bluuebunny/crossref_metadata_2025 huggingface dataset:
Modified low density lipoproteins activate human macrophages to secrete immunoreactive endothelin
Authors: Martin-Nizard Françoise , Houssaini Hayat Sqalli , Lestavel-Delattre Sophie , Duriez Patrick , Fruchart Jean-Charles
Published: July 2002

This study attempted to determine if low density lipoproteins (LDL) induce the production of endothelins (ET) by human macrophages. Non‐protected LDL from macrophage induced oxidation (n‐LDL), copper‐oxidized LDL (Ox‐LDL), acetylated‐LDL (Ac‐LDL), butylated hydroxytoluene‐LDL (BHT‐LDL), BHT‐Ac‐LDL, polyinosinic acid (PiA, 1.5μg/ml), phorbol myristate acetate (PMA; 0.5 μM) and BHT alone (20 μM) were studied. The different compounds had the following potency to stimulate the ET secretion: PMA>Ox‐LDL>Ac‐LDL>n‐LDL>BHT‐LDL>PiA>PiA+Ac‐LDL>BHT. In conclusion, mod stimulated ET secretion by human macrophages.
Cysteine residues are not essential for the catalytic activity of human class Mu glutathione transferase M1a‐1a
Authors: Widersten Mikael , Holmström Eva , Mannervik Bengt
Published: July 2002

To investigate the possible involvement of a Cys thiol in the catalysis of the human glutathione transferase M1a‐1a, we constructed mutants of this enzyme wherein the four Cys residues present in the native enzyme where replaced by Ala residues. Three mutants, one where all four Cys residues had been replaced and two mutants where three out of four Cys residues were changed into Ala, were characterized regarding their catalytic activities with three different substrates as well as by their binding of three different inhibitors. All three Cys‐deficient mutant forms of glutathione transferase M1a‐1a were catalytically active with the tested substrates and their binding of inhibitors, measured by l
50, were not significantly different from the values previously obtained for the wild‐type enzyme. We therefore conclude that none of the Cys residues in this class Mu glutathione transferase are directly involved in the catalysis performed by this enzyme.
Magnetic circular dichroism spectroscopy as a probe of axial heme ligand replacement in semisynthetic mutants of cytochrome c
Authors: Rux John J. , Dawson John H.
Published: July 2002

Horse heart cytochrome c with either histidine or cysteine replacing the endogenous axial methionine ligand at position 80 has been characterized with magnetic circular dichroism (MCD) spectroscopy in the UV‐visible region. Comparison of the MCD spectra of the mutant proteins in the ferric state to those of authentic bis‐imidazole‐ and imidazole/thiolate‐ligated ferric heme proteins clearly shows that the histidine‐imidazole and cysteine‐thiolate groups of the replacement amino acids at position 80 are coordinated to the heme iron in the mutant proteins. This study demonstrates the power of MCD spectroscopy in identifying axial ligands in mutant heme proteins. Accurate axial ligand assignment is essential for proper interpretation of the altered properties of such novel proteins.
Trivaline ‘catalyzes’ 5′‐pdGTT oligomerization in solution
Authors: Streltsov S.A. , Khorlin A.A. , Victorova L.S. , Kochetkova S.V. , Tsilevich T.L. , Florentiev V.L.
Published: July 2002

We have found that the 5′‐pdGTT molecules at a concentration of 10−4 M are oligomerized in solution in the presence of 10−4 M tripeptide ‐ (L‐Val)3‐NH‐NH‐DNS‐CF3COOH and the condensation reagents (carbodiimide and imidazole). Oligonucleotides not less than 12 bases long were formed in the yield which was over 15%. It is known that in the absence of peptide 10−2 M mono‐ or dinucleotides are required. Thus trivaline can be considered as one of the simplest enzymes. This oligomerization seems to be an essential way for the synthesis of long enough oligonucleotides of the random GC‐sequence, which could be used at the earliest steps of evolution.
Site‐specific amino‐terminal mutants of yeast‐expressed β‐actin Characterization of the interaction with myosin and tropomyosin
Authors: Aspenström Pontus , Lindberg Uno , Karlsson Roger
Published: July 2002

Neutral or charge‐shifting, mutagenesis of β‐actin at positions 3 and 4 strongly influenced the actomyosin interaction under non‐rigor conditions. The polymerization behaviour and tropomyosin binding properties on the other hand remained unaffected.
Protein‐tyrosine kinase activity of alternate protien products of the Drosophila Dsrc28C locus
Authors: Theroux Steven J. , Wadsworth Samuel C.
Published: July 2002

The Dsrc28C gene is a unique member of the extensive tyrosine kinase family. Two proteins, p66Dsrc28C and p55Dsrc28C, are encoded by the gene. Each contains a highly conserved tyrosine kinase domain and each lacks the usual amino‐terminal myristylation signal. The protein‐tyrosine kinase activity of the two proteins was investigated through a recombiaant baculovirus expression system. p66Dsrc28C expressed from a recombinant baculovirus phosphorylated a large number of Sf9 cell proteins on tyrosine. A group of proteins of approximately 100 kDa were the preferred substrates. No evidence of p66Dsrc28C autophosphorylation was found. In contrast to p66Dsrc28C, p55Dsrc28C did not exhibit protein‐tyrosine kinase activity when expressed from a recombinant baculovirus. A deletion derivative of p66Dsrc28C lacking the SH3 and SH2 domains also failed to phosphorylate Sf9 cell proteins. These results suggest that the protein‐tyrosine kinase activity of Dsrc28C proteins is tightly regulated.
Variability patterns of dehydrogenases versus peptide hormones and proteases/antiproteases
Authors: Jörnvall Henrik , Persson Bengt , Jörnvall Hans
Published: July 2002

Comparisons of human/rat species variants of 23 dehydrogenases versus 41 entries for peptide hormones and 41 for proteases/antiproteases reveal characteristic patterns. Dehydrogenases are ‘constant’ (2–8% residue differences between homologues) or ‘variable’ (12–40% differences). Peptide hormones exhibit a wider range, with many ‘strictly conserved’ and others spreading upscale, while proteases/antiproteases are overall shifted upscale, with few ‘constant’ and many ‘variable’. Overall, the influence of polypeptide size and function is confirmed, while for the dehydrogenases, the data highlight the ‘constant’ pattern as the one with high frequency values and suggest for classic liver alcohol dehydrogenase the ‘variable’ pattern to correlate with emerging functions.
Inactivation of α‐ketoglutarate dehydrogenase during oxidative decarboxylation of α‐ketoadipic acid
Authors: Bunik V.I. , Pavlova O.G.
Published: July 2002

α‐Ketoglutarate dehydrogenase was inactivated irreversibly and completely during oxidation of α‐ketoadipic acid. The inactivation was revealed both in the model system with ferricyanide and in the overall reaction catalyzed by the α‐ketoglutarate dehydrogenase complex. Neither substrate depletion nor product accumulation induced the inactivation. The results obtained were compared with recent data on the enzyme inactivation during oxidation of α‐ketoglutaric acid. The differences in the inactivation kinetics observed with the two substrates of the enzyme were analyzed. They seem not to reflect the different mechanisms of the inactivation, but, rather, depend on the changes in the rates of the individual stages of the process.
A 31P NMR study of the interaction of the antitumor active metallocene Cp2MoCl2 with calf thymus DNA
Authors: Harding Margaret M. , Harden Grahame J. , Field Leslie D.
Published: July 2002

Treatment of sonicated calf thymus DNA with the antitumor active metallocene Cp2MoCl2 afforded a metallocene‐DNA complex which was characterized by 31P NMR spectroscopy. In addition to the resonance for the phosphate backbone (δ −1.6), the spectrum contained 2 signals assigned to a phosphate bound Mo—DNA complex(es) (δ 37.2, 36.5) and a broad signal at δ 6.2 ppm. This result suggests that covalent attachment of the metallocene Cp2MoCl2 occurs via phosphate(O) coordination and is accompanied by local distortion of the DNA backbone. This result supports recent ICP studies with Cp2TiCl2 that have DNA detected DNA—metallocene adducts.
Bacterial protein toxins penetrate cells via a four‐step mechanism
Authors: Montecucco Cesare , Papini Emanuele , Schiavo Giampietro
Published: July 2002

Bacteria produce several protein toxins that act inside cells. These toxins bind with high affinity to glycolipid or glycoprotein receptors present on the cell surface. Binding is followed by endocytosis and intracellular trafficking inside vesicles. Different toxins enter different intracellular routes, but have the common remarkable property of being able to translocate their catalytic subunit across a membrane into the cytosol. Here, a toxin modifies a specific target with ensuing cell alterations, necessary for the survival and diffusion strategies of the toxin producing bacterium.
Real‐time analysis of the calcium‐dependent interaction between calmodulin and a synthetic oligopeptide of calcineurin by a surface plasmon resonance biosensor
Authors: Takano Emiko , Hatanaka Masakazu , Maki Masatoshi
Published: July 2002

The calcium‐dependent interaction between calmodulin (CaM) and the synthetic oligopeptide of a predicted CaM‐binding region of human calcineurin A‐2 was analysed with an automated surface plasmon resonance biosensor, BIAcore. The oligopeptide was immobilized to a biosensor chip via the amino‐terminal cysteine residue by a thioldisulphide exchange method. The biosensor chip was regenerated by an EGTA‐containing buffer after each analysis. Kinetics experiments showed that CaM bound with a high affinity to the oligopeptide in a Ca2+‐dependent manner. The estimated rate constants of association (k
ass) and dissociation (k
diss) were 2.3 × 1O5 M−1·s−1 and 3.9 × 10−3 s−1, respectively. The ratio of k
diss/k
ass, 1.7 × 10−8 M, was in good agreement with the dissociation constant (K
d) of 2.4 × 10−8 M determined from the equilibrium phase.
Inositol phospholipid metabolism in Xenopus oocytes mediated by endogenous Go and Gi proteins
Authors: Kasahara Jiro , Sugiyama Hiroyuki
Published: July 2002

To characterize G‐proteins which mediate the signal transduction from ligand stimulated receptor to phospholipase C (PLC), we injected antisense DNAs complementary to Xenopus Goα or Gi‐1α to suppress these endogenous G‐proteins, together with the mRNAs encoding metabotropic glutamate receptor 1 (mGluR1), 5 (mGluR5) or with M1 type muscarinic receptor into oocytes. Receptor‐stimulated chloride current responses were reduced by the suppression of Xenopus Goα regardless of the types of receptors. However, injection of Gi‐1 antisense DNA resulted in the reduction of M1‐stimulated responses but not mGluR‐stimulated responses. These results suggested that all these receptors could use Goα and M1 receptors, but not mGluRs, could also use Gi‐1 proteins, to activate PLC in Xenopus oocytes.
Mapping the binding surface of interleukin‐8 complexed with an N‐terminal fragment of the Type 1 human interleukin‐8 receptor
Authors: Clubb Robert T. , Omichinski James G. , Clore G.Marius , Gronenborn Angela M.
Published: July 2002

Interleukin‐8 and its receptors are key mediators of immune and inflammatory responses. Heteronuclear NMR spectroscopy has been utilized to map the binding surface on interleukin‐8 (IL‐8) for an N‐terminal fragment of the human Type‐1 IL‐8 receptor. A peptide corresponding to residues 1–40 of the IL‐8 type 1 receptor (IL8‐rl) was titrated into a sample of uniformly 15N‐labeled IL‐8. IL8‐rl binds to IL‐8 with a dissociation constant of 170 ± 50 μM assuming the peptide binds with a stoichiometry of one peptide per IL‐8 monomer, exchanges rapidly (> 900 s−1) between free and bound states, and selectively perturbs the chemical environment of several IL‐8 residues. The binding surface on IL‐8 suggested by our results is comprised of residues in strand β3 of the β‐sheet (Glu48 to Cys50), the turn preceding β3 (Ser44), the C‐terminal α‐helix (Val61) and the irregular N‐terminal loop region (Thr123, Lys15, Phe17, His18, Lys20 and Phe21). The IL‐8 dimer appears to present two symmetrical binding surfaces for the IL8‐rl peptide, suggesting two receptor peptides may bind per dimer.
Stereochemical course of the hydrolysis reaction catalyzed by chitinases Al and D from Bacillus circulans WL‐12
Authors: Armand S. , Tomita H. , Heyraud A. , Gey C. , Watanabe T. , Henrissat B.
Published: July 2002

Chitinases A1 and D were purified from the periplasmic proteins produced by Escherichia coli HB101 harbouring recombinant plasmids carrying respectively the chiA and chiD genes of Bacillus circulans WL‐12. HPLC analysis indicated that during the hydrolysis of chitotriose, both chitinases initially produce N‐acetylglucosamine and only one anomer of chitobiose. 1H NMR spectroscopy of the hydrolysis of chitotetraitol showed that this anomer corresponds to β‐chitobiose, demonstrating that chitinases Al and D act by a molecular mechanism that retains the anomeric configuration. This mechanism is similar to that of lysozymes although both chitinases belong to a family of proteins sharing no demonstrable amino acid sequence similarity with lysozymes.
Mutation of serine‐516 in human prostaglandin G/H synthase‐2 to methionine or aspirin acetylation of this residue stimulates 15‐R‐HETE synthesis
Authors: Mancini Joseph A. , O'Neill Gary P. , Bayly Christopher , Vickers Philip J.
Published: July 2002

Prostaglandin G/H synthase (PGHS) is a key enzyme in cellular prostaglandin (PG) synthesis and is the target of non‐steroidal anti‐inflammatory agents. PGHS occurs in two isoforms, termed PGHS‐1 and PGHS‐2. These isoforms differ in several respects, including their enzymatic activity following acetylation by aspirin. While PG synthesis by both isoforms is inhibited by aspirin, 15‐R‐hydroxyeicosatetraenoic acid (15‐R‐HETE) synthesis by PGHS‐2, but not PGHS‐1, is stimulated by preincubation with aspirin. We have mutated the putative aspirin acetylation site of hPGHS‐2, and expressed the mutants in COS‐7 cells using recombinant vaccinia virus. Enzyme activity and inhibitor sensitivity studies provide evidence that Ser516 is the aspirin acetylation site of human PGHS‐2 and that substitution of a methionine residue at this position can mimic the effects of aspirin acetylation on enzyme activity.
Production of bioactive gastrin from the non‐endocrine cell lines CHO and COS‐7
Authors: Hayashi Naoki , Kayo Tsuyoshi , Sugano Kentaro , Takeuchi Toshiyuki
Published: July 2002

We made a mutated progastrin cDNA construct that contains a cleavage site (‐Arg−4‐Arg−3‐Lys−2 ‐Arg−1) specific for the Kex2‐like endoprotease furin, located ahead of the bioactive gastrin. For expressing the mutated progastrin cDNA, we used two non‐endocrine cell lines, CHO and COS‐7. CHO cells exhibit amidating enzyme activity and levels of amidation enzyme mRNA as high as those in the pituitary‐derived endocrine cell line GH3, whereas COS‐7 cells have far less amidating activity and lower amounts of mRNA. Mutant progastrin‐expressing CHO cells produced mostly amidated gastrin. Gel filtration showed the size of this gastrin corresponded to that of the synthetic human gastrin‐17. In contrast, COS‐7 cells produced glycine‐extended gastrin and only a small amount of amidated gastrin. The difference in the amount of amidated gastrin products produced by the two non‐endocrine cell lines is due to differing amounts of the amidation enzyme contained in each cell line.
Ovarian tumors associated with pregnancy
Authors: Ueda M. , Ueki M.
Published: July 2002

Objective: The clinical characteristics of ovarian tumors associated with pregnancy were investigated. Method: A series of 106 cases of ovarian tumor surgically resected during pregnancy was investigated with respect to incidence, clinicopathologic features and outcome. Results: Among 106 cases undergoing ovarian surgery, 31 (29.2%), 70 (66%) and five (4.7%) were diagnosed as physiologic, benign and malignant, respectively. The incidence of benign neoplastic tumor was 1:112 deliveries and that of malignant neoplastic tumor was 1:1684 deliveries. Dermoid cyst was the most common lesion found. Of the 70 benign tumors, 51 (72.9%) were greater than 8 cm in diameter and 55 (78.6%) were preoperatively diagnosed before the 10th gestational week; 44 (62.9%) were operated before the 15th gestational week. The spontaneous abortion rate in 80 cases followed up after surgery was only 10%, 61 patients (76.3%) progressing to full‐term delivery. Five malignant tumors included three epithelial carcinomas, one embryonal carcinoma and one dysgerminoma. Conclusion: Ovarian surgery in the first trimester for persistent or enlarging masses is important to obtain a final histologic diagnosis and rule out malignancy.
CNS demyelination in monoclonal gammopathy of undetermined significance (MGUS): possible cause of a dementia syndrome
Authors: Hampel H , Schneider C , Hock C , Müller-Spann F , Ackenheil M
Published: July 2002

In acquired peripheral demyelinating disease only few publications point out the possibility of simultaneous involvement of the CNS. We describe two patients with chronic polyneuropathy and monoclonal gammopathy of undetermined significance (MGUS) developing a progressive dementia syndrome with extensive cerebral white matter alterations.
Burden on the families of patients with obsessive-compulsive disorder: a pilot study
Authors: Magliano L , Tosini P , Guarneri M , Marasco C , Catapano F
Published: July 2002

This study aimed to explore the burden on the key-relatives of a sample of patients with obsessive-compulsive disorder and a control sample of patients with major depression. The level of burden of relatives of obsessive-compulsive patients was moderate to severe in most of the explored situations, and did not differ significantly from that of the relatives of depressives. Social relationships were compromised in 74% of cases, and feelings of depression were recorded in 84% of cases. The level of subjective burden changed according to the relatives' sex, age and position in the family. The degree of objective burden correlated with the level of patients' disability, whereas that of subjective burden correlated with the severity of patients' obsessive-compulsive symptomatology.
Targeting of proteins to mitochondria
Authors: Lithgow Trevor
Published: July 2002

A clear picture has emerged over the past years on how a ‘classic’ mitochondrial protein, like subunit IV of cytochrome c oxidase, might be targeted to mitochondria. The targeting and subsequent import process involves the commitment of the TOM (ranslocase in the uter itochondrial membrane) receptor complex on the mitochondrial surface, a TIM (ranslocase in the nner itochondrial membrane) translocation complex in the mitochondrial inner membrane, and assorted chaperones and processing enzymes within the organelle. Recent work suggests that while very many mitochondrial precursor proteins might follow this basic targeting pathway, a large number have further requirements if they are to be successfully imported. These include ribosome‐associated factors and soluble factors in the cytosol, soluble factors in the mitochondrial intermembrane space, an additional TIM translocase in the inner membrane and a range of narrow specificity assembly factors in the inner membrane. This review is focused on the targeting of proteins up to the stage at which they enter the TOM complex in the outer membrane.
Caenorhabditis elegans Chk2‐like gene is essential for meiosis but dispensable for DNA repair
Authors: Higashitani Atsushi , Aoki Hidetoshi , Mori Akiyuki , Sasagawa Yohei , Takanami Takako , Takahashi Hideyuki
Published: July 2002

A Chk2‐like gene was identified in the genome of Caenorhabditis elegans. The putative gene product, termed Ce‐chk‐2 consists of 450 amino acid residues, and shows good homology with the Chk2/Cds1 gene family. The results of RNA‐mediated interference (RNAi) indicated that the F1 generation from dsRNA injected animals grew to adulthood, but approximately 95% of their eggs (F2) died during early embryogenesis. Among the few surviving progeny, males (XO animals) arose at an abnormally high frequency (30%). In addition, 12 univalents were observed in full grown oocytes of the F1, while six bivalents were normally observed in wild‐type oocytes. Ce‐chk‐2 gene expression increased in the adult stage, and their expression level decreased in the glp‐4 mutant, which is defective in germ line proliferation. The radiation sensitivity of F1 embryos carrying Ce‐chk‐2 RNAi was not significantly affected.
Genomic Exploration of the Hemiascomycetous Yeasts: 6. Saccharomyces exiguus
Authors: Bon Elisabeth , Neuvéglise Cécile , Lépingle Andrée , Wincker Patrick , Artiguenave François , Gaillardin Claude , Casaregola Serge
Published: July 2002

Random sequence tags were obtained from a genomic DNA library of Saccharomyces exiguus. The mitochondrial genome appeared to be at least 25.7 kb in size, with a different organization compared to Saccharomyces cerevisiae. An unusual putative 953 bp long terminal repeated element associated to Ty3 was found. A set of 1451 genes was identified homologous to S. cerevisiae open reading frames. Only five genes were identified outside the S. cerevisiae taxon, confirming that S. exiguus is phylogenetically closely related to S. cerevisiae. Unexpectedly, numerous duplicated genes were found whereas they are unique in S. cerevisiae. The sequences are deposited at EMBL under the accession numbers: AL407377–AL409955.
Membrane distal cytokine binding domain of LIFR interacts with soluble CNTFR in vitro
Authors: He Wei , Gong Ke , Zhu Guang , Smith David K , Ip Nancy Y
Published: July 2002

Ciliary neurotrophic factor (CNTF) is a member of the gp130 family of cytokines. The functional receptor complex of CNTF is composed of the CNTF receptor α (CNTFR), gp130 and the leukemia inhibitory factor receptor (LIFR). Three regions on CNTF have been identified as binding sites for its receptors. The ligand–receptor interactions are mediated through the cytokine binding domains (CBDs) and/or the immunoglobulin‐like domains of the receptors. However, in the case of CNTF, the precise nature of the protein–protein contacts in the signaling complex has not yet been resolved. In this study, we provide the first demonstration that the membrane distal CBD (CBD1) of LIFR associates in vitro with soluble CNTFR in the absence of CNTF. Moreover, purified CBD1 partially blocks CNTF signaling, but not that of interleukin‐6 or LIF, in human embryonal carcinoma cell line Ntera/D1 cells. These data raise the possibility that LIFR has the capability to form a ligand‐free complex with CNTFR.
Evidence for an actin binding helix in gelsolin segment 2; have homologous sequences in segments 1 and 2 of gelsolin evolved to divergent actin binding functions?
Authors: Van Troys M. , Dewitte D. , Goethals M. , Vandekerckhove J. , Ampe C.
Published: July 2002

Gelsolin is built up of six homologous segments that perform different functions on actin. Segments 1 and 2, which are suggested to be highly similar in their overall folds, bind monomeric and filamentous actin respectively. A long α‐helix in segment 1 forms the major contact site of this segment with actin. We show that sequence 197–226 of segment 2, equivalent to the region around the actin binding helix in segment 1, contains F‐actin binding activity. Consequently, positionally similar parts of segment 1 and 2 are implicated in the actin contact and solvent exposed residues in these parts must have evolved differentially to meet their different actin binding properties.
Etomoxir, sodium 2‐[6‐(4‐chlorophenoxy)hexyl]oxirane‐2‐carboxylate, inhibits triacylglycerol depletion in hepatocytes and lipolysis in adipocytes
Authors: Spurway Tracey D , Pogson Christopher I , Sherratt H.Stanley A , Agius Loranne
Published: July 2002

© 1997 Federation of European Biochemical Societies.
A Mox homeobox gene in the gastropod molluscHaliotis rufescensis differentially expressed during larval morphogenesis and metamorphosis
Authors: Degnan Bernard M. , Degnan Sandie M. , Fentenany Gabriel , Morse Daniel E.
Published: July 2002

We have isolated a homeobox‐containing cDNA from the gastropod molluscHaliotis rufescensthat is most similar to members of the Mox homeobox gene class. The derivedHaliotishomeodomain sequence is 85% identical to mouse and frog Mox‐2 homeodomains and 88.9% identical to the partial cnidarian cnox5‐Hm homeodomain. Quantitative reverse transcription‐polymerase chain reaction analysis of mRNA accumulation reveals that this gene, calledHruMox, is expressed in the larva, but not in the early embryo. Transcripts are most prevalent during larval morphogenesis from trochophore to veliger. There are also transient increases in transcript prevalence 1 and 3 days after the intitiation of metamorphosis from veliger to juvenile. The identification of a molluscan Mox homeobox gene that is more closely related to vertebrate genes than other protostome (e.g.Drosophila) genes suggests the Mox class of homeobox genes may consist of several different families that have been conserved through evolution.
Deletions of the N‐terminal regions of the human melanocortin receptors
Authors: Schiöth Helgi B , Petersson Susanna , Muceniece Ruta , Szardenings Michael , Wikberg Jarl E.S
Published: July 2002

The non‐homologous N‐terminal regions of four human melanocortin (MC) receptors were truncated in order to investigate their putative participation in ligand binding. Eleven constructs were made, where different numbers of residues from the N terminus were deleted. These constructs were used for transient expression experiments in COS cells and analysed by ligand binding. The results show that 27, 25, 28, and 20 amino acids could be deleted from the N terminus of the human MC1, MC3, MC4 and MC5 receptors, respectively, including all potential N‐terminal glycosylation sites in the MC1 and the MC4 receptors, without affecting ligand binding or expression levels. The results indicate that the N‐terminal regions of the human MC1, MC3, MC4 and MC5 receptors, do not play an important role for the ligand binding properties of these receptors.
Mechanism of gluconate synthesis in Rhizobium meliloti by using in vivo NMR
Authors: Portais Jean-Charles , Tavernier Patricia , Besson Isabelle , Courtois Josiane , Courtois Bernard , Barbotin Jean-Noël
Published: July 2002

The dehydrogenation of [1‐13C]‐ and [2‐13C]glucose into gluconate was monitored by NMR spectroscopy in living cell suspensions of two Rhizobium meliloti strains. The synthesis of gluconate was accompanied, in the cellular environment, by the formation of two gluconolactones, a γ‐lactone being detected in addition to the expected δ‐lactone. These lactones – as well as the gluconate – could be further metabolized by the cells. The δ‐lactone was utilized faster than the γ‐lactone. The presence – in significant amounts – and the relative stability of the lactones raise the question of their possible physiological significance.
An electrospray ionisation mass spectrometry (ESI‐MS) study to probe the metal ion binding site in the DNA binding domain of the yeast transcriptional activator GAL4
Authors: Gadhavi Paresh L
Published: July 2002

Electrospray ionisation mass spectrometry (ESI‐MS) is used to detect metal ions and their stoichiometry of binding in the DNA binding domain of GAL4. In this analysis, the mass spectra of the apo‐ and metallo‐proteins differ by both mass and charge, precluding the possibility of random adduct formation. Deuterium exchange NMR experiments of Zn(II)‐GAL4(7–49) indicate that the binuclear metal ion structure, which is shown to have a net negative charge of −2, is the recipient of several hydrogen bonds, notably from the main‐chain amide protons of the ligating cysteine residues, indicating the charge is stabilised in this manner.
Cloning and transcriptional regulation of the gene encoding the vacuolar/H+ ATPase B subunit of Dictyostelium discoideum
Authors: Bracco Enrico , Peracino Barbara , Noegel Angelika A , Bozzaro Salvatore
Published: July 2002

The main function of vacuolar H+ ATPases in eukaryotic cells is to generate proton and electrochemical gradients across the membrane of inner compartments. We have isolated the gene encoding the B subunit of Dictyostelium discoideum vacuolar H+ ATPase (vatB) and analyzed its transcriptional regulation. The deduced protein comprises 493 amino acids with a calculated molecular mass of 54 874 Da. The predicted protein sequence is highly homologous to previously determined V/H+ ATPase B subunit sequences. The protein is encoded by a single gene in the Dictyostelium genome. The gene is maximally expressed during growth and it decreases during the first hours of development. Gene expression is rapidly enhanced by phagocytosis, but not by fluid‐phase endocytosis. Acidic and alkaline conditions affect vatB gene expression differently.
Regulation of a promoter from the mouse insulin like growth factor II gene by glucocorticoids
Authors: Dell Ghislane , Ward Andrew , Engström Wilhelm
Published: July 2002

We have microinjected constructs containing the murine IG II P3 promoter linked to different flanking sequences and a luciferase reporter gene into mouse pronuclei to establish transgenic lines of mice. The offspring was used as a source of embryonic fibroblast cultures and the effect of exogenous addition of glucocorticoids on transgene expression was analysed. It was found that both dexamethasone and hydrocortisone gave rise to a functional stimulation of the IGF II P3 promoter when the construct also contained other elements. This study demonstrates for the first time that there is an effect of glucocorticoids on the activation of an embryonic IGF II promoter, thus providing a molecular rationale for previous findings that glucocorticoids can under certain circumstances give rise to an increased transcriptional activity of the IGF II gene.
Effect of point mutations at position 89 of theE. coli5S rRNA on the assembly and activity of the large ribosomal subunit
Authors: Zvereva Maria I , Shpanchenko Olga V , Dontsova Olga A , Nierhaus Knud H , Bogdanov Alexey A
Published: July 2002

Nucleotide residue U89 in the D loop ofEscherichia coli5S rRNA is adjacent to two domains of 23S rRNA in the large ribosomal subunit [Dokudovskaya et al., RNA 2 (1996) 146–152]. 50S ribosomal subunits were reconstituted containing U89(C, G or A) mutants of 5S rRNAs and the activities of the corresponding 70S ribosomes were studied. The U89C mutant behaves similarly to the wild‐type 5S rRNA. Replacement of the pyrimidine base at position U89 by more bulky purine bases impairs the incorporation of 5S rRNA into 50S subunits, whereas the particles formed showed full activities in poly(U)‐dependent poly(Phe) synthesis in the presence of either U89G or U89A 5S rRNA mutants. The activity of the reconstituted particles depends on the incorporation of 5S rRNA in agreement with early observations.
Evidence against structural and functional identity of microtubule‐associated protein 1B and proteoglycan claustrin
Authors: Tögel Martin , Wiche Gerhard , Propst Friedrich
Published: July 2002

Recently, the concept of microtubule‐associated protein 1B as an intracellular 2460 amino acid protein was challenged by the suggestion that only the N‐terminal 1022 codons are utilized and encode the core protein of the extracellular proteoglycan claustrin (Burg and Cole (1994) J. Neurobiol. 25, 1–22). We expressed this N‐terminal MAP1B fragment in tissue culture cells and found that it bound to microtubules and was not localized in the extracellular matrix. In addition, epitope mapping demonstrated that MAP1B consisted of more than 1022 amino acids and that the reported cDNA of claustrin is incomplete.
Plasmodium falciparum glutathione metabolism and growth are independent of glutathione system of host erythrocyte
Authors: Ayi Kodjo , Cappadoro Marina , Branca Mario , Turrini Franco , Arese Paolo
Published: July 2002

Plasmodium falciparum parasites grew normally in glutathione (GSH)‐depleted normal and G6PD‐deficient (Mediterranean variant) erythrocytes (RBC). Growth inhibition was observed only at less than approximately 6–12% residual GSH. Parasites studied separately with the Sendai virus technique synthesized GSH de novo and regenerated reduced GSH 10–20 times faster than non‐parasitized RBC. Electron spin resonance measurement of Tempol reduction indicated that the ability to reduce free radicals was restricted to the parasite. The marked efflux of oxidized GSH was mainly derived from the parasite. In conclusion, parasites are endowed with powerful and host‐independent mechanisms which de novo synthesize or regenerate GSH and allow undisturbed parasite development in GSH‐depleted RBC.
Acceleration of virus‐induced apoptosis by tumor necrosis factor
Authors: Koyama A.Hajime , Arakawa Tsutomu , Adachi Akio
Published: July 2002

The multiplication of vesicular stomatitis virus in HeLa cells was inhibited by treating the cells with tumor necrosis factor (TNF). Comparison of the kinetics of virus multiplication and that of virus‐induced apoptosis in the TNF‐treated cells revealed that the antiviral effect of TNF is accompanied by a rapid induction of apoptosis in the cells upon infection, suggesting that TNF can inhibit virus multiplication by accelerating an apoptotic response in the infected cells.
Unique chemical reactivity of His‐21 of CRM‐197, a mutated diphtheria toxin
Authors: Chang Jui-Yoa , Ramseier Ueli , Hawthorne Tom , O'Reilly Terry , van Oostrum Jan
Published: July 2002

CRM‐197 is a mutated diphtheria toxin (63 000 Da) widely used as a carrier protein of conjugated vaccines. Among the 14 histidines of CRM‐197, His‐21 was found to be modified selectively with iodoacetamide based reagents. This finding suggests a simplified method for the preparation of conjugate vaccines crosslinked to CRM‐197. A bifunctional iodoacetamide, N,N′‐(2‐hydroxy‐1,3‐propanediyl)‐bis‐[2‐iodoacetamide] (I‐CH2‐CONH‐CH2‐CH(OH)‐CH2‐NHCO‐CH2‐I) (HPBIA), was synthesized and allowed to react with CRM‐197. In the alkaline buffer of pH 8.0–8.4, HPBIA was shown to react and intra‐bridge His‐21 and Lys‐24 of CRM‐197 sequentially. At lower pH (7.1–7.5) in the phosphate buffer, the reactivity of Lys‐24 toward HPBIA was suppressed drastically. Under these conditions, His‐21 could be specifically labeled with HPBIA. Initial experiments have demonstrated that HPBIA modified CRM‐197 is able to crosslink to a cysteine‐containing peptide. These results offer a potential route for improving the homogeneity of CRM‐197 based protein‐peptide as well as protein‐polysaccharide conjugates.
Structural stabilization of botulinum neurotoxins by tyrosine phosphorylation
Authors: Encinar José A , Fernández Asia , Ferragut José A , González-Ros José M , DasGupta Bibhuti R , Montal Mauricio , Ferrer-Montiel Antonio
Published: July 2002

Tyrosine phosphorylation of botulinum neurotoxins augments their proteolytic activity and thermal stability, suggesting a substantial modification of the global protein conformation. We used Fourier‐transform infrared (FTIR) spectroscopy to study changes of secondary structure and thermostability of tyrosine phosphorylated botulinum neurotoxins A (BoNT A) and E (BoNT E). Changes in the conformationally‐sensitive amide I band upon phosphorylation indicated an increase of the α‐helical content with a concomitant decrease of less ordered structures such as turns and random coils, and without changes in β‐sheet content. These changes in secondary structure were accompanied by an increase in the residual amide II absorbance band remaining upon H‐D exchange, consistent with a tighter packing of the phosphorylated proteins. FTIR and differential scanning calorimetry (DSC) analyses of the denaturation process show that phosphorylated neurotoxins denature at temperatures higher than those required by non‐phosphorylated species. These findings indicate that tyrosine phosphorylation induced a transition to higher order and that the more compact structure presumably imparts to the phosphorylated neurotoxins the higher catalytic activity and thermostability.
Functional analyses of the Dof domain, a zinc finger DNA‐binding domain, in a pumpkin DNA‐binding protein AOBP
Authors: Shimofurutani Naoko , Kisu Yasutomo , Suzuki Masashi , Esaka Muneharu
Published: July 2002

AOBP, a DNA‐binding protein in pumpkin, contains a Dof domain that is composed of 52 amino acid residues and is highly conserved in several DNA‐binding proteins of higher plants. The Dof domain has a significant resemblance to Cys2/Cys2 zinc finger DNA‐binding domains of steroid hormone receptors and GATA1, but has a longer putative loop where an extra Cys residue is conserved. We show that the Dof domain in AOBP functions as a zinc finger DNA‐binding domain and suggest that the Cys residue uniquely conserved in the putative loop might negatively regulate the binding to DNA.
Conformational preferences in the Ser133‐phosphorylated and non‐phosphorylated forms of the kinase inducible transactivation domain of CREB
Authors: Radhakrishnan Ishwar , Pérez-Alvarado Gabriela C , Dyson H.Jane , Wright Peter E
Published: July 2002

Phosphorylation of Ser133 within the kinase inducible transactivation domain (KID) of the transcription factor CREB potentiates interaction with the KIX domain of coactivator CBP. Heteronuclear NMR spectroscopic analyses reveal that the KID domain is largely unstructured except for residues that comprise the αA helix in the pKID‐KIX complex, which populate helical conformations to a significant extent (>50%). The helical content in the αB region is very small in the non‐phosphorylated form (∼10%) although a small increase is detected upon Ser133 phosphorylation. The intrinsic bias towards helical conformations probably facilitates folding of the KID domain upon binding to KIX while the principal role of the phosphate group appears to be largely in mediating the intermolecular interactions in the pKID‐KIX complex.
Isolation of human intestinal defensins from ileal neobladder urine
Authors: Porter Edith Martin , Poles Michael A , Lee Jenny S , Naitoh John , Bevins Charles L , Ganz Tomas
Published: July 2002

We describe the isolation of naturally occurring human intestinal defensins HD‐5 and HD‐6 from ileal neobladder urine and ileal mucosa. Using an antibody‐based detection assay, we found multiple N‐terminally processed forms of HD‐5. The predominant HD‐5 forms in tissue were longer than those in neobladder urine (amino acid (aa) 23–94 and 29–94 versus aa 36–94, 56–94 and 63–94) suggesting that Paneth cells store prodefensin that is processed to mature defensin during or after degranulation. Search for mature HD‐6 yielded aa 69–100 as the predominant form in both sources. The ileal neobladder is a promising model to study human Paneth cell secretion.
The intramolecular electron transfer between copper sites of nitrite reductase: a comparison with ascorbate oxidase
Authors: Farver Ole , Eady Robert R. , Abraham Zelda H.L. , Pecht Israel
Published: July 2002

The intramolecular electron transfer (ET) between the type 1 Cu(I) and the type 2 Cu(II) sites of Alcaligenes xylosoxidans dissimilatory nitrite reductase (AxNiR) has been studied in order to compare it with the analogous process taking place in ascorbate oxidase (AO). This internal process is induced following reduction of the type 1 Cu(II) by radicals produced by pulse radiolysis. The reversible ET reaction proceeds with a rate constant k
ET=k
1→2+k
2→1 of 450±30 s−1 at pH 7.0 and 298 K. The equilibrium constant K was determined to be 0.7 at 298 K from which the individual rate constants for the forward and backward reactions were calculated to be: k
1→2=185±12 s−1 and k
2→1265±18 s−1. The temperature dependence of K allowed us to determine the ΔH° value of the ET equilibrium to be 12.1 kJ mol−1. Measurements of the temperature dependence of the ET process yielded the following activation parameters: forward reaction, ΔH
≠=22.7±3.4 kJ mol−1 and ΔS
≠=−126±11 J K−1 mol−1; backward reaction, ΔH
≠=10.6±1.7 kJ mol−1 and ΔS
≠=−164±15 J K−1 mol−1. X‐ray crystallographic studies of NiRs suggest that the most probable ET pathway linking the two copper sites consists of Cys136, which provides the thiolate ligand to the type 1 copper ion, and the adjacent His135 residue with its imidazole being one of the ligands to the type 2 Cu ion. This pathway is essentially identical to that operating between the type 1 Cu(I) and the trinuclear copper centre in ascorbate oxidase, and the characteristics of the internal ET processes of these enzymes are compared. The data are consistent with the faster ET observed in nitrite reductase arising from a more advantageous entropy of activation when compared with ascorbate oxidase.
Thyroid stimulating hormone and selenium supply interact to regulate selenoenzyme gene expression in thyroid cells (FRTL‐5) in culture
Authors: Villette Stéphane , Bermano Giovanna , Arthur John R. , Hesketh John E.
Published: July 2002

In the absence of a sodium selenite supplement, FRTL‐5 cells showed a reduced activity of cytosolic glutathione peroxidase (cGSH‐Px), a marker of selenium status, indicating the cells were Se‐deficient. Se‐deficient cells showed a 65% reduction in cGSH‐Px mRNA abundance but little change in abundance of either phospholipid hydroperoxide glutathione peroxidase or type 1 deiodinase (IDI) mRNA. In Se‐replete cells increased thyroid stimulating hormone (TSH) caused a small decrease in IDI abundance but in Se‐deficient cells TSH caused a large increase. The results indicate an interaction between TSH and Se status in the regulation of thyroid selenoenzyme synthesis.
Sequence analysis of a monoclonal antibody specific for the preS2 region of hepatitis B surface antigen, and the cloning, expression and characterisation of its single‐chain Fv construction
Authors: Passafiume Maria , Vulliez-le Normand Brigitte , Riottot Marie-Madeleine , Bentley Graham A.
Published: July 2002

The nucleotide sequence of the monoclonal antibody F124, specific for the preS2 region of the surface antigen of hepatitis B virus, has been determined and an single‐chain Fv fragment (scFv) recombinant construction has been cloned and expressed into the periplasmic region ofEscherichia coli. The recombinant antibody fragment contains a (Gly4Ser)3linker connecting the C‐terminus of the heavy chain variable region (VH) domain to the N‐terminus of the light chain variable region (VL) domain. A 23‐residue peptide segment, containing a c‐mycmarker for immunochemical detection of the scFv and hexahistidine tag to facilitate its purification, was added C‐terminal to the VLdomain. The scFv mimics the antibody in binding to the native antigen in the form of recombinant hepatitis B surface antigen (HBsAg) particles as well as to peptide fragments carrying the viral epitope.
Eos: a novel member of the Ikaros gene family expressed predominantly in the developing nervous system
Authors: Honma Yutaka , Kiyosawa Hidenori , Mori Tetsuji , Oguri Atsushi , Nikaido Takuya , Kanazawa Ken-ya , Tojo Michiko , Takeda Junko , Tanno Yoshihiro , Yokoya Sachihiko , Kawabata Isao , Ikeda Hisami , Wanaka Akio
Published: July 2002

We identified a novel member of the Ikaros gene family, which has critical roles in the development of lymphoid lineages. This gene, which we named Eos, was expressed predominantly in the developing central and peripheral nervous system. Eos protein could interact with itself and Ikaros protein through its C‐terminal portion in the yeast two hybrid assay. These findings suggested that Eos may have important roles in neural development similarly to the Ikaros family in the development of hemolymphoid tissue.
R.SspD5I is a neoschizomer ofHphI producing blunt end DNA fragments
Authors: Zheleznaya Lyudmila , Shiryaev Sergey , Zheleznyakova Elena , Nadezhda Matvienko  , Matvienko Nicholas
Published: July 2002

The strainStaphylococcusspecies D5 produces a restriction enzyme. It is the neoschizomer ofHphI endonuclease, which cleaves DNA at a distance of eight nucleotides from the recognition sequences producing blunt end DNA fragments: 5′‐GGTGA8N↓‐3′ and 3′‐CCACT8N↑‐5′.
Oleamide‐mediated sleep induction does not depend on perturbation of membrane homeoviscosity
Authors: Gobbi Marco , Mennini Tiziana , Dalla Valle Fabio , Cervo Luigi , Salmona Mario , Diomede Luisa
Published: July 2002

To verify whether the sleep‐inducing properties of oleamide were related to its ability to perturb membrane homeoviscosity, affecting 5‐HT2A receptors, we compared the effects of oleamide and oleic acid, the latter lacking both the sleep‐inducing effect and the action on 5‐HT2A receptors. In binding studies the two compounds did not directly interact with rat brain cortex 5‐HT2A receptors, nor did they increase the affinity of a 5‐HT2A agonist, either in vitro or ex vivo. They had similar fluidizing effects, in vitro at high concentrations (≥10 μM), and ex vivo after a dose of 100 mg/kg, and they reduced locomotor activity with similar potency. There thus appears to be no causal relationship between the fluidizing effects of oleamide and its sleep‐inducing properties.
Importance of the G protein γ subunit in activating G protein‐coupled inward rectifier K+ channels
Authors: Kawano Takeharu , Chen Lei , Watanabe Shinju Y. , Yamauchi Junji , Kaziro Yoshito , Nakajima Yasuko , Nakajima Shigehiro , Itoh Hiroshi
Published: July 2002

The G protein‐coupled inward rectifier K+ channel (GIRK) is activated by direct interaction with the heterotrimeric GTP‐binding protein βγ subunits (Gβγ). However, the precise role of Gβ and Gγ in GIRK activation remains to be elucidated. Using transient expression of GIRK1, GIRK2, Gβ1, and Gγ2 in human embryonic kidney 293 cells, we show that C‐terminal mutants of Gβ1, which do not bind to Gγ2, are still able to associate with GIRK, but these mutants are unable to induce activation of GIRK channels. In contrast, other C‐terminal mutants of Gβ1 that bind to Gγ2, are capable of activating the GIRK channel. These results suggest that Gγ plays a more important role than that of an anchoring device for the Gβγ‐induced GIRK activation.
Women and reproductive health in a graying world
Authors: Senanayake P
Published: July 2002

Gender differences in longevity suggest that the health problems in both developed and developing countries will largely be the problems of older women. As life expectancy increases, the important causes of death and ill health change. Older women's health issues and reproductive health needs are different from those of younger women. Biological susceptibility to various conditions also differs between the sexes. Monitoring of differences in incidence can better direct preventive efforts and contribute to improving the quality of life for aging women worldwide.
The role of FIGO in the advancement of women's health — in the year 2000
Authors: Seppälä M
Published: July 2002

The paper reviews the role and activities of FIGO as a global federation with a mission to promote the health and well‐being of women and to improve the practice of gynecology and obstetrics.
Consanguinity and the age of menopause in the United Arab Emirates
Authors: Bener A , Rizk D.E , Ezimokhai M , Hassan M , Micallef R , Sawaya M
Published: July 2002

Objective: The study examined the association between some biosocial factors, consanguinity and age at natural menopause in the United Arab Emirates (UAE). Method: A cross‐sectional population‐based study using a multi‐stage sampling design and face‐to‐face interview. Results: In a sample of 800 UAE females aged 40 years and above, there were 742 (85.8%) respondents. The median age of natural menopause was 48 years. The bodyweight, parity number, occupation, smoking habits and consanguinity in marriage were the significant variables associated with the age at natural menopause. There were statistically significant differences between women in consanguineous and non‐consanguineous marriages with regard to BMI (P<0.002), occupation (P<0.008), weight (P<0.0001), age (P<0.03), age of menopause (P<0.005), parity (P<0.0001), mother's age at menopause (P<0.007) and sister's age at menopause (P<0.002). Conclusion: The study showed that among UAE women, consanguinity of marriage, maternal and sister's age at menopause, BMI, parity number and smoking habits significantly influence the natural age of menopause.