bluuebunny/crossref_metadata_2025 huggingface dataset:
Cross‐talk between posttranscriptionally silenced neomycin phosphotransferase II transgenes
Authors: Van Houdt Helena , Kovařı́k Ales , Van Montagu Marc , Depicker Anna
Published: July 2002

Tobacco plants containing a transgene locus with two chimeric neomycin phosphotransferase II (nptII) genes in tail‐to‐tail orientation (locus 1) show posttranscriptional gene silencing. The silenced nptII transgenes of locus 1 can downregulate the expression of homologous nptII transgenes in hybrid plants. The 3′ region of the silenced nptII genes located in the center of the inverted repeat locus 1 is extensively methylated. Moreover, 3′ segments of in trans‐inactivated transgenes also become methylated, suggesting cross‐talk between homologous posttranscriptionally silenced genes. Our results are in accordance with the hypothesis that this cross‐talk can be mediated by specially featured RNAs.
Retinoic acid stimulates HIV‐1 transcription in human neuroblastoma SH‐SY5Y cells
Authors: Recio Juan A , Martı́nez de la Mata Jorge , Martı́n-Nieto José , Aranda Ana
Published: July 2002

Although the brain is an important target for the human immunodeficiency virus type 1 (HIV) and viral infection causes neuronal degeneration and dementia, the mechanisms responsible for HIV transcription in neuronal cells are largely unknown. We show here that retinoic acid (RA) stimulates HIV transcription in human neuronal SH‐SY5Y cells. The steroid receptor coactivator 1 (SRC‐1) enhances the transcriptional response to RA, and the viral protein Tat cooperates with RA and SRC‐1 to induce a strong transactivation. These results suggest that retinoid receptors could play an important role as activators of viral gene expression in the human brain.
The HslU ATPase acts as a molecular chaperone in prevention of aggregation of SulA, an inhibitor of cell division in Escherichia coli
Authors: Seong Ihn Sik , Oh Ji Yeon , Lee Jung Wook , Tanaka Keiji , Chung Chin Ha
Published: July 2002

HslVU is an ATP‐dependent protease consisting of two multimeric components: the HslU ATPase and the HslV peptidase. SulA, which is an inhibitor of cell division and has high tendency of aggregation, is degraded by HslVU protease. Here we show that HslU plays a role not only as a regulatory component for the HslV‐mediated proteolysis but also as a molecular chaperone. Purified HslU prevented aggregation of SulA in a concentration‐dependent fashion. This chaperone activity required oligomerization of HslU subunits, which could be achieved by ATP‐binding or in the presence of high HslU protein concentrations. hsl mutation reduced the SulA‐mediated inhibition of cell growth and this effect could be reversed upon overproduction of HslU, suggesting that HslU promotes the ability of SulA to block cell growth through its chaperone function. Thus, HslU appears to have two antagonistic functions: one as a chaperone for promotion of the ability of SulA in cell growth inhibition by preventing SulA aggregation and the other as the regulatory component for elimination of SulA by supporting the HslV‐mediated degradation.
The Yersinia Ser/Thr protein kinase YpkA/YopO directly interacts with the small GTPases RhoA and Rac‐1
Authors: Barz Corinna , Abahji Thomas N , Trülzsch Konrad , Heesemann Jürgen
Published: July 2002

Pathogenic bacteria of the genus Yersinia counteract host defense by interfering with eukaryotic signal transduction pathways. YpkA of Yersinia pseudotuberculosis shares significant homology with eukaryotic Ser/Thr protein kinases, is translocated into the host cell and has been shown to be an essential virulence factor in a mouse infection model. In this study, we identify the small GTPases RhoA and Rac‐1 as eukaryotic binding partners of YpkA and its homolog YopO of Yersinia enterocolitica. We demonstrate that the interaction is independent of phosphorylation of YpkA and nucleotide loading state of the GTPases. The interaction with RhoA and Rac‐1 might provide an important clue to how YpkA interferes with eukaryotic signaling on a molecular level.
A novel peptide, THALWHT, for the targeting of human airway epithelia
Authors: Jost P.J. , Harbottle R.P. , Knight A. , Miller A.D. , Coutelle C. , Schneider H.
Published: July 2002

Targeting gene vectors to human airway epithelial cells may help to overcome the current inefficiency of gene transfer as the major problem confronting cystic fibrosis gene therapy. To elucidate novel ligands targeting abundant, apically located receptors on airway epithelial cells, a phage display library was screened for peptides binding with high affinity to such cells. This screening yielded a selectively enriched amino acid sequence, Thr‐His‐Ala‐Leu‐Trp‐His‐Thr (THALWHT). Subsequent binding studies confirmed that THALWHT‐displaying phages bound much stronger than phages displaying control peptides to human airway epithelial cells. In contrast, no significant binding differences were observed on a variety of non‐airway‐derived human cell lines suggesting selective binding of the THALWHT motif to airway epithelia. Confocal microscopy of such cells after exposure to labelled synthetic THALWHT peptide indicated that its binding is followed by specific internalisation via endocytosis. A synthetic peptide comprising a cyclic CTHALWHTC domain and a DNA binding moiety enabled efficient targeted gene delivery into human airway epithelial cells. Competition assays with free THALWHT peptide confirmed the specificity of gene delivery. Thus, the THALWHT motif may prove a useful targeting moiety for both non‐viral and viral gene therapy vectors.
Negative regulation of receptor tyrosine kinase signals
Authors: Fiorini Monia , Alimandi Maurizio , Fiorentino Loredana , Sala Gianluca , Segatto Oreste
Published: July 2002

In Metazoans a number of cellular functions are controlled by receptor tyrosine kinases (RTKs) during development and in postnatal life. The execution of these programs requires that signals of adequate strength are delivered for the appropriate time within precise spatial boundaries. Several RTK inhibitors have been identified in invertebrate and mammalian organisms. Because they are involved in tuning and termination of receptor signals, negative regulators of RTK activity fulfill a fundamental function in the control of receptor signaling.
MDM2 and MDMX can interact differently with ARF and members of the p53 family
Authors: Wang XiaoQi , Arooz Talha , Siu Wai Yi , Chiu Clarissa H.S , Lau Anita , Yamashita Katsumi , Poon Randy Y.C
Published: July 2002

Members of the p53 family of transcription factors have essential roles in tumor suppression and in development. MDM2 is an essential regulator of p53 that can inhibit the transcriptional activity of p53, shuttle p53 out of the nucleus, and target p53 for ubiquitination‐mediated degradation. Little is known about the interaction and selectivity of different members of the p53 family (p53, p63, and p73) and the MDM2 family (MDM2 and MDMX). Here we show that the transcriptional activities of p53 and p73, but not that of p63, were inhibited by both MDM2 and MDMX. Consistent with these, we found that MDMX can physically interact with p53 and p73, but not with p63. Moreover, ectopically expressed MDM2 and MDMX could induce alterations in the subcellular localization of p73, but did not affect the subcellular localization of p53 and p63. Finally, we demonstrate that while ARF can interact with MDM2 and inhibit the regulation of p53 by MDM2, no interaction was found between ARF and MDMX. These data reveal that significant differences and selectivity exist between the regulation of different members of the p53 family by MDM2 and MDMX.
Five isoforms of the phosphatidylinositol 3‐kinase regulatory subunit exhibit different associations with receptor tyrosine kinases and their tyrosine phosphorylations
Authors: Inukai Kouichi , Funaki Makoto , Anai Motonobu , Ogihara Takehide , Katagiri Hideki , Fukushima Yasushi , Sakoda Hideyuki , Onishi Yukiko , Ono Hiraku , Fujishiro Midori , Abe Miho , Oka Yoshitomo , Kikuchi Masatoshi , Asano Tomoichiro
Published: July 2002

There are five isoforms of the regulatory subunit for the heterodimeric type of phosphatidylinositol 3‐kinase. These five regulatory subunit isoforms were overexpressed using an adenovirus transfection system, and their own tyrosine phosphorylations and associations with various tyrosine kinase receptors were investigated. When overexpressed in CHO‐PDGFR cells, the associations of these regulatory subunit isoforms with the platelet‐derived growth factor receptor were similar. However, when overexpressed in CHO‐IR cells, p55γ exhibited a significantly lower ability to bind with IRS‐1 upon insulin stimulation, as compared with other regulatory subunit isoforms. Furthermore, p55α and p55γ were found to be tyrosine‐phosphorylated. Finally, interestingly, when overexpressed in CHO‐EGFR cells or A431 cells and stimulated with epidermal growth factor (EGF), phosphorylated EGF receptor was detected in p85α, p85β and p50α immunoprecipitates, but not in p55α and p55γ immunoprecipitates. In addition, EGF‐induced tyrosine phosphorylation was observed in p85α, p85β, p55α and p55γ, but not in p50α, immunoprecipitates. Thus, each regulatory subunit exhibits specific responses regarding both the association with tyrosine‐phosphorylated substrates and its own tyrosine phosphorylation. These results suggest that each isoform possesses specific roles in signal transduction, based on its individual tyrosine kinase receptor.
Photoactivation and calcium sensitivity of the fluorescent NO indicator 4,5‐diaminofluorescein (DAF‐2): implications for cellular NO imaging
Authors: Broillet Marie-Christine , Randin Olivier , Chatton Jean-Yves
Published: July 2002

The fluorescent indicator of nitric oxide (NO), 4,5‐diaminofluorescein (DAF‐2), and its membrane‐permeable derivative (DAF‐2 diacetate) have been recently developed to perform real‐time biological imaging of NO. In this study, we show that DAF‐2 is strongly influenced by factors other than the concentration of NO itself. Using measurements with a fluorimeter as well as fluorescence microscopy, we found that the divalent cation concentration in the medium, as well as the incident light, strongly affects the ability of DAF‐2 to detect NO. Calcium, in particular, enhanced the signal detection of NO released by NO donors by up to 200 times. With multiple and longer exposures to light, no bleaching of the dye was observed but, instead, a potentiation of the fluorescence response could be measured. While these two properties will affect the use and interpretation of the hitherto acquired data with this fluorescent compound, they may also open up new possibilities for its application.
Nuclear localisation of calreticulin in vivo is enhanced by its interaction with glucocorticoid receptors
Authors: Roderick H.Llewelyn , Campbell Anthony K , Llewellyn David H
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Sphingosine induces apoptosis in androgen‐independent human prostatic carcinoma DU‐145 cells by suppression of bcl‐XL gene expression
Authors: Shirahama Tsutomu , Sakakura Chohei , Sweeney Elizabeth A. , Ozawa Masayuki , Takemoto Masakazu , Nishiyama Kenryu , Ohi Yoshitada , Igarashi Yasuyuki
Published: July 2002

Our recent studies have suggested that sphingosine, an endogenous protein kinase C (PKC) inhibitor, may mediate apoptosis induced by a phorbol ester (PMA) in human promyelocytic leukemia HL‐60 cells [Ohta et al. Cancer Res. 1995;55:691–697], and that the apoptotic induction by both PMA and sphingosine is accompanied by down‐regulation of bcl‐2, a gene which acts to prevent apoptotic cell death [Sakakura et al. FEBS Lett. 1996;397:177–180]. In this study, we examined the sphingosine‐induced apoptosis of the androgen‐independent human prostatic carcinoma cell line DU‐145, which expresses bcl‐XL and Bax but not bcl‐2, and found that treatment of DU‐145 cells with sphingosine suppressed bcl‐XL in both mRNA and protein levels but did not change bax expression at all. In contrast, in apoptotic cells treated with a PKC inhibitor, staurosporine, no effect on bcl‐XL or bax expression was observed. The initial metabolites of sphingosine in the cells, ceramide and sphingosine 1‐phosphate, failed to induce apoptosis. These results indicate that, in DU‐145 cells, sphingosine, but not its metabolites, induces apoptosis through down‐regulation of bcl‐XL, independently of PKC inhibition. Our present results, together with previous observations, strongly suggest that apoptosis regulatory genes differ according to cell type and apoptosis induction through sphingosine is accompanied by inhibition of either bcl‐2 or bcl‐XL activity in these cells.
The post‐translational modifications of Ral and Rac1 are important for the action of Ral‐binding protein 1, a putative effector protein of Ral
Authors: Matsubara Kenji , Hinoi Takao , Koyama Shinya , Kikuchi Akira
Published: July 2002

Ral‐binding protein 1 (RalBP1) is a putative effector protein of Ral and possesses the GTPase‐activating activity for Rac1 and CDC42. We examined the roles of the post‐translational modifications of Ral and Rac1 for the action of RalBP1. In COS cells, RalG23V, a constitutively active form, was mainly detected in the membrane fraction while most of RalG23V/C203S, a Ral mutant which is not post‐translationally modified, was found in the cytosol fraction. When RalBP1 was expressed alone in COS cells, it was found in the cytosol but not in the membrane fraction. When RalBP1 was coexpressed with RalG23V, a part of RalBP1 was found in the membrane fraction. However, when RalBP1 was coexpressed with RalG23V/C203S, all of RalBP1 was recovered in the cytosol fraction. Although Ral bound to RalBP1 at a molar ratio of 1:1, the interaction of Ral with RalBP1 did not affect the GTPase‐activating activity of RalBP1 for Rac1. Furthermore, RalBP1 was more active on the post‐translationally modified form of Rac1 and CDC42 than the unmodified form. These results suggest that the post‐translational modification of Ral is important for the subcellular localization of RalBP1 and that the interaction of Ral with RalBP1 is not essential for the activity of RalBP1 but plays a role in recruiting RalBP1 to the membrane where its substrates, Rac1 and CDC42, reside.
Metabolic analysis of S. cerevisiae strains engineered for malolactic fermentation
Authors: Bony M , Bidart F , Camarasa C , Ansanay V , Dulau L , Barre P , Dequin S
Published: July 2002

A complete malolactic fermentation was achieved using Saccharomyces cerevisiae strains coexpressing the genes mleS and mae1 coding for the Lactococcus lactis malolactic enzyme and the Schizosaccharomyces pombe malate permease under the control of yeast promoters. The expression level of mae1 greatly influences the kinetics of the reaction by controlling the rate of malate uptake meanwhile a high expression level of mleS induces a partial consumption of malate derived from glucose by the malolactic enzyme. A strain expressing several copies of mae1 and one copy of mleS degrades 3 g/l of malate almost exclusively through the malolactic pathway in 4 days under enological conditions, without metabolic side effects.
Mutational analysis of human prothymosin α reveals a bipartite nuclear localization signal
Authors: Rubtsov Yuri P. , Zolotukhin Andrei S. , Vorobjev Ivan A. , Chichkova Nina V. , Pavlov Nickolay A. , Karger Elena M. , Evstafieva Alexandra G. , Felber Barbara K. , Vartapetian Andrey B.
Published: July 2002

Mutants of human prothymosin α with impaired ability to inhibit yeast Saccharomyces cerevisiae. cerevisiae cell growth were characterized. Two types of prothymosin α‐inactivating mutations were observed. Mutations that belong to the first type compromised the nuclear entry of prothymosin α by affecting its nuclear localization signal. Analysis of subcellular distribution of GFP‐prothymosin α fusions revealed a bipartite nuclear localization signal that is both necessary and sufficient for nuclear import of the protein in human cells. Mutations of the second type abrogated the inhibitory action of prothymosin α through an unknown mechanism, without influencing the nuclear import of the protein.
Novel components and enzymatic activities of the human erythrocyte plasma membrane calcium pump
Authors: Reusch Rosetta N , Huang Ruiping , Kosk-Kosicka Danuta
Published: July 2002

The plasma membrane Ca2+ pump is essential for the maintenance of cystolic calcium ion concentration levels in eukaryotes. Here we show that the Ca2+‐ATPase, purified from human erythrocytes, contains two homopolymers, poly(3‐hydroxybutyrate) (PHB) and inorganic polyphosphate (polyP), which form voltage‐activated calcium channels in the plasma membranes of Escherichia coli and other bacteria. Furthermore, we demonstrate that the plasma membrane Ca2+‐ATPase may function as a polyphosphate kinase, i.e. it exhibits ATP‐polyphosphate transferase and polyphosphate‐ADP transferase activities. These findings suggest a novel supramolecular structure for the functional Ca2+‐ATPase, and a new mechanism of uphill Ca2+ extrusion coupled to ATP hydrolysis.
Evidence for protein dolichylation
Authors: Hjertman Magnus , Wejde Johan , Dricu Anica , Carlberg Magdalena , Griffiths William J. , Sjövall Jan , Larsson Olle
Published: July 2002

Labeling of human colon carcinoma cells with [3H]dol, followed by extensive delipidation and removal of dol‐P oligosaccharides, showed that dol are bound to cellular proteins with sizes of 5, 10, 27, 75 and >140 kDa. HPLC purification of proteolytic products of [3H]dol‐ and [35S]cys‐labeled proteins revealed a hydrophobic peak containing both dol and cysteine. The dol/cys‐labeled products were clearly separated from GG‐cys, and exhibited a hydrophobicity between that of dol‐P and dol. In another set of experiments delipidated proteins were treated with methyl iodide, which cleaves thioether bonds. After HPLC purification of released dol‐like lipids, these were subjected to mass spectrometry. This demonstrated molecular ions with the same mass as that of dol. Taken together our data provide evidence for the existence of proteins covalently modified with dol.
Fostriecin, an antitumor antibiotic with inhibitory activity against serine/threonine protein phosphatases types 1 (PP1) and 2A (PP2A), is highly selective for PP2A
Authors: Walsh Aimée H. , Cheng Aiyang , Honkanen Richard E.
Published: July 2002

Fostriecin, an antitumor antibiotic produced by Streptomyces pulveraceus, is a strong inhibitor of type 2A (PP2A; IC50 3.2 nM) and a weak inhibitor of type 1 (PP1; IC50 131 μM) serine/threonine protein phosphatases. Fostriecin has no apparent effect on the activity of PP2B, and dose‐inhibition studies conducted with whole cell homogenates indicate that fostriecin also inhibits the native forms of PP1 and PP2A. Studies with recombinant PP1/PP2A chimeras indicate that okadaic acid and fostriecin have different binding sites.
Evidence for intracellular generation of angiotensin II in rat juxtaglomerular cells
Authors: Mercure Chantal , Ramla Djamel , Garcia Raul , Thibault Gaétan , Deschepper Christian F , Reudelhuber Timothy L
Published: July 2002

The formation of the vasoactive peptide angiotensin II (AII) is dependent on the sequential action of two enzymes, renin and angiotensin converting enzyme (ACE), on the substrate angiotensinogen. Although the renin‐producing cells of the kidney do not express angiotensinogen, they contain large amounts of AII in the same storage granules that contain renin. When renin expression is suppressed in these cells, AII also disappears. In the current study, we have tested whether the renin‐associated disappearance of AII in renal juxtaglomerular (JG) cells is due to a renin‐dependent down‐regulation of granule biosynthesis and whether receptor‐mediated internalization of AII could account for its concentration in these cells. Our results support a model whereby AII peptides are generated within JG cells, presumably by a mechanism which involves the action of endogenous renin on internalized, exogenous angiotensinogen.
The solution structure of DNA decamer duplex containing the Dewar product of thymidylyl(3′→5′)thymidine by NMR and full relaxation matrix refinement
Authors: Lee Joon-Hwa , Hwang Geum-Sook , Kim Jong-Ki , Choi Byong-Seok
Published: July 2002

The (6‐4) adducts and their Dewar isomers play an important role in cytotoxicity and mutation in skin cells exposed to sunlight. Structural study of the DNA duplex containing a site‐specific photoproduct is an essential step toward understanding the molecular mechanism of the mutagenesis and the repair activity of UV‐irradiated DNA. Here we use 1H NMR spectroscopy and full relaxation matrix refinement to investigate the solution structure of the duplex Dewar decamer. We find that the isomerization of the (6‐4) adduct to its Dewar form induces a substantial change in overall structure of the oligonucleotide duplex. Contrasting base stacking of two lesion sites results in a large difference in the structural impacts induced by the two photoproducts, such as differential disruption of hydrogen bonding at the lesion sites and overall helical bending of 44° by the (6‐4) lesion and 21° by the Dewar lesion.
Cloning and characterization of the gene encoding the human peroxisomal assembly protein Pex3p1
Authors: Kammerer Stefan , Holzinger Andreas , Welsch Ulrich , Roscher Adelbert A
Published: July 2002

Proteins essential for the assembly of functional peroxisomes are designated peroxins and are encoded by PEX genes. In yeast, Pex3p was previously identified as a peroxisomal integral membrane protein indispensable for peroxisome biogenesis and integrity. Here we report the cloning of the orthologous human PEX3 gene. It encodes a polypeptide of 373 amino acids (42 kDa) and is expressed in all tissues examined. As shown by transfection of epitope tagged constructs and immunofluorescence analysis, human Pex3p is localized at the peroxisome. The N‐terminal 40 amino acids were revealed to be sufficient to target a GFP reporter protein to the peroxisome. A positively charged five amino acid sequence within this N‐terminal region is highly conserved from yeast to human Pex3p. Overexpression of human Pex3p leads to proliferation of ER membranes in COS7 cells. Since disruption of human peroxins has been shown to result in peroxisomal biogenesis disorders, PEX3 is another candidate gene being involved in this disease group.
Photosynthetic reaction centers
Authors: Allen J.P. , Williams J.C.
Published: July 2002

The reaction center is the key component for the primary events in the photochemical conversion of light into chemical energy. After excitation by light, a charge separation that spans the cell membrane is formed in the reaction center in a few hundred picoseconds with a quantum yield of essentially one. A conserved pattern in the cofactors and core proteins of reaction centers from different organisms can be defined based on comparisons of the three dimensional structure of two types of reaction centers. Different functional aspects of the reaction center are discussed, including the properties of the bacteriochlorophyll or chlorophyll dimer that constitutes the primary electron donor, the pathway of electron transfer, and the different functional roles of the electron acceptors. The implication of these results on the evolution of the reaction center is presented.
Identification of the active site of legumain links it to caspases, clostripain and gingipains in a new clan of cysteine endopeptidases
Authors: Chen Jinq-May , Rawlings Neil D. , Stevens Richard A.E. , Barrett Alan J.
Published: July 2002

We show by site‐directed mutagenesis that the catalytic residues of mammalian legumain, a recently discovered lysosomal asparaginycysteine endopeptidase, form a catalytic dyad in the motif His‐Gly‐spacer‐Ala‐Cys. We note that the same motif is present in the caspases, aspartate‐specific endopeptidases central to the process of apoptosis in animal cells, and also in the families of clostripain and gingipain which are arginyl/lysyl endopeptidases of pathogenic bacteria. We propose that the four families have similar protein folds, are evolutionarily related in clan CD, and have common characteristics including substrate specificities dominated by the interactions of the S1 subsite.
Hepatitis B core particles as a universal display model: a structure‐function basis for development
Authors: Pumpens Paul , Grens Elmars
Published: July 2002

Because it exhibits a remarkable capability to accept mutational intervention and undergo correct folding and self‐assembly in all viable prokaryotic and eukaryotic expression systems, hepatitis B core (HBc) protein has been favored over other proposed particulate carriers. Structurally, the unusual α‐helical organization of HBc dimeric units allows introduction of foreign peptide sequences into several areas of HBc shells, including their most protruding spikes. Progress toward full resolution of the spatial structure as well as accumulation of chimeric HBc‐based structures has brought closer the knowledge‐based design of future vaccines, gene therapy tools and other artificial particulate objects.
Inhibition of platelet aggregation by S‐nitroso‐cysteine via cGMP‐independent mechanisms: evidence of inhibition of thromboxane A2 synthesis in human blood platelets
Authors: Tsikas Dimitrios , Ikic Milos , Tewes Kathrin S. , Raida Manfred , Frölich Jürgen C.
Published: July 2002

S‐Nitroso‐cysteine (SNC), a putative endothelium‐derived relaxing factor, potently inhibited collagen‐ and arachidonic acid‐induced platelet aggregation (IC50=100 nM) and thromboxane A2 (TxA2) synthesis of human blood platelets. ODQ, a selective inhibitor of the soluble guanylyl cyclase, inhibited SNC‐induced formation of cGMP but did not reverse inhibition by SNC of collagen‐ and arachidonic acid‐induced platelet aggregation. Combination of ODQ with SQ‐29548, a specific platelet TxA2 receptor antagonist, did not modify the antiaggregatory action of SNC. Our study shows that SNC inhibits platelet aggregation by cGMP‐independent mechanisms that may involve inhibition of TxA2 synthesis in human platelets.
Purification and characterization of hydrogenase from the marine green alga, Chlorococcum littorale
Authors: Ueno Yoshiyuki , Kurano Norihide , Miyachi Shigetoh
Published: July 2002

Hydrogenase from the marine green alga, Chlorococcum littorale, was purified 1485‐fold, resulting in a specific activity for hydrogen evolution of 75.7 μmol/min/mg of protein at 25°C, using reduced methyl viologen as an electron donor. The K
m value for methyl viologen was 0.5 mM. The purity of the enzyme was judged by native PAGE. The molecular weight was estimated to be 55 kDa by SDS‐PAGE, and 57 kDa by gel filtration. The optimum temperature and pH value for hydrogen evolution were 50°C and 7.5, respectively. The partially purified hydrogenase catalyzed hydrogen evolution from ferredoxin that had been isolated from the same cells, but not from NADH or NADPH. The K
m value for ferredoxin was 0.68 μM. The enzyme was extremely oxygen sensitive, losing over 95% of its activity upon exposure to air within minutes, even at 4°C. Two peptide fragments were obtained from the hydrogenase protein digested enzymatically, and their amino acid sequences were determined. No significant homology was found to any other known sequences of hydrogenases.
Growth phase‐dependent subcellular localization of nitric oxide synthase in maize cells
Authors: Ribeiro Euripedes A , Cunha Fernando Q , Tamashiro Wirla M.S.C , Martins Ione S
Published: July 2002

A protein band of approximately 166 kDa was detected in the soluble fraction of root tips and young leaves of maize seedlings, based on Western blot analysis using antibodies raised against mouse macrophage nitric oxide synthase (NOS) and rabbit brain NOS. NOS activity was present in these soluble fractions, as determined by l‐[U‐14C]citrulline synthesis from l‐[U‐14C]arginine. Immunofluorescence showed that the maize NOS protein is present in the cytosol of cells in the division zone and is translocated into the nucleus in cells in the elongation zone of maize root tips. These results indicate the existence of a NOS enzyme in maize tissues, with the localization of this protein depending on the phase of cell growth.
Differential subcellular localization of endogenous and transfected soluble epoxide hydrolase in mammalian cells: evidence for isozyme variants
Authors: Mullen Robert T , Trelease Richard N , Duerk Heike , Arand Michael , Hammock Bruce D , Oesch Franz , Grant David F
Published: July 2002

Endogenous, constitutive soluble epoxide hydrolase in mice 3T3 cells was localized via immunofluorescence microscopy exclusively in peroxisomes, whereas transiently expressed mouse soluble epoxide hydrolase (from clofibrate‐treated liver) accumulated only in the cytosol of 3T3 and HeLa cells. When the C‐terminal Ile of mouse soluble epoxide hydrolase was mutated to generate a prototypic putative type 1 PTS (‐SKI to ‐SKL), the enzyme targeted to peroxisomes. The possibility that soluble epoxide hydrolase‐SKI was sorted slowly to peroxiosmes from the cytosol was examined by stably expressing rat soluble epoxide hydrolase‐SKI appended to the green fluorescent protein. Green fluorescent protein soluble epoxide hydrolase‐SKI was strictly cytosolic, indicating that ‐SKI was not a temporally inefficient putative type 1 PTS. Import of soluble epoxide hydrolase‐SKI into peroxisomes in plant cells revealed that the context of ‐SKI on soluble epoxide hydrolase was targeting permissible. These results show that the C‐terminal ‐SKI is a non‐functional putative type 1 PTS on soluble epoxide hydrolase and suggest the existence of distinct cytosolic and peroxisomal targeting variants of soluble epoxide hydrolase in mouse and rat.
The induction of GSH synthesis by nanomolar concentrations of NO in endothelial cells: a role for γ‐glutamylcysteine synthetase and γ‐glutamyl transpeptidase
Authors: Moellering Douglas , Mc Andrew Joanne , Patel Rakesh P , Forman Henry Jay , Mulcahy R.Timothy , Jo Hanjoong , Darley-Usmar Victor M
Published: July 2002

Nitric oxide protects cells from oxidative stress through a number of direct scavenging reactions with free radicals but the effects of nitric oxide on the regulation of antioxidant enzymes are only now emerging. Using bovine aortic endothelial cells as a model, we show that nitric oxide, at physiological rates of production (1–3 nM/s), is capable of inducing the synthesis of glutathione through a mechanism involving γ‐glutamylcysteine synthetase and γ‐glutamyl transpeptidase. This novel nitric oxide signalling pathway is cGMP‐independent and we hypothesize that it makes an important contribution to the anti‐atherosclerotic and antioxidant properties of nitric oxide.
Absorption and metabolism of cyanidin 3‐O‐β‐D‐glucoside in rats
Authors: Tsuda Takanori , Horio Fumihiko , Osawa Toshihiko
Published: July 2002

We have clarified for the first time how cyanidin 3‐O‐β‐D‐glucoside (C3G), which is a potent antioxidant anthocyanin, is absorbed and metabolized in vivo. Rats were orally administered C3G (0.9 mmol/kg body weight), and C3G rapidly appeared in the plasma. However, the aglycon of C3G (cyanidin; Cy) was not detected, although it was present in the jejunum. Protocatechuic acid (PC), which may be produced by degradation of Cy, was present in the plasma and the concentration was 8‐fold higher than that of C3G. These results suggest that plasma PC and C3G may contribute to the antioxidant activity of the plasma. In the liver and kidney, C3G was metabolized to methylated C3G (methyl‐C3G), suggesting that C3G and/or methyl‐C3G act as antioxidants in the tissues.
Spermine triggers the activation of caspase‐3 in a cell‐free model of apoptosis
Authors: Stefanelli Claudio , Bonavita Francesca , Stanic’ Ivana , Pignatti Carla , Flamigni Flavio , Guarnieri Carlo , Caldarera Claudio M
Published: July 2002

Polyamines are ubiquitous organic cations required for cell proliferation. However, some evidence suggested that their excessive accumulation can induce apoptosis. We show here that, in a post‐nuclear extract from U937 cells, the addition of spermine triggers the death program, represented by cytochrome c exit from mitochondria, the dATP‐dependent processing of pro‐caspase‐3 and the onset of caspase activity. Spermine is more effective than spermidine, whereas putrescine has no effect. Polyamine acetylation abolishes their pro‐apoptotic power. These data demonstrate a direct mechanism responsible for polyamine toxicity and also suggest that an excessive elevation of free polyamines could be involved in the transduction of a death signal.
Identification of a novel gene of the X,K‐ATPase β‐subunit family that is predominantly expressed in skeletal and heart muscles1
Authors: Pestov Nikolay B. , Adams Gail , Shakhparonov Mikhail I. , Modyanov Nikolai N.
Published: July 2002

We have identified the fifth member of the mammalian X,K‐ATPase β‐subunit gene family. The human and rat genes are largely expressed in skeletal muscle and at a lower level in heart. The deduced human and rat proteins designated as βmuscle (βm) consist of 357 and 356 amino acid residues, respectively, and exhibit 89% identity. The sequence homology of βm proteins with known Na,K‐ and H,K‐ATPase β‐subunits are 30.5–39.4%. Unlike other β‐subunits, putative βm proteins have large N‐terminal cytoplasmic domains containing long Glu‐rich sequences. The data obtained indicate the existence of hitherto unknown X,K‐ATPase (most probably Na,K‐ATPase) isozymes in muscle cells.
Identification of a nuclear localization signal, RRMKWKK, in the homeodomain transcription factor PDX‐1
Authors: Moede Tilo , Leibiger Barbara , Pour Hamedeh Ghanaat , Berggren Per-Olof , Leibiger Ingo B
Published: July 2002

Pancreatic duodenal homeobox‐containing transcription factor 1 (PDX‐1) plays a crucial role in pancreas development and β‐cell gene regulation. Absence of PDX‐1 leads to pancreas agenesis and its malfunction causes MODY4 diabetes mellitus. PDX‐1 has been suggested to be involved in the glucose‐dependent regulation of insulin gene transcription. Whereas DNA‐binding and transactivation domains of PDX‐1 are in the process of being characterized, protein sequences responsible for its nuclear translocation remain unknown. By combining site‐directed mutagenesis of putative phosphorylation sites and nuclear localization signal (NLS) motifs with on‐line monitoring of GFP‐tagged PDX‐1 translocation, we demonstrate that the NLS motif RRMKWKK is necessary and in conjunction with the integrity of the ‘helix 3’ domain of the PDX‐1 homeodomain is sufficient for the nuclear import of PDX‐1. Furthermore, we show that there is no glucose‐dependent cytoplasmic‐nuclear cycling of PDX‐1.
Evidence for a crucial role of neutrophil‐derived serine proteases in the inactivation of interleukin‐6 at sites of inflammation
Authors: Bank U. , Küpper B. , Reinhold D. , Hoffmann T. , Ansorge S.
Published: July 2002

The bioactivity of interleukin‐6 (IL‐6) was found to be dramatically reduced in fluids from sites of inflammation. Here, we provide evidence that the neutrophil‐derived serine proteases elastase, proteinase 3 and cathepsin G are mainly involved in its degradation and subsequent inactivation. The initially hydrolyzed peptide bonds were detected to be Val11‐Ala12 and Leu19‐Thr20 (elastase), Phe78‐Asn79 (cathepsin G) and Ala145‐Ser146 (proteinase 3). The soluble IL‐6 receptor elicits a protective effect against the IL‐6 inactivation by cathepsin G only. The inactivation of IL‐6 by neutrophil‐derived serine proteases might act as a feedback mechanism terminating the IL‐6‐induced activation of neutrophils.
Induced expression of adipophilin mRNA in human macrophages stimulated with oxidized low‐density lipoprotein and in atherosclerotic lesions
Authors: Wang Xinkang , Reape Theresa J. , Li Xiang , Rayner Kim , Webb Christine L. , Burnand Kevin G. , Lysko Paul G.
Published: July 2002

Oxidized low‐density lipoprotein (OxLDL) plays a critical role in foam cell formation and atherosclerogenesis. A cDNA encoding adipophilin was identified in cultured human macrophages stimulated with OxLDL using mRNA differential display. Adipophilin is a 50 kDa protein known to be a specific marker for adipocyte cell differentiation and lipid accumulation in a variety of cells. The time‐dependent induction of adipophilin mRNA in macrophages was specific to OxLDL but not native LDL, and not to various cytokines and serum. In human atherosclerotic lesions, adipophilin mRNA expression was localized in a subset of lipid‐rich macrophages. These data suggest that adipophilin‐expressing macrophages may represent foam cells and this gene expression is likely to be associated with the lipid accumulation in foam cells of the atherosclerotic lesions.
AATF, a novel transcription factor that interacts with Dlk/ZIP kinase and interferes with apoptosis1
Authors: Page Grit , Lödige Inga , Kögel Donat , Scheidtmann Karl Heinz
Published: July 2002

Dlk, also known as ZIP kinase, is a serine/threonine kinase that is tightly associated with nuclear structures. Under certain conditions, which require cytoplasmic localization, Dlk can induce apoptosis. In search for interaction partners that might serve as regulators or targets of this kinase we identified apoptosis antagonizing transcription factor (AATF), a nuclear phosphoprotein of 523 amino acids. The 1.8 kb mRNA seems to be ubiquitously expressed. AATF contains an extremely acidic domain and a putative leucine zipper characteristic of transcription factors. Indeed, a Gal4‐BD‐AATF fusion protein exhibited strong transactivation activity. Interestingly, AATF interfered with Dlk‐induced apoptosis.
Absolute concentrations of mRNA for type I and type VI collagen in the canine meniscus in normal and ACL‐deficient knee joints obtained by RNase protection assay
Authors: Wildey G. M. , Billetz A. C. , Matyas J. R. , Adams M. E. , McDevitt C. A.
Published: July 2002

Relatively little is known about the cellular and molecular responses of the knee joint meniscus to joint injury, despite the functional importance of the tissue. We investigated how meniscus cells respond to joint injury in the early stages of post‐traumatic osteoarthritis by characterizing the changes in matrix gene expression in menisci at 3 and 12 weeks post‐surgery in dogs in which the anterior cruciate ligament (ACL) in one joint was transected and the other unoperated joint served as a control. Changes in the total RNA and DNA concentrations of the menisci were determined. Absolute concentrations of the mRNA of the COL1A1 gene of type 1 collagen, the major fibrillar collagen of the meniscus, and the COL6A3 gene of type VI collagen, a major repair molecule, were determined by quantitative ribonuclease (RNase) protection assay. The concentration of total RNA in medial and lateral menisci increased from 40 to 60 μg RNA/g wet wt in unoperated, control joints to 200—350 μg RNA/g wet wt in ACL‐deficient joints. No significant changes were detected in the concentration of DNA (900—1200 μg DNA/g wet wt). Low concentrations of COL1A1 (2—3 pmol mRNA/g DNA) and COL6A3 (0.3–0.6 pmol mRNA/g DNA) mRNA transcripts were measured in normal menisci. ACL‐deficiency induced a 20–38 fold increase in COL1A1 and COL6A3 mRNA concentration at 3 weeks, and an 11–19 fold increase at 12 weeks post‐surgery. In general, the increase in COL1A1 and COL6A3 mRNA concentrations was greater in medial menisci than in lateral menisci. These results demonstrate that the menisci initiate a vigorous biosynthetic response to transection of the ACL. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
No effect of verapamil on the local bone response to in vivo mechanical loading
Authors: Samnegård Eva , Cullen Diane M. , Akhter Mohammed P. , Kimmel Donald B.
Published: July 2002

Verapamil, a calcium channel blocker, alters the intracellular calcium concentration in bone cells in vitro, while mechanical loading stimulates calcium channels. The purpose of this study was to examine the effect of systemic verapamil treatment on the bone response to in vivo external mechanical loading. Female rats (age 5–6 months) were divided into six groups. Half were verapamil treated (0.75 mg/ml drinking water) for 12 weeks. After 8 weeks of treatment, the right tibia was loaded by a four‐point bending device. In one set of verapamil and control groups, the right tibia was loaded at 31.8 ± 0.2 N (36 cycles, 2 Hz, 3 d/wk) for four weeks. A second set was loaded at 40.1 plusmn; 0.3 N and the third set remained nonloaded. Tibial cortical bone formation and femur bone mineral density (BMD) were evaluated. With loading, bone formation was similarly elevated in loaded tibia of verapamil and control rats (P < 0.003). However, periosteal bone formation (P < 0.001) in the nonloaded tibia, and femoral diaphysis BMD (P < 0.04) were greater in verapamil rats than in controls.

We conclude that verapamil, in the dose given, does not interfere with mechanical loading (30, 40 N) at the loaded site and that the voltage‐dependent calcium channels, blocked by verapamil, are not significantly involved in the local bone response to increased strain in female rats. However, verapamil increased bone formation and BMD at nonloaded sites of loaded rats. Previously unknown systemic or regional factors associated with loading may explain the potential mechanisms for this interaction and need further investigation. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Brain‐derived neurotrophic factor and neurotrophin‐3 levels in Alzheimer's disease brains
Authors: Durany Nuria , Michel Tanya , Kurt Jellinger , Cruz‐Sánchez Felix F. , Cervós‐Navarro Jorge , Riederer Peter
Published: July 2002

In the present study, we investigated the levels of brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) in post‐mortem brain tissue of Alzheimer's disease (AD) patients, and we observed a significant increase of BDNF concentration in hippocampus and parietal cortex of AD patients, as well as a negative correlation between NT‐3 levels and age in hippocampus and putamen of control subjects, and for BDNF in frontal cortex. A defining feature of AD is the post‐mortem identification of neuritic plaques and neurofibrillary tangles in the brain, however, a more significant neuropathological finding is the degeneration of cholinergic neurones of the basal forebrain, critically involved in memory and cognition. Neurotrophic factors are partly responsible for the maintenance of neuronal function and structural integrity in the adult brain. Our results provide, therefore, evidence that, under conditions of progressive neurodegeneration the brain stimulates the over expression of certain neurotrophic factors as a possible mechanisms of compensation, and that during senescence the expression of these molecules is regulated.
Pain modulation role of melatonin in eating disorders
Authors: Papezová H. , Yamamotová A. , Nedvídková J.
Published: July 2002

The objective of this study was to test the effect of melatonin on thermal pain threshold in female patients with eating disorders. Fourteen patients were included in the study. We found a parabolic relation between pain threshold and the content of urine sulfatoxymelatonin (r = 0.6299, P < 0.05). We can speculate that increase in severity of eating disorder pathology may decrease both the melatonin level and pain sensitivity. In contrast with expected melatonin analgesic effect, our results showed its possible normalizing influence as well on pathologically decreased pain sensitivity.
Attitude towards psychiatric patients: a pilot study in a northern Italian town
Authors: Vezzoli R. , Archiati L. , Buizza C. , Pasqualetti P. , Rossi G. , Pioli R.
Published: July 2002

The stigmatisation of the psychiatric patient is still a strong barrier to the integration process of these subjects in our society. Therefore, it is necessary to identify what types of prejudices exist and the types of variables with which they are linked, in order to plan strategies to reduce them. In this exploratory study we administered a semi-structured interview to 303 subjects in order to examine the relationships between social attitudes towards mental disorders and some social demographic variables, the information about mental disorders and the previous ‘contacts’ with psychiatric patients.

The data analysis shows that there is a relationship between knowledge of psychiatric patients and a more positive attitude towards them (i.e., less fear, more integration and work opportunities). This study is to be considered preliminary as far as people’s attitudes towards psychiatric patients are concerned. This result encourages the planning and implementation of sensitisation and information programs concerning mental disorders, in the sense that increasing the knowledge of mental disorders could lead to significant achievements in the important fight against the stigma surrounding psychiatric patients.
Follow-up study of 67 first episode schizophrenic patients and their involvement in religious activities
Authors: Huguelet P , Binyet-Vogel S , Gonzalez C , Favre S , McQuillan A
Published: July 2002

We studied the characteristics of religious practice in a cohort of 67 first admission schizophrenic patients over 5 years. Thirty percent of these patients were involved in religious activities, either with an established religion or in a marginal group. They were mostly women, who had a good premorbid psychosocial adaptation and tended not to be substance abusers. Their social adaptation was improved at year 5. They were as compliant with their ambulatory treatment as the other patients. However, when controlling for the inclusion characteristics, a similar outcome was shown between the group of practicing patients and the nonpracticing group. Religious activity may not be by itself the cause of this favorable outcome, as it is probable that only the patients who are less symptomatic and relatively well adapted could actually join a religious movement. The fact that many schizophrenic patients find an occupation and relationships in religious activities that they would not find elsewhere should encourage the psychiatric community target its occupational goals.
Stock selection based on Morningstar’s ten-year, five-star general equity mutual funds
Authors: Loviscek Anthony L. , Jordan W. John
Published: July 2002

Recent research suggests that the individual investor can build stock portfolios that outperform broad market indices. Based on this research and on evidence supporting the persistence of mutual fund performance, we test whether or not the individual investor can build market-superior portfolios from stocks selected from the top holdings of Morningstar’s ten-year, five-star general equity mutual funds. We use modern portfolio theory to construct the portfolios. Although the portfolios tend to outperform the S&P 500 for the 1990s, we conclude that the evidence is not strong enough to recommend this stock selection strategy to the individual investor. © 2000 Elsevier Science Inc. All rights reserved.