bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequence analysis and modular organization of threonyl‐tRNA synthetase from Thermus thermophilus and its interrelation with threonyl‐tRNA synthetases of other origins
Authors: Cura Vincent , Moras Dino , Kern Daniel 
Published: March 2003

The gene encoding threonyl‐tRNA synthetase (Thr‐tRNA synthetase) from the extreme thermophilic eubacterium Thermus thermophilus HB8 has been cloned and sequenced. The ORF encodes a polypeptide chain of 659 amino acids (Mr 75 550) that shares strong similarities with other Thr‐tRNA synthetases. Comparative analysis with the three‐dimensional structure of other subclass IIa synthetases shows it to be organized into four structural modules: two N‐terminal modules specific to Thr‐tRNA synthetases, a catalytic core and a C‐terminal anticodon‐binding module. Comparison with the three‐dimensional structure of Escherichia coli Thr‐tRNA synthetase in complex with tRNAThr enabled identification of the residues involved in substrate binding and catalytic activity. Analysis by atomic absorption spectrometry of the enzyme overexpressed in E. coli revealed the presence in each monomer of one tightly bound zinc atom, which is essential for activity.Despite strong similarites in modular organization, Thr‐tRNA synthetases diverge from other subclass IIa synthetases on the basis of their N‐terminal extensions. The eubacterial and eukaryotic enzymes possess a large extension folded into two structural domains, N1 and N2, that are not significantly similar to the shorter extension of the archaebacterial enzymes. Investigation of a truncated Thr‐tRNA synthetase demonstrated that domain N1 is not essential for tRNA charging. Thr‐tRNA synthetase from T. thermophilus is of the eubacterial type, in contrast to other synthetases from this organism, which exhibit archaebacterial characteristics. Alignments show conservation of part of domain N2 in the C‐terminal moiety of Ala‐tRNA synthetases. Analysis of the nucleotide sequence upstream from the ORF showed the absence of both any anticodon‐like stem‐loop structure and a loop containing sequences complementary to the anticodon and the CCA end of tRNAThr. This means that the expression of Thr‐tRNA synthetase in T. thermophilus is not regulated by the translational and trancriptional mechanisms described for E. coli thrS and Bacillus subtilis thrS and thrZ. Here we discuss our results in the context of evolution of the threonylation systems and of the position of T. thermophilus in the phylogenic tree.
Structure of the O polysaccharide and serological classification of Pseudomonas syringae pv. ribicola NCPPB 1010
Authors: Ovod Vladimir V. , Zdorovenko Evelina L. , Shashkov Aleksander S. , Kocharova Nina A. , Knirel Yuriy A. 
Published: March 2003

The O polysaccharide (OPS) moiety of the lipopolysaccharide (LPS) of a phytopathogenic bacterium Pseudomonas syringae pv. ribicola NCPPB 1010 was studied by sugar and methylation analyses, Smith degradation, and 1H‐ and 13C‐NMR spectroscopy, including 2D COSY, TOCSY, NOESY and H‐detected 1H,13C HMQC experiments. The OPS structure was elucidated, and shown to be composed of branched pentasaccharide repeating units (O repeats) of two types, major (1) and minor (2), differing in the position of substitution of one of the rhamnose residues. Both O repeats form structurally homogeneous blocks within the same polysaccharide molecule.Although P. syringae pv. ribicola NCPPB 1010 demonstrates genetic relatedness and similarity in the OPS chemical structure to some other P. syringae pathovars, it did not cross‐react with any OPS‐specific mAbs produced against heterologous P. syringae strains. Therefore, we propose to classify P. syringae pv. ribicola NCPPB 1010 in a new serogroup, O8.
Enzyme‐mediated sulfide production for the reconstitution of [2Fe−2S] clusters into apo‐biotin synthase of Escherichia coli
Authors: Tse Sum Bui Bernadette , Escalettes Franck , Chottard Geneviève , Florentin Dominique , Marquet Andrée 
Published: March 2003

We previously showed that biotin synthase in which the (Fe–S) cluster was labelled with 34S by reconstitution donates 34S to biotin [B. Tse Sum Bui, D. Florentin, F. Fournier, O. Ploux, A. Méjean & A. Marquet (1998) FEBS Lett. 440, 226–230]. We therefore proposed that the source of sulfur was very likely the (Fe–S) centre. This depletion of sulfur from the cluster during enzymatic reaction could explain the absence of turnover of the enzyme which means that to restore a catalytic activity, the clusters have to be regenerated.In this report, we show that the NifS protein from Azotobacter vinelandii and C‐DES from Synechocystis as well as rhodanese from bovine liver can mobilize the sulfur, respectively, from cysteine and thiosulfate for the formation of a [2Fe−2S] cluster in the apoprotein of Escherichia coli biotin synthase. The reconstituted enzymes were as active as the native enzyme.When [35S]cysteine was used during the reconstitution experiments in the presence of NifS, labelled (Fe35S) biotin synthase was obtained. This enzyme produced [35S]biotin, confirming the results obtained with the 34S‐reconstituted enzyme.NifS was also effective in mobilizing selenium from selenocystine to produce an (Fe–Se) cluster.However, though NifS could efficiently reconstitute holobiotin synthase from the apoform, starting from cysteine, these two effectors had no significant effect on the turnover of the enzyme in the in vitro assay.
Murine FATP alleviates growth and biochemical deficiencies of yeast fat1Δ strains
Authors: DiRusso Concetta C. , Connell Elise J. , Færgeman Nils J. , Knudsen Jens , Hansen Jan K. , Black Paul N. 
Published: March 2003

Saccharomyces cerevisiae is an ideal model eukaryote for studying fatty‐acid transport. Yeast are auxotrophic for unsaturated fatty acids when grown under hypoxic conditions or when the fatty‐acid synthase inhibitor cerulenin is included in the growth media. The FAT1 gene encodes a protein, Fat1p, which is required for maximal levels of fatty‐acid import and has an acyl CoA synthetase activity specific for very‐long‐chain fatty acids suggesting this protein plays a pivotal role in fatty‐acid trafficking. In the present work, we present evidence that Fat1p and the murine fatty‐acid transport protein (FATP) are functional homologues. FAT1 is essential for growth under hypoxic conditions and when cerulenin was included in the culture media in the presence or absence of unsaturated fatty acids. FAT1 disruptants (fat1Δ) fail to accumulate the fluorescent long‐chain fatty acid fatty‐acid analogue 4,4‐difluoro‐5‐methyl‐4‐bora‐3a,4a‐diaza‐s‐indacene‐3‐dodecanoic acid (C1‐BODIPY‐C12), have a greatly diminished capacity to transport exogenous long‐chain fatty acids, and have very long‐chain acyl CoA synthetase activities that were 40% wild‐type. The depression in very long‐chain acyl CoA synthetase activities were not apparent in cells grown in the presence of oleate. Additionally, β‐oxidation of exogenous long‐chain fatty acids is depressed to 30% wild‐type levels. The reduction of β‐oxidation was correlated with a depression of intracellular oleoyl CoA levels in the fat1Δ strain following incubation of the cells with exogenous oleate. Expression of either Fat1p or murine FATP from a plasmid in a fat1Δ strain restored these phenotypic and biochemical deficiencies. Fat1p and FATP restored growth of fat1Δ cells in the presence of cerulenin and under hypoxic conditions. Furthermore, fatty‐acid transport was restored and was found to be chain length specific: octanoate, a medium‐chain fatty acid was transported in a Fat1p‐ and FATP‐independent manner while the long‐chain fatty acids myristate, palmitate, and oleate required either Fat1p or FATP for maximal levels of transport. Lignoceryl CoA synthetase activities were restored to wild‐type levels in fat1Δ strains expressing either Fat1p or FATP. Fat1p or FATP also restored wild‐type levels of β‐oxidation of exogenous long‐chain fatty acids. These data show that Fat1p and FATP are functionally equivalent when expressed in yeast and play a central role in fatty‐acid trafficking.
α3‐Galactosylated glycoproteins can bind to the hepaticasialoglycoprotein receptor
Authors: Joziasse David H. , Lee Reiko T. , Lee Yuan C. , Biessen Erik A. L. , Schiphorst Wietske E. C. M. , Koeleman Carolien A. M. , van den Eijnden Dirk H. 
Published: March 2003

In mammals, clearance of desialylated serum glycoproteins to the liver is mediated by a galactose‐specific hepatic lectin, the ‘asialoglycoprotein receptor’. In humans, serum glycoprotein glycans are usually capped with sialic acid, which protects these proteins against hepatic uptake. However, in most other species, an additional noncharged terminal element with the structure Galα1→3Galβ1→4R is present on glycoprotein glycans. To investigate if α3‐galactosylated glycoproteins, just like desialylated glycoproteins, could be cleared by the hepatic lectin, the affinities of α3‐galactosylated compounds towards this lectin were determined using an in vitro inhibition assay, and were compared with those of the parent compounds terminating in Galβ1→4R. Diantennary, triantennary and tetraantennary oligosaccharides that form part of N‐glycans were α3‐galactosylated to completion by use of recombinant bovine α3‐galactosyltransferase. Similarly, desialylated α1‐acid glycoprotein (orosomucoid) was α3‐galactosylated in vitro. The α3‐galactosylation of a branched, Galβ1→4‐terminated oligosaccharide lowered its affinity for the membrane‐bound lectin on whole rat hepatocytes 50–250‐fold, and for the detergent‐solubilized hepatic lectin 7–50‐fold. In contrast, α3‐galactosylation of asialo‐α1‐acid glycoprotein caused only a minor decrease in affinity, increasing the IC50 from 5 to 15 nm.Fully α3‐galactosylated α1‐acid glycoprotein, intravenously injected into the mouse, was rapidly cleared from the circulation, with a clearance rate close to that of asialo‐α1‐acid glycoprotein (t1/2 of 0.42 min vs. 0.95 min). Its uptake was efficiently inhibited by pre‐injection of an excess asialo‐fetuin. Organ distribution analysis showed that the injected α1‐acid glycoprotein accumulated predominantly in the liver. Taken together, these observations suggest that serum glycoproteins that are heavily α3‐galactosylated will be rapidly cleared from the bloodstream via the hepatic lectin. It is suggested that glycosyltransferase expression in murine hepatocytes is tightly regulated in order to prevent undesired uptake of hepatocyte‐derived, circulating glycoproteins.
Effects of proline mutations in the major house dust mite allergen Der f 2 on IgE‐binding and histamine‐releasing activity
Authors: Takai Toshiro , Ichikawa Saori , Hatanaka Hideki , Inagaki Fuyuhiko , Okumura Yasushi 
Published: March 2003

Der f 2 is the major group 2 allergen from house dust mite Dermatophagoides farinae and is composed of 129 amino‐acid residues. Wild‐type and six proline mutants of Der f 2 (P26A, P34A, P66A, P79A, P95A, and P99A) expressed in Escherichia coli were refolded and purified. Formations of intramolecular disulfide bonds in the purified proteins were confirmed correct. The apparent molecular masses analyzed by gel‐filtration were 14–15 kDa. The IgE‐binding capacity in the sera of seven mite‐allergic patients, inhibitory activity for IgE‐binding to immobilized wild‐type Der f 2, and activity to stimulate peripheral blood basophils to release histamine in two volunteers were analyzed. P95A and P99A, which slightly differed from the wild‐type Der f 2 in their CD spectrum, showed reduced IgE‐binding, reduced inhibitory activity, and less histamine‐releasing activity than the wild‐type. P34A also showed reduced allergenicity. Considering that Pro95, Pro99 and Pro34 are closely located in loops at one end of the tertiary structure of Der f 2, we concluded that these loop regions included an IgE‐binding site common to all tested patients. P66A showed reduced IgE‐binding in two sera out of seven. P26A and P79A showed no reduced allergenicity. However, in immunoblot analysis after SDS/PAGE under reduced conditions, P79A showed no or markedly reduced IgE‐binding while the other mutants showed IgE‐binding corresponding to that in the assay using correctly refolded proteins. This suggests that Pro79 is involved in refolding of Der f 2. The findings in this study are important for the understanding of the antigenic structure of mite group 2 allergens and for manipulation of the allergens for specific immunotherapy.
Effects of pressure on the activity and spectroscopic properties of carboxyl proteinases
Authors: Fujiwara Satoshi , Kunugi Shigeru , Oyama Hiroshi , Oda Kohei 
Published: March 2003

The pressure dependence of the activity and spectroscopic properties of four carboxyl proteinases were investigated. Two were pepstatin‐sensitive carboxyl proteinases (porcine pepsin and proteinase A from baker's yeast) and two were pepstatin‐insensitive carboxyl proteinases (from Pseudomonas sp. 101 (pseudomonapepsin; PCP) and Xanthomonas sp. T‐22 (xanthomonapepsin; XCP)). The specificity constant [kcat/Km(app)] of PCP and XCP for a synthetic peptide substrate showed only a slight decrease with increasing pressure, whereas pepsin and proteinase A showed substantial disactivation at higher pressures. The calculated apparent activation volume (ΔVkcat/Km) was about 1, 3, 13, and 14 mL·mol−1 for PCP, XCP, pepsin, and proteinase A, respectively. The hydrolysis of acid‐denatured myoglobin by the four carboxyl proteinases was only slightly affected by high pressure (except for proteinase A at 400 MPa), in contrast to the results for the peptide hydrolysis. In fact, PCP, XCP, and proteinase A actually showed slightly higher degradations of acid‐denatured myoglobin at higher pressures. The residual activities of these enzymes after the incubation at high pressures implied a pressure‐induced stabilization towards autolysis. The changes in the fourth derivative near‐UV absorbance spectrum of the four enzymes in aqueous solution were measured at various pressures from 0.1 to 400 MPa. Upon an increase in pressure, the peaks from PCP and XCP red‐shifted slightly, whereas pepsin and proteinase A blue‐shifted substantially, thus indicating a more polar environment. The intrinsic fluorescence also decreased upon increasing pressure. However, the change for XCP was rather small, but the change for the other three was very large. The changes in the peak wavelength for pepsin and proteinase A were characteristic, and also indicated a more polar environment under high pressure. An analysis by the center of spectra mass (CSM) gave the ΔG and ΔV of transition as 9.8 kJ·mol−1 and −24 mL·mol−1 (pepsin) and 11.7 kJ·mol−1 and −43 mL·mol−1 (proteinase A), respectively, by assuming a simple two‐state transition. The circular dichroism (CD) showed relatively small changes after 1‐h incubations at 400 MPa, indicating that the secondary structures were largely maintained.
Targeting neighbouring poly(purine·pyrimidine) sequences located in the human bcr promoter by triplex‐forming oligonucleotides
Authors: Xodo Luigi E. , Rathinavelan Thenmalarchelvi , Quadrifoglio Franco , Manzini Giorgio , Yathindra Narayanarao 
Published: March 2003

Most poly(purine·pyrimidine) [poly(R·Y)] sequences in eukaryotic genomes are interrupted by one or more base pair inversions. When the inversions are centrally located, the poly(R·Y) sequences can be regarded as the sum of two abutting sites, each potentially capable of forming a triple helix. Employing band‐shift, footprinting and modeling methods we examined the formation of triple helices at a critical 27 bp poly(R·Y) sequence interrupted by two adjacent CG inversions, and located in the promoter of the human bcr gene at transcription initiation. We designed several 13‐mer and 14‐mer triplex‐forming oligonucleotides (TFOs) capable of binding the bcr abutting sites, thereby generating different base juxtapositions at the triple helical junction, to examine whether triplex formation occurs in a cooperative manner. It is found that in 50 mm Tris/HCl, pH 7.4, 10 mm MgCl2, 2 mm spermine, 37 °C, the 13‐and the 14‐mer TFOs bind to one half of the bcr site with ΔG between −30 and −35 kJ·mol−1. However, when different 13‐mer/14‐mer combinations of TFOs were directed against the abutting poly(R·Y) sites, triplex formation has been found to be enhanced only for the triple helical junction formed by the 5′‐A‐T‐3′ base juxtaposition, in keeping with a partial stacking suggested from modeling analysis. On the other hand, a longer 24‐mer TFO, binding noncooperatively to the same abutting sites, forms a much more stable triplex (ΔG = −51 kJ·mol−1), notwithstanding the two T·CG triads in the middle. Modeling investigations reveal that there is no continuity or propagation of base stacking involving adjacent bases of the third strand at the site of base inversion as well as on the 5′ side. The data indicate that the entropy penalty of forming a triplex with two oligonucleotides is much higher than the energy gained from base stacking interactions at the triplex junction formed between the two TFOs.
A polymorphic GT repeat from the human cardiac Na+Ca2+ exchanger intron 2 activates splicing
Authors: Gabellini Nadia 
Published: March 2003

The sequence analysis of the human intron 2 from the Na+/Ca2+ exchanger 1 (NCX1) gene has revealed a GT repeat of variable length (10–16). The 5′ sequence of intron 2 exhibited significant homology (65–70%) with other minisatellite sequences. DNA segments at the 5′ end of intron 2 were inserted in the NCX1 cDNA (3.7 kb) to reconstruct the exon 2/intron 2 junction. Transient expression of these constructs in HEK293 cells generated shortened mRNAs (≈ 2.5 kb). RT‐PCR and ribonuclease protection analysis of the 3′ end of the short transcripts indicated a splicing event at the intron 2/exon 2 junction (5′ site) and in the vector sequence downstream of the NCX1 insert (3′ site). Molecular dissection of the 5′‐intron 2 sequence showed that the GT repeat was required for splicing activation, whereas the remainder of the 5′‐intron 2 segment was completely inactive. The results indicate that the GT repeat is a strong intronic splicing enhancer that could be involved in the regulation of NCX1 expression, possibly mediating tissue‐specific alternative splicing of the mutually exclusive exons 3 and 4, and/or exon‐2 circularization.
FeMo cofactor biosynthesis in a nifE − mutant of Rhodobacter capsulatus
Authors: Siemann Stefan , Schneider Klaus , Behrens Kai , Knöchel Arndt , Klipp Werner , Müller A. 
Published: March 2003

In all diazotrophic micro‐organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase. In this study, we have found that strains of the phototrophic non‐sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild‐type strain. The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R. capsulatus. The in vivo activity, as measured by the routine acetylene‐reduction assay, was strictly Mo‐dependent. Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20–50 µm). Moreover, in these strains a high proportion of ethane was produced from acetylene (≈ 10% of ethylene) in vivo. However, in in vitro measurements with cell‐free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1%. The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q‐Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer). On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration. It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco‐containing MoFe protein molecules. The implications of our findings for the biosynthesis of the FeMoco will be discussed.
Protein kinase C regulates transcription of the human guanylate cyclase C gene
Authors: Roy Nivedita , Guruprasad Medigeshi R. , Kondaiah Paturu , Mann Elizabeth A. , Giannella Ralph A. , Visweswariah Sandhya S. 
Published: March 2003

Guanylate cyclase C is the receptor for the bacterial heat‐stable enterotoxins and guanylin family of peptides, and mediates its action by elevating intracellular cGMP levels. Potentiation of ligand‐stimulated activity of guanylate cyclase C in human colonic T84 cells is observed following activation of protein kinase C as a result of direct phosphorylation of guanylate cyclase C. Here, we show that prolonged exposure of cells to phorbol esters results in a decrease in guanylate cyclase C content in 4β‐phorbol 12‐myristate 13‐acetate‐treated cells, as a consequence of a decrease in guanylate cyclase C mRNA levels. The reduction in guanylate cyclase C mRNA was inhibited when cells were treated with 4β‐phorbol 12‐myristate 13‐acetate (PMA) in the presence of staurosporine, indicating that a primary phosphorylation event by protein kinase C triggered the reduction in RNA levels. The reduction in guanylate cyclase C mRNA levels was not due to alterations in the half‐life of guanylate cyclase C mRNA, but regulation occurred at the level of transcription of guanylate cyclase C mRNA. Expression in T84 cells of a guanylate cyclase C promoter‐luciferase reporter plasmid, containing 1973 bp of promoter sequence of the guanylate cyclase C gene, indicated that luciferase activity was reduced markedly on PMA treatment of cells, and the protein kinase C‐responsive element was present in a 129‐bp region of the promoter, containing a HNF4 binding element. Electrophoretic mobility shift assays using an oligonucleotide corresponding to the HNF4 binding site, indicated a decrease in binding of the factor to its cognate sequence in nuclear extracts prepared from PMA‐treated cells. We therefore show for the first time that regulation of guanylate cyclase C activity can be controlled at the transcriptional level by cross‐talk with signaling pathways that modulate protein kinase C activity. We also suggest a novel regulation of the HNF4 transcription factor by protein kinase C.