we demonstrate, for the first time, that tyrosine residues of human inducible nitric oxide synthase (NOS2) can be nitrated by peroxynitrite in vitro, leading to a decreased activity. Moreover, we show that NOS2 expressed in a skeletal muscle from septic patients is nitrated on selective tyrosine residues belonging to a canonic sequence. This phenomenon could be an endogenous mechanism of in vivo modulation of NOS2 enzymic activity.
New perspectives in the regulation of hepatic glycolytic and lipogenic genes by insulin and glucose: a role for the transcription factor sterol regulatory element binding protein-1c
Authors: FOUFELLE Fabienne , FERRÉ Pascal
Published: September 2002

The regulation of hepatic glucose metabolism has a key role in whole-body energy metabolism, since the liver is able to store (glycogen synthesis, lipogenesis) and to produce (glycogenolysis, gluconeogenesis) glucose. These pathways are regulated at several levels, including a transcriptional level, since many of the metabolism-related genes are expressed according to the quantity and quality of nutrients. Recent advances have been made in the understanding of the regulation of hepatic glycolytic, lipogenic and gluconeogenic gene expression by pancreatic hormones, insulin and glucagon and glucose. Here we review the role of the transcription factors forkhead and sterol regulatory element binding protein-1c in the inductive and repressive effects of insulin on hepatic gene expression, and the pathway that leads from glucose to gene regulation with the recently discovered carbohydrate response element binding protein. We discuss how these transcription factors are integrated in a regulatory network that allows a fine tuning of hepatic glucose storage or production, and their potential importance in metabolic diseases.
A novel form of the RelA nuclear factor κB subunit is induced by and forms a complex with the proto-oncogene c-Myc
Authors: CHAPMAN Neil R. , WEBSTER Gill A. , GILLESPIE Peter J. , WILSON Brian J. , CROUCH Dorothy H. , PERKINS Neil D.
Published: September 2002

Members of both Myc and nuclear factor κB (NF-κB) families of transcription factors are found overexpressed or inappropriately activated in many forms of human cancer. Furthermore, NF-κB can induce c-Myc gene expression, suggesting that the activities of these factors are functionally linked. We have discovered that both c-Myc and v-Myc can induce a previously undescribed, truncated form of the RelA(p65) NF-κB subunit, RelA(p37). RelA(p37) encodes the N-terminal DNA binding and dimerization domain of RelA(p65) and would be expected to function as a trans-dominant negative inhibitor of NF-κB. Surprisingly, we found that RelA(p37) no longer binds to κB elements. This result is explained, however, by the observation that RelA(p37), but not RelA(p65), forms a high-molecular-mass complex with c-Myc. These results demonstrate a previously unknown functional and physical interaction between RelA and c-Myc with many significant implications for our understanding of the role that both proteins play in the molecular events underlying tumourigenesis.
A novel pressure-jump apparatus for the microvolume analysis of protein–ligand and protein–protein interactions: its application to nucleotide binding to skeletal-muscle and smooth-muscle myosin subfragment-1
Authors: PEARSON David S. , HOLTERMANN Georg , ELLISON Patricia , CREMO Christine , GEEVES Michael A.
Published: September 2002

Reactions involving proteins frequently involve large changes in volume, which allows the equilibrium position to be perturbed by changes in pressure. Rapid changes in pressure can thus be used to initiate relaxation in pressure; however, this approach is seldom used, because it requires specialized equipment. We have built a microvolume (50μl) pressure-jump apparatus, powered by a piezoelectric actuator, based on the original design of Clegg and Maxfield [(1976) Rev. Sci. Instrum. 47, 1383–1393]. This equipment can apply pressure changes of ±20MPa (maximally) in time periods as short as 80μs and follow the resulting change in fluorescence signals. The system is relatively simple to use with fast (approx. 1min) exchange of samples. In the present study, we show that this system can perturb the binding of 2′(3′)-O-(N-methylanthraniloyl)-ADP to myosin subfragment-1(S1) from skeletal and smooth muscles. The kinetic data are consistent with previous work, and in addition show that, although 2′(3′)-O-(N-methylanthraniloyl)-ADP binds with a similar affinity to both proteins, the increase in molar volume for the skeletal-muscle S1 binding to ADP is half of that for the smooth-muscle protein. This high-volume change for smooth-muscle S1 may be related to the ability of ADP to induce a 23° tilt in the tail of S1 bound to actin.
Lysosomal degradation of cholecystokinin-(29–33)-amide in mouse brain is dependent on tripeptidyl peptidase-I: implications for the degradation and storage of peptides in classical late-infantile neuronal ceroid lipofuscinosis
Authors: BERNARDINI Francesca , WARBURTON Michael J.
Published: September 2002

Tripeptidyl peptidase-I (TPP-I) is a lysosomal exopeptidase which removes tripeptides from the N-terminus of small peptides. Mutations in the TPP-I gene result in a lethal neurodegenerative disease, classical late-infantile neuronal ceroid lipofuscinosis (CLN2). This disease is characterized by the accumulation of proteinaceous and autofluorescent material within the lysosomes of neurons, which undergo massive cell death during the course of the disease. The absence of TPP-I may result in the lysosomal accumulation of small peptides and proteins, which eventually compromises lysosomal functions critical to the survival of neurons. To investigate the metabolism of small peptides, we have studied the degradation of cholecystokinin-(29–33)-amide (GWMDF-NH2; cholecystokinin C-terminal pentapeptide) by lysosomal fractions isolated from mouse brain and several other tissues. GWMDF-NH2 is cleaved at only one peptide bond by brain lysosomes, to produce GWM and DF-NH2. Inhibitor studies demonstrate that this reaction is catalysed by TPP-I. In contrast, lysosomal fractions from other mouse tissues additionally cleave a second peptide bond to produce GW and MDF-NH2. Inhibitor studies indicate that this reaction is catalysed by dipeptidyl peptidase-I (DPP-I; cathepsin C). Inhibitors of TPP-I are sufficient to completely block the degradation of GWMDF-NH2 by brain, but inhibitors of both TPP-I and DPP-I are required to completely inhibit the degradation of GWMDF-NH2 by other mouse tissues. Enzyme assays confirm the low activity of DPP-I in brain. An unrelated neuropeptide, neuromedin B, is degraded by a pathway that is partially dependent on TPP-I. These results indicate that TPP-I is required for the partial or complete digestion of certain neuropeptides by brain lysosomes. In the absence of TPP-I, neuropeptides or their degradation products will accumulate in brain lysosomes and may contribute to the pathogenesis of CLN2. Other tissues are spared because they express another peptidase, DPP-I, which has extensive activity on peptides and can compensate for the loss of TPP-I.
Nucleotide binding to human uncoupling protein-2 refolded from bacterial inclusion bodies
Authors: JEKABSONS Mika B. , ECHTAY Karim S. , BRAND Martin D.
Published: September 2002

Experiments were performed to test the hypothesis that recombinant human uncoupling protein-2 (UCP2) ectopically expressed in bacterial inclusion bodies binds nucleotides in a manner identical with the nucleotide-inhibited uncoupling that is observed in kidney mitochondria. For this, sarkosyl-solubilized UCP2 inclusion bodies were treated with the polyoxyethylene ether detergent C12E9 and hydroxyapatite. Protein recovered from hydroxyapatite chromatography was approx. 90% pure UCP2, as judged by Coomassie Blue and silver staining of polyacrylamide gels. Using fluorescence resonance energy transfer, N-methylanthraniloyl-tagged purine nucleoside di- and tri-phosphates exhibited enhanced fluorescence with purified UCP2. Dissociation constants determined by least-squares non-linear regression indicated that the affinity of UCP2 for these fluorescently tagged nucleotides was 3–5μM or perhaps an order of magnitude stronger, depending on the model used. Competition experiments with [8-14C]ATP demonstrated that UCP2 binds unmodified purine and pyrimidine nucleoside triphosphates with 2–5μM affinity. Affinities for ADP and GDP were approx. 10-fold lower. These data indicate that: UCP2 (a) is at least partially refolded from sarkosyl-solubilized bacterial inclusion bodies by a two-step treatment with C12E9 detergent and hydroxyapatite; (b) binds purine and pyrimidine nucleoside triphosphates with low micromolar affinity; (c) binds GDP with the same affinity as GDP inhibits superoxide-stimulated uncoupling by kidney mitochondria; and (d) exhibits a different nucleotide preference than kidney mitochondria.