bluuebunny/crossref_metadata_2025 huggingface dataset:
La imagen de la mujer en el patrimonio urbano de Granada. El espacio público de la ciudad como «escenario comunicativo»
Authors: Romero-Sánchez Guadalupe , Marfil-Carmona Rafael
Published: April 2020

Purpose: To establish an analysis of the presence of women in public spaces of Granada through movable heritage of an urban nature, deepening an analysis from a gender perspective and the contribution of these heritage elements to the main identity features of the city of Granada (Spain).

Design / Methodology / Approach: Selection of works related to women in the public space of this city, following a methodology of content analysis in the development of a case study. The following classification of sculptures has been applied: those that represent female historical figures: works where the image of the woman appears in a generic way as a symbol or resource, and the works that refer us to stories with a feminine «spirit». Public sculpture and heritage elements are considered in the broader context that constitutes the identity and brand of a city. Heritage is valued from its influence and communicational role, assessed from a gender perspective.

Results: Low presence of women in their public space and predominance of values and attributes linked to history, culture and university in the works analyzed.

Limitations / Implications: Those of a case study, which can be enriched with a comparative study between different cities and contexts. Utility when thinking about public space and establishing new strategies in its management, linking the city with the plural and open society of the 21st century.

Originality/value: Information is provided on the works studied, the women represented and their links with the city, considering urban heritage as a communication scenario, where the history, memory and current affairs of the city are present. Some identifying traits linked to the city of Granada are offered.
Extracting and Studying the Factors Affecting the Tanning Material in the Garad Pods
Authors: Eltom Egbal , Abdalla Gurashi
Published: April 2020

In this study the chemical structure of Garad tan pods (acacia – nilotica) were determined and investigated. It was found that Garad tan is a polyphenol type of structure and consequently it has an antiseptic effect, it is also used as pretaning and retaning agent.

Garad pods need to be disintegrated and ground into fine grains of very small diameter. In its powder form it cannot be used in tannage as the grains may block the sewage, hence, it has to be leached, concentrated and spray dried.

The factors that affect leaching of the tannins from Garad pods were studied, there are: Temperature, solvent to solid ratio, degree of mixing and time were specified and factorial experiments were designed to study the effect and significance of these factors. The levels of these factors were (30 and 50°C), (1:5 and 1:7), (with and without) mixing and (25 and 50) minutes respectively. All these factors were found to be very significant.

Having determined the optimum factors their levels trials were carried out at the following levels:

Temperature: 50°C

Solid to solvent ratio: 1:7

Degree of mixing: With mixing

Time: 50 minutes

The trials made at these conditions were found to be efficient with high yield of the extract. The extract produced was concentration which makes it ready for use in pertannage or retannage or for spray drying to powder.
Nature of the stacking interaction of nucleotide bases in water: a Monte Carlo study of the hydration of uracil molecule associates
Authors: Danilov V.I. , Tolokh I.S. , Poltev V.I. , Malenkov G.G.
Published: July 2002

The results of a Monte Carlo study of the hydration of a uracil molecule, its stacked and H‐bonded dimers are presented. It is shown that the formation of the stacked dimer is mainly governed by the energetically favourable change of water structure during the transition of monomers to dimer. The formation of the coplanar base pair is less favourable than that of the stack.
A Monte Carlo study of the hydration of thymine molecule associates
Authors: Danilov V.I. , Tolokh I.S. , Poltev V.I.
Published: July 2002

The results of a Monte Carlo study of the hydration of a thymine molecule, its stacked and H‐bonded dimers are presented. It is shown that the stacking interactions of thymine molecules in water arise mainly due to the increase in the water—water interaction during the transition from monomers to dimer. It has been found out that stacked base associates are more preferable than H‐bonded dimers in water. This preference is mainly due to the energetically more favourable structure of water around the stack.
New fluorescence of nonenzymatically glucosylated human serum albumin
Authors: Sakurai Tamiko , Takahashi Hiroshi , Tsuchiya Seishi
Published: July 2002

Glucosylated human serum albumin (G‐HSA) obtained under incubation with glucose at 37° C for 8 days showed a new fluorescence with a maximum at 430 nm, resulting in quenching of the fluorescence of only one tryptophan residue on HSA. The quantum yield of new fluorescence is 0.024 at 25° C. The analysis of the excitation spectra allowed us to conclude the absence of energy transfer. In G‐HSA, non‐disulfide cross‐linking hexamer was confirmed by SDS‐PAGE.
Acetyl xylan esterases in fungal cellulolytic systems
Authors: Biely Peter , Puls Jürgen , Schneider Henry
Published: July 2002

Xylan of several tree and other plant species is acetylated, and the enzymology of its degradation is unknown. The present study shows that enzymes liberating acetic acid from an acetylated xylan occur in several fungal cellulolytic systems. Consequently, an additional step will have to be considered in current concepts for the degradation of hemicellulose by xylanases and xylosidases.
cAMP mediated proteolysis of the catalytic subunit of cAMP‐dependent protein kinase
Authors: Hemmings Brian A.
Published: July 2002

The cAMP‐dependent protein kinase from LLC‐PK1 cells can be activated in vivo by calcitonin and vasopressin, or forskolin. Continuous treatment of cells with these agents results in a decrease of total cAMP‐PK activity. The loss of kinase activity was enhanced when either of these three agents was incubated in the presence of isobutylmethylxanthine. Results obtained using affinity purified antibodies to the catalytic subunit show that the loss of kinase was due to specific proteolysis of this subunit.
Aliphatic side chain of catecholamine potentiates the stimulatory effect of the catechol part on the synthesis of nerve growth factor
Authors: Furukawa Yoshiko , Furukawa Shoei , Ikeda Fumiaki , Satoyoshi Eijiro , Hayashi Kyozo
Published: July 2002

Catecholamines are potent in stimulating nerve growth factor (NGF) synthesis in mouse L‐M cells. The relationship between the structure of catecholamines and their stimulatory effect on NGF synthesis has been studied using various 3,4‐dihydroxyphenyl derivatives or their analogues. All 3,4‐dihydroxyphenyl derivatives with two saturated carbons on the side chain were potent stimulators, whereas those with only one carbon on the side chain were weak stimulators. Drugs lacking the catechol ring were not effective. These results suggest that the catechol part of catecholamines is essential for the stimulatory effect and that the aliphatic side chain potentiates this effect. The present results also suggest the terminal amino residue on the side chain is not critical for the effect.
Complete primary structure of the α1‐chain of human basement membrane (type IV) collagen
Authors: Raija Soininen , Haka-Risku Tuula , Prockop Darwin J. , Tryggvason Karl
Published: July 2002

We have determined the primary structure of the α1(IV)‐chain of human type IV collagen by nucleotide sequencing of overlapping cDNA clones that were isolated from a human placental cDNA library. The present data provide the sequence of 295 amino acids not previously determined. Altogether, the α1(IV)‐chain contains 1642 amino acids and has a molecular mass of 157625 Da. There are 1413 residues in the collagenous domain and 229 amino acids in the carboxy‐terminal globular domain. The human α1(IV)‐chain contains a total of 21 interruptions in the collagenous Gly‐X‐Y repeat sequence. These interruptions vary in length between two and eleven residues. The α1(IV)‐chain contains four cysteine residues in the triple‐helical domain, four cysteines in the 15‐residue long noncollagenous sequence at the amino‐terminus and 12 cysteines in the carboxy‐terminal NC‐domain
Receptors on phaeochromocytoma cells for two members of the PP‐fold family — NPY and PP
Authors: Schwartz Thue W. , Sheikh Søren P. , O'Hare Mairead M.T.
Published: July 2002

Pancreatic polypeptide (PP) and neuropeptide Y (NPY) belong to a family of regulatory peptides which hold a distinct tertiary structure, the PP‐fold, even in dilute aqueous solution. High‐affinity receptors, specific for both PP and NPY, are described on the rat phaeochromocytoma cell line, PC‐12. The binding of [125I‐Tyr36]PP to PC‐12 cells was inhibited by concentrations of unlabeled PP which correspond to physiological concentrations of the hormone, 10−11‐10−9 mol/1. The affinity of the receptor for the neuropeptide, NPY, was 102‐times lower than that of the PP receptor. C‐terminal fragments of both PP (PP24–36) and NPY (NPY13–36) were between 102 ‐ and 103‐times less potent in displacing the radiolabeled 36‐amino‐acid peptides from their respective receptors. It is concluded that PC‐12 cells are suited for structure‐function studies of the PP‐fold peptides and studies on the cellular events following cellular binding of PP‐fold peptides.
Botulinum neurotoxin inhibits depolarization‐stimulated protein phosphorylation in pure cholinergic synaptosomes
Authors: Guitart Xavier , Egea Gustau , Solsona Carles , Marsal Jordi
Published: July 2002

Botulinum neurotoxin, a strong blocker of acetylcholine release at peripheral cholinergic synapses, inhibits depolarization‐stimulated protein phosphorylation in pure cholinergic synaptosomes isolated from the electric organ of Torpedo marmorata. Moreover, tetrodotoxin has the same effect on protein phosphorylation when cholinergic synaptosomes are depolarized by veratridine. Correlation between presynaptic protein phosphorylation and acetylcholine release is suggested by the fact that botulinum neurotoxin blocks specifically neurotransmitter release without affecting membrane depolarization and calcium fluxes in our synaptosomal preparation.
The high‐molecular‐mass kininogen deficient rat expresses all kininogen mRNA species, but does not export the high‐molecular‐mass kininogen synthesized
Authors: Lattion Anne-Laure , Baussant Thierry , Alhenc-Gelas François , Seidah Nabil G. , Corvol Pierre , Soubrier Florent
Published: July 2002

The Katholiek substrain of Brown Norway (BN/Kat) rats exhibits a very low level of circulating high‐molecular‐mass (HMW) kininogen and a partial deficiency in plasma prekallikrein. Northern blot analysis of liver RNA revealed that HMW kininogen and prekallikrein mRNAs are present in these rats with a similar size and abundance compared to control Brown Norway (BN/Orl) rats. The low‐molecular‐mass kininogen mRNA, encoded by the same kininogen gene as HMW kininogen MRNA by alternative splicing, is detected in both strains by dideoxynucleotide limited primer extension analysis. Measurement of HMW kininogen by radioimmunoassay was performed in liver subcellular fractions. It reveals that, in contrast to its absence in the cytosolic fraction, HMW kininogen in deficients rats is slightly more abundant in the microsomal fraction, than in control rats. These observations exclude both an abnormality at the level of gene transcription and a major structural modification of the transcribed RNA and of the synthesized HMW kininogen. They favour the hypothesis of an abnormal intracellular transport of the HMW kininogen in deficient rats.
In vivo release of CCK‐8 from the dorsal horn of the rat: Inhibition by DAGOL
Authors: Rodríguez R.E. , Sacristán M.P.
Published: July 2002

There is evidence that CCK‐8 may interact with opioids and that both systems are probably implicated in pain modulation. In order to elucidate this relationship we sought to examine factors governing the movement of CCK‐8 from the spinal cord into the extracellular space. We report that CCK‐8 like immunoreactivity, as measured by RIA, is released from the spinal cord of the rat in vivo, following potassium stimulation and by direct activation of high threshold peripheral afferents by stimulation of the sciatic nerve. Also, we show that CCK‐8 release is inhibited by the μ‐selective opioid receptor agonist DAGOL. Naloxone totally reversed the effect produced by DAGOL, implying an opiate mediated mechanism.
Mechanism of biosynthesis of 11R‐ and 12R‐hydroxyeicosatetraenoic acids by eggs of the sea urchin Strongylocentrotus purpuratus
Authors: Hawkins Dan J. , Brash Alan R.
Published: July 2002

11(R)‐Hydroxyeicosatetraenoic acid [11(R)‐HETE] and 12(R)‐HETE are biosynthesized by eggs of the sea urchin S. purpuratus. We report here the isolation of the 11(R)‐ and 12(R)‐hydroperoxy‐eicosanoids from incubations of the desalted 30–50% (NH4)2SO4 fraction of the egg homogenate; biosynthesis required the addition of calcium but not NADPH. Egg 11‐ and 12‐HETE were formed from octadeuterated arachidonic acid without loss of geminal 2H from C11 or C12, thus revealing that 11‐ or 12‐keto intermediates are not involved in the biosynthesis. The results support the conclusion that egg 11(R)‐ and 12(R)‐HETE are synthesized by a lipoxygenase and not by an NADPH‐dependent cytochrome P450 monooxygenase mechanism.
Cell surface sialic acid affects immunoglobulin binding to macrophages
Authors: Gorczyca Wojciech , Wieczorek Zbigniew , Lisowski Józef
Published: July 2002

We demonstrate that guinea pig peritoneal macrophages pretreated with neuraminidase from Vibrio cholerae bind more 125I‐IgG than non‐treated cells. Estimation of binding constants (K
a and B
max) shows that the elevation of binding is the result of an increase in affinity and not in the number of receptors for IgG. The change of affinity is proportional to amounts of sialic acid liberated from the cells by increasing doses of neuraminidase. It is also shown that affinity of interactions of IgG with the macrophage receptor is pH dependent. These results indicate that electrostatic forces are important for IgG binding to the macrophage FcγR. The IgG‐FcγR interaction can be modulated by changing the degree of sialylation of the macrophage surface glycoconjugates.
Identification of sites on chromosomal protein HMG‐I phosphorylated by casein kinase II
Authors: Palvimo Jorma , Linnala-Kankkunen Annikka
Published: July 2002

High‐mobility‐group protein; Casein kinase II; Protein phosphorylation; Phosphopeptide; Chromatin
Time‐dependent inhibition of porcine kidney trehalase by aminosugars
Authors: Salleh Hamzah Mohd. , Honek John F.
Published: July 2002

The inhibitory effects of various nitrogen‐containing sugars on porcine kidney trehalase were studied. Validamycin A, validoxylamine A and MDL 25637 were found to be potent, time‐dependent inhibitors of the enzyme in vitro. The validoxylamine A‐inhibited enzyme showed slow but reversible reactivation over time (t

= 1.2 h). To our knowledge, this is the first report of time‐dependent inhibition exhibited by either these particular amino‐sugars or a trehalase.
Cellular mono(ADP‐ribosyl) transferase inhibits protein synthesis
Authors: Iglewski Wallace J. , Dewhurst Stephen
Published: July 2002

A reticulocyte translation system was depleted of functional EF‐2 by treatment with diphtheria toxin (DT) fragment A and NAD. After dialysis to remove NAD, the system was reconstituted using preparation of EF‐2 derived from pyBHK cells. Untreated and reconstituted lysates permitted similar rates of translation. As expected, when DT‐treated EF‐2 was used to reconstitute the system, no translation occurred. Furthermore EF‐2, reacting with the endogenous ADP‐ribosyl transferase from pyBHK cells, was also unable to restore protein synthesis in the reconstituted system. These studies suggest that eukaryotic cellular ADP‐ribosyl transferases may play a role in regulating protein synthesis.
A 64 kDa protein from Mycobacterium bovis BCG shares the same antigenic determinants with line 10 hepatoma cells and has anti‐line 10 tumor activity
Authors: Ahsan C.R. , Sasaki J.
Published: July 2002

A 64 kilodalton (kDa) surface protein was isolated from the water‐extracted materials from Mycobacterium bovis strain BCG, the determinants of which are antigenically shared by a 64 kDa major surface antigenic component of line 10 hepatoma cells. The 64 kDa protein showed anti‐line 10 tumor activity in pre‐immunized guinea pigs, and this suggests that the BCG 64 kDa protein is probably identical with the tumor specific antigen.
Evidence for a new extracellular peroxidase Manganese‐inhibited peroxidase from the white‐rot fungus Bjerkandera sp. BOS 55
Authors: de Jong Ed , Field Jim A. , de Bont Jan A.M.
Published: July 2002

A novel enzyme activity was detected in the extracellular fluid of Bjerkandera sp, BOS 55. The purified enzyme could oxidize several compounds, such as Phenol red, 2,6‐dimethoxyphenol (DMP), Poly R‐478, ABTS and guaiacol, with H2O2 as an electron acceptor. In contrast, veratryl alcohol was not a substrate. This enzyme also had the capacity to oxidize DMP in the absence of H2O2. With some substrates, a strong inhibition of the peroxidative activity by Mn2+ was observed. Phenol red oxidation was inhibited by 84% with only 1 mM of this metal ion. Because DMP oxidation by this enzyme is only slightly inhibited by Mn2+, this substrate should not be used in assays to detect manganese peroxidase. The enzyme is tentatively named ‘Manganese‐Inhibited Peroxidase’.
Chimeric study of sodium channels from rat skeletal and cardiac muscle
Authors: Chen L.-Q. , Chahine M. , Kallen R.G. , Barchi R.L. , Horn R.
Published: July 2002

Two isoforms of voltage‐dependent Na channels, cloned from rat skeletal muscle, were expressed in Xenopus oocytes. The currents of rSkM1 and rSkM2 differ functionally in 4 properties: (i) tetrodotoxin (TTX) sensitivity, (ii) μ‐conotoxin (μ‐CTX) sensitivity, (iii) amplitude of single channel currents, and (iv) rate of inactivation. rSkM1 is sensitive to both TTX and μ‐CTX. rSkM2 is resistant to both toxins. Currents of rSkM1 have a higher single channel conductance and a slower rate of inactivation than those of rSkM2. We constructed (i) chimeras by interchanging domain 1 (D1) between the two isoforms, (ii) block mutations of 22 amino acids in length that interchanged parts of the loop between transmembrane segments S5 and S6 in both D1 and D4, and (iii) point mutations in the SS2 region of this loop in D1. The TTX sensitivity could be switched between the two isoforms by the exchange of a single amino acid, tyrosine‐401 in rSkM1 and cysteine‐374 in rSkM2 in SS2 of D1. By contrast most chimeras and point mutants had an intermediate sensitivity to μ‐CTX when compared with the wild‐type channels. The point mutant rSkM1 (Y401C) had an intermediate single‐channel conductance between those of the wild‐type isoforms, whereas rSkM2 (C374Y) had a slightly lower conductance than rSkM2. The rate of inactivation was found to be determined by multiple regions of the protein, since chimeras in which D1 was swapped had intermediate rates of inactivation compared with the wild‐type isoforms.
Noradrenaline release from permeabilized synaptosomes is inhibited by the light chain of tetanus toxin
Authors: Stecher Brigitte , Hens Jacques J.H. , Weller Ulrich , Gratzl Manfred , Gispen Willem-Hendrik , De Graan Pierre N.E.
Published: July 2002

Noradrenaline release from rat brain cortical synaptosomes permeabilized with streptolysin O can be triggered by μM concentrations of free Ca2+. This process was inhibited within minutes by tetanus toxin and its isolated light chain, but not by its heavy chain. The data demonstrate that the effect of tetanus toxin on NA release from purified synaptosomes is caused by the intraterminal action of its light chain.
Light‐induced degradation of D2 protein in isolated photosystem II reaction center complex
Authors: Barbato Roberto , Friso Giulia , de Laureto Patrizia Polverino , Frizzo Annalisa , Rigoni Fernanda , Giacometti Giorgio M.
Published: July 2002

When isolated photosystem II reaction centers from spinach are exposed to photoinhibitory light in the presence of an electron acceptor, breakdown products of the D2 protein at 28, 25, 23, 18, 9, 5 and 4.5 kDa are detected by immunoblotting with a monospecific anti‐D2 polyclonal antibody. In a time—course experiment the 23 and 4.5 kDa fragments show a transient appearance, whilst the others are photoaccumulated. The regions of the D2 protein containing the cleavage sites for the 28 and 18 kDa photoinduced fragments have been identified. Significant degradation of D2 takes place only in the presence of an electron acceptor, and breakdown of the protein is partially prevented by serine‐type protease inhibitors.
Cytotoxic effect on lymphocytes of Tat from human immunodeficiency virus (HIV‐1)
Authors: Benjouad Abdelaziz , Mabrouk Kamel , Moulard Maxime , Gluckman Jean-Claude , Rocht Hervé , Van Rietschoten Jurphaas , Sabatier Jean-Marc
Published: July 2002

The human immunodeficiency virus type 1 (HIV‐1) genome codes for trans‐activator Tat, an 86‐residue protein whose expression is critical for viral replication. Full‐length Tat and Tat peptides from HIV‐1 were chemically synthesized using optimized solid phase technique. Synthetic Tat
2 in86, was found not only to inhibit antigen‐induced human peripheral blood lymphocyte (PBL) proliferation in vitro, as described by Viscidi et al. [1989, Science 246, 1606‐1608], but also mitogen‐induced PBL proliferation, with 50% inhibition obtained at 0.9 and 8 μ;M, respectively. To assess the mechanism by which Tat exert its inhibitory effect, we analysed its interaction and effect on CD4+‐cells. Direct fluorescence and indirect immunofluorescence assays analysed by flow cytometry showed that fluorescein isothiocyanate‐labeled and ‐unlabeled Tat interact (>0.2 μ;M) with CD4‐expressing lymphoid cells (CEM cell line). Experiments of chromium‐51 release and Trypan blue exclusion on these tumor cells in vitro have demonstrated the capacity of Tat to modify cellular membrane permeability and cell viability, in a dose‐dependent manner. The use of Tat peptides revealed that those containing the Tat basic region from 49 to 57 were able to bind to the cell membrane and to exhibit a cytotoxic activity on lymphocytes. Together, the data suggest that the potential cytotoxicity of Tat on lymphocytes could be directly implicated in virus‐induced immune dysfunction observed in HIV‐1 infected patients.
Synaptotagmin is endogenously phosphorylated by Ca2+/calmodulin protein kinase II in synaptic vesicles
Authors: Popoli Maurizio
Published: July 2002

The cytoplasmic domain of synaptotagmin (a synaptic vesicle‐specific protein) has a high degree of homology with the Ca2+‐phospholipid binding domain of protein kinase C. The Ca2+‐phospholipid binding activity of synaptotagmin has been implicated in the docking and fusion of synaptic vesicles with the presynaptic membrane during Ca2+‐induced exocytosis. The protein sequence contains potential phosphorylation sites for various protein kinases which could modulate its binding activity. At present there is no clear evidence that the protein is endogenously phosphorylated in intact vesicles. Here it is reported that phospho‐synaptotagmin was immunoprecipitated from endogenously phosphorylated synaptic vesicles. The conditions used indicate that synaptotagmin, as synapsin I, is phosphorylated by Ca2+/calmodulin‐dependent protein kinase II.
Expression and regulation of human and rat phosphodiesterase type IV isogenes
Authors: Engels Peter , Fichtel Katharina , Lübbert Hermann
Published: July 2002

Type IV phosphodiesterases (PDE IV) specifically hydrolyze cAMP and are inhibited by rolipram. RT‐PCR was applied to analyze the expression patterns of mRNAs for four cloned human and rat phosphodiesterase type IV isogenes (PDE IV‐A, ‐B, ‐C and ‐D). Although these patterns were mostly coincident for the human and rat PDE IV genes, some differences were found between the two species. PDE IV‐A expression was detectable in human blood but not in rat blood, suggesting a species‐specific difference in the expression of this PDE IV isogene. PDE IV‐C was neither detected in human or rat blood nor in different cell populations of the human immune system. It is further demonstrated that the PDE IV isogene expression is differentially regulated by cAMP in different cell types.
ARF1‐regulated phospholipase D in human neutrophils is enhanced by PMA and MgATP
Authors: Whatmore Jacqueline , Cronin Paul , Cockcroft Shamshad
Published: July 2002

Human neutrophil PLD activity stimulated with GTP‐γ‐S was reconstituted with recombinant ARF1 in cytosol‐depleted cells. PMA‐pretreatment of intact cells greatly enhanced the subsequent reconstitution of the ARF1‐regulated PLD activity. This enhancement was only observed provided that the intact cells were pretreated with PMA, suggesting the stable recruitment of a cytosolic component, presumably protein kinase C, to the membranes. rARF1‐reconstituted PLD activity was not dependent on MGATP, but could be considerably enhanced by MgATP. Maximal effects of MgATP were seen at 1 mM. This enhancement by MgATP could not be attributed to protein kinase C. Neomycin was found to inhibit ARF1‐regulated PLD activity suggesting the requirement for polyphosphoinositides. We conclude: (i) that many of the observed effects of PMA may be dependent on the presence of the small GTP‐binding protein, ARF, and (ii) polyphosphoinositides are required for ARF1‐stimulated PLD activity.
The scanning force microscopy of DNA in air and in n‐propanol using new spreading agents
Authors: Schaper Achim , Starink J.P.Pascual , Jovin Thomas M.
Published: July 2002

We present recent advances in DNA specimen preparation technique for scanning force microscopy (SFM) based on spreading on mica in the presence of cationic and non‐ionic detergents. Reproducible DNA imaging in air and in n‐propanol has been achieved in the presence of the non‐ionic detergent 2,4,6‐tris(dimethylaminomethyl) phenol (DMP‐30) or the cationic detergent cetylpyridinium chloride (CP) in a microdrop containing nanograms of DNA. In an alternative procedure, a microdrop of detergent is applied to the surface just prior to the DNA. Quantitative image analysis yields as the apparent molecular dimensions of the DNA a width of ∼ 7 nm and a height of ∼ 0.7 nm, and delineates the problems of DNA metrology by SFM.
Fourier transform infrared spectrum of the radical cation of β‐carotene photoinduced in photosystem II
Authors: Noguchi Takumi , Mitsuka Tomoya , Inoue Yorinao
Published: July 2002

A Fourier‐transform infrared (FTIR) spectrum of the radical cation of β‐carotene photoinduced in photosystem II (PSII) membranes was obtained at 80K under oxidizing conditions, by utilizing the light‐induced FTIR difference technique. Formation of the β‐carotene cation was monitored with the electronic absorption band at 993 nm. An FTIR spectrum of a chemically‐generated β‐carotene cation in chloroform was also measured and compared with the spectrum of PSII. Since the FTIR bands of carotenoid cation have characteristic features with strong intensities, they can be useful markers in studying the reaction of carotenoid in PSII.
Constitutive activation of cyclic AMP but not phosphatidylinositol signaling caused by four mutations in the 6th transmembrane helix of the human thyrotropin receptor
Authors: Kosugi Shinji , Shenker Andrew , Mori Toru
Published: July 2002

Four different somatic mutations (F631C, T632I, D633E, and D633Y) in the putative 6th transmembrane helix of the human thyrotropin receptor (TSHR) were recently described in hyperfunctioning thyroid adenomas [Porcellini et al. (1994) J. Clin. Endocrinol. Metab. 79, 657‐661]. We transiently expressed these mutant receptors in Cos‐7 cells and measured [125I]TSH binding, basal and TSH‐stimulated cAMP production, and phosphatidylinositol hydrolysis. The concentration of receptors expressed at the cell surface was lower for the mutants than for the wild type (WT) TSHR. Compared to the WT, all four mutant receptors caused a marked increase in basal cAMP levels, but did not increase basal production of inositol phosphates. This suggests that autonomous thyroid function and adenoma formation may be related to constitutive activation of the cAMP pathway alone. A cluster of conserved residues at the base of the 6th transmembrane helix of the TSHR and other glycoprotein hormone receptors appears important for maintaining an inactive receptor conformation.
The first cytoplasmic loop of the thyrotropin receptor is important for phosphoinositide signaling but not for agonist‐induced adenylate cyclase activation
Authors: Kosugi Shinji , Mori Toru
Published: July 2002

We investigated the role of the 1st cytoplasmic loop of the thyrotropin receptor (TSHR) on signal transduction using mutants as we did the 2nd and 3rd cytoplasmic loops [Kosugi S. et al. (1994) Mol. Endocrinol., in press; and 7, 1009‐1020]. Five substitution mutants involving the first cytoplasmic loop showed a TSH‐ or Graves' IgG‐stimulated cAMP response despite the low TSH binding B
max. All the mutants completely lost or markedly decreased the TSH‐ or Graves' IgG‐stimulated inositol phosphate increase. These findings suggest that the l st cytoplasmic loop of the TSHR does not play a crucial role in agonist‐induced adenylate cyclase activation but that it is important for phosphoinositide signaling.
Crystallisation of the Bacillus subtilis sporulation inhibitor SinR, complexed with its antagonist, Sinl
Authors: Lewis Richard J. , Brannigana James A. , Smith Issar , Wilkinson Anthony J.
Published: July 2002

The transcription factor SinR, a pleiotropic regulator of late growth processes in Bacillus subtilis, has been crystallised as a complex with its antagonist Sinl, in a form suitable for structural analysis. The Sinl : SinR crystals diffract X‐rays generated from a rotating copper anode source to 2.3 Å spacing and a complete native dataset has been collected to this resolution limit. The space group of the crystals is P3121 (or its enantiomorph P3221) with cell dimensions . Assuming that there is a single Sinl: SinR heterodimer in the asymmetric unit, the crystals have a V

m
of 2.53 Å3 · Da−1.
Endosomal proteolysis of internalized proteins
Authors: Authier François , Posner Barry I. , Bergeron John J.M.
Published: July 2002

Endosomal proteases have been implicated in the DAgradation of internalized regulatory peptides involved in the control of metabolic pathways and in the processing of intracellular antigens for cytolytic immune responses. Processing in the endocytic vesicles is regulated by changes in endosomal acidity due to the presence of an ATP‐DApenDAnt proton pump which modulates protease activity, protein unfolding and receptor‐ligand interactions. A limited number of proteases appear to reside in endosomes which do not contain the full complement of active proteases capable of completely DAgrading all internalized polypeptides. Retention of some acid hydrolases in endosomes is apparently related to their association with unDAfined endosomal membrane receptors. The limited number of proteases and the pH gradient from neutral to acidic (pH 7 to 5) within endosomes make possible a selective and controlled processing environment in comparison to lysosomes. The full set of endo‐ and exopeptidases that break down proteins to amino acids are active later in the pathway in lysosomes.
Evidence that Arg‐295, Glu‐378, and Glu‐380 are active‐site residues of the ADP‐ribosyltransferase activity of iota toxin
Authors: Perelle Sylvie , Domenighini Mario , Popoff Michel R
Published: July 2002

The active site of the enzymatic component (Ia) of the Clostridium perfringens iota toxin has been studied by site‐directed mutagenesis. Sequence alignment showed that Ia and C3 enzymes display a segment in their C‐terminal part which is homologous to that forming the active domain of pertussis toxin, cholera toxin, and Escherichia coli thermolabile toxins. This structure consists of a β‐strand and an α‐helix which forms the NAD‐binding cavity and which is flanked by two catalytic spatially conserved residues involved in catalysis [Domenighini et al. (1994) Mol. Microbiol. 14, 41–50]. Substitutions (Arg‐295‐Lys, Glu‐378‐Ala, Glu‐380‐Asp, and Glu‐380‐Ala) induced a drastic decrease in ADP‐ribosylation and cytotoxic activities, while substitution of the adjacent Arg (Arg‐296‐Lys) only partially affected the enzymatic activity and cytotoxicity. These results indicate that Arg‐295, Glu‐378 and Glu‐380 of Ia are involved in the ADP‐ribosylation activity which is essential for the morphological changes of cells treated with iota toxin.
Functional expression of human cone pigments using recombinant baculovirus: compatibility with histidine tagging and evidence for N‐glycosylation
Authors: Vissers Peter M.A.M. , DeGrip Willem J.
Published: July 2002

Mammalian color vision is mediated by light‐sensitive pigments in retinal cone cells. Biochemical studies on native mammalian cone visual pigments are seriously hampered by their low levels and instability. We describe a novel approach for their functional expression, employing the baculovirus system in combination with histidine tagging to allow future purification and structural analysis. The human red and green cone pigments are produced in relatively large amounts and can be detected by immunocytochemistry as well as by immunoblotting. Histidine tagging has no significant effect on the absorbance maxima. The first evidence is presented that these pigments are N‐glycosylation.
PDGF‐induced Akt phosphorylation does not activate NF‐κB in human vascular smooth muscle cells and fibroblasts
Authors: Rauch Bernhard H. , Weber Artur-Aron , Braun Marina , Zimmermann Norbert , Schrör Karsten
Published: July 2002

A recent report suggested that platelet‐derived growth factor (PDGF) activates nuclear factor‐κB (NF‐κB) by phosphorylation of the protein kinase Akt [Romashkova and Makarov, Nature 401 (1999) 86–90]. The present study investigates the role of Akt in the activation of NF‐κB by tumor necrosis factor‐α (TNFα, 10 ng/ml) and PDGF‐BB (20 ng/ml) in human vascular smooth muscle cells (SMC), skin and foreskin fibroblasts. TNFα stimulated serine phosphorylation and degradation of the inhibitory protein IκBα and strongly induced nuclear NF‐κB translocation and binding activity. PDGF did not induce serine phosphorylation or degradation of IκBα and did not enhance binding activity of NF‐κB. In contrast, stimulation with PDGF resulted in a marked phosphorylation of Akt, but no Akt phosphorylation occurred after stimulation with TNFα. These data suggest that Akt phosphorylation is not involved in NF‐κB activation in human SMC and fibroblasts.
Hepatoma cell migration through a mesothelial cell monolayer is inhibited by cyclic AMP‐elevating agents via a Rho‐dependent pathway
Authors: Mukai Mutsuko , Nakamura Hiroyuki , Tatsuta Masaharu , Iwasaki Teruo , Togawa Atsushi , Imamura Fumio , Akedo Hitoshi
Published: July 2002

1‐Oleoyl lysophosphatidic acid (LPA) induces transmonolayer migration (in vitro invasion) of rat ascites hepatoma MM1 cells and their morphological changes leading to the migration. We have previously shown that an LPA analog, palmitoyl cyclic phosphatidic acid (Pal‐cPA), suppresses transmonolayer migration of MM1 cells by rapidly increasing the intracellular cyclic AMP (cAMP) concentration. We report here that various cAMP‐elevating agents, including dibutyryl cAMP, forskolin, cholera toxin and 3‐isobutyl‐1‐methylxanthine, consistently inhibited LPA‐induced transmonolayer migration of MM1 cells. Moreover, pull‐down assays for GTP‐bound, active RhoA demonstrated that the blockage by cAMP‐elevating agents of morphological changes leading to the migration was probably mediated through inhibiting RhoA activation.
Limited proteolysis of the coxsackievirus and adenovirus receptor (CAR) on HeLa cells exposed to trypsin
Authors: Carson Steven D.
Published: July 2002

Trypsin treatment of HeLa cells results in a limited proteolysis of the coxsackievirus and adenovirus receptor (CAR) after which the cleaved CAR remains cell‐associated and tryptic peptides remain associated through disulfide bonds. Trypsin‐treated HeLa cells remain susceptible to infection with coxsackievirus B and produce progeny virus at 8 h post‐infection in amounts comparable to cells with intact CAR. HeLa cells remove the proteolysed CAR within 15 h and require over 24 h to restore intact CAR to control levels. As turnover is relatively slow, physiological functions that require intact CAR protein may be compromised for more than 24 h following trypsin treatment. Moreover, since removal of proteolysed CAR proceeds at more than twice the replacement rate, trypsin treatment disrupts the receptor‐per‐cell steady state for at least 24 h.
Serotonin transporter phosphorylation modulated by tetanus toxin
Authors: Najib Abderrahim , Pelliccioni Patricia , Gil Carles , Aguilera José
Published: July 2002

Tetanus toxin (TeTx) modifies Na+‐dependent, high‐affinity 5‐hydroxytryptamine (5‐HT, serotonin) uptake in a synaptosomal‐enriched P2 fraction from rat brain. The effect corresponds to a rapid and non‐competitive uptake inhibition, and it is preceded by induction of phospholipase C (PLC) activity and translocation and down‐regulation of the classical protein kinase C (PKC‐α, ‐β and ‐γ) isoforms. The effects on serotonin transport and on cPKC activation were similar to the effects exhibited by phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate (TPA). Moreover, after treatment with TeTx, an increase in Ser‐ and Tyr‐specific phosphorylation was found. Activation of PKC by both TeTx and TPA results in a loss of transport capacity and serotonin transporter (SERT) phosphorylation, which are abolished by coapplication of the specific PKC inhibitor bisindolylmaleimide‐1. Since a specific PLCγ1 phosphorylation prior to TeTx's inducing SERT phosphorylation was found, the studies suggest that part of the action of TeTx consists of modifying the signal cascade initiated in tyrosine kinase receptors on nerve tissue previous to its cellular internalization, resulting in transporter phosphorylation.
Functional characterization of novel human ARFGAP3
Authors: Liu Xiaoqin , Zhang Chenggang , Xing Guichun , Chen Qingtang , He Fuchu
Published: July 2002

ADP ribosylation factors (ARFs) are critical in the vesicular trafficking pathway. ARF activity is controlled by GTPase‐activating proteins (GAPs). We have identified recently a novel tentative ARF GAP derived from human fetal liver, ARFGAP3 (originally named as ARFGAP1). In the present study, we demonstrated that ARFGAP3 had GAP activity in vitro and remarked that the GAP activity of ARFGAP3 was regulated by phospholipids, i.e. phosphatidylinositol 4,5‐diphosphate as agonist and phosphatidylcholine as antagonist. ARFGAP3 is a predominantly cytosolic protein, and concentrated in the perinuclear region. Its transient ectopic overexpression in cultured mammalian cells reduced the constitutive secretion of secreted alkaline phosphatase, indicating that ectopic overexpression of ARFGAP3 inhibits the early secretory pathway of proteins in vivo. These results demonstrated that ARFGAP3 is a novel GAP for ARF1 and might be involved in intracellular traffic of proteins and vesicular transport as predicted.
Cracking the folding code. Why do some proteins adopt partially folded conformations, whereas other don't?
Authors: Uversky Vladimir N
Published: July 2002

Many, but not all, globular proteins have been shown to have compact intermediate state(s) under equilibrium conditions in vitro, giving rise to the question: why do some proteins adopt partially folded conformations, whereas other do not? Here we show that charge to hydrophobicity ratio of a polypeptide chain may represent a key determinant in this respect, as proteins known to form equilibrium partially folded intermediates are specifically localized within a unique region of charge–hydrophobicity space. Thus, the competence of a protein to form equilibrium intermediate(s) may be determined by the bulk content of hydrophobic and charged amino acid residues rather than by the positioning of amino acids within the sequence.
The involvement of the Saccharomyces cerevisiae multidrug resistance transporters Pdr5p and Snq2p in cation resistance
Authors: Miyahara Kohji , Mizunuma Masaki , Hirata Dai , Tsuchiya Eiko , Miyakawa Tokichi
Published: July 2002

The ATP‐binding cassette superfamily proteins Pdr5p and Snq2p of Saccharomyces cerevisiae are implicated in multidrug resistance. Here, we show that these transporters are also involved in cation resistance. Null mutants of PDR5 and SNQ2 genes exhibit increased sensitivity to NaCl, LiCl and MnCl2. The mutant cells grown in the presence of high concentrations of these metal salts contain higher levels of the metals than wild‐type cells. The expression of PDR5 and SNQ2 is induced by the metal salts. These results provide evidence that the yeast drug transporters contribute to cation resistance by regulating cellular cation homeostasis under ionic stress conditions.
Tyrosine phosphorylation and Fcγ receptor‐mediated phagocytosis
Authors: Strzelecka Agnieszka , Kwiatkowska Katarzyna , Sobota Andrzej
Published: July 2002

Phagocytosis of IgG‐opsonized particulate material in hematopoietic cells is mediated by Fcγ receptors (FcγRs). Interaction of the receptors with Fc domains of IgG triggers transduction of phagocytic signal in which a key role is played by phosphorylation of tyrosine residues of the receptors. These residues are arranged into a specific motif (immunoreceptor tyrosine‐based activation motif; ITAM) which is located either in the cytoplasmic part of FcγRIIA or in γ chains associated with FcγRI and FcγRIIIA. The conserved tyrosine residues are phosphorylated by, and associate with, tyrosine kinases of Src and Syk families. Coordinated action of these components initiates numerous intracellular events leading finally to local rearrangement of the actin‐based cytoskeleton and internalization of the particles.
Identification of a conserved phosphorylation site modulating nuclear lamin polymerization
Authors: Stuurman Nico
Published: July 2002

Mitotic lamin disassembly results from phosphorylation at specific sites. In vitro, lamins can form head‐to‐tail polymers that disassemble upon phosphorylation by cdc2 kinase. A co‐immunoprecipitation assay, employing Drosophila nuclear lamin Dm0 fragments was used to study the effect of phosphorylation on head‐to‐tail binding. Phosphorylation of serine‐50 by cAMP‐dependent kinase inhibited head‐to‐tail binding in the same manner as phosphorylation of serine‐42 by cdc2 kinase. Results suggest that multiple pathways may be employed to disassemble nuclear lamins in vivo.
Participation of the import receptor Tom20 in protein import into mammalian mitochondria: analyses in vitro and in cultured cells
Authors: Terada Kazutoyo , Kanazawa Masaki , Yano Masato , Hanson Brendon , Hoogenraad Nicholas , Mori Masataka
Published: July 2002

© 1997 Federation of European Biochemical Societies.
High affinity interaction of mouse DNA topoisomerase I with di‐ and trinucleotides corresponding to specific sequences of supercoiled DNA cleaved chain
Authors: Bugreev Dmitry V. , Vasyutina Elena L. , Buneva Valentina N. , Yasui Yoshihiro , Nishizawa Miwako , Andoh Toshiwo , Nevinsky Georgy A.
Published: July 2002

Recently mouse DNA topoisomerase I (topo) was shown to possess high affinity for a single‐stranded AAGACTTAG nonanucleotide (K
i=2.0 μM) corresponding to the scissile strand of the minimal DNA duplex, which is necessary for cleavage of supercoiled DNA. In order to determine the most important part of the above sequence for the DNA recognition by topo, the interactions of the enzyme with a set of extremely short (2–5 nucleotides in length) oligonucleotides corresponding to different parts of the nonanucleotide have been investigated. The affinities of different oligonucleotides corresponding to the CTTAG part of the sequence (K
i=0.13–0.92 mM) were shown to be significantly lower than that for the AAGA tetranucleotide (K
i=9.0 μM). Topo effectively recognized even short oligonucleotides containing only two or three bases (AGA and pAG, K
i=20 and 50 μM). We suppose that oligonucleotides having a high affinity to the enzyme can offer a unique opportunity for the rational design of topoisomerase‐targeting drugs.
A putative cytochrome c biogenesis gene in Synechocystis sp. PCC 68031
Authors: Hübschmann Thomas , Wilde Annegret , Elanskaya Irina , Shestakov Sergey V , Börner Thomas
Published: July 2002

A gene (orf334) with homology to chloroplast ycf5 (ccsA) was isolated from the cyanobacterium Synechocystis PCC 6803. The mRNA level of orf334 decreases in the dark and increases rapidly upon illumination. Transcription is initiated 69 nucleotides upstream of the start site of translation. The deduced amino acid sequence of orf334 has limited identity with bacterial proteins involved in cytochrome c biogenesis. Sequence comparison indicates differing pathways of cytochrome c biogenesis in cyanobacteria/chloroplasts and Gram− positive bacteria versus proteobacteria and mitochondria. Insertional inactivation of the orf334 gene gave rise to a heterozygous mutant, i.e. complete absence of the orf334 product seems to be lethal to the cell.
Modulation of cellular AP‐1 DNA binding activity by heat shock proteins
Authors: Carter David A.
Published: July 2002

Recent studies have indicated that ubiquitously expressed molecular chaperones of the heat shock protein (Hsp) class may have an additional, nuclear, role in the regulation of gene expression. Experiments on cellular transcription factors derived from the rat adrenal gland have now shown that Hsps modulate in vitro DNA binding activity of the AP‐1 factor. Both Hsc70 (p73) and Hsp70 (p72) were demonstrated to exert this effect through a mechanism that appears to be independent of both redox, and phosphorylation state. Further studies on the effect of Hsps on recombinant Fos/Jun protein binding activity indicated that the mechanism of action involves a selective attenuation of high affinity c‐Fos:c‐Jun binding as compared with c‐Jun homodimer binding activity. Because cellular and physiological stress are associated with the induction of both AP‐1 and Hsps it is apparent that Hsps may play a modulatory role in the regulation of AP‐1 responsive genes.
Synergistic action of R‐Ras and IGF‐1 on Bcl‐xL expression and caspase‐3 inhibition in BaF3 cells: R‐Ras and IGF‐1 control distinct anti‐apoptotic kinase pathways
Authors: Suzuki Jotaro , Kaziro Yoshito , Koide Hiroshi
Published: July 2002

R‐Ras and insulin‐like growth factor‐1 (IGF‐1) synergistically inhibit apoptosis of BaF3 cells upon interleukin‐3 deprivation. To characterize the mechanism of this synergistic inhibition, we examined the effect of R‐Ras and IGF‐1 on several apoptosis‐related proteins. Extracellular signal‐regulated kinase (ERK) was activated by IGF‐1, but not by R‐Ras. In contrast, Akt was activated strongly by R‐Ras, but weakly by IGF‐1. It was also found that R‐Ras and IGF‐1 cooperatively induced Bcl‐xL expression and inhibited caspase‐3 activation.
Inactivation of bacterial respiratory chain enzymes by singlet oxygen
Authors: Tatsuzawa Hidetaka , Maruyama Tadashi , Misawa Norihiko , Fujimori Ken , Hori Kazutoshi , Sano Yosiaki , Kambayashi Yasuhiro , Nakano Minoru
Published: July 2002

To distinguish the bactericidal action of singlet oxygen (1O2) from hypohalous acids, wild‐type and lycopene transformant E. coli strains were exposed to each of the oxidants and then bacterial viability was investigated. 1O2 was generated by chemical and enzymatic systems at pH 4.5. Exposure of wild‐type E. coli to 1O2 caused a significant loss of E. coli viability due to inactivation of membrane respiratory chain enzymes by 1O2. This action of 1O2 could be attenuated by lycopene in the bacterial cell membrane. In the lycopene transformant strain of E. coli, inactivation of NADH oxidase and succinate oxidase by hypohalous acids were significantly suppressed, but E. coli viability was unaffected. Based on these findings, we suggest that phagocytic leukocytes produce 1O2 as a major bactericidal oxidant in the phagosome.