bluuebunny/crossref_metadata_2025 huggingface dataset:
İron binding to microsomes and liposomes in relation to lipid peroxidation
Authors: Vile Glenn F. , Winterbourn Christine C.
Published: July 2002

The effects of ADP, ATP, citrate and EDTA on iron‐dependent microsomal and liposomal lipid peroxidation, and on 59FeCl3 binding to the lipid membranes were measured. The aim was to test if initiation of lipid peroxidation is a site‐specific mechanism requiring bound iron. In the absence of chelator, iron was bound to both membranes. EDTA and citrate removed the iron and inhibited peroxidation. ATP and ADP stimulated peroxidation, but whereas ADP allowed only half of the iron to remain bound, all was removed by ATP. Chelators, therefore, cannot be simply influencing a site‐specific mechanism. Their effects must relate to the reactivities of the different iron chelates as initiators of lipid peroxidation.
Maintenance of membrane phospholipid asymmetry Lipid‐cytoskeletal interactions or lipid pump?
Authors: Williamson Patrick , Antia Rustom , Schlegel Robert A.
Published: July 2002

Two models for the mechanism of maintenance of lipid asymmetry in erythrocytes are considered: binding of internal lipids to cytoskeletal proteins, and pumping of internal lipids from the outside to the inside of the cell. Analysis of the kinetics of lipid internalization suggests that the first model is more likely, and that the apparent pumping of lipids represents the activity of an ATP‐dependent lipid flip/flop catalyst.
The phosphorylation of nucleoplasmin by casein kinase‐2 is resistant to heparin inhibition
Authors: Taylor Aída , Allende Catherine C. , Weinmann Roberto , Allende Jorge E.
Published: July 2002

Highly purified preparations of casein kinase‐2 from the nuclei of Xenopus laevis oocytes and from calf thymus can phosphorylate in vitro purified nucleoplasmin from X. laevis oocytes and eggs. The phosphorylation of nucleoplasmin by both kinase preparations is quite insensitive to heparin in contrast with casein phosphorylation which is completely abolished by heparin concentrations above 10 μg/ml. However, the phosphorylation of nucleoplasmin and casein are inhibited in a very similar fashion by 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB), a well characterized specific inhibitor of casein kinase‐2. Similarly, nucleoplasmin phosphorylation by the oocyte enzyme can be stimulated several‐fold by spermine, another characteristic of this enzyme. These findings indicate that the phosphorylation of nucleoplasmin by purified casein kinase‐2, while showing typical response to DRB and spermine, exhibits anomalous behavior in its resistance to heparin inhibition. It is possible that the large clusters of acidic amino acids in nucleoplasmin permit this substrate to interact with the enzyme more efficiently than other protein substrates. Heparin is generally considered a potent and specific inhibitor of casein kinase‐2. This study, however, questions the validity of utilizing heparin inhibition as a criterion for casein kinase‐2 involvement.
Molecular cloning of a new human G protein Evidence for two Giα‐like protein families
Authors: Didsbury John R. , Snyderman Ralph
Published: July 2002

The amino acid sequence of a novel G protein α subunit (Gxα) has been deduced from the nucleotide sequence of a human cDNA clone isolated from a differentiated HL‐60 cDNA library. The cDNA encodes a polypeptide of 354 amino acids (M
r 40 519) which is closely related to Giα proteins. The amino acid sequence homology between Gxα and human myeloid Giα is 86% with 15 nonconservative substitutions. Gxα also shares 86% homology with both rat brain and mouse macrophage Giα but is more homologous (94%) to bovine brain Giα with only 5 nonconservative amino acid differences. G proteins previously termed Giα may fall into at least two distinct groups, with one including human myeloid Giα, rat brain Giα and mouse macrophage Giα; and other Gxα; and bovine brain Giα. One group probably contains true Gi and the other a new class of G protein whose function remains to be determined.
Enzymatic activity and filament assembly of Acanthamoeba myosin II are regulated by adjacent domains at the end of the tail
Authors: Atkinson Mark A.L. , Appella Ettore , Corigliano-Murphy M.Angela , Korn Edward D.
Published: July 2002

Polyclonal antibodies raised against a synthetic peptide consisting of the last 19 amino acids at the end of the coiled‐coil region of the heavy chains inhibited the actin‐activated Mg2+‐ATPase activity of myosin II and its ability to form filaments. Antibodies against a synthetic peptide corresponding to the 21 adjacent amino acids at the beginning of the non‐helical tailpiece, which include the three regulatory phosphorylatable serines, had no effect on either activity.
Chemiosmotic H+ cycling across the plasma membrane of the thermoacidophilic archaebacterium Sulfolobus acidocaldarius
Authors: Moll Ralf , Schäfer Günter
Published: July 2002

Transitory H+ ejection from Sulfolobus acidocaldarius cells induced by oxygen pulses, and anaerobic H+ backflow were investigated. Aerobic proton extrusion is inhibited by protonophores, by nigericin and by inhibitors of respiratory electron transport; it is stimulated by DCCD. In contrast, DCCD inhibits the rate of anaerobic H+ backflow. Aerobic proton extrusion is significantly enhanced by K+/valinomycin. Apparent H+/O ratios of 2.5–3 are measured. Proton extrusion generates large pH gradients (3–4 units) representing the major contribution to the total proton motive force across the plasma membrane of this thermoacidophilic archaebacterium.
Diffusion of fluorescein‐labelled molecules in suspensions of erythrocyte ghosts
Authors: Järnefelt Johan , Laurent Torvard , Rigler Rudolf
Published: July 2002

The diffusion of fluorescein isothiocyanate‐labelled dextran molecules in suspensions of centrifugally, tightly packed, erythrocyte ghosts was measured by fluorescence recovery after photobleaching. In comparison with diffusion in aqueous solution, the diffusion coefficients for probe molecules of varying size were about two orders of magnitude smaller. It was established that the dextran molecules remained in the space between the ghosts. Since crosslinking membrane surface carbohydrates with antibodies further inhibits diffusion, it is assumed that interactions between surface carbohydrates and the probe molecules are the cause of slow diffusion. Two alternative models are discussed.
Iron chelators inhibit human platelet aggregation, thromboxane A2 synthesis and lipoxygenase activity
Authors: Barradas Manuel A. , Jeremy Jamie Y. , Kontoghiorghes George J. , Mikhailidis Dimitri P. , Hoflbrand A.Victor , Dandona Paresh
Published: July 2002

The iron chelators desferrioxamine and 1,2‐dimethyl‐3‐hydroxypyrid‐4‐one (L1) inhibited human platelet aggregation in vitro as well as thromboxane A2 synthesis and conversion of arachidonate to lipoxygenase‐derived products. Non‐chelating compounds related to L1 were without effect on cyclooxygenase or lipoxygenase activity. Since both cyclooxygenase and lipoxygenase are iron‐containing enzymes, it is suggested that the inhibition of platelet function by these iron chelators may be related to the removal or binding of iron associated with these enzymes. These iron chelators may therefore be of potential therapeutic value as platelet antiaggregatory agents and of possible use in the treatment of atherosclerotic and inflammatory joint diseases.
Primary structure of human proacrosin deduced from its cDNA sequence
Authors: Baba Tadashi , Watanabe Ken , Kashiwabara Shin-ichi , Arai Yuji
Published: July 2002

cDNA clones encoding proacrosin, the zymogen of acrosin, were isolated from a human testis cDNA library by using a fragment of boar acrosin cDNA as a probe. Nucleotide sequencing of the longest cDNA clone has predicted that human proacrosin is synthesized with a 19‐amino‐acid signal peptide at the N‐terminus. The cleavable signal sequence is followed by a 23‐residue segment corresponding to the light chain and then by a 379‐residue stretch that constitutes the heavy chain containing the catalytic site of the mature protease. The C‐terminal portion of the deduced sequence for the heavy chain is very rich in proline residues, most of which are encoded by a unique repeat of CCCCCA. The active‐site residues including histidine, aspartic acid, and serine are also predicted to be located at residues 69, 123, and 221, respectively.
The neuroleptic activity of haloperidol increases after its solubilization in surfactant micelles
Authors: Kabanov A.V. , Chekhonin V.P. , Alakhov V.Yu. , Batrakova E.V. , Lebedev A.S. , Melik-Nubarov N.S. , Arzhakov S.A. , Levashov A.V. , Morozov G.V. , Severin E.S. , Kabanov V.A.
Published: July 2002

It has been suggested to use surfactant micelles as microcontainers for increasing the efficiency of neuroleptic targeting from blood flow into the brain. The neuroleptic action of haloperidol, intraperitoneally injected into mice in micellar solution of non‐ionic block copolymer surfactant (pluronic P‐85) in water, increased several‐fold if compared with that observed for haloperidol aqueous solution. Incorporation of brain‐specific antibodies into haloperidol‐containing micelles resulted in additional drastic increase (more than by 2 orders of magnitude) in the drug effect.
Structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae
Authors: Ballio A. , Bossa F. , Collina A. , Gallo M. , Iacobellis N.S. , Paci M. , Pucci P. , Scaloni A. , Segre A. , Simmaco M.
Published: July 2002

The covalent structure of syringotoxin, a bioactive metabolite of Pseudomonas syringae pv. syringae isolates, pathogenic on various species of citrus trees, has been deduced from ID and 2D 1H‐ and 13C‐NMR spectra combined with extensive FAB‐MS data and results of some chemical reactions. Similarly to syringomicins and syringostatins, produced by other plant pathogenic strains of P. syringae pv. syringae, syringotoxin is a lipodep‐sinonapeptide. Its peptide moiety corresponds to Ser‐Dab‐Gly‐Hse‐Om‐aThr‐Dhb‐(3‐OH)Asp‐(4‐Cl)Thr with the terminal carboxy group closing a macrocyclic ring on the OH group of the N‐terminal Ser, which in turn is N‐acetylated by 3‐hydroxytetradecanoic acid.
Identification of creatine kinase isoenzymes in the guinea‐pig presence of mitochondrial creatine kinase in smooth muscle
Authors: Ishida Yukisato , Wyss Markus , Hemmer Wolfram , Wallimann Theo
Published: July 2002

Isoenzymes of creatine kinase (CK, EC 2.7.3.2) in guinea‐pig smooth, cardiac and skeletal muscles as well as in brain were analyzed in cellulose acetate electrophoresis and FPLC gel permeation chromatography. In crude tissue extracts of smooth muscles brain type BB‐CK and the hybrid form MB‐CK were detected, but in enriched mitochondrial fractions from different quinea‐pig smooth muscles, mitochondrial type Mi‐CK was unambiguously identified. Smooth muscle Mi‐CK displayed the same electrophoretic mobility as Mi‐CK from brain, which migrates slower than cardiac Mi‐CK. Identical to parallel experiments with Mi‐CK from cardiac muscle and brain, smooth muscle Mi‐CK could be resolved into dimeric and octameric species, the latter being remarkably stable. In contrast to muscles, Mi‐CK was not detected in chicken gizzard tissue extracts nor in enriched mitochondrial fractions thereof. The presence of Mi‐CK, predominantly in octameric form, in guinea‐pig smooth muscles, but not in chicken gizzard, may represent a clue for the different physiological properties of these muscles and may provide the molecular basis for the dependence of the PCr production on oxidative metabolism observed in the guinea‐pig taenia cacci.
Apolipoprotein D transcription occurs specifically in nonproliferating quiescent and senescent fibroblast cultures
Authors: Provost Pierre R. , Marcel Yves L. , Milne Ross W. , Weech Philip K. , Rassart Eric
Published: July 2002

We studied apolipoprotein D (apoD) mRNA in primary cultures of human diploid fibroblasts (HDF). In early‐passage HDF no apoD mRNA was detected in replicating cells in sparse culture, but the gene was expressed in quiescent cells in confluent and in serum‐starved cultures. In contrast, late‐passage HDF expressed apoD mRNA in sparse culture, but the level increased after attainment of confluence. Thus fibroblasts, the common cell‐type expressing apoD mRNA in vivo, express this characteristic following growth‐arrest. The same pattern of activation was found in another fibroblast cell line deficient in apoB/E (LDL) receptors, excluding a role for cellular cholesterol delivery by the LDL‐receptor pathway controlling apoD expression
Matrix assembly of recombinant fibronectin polypeptide consisting of amino‐terminal 70 kDa and carboxyl‐terminal 37 kDa regions
Authors: Ichihara-Tanaka Keiko , Maeda Toshinaga , Titani Koiti , Sekiguchi Kiyotoshi
Published: July 2002

Three different forms of recombinant human fibronectin polypeptides consisting of the amino‐terminal 70 kDa region, the carboxyl‐terminal 37 kDa region, or both, were expressed in mouse L cells. Although either the amino‐terminal or the carboxyl‐terminal region alone was only poorly incorporated into the extracellular matrix, the fused form of the polypeptide was highly capable of assembling into the matrix. These results indicate that matrix assembly of fibronectin requires both regions and can proceed in the absence of most of the type III repeats including the one containing the cell adhesive Arg‐Gly‐Asp sequence.
Quercetin, a bioflavonoid, inhibits the increase of human multidrug resistance gene (MDR1) expression caused by arsenite
Authors: Kioka Noriyuki , Hosokawa Nobuko , Komano Tohru , Hirayoshi Kazunori , Nagate Kazuhiro , Ueda Kazumitsu
Published: July 2002

Expression of the MDR1 gene, which encodes P‐glycoprotein, is increased under some stress conditions. We have reported that quercetin, a bioflavonoid, inhibits the expression of heat‐shock proteins. We have identified the effects of quercetin on the MDR1 gene expression in the human hepatocarcinoma cells line, HepG2. The increase of P‐glycoprotein synthesis and MDR1 mRNA accumulation caused by exposure to arsenite were inhibited by quercetin. The CAT assay suggested that quercetin suppressed the transcriptional activation of the MDR1 gene after exposure to arsenite. Although many drugs that prevent the P‐glycoprotein function have been reported, this is the first report to describe the inhibition of MDR1 expression by a reagent.
X‐Ray studies reveal lanthanide binding sites at the A/B5 interface of E. coli heat labile enterotoxin
Authors: Sixma Titia K. , Terwisscha van Scheitinga Anke C. , Kalk Kor H. , Zhou Kangjing , Wartna Ellen S. , Hol Wim G.J.
Published: July 2002

The crystal structure determination of heat labile enterotoxin (LT) bound to two different lanthanide ions, erbium and samarium, revealed two distinct ion binding sites in the interface of the A subunit and the B pentamer of the toxin. One of the interface sites is conserved in the very similar cholera toxin sequence. These sites may be potential calcium binding sites. Erbium and samarium binding causes a change in the structure of LT: a rotation of the A1 subunit of up to two degrees relative to the B pentamer.
Direct transfer of molybdopterin cofactor to aponitrate reductase from a carrier protein in Chlamydomonas reinhardtii
Authors: Aguilar Miguel , Kalakoutskii Kyrill , Cárdenas Jacobo , Fernández Emilio
Published: July 2002

A Chlamydomonas reinhardtii molybdenum cofactor (MoCo)‐carrier protein (CP), capable of reconstituting nitrate reductase activity with apoprotein from the Neurospora crassa mutant nit‐1, was subjected to experiments of diffusion through a dialysis membrane and gel filtration. CP bonded firmly MoCo and did not release it efficiently unless aponitrate reductase was present in the incubation mixture, Stability of MoCo bound to CP against air and heat was very similar to that of free‐MoCo released from milk xanthine oxidase. Our data strongly suggest that MoCo is directly transferred from CP to aponitrate reductase to form an active enzyme.
Endothelial nitric oxide synthase is myristylated
Authors: Pollock Jennifer S. , Klinghofer Vered , Förstermann Ulrich , Murad Ferid
Published: July 2002

The enzyme responsible for the synthesis of endothelium‐derived relaxing factor and/or nitric oxide in the endothelium has been described as a particulate enzyme, whereas other isoforms of nitric oxide synthase are soluble enzymes. Here we are reporting that endothelial cells metabolically incorporate myristate (C14), but not palmitate (C16), into nitric oxide synthase. We are postulating that the endothelial‐derived nitric oxide synthase is a particulate enzyme because of the fatty acid acylation of the protein which ‘anchors’ the enzyme into the membrane either directly or via another membrane‐bound protein.
Transport of proteins into the various subcompartments of mitochondria
Authors: Segui-Real Bartolome , Stuart Rosemary A. , Neupert Walter
Published: July 2002

The import of proteins into mitochondria is an intricate process comprised of multiple steps. The first step involves the sorting of cytosolically synthesized precursor proteins to the mitochondrial surface. There precursor proteins are recognized by specific receptors which deliver them to the general import site present in the outer membrane. The second stage of import involves a series of complex intraorganelle sorting events which results in the delivery of the proteins to one of the four possible submitochondrial destinations, namely the outer and inner membranes, the matrix and intermembrane space. Here in this review, we discuss the current knowledge on these intramitochondrial sorting events. We especially focus on targetting of proteins to the intermembrane space. Sorting to the intermembrane space represents a particularly interesting situation, as at least three separate targetting pathways to this subcompartment are known to exist.
Primary structure of frog rhodopsin
Authors: Pittler Steven J. , Fliesler Steven J. , Baehr Wolfgang
Published: July 2002

Amphibians have been employed extensively to study the anatomy, physiology, biochemistry, and cell biology of the visual system for decades, yet there have been no reports concerning the primary structure of amphibian visual transduction components. Thus, we have determined the entire nucleotide sequence of frog (Rana pipiens) rhodopsin cDNA, including a putative transcription start point and poly A tail, by sequence analysis of PCR products and mRNA. The open reading frame predicts an opsin of 354 residues, six residues longer than the mammalian rod opsins, containing 11 potential phosphorylation sites in the C‐terminal domain. RNA blot analysis revealed two transcripts of ca. 1.7 and 3.1 kb. Frog rhodopsin exhibits ∼85% identity to mammalian rhodopsin at the amino acid level. Sequence analysis of additional components will produce the Framework from which a more detailed understanding of amphibian phototransduction can emerge.
Different thyroid hormone‐deiodinating enzymes in tilapia (Oreochromis niloticus) liver and kidney
Authors: Mol Koen , Kaptein Ellen , Darras Veerle M. , de Greef Wim J. , Kühn Eduard R. , Visser Theo J.
Published: July 2002

Enzymes catalyzing the outer ring deiodination (ORD) of iodothyronines are important for the regulation of thyroid hormone bioactivity. We have studied ORD of thyroxine (T4) and 3,3',5'‐triiodothyronine (rT3) in liver and kidney microsomes offish, i.e. tilapia (Oreochromis niloticus). Tilapia kidney contains an enzyme which resembles the mammalian selenoenzyme type I iodothyronine deiodinase (ID‐I) with respect to substrate preference (rT3 > T4) and high (≈ μM) K
m values, but is much less sensitive to selenocysteine (Sec)‐targeted inhibitors, including 6‐propyl‐2‐thiouracil (PTU). In contrast, tilapia liver contains an enzyme very similar to mammalian type II deiodinase (ID‐II) with respect to substrate preference (T4 > rT3), low (≈ nM) K
m values, and lack of sensitivity to Sec inhibitors.
rab3‐Peptide stimulates exocytosis from mast cells via a pertussis toxin‐sensitive mechanism
Authors: Law G.J. , Northrop A.J. , Mason W.T.
Published: July 2002

Synthetic peptides of the putative effector domain of members of the rab3 gene family of small GTP‐binding proteins have been shown to have potent actions on vesicular transport and exocytosis [1,2]. Here, we use similar rab3‐effector domain peptides to study their role in intracellular signalling in mast cells. We find that rab3‐like peptides stimulate exocytosis and decrease cyclic 3',5'‐AMP levels in these cells when applied extracellularly. Cells pretreated with pertussis toxin (PtX) to selectively uncouple αi/α0 type G proteins from their biological activators, however, did not respond to rab3 peptides. rab3‐like peptides also induce a Ca2+ transient in mast cells. These observations provide evidence for functional coupling between an effector domain peptide sequence of rab3 protein and a PtX‐sensitive G protein substrate.
A novel form of FGF receptor‐3 using an alternative exon in the immunoglobulin domain III
Authors: Avivi Aaron , Yayon Avner , Givol David
Published: July 2002

Four distinct FGF receptors were cloned and characterized and it was demonstrated that the ligand binding site of FGF receptors is confined to the extracellular immunoglobulin‐like (Ig)‐domain 2 and 3. The Ig‐domain 3 is encoded by two separate exons: exon IIIa encodes the N‐terminal half, and the C‐terminal half is encoded by either exon IIIb or IIIc in FGFR1 and FGFR2, whereas FGFR4 is devoid of exon IIIb. Alternative usage of exons IIIb and IIIc determine the ligand binding specificity of the receptor. To analyze the arrangement of these exons in FGFR3 we cloned the genomic sequence between exon IIIa and IIIc of FGFR3 and identified an alternative exon, corresponding to exon IIIb of the FGFR1 and FGFR2. The sequence of this exon shows Ig‐domain hallmarks, 44% identity with exon IIIb of other FGF receptors and 36% identity with exon IIIc of FGFR3. Using this exon as a probe for mouse RNA as well as PCR analysis, demonstrated that exon IIIb encodes an authentic form of FGFR3 that is expressed in mouse embryo, mouse skin and mouse epidermal keratinocytes. The results demonstrate that the presence of alternative exons for Ig‐domain 3 is a general phenomena in FGFR1, 2 and 3, and represents a novel genetic mechanism for the generation of receptor diversity.
Characterization of a tropomyosin cDNA from the hydrozoan Podocoryne carnea
Authors: Baader Christine D. , Schmid Volker , Schuchert Peter
Published: July 2002

A cDNA clone from the hydrozoan Podocoryne carnea was characterized. It consists of an open reading frame of 726 nt flanked by a 84 nt 5' and a 307 nt 3' untranslated region. The corresponding gene exists apparently as a single copy. The transcript is ubiquitously expressed in the polyp and the medusa stage. Several features of the predicted peptide sequence indicate a relationship to tropomyosins. At the amino acid level it shares 26–30% identical residues with other invertebrate and vertebrate tropomyosin sequences.
A dynamical model for post‐translational modifications of microtubules
Authors: Laurent Michel , Fleury Anne
Published: July 2002

In many cases, post‐translational modifications constitute indirect markers of non‐dynamic microtubules. The correlation between stability of microtubular systems and post‐translational modifications suggests that they may act as a signal in patterning mechanisms. However, a crucial question remains as to how the repertoire of distinctly modified microtubules is generated. We propose here an ubiquitous mechanism of spatial and temporal differentiation of microtubules. In this model, the diversity of post‐translational modifications results from a dynamical pathway separation which is ensured by biochemical switches between self‐regulated mechanisms of differentiation. Thus, it does not require any hypothetical subcellular compartmentalization of enzymatic activities responsible for the various post‐translational modifications. These results are discussed in relation to experimental evidences for a temporal and spatial regulation of microtubule modifications.
High levels of profilin suppress the lethality caused by overproduction of actin in yeast cells
Authors: Magdolen Viktor , Drubin David G. , Mages Günter , Bandlow Wolfhard
Published: July 2002

Overproduction of actin is lethal to yeast cells. In contrast, overexpression of the profilin gene, PFYI, encoding an actin‐binding protein, leads to no very obvious phenotype. Interestingly, profilin over‐production can compensate for the deleterious effects of too much actin in a profilin concentration‐dependent manner. Our results, thus, document that actin and profilin interact in vivo. Immunofluorescence studies suggest that suppression works by reducing actin assembly. We observed, however, that even massive overproduction of profilin fails to fully restore the wild‐type phenotype (e.g. the wild‐type appearance of the actin microfilament system). This may indicate that actin monomer sequestration is not the only mechanism by which the balance of actin polymerization is controlled.
MFH‐1, a new member of the fork head domain family, is expressed in developing mesenchyme
Authors: Miura Naoyuki , Wanaka Akio , Tohyama Masaya , Tanaka Kiyoji
Published: July 2002

We have isolated a novel mouse gene, MFH‐1 (mesenchyme fork head 1) that is related to the Drosophila fork head and rat HNF3 genes. MFH‐1 encodes a distinct fork head domain that is classified into a distinct subfamily. A recombinant MFH‐1 protein could bind to the HNF3 binding site. MFH‐1 is expressed temporally in developing embryos, first in the non‐notochordal mesoderm and later in areas of mesenchymal condensation in the trunk, head, and limbs. Our results suggest that MFH‐1 might be involved in the formation of special mesenchymal tissues.
Peroxynitrite‐mediated oxidation of albumin to the protein‐thiyl free radical
Authors: Gatti Reynaldo M. , Radi Rafael , Augusto Ohara
Published: July 2002

Nitric oxide reacts with superoxide to produce peroxynitrite, which may be an important mediator of oxidant‐induced cellular injury. Here we report that peroxynitrite is able to oxidize a protein, bovine serum albumin (BSA), to the corresponding protein‐thiyl free radical as demonstrated by electron paramagnetic resonance (EPR)‐spin‐trapping experiments with both α‐phenyl‐N‐tert‐butyl nitrone (PBN) and 5,5‐dimethyl‐1 ‐pyrroline‐ N‐oxide (DMPO). BSA radical adduct yields increased with pH indicating peroxynitrite anion as its main forming agent. Reaction with peroxynitrite may be another aspect of the antioxidant action of albumin in extracellular fluids.
Multiple nuclear localization signals of the B‐myb gene product
Authors: Takemoto Yoshihiro , Tashiro Shigeki , Handa Hiroshi , Ishii Shunsuke
Published: July 2002

Nuclear entry of the B‐myb gene product (B‐Myb) is dependent on multiple nuclear localization signals (NLS's). Mutagenesis of the putative NLS's of B‐Myb has identified two separate NLS's, NLS1 and NLS2. Each of the two NLS's is essential for efficient nuclear targeting. NLS2 contains two interdependent basic domains separated by 8 intervening spacer amino acids, and both basic domains are required for nuclear entry. Thus, NLS2 belongs to a class of bipartite NLS's. Like the NLS's in yeast transcription factor SW15, NLS2 contains a putative cdc2 kinase site. However, unlike the case of SWI5, phosphorylation at this site did not affect the nuclear targeting of B‐Myb.
Surface labeling of key residues during assembly of the transmembrane pore formed by staphylococcal α‐hemolysin
Authors: Krishnasastry Musti , Walker Barbara , Braha Orit , Bayley Hagan
Published: July 2002

Structural changes in staphylococcal α‐hemolysin (αHL) that occur during oligomerization and pore formation on membranes have been examined by using a simple gel‐shift assay to determine the rate of modification of key single‐cysteine mutants with the hydrophilic sulfhydryl reagent, 4‐acetamido‐4′‐((iodoacetyl)amino)stilbene‐2,2′‐disulfonate (IASD). The central glycine‐rich loop of αHL lines the lumen of the transmembrane channel. A residue in the loop remains accessible to IASD after assembly, in keeping with the ability of the pore to pass molecules of 1̃000 Da. By contrast, residues near the N‐terminus, which are critical for pore function, become deeply buried during oligomerization, while a residue at the extreme C‐terminus increases in reactivity after assembly, consistent with a location in the part of the pore that projects from the surface of the lipid bilayer.
Mutations in RCA1 and AFG3 inhibit F1‐ATPase assembly in Saccharomyces cerevisiae
Authors: Paul Marie-Françoise , Tzagoloff Alexander
Published: July 2002

The RCA1 (YTA12) and AFG3 (YTA10) genes of Saccharomyces cerevisiae code for homologous mitochondrial proteins that belong to the recently described AAA protein‐family [Kunau et al. (1993) Biochimie 75, 209–224]. Mutations in either gene have been shown to induce a respiratory defect. In the case of rca1 mutants this phenotype has been ascribed to defective assembly of cytochrome oxidase and ubiquinol‐cytochrome c reductase. In the present study we show that the respiratory defect of afg3 mutants, like that of rca1 mutants, is also caused by an arrest in assembly of cytochrome oxidase and ubiquinol‐cytochrome c reductase. In addition to the absence of the respiratory complexes, rca1 and afg3 mutants exhibit reduced mitochondrial ATPase activity. As a first step to an understanding of the biochemical basis for the ATPase defect we have examined the assembly of the F1 and F0 constituents of the ATPase complex. We present evidence that the ATPase lesion stems at least in part from the failure of rca1 and afg3 mutants to assemble F1. Although the mutants also display lower steady‐state concentrations of some F0 subunits, this could be a secondary effect of defective F1 assembly.
Membrane‐anchoring domains of Cdc25p, a Saccharomyces cerevisiae ras exchange factor
Authors: Garreau Herve´ , Geymonat Marco , Renault Georges , Jacquet Michel
Published: July 2002

Summry— The CDC25 gene product from Saccharomyces cerevisiae, the prototype of the family of ras guanine nucleotide exchange factors, is expressed as a 180‐kDa polypeptide, tightly bound to a membrane fraction. The ability to complement a cdc25 defect is located in the 3′ part of the gene (codons 877–1589). Sequence analysis reveals only a short hydrophobic domain (residues 1459–1471) and no consensus sequence for post‐translational acylation. The SH3 domain present in the N‐terminal part of Cdc25p is not involved nor required for membrane localization, since the N‐terminal part of Cdc25p did not fractionate with a membrane pellet. In contrast, the C‐terminal part was attached to a 18000 g pellet after subcellular fractionation and immunoblotting. This subcellular localization was conserved in a ras1ras2 double disruption mutant and in a ira2 disruption mutant. Immunofluorescence analysis showed a patchy staining, mainly at the periphery of the cells. These patches were quite distinct from actin patches by double immunolabeling. By analysing a set of truncated derivatives, the elements required for a particulate localization were restricted to residues 1441–1552.
Age‐related changes in antioxidant enzymes, superoxide dismutase, catalase, glutathione peroxidase and glutathione in different regions of mouse brain
Authors: Hussain S. , Slikker W. , Ali S.F.
Published: July 2002

It has been proposed that neurodegenerative processes of aging are associated with the generation of reactive oxygen species (ROS) during cellular metabolism. These reactive oxygen species are scavenged by antioxidant enzymes in biological systems. The present study was designed to determine the selective distribution of the antioxidant enzymes superoxide dismutase, catalase and glutathione peroxidase activity and reduced glutathione (GSH) levels in different regions of the C57BL/6N mouse brain and to determine if any alterations occurred with age. Catalase activity did not show any significant change except in cerebellum. Activity of superoxide dismutase was increased with age in all regions of the brain except in hippocampus of 2‐yr‐old mice. The glutathione peroxidase activity in the caudate nucleus increased in all regions of the brain, however, the activity did not change at one, six and 12 months. A significant increasing pattern of glutathione content was found in the cerebellum and brain stem with age. These data demonstrate that although the level of antioxidant enzymes varied in different regions of the brain, overall the enzyme activities tend to increase with age.
The active‐site residue Cys‐29 is responsible for the neutral‐pH inactivation and the refolding barrier of human cathepsin B
Authors: Song Jianxing , Xu Ping , Xiang Hui , Su Zhengding , Storer Andrew C. , Ni Feng
Published: July 2002

Human cathepsin B, the most abundant lysosomal cysteine protease, has been implicated in a variety of important physiological and pathological processes. It has been known for a long time that like other lysosomal cysteine proteases, cathepsin B becomes inactivated and undergoes irreversible denaturation at neutral or alkaline pH. However, the mechanism of this denaturation process remains mostly unknown up to this day. In the present work, nuclear magnetic resonance spectroscopy was used to characterize the molecular origin of the neutral‐pH inactivation and the refolding barrier of human cathepsin B. Two forms of human cathepsin B, the native form with Cys‐29 at the active site and a mutant with Cys‐29 replaced by Ala, were shown to have well‐folded structures at the active and slightly acidic condition of pH 5. Surprisingly, while the native cathepsin B irreversibly unfolds at pH 7.5, the C29A mutant was found to maintain a stable three‐dimensional structure at neutral pH conditions. In addition, replacement of Cys‐29 by Ala renders the process of the urea denaturation of human cathepsin B completely reversible, in contrast to the opposite behavior of the wild‐type cathepsin B. These results are very surprising in that replacement of one single residue, the active‐site Cys‐29, can eliminate the neutral‐pH denaturation and the refolding barrier. We speculate that this finding may have important implications in understanding the process of pH‐triggered inactivation commonly observed for most lysosomal cysteine proteases.
Ligand–protein interactions in nuclear receptors of hormones
Authors: Egea Pascal F , Klaholz Bruno P , Moras Dino
Published: July 2002

Nuclear hormone receptors are transcription factors regulated by lipophilic ligands. These hormones bind to their nuclear receptor targets using an induced fit mechanism that triggers a large conformational change and generates the proper surface for the binding of protein coactivators. The molecular details of the various steps of this activation process or its inhibition by antagonists are now understood for several nuclear receptors.
The humanPER1gene is transcriptionally regulated by multiple signaling pathways
Authors: Motzkus Dirk , Maronde Erik , Grunenberg Uwe , Lee Cheng Chi , Forssmann Wolf-Georg , Albrecht Urs
Published: July 2002

The mammalian period (Per) genes are components of the circadian clock and appear to be regulated via an autoregulatory feedback loop. Here we show that the humanPER1(hPER1) gene is synergistically activated by protein kinases A and C (PKA, PKC) and cAMP responsive element binding protein. Activators and inhibitors of PKA as well as PKC modulate endogenoushPER1expression andhPER1promoter‐driven reporter gene activity in a dose‐dependent manner. Our results suggest that thehPER1promoter acts as a sensor for multiple signaling molecules thereby integrating different physiological parameters. This regulation ofhPER1appears to be significant for rapid adaptation to changing environmental conditions.
Expression of telomerase inhibits hydroxyl radical‐induced apoptosis in normal telomerase negative human lung fibroblasts
Authors: Ren Jian-Guo , Xia Hui-Li , Tian Yan-Mei , Just Tom , Cai Guo-Ping , Dai Yao-Ren
Published: July 2002

In tumor cells telomerase activity is associated with resistance to apoptosis and the introduction of the human telomerase reverse transcriptase (hTERT) subunit into normal human cells is associated with life span extension of the cells. To determine the role of telomerase in regulating apoptosis, telomerase negative human embryo lung fibroblasts were transfected with the hTERT gene. Unlike the control fibroblasts, the telomerase‐expressing cells had elongated telomeres and were resistant to apoptosis induced by hydroxyl radicals. The results indicate that expression of telomerase and, thus, the maintenance of telomere length in normal human somatic cells caused resistance to not only cellular senescence but also apoptosis. Moreover, we found that hydroxyl radical‐induced apoptosis in telomerase‐expressing and control fibroblasts was caspase‐3 independent. These findings have revealed a new type of interrelation between telomerase and caspase‐3, which may indicate that in this case the expressed telomerase may inhibit apoptosis at a site not related to the caspase‐3 cascade.
Annexin A5 D226K structure and dynamics: identification of a molecular switch for the large‐scale conformational change of domain III
Authors: Sopkova-De Oliveira Santos Jana , Vincent Michel , Tabaries Sébastien , Chevalier Anne , Kerbœuf Daniel , Russo-Marie Françoise , Lewit-Bentley Anita , Gallay Jacques
Published: July 2002

The domain III of annexin 5 undergoes a Ca2+‐ and a pH‐dependent conformational transition of large amplitude. Modeling of the transition pathway by computer simulations suggested that the interactions between D226 and T229 in the IIID–IIIE loop on the one hand and the H‐bond interactions between W187 and T224 on the other hand, are important in this process [Sopkova et al. (2000) Biochemistry 39, 14065–14074]. In agreement with the modeling, we demonstrate in this work that the D226K mutation behaves as a molecular switch of the pH‐ and Ca2+‐mediated conformational transition. In contrast, the hydrogen bonds between W187 and T224 seem marginal.
Two splicing variants of a new inhibitor of apoptosis gene with different biological properties and tissue distribution pattern
Authors: Ashhab Yaqoub , Alian Akram , Polliack Aaron , Panet Amos , Yehuda Dina Ben
Published: July 2002

Using homology searches, we identified a novel human inhibitor of apoptosis (IAP) gene. This gene has two splicing variants that contain open reading frames of 298 and 280 amino acids and both contained a single copy of baculovirus IAP repeat (BIR) and RING domain. We refer here to the longer and shorter variants as Livin α and β, respectively. Semiquantitative reverse transcriptase‐polymerase chain reaction demonstrated a tissue‐specific and non‐correlated expression pattern in both adult and fetal tissues. Both mRNA variants were detected in various transformed cell lines. Despite their very close similarity, the two isoforms have different antiapoptotic properties. Both isoforms have a significant antiapoptotic activity in the Jurkat T cell line after triggering apoptosis via tumor necrosis factor and CD95 receptors. The Livin α but not β protects cells from apoptosis induced by staurosporine, but in contrast, apoptosis initiated by etoposide was blocked only by the β isoform. This difference in biological activities may indicate the presence of critical amino acids outside the BIR and RING domains. These functional and tissue distribution differences of Livin α and β suggest that Livin may play a complex role in the regulation of apoptosis.
Effects of ‘hydrophobic mismatch’ on the location of transmembrane helices in the ER membrane
Authors: Monné Magnus , von Heijne Gunnar
Published: July 2002

We have studied the effects of ‘hydrophobic mismatch’ between a poly‐Leu transmembrane helix (TMH) and the ER membrane using a glycosylation mapping approach. The simplest interpretation of our results is that the lumenal end of the TMH is located deeper in the membrane for both short (negative mismatch) and long (positive mismatch) TMHs than for poly‐Leu segments of intermediate length. We further find that the position‐specific effect of Lys residues on the location of short TMHs in the membrane varies with an apparent helical periodicity when the Lys residue is moved along the poly‐Leu stretch. We discuss these findings in the context of models for peptide–lipid interactions during hydrophobic mismatch.
Glutamate neurotoxicity, oxidative stress and mitochondria
Authors: Atlante Anna , Calissano Pietro , Bobba Antonella , Giannattasio Sergio , Marra Ersilia , Passarella Salvatore
Published: July 2002

The excitatory neurotransmitter glutamate plays a major role in determining certain neurological disorders. This situation, referred to as ‘glutamate neurotoxicity’ (GNT), is characterized by an increasing damage of cell components, including mitochondria, leading to cell death. In the death process, reactive oxygen species (ROS) are generated. The present study describes the state of art in the field of GNT with a special emphasis on the oxidative stress and mitochondria. In particular, we report how ROS are generated and how they affect mitochondrial function in GNT. The relationship between ROS generation and cytochrome c release is described in detail, with the released cytochrome c playing a role in the cell defense mechanism against neurotoxicity.
A calmodulin‐stimulated Ca2+‐ATPase from plant vacuolar membranes with a putative regulatory domain at its N‐terminus1
Authors: Malmström Susanna , Askerlund Per , Palmgren Michael G.
Published: July 2002

A cDNA, BCA1, encoding a calmodulin‐stimulated Ca2+‐ATPase in the vacuolar membrane of cauliflower (Brassica oleracea) was isolated based on the sequence of tryptic peptides derived from the purified protein. The BCA1 cDNA shares sequence identity with animal plasma membrane Ca2+‐ATPases and Arabidopsis thaliana ACA1, that encodes a putative Ca2+ pump in the chloroplast envelope. In contrast to the plasma membrane Ca2+‐ATPases of animal cells, which have a calmodulin‐binding domain situated in the carboxy‐terminal end of the molecule, the calmodulin‐binding domain of BCA1 is situated at the amino terminus of the enzyme.
A new model for the membrane topology of glucose‐6‐phosphatase: the enzyme involved in von Gierke disease
Authors: Hemrika Wieger , Wever Ron
Published: July 2002

Very recently we have proposed [Hemrika et al. (1997) Proc. Natl. Acad. Sci. USA 94, 2145–2149] that the active site of the vanadate‐containing chloroperoxidase from the fungus Curvularia inaequalis, of which the tertiary structure is known, is structurally very similar to that of the membrane‐bound mammalian glucose‐6‐phosphatases for which no structural data are available. The proposed active site of glucose‐6‐phosphatase, however, is incompatible with the six transmembrane–helix topology model that is currently used. Here we present a new topology model for glucose‐6‐phosphatase which is in agreement with all available data.
Human recombinant tissue factor pathway inhibitor induces apoptosis in cultured human endothelial cells
Authors: Hamuro Tsutomu , Kamikubo Yu-ichi , Nakahara Yo , Miyamoto Seiji , Funatsu Akinobu
Published: July 2002

Tissue factor pathway inhibitor (TFPI) is mainly synthesized in vascular endothelial cells and exhibits a strong and specific inhibitory activity against tissue factor‐mediated blood coagulation. In the present study, we demonstrate that human recombinant TFPI (h‐rTFPI) inhibits the growth of cultured human umbilical vein endothelial cells (HUVECs) by inducing apoptosis. In a growth‐rate assay of HUVECs, the growth of the cultured HUVECs is completely abolished by the addition of 1 μM h‐rTFPI to the culture medium containing fetal bovine serum (FBS), basic fibroblast growth factor, and epidermal growth factor. In addition, h‐rTFPI and h‐rTFPI‐C which lacks the carboxyl‐terminal basic region prevent the survival of growth‐arrested HUVECs which are starved in a medium containing 2% FBS alone, suggesting that h‐rTFPI directly induces the death of these HUVECs. This hypothesis is supported by the finding that h‐rTFPI does not inhibit the synthesis of DNA in HUVECs during proliferation, as shown by a 5‐bromo‐2′‐deoxyuridine (BrdU) incorporation assay. Furthermore, Giemsa staining and a gel electrophoretic analysis of DNA fragmentation show that the HUVEC death mediated by h‐rTFPI has the typical characteristics of apoptosis. However, the apoptosis in HUVECs is considerably inhibited in the presence of 1 μg/ml of the protein synthesis inhibitor, cycloheximide. Therefore, the process of apoptosis triggered by h‐rTFPI is, at least in part, actively conducted by the cells.
Regulated expression of mature human insulin in the liver of transgenic mice
Authors: Mitanchez Delphine , Chen Ruihuan , Massias Jean-Francis , Porteu Arlette , Mignon Alexandre , Bertagna Xavier , Kahn Axel
Published: July 2002

Transgenic mice expressing either human proinsulin cDNA or mutated proinsulin cDNA in the liver were created. The human proinsulin cDNA was mutated to generate a protein cleavable by the ubiquitous prohormone convertase furin, thus leading to mature insulin peptide. All transgenic lines expressed human C‐peptide in the blood, whose level varied according to nutritional conditions. High performance liquid chromatography fractionation of mouse serum revealed that mutant proinsulin was effectively processed into mature insulin in vivo. This transgenic mouse model provides a useful tool for further prospects of gene therapy of insulin‐dependent diabetes mellitus.
Role of UEV‐1A, a homologue of the tumor suppressor protein TSG101, in protection from DNA damage
Authors: Thomson Timothy M , Khalid Hajji , Lozano Juan José , Sancho Elena , Ariño Joaquı́n
Published: July 2002

The open reading frame YGL087c in the budding yeast Saccharomyces cerevisiae genome encodes a polypeptide highly similar to the human UEV (ubiquitin‐conjugating E2 enzyme variant) proteins, which have been proposed to belong to a family of putative dominant negative ubiquitin regulators. Deletion of the YGL087c open reading frame yields viable cells which are sensitive to UV irradiation or methyl methanesulfonate, but not to hydroxyurea. This phenotype is reminiscent of that of rad mutants and suggests that the YGL087c‐encoded protein functions in a process related to tolerance to DNA damage. We also show that the mutant phenotype is fully complemented by expression of the human UEV‐1A cDNA and we propose that UEV‐1 proteins could also have a role in protecting higher eukaryotic cells from DNA damaging agents.