bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein flexibility and aggregation state of human epidermal growth factor
Authors: GALLAY Jacques , VINCENT Michel , Li de la SIERRA Ines M. , ALVAREZ Julio , UBIETA Raimundo , MADRAZO Juan , PADRON Gabriel
Published: March 2005

A time‐resolved fluorescence spectroscopic study of the recombinant human epidermal growth factor (hEGF), a bis(tryptophan)‐containing protein (Trp49‐Trp50), and of the two single‐tryptophan‐containing engineered mutants with Trp49 or Trp50 replaced by Phe ([W49F]hEGF, [W50F]hEGF), was undertaken in order to gain insight into the conformational dynamics of the C‐terminal region. Quite different position‐dependent microenvironments for the two Trp residues are shown by comparing the fluorescence intensity decay of both mutants. Trp50 in the single‐tryptophan mutant [W49F]EGF probably undergoes a dominant interaction with the solvent. A more heterogeneous environment of Trp49 in the [W50F]hEGF mutant is found. Moreover, the fluorescence decay of the native hEGF is not simply the additive result of the decays of both mutants: the Trp2 sequence confers a conformation of the C‐terminal sequence which is more in contact with the rest of the protein molecule. By contrast, the fluorescence anisotropy decay of the native protein is quite similar to that of the single‐tryptophan mutants. A high degree of rotational freedom in the C‐terminal region of the protein is demonstrated. The resonance energy transfer, which could contribute to the anisotropy decay, appears therefore not to be highly efficient with respect to the depolarization motions. In addition to these local conformational and dynamic aspects of the hEGF C‐terminal sequence, the fluorescence anisotropy decay data demonstrate the existence of a dimerization process of the native protein which is dependent on pH and protein concentration. This phenomenon influences the excited‐state lifetime profiles and, therefore, the local conformational equilibrium of the C‐terminal region.
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.
Chemical assay for cyst(e)ine‐rich peptides detects a novel intestinal peptide ZF‐1, homologous to a single zinc‐finger motif
Authors: SILLARD Rannar , JÖRNVALL Hans , CARLQUIST Mats , MUTT Viktor
Published: March 2005

Cysteine is a relatively infrequent constituent of proteins, which in its thiol or half‐cystine form contributes in a special manner to their three‐dimensional structure. We show that in small cystine‐containing peptides, the Cys content is always higher than the average in proteins in general. This observation makes it possible to search for new peptides by monitoring only their Cys content. We have developed a chemical assay for the detection of cyst(e)ine‐rich peptides in tissue extracts. Using this assay we have isolated from porcine intestine a novel cysteine‐rich peptide, which we denote ZF‐1. ZF‐1 is homologous to a single zinc‐finger motif and has an acetylated N‐terminus. This is the first demonstration of the existence of a processed single zinc‐finger‐like structure. The structural homology of ZF‐1 to the zinc‐finger motif, present in several metal‐binding and DNA‐binding proteins, suggests an important role of this peptide in metal transport and/or modulation of gene expression.
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.
Acetylcholinesterase and Butyrylcholinesterase Expression in Adult Rabbit Tissues and During Development
Authors: Jbilo Omar , L'hermite Yann , Talesa Vincenzo , Toutant Jean‐Pierre , Chatonnet Arnaud
Published: March 2005

A large cDNA fragment covering the complete sequence of the mature catalytic subunit of rabbit acetylcholinesterase (AChE) has been cloned and sequenced. This sequence was compared to that of rabbit butyrylcholinesterase [BChE; Jbilo, O. & Chatonnet, A. (1990) Nucleic Acids Res. 18, 3990]. Amino acid sequences of AChE and BChE have 51% identity. They both possessed a choline‐binding site W84, a catalytic triad S200‐H440‐E327 and six cysteine residues (positions 67–94, 254–265, 402–521) in conserved sequence positions to those that form three intrachain disulfide bonds in all cholinesterases (by convention, numbering of amino acids is that used for Torpedo AChE). Rabbit AChE had a larger number of aromatic residues lining the active‐site gorge than rabbit BChE (14 compared to 8, respectively) and a smaller number of potential N‐glycosylation sites (3 compared to 8, respectively). Both catalytic subunits have a hydrophilic C‐terminus (catalytic subunits of type T). Expression of acetycholinesterase and butyrylcholinesterase genes (ACHE and BCHE) was studied in rabbit tissues and during development by a correlation of Northern‐blot analysis and enzymic activities. This correlation was rendered difficult by the presence of an eserine‐resistant esterase active on butyrylthiocholine in serum, liver and lung. When the contribution of this carboxylesterase was taken into account, brain was found as the richest source of BChE followed by lung and heart. Rabbit liver had a very low content of BChE that correlated with the low BChE activity in plasma. During development, BCHE transcripts were detected as early as day 10 post coitum, whereas ACHE transcripts appeared only on day 12.
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).
Expression and Biochemical Characterization of Human Protein Kinase C‐θ
Authors: Baier Gottfried , Baier‐Bitterlich Gabriele , Meller Nahum , Coggeshall K. Mark , Giampa Leslie , Telford David , Isakov Noah , Altman Amnon
Published: March 2005

In this study, the recently identified human protein kinase C‐θ(PKC‐θ) isoform has been biochemically characterized in detail. An antiserum raised against the unique V3 domain of PKC‐θ identified an 80‐kDa protein in all human T‐cell lines tested, in erythroleukemia K562 cells and in histiocytic lymphoma U‐937 cells, but not in a B‐lymphoma line (Raji) or in several melanoma, carcinoma, schwanoma or astrocytoma lines, confirming, at the protein level, its predominant expression in hematopoietic cell lines, in particular T cells. Immunoreactive PKC‐θ was detected almost exclusively in the cytosolic compartment of unstimulated Jurkat T cells. Stimulation with phorbol ester, however, caused rapid translocation to the membrane. In order to compare the properties of PKC‐θ with a representative member of the Ca2+‐dependent PKC enzymes, full‐length cDNAs encoding PKC‐θ or PKC‐α were transiently expressed in COS‐1 cells, and recombinant enzymes were partially purified via a six‐histidine peptide tag. The catalytic activity of these PKC enzymes was assayed against distinct substrates in the absence and presence of known PKC cofactors. Significant differences were found with respect to activation requirements and substrate preferences between PKC‐θ and PKC‐α. Both enzymes were stimulated by phospholipid and phorbol ester, and were active towards a PKC‐derived substrate peptide corresponding to the pseudosubstrate site of PKC. In contrast to PKC‐α, however, full activation of PKC‐θ did not require Ca2+, and its basal activity towards histone H1 was not stimulated by lipid cofactors. Additionally, a myelin‐basic‐protein‐(MBP)‐derived peptide, which was readily phosphorylated by PKC‐α, was a poor substrate for PKC‐θ. Similar to PKC‐α, transient PKC‐θ overexpression in murine EL4 thymoma cells caused an approximately 2.5‐fold increase in the phorbol‐12‐myristate‐13‐acetate‐induced transcriptional activation of an interleukin‐2 promoter‐reporter gene construct. The unique expression and functional properties of PKC‐θ suggest that it may play a specialized role in T‐cell signaling pathways.
Oligosaccharides Derived by Keratanase II Digestion of Bovine Articular Cartilage Keratan Sulphates
Authors: Brown Gavin M. , Huckerby Thomas N. , Nieduszynski Ian A.
Published: March 2005

Alkaline borohydride‐reduced keratan sulphate chains from bovine articular cartilage (6–8‐year‐old animals) were subjected to a limit digest with the enzyme keratanase II. Using 1H‐NMR spectroscopy, 25 reduced oligosaccharides deriving from keratan sulphate were shown to have the following structures [GlcNAc(6S)‐ol represents N ‐acetylglucosaminitol 6‐O ‐sulphate]: Galβ1–4‐GlcNAc(6S)‐ol, Galβ1‐4GlcNAc(6S) β1–3Galβ1–4GlcNAc(6S)‐ol, Gal(6S) β1–4GlcNAc(6S)‐ol, Gal‐(6S) β1–4GlcNAc(6S) β‐1–3 Galβ‐4GlcNAc(6S)‐ol, Galβ1–4GlcNAc(6S) β1–3Gal(6S) β1–4GlcNAc(6S)‐ol, Gal(6S) β1–4GlcNAc(6S) β1–3Gal(6S)1–4GlcNAc(6S)‐ol, Galβ1–4(Fuca1–3)GlcNAc(6S)‐ol, Galβ–4‐(Fucα1–3)GlcNAc(6S) β1–3Galβ1–4(Fcα1–3)G1cNAc(6S)‐ol, Galβ1–4GlcNAc(6S) β1–3Galβ1–4(Fucα1–3)‐GlcNAc(6S)‐ol, Galβ1–3(Fucα1–3)GlcNAc(6S) β1–3Galβ1–4GlcNAc(6S)‐ol, Gal(6S) β1–4GlcNAc‐(6S) β1–3Galβ1–4(Fucα1–3)GlcNAc(6S)‐ol, Galβ1–4(Fucα1–3)GlcNAc(6S) β1–3Gal(6S) β1–4GlcNAc(6S)‐ol, Galβ1–4 GlcNAc(6S) β1–6(Galβ1–3)GalNAc‐ol, Galβ1–4 GlcNAc(6S) β1–6(NeuAc2–3Galβ1–3)Gal‐Nac‐ol, Galβ1–4GclNAc(6S) β1–3Galβ1–4GlcNAc(6S) β1–4GlcNAc(6S) β1–6(Galβ1–3)GalNAc‐ol, Gal(6S) β1–4GlcNAc‐(6S) β1–6(Galβ1–3)GalNAc‐ol. Galβ1–4GlcNAc(6S) β1–3Galβ1–4GlcNAc (6S) β1–6(NeuAc2–3Galβ1–3)‐GalNAc‐ol. Gal(6S) β1–4GlcNAc‐(6S) β1–6(NeuAcα2–3Galβ1–3)GalNAc‐ol. Gal(6S) β1–4GlcNAc‐(6S) β1–3Galβ1–4GlcNAc(6S) β1–6(Galβ1–3)GalNAc‐ol, Gal(6S) β1–4GlcNAc(6S)β1–3Galβ1–4GlcNAc‐ (6S) β1–6(NeuAcα2–3Galβ1–3)GalNAc‐ol,NeuAcα2–6Galβ1–4 GalNAc(6S) β1–3Galβ1–4 GlcNAc(6s)‐ol, NecAcα2–3Galβ1–4GlcNAC(6s)β1–3Galβ1–4GlcNAc(6S)‐ol, NeuAcα2–6Galβ1–4GlcNAc(6S)β1–3Gal‐(6S)β1–4GlcNAc(6S)‐ol, NeuAcα2–3Galβ1–4GlcNAc(6S) β1–3Gal(6S)β1–4GlcNAc(6S)‐ol and Neu‐Acα2–3Gal(6S) β1–4GlcNAc(6S)β1–3Gal(6Sβ)1–4GlcNAc(6S)‐ol. Proton chemical shifts for these oligosaccharides were assigned using one‐ and two‐dimensional NMR spectroscopic methods.

These results confirm the findings of Nakazawa et al. [Nakazawa, K., Ito, M., Yamagata, T. and Suzuki, S. (1989) in Keratan sulphate: chemistry, biology and chemical pathology (Greiling, H. and Scott, J. E., eds) pp. 99–110, The Biochemical Society, London], namely that keratanase II cleaves the O ‐glycosidic bond of a β (1–3)‐linked 6‐O ‐sulphated N ‐acetylglucosamine.

However, the internal sulphated N ‐acetylglucosamine in the sialylated capping oligosaccharides is not cleaved because of the proximity of the sialic acid residue. In addition, keratanase II is the only degradative method examined so far which can cleave the glycosidic bond of a fucosylated N ‐acetylglucosamine residue as fucose residues confer resistance to both keratanase and hydrazino‐lysisnitrous acid fragmentation.
Bradykinin Induces Translocation of the Protein Kinase C Isoforms α, ɛ, and ζ
Authors: Tippmer Stefanie , Quitterer Ursula , Kolm Verena , Faussner Alexander , Roscher Adalbert , Mosthaf Luitgard , Müller‐Esterl Werner , Häring Hans
Published: March 2005

Bradykinin exerts a broad spectrum of cellular effects on different tissues. It is believed that these effects are predominantly mediated by the recently cloned B2 receptor. The mechanism of post‐receptor signal transduction is not known in detail. Involvement of protein kinase C (PKC) was suggested and activation of the classical PKC isoforms α and β was recently demonstrated. The aim of the present study was to investigate whether the B2 receptor also activates new (δ, ɛ) and atypical (ζ) PKC isoforms. To investigate this, Chinese hamster ovary (CHO) cells, stably transfected with human B2 receptor, were used. In these cells the PKC isoforms α, δ, ɛ and ζ were detected by immunoblotting with specific antibodies. To monitor hormone‐induced PKC translocation plasma membranes were prepared. Stimulation of the cells with bradykinin resulted in a rapid (30–60 s) translocation of the PKC isoforms α, ɛ, and ζ Translocation of PKC δ was not detected. The effect of bradykinin was reduced by simultaneous addition of the receptor antagonist HOE 140, a bradykinin‐related decapeptide. The data show that the B2 receptor in this cell model is able to activate, in addition to the classical PKC isoform α, the new PKC isoform ɛ and the atypical PKC isoform ζ. To test whether these effects are as well observed in a non‐transfected cell, the experiments were repeated in human foreskin fibroblasts which naturally express high levels of B2 receptors. In this cell system similar results on PKC α, ɛ, and ζ were observed, suggesting that all three PKC isoforms are involved in signal transduction of the B2 receptor.
Translocation, Activation and Association of Protein‐tyrosine Kinase (p72syk) with Phosphatidylinositol 3‐Kinase are Early Events During Platelet Activation
Authors: Yanagi Shigeru , Sada Kiyonao , Tohyama Yumi , Tsubokawa Misao , Nagai Katsuya , Yonezawa Kazuyoshi , Yamamura Hirohei
Published: March 2005

We have previously reported that a non‐receptor‐type protein‐tyrosine kinase p72syk, exists in both membrane and cytosolic fractions in porcine platelets and is activated after thrombin stimulation. To facilitate the understanding of the function of p72syk, we have investigated the topological features, kinase activities and the interaction with another signal‐transducing molecule, namely phosphatidylinositol 3‐kinase, during platelet activation. Membrane and cytosolic fractions were separated from thrombin‐treated porcine platelets, and the amount of p72syk was quantified by the immunoblot technique or the kinase activity of each fraction was determined by an immunoprecipitation kinase assay. After stimulation by thrombin, cytosolic p72syk rapidly translocated to the membrane fraction within 10 s and there was also a significant increase in the amount of p72syk in the cytoskeletal fraction. The autophosphorylation activity of membrane‐associated p72syk significantly increased approximately tenfold and reached a maximum at 10 s; the activity subsequently decreased to almost the basal level within 120 s. For similar time courses, association of p72syk with phosphatidylinositol 3‐kinase and tyrosine phosphorylation of p72syk were observed. These results suggest that translocation, activation, and association of p72syk with transducing molecules such as phosphatidylinositol 3‐kinase, events which occur during platelet activation, may participate in early signal‐transduction events.
Trans‐sialidase and Sialidase Activities Discriminate between Morphologically Indistinguishable Trypanosomatids
Authors: Medina‐Acosta Enrique , Franco Antonia M. R. , Jansen Ana Maria , Sampol Marcos , Nevés Neuza , Pontes‐De‐Carvalho Lain , Grimaldi Gabriel , Nussenzweig Victor
Published: March 2005

The expression of trans‐sialidase and sialidase activities in the kinetoplastid protozoa was explored as a potential marker to discriminate between the morphologically indistinguishable flagellates isolated from human, insects and vertebrate reservoir hosts. By virtue of the differences observed in the ratios of these enzyme activities, a collection of 52 species and strains comprising the major taxa of these parasites could be separated into four expression types. Type‐I parasites express comparable levels of both trans‐sialidase and sialidase activities (Endotrypanum species and Trypanosoma lewisi). Type‐II parasites express predominantly trans‐sialidase activity (Trypanosoma cruzi and Trypanosoma conorhini).Type‐III parasites express sialidase activity exclusively (Trypanosoma rangeli and Trypanosoma leeuwenhoeki). Type‐IV parasites do not express either activity (Leishmania species and Trypanoplasma borreli). The measurement of trans‐sialidase and sialidase activities thus permits the differentiation of parasites frequently found in the same insect vectors that are difficult to distinguish, such asT. cruzi and T. rangeli, or in the same sylvatic vertebrate and invertebrate hosts, such as Leishmania and Endotrypanum.
Further Oxidation of Hydroxycalcidiol by Calcidiol 24‐Hydroxylase
Authors: Akiyoshi‐Shibata Megumi , Sakaki Toshiyuki , Ohyama Yoshihiko , Noshiro Mitsuhide , Okuda Kyuichiro , Yabusaki Yoshiyasu
Published: March 2005

The coding region of the cDNA for rat kidney calcidiol 24‐hydroxylase (P450cc24), which is involved in calcium homeostasis in animals, was inserted into an expression vector pKK223‐3. The recombinant plasmid was formed in a specific manner without deletion or substitution of any parts of the coding region of the cDNA. When the resulting plasmid was introduced into Escherichia coli JM109, the recombinant cells produced a protein which was immunoreactive to an antibody against P450cc24. When the cell‐free extract of the transformed cells was incubated with calcidiol together with bovine adrenodoxin and NADPH‐adrenodoxin reductase, not only hydroxycalcidiol but also other metabolites such as oxocalcidiol and oxohydroxycalcidiol were produced. Similarly, calcitriol was converted not only to calcitetrol but also to oxocalcitriol and oxohydroxycalcitriol. These results indicate that a single enzyme expressed in the bacteria is responsible for all these successive reactions.
Thermal Uncoupling of the Ca2+‐transporting ATPase in Sarcoplasmic Reticulum
Authors: Geimonen Erika , Batrukova Marina A. , Rubtsov Alexander M.
Published: March 2005

It is known that the light fraction of rabbit skeletal muscle sarcoplasmic reticulum vesicles can release Ca2+ from the intravesicular space, although the Ca2+‐conductive channels are present only in the heavy fraction of sarcoplasmic reticulum vesicles. To study the possible pathways of the Ca2+ leakage from light vesicles we have used a short‐term treatment for 4.5 min at 45°C which quickly decreases the efficiency of Ca2+‐transporting ATPase operation without any visible effects on the hydrolytic activity of the Ca2+‐ATPase in the membranes. The treatment of the vesicles decreased the negative membrane surface potential created by the Ca2+‐ATPase. Comparative titration of control and heat‐treated preparations of light sarcoplasmic reticulum vesicles by K+, Na+, Mg2+, and Ca2+ revealed clear differences in their surface properties. The short‐term heating resulted in release of Ca2+ from the vesicles previously loaded with 45Ca2+, which indicates an increase in passive membrane permeability to Ca2+. Study of Ca2+‐ATPase protein arrangement in the membrane indicated that the heat treatment induced protein oligomerization and some of the Ca2+‐ATPase molecules acquired intermolecular and intramolecular covalent bonds. From these data, we have concluded that the changes in the surface and structure properties of the vesicle membranes after the short‐term heat treatment were the result of clustering of the Ca2+‐ATPase molecules. This protein rearrangement may create channels for calcium leakage from light sarcoplasmic reticulum vesicles.
A Quantitative Test of Long‐range Correlations and Compositional Fluctuations in DNA Sequences
Authors: Chatzidimitriou‐Dreismann C. Aris , Streffer R. M. Friedrich , Larhammar Dan
Published: March 2005

Recent findings concerning long‐range correlations and fractals in intron‐containing DNA sequences of living organisms are tested qualitatively and quantitatively. Extending previous studies, we demonstrate that these findings are trivially equivalent to variations of the base‐pair composition in different regions of a DNA sequence. It is shown explicitly that a well‐defined scaling or fractal exponent does not exist anywhere. Comparisons of natural DNAs with computer‐generated, artificial sequences are made. The present study reveals that certain natural DNA sequences (especially those with compact genomes) do have stochastic characteristics which are intrinsically different from artificial sequences. The results for 21 DNA sequences of various types from widely different taxa are reported.
Purification and Characterisation of an Extracellular β‐Glucosidase with Transglycosylation and Exo‐glucosidase Activities from Fusarium oxysporum
Authors: Christakopoulos Paul , Goodenough Peter W. , Kekos Dimitris , Macris Basil J. , Claeyssens Marc , Bhat Mahalingeshwara K.
Published: March 2005

An extracellular β‐glucosidase from Fusarium oxysporum was purified to homogeneity by gel‐filtration and ion‐exchange chromatographies. The enzyme, a monomeric protein of 110 kDa, was maximally active at pH 5.0–6.0 and at 60°C. It hydrolysed 1→4‐linked aryl‐β‐glucosides and 1→4‐linked, 1→3‐linked and 1→6–linked β‐glucosides. The apparent Km and kcat values for p ‐nitrophenyl β‐d‐glucopyranoside (4‐NpGlcp) and cellobiose were 0.093 (Km), 1.07 mM (kcat) and 1802 (Km), 461.5 min‐1 (kcat), respectively. Glucose and gluconolactone inhibited the enzyme competitively with Ki values of 2.05 mM and 3.03 μM, respectively. Alcohols activated the enzyme; butanol showed maximum effect (2.2‐fold at 0.5 M) while methanol increased the activity by 1.4‐fold at 1 M. The enzyme catalysed the synthesis of methylglucosides, ethylglucoside and propylglucosides, as well as trisaccharides in the presence of different alcohols and disaccharides, respectively. In addition, the enzyme hydrolysed the unsubstituted and methylumbelliferyl cello‐oligosaccharides [MeUmb(Glc)n] but the rate of hydrolysis decreased with increasing chain length. Analysis of products released from MeUmb(Glc)n as a function of time revealed that the enzyme attacked these substrates in a stepwise manner and from both ends. Thus, β‐glucosidase from F. oxysporum, with the above interesting properties, could be of commercial interest.
The phospho‐opsin Phosphatase from Bovine Rod Outer Segments
Authors: King Alastair J. , Andjelkovic Nataša , Hemmings Brian A. , Akhtar Muhammad
Published: March 2005

The vertebrate visual transduction system involves a cycle of phosphorylation and dephosphorylation of a transmembranous photoreceptor (rhodopsin). Upon illumination, the activated photoreceptor (metarhodopsin‐II) is phosphorylated by a specific kinase on up to seven serine and threonine residues. A dephosphorylation process must then be undertaken to return the photoreceptor to its ground state. Initial work, along with studies using the rabbit skeletal muscle catalytic subunit of protein phosphatase 2A, indicated that the phosphatase responsible was a member of the type‐2 family. The work has been further extended and using 1000 bovine retinae, the catalytic subunit and a holoenzyme form of phospho‐opsin phosphatase were purified 2100‐fold and 550‐fold respectively. The stimulation of the activities of both these fractions with protamine sulphate and the inhibition by okadaic acid are consistent with the fact that these phosphatases belong to the type‐2A family. Western blotting using a variety of specific antibodies established that the catalytic subunit (36 kDa, C subunit) was indeed of type 2A, while the holoenzyme was a heterotrimer comprising the preceding catalytic subunit complexed to two other polypeptides of 55 kDa (B sub‐unit) and 65 kDa (A subunit), both of which were of α subtype; phospho‐opsin phosphatase may thus be described as a trimeric enzyme containing the ABC subunits of type‐2A protein phosphatase, i.e. PP2A1. The dephosphorylation of phospho‐opsin by both fractions was found to be stimulated (4–8‐fold) by the presence of protamine sulphate (250 μg/ml; 50 μM). However, when phospho‐peptides corresponding to the C‐terminal region of opsin were used, these were maximally dephosphorylated without requiring the presence of protamine; at equivalent concentrations of substrates the phospho‐peptides were dephosphorylated (in the absence of protamine) at rates which were approximately equal to those obtained with phospho‐opsin (in the presence of protamine). It was shown that type‐1 phosphatases had little activity against these phospho‐peptides. Furthermore, if phospho‐opsin was treated with protamine, the activity of the phosphatase assumed an elevated level and was not significantly stimulated by the addition of exogenous protamine. This effect could be reversed by washing the protamine‐treated substrate with 1 M NaCl, whence the protamine‐dependent stimulation returned to normal levels. To this end, studies revealed that protamine was binding to the particulate substrate in a ratio of protamine/opsin of 0.7:1. The cumulative finding may be rationalised by suggesting that the effect of protamine is a substrate‐directed phenomenon and a hypothetical mechanism for this effect is considered.
Sequence Variation of the Hydroxymethyldihydropterin Pyrophosphokinase: Dihydropteroate Synthase Gene in Lines of the Human Malaria Parasite, Plasmodium falciparum, with Differing Resistance to Sulfadoxine
Authors: Brooks Darren R. , Wang Ping , Read Martin , Watkins William M. , Sims Paul F. G. , Hyde John E.
Published: March 2005

Dihydropteroate synthase (H2Pte synthase) is the target of the sulfur‐based antimalarial drugs, which are frequently used in synergistic combination with inhibitors of dihydrofolate reductase (H2folate reductase) to combat chloroquine‐resistant malaria. We have isolated the H2Pte synthase coding sequence of the most pathogenic human parasite Plasmodium falciparum. It forms part of a longer coding sequence, located on chromosome 8, that also specifies 6‐hydroxymethyl‐7,8‐dihydropterin pyrophosphokinase (CH2OH‐H2pterinPP kinase) at its 5′ proximal end. This domain is unusually large, with two long insertions relative to other CH2OH‐H2pterinPP kinase molecules. To investigate a possible genetic basis for clinical resistance to sulfa drugs, we sequenced the complete H2Pte synthase domains from eleven isolates of P. falciparum with diverse geographical origins and levels of sulfadoxine resistance. Overall, point mutations in five positions were observed, affecting four codons. Parasite lines exhibiting high‐level resistance were found to carry either a double mutation, altering both Ser436 and Ala613, or a single mutation affecting Ala581. The mutations at positions 436 and 581 have the same location relative to each of two degenerate repeated amino acid motifs that are conserved across all other known H2Pte synthase molecules. The amino acid alteration at residue 613 is identically positioned relative to a different conserved motif. The fourth amino acid residue (437) affected by mutation, though adjacent to the apparently crucial residue 436, shows no obvious correlation with resistance. Although these mutations have no exact counterparts in any other organism, that at position 581 falls within a region of three amino acids where H2Pte synthase is modified in various ways in a number of sulfonamide‐resistant pathogenic bacteria. Copy‐number analysis indicated that there was no amplification of the H2Pte synthase domain in resistant parasite lines of P. falciparum, compared to sensitive lines.
Chloroplast Protein Import
Authors: Endo Toshiya , Kawakami Makiko , Goto Akira , America Twan , Weisbeek Peter , Nakai Masato
Published: March 2005

Proteins have to be at least partially unfolded upon passage through the biological membranes. Previous studies with a dihydrofolate reductase fusion protein containing a chloroplast transit peptide showed that stabilization of the tertiary structure of the fusion protein by binding of a ligand, methotrexate, failed to block its translocation across the envelopes, suggesting that chloroplast envelopes have strong activity to unfold proteins [America, T., Hageman, J., Guéra, A., Rook, F., Archer, K., Keegstra, K. & Weisbeek, P. (1994) Plant Mol. Biol. 24, 283–294]. In the present study, we have analyzed in vitro translocation of a fusion protein consisting of the entire plastocyanin precursor and dihydrofolate reductase across the chloroplast envelope membranes and the thylakoid membrane. In the presence of methotrexate, the fusion protein was imported into the stroma but its translocation across the thylakoid membrane was blocked. The fusion protein that bound to the envelope became susceptible to digestion by thermolysin. These results suggest that, while the envelope membranes can unfold the methotrexate‐bound fusion protein to allow its passage, the thylakoid membrane cannot unfold the fusion protein that has re‐bound to methotrexate in the stroma.
NAD+‐Dependent d‐2‐Hydroxyisocaproate Dehydrogenase of Lactobacillus Delbrueckii subsp. Bulgaricus
Authors: Bernard Nathalie , Johnsen Keyji , Ferain Thierry , Garmyn Dominique , Hols Pascal , Holbrook J. John , Delcour Jean
Published: March 2005

A genomic library from Lactobacillus delbrueckii subsp. bulgaricus was used to complement an Escherichia coli mutant strain deficient for both lactate dehydrogenase and pyruvate formate lyase, and thus unable to grow anaerobically. One recombinant clone was found to display a broad specificity NAD+‐dependent d‐2‐hydroxyacid dehydrogenase activity. The corresponding gene (named hdhD) was subcloned and sequenced. The deduced amino acid sequence of the encoded enzyme indicates a 333‐residue protein closely related to D‐2‐hydroxyisocaproate (i.e. 2‐hydroxy‐4‐methyl‐pentanoate) dehydrogenase (d‐HO‐HxoDH) of Lactobacillus casei and other NAD+‐dependent d‐lactate dehydrogenases (d‐LDH) from several other bacterial species. The hdhD gene was overexpressed under the control of the lambda phage PL promoter and the enzyme was purified with a two‐step method. The L. delbrueckii subsp. bulgaricus enzyme, like that of L. casei, was shown to be active on a wide variety of 2‐oxoacid substrates except those having a branched β‐carbon.
Agonist‐induced Transfer of the α Subunits of the Guanine‐nucleotide‐binding Regulatory Proteins Gq and G11, and of Muscarinic m1 Acetylcholine Receptors from Plasma Membranes to a Light‐vesicular Membrane Fraction
Authors: Svoboda Petr , Milligan Graeme
Published: March 2005

A clone of a Chinese hamster ovary (CHO) cell line expressing high levels of the human muscarinic M1 acetylcholine (Hm1) receptor undergoes a substantial agonist‐specific down‐regulation of both Hm1 receptors and the α subunits of the guanine‐nucleotide‐binding (G)‐proteins Gq and G11 which is accompanied by a desensitization of inositol‐phospholipid‐specific‐phospholipase‐C response [Mullaney, I., Dodd, M. W., Buckley, N. J. & Milligan, G. (1993) Biochem. J. 289, 125–131]. To examine early events in this process, the effect of agonist on subcellular distribution of Gqα and G11α and of Hm1 receptors was assessed after short‐term and long‐term treatment with carbachol.

Short‐term (30 min) incubation with carbachol (1 mM) induced a simultaneous transfer of a proportion of both Gqα and G11α and Hm1 receptors from plasma membranes to distinct light vesicular membranes. The total number of receptors and of Gqα and G11α in each cell remained unchanged under these conditions. A similar transfer was noted for the G‐protein Gsα but not for intrinsic plasma membrane markers. The plasma membrane, as well as light vesicular membrane, pool of Gsα subunit was unaffected by further sustained incubation with carbachol (16 h), whereas Hm1 receptors and both Gqα and G11α proteins were down‐regulated to 25% and 40%, respectively, when compared with untreated cells.

Such observations support the idea that down‐regulation of both the Hm1 receptor and its associated inositol‐phospholipid‐specific‐phospholipase‐C‐linked G‐proteins is produced by two sequential steps. The first is a transfer of signal‐transducing polypeptides from the plasma membrane to a non‐plasma membrane light vesicle fraction. The second step is represented by an agonist‐specific down‐regulation pathway. Both the Hm1 receptor and Gqα/G11α appear to follow similar sequestration and down‐regulation patterns.
Heterotropic Modulation of the Protease‐Inhibitor‐Recognition Process
Authors: Fioretti Evandro , Angeletti Mauro , Lupidi Giulio , Coletta Massimo
Published: March 2005

The effect of calcium and lanthanide ions (e.g. terbium) on the binding properties of α‐chymotrypsin has been studied focussing on the modulation exerted by cations on the interaction of the enzyme with the bovine pancreatic trypsin inhibitor (BPTI or Kunitz inhibitor). The results obtained indicate that the cation binding induces conformational transitions, on the enzyme molecule, which destabilize the enzyme‐inhibitor complex formation affecting the interaction of the inhibitor with the secondary specificity site of the proteolytic enzyme. This negative heterotropic effect can be observed only with macromolecular inhibitors (or substrates), displaying an extended interacting surface with the enzyme, and it seems linked to the number of positive charges carried by the cations. Thus, owing to the large conformational changes induced by the binding of trivalent cations, the divalent ones (e.g. calcium) appear to be more suitable for a fine regulation of the enzyme activity. The mutual correlation between inhibitors binding to (and calcium release by) the proteolytic enzymes (and vice versa) could assume an important physiological significance linking parameters, such as calcium concentration and the activity levels of proteolytic enzymes, which are both of great importance for the cell life.
The Modulation of Apolipoprotein E Gene Expression by 3,3′‐5‐triiodothyronine in HepG2 Cells Occurs at Transcriptional and Post‐transcriptional Levels
Authors: Vandenbrouck Yves , Janvier Brigitte , Loriette Claude , Bereziat Gilbert , Mangeney‐Andreani Marise
Published: March 2005

The regulation of the synthesis and secretion of apolipoprotein E (apoE) is incompletely understood. This study examines the mechanisms responsible for regulating apoE gene expression in HepG2 cells by thyroid hormone (3,3′‐5‐triiodothyronine). The secretion rate of apoE was by thyroid hormone increased (1.5–1.8‐fold) in pulse/chase experiments. Thyroid hormone doubled apoE mRNA concentration as determined by Northern‐blot analysis. Inhibition of protein synthesis by cycloheximide increased the thyroid‐hormone‐induced stimulation of apoE mRNA. This suggests that the synthesis of new protein is not required for thyroid hormone to stimulate apoE mRNA. Actinomycin D was used to inhibit new transcription; there was a more rapid degradation of mature apoE mRNA in thyroid hormone‐treated HepG2 cells than in control cells, suggesting that thyroid hormone acts post‐transcriptionally to regulate apoE gene expression. Cycloheximide blocked the action of thyroid hormone, suggesting that thyroid hormone regulates the turnover of apoE mRNA via the synthesis of de novo protein. Nuclear run‐on transcription assays demonstrated that thyroid hormone stimulated apoE gene transcription threefold in 24 h. These findings indicate that the expression of the apoE gene is controlled at both transcriptional and post‐transcriptional loci by the thyroid hormone.
Stable Expression of Human H1‐histamine‐receptor cDNA in Chinese Hamster Ovary Cells
Authors: Moguilevsky Nicole , Varsalona Francesca , Noyer Michel , Gillard Michel , Guillaume Jan‐Paul , Garcia Lida , Szpirer Claude , Szpirer Josiane , Bollen Alex
Published: March 2005

A cDNA clone for the histamine H1 receptor was isolated from a human lung cDNA library; it encoded a protein of 487 amino acids which showed characteristic features of G‐protein‐coupled receptors. The percentages of identity of the deduced amino acid sequence with bovine, rat and guinea pig H1 histamine receptors were 82.6%, 79.4% and 73.3%, respectively, whereas these percentages decreased to 74.6%, 66% and 56.7% for the amino acid sequence of the third intracellular loop. The human H1‐receptor cDNA was transfected into Chinese hamster ovary cells (CHO) via an eukaryotic expression vector; the receptor protein present on cell membranes specifically bound [3H]mepyramine with a Kd of 3.7 nM. The binding was displaced by H1‐histamine‐receptor antagonists and histamine. Northern blot analysis indicated the presence of two histamine H1 receptor mRNAs of 3.5 kb and 4.1 kb in various human tissues and an additional mRNA of 4.8 kb restricted to the human brain. Finally, by means of somatic cell hybrids segregating either human or rat chromosomes, the gene for histamine H1 receptor was found to reside on human chromosome 3 and rat chromosome 4.
Recombinant expression and properties of the Kunitz‐type protease‐inhibitor module from human type VI collagen α3(VI) chain
Authors: Mayer Ulrike , PÖSchl Ernst , Nischt Roswitha , Specks Ulrich , Pan Te‐Cheng , Chip Mon‐Li , Timpl Rupert
Published: March 2005

The Kunitz‐type inhibitor motif (domain C5) present at the C‐terminus of the human collagen α3(VI) chain was prepared in a recombinant form from the culture medium of stably transfected kidney cell clones. The 76‐residue protein was disulfide bonded and showed a high stability against protease treatment. The recombinant protein lacked, however, any inhibitory activity for trypsin, thrombin, kallikrein and several other proteases, which could be due to a few unusual substitutions in the region crucial for inhibitor binding. A sensitive radioimmunoassay detected low concentrations of C5 epitopes in normal human serum and fibroblast culture medium and showed a lack of cross‐reaction with aprotinin. Antibodies against C5 immunoprecipitated collagen VI obtained from fibroblast medium. The C5 epitopes could not be detected on intact collagen VI purified from guanidine extracts of human placenta. Collagen VI was shown to possess several α3(VI) chain bands (approximately 200 kDa) and reacted strongly with antibodies to an N‐terminal recombinant fragment. Immunofluorescence with anti‐C5 antibodies failed to stain several human tissues but produced a distinct intracellular staining of cultured fibroblasts. The data indicate the rapid loss of the C5 domain after biosynthesis of collagen VI.