bluuebunny/crossref_metadata_2025 huggingface dataset:
Occurrence of ceramides and neutral glycolipids with unusual long‐chain base composition in purified rat liver mitochondria
Authors: Ardail D , Popa I , Alcantara K , Pons A , Zanetta J.P , Louisot P , Thomas L , Portoukalian J
Published: July 2002

The free ceramide content of rat liver mitochondria was found to be 1.7 nmol/mg protein and outer membranes contained a three‐fold higher concentration than inner membranes. The mitochondrial content in neutral glycolipids was 0.6 nmol/mg protein. The long‐chain bases found in free ceramides were d18:1 sphingosine, d18:0 3‐ketosphinganine and t21:1 phytosphingosine in increasing order. In contrast, 3‐ketosphinganine was the only base of glucosylceramide and lactosylceramide of inner membranes, whereas d18:1 sphingosine was the major long‐chain base of glucosylceramide of outer membranes.
Cystatin inhibition of cathepsin B requires dislocation of the proteinase occluding loop. Demonstration by release of loop anchoring through mutation of His110
Authors: Pavlova Alla , Krupa Joanne C , Mort John S , Abrahamson Magnus , Björk Ingemar
Published: July 2002

Cystatins A and C were both shown to inhibit cathepsin B by a two‐step mechanism, involving an initial weak interaction followed by a conformational change. Disruption of the major salt bridge anchoring the occluding loop of cathepsin B to the main body of the enzyme by mutation of His110 to Ala converted the binding to an apparent one‐step reaction. The second step of cystatin binding to cathepsin B must therefore be due to the inhibitor having to alter the conformation of the enzyme by displacing the occluding loop to allow a tight complex to be formed. Cystatin A was appreciably less effective in displacing the loop than cystatin C, resulting in a considerably lower overall inhibition rate constant.
Anti‐amyloidogenic activity of tetracyclines: studies in vitro
Authors: Forloni Gianluigi , Colombo Laura , Girola Laura , Tagliavini Fabrizio , Salmona Mario
Published: July 2002

Cerebral deposition of β‐amyloid is a major neuropathological feature in Alzheimer's disease. Here we show that tetracyclines, tetracycline and doxycycline, classical antibiotics, exhibit anti‐amyloidogenic activity. This capacity was determined by the exposure of β 1‐42 amyloid peptide to the drugs followed by the electron microscopy examination of the amyloid fibrils spontaneously formed and quantified with thioflavine T binding assay. The drugs reduced also the resistance of β 1‐42 amyloid fibrils to trypsin digestion. Tetracyclines not only inhibited the β‐amyloid aggregates formation but also disassembled the pre‐formed fibrils. The results indicate that drugs with a well‐known clinical profile, including activity in the central nervous system, are potentially useful for Alzheimer's therapy.
Ser1901 of α1C subunit is required for the PKA‐mediated enhancement of L‐type Ca2+ channel currents but not for the negative shift of activation
Authors: Naguro Isao , Nagao Taku , Adachi-Akahane Satomi
Published: July 2002

Cardiac L‐type Ca2+ channel is facilitated by protein kinase A (PKA)‐mediated phosphorylation. Here, we investigated the role of Ser1901, a putative phosphorylation site in the carboxy‐terminal of rat brain type‐II α1C subunit (rbCII), in the PKA‐mediated regulation. Forskolin (3 μM) enhanced Ca2+ channel currents (I
Ca) and shifted the activation curve to negative voltages, which were abolished by protein kinase inhibitor. Replacement of Ser1901 of rbCII by Ala abolished the enhancement of I
Ca by forskolin but not the shift of the activation curve. These results indicate that Ser1901 is required for the PKA‐mediated enhancement of I
Ca, and that the voltage‐dependence of the activation of I
Ca appears to be modulated via another PKA phosphorylation site.
Schizosaccharomyces pombe och1+ encodes α‐1,6‐mannosyltransferase that is involved in outer chain elongation of N‐linked oligosaccharides
Authors: Yoko-o Takehiko , Tsukahara Kappei , Watanabe Tatsuo , Hata-Sugi Naoko , Yoshimatsu Kentaro , Nagasu Takeshi , Jigami Yoshifumi
Published: July 2002

The fission yeast Schizosaccharomyces pombe attaches an outer chain containing mannose and galactose to the N‐linked oligosaccharides on many of its glycoproteins. We identified an S. pombe och1 mutant that did not synthesize the outer chains on acid phosphatase. The S. pombe och1
+ gene was a functional homolog of Saccharomyces cerevisiae OCH1, and its gene product (SpOch1p) incorporated α‐1,6‐linked mannose into pyridylaminated Man9GlcNAc2, indicating that och1
+ encodes an α‐1,6‐mannosyltransferase. Our results indicate that SpOch1p is a key enzyme of outer chain elongation. The substrate specificity of SpOch1p was different from that of S. cerevisiae OCH1 gene product (ScOch1p), suggesting that SpOch1p may have a wider substrate specificity than that of ScOch1p.
Kcc4 associates with septin proteins of Saccharomyces cerevisiae
Authors: Okuzaki Daisuke , Nojima Hiroshi
Published: July 2002

Kcc4, a kinase of the budding yeast Saccharomyces cerevisiae, is homologous to the bud neck protein kinases Hsl1/Nik1 and Gin4. We report here that a GFP‐Kcc4 fusion protein is localized at the bud neck and that the non‐kinase domain is required for this localization. We also demonstrate that Kcc4 associates with septin proteins in vitro and in vivo by two‐hybrid analysis, GST pull‐down experiments, immunoprecipitation, and analysis of direct association with affinity‐purified GST‐Kcc4 and MBP‐Septin proteins. From the results obtained here, we suggest that Cdc11 is the primary association partner of Kcc4.
Inhibition of PP‐2A upregulates CaMKII in rat forebrain and induces hyperphosphorylation of tau at Ser 262/356
Authors: Bennecib Malika , Gong Cheng-Xin , Grundke-Iqbal Inge , Iqbal Khalid
Published: July 2002

The regulation of the activity of CaMKII by PP‐1 and PP‐2A, as well as the role of this protein kinase in the phosphorylation of tau protein in forebrain were investigated. The treatment of metabolically active rat brain slices with 1.0 μM okadaic acid (OA) inhibited ∼65% of PP‐2A and had no significant effect on PP‐1 in the 16 000×g tissue extract. Calyculin A (CL‐A), 0.1 μM under the same conditions, inhibited ∼50% of PP‐1 and ∼20% of PP‐2A activities. In contrast, a mixture of OA and CL‐A practically completely inhibited both PP‐2A and PP‐1 activities. The inhibition of the two phosphatase activities or PP‐2A alone resulted in an ∼2‐fold increase in CaMKII activity and an ∼8‐fold increase in the phosphorylation of tau at Ser 262/356 in 60 min. Treatment of the brain slices with KN‐62, an inhibitor of the autophosphorylation of CaMKII at Thr 286/287, produced ∼60% inhibition in CaMKII activity and no significant effect on tau phosphorylation at Ser 262/356. The KN‐62‐treated brain slices when further treated with OA and CL‐A did not show any change in CaMKII activity. In vitro, both PP‐2A and PP‐1 dephosphorylated tau at Ser 262/356 that was phosphorylated with purified CaMKII. These studies suggest (i) that in mammalian forebrain the cytosolic CaMKII activity is regulated mainly by PP‐2A, (ii) that CaMKII is the major tau Ser 262/356 kinase in brain, and (iii) that a decrease in PP‐2A/PP‐1 activities in the brain leads to hyperphosphorylation of tau not only by inhibition of its dephosphorylation but also by promoting the CaMKII activity.
Activation of ion‐conducting pathways in the inner mitochondrial membrane – an unrecognized activity of fatty acid?
Authors: Schönfeld P , Schlüter T , Schüttig R , Bohnensack R
Published: July 2002

The effect of non‐esterified myristate (C14:0) or dodecyl sulfate was studied on passive swelling of rat liver mitochondria suspended in hypotonic alkaline KCl medium in the absence of the potassium ionophore valinomycin. Both compounds rapidly initiated large‐amplitude swelling. However, they failed to initiate swelling when the mitochondria were suspended in hypotonic alkaline sucrose medium. In contrast to myristate or dodecyl sulfate, the non‐ionic detergent Triton X‐100 initiated swelling of mitochondria in both of the media. The following findings indicate that the inner mitochondrial membrane (IMM) is permeabilized by myristate to K+ and Cl− in a specific manner. (i) Swelling initiated by myristate did not respond to cyclosporin A, (ii) the protonophoric uncoupler FCCP was unable to mimic the myristate effect on swelling, and (iii) myristate‐induced Cl−‐permeation (measured with KCl medium plus valinomycin) was inhibited by N,N′‐dicyclohexylcarbodiimide, quinine or ATP. Myristate‐ or dodecyl sulfate‐initiated swelling was paralleled by the lowering of endogenous Mg2+ content. Both effects, stimulation of swelling and depletion of endogenous Mg2+ are correlated with each other. Similar effects have been reported previously for the carboxylic divalent cation ionophore calcimycin (A23187). The A23187‐induced swelling has identical inhibiting characteristics on Cl−‐permeation with respect to N,N′‐dicyclohexylcarbodiimide, quinine and ATP as the myristate‐stimulated swelling. Therefore, we conclude that non‐esterified fatty acids increase the permeability of mitochondria to K+ and Cl− at alkaline pH by activating Mg2+‐dependent ion‐conducting pathways in IMM.
Epidermal growth factor receptor induced apoptosis: potentiation by inhibition of Ras signaling
Authors: Högnason Thorbergur , Chatterjee Sukalyan , Vartanian Timothy , Ratan Rajiv R , Ernewein Krista M , Habib Amyn A
Published: July 2002

Previous studies have shown that certain tumor cell lines which naturally express high levels of the epidermal growth factor receptor (EGFR) undergo apoptosis when exposed to epidermal growth factor. Whether this phenomenon is a direct result of receptor overexpression or some other genetic alteration renders these cells sensitive to apoptosis is yet to be established. We show that experimentally increasing the level of EGFR expression predictably leads to apoptosis in a variety of cell types which requires an active tyrosine kinase but not EGFR autophosphorylation sites. Expression of a dominant negative Ras mutant in EGFR overexpressing cells results in a significant potentiation of EGFR induced apoptosis suggesting that Ras activation is a key survival signal generated by the EGFR. We propose that potentiation of EGFR induced apoptosis by dominant negative Ras results, at least in part, by a block of Akt activation.
Membrane type 1 matrix metalloproteinase and gelatinase A synergistically degrade type 1 collagen in a cell model
Authors: Atkinson Susan J. , Patterson Margaret L. , Butler Michael J. , Murphy Gillian
Published: July 2002

A fibrosarcoma cell line transfected with the matrix metalloproteinase MT1 MMP showed an enhanced ability to degrade 14C‐labelled collagen films. As previously shown for proMMP 2 activation, TIMP 1 was an ineffective inhibitor of the process of collagenolysis whereas TIMP 2 was efficient and completely prevented collagen degradation. In the presence of the calcium ionophore, ionomycin, proteolytic processing of MT1 MMP was restricted and collagenolysis did not occur indicating that the 63 kDa form of the enzyme is not a functional collagenase. The collagenolytic activity of MT1 MMP was shown to be enhanced by the addition of proMMP 2, but TIMP 1 inhibition remained poor relative to that of TIMP 2. The study demonstrated that synergy between two non‐conventional collagenases effectively degrades insoluble pericellular collagen. Due to the membrane localisation of MT1 MMP, this could potentially occur in a highly localised manner.
Hematopoietic cell‐specific adapter proteins, SLP‐76 and BLNK, effectively activate NF‐AT as well as NF‐κB by Syk and Tec PTKs in non‐lymphoid cell lines
Authors: Yamamoto Tetsuya , Matsuda Tadashi , Junicho Akira , Kishi Hiroyuki , Yoshimura Akihiko , Muraguchi Atsushi
Published: July 2002

To investigate the roles of various hematopoietic cell‐specific adapter proteins in T cell receptor (TCR)‐signaling leading to nuclear factor of activated T cell (NF‐AT) and nuclear factor of κB (NF‐κB) activation, we reconstituted TCR‐signaling with CD8/ζ, various protein tyrosine kinases (PTKs), and adapter proteins in a non‐lymphoid cell line, 293T. We show that SLP‐76 and BLNK, but not LAT, effectively co‐operated with Syk and Tec family PTKs to activate NF‐AT and NF‐κB. We also show that Tec family PTKs enhanced endogenous phospholipase C (PLC)‐γ1 phosphorylation induced by CD8/ζ and Syk in 293T cells. These results imply that PLC‐γ1 may play a critical role in a hematopoietic cell‐specific adapter protein‐mediated NF‐AT and NF‐κB activation in a non‐lymphoid cell.
ctr1, a gene involved in a signal transduction pathway of the gliding motility in the cyanobacterium Synechocystis sp. PCC 6803
Authors: Chung Young-Ho , Cho Mi-Sun , Moon Yoon-Jung , Choi Jong-Soon , Yoo Yong-Cheol , Park Youn-Il , Lee Kyun-Min , Kang Kye-Won , Park Young Mok
Published: July 2002

We generated random Tn5 mutations in Synechocystis sp. PCC 6803 in search for genes involved in the signal transduction cascade for the cyanobacterial gliding motility. One of the non‐gliding Tn5 mutants, S1‐105, had an insertional inactivation in the slr1044 gene encoding a putative methyl‐accepting chemotaxis protein. Interposon mutation on the slr1044 (named ctr1) in the bacterium also eliminated gliding motility. In the interposon mutant, the expression of pilA1 was 5‐fold decreased compared with that of wild‐type and thick pili, that are believed to be the motor for gliding, could not be observed by an electron microscope. Therefore, we suggest that the Ctr1 protein functions as a transducer that regulates the expression of pilA1, and thus is required for the biogenesis of thick pili.
The TIM‐barrel fold: a versatile framework for efficient enzymes
Authors: Wierenga R.K
Published: July 2002

Recent studies on triosephosphate isomerase (TIM)‐barrel enzymes highlight the remarkable versatility of the TIM‐barrel scaffold. At least 15 distinct enzyme families use this framework to generate the appropriate active site geometry, always at the C‐terminal end of the eight parallel β‐strands of the barrel. Sequence and structure comparisons now suggest that many of the TIM‐barrel enzymes are evolutionarily related. Common structural properties of TIM‐barrel enzymes are discussed.
Involvement of mitochondrial ferredoxin and Cox15p in hydroxylation of heme O
Authors: Barros Mario H. , Carlson Christopher G. , Glerum D.Moira , Tzagoloff Alexander
Published: July 2002

Cox15p is essential for the biogenesis of cytochrome oxidase [Glerum et al., J. Biol. Chem. 272 (1997) 19088–19094]. We show here that cox15 mutants are blocked in heme A but not heme O biosynthesis. In Schizosaccharomyces pombe COX15 is fused to YAH1, the yeast gene for mitochondrial ferredoxin (adrenodoxin). A fusion of Cox15p and Yah1p in Saccharomyces cerevisiae rescued both cox15 and yah1 null mutants. This suggests that Yah1p functions in concert with Cox15p. We propose that Cox15p functions together with Yah1p and its putative reductase (Arh1p) in the hydroxylation of heme O.
Tau‐tubulin kinase phosphorylates tau at Ser‐208 and Ser‐210, sites found in paired helical filament‐tau
Authors: Tomizawa Kayoko , Omori Akira , Ohtake Atsuko , Sato Kazuki , Takahashi Miho
Published: July 2002

Hyperphosphorylated tau protein is known to be a major component of the paired helical filaments (PHFs) that accumulate in the brain of Alzheimer's patients. The kinase that phosphorylated Ser‐208 and Ser‐210 in PHF‐tau had remained unknown. We used anti‐pS208 and anti‐pS210 antibodies and Western blots to confirm that the tau‐tubulin kinase (TTK) phosphorylates tau at Ser‐208 and at Ser‐210. Using partial amino acid sequences of purified bovine brain TTK, a mouse cDNA of TTK was isolated and the sequence was determined. Its 963 bp coding region is composed of 320 amino acids and encodes a 36 kDa protein indistinguishable in size from authentic bovine brain TTK. Our immunoblot analysis demonstrated that TTK is ubiquitously distributed in the rat tissues, and that it is developmentally regulated in the rat brain.
Development and application of bioluminescent Caenorhabditis elegans as multicellular eukaryotic biosensors
Authors: Lagido C. , Pettitt J. , Porter A.J.R. , Paton G.I. , Glover L.A.
Published: July 2002

We describe a novel approach to assess toxicity to the free‐living nematode Caenorhabditis elegans that relies on the ability of firefly luciferase to report on endogenous ATP levels. We have constructed bioluminescent C. elegans with the luc gene under control of a constitutive promoter. Light reduction was observed in response to increasing temperature, concentrations of copper, lead and 3,5‐dichlorophenol. This was due to increased mortality coupled with decreased metabolic activity in the surviving animals. The light emitted by the transgenic nematodes gave a rapid, real‐time indication of metabolic status. This forms the basis of rapid and biologically relevant toxicity tests.
A sequence motif responsible for ER export and surface expression of Kir2.0 inward rectifier K+ channels
Authors: Stockklausner C , Ludwig J , Ruppersberg J.P , Klöcker N
Published: July 2002

Integral membrane proteins are sorted via the secretory pathway. It was proposed that this pathway is non‐selective provided that the cargo protein is properly assembled and lacks an endoplasmic reticulum (ER) retention signal. However, recent experimental evidence suggests that efficient export of proteins from the ER to the Golgi complex is not simply a default pathway. Here we demonstrate a novel sequence motif (FxYENEV) in the cytoplasmic C‐terminus of mammalian inward rectifier potassium (Kir) channels which determines ER export. This motif is found to be both necessary and sufficient for efficient export from the ER that eventually leads to efficient surface expression of Kir2.1 channels.
Contribution of the DDDD motif of H. influenzae e (P4) to phosphomonoesterase activity and heme transport
Authors: Reilly Thomas J , Green Bruce A , Zlotnick Gary W , Smith Arnold L
Published: July 2002

Haemophilus influenzae lipoprotein e (P4) is a member of the DDDD phosphohydrolase superfamily and mediates heme transport. Each of the aspartate residues of the signature motif is required for phosphomonoesterase activity, as none of the e (P4) single D mutants (D64A, D66A, D181N, and D185A) possessed detectable phosphomonoesterase activity. These results suggest that the signature motif is essential to the phosphomonoesterase activity of lipoprotein e (P4). When assessed for phosphomonoesterase‐dependent heme transport activity in Escherichia coli hemA strains, plasmids containing D181N and D185A retained heme transport as indicated by aerobic growth while D64A and D66A did not. We conclude that phosphomonoesterase activity is not required for heme transport.
Trp122 and Trp134 on the surface of the catalytic domain are essential for crystalline chitin hydrolysis by Bacillus circulans chitinase A1
Authors: Watanabe Takeshi , Ishibashi Asuka , Ariga Yumiko , Hashimoto Masayuki , Nikaidou Naoki , Sugiyama Junji , Matsumoto Takuo , Nonaka Takamasa
Published: July 2002

From the 3D‐structural analysis of the catalytic domain of chitinase A1, two exposed tryptophan residues (W122 and W134) are proposed to play an important role in guiding a chitin chain into the catalytic cleft during the crystalline chitin hydrolysis. Mutation of either W122 or W134 to alanine significantly reduced the hydrolyzing activity against highly crystalline β‐chitin microfibrils. Double mutation almost completely abolished the hydrolyzing activity. On the other hand, the hydrolyzing activity against either soluble or amorphous substrate was not reduced. These mutations slightly impaired the binding activity of this enzyme. These results clearly demonstrated that the two exposed aromatic residues play a critical role in hydrolyzing the chitin chain in crystalline chitin.
Inhibition of ADAMTS4 (aggrecanase‐1) by tissue inhibitors of metalloproteinases (TIMP‐1, 2, 3 and 4)
Authors: Hashimoto Gakuji , Aoki Takanori , Nakamura Hiroyuki , Tanzawa Kazuhiko , Okada Yasunori
Published: July 2002

ADAMTS4 (aggrecanase‐1) is considered to play a key role in the degradation of aggrecan in arthritides. The inhibitory activity of tissue inhibitors of metalloproteinases (TIMPs) to ADAMTS4 was examined in an assay using aggrecan substrate. Among the four TIMPs, TIMP‐3 inhibited the activity most efficiently with an IC50 value of 7.9 nM, which was at least 44‐fold lower than that of TIMP‐1 (350 nM) and TIMP‐2 (420 nM) and >250‐fold less than that of TIMP‐4 (2 μM for 35% inhibition). These results suggest that TIMP‐3 is a potent inhibitor against the aggrecanase activity of ADAMTS4 in vivo.
Functional disruption of IEX‐1 expression by concatemeric hammerhead ribozymes alters growth properties of 293 cells
Authors: Grobe Olaf , Arlt Alexander , Ungefroren Hendrik , Krupp Guido , Fölsch Ulrich R. , Schmidt Wolfgang E. , Schäfer Heiner
Published: July 2002

The early response gene IEX‐1 modulates apoptosis and cell growth in a poorly defined fashion. Here, we describe the effect of hammerhead ribozymes specifically disrupting IEX‐1 expression in 293 cells. Compared to vector control, 293 cells exhibit a reduced growth rate and a slowed cell cycle progression, when stably transfected with a concatemeric ribozyme construct. In addition, these 293 cells were much less sensitive to apoptosis induced by an activating Fas/CD95 antibody or by the anti‐cancer drugs etoposide and doxorubicin. By modulating the cell cycle, IEX‐1 might be part of a growth signal if favourable growth conditions prevail, whereas under unfavourable conditions, i.e. death receptor activation, IEX‐1 facilitates apoptosis.
Molecular characterization of human UDP‐glucuronic acid/UDP‐N‐acetylgalactosamine transporter, a novel nucleotide sugar transporter with dual substrate specificity
Authors: Muraoka Masatoshi , Kawakita Masao , Ishida Nobuhiro
Published: July 2002

A novel human nucleotide sugar transporter (NST) which transports both UDP‐glucuronic acid (UDP‐GlcA) and UDP‐N‐acetylgalactosamine (UDP‐GalNAc) has been identified, cloned and characterized. The strategy for the identification of the novel NST involved a search of the expressed sequence tags database for genes related to the human UDP‐galactose transporter‐related isozyme 1, followed by heterologous expression of a candidate gene (hUGTrel7) in Saccharomyces cerevisiae and biochemical analyses. Significantly more UDP‐GlcA and UDP‐GalNAc were translocated from the reaction medium into the lumen of microsomes prepared from the hUGTrel7‐expressing yeast cells than into the control microsomes from cells not expressing hUGTrel7. The possibility that this transporter participates in glucuronidation and/or chondroitin sulfate biosynthesis is discussed.
Stereochemistry of family 52 glycosyl hydrolases: a β‐xylosidase from Bacillus stearothermophilus T‐6 is a retaining enzyme
Authors: Bravman Tsafrir , Zolotnitsky Gennady , Shulami Smadar , Belakhov Valery , Solomon Dmitry , Baasov Timor , Shoham Gil , Shoham Yuval
Published: July 2002

A β‐xylosidase from Bacillus stearothermophilus T‐6 assigned to the uncharacterized glycosyl hydrolase family 52 was cloned, overexpressed in Escherichia coli and purified. The enzyme showed maximum activity at 65°C and pH 5.6–6.3. The stereochemistry of the hydrolysis of p‐nitrophenyl β‐D‐xylopyranoside was followed by 1H‐nuclear magnetic resonance. Time dependent spectrum analysis showed that the configuration of the anomeric carbon was retained, indicating that a retaining mechanism prevails in family 52 glycosyl hydrolases. Sequence alignment and site‐directed mutagenesis enabled the identification of functionally important amino acid residues of which Glu337 and Glu413 are likely to be the two key catalytic residues involved in enzyme catalysis.
Duffy‐null promoter heterozygosity reduces DARC expression and abrogates adhesion of the P. vivax ligand required for blood‐stage infection
Authors: Michon Pascal , Woolley Ian , Wood Erica M , Kastens W , Zimmerman Peter A , Adams John H
Published: July 2002

The Duffy blood group antigen is an essential receptor for Plasmodium vivax entry into erythrocytes in a process mediated by the parasite ligand, the Duffy binding protein (DBP). Recently, individuals living in a malaria endemic region of Papua New Guinea were identified as heterozygous for a new allele conferring Duffy negativity, which results in 50% less Duffy antigen on their erythrocytes. We demonstrate that DBP adherence to erythrocytes is significantly reduced for erythrocytes from heterozygous individuals who carry one Duffy antigen negativity allele. These data provide evidence that emergence of this new allelic form of Duffy negativity is correlated with resistance against vivax malaria.
Curcumin induces the mitochondrial permeability transition pore mediated by membrane protein thiol oxidation
Authors: Morin Didier , Barthélémy Sophie , Zini Roland , Labidalle Serge , Tillement Jean-Paul
Published: July 2002

Curcumin is a natural compound showing antiproliferative properties. Recent studies suggest that these properties might be due to its ability to induce apoptosis in tumor cells. As mitochondria play a pivotal role in the induction of the apoptotic process, we analyzed the effect of curcumin on mitochondrial function. Curcumin induced an increase in rat liver mitochondrial membrane permeability, resulting in swelling, loss of membrane potential and inhibition of ATP synthesis. These effects were mediated by the opening of the permeability transition pore. Curcumin pore induction involved the oxidation of membrane thiol functions and required the presence of low Ca2+ concentrations. These data suggest that mitochondria might be a target by which curcumin induces apoptosis of tumor cells.
Conventional protein kinase C isoforms and cross‐activation of protein kinase A regulate cardiac Na+ current
Authors: Shin Hyeon-Gyu , Murray Katherine T
Published: July 2002

We tested the hypothesis that specific isoforms of protein kinase C (PKC) are responsible for modulation of Na+ current (I
Na) derived from the human cardiac Na+ channel using activators and inhibitors selective for specific PKCs. Experimental results demonstrated that I
Na suppression was mediated by activation of conventional PKCs (cPKCs) and possibly resulted from channel internalization. In the presence of cPKC inhibition, phorbol ester application unexpectedly increased Na+ current, an effect eliminated by inhibition of protein kinase A. These findings demonstrate complex modulation of cardiac I
Na by protein kinases and provide further evidence that PKC isoforms have distinct protein targets.
Effect of divalent cations on antiparallel G‐quartet structure of d(G4T4G4)
Authors: Miyoshi Daisuke , Nakao Akihiro , Toda Takeshi , Sugimoto Naoki
Published: July 2002

The thermodynamic parameters of an antiparallel G‐quartet formation of d(G4T4G4) with 1 mM divalent cation (Mg2+, Ca2+, Mn2+, Co2+, and Zn2+) were obtained. The thermodynamic parameters showed that the divalent cation destabilizes the antiparallel G‐quartet of d(G4T4G4) in the following order: Zn2+>Co2+>Mn2+>Mg2+>Ca2+. In addition, a higher concentration of a divalent cation induced a transition from an antiparallel to a parallel G‐quartet structure. These results indicate that these divalent cations are a good tool for regulating the G‐quartet structures.
Design and production of genetically modified soybean protein with anti‐hypertensive activity by incorporating potent analogue of ovokinin(2–7)
Authors: Matoba Nobuyuki , Doyama Naomi , Yamada Yuko , Maruyama Nobuyuki , Utsumi Shigeru , Yoshikawa Masaaki
Published: July 2002

The potent anti‐hypertensive peptide, RPLKPW, has been designed based on the structure of ovokinin(2–7). The sequence encoding this peptide was introduced into three homologous sites in the gene for soybean β‐conglycinin α′ subunit. The native α′ subunit as well as the modified, RPLKPW‐containing α′ subunit were expressed in Escherichia coli, recovered from the soluble fraction and then purified by ion‐exchange chromatography. The RPLKPW peptide was released from recombinant RPLKPW‐containing α′ subunit after in vitro digestion by trypsin and chymotrypsin. Moreover, the undigested RPLKPW‐containing α′ subunit given orally at a dose of 10 mg/kg exerted an anti‐hypertensive effect in spontaneously hypertensive rats, unlike the native α′ subunit. These results provide evidence for the first time that a physiologically active peptide introduced into a food protein by site‐directed mutagenesis could practically function in vivo even at a low dose.
Involvement of the NF‐κB pathway in the transforming properties of the TEL–Jak2 leukemogenic fusion protein
Authors: Constantino Rosa Santos Susana , Monni Richard , Bouchaert Isabelle , Bernard Olivier , Gisselbrecht Sylvie , Gouilleux Fabrice , Penard-Lacronique Virginie
Published: July 2002

Constitutively active tyrosine kinases are frequently expressed in various types of human leukemias as the result of chromosomal translocations. The TEL–Jak2 fusion oncoprotein possesses transforming properties in both animal and cellular models, that are tightly dependent on Stat5 activation. In the IL‐3‐independent TEL–Jak2‐transformed Ba/F3 cells, activation of the PI‐3K/Akt pathway appears essential to cell proliferation. Here we report a sustained activation of NF‐κB factors in Ba/F3 cells, which inhibition dramatically impairs cell viability, indicating that NF‐κB signaling exerts a major role in the anti‐apoptotic activities of TEL–Jak2 oncoprotein.
Endocytosis and signaling cascades: a close encounter
Authors: Cavalli Valeria , Corti Michela , Gruenberg Jean
Published: July 2002

Internalization of receptors and other cell surface components is well known to occur via clathrin‐mediated endocytosis, although other less well characterized pathways are also involved. Internalized receptors are then delivered to early endosomes, where they are sorted to be recycled back to the plasma membrane for reutilization or transported to late endosomes/lysosomes for degradation. Endocytosis has long been considered as a constitutive, housekeeping function of animal cells that occurs independently of the cellular environment in contrast to regulated secretion. Here, we will discuss recent studies that are uncovering the existence of cross‐talk between signaling molecules and components of the transport machinery, indicating that endocytosis can be modulated by signaling pathways.
Expanding coincident signaling by PTEN through its inositol 1,3,4,5,6‐pentakisphosphate 3‐phosphatase activity
Authors: Caffrey James J. , Darden Tom , Wenk Markus R. , Shears Stephen B.
Published: July 2002

PTEN, a tumor suppressor among the most commonly mutated proteins in human cancer, is recognized to be both a protein phosphatase and a phosphatidylinositol 3,4,5‐trisphosphate (PtdIns(3,4,5)P3) 3‐phosphatase. Previous work [Maehama and Dixon, J. Biol. Chem. 273 (1998) 13375–13378] has led to a consensus that inositol phosphates are not physiologically relevant substrates for PTEN. In contrast, we demonstrate that PTEN is an active inositol 1,3,4,5,6‐pentakisphosphate (Ins(1,3,4,5,6)P5) 3‐phosphatase when expressed and purified from bacteria or HEK cells. Kinetic data indicate Ins(1,3,4,5,6)P5 (K
m=7.1 μM) and PtdIns(3,4,5)P3 (K
m=26 μM) compete for PTEN in vivo. Transient transfection of HEK cells with PTEN decreased Ins(1,3,4,5,6)P5 levels. We discuss the physiological significance of these studies in relation to recent work showing that dephosphorylation of Ins(1,3,4,5,6)P5 to inositol 1,4,5,6‐tetrakisphosphate is a cell signaling event.
Primary sequence requirements for S‐acylation of β2‐adrenergic receptor peptides
Authors: Bélanger Charlène , Ansanay Hervé , Qanbar Riad , Bouvier Michel
Published: July 2002

Palmitoylation is a post‐translational modification that occurs on selected cysteines of many proteins. Since a high proportion of basic and hydrophobic residues is often found near the palmitoylated cysteine, the role of these residues in the selection of specific palmitoylation sites was assessed. Short peptides derived from the β2‐adrenergic receptor sequence, modified to present different proportions of basic, acidic and hydrophobic residues, were tested in an in vitro S‐acylation assay. Basic residues proved to be essential, whereas hydrophobic residues greatly enhanced S‐acylation and acidic residues inhibited it. Taken together, these results show that short peptides contain the required molecular determinants leading to selective S‐acylation. Whether or not these sequence characteristics also contribute to the selectivity of palmitoylation in vivo will need to be further investigated.
Identification of human endomucin‐1 and ‐2 as membrane‐bound O‐sialoglycoproteins with anti‐adhesive activity1
Authors: Kinoshita Makoto , Nakamura Tomoyuki , Ihara Masafumi , Haraguchi Tokuko , Hiraoka Yasushi , Tashiro Kei , Noda Makoto
Published: July 2002

Using a signal sequence trap method and database search, we identified a series of human cDNAs encoding two structurally related type I membrane proteins of ∼25 kDa with multiple glycosylation motifs. These genes, termed endomucin‐1/‐2, are expressed in several human tissues including heart, kidney, and lung. Exogenously expressed human endomucin‐1/‐2 proteins were modified into 80–120 kDa glycoproteins, which were susceptible to O‐sialoglycoprotein endopeptidase digestion. Transient overexpression of endomucin‐1/‐2 reduced the number of adhesion plaques and reduced cell attachment to the substrate. This phenotype was suppressed by laminin or the protein kinase inhibitor staurosporine. Our findings suggest that human endomucin‐1/‐2 negatively regulate cell adhesion to the extracellular matrix.
Phosphorylation of plant actin‐depolymerising factor by calmodulin‐like domain protein kinase
Authors: Allwood Ellen G , Smertenko Andrei P , Hussey Patrick J
Published: July 2002

The actin‐depolymerising factor (ADF)/cofilin group of proteins are stimulus‐responsive actin‐severing proteins, members of which are regulated by reversible phosphorylation. The phosphorylation site on the maize ADF, ZmADF3, is Ser‐6 but the kinase responsible is unknown [Smertenko et al., Plant J. 14 (1998) 187–193]. We have partially purified the ADF kinase(s) and found it to be calcium‐regulated and inhibited by N‐(6‐aminohexyl)‐[3H]5‐chloro‐1‐naphthalenesulphonamide. Immunoblotting reveals that calmodulin‐like domain protein kinase(s) (CDPK) are enriched in the purified preparation and addition of anti‐CDPK to in vitro phosphorylation assays results in the inhibition of ADF phosphorylation. These data strongly suggest that plant ADF is phosphorylated by CDPK(s), a class of protein kinases unique to plants and protozoa.
CSN3 interacts with IKKγ and inhibits TNF‐ but not IL‐1‐induced NF‐κB activation
Authors: Hong Xia , Xu Liang-Guo , Li Xiaoyan , Zhai Zhonghe , Shu Hong-Bing
Published: July 2002

The transcription factor nuclear factor κB (NF‐κB) plays a pivotal role in immune and inflammatory responses. Activation of NF‐κB requires the activity of IKK, a kinase complex that contains two catalytic subunits, IKKα and IKKβ, and a regulatory subunit IKKγ. To understand how IKK activity is regulated, we searched for IKKγ‐interacting proteins by the yeast two‐hybrid system. These screenings identified CSN3, a component of the COP9 signalsome, as a protein specifically interacting with IKKγ. Overexpression of CSN3 inhibits NF‐κB activation triggered by tumor necrosis factor (TNF), but not interleukin‐1 (IL‐1). Moreover, overexpression of CSN3 also inhibits NF‐κB activation triggered by proteins involved in TNF signaling, including TNF‐R1, TRAF2, RIP, and NIK, but not by TRAF6, a protein involved in IL‐1 signaling. These data suggest that CSN3 is a specific negative regulator of TNF‐ but not IL‐1‐induced NF‐κB activation pathways.
Cg‐TIMP, an inducible tissue inhibitor of metalloproteinase from the Pacific oyster Crassostrea gigas with a potential role in wound healing and defense mechanisms1
Authors: Montagnani Caroline , Le Roux Frédérique , Berthe Franck , Escoubas Jean-Michel
Published: July 2002

We have cloned and characterized a cDNA encoding Cg‐TIMP, the first tissue inhibitor of metalloproteinase identified in mollusks. The isolated cDNA encodes a protein of 221 residues that has a domain organization similar to that of vertebrate TIMPs including a signal sequence, and the 12 cysteines characteristic of the TIMP signature. Analysis of Cg‐TIMP expression in adult oyster tissues, by Northern blot and in situ hybridization, indicates that Cg‐TIMP was only expressed in hemocytes which are the key components of defense mechanisms in mollusks. We also observed that Cg‐TIMP mRNA accumulated during shell damage and bacterial challenge. This pattern of expression suggests that Cg‐TIMP may be an important factor in wound healing and defense mechanisms.
Transmembrane TNF (pro‐TNF) is palmitoylated
Authors: Utsumi Toshihiko , Takeshige Tomonori , Tanaka Kenji , Takami Kenji , Kira Yukimi , Klostergaard Jim , Ishisaka Rumi
Published: July 2002

Human transmembrane tumor necrosis factor (pro‐TNF) was examined for protein acylation. The cDNA encoding pro‐TNF was expressed in both COS‐1 cells and Sf9 cells and metabolic labeling with [3H]myristic or [3H]palmitic acid was attempted. The 17 kDa mature TNF secreted from the transfected cells was not labeled, whereas the 26 kDa pro‐TNF was specifically labeled with [3H]palmitic acid. The [3H]palmitic acid labeling of pro‐TNF was eliminated by treatment with hydroxylamine, indicating that the labeling was due to palmitoylation of a cysteine residue via a thioester bond. Site‐directed mutagenesis of the two cysteine residues residing in the leader sequence of pro‐TNF demonstrated that palmitoylation of pro‐TNF occurs solely at Cys‐47, located at the boundary between the transmembrane and cytoplasmic domains of pro‐TNF. Thus, pro‐TNF interacts with the plasma membrane via both its proteinaceous transmembrane domain and a lipid anchor.
Disulfide bonds stabilize JC virus capsid‐like structure by protecting calcium ions from chelation
Authors: Chen Pei-Lain , Wang Meilin , Ou Wei-Chih , Lii Chong-Kuei , Chen Li-Sheng , Chang Deching
Published: July 2002

To investigate the role of disulfide bonds in the capsid structure, a recombinant JC virus‐like particle (VLP) was used. The major capsid protein, VP1, of the JC virus was expressed in yeast cells. The yeast‐expressed VP1 was self‐assembled into a VLP. Disulfide bonds were found in the VLP which caused dimeric and trimeric VP1 linkages as demonstrated by non‐reducing SDS–PAGE. The VLP remained intact when disulfide bonds were reduced by dithiothreitol. The VLP without disulfide bonds could be disassembled into capsomeres by EGTA alone, but those with disulfide bonds could not be disassembled by EGTA. Capsomeres were reassembled into VLPs in the presence of calcium ions. Capsomeres formed irregular aggregations instead of VLPs when treated with diamide to reconstitute the disulfide bonds. These results indicate that disulfide bonds play an important role in maintaining the integrity of the JC VLP by protecting calcium ions from chelation.
Properties of the Escherichia coli rhodanese‐like protein SseA: contribution of the active‐site residue Ser240 to sulfur donor recognition
Authors: Colnaghi Rita , Cassinelli Gabriela , Drummond Martin , Forlani Fabio , Pagani Silvia
Published: July 2002

The product of Escherichia coli sseA gene (SseA) was the subject of the present investigation aimed to provide a tool for functional classification of the bacterial proteins of the rhodanese family. E. coli SseA contains the motif CGSGVTA around the catalytic cysteine (Cys238). In eukaryotic sulfurtransferases this motif discriminates for 3‐mercaptopyruvate:cyanide sulfurtransferase over thiosulfate:cyanide sulfurtransferases (rhodanese). The biochemical characterization of E. coli SseA allowed the identification of the first prokaryotic protein with a preference for 3‐mercaptopyruvate as donor substrate. Replacement of Ser240 with Ala showed that the presence of a hydrophobic residue did not affect the binding of 3‐mercaptopyruvate, but strongly prevented thiosulfate binding. On the contrary, substitution of Ser240 with an ionizable residue (Lys) increased the affinity for thiosulfate.
A new class of scorpion toxin binding sites related to an A‐type K+ channel: pharmacological characterization and localization in rat brain
Authors: Vacher Hélène , Romi-Lebrun Régine , Mourre Christiane , Lebrun Bruno , Kourrich Said , Masméjean Frédérique , Nakajima Terumi , Legros Christian , Crest Marcel , Bougis Pierre E. , Martin-Eauclaire Marie-France
Published: July 2002

A new scorpion toxin (3751.8 Da) was isolated from the Buthus martensi venom, sequenced and chemically synthesized (sBmTX3). The A‐type current of striatum neurons in culture completely disappeared when 1 μM sBmTX3 was applied (K
d=54 nM), whereas the sustained K+ current was unaffected. 125I‐sBmTX3 specifically bound to rat brain synaptosomes (maximum binding=14 fmol mg−1 of protein, K
d=0.21 nM). A panel of toxins yet described as specific ligands for K+ channels were unable to compete with 125I‐sBmTX3. A high density of 125I‐sBmTX3 binding sites was found in the striatum, hippocampus, superior colliculus, and cerebellum in the adult rat brain.
Use of amphipathic polymers to deliver a membrane protein to lipid bilayers
Authors: Nagy Joanna K , Kuhn Hoffmann Amy , Keyes Melvin H , Gray Don N , Oxenoid Kirill , Sanders Charles R
Published: July 2002

Data are presented which suggest that a class of amphiphilic polymers known as ‘amphipols’ may serve as a vehicle for delivering complex integral membrane proteins into membranes. The integral membrane protein diacylglycerol kinase (DAGK) was maintained in soluble form by either of two different amphipols. Small aliquots of these solutions were added to pre‐formed lipid vesicles and the appearance of DAGK catalytic activity was monitored as an indicator of the progress of productive protein insertion into the bilayers. For one of the two amphipols tested, DAGK was observed to productively transfer from its amphipol complex into vesicles with moderate efficiency. Results were not completely clear for the other amphipol.
The cAMP response element binding protein‐2 (CREB‐2) can interact with the C/EBP‐homologous protein (CHOP)
Authors: Gachon Frédéric , Gaudray Gilles , Thébault Sabine , Basbous Jihane , Koffi Joseph Aman , Devaux Christian , Mesnard Jean-Michel
Published: July 2002

cAMP response element binding protein‐2 (CREB‐2) is a basic leucine zipper (bZIP) factor that was originally described as a repressor of CRE‐dependent transcription but that can also act as a transcriptional activator. Moreover, CREB‐2 is able to function in association with the viral Tax protein as an activator of the human T‐cell leukemia virus type I (HTLV‐I) promoter. Here we show that CREB‐2 is able to interact with C/EBP‐homologous protein (CHOP), a bZIP transcription factor known to inhibit CAAT/enhancer‐dependent transcription. Cotransfection of CHOP with CREB‐2 results in decreased activation driven by the cellular CRE motif or the HTLV‐I proximal Tax‐responsive element, confirming that CREB‐2 and CHOP can interact with each other in vivo.
Down modulation of IL‐18 expression by human papillomavirus type 16 E6 oncogene via binding to IL‐18
Authors: Cho Young-Sik , Kang Jeong-Woo , Cho MinChul , Cho Cheong-Weon , Lee ShinJe , Choe Yong-Kyung , Kim YongMan , Choi InPyo , Park Sue-Nie , Kim SooHyun , Dinarello Charles A. , Yoon Do-Young
Published: July 2002

To understand modulation of a novel immune‐related cytokine, interleukin‐18, by human papillomavirus type (HPV) 16 oncogenes, HaCaT, normal keratinocyte cell line, and C‐33A, HPV‐negative cervical cancer cell line, were prepared to establish stable cell lines expressing E6, E6 mutant (E6m), E6E7, or E7 constitutively. Expressions of various HPV oncogene transcripts were identified by RT‐PCR. Expression of HPV oncogene E6 was reversely correlated to the expression of interleukin‐18, a novel pro‐inflammatory cytokine. The expression of E6 in C‐33A, independent of E6 splicing, resulted in decreased IL‐18 expression and that of IL‐18 was also significantly reduced in HaCaT cells expressing E6. The level of p53 was reduced in C‐33A cells expressing E6 whereas not altered in HaCaT cells expressing E6, suggesting that E6 downregulated IL‐18 expression via an independent pathway of p53 degradation in HaCaT cells which have a mutated p53 form. However, E7 did not affect IL‐18 expression significantly in both C‐33A and HaCaT cells. Cotransfection experiments showed that E6 oncogene did not inhibit the activities of IL‐18 promoter P1 and P2, suggesting that E6 oncogene indirectly inhibited IL‐18 expression. Taken together, E6, E6m and E6/E7 inhibited IL‐18 expression with some variation, assuming that cells expressing E6 oncogene can evade immune surveillance by downregulating the expression of immune stimulating cytokine gene, IL‐18, and inhibiting the cascade of downstream effects that follow activation of the IL‐18 receptor.
Using an in vivo phagemid system to identify non‐compatible loxP sequences
Authors: Siegel Robert W , Jain Raj , Bradbury Andrew
Published: July 2002

The site‐specific recombination system of bacteriophage P1 is composed of the Cre recombinase that recognizes a 34‐bp loxP site. The Cre/loxP system has been extensively used to manipulate eukaryotic genomes for functional genomic investigations. The creation of additional heterologous loxP sequences potentially expands the utility of this system, but only if these loxP sequences do not recombine with one another. We have developed a stringent in vivo assay to examine the degree of recombination between all combinations of each previously published heterologous loxP sequence. As expected, homologous loxP sequences efficiently underwent Cre‐mediated recombination. However, many of the heterologous loxP pairs were able to support recombination with rates varying from 5 to 100%. Some of these loxP sequences have previously been reported to be non‐compatible with one another. Our study also confirmed other heterologous loxP pairs that had previously been shown to be non‐compatible, as well as defined additional combinations that could be used in designing new recombination vectors.
Inhibition of lipoprotein‐associated phospholipase A2 diminishes the death‐inducing effects of oxidised LDL on human monocyte‐macrophages
Authors: Carpenter Keri L.H. , Dennis Ian F. , Challis Iain R. , Osborn David P. , Macphee Colin H. , Leake David S. , Arends Mark J. , Mitchinson Malcolm J.
Published: July 2002

The death of macrophages contributes to atheroma formation. Oxidation renders low‐density lipoprotein (LDL) cytotoxic to human monocyte‐macrophages. Lipoprotein‐associated phospholipase A2 (Lp‐PLA2), also termed platelet‐activating factor acetylhydrolase, hydrolyses oxidised phospholipids. Inhibition of Lp‐PLA2 by diisopropyl fluorophosphate or Pefabloc (broad‐spectrum serine esterase/protease inhibitors), or SB222657 (a specific inhibitor of Lp‐PLA2) did not prevent LDL oxidation, but diminished the ensuing toxicity and apoptosis induction when the LDL was oxidised, and inhibited the rise in lysophosphatidylcholine levels that occurred in the inhibitors’ absence. Hydrolysis products of oxidised phospholipids thus account for over a third of the cytotoxic and apoptosis‐inducing effects of oxidised LDL on macrophages.
AS‐48: a circular protein with an extremely stable globular structure
Authors: Cobos E.S , Filimonov V.V , Gálvez A , Maqueda M , Valdı́via E , Martı́nez J.C , Mateo P.L
Published: July 2002

The unfolding thermodynamics of the circular enterocin protein AS‐48, produced by Enterococcus faecalis, has been characterized by differential scanning calorimetry. The native structure of the 70‐residue protein is extremely thermally stable. Thus, at pH 2.5 and low ionic strength thermal denaturation occurs under equilibrium at 102°C, while the unfolded state irreversibly aggregates at neutral and alkaline pH. Calorimetric data analysis shows that the specific enthalpy change upon unfolding is unusually small and the heat capacity change is quite normal for a protein of this size, whereas the Gibbs energy change at 25°C is relatively high. At least part of this high stability might be put down to entropic constraints induced by the circular organization of the polypeptide chain.