bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of exonucleolytic activities of herpes simplex virus type‐1 DNA polymerase and DNase
Authors: KNOPF Charles W. , WEISSHART Klaus 
Published: March 2005

The exonucleolytic activities associated with herpes simplex virus type‐1 (HSV‐1) DNA polymerase and DNase were compared. The unique properties of these nucleases were assessed by applying biochemical and immunological methods as well as by genetics. In contrast to the viral DNA polymerase, HSV DNase is equipped with a 5′–3′‐exonuclease activity. Under reaction conditions optimal for HSV DNA polymerase, i.e. at high ionic strength, HSV DNase exhibited only limited endonucleolytic activity and degraded double‐stranded DNA in a very processive manner and exclusively in the 5′–3′ direction, producing predominantly mononucleotides. Both viral enzymes displayed significant RNase activity which could be correlated with the endogenous endonucleolytic and 5′–3′‐exonucleolytic activities of the DNase and the polymerase‐associated 3′–5′ exonuclease.The tight linkage of polymerizing and exonucleolytic functions of the viral DNA polymerase was demonstrated by their identical response to (a) thermal inactivation, (b) drug inhibition and (c) neutralization by polyclonal antibodies reacting specifically with the N‐terminal, central and C‐terminal polypeptide domains of HSV‐1 DNA polymerase. From the data presented it can be concluded that the cryptic 3′–5′ exonuclease is the only exonucleolytic activity associated with the viral DNA polymerase.
A 19F‐NMR study of 3‐fluoro‐4‐demethoxydaunomycin intercalation complexes with the hexanucleotide d(CTGCAG)2
Authors: HAMMER Brendon C. , RUSSELL Richard A. , WARRENER Ronald N. , COLLINS J. Grant 
Published: March 2005

Equilibrium systems containing intercalation complexes formed between the novel anthracycline drug, 3‐fluoro‐4‐demethoxydaunomycin (3FD), and the hexanucleotide duplex d(CTGCAG)2 have been studied by 19FNMR spectroscopy. Solutions containing a 1:1 molar ratio of 3FD/d(CTGCAG)2 gave four 19F signals which have been assigned to each of four possible intercalation isomers for the 1:1 3FD · d(CTGCAG)2 complex, which we denote by [d(CTGCAG)2][3FD]; these were where 3FD bound between the 5′‐CT‐3′,5′‐TG‐3′, 5′‐GC‐3′ or 5′‐CA‐3′ base sequences, with the drug sugar moiety lying in the minor groove and pointed in the 3′ direction in each case. Changes in temperature and NaCl concentration affecting the equilibrium distribution of these isomers were studied and indicated that no overriding binding site preference prevailed under standard biochemical conditions.Formation of some of the 2:1 3FD · d(CTGCAG)2 complex occurred when a solution of [d(CTGCAG)2][3FD] was exposed to excess 3FD; however, this complex was unstable to gel filtration and no co‐operative binding of the second 3FD molecule was observed.
Nucleotide sequence, organisation and structural analysis of the products of genes in the nirB–cysG region of the Escherichia coli K‐12 chromosome
Authors: PEAKMAN Timothy , CROUZET Joel , MAYAUX Jean Francois , BUSBY Stephen , MOHAN Sudesh , HARBORNE Nerina , WOOTTON John , NICOLSON Rachel , COLE Jeffrey 
Published: March 2005

The DNA sequence and derived amino‐acid sequence of a 5618‐base region in the 74‐min area of the Escherichia coli chromosome has been determined in order to locate the structural gene, nirB, for the NADH‐dependent nitrite reductase and a gene, cysG, required for the synthesis of the sirohaem prosthetic group. Three additional open reading frames, nirD, nirE and nirC, were found between nirB and cysG.Potential binding sites on the NirB protein for NADH and FAD, as well as conserved central core and interface domains, were deduced by comparing the derived amino‐acid sequence with those of database proteins. A directly repeated sequence, which includes the motif ‐Cys‐Xaa‐Xaa‐Cys‐, is suggested as the binding site for either one [4Fe‐4S] or two [2Fe‐2S] clusters.The nirD gene potentially encodes a soluble, cytoplasmic protein of unknown function. No significant similarities were found between the derived amino‐acid sequence of NirD and either NirB or any other protein in the database. If the nirE open reading frame is translated, it would encode a 33‐amino‐acid peptide of unknown function which includes 8 phenylalanyl residues.The product of the nirC gene is a highly hydrophobic protein with regions of amino‐acid sequence similar to cytochrome oxidase polypeptide 1.
The measurement of the rotational diffusion coefficient of bovine plasma fibronectin by electric birefringence technique
Authors: VUILLARD Laurent , ROUX Bernard , MILLER Andrew 
Published: March 2005

Although the conformation of fibronectin has been widely investigated by various techniques, there has not yet been any determination of its rotational diffusion coefficient. We report here this determination by the transient electric birefringence study of solutions of bovine plasma fibronectin at physiological ionic strength.The solutions showed a positive birefringence. A linear relationship was observed between the intensity of the birefringence at equilibrium and the square of the electric field within the range of fields applied (up to 12.5 kV · cm−1). The field‐independent decay of the induced birefringence was described by a single exponential with a relaxation time of 0.76 (±0.08) μs at 23°C. This establishes fibronectin in solution as a globally rigid structure with a rotational diffusion coefficient, at 20°C, of 202000 s−1.This result allows the first rigorous determination of the low‐resolution structure of fibronectin. It is important to notice that the analysis combines only results obtained in physiological conditions on native molecules and follows a strict hydrodynamic interpretation. The conclusion of this work is that a hollow sphere of about 20 nm external diameter can be proposed as a model for the three‐dimensional structure of the fibronectin molecule in solution. This new model suggests the fibronectin could have the structure of a carrier protein.
Glycosphingolipids in insects
Authors: WESKE Bianka , DENNIS Roger D. , HELLING Friedhelm , KELLER Martin , NORES Gustavo A. , PETER‐KATALINIC Jasna , EGGE Heinz , DABROWSKI Ursula , WIEGANDT Herbert 
Published: March 2005

The two major components of the acidic glycolipid fraction from the pupae of Calliphora vicina were isolated using high‐performance liquid chromatography. The acidic moiety was identified as glucuronic acid by β‐glucuronidase cleavage and gas chromatographic analysis as the pentafluoropropionyl derivative. The structures of the carbohydrate moiety were elucidated by peracetylation, methylation, exoglycosidase cleavage, fast‐atom‐bombardment mass spectrometric and 1H‐nuclear magnetic resonance spectroscopic analysis. The only difference between the two hexasaccharide variants was the presence, in one of them, of a phosphoethanolamine (AeP) sidechain on the third sugar of the sequence, i. e. N‐acetylglucosamine. The composition of the ceramide moiety was dominated by a C20:0 fatty acid (arachidic acid) and a C14:1 sphingoid base (tetradecasphing‐4‐enine). The chemical structures of the two insect acidic glycosphingolipids were determined to be: GlcA(β‐3)Gal‐(β1‐3)GalNAc(β1 ‐4)GlcNAc(β1 ‐3)Man(β1‐4)Glc(β ‐1)Cer; GlcA(β1‐3)Gal(β1‐3)GalNAc(β1 ‐4)[2AeP‐6]‐GlcNAc(β1‐3)Man(β1 ‐4)Glc(β1‐1)Cer.Such glucuronic‐acid‐containing insect glycosphingolipids have been given the generic name arthrosides, with the implied synonymity to the gangliosides.
The mechanism by which rat liver glucokinase is inhibited by the regulatory protein
Authors: VANDERCAMMEN Annick , van SCHAFTINGEN Emile 
Published: March 2005

The fructose‐6‐phosphate‐sensitive and fructose‐1‐phosphate‐sensitive protein that inhibits rat liver glucokinase [Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179–184] was purified close to homogeneity by a procedure involving poly(ethyleneglycol) precipitation, chromatography on anion‐exchangers and hydroxylapatite, gel filtration and chromatography on Mono S, a cation exchanger. In the last chromatographic step, the regulatory protein coeluted with a 62 kDa peptide. From the elution volume of the gel‐filtration column a molecular mass of 60 kDa was determined, allowing the conclusion that the regulator is a monomer.The decrease in the apparent affinity of glucokinase for glucose, which the regulator induced, disappeared upon separation of the two proteins. Furthermore, the regulator did not appear to catalyse the formation of a heat‐stable or a trypsin‐resistant inhibitor of glucokinase. Finally, the inhibition exerted by the regulatory protein reached a steady value 1–2 min after the addition of the regulator. These results indicate that the regulator does not act by causing a covalent modification of glucokinase nor by catalysing the formation of a low‐molecular‐mass inhibitor.Raising the concentration of glucokinase in the assay from 6 mU/ml to 120 mU/ml caused a 2.5‐fold increase in the concentration of regulator required to half‐maximally inhibit the enzyme. The apparent mass of glucokinase, as determined by centrifugation in isokinetic sucrose gradient, was 55 kDa, and this value was unaffected by the separate presence of fructose 6‐phosphate or of the regulatory protein. However, the apparent mass of the enzyme increased to 105 kDa when glucokinase was centrifuged together with both fructose 6‐phosphate and the regulatory protein, although not when fructose 1‐phosphate was also present. Conversely, the presence of glucokinase increased the apparent molecular mass of the regulator in the presence of fructose 6‐phosphate. From these results, it is concluded that the regulatory protein inhibits glucokinase by forming a complex with this enzyme in the presence of fructose 6‐phosphate, and that fructose 1‐phosphate antagonises this inhibition by preventing the formation of the complex.
Phosphorothioate analogs of ATP as the substrates of dynein and ciliary or flagellar movement
Authors: SHIMIZU Takashi , OKUNO Makoto , MARCHESE‐RAGONA Silvio P. , JOHNSON Kenneth A. 
Published: March 2005

The phosphorothioate analog of ATP has a sulfur atom replacing a non‐bridging oxygen atom of the triphosphate moiety of ATP. Due to the tetrahedral nature of the phosphorus atom, stereoisomers are known to exist, designated as the Sp and Rp isomers. We have reported [Shimizu & Furusawa (1986) Biochemistry 25, 5787] on the hydrolytic activity of the 22S dynein from Tetrahymena cilia towards the phosphorothioate analogs of ATP. In this paper, we extend our study and report on the microtubule‐dynein dissociation by these analogs and on their ability to support sea urchin flagellar dynein enzymatic activity as well as ciliary or flagellar motility.It has been shown that the microtubule–22S‐dynein complex is dissociated by the binding of ATP to the dynein enzymatic sites [Porter & Johnson (1983) J. Biol. Chem. 258, 6575]. We studied the dissociation by adenosine 5′‐[α‐thio]triphosphate (ATP[αS]), Sp or Rp, by light‐scattering stopped‐flow methods. The dissociation by (Sp)ATP[αS] was rapid and the rate of the light‐scattering change by (Sp)ATP[αS] was a hyperbolic function of the nucleotide concentration, indicating that dissociation was a two‐step process. On the other hand, (Rp)ATP[αS] up to 2 mM induced only slow and partial dissociation of the complex, while, in the presence of vanadate, it induced complete dissociation with a slightly higher rate (0.5 s−1). The adenosine 5′‐[β‐thio]triphosphate (ATP[βS]) isomers did not induce dissociation.The hydrolytic activity of the outer arm dynein from sea urchin sperm flagella towards these analogs was similar to that of 22S dynein. The ratios of Vmax (nmol · mg protein−1· min−1)/apparent Km (μM) of this dynein were 400–720, 53, 9.7, 0.62 and 0.028 for ATP, ATP[αS] (Sp or Rp), ATP[βS] (Sp or Rp), respectively, in the presence of Mg2+ as the supporting cation. This dynein exhibited the same stereospecificity at β phosphate as the 22S dynein or myosin.The detergent models of Tetrahymena or sea urchin spermatozoa were reactivated only by ATP or (Sp)ATP[αS] while other analogs were ineffective. The maximal beat frequency of the cilia or flagella reactivated by (Sp)ATP[αS] was one‐quarter to one‐half of that produced by ATP reactivation.
Characterisation of tissue‐specific trans‐acting factor binding to a proximal element in the rat growth hormone gene promoter
Authors: LARKIN Sarah , TAIT Sarah , TREACY Maurice , MARTIN Finian 
Published: March 2005

Using an exonuclease III protection assay, tissue‐specific binding of rat pituitary tumour cell (GH3 cell) nuclear factors to a proximal region (−68 to −138) of the rat growth hormone gene promoter has been detected. The binding is particularly strong between the borders −68 to −102. The binding is eliminated in the presence of excess unlabelled rat growth hormone gene promoter sequences but also by proximal (−423 to +38) or distal (−1960 to −1260) rat prolactin gene promoter sequences and simian virus 40 enhancer/promoter sequences. Extracts of rat pituitaries showed identical binding characteristics. Methylation interference analysis indicated that the contact points between the pituitary‐specific factor and the proximal rat growth hormone gene promoter‐binding element (−65 to −95) are over a conserved sequence which occurs twice in the rat growth hormone gene promoter and at least eight times in the rat prolactin gene 5′‐flanking sequences. This sequence has previously been proposed to constitute the binding site for the somatotroph/lactotroph tissue‐specific transcription factor.Gel‐retardation and exonuclease III competition analysis showed that three of the rat prolactin gene promoter elements (−46 to −71, −156 to −180 and −174 to −204) share the ability to bind the pituitary‐specific factor. The binding to the most proximal rat prolactin gene promoter element (−46 to −71) was clearly more avid than to the rat growth hormone gene promoter (−65 to −95) proximal element. However, both these elements displayed the formation of two gel‐retarded complexes while the more distal rat prolactin gene binding elements (−156 to −180 and −174 to −204) formed only the smaller of the two complexes.Finally, we demonstrated by co‐transfection competition analysis that plasmids containing the most proximal rat prolactin gene promoter binding element completely inhibited transcription from the rat growth hormone gene promoter while rat growth hormone gene promoter sequences only partially inhibited transcription from the rat prolactin gene promoter. This suggests that the higher affinity for factor binding displayed by the proximal rat prolactin gene promoter binding site in vitro is reflected in factor binding activity in vivo.
Function of oligosaccharide modification in glucocerebrosidase, a membrane‐associated lysosomal hydrolase
Authors: VAN WEELY Sonja , AERTS Johannes M. F. G. , VAN LEEUWEN Marinella B. , HEIKOOP Judith C. , DONKER‐KOOPMAN Wilma E. , BARRANGER John A. , TAGER Joseph M. , SCHRAM André W. 
Published: March 2005

The nature and function of oligosaccharide modification in glucocerebrosidase, a membrane‐associated lysosomal hydrolase, have been investigated in cultured human skin fibroblasts.Glucocerebrosidase is synthesised as a 62.5‐kDa precursor with high‐mannose‐type oligosaccharide chains and an apparent native isoelectric point of 6.0–7.0. Subsequent processing of the oligosaccharide moieties to sialylated complex‐type structures results in formation of 65–68‐kDa forms of the enzyme with apparent native isoelectric points of 4.3–5.0. These forms are transported to lysosomes and subsequently modified by the sequential action of lysosomal exoglycosidases, finally resulting in a 59‐kDa form with an isoelectric point near neutrality. The existence of oligosaccharide modification of the enzyme in the lysosomes is illustrated by the accumulation of different intermediate forms of glucocerebrosidase in mutant cell lines deficient in lysosomal exoglycosidases. The enzyme does not undergo proteolytic modification during maturation.The possible physiological relevance of the oligosaccharide modification of glucocerebrosidase in the lysosomes was investigated by studying the properties of the enzyme in fibroblasts deficient in lysosomal exoglycosidases, and also the properties of homogeneous pure glucocerebrosidase from placenta, modified in the oligosaccharide moieties by digestion in vitro with glycosidases. Modification of the oligosaccharide moieties of glucocerebrosidase had no significant effect on the catalytic activity of the enzyme as measured with either artificial or natural substrates in the presence of artificial or natural activators. There was also no effect of modification of the oligosaccharide chains on the intracellular stability of the enzyme or on its apparent hydrophobicity. We conclude that oligosaccharide modification of glucocerebrosidase in the lysosomes simply reflects further maturation of the enzyme in the lysosome and is of no importance to its function.
The oligosaccharides of the glycoprotein pheromone of Volvox carteri f. nagariensis Iyengar (Chlorophyceae)
Authors: BALSHÜSEMANN Dirk , JAENICKE Lothar 
Published: March 2005

The sexuality‐inducing glycoprotein of Volvox carteri f. nagariensis was purified from supernatants of disintegrated sperm packets of the male strain IPS‐22 and separated by reverse‐phase HPLC into several isoforms which differ in the degree of O‐glycosylation. Total chemical deglycosylation with trifluoromethanesulphonic acid yields the biologically inactive core protein of 22.5 kDa. This core protein possesses three putative binding sites for N‐glycans which are clustered in the middle of the polypeptide chain. The N‐glycosidically bound oligosaccharides were obtained by glycopeptidase F digestion and were shown by a combination of exoglycosidase digestion, gas‐chromatographic sugar analysis and two‐dimensional HPLC separation to possess the following definite structures:

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Two of the three N‐glycosidic binding sites carry one B and one D glycan. The A and C glycans are shared by the third N‐glycosylation site. The O‐glycosidic sugars, which make up 50% of the total carbohydrate, are short (up to three sugar residues) chains composed of Ara, Gal and Xyl and are exclusively bound to Thr residues.
Biochemical and immunochemical studies of proteolytic fragments of exotoxin A from Pseudomonas aeruginosa
Authors: BOURDENET Sylviane , VACHERON Marie‐Jeanne , GUINAND Micheline , MICHEL Georges , ARMINJON François 
Published: March 2005

Limited proteolysis of Pseudomonas aeruginosa exotoxin A by four proteases (chymotrypsin, Staphylococcal serine proteinase, pepsin A and subtilisin) resulted in the formation of polypeptides having a molecular mass of approximately 25 kDa. They possessed both enzymatic activity and residual antigenicity. Their N‐terminal sequence analysis showed that the different proteases cleaved exotoxin A in a very restricted area within domain Ib (amino acids 365–404). As a result, the polypeptides contained a large portion (13–34 amino acids) of domain Ib linked to the adjacent C‐terminal domain III (amino acids 405–613). The major fragment derived from subtilisin cleavage, at a final yield of 35% (S‐fragment; residues 392–613; 24201 Da; pI 4.7) possessed the same level of ADP‐ribosyltransferase activity as uncleaved exotoxin A (by mass), and a 37‐fold higher NAD‐glycohydrolase activity. Polyclonal antibodies from rabbits against exotoxin A completely inhibited the ADP‐ribosyltransferase activity of both exotoxin A and the S‐fragment, but not the NAD‐glycohydrolase activity of the S‐fragment. Antibodies against the S‐fragment neutralized the ADP‐ribosyltransferase activity of exotoxin A.These data determine the primary proteolytic cleavage site of exotoxin A, suggest that some residues in the amino acid sequence 392–404 of exotoxin A seem to have a role in binding or positioning elongation factor 2 (EF‐2) and show that antibodies recognize the EF‐2‐binding site but not the NAD+‐binding site.
Inhibition of inositol 1,4,5‐trisphosphate metabolism in permeabilised SH‐SY5Y human neuroblastoma cells by a phosphorothioate‐containing analogue of inositol 1,4,5‐trisphosphate
Authors: WOJCIKIEWICZ Richard J. H. , COOKE Allan M. , POTTER Barry V. L. , NAHORSKI Stefan R. 
Published: March 2005

Electrically permeabilised [3H]inositol‐labelled SH‐SY5Y human neuroblastoma cells were employed to examine the effects of two synthetic, phosphatase‐resistant analogues of inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] on the metabolism of cell membrane‐derived [3H]Ins(1,4,5)P3 or exogenous [5‐32P]Ins(1,4,5)P3.Incubation of permeabilised SH‐SY5Y cells for 5 min at 37°C with carbachol and guanosine 5′‐[γ‐thio]triphosphate caused a decrease in [3H]phosphoinositol phospholipid levels and an increase in [3H]inositol phosphate accumulation with inositol 4‐phosphate, inositol 1,4‐bisphosphate, Ins(1,4,5)P3 and inositol 1,3,4,5‐tetrakisphosphate comprising approximately 79%, 16%, 3% and 2%, respectively, of the increase. Inositol 1‐phosphate levels did not increase upon stimulation, nor was inositol 4‐phosphate converted rapidly to inositol.In parallel incubations, the analogues, DL‐inositol 1,4,5‐trisphosphorothioate (DL‐InsP3S3) and DL‐inositol 1,4‐bisphosphate 5‐phosphorothioate (DL‐InsP3S), and synthetic racemic Ins(1,4,5)P3 (DL‐InsP3), altered the profile of the [3H]inositol phosphates recovered and led, at millimolar concentrations, to a 10–15‐fold increase in [3H]Ins(1,4,5)P3. The extent of inhibition of [3H]Ins(1,4,5)P3 metabolism was, however, greatest in the presence of synthetic D‐Ins(1,4,5)P3 (5 mM), when [3H]Ins(1,4,5)P3 comprised approximately 50% of the increase in total [3H]inositol phosphates. Thus, under these conditions, at least 50% of [3H]inositol phosphates were derived from [3H]phosphatidylinositol 4,5‐bisphosphate. [32P]Pi release from exogenous [5‐32P]Ins(1,4,5)P3 was also inhibited by DL‐InsP3S3, DL‐InsP3S and DL‐InsP3, with half‐maximal inhibition at approximately 50 μM, 160 μM and 240 μM respectively. These actions were approximately ten times more potent than the effects of these compounds on [3H]inositol phosphate accumulation, indicating that homogenous mixing of exogenous and cellmembrane‐derived Ins(1,4,5)P3 does not occur.These findings indicate that DL‐InsP3S3 and DL‐InsP3S inhibit Ins(1,4,5)P3 5‐phosphatase. In contrast. the effects of synthetic DL‐InsP3 and d‐Ins(1,4,5)P3 are due to isotopic dilution. Whilst DL‐InsP3S3 was the most potent inhibitor of dephosphorylation of exogenous or cell‐membrane‐derived Ins(1,4,5)P3, it was the weakest inhibitor of 3‐kinase‐catalysed Ins(1,4,5)P3 phosphorylation. Similarly, although approximately 50 times less potent than DL‐InsP3S3, 2,3‐diphosphoglycerate inhibited Ins(1,4,5)P3 5‐phosphatase activity and was apparently without effect of Ins(1,4,5)P3 3‐kinase activity.These data show that both DL‐InsP3S3 and DL‐InsP3S are potent inhibitors of Ins(1,4,5)P3 5‐phosphatase. In contrast, phosphorylation of Ins(1,4,5)P3 was inhibited potently only by DL‐InsP3S.
Plasma membrane Fe2‐transferrin reductase and iron uptake in K562 cells are not directly related
Authors: GOLDENBERG Hans , DODEL Beate , SEIDL Dagmar 
Published: March 2005

Receptor‐mediated endocytosis and recycling of transferrin is partly inhibited by the ferrous iron chelator bipyridine, which almost completely blocks iron uptake. Bipyridine causes iron release at the cell surface, but inhibition of iron uptake is also due to a blockage of its passage through the endosomal membrane. The rate of release of iron to bipyridine is decreased by the competing electron acceptor ferricyanide and by amiloride, but not by iron uptake inhibiting acidotropic amines.Transferrin reduction at the plasma membrane may be artificially induced by presence of a ferrous chelator and caused by low‐affinity transmembrane NAD(P)H oxidoreductase.