bluuebunny/crossref_metadata_2025 huggingface dataset:
Folding topology and DNA binding of the N‐terminal fragment of ada protein
Authors: Sakashita Hitoshi , Sakuma Takahiko , Ohkubo Tadayasu , Kainosho Masatsune , Sakumi Kunihiko , Sekiguchi Mutsuo , Morikawa Kosuke
Published: July 2002

Three amino terminal fragments of Escherichia coli Ada protein (39 kDa) with different molecular masses (14 kDa, 16 kDa and 20 kDa) were prepared in large quantities from an E. coli strain harboring plasmids constructed for the overproduction of the truncated proteins. The three fragments can be methylated to an extent similar to that of the intact molecule. The methylated 16 kDa fragment specifically binds to the ada box on a DNA duplex. NMR analyses revealed that the 14 kDa fragment comprises two α‐helices and a β‐sheet with parallel and anti‐parallel mixed strands. A comparison of the 15N‐1H HMQC spectra of the fragments has led to the conclusion that this tertiary structure within the 14 kDa fragment is retained in the larger 16 kDa and 20 kDa fragments.
Purification and molecular cloning of chymase from human tonsils
Authors: Sukenaga Yoshikazu , Kido Hiroshi , Neki Akio , Enomoto Mitsuo , Ishida Koichi , Takagi Kenkichi , Katunuma Nobuhiko
Published: July 2002

A chymotrypsin‐like protease was purified to homogeneity from human tonsils by a series of Chromatographie procedures. The purified enzyme gave a single protein band with an apparent molecular mass of 30 kDa on SDS‐PAGE. The sequence of the first 21 amino acids at the N‐terminus of the enzyme was determined. A cDNA for the enzyme was cloned by PCR amplification from extracted tonsillar mRNA using a supposed N‐terminal oligonucleotide primer and a conserved C‐terminal primer of the chymase family. The deduced amino acid sequence of the isolated clone was identical to that of human chymase in connective tissue‐type mast cells from heart except for a Ser instead of a Cys at the N‐terminal 7th position.
The HOX complex neighbored by the EVX gene, as well as two other homeobox‐containing genes, the GBX‐class and the EN‐class, are located on the same chromosomes 2 and 7 in humans
Authors: Matsui Toshikazu , Hirai Momoki , Hirano Masami , Kurosawa Yoshikazu
Published: July 2002

Two newly identified human homeobox‐containing genes, GBX1 and GBX2, are closely related genes, as are members of the other homeobox genes, EN‐1 and EN‐2. GBX1 and EN‐2 have been mapped to chromosome 7q36. The present study shows that GBX2
was mapped to chromosome 2q37. EN‐1 was mapped to chromosome 2q14. Moreover, two HOX complexes neighbored by the EVX gene, HOXA and HOXD, are located at chromosome 7p15‐p14 and 2q31‐q37, respectively. Thus, it is possible that these homeobox genes were linked to each other on an ancestral genome and that the ancestral chromosome segment was duplicated during evolution.
Thromboxane A2analogue U 46619 enhances tumour cell proliferation in HeLa cells via specific receptors which are apparently distinct from TXA2 receptors on human platelets
Authors: Nigam Santosh , Eskafi Saeed , Roscher Almut , Weitzel Hans
Published: July 2002

In this study, we have demonstrated for the first time by using U 46619, a stable analogue of thromboxane A2 (TXA2), that TXA2 exerts a cell proliferative effect on HeLa cells which is mediated by specific TXA2 receptors, inasmuch as the cell proliferation could be dose‐dependently suppressed by TXA2 receptor antagonist BM 13177. The investigation of the phospholipase C pathway by U 46619 and prostaglandin H2(PGH2) in the presence and absence of BM 13177 in cells with or without pertussis toxin pretreatment, as well as radioligand receptor binding studies, revealed that, in contrast to TXA2 receptors on human platelets, where TXA2 and PGH2 share the same receptor binding sites, HeLa cells possess distinct receptors for TXA2 and PGH2.
Human cathepsin E produced in E. coli
Authors: Hill Jeffrey , Montgomery Douglas S. , Kay John
Published: July 2002

A cDNA for procathepsin E was generated from human gastric adenocarcinoma (AGS) cells, amplified by PCR and inserted into the T7 dependent vector pET 22b for expression in E. coli. Purification of the resultant product was accomplished simply, without the need to resort to column chromatography. The recombinant protein displayed comparable properties to those of its naturally occurring counterpart. The yield of homogeneous active enzyme obtained was ≈ 3 mg per 40 g of cells. This is sufficient to permit crystallisation and structural analysis to begin and a mutagenesis programme to examine structure/activity relationships now to be undertaken.
Diabetes induces selective alterations in the expression of protein kinase C isoforms in hepatocytes
Authors: Tang Eric Y. , Parker Peter J. , Beattie James , Houslay Miles D.
Published: July 2002

Membrane and cytosol fractions from hepatocytes of both normal and streptozotocin‐induced diabetic animals were probed with a panel of polyclonal anti‐peptide antiscra in order to identify protein kinase C (PKC) isoforms. Immunoreactive species were noted with antisera specific for α (∼ 81 kDa), β‐II (∼ 82 kDA), ε (∼ 95 kDa) and ξ (∼ 79 kDa). In addition, a species migrating with an apparent size of ∼ 94 kDa was also detected in cytosol fractions using an antiserum specific for PKC‐α. Each of these species was specifically displaced when the PKC‐isoform specific peptide was included in the immunodetection system. No immunoreactive species consistent with the presence of the β‐I, λ, δ and η isoforms of protein kinase C was observed. Induction of diabetes using streptozotocin invoked selective alterations in the expression of PKC isoforms which were reversed upon insulin therapy. In the cytosol fraction, marked increases of ∼ 3‐fold occurred in levels of the β‐II isoform and the ∼ 90 kDa (upper) form of PKC‐α, with no apparent/little change in the levels of the ∼ 81 kDa (lower) form of PKC‐α and those of PKC‐ξ. Diabetes induction also appeared to have elicited the translocation of PKC‐β‐II and the ∼ 81 kDa (lower) form of PKC‐α to the membrane fraction where immunoreactivity for these species was now apparent. The level of PKC‐ϵ, which was noted only in membrane fractions, was also increased upon induction of diabetes. It is suggested that the selective alterations in the expression of PKC isoforms occurring upon streptozotocin‐induced diabetes may lead to altered cellular functioning and underly defects in inhibitory G‐protein functioning and insulin action which characterise this animal model of diabetes.
Native cytosolic protein phosphatase‐1 (PP‐1S) containing modulator (inhibitor‐2) is an active enzyme
Authors: Bollen Mathieu , DePaoli-Roach Anna A. , Stalmans Willy
Published: July 2002

In vitro, the modulator protein (inhibitor‐2) slowly converts the catalytic subunit of protein phosphatase‐1 (PP‐1c) into an inactive ‘MgATP‐dependent form’ that can be reactivated by the transient phosphorylation of modulator with GSK‐3/FA. We report here that this modulator‐induced inactivation of PP‐1C can be blocked by addition (at pH 7.5) of either 0.3 mM NaF or 150 mM NaCl, or by raising the pH to 8.5. Making use of a combination of the latter conditions, we have partially purified a soluble modulator‐associated form of PP‐1 (PP‐1S) from rabbit skeletal muscle as a spontaneously active enzyme that cannot be further activated by kinase GSK‐3/FA. These observations argue against a role for the ‘MgATP‐dependent’ form of PP‐1S as an inactive reservoir of PP‐1C. PP‐1S was separated on aminohexyl Sepharose from another active, cytosolic species of PP‐1, which appears to be a proteolytic product of the glycogen‐bound PP‐1G.
Ligand—receptor interactions in the nicotinic acetylcholine receptor probed using multiple substitutions at conserved tyrosines on the α subunit
Authors: Aylwin Maria L. , White Michael M.
Published: July 2002

Affinity labeling studies have identified several conserved tyrosine residues in the α subunit of the nicotinic acetylcholine receptor (αY93, αY190, and αY198) as being in or near the ligand binding site. Mutagenesis studies from several laboratories have shown that substitution of phenylalanine for tyrosine at these positions reduces the apparent affinity for ACh. We have examined this apparent reduction in affinity further through the use of multiple substitutions at each position. Substitution of either phenylalanine, tryptophan, or serine resulted in an apparent decrease in agonist affinity, but the degree of reduction depended on both the position and the nature of the substitution. Analysis of the effects of each substitution suggests that each residue interacts with the quaternary N of ACh, and that each residue may make a different type of interaction with the agonist.
Posttranslational processing of a carboxy‐terminal propeptide containing a KDEL sequence of plant vacuolar cysteine endopeptidase (SH‐EP)
Authors: Okamoto Takashi , Nakayama Hiroshi , Seta Kazuo , Isobe Toshiaki , Minamikawa Takao
Published: July 2002

A plant cysteine endopeptidase, designated SH‐EP, is a major protease occurring in cotyledons of Vigna mungo seedlings, and acts to degrade seed globulin stored in protein bodies. Here we show that the 43 kDa intermediate of SH‐EP formed in the endoplasmic reticulum is transported to protein bodies and processed to the 33 kDa mature form during transport or thereafter, and that the COOH‐terminal propeptide of 10 amino acid residues containing a KDEL sequence, which is known as a retention signal for the endoplasmic reticulum lumen, is processed to form the mature SH‐EP.
Kinetic analyses of DNA‐linked ribonucleases H with different sizes of DNA
Authors: Kanaya Eiko , Uchiyama Yohtaro , Ohtsuka Eiko , Ueno Yoshio , Ikehara Morio , Kanaya Shigenori
Published: July 2002

A series of DNA‐linked ribonucleases H with DNA adducts varying in size and sequence, ranging from heptamer to nonamer, were constructed and examined for their ability to cleave the 12‐base RNA (5′‐CGGAGAUGACGG‐3′) site‐specifically. The DNA‐linked RNase H with the 9‐base DNA (5′‐GTCATCTCC‐3′) cleaved the 12‐base RNA specifically at A6‐U7. Kinetic studies revealed that the DNA‐linked RNase H with the 8‐base DNA (5′‐TCATCTCC‐3′) cleaved it slightly more effectively than that with the 9‐base DNA. Factors that may affect the specificity and catalytic efficiency of a DNA‐linked RNase H are described.
A 1H NMR NOE study on Co(II) stellacyanin: Some clues about the structure of the metal site
Authors: Vila Alejandro J.
Published: July 2002

The 1H NMR spectrum of Co(II) stellacyanin is reported, in which four signals not previously observed have been detected. NOE experiments were performed to assign the hyperfine shifted signals corresponding to a Cys and two His residues. Both His residues are solvent‐accessible and are shown to bind the metal ion through their Nδ1 atoms. The β‐CH2 Cys proton shifts indicate the presence of a strong axial ligand.
Ha‐Ras stimulates uptake and phosphorylation of ethanolamine: inhibition by wortmannin
Authors: Kiss Zoltan , Crilly Karan S.
Published: July 2002

Transformation of NIH 3T3 fibroblasts by Ha‐Ras resulted in large increases in the phosphorylation of both [14C]ethanolamine (Etn) and [14C]choline (Cho) when these precursors were added to the medium. Wortmannin, an inhibitor of phosphatidylinositol 3‐kinase (P13K), preferentially decreased phosphorylation of externally added Etn in the Ha‐Ras transformed, but not in the untransformed, fibroblasts. However, wortmannin had no effect on the phosphorylation of Etn formed endogenously by phorbol ester‐stimulated hydrolysis of phosphatidylethanolamine. Data suggest that interaction of mutated Ras with PI3K leads to specific stimulation of Etn uptake, followed by nearly quantitative phosphorylation of Etn by a Ras‐activated Cho/Etn kinase.
Polarity conserved positions in transmembrane domains of G‐protein coupled receptors and bacteriorhodopsin
Authors: Zhang Daqun , Weinstein Harel
Published: July 2002

The polarity of residues at certain positions in the transmembrane domains of G‐protein coupled receptors (GPCR) is found to be conserved, and to indicate the pattern of specific helix‐helix packing of the helices. A concept of polarity conserved positions (PCP) is proposed to describe this conserved property, and is applied to obtain insight into the structural features of the transmembrane proteins. The common pattern of PCPs for GPCRs indicates that they share a similar packing arrangement of their transmembrane helix bundles. For proteins in the bacteriorhodopsin family the PCP pattern suggests a common packing arrangement that differs from that of GPCRs, in agreement with experimental data. This difference in the packing arrangement underscores the shortcomings of a BR template for the construction of molecular models of GPCRs.
Tris: an allosteric effector of tarantula haemocyanin
Authors: Sterner R. , Bardehle K. , Paul R. , Decker H.
Published: July 2002

The effect of the chemical buffering component Tris (hydroxy‐methyl‐amino‐methane) and of chloride ions on the oxygen binding of tarantula hemocyanin was studied at constant pH. It revealed that Tris at micromolar concentrations decreases the oxygen pressure at half‐saturation (P50) by a factor of more than two, whereas chloride does not influence oxygen affinity. A thermodynamic analysis in terms of the nested model of allostery [(1987) Proc. Natl. Acad. Sci. 84, 1891‐1895] indicated that Tris acts a an allosteric activator of oxygen binding by influencing the interaction between the 12‐meric half‐molecules of the 24‐meric tarantula haemocyanin.
The intrinsic 22 kDa protein is a chlorophyll‐binding subunit of photosystem II
Authors: Funk Christiane , Schröder Wolfgang P. , Green Beverley R. , Renger Gernot , Andersson Bertil
Published: July 2002

The intrinsic 22 kDa polypeptide associated with photosystem II (psbS protein) was found to be able to bind chlorophyll. Extraction of isolated photosystem II membranes with octyl‐thioglucopyranoside, followed by repetitive electrophoresis under partially denaturing conditions gave only one green band. It contained both chlorophyll a and chlorophyll b, exhibited an absorption maximum at 674 nm and a 77 K fluorescence peak at 675 nm. The chlorophyll‐protein band contained a single polypeptide of 22 kDa. Based on these results and on previous protein sequence comparisons, it is suggested that the psbS protein is a chlorophyll a/b binding polypeptide and should thus be denoted CP22.
Stimulation of MAP kinase by v‐raf transformation of fibroblasts fails to induce hyperphosphorylation of transfected tau
Authors: Latimer Donna A. , Gallo Jean-Marc , Lovestone Simon , Miller Christopher C.J. , Hugh Reynolds C. , Marquardt Betina , Stabel Silvia , Woodgett James R. , Anderton Brian H.
Published: July 2002

A proportion of the microtubule‐associated protein, tau, is in an elevated state of phosphorylation in foetal and adult brain whereas all of the tau in paired helical filaments, which are characteristic of Alzheimer's disease is hyperphosphorylated; it is important therefore to elucidate the mechanisms that regulate tau phosphorylation. Here we describe results that show that although MAP kinase can hyperphosphorylate tau in vitro, activation of MAP kinase in transformed fibroblasts does not result in hyperphosphorylation of transfected tau, whereas glycogen synthase kinase‐3β (GSK‐3β) when co‐transfected with tau does result in tau hyperphosphorylation. The findings imply that GSK‐3β may be a stronger candidate than MAP kinase for inducing tau hyperphosphorylation in vivo.
The depolarization response element in acetylcholine receptor genes is a dual‐function E box
Authors: Su Ching-Tien , Huang Chang-Fen , Schmidt Jakob
Published: July 2002

All acetylcholine receptor subunit genes contain E boxes and are blocked by membrane depolarization. We have used transfected C2C12 myogenic cells to investigate the response, to electrical stimulation and KCl, of wildtype and mutant regulatory regions of the chick acetylcholine receptor α, γ and δ subunit, and the mouse MLC genes. Point mutations revealed that E boxes function as activating elements targeted by the depolarization signal. These experiments suggest, and insertion of a depolarization response element into an unrelated promoter confirms, that plasma membrane depolarization switches the depolarization response element from an activating to a repressive mode.
Rabbit translation elongation factor 1α stimulates the activity of homologous aminoacyl‐tRNA synthetase
Authors: Negrutskii Boris S , Budkevich Tatyana V , Shalak Vyacheslav F , Turkovskaya Galina V , El'Skaya Anna V
Published: July 2002

Functional and structural sequestration of aminoacyl‐tRNA has been recently found in eukaryotic cells and the aminoacyl‐tRNA channeling has been suggested [B.S. Negrutskii et al., Proc. Natl. Acad. Sci. 91 (1994) 964–968], but molecular details and mechanism of the process remained unclear. In this paper we have verified a possible interaction between rabbit aminoacyl‐tRNA synthetase and homologous translation elongation factor 1α (EF‐1α), the proteins which may play a role of sequential components involved into the transfer of the aminoacyl‐tRNA along the protein synthetic metabolic chain. The stimulation of the phenylalanyl‐tRNA synthetase activity by EF‐1α is found. The effect is shown to be specific towards the origin of tRNA and elongation factor molecules. The data obtained favor the direct transfer mechanism of the aminoacyl‐tRNA channeling process during eukaryotic protein synthesis.
Conserved secondary structure in the actinorhodin polyketide synthase acyl carrier protein from Streptomyces coelicolor A3(2) and the fatty acid synthase acyl carrier protein from Escherichia coli
Authors: Crump Matthew P. , Crosby John , Dempsey Christopher E. , Murray Martin , Hopwood David A. , Simpson Thomas J.
Published: July 2002

The acyl carrier protein (ACP) of Streptomyces coelicolor A3(2) functions as a molecular chaperone during the biosynthesis of the polyketide actinorhodin (act). Here we compare structural features of the polyketide synthase (PKS) ACP, determined by two‐dimensional 1H‐NMR, with the Escherichia coli fatty acid synthase (FAS) ACP. The PKS ACP contains four helices (residues 7–16 [A], 42–53 [B], 62–67 [C], 72–86 [D]), and a large loop (residues 17–41) having no defined secondary structure with the exception of a turn between residues 21 and 24. The act ACP shows 47% sequence similarity with the E. coli FAS ACP and the results demonstrate that the sequence homology is extended to the secondary structure of the proteins.
Reinitiation of protein synthesis in Escherichia coli can be induced by mRNA cis‐elements unrelated to canonical translation initiation signals
Authors: Andrè Alessandra , Puca Antimina , Sansone Federica , Brandi Anna , Antico Giovanni , Calogero Raffaele A.
Published: July 2002

In Eubacteria, de novo translation of some internal cistrons may be inefficient or impossible unless the 5′ neighboring cistron is also translated (translational coupling). Translation reinitiation is an extreme case of translational coupling in which translation of a message depends entirely on the presence of a nearby terminating ribosome. In this work, the characteristics of mRNA cis‐elements inducing the reinitiation process in Escherichia coli have been investigated using a combinatorial approach. A number of novel translational reinitiation sequences (TRSs) were thus identified, which show a wide range of reinitiation activities fully dependent on a translational coupling event and unrelated to the presence/absence of secondary structure or mRNA stability. Moreover, some of the isolated TRSs are similar to intercistronic sequences present in the E. coli genome.
Selective inhibition of DNA gyrase in vitro by a GC specific eight‐ring hairpin polyamide at nanomolar concentration
Authors: Simon H. , Kittler L. , Baird E. , Dervan P. , Zimmer C.
Published: July 2002

The influence of an eight‐ring hairpin DNA minor groove binder on the gyrase mediated DNA supercoiling and cleavage reaction step of the enzyme was investigated. The results demonstrate that supercoiling is affected by the hairpin polyamide in the millimolar concentration range while the enzyme catalyzed cleavage of a 162 bp fragment of pBR322 containing a single strong gyrase site is effectively inhibited at nanomolar concentration. As demonstrated by footprint analysis the latter effect is caused by a specific binding of the hairpin forming polyamide to the enzyme recognition site (GGCC), which indicates that the gyrase activity to produce a double strand break is blocked at this site. The pyrrole–imidazole hairpin polyamide is the most potent inhibitor of the gyrase mediated cleavage reaction compared to other known anti‐gyrase active DNA binding agents.
Transient kinetics of ligand binding and role of the C‐terminus in the dUTPase from equine infectious anemia virus
Authors: Nord Johan , Kiefer Martin , Adolph Hans-Werner , Zeppezauer Michael M. , Nyman Per-Olof
Published: July 2002

Transient kinetics of the equine infectious anemia virus deoxyuridine 5′‐triphosphate nucleotide hydrolase were characterized by monitoring the fluorescence of the protein. Rate constants for the association and dissociation of substrate and inhibitors were determined and found to be consistent with a one‐step mechanism for substrate binding. A C‐terminal part of the enzyme presumed to be flexible was removed by limited trypsinolysis. As a result, the activity of the dUTPase was completely quenched, but the rate constants and fluorescent signal of the truncated enzyme were affected only to a minor degree. We conclude that the flexible C‐terminus is not a prerequisite for substrate binding, but indispensable for catalysis.
Human acylation stimulating protein enhances triacylglycerol biosynthesis in plant microsomes
Authors: Weselake Randall J. , Kazala E.Chris , Cianflone Katherine , Boehr David D. , Middleton Cameron K. , Rennie Courtney D. , Laroche André , Recnik Ingrid
Published: July 2002

Diacylglycerol acyltransferase has a universal role in catalyzing the acyl‐CoA‐dependent formation of triacylglycerol in microorganisms, animals and plants. Acylation stimulating protein, from human blood, is known to enhance diacylglycerol acyltransferase activity and triacylglycerol biosynthesis in human adipocytes. In the current study, acylation stimulating protein was also shown to enhance diacylglycerol acyltransferase activity in microsomes from cell suspension cultures of oilseed rape. Enzyme stimulation occurred over the pH range of 6–9 but the degree of stimulation decreased with increasing ionic strength at pH 7.4. Varying acyl‐CoA concentration did not affect the degree of stimulation. Membranes from triacylglycerol producing cells in plants and humans may have similar binding sites for acylation stimulating protein which have been preserved during molecular evolution. The results suggest that human acylation stimulating protein may be useful in modifying lipid biosynthesis in plants.
Triiodothyronine downregulates the periportal expression of α class glutathione S‐transferase in rat liver
Authors: Vanhaecke T. , Lindros K.O. , Oinonen T. , Vercruysse A. , Rogiers V.
Published: July 2002

Most drug‐metabolizing phase I and phase II enzymes, including the glutathione S‐transferases (GST), exhibit a zonated expression in the liver, with lower expression in the upstream, periportal region. To elucidate the involvement of pituitary‐dependent hormones in this zonation, the effect of hypophysectomy and 3,3′,5‐triiodo‐L‐thyronine (T3) on the distribution of GST was studied in rats. Hypophysectomy increased total GST activity both in the periportal and perivenous liver region. Subsequent T3 treatment counteracted this effect in the perivenous zone. However, analysis for either μ class M1/M2‐specific (1,2‐dichloro‐4‐nitrobenzene) or α class A1/A2‐specific (7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole) GST activity revealed that T3 treatment did not significantly affect the perivenous activity of these GST classes. In contrast, T3 was found to significantly counteract the increase of α class GST activity caused by hypophysectomy in the periportal zone. To establish whether this effect was T3‐specific, hepatocytes were isolated from either the periportal and perivenous zone by digitonin/collagenase perfusion and cultured either as pyruvate‐supplemented monolayer or as co‐culture with rat liver epithelial cells. Only in the latter it was found that T3 suppressed the A1/A2‐specific GST activity and α class proteins predominantly in periportal cells. The data demonstrate that T3 is an important factor responsible for the low expression of α GST in the periportal region. T3 may be involved in the periportal downregulation of other phase I and II enzymes as well.
Activity and mRNA abundance of Δ‐5 and Δ‐6 fatty acid desaturases in two human cell lines
Authors: de Antueno Roberto J. , Allen Stephen J. , Ponton André , Winther Michael D.
Published: July 2002

We analyzed fatty acid biosynthesis in Chang and ZR‐75‐1 cells. Both cell lines could desaturate and further elongate substrates for Δ‐5 desaturase. ZR‐75‐1 but not Chang cells showed Δ‐6 desaturation of 18:2n‐6, 18:3n‐3, 24:4n‐6 and 24:5n‐3. In both cell lines, the mRNA abundance can be related to Δ‐5 or Δ‐6 fatty acid desaturase activities. These results suggest that desaturase genes could have, at least in part, independent control mechanisms and that Δ‐6 desaturase impairment is not specific to any particular step of the fatty acid metabolic pathways, which may diminish the rationale for the existence of at least two distinct enzymes.
Sphingolipids of the mycopathogen Sporothrix schenckii: identification of a glycosylinositol phosphorylceramide with novel core GlcNH2α1→2Ins motif
Authors: Toledo Marcos S. , Levery Steven B. , Straus Anita H. , Takahashi Helio K.
Published: July 2002

Acidic glycosphingolipid components were extracted from the yeast form of the dimorphic mycopathogen Sporothrix schenckii. Two minor and the major fraction from the yeast form (Ss‐Y1, ‐Y2, and ‐Y6, respectively) have been isolated. By a combination of 1‐ and 2‐D 1H‐nuclear magnetic resonance (NMR) spectroscopy, electrospray ionization mass spectrometry (ESI‐MS), and gas chromatography/mass spectrometry (GC/MS), Ss‐Y6 was determined to be triglycosylinositol phosphorylceramide with a novel glycan structure, Manα1→3Manα1→6GlcNH2α1→2Ins1‐P‐1Cer (where Ins=myo‐inositol, P=phosphodiester). While the GlcNH2α1→
6
Ins1‐P‐ motif is found widely distributed in eukaryotic GPI anchors, the linkage GlcNH2α1→
2
Ins1‐P‐ has not been previously observed in any glycolipid. Ss‐Y1 and Ss‐Y2 were both found to have the known glycan structure Manα1→3Manα1→2Ins1‐P‐1Cer. Together with the results of a prior study [Toledo et al. (2001) Biochem. Biophys. Res. Commun. 280, 19–24] which showed that the mycelium form expresses GIPCs with the structures Manα1→6Ins1‐P‐1Cer and Manα1→3Manα1→6Ins1‐P‐1Cer, these results demonstrate that S. schenckii can synthesize glycosylinositol phosphorylceramides with at least three different core linkages.
Up‐regulation of uncoupling proteins by β‐adrenergic stimulation in L6 myotubes
Authors: Nagase Itsuro , Yoshida Toshihide , Saito Masayuki
Published: July 2002

Catecholamine‐induced and β‐adrenergic receptor (β‐AR)‐mediated thermogenesis in skeletal muscle is a significant component of whole‐body energy expenditure. Skeletal muscle expresses uncoupling protein (UCP) 2 and UCP3, which can dissipate the transmitochondrial electrochemical gradient and thereby may be involved in regulation of energy metabolism. We investigated the effects of β‐AR stimulation on UCP2 and UCP3 expression in L6 myotubes. Stimulation of the cells with epinephrine increased the UCP3 mRNA level transiently at 6 h, and also the UCP2 mRNA level at 6–24 h. The stimulatory effects of epinephrine were also observed in the presence of carbacyclin and 9‐cis retinoic acid, and mimicked by isoproterenol and salbutamol (β2‐AR agonists), but abolished by propranolol and ICI‐118,551 (β2‐AR antagonists). Pharmacological and mRNA analyses revealed the existence of β2‐AR, but not β1‐ and β3‐ARs, in L6 myotubes. These results suggested that catecholamines up‐regulate UCP2 and UCP3 expression through direct action on the β2‐AR in skeletal muscle.
Complete synthesis of 3′‐sialyl‐N‐acetyllactosamine by regioselective transglycosylation
Authors: Vetere Amedeo , Paoletti Sergio
Published: July 2002

Transglycolytic synthesis of 3′‐sialyl‐N‐acetyllactosamine by sequential use of β‐galactosidase from Bacillus circulans and trans‐sialidase from Trypanosoma cruzi was described. These reactions depicted the first complete synthesis of a biologically important oligosaccharide with high regioselectivity avoiding use of glycosyltransferases and NDP sugars.
Evidence for the formation of an unusual ternary complex of rabbit liver EF‐1α with GDP and deacylated tRNA
Authors: Petrushenko Zoya M. , Negrutskii Boris S. , Ladokhin Alexey S. , Budkevich Tat'yana V. , Shalak Vyacheslav F. , El'skaya Anna V.
Published: July 2002

Eukaryotic translation elongation factor 1α is known to interact in GTP‐bound form with aminoacyl‐tRNA promoting its binding to the ribosome. In this paper another ternary complex [EF‐1α*GDP*deacylated tRNA], never considered in widely accepted elongation schemes, is reported for the first time. The formation of this unusual complex, postulated earlier (FEBS Lett. (1996) 382, 18–20), has been detected by four independent methods. [EF‐1α*GDP]‐interacting sites are located in the acceptor stem, TψC stem and TψC loop of tRNAPhe and tRNALeu molecules. Both tRNA and EF‐1α are found to undergo certain conformational changes during their interaction. The ability of EF‐1α to form a complex with deacylated tRNA indicates that the factor may perform an important role in tRNA and aminoacyl‐tRNA channeling in higher eukaryotic cells.
Demonstration that the BchH protein of Rhodobacter capsulatus activates S‐adenosyl‐l‐methionine:magnesium protoporphyrin IX methyltransferase
Authors: Hinchigeri Shivayogeppa B , Hundle Bhupinder , Richards William R
Published: July 2002

The bchH gene of Rhodobacter capsulatus has been cloned into an expression strain of Escherichia coli. Following induction of expression of the BchH protein, it was found that the E. coli strain also accumulated porphyrins with the fluorescence properties of protoporphyrin and zinc protoporphyrin. It was also found that the soluble BchH protein increased the activity of S‐adenosyl‐l‐methionine:magnesium protoporphyrin IX methyltransferase, when mixed with membranes of an expression strain of E. coli into which the bchM gene (which encodes the methyltransferase) had been cloned, as well as membranes of a bchH mutant of R. capsulatus.
Phosphatidylinositol 4,5‐bisphosphate stimulates phosphorylation of the adaptor protein Shc by c‐Src
Authors: Sato Ken-ichi , Yamamoto Hideki , Otsuki Tetsuji , Aoto Mamoru , Tokmakov Alexander A , Hayashi Fumio , Fukami Yasuo
Published: July 2002

The adaptor protein Shc was prepared as glutathione S‐transferase fusion proteins (GST–Shc) and used as in vitro substrate for c‐Src. Since phosphotyrosine‐binding domain of Shc has been shown to bind phosphatidyl‐inositol 4,5‐bisphosphate (PtdIns(4,5)P2) [Zhou et al. (1995) Nature 378, 584–592], effect of PtdIns(4,5)P2 on the phosphorylation of GST–Shc by c‐Src was examined. PtdIns(4,5)P2 stimulated the phosphorylation of GST–Shc without any effect on the c‐Src activity as judged by both its autophosphorylation and phosphorylation of exogenous substrate, Cdc2 peptide. On the other hand, phosphatidylserine, phosphatidic acid, phosphatidylinositol, and phosphatidylinositol 4‐phosphate but not phosphatidylcholine stimulated the c‐Src activity itself. K
m for GST–Shc in the presence of 1 μM PtdIns(4,5)P2 was calculated to be 90 nM. The PtdIns(4,5)P2‐dependent phosphorylation of GST–Shc was inhibited by a GST–fusion protein containing the phosphotyrosine‐binding domain of Shc. These results suggest that PtdIns(4,5)P2 can act as a regulator of phosphorylation of Shc by c‐Src through its binding to Shc.
9,13‐di‐cis‐Retinoic acid induces the production of tPA and activation of latent TGF‐β via RARα in a human liver stellate cell line, LI90
Authors: Imai Shoko , Okuno Masataka , Moriwaki Hisataka , Muto Yasutoshi , Murakami Kazuhiro , Shudo Koichi , Suzuki Yasuhiro , Kojima Soichi
Published: July 2002

We studied the mechanism by which 9,13‐di‐cis‐retinoic acid (9,13dcRA), a novel and endogenous stereoisomer of all‐trans‐RA, induces TGF‐β formation in a human liver stellate cell line, LI90. 9,13dcRA induced the expression of RARα and RARβ, enhanced the production of tissue‐type plasminogen activator (tPA), thereby, surface plasmin levels, and induced the activation of latent TGF‐β. Similar effects were obtained with RARα‐selective retinoid, but not with RARβ‐ or RARγ‐selective retinoid, and the induction was inhibited by RARα‐selective antagonist. These results suggest that 9,13dcRA up‐regulates tPA expression, resulting in the formation of TGF‐β by LI90 cells, at least in part, via induction and activation of RARα.
High‐level expression of human c‐Src can cause a spherical morphology without loss of anchorage‐dependent growth of NIH 3T3 cells
Authors: Kato Goro , Maeda Shuichiro
Published: July 2002

To investigate whether overexpression of human c‐Src leads to cell rounding in anchorage‐dependent NIH 3T3 fibroblasts, we have established c‐Src‐inducible cell lines using a lac repressor–operator system. RN1 cells, which expressed c‐Src at a high level after induction, exhibited a spherical morphology and ceased to grow in monolayer culture. RN1 cells, however, exhibited neither focus‐forming ability nor anchorage‐independent growth potential with or without induction. Induced RN1 c‐Src was phosphorylated at Ser75, a previously reported spherical cell‐associated site, and at Tyr419. These data demonstrated for the first time that highly elevated human c‐Src tyrosine kinase activity can cause NIH 3T3 cells to have a spherical morphology without loss of anchorage‐dependent growth. The inducible cell line should be useful to study the mechanism for cell rounding by c‐Src.
The complete mature bovine prion protein highly expressed in Escherichia coli: biochemical and structural studies
Authors: Negro Alessandro , De Filippis Vincenzo , Skaper Stephen D , Peter James  , Sorgato M.Catia
Published: July 2002

According to the `protein only' hypothesis, modification of the 3‐dimensional fold of the constituent cellular protein, PrPC, into the disease‐associated isoform, PrPSc, is the cause of neurodegenerative diseases in animals and humans. Here we describe the high‐level synthesis in Escherichia coli, and purification in the monomeric form, of a histidine‐tagged full‐length mature PrP (25–249) of bovine brain, termed His‐PrP. Based on biochemical and spectroscopic data, His‐PrP displays characteristics expected for the PrPC isoform. The reported expression system should allow the production of quantities of bovine PrPC sufficient to permit 3‐dimensional structure determinations.
A cytoplasmic peptide of the neurotrophin receptor p75NTR: induction of apoptosis and NMR determined helical conformation
Authors: Hileman Matthew R. , Chapman Barbara S. , Rabizadeh S. , Krishnan V.V. , Bredesen Dale , Assa-Munt Nuria , Plesniak Leigh A.
Published: July 2002

The neurotrophin receptor (NTR) and tumor necrosis factor receptor family of receptors regulate apoptotic cell death during development and in adult tissues [Beutler and van Huffel, Science 264 (1994) 667–668]. We have examined a fragment of p75NTR from the carboxyl terminus of the receptor and a variant form of this peptide via NMR techniques and in vitro assays for apoptotic activity. The wild type peptide induces apoptosis and adopts a helical conformation oriented parallel to the surface of lipid micelles, whereas the variant form adopts a non‐helical conformation in the presence of lipid and shows no activity. These experiments suggest a link between structure and function of the two peptides.
Distinct patterns of all‐trans retinoic acid dependent expression of HOXB and HOXC homeogenes in human embryonal and small‐cell lung carcinoma cell lines
Authors: Flagiello Domenico , Gibaud Anne , Dutrillaux Bernard , Poupon Marie-France , Malfoy Bernard
Published: July 2002

The expression patterns of the class I homeogenes HOXB and HOXC clusters in the presence of retinoic acid (RA) were studied in two human small‐cell lung cancer (SCLC) cell lines and compared to that of NT2/D1 embryonal carcinoma cells. Contrasting with the sequential 3′‐5′ induction of the HOX genes observed after RA treatment of embryonic NT2/D1 cells, in the SCLC cells the responding genes (induced or down‐regulated) were interspersed with insensitive genes (expressed or unexpressed), while no genomic alteration affected the corresponding clusters. These findings imply that HOX gene regulatory mechanisms are altered in non‐embryonic SCLC cells, perhaps reflecting their ability to respond to more diversified stimuli, in relation with their origin from adult tissues.
Urinary trypsin inhibitor suppresses the production of interstitial procollagenase/proMMP‐1 and prostromelysin 1/proMMP‐3 in human uterine cervical fibroblasts and chorionic cells
Authors: Imada Keisuke , Ito Akira , Kanayama Naohiro , Terao Toshihiko , Mori Yo
Published: July 2002

The mechanisms by which urinary trypsin inhibitor (UTI) prevents preterm premature rupture of fetal membrane and premature cervical ripening were investigated. We, therefore, examined the effects of UTI on the production of matrix metalloproteinases (MMPs) which closely participate in the breakdown of extracellular matrix in cultured human uterine cervical fibroblasts and human chorionic cells. UTI suppressed specifically the production of interstitial procollagenase/proMMP‐1 and prostromelysin 1/proMMP‐3 from both cells in a dose‐dependent manner (0.32‐1.28 μM). This suppression was accompanied by a decrease in steady‐state levels of their mRNAs. These results indicate for the first time that UTI down‐regulates the production of proMMP‐1 and proMMP‐3 accompanying with the decrease in the expression of their mRNAs, and therefore UTI actually participates in the maintenance of fetal membranes and/or uterine cervix by overall suppression of MMP production along with the known inhibitory actions towards serine proteinases.
Mitochondrial respiratory chain deficiency leads to overexpression of antioxidant enzymes
Authors: Brambilla Liliana , Cairo Gaetano , Sestili Piero , O'Donnel Valery , Azzi Angelo , Cantoni Orazio
Published: July 2002

U937 cell growth in the presence of either chloramphenicol or ethidium bromide rapidly leads to respiratory deficiency. The novel finding of this report is that this response is paralleled by a specific increase in Se‐dependent and ‐independent glutathione peroxidase activities as well as of glutathione peroxidase and heme oxygenase mRNAs. Under the same experimental conditions, catalase activity and catalase mRNA do not show appreciable changes. These results can be explained by an increased formation of H2O2 at the early times of development of respiratory deficiency followed by induction of antioxidant enzymes.
Aminoacylation of tRNA gene transcripts is strongly affected by 3′‐extended and dimeric substrate RNAs
Authors: Kholod Natalya , Pan'kova Natalya , Ksenzenko Vladimir , Kisselev Lev
Published: July 2002

Kinetic parameters of aminoacylation by E. coli phenylalanyl‐tRNA synthetase vary for phage T5 tRNAPhe gene transcript from 0.950 to 2.545 μM for K
m and from 550 to 400 min−1 for k
cat. To reveal the source of this variability for various RNA preparations, homogeneity of the transcripts has been examined. Presence of 3′ extensions and dimer formation in transcript preparations reduced the catalytic efficiency k
cat/K
m several‐fold. We have shown that the proportion of dimers and 3′‐extended transcripts in tRNA preparations is sensitive to single‐base substitutions in tRNA. While wild‐type phage T5 tRNAPhe gene transcript contains about half of dimeric molecules, for some mutants this value increases up to 90% or drops to 0%. Phage T5 tRNAPhe gene with anticodon stem nucleotide substitutions used as a template in run‐off transcription produces 5 times less 3′‐extended molecules than the wild‐type gene. In view of all these results kinetic parameters of aminoacylation reaction for many wild‐type and mutant tRNA gene transcripts should be reevaluated.
15‐Lipoxygenation of phospholipids may precede the sn‐2 cleavage by phospholipases A2: reaction specificities of secretory and cytosolic phospholipases A2 towards native and 15‐lipoxygenated arachidonoyl phospholipids
Authors: Chaitidis Pavlos , Schewe Tankred , Sutherland Mark , Kühn Hartmut , Nigam Santosh
Published: July 2002

Reticulocyte‐type 15‐lipoxygenase is known to dioxygenate phospholipids without preceding action of phospholipases A2 (PLA2). Therefore we studied the reaction of the secretory PLA2s (sPLA2) from pancreas and snake venom, and of the human cytosolic PLA2 (cPLA2) with 1‐palmitoyl‐2‐arachidonoyl phosphatidylcholine (PAPC) and their 15‐lipoxygenated species (PAPC‐OOH and PAPC‐OH) either alone or as equimolar mixtures. These PLA2s cleaved PAPC‐O(O)H with higher (sPLA2) or similar rates (cPLA2) as compared with native PAPC. In mixtures, however, PAPC proved to be the preferred, albeit not exclusive substrate for all three PLA2s. Thus, partial 15‐lipoxygenation of phospholipids may also trigger liberation of arachidonic acid.
Characterization of the bleomycin resistance determinant encoded on the transposon Tn5
Authors: Kumagai Takanori , Nakano Tetsuji , Maruyama Masafumi , Mochizuki Hiroshi , Sugiyama Masanori
Published: July 2002

The transposon Tn5 carries a gene, ble, which confers resistance to bleomycin (Bm) and gives a survival advantage to its host cell. We found that the ble gene product, designated BLMT, is a binding protein with a strong affinity for Bm. BLMT quenched both the antibacterial and DNA‐cleaving activities of Bm, when incubated with the antibiotic. An electron spin resonance spin‐trapping analysis showed that BLMT inhibits the generation of Bm‐induced hydroxyl radical, by trapping Bm but not the hydroxyl radical. Western blot analysis using an anti‐BLMT monoclonal antibody revealed that BLMT is immunologically distinct from Bm‐binding proteins from Streptomyces verticillus, Staphylococcus aureus and Streptoalloteichus hindustanus. Escherichia coli, transformed with a mutant ble having leucine instead of proline at N‐terminal amino acid position 7, lost resistance to Bm, although the cell maintained the survival benefit. This suggests that the Bm resistance mediated by ble is independent of its ability to give a survival advantage to the host bacterium.
pH‐dependent thermal transitions of lentil lectin
Authors: Marcos Marı́a J. , Chehı́n Rosana , L. Arrondo Jose , Zhadan Galina G. , Villar Enrique , L. Shnyrov Valery
Published: July 2002

The thermal stability of lentil lectin in the 5.0–10.0 pH range was studied by high‐sensitivity differential scanning calorimetry and infrared spectroscopy. The thermally induced transitions for protein were irreversible and strongly dependent upon the scan rate at all pH values, suggesting that the denaturation is under kinetic control. It is shown that process of lentil lectin denaturation can be interpreted with sufficient accuracy in terms of the simple kinetic scheme, , where k is a first‐order kinetic constant that changes with temperature, as given by the Arrhenius equation, N is the native state, and D is the denatured state. On the basis of this model, the parameters of the Arrhenius equation were calculated.
Expression and a role of functionally coupled P2Y receptors in human dendritic cells
Authors: Liu Q.H. , Bohlen H. , Titzer S. , Christensen O. , Diehl V. , Hescheler J. , Fleischmann B.K.
Published: July 2002

We investigated the physiology and function of P2Y receptors expressed in human dendritic cells (DCs) differentiated in vitro from CD14+ cells (DC‐14). These were obtained after a 10 day stimulation period in GM‐CSF, IL‐4 and monocyte conditioned medium. DC‐14 were found to express high amounts of MHC class II, B7, CD40 as well as CD83. The functional analysis, using single cell Ca2+ imaging, demonstrated the expression of at least three subtypes of P2Y receptors. We further found using patch‐clamp measurements that ATP evoked a pertussis toxin insensitive non‐selective cation current with a peak current amplitude of −276±43 pA (holding potential −80 mV, n=23). This current was not Ca2+‐activated, since it was still observed under conditions of high intracellular Ca2+ buffering and could be blocked by Gd3+ (0.5 mM). In addition, intracellular application of GTP‐γ‐S (0.3 mM) also activated the current. Interestingly, DC‐14 redirected the orientation of their dendrites as well as cell shape towards a pipette containing ATP as observed with time lapse microscopy. These data suggest that in human DCs, ATP acts via P2Y receptors and induces chemokine effects.
NMR characterization of the NADP(H)‐binding domain of Escherichia coli transhydrogenase: sequential assignment and global fold
Authors: Johansson Carina , Bergkvist Anders , Fjellström Ola , Rydström Jan , Karlsson B.Göran
Published: July 2002

The soluble NADP(H)‐binding domain of Escherichia coli transhydrogenase (186 amino acids, 20.4 kDa, rotational correlation time 14 ns) was characterized using NMR techniques. The global fold is similar to that of a classical dinucleotide‐binding fold with six parallel β‐strands in a central sheet surrounded by helices and irregular structures, but is lacking both αD and αE. The substrate is bound in an extended conformation at the C‐terminal end of the parallel β‐sheet and our data support the notion of a redox dependent structural rearrangement.
In vitro interactions of Caenorhabditis elegans dystrophin with dystrobrevin and syntrophin
Authors: Gieseler Kathrin , Abdel-Dayem Manal , Ségalat Laurent
Published: July 2002

Dystrophin, the product of the gene mutated in Duchenne muscular dystrophy (DMD) is bound by its C‐terminus to a protein complex including the related protein dystrobrevin. Both proteins contain a putative coiled‐coil domain consisting of two α‐helices. It has been reported that the two proteins bind to each other by the first one of the two α‐helices. We have revisited this question using the Caenorhabditis elegans homologs of dystrophin and dystrobrevin. In vitro interaction occurs through the more conserved second helix. We propose a new model of dystrophin interactions with associated proteins.