bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of Mutant Bordetella Pertussis Adenylate Cyclase Toxins with Reduced Affinity for Calmodulin
Authors: Heveker Nikolaus , Ladant Daniel
Published: July 2004

Bordetella pertussis secretes a calmodulin‐stimulated adenylate cyclase toxin (CyaA) that is one of the major virulence factors of this organism. The toxin is able to enter various types of eukaryotic cells where, upon activation by calmodulin, it catalyzes the production of non‐physiological amounts of cyclic AMP. The mechanism of toxin entry into target cells is unknown, although it has been shown that it does not involve receptor‐mediated endocytosis. The adenylate cyclase toxin exhibits a very high affinity for calmodulin, and it has been proposed that the energy of calmodulin‐binding to CyaA might be required for the entry of the toxin into the target cells [Oldenburg, D. J., Gross, M. K., Wong, C. S. & Storm, D. R. (1992) Biochemistry 31, 8884–88911. In the present study, we have reexamined this issue by analyzing the cytotoxicity of various modified CyaA toxins that have altered calmodulin affinity. We show that despite their low affinity for calmodulin (at least 1000‐times less than that of the wild type CyaA), these toxins were able to efficiently deliver their catalytic domain into the cytoplasm of the target cells, erythrocytes. These results demonstrate that high‐affinity calmodulin binding is not required for the entry of B. pertussis adenylate cyclase into eukaryotic cells. However, the high‐affinity of CyaA for calmodulin is crucial for an efficient synthesis of CAMP within the target cells.
Evaluation of Subunit Truncation and the Nature of the Spacer for Single Chain Human Gonadotropins
Authors: Heikoop Judith C. , Van Beuningen‐De Vaan Miranda M. J. A. C. M. , Van Den Boogaart Paul , Grootenhuis Peter D. J.
Published: July 2004

Three single chain gonadotropins were designed based on the three‐dimensional‐structure of human choriogonadotropin and structure/activity relationships of the glycoprotein hormones. In each single chain, the C‐terminal end of the human choriogonadotropin β subunit is connected via Ser‐Gly repeats to the N‐terminal end of the a subunit. In addition, two of the single chains have truncated subunits. The three mutants were expressed in CHO cells. In vitro binding of two of the three mutants to the human lutropinl choriogonadotropin receptor was found to be comparable to wild‐type lutropin. In contrast, both the receptor binding and the in vitro bioactivity of the mutant with truncated a and β subunits in which the β:26‐110 disulphide bond cannot be formed, are lowered relative to wild‐type lutropin. The fact that this mutant still displays biological activity shows that the seat‐belt arrangement proposed by Isaacs and coworkers [Lapthorn, A. J., Harris, D. C., Littlejohn, A., Lustbader, J. W., Canfield, R. E., Machin, K. J., Morgan, F. J. & Isaacs, N. W. (1994)Nuture 369, 455‐4611 is important but not essential for receptor binding and biological activity in the context of single chain gonadotropins. Single chains in which Ser‐Gly spacers are combined with truncated subunits, provide an attractive approach towards the design and generation of novel, biologically active gonadotropins.
Effects of Tyrosine Ring Fluorination on Rates and Equilibria of Formation of Intermediates in the Reactions of Carbon‐Carbon Lyases
Authors: Phillips Robert S. , Von Tersch Robert L. , Secundo Francesco
Published: July 2004

The interactions of ring fluorinated analogs of tyrosine with tyrosine phenol‐lyase and tryptophan indole‐lyase (tryptophanase) were studied by rapid‐scanning stopped‐flow spectrophotometry. The reaction of L‐tyrosine with tyrosine phenol‐lyase resulted in rapid formation of a small absorbance peak at 500 nm, attributed to a quinonoid intermediate. The reaction of 3‐fluoro‐L‐tyrosine with tyrosine phenol‐lyase resulted in a peak at 500 nm with much higher absorbance, as did the reaction of 3,5‐difluoro‐L‐tyrosine, due to increased accumulation of quinonoid intermediates. In constrast, complexes with 2‐fluoro‐L‐tyrosine, 2,3‐difluoro‐L‐tyrosine, 2,5‐difluoro‐L‐tyrosine, and 2,6‐difluoro‐L‐tyrosine exhibited much lower absorbance intensity at 500 nm. The rate constant for quinonoid intermediate formation from 3‐fluoro‐L‐tyrosine was comparable to that for L‐tyrosine. However, 3,5‐difluoro‐L‐tyrosine reacted to form a quinonoid intermediate at about half the rate of L‐tyrosine, while 2,3‐difluoro‐L‐tyrosine reacted at twice the rate of L‐tyrosine. In addition, the 2‐substituted difluorotyrosines exhibited an intermediate, which was formed rapidly, absorbing strongly at about 340 nm, which is likely due to a gem‐diamine intermediate. Tyrosine is not a substrate for tryptophan indole‐lyase; the reaction of tryptophan indole‐lyase with L‐tyrosine resulted in formation of external aldimine, which absorbed at 420 nm, and a very small absorbance peak at 500 nm. 3‐Fluoro‐L‐tyrosine reacted with tryptophan indole‐lyase to produce a prominent quinonoid absorbance peak at 500 nm, whereas L‐tyrosine, 2‐fluoro‐L‐tyrosine, and all difluoro‐L‐tyrosines, had a much reduced intensity for this peak. Thus, the presence of ring fluorine substituents in L‐tyrosine that are remote from the site of the chemical transformation has significant effects on the rates and equilibria of intermediate formation in the reactions with both tyrosine phenol‐lyase and tryptophan indole‐lyase. Although it is commonly thought that fluorine substitution will not result in any significant steric effects, our results suggest that the effects of fluorine substitution in the reactions of fluorinated tyrosines with tyrosine phenol‐lyase and tryptophan indole‐lyase are due to a combination of steric and electronic effects.
Ca2+ and its Substitutes have Two Different Binding Sites and Roles in Soluble, Quinoprotein (Pyrroloquinoline‐Quinone‐Containing) Glucose Dehydrogenase
Authors: Olsthoorn Arjen J. J. , Otsuki Tetsuo , Duine Johannis A.
Published: July 2004

To investigate the mode of binding and the role of Ca2+ in soluble, pyrroloquinoline‐quinone (PQQ)‐containing glucose dehydrogenase of the bacterium Acinetobacter calcoaceticus (sGDH), the following enzyme species were prepared and their interconversions studied: monomeric apoenzyme (M); monomer with one firmly bound Ca2+ ion (M*); dimer consisting of 2 M* (D); dimer consisting of 2 M and 2 PQQ (Holo‐Y); dimer consisting of D with 2 PQQ (Holo‐X); fully reconstituted enzyme consisting of Holo‐X with two extra Ca2+ ions (Holo) or substitutes for Ca2+ (hybrid Holo‐enzymes). D and Holo are very stable enzyme species regarding monomerization and inactivation by chelator, respectively, the bound Ca2+ being locked up in such a way that it is not accessible to chelator. D can be converted into M* by heat treatment and the tightly bound Ca2+ can be removed from M* with chelator, transforming it into M. Reassociation of M* to D occurs spontaneously at 20°C; reassociation of M to D occurs by adding a stoichiometric amount of Ca2+. Synergistic effects were exerted by bound Ca2+ and PQQ, each increasing the affinity of the protein for the other component. Dimerization of M to D occurred with Ca2+, Cd2+, Mn2+, and Sr2+ (in decreasing order of effectiveness), but not with Mg2+, Ba2+, Co2+, Ni2+, Zn2+, or monovalent cations. Conversion of inactive Holo‐X into active Holo, was achieved with Ca2+or metal ions effective in dimerization. Although it is likely that activation of Holo‐X involves binding of metal ion to PQQ, the spectral and enzymatic activity differences between normal Holo‐ and hybrid Holo‐enzymes are relatively small. Titration experiments revealed that the two Ca2+ ions required for activation of Holo‐X are even more firmly bound than the two required for dimerization of M and anchoring of PQQ. Although the two binding sites related with the dual function of Ca2+ show similar metal ion specificity, they are not identical. The presence of two different sites in sGDH appears to be unique because in other PQQ‐containing dehydrogenases, the PQQ‐containing subunit has only one site. Given the broad spectrum of bivalent metal ions effective in reconstituting quinoprotein dehydrogenase apoenzymes to active holoenzymes, but the limited spectrum for an individual enzyme, the specificity is not so much determined by PQQ but by the variable metal‐ion‐binding sites.
A Determination of the Solution Conformation of Secretoneurin, a Neuropeptide Originating from the Processing of Secretogranin II, by 1H‐NMR and Restrained Molecular Dynamics
Authors: Dulyadi Hassan , Davoust Daniel , Vaudry Hubert
Published: July 2004

Secretoneurin is a 33‐amino‐acid polypeptide generated by proteolytic cleavage of secretogranin II at paired dibasic sequences. It has recently been shown that secretoneurin exerts biological activities such as stimulation of dopamine release from striatal neurons and activation of monocyte migration, suggesting that the peptide may modulate both neurotransmission and inflammatory response. In the present study, we have investigated the conformation of synthetic secretoneurin in methanol solution by two‐dimensional 1H‐NMR, circular dichroism and molecular modeling. Using sequential information, specific assignments have been made for resonances arising from all protons, except for the labile proton of the N‐terminal Thr of the peptide. The solution structure of secretoneurin has been determined by distance geometry and restrained molecular dynamics, using distance and dihedral constraints derived from the NMR data. The conformation obtained is composed of two contiguous a‐helices comprising residues Glu3–Gln8 and Pro11–Gly25. An excellent concordance was observed between these conformational data and prediction with the AGADIR program for the location for the helices in the sequence. These conformational data should help to elucidate the involvement of the tertiary interactions and to design secretoneurin analogs.
Mutational Studies on Conserved Histidine Residues in the Chlorophyll‐Binding Protein CP43 of Photosystem II
Authors: Manna Pradip , Vermaas Wim
Published: July 2004

Two chlorophyll‐binding antenna proteins in the photosystem II core, CP43 and CP47, are structurally similar and are thought to have evolved from a common ancestor. Several conserved histidine residues in hydrophobic regions of CP47 have been shown to be important for photosystem I1 structure, function, and energy transfer. The purpose of this study was to determine whether similarly located histidine residues in CP43 function in a similar way. Three conserved histidine residues in presumed membrane‐spanning regions of CP43, His40, His105, and Hisll9, were mutated to glutamine (Q) and tyrosine (Y). The strains H105Q, H119Q, and H119Y were photoautotrophs whereas H40Q, H40Y, and H105Y were obligate photoheterotrophs. The H40Y and H105Y strains lacked detectable amounts of photosystem II reaction centers and hence could not evolve oxygen whereas H40Q retained a significant amount of photosystem II and oxygen evolution capacity. The observation that mutation of histidine residues to tyrosine has more drastic effects than mutation of these residues to glutamine is in agreement with results obtained for CP47 and suggests the involvement of these residues in chlorophyll binding. The drastic functional changes observed upon mutating His40 and His105 of CP43 are similar to those observed when mutating the corresponding histidine residues in CP47, thus suggesting that the similarity between CP43 and CP47 extends to the relative importance of functionally relevant residues. Interestingly, the His40 → Gln mutation in CP43 had significant effects on photosystem II electron transfer in that it affected the thermodynamics of QA oxidation by QB and increased the charge recombination rate between QA and donor side components. This indicates that relatively minor changes in CP43 can significantly impact the properties of the photosystem II reaction center. The implications of this finding are discussed.
Structural Studies of the O‐Specific Chains of Hafnia Alvei Strains 744, PCM 1194 and PCM 1210 Lipopolysaccharides
Authors: Petersson Carl , Jachymek Wojciech , Klonowska Agnieszka , Lugowski Czeslaw , Niedziela Tomasz , Kenne Lennart
Published: July 2004

The structures of the O‐specific chains of the Hafnia alvei strain 744 and PCM strains 1194 and 1210 lipopolysaccharides have been investigated. Methylation analysis, dephosphorylation, NMR spectroscopy, matrix‐assisted laser‐desorption ionisation time‐of‐flight mass spectrometry and fast‐atom‐bombardment mass spectrometry were the principal methods used. It was concluded that the polysaccharides of strains 744 and PCM 1194 are composed of the same pentasaccharide repeating unit having the following structure:

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and that the polysaccharide of strain PCM 1210 is composed of a pentasaccharide repeating unit having the following structure:

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Repair Pattern in the β‐Globin Gene Cluster of Human Fibroblasts after Ultraviolet Irradiation
Authors: Chakarov Stoyan , Stoilov Peter , Alexandrov Alexander , Russev George
Published: July 2004

We have developed a novel technique to determine repair of structurally different DNA lesions. It was used to address the question of whether DNA repair in the absence of transcription occurs in a uniformly random manner or with preferences for certain regions. Human fibroblasts were exposed to ultraviolet light (3–10 J/m2) and treated with 7.5 mM hydroxyurea to inhibit replicative DNA synthesis. During the first hours after irradiation cells were treated with 5‐bromodeoxyuridine to label the regions undergoing repair, with the presumption that the regions that have been more efficiently repaired would incorporate more of the nucleoside. A 155‐kb DNA sequence containing the entire human β‐globin do‐main was reconstructed using sequences deposited in the EMBL gene bank. Twelve uniformly long single‐copy RNA probes spanning the β‐globin cluster were synthesised In vitro and immobilized on microtiter plates. They were hybridized with DNA from the irradiated cells. The amount of 5‐bromodeoxyuridine, incorporated as a result of repair in the DNA fractions hybridized to the different RNA probes, was determined immunochemically using antibody to this nucleoside. By this technique we registered increased repair efficiency in the zone of the permanent scaffold attachment region at the 5′‐end of the β‐globin domain during the first hours after ultraviolet irradiation. This result was confirmed and by the more conventional T4 endonuclease V technique detecting the removal of cyclobutane pyrimidine dimers.
Isolation and Properties of an ATP Transporter from a Strain of Aspergillus Niger
Authors: Chowdhury Bhabadeb , Bose Swagata K. , Bhaduri Saswata , Bose Susil K.
Published: July 2004

A purified ATP transporter from Aspergillus niger did not show release or uptake for any of the nucleotides (ADP or UTP) except ATP. The release and uptake did not result from non‐specific binding, but appeared to be concentration‐dependent processes. ATP was shown by a double‐isotopic technique to be transported across membrane vesicles without degradation. The ATP‐transport protein was purified to near homogeneity from the membrane vesicles of a strain of A. niger and its apparent M, was approximately 60000. The purified protein showed the properties of a membrane‐bound protein in that the carrier protein was shown, during the liposome‐preparative process, to translocate from the aqueous phase into the lipid bilayer of the liposome, unlike the cytosolic protein glucose‐6‐phosphate dehydrogenase, which remained confined to the aqueous compartment. Mycobacillin, a lipid‐reactive antibiotic, was bound to the transport protein at a site other than the ATP‐binding site, leading to its enhanced release or uptake, which was very feeble in absence of the antibiotic.
Changes in the Electronic Structure Around Ni in Oxidized and Reduced Selenium‐Containing Hydrogenases from Methanococcus Voltae
Authors: Sorgenfrei Oliver , Duin Evert C. , Klein Albrecht , Albracht Simon P. J.
Published: July 2004

The selenium‐containing F420‐reducing hydrogenase from Methanococcus voltae was anaerobically purified to a specific hydrogen‐uptake activity of 350 U/mg protein as determined with the natural electron acceptor. The concentrated enzyme was used for EPR‐spectroscopic investigations. As isolated, the enzyme showed an EPR spectrum with gnyL values of 2.21, 2.15 and 2.01. Illumination of such samples at low temperatures led to an EPR spectrum with gxyz values of 2.05, 2.11 and 2.29. These spectra are typical for [NiFe]hydrogenases in the active state. Spectra of samples enriched in 77Se showed a hyperfine interaction between the unpaired spin of the nickel ion and the nuclear spin of one 77Se atom before and after illumination. A 900 flip of the electronic z‐axis is proposed to explain the hyperfine interaction in both states. This has been demonstrated previously only for the F420‐non‐reducing hydrogenase from M. voltue, where the selenium atom is present as a selenocysteine residue on an unusually small seperate subunit [Sorgenfrei, O., Klein, A. & Albracht, S. P. J. (1993) FEBS Lett. 332, 291–2971. The results demonstrate that the three‐dimensional structures of the active sites in the selenium‐containing F420‐reducing and F420‐non‐reducing hydrogenases from M. voltue are highly similar and hence are not influenced by the unusual subunit structure of the latter enzyme. Oxidized samples containing either natural selenium or 77Se were prepared from the F420‐reducing and the selenium‐containing F420‐non‐reducing hydrogenase. Both enzymes exhibited EPR spectra typical for [NiFe]hydrogenases in the inactive ‘ready’ state. In contrast to the reduced form, no splitting of the nickel‐derived signal due to the nuclear spin of 77Se was observed in the oxidized state, indicating that the electronic z‐axis is perpendicular to the Ni‐Se direction.
Inert Chromium and Cobalt Complexes as Probes of Magnesium‐Dependent Enzymes
Authors: Black Christopher B. , Cowan James A.
Published: July 2004

An investigation of the metal ion dependence of Escherichia coli exonuclease III, 3'‐5'‐exonuclease and exoribonuclease H activities is reported. Catalytic activation of E. coli exonuclease III has been examined for a series of inert chromium complexes Cr(NH3)6‐x(H2O)3+x (x = 0–6) that bear water and ammine ligands in well defined inner‐sphere geometries. The importance of hydrogen bonding and electrostatic stabilization for catalysis of this reaction were quantitatively evaluated. Catalytic activation by the essential metal cofactor appears to be mediated through transition‐state stabilization by outer‐sphere complex formation with substrate. Hydrogen bonding to metal‐bound water molecules is the dominant stabilizing interaction.
The Contact Zones in Human α2‐Macroglobulin
Authors: Shanbhag Vithaldas P. , Stigbrand Torgny , Jensen Poul Erik H.
Published: July 2004

A functional domain termed the contact zone, which is the region of a subunit interacting with another non‐covalently bound subunit, is suggested to play a decisive role in the trapping mechanism of human α‐macroglobulin. Tetrameric α2‐macroglobulin can be dissociated into stable dimers with intact thiol esters by sodium thiocyanate, whereby the contact zones are disrupted. The dissociation leads to significant conformational changes, as studied by ultraviolet‐difference spectroscopy, CD, fluorescence and affinity partitioning. The conformation of the dimers is similar to that of MeNH2‐treated α2‐macroglobulin, in which the thiol esters are cleaved, a conformational state with a closed trap occurs, and receptor‐recognition sites are exposed. The receptor‐binding domain is at least partly exposed in the dimer, as judged by binding of specific mAbs. The bait region in the dimers can be cleaved by proteases, and activation of the thiol esters ensues without binding of the protease. When the dimers were treated with MeNH2, no conformational changes could be detected by ultraviolet‐difference spectroscopy or CD. The conformational changes occurring on dissociation into dimers are suggested to be related to trap closure and receptor‐recognition‐site exposure without cleavage of the thiol esters. The model presented here suggests that two separate conformational changes occur in α‐macroglobulin upon activation. The first involves changes at the contact zones as a result of the thiol‐ester cleavage, and the second causes exposure of the receptor‐recognition sites and closure of the trap.
Molecular Cloning and Characterization Of β‐1,4‐Galactosyltransferase Expressed in Mouse Testis
Authors: Uehara Kazuyoshi , Muramatsu Takashi
Published: July 2004

Using an affinity‐purified antibody against β‐1,4‐galactosyltransferase purified from F9 embryonal carcinoma cells, we screened a λgt11 expression library constructed from the same cell line. The cDNA clone obtained encoded a 46‐kDa protein. Among adult mouse organs, the testis was found to express large amounts of the corresponding mRNA. An antibody against the 46‐kDa protein was raised in a rabbit by immunization with a maltose‐binding‐protein fusion protein produced in Escherichia coli. The affinity‐purified antibody against the cloned 46‐kDa protein reacted with a 59‐kDa protein in sperm extract on western blotting. Among the proteins in the F9 β‐1,4‐galactosyltransferase preparation, which were mostly 68‐kDa and 59‐kDa species, 59‐kDa and 50‐kDa bands reacted with the antibody against the cloned 46‐kDa protein. The cloned 46‐kDa protein had type‐II transmembrane topology and some blocks of sequence similarity with the known β‐1,4–galactosyltransferase. Furthermore, predicted secondary structures were similar over large portions of the two proteins. The histidine‐tagged 46‐kDa protein produced in E. coli was partially adsorbed onto a N‐acetylglucosamine‐Affi‐Gel column and eluted by 20 mM N‐acetylglucosamine. The histidine‐tagged 46‐kDa protein, which was purified to homogeneity, had a small, but significant amount of β‐1,4‐galactosyltransferase activity. These results strongly suggest that the 46‐kDa protein is a β‐1,4‐galactosyltransferase.
Modulation of the Mitochondrial Permeability Transition by Nitric Oxide
Authors: Balakirev Maxim Yu. , Khramtsov Valery V. , Zimmer Guido
Published: July 2004

The influence of nitric oxide on mitochondrial permeability transition (MPT) phenomenon was studied. NO was generated by photolysis of S‐nitroso‐N‐acetylcysteine, AcCys(NO), with green light (λ= 550 nm). Two distinct effects of nitric oxide on rat liver mitochondria were identified. First, NO accelerated an onset of swelling in Ca2+‐loaded mitochondria in a cyclosporin‐A‐sensitive manner acting as an inducer of permeability transition. This was, apparently, a result of irreversible alteration of mitochondrial function accompanying the inhibition of respiratory chain in the presence of calcium. Formation of ESR‐visible iron‐sulfur dinitrosyl complexes (g= 2.041) could also contribute to the irreversible changes resulting in MPT induction. Second, NO changed significantly the response of mitochondria to Ca2+/phosphate‐induced MPT, acting as a regulator of permeability transition. In this case the action of nitric oxide led to division of the mitochondria into two subpopulations: one which underwent the rapid permeability transition and another in which the MPT was inhibited. The effect of NO on Ca2+/Piinduced MPT was transient and resulted from reversible inhibition of cytochrome oxidase followed by the changes in transmembrane potential and Ca2+ distribution. The characteristic time of duration of these NO modulated effects depended on nitric oxide as well as on oxygen concentrations. With increasing NO at fixed oxygen concentrations, this time levelled off to reach a maximum value which was inversely related to the oxygen concentration. It is concluded that under physiological condition the duration of reversible NO effects on mitochondrial function could be determined by oxygen concentration.
Thermochemical and Kinetic Evidence for Nucleotide‐Sequence‐Dependent RECA‐DNA Interactions
Authors: Wittung Pemilla , Ellouze Christine , Maraboeuf Fabrice , Takahashi Masayuki , Nordèn Bengt
Published: July 2004

RecA catalyses homologous recombination in Escherichia coli by promoting pairing of homologous DNA molecules after formation of a helical nucleoprotein filament with single‐stranded DNA. The primary reaction of RecA with DNA is generally assumed to be unspecific. We show here, by direct measurement of the interaction enthalpy by means of isothermal titration calorimetry, that the polymerisation of RecA on single‐stranded DNA depends on the DNA sequence, with a high exothermic preference for thymine bases. This enthalpic sequence preference of thymines by RecA correlates with faster binding kinetics of RecA to thymine DNA. Furthermore, the enthalpy of interaction between the RecA DNA filament and a second DNA strand is large only when the added DNA is complementary to the bound DNA in RecA. This result suggests a possibility for a rapid search mechanism by RecA DNA filaments for homologous DNA molecules.
Chloroplast SecA Functions as a Membrane‐Associated Component of the Sec‐Like Protein Translocase of Pea Chloroplasts
Authors: Haward Sam R. , Napter Johnathan A. , Gray John C.
Published: July 2004

Protein cross‐linking studies with a thylakoid membrane translocation intermediate were used to demonstrate that chloroplast SecA functions as a membrane‐associated component of the Sec‐like ATP dependent protein translocase of pea chloroplasts. In assays with isolated thylakoids, it was observed that translocation of the 33‐kDa protein of the oxygen‐evolving complex of photosystem 11 (OE33) decreased when the ATP concentration was low, and that the protein accumulated as a bound precursor. The bound precursor was able to be translocated into the lumen when the ATP concentration was raised, indicating that the precursor was bound to the translocation apparatus. Inclusion of apyrase in the import reaction prevented translocation but did not affect precursor binding to the membrane. When this translocation intermediate was treated with the cross‐linking agent disuccinimidyl suberate, a single predominant cross‐linked product of 120 kDa was produced. This conjugate could be immunoprecipitated with antibodies to pea chloroplast SecA, identifying the cross‐linking partner as SecA. This provides direct evidence for a functional interaction between a thylakoid precursor protein and a component of the thylakoid proteintranslocation apparatus.
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.
Mating and Sex Peptide Stimulate the Accumulation of Yolk in Oocytes of Drosophila Melanogaster
Authors: Soller Matthias , Bownes Mary , Kubli Eric
Published: July 2004

Mating elicits two reactions in many insect females: egg deposition is increased and receptivity to males is reduced. Central to the control of receptivity and oviposition in Drosophila melanogaster is the sex peptide (SP), a 36‐amino‐acid peptide sex pheromone synthesized in the male accessory glands and transferred to the female during copulation. To identify regulatory mechanisms involved in the maintenance of the oviposition response, we have compared the effects of mating and SP application with respect to oogenesis. The distribution of the various stages of oogenesis in the ovary, yolk protein (YP) synthesis by the fat body, as well as YP content, uptake and synthesis by the ovary were investigated. Transcripts of the yolk protein genes (yp) were quantified by Northern blotting. Based on our results, we conclude that mating and SP injection into virgin females stimulate yp gene transcription in the fat body only moderately above the background level. However, uptake into the ovary and transcription of the yp genes in the ovary is strongly enhanced after either mating or SP injection. These data are supported by the finding that the abundance of the vitellogenic stage 10 oocytes is also increased. In contrast, early vitellogenic stages 8 and 9 of oogenesis are present in the same numbers in virgin, mated, and SP‐injected females, which suggests a control point at about stage 9 determining vitellogenic oocyte progression. The finding that SP can elicit equally all changes observed after copulation suggests that in the sexually mature female it is the major component controlling and stimulating oogenesis after mating.
Formation of the Early‐Region‐2 Transcription‐Factor‐1‐Retinoblastoma‐Protein (E2F‐1‐RB) TransRepressor and Release of the Retinoblastoma Protein from Nuclear Complexes Containing Cyclin A is Induced by Interferon α in U937V Cells but not in Interferon‐α‐Resistant U937VR Cells
Authors: Kirch Hans‐Christoph , Putzer Brigitte , Brockmann Dieter , Esche Helmut , Kloke Otto
Published: July 2004

We have analysed the different regulation of cell‐cycle‐relevant proteins by interferon α (IFNα) in IFNα‐sensitive and resistant U937 leukemic cell lines. In contrast to the IFNα‐sensitive U937 variant cell line U937V, the IFNα‐resistant derivative (U937VR) is insensitive to the antiproliferative activity of IFNα. As we found no differences between these cell lines concerning the induction by IFNα of the pathway involving tyrosine‐protein kinases and the signal transducer and activator of transcription (Jak‐Stat), we examined whether cell‐cycle‐regulating proteins are differently affected by IFNα in U937V and U937VR cells. In U937V cells IFNα induced the formation of the complex between early‐region‐2 transcription factor 1 (E2F‐1) and retinoblastoma protein (RB) which is known to repress transcription of E2F‐1‐inducible genes, necessary for cell cycle progression. Formation of this complex was not inducible by IFNα in U937VR cells, although the suitable binding partners (E2F‐1 and under‐phosphorylated RB) were present. Interestingly, treatment of nuclear extracts from logarithmically growing U937V and U937VR cells with an antiserum against cyclin A that disrupts cyclin‐A‐containing complexes, led to the formation of the E2F‐1‐RB complex, suggesting the presence of under‐phosphorylated (active) RB, trapped in nuclear complexes that contain cyclin A. This suggestion was supported by combined immuno‐precipitation/western blot experiments that revealed a physical interaction between phosphorylated as well as under‐phosphorylated forms of RB and cyclin A complex(es) in U937V and U937VR cells. RB, especially the under‐phosphorylated form, was released by treatment with IFNα from this complex(es) in the case of U937V cells but not U937VR cells. We conclude that the missing induction of the E2F‐1‐RB transrepressor by IFNα and the failure to release RB from cyclin‐A‐containing complexes might contribute to the resistance of U937VR cells to the antiproliferative effects of IFNα.
Characterization of the Molecular‐Chaperone Function of the Heat‐Shock‐Cognate‐70–Interacting Protein
Authors: Bruce Barry D. , Churchich Jorge
Published: July 2004

A histidine‐tagged form of the recently discovered molecular chaperone, 70‐kDa heat‐shock cognate (Hsc70)‐interacting protein (Hip), has been expressed in Escherichia coli and purified to near homogeneity. This protein remains soluble when expressed in E. coli. Several important properties of this chaperone have been investigated. HPLC size‐exclusion chromatography indicates that the chaperone forms a tetramer similar to what has been reported for the native protein from rat liver cytosol. The recombinant form of Hip did not catalyze the hydrolysis of ATP and ATP analogs, although fluorescence measurements indicated that the chaperone recognizes anthraniloyl‐dATP, anthraniloyl‐ADP, and 2′‐O‐trinitrophenyl‐ATP. The role of Hip as a molecular chaperone has been confirmed by its ability to strongly bind to the reduced, carboxymethylated form of α‐lactalbumin. This interaction is specific for non‐native domains since native α‐lactalbumin fails to interact with Hip. Fluorescence‐anisotropy measurements indicate that reduced, carboxymethylated lactalbumin binds Hip with a Kd of 5 μM. Although Hip appears to be able to bind nucleotides and non‐native proteins, it is unable to facilitate the refolding of two denatured proteins, E. coli alkaline phosphatase and mitochondrial malate dehydrogenase. Hip inhibited the refolding of alkaline phosphatase and malic dehydrogenase. Inhibition occurred at near stoichiometric levels of Hip and could not be reversed by the addition of ATP. These results suggest that Hip may regulate the function of the Hsp70 molecular chaperone complex in vivo and play a critical role in protein folding in the eukaryotic cytoplasm.
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.
Self‐Cleaving Ribozymes of Hepatitis Delta Virus RNA
Authors: Been Michael D. , Wickham Gene S.
Published: July 2004

Hepatitis delta virus (HDV) is a small single‐straded RNA satellite of hepatitis B virus. Although it is a human pathogen, it shares a number of features with a subset of the small plant satellite RNA viruses, including self‐cleaving sequences in the genomic and antigenomic sequences of the viral RNA. The self‐cleaving sequence is critical to viral replication and is thought to function as a ribozyme in vivo to process the products of rolling‐circle replication to unit‐length molecules. A divalent cation is required for cleavage and while a structural role is implicated for metal ions, a more direct role for a metal ion in catalysis has not yet been proven. A minimal natural ribozyme sequence with proficient in vitro self‐cleavage activity is about 85 nucleotides long and adopts a secondary structure with four paired regions (P1–P4). The two pairings that define the 5′ and 3′ boundaries of the ribozyme, P1 and P2, form an atypical pseudoknot arrangement. This secondary structure places a number of constraints on the possible tertiary folding of the sequence, which together with chemical probing, photo‐cross‐linking, mutagenesis and computer‐assisted modeling provides clues to the three‐dimensional structure. The data are consistent with a model in which the cleavage site, located at the 5′ end of P1, is in close proximity to three single‐stranded regions, consisting of a hairpin loop at the end of P3 and two sequences joining P1 to P4 and P4 to P2. While the natural forms of the HDV ribozymes appear to be prone to misfolding, biochemical and mutagenesis studies from a number of laboratories has allowed the production of trans‐acting ribozymes and smaller more active cis‐acting ribozymes, both of which will aid in further mechanistic and structural studies of this RNA.
Binding Kinetics of Bacillus Sphaericus Binary Toxin to Midgut Brush‐Border Membranes of Anopheles and Culex sp. Mosquito Larvae
Authors: Silva‐Filha Maria Helena , Nielsen‐Leroux Christina , Charles Jean‐François
Published: July 2004

Direct‐binding assays and homologous‐competition assays were used to identify specific binding between the radiolabelled toxin of Bacillus sphaericus and brush‐border membrane fractions (BBMF) from Anopheles gambiae and Anopheles stephensi, obtained from whole larvae preparations. In both species, the toxin bound to a single class of receptors. BBMF of A. gunzbiae had the highest binding affinity for the toxin of the species tested, with a dissociation constant (Kd of 30 ± 15 nM and a maximum receptor concentration of 5 ± 1 pmol/mg. Toxin binding to A. gambiae BBMF was compared with that to BBMF from B. sphaericus‐susceptible (IP) and B. sphaericus‐resistant (SPHAE) Culex pipiens populations. BBMF toxin binding was slower in A. gambiae than in the C. pipiens populations. The BBMF of the B. sphaericus‐resistant population of C. pipiens had an association profile that was similar to the susceptible population, despite of the lack of susceptibility in vivo. No relationship between toxicity and irreversibility of toxin binding was detected. On the contrary, toxin dissociation from BBMF was fast and almost complete in BBMF of all species studied.
Sequence and Expression of an Eisenia‐Fetida‐Derived cDNA Clone That Encodes the 40‐kDa Fetidin Antibacterial Protein
Authors: Lassèues Maguy , Milochau Alexandra , Doignon François , Du Pasquier Louis , Valembois Pierre
Published: July 2004

Fetidins are 40‐kDa and 45‐kDa hemolytic and antibacterial glycoproteins present in the coelomic fluid of the earthworm Eisenia fetida andrei. By screening a cDNA library with a polyclonal anti‐fetidin serum, we have cloned a cDNA that encoded the 40‐kDa fetidin. The clone contains an insert of 1.44 kb encoding a protein of 34 kDa, which corresponds to the size of deglycosylated fetidins. The recombinant protein inhibits Bacillus megaterium growth. Restriction fragment polymorphisms were observed on Southern blots and correspond to a known protein polymorphism. The sequence of the cDNA contains a peroxidase signature and fetidins from earthworm coelomic fluid have peroxidase activity. The 40‐kDa and 45‐kDa fetidins therefore represent two related polymorphic defence factors in invertebrates.
Structural and Serological Characterisation of the O‐Antigenic Polysaccharide of the Lipopolysaccharide from Acinetobacter Haemolyticus Strain ATCC 17906
Authors: Haseley Simon R. , Holst Otto , Brade Helmut
Published: July 2004

A polysaccharide containing 2‐acetamido‐2‐deoxy‐d‐galacturonic acid (GalNAcA), 2,4‐diacetamido‐2,4,6‐trideoxy‐d‐glucose (QuiNAc4NAc), and d‐alanine (Ala) was isolated from the water‐soluble lipopolysaccharide (LPS) originating from the reference strain for Acinetobacter haemolyticus (DNA group 4) strain ATCC 17906. The polysaccharide, characterised by means of monosaccharide analyses and NMR studies, was shown to be based on a linear trisaccharide repeating unit, as shown below, with the alanine group amide‐bound to position 6 of one GalNAcA residue. It was specifically recognised in western blots by polyclonal rabbit antisera.
Role of Escherichia Coli Histone‐Like Nucleoid‐Structuring Protein in Bacterial Metabolism and Stress Response
Authors: Laurent‐Winter Christine , Ngo Saravuth , Danchin Antoine , Bertin Philippe
Published: July 2004

The histone‐like nucleoid‐structuring protein, H‐NS, is a major bacterial chromatin component which influences DNA structure and gene expression. Mutations in hns, the structural gene of H‐NS protein, have been shown to result in highly pleiotropic effects in Escherichia coli cells. In this study, we have initiated an index of the proteins whose synthesis is, directly or indirectly regulated by H‐NS. Using two‐dimensional gel electrophoresis, we have examined the global changes in gene expression which occured in an hns background compared with its wild‐type parent. In addition, we analysed the effects of mutations in two other genes i.e. Irp and pta, which are also involved in global regulatory pathways. Although these comparative analyses revealed several common differences, thus suggesting possible interactions between these regulatory mechanisms, i.e. H‐NS, Lip (leucine‐responsive regulatory protein) and acetylphosphate, the most extensive modifications occurred in an hns mutant. Among the polypeptides whose level of synthesis was specifically altered in an hns mutant, several corresponded to H‐NS targets previously identified by classical selection methods. Moreover, the present study allows us to characterize several H‐NS targets, which were identified either by comparison with the E. coli two‐dimensional reference maps or by microsequencing procedure. Many of these newly identified polypeptides are involved in adaptation of E. coli cells to environmental challenges, and one of them could be involved in bacterial virulence. Finally, synthesis of several proteins belonging to the heat‐shock regulon, more particularly molecular chaperones, was induced in an hns mutant.
Characterization of a Promoter within the First Intron of the Human CD4 Gene
Authors: Rushton John J. , Zorich George P. , Stolc Victor , Neudorf Steven M. L.
Published: July 2004

The CD4 molecule is subject to complex regulation during T cell differentiation and activation. The elements regulating CD4 gene expression have only partially been defined. In this report, we identified a promoter element located in the first intron of the CD4 gene. This promoter preferentially functions in T cell lines and is preferentially active in CD4+, CD8+ cells. These findings are similar to other systems in which multiple promoters define tissue‐ and developmental‐specific patterns of expression. Through a series of deletions, electrophoretic mobility shift assays and exonuclease III protection assays, we localized the basal promoter element to a 32‐bp fragment. This element lacks potential binding domains for rnyb and ets, both of which have previously been shown to be involved in the function of the 5′ murine and human CD4 promoter, and this suggests the presence of a novel, T‐cell‐specific transcription factor. These results also suggest that the CD4 expression requires the use of multiple regulatory elements located throughout the CD4 gene.
Renal Expression of Serum‐Type Mannan‐Binding Protein in Rat
Authors: Morio Hiroki , Kurata Hiroshi , Katsuyama Ryosuke , Oka Shogo , Kozutsumi Yasunori , Kawasaki Toshisuke
Published: July 2004

Mannan‐binding proteins (MBPs) occur as liver and serum forms, which are encoded by separate genes and synthesized by the liver. Serum MBP plays a role in the first defense against a variety of pathogens. We found in this study, by reverse‐transcriptional PCR, that rat serum MBP mRNA but not liver MBP is also expressed in the kidney. The level of the serum mRNA expression in the kidney as estimated by competitive PCR was about one‐eighth that of the liver. Transcripts of serum MBP were detected in the renal corpuscles and distal convolutions by in situ hybridization. Affinity chromatography of the extract of rat kidney on a column of Sepharose‐4B – mannan isolated a 29‐kDa protein. These results indicate that serum MBP is synthesized in the kidney as well as in the liver. The kidney protein may be associated with local host defense around the kidney.
Characterisation of the Ultraviolet‐Sensitive Opsin Gene in the Honey Bee, Apis Mellifera
Authors: Bellingham James , Wilkie Susan E. , Morris Alex G. , Bowmaker James K. , Hunt David M.
Published: July 2004

The cDNA sequence of the ultraviolet‐sensitive opsin in the honey‐bee, Apis mellifera, with associated 5′ and 3′ untranslated regions, is presented. The analysis of genomic structure reveals seven introns in the coding region of the gene, with six at novel positions for an insect opsin gene. The equivalent site to the counterion in vertebrate opsins is occupied by a Tyr residue. This contrasts with the presence of Phe at this site in the ultraviolet‐sensitive opsins of Drosophila sps. A comparison of the amino acid sequence within the seven α‐helical transmembrane regions of insect ultraviolet/blue‐sensitive opsins identifies substitution at five sites that involve either replacement of a polar with a non‐polar residue, or a change in charge. Such changes are known to result in spectral shifts in vertebrate pigments. Phylogenetic analysis indicates that the ultraviolet‐sensitive pigments represent an ancient class of insect opsins.