bluuebunny/crossref_metadata_2025 huggingface dataset:
Origin of the carbon atoms of biotin
Authors: IFUKU Ohji , MIYAOKA Hiroaki , KOGA Nobuyoshi , KISHIMOTO Jiro , HAZE Shin‐ichirou , WACHI Youji , KAJIWARA Masahiro
Published: March 2005

The origin of the carbon atoms of pimeloyl‐CoA, the earliest known precursor in the pathway of de novo biotin biosynthesis in Escherichia coli, was investigated by 13C‐NMR spectroscopy.

In fermentation of the biotin‐overproducing DRK332/pXBA312 strain of Escherichia coli (a repressor mutant carrying a biotin operon fragment in the plasmid), a high dose of L‐alanine (8 g/l) stimulated dethiobiotin and biotin accumulation. Although L‐alanine is a known precursor of 7‐keto‐8‐aminopelargonic acid in biotin biosynthesis, the 13C‐NMR spectrum of dethiobiotin showed that the C‐3 of L‐[3‐13C]alanine was incorporated into not only the methyl carbon (C‐9) but also alternate carbons (C‐2, C‐4, C‐6) of the side chain, and these latter positions are the same as those labeled with D‐[1‐13C]glucose. These data indicate that L‐alanine can act as an alternative carbon source, suggesting that acetyl‐CoA is a possible precursor for pimeloyl‐CoA synthesis. In accordance with this hypothesis, the C‐1 of sodium (1‐13C)acetate and the C‐2 of sodium (2‐13C)acetate were incorporated into alternate carbons in the side chain of dethiobiotin, i.e., (C‐1, C‐3, C‐5, C‐7) and (C‐1, C‐2, C‐4, C‐6), respectively. These results suggested firstly that in E. coli pimeloyl‐CoA is biosynthesized from L‐alanine and/or acetate via acetyl‐CoA, but not via pimelic acid, which has been suggested as a biotin precursor in other species, and secondly that the carboxyl group of biotin originates from carbon dioxide produced through the tricarboxylic acid cycle.
High‐level expression of the trucated α chain of Human high‐affinity receptor for IgE as a soluble form by baculovirus‐infected insect cells
Authors: YAGI Shintaro , YANAGIDA Mitsuaki , HASEGAWA Akira , OKUMURA Ko , RA Chisei
Published: March 2005

The binding subunit of human high‐affinity receptor for IgE (FcɛRI α) was efficiently expressed as a truncated form in insect cells. The soluble (s)FcɛRI α purified from culture medium by affinity chromatography with an anti‐(α chain) mAb was nearly homogeneous and had an IgE‐binding activity. The amino acid composition and the revealed N‐terminal amino acid sequence of sFcɛRI α suggested that it was properly processed in insect cells. The apparent molecular mass (35 kDa) of purified sFcɛRI α was smaller than that of sFcɛRI α produced by CHO transfectants. The reduction of the apparent molecular mass after N‐glycanase treatment showed the recombinant product was N‐glycosylated. Peptide mapping of native and deglycosylated sFcɛRI α indicated that three Asn residues (Asn21, Asn42 and Asn 166) should be almost fully glycosylated, and that two Asn residues (Asn 74 and Asn 135) were partially glycosylated.
Cloning and functional expression of poly(ADP‐ribose) polymerase cDNA from Sarcophaga peregrina
Authors: MASUTANI Mitsuko , NOZAKI Tadashige , HITOMI Yoshiaki , IKEJIMA Miyoko , NAGASAKI Koichi , DE PRATI Alessandra Carcereri , KURATA Shoichi , NATORI Shunji , SUGIMURA Takashi , ESUMI Hiroyasu
Published: March 2005

A cDNA spanning the entire coding region for poly(ADP‐ribose) polymerase (PARP) of Sarcophaga peregrina was isolated and the nucleotide sequence was determined. The longest open reading frame encodes a polypeptide of 996 amino acid residues with a molecular mass of 113033 Da. The similarities to the human PARP in amino acid sequence were relatively low in the DNA‐binding and auto‐modification domains, but very high in the C‐terminal catalytic domain: identity of amino acids is 34% in the N‐terminal DNA‐binding domain (residues 1–369), 27% in the auto‐modification domain (residues 370–507), and 56% in the C‐terminal NAD‐binding domain (residues 508–996). Two zinc‐fingers (C‐X2‐C‐X28‐H‐X2‐C and C‐X2‐C‐X31‐H‐X2‐C)2 and a basic region in the N‐terminal DNA‐binding domain recognized in other PARP are conserved. Downstream of the basic region, another cysteine‐rich motif (C‐X2‐C‐X13‐C‐X9‐C), a putative zinc‐finger, was found to be well conserved in the PARP of Sarcophaga, Drosophila and human. A leucine‐zipper motif (l‐X6‐l‐X6‐l‐X6‐L) which was found in the auto‐modification domain of Drosophila PARP, is disrupted in the Sarcophaga enzyme: the second leucine is replaced by proline, and the third leucine by valine. Full‐length cDNA for Sarcophaga PARP was cloned into an expression plasmid and expressed in Escherichia coli. A lysate of E. coli cells containing expressed protein reacted with antibody against Sarcophaga PARP, and PARP activity was detected. Thus, we conclude that isolated cDNA encodes a functional Sarcophaga PARP cDNA.
Molecular cloning and nucleotide sequence of chicken avidin‐related genes 1–5
Authors: KEINÄNEN Riitta A. , WALLÉN Mika J. , KRISTO Paula A. , LAUKKANEN Mikko O. , TOIMELA Tarja A. , HELENIUS Merja A. , KULOMAA Markku S.
Published: March 2005

Using avidin cDNA as a hybridisation probe, we detected a gene family whose putative products are related to the chicken egg‐white avidin. Two overlapping genomic clones were found to contain five genes (avidin‐related genes 1–5, avrl‐avr5), which have been cloned, characterized and sequenced. All of the genes have a four‐exon structure with an overall identity with the avidin cDNA of 88–92%. The genes appear to have no pseudogenic features and, in fact, two of these genes have been shown to be transcribed. The putative proteins share a sequence identity of 68–78% with avidin. The amino acid residues responsible for the biotin‐binding activity of avidin and the bacterial biotin‐binding protein, streptavidin, are highly conserved. Since avidin is induced in both a progesterone‐specific manner and in connection with inflammation, these genes offer a valuable tool to study complex gene regulation in vivo.
Structure and expression of the gene for the prothoracicotropic hormone of the silkmoth Bombyx mori
Authors: ADACHI‐YAMADA Takashi , IWAMI Masafumi , KATAOKA Hiroshi , SUZUKI Akinori , ISHIZAKI Hironori
Published: March 2005

We cloned and characterized two allelic variants of the gene for the Bombyx mori prothoracicotropic hormone (PTTH), a homodimeric 30‐kDa brain secretory protein. These PTTH genes contain five exons that encode a precursor protein consisting of 224 amino acid residues whose C ‐terminal 109 residues represent the PTTH expression is the brain but expression at a very low level occurs in the gut. One Bombyx brain at day 0 of the fifth larval instar contained 2.4–2.8 pg PTTH mRNA, and this amount did not change markedly during larval‐pupal development.
X‐ray crystal structures of cytosolic glutathione S‐transferases
Authors: DIRR Heini , REINEMER Peter , HUBER Robert
Published: March 2005

Crystal structures of cytosolic glutathione S‐transferases (EC 2.5.1.18), complexed with glutathione or its analogues, are reviewed. The atomic models define protein architectural relationships between the different gene classes in the superfamily, and reveal the molecular basis for substrate binding at the two adjacent subsites of the active site. Considerable progress has been made in understanding the mechanism whereby the thiol group of glutathione is destabilized (lowering its pKa) at the active site, a rate‐enhancement strategy shared by the soluble glutathione S‐transferases.
Redox control of β‐oxidation in rat liver mitochondria
Authors: EATON Simon , TURNBULL Douglass M. , BARTLETT Kim
Published: March 2005

Coupled rat liver mitochondria were incubated with [U‐14C]hexadecanoate and carnitine which resulted in the formation of acyl‐, 2‐enoyl‐ and 3‐hydroxyacyl‐CoA and carnitine esters. The production of 2‐enoyl‐CoA and 3‐hydroxyacyl‐CoA esters was associated with a significant lowering of the NAD+/NADH ratio, in contrast to rat muscle mitochondria [Eaton, S., Bhuiyan, A. K. M. J., Kler, R. S., Turnbull, D. M. & Bartlett, K. (1993) Biochem. J. 289, 161–172], suggesting that control by the respiratory chain is important under normal conditions. When NAD+/NADH ratios were held low by succinate‐induced reverse electron flow, 3‐enoyl‐CoA esters were also detected, probably formed by the action of 3,2‐enoyl‐CoA isomerase. Measurement of the flux of ß‐oxidation at different osmolalities showed that flux was strongly dependent on osmolality changes in the physiological range. Measurement of the CoA and carnitine esters resulting from incubations made at different osmolalities showed that there was an increase in the amounts of the saturated acyl‐CoA esters with respect to 2‐enoyl‐CoA and 3‐hydroxyacyl‐CoA esters, consistent with control by the electron‐transfer flavoprotein‐ubiquinone segment [Halestrap, A. P. & Dunlop, J. L. (1986) Biochem. J. 239, 559–565]. This however could not be the only factor operating as indicated by the continued presence of 2‐enoyl‐CoA and 3‐hydroxyacyl‐CoA esters at high osmolalities.
Threonyl‐tRNA synthetase from yeast Discrimination of 19 amino acids in aminoacylation of tRNAThr‐C‐C‐A and tRNAThr‐C‐C‐A(2′NH2).
Authors: FREIST Wolfgang , STERNBACH Hans , CRAMER Friedrich
Published: March 2005

For discrimination between threonine and 18 other naturally occuring non‐cognate amino acids by the class II aminoacyl‐tRNA synthetase specific for threonine, discrimination factors (D) have been determined from kcat and Km values. The lowest values were found for Cys, Met, Val (D= 70–280), indicating that threonine is only 70–280‐times more often esterified to tRNAThr‐C‐C‐A than are these non‐cognate compounds at the same amino acid concentrations. The highest D values have been observed for Gly, Pro, Gln, Leu, Phe, and Lys (D= 1000–2000), for the other non‐cognate amino acids D values are in the medium range 300–1000. Generally, threonyl‐tRNA synthetase is less specific than the class I enzymes specific for Ile, Val, Tyr, Arg, but more specific than the only investigated class II enzyme specific for Lys.

In aminoacylation of tRNAThr‐C‐C‐A(2′NH2) discrimination factors D1, are in the range 2–170. From D1, values and AMP‐formation stoichiometry, pre‐transfer proof‐reading factors II1, were determined; post‐transfer proof‐reading factors II2, were determined from D values and AMP‐formation stoichiometry in acylation of tRNAThr‐C‐C‐A. II1, values are in the range 1.8–33, II2, values in the range 1.4–22, thus threonyl‐tRNA synthetase shows the highest post‐transfer proof‐reading activity of six investigated synthetases (specific for Ile, Val, Tyr, Arg, Lys).

Initial discrimination factors caused by differences in Gibbs free energies of binding between threonine and non‐cognate amino acids have been calculated from discrimination and proof‐reading factors. Assuming a two‐step binding process, two factors (I1, and I2) have been determined which can be related to hydrophobic interaction forces depending on accessible surface areas of the amino acids. The threonine side chain must be bound by hydrophobic forces and two hydrogen bonds. In contrast to proof‐reading factors obtained with the synthetases specific for Ile, Val, Tyr, Arg, and Lys, proof‐reading factors II1, and II2, obtained with threonyl‐tRNA synthetase are also related to hydrophobic interaction of the amino acid side chains and the enzyme. Threonyl‐tRNA synthetase examines side chain structures of amino acids in the four postulated recognition steps, for each step the enzyme uses special distinct structures or conformations of the binding cleft.
A human POU domain gene, mPOU, is expressed in developing brain and specific adult tissues
Authors: WEY Eva , LYONS Gary E. , SCHÄFER Beat W.
Published: March 2005

POU domain genes constitute a family of transcription factors that exhibit distinct temporal and spatial patterns of expression. To investigate the possible functions that POU proteins may have in muscle development we have isolated four novel POU‐domain‐encoding sequences from human muscle tissue. One of these sequences, referred to as mPOU, encodes a new member of subclass VI of the POU family. In the embryo, mPOU is expressed exclusively in the developing brain, whereas in the adult its expression is restricted to brain, heart, skeletal muscle and lung. In the brain, the highest expression levels were found in specific cell layers of the cortex, the olfactory bulb, the hippocampus and the cerebellum. mPOU is shown to bind to DNA sequences containing the octamer motif and other POU factor target sites. The distinct expression pattern and divergent DNA‐binding characteristics indicate that mPOU may regulate a distinct set of genes.
The nuclear‐magnetic‐resonance solution structure of the mutant α‐amylase inhibitor [R19L]Tendamistat and comparison with wild‐type Tendamistat
Authors: O'CONNELL John F. , BENDER Rudolf , ENGELS Joachim W. , KOLLER Klaus‐P. , SCHARF Mathias , WÜTHRICH Kurt
Published: March 2005

The recombinant mutant α‐amylase inhibitor [R19L]Tendamistat, with Arg19 replaced by Leu, was prepared and its NMR solution structure determined. Based on complete sequence‐specific 1H‐NMR assignments, 845 nuclear Overhauser effect upper‐distance constraints and 156 dihedral angle constraints were collected using two‐dimensional homonuclear 1H‐NMR experiments. The structure was calculated with the program DIANA, using the redundant dihedral angle constraints strategy for improved convergence. For restrained energy minimization, the programs FANTOM and AMBER were used. The wild‐type NMR solution structure was similarly recalculated from the previously published input of conformational constraints [Kline, A., Braun, W. & Wüthrich, K. (1988) J. Mol. Biol. 204, 675–724]. For each protein a group of 20 conformers represents a well‐defined solution structure, with average root‐mean‐square‐distance values for the backbone atoms of the individual conformers relative to the mean coordinates of 50 pm. The two structures are nearly identical to each other and to the previously published Tendamistat structures in solution and in crystals. The only significant difference is strictly localized near the single amino acid substitution in the presumed active site ‐Trp18‐Arg(Leu)‐Tyr‐, i.e. Leu19 and Tyr20 are more precisely defined in the solution structure of [R19L]Tendamistat than the corresponding residues Arg19 and Tyr20 in wild‐type Tendamistat.
pH‐induced structural changes in human serum apotransferrin pKa values of histidine residues and N‐terminal amino group determined by 1H‐NMR spectroscopy
Authors: KUBAL Gina , SADLER Peter J. , TUCKER Alan
Published: March 2005

The binding of apotransferrin (80 kDa) to the transferrin receptor is known to be highly pH‐dependent. We have investigated pH‐induced structural changes in human serum apotransferrin over the pH* (meter reading in D2O solutions) range 2.5–11 using 1H‐NMR spectroscopy. The pKa values of 14 (possibly 15) of the 19 His residues in the protein have been determined as well as that of the terminal amino group (Val1, 7.75). About eight His residues deprotonate when the pH* is raised from the endosomal value of about 5.5 to the blood plasma value (7.4). Four His residues have pKa < 6. Sharp discontinuities in the His titration curves were observed below pH 4.3 and at pH 3.5 molten globule states were detected.
Purification and partial characterization of a putative thymidylate synthase from Methanobacterium thermoautotrophicum
Authors: KRONE Ute E. , McFARLAN Sara C. , HOGENKAMP Harry P. C.
Published: March 2005

A protein catalyzing the tritium exchange of [5‐3H]deoxyuridine monophosphate ([5‐3H]dUMP) for solvent protons and the dehalogenation of 5‐bromo‐deoxyuridine monophosphate (Br‐dUMP) has been isolated from the methanogenic archaea Methanobacterium thermoautotrophicum. These two activities are well‐established side reactions of thymidylate synthase and do not require cofactors. Sodium dodecylsulfate/polyacrylamide gel electrophoresis of the purified enzyme showed a single band with a molecular mass of 27 kDa. The suggested molecular mass of the native protein calculated from sedimentation equilibrium experiments was 33.5 kDa, indicating that the enzyme is a monomer. The pH optima were 9.0 and 7.0 for the exchange reaction and the dehalogenation, respectively. The effects of temperature, salt, reducing agent and inhibitors were determined. The apparent Km for the tritium exchange from [5‐3H]dUMP was 7 μM and for the dehalogenation of Br‐dUMP was 14 μM. However, thus far, the conditions for dTMP synthesis from dUMP have not yet been established. Incubation of the enzyme with dUMP, tetrahydromethanopterin, a folate analog present in methanogens, and formaldehyde did not yield dTMP. The first 30 amino acids of the amino terminus have been sequenced. However, there is no similarity with any of the thymidylate synthases. Surprisingly, the protein from M. thermoautotrophicum appears to be related to chitin synthases from several organisms.
In vivo and in vitro photoinhibition reactions generate similar degradation fragments of D1 and D2 photosystem‐II reaction‐centre proteins
Authors: SHIPTON Catherine Ann , BARBER James
Published: March 2005

Isolation of photosystem‐II reaction centres from pea leaves after photoinhibitory treatment at low temperature (0–1°C) has provided evidence for the mechanism of degradation of the D1 protein in vivo. These isolated reaction centres did not appear to be spectrally distinct from preparations obtained from control leaves that had not been photoinhibited. Breakdown fragments of both the D1 and D2 proteins were, however, found in preparations isolated from photoinhibited leaves, and showed similarities with those detected when isolated reaction centres were exposed to acceptorside photoinhibition. Analyses of the origin of D1 fragments indicated that the primary cleavage site of this protein was between transmembrane helices IV and V indicative of the acceptor‐side mechanism for photoinhibition. The origins of other D1 protein fragments indicate that some donorside photoinhibition may also have occurred in vivo under the conditions employed. We have shown that the spectral and functional integrating of the isolated photosystem II reaction centre complex is resistant to proteolytic cleavage by trypsin. Use of a more non‐specific protease (subtilisin), however, caused significant destabilisation of the special pair of chlorophylls constituting the primary electron donor, P680, with a consequential loss of functional activity. Thus, it is possible that specific cleavage of photosystem‐II reaction‐centre proteins may occur in vivo following photoinhibitory damage without a significant change in structural integrity, a conclusion supported by the finding that photodamaged and normal reaction centres were isolated together.
The type‐III Fe receptor from Streptococcus dysgalactiae is also an α2‐macroglobulin receptor
Authors: JONSSON Hans , MÜLLER Hans‐Peter
Published: March 2005

We have cloned and sequenced the gene for a type‐III Fe receptor from a Streptococcus dysgalactiae strain isolated from bovine mastitis and report here the complete sequence of the gene. The 1992‐nucleotides‐long open reading frame codes for a protein of 664 amino acids with a predicted molecular mass of 72 kDa (including the signal peptide). The C‐terminal part of the protein is very similar to other streptococcal type‐III receptors but contains five homologous IgG‐binding domains compared to two or three in earlier reported Fe receptors of this type. Upstream of the IgG‐binding domains, there is a unique sequence containing short repeated motifs. Subeloning different parts of the gene and expressing them as maltose‐binding fusion proteins revealed that the upstream part is mediating binding to the plasma proteinase inhibitor α2‐macroglobulin after its complex formation with proteases. In contrast to earlier described type‐III Fe receptors, this molecule does not bind human serum albumin.
The I‐Ceu I endonuclease: purification and potential role in the evolution of Chlamydomonas group I introns
Authors: MARSHALL Philip , DAVIS Ted B. , LEMIEUX Claude
Published: March 2005

During genetic crosses between the interfertile green algae Chlamydomonas eugametos and Chlamydomonas moewusii, the I‐Ceul endonuclease encoded by the fifth group I intron (CeLSU · 5) in the C. eugametos chloroplast large subunit rRNA gene mediates the mobility of this intron by introducing a double‐strand break near the insertion site of the intron in the corresponding C. moewusii intronless allele. To characterize the biochemical properties of this endonuclease, we have purified I‐CeuI and determined the optimal reaction conditions for cleavage. I‐CeuI activity is maximal at 50°C, pH 10.0, 2.5 mM MgCl2 and in the absence of NaCl. Unlike the well‐characterized I‐SceI endonuclease, I‐CeuI remains stable following preincubation in the absence of substrate. We discuss the role that homing endonucleases may have played in the evolution of Chlamydomonas chloroplast group I introns.
Structural analysis of the functional gene and pseudogene encoding the murine granulocyte colony‐stimulating‐factor receptor
Authors: ITO Yasuhiko , SETO Yoshiyuki , BRANNAN Camilynn I. , COPELAND Neal G. , JENKINS Nancy A. , FUKUNAGA Rikiro , NAGATA Shigekazu
Published: March 2005

Granulocyte colony‐stimulating factor is a cytokine which specifically regulates the production of neutrophilic granulocytes. The granulocyte colony‐stimulating‐factor receptor (GCSFR) is mainly expressed in neutrophils and their precursor cells. In this study, we isolated the chromosomal gene for murine GCSFR and determined its structure. Like the human GCSFR gene homolog, it consists of 17 exons. The exon‐intron organization of the murine and human GCSFR‐encoding genes are very similar, except that exon 14 and exon 15 in the murine gene are interrupted by a larger intron (greater than 10 kbp) than that found in the human gene (128 bp). This GCSFR‐encoding functional gene (Csfgr) was localized to the distal region of murine chromosome 4 by interspecific backcross mapping.

A comparison of the 5′ flanking sequence of murine and human Csfgr revealed that a sequence of approximately 300 bp upstream from the cap site is highly conserved. Within this region, an 18‐nucleotide element conserved in the promoter of the genes for neutrophil‐specific enzymes, was found approximately 140 bp upstream from the cap site, suggesting an involvement of this element in the specific expression of GCSFR in neutrophilic granulocytes.

In addition to the functional GCSFR‐encoding gene, we isolated a pseudogene for GCSFR, which is flanked by a 15‐bp direct repeat at the 5′ and 3′ ends, and lacks all introns, exons 1–3 and exons 7–8 of the functional gene. The processed pseudogene has, in its most 5′ region, a sequence of approximately 200 bp that is highly related to the DNA sequence approximately 1.2 kbp upstream of the cap site of the functional gene.
Coordination of catalytic activities within enzyme complexes
Authors: KELLERSHOHN Nicolas , RICARD Jacques
Published: March 2005

If two enzymes are physically and permanently associated as a bi‐enzyme complex and if these enzymes catalyze non‐consecutive chemical reactions, either of these reactions may inhibit or activate the other. If these reactions belong to two different metabolic cycles, the functioning of one of these cycles will control the fine tuning of the other.

Thus simple kinetic considerations lead to the conclusion that, owing to the spatial organization of enzymes as multimolecular complexes, a fine tuning and a coordination of different metabolic networks, or cycles, may be exerted. It thus appears that channelling of reaction intermediates within a multienzyme complex does not represent the only functional advantage brought about by this type of spatial molecular organization.
Activation of the retinal cGMP‐specific phosphodiesterase by the GDP‐loaded α‐subunit of transducin
Authors: KUTUZOV Mikhail , PFISTER Claude
Published: March 2005

The interaction of the GDP‐bound form of the α‐subunit of transducin (TαGDP) with the cGMP‐specific phosphodiesterase, the effector enzyme in the visual system, has been studied. TαGDP is demonstrated to be able to activate the phosphodiesterase: (a) the basal activity in suspensions of dark‐adapted retinal rod outer segments, examined in the absence of GTP, was found to be inhibited by binding of transducin to activated rhodopsin (Rh*) and by the complex of the β‐and γ‐subunits of transducin (Tβγ); (b) purified TαGDP is able to activate phosphodiesterase in the presence of membranes; (c) no activation is obtained either with holotransducin (TαGDPTβγ) or with TαGDP in the presence of excess Tβγ to prevent dissociation of TGDP. The maximal level of phosphodiesterase activation reached with TαGDP (about 1500 mol cGMP/mol phosphodiesterase−1· s−1) is similar to that obtained through the ‘classical’ activation by TαGTP, whereas the apparent affinity of TαGDP for phosphodiesterase (Kd about 50 μM) is much lower than that of TαGTP. Our data suggest that GTP hydrolysis itself does not inactivate Tα. The role of Tβγ to sequester Tα is therefore of critical importance for phosphodiesterase inactivation. Our results support observations on the regulation of adenylyl cyclase by G‐proteins, which suggested the ability of the free α‐subunits loaded with GDP to activate their effectors.
Cross‐reactivity between the mannan of Candida species, Klebsiella K24 polysaccharide and Salmonella C1 and E O‐antigens is mediated by a terminal non‐reducing β‐mannosyl residue
Authors: NNALUE Ndubisi A. , WEINTRAUB Andrej , OSCARSON Stefan , LINDBERG Alf A.
Published: March 2005

Rat monoclonal antibody MASC1‐MR9 (MR9) binds to a mannan of Candida species and the O‐antigenic polysaccharides of Salmonella bacteria of serogroups C1 (CO) and E (EO). Mannan and glycoconjugates comprising BSA and O‐antigen polysaccharides, decasaccharide‐BSA (CO‐BSA) or trisaccharide‐BSA (EO‐BSA), inhibited each other's reactivity with MR9. The saccharides β‐d‐Manp‐(1→6)‐α‐d‐Manp‐1‐OMe, β‐d‐Manp(1→3)‐α‐d‐Manp‐1‐OMe, β‐d‐Manp(1→2)‐α‐d‐Manp‐1‐OMe (corresponds to the terminal non‐reducing end of Salmonella serogroup C1 O‐antigen) and d‐Manp(1→4)‐α‐l‐Rhap(1→3)‐α ‐d‐Galp‐1‐O‐p‐trifluoroacetamido aniline (corresponds to the backbone of Salmonella serogroup E O‐antigen) inhibited the binding of MR9 to these antigens whereas α‐d‐Manp(1→3)‐α‐d‐Manp‐1‐OMe and α‐d‐Manp(1→4)‐α‐l‐Rhap‐1‐O‐p‐ nitrophenyl did not. Saccharides (3–10 residues) of mammalian origin with terminal and internal Manpα‐1→2, Manpα‐1→3 and Manpα‐1→6 residues also failed to inhibit at any concentration. None of the saccharides with internal β‐mannosyl residue was able to inhibit the MR9 antibody. Monosaccharides d‐mannose, β‐d‐Manp‐1‐OMe and 1,5 anhydro‐d‐mannitol inhibited the MR9 monoclonal antibody whereas α‐d‐Manp‐1‐OMe, β‐d‐Glcp‐1‐OMe, and β‐d‐Galp‐1‐OMe did not. In addition a Klebsiella K24 capsular polysaccharide containing a β‐d‐Manp(1→4)‐α‐d‐G1cA, glucuronic acid) as a structural element possessed an inhibitory activity. MR9 therefore recognizes an epitope within β‐mannose monosaccharide residues at the terminal non‐reducing ends of carbohydrate chains in mannan, and polysaccharides in Salmonella serogroups CO and EO and Klebsiella K24.
Primary sequence, oxidation‐reduction potentials and tertiary‐structure prediction of Desulfovibrio desulfuricans ATCC 27774 flavodoxin
Authors: CALDEIRA Jorge , PALMA P. Nuno , REGALLA Manuela , LAMPREIA Jorge , CALVETE Juan , SCHÄFER Wolfram , LEGALL Jean , MOURA Isabel , MOURA José J. G.
Published: March 2005

Flavodoxin was isolated and purified from Desulfovibrio desulfuricans ATCC 27774, a sulfate‐reducing organism that can also utilize nitrate as an alternative electron acceptor. Mid‐point oxidation‐reduction potentials of this flavodoxin were determined by ultraviolet/visible and EPR methods coupled to potentiometric measurements and their pH dependence studied in detail. The redox potential E2, for the couple oxidized/semiquinone forms at pH 6.7 and 25°C is –40 mV, while the value for the semiquinone/hydroquinone forms (E1), at the same pH, –387 mV. E2 varies linearly with pH, while E1 is independent of pH at high values. However, at low pH (< 7.0), this value is less negative, compatible with a redox‐linked protonation of the flavodoxin hydroquinone. A comparative study is presented for Desulfovibrio salexigens NCIB 8403 flavodoxin [Moura, I., Moura, J. J. G., Bruschi, M. & LeGall, J. (1980) Biochim. Biophys. Acta 591, 1–8].

The complete primary amino acid sequence was obtained by automated Edman degradation from peptides obtained by chemical and enzymic procedures. The amino acid sequence was confirmed by FAB/MS.

Using the previously determined tridimensional structure of Desulfovibrio vulgaris flavodoxin as a model [similarity, 48.6%; Watenpaugh, K. D., Sieker, L. C., Jensen, L. H., LeGall, J. & Dubourdieu M. (1972) Proc. Natl Acad. Sci. USA 69, 3185–3188], the tridimensional structure of D. desulfuricans ATCC 27774 flavodoxin was predicted using AMBER force‐field calculations.
RNA editing in trypanosomes
Authors: BENNE Rob
Published: March 2005

The nucleotide sequence of mitochondrial pre‐mRNAs in trypanosomes is posttranscriptionally edited by the insertion and deletion of uridylate (U) residues. In some RNAs editing is limited to small sections but in African trypanosomes, such as Trypanosoma brucei, 9 of the 18 known mitochondrial mRNAs are created by massive editing which can produce more than 50% of the coding sequence. In all cases, however, RNA editing is a key event in gene expression during which translatable RNAs are generated. The information for the editing process and possibly also the inserted Us are provided by small guide RNAs, which are encoded in both the maxicircle and minicircle components of the trypanosome mitochondrial DNA. Current models of editiong are largely based on the characteristics of partially edited RNAs and on the occurrence in vivo and the possibility of synthesis in vitro of chimeric molecules in which a guide RNA is covalently linked through its 3′ oligo(U) tail to an editing site in pre‐mRNA. In this paper, I will review the research in this rapidly growing field and illustrate how different interpretations of the available data can lead to different views of the mechanism and the biochemistry of the editing process.
The protective role of high‐density lipoprotein on oxidized‐low‐density‐lipoprotein‐induced U937/endothelial cell interactions
Authors: MAIER Jeanette Anne Marie , BARENGHI Livia , PAGANI Franco , BRADAMANTE Silvia , COMI Paola , RAGNOTTI Giovanni
Published: March 2005

The adherence of monocytes to the endothelium is an early event in atherogenesis. We have investigated this process by examining whether native and oxidized low‐density and high‐density lipoproteins could modulate this process. Only oxidized low‐density lipoprotein caused a significant dose‐dependent and time‐dependent increase in U937 monocyte‐like cell line binding to human endothelial cells, by a process which required de novo protein synthesis. Interstingly, E‐selectin, intercellular adhesion molecule‐1, vascular cell‐adhesion molecule or P‐selectin induction was not apparent in this system suggesting the presence of an alternative system for the interaction of endothelial cells with monocyte‐like cells in response to oxidized low‐density lipoprotein. Highdensity lipoprotein completely suppressed oxidized low‐density‐lipoprotein‐induced adhesion of U937 cells to the endothelial monolayer, while oxidized high‐density lipoprotein did not. These data suggest that the balance between native and oxidized lipoproteins may play a role in the formation of the atherosclerotic lesion by modulating monocyte endothelial interactions.
Cis‐acting elements reinforcing the activity of the estrogen‐response element in the very‐low‐density apolipoprotein II gene promoter
Authors: SCHIPPERS Ingrid J. , KLOPPENBURG Mariska , van WAARDENBURG Robert , AB Geert
Published: March 2005

The gene coding for chicken very low density apolipoprotein II (apoVLDLII) is expressed exclusively in liver in response to estrogen. Previous work in our laboratory identified several protein binding sites, identified by the letters A to F, and their cognate factors within the first 300 bp flanking the gene. Here we present an extensive functional analysis of the apoVLDLII promoter by gene transfer experiments using a chicken hepatoma cell line and cultured non‐hepatic cells. Deletion analysis revealed that the −301 to −163‐bp promoter region, comprising elements E1, E2 and F, is sufficient for strong estrogen‐dependent expression. Mutation analysis demonstrated that efficient transcription requires the interplay of the major estrogen response element E1 with several other cis‐acting elements. Analysis of individual protein binding sites showed that element E1 is sufficient by itself to confer weak estrogen‐induced transcription from the apoVLDLII promoter, and that additional promoter elements are required for full estrogen‐responsiveness. Elements F and B1 were capable of strongly potentiating the activity of element E1. In general, the activity of certain cis‐acting elements appeared to be strongly promoter‐context dependent. Cultured non‐liver cells expressed transfected VLDL‐CAT reporter plasmids in the presence of cotransfected estrogen receptor expression vector in a hormone‐dependent way, indicating that for the control of tissue specificity the 5′‐proximal promoter region is not sufficient.
Overexpression in Escherichia coli, purification and characterization of the molecular chaperone HSC70
Authors: BENAROUDJ Nadia , FANG Bo , TRINIOLLES Françoise , GHELIS Charis , LADJIMI Moncef M.
Published: March 2005

The 70‐kDa heat‐shock cognate protein (HSC70), a constitutively expressed protein in mammalian cells, plays a major role in several cellular processes such as protein folding and assembly, uncoating of clathrin‐coated vesicles and transport of protein through membranes. HSC70 has been overexpressed in Escherichia coli in a soluble form using a designed two‐cistron expression vector, and purified to homogeneity in a two‐step procedure involving ion‐exchange and affinity chromatography. Up to 20 mg of pure protein could be obtained from 1 l of cell culture. Amino‐terminal sequencing of the recombinant protein gives the expected sequence, and non‐denaturing gel electrophoresis as well as gel filtration analysis reveal the presence of self‐associating species that could be dissociated by ATP. Crosslinking studies confirm the presence of multiple species and the dissociating effect of ATP. Temperatures above 42°C induce the aggregation of HSC70; ATP shifts this effect to higher temperatures. The recombinant protein displays a low intrinsic ATPase activity that can be stimulated about threefold by binding to apocytochrome c, a permanently unfolded protein, while native cytochrome c has no effect on the ATPase activity indicating that recombinant HSC70 binds specifically unfolded protein but not their native counterpart. Thus, efficient production of recombinant HSC70 having structural and functional properties comparable to those of the natural protein could be achieved, thereby allowing the molecular basis of the chaperone function and its regulation through ATP hydrolysis to be probed.
Cloning, amino acid sequence and tissue distribution of porcine thimet oligopeptidase
Authors: KATO Akira , SUGIURA Naoaki , HAGIWARA Hiromi , HIROSE Shigehisa
Published: March 2005

We have previously determined the amino acid sequence of porcine soluble angiotensin‐binding protein (sABP) by cDNA cloning and sequencing. In this study, we have cloned a sABP homologue (PABH) from the same porcine cDNA libraries used for sABP cloning. PABH and sABP have 65% sequence identity. Sequence comparisons with other proteins revealed very high similarities between porcine PABH and rat thimet oligopeptidase (90%), and between porcine sABP and rabbit microsomal endopeptidase (93%). This suggests that PABH and thimet oligopeptidase are identical and that sABP and microsomal endopeptidase are also the same. Indeed, sABP was shown to have a peptidase activity that is sensitive to the metal‐chelating agents EDTA and 1,10‐phenanthroline; sABP was also sensitive to the thiol reagent p‐chloromercuriphenylsulfonic acid. RNase‐protection assays, using RNA preparations from various porcine tissues, indicated that thimet oligopeptidase mRNA is ubiquitously expressed whereas sABP mRNA is predominantly expressed in the liver, kidney and adrenal gland. This assay also revealed tissue‐specific alternative splicing of the sABP‐encoding message.
Biochemical characterisation of purified human wild‐type p53 overexpressed in insect cells
Authors: CHALKLEY Gillian E. , KNOWLES Phillip P. , WHITEHEAD Philip C. , COFFER Arnold I.
Published: March 2005

Conditions for the overexpression of human wild‐type p53 using a baculovirus construct were optimised in insect cells which produced up to 20 mg p53/l culture. Milligram amounts of p53 were purified to apparent homogeneity using chromatography on double‐stranded DNA‐cellulose (∼58% yield) followed by immunoaffinity chromatography with an epitope elution step (up to 48% yields) at 4°C. The Mr of extracted p53 both from insect cell lysates and after purification was 54000 by SDS/PAGE. Isoelectric focusing showed recombinant p53 to be an acidic protein, focusing at pI 6.0 under non‐denaturing conditions. Expressed p53 at all stages of purification reacted by immunoblotting with specific p53 monoclonal antibodies, indicating the presence of intact epitopes at the C‐terminus, N‐terminus and central region of the protein. From ultracentrifugation studies, pure p53 exhibited significant oligomerisation, and sedimented broadly within the 7–12‐S region of sucrose gradients. Pure p53 slowly precipitated out of solution at concentrations between 1–6 mg/ml even in the presence of 1% detergent. Using metal affinity chromatography, we have established that pure p53 binds the immobilised divalent ions Zn2+, Ni2+ and Co2+ with high affinity.
Homeobox‐containing genes in the most primitive metazoa, the sponges
Authors: SEIMIYA Makiko , ISHIGURO Hiroshi , MIURA Keiji , WATANABE Yoko , KUROSAWA Yoshikazu
Published: March 2005

The porifera represent the most primitive phylum of the metazoa. We identified three homeobox‐containing genes in the freshwater sponge (Ephydatia fluviatilis). Genomic DNA of the sponge was subjected to amplification by PCR with two primers that corresponded to the helix‐1 and helix‐3 regions of the homeodomain. Using the amplified products as probes, we isolated two homeobox genes, designated proxl and prox2. The amino acid sequences of the homeodomains of prox1 and prox2 were 72% and 62% identical to those of the NK‐3 and Om(1 D) genes of Drosophila, respectively. Screening of a sponge genomic library with degenerate oligonucleotides that corresponded to helix 3 further revealed the presence of one more homeobox gene, prox3. The amino acid sequence of the homeodomain of the prox3 product was 77% identical to that of the msh gene product of human. These results indicate that, when the metazoa appeared during the course of evolution, the multiple and distinct classes of homeobox‐containing genes that have been identified in higher organisms already existed.
Differential effect of p53 on the promoters of mouse DNA polymerase β gene and proliferating‐cell‐nuclear‐antigen gene
Authors: YAMAGUCHI Masamitsu , HAYASHI Yuko , MATSUOKA Shuhei , TAKAHASHI Takashi , MATSUKAGE Akio
Published: March 2005

A plasmid carrying the 5′ flanking region of the mouse proliferating‐cell‐nuclear‐antigen (PCNA) gene or DNA polymerase β gene was fused with the chloramphenicol acetyltransferase (CAT) gene, then cotransfected into mouse N18TG2 cells with the expression plasmid for the p53 gene. Expression of the wild‐type p53 repressed the CAT expression directed by the PCNA gene promoter, while it had little effect on the DNA polymerase β gene promoter. RNase protection analysis revealed that the repression of the PCNA gene promoter by p53 was at the transcription step. Analysis with various deletion mutants in the PCNA gene promoter revealed that a specific sequence is not required for the repression, suggesting that p53 represses the PCNA gene promoter by interacting with some components of the basic transcription machinery. By analysis with various deletion mutants in the DNA polymerase β gene promoter, we identified the unique 10‐bp palindromic sequence (−24 to −15), in the presence of which p53 was not able to repress the promoter activity. This sequence conferred resistance to p53 repression onto the PCNA gene promoter, when it was placed 21‐bp upstream from the transcription‐initiation site.
Role of the C‐terminus of Saccharomyces cerevisiae ubiquitin‐conjugating enzyme (Rad6) in substrate and ubiquitin‐protein‐ligase (E3‐R) interactions
Authors: RABOY Bilha , KULKA Richard G.
Published: March 2005

The product of the RAD6 (UBC2) gene of Saccharomyces cerevisiae is a ubiquitin‐conjugating enzyme (Rad6) which is implicated in DNA repair, induced mutagenesis, retrotransposition, sporulation and the degradation of proteins with destabilizing N‐terminal amino acid residues. Deletion of the 23‐residue acidic C‐terminus of Rad6 impairs sporulation and N‐end rule protein degradation in vivo but does not affect other functions such as DNA repair and induced mutagenesis. We have investigated the role of the C‐terminus of Rad6 in in vitro interactions with various substrates and with a putative ubiquitin‐protein ligase, E3‐R. The removal of the Rad6 C‐terminus had significant different effects on enzyme activity for individual substrates. Although the 23‐residue truncated Rad6‐149 protein had markedly impaired activity for histone H2B and micrococcal nuclease, the activity for cytochrome c was the same as that of the intact Rad6 protein. Similarly, truncation of Rad6 had no effect on its activity for several poor substrates, namely, β‐casein, β‐lactoglobulin and oxidized RNase. E3‐R stimulated the activities of both Rad6 and Rad6‐149 for the latter three substrates to similar degrees. E3‐R appears to act by enhancing the low intrinsic affinity of Rad6 and Rad6‐149 for these substrates. Thus Rad6 can act in three different modes in vitro depending on the substrate, namely unassisted C‐terminus‐dependent, unassisted C‐terminus‐independent and E3‐R‐assisted C‐terminus‐independent modes. We also examined the results of removing the C‐terminal acidic region of Cdc34 (Ubc3), a ubiquitin‐conjugating enzyme closely related to Rad6. Truncation of Cdc34 like that of Rad6 had no effect on activity for β‐casein, β‐lactoglobulin or oxidized RNase in the presence or absence of E3‐R.
Three‐dimensional structure of the higher‐plant photosystem II reaction centre and evidence for its dimeric organization in vivo
Authors: SANTINI Caterina , TIDU Valentino , TOGNON Giuseppe , GHIRETTI MAGALDI Anna , BASSI Roberto
Published: March 2005

The three‐dimensional structure of photosystem II (PSII) has been determined by conventional transmission electron microscopy and computerized three‐dimensional reconstruction. Both the complete system and that lacking the oxygen‐evolving complex have been analyzed. The PSII complex has a four‐lobed structure with twofold symmetry. An estimate of the molecular mass and the results of Deriphat/PAGE analysis suggest that a reaction centre is present in each half of the structure resolved by electron microscopy. Stepwise removal of components of the complex showed that the removal of CP47 (a 47‐kDa chlorophyll‐protein complex) induces monomerization of PSII, which indicates the importance of this subunit for the dimeric structure.
The serum‐induced phosphorylation of mammalian hsp27 correlates with changes in its intracellular localization and levels of oligomerization
Authors: MEHLEN Patrick , ARRIGO André‐Patrick
Published: March 2005

The oligomeric small heat‐shock protein hsp27, also denoted hsp28, is constitutively expressed in several mammalian cells and displays a phosphorylation status that is related to cellular growth and differentiation. This protein is related to α‐crystallin and has strong sequence similarity with an in vitro inhibitor of actin polymerization. Here, we have analyzed hsp27 phosphorylation, cellular localization and structural organization following serum stimulation of serum‐starved HeLa cells. hsp27 is dephosphorylated in starved cells and quantitatively recovered in the form of small structures (<200 kDa) present in the soluble phase of the cytoplasm. Immediately after the addition of serum to starved cells, a rapid phosphorylation and complex changes in the intracellular distribution and structural organization of hsp27 are observed. Phosphorylation essentially occurs at the level of small hsp27 structures (<200 kDa) and is concomitant with the increased molecular mass (up to 700 kDa) of a fraction of this protein. Serum treatment also induced the detergent‐sensitive association of another fraction of hsp27, still in the form of small and dephosphorylated structures, with cellular particulate fractions. Contrasting with these observations, hsp70 had the tendency to concentrate into nucleoli during serum starvation.
The multidrug‐resistance‐reverser verapamil interferes with cellular P‐glycoprotein‐mediated pumping of daunorubicin as a non‐competing substrate
Authors: SPOELSTRA Ellen C. , WESTERHOFF Hans V. , PINEDO Herbert M. , DEKKER Henk , LANKELMA Jan
Published: March 2005

We examined P‐glycoprotein‐mediated verapamil transport, using two drug‐sensitive and multidrug resistant cell‐line couples, i.e. A2780, 2780AD and SW‐1573, SW‐1573/1R500. The interaction of 3H‐labeled verapamil with cells was measured using a flow‐through system. The verapamil‐containing medium was pumped over the cells and monitored on‐line for radioactivity. In the P‐glycoprotein‐expressing cells, verapamil accumulation was increased by vinblastine and some known multidrug resistant (MDR) modifiers. Subsequent removal of these modifiers caused release of verapamil into the medium against a verapamil concentration gradient. In this manner, we obtained evidence that verapamil is actively transported by the MDR‐related P‐glycoprotein.

Using the flow‐through system, we also exposed the cells to flowing culture medium containing daunorubicin, and measured the inhibition of daunorubicin efflux by verapamil. We found that, although the active efflux of daunorubicin was maximally blocked by verapamil short‐term, longer‐term active efflux of daunorubicin resumed. At a daunorubicin concentration in the flowing medium of 5 μM, increasing the verapamil concentration resulted in the same short‐term effects, but in a significantly longer period of a maximal inhibition of daunorubicin efflux from the cells. At a daunorubicin concentration of 20 μM, increasing the verapamil concentration affected neither the short‐term nor the long‐term effects. These and other observations are in agreement with a model in which daunorubicin and verapamil are non‐competing substrates for P‐glycoprotein.

In conclusion, we obtained evidence that verapamil is actively transported by the MDR‐related P‐glycoprotein and that verapamil and daunorubicin are non‐competing substrates for P‐glycoprotein. Consequently, the effectiveness of verapamil as an MDR antagonist may be compromised because it is extruded by P‐glycoprotein.