bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid up‐ and down‐regulation of hexokinase II in rat skeletal muscle in response to altered contractile activity
Authors: Weber Franz E. , Pette Dirk
Published: July 2002

An up to 14‐fold increase in total hexokinase activity induced by low‐frequency stimulation in rat fast‐twitch muscle was followed by a rapid decay in enzyme activity after cessation of stimulation. In vivo labeling revealed that these alterations were related to rapid changes in [35S]methionine incorporation into hexokinase II. A recovery period of 15 h after cessation of stimulation was sufficient to normalize the approximately 30‐fold elevated [35S]methionine incorporation.
Differential regulation of prostatic protein kinase C isozymes by androgens
Authors: Goueli Said A.
Published: July 2002

Multiple isozymes of Ca2+/phospholipid‐dependent protein kinase (PKC) were isolated from the rat ventral prostate. The enzyme exists mainly as type II (β), and type III (α) forms, and it is possible that type II isozyme may comprise the subspecies β1 and β2. The total and specific activities of prostatic PKC isoforms were reduced in castrated animals; this decrease was specific since administration of androgens to castrated animals reversed such a decline. Also, there was a differential response to androgen deprivation so that type III isozyme declined at a faster rate than that of type II. Thus, our studies show for the first time that PKC of the rat ventral prostate comprises multiple isozymes, and that the activity of these various forms are differentially regulated by androgens.
Severin is a gelsolin prototype
Authors: Yin H.L. , Janmey P.A. , Schleicher M.
Published: July 2002

A number of Ca2+ ‐activated actin filament severing proteins have been identified in eukaryotic cells of diverse lineages. Gelsolin and villin, with molecular mass of about 80–90 kDa, and severin and fragmin, with molecular mass of about 40 kDa, have been isolated from vertebrates and invertebrates, respectively. We report here a direct comparison of the functional properties of gelsolin and severin, and the finding that the actin filament severing activity of severin, like that of gelsolin, is inhibited by polyphosphoinositides. However, severin does not nucleate actin filament assembly as well as gelsolin. These characteristics are very similar to those ascribed to the NH2,‐terminal half of gelsolin, supporting the idea that they are evolutionarily related. Regulation of severin by polyphospholipids raises the possibility that it may participate in agonist‐stimulated regulation of the actin cytoskeleton in Dictyostelium discoideum.
Extracellular ATP activates receptor‐operated cation channels in mouse lacrimal acinar cells to promote calcium influx in the absence of phosphoinositide metabolism
Authors: Sasaki T. , Gallacher D.V.
Published: July 2002

In exocrine acinar cells a variety of neurotransmitters (e.g. acetylcholine) stimulate phosphatidylinositol 4,5‐bisphosphate hydrolysis elevating intracellular calcium to activate calcium‐dependent membrane currents (outward K+ and inward Cl−). This study shows that in lacrimal acinar cells extracellular application of ATP is also associated with outward and inward current responses; these, however, are not the result of phosphoinositide metabolism. ATP directly activates receptor‐operated cation channels which permit influx of Na+ and Ca+ (the inward current). The elevation in [Ca2+], which results is sufficient to activate the outward K+ current. ATP thus promotes Ca+ influx in the absence of phosphoinositide metabolism.
Carbon‐13 labelled biotin ‐ a new probe for the study of enzyme catalyzed carboxylation and decarboxylation reactions
Authors: Bendrat K. , Berger S. , Buckel W. , Etzel W.A. , Röhm K.-H.
Published: July 2002

[2'‐13C]Biotin was incorporated into avidin (egg white), glutaconyl‐CoA decarboxylase (EC 4.1.1.70) from Acidaminococcus fermentans and the biotin carrier of transcarboxylase from Propionibacterium freudenreichii (EC 2.1.3.1). 13C‐NMR measurements showed an upfield shift of the carbonyl carbon of 3.1 and 2.0 ppm for both enzymes, whereas binding to avidin induced no significant change of the chemical shift as compared to free biotin. The data indicate that the enzymes provide an electronic environment for the covalently bound biotin which favours carboxylation. In addition it was demonstrated by NMR‐measurements that glutaconyl‐CoA decarboxylase, from which the hydrophobic carboxy‐lyase subunit (β) was removed, could carboxylate free biotin.
Ins(1,4,5)P3 formation and fluctuating chloride current response induced by external ATP in Xenopus oocytes injected with embryonic guinea pig brain mRNA
Authors: Fournier Franck , Honoré Eric , Collin Thibault , Guilbault Pierre
Published: July 2002

In voltage‐clamped Xenopus oocytes injected with embryonic guinea pig mRNA, effective concentrations of extracellular ATP elicited an inward fluctuating current. This current, carried by Cl‐ions, was mainly dependent upon liberation of Ca2+ ions from stores as demonstrated by experiments using intracellular EGTA loading and TMB‐8 superfusion. Neomycin inhibited these fluctuating currents indicating that the transplanted purinoceptor is linked to phospholipase C activity and triggers Ins(1,4,5)P3 formation. Ins(1,4,5)P3 production evoked by external ATP was clearly demonstrated by directly measuring the water‐soluble Ins(1,4,5)P3 level in injected oocytes. Finally, it is suggested that the ATP effect was mediated by a Ca2+ release from Ins(1,4,5)P3 sensitive pools since heparin blocked the ATP responsiveness. The acquired purinoceptor may be made apparent to a P2 subtype since ATP and ADP were equipotent in eliciting Cl− current while AMP and Adenosine were ineffective in injected oocytes.
Inositol 1,4,5‐trisphosphate activates two types of Ca2+‐permeable channels in human carcinoma cells
Authors: Mozhayeva Galina N. , Naumov Alexander P. , Kuryshev Yuri A.
Published: July 2002

Ca2+‐permeable channels in human carcinoma A431 cells were studied using the patch clamp technique. We have found two types of Ca2+‐penneable channels which are activated by inositol 1,4,5‐trisphosphate (IP3) applied to the intracellular side of the plasma membrane. Unitary conductances of these channels are 3.7 and 13 pS (105 mM Ca2+ in recording pipette, 30–33°C). From the extracellular side of the membrane the channels are activated by EGF. It is assumed that extracellular agonists open both channel types by stimulating the release of IP3 from the membrane.
The involvement of fatty acid binding protein in peroxisomal fatty acid oxidation
Authors: Reubsaet F.A.G. , Veerkamp J.H. , Brückwilder M.L.P. , Trijbels J.M.F. , Monnens L.A.H.
Published: July 2002

Rat liver fatty acid‐binding protein (FABP) can function as a fatty acid donor protein for both peroxisomal and mitochondrial fatty acid oxidation, since 14C‐labeled palmitic acid bound to FABP is oxidized by both organelles. FABP is, however, not detected in peroxisomes and mitochondria of rat liver by ELISA. Acyl‐CoA oxidase activity of isolated peroxisomes was not changed by addition of FABP or flavaspidic acid, an inhibitor of fatty acid binding to FABP, nor by disruption of the peroxisomal membranes. These data indicate that FABP may transfer fatty acids to peroxisomes, but is not involved in the transport of acyl‐CoA through the peroxisomal membrane.
Activation of single Ca2+‐dependent K+ channel by external ATP in mouse macrophages
Authors: Hara N. , Ichinose M. , Sawada M. , Imai K. , Maeno T.
Published: July 2002

Single Ca2+‐dependent K+ currents responding to external ATP were recorded from cell‐attached patches on mouse peritoneal macrophages. Extracellularly applied ATP activated an inward single‐channel current with a conductance of 25 pS and a reversal potential of −79 mV (pipette potential, V
p) when the pipette contained a 145 mM KC1 solution. The reversal potential was shifted 56 mV positive by a 10‐fold reduction in external (pipette) K+ concentration. The effect of ATP was abolished by either removal of external Ca2+ or treatment with an intracellular Ca2+ chelator, the acetoxymethyl ester of 1,2‐bis (2‐aminophenoxy)ethane‐N,N,N',N'‐tetraacetic acid (BAPTA‐AM). This channel has a mean open time of 9.1 ms and open probability was not strongly dependent on V
p in the range tested (+ 120 to −30 mV).
Expression and characterization of human lamin C
Authors: Moir Robert D. , Quinlan Roy A. , Stewart Murray
Published: July 2002

We have expressed human lamin C cDNA in E. coli using a modification of the pLcII vector system. Protein produced in this way had seven additional amino acids at its N‐terminus, but retained key lamin structural and assembly properties. The modified vector we produced may prove useful when difficulties are encountered in removal of the cII fusion peptide by factor X cleavage in the pLcII system. Shadowed preparations of expressed lamin C showed the presence of 50‐nm rod‐like particles that closely resembled those observed for native material. Isolated molecules had two globular domains at one end, indicating that they were constructed from two parallel polypeptide chains. The expressed material also formed paracrystals with a characteristic 22.5 nm axial repeat, indicating that its assembly properties had also been retained. We also used site‐specific mutagenesis to engineer a lamin fragment that lacked the C‐tenninal non‐helical domain of the molecule. This material formed paracrystals similar to those obtained with the intact molecule, indicating that the large C‐terminal non‐helical domain did not contain information vital for lamin assembly.
Functional expression of the rat heart I Na+ channel isoform Demonstration of properties characteristic of native cardiac Na+ channels
Authors: Cribbs Leanne L. , Satin Jonathan , Fozzard Harry A. , Rogart Richard B.
Published: July 2002

We describe the expression of functional Na+ channel in Xenopus oocytes injected with cRNA transcribed from the rat heart 1 cDNA clone. The expressed rat heart 1 Na+ currents show kinetic properties and resistance to tetrodotoxin and saxitoxin which are characteristic of native cardiac Na+ currents. The primary amino acid sequence of the rat heart 1 α‐subunit is therefore sufficient for expression of of tetrodotoxin resistance, and the rat heart 1 clone is likely to account for the tetrodotoxin‐resistant phenotype of cardiac and denervated skeletal muscle.
Characterization of potassium channels in pancreatic β cells from ob/ob mice
Authors: Kukuljan Manuel , Li Min Yi , Atwater Illani
Published: July 2002

The patch‐clamp technique in the cell‐attached mode was used to study the K channels present in the membrane of cultured pancreatic β cells from ob/ob mice. Three types of K+ channels were regularly observed, with conductances of 64, 20 and 146 pS. The conduction and kinetic properties of the 64 pS channel were similar to those of the ATP‐sensitive potassium channel from normal β cells. Furthermore, glucose blocked the activity of this channel at the same concentrations as that reported for normal cells. The 20 pS and the 146 pS were insensitive to glucose. The latter K+ channel appears to be similar to the large conductance voltage‐activated potassium channels described in normal rodent β cells. Thus, potassium channels in ob/ob pancreatic β cells in culture are in most respects normal. Other factors may account for the abnormal electrical response to glucose of ob/ob pancreatic islets, such as reversible impairment of their function in vivo or defects not related to potassium permeability.
Molecular cloning of the Ecotin gene in Escherichia coli
Authors: Lee Hae Ryun , Seol Jae Hong , Kim Ok Mae , Lee Cheol Soon , Suh Se Won , Hong Yeong-Man , Tanaka Keiji , Ichihara Akira , Ha Doo Bong , Chung Cin Ha
Published: July 2002

The nucleotide sequence of a 876 bp region in E. coli chromosome that encodes Ecotin was determined. The proposed coding sequence for Ecotin is 486 nucleotides long, which would encode a protein consisting of 162 amino acids with a calculated molecular weight of 18 192 Da. The deduced primary sequence of Ecotin includes a 20‐residue signal sequence, cleavage of which would give rise to a mature protein with a molecular weight of 16 099 Da. Ecotin does not contain any consensus reactive site sequences of known serine protease inhibitor families, suggesting that Ecotin is a novel inhibitor.
Cloning and functional expression of a TEA‐sensitive A‐type potassium channel from rat brain
Authors: Schro̊ter Klaus-Hasso , Ruppersberg Johann Peter , Wunder Frank , Rettig Jens , Stocker Martin , Pongs Olaf
Published: July 2002

A rat brain cDNA (Raw3) related to the Drosophila Show K* channel family has been characterized, Raw3 cRNA leads to the formation of TEA‐insensitive, fast inactivating (A‐type) K* channels when injected into Xenopus leavis oocytes. Raw3 channels have markedly different properties from the previously cloned rat A‐type K* channel RCK4, Raw3 channels operate in the positive voltage range.
Active oxygen induced protein ubiquitination in Chlamydomonas
Authors: Shimogawara Kousuke , Muto Shoshi
Published: July 2002

When methylviologen‐treated Chlamydomonas cells were exposed to light, the amount of ubiquitinated proteins with molecular masses of 28‐ and 31‐kDa were drastically changed, i.e. the former increased within 20 min illumination, while the latter decreased. Since these changes are completely dependent on illumination and suppressed by adding 3‐(3,4‐dichlorophenyl)‐1, 1‐dimethylurea, it was concluded that these changes were caused by active oxygen stress. Treating cells with hydrogen peroxide did not cause such changes of ubiquitination, suggesting that the molecular species of active oxygen is a superoxide anion.
Phorbol‐ester‐induced phosphorylation of the β2‐adrenergic receptor decreases its coupling to Gs
Authors: Bouvier Michel , Guilbault Nathalie , Bonin Hélène
Published: July 2002

Phorbol‐esters have been shown to modulated the β‐adrenergie‐stimulated adenylyl cyclase in a number of cell lines. Here, using site directed mutagenesis, we investigate the role of the β‐adrenergic receptor phosphorylation by protein kinase C in this regulatory process. Mutation of the serine‐261, ‐262, ‐344 and ‐345 of the β2‐adrenergic receptor prevented the phorbol‐ester‐induced phosphorylation of the receptor. This mutation also abolished the phorbol‐ester‐induced decrease in high‐affinity agonist binding and potency of the β2adrenergic receptor. We suggest that protein kinase C mediated phosphorylation of the receptor promotes its functional uncoupling.
A 28 kDa mitochondrial protein is radiolabelled by crosslinking with a 123I‐labelled presequence
Authors: Font B. , Goldschmidt D. , Chich J.F. , Thieffry M. , Henry J.P. , Gautheron D.C.
Published: July 2002

A 13‐residue peptide containing the first 12 amino acids of the N‐terminal part of the signal sequence of yeast cytochrome ???oxidase subunit IV is shown by chemical crosslinking to interact with a mitochondrial protein. This result is obtained with mitochondria from four different origins. Submitochondrial localization experiments suggest that the 28 kDa labelled component is present on the outer face of the inner membrane. Since such addressing peptides are imported into mitochondria through the same machinery as protein precursors, the 28 kDa protein might be a component of the translocation apparatus.
ICAM‐2 peptides mediate lymphocyte adhesion by binding to CD11a/CD18 and CD49d/CD29 integrins
Authors: Seth R. , Salcedo R. , Patarroyo M. , Makgoba M.W.
Published: July 2002

Three fifteen‐amino‐acid polypeptides designated peptides 1, 2 and 3 were synthesised as likely candidates for mimicking the role of ICAM‐2 as a ligand. The ability of each peptide to bind lymphoid cells was tested. Peptide 2 largely mediated cell attachment of unstimulated cells and this binding was only marginally increased by stimulating the cells with phorbol dibutyrate (P(Bu)2), Peptide 3 mediated minimal spontaneous cell attachment, but this binding was significantly enhanced following P(Bu)2 stimulation. Peptide I had no effect on cell attachment with or without stimulation. The cell attachment to peptide 2 was both temperature‐ and cation‐dependent. Studies using specific monoclonal antibodies showed that with unstimulated cells, anti‐VLA‐4α(CD49d) or β chain (CD29) antibodies (KD4‐13 and 4B4) and anti‐CD18 (1B4) each partially inhibited the cell binding. Monoclonal antibodies against CD54 (ICAM‐1; 84H10 or LB2). MHC class I (W6/32) and control mouse 1gG had no effect. When anti‐CD29 and anti‐CD18 monoclonal antibodies were used concurrently, there was almost complete inhibition of the cell attachment. These observations indicated that cell adhesion via ICAM‐2 is mediated: (i) predominantly by peptide 2 in unstimulated and P(Bu)2‐stimulated cells, and also, to some extent, by peptide 3 in P(Bu)2‐stimulated cells and (ii) by binding to both CD11/CD18 and CD49d/CD29 integrins.
Regulation of acetylcholine receptor gene expression in rats treated with α‐bungarotoxin
Authors: Asher Orna , Provenzano Carlo , Fuchs Sara
Published: July 2002

Regulation of acetylcholine receptor (AChR) gene expression was analyzed in α‐bungarotoxin (α‐BTX) treated rats. A reduction in available 125l‐α‐BTX binding sites was accompanied by an increase in the various AChR transcripts. The increase in the AChR α‐, β‐ c‐ and δ‐subunit mRNAs was similar to that observed in rats with experimental autoimmune myasthenia gravis (EAMG). Unlike in EAMG, the γ‐subunit transcripts reappeared following α‐BTX treatment. The quantitative differences in the levels of AChR transcripts between α‐BTX treatment and EAMG on one hand and denervation on the other hand, support the notion that the regulation of AChR gene expression is controlled by muscle activity and by neuronal factors as well. We also demonstrate in this report that myogenin transcripts increase following α‐BTX treatment as well as following denervation, whereas MyoD1 transcripts remain stable.
Changes in the kinetics of inositol transport during TPA‐induced differentiation of HL60 cells towards monocytes
Authors: Grafton G. , Bunce C.M. , Sheppard M.C. , Brown G. , Baxter M.A.
Published: July 2002

When exposed to the phorbol ester TPA, HL60 cells undergo growth arrest and differentiate towards monocytes. During TPA‐induced differentiation there was a 2.6‐fold increase in the rate of inositol transport (V
max), a 2.1‐fold increase in intracellular inositol and a 1.5‐fold increase in inositol lipid. An increase in the V
max of inositol transport did not occur when the variant cell line HL60Ast3 was exposed to TPA, which has been shown in this cell line to induce growth arrest but not differentiation. This observation suggests that the change in inositol transport during HL60 monocyte differentiation is specifically associated with the process of cell differentiation as opposed to growth arrest.
Inhibition of fetal rat pancreatic β‐cell replication by interleukin‐1β in vitro is not mediated through pertussis toxin‐sensitive G‐proteins, a decrease in cyclic AMP, or protease activation
Authors: Sjöholm Åke
Published: July 2002

It has been proposed that the cytokine interleukin‐1β (IL‐1β), secreted by islet‐infiltrating macrophages, may be involved in the pathogenesis of insulin‐dependent diabetes mellitus by participation in β‐cell destruction. Addition of IL‐ 1β to isolated pancreatic islets in vitro results in cytotoxic effects on β‐cell function, but there is little information on the intracellular events that convey the actions of the cytokine. In the present study, fetal rat pancreatic islets containing a high fraction of β‐cells were exposed in culture to IL‐1β. It was found that IL‐1β markedly decreased β‐cell DNA synthesis, insulin secretion and cyclic AMP content, In order to explore whether the decrease in cAMP resulted from IL‐1β interaction with GTP‐binding proteins coupled to adenylyl cyclase, islets were treated for 24h with pertussis toxin prior to addition of cytokine. While this treatment restored the decrease in cAMP, the reduced DNA synthesis and insulin secretion persisted. Pertussis toxin treatment without the addition of IL‐1β resulted in increases in cAMP, DNA synthesis and insulin secretion. Addition of the stimulatory cAMP analog Sp‐cAMPS also increased DNA synthesis and insulin secretion, but failed to affect the decrease in these functions evoked by IL‐1β. The protease inhibitor Nα‐p‐tosyl‐L‐lysine chloromethyl ketone, recently shown to protect completely against IL‐1β‐induced suppression of insulin production and secretion, was found to markedly reduce DNA synthesis without affecting insulin secretion. When the protease inhibitor was combined with IL‐ 1β, the suppressed secretion was counteracted while DNA synthesis inhibition was not. It is concluded that cAMP stimulates DNA synthesis and insulin secretion in β‐cells, but that the inhibitory effect of IL‐1β on these functions cannot be ascribed to the decrease in cAMP evoked by the cytokine. However, the repressive effect of the cytakine on insulin secretion, but not DNA synthesis, may be prevented by protcase inhibition.
Effects of anti‐triadin antibody on Ca2+ release from sarcoplasmic reticulum
Authors: Brandt Neil R. , Caswell Anthony H. , Brunschwig J.-P. , Kang Jaw-Jou , Antoniu Bozena , Ikemoto Noriaki
Published: July 2002

The monoclonal antibody, mAb GE 4.90, raised against triadin, a 95 kDa protein of sarcoplasmic reticulum (SR), inhibits the slow phase of Ca2+ release from SR following depolarization of the T‐tubule moiety of the triad. The antibody has virtually no effect on the fast phase of depolarization‐induced Ca2+ release nor on caffeine‐induced Ca2+ release. Since the slow phase of depolarization‐induced Ca2+ release is also inhibited by dihydropyridines (DHP), these results suggest that triadin may be involved in the functional coupling between the DHP receptor and the SR Ca2+ channel.
Voltage‐dependent calcium entry in confluent bovine capillary endothelial cells
Authors: Bossu J.L. , Elhamdani A. , Feltz A.
Published: July 2002

Confluent bovine capillary endothelial cells display, when examined for voltage‐dependent calcium entries using cell‐attached channel recordings, two types of Ca2+ channels (4 and 23.5 pS in 110 mM Ba2+) both sensitive to the dihydropyridine Ca agonist BAY K 8644. In contrast to isolated cells, confluent cells display no T‐type, low threshold activity, and Ca currents were typically only elicited at very depolarized potentials. In these cells, voltage‐dependent calcium entries will only be made operative by substances able to shift their activation towards the resting potential.
Phorbol ester‐induced secretion of human hepatocyte growth factor by human skin fibroblasts and its inhibition by dexamethasone
Authors: Gohda Eiichi , Kataoka Hirotoshi , Tsubouchi Hirohito , Daikilara Yasushi , Yamamoto Itaru
Published: July 2002

Human skin fibroblasts secreted a certain amount of human hepatocyte growth factor (hHGF), as determined by an enzyme‐linked immunosorbent assay for hHGF. This hHGF secretion was remarkably stimulated by protein kinase C (PKC)‐activating phorbol esters, which was inhibited by the simultaneous addition of dexamethasone. Pretreatment with phorbol 12‐myristate 13‐acetate (PMA) caused a down‐regulation in hHGF secretion. hHGF secreted by the PMA‐treated cells showed a potent hepatocyle growth‐promoting activity which was neutralized by an anti‐hHGF antiserum. These results indicate both that PMA‐treated human skin fibroblasts produce biologically active hHGF and the possible involvement of PKC activation in this process.
A possible biological role of the electron transfer between tyrosine and tryptophan
Authors: Lee Chyuan-Yih
Published: July 2002

Experiments have demonstrated that four tryptophan residues are located near the tetrodotoxin binding site in Na+ channels, and that conserved tyrosine and tryptophan residues are located in the pore‐forming region of voltage‐sensitive K+ channels. This paper proposes an activation mechanism involving electron transfer between these residues. The K+ channel may be closed by four tyrosine residues forming hydrogen bonds with each other. After electron transfer, these hydrogen bonds will be broken, thereby opening the channel. The Na+ channel could be activated by a similar mechanism. This idea can be tested directly by observing tyrosine or tryptophan radicals when the channels are in the open state.
Expression of c‐met proto‐oncogene in COS cells induces the signal transducing high‐affinity receptor for hepatocyte growth factor
Authors: Higuchi Osamu , Mizuno Kensaku , Vande Woude George F. , Nakamura Toshikazu
Published: July 2002

By transfection of the expression plasmid containing a human c‐met cDNA into COS‐7 cells, high‐affinity binding sites specific for HGF with a K
d value of 30 pM were newly detected. Furthermore, only in the c‐met transfected COS‐7 cells, but not in the control COS‐7 cells, DNA synthesis was markedly induced in response to HGF. Thus, transient expression of exogenous c‐met cDNA resulted in the appearance of high‐affinity receptor for HGF and conversion of the normally non‐responsive COS‐7 cells into the HGF‐responsive cells. These results provide evidence for identifying the c‐met product as a signal transducing high‐affinity receptor for HGF.
An antipodean perception of the mode of action of glycoprotein hormones
Authors: Renwick Alistair , Wiggins Philippa
Published: July 2002

Continuance of vertebrate species and maintenance of metabolism have an absolute requirement for the glycoprotein hormones of the anterior pituitary gland. It is now firmly accepted that the N‐glycans of these and the related placental hormone, chorionic gonadotropin, have essential if undefined roles in their mechanism of action. However, recent investigations by Weisshaar and his colleagues on the oligosaccharides of human luteinizing hormone and chorionic gonadotropin, do not support the generally accepted view of carbohydrates in hormone—receptor interactions and a further concept is proposed that invokes negative charges and changes in structured water.
Different sequence environments of cysteines and half cystines in proteins Application to predict disulfide forming residues
Authors: Fiser András , Cserzö Miklós , Tüdös Éva , Simon István
Published: July 2002

Protein sequences are often derived by translating genetic information, rather than by classical protein sequencing. At the DNA level cysteines and half cystines are indistinguishable. Here we show that the sequential environments of ‘free’ cysteine and half cystine are different. A possible origin of this difference is discussed and a simple method to predict cysteines and half cystines from the amino acid sequence is also presented.
ATP and GTP as alternative energy sources for vinblastine transport by P‐170 in KB‐V1 plasma membrane vesicles
Authors: Lelong Isabelle H. , Padmanabhan Raji , Lovelace Elizabeth , Pastan Ira , Gottesman Michael M.
Published: July 2002

Purified plasma membrane vesicles isolated from multidrug‐resistant human KB‐V1 cells accumulate [3H]vinblastine in an energy‐dependent manner. The accumulation of [3H]vinblastine in the presence of ATP is a saturable process. ATP can be replaced by other purine nucleotide triphosphates, of which GTP is the most efficient.
Copper(II)‐substituted horse liver alcohol dehydrogenase: Structure of the minor species
Authors: Formicka Grazyna , Zeppezauer Michael , Fey Frunk , Hüttermann Jürgen
Published: July 2002

Oxygen treatment of horse liver alcohol dehydrogenase EE isoenzyme substituted with Cu(II) at the catalytic site leads to bleaching with concomitant reduction to Cu(I) of‐90% of total Cu(II). The Cu(II) of the remaining ‘minor species’ cannot be reduced nor does it interact with exogenous ligands, e.g. 2‐mercaploethanol, imidazole, purazole, or the azide ions. The EPR spectrum is axial with a super‐hyperfine splitting of 15.6G indicating binding of one nitrogen atom to Cu(II). These data as well as the energies and intensities of the absorption and CD spectra suggest the Cu(II) ion of the minor species to be located in the catalytic site of HLADH in a position and geometry different from that of the major species.
Site‐directed mutagenesis of active‐site‐related residues in Torpedo acetylcholinesterase Presence of a glutamic acid in the catalytic triad
Authors: Duval Nathalie , Bon Suzanne , Silman Israel , Sussman Joel , Massoulié Jean
Published: July 2002

Site‐directed mutagenesis was used to investigate the role of acidic amino acid residues close to the active site of Torpedo acetylcholinesterass. The recently determined atomic structure of this enzyme shows the conserved Glu‐327, together with His‐440 and Ser‐200 as forming a catalytic triad, while the adjacent conserved Asp‐326 points away from the active site. Transfection of appropriately mutated DNA into COS cells showed that the mutation of Asp‐326 → Asn had little effect on catalytic activity or the molecular forms expressed, suggesting no crucial structural or functional role for this residue. Mutation of Glu‐327 to Gin or to Asp led to an inactive product. These results support the conclusions of the structural analysis for the two acidic residues.
Modulation of cardiac Ca2+ channels in Xenopus oocytes by protein kinase C
Authors: Singer-Lahat Dafna , Gershon Eli , Lotan Ilana , Hullin Roger , Biel Martin , Flockerzi Veit , Hofmann Franz , Dascal Nathan
Published: July 2002

L‐Type calcium channel was expressed in Xenopus laevis oocytes injected with RNAs coding for different cardiac Cu2+ channel subunits, or with total heart RNA. The effects of activation of protein kinase C (PKC) by the phorbol ester PMA (4β‐phorbol 12‐myristate 13‐acetate) were studied. Currents through channels composed of the main (α1) subunit alone were initially increased and then decreased by PMA. A similar biphasic modulation was observed when the α1 subunit was expressed in combination with α2/δ, β and/or γ subunits, and when the channels were expressed following injection of total rat heart RNA. No effects on the voltage dependence of activation were observed. The effects of PMA were blocked by staurosporine, a protein kinase inhibitor. β subunit moderated the enhancement caused by PMA. We conclude that both enhancement and inhibition of cardiac L‐type Ca2+ currents by PKC are mediated via an effect on the α1 subunit, while the β subunit may play a mild modulatory role.
Molecular cloning of a novel ras‐like protein from chicken
Authors: Trueb Judith , Trueb Beat
Published: July 2002

We have isolated a cDNA clone from a chicken DNA expression library which codes for a ras‐like polypeptide of 216 amino acid residues. This polypeptide is closely related to the human protein TC4 and to the yeast protein Spil, two novel proteins that may be involved in the coordination of the cell cycle. In the amino‐terminal region, the three polypeptides possess a P‐loop motif characteristic of GTP‐binding proteins. At the carboxy‐terminal end, however, they lack the typical CAAX‐box which is usually responsible for membrane anchorage of ras‐like proteins. It is therefore likely that the three polypeptides define a new subclass of GTP‐binding proteins within the ras‐like superfamily.
Modulation of expression of multidrug resistance gene (mdr‐1) by adriamycin
Authors: Kato Shoko , Nishimura Junji , Yufu Yuji , Ideguchi Hiroshi , Umemura Tsukuru , Nawata Hajime
Published: July 2002

The acquired resistance to various drugs in cancer is mediated by P‐glycoprotein (P‐gp) which is encoded by the mdr‐1 gene. An increased level of mdr‐1/P‐gp was demonstrated after chemotherapy administered to treat cancer in humans. To clarify the direct effect of anticancer drugs on mdr‐1/P‐gp expression, we investigated the change in transport of adriamycin (ADR), and the expression of the mdr‐1 gene and P‐gp in an ADR‐treated, multidrug‐resistant leukemic cell line (K562/ADR500). The addition of ADR induced the over‐expression of mdr‐1/P‐gp, which led to a transient decrease in the intracellular accumulation of ADR although the difference was not statistically significant. A maximal effect was observed after 4 h incubation, returning to the baseline level after further incubation for 12–24 h. The phosphorylation of P‐gp was inversely correlated with the levels of P‐gp. These observations suggest that ADR itself modulates both the expression and function of P‐gp. Determination of the optimal schedule for administering adriamycin is essential to achieving the optimal effect in treating cancer.
Troponin replacement in permeabilized cardiac muscle Reversible extraction of troponin I by incubation with vanadate
Authors: Strauss John D. , Zeugner Claudia , Van Eyk Jennifer E. , Bletz Christel , Troschka Monika , Rüegg J.Caspar
Published: July 2002

Calcium‐dependent regulation of tension and ATPase activity in permeabilized porcine ventricular muscle was lost after incubation with 10 mM vanadate. After transfer from vanadate to a vanadate‐free, low‐Ca2+ solution (pCa > 8), the permeabilized muscle produced 84.8% ± 20.1% (± S.D., n=98) of the isometric force elicited by high Ca22+ (pCa ∼4.5 prior to incubation with vanadate. Transfer back to a high Ca2+ solution elicited no additional force (83.2% ± 18.7% of control force). SDS‐PAGE and immunoblot analysis of fibers and solutions demonstrated substantial extraction (>90%) of Troponin I (TnI). Calcium dependence was restored after incubation with solutions containing either whole cardiac troponin or a combination of TnI and troponin C subunits. This reversible extraction of troponin directly demonstrates the role of TnI in the regulation of striated muscle contractility and permits specific substitution of the native TnI with exogenously supplied protein.
Muscarinic acetylcholine receptor produced in recombinant baculovirus infected Sf9 insect cells couples with endogenous G‐proteins to activate ion channels
Authors: Vasudevan Subhash , Premkumar Louis , Stowe Sally , Gage Peter W. , Reiländer Helmut , Chung Shin-Ho
Published: July 2002

Following the infection of insect ovarian cells (Sf9) with recombinant baculovirus bearing the cDNA coding for the rat muscarinic acetylcholine (ACh) receptor subtype m3, ionic flux across the membrane in response to the application of ACh was examined electrophysiologically. We show that ACh activates potassium currents. The response is abolished when cells are treated with pertussis toxin. No ACh‐induced currents are observed from uninfected cells or cells infected with virus which do not contain the cDNA coding for ACh receptors in its genome. The characteristics of single channel currents show time‐dependent changes following the application of ACh. Initially, ACh activates brief channel currents with a conductance of about 5 pS. The conductance level of channels gradually increases in steps to 10 pS and then to 20 pS and 40 pS. At the same time, channel open probability also increases. Thereafter, additional channels appear, opening and closing independently of, or at times in synchrony with, the original channel.
Effect of modulation of protein kinase C on the cAMP‐dependent chloride conductance in T84 cells
Authors: Dechecchi Maria Cristina , Rolfini Rossella , Tamanini Anna , Gamberi Chiara , Berton Giorgio , Cabrini Giulio
Published: July 2002

The regulation of chloride conductance was investigated in the T84 human colon carcinoma cell line by the quenching of the fluorescent probe 6‐methoxy‐N‐(3‐sulfopropyl)quinolinium. The permeable cAMP analog 8‐Br‐cAMP (100 μ) and the calcium ionophore ionomycin (1 μM) activate a chloride conductance. A prolonged (4 h) preincubation of cells with phorbol 12‐myristate 13‐acetate (100 nM) or with the diacylglycerol analog 1‐oleoyl‐2‐acetyl‐glycerol (100 μM): (1) down‐modulates to almost zero the protein kinase C activity in the membranes; (ii) inhibits the activation of the chloride conductance mediated by 8‐Br‐cAMP but not by calcium; (iii) reduces the mRNA without changing the expression of the protein product of the cystic fibrosis gene. The data suggest that PKC is essential for the activation of the cAMP‐dependt chloride conductance in T84 cells.
Altered expression of G‐protein mRNA in spontaneously hypertensive rats
Authors: Thibault Christelle , Anand-Srivastava Madhu B.
Published: July 2002

We have recently demonstrated that the decreased ability of hormones, forskolin and GTP to stimulate adenylate cyclase in heart and aorta from spontaneously hypertensive rats (SHR), as compared to their age‐matched Wistar—Kyoto control rats (WKY), was associated with enhanced levels of Gi‐ and not with Gs‐regulatory proteins. In the present studies we have investigated the expression of Gi‐regulatory proteins at the mRNA level by Northern blotting. Total RNA of heart ventricle and aorta from WKY and SHR was probed with radiolabeled cDNA inserts encoding Giα‐2 and Giα‐3. The Giα‐2 and Giα‐3 probes detected a message of 2–3 and 3–5 kb, respectively, in both WKY and SHR, however, the message was significantly enhanced in SHR, as compared by WKY. On the other hand the cDNA probe encoding Gsα detected a message of 1.8 kb in heart and aorta from both WKY and SHR, however, no difference in the levels of Gsα mRNA was detected in SHR and WKY tissues. These results indicate that the mRNA levels of Giα‐2 and Giα‐3 and not of Gs are overexpressed in heart and aorta from SHR, which may be responsible for the increased levels of Gi as shown earlier by immunoblotting techniques. It may be suggested that the enhanced vascular tone and impaired cardiac contractility in hypertension may partly be the consequences of increased levels of Gi in heart and aorta.
Functional consequences of alterations to Pro328 and Leu332 located in the 4th transmembrane segment of the α‐subunit of the rat kidney Na+,K+‐ATPase
Authors: Vilsen Bente
Published: July 2002

Site‐specific mutagenesis was used to analyse the functional roles of the residues Pro328 and Leu332 located in the conserved PEGLL motif of the predicted transmembrane helix M4 in the α1‐subunit of the ouabain resistant rat kidney Na+,K+‐ATPase. cDNAs encoding either of the Na+,K+‐ATPase mutants Pro328→Ala and Leu332→Ala, and wild type, were cloned into the expression vector pMT2 and transfected into COS‐1 cells. Ouabain‐resistant clones growing in the presence of 10 μM ouabain were isolated, and the Na+, K+, ATP and pH dependencies of the Na+,K+‐ATPase activity measured in the presence of 10 μM ouabain were analysed. Under these conditions the exogenous expressed Na+,K+‐ATPase contributed more than 95% of the Na+,K+‐ATPase activity. The Pro328→Ala mutant displayed a reduced apparent affinity for Na+ (K
0.5 (Na+) 13.04 mM), relative to the wild type (K
0.5 (Na+) 7.13 mM). By contrast, the apparent affinity for Na+ displayed by the Leu 332→Ala mutant was increased (K
0.5 (Na+) 3.92 mM). Either of the mutants exhibited lower apparent affinity for K+ relative to the wild type (K
0.5 (K+) 2.46 mM for Pro328→Ala and 1.97 mM for Leu332→Ala, compared with 0.78 mM for wild type). Both mutants exhibited higher apparent affinity for ATP than the wild type (K
0.5 (ATP) 0.086 mM for Pro328→Ala and 0.042 mM for Leu332→Ala, compared with 0.287 mM for wild type). The influence of pH was in accordance with an acceleration of the E2 (K)→E1 transition in the mutants relative to the wild type. These data are consistent with a role of Pro328 and Leu332 in the stabilization of the E2 form and of Pro328 in Na+ binding. The possible role of the mutated residues in K+ binding is discussed.
Evidence for receptor‐linked activation of phospholipase D in rat parotid glands Stimulation by carbamylcholine, PMA and calcium
Authors: Guillemain Isabelle , Rossignol Bernard
Published: July 2002

In order to test if phospholipase D (PLD) activity exists in the rat parotid gland, we took advantage of the fact that, in the presence of ethanol, PLD generates phosphatidylethanol (PEth) via a transphosphatidylation reaction. Lipid extracts of parotid acini prelabelled with [3H]myristic acid were analyzed by thin layer chromatography to determine [3H]phosphatidylethanol ([3H]PEth) formation. Carbamylcholine (1 mM) stimulated [3H]PEth formation in the presence of 2% ethanol, this effect was completely inhibited by atropine (10 μM), PMA (0.1–1 μM) and ionomycine (10 μM) also caused [3H]PEth generation. We conclude that a phospholipase D activity is present in the rat parotid gland and is regulated by muscarinic cholinergic receptors. Protein kinase C and calcium could also modulate this activity. This report provides the first evidence for the existence and receptor‐linked regulation of phospholipase D in an exocrine gland, the rat parotid gland.
Stimulation of insulin release from permeabilized HIT‐T15 cells by a synthetic peptide corresponding to the effector domain of the small GTP‐binding protein rab3
Authors: Li Guodong , Regazzi Romano , Balch William E. , Wollheim Claes B.
Published: July 2002

A synthetic peptide (rab3AL) corresponding to the effector domain of rab3, a small GTP‐binding protein, stimulated basal and potentiated Ca2+‐as well as GTPγS‐evoked insulin secretion about 2‐fold from streptolysin‐O permeabilized HIT cells. This effect was specific, since the analogous peptides of ras or rab1 did not affect the exocytotic event. The more than additive effect of rab3AL on Ca2+ or GTPγS stimulation indicates a distinct mode of action of the peptide. The partial loss of cytosolic proteins from permeabilized cells was accompanied by a faster run‐down of the secretory response to Ca2+ than the one to GTPγS. The persistent effect of rab3AL under these conditions points to a membrane localization of its target. These results suggest that rab3 and its effector are involved in the regulation of insulin secretion.
Induction of stathmin expression during liver regeneration
Authors: Koppel Juraj , Loyer Pascal , Maucuer Alexandre , Rehák Pavol , Manceau Valérie , Guguen-Guillouzo Christiane , Sobel André
Published: July 2002

Stathmin is a 19 kDa cytoplasmic phosphoprotein proposed to act as a relay for signals activating diverse intracellular regulatory pathways. After two‐thirds partial hepatectomy, the concentration of stathmin reached a peak between 48 and 72 hours, comparable to the levels observed in neonatal liver, at about 10 times the basal adult level. Stathmin then decreased to basal levels within 7 days, more rapidly than during postnatal tissue development (7 weeks), with no detectable change in its phosphorylation state. Interestingly, the mRNA for stathmin reached a peak much earlier than the protein, at 24 hours posthepatectomy, and decreased to a still detectable level until 96 hours after hepatectomy. Altogether, the present results further support the generatility of the implication of stathmin in regulatory pathways of cell proliferation and differentation during normal tissue development and posttraumatic regeneration.
Bovine inositol monophosphatase
Authors: Greasley P.J. , Gore M.G.
Published: July 2002

Rapid equilibrium dialysis has been used to show that recombinant bovine brain inositol monophosphatase binds one equivalent of Pi per subunit of enzyme. Pi is only bound in the presence of Mg2+ ions. The dissociation constant for the equilibrium is approximately 50 μM. This value of K
d is independent of the concentration of the Mg2+ ions and of the presence or absence of Li+ ions. Lithium ions which inhibit the enzyme uncompetitively are not able to support the binding of the Pi to the enzyme. The observation that Pi only binds in the presence of Mg2+ ions supports similar conclusions made in experiments which studied the protection of the enzyme from proteolytic degradation and chemical modification.
Characterisation of protein kinase C isoforms and enzymic activity from the αT3‐1 gonadotroph‐derived cell line
Authors: Johnson Melanie S. , MacEwan David J. , Simpson James , Mitchell Rory
Published: July 2002

Western blots of αT3‐1 cell extracts were immunostained with antibodies specific for various protein kinase C (PKC) isoforms. These revealed the presence of PKC types α, ϵ and ζ, but β, γ, δ and η were not detected. The potency with which partially‐purified cytosolic PKC from αT3‐1 cells was activated by phorbol 12,13‐dibutyrate (PDBu), mezerein and 1,2‐dioctanoyl‐sn‐glycerol was assessed in the presence and absence of Ca2+ The inhibitors staurosporine, K252a, H7, GF109203X and Ro 31‐8220 were tested on basal activity, PDBu‐induced activity and Ca2++ PDBu‐induced kinase activity. Each inhibitor showed distinct differences in their IC50 values under the three conditions, suggesting that these inhibitors may exhibit different potencies on the PKC isoforms present in αT3‐1 cells. Although histone IIIs was used as the phosphate acceptor for most of these experiments, the efficiency of α, ϵ and ζ, peptide and GS peptide substrates were also determined, with ϵ peptide giving the greatest activity in the presence of PDBu or Ca2+. Each substrate displayed a different pattern of activation under the conditions tested. Overall, the findings suggest that 3 or more PKC isoforms with varying specificities are present in gonadotroph‐derived αT3‐1 cells and that the contribution of each isofonn should be considered when these cells are used in models of anterior pituitary cell function where PKC is involved.
Cyclic ADP‐ribose‐induced Ca2+ release from rat brain microsomes
Authors: White Alison M. , Watson Stephen P. , Galione Antony
Published: July 2002

Cyclic ADP‐ribose (cADPR), an endogenous NAD+ metabolite in many mammalian and invertebrate tissues, is a potent mediator of calcium mobilization in sea urchin eggs. Our results show that cADPR also stimulates calcium release from rat brain microsomes, marked release occurring over the concentration range 10–250 nM. This is not inhibited by concentrations of heparin which completely abolish inositol 1,4,5‐trisphosphate (IP3)‐induced Ca2+ release. Ryanodine (100μM) inhibits the cADPR response. Our results are consistent with cADPR being an endogenous messenger mediating Ca2+ release from ryanodine‐sensitive pools in brain.
The IP3‐sensitive calcium store of HIT cells is located in a surface‐derived vesicle fraction
Authors: Lange Klaus , Brandt Ursula
Published: July 2002

Electron microscopic and biochemical techniques were used to study the cellular localization of the ATP‐dependent, IP3‐sensitive, Ca2+ store in the glucose‐ and phosphatidylinositol(PI) agonist‐sensitive hamster insulinoma cell line HIT‐T15. Scanning electron microscopy revealed conspicuous shape changes of the microvilli following stimulation of these cells with bombesin or thapsigargin. These changes closely resemble those previously shown to accompany stimulation of hexose transport in adipocytes with insulin [J. Cell. Physiol. 142 (1990) 1‐14]. Using a hydrodynamic shearing technique for the isolation of microvilli, two cell surface‐derived vesicle fractions were prepared containing 80% of the total cellular Ca2+‐storing activity. In contrast, subcellular fractionation using normal homogenization with a glass/teflon homogenizer yielded the well‐known distribution of the Ca2+‐storing activity which is then predominantly recovered within the microsomal fraction. The surface‐derived vesicle fraction was clearly distinguished from the microsomal fraction by its high content of Na+/K+‐ATPase and an immunoreactive fragment of the GluT‐1 glucose transporter isoform which both are not detectable in the microsomal fraction isolated from homogenates from sheared cells. The Ca2+ uptake properties of the cell surface‐derived vesicle fractions including the vanadate, A23187, and thapsigargin sensitivity were found to be identical with those described for the microsomal Ca2+ stores of various cell types. Inositol 1,4,5‐trisphosphate (IP3) at 1 μM induced a maximal release of 35–40% of the stored Ca2+ from these vesicles.