bluuebunny/crossref_metadata_2025 huggingface dataset:
Streptozotocin, an inducer of NAD+ decrease, attenuates M‐potassium current inhibition by ATP, bradykinin, angiotensin II, endothelin 1 and acetylcholine in NG108‐15 cells
Authors: Higashida Haruhiro , Egorova Alla , Hoshi Naoto , Noda Mami
Published: April 2003

The M‐potassium current was inhibited by bath application of 100 μM ATP, 10 nM bradykinin, 100 nM angiotensin II and 100 nM endothelin 1 as well as by 10 μM acetylcholine in an ml‐muscarinic acetylcholine receptor‐transformed NG108‐15 cell line. The inhibition of M‐current was attenuated in cells pretreated with 5 mM streptozotocin for 5–15 h and restored by simultaneous incubation with 5 mM nicotinamide. The results suggest that signal transduction from these five different receptors to M channels shares a common pathway which is susceptible to a streptozotocin‐induced decrease in cellular NAD+ content.
Characterization of the binding of Fe(III) to F1ATPase from bovine heart mitochondria
Authors: Lippe Giovanna , Polizio Francesca , Di Pancrazio Francesca , Dabbeni-Sala Federica , Bortolotti Nadia , Desideri Alessandro , Mavelli Irene
Published: April 2003

The binding of Fe(III) to F1ATPase purified from beef heart mitochondria has been characterized by chemical analyses and EPR spectroscopy. F1ATPase binds 2 of Fe(III)/mol of protein selectively in the presence of saturating concentrations of ATP. In the absence of nucleotides or in the presence of either saturating ADP or limiting ATP concentrations, the enzyme binds 1 equivalent of Fe(III). F1ATPase pretreated with 5′‐p‐fluorosulfonylbenzoyladenosine, that selectively modifies the noncatalytic sites, binds only 1 mol of Fe(III)/mol of protein in the presence of either saturating ATP or ADP. Fe(III)‐loaded F1ATPase containing either 1 or 2 equivalents of Fe(III) show identical EPR signals at g = 4.3. The signals are not perturbed by the binding of nucleotides to the enzyme while they are altered by phosphate addition. These results indicate that F1ATPase contains two distinct Fe(III)‐binding sites, which differ from nucleotide‐binding sites, and that one of these sites is opened up for Fe(III) uptake by conformational changes induced by binding of ATP to the loose non‐catalytic site.
Differential distribution of classical inwardly rectifying potassium channel mRNAs in the brain: comparison of IRK2 with IRK1 and IRK3
Authors: Horio Yoshiyuki , Morishige Ken-ichirou , Takahashi Naohiko , Kurachi Yoshihisa
Published: April 2003

Distribution of IRK2 inwardly rectifying potassium channel mRNA in the mouse brain was studied using in situ hybridization histochemistry and compared with those of other classical inwardly rectifying potassium channel (IRK1 and IRK3) mRNAs. All these IRK channel mRNAs were detected in neurons, but not in glial cells. Their distribution patterns in the brain were, however, quite divergent: IRK2 mRNA was detected extremely high in granule cells of cerebellum, relatively high in motor trigeminal nucleus and moderate in olfactory bulb, piriform cortex, cerebral cortex, CA1 through CA3 regions of hippocampus, dentate gyrus and pontine nucleus. On the other hand, IRK1 mRNA was expressed throughout whole brain but in particular subsets of neurons, and IRK3 mRNA was in forebrain. Expression of these three IRK mRNAs overlapped in hippocampus, olfactory bulb, and cerebral cortex. This differential distribution of IRK mRNAs suggests that each of these channels has its specific function in regulation of the excitability of brain neurons.
Cellular processing of the neurotrophin precursors of NT3 and BDNF by the mammalian proprotein convertases
Authors: Seidah Nabil G. , Benjannet Suzanne , Pareek Sangeeta , Chrétien Michel , Murphy Richard A.
Published: April 2003

In order to define the enzymes responsible for the maturation of the precursors of brain‐derived neurotrophic factor (proBDNF) and neurotrophin‐3 (proNT3), we have analysed their biosynthesis and intracellular processing by the proprotein convertases furin, PC1, PC2, PACE4, PC5 and its isoform PC5/6‐B. In these studies, we utilized a vaccinia virus expression system in either BSC40 or the furin activity‐deficient LoVo cells. Results demonstrated that in both cells furin and, to a lesser extent, PACE4 and PC5/6‐B effectively process proBDNF and proNT3. Furthermore, we have determined that human proNT3 is sulfated, suggesting that processing of proNT3 occurs following the arrival of the precursor to the Trans Golgi Network.
Ozone induction of cytokine‐induced neutrophil chemoattractant (CINC) and nuclear factor‐κb in rat lung: inhibition by corticosteroids
Authors: Haddad E.-B. , Salmon M. , Koto H. , Barnes P.J. , Adcock I. , Chung K.F.
Published: April 2003

We determined in rat lung whether ozone exposure was associated with the expression of the chemokine, cytokine‐induced neutrophil chemoattractant (CINC), and of the transcription factor, NF‐κB. CINC mRNA expression peaked at 2 h after cessation of ozone exposure, and returned to basal levels by 24 h. DNA‐binding activity of NF‐κB showed a marked increase after ozone, maximal at 2 h. Dexamethasone inhibited CINC mRNA and NF‐κB expression, together with neutrophilic inflammation. Our data supports the concept that ozone leads to NF‐κB activation which increases CINC mRNA expression. These series of events could lead to neutrophilic inflammation.
DNA recognition by two mitoxantrone analogues: Influence of the hydroxyl groups
Authors: Bailly Christian , Routier Sylvain , Bernier Jean-Luc , Waring Michael J.
Published: April 2003

The clinically useful anticancer drug mitoxantrone intercalates preferentially into 5′‐(A/T)CG and 5′‐(A/T)CA sites on DNA. The 5,8 hydroxyl substituents on its anthracenedione chromophore are available to interact with the double helix. Footprinting experiments with two anthraquinone derivatives structurally related to mitoxantrone and ametantrone have been undertaken to assess the influence of the hydroxyl groups on the DNA recognition process. The results confirm that they do play a role in the recognition of preferred nucleotide sequences and suggest that the binding of anthraquinones to a 5′‐(A/T)CG site is dependent on the presence of the 5,8 hydroxyl substituents whereas binding to 5′‐(A/T)CA sites appears to proceed just as well without them.
Antibacterial activity of secretolytin, a chromogranin B‐derived peptide (614–626), is correlated with peptide structure
Authors: Strub Jean-Marc , Hubert Pierre , Nullans Gérard , Aunis Dominique , Metz-Boutigue Marie-Hélène
Published: April 2003

Amongst the chromogranin B (CGB) derived fragments naturally generated in bovine chromaffin granules and detected in the extracellular space, we recently identified a major peptide corresponding to the 614–626 sequence of CGB. This peptide, named secretolytin, shared an interesting sequence homology with the lytic domain of cecropins and displayed a potent antibacterial activity. The aim of the present study was to determine the structural features of secretolytin necessary for this biological activity. Our results suggest that an α‐helical amphipathic structure common to secretolytin, cecropins and pig myeloid antibacterial peptide may account for the antibacterial activity.
Identification of critical amino acids involved in α1‐β interaction in voltage‐dependent Ca2+ channels
Authors: De Waard Michel , Scott Victoria E.S. , Pragnell Marlon , Campbell Kevin P.
Published: April 2003

In voltage‐dependent Ca2+ channels, the α
1 and β subunits interact via two cytoplasmic regions defined as the Alpha Interaction Domain (AID) and Beta Interaction Domain (BID). Several novel amino acids for that interaction have now been mapped in both domains by point mutations. It was found that three of the nine amino acids in AID and four of the eight BID amino acids tested were essential for the interaction. Whereas the important AID amino acids were clustered around five residues, the important BID residues were more widely distributed within a larger 16 amino acid sequence. The affinity of the AIDA GST fusion protein for the four interacting β
1b
BID mutants was not significantly altered compared with the wild‐type β
1b
despite the close localization of mutated residues to disruptive BID amino acids. Expression of these interactive β mutants with the full‐length α
1A
subunit only slightly modified the stimulation efficiency when compared with the wild‐type β
1b
subunit. Our data suggest that non‐disruptive BID sequence alterations do not dramatically affect the β subunit‐induced current stimulation.
Formation and characterization of a transition state complex of Azotobacter vinelandii nitrogenase
Authors: Duyvis Martina G. , Wassink Hans , Haaker Huub
Published: April 2003

A stable complex is formed between the nitrogenase proteins of Azotobacter vinelandii, aluminium fluoride and MgADP. All nitrogenase activities are inhibited. The complex formation was found to be reversible. An incubation at 50°C recovers nitrogenase activity. The complex has been characterized with respect to protein and nucleotide composition and redox state of the metal‐sulphur clusters. Based on the inhibition by aluminium fluoride together with MgADP, it is proposed that a stable transition state complex of nitrogenase is isolated.
Adenosine deaminase affects ligand‐induced signalling by interacting with cell surface adenosine receptors
Authors: Ciruela Francisco , Saura Carles , Canela Enric I. , Mallol Josefa , Lluis Carmen , Franco Rafael
Published: April 2003

Adenosine deaminase (ADA) is not only a cytosolic enzyme but can be found as an ecto‐enzyme. At the plasma membrane, an adenosine deaminase binding protein (CD26, also known as dipeptidylpeptidase IV) has been identified but the functional role of this ADA/CD26 complex is unclear. Here by confocal microscopy, affinity chromatography and coprecipitation experiments we show that A1 adenosine receptor (A1R) is a second ecto‐ADA binding protein. Binding of ADA to A1R increased its affinity for the ligand thus suggesting that ADA was needed for an effective coupling between A1R and heterotrimeric G proteins. This was confirmed by the fact that ASA, independently of its catalytic behaviour, enhanced the ligand‐induced second messenger production via A1R. These findings demonstrate that, apart from the cleavage of adenosine, a further role of ecto‐adenosine deaminase on the cell surface is to facilitate the signal transduction via A1R.
Stable expression of insect GABA receptors in insect cell lines promoters for efficient expression of Drosophila and mosquito Rdl GABA receptors in stably transformed mosquito cell lines
Authors: Shotkoski Frank , Zhang Hai-Guang , Jackson Meyer B. , ffrench-Constant Richard H.
Published: April 2003

We are interested in establishing stably transformed insect cell lines efficiently expressing the insect γ‐aminobutyric acid (GABA) receptor subunit gene Resistance to dieldrin or Rdl. In order to facilitate this we utilized a system based on stable transformation of Aedes albopictus mosquito cell lines using the dihydrofolate reductase (dhfr) gene as a selectable marker. Here we report the production of stable mosquito cell lines carrying high copy numbers of Rdl genes from both Drosophila and Aedes aegypti mosquitoes and the subsequent high efficiency expression of functional GABA gated chloride ion channels. We also used this system to compare the activity of a range of immediate early baculovirus promoters in mosquito cell culture and demonstrate that IE1 promoter constructs work efficiently across insect species. Results are discussed in relation to the potential use of these constructs in the genetic transformation of non‐Drosophilid insects.
Functional expression of the plant K+ channel KAT1 in insect cells
Authors: Marten Irene , Gaymard Frederic , Lemaillet Guy , Thibaud Jean-Baptiste , Sentenac Hervé , Hedrich Rainer
Published: April 2003

Following the biophysical analysis of plant K+ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1 and KST1 have recently been identified on the molecular level. Among them, we focused on the expression and characterization of the Arabidopsis thaliana K+ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1‐recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward‐rectifying, K+‐selective (impermeable to Na+ and even NH4
+) ionic conductance in whole‐cell patch‐clamp recordings. A voltage threshold as low as −60 to −80 mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca2+ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post‐translational modification and assembly of a green inward rectifier.
Antioxidants differentially affect nuclear factor κB‐mediated nitric oxide synthase expression in vascular smooth muscle cells
Authors: Hecker Markus , Preiβ Christiane , Schini-Kerth Valérie B. , Busse Rudi
Published: April 2003

Increased ‘oxidative stress’ resulting in the activation of nuclear factor κB (NF‐κB) is thought to play a crucial role in the cytokine‐mediated expression of the inducible isoform of nitric oxide synthase (iNOS) in different cell types. Therefore, the effects of four different antioxidants, carbocromen, chrysin, 3,4‐dichloroisocoumarin (DCI) and N‐acetylserotonin (NAS), on iNOS expression were investigated in vascular smooth muscle cells (VSMC). All antioxidants strongly reduced the phorbol ester‐stimulated superoxide anion formation in native VSMC. Carbocromen (200 μM) and chrysin (50 μM) had no effect, while NAS (1 mM) abolished the increase in nitrine production and iNOS protein abundance in cultured VSMC exposed to interleukin‐1β (IL‐1β, 60 U/ml) or the adenylyl cyclase activator forskolin (10 μM). DCI also revealed a marked inhibitory effect in IL‐1β‐stimulated VSMC, but was less effective in cells treated with forskolin. DCI, but not NAS, also suppressed the activation of NF‐κB in VSMC exposed to IL‐1β, while no significant NF‐κB activation was detected in forskolin‐treated cells. These findings demonstrate that antioxidants differentially affect iNOS expression in VSMC both at the transcriptional level by preventing the activation of NF‐κB and at the post‐transcriptional level, presumably by promoting iNOS mRNA or protein degradation. They also suggest that reactive oxygen intermediates do not play a role in the activation of NF‐κB by IL‐1β in VSMC, and that transcripton factors other than NF‐κB mediate the induction of iNOS expression by elevating the intracellular concentration of cyclic AMP.
Calcium‐dependent inhibition of in vitro thin‐filament motility by native titin
Authors: Kellermayer Miklós S.Z. , Granzier Henk L.
Published: April 2003

Titin (also known as connectin) is a giant filamentous protein that spans the distance between the Z‐ and M‐lines of the vertebrate muscle sarcomere and plays a fundamental role in the generation of passive tension. Titin has been shown to bind strongly to myosin, making it tightly associated to the thick filament in the sarcomere. Recent observations have suggested the possibility that titin also interacts with actin, implying further functions of titin in muscle contraction. We show — using in vitro motility and binding assays — that native titin interacts with both filamentous actin and reconstituted thin filaments. The interaction results in the inhibition of the filaments' in vitro motility. Furthermore, the titin‐thin filament interaction occurs in a calcium‐dependent manner: increased calcium results in enhanced binding of thin filaments to titin and greater suppression of in vitro motility.
Surfactosomes: a novel approach to the reconstitution and 2‐D crystallisation of membrane proteins
Authors: Reviakine Ilya , Stoylova Svetla , Holzenburg Andreas
Published: April 2003

The formation of vesicle‐like structures (termed surfactosomes) and lamellar sheets from solutions containing ammonium perfluoroocanoate (APFO) is illustrated using conventional and cryo‐transmission electron microscopy. It is shown how this detergent can be used for the solubilisation, reconstitution, and 2‐D crystallisation of membrane proteins as demonstrated for the major protein of the membrane sector of the V‐type H+‐ATPase (16‐kDa protein). Electron microscopical analysis of 2‐D crystals of the 16‐kDa protein (a = b = 13.0 ± 0.2 nm withγ= 90° and p4 projection symmetry) revealed a unit cell comprising four dimeric complexes of the 16‐kDa protein the significance of which is discussed.
Prostaglandin E receptor EP3γ isoform, with mostly full constitutive Gi activity and agonist‐dependent Gs activity
Authors: Negishi Manabu , Hasegawa Hiroshi , Ichikawa Atsushi
Published: April 2003

We recently demonstrated that two exclusively Gi‐coupled isoforms of the mouse EP3 receptor, EP3α and β, with different carboxyl‐terminal tails, differed in agonist‐independent constitutive Gi activity, and the carboxyl‐terminal tail‐truncated receptor showed full constitutive activity (Hasegawa, H., Negishi, M., and Ichikawa, A. (1996) J. Biol. Chem. 271, 1857–1860). Here we further examined Gi and Gs activities of the third isoform, EP3γ, coupled to both Gi and Gs. The EP3γ receptor showed mostly full constitutive Gi activity and agonist‐dependent Gs activity. The truncated receptor also showed agonist‐dependent Gs activity, but the level was lower than that of the EP3γ receptor. Thus, the carboxyl‐terminal tail would differentially regulate Gi and Gs activities of the EP3 receptor.
Differential regulation of Raf isozymes by growth versus differentiation inducing factors in PC12 pheochromocytoma cells
Authors: Wixler Viktor , Smola Ute , Schuler Manuela , Rapp Ulf
Published: April 2003

PC12 pheochromocytoma cells possess four known MEK activators: A‐, B‐, c‐Raf‐1 and MEKK. In order to examine whether differentiation factors or growth factors have a Raf isozyme preference for activation of the mitogenic cytoplasmic Raf‐MEK‐MAPK protein kinase cascade, the activation kinetics of these enzymes in response to epidermal growth factor (EGF) and nerve growth factor (NGF) were compared. An initial activation of all three Raf kinases was noticed, but only A‐ and B‐Raf showed sustained activation by NGF, which was not seen after EGF treatment. Furthermore, expression of oncogenic versions of all three Raf kinases as well as a potentially Raf‐independent MEK activator, v‐Mos, leads to activation of MAPK and to differentiation of PC12 cells. These data suggest a differential regulation of Raf kinases and that probably no alternative Raf substrates are involved in differentiation processes of PC12 cells.
Human guanylate kinase (GUK1): cDNA sequence, expression and chromosomal localisation
Authors: Fitzgibbon Jude , Katsanis Nicholas , Wells Dagan , Delhanty Joy , Vallins William , Hunt David M.
Published: April 2003

Guanylate kinase (GK) catalyses the conversion of GMP to GTP as part of the cGMP cycle. In mammalian phototransduction, this cycle is essential for the regeneration of cGMP following its hydrolysis by phosphodiesterase. Mutations in different parts of this signalling cascade lead to retinal degeneration in humans. Protein studies have localized a locus for GK to a region of human chromosome 1 that also contains an autosomal recessive form of retinitis pigmentosa (RP12) and Usher's type 11a (USH2A). We report the sequence of this human GK (GUK1) and a further refinement of its localization to 1q32‐41, placing it in the same interval as USH2A.
Specific transformation abolishes cyclin D1 fluctuation throughout the cell cycle
Authors: Pusch Oliver , Soucek Thomas , Wawra Edgar , Hengstschläger-Ottnad Elke , Bernaschek Gerhard , Hengstschläger Markus
Published: April 2003

We analysed cyclin D1 mRNA and protein expression in several different cell types after separating these cells according to their different cell cycle phases by centrifugal elutriation. In normal human and rat fibroblasts cyclin D1 expression is high in early to mid G1 and decreases about 6–7 fold before onset of replication. It has been demonstrated that specific transforming events, such as loss of functional retinoblastoma protein, overexpression of c‐myc, and transfection with the human papillomavirus oncoproteins E6 and E7 cause transcriptional downregulation of cyclin D1 expression in logarithmically growing cells. We found that such transformed cells exhibit loss of the cell cycle‐dependent cyclin D1 fluctuation accompanied with reduced upregulation of cyclin D1 in G1 phase. The data presented here provide the experimental support for a recently suggested model involving the function of the retinoblastoma protein in cyclin D1 cell cycle regulation.
Circadian rhythm in the Ca2+‐inhibitable adenylyl cyclase activity of the rat striatum
Authors: Chern Yijuang , Lee Eminy H.Y. , Lai Hsing-Lin , Wang Hai-Long , Lee Yi-Chao , Ching Yung-Hao
Published: April 2003

In the present study, we demonstrate that the Ca2+‐inhibitable adenylyl cyclase (AC) activity in the striatum exhibits a daily oscillation with a peak occurring around 10:00 h. A circadian fluctuation of the AC activity evoked by an A2a adenosine‐selective agonist was also observed. Intrastriatal injection of an A2a‐selective adenosine agonist or antagonist during the interval in which the Ca2+‐inhibitable AC activity was at its peak resulted in a more significant alteration of locomotor activity than those observed at a later interval. The marked circadian variation in the Ca2+‐inhibitable AC activity in the striatum appears to cause a circadian fluctuation in the action of at least one neuromodulator.
Stimulation of mitogen activated protein kinase by LDL and oxLDL in human U‐937 macrophage‐like cells
Authors: Deigner Hans-Peter , Claus Ralf
Published: April 2003

Mitogen activated protein kinase in extracts of U‐937 macrophage‐like cells was stimulated by LDL and oxLDL. A maximum value (161% of the basal phosphotransferase activity) was obtained after 6 min exposure to oxidized LDL (27 μ/ml) using APRTPGGRR peptide substrate. The activatory effect was more pronounced (LDL 181%, oxLDL 201%) when MAPK of stimulated cells was immunoprecipitated with anti‐p42MAPK antibodies and phosphotransferase activity was assayed in immune complexes. Stimulation produced by oxLDL was inhibited by poly I, fucoidan, dextran sulfate and by the MAPKK inhibitor PD 098059 but not by PMA‐mediated depletion of PKC or by pre‐treatment with chloroquine or with pertussis toxin. These results suggest a direct mitogenic effect of LDL which, in the case of oxLDL, is dependent on scavenger receptor ligation but not on G‐protein mediated or PKC‐dependent signal transduction.
Shortcut of the photosynthetic electron transfer in a mutant lacking the reaction center‐bound cytochrome subunit by gene disruption in a purple bacterium, Rubrivivax gelatinosus
Authors: Nagashima Kenji V.P. , Shimada Keizo , Matsuura Katsumi
Published: April 2003

A mutant lacking the reaction center‐bound cytochrome subunit was constructed in a purple photosynthetic bacterium, Rubrivivax gelatinosus IL144, by inactivation of the cytochrome gene. Photosynthetic growth of the C244 mutant strain occurred at approximately half the rate of the wild‐type strain. Although mutagenesis resulted in a greatly reduced amount of membrane‐bound cytochromes c, illumination induced cyclic electron transfer and the generation of membrane potential in the mutant as observed in the wild‐type strain. These findings are consistent with previous observations that the cytochrome subunit is absent in the reaction center complex in some species of purple bacteria and that the biochemical removal of the subunit did not significantly affect the in vitro electron transfer from the soluble cytochrome c to the photosynthetic reaction center. These results suggest that the cytochrome subunit in purple bacteria is not essential for photosynthetic electron transfer and growth, even in those species generally containing the subunit.
Keratin 9 point mutation in the pedigree of epidermolytic hereditary palmoplantar keratoderma perturbs keratin intermediate filament network formation
Authors: Kobayashi Setsu , Tanaka Toshihiro , Matsuyoshi Norihisa , Imamura Sadao
Published: April 2003

Keratins form an intracellular keratin filament network in keratinocytes. Point mutations in the epidermal keratins could lead to the disruption of keratin filament formation, developing skin diseases such as epidermolytic hereditary palmoplantar keratoderma (EHPPK). We found a G to A transition in keratin 9 (K9) cDNA, resulting in the substitution of glutamine for arginine at 162, in all patients of a pedigree of EHPPK. Transfection into MDCK cells and DJM‐1 cells revealed that the plasmid CMX vector containing normal keratin 9 cDNA showed normal keratin network formation, whereas the vector with a G to A point mutated keratin 9 cDNA showed disrupted keratin filaments with droplet formation in the cells. These results indicate that the point mutation seen in our patients had a dominant‐negative effect on keratin network formation.
Molecular cloning of a levocabastine‐sensitive neurotensin binding site
Authors: Chalon Pascale , Vita Natalio , Kaghad Mourad , Guillemot Marie , Bonnin Jacques , Delpech Bruno , Le Fur Gerard , Ferrara Pascual , Caput Daniel
Published: April 2003

A search for sequences homologous to the neurotensin receptor cDNA in a rat hypothalamic library has identified a novel neurotensin receptor (NTR‐2). The 1539 bp cDNA encodes a 416 amino acid protein and shows highest homology to the previously cloned neurotensin receptor (NTR‐1) (64% homology and 43% identity). Binding and pharmacological studies demonstrate that NTR‐2 expressed in COS cells recognizes neurotensin (NT) with high affinity as well as several other agonists and antagonists. However, a fundamental difference was found; unlike NTR‐1, NTR‐2 recognizes, with high affinity, levocabastine, a histamine H1 receptor antagonist previously shown to compete with NT for low‐affinity binding sites in brain.
Phosphorylation of the N‐terminal domain of Xenopus TATA‐box binding protein by DNA‐dependent protein kinase depends on the C‐terminal core domain
Authors: Labhart Paul
Published: April 2003

DNA‐dependent protein kinase (DNA‐PK) has been shown to phosphorylate several transcription factors in vitro, suggesting that this nuclear enzyme — in addition to its role in DNA repair and recombination — may be involved in transcriptional regulation. In the typical mechanism the DNA‐bound kinase phosphorylates a substrate that is bound to the same DNA molecule. Here I report that the Xenopus TATA‐box binding protein (xTBP) is hyperphosphorylated by DNA‐PK in vitro. The phosphorylation is in the N‐terminal domain of the protein but depends fully on the presence of the C‐terminal core domain.
Substrate residues N‐terminal to the cleavage site of botulinum type B neurotoxin play a role in determining the specificity of its endopeptidase activity
Authors: Wictome Matthew , Rossetto Ornella , Montecucco Cesare , Shone Clifford C.
Published: April 2003

Clostridium botulinum type B neurotoxin is a highly specific zinc‐endopeptidase which cleaves vesicle‐associated membrane protein (VAMP/synaptobrevin), a critical component of the vesicle docking/fusion mechanism. In this study, substrate residues flanking the N‐terminal side of the cleavage site are shown to play a key role in enzyme substrate recognition. Two aspartate residues in this region are identified as critical determinants of the neurotoxin's specificity. These findings are discussed in relation to the mechanism by which botulinum type B neurotoxin cleaves its substrate.
Possible nystatin‐protein interaction in yeast plasma membrane vesicles in the presence of ergosterol. A Förster energy transfer study
Authors: Opekarová Miroslava , Urbanová Petra , Konopásek Ivo , Kvasnička Peter , Strzalka Kazimierz , Sigler Karel , Amler Evžen
Published: April 2003

The Förster energy transfer from tryptophan residues of membrane proteins to nystatin was measured in reconstituted yeast plasma membrane vesicles free of, or doped with, ergosterol. We wanted to elucidate whether the functional change of membrane transport proteins from H+ symporters to facilitators, observed in ergosterol‐containing plasma membrane vesicles on addition of nystatin [Opekarová and Tanner (1994) FEBS Lett. 350, 46–50], is reflected in altered protein‐nystatin relations within the membrane. Both frequency‐domain and time‐domain time‐resolved fluorescence spectroscopy showed that in the presence of ergosterol nystatin is located much closer to membrane proteins than in its absence.
Very low density lipoprotein receptor binds apolipoprotein E2/2 as well as apolipoprotein E3/3
Authors: Takahashi Sadao , Oida Koji , Ookubo Minoru , Suzuki Jinya , Kohno Mitsuyuki , Murase Toshio , Yamamoto Tokuo , Nakai Tsuguhiko
Published: April 2003

The VLDL receptor, a newly identified lipoprotein receptor, recognizes apoE containing lipoproteins. The human VLDL receptor was overexpressed in ldlA‐7, a mutant Chinese hamster ovary cells lacking LDL receptors. Each VLDL obtained from a normolipidemic subject with two ε3 or ε2 alleles similarly competed for the binding of radiolabeled rabbit β‐VLDL to the VLDL receptors. The anti‐apoE monoclonal antibody 1D7, which inhibited binding of apoE3 to the LDL receptors, failed to compete for the binding of VLDL (apoE3 or apoE2) to the VLDL receptors. Results indicate that the binding site of apoE on the VLDL receptor may differ from its binding site on the LDL receptor.
The stiffness of the crossbridge is a function of the intrinsic protein osmotic pressure generated by the crossbridge itself
Authors: Grazi Enrico , Magri Ermes , Schwienbacher Christine , Trombetta Giorgio
Published: April 2003

A model is presented that makes it possible to determine the stiffness of the crossbridge from protein osmotic stress experiments. The model was elaborated while studying the osmotic properties of F‐actin and of myosin subfragment‐1 F‐actin. These studies showed that the elastic modulus by bending of the monomer is directly related to the intrinsic protein osmotic pressure of the system. At a protein osmotic pressure of 1.8 × 105dynes/cm2, the physiological protein osmotic pressure of frog skeletal muscle, it was found that the elastic moduli by bending of the monomer in F‐actin and in the myosin subfragment‐1 decorated F‐actin are 6.5 × 107 and 3.3 × 108 dynes/cm2, respectively. The value of the elastic modulus by bending of the monomer in the myosin subfragment‐1 decorated F‐actin compares favorably with the values of the elastic modulus by stretching determined in skeletal muscle fibres.
Calcium/calmodulin‐dependent protein kinase II phosphorylates tau at Ser‐262 but only partially inhibits its binding to microtubules
Authors: Singh Toolsee J. , Wang Jian-Zhi , Novak Michal , Kontzekova Eva , Grundke-Iqbal Inge , Iqbal Khalid
Published: April 2003

PHF‐tau, which is phosphorylated at 10 Ser/Thr‐Pro and 11 non‐Ser/Thr‐Pro sites, is unable to promote microtubule assembly. Phosphorylation of the non‐Ser/Thr‐Pro site, Ser‐262, is reported to be primarily responsible for this. The identities of kinase(s) responsible for Ser‐262 phosphorylation are still to be clarified. In this study we have used the monoclonal antibody 12E8, which recognizes P‐Ser‐262 and P‐Ser‐356 on tau, to survey different kinases for their abilities to phosphorylate Ser‐262 on human tau 3L (tau410). In decreasing order of effectiveness we found that Ser‐262 and Ser‐356 phosphorylation can be catalyzed by CaM kinase II ⪢ C‐kinase ⪢ GSK‐3 ≌ A‐kinase ⪢ CK‐1. CaM kinase II and C‐kinase were shown to phosphorylate both Ser‐262 and Ser‐356. The binding of tau to taxol‐stabilized microtubules was decreased by 35 and 42% after phosphorylation by CaM kinase II and C‐kinase, respectively. Of the fraction of tau that bound to microtubules, about 50% was phosphorylated at Ser‐262 and Ser‐356. These results suggest that Ser‐262 and Ser‐356 are very good substrates for CaM kinase II but their phosphorylations are not sufficient to achieve maximal inhibition of tau binding to microtubules.
Single point mutations in Met4p impair the transcriptional repression of MET genes in Saccharomyces cerevisiae
Authors: Omura Fumihiko , Fujita Atsushi , Shibano Yuji
Published: April 2003

Transcription of MET genes in Saccharomyces cerevisiae depends on a transcriptional activator, the MET4 gene product (Met4p). Using in vitro mutagenesis, we isolated two mutant MET4 alleles encoding [Pro215]Met4p and [Ser156]‐Met4p. These mutations impeded Met4p's responsiveness to methionine in the media, and yeast cells carrying mutant alleles exhibited enhanced transcription of MET genes under repressing conditions. The enhanced transcription was dependent on the CBF1 gene, but did not compete with an excess of wild‐type Met4p, suggesting that some changes in the affinity of Met4p to other factors might be involved in S‐adenosylmethionine‐mediated transcriptional regulation
The putatively protective Onchocerca volvulus neuronal protein E1 is a member of the death domain protein family
Authors: Erttmann K.D. , Domeyer A. , Gallin M.
Published: April 2003

Here we show that E1, an ankyrin‐related, potentially protective, neuronal protein of the human filarial nematode Onchocerca volvulus contains a death domain (DD), most similar to that of human Mort1/FADD (39% identity). In addition, sequence comparison of E1 to its homologue from Litomosoides sigmodontis and to Caenorhabditis elegans ankyrin defines two further putative functional domains. One represents the end of the spectrin‐binding domain of ankyrins, the other an unique domain, most highly conserved between these nematodes, containing a calpain sequence motif. Thus, E1 may be involved in apoptosis, raising the possibility that protection against this parasitic helminth may be induced by apoptotic processes.
Cloning and expression of human mitochondrial deoxyguanosine kinase cDNA
Authors: Wang Liya , Hellman Ulf , Eriksson Staffan
Published: April 2003

Mammalian mitochondrial deoxyguanosine kinase (dGK) is responsible for phosphorylation of purine deoxyribonucleosides in the mitochondrial matrix. Using a RT‐PCR‐generated probe, based on amino acid sequence information from proteolytic fragments of purified bovine dGK, we have cloned a cDNA from a human brain cDNA library that encodes a 30 kDa protein. The deduced amino acid sequence of this protein included the sequence of all six peptides isolated and sequenced from purified dGK. Expression and purification of recombinant protein from induced Escherichia coli extracts revealed that it catalyses efficient phosphorylation of dGuo, arabinosyl guanine, dAdo, 2‐chloro‐2′‐deoxyadenosine and dIno similar to purified dGK. Northern blot analysis demonstrated one dominant positive mRNA of 1.35 kb and it was found in several tissues at similar levels. The coding sequence of dGK showed 46% identity to the coding sequence of cytosolic deoxycytidine kinase, and conserved sequence motifs among the known deoxynucleoside kinase were identified.
The construction and characterization of an effective transpositional system based on IS30
Authors: Farkas Tibor , Kiss János , Olasz Ferenc
Published: April 2003

We constructed an in vivo system to detect transpositional rearrangements induced by the insertion sequence IS30. The transposase protein expressed from the transposase producer plasmids catalyzed rearrangements on different target sequences presented in trans. High yields, up to 83%, of transpositional frequencies were observed. The frequency of rearrangements correlated with the amount of transposase protein produced and the attractivity of the target sequences. Alteration in the frequency of transposition was observed in the recA
− E. coli strains JM109 and TG2. Remarkable structural and functional analogy was found with site‐specific recombination systems.
An electrophysiological study of calcium entry during normal human T‐lymphocyte activation
Authors: Ricci Riccardo , Buffelli Mario , Riviera Anna P. , Cangiano Alberto
Published: April 2003

Our aim was to observe whether normal human T‐cells respond to mitogenic stimulation with large whole‐cell inward currents (composed of identifiable single‐channel contributions) when [Ca2+]i is not markedly lowered but instead kept normal or moderately low, as has been reported in human leukaemic Jurkat T‐cell line and T‐cell clones [Kuno et al. (1986) Nature 323, 269–273; Kuno and Gardner (1987) Nature 326, 301–304; Gardner (1990) Annu. Rev. Immunol. 8, 231–252]. Whole‐cell patch recordings showed no such currents in cells otherwise normally responding to depolarisation with the macroscopic I
K described in T‐lymphocytes and thus deemed viable, in agreement with the notion that Ca2+ influx in normal T‐cells enterily depends on depletion of internal stores [Putney (1986) Cell Calcium 7, 1–12; Putney (1990) Cell Calcium 11, 611–624].
The weaver mutation changes the ion selectivity of the affected inwardly rectifying potassium channel GIRK2
Authors: Tong Yanhe , Wei Jianjun , Zhang Shengwen , Strong Judith A. , Dlouhy Stephen R. , Hodes M.E. , Ghetti Bernardino , Yu Lei
Published: April 2003

The weaver mutation in mice has recently been identified as a single base‐pair mutation in the Girk2 gene, which encodes a G‐protein‐activated inwardly rectifying potassium channel, GIRK2. The mutation results in a Gly to Ser substitution at residue 156, in the putative pore‐forming region of the potassium channel. In the present study, we used Xenopus oocytes to express mutant GIRK2, and to characterize the effects of the mutation on the channel. The mutation results in a loss of the normal high selectivity for K+ over Na+, with little effect on other channel properties such as activation by the mu opioid receptor. The resulting increase in basal Na+ permeability causes a marked depolarization of oocytes expressing the mutant GIRK2 protein. This result was observed even when the mutant GIRK2 was coexpressed with GIRK1, a situation more analogous to that seen in vivo. Thus, the increased Na+ permeability and resulting depolarization may contribute to the pathology of cerebellar granule cells and substantia nigra dopaminergic neurons observed in the weaver mice.
Characterization of an Arabidopsis thaliana cDNA encoding an S‐adenosylmethionine‐sensitive threonine synthase Threonine synthase from higher plants
Authors: Curien Gilles , Dumas Renaud , Ravanel Stéphane , Douce Roland
Published: April 2003

An Arabidopsis thaliana cDNA encoding an Sadenosylmethionine‐sensitive threonine synthase (EC 4.2.99.2) has been isolated by functional complementation of an Escherichia coli mutant devoid of threonine synthase activity. Threonine synthase from A. thaliana was shown to be synthesized with a transit peptide. The recombinant protein is activated by Sadenosylmethionine in the same range as the plant threonine synthase and evidence is presented for an involvement of the N‐terminal part of the mature enzyme in the sensitivity to Sadenosylmethionine.
Prevention of necrosis and activation of apoptosis in oxidatively injured human myeloid leukemia U937 cells
Authors: Palomba Letizia , Sestili Piero , Cattabeni Flaminio , Azzi Angelo , Cantoni Orazio
Published: April 2003

A 3 h exposure to 1 mM H2O2 followed by 6 h post‐challenge growth in peroxide‐free medium induces necrosis in U937 cells. Addition of the poly(ADP‐ribose)polymerase inhibitor 3‐aminobenzamide during recovery prevents necrosis and triggers apoptosis, as shown by the appearance of apoptotic bodies, extensive blebbing and formation of multimeric DNA fragments as well as 50 kb double stranded DNA fragments. Thus, the same initial damage can be a triggering event for both apoptotic and necrotic cell death. Furthermore, necrosis does not appear to be a passive response to overwhelming damage.
Unassembled subunits of the photosynthetic oxygen‐evolving complex present in the thylakoid lumen are long‐lived and assembly‐competent
Authors: Akiko Hashimoto , Yasusi Yamamoto , Theg Steven M.
Published: April 2003

Physiologically healthy pea chloroplasts contain unassembled, soluble subunits of the oxygen‐evolving complex in the thylakoid lumen. We report that the lifetimes of two of these subunits, both on and off the membrane, exceed 8 h in vitro. We also demonstrate that each of the subunits present in the thylakoid lumen is competent for assembly into the membrane‐bound complex. These data are consistent with the postulate that the soluble lumen‐resident subunits play a role in photosystem II homeostasis. We also demonstrate that the reconstitution of the 33 kDa subunit is inhibited by extremely low concentrations of Triton X‐100, suggesting that hydrophobic interactions are involved in the binding of this subunit to the photosystem II reaction center.
Nitric oxide donor induces HSP70 accumulation in the heart and in cultured cells
Authors: Malyshev Igor Yu. , Malugin Alexander V. , Golubeva Lidia Yu. , Zenina Tatiyana A. , Manukhina Eugenia B. , Mikoyan Vasak D. , Vanin Anatoly F.
Published: April 2003

As our group has shown, the NO‐synthase inhibitor L‐NNA decreased 2–3 times heat shock‐induced synthesis of the heat shock protein HSP70 (FEBS Lett. 370 (1995) 159–162). It was suggested that NO is involved in such induction. In the present study, it was found that (1) injection of the NO donor dinitrosyl iron complex (DNIC) into rats results in accumulation of HSP70 in the heart; (2) heat shock is accompanied by increased generation of NO (EPR assay) and HSP70 accumulation in cultured cells; (3) DNIC induces HSP70 accumulation in cultured cells not exposed to heat shock.
Cell type specific regulation of COUP‐TF II promoter activity
Authors: Soosaar A. , Neuman K. , Nornes H.O. , Neuman T.
Published: April 2003

COUP‐TF family orphan receptors regulate activity of ligand‐activated nuclear hormone receptors or function independently in the regulation of gene expression. COUP‐TF II has a complex expression pattern suggesting that different mechanisms are involved in the regulation of its expression. We isolated the 5′ regulatory region of the mouse COUP‐TF II gene and demonstrated that the basal promoter is localized in a ‐200 by region 5′ from the transcription start site. All‐trans retinoic acid and dibutyryl cyclic AMP have cell type specific effects on COUP‐TF II promoter activity. The effect of cyclic AMP is mediated by the cyclic AMP response element that is localized 74 nucleotides upstream from the major transcriptional start. In vitro promoter analyses also demonstrated that the effect of all‐trans RA is not directly mediated by the binding of RARs or RXRs to the promoter sequence.
Effects of sequence contexts on misincorporation of nucleotides opposite 2‐hydroxyadenine
Authors: Kamiya Hiroyuki , Kasai Hiroshi
Published: April 2003

Twelve oligonucleotides containing 2‐hydroxyadenine (2‐OH‐Ade) with different neighboring bases were used as templates in DNA polymerase reactions, and the effects of the sequence contexts were investigated. DNA polymerases α and β inserted dTMP and dCMP opposite 2‐OH‐Ade in most of the oligonucleotides tested. The Klenow fragment of DNA polymerase I primarily incorporated dTMP and dGMP. Effects of the 5′‐flanking base of 2‐OH‐Ade was found when the 3′‐flanking base of 2‐OH‐Ade was A or C. Incorporation of dAMP occurred when the oxidized base was located in a 5′ ‐TA∗A‐ 3′ (A∗ represents 2‐OH‐Ade) sequence. These results suggest that the formation of 2‐OH‐Ade in DNA may induce all the mutations involving A (A → G transition, and A → T and A → C transversions) in cells.
Cyclic GMP potentiates phenylephrine but not cyclic ADP‐ribose‐evoked calcium release from rat lacrimal acinar cells
Authors: Jorgensen Tino D. , Dissing Steen , Gromada Jesper
Published: April 2003

In the present study, we describe a role for cyclic GMP (cGMP) in the signalling pathway that leads from α‐adrenergic receptor activation to intracellular Ca2+ mobilization in rat lacrimal acinar cells. The α‐adrenergic agonist, phenylephrine, stimulates intracellular Ca2+ release which is blocked by inhibitors of guanylate cyclase and cGMP‐dependent protein kinase Ia. The membrane‐permeable cGMP analogues, dibutyryl‐cGMP and 8‐bromo‐cGMP, potentiate (≈5‐fold) the Ca2+ response to submaximal phenylephrine stimulation. In contrast, the same cGMP analogues have no effect on cyclic ADP‐ribose‐evoked Ca2+ release from permeabilized lacrimal acinar cells. Collectively, these findings suggest that cGMP, via cGMP‐dependent protein kinase Iα, is required for intracellular Ca2+ release following α‐adrenergic receptor activation in lacrimal acinar cells.
A fungal metabolite mediates degradation of non‐phenolic lignin structures and synthetic lignin by laccase
Authors: Eggert Claudia , Temp Ulrike , Dean Jeffrey F.D. , Eriksson Karl-Erik L.
Published: April 2003

Lignin peroxidase is generally considered to be a primary catalyst for oxidative depolymerization of lignin by white‐rot fungi. However, some white‐rot fungi lack lignin peroxidase. Instead, many produce laccase, even though the redox potentials of known laccases are two to directly oxidize non‐phenolic components of lignin. Pycnoporus cinnabarinus is one example of a laccase‐producing fungus that degrades lignin very efficiently. To overcome the redox potential barrier, P. cinnabarinus produces a metabolite, 3‐hydroxyanthranilate that can mediate the oxidation of non‐phenolic substrates by laccase. This is the first description of how laccase might function in a biological system for the complete depolymerization of lignin.
A structural tree for α‐helical proteins containing α‐α‐corners and its application to protein classification
Authors: Efimov A.V.
Published: April 2003

A structural tree for α‐helical proteins and domains including α‐α‐corners has been constructed. The α‐α‐corner is taken as a root structure of the tree. The larger protein structures are obtained by stepwise addition of a‐helices to the root α‐α‐corner taking into account a restricted set of rules inferred from known principles of protein structure. The protein structures that can be obtained in this way are grouped into one structural class and those found in branches of the tree into subclasses.
Levels of endogenous cytokinins, indole‐3‐acetic acid and abscisic acid during the cell cycle of synchronized tobacco BY‐2 cells
Authors: Redig Pascale , Shaul Orit , Inzé Dirk , Van Montagu Marc , Van Onckelen Harry
Published: April 2003

Correlation between cell cycle progression and endogenous levels of plant hormones was studied in synchronized tobacco BY‐2 cell suspension cultures. Sixteen different cytokinins, indole‐3‐acetic acid (IAA) and abscisic acid (ABA) were extracted using solid‐phase anion exchange chromatography in combination with immunoaffinity purification, and quantified by mass spectrometry. No significant correlation could be identified for IAA and ABA. In contrast, there were sharp peaks in the levels of specific cytokinins (zeatin‐ and dihydrozeatin‐type) at the end of the S phase and during mitosis. The levels of other cytokinins analyzed, including zeatins N‐ and O‐glucosides, remained low, suggesting that the increased amounts of their corresponding non‐glucosylated from resulted from de novo synthesis. These findings suggest that zeatin‐ and dihydrozeatin‐type cytokinins might play a specific regulatory role in the progression of the plant cell cycle. One hypothesis to explain cytokinin action is based on a specific interaction with kinases that regulate cell cycle progression, as has been recently shown for the cytokinin analogue olomoucine.