bluuebunny/crossref_metadata_2025_split huggingface dataset:
Influence of the supramolecular structure of free lipid A on its biological activity
Authors: BRANDENBURG Klaus , MAYER Hubert , KOCH Michel H. J. , WECKESSER Jürgen , RIETSCHEL Ernst Th. , SEYDEL Ulrich 
Published: March 2005

The three‐dimensional supramolecular structures and the states of order of the acyl chains of lipid A from different Gram‐negative species were investigated at 40°C, high water content (80–90%), and different [lipid A]/[Mg2+] molar ratios using synchrotron radiation X‐ray diffraction and Fourier‐transform infrared spectroscopy. Measurements were made on free lipid A from Salmonella minnesota R595, mono‐ and bi‐phosphoryl, as well as those from the non‐enterobacterial strains Rhodobacter capsulatus 37 b4, Rhodopseudomonas viridis F, and Rhodocyclus gelatinosus 29/1. Parallel to differences in their chemical primary structure, the structural polymorphisms and states of order at 37°C of the non‐enterobacterial lipid A were found to be different from those of enterobacterial lipid A. A clear correlation between the supramolecular structure and previously determined biological activities was found. Lipid A with a strong preference for lamellar structures (Rb. capsulatus and Rp. viridis) are endotoxically inactive and lack cytokine‐inducing capacity; the compounds assuming a mixed lamellar/nonlamellar structure (monophosphoryl lipid A from S. minnesota) are of lower toxicity in vivo, but may induce cytokines in vitro; those lipid A with a strong tendency to form non‐lamellar inverted structures (lipid A from S. minnesota and Rc. gelatinosus) exhibit full endotoxicity in vitro and in vivo. In contrast, anti‐complementary activity is most pronounced for compounds with lamellar and least expressed for those with inverted structures. The states of order at 37°C vary non‐systematically, exhibiting the highest values for lipid A of S. minnesota and the lowest for that of Rc. gelatinosus.We propose to extend the term ‘endotoxic conformation’, which is used to describe the conformation of a single lipid A molecule required for optimal triggering of biological effects, to ‘endotoxic supramolecular conformation’ which denotes the particular organization of lipid A aggregates in physiological fluids causing biological activity.
Gene structure of CYP3A4, an adult‐specific form of cytochrome P450 in human livers, and its transcriptional control
Authors: HASHIMOTO Hisashi , TOIDE Kenji , KITAMURA Ryuji , FUJITA Masako , TAGAWA Sanae , ITOH Susumu , KAMATAKI Tetsuya 
Published: March 2005

CYP3A4 is the adult‐specific form of cytochrome P450 in human livers [Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K. & Kamataki, T. (1990) Biochemistry 29, 4430–4433]. The sequences of three genomic clones for CYP3A4 were analyzed for all exons, exon‐intron junctions and the 5′‐flanking region from the major transcription site to nucleotide position ‐1105, and compared with those of the CYP3A7 gene, a fetal‐specific form of cytochrome P450 in humans. The results showed that the identity of 5′‐flanking sequences between CYP3A4 and CYP3A7 genes was 91%, and that each 5′‐flanking region had characteristic sequences termed as NFSE (P450NF‐specific element) and HFLaSE (P450HFLa specific element), respectively. A basic transcription element (BTE) also lay in the 5′‐flanking region of the CYP3A4 gene as seen in many CYP genes [Yanagida, A., Sogawa, K., Yasumoto, K. & Fujii‐Kuriyama, Y. (1990) Mol. Cell. Biol. 10, 1470–1475]. The BTE binding factor (BTEB) was present in both adult and fetal human livers.To examine the transcriptional activity of the CYP3A4 gene, DNA fragments in the 5′‐flanking region of the gene were inserted in front of the simian virus 40 promoter and the chloramphenicol acetyltransferase structural gene, and the constructs were transfected in HepG2 cells. The analysis of the chloramphenicol acetyltransferase activity indicated that (a) specific element(s) which could bind with a factor(s) in livers was present in the 5′‐flanking region of the CYP3A4 gene to show the transcriptional activity.
Effect of proglycosyn and other phenolic compounds on glycogen metabolism in isolated hepatocytes
Authors: VAN SCHAFTINGEN Emile , DE HOFFMANN Edmond 
Published: March 2005

The mechanism by which proglycosyn (LY 177 507) stimulates glycogen synthesis in isolated hepatocytes [Harris, R. A., Yamanouchi, K., Roach, P. J., Yen, T. T., Dominiani, S. J. & Stephens, T. W. (1989) J. Biol. Chem. 264, 13674–13680] has been investigated. When incubated in the presence of hepatocytes, proglycosyn was metabolized to an O‐demethylated glucuronidated derivative, as determined by fast‐atom‐bombardment mass spectrometry and enzymic analysis. This metabolite accumulated almost linearly inside the cells to reach a concentration of approximately 3 μmol/g protein after 50 min, without apparent release into the medium. In confirmation of previous work, proglycosyn decreased the level of phosphorylase a and increased that of synthase a in hepatocytes. Washing of cells incubated with proglycosyn for 30 min considerably decreased the concentration of the drug without significantly modifying the intracellular concentration of the metabolite and the activation state of glycogen synthase.Several compounds bearing structural analogy with proglycosyn were also tested for their effect on glycogen metabolism. At millimolar or submillimolar concentrations, resorcinol, m‐anisidine, phenol, 3‐hydroxyacetophenone, and 3‐acetamidophenol, although not 4‐acetamidophenol, stimulated the incorporation of [14C]glucose into glycogen, decreased the level of phosphorylase a and increased the level of synthase a. In the case of phenol, the effect on the glycogen enzymes paralleled the intracellular accumulation of phenylglucuronide. Furthermore, ethanol and D‐galactosamine, which decreased the conversion of phenol to phenylglucuronide and the intracellular concentration of phenylglucuronide, counteracted the effect of phenol on the synthase and on the phosphorylase.From these results, it is suggested that the effect of proglycosyn and of simpler phenol derivatives is mediated by glucuronidated metabolites, which act on an intracellular target.
Structures of the N‐linked oligosaccharides on porcine plasma vitronectin
Authors: YONEDA Atsuko , OGAWA Haruko , MATSUMOTO Isamu , ISHIZUKA Ineo , HASE Sumihiro , SENO Nobuko 
Published: March 2005

The structures of N‐linked oligosaccharides, especially the distribution of sialic acid species, present on porcine plasma vitronectin were elucidated. Oligosaccharides were released from the vitronectin by N‐glycosidase F digestion and tagged with 2‐aminopyridine, and the pyridylamino‐oligosaccharides were fractionated by anion‐exchange and reverse‐phase HPLC. Nine major pyridylamino‐oligosaccharides were isolated. The linkages and locations of sialic acids were determined by a novel approach involving desialylation with Salmonella sialidase in combination with acid desialylation. After desialylation, the asialo‐forms were analyzed by two‐dimensional sugar mapping, component sugar analysis and 400‐MHz 1H‐NMR spectroscopy.The major oligosaccharides of porcine vitronectin were of the fucosylated biantennary type, with a small amount of the triantennary N‐acetyllactosamine type, to which 1–3 mol sialic acids was linked. Sialic acids were linked predominantly through α2–6 linkages, although α2–3 linkages were also present, and fucose was linked to the innermost N‐acetylglucosamine through an α1–6 linkage. It was found that every pyridylamino‐oligosaccharide population contained N‐glycolylneuraminic acid and N‐acetylneuraminic acid in a molar ratio of 1:2–9, and that N‐glycolylneuraminic acids were located predominantly on the Man α1–6 arm.
Comparison of the dynamic structure of α‐chymotrypsin in aqueous solution and in reversed micelles by fluorescent active‐site probing
Authors: DOROVSKA‐TARAN Victoria N. , VEEGER Cees , VISSER Antonie J. W. G. 
Published: March 2005

A highly fluorescent anthraniloyl (Ant) group was covalently attached to the active site of α‐chymotrypsin (CT), probably at Ser195. Ant‐CT is stable at neutral pH for months, allowing a detailed fluorescence study of Ant‐CT as a model protein to investigate its physical properties in 0.1 M Tris/HCl, pH 8.2, and in reversed micelles of n‐octane, 0.1 M Tris/HCl, pH 8.2, and sodium bis(2‐ethylhexyl)sulfosuccinate (AOT).Steady‐state fluorescence measurements of the progressive red‐shift of the center of gravity of the emission band as function of degree of hydration, wo, defined as [H2O]/[AOT], indicate that the average polarity in the vicinity of the probe is approaching that of bulk water at wo > 12. Time‐resolved fluorescence measurements of Ant‐CT in water and in reversed micelles showed that the active site has different properties in reversed micelles compared to those in water. Some specific changes at very low water content (0.6 < wo < 5) can be observed, which correlate with enzyme activity measurements in the same wo region (unpublished results). These effects are, for instance, significant changes in the average fluorescence lifetime and the internal flexibility of the probe. The overall rotational‐correlation time of the enzyme in AOT reversed micelles seems to be independent on wo (5 < wo < 29), which suggests that the enzyme creates its own micelle.
Purification and characterization of ribonucleoproteins from pea chloroplasts
Authors: SUBBAIAH Chalivendra Chenchu , TEWARI Krishna K. 
Published: March 2005

RNA‐binding proteins are known to mediate the post‐transcriptional regulation of genes in many organisms. Recently they have been found to be important in the expression of plastid genes.We have purified a group of three single‐stranded nucleic‐acid‐specific acidic proteins (33, 30 and 28 kDa) from chloroplast extracts of pea (Pisum sativum L.), using single‐stranded DNA affinity chromatography. All of them have acidic amino termini but the amino acid sequences are unique to each polypeptide, with partial similarities to the recently reported ribonucleoproteins from tobacco chloroplasts. The pea proteins are also antigenically distinct, as shown by Western blot analysis using polyclonal antisera for purified proteins. Further, from their large nucleic‐acid‐binding domains and the polynucleotide substrate affinities, they are predicted to belong to a family of pea plastid ribonucleoproteins. In vivo radiolabeling of proteins in the presence of translational inhibitors as well as in vitro translation of leaf tissue RNA suggest that these proteins are encoded in the nucleus. Antibody cross‐reactivity experiments reveal that their genes are conserved during plastid evolution.
Differential insertion of insulin receptor complexes into Triton X‐114 bilayer membranes
Authors: FLÖRKE Ralf‐Rüdiger , KLEIN Helmut W. , REINAUER Hans 
Published: March 2005

In the present study, the Triton X‐114 phase‐separation system has been used to characterize molecular properties of the membrane‐exposed domain of an integral‐membrane hormone receptor. This approach provides novel details of the structure/function relationship of insulin receptors.Upon raising the temperature of a micellar Triton X‐114 solution above the cloud‐point, a detergent enriched phase pellets and coprecipitates 95% of the purified insulin‐free (αβ)2 receptors. In contrast, 83% of the hormone bound (αβ)2 receptor complexes prefer the detergent‐depleted phase, exhibiting prominent properties of non‐membraneous proteins. Kinetic studies show that, following insulin binding, the amphiphilicity of the receptor complexes is immediately altered. Only monodisperse (αβ)2 complexes were detected when receptor/insulin complexes of the detergent‐depleted phase were analyzed by detergent‐free sucrose density centrifugation in the presence of 10 nM insulin. These results can be explained in the light of the lipid‐bilayer‐like organization of the precipitating Triton X‐114; hormone‐induced intramolecular alterations of (αβ)2 receptors appear to fundamentally restrict access to the membrane‐exposed receptor domain.Basically, different molecular properties are found for αβ receptors. Only 67% of the insulin‐free αβ receptors coprecipitate with the Triton‐X‐114‐enriched phase; following insulin binding the coprecipitation is only decreased to 42%. In contrast to (αβ)2 receptors, formation of noncovalently aggregated receptor complexes, which are detected by sucrose density centrifugation, could account for the exclusion of αβ receptor species from Triton X‐114 membranes.
cDNA cloning, structural features, and eucaryotic expression of human TAG‐1/axonin‐1
Authors: HASLER Thomas H. , RADER Christoph , STOECKLI Esther T. , ZUELLIG Richard A. , SONDEREGGER Peter 
Published: March 2005

Axonal surface glycoproteins, composed of repeated immunoglobulin‐like and fibronectin‐type‐III(FNIII)‐like domains, mediate adhesion between axons or between axons and non‐neuronal cells or extracellular matrix proteins. Several representatives of this group promote neurite outgrowth, when presented as substratum to neurons in culture, and have been implicated in axonal guidance mechanisms. TAG‐1 and axonin‐1 are presumptive species homologues of the rat and the chick, respectively; together with F11/F3, they form a subgroup of Ig/FNIII‐like molecules containing a glycosyl‐PtdIns membrane anchor.Recent reports on tumor suppressor genes encoding Ig‐like and FNIII‐like sequences prompted us to isolate the human homologue to TAG‐1 and axonin‐1. Polymerase chain reaction (PCR) primers were designed to regions conserved in both TAG‐1 and axonin‐1 using deoxyinosine at ambiguous positions. An expected 1000‐bp fragment was obtained from cDNA derived from adult human cerebellum. Using this PCR fragment as a probe, several clones were isolated from a human fetal brain cDNA library. Nucleotide sequence analysis of a full‐length clone, as expected, revealed a high degree of similarity to rat TAG‐1 (91% identity) and chicken axonin‐1 (75% identity) at the amino acid level. The encoded protein was then transiently expressed in monkey COS1 cells, and a stable mouse myeloma cell line was established expressing human TAG‐1/axonin‐1. The transfected COS1 and myeloma cells showed immunoreactivity on the cell surface with polyclonal anti‐(chicken axonin‐1) serum. On Western blots, the same antibodies recognized the recombinant protein migrating slightly slower on SDS/PAGE than chicken axonin‐1. A comparison of chicken and human braintissue proteins by Western‐blot analysis revealed a similar apparent molecular mass difference between the two species, which might be due to three additional N‐glycosylation sites present on human TAG‐1/axonin‐1. Immunostaining of cryostat sections of embryonic retinas with polyclonal anti‐(axonin‐1) serum showed similar expression patterns in chicken and human samples at corresponding developmental stages. An additional shared feature of human TAG‐1/axonin‐1, rat TAG‐1 and chick axonin‐1 is their attachment to the cell membrane with a glycosyl‐PtdIns anchor.
Structure and Modifications of the Junior Chaperone α‐Crystallin
Authors: Groenen Patricia J. T. A. , Merck Karin B. , De Jong Wilfried W. , Bloemendal Hans 
Published: March 2005

α‐Crystallin is a high‐molecular‐mass protein that for many decades was thought to be one of the rare real organ‐specific proteins. This protein exists as an aggregate of about 800 kDa, but its composition is simple. Only two closely related subunits termed αA‐ and αB‐crystallin, with molecular masses of approximately 20 kDa, form the building blocks of the aggregate.The idea of organ‐specificity had to be abandoned when it was discovered that α‐crystallin occurs in a great variety of nonlenticular tissues, notably heart, kidney, striated muscle and several tumors. Moreover αB‐crystallin is a major component of ubiquinated inclusion bodies in human degenerative diseases.An earlier excitement arose when it was found that αB‐crystallin, due to its very similar structural and functional properties, belongs to the heat‐shock protein family. Eventually the chaperone nature of α‐crystallin could be demonstrated unequivocally.All these unexpected findings make α‐crystallin a subject of great interest far beyond the lens research field.A survey of structural data about α‐crystallin is presented here. Since α‐crystallin has resisted crystallization, only theoretical models of its three‐dimensional structure are available. Due to its long life in the eye lens, α‐crystallin is one of the best studied proteins with respect to post‐translational modifications, including age‐induced alterations. Because of its similarities with the small heat‐shock proteins, the findings about α‐crystallin are illuminative for the latter proteins as well.This review deals with: structural aspects, post‐translational modifications (including deamidation, racemization, phosphorylation, acetylation, glycation, age‐dependent truncation), the occurrence outside of the eye lens, the heat‐shock relation and the chaperone activity of α‐crystallin.
Protein Structure of Fetal Antigen 1 (FA1)
Authors: Jensen C. Harken , Krogh Thomas N. , Højrup Peter , Clausen Per P. , Skjødt Karsten , Larsson Lars‐Inge , Enghild Jan J. , Teisner Børge 
Published: March 2005

The present paper describes the primary structure, glycosylation and tissue localization of fetal antigen 1 (FA1) isolated from second‐trimester human amniotic fluid. FA1 is a single‐chained, heterogeneous glycoprotein of 225–262 amino acid residues. FA1 has six well conserved epidermal‐growth‐factor motifs and contains up to ten O ‐glycosylation and N‐glycosylation sites, six of which are differentially glycosylated. Alignment to the translated sequences of Mus. musculus dlk and human dlk revealed 86% and 99% identity, respectively, to a 259‐amino‐acid residue overlap, and this high similarity extends with minor corrections to the human adrenal‐specific mRNA, pG2 as well. Immunohistochemical analysis demonstrated the presence of FA1 in 10 out of 14 lung tumors containing neuroendocrine elements, and in the placental villi where FA1 was exclusively seen in stromal cells in close contact to the vascular structure. In the pancreas, FA1 co‐localized with insulin in the insulin secretory granules of the β cells within the islets of Langerhans.Our findings suggest that FA1 is synthesized as a membrane anchored protein and released into the circulation after enzymic cleavage, and that circulating FA1 represents the post‐translationally modified gene product of human dlk which, in turn, is identical to human adrenal‐specific mRNA pG2.
GTP‐blot Analysis of Small GTP‐binding Proteins
Authors: Klinz Franz‐Josef 
Published: March 2005

Recombinant c‐Ha‐ras, ralA and rap2, but not rap1A or rap1B proteins retained their ability to bind [α‐32P] GTP after SDS/PAGE and transfer to nitrocellulose. Recombinant c‐Ha‐ras missing the C‐terminal 23 amino acid residues failed to bind [α‐32P] GTP after the blot, and the ability of recombinant ralA missing the C‐terminal 28 amino acid residues to bind [α‐32P] GTP was decreased manyfold. The presence of nonionic detergents of the polyoxyethylene type such as Tween 20, Triton X‐100, Nonidet P40 or Lubrol PX in the incubation buffer was necessary to induce renaturation of blotted recombinant c‐Ha‐ras protein, whereas other types of detergents were ineffective. We propose that detergents of the polyoxyethylene type induce the refolding of some types of blotted small GTP‐binding proteins and that the C‐terminus is involved in the refolding process.Membranes from NIH3T3 fibroblasts overexpressing c‐Ha‐ras protein showed much weaker binding of [α‐32P] GTP as expected from the level of ras immunoreactivity. Treatment of fibroblasts with lovastatin, an inhibitor of hydroxymethylglutaryl‐coenzyme A reductase, caused the accumulation of the unfarnesylated form of c‐Ha‐ras in the cytosol. Examination of [α‐32P] GTP‐binding and immunoreactivity for cytosolic and membrane‐bound c‐Ha‐ras revealed that binding of [α‐32P] GTP to unprocessed c‐Ha‐ras was increased about threefold compared to the same amount of processed c‐Ha‐ras.Our results demonstrate that detection and quantification of small GTP‐binding proteins in eukaryotic cells by GTP‐blot analysis is hampered by the fact that these proteins differ strongly in their ability to renature after blotting to nitrocellulose.
The Stability and Steady‐State Kinetics of Vanadium Chloroperoxidase from the Fungus Curvularia Inaequalis
Authors: Van Schijndel Johannes W. P. M. , Barnett Philip , Roelse Jeanet , Vollenbroek Esther G. M. , Wever Ronald 
Published: March 2005

In this article we report on the steady‐state kinetics of the chlorination and the stability of the vanadium chloroperoxidase from the fungus Curvularia inaequalis. The data show that the kinetics of this enzyme resemble that of the vanadium bromoperoxidase from the seaweed Ascophyllum nodosum. At low pH, chloride inhibited the enzyme, but the inhibition was of a dual nature. At pH 4.1 a mixed type of inhibition by chloride with respect to hydrogen peroxide was observed whereas at pH 3.1 the nature of the inhibition became competitive. The log Km, for hydrogen peroxide decreased linearly with pH with a slope of –1 in the pH range 3–5. A reaction mechanism is presented to explain the observed data.We also showed that this class of enzymes is inhibited by nitrate. At pH 5.5, nitrate inhibits the chlorination reaction competetively with respect to chloride (Ki= 2 mM) and uncompetetively with respect to hydrogen peroxide. Furthermore, we showed that the enzyme produces HOCl as a reaction product. The enzyme exhibited a high thermostability (tm of 90°C) and displayed high stability in organic solvents (solutions of 40% methanol, ethanol or 2‐propanol) and moderate stability in the presence of the chaotropic agent guanidine/HCl (G1/2, the concentration of guanidine/HCl at which the enzyme activity was half the original activity was 3.7 M).