bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of type‐1 protein phosphatase by cdc2 kinase
Authors: Villa-Moruzzi Emma
Published: July 2002

Purified cdc2 or cdc2 obtained from HeLa cells in association with p131sucl activate inactive type‐1 protein phosphatase (PP1) (catalytic subunit · inhibitor‐2 complex, purified from skeletal muscle). Likewise in the case of PP1 activation by FA/GSK3, activation by cdc2 is accompanied by phosphorylation of inhibitor‐2 (I2) and free I2 can be phosphorylated as well. Correlation between PP1 activation and 12 phosphorylation is suggested by the fact that both activation and phosphorylation (a) increase in parallel during incubation with cdc2, (b) decrease in parallel upon subsequent cdc2 inhibition by EDTA, and (c) are inhibited by the cdc2 inhibitor 5,6‐dichlorobenzimidazole riboside. cdc2 also phosphorylates the catalytic subunit of PP1, whether in the complex with I2 or as free molecule. The activation of PPI by cdc2 and by FA/GSK3 is compared.
Acidic pH induces fusion of cells infected with baculovirus to form syncytia
Authors: Leikina Evgenia , Onaran H.Ongun , Zimmerberg Joshua
Published: July 2002

The enveloped baculovirus⧹insect cell system has been used extensively for expression of recombinant proteins. including viral fusion proteins. We tested wild‐type baculovirus for endogenous fusion protein activity. Syncytia formation, dye transfer, and capacitance changes were observed after incubating infected Spodoptera frugiperda cells in acidic media, consistent with fusion protein activity. Only a short acidic pulse or 10 s is needed to trigger syncytia formation. Identical results were obtained with recombinant baculovirus. This new system convenient for studying pH activated cell‐cell fusion. However, using this enveloped virus to study the mechanism of recombinant fusion proteins requires caution.
Synthetic peptides of the effector‐binding domain of rab enhance secretion from digitonin‐permeabilized chromaffin cells
Authors: Senyshyn Jan , Balch William E. , Holz Ronald W.
Published: July 2002

There is evidence that the rab class of low molecular weight GTP‐binding proteins is involved in vesicular transfer from endoplasmic reticulum to Golgi and between Golgi cisternae. To determine whether similar proteins play a role in regulated exocytosis, the effects of synthetic peptides derived from low molecular weight GTP‐binding proteins on catecholamine secretion from digitonin‐permeabilized chromaffin cells were investigated. The synthetic peptides represent the putative effector‐binding domains of the rab, ras and ral classes of low molecular weight GTP‐binding proteins and correspond to ras(33–48). Two rab peptides but neither a ras nor a ral peptide enhanced Ca2+‐dependent secretion by approximately 30%. Maximal secretion in response to Ca2+ was increased. The enhancement was not blocked by the pseudosubstrate inhibitor of protein kinase C, PKC(19–31), thus indicating that activation of protein kinase C was not responsible for the enhancement of secretion. Similarly a rab peptide but neither a ras nor a ral peptide enhanced CppNHp‐induced secretion 30–70%. The peptides had little or no effet in the absence of Ca2+ or GppNHp. The data are consistent with a protein of the rab class playing a role in regulated exocytosis.
Molecular cloning and functional expression of the second mouse nm23/NDP kinase gene, nm23‐M2
Authors: Urano Takeshi , Takamiya Kogo , Furukawa Koichi , Shiku Hiroshi
Published: July 2002

A new murine cDNA of nm23/NDP kinase was isolated. A RT‐PCR product was obtained from the normal mouse liver mRNA with primers designed for the human nm23‐H2 gene. The product was used as a probe to screen a cDNA library from the murine melanoma cell line. B16, and two clones containing the entire open reading frame were obtained. It was predicted that the DNA sequence encoded 152 amino acids which was 98% identical to the nm23‐H2 protein. The entire nm23‐M1 and ‐M2 gene‐coding regions were translated as fusion proteins with a glutathione S‐transferase. These fusion proteins displayed NDP kinase activities.
Protein kinase C mutants in the auto‐inhibitory region exhibit two distinct properties
Authors: Muramatsu Masa-aki , Kaibuchi Kozo , Arai Ken-ichi
Published: July 2002

To define the role of the auto‐inhibitory region of protein kinase C (PKC),
22‐
23‐Gly24‐Ala25‐Leu26‐Arg27, site‐directed mutations were introduced into the basic residues. Three mutants, PKCAla22,23, PKCAla27 and PKCAla22,23,27, apparently fell into two distinct types with regard to their biochemical properties and biological activities, as judged by the enhancement of a c‐fos promoter in Jurkat cells and by the initiation of germinal vesicle breakdown (GVBD) in Xenopus laevis oocytes. (i) PKCAlu22,23 and PKCAla27 had activators independent in vitro kinase activity, high phosphorylation levels in vivo, and localized in both cytosolic and particulate fractions. These mutants were not fully biologically active. (ii) PKCAla22,23,27 had a low phosphorylation level in vivo, was found predominantly in the particulate fraction and was the most biologically active. These results suggest that basic residues in the auto‐inhibitory domain account for the regulation of kinase activity and the cytosolic retention of PKC. The particulate association or the cytosolic clearance of PKC may facilitate signal transduction in the cell.
Inhibition of calcineurin by cyclosporin A‐cyclophilin requires calcineurin B
Authors: Haddy Alice , Swanson Selene K.-H. , Born Timothy L. , Rusnak Frank
Published: July 2002

The interaction of the immunosuppressive complex cyclosporin A‐cyclophilin (CsA‐CyP) with the Ca2+/calmodulin‐dependent protein phosphatase calcineurin is investigated using a recombinant form of the A subunit of calcineurin (rCNA). Only in the presence of purified calcineurin B (CNB) does rCNA show the response of native calcineurin, i.e. 50% inhibition of rCNA phosphatase activity at 6 nM human cyclophilin B and 0.6 μM human cyclophilin A using [32P]casein as substrate, yet stimulation of activity with p‐nitrophenyl phosphate as substrate. This study demonstrates that the B subunit is necessary to confer sensitivity of calcineurin to CsA‐CyP.
The projection structure of Perfringolysin O (Clostridium perfringens θ‐toxin)
Authors: Olofsson Anders , Hebert Hans , Thelestam Monica
Published: July 2002

The cytolysin Perfringolysin O was applied to lipid layers and the obtained ring‐shaped oligomers analyzed by electron microscopy and image processing. The final result shows the periodic repeat of 2.4 nm along the outer rim of the ring. The asymmetric protein unit, corresponding to one monomer, spans the ring from the convex to the concave surface. It shows a clear protein peak close to the outer radius and less density in the middle of the oligomer. The number of monomers in the average ring is 50, and the inner radius of the aggregate is approximately 15 nm.
The protein kinase mos activates MAP kinase kinase in vitro and stimulates the MAP kinase pathway in mammalian somatic cells in vivo
Authors: Nebreda Angel R. , Hill Caroline , Gomez Nestor , Cohen Philip , Hunt Tim
Published: July 2002

The mos protooncogene encodes a serine/threonine protein kinase that is only expressed at significant levels in germ cells. Recombinant malE‐mos protein (Xenopus mos protooncogene fused in frame to the maltose binding protein of E. coli) activates MAP kinase in cell‐free extracts prepared from Xenopus oocytes and eggs. Here we show that malE‐mos immunoprecipitates from Xenopus extracts phosphorylate and activate MAP kinase kinase in vitro, indicating that mos can function as a MAP kinase kinase kinase. Moreover, ectopic expression of mos in mammalian somatic cells, that lack any endogenous mos protein, triggers the activation of MAP kinase in vivo. These results identify the mos protooncogene as a direct activator of the MAP kinase pathway, with the potential to activate this kinase cascade even in cells where normally there is no expression of mos.
Basic fibroblast growth factor protects cerebellar neurons in primary culture from NMDA and non‐NMDA receptor mediated neurotoxicity
Authors: Fernández-Sánchez M.Teresa , Novelli Antonello
Published: July 2002

We have investigated the ability of bFGF to protect cerebellar neurons from neurotoxicity by excitatory amino acids. We have found that preincubation with 1–2.5 nM bFGF for 1–6 days significantly protected neurons from excitotoxic damage via NMDA receptors as well as ionotropic non‐NMDA receptors. bFGF neuroprotection appeared not to be dependent upon neuronal differentiation and was not mimicked by other neurotrophins including BDNF, NT‐3 and NGF. A greater rise in extracellular calcium‐dependent cGMP formation, following either depolarization or excitatory amino acid receptor activation was observed in bFGF‐pretreated neurons. We suggest that neuroprotection from excitotoxicity following bFGF treatment may be associated to the modulation of neurochemical pathways dependent upon extracellular calcium influx.
Conventional protein kinase C isoforms are not essential for cellular proliferation of a T cell lymphoma line
Authors: Long Aideen , Kelleher Dermot
Published: July 2002

The role of conventional protein kinase C (PKC) isoforms in the proliferation of a T cell lymphoma line was investigated. PKC isozyme‐deficient cell lines were generated from HuT 78 which expresses PKC isoforms α, β, ϵ and ζ. The first of these, K‐4, expresses PKC α, ϵ and ζ but not β. L‐2, a mutant of K‐4, expresses PKC α and ζ, but expresses neither PKC α, β, γ, ϵ nor ζ when cultured continuously in PMA (L‐2/PMA). All four cell lines continued to proliferate although at reduced rates in the presence of the PKC inhibitors staurosporine and H‐7. K‐4 and HuT 78 undergo growth arrest when activated with PMA (rate of proliferation decreases by 76 and 95%, respectively). While PMA‐induced growth arrest occurs in L‐2 cells which possess PKC α and ζ, PMA‐induced growth arrest does not occur in L‐2/PMA which is deficient in these isoforms. As these cell lines continue to proliferate in the presence of PKC inhibitors and since proliferation occurs in the absence of PKC isoforms (L‐2/PMA), the results suggest that neither conventional PKC nor the ϵ and ζ isoforms are essential for proliferation of these cells. These data further suggest that PKC α and/or ζ may be involved in PMA‐induced growth arrest.
Annexin 1 is present in different molecular forms in rat cerebral cortex
Authors: Pradel L.A. , Rendon A.
Published: July 2002

Previously we have purified annexin 1 [J. Neurochem. 56 (1991) 1985‐1986] from pig cerebral cortex as a monomeric protein of 37 kDa. Here, the localization of annexin 1 was investigated in subcellular fractionations of rat cerebral cortex using immunodetection by a specific antibody. In contrast to synaptophysin, a specific synaptic vesicle integral membrane protein, annexin 1 is located in the synaptic plasma membrane fraction where it appears on SDS‐PAGE as a polypeptide of 74 kDa. Annexin 1 is extracted also as a 74 kDa polypeptide from the purified synaptic plasma membranes. These results suggest for the 74 kDa molecular form an enzymatic dimerization of annexin 1 when associated to the membrane.
N‐Acetylcysteine inhibits apoptosis and decreases viral particles in HIV‐chronically infected U937 cells
Authors: Malorni Walter , Rivabene Roberto , Teresa Santini Maria , Donelli Gianfranco
Published: July 2002

Apoptosis or programmed cell death (PCD) is a type of death occurring in various physiological processes. Several data suggest that: (1) apoptosis may play a critical role in AIDS pathogenesis; (2) an increase of endocellular free radical levels can be associated with activation of previously latent HIV virus. Tumor necrosis factor (TNF), a cytokine capable of inducing oxygen free radicals and apoptosis, appears also to be involved in HIV activation. The present findings, which elucidate a relationship between the percentage of apoptotic cells, reduced glutathione (GSH) depletion and an increase of p24 antigenemia, suggest that pretreatment with N‐acetylcysteine (NAC) is capable of decreasing the above‐mentioned phenomena in HIV‐infected U937 cells.
Monoclonal antibodies targeted against the C‐terminal domain of dystrophin or utrophin
Authors: Fabbrizio Eric , Léger Jocelyne , Anoal Monique , Léger Jean J. , Mornet Dominique
Published: July 2002

The structure‐function relationships of dystrophin, a protein which is absent or defective in patients with Duchenne or Becker muscular dystrophies, and utrophin can only be compared if specific antibodies are produced. We expressed C‐terminal parts of dystrophin and utrophin in expression vectors. Mice were immunized with recombinant proteins and 26 monoclonal antibodies were produced and analyzed. Their respective epitopes were determined using other overlapping recombinant products. We observed antibody specificity towards 400 kDa dystrophin and/or utrophin protein bands, either by Western blot analysis or immunodetection in human skeletal (quadriceps) and smooth (uterus) muscles. These antibodies have been used to compare the relative abundance of both dystrophin and utrophin relative to the structures analyzed.
X‐ray crystallographic structure of a papain‐leupeptin complex
Authors: Schröder Ewald , Phillips Christopher , Garman Elspeth , Harlos Karl , Crawford Catherine
Published: July 2002

The three‐dimensional structure of the papain‐leupeptin complex has been determined by X‐ray crystallography to a resolution of 2.1 Å (overall R‐factor = 19.8%). The structure indicates that: (i) leupeptin contacts the S subsites of the papain active site and not the S'subsites; (ii) the ‘carbonyl’ carbon atom of the inhibitor is covalently bound by the Cys‐25 sulphur atom of papain and is tetrahedrally coordinated; (iii) the ‘carbonyl’ oxygen atom of the inhibitor faces the oxyanion hole and makes hydrogen bond contacts with Gln‐19 and Cys‐25.
Binding of an α scorpion toxin to insect sodium channels is not dependent on membrane potential
Authors: Gordon Dalia , Zlotkin Eliahu
Published: July 2002

The insect‐specific LqhαIT toxin resembles α scorpion toxins affecting mammals by its amino acid sequence and effects on sodium conductance. The present study reveals that LqhαIT does not bind to rat brain membranes and possesses in locust neuronal membranes a single class of high affinity (K
d = 1.06 ± 0.15 nM) and low capacity (B
max = 0.7 ± 0.19 protein) binding sites. The latter are: (1) distinct from binding sites of other sodium channel neurotoxins; (2) inhibited by sea anemone toxin II; (3) cooperatively interacting with veratridine; (4) not dependent on membrane potential, in contrast to the binding sites of α toxins in vertebrate systems. These data suggest the occurrence of (a) conformational‐structural differences between insect and mammal sodium channels and (b) the animal group specificity and pharmacological importance of the α scorpion toxins.
FVIIa derivatives obtained by autolytic and controlled cathepsin G mediated cleavage
Authors: Nicolaisen Else Marie , Thim Lars , Jacobsen Jes Kristian , Nielsen Per Franklin , Mollerup Inger , Jørgensen Tony , Hedner Ulla
Published: July 2002

The heavy chain of coagulation factor VII contains a serine esterase entity. A partial cleavage in the heavy chain occurs during purification and activation of the single‐chain zymogen, presumably as a result of autolysis. Neutrophil cathepsin G initially generates a Gla‐domainless FVIIa without coagulant activity. However, on extended exposure cleavage also occurs in the heavy chain, resulting in a complete loss of enzyme activity. Four cleavage sites on the heavy chain, two susceptible to trypsin‐like autolysis and two susceptible to chymotrypsin‐like cathepsin G‐mediated catalysis have been identified. The hydrolysis of peptide bonds in the heavy chain might contribute to regulation of the coagulation process in vivo.
Protein phosphatase inhibitor calyculin A induces hyperphosphorylation of cytokeratins and inhibits amylase exocytosis in the rat parotid acini
Authors: Takuma Taishin , Ichida Tokuro , Okumura Kazuhiko , Kanazawa Masaaki
Published: July 2002

Calyculin A, a protein phosphatase inhibitor with a chemical structure completely different from that of okadaic acid, reproduced the inhibitory effect of okadaic acid on cyclic AMP‐mediated amylase release from rat parotid acinar cells. Calyculin A markedly enhanced phosphorylation of cytokeratins in the cytoskeletal fraction of the cells, whereas cAMP had apparently no effect on the phosphorylation. Microscopic observations showed that parotid acini incubated with 100 nM calyculin A for 15 min had large vacuoles in the cytoplasm and conspicuous blebs on the basal plasma membrane. K252a, a nonselective protein kinase inhibitor, clearly reduced calyclin A‐induced phosphorylation of cytokeratins, and it markedly blocked the inhibition of amylase release and morphological changes evoked by calyculin A. These results suggest that hyperphosphorylation of cytokeratins profoundly affects the morphology and secretory activity of parotid acinar cells.
On the two iron centers of desulfoferrodoxin
Authors: Verhagen Marc F.J.M. , Voorhorst Wilfried G.B. , Kolkman Joost A. , Wolbert Ronnie B.G. , Hagen Wilfred R.
Published: July 2002

Desulfoferrodoxin from Desulfovibrio vulgaris, strain Hildenborough, is a homodimer of 28 kDa; it contains two Fe atoms per 14.0 kDa subunit. The N‐terminal amino‐acid sequence is homogeneous and corresponds to the previously described Rbo gene, which encodes a highly charged 14 kDa polypeptide without a leader sequence. Although one of the two iron centers, FeA, has previously been described as a ‘strained rubredoxin‐like’ site, EPR of the ferric form proves very similar to that of the pentagonal bipyramidally coordinated iron in ferric complexes of DTPA, diethylenetriaminepentaacetic acid: both systems have spin S = and rhombicity E/D = 0.08. Unlike the Fe site in rubredoxin the FeA site in desulfoferrodoxin has a pH dependent midpoint potential with pK
ox= 9.2 and pK
red = 5.3. Upon reduction (E
m,7.5 = +2 mV) FeA exhibits an unusually sharp S = 2 resonance in parallel‐mode EPR. The second iron, FeB, has S = and E/D = 0.33; upon reduction (E
m,7.5 = +90 mV) FeB turns EPR‐silent.
Functional characterization of the catalytic site of the tetanus toxin light chain using permeabilized adrenal chromaffin cells
Authors: Höhne-Zell Barbara , Stecher Brigitte , Gratzl Manfred
Published: July 2002

The molecular events underlying the inhibition of exocytosis by tetanus toxin were investigated in permeabilized adrenal chromaffin cells. We found that replacement of amino acid residues within the putative zinc binding domain of the tetanus toxin light chain such as of histidine (position 233) by cysteine or valine, or of glutamate (position 234) by glutamine completely abolished the effect of the light chains on Ca2+ induced catecholamine release. Dipicolinic acid, a strong chelating agent for zinc, also prevented the effect of the tetanus toxin light chain. Zn2+ and, less potently Cu2+ and Ni2+, but not Cd2+ and Co2+, restored the activity of the neurotoxin. These data show that zinc and the putative zinc binding domain constitute the active site of the tetanus toxin light chain. Neither captopril, an inhibitor of synaptobrevin cleavage nor peptides spanning the site of synaptobrevins cleaved by the tetanus toxin in neurons, prevented the inhibition of Ca2+ induced catecholamine release by the tetanus toxin light chain. This suggests that synaptobrevins are not a major target of tetanus toxin in adrenal chromaffin cells.
Amino acids 327–350 of the human C5a‐receptor are not essential for [125I]C5a binding in COS cells and signal transduction in Xenopus oocytes
Authors: Klos A. , Mätje C. , Rheinheimer C. , Bautsch W. , Köhl J. , Martin U. , Burg M.
Published: July 2002

The anaphylatoxic peptide C5a is an important inflammatory mediator of the complement system. We have generated human C5a‐receptor (hC5aR) mutants with truncation of its cytosolic carboxyl‐terminus (C‐terminus). Both mutants were analysed for C5a‐binding in transiently expressing COS cells, and one mutant additionally for GTP‐binding regulatory protein (G‐protein) coupling in cRNA‐injected Xenopus oocytes. Our data suggest that (a) amino acids (aa) 314 to 326 as part of the C‐terminus are necessary for proper receptor folding or expression and (b) the receptor C‐terminus distal from position 327 is not critical for receptor expression, folding, binding and G‐protein coupling.
Pattern recognition and self‐correcting distance geometry calculations applied to myohemerythrin
Authors: Hänggi G. , Braun W.
Published: July 2002

A topological list, consisting of segments of regular secondary structures and a list of buried and solvent accessible residues, is automatically predicted from multiple aligned sequences in a protein family. This topological list is translated into geometric constraints for distance geometry calculation in torsion angle space. A new self‐correcting distance geometry method detects and eliminates false distance constraints. In an application to the four‐helix bundle protein, myohem‐erythrin, the right‐handed global fold was correctly reproduced with a root‐mean‐square deviation of 2.6 Å, when the topological list was derived from the X‐ray structure. A predicted topological list, coupled with constraints from the residues in the active site of myohemerythrin, predicted the correct fold with a root‐mean‐square deviation of 4 Å for backbone atoms.
The studies of cooperative regions in T7 RNA polymerase
Authors: Protasevich I.I. , Memelova L.V. , Kochetkov S.N. , Makarov A.A.
Published: July 2002

The heat denaturation of bacteriophage T7 RNA polymerase (T7RNAP) was studied by scanning microcalorimetry. The thermodynamic parameters of the denaturation were estimated within the pH range 6–9. The analysis of the denaturation curves showed the presence of two cooperative parts of the T7RNAP molecule melting according to the ‘all‐or‐none’ principle. The molecular masses of these parts were determined as 22 and 77 kDa. These values are close to the molecular masses of protein domains obtained from X‐ray diffraction and limited trypsinolysis data. The smaller N‐terminal domain was shown to increase the thermostability of the ‘catalytic’ C‐terminal domain within the intact T7RNAP molecule.
RNA synthesis inhibition stabilises urokinase mRNA in macrophages
Authors: Stacey Katryn J. , Nagamine Yoshikuni , Hume David A.
Published: July 2002

Urokinase‐type plasminogen activator (uPA) mRNA is induced in macrophages by the lineage specific growth factor CSF‐1. Upon removal of CSF‐1 from bone marrow‐derived macrophages (BMM), uPA mRNA decayed with a half‐life of 2 h. If RNA synthesis inhibitors actinomycin D, 5,6‐dichloro‐1‐β‐ribofuranosyl benzimidazole (DRB) or α‐amanitin were added at the time as CSF‐1 removal, the uPA message was stabilised. This was not a general effect on CSF‐1 responsive mRNAs, as c‐myc mRNA decayed with normal kinetics in the presence of inhibitors. The requirement for ongoing RNA synthesis for the degradation of uPA mRNA in BMM suggests that a component of the degradative pathway may be induced following removal of CSF‐1.
Cloning of the classical guinea pig pancreatic lipase and comparison with the lipase related protein 2
Authors: Carrière Frédéric , Thirstrup Kenneth , Hjorth Siv , Boel Esper
Published: July 2002

Starting from total pancreatic mRNAs, the classical guinea pig pancreatic lipase was cloned using rapid amplification of 3' and 5' cDNA ends. Internal oligonucleotide primers were designed from a partial cDNA clone including the region coding for the lid domain. Using this strategy, we did not amplify the cDNA corresponding to the pancreatic lipase related protein 2 in which the lid domain is deleted. Amino acid sequences of the classical guinea pig pancreatic lipase and the related protein 2 were compared based on the primary and tertiary structures of the classical human pancreatic lipase. Their distinct physiological roles are discussed in the light of functional amino acid differences.
Tumor necrosis factor is markedly synergistic with interleukin 1 and interferon‐γ in stimulating the production of nerve growth factor in fibroblasts
Authors: Hattori Akira , Iwasaki Shoji , Murase Katsuhito , Tsujimoto Masafumi , Sato Masahiro , Hayashi Kyozo , Kohno Michiaki
Published: July 2002

A possible interaction between tumor necrosis factor‐α (TNF) and other cytokines/growth factors in stimulating the production of nerve growth factor (NGF) in Swiss 3T3 cells was studied. TNF's stimulatory activity on fibroblast NGF production was synergized by interleukin‐1α (IL‐1α), IL‐1β and interferon‐γ (IFN‐γ), but was antagonized by transforming growth factor‐β (TGF‐β). The most remarkable synergistic effect was observed between TNF and IL‐1α/β; as little as 0.003 of IL‐1β markedly enhanced TNF's stimulatory activity on NGF production in the cells. These findings reinforce the idea that TNF, in concert with IL‐1α/β, plays an essential role in regulating the regeneration of peripheral nerves following injury through an indirect mechanism by which it stimulates NGF production in fibroblasts.
Serotonin release and cell proliferation are under the control of α‐bungarotoxin‐sensitive nicotinic receptors in small‐cell lung carcinoma cell lines
Authors: Codignola A. , Tarroni P. , Cattaneo M.G. , Vicentini L.M. , Clementi F. , Sher E.
Published: July 2002

Neuronal type nicotinic acetylcholine receptors (nAchRs) have recently been identified in small‐cell lung carcinoma. We here show that both nicotine and cytisine stimulate [3H]serotonin release in a dose‐dependent manner; this effect is antagonized by α‐bungarotoxin (αBgtx) and α‐conotoxin MI (αCtx). Nicotine and cytisine stimulate in vitro SCLC proliferation and this effect is completely antagonized by both αBgtx and αCtx. By PCR analysis, we demonstrate the presence in SCLC of both the α7 and the β2 nAchR subunits mRNA. These data show that nAchRs play an important role in the biology of SCLC, and that αBgtx‐sensitive receptors of the α7 subtype are crucially involved in both the secretagogue and mitogenic effects of nicotinic agonists.
Effects of TGF‐β1 (transforming growth factor‐β1) on the cell cycle regulation of human breast adenocarcinoma (MCF‐7) cells
Authors: Mazars Philippe , Barboule Nadia , Baldin Véronique , Vidal Simone , Ducommun Bernard , Valette Annie
Published: July 2002

The antiproliferative effects of TGF‐β1 were investigated in a human breast adenocarcinoma cell line (MCF‐7). We report that TGF‐β1 inhibits proliferation through cell cycle arrest in G1. A MCF ‐7 cell subline (MCF‐7(‐)), in which the type II TGF‐β receptor is not detected, was shown to be resistant to TGF‐β1 growth inhibitory effect. Cdk2 kinase activity was inhibited in the MCF‐7 sensitive cell subline in parallel with the inhibition of cell cycle progression. In both sensitive and resistant cell lines, TGF‐β1 treatment did not affect cdk2, cdk4, cyclin E and cyclin D1 mRNA and protein levels. However, in the MCF‐7 sensitive cell subline, a time‐dependent increase in cells positive for p21WAF1/CIP1 nuclear localization was observed after TGF‐β1 treatment. These findings suggest that TGF‐β1 inhibition of MCF‐7 cell proliferation is achieved through a type II receptor‐dependent down‐regulation of Cdk2 kinase activity without modification of Cdk and cyclin expression, but correlated with an increase in p21WAF1/CIP1 nuclear accumulation.
Energy‐dependent Complex I‐associated ubisemiquinones in submitochondrial particles
Authors: Vinogradov A.D. , Sled V.D. , Burbaev D.S. , Grivennikova V.G. , Moroz I.A. , Ohnishi T.
Published: July 2002

Two distinct species of Complex I‐associated ubisemiquinones (SQNf and SQNs) were detected by cryogenic EPR analysis of tightly coupled submitochondrial particles oxidizing NADH or succinate under steady‐state conditions. The g = 2.00 signals from both fast‐relaxing at 40 K) and slow‐relaxing ) are sensitive to uncouplers, rotenone and thermally induced deactivation of Complex I. At higher temperatures the SQNf signal is broadened and only the SQNs, signal is seen (). The spin‐spin interaction between SQNf and the iron‐sulfur cluster N2 was detected as split peaks of the 2.05 signal with a coupling constant of 1.65 mT, revealing their mutual distance of 8–11 Å. The data obtained are consistent with a model in which N2 and two interacting bound ubisemiquinone species are spatially arranged within the hydrophobic domain of Complex I, participating in the vectorial proton translocation.
Expression of serotonin receptor mRNAs in blood vessels
Authors: Ullmer Christoph , Schmuck Karin , Kalkman Hans O. , Lübbert Hermann
Published: July 2002

Using RT‐PCR we distinguished mRNAs for all known G‐protein coupled serotonin receptors expressed in various rat and porcine blood vessels. Nearly all vessels expressed 5HT1
dβ, 5‐HT2A, 5‐HT2B, 5‐HT4, and 5‐Ht7 receptor mRNA to different extents. New splice variants of the porcine 5‐HT4 receptor were observed. Similar PCR assays were performed with endothelial and smooth muscle cells from human pulmonary artery, aorta, and with endothelial cells from human coronary artery and umbilical vein. All endothelial cells expressed 5‐HT1dβ, 5‐HT2b, and 5‐HT4 receptor mRNA, whereas in smooth muscle cells 5‐HT1dβ, 5‐HT2A, 5‐HT7, and in some experiments 5‐HT2B receptor mRNA were found. A model for the regulation of vascular tone by different 5‐HT receptors is proposed.
Sequencing and functional analysis of the genome of Bacillus subtilis strain 168
Authors: Harwood Colin R. , Wipat Anil
Published: July 2002

The international programme to sequence the 4.2 Mb genome of Bacillus subtilis, a model Gram‐positive bacterium, is a joint project involving European, Japanese and US research groups. To date ca. 3.0 Mb of the genome has been sequenced, with the remaining 1.2 Mb expected to be completed in 1997. The amenability of B. subtilis to genetic manipulation, combined with the availability of extensive expertise on its biochemistry and physiology, makes this bacterium a valuable organism in which to investigate the properties of genes for which functions cannot be readily ascribed by standard methods.
Determination of relative abundance of splicing variants of Oreochromis glutamate receptors by quantitative reverse‐transcriptase PCR
Authors: Wu Yi-Mi , Kung Shieh-Shiuh , Chow Wei-Yuan
Published: July 2002

In this study, the relative abundance of splicing variants of Oreochromis non‐NMDA subtype glutamate receptors was studied by quantitative reverse‐transcriptase PCR (RT‐PCR). The relative expression level between the flip and flop transcripts of fGluR2α determined by quantitative RT‐PCR is apparently much higher than that estimated by sequence analysis of the cloned RT‐PCR products. Control studies were performed to demonstrate the accuracy of the application of quantitative RT‐PCR analysis in studying the relative abundance between the flip and flop transcripts of glutamate receptors.
Early de novo ovarian cancer and cancer developing in benign ovarian lesions
Authors: Scully R.E.
Published: July 2002

Ovarian carcinoma can arise directly from the ovarian surface epithelium or its intraparenchymal inclusions or from pre‐existing benign ovarian lesions such as endometriosis and epithelial tumors.
Evaluation of the endometrial cavity during menopause
Authors: Maia H. , Barbosa I.C. , Farias J.P. , Ladipo O.A. , Coutinho E.M.
Published: July 2002

Objective: To compare transvaginal sonography (TVS), hysteroscopy and suction curettage in the evaluation of uterine bleeding during the menopause. Methods: Forty‐seven patients who presented with either postmenopausal bleeding (31 cases) or sonographic endometrial abnormalities at menopause (16 cases) were evaluated using TVS, hysteroscopy, and curettage with a Karman curette. Results: When endometrial thickness measured by TVS was <4 mm, there was no endometrial pathology. However TVS could not differentiate accurately between hyperplasia, polyps or endometrial carcinoma. In these cases, endometrial thickness was invariably greater than 5 mm. Conclusion: Hysteroscopy proved superior to curettage in the diagnosis of endometrial polyps.
The impact of therapeutic relatives’ groups on the course of illness of schizophrenic patients
Authors: Buchkremer G , Schulze Mönking H , Holle R , Hornung WP
Published: July 2002

This study considers the question of whether relapse rates among schizophrenic patients can be reduced by means of relatives' groups. In a randomized, controlled intervention study, two therapeutic strategies (therapeutic relatives' groups, initiated relatives' self-help groups) were compared with each other and with a control group. Interventions were confined to the relatives, with the patients continuing their standard treatment. The study involved 151 relatives of 99 chronic DSMIII schizophrenics. Data were collected before and after a 1-year intervention phase and in a 2-year follow-up. No difference existed between the groups with respect to rehospitalization data. However, numerous differences recorded in the psychopathological findings and in living and working circumstances suggest that therapeutic work with relatives is of clinically significant benefit.
Sensation seeking, alcoholism and dopamine activity
Authors: Wiesbeck GA , Wodarz N , Mauerer C , Thome J , Jakob F , Boening J
Published: July 2002

Sensation seeking scale (SSS) scores were determined in 15 alcohol dependent men with a positive family history for alcoholism (FHP), in 15 alcohol dependent men with a negative family history for alcoholism (FHN) and in 15 well-matched healthy male controls (CONTR). Both FHPs and FHNs suffered from longlasting alcohol dependence meeting ICD-10 and DSM-III-R diagnostic criteria. Dopamine activity was neuroendocrinologically assessed by measuring the amount of growth hormone released after stimulation with the dopamine receptor agonist apomorphine. Planned comparisons within a one-way ANOVA yielded significantly elevated levels of boredom susceptibility (BOS) in both FHPs and FHNs against CONTRs. SSS total scores, while approaching statistical significance, were elevated in FHPs only. Partial correlations (controlling for age, body weight, alcohol intake and duration of dependence) were calculated to examine the relationship between SSS and dopamine activity. Among the SSS subtraits, BOS revealed the highest correlation in each group. However, only in CONTRs did the relationship between BOS and dopamine activity reach statistical significance.
A family of γ‐like calcium channel subunits
Authors: Klugbauer Norbert , Dai Shuiping , Specht Verena , Lacinová Lubica , Marais Elsé , Bohn Georg , Hofmann Franz
Published: July 2002

The γ subunit was initially identified as an auxiliary subunit of the skeletal muscle calcium channel complex. Evidence for the existence of further γ subunits arose following the characterization of a genetic defect that induces epileptic seizures in stargazer mice. We present here the first account of a family of at least five putative γ subunits that are predominantly expressed in brain. The γ‐2 and γ‐4 subunits shift the steady‐state inactivation curve to more hyperpolarized potentials upon coexpression with the P/Q type α1A subunit. The coexpression of the γ‐5 subunit accelerates the time course of current activation and inactivation of the α1G T‐type calcium channel.
Automated extraction of information in molecular biology
Authors: Andrade Miguel A. , Bork Peer
Published: July 2002

We review data mining techniques in molecular biology, specifically those that extract information from the scientific literature itself. As more of the biological literature is published electronically, there is an opportunity, and even a need, to automatically summarize the literature in a customized way, for example by associating keywords to a topic. These keywords can be extracted from relevant publications. The process of keyword extraction can be automated and optimized to keep literature pointers automatically up‐to‐date or to filter relevant information from the literature. To illustrate these points, OMIM (Online Mendelian Inheritance in Man), a database of human inherited diseases, was linked to the literature and keywords were derived that covered distinct aspects such as genetic information on the one hand and disease‐specific protein and phenotypic information on the other. They were used to extract information that is helpful for keeping entries about disease up‐to‐date.
Effects of mutation at a conserved N‐glycosylation site in the bovine retinal cyclic nucleotide‐gated ion channel
Authors: Rho Seong-hwan , Lee Han Mi , Lee Kyunglim , Park Chul-Seung
Published: July 2002

Bovine retinal cyclic nucleotide‐gated (CNG) ion channel contains an evolutionary conserved N‐glycosylation site in the external loop between the fifth transmembrane segment and the pore‐forming region. The effect of tunicamycin treatment and the site‐specific mutation suggested that the channel is glycosylated when expressed in Xenopus oocytes. To test the role of glycosylation in this channel, N‐glycosylation was abolished by mutation, and the detailed permeation and the gating characteristics of the mutant channel were investigated. The charge contribution turned out to be detectable, although the mutation of the N‐glycosylation site did not affect expression and functionality of the CNG channel in oocytes.
Protein aggregation as bacterial inclusion bodies is reversible
Authors: Carrió M.Mar , Villaverde Antonio
Published: July 2002

Inclusion bodies are refractile, intracellular protein aggregates usually observed in bacteria upon targeted gene overexpression. Since their occurrence has a major economical impact in protein production bio‐processes, in vitro refolding strategies are under continuous exploration. In this work, we prove spontaneous in vivo release of both β‐galactosidase and P22 tailspike polypeptides from inclusion bodies resulting in their almost complete disintegration and in the concomitant appearance of soluble, properly folded native proteins with full biological activity. Since, in particular, the tailspike protein exhibits an unusually slow and complex folding pathway involving deep interdigitation of β‐sheet structures, its in vivo refolding indicates that bacterial inclusion body proteins are not collapsed into an irreversible unfolded state. Then, inclusion bodies can be observed as transient deposits of folding‐prone polypeptides, resulting from an unbalanced equilibrium between in vivo protein precipitation and refolding that can be actively displaced by arresting protein synthesis. The observation that the formation of big inclusion bodies is reversible in vivo can be also relevant in the context of amyloid diseases, in which deposition of important amounts of aggregated protein initiates the pathogenic process.
β‐Adrenergic agonists increase phosphorylation of elongation factor 2 in cardiomyocytes without eliciting calcium‐independent eEF2 kinase activity
Authors: McLeod Laura E , Wang Lijun , Proud Christopher G
Published: July 2002

The β‐adrenergic agonist isoproterenol increased the phosphorylation of elongation factor eEF2 in ventricular cardiomyocytes from adult rats (ARVC). Phosphorylation of eEF2 inhibits its activity, and protein synthesis was inhibited in ARVC concomitantly with increased eEF2 phosphorylation. eEF2 kinase activity in ARVC extracts was completely dependent upon Ca2+/calmodulin. In contrast to other cell types, however, treatments designed to raise intracellular cAMP failed to induce Ca2+/calmodulin‐independent activity. Instead, they increased maximal eEF2 kinase activity. Similar data were obtained when partially purified ARVC eEF2 kinase was treated with cAMP‐dependent protein kinase in vitro. These data suggest that ARVC possess a distinct isoform of eEF2 kinase.
Binding and regulation of the transcription factor NFAT by the peptidyl prolyl cis–trans isomerase Pin1
Authors: Liu Weifeng , Youn Hong-Duk , Zhou Xiao Zhen , Lu Kun Ping , Liu Jun O.
Published: July 2002

Nuclear factor of activated T cells (NFAT) plays a key role in T cell activation. The activation of NFAT involves calcium‐ and calcineurin‐dependent dephosphorylation and nuclear translocation from the cytoplasm, a process that is opposed by protein kinases. We show here that the peptidyl prolyl cis–trans isomerase Pin1 interacts specifically with the phosphorylated form of NFAT. The NFAT–Pin1 interaction is mediated through the WW domain of Pin1 and the serine–proline‐rich domains of NFAT. Furthermore, binding of Pin1 to NFAT inhibits the calcineurin‐mediated dephosphorylation of NFAT in vitro, and overexpression of Pin1 in T cells inhibits calcium‐dependent activation of NFAT in vivo. These results suggest a possible role for Pin1 in the regulation of NFAT in T cells.
Global gene expression during short‐term ethanol stress in Saccharomyces cerevisiae
Authors: Alexandre H , Ansanay-Galeote V , Dequin S , Blondin B
Published: July 2002

DNA microarrays were used to investigate the expression profile of yeast genes in response to ethanol. Up to 3.1% of the genes encoded in the yeast genome were up‐regulated by at least a factor of three after 30 min ethanol stress (7% v/v). Concomitantly, 3.2% of the genes were down‐regulated by a factor of three. Of the genes up‐regulated in response to ethanol 49.4% belong to the environmental stress response and 14.2% belong to the stress gene family. Our data show that in addition to the previously identified ethanol‐induced genes, a very large number of genes involved in ionic homeostasis, heat protection, trehalose synthesis and antioxidant defence also respond to ethanol stress. It appears that a large number of the up‐regulated genes are involved in energy metabolism. Thus, ‘management’ of the energy pool (especially ATP) seems to constitute an ethanol stress response and to involve different mechanisms.
NADPH‐dependent glutathione peroxidase‐like proteins (Gpx‐1, Gpx‐2) reduce unsaturated fatty acid hydroperoxides in Synechocystis PCC 6803
Authors: Gaber Ahmed , Tamoi Masahiro , Takeda Toru , Nakano Yoshihisa , Shigeoka Shigeru
Published: July 2002

Here we isolated and characterized two genes (slr1171, slr1992) designated gpx‐1 and gpx‐2, respectively, encoding glutathione peroxidase (GPX)‐like proteins (Gpx‐1, Gpx‐2) from Synechocystis PCC 6803. The deduced amino acid sequences for gpx‐1 and gpx‐2 showed high similarity to those of GPX‐like proteins from higher plants and mammalian GPXs, respectively. Surprisingly, both recombinant proteins in Escherichia coli were able to utilize NADPH, but not reduced glutathione, as an electron donor and unsaturated fatty acid hydroperoxides or alkyl hydroperoxides as an acceptor. It seems accurate to refer to Gpx‐1 and Gpx‐2 as NADPH‐dependent GPX‐like proteins that serve as a new defense system for the reduction of unsaturated fatty acid hydroperoxides.
Phosphatidylinositol transfer proteins: requirements in phospholipase C signaling and in regulated exocytosis
Authors: Cockcroft Shamshad
Published: July 2002

Phosphatidylinositol transfer proteins (PITP) are abundant cytosolic proteins originally identified because of their ability to act in vitro as specific transporters of phosphatidylinositol or phosphatidylcholine between membranes. However, the cellular function of mammalian PITP has remained enigmatic till recently. Due to the development of reconstitution assays in cytosol‐depleted cells, PITP was found to be an essential component for phospholipase C‐mediated hydrolysis of PIP2 and for regulated exocytosis. The exact mechanism how PITP exerts its effects is not known but the PI binding/transfer activity of PITP can partly explain its cellular function. PITP would enable the local synthesis of PIP2 by delivering PI to specialized signaling sites.
Induction of chymase that forms angiotensin II in the monkey atherosclerotic aorta
Authors: Takai Shinji , Shiota Naotaka , Kobayashi Shigeyoshi , Matsumura Eiko , Miyazaki Mizuo
Published: July 2002

Chymase shows a catalytic efficiency in the formation of angiotensin (Ang) II. In the present study, the characterization and primary structure of monkey chymase were determined, and the pathophysiological role of chymase was investigated on the atherosclerotic monkey aorta. Monkey chymase was purified from cheek pouch vascular tissue using heparin affinity and gel filtration columns. The enzyme rapidly converted Ang I to Ang II (K
m=98 μM, k
cat=6203/min) but did not degrade several peptide hormones such as Ang II, substance P, vasoactive intestinal peptide and bradykinin. The primary structure, which was deduced from monkey chymase cDNA, showed a high homology to that of human chymase (98%). The mRNA levels of the aorta chymase were significantly increased in the atherosclerotic aorta of monkeys fed a high‐cholesterol diet. These results indicate that monkey chymase has a highly specific Ang II‐forming activity and may be related to the pathogenesis of atherosclerosis.
Characterization of three cDNA species encoding plastid RNA polymerase sigma factors in Arabidopsis thaliana: evidence for the sigma factor heterogeneity in higher plant plastids
Authors: Tanaka Kan , Tozawa Yuzuru , Mochizuki Nobuyoshi , Shinozaki Kazuo , Nagatani Akira , Wakasa Kyo , Takahashi Hideo
Published: July 2002

By database search analysis, we identified three Arabidopsis EST (Expression Sequence Tag) entries having similarity to eubacterial RNA polymerase sigma factors. cDNA clones corresponding to these partial sequences were isolated, and the complete nucleotide sequences were determined. All three sequences encode proteins highly homologous to cyanobacterial and plastid sigma factors, and the gene products have N‐terminal extensions which are assumed to function as plastid‐targeting transit peptides. Thus we have concluded that the gene products are RNA polymerase sigma factors of plastids, and named sigA, sigB and sigC, respectively. Expression of these genes was analyzed by RNA gel‐blot analysis and shown to be induced by illumination after a short‐term dark adaptation. This strongly suggests that light regulation of the nuclear encoded sigma factor genes is involved in light‐dependent activation of plastid promoters.
Missense mutations affecting a conserved cysteine pair in the TH domain of Btk
Authors: Vihinen Mauno , Nore Beston F. , Mattsson Pekka T. , Bäckesjö Carl-Magnus , Nars Martin , Koutaniemi Sanna , Watanabe Chiaki , Lester Tracy , Jones Allison , Ochs Hans D. , Smith C.I.Edvard
Published: July 2002

Tec family protein tyrosine kinases have in their N‐terminus two domains. The PH domain is followed by Tec homology (TH) domain, which consists of two motifs. The first pattern, Btk motif, is also present in some Ras GAP molecules. C‐terminal half of the TH domain, a proline‐rich region, has been shown to bind to SH3 domains. Mutations in Bruton's tyrosine kinase (Btk) belonging to the Tec family cause X‐linked agammaglobulinemia (XLA) due to developmental arrest of B cells. Here we present the first missense mutations in the TH domain. The substitutions affect a conserved pair of cysteines, residues 154 and 155, involved in Zn2+ binding and thereby the mutations alter protein folding and stability.
Activation of sphingosine kinase in pheochromocytoma PC12 neuronal cells in response to trophic factors
Authors: Rius Ricardo A , Edsall Lisa C , Spiegel Sarah
Published: July 2002

Nerve growth factor (NGF), basic fibroblast growth factor (bFGF), dibutyryl cAMP and forskolin, known differentiating agents for pheochromocytoma PC12 cells, induced sustained activation of sphingosine kinase, the enzyme responsible for the formation of the sphingolipid second messenger, sphingosine‐1‐phosphate, which mediates the mitogenic effects of certain growth factors. In contrast, epidermal growth factor and insulin‐like growth factor‐1, which stimulate proliferation of PC12 cells, induced only small and transient increases in sphingosine kinase activity. Of the growth factors examined, NGF was the most potent activator of sphingosine kinase, inducing a 4‐fold increase inVmax. Sphingosine kinase activity induced by NGF, but not FGF, was blocked by the protein kinase inhibitor K252a when added simultaneously, with minimal effect when added after 60 min. Thus, activation of sphingosine kinase may have an important role in neural differentiation.