bluuebunny/crossref_metadata_2025 huggingface dataset:
Intracellular trafficking of glycosphingolipids: Role of sphingolipid activator proteins in the topology of endocytosis and lysosomal digestion
Authors: Sandhoff K. , Klein A.
Published: July 2002

Glycosphingolipids (GSL) are components of the outer leaflet of the plasma membrane (PM) of vertebrate tissues. Our current knowledge of GSL metabolism and their intracellular traffic has been derived from metabolic studies but the exact mechanisms by which GSLs are transported from sites of synthesis (endoplasmic reticulum and Golgi) to the sites of residence (PM) and degradation (lysosomes) have not been clearly defined. It is now established that components of the PM reach the lysosomal compartment mainly by endocytic membrane flow. According to a new model, GSLs derived from the PM are thought to end up in intra‐endosomal vesicles which could be delivered, by successive processes of membrane fission and fusion, along the endocytic pathway directly into the lumen of the lysosomes. Here the GSLs are degraded in a step‐wise manner by exohydrolases. However, the catabolism of membrane‐bound GSLs with short hydrophilic head groups needs the assistance of sphingolipid activator proteins (SAPs), which lift the GSLs from the plane of the membrane and present them for degradation to the lysosomal exohydrolases, which are usually water‐soluble. The inherited deficiency of one of these enzymes or SAPs causes the lysosomal storage of their respective GSL substrates. In the case of the simultaneous deficiency of all 4 different SAPs the storage of all GSLs with short hydrophilic head groups occurs within multivesicular bodies and/or intra‐lysosomal vesicles.
The crystal structures of reduced pseudoazurin from Alcaligenes faecalis S‐6 at two pH values
Authors: Vakoufari Eleni , Wilson Keith S. , Petratos Kyriacos
Published: July 2002

The structures of the reduced (Cu1+) blue‐copper protein pseudoazurin from Alcaligenes faecalis strain S‐6 are refined at pH 7.8 and 4.4 using X‐ray diffraction data to 1.8 Å resolution. The final R‐factors for the high and low pH structures are 0.178 and 0.177, respectively. Comparing the reduced pseudoazurin at pH 7.8 with the oxidised (Cu2+) molecule, small changes are observed in the vicinity of the copper site and on the protein surface. At pH 4.4 the copper substituent imidazole of His81 rotates away from the metal with a concurrent movement of the latter towards the plane of the remaining three ligands (Sγ‐Cys78, Nδl‐His40 and Sδ‐Met86) thus the geometry of the copper site becomes planar trigonal.
Mitochondrial calcium spiking: A transduction mechanism based on calcium‐induced permeability transition involved in cell calcium signalling
Authors: Ichas François , Jouaville Laurence S. , Sidash Sergueï S. , Mazat Jean-Pierre , Holmuhamedov Ekhson L.
Published: July 2002

We report reversible Ca2+‐induced Ca2+ release from mitochondria, which takes the form of Ca2+ spikes. Mitochondrial Ca2+ spiking is an all‐or‐none process with a threshold dependence on both the frequency and the amplitude of the Ca2+ pulses used as stimuli. This spiking relies on the transient operation of the mitochondrial permeability transition pore, and is initiated — in a threshold‐dependent manner — with inositol‐triphosphate‐mediated Ca2+ responses in permeabilized cells. Evidence that mitochondrial Ca2+‐induced Ca2+ release contributes to inositol‐triphosphate‐mediated Ca2+ responses in intact cells is also reported.
Generation of the topa quinone cofactor in bacterial monoamine oxidase by cupric ion‐dependent autooxidation of a specific tyrosyl residue
Authors: Matsuzaki Ryuichi , Fukui Toshio , Sato Hidetoshi , Ozaki Yukihiro , Tanizawa Katsuyuki
Published: July 2002

The quinone of 2,4,5‐trihydroxyphenylalanine (topa), recently identified as the covalently bound redox cofactor in copper amine oxidases, is encoded by a specific tyrosine codon. To elucidate the mechanism of its formation, the recombinant phenylethylamine oxidase of Arthrobacter globiformis has been overproduced in Escherichia coli and purified in a Cu2+‐deficient form. The inactive precursor enzyme thus obtained was dramatically activated upon incubation with Cu2+, concomitantly with the formation of the topa quinone at the position corresponding to Tyr382, occurring in the tetrapeptide sequence highly conserved in this class of enzymes. The topa quinone was produced only under aerobic conditions, but its formation required no external enzymatic systems. These findings demonstrate the Cu2+‐dependent autooxidation of a specific tyrosyl residue to generate the topa quinone cofactor.
Effect of netropsin, distamycin A and chromomycin A3 on the binding and cleavage reaction of DNA gyrase
Authors: Simon H. , Wittig B. , Zimmer C.
Published: July 2002

The influence of netropsin (Nt), distamycin A (Dst‐3) and chromomycin A3 (CHR) on the binding of gyrase from Streptomyces noursei to an 162 bp‐fragment of pBR 322 containing a strong gyrase cleavage site and on the gyrase mediated cleavage of this fragment was analyzed. Binding of the enzyme to the fragment is effectively inhibited by the GC‐specific drug CHR, but poorly influenced by Dst‐3, while Nt is ineffective. Cleavage of the fragment catalysed by the enzyme is inhibited by all three ligands but to different extent. Dst‐3 and Nt inhibit the enzyme cleavage reaction at 20‐ or 250‐fold higher concentration than that required for CHR. The inhibitory mechanism of CHR on gyrase‐DNA binding and cleavage may be related to a competitive interaction of the ligand to GC sequences located at and around the gyrase cleavage site. The fact that AT‐specific minor groove binders Dst‐3 and Nt poorly inhibit the binding of gyrase to the fragment due to the low amount of the AT basepair sequences contained in the fragment and their inhibitory influence on the cleavage step underlines the role of the DNA minor groove during enzyme action.
Selective inactivity of TGF‐β/decorin complexes
Authors: Hausser Heinz , Gröning Arndt , Hasilik Andrej , Schönherr Elke , Kresse Hans
Published: July 2002

Previous studies had shown that binding of TGF‐β to the small proteoglycan decorin results in its inactivation. Indeed, in osteosarcoma cells the addition of decorin prevented the TGF‐β1‐mediated up‐regulation of biglycan synthesis. However, the down‐regulation of proteoglycan‐100 remained unaltered. Even in the presence of a 100,000‐fold molar excess of decorin, TGF‐β1 was fully active in U937 monocytes with respect to the inhibition of cell proliferation. There was no inhibition of the TGF‐β‐mediated stimulation of the retraction of fibroblast‐populated collagen lattices. Thus, the formation of TGF‐β/decorin complexes leads to the neutralization of distinct effects only.
Succinic acid monomethyl ester protects rat pancreatic islet secretory potential against interleukin‐1β (IL‐1β) without affecting glutamate decarboxylase expression or nitric oxide production
Authors: Eizirik Décio L. , Welsh Nils , Niemann Audrey , Velloso Licio A. , Malaisse Willy J.
Published: July 2002

Rat pancreatic islets exposed to interleukin‐1β (IL‐1β) in the presence of succinic acid monomethyl ester (SAM) have a higher insulin release in response to glucose and higher glucose oxidation rates, as compared to islets exposed to IL‐1β alone. These beneficial effects of SAM were not accompanied by any decrease in IL‐1β‐induced nitric oxide (NO) production nor inhibition of aconitase activity. Moreover, SAM did not increase biosynthesis of glutamate decarboxylase. SAM apparently improves β‐cell function mostly by increasing the capacity of these cells to endure NO exposure and partial blockage of the Krebs cycle.
The rapid and reversible association of phosphofructokinase with myocardial membranes during myocardial ischemia
Authors: Hazen Stanley L. , Wolf Matthew J. , Ford David A. , Gross Richard W.
Published: July 2002

Myocardial calcium‐independent phospholipase A2 (PLA2) activity is mediated by a 400 kDa catalytic complex comprised of a tetramer of phosphofructokinase (PFK) and a 40 kDa catalytic subunit [1,2]. During myocardial ischemia, calcium‐independent PLA2 activity rapidly and reversibly translocates from the cytosol to a membrane‐associated compartment where it has been implicated as a mediator of ischemic damage [3,4]. Herein we demonstrate that the majority of both PFK mass and activity is translocated from the cytosol to a membrane‐associated compartment prior to the onset of irreversible myocytic injury and that translocated PFK is catalytically inactive while membrane‐associated. Furthermore, reperfusion of ischemic myocardium, or treatment of membranes derived from ischemic myocardium with ATP results in the conversion of both PFK mass and activity from its membrane‐associated state to a soluble, catalytically‐competent form. Collectively, these studies demonstrate that the concomitant changes in glycolysis and phospholipid hydrolysis during early myocardial ischemia result, at least in part, from the translocation of a common regulatory polypeptide critical in both processes.
Preference of calcium‐dependent interactions between calmodulin‐like domains of calpain and calpastatin subdomains
Authors: Takano Emiko , Ma Hong , Yang Hong Qiong , Maki Masatoshi , Hatanaka Masakazu
Published: July 2002

Calpastatin molecule contains four repeated inhibition domains, each having highly conserved internal regions A, B and C. The synthetic oligopeptides of regions A and C had no calpain inhibition activity while region B oligopeptide showed weak inhibition activity. Real‐time biomolecular interaction analysis using a BIAcore instrument revealed that the bacterially expressed calmodulin‐like domain of the calpain large subunit (L‐CaMLD) and that of the small subunit (S‐CaMLD) interacted, in a Ca2+‐dependent fashion, preferentially with the immobilized synthetic oligopeptide of region A and that of region C, respectively. Calmodulin showed no specific binding to these oligopeptides. The tripartite structure of the calpastatin functional domain may confer the specific interactions with the protease domain and the two CaMLDs of calpain.
Multiple transduction pathways regulate the sodium‐extrusion gene PMR2/ENA1 during salt stress in yeast
Authors: Märquez JoséA , Serrano Ramón
Published: July 2002

The yeast PMR2/ENA1 gene encodes an ATPase involved in sodium extrusion and induced by NaCl. At low salt concentration (0.3 M) induction is mediated by the HOG‐MAP kinase pathway, a system activated by non‐specific osmotic stress. At high salt concentrations (0.8 M) induction is mediated by the protein phosphatase calcineurin and is specific for sodium. Protein kinase A and Sis2p/Hal3p modulate PMR2/ENA1 expression as negative and positive factors, respectively but Sis2p/Hal3p does not participate in the transduction of the salt signal. Salt stress decreases the level of cAMP and the resulting decrease in protein kinase A activity may contribute to HOG‐mediated induction.
Heat‐labile uracil‐DNA glycosylase: purification and characterization
Authors: Sobek H. , Schmidt M. , Frey B. , Kaluza K.
Published: July 2002

A uracil‐DNA glycosylase (UNG) from a psychrophilic marine bacterium (BMTU 3346) has been purified to apparent homogeneity. The enzyme has a molecular weight of 23 400 Da. It is stable in complex buffers (containing glycerol/BSA), whereas it is heat‐labile in dilute buffers (free of stabilizers) with a half‐life of 2 min at 40°C. Due to the thermolability, uracil‐DNA glycosylase is suitable for application in the carryover prevention technique showing less residual activity and/or a slower reactivation rate than the usually applied UNG from Escherichia coli.
The resource constrained time/cost tradeoff project scheduling problem with discounted cash flows
Authors: Icmeli Oya , Erenguc S.Selcuk
Published: July 2002

Many of the recent studies on Project Scheduling focus on maximizing the Net Present Value (NPV) of cash flows that occur during the implementation of a project in the presence of precedence and resource constraints. The literature contains several integer programming formulations, heuristic procedures and their implementations. In most of these formulations, activity durations are assumed to be fixed. In this paper, we study a practical extension of the problem where the activity durations can be reduced from their normal durations by allocating more resources. Costs associated with such reductions are referred to as crashing costs. The problem under consideration involves determining the timing and duration of activities such that the NPV of all cash flows is maximized in the presence of precedence and resource constraints. We suggest a heuristic procedure for obtaining “good” feasible solutions. We compare the performance of three priority rules embedded in the proposed procedure. Two of these priority rules have been shown to be the most effective in the literature, the third is a new rule developed in this paper. Extensive computational experiments with the heuristic procedure on a total of 380 test problems are reported. The heuristic solutions are compared to benchmark solutions obtained from the best of 50‐randomly generated solutions for each test problem. In addition, upper bounds on the optimal values are obtained by performing Subgradient optimization on a Lagrangian Relaxation of the problem. These upper bounds are used to assess the quality of the heuristic solutions. In general, none of the priority rules produced consistently better results for all the test problems. However, all three priority rules provide “good” quality solutions with modest computational effort. The Lagrangian approach is found to provide tight bounds on the optimal values.
Ganglioside GM1 and GM3 in early human brain development: An immunocytochemical study
Authors: Stojiljković M. , Blagojević T. , Vukosavić S. , Zvezdina N.D. , Peković S. , Nikezić G. , Rakić L.
Published: July 2002

The distribution of GM1 and GM3 gangliosides in human brain development between gestational week (g.w.) 6 and 15 was demonstrated by an immunocytochemical approach using polyclonal anti‐GM1 and anti‐GM3 antibodies. The first appearance of GM1‐ and GM3‐positive cells was recorded as early as in g.w. 6. Both antibodies labeled the cells in the ventricular zone of the telencephalic wall, with radially oriented fibers toward the pial surface, which represent radial glia cells with glia fibers. The intensive GM3 immunoreactivity was also exhibited in proliferating cells in the ventricular zone between g.w. 6 and 12. During the period from g.w. 12 to 15, characterized by a rapid multiplication of neurons and glia cells, an increased number of GM1‐ and GM3‐positive cells was observed. Prominent GM1 ganglioside staining was observed at the surface of the cell bodies in the ventricular zone. Besides surface labeling in migrating cells, GM1 immunoreactivity was identified inside the soma in the regions of cortical plate and subplate. GM1 immunoreactivity was more pronounced on the membrane of neuronal cells migrating along radial glia fibers, especially at the contact site between neuronal and glial cells. The GM3 ganglioside was localized mostly inside the soma, showing a granular immunoreactivity pattern.

Our observations confirm the presence of GM1 and GM3 gangliosides in neuronal and glial cells in early human brain development. The involvement, especially of GM1 ganglioside in glia‐neuronal contacts during migration of neuroblasts to their final destination, as well as the presence of GM3 ganglioside in proliferative cells in the ventricular zone of the telencephalic wall was also recorded.
Activation of a pro‐apoptotic amplification loop through inhibition of NF‐κB‐dependent survival signals by caspase‐mediated inactivation of RIP
Authors: Martinon Fabio , Holler Nils , Richard Christelle , Tschopp Jürg
Published: July 2002

Death domain containing members of the tumor necrosis factor receptor (TNFR) superfamily can induce apoptosis or cell activation. However, the mechanisms by which these opposing programs are selected remain unclear. Frequently, NF‐κB activation conveys protection against cell death. We show that the serine/threonine kinase RIP that is required for TNF‐induced NF‐κB activation is processed by caspase‐8 into a dominant‐negative (DN) fragment during death receptor‐induced apoptosis, thereby leading to a blockade of NF‐κB‐mediated anti‐apoptotic signals. Our results suggest that cleavage of RIP is part of an amplification loop which is triggered by Fas and most likely by other death receptors.
The heterotrimeric Thermus thermophilus Asp‐tRNAAsn amidotransferase can also generate Gln‐tRNAGln
Authors: Becker Hubert D. , Min Bokkee , Jacobi Carsten , Raczniak Gregory , Pelaschier Joanne , Roy Hervé , Klein Sylvain , Kern Daniel , Söll Dieter
Published: July 2002

Thermus thermophilus strain HB8 is known to have a heterodimeric aspartyl‐tRNAAsn amidotransferase (Asp‐AdT) capable of forming Asn‐tRNAAsn [Becker, H.D. and Kern, D. (1998) Proc. Natl. Acad. Sci. USA 95, 12832–12837]. Here we show that, like other bacteria, T. thermophilus possesses the canonical set of amidotransferase (AdT) genes (gatA, gatB and gatC). We cloned and sequenced these genes, and constructed an artificial operon for overexpression in Escherichia coli of the thermophilic holoenzyme. The overproduced T. thermophilus AdT can generate Gln‐tRNAGln as well as Asn‐tRNAAsn. Thus, the T. thermophilus tRNA‐dependent AdT is a dual‐specific Asp/Glu‐AdT resembling other bacterial AdTs. In addition, we observed that removal of the 44 carboxy‐terminal amino acids of the GatA subunit only inhibits the Asp‐AdT activity, leaving the Glu‐AdT activity of the mutant AdT unaltered; this shows that Asp‐AdT and Glu‐AdT activities can be mechanistically separated.
Incorporation of N‐propanoylneuraminic acid leads to calcium oscillations in oligodendrocytes upon the application of GABA
Authors: Schmidt Carolin , Ohlemeyer Carsten , Kettenmann Helmut , Reutter Werner , Horstkorte Rüdiger
Published: July 2002

Sialylation of glycoproteins and glycolipids plays an important role during development, regeneration and pathogenesis. It has been shown that unnatural sialylation within glial cell cultures can have distinct effects on their proliferation and antigenic profiles. These cultures metabolize N‐propanoylmannosamine (N‐propanoylneuraminic acid precursor=P‐NAP), a synthetic non‐physiological precursor of neuraminic acid, resulting in the expression of N‐propanoylneuraminic acid in glycoconjugates of their cell membranes [Schmidt, C., Stehling, P., Schnitzer, J., Reutter, W. and Horstkorte, R. (1998) J. Biol. Chem. 273, 19146–19152]. To determine whether these biochemically engineered sialic acids influence calcium concentrations in cells of the oligodendrocyte lineage, mixed glial cultures of oligodendrocytes growing on top of an astrocyte monolayer were exposed to glutamate, histamine, adrenaline, γ‐aminobutyric acid (GABA), high potassium (high K+) and ATP. Calcium responses in P‐NAP‐treated oligodendrocytes were determined by confocal microscopy with the calcium indicator fluo‐3 AM, and compared with control cultures. We showed that P‐NAP differentially modulated the calcium responses of individual oligodendrocytes when GABA was applied. GABA induced calcium oscillations with up to four spikes per min in 60% of oligodendrocytes when treated with P‐NAP.
Role of Thr11 in the binding of ω‐conotoxin MVIIC to N‐type Ca2+ channels
Authors: Minami Kazushi , Raymond Cecile , Martin-Moutot Nicole , Ohtake Atsuko , Van Renterghem Catherine , Takahashi Masami , Seagar Michael J. , Mori Yasuo , Sato Kazuki
Published: July 2002

As replacement of Thr11 of ω‐conotoxin MVIIC with Ala significantly reduced the affinity for both N‐ and P/Q‐type calcium channels, we examined the effect of substitution at this position with other residues. Binding assays using rat cerebellar P2 membranes showed that the affinity is in the order of Leu>Val, aminobutyric acid, Thr>Asn≫Ser, Ala, Asp, Phe, Tyr for N‐type channels and Thr>Leu, Val, aminobutyric acid, Asn, Ser>Ala≫Asp, Phe, Tyr for P/Q‐type channels, suggesting that aliphatic amino acids with longer side chains are favorable for block of N‐type channels. The effects of substitution were examined electrophysiologically in BHK cells expressing N‐type Ca2+ channels. Inhibition of Ba2+ current by the analogs did not completely correlate with binding affinity, although binding to BHK cells was comparable to rat cerebellar membranes.
Study of the cytolethal distending toxin (CDT)‐activated cell cycle checkpoint
Authors: Alby Frédéric , Mazars Raoul , de Rycke Jean , Guillou Emmanuelle , Baldin Véronique , Darbon Jean-Marie , Ducommun Bernard
Published: July 2002

The bacterial cytolethal distending toxin (CDT) triggers a G2/M cell cycle arrest in eukaryotic cells by inhibiting the CDC25C phosphatase‐dependent CDK1 dephosphorylation and activation. We report that upon CDT treatment CDC25C is fully sequestered in the cytoplasmic compartment, an effect that is reminiscent of DNA damage‐dependent checkpoint activation. We show that the checkpoint kinase CHK2, an upstream regulator of CDC25C, is phosphorylated and activated after CDT treatment. In contrast to what is observed with other DNA damaging agents, we demonstrate that the activation of CHK2 can only take place during S‐phase. Use of wortmannin and caffeine suggests that this effect is not dependent on ATM but rather on another as yet unidentified PI3 kinase family member. These results confirm that the CDT is therefore responsible for specific genomic injuries that block cell proliferation by activating a cell cycle checkpoint.
Domain assembly, surface accessibility and sequence conservation in full length HIV‐1 Nef
Authors: Geyer Matthias , Peterlin B.Matija
Published: July 2002

The accessory Nef protein from human and simian immunodeficiency viruses is critical for efficient viral replication and pathogenesis. Here we present an assembly of the full length structure of HIV‐1 Nef, allele NL4‐3, based on the previously solved anchor and core domain structures. The center part of the 33 residue encompassing flexible loop at the C‐terminus of Nef, involved in Nef internalization and CD4 endocytosis, has been modelled. The degree of sequence conservation in HIV‐1 Nef proteins was determined using a total of 186 different strains from five different subtypes. The sequence conservation has been correlated with the accessible surface area and with secondary structure features for individual residues. The high amount of flexible regions in Nef accounts for the large surface and the multiple interaction sites the protein exhibits.
Antibiotic activities of peptides, hydrogen peroxide and peroxynitrite in plant defence
Authors: Garcı́a-Olmedo Francisco , Rodrı́guez-Palenzuela Pablo , Molina Antonio , Alamillo Josefa M , López-Solanilla Emilia , Berrocal-Lobo Marta , Poza-Carrión César
Published: July 2002

Genes encoding plant antibiotic peptides show expression patterns that are consistent with a defence role. Transgenic over‐expression of defence peptide genes is potentially useful to engineer resistance of plants to relevant pathogens. Pathogen mutants that are sensitive to plant peptides in vitro have been obtained and a decrease of their virulence in planta has been observed, which is consistent with their hypothetical defence role. A similar approach has been followed to elucidate the potential direct anti‐microbial role of hydrogen peroxide. Additionally, a scavenger of peroxynitrite has been used to investigate its involvement in plant defence.
Homeodomain proteins Mox1 and Mox2 associate with Pax1 and Pax3 transcription factors
Authors: Stamataki Despina , Kastrinaki Maria-Christina , Mankoo Baljinder S. , Pachnis Vassilis , Karagogeos Domna
Published: July 2002

Mox1 and Mox2 homeobox genes have been shown to be critical in axial skeleton and in limb muscle development respectively. Pax1 and Pax3 gene products are also implicated in these processes. Mox and Pax expression patterns are highly overlapping both spatially and temporally during embryonic development. We show here for the first time that Mox proteins physically interact with Pax1 and Pax3 using the yeast two‐hybrid protein interaction assay as well as in vitro biochemical assays. There is a strong preference of Mox1 to associate with Pax1 rather than Pax3 and of Mox2 to associate with Pax3 rather than Pax1. The observed interactions are mediated through the homeodomain of Mox.
One‐electron reduction potentials of dietary carotenoid radical cations in aqueous micellar environments
Authors: Burke Marc , Edge Ruth , Land Edward J. , McGarvey David J. , Truscott T.George
Published: July 2002

The one‐electron reduction potentials of the radical cations of five dietary carotenoids (β‐carotene, canthaxanthin, zeaxanthin, astaxanthin and lycopene) in aqueous micellar environments have been obtained from a pulse radiolysis study of electron transfer between the carotenoids and tryptophan radical cations as a function of pH, and lie in the range of 980–1060 mV. These values are consistent with our observation that the carotenoid radical cations oxidise tyrosine and cysteine. The decays of the carotenoid radical cations in the absence of added reactants suggest a distribution of exponential lifetimes. The radicals persist for up to about 1 s, depending on the medium.
15‐Deoxy‐Δ12,14‐PGJ2, but not troglitazone, modulates IL‐1β effects in human chondrocytes by inhibiting NF‐κB and AP‐1 activation pathways
Authors: Boyault Sandrine , Simonin Marie-Agnès , Bianchi Arnaud , Compe Emmanuel , Liagre Bertrand , Mainard Didier , Bécuwe Philippe , Dauça Michel , Netter Patrick , Terlain Bernard , Bordji Karim
Published: July 2002

The activation of peroxisome proliferator‐activated receptor γ (PPARγ) has been shown to inhibit the production and the effects of proinflammatory cytokines. Since interleukin‐1β (IL‐1β) directly mediates cartilage degradation in osteoarthritis, we investigated the capability of PPARγ ligands to modulate IL‐1β effects on human chondrocytes. RT‐PCR and Western blot analysis revealed that PPARγ expression was decreased by IL‐1β. 15‐Deoxy‐Δ12,14‐prostaglandin J2 (15d‐PGJ2), in contrast to troglitazone, was highly potent to counteract IL‐1β‐induced cyclooxygenase‐2 and inductible nitric oxide synthase expression, NO production and the decrease in proteoglycan synthesis. Western blot and gel‐shift analyses demonstrated that 15d‐PGJ2 inhibited NF‐κB activation, while troglitazone was ineffective. Although 15d‐PGJ2 attenuated activator protein‐1 binding on the DNA, it potentiated c‐jun migration in the nucleus. The absence or the low effect of troglitazone suggests that 15d‐PGJ2 action in human chondrocytes is mainly PPARγ‐independent.
Regulation of maltose utilization in Saccharomyces cerevisiae by genes of the RAS/protein kinase A pathway1
Authors: Wanke Valeria , Vavassori Monica , Thevelein Johan M , Tortora Paolo , Vanoni Marco
Published: July 2002

In Saccharomyces cerevisiae maltose utilization requires a functional MAL locus, each composed of three genes: MALR (gene 3) encoding a regulatory protein, MALT (gene 1) encoding maltose permease and MALS (gene 2) encoding maltase. We show that constitutive activation of the RAS/protein kinase A pathway severely reduces growth of MAL1 strains on maltose. This may be a consequence of reduction in MALT mRNA, reduced V
max and increased catabolite inactivation of the MALT‐encoded maltose transporter in the MAL1 strain. Mutations in the GGS1/TPS1 gene, which restricts glucose influx and possibly affects signalling, relieve carbon catabolite repression on both maltase and maltose permease and reduce maltose permease inactivation.
A‐Raf kinase is a new interacting partner of protein kinase CK2 β subunit
Authors: Boldyreff Brigitte , Issinger Olaf-Georg
Published: July 2002

© 1997 Federation of European Biochemical Societies.
The regulatory subunit of protein kinase CK2 is a specific A‐Raf activator
Authors: Hagemann Carsten , Kalmes Andreas , Wixler Viktor , Wixler Ludmilla , Schuster Tillman , Rapp Ulf R
Published: July 2002

Two protein kinases that are involved in proliferation and oncogenesis but so far were thought to be functionally independent are Raf and CK2. The Raf signaling pathway is known to play a critical role in such fundamental biological processes as cellular proliferation and differentiation. Abnormal activation of this pathway is potentially oncogenic. Protein kinase CK2 exhibits enhanced levels in solid human tumors and proliferating tissue. In a two‐hybrid screen of a mouse‐embryo cDNA library we detected an interaction between A‐Raf and CK2β subunit. This binding was specific, as no interaction between CK2β and B‐Raf or c‐Raf‐1 was observed. Regions critical for this interaction were localized between residues 550 and 569 in the A‐Raf kinase domain. A‐Raf kinase activity was enhanced 10‐fold upon coexpression with CK2β in Sf9 cells. The α subunit of CK2 abolishes this effect. This is the first demonstration of both a direct Raf‐isoform‐specific activation and a regulatory role for CK2β independent of the CK2α subunit. The present data thus link two different protein kinases that were thought to work separately in the cell.© 1997 Federation of European Biochemical Societies.
Human cathepsin W, a putative cysteine protease predominantly expressed in CD8+ T‐lymphocytes
Authors: Linnevers C , Smeekens S.P , Brömme D
Published: July 2002

A 750‐bp fragment of a novel human cysteine protease has been identified from the dbEST databank. PCR cloning and DNA sequencing yielded a 1.38‐kb full‐length cDNA which encodes a polypeptide of 376 amino acids. The protein consists of a putative 21‐residue signal peptide, a 106‐residue propeptide and a 252‐residue mature protein. The deduced amino acid sequence contains the highly conserved residues of the catalytic triad of papain‐like cysteine proteases: cysteine, histidine, and asparagine. Furthermore, the protein sequence possesses two potential N‐glycosylation sites: one in the propeptide and one in the mature protein. Comparison of the amino acid sequence of human cathepsin W with other human thiol‐dependent cathepsins revealed a relatively low degree of similarity (21–31%). In contrast to cathepsins L, S, K, B, H and O, cathepsin W contains a 21‐amino acid peptide insertion between the putative active site histidine and asparagine residues and an 8‐amino acid C‐terminal extension. This unique sequence may indicate that cathepsin W belongs in a novel subgroup of papain‐like proteases distinct from that of cathepsin L‐ and B‐like proteases. Northern blot analysis indicates a specific expression of cathepsin W in lymphatic tissues. Further analysis revealed predominant levels of expression in T‐lymphocytes, and more specifically in CD8+ cells. The expression of the protease in cytotoxic T‐lymphocytes may suggest a specific function in the mechanism or regulation of T‐cell cytolytic activity.
Correlated bond rotations in interactions of arginine residues with ligand carboxylate groups in protein ligand complexes
Authors: Nieto Pedro M , Birdsall Berry , Morgan William D , Frenkiel Thomas A , Gargaro Angelo R , Feeney James
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Over‐expression of plant 14‐3‐3 proteins in tobacco: enhancement of the plasmalemma K+ conductance of mesophyll cells12
Authors: Saalbach Gerhard , Schwerdel Marc , Natura Gabriel , Buschmann Peter , Christov Veselin , Dahse Ingo
Published: July 2002

Two cDNA clones encoding 14‐3‐3 homologous proteins were isolated from Vicia faba. Deduced amino acid sequences share different degrees of homology with other plant 14‐3‐3 proteins. Both clones, under the control of the CaMV 35S promoter, were transformed into tobacco plants. Immunoblotting showed three different forms of ca. 31, 34, and 37 kDa, indicating a covalent modification of the expressed 14‐3‐3 proteins. These forms were mainly present in the microsomal fraction. Patch‐clamp studies of mesophyll protoplasts of the transformants revealed a strongly enhanced K+ conductance compared to the wild type. This indicates the involvement of 14‐3‐3 proteins in ion channel regulation, presumably by modulating kinase activities or binding the channel.
Ether lipid biosynthesis: isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase
Authors: Thai Thanh-Phuong , Heid Hans , Rackwitz Hans-Richard , Hunziker Andreas , Gorgas Karin , Just Wilhelm W
Published: July 2002

In this paper we describe isolation and molecular characterization of human dihydroxyacetonephosphate acyltransferase (DAP‐AT). The enzyme was extracted from rabbit Harderian gland peroxisomes and isolated as a trimeric complex by sucrose density gradient centrifugation. From peptide sequences matching EST‐clones were obtained which allowed cloning and sequencing of the cDNA from a human cDNA library. The nucleotide‐derived amino acid sequence revealed a protein consisting of 680 amino acid residues of molecular mass 77 187 containing a C‐terminal type 1 peroxisomal targeting signal. Monospecific antibodies raised against this polypeptide efficiently immunoprecipitated DAP‐AT activity from solubilized peroxisomal preparations, thus demonstrating that the cloned cDNA codes for DAP‐AT.
Acetyl xylan esterase II from Penicillium purpurogenum is similar to an esterase from Trichoderma reesei but lacks a cellulose binding domain
Authors: Gutiérrez Rodrigo , Cederlund Ella , Hjelmqvist Lars , Peirano Alessandra , Herrera Francisco , Ghosh Debashis , Duax William , Jörnvall Hans , Eyzaguirre Jaime
Published: July 2002

Penicillium purpurogenum produces at least two acetyl xylan esterases (AXE I and II). The AXE II cDNA, genomic DNA and mature protein sequences were determined and show that the axe 2 gene contains two introns, that the primary translation product has a signal peptide of 27 residues, and that the mature protein has 207 residues. The sequence is similar to the catalytic domain of AXE I from Trichoderma reesei (67% residue identity) and putative active site residues are conserved, but the Penicillium enzyme lacks the linker and cellulose binding domain, thus explaining why it does not bind cellulose in contrast to the Trichoderma enzyme. These results point to a possible common ancestor gene for the active site domain, while the linker and the binding domain may have been added to the Trichoderma esterase by gene fusion.
Retinal dehydrogenase gene expression in stomach and small intestine of rats during postnatal development and in vitamin A deficiency
Authors: Bhat Pangala V.
Published: July 2002

Retinal dehydrogenase (RALDH) catalyzes the oxidation of retinal to all‐trans and 9‐cis retinoic acid, which function as ligands controlling RAR and RXR nuclear receptor‐signaling pathways. We have recently shown the expression of RALDH transcript in the stomach and small intestine by reverse transcription polymerase chain reaction [Bhat, P.V., Labrecque J., Dumas, F., Lacroix, A. and Yoshida, A. (1995) Gene 166, 303–306]. We have examined RALDH expression in the stomach and small intestine before and during postnatal development and in vitamin A deficiency by assaying for mRNA levels and protein as well as for enzyme activity. In −2 day fetuses, RALDH expression was high in the small intestine, whereas RALDH protein was not detectable in the stomach. However, expression of RALDH was seen in the stomach after birth, and gradually increased with age and reached the highest level at postnatal day 42. In the intestine, RALDH expression decreased postnatally. Vitamin A deficiency up‐regulated RALDH expression in the stomach and small intestine, and administration of retinoids down‐regulated the RALDH expression in these tissues. These results show the differential expression of RALDH in the stomach and small intestine during postnatal development, and that vitamin A status regulates the expression of RALDH gene in these tissues.
Promoter elements required for sugar‐repression of the RAmy3D gene for α‐amylase in rice
Authors: Toyofuku Kyoko , Umemura Taka-aki , Yamaguchi Junji
Published: July 2002

There is increasing evidence showing that cereal α‐amylase gene expression is controlled not only by the classical hormonal regulation, but also by feed‐back sugar repression. We demonstrated by in situ hybridization that the sugar repression of rice α‐amylase gene RAmy3D takes place in scutellar epithelium cells of callus‐forming rice embryos. We also used a transient expression system to study the cis‐acting elements involved in the sugar repression of the RAmy3D promoter activity. Site‐directed mutagenesis of the 50‐bp nucleotide sequence from −172 to −123 revealed that consensus sequences of G motif (TACGTA) and TATCCA T/C motif (GATA motif as its antisense sequence) are responsible for sugar repression. The promoter sequences required for sugar repression are reported and discussed.
Glycosylation of bovine pulmonary angiotensin‐converting enzyme modulates its catalytic properties
Authors: Orth Tatiana , Voronov Sergei , Binevski Petr , Saenger Wolfram , Kost Olga
Published: July 2002

To study the role of the oligosaccharide moiety in the catalytic properties of angiotensin‐converting enzyme (ACE), we obtained asialo‐ and partially deglycosylated ACE by enzymatic treatment of two‐domain somatic enzyme from bovine lung. Treated enzymes demonstrated appreciable, but different changes of catalytic properties in the reaction of the hydrolysis of N‐substituted tripeptides, C‐terminal analogs of angiotensin I and bradykinin among them, compared to those for native enzyme. Deglycosylation also altered the catalytic properties of a single N domain of bovine ACE. So, various patterns of glycosylation modulate substrate specificity of somatic ACE and may be the reason for functional heterogeneity of the enzyme.
The HIV‐1 Vpr displays strong anti‐apoptotic activity
Authors: Fukumori Tomoharu , Akari Hirofumi , Iida Shinya , Hata So , Kagawa Susumu , Aida Yoko , Koyama A.Hajime , Adachi Akio
Published: July 2002

Mutations in the human immunodeficiency virus type 1 (HIV‐1) vpr gene only slightly reduce the replication rate of the virus. To study the role of HIV‐1 Vpr in biological effects on cells, HEp‐2 cells, which express HIV‐1 Vpr constitutively but at a low level, were established. While control HEp‐2 cells underwent apoptosis when incubated with sorbitol, the morphological and biochemical apoptotic changes were inefficiently induced in the HIV‐1 Vpr‐expressing cells by the same treatment. These results clearly indicate that HIV‐1 Vpr has anti‐apoptotic activity, and raise the possibility that Vpr acts as a weak activator of virus replication through anti‐apoptosis.
Cloning of a human ether‐a‐go‐go potassium channel expressed in myoblasts at the onset of fusion
Authors: Occhiodoro Teresa , Bernheim Laurent , Liu Jian-Hui , Bijlenga Philippe , Sinnreich Michael , Bader Charles R , Fischer-Lougheed Jacqueline
Published: July 2002

An early sign of human myoblast commitment to fusion is the expression of a non‐inactivating delayed rectifier K+ current, IK(NI), and an associated membrane potential hyperpolarization. We have isolated the full‐length coding region of a human ether‐a‐go‐go K+ channel (h‐eag) from myoblasts undergoing differentiation. The h‐eag gene was localized to chromosome 1q32–41, and is expressed as a ∼9 kb transcript in myogenic cells and in adult brain tissue. Forced expression of h‐eag in undifferentiated myoblasts generates a current with remarkable similarity to IK(NI) indicating that h‐eag constitutes the channel responsible for this current in vivo.
Mono‐ and binuclear Zn‐β‐lactamase from Bacteroides fragilis: catalytic and structural roles of the zinc ions
Authors: Paul-Soto Raquel , Hernandez-Valladares Maria , Galleni Moreno , Bauer Rogert , Zeppezauer Michael , Frère Jean-Marie , Adolph Hans-Werner
Published: July 2002

The Bacteroides fragilis Zn‐β‐lactamase is active with a mono‐ and a binuclear zinc site. The apoenzyme produced by removal of both Zn ions does not recover full activity upon readdition of Zn2+ in contrast to an active mono‐Zn form prepared at pH 6.0. Differences in k
cat values observed are substrate‐dependent implying distinct mechanisms for the mono‐ and binuclear species. The substrate profile of a Zn,Cd hybrid obtained by selective exchange of one zinc ion is different from that of the Zn2 enzyme with a remarkable 15‐fold increased activity with cefoxitin as substrate.
Biodegradative mechanism of the brown rot basidiomycete Gloeophyllum trabeum: evidence for an extracellular hydroquinone‐driven fenton reaction
Authors: Kerem Zohar , Jensen Kenneth A , Hammel Kenneth E
Published: July 2002

We have identified key components of the extracellular oxidative system that the brown rot fungus Gloeophyllum trabeum uses to degrade a recalcitrant polymer, polyethylene glycol, via hydrogen abstraction reactions. G. trabeum produced an extracellular metabolite, 2,5‐dimethoxy‐1,4‐benzoquinone, and reduced it to 2,5‐dimethoxyhydroquinone. In the presence of 2,5‐dimethoxy‐1,4‐benzoquinone, the fungus also reduced extracellular Fe3+ to Fe2+ and produced extracellular H2O2. Fe3+ reduction and H2O2 formation both resulted from a direct, non‐enzymatic reaction between 2,5‐dimethoxyhydroquinone and Fe3+. polyethylene glycol depolymerization by G. trabeum required both 2,5‐dimethoxy‐1,4‐benzoquinone and Fe3+ and was completely inhibited by catalase. These results provide evidence that G. trabeum uses a hydroquinone‐driven Fenton reaction to cleave polyethylene glycol. We propose that similar reactions account for the ability of G. trabeum to attack lignocellulose.
Expression of prokaryotic 1‐deoxy‐d‐xylulose‐5‐phosphatases in Escherichia coli increases carotenoid and ubiquinone biosynthesis
Authors: Harker M. , Bramley P.M.
Published: July 2002

Isopentenyl diphosphate (IPP) acts as the common, five‐carbon building block in the biosynthesis of all isoprenoids. The first reaction of IPP biosynthesis in Escherichia coli is the formation of 1‐deoxy‐d‐xylulose‐5‐phosphate, catalysed by 1‐deoxy‐d‐xylulose‐5‐phosphate synthase (DXPS). E. coli engineered to produce lycopene, was transformed with dxps genes cloned from Bacillus subtilis and Synechocystis sp. 6803. Increases in lycopene levels were observed in strains expressing exogenous DXPS compared to controls. The recombinant strains also exhibited elevated levels of ubiquinone‐8. These increases corresponded with enhanced DXP synthase activity in the recombinant E. coli strains.
UDP‐N‐acetylglucosamine:N‐acetylmuramoyl‐(pentapeptide) pyrophosphoryl undecaprenol N‐acetylglucosamine transferase from Escherichia coli: overproduction, solubilization, and purification
Authors: Crouvoisier Muriel , Mengin-Lecreulx Dominique , van Heijenoort Jean
Published: July 2002

Plasmids for the high‐level overproduction of wild‐type, and C‐ and N‐terminal His‐tagged MurG N‐acetylglucosaminyl transferase from Escherichia coli were constructed. In complementation tests the three forms were active in vivo. After IPTG induction, growth, spheroplast formation and lysis, overproduced MurG proteins were mainly present (90%) in the particulate fraction. Readily solubilized by CHAPS, they were purified without any detergent to over 80% purity for both His‐tagged forms but only up to 20% for the wild‐type form. The enzymatic activity of each purified MurG protein was determined and found to be inhibited to the same extent by ramoplanin.
Knock‐out of the cyaY gene in Escherichia coli does not affect cellular iron content and sensitivity to oxidants
Authors: Li Dong Sheng , Ohshima Keiichi , Jiralerspong Sarn , Bojanowski Michel W. , Pandolfo Massimo
Published: July 2002

Friedreich ataxia is a recessively inherited neurodegenerative disease caused by deficiency of a highly conserved mitochondrial protein, frataxin. Frataxin deficiency results in mitochondrial iron accumulation and oxidative stress. Frataxin shows homology with the CyaY proteins of γ‐purple bacteria, whose function is unknown. We knocked out the CyaY gene in Escherichia coli MM383 by homologous recombination and we generated an E. coli MM383 strain overexpressing CyaY. Bacterial growth, iron content and survival after exposure to H2O2 did not differ among these strains, suggesting that, despite structural similarities, cyaY proteins in bacteria may have a different function from frataxin homologues in mitochondria.
Glucokinase regulatory protein is essential for the proper subcellular localisation of liver glucokinase
Authors: de la Iglesia Núria , Veiga-da-Cunha Maria , Van Schaftingen Emile , Guinovart Joan J. , Ferrer Juan C.
Published: July 2002

Glucokinase (GK), a key enzyme in the glucose homeostatic responses of the liver, changes its intracellular localisation depending on the metabolic status of the cell. Rat liver GK and Xenopus laevis GK, fused to the green fluorescent protein (GFP), concentrated in the nucleus of cultured rat hepatocytes at low glucose and translocated to the cytoplasm at high glucose. Three mutant forms of Xenopus GK with reduced affinity for GK regulatory protein (GKRP) did not concentrate in the hepatocyte nuclei, even at low glucose. In COS‐1 and HeLa cells, a blue fluorescent protein (BFP)‐tagged version of rat liver GK was only able to accumulate in the nucleus when it was co‐expressed with GKRP‐GFP. At low glucose, both proteins concentrated in the nuclear compartment and at high glucose, BFP‐GK translocated to the cytosol while GKRP‐GFP remained in the nucleus. These findings indicate that the presence of and binding to GKRP are necessary and sufficient for the proper intracellular localisation of GK and directly involve GKRP in the control of the GK subcellular distribution.
Binding of Bacillus thuringiensis Cry1Ac toxin to Manduca sexta aminopeptidase‐N receptor is not directly related to toxicity
Authors: Jenkins Jeremy L. , Lee Mi Kyong , Sangadala Sreedhara , Adang Michael J. , Dean Donald H.
Published: July 2002

Bacillus thuringiensis Cry1Ac δ‐endotoxin specifically binds a 115‐kDa aminopeptidase‐N purified from Manduca sexta midgut. Cry1Ac domain III mutations were constructed around a putative sugar‐binding pocket and binding to purified aminopeptidase‐N and brush border membrane vesicles (BBMV) was compared to toxicity. Q509A, R511A, Y513A, and 509–511 (QNR‐AAA) eliminated aminopeptidase‐N binding and reduced binding to BBMV. However, toxicity decreased no more than two‐fold, indicating activity is not directly correlated with aminopeptidase‐N binding. Analysis of toxin binding to aminopeptidase‐N in M. sexta is therefore insufficient for predicting toxicity. Mutants retained binding, however, to another BBMV site, suggesting alternative receptors may compensate in vivo.
Illegal abortion: consequences for women's health and the health care system
Authors: Faúndes A. , Hardy E.
Published: July 2002

Illegal abortion is responsible for up to half of maternal deaths and consumes a large proportion of health resources in many developing countries, particularly in Africa and Latin America. The legal situation of abortion in a country does not influence the abortion rate, but illegality is associated with a much greater risk of complications and death. To make abortion legal is not enough. Access to safe abortion strongly depends on the capacity and willingness of physicians and the health system to provide safe services, which sometimes are made available in spite of restrictive laws. The abortion rate will drop and the safety of the procedure will improve, parallel to the position women occupy in a given society, and to the level of recognition of their sexual and reproductive rights. The medical profession, and FIGO in particular, has a great role to play in implementing initiatives that will reduce the consequences of illegal abortion for women and society.