bluuebunny/crossref_metadata_2025_split huggingface dataset:
Dynamic redistribution of STAT1 protein in IFN signaling visualized by GFP fusion proteins
Authors: Köster Mario , Hauser Hansjörg 
Published: March 2003

STAT proteins (signal transducers and activators of transcription) are a family of transcription factors which are used by many cytokines and cell growth factors for initiating gene expression. They are activated by tyrosine phosphorylation through the cytoplasmic domain of stimulated receptors. Upon phosphorylation STAT proteins dimerize, translocate to the nucleus and activate transcription by binding to specific recognition sites. Different cytokines activate different subsets of STATs and other signaling proteins. We have made use of green fluoresencent protein (GFP) fusion proteins to visualize the subcellular localization and trafficking of STAT1, STAT2 and p48 during interferon (IFN) stimulation and have analysed in detail STAT1‐GFP trafficking in living cells. Analysis of GFP fusion proteins allowed the determination of time kinetics of subcellular trafficking in individual living cells. STAT1‐GFP is indistinguishable from its wild‐type protein displaying strong activity as transcriptional activator as well as the same time kinetics of transport to the nucleus and retreat to the cytoplasm. After prolonged exposure to IFN, STAT1‐GFP is no longer retained in the nucleus and relocation to the cytoplasm is observed. Restimulation with the same type of IFN does not lead to repeated nuclear translocation of STAT1‐GFP. STAT1 is not subject of inhibition, as restimulation with another type of IFN allows immediate reuse of previously activated STAT1‐GFP. However, restimulation with the same type of IFN can be achieved when the primary stimulus is removed after a short induction period. This method of visualizing signal transduction reveals a considerable inhomogeneity with respect to the extent of STAT1‐GFP shuttling within a clonal cell population, indicating that competence for full‐blasted IFN response is restricted to a cellular subpopulation whereas other cells respond incompletely, retarded or not at all.
Purification, redox and spectroscopic properties of the tetraheme cytochrome c isolated from Rubrivivax gelatinosus
Authors: Agalidis Ileana , Othman Samya , Boussac Alain , Reiss‐Husson Françoise , Desbois Alain 
Published: March 2003

The tetraheme cytochrome c subunit of the Rubrivivax gelatinosus reaction center was isolated in the presence of octyl β‐d‐thioglucoside by ammonium sulfate precipitation and solubilization at pH 9 in a solution of Deriphat 160. Several biochemical properties of this purified cytochrome were characterized. In particular, it forms small oligomers and its N‐terminal amino acid is blocked. In the presence or absence of diaminodurene, ascorbate and dithionite, different oxidation/reduction states of the isolated cytochrome were studied by absorption, EPR and resonance Raman spectroscopies. All the data show two hemes quickly reduced by ascorbate, one heme slowly reduced by ascorbate and one heme only reduced by dithionite. The quickly ascorbate‐reduced hemes have paramagnetic properties very similar to those of the two low‐potential hemes of the reaction center‐bound cytochrome (gz = 3.34), but their α band is split with two components peaking at 552 nm and 554 nm in the reduced state. Their axial ligands did not change, being His/Met and His/His, as indicated by the resonance Raman spectra. The slowly ascorbate‐reduced heme and the dithionite‐reduced heme are assigned to the two high‐potential hemes of the bound cytochrome. Their α band was blue‐shifted at 551 nm and the gz values decreased to 2.96, although the axial ligations (His/Met) were conserved. It was concluded that the estimated 300 mV potential drop of these hemes reflected changes in their solvent accessibility, while the reduction in gz indicates an increased symmetry of their cooordination spheres. These structural modifications impaired the cytochrome’s essential function as the electron donor to the photooxidized bacteriochlorophyll dimer of the reaction center. In contrast to its native state, the isolated cytochrome was unable to reduce efficiently the reaction center purified from a Rubrivivax gelatinosus mutant in which the tetraheme was absent. Despite the conformational changes of the cytochrome, its four hemes are still divided into two groups with a pair of low‐potential hemes and a pair of high‐potential hemes.
Effect of gel re‐organization and tensional forces on α2β1 integrin levels in dermal fibroblasts
Authors: Jenkins Gail , Redwood Katherine L. , Meadows Lisa , Green Martin R. 
Published: March 2003

Mechanical forces are known to play an important role in regulating cell function in a wide range of biological systems. This is of particular relevance to dermal fibroblast function, given that the skin is known to be held under an intrinsic natural tension. To understand more about the generation of force by dermal fibroblasts and their ability to respond to changes in it, we have studied the role of the β1 integrin receptors expressed by dermal fibroblasts in their ability to generate tensional forces within a collagen type I matrix and the effect of altered tensional force on integrin expression by dermal fibroblasts. Using a purpose‐built culture force monitor, function‐blocking antibodies directed towards the β1 receptors dramatically reduced the tensional forces generated by dermal fibroblasts in a 3D collagen I matrix. However, the specific involvement of α1 or α2 subunits could not be demonstrated. Analysis of cellular response demonstrated that cells isolated from contracting collagen gels expressed fourfold higher levels of α2 mRNA than cells isolated from fully restrained gels. The levels of β1 messenger RNA were relatively unaffected by reductions in force. Cells exposed to single reductions in force, however, did not exhibit alterations in either α1 or β1 mRNA levels. We propose, therefore that α2β1 integrin receptor levels in dermal fibroblasts are not altered in response to single reductions of gel tension, but do change following a continual change in force and associated matrix re‐organization
Roles of Gln81 and Cys80 in catalysis by glycosylphosphatidylinositol‐phospholipase C from Trypanosoma brucei
Authors: Rashid Mohammad B. , Russell Malissa , Mensa‐Wilmot Kojo 
Published: March 2003

Glycosylphosphatidylinositol‐specific phospholipase C (GPtdIns‐PLC) is found in the protozoan parasite Trypanosoma brucei. A region of protein sequence similarity exists between the protozoan enzyme and eubacterial phosphatidylinositol‐phospholipases C. The functional relevance of Cys80 and Gln81 of GPtdIns‐PLC, both in this region, was tested with a panel of mutations at each position. Gln81Glu, Gln81Ala, Gln81Gly, Gln81Lys and Gln81Leu mutants were inactive. Cleavage of GPtdIns was detectable in Gln81Asn, although the specific activity decreased 500‐fold, and kcat was reduced 50‐fold. Thus an amide side‐chain at residue 81 is essential for catalysis by GPtdIns‐PLC. Sulfhydryl reagents inactivate GPtdIns‐PLC, suggesting that a Cys could be close to the enzyme active site. Surprisingly, p‐chloromercuriphenyl sulfonate (p‐CMPS) is significantly more potent than N‐ethylmaleimide, the less bulky compound. This knowledge prompted us to test whether replacement of Cys80 with an amino acid possessing a bulky side‐chain would inactivate GPtdIns‐PLC: Cys80Ala, Cys80Thr, Cys80Phe, Cys184Ala, and Cys269‐270‐273Ser were constructed for that purpose. Cys80Phe lacked enzyme activity, while Cys80Ala, Cys80Thr and Cys269‐270‐273Ser retained 33 to 100% of wild‐type activity. Interestingly, the Cys80Ala and Cys80Thr mutants became resistant to p‐CMPS, as predicted if the sulfhydryl reagent reacted with Cys80 in the wild‐type enzyme to form a cysteinyl mercurylphenylsulfonate moiety, a bulky adduct that inactivated GPtdIns‐PLC, similar to the Cys80Phe mutation. We conclude that a bulky side‐chain (or adduct) at position 80 of GPtdIns‐PLC abolishes enzyme activity. Together, these observations place Cys80 and Gln81 at, or close to, the active site of GPtdIns‐PLC from T. brucei.
Structure–activity relationship and site of binding of polyamine derivatives at the nicotinic acetylcholine receptor
Authors: Bixel M. Gabriele , Krauss Michael , Liu Ying , Bolognesi Maria L. , Rosini Michela , Mellor Ian S. , Usherwood Peter N. R. , Melchiorre Carlo , Nakanishi Koji , Hucho Ferdinand 
Published: March 2003

Several wasp venoms contain philanthotoxins (PhTXs), natural polyamine amides, which act as noncompetitive inhibitors (NCIs) on the nicotinic acetylcholine receptor (nAChR). Effects of varying the structure of PhTXs and poly(methylene tetramine)s on the binding affinity have been investigated. Using the fluorescent NCI ethidium in a displacement assay Kapp values of these compounds have been determined. We found that an increase in size of the PhTX's hydrophobic head group significantly increased the binding affinity, while inserting positive charge almost completely destroyed it. Elongating the PhTX polyamine chain by introducing an additional aminomethylene group decreased the binding affinity, whereas a terminal lysine improved it. In general, poly(methylene tetramine)s showed higher binding affinities than PhTX analogues.The stoichiometry of PhTX binding was determined to be two PhTX molecules per receptor monomer. PhTXs appeared to bind to a single class of nonallosterically interacting binding sites and bound PhTX was found to be completely displaced by well‐characterized luminal NCIs. To elucidate the site of PhTX binding, a photolabile, radioactive PhTX derivative was photocross‐linked to the nAChR in its closed channel conformation resulting in labeling yields for the two α and the β, γ and δ subunits of 10.4, 11.1, 4.0 and 7.4%, respectively.Based on these findings we suggest that PhTXs and poly(methylene tetramine)s enter the receptor's ionic channel from the extracellular side. The hydrophobic head groups most likely bind to the high‐affinity NCI site, while the positively charged polyamine chains presumably interact with the negatively charged selectivity filter located deep in the channel lumen.
Kinetic properties of the 2‐oxoglutarate dehydrogenase complex from Azotobacter vinelandii
Authors: Bunik Victoria , Westphal Adrie H. , de Kok Aart 
Published: March 2003

The 2‐oxoglutarate dehydrogenase complex was purified from Azotobacter vinelandii. The complex consists of three components, 2‐oxoglutarate dehydrogenase/decarboxylase (E1o), lipoate succinyltransferase (E2o) and lipoamide dehydrogenase (E3). Upon purification, the E3 component dissociates partially from the complex. From reconstitution experiments, the Kd for E3 was found to be 26 nm, about 30 times higher than that for the pyruvate dehydrogenase complex. The Km values for the substrates 2‐oxoglutarate, CoA and NAD+ were found to be 0.15, 0.014 and 0.17 mm, respectively. The system has a high specificity for 2‐oxoglutarate, which is determined by the action of both E1o and E2o. Above 4 mm substrate inhibition is observed.From steady‐state inhibition experiments with substrate analogs, two substrate‐binding modes are revealed at different degrees of saturation of the enzyme with 2‐oxoglutarate. At low substrate concentrations (10−6 to 10−5 m), the binding mainly depends on the interaction of the enzyme with the substrate carboxyl groups. At a higher degree of substrate saturation (10−4 to 10−3 m) the relative contribution of the 2‐oxo group in the binding increases.A kinetic analysis points to a single binding site for a substrate analog under steady state conditions. Saturation of this site with an analog indicates that two kinetically different complexes are formed with 2‐oxoglutarate in the course of catalysis. From competition studies with analogs it is concluded that one of these complexes is formed at the site that is sterically identical to the substrate inhibition site.The data obtained are represented by a minimal scheme that considers formation of a precatalytic complex SE between the substrate and E1o before the catalytic complex ES, in which the substrate is added to the thiamin diphosphate cofactor, is formed. The incorrect orientation of the substrate molecule in SE or the occupation of this site by analogs is supposed to cause substrate or analog inhibition, respectively.
Isolation and characterization of RNA aptamers specific for the hepatitis C virus nonstructural protein 3 protease
Authors: Fukuda Kotaro , Vishnuvardhan Daesety , Sekiya Satoru , Hwang Joonsung , Kakiuchi Nobuko , Taira Kazunari , Shimotohno Kunitada , Kumar Penmetcha K. R. , Nishikawa Satoshi 
Published: March 2003

Nonstructural protein 3 (NS3) from hepatitis C virus (HCV) is a serine protease that provides an essential function in maturation of the virus by cleaving the nonstructural regions of the viral polyprotein. The goal of this work was to isolate RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide ΔNS3. RNA aptamers were selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). The RNA pool for SELEX had a 30‐nucleotide randomized core region. After nine selection cycles, a pool of ΔNS3‐specific RNA aptamers were obtained. This RNA pool included 45 clones that divided into three main classes (G9‐I, II and III). These classes include the conserved sequence GA(A/U)UGGGAC. These aptamers bind to ΔNS3 with a binding constant of about 10 nm and inhibit approximately 90% of the protease activity of ΔNS3 and MBP‐NS3 (full‐length of NS3 fused with maltose binding protein). In addition, these aptamers inhibited approximately 70% of the MBP‐NS3 protease activity in the presence of the NS4A peptide P41. G9‐I aptamer appeared to be a noncompetitive inhibitor for ΔNS3 with a Ki ≈ 100 nm in the presence of P41. These results suggest that the pool of selected aptamers have potential as anti‐HCV compounds. Mutational analysis of the G9‐I aptamer demonstrated that the sequences required for protease inhibition are in stem I, stem III and loop III of the aptamer. These regions include the conserved sequence GA(A/U)UGGGAC.
Mechanism of reaction of myeloperoxidase with hydrogen peroxide and chloride ion
Authors: Furtmüller Paul G. , Obinger Christian , Hsuanyu Yuchiong , Dunford H. Brian 
Published: March 2003

The reaction of myeloperoxidase compound I (MPO‐I) with chloride ion is widely assumed to produce the bacterial killing agent after phagocytosis. Two values of the rate constant for this important reaction have been published previously: 4.7 × 106 m−1·s−1 measured at 25 °C [Marquez, L.A. and Dunford, H.B. (1995) J. Biol. Chem.270, 30434–30440], and 2.5 × 104 m−1·s−1 at 15 °C [Furtmüller, P.G., Burner, U. & Obinger, C. (1998) Biochemistry37, 17923–17930]. The present paper is the result of a collaboration of the two groups to resolve the discrepancy in the rate constants. It was found that the rate constant for the reaction of compound I, generated from myeloperoxidase (MPO) and excess hydrogen peroxide with chloride, decreased with increasing chloride concentration. The rate constant published in 1995 was measured over a lower chloride concentration range; the 1998 rate constant at a higher range. Therefore the observed conversion of compound I to native enzyme in the presence of hydrogen peroxide and chloride ion cannot be attributed solely to the single elementary reaction MPO‐I + Cl− → MPO + HOCl. The simplest mechanism for the overall reaction which fit the experimental data is the following:
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where MPO‐I–Cl− is a chlorinating intermediate. We can now say that the 1995 rate constant is k2 and the corresponding reaction is rate‐controlling at low [Cl−]. At high [Cl−], the reaction with rate constant k3 is rate controlling. The 1998 rate constant for high [Cl−] is a composite rate constant, approximated by k2k3/k−2. Values of k1 and k−1 are known from the literature. Results of this study yielded k2 = 2.2 × 106 m−1·s−1, k−2 = 1.9 × 105 s−1 and k3 = 5.2 × 104 s−1. Essentially identical results were obtained using human myeloperoxidase and beef spleen myeloperoxidase.
Functional characterization of green fluorescent protein–profilin fusion proteins
Authors: Wittenmayer Nina , Rothkegel Martin , Jockusch Brigitte M. , Schlüter Kathrin 
Published: March 2003

To clarify the role of profilins in cells, fusion proteins constructed with green fluorescent protein (GFP) should be extremely helpful. As profilins are considerably smaller than the GFP fusion partner (14–17 kDa compared with 27 kDa, respectively), we characterized the fusion proteins in vitro, to ascertain their biological function.We fused mouse profilin I and II to either the C‐terminus or N‐terminus of GFP. These fusion proteins were expressed in Escherichia coli and affinity‐purified on polyproline–Sepharose. Interaction with vasodilator‐stimulated phosphoprotein, a proline‐rich ligand of profilin, was investigated by ELISA, as was binding to PtdIns(4,5)P2. The affinity for actin was quantitatively determined in polymerization assays. Our results show that fusion of GFP to the C‐terminus of profilin I abolishes polyproline binding. In contrast, the other fusion proteins bound to polyproline–Sepharose and VASP. Binding to PtdIns(4,5)P2 was not significantly altered. Furthermore, fusion of either isoform with GFP did not decrease the affinity for actin. In localization studies with mammalian cells, all fusion proteins showed the localization expected for profilin in areas of high actin dynamics, such as leading lamellae and ruffles induced by epidermal growth factor. However, with regard to our in vitro data, we suspect that only a minor fraction of profilin I carrying the GFP at the C‐terminus can target these sites. Therefore, other constructs should be preferred for further in vivo studies.
Molecular heterogeneity of the hemocyanin isolated from the king crab Paralithodes camtschaticae
Authors: Molon Annamaria , Di Muro Paolo , Bubacco Luigi , Vasilyev Vadim , Salvato Benedetto , Beltramini Mariano , Conze Werner , Hellmann Nadja , Decker Heinz 
Published: March 2003

Native Paralithodes camtschaticae hemocyanin is found as a mixture of dodecamers (24S; 80%) and hexamers (16S; 20%). Removal of Ca2+ ions by dialysis against EDTA‐containing buffer solution at neutral pH induces complete dissociation of the 24S form into the 16S form. Under these conditions, a further increase in pH to 9.2 produces complete dissociation of the hexamers into monomers (5S). In both cases, the dissociation process is reversible. The dodecamer (24S) is composed of two different hexamers which can be discriminated only by ion‐exchange chromatography in the presence of Ca2+ ions. At alkaline pH and in the presence of EDTA, two major monomeric fractions can be separated by ion‐exchange chromatography: ParcI (60%) and ParcII (40%). The reassociation properties of the two fractions were studied separately to define their ability to form hexamers and dodecamers.The oxygen‐binding properties of the different aggregation states were investigated. Native hemocyanin binds O2 co‐operatively (nH = 3) and with low affinity (p50 ≈ 103 Torr). The two monomeric fractions, ParcI and ParcII, are not co‐operative and the affinity is twice that of the native protein (p50 ≈ 65 and 52 Torr).Oxygen‐binding measurements of native hemocyanin carried out at different pH values indicate a strong positive Bohr effect within the pH range 6.5–8.0 and an increase in oxygen affinity at pH below 6.5.
Expression of glucagon‐like peptide‐1 (GLP‐1) receptor and the effect of GLP‐1‐(7–36) amide on insulin release by pancreatic islets during rat ontogenic development
Authors: García‐Flores Marta , Zueco José A. , Álvarez Elvira , Blázquez Enrique 
Published: March 2003

The expression of glucagon‐like peptide‐1 (GLP‐1) receptor and the effects of GLP‐1‐(7–36) amide (t‐GLP‐1) on glucose metabolism and insulin release by pancreatic islets during rat development were studied. GLP‐1 receptor mRNA was found in significant amounts in pancreatic islets from all age groups studied, GLP‐1 receptor expression being maximal when pancreatic islets were incubated at physiological glucose concentration (5.5 mm), but decreasing significantly when incubated with either 1.67 or 16.7 mm glucose. Glucose utilization and oxidation by pancreatic islets from fetal and adult rats rose as a function of glucose concentration, always being higher in fetal than in adult islets. The addition of t‐GLP‐1 to the incubation medium did not modify glucose metabolism but gastric inhibitory polypeptide and glucagon significantly increased glucose utilization by fetal and adult pancreatic islets at 16.7 mm glucose. At this concentration, glucose produced a significant increase in insulin release by the pancreatic islets from 10‐day‐old and 20‐day‐old suckling rats and adult rats, whereas those from fetuses showed only a significant increase when glucose was raised from 1.67 to 5.5 mm. t‐GLP‐1 elicited an increase in insulin release by pancreatic islets from all the experimental groups when the higher glucose concentrations were used. Our findings indicate that GLP‐1 receptors and the effect of t‐GLP‐1 on insulin release are already present in the fetus, and they therefore exclude the possibility that alterations in the action of t‐GLP‐1 are responsible for the unresponsiveness of pancreatic β cells to glucose in the fetus, but stimulation of t‐GLP‐1 release by food ingestion in newborns may partially confer glucose competence on β cells.