bluuebunny/crossref_metadata_2025_split huggingface dataset:
Redox‐Mediated Regulation of p21Waf1/Cip1 Expression Involves a Post‐Transcriptional Mechanism and Activation of the Mitogen‐Activated Protein Kinase Pathway
Authors: Esposito Franca , Cuccovillo Franca , Vanoni Marco , Cimino Filiberto , Anderson Carl W. , Appella Ettore , Russo Tommaso 
Published: July 2004

p21waf/cip1gene expression is induced by DNA damage in cells with wild‐type p53 and contributes to the arrest of cell growth. It was demonstrated that under many experimental conditions, including oxidative stress, p21waf/cip1 expression can be induced through p53‐independent pathways. Since most of these experimental conditions induce the phosphorylation of mitogen‐activated protein kinase (MAPK) and thus its activation, we evaluated p21waf/cip1 mRNA levels in cells exposed to an oxidative stress, induced by diethylnialeate (Et2Mal), and in which the MAPK pathway was blocked. The expression of a dominant‐negative mutant of MEK, the MAPK kinase that phosphorylates and activates MAPK, and of a dominant‐negative [Asn17]Ras mutant prevented the EtZMal‐induced accumulation of p21waf/cipl mRNA. Similarly, the expression of MEK−and of [Asn17]Ras mutants decreased the 12‐O‐tetradecanoyl‐phorbol 13‐acetate (TPA)‐mediated p21waf1/cip1induction. Furthermore, TPA‐induced and serum‐induced p21waf1/cip1mRNA accumulation was blocked by pretreating the cells with the antioxidant compound N‐acetylcysteine, suggesting that oxidative stress is involved in these responses.p21waf1/cipl mRNA levels reached a maximum within 2 h of adding Et2Mal or TPA; however, the rate of transcription from a p21waf1/cip1‐promoter construct did not increase during this period. In contrast, cells treated with actinomycin D show an increase of p21waf1/cip1 mRNA stability after Et2 Mal treatment.This result suggests that the increase in p21waf1/cip1 mRNA at early times results from post‐transcriptional regulatory events. Longer exposure to TPA may activate p21waf1/cip1 gene transcription through an Sp1‐dependent mechanism, while Et2Mal treatment gradually inhibits p21waf1/cipgene transcription through oxidative changes that affect Sp1 binding to DNA.
Cloning, Sequencing and Functional Expression of Dihydrolipoamide Dehydrogenase from the Human Pathogen Trypanosoma Cruzi
Authors: SchÖneck Ralf , Billaut‐Mulot Odile , Numrich Petra , Ouaissi M. Ali , Krauth‐Siegel R. Luke 
Published: July 2004

This work presents the complete sequences of a cDNA and the two allelic genes of dihydrolipoamide dehydrogenase (LipDH) from Trypanosoma cruzi, the causative agent of Chagas' disease (American trypanosomiasis). The full‐length cDNA has an ORF of 1431 bp and encodes a protein of 477 amino acid residues. LipDH is a homodimeric protein with FAD as prosthetic group. The calculated molecular mass of the subunit of the mature protein with bound FAD is 50066. Comparison of the deduced amino acid sequence of LipDH from T. cruzi with that of Trypanosoma brucei and man shows identities of 81% and 50%, respectively.An N‐terminal nonapeptide, not present in the mature enzyme, represents a mitochondrial targeting sequence so far found only in trypanosomatids. The gene lpd1 of T. cruzi LipDH was expressed without the targeting sequence in Escherichia coli JRG1342 cells which are deficient for LipDH. For this purpose an ATG codon was introduced directly upstream the codon for Asn10 which represents the N‐terminus of the mature protein. This system allowed the synthesis of 1000U T. cruzi LipDH/1 bacterial cell culture. The recombinant protein was purified to homogeneity by (NH4)2SO4‐precipitation and affinity chromatography on 5′ AMP‐Sepharose. The Km, values for NAD+, NADH, lipoamide and dihydrolipoamide are identical with those of the enzyme isolated from the parasite.LipDH is present in all major developmental stages of T. cruzi as shown by northern and western blot analyses. This finding is in agreement with the citric acid cycle being active throughout the whole life cycle of the parasite. In vitro studies on a mammalian LipDH revealed the ability of the flavoenzyme to catalyze the redox‐cycling and superoxide anion production of nitrofuran derivatives including the antitrypanosomal drug Nifurtimox. For that reason T. cruzi LipDH is regarded as a promising target for the structure‐based development of new antiparasitic drugs. The bacterial expression system for the parasite enzyme will now allow the study of the role of T. cruzi LipDH in drug activation and the crystallization of the protein.
Molecular Characterization of Fdx1, a Putidaredoxin‐Type [2Fe‐2S] Ferredoxin Able to Transfer Electrons to the Dioxin Dioxygenase of Sphingomonas sp. RW1
Authors: Armengaud Jean , Timmis Kenneth N. 
Published: July 2004

Bacterium Sphingomonas sp. strain RW1 is, under aerobic conditions, able to degrade dibenzofuran and dibenzo‐p‐dioxin. The first step of the pathway is performed by a ring‐dihydroxylating enzyme. Bünz and Cook have reported the purification and characterization of this dioxin dioxygenase and a ferredoxin able to transfer electrons to the dioxygenase [Bünz, P. V. & Cook, A. M. (1993) J. Bacteriol. 175, 6467–64751. The gene encoding this [2Fe‐2S] ferredoxin was identified by screening a genomic library constructed in pLAFR3 with a probe generated by a nested‐PCR amplification. Primers for the amplification were designed based on the N‐terminus sequence of the purified ferredoxin and on sequence comparisons with related proteins, Several cosmids were obtained and the ferredoxin gene, fdxl, was subcloned from one of them. The nucleotide sequence of a 4.6‐kb DNA fragment encompassing the ferredoxin gene was determined. While in the case of all known multi‐component dioxygenases, genes encoding the a and β subunits are found to be contiguous with the gene of the specific electron carrier, the fdxl gene in Sphingomonas sp. RW1 does not appear to be directly linked with the dioxin dioxygenase genes. Rather, it is clustered with genes apparently encoding two atypical decarboxylaseslisomerases and a glutathione S‐transferase.The ferredoxin gene was hyperexpressed and the recombinant ferredoxin was purified. Spectroscopic characterization of Fdx1 demonstrated the presence of a putidaredoxin‐type [2Fe‐2S] cluster in this protein. Its redox potential was determined to be ‐245 (±5) mV versus the normal hydrogen electrode at 25°C, pH 8.0. Therefore, the protein is closely related to [2Fe‐2S] ferredoxins known to be electron donors to monooxygenases involved in hydroxylation of aromatic compounds. Thus, this report provides clear evidence that a putidaredoxin‐type [2Fe‐2S] ferredoxin, namely Fdxl, is able to transfer electrons to the dioxin dioxygenase of Sphingomonas sp. RW1.
Characterization of the Embryonic Globin Chains of the Marsupial Tammar Wallaby, Macropus Eugenii
Authors: Holland Robert A. B. , Gooley Andrew A. 
Published: July 2004

The embryonic hemoglobins of the marsupial Tammar wallaby (Macropus eugenii) are known to aggregate, which was shown by the finding that the Hill coefficient, h, was greater than 4.0 in the upper part of the oxygen equilibrium curve. Here, we have undertaken a detailed primary structure analysis of the Tammar wallaby pouch young hemoglobin complement, which we hoped might provide clues into the residues that cause aggregation and a high embryonic h.The Tammar wallaby embryonic hemoglobin complement is principally four major hemoglobins each with a different isoelectric point. Two early expressed hemoglobins contain the same embryonic β‐like chain, epsilon (ɛ), but two separate α‐like chains, termed zeta and zeta prime (ζ and ζ′)both of which are N‐terminally blocked. The later two expressed hemoglobins contain the same adult α‐chain, but different β‐like chains. The latest expressed hemoglobin contains the same β‐like chain, ɛ, as the two early expressed forms, but the third expressed hemoglobin contains a unique β‐like chain which we have termed omega (ω). A protein database similarity search using the first 54 N‐terminal amino acids of the ω‐chain showed a range of sequence identities of 57–72% to all known mammalian β‐like chains, including the other marsupial ɛ‐chains. The closest identity, reflected by both the highest percentage identity and Smith‐Waterman score, was with the embryonic β‐chains of the aves.While the primary structures of the hemoglobins reported here do not explain the low hemoglobin‐oxygen affinity in embryonic marsupial blood, the finding of the similarity with the bird globin‐like sequence with one of the marsupial chains has implications on mammalian globin evolution. How many other marsupials and placental mammals are harboring a bird‐like globin in their embryos?
Small‐Angle X‐Ray Scattering Analysis of Pneumococcal Lipoteichoic Acid Phase Structure
Authors: Fischer Werner , Markwitz Sabine , Labischinski Harald 
Published: July 2004

X‐Ray‐scattering analysis was performed on micelles of lipoteichoic acid from Streptococcus pneumoniae. This lipoteichoic acid differs from poly(glycerophosphate) and poly(glycosylglycerophosphate) lipoteichoic acids by a unique positively charged diacylglyceroglycolipid anchor and a complex structure of the hydrophilic chain which is composed of zwitterionic tetrasaccharide ribitol phosphate repeats each carrying two zwitterionic phosphocholine substituents. The size distribution of the lipoteichoic acid micelles was sufficiently homogenous to determine their size and some related molecular parameters by small‐angle scattering analysis. Nearly independent on the ionic strength of the aqueous dispersion, an average micelle contained about 150 lipoteichoic acid molecules arranged in a spherical assembly with a diameter of about 23 nm, whereby the hydrophilic region occupied an outer shell of about 8.5 nm thickness. Taken this as the average chain length of LTA in the micelle and 7.2 repeats per chain, each repeat contributed about 1.2nm to the thickness of the hydrophilic shell as compared to 2.4 nm for a fully extended chain conformation and 1.1 nm estimated for a more helical arrangement [Klein, R. A., Hartmann, R., Egge, H., Behr, T. & Fischer, W. (1994) Carbohydr. Res. 256, 189‐222].A comparison with the micelle of poly(glycerophosphate) lipoteichoic acid of Staphylococcus aureus suggests that the supramolecular structure is largely independent of the structure of the hydrophilic chain and solely dictated by the small cross‐sectional area of the diacylglycerol moiety common to all lipoteichoic acids and lipoglycans of gram‐positive bacteria.
Structural Aspects of the Interaction of peptidyl‐glycylleucine‐carboxyamide, a Highly Potent Antimicrobial Peptide from Frog Skin, with Lipids
Authors: Latal Angelika , Degovics Gabor , Epand Raquel F. , Epand Richard M. , Lohner Karl 
Published: July 2004

The interaction of PGLa (peptidyl‐glycylleucine‐carboxyamide), a 21‐amino‐acid residue cationic peptide, isolated from the skin of the South African clawed frog, Xenopus laevis, with model membrane systems was investigated. Our studies focussed on the importance of the difference in the phospholipid composition of bacterial and erythrocyte membranes. This is of particular interest to gain information on the specificity of membranolysis exhibited by this peptide against bacteria but not against erythrocytes.In phosphate buffer at physiological pH, as well as in the presence of the zwitterionic phosphatidylcholine and sphingomyelin, the peptide had a random structure but it adopted an α‐helical conformation in the presence of negatively charged lipids. Furthermore, calorimetric experiments showed that PGLa had no effects on the thermotropic phase behavior of liposomes composed of the choline phosphatides, while separation of a distinct peptide‐rich domain was observed for phosphatidylglycerol liposomes. In addition to the main transition of pure 1,2‐dipalmitoylglycerophosphoglycerol at 40°C a second transition owing to the peptide‐perturbed lipid domains was found at 41°C. This conclusion is supported by X‐ray diffraction experiments which indicated that PGLa penetrates into the hydrophobic core of the bilayer inducing an untilting of the hydrocarbon chains as observed in the gel phase of the pure lipid. These results demonstrate that this antibacterial peptide specifically interacts with negatively charged lipid membranes, which are characteristic of bacterial membranes. This can be explained based on the structural features of PGLa.
Heparin‐Induced Structural Modifications and Oxidative Cleavage of Human Serum Albumin in the Absence and Presence of Glucose
Authors: Finotti Paola , Pagetta Andrea 
Published: July 2004

Both unfractionated and fractionated, low‐molecular‐mass heparins were tested on human serum albumin in the absence and presence of glucose at concentrations similar to those frequently found in diabetic hyperglycaemic patients, to ascertain whether heparin and glucose interfered with each other in affecting the conformation of albumin. Reproducible results were obtained with both heparins when used at equal masses, but not when used at equal molar concentrations, suggesting a crucial role of the amount of the saccharide units in determining the observed effects. Spectroscopic studies showed that the binding sites of glucose and heparin on albumin do not overlap and that changes in protein structure depend on complex and mutual interference of glucose and heparin with the protein, although the effects of heparin in modifying the chromophore environment and increasing the ordered structure of the protein also prevailed in the presence of glucose.Heparin binding to albumin rapidly gave rise to oxidative reactions, which were responsible for the increase in the carbonyl content of the protein together with its higher susceptibility to tryptic digestion. Glucose enhanced and prolonged the production of heparin‐induced oxidants. Oxidation caused peptide bond cleavage at Lys323 in the primary structure of albumin, yielding two large fragments of 27.5 kDa and 35 kDa which aggregated to form disulphide‐linked homodimers visible in SDS/PAGE as two new bands of 54 kDa and 74 kDa, respectively. This was accompanied with a reduction in Val, Glu, and Gly residues, only partially counterbalanced by an increase in Thr and Ser residues. While only a small percentage of albumin molecules underwent fragmentation in the presence of heparin with glucose, albumin turned out to display in an even higher proportion structural modifications consistent with a higher degree of ordered structure. The mechanism(s) underlying this heparin‐driven effect and possible physio‐pathological implications in vivo are discussed.
The Bioactive Conformation of Neuropeptide Y Analogues at the Human Y2‐Receptor
Authors: Rist Beate , Ingenhoven Nikolaus , Scapozza Leonardo , Schnorrenberg Gerd , Gaida Wolfram , Wieland Heike A. , Beck‐Sickinger Annette G. 
Published: July 2004

Several attempts to investigate the bioactive conformation of neuropeptide Y have been made so far. As cyclic peptides are much more rigid than linear ones, we decided to synthesise cyclic analogues of the C‐terminal dodekapeptide amide neuropeptide Y Ac‐25‐36. Cyclisation was performed by side chain lactamisation of ornithine or lysine and glutamic or aspartic acid. The affinity of the 19 peptides ranged from K1 0.6 nM to greater than 10000 nM. We found that the size, position, orientation, configuration, and the location of the cycle plays an important role for receptor recognition. Circular dichroic studies have been performed to characterise the secondary structure of each peptide. Receptor binding studies were carried out on human neuroblastoma cell lines SK‐N‐MC (Y1) and SMS‐KAN (Y2), and on rabbit kidney membranes (Y2).The pharmacological and spectral data showed that the α‐helix content was not the predominant factor for high Y1‐receptor affinity. Instead, the location and the size of the hydrophobic lactam bridge, and the conserved C‐terminal tetrapeptide (Arg‐Glu‐Arg‐Tyr) seemed to be the main parameters.Using molecular dynamics, the structures of four cyclic peptides (i,i+4) have been investigated and compared with the previously published NMR structure of one of the cyclic peptide analogues. Significant differences have been found in the overall three‐dimensional fold of the peptides. The distances between the N‐ and the C‐terminus allow discrimination between peptides with high binding affinity and those with low binding affinity, because of the correlation that was found with the measured affinity. Thus, this study suggests that a turn‐like structure and the orientation of the C‐terminus towards the N‐terminus play major roles for high affinity binding of cyclic dodecapeptides to the Y2‐receptor.None of the cyclic segments exhibits significant affinity to the Y1‐receptor. Thus, these results support the hypothesis of a discontinuous binding site of neuropeptide Y at the Y1‐receptor.
The Regulatory Functions of the γ and ɛ Subunits from Chloroplast CF1 are Transferred to the Core Complex, α3β3, from Thermophilic Bacterial F1
Authors: Hisabori Toru , Kato Yasuyuki , Motohashi Ken , Kroth‐Pancic Peter , Strotmann Heinrich , Amano Toyoki 
Published: July 2004

The expression plasmids for the subunit γ (γc) and the subunit ɛ (ɛc) of chloroplast coupling factor (CF1) from spinach were constructed, and the desired proteins were expressed in Escherichia coli. Both expressed subunits were obtained as inclusion bodies. When recombinant γc was mixed with recombinant α and β subunits of F1 from thermophilic Bacillus PS3 (TF1), a chimeric subunit complex (α3β3γc) was reconstituted and it showed significant ATP hydrolysis activity. The ATP hydrolysis activity of this complex was enhanced in the presence of dithiothreitol and suppressed by the addition of CuCI2, which induces formation of a disulfide bond between two cysteine residues in γc. Hence, this complex has similar modulation characteristics as CF1.The effects of recombinant ɛc, and ɛ subunit from TF1 (ɛ1) on α3β3γc were also investigated. ɛc, strongly inhibited the ATP hydrolysis activity of chimeric α3β3γc complex but ɛ1 did not. The inhibition was abolished and the ATP hydrolysis activity was recovered when methanol was added to the assay medium. The addition of ɛc, or ɛ1, to the α3β3γt complex, which is the authentic subunit complex from TF1, resulted in weak stimulation of the ATP hydrolysis activity.These results suggest that (a) the specific regulatory function of γc can be transferred to the bacterial subunit complex; (b) the interaction between the γc subunit and ɛc, strongly affects the enzyme activity, which was catalyzed at the catalytic sites that reside on the α3β3 core.
The Phosphatidylinositol 3‐Kinase Inhibitors Wortmannin and LY294002 Inhibit Autophagy in Isolated Rat Hepatocytes
Authors: Blommaart Edward F. C. , Krause Ulrike , Schellens Jacques P. M. , Vreeling‐Sindelárová Heleen , Meijer Alfred J. 
Published: July 2004

Recent studies indicate that phosphatidylinositol 3‐kinase is essential in the regulation of many processes dependent on membrane flow. Autophagy is a complex pathway in which cell material, including proteins, can be degraded. Membrane flow plays a pivotal role in this process. To find out whether phosphatidylinositol 3‐kinase is also required for autophagy, we tested the effects on autophagy of two structurally unrelated phosphatidylinositol 3‐kinase inhibitors, wortmannin and 2‐(4‐morpholinyl)‐8‐phe‐nylchromone (LY294002).The addition of low concentrations of each of these inhibitors to incubations of hepatocytes in the absence of amino acids resulted in a strong inhibition of proteolysis. The antiproteolytic effect of wortmannin (IC50, 30 nM) and LY294002 (IC50 10 μM) was accompanied by inhibition of autophagic sequestration and not by an increase in lysosomal pH or a decrease in intracellular ATP. No further inhibition of proteolysis by the two compounds was observed when autophagy was already maximally inhibited by high concentrations of amino acids.3‐Methyladenine, which is commonly used as a specific inhibitor of autophagic sequestration, was an inhibitor of phosphatidylinositol 3‐kinase, thus providing a target for its action.It is proposed that phosphatidylinositol 3‐kinase activity is required for autophagy. 3‐Methyladenine inhibits autophagy by inhibition of this enzyme.
Recombinant [Pheβ63]Hemoglobin Shows Rapid Oxidation of the β Chains and Low‐Affinity, Non‐Cooperative Oxygen Binding to the α Subunits
Authors: Kiger Laurent , Baudin Véronique , Desbois Alain , Pagnier Josée , Kister Jean , Griffon Nathalie , Henry Yann , Poyart Claude , Marden Michael C. 
Published: July 2004

We have engineered αβ2 [Phe63]hemoglobin by changing the highly conserved distal histidine of the β chains to a phenylalanine. The mutant tetramer binds four high‐affinity ligands, such as CO or NO, to the ferrous form, or CN to the oxidized iron; however, it binds only two low‐affinity ligands, oxygen and azide. The absorption spectrum of the ferrous deoxy or ferric forms are not normal, displaying an enhanced absorption of the visible band near 560 nm. Half of the autooxidation process, attributed to the mutated β subunits, is over 1000‐fold faster than for Hb A.The mutant Hb exhibits non‐cooperative binding of two oxygens with an affinity about fivefold lower than those of HbA valency hybrids (αmetβ)2. Functional properties of this mutant Hb resemble those of Hb Saskatoon ([Tyr63]Hb) [Suzuki, T., Hayashi, A., Shimizu, A. & Yamamura, Y. (1966) Biochim. Biophys. Acta 127, 280–2821, Flash‐photolysis experiments also indicate non‐cooperative behaviour: the CO‐recombination kinetics were independent of the fraction dissociated. Furthermore, the amplitude of the CO bimolecular phase was the same for the (αcometβ), valency hybrid or the (αcoβco), form, suggesting mainly geminate CO‐recombination kinetics to the β chains.EPR and Resonance Raman spectra did not show evidence for a hemichrome, normally considered as a six‐coordinated iron with low‐spin character. The EPR and resonance Raman spectra for the mutated β subunits demonstrate the presence of a high‐spin compound in the ferric and deoxy ferrous forms. In particular, the ferrous mutated β subunits are penta‐coordinated. The abnormal absorption spectra are possibly due to an interaction between the porphyrin and the phenyl ring in the distal position rather than to direct binding to the iron.