bluuebunny/crossref_metadata_2025_split huggingface dataset:
The HIV‐1Lai RNA dimerization
Authors: Theilleux‐Delalande Valérie , Girard Frédéric , Huynh‐Dinh Tam , Lancelot Gérard , Paoletti Jacques 
Published: March 2003

Retroviruses contain dimeric RNA consisting of two identical copies of the genomic RNA. The interaction between these two RNA molecules occurs near their 5′ ends. A region upstream from the splice donor comprising an auto‐complementary sequence has been identified as being responsible for the initiation of the formation of dimeric HIV‐1Lai RNA. This region (SL1), part of the PSI encapsidation domain, can adopt a stem–loop structure. It has already been shown that this stem–loop structure can initiate the formation of two distinct dimers differing in their thermostability: a loop–loop dimer or ‘kissing complex’ and an extended dimer.We report here a study using UV and 1D NMR spectroscopy of the dimerization of a short oligoribonucleotide (23 nucleotides) spanning nucleotides 248–270 of the HIV‐1Lai SL1 sequence, in order to derive the thermodynamic parameters associated with the transition from the loop–loop complex to the extended dimer. The temperature dependence of the UV absorbency shows an hypochromicity for this transition with a small enthalpy change equal to – 29.4 ± 5 kcal·mol−1, together with a concentration independent transition which implies a monomolecular reaction. On the other hand, our NMR results don’t indicate a dissociation of the GCGCGC sequence engaged in the loop–loop interaction during the rearrangement of the loop–loop complex into the extended dimer. Our data suggest that the loop–loop interaction is maintained during the temperature dependent conformational change while the intramolecular base‐pairing of the stems is disrupted and then reconstituted to form an intermolecular base‐pairing leading to an extended dimer.
Solution structure of a neurotrophic ligand bound to FKBP12 and its effects on protein dynamics
Authors: Sich Christian , Improta Sabina , Cowley David John , Guenet Chantal , Merly Jean‐Pierre , Teufel Michael , Saudek Vladimir 
Published: March 2003

The structure of a recently reported neurotrophic ligand, 3‐(3‐pyridyl)‐1‐propyl(2S)‐1‐(3,3‐dimethyl‐1,2‐dioxopentyl)‐2‐pyrrolidinecarboxylate, in complex with FKBP12 was determined using heteronuclear NMR spectroscopy. The inhibitor exhibits a binding mode analogous to that observed for the macrocycle FK506, used widely as an immunosuppressant, with the prolyl ring replacing the pipecolyl moiety and the amide bond in atransconformation. However, fewer favourable protein–ligand interactions are detected in the structure of the complex, suggesting weaker binding compared with the immunosuppressant drug. Indeed, a micromolar dissociation constant was estimated from the NMR ligand titration profile, in contrast to the previously published nanomolar inhibition activity. Although the inhibitor possesses a remarkable structural simplicity with respect to FK506,15N relaxation studies show that it induces similar effects on the protein dynamics, stabilizing the conformation of solvent‐exposed residues which are important for mediating the interaction of immunophilin/ligand complexes with molecular targets and potentially for the transmission of the neurotrophic action of FKBP12 inhibitors.
Structural analysis of the lipopolysaccharide from Chlamydophila psittaci strain 6BC
Authors: Rund Sabine , Lindner Buko , Brade Helmut , Holst Otto 
Published: March 2003

The lipopolysaccaride of Chlamydophila psittaci 6BC was isolated from tissue culture‐grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. Compositional analyses indicated the presence of 3‐deoxy‐
dmanno‐oct‐2‐ulosonic acid, GlcN, organic bound phosphate and fatty acids in a molar ratio of ≈ 3.3 : 2 : 1.8 : 4.6. Deacylated lipopolysaccharide was obtained after successive microscale treatment with hydrazine and potassium hydroxide, and was then separated by high performance anion‐exchange chromatography into two major fractions, the structures of which were determined by 600 MHz NMR spectroscopy as α‐Kdo‐(2→8)‐α‐Kdo‐(2→4)‐α‐Kdo‐(2→6)‐β‐
d‐GlcpN‐(1→6)‐α‐
d‐GlcpN 1,4′‐bisphosphate and α‐Kdo‐(2→4)‐[α‐Kdo‐(2→8)]‐α‐Kdo‐(2→4)‐α‐Kdo‐(2→6)‐β‐
d‐GlcpN‐(1→6)‐α‐
d‐GlcpN 1,4′‐bisphosphate. The distribution of fatty acids in lipid A was determined by compositional analyses and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry experiments on lipid A and de‐O‐acylated lipid A. It was shown that the carbohydrate backbone of lipid A is replaced by a complex mixture of fatty acids, including long‐chain and branched (R)‐configured 3‐hydroxy fatty acids, the latter being exclusively present in an amide linkage.
Evidence for an evolutionary conserved role of bone morphogenetic protein growth factors and Phox2 transcription factors during noradrenergic differentiation of sympathetic neurons
Authors: Ernsberger Uwe 
Published: March 2003

The noradrenergic transmitter phenotype in postganglionic sympathetic neurons is induced early during embryonic development in avian and mammalian primary sympathetic ganglia. The simultaneous expression of tyrosine hydroxylase and dopamine β‐hydroxylase, enzymes of the noradrenaline biosynthesis pathway, indicates that different genes contributing to the noradrenergic transmitter phenotype are regulated as a synexpression group. This conclusion is supported by the demonstration of bone morphogenetic protein (BMP) growth factors and Phox2 transcription factors being necessary for the expression of both tyrosine hydroxylase and dopamine β‐hydroxylase in differentiating sympathetic neurons. The close similarity in the expression patterns of the relevant genes as well as in the function of BMPs and Phox2s between avian and mammalian embryos strongly suggests that noradrenergic induction occurs along a conserved signalling pathway in these vertebrate classes.
Three‐dimensional structure of the histidine‐containing phosphocarrier protein (HPr) from Enterococcus faecalis in solution
Authors: Maurer Till , Döker Rolf , Görler Adrian , Hengstenberg Wolfgang , Kalbitzer Hans R. 
Published: March 2003

The histidine‐containing phosphocarrier protein (HPr) transfers a phosphate group between components of the prokaryotic phosphoenolpyruvate‐dependent phosphotransferase system (PTS), which is finally used to phosphorylate the carbohydrate transported by the PTS through the cell membrane. Recently it has also been found to act as an intermediate in the signaling cascade that regulates transcription of genes related to the carbohydrate‐response system. Both functions involve phosphorylation/dephosphorylation reactions, but at different sites. Using multidimensional 1H‐NMR spectroscopy and angular space simulated annealing calculations, we determined the structure of HPr from Enterococcus faecalis in aqueous solution using 1469 distance and 44 angle constraints derived from homonuclear NMR data. It has a similar overall fold to that found in HPrs from other organisms. Four β strands, A, B, C, D, encompassing residues 2–7, 32–37, 40–42 and 60–66, form an antiparallel β sheet lying opposite the two antiparallel α helices, a and c (residues 16–26 and 70–83). A short α helix, b, from residues 47–53 is also observed. The pairwise root mean square displacement for the backbone heavy atoms of the mean of the 16 NMR structures to the crystal structure is 0.164 nm. In contrast with the crystalline state, in which a torsion angle strain in the active‐center loop has been described [Jia, Z., Vandonselaar, M., Quail, J.W. & Delbaere, L.T.J. (1993) Nature (London) 361, 94–97], in the solution structure, the active‐site His15 rests on top of helix a, and the phosphorylation site Nδ1 of the histidine ring is oriented towards the surface, making it easily accessible to the solvent. Back calculation of the 2D NOESY NMR spectra from both the NMR and X‐ray structures shows that the active‐center structure derived by X‐ray crystallography is not compatible with experimental data recorded in solution. The observed torsional strain must either be a crystallization artefact or represents a conformational state that exists only to a small extent in solution.
Analogs of the antimicrobial peptide trichogin having opposite membrane properties
Authors: Epand Raquel F. , Epand Richard M. , Formaggio Fernando , Crisma Marco , Wu Huiyuan , Lehrer Robert I. , Toniolo Claudio 
Published: March 2003

Four analogs of the antimicrobial peptide trichogin GA IV were studied. Their sequences are as follows: GT, n‐octanoyl‐Aib‐Gly‐Leu‐Aib‐Gly‐Gly‐Leu‐Aib‐Gly‐Ile‐Leu‐OMe; ST, n‐octanoyl‐Aib‐Ser‐Leu‐Aib‐Ser‐Ser‐Leu‐Aib‐Ser‐Ile‐Leu‐OMe; BT, n‐octanoyl‐Aib‐Ser(tBu)‐Leu‐Aib‐Ser(tBu)‐Ser(tBu)‐Leu‐Aib‐Ser(tBu)‐Ile‐Leu‐OMe; and DT, n‐octanoyl‐Aib‐Ser(tBu)‐Leu‐Aib‐Ser(tBu)‐Ser(tBu)‐Leu‐Aib‐Ser(tBu)‐Ile‐Leu‐Aib‐Ser(tBu)‐Leu‐Aib‐Ser(tBu)‐Ser(tBu)‐Leu‐Aib‐Ser(tBu)‐Ile‐Leu‐OMe. The trichogin GA IV differs from GT only in the nature of the C‐terminal residue, being a 1,2 aminoalcohol (leucinol) in the case of the parent peptide. Compared with GT, ST has an increased amphiphilicity. In contrast, BT has little amphiphilicity being composed only of hydrophobic amino acids. DT is an octanoylated head‐to‐tail dimer of BT. We show that BT and DT lower the bilayer‐to‐hexagonal phase transition temperature (TH) of dipalmitoleoylphosphatidylethanolamine, indicating that the peptides promote negative curvature. These two peptides, composed of only hydrophobic amino acids, have their bulkier groups on one face of the helix, suggesting that they may penetrate membranes at an oblique angle. In contrast, GT and ST, like trichogin itself, increase TH, promoting positive curvature. These peptides have contrasting membrane lytic activities. Whereas DT and BT did not produce leakage of aqueous contents, GT and ST, like trichogin, did cause rapid leakage. The leakage activity with liposomes also correlates with the greater potency of GT and ST, compared with the hydrophobic analogs, in their hemolytic and bacteriostatic action. ST has greater lytic ability than GT in liposomal leakage as well as hemolysis. We also measured the rate of peptide‐promoted lipid mixing as an indication of membrane fusion. BT produced lipid mixing only with large unilamellar vesicles enriched with dioleoylphosphatidylethanolamine; ST did not produce lipid mixing, as its apparent reduction of energy transfer proved to be artifactual. Quasi‐elastic light scattering of large unilamellar vesicles was also carried out after adding ST and BT. Peptide BT, but not ST, was able to aggregate large unilamellar vesicles. Thus, one of the properties of BT that leads to the induction of lipid mixing is that it is able to aggregate vesicles, placing the bilayers in juxtaposition. Thus, the two pairs of peptides, BT and DT vs GT and ST, exhibit contrasting behaviour with respect to a number of membrane biophysical properties. This occurs despite the fact that the chemical structures of the peptides are rather similar. Such distinct behavior is also reflected in their hemolytic and bacteriostatic actions.
Mutagenesis and modelling of linoleate‐binding to pea seed lipoxygenase
Authors: Hughes Richard K. , Lawson David M. , Hornostaj Andrzej R. , Fairhurst Shirley A. , Casey Rod 
Published: March 2003

We have produced a model to define the linoleate–binding pocket of pea 9/13‐lipoxygenase and have validated it by the construction and characterization of eight point mutants. Three of the mutations reduced, to varying degrees, the catalytic centre activity (kcat) of the enzyme with linoleate. In two of the mutants, reductions in turnover were associated with changes in iron‐coordination. Multiple sequence alignments of recombinant plant and mammalian lipoxygenases of known positional specificity, and the results from numerous other mutagenesis and modelling studies, have been combined to discuss the possible role of the mutated residues in pea 9/13‐lipoxygenase catalysis. A new nomenclature for recombinant plant lipoxygenases based on positional specificity has subsequently been proposed. The null‐effect of mutating pea 9/13‐lipoxygenase at the equivalent residue to that which controlled dual positional specificity in cucumber 13/9‐lipoxygenase, strongly suggests that the mechanisms controlling dual positional specificity in pea 9/13‐lipoxygenase and cucumber 13/9‐lipoxygenase are different. This was supported from modelling of another isoform of pea lipoxygenase, pea 13/9‐lipoxygenase. Dual positional specificity in pea lipoxygenases is more likely to be determined by the degree of penetration of the methyl terminus of linoleate and the volume of the linoleate‐binding pocket rather than substrate orientation. A single model for positional specificity, that has proved to be inappropriate for arachidonate‐binding to mammalian 5‐, 12‐ and 15‐lipoxygenases, would appear to be true also for linoleate‐binding to plant 9‐ and 13‐lipoxygenases.
Regulation of mouse kidney tubular epithelial cell‐specific expression of core 2 GlcNAc transferase
Authors: Sekine Michiko , Taya Choji , Kikkawa Yoshiaki , Yonekawa Hiromichi , Takenaka Masaru , Matsuoka Yasuko , Imai Enyu , Izawa Mineko , Kannagi Reiji , Suzuki Akemi 
Published: March 2003

A mouse gene, Gsl5, controls the expression of Galβ1–4(Fucα1–3)GlcNAcβ1–6(Galβ1–3)Gb4Cer and its precursor glycolipids in the kidney by regulating transcription of β‐1,6‐GlcNAc transferase. Here we report that Gsl5 controls the expression of the core 2 structure [GlcNAcβ1–6(Galβ1–3)GalNAcα1‐Ser/Thr] of glycoproteins as well as the glycolipid, GlcNAcβ1–6(Galβ1–3)GalNAcβ1–3Galα1–4Galβ1–4Glcβ1‐ceramide. Immunohistochemical studies using an anti‐(core 2‐Lex) monoclonal antibody demonstrated that lysosome‐like vesicles of proximal tubule cells were clearly stained in a Gsl5 wild type mouse, but not in a Gsl5 mutant strain of mice. Western blotting of microsomal fractions of kidney tissue with the same antibody confirmed the histological findings. In situ hybridyzation with an antisense probe to the kidney‐specific mRNA demonstrated that the mRNA is localized at proximal tubule‐cells in the cortex adjacent to the medulla, but not detected in glomeruli nor in collecting duct cells in the medulla. The results obtained by immunohistological staining and in situ hybridyzation are compatible and lead to the conclusion that the kidney specific mRNA is expressed in a proximal tubular cell specific manner and produces core 2 GlcNAc transferase responsible for the production of glycoproteins localized at vesicles in the proximal tubular cells. Glycosylation regulated by Gsl5 gene may modify functions of membrane glycoproteins in proximal tubular cells.
Cloning and expression analysis of rainbow trout Oncorhynchus mykiss tumour necrosis factor‐α
Authors: Laing Kerry J. , Wang Tiehui , Zou Jun , Holland Jason , Hong Suhee , Bols Niels , Hirono Ikuo , Aoki Takashi , Secombes Christopher J. 
Published: March 2003

A rainbow trout (Oncorhynchus mykiss) gene for tumor necrosis factor (TNF) has been cloned and sequenced. The cDNA contains an open reading frame of 738 nucleotides that translate into a 246 amino‐acid putative peptide, with a 5′ untranslated region (UTR) of 140 bp and a 3′ UTR of 506 bp. Two potential N‐linked glycosylation sites exist in the translation. The genomic sequence measures 2007 bp and contains three introns that intercept four coding exons. Expression studies using RT‐PCR have shown that the trout TNF gene is constitutively expressed in the gill and kidney of unstimulated fish. Trout TNF expression could be up‐regulated by stimulation of isolated head kidney leucocytes with lipopolysaccharide (LPS). Similarly, stimulation of a trout macrophage cell line (RTS11) with LPS resulted in an increased transcript level, as did incubation with recombinant trout interleukin (IL)‐1β. The optimal timing for induction of TNF expression in trout macrophages was determined using recombinant trout IL‐1β, where a clear induction was apparent by 2 h and peaked at 4 h. Evidence that this TNF gene is equivalent to mammalian TNF‐α is discussed.
Analysis of KdpC of the K+‐transporting KdpFABC complex of Escherichia coli
Authors: Gaßel Michael , Altendorf Karlheinz 
Published: March 2003

The Kdp complex, a high affinity ATP‐driven K+ transport system of Escherichia coli, is composed of the four membrane‐bound subunits KdpF, KdpA, KdpB and KdpC. Whereas the role of KdpB (catalytical subunit), KdpA (K+‐translocating subunit) and KdpF (stabilizing peptide) is well understood, the function of KdpC is still unknown. Therefore, a kdpC deletion strain was constructed and complementation experiments were performed using different kdpC constructs. Truncations of the kdpC gene revealed that only one derivative, which lacks base pairs coding for the four C‐terminal amino acids, was able to complement the chromosomal deletion of kdpC. Furthermore, complementation was also observed with kdpC of Mycobacterium tuberculosis, but not with kdpC from Clostridium acetobutylicum or Synechocystis sp. PCC6803. Sequence alignment of 17 different KdpC proteins led to the construction of hybrids between kdpC of E. coli and that of C. acetobutylicum. Complementation revealed that the N‐terminal transmembrane segment as well as the C‐terminal‐third of the protein can be exchanged between both species, but only one after the other. A simultaneous substitution of both regions was not possible.
A third crystal form of Wolinella succinogenes quinol:fumarate reductase reveals domain closure at the site of fumarate reduction
Authors: Lancaster C. Roy D. , Groß Roland , Simon Jörg 
Published: March 2003

Quinol:fumarate reductase (QFR) is a membrane protein complex that couples the reduction of fumarate to succinate to the oxidation of quinol to quinone. Previously, the crystal structure of QFR from Wolinella succinogenes was determined based on two different crystal forms, and the site of fumarate binding in the flavoprotein subunit A of the enzyme was located between the FAD‐binding domain and the capping domain [Lancaster, C.R.D., Kröger, A., Auer, M., & Michel, H. (1999) Nature402, 377–385]. Here we describe the structure of W. succinogenes QFR based on a third crystal form and refined at 3.1 Å resolution. Compared with the previous crystal forms, the capping domain is rotated in this structure by approximately 14° relative to the FAD‐binding domain. As a consequence, the topology of the dicarboxylate binding site is much more similar to those of membrane‐bound and soluble fumarate reductase enzymes from other organisms than to that found in the previous crystal forms of W. succinogenes QFR. This and the effects of the replacement of Arg A301 by Glu or Lys by site‐directed mutagenesis strongly support a common mechanism for fumarate reduction in this superfamily of enzymes.
Nucleotide sequence analysis of 5′‐flanking region of salicylate hydroxylase gene, and identification and purification of a LysR‐type regulator, SalR
Authors: Sato Hiroyuki , Kudo Sayumi , Ohnishi Kuniharu , Mizuguchi Mitsuo , Goto Eiji , Suzuki Kenzi 
Published: March 2003

The sal gene comprised of 1266 nucleotides encoding salicylate hydroxylase was cloned from the chromosomal DNA of Pseudomonas putida S‐1 and sequenced [Suzuki, K., Mizuguchi, M., Ohnishi, K. and Itagaki, E. (1996) Biochim. Biophys. Acta1275, 154–156]. Here, we describe the nucleotide sequences of the regulatory region of the sal gene and an ORF (salR gene) divergently oriented from the sal gene, which encodes the protein SalR. This gene product positively controls sal gene expression at the transcriptional level. The salR gene consists of 930 base pairs starting from a GTG codon and encodes a protein of 309 amino acids with a molecular mass of 34 542 Da. The amino‐acid sequence is homologous to LysR‐family regulatory proteins such as CatR of P. putida RB1 and has helix‐turn‐helix DNA binding motif near its N‐terminal. Transcription start sites of sal and salR genes were determined to lie 30‐ and 24‐bp upstream of the respective initiation codons and separated from each other by 78 nucleotides. A Shine–Dalgarno sequence and the putative promoter sequences containing −10 and −35 sequences were seen in the sal and salR genes. Expression of the salR gene on a plasmid in Escherichia coli cells was confirmed by DNA mobility shift assay. For the overexpression of the salR gene, it was cloned to pET28a (pSAHR) which was transferred to E. coli BL21 (E. coli BL21/pSAHR), and expressed by an inducer, isopropyl thio‐β‐d‐galactoside. SalR was further purified to homogeneity from the cell‐free extracts in yields of approximately 3 mg·L−1 culture volume. The molecular mass was determined to be 33 kDa and the N‐terminal amino‐acid sequence was the same as that deduced from the nucleotide sequence of salR gene. Native SalR was also purified to homogeneity from P. putida S‐1 with very low contents. The properties of the protein were similar to those of SalR expressed in E. coli.
Insight into thyrotropin receptor cleavage by engineering the single polypeptide chain luteinizing hormone receptor into a cleaving, two subunit receptor
Authors: Chazenbalk Gregorio D. , McLachlan Sandra M. , Chen Chun‐Rong , Rapoport Basil 
Published: March 2003

To gain insight into the thyrotropin hormone (TSH) receptor (TSHR) cleavage, we sought to convert the noncleaving luteinizing hormone (LH) receptor (LHR) into a cleaved, two‐subunit molecule. For this purpose, we generated a series of LHR mutants and chimeric LH‐TSH receptors. Cleavage of mature, ligand binding receptors on the cell surface was determined by covalent 125I‐labeled hCG crosslinking to intact, stably transfected mammalian cells. We first targeted a cluster of three N‐linked glycans in the LHR (N295, N303, N317) in a region corresponding to the primary TSHR cleavage site, which has only one N‐linked glycan. Elimination by mutagenesis of the most strategic N‐linked glycan (LHR‐N317Q) generated only a trace amount of LHR cleavage. Removal of the other N‐linked glycans had no additive effect. A much greater degree of cleavage (≈ 50%) was evident in a chimeric LH‐TSHR in which the juxtamembrane segment of the LHR (domain E; amino acids 317–367) was replaced with the corresponding domain of the TSHR (residues 363–418). Similarly cleaving LHR were created using a much smaller component within this region, namely LHR‐NET317–319 replaced with TSHR‐GQE367–369, or by substitution of the same three amino‐acid residues with AAA (LHR‐NET317–319AAA). In summary, our data alter current concepts regarding TSHR cleavage by suggesting limited (not absent) amino‐acid specificity in a region important for TSHR cleavage (GQE367–369). The data also support the concept of a separate and distinct downstream cleavage site 2 in the TSHR.