bluuebunny/crossref_metadata_2025_split huggingface dataset:
Antitermination in bacteriophage λ
Authors: Schärpf Manuela , Sticht Heinrich , Schweimer Kristian , Boehm Markus , Hoffmann Silke , Rösch Paul 
Published: March 2003

The solution structure of a 15‐mer nutRboxBRNA hairpin complexed with the 36‐mer N‐terminal peptide of the N protein (N36) from bacteriophage λ was determined by 2D and 3D homonuclear and heteronuclear magnetic resonance spectroscopy. These 36 amino acids include the arginine‐rich motif of the N protein involved in transcriptional antitermination of phage λ. Upon complex formation withboxBRNA, the synthetic N36 peptide binds tightly to the major groove of theboxBhairpin through hydrophobic and electrostatic interactions forming a bent α helix. Four nucleotides of the GAAAA pentaloop of theboxBRNA adopt a GNRA‐like tetraloop fold in the complex. The formation of a GAAA tetraloop involves a loop‐closing sheared base pair (G6‐A10), base stacking of three adenines (A7, A8, and A10), and extrusion of one nucleotide (A9) from the loop, as observed previously for the complex of N(1–22) peptide and the nutLboxBRNA [Legault, P., Li, J., Mogridge, J., Kay, L.E. & Greenblatt, J. (1998)Cell93, 289–299]. Stacking of the bases is extended by the indole‐ring of Trp18 which also forms hydrophobic contacts to the side‐chains of Leu24, Leu25, and Val26.Based on the structure of the complex, three mutant peptides were synthesized and investigated by CD and NMR spectroscopy in order to determine the role of particular residues for complex formation. These studies revealed very distinct amino‐acid requirements at positions 3, 4, and 8, while replacement of Trp18 with tyrosine did not result in any gross structural changes.
Aspartate aminotransferase from the Antarctic bacterium Pseudoalteromonas haloplanktis TAC 125
Authors: Birolo Leila , Tutino M. Luisa , Fontanella Bianca , Gerday Charles , Mainolfi Katia , Pascarella Stefano , Sannia Giovanni , Vinci Floriana , Marino Gennaro 
Published: March 2003

The gene encoding aspartate aminotransferase from the psychrophilic bacterium Pseudoalteromonas haloplanktis TAC 125 was cloned, sequenced and overexpressed in Escherichia coli. The recombinant protein (PhAspAT) was characterized both at the structural and functional level in comparison with the E. coli enzyme (EcAspAT), which is the most closely related (52% sequence identity) bacterial counterpart. PhAspAT is rapidly inactivated at 50 °C (half‐life = 6.8 min), whereas at this temperature EcAspAT is stable for at least 3 h. The optimal temperature for PhAspAT activity is ≈ 64 °C, which is some 11 °C below that of EcAspAT. The protein thermal stability was investigated by following changes in both tryptophan fluorescence and amide ellipticity; this clearly suggested that a first structural transition occurs at ≈ 50 °C for PhAspAT. These results agree with the expected thermolability of a psychrophilic enzyme, although the observed stability is much higher than generally found for enzymes isolated from cold‐loving organisms. Furthermore, in contrast with the higher efficiency exhibited by several extracellular psychrophilic enzymes, both kcat and kcat/Km of PhAspAT are significantly lower than those of EcAspAT over the whole temperature range. This behaviour possibly suggests that the adaptation of this class of endocellular enzymes to a cold environment may have only made them less stable and not more efficient.The affinity of PhAspAT for both amino‐acid and 2‐oxo‐acid substrates decreases with increasing temperature. However, binding of maleate and 2‐methyl‐l‐aspartate, which both inhibit the initial steps of catalysis, does not change over the temperature range tested. Therefore, the observed temperature effect may occur at any of the steps of the catalytic mechanism after the formation of the external aldimine.A molecular model of PhAspAT was constructed on the basis of sequence homology with other AspATs. Interestingly, it shows no insertion or extension of loops, but some cavities and a decrease in side chain packing can be observed.
Phytanoyl‐CoA hydroxylase activity is induced by phytanic acid
Authors: Zomer Anna W. M. , Jansen Gerbert A. , van der Burg Bart , Verhoeven Nanda M. , Jakobs Cornelis , van der Saag Paul T. , Wanders Ronald J. A. , Poll‐The Bwee Tien 
Published: March 2003

Phytanic acid (3,7,11,15‐tetramethylhexadecanoic acid) is a branched‐chain fatty acid present in various dietary products such as milk, cheese and fish. In patients with Refsum disease, accumulation of phytanic acid occurs due to a deficiency of phytanoyl‐CoA hydroxylase, a peroxisomal enzyme containing a peroxisomal targeting signal 2. Recently, phytanoyl‐CoA hydroxylase cDNA has been isolated and functional mutations have been identified. As it has been shown that phytanic acid activates the nuclear hormone receptors peroxisome proliferator‐activated receptor (PPAR)α and all three retinoid X receptors (RXRs), the intracellular concentration of this fatty acid should be tightly regulated.When various cell lines were grown in the presence of phytanic acid, the activity of phytanoyl‐CoA hydroxylase increased up to four times, depending on the particular cell type. In one cell line, HepG2, no induction of phytanoyl‐CoA hydroxylase activity was observed. After addition of phytanic acid to COS‐1 cells, an increase in phytanoyl‐CoA hydroxylase activity was observed within 2 h, indicating a quick cell response. No stimulation of phytanoyl‐CoA hydroxylase was observed when COS‐1 cells were grown in the presence of clofibric acid, 9‐cis‐retinoic acid or both ligands together. This indicates that the activation of phytanoyl‐CoA hydroxylase is not regulated via PPARα or RXR. However, stimulation of PPARα and all RXRs by clofibric acid and 9‐cis‐retinoic acid was observed in transient transfection assays. These results suggest that the induction of phytanoyl‐CoA hydroxylase by phytanic acid does not proceed via one of the nuclear hormone receptors, RXR or PPARα.
Heme‐(hydro)peroxide mediated O‐ and N‐dealkylation
Authors: Boersma Marelle G. , Primus Jean‐Louis , Koerts Janneke , Veeger Cees , Rietjens Ivonne M. C. M. 
Published: March 2003

The mechanism of microperoxidase‐8 (MP‐8) mediated O‐ and N‐dealkylation was investigated. In the absence of ascorbate (peroxidase mode), many unidentified polymeric products are formed and the extent of substrate degradation correlates (r = 0.94) with the calculated substrate ionization potential, reflecting the formation of radical intermediates.In the presence of ascorbate (P450 mode) formation of polymeric products is largely prevented but, surprisingly, dealkylation is not affected. In addition, aromatic hydroxylation and oxidative dehalogenation is observed. The results exclude a radical mechanism and indicate the involvement of a (hydro)peroxo‐iron heme intermediate in P450‐type of heteroatom dealkylation.
Scots pine expresses short‐root‐specific peroxidases during development
Authors: Tarkka Mika T. , Nyman Tuula A. , Kalkkinen Nisse , Raudaskoski Marjatta 
Published: March 2003

Nine short‐root‐specific proteins from Scots pine (Pinus sylvestris L.) detected and isolated as individual spots by 2D‐PAGE were identified. The similar peptide mass maps obtained for all nine polypeptide spots together with lectin‐blotting results suggest that they represent forms of the same modified protein. N‐Terminal sequence analysis of two of the peptides showed high similarity to peroxidases. RT‐PCR with oligonucleotide primers corresponding to determined peptide sequences and conserved regions in plant peroxidases led to isolation of Psyp1 cDNA which is most abundantly expressed in short roots.Psyp1 is the first peroxidase cDNA to be isolated from the genus Pinus. It encodes a 363‐amino‐acid class‐III peroxidase with a calculated molecular mass of 35.7 kDa and theoretical pI of 4.74. The predicted PSYP1 amino‐acid sequence is grouped with other class‐III peroxidases in phylogenetic analyses, but it has a unique amino‐acid sequence which may be associated with its function in short roots or with its phylogenetic group. The presence of a signal sequence for extracellular transport indicates that PSYP1 belongs to the group of secreted class‐III peroxidases. The presence of 10 tyrosine residues and putative auxin‐binding regions in PSYP1 suggests that the function of the enzyme is associated with cell‐wall formation in short roots. The downregulation of Psyp1 expression in symbiotic short roots hosting the ectomycorrhizal fungus Suillus bovinus is perhaps related to the change in cell‐wall structure necessary for ectomycorrhizal development.
Mapping DNA‐binding sites of HIV‐1 integrase by protein footprinting
Authors: Dirac Annette M. G. , Kjems Jørgen 
Published: March 2003

The HIV‐1 integrase protein catalyzes integration of the viral genome into host cell DNA. Whereas the structures of the three domains of integrase have been solved separately, both the structural organization of the full‐length protein and its interaction with DNA remain unresolved. A protein footprinting approach was employed to investigate the accessibility of residues in the full‐length soluble integrase mutant, INF185K,C280S, to proteolytic attack in the absence and presence of DNA. The N‐terminal and C‐terminal domains were relatively more accessible to proteolytic attack than the core domain. The susceptibility to proteolytic attack was specifically affected by DNA at residues Lys34, in the N‐terminal domain, Lys111, Lys136, Glu138, Lys156–Lys160, Lys185–Lys188, in the core domain, and Asp207, Lys 215, Glu246, Lys258 and Lys273 in the linker and C‐terminal domain, suggesting that these regions are involved in, or shielded by, DNA binding. Lys34 is positioned in a putative dimerization domain, consistent with the notion that DNA stabilizes the dimeric state of integrase.
Phosphatidylinositol 3‐kinases in tumor progression
Authors: Roymans Dirk , Slegers Herman 
Published: March 2003

Many cellular processes have been identified in which phosphatidylinositol 3‐kinase has a key regulatory function. As an oncogene, it is also involved in the development of cancer. The transformation and progression of normal cells towards an advanced stage tumor and/or towards metastatic lesions involves a complex series of events, including genetic alterations, leading to aberrant cell cycle progression, altered adhesion and motility characteristics, inhibition of apoptosis and induction of angiogenesis. This review highlights the processes involved in the pathogenesis of cancer in which phosphatidylinositol 3‐kinase is involved and provides an overview of the possible mechanisms by which the enzyme exerts its oncogenic action.
Hydrolysis of glycine‐containing elastin pentapeptides by LasA, a metalloelastase from Pseudomonas aeruginosa
Authors: Vessillier Sandrine , Delolme Frédéric , Bernillon Jacques , Saulnier Joëlle , Wallach Jean 
Published: March 2003

Pseudomonas aeruginosa is an opportunistic pathogen that causes severe infections in vulnerable hosts. It may produce various virulence factors including proteases. Among them, LasA possesses both elastolytic and staphylolytic (hydrolysis of pentaglycine cross‐links in the cell wall peptidoglycan) activities. To understand if its elastolytic activity results from a preference for glycine‐rich substrates, we studied its ability to hydrolyse the 65 pentapeptides of human tropoelastin containing at least three glycines. As demonstrated by capillary electrophoresis (CE), 22 of these peptides were hydrolysed by LasA, generally at a single peptide bond and the catalytic ratio kcat/KM was determined for most of them. The highest value was obtained for LGGGA, 59 ± 9 min−1·mmol−1·L. The specificity of hydrolysis was elucidated by CE, liquid secondary ion mass spectrometry and, in some cases, collision activated dissociation‐mass analysis of ion kinetic energy. The preferred cleavage sites are GG and GA peptide bonds, the sequence GG↓A being especially sensitive to hydrolysis. Both positions P2 and P′2 must be occupied for hydrolysis and the presence of an amino acid in P3 (but not in P′3) significantly increases the catalytic ratio. Considering these results, about 30 GGX sequences (X: G, A or Y) of human tropoelastin could be susceptible to LasA elastolysis.
Different sensitivity of rabbit heart and skeletal muscle to endotoxin‐induced impairment of mitochondrial function
Authors: Trumbeckaite Sonata , Opalka Jens R , Neuhof Christiane , Zierz Stephan , Gellerich Frank N 
Published: March 2003

The involvement of mitochondrial dysfunction in septic disturbances of tissues is controversial. The aim of this study was to investigate the effects of endotoxin‐induced sepsis on the function of heart and skeletal muscle mitochondria. Rabbits were made septic by subcutaneous injection of endotoxin (lipopolysaccharide, LPS) from Escherichia coli at concentrations of 100 or 150 µg LPS·kg−1 24 h prior to the experiments. Mitochondrial respiration was measured in saponin‐skinned muscle fibers and compared with photometrically detected activities of respiratory chain enzymes as well as with function of perfused hearts. In heart fibers a dosage of 100 µg LPS·kg−1 caused a significant decrease of state 3‐respiration for the substrates pyruvate (−38%), octanoyl‐carnitine (−38%) and succinate (−30%) with correspondingly decreased respiratory control indexes (RCI). In addition, endotoxin caused a decreased temporal stability of the rate of state 3‐respiration. At least in part these changes can be attributed to a reduced activity of complex I + III (−50%) of the respiratory chain. State 4‐respiration rates were not significantly altered. The lowered state 3‐respiration in heart mitochondria seems to contribute to the impairment of heart muscle function as detected by an increase of coronary vascular resistance (CVR) in endotoxin‐treated hearts. Functional properties of mitochondria from M. Vastus lasteralis were not affected by 100 µg LPS·kg−1 but a higher dosage of 150 µg LPS·kg−1 caused decreased RCI for the substrates pyruvate (−29%) and octanoyl‐carnitine (−32%). Also the activity of complex I + III was not significantly affected at lower dose of endotoxin but decreased (−42%) after treatment with 150 µg LPS·kg−1. Results demonstrate the involvement of impaired mitochondria in the pathophysiology of septic organ failure and a tissue specifity of endotoxaemia.
Extensive deproteinization of Dictyostelium discoideum RNase P reveals a new catalytic activity
Authors: Stathopoulos Constantinos , Tekos Apostolos , Zarkadis Ioannis K. , Drainas Denis 
Published: March 2003

Nuclear Dictyostelium discoideum RNase P was subjected to vigorous deproteinization procedures. After treatment with proteinase K followed by phenol extraction of samples containing D. discoideum RNase P activity, a new enzymatic activity was recovered. The proteinase K/phenol/SDS treated enzyme cleaves Schizossacharomyces pombe tRNAser (supS1), D. discoideum tRNASer and tRNALeu precursors several nucleotides upstream of the cleavage site of RNase P, liberating products with 5′‐hydroxyl ends. This activity seems to be associated with one or two RNA molecules copurifying with D. discoideum RNase P activity as judged by its inhibition in the presence of micrococcal nuclease, which is in contrast to its resistance to proteinase K/phenol/SDS treatment.
Specific inhibition of transforming growth factor‐β2 expression in human osteoblast cells by antisense phosphorothioate oligonucleotides
Authors: Shen Zhong‐Jian , Kim Sang Kook , Kwon Oh‐Shin , Lee Young Sup , Moon Byung Jo 
Published: March 2003

To elucidate the role of endogenous transforming growth factor (TGF)‐β2 on human osteoblast cell, antisense phosphorothioate oligonucleotides (S‐ODNs) complementary to regions in mRNA of TGF‐β2 were synthesized and examined their effects on TGF‐β2 production and cell proliferation in a human osteoblast cell line ROS 17/2. Antisense S‐ODNs were designated for three different target regions in the mRNA of TGF‐β2. Among several antisense S‐ODN analyzed, an oligonucleotide (AS‐11) complementary to the translation initiation site of mRNA of TGF‐β2 demonstrated a selective and strong inhibitory effect on TGF‐β2 production in osteoblast cells. Other antisense S‐ODNs which were designated for other regions in mRNA of TGF‐β2 and one‐ or three‐base mismatched analogs of AS‐11 showed little or much less antisense activities than AS‐11. Therefore, the most effective target site in mRNA of TGF‐β2 is at the initiation codon region. The antisense effects of AS‐11 were observed without reduction of levels of mRNA of TGF‐β2. Furthermore, the inhibition of TGF‐β2 expression by antisense S‐ODN appeared to enhance cell proliferation, demonstrating the growth inhibitory effect of autocrine TGF‐β2 in osteoblast cells.
A novel RNA polymerase binding site upstream of the galactose promoter in Escherichia coli exhibits promoter‐like activity
Authors: Sur Runa , Debnath Dipanwita , Mukhopadhyay Jayanta , Parrack Pradeep 
Published: March 2003

RNA polymerase is known to bind and utilize the overlapping promoters P1 and P2 in Escherichia coli galactose operon. We have identified an additional specific site upstream of P2, where RNA polymerase binds in a heparin‐resistant manner. Binding of polymerase to this site, termed P3, occurs simultaneous to its binding at P1/P2. We have located this P3 site by DNase I footprinting. A 63 base pair region centered around position − 100 with respect to galP1 is protected by polymerase. Interestingly, a Pribnow box TATAAT is present within this protected region (−103 to −108). We have shown that transcription occurs from P3 in vitro. Primer extension analysis provides direct evidence that P3 is transcribed in vivo. The start site of transcription has been mapped at −96 position relative to galP1. β‐galactosidase assays with different gal promoter constructs reveal that while P3 alone functions as a weak in vivo promoter, it has a synergistic effect on transcription from the gal operon, since deletion of P3 or specifically mutating its −10 region result in a substantial reduction in the gal promoter activity.
Mechanism of nitrite‐stimulated catalysis by lactoperoxidase
Authors: Brück T. B. , Fielding R. J. , Symons M. C. R. , Harvey P. J. 
Published: March 2003

The reactions of lactoperoxidase (LPO) intermediates compound I, compound II and compound III, with nitrite ( ) were investigated. Reduction of compound I by

was rapid (k2 = 2.3 × 107 m−1·s−1; pH = 7.2) and compound II was not an intermediate, indicating that

radicals are not produced when

reacts with compound I. The second‐order rate constant for the reaction of compound II with

at pH = 7.2 was 3.5 × 105 m−1·s−1. The reaction of compound III with

exhibited saturation behaviour when the observed pseudo first‐order rate constants were plotted against

concentrations and could be quantitatively explained by the formation of a 1 : 1 ratio compound III/NO2− complex. The Km of compound III for

was 1.7 × 10−4 m and the first‐order decay constant of the compound III/ complex was 12.5 ± 0.6 s−1. The second‐order rate constant for the reaction of the complex with

was 3.3 × 103 m−1·s−1. Rate enhancement by

does not require

as a redox intermediate.

accelerates the overall rate of catalysis by reducing compound II to the ferric state. With increasing levels of H2O2, there is an increased tendency for the catalytically dead‐end intermediate compound III to form. Under these conditions, the ‘rescue’ reaction of

with compound III to form compound II will maintain the peroxidatic cycle of the enzyme.
Regulation of laminin β2 chain gene expression in human cancer cell lines
Authors: Durkin Marian E. , Nielsen Finn C. , Loechel Frosty , Albrechtsen Reidar , Wewer Ulla M. 
Published: March 2003

The laminin β2 chain is a basement membrane component expressed in a tissue‐ and developmental stage‐specific manner. In this report we have examined the transcriptional and post‐transcriptional regulation of the human laminin β2 chain in human tumor cell lines. Both the A204 rhabdomyosarcoma and clone A colon carcinoma cells express the laminin β2 chain mRNA, but only the A204 cells secrete laminin heterotrimers containing the β2 chain. Segments of the β2 chain gene promoter region were cloned into luciferase reporter vectors, and their ability to stimulate transcription was tested by transient transfection. Sequences downstream of the transcription start site between nucleotides +91 and +120 were found to be essential for luciferase activity in the two cell lines. Additional positive regulatory regions were present further upstream, between nucleotides −164 to −667 and between nucleotides −667 to −1724. Genomic DNA at the 3′ end of the gene also appeared to have enhancer activity, as a 1.1‐kb fragment located downstream of the last exon stimulated the luciferase activity of the nucleotides −667/+297 promoter segment ≈ threefold. Alternative splicing of the first intron of the human laminin β2 chain gene generates two isoforms of the 5′ untranslated region of the β2 chain mRNA. The translational efficiencies of the two laminin β2 chain leaders did not differ significantly, when assayed by polysome profile analysis of endogenous clone A cell β2 chain mRNA, transient transfection of chimeric β2 chain leader/luciferase expression plasmids in clone A cells, and translation of in vitro synthesized RNAs in rabbit reticulocyte lysates.
The contribution of metal ions to the conformational stability of ribonuclease T1
Authors: Deswarte Joeri , De Vos Stefan , Langhorst Ulrike , Steyaert Jan , Loris Remy 
Published: March 2003

In the crystalline state, ribonuclease T1 binds calcium ions at different lattice‐dependent positions. In solution, its conformational stability is also remarkably increased in the presence of divalent metal ions. Combining urea unfolding studies and X‐ray crystallography, we compared the presence of several metal ions at specific sites in the protein to their contribution to the overall stabilizing effect in solution. We constructed thermodynamic cycles involving particular metal ions and specific carboxylate functions. The resulting coupling energies indicate that some (but not all) metal ions found at lattice contacts in crystal structures may indeed significantly contribute to stability enhancement in the presence of metal ions in solution.
The hemoglobin system of the brown moray Gymnothorax unicolor
Authors: Tamburrini Maurizio , Verde Cinzia , Olianas Alessandra , Giardina Bruno , Corda Marcella , Sanna Maria T. , Fais Antonella , Deiana Anna M. , di Prisco Guido , Pellegrini Mariagiuseppina 
Published: March 2003

The Gymnothorax unicolor hemoglobin system is characterized by two components, called cathodic and anodic on the basis of their isoelectric point, which were separated by ion‐exchange chromatography. The oxygen‐binding properties of the purified components were studied in the absence and presence of chloride and/or GTP or ATP in the pH range 6.5–8.0. Stripped cathodic hemoglobin showed a small reverse Bohr effect, high oxygen affinity, and low co‐operativity; the addition of chloride only caused a small decrease in oxygen affinity. In the presence of GTP or ATP, the oxygen affinity was dramatically reduced, the co‐operativity increased, and the reverse Bohr effect abolished. Stripped anodic hemoglobin is characterized by both low oxygen affinity and co‐operativity, and displayed a normal Bohr effect; the addition of chloride increased co‐operativity, whereas ATP and GTP significantly modulated oxygen affinity at acidic pH values, enhancing the Bohr effect and giving rise to the Root effect. The complete amino‐acid sequences of the α and β chains of both hemoglobins were established; the molecular basis of the functional properties of the hemoglobins is discussed in the light of the primary structure and compared with those of other fish hemoglobins.