bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of HIV‐1 integrase by modified oligonucleotides derived from U5′ LTR
Authors: Snášel Jan , Rejman Dominik , Liboska Radek , Točík Zdenčk , Ruml Tomáš , Rosenberg Ivan , Pichová Iva
Published: March 2003

Retroviral integrase catalyzes integration of double‐stranded viral DNA into the host chromosome by a process that has become an attractive target for drug design. In the 3′ processing reaction, two nucleotides are specifically cleaved from both 3′ ends of viral DNA yielding a 5′ phosphorylated dimer (pGT). The resulting recessed 3′ hydroxy groups of adenosine provide the attachment sites to the host DNA in the strand transfer reaction. Here, we studied the effect of modified double‐stranded oligonucleotides mimicking both the unprocessed (21‐mer oligonucleotides) and 3′ processed (19‐mer oligonucleotides) U5 termini of proviral DNA on activities of HIV‐1 integrase in vitro. The inhibitions of 3′ processing and strand transfer reactions were studied using 21‐mer oligonucleotides containing isopolar, nonisosteric, both conformationally flexible and restricted phosphonate internucleotide linkages between the conservative AG of the sequence CAGT, and using a 21‐mer oligonucleotide containing 2′‐fluoroarabinofuranosyladenine. All modified 21‐mer oligonucleotides competitively inhibited both reactions mediated by HIV‐1 integrase with nanomolar IC50 values. Our studies with 19‐mer oligonucleotides showed that modifications of the 3′ hydroxyl significantly reduced the strand transfer reaction. The inhibition of integrase with 19‐mer oligonucleotides terminated by (S)‐9‐(3‐hydroxy‐2‐phosphonomethoxypropyl)adenine, 9‐(2‐phosphonomethoxyethyl)adenine, and adenosine showed that proper orientation of the 3′ OH group and the presence of the furanose ring of adenosine significantly influence the strand transfer reaction.
Ectopic expression of the cAMP‐responsive element binding protein inhibits phorbol ester‐mediated induction of tissue‐type plasminogen activator gene expression
Authors: Costa Magdaline , Medcalf Robert L.
Published: March 2003

The human tissue‐type plasminogen activator (t‐PA) gene is regulated in a cell‐type dependent manner. The t‐PA gene is transcriptionally induced by the phorbol ester PMA in HeLa cells, but suppressed by PMA in HT‐1080 cells. A cAMP responsive element (tPACRE) and a Sp‐1 site located within the proximal t‐PA gene promoter are functionally important in both cell systems. HeLa and HT‐1080 cells contain a different repertoire of factors that associate with the tPACRE. In HT‐1080 cells, CREB and c‐Jun are the two major t‐PACRE binding proteins identified, while activating transcription factor 2 (ATF‐2) is a predominant t‐PACRE binding protein in HeLa cells. To determine whether alteration in the distribution of tPACRE binding proteins would influence the differential regulation of the t‐PA gene in these cells, the tPACRE binding profiles in these two cell systems were manipulated by over expressing ATF‐2 in HT‐1080 cells and CREB in HeLa cells. Supershift experiments confirmed that the overexpression of these factors resulted in binding to the tPACRE site. However, the presence of ATF‐2 in HT‐1080 cells did not affect either constitutive or PMA‐mediated suppression of the endogenous t‐PA gene. In contrast, enforced tPACRE‐binding activity of CREB in HeLa cells significantly reduced the magnitude of PMA‐mediated induction of t‐PA mRNA in HeLa cells. These results indicate that the introduction of CREB into HeLa cells disrupts the regulation of the t‐PA gene.
P‐Glycoprotein preferentially effluxes anthracyclines containing free basic versus charged amine
Authors: Frézard Frédéric , Pereira‐Maia Elene , Quidu Patricia , Priebe Waldemar , Garnier‐Suillerot Arlette
Published: March 2003

The multidrug resistant (MDR) tumor phenotype, characterized by a decreased cellular drug accumulation is achieved by ATP‐dependent extrusions of drugs from cells by P‐glycoprotein (P‐gp) and/or by multidrug resistance protein (MRP1). Despite the huge amount of research that has been performed on the mechanisms of P‐gp‐mediated efflux of drug, it is not yet known what the molecular parameters are required for a molecule to be recognized and pumped out by P‐gp. Anthracyclines are weak bases and, depending on the pH, can exist either in the neutral or in the positively charged form. The aim of the work reported here was to determine which molecular form is actively pumped out by P‐gp (the neutral form, the protonated form, or both), and if both, the relative efficiencies of pumping. We used spectrofluorometric methods to determine the efflux of anthracyclines in K562/Adr cells, at different intracellular and extracellular pH levels. Using 3′‐deamino, 3′‐hydroxyl doxorubicin (OH‐DOX), which is permanently neutral, we first verified that our methodologies were accurate and that the P‐gp‐mediated efflux of OH‐DOX would not depend on the pH being in the range 6.6–8.4. The P‐gp‐mediated efflux of daunorubicin (DNR) and 3′‐hydroxy‐4‐amino (WP608) was determined at different pH values. These two drugs were chosen because: (a) the lipophilicity of the neutral forms of these two molecules is so similar that any difference in the P‐gp‐mediated efflux cannot be assigned to lipohilicity variation, and (b) their pKa values are different (8.4 and 7.7 for DNR and WP608, respectively), which makes it easy to obtain a large variation in the proportions of the neutral and positively charged forms. Our data show that both forms are recognized by P‐gp but the neutral form is pumped about three times more efficiently than the charged form. This is corroborated by results showing the active efflux (checked at pHi 7.3 only) of five other anthracycline containing a basic center. We interpret these data to mean that: (a) the positive charge of anthracycline is not a necessary requirement for P‐gp recognition, but that (b) the presence of a protonable basic nitrogen facilitates the processing of these compounds by MDR efflux system.
High levels of ceruloplasmin in the serum of transgenic mice developing hepatocellular carcinoma
Authors: Pousset Dominique , Piller Véronique , Bureaud Nicole , Piller Friedrich
Published: March 2003

Transgenic mice expressing the Simian virus 40 large T antigen under the control of the liver‐specific human antithrombin‐III promoter all develop well‐differentiated hepatocellular carcinoma. During tumour development serum ceruloplasmin (Cp) increases gradually until it reaches 30 times control levels in all transgenic mice at 6 months of age. The accumulation of Cp in the serum is due to the increased transcription of theCpgene as well as to the increase inCpmRNA stability in the livers of the transgenic mice. One‐half of the overproduced Cp is charged with copper and Cp‐associated serum oxidase activity increases in parallel with the holo‐Cp concentration. Through its ferroxidase activity Cp is involved prominently in iron metabolism. Analysis of copper and iron in serum and liver revealed increased copper levels in the serum of tumour‐bearing animals and which increased in parallel with Cp concentration; the amounts of copper in the liver were unchanged. In contrast, serum iron remained constant during tumour development whereas the iron concentration in the livers of the transgenic mice decreased.
Oxidation of polychlorinated benzenes by genetically engineered CYP101 (cytochrome P450cam)
Authors: Jones Jonathan P. , O'Hare Ellen J. , Wong Luet‐Lok
Published: March 2003

Polychlorinated benzenes are recalcitrant environmental pollutants primarily because they are resistant to attack by dioxygenases commonly used by micro‐organisms for the biodegradation of aromatic compounds. We have investigated the oxidation of polychlorinated benzenes by mutants of the haem mono‐oxygenase CYP101 (cytochrome P450cam) from Pseudomonas putida with the aim of generating novel systems for their biodegradation. Wild‐type CYP101 had low activity for the oxidation of dichlorobenzenes and trichlorobenzenes to the chlorophenols, but no products were detected for the heavily chlorinated benzenes. Increasing the active‐site hydrophobicity with the Y96F mutation increased the activity up to 100‐fold, and both pentachlorobenzene and hexachlorobenzene were oxidized slowly to pentachlorophenol. Decreasing the space available at the top of the active site with the F87W mutation to force the substrate to be bound closer to the haem resulted in a further 10‐fold increase in activity with most substrates. Introducing the F98W mutation, also at the top of the active site, decreased the NADH‐turnover rates but increased the coupling efficiencies, and > 90% coupling was observed for 1,3‐dichlorobenzene and 1,3,5‐trichlorobenzene with the F87W–Y96F–F98W mutant. The V247L mutation generally increased the NADH‐turnover rates, and the F87W–Y96F–V247L mutant showed reasonably fast NADH turnover (229 min−1) with the highly insoluble pentachlorobenzene without the need for surfactants or organic cosolvents. As all chlorophenols are degraded by micro‐organisms, novel biodegradation systems could be constructed in which CYP101 mutants convert the inert polychlorinated benzenes to the phenols, which are then readily degraded by natural pathways.
Characterization of carbonic anhydrase from Neisseria gonorrhoeae
Authors: Elleby Björn , Chirica Laura C. , Tu Chingkuang , Zeppezauer Michael , Lindskog Sven
Published: March 2003

We have investigated the steady state and equilibrium kinetic properties of carbonic anhydrase from Neisseria gonorrhoeae (NGCA). Qualitatively, the enzyme shows the same kinetic behaviour as the well studied human carbonic anhydrase II (HCA II). This is reflected in the similar pH dependencies of the kinetic parameters for CO2 hydration and the similar behaviour of the kinetics of 18O exchange between CO2 and water at chemical equilibrium. The pH profile of the turnover number, kcat, can be described as a titration curve with an exceptionally high maximal value of 1.7 × 106 s−1 at alkaline pH and a pKa of 7.2. At pH 9, kcat is buffer dependent in a saturable manner, suggesting a ping‐pong mechanism with buffer as the second substrate. The ratio kcat/Km is dependent on two ionizations with pKa values of 6.4 and 8.2. However, an 18O‐exchange assay identified only one ionizable group in the pH profile of kcat/Km with an apparent pKa of 6.5. The results of a kinetic analysis of a His66→Ala variant of the bacterial enzyme suggest that His66 in NGCA has the same function as a proton shuttle as His64 in HCA II. The kinetic defect in the mutant can partially be overcome by certain buffers, such as imidazole and 1,2‐dimethylimidazole. The bacterial enzyme shows similar Ki values for the inhibitors NCO−, SCN− and N3− as HCA II, while CN− and the sulfonamide ethoxzolamide are considerably weaker inhibitors of the bacterial enzyme than of HCA II. The absorption spectra of the adducts of Co(II)‐substituted NGCA with acetazolamide, NCO−, SCN−, CN− and N3− resemble the corresponding spectra obtained with human Co(II)‐isozymes I and II. Measurements of guanidine hydrochloride (GdnHCl)‐induced denaturation reveal a sensitivity of the CO2 hydration activity to the reducing agent tris(2‐carboxyethyl)phosphine (TCEP). However, the A292/A260 ratio was not affected by the presence of TCEP, and a structural transition at 2.8–2.9 m GdnHCl was observed.
ATP‐citrate lyase from the green sulfur bacterium Chlorobium limicola is a heteromeric enzyme composed of two distinct gene products
Authors: Kanao Tadayoshi , Fukui Toshiaki , Atomi Haruyuki , Imanaka Tadayuki
Published: March 2003

The reductive tricarboxylic acid cycle functions as a carbon dioxide fixation pathway in the green sulfur bacterium, Chlorobium limicola. ATP‐citrate lyase, one of the key enzymes of this cycle, was partially purified from C. limicola strain M1 and the N‐terminal sequence of a 65‐kDa protein was found to show similarity toward eukaryotic ATP‐citrate lyase. A DNA fragment was amplified with primers designed from this sequence and an internal sequence highly conserved among eukaryotic enzymes. Using this fragment as a probe, we isolated a DNA fragment containing two adjacent open reading frames, aclB (1197 bp) and aclA (1827 bp), whose products showed significant similarity to the N‐ and C‐terminal regions of the human enzyme, respectively. Heterologous expression of these genes in Escherichia coli showed that both gene products were essential for ATP‐citrate lyase activity. The recombinant enzyme was purified from the cell‐free extract of E. coli harboring aclBA for further characterization. The molecular mass of the recombinant enzyme was determined to be approximately 532–557 kDa by gel‐filtration. The enzyme catalyzed the cleavage of citrate in an ATP‐, CoA‐ and Mg2+‐dependent manner, where ATP and Mg2+ could be replaced by dATP and Mn2+, respectively. ADP and oxaloacetate inhibited the reaction. These properties suggested that ATP‐citrate lyase from C. limicola controlled the cycle flux depending on intracellular energy conditions. This paper provides the first direct evidence that a bacterial ATP‐citrate lyase is a heteromeric enzyme, distinct from mammalian enzymes.
Active oligomeric states of pyruvate decarboxylase and their functional characterization
Authors: Killenberg‐Jabs Margrit , Jabs Andreas , Lilie Hauke , Golbik Ralph , Hübner Gerhard
Published: March 2003

Homomeric pyruvate decarboxylase (E.C 4.1.1.1) from yeast consists of dimers and tetramers under physiological conditions, a Kd value of 8.1 µm was determined by analytical ultracentrifugation. Dimers and monomers of the enzyme could be populated by equilibrium denaturation using urea as denaturant at defined concentrations and monitored by a combination of optical (fluorescence and circular dichroism) and hydrodynamic methods (analytical ultracentrifugation). Dimers occur after treatment with 0.5 m urea, monomers with 2.0 m urea independent of the protein concentration. The structured monomers are catalytically inactive. At even higher denaturant concentrations (6 m urea) the monomers unfold. The contact sites of two monomers in forming a dimer as the smallest enzymatically active unit are mainly determined by aromatic amino acids. Their interactions have been quantified both by structure‐theoretical calculations on the basis of the X‐ray crystallography structure, and experimentally by binding of the fluorescent dye bis‐ANS. The contact sites of two dimers in tetramer formation, however, are mainly determined by electrostatic interactions. Homomeric pyruvate decarboxylase (PDC) is activated by its substrate pyruvate. There was no difference in the steady‐state activity (specific activity) between dimers and tetramers. The activation kinetics of the two oligomeric states, however, revealed differences in the dissociation constant of the regulatory substrate (Ka) by one order of magnitude. The tetramer formation is related to structural consequences of the interaction transfer in the activation process causing an improved substrate utilization.
Processing of tumor necrosis factor by the membrane‐bound TNF‐α‐converting enzyme, but not its truncated soluble form
Authors: Itai Toshimitsu , Tanaka Masato , Nagata Shigekazu
Published: March 2003

Tumour necrosis factor (TNF)‐α‐converting enzyme (TACE) is a membrane protein belonging to the ADAM (a disintegrin and metalloproteinase) family that cleaves various membrane proteins, including the proform of TNF‐α. In this study, we constructed expression vectors for the membrane‐bound full‐length TACE (mTACE) and its truncated soluble form (sTACE). When a human TNF‐α expression vector was introduced into human 293 cells, processing of TNF‐α to its mature form was enhanced by coexpressing mTACE, and this processing was inhibited by a metalloproteinase inhibitor. On the other hand, coexpression of sTACE had no effect on the processing of TNF‐α, although the culture medium of sTACE‐transfected cells could cleave a peptide containing the TNF‐α cleavage site. Fas ligand (FasL)‐transfected 293 cells released a considerable amount of soluble FasL, and coexpression of neither mTACE nor sTACE enhanced this shedding. Immunoprecipitation and Western blotting analysis with cells that were cotransfected with TACE and TNF‐α indicated that both mTACE and sTACE could interact with the proform of TNF‐α. In the same assay, neither mTACE nor sTACE interacted with FasL. The catalytic domain‐lacking TACE mutant, which could also interact TNF‐α, showed a dominant negative effect on not only TNF‐α secretion but also FasL secretion. These results suggest that binding of the membrane‐anchored but not the soluble form of TACE to TNF‐α results in efficient ectodomain shedding, and that FasL secretase is a metalloproteinase similar, but not identical, to TACE.
Transport of L‐carnitine in isolated cerebral cortex neurons
Authors: Wawrzeńczyk Agnieszka , Sacher Ayelet , Mac Magdalena , Nałecz Maciej J. , Nałecz Katarzyna A.
Published: March 2003

The accumulation of carnitine was measured in cerebral cortex neurons isolated from adult rat brain. This process was found to be lowered by 40% after preincubation with ouabain and with SH‐group reagents (N‐ethylmaleimide and mersalyl). The initial velocity of carnitine transport was found to be inhibited by 4‐aminobutyrate (GABA) in a competitive way (Ki = 20.9 ± 2.4 mm). However, of various inhibitors of GABA transporters, only nipecotic acid and very high concentrations of 1‐[2‐([(diphenylmethylene)amino]oxy)ethyl]‐1,2,5,6‐tetrahydro‐3‐pyridinecarboxylic acid hydrochloride (NO‐711) acid decreased carnitine accumulation while betaine, taurine and β‐alanine had no effect. The GABA transporters expressed inXenopus laevisoocytes did not transport carnitine. Moreover, carnitine was not observed to diminish the accumulation of GABA in cerebral cortex neurons, which further excluded a possible involvement of the GABA transporter GAT1 in the process of carnitine accumulation, despite the expression of this protein in the cells under study. The absence of carnitine transporter OCTN2 in rat cerebral cortex neurons (K. A. Nałecz, D. Dymna, J. E. Mroczkowska, A. Broër, S. Broër, M. J. Nałecz and R. Cecchelli, unpublished results), together with the insensitivity of carnitine accumulation towards betaines, implies that a novel transporting protein is present in these cells.
Identification of the rat adrenal zona fasciculata/reticularis specific protein, inner zone antigen (IZAg), as the putative membrane progesterone receptor
Authors: Raza Farah S. , Takemori Hiroshi , Tojo Hiromasa , Okamoto Mitsuhiro , Vinson Gavin P.
Published: March 2003

Using immunological methods, a protein specific to the inner zones of the rat adrenal cortex, and called inner zone antigen (IZAg), was previously shown to have two interrelated forms of 26 kDa (IZAg1) and 55–60 kDa (IZAg2), and to have an action on steroid hydroxylation. After two‐dimensional gel electrophoresis, and immunoaffinity column purification, N‐terminal amino‐acid analysis showed that the first 12 amino acids were identical to those of a recently described putative membrane located progesterone receptor (PPMR). RT‐PCR was then used to generate the cDNA of this protein, using RNA extracted from rat adrenals. A glutathione S‐transferase (GST)‐fusion construct was expressed in Escherichia coli, and shown to generate an immunoreactive product of molecular mass consistent with its identification as IZAg1. More detailed examination of the distribution of this protein, not only in the zona fasciculata/reticularis of the adrenal cortex, but also in the Leydig cell, kidney and liver, suggest it may have a role in steroid hormone synthesis and/or metabolism.
Mini‐plasmin found in the epithelial cells of bronchioles triggers infection by broad‐spectrum influenza A viruses and Sendai virus
Authors: Murakami Meiko , Towatari Takae , Ohuchi Masanobu , Shiota Mayumi , Akao Miyoko , Okumura Yuushi , Parry Marina A. A. , Kido Hiroshi
Published: March 2003

Extracellular cleavage of virus envelope fusion glycoproteins by host cellular proteases is a prerequisite for the infectivity of mammalian and nonpathogenic avian influenza viruses, and Sendai virus. Here we report a protease present in the airway that, like tryptase Clara, can process influenza A virus haemagglutinin and Sendai virus envelope fusion glycoprotein. This protease was extracted from the membrane fraction of rat lungs, purified and then identified as a mini‐plasmin. Mini‐plasmin was distributed predominantly in the epithelial cells of the upward divisions of bronchioles and potentiated the replication of broad‐spectrum influenza A viruses and Sendai virus, even that of the plasmin‐insensitive influenza A virus strain. In comparison with plasmin, its increased hydrophobicity, leading to its higher local concentrations on membranes, and decreased molecular mass may enable mini‐plasmin to gain ready access to the cleavage sites of various haemagglutinins and fusion glycoproteins after expression of these viral proteins on the cell surface. These findings suggest that mini‐plasmin in the airway may play a pivotal role in the spread of viruses and their pathogenicity.
The chondroitin sulfate chain of bikunin‐containing proteins in the inter‐α‐inhibitor family increases in size in inflammatory diseases
Authors: Mizon Charlotte , Mairie Christophe , Balduyck Malika , Hachulla Eric , Mizon Jacques
Published: March 2003

Inter‐α‐inhibitor (IαI) and pre‐α‐inhibitor (PαI) are the main members of a set of multichain serine proteinase inhibitors. Present in human plasma, they may be involved in control of the inflammatory process. They are composed of homologous heavy chains (H1 and H2 for IαI; H3 for PαI) covalently linked by a protein–glycosaminoglycan–protein cross‐link to bikunin, which is a chondroitin 4‐sulfate proteoglycan. During the acute‐phase response, biosynthesis of IαI and PαI is downregulated and upregulated, respectively. In this work, we provide evidence that, in inflammatory diseases, the chondroitin sulfate chain of bikunin increases in size proportionally to the severity of the inflammatory response. As a consequence, all IαI‐related components that contain bikunin are structurally modified. Therefore, the changes in glycosylation of the acute‐phase proteins are not restricted to N‐linked glycans but also affect glycosaminoglycans. The implications of these findings are discussed with regard to biosynthesis and biological role, especially the anti‐inflammatory effects of IαI‐related proteinase inhibitors.
Subsite specificity of trypanosomal cathepsin L‐like cysteine proteases
Authors: Lecaille Fabien , Authié Edith , Moreau Thierry , Serveau Carole , Gauthier Francis , Lalmanach Gilles
Published: March 2003

The S2 subsite of mammalian cysteine proteinases of the papain family is essential for specificity. Among natural amino acids, all these enzymes prefer bulky hydrophobic residues such as phenylalanine at P2. This holds true for their trypanosomal counterparts: cruzain from Trypanosoma cruzi and congopain from T. congolense. A detailed analysis of the S2 specificity of parasitic proteases was performed to gain information that might be of interest for the design of more selective pseudopeptidyl inhibitors. Nonproteogenic phenylalanyl analogs (Xaa) have been introduced into position P2 of fluorogenic substrates dansyl‐Xaa‐Arg‐Ala‐Pro‐Trp, and their kinetic constants (Km, kcat/Km) have been determined with congopain and cruzain, and related host cathepsins B and L. Trypanosomal cysteine proteases are poorly stereoselective towards d/l‐Phe, the inversion of chirality modifying the efficiency of the reaction but not the Km. Congopain binds cyclohexylalanine better than aromatic Phe derivatives. Another characteristic feature of congopain compared to cruzain and cathepsins B and L was that it could accomodate a phenylglycyl residue (kcat/Km = 1300 mm−1·s−1), while lengthening of the side chain by a methylene group only slightly impaired the specificity constant towards trypanosomal cysteine proteases. Mono‐ and di‐halogenation or nitration of Phe did not affect Km for cathepsin L‐like enzymes, but the presence of constrained Phe derivatives prevented a correct fitting into the S2 subsite. A model of congopain has been built to study the fit of Phe analogs within the S2 pocket. Phe analogs adopted a positioning within the S2 pocket similar to that of the Tyr of the cruzain/Z‐Tyr‐Ala‐fluoromethylketone complex. However, cyclohexylalanine has an energetically favorable chair‐like conformation and can penetrate deeper into the subsite. Fitting of modeled Phe analogs were in good agreement with kinetic parameters. Furthermore, a linear relationship could be established with logP, supporting the suggestion that fitting into the S2 pocket of trypanosomal cysteine proteases depends on the hydrophobicity of Phe analogs.
CCR2 and CCR5 receptor‐binding properties of herpesvirus‐8 vMIP‐II based on sequence analysis and its solution structure
Authors: Shao Weiping , Fernandez Elias , Sachpatzidis Aristidis , Wilken Jill , Thompson Darren A. , Schweitzer Barry I. , Lolis Elias
Published: March 2003

Human herpesvirus‐8 (HHV‐8) is the infectious agent responsible for Kaposi’s sarcoma and encodes a protein, macrophage inflammatory protein‐II (vMIP‐II), which shows sequence similarity to the human CC chemokines. vMIP‐II has broad receptor specificity that crosses chemokine receptor subfamilies, and inhibits HIV‐1 viral entry mediated by numerous chemokine receptors. In this study, the solution structure of chemically synthesized vMIP‐II was determined by nuclear magnetic resonance. The protein is a monomer and possesses the chemokine fold consisting of a flexible N‐terminus, three antiparallel β strands, and a C‐terminal α helix. Except for the N‐terminal residues (residues 1–13) and the last two C‐terminal residues (residues 73–74), the structure of vMIP‐II is well‐defined, exhibiting average rmsd of 0.35 and 0.90 Å for the backbone heavy atoms and all heavy atoms of residues 14–72, respectively. Taking into account the sequence differences between the various CC chemokines and comparing their three‐dimensional structures allows us to implicate residues that influence the quaternary structure and receptor binding and activation of these proteins in solution. The analysis of the sequence and three‐dimensional structure of vMIP‐II indicates the presence of epitopes involved in binding two receptors CCR2 and CCR5. We propose that vMIP‐II was initially specific for CCR5 and acquired receptor‐binding properties to CCR2 and other chemokine receptors.
Cloning and functional characterization of the 5′ flanking region of the human mitochondrial malic enzyme gene
Authors: Butta Nora , González‐Manchón Consuelo , Arias‐Salgado Elena G. , Ayuso Matilde S. , Parrilla Roberto
Published: March 2003

This work reports the molecular cloning and functional characterization of the 5′ flanking region of the human mitochondrial malic enzyme (mME) gene. The proximal promoter region has features of housekeeping genes like high G + C‐content and absence of TATA or CCAAT boxes. Deletion analysis of the 5′ region of the mME showed that maximal transcriptional activity is located within the −205/+86 region. Footprinting analysis showed two protected regions, one comprising potential overlapped AP‐1, CREB, and AP‐4 sites and a second one encompassing AP‐2 and several Sp1 ci‐acting elements. Mutation of putative AP‐1/AP‐4/CREB sites reduced basal promoter activity to less than 50%. Supershift assays demonstrated the specific binding of Sp1 and AP‐2 proteins. Moreover, experiments in Drosophila SL2 cells lacking endogenous Sp1 demonstrated that the Sp1 site(s) is essential to maintain a normal basal rate of transcription of this gene. A low‐level expression of AP‐2 enhanced the activity of a mME promoter construct in HepG2 cells and this effect was prevented by disruption of the putative AP‐2 element. In contrast, higher levels of expression of AP‐2 induced a DNA‐independent inhibitory response. A biphasic regulation of endogenous mME gene is also shown in HepG2 cells transfected with an AP‐2 expression plasmid, suggesting that availability of AP‐2 protein may control this gene under physiological conditions. A recombinant λ genomic clone containing a mME pseudogene was also isolated. The high degree of sequence conservation seems to indicate a recent emergency of this human pseudogene.
Inhibitory effect of a presenilin 1 mutation on the Wnt signalling pathway by enhancement of β‐catenin phosphorylation
Authors: Kawamura Yuuki , Kikuchi Akira , Takada Ritsuko , Takada Shinji , Sudoh Shinji , Shibamoto Sayumi , Yanagisawa Katsuhiko , Komano Hiroto
Published: March 2003

Mutations in the presenilin 1 (PS1) gene are the most common genetic factor underlying the development of early onset familial Alzheimer's disease (FAD). Accumulating evidence has shown that FAD‐linked mutations of PS1 enhance the generation of amyloid‐β (1–42) protein. Recently, β‐catenin has been shown to interact with PS1. β‐catenin is essential for the Wnt signalling pathway. However, the biological significance of the interaction between β‐catenin and PS1 in this signalling pathway remains to be clarified. In this study, we investigated the effect of FAD‐linked PS1 (M146L) mutation in the Wnt signalling pathway using the conditioned medium containing Wnt‐3A. The expression of mutated PS1 inhibited the Wnt‐3A‐induced accumulation of β‐catenin. Chase analysis of β‐catenin in Wnt‐3A‐stimulated cells following cycloheximide treatment revealed that PS1 mutation enhanced the generation of the higher molecular mass form of β‐catenin, most likely, ubiquitinated β‐catenin. In addition, the expression of mutated PS1 elevated the level of phosphorylated β‐catenin, which is targeted to the ubiquitin/proteasome pathway. Thus, it appears that PS1 (M146L) mutation down‐regulates the Wnt‐3A‐induced accumulation of β‐catenin due to an increase in the level of phosphorylated β‐catenin.
De novo identification of cell‐type specific antibody–antigen pairs by phage display subtraction
Authors: Stausbøl‐Grøn Brian , Jensen Kim Bak , Jensen Kristian Hobolt , Jensen Morten Østergaard , Clark Brian F. C.
Published: March 2003

The aim of this study was to identify novel antibodies directed against cytosolic keratinocyte‐specific antigens from a phage display antibody repertoire by using phage display subtraction. Phage display is a method of displaying foreign molecules on the surface of filamentous bacteriophage particles. It allows the interaction between two cognate molecules to be analysed through affinity selections. Recently, large repertoires of phage displayed human antibody fragments have been constructed. From such repertoires, antibodies can be obtained in vitro without the need for immunization or the hybridoma technology. A novel subtractive strategy for selecting antibodies from phage libraries was applied. Phage antibodies were selected against immobilized crude lysates of cultured human keratinocytes, the target antigens being unknown beforehand. A competing cell lysate was used to reduce retrieval of phage antibodies with specificities to commonly nondifferentially expressed antigens. A monoclonal single chain fragment variable (scFv) with specificity for crude lysates of cultured human keratinocytes was identified as demonstrated by ELISA assays and immunoblotting analysis. The cognate keratinocyte antigen was shown to be keratin 14 (K14) by using immunoblotting based on 2D PAGE and a corresponding 2D PAGE protein database. In accordance with the expected tissue localization of K14, the identified scFv stained the basal layer of human epidermis by indirect immunofluorescence analysis. Starting with crude cell lysates, phage display subtraction in combination with 2D PAGE and 2D PAGE protein databases can be used to identify antibody–antigen pairs that characterize a specific cell type.
Ets1 is a common element in directing transcription of the α and β genes of human protein kinase CK2
Authors: Krehan Andreas , Schmalzbauer Rüdiger , Böcher Oliver , Ackermann Karin , Wirkner Ute , Brouwers Stefan , Pyerin Walter
Published: March 2003

Protein kinase CK2 is a conserved and vital Ser/Thr phosphotransferase with various links to malignant diseases, occurring as a tetramer composed of two catalytically active (CK2α and/or CK2α′) and two regulatory subunits (CK2β). There is balanced availability of CK2α and CK2β transcripts in proliferating and differentiating cultured cells. Examination of the human CK2β gene for transcriptionally active regions by systematic deletions and reporter gene assays indicates strong promoter activity at positions −42 to 14 and 12 to 72 containing transcription start sites 1 and 2 of the gene (positions +1 and 33), respectively, an upstream and a downstream enhancer activity at positions −241 to −168 and 123 to 677, respectively, and silencer activity at positions −241 to −261. Of the various transcription factor binding motifs present in those regions, Ets1 and CAAT‐related motifs turned out to be of particular importance, Ets1 for promoter activation and CAAT‐related motifs for enhancer activation. In addition, there are contributions by Sp1. Most strikingly, the Ets1 region representing two adjoining consensus motifs also occurs with complete identity in the recently characterized promoter of the CK2α gene [Krehan, A., Ansuini, H., Böcher, O., Grein, S., Wirkner, U. & Pyerin, W. (2001) J. Biol. Chem.275, 18327–18336], and affects comparably, when assayed in parallel, the promoters of both CK2 genes, both by motif mutations and by Ets1 overexpression. The data strongly support the hypothesis that Ets1 acts as a common regulatory element of the CK2α and CK2β genes involved in directing coordinate transcription and contributing to the balanced availability of transcripts.
Site‐directed mutagenesis of the ionizable groups in the active site of Zymomonas mobilis pyruvate decarboxylase
Authors: Huang Chang‐Yi , Chang Alan K. , Nixon Peter F. , Duggleby Ronald G.
Published: March 2003

Pyruvate decarboxylase (PDC, EC 4.1.1.1) is a thiamin diphosphate‐dependent enzyme about which there is a large body of structural and functional information. The active site contains several absolutely conserved ionizable groups and all of these appear to be important, as judged by the fact that mutation diminishes or abolishes catalytic activity. Previously we have shown [Schenk, G., Leeper, F.J., England, R., Nixon, P.F. & Duggleby, R.G. (1997) Eur. J. Biochem.248, 63–71] that the activity is pH‐dependent due to changes in kcat/Km while kcat itself is unaffected by pH. The effect on kcat/Km is determined by a group with a pKa of 6.45; the identity of this group has not been determined, although H113 is a possible candidate. Here we mutate five crucial residues in the active site with ionizable side‐chains (D27, E50, H113, H114 and E473) in turn, to residues that are nonionizable or should have a substantially altered pKa. Each protein was purified and characterized kinetically. Unexpectedly, the pH‐dependence of kcat/Km is largely unaffected in all mutants, ruling out the possibility that any of these five residues is responsible for the observed pKa of 6.45. We conjecture that the kcat/Km profile reflects the protonation of an alcoholate anion intermediate of the catalytic cycle.
Expression and functional analysis of a gene cluster involved in the synthesis of decaprenoxanthin reveals the mechanisms for C50 carotenoid formation
Authors: Krubasik Philipp , Kobayashi Miki , Sandmann Gerhard
Published: March 2003

Corynebacterium glutamicum accumulates the C50 carotenoid decaprenoxanthin. Rescued DNA from transposon color mutants of this Gram‐positive bacterium was used to clone the carotenoid biosynthetic gene cluster. By sequence comparison and functional complementation, the genes involved in the synthesis of carotenoids with 50 carbon atoms were identified. The genes crtE, encoding a geranylgeranyl pyrophosphate synthase, crtB, encoding a phytoene synthase, and crtI, encoding a phytoene desaturase, are responsible for the formation of lycopene. The products of three novel genes, crtYe and crtYf, with sequence similarities to heterodimeric lycopene cyclase crtYc and crtYd, together with crtEb which exhibits a prenyl transferase motif, were involved in the conversion of C40 acyclic lycopene to cyclic C50 carotenoids. Using functional complementation in Escherichia coli, it could be shown that the elongation of lycopene to the acyclic C50 carotenoid flavuxanthin by the addition of C5 isoprenoid units at positions C‐2 and C‐2′ is catalyzed by the crtEb gene product. Subsequently, the gene products of crtYe and crtYf in a concerted action convert the acyclic flavuxanthin into the cyclic C50 carotene, decaprenoxanthin, forming two ε‐ionone groups. The mechanisms, involving two individual steps for the formation of cyclic C50 carotenoids from lycopene, are proposed on the basis of these results.
Properties of the C‐terminal domain of 4.1 proteins
Authors: Scott Catherine , Phillips Gareth W. , Baines Anthony J.
Published: March 2003

At the C‐terminus of all known 4.1 proteins is a sequence domain unique to these proteins, known as the C‐terminal domain (CTD). Mammalian CTDs are associated with a growing number of protein–protein interactions, although such activities have yet to be associated with invertebrate CTDs. Mammalian CTDs are generally defined by sequence alignment as encoded by exons 18–21. Comparison of known vertebrate 4.1 proteins with invertebrate (Caenorhabditis elegans and Drosophila melanogaster) 4.1 proteins indicates that mammalian 4.1 exon 19 represents a vertebrate adaptation that extends the sequence of the CTD with a Ser/Thr‐rich sequence. The CTD was first described as a 22/24‐kDa domain by chymotryptic digestion of erythrocyte 4.1 (4.1R) [Leto, T.L. & Marchesi, V.T. (1984) J. Biol. Chem.259, 4603–4608]. Here we show that in 4.1R the 22/24‐kDa fragment is not stable but rapidly processed to a 15‐kDa fragment by chymotrypsin. The 15‐kDa fragment is extremely stable, being resistant to overnight digestion in chymotrypsin on ice. Analysis of this fragment indicates that it is derived from residues 709–858 (SwissProt accession no. P48193), and represents the CTD of 4.1R. The fragment behaves as a globular monomer in solution. Secondary‐structure predictions indicate that this domain is composed of five or six β strands with an α helix before the most C‐terminal of these. Together these data indicate that the CTD probably represents an independent folding structure which has gained function since the divergence of vertebrates from invertebrates.
The physico‐chemical characteristics of the phosphocholine‐containing glycoglycerolipid MfGL‐II govern the permeability properties of Mycoplasma fermentans
Authors: Ben‐Menachem Gil , Byström Tomas , Rechnitzer Hagai , Rottem Shlomo , Rilfors Leif , Lindblom Göran
Published: March 2003

Mycoplasma fermentans seems to be involved in several pathogenic condtions in humans, and is among other things capable of fusing with T‐cells and lymphocytes. The choline‐containing phosphoglycolipid 6′‐O‐(3″‐phosphocholine‐2″‐amino‐1″‐phospho‐1″,3″‐propanediol)‐α‐d‐glucopyranosyl‐(1′→3)‐1,2‐diacylglycerol (MfGL‐II) in the membrane of M. fermentans has been suggested to enhance the fusion process, and the characteristics of MfGL‐II were therefore investigated. When a cell culture ages the fraction of MfGL‐II increases, and the fraction of the other major membrane lipid, phosphatidylglycerol (PtdGro), decreases concomitantly. Swelling experiments showed that the permeability and osmotic fragility are markedly reduced in aged cells. MfGL‐II is selectively released into the surrounding medium when aged M. fermentans cells are incubated in buffer containing EDTA. The physico‐chemical properties of MfGL‐II were studied by NMR spectroscopy and differential scanning calorimetry, and they can explain the biochemical results. The temperature for the transition between gel and lamellar liquid crystalline (Lα) phases is 35–45 °C higher for MfGL‐II than for PtdGro, which most probably gives rise to the reduced permeability in aged cells. At high water contents MfGL‐II forms an Lα phase and isotropic aggregates which were interpreted to be vesicles with a radius of ≈ 450 Å. It is proposed that MfGL‐II forms vesicles in the surrounding medium when it is released from the cell membrane. Neither EDTA nor Ca2+ ions have a significant influence on the aggregate structures formed by MfGL‐II. Our results indicate that MfGL‐II has no fusogenic properties. It is more probable that a recently identified lysolipid in the M. fermentans membrane acts as a fusogen.
Factor VII and single‐chain plasminogen activator‐activating protease
Authors: Kannemeier Christian , Feussner Annette , Stöhr Hans‐Arnold , Weisse Jörg , Preissner Klaus T. , Römisch Jürgen
Published: March 2003

Structural and biological characteristics of a recently described plasma serine protease, which displayed factor VII as well as pro‐urokinase‐activating properties in vitro, indicated a dual role for this factor VII‐activating protease (FSAP) in hemostasis. Only the active protease (two‐chain FSAP) has been isolated from plasma and from a prothrombin complex concentrate, whereas activators of the proenzyme have not been identified so far. After purification of the FSAP proenzyme from cryo‐poor plasma by adsorption to an immobilized mAb and subsequent ion‐exchange chromatography, activation to generate two‐chain FSAP was followed by a direct chromogenic assay as well as by the ability of two‐chain FSAP to activate pro‐urokinase. Purified single‐chain FSAP underwent autoactivation leading to the typical protease two‐chain pattern and subsequent degradation products, as demonstrated by Western‐blotting analysis using a site‐specific mAb. This autoactivation was significantly enhanced in the presence of heparin, whereas Ca2+ ions stabilized single‐chain FSAP (the proenzyme) resulting in slower autoactivation kinetics. Correspondingly, the heparin‐augmented reaction, which was associated with autodegradation particularly of the protease domain, was slowed down by co‐incubation with Ca2+. Of the other proteases and cofactors tested, only urokinase (uPA) was able to generate the typical two‐chain FSAP pattern. Studies with different forms of uPA suggest that the catalytic activity of pro‐urokinase/uPA is needed to activate single‐chain FSAP, indicating that it is the only hemostatic protease that can act as a physiological activator of FSAP.
Molecular characterization of Escherichia coli NAD kinase
Authors: Kawai Shigeyuki , Mori Shigetarou , Mukai Takako , Hashimoto Wataru , Murata Kousaku
Published: March 2003

NAD kinase was purified to homogeneity from Escherichia coli MG1655. The enzyme was a hexamer consisting of 30 kDa subunits and utilized ATP or other nucleoside triphosphates as phosphoryl donors for the phosphorylation of NAD, most efficiently at pH 7.5 and 60 °C. The enzyme could not use inorganic polyphosphates as phosphoryl donors and was designated as ATP–NAD kinase. The N‐terminal amino‐acid sequence of the purified enzyme was encoded by yfjB, which had been deposited as a gene of unknown function in the E. coli whole genomic DNA sequence database. yfjB was cloned and expressed in E. coli BL21(DE3)pLysS. The purified product (YfjB) showed NAD kinase activity, and was identical to ATP–NAD kinase purified from E. coli MG1655 in molecular structure and other enzymatic properties. The deduced amino‐acid sequence of YfjB exhibited homology with that of Mycobacterium tuberculosis inorganic polyphosphate/ATP–NAD kinase [Kawai, S., Mori, S., Mukai, T., Suzuki, S., Hashimoto, W., Takeshi, Y. & Murata, K. (2000) Biochem. Biophys. Res. Commun. 276, 57–63], and those of many hypothetical proteins for which functions have not yet been revealed. The YfjB homologues were considered to be NAD kinases and alignment of their sequences revealed highly conserved regions, XXX‐XGGDG‐XL and DGXXX‐TPTGSTAY, where X represents a hydrophobic amino‐acid residue.
Characterization of PSA‐RP2, a protein related to prostate‐specific antigen and encoded by alternative hKLK3 transcripts
Authors: Heuzé‐Vourc'H Nathalie , Leblond Valérie , Olayat Sophie , Gauthier Francis , Courty Yves
Published: March 2003

Despite the wide use of prostate‐specific antigen (PSA) as a marker of prostate cancer, analysis of its gene products has not yet been completed. The structure of two alternative mRNAs (0.9 and 1.65 kb) of the hKLK3 gene that retain the third intron is reported here. These partially spliced transcripts were detected by hybridization or RT‐PCR in normal prostate tissue, benign prostate hyperplasia (BPH) and cancerous prostate tissues, and also in the prostate LNCaP cell line. Insertion of the unspliced intron creates an in‐frame stop codon and results in a truncated prepro PSA variant of 180 amino‐acid residues. This novel variant, designated PSA‐RP2, has an alternate C‐terminal tail and lacks the serine residue essential for the catalytic activity of PSA. Prepro PSA‐RP2 was transiently produced in COS‐7 cells and detected in the spent medium using an anti‐PSA serum. Secreted PSA‐RP2 was glycosylated with an apparent molecular mass of 25 kDa. Our findings suggest that PSA‐RP2 contributes to the molecular heterogeneity of free‐PSA in the serum of patients with benign or malignant prostate tumors.
A mouse in vitro transcription system reconstituted from highly purified RNA polymerase II, TFIIH and recombinant TBP, TFIIB, TFIIE and TFIIF
Authors: Kotova Irina , Chabes Anna Lena , Segerman Bo , Flodell Sara , Thelander Lars , Björklund Stefan
Published: March 2003

Unregulated transcription of protein‐encoding genes in vitro is dependent on 12‐subunit core RNA polymerase II and five general transcription factors; TATA binding protein (TBP), transcription factor (TF)IIB, TFIIE, TFIIF, and TFIIH. Here we describe cloning of the mouse cDNAs encoding TFIIB and the small and large TFIIE and TFIIF subunits. The cDNAs have been used to express the corresponding proteins in recombinant form in Escherichia coli and in Sf21 insect cells, and all proteins have been purified to > 90% homogeneity. We have also purified a recombinant His6‐tagged mouse TBP to near homogeneity and show that it is active in both a reconstituted mouse in vitro transcription system and a TBP‐dependent in vitro transcription system from Saccharomyces cerevisiae. The more complex general transcription factors, TFIIH and RNA polymerase II, were purified more than 1000‐fold and to near homogeneity, respectively, from tissue cultured mouse cells. When combined, the purified factors were sufficient to initiate transcription from different promoters in vitro. Functional studies of the S‐phase‐specific mouse ribonucleotide reductase R2 promoter using both the highly purified system described here (a mouse cell nuclear extract in vitro transcription system) and in vivo R2‐promoter reporter gene assays together identify an NF‐Y interacting promoter proximal CCAAT‐box as being essential for high‐level expression from the R2 promoter.
Structural analysis of the polysaccharide from the lipopolysaccharide of Porphyromonas gingivalis strain W50
Authors: Paramonov Nikolay , Bailey David , Rangarajan Minnie , Hashim Ahmed , Kelly Geoff , Curtis Michael A. , Hounsell Elizabeth F.
Published: March 2003

The lipopolysaccharide (LPS) of Porphyromonas gingivalis is an important pro‐inflammatory molecule in periodontal disease and a significant target of the host's specific immune response. In addition, we recently demonstrated using monoclonal antibodies that the Arg‐gingipains of P. gingivalis are post‐translationally modified with glycan chains that are immunologically related to an LPS preparation from this organism. In the present investigation, we determined the structure of the O‐polysaccharide of P. gingivalis W50 that was fully characterized on the basis of 1D and 2D NMR (DQF‐COSY, TOCSY, NOESY, ROESY, 1H‐13C HSQC and 1H‐31P HXTOCSY) and GC‐MS data. These data allowed us to conclude that the O‐polysaccharide is built up of the tetrasaccharide repeating sequence: →6)‐α‐d‐Glcp‐(1→4)‐α‐l‐Rhap‐(1→3)‐β‐d‐GalNAc‐(1→3)‐α‐d‐Galp‐(1→ and carries a monophosphoethanolamine residue at position C‐2 of the α‐rhamnose residue in a nonstoichiometric (approximately 60%) amount. These data indicate that the O‐polysaccharide of P. gingivalis LPS is composed of an unusually modified tetrasaccharide repeating unit.