bluuebunny/crossref_metadata_2025 huggingface dataset:
Contos de fada e pós-modernidade: Reflexões sobre o oximoro do reencantamento desencantado // Fairy tales and postmodernity: Reflections about the disenchanted reenchantment oxymoron
Authors: Tonin Juliana , Azubel Larissa
Published: July 2021

RESUMO

O presente artigo busca compreender as versões cinematográficas infantis de contos de fada, lançadas no Brasil entre 2000 e 2012, como sintomas de uma época marcada, principalmente, pelas noções do encantamento e do desencantamento. A estética do realismo adentra os contos e promove metamorfoses, demonstrando uma migração das histórias do contexto moderno para o pós-moderno: as expectativas se apagam e os desejos se voltam às esferas do possível, do tangível e do concreto, daquilo que os seres ordinários também podem almejar. Procura-se, portanto, analisar o oximoro desencantamento/reencantamento. Para tanto, selecionou-se cinco filmes: Deu a Louca na Chapeuzinho, Deu a Louca na Cinderela, A Verdadeira História do Gato de Botas, Deu a Louca na Branca de Neve e Deu a Louca na Chapeuzinho 2. Sua escolha justifica-se por enunciarem as mudanças nos títulos e em função dos personagens clássicos.

ABSTRACT

This paper intends to understand the children's film versions of fairy tales, launched in Brazil, between 2000 and 2012, as symptoms of an age marked mainly by the notions of enchantment and disenchantment. The aesthetics of realism enters into the tales and promotes metamorphosis, demonstrating a migration of the stories of the modern context to the postmodern: the expectations are erased and the desires migrate to the spheres of possible, tangible and concrete, to what ordinary beings can aspire too. This study aims, therefore, analyze the oxymoron disenchantment/reenchantment. For that, we selected five films: Hoodwinked; Happily N’Ever After; The True Story of Puss in Boots, Happily N’Ever After 2 (Snow White – Another Bite at the Apple) and Hoodwinked Too. This choice is justified by showing changes in the titles and because of the classic characters.
Narrativas Juvenis: As apropriações da rua e da internet no consumo e produção de graffitis em Belo Horizonte // Youth narratives: street and Internet’s appropriations in the consumption and production of graffitis in Belo Horizonte
Authors: Tranquilin-Silva Josefina De Fátima , Migliano Milene
Published: July 2021

Este artigo congrega duas pesquisas anteriores: a primeira, já finalizada no mestrado , buscou compreender percursos simbólicos de criação de situações comunicativas por meio de táticas que produzem narrativas inscritas nos muros, na região central de Belo Horizonte/Minas Gerais. A segunda se desenvolve em um pós-doutorado, que contempla as análises sobre narratividades juvenis e redes sociais e, aqui, objetiva trabalhar teoricamente o conceito de juventudes e os contextos do ambiente digital e das cidades contemporâneas. A partir de duas pesquisas diferentes, porém complementares em termos teóricos, temos como objetivo, neste artigo, analisar como os jovens grafiteiros de Belo Horizonte se apropriam do espaço urbano e do espaço digital, usando como recorte o Mapa dos Graffitis, para se mostrarem resistentes às políticas hegemônicas de espetacularização dos espaços públicos e, assim, promover novas sociabilidades e outros processos comunicacionais
On the geometry of leukocyte NADPH‐oxidase, a membrane flavoenzyme
Authors: Schirmer R.Heiner , Schulz Georg E. , Untucht-Grau Renate
Published: July 2002

Using the structure of glutathione reductase as a model, we suggest the following topography for leukocyte NADPH‐oxidase: The binding sites of NADPH and O2 are separated from each other by the flavin ring and are thus exposed to opposite sides of the plasma membrane. This model supports the concept that O−
2 is formed at the membrane facing the extracellular or phagosomal space, respectively. The fate of the proton produced in the reaction NADPH + 2 O2 → NADP + 2 O−
2 + H+ is also discussed in light of our model. NAD(P)H‐oxidases possessing the topography of glutathione reductase may establish transmembrane proton gradients. Consequently our model suggests that leukocyte NADPH‐oxidase produces not only the O−
2 burst but also a proton burst.
Purification and properties of the β‐fructofuranosidase from Kluyveromyces fragilis
Authors: Workman Wesley E. , Day Donal F.
Published: July 2002

The β‐fructofuranosidase from Kluyveromyces fragilis was purified to one band on electrophoresis by 3 different methods. Two of the preparations were found to be impure by isoelectric focusing. This demonstrates the need for more than one criteria of homogeneity when purifying this enzyme. The enzyme was found to be a glycoprotein, stable at 50°C, with a pH optimum of 4.5. The cations Hg2+, Ag+, Cu2+ and Cd2+ exhibited a marked inhibition of the enzyme. Competitive inhibition was observed with the fructose analog 2,5‐anhydro‐D‐mannitol suggesting that the enzyme is inhibited by the furanose form of fructose.
Monoclonal antibodies which prevent experimental lung metastases
Authors: Vollmers H.P. , Imhof B.A. , Braun S. , Waller C.A. , Schirrmacher V. , Birchmeier W.
Published: July 2002

Cellular adhesion is important during metastasis, as metastatic cells must escape from the primary site into lymph and blood systems, there to adhere specifically to sites in distant organs. We have recently selected monoclonal antibodies which prevent adherence of B 16 mouse melanoma cells to tissue culture dishes, and also markedly reduce experimental lung metastasis in mice when injected before or with the tumour cells. Here, we investigated which step in the metastatic process may be affected by the antibodies. The possible inhibitory effect of antibody on tumour cell adherence to vascular endothelial monolayers and to purified components of the underlying extracellular matrix‐fibronectin, laminin and collagen type IV ‐ was studied using in vitro assays. We found that the antibodies significantly blocked attachment to laminin, suggesting that specific basement membrane components play an important role in attracting or otherwise modifying the behaviour of metastatic tumour cells.
Analysis of an inhibin preparation reveals apparent identity between a peptide with inhibin‐like activity and a sperm‐coating antigen
Authors: Johansson Jan , Sheth Anil , Cederlund Ella , Jörnvall Hans
Published: July 2002

The amino acid sequence of a large form of inhibin‐like peptide in human seminal plasma was determined, and compared with structures reported for similar inhibin preparations and a seminal plasma globulin. The data confirm and correlate previous reports on this form of inhibin‐like peptide. The structural comparisons further suggest that the peptide is closely similar to or possibly identical to a sperm‐coating antigen reported to be synthesized from prostatic epithelium. This may correlate with non‐gonadal origins of inhibin‐like material and will help to elucidate the biological roles of inhibin(s).
Identification of γ‐type hordeins in barley
Authors: Shewry P.R. , Kreis M. , Parmar S. , Lew E.J.-L. , Kasarda D.D.
Published: July 2002

The barley mutant Risϕ 56 has a deletion of at least 85 kb of DNA at the Hor 2 locus, resulting in the absence from the seeds of almost all of the major B hordein group of storage proteins. We have purified two fractions containing the three remaining ‘B hordein‐like’ bands and determined their N‐terminal amino acid sequences. One fraction, containing the two bands with higher M
r, gave a single major N‐terminal sequence which was closely related to those reported for γ‐type prolamins of wheat and rye. The sequence of the third band was also homologous with those of the γ‐type prolamins, but less closely so. We consider that the γ‐type prolamins are most closely related to the ancestral S‐rich prolamins of the Triticeae, and this is the first report of their presence in barley.
Dihydropyridine Ca2+ agonists and channel blockers interact in the opposite manner with photogenerated unpaired electrons
Authors: Marinov Beniamin S. , Saxon Maya E.
Published: July 2002

Interaction of Ca2+‐channel antagonists (felodipine, ryocidil, verapamil, diltiazem) and agonists (dihydropyridine derivatives Bay K 8644 and CGP 28392) was studied by the methods of absorption spectroscopy. Ca2+‐channel antagonists were found to act as electron donors, the agonists being electron acceptors in the interaction with dye free radicals in solution. Redox transitions in channel‐forming protein were proposed as a possible mechanism of the modulation of channel activity by the compounds tested.
A new class of inhibitors of human leucocyte elastase
Authors: Hassall Cedric H. , Johnson William H. , Kennedy Anthony J. , Roberts Noel A.
Published: July 2002

Studies of the inhibition of elastases at a molecular level have resulted in the identification of protected dipeptides which are reversible and highly specific inhibitors of human leucocyte elastase (HLE). These have been further developed by increasing their hydrophilicity and potency to give a new family of elastase inhibitors, typicaly Nα
‐(1‐adamantanesulphonyl)‐Nϵ
‐(4‐carboxybenzoyl)‐L‐lysyl‐L‐alanyl‐L‐valinal. These compounds are active in pharmacological models designed to detect compounds of potential therapeutic value in the treatment of emphysema.
Effects of substitution of putative transmembrane segments on nicotinic acetylcholine receptor function
Authors: Tobimatsu Takamasa , Fujita Yoshihiko , Fukuda Kazuhiko , Tanaka Ken-ichi , Mori Yasuo , Konno Takashi , Mishina Masayoshi , Numa Shosaku
Published: July 2002

Mutants of the Torpedo nicotinic acetylcholine receptor in which each of the putative transmembrane segments of the α‐subunit is replaced by the hydrophobic transmembrane segment of the vesicular stomatitis virus glycoprotein or of the human interleukin‐2 receptor have been produced in Xenopus oocytes by cDNA manipulations. Functional analysis of these mutants shows that the hydrophobic segment M4 can be replaced by foreign transmembrane sequences without loss of channel activity. It is also suggested that the hydrophobic segments M1, M2 and M3 and the amphipathic segment MA are important for efficient expression of the acetylcholine receptor on the cell surface and that the specific amino acid sequence of segment M2 may be involved in channel activity.
Oxidation of uroporphyrinogens by hydroxyl radicals Evidence for nonporphyrin products as potential inhibitors of uroporphyrinogen decar☐ylase
Authors: Francis Jean E. , Smith Andrew G.
Published: July 2002

A hydroxyl radical‐generating system, hypoxanthine/xanthine oxidase/Fe‐EDTA, oxidises uroporphyrinogens to uroporphyrins and to more polar nonporphyrin products. The evidence suggests that the nonporphyrin products have inhibitory activity towards mouse liver uroporphyrinogen decar☐ylase which could explain some forms of human and experimental porphyrias.
The multicatalytic proteinase (prosome) is ubiquitous from eukaryotes to archaebacteria
Authors: Dahlmann Burkhardt , Kopp Friedrich , Kuehn Lothar , Niedel Barbara , Pfeifer Günther , Hegerl Reiner , Baumeister Wolfgang
Published: July 2002

From the thermoacidophilic archaebacterium, Thermoplasma acidophilum, a proteolytically active particle has been isolated which is almost identical in size and shape with the multicatalytic proteinase (prosome) from rat. This result indicates that prosomes have been developed early in evolution and that they possibly serve functions common to all living cells.
Investigation of models for photosynthetic electron acceptors
Authors: Bauscher M. , Nabedryk E. , Bagley K. , Breton J. , Mäntele W.
Published: July 2002

In order to gain more information about the binding and interaction of quinones acting as electron acceptors in photosynthetic reaction centers, infrared spectra of ‘model” quinones and their reduced species (semiquinones, quinone dianions and quinols) were recorded in a spectroelectrochemical cell. The frequencies and the extinction coefficients of the quinone C = O and C = C stretching vibrations which are shifted upon reduction were obtained in different solvents. These spectra are used to estimate the contribution of quinone reduction to light‐induced infrared difference spectra between the charge‐separated and the relaxed state of photosynthetic reaction centers.
A chimeric D2 dopamine/ml muscarinic receptor with D2 binding specificity mobilizes intracellular calcium in response to dopamine
Authors: England Bruce P. , Ackerman Martha S. , Barrett Ronald W.
Published: July 2002

Using PCR methodology, a chimeric receptor cDNA was constructed in which the entire third cytoplasmic loop of the human D2 dopamine receptor was replaced by the analogous portion of the human ml muscarinic receptor. When expressed in CHO cells, the chimeric D2/ml receptor bound dopaminergic ligands with affinities similar to the native D2(414) receptor. Intracellular calcium levels (measured with Furu‐2) were not altered when CHO cells expressing the D2(414) receptor were exposed to dopamine. In contrast, dopamine elevated intracellular calcium levels in cells expressing the D2/ml chimeric receptor in a dose‐dependent manner which was blocked by the D2 antagonist, fluphenazine. The ability to construct G‐protein‐linked receptor chimeras which mobilize calcium with nearly unaltered pharmacologic specificity raises the possibility of a generic strategy for creating non‐radioisotopic reporter systems for use in drug discovery.
Differential regulation of the two mRNA species of the rodent negative acute phase protein α1‐inhibitor 3
Authors: Regler Roger , Sickinger Stefan , Schweizer Michael
Published: July 2002

Screening of two rat liver cDNA libraries, one of which was constructed using an α1‐inhibitor 3 (α1‐I3) specific primer, yielded overlapping cDNA clones which correspond to the full length cDNA for α1‐I3 mRNA. On the basis of sequence microheterogeneity existing throughout the cDNA sequence we identified two α1‐I3 mRNA species whose sequences are so grossly different in their bait regions that the amino acid homology therein is only 30%. Using oligonucleotide probes derived from their respective bait regions we investigated the regulation of the two α1 I3 mRNA species and demonstrated that only one of them, α1‐I3 variant I, is regulated pretranslationally following experimentally induced inflammation.
Bidirectional effects of Kupffer cells on hepatocyte proliferation in vitro
Authors: Meyer Dieter H. , Bachem Max G. , Gressner Axel M.
Published: July 2002

The control of rat hepatocyte DNA synthesis in vitro by Kupffer cells and transformed perisinusoidal lipocytes, i.e. myofibroblast‐like cells was studied. Conditioned media from Kupffer cells inhibit the replicative (hydroxyurea‐sensitive) DNA synthesis dose‐dependently in primary cultures of hepatocytes stimulated by epidermal growth factor (EGF). The cytokine responsible for the inhibition was identified as transforming growth factor β (TGFβ). After neutralization of activated TGFβ in these media. DNA synthesis is stimulated in quiescent hepatocytes via transforming growth factor α (TGFα) demonstrated by competitive TGFα/EGF‐receptor blocking on hepatocytes. Results similar to those obtained with Kupffer cells were found with conditioned media of myofibroblast‐like cells. Northern blot hybridization confirms the expression of both TGFβ and TGFα in Kupffer cells and myofibroblast‐like cells, respectively. These findings support the notion that Kupffer cells and myofibroblast‐like cells might regulate in both directions liver regeneration depending on the proportion of secreted TGFα and TGFβ and on the activation status of TGFβ, of which a significant fraction is secreted in the latent form.
X‐Ray structure of the antibiotic bacitracin A
Authors: Pfeffer Sabine , Höhne Wolfgang , Branner Sven , Wilson Keith , Betzel Christian
Published: July 2002

Bacitracins are a group of widely used peptide antibiotics. There has been interest in determining the three‐dimensional structure of the bacitracins. However, solution studies indicate significant flexibility in their structure and to date native bacitracins have resisted attempts at crystallisation despite considerable efforts over a number of years by several groups. Here we report the first three‐dimensional X‐ray structure of a bacitracin, complexed to a subtilisin proteinase. X‐Ray diffraction data were collected using synchrotron radiation in combination with the Image Plate Scanner system. The complex structure including two enzymes, two bacitracins, 220 water molecules and two Ca2+ ions was refined by restrained least‐squares to a crystallographic R factor (=Σ{{F
o‐F
c}}/Σ{F
o}}) of 16.3% at 2.0 Å.
Cloning and sequencing of the 23 kDa mouse photoreceptor cell‐specific protein
Authors: McGinnis James F. , Stepanik Phillip L. , Baehr Wolfgang , Subbaraya Iswari , Lerious Valentine
Published: July 2002

The 23 kDa protein was localized by immunocytochemistry to photoreceptor cells of the mouse retina, and bovine and mouse cDNA clones were isolated and sequenced. The deduced amino acid sequences showed that the mouse 23 kDa protein is 91% identical to the bovine protein, and is the same as S‐modulin, the CAR (cancer‐associated retinopathy) protein and recoverin, the Ca2+‐dependent activator of photoreceptor guanylate cyclase. The amino acid sequence reveals two Ca2+ binding sites, no internal repeats, 59% homology to the chicken visinin protein and 40% homology to calmodulin while Northern analysis demonstrated a single 1.0 kb mRNA species in bovine and mouse retina.
Role of leucine residues in the C‐terminal region of human interleukin‐6 in the biological activity
Authors: Nishimura Chiaki , Ekida Teiji , Nomura Kazuhide , Sakamoto Koji , Suzuki Hiroshi , Yasukawa Kiyoshi , Kishimoto Tadamitsu , Arata Yoji
Published: July 2002

Site‐directed mutagenesis of two sets of three periodic leucine residues which appear at every seventh position in the C‐terminal region of human interleukin‐6 (IL‐6) was performed. Both receptor‐binding and immunoglobulin (Ig)‐induction activities of a triple mutant Leu168,175,182→Val were only 1% compared with those of wild‐type IL‐6. However, the mutant Leu152,159,166→Val had 13% receptor‐binding and 2% Ig‐induction activities of those of wild‐type IL‐6. In order to obtain more direct information on the receptor‐binding region, we prepared two synthetic peptides. A significant binding activity was observed for the peptide Leu168‐Met185, but not for the peptide Leu152‐Arg169. These results indicate that leucine residues in the C‐terminal region, especially Leu168, Leu175, and Leu182, play an important role in the receptor‐binding and Ig‐induction activities.
Specific cleavage of DNA at CG sites by Co(III) and Ni(II) desferal complexes
Authors: Joshi Rajendra R. , Ganesh K.Nagappa
Published: July 2002

It is demonstrated that Co(III) and Ni(II) complexes of desferal, a siderophore chelating drug, show specificity for cleavage of DNA at CG sites, while the corresponding Cu(II) complex cleaves DNA sequences at both CG and AT sites.
On the localization of FKBP25 in T‐lymphocytes
Authors: Rivière Sylvie , Ménez André , Galat Andrzej
Published: July 2002

Using polyclonal rabbit antibodies against bovine FKBP25, NEPHGE/SDS‐PAGE and Western blotting we demonstrate that the rapamycin‐specific immunophilin FKBP25 is present in T‐lymphoma Jurkat cells. Subsequent fractionations of the soluble Jurkat cell proteins have revealed that FKBP25 predominantly occurs in the nuclear fraction. FKBP25 has the ability to bind to DNA. The FKBP25/DNA complex can be dissociated in the presence of a high salt concentration. FKBP12, which shares high amino acid sequence homology to the C‐tenninal domain of FKBP25, has no tendency to bind to DNA. CD‐constrained predictions of the secondary structures in FKBP25 suggest that an amphipathic helix‐loop‐helix occurs in the N‐terminal part of the protein and may account for its binding to DNA.
GTP‐binding protein‐activator sequences in the insulin receptor
Authors: Okamoto Toshimi , Okamoto Takashi , Murayama Yoshitake , Hayashi Yujiro , Ogata Etsuro , Nishimoto Ikuo
Published: July 2002

Some functions of the insulin receptor (insR) are assumed to be mediated by pertussis toxin‐sensitive Gi/Go. proteins. Here we have located G‐protein‐activator domains in the cytoplasmic region of the human insR. We searched the sequence of insR and found three candidate regions at residues 1039‐1061, 1147‐1168 and 1325‐1345, referred to as ISRP 1, ISRP2 and ISRP3, respectively. Among them, the Gi/Go‐activating function was observed only in peptide ISRP3. ISRP1 specifically activated G3, whereas ISRP2 had no effect on G proteins. ISRP2 and ISRP3 contained five of six autophosphorylated tyrosine residues in insR. After tyrosine phosphorylation, ISRP2 showed specific G1‐activating function, and ISRP3 potentiated its ability and became capable of activating G proteins generally. This is the first study that specifies G‐protein‐activator domains in insR and describes their modification by autophosphorylation.
The Pseudomonas phytotoxin coronatine mimics octadecanoid signalling molecules of higher plants
Authors: Weiler E.W. , Kutchan T.M. , Gorba T. , Brodschelm W. , Niesel U. , Bublitz F.
Published: July 2002

The phytotoxic principle, coronatine, which is present in several pathovars of the plant pathogen, Pseudomonas syringae was shown to be highly active in completely different, jasmonate‐selective bioassays. At nanomolar to micromolar concentrations, coronatine induced the accumulation of defense‐related secondary metabolites in several plant cell cultures, induced transcript accumulation of the elicitor‐responsive gene encoding the berberine bridge enzyme of Eschscholtzia californica, as well as the coiling response of Bryonia dioica tendrils. Biological activity critically depended upon the structure of coronatine, and slight modifications, such as methylation of the carboxyl moiety or reduction of the carbonyl group, rendered the molecules almost inactive. Coronafacic acid, obtained by hydrolysis of coronatine, was also nearly inactive. Coronatine did not elicit the accumulation of endogenous jasmonic acid in the systems analyzed. While coronafacic acid is similar in structure to jasmonic acid, we found coronatine to be a close structural analogue of the cyclic C18‐precursor of jasmonic acid, 12‐oxo‐phytodienoic acid. The phytotoxic symptoms produced by coronatine can now be understood on the basis of the toxin's action as a mimic of the octadecanoid signalling molecules of higher plants.
Cleavage of proalbumin peptides by furin reveals unexpected restrictions at the P2 and P′1 sites
Authors: Brennan Stephen O. , Nakayam Kazuhisa
Published: July 2002

Proalbumin is the principal substrate of the in situ hepatic convertase. Here we investigated the specificity of furin using synthetic peptides based on the N‐terminal sequence of human proalbumin. The propeptide was rapidly cleaved from the normal ((−6)RGVFRR(−1)DAHKSEAVW(+9)) peptide but as expected, there was no cleavage of the proalbumin Lille analogue with a −2 His (−2H). Surprisingly, the effect of this substitution could not be corrected by introducing a −4 Arg (−4R−2H). In contrast, the peptide −4R−2A was an excellent substrate being cleaved five times faster than normal, indicaabting that His is not allowed as an P2 residue. Replacement of the −4 Val by Glu supported the expected importance of a positive charge at P4 as the cleavage rate dropped to 10% of normal after this substitution. The −6 Arg makes a small contribution to cleavage, its replacement by Ala decreased the cleavage rate to 60% of normal. The Lys‐Arg propeptide was almost as good a substrate as the normal Arg‐Arg peptide, but the introduction of a Lys at P′1 totally abolished processing. The exclusion of P′1 positive charges would be an important requirement for preventing aberrant cleavage in the middle of tetrabasic sequences.
Catabolite inactivation of fructose‐1,6‐bisphosphatase in yeast is mediated by the proteasome
Authors: Schork Stefan M. , Bee Gisela , Thumm Michael , Wolf Dieter H.
Published: July 2002

Fructose‐1,6‐bisphosphatase, a key enzyme in gluconeogenesis, undergoes catabolite inactivation when glucose is added to gluconeogenetically active cells of the yeast Saccharomyces cerevisiae. Phosphorylation of the enzyme is followed by rapid degradation. To elucidate the cellular proteolytic system involved in catabolite‐triggered degradation of fructose‐1,6‐bisphosphatase this event was followed in different protease‐deficient yeast mutants. In a mutant defective in the proteolytic function of the vacuole the degradation rate of the enzyme is not diminished. In contrast mutants defective in the proteolytic activity of the proteasome exhibit a strongly reduced glucose‐induced degradation of fructose‐1,6‐bisphosphatase as compared to their isogenic wild‐type counterparts. Our studies suggest that catabolite inactivation of fructose‐1,6‐bisphosphatase occurs in the cytosol, the degradation event being mediated by the proteasome. An explanation is presented which tries to resolve the formerly conflicting results, which suggested glucose‐triggered uptake of fructose‐1,6‐bisphosphatase into the vacuole followed by vacuolar proteolysis.
Multi‐step processing of procathepsin L in vitro
Authors: Ishidoh Kazumi , Kominami Eiki
Published: July 2002

The proteolytic processes involved in the conversion of procathepsin L to cathepsin L on a negatively charged surface, dextran sulfate, were studied. Upon incubation for 30 min at 37°C, pH 5.5 with dextran‐sulfate and dithiothreitol, purified procathepsin L showed maximal activation and, correspondingly, the complete conversion to the 30 kDa, single chain mature form of enzyme was observed. In contrast, incubation under the same conditions on ice rather than at 37°C for 30 or 60 min resulted in partial proteolysis to produce a 31 kDa form without a significant increase in activity. Amino terminal amino acid sequence analyses showed that the 30 kDa form obtained by incubation at 37°C corresponds to the purified form of mature cathepsin L with a 2 amino acid extension at the amino terminal, and that the 31 kDa form generated by incubation on ice possesses a 6 amino acid amino terminal extension, suggesting that the activation and processing of procathepsin L are different processes, and that 4 amino acid residues (Glu‐Pro‐Leu‐Met) at the carboxyterminal in the propeptide function to prevent the activation of processed cathepsin L.
Production of platelet‐derived growth factor receptor (PDGFR‐β) in E. coli
Authors: Rooney B.C. , Hosang M. , Hunziker W.
Published: July 2002

Portions of the extracellular domain of the platelet‐derived growth factor receptor β (PDGFR‐β) were expressed as fusion proteins with a hexa His tag in E. coli. Following purification by Ni chelate chromatography, the recombinant receptors were tested in cross‐competition studies with 125I‐Iabelled PDGF‐AA and ‐BB. Although of lower affinity than the native receptor (IC50 values of 10−8 M) the recombinant molecules retained ligand binding specificity and neutralised the mitogenic effect of PDGF‐BB. These data indicate that the ligand binding region lies within the first four immunoglobulin‐like domains on PDGFR‐β. This E. coli expression system could be further used as a rapid and economical means to produce mutated receptors and map the ligand binding domain.
Unfarnesylated transforming Ras mutant inhibits the Ras‐signaling pathway by forming a stable Ras·Raf complex in the cytosol
Authors: Miyake Mika , Mizutani Shin , Koide Hiroshi , Kaziro Yoshito
Published: July 2002

Farnesyltransferase inhibitors cause the growth arrest of ras‐transformed cells, but not that of normal cells. To elucidate the mechanism of this differential effect, we examined the effect of accumulation of unfarnesylated Ras in the cytosol by using RasG12V,C186S and RasC186S, which mimic unfarnesylated form of the oncogenic and the normal Ras, respectively. We found that RasG12,C186S inhibited activation and membrane translocation of Raf by forming a stable complex with Raf in the cytosol. In contrast, RasC186S showed inhibitory effect on neither Raf activation nor Raf translocation. These results indicate that unfarnesylated oncogenic Ras interacts with Raf in the cytosol and inhibits its membrane translocation, a crucial step for the Raf activation, while unfarnesylated normal Ras does not.
Identification and characterization of the Drosophila histone H4 replacement gene
Authors: Akhmanova Anna , Miedema Koos , Hennig Wolfgang
Published: July 2002

Replacement variant genes for different histones have been described in most higher eukaryotes. However, so far no such gene has been found for histone H4. We have isolated from both Drosophila melanogaster and D. hydei a novel histone H4 encoding gene, H4r, which displays all the properties of a histone replacement variant gene: it contains introns, generates polyadenylated mRNA, represents the predominant H4 transcript in non‐dividing tissues and is present in the genome as a single copy. The encoded polypeptide is identical to the Drosophila cell‐cycle regulated histone H4. The fact that it is a single copy gene makes it prone to genetic analysis and it might be a useful tool for studying nucleosome structure and function.
Structure‐activity relationships in the peptide antibiotic nisin: antibacterial activity of fragments of nisin
Authors: Chan W.C. , Leyland M. , Clark J. , Dodd H.M. , Lian L.-Y. , Gasson M.J. , Bycroft B.W. , Roberts G.C.K.
Published: July 2002

The post‐translationally modified peptide antibiotic nisin has been cleaved by a number of proteases and the fragments produced purified, characterised chemically, and assayed for activity in inhibiting the growth of Lactococcus lactis MG1614 and Micrococcus luteus NCDO8166. These results provide information on the importance of different parts of the nisin molecule for its growth‐inhibition activity. Removal of the C‐terminal five residues leads to approximately a 10‐fold decrease in potency, while removal of a further nine residues, encompassing two of the lanthionine rings, leads to a 100‐fold decrease. There are some differences between analogous fragments of nisin and subtilin, suggesting possible subtle differences in mode of action. Cleavage within, or removal of, lanthionine ring C essentially abolishes the activity of nisin. The fragment nisin1−12 is inactive itself, and specifically antagonises the growth‐inhibitory action of nisin. These results are discussed in terms of current models for the mechanism of action of nisin.
Arabidopsis loss‐of‐function mutant in the lysine pathway points out complex regulation mechanisms
Authors: Craciun Adrian , Jacobs Michel , Vauterin Marc
Published: July 2002

In plants, the amino acids lysine, threonine, methionine and isoleucine have L‐aspartate‐β‐semialdehyde (ASA) as a common precursor in their biosynthesis pathways. How this ASA precursor is dispersed among the different pathways remains vague knowledge. The proportional balances of free and/or protein‐bound lysine, threonine, isoleucine and methionine are a function of protein synthesis, secondary metabolism and plant physiology. Some control points determining the flux through the distinct pathways are known, but an adequate explanation of how the competing pathways share ASA in a fine‐tuned amino acid biosynthesis network is yet not available. In this article we discuss the influence of lysine biosynthesis on the adjacent pathways of threonine and methionine. We report the finding of an Arabidopsis thaliana dihydrodipicolinate synthase T‐DNA insertion mutant displaying lower lysine synthesis, and, as a result of this, a strongly enhanced synthesis of threonine. Consequences of these cross‐pathway regulations are discussed.
Nucleotide‐induced conformational changes in the human multidrug resistance protein MRP1 are related to the capacity of chemotherapeutic drugs to accumulate or not in resistant cells
Authors: Manciu Liliana , Chang Xiu-Bao , Riordan John R. , Buyse Frédéric , Ruysschaert Jean Marie
Published: July 2002

Intracellular accumulation of anthracycline derivatives was measured in a human embryonic kidney cell line (HEK) and a resistant subline (HEK/multidrug resistance protein (MRP1)) overexpressing MRP1 at the plasma membrane surface. Two compounds (daunorubicin and doxorubicin) were rejected outside the multidrug‐resistant cells. On the contrary, three compounds (4′‐deoxy‐4′‐iodo‐doxorubicin, 4‐demethoxy‐daunorubicin and 3′‐(3‐methoxymorpholino)doxorubicin) accumulated equally within sensitive HEK cells and resistant HEK/MRP1 cells. Our main objective here was to characterize the MRP1 conformational changes mediated by the binding of these anthracycline derivatives and to determine whether these conformational changes are related to MRP1‐mediated drug transport. MRP1 was reconstituted in lipid vesicles as previously described [Manciu, L., Chang, X.B., Riordan, J.R. and Ruysschaert, J.‐M. (2000) Biochemistry 39, 13026–13033]. The reconstituted protein was shown to conserve its ATPase and drug transport activity. Acrylamide quenching of Trp fluorescence was used to monitor drug‐dependent conformational changes. Binding of drugs (4‐demethoxy‐daunorubicin and 3′‐(3‐methoxymorpholino)doxorubicin) which accumulate in resistant cells immobilizes MRP1 in a conformational state that is insensitive to ATP binding whereas drugs rejected outside the resistant cells (daunorubicin, doxorubicin) favor a conformational change which may be a required step in the transport process.
Overexpressed human survival motor neurone isoforms, SMNΔexon7 and SMN+exon7, both form intranuclear gems but differ in cytoplasmic distribution
Authors: Dodds Emma , Dunckley Matthew G. , Roberts Roland G. , Muntoni Francesco , Shaw Christopher E.
Published: July 2002

Homozygous mutations of the telomeric survival motor neurone gene (SMN1) cause spinal muscular atrophy (SMA). The centromeric copy gene (SMN2) generally skips exon 7 during splicing and fails to compensate for SMN1 deficits, so SMA cells have reduced SMN protein and few nuclear gems. To investigate the role of exon 7 in SMN localisation, cDNAs for full‐length SMN and SMNΔexon 7 were overexpressed in COS cells, neurones and SMA fibroblasts. Both constructs formed discrete intranuclear bodies colocalising with p80‐coilin, but produced more cytoplasmic aggregates in cells overexpressing exon 7. Hence, the exon 7 domain enhances SMN aggregation but is not critical for gem formation.
Overlapping CRE and E‐box promoter elements can independently regulate COX‐2 gene transcription in macrophages
Authors: Mestre Juan R. , Rivadeneira David E. , Mackrell Peter J. , Duff Michael , Stapleton Philip P. , Mack-Strong Vivian , Maddali Sirish , Smyth Gordon P. , Tanabe Tadashi , Daly John M.
Published: July 2002

Macrophage cyclooxygenase‐2 (COX‐2) transcription is mediated through the collaboration of different promoter elements. Here, the role of an overlapping cyclic AMP responsive element (CRE)/E‐box was investigated. Nuclear proteins bound both the CRE and E‐box, which synergized with other promoter elements to induce COX‐2 transcription. Endotoxin induced binding of nuclear proteins to the CRE and E‐box and each element independently induced higher COX‐2 transcription levels than the overlapping CRE/E‐box. Transcription factors associated with the CRE binding complex included c‐Jun and CRE binding protein and with the E‐box binding complex USF‐1; their overexpression significantly induced COX‐2 transcription. Therefore, both CRE and E‐box promoter elements regulate COX‐2 transcription in macrophages.
A simple determination of steroidal alkaloid glycosides by thin‐layer chromatography immunostaining using monoclonal antibody against solamargine
Authors: Tanaka Hiroyuki , Putalun Waraporn , Tsuzaki Chiyumi , Shoyama Yukihiro
Published: July 2002

© 1997 Federation of European Biochemical Societies.
A domain containing the Cdc42/Rac interactive binding (CRIB) region of p65PAK inhibits transcriptional activation and cell transformation mediated by the Ras‐Rac pathway
Authors: Osada Shin-ichi , Izawa Masaki , Koyama Tatsunobu , Hirai Syu-ichi , Ohno Shigeo
Published: July 2002

© 1997 Federation of European Biochemical Societies.
The cytokine interleukin‐5 (IL‐5) effects cotransport of its receptor subunits to the nucleus in vitro
Authors: Jans David A , Briggs Lyndall J , Gustin Sonja E , Jans Patricia , Ford Sally , Young Ian G
Published: July 2002

Interleukin (IL)‐5 is central in regulating eosinophilia in allergic disease and parasitic infections. We have recently shown that human (h) IL‐5 both possesses a functional nuclear localization signal capable of targeting a heterologous protein to the nucleus and localises to the nucleus of intact receptor‐expressing cells. In this study, the extracellular domains of the hIL‐5 α‐ and β‐receptor subunits were expressed in baculovirus, fluorescently labelled and assayed for nuclear targeting in vitro in the absence and presence of IL‐5. The β‐subunit, which lacks IL‐5 binding activity, only accumulated in the nucleus in the presence of both the hIL‐5 binding α‐subunit and hIL‐5. The IL‐5‐binding α‐subunit showed similar results. IL‐5 thus effected nuclear transport of its α‐ and β‐receptor subunits apparently through a ‘piggy back’ mechanism, raising the possibility that IL‐5's nuclear signalling role may be to cotarget its receptor subunits to the nucleus. This is the first demonstration of nuclear protein piggy back transport in vitro.
The 58 kDa mouse selenoprotein is a BCNU‐sensitive thioredoxin reductase
Authors: Gromer Stephan , Schirmer R.Heiner , Becker Katja
Published: July 2002

The flavoprotein thioredoxin reductase [EC 1.6.4.5] (NADPH+H++thioredoxin‐S2→NADP++thioredoxin‐(SH)2) was isolated from mouse Ehrlich ascites tumour (EAT) cells. Like the counterpart from human placenta but unlike the known thioredoxin reductases from non‐vertebrate organisms, the mouse enzyme was found to contain 1 equivalent of selenium per subunit of 58 kDa. The K
M values were 4.5 μM for NADPH, 480 μM for DTNB and 36 μM for Escherichia coli thioredoxin, the turnover number with DTNB being ≈40 s−1. As mouse is a standard animal model in cancer and malaria research, thioredoxin reductase and glutathione reductase [EC 1.6.4.2] from EAT cells were compared with each other. While both enzymes in their 2‐electron reduced form are targets of the cytostatic drug carmustine (BCNU), no immunologic cross‐reactivity between the two mouse disulfide reductases was observed.
PP2Cγ: a human protein phosphatase with a unique acidic domain1
Authors: Travis Sue M , Welsh Michael J
Published: July 2002

We have cloned a novel cDNA from human skeletal muscle which encodes a protein phosphatase with a unique acidic domain. It is 34% identical to mammalian PP2Cα and PP2Cβ, and we call it PP2Cγ. It more closely resembles PP2Cs from Paramecium tetraurelia and Schizosaccharomyces pombe than mammalian PP2Cs. Northern blot analysis shows that PP2Cγ is widely expressed, and is most abundant in testis, skeletal muscle, and heart. Like known PP2Cs, recombinant PP2Cγ requires Mg2+ or Mn2+ for activity. Unlike any other known phosphatase, PP2Cγ has a highly acidic domain: 75% of the 54 residues are glutamate or aspartate.
Two regions in c‐myb proto‐oncogene product negatively regulating its DNA‐binding activity
Authors: Tanaka Yasunori , Nomura Teruaki , Ishii Shunsuke
Published: July 2002

The c‐myb proto‐oncogeneproduct (c‐Myb) is a transcriptional regulator that binds to the specific DNA sequence. Deletion of the negative regulatory domain (NRD) in the carboxyl‐proximal region of c‐Myb results in both increased trans‐activating capacity and oncogenic activation. One possible mechanism to modulate c‐Myb activity is a regulation of DNA‐binding activity. However, it is not known whether any region in NRD affects the in vivo DNA‐binding activity of c‐Myb. Using the highly transfectable cell line 293T, we developed a system to precisely measure the DNA‐binding activity of Myb expressed in mammalian cells. Using this system, two regions in NRD were shown to repress DNA‐binding activity. These results suggest that DNA‐binding activity of c‐Myb is independently regulated by multiple mechanisms through these subdomains.
Thiols and disulphides can aggravate peroxynitrite‐dependent inactivation of α1‐antiproteinase
Authors: Whiteman Matthew , Halliwell Barry
Published: July 2002

Peroxynitrite (ONOO−) is a cytotoxic species formed in vivo. There is considerable interest in the development of ONOO− `scavengers' as therapeutic agents; several thiols have been suggested to fulfil this role. One protein inactivated by ONOO− is α1‐antiproteinase (α1AP), the major inhibitor of serine proteinases in human body fluids. At low thiol:ONOO− concentration ratios, several thiols (captopril, penicillamine, cysteine, cystine and penicillamine disulphide) aggravated inactivation of α1AP by ONOO−, whereas GSH, GSSG, homocysteine, ergothioneine, N‐acetylcysteine, lipoate and dihydrolipoate did not. We suggest that sulphur‐containing radicals are produced by reaction of certain thiols/disulphides with ONOO− or ONOO−‐derived products and could mediate biological damage, including inactivation of α1AP. This must be considered in attempts to use thiols as `peroxynitrite scavengers'.
Elevation of apolipoprotein E in the CSF of cattle affected by BSE
Authors: Hochstrasser Denis F. , Frutiger Séverine , Wilkins Marc R. , Hughes Graham , Sanchez Jean-Charles
Published: July 2002

The cerebrospinal fluid (CSF) of patients suffering from Creutzfeldt‐Jakob disease (CJD) display two unique polypeptide chains by two‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE). In the absence of a well‐defined ante‐mortem diagnostic test for bovine spongiform encephalopathy (BSE), spinal fluid samples of eight normal cows and eight cows known to carry BSE by post‐mortem histological analysis were investigated to verify if equivalent polypeptides were present. Proteins with similar migration to human CJD polypeptides were not detected. But surprisingly, a cluster of polypeptide spots that was faint or not detected in normal bovine CSF samples was found to be elevated or massively increased in BSE CSF samples (more than 10‐fold increase). These elevated polypeptide chains were identified as apolipoprotein E.
The ABC transporter Atm1p is required for mitochondrial iron homeostasis
Authors: Kispal Gyula , Csere Peter , Guiard Bernard , Lill Roland
Published: July 2002

The function of the ABC transporter Atm1p located in the mitochondrial inner membrane is not yet known. To study its cellular role, we analyzed a mutant in which ATM1 was disrupted. Δatm1 cells are deficient in the holoforms, but not the apoforms of heme‐carrying proteins both within and outside mitochondria, yet both synthesis and transport of heme are functional. Δatm1 cells are hypersensitive for growth in the presence of oxidative reagents, and they contain increased levels of the antioxidant glutathione, in particular of its oxidized form. Mitochondria deficient in Atm1p accumulate 30‐fold higher levels of free iron as compared to wild‐type organelles, i.e. three‐fold more than mitochondria deficient in frataxin, the protein mutated in Friedreich's ataxia. The increased mitochondrial iron content may be causative of the oxidative damage of heme‐containing proteins in Δatm1 cells. Our data assign an important function to Atm1p in mitochondrial iron homeostasis.
Quenching of 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole‐modified Na+/K+‐ATPase reveals a higher accessibility of the low‐affinity ATP‐binding site
Authors: Linnertz Holger , Urbanova Petra , Amler Evzen
Published: July 2002

7‐Chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole (NBD‐Cl) labeled Na+/K+‐ATPase covalently with two different inactivation constants (K
i=2.5 μM; K
i′=10 μM). It apparently modified the two different ATP‐binding sites of the enzyme since it decreased the activity of the E2ATP site, i.e. the K+‐activated para‐nitrophenylphosphatase activity, in an enzyme whose high‐affinity E1ATP site had been blocked by fluorescein 5′isothiocyanate (FITC). It also reduced the activity of the E1ATP site, i.e. the Na+‐activated protein phosphorylation, in an enzyme whose low‐affinity E2ATP site had been blocked by Co(NH3)4PO4. Fluorescence quenching experiments with KI, CsCl and MnCl2 of the NBD‐Cl‐labeled Na+/K+‐ATPase revealed two differently accessible types of fluorophores depending on the ATP site: The E2ATP site apparently differs from the E1ATP site in that it is more open because the fluorophore labeling in the E2ATP site was sterically better accessible for quenchers.
Molecular diversity of sterol 14α‐demethylase substrates in plants, fungi and humans
Authors: Lamb David C , Kelly Diane E , Kelly Steven L
Published: July 2002

Metabolism of lanosterol (LAN), 24‐methylene‐24,25‐dihydrolanosterol (24‐methyleneDHL), dihydrolanosterol (DHL) and obtusifoliol (OBT) by purified human, plant (Sorghum bicolor) and fungal (Candida albicans) sterol 14α‐demethylase (CYP51; P45014DM) reconstituted with NADPH cytochrome P450 reductases was studied in order to elucidate the substrate specificity and sterol stereo‐ and regio‐structural requirements for optimal CYP51 activity. Both human and C. albicans CYP51 could catalyse 14α‐demethylation of each substrate with varying levels of activity, but having slightly higher activity for their respective endogenous substrates in vivo, dihydrolanosterol for human CYP51 (V
max=0.5 nmol/min/nmol CYP51) and 24‐methylene‐24,25‐dihydrolanosterol for C. albicans CYP51 (V
max=0.3 nmol/min/nmol CYP51). In contrast, S. bicolor CYP51 showed strict substrate specificity and selectivity towards its own endogenous substrate, obtusifoliol (V
max=5.5 nmol/min/nmol CYP51) and was inactive towards 14α‐demethylation of lanosterol, 24‐methylene‐24,25‐dihydrolanosterol and dihydrolanosterol. These findings confirm that the presence of the 4β‐methyl group in the sterol molecule renders the plant CYP51 incapable of 14α‐demethylation thus revealing the strict active site conservation of plant CYP51 during evolution.
Cell surface expression of the metabotropic glutamate receptor type 1α is regulated by the C‐terminal tail
Authors: Ciruela F. , Soloviev M.M. , McIlhinney R.A.J.
Published: July 2002

The cell surface expression of metabotropic glutamate receptor type 1 splice variants has been studied using cell surface biotinylation. Co‐expression of the last 86 residues of the C‐terminal tail of mGluR1α (F2‐protein) with mGluR1α caused a significant reduction of the amount of the cell surface receptor when compared to that in cells transfected with mGlur1α alone, and this was accompanied by a reduction in the production of inositol following agonist stimulation of the cells. In contrast, cell surface expression of mGluR1β was unaltered by co‐expression with the F2‐protein. These results suggest that the C‐terminal tail of mGluR1α regulates cell surface expression of the receptor.
Inhibition of virulence factor expression in Staphylococcus aureus by the Staphylococcus epidermidis agr pheromone and derivatives
Authors: Otto Michael , Süßmuth Roderich , Vuong Cuong , Jung Günther , Götz Friedrich
Published: July 2002

The agr quorum‐sensing system in Staphylococci controls the production of surface proteins and exoproteins. In the pathogenic species Staphylococcus aureus, these proteins include many virulence factors. The extracellular signal of the quorum‐sensing system is a thiolactone‐containing peptide pheromone, whose sequence varies among the different staphylococcal strains. We demonstrate that a synthetic Staphylococcus epidermidis pheromone is a competent inhibitor of the Staphylococcus aureus agr system. Derivatives of the pheromone, in which the N‐terminus or the cyclic bond structure was changed, were synthesized and their biological activity was determined. The presence of a correct N‐terminus and a thiolactone were absolute prerequisites for an agr‐activating effect in S. epidermidis, whereas inhibition of the S. aureus agr system was less dependent on the original structure. Our results show that effective quorum‐sensing blockers that suppress the expression of virulence factors in S. aureus can be designed based on the S. epidermidis pheromone.