bluuebunny/crossref_metadata_2025 huggingface dataset:
A single‐stranded telomere binding protein in the nematode Caenorhabditis elegans
Authors: Yi Soo Young , Joeng Kyu Sang , Kweon Jai Uk , Cho Jin Won , Chung In Kwon , Lee Junho
Published: October 2002

We identified and characterized a protein (STB‐1) from the nuclear extract of Caenorhabditis elegans that specifically binds single‐stranded telomere DNA sequences, but not the corresponding RNA sequences. STB‐1 binding activity is specific to the nematode telomere, but not to the human or plant telomere. STB‐1 requires the core nucleotides of GCTTAGG and three spacer nucleotides in front of them for binding. While any single nucleotide change in the core sequence abolishes binding, the spacer nucleotides tolerate substitution. STB‐1 was determined to be a basic protein of 45 kDa by Southwestern analyses. STB‐1 forms a stable complex with DNA once bound to the telomere.
Nitric oxide and its decomposed derivatives decrease the binding of extracellular‐superoxide dismutase to the endothelial cell surface
Authors: Yamamoto Masayuki , Hara Hirokazu , Adachi Tetsuo
Published: October 2002

Extracellular‐superoxide dismutase (EC‐SOD) is bound to the vascular endothelial cell surface with an affinity for heparan sulfate proteoglycan. The binding of EC‐SOD to the human umbilical vein endothelial cell (HUVEC) and bovine aortic endothelial cell surface proteoglycans was significantly decreased by the incubation with S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) and (±)‐N‐[(E)‐4‐ethyl‐2‐[(Z)‐hydroxyimino]‐5‐nitro‐3‐hexene‐1‐yl]‐3‐pyridine carboxamide (NOR4), potent nitric oxide (NO) donors. NO derived from lipopolysaccharide‐stimulated J774 A‐1 cells also decreased the binding of EC‐SOD to HUVEC, and this decrease was blocked by N
G‐nitro‐L‐arginine, a nitric oxide synthase inhibitor. SNAP and NOR4 also decreased the binding of EC‐SOD to immobilized heparin. Furthermore, the decomposed derivatives of NO donors and sodium nitrite decreased the binding of EC‐SOD. These observations suggest that excess NO produced in the inflammatory conditions decreases the binding of EC‐SOD to the vascular endothelial cell surface, which results in a loss of the ability to protect the endothelial cell surface from oxidative stress.
Cellular strategies for proteolytic targeting during migration and invasion
Authors: Ellis Vincent , Murphy Gillian
Published: October 2002

Cell migration over or through the extracellular matrix (ECM) is an integral feature of both physiological and pathological processes. Regulation of the changing cell–ECM interactions involved can be effected by proteolysis and requires strict spatial and temporal targeting of proteinase activity. The versatile use of different proteinase systems, with a variety of localisation mechanisms and cleavage targets, is being revealed by a plethora of studies using in vitro models. This mini review reflects the status of our knowledge of strategies for the localisation of proteolytic activity effected during cell migration.
Replication of the rat aldolase B locus differs between aldolase B‐expressing and non‐expressing cells
Authors: Miyagi Satoru , Zhao Yung-peng , Saitoh Yasushi , Tamai Katsuyuki , Tsutsumi Ken-ichi
Published: October 2002

We previously reported a rat chromosomal origin of DNA replication (oriA1) that encompassed the aldolase B (AldB) gene promoter. Here, we examined utilization of oriA1 in AldB‐expressing and non‐expressing cells. The results suggested the occurrence of mutually exclusive regulation between DNA replication and transcription. Nascent strand abundance as assayed by competitive polymerase chain reaction using bromodeoxyuridine‐labeled nascent DNA indicated that oriA1 is not utilized in AldB‐expressing cells, while it is fired in non‐expressing cells. In the latter non‐expressing cells, the replication fork seemed to slow at 20–22 kb downstream of oriA1.
Ca2+ and H+ homeostasis in fission yeast: a role of Ca2+/H+ exchange and distinct V‐H+‐ATPases of the secretory pathway organelles
Authors: Okorokov Lev A , Silva Flavia E , Okorokova Façanha Anna L
Published: October 2002

We determined the H+ and Ca2+ uptake by fission yeast membranes separated on sucrose gradient and found that (i) Ca2+ sequestering is due to Ca2+/H+ antiporter(s) localized to secretory pathway organelles while Ca2+‐ATPase activity is not detectable in their membranes; (ii) immunochemically distinct V‐H+‐ATPases acidify the lumen of the secretory pathway organelles. The data indicate that the endoplasmic reticulum, Golgi and vacuole form a network of Ca2+ and H+ stores in the single cell, providing favorable conditions for such key processes as protein folding/sorting, membrane fusion, ion homeostasis and Ca2+ signaling in a differential and local manner.
The chaperone activity of trigger factor is distinct from its isomerase activity during co‐expression with adenylate kinase in Escherichia coli
Authors: Li Zhen-Yu , Liu Chuan-Peng , Zhu Liu-Qin , Jing Guo-Zhong , Zhou Jun-Mei
Published: October 2002

To investigate the molecular chaperone function of trigger factor (TF) and its relationship with isomerase activity in vivo, the assisted folding of adenylate kinase (AK) by TF in Escherichia coli was examined by measuring the amounts of soluble AK produced during co‐expression. When the mutant of chicken AK, P17G, is expressed in plasmid pBVAK, 95% of the protein is found in inclusion bodies. Co‐expression of AK with TF was achieved using a plasmid pBVAT that allowed expression of TF and AK in the same plasmid under separate control. Co‐expression with TF resulted in an increase in the amount of soluble AK, with a higher increase when TF was expressed at higher levels in the cell. Co‐expression of AK with the two TF mutants, Y221G and F233Y, in which peptidyl‐prolyl cis/trans isomerase activity was 1% of wild‐type, gave the same results as wild‐type TF. This provides in vivo evidence that the molecular chaperone activity of TF is distinct from its isomerase activity.
Functional characterization of the Bag7, Lrg1 and Rgd2 RhoGAP proteins from Saccharomyces cerevisiae
Authors: Roumanie Olivier , Weinachter Caroline , Larrieu Isabelle , Crouzet Marc , Doignon François
Published: October 2002

Rho proteins are down‐regulated in vivo by specific GTPase activating proteins (RhoGAP). We have functionally studied three Saccharomyces cerevisiae putative RhoGAP. By first identifying Rho partners with a systematic two‐hybrid approach and then using an in vitro assay, we have demonstrated that the Bag7 protein stimulated the GTPase activity of the Rho1 protein, Lrg1p acted on the Cdc42 and Rho2 GTPases and we showed that Rgd2p has a GAP activity on both Cdc42p and Rho5p. In addition, we brought the first evidence for the existence of a sixth functional Rho in yeast, the Cdc42/Rac‐like GTPase Rho5.
Tetrameric dipeptidyl peptidase I directs substrate specificity by use of the residual pro‐part domain
Authors: Olsen Johan Gotthardt , Kadziola Anders , Lauritzen Conni , Pedersen John , Larsen Sine , Dahl Søren Weis
Published: October 2002

The crystal structure of mature dipeptidyl peptidase I reveals insight into the unique tetrameric structure, substrate binding and activation of this atypical papain family peptidase. Each subunit is composed of three peptides. The heavy and light chains form the catalytic domain, which adopts the papain fold. The residual pro‐part forms a β‐barrel with the carboxylate group of Asp1 pointing towards the substrate amino‐terminus. The tetrameric structure appears to stabilize the association of the two domains and encloses a 12 700 Å3 spherical cavity. The tetramer contains six chloride ions, one buried in each S2 pocket and two at subunit interfaces.
Biliary excretion of a stretched bilirubin in UGT1A1‐deficient (Gunn) and Mrp2‐deficient (TR−) rats
Authors: McDonagh Antony F. , Lightner David A. , Nogales Daniel F. , Norona Wilma S.
Published: October 2002

The metabolism and biliary excretion of a stretched bilirubin analog with a p‐xylyl group replacing the central CH2 hinge were investigated in normal rats, Gunn rats deficient in bilirubin conjugation, and TR− rats deficient in bilirubin glucuronide hepatobiliary transport. Unlike bilirubin, the analog was excreted rapidly in bile unchanged in all three rat strains after intravenous administration. In TR− rats biliary excretion of the analog was diminished, but still substantial, demonstrating that the ATP‐binding cassette transporter Mrp2 is not required for its hepatic efflux. These effects are attributable to differences in the preferred conformations of bilirubin and the analog.
In silico analysis of the tRNA:m1A58 methyltransferase family: homology‐based fold prediction and identification of new members from Eubacteria and Archaea
Authors: Bujnicki Janusz M
Published: October 2002

The amino acid sequences of Gcd10p and Gcd14p, the two subunits of the tRNA:(1‐methyladenosine‐58; m1A58) methyltransferase (MTase) of Saccharomyces cerevisiae, have been analyzed using iterative sequence database searches and fold recognition programs. The results suggest that the ‘catalytic’ Gcd14p and ‘substrate binding’ Gcd10p are related to each other and to a group of prokaryotic open reading frames, which were previously annotated as hypothetical protein isoaspartate MTases in sequence databases. It is predicted that the prokaryotic proteins are genuine tRNA:m1A MTases based on similarity of their predicted active site to the Gcd14p family. In addition to the MTase domain, an additional domain was identified in the N‐terminus of all these proteins that may be involved in interaction with tRNA. These results suggest that the eukaryotic tRNA:m1A58 MTase is a product of gene duplication and divergent evolution of a possibly homodimeric prokaryotic enzyme.
Inhibitors can arrest the membrane activity of human islet amyloid polypeptide independently of amyloid formation
Authors: Harroun Thad A. , Bradshaw Jeremy P. , Ashley Richard H.
Published: October 2002

Human islet amyloid polypeptide (hIAPP), co‐secreted with insulin from pancreatic β cells, misfolds to form amyloid deposits in non‐insulin‐dependent diabetes mellitus (NIDDM). Like many amyloidogenic proteins, hIAPP is membrane‐active: this may be significant in the pathogenesis of NIDDM. Non‐fibrillar hIAPP induces electrical and physical breakdown in planar lipid bilayers, and IAPP inserts spontaneously into lipid monolayers, markedly increasing their surface area and producing Brewster angle microscopy reflectance changes. Congo red inhibits these activities, and they are completely arrested by rifampicin, despite continued amyloid formation. Our results support the idea that non‐fibrillar IAPP is membrane‐active, and may have implications for therapy and for structural studies of membrane‐active amyloid.
GSK‐3 inhibition by adenoviral FRAT1 overexpression is neuroprotective and induces Tau dephosphorylation and β‐catenin stabilisation without elevation of glycogen synthase activity
Authors: Culbert Ainsley A. , Brown Murray J. , Frame Sheelagh , Hagen Thilo , Cross Darren A.E. , Bax Benjamin , Reith Alastair D.
Published: October 2002

Glycogen synthase kinase 3 (GSK‐3) has previously been shown to play an important role in the regulation of apoptosis. However, the nature of GSK‐3 effector pathways that are relevant to neuroprotection remains poorly defined. Here, we have compared neuroprotection resulting from modulation of GSK‐3 activity in PC12 cells using either selective small molecule ATP‐competitive GSK‐3 inhibitors (SB‐216763 and SB‐415286), or adenovirus overexpressingrequentlyearranged indvanced‐cell lymphomas 1 (FRAT1), a protein proposed as a negative regulator of GSK‐3 activity towards Axin and β‐catenin. Our data demonstrate that cellular overexpression of FRAT1 is sufficient to confer neuroprotection and correlates with inhibition of GSK‐3 activity towards Tau and β‐catenin, but not modulation of glycogen synthase (GS) activity. By comparison, treatment with SB‐216763 and SB‐415286 proved more potent in terms of neuroprotection, and correlated with inhibition of GSK‐3 activity towards GS in addition to Tau and β‐catenin.
Involvement of B‐Raf in Ras‐induced Raf‐1 activation
Authors: Mizutani Shin , Inouye Kaoru , Koide Hiroshi , Kaziro Yoshito
Published: October 2002

The mechanism of Ras‐induced Raf‐1 activation is not fully understood. Previously, we identified a 400‐kDa protein complex as a Ras‐dependent Raf‐1 activator. In this study, we identified B‐Raf as a component of this complex. B‐Raf was concentrated during the purification of the activator. Immunodepletion of B‐Raf abolished the effect of the activator on Raf‐1. Furthermore, B‐Raf and Ras‐activated Raf‐1 co‐operatively, when co‐transfected into human embryonic kidney 293 cells. On the other hand, Ras‐dependent extracellular signal‐regulated kinase/mitogen‐activated protein kinase kinase stimulator (a complex of B‐Raf and 14‐3‐3) failed to activate Raf‐1 in our cell‐free system. These results suggest that B‐Raf is an essential component of the Ras‐dependent Raf‐1 activator.
Phosphoenolpyruvate carboxylase kinase involved in C4 photosynthesis in Flaveria trinervia: cDNA cloning and characterization1
Authors: Tsuchida Yuhei , Furumoto Tsuyoshi , Izumida Atsushi , Hata Shingo , Izui Katsura
Published: October 2002

In C4 plants, phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31), a key enzyme in C4 photosynthesis, is controlled by reversible phosphorylation of a conserved Ser residue near the N‐terminus. We now report the first cloning of a cDNA from a C4 plant, Flaveria trinervia, which encodes the specific protein kinase (FtPEPC‐PK) involved in the phosphorylation of C4‐form PEPC. Several lines of supportive evidence are: strict substrate specificity of the recombinant enzyme, prominent light/dark response of the transcript level and abundant expression in leaves of C4 plant (F. trinervia) but very low expression in a C3 plant of the same genus (Flaveria pringlei). We also discuss the possibility that the FtPEPC‐PK gene has co‐evolved with the PEPC gene to participate in C4 photosynthesis.
Inhibition of isoprenoid biosynthesis causes insulin resistance in 3T3‐L1 adipocytes
Authors: Chamberlain Luke H
Published: October 2002

Lovastatin treatment caused down‐regulation of the insulin‐responsive glucose transporter 4 (Glut4) and up‐regulation of Glut1 in 3T3‐L1 adipocytes. These changes in protein expression were associated with a marked inhibition of insulin‐stimulated glucose transport. Lovastatin had no effect on cell cholesterol levels, but its effects were reversed by mevalonate, demonstrating that inhibition of isoprenoid biosynthesis causes insulin resistance in 3T3‐L1 adipocytes. These findings support the notion that whole body insulin resistance may arise as a result of perturbations in general biochemical pathways, rather than primary defects in insulin signalling.
Effect of precursor protein phosphorylation on import into isolated chloroplasts from Chlamydomonas
Authors: Su Qingxiang , Schmid Katharina , Schild Christof , Boschetti Arminio
Published: October 2002

In higher plants, chloroplast‐destined precursor proteins are thought to be phosphorylated. Mediated by a specific 14‐3‐3 protein, these phosphorylated proteins bind to the chloroplast surface and are subsequently imported into the chloroplast. We demonstrate that also in the green alga Chlamydomonas reinhardtii the precursor of the small subunit of ribulose‐1,5‐bisphosphate carboxylase becomes phosphorylated by a plant protein kinase and that the phosphorylation site is located in the transit peptide. The phosphorylation status of the precursor protein regulates its import into chloroplasts especially at an early step during this process. The possible physiological function is discussed.
A pathogen‐inducible divinyl ether synthase (CYP74D) from elicitor‐treated potato suspension cells1
Authors: Stumpe Michael , Kandzia Romy , Göbel Cornelia , Rosahl Sabine , Feussner Ivo
Published: October 2002

In elicitor‐treated potato cells, 9‐lipoxygenase‐derived oxylipins accumulate with the divinyl ether colneleic acid as the major metabolite. Here, the identification of a potato cDNA is described, whose predicted amino acid sequence corresponds to divinyl ether synthases, belonging to the recently identified new P450 subfamily CYP74D. The recombinant protein was expressed in Escherichia coli and shown to metabolize 9‐hydroperoxy linoleic acid to colneleic acid at pH 6.5. This fatty acid derivative has been implicated in functioning as a plant antimicrobial compound. RNA blot analyses revealed accumulation of divinyl ether synthase transcripts both upon infiltration of potato leaves with Pseudomonas syringae and after infection with Phytophthora infestans.
Retina‐specific cis‐elements and binding nuclear proteins of carp rhodopsin gene
Authors: Ma Gwo-Chin , Wang Tzu-Ming , Su Chih-Ying , Wang Yi-Lei , Chen Shiming , Tsai Huai-Jen
Published: October 2002

The upstream cis‐elements controlling the retina‐specific expression of carp rhodopsin gene were fully characterized in vivo. Transgenic studies demonstrated that both carp neural retina leucine zipper response‐like element (cNRE, within nucleotides (nt) −63 to −75) and carp‐specific element (CSE, nt −46 to −52) were crucial to reporter gene expression in medaka retinae. The retina‐specific expression rates of embryos injected with nt −1 to −641 and longer fragments were much higher than those of embryos injected with nt −1 to −138 and shorter fragments, indicating that an enhancer is located in the nt −138 to −641 region. Retinal extracts and the probe BAT‐1 (nt −90 to −120) formed two DNA–protein complexes, B1 and B2. Retinal extracts and the probes cNRE and CSE formed the complexes N1 and C1, respectively. The protein factors in B1 and C1 were mammal‐like cone‐rod homeobox proteins.
Transformation of acridone synthase to chalcone synthase
Authors: Lukačin Richard , Schreiner Stephan , Matern Ulrich
Published: October 2002

Acridone synthase (ACS) and chalcone synthase (CHS) catalyse the pivotal reactions in the formation of acridone alkaloids or flavonoids. While acridone alkaloids are confined almost exclusively to the Rutaceae, flavonoids occur abundantly in all seed‐bearing plants. ACSs and CHSs had been cloned from Ruta graveolens and shown to be closely related polyketide synthases which use N‐methylanthraniloyl‐CoA and 4‐coumaroyl‐CoA, respectively, as the starter substrate to produce the acridone or naringenin chalcone. As proposed for the related 2‐pyrone synthase from Gerbera, the differential substrate specificities of ACS and CHS might be attributed to the relative volume of the active site cavities. The primary sequences as well as the immunological cross reactivities and molecular modeling studies suggested an almost identical spatial structure for ACS and CHS. Based on the Ruta ACS2 model the residues Ser132, Ala133 and Val265 were assumed to play a critical role in substrate specificity. Exchange of a single amino acid (Val265Phe) reduced the catalytic activity by about 75% but grossly shifted the specificity towards CHS activity, and site‐directed mutagenesis replacing all three residues by the corresponding amino acids present in CHS (Ser132Thr, Ala133Ser and Val265Phe) fully transformed the enzyme to a functional CHS with comparatively marginal ACS activity. The results suggested that ACS divergently has evolved from CHS by very few amino acid exchanges, and it remains to be established why this route of functional diversity has developed in the Rutaceae only.
The triiodothyronine nuclear receptor c‐ErbAα1 inhibits avian MyoD transcriptional activity in myoblasts
Authors: Daury Laetitia , Busson Muriel , Casas François , Cassar-Malek Isabelle , Wrutniak-Cabello Chantal , Cabello Gérard
Published: October 2002

Thyroid hormone stimulates myoblast differentiation, through an inhibition of AP‐1 activity occurring at the onset of differentiation. In this study we found that the T3 nuclear receptor c‐ErbAα1 (T3Rα1) is involved in a mechanism preserving the duration of myoblast proliferation. Independently of the hormone presence, T3Rα1 represses avian MyoD transcriptional activity. Using several mutants of T3Rα1, we found that the hinge region plays a crucial role in the inhibition of MyoD activity. In particular, mutations of two small basic sequences included in α helices abrogate the T3Rα1/MyoD functional interaction. Similarly, the T3 receptor also represses myogenin transcriptional activity. Therefore, despite stimulating avian myoblast differentiation by a T3‐dependent pathway not involving myogenic factors, T3Rα1 contributes to maintain an optimal myoblast proliferation period by inhibiting MyoD and myogenin activity.
The antimicrobial peptides lactoferricin B and magainin 2 cross over the bacterial cytoplasmic membrane and reside in the cytoplasm
Authors: Haukland H.H. , Ulvatne H. , Sandvik K. , Vorland L.H.
Published: October 2002

The localization of immunolabelled antimicrobial peptides was studied using transmission electron microscopy. Staphylococcus aureus and Escherichia coli were exposed to lactoferricin B (17–41), lactoferricin B (17–31) and D‐lactoferricin B (17–31). E. coli was also exposed to cecropin P1 and magainin 2. The lactoferricins were found in the cytoplasm of both bacteria. In S. aureus the amount of cytoplasmic lactoferricin B (17–41) was time‐ and concentration‐dependent, reaching a maximum within 30 min. Cecropin P1 was confined to the cell wall, while magainin 2 was found in the cytoplasm of E. coli. The finding of intracellularly localized magainin is not reported previously.
Drosophila Chk2 is required for DNA damage‐mediated cell cycle arrest and apoptosis
Authors: Xu Jinhua , Xin Shijie , Du Wei
Published: October 2002

Chk2 is a major target of ataxia telangiectasia‐mutated (ATM) and ATM‐ and Rad3‐related (ATR). Germline mutations in Chk2 have been identified in a subset of patients with Li–Fraumeni syndrome, suggesting that Chk2 is a tumor suppressor gene. To investigate the role of Chk2 in multicellular organisms, a Drosophila chk2 (Dmchk2) mutant was generated. Dmchk2 mutants are viable but show defects in maintaining genome stability and are highly sensitive to ionizing radiation. Interestingly, mutating Dmchk2 completely blocks DNA damage‐induced apoptosis and partially blocks DNA damage‐induced cell cycle arrest. These results indicate that Chk2 protein plays a crucial role in the DNA damage response pathway mediating cell cycle arrest and apoptosis, and that the ATM‐Chk2 pathway is likely conserved in Drosophila.
K+ channel profile and electrical properties of Arabidopsis root hairs
Authors: Ivashikina Natalya , Becker Dirk , Ache Peter , Meyerhoff Oliver , Felle Hubert H. , Hedrich Rainer
Published: October 2002

Ion channels and solute transporters in the plasma membrane of root hairs are proposed to control nutrient uptake, osmoregulation and polar growth. Here we analyzed the molecular components of potassium transport in Arabidopsis root hairs by combining K+‐selective electrodes, reverse transcription‐PCR, and patch‐clamp measurements. The two inward rectifiers AKT1 and ATKC1 as well as the outward rectifier GORK dominated the root hair K+ channel pool. Root hairs of AKT1 and ATKC1 loss‐of‐function plants completely lack the K+ uptake channel or exhibited altered properties, respectively. Upon oligochitin‐elicitor treatment of root hairs, transient changes in K+ fluxes and membrane polarization were recorded in wild‐type plants, while akt1‐1 root hairs showed a reduced amplitude and pronounced delay in the potassium re‐uptake process. This indicates that AKT1 and ATKC1 represent essential α‐subunits of the inward rectifier. Green fluorescent protein (GFP) fluorescence following ballistic bombardment with GORK promoter‐GFP constructs as well as analysis of promoter‐GUS lines identified this K+ outward rectifier as a novel ion channel expressed in root hairs. Based on the expression profile and the electrical properties of the root hair plasma membrane we conclude that AKT1‐, ATKC‐ and GORK‐mediated potassium transport is essential for osmoregulation and repolarization of the membrane potential in response to elicitors.
Trimethylamine‐N‐oxide‐induced folding of α‐synuclein
Authors: Uversky Vladimir N. , Li Jie , Fink Anthony L.
Published: October 2002

The effect of the natural osmolyte trimethylamine‐N‐oxide (TMAO) on the structural properties and fibril formation of the natively unfolded protein human α‐synuclein was studied using several physico‐chemical methods. TMAO induced folding of α‐synuclein: at moderate concentrations, a partially folded intermediate with enhanced propensity for fibrillation accumulated; at higher concentrations, α‐synuclein was tightly folded and underwent self‐association to form oligomers. The latter conformation was significantly helical and probably represents the physiologically folded form of the protein.
Mutational analysis of the iron binding site of Saccharomyces cerevisiae ferroxidase Fet3. An in vivo study
Authors: Bonaccorsi di Patti Maria Carmela , Paronetto Maria Paola , Dolci Valeria , Felice Maria Rosa , Lania Amalia , Musci Giovanni
Published: October 2002

The role of residues predicted to be involved in the binding of iron by the yeast ferroxidase Fet3 has been studied by site‐directed mutagenesis. The effect of Fet3 mutations E185A, E185Q, Y354F, D409V and H489D has been investigated in vivo by kinetic analyses of high affinity iron uptake. Our results indicate that Glu‐185 is critical for the binding of iron, since substitution of this residue with Ala or Gln strongly affects both growth and the kinetic parameters of high affinity iron uptake, greatly increasing K
m. Mutations Y354F and D409V result in less severe alteration of high affinity iron uptake, while mutant H489D is unable to grow under conditions of iron limitation.
Vacuolation induced by VacA toxin of Helicobacter pylori requires the intracellular accumulation of membrane permeant bases, Cl− and water
Authors: Morbiato Laura , Tombola Francesco , Campello Silvia , Del Giudice Giuseppe , Rappuoli Rino , Zoratti Mario , Papini Emanuele
Published: October 2002

The protein vacuolating toxin A (VacA) of Helicobacter pylori converts late endosomes into large vacuoles in the presence of permeant bases. Here it is shown that this phenomenon corresponds to an accumulation of permeant bases and Cl− in HeLa cells and requires the presence of extracellular Cl−. The net influx of Cl− is due to electroneutral, Na+, K+, 2Cl− cotransporter‐mediated transport. Cell vacuolation leads to cell volume increase, consistent with water flux into the cell, while hyper‐osmotic media decreased vacuole formation. These data represent the first evidence that VacA‐treated cells undergo an osmotic unbalance, reinforcing the hypothesis that the VacA chloride channel is responsible for cell vacuolation.
Domain IVa of laminin α5 chain is cell‐adhesive and binds β1 and αVβ3 integrins through Arg‐Gly‐Asp
Authors: Sasaki Takako , Timpl Rupert
Published: October 2002

The globular domain IVa from the short arm region of mouse laminin α5 chain was obtained by recombinant production and shown to be a cell‐adhesive substrate and to bind αVβ3 integrin in solid‐phase assays. These interactions were blocked by RGD peptides and a restricted panel of anti‐integrin antibodies. The two RGD sequences present in α5IVa were shown by site‐directed mutagenesis to make different contributions to cell adhesion but were equivalent in binding αVβ3 integrin. A quantitative radioimmuno‐inhibition assay was established based on domain α5IVa which demonstrated distinct amounts of α5 chain in various tissues, particularly in vessel walls. There it could play a role in angiogenesis steps requiring RGD‐dependent integrins.
Depolarisation induces rapid and transient formation of intracellular sphingosine‐1‐phosphate
Authors: Alemany Regina , Kleuser Burkhard , Ruwisch Lars , Danneberg Kerstin , Lass Holger , Hashemi Rozita , Spiegel Sarah , Jakobs Karl H. , Meyer zu Heringdorf Dagmar
Published: October 2002

Formation of sphingosine‐1‐phosphate (SPP) by sphingosine kinase serves as a signalling pathway for various membrane receptors. Here, we show that membrane depolarisation is another mechanism by which this pathway can be activated. Formation of [3H]SPP as well as levels of endogenous SPP were rapidly and transiently increased in PC12 pheochromocytoma cells depolarised with high KCl. Time course and maximum were similar to those induced by bradykinin. Depolarisation‐induced SPP production was also observed in RINm5F insulinoma cells, dependent on extracellular Ca2+ and fully suppressed by verapamil, thus apparently caused by Ca2+ influx via voltage‐gated Ca2+ channels. Studies with sphingosine kinase inhibitors and overexpression of sphingosine kinase revealed a partial contribution of this pathway to depolarisation‐induced noradrenaline release and Ca2+ increase.
HuR binds a cyclic nucleotide‐dependent, stabilizing domain in the 3′ untranslated region of Na+/glucose cotransporter (SGLT1) mRNA
Authors: Loflin Paul , Lever Julia E
Published: October 2002

Differentiation‐dependent expression of the Na+/glucose cotransporter (SGLT1) is accompanied by a large, cAMP‐dependent increase in stability of its mRNA. Stabilization is mediated by protein binding to a critical uridine‐rich element (URE) in its 3′ untranslated region. In the present study, we demonstrate that HuR, an RNA binding protein of the embryonic lethal abnormal vision family, binds the SGLT1 URE. HuR binding was increased after elevation of intracellular cAMP levels and was dependent on protein phosphorylation. This interaction was prevented by a substitution mutation previously shown to block cAMP‐dependent reporter message stabilization. These results implicate HuR as a key mediator of cAMP‐dependent SGLT1 mRNA stabilization.
Delayed oxidant‐induced cell death involves activation of phospholipase A2
Authors: Zhao Ming , Brunk Ulf T. , Eaton John W.
Published: October 2002

Short‐term (1 h) exposure of cells to a low steady‐state concentration of H2O2 causes no immediate cell death but apoptosis occurs several hours later. This delayed cell death may arise from activation of phospholipases, in particular phospholipase A2 (PLA2), which may destabilize lysosomal and mitochondrial membranes. Indeed, the secretory PLA2 (sPLA2) inhibitor 4‐bromophenacyl bromide diminishes both delayed lysosomal rupture and apoptosis. Furthermore, sPLA2 activation by mellitin, or direct micro‐injection of sPLA2, causes lysosomal rupture and apoptosis. Finally, B‐cell leukemia/lymphoma 2 (Bcl‐2) over‐expression prevents oxidant‐induced activation of PLA2, delayed lysosomal destabilization and apoptosis. This supports a causal association between PLA2 activation and delayed oxidant‐induced cell death and suggests that Bcl‐2 may suppress apoptosis by preventing PLA2 activation.
Golgi retention of human protein NEFA is mediated by its N‐terminal Leu/Ile‐rich region
Authors: Nesselhut Jan , Jurgan Ulrich , Onken Elke , Götz Hilde , Barnikol Heinz Ulrich , Hirschfeld Gregor , Barnikol-Watanabe Shitsu , Hilschmann Norbert
Published: October 2002

The subcellular localization of the human Ca2+‐binding EF‐hand/leucine zipper protein NEFA was studied in HeLa cells by immunofluorescence microscopy. Double immunostaining using mouse anti‐NEFA monoclonal antibody 1H8D12 and rabbit anti‐ERD2 polyclonal antibody proved that NEFA is localized in the Golgi apparatus. The result was confirmed by the expression of NEFA–green fluorescent protein (GFP) fusion protein in the Golgi in the same cell line. Cycloheximide treatment proved NEFA to be a Golgi‐resident protein. Seven NEFA deletion mutants were constructed to ascertain the peptide region relevant for Golgi retention. The expression of each NEFA–GFP variant was detected by fluorescence microscopy and immunoblotting. Only the ΔN mutant, lacking the N‐terminal Leu/Ile‐rich region, failed to be retained in the Golgi after cycloheximide treatment. The other six deletion mutants in which either the basic region, the complete EF‐hand pair domain, the two EF‐hand motifs separately, the leucine zipper and the leucine zipper plus the C‐terminal region is deleted, were localized to the Golgi. The peptide sequence within the Leu/Ile‐rich region is discussed as a novel Golgi retention motif.
Simple shifts in redox/thiol balance that perturb blood coagulation
Authors: Bayele Henry K. , Murdock Paul J. , Perry David J. , Pasi K.John
Published: October 2002

The biological chemistry that underlies and regulates the blood coagulation cascade is not fully understood. To begin to understand this, we performed clotting assays under various redox conditions. By varying the amount of oxidant and/or antioxidant in these assays, we observed that both the intrinsic/tenase complex and the extrinsic pathways were susceptible to shifts in the thiol/redox balance. We established a dichotomy where blood clotting via the intrinsic pathway was sensitive to oxidation whereas the tissue factor or extrinsic pathway was more sensitive to reduction. These differential inhibitory effects present a conceptual mechanism for selective modulation of the activities of clotting factors specific for the respective pathways. These data also suggest that blood clotting may be influenced by unidentified redox or thiol equilibria.
Classification of PDZ domains
Authors: Bezprozvanny Ilya , Maximov Anton
Published: October 2002

A diverse family of PDZ domains has been identified, but the rules that govern their ligand specificity are not clear. Here we propose a novel classification of PDZ domains based on the nature of amino acids in the two critical positions in the PDZ domain fold. Using these principles, we classified PDZ domains present in the SMART database. Using yeast two‐hybrid, in vitro pull‐down and plasmon surface resonance assays, we demonstrated that in agreement with their position in the proposed classification the Mint1‐1, hINADL‐5, and PAR6 PDZ domains display similar dual ligand specificity. The proposed classification helps to organize PDZ domain containing proteins.
A study on permeability transition pore opening and cytochrome c release from mitochondria, induced by caspase‐3 in vitro
Authors: Xia Tian , Jiang Chunsun , Li Linjiang , Wu Caihong , Chen Quan , Liu Shu-sen
Published: October 2002

We recently described that there is a feedback amplification of cytochrome c release from mitochondria by caspases. Here we investigated how caspases impact on mitochondria to induce cytochrome c release and found that recombinant caspase‐3 induced opening of permeability transition pore and reduction of membrane potential in vitro. These events were inhibited by Bcl‐xL, cyclosporin A and z‐VAD.fmk. Moreover, caspase‐3 stimulated the rate of mitochondrial state 4 respiration, superoxide production and NAD(P)H oxidation in a Bcl‐xL‐ and cyclosporin A‐inhibitable manner. These results suggest that caspase‐3 induces cytochrome c release by inducing permeability transition pore opening which is associated with changes in mitochondrial respiration and redox potential.
Functional plasticity of CH domains
Authors: Gimona Mario , Djinovic-Carugo Kristina , Kranewitter Wolfgang J. , Winder Steven J.
Published: October 2002

With the refinement of algorithms for the identification of distinct motifs from sequence databases, especially those using secondary structure predictions, new protein modules have been determined in recent years. Calponin homology (CH) domains were identified in a variety of proteins ranging from actin cross‐linking to signaling and have been proposed to function either as autonomous actin binding motifs or serve a regulatory function. Despite the overall structural conservation of the unique CH domain fold, the individual modules display a quite striking functional variability. Analysis of the actopaxin/parvin protein family suggests the existence of novel (type 4 and type 5) CH domain families which require special attention, as they appear to be a good example for how CH domains may function as scaffolds for other functional motifs of different properties.
PSI‐O, a new 10‐kDa subunit of eukaryotic photosystem I
Authors: Knoetzel Jürgen , Mant Alexandra , Haldrup Anna , Jensen Poul Erik , Scheller Henrik Vibe
Published: October 2002

A novel polypeptide with an apparent molecular mass of 9 kDa was detected after sodium dodecyl sulphate–polyacrylamide gel electrophoresis of Arabidopsis photosystem I (PSI) and was N‐terminally sequenced. Corresponding cDNA clones encode a precursor protein of 140 amino acid residues which was imported into isolated intact chloroplasts and processed to the mature protein, designated PSI‐O. The mature protein has two transmembrane helices and a calculated mass of 10 104 Da. The PSI‐O protein was also shown to be present in PSI isolated from barley and spinach, and was essentially absent in chloroplast grana. Expressed sequences encoding similar proteins are available from many species of plants and green algae.
Discriminating small molecule DNA binding modes by single molecule force spectroscopy
Authors: Krautbauer Rupert , Pope Lisa H. , Schrader Tobias E. , Allen Stephanie , Gaub Hermann E.
Published: October 2002

Drugs may interact with double stranded DNA via a variety of binding modes, each mode giving rise to a specific pharmacological function. Here we demonstrate the ability of single molecule force spectroscopy to discriminate between different interaction modes by measuring the mechanical properties of DNA and their modulation upon the binding of small molecules. Due to the unique topology of double stranded DNA and due to its base pair stacking pattern, DNA undergoes several well‐characterised structural transitions upon stretching. We show that small molecule binding markedly affects these transitions in ways characteristic to the binding mode and that these effects can be detected at the level of an individual molecule. The minor groove binder berenil, the crosslinker cisplatin and the intercalator ethidium bromide are compared.
Observation of positive selection within hypervariable regions of a newly identified DNA virus (SEN virus)1
Authors: Umemura Takeji , Tanaka Yasuhito , Kiyosawa Kendo , Alter Harvey J. , Shih J.Wai-Kuo
Published: October 2002

To elucidate the evolution of SEN virus (SEN‐V), serial sequences of chronically SEN‐V‐infected patients were analyzed. In the hypervariable regions, non‐synonymous substitutions significantly predominated. This could be attributed to positive selection in evading immune surveillance of the hosts and to establish a persistent infection. On the basis of the sequences in the two open reading frames of SEN‐V DNA, the rate of synonymous substitutions was 7.32×10−4 per site per year. Since this rate is close to RNA viruses and higher than other DNA viruses, the SEN‐V might be replicated by machinery with poor or no proofreading function.
A non‐classical ISRE/ISGF3 pathway mediates induction of RANTES gene transcription by type I IFNs
Authors: Cremer Isabelle , Ghysdael Jacques , Vieillard Vincent
Published: October 2002

RANTES (regulated upon activation normal T cell expressed and secreted) is a chemoattractant cytokine important in the generation of inflammatory responses and human immunodeficiency virus resistance. In hematopoietic cells, RANTES is over‐expressed by type I interferons (IFN‐α and IFN‐β). The upstream region of the RANTES gene promoter contains a distal low affinity IFN‐stimulated response element (ISRE). Specific mutagenesis in this ISRE‐like motif abolished the activation of RANTES transcription by type I IFNs. Examination of the ISRE binding factors strongly suggested that signal transducer and activator of transcription (Stat)‐2 and p48/IFN‐stimulated gene factor 3γ (ISGF3γ) are not required for the induction of RANTES by type I IFNs. The specific requirement of Stat‐1 was demonstrated using Stat‐1‐deficient U3A cells. These results revealed a non‐classical ISRE/ISGF3 signal transduction pathway for the induction of RANTES by type I IFNs.
A DMSO‐sensitive conditional mutant of the fission yeast orthologue of the Saccharomyces cerevisiae SEC13 gene is defective in septation
Authors: Poloni Deborah , Simanis Viesturs
Published: October 2002

Dissection of complex processes using model organisms such as yeasts relies heavily upon the use of conditional mutants. We have generated a collection of fission yeast mutants sensitive to dimethylsulphoxide (DMSO). Among these we have found a mutant in the Schizosaccharomyces pombe orthologue of the Saccharomyces cerevisiae SEC13 gene, which fails to cleave the division septum. Generation of a null allele demonstrates that the S. pombe sec13 gene is essential.
Regulated and unregulated mitochondrial permeability transition pores: a new paradigm of pore structure and function?
Authors: He Lihua , Lemasters John J
Published: October 2002

Cyclosporin A (CsA) inhibits the mitochondrial permeability transition (MPT), but not always. To characterize the CsA‐sensitive and ‐insensitive MPT, rat liver mitochondria were exposed to low and high doses of various MPT inducers. Mitochondrial swelling, cyclophilin D membrane binding and permeability transition (PT) pore diameter were measured. The results indicate two conductance modes for the PT pore: one activated by Ca2+ and inhibited by CsA and Mg2+ and the other unregulated. We propose a new model of pore formation and gating in which PT pores form by aggregation of misfolded integral membrane proteins damaged by oxidant and other stresses. Chaperone‐like proteins initially block conductance through these misfolded protein clusters; however, increased Ca2+ opens these regulated PT pores, an effect blocked by CsA. When protein clusters exceed chaperones available to block conductance, unregulated pore opening occurs.
Charge conversion enables quantification of the proximity between a normally‐neutral μ‐conotoxin (GIIIA) site and the Na+ channel pore
Authors: Li Ronald A , Sato Kazuki , Kodama Kyoko , Kohno Toshiyuki , Xue Tian , Tomaselli Gordon F , Marbán Eduardo
Published: October 2002

μ‐Conotoxin (μ‐CTX) inhibits Na+ flux by obstructing the Na+ channel pore. Previous studies of μ‐CTX have focused only on charged toxin residues, ignoring the neutral sites. Here we investigated the proximity between the C‐terminal neutral alanine (A22) of μ‐CTX and the Na+ channel pore by replacing it with the negatively charged glutamate. The analog A22E and wild‐type (WT) μ‐CTX exhibited identical nuclear magnetic resonance spectra except at the site of replacement, verifying that they have identical backbone structures. A22E significantly reduced μ‐CTX affinity for WT μ1 Na+ channels (90‐fold↓), as if the inserted glutamate repels the anionic pore receptor. We then looked for the interacting partner(s) of residue 22 by determining the potency of block of Y401K, Y401A, E758Q, D762K, D762A, E765K, E765A and D1241K channels by WT μ‐CTX and A22E, followed by mutant cycle analysis to assess their individual couplings. Our results show that A22E interacts strongly with E765K from domain II (DII) (ΔΔG=2.2±0.1 vs. <1 kcal/mol for others). We conclude that μ‐CTX residue 22 closely associates with the DII pore in the toxin‐bound channel complex. The approach taken may be further exploited to study the proximity of other neutral toxin residues with the Na+ channel pore.
Lectin‐like oxidized low density lipoprotein receptor‐1 (LOX‐1) serves as an endothelial receptor for advanced glycation end products (AGE)
Authors: Jono Tadashi , Miyazaki Akira , Nagai Ryoji , Sawamura Tatsuya , Kitamura Toshinori , Horiuchi Seikoh
Published: October 2002

Advanced glycation end products (AGE) are known to serve as ligands for the scavenger receptors such as SR‐A, CD36 and SR‐BI. In the current study, we examined whether AGE is recognized by lectin‐like oxidized low density lipoprotein receptor‐1 (LOX‐1). Cellular binding experiments revealed that AGE‐bovine serum albumin (AGE‐BSA) showed the specific binding to CHO cells overexpressing bovine LOX‐1 (BLOX‐1), which was effectively suppressed by an anti‐BLOX‐1 antibody. Cultured bovine aortic endothelial cells also showed the specific binding for AGE‐BSA, which was suppressed by 67% by the anti‐BLOX‐1 antibody. Thus, LOX‐1 is identified as a novel endothelial receptor for AGE.
Role of glycosylation in hyperphosphorylation of tau in Alzheimer's disease
Authors: Liu Fei , Zaidi Tanweer , Iqbal Khalid , Grundke-Iqbal Inge , Merkle Roberta K. , Gong Cheng-Xin
Published: October 2002

In Alzheimer's disease (AD) brain, microtubule‐associated protein tau is abnormally modified by hyperphosphorylation and glycosylation, and is aggregated as neurofibrillary tangles of paired helical filaments. To investigate the role of tau glycosylation in neurofibrillary pathology, we isolated various pools of tau protein from AD brain which represent different stages of tau pathology. We found that the non‐hyperphosphorylated tau from AD brain but not normal brain tau was glycosylated. Monosaccharide composition analyses and specific lectin blots suggested that the tau in AD brain was glycosylated mainly through N‐linkage. In vitro phosphorylation indicated that the glycosylated tau was a better substrate for cAMP‐dependent protein kinase than the deglycosylated tau. These results suggest that the glycosylation of tau is an early abnormality that can facilitate the subsequent abnormal hyperphosphorylation of tau in AD brain.
Characterization of a monomeric disintegrin, ocellatusin, present in the venom of the Nigerian carpet viper, Echis ocellatus1
Authors: Smith J.Bryan , Theakston R.David G , Coelho Ana Lucia J , Barja-Fidalgo Christina , Calvete Juan J , Marcinkiewicz Cezary
Published: October 2002

Ocellatusin is a new RGD‐containing short monomeric disintegrin. It is a better inhibitor of α5β1 integrin and a more potent inducer of the expression of a ligand‐induced binding site epitope on β1 integrin subunit than echistatin. In further contrast to echistatin, ocellatusin has a direct chemotactic stimulus on human neutrophils in vitro. The distinct effects of these two close evolutionarily related disintegrins might be explained by the presence of methionine‐22 and histidine‐29 in the RGD loop of ocellatusin, which are arginine and aspartic acid, respectively, in echistatin. These mutations may modulate the conformation and/or recognition properties of the integrin‐binding loop of ocellatusin.
Hydroxylation of fatty acids by microsomal and reconstituted cytochrome P450 2B1
Authors: Alterman Michail A. , Hanzlik Robert P.
Published: October 2002

Understanding the mechanisms by which cytochrome(s) P450 (CYP) discriminate good from poor substrates, and orient them for highly regio‐ and stereoselective oxidation, has considerable intrinsic and practical importance. Here we present results of a study of fatty acid hydroxylation by CYP2B1 in a reconstituted system and in microsomes from phenobarbital‐pretreated rats. The results indicate that 2B1 prefers decanoic acid as the optimum fatty acid substrate (among C8–C16) and that it hydroxylates positions five or more methylene groups distant from the carboxylate carbon. That hydroxylation does not occur at carbon atoms closer to the carboxyl group than the C6 position suggests that these carbons may not reach the ferryl oxygen because the carboxyl group is anchored to a specific site at a fixed distance from the heme iron.