bluuebunny/crossref_metadata_2025 huggingface dataset:
hBD‐1: a novel β‐defensin from human plasma
Authors: Bensch Klaus W. , Raida Manfred , Mägert Hans-Jürgen , Schulz-Knappe Peter , Forssmann Wolf-Georg
Published: April 2003

We report the isolation and characterization of a novel peptide with significant sequence homology to β‐defensins from human blood filtrate. The human β‐defensin‐1 (hBD‐1) is a short basic peptide of 36 amino acid residues. It contains six cysteines forming three intramolecular disulfide bonds. The molecular mass of hBD‐1 is 3928.6 Da. Cloning of the specific cDNA confirmed the amino acid sequence of the native peptide. hBD‐1 shares the nine conserved amino acids characteristic for β‐defensins from respiratory epithelial cells and neutrophils of cattle and chicken leukocytes. hBD‐1 is present in nanomolar concentration in human plasma.
Angiotensin II transduces its signal to focal adhesions via angiotensin II type 1 receptors in vascular smooth muscle cells
Authors: Okuda Masanori , Kawahara Yasuhiro , Nakayama Ichiro , Hoshijima Masahiko , Yokoyama Mitsuhiro
Published: April 2003

In cultured vascular smooth muscle cells (VSMCs), angiotensin II (Ang II) stimulated tyrosine phosphorylation of several proteins including a cluster of 70–80‐kDa proteins as assessed by anti‐phosphotyrosine immunoblotting. These 70–80‐kDa proteins were identified as a focal adhesion‐associated protein, paxillin, by anti‐paxillin immunoprecipitation. Ang II‐stimulated tyrosine phosphorylation of paxillin was detectable within 1 min and maximal at around 10 min and was concentration dependent (half‐maximal effect at around 1 nM). Ang II also stimulated tyrosine phosphorylation of focal adhesion kinase in a time‐ and concentration‐dependent manner. The Ang II type 1 (AT1) receptor antagonist, CV‐11974, but not the Ang II type 2 receptor antagonist, PD123319, inhibited these reactions. These results indicate that Ang II transduces its signal to focal adhesions via AT1 receptors in cultured VSMCs.
Sphingomyelin is synthesized at the plasma membrane of oligodendrocytes and by purified myelin membranes: a study with fluorescent‐ and radio‐labelled ceramide analogues
Authors: Vos Jan P. , Giudici M.Luisa , van der Bijl Petra , Magni Paola , Marchesini Sergio , van Golde Lambert M.G. , Lopes-Cardozo Matthijs
Published: April 2003

In most cell types sphingomyelin is synthesized predominantly in the cis‐medial compartments of the Golgi stacks whereas the contribution of the plasma membrane is much lower. The aim of this study was to assess the contribution of both compartments to the synthesis of sphingomyelin in myelinating cells. Therefore, oligodendrocytes from rat spinal cord were incubated in culture with fluorescently‐ or radiolabelled ceramides, and the effects of a block in the vesicular flow (monensin, brefeldin A, low temperature) on surface synthesis of sphingomyelin were evaluated. The results indicate that ≈50% of the sphingomyelin synthase is present at the plasma and myelin membranes of oligodendrocytes.
Repression of transcriptional activity by heterologous KRAB domains present in zinc finger proteins
Authors: Vissing H. , Meyer W.K.-H. , Aagaard L. , Tommerup N. , Thiesen H.-J.
Published: April 2003

We report the characterization of three novel members of the KRAB‐domain‐containing C2‐H2 zinc finger family (ZNF133, 136 and 140). KRAB (Krüppel‐associated box) is an evolutionarily conserved protein domain found N‐terminally with respect to the zinc finger repeats that encodes the DNA binding domain. ZNF133 and ZNF140 have both the KRAB A‐ and KRAB B‐boxes present at their N‐terminus, whereas ZNF136 contains only the KRAB A‐box. We have previously demonstrated that the KRAB domains derived from ZNF133 and ZNF140 are potent transcriptional repression domains [Margolin et al. (1994) Proc. Natl. Acad. Sci. USA 91, 4509–4513]. The KRAB domain from ZNF136, containing only subdomain A, is a considerable weaker suppression domain; however, when fused to the heterologous KRAB B subdomain of ZNF10 (KOX1) the two subdomains form a KRAB domain which induces repression as potently as previously reported KRAB domains.
Influence of synthetic peptides, corresponding to fragments of the human α‐2 interferon molecule, on the proliferation of lymphoblastoid cells in vitro. Growth inhibition and receptor binding
Authors: Danilkovich A. , Freze K. , Romashkova J. , Valujskikh A. , Makarov E. , Targoni O. , Makarova N. , Kushch A.
Published: April 2003

Influence of nine synthetic peptides from the C‐terminal part of human α‐2 interferon (EN) molecule on growth of a human T‐lymphoblastoid cell line MT‐4 was investigated. It was shown that some peptides inhibited the proliferation of MT‐4 cells. It was also found that MT‐4 cells expressed specific receptors on the outer surface of the plasma membrane. All studied peptides competed for binding to a common binding site. Antiproliferative activity of the peptides correlated with their length and affinity to receptors on the outer membrane of MT‐4 cells.
Biosynthesis and apical localization of the urokinase receptor in polarized MDCK epithelial cells
Authors: Limongi Paola , Resnati Massimo , Hernandez-Marrero Luciano , Cremona Ottavio , Blasi Francesco , Fazioli Francesca
Published: April 2003

The biosynthesis and the surface localization of the urokinase plasminogen activator receptor (uPAR) were analysed in MDCK epithelial cells and in unpolarized fibroblasts. No differences were observed with respect to rate of synthesis, nature of precursors and time of surface appearance. uPAR was localized particularly at the focal and cell‐cell contacts when expressed in fibroblasts. On the contrary, in MDCK cells uPAR was found mostly on the apical surface; in agreement with its localization, down‐regulation of uPAR by the uPA‐PAI‐1 complex was observed only from the apical membrane.
Evidence for, and characterization of, a lipopolysaccharide‐inducible adenosine A2 receptor in human tracheal gland serous cells
Authors: Merten M.D. , Kammouni W. , Figarella C.
Published: April 2003

Human tracheal glands are considered as the principle secretory structures in the bronchotracheal tree. In earlier studies, we successfully performed primary cultures of human tracheal gland (HTG) serous cells and noted that these cells were responsive to many secretagogues including purinergic agonists but not to the inflammatory mediator adenosine. In this study, we demonstrate that adenosine was capable of inducing stimulation of protein secretion by HTG serous cells which had previously been cultured in pro‐inflammatory conditions (induced by lipopolysaccharide (LPS)). This stimulation was inhibited by 8‐phenyltheophylline but not by dipyridamole, which is indicative of a P1 purinoceptor. This inducible receptor is of the adenosine A2 subtype [rank potency order: 5′‐(N‐ethyl)‐carboxamidoadenosine (NECA) > adenosine > N
6‐(phenylisopropyl)‐adenosine (PIA); and stimulation of adenylyl cyclase]. The adenosine‐induced protein secretion was concentration‐dependent, however, increased intracellular cyclic adenosine monophosphate (cAMP) was not dependent on the concentration of adenosine. The adenosine‐induced secretion and the ATP‐induced secretion were shown to be additive. This study concludes that there is evidence of a LPS‐inducible adenosine A2 receptor in human tracheal gland serous cells.
On the physical basis for the cis‐positive rule describing protein orientation in biological membranes
Authors: Krishtalik L.I. , Cramer W.A.
Published: April 2003

The topology of hydrophobic intramembrane proteins is characterized by a statistical asymmetry in the distribution of positively‐charged residues on the two sides of the membrane, the ‘inside‐ or cis‐positive rule’. A mechanism is proposed involving only neutral residue transfer. For a tightly bound polypeptide adsorbed on the membrane and not at equilibrium, the pK values of the ionic residues related to dissociation of the proton into the aqueous phase bulk are increased because of interaction with the negative charges at the membrane surface. The pK shift would selectively neutralize aspartate and glutamate residues, favoring their translocation across the membrane, while stabilizing the impermeant positively charged state of lysine and arginine residues.
Interaction of DNA binding domain of HNF‐3α with its transferrin enhancer DNA specific target site
Authors: Terenzi Hernán , Petropoulos Isabelle , Ellouze Christine , Takahashi Masayuki , Zakin Mario M.
Published: April 2003

Transferrin hepato‐specific gene enhancer, associated with the liver‐enriched HNF‐3α transcriptional factor and ubiquitous proteins, is a complex molecular edifice maintained through DNA‐protein and protein‐protein interactions. As a first step to understand the mechanisms responsible for its organization and activity, we have analyzed the interaction of the DNA binding domain of HNF‐3α (HDBD) with a specific DNA segment present in the transferrin enhancer by different biophysical techniques. The kinetic constants of this interaction were measured using surface plasmon resonance. The HDBD‐DNA interaction was also characterized by circular dichroism and fluorescence spectroscopy. HDBD binds to its specific DNA site with high affinity (). The affinity is reduced after sequence modification of the target DNA. Size exclusion chromatography and binding stoichiometry determined by fluorescence measurements indicate that the protein is present in a monomeric form before and after interaction with the DNA. The secondary structure of the protein was not significantly altered upon binding to specific DNA. By contrast, a structural change of DNA by interaction with HDBD seems to occur.
The linoleic acid metabolite 13‐HODE modulates degranulation of human polymorphonuclear leukocytes
Authors: van de Velde Marco J. , Engels Ferdi , Henricks Paul A.J. , Bloemen Pauline G.M. , Nijkamp Frans P.
Published: April 2003

The effect of the linoleic acid metabolite 13‐hydroxyoctadecadienoic acid (13‐HODE) on degranulation of human polymorphonuclear leukocytes (PMNs) was investigated by measuring the expression of CD11b and CD67 on the plasma membrane. 13‐HODE (5 μM) by itself induced degranulation of PMNs, but to a lesser extent as compared to PAF and fMLP. In addition, 13‐HODE was found to inhibit the PAF‐induced degranulation whereas an additive effect on the fMLP‐induced PMNs degranulation was observed. These results indicate that 13‐HODE can play a modulatory role in degranulation of PMNs.
Scanning tunnelling microscopy studies of β‐amyloid fibril structure and assembly
Authors: Shivji A.P. , Brown F. , Davies M.C. , Jennings K.H. , Roberts C.J. , Tendler S.J.B. , Wilkinson M.J. , Williams P.M.
Published: April 2003

Alzheimer's disease is in part characterised by the deposit of β‐amyloid peptide in the form of fibrils in the brain. In this study, the scanning tunnelling microscope (STM) has been used to provide high resolution images of synthetic fibril structure and formation as a function of time. Short fibrils are observed following brief peptide incubation times. At longer incubation periods ribbon like filaments were observed. These results suggest that β‐amyloid self‐assembly is an ordered process, with a correlation between time of incubation and length of β‐amyloid filament growth.
Regulation by extracellular Na+ of cytosolic Mg2+ concentration in Mg2+‐loaded rat sublingual acini
Authors: Zhang Guo H. , Melvin James E.
Published: April 2003

The regulation of cytosolic free Mg2+ concentration ([Mg2+]i) in Mg2+‐loaded rat sublingual mucous acini was examined using the Mg2+‐sensitive fluorescent indicator mag‐fura‐2. Loading sublingual acini with 5 mM Mg2+ elevated the [Mg2+]i from 0.35 ± 0.01 mM to 0.66 ± 0.01 mM. Removal of extracellular Mg2+ resulted in a significantly faster [Mg2+]i decrease in Mg2+‐loaded rcini than in unloaded acini. Membrane depolarization with high extracellular [K+] and inhibition of P‐type ATPases by vanadate did not alter the [Mg2+]i decrease, indicating that the Mg2+ efflux mechanism is not electrogenic. Na+‐free medium inhibited 80% of the [Mg2+]i decrease suggesting that a Na+‐dependent Mg2+ efflux pathway mediates the [Mg2+]i decrease. Accordingly, the Na+‐dependent antiport inhibitor quinidine reduced > 80% of the [Mg2+]i decrease, suggesting that the Na+‐dependent Mg+ efflux was also partly driven by K+. The [Mg+]i system. Mg2+ efflux was also partly driven by K+. The [Mg2+]i decrease was significantly inhibited by carbachol, a muscarinic agonist, but not by cAMP. These results indicate that in sublingual acinar cells a Na+‐dependent pathway mediates Mg2+ efflux and that muscarinic stimulation may regulate Mg2+ extrusion.
Self‐association of plasma membrane Ca2+‐ATPase by volume exclusion
Authors: Kosk-Kosicka Danuta , Lopez Maria M. , Fomitcheva Ioulia , Lew Virgilio L.
Published: April 2003

At enzyme concentrations above 40 nM the configuration of the purified plasma membrane Ca2+‐ATPase is that of calmodulin‐insensitive dimers. Dilution of the enzyme generates progressively higher proportions of calmodulin‐sensitive monomers with lower V
max and Ca2+ sensitivity than the dimeric enzyme. Dimerization from monomeric state had not been documented before. We investigated whether concentration by volume exclusion, obtained by addition of a large molecular weight dextran to a monomeric Ca2+‐ATPase would elicit dimer‐like behavior. Dextran induced self‐association of monomers, as monitored by fluorescence energy transfer, but the Ca2+ sensitivity of the re‐associated monomers was lower than that of the native dimers. These results suggest that the self‐association reaction is structurally but not functionally reversible, and also document the existence of a hitherto unknown kinetic state of the oligomerized Ca2+‐ATPase, with high V
max but low Ca2+‐sensitivity.
Abscisic acid induced protection against photoinhibition of PSII correlates with enhanced activity of the xanthophyll cycle
Authors: Ivanov A.G. , Krol M. , Maxwell D. , Huner N.P.A.
Published: April 2003

The exogenous application of abscisic acid (ABA) to barley seedlings resulted in partial protection of the PSII photochemistry against photoinhibition at low temperature, the effect being most pronounced at 10−5 M ABA. This was accompanied by higher photochemical quenching (qP) in ABA‐treated leaves. A considerable increase (122%) in the amount of total carotenoids and xanthophylls (antheraxanthin, violaxanthin and zeaxanthin) was also found in the seedlings subjected to ABA. The activity of the xanthophyll cycle measured by the epoxidation state of xanthophylls under high‐light treatment was higher in ABA‐treated plants compared with the control. This corresponds to a higher value (0.411) of non‐photochemical quenching (qNP) observed in ABA‐treated than in control (0.306) leaves.
Specific high affinity binding of human interleukin 1β by Caf1A usher protein of Yersinia pestis
Authors: Zav'yalov Vladimir P. , Chernovskaya Tatiana V. , Navolotskaya Elena V. , Karlyshev Andrey V. , MacIntyre Sheila , Vasiliev Anatoly M. , Abramov Vyacheslav M.
Published: April 2003

Understanding the interaction of Yersinia pestis with the key components of the immune system is important for elucidation of the pathogenesis of bubonic plague, one of the most severe and acute bacterial diseases. Here we report the specific, high affinity binding (K
d = 1.40 × 10−10 M ± 0.14 × 10−10) of radiolabelled human interleukin 1β (hIL‐1β) to E. coli cells carrying the capsular fl operon of Y. pestis. Caf1A outer membrane usher protein was isolated to greater than 98% purity. Competition studies with purified Caf1, together with immunoblotting studies, identified Caf1A as the hIL‐1β receptor. Competition between Caf1 subunit and hIL‐1β for the same or an overlapping binding site on CaflA was demonstrated. Relevance of these results to the pathogenesis of Y. pestis and other Gram negative bacterial pathogens with homologous outer membrane usher proteins is discussed.
Divergence of insulin‐like growth factors I and II in the elasmobranch, Squalus acanthias
Authors: Duguay Stephen J. , Shu Jin Chan  , Mommsen Thomas P. , Steiner Donald F.
Published: April 2003

Recent studies have shown that vertebrates, including teleostean fishes, amphibians, birds and mammals, contain two distinct insulin‐like growth factor (IGF) genes. In contrast agnathans, represented by hagfish, apparently have only one IGF that has features characteristic of both IGF‐I and IGF‐II. Between these groups the elasmobranchs occupy a critical position in terms of the phylogeny of IGFs. We sought to determine if gene duplication and divergence of IGF‐I and IGF‐II occurred before or after divergence of elasmobranchs from other vertebrates by cloning IGF‐like molecules from Squalus acanthias. Our analysis shows that Squalus liver produces two distinct IGF‐like molecules. One has greater sequence identity to, and conserved features characteristic of, known IGF‐I molecules, while the other is more IGF‐II like. These results suggest that the prototypical IGF molecule duplicated and diverged in an ancestor of the extant gnathostomes.
Subunit organisation and symmetry of pore‐forming, oligomeric pneumolysin
Authors: Morgan Peter J. , Hyman Stefan C. , Rowe Arthur J. , Mitchell Timothy J. , Andrew Peter W. , Saibil Helen R.
Published: April 2003

We present a detailed analysis of the oligomeric subunit organisation of pneumolysin by the use of negative stain electron microscopy and image processing to produce a projection density map. Analysis of the rotational symmetry has revealed a large and variable subunit number, between 40–50. The projected subunit density by rotational averaging shows at least two distinct subunit domains at different radial positions. Side views of the rings reveal further details concerning the dimensions of the oligomer in the membrane. On the basis of these observations and our previous knowledge of the monomer domain structure we propose that the 4‐domain subunits are packed in a square planar arrangement to form the pneumolysin oligomer.
A rapid and non‐invasive selection of transgenic embryos before implantation using green fluorescent protein (GFP)
Authors: Ikawa Masahito , Kominami Katsuya , Yoshimura Yasuhide , Tanaka Keiichi , Nishimune Yoshitake , Okabe Masaru
Published: April 2003

Non‐invasive selection of transgenic mice was performed at the stage of preimplantation embryos. The morulae collected from wild female mated with hemizygous transgenic male expressing Aequorea victoria green fluorescent protein (GFP) under chicken β‐actin promoter could be classified as green or non‐green under a fluorescent microscope. All the green embryos were shown to carry the transgene by PCR analysis. Taking advantage of the detection of GFP expression can be done non‐invasively, the selected embryos were demonstrated to be able to developed to term with 100% of accuracy of the selection.
Predicted structure and possible ionmotive mechanism of the sodium‐linked NADH‐ubiquinone oxidoreductase of Vibrio alginolyticus
Authors: Rich Peter R. , Meunier Brigitte , Ward F.Bruce
Published: April 2003

Two groups have now published sequences of the six genes contained in the operon coding for the sodium‐linked NADH‐ubiquinone oxidoreductase of Vibrio alginolyticus. Sequence analyses indicate that this enzyme is unrelated to other known respiratory NADH dehydrogenases. A search for cofactor motifs suggests that the enzyme contains only one FAD, a ferredoxin‐type iron sulphur centre, and the NADH‐binding site. These are all located on NqrF, a subunit that can be recognized as a new member of a large diverse family of NAD(P)H‐oxidizing flavoenzymes. A possible model of ion‐coupling is presented, based upon this new information.
Inverse deuterium kinetic isotope effect for peroxidation in human low‐density lipoprotein (LDL): a simple test for tocopherol‐mediated peroxidation of LDL lipids
Authors: Witting Paul K. , Bowry Vincent W. , Stocker Roland
Published: April 2003

α‐Tocopherol (α‐TOH) can act as a pro‐ or anti‐oxidant for isolated ubiquinol‐10‐free human low density lipoprotein (LDL). We demonstrate that α‐TOH is a more potent pro‐oxidant than other forms of vitamin E for LDL peroxidation initiated by mild fluxes of aqueous peroxyl radicals and low concentrations of Cu2+. A simple deuterium exchange test shows that α‐TOH switches from pro‐ to anti‐oxidant at Cu2+: LDL ratios 2.5. The results suggest that this test may be useful to distinguish ‘inhibited’ perioxidation of emulsion lipids propagated via the lipid peroxyl radical from that mediated via the antioxidant radical.
Direct binding of Torpedo syntrophin to dystrophin and the 87 kDa dystrophin homologue
Authors: Dwyer Timothy M. , Froehner Stanley C.
Published: April 2003

Syntrophin, a 58‐kDa membrane‐associated protein, is one component of a protein complex associated with dystrophin and other members of the dystrophin family, including the 87‐kDa homologue (87K protein). To characterize interactions between syntrophin and 87K protein, we used an in vitro overlay binding assay. We demonstrate that purified Torpedo syntrophin binds directly to dystrophin and 87K. By expressing overlapping regions of the 87K protein as bacterial fusion proteins for binding targets, we show that a 52‐amino acid region of 87K (residues 375–426) is sufficient for binding syntrophin.
Distribution of isoforms of the microtubule‐associated protein tau in grey and white matter areas of human brain: A two‐dimensional gelelectrophoretic analysis
Authors: Janke Carsten , Holzer Max , Klose Joachim , Arendt Thomas
Published: April 2003

The microtubule‐associated protein tau in human brain consists of six molecular isoforms derived from a single gene by alternative mRNA‐splicing and further modified by posttranslational processing. In the present study, the distribution of tau isoforms in grey and white matter of human temporal cortex was investigated by two‐dimensional gelelectrophoresis. More than 80 isoforms were detected. The pattern of isoforms obtained after treatment with alkaline phosphatase was still more complex than those of recombinant tau, indicating that posttranslational modifications other than phosphorylation contribute to the molecular heterogeneity of tau. The tau isoform D according to Goedert [1] containing fout tubulin‐binding regions shown to promote tubulin polymerisation most efficiently was present in higher amounts in white as compared to grey matter. The pattern of isoform distribution was not significantly altered in Alzheimer's disease. It is concluded that molecular isoforms that differ in their tubulin‐binding characteristics are differentially distributed in subcellular neuronal compartments and/or neuronal types.
Activation of p70 S6 protein kinase is necessary for angiotensin II‐induced hypertrophy in neonatal rat cardiac myocytes
Authors: Takano Hiroyuki , Komuro Issei , Zuu Yunzeng , Kudoh Sumiyo , Yamazaki Tsutomu , Yazaki Yoshio
Published: April 2003

Although many lines of evidence have suggested that angiotensin II (Ang II) plays an important role in development of cardiac hypertrophy, the mechanism by which Ang II increases protein synthesis in cardiac myocytes remains unclear. It has been reported that the phosphorylation of S6 protein in 40 S ribosome is correlated to the efficiency of protein synthesis. In the present study, we have examined whether Ang II activates p70 S6 kinase (p70S6K), which has been reported to phosphorylate S6 protein. Ang II activated p70S6K through AT1 receptor. An immunosuppressant agent, rapamycin, inhibited Ang II‐induced p70S6K activation but not the activation of MAP kinases or the induction of c‐fos gene expression. Rapamycin also abolished Ang II‐induced increase in protein synthesis. These results suggest that Ang II induces cardiac hypertrophy by activating p70S6K.
TPA and cycloheximide modulate the activation of NF‐κB and the induction and stability of nitric oxide synthase transcript in primary neonatal rat hepatocytes
Authors: Menegazzi Marta , Guerriero Chiara , Carcereri de Prati Alessandra , Cardinale Caterina , Suzuki Hisanori , Armato Ubaldo
Published: April 2003

12‐O‐Tetradecanoylphorbol 13‐acetate (TPA) elicited a transient increase in the transcription of the inducible nitric oxide synthase (iNOS) gene coupled with a shortening of the half‐life of its mRNA in primary neonatal rat hepatocytes. These effects of TPA were preceded by a surge in nuclear translocation of the transcription factor NF‐κB, and followed by a mounting accumulation of NO2
− in the growth medium. Even cycloheximide (CHX) added by itself elicited an early, sustained activation of NF‐κB followed by an intense induction of iNOS gene expression, irrespective of what degree of protein synthesis inhibition was brought about by the several concentrations tested. When given together, TPA and CHX exerted additive effects on hepatocellular iNOS mRNA levels. These results suggest the likelihood of an ordered sequence of events by which an activated NF‐κB mediates the induction of iNOS gene expression in TPA‐ and/or CHX‐treated primary hepatocytes.
Analysis of the catalytic site of the actin ADP‐ribosylating Clostridium perfringens iota toxin
Authors: van Damme Josef , Jung Martin , Hofmann Fred , Just Ingo , Vandekerckhove Joel , Aktories Klaus
Published: April 2003

The enzyme component of the actin ADP‐ribosylating Clostridium perfringens iota toxin was affinity labelled by UV irradiation in the presence of [carbonyl‐14C]NAD. A peptide containing the radiolabel was generated by CNBr cleavage and subsequent proteolysis with trypsin. Its amino acid sequence is Gly‐Ser‐Pro‐Gly‐Ala‐Tyr‐Leu‐Ser‐Ala‐Ile‐Pro‐Gly‐Tyr‐Ala‐Gly‐X‐Tyr‐Glu‐Val‐Leu‐Leu‐Asn‐His‐Gly‐Ser‐Lys corresponding with the region Gly‐363 through Lys‐388 in the C. perfringens iota toxin. Mass spectrometric data as well as the results of the PTH‐amino acid analysis are in line with a modification of a glutamic acid side chain located at position 378. Therefore, in addition to Glu‐380, as could be concluded by analogy with other ADP‐ribosyltransferases, Glu‐378 may play a pivotal role in the active site of C. perfringens iota toxin.
Similar Ca2+ dependences of [3H]ryanodine binding to α‐ and β‐ryanodine receptors purified from bullfrog skeletal muscle in an isotonic medium
Authors: Murayama Takashi , Ogawa Yasuo
Published: April 2003

To understand the functions of the two ryanodine receptor isoforms (α‐ and β‐RyRs) in nonmammalian skeletal muscles, we determined [3H]ryanodine binding to these isoforms purified from bullfrog skeletal muscle. In 0.17 M‐NaCl medium, both isoforms demonstrated similar Ca2+ dependent ryanodine binding activities, while the Ca2+ sensitivity for activation of β‐RyR was increased in 1 M‐NaCl medium. This enhancement in Ca2+ sensitivity depended on the kind of salts used. These results imply that α‐ and β‐RyRs may have similar properties as Ca2+‐induced Ca2+ release channels in bullfrog skeletal muscle.
Modified 2S albumins with improved tryptophan content are correctly expressed in transgenic tobacco plants
Authors: Marcellino Lucilia H. , Neshich Goran , Grossi de Sá Maria F. , Krebbers Enno , Gander Eugen S.
Published: April 2003

Brazil nut 2S albumins lack the essential amino acid tryptophan. In order to improve the protein's nutritional value and create a basis for structural investigations, three separate modified Brazil nut 2S albumin genes were constructed. The first mutant contains five consecutive tryptophan codons, while the other two modified genes encode proteins carrying single tryptophan residues at sites that will allow confirmation of the predicted protein structure through fluorescence quenching techniques. The modified genes, under the regulation of the CaMV 35S promoter, were introduced into Nicotiana tabacum. All three modified genes were correctly transcribed and the 2S albumin accumulated in the seeds of transgenic plants.
Sucrose protects cell wall invertase but not vacuolar invertase against proteinaceous inhibitors
Authors: Sander Andreas , Krausgrill Silke , Greiner Steffen , Weil Marion , Rausch Thomas
Published: April 2003

Vacuolar (VI) and cell wall invertases (CWI) of higher plants can be inactivated in vitro and, possibly, in vivo by proteinaceous inhibitors. The respective mechanisms have not yet been compared. Therefore, partially purified CWI from transformed tobacco cells and VI from tomato fruit were preincubated with invertase‐inhibitor fractions isolated from the same tissues. Both inhibitors were able to inhibit both invertases. However, VI was fully inhibited within less than 1 min by both inhibitors, whereas inactivation of CWI was much slower. Furthermore, CWI, but not VI, was strongly protected against inhibition by sucrose. A polyclonal antiserum directed against the tobacco inhibitor (INT) cross‐reacted with a 19 kDa polypeptide in the partially purified tomato inhibitor (ILE) fraction. The results indicate that INT and ILE have similar structural properties, whereas the mechanism of inactivation is clearly different for CWI and VI.
First‐report of the inhibition of arbuscular mycorrhizal infection of Pisum sativum by specific and irreversible inhibition of polyamine biosynthesis or by gibberellic acid treatment
Authors: El Ghachtouli N. , Martin-Tanguy J. , Paynot M. , Gianinazzi S.
Published: April 2003

DFMO (α‐dl‐difluoromethylornithine), a specific irreversible inhibitor of ornithine decarboxylase (ODC), a polyamine biosynthetic pathway enzyme, strongly inhibits root growth and arbuscular mycorrhizal infection of Pisum sativum (P56 myc+, isogenic mutant of cv. Frisson). This inhibition is reversed when exogenous polyamine (putrescine) is included in the DFMO treatment, showing that the effect of DFMO on arbuscular mycorrhizal infection is indeed due to putrescine limitation and suggesting that ODC may have a role in root growth and mycorrhizal infection. However, treatment with gibberellic acid (GA3) which increased root titers of polyamines strongly inhibited arbuscular mycorrhizal development. The possible role of polyamines in the regulation of the development of arbuscular mycorrhizal infection is discussed.
CMP‐N‐acetylneuraminic acid hydroxylase: the first cytosolic Rieske iron‐sulphur protein to be described in Eukarya
Authors: Schlenzka Wiebke , Shaw Lee , Kelm Soerge , Schmidt Christian L. , Bill Eckhard , Trautwein Alfred X. , Lottspeich Friedrich , Schauer Roland
Published: April 2003

Electron paramagnetic resonance (EPR) spectroscopy and analysis of the primary structure of the CMP‐N‐acetylneuraminic acid hydroxylase revealed that this enzyme is the first iron‐sulphur protein of the Rieske type to be found in the cytosol of Eukarya. The dithionite‐reduced hydroxylase exhibited an EPR signal known to be characteristic for a Rieske iron‐sulphur centre (2Fe‐2S), the g‐values being 1.78, 1.91 and 2.01, respectively. An analysis of the primary structure of the hydroxylase led to the identification of an amino acid sequence, known to be characteristic for Rieske proteins. Furthermore, possible binding sites for cytochrome b
5, the substrate CMP‐Neu5Ac and a mononuclear iron centre were also identified.
Cell surface binding and activation of gelatinase A induced by expression of membrane‐type‐1‐matrix metalloproteinase (MT1‐MMP)
Authors: Sato Hiroshi , Takino Takahisa , Kinoshita Takeshi , Imai Kazushi , Okada Yasunori , Stetler Stevenson William G. , Seiki Motoharu
Published: April 2003

Gelatinase A is secreted as a proenzyme (progelatinase A) which is activated and bound on the surface of tumor and normal cells. We have reported that the expression of a membrane‐type‐1‐matrix metalloproteinase (MT1‐MMP) induces activation of progelatinase A. Here we demonstrate that the expression of MT1‐MMP in COS‐1 cells induces cell‐surface binding of progelatinase A which is consequently processed to an intermediate form. Processing from the intermediate to the fully active form is dependent on the gelatinase A concentration. These results suggest that the cell‐surface binding concentrates the gelatinase A intermediate form locally to allow autoproteolytic processing to the fully active form.
Site‐directed mutagenesis but not γ‐carboxylation of Glu‐35 in factor VIIa affects the association with tissue factor
Authors: Persson Egon , Nielsen Lars S.
Published: April 2003

Factor VIIa is a vitamin K‐dependent enzyme whose γ‐carboxyglutamic acid (Gla)‐containing domain is important for calcium ion‐dependent binding to the cofactor tissue factor and membrane surfaces. This domain contains 10 Gla residues, the individual roles and importance of which are not known. Comparisons with the homologous protein C, factor IX and prothrombin may provide functional information on the first nine Gla residues, whereas no data can be extrapolated to Gla‐35 in factor VIIa. Therefore, the effects of posttranslational γ‐carboxylation and site‐directed mutagenesis of Glu‐35 were investigated. Mutations to Asp, Gln or Val all lead to a lower affinity for tissue factor by decreasing the rate of association, in the case of the Val mutant by a factor of 200, as measured by surface plasmon resonance. In contrast, Glu or Gla side chains at position 35 appear to fulfil the functional roles equally well.
RNGB: a Dictyostelium RING finger protein that is specifically located in maturing spore cells
Authors: Kawata Takefumi , Steel Jane B. , Williams Jeffrey G.
Published: April 2003

The RING finger is a form of zinc finger motif found in proteins of widely varying biological function. The Dictyostelium RNGB protein contains a RING finger and also a K‐box, a sequence motif found in several plant homeotic proteins. The rngB mRNA is present at low concentration in growing cells and gradually increases in abundance throughout development. However, the RNGB protein is not detected until culmination and we present evidence that suggests it is translationally regulated. The protein is specifically localised in maturing spore cells and is cytoplasmic, suggesting that the RING finger does not function as a DNA binding domain.
Ligation of CD40 rescues Ramos‐Burkitt lymphoma B cells from calcium ionophore‐ and antigen receptor‐triggered apoptosis by inhibiting activation of the cysteine protease CPP32/Yama and cleavage of its substrate PARP
Authors: An Sungkwan , Knox Kirstine A.
Published: April 2003

The new and growing family of interleukin‐1β‐converting enzyme (ICE) cysteine proteases are now recognised to be major effectors of cellular death by apoptosis. Like other members of this family, the CPP32/Yama proform is activated by processing to its active heterodimeric enzyme or apopain when it likely contributes to the process of apoptosis by cleaving poly(ADP‐ribose) polymerase (PARP) and thereby inhibiting much of its DNA repair activity. Apoptosis plays a fundamental role in the regulation of the immune system where it is involved in the selection of both T and B lymphocytes bearing antigen receptor (AgR) for non‐self. Cells of the Ramos Epstein‐Barr virus (EBV)‐genome‐negative Burkitt lymphoma (BL) B cell line (Ramos‐BL) can be triggered into growth arrest and apoptosis by treating with the calcium ionophore ionomycin or by crosslinking their surface AgR with antibodies directed against immunoglobulin (Ig)M (anti‐IgM). Ionomycin‐ and AgR‐triggered growth arrest and apoptosis are arrested by signals transduced through the surface CD40 of Ramos‐BL B cells. Both ionomycin and anti‐IgM trigger activation of CPP32 and cleavage of PARP prior to the onset of apoptosis; this process is abrogated by treatment with anti‐CD40 and is independent of Bcl‐2 expression. A tripeptide inhibitor of ICE family cysteine proteases, Z‐Val‐Ala‐Asp‐fluoromethylketone (zVAD‐fmk) inhibits ionomycin‐ and AgR‐triggered CPP32 activation, PARP cleavage and apoptosis, but not growth arrest, in Ramos‐BL B cells. Thus, in this report we demonstrate that in a physiological system, activation of endogenous members of the ICE family, including CPP32, and cleavage of the death substrate PARP act as major effectors of apoptotic death.
Botulinum neurotoxin light chains inhibit both Ca2+‐induced and GTP analogue‐induced catecholamine release from permeabilised adrenal chromaffin cells
Authors: Glenn Daphne E. , Burgoyne Robert D.
Published: April 2003

Using digitonin‐permeabilised bovine adrenal chromaffin cells, the effects of botulinum neurotoxin light chains on exocytosis triggered by Ca2+ or by GppNHp were examined. Botulinum neurotoxin D light chain, prepared as a His6‐tagged recombinant protein, cleaved VAMP and substantially inhibited catecholamine release due to Ca2+ and GppNHp. Botulinum neurotoxin C1 and E light chains produced partial inhibition of both Ca2+‐ and GppNHp‐induced catecholamine release. These results suggest that Ca 2+‐dependent exocytosis and Ca2+‐independent exocytosis triggered by a non‐hydrolysable GTP analogue occurs via a SNARE‐dependent mechanism in chromaffin cells.
Acetylcholinesterase from Bungarus venom: a monomeric species
Authors: Cousin Xavier , Créminon Christophe , Grassi Jacques , Méflah Khaled , Cornu Gur , Saliou Bernard , Bon Suzanne , Massoulié Jean , Bon Cassian
Published: April 2003

The venom of Bungarus fasciatus, an Elapidae snake, contains a high level of AChE activity. Partial peptide sequences show that it is closely homologous to other AChEs. Bungarus venom AChE is a non‐amphiphilic monomeric species, a molecular form of AChE which has not been previously found in significant levels in other tissues. The composition of carbohydrates suggests the presence of N‐glycans of the ‘complex’ and ‘hybrid’ types. Ion exchange chromatography, isoelectric focusing and electrophoresis in non‐denaturing and denaturing conditions reveal a complex microheterogeneity of this enzyme, which is partly related to its glycosylation.
Direct involvement of hydrogen peroxide in bacterial α‐hydroxylation of fatty acid
Authors: Matsunaga Isamu , Yamada Maki , Kusunose Emi , Nishiuchi Yukiko , Yano Ikuya , Ichihara Kosuke
Published: April 2003

We have reported that fatty‐acid α‐hydroxylase partially purified from Sphingomonas paucimobilis required NADH and molecular oxygen. In this study, we found that the reaction was greatly inhibited by catalase. Glutathione and glutathione peroxidase also inhibited α‐hydroxylation, but superoxide dismutase and mannitol did not. Replacement of NADH and molecular oxygen by hydrogen peroxide increased the α‐hydroxylation activity. In the presence of hydrogen peroxide, molecular oxygen was not required for the activity. These findings suggest that hydrogen peroxide was essential for bacterial α‐hydroxylase.
Structures of new acidic O‐specific polysaccharides of the bacterium Proteus mirabilis serogroups O26 and O30
Authors: Shashkov Alexander S. , Toukach Filip V. , Paramonov Nikolay A. , Ziolkowski Andrzej , Senchenkova Sof'ya N. , Kaca Wieslaw , Knirel Yuriy A.
Published: April 2003

The polysaccharide chains of the lipopolysaccharides of the Proteus mirabilis serogroups O26 and O30 were studied using sugar and methylation analysis and 1H and 13C NMR spectroscopy, including two‐dimensional correlation spectroscopy and rotating‐frame NOE spectroscopy. The polysaccharides were found to be acidic due to the presence of d‐galacturonic acid and its amide with l‐lysine in serogroup O26 or d‐glucuronic acid in serogroup O30, and the structures of their tetrasaccharide repeating units were established. The O26‐specific polysaccharide is structurally and serologically related to the O‐specific polysaccharide of P. mirabilis O28, which includes amides of d‐GalA with l‐lysine and l‐serine [Radziejewska‐Lebrecht, J. et al. (1995) Eur. J. Biochem. 230, 705–712].
Interferon‐γ inhibits 35S incorporation in heparan sulfate synthesized by human skin fibroblasts
Authors: Praillet Christel , Lortat-Jacob Hugues , Grimaud Jean-Alexis
Published: April 2003

Glycosaminoglycans synthesized by human skin fibroblasts were simultaneously radiolabelled with d‐[1‐3H]glucosamine and Na2
35SO4. Considering 3H incorporation, we found that IFNγ increased the production of glycosaminoglycan synthesis, including hyaluronic acid, heparan and chondroitin/dermatan sulfate. In contrast, the production of heparan and chondroitin/dermatan sulfate was slightly decreased on the basis of the 35S signal. Furthermore, when heparan sulfate was treated with nitrous acid, the release of free 35S was greater in control than in treated cells, although the 3H patterns of depolymerization with this agent were similar. These data demonstrate that IFNγ inhibits the incorporation of sulfate from extracellular medium into heparan sulfate.
Comparison of proline and N‐methylnorleucine induced conformational equilibria in cyclic pentapeptides
Authors: Weiβhoff Hardy , Wieprecht Torsten , Henklein Peter , Frömmel Cornelius , Antz Christof , Mügge Clemens
Published: April 2003

The cyclic, imido acid containing pentapeptides cyclo(Asp‐Trp‐(NMe)Nle‐Asp‐Phe) (cpp[NMeNle3]) and cyclo(Asp‐Trp‐Pro‐Asp‐Phe) (cpp[Pro3]) have been investigated by 1H‐NMR spectroscopy in DMSO and by restrained molecular dynamics methods. The spectra indicate the existence of at least four cis/trans isomers for cpp[NMeNle3] and two cis/trans isomers for cpp[Pro3]. In addition to the imido peptide bonds, cpp[NMeNle3] shows cis/trans isomerization of the Asp4‐Phe5 and Phe5‐Asp1 peptide bonds whereas only the Phe5‐Asp1 peptide bond isomerizes in the Pro‐containing peptide. In cpp[Pro3] all cis bonds are centred in βVIb turns. Also, cpp[NMeNle3] prefers backbone angles around the cis bonds which are rather similar to the angles of a βVIb turn. The higher number of cis/trans isomers and slight deviations in the backbone angles of comparable isomers of both peptides are caused by an enhanced flexibility of cpp[NMeNle3] due to the possibility of the φ‐(NMe)Nle rotation.
Oxidant‐mediated proteolytic activation of Ca2+‐ATPase in microsomes of pulmonary smooth muscle
Authors: Ghosh Salil K. , Chakraborti Tapati , Michael John R. , Chakraborti Sajal
Published: April 2003

Treatment of bovine pulmonary artery smooth muscle tissue microsomes with H2O2 (1 mM) markedly stimulated protease activity tested with a synthetic substrate
p‐nitroanilide (BAPNA), and also enhanced Ca2+‐ATPase activity. ATP‐dependent Ca2+ uptake was found to be stimulated upon treatment of the microsomes with H2O2. Pretreatment of the microsomes with vitamin E and aprotinin prevented the H2O2‐induced stimulation of Ca2+‐ATPase activity and also ATP‐dependent Ca2+ uptake. In contrast, H2O2‐induced inhibition of Na+‐dependent Ca2+ uptake was reversed by vitamin E and aprotinin.
The irreversible inactivation of ribonucleotide reductase from Escherichia coli by superoxide radicals
Authors: Gaudu Philippe , Nivière Vincent , Pétillot Yves , Kauppi Björn , Fontecave Marc
Published: April 2003

The expression of superoxide dismutase in all aerobic living organisms supports the concept that superoxide radicals are toxic species. However, because of the limited chemical reactivity of superoxide, the mechanisms of this toxicity are still uncertain. Protein R2, the small component of ribonucleotide reductase, a key enzyme for DNA synthesis, is shown here to be irreversibly inactivated during incubation with an enzymatic generator of superoxide radicals, at neutral pH. During inactivation the essential tyrosyl radical of protein R2 is irreversibly destroyed. Full protection is afforded by superoxide dismutase. It is proposed that coupling between superoxide radicals and the radical protein R2 generates oxidized forms of tyrosine, tyrosine peroxide and 3,4‐dihydroxyphenylalanine.
Molecular cloning of a mouse homologue for the Drosophila splicing regulator Tra2
Authors: Segade Fernando , Hurlé Belén , Claudio Estefanía , Ramos Sofía , Lazo Pedro S.
Published: April 2003

We report the identification of a mouse cDNA, SIG41, encoding a protein of 288 amino acids that is 45% identical (58% similar) to the Drosophila splicing regulator Tra2. SIG41 cDNA contains four polyadenylation signals whose alternative use gives rise to four types of transcripts (2.1, 2.0, 1.5, and 1.4 kb) in mouse cells. Northern analysis and RT‐PCR assays showed that SIG41 mRNA is present in virtually all the cell lines and tissues studied, with remarkable levels of expression in uterus and brain tissues. Differential stability of the SIG41 mRNAs was detected in mouse macrophage cells.
Characterization of a new peptide from Tityus serrulatus scorpion venom which is a ligand of the apamin‐binding site
Authors: Legros Christian , Oughuideni Razika , Darbon Hervé , Rochat Hervé , Bougis Pierre E. , Martin-Eauclaire Marie-France
Published: April 2003

A new ligand (Ts κ) of the apamin binding site on rat brain synaptosomes (K
0.5 = 300 pM) was purified and characterized from the venom of Tityus serrulatus. It is a polypeptide toxin of 35 amino acid residues, with three disulfide bridges. Its cDNA was amplified from a venom gland cDNA library and the nucleotide sequence determined. A model of Ts κ was constructed by amino acid replacement using charybdotoxin structure as determined by 1H nuclear magnetic resonance as starting model.
A possible role for CYP27 as a major renal mitochondrial 25‐hydroxyvitamin D3 1α‐hydroxylase
Authors: Araya Zufan , Norlin Maria , Postlind Hans
Published: April 2003

A mitochondrial cytochrome P450 fraction catalyzing 1α‐ and 27‐hydroxylation but not 24‐hydroxylation of 25‐hydroxyvitamin D3 was purified from pig kidney. The ratio between the 1α‐ and 27‐hydroxylase activities was the same in all purification steps including a side fraction. Attempts to separate the 1α‐ and 27‐hydroxylase activities were unsuccessful. A monoclonal antibody directed against purified pig liver CYP27 recognized a protein of the same apparent M
r and immunoprecipitated both the 1α‐ and 27‐hydroxylase activities towards 25‐hydroxyvitamin D3 in the purified kidney enzyme fraction as well as in a solubilized, crude cytochrome P450 extract considered to represent the major part of the 25‐hydroxyvitamin D3 hydroxylases in kidney mitochondria. Taken together, the results from the purification and the experiments with CYP27 antibody, substrate inhibition, and recombinant expressed human liver CYP27 strongly indicate that CYP27 is able to catalyze 1α‐hydroxylation but not 24‐hydroxylation of 25‐hydroxyvitamin D3 in kidney. In conclusion, the results provide evidence for a role for CYP27 as a major renal mitochondrial 25‐hydroxyvitamin D3 1α‐hydroxylase.
Antimicrobial activity of a 13 amino acid tryptophan‐rich peptide derived from a putative porcine precursor protein of a novel family of antibacterial peptides
Authors: Lawyer C. , Pai S. , Watabe M. , Borgia P. , Mashimo T. , Eagleton L. , Watabe K.
Published: April 2003

It has long been speculated that porcine cathelin is an N‐terminal fragment of a longer precursor protein which possesses antimicrobial activity. In an attempt to find such a precursor, a cDNA clone was recently isolated and sequenced by screening a cDNA library from porcine bone marrow. In order to identify the functional activity of the putative protein encoded by an open reading frame, we have synthesized various lengths of peptides that correspond to the C‐terminal region of the protein and examined them for their antimicrobial activities. We found that a 13 amino acid tryptophan‐rich region with the sequence of VRRFPWWWPFLRR had strong antimicrobial activity with a wide spectrum. It showed potency against Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Staphylococcus epidermidis, Proteus mirabilis, and Streptococcus group D as well as Aspergillus fumigatus. The action of this peptide is bactericidal rather than bacteriostatic and this activity is completely inhibited by 2 mM MgCl2. Our results indicate that the previously identified putative precursor encoded by the isolated cDNA indeed possesses a potent antimicrobial activity and that this 13 amino acid synthetic peptide is considered to be a potentially effective drug against various infectious agents.