bluuebunny/crossref_metadata_2025 huggingface dataset:
NGF‐dependent and tissue‐specific transcription of vgf is regulated by a CREB–p300 and bHLH factor interaction
Authors: Mandolesi Georgia , Gargano Silvia , Pennuto Maria , Illi Barbara , Molfetta Rosa , Soucek Laura , Mosca Laura , Levi Andrea , Jucker Richard , Nasi Sergio
Published: October 2002

Neurotrophins support neuronal survival, development, and plasticity through processes requiring gene expression. We studied how vgf target gene transcription is mediated by a critical promoter region containing E‐box, CCAAT and cAMP response element (CRE) sites. The p300 acetylase was present in two distinct protein complexes bound to this region. One complex, containing HEB (ubiquitous basic helix–loop–helix (bHLH)), bound the promoter in non‐neuronal cells and was involved in repressing vgf expression. Neurotrophin‐dependent transcription was mediated by the second complex, specific for neuronal cells, which included CRE binding protein and MASH1 (neuro‐specific bHLH), bound the CCAAT motif, and was target of neurotrophin signalling. The interaction, mediated by p300, of different transcription factors may add specificity to the neurotrophin response.
Participation of the mitochondrial permeability transition pore in nitric oxide‐induced plant cell death
Authors: Saviani Elzira E. , Orsi Cintia H. , Oliveira Jusceley F.P. , Pinto-Maglio Cecı́lia A.F. , Salgado Ione
Published: October 2002

In the present study, we investigated the involvement of the mitochondrial permeability transition pore (PTP) in nitric oxide (NO)‐induced plant cell death. NO donors such as sodium nitroprusside (SNP) and S‐nitroso‐N‐acetylpenicillamine inhibited growth and caused death in suspension‐cultured cells of Citrus sinensis. Cells treated with SNP showed chromatin condensation and fragmentation, characteristic of apoptosis. SNP caused loss of the mitochondrial membrane electrical potential, which was prevented by cyclosporin A (CsA), a specific inhibitor of PTP formation. CsA also prevented the nuclear apoptosis and subsequent Citrus cell death induced by NO. These findings indicate that mitochondrial PTP formation is involved in the signaling pathway by which NO induces apoptosis in cultured Citrus cells.
Anti‐angiogenic activity of a novel multi‐substrate analogue inhibitor of thymidine phosphorylase
Authors: Liekens Sandra , Bilsen Filip , De Clercq Erik , Priego Eva Marı́a , Camarasa Marı́a-José , Pérez-Pérez Marı́a-Jesús , Balzarini Jan
Published: October 2002

7‐Deazaxanthine (7‐DX) was recently identified as the first purine derivative with pronounced inhibitory activity against Escherichia coli thymidine phosphorylase (TP) and angiogenesis. In order to ‘freeze’ the enzyme in an open, inactive conformation, a novel multi‐substrate analogue inhibitor of TP, containing an alkyl phosphonate moiety covalently linked to 7‐DX, was synthesized. The prototype compound TP65 (9‐(8‐phosphonooctyl)‐7‐deazaxanthine) (at 250 μM) completely inhibited TP‐induced formation of microvascular sprouts from endothelial cell aggregates in a three‐dimensional fibrin gel. In the chick chorioallantoic membrane assay, TP caused a dose‐dependent stimulation of angiogenesis, which was completely inhibited by 250 nmol TP65. This dose proved to be non‐toxic for the developing chick embryo. TP65 thus emerges as a potent and specific inhibitor of TP and TP‐induced angiogenesis, which opens new perspectives for multi‐substrate analogue inhibitors of TP as potential anti‐cancer agents and as inhibitors of angiogenesis and of diseases with enhanced expression of TP.
Directional degradation of β‐chitin by chitinase A1 revealed by a novel reducing end labelling technique
Authors: Imai Tomoya , Watanabe Takeshi , Yui Toshifumi , Sugiyama Junji
Published: October 2002

A novel procedure for labelling the molecular ends of β‐chitin crystals has been established. By introducing a hydrazide derivative of biotin at the reducing end of a chitin chain, followed by a specific interaction between biotin and streptavidin coupled with a colloidal gold particle, the chain directionality of β‐chitin microcrystals could be directly visualized by transmission electron microscopy. This method allowed to certify the parallelism of the chitin chains in the β‐chitin microcrystals, and also to label the reducing tips of β‐chitin microcrystals degraded by Bacillus circulans chitinase A1. With these substrates, the labelling occurred only at their tapered tip, which indicates that the digestion of these crystals proceeded from their reducing end. The generalization of this new labelling method to other polysaccharide crystals is discussed.
Flow cytometry of isolated mitochondria during development and under some pathological conditions
Authors: Medina José M , López-Mediavilla Casilda , Orfao Alberto
Published: October 2002

Mitochondria play an essential role in the generation of the energy needed for eukaryotic cell life and in the release of molecules involved in initiation of cell death. Here we review the changes in isolated mitochondrial fluorescent populations as distinguished by flow cytometry during postnatal development and their regulation by thyroid hormones and catecholamines. The use of flow cytometry in the study of mitochondrial changes occurring under hypothyroidism, alcohol abuse and aging is also reviewed.
Exploring the molecular nature of alternative oxidase regulation and catalysis
Authors: Affourtit Charles , Albury Mary S , Crichton Paul G , Moore Anthony L
Published: October 2002

Plant mitochondria contain a non‐protonmotive alternative oxidase (AOX) that couples the oxidation of ubiquinol to the complete reduction of oxygen to water. In this paper we review theoretical and experimental studies that have contributed to our current structural and mechanistic understanding of the oxidase and to the clarification of the molecular nature of post‐translational regulatory phenomena. Furthermore, we suggest a catalytic cycle for AOX that involves at least one transient protein‐derived radical. The model is based on the reviewed information and on recent insights into the mechanisms of cytochrome c oxidase and the hydroxylase component of methane monooxygenase.
The N‐terminal domain of SV40 large T antigen represses the HER2/neu‐mediated transformation and metastatic potential in breast cancers
Authors: Chuang Tzu-Chao , Yu Yuh-Hua , Lin Yen-Shing , Wang Shan-Shue , Kao Ming-Ching
Published: October 2002

HER2/neu is known to be overexpressed in approximately 40% of human breast and ovarian cancers and it is associated with increased metastasis and poor prognosis. We have shown previously that the N‐terminal domain of simian virus 40 large T antigen (LT425) can act as a transforming suppressor of the HER2/neu oncogene in human ovarian cancer. In the present study, we demonstrate that LT425 can also repress the transforming properties of HER2/neu‐overexpressing human breast cancer cells. In addition, the results of a chemotaxis assay and an in vitro chemoinvasion assay further suggest that LT425 can also suppress the metastatic potential of the HER2/neu‐transformed breast cancer cells. Taken together, these data clearly suggest that the inhibition of the expression of p185HER2/neu tyrosine kinase by LT425 is capable of suppressing the HER2/neu‐mediated transformation and metastatic potential in breast cancers.
A compact view of isochores in the draft human genome sequence
Authors: Pavlı́ček Adam , Pačes Jan , Clay Oliver , Bernardi Giorgio
Published: October 2002

Prior to genome sequencing, information on base composition (GC level) and its variation in mammalian genomes could be obtained using density gradient ultracentrifugation. Analyses using this approach led to the conclusion that mammalian genomes are organized into mosaics of fairly homogeneous regions, called isochores. We present an initial compositional overview of the chromosomes of the recently available draft human genome sequence, in the form of color‐coded moving window plots and corresponding GC level histograms. Results obtained from the draft human genome sequence agree well with those obtained or deduced earlier from CsCl experiments. The draft sequence now permits the visualization of the mosaic organization of the human genome at the DNA sequence level.
Interaction domains: from simple binding events to complex cellular behavior
Authors: Pawson Tony , Raina Monica , Nash Piers
Published: October 2002

Many of the signaling pathways and regulatory systems in eukaryotic cells are controlled by proteins with multiple interaction domains that mediate specific protein–protein and protein–phospholipid interactions, and thereby determine the biological output of receptors for external and intrinsic signals. Here, we discuss the basic features of interaction domains, and suggest that rather simple binary interactions can be used in sophisticated ways to generate complex cellular responses.
The ENTH domain
Authors: De Camilli Pietro , Chen Hong , Hyman Joel , Panepucci Ezequiel , Bateman Alex , Brunger Axel T
Published: October 2002

The epsin NH2‐terminal homology (ENTH) domain is a membrane interacting module composed by a superhelix of α‐helices. It is present at the NH2‐terminus of proteins that often contain consensus sequences for binding to clathrin coat components and their accessory factors, and therefore function as endocytic adaptors. ENTH domain containing proteins have additional roles in signaling and actin regulation and may have yet other actions in the nucleus. The ENTH domain is structurally similar to the VHS domain. These domains define two families of adaptor proteins which function in membrane traffic and whose interaction with membranes is regulated, in part, by phosphoinositides.
Functional properties of ryanodine receptors from rat dorsal root ganglia
Authors: Lokuta Andrew J. , Komai Hirochika , McDowell Thomas S. , Valdivia Héctor H.
Published: October 2002

The properties of ryanodine receptors (RyRs) from rat dorsal root ganglia (DRGs) have been studied. The density of RyRs (B
max) determined by [3H]ryanodine binding was 63 fmol/mg protein with a dissociation constant (K
d) of 1.5 nM. [3H]Ryanodine binding increased with caffeine, decreased with ruthenium red and tetracaine, and was insensitive to millimolar concentrations of Mg2+ or Ca2+. DRG RyRs reconstituted in planar lipid bilayers were Ca2+‐dependent and displayed the classical long‐lived subconductance state in response to ryanodine; however, unlike cardiac and skeletal RyRs, they lacked Ca2+‐dependent inactivation. Antibodies against RyR3, but not against RyR1 or RyR2, detected DRG RyRs. Thus, DRG RyRs are immunologically related to RyR3, but their lack of divalent cation inhibition is unique among RyR subtypes.
Inhibitory actions of the selective serotonin re‐uptake inhibitor citalopram on HERG and ventricular L‐type calcium currents
Authors: Witchel Harry J , Pabbathi Vijay K , Hofmann Giovanna , Paul Ashok A , Hancox Jules C
Published: October 2002

Using whole‐cell patch clamp recording of heterologous HERG‐mediated currents in transfected mammalian cells, we observed that the selective serotonin re‐uptake inhibitor citalopram blocks HERG with an IC50 of 3.97 μM. This is slightly less potent than fluoxetine in our system (IC50 of 1.50 μM). In isolated guinea pig ventricular cardiomyocytes citalopram inhibited L‐type calcium current (I
Ca,L). The voltage dependence of I
Ca,L inactivation in the presence of 100 μM citalopram was shifted significantly leftward. As a result, the I
Ca,L ‘window’ in citalopram was found to be (a) smaller and (b) leftward‐shifted compared to control. The effects of citalopram on both calcium current amplitude and the I
Ca,L ‘window’ may help to explain citalopram's good cardiac safety profile, given its propensity to block HERG at excessive dosages.
Identification of functional type 1 ryanodine receptors in mouse dendritic cells
Authors: O'Connell Peta J. , Klyachko Vitaly A. , Ahern Gerard P.
Published: October 2002

Ca2+ signaling plays an important role in the function of dendritic cells (DC), the specialized antigen‐presenting cells of the immune system. Here we describe functional ryanodine receptor (RyR) Ca2+ release channels in murine, bone marrow‐derived DC. RT‐PCR analysis identified selective expression of the type 1 RyR, with higher levels detected in immature rather than mature DC. The RyR activators caffeine, FK506, ryanodine and 4‐chloro‐m‐cresol mobilized Ca2+ in DC, and responses to 4‐chloro‐m‐cresol were inhibited by dantrolene. Furthermore, activation of RyRs both inhibited subsequent inositol trisphosphate‐mediated Ca2+ release and provoked store‐operated Ca2+ entry, suggesting a functional interaction between these intracellular Ca2+ channels. Thus, the RyR1 channel may play an intrinsic role in Ca2+ signaling in DC.
N‐linked oligosaccharide chains of Sendai virus fusion protein determine the interaction with endoplasmic reticulum molecular chaperones
Authors: Tamura Taku , Yamashita Tetsuro , Segawa Hiroaki , Taira Hideharu
Published: October 2002

The selectivity and individual roles of the N‐linked oligosaccharide chains of Sendai virus fusion protein (F protein) in the interaction with endoplasmic reticulum molecular chaperones were investigated by analyses of transient expression of single N‐glycosylation mutants and sequential immunoprecipitation. We demonstrated differential interactions depending on the location of the N‐linked oligosaccharide chain, and showed that these interactions were correlated with the folding and transport of F proteins. Moreover, mutant F proteins that lacked the specific N‐linked oligosaccharide chains required for disulfide bond formation showed increased association with ERp57.
Proteasome inhibitors prevent cytochrome c release during apoptosis but not in excitotoxic death of cerebellar granule neurons
Authors: Bobba Antonella , Canu Nadia , Atlante Anna , Petragallo Vito , Calissano Pietro , Marra Ersilia
Published: October 2002

In order to find out whether and how proteasomes participate in the processes leading cerebellar granule cells to death either in necrosis, due to glutamate neurotoxicity, or in apoptosis, due to K+ shift, we measured the three proteasome activities by using specific fluorescent probes and investigated the effect of several proteasome inhibitors, including MG132, on the cytochrome c release taking place in the early phase of both apoptosis and necrosis. We show that differently from apoptosis, the early phase of necrosis does not require proteasome activation. Inhibition of proteasome activity can prevent cytochrome c release in cerebellar granule cells undergoing apoptosis, thus improving cell survival, but not necrosis. These findings show that proteasomes play an important role in the early phase of apoptosis but not that of necrosis, and that these two types of cell death differ from each other in their mechanism of cytochrome c release.
8‐Chloro‐dGTP, a hypochlorous acid‐modified nucleotide, is hydrolyzed by hMTH1, the human MutT homolog
Authors: Fujikawa Katsuyoshi , Yakushiji Hiroyuki , Nakabeppu Yusaku , Suzuki Toshinori , Masuda Mitsuharu , Ohshima Hiroshi , Kasai Hiroshi
Published: October 2002

The human mutT homolog, hMTH1, suppresses spontaneous mutations by degrading the endogeneous mutagen, 8‐hydroxy‐dGTP. We previously reported the broad substrate specificity of hMTH1, which also degrades the oxidatively damaged purine nucleotides, 2‐hydroxy‐dATP, 8‐hydroxy‐dATP, 2‐hydroxy‐ATP, and 8‐hydroxy‐GTP, in addition to 8‐hydroxy‐dGTP. In this paper, we describe the hMTH1 activity for 8‐chloro‐dGTP, which could be formed in inflamed tissue by the reaction of dGTP with hypochlorous acid, a product of myeloperoxidase from activated human neutrophils. The hMTH1 protein was mixed with 1–20 μM of 8‐chloro‐dGTP and 8‐hydroxy‐dGTP, and the reaction products were quantified by anion‐exchange HPLC to measure the pyrophosphatase reaction rate. The kinetic parameters revealed that 8‐chloro‐dGTP was degraded by hMTH1 with 50% efficiency as compared with that of 8‐hydroxy‐dGTP. This result suggests that 8‐chloro‐dGTP is an intrinsic substrate for hMTH1.
Normoxic induction of the hypoxia‐inducible factor 1α by insulin and interleukin‐1β involves the phosphatidylinositol 3‐kinase pathway
Authors: Stiehl Daniel P , Jelkmann Wolfgang , Wenger Roland H , Hellwig-Bürgel Thomas
Published: October 2002

Hypoxia‐inducible factor 1 (HIF‐1) is a heterodimeric DNA‐binding complex of the subunits α and β with relevance in O2 and energy homeostasis. The labile component, HIF‐1α, is not only activated by hypoxia but also by peptides such as insulin and interleukin‐1 (IL‐1) in normoxia. We investigated whether inhibitors of mitogen‐activated protein kinase kinases (MAPKKs: PD 98059, U0126) and phosphatidylinositol 3‐kinase (PI3K: LY 294002) do not only lower the hypoxia‐induced, but also the insulin‐ and IL‐1‐induced HIF‐1α accumulation and HIF‐1 DNA‐binding in human hepatoma cell cultures (line HepG2). The results show that LY 294002 suppressed HIF‐1 activation in a dose‐dependent manner irrespective of the stimulus. With respect to target proteins controlled by HIF‐1, the production of erythropoietin was fully blocked and that of vascular endothelial growth factor reduced following inhibition of the PI3K pathway. The role of MAPKKs in this process remained in question, because PD 98059 and U0126 did not significantly reduce HIF‐1α levels at non‐toxic doses. We propose that PI3K signaling is not only important in the hypoxic induction of HIF‐1 but it is also crucially involved in the response to insulin and IL‐1.
Murine T cells expressing high activity of prolyl endopeptidase are susceptible to activation‐induced cell death
Authors: Odaka Chikako , Mizuochi Toshiaki , Shirasawa Takuo , Morain Philippe , Checler Fredéric
Published: October 2002

Prolyl endopeptidase (PEP) is widely distributed and thought to play an important role in the degradation of peptide hormones and neuropeptides, but its biological role is totally unknown. In this study, we examined PEP activity in subpopulations of murine T cells and found that PEP activity was significantly higher in immature thymocytes than in mature thymocytes or in peripheral T cells. Stimulation of murine peripheral T cells time‐dependently increased PEP activity. Although murine T cell hybridomas exhibited high PEP activity, the PEP activity was fully inhibited by treatment with PEP inhibitor. The pretreated T cells were found to be resistant to activation‐induced cell death (AICD). Similar results were obtained in murine thymocytes as well as in activated peripheral T cells. PEP activity in T cell hybridomas remained unchanged during AICD. These results suggest that T cells expressing high PEP activity are susceptible to ACID.
Three‐dimensional architecture of the eukaryotic multisynthetase complex determined from negatively stained and cryoelectron micrographs
Authors: Norcum Mona T , Boisset Nicolas
Published: October 2002

This study provides the first description of the three‐dimensional architecture of the multienzyme complex of aminoacyl‐tRNA synthetases. Reconstructions were calculated from electron microscopic images of negatively stained and frozen hydrated samples using three independent angular assignment methods. In all cases, volumes show an asymmetric triangular arrangement of protein domains around a deep central cavity. The structures have openings or indentations on most sides. Maximum dimensions are ca. 19×16×10 nm. The central cavity is 4 nm in diameter and extends two‐thirds of the length of the particle.
A novel rat hypothalamic RFamide‐related peptide identified by immunoaffinity chromatography and mass spectrometry
Authors: Ukena Kazuyoshi , Iwakoshi Eiko , Minakata Hiroyuki , Tsutsui Kazuyoshi
Published: October 2002

Recently, cDNAs encoding novel RFamide‐related peptides (RFRPs) have been reported in the mammalian brains by a gene database search and the deduced RFRPs have been suggested to participate in neuroendocrine and pain mechanisms in the rat. Two peptides have been predicted to be encoded in the cDNA of rodent RFRPs. To assess precise functions of rodent RFRPs in the brain, in the present study we identified a naturally occurring RFRP in the rat hypothalamus by immunoaffinity purification combined with mass spectrometry (MS). The affinity chromatography showed that the rat hypothalamus contained RFRP‐like immunoreactivity. The immunoreactive material was analyzed by a nanoflow electrospray ionization time‐of‐flight MS followed by tandem MS analysis. The mass peak corresponding to octadecapeptide was detected at 1010.54 m/z ([M+2H]2+) and its sequence, ANMEAGTMSHFPSLPQRF‐NH2, was revealed by the fragmentation, showing a mature form encoded in the cDNA sequence of RFRPs. The identified endogenous RFRP will aid not only in defining its physiological roles but also facilitate the development of its agonists and antagonists in the rodent brain.
Heterodisulfide reductase from Methanothermobacter marburgensis contains an active‐site [4Fe–4S] cluster that is directly involved in mediating heterodisulfide reduction
Authors: Duin Evert C , Madadi-Kahkesh Shahla , Hedderich Reiner , Clay Michael D , Johnson Michael K
Published: October 2002

Heterodisulfide reductases (HDRs) from methanogenic archaea are iron–sulfur flavoproteins or hemoproteins that catalyze the reversible reduction of the heterodisulfide (CoM‐S–S‐CoB) of the methanogenic thiol coenzymes, coenzyme M (CoM‐SH) and coenzyme B (CoB‐SH). In this work, the ground‐ and excited‐state electronic properties of the paramagnetic Fe–S clusters in Methanothermobacter marburgensis HDR have been characterized using the combination of electron paramagnetic resonance and variable‐temperature magnetic circular dichroism spectroscopies. The results confirm multiple S=1/2 [4Fe–4S]+ clusters in dithionite‐reduced HDR and reveal spectroscopically distinct S=1/2 [4Fe–4S]3+ clusters in oxidized HDR samples treated separately with the CoM‐SH and CoB‐SH cosubstrates. The active site of HDR is therefore shown to contain a [4Fe–4S] cluster that is directly involved in mediating heterodisulfide reduction. The catalytic mechanism of HDR is discussed in light of the crystallographic and spectroscopic studies of the related chloroplast ferredoxin:thioredoxin reductase class of disulfide reductases.
Quinones in long‐lived clk‐1 mutants of Caenorhabditis elegans
Authors: Miyadera Hiroko , Kano Kenji , Miyoshi Hideto , Ishii Naoaki , Hekimi Siegfried , Kita Kiyoshi
Published: October 2002

Ubiquinone (UQ) (coenzyme Q) is a lipophilic redox‐active molecule that functions as an electron carrier in the mitochondrial electron transport chain. Electron transfer via UQ involves the formation of semiubiquinone radicals, which causes the generation of superoxide radicals upon reaction with oxygen. In the reduced form, UQ functions as a lipid‐soluble antioxidant, and protects cells from lipid peroxidation. Thus, UQ is also important as a lipophilic regulator of oxidative stress. Recently, a study on long‐lived clk‐1 mutants of Caenorhabditis elegans demonstrated that biosynthesis of UQ is dramatically altered in mutant mitochondria. Demethoxy ubiquinone (DMQ), that accumulates in clk‐1 mutants in place of UQ, may contribute to the extension of life span. Here we elucidate the possible mechanisms of life span extension in clk‐1 mutants, with particular emphasis on the electrochemical property of DMQ. Recent findings on the biochemical function of CLK‐1 are also discussed.
Sucrose is involved in the diazotrophic metabolism of the heterocyst‐forming cyanobacterium Anabaena sp
Authors: Curatti Leonardo , Flores Enrique , Salerno Graciela
Published: October 2002

Sucrose synthase (SuS) expression was studied in the filamentous, nitrogen‐fixing cyanobacterium Anabaena sp. PCC 7119. SuS activity, SusA polypeptide, and susA mRNA levels were lower in cells cultured diazotrophically than in the presence of combined nitrogen. An insertional susA mutant presented a dramatic increase in sucrose levels, whereas the disaccharide was not detectable in a susA overexpressing strain, indicating that SusA is involved in the cleavage of sucrose in vivo. Diazotrophic growth was impaired in the susA overexpressing strain, suggesting a role for sucrose in diazotrophic metabolism and the involvement of SusA in the control of carbon flux in the N2‐fixing filament.
Cellular prion protein: on the road for functions
Authors: Martins Vilma R , Linden Rafael , Prado Marco A.M , Walz Roger , Sakamoto Américo C , Izquierdo Ivan , Brentani Ricardo R
Published: October 2002

Cellular prion (PrPc) is a plasma membrane glycosyphosphatidylinositol‐anchored protein present in neurons but also in other cell types. Protein conservation among species suggests that PrPc may have important physiological roles. Cellular and molecular approaches have established several novel features of the regulation of PrPc expression, cellular trafficking as well as its participation in copper uptake, protection against oxidative stress, cell adhesion, differentiation, signaling and cell survival. It is therefore likely that PrPc plays pleiotropic roles in neuronal and non‐neuronal cells, and as such the loss of function of PrPc may be an important component of various diseases.
Catalytic site occupancy during ATP synthase catalysis
Authors: Boyer Paul D
Published: October 2002

An early proposal was that for rapid ATP synthesis by the rotational ATP synthase, a specific second site must bind ADP and Pi, and for rapid ATP hydrolysis a different second site must bind ATP. Such bi‐site activation was considered to occur whether or not an ADP or ATP was at a third site. In contrast, a more recent proposal is that rapid ATP hydrolysis requires that all three sites have bound ADP or ATP present. However, discovery that one second site binds ADP better than ATP, together with other data and considerations support the earlier proposal. The retention or rebinding of ADP can explain why three sites fill during hydrolysis as ATP concentration is increased although bi‐site activation still prevails.
Functional evidence for D‐ and T‐loop interactions in tmRNA
Authors: Barends Sharief , Björk Karl , Gultyaev Alexander P. , de Smit Maarten H. , Pleij Cornelis W.A. , Kraal Barend
Published: October 2002

During bacterial protein synthesis, stalled ribosomes can be rescued by tmRNA, a molecule with both tRNA and mRNA features. The tRNA region of tmRNA has sequence similarity with tRNAAla and also has a clover‐leaf structure folded similarly as in canonical tRNAs. Here we propose the L‐shape of tmRNA to be stabilized by two tertiary interactions between its D‐ and T‐loop on the basis of phylogenetic and experimental evidence. Mutational analysis clearly demonstrates a tertiary interaction between G13 and U342. Strikingly, this in evolution conserved interaction is not primarily important for tmRNA alanylation and for binding to elongation factor Tu, but especially for a proper functioning of SmpB.
How does tmRNA move through the ribosome?
Authors: Ivanov Pavel V , Zvereva Maria I , Shpanchenko Olga V , Dontsova Olga A , Bogdanov Alexey A , Aglyamova Galina V , Lim Valery I , Teraoka Yoshika , Nierhaus Knud H
Published: October 2002

To test the structure of tmRNA in solution, cross‐linking experiments were performed which showed two sets of cross‐links in two different domains of tmRNA. Site‐directed mutagenesis was used to search for tmRNA nucleotide bases that might form a functional analogue of a codon–anticodon duplex to be recognized by the ribosomal A‐site. We demonstrate that nucleotide residues U85 and A86 from tmRNA are significant for tmRNA function and propose that they are involved in formation of a tmRNA element playing a central role in A‐site recognition. These data are discussed in the frame of a hypothetical model that suggests a general scheme for the interaction of tmRNA with the ribosome and explains how it moves through the ribosome.
Induction of inducible nitric oxide synthase expression by 18β‐glycyrrhetinic acid in macrophages
Authors: Jeong Hye Gwang , Kim Ji Young
Published: October 2002

Glycyrrhizin (GL), a triterpenoid saponin fraction of licorice, is reported to have anti‐viral and anti‐tumor activities and is metabolized to 18β‐glycyrrhetinic acid (GA) in the intestine by intestinal bacteria. However, the mechanism underlying its effects is poorly understood. To further elucidate the mechanism of GA, the aglycone of GL, we investigated the effects of GA on the release of nitric oxide (NO) and at the level of inducible NO synthase (iNOS) gene expression in mouse macrophages. We found that GA elicited a dose‐dependent increase in NO production and in the level of iNOS mRNA. Since iNOS transcription has been shown to be under the control of the transcription factor nuclear factor κB (NF‐κB), the effects of GA on NF‐κB activation were examined. Transient expression assays with NF‐κB binding sites linked to the luciferase gene revealed that the increased level of iNOS mRNA, induced by GA, was mediated by the NF‐κB transcription factor complex. By using DNA fragments containing the NF‐κB binding sequence, GA was shown to activate the protein/DNA binding of NF‐κB to its cognate site, as measured by electrophoretic mobility shift assay. These results demonstrate that GA stimulates NO production and is able to up‐regulate iNOS expression through NF‐κB transactivation in macrophages.
Vav‐induced activation of the human IFN‐γ gene promoter is mediated by upregulation of AP‐1 activity
Authors: Kaminuma Osamu , Elly Chris , Tanaka Yoshihiko , Mori Akio , Liu Yun-Cai , Altman Amnon , Miyatake Shoichiro
Published: October 2002

The role of Vav in the transcriptional regulation of the human interferon‐γ (IFN‐γ) promoter was investigated. Overexpression of Vav in Jurkat‐TAg cells enhanced T cell receptor (TCR)‐induced activation of a luciferase (Luc) reporter gene construct driven by cis‐regulatory element of the IFN‐γ gene (−346 to +7). Electrophoresis mobility shift and Luc reporter assays demonstrated that the DNA‐binding and transcriptional activity of the proximal AP‐1‐dependent NFAT site (positions −172 to −138), the AP‐1/Ying‐Yang 1 (YY1)‐binding site (−209 to −184), and a consensus AP‐1‐binding site were upregulated by Vav. Vav enhanced TCR‐induced activation of c‐Jun N‐terminal kinase (JNK) and its upstream regulator, Rho family GTPases. Finally, coexpression of a dominant‐negative Rac1 mutant suppressed Vav‐mediated upregulation of the transcriptional and DNA‐binding activity of the proximal NFAT/AP‐1 site and the AP‐1/YY1 site, as well as the complete IFN‐γ promoter activity. Vav activates the IFN‐γ promoter via upregulation of AP‐1‐binding through a Rac1/JNK pathway.
Evidence for an operative glyoxylate cycle in the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius
Authors: Uhrigshardt Helge , Walden Michael , John Harald , Petersen Arnd , Anemüller Stefan
Published: October 2002

Both key enzymes for the glyoxylate cycle, isocitrate lyase (EC 4.1.3.1) and malate synthase (EC 4.1.3.2), were purified and characterized from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius. Whereas the former enzyme was copurified with the aconitase, the latter enzyme could be enriched to apparent homogeneity. Amino acid sequencing of three internal peptides of the isocitrate lyase revealed the presence of highly conserved residues. With respect to cofactor requirement and quarternary structure the crenarchaeal malate synthase might represent a novel type of this enzyme family. High activities of both glyoxylate cycle enzymes could already be detected in extracts of glucose grown cells and both increased about two‐fold in extracts of acetate grown cells.
Molecular basis for exaggerated sensitivity to mexiletine in the cardiac isoform of the fast Na channel
Authors: Kawagoe Hiroyuki , Yamaoka Kaoru , Kinoshita Eiji , Fujimoto Yoshinori , Maejima Hiroshi , Yuki Tsunetsugu , Seyama Issei
Published: October 2002

Cardiac sodium channels have been shown to have a higher sensitivity to local anesthetic agents, such as lidocaine, than the sodium channels of other tissues. To examine if this is also true for mexiletine, we have systematically measured mexiletine sensitivity of the Na channel isoforms, rH1, μ1, and rBII, which were transiently expressed in human embryonic kidney (HEK) 293 cells. We confirmed that the cardiac isoform rH1 exhibited the highest sensitivity among the three tested channel isoforms. In rH1, μ1, and rBII, the respective IC50 values were 62, 294, and 308 μM mexiletine, in regard to tonic block, and 18, 54, and 268 μM mexiletine, in relation to use (8 Hz)‐dependent block. The relatively high drug sensitivity of rH1 was an invariant finding, irrespective of channel state or whether channels were subjected to infrequent or frequent depolarizing stimuli. Mutating specific amino acids in the skeletal muscle isoform μ1 (namely, μ1‐I433V and μ1‐S251A) to those of the cardiac isoform at putative binding sites for local anesthetic agents revealed that only one of the point mutations (μ1‐S251A) has relevance to the high cardiac drug sensitivity, because mexiletine produced significantly more use‐dependent and tonic block in μ1‐S251A than wild‐type μ1.
Translocation of tRNA during protein synthesis
Authors: Noller Harry F. , Yusupov Marat M. , Yusupova Gulnara Z. , Baucom Albion , Cate J.H.D.
Published: October 2002

Coupled translocation of tRNA and mRNA in the ribosome during protein synthesis is one of the most challenging and intriguing problems in the field of translation. We highlight several key questions regarding the mechanism of translocation, and discuss possible mechanistic models in light of the recent crystal structures of the ribosome and its subunits.
The role of SmpB protein in trans‐translation
Authors: Shimizu Yoshihiro , Ueda Takuya
Published: October 2002

The function of SmpB protein in the trans‐translation system was evaluated using the well‐defined cell‐free translation system consisting of purified ribosome, alanyl‐tRNA synthetase and elongation factors. The analysis showed that SmpB protein enhances alanine‐accepting activity of tmRNA and that SmpB protein and tmRNA are sufficient to complete the trans‐translation process in the presence of translational components. Moreover, SmpB is indispensable in the addition of tag‐peptide onto ribosomes by tmRNA. In particular, the A‐site binding of tmRNA is inhibited in the absence of SmpB.
Kinetic mechanism and order of substrate binding for sn‐glycerol‐3‐phosphate acyltransferase from squash (Cucurbita moschata)
Authors: Hayman Matthew W , Fawcett Tony , Slabas Antoni R
Published: October 2002

sn‐Glycerol‐3‐phosphate acyltransferase (G3PAT, EC 2.3.1.15), a component of glycerolipid biosynthesis, is an important enzyme in chilling sensitivity in plants. The three‐dimensional structure of the enzyme from squash (Cucurbita moschata), without bound substrate, has been determined [Turnbull et al. (2001) Acta Crystallogr. D 57, 451–453; Turnbull et al. (2001) Structure 9, 347–353]. Here we report the kinetic mechanism of plastidial G3PAT from squash and the order of substrate binding using acyl‐acyl carrier protein (acyl‐ACP) substrates. The reaction proceeds via a compulsory‐ordered ternary complex with acyl‐ACP binding before glycerol‐3‐phosphate. We have also determined that the reaction will proceed with C4:0‐CoA, C6:0‐CoA and C12:0‐ACP substrates, allowing a wider choice of acyl groups for future co‐crystallisation studies.
Structural characterization of a protein pheromone from a cold‐adapted (Antarctic) single‐cell eukaryote, the ciliate Euplotes nobilii
Authors: Alimenti Claudio , Ortenzi Claudio , Carratore Vito , Luporini Pierangelo
Published: October 2002

Free‐living species of ciliated Protozoa control their vegetative (mitotic) proliferation and mating (sexual) processes by diffusible, cell type‐specific protein signals (pheromones). One of these molecules, designated En‐2, was isolated from a species, Euplotes nobilii, living in the stably cold marine waters of Antarctica, and its complete amino acid sequence of 60 residues was determined by automated Edman degradation of the whole protein and peptides generated by trypsin digestion. The proposed sequence is: DIEDFYTSETCPYKNDSQLA20WDTCSGGTGNCGTVCCGQCF40SFPVSQSCAGMADSNDCPNA60. The En‐2 structure appears to be characterized by an adaptive insertion of a glycine‐rich motif potentially capable to confer more flexibility to a functionally critical region of the molecule.
A rapid cytokinin response assay inArabidopsisindicates a role for phospholipase D in cytokinin signalling
Authors: Romanov Georgy A , Kieber Joseph J , Schmülling Thomas
Published: October 2002

Seedlings ofArabidopsis thalianaharboring a fusion of the cytokinin‐responsiveARR5gene promoter and theGUSreporter gene were used for a pharmacological approach to study cytokinin signal transduction. The assay was shown to be rapid, sensitive, dose‐dependent and highly specific for cytokinins, both adenine and phenylurea derivatives. Numerous inhibitors of known signalling pathways were tested and some were shown to suppress reporter gene induction. Particularly, primary alcohols that specifically inhibit phospholipase D (PLD) partially prevented cytokinin‐induced GUS activity and reduced the accumulation ofARR5gene transcripts. This indicates a role for PLD early during cytokinin signalling.
Inhibition of mitochondrial and plastid activity of Plasmodium falciparum by minocycline
Authors: Lin Qinghua , Katakura Ken , Suzuki Mamoru
Published: October 2002

We previously reported the superior effect of minocycline against drug‐resistant Plasmodium falciparum in vitro. Here, we report that RT‐PCR for falciparum parasites treated with minocycline revealed reduced levels of RNA transcripts of the mitochondrion‐encoded genes such as the COI and Cyb genes, as well as the plastid‐encoded RNA polymerase subunit (rpoB/C) gene. However, we detected no apparent effects of the antibiotic on the transcription of merozoite surface antigen and small subunit rRNA genes encoded by the nucleus. In addition, treatment with chloroquine and pyrimethamine showed no substantial reduction of any RT‐PCR products. These findings suggest that tetracycline antibiotics selectively inhibit both mitochondrial and plastid activity.
Identification of the novel proteins Yip4p and Yip5p as Rab GTPase interacting factors
Authors: Calero Monica , Winand Nena J. , Collins Ruth N.
Published: October 2002

The Rab GTPases are key regulators of membrane traffic. Yip1p is a membrane protein of unknown function that has been reported to interact with the Rabs Ypt1p and Ypt31p. In this study we identify Yif1p, and two unknown open reading frames, Ygl198p and Ygl161p, which we term Yip4p and Yip5p, as Yip1p‐related sequences. We demonstrate that the Yip1p‐related proteins possess several features: (i) they have a common overall domain topology, (ii) they are capable of biochemical interaction with a variety of Rab proteins in a manner dependent on C‐terminal prenylation, and (iii) they share an ability to physically associate with other members of the YIP1 family.
Mutations in the S4–H2 loop of eIF4E which increase the affinity for m7GTP
Authors: Spivak-Kroizman Taly , Friedland Diana E , De Staercke Christine , Gernert Kim M , Goss Dixie J , Hagedorn Curt H
Published: October 2002

Eukaryotic initiation factor 4E (eIF4E) binds the 5′‐cap of eukaryotic mRNAs and overexpression of eIF4E in epithelial cell cancers correlates with the metastases/tissue invasion phenotype. Photolabeling of eIF4E with [γ‐32P]8‐azidoguanosine 5′‐triphosphate (8‐N3GTP) demonstrated cross‐linking at Lys‐119 in the S4–H2 loop which is distant from the m7GTP binding site [Marcotrigiano et al. (1997) Cell 89, 951–961; Friedland et al. (1997) Protein Sci. 6, 125–131]. Modeling studies indicate that 8‐N3GTP cross‐linked with Lys‐119 because it binds a site that is occupied by the second nucleotide of a bound mRNA. Mutagenesis of the S4–H2 loop produced proteins with a 5–10‐fold higher affinity for m7GTP than wild‐type eIF4E. These mutants of eIF4E may have uses in selectively purifying mRNAs with intact 5′‐ends or in determining how the promyelocytic leukemia protein decreases the affinity of eIF4E for mRNA caps.
The reaction order of the dissociation reaction of the B820 subunit of Rhodospirillum rubrum light‐harvesting I complex
Authors: Arluison Véronique , Seguin Jérôme , Robert Bruno
Published: October 2002

We have studied the equilibrium between the dissociated B777 form (absorbing at 777 nm) of the light‐harvesting complex of Rhodospirillum rubrum and the oligomeric B820 form. Analysis of the reaction order for the B820 dissociation reaction to form B777 shows that this reaction depends on the concentration of octylglucoside detergent (n‐octyl‐β‐D‐glucopyranoside (βOG)) present in the sample. At low βOG concentrations (less than 1.2%) this reaction requires two components, presumably one α‐B777 and one β‐B777, implying that the B820 subunit is a dimer. At higher βOG concentrations this reaction requires four components, implying that B820 is a tetramer. These results partly explain the discrepancies in the literature about the stoichiometry of B820 and open an original way for studying protein–detergent interactions.
β‐Arrestin is involved in the desensitization but not in the internalization of the somatostatin receptor 2A expressed in CHO cells
Authors: Brasselet Sabrina , Guillen Stéphanie , Vincent Jean-Pierre , Mazella Jean
Published: October 2002

The interaction of β‐arrestin‐1 with the somatostatin receptor type 2A (sst2A) was monitored using both biochemical and confocal imaging approaches. We show that, using transient transfection of either β‐arrestin‐1 or its dominant negative Δ‐arrestin‐1 in CHO cells stably transfected with the sst2A, β‐arrestin‐1 is colocalized with the receptor in endosomal vesicles after somatostatin‐induced sequestration. However, this interaction leads to a role of β‐arrestin‐1 in the desensitization of the sst2A rather than in the internalization process of the receptor–ligand complex.
A new RNase‐based immunoconjugate selectively cytotoxic for ErbB2‐overexpressing cells
Authors: De Lorenzo Claudia , Nigro Alessandra , Piccoli Renata , D'Alessio Giuseppe
Published: October 2002

We report a new tumor‐directed immunoRNase, a chimeric protein made up of an antibody fragment (single‐chain Fv fragment) directed to ErbB2, a cell surface receptor, and a non‐toxic, human ribonuclease, which upon cell internalization becomes cytotoxic. The immunoRNase is active as a ribonuclease, specifically binds and selectively kills ErbB2‐positive cells. ErbB2 is one of the most specific tumor‐associated antigens identified so far, overexpressed on tumor cells of different origin. Its choice as target antigen and that of a non‐toxic, human RNase as the killer moiety makes this immunoRNase a new, potentially attractive anticancer agent.
Der f 16: a novel gelsolin‐related molecule identified as an allergen from the house dust mite, Dermatophagoides farinae1
Authors: Kawamoto Seiji , Suzuki Takayuki , Aki Tsunehiro , Katsutani Takashi , Tsuboi Shinji , Shigeta Seiko , Ono Kazuhisa
Published: October 2002

Allergen from the house dust mite (Dermatophagoides sp.) is a major trigger factor of allergic disorders, and its characterization is crucial for the development of specific diagnosis or immunotherapy. Here we report the identification of a novel dust mite (Dermatophagoides farinae) antigen whose primary structure belongs to the gelsolin family, a group of actin cytoskeleton‐regulatory proteins. Isolated mite cDNA, termed Der f 16, encodes 480 amino acids comprising a four‐repeated gelsolin‐like segmental structure, which is not seen in conventional gelsolin family members. Enzyme immunoassay indicated that recombinant Der f 16 protein, prepared using an Escherichia coli expression system, bound IgE from mite‐allergic patients at 47% (8/17) frequency. This is the first evidence that the gelsolin family represents a new class of allergen recognizable by atopic patient IgE.
Primary sequence determinants responsible for site‐selective dephosphorylation of the PDGF β‐receptor by the receptor‐like protein tyrosine phosphatase DEP‐1
Authors: Persson Camilla , Engström Ulla , Mowbray Sherry L , Östman Arne
Published: October 2002

Site‐selective dephosphorylation of receptor tyrosine kinases contributes to receptor regulation. The receptor‐like protein tyrosine phosphatase DEP‐1 site‐selectively dephosphorylates the PDGF β‐receptor. DEP‐1 dephosphorylation of original and chimeric phospho‐peptides spanning the preferred pY1021 and the less preferred pY857 and pY562 sites was analyzed. Double substitutions of basic residues at −4 and +3 of pY857 and pY562 peptides improved affinity. Substitutions of single amino acids indicated preference for an acidic residue at position −1 and a preference against a basic residue at position +3. DEP‐1 site‐selective dephosphorylation of PDGF β‐receptor is thus determined by the primary sequence surrounding phosphorylation sites and involves interactions with residues spanning at least between positions −1 and +3.
WARP is a new member of the von Willebrand factor A‐domain superfamily of extracellular matrix proteins
Authors: Fitzgerald Jamie , Tay Ting Su , Bateman John F.
Published: October 2002

We report a new member of the von illebrand factor A‐domain protein superfamily, WARP (for von illebrand factor ‐domain‐elated rotein). The full‐length mouse WARP cDNA is 2.3 kb in size and predicts a protein of 415 amino acids which contains a signal sequence, a VA‐like domain, two fibronectin type III‐like repeats, and a short proline‐ and arginine‐rich segment. WARP mRNA was expressed predominantly in chondrocytes and in vitro expression experiments in transfected 293 cells indicated that WARP is a secreted glycoprotein that forms disulphide‐bonded oligomers. We conclude that WARP is a new member of the von Willebrand factor A‐domain (VA‐domain) superfamily of extracellular matrix proteins which may play a role in cartilage structure and function.
Cold stress decreases the capacity for respiratory NADH oxidation in potato leaves
Authors: Svensson Å.Staffan , Johansson Fredrik I , Møller Ian M , Rasmusson Allan G
Published: October 2002

Cold stress effects on the expression of genes for respiratory chain enzymes were investigated in potato (Solanum tuberosum L., cv. Desiree) leaves. The nda1 and ndb1 genes, homologues to genes encoding the non‐proton‐pumping respiratory chain NADH dehydrogenases of Escherichia coli and yeast, were compared to genes encoding catalytic subunits of the proton‐pumping NADH dehydrogenase (complex I). Using a real‐time PCR system, we demonstrate a specific and gradual decrease of the NDA1 transcript after exposing the plants to 5°C. After 6 days of cold treatment the NDA1 transcript abundance is 10% of the original level. This decrease is accompanied by specific decreases of immunodetected NDA protein and internal rotenone‐insensitive NADH oxidation in mitochondria isolated from cold‐treated plants. The alternative oxidase is not cold‐induced neither at the protein nor at the activity level. The results are discussed in relation to the recent finding that the nda1 gene expression is completely light‐dependent.
Tumor necrosis factor α stimulates taurine uptake and transporter gene expression in human intestinal Caco‐2 cells
Authors: Mochizuki Tetsunosuke , Satsu Hideo , Shimizu Makoto
Published: October 2002

The effect of cytokines on the taurine uptake by human intestinal epithelial Caco‐2 cells was investigated. Among the various cytokines tested, tumor necrosis factor α (TNF‐α) markedly increased the taurine uptake by Caco‐2 cells, resulting in an increase in the intracellular taurine level. TNF‐α did not induce up‐regulation of the taurine uptake in hepatic HepG2, renal human embryo kidney 293, and macrophage‐like THP‐1 cells. The uptake of glycine,L‐leucine, andL‐glutamic acid by Caco‐2 cells was not affected by TNF‐α. A kinetic analysis of the taurine uptake by TNF‐α‐treated Caco‐2 cells suggests that this up‐regulation was associated with both an increase in the amount of the taurine transporter (TAUT) and an increase in its affinity. TNF‐α‐treated cells showed a higher mRNA level of the TAUT than did the control cells.