bluuebunny/crossref_metadata_2025 huggingface dataset:
Heterologous Expression of the Human D2S Dopamine Receptor in Protease‐Deficient Saccharomyces cerevisiae Strains
Authors: Sander Peter , Grünewald Sylvia , Bach Marion , Haase Winfried , Reiländer Helmut , Michel Hartmut
Published: March 2005

The cDNA for the human D2S dopamine receptor has been functionally expressed in the unicellular yeast Saccharomyces cerevisiae. The original D2S gene and an elongated D2S gene with an N‐terminal fusion to the first 24 amino acids of the STE2 gene from S. cerevisiae were introduced into the episomal yeast expression vector YEP51 under the control of the GAL10 promoter. Expression studies performed in a wild‐type strain and in two protease‐deficient strains of S. cerevisiae revealed that the receptor was functionally expressed with respect to its ligand‐binding properties. The KD values for the binding of the dopamine antagonist [3H]spiperone were calculated to be 1.6 nM for the D2S receptor alone and 1.9 nM for the STE2‐D2S chimaera. Both membrane proteins could be further characterized by ligand‐displacement studies using certain dopamine agonists and antagonists. D2S dopamine‐receptor‐specific polyclonal antibodies were used to monitor the heterologous expression of the receptor. Western‐blot analysis of membranes prepared from transformed yeast cells producing either the receptor protein alone or the receptor fusion protein revealed apparent molecular masses of 40 kDa (D2S receptor alone) and 42 kDa (STE2/D2S receptor fusion protein). It could be shown that, in comparison to the expression in a wild‐type S. cerevisiae strain, the amount of receptor degradation was drastically reduced in the protease‐deficient strains. The localizations of the heterologously produced dopamine receptor and of the chimaera in the recombinant yeast were studied by immunogold electron microscopy and were found to be restricted mainly to the vacuole of the cells.
Arrangement of mRNA at the Decoding Site of Human Ribosomes
Authors: Malygin Alexey A. , Graifer Dmitri M. , Bulygin Konstantin N. , Zenkova Marina A. , Yamkovoy Vitaly I. , Stahl Joachim , Karpova Galina G.
Published: March 2005

Affinity labeling of human placental 80S ribosomes with mRNA analogs of up to 12 uridyl residues, i.e. alkylating derivatives of oligouridylates bearing either 4‐(N‐2‐chloroethyl‐N‐methyl‐amino)benzylmethylphosphamide group at the 5′‐termini or 2′,3′‐O‐[4‐(N‐2‐chloroethyl‐N‐methyl‐amino)]benzylidene residue attached to the 3′‐termini, in the presence of cognate Phe‐tRNAPhe has been investigated. All the mRNA analogs modified only the 40S subunit. The fraction of 18S rRNA modified by the mRNA analogs with the alkylating group at the 5′‐end decreased dramatically with extension of the reagent oligouridylate moiety. Nucleotides of 18S rRNA alkylated with the mRNA analogs were determined using a reverse transcription technique. For the mRNA analogs with the alkylating groups at the 3′‐termini, G1702 and G1763/G1764 were identified as the cross‐linking sites. The intensities of the bands corresponding to reverse transcriptase stops depended on the length of the reagent oligouridylate moieties. Cross‐linking sites of the mRNA analogs with the alkylating group at the 5′‐termini on 18S rRNA were A1023, C1026, C1057 and A1058 for the (pU)3 and (pU)4 derivatives and a single nucleotide C1057 for the (pU)6 one. Ribosomal protein S26 was found as the main target of modification with the same derivatives of (pU)6 and (pU)12.
Selective Activation and Inhibition of Calmodulin‐Dependent Enzymes by a Calmodulin‐Like Protein Found in Human Epithelial Cells
Authors: Edman Carl F. , George Samuel E. , Means Anthony R. , Schulman Howard , Yaswen Paul
Published: March 2005

A calmodulin‐like protein, which is identical in size and 85% identical to vertebrate calmodulin, was recently identified by ‘subtractive hybridization’ comparison of transcripts expressed in normal versus transformed human mammary epithelial cells. Unlike the ubiquitous distribution of calmodulin, calmodulin‐like protein expression is restricted to certain epithelial cells, and appears to be modulated during differentiation. In addition, calmodulin‐like protein levels are often significantly reduced in malignant tumor cells as compared to corresponding normal epithelial cells. The current studies compare calmodulin‐like protein functions with those of calmodulin. We find that calmodulin‐like protein activation of multifunctional Ca2+/calmodulin‐dependent protein kinase II (calmodulin kinase II) is equivalent to activation by calmodulin, but that four other calmodulin‐dependent enzymes, cGMP phosphodiesterase, calcineurin, nitric‐oxide synthase, and myosin‐light‐chain kinase, display much weaker activation by calmodulin‐like protein than by calmodulin. In the case of myosin‐light‐chain kinase, calmodulin‐like protein competitively inhibits calmodulin activation of the enzyme with a Ki value of 170 nM. Thus, calmodulin‐like protein may have evolved to function as a specific agonist of certain calmodulin‐dependent enzymes, and/or as a specific competitive antagonist of other calmodulin‐dependent enzymes.
The Interaction of Synthetic Templates with Eukaryotic DNA Primase
Authors: Anarbaev Rashid O. , Vladimirova Olga V. , Lavrik Olga I.
Published: March 2005

The template interaction of calf thymus and human placenta DNA primases has been investigated. Using oligothymidylates, we showed that a template consisting of ten monomeric units was the critical size for interaction with the enzyme. The hydrophobic effect is likely to be a major factor determining template recognition by the DNA primase. The correlation between the template affinity with the enzyme and the octanol‐water hydrophobic scale confirms this suggestion. In addition, the electrostatic interaction between the phosphate group of the template and side chains on the enzyme probably increases template affinity. Using methylated poly(dA), we found that the first nucleotide base of the primer should be more hydrophobic than the corresponding nucleotide base of the template. A model for the mechanism of action of DNA primase is suggested on the basis of data presented in this study and previous findings. According to this model, (a) DNA primase binds ten nucleotides of the template; (b) the synthesis of primer up to the formation of a decamer occurs processively and competes with template binding of the enzyme; (c) the conditions under which the nucleotides are incorporated into the RNA product change during the extension of the primer, and these changes are responsible for switching from primase to polymerase activity. The template specificity of DNA primase is likely to play an important role in the initiation and regulation of DNA replication.
Recombinant Chicken Interferon from Escherichia coli and Transfected COS Cells is Biologically Active
Authors: Schultz Ursula , Rinderle Conny , Sekellick Margaret J. , Marcus Philip I. , Staeheli Peter
Published: March 2005

We have expressed a cDNA for virus‐induced chick interferon in Escherichia coli. The product, a 19‐kDa protein lacking the signal peptide, was purified to homogeneity from the bacterial inclusion bodies. Proteins in the insoluble fraction of bacterial lysates were dissolved in guanidine hydrochloride and subjected to chromatography on Q‐Sepharose and MonoS columns. Purified recombinant chick interferon has a specific antiviral activity of approximately 108 IU/mg and is a powerful inducer of the interferon‐responsive promoter of the chicken mx gene. Culture medium of transfected COS cells expressing full‐length chick interferon cDNA contained up to 5 × 104 IU antiviral activity/ml that could be neutralized by antibodies to purified recombinant chick interferon. The antibodies precipitated proteins of 23–28 kDa from the supernatants of transfected COS cells. Treatment with endoglycosidase F reduced the size of the immunoprecipitated proteins to approximately 20 kDa, demonstrating that chick interferon is a glycoprotein.
Arg21 is the Preferred Kexin Cleavage Site in Parathyroid‐Hormone‐Related Protein
Authors: Diefenbach‐Jagger Hanne , Brenner Charles , Kemp Bruce E. , Baron Will , Mclean John , Martin T. John , Moseley Jane M.
Published: March 2005

Parathyroid‐hormone‐related protein (PTHrP) contains several potential sites for proteolytic processing. Although there is considerable evidence for the existence of cleaved products in vivo, little is known about the post‐translational processing of PTHrP. We have used purified kexin (Kex2) protease to identify which cleavage sites in recombinant PTHrP(1–141) might be of physiological significance. Cleavage products were identified by N‐terminal sequencing. Kex2 preferentially cleaved PTHrP(l‐141) carboxy to the triplet arginine site Arg‐Arg‐Arg21 with a Km of 3.3±1.7 μM and a kcatof 6±1.2 s−1. Substitution of alanine for Arg19 resulted in substantially reduced conversion, while no detectable cleavage occurred when alanine was substituted for either Arg20 or Arg21. In contrast, the degree of Kex2 cleavage at Arg21 in PTHrP(l‐34) was lower. No detectable cleavage occurred in an unrelated synthetic peptide containing both double and triple arginine sites. Low levels of cleavage also took place carboxy to Lys‐Arg97, Lys‐Arg105, Arg‐Arg106 and Thr‐Arg108. Cleavage carboxy to Lys‐Arg105, the best of these minor sites, occurred with a Km of 8.4±2.7 μM and a kcat of 0.8±0.2 s−1. These studies indicate that the preferred Kex2 cleavage site in PTHrP(l ‐141) is carboxy to Arg‐Arg‐Arg21, which effectively destroys its parathyroid‐hormone‐like biological activity. Cleavage of this site by Kex2‐related mammalian convertases in vivo may be an important mechanism for full elaboration of the non‐parathyroid‐hormone‐like paracrine actions of PTHrP in a tissue‐specific manner.
Two Distinct Regions of Ras Participate in Functional Interaction with GDP‐GTP Exchangers
Authors: Segal Marisa , Marbach Irith , Willumsen Berthe M. , Levitzk Alexander
Published: March 2005

We have previously implemented a combined genetic/biochemical approach, for analysis of insertion‐deletion mutants, to identify sites of Harvey‐Ras participating in the interaction with guanine nucleotide exchangers, using the yeast Cdc25 as a model exchanger. We showed that positions 101–106 may be required for catalyzed exchange. We here present a further improved strategy to define more precisely the residues on Ras participating in this interaction. Non‐conservative replacements at positions 103 or 105 abolished response to Cdc25 while substitutions at positions 102 or 104 were partially affected. The same substitutions had no effect on coupling to adenylyl cyclase. Since the strategy enables us to assess Ras functional interaction with both the exchanger and effector simultaneously, we have also examined the effect of substitutions in the distal part of the switch II region (amino acids 69–78). In contrast to other reports, substitutions at positions 69 or 73 prevented Cdc25 response while mutations at position 74 did not prevent this interaction. However, all these substitutions partly affected cyclase activation. These findings establish the crucial role of the 102–105 region in the catalyzed exchange reaction and suggest that the 69–74 area would be required for the functional interaction with both exchangers and effector molecules.
GTP Hydrolysis by HypB is Essential for Nickel Insertion into Hydrogenases of Escherichia Coli
Authors: Maier Thomas , Lottspeich Friedrich , Böck August
Published: March 2005

The product of the hypB gene, which is required for the maturation of the three [NiFe]hydrogenases of Escherichia coli, is a member of the GTPase family and exhibits a low intrinsic GTPase activity. It was studied whether or not GTP hydrolysis by HypB is coupled to nickel insertion into hydrogenases and to maturation of hydrogenases. Mutations were introduced into the hypB gene at sites expected to code for amino acids involved in guanine‐nucleotide binding. Lys117 of G‐motif 1, as well as Asp241 of G‐motif 4 were substituted by asparagine residues. The purified mutant HypB proteins showed strongly reduced, but still significant, GTPase activity. In the case of [D241N]HypB, the kcat/Km value was lowered by a factor of 85 and the specificity of the enzyme for GTP was apparently lost, with other nucleoside triphosphates including XTP becoming compatible substrates. The decrease in GTPase activity was even more pronounced for [K117N]HypB. To assess the functionality of these HypB proteins in vivo, the wild‐type hypB gene in the chromosome of E. coli was replaced by the mutant alleles. The resulting mutant strains BKN117 and BDN241 were affected in hydrogen metabolism under fermentative conditions. BKN117 did not display hydrogenase activity due to a loss of nickel incorporation into the large subunit. BDN241 exhibited a reduction of hydrogenase activity by 44% and only a portion of the hydrogenase 3 large subunit was in the mature nickel‐containing form. From these results, it is concluded that GTP hydrolysis catalysed by HypB is an integral process in nickel incorporation into hydrogenases.
Synthesis, Three‐Dimensional Structure, and Specific 15N‐Labelling of the Streptococcal Protein G B1‐Domain
Authors: Boutillon Christophe , Wintjens René , Lippens Guy , Drobecq Hervé , Tartar André
Published: March 2005

The 55‐amino‐acid B1‐domain of the streptococcal protein G shows a high binding affinity to IgG isolated from a wide range of mammalian species. Since the B1‐domain forms an extremely stable globular folding unit containing the major secondary structure elements and is devoid of proline residues and disulfide bridges, it is also a useful tool for protein folding and stability studies. Its small size makes this protein an ideal candidate for production by chemical synthesis, allowing incorporation of non‐natural amino acids with the possibility of assessing the influence of such residues on both the functional and structural characteristics of proteins.

In this study, we enployed three successive chemical syntheses of the B1‐domain in order to define the optimal conditions of coupling and protection. The stepwise solid‐phase methodology using the tert ‐butyloxycarbonyl/benzyl strategy was used for this purpose.

First, the sequence assembly difficulties were evaluated. After analyzing of the problems found during assembly, a second optimized synthesis was performed leading to formation of a synthetic B1‐domain with a higher yield; the synthetic B1‐domain was completely functional in its binding properties to IgG. Three orthogonal purification steps (gel‐permeation, reverse‐phase and ion‐exchange HPLC) were required to obtain a sample suitable for structural analysis by high‐resolution NMR. This study led to the conclusion that the synthetic B1‐domain adopts a three‐dimensional structure identical to that of the molecule obtained by recombinant techniques [Gronenborn, A. M., Filpula, D. R., Essig, N. Z., Achari, A., Whitlow, M., Wingfield, P. T. & Clore, G. M. (1991) Science 253, 657–661].

To demonstrate the usefulness of the chemical approach for the specific introduction of labelled amino acids in the primary structure, fourteen α‐15N‐labelled amino acids were incorporated at selected critical positions during the third synthesis. This analog is the first in a series of molecules planned to study in detail the folding dynamics of the B1‐domain.
The Expression of the Avian Clusterin Gene can be Driven by two Alternative Promoters with Distinct Regulatory Elements
Authors: Michel Denis , Chatelain Gilles , Herault Yann , Brun Gilbert
Published: March 2005

Clusterin cDNA has been isolated as a copy of a mRNA overexpressed in a wide variety of biological disorders, including tissue regression, brain injuries and oncogenic cell transformation. While the molecular cloning of the rat and the human clusterin genes has revealed a high degree of conservation of the genomic organization between mammals, the avian locus described here illustrates several divergent features. The avian gene has the particularity to be transcribed from at least two different promoters, both of which are active in transient expression assays using the quail QT6 transformed cell line. The detection of the two clusterin mRNA species by reverse‐transcription‐mediated PCR reveals a coordinated initiation of transcription from both promoters in all organs tested. In possible relation to the bipartite organization of the avian regulatory region, the putative cis‐elements described in the unique mammalian promoters appear divided among the two avian promoters. In addition, the sequence comparison of avian and mammalian regulatory sequences has allowed the identification of a conserved putative cis‐element which appears to be the target for specific DNA‐binding factors.
Structural and Functional Characterization and Cloning ofXenopusFOSP‐1 (Frog Oviduct‐Specific Protein‐1) Genes
Authors: Mehta Raj J. , Tata Jamshed R.
Published: March 2005

As a model for tissue‐specific gene expression, our laboratory has been studying the expression of vitellogenin and FOSP‐1 (frog oviduct‐specific protein‐1) genes inXenopus laeviswhich are expressed exclusively in the liver and oviduct, respectively, both strictly regulated by estrogen. Whereas the structure and function ofXenopusvitellogenin mRNAs and the upstream regulatory sequences (URS) of their genes are well established, little or no similar information is available for FOSP‐1 genes. In this study, using a combination of 5′ rapid amplification of cDNA ends (RACE) and reverse‐transcriptase PCR, we have identified two gene copies of FOSP‐1, termed FOSP‐1A and FOSP‐IB. Comparison of the sequences of full‐length FOSP‐1A and partial FOSP‐IB cDNAs revealed a high degree of similarity at the 5′ end. We next isolated FOSP‐1A and FOSP‐IB genomic clones. Dot‐plot comparison of their URS showed both similarities and differences. Two estrogen‐responsive elements (EREs), termed proximal (pERE) and distal (dERE), were identified at ‐1070/‐1082 and ‐1167/‐1179, respectively, in FOSP‐IB, but not FOSP‐1A, URS. Quantitative electrophoretic mobility shift assay (EMSA) and DNA foot‐printing with recombinantXenopusestrogen receptor (xER) expressed in insect Sf9 cells, showed that xER interacted with a higher affinity with dERE than pERE in a hormone‐independent manner, and that the two EREs do not act cooperatively. Functional studies involving transient transfection of human MCF‐7 cells with a FOSP‐IB URS‐tkCAT construct confirmed that both EREs act as hormone‐induciblecis‐acting elements. These studies now pave the way for analysis of tissue specificity of estrogen‐inducible gene expression inXenopusliver and oviduct.
Mutational Effects on the Spectroscopic Properties and Biological Activities of Oxidized Bovine Adrenodoxin, and Their Structural Implications
Authors: Beckert Vita , Schrauber Hannelore , Bernhardt Rita , Van Dijk Alard A. , Kakoschke Christel , Wray Victor
Published: March 2005

Of the aromatic 1H‐NMR signals of oxidized bovine adrenodoxin only those of His56 showed intrinsic chemical shift changes upon replacement of Tyr82 by Ser or Leu, that must arise from a loss of a through‐space ring‐current effect of the tyrosine ring in these mutants. Thus, of the three His residues contained in adrenodoxin, His56 is closest to Tyr82, and hence to the highly acidic determinant region of adrenodoxin that is the interaction site for adrenodoxin reductase and P‐450. The strong dependence of the fluorescence intensity of Tyr82 on the residue in position 56 supported this observation.

As a consequence of this, the effects of replacement of His56 by Gln or Thr on cytochrome c reduction and cytochromes P‐45O11β (CYP11B1)‐dependent and P ‐450scc (CYP11A1)‐dependent substrate conversions were studied. No influence on Vmax values was observed for all reactions mediated by the mutants, implying His56 does not play a decisive role in the intramolecular or intermolecular electron transfer. In contrast, the Km values were increased, as was the Ks value for binding of CYP11A1 to the [H56T]adrenodoxin.

The secondary structure deduced from further NMR data of adrenodoxin was compared with that of other ferredoxins. Tyr82 is in a region of the molecule containing no secondary‐structure elements. The data for Tyr82 are in keeping with the biological activities and suggests it is in a flexible, solvent‐exposed region of the molecule.
Expression, Purification and Characterisation of the Product from the Bacillus Subtilis hemD gene, Uroporphyrinogen III Synthase
Authors: Stamford N. Patrick J. , Capretta Alfredo , Battersby Alan R.
Published: March 2005

Uroporphyrinogen III synthase, the product of the hemD gene, is the enzyme responsible for the cyclisation of the linear tetrapyrrole, hydroxymethylbilane. The hemD gene isolated from Bacillus subtilis was manipulated by PCR to enable direct cloning behind a synthetic ribosome‐binding site downstream of tandem bacteriophage λPR and PL promoters in a pCE30‐derived vector. Following thermal induction of transcription, the resulting plasmid (pPS21) directed the synthesis of uroporphyrinogen III synthase. The protein produced was soluble and was readily purified. Pure uroporphyrinogen III synthase is monomeric with an isoelectric point of 4.1 and an optimum pH for activity of 8.3. Its specific activity by assay using synthetic hydroxymethylbilane as substrate is 565 units mg−1 and the Km for this substrate is 330 ± 30 nM. The N ‐terminal sequence of the enzyme is Met‐Glu‐Asn‐Asp‐Phe‐Pro‐Leu, in agreement with the gene‐derived sequence. Studies based on amino acid modifications suggest that arginine, lysine and probably histidine residues are essential for the activity of uroporphyrinogen III synthase. Significantly, this synthase from B. subtilis is substantially more thermostable than the enzymes from previously studied sources.
Degradation of Ornithine Decarboxylase by the Mammalian and Yeast 26S Proteasome Complexes Requires all the Components of the Protease
Authors: Elias Sarah , Bercovich Beatrice , Kahana Chaim , Coffino Philip , Fischer Michael , Hilt Wolfgang , Wolf Dieter H. , Ciechanover Aaron
Published: March 2005

Ornithine decarboxylase (ODC), a key enzyme in the biosynthesis of polyamines, is an extremely short‐lived protein. This attribute is important for the regulation of the activity of the enzyme and implies that the mechanisms involved in its degradation play an important role in the control of the cellular processes in which the enzyme is involved. Recently, it has been shown that ODC is degraded by the 26S proteasome complex in a process that requires antizyme, but not ubiquitin. With one reported exception, ODC, the 26S complex recognizes and degrades specifically ubiquitinated proteins. Their unconjugated counterparts are not targeted. The 26S complex is composed of a core catalytic unit, the 20S proteasome complex, and two additional, and apparently distinct, subcomplexes. The two additional sub‐complexes are regulatory subunits that are required in order to confer specificity and control. In this study, we demonstrate that, like the degradation of ubiquitin‐conjugated proteins, ubiquitin‐independent degradation of ODC also requires prior assembly of the mammalian 26S proteasome from all the three subunits, the 20S proteasome and the two subcomplexes. The combination of any two subunits does not support generation of a proteolytically active complex. This is also true for the yeast 26S complex. Like the mammalian 20S proteasome, the yeast 20S complex can cleave short peptides in an ATP‐independent mode, but cannot degrade ODC or ubiquitin‐conjugated proteins. These proteins are degraded only following addition of the regulatory subunits and generation of the high‐molecular‐mass 26S complex. In a distinct, but related, set of experiments, we demonstrate that the degradation of ODC by the assembled 26S proteasome in vitro reproduces faithfully proteolysis of the enzyme in the intact cell. Namely, (a) a C‐terminal‐deleted mouse ODC and trypanosome ODC are stable both in vitro and in vivo, and (b) like proteolysis in the intact cell, degradation in the reconstituted cell‐free system is also dependent upon the addition of antizyme.
A Putative Lipophilic Stimulant Carrier Protein Commonly Found in the Taste and Olfactory Systems
Authors: Ozaki Mamiko , Morisaki Kazuyo , Idei Wataru , Ozaki Koichi , Tokunaga Fumio
Published: March 2005

In chemosensory systems, a variety of lipophilic ligand‐binding proteins have been found in saliva or nasal mucus. Lipophilic stimulants reach the receptor membrane, carried by these proteins. An acidic 14‐kDa protein purified in the blowfly, Phormia regina, belongs to the insect pheromone‐binding protein superfamily, but unlike other lipohilic ligand‐binding proteins in insect or vertebrate chemosensory systems, it was distributed in both taste and olfactory organs. A similar protein was also isolated in Drosophila melanogaster. Considering their distributions, cDNA sequences and structural features, we concluded that these proteins belong to a unique subfamily whose members have convergently evolved for a common function required for both senses of taste and olfaction. By an electrophysiological experiment using antiserum, we also suggested that these proteins carry fragrant components of natural foods in taste systems as well as in olfactory systems.
Induction of Fatty‐Acid‐Synthase Gene Expression by Glucose in Primary Culture of Rat Hepatocytes
Authors: Prip‐Buus Carina , Perdereau Dominique , Foufelle Fabienne , Maury Jocelyne , Ferré Pascal , Girard Jean
Published: March 2005

Fatty acid synthase (FAS) expression is low in liver and adipose tissue of suckling rats and increases markedly after weaning on to a high‐carbohydrate low‐fat diet. It has been shown previously that glucose alone, via an increase in intracellular glucose‐6‐phosphate level, stimulated the accumulation of FAS mRNA in cultured white adipose tissue of suckling rats. The regulation of FAS expression by glucose and hormones (insulin, dexamethasone and triiodothyronine) was studied in cultured hepatocytes from suckling rats. In hepatocytes cultured for 48 h in the absence of hormones and glucose, FAS mRNA, as well as glucokinase mRNA, levels remained undetectable. Glucose alone was unable to stimulate FAS expression. The combination of hormones, in the absence of glucose, has a marginal effect on FAS mRNA levels. However, FAS mRNA levels were increased in the presence of both glucose and the combination of hormones. This demonstrated that the hormonal induction of FAS mRNA was dependent on the presence of glucose in the culture medium. We have then investigated if glucokinase expression could be a prerequisite for the stimulation of FAS expression in response to glucose. Hepatocytes were cultured for 48 h in the absence of glucose but in the presence of insulin, dexamethasone and triiodothyronine. In these conditions, glucokinase mRNA and activity were markedly increased but there was no accumulation of FAS mRNA. When these hepatocytes were then exposed to various levels of glucose, FAS mRNA rapidly accumulated. Glucose stimulation of FAS expression was observed only in hepatocytes which expressed glucokinase activity. The importance of glucokinase expression for the induction of FAS mRNA by glucose is supported by the striking correlation between glucose‐6‐phosphate concentrations and the levels of FAS mRNA. This study clearly demonstrates that: (a) glucose metabolism is directly involved in the regulation of FAS gene expression; (b) the effect of hormones is partly due to their capacity to induce in the hepatocytes the capacity for glucose phosphorylation.
Influence of Peroxisome Proliferators on Phosphoprotein Levels in Human and Rat Hepatic‐Derived Cell Lines
Authors: Passilly Patricia , Jannin Brigitte , Latruffe Norbert
Published: March 2005

To elucidate the effect of peroxisome proliferators on the signal‐transduction pathway, we have compared the effect of ciprofibrate, an hypolipaemic agent, on the overall phosphoprotein level between rat and human well differentiated hepatic derived cell lines. The phosphorylation status of several phosphoproteins in the rat Fao cell line was increased by the drug while no changes were observed in the human HepG2 cell line. In rat Fao cells, this increase, which is concentration and time dependent, can be as much as eightfold for 20‐kDa and 22‐kDa proteins. Wy‐14,643, a non‐fibrate molecule and a more potent peroxisome proliferator than ciprofibrate, increased the phosphorylation status of the same phosphoproteins. Peroxisome proliferators may act by activating kinases inactive in control cells, by amplifying kinases already active in control cells or by inactivating phosphatases. The phosphoamino acid residues affected are essentially serine and threonine. This modification of the signal‐transduction pathway by the peroxisome proliferators in rodent cells appears to be an early event or an independent mechanism of the peroxisome proliferation. These results support the accumulating evidence that the perturbation of this pathway may be a major cause of the hepatomegaly and the hepatocarcinogenesis induced by peroxisome proliferators in rodent species. In contrast, the lack of phosphorylation changes in the human HepG2 cell line supports the non‐toxic effect of peroxisome proliferators also used as hypolipaemic agents in humans.
The Cloning of a cDNA Encoding a Protein (Latrodectin) which Co‐purifies with the α‐latrotoxin from the Black Widow Spider Latrodectus tredecimguttatus (Theridiidae)
Authors: Pescatori Mario , Bradbury Andrew , Bouet Francoise , Gargano Nicola , Mastrogiacomo Alessandro , Grasso Alfonso
Published: March 2005

A cDNA encoding a polypeptide of 88 amino acids was cloned following the rapid amplification of cDNA ends (RACE) procedure using mRNA isolated from the venom glands of the Mediterranean black widow spider (Latrodectus tredecimguttatus) and oligonucleotides based on the sequence of a tryptic fragment putatively from α‐latrotoxin. Apart from a potential signal peptide, the rest of this small protein, named latrodectin, was highly hydrophilic, having a calculated molecular mass of 7945 Da and a pI of 4.3. Northern‐blot analysis showed that the mRNA was specifically expressed in the venom gland of L. tredecimguttatus and that it was well conserved between two geographically remote species (L. geometricus and L. indistinctus). A polyclonal serum raised in rabbits against the C‐terminal sequence of latrodectin detected cross‐reactive proteins in the venom fluid, venom gland extracts, and in purified α‐latrotoxin, suggesting that latrodectin is intimately associated with α‐latrotoxin. Finally, we produced a recombinant protein in a cell system infected with baculovirus and developed an immunoaffinity purification procedure for latrodectin to facilitate further structural and functional analyses of the molecule.
In Chinese Hamster Ovary K1 Cells Dog and Human Thyrotropin Receptors Activate Both the Cyclic AMP and the Phosphatidylinositol 4,5‐Bisphosphate Cascades in the Presence of Thyrotropin and the Cyclic AMP Cascade in Its Absence
Authors: Van Sande Jacqueline , Swillens Stéphane , Gerard Catherine , Allgeier Anouk , Massart Claude , Vassart Gilbert , Dumont Jacques E.
Published: March 2005

Thyrotropic hormone, through its human thyrotropin receptor, activates both the cyclic AMP and the phosphatidylinositol 4,5‐bisphosphate‐phospholipase‐C cascades in human thyroid cells and in Chinese hamster ovary cells (CHO‐K1) expressing this receptor. However, thyrotropin only activates the cyclic ‐AMP cascade in dog thyroid cells. In order to establish whether this different pattern of responses reflects a different structure of the human and dog thyrotropin receptors, CHO‐K1 cells were permanently transfected with a plasmid coding for one or the other receptor. For various levels of receptor expression, CHO‐K1 cells expressing either receptor presented qualitatively similar cyclic AMP and inositol phosphates responses to thyrotropin. This suggests that the difference in the response of the dog and human thyroid to thyrotropin involves elements of the phosphatidylinositol 4,5‐bisphosphate cascade downstream of the receptor. In CHO‐K1 cells overexpressing the thyrotropin receptor, the basal level of cyclic AMP was raised, suggesting a constitutive activity of the wild‐type receptor. This was confirmed in COS‐7 cells transiently expressing the human or dog thyrotropin receptors, the basal cyclic AMP levels of these cells increased in parallel with thyrotropin binding. This spontaneous activity of the thyrotropin receptor may have physiological and pathological consequences.
The Influence of Thyroid State on Hepatic Glycolysis
Authors: Gregory Roland B. , Berry Michael N.
Published: March 2005

The effects of thyroid status on glycolysis using 10, 20, and 40 mM glucose have been examined in hepatocytes derived from hypothyroid, euthyroid, and hyperthyroid rats. For any given concentration of added glucose, total glycolytic rates, as measured by the release of tritium from [6‐3H]glucose, were similar in all thyroid states. The aerobic component of glycolysis, where cytoplasmically generated reducing equivalents are transferred to the mitochondria for oxidation, was the major component in the hyper‐thyroid state, at all concentrations of glucose. In contrast, the aerobic proportion of glycolysis in the hypothyroid and euthyroid states decreased with increasing concentration of added glucose and the anaerobic component became dominant above 20 mM glucose. Cytoplasmic reducing equivalents generated during aerobic glycolysis were transferred to the mitochondria via both the glycerol 1‐phosphate and malate/aspartate shuttles in each thyroid state, even though the former shuttle was considerably depressed in the livers of hypothyroid rats. Both asparagine and aminooxyacetate had only minor effects on the rate of glycolysis, but aminooxyacetate depressed the contribution of aerobic glycolysis whereas asparagine had relatively little influence. The respiration rate in the presence of 40 mM glucose was twice as high in hepatocytes from hyperthyroid rats as in cells from hypothyroid animals, and 1.4 times as high as in hepatocytes from euthyroid rats. Smaller stimulations were observed with lower concentrations of added glucose. Furthermore, the increase in respiratory rate over the endogenous value, induced by 10 mM glucose, was six times higher in cells from hyperthyroid rats than in hepatocytes from hypothyroid animals and 2.7 times higher than that observed with cells from euthyroid rats.

The insensitivity of glycolysis to thyroid status in contrast to the marked response of respiration provides additional support for the view that the stimulation of metabolism by thyroid hormone is mediated primarily by its action on mitochondrial processes.
The Structure of Ribosomal RNA: A Three‐Dimensional Jigsaw Puzzle
Authors: Brimacombe Richard
Published: March 2005

CONTENTS–The functional centre of the ribosomal RNA–Site‐directed cross‐linking – Cross‐links to tRNA – Cross‐links to mRNA – Cross‐links to the growing peptide chain – Other data at the functional centre – Methodology for localizing site‐directed cross‐links on ribosomal RNA–Topographical data for the complete 16S and 23S RNA molecules–16S RNA – What is nucleotide resolution?– 23S RNA–Ribosomal morphology–Electron microscopy of ribosomes and subunits – The R versus S configuration of two tRNA molecules–Problems with proteins–Uncertainties in fitting the 16S RNA to the 30S protein distribution – The 530 loop controversy–Our new 16S RNA model – Future prospects
Molecular Cloning and Nuclear Localization of ATBP, A Novel (A+T)‐Stretch‐binding Protein of Sarcophaga peregrina (Flesh Fly)
Authors: Nakanishi‐Matsui Mayumi , Kubo Takeo , Natori Shunji
Published: March 2005

Previously, we purified a DNA‐binding protein, named ATBP, from cultured Sarcophaga cells. This protein was found to bind specifically to (A+T) stretches that locate in the 5′‐upstream region and the intron of the Sarcophaga lectin gene [Matsui, M., Kobayashi, A., Kubo, T. & Natori, S. (1994) Eur. J. Biochem. 219, 449–454]. Here, we report molecular cloning of a cDNA for ATBP. The cDNA encoded a protein with two zinc‐finger motifs, an acidic domain, two glutamine‐rich domains and a glutamine run. Immunofluorescence study revealed that ATBP was localized in the nuclei. Thus, ATBP is possibly a transcription factor for genes having (A+T) stretches in their promoter and/or enhancer regions.
Replacement of Tryptophan Residues in Haloalkane Dehalogenase Reduces Halide Binding and Catalytic Activity
Authors: Kennes Christian , Pries Frens , Krooshof Geja H. , Bokma Evert , Kingma Jaap , Janssen Dick B.
Published: March 2005

Haloalkane dehalogenase catalyzes the hydrolytic cleavage of carbon‐halogen bonds in short‐chain haloalkanes. Two tryptophan residues of the enzyme (Trp125 and Trp175) form a halide‐binding site in the active‐site cavity, and were proposed to play a role in catalysis. The function of these residues was studied by replacing Trp125 with phenylalanine, glutamine or arginine and Trp175 by glutamine using site‐directed mutagenesis. All mutants except Trp1l25→Phe showed a more than 10‐fold reduced Kcatand much higher Km values with 1,2‐dichloroethane and 1,2‐dibromoethane than the wild‐type enzyme. Fluorescence quenching experiments showed a decrease in the affinity of the mutant enzymes for halide ions. The 2H kinetic isotope effect observed with the wild‐type enzyme in deuterium oxide was lost in the active mutants, except the Trp125→Phe enzyme. The results indicate that both tryptophans are involved in stabilizing the transition state during the nucleophilic substitution reaction that causes carbon‐halogen bond cleavage.
Sequence Dependent Effects of CpG Cytosine Methylation
Authors: Lefebvre Anne , Mauffret Olivier , Antri Saïd El , Monnot Monique , Lescot Elie , Fermandjian Serge
Published: March 2005

The impact of cytosine methylation in the central CpG step of two closely related octanucleotide duplexes d(CATCGATG)2 and d(CTTCGAAG)2 was examined by 1H‐NMR and 31P‐NMR experiments, and a quantitative structural analysis was performed using the NOE‐derived distances, the sugar puckers and the ε torsion angles. The two starting oligonucleotides displayed a B‐DNA conformation with, however, significant local structure differences. Although the methylated oligonucleotides retained their B‐DNA conformation, different structural and thermal stability effects were observed. The magnitude of the methylation effects was to depend on the initial conformation of the CpG site, which is governed by the nature of the dinucleotide AT or TT located on the CpG flanks. As an example of sequence dependence, the methylation of CpG entailed larger conformational variation in d(CATCGATG)2 than in d(CTTCGAAG)2. In this study, the 1H and 31P chemical‐shift parameters averred as extremely sensitive probes for detecting subtle conformational changes. Finally, our comparative results may aid our understanding of the structural and related biological effects produced by cytosine methylation in DNA.
Mechanisms of Nuclear Signalling by Vitamin D3
Authors: Carlberg Carsten
Published: March 2005

Hormones that act through nuclear receptors contribute to vertebrate development and homeostasis by serving as biological signals to control cellular growth and differentiation. Their response pathways must meet two requirements, which can be contradictory: they must be highly specific for a given signal, but at the same time they have to be flexible, allowing different responses under different physiological conditions. The current model for nuclear signalling of vitamin D3 (calcitriol), thyroid hormone and retinoic acid seems to be too simple to solve the specificity puzzle. Therefore, an advanced model is proposed that combines recent findings on the structure of hormone response elements, the promiscuous dimerization of the receptors for vitamin D3, thyroid hormone and retinoids and the heterodimer polarity. Special attention is taken on the mechanism of vitamin D3 signalling, where at least 14 different pathways are differentiated.
Molecular Cloning and DNA Sequence Analysis of Pepl, a Leucyl Aminopeptidase Gene from Lactobacillus delbrueckii Subsp. Lactis DSM7290
Authors: Klein Jürgen Robert , Dick Andrea , Schick Joachim , Matern Hugo Tiberius , Henrich Bernhard , Plapp Roland
Published: March 2005

A genomic library of Lactobacillus delbrueckii subsp. lactis DSM7290 DNA fragments from a Sau 3A partial digestion in the low‐copy‐number vector pLG339, was used to screen Escherichia coli for the presence of peptidases. Using the chromogenic substrate leucine‐β‐naphthylamide (Leu‐NH‐Nap) and E. coli strain CM89 lacking the corresponding enzyme activity in an enzymic plate assay, allowed the isolation of two peptidase genes; the newly described pepL and the recently cloned and sequenced pepN. Clones could be distinguished not only by the restriction pattern of isolated plasmids but also by the rate and intensity of their colour reaction with Leu‐NH‐Nap. Three out of five clones were identified to express the Lactobacillus pepN gene; the others were shown to express a second aminopeptidase gene, designated pepL. This gene, together with 200 bp upstream of the proposed AUG initiation codon, was further subcloned and sequenced. The corresponding open reading frame of 897 nucleotides is predicted to encode a protein of 299 amino acids (34541 Da). Searching the EMBL database revealed similarity to the prolinase of Lactobacillus helveticus (45.8% identity), to the iminopeptidases of Lb. delbrueckii subsp. lactis and Lb. delbrueckii subsp. bulgaricus (25.5%), and to the Bacillus coagulans prolinase (21.5%). Minor similarities were detected for hydrolytic enzymes with serine active sites. The product encoded by the pepL gene was functional but could not be visualized on Coomassie‐blue‐stained polyacrylamide gels. High level expression of peptidase L in E. coli was achieved by placing the gene under the control of the T7 promoter.
Immunochemistry of Group A and Inaba C Antigen Factors Constituting the O Antigen of Ol Vibrio cholerae
Authors: Isshiki Yasunori , Haishima Yuji , Kondo Seiichi , Hisatsune Kazuhito
Published: March 2005

Serological cross‐reactivity among intact lipopolysaccharides (LPS) from Ol Vibrio cholerae Inaba O‐form (Inaba), Yersinia enterocolitica 09 (09), non‐Ol V cholerae serogroup Hakata (Hakata) and Vibrio bio‐serogroup 1875 Variant (1875 Variant) (all of which share Inaba antigen factor C), as well as a total of six kinds of chemically modified LPS (three from 09 and three from Inaba) was demonstrated by passive hemolysis and passive hemolysis inhibition by using these LPS as antigen for sensitizing sheep red blood cells and as inhibitor. These intact as well as chemically modified LPS contained, in their O polysaccharide chain, α:(1→2)‐linked linear perosamine (4‐amino‐4,6‐dideoxy‐D‐manno‐pyra‐nose) homopolymers with different Af‐acyl groups: their acyl groups comprise 3‐deoxy‐L‐gfycero‐tetronyl (Inaba LPS), formyl (09 LPS), 3‐hydroxypropionyl (1875 Variant LPS), acetyl (Hakata LPS and artificially introduced into Inaba and 09 LPS), propionyl and butyryl (both artificially introduced into Inaba and 09 LPS) groups. N‐Deacylation of the a(l→2)‐linked Af‐(3‐deoxy‐L‐gfycero‐tetronyl)perosarnine ho‐mopolymer of Inaba and the JV‐formyl one of 09 LPS resulted in virtual elimination of their serological reactivity with both homologous and heterologous antisera. Furthermore, when the resultant NH2 groups of the N‐deacylated perosamine homopolymers of both LPS were 7V‐acylated with acetyl, propionyl or butyryl groups, they markedly recovered both of their serological reactivities. These results are compatible with the interpretation that the Inaba antigen factor C possessed by the four bacteria is substantially related to the common presence of Af‐acyl groups, regardless of their identity, residing in the perosamine residues constituting the O polysaccharide chain of their LPS. It was also indicated that the group antigen factor A of Ol V. cholerae is substantially related to the 3‐deoxy‐L‐glycero ‐tetronyl groups residing in the perosamine homopolymer of Inaba LPS.
The Function of Wolinella succinogenes psr Genes in Electron Transport with Polysulphide as the Terminal Electron Acceptor
Authors: Krafft Torsten , Gross Roland , Kröger Achim
Published: March 2005

The membrane‐integrated polysulphide reductase (Psr) of Wolinella succinogenes is part of the electron transport chain catalyzing polysulphide reduction by formate or hydrogen. The isolated enzyme catalyzes sulphide oxidation by dimethylnaphthoquinone. The two hydrophilic subunits, PsrA and PsrB of the enzyme, are encoded by genes that form an apparent operon psrABC together with a third gene. Using homologous recombination, three deletion mutants of W. succinogenes were constructed that lack psrC, psrBC or the whole psr operon. The mutants grown with formate and fumarate were fractionated, and the cell fractions were analyzed for the presence of PsrA and enzyme activity. It was concluded that: (a) polysulphide reductase is a constituent of the wild‐type chain catalyzing electron transport from formate to polysulphide; (b) the gene psrC encodes a subunit that anchors the enzyme in the membrane and is required for electron transport; (c) PsrA which probably carries the substrate site, is exposed to the bacterial periplasm; (d) PsrA and PsrB are required for the activity of sulphide oxidation with 2,3‐dimethyl‐1,4‐naphthoquinone. Surprisingly, the psrABC mutant could grow with formate and polysulphide. The membrane fraction of the mutant grown under these conditions contained an enzyme that replaced polysulphide reductase in electron transport, and catalyzed sulphide oxidation with 2,3‐dimethyl‐1,4‐naphthoquinone.
Transcriptional Repression, a Novel Function for 3′ Untranslated Regions
Authors: Le Cam Alphonse , Legraverend Catherine
Published: March 2005

The transcription rates of the rat serine protease inhibitor 2.3 and 2.1 genes (spi 2.3 and spi 2.1), which are normally very low and high, respectively, are inversely modulated during inflammation. Two growth‐hormone‐response elements (GHRE‐I and GHRE‐II) maintain the spi 2.1 gene under the stringent control of growth hormone [Le Cam, A., Pantescu, V., Paquereau, L., Legraverend, C., Fauconnier, G. & Asins, G. (1994) J. Biol. Chem. 269, 21532–21539], whereas spi 2.3 appears to escape control by this hormone, despite the presence in its promoter of a functional GHRE‐1. A major difference between these two otherwise very similar genes is the presence in spi 2.3 of a specific 348‐bp extension of the 3′ untranslated region (3′ UTR). Inserting this 3′ UTR element downstream of the polyadenylation signal or upstream of the spi 2.3 promoter in constructs containing the chloramphenicol acetyltransferase gene strongly decreases basal transcription and inhibits growth‐hormone‐stimulated transcription, but poorly affects transcriptional stimulation by dexamethasone or interleukin‐6. The spi 2.3 3′ UTR extension also inhibits, basal and growth‐hormone‐induced transcription from the spi 2.1 promoter. Repressor activity appears to be distributed throughout the specific extension of the 3′ UTR and seems to involve interactions with two types of 5′cis ‐acting promoter elements. The first is the GAGA box, a key control spi promoter element, whose mutation faithfully reproduces the effects of the 3′ UTR silencer on spi 2.1 and spi 2.3 promoters. The second is represented by CCAAT enhancer‐binding‐protein‐(C/EBP)‐binding sites, whose functions are severely impaired by the spi 2.3‐specific 3′ UTR extension. The presence of this silencer in the spi 2.3 gene very likely accounts for the lack of basal of transcription in vivo and for induction of the gene during acute inflammation.