bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation, crystallization and X‐ray analysis of the quaternary complex of Phe‐tRNAPhe, EF‐Tu, a GTP analog and kirromycin
Authors: Kristensen Ole , Reshetnikova Ludmila , Nissen Poul , Siboska Gunhild , Thirup Søren , Nyborg Jens
Published: July 2002

Kirromycin inhibits bacterial protein synthesis by acting on elongation factor Tu (EF‐Tu). Complexes of the antibiotic, Phe‐tRNAPhe, the guanosine triphosphate analog GDPNP, and mesophilic (Escherichia coli), as well as thermophilic (Thermus thermophilus) EF‐Tu were isolated. Crystallization was achieved at 4°C, pH 6.4, using ammonium sulphate as precipitant. Crystallographic data were recorded at cryogenic temperature on crystals exposed to synchrotron radiation. Crystals of the thermophilic complex are based on a rhombohedral lattice with cell dimensions of 137.3 Å, and angles of 54.0°. Although related, these cell parameters are different from those found in the crystals of the recently solved structure of the ternary complex of Phe‐tRNAPhe, GDPNP, and Thermus aquaticus EF‐Tu (Nissen, P., Kjeldgaard, M., Thirup, S., Polekhina, G., Reshetnikova, L., Clark, B.F. and Nyborg, J. (1995) Science 270, 1464–1472 [1]), possibly indicating some allosteric effect caused by kirromycin. Crystals of the mesophilic complex belong to the cubic space P432, with cell axis of 196.26 Å. In both cases, the crystals contain one complex per asymmetric unit.
Toxicity of expanded polyglutamine‐domain proteins in Escherichia coli
Authors: Onodera Osamu , Rose Allen D. , Tsuji Shoji , Vance Jeffery M. , Strittmatter Warren J. , Burke James R.
Published: July 2002

Five neurodegenerative diseases are caused by proteins with expanded polyglutamine domains. Toxicity of these proteins has been previously identified only in mammals, and no simple model systems are available. In this paper, we demonstrate in E. coli that long polyglutamine domains (59–81 residues) as GST‐fusion proteins inhibit growth while smaller glutamine (10–35 residues) or polyalanine (61 residues) domains have no effect. Analogously in humans, polyglutamine repeats less than 35–40 glutamines produce a normal phenotype, while expansion greater than 40 glutamines is always associated with disease. Expression of polyglutamine proteins in E. coli may help identify the molecular mechanism of pathogenesis of CAG trinucleotide repeat diseases and be a useful screen to identify potential therapeutic compounds.
Lipopolysaccharide‐binding protein mediates CD14‐independent intercalation of lipopolysaccharide into phospholipid membranes
Authors: Schromm Andra B. , Brandenburg Klaus , Rietschel Ernst Th. , Flad Hans-Dieter , Carroll Stephen F. , Seydel Ulrich
Published: July 2002

Lipopolysaccharides (LPS, endotoxin) stimulate mononuclear cells to release cytokines which initiate endotoxic effects. Interaction of LPS at low concentrations with target cells is CD14‐independent whereas at high LPS concentrations it is CD14‐independent. Here, we demonstrate by resonance energy transfer (RET) technique that nonspecific, CD14‐independent intercalation of LPS into membrane systems can be mediated by lipopolysaccharide‐binding protein (LBP). It is proposed that in this pathway, LBP breaks down LPS aggregates, transports the smaller units to and inserts them into the phospholipid cell matrix. We furthermore show that LBP also mediates the intercalation of other negatively charged amphiphilic molecules. We propose a model explaining CD14‐independent cell activation at high endotoxin concentrations.
Osmosignalling in C6 glioma cells
Authors: Sinning Ralf , Schliess Freimut , Kubitz Ralf , Häussinger Dieter
Published: July 2002

The influence of aniso‐osmolarity on the activity of the MAP kinases Erk‐1 and Erk‐2 was studied in C6 glioma cells. Hypo‐osmotic treatment (205 mosmol/l) led to an increased activity of Erk‐1 and Erk‐2 within 3 min, which became maximal at 10 min and returned to basal level within 120 min. In contrast, Erk activity was reduced under hyper‐osmotic conditions (405 mosmol/l), compared to the normo‐osmotic control (305 mosmol/l). Erk activation was accompanied by a mobility shift of Raf‐1. Hypo‐osmotic exposure increased the cytosolic Ca2+ concentration ([Ca2+]i). Absence of extracellular Ca2+ largely abolished the [Ca2+]i response to hypo‐osmolarity, whereas Erk activation following hypo‐osmotic stimulation remained unaffected, suggesting a Ca2+ independence of the osmosignalling pathway to the MAP kinases. Both the Ca2+ response as well as the Erk activation following hypo‐osmotic exposure were maintained in the presence of the phospholipase C inhibitor U73122. Application of 8‐CPT cAMP, forskolin/isobutylmethylxanthine or isoproterenol blocked Erk activation following hypo‐osmotic treatment of the cells, suggesting a role of the Ras/Raf pathway upstream from Erk‐1 and Erk‐2. Protein kinase C (PKC) is unlikely to play a role in the hypo‐osmolarity‐ induced signalling towards MAP kinases, as revealed by inhibition of PKC with Gö6850. Inhibition of pertussis‐ or cholera toxin‐sensitive G‐proteins as well as inhibition of tyrosine kinases with genistein and of PI3 kinase by wortmannin had no effect on the Erk response to hypo‐osmolarity. It is concluded that osmosignalling in C6 glioma cells differs upstream of the MAP kinases from that observed in primary rat astrocytes, H4IIE rat hepatoma cells and isolated rat hepatocytes.
Murine tissue inhibitor of metalloproteinases—4 (Timp—4): cDNA isolation and expression in adult mouse tissues1
Authors: Leco Kevin J. , Apte Suneel S. , Taniguchi Gary T. , Hawkes Susan P. , Khokha Rama , Schultz Gilbert A. , Edwards Dylan R.
Published: July 2002

We have isolated cDNA clones corresponding to a new member of the murine tissue inhibitor of metalloproteinase (TIMP) family, designated Timp‐4. The nucleotide sequence predicts a protein of 22 609 Da that contains the characteristic 12 cysteine TIMP signature. TIMP‐4 is more closely related to TIMP‐2 and TIMP‐3 than to TIMP‐1 (48%, 45% and 38% identity, respectively). Analysis of Timp‐4 mRNA expression in adult mouse tissues indicated a 1.2 kb transcript in brain, heart, ovary and skeletal muscle. This pattern of expression distinguishes Timp‐4 from other Timps, suggesting that the TIMP‐4 protein may be an important tissue‐specific regulator of extracellular matrix remodelling.
Calcium mediates the activation of the inhibitory current induced by odorants in toad olfactory receptor neurons
Authors: Morales Bernardo , Madrid Rodolfo , Bacigalupo Juan
Published: July 2002

In toad olfactory neurons, a putrid odorant mixture inducing inhibitory responses increases Ca2+‐activated K+ conductance, developing a hyperpolarizing receptor potential. Removal of extracellular Ca2+ or exposure to nifedipine reversibly reduced the inhibitory response, suggesting that odorants induce a Ca2+ influx. We show evidence for an odorant‐induced Ca2+ current. Using confocal microscopy, it is shown that odorants induce a nifedipine‐sensitive elevation of Ca2+ in the apical end of the cell. These results suggest an inhibitory mechanism in which an apical Ca2+ influx causes an increase in internal Ca2+, opening Ca2+‐activated K+ channels that lead to membrane hyperpolarization. © 1997 Federation of European Biochemical Societies. All rights reserved.
Capacitative calcium entry is inhibited in vascular endothelial cells by disruption of cytoskeletal microfilaments
Authors: Holda Jaclyn R , Blatter Lothar A
Published: July 2002

© Federation of European Biochemical Societies.
Increased stress parameter synthesis in the yeast Saccharomyces cerevisiae after treatment with 4‐hydroxy‐2‐nonenal1
Authors: Wonisch Willibald , Hayn Marianne , Schaur R.Jörg , Tatzber Franz , Kranner Ilse , Grill Dieter , Winkler Rudolf , Bilinski Tomasz , Kohlwein Sepp D , Esterbauer Hermann
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Nitric oxide protects endothelial cells from tumor necrosis factor‐α‐mediated cytotoxicity: possible involvement of cyclic GMP
Authors: Polte Tobias , Oberle Stefanie , Schröder Henning
Published: July 2002

In cultured endothelial cells, incubation with TNF‐α (50 ng/ml) for 48 h markedly reduced viability of endothelial cells. A 6 h preincubation with Sper/NO (0.03‐−1 μM) protected endothelial cells in a concentration‐dependent manner and increased viability by 63% of control. The NO scavenger PTIO (30 μM) completely abolished cytoprotection by Sper/NO. A cytoprotective effect comparable to Sper/NO was observed when preincubating the cells with 8‐bromo cyclic GMP (1–10 μM). Moreover, no protection by Sper/NO occurred in the presence of ODQ (0.1 μM), a selective inhibitor of soluble guanylyl cyclase. Our results demonstrate that NO produces a long‐term endothelial protection against cellular injury by TNF‐α, presumably via a cyclic GMP‐dependent pathway.
The hydrophobic probe 4,4′‐bis(1‐anilino‐8‐naphthalene sulfonic acid) is specifically photoincorporated into the N‐terminal domain of αB‐crystallin
Authors: Smulders Ronald H.P.H , de Jong Wilfried W
Published: July 2002

Photoincorporation of the fluorescent probe 4,4′‐bis(1‐anilino‐8‐naphthalene sulfonic acid) (bis‐ANS) can be used to locate solvent‐exposed hydrophobic regions in proteins. We show that bis‐ANS is specifically incorporated into the putative N‐terminal domain of αB‐crystallin. This incorporation diminishes the chaperone‐like activity of αB‐crystallin, suggesting that hydrophobic surfaces in the N‐terminal domain are involved in the binding of unfolding proteins.
Molecular cloning and expression of a bovine endothelial inward rectifier potassium channel1
Authors: Forsyth Scott E , Hoger Anne , Hoger Jeff H
Published: July 2002

A 5.1 kb cDNA encoding an inward rectifier K+ channel (BIK) was isolated from a bovine aortic endothelial cell library. The cDNA codes for a 427‐amino‐acid protein with two putative transmembrane regions. Sequence analysis reveals that BIK is a member of the Kir2.1 family of inward rectifier K+ channels. Expression in Xenopus oocytes showed that BIK is a K+‐specific strong inward rectifier channel that is sensitive to extracellular Ba2+, Cs+, and a variety of anti‐arrhythmic agents. Northern analysis revealed that endothelial cells express a 5.5 kb BIK mRNA that is sensitive to shear stress.
Structure of KW‐2228, a tailored human granulocyte colony‐ stimulating factor with enhanced biological activity and stability
Authors: Fujii Isao , Nagahara Yoshitomo , Yamasaki Motoo , Yokoo Yoshiharu , Itoh Seiga , Hirayama Noriaki
Published: July 2002

KW‐2228 is a tailored human granulocyte colony‐stimulating factor (hG‐CSF) which has more potent granulopoietic activity and is more stable than wild‐type hG‐CSF. Analysis of the 2.3 Å resolution crystal structure of KW‐2228 unambiguously revealed a four‐α‐helix bundle motif with up‐up‐down‐down connectivity. The structures of long overhand loops connecting the helices and the N‐terminus have been definitively determined. The present analysis has clearly revealed that substituted residues play important roles in fastening a long overhand loop to the N‐ and C‐termini to fix the conformation. This conformation should be responsible for a substantial enhancement of the biological activity and stability.
Inhibition of protein tyrosine phosphatases causes phosphorylation of tyrosine‐331 in the p60 TNF receptor and inactivates the receptor‐associated kinase
Authors: Darnay Bryant G , Aggarwal Bharat B
Published: July 2002

Inhibition of protein tyrosine phosphatases blocks tumor necrosis factor (TNF)‐induced growth modulation and NF‐κB activation, both mediated primarily through the p60 TNF receptor. How inhibition of the phosphatases affects the p60 TNF receptor or the recently described receptor‐associated serine/threonine kinase (p60TRAK) is not known. In this report, we show that this inhibition, when induced by pervanadate, caused the tyrosine phosphorylation of the cytoplasmic domain (CD) of the p60 receptor, as revealed by phosphoamino acid analysis. Furthermore, site‐directed mutagenesis indicated that pervanadate specifically induced the phosphorylation of tyrosine‐331, which is located in the death domain of the TNF receptor, a domain to which p60TRAK binds. This tyrosine residue was also phosphorylated by purified, recombinant pp60Src in vitro. Inhibition of protein tyrosine phosphatases by pervanadate also led to the inactivation of p60TRAK. In contrast, okadaic acid, a specific inhibitor of protein serine/threonine phosphatase, increased p60TRAK activity. Taken together, these results suggest that protein tyrosine phosphatases play an essential role in phosphorylation of the cytoplasmic domain of the TNF receptor and in regulation of the receptor‐associated kinase, and this in turn may play a role in TNF‐mediated growth modulation and NF‐κB activation.
Ca‐dependent regulation of Na+‐selective channels via actin cytoskeleton modification in leukemia cells
Authors: Maximov Anton V , Vedernikova Elena A , Hinssen Horst , Khaitlina Sofia Y , Negulyaev Yuri A
Published: July 2002

With the use of the patch‐clamp technique, physiological mechanisms of Na+ channel regulation involving submembranous actin rearrangements were examined in human myeloid leukemia K562 cells. We found that the actin‐severing protein gelsolin applied to cytoplasmic surface of membrane fragments at a high level of [Ca2+]i (1 μM) increased drastically the activity of Na‐selective channels of 12 pS unitary conductance. In the experiments on intact cells, the elevation of [Ca2+]i using the ionophore 4Br‐A23187 also resulted in Na+ channel activation. Addition of actin to the cytoplasmic surface of membrane patches reduced this activity to background level, likely due to actin polymerization. Our data imply that Ca‐dependent modulations of the actin cytoskeleton may represent one of the general mechanisms of channel regulation and cell signalling.
Cyclosporine A is an uncompetitive inhibitor of proteasome activity and prevents NF‐κB activation
Authors: Meyer Stephanie , Kohler N.Gail , Joly Alison
Published: July 2002

Cyclosporine A is an immunosuppressive agent that is used clinically in the prevention of transplant rejection and development of graft‐versus‐host disease. Recently, cyclosporine A has been shown to possess anti‐inflammatory properties and is capable of inhibiting lipopolysaccharide‐induced NF‐κB activation. Ubiquitin‐mediated proteasomal proteolysis plays a critical role in signal‐induced NF‐κB activation since it regulates both IκB degradation and p105 processing, it is also involved in the production of peptides for the assembly of MHC class I molecules. We report here that cylcosporine A acts as an uncompetitive inhibitor of the chymotrypsin‐like activity of the 20S proteasome in vitro and that it suppresses lipopolysaccharide‐induced IκB degradation and p105 processing in vivo demonstrating that inhibition of proteasome proteolysis is the mechanism by which cyclosporine A prevents NF‐κB activation. A structurally unrelated immunosuppressant, rapamycin, did not inhibit the 20S proteasome in vitro.
New biomarker evidence of oxidative DNA damage in patients with non‐insulin‐dependent diabetes mellitus
Authors: Leinonen Janne , Lehtimäki Terho , Toyokuni Shinya , Okada Kunihiko , Tanaka Tomoyuki , Hiai Hiroshi , Ochi Hirotomo , Laippala Pekka , Rantalaiho Vappu , Wirta Ole , Pasternack Amos , Alho Hannu
Published: July 2002

Urinary 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) has been reported to serve as a sensitive biomarker of oxidative DNA damage and also of oxidative stress. We have investigated oxidative DNA damage in patients with non‐insulin‐dependent diabetes mellitus (NIDDM) by urinary 8‐OHdG assessments. We determined the total urinary excretion of 8‐OHdG from 24 h urine samples of 81 NIDDM patients 9 years after the initial diagnosis and of 100 non‐diabetic control subjects matched for age and gender. The total 24 h urinary excretion of 8‐OHdG was markedly higher in NIDDM patients than in control subjects (68.2±39.4 μg vs. 49.6±37.7 μg, P=0.001). High glycosylated hemoglobin was associated with a high level of urinary 8‐OHdG. The increased excretion of urinary 8‐OHdG is seen as indicating an increased systemic level of oxidative DNA damage in NIDDM patients.
Characterisation of the rat and mouse homologues of gC1qBP, a 33 kDa glycoprotein that binds to the globular `heads' of C1q
Authors: Lynch Nicholas J , Reid Kenneth B.M , van den Berg Rocco H , Daha Mohamed R , Leigh Leonora A.E , Ghebrehiwet Berhane , Lim Wallace B.L , Schwaeble Wilhelm J
Published: July 2002

gC1qBP is a 33 kDa glycoprotein that binds to the globular `heads' of C1q. We have cloned cDNAs encoding the rat and mouse homologues of gC1qBP. Comparison of the cDNA‐derived amino acid sequences of gC1qBP reveals that either of the rodent sequences is 89.9% identical to the reported human sequence. Recombinant rat gC1qBP binds avidly to human C1q. gC1qBP mRNA is abundantly expressed in every rat and mouse tissue analysed. Rat mesangial cells synthesise gC1qBP, but do not express gC1qBP on the cell surface. In rat serum, gC1qBP is present at low levels.
Purification and characterization of 25‐hydroxyvitamin D3 1α‐hydroxylase from rat kidney mitochondria
Authors: Nakamura Yoshito , Eto Tada-Aki , Taniguchi Tomotaka , Miyamoto Koji , Nagatomo Junji , Shiotsuki Hironori , Sueta Hideto , Higashi Shushi , Okuda Kyu-Ichiro , Setoguchi Toshiaki
Published: July 2002

We purified extensively 25‐hydroxyvitamin D3 1α‐hydroxylase (calcidiol, NADPH: oxygen oxidoreductase (1‐hydroxylating), EC 1.14.13.13) from kidney mitochondria of rachitic rats and disclosed its peculiar properties as a P450. The final preparation was identified as a 55 kDa protein having an intense absorption at 417 nm characteristic of P450. The specific activity was 4.8 nmol/min/mg of protein indicating a 350‐fold purification. Specific content of P450 was 1.1 nmol/mg of protein and turnover number was 4.4 min−1.
Activation of myocardial cAMP‐dependent protein kinase by lysoplasmenylcholine
Authors: Williams Scott D , Ford David A
Published: July 2002

Plasmalogen‐specific, calcium‐independent phospholipase A2 (iPLA2) is activated during myocardial ischemia. Accordingly, we have assessed the activation of myocardial protein kinases by the iPLA2 product, lysoplasmenylcholine. Lysoplasmenylcholine‐activated protein kinase activity from heart cytosol fractionated on a DE‐52 column was identified as cAMP‐dependent protein kinase (PKA) based on the following: (1) protein kinase activity stimulated by cAMP and lysoplasmenylcholine co‐eluted on sequential chromatographic steps; (2) lysoplasmenylcholine‐activated protein kinase activity was inhibited by the PKA inhibitor, PKI; and (3) the unprimed PKA form generated from the primed form of PKA was activated by cAMP and lysoplasmenylcholine. These results demonstrate a novel mechanism for PKA activation by lysoplasmenylcholine.
Stathmin overexpression in 293 cells affects signal transduction and cell growth
Authors: Lawler Sean , Gavet Olivier , Rich Tina , Sobel André
Published: July 2002

Stathmin is a ubiquitous cytoplasmic protein whose phosphorylation state changes markedly in response to extracellular signals, and during the cell cycle. To clarify the function of stathmin, its four phosphorylation sites were mutated to either alanines (4A‐stathmin) or glutamates (4E‐stathmin). In transfected cells, 4A‐stathmin caused a strong G2/M block and also inhibited the responsiveness of a co‐transfected fos promoter/luciferase reporter plasmid to serum stimulation, whereas wild type and 4E‐stathmin had relatively minor effects. These results support the idea that stathmin plays a role in multiple cellular processes and indicate that the regulation of the phosphorylation state of stathmin is likely to determine its action.
Expression of the potassium channel KV3.4 in mouse skeletal muscle parallels fiber type maturation and depends on excitation pattern
Authors: Vullhorst Detlef , Klocke Rainer , Bartsch Jörg W , Jockusch Harald
Published: July 2002

We report the detailed expression pattern of the voltage‐dependent potassium channel KV3.4 (rat homologue, Raw3) in mouse skeletal muscle. Using semi‐quantitative RT‐PCR, we show that its expression is detectable at embryonic day 17 and rises to adult levels within 2 weeks after birth. Expression is fiber type‐dependent, with mRNA levels being 5–6‐fold lower in the mixed slow/fast soleus muscle than in the fast tibialis anterior and extensor digitorum longus muscles. Fast muscles from myotonic mice exhibit low KV3.4 mRNA levels similar to those of wild‐type soleus. In denervated extensor digitorum longus, KV3.4 expression declines to perinatal levels. We conclude that KV3.4 expression in mouse skeletal muscle is regulated by the pattern of excitation.
Long lasting facilitation of the rabbit cardiac Ca2+ channel: correlation with the coupling efficiency between charge movement and pore opening
Authors: Costantin James L , Qin Ning , Zhou Jianming , Platano Daniela , Birnbaumer Lutz , Stefani Enrico
Published: July 2002

Facilitation of Ca2+ entry into cells enhances Ca2+‐activated events such as transmitter release and stimulation of second messenger systems. We have found a long lasting prepulse facilitation (up to 3‐fold) of the cardiac Ca2+ channel α1C+β1b that lasts for tens of seconds without altering current kinetics. The voltage‐ and time‐dependence of the installation of facilitation was characterized as well as the time‐ and use‐dependence of the decay of facilitation. The degree of facilitation was correlated with the coupling efficiency between the charge movement and pore opening channels that were poorly coupled prior to facilitation exhibited the largest facilitation.
Two high conductance channels of the mitochondrial inner membrane are independent of the human mitochondrial genome
Authors: Murphy Robert C , Diwan Joyce J , King Michael , Kinnally Kathleen W
Published: July 2002

Patch‐clamp techniques were used to characterize the channel activity of mitochondrial inner membranes of two human osteosarcoma cell lines: a mitochondrial genome‐deficient (ρ0) line and its corresponding parental (ρ+) line. Previously, two high conductance channels, mitochondrial Centum picoSiemen (mCS) and multiple conductance channels (MCC), were detected in murine mitochondria. While MCC was assigned to the protein import in yeast mitochondria, the role of mCS is unknown. This study demonstrates that mCs and MCC activities from mouse mitochondria are indistinguishable from those of human mitochondria. The channel activities and their functional expression levels are not altered in cells lacking mtDNA. Hence, ρ0 cells may provide a model system for elucidating the role of mitochondrial channels in disease processes and apoptosis.
Very‐long‐chain acyl‐CoA dehydrogenase subunit assembles to the dimer form on mitochondrial inner membrane
Authors: Souri Masayoshi , Aoyama Toshifumi , Hoganson George , Hashimoto Takashi
Published: July 2002

This paper describes the process of dimer assembly of mitochondrial very‐long‐chain acyl‐CoA dehydrogenase (VLCAD) subunit. Mature VLCAD is a homodimer of a 70‐kDa protein associated with the mitochondrial membrane. Newly synthesized VLCAD was present as a monomer and the major fraction was associated with the mitochondrial inner membrane. The association of VLCAD subunit with the mitochondrial membrane was observed early during dimer formation. In contrast, a VLCAD monomeric mutant S583W, a novel mutation identified from a patient with VLCAD deficiency, did not associate with the mitochondrial membrane after import and the major fraction remained in the mitochondrial matrix. These results suggest that association of VLCAD protein with mitochondrial inner membrane is necessary for dimer assembly and formation of mature VLCAD.
Identification of an uncoupling mutation affecting the b subunit of F1F0 ATP synthase in Escherichia coli
Authors: Caviston Tamara L , Ketchum Christian J , Sorgen Paul L , Nakamoto Robert K , Cain Brian D
Published: July 2002

A specific b subunit arginine, b
Arg‐36 in Escherichia coli, displays evolutionary conservation among bacterial F1F0 ATP synthases. Site‐directed mutagenesis was used to generate a collection of mutations affecting b
Arg‐36. The phenotype differed depending upon the substitution, and the b
Arg‐36→Glu and b
Arg‐36→Ile substitutions virtually abolished enzyme function. Although the total amounts of F1F0 ATP synthase present in the membranes prepared from mutant strains were reduced, the primary effect of the b
Arg‐36 substitutions was on the activities of the intact enzyme complexes. The most interesting result was that the b
Arg‐36→Glu substitution results in the uncoupling of a functional F0 from F1 ATP hydrolysis activity.
Oligomerization properties of ERp29, an endoplasmic reticulum stress protein
Authors: Mkrtchiana Souren , Baryshev Mikhail , Matvijenko Olga , Sharipo Anatoly , Sandalova Tatyana , Schneider Gunter , Ingelman-Sundberg Magnus
Published: July 2002

ERp29, a novel and ubiquitously expressed endoplasmic reticulum (ER) stress‐inducible protein, was recently isolated and cDNA cloned in our laboratory. Using size exclusion chromatography and chemical cross‐linking we have assessed the oligomerization properties of ERp29. Purified ERp29 in solution as well as in rat hepatoma cells self‐associates predominantly into homodimers. Labeling of the cells with [35S]methionine with subsequent cross‐linking and immunoprecipitation showed that ERp29 interacts with a number of ER proteins, one of which was previously identified as BiP/GRP78. Secondary structure prediction and fold recognition methods indicate that the native conformation of ERp29 resembles the thioredoxin fold, a structural motif characteristic of a number of enzymes with the redox function, including protein disulfide isomerase (with which ERp29 shares limited sequence similarity). Dimerization of the protein is suggested to be advantageous for the protein binding potential of ERp29.
Ca2+ is released from the nuclear tubular structure into nucleoplasm in C6 glioma cells after stimulation with phorbol ester
Authors: Lui P.P.Y. , Lee C.Y. , Tsang D. , Kong S.K.
Published: July 2002

It is well established that cellular Ca2+ is an important messenger that controls many nuclear functions but the source of nuclear Ca2+ is far from clear. It has long been thought that Ca2+ is translocated from the cytosol over a long distance to activate the nuclear transcription machinery. However, this model is at best an incomplete one. With the aid of confocal microscopy, we observed tubules extended deep inside the nucleus of C6 cells in agreement with previous studies (Fricker et al. (1997) J. Cell Biol. 136, 531–544). When cells were stimulated with phorbol 12‐myristate 13‐acetate or phorbol 12,13‐diacetate, Ca2+ was released from these tubules. DiOC6(3), a vital marker for intracellular membranes, stained the tubule in the nucleus of the same cell used for Ca2+ imaging. Moreover, results from labelling the cells with rhodamine 123 further indicate that the tubule was formed by a double‐membraned invagination with mitochondria inside. Studies with acridine orange showed that chromatin was excluded from the tubules. Taken together, our results demonstrate that the nuclear tubule is a structural entity responsible for the release of Ca2+ into the nucleoplasm after stimulation with phorbol ester.
Modulation of interleukin‐8 activity by gingipains from Porphyromonas gingivalis: implications for pathogenicity of periodontal disease
Authors: Mikolajczyk-Pawlinska Jowita , Travis James , Potempa Jan
Published: July 2002

Gingipains are the major cysteine proteinases synthesized by Porphyromonas gingivalis which, in soluble form, are able to initially convert IL‐8 (77 amino acid residues) to a more potent species truncated at the amino terminus, followed by slow degradation and destruction of chemokine biological activity. In contrast, the same enzymes when associated with bacterial outer‐membrane blebs (vesicles), instantly degrade this chemokine. This division of enhancing and inactivating activity between soluble and membrane‐bound gingipains can cause the compartmentalization of pro‐ and anti‐inflammatory reactions to distal and proximal positions from bacterial plaque, respectively, which may explain why, despite the massive neutrophil accumulation at periodontitis sites, there is no elimination of infection.
Dexamethasone modulation of multidrug transporters in normal tissues
Authors: Demeule Michel , Jodoin Julie , Beaulieu Édith , Brossard Mathieu , Béliveau Richard
Published: July 2002

The expression of P‐glycoprotein (P‐gp) and canalicular multispecific organic anion transporter (cMOAT or Mrp2) was evaluated by Western blotting analysis of rat tissues isolated following daily administration (1 mg kg−1 day−1) of dexamethasone over 4 days. Dexamethasone rapidly increased P‐gp expression more than 4.5‐ and 2‐fold in liver and lung, respectively, while it was decreased 40% in kidney. cMOAT expression was increased 2‐fold in liver and kidney following dexamethasone treatment. The levels of both proteins returned to control values by 6 days after the conclusion of dexamethasone administration. These results indicate that dexamethasone can modulate P‐gp and cMOAT expression in specific rat tissues and may have significant relevance for patients treated with dexamethasone as a single agent or in combination therapy with other drugs.
Nuclear translocation of green fluorescent protein‐nuclear factor κB with a distinct lag time in living cells
Authors: Tenjinbaru Kazuyoshi , Furuno Tadahide , Hirashima Naohide , Nakanishi Mamoru
Published: July 2002

A highly fluorescent mutant form of the green fluorescent protein (GFP) has been fused to the human nuclear factor κB (NF‐κB) p50 and p105 (p50/IκBγ), a precursor protein of NF‐κB p50. GFP‐p50 and GFP‐p105 were expressed in monkey COS‐7 cells and human HeLa cells. Translocation of these chimeric proteins was observed by confocal laser scanning microscopy. GFP‐p50 (without IκBγ) in the transfected cells resided in the nucleus. On the other hand, GFP‐p105 (GFP‐p50 with IκBγ) localized only in the cytoplasm before stimulation and translocated to the nucleus with stimulant specificity similar to that of native NF‐κB/IκB. In addition, the translocation of NF‐κB to the nucleus had a distinct lag time (a quiescent time) in the target cells. The lag time lasted 10–20 min after stimulation with hydrogen peroxide or tumor necrosis factor α. It was suggested that this might be due to the existence of a limiting step where NF‐κB is released from NF‐κB/IκB by the proteasome.
Localization of curved DNA and its association with nucleosome phasing in the promoter region of the human estrogen receptor α gene
Authors: Wada-Kiyama Yuko , Kuwabara Kentaro , Sakuma Yasuo , Onishi Yoshiaki , Trifonov Edward N , Kiyama Ryoiti
Published: July 2002

We determined DNA bend sites in the promoter region of the human estrogen receptor (ER) gene by the circular permutation assay. A total of five sites (ERB−4 to −1, and ERB+1) mapped in the 3 kb region showed an average distance of 688 bp. Most of the sites were accompanied by short poly(dA)·poly(dT) tracts including the potential bend core sequence A2N8A2N8A2 (A/A/A). Fine mapping of the ERB−2 site indicated that this A/A/A and the 20 bp immediate flanking sequence containing one half of the estrogen response element were the sites of DNA curvature. All of the experimentally mapped bend sites corresponded to the positions of DNA curvature as well as to nucleosomes predicted by computer analysis. In vitro nucleosome mapping at ERB−2 revealed that the bend center was located 10–30 bp from the experimental and predicted nucleosome dyad axes.
Stimulation of ultraviolet‐induced apoptosis of human fibroblast UVr‐1 cells by tyrosine kinase inhibitors
Authors: Hiwasa Takaki , Arase Yoshiko , Chen Zheng , Kita Kazuko , Umezawa Kazuo , Ito Hisao , Suzuki Nobuo
Published: July 2002

Damnacanthal is an anthraquinone compound isolated from the root of Morinda citrifolia and was reported to have a potent inhibitory activity towards tyrosine kinases such as Lck, Src, Lyn and EGF receptor. In the present study, we have examined the effects of damnacanthal on ultraviolet ray‐induced apoptosis in ultraviolet‐resistant human UVr‐1 cells. When the cells were treated with damnacanthal prior to ultraviolet irradiation, DNA fragmentation was more pronounced as compared to the case of ultraviolet irradiation alone. The other tyrosine kinase inhibitors, herbimycin A and genistein, also caused similar effects on ultraviolet‐induced apoptosis but to a lesser extent. Serine/threonine kinase inhibitors, K252a, staurosporine and GF109203X, rather suppressed the ultraviolet‐induced DNA cleavage. Immunoblot analysis showed that pretreatment with damnacanthal followed by ultraviolet irradiation increased the levels of phosphorylated extracellular signal‐regulated kinases and stress‐activated protein kinases. However, the other tyrosine kinase inhibitors did not increase the phosphorylation of extracellular signal‐regulated kinases but stimulated phosphorylation of stress‐activated protein kinases. Consequently, the ultraviolet‐induced concurrent increase in both phosphorylated extracellular signal‐regulated kinases and stress‐activated protein kinases after pretreatment with damnacanthal might be characteristically related to the stimulatory effect of damnacanthal on ultraviolet‐induced apoptosis.
Inhibition of insulin‐like growth factor‐I mitogenic action by zinc chelation is associated with a decreased mitogen‐activated protein kinase activation in RAT‐1 fibroblasts
Authors: Lefebvre Donatienne , Boney Charlotte M. , Ketelslegers Jean-Marie , Thissen Jean-Paul
Published: July 2002

The mechanisms responsible for the resistance to the anabolic actions of IGF‐I induced by zinc deficiency are not understood. We showed that zinc chelation by DTPA (diethylenetriaminepenta‐acetic acid) inhibits [3H]thymidine incorporation stimulated by IGF‐I in Rat‐1 fibroblasts. This inhibition was specific of zinc chelation since it was prevented by the addition of zinc to DTPA. The stimulation of MAPK, which is crucial for the [3H]thymidine incorporation induced by IGF‐I in Rat‐1 cells, was partially blunted by DTPA. Therefore, the inhibition of the mitogenic action of IGF‐I in Rat‐1 fibroblasts by DTPA is potentially caused by decreased MAPK activation by IGF‐I.
Modelling charge interactions in the prion protein: predictions for pathogenesis
Authors: Warwicker James
Published: July 2002

Calculations are presented for the pH‐dependence of stability and membrane charge complementarity of prion protein fragments. The theoretical results are compared with reported characterisations of prion protein folding in vitro. Discussion of models for conformational change and pathogenesis in vivo leads to the prediction of amino acids that could mediate sensitivity to the endosomal pH and to a design strategy for recombinant prion proteins with an increased susceptibility to prion proteinSc‐like properties in vitro. In this model, the protective effect of certain basic polymorphisms can be interpreted in terms of oligomerisation on a negatively‐charged surface.
Di‐, tri‐ and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding
Authors: Le Gall Fabrice , Kipriyanov Sergey M. , Moldenhauer Gerhard , Little Melvyn
Published: July 2002

Single chain variable fragments (scFv) of the murine monoclonal antibody HD37 specific to human B‐cell antigen CD19 were constructed by joining the VH and VL domains with linkers of 18, 10, 1 and 0 residues. ScFv‐18 formed monomers, dimers and small amounts of tetramers; scFv‐10 formed dimers and small amounts of tetramers; scFv‐1 formed exclusively tetramers; scFv‐0 formed exclusively trimers. The affinities of the scFv‐10 (diabody) and scFv‐1 (tetrabody) were approximately 1.5‐ and 2.5‐fold higher, respectively, than that of the scFv‐0 (triabody). The tetrabody displayed a significantly prolonged association with cell‐bound antigen (t
1/2 cell surface retention at 37°C of 26.6 min) compared to both the diabody (13.3 min) and triabody (6.7 min). This increase in avidity of the tetrabody combined with its larger size could prove to be particularly advantageous for imaging and the immunotherapy of B‐cell malignancies.
Prediction of the maximal stability temperature of monomeric globular proteins solely from amino acid sequence
Authors: Ganesh C , Eswar Narayanan , Srivastava Sarika , Ramakrishnan Chandrasekharan , Varadarajan Raghavan
Published: July 2002

Globular protein thermostability is characterized the cold denaturation, maximal stability (T
ms) and heat denaturation temperatures. For mesophilic globular proteins, T
ms typically ranges from −25°C to +35°C. We show that the indirect estimate of T
ms from calorimetry and the direct estimate from chemical denaturation performed in a range of temperatures are in close agreement. The heat capacity change of unfolding per mol residue (ΔC
p) alone is shown to accurately predict T
ms. ΔC
p and hence T
ms can be predicted solely from the protein sequence. The average difference in free energy of unfolding at the observed and predicted values of T
ms is 1.0 kcal mol−1, which is small compared to typical values of the total free energy of unfolding.
Phosphorylation of p125FAK and paxillin focal adhesion proteins in src‐transformed cells with different metastatic capacity
Authors: Rodina Anna , Schramm Kira , Musatkina Elena , Kreuser Ernst-Dietrich , Tavitian Armand , Tatosyan Alexander
Published: July 2002

Hamster fibroblasts transformed by Rous sarcoma virus (RSV) display different metastatic potentials that are associated with specific structural features of the v‐src oncoprotein. This diverse metastatic activity could be due to various tyrosine phosphorylation levels of specific src protein substrates. To check this hypothesis, phosphorylation of the FAK and paxillin proteins, involved in signal transduction pathways and known as src protein substrates, was tested. It was shown that FAK and paxillin are hyperphosphorylated in the high metastatic cell lines as compared with the phosphotyrosine level of these proteins found in the low metastatic cell lines. In addition, our data confirm that v‐src protein plays a direct role in paxillin phosphorylation.
Deoxygenation and elevation of intracellular magnesium induce tyrosine phosphorylation of band 3 in human erythrocytes
Authors: Barbul Alexander , Zipser Yehudit , Nachles Alexander , Korenstein Rafi
Published: July 2002

Deoxygenation increases the level of tyrosine phosphorylation of band 3 by ∼25% in human red blood cells (RBCs), as determined by Western blotting. The effect is much more pronounced in osmotically shrunken RBCs or in the presence of vanadate. When the rise in intracellular free Mg2+ concentration in deoxygenated RBCs is simulated via clamping of the intracellular magnesium in oxygenated RBCs by ionomycin, band 3 phosphorylation is elevated by up to 10‐fold. Phosphorylated band 3 is preferentially retained by RBC skeletons, after mild extraction with Triton X‐100. Elevation of intracellular free Mg2+ leads to band 3 phosphorylation and is accompanied by rigidification of the membrane skeleton as determined by analysis of RBC membrane mechanical fluctuations. These findings suggest that the visco‐elastic properties of human erythrocytes may be regulated by band 3 tyrosine phosphorylation.
Identification of a multidrug resistance‐like system in Tetrahymena pyriformis: evidence for a new detoxication mechanism in freshwater ciliates
Authors: Bamdad Mahchid , Brousseau Pauline , Denizeau Francine
Published: July 2002

The freshwater ciliate Tetrahymena pyriformis is an ubiquitous organism that is present in all aquatic ecosystems. This protozoan showed a clear resistance against some polycyclic aromatic hydrocarbons which can be attributed to an efflux pump probably of the multidrug resistance (MDR) type. Immunocytochemical detection showed a positive stain of ciliate cells using the monoclonal antibodies 4E3, raised against P‐glycoprotein (P‐gp). The kinetics of P‐gp expression were studied for control cultures and cultures treated with 15 μM benzo(a)pyrene. Western blot analysis using the Ab1, anti‐P‐gp polyclonal antibodies indicates the presence of two bands of 66 and 96 kDa of which the intensity increased with time in benzo(a)pyrene‐treated ciliates. Uptake experiments with target compounds for the MDR pump, namely adriamycin, rhodamine 123 and two polycyclic aromatic hydrocarbons, benzo(a)pyrene and 7,12‐dimethylbenzanthracene, were carried out by flow cytometry, in the presence or absence of cyclosporin (an inhibitor of the multidrug resistant pump). The data indicate that the accumulation of these compounds by ciliate cells is significantly enhanced in the presence of cyclosporin. This suggests that Tetrahymena is provided with a P‐gp‐like system that is functionally active in a way similar to that of the mammalian P‐gp.
Spatial structure of the M2 transmembrane segment of the nicotinic acetylcholine receptor α‐subunit
Authors: Pashkov Vladimir S , Maslennikov Innokenty V , Tchikin Leonid D , Efremov Roman G , Ivanov Vadim T , Arseniev Alexander S
Published: July 2002

A synthetic peptide corresponding to the transmembrane segment M2 (residues 236–267) of the α‐subunit of the nicotinic acetylcholine receptor from Torpedo californica has been studied by two dimensional 1H‐NMR spectroscopy in a chloroform‐methanol (1:1) mixture containing 0.1 M LiClO4. Reconstruction of the spatial structure of M2 from the NMR data resulted in an α‐helix formed by residues 241–263. Distribution of the molecular hydrophobicity potential on the helix surface is very similar to that in five‐helix bundles of proteins with a known three dimensional structure: two hydrophilic bands located on the opposite helix sides separated by strong hydrophobic zones.
Overexpression of the hereditary hemochromatosis protein, HFE, in HeLa cells induces an iron‐deficient phenotype
Authors: Corsi Barbara , Levi Sonia , Cozzi Anna , Corti Angelo , Altimare Domenico , Albertini Alberto , Arosio Paolo
Published: July 2002

A transfectant HeLa cell clone expressing HFE under the control of a tetracycline‐repressible promoter was generated. HFE expression was fully repressed by the presence of doxycycline, while it was strongly induced by growth in the absence of doxycycline. HFE accumulation was accompanied by a large (∼10‐fold) decrease in H‐ and L‐ferritin levels, by a ∼3–4‐fold increase in transferrin receptor, and a ∼2‐fold increase in iron regulatory protein activity. These indices of cellular iron deficiency were reversed by iron supplementation complexes. The overexpressed HFE immunoprecipitated together with transferrin receptor, indicating a physical association which is the likely cause for the observed ∼30% decrease in 55Fe‐transferrin incorporation after 18 h incubation. In the HFE‐expressing cells the reduction in transferrin‐mediated iron incorporation was partially compensated by a ∼30% increase in non‐transferrin iron incorporation from 55Fe‐NTA, evident after prolonged, 18 h, incubations. The findings indicate that HFE binding to transferrin receptor reduces cellular iron availability and regulates the balance between transferrin‐mediated and non‐transferrin‐mediated cellular iron incorporation.
TRAF1 is a TNF inducible regulator of NF‐κB activation
Authors: Carpentier Isabelle , Beyaert Rudi
Published: July 2002

Tumor necrosis factor receptor (TNFR)‐associated factor TRAF1 was first identified as a component of the TNFR2 signalling complex. Unlike the other members of the TRAF family, TRAF1 lacks the N‐terminal ring finger motif and has a tissue specific expression. Here we demonstrate that expression of TRAF1 is induced by TNF and the protein kinase C (PKC) activator PMA, but not by interleukin‐1 (IL‐1). TNF‐induced upregulation of TRAF1 could be prevented by pretreatment of the cells with the proteasome inhibitor MG‐132, whereas the PKC inhibitor Ro31‐8220 was without effect. Interestingly, overexpression of TRAF1 in HEK293T completely prevented NF‐κB activation induced by TNF, IL‐1, or overexpression of TRAF2 or TRAF6. These data suggest that inducible expression of TRAF1 may serve a negative regulatory function in NF‐κB signalling pathways.
Bruton's tyrosine kinase (Btk) associates with protein kinase C μ
Authors: Johannes Franz-Josef , Hausser Angelika , Storz Peter , Truckenmüller Lars , Link Gisela , Kawakami Toshiaki , Pfizenmaier Klaus
Published: July 2002

Bruton's tyrosine kinase (Btk) is considered an essential signal transducer in B‐cells. Mutational defects are associated with a severe immunodeficiency syndrome, X‐chromosome linked agammaglobulinemia (XLA). Here we show by coimmunoprecipitation that a member of the protein kinase C (PKC) family, PKCμ, is constitutively associated with Btk. Neither antigen receptor (Ig) crosslinking nor stimulation of B‐cells with phorbol ester or H2O2 affected Btk/PKCμ interaction. GST precipitation analysis revealed association of the Btk pleckstrin/Tec homology domain with PKCμ. Transient overexpression of PKCμ deletion mutants as well as expression of selected PKCμ domains in 293T cells revealed that both the kinase domain and the regulatory C1 region are independently capable of binding to the Btk PH‐TH domain. These data show the existence of a PKCμ/Btk complex in vivo and identify two PKCμ domains that participate in Btk interaction.
Expression of E16/CD98LC/hLAT1 is responsive to 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin
Authors: Sarkar Devanand , Kambe Fukushi , Hirata Akihiro , Iseki Atsushi , Ohmori Sachiko , Seo Hisao
Published: July 2002

We employed cDNA representational difference analysis to identify new genes that are upregulated by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) in a human hepatoblastoma cell line HepG2. We isolated several TCDD‐responsive cDNAs. Sequence analysis revealed that one of them encodes E16/CD98LC/hLAT1, an integral membrane protein involved in multiple cellular functions including cellular transport of L‐type amino acids. Northern blot analysis confirmed the TCDD‐dependent upregulation of the mRNA. Induction of E16/CD98LC/hLAT1 mRNA by TCDD did not require de novo protein synthesis as revealed by the experiment using cycloheximide. Consistent with the changes at mRNA level, the transport of 3H‐leucine into HepG2 cells was significantly increased by TCDD treatment. These findings provide a novel aspect of biological effects of TCDD on human hepatocytes.
The central interactive region of human MxA GTPase is involved in GTPase activation and interaction with viral target structures
Authors: Flohr Felix , Schneider-Schaulies Sibylle , Haller Otto , Kochs Georg
Published: July 2002

To define domains of the human MxA GTPase involved in GTP hydrolysis and antiviral activity, we used two monoclonal antibodies (mAb) directed against different regions of the molecule. mAb 2C12 recognizes an epitope in the central interactive region of MxA, whereas mAb M143 is directed against the N‐terminal G domain. mAb 2C12 greatly stimulated MxA GTPase activity, suggesting that antibody‐mediated crosslinking enhances GTP hydrolysis. In contrast, monovalent Fab fragments of 2C12 abolished GTPase activity, most likely by blocking intramolecular interactions required for GTPase activation. Interestingly, intact IgG molecules and Fab fragments of 2C12 both prevented association of MxA with viral nucleocapsids and neutralized MxA antiviral activity in vivo. mAb M143 had no effect on MxA function, indicating that this antibody binds outside functional regions. These data demonstrate that the central region recognized by 2C12 is critical for regulation of GTPase activity and viral target recognition.