bluuebunny/crossref_metadata_2025 huggingface dataset:
Thymosins β-4 and β-10 are expressed in bovine ovarian follicles and upregulated in cumulus cells during meiotic maturation
Authors: Salhab Mohamad , Papillier Pascal , Perreau Christine , Guyader-Joly Catherine , Dupont Joelle , Mermillod Pascal , Uzbekova Svetlana
Published: October 2010

β-Thymosins are small proteins that regulate the actin cytoskeleton and are involved in cell motility, differentiation, the induction of metalloproteinases, in anti-inflammatory processes and tumourigenesis. However, their roles in the ovary have not yet been elucidated. Using transcriptomics and real time reverse transcription–polymerase chain reaction validation, the present study demonstrates that thymosin β-4 (TMSB4) and thymosin β-10 (TMSB10) are upregulated in bovine cumulus cells (CCs) during in vitro maturation of cumulus–oocyte complexes (COCs) in parallel with an increase in mRNA expression of HAS2, COX2 and PGR genes. Using immunocytochemistry, both proteins were found to be localised mainly in granulosa cells, CCs and oocytes, in both the cytoplasm and nucleus, as well as being colocalised with F-actin stress fibres in CCs. Using different maturation mediums, we showed that the expression of TMSB10, but not TMSB4, was positively correlated with COC expansion and progesterone secretion and negatively correlated with apoptosis. Immunofluorescence, coupled with terminal deoxyribonucleotidyl transferase-mediated dUTP–digoxigenin nick end-labelling (TUNEL), demonstrated the absence of TMSB4 and/or TMSB10 in apoptotic cells. TMSB10 expression was higher in COCs matured in vivo than in vitro, and differences related to the age of the animal were observed. TMSB4 and/or TMSB10 expression was unchanged, whereas HAS2 overexpressed in CCs from oocytes that developed to the blastocyst stage in vitro compared with those that did not. Thus, TMSB4 and/or TMSB10 ovarian expression patterns suggest that these two thymosins may be involved in cumulus modifications during maturation.
Asymmetrical allocation of mitochondrial DNA to blastomeres during the first two cleavages in mouse embryos
Authors: Kameyama Yuichi , Ohnishi Hidehisa , Shimoi Gaku , Hashizume Ryoichi , Ito Masao , Smith Lawrence C.
Published: October 2010

A recent report showed higher oxygen consumption, adenosine triphosphate (ATP) production and mitochondrial localisation in trophectoderm cells than in the inner cell mass of mouse blastocysts. We hypothesised that this phenomenon was due to the asymmetrical distribution of mitochondria in the blastomeres during the earlier stages. Oocytes, 2-cell embryos and 4-cell embryos were analysed to determine the volume, ATP content and mitochondrial DNA (mtDNA) copy number in the whole egg and individual blastomeres. Significant differences were detected in the volumes of cytoplasm and ATP contents between blastomeres from the 2-cell and 4-cell embryos. Moreover, whilst remaining stable in whole embryos, mtDNA copy number differed between blastomeres, indicating that mitochondria in oocytes are unevenly delivered into the daughter blastomeres during the first two cleavages. Although their volume and ATP content were not correlated, there was a significant correlation between volume and mtDNA copy number in 2- and 4-cell blastomeres. These results indicate that the number of mitochondria delivered to blastomeres during early cleavage is not precisely equal, suggesting that the allocation of mitochondria into daughter blastomeres is affected by uneven cytoplasmic distribution during cytokinesis in the oocyte and mother blastomeres.
Sildenafil increases chemotherapeutic efficacy of doxorubicin in prostate cancer and ameliorates cardiac dysfunction
Authors: Das Anindita , Durrant David , Mitchell Clint , Mayton Eric , Hoke Nicholas N. , Salloum Fadi N. , Park Margaret A. , Qureshi Ian , Lee Ray , Dent Paul , Kukreja Rakesh C.
Published: October 2010

We have shown that the potent phosphodiesterase-5 (PDE-5) inhibitor sildenafil (Viagra) induces a powerful effect on reduction of infarct size following ischemia/reperfusion injury and improvement of left ventricular dysfunction in the failing heart after myocardial infarction or doxorubicin (DOX) treatment. In the present study, we further investigated the potential effects of sildenafil on improving antitumor efficacy of DOX in prostate cancer. Cotreatment with sildenafil enhanced DOX-induced apoptosis in PC-3 and DU145 prostate cancer cells, which was mediated by enhanced generation of reactive oxygen species, up-regulation of caspase-3 and caspase-9 activities, reduced expression of Bcl-xL, and phosphorylation of Bad. Overexpression of Bcl-xL or dominant negative caspase 9 attenuated the synergistic effect of sildenafil and DOX on prostate cancer cell killing. Furthermore, treatment with sildenafil and DOX in mice bearing prostate tumor xenografts resulted in significant inhibition of tumor growth. The reduced tumor size was associated with amplified apoptotic cell death and increased expression of activated caspase 3. Doppler echocardiography showed that sildenafil treatment ameliorated DOX-induced left ventricular dysfunction. In conclusion, these results provide provocative evidence that sildenafil is both a powerful sensitizer of DOX-induced killing of prostate cancer while providing concurrent cardioprotective benefit.
Natural Selection, Causality, and Laws: What Fodor and Piatelli-Palmarini Got Wrong
Authors: Sober Elliott
Published: October 2010

In their bookWhat Darwin Got Wrong, Jerry Fodor and Massimo Piattelli-Palmarini construct an a priori philosophical argument and an empirical biological argument. The biological argument aims to show that natural selection is much less important in the evolutionary process than many biologists maintain. The a priori argument begins with the claim that there cannot be selection for one but not the other of two traits that are perfectly correlated in a population; it concludes that there cannot be an evolutionary theory of adaptation. This article focuses mainly on the a priori argument.
Validation of the ITER-relevant passive-active-multijunction LHCD launcher on long pulses in Tore Supra
Authors: Ekedahl A. , Delpech L. , Goniche M. , Guilhem D. , Hillairet J. , Preynas M. , Sharma P.K. , Achard J. , Bae Y.S. , Bai X. , Balorin C. , Baranov Y. , Basiuk V. , Bécoulet A. , Belo J. , Berger-By G. , Brémond S. , Castaldo C. , Ceccuzzi S. , Cesario R. , Corbel E. , Courtois X. , Decker J. , Delmas E. , Ding X. , Douai D. , Goletto C. , Gunn J.P. , Hertout P. , Hoang G.T. , Imbeaux F. , Kirov K.K. , Litaudon X. , Magne R. , Mailloux J. , Mazon D. , Mirizzi F. , Mollard P. , Moreau P. , Oosako T. , Petrzilka V. , Peysson Y. , Poli S. , Prou M. , Saint-Laurent F. , Samaille F. , Saoutic B.
Published: October 2010

A new ITER-relevant lower hybrid current drive (LHCD) launcher, based on the passive-active-multijunction (PAM) concept, was brought into operation on the Tore Supra tokamak in autumn 2009. The PAM launcher concept was designed in view of ITER to allow efficient cooling of the waveguides, as required for long pulse operation. In addition, it offers low power reflection close to the cut-off density, which is very attractive for ITER, where the large distance between the plasma and the wall may bring the density in front of the launcher to low values. The first experimental campaign on Tore Supra has shown extremely encouraging results in terms of reflected power level and power handling. Power reflection coefficient <2% is obtained at low density in front of the launcher, i.e. close to the cut-off density, and very good agreement between the experimental results and the coupling code predictions is obtained. Long pulse operation at ITER-relevant power density has been demonstrated. The maximum power and energy reached so far is 2.7 MW during 78 s, corresponding to a power density of 25 MW m−2, i.e. its design value at f = 3.7 GHz. In addition, 2.7 MW has been coupled at a plasma–launcher distance of 10 cm, with a power reflection coefficient <2%. Finally, full non-inductive discharges have been sustained for 50 s with the PAM.
Geoffrey Dearnaley. 22 June 1930 — 5 May 2009
Authors: Stoneham Marshall , Buckley-Golder Ian
Published: October 2010

Geoffrey Dearnaley was distinguished for seminal contributions to the fields of low-energy nuclear physics, semiconductor detectors for nuclear radiations, and ion channelling at megaelectronvolt energies. Especially significant was his pioneering research on the ion implantation of semiconductor materials, ion implantation of metals, and ion-assisted coatings. He had a special skill in translating complex scientific concepts into industrially exploitable technologies, so his fundamental research contributions were taken up by industry in the automotive sector, electronics, and engineering. His innovations were seminal in the designs of several ion implantation systems, such as the Lintott implanter, that remained a major force in semiconductor implantation after many years. Later, he led the way with his second-generation ‘Blue Tank’ facility. It was his idea to improve the wear and oxidation resistance of metals by ion implantation, leading to successes as varied as improved resistance to high-temperature corrosion of nuclear fuel cladding and to notable reductions in wear of replacement hip joints. Geoffrey Dearnaley was a fundamental physicist who made significant original contributions to many scientific fields, and his understanding of both science and technology successfully showed industry how to exploit them.
Pathogens and symbionts in ticks: a survey on tick species distribution and presence of tick-transmitted micro-organisms in Sardinia, Italy
Authors: Satta Giuseppe , Chisu Valentina , Cabras Pierangela , Fois Francesco , Masala Giovanna
Published: October 2010

A total of 1485 adult ticks were collected from mammalian hosts in south-eastern Sardinia, Italy, during the years 2007–2008. Ticks were identified and tested by PCR analysis for presence of Rickettsia species of the spotted fever group, Ehrlichia canis, Anaplasma phagocytophilum, Coxiella burnetii, Bartonella species and Leishmania species. Among all tick species examined (Rhipicephalus sanguineus, Rhipicephalus turanicus, Rhipicephalus bursa, Rhipicephalus pusillus, Hyalomma marginatum marginatum, Haemaphysalis sulcata and Dermacentor marginatus), only Hyalomma marginatum marginatum produced negative results. A total of 22 pools belonging to the three tick species Rhipicephalus sanguineus (0.9 %), Rhipicephalus turanicus (4.5 %) and Rhipicephalus pusillus (100 %) were positive for Rickettsia species, while a total of five pools belonging to Rhipicephalus sanguineus (0.09 %), Haemaphysalis sulcata (16.7 %) and D. marginatus (7.8 %) were positive for E. canis. Five pools of Rhipicephalus turanicus (1.8 %) were positive for A. phagocytophilum. Positivity for C. burnetii was found in seven pools belonging to three tick species: Rhipicephalus sanguineus (0.5 %), Rhipicephalus turanicus (0.3 %) and Haemaphysalis sulcata (4.4 %). Finally, four pools belonging to Rhipicephalus sanguineus (0.09 %), Rhipicephalus turanicus (0.7 %) and Rhipicephalus bursa (1.1 %) were positive for Bartonella species. Leishmania species DNA was not detected in any of the tick pools examined. Data presented here increase our knowledge on tick-borne diseases in Sardinia, and provide a useful contribution to understanding their epidemiology.
Growth- and substrate-dependent transcription of formate dehydrogenase and hydrogenase coding genes in Syntrophobacter fumaroxidans and Methanospirillum hungatei
Authors: Worm Petra , Stams Alfons J. M. , Cheng Xu , Plugge Caroline M.
Published: October 2010

Transcription of genes coding for formate dehydrogenases (fdhgenes) and hydrogenases (hydgenes) inSyntrophobacter fumaroxidansandMethanospirillum hungateiwas studied following growth under different conditions. Under all conditions tested, allfdhandhydgenes were transcribed. However, transcription levels of the individual genes varied depending on the substrate and growth conditions. Our results strongly suggest that in syntrophically grownS. fumaroxidanscells, the [FeFe]-hydrogenase (encoded by Sfum_844-46), FDH1 (Sfum_2703-06) and Hox (Sfum_2713-16) may confurcate electrons from NADH and ferredoxin to protons and carbon dioxide to produce hydrogen and formate, respectively. Based on bioinformatic analysis, a membrane-integrated energy-converting [NiFe]-hydrogenase (Mhun_1741-46) ofM. hungateimight be involved in the energy-dependent reduction of CO2to formylmethanofuran. The best candidates for F420-dependentN5,N10-methyl-H4MPT andN5,N10,-methylene-H4MPT reduction are the cytoplasmic [NiFe]-hydrogenase and FDH1. 16S rRNA ratios indicate that in one of the triplicate co-cultures ofS. fumaroxidansandM. hungatei, less energy was available forS. fumaroxidans.This led to enhanced transcription of genes coding for the Rnf-complex (Sfum_2694-99) and of severalfdhandhydgenes. The Rnf-complex probably reoxidized NADH with ferredoxin reduction, followed by ferredoxin oxidation by the induced formate dehydrogenases and hydrogenases.
Dur3 is the major urea transporter in Candida albicans and is co-regulated with the urea amidolyase Dur1,2
Authors: Navarathna Dhammika H. M. L. P. , Das Aditi , Morschhäuser Joachim , Nickerson Kenneth W. , Roberts David D.
Published: October 2010

Hemiascomycetes, including the pathogenCandida albicans, acquire nitrogen from urea using the urea amidolyase Dur1,2, whereas all other higher fungi use primarily the nickel-containing urease. Urea metabolism via Dur1,2 is important for resistance to innate host immunity inC. albicansinfections. To further characterize urea metabolism inC. albicanswe examined the function of seven putative urea transporters. Gene disruption established that Dur3, encoded by orf 19.781, is the predominant transporter. [14C]Urea uptake was energy-dependent and decreased approximately sevenfold in adur3Δ mutant.DUR1,2andDUR3expression was strongly induced by urea, whereas the other putative transporter genes were induced less than twofold. Immediate induction ofDUR3by urea was independent of its metabolism via Dur1,2, but further slow induction ofDUR3required the Dur1,2 pathway. We investigated the role of the GATA transcription factors Gat1 and Gln3 inDUR1,2andDUR3expression. Urea induction ofDUR1,2was reduced in agat1Δ mutant, strongly reduced in agln3Δ mutant, and abolished in agat1Δgln3Δ double mutant. In contrast,DUR3induction by urea was preserved in both single mutants but reduced in the double mutant, suggesting that additional signalling mechanisms regulateDUR3expression. These results establish Dur3 as the major urea transporter inC. albicansand provide additional insights into the control of urea utilization by this pathogen.
Cytidine deaminases: AIDing DNA demethylation?
Authors: Fritz Eric L. , Papavasiliou F. Nina
Published: October 2010

The presence of 5-methylcytosine (5-mC) in DNA is a vital epigenetic mark in vertebrates. While the enzymes responsible for methylating DNA in vertebrates have been identified, the means by which this mark can be removed are still unclear. Recently, it has been shown that activation-induced cytidine deaminase (AID) contributes to the demethylation of DNA in certain systems. This enzyme has been intensely studied in its role as a key driver of antibody diversification in B cells, but recent observations from early development in zebrafish and mice as well as heterokaryons point to a role beyond immunology. This review takes stock of the reports linking AID and related deaminases to DNA demethylation, and describes the many important questions left to be answered in this field.
Preparation of Late Drosophila Embryonic Fillets
Authors: Ramachandran Preethi , Budnik Vivian
Published: October 2010

INTRODUCTION

Beyond the age of ~15 h (at 25°C), Drosophila embryos become impenetrable to aqueous fixatives and staining methods as a result of cuticle deposition. This problem can be overcome using flat or fillet preparations, as described in this protocol.
Preparation of Microsphere Tips for Atomic Force Microscopy (AFM): Figure 1.
Authors: Plodinec Marija , Loparic Marko , Aebi Ueli
Published: October 2010

INTRODUCTION

This protocol describes the preparation and use of spherical indenters for micrometer-scale imaging and mechanical testing with atomic force microscopy (AFM). A spherical indenter is prepared by gluing a hard borosilicate sphere to a tipless cantilever. For this purpose, a stereomicroscope with a micromanipulator attachment is employed. The spheres are cleaned prior to gluing to remove any contamination from their surfaces. Once they are cleaned, the borosilicate spheres are stored in absolute ethanol in a glass tube.
Detection of Cytosine Methylation in RNA Using Bisulfite Sequencing
Authors: Pollex Tim , Hanna Katharina , Schaefer Matthias
Published: October 2010

INTRODUCTION

Post-transcriptional RNA modifications are a characteristic feature of noncoding RNAs and have been described for ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), and various other small RNAs. However, the biological function of most of these modifications remains uncharacterized. Cytosine-5 methylation (5mC) has been detected in abundant and long-lived RNA molecules such as rRNAs and tRNAs, but, because of technical limitations, the occurrence of base-methylated cytosines in other RNAs is not known. To facilitate the detection of RNA methylation, we have established a method for analyzing base-methylated cytosines in RNA using bisulfite sequencing. Treatment of RNA with bisulfite causes the chemical deamination of nonmethylated cytosines to uracil, while methylated cytosines remain unaffected. cDNA synthesis followed by polymerase chain reaction (PCR) amplification and DNA sequencing allows investigators to reproducibly and quantitatively distinguish unmethylated cytosines (as thymines) from methylated cytosines in tRNAs and rRNAs. Using high-throughput sequencing approaches, this protocol should enable the characterization of 5mC methylation patterns in any RNA molecule, including low abundance RNAs.
Functional Analysis of Genes in Aedes aegypti Embryos
Authors: Clemons Anthony , Haugen Morgan , Severson David , Duman-Scheel Molly
Published: October 2010

INTRODUCTION

Blood-feeding mosquitoes, including the dengue and yellow fever vector Aedes aegypti, transmit many of the world’s deadliest diseases. Such diseases have resurged in developing countries and pose clear threats for epidemic outbreaks in developed countries. Recent mosquito genome projects have stimulated interest in the potential for arthropod-borne disease control by genetic manipulation of vector insects, and genes that regulate development are of particular interest. In recent years, RNA interference (RNAi) has proven to be an effective strategy for inhibiting gene function in many organisms. This protocol describes a method for knockdown of embryonic genes in Ae. aegypti embryos by microinjection of small interfering RNA (siRNA) designed to target a specific gene of interest. The procedure includes a strategy for siRNA design, microinjection, and measurement of knockdown effectiveness.
The exo‐ or endonucleolytic preference of bovine pancreatic ribonuclease A depends on its subsites structure and on the substrate size
Authors: Cuchillo Claudi M. , Moussaoui Mohamed , Barman Tom , Travers Franck , Nogués M. Victòria
Published: October 2010

The cleavage pattern of oligocytidylic acid substrates by bovine pancreatic ribonuclease A (RNase A) was studied by means of reversed‐phase HPLC. Oligocytidylic acids, ranging from dinucleotides to heptanucleotides, were obtained by RNase A digestion of poly(C). They were identified by MALDI‐TOF mass spectrometry; it was confirmed that all of them corresponded to the general structure (Cp)nC>p, in which C>p indicates a 2′,3′‐cyclic phosphate. This is a confirmation of the proposed mechanism for RNase A, wherein the so‐called hydrolytic (or second) step is in fact a special case of the reverse of transphosphorylation (first step). The patterns of cleavage for the oligonucleotide substrates show that the native enzyme has no special preference for endonucleolytic or exonucleolytic cleavage, whereas a mutant of the enzyme (K7Q/R10Q‐RNase A) lacking p2 (a phosphate binding subsite adjacent, on the 3′ side, to the main phosphate binding site p1) shows a clear exonucleolytic pattern; a mutant (K66Q‐RNase A) lacking p0 (a phosphate binding subsite adjacent, on the 5′ side, to the main phosphate binding site p1) shows a more endonucleolytic pattern. This indicates the important role played by the subsites on the preference for the bond cleaved. Molecular modeling shows that, in the case of the p2 mutant, the amide group of glutamine can form a hydrogen bond with the 2′,3′‐cyclic terminal phosphate, whereas the distance to a 3′,5′‐phosphodiester bond is too long to form such a hydrogen bond. This could explain the preference for exonucleolytic cleavage shown by the p2 mutant.
15N nuclear magnetic resonance relaxation studies on rat β‐parvalbumin and the pentacarboxylate variants, S55D and G98D
Authors: Henzl Michael T. , Wycoff Wei G. , Larson John D. , Likos John J.
Published: October 2010

15N relaxation data for Ca2+‐bound rat β‐parvalbumin (a.k.a. oncomodulin) were analyzed using the Lipari‐Szabo formalism and compared with existing data for rat α‐parvalbumin. Although the averageS2values for the two proteins are very similar (0.85 for α, 0.84 for β), residue‐by‐residue inspection reveals systematic differences. α tends to have the lowerS2value in helical regions; β tends to have the lower value in the loop regions. Rat β was also examined in the Ca2+‐free state. The 59 assigned residues displayed an average order parameter (0.90) significantly greater than the corresponding residues in the Ca2+‐loaded form. The pentacarboxylate variants of rat β—S55D and G98D—also were examined in the Ca2+‐bound state. Although both mutations significantly heighten Ca2+affinity, they utilize distinct energetic strategies. S55D improves the Ca2+‐binding enthalpy; G98D improves the binding entropy. They also show disparate peptide backbone dynamics. Whereas β G98D displays an average order parameter (0.87) slightly greater than that of the wild‐type protein, β S55D displays an average order parameter (0.82) slightly lower than wild‐type β. Furthermore, whereas just two backbone N‐H bonds in β G98D show internal motion on the 20–200‐psec timescale, fully 52 of the 93 residues analyzed in β S55D show this behavior. These findings suggest that the increased electrostatic repulsion attendant to introduction of an additional carboxylate into the CD site ligand array impedes backbone vibrational motion throughout the molecule.
Structure‐based prediction of modifications in glutarylamidase to allow single‐step enzymatic production of 7‐aminocephalosporanic acid from cephalosporin C
Authors: Fritz‐Wolf Karin , Koller Klaus‐Peter , Lange Gudrun , Liesum Alexander , Sauber Klaus , Schreuder Herman , Aretz Werner , Kabsch Wolfgang
Published: October 2010

Glutarylamidase is an important enzyme employed in the commercial production of 7‐aminocephalosporanic acid, a starting compound in the synthesis of cephalosporin antibiotics. 7‐aminocephalosporanic acid is obtained from cephalosporin C, a natural antibiotic, either chemically or by a two‐step enzymatic process utilizing the enzymes D‐amino acid oxidase and glutarylamidase. We have investigated possibilities for redesigning glutarylamidase for the production of 7‐aminocephalosporanic acid from cephalosporin C in a single enzymatic step. These studies are based on the structures of glutarylamidase, which we have solved with bound phosphate and ethylene glycol to 2.5 Å resolution and with bound glycerol to 2.4 Å. The phosphate binds near the catalytic serine in a way that mimics the hemiacetal that develops during catalysis, while the glycerol occupies the side‐chain binding pocket. Our structures show that the enzyme is not only structurally similar to penicillin G acylase but also employs essentially the same mechanism in which the α‐amino group of the catalytic serine acts as a base. A subtle difference is the presence of two catalytic dyads, His B23/Glu B455 and His B23/Ser B1, that are not seen in penicillin G acylase. In contrast to classical serine proteases, the central histidine of these dyads interacts indirectly with the Oγ through a hydrogen bond relay network involving the α‐amino group of the serine and a bound water molecule. A plausible model of the enzyme–substrate complex is proposed that leads to the prediction of mutants of glutarylamidase that should enable the enzyme to deacylate cephalosporin C into 7‐aminocephalosporanic acid.
Use of electrospray ionization mass spectrometry to study binding interactions between a replication terminator protein and DNA
Authors: Kapur Amit , Beck Jennifer L. , Brown Susan E. , Dixon Nicholas E. , Sheil Margaret M.
Published: October 2010

Tus protein binds tightly to specific DNA sequences (Ter) on the Escherichia coli chromosome halting replication. We report here conditions for detecting the 1 : 1 Tus–Ter complex by electrospray ionization mass spectrometry (ESI‐MS). ESI mass spectra of a mixture of Tus and nonspecific DNA showed ions predominantly from uncomplexed Tus protein, indicating that the Tus–Ter complex observed in the gas phase was the result of a specific interaction rather than nonspecific associations in the ionization source. The Tus–Ter complex was very stable using a spray solvent of 10 mM ammonium acetate at pH 8.0, and initial attempts to distinguish binding affinities of Tus and mutant Tus proteins for Ter DNA were unsuccessful. Increasing the ammonium acetate concentration in the electrospray solvent (800 mM at pH 8.0) increased the dissociation constants sufficiently such that relative orders of binding affinity for Tus and various mutant Tus proteins for various DNA sequences could be determined. These were in agreement with the dissociation constants determined in solution studies. A dissociation constant of 700 × 10−9 M for the binding of the mutant Tus protein A173T (where residue 173 is changed from alanine to threonine) to Ter DNA was estimated, compared with a value of ≤2 × 10−9 M for Tus where A173 was unchanged. This is the first example in which ESI‐MS has been used to compare binding affinities of a DNA‐binding protein with mutant proteins for specific DNA recognition sequences. It was also possible to estimate the strength of the interaction between Tus and a DNA sequence (TerH) that had been identified by database searching.
Hemoglobin Porto Alegre forms a tetramer of tetramers superstructure
Authors: Baudin‐Creuza Véronique , Fablet Christophe , Zal Franck , Green Brian N. , Promé Danielle , Marden Michael C. , Pagnier Josée , Wajcman Henri
Published: October 2010

The effects of the mutation β9(A6)Ser → Cys on the interactions between the human hemoglobin molecules were investigated, and comparisons were made with other variants having an additional cysteine residue. In hemoglobin Porto Alegre (PA), the β9 mutation induces polymerization by forming interchain disulfide bonds via the extra cysteine. The hemolysate from a heterozygote was separated by gel filtration into a tetrameric fraction and a higher‐molecular‐weight oligomeric fraction (30%). Reversed‐phase high‐performance liquid chromatography and electrospray ionization mass spectrometry (ESI‐MS) under denaturing conditions showed that the tetrameric fraction contained only normal α‐ and β‐chains, whereas the oligomeric fraction contained only normal α‐chain and disulfide‐linked βPA dimer. Under native conditions, ESI‐MS of the oligomeric fraction revealed a principal complex of mass 258,400 Da corresponding to a tetramer of tetramers, and 10% of minor components. Transmission electron microscopy corroborated this structure by showing four spheres of 140 Å diameter surrounding a central cavity. Equilibrium experiments on the oligomer at different concentrations, using gel filtration and dimer exchange experiments with metHbA‐CN, showed that the tetramer of tetramers dissociates into smaller species, probably by breaking the dimer–dimer allosteric interface. None of the other variants investigated formed such a large oligomer.
Identification of Tyr438 as the major in vitro c‐Src phosphorylation site in human gelsolin: A mass spectrometric approach
Authors: Corte Veerle De , Demol Hans , Goethals Mark , Damme Jozef Van , Gettemans Jan , Vandekerckhove Joël
Published: October 2010

Gelsolin is an actin‐binding protein (82 kDa) consisting of six repeated segments (S1—S6), each approximately 120 residues long. It interacts with phospholipids and we previously showed that phosphatidylinositol 4,5‐bisphosphate promotes phosphorylation of gelsolin by the tyrosine kinase c‐Src. We used a combination of different methods, such as thin‐layer chromatography and anti‐phosphotyrosine‐agarose immunoprecipitation of phosphopeptides combined with matrix assisted laser desorption ionization‐mass spectrometry (MALDI‐MS) and post source decay (PSD) analysis, to identify the phosphorylation sites in gelsolin. The major phosphorylation site (Tyr438) was located in subdomain 4 (S4). Phosphorylation of gelsolin in the gelsolin‐actin2 complex was inhibited by 90%. Gelsolin phosphorylation by c‐Src in the presence of lysophosphatidic acid also revealed Tyr438 as the most prominent site. Additional minor sites were found using the anti‐phosphotyrosine bead immunoprecipitation method followed by MALDI‐MS and PSD analysis. These sites, representing ∼5% of the total phosphate incorporation, were identified as Tyr59, Tyr382, Tyr576, and Tyr624. Based on these results we generated antibodies which specifically recognize Tyr438 phosphorylated gelsolin.
Comparison of anionic and cationic trypsinogens: The anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure
Authors: Pasternak Annette , Ringe Dagmar , Hedstrom Lizbeth
Published: October 2010

Unlike bovine cationic trypsin, rat anionic trypsin retains activity at high pH. This alkaline stability has been attributed to stabilization of the salt bridge between the N‐terminal Ile16 and Asp194 by the surface negative charge (Soman K, Yang A‐S, Honig B, Fletterick R., 1989, Biochemistry 28:9918—9926). The formation of this salt bridge controls the conformation of the activation domain in trypsin. In this work we probe the structure of rat trypsinogen to determine the effects of the surface negative charge on the activation domain in the absence of the Ile16—Asp194 salt bridge. We determined the crystal structures of the rat trypsin‐BPTI complex and the rat trypsinogen‐BPTI complex at 1.8 and 2.2 å, respectively. The BPTI complex of rat trypsinogen resembles that of rat trypsin. Surprisingly, the side chain of Ile16 is found in a similar position in both the rat trypsin and trypsinogen complexes, although it is not the N‐terminal residue and cannot form the salt bridge in trypsinogen. The resulting position of the activation peptide alters the conformation of the adjacent autolysis loop (residues 142—153). While bovine trypsinogen and trypsin have similar CD spectra, the CD spectrum of rat trypsinogen has only 60% of the intensity of rat trypsin. This lower intensity most likely results from increased flexibility around two conserved tryptophans, which are adjacent to the activation domain. The NMR spectrum of rat trypsinogen contains high field methyl signals as observed in bovine trypsinogen. It is concluded that the activation domain of rat trypsinogen is more flexible than that of bovine trypsinogen, but does not extend further into the protein core.
On the role of the cis‐proline residue in the active site of DsbA
Authors: Charbonnier Jean‐Baptiste , Belin Pascal , Moutiez Mireille , Stura Enrico A. , Quéméneur Eric
Published: October 2010

In addition to the Cys‐Xaa‐Xaa‐Cys motif at position 30—33, DsbA, the essential catalyst for disulfide bond formation in the bacterial periplasm shares with other oxidoreductases of the thioredoxin family a cis‐proline in proximity of the active site residues. In the variant DsbAP151A, this residue has been changed to an alanine, an almost isosteric residue which is not disposed to adopt the cis conformation. The substitution strongly destabilized the structure of DsbA, as determined by the decrease in the free energy of folding. The pKa of the thiol of Cys30 was only marginally decreased. Although in vivo the variant appeared to be correctly oxidized, it exhibited an activity less than half that of the wild‐type enzyme with respect to the folding of alkaline phosphatase, used as a reporter of the disulfide bond formation in the periplasm. DsbAP151A crystallized in a different crystal form from the wild‐type protein, in space group P21 with six molecules in the asymmetric unit. Its X‐ray structure was determined to 2.8 Å resolution. The most significant conformational changes occurred at the active site. The loop 149—152 adopted a new backbone conformation with Ala151 in a trans conformation. This rearrangement resulted in the loss of van der Waals interactions between this loop and the disulfide bond. His32 from the Cys‐Xaa‐Xaa‐Cys sequence presented in four out of six molecules in the asymmetric unit a gauche—‐conformation not observed in the wild‐type protein. The X‐ray structure and folding studies on DsbAP151A were consistent with the cis‐proline playing a major role in the stabilization of the protein. A role for the positioning of the substrate is discussed. These important properties for the enzyme function might explain the conservation of this residue in DsbA and related proteins possessing the thioredoxin fold.
Crystal structure of human muscle aldolase complexed with fructose 1,6‐bisphosphate: Mechanistic implications
Authors: Dalby Andrew , Dauter Zbigniev , Littlechild Jennifer A.
Published: October 2010

Fructose 1,6‐bisphosphate aldolase catalyzes the reversible cleavage of fructose 1,6‐bisphosphate and fructose 1‐phosphate to dihydroxyacetone phosphate and either glyceraldehyde 3‐phosphate or glyceraldehyde, respectively. Catalysis involves the formation of a Schiff's base intermediate formed at the ϵ‐amino group of Lys229. The existing apo‐enzyme structure was refined using the crystallographic free‐R‐factor and maximum likelihood methods that have been shown to give improved structural results that are less subject to model bias. Crystals were also soaked with the natural substrate (fructose 1,6‐bisphosphate), and the crystal structure of this complex has been determined to 2.8 å. The apo structure differs from the previous Brookhaven‐deposited structure (1ald) in the flexible C‐terminal region. This is also the region where the native and complex structures exhibit differences. The conformational changes between native and complex structure are not large, but the observed complex does not involve the full formation of the Schiff's base intermediate, and suggests a preliminary hydrogen‐bonded Michaelis complex before the formation of the covalent complex.
Thermodynamic analysis of halide binding to haloalkane dehalogenase suggests the occurrence of large conformational changes
Authors: Krooshof Geja H. , Floris René , Tepper Armand W.J.W. , Janssen Dick B.
Published: October 2010

Haloalkane dehalogenase (DhlA) hydrolyzes short‐chain haloalkanes to produce the corresponding alcohols and halide ions. Release of the halide ion from the active‐site cavity can proceed via a two‐step and a three‐step route, which both contain slow enzyme isomerization steps. Thermodynamic analysis of bromide binding and release showed that the slow unimolecular isomerization steps in the three‐step bromide export route have considerably larger transition state enthalpies and entropies than those in the other route. This suggests that the three‐step route involves different and perhaps larger conformational changes than the two‐step export route. We propose that the three‐step halide export route starts with conformational changes that result in a more open configuration of the active site from which the halide ion can readily escape. In addition, we suggest that the two‐step route for halide release involves the transfer of the halide ion from the halide‐binding site in the cavity to a binding site somewhere at the protein surface, where a so‐called collision complex is formed in which the halide ion is only weakly bound. No large structural rearrangements are necessary for this latter process.
Identification of a novel domain shared by putative components of the endocytic and cytoskeletal machinery
Authors: Kay Brian K. , Yamabhai Montarop , Wendland Beverly , Emr Scott D.
Published: October 2010

We have identified a ∼140 amino acid domain that is shared by a variety of proteins in budding and fission yeast, nematode, rat, mouse, frog, oat, and man. Typically, this domain is located within 20 residues of the N‐terminus of the various proteins. The percent identity among the domains in the 12 proteins ranges from 42 to 93%, with 16 absolutely conserved residues: N‐x11–13‐V‐x2‐A‐T‐x34–36‐R‐x7–8‐W‐R‐x3‐K‐x12‐G‐x‐E‐x15‐L‐x11–12‐D‐x‐G‐R‐x11‐D‐x7‐R. Even though these proteins share little beyond their segment of homology, data are emerging that several of the proteins are involved in endocytosis and or regulation of cytoskeletal organization. We have named this protein segment the ENTH domain, for Epsin N‐terminal Homology domain, and hypothesize that it is a candidate for binding specific ligands and/or enzymatic activity in the cell.
The solution structure of a superpotent b‐chain‐shortened single‐replacement insulin analogue
Authors: Kurapkat Günther , Siedentop Michael , Gattner Hans‐Gregor , Hagelstein Michael , Brandenburg Dietrich , Grötzinger Joachim , Wollmer Axel
Published: October 2010

This paper reports on an insulin analogue with 12.5‐fold receptor affinity, the highest increase observed for a single replacement, and on its solution structure, determined by NMR spectroscopy.

The analogue is [D‐AlaB26]des‐(B27‐B30)‐tetrapeptide‐insulin‐B26‐amide. C‐terminal truncation of the B‐chain by four (or five) residues is known not to affect the functional properties of insulin, provided the new carboxylate charge is neutralized. As opposed to the dramatic increase in receptor affinity caused by the substitution of D‐Ala for the wild‐type residue TyrB26 in the truncated molecule, this very substitution reduces it to only 18% of that of the wild‐type hormone when the B‐chain is present in full length.

The insulin molecule in solution is visualized as an ensemble of conformers interrelated by a dynamic equilibrium. The question is whether the “active” conformation of the hormone, sought after in innumerable structure/function studies, is or is not included in the accessible conformational space, so that it could be adopted also in the absence of the receptor. If there were any chance for the active conformation, or at least a predisposed state to be populated to a detectable extent, this chance should be best in the case of a superpotent analogue. This was the motivation for the determination of the three‐dimensional structure of [D‐AlaB26]des‐(B27‐B30)‐tetrapeptide‐insulin‐B26‐amide. However, neither the NMR data nor CD spectroscopic comparison of a number of related analogues provided a clue concerning structural features predisposing insulin to high receptor affinity. After the present study it seems more likely than before that insulin will adopt its active conformation only when exposed to the force field of the receptor surface.
Domain exchange experiments in duck d‐crystallins: Functional and evolutionary implications
Authors: Sampaleanu Liliana M. , Davidson Alan R. , Graham Caroline , Wistow Graeme J. , Howell P. Lynne
Published: October 2010

δ‐Crystallin, the major soluble protein component of the avian and reptilian eye lens, is homologous to the urea cycle enzyme argininosuccinate lyase (ASL). In duck lenses there are two δ crystallins, denoted δ1 and δ2. Duck δ2 is both a major structural protein of the lens and also the duck orthologue of ASL, an example of gene recruitment. Although 94% identical to δ2/ASL in the amino acid sequence, δ1 is enzymatically inactive. A series of hybrid proteins have been constructed to assess the role of each structural domain in the enzymatic mechanism. Five chimeras—221, 122, 121, 211, and 112, where the three numbers correspond to the three structural domains and the value of 1 or 2 represents the protein of origin, δ1 or δ2, respectively—were constructed and thermodynamically and kinetically analyzed. The kinetic analysis indicates that only domain 1 is crucial for restoring ASL activity to d 1 crystallin, and that amino acid substitutions in domain 2 may play a role in substrate binding. These results confirm the hypothesis that only one domain, domain 1, is responsible for the loss of catalytic activity in d 1. The thermodynamic characterization of human ASL (hASL) and duck δ1 and δ2 indicate that δ crystallins are slightly less stable than hASL, with the δ1 being the least stable. The DGs of unfolding are 57.25, 63.13, and 70.71 kcal mol–1 for δ1, δ2, and hASL, respectively. This result was unexpected, and we speculate that δ crystallins have adapted to their structural role by adopting a slightly less stable conformation that might allow for enhanced protein‐protein and protein‐solvent interactions.
A survey of left‐handed polyproline II helices
Authors: Stapley Benjamin J. , Creamer Trevor P.
Published: October 2010

Left‐handed polyproline II helices (PPII) are contiguous elements of protein secondary structure in which the ø and Ψ angles of constituent residues are restricted to around –75° and 145°, respectively. They are important in structural proteins, in unfolded states and as ligands for signaling proteins. Here, we present a survey of 274 nonhomologous polypeptide chains from proteins of known structure for regions that form these structures. Such regions are rare, but the majority of proteins contain at least one PPII helix. Most PPII helices are shorter than five residues, although the longest found contained 12 amino acids. Proline predominates in PPII, but Gln and positively charged residues are also favored. The basis of Gln's prevalence is its ability to form an i, i + 1 side‐chain to main‐chain hydrogen bond with the backbone carbonyl oxygen of the proceeding residue; this helps to fix the Ψ angle of the Gln and the ø and Ψ of the proceeding residue in PPII conformations and explains why Gln is favored at the first position in a PPII helix. PPII helices are highly solvent exposed, which explains why apolar amino acids are disfavored despite preferring this region of ø/Ψ space when in isolation. PPII helices have perfect threefold rotational symmetry and within these structures we find significant correlation between the hydrophobicity of residues at i and i + 3; thus, PPII helices in globular proteins can be considered to be amphipathic.
The mechanism of sugar phosphate isomerization by glucosamine 6‐phosphate synthase
Authors: Teplyakov Alexei , Obmolova Galya , Badet‐Denisot Marie‐Ange , Badet Bernard
Published: October 2010

Glucosamine 6‐phosphate synthase converts fructose‐6P into glucosamine‐6P or glucose‐6P depending on the presence or absence of glutamine. The isomerase activity is associated with a 40‐kDa C‐terminal domain, which has already been characterized crystallographically. Now the three‐dimensional structures of the complexes with the reaction product glucose‐6P and with the transition state analog 2‐amino‐2‐deoxyglucitol‐6P have been determined. Glucose‐6P binds in a cyclic form whereas 2‐amino‐2‐deoxyglucitol‐6P is in an extended conformation. The information on ligand‐protein interactions observed in the crystal structures together with the isotope exchange and site‐directed mutagenesis data allow us to propose a mechanism of the isomerase activity of glucosamine‐6P synthase. The sugar phosphate isomerization involves a ring opening step catalyzed by His504 and an enolization step with Glu488 catalyzing the hydrogen transfer from C1 to C2 of the substrate. The enediol intermediate is stabilized by a helix dipole and the ‐amino group of Lys603. Lys485 may play a role in deprotonating the hydroxyl O1 of the intermediate.
Covalent modification of lys19 in the ctp binding site of cytidine 5′‐monophosphate n‐acetylneuraminic acid synthetase
Authors: Tullius Michael V. , Vann Willie F. , Gibson Bradford W.
Published: October 2010

Periodate oxidized CTP (oCTP) was used to investigate the importance of lysine residues in the CTP binding site of the cytidine 5′‐monophosphate N‐acetylneuraminic acid (CMP‐NeuAc) synthetase (EC 2.7.7.43) from Haemophilus ducreyi. The reaction of oCTP with the enzyme follows pseudo‐first‐order saturation kinetics, giving a maximum rate of inactivation of 0.6 min–1 and a KI of 6.0 mM at pH 7.1. Mass spectrometric analysis of the modified enzyme provided data that was consistent with β‐elimination of triphosphate after the reaction of oCTP with the enzyme. A fully reduced enzyme‐oCTP conjugate, retaining the triphosphate moiety, was obtained by inclusion of NaBH3CN in the reaction solution. The β‐elimination product of oCTP reacted several times more rapidly with the enzyme compared to equivalent concentrations of oCTP This compound also formed a stable reduced morpholino adduct with CMP‐NeuAc synthetase when the reaction was conducted in the presence of NaBH3CN, and was found to be a useful lysine modifying reagent. The substrate CTP was capable of protecting the enzyme to a large degree from inactivation by oCTP and its β‐elimination product. Lys19, a residue conserved in CMP‐NeuAc synthetases, was identified as being labeled with the β‐elimination product of oCTP
Common structural features of mapeg—a widespread superfamily of membrane associated proteins with highly divergent functions in eicosanoid and glutathione metabolism
Authors: Jakobsson Per‐Johan , Morgenstern Ralf , Mancini Joseph , Ford‐Hutchinson Anthony , Persson Bengt
Published: October 2010

A novel superfamily designated MAPEG (Membrane Associated Proteins in Eicosanoid and Glutathione metabolism), including members of widespread origin with diversified biological functions is defined according to enzymatic activities, sequence motifs, and structural properties. Two of the members are crucial for leukotriene biosynthesis, and three are cytoprotective exhibiting glutathione S‐transferase and peroxidase activities. Expression of the most recently recognized member is strongly induced by p53, and may therefore play a role in apoptosis or cancer development. In spite of the different biological functions, all six proteins demonstrate common structural characteristics typical of membrane proteins. In addition, homologues are identified in plants, fungi, and bacteria, demonstrating this superfamily to be generally occurring.
In vitro evolution of thermostable p53 variants
Authors: Matsumura Ichiro , Ellington Andrew D.
Published: October 2010

The tumor suppressor p53 is conformationally unstable at physiological temperature. Even the activated p53Δ30 variant, which lacks the self‐inhibiting carboxy terminal domain, has a half‐life of only 8 min at 37°C in vitro. We have developed a genetic approach to identify p53 variants that stabilize the active conformation. The human p53Δ30 gene was randomly mutated, and the resulting library was expressed in Escherichia coli under conditions that apparently denatured the parental protein. Stable p53 variants were identified based on their ability to specifically bind a p53 consensus site. The initial thermostable variants were randomly recombined by DNA shuffling, and substitutions that were functionally additive or synergistic were identified in a second more stringent round of screening. The DNA binding activity of N239Y/N268D/E336V p53Δ30 variant has a half‐life of 100 min at 37 °C, 12 times longer than that of the parental protein. The thermostable variants should be more amenable to crystallographic studies and more effective in gene therapies than the wild‐type protein.