bluuebunny/crossref_metadata_2025_split huggingface dataset:
The interferon‐stimulable response elements of two human genes detect overlapping sets of transcription factors
Authors: PARRINGTON John , ROGERS Neil C. , GEWERT Dirk R. , PINE Richard , VEALS Susan A. , LEVY David E. , STARK George R. , KERR Ian M. 
Published: March 2005

We have previously reported three types of DNA‐protein complexes, formed specifically with the interferon‐stimulable response elements (ISRE) in the 5′ flanking DNA of the interferon‐inducible 6–16 and 9–27 genes, a type‐I interferon‐Inducible early complex involving factor E (ISGF3), M and G complexes induced more slowly in response to type‐I and type‐II interferons, respectively and C1/C2, a constitutive complex(s). Similar complexes have been reported by others. The operationally defined band‐shift complexes M, G and C1/C2 are shown here to be heterogeneous and to differ in their factor content, depending on the ISRE probe. With a 9–27 ISRE probe the M, G and C1/C2 complexes all contain the γ subunit of ISGF3, which is present constitutively but is induced in response to IFN‐α (to yield M) or IFN‐γ (to yield G). In contrast, a 6–16 ISRE probe forms band‐shift complexes with IFN‐α‐inducible and IFN‐γ‐inducible IRF1 and IRF2. With a 6–16 ISRE probe, therefore, M and G each correspond to two complexes which co‐migrate in band‐shift assays, one corresponding to IRF1, the other to IRF2. With this probe, the constitutive complex C1/C2 corresponds predominantly to IRF2. Consistent with this, IRF1 and IRF2 have lower affinity for the 9–27 ISRE than the 6–16 ISRE, whereas the reverse is true for E (ISGF3) and its γ subunit. Relatively small differences in affinity appear sufficient to determine whether or not a band‐shift complex is detected. In the case of IRF1 and IRF2, the different affinities for the 6–16 and 9–27 probes are dominated by a dinucleotide sequence in the centre of the 14‐nucleotide ‘core’ ISRE. In contrast, preferential binding of E (ISGF3) by the 39‐nucleotide 9–27 ISRE‐containing sequence, although ISRE dependent, appears to be mediated by sequences 3′ of the ‘core’ ISRE. Accordingly, these complexes can be simultaneously assayed using a hybrid probe consisting of the 5′ flanking region and ‘core’ ISRE sequences from the 6–16 gene and sequences immediately 3′ of the ‘core’ 9–27 ISRE sequence. No evidence was obtained for a modulatory role in factor binding for a pseudo‐ISRE sequence close to ISRE in the 9–27 gene.The precise roles of IRF1 and IRF2 in the induction of IFN‐β and the control of interferon‐inducible gene expression remain to be established. Results from the analysis of the expression of IRF1 and IRF2 in wild‐type and mutant cells argue against a dominant negative role for unmodified IRF2.
Chemical structure of the lipid A of Escherichia coli J‐5
Authors: HOLST Otto , MÜLLER‐LOENNIES Sven , LINDNER Buko , BRADE Helmut 
Published: March 2005

The lipopolysaccharide, and particularly its lipid A moiety, of the J‐5 mutant of Escherichia coli O111 plays a central role in studies on potential induction of cross‐reactive and cross‐protective antibodies, however, its chemical and antigenic structure was hitherto unknown. Here, the chemical structure of the J‐5 lipid A is reported. It is composed of the bisphosphorylated disaccharide β‐d‐GlcpN‐4‐P‐(1‐6)‐α‐d‐GlcpN‐1‐P which carries four residues of 3‐hydroxytetradecanoic acid, one each at positions 2, 3, 2′, and 3′. The hydroxyl groups of the acyl residues at 2′ and 3′ are esterified with dodecanoic and tetradecanoic acid, respectively. The hydroxyl group at C‐6′ functions in the lipopolysaccharide as the attachment site of the core oligosaccharide.Furthermore, a new method to isolate the hydrophilic backbone, i.e. the 1,4′‐bisphosphorylated glucosamine disaccharide, and its structural analysis by 1H‐, 13C‐, and 31P‐NMR spectroscopy, are described, leading to a new and easier strategy in structural analysis of lipid A from bacterial lipopolysaccharides.
Single amino‐acid replacement is responsible for the stabilization of ornithine decarboxylase in HMOA cells
Authors: MIYAZAKI Youichi , MATSUFUJI Senya , MURAKAMI Yasuko , HAYASHI Shin‐ichi 
Published: March 2005

The half‐life of ornithine decarboxylase (ODC) in HMOA cells, a variant cell line derived from hepatoma tissue culture (HTC) cells, is markedly increased compared with that in the parental cell line. In the present study, we examined which of the three relevant factors is responsible for the ODC stabilization in HMOA cells, namely ODC itself, a regulatory protein antizyme and an ODC‐degrading activity.SDS/PAGE analysis of radiolabeled ODC revealed that ODC from HMOA cells migrated somewhat faster than that from HTC cells, suggesting that HMOA ODC was structurally altered. Direct sequencing of reverse‐transcription/polymerase‐chain‐reaction (RT‐PCR) products of ODC mRNA from HMOA cells revealed a T to G replacement, causing a Cys441→Trp replacement near the C‐terminus. No alteration was found in the whole coding region of antizyme mRNA. An authentic mutant ODC cDNA with the same replacement was transfected and expressed in C55.7 ODC‐deficient Chinese hamster ovary cells. Upon cycloheximide treatment, the mutant ODC activity did not decrease appreciably for at least 3 h, whereas wild‐type ODC activity decreased with a half‐life of 1 h. In‐vitro‐synthesized mutant ODC with the Cys441→Trp (or Ala) replacement was also stable in a reticulocyte‐lysate ODC‐degradation system. Metabolically labeled and purified mouse ODC was degraded in HMOA cell extracts in the presence of ATP and antizyme as rapidly as in HTC cell extracts, indicating that HMOA cells have a normal ODC degrading activity. These results indicated that the single amino acid replacement, Cys441→Trp, is responsible for the stabilization of ODC in HMOA cells and that Cys441 is important for rapid ODC turnover.
The glucagon‐insulin antagonism in the regulation of cytosolic protein binding to the 3′ end of phosphoenolpyruvate carboxykinase mRNA in cultured rat hepatocytes Possible involvement in the stabilization of the mRNA
Authors: CHRIST Bruno , NATH Annegret 
Published: March 2005

Since protein binding to the 3′ end of mRNA is believed to be involved in the control of mRNA stability, the time course of alterations in glucagon‐induced phosphoenolpyruvate‐carboxykinase‐mRNA (PCK) levels, in the absence and presence of insulin, was correlated with the time course of changes in the binding of cytosolic protein from 24‐h cultured rat hepatocytes to the 3′ end of PCK mRNA. PCK‐mRNA levels were monitored by Northern blot analysis and protein binding was analyzed by an electrophoretic mobility‐shift assay.In 24‐h cultured rat hepatocytes, binding of cytosolic protein to the PCK‐mRNA 3′ end and PCK‐mRNA levels were increased to a transient maximum at 2 h and 2–4 h, respectively, by a 1‐nM glucagon treatment, added with a change of medium. 100 nM insulin, added simultaneously with glucagon, reduced the glucagon‐induced maximum of protein binding by 80% and the increase of PCK mRNA by about 30%. In controls without hormonal treatment protein binding at 1 h was also increased; this increase was prevented by insulin.100 nM insulin, added 1 h after glucagon, reversed protein binding to the 3′ end of PCK mRNA to nearly initial levels within 1 h and impaired the glucagon‐induced increase in PCK‐mRNA levels by 30%.The transcriptional inhibitor cordycepin, added 1 h after glucagon, did not prevent the further increase in glucagon‐enhanced protein binding nor its reversal by insulin. It did, however, prevent a further significant increase in PCK mRNA.Hormonally regulated protein binding could be localized to the 256 distal bases of the PCK‐mRNA 3′ end. The proximal 466 bases of the PCK‐mRNA 3′ end as well as the 1050 bases of the histone‐H1°‐mRNA 3′ end and the 1200 bases of the arylsulfatase‐A‐mRNA 3′ end also bound cytosolic protein(s), but this protein binding was not altered by treatment with glucagon or insulin.The 3′ end of PCK, arylsulfatase A and H1° mRNA exhibited strong binding of cytosolic protein(s) from diverse rat tissues such as heart, liver and lung as well as Fao rat hepatoma cells. Cytosolic protein(s) from spleen showed weak binding and proteins from HeLa and U937 tumor cells did not bind. Protein binding was most prominent with the 3′ end of PCK mRNA and cytosolic extracts from liver.The results support the hypothesis that the induction by glucagon and reversal by insulin of both protein binding to the 3′ end of PCK mRNA and the increase in PCK‐mRNA levels are correlated. This correlation suggests a functional relationship, possibly the regulation of mRNA stability by protein/mRNA interactions. Regulation of protein binding by glucagon and insulin is specific for the 3′ end of PCK mRNA and occurs possibly via covalent modification of the binding protein.
Cholesterol 7α‐hydroxylase is up‐regulated by the competitive inhibitor 7‐oxocholesterol in rat liver
Authors: BREUER Olof , SUDJANA‐SUGIAMAN Elly , EGGERTSEN Gösta , CHIANG John Y. L. , BJÖRKHEM Ingemar 
Published: March 2005

Rats of the Sprague‐Dawley strain were infused intravenously with a fat emulsion (Intralipid, trademark of Kabi Pharmacia, Uppsala, Sweden) containing 7‐oxocholesterol. This resulted in an increased cholesterol 7α‐hydroxylase activity in liver microsomes as compared to controls and was accompanied by increased levels of cholesterol 7α‐hydroxylase mRNA and microsomal cholesterol 7α‐hydroxylase protein. Rats were also fed a cholestyramine‐supplemented diet and infused with 7‐oxocholesterol. These animals excreted about half as much bile acids in faeces as cholestyraminefed controls.Addition of 7‐oxocholesterol to liver microsomes from normal rats in amounts corresponding to those present in microsomes from 7‐oxocholesterol‐treated rats inhibited the cholesterol 7α‐hydroxylase activity by about 75%.Cholesterol induced a type‐I binding spectrum when added to a purified bacterial‐expressed cholesterol 7α‐hydroxylase (P‐450c72–24). 7‐Oxocholesterol competitively inhibited the cholesterol binding spectrum, while 7β‐hydroxycholesterol did not interfere with binding of cholesterol to the enzyme.It is concluded that treatment with the competitive inhibitor 7‐oxocholesterol leads to a reduced bile acid biosynthesis and, as a consequence of reduced bile acid inhibition, a compensatory increase in cholesterol 7α‐hydroxylase synthesis. The high enzyme activity measured in microsomal preparations from 7‐oxocholesterol‐treated rats may be due to a continuous conversion of 7‐oxocholesterol into less inhibitory metabolites, e.g. 7β‐hydroxycholesterol. The latter compound was found in high concentrations in liver microsomes from rats treated with 7‐oxocholesterol.The physiological importance of these results is discussed in relation to the previous findings that 7‐oxocholesterol is accumulated in liver after cholesterol feeding and that 7‐oxocholesterol is formed from cholesterol during lipid peroxidation.
Human α and β parvalbumins
Authors: FÖHR Ursula G. , WEBER Barbara R. , MÜNTENER Markus , STAUDENMANN Werner , HUGHES Graham J. , FRUTIGER Séverine , BANVILLE Denis , SCHÄFER Beat W. , HEIZMANN Claus W. 
Published: March 2005

α and β parvalbumins are Ca2+‐binding proteins of the EF‐hand type. We determined the protein sequence of human brain α parvalbumin by mass spectrometry and cloned human β parvalbumin (or oncomodulin) from genomic DNA and preterm placental cDNA. β parvalbumin differs in 54 positions from α parvalbumin and lacks the C‐terminal amino acid 109. From MS analyses of α and β parvalbumins we conclude that parvalbumins generally lack posttranslational modifications.α and β parvalbumins were differently expressed in human tissues when analyzed by immunoblotting and polymerase‐chain‐reaction techniques. Whereas α parvalbumin was found in a number of adult human tissues, β parvalbumin was restricted to preterm placenta. The pattern of α parvalbumin expression also differs in man compared to other vertebrates. For example, in rat, α parvalbumin was found in extrafusal and intrafusal skeletal‐muscle fibres whereas, in man, α parvalbumin was restricted to the muscle spindles. Different functions for α and β parvalbumins are discussed.
The anatomy of a kinase and the control of phosphate transfer
Authors: JOÃO Heidi C. , WILLIAMS Robert J. P. 
Published: March 2005

Table of contents Basic features of phosphoglycerate kinase (PGK), The sequences and crystal structures of PGK NMR studies of the structure of the protein PGK, Thermal analysis of yeast PGK, Additional data from NMR studies Substrate binding, Nucleotide binding site: MgATP and MgADP, The function of magnesium, The triose substrate binding site, NMR analysis of substrate binding: summary, General anion‐binding site The conformation change, Kinetic studies of the reaction, Site‐specific mutagenesis studies Effects of mutations on protein structure in the absence of substrates, Mutations in the interdo‐main region, Mutations in the basic patch Effect of Gri3P binding on mutant proteins, Mutations in the interdomain region, Mutations in the basic patch Effects of mutations on kinetics of PGK, Mutations in the interdomain region, Mutations in the basic patchConclusions from experiments with mutants Kinetic effects of low sulphate concentration, Interdomain region, Basic patch region The so‐called hinge of proteins, Features of PGK Common features with other NTP‐utilising systems, The nucleotide‐binding site, The catalytic loop, Domain closure Comments on mechanical devices in multi‐protein enzymes
Sweet potato β‐amylase
Authors: TODA Hiroko , NITTA Yasunori , ASANAMI Shogo , KIM Jun Pyong , SAKIYAMA Fumio 
Published: March 2005

The complete amino acid sequence of a subunit of sweet potato β‐amylase, a homotetramer, was established by sequence analysis of peptides obtained by digestions with Achromobacter protease I and Staphylococcus aureus V8 protease and by cyanogen bromide cleavage of the S‐carboxyme‐thylated subunit. The subunit of the enzyme is a single polypeptide consisting of 498 amino acid residues. It showed 50–60% identity in the amino acid sequence with those of β‐amylases from soybean and barley, while it about 25% with those of three bacterial β‐amylases deduced from the cDNA sequences.Sweet potato β‐amylase was completely inactivated with 2,3‐epoxypropyl α‐d‐[U‐14C]glucopyranoside. Sequence analysis of the inactivated enzyme revealed that Glu187 was specifically esterified by the affinity labeling with the above reagent, proposing that Glu187 is a potent candidate involved directly in the catalysis with this plant β‐amylase.
Prosomatostatin processing in Neuro2A cells
Authors: BRAKCH Noureddine , BOILEAU Guy , SIMONETTI Mario , NAULT Christiane , JOSEPH‐BRAVO Patricia , RHOLAM Mohamed , COHEN Paul 
Published: March 2005

Proline residues located near the processing sites of human prosomatostatin were previously shown to be important for cleavage of the precursor into somatostatin 28 and somatostatin 14 [Gomez, S., Boileau, G., Zollinger, L., Nault, C., Rholam, M. & Cohen, P. (1989) EMBO J. 8, 2911–2916]. In this study, site‐directed and regional mutagenesis of the human prosomatostatin cDNA coupled with analysis by circular‐dichroism and Fourier‐transform‐infrared spectroscopies of the native and mutated peptide sequences were used to elucidate the role of proline in proteolytic processing. Glycine was substituted for proline a position –5 and the β‐turn‐promoting sequence Pro‐Arg‐Glu‐Arg, located near the somatostatin‐14 cleavage site and predicted to form a β‐turn structure, was replaced by Ser‐Ser‐Asn‐Arg or Tyr‐Lys‐Gly‐Arg, which have been shown by X‐ray diffraction to form β turns in other proteins. Analysis of the prosomatostatin‐derived peptides produced by expression of the mutated cDNA species in Neuro2A cells indicated that while Pro‐5 → Ala abolished cleavage at the dibasic site, the formation of mutants [Gly−5] prosomatostatin, [Ser−5, Ser−4, Arg−3] prosomatostatin and [Tyr−5, Lys−4, Gly−3] prosomatostatin did not affect cleavage at the dibasic site but produced modifications in both the relative proportions of the generated hormones and in precursor processing efficiency. Moreover, spectroscopical analysis showed that whereas these substitutions did not modify the presence of a β turn structure in the corresponding peptide sequences, replacement of Pro‐5 Ala resulted in a dramatic increase in α‐helix accompanied by the significant decrease of other structures including β turn.The data support the hypothesis that the proline residue near the processing site for somatostatin‐14 production is an important structural feature for conferring on the cleavage domain the adequate conformation for accessibility to processing enzymes and permitting production of equivalent amounts of both hormones.
Basic features of class‐I alcohol dehydrogenase: variable and constant segments coordinated by inter‐class and intra‐class variability
Authors: PERSSON Bengt , BERGMAN Tomas , KEUNG Wing Ming , WALDENSTRÖM Ulrika , HOLMQUIST Barton , VALLEE Bert L. , JÖRNVALL Hans 
Published: March 2005

The enzymatic and structural properties of alligator liver alcohol dehydrogenase have been determined. Aliphatic and alicyclic alcohols serve as substrates for this first reptilian form of the enzyme characterized, with Km values decreasing rapidly from methanol to hexanol, as for the human class I enzymes, and a Km of 1.2 mM for ethanol at pH 9.9. The N‐terminus of the 374‐residue protein chain is acetyl‐blocked.The enzyme is related in descending order to class I > III > V > II of the structurally characterized mammalian alcohol dehydrogenases. This observation is compatible with the presence of a I/III ancestral line. Differences of the enzyme classes exceed those of the species, suggesting an early origin of the classes. Within its enzyme class, the reptilian protein is most closely related to the avian from (82% residue identities), and is closer to the human than to the amphibian form (76%, versus 69%, respectively). This establishes class I alcohol dehydrogenase as an enzyme having fairly constant rate of change during much of vertebrate evolution, ∼10% residue differences/100 million years of separation between pairs compared.Residues interacting with the substrate and coenzyme are largely conserved. In the alligator enzyme, there are only four replacements in the substrate pocket compared with the human class I γ subunit, and those are not known to have functional roles. These properties account for the kinetic parameters, and suggest distinct metabolic functions for the class I enzyme in vertebrates.Comparisons of the enzymes of the different vertebrate lines reveal that segment patterns are characteristic features of the class I enzymes. Three segments are ‘variable’, while two are ‘constant’, and both these types of segment are identical with those of the classes. There is extensive variability in close proximity to the active site of the enzyme and this appears to constitute a fundamental property of class I liver alcohol dehydrogenases in general.
The proteolytic susceptibility of specific sites in myosin light chains is modulated by the filament conformation
Authors: ROULET Agnès , BURGAT Jean‐Marc , CARDINAUD Robert 
Published: March 2005

The proteolytic susceptibilities of specific sites in the LC1 and LC2 N‐termini were modulated by ionic strength in myosin (a species able to form filaments) but not in S1. (a) In the presence of Ca2+ or Mg2+, the proteolytic susceptibility (apparent initial reaction rate) showed a sharp discontinuity at a critical ionic concentration similar for LC1′, LC2′ and LC2” cleavages. (b) The susceptibility of LC1′ and LC2” was higher at low ionic concentration in the more compact structure of the filament than in the dissociated form at high ionic concentration. (c) The ionic concentration effect was no longer observed with species unable to form filaments. (d) This effect occurred at a critical ionic concentration markedly different from the critical concentration at which the monomer—filament equilibrium was found.These observations lead to the following conclusions. (a) The ionic concentration effect is an attribute of the filament structure. (b) In the filament the faster cleavage at sites (LC1′ and LC2”) near the LC1 and LC2 N‐termini are due to an extended configuration of the N‐terminal segment binding to a site in the filament structure. (c) The slower rate of formation of LC2′ in the filament indicates that the N‐terminal segment of LC2 binds more tightly to the structure than that of LC1. (d) The critical ionic concentration is not that of the filament—monomer equilibrium but corresponds to the order—disorder transition of the heads in the filament. These results suggest that the N‐termini of the light chains (here in striated muscles) play a role in a secondary regulatory mechanism. The analysis of these regions may contribute to our understanding of the altered activity and regulation seen in such diseases as idiopathic dilated cardiomyopathy [Margossian, S. S., White, H. D., Caulfield, J. B., Norton, P., Taylor, S. & Slayter, H. S. (1992) Circulation 85, 1720–1733].
Role of Tyr201 and Tyr385 in substrate activation by p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens
Authors: ESCHRICH Klaus , van der BOLT Frank J. T. , de KOK Arie , van BERKEL Willem J. H. 
Published: March 2005

The crystal structure of the enzyme‐substrate complex of p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens shows that the hydroxyl group of 4‐hydroxybenzoate interacts with the side chain of Tyr201, which is in close contact with the side chain of Tyr385. The role of this hydrogen bonding network in substrate activation was studied by kinetic and spectral analysis of Tyr→Phe mutant enzymes.The catalytic properties of the enzymes with Tyr201 or Tyr385 replaced by Phe (Tyr201→Phe and Tyr385→Phe) with the physiological substrate are comparable with those of the corresponding mutant proteins of p‐hydroxybenzoate hydroxylase from P. aeruginosa [Entsch, B., Palfey, B. A., Ballou, D. P. & Massey, V. (1991) J. Biol. Chem. 266, 17341–17349]. Enzyme Tyr201→Phe has a high Km for NADPH and produces only 5% of 3,4‐dihydroxybenzoate/catalytic cycle. Uniike the wild‐type enzyme, the Tyr201→Phe mutant does not stabilize the phenolate form of 4‐hydroxybenzoate. With enzyme Tyr385→Phe, flavin reduction is rate‐limiting and the turnover rate is only 2% of wild type. Despite rather efficient hydroxylation, and deviating from the description of the corresponding P. aeruginosa enzyme, mutant Tyr385→Phe prefers the binding of the phenolic form of 4‐hydroxybenzoate.Studies with substrate analogs show that both tyrosines are important for the fine tuning of the effector specificity. Binding of 4‐fluorobenzoate differentially stimulates the stabilization of the 4a‐hydroperoxyflavin intermediate. Unlike wild type, both Tyr mutants produce 3,4,5‐trihydroxybenzoate from 3,4‐dihydroxybenzoate. The affinity of enzyme Tyr201→Phe for the dianionic substrate 2,3,5,6‐tetrafluoro‐4‐hydroxybenzoate is very low, probably because of repulsion of the substrate phenolate in a more nonpolar microenvironment.In contrast to data reported for p‐hydroxybenzoate hydroxylase from P. aeruginosa, binding of the inhibitor 4‐hydroxycinnamate to wild‐type and mutant proteins is not simply described by binary complex formation. A binding model is presented, including secondary binding of the inhibitor. Enzyme Tyr201→Phe does not stabilize the phenolate form of the inhibitor. In enzyme Tyr385→Phe, the phenolic pKa of bound 4‐hydroxycinnamate is increased with respect to wild type. It is proposed that Tyr385→Phe is involved in substrate activation by facilitating the deprotonation of Tyr201.