bluuebunny/crossref_metadata_2025 huggingface dataset:
A Plant Acyltransferase Involved in Triacylglycerol Biosynthesis Complements an Escherichia Coli sn‐1‐acylglycerol‐3‐phosphate Acyltransferase Mutant
Authors: Hanke Christiane , Wolter Frank P. , Coleman Jack , Peterek Gabriele , Frentzen Margrit
Published: July 2010

The second acylation reaction in glycerolipid biosynthesis is catalyzed by an sn‐1‐acylglycerol‐3‐phosphate acyltransferase. The enzyme of Limnanthes douglusii involved in triacylglycerol synthesis has an unusual specificity for very long chain acyl groups in both of its substrates, namely acyl‐CoA and sn‐1‐acylglycerol‐3‐phosphate, and causes the enrichment of erucoyl groups in the sn‐2 position of the seed oil of this plant species. We have isolated a cDNA clone encoding this embryo‐specific, microsomal acyltransferase via heterologous complementation of an Escherichia coli mutant deficient in sn ‐1‐acyl‐glycerol‐3‐phosphate acyltransferase activity. The open reading frame of the cDNA insert encodes a protein with a length of 281 amino acids, with three predicted membrane‐spanning domains and of about 31.7 kDa. The sequence exhibits substantial sequence similaritiy to the sn‐1‐acylglycerol‐3‐phosphate acyltransferase of E. coli. The corresponding transcript was detectable in developing embryos but not in leaves of L. douglasii, and expression of the open reading frame in E. coli caused sn‐1‐acylglycerol‐3‐phosphate acyltransferase activity which showed properties different from those of the bacterial acyltransferase but typical of the L. douglusii enzyme involved in triacylglycerol biosynthesis.
The Human Inter‐α‐Trypsin Inhibitor Genes Respond Differently to Interleukin‐6 in HepG2 Cells
Authors: Sarafan Nasrin , Martin Jean‐Pierre , Bourguignon Jeannette , Borghi Hélè , Callé Aleth , Sesboüé Richard , Diarra‐Mehrpour Maryam
Published: July 2010

The effects of interleukin 6 (IL‐6), the major inducer of the acute‐phase reaction, on the expression of inter‐α‐trypsin inhibitor (ITI) genes were examined using human HepG2 hepatoma cells. The three ITI heavy chain genes H1, H2 and H3 were transcriptionally regulated by IL‐6 in a dose‐ and time‐dependent manner. The treatment of HepG2 cells with IL‐6 resulted in an increase of H1 and H3 mRNA levels and a decrease of H2 and L mRNA levels. Actinomycin D blocked the action of IL‐6, suggesting that IL‐6 regulated the H1, H2, H3 gene expression. Moreover, the kinetics of the ITI mRNA degradation in untreated and IL‐6‐treated cells confirmed these data. The nuclear run‐on assay supports the regulatory effect of IL‐6 at the transcription level of the L and H2 genes. Primer extension experiments showed that the effect of IL‐6 on L, H2 and H3 mRNA synthesis was not related to the transcription starting point. Although H1, H2, H3 and L gene products are supposedly present in similar amounts in the ITI and pre‐α‐trypsin inhibitor molecules, the present work shows that these genes are regulated in a different manner, at least under the influence of IL‐6.
Substitution of Arg230 and Arg233 in Escherichia coli Elongation Factor Tu Strongly Enhances Its Pulvomycin Resistance
Authors: Boon Kathy , Krab Ivo , Parmeggiani Andrea , Bosch Leendert , Kraal Barend
Published: July 2010

Pulvomycin is a strong inhibitor of protein synthesis, known to prevent the binding of aminoacyl‐tRNA to elongation factor Tu · GTP (EF‐Tu · GTP). Recently, three pulvomycin‐resistant mutant strains have been isolated by targeted mutagenesis of the tufA gene resulting in EF‐Tu substitutions at positions 230, 333 or 334. In order to analyze the functions of arginine residues located in domain II, with respect to pulvomycin resistance and the interaction with aminoacyl‐tRNA, we have investigated the effect of the substitutions of the highly conserved residues Arg230 and Arg233 by site‐directed mutagenesis. We have purified two mutants species, [R233S]EF‐TuHis and [R230V, R233F]EF‐TuHis, both with a C‐terminal histidine extension to enable purification by Ni2+ affinity chromatography. In this study, we describe the in vitro characterization of these mutant proteins. The results show that the concomitant substitution of residues at positions 230 and 233, dramatically increases the pulvomycin resistance. Preliminary evidence is presented that protein synthesis is inhibited by an EF‐Tu · GDP · pulvomycin complex rather than by EF‐Tu · GTP · pulvomycin. Moreover, the mutant [R230V, R233F]EF‐TuHis shows a stronger protection of the ester bond of aminoacyl‐tRNA than wild‐type EF‐Tu.
pH‐Induced Conformational Perturbation in Horseradish Peroxidase
Authors: Das Tapan Kanti , Mazumdar Shyamalava
Published: July 2010

The fluorescence‐decay characteristics of the single tryptophan present in horseradish peroxidase (HRP) have been studied using dye laser pulses and single‐photon counting techniques. The decay was found to be dominated by a picosecond‐lifetime component, with small contributions from two other lifetime components in the nanosecond range. The distance of the tryptophan residue was estimated from the fluorescence‐energy transfer to the heme moiety using Förster's theory. The tryptophan residue was found to be approximately 1.2 nm from the heme moiety at neutral pH. Detailed analysis of the fluorescence‐decay profiles using the maximum‐entropy method (MEM) has been carried out. The results of the MEM analysis also showed a maximum amplitude peak at approximately 45 ps (at pH ? 7) with a very small (< 5%) contribution from two other components. Similar results were obtained with the cyanide derivative of the enzyme (HRPCN) where the major lifetime component was found to be 58 ps at neutral pH. The picosecond component of fluorescence lifetimes of native HRP as well as of HRPCN were found to increase with decrease in pH in the range pH 6–3.5. Moreover, the native enzyme showed significant increase in the magnitude of this fast lifetime component at pH above 8. Such increase in the major lifetime component possibly indicated a conformational perturbation caused by pH change in the enzyme. However, the pH dependence of HRPCN, which is devoid of alkaline transition, showed that the shortest lifetime component remains almost unchanged over the pH range 6–11. This result showed that the alkaline transition in native HRP is associated with a structural change in the distal region of the heme center, which is absent in the cyanide‐ligated enzyme. The results have been discussed with respect to understanding the pH‐induced effects associated with salt bridge and hydrogen‐bonding network in HRP.
Refined Crystal Structure of the Interleukin‐1 Receptor Antagonist
Authors: Schreuder Herman A. , Rondeau Jean Michel , Tardif Chantal , Soffientini Adolfo , Sarubbi Edoardo , Akeson Ann , Bowlin Terry L. , Yanofsky Stephen , Barrett Ronald W.
Published: July 2010

Interleukin‐I (IL‐1) molecules are cytokines involved in the acute‐phase response against infection and injury. Three naturally occurring IL‐1 molecules are known, two agonists: IL‐1α and IL‐1β, and one antagonist, the 1L‐1 receptor antagonist (IL‐1ra). Although IL‐1 action protects the organism by enhancing the response to pathogens, its overproduction can lead to pathology and has been implicated in disease states that include septic shock, rheumatoid arthritis, graft versus host disease and certain leukemias. The crystal structure of IL‐1ra has been solved at 0.21‐nm resolution by molecular replacement using the IL‐1β structure as a search model. The crystals contain two independent IL‐1ra molecules which are very similar, IL‐1ra has the same fold as IL‐1α and IL‐1β. The fold consists of twelve β‐strands which form a six‐stranded β‐barrel, closed on one side by three β‐hairpin loops. Cys69 and Cys116 are linked via a disulfide bond and Pro53 has been built in the cisconformation. Comparison of the IL‐1ra structure with the IL‐1α and IL‐1β structures present in the Protein Data Bank shows that a putative receptor interaction region, involving the N‐terminus up to the beginning of strand β1 and the loops D and G, is very different in the three IL‐1 molecules. Other putative interaction regions, as identified with mutagenesis studies, are structurally conserved and rigid, allowing precise and specific interactions with the IL‐1 receptor.
Abnormally High pKa of an Active‐Site Glutamic Acid Residue in Bacillus Circulans Xylanase
Authors: Davoodi Jamshid , Wakarchuk Warren W. , Campbell Robert L. , Carey Paul R. , Surewicz Witold K.
Published: July 2010

The active site of Bacillus circulans xylanase (1,4‐β‐D‐xylanohydrolase, EC 3.2.1.8) contains two glutamic acid residues, Glu78 and Glu172, which are crucial for the catalytic activity of the enzyme. Fourier‐transform infrared spectroscopy was used to determine the ionization state of these residues as a function of pH. For the wild‐type enzyme, titration of one of the carboxylate groups occurs at pH 6.8. This titration is absent in the Glu78→Gln and Glu172→Gln variants of the enzyme. This, together with crystallographic data, indicates that Glu172 has an abnormally high pKa of 6.8, caused largely by electrostatic interactions of this residue with the proximal Glu78. Differential scanning calorimetry experiments with the wild‐type xylanase and a number of its mutants have shown that the presence of two nearby carboxyl groups results in a pH‐dependent destabilization of the protein structure.
Interaction of a Fluorescent Analog of N ‐Deacetyl‐N ‐Methyl‐Colchicine (Colcemid) with Liver Alcohol Dehydrogenase
Authors: Sengupta Suparna , Mahapatra Pradip K. , Chakrabarti Gopal , Roy Siddhartha , Bhattacharyya Bhabatarak
Published: July 2010

The evidence for specific binding of N‐(7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl)‐colcemid (NBD‐colcemid), a fluorescent analog of colcemid (N‐deacetyl‐N‐methyl‐colchicine), to liver alcohol dehydrogenase is presented. Alcohol dehydrogenase bound NBD‐colcemid in a time‐dependent manner, enhanced the fluorescence intensity, and caused a large blue shift of the emission maximum of the free drug. The specificity of binding was determined for both the colchicine nucleus and the NBD moiety. The binding was not affected by the presence of alcohol or NAD in the reaction mixture. Preincubation of horse liver alcohol dehydrogenase with colcemid inhibited the binding to a considerable extent. NBD‐colcemid inhibited the enzymic activity of alcohol dehydrogenase in a mixed‐type noncompetitive mode with a Ki value of 32 μM, whereas colcemid showed noncompetitive inhibition with a Ki of 100 μM. The association rate constant of NBD‐colcemid binding with liver alcohol dehydrogenase was 587 M‐1 s‐1 at 25 °C. The stoichiometry and dissociation constant of the binding reaction were 0.62/dimer and 12 μM, respectively. Donor quenching experiments showed that both tryptophans of alcohol dehydrogenase transferred energy to the bound NBD‐colcemid. Thus, this study reports the binding of a colchicine analog to a protein other than tubulin with high affinity. It is concluded that NBD‐colcemid binding to dehydrogenases is a general phenomenon, but the common structural element(s) that is responsible for the binding activity, and which exists among tubulin and dehydrogenases, has yet to be determined.
The Thermosome of Thermoplasma acidophilum and Its Relationship to the Eukaryotic Chaperonin TRiC
Authors: Waldmann Thomas , Nimmesgern Elmar , Nitsch Michael , Peters Jürgen , Pfeifer Günter , Müller Shirley , Kellermann Joseph , Engel Andreas , Hartl Franz‐Ulrich , Baumeister Wolfgang
Published: July 2010

A high molecular‐mass protein complex from the archaebacterium Thermoplasma acidophilum, referred to here as the ‘thermosome’, is built from two subunits (Mr 58 and 60). The thermosome has been purified to homogeneity. The molecular mass of the native complex was determined to be 1061 ± 30 Da by scanning transmission electron microscopy. It shows a weak ATPase activity and is able to bind denatured polypeptides. Averages obtained from electron micrographs of negatively stained molecules in the end‐on and side‐on orientations, respectively, were compared with those of the t‐complex polypeptide 1 ring complex (TRiC), isolated from bovine testes. Both molecules consist of two stacked pseudo eightfold symmetric rings which build up a cylindrical particle with a large cavity in the center. Sequence alignments of peptides generated from both subunits of the thermosome and different subunits of TRiC reveal a high partial similarity to each other and to the archaebacterial chaperonin thermophilic factor 55 from Sulfolobus shibatae as well as to eukaryotic TCP1 proteins. These striking structural similarities confirm the proposition that all these molecules belong to a single protein family which is structurally and functionally related to the GroEL class of molecular chaperones.
Functional Characterization of Black Widow Spider Neurotoxins Synthesised in Insect Cells
Authors: Kiyatkin Nikita I. , Kulikovskaya Irina M. , Grishin Eugene V. , Beadle David J. , King Linda A.
Published: July 2010

α‐latrotoxin, α‐latroinsectotoxin and the low‐molecular‐mass protein from black widow spider venom were synthesised in insect cells using the baculovirus expression system. SDS/PAGE analysis of recombinant‐virus‐infected cells revealed novel proteins that migrated with sizes similar to those of the neurotoxins from spider venom. The identities of these proteins as α‐latrotoxin, α‐latroinsectotoxin or the low‐molecular‐mass protein were confirmed by immunoblot analysis of infected cells with anti‐(α‐latrotoxin), anti‐(α‐latroinsectotoxin) or anti‐(low‐molecular‐mass protein) IgG. Neither the low‐molecular‐mass protein nor α‐latrotoxin were toxic upon injection into Trichoplusia ni larvae or upon virus‐derived synthesis directly in the cytoplasm of the target tissue. Analysis of the biological activity of the recombinant virus encoding α‐latroinsectotoxin, however, revealed a strong toxic effect on the T. ni larvae. These data indicate that the toxic effect of the native insectotoxin may be promoted by the α‐latroinsectotoxin subunit alone and provides evidence that the mechanism of action of α‐latroinsectotoxin may be mediated by internalisation of part of the neurotoxin α‐subunit molecule.
The Roles of Tyr70 and Tyr225 in Aspartate Aminotransferase Assessed by Analysing the Effects of Mutations on the Multiple Reactions of the Substrate Analogue Serine O‐Sulphate
Authors: Birolo Leila , Sandmeier Erika , Christen Philipp , John Robert A.
Published: July 2010

Aspartate aminotransferase catalyses multiple reactions of the glutamate analogue, serine O‐sulphate. The predominant reaction is β‐elimination of sulphate to give aminoacrylate (kcat= 13 s−1 for the Escherichia coli enzyme) which may either hydrolyse to pyruvate and ammonia, or react covalently with the enzyme and inactivate it (kinact= 1.1×10‐3 s−1). Serine O‐sulphate also undergoes a transamination reaction that converts the enzyme to its pyridoxamine form (kcat= 0.11 s−1). Tyr70 and Tyr225, each of which forms a hydrogen bond with the coenzyme, were substituted with methionine and phenylalanine, respectively. The Y225F mutation does not affect β‐elimination but reduces the rates of transamination and inactivation about 70‐fold and 3‐fold, respectively. Apparently, Tyr225 is not essential for the steps leading to and including abstraction of the proton from Cα of the substrate. It is argued that the Y225F mutation interferes with ketimine hydrolysis. The Y70M mutation affects all three reactions, β‐elimination being about fourfold slower, transamination 340‐fold slower, and inactivation being 1,4 times faster than in the wild‐type enzyme. It is proposed that a hydrogen bond from Tyr70 positions Lys258 for protonation of the quinonoid intermediate at C4′ and that, although the full kinetic contribution of this interaction is only revealed in the multiple reactions of serine O‐sulphate, the same interaction is equally important in increasing the reaction specificity for transamination of the natural substrates.
Mutational Analysis of Mau Genes Involved in Methylamine Metabolism in Paracoccus Denitrificans
Authors: Van Der Palen Carol J. N. M. , Slotboom Dirk‐Jan , Jongejan Laurian , Reijnders Willem N. M. , Harms Nellie , Duine Johannis A. , Van Spanning Rob J. M.
Published: July 2010

A chromosomal fragment containing DNA downstream from mauC was isolated from Paracoccus denitrificans. Sequence analysis of this fragment revealed the presence of four open reading frames, all transcribed in the same direction. The products of the putative genes were found to be highly similar to MauJ, MauG, MauM and MauN of Methylobacterium extorquens AM1. Using these four mau genes, 11 mau genes have been cloned from P. denitrificans to date. The gene order is mauRFBEDACJGMN, which is similar to that in M. extorquens AM1. mauL, present in M. extorquens AM1, seems to be absent in P. denitrificans.

MauJ is predicted to be a cytoplasmic protein, and MauG a periplasmic protein. The latter protein contains two putative heme‐binding sites, and has some sequence resemblance to the cytochrome c peroxidase from Pseudomonas aeruginosa. MauM is also predicted to be located in the periplasm, but MauN appears to be membrane associated. Both resemble ferredoxin‐like proteins and contain four and two motifs, respectively, characteristic for [4Fe‐4S] clusters.

Inactivation of mauA, mauJ, mauG, mauM and mauN was carried out by introduction of unmarked mutations in the chromosomal copies of these genes. mauA and mauG mutant strains were unable to grow on methylamine. The mauJ mutant strain had an impaired growth rate and showed a lower dye‐linked methylamine dehydrogenase (MADH) activity than the parent strain. Mutations in mauM and mauN had no effect on methylamine metabolism. The mauA mutant strain specifically lacked the β subunit of MADH, but the α subunit and amicyanin, the natural electron acceptors of MADH, were still produced. The mauG mutant strain synthesized the α and β subunits of MADH as well as amicyanin. However, no dye‐linked MADH activity was found in this mutant strain. In addition, as the wild‐type enzyme displays a characteristic fluorescence emission spectrum upon addition of methylamine, this property was lost in the mauG mutant strain. These results clearly show that MauG is essential for the maturation of the β subunit of MADH, presumably via a step in the biosynthesis of tryptophan tryptophylquinone, the cofactor of MADH.

The mau gene cluster mauRFBEDACJGMN was cloned on the broad‐host vector pEG400. Transfer of this construct to mutant strains which were unable to grow on methylamine fully restored their ability to grow on this compound. A similar result was achieved for the closely related bacterium Thiosphaera pantotropha, which is unable to utilize methylamine as the sole sources of carbon and energy.
Effect of 1‐O ‐Octadecyl‐2‐O ‐Methyl‐Glycerophosphocholine on Phosphatidylcholine and Phosphatidylethanolamine Synthesis in MCF‐7 and A549 Cells and its Relationship to Inhibition of Cell Proliferation
Authors: Zhou Xi , Arthur Gilbert
Published: July 2010

The role of perturbation of lipid synthesis in the inhibition of cell proliferation by OctMeGroP Cho was investigated with sensitive (MCF‐7) and resilient (A549) cell lines. It inhibited de novo synthesis of phosphatidylcholine in both cells but increased triacylglycerol synthesis in A549 cells and phosphatidylethanolamine, phosphatidic acid and diacylglycerol synthesis in MCF‐7 cells. The inhibition of synthesis of CDP‐choline metabolites in MCF‐7 cells and phosphatidylcholine biosynthetic enzyme activities in vitro by OctMeGroP Cho suggests that direct inhibition of phosphocholine cytidylyltransferase may contribute to the observed inhibition of phosphatidylcholine synthesis. The activation of phosphoethanolamine cytidylyltransferase and ethanolamine phosphotransferase activities by OctMeGroP Cho in vitro and increased production of CDP‐ethanolamine suggest that stimulation of the above enzymes by OctMeGroPCho in the cells is responsible for the increased phosphatidylethanolamine synthesis. The apparent effect of OctMeGroP Cho on intracellular lipid‐metabolising enzymes is a strong indication that it may be widely distributed intracellularly and not just confined to the plasma membrane. The decrease in phosphatidylcholine synthesis by OctMeGroP Cho in MCF‐7 cells was prevented by co‐incubation with oleic acid without any effect on the inhibition of cell growth. Although OctMeGroP Cho resulted in similar decreases in phosphatidylcholine content in both cells, this did not affect the proliferation of A549 cells. The above results indicate that, although OctMeGroP Cho has profound effects on lipid metabolism, these changes are not responsible for the inhibition of proliferation observed in MCF‐7 cells.
3‐Deoxy‐Octulosonic‐Acid‐Containing Hexasaccharide Fragment of Unusual Core Type Isolated from Hafnia alvei 2 Lipopolysaccharide
Authors: Ravenscroft Neil , Gamian Andrzej , Romanowska Elżbieta
Published: July 2010

The hexasaccharide containing 3‐deoxy‐octulosonic acid (Kdo) was isolated from the carbohydrate material obtained after the mild acid hydrolysis of lipopolysaccharide of Hafnia alvei strain 2. The hexasaccharide was purified by gel filtration on Bio‐Gel P‐4 and P‐2 columns. On the basis of sugar and methylation analyses, one‐ and two‐dimensional NMR spectroscopic methods, the hexasaccharide was identified as
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The tetrasaccharide linked to position 7 of Kdo is also part of the sialic‐acid‐containing O‐specific unit. Di‐substitution of Kdo at positions 7 and 8 has not been previously reported.
Studies on a Stabilisation of Ubisemiquinone by Escherichia coli Quinol Oxidase, Cytochrome bo
Authors: Ingledew W. John , Ohnishi Tomoko , Salerno John C.
Published: July 2010

The Escherichia coli quinol oxidase, cytochrome bo, is closely related to the cytochrome c oxidase, cytochrome aa3 in all aspects of its structure and function except for the replacement of the cytochrome‐c ‐binding site and its attendant CuA prosthetic group with a quinone‐binding site. The putative oxidation of quinol by ferrihaem (cytochrome b) at this site in sequential one‐electron steps requires the stabilisation of semiquinone. We have observed, by electron paramagnetic resonance, the properties of a ubisemiquinone radical in appropriately poised samples of purified enzyme reconstituted with excess ubiquinone. The ubisemiquinone is highly stabilised with respect to free ubisemiquinone; significant free radical can be observed even at pH 7.0, while at pH 9.0 the stability constant is 5–10. The pH dependence of the stability constant indicates that the anionic form of the semiquinone predominates above pH 7.5. The two‐electron couple has an Em7 of approximately 70 mV. Below pH 9, the pH dependence of the two‐electron couple is –60 mV/pH, indicative of a 2H+/2e‐ reaction.

The line width of the EPR spectrum is approximately 0.9 mT, which is consistent with a ubisemiquinone anion. In comparison with other respiratory chain Q˙‐ species that have been described, the relaxation rate in the presence of reduced haems appears comparable to magnetically isolated Q˙‐ radicals. Partially resolved splittings of approximately 0.4 mT can be observed in the spectrum of Q˙‐bo (QH˙bo).
Human Diseases with Defects in Oxidative Phosphorylation
Authors: Bentlage Herman , Coo René de , ter Laak Henk , Sengers Rob , Trijbels Frans , Ruitenbeek Wim , Schlote Wolfgang , Pfeiffer Kathy , Gencic Simonida , von Jagow Gebhard , Schägger Hermann
Published: July 2010

The amount of oxidative phosphorylation enzymes in mitochondrial encephalomyopathy patients has been studied by two‐dimensional electrophoresis (blue native PAGE/Tricine‐SDS‐PAGE). Only 20 mg muscle was required to identify and analyse complexes I, III, IV, and V after Coomassie staining. In most cases reduced amounts of the involved complex(es) correlated well with decreased enzyme activities. The reliability of the method was reflected by the constant mutual ratio of the complexes found in all controls. Deviations from normal ratios were found to be more sensitive indicators for a defect than the absolute quantities, which varied considerably within the control group both in the enzymic and in the electrophoretic analysis. The effect of the mitochondrial tRNALeu(UUR) mutation in mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke‐like episodes on the amount of oxidative phosphorylation complexes was demonstrated for the first time directly on the protein level. In patients without known DNA mutations, specific defects of single complexes were identified. The new technique is a sensitive method for the identification of oxidative phosphorylation defects, complementary to enzymic measurements.
Human Diseases with Defects in Oxidative Phosphorylation
Authors: Schägger Hermann , Ohm Thomas Georg
Published: July 2010

F1F0 ATP‐synthase (complex V) deficiencies in Alzheimer's disease are reported. Tissue specimens from the hippocampus of brains from patients with Alzheimer's disease were screened by blue native electrophoresis for alterations of the proteins of oxidative phosphorylation. Ubiquinol:cytochrome‐c reductase (complex III) and cytochrome‐c oxidase (complex IV) were found to be present at almost normal concentrations, however, complex V was substantially reduced in most cases studied. The specific reduction of complex V and the absence of electrophoretically detectable degradation products do not exclude a secondary defect of complex V, but should stimulate the search for genetic defects related to protein subunits of complex V.
Structure and Expression of Hemolin, an Insect Member of the Immunoglobulin Gene Superfamily
Authors: Lindström‐Dinnetz Ingrid , Sun Shao‐Cong , Faye Ingrid
Published: July 2010

Hemolin is an insect protein which belongs to the immunoglobulin superfamily and is strongly induced upon bacterial infection. It has been isolated from two moths, Hyalophora cecropia and Manduca sexta. We have isolated and sequenced a genomic clone for hemolin in H. cecropia, in order to resolve its organization and as a basis for investigating hemolin gene regulation. According to Southern‐blot analysis, hemolin is encoded by a single gene, Hemolin. It contains six exons ranging over 32–603 bp. The introns are positioned both within and between the immunoglobulin‐like domains, a feature typical for cell‐adhesion molecules belonging to the immunoglobulin superfamily. By an RNase protection assay, we show that the Hemolin transcript is strongly induced not only by bacteria, but also by lipopolysaccharide and phorbol 12‐myristate 13‐acetate. Analysis of the upstream region and introns revealed potential binding sites for the Cecropia immunoresponsive factor (CIF), which recognizes the κB‐like consensus GGGRA YYYYY.
Binding of Thyroxine to Pig Transthyretin, its cDNA Structure, and Other Properties
Authors: Duan Wei , Richardson Samantha J. , Köhrle Josef , Chang Linus , Southwell Bridget R. , Harms Paul J. , Brack Charlotte M. , Pettersson Tom M. , Schreiber Gerhard
Published: July 2010

Thyroxine binding to proteins in pig plasma during electrophoresis was observed in the albumin, but not in the prealbumin and post‐albumin regions.

Transthyretin could be identified in medium from in vitro pig choroid plexus incubations by size and number of subunits and a very high rate of synthesis and secretion. Its electrophoretic mobility was intermediate between that of thyroxine‐binding globulin and albumin. It bound thyroxine, retinol‐binding protein, anti‐(rat transthyretin) antibodies and behaved similarly to transthyretins from other vertebrate species when plasma was extracted with phenol. Inhibition experiments with the synthetic flavonoid F 21388, analysing the binding of thyroxine, suggested that transthyretin is not a major thyroxine carrier in the bloodstream of pigs.

Cloning and sequencing of transthyretin cDNA from both choroid plexus and liver showed that the same transthyretin mRNA is expressed in pig choroid plexus and liver. The amino acid sequence derived from the nucleotide sequence revealed that pig transthyretin differs from the transthyretins of all other studied vertebrate species by an unusual C‐terminal extension consisting of the amino acids glycine, alanine and leucine. This extension results from the mutation of a stop codon into a codon for glycine. The unusual C‐terminal extensions do not seem to interfere with the access of thyroxine to its binding site in the central channel of transthyretin.
NMR Studies and Redox Titration of the Tetraheme Cytochrome c3 from Desulfomicrobium baculatum
Authors: Coutinho Isabel B. , Turner David L. , Legall Jean , Xavier António V.
Published: July 2010

The tetraheme cytochromes c3 isolated from two strains of Desulfomicrobium baculatum were studied by monitoring the spectral changes undergone during redox titrations followed by 1H NMR. The evolution of the three‐proton intensity signals at low field allowed the partial identification of the heme methyl resonances in the spectrum of the fully oxidized state. The chemical shift variation shown by the protons of the aromatic sidechains as well as of the substituents of the higher‐potential heme HIII [Coutinho, I. B., Turner, D. L., LeGall, J. & Xavier, A. V. (1993) Biochem. J. 294, 899–908] yielded the assignment of the lower midpoint redox potential to heme HII in the three‐dimensional structure. This cross‐assignment is achieved by comparing the chemical shifts of the resonances in the spectra obtained at intermediate oxidation levels with the pseudocontact shifts predicted to arise from the three lower‐potential hemes. The cross‐assignment for the cytochromes from these two strains is different from that of the cytochromes from Desulfovibrio vulgaris and Desulfovibrio gigas.
Cloning, Sequencing, and Determination of the Sites of Expression of Mouse Sorbitol Dehydrogenase cDNA
Authors: Lee Fuk Ki , Lee Alan Yiu Wah , Lin Colin Xiao Feng , Chung Stephen Sum‐Man , Chung Sookja Kim
Published: July 2010

Sorbitol dehydrogenase is one of the enzymes in the polyol pathway, which is thought to be implicated in the pathogenesis of diabetic complications. The cDNA encoding mouse sorbitol dehydrogenase was cloned from a liver library and its sequence was determined. The open reading frame encodes a product of 356 amino acids that shares high similarity with the human and rat liver sorbitol dehydrogenases (83% and 93% identity, respectively). The 3′‐untranslated region contains a truncated L1Md repeat element inserted in reverse relative to the sorbitol dehydrogenase cDNA. Northern‐blot hybridization showed that the testis has the highest level of expression, followed by kidney, liver, and lung. Low levels of expression were also observed in lens, brain, and skeletal muscle. In situ hybridization revealed that in the kidney, the highest concentration of sorbitol dehydrogenase mRNA is observed in the cortex, but is absent from the inner medulla. The parenchymal cells of the liver showed strong expression while the cells of the hepatic vasculature did not hybridize. The sorbitol dehydrogenase expression in the seminiferous tubules was mostly associated with the mature cells of the developing germ cells, confirming the usefulness of sorbitol dehydrogenase as an enzyme indicator for sexual maturation. The seminal vesicle, where most of the seminal fructose is produced, also showed a high level of expression in the epithelial cells. The mouse sorbitol dehydrogenase cDNA will be useful in the studies of the involvement of the polyol pathway in diabetic complications.
A Human Homologue of the Yeast Replication Protein Cdc21
Authors: Musahl Christine , Schulte Dorothea , Burkhart Roland , Knippers Rolf
Published: July 2010

We present the amino acid sequence of the human homologue of the yeast replication protein Cdc21, a member of the Mcm family of nuclear proteins. Specific antibodies, raised against protein hCdc21, were used to investigate the expression of the protein through the cell cycle. The protein is highly phosphorylated in mitotic cells. The phosphorylated form of protein hCdc21 appears to be less tightly bound to nuclear structures than the underphosphorylated form suggesting that phosphorylation/dephosphorylation reactions may determine the nuclear distribution of the protein. Protein hCdc21 forms a stable trimeric complex with two novel human Mcm proteins, p85Mcm and p105Mcm. Protein BM28/Mcm2 is more loosely associated with the trimeric hCdc21 complex.
Transferrin, a Mechanism for Iron Release
Authors: Chahine Jean‐Michel El Hage , Pakdaman Rowchanak
Published: July 2010

Iron release from transferrin has been investigated in mildly acidic and acidic media in the presence of formate, acetate and citrate. It occurs first from the N‐terminal iron‐binding site (N‐site) of the holoprotein. It is independent of the nature and the concentration of competing ligands and is controlled by a slow proton transfer; second‐order rate constant k1= (7.4 ± 0.5) × 104 M−1 s−1 which can be attributed to a rate‐limiting slow proton gain by a protein ligand subsequent to a fast decarbonation of the N‐site. Iron loss from the C‐terminal iron‐binding site (C‐site) is slower than that from the N‐site and occurs by two pathways. The first is favoured below pH 4 and does not involve the formation of an intermediate ternary complex. It can be controlled by a rate‐limiting slow proton‐triggered decarbonation of the binding site; second‐order rate constant k3= (2.25 ± 0.05) × 104 M−1 s−1. The second pathway is favoured above pH 4 and involves a mixed protein‐ligand iron complex. It takes place through the slow protonation of the mixed ternary complex and depends on the nature of the competing ligand. It is faster in the presence of citrate than in that of acetate; second‐order rate constant k4= (1.75 ± 0.10) × 103 M−1 s−1 for citrate and (85 ± 5) M−1 s−1 for acetate. All these phenomena can possibly describe proton‐triggered changes of conformation of the binding sites.
Characterization of Mutations in the Mitochondrial Cytochrome b Gene of Saccharomyces Cerevisiae Affecting the Quinone Reductase Site (QN)
Authors: Brasseur Gaël , Brivet‐Chevillotte Paule
Published: July 2010

The revertant [G33A]cytochrome b recently isolated from the [G33D]cytochrome b mutant [Coppée, J.Y., Tokutake, N., Marc, D., di Rago, J.‐R, Miyoshi, H. & Colson, A.‐M. (1994) FEBS Lett. 339, 1–6] exhibits cross resistance to center‐N inhibitors 2‐heptyl‐4‐hydroxyquinoline N‐oxide (HQNO) and funiculosin and a spectral shift in the cytochrome b562 heme. This indicates that the conserved G33 residue is in the vicinity of this heme, and thus agrees with the previous suggestion that glycine may play a role in the helix packing around the hemes. The [S206L]cytochrome b and [M221K]cytochrome b respiratory‐growth‐deficient mutants [Lemesle‐Meunier, D., Brivet‐Chevillotte, P., di Rago, J. P., Slonimski, P. P., Bruel, C., Tron, T. & Forget, N. (1993) J. Biol. Chem. 268, 15626–15632], which synthesize cytochrome b and retain little or no bc1 complex activity, show no change in the reduction kinetics of cytochrome b via center P, which suggests that the oxidizing site is functional. Impairment of both the reduction and oxidation of heme b562 at the ubiquinone reduction center of the mitochondrial ubiquinone‐cytochrome‐c oxidoreductase site is, therefore, responsible for the deficient catalytic activity and respiratory growth in these strains.
Transitions of gene expression induced by short‐term blue light
Authors: Lehmann P. , Nöthen J. , Schmidt von Braun S. , Bohnsack M. T. , Mirus O. , Schleiff E.
Published: July 2010

Blue light modulates many processes in plants and plant cells. It influences global and long‐term responses, such as seedling development and phototropism, and induces short‐term adaptations like stomatal opening and chloroplast movement. Three genes were identified as important for the latter process, namely PHOT1, PHOT2 and CHUP1. The former two phototropin blue light receptors act in perception of the blue light signal. The protein CHUP1 is localised to the outer envelope membrane of chloroplasts and is involved in chloroplast movement. To explore whether short‐term reactions required for chloroplast movement are under transcriptional control, we analysed the transcriptome in wild‐type Arabidopsis thaliana, phot1, phot2 and chup1 with different blue light treatments for 5 or 30 min. Blue light‐induced changes in transcription depended on illumination time and intensity. Illumination with 100 μmol·m−2·s−1 blue light induced down‐regulation of several genes and might point to cascades that could be important for sensing low levels of blue light. Analysis of the transcriptome of the mutants in response to the different light regimes suggests that the transcriptional response to blue light in the wild‐type can be attributed to phot1 rather than phot2, suggesting that blue light‐induced alteration of expression is a function of phot1. In contrast, the blue light response at the transcriptional level of chup1 plants was unique, and confirmed the higher light sensitivity of this mutant.
OCT4 Expression in Outgrowth Colonies Derived from Porcine Inner Cell Masses and Epiblasts
Authors: Wolf XA , Rasmussen MA , Schauser K , Jensen AT , Schmidt M , Hyttel P
Published: July 2010

Contents

The present study was conducted to test different methods for porcine inner cell mass (ICM) and epiblast isolation and to evaluate the morphology and expression of pluripotency genes in ICM‐ and epiblast‐derived outgrowth colonies (OCs) and passages thereof with particular attention on the relationship between OCT4 expression and embryonic stem cell (ESC)‐like morphology. A total of 104 zona pellucida‐enclosed and 101 hatched blastocysts were subjected to four different methods of ICM and epiblast isolation, respectively: Manual isolation, immunosurgery, immunosurgery with manual cleaning, or whole blastocyst culture. OCs were established on mouse embryonic fibroblast (MEF) cells and categorized according to morphology and OCT4 staining. Although all isolation methods resulted in ESC‐like OCs, immunosurgery with manual cleaning yielded significantly higher rates of ICM/epiblast attachment and subsequent ESC‐like morphology, whereas no significant difference was found between ICM and epiblasts with respect to these characteristics. All ESC‐like OCs showed nuclear OCT4 staining and expression of OCT4, NANOG and SOX2 as evaluated by RT‐PCR. Upon initial passages, the expression of pluripotency markers was, however, gradually lost in spite of maintained ESC‐like morphology. In conclusion, we have established a robust system for derivation of ESC‐like OCs from porcine ICM and epiblasts and we have shown that localization of OCT4 is associated with an ESC‐like morphology although this relationship is lost during early passages.
Effect of mSOF and G1.1/G2.2 Media on the Developmental Competence of SCNT‐Derived Bovine Embryos
Authors: Wang YS , Tang S , An ZX , Li WZ , Liu J , Quan FS , Hua S , Zhang Y
Published: July 2010

Contents

The objective of this study was to compare the effect of two culture media: modified synthetic oviductal fluid (mSOF) and G1.2/G2.2, on the developmental competence of bovine somatic cell–cloned embryos. Cloned embryos were produced by transferring adult skin fibroblasts into enucleated MII oocytes. After activation, the reconstructed embryos were randomly allotted to either mSOF or G1.2/G2.2 for culture (the embryos were transferred from G1.2 to G2.2 on days 3 of culture). The development competence of cloned embryos in these two culture systems was compared in terms of cleavage rate, blastocyst formation rate and apoptosis cell number in day 7 blastocyts. To investigate the in vivo developmental competence of cloned embryos in the two culture systems, a total of 87 and 104 blastocysts derived from mSOF and G1.2/G2.2 medium groups were transferred individually to recipient Angus cows, respectively. No differences were observed in terms of cleavage rate, day 7 blastocyst rate and blastocyst cell number between these two culture systems. However, the day 6 blastocyst formation rate was significantly higher in G1.2/G2.2 than that in mSOF. In addition, blastocysts cultured in mSOF have a higher percentage of apoptotic blastomeres compared to those in G1.2/G2.2 (8.5 ± 1.2 vs 16.8 ± 1.5, p < 0.05). Although difference in pregnancy rate was not observed 40 days after embryo transfer, significantly higher pregnancy rate was observed in G1.2/G2.2 group after 90 days of embryo transfer (12.4% vs 37.5%, p < 0.05). Moreover, calving rate was significantly improved in G1.2/G2.2 group compared to mSOF group (27.9% vs 6.7%, p < 0.05). In conclusion, our results indicate that G1.2/G2.2 can improve developmental competence of bovine SCNT embryos both in vitro and in vivo, which is more suitable for culture of bovine SCNT embryos than mSOF medium.
Lilliputians get into the limelight: Novel class of small peptide genes in morphogenesis
Authors: Hashimoto Yoshiko , Kondo Takefumi , Kageyama Yuji
Published: July 2010

Generally, bioactive small peptides are derived from precursors with signal sequences at their N‐terminal ends, which undergo modification and proteolysis through a secretory pathway. By contrast, small peptides encoded in short open reading frames (sORF) lack signaling sequences and therefore are released into the cytoplasm, which may result in their having functions distinct from those of secreted peptides. Several small peptides encoded by sORF are involved in the morphogenesis of multicellular organisms. POLARIS, ROTUNDIFOLIA4, and Enod40 are plant peptides that are involved, respectively, in root formation, leaf shape control, and cortical cell division during nodule formation. Brick1/HSPC300 is an evolutionarily conserved component of the actin reorganization complex. polished rice/tarsal‐less and mille‐pattes encode related small peptides that are required for epithelial morphogenesis in Drosophila and segmentation in Tribolium. There are only a few known examples of small peptides encoded by sORF, and their molecular functions are still largely obscure. Nevertheless, an increasing number of sORF genes is being identified, and further research should reveal their roles in novel molecular mechanisms underlying developmental events.
Regulation of oocyte maturation in fish
Authors: Nagahama Yoshitaka , Yamashita Masakane
Published: July 2010

A period of oocyte growth is followed by a process called oocyte maturation (the resumption of meiosis) which occurs prior to ovulation and is a prerequisite for successful fertilization. Our studies using fish models have revealed that oocyte maturation is a three‐step induction process involving gonadotropin (LH), maturation‐inducing hormone (MIH), and maturation‐promoting factor (MPF). LH acts on the ovarian follicle layer to produce MIH (17α, 20β‐dihydroxy‐4‐pregnen‐3‐one, 17α, 20β‐DP, in most fishes). The interaction of ovarian thecal and granulosa cell layers (two‐cell type model), is required for the synthesis of 17α,20β‐DP. The dramatic increase in the capacity of postvitellogenic follicles to produce 17α,20β‐DP in response to LH is correlated with decreases in P450c17 (P450c17‐I) and P450 aromatase (oP450arom) mRNA and increases in the novel form of P450c17 (P450c17‐II) and 20β‐hydroxysteroid dehydrogenase (20β‐HSD) mRNA. Transcription factors such as Ad4BP/SF‐1, Foxl2, and CREB may be involved in the regulation of expression of these steroidogenic enzymes. A distinct family of G‐protein‐coupled membrane‐bound MIH receptors has been shown to mediate non‐genomic actions of 17α, 20β‐DP. The MIH signal induces the de novo synthesis of cyclin B from the stored mRNA, which activates a preexisting 35 kDa cdc2 kinase via phosphorylation of its threonine 161 by cyclin‐dependent kinase activating kinase, thus producing the 34 kDa active cdc2 (active MPF). Upon egg activation, MPF is inactivated by degradation of cyclin B. This process is initiated by the 26S proteasome through the first cut in its NH2 terminus at lysine 57.
Mechanisms of trophectoderm fate specification in preimplantation mouse development
Authors: Sasaki Hiroshi
Published: July 2010

During preimplantation mouse development, embryos establish two distinct cell lineages by the time of blastocyst formation: trophectoderm (TE) and inner cell mass (ICM). To explain the mechanism of this cell fate specification, two classical models, namely the inside–outside model and polarity model have been proposed based on experimental manipulation studies on embryos. This review summarizes recent findings on the molecular mechanisms of fate specification, and discusses how these findings fit into the classical models. TE development is regulated by a transcription factor cascade, the core transcription factors of which are Tead4 and Cdx2. The transcriptional activity of Tead4 is regulated by the position‐dependent Hippo signaling pathway, thus supporting the inside–outside model. In contrast, several findings support the polarity model; some other findings suggest different mechanisms. We also discuss how the two classical models could be further developed in the light of recent molecular findings.
Role of the polycomb group proteins in hematopoietic stem cells
Authors: Konuma Takaaki , Oguro Hideyuki , Iwama Atsushi
Published: July 2010

Polycomb group (PcG) proteins play a role in the transcriptional repression of genes through histone modifications. Recent studies have clearly demonstrated that PcG proteins are required for the maintenance of embryonic as well as a broad range of adult stem cells, including hematopoietic stem cells (HSCs). PcG proteins maintain the self‐renewal capacity of HSCs by repressing tumor suppressor genes and keep differentiation programs poised for activation in HSCs by repressing a cohort of hematopoietic developmental regulator genes via bivalent chromatin domains. Enforced expression of one of the PcG genes, Bmi1, augments the self‐renewal capacity of HSCs. PcG proteins also maintain redox homeostasis to prevent premature loss of HSCs. These findings established PcG proteins as essential regulators of HSCs and underscored epigenetics as a new field of HSC research. In this review, we focus on the role of PcG proteins in the epigenetic regulation of the self‐renewal capacity and multipotency of HSCs.
CLINICAL CHARACTERISTICS AND TREATMENT FOR PATIENTS PRESENTING WITH BLEEDING DUODENAL VARICES
Authors: Kakizaki Satoru , Toyoda Mitsuo , Ichikawa Takeshi , Sato Ken , Takagi Hitoshi , Arai Hirotaka , Sohara Naondo , Iizuka Haruhisa , Onozato Yasuhiro , Mori Masatomo
Published: July 2010

Background and Aim:  Bleeding from ectopic varices, including duodenal varices, is uncommon, but it can be difficult to manage. The clinical data of patients diagnosed and treated for duodenal varices were reviewed to investigate the strategy for treatment.

Methods:  The present study reviewed the clinical data of 10 patients with duodenal varices (mean age, 58.2 ± 15.6 years) at our associated institutes during the period between January 1996 and December 2008.

Results:  Nine patients had duodenal varices located in the second portion, whereas in one case they were located in the duodenal bulbus. The underlying diseases included liver cirrhosis in eight patients, and extrahepatic portal vein obstruction in two patients. The lesions were identified with bleeding from varices in eight of 10 patients. Initial hemostasis was achieved in all eight patients. However, among four patients treated endoscopically only, two patients died from rebleeding from varices and two died from hepatic failure resulting from variceal bleeding. Additional interventional radiology (IVR) was used in three patients and additional surgery was carried out in one case. One patient who was treated with balloon‐occluded retrograde transvenous obliteration rebled during IVR and died from bleeding. Two patients who underwent double balloon‐occluded embolotherapy and one case who had surgery achieved good clinical outcomes.

Conclusions:  Although endoscopic treatment is useful for initial hemostasis of hemorrhagic duodenal varices, the patients who underwent additional IVR after endoscopic treatment achieved good outcomes.