bluuebunny/crossref_metadata_2025 huggingface dataset:
Human liver and human placenta both contain CMP‐NeuAc:Galβ1→4GlcNAc‐R α2→3‐ as well as α2→6‐sialyltransferase activity
Authors: Nemansky Martin , Schiphorst Wietske E.C.M. , Koeleman Carolien A.M. , Van den Eijnden Dirk H.
Published: July 2002

A high pH anion exchange chromatographic (HPAEC) system for the separation of isomeric sialo‐oligosaccharide products was developed. Employing this system, using Galβ1→4GlcNAcβ1→2Manα1→6Manβ1→4GlcNAc as a substrate, a Galβ1→4GlcNAc‐R α2→3‐sialyltransferase activity was detected for the first time in human liver. This activity is expressed together with the prevalent α2→6‐sialyltransferase. Furthermore, in addition to the major α2→3‐sialyltransferase, a low but distinct activity of α2→6‐sialyltransferase was detected in human placenta. This activity could not be found by methods based on methylation analysis or high resolution NMR spectroscopy. It is concluded that HPAEC, in combination with the use of the pentasaccharide as an acceptor substrate, is suited for the specific detection of minor, Galβ1→4GlcNAc‐specific sialyltransferase activities.
Dissociation of store release from transmembrane influx of calcium in human neutrophils
Authors: Davies E.V. , Campbell A.K. , Hallett M.B.
Published: July 2002

Release of Ca2+ from intracellular stores was visualised in individual neutrophils in the presence of the Mn2+ or SKF 96365, Influx of Mn2+ quenched fura‐2 close to the plasma membrane but did not quench fura‐2 at the site of store release. The size and location of the ‘cloud’ of elevated Ca2+ was unaffected by the channel blocker SKF 96365. Furthermore, the size and location was unaffected by the presence of extracellular Ca2+. This dissociation of transmembrane influx from store release demonstrates that the entry of Ca2+ into the cytosol of neutrophils occurs directly into the cytosol and not via the store site.
Inhibition of actin‐activated myosin Mg2+‐ATPase in smooth muscle by Ruthenium red
Authors: Nakamura Fumiaki , Naka Michiko , Tanaka Toshio
Published: July 2002

Ruthenium red was found to inhibit actin‐activated myosin Mg2+‐ATPase in smooth muscle and to bind to myosin heavy chain, but not to F‐actin. The inhibition by Ruthenium red of actin‐activated Mg2+‐ATPase was of the competitive type with respect to actin (K
i 4.4μM) and of the non‐competitive type with respect to ATP (K
i 6.6μM). However, Ruthenium red scarcely dissociated the acto‐heavy meromyosin complex during the ATPase reaction. These results suggest that Ruthenium red interacts directly with the binding site for F‐actin on the myosin heavy chain. This site is considered to be necessary not for maintaining the binding affinity of myosin for F‐actin, but for activation of the Mg2+‐ATPase.
A novel nuclear 42‐kDa casein kinase identified in Chironomus tentans
Authors: Stigare J. , Kovacs J. , Buddelmeijer N. , Egyhazi E.
Published: July 2002

We have purified and characterised an apparently novel nuclear 42‐kDa casein kinase from epithelial cells of Chironomus tentans which comigrates with a phosphoprotein associated with transcriptionally active salivary gland genes. The protein kinase promotes phosphorylation of casein and phosvitin, using either ATP or GTP as phosphate donors, and undergoes autophosphorylation. The casein kinase activity of the 42‐kDa protein sensitive to heparin, 5,6‐dichloro‐1‐β‐d‐ribofuranosylbezimidazole (DRB), spermine and spermidine indicating that it is a novel enzyme with similar but not identical properties to casein kinase II or nuclear protein kinase NII.
Studies of oxidative stress in cellular systems The interaction of monocytes and erythrocytes
Authors: McGowan P.E. , Reglinski J. , Smith W.E. , Wilson R. , Sturrock R.D.
Published: July 2002

1H spin echo NMR spectroscopy is used to follow the interaction of intact and viable erythrocytes and monocytes obtained from different sources in mixed cultures. After a lag time (270 min) erythrocyte glutathione is observed to become more oxidised. This result is believed to occur as a consequence of monocyte activation generating hydrogen peroxide or hypochlorous acid, which is targeted at the erythrocyte. The red cell in turn employs its sulphydryl system as an anti‐oxidant defence.
The binding of the fluorescent ATP analogue 2′(3′)‐trinitrophenyladenosine‐5′‐triphosphate to rat liver fatty acid‐binding protein
Authors: Sheridan Mary , Wilton David C.
Published: July 2002

The less polar fluorescent analogue of ATP, 2′(3′)‐trinitrophenyl‐5′‐triphosphate bound to rat liver fatty acid‐binding protein with high affinity (K
d 6.3 × 10−6 M) and 1:1 molar stoichiometry. This probe bound to the fatty acid binding site of the protein and was displaced by oleic acid and oleoyl CoA. High concentrations of ATP did not cause significant displacement of the fluorescent ATP analogue. Since the anionic part of this molecule is the triphosphate group it is difficult to envisage this group being accommodated at an anion binding site within the non‐polar core of this protein as is the case with other fatty acid binding proteins. Therefore it is anticipated that the ligand must bind to liver fatty acid‐binding protein with these triphosphate group surface exposed. Caution must be exercised when using the more hydrophobic fluorescent analogue of ATP to investigate the ATP binding properties of proteins.
Interaction of carbonyl cyanide m‐chlorophenylhydrazone with the photosystem II acceptor side
Authors: Samuilov Vitaly D. , Barsky Eugene L.
Published: July 2002

We show that CCCP, known as an uncoupler of photophosphorylation and an ADRY agent, inhibits FeCy photoreduction and coupled O2 evolution by isolated chloroplasts equally (150 ∼ 2μM), but is practically without effect on the O2 evolution coupled with SiMo reduction within the 0.2–10μM concentration range. CCCP has no effect on the nanosecond chlorophyll fluorescence in chloroplasts incubated at low light intensity, but decreases it at high light intensity. The electron transfer from reduced TMPD or duroquinol to methylviologen is resistant to CCCP. The efficiency of the CCCP inhibitory action on the FeCy photoreduction depends on the rate of electron flow, which is controlled by the light intensity. The data obtained show that CCCP is oxidized by the photosystem II donor side and is reduced by Qp, competing for electrons with FeCy and the cytochrome blf complex.
Molecular cloning of a mouse myosin I expressed in brain
Authors: Koslovsky Janet S. , Qian Chungeng , Jiang Xin , Mercer John A.
Published: July 2002

We have isolated two cDNAs that encode putative myosin I heavy chains by polymerase chain reaction amplification of brain cDNA with degenerate oligodeoxynucleotide primers representing myosin I‐speeific conserved amino acid sequences. We report the complete deduced amino acid sequence of one of these cDNAs. The sequence is most similar to those of the avian and bovine brush border myosin Is, with five putative calmodulin‐binding repeats at the head‐tail junction. Northern analysis demonstrates that this myosin heavy chain, unlike the brush border myosins, is expressed in many tissues.
Multiple cell cycle access to the apoptotic death programme in human neuroblastoma cells
Authors: Piacentini Mauro , Fesus Laszlo , Melino Gerry
Published: July 2002

We report the induction of apoptosis in a human neuroblastoma cell line SK‐N‐BE(2) by cisplatin or retinoic acid, and its relation to cell cycle. Apoptosis was monitored by counting apoptotic bodies and evaluating the activity of ‘tissue’ transglutaminase (EC 2.3.2.13), one of the genes specifically expressed in apoptotic cells. Data indicate that both agents enhance apoptosis, even though cells arrest at different cell cycle phases. In fact, retinoic acid causes accumulation in G1, whilst cisplatin induces accumulation of cells in the G2/M phase. This evidence suggests the presence of multiple start points for the apoptotic death programme within the cell cycle of human neuroblastoma cells.
Expression in E. coli and purification of a chimeric p22‐NS3 recombinant antigen of Hepatitis C Virus (HCV)
Authors: Osborne S. , Cecconato E. , Griva S. , Garetto F. , Calogero R. , Rosa C. , Bonelli F.
Published: July 2002

A recombinant antigen (p22‐NS3), possessing putative HCV nucleocapsid protein (p22) and non‐structural protein 3 (NS3) epitopes, was heavily expressed in E. coli and purified. The p22‐NS3 purified recombinant antigen strongly reacts with sera containing human antibodies directed against p22 and NS3 providing a starting point for the design of an HCV single all‐encompassing antigen for a blood screening assay.
Phospholipase D activation regulates endothelin‐1 stimulation of phosphoinositide‐specific phospholipase C in SK‐N‐MC cells
Authors: Challiss R.A.John , Wilkes Lesley C. , Patel Viral , Purkiss John R. , Boarder Michael R.
Published: July 2002

Endothelin‐1 (ET‐1) is known to stimulate phospholipase C (PLC) activity in SK‐N‐MC human neuroblastoma/epithelioma cells: here we show that phospholipase D(PLD) is also stimulated. The generation of inositol 1,4,5‐trisphosphate (Ins(1,4,5)P3) by ET‐1‐stimulated PLC was attenuated by protein kinase C (PKC) activation and enhanced by PKC inhibition. An enhancement of ET‐1‐stimulated Ins(1,4,5)P3 accumulation was also seen when the product of PLD activity was either diverted into phosphatidyl butanol in the presence of butanol, or phosphatidate phosphohydrolase (PPH) activity was inhibited by dl‐propranolol. We conclude that there is an inhibitory, PKC‐mediated, feedback loop in these cells which is dependent, in part, on the activation of PKC by product(s) of the PLD/PPH pathway. This provides a novel role for agonist‐stimulated PLD activation.
Rapid binding of synapsin I to F‐ and G‐actin
Authors: Ceccaldi P.E. , Benfenati F. , Chieregatti E. , Greengard P. , Valtorta F.
Published: July 2002

Synapsin I is a nerve terminal phosphoprotein which interacts with synaptic vesicles and actin in a phosphorylation‐dependent manner. By using fluorescence resonance energy transfer between purified components labeled with fluorescent probes, we now show that the binding of synapsin I to actin is a rapid phenomenon. Binding of synapsin I to actin can also be demonstrated when synaptic vesicles are present in the medium and appears to be modulated by ionic strength and synapsin I phosphorylation.
Replacement of His23 by Cys in a zinc finger of HIV‐1 NCp7 led to a change in 1H NMR‐derived 3D structure and to a loss of biological activity
Authors: Julian N. , Demene H. , Morellet N. , Maigret B. , Roques B.P.
Published: July 2002

The nucleocapsid protein NCp7 of human immunodeficiency virus type 1 (HIV‐1), which is necessary for the formation of infectious virions, contains two zinc fingers of the Cys‐X2‐Cys‐X4‐His‐X4‐Cys form. To elucidate the importance of this particular motif, well conserved in retroviruses and retroelements, we substituted the histidine residue by a cysteine in the first zinc binding domain 13VKCFNCGKEGHTARNCRA30. The structures of the mutated and native zinc complexed peptides were studied by two‐dimensional 600 MHz 1H nuclear magnetic resonance (NMR) in aqueous solution. The nuclear Overhauser effects were used as constraints to determine the solution structures using DIANA software followed by AMBER energy refinement. The results show that native and mutant peptides fold into non‐identical three‐dimensional structures, probably accounting for the loss of retrovirus infectivity following the His‐Cys point mutation.
ras oncogene‐induced transformation of a rat seminal vesicle epithelial cell line produces a marked increase of adenylate cyclase and protein kinase C activities
Authors: Spina A.M. , Chiosi E. , Naviglio S. , Valente F. , Marchese M. , Furgi A. , Metafora S. , Illiano G.
Published: July 2002

Cells transformed by Kirsten murine sarcoma virus (Ki‐MSV) have basal adenylate cyclase activity (AC) higher than control cells and comparable level of forskolin‐stimulated AC activity. Moreover, a higher protein kinase C (PKC) activity was found to be present in the transformed cells. The molecular mechanism underlying the increase of AC activity was investigated. Our findings strongly suggest that this biochemical event is due to a marked decrease of the αi negative control of the enzyme, even though the αi of transformed cells appears to possess fully functional domains interacting with both the effector enzyme and the agonist‐activated receptor.
Peroxisome proliferators and T3 operate by way of distinct receptors
Authors: Castelein Hilde , Declercq Peter E. , Mannaerts Guy P. , Baes Myriam I.
Published: July 2002

Peroxisome proliferators and thyroid hormones have a number of common metabolic effects. The possibility that the signal transduction pathways of both groups of effectors converge at the receptor level was investigated. It was shown that T3, specifically bound to the rat thyroid β‐receptor, was not displaced to a significant extent by ciprofibrate or bezafibrate. No specific binding of T3 to the mouse peroxisome proliferator activated receptor could be demonstrated. In transactivation experiments peroxisome proliferators were unable to activate the thyroid receptor and T3 did not activate a chimeric receptor containing the ligand binding domain of the peroxisome proliferator activated receptor. It is concluded that peroxisome proliferators and thyroid hormone do not cross‐react at the level of their nuclear receptors.
The ligand binding domain of the nicotinic acetylcholine receptor
Authors: Kachalsky Sylvia G. , Aladjem Mirit , Barchan Dora , Fuchs Sara
Published: July 2002

The interaction of the acetylcholine receptor (AChR) binding site domain with specific antibodies and with α‐bungarotoxin (α‐BTX) has been compared. The cloned and expressed ligand binding domain of the mouse AChR α‐subunit binds α‐BTX, whereas the mongoose‐expressed domain is not recognized by α‐BTX. On the other hand, both the mouse and mongoose domains bind to the site‐specific monoclonal antibody 5.5. These results demonstrate that the structural requirements for binding of α‐BTX and mcAb 5.5, both of which interact with the AChR binding site, are distinct from each other.
Reverse transcriptase families and a copia‐like retrotransposon, Osser, in the green alga Volvox carteri
Authors: Lindauer Andreas , Fraser David , Brüderlein Martina , Schmitt Rüdiger
Published: July 2002

By using the polymerase chain reaction (PCR) we have isolated and sequenced two distinct families of reverse transcriptase (RT) sequences from the genome of the colonial alga, Volvox carteri. Probing a genomic library with these RT clones revealed copia‐like retrotransposons. One of these elements, named Osser, is 4,875 bp long, bordered by 197‐bp identical long terminal repeats (LTRs), and shows the typical organization of retrotransposons belonging to the copia‐Tye group. This is the first complete copra‐like retrotransposon sequence described in a green alga.
Expression of Saccharomyces cerevisiae inorganic pyrophosphatase in Escherichia coli
Authors: Kurilova Svetlana A. , Vorobjeva Natalia N. , Nazarova Tatiana I. , Avaeva Svetlana M.
Published: July 2002

A segment of DNA encoding Saccharomyces cerevisiae inorganic pyrophosphatase (ppa gene) was amplified by the polymerase chain reaction. The pSCH1 and pSCB6 plasmids containing the ppa gene were obtained. Transformation of the E. coli BL21 strain with the resulting recombinant plasmids and selection of clones having extremely high expression of inorganic pyrophosphatase (PPase) were carried out. Superproduction of recombinant S. cerevisiae PPase up to 50% of the total bacterial protein was achieved. The enzyme was readily obtained and purified to homogeneity with the use of a simple purification technique. This work is the first description of S. cerevisiae PPase superproducer creation.
Selectivity of phospholipase C isozymes in growth factor signaling
Authors: Cho Yee-Sook , Han Myung-Kwan , Chae Soo-Wan , Park Chung-Ung , Kim Uh-Hyun
Published: July 2002

Xenopus laevis oocytes were injected with mRNA extracted from growth factor‐responsive CCL39, Chinese hamster lung fibroblasts. The expression of functional growth factor receptors on the oocytes was demonstrated by growth factor‐induced 45Ca2+ efflux. To determine the isozyme(s) of phospholipase C (PLC) coupled to growth factor receptors, growth factor‐induced 45Ca2+ efflux were measured following coinjection of mRNA from CCL39 cells with PLC antibodies. PLC‐γ1 antibody did not lead to loss of 45Ca2+ efflux induced by thrombin but resulted in loss of that induced by platelet‐derived growth factor (PDGF). In contrast, PLC‐δ1 antibody did not block PDGF‐induced 45Ca2+ efflux but led to inhibition of thrombin‐induced 45Ca2+ efflux. PLC‐β1 antibody did not affect Ca2+ efflux by the treatment of either thrombin or PDGF. These results suggest that these growth factor receptors are coupled to specific effectors, i.e. thrombin receptor to PLC‐δ1 and PDGF receptor to PLC‐γ1.
Novel surface functional properties of polymannosyl lysozyme constructed by genetic modification
Authors: Nakamura Soichiro , Kobayashi Kunihiko , Kato Akio
Published: July 2002

The surface functional properties of glycosylated lysozyme were investigated by using polymannosyl and oligomannosyl enzymes at the position 49 by genetic modification [Nakamura S. et al. (1993) J. Biol. Chem. 268, in press]. The polymannosyl lysozyme exhibited excellent emulsifying properties superior to those of commercial emulsifiers in addition to heat stability, while the oligomannosyl lysozyme did not. The surface tension of the polymannosyl lysozyme was greatly decreased correspondingly to the enhanced emulsifying properties, although that of oligomannosyl protein was not. The emulsifying activity and the emulsion stability of the polymannosyl lysozyme were stable in acidic pH or high salt conditions; in addition, they were greatly enhanced also by preheating the polymannosyl lysozyme. Thus novel surface functional properties of polymannosyi lysozyme in addition to heat stability suggest the direction of the design of new functional proteins by genetic modification.
The regulation of gene expression by insulin is differentially impaired in the liver of the genetically obese‐hyperglycemic Wistar fatty rat
Authors: Noguchi Tamio , Matsuda Tamiko , Tomari Yoshiaki , Yamada Kazuya , Imai Enyu , Wang Ziyuan , Ikeda Hitoshi , Tanaka Takehiko
Published: July 2002

The regulation by insulin and carbohydrates of the gene expression of three key enzymes involved in glucose metabolism was studied in the liver of the Wistar fatty rat, a model of obese non‐insulin‐dependent diabetes mellitus. A high glucose or fructose diet, or insulin administration caused a similar magnitude of increase in the level of L‐type pyruvate kinase mRNA in the liver of Wistar fatty rats and their lean littermates. However, the induction of glucokinase mRNA and repression of phosphoenolpyruvate carboxykinase mRNA by dietary glucose or insulin were impaired in the fatty rats, whereas fructose caused a similar decrease in phosphoenolpyruvate carboxykinase mRNA in both types of rats. These results indicate that the regulation of gene expression of glucokinase and phosphoenolpyruvate carboxykinase, but not of L‐type pyruvate kinase, by insulin is impaired in the liver of the Wistar fatty rat.
Identification of a new 80 k isoform of phosphatidylinositol 4‐phosphate 5‐kinase from bovine brain
Authors: Schmidt Martina , Varsányi Magdolna , Thieleczek Rolf , Heilmeyer Ludwig M.G.
Published: July 2002

Phosphatidylinositol 4‐phosphate 5‐kinase is associated with bovine brain microsomes to an extent of approximately 65% of the total cellular enzyme activity. This membrane‐associated kinase activity can be solubilized with Triton X‐114. Following polyacrylamide gel electrophoresis in the presence of SDS the enzyme can be renaturated from gel slices in the presence of desoxycholate and Triton X‐100. Catalytic activity appears at an apparent molecular weight of 80 k. Analysis of the reaction product formed by the 80 k protein reveals the existence of a 5‐phosphotransferase, identifying the 80 k polypeptide as a new phosphatidylinositol 4‐phosphate 5‐kinase isoform.
Continuous presence of phorbol ester is required for its IL‐1β mRNA stabilizing effect
Authors: Siljander Pia , Hurme Mikko
Published: July 2002

The protein kinase C (PKC) activating phorbol esters are known to prevent the decay of mRNA of several cytokines and proto‐oncogenes. To examine whether the phorbol ester signal is continuously required for this stabilizing effect, THP‐1 monocytic cells were stimulated either with phorbol 12,13‐dibutyrate (PDBu), which can be removed from the cells by washings, or with the more hydrophobic phorbol 12‐myristate 13‐acetate (PMA). Both of these stimuli induced high levels of interleukin‐1β (IL‐1β) mRNA. When the cells were washed at the peak of the IL‐1β mRNA expression, this mRNA decayed rapidly in the PDBu stimulated cells while in PMA stimulated cells the mRNA levels were not affected. Moreover, this mRNA degradation induced by the removal of PDBu could be inhibited by readdition of the phorbol ester. This restabilization could be prevented by pharmacologie inhibitors of PKC, but not by inhibiting protein or RNA synthesis. Thus these data suggest that the phorbol ester must be continuously present to exert its mRNA stabilizing effect and that its effect is PKC‐mediated but does not require active protein or RNA synthesis.
Identification and characterization of an anti‐tyrosine kinase factor in cystic gliomas
Authors: Berger F. , Laine M. , Amalfitano G. , Brunet J.F. , Verna J.M. , Nissou M.F. , Benabid A.L.
Published: July 2002

In view of the frequent activation of the epidermal growth factor receptor (EGF‐R) in gliomas and autocrine hypothesis, we searched for ‘EGF‐like’ factor(s) in cystic fluids (CFs) associated with gliomas. Membranes of A431 cells, which overexpress EGF‐R, were used to explore such activity in 20 CFs. In all cases CFs induced inhibition of EGF‐R phosphorylation. Biochemical analysis revealed an anti‐tyrosine kinase activity which was identified as a 18 kDa proteic factor. Effectiveness at high dilution and anti‐proliferative effect on living cells in culture suggest that this factor may be involved in the negative regulation of glial oncogenesis.
Effect of platelet activating factor on the kinetics of LDL oxidation in vitro
Authors: Deigner Hans-Peter , Dresel Hans Alois
Published: July 2002

Oxidatively modified low‐density‐lipoprotein (LDL) might contribute to the atherosclerotic process. This study was performed to examine an effect of platelet‐activating factor (PAF) and of synthetic PAF analogs on Cu(II) induced oxidation of LDL in vitro: The d‐ and l‐isomers of PAF and analogs with short‐chain sn‐2‐substituents, 1‐O‐alkyl‐2‐butyryl‐sn‐glycero‐3‐phosphocholine and 1‐O‐alkyl‐2‐heptanoyl‐sn‐glycero‐3‐phosphocholine, were found to be the most effective inhibitors of LDL oxidation. Oxidation was inhibited completely at PAF concentrations above 100 μM. Lyso‐PAF and analogs carrying longer chains at the sn‐2 position were less effective. These results thus provide evidence for the involvement of other parameters in LDL oxidation beyond the content of natural antioxidants like vitamin E and β‐carotene.
Characterization of the methylenediphosphonate transport system in Arthrobacter sp. GLP‐1 using the novel tritium‐labelled derivative
Authors: Yakovleva G.M. , Blackburn G.M.
Published: July 2002

The novel tritium‐labelled derivative of methylenediphosphonate (MDP) was used in uptake studies ofArthrobacter sp. GLP‐1 capable of utilizing a wide range of organophosphonates as its sole source of phosphorus. The MDP uptake was greatly stimulated upon phosphate deprivation. The uptake obeys Michaelis‐Menten kinetics with respective K
m and V
max values of 33 μM and 0.3 nmol·min−1·mg−1 fr.wt. Glyphosate and pyrophosphate were competitive inhibitors of MDP uptake. The effect of orthophosphate was more complex than a mere inhibition of MDP uptake since activation occurred at low concentrations. The uptake of MDP by Arthrobacter sp. strain GLP‐1 appears to be mediated by a transport system different from the glyphosate uptake system operating in the same cells. The driving force for MDP uptake by Arthrobacter sp. GLP‐1 may be a proton gradient across the cell membrane.
Phase transitions of Acholeplasma laidlawii membranes The involvement of Mg2+‐ATPase in the C transition
Authors: Chen Jian-Wen , Hu Liao-Yuan , Hwang Fen
Published: July 2002

Highly sensitive differential scanning calorimetry has been employed to study the phase transitions of A. laidlawii membrane. The DSC curves obtained show five distinct transitions between 20 and 80°C which contain a reversible lipid thermotropic transition at about 37°C and four irreversible denaturation transitions of the membrane proteins occurred at about 44°C, 52°C, 62°C and 67°C, respectively. Total enthalpy of the thermal denaturation of membrane proteins is 3.4 ± 0.5 cal/g. Further study of A. laidlawii membrane preparations by means of thermal gel analysis and enzyme acitvity measurements at various temperatures provided information that the third peak (C transition) of the DSC curve involved primarity with Mg2+‐ATPase on A. laidlawii membranes.
Doxorubicin‐induced oxygen free radical formation in sensitive and doxorubicin‐resistant variants of rat glioblastoma cell lines
Authors: Benchekroun M.Nabil , Sinha Birandra K. , Robert Jacques
Published: July 2002

We have studied the formation of hydroxyl radical (OH•) induced by doxorubicin in a series of doxorubicin‐ or vincristine‐selected variants of C6 rat glioblastoma cells in culture by electron‐spin resonance spectroscopy using 5,5′‐dimethyl‐1‐pyrroline‐1‐oxide as a spin trap. Wild‐type cells, sensitive to doxorubicin, exhibited in the presence of this drug a concentration‐dependent OH• formation which could be inhibited by preincubation with superoxide dismutase, catalase or an antibody against cytochrome P450‐reductase. In highly doxorubicin‐resistant cells, OH• formation was reduced to about 20% of the level obtained in sensitive cells. In cells presenting a very low level of resistance to doxorubicin or in cells selected with vincristine, both presenting a pure multidrug‐resistant phenotype, OH• formation was identical to that obtained in sensitive cells. In cells of intermediate resistance or in revertant cells, intermediate levels of OH• formation were obtained. Protection against OH• formation and action can be identified at the levels of superoxide dismutase and glutathione peroxidase activities, which are both enhanced in the resistant cells.
Electron microscopic study of compaction of individual DNA molecules with histone H1 in surface films
Authors: Vengerov Yuri Yu. , Martinkina Larissa P. , Semenov Timur E.
Published: July 2002

Interaction of individual spread DNA molecules with histone H1 was studied by electron microscopy using the protein‐free spreading technique. It was shown that in the presence of 0.2–5 μg/ml of histone H1 in hypophase there are three types of structures in electron‐microscopic preparations: fibres of non‐compacted DNA, compact fibres with twisted strands of duplex DNA and compacted rod‐like and circular structures where the separate fibres of duplex DNA could not be distinguished. The study of compact structure morphology allows us to conclude that they are formed by side‐by‐side association of DNA fibres. At an increase of ionic strength there is a tendency for transition from the second to the third type structures. The latter can be explained by transition from non‐cooperative to cooperative binding of histone H1 to DNA. Regularities established for interaction between DNA and histone H1 can be useful for understanding the role of histone H1 in the higher order structure organization of chromatin.
mRNA amplification system by viral replicase in transgenic plants
Authors: Mod Masashi , Kaido Masanori , Okuno Tetsuro , Furusawa Iwao
Published: July 2002

We have constructed transgenic tobacco plants (M1×2‐FCP2IFN plants) expressing viral RNA replication genes of brome mosaic virus (BMV) and BMV RNA3 derivative (FCP2IFN) carrying the human gamma Interferon (IFN‐γ) gene. In M1×2‐FCP2IFN plants the RNA3 derivative expressed from the integrated cDNA was replicated and subgenomic RNA (i.e. mRNA of IFN‐γ) was produced by BMV replicase. The accumulation level of the mRNA of IFN‐γ was approximately 5‐fold higher than that by the cauliflower mosaic virus (CaMV) 35S RNA promoter. In addition IFN‐γ accumulated in M1×2‐FCP2IFN plants.
Enzymatic oxidation of the bifunctional wheat inhibitor of subtilisin and endogenous α‐amylase
Authors: Gvozdeva Ekaterina L. , Valueva Tatyana A. , Mosolov Vladimir V.
Published: July 2002

Oxidation of the bifunctional wheat inhibitor of subtilisin and endogenous α‐amylase catalyzed by horseradish peroxidase results in the loss of the inhibitory activity against both enzymes. The enzymatic oxidation is accompanied by modification of one methionine and two tryptophan residues in the protein. The results obtained, together with data on chemical modification and limited proteolysis, allow us to conclude that Met34‐Ala35 is the reactive site of the inhibitor responsible for the interaction with subtilisin. It is supposed that the reactive site of the inhibitor responsible for the interaction with α‐amylase contains one or two tryptophan residues.
Kinetic properties of pure overproduced Bacillus subtilis phenylalanyl‐tRNA synthetase do not favour its in vivo inhibition by ochratoxin A
Authors: Roth A. , Eriani G. , Dirheimer G. , Gangloff J.
Published: July 2002

Ochratoxine A (OTA) inhibits growth of Bacillus subtilis at pHs below 7. Since OTA is a phenylalanine analogue, this effect could be due to inhibition of phenylalanine‐tRNA synthetase (PheRS) by competition of this mycotoxin with the amino acid. Homogeneous PheRS was purified from Bacillus subtilis and from E. coli transformed with the PheRS gene. The latter produced about 40 times more PheRS than B. subtilis. The Km
and Ki
values of PheRS, respectively, for phenylalanine and OTA were measured and their concentrations within the cell determined. It appears that the concentration of OTA in the cell, in spite of a 25‐fold accumulation, remained too low to significantly compete with phenylalanine. This does not suggest PheRS to be the target of OTA in cell growth and protein synthesis inhibition in Bacillus subtilis. It was also shown that the 2–3‐fold increase of PheRS in OTA‐treated cells is not due to phenylalanine‐controlled attenuation regulation.
Inhibition of pituitary hormone exocytosis by a synthetic peptide related to the rab effector domain
Authors: Davidson J.S. , Eales A. , Roeske R.W. , Millar R.P.
Published: July 2002

GTP‐binding proteins of the rab family are believed to function at several steps in intracellular vesicular transport. We examined the effects of a rab‐related peptide in permeabilized pituitary cells, in which exocytosis can be triggered by distinct Ca2+‐dependent or Ca2+‐independent pathways. We report that a synthetic peptide of 18 amino acids related to the rab effector domain, rab3AL(30–47) inhibited luteinizing hormone (LH) and growth hormone (GH) exocytosis triggered by either pathway. Ca2+‐stimulated LH and GH release were inhibited by more than 80% and 50%, respectively, by 100 μM peptide. The peptide (100 μM) also inhibited LH and GH exocytosis stimulated by phorbol myristate acetate plus cAMP by more than 45% and 80%, respectively. The effect was sequence‐specific since a second peptide, lacking the first 3 amino acids but otherwise identical failed to inhibit exocytosis. These results suggest that a protein of the rab family is involved in regulated pituitary hormone exocytosis, and they identify 3 amino acids of the putative rab effector domain which may be functionally important in exocytosis.
Vitamin D status modulates rat colonic M3 muscarinic receptor characteristics and coupling to guanylate cyclase
Authors: Khare Sharad , Tien Xiao-Ying , Wilson David , Wali Ramesh K. , Bissonnette Bruce M. , Sitrin Michael D. , Brasitus Thomas A.
Published: July 2002

The present studies were conducted to determine whether [3H]quinuclidinyl benzilate binding in rat colonic membranes and/or carbachol‐mediated stimulation of particulate guanylate cyclase were altered by changes in vitamin D status. EC50 values for the stimulation of colonic guanylate cyclase by carbachol were found to be significantly greater in vitamin D‐deficient rats compared to their D‐sufficient counterparts. Concomitantly, the density of receptors (B
max) were significantly lower, and dissociation constants (K
d) were significantly higher in D‐deficient colonic membranes. In vitamin D‐repleted animals, moreover, all of these aforementioned alterations were at least partially corrected.
Parallel changes in Glut 4 and Rab4 movements in two insulin‐resistant states
Authors: Ricort Jean-Marc , Tanti Jean-François , Cormont Mireille , Van Obberghen Emmanuel , Le Marchand-Brustel Yannick
Published: July 2002

Insulin‐induced Glut 4 and Rab4 movements were studied in two insulin‐resistant states. In adipocytes from streptozotocin diabetic rats, the amount of Glut 4 was decreased by 60%. The remaining Glut 4 molecules were translocated in response to insulin, and in parallel, Rab4 left the intracellular compartment. In contrast, in 3T3‐L1 adipocytes rendered insulin‐resistant by a prolonged insulin treatment, both Rab4 and Glut 4 remained in the intracellular compartment following an acute insulin stimulation. Those results illustrate a similar behavior of Glut 4 and Rab4 in two situations where insulin resistance results from different mechanisms, and add further support for a role of Rab4 in Glut 4 transiocation.
Human chromosomal centromere (AATGG)n sequence forms stable structures with unusual base pairs
Authors: Jaishree T.N. , Wang Andrew H.-J.
Published: July 2002

Nine DNA sequences related to the purine strand of the human centromeric satellite (AATGG)n·(CCAAT)n repeat have been studied by two‐dimensional nuclear magnetic resonance spectroscopy. Earlier studies have suggested that the structure of (AATGG)n sequence has an equilibrium between the duplex form and a fold‐back form. Structural refinement of d(CAATGG) and its related sequences by an NOE‐constrained simulated annealing procedure reveals that the duplex form incorporates dynamic type‐I G‐A base pairs. 1D exchangeable proton NMR data support this model. The reverse sequence motif (GGTAA) destabilizes the structure.
Yeast serine isoacceptor tRNAs: Variations of their content as a function of growth conditions and primary structure of the minor tRNASerGCU
Authors: Heyman Thérèse , Agoutin Bernadette , Fix Christine , Dirheimer Guy , Keith Gérard
Published: July 2002

The primary structure of Saccharomyces cerevisiae tRNASerGCU is presented (EMBL database accession No. X74268 S. cerevisiae tRNA‐Ser). In addition, quantitation of the relative amounts of serine isoaccepting tRNAs in yeast grown on different media showed that the minor tRNASerGCU decreased while the major tRNASerAGA increased as the growth rate and the cellular protein content increased. The minor species, tRNASerCGA and tRNASerUGA, were not separated by our gel system, however, taken together they appeared to vary in the same way as TRNASerGCU. These data suggest a growth rate dependence of yeast tRNAs similar to that previously described for E. coli tRNAs.
The presence but not the sequence of the N‐terminal peptide in cardiac TnC is important for function
Authors: Liu Wen , Dotson Darrell G. , Lin Xin , Mullen James J. , Gonzalez-Garay Manuel L. , Lu Qingxian , Putkey John A.
Published: July 2002

The most diverged region of the primary amino acid sequence between cardiac (cTnC) and fast skeletal troponin C is the N‐terminal ten amino acids. We report here that major changes in the primary sequence of this region in cTnC had a minimal effect on the ability of the mutant proteins to recover maximal activity in TnC‐extracted cardiac and fast skeletal muscle myofibrils. However, deletion of the N‐terminal nine amino acids resulted in a 60% decrease in maximal Ca2+‐dependent ATPase activity with only a small change in the pCa50 of activation. Deletion of the N‐terminal peptide did not appear to appreciably affect the Ca2+‐binding properties of cTnC, but it did alter the interaction with hydrophobic fluorescent probes. Thus, the presence but not the sequence, of the N‐terminal extension is important for the maximal activity of cTnC. The N‐terminal helix may function in a relatively non‐specific manner to prevent unfavorable interactions between domains in cTnC or between cTnC and other troponin subunits.
Protein kinase C rapidly up‐regulates the number of AT1 angiotensin receptors on cultured rat intestinal epithelial (RIE‐1) cells
Authors: Smith Roger D.
Published: July 2002

Addition of 12‐O‐tetradecanoylphorbol‐13‐acetate to RIE‐1 rat intestinal epithelial cells stimulated a rapid (mean 3‐fold) increase in the subsequent binding of 125 I‐labelled angiotensin II which was reversed or prevented when cellular protein kinase C was depleted. The increased binding was due, in part, to an up‐regulation in the number of AT1 angiotensin receptors on RIE‐1 cells, without any significant change in their binding affinity. Since this rapid up‐regulation was independent of receptor synthesis, it may result from an increased availability (to extracellular ligand) of preformed, but previously ‘cryptic’, AT1 angiotensin receptors.
Transfer RNA binding protein in the nucleus of Saccharomyces cerevisiae
Authors: Wilhelm M.L. , Reinbolt J. , Gangloff J. , Dirheimer G. , Wilhelm F.X.
Published: July 2002

A yeast nuclear protein that binds to tRNA was identified using a RNA mobility shift assay. Northwestern blotting and N‐terminal sequencing experiments indicate that this tRNA‐binding protein is identical to zuotin which has previously been shown to bind to Z‐DNA [(1992) EMBO J. 11, 3787–37961. Labeled TRNA and poly(dG‐m5dC) stabilized in the Z‐DNA form identify the same protein on a Northwestern blot. In a gel retardation assay poly(dG‐m5dC) in the Z‐form strongly diminishes the binding of tRNA to zuotin. These studies establish that zuotin is able to bind to both tRNA and Z‐DNA. Zuotin may be transiently associated with tRNA in the nucleus of yeast cells and play a role in its processing or transport to the cytoplasm.
Renaturation of the mature subtilisin BPN' immobilized on agarose beads
Authors: Hayashi Tetsuya , Matsubara Mamoru , Nohara Daisuke , Kojima Shuichi , Miura Kin-ichiro , Sakai Tomoya
Published: July 2002

We report here another example of renaturation of subtilisin BPN′(Sbtl) by using an immobilized preparation instead of applying a digestible mutant of Streptomyces subtilisin inhibitor (SSI), a proteinaceous inhibitor of Sbtl [M. Matsubara et al. (1994) FEBS Letters 342, 193–196]. The mature Sbtl was immobilized on agarose beads employing the amino group of the protein. After thorough washing, the immobilized Sbtl was subjected to denaturation in 6 M guanidine hydrochloride (GdnHCl) at pH 2.4 for 4 h, followed by renaturation in 2 M potassium acetate at pH 6.5 for 24 h. This denaturation/renaturation cycle was repeated five times. The recovered activity of the renatured immobilized Sbtl settled at a constant level after the third denaturation/renaturation cycle, demonstrating that almost 100% renaturation was attained by use of the immobilized Sbtl. This immobilized Sbtl preparation could well be utilized for the mechanistic study of protein folding. We then found that 2 M potassium acetate was superior to 2 M potassium chloride as a refolding medium and that the ability of SSI to induce the correct shape of the mature Sbtl was lacking in several refolding media in both thermodynamic and kinetic criteria. Thus the main cause for the increase of refolding yield of Sbtl by coexistence of SSI was prevention of the autolysis of Sbtl.
Activation of gastrin gene transcription in islet cells by a RAP1‐like cis‐acting promoter element
Authors: Simon Babette , Tillotson Loyal , Brand Stephen J.
Published: July 2002

Gastrin transcription in islet cells is activated by a cis‐regulatory sequence containing a binding site for the yeast transcription factor RAP1. The DNA—protein interactions between RAP1 protein and the gastrin DNA element determined by methylation interference assays are identical to those of RAP1 and yeast genes. Point mutations in the gastrin RAP1 binding site, which abolished RAP1 binding, decreased transcriptional activation by this sequence. Islet cells revealed a DNA binding protein with RAP1 ‐like binding specificity. These findings support the conclusion that gastrin transcription is activated in mammalian cells by a RAP1 ‐like transcription factor.
Autophosphorylation of nucleoside diphosphate kinase on non‐histidine residues
Authors: Bominaar Anthony A. , Tepper Annemiek D. , Véron Michel
Published: July 2002

Recently, several reports appeared which described auto‐phosphorylation of NDP kinase on residues different from the active‐site histidine. Based on these findings conclusions were drawn with respect to a regulation of enzyme activity and to a possible role as a metastasis suppressor. In this paper we show that although non‐histidine autophosphorylation occurs on NDP kinases from mammals, lower eukaryotes and bacteria, less than 0.2% of the subunits are phosphorylated. Using site‐directed mutagenesis, we show that the active site histidine is essential for non‐histidine autophosphorylation. The low stoichiometry of phosphate incorporation excludes a role of autophosphorylation in regulating overall enzyme activity.
Energy migration in allophycocyanin‐B trimer with a linker polypeptide: Analysis by the principal multi‐component spectral estimation (PMSE) method
Authors: Yamazaki Tomoko , Nishimura Yoshinobu , Yamazaki Iwao , Hirano Masahiko , Matsuura Katsumi , Shimada Keizo , Mimuro Mamoru
Published: July 2002

Energy migration processes in allophycocyanin‐B trimer with a linker polypeptide were analyzed using the principal multi‐component spectral estimation (PMSE) method, which does not require assumption of component number, decay function, or the spectral band shape. We determined the number of spectral components showing independent kinetic behavior by the eigenvalue of an auto‐correlation matrix, and further the spectra of the components and their rise and decay curves. Two decay components were resolved at 20°C: One corresponded to the decay of one type of β‐84 chromophore, and the other to the decay from the thermally equilibrated state between another type of β‐84 chromophore and the α‐allophycocyanin B chromophore. An additional slow decay process was resolved at ‐ 196°C. We also compared the component spectra obtained using the PMSE method with the decay‐associated spectra obtained using the global analysis.
Isolation and characterization of mutants of human mitogen‐activated protein kinase (ERK2)
Authors: Krishnan Indira S. , Kovacs Eleonora D. , Sass Philip M.
Published: July 2002

Site directed mutagenesis/charged‐to‐alanine scanning mutagenesis of the amino terminal portion of human ERK2 (from amino acids 1 to 150) purified as a glutathione‐S‐transferase fusion protein (GST‐ERK2) from E. coli has been done to determine regions/amino acids important for activation by rabbit skeletal muscle MAP kinase kinase (rMEK) and kinase activity towards myelin basic protein (MBP). Five classes of mutants have been isolated. The first class of mutants comprises of G30A/G32A, A50D and R65A/R68A/E69A, that can be phosphorylated by rMEK and have no kinase activity towards MBP, the second class includes mutants D122A/H123A and N142A which have lower kinase activities but no change in their activation by rMEK; third class being Y34A, E58A/H59A, which have neutral effect towards either activity, the fourth class that includes completely inactive mutants D42A/K46A/R48A, the deletion mutant in the same region (−9aa[40–48]) and D104A/E107A/D109A and finally the fifth class that include K53A, E94A/K97A/D99A, K112A/K115A and R133A/K136A that are phosphorylated 140–240% but with kinase activity toward MBP ranging from 50–100% of the wild type.
Equivalent regulation of wild type and an epitope‐tagged variant of Gsα by the IP prostanoid receptor following expression in neuroblastoma × glioma hybrid, NG108‐15, cells
Authors: Mullaney Ian , Milligan Graeme
Published: July 2002

NG108‐15 cells were transfected to stably express a haemagglutinin epitope‐tagged variant of the long isoform of Gsα. Clone BST15 expressed this polypeptide at similar levels to the endogenous long isoform of Gsα. Treatment of clone BST15 with the IP prostanoid receptor agonist iloprost resulted in down‐regulation of both forms of Gsα with both dose—effect curves to iloprost and time courses of loss of the two forms of Gsα being indistinguishable. These results demonstrate that the IP prostanoid receptor interacts with and regulates the epitope‐tagged variant of Gsα in an equivalent manner to the unmodified protein and indicates that the epitope‐tagged polypeptide can be used to analyse mechanisms of receptor regulation of cellular G‐protein levels.
Production and characterization of monoclonal anti‐idiotypic antibodies to muramylpeptide
Authors: Mareeva T.Yu. , Kotova O.V. , Golovina T.N. , Nesmeyanov V.A.
Published: July 2002

Hybridomas producing monoclonal anti‐idiotypic antibodies (anti‐id MAbs) to N‐acetylglucosaminyl‐β1‐4‐N‐acetylmuramyl‐alanyl‐d‐isoglutamine (GMDP) were developed. Three clones of hybridomas demonstrated the properties characteristic for the Ab2β type of anti‐id antibody: they bound to Fab‐fragments of high‐affinity MAb to GMDP; dose‐dependent inhibition of this binding by GMDP was observed; immunization of mice with these MAbs resulted in production of GMDP‐specific antibodies. When these antibodies were used to stain blots from SDS‐PAGE of macrophage lysate, the same receptor proteins were specifically stained as upon staining with 125I‐labelled GMDP derivative.
Transient induction of fatty acid synthase in rat liver after removal of a peroxisome proliferator
Authors: Motojima Kiyoto , Isaka Masanori , Takino Yasuko , Goto Sataro
Published: July 2002

Removal of a peroxisome proliferator from the diet triggered the degradation of peroxisomes and induced the transient expression of a 220 kDa soluble protein in rat liver. The 220 kDa protein was purified by conventional methods and analyzed by amino acid sequencing. A total of 99 amino acid residues in 4 lysylendopeptidase‐digested peptides completely matched those in rat fatty acid synthase. The transient induction of fatty acid synthase mRNA during peroxisome degradation was confirmed by Northern blotting.
The number of amino acid residues in hydrophilic loops connecting transmembrane domains of the GABA transporter GAT‐1 is critical for its function
Authors: Kanner Baruch I. , Bendahan Annie , Pantanowitz Shifrah , Su Hailing
Published: July 2002

Transporter proteins consist of multiple transmembrane domains connected by hydrophillic loops. As the importance of these loops in transport processes is poorly understood, we have studied this question using the cDNA coding for GAT‐1, a Na+/Cl−‐coupled γ‐aminobutyric acid transporter from rat brain. Deletions of randomly picked non‐conserved single amino acids in the loops connecting helices 7 and 8 or 8 and 9 result in inactive transport upon expression in HeLa cells. However, transporters where these amino acids are replaced with glycine retain significant activity. The expression levels of the inactive mutant transporters was similar to that of the wild‐type, but one of these, ΔVal‐348, appears to be defectively targetted to the plasma membrane. Our data are compatible with the idea that a minimal length of the loops is required, presumably to enable the transmembrane domains to interact optimally with each other.