bluuebunny/crossref_metadata_2025 huggingface dataset:
Paxillin association in vitro with integrin cytoplasmic domain peptides
Authors: Tanaka Toshiki , Yamaguchi Ryuji , Sabe Hisataka , Sekiguchi Kiyotoshi , Healy Judith M.
Published: July 2002

Short cytoplasmic domains of integrin heterodimers are crucial for transduction of signals generated by adhesion of cells to the extracellular matrix. Here, we describe the use of peptides mimicking the intracellular tails of integrin α
5
β
1 to assay in vitro associations with cytoskeletal proteins. Our results suggest that the focal adhesion protein, paxillin, may interact directly with the intracellular region of the integrin β
1 subunit. Paxillin is known to form stable complexes with several signaling molecules, including focal adhesion kinase. Physical interaction between paxillin and the β
1 cytoplasmic domain suggests a model in which paxillin may function as a key intermediary in integrinmediated signal transduction.
Distinct roles of mouse laminin β1 long arm domains for α1β1γ1 trimer formation
Authors: Niimi Tomoaki , Kitagawa Yasuo
Published: July 2002

Mouse embryonal carcinoma F9 cells expressing partial mouse laminin β1 covering either the C‐terminal end (Δβ1S) or the whole (Δβ1L) of the long arm were established to study the assembly and interchain disulfide‐bonding of β1 to endogenous laminin α1 and γ1. Both Δβ1S and Δβ1L were disulfide‐bonded to γ1 but only Δβ1Lγ1 dimer formed a disulfide‐bonded α1Δβ1Lγ1 trimer which was actively secreted into the medium. Meanwhile, in the cells producing Δβ1Sγ1 dimer, the level of endogenous α1β1γ1 was reduced but the level of monomeric α1 was increased, suggesting that α1 was recruited to trimer formation with the Δβ1Sγ1 dimer without disulfide‐bonding. This shows that the Δβ1Sγ1 dimer can associate with α1 but not support the disulfide‐bonding at the N‐terminus of the long arm of α1. While control cells secrete neither monomeric α1 nor the β1γ1 dimer into the medium, the Δβ1Sγ1 producing cells probably do as α1Δβ1γ1 trimer. We thus propose that the N‐ and C‐termini of the long arm of laminin β1 have distinct roles for trimer formation.
Z,E isomerization of the α‐84 phycoviolobilin chromophore of phycoerythrocyanin from Mastigocladus laminosus investigated by Fourier‐transform infrared difference spectroscopy
Authors: Foerstendorf H , Parbel A , Scheer H , Siebert F
Published: July 2002

The photoreaction of the phycoviolobilin (PVB) chromophore‐containing part of phycoerythrocyanin (PEC) from Mastigocladus laminosus was investigated by Fourier‐transform infrared spectroscopy (FT‐IR). Difference spectra between the parent states P566 and P507 were obtained in 1H2O and 2H2O for the first time. The spectra are generally characterised by large changes in the range between 1710 and 1590 cm−1 and by a strong difference band around 1270 cm−1. In order to study the influence on the PVB chromophore upon aggregation, spectra of the α‐subunit and the (αβ)3 trimer are compared, showing distinct differences which may be of relevance for the chromophore‐protein and protein‐protein interactions. The difference spectra demonstrate many similarities to the spectra recently obtained for the Pr→meta‐Rc transition of phytochrome [Foerstendorf et al. (1996) Biochemistry 35, 10793–10799]. In particular, a band around 1710 cm−1, which was tentatively assigned to the C=O stretch of ring D is also observed in the spectra of PEC. It strongly supports this identification and the deduced molecular interpretation on the protonation state of the chromophore.
In vitro interaction of U2 snRNA with cytoplasmic 6S protein complexes
Authors: Fury Matthew G. , Andersen Janet
Published: July 2002

© 1997 Federation of European Biological Societies.
Mutagenesis of firefly luciferase shows that cysteine residues are not required for bioluminescence activity
Authors: Ohmiya Yoshihiro , Tsuji Frederick I.
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Fenofibrate differently alters expression of genes encoding ATP‐binding transporter proteins of the peroxisomal membrane
Authors: Albet Sabrina , Causeret Catherine , Bentejac Marc , Mandel Jean-Louis , Aubourg Patrick , Maurice Bugaut
Published: July 2002

The 70‐kDa peroxisomal membrane protein (PMP 70), adrenoleukodystrophy protein (ALDP) and adrenoleukodystrophy‐related protein (ALDRP) belong to the ATP‐binding transporter family, share a structure of half‐transporters and are localized in the peroxisomal membrane of mammals. It was suggested that these proteins may heterodimerize to form functional transporters. The expression of the three genes was examined in various tissues of control or fenofibrate (a peroxisome proliferator)‐treated rats using Northern and immuno‐blotting techniques. The patterns of tissue expression were distinct for the three genes. Upon treatment, expression of the ALD gene was not altered while that of the PMP 70 and ALDR genes was strongly increased in intestine and liver, respectively. The absence of coordinated expression excludes that the three transporters function as exclusive and obligatory partners. We also report for the first time that the ALDR gene is inducible in rodents and that the corresponding mRNA is different in length in rat (3.0 and 5.5 kb) and in mouse and human (4.2 kb).
Mechanical stability of compact modules of barnase
Authors: Takahashi Ken-ichi , Oohashi Michita , Noguti Tosiyuki , Gō Mitiko
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Characterization of a marsupial glutathione transferase, a class Alpha enzyme from Brown Antechinus (Antechinus stuartii)1
Authors: Bolton Robyn M , Curstedt Linda , Cederlund Ella , Hjelmqvist Lars , Mannervik Bengt , Ahokas Jorma T , Jörnvall Hans
Published: July 2002

The major form of glutathione transferase from the marsupial Antechinus stuartii has been purified and characterized as an Alpha class enzyme (Ast GST A1‐1) with distant sequence relationships to other class Alpha sublines, compatible with the early origin of marsupials. Amino acid replacements toward the closest enzyme characterized (chicken, form A3) involve no less than 79 positions (36%). At the active site, as deduced from comparisons with the known tertiary structure of the corresponding human enzyme, over half of the residues (8 of 15) ascribed to substrate binding interactions are exchanged although the general character of that site is conserved, while only 1 of 11 positions ascribed to interactions with GSH is exchanged. Class variability and species variability appear to coincide, with divergent segments centering around positions 33–49, 103–130 and 205–222. The pattern is reminiscent of that in similarly multiple MDR alcohol dehydrogenases. Both these enzyme families involved in cellular defense reactions have diverged considerably.
Partial purification and characterization of the DNA polymerase from the cyanelles of Cyanophora paradoxa
Authors: White Melissa A , Bailey II J.Clinton , Cannon Gordon C , Heinhorst Sabine
Published: July 2002

A DNA polymerase was partially purified and characterized from the photosynthetic organelles (cyanelles) of the protist, Cyanophora paradoxa. While cyanelles have several cyanobacterial features, such as a lysozyme‐sensitive cell wall, unstacked thylakoids and light harvesting phycobilisomes, their genome size and structure resemble those of chloroplasts, suggesting that cyanelles occupy a unique intermediate position between chloroplasts and their phylogenetic ancestors, the cyanobacteria. When comparing the biochemical characteristics of the cyanelle DNA polymerase to those of its counterparts from higher plant chloroplasts and from a cyanobacterium, it is clear that the cyanelle enzyme resembles chloroplast DNA polymerases which are eukaryotic γ‐type enzymes.
The active site region of the vitamin K‐dependent carboxylase includes both the amino‐terminal hydrophobic and carboxy‐terminal hydrophilic domains of the protein
Authors: Maillet Magali , Morris Daniel , Gaudry Michel , Marquet André
Published: July 2002

In order to localize the active site of the vitamin K‐dependent carboxylase, we developed an affinity probe containing the propeptide and the first two carboxylatable glutamate residues conserved in many native substrates. This probe crosslinked to both the hydrophobic amino‐terminal and hydrophilic carboxy‐terminal domains of the carboxylase, in contrast with previous work which localized both the catalytic and the propeptide binding site within the amino‐terminal hydrophobic domain. Amino acid analysis revealed that the mass of an amino‐terminal fragment is seriously underestimated by SDS‐PAGE. Reanalysis of the published data in light of this information suggests that a portion of the propeptide binding site resides within the carboxy‐terminal hydrophilic domain.
Mutagenesis of residues 27 and 78 modulates heme orientation in cytochrome b5
Authors: Mortuza Gulnahar B , Whitford David
Published: July 2002

A comparison of the primary sequences of the heme binding domains of bovine and rat microsomal cytochrome reveal differences at only six residues. These residues must therefore provide the origin for the observed variation in the ratio of the heme orientational isomers, the equilibrium constant of which ranges from ∼9 in the bovine protein to ∼1.6 for rat cytochrome b
5. Residues 7, 20, 21, and 30 are distant from the exposed heme edge whilst Leu27 and Phe78 are located close to different parts of the porphyrin macrocycle. 1H NMR spectra of the heme and heme ligand resonances of a recombinant tobacco cytochrome b
5 extending from Gly1 to Lys89 suggest, in combination with NMR data acquired for other forms of cytochrome b
5 and an inspection of their sequence homology, that the identity of residue 78 influences the relative ratios of heme isomers. The Gly1‐Lys89 domain of tobacco cytochrome b
5 has two equally abundant heme orientational isomers but retains the leucine side chain at position 27 whilst phenylalanine 78 is replaced by tyrosine. A more direct role for residue 78 in modulating the heme ratio is shown by site directed mutagenesis of bovine microsomal cytochrome b
5 where the mutation Phe78>Tyr shifts the equilibrium constant for the heme orientational isomers from 9 to 3.5. Whilst the ratio is clearly shifted towards that exhibited by the rat protein the incomplete transition suggested the involvement of other residues. The mutation of Leu27>Val was shown to result in a slightly smaller change in ratios of each isomer (from 9 to 4.0). Together these results point to the importance of these residues in modulating the ratio of heme isomers.
Differential resistance to proteinase K digestion of the yeast prion‐like (Ure2p) protein synthesized in vitro in wheat germ extract and rabbit reticulocyte lysate cell‐free translation systems
Authors: Komar Anton A. , Lesnik Thierry , Cullin Christophe , Guillemet Elisabeth , Ehrlich Ricardo , Reiss Claude
Published: July 2002

The Ure2p yeast prion‐like protein was translated in vitro in the presence of labeled [35S]methionine in either rabbit reticulocyte lysate (RRL) or wheat germ extract (WGE) cell‐free systems. When subjected to proteinase K digestion, the Ure2p protein synthesized in WGE was proteolysed much more slowly compared to that synthesized in RRL; this displays fragments of about 31–34 kDa, persisting over 8 min. Thus, the digestion rate and pattern of the protein synthesized in WGE, unlike that synthesized in RRL, revealed characteristic features of the [URE3] prion‐like isoform of the Ure2p protein [Masison, D.C. and Wickner, R.B. (1995) Science 270, 93–95]. Chloramphenicol acetyltransferase, synthesized under the same conditions, differed fundamentally in its proteolytic sensitivity toward proteinase K (PK); in the RRL system it was more slowly digested than in WGE, proving specific PK inhibitors to be absent in both systems. Posttranslational addition of the WGE to the RRL‐synthesized Ure2p does not protect Ure2p from efficient PK degradation either. The differences in Ure2p degradation may be ascribed to a specific structure or specific states of association of Ure2p synthesized in WGE; obviously, they yield a protein that mimics the behavior of the Ure2p in [URE3] yeast strains. The present data suggest that particular conditions of the Ure2p protein translation and/or certain cellular components (accessory proteins and extrinsic factors), as well as the nature of the translation process itself, could affect the intracellular folding pathway of Ure2p leading to the de novo formation of the prion [URE3] isoform.
Disturbed progastrin processing in carboxypeptidase E‐deficient fat mice
Authors: Lacourse Karen A , Friis-Hansen Lennart , Rehfeld Jens F , Samuelson Linda C
Published: July 2002

The fat mouse strain exhibits a late‐onset obesity syndrome associated with a mutation in the gene encoding carboxypeptidase E (CPE). Since CPE plays a central role in the biosynthesis of a number of regulatory peptides, including gastrin, we examined the biogenesis and processing of progastrin in fat/fat mice by measuring gastrin mRNA, carboxyamidated gastrin and its processing intermediates in the stomach. The tissue concentration of carboxyamidated (i.e. bioactive) gastrin was only slightly reduced (601±28 pmol/g in fat/fat mice vs. 715±43 pmol/g in wild‐type controls). However, progastrin processing intermediates accumulated excessively with an 86‐fold increase in the concentration of the CPE substrate, glycyl‐arginine extended gastrin, and a seven‐fold increase in the concentration of glycine‐extended gastrin. Accordingly, the total progastrin product was doubled, as was the concentration of gastrin mRNA. Plasma concentrations of carboxyamidated gastrin were, however slightly reduced both in fasted fat/fat mice and postprandially. The results show that the CPE mutation diminishes the efficiency of progastrin processing, but gastrin synthesis is nevertheless increased to maintain an almost normal production of bioactive gastrins. By comparison with other neuroendocrine prohormones, progastrin processing in CPE‐deficient mice is unique. Hence, the increase of glycine‐extended gastrin in combination with normal levels of carboxyamidated gastrin suggests that G‐cells may have another biosynthetic pathway for gastrin.
Characterization of nuclear tRNATyr introns: their evolution from red algae to higher plants
Authors: Akama Kazuhito , Naß Armin , Junker Volker , Beier Hildburg
Published: July 2002

We have previously isolated numerous intron‐containing nuclear tRNATyr genes derived from either monocotyledonous (Triticum) or dicotyledonous (Arabidopsis, Nicotiana) plants by screening the corresponding genomic phage libraries with a synthetic tRNATyr‐specific oligonucleotide. Here we have characterized additional tRNATyr genes from phylogenetically divergent plant species representing red algae (Champia), brown algae (Cystophyllum), green algae (Ulva), stonewort (Chara), liverwort (Marchantia), moss (Polytrichum), fern (Rumohra) and gymnosperms (Ginkgo) using amplification of the coding sequences from the corresponding genomic DNAs by polymerase chain reaction (PCR). All novel tRNATyr genes contain intervening sequences of variable sequence and length ranging in size from 11 to 21 bp. However, two features are conserved in all plant pre‐tRNATyr introns: they possess a uridine and less frequently an adenosine at the 5′ boundary and can adopt similar intron secondary structures in which an extended anticodon helix of 4–5 bp is formed by base‐pairing between nucleotides of the intron and the anticodon loop. In order to elucidate the potential role of the highly conserved uridine at the first intron position, we have replaced it by all other nucleosides in an Arabidopsis pre‐tRNATyr and have studied in wheat germ extract its effect on splicing and on conversion of U to Ψ in the GΨA anticodon. Furthermore, we discuss the putative acquisition of tRNATyr introns at an early step of evolution after the separation of Archaea and Eucarya.
Chemical shift dispersion and secondary structure prediction in unfolded and partly folded proteins
Authors: Yao Jian , Dyson H.Jane , Wright Peter E
Published: July 2002

The intrinsic chemical shift dispersion for 15N, 1HN, 13Cα, 1Hα, 13Cβ and 13CO resonances has been evaluated utilizing complete resonance assignment data for unfolded apomyoglobin, together with two other unfolded and five folded proteins, obtained from the literature. The dispersion of 13Cα, 1Hα, and 13Cβ resonances for the unfolded proteins is poor, whereas the dispersion of 15N, 1HN and 13CO is much greater, reflecting the sensitivity of these nuclei to the nature of the neighboring amino acid in the primary sequence. By contrast, the dispersion of the 13Cα, 1Hα, and 13Cβ nuclei are much greater in the folded proteins, reflecting the well‐known dependence of the environments of these nuclei on secondary and tertiary structure. These differences in chemical shift dispersion dictate differences in strategies for resonance assignment in unfolded proteins compared with those most commonly used for folded proteins. Strategies utilizing the superior chemical shift dispersion of the 15N, 1HN and, in particular, the 13CO nuclei, are indicated for use with unfolded or partially folded proteins.
The carboxyl‐terminal domain of insulin‐like growth factor‐I receptor interacts with the insulin receptor and activates its protein tyrosine kinase
Authors: Li Shu-lian , Termini John , Hayward Amanda , Siddle Ken , Zick Yehiel , Koval Anatolii , LeRoith Derek , Fujita-Yamaguchi Yoko
Published: July 2002

Receptors for insulin and insulin‐like growth factor‐I (IR and IGFIR) consisting of the α2β2 structure are protein tyrosine kinases (PTKs). Carboxyl‐terminal (CT) domains of their β subunits are structurally diverse while the PTK domains share the highest homology. Interactions between CT and PTK domains of IR and IGFIR were studied by means of PTK activity, fluorescence energy transfer or surface plasmon resonance using BIAcore. We present evidence that IGFIR CT directly interacts with both IGFIR and IR. Although binding to both receptors, stimulation of PTK activity only occurs with IR but not IGFIR.
Oct‐1 promoter region contains octamer sites and TAAT motifs recognized by Oct proteins
Authors: Pankratova E.V , Polanovasky O.L
Published: July 2002

The 5′‐upstream region (1.3 kb) of the gene encoding the POU domain transcription factor Oct‐1 was cloned and sequenced. CAT reporter gene analysis of this region has detected a functionally active promoter. This region contains 24 TAAT‐core sites, arranged in five clusters (four to six sites in one cluster); two octamer sites (ATGCAAAT) are located in the first and second clusters; in the second one the CCAAT‐box adjacent to the octamer overlaps with the TAAT‐core site. As shown by gel retardation assay, Oct‐1, Oct‐2, and some unknown proteins from myeloma cell line NS/0 interact with the TAAT‐core sites of these clusters. The results suggest autoregulation of Oct‐1 gene expression that may also be controlled by other POU proteins, homeodomain proteins and CCAAT trans‐action factors.
Three‐stepped rotation of subunits γ and ϵ in single molecules of F‐ATPase as revealed by polarized, confocal fluorometry
Authors: Häsler Katrin , Engelbrecht Siegfried , Junge Wolfgang
Published: July 2002

The proton translocating ATP synthase is conceived as a rotatory molecular engine. ATP hydrolysis by its headpiece, CF1, drives the rotation of subunit γ relative to the hexagonally arranged large subunits, (αβ)3. We investigated transition states of the rotatory drive by polarized confocal fluorometry (POCOF) as applied to single molecules of engineered, immobilized and load‐free spinach‐CF1. We found that the hydrolysis of ATP caused the stepped and sequential progression of subunit γ through three discrete angular positions, with the transition states of γ being too shortlived for detection. We also observed the stepped motion of ϵ, whereas δ was immobile as (αβ)3.
Requirement of membrane‐proximal amino acids in the carboxyl‐terminal tail for expression of the rat AT1a angiotensin receptor
Authors: Gáborik Zsuzsanna , Mihalik Balázs , Jayadev Suman , Jagadeesh Gowraganahalli , Catt Kevin J. , Hunyady László
Published: July 2002

A series of deletion mutants was created to analyze the function of the membrane‐proximal region of the cytoplasmic tail of the rat type 1a (AT1a) angiotensin receptor. In transiently transfected COS‐7 cells, the truncated mutant receptors showed a progressive decrease in surface expression, with no major change in binding affinity for the peptide antagonist, [Sar1,Ile8]angiotensin II. In parallel with the decrease in receptor expression, a progressive decrease in angiotensin II‐induced inositol phosphate responses was observed. Alanine substitutions in the region 307–311 identified the highly conserved phenylalanine309 and adjacent lysine residues as significant determinants of AT1a receptor expression.
The mating factor response pathway regulates transcription of TEC1, a gene involved in pseudohyphal differentiation of Saccharomyces cerevisiae
Authors: Oehlen L , Cross F.R
Published: July 2002

The transcription factor Tec1 is involved in pseudohyphal differentiation and agar‐invasive growth of Saccharomyces cerevisiae cells. The sole element in the TEC1 promoter that has thus far been shown to control Tec1 function is the filament response element. We find that the TEC1 promoter also contains several pheromone response element sequences which are likely to be functional: TEC1 transcription is induced by mating factor, cell cycle regulated and dependent on the Ste4, Ste18 and Ste5 components of the mating factor signal transduction pathway. Using alleles of the transcription factor Ste12 that are defective in DNA binding, transcriptional induction or cooperativity with other transcription factors, we find little correlation between TEC1 transcript levels and agar‐invasive growth.
Biochemical and functional analysis of highly phosphorylated forms of c‐Jun protein
Authors: Ui Motoyasu , Sonobe Martha H. , Ito Taiji , Murakami Masao , Okazaki Satoshi , Takada Mizuho , Sato Toshitaka , Iba Hideo
Published: July 2002

We report here that, upon UV irradiation or growth stimulation, endogenous c‐Jun (40 kDa) in chicken embryo fibroblasts (CEF) is converted into several forms with apparently higher molecular weights in SDS‐polyacrylamide gel electrophoresis (45, 44, 42 kDa). Two of the bands (44 and 45 kDa) were transient after growth stimulation, but were much more persistent after UV irradiation. In both cases, the drastic mobility shifts were accompanied with the activation of endogenous JNK activity but not of MAPK activity, and the bands were shown to represent different phosphorylation states of c‐Jun rather than ubiquitinated c‐Jun. Biochemical analysis indicated that phosphorylation at Ser63 and Ser73 was not sufficient to produce these drastic mobility shifts, which additionally required phosphorylation at Thr91 and Thr93. Substitution of both Ser63 and Ser73 with either Ala or Asp had no significant effect on the transforming activity of c‐Jun, but the mutants failed to show drastic mobility shifts even after UV irradiation. These results indicate that Ser63 and Ser73 are essential for the drastic mobility shifts and further suggest that the highly phosphorylated forms of c‐Jun are not directly involved in cellular transformation.
Modification of tissue factor by peroxynitrite influences its procoagulant activity
Authors: Adam J.M. , Ettelaie C. , Naseem K.M. , James N.J. , Bradley N.J. , Bruckdorfer K.R.
Published: July 2002

Peroxynitrite, a reactive oxidising species resulting from a reaction between nitric oxide and the superoxide anion, modifies proteins by nitration of certain amino acids such as tyrosine. Tissue factor (TF), a transmembrane protein, is expressed on cells under inflammatory conditions and initiates the coagulation cascade. The extracellular domain of TF is rich in tyrosine. Exposure of recombinant TF and cellular TF to peroxynitrite was associated with a reduction in procoagulant activity. This was accompanied by an elevated level of nitrotyrosine residues. Peroxynitrite may have a protective role by attenuation of the thrombotic properties of TF.
The multidrug resistance modulator SDZ PSC 833 is a potent activator of cellular ceramide formation
Authors: Cabot Myles C , Han Tie-Yan , Giuliano Armando E
Published: July 2002

In this study we demonstrate that the multidrug resistance (MDR) modulator PSC 833 is a potent agonist of ceramide metabolism. When added with [3H]serine or [3H]palmitic acid to the culture medium of MCF‐7 cells, PSC 833, in a dose‐responsive fashion (1–10 μM), increased the levels of [3H]ceramide as much as 16‐fold over control. The actual increase in ceramide mass was verified by thin‐layer chromatographic chars. Cellular sphingomyelin radioactivity did not decrease during treatment, indicating that PSC 833 does not elicit ceramide formation through a sphingomyelinase pathway. Inclusion of fumonisin B1, an inhibitor of ceramide synthase, blocked formation of ceramide by PSC 833. The results of cell proliferation assays demonstrated a clear correlation between PSC 833 elicitation of ceramide formation and increased cytotoxicity. The MDR modulator and chemical cousin of PSC 833, cyclosporin A, had little impact on cellular ceramide formation. At a concentration of 2.5 μM, cyclosporin A and PSC 833 treatment increased ceramide formation by 20% and 7.5‐fold, respectively. These results reveal a new action of PSC 833 which may contribute to its potency as a drug resistance modulator.
Mitochondrial formation of hydrogen peroxide is causally linked to the antimycin A‐mediated prevention of tert‐butylhydroperoxide‐induced U937 cell death
Authors: Brambilla Liliana , Cantoni Orazio
Published: July 2002

Antimycin A and 2‐heptyl‐4‐hydroxyquinoline N‐oxide (HQNO), both of which bind to the same site of complex III, prevented U937 cell killing promoted by tert‐butylhydroperoxide (tB‐OOH). This cytoprotection was not directly caused by inhibition of electron transport or reduced formation of tB‐OOH‐derived toxic species, but rather appeared to be the consequence of a mechanism involving mitochondrial formation of hydrogen peroxide. Ubisemiquinone was most likely the electron donor allowing the formation of superoxides and, as a consequence, of hydrogen peroxide.
Membrane fusion plays an important role in gene transfection mediated by cationic liposomes
Authors: Noguchi Ari , Furuno Tadahide , Kawaura Chiyo , Nakanishi Mamoru
Published: July 2002

By confocal laser scanning microscopy (CLSM) we have studied the membrane fusion between cationic liposomes and the endosome membranes involved in gene transfection mediated by cationic liposomes. Antisense oligonucleotides were transferred by cationic liposomes with a cationic cholesterol derivative, cholesteryl‐3β‐carboxyamidoethylenedimethylamine (I). Cationic liposomes were made by a mixture of the derivative I and DOPE. The intracellular distribution of fluorescein‐conjugated antisense oligonucleotides (phosphorothioate) was studied by CLSM. The images showed that the antisense oligonucleotides were preferentially transferred into the nucleus of target cells (NIH3T3, COS‐7 and HeLa cells) by the liposomes with derivative I. However, their transfection was completely blocked by nigericin which was able to dissipate the pH gradient across the endosome membranes, although the liposome/DNA complex was found in the cytoplasm of the target cells. This was quite in contrast with the fluorescence images of the target cells treated with wortmannin, an inhibitor of endocytosis. The results suggest that at least two steps are effective for gene transfection mediated by the cationic liposomes with cationic cholesterol derivatives. One is the endocytosis of the liposome/DNA complex into the target cells and the other is the removal of antisense oligonucleotides (plasmid DNAs) from the complex in the endosomes. The latter step was preferentially preceded by the membrane fusion between the cationic liposomes and the endosome membranes at around pH 5.0.
Incorporation of monoclonal antibodies in living rat pheochromocytoma PC12 cells
Authors: Alexandrova N.A , Sveshnikov P.G , Nagradova N.K , Grozdova I.D
Published: July 2002

PC12 cells permeabilized with a low concentration of digitonin (5 μM) under controlled conditions were loaded with monoclonal antibodies (MoAb) against the regulatory subunit type II (RII) of cAMP‐dependent protein kinase. After digitonin removal from the nutrient medium (DMEM) the loaded cells repaired within 20–30 min and recontinued growth. The inserted MoAb stayed in the repaired cells at least for several hours. MoAb inhibiting the cAMP binding activity of neural RII [Grozdova et al. (1992) Biochem. Int. 27, 811–822; Sveshnikova et al. (1996) Biochem. Int. 39, 1063–1070] were shown to bind the target antigen inside the cells and influence the properties of intracellular protein kinases.
DNA binding of NF‐Y: the effect of HMGI proteins depends upon the CCAAT box
Authors: Liberati Chiara , Sgarra Riccardo , Manfioletti Guidalberto , Mantovani Roberto
Published: July 2002

NF‐Y is a conserved sequence‐specific transcription factor binding to CCAAT boxes. The chromatin‐associated HMGI proteins influence promoter activities through positive and negative effects on binding of transcription factors. It was previously shown that HMGI(Y) synergizes the binding of NF‐Y to the α2‐collagen CCAAT box [Currie, R.A. (1997) J. Biol Chem. 272, 30880–30888]. Using recombinant proteins, we confirm that at low concentrations of NF‐Y, HMGI(Y) acts synergistically on the α2‐collagen CCAAT and we extend this observation to HMGI and HMGI‐C. However, enhancement of DNA binding to γ‐globin, α‐globin and MHC class II Ea CCAAT boxes was not observed. At high concentrations, HMGI proteins inhibit binding to α2‐collagen and to γ‐globin, but not to high affinity Ea or α‐globin CCAAT. In none of our experiments did we see a ternary complex between NF‐Y, HMGI(Y) and DNA. In protein competition experiments, NF‐Y affinity was at least two orders of magnitude higher, even in the context of the suboptimal γ‐globin CCAAT. Our data prove that HMGI proteins have complex positive and negative effects on NF binding to some, but not to all CCAAT boxes, suggesting that this phenomenon is dictated by the sequences flanking the pentanucleotide rather than direct protein‐protein interactions.
τ is phosphorylated by GSK‐3 at several sites found in Alzheimer disease and its biological activity markedly inhibited only after it is prephosphorylated by A‐kinase
Authors: Wang Jian-zhi , Wu Qiongli , Smith Alan , Grundke-Iqbal Inge , Iqbal Khalid
Published: July 2002

Alzheimer disease is characterized by a specific type of neuronal degeneration in which the microtubule associated protein τ is abnormally hyperphosphorylated causing the disruption of the microtubule network. We have found that the phosphorylation of human τ (τ3L) by A‐kinase, GSK‐3 or CK‐1 inhibits its microtubule assembly‐promoting and microtubule‐binding activities. However, the inhibition of these activities of τ by GSK‐3 is significantly increased if τ is prephosphorylated by A‐kinase or CK‐1. The most potent inhibition is observed by combination phosphorylation of τ with A‐kinase and GSK‐3. Under these conditions, only very few microtubules are seen by electron microscopy. Sequencing of 32P‐labeled trypsin phosphopeptides from τ prephosphorylated by A‐kinase (using unlabeled ATP) and further phosphorylated by GSK‐3 in the presence of [γ‐32P]ATP revealed that Ser‐195, Ser‐198, Ser‐199, Ser‐202, Thr‐205, Thr‐231, Ser‐235, Ser‐262, Ser‐356 and Ser‐404 are phosphorylated, whereas if τ is not prephosphorylated by A‐kinase, GSK‐3 phosphorylates it at Thr‐181, Ser‐184, Ser‐262, Ser‐356 and Ser‐400. These data suggest that (i) prephosphorylation of τ by A‐kinase makes additional and different sites accessible for phosphorylation by GSK‐3; (ii) phosphorylation of τ at these additional sites further inhibits the biological activity of τ in its ability to bind to microtubules and promote microtubule assembly. Thus a combined role of A‐kinase and GSK‐3 should be considered in Alzheimer neurofibrillary degeneration.
HIV/gp120 and PMA/ionomycin induced apoptosis but not activation induced cell death require PKC for Fas‐L upregulation
Authors: Accornero Paola , Radrizzani Marina , Carè Alessandra , Mattia Gianfranco , Chiodoni Claudia , Kurrle Roland , Colombo Mario P
Published: July 2002

HIV protein gp120 in combination with T cell antigen receptor (TCR) triggering induces apoptosis (gp120‐apoptosis) in Th1 cells. Gp120‐apoptosis occurs by induction of Fas‐L and subsequent triggering of the Fas apoptotic pathway. Here, through the use of several compounds inhibiting induction of Fas‐L, we show that, in a Th1 clone, a protein kinase C (PKC) independent pathway activated by TCR stimulation is distinguishible from a PKC dependent pathway activated by either phorbol 12‐myristate 13‐acetate (PMA)/ionomycin or asynchronous stimulation of TCR and CD4 as occurs in gp120‐apoptosis.
Binding of α‐tocopherylquinone, an oxidized form of α‐tocopherol, to glutathione‐S‐transferase in the liver cytosol
Authors: Arita Makoto , Sato Yuji , Arai Hiroyuki , Inoue Keizo
Published: July 2002

α‐Tocopherol (vitamin E) is an important fat‐soluble antioxidant in biological systems and, as a result of scavenging reactive oxygen, it is converted to α‐tocopherylquinone. α‐Tocopherol binds to α‐tocopherol transfer protein (αTTP) in the liver cytosol, whereas α‐tocopherylquinone does not. We found that α‐tocopherylquinone binds to a liver protein with a molecular mass of about 40 kDa that is distinct from αTTP. This α‐tocopherylquinone binding protein was purified further by multiple‐step column chromatography. Sodium dodecylsulfate‐polyacrylamide gel electrophoresis of the final preparation yielded a single band with an apparent molecular mass of 25 kDa, which microsequencing revealed was identical to glutathione‐S‐transferase (GST). The GST activity was inhibited in the presence of α‐tocopherylquinone, as it is by other non‐substrate ligands for GST, confirming that GST and α‐tocopherylquinone interact directly. α‐Tocopherylquinone binds to GST and may be transported to the site of metabolism or excreted in the bile as other non‐substrate ligands for GST.
GATA‐3 represses gp91phox gene expression in eosinophil‐committed HL‐60‐C15 cells
Authors: Anowar Sadat Mohammed , Kumatori Atsushi , Suzuki Shoichi , Yamaguchi Yuji , Tsuji Yoshiro , Nakamura Michio
Published: July 2002

To study the regulatory mechanism of gp91phox gene expression in eosinophils, we transiently transfected eosinophil‐committed HL‐60‐C15 cells with gp91phox promoter constructs, and identified a negative element from bp −267 to −246 of the gp91phox gene, the deletion of which caused an 83% increase in promoter activity. Electrophoresis mobility shift assays demonstrated GATA‐3 binds to the GATA consensus site from bp −256 to −250. An 81% increment in promoter activity was obtained when a mutation was introduced in the GATA‐3 binding site of the bp −267 to +12 construct, which is comparable to that of the bp −245 to +12 construct. We therefore conclude that GATA‐3 specifically binding to the GATA site negatively regulates the expression of the gene in HL‐60‐C15 cells.
The Pur10 protein encoded in the gene cluster for puromycin biosynthesis of Streptomyces alboniger is an NAD‐dependent ATP dehydrogenase
Authors: Rubio Miguel Angel , Espinosa Juan Carlos , Tercero José Antonio , Jiménez Antonio
Published: July 2002

The pur10 gene of the puromycin (pur) cluster of Streptomyces alboniger is essential for the biosynthesis of this antibiotic. Highly purified Pur10 protein, obtained in Escherichia coli as a recombinant protein fused to a histidine tail, had an NAD‐dependent ATP dehydrogenase activity. The K
m and V
max values for ATP were 0.49 mM and 14.5 nmol/min and for NAD 0.53 mM and 15.2 nmol/min, respectively. The ATP‐derived product of the reaction apparently decomposed producing a triphosphorylated compound plus an adenine derivative. These and previous results suggested that Pur10 carries out the first step of the puromycin biosynthetic pathway, namely, conversion of ATP into 3′‐keto‐3′‐deoxyATP.
BCL‐2 promotes migration and invasiveness of human glioma cells
Authors: Wick Wolfgang , Wagner Sven , Kerkau Siglinde , Dichgans Johannes , Tonn Jörg C , Weller Michael
Published: July 2002

Malignant progression in gliomas is correlated with increased migratory capacity which involves metalloproteolytic activity. Here, we report that ectopic expression of BCL‐2 in two malignant glioma sublines markedly promoted glioma cell migration from spheroids and invasion into Matrigel‐coated membranes. Invasion of fetal rat‐brain aggregates was enhanced by BCL‐2. Zymography revealed activation of matrix metalloproteinase‐2 (MMP‐2) in BCL‐2‐expressing cells. BCL‐2 expressing cells showed an increase in MMP‐2/‐3/‐12 (LN‐18), and MMP‐9/‐12 and cell surface urokinase‐type plasminogen activator (u‐PA) (LN‐229) mRNA and a reduction in tissue inhibitors of metalloproteinases (TIMP)‐2 mRNA (LN‐229). Taken together, we propose a novel function for BCL‐2 in the malignant phenotype of glioma cells, that is, to enhance migration and invasion by altering the expression of a set of metalloproteinases and their inhibitors.
The C2 cytosolic loop of adenylyl cyclase interacts with the activated form of Gαs
Authors: Torrejón Marcela , Genevière Anne-Marie , Echeverrı́a Valentina , Guzmán Leonardo , Hinrichs Marı́a Victoria , Olate Juan
Published: July 2002

Using the yeast two‐hybrid system, we studied the physical interaction between the complete C1 and C2 cytosolic domains of Xenopus laevis type 9 (xl9C1, xl9C2) and the C2 domain of rat type 6 (r6C2) adenylyl cyclase (AC). Heterodimerization between xl9C1 and xl9C2 and homodimerization between C2 (but not C1) domains was observed. Interaction between C2 and human Gαs (hGαs) was also detected and was dependent on Gαs activation. In contrast X. laevis Gαs (xlGαs), which is 92% identical to hGαs, was unable to interact with any of the three AC cytosolic domains tested, corroborating previous findings that showed no effector activation. Through the construction of chimeras, we demonstrated that the amino‐terminal half of xlGαs was responsible for the lack of interaction with AC. Chimeras between mouse Gαi2 and Gαs (N‐mGαi2/C‐Gαs), that have previously shown to activate AC to a higher extent than wild‐type Gαs, also interacted with the C2 cytosolic domain and with a higher affinity. Interestingly, N‐mGαi2/C‐xlGαs chimera was not only able to interact with C2 but also with the C1 cytosolic domain.
Multiple regulatory elements control the expression of the yeast ACR1 gene
Authors: Redruello Begoña , Valdes Eva , Luz Lopez M. , Rodicio Rosaura
Published: July 2002

The ACR1 gene, encoding a succinate‐fumarate transporter, is required by the yeast Saccharomyces cerevisiae for ethanol utilization. Accordingly, gene expression is induced by ethanol and repressed by glucose. Here, we investigated three carbon source response elements present in its promoter region. Specific deletions as well as functional analysis of the elements in a heterologous promoter confirmed their role in transcriptional regulation. Protein binding to carbon source response elements of the ICL1 promoter was competed by all three elements to various extents by the respective ACR1 sequences. In addition, two putative stress response promoter elements present in the ACR1 promoter were investigated in deletion analyses and shown to contribute to gene expression.
A pseudoknot‐compatible universal site is located in the large ribosomal RNA in the peptidyltransferase center
Authors: Ivanov V.I , Bondarenko S.A , Zdobnov E.M , Beniaminov A.D , Minyat E.E , Ulyanov N.B
Published: July 2002

The RNA secondary structure is not confined to a system of the hairpins and can contain pseudoknots as well as topologically equivalent slipped‐loop structure (SLS) conformations. A specific primary structure that directs folding to the pseudoknot or SLS is called SL‐palindrome (SLP). Using a computer program for searching the SLP in the genomic sequences, 419 primary structures of large ribosomal RNAs from different kingdoms (prokaryota, eukaryota, archaebacteria) as well as plastids and mitochondria were analyzed. A universal site was found in the peptidyltransferase center (PTC) capable of folding to a pseudoknot of 48 nucleotides in length. Phylogenetic conservation of its helices (concurrent replacements with no violation of base pairing, covariation) has been demonstrated. We suggest the reversible folding‐unfolding of the pseudoknot for certain stages of the ribosome functioning.
Gender modulates the energy cost of muscle contraction in untrained healthy subjects. A 31P magnetic resonance spectroscopy analysis
Authors: Mattei Jean P , Bendahan David , Roussel Magali , Lefur Yann , Cozzone Patrick J
Published: July 2002

The forearm flexor muscles of 56 untrained volunteers (26 women and 30 men) were examined by 31P magnetic resonance spectroscopy, during a rest‐exercise‐recovery protocol, in order to document the impact of gender on muscle energetics. Absolute concentrations of high‐energy phosphate compounds, intracellular pH and rates of aerobic and anaerobic ATP production were calculated. An inverse correlation was found between body mass index (BMI) and power output in women but not in men. After correcting for power output and BMI, the measured energy cost of contraction was twice larger for women than for men. This increase was also reflected in larger ATP production from aerobic and anaerobic pathways. This higher energy cost might be explained in part by differences in local muscle mass, a higher impact of fatness, but also by a reduced metabolic efficiency of muscle fibers in untrained women.
Mutation patterns for two flaviviruses: hepatitis C virus and GB virus C/hepatitis G virus
Authors: Mizokami Masashi , Imanishi Tadashi , Ikeo Kazuho , Suzuki Yoshiyuki , Orito Etsuro , Kumada Takashi , Ueda Ryuzo , Iino Shiro , Nakano Tatsunori
Published: July 2002

We studied the mutation patterns of hepatitis C virus (HCV) and GB virus C/hepatitis G virus (HGV). Although the mutation patterns of the two viruses were similar to each other, they were quite different from that of HIV. In particular, the similarity of the patterns between HCV or HGV and human nuclear pseudogenes was statistically significant whereas there was no similarity between HIV and human nuclear pseudogenes. This finding suggests that the mutation patterns of HCV and HGV are similar to the patterns of spontaneous substitution mutations of human genes, implying that nucleotide analogues which are effective against HCV and HGV may have a side effect on the normal cells of humans.
N‐terminal domain, residues 1–91, of ribosomal protein TL5 from Thermus thermophilus binds specifically and strongly to the region of 5S rRNA containing loop E
Authors: Gongadze George M. , Meshcheryakov Vladimir A. , Serganov Alexander A. , Fomenkova Natalia P. , Mudrik Elena S. , Jonsson Bengt-Harald , Liljas Anders , Nikonov Stanislav V. , Garber Maria B.
Published: July 2002

In this work we show for the first time that the overproduced N‐terminal fragment (residues 1–91) of ribosomal protein TL5 binds specifically to 5S rRNA and that the region of this fragment containing residues 80–91 is a necessity for its RNA‐binding activity. The fragment of Escherichia coli 5S rRNA protected by TL5 against RNase A hydrolysis was isolated and sequenced. This 39 nucleotides fragment contains loop E and helices IV and V of 5S rRNA. The isolated RNA fragment forms stable complexes with TL5 and its N‐terminal domain. Crystals of TL5 in complex with the RNA fragment diffracting to 2.75 Å resolution were obtained.
Probing the reactivity of nucleophile residues in human 2,3‐diphosphoglycerate/deoxy‐hemoglobin complex by aspecific chemical modifications
Authors: Scaloni Andrea , Ferranti Pasquale , De Simone Giuseppina , Mamone Gianfranco , Sannolo Nicola , Malorni Antonio
Published: July 2002

The use of aspecific methylation reaction in combination with MS procedures has been employed for the characterization of the nucleophilic residues present on the molecular surface of the human 2,3‐diphosphoglycerate/deoxy‐hemoglobin complex. In particular, direct molecular weight determinations by ESMS allowed to control the reaction conditions, limiting the number of methyl groups introduced in the modified globin chains. A combined LCESMS‐Edman degradation approach for the analysis of the tryptic peptide mixtures yielded to the exact identification of methylation sites together with the quantitative estimation of their degree of modification. The reactivities observed were directly correlated with the pK
a and the relative surface accessibility of the nucleophilic residues, calculated from the X‐ray crystallographic structure of the protein. The results here described indicate that this methodology can be efficiently used in aspecific modification experiments directed to the molecular characterization of the surface topology in proteins and protein complexes.
Transcriptional regulation of leptin gene promoter in rat
Authors: Fukuda Hitomi , Iritani Nobuko
Published: July 2002

To investigate the DNA regulatory sequences required for stimulation and suppression of leptin gene expression, primary cultured hepatocytes and adipocytes of rats were transfected with plasmids containing the 5′‐flanking sequences of the rat leptin gene fused to the luciferase gene. When two copies of the sequences spanning nucleotides −101 to −83 of the leptin promoter were used for transfection, the reporter activity significantly increased in the presence of glucose/insulin in comparison with glucose alone. The glucose/insulin stimulation of the transcription was inhibited by addition of polyunsaturated fatty acids. These results were similar to those found earlier for the transcription of the fatty acid synthase, FAS(−57/−35) and ATP citrate‐lyase, ACL(−64/−41) genes. Cotransfection studies in the cells with a Sp1 expression vector and leptin (−101/−83) constructs showed the inactivation of the leptin promoter by Sp1. Gel mobility shift assays using an end‐labeled leptin(−101/−83) construct as a probe revealed that nuclear factor(s) from rat liver or adipose tissue specifically formed complexes with the sequence. The DNA‐protein complexes were common to the glucose/insulin‐responsive regions of the leptin, ACL and FAS genes, suggesting that these genes are coordinately regulated. In addition, by antibody supershift assays, the transcription factor Sp1 was found to bind the GC‐rich region located between nucleotides −101 and −83 of the leptin gene. Mutational analysis of this region showed that the sequence of the region was critical for glucose/insulin stimulation of transcription. Thus, we postulated that the region from −101 to −83 of the leptin gene is responsible for glucose/insulin stimulation of transcription, and that Sp1 is somehow involved in this regulation.
The X gene of hepatitis B virus shows a high level stimulation of the Rous sarcoma virus long terminal repeat in the methylotropic yeast, Pichia pastoris
Authors: Lal Sunil K. , Kumar Vijay
Published: July 2002

In order to study the transactivational property of the X gene in the methylotropic yeast Pichia pastoris, a Rous sarcoma virus‐chloramphenicol acetyltransferase (RSV‐CAT) cassette was co‐transformed and integrated into the host yeast strain as a reporter which showed an overwhelming CAT activity. Immunoprecipitation of the yeast cell extracts with an X‐specific monoclonal antibody, however, showed a low level expression of the X gene. Therefore besides a trans‐effect of the X protein, the enhanced reporter activity could be a manifestation of a cis‐effect of the X gene sequences also. Therefore, unlike the transactivation studies with X gene in animal cells where limited functional activity is observed, P. pastoris appears to be an excellent system to study cis‐ and trans‐aspects of gene regulation by the X gene.