bluuebunny/crossref_metadata_2025 huggingface dataset:
Insufficient hydrogen‐bond desolvation and prion‐related disease
Authors: Fernández Ariel
Published: March 2003

A structuring and eventual exclusion of water surrounding backbone hydrogen bonds takes place during protein folding as hydrophobic residues cluster around such bonds. Taken as an average over all hydrogen bonds, the extent of desolvation is nearly a constant of motion, as revealed by re‐examination of the longest all‐atom trajectory with explicit solvent [Y. Duan & P. A. Kollman (1998) Science 282, 740]. Furthermore, this extent of desolvation is preserved across native soluble proteins, except for cellular prion proteins. Thus, a physico‐chemical picture of prion‐related disease emerges. The epitope for protein‐X binding, the region undergoing vast conformational change and the trigger and locker for this change are inferred from the location of under‐desolvated hydrogen bonds in the cellular prion protein.
Polyamines interact with DNA as molecular aggregates
Authors: D'Agostino Luciano , Di Luccia Aldo
Published: March 2003

New compounds, named nuclear aggregates of polyamines, having a molecular mass of 8000, 4800 and < 1000 Da, were found in the nuclear extracts of several replicating cells. Their molecular structure is based on the formation of ionic bonds between polyamine ammonium and phosphate groups. The production of the 4800 Da compound, resulting from the aggregation of five or more < 1000 Da units, was increased in Caco‐2 cells treated with the mitogen gastrin. Dissolving single polyamines in phosphate buffer resulted in the in vitro aggregation of polyamines with the formation of compounds with molecular masses identical to those of natural aggregates. After the interaction of the 4800 Da molecular aggregate with the genomic DNA at 37 °C, both the absorbance of DNA in phosphate buffer and the DNA mobility in agarose gel increased greatly. Furthermore, these compounds were able to protect the genomic DNA from digestion by DNase I, a phosphodiesterasic endonuclease. Our data indicate that the nuclear aggregate of polyamines interacts with DNA phosphate groups and influence, more efficaciously than single polyamines, both the conformation and the protection of the DNA.
The crystal structure of coenzyme B12‐dependent glycerol dehydratase in complex with cobalamin and propane‐1,2‐diol
Authors: Yamanishi Mamoru , Yunoki Michio , Tobimatsu Takamasa , Sato Hideaki , Matsui Junko , Dokiya Ayako , Iuchi Yasuhiro , Oe Kazunori , Suto Kyoko , Shibata Naoki , Morimoto Yukio , Yasuoka Noritake , Toraya Tetsuo
Published: March 2003

Recombinant glycerol dehydratase of Klebsiella pneumoniae was purified to homogeneity. The subunit composition of the enzyme was most probably α2β2γ2. When (R)‐ and (S)‐propane‐1,2‐diols were used independently as substrates, the rate with the (R)‐enantiomer was 2.5 times faster than that with the (S)‐isomer. In contrast to diol dehydratase, an isofunctional enzyme, the affinity of the enzyme for the (S)‐isomer was essentially the same or only slightly higher than that for the (R)‐isomer (Km(R)/Km(S) = 1.5). The crystal structure of glycerol dehydratase in complex with cyanocobalamin and propane‐1,2‐diol was determined at 2.1 Å resolution. The enzyme exists as a dimer of the αβγ heterotrimer. Cobalamin is bound at the interface between the α and β subunits in the so‐called ‘base‐on’ mode with 5,6‐dimethylbenzimidazole of the nucleotide moiety coordinating to the cobalt atom. The electron density of the cyano group was almost unobservable, suggesting that the cyanocobalamin was reduced to cob(II)alamin by X‐ray irradiation. The active site is in a (β/α)8 barrel that was formed by a central region of the α subunit. The substrate propane‐1,2‐diol and essential cofactor K+ are bound inside the (β/α)8 barrel above the corrin ring of cobalamin. K+ is hepta‐coordinated by the two hydroxyls of the substrate and five oxygen atoms from the active‐site residues. These structural features are quite similar to those of diol dehydratase. A closer contact between the α and β subunits in glycerol dehydratase may be reminiscent of the higher affinity of the enzyme for adenosylcobalamin than that of diol dehydratase. Although racemic propane‐1,2‐diol was used for crystallization, the substrate bound to glycerol dehydratase was assigned to the (R)‐isomer. This is in clear contrast to diol dehydratase and accounts for the difference between the two enzymes in the susceptibility of suicide inactivation by glycerol.
The molecular surface of proteolytic enzymes has an important role in stability of the enzymatic activity in extraordinary environments
Authors: Yamagata Youhei , Maeda Hiroshi , Nakajima Tasuku , Ichishima Eiji
Published: March 2003

It is scientifically and industrially important to clarify the stabilizing mechanism of proteases in extraordinary environments. We used subtilisins ALP I and Sendai as models to study the mechanism. Subtilisin ALP I is extremely sensitive to highly alkaline conditions, even though the enzyme is produced by alkalophilic Bacillus, whereas subtilisin Sendai from alkalophilic Bacillus is stable under conditions of high alkalinity. We constructed mutant subtilisin ALP I enzymes by mutating the amino acid residues specific for subtilisin ALP I to the residues at the corresponding positions of amino acid sequence alignment of alkaline subtilisin Sendai. We observed that the two mutations in the C‐terminal region were most effective for improving stability against surfactants and heat as well as high alkalinity. We predicted that the mutated residues are located on the surface of the enzyme structures and, on thebasis of three‐dimensional modelling, that they are involved in stabilizing the conformation of the C‐terminal region. As proteolytic enzymes frequently become inactive due to autocatalysis, stability of these enzymes in an extraordinary environment would depend on the conformational stability of the molecular surface concealing scissile peptide bonds. It appeared that the stabilization of the molecular surface structure was effective to improve the stability of the proteolytic enzymes.
Casein kinase 2 specifically binds to and phosphorylates the carboxy termini of ENaC subunits
Authors: Shi Haikun , Asher Carol , Yung Yuval , Kligman Luba , Reuveny Eitan , Seger Rony , Garty Haim
Published: March 2003

A number of findings have suggested the involvement of protein phosphorylation in the regulation of the epithelial Na+ channel (ENaC). A recent study has demonstrated that the C tails of the β and γ subunits of ENaC are subject to phosphorylation by at least three protein kinases [Shi, H., Asher, C., Chigaev, A., Yung, Y., Reuveny, E., Seger, R. & Garty, H. (2002) J. Biol. Chem. 277, 13539–13547]. One of them was identified as ERK which phosphorylates βT613 and γT623 and affects the channel interaction with Nedd4. The current study identifies a second protein kinase as casein kinase 2 (CK2), or CK‐2‐like kinase. It phosphorylates βS631, a well‐conserved serine on the β subunit. Such phosphorylation is observed both in vitro using glutathione‐S‐transferase–ENaC fusion proteins and in vivo in ENaC‐expressing Xenopus oocytes. The γ subunit is weakly phosphorylated by this protein kinase on another residue (γT599), and the C tail of α is not significantly phosphorylated by this kinase. Thus, CK2 may be involved in the regulation of the epithelial Na+ channel.
Novel complexes of mammalian translation elongation factor eEF1A·GDP with uncharged tRNA and aminoacyl‐tRNA synthetase
Authors: Petrushenko Zoya M. , Budkevich Tatyana V. , Shalak Vyacheslav F. , Negrutskii Boris S. , El'skaya Anna V.
Published: March 2003

Multimolecular complexes involving the eukaryotic elongation factor 1A (eEF1A) have been suggested to play an important role in the channeling (vectorial transfer) of tRNA during protein synthesis [Negrutskii, B.S. & El'skaya, A.V. (1998) Prog. Nucleic Acids Res. Mol. Biol. 60, 47–78]. Recently we have demonstrated that besides performing its canonical function of forming a ternary complex with GTP and aminoacyl‐tRNA, the mammalian eEF1A can produce a noncanonical ternary complex with GDP and uncharged tRNA [Petrushenko, Z.M., Negrutskii, B.S., Ladokhin, A.S., Budkevich, T.V., Shalak, V.F. & El'skaya, A.V. (1997) FEBS Lett. 407, 13–17]. The [eEF1A·GDP·tRNA] complex has been hypothesized to interact with aminoacyl‐tRNA synthetase (ARS) resulting in a quaternary complex where uncharged tRNA is transferred to the enzyme for aminoacylation. Here we present the data on association of the [eEF1A·GDP·tRNA] complex with phenylalanyl‐tRNA synthetase (PheRS), e.g. the formation of the above quaternary complex detected by the gel‐retardation and surface plasmon resonance techniques. To estimate the stability of the novel ternary and quaternary complexes of eEF1A the fluorescence method and BIAcore analysis were used. The dissociation constants for the [eEF1A·GDP·tRNA] and [eEF1A·GDP·tRNAPhe·PheRS] complexes were found to be 20 nm and 9 nm, respectively. We also revealed a direct interaction of PheRS with eEF1A in the absence of tRNAPhe (Kd = 21 nm). However, the addition of tRNAPhe accelerated eEF1A·GDP binding to the enzyme. A possible role of these stable novel ternary and quaternary complexes of eEF1A·GDP with tRNA and ARS in the channeled elongation cycle is discussed.
The glucose‐specific carrier of the Escherichia coli phosphotransferase system
Authors: García‐Alles Luis Fernando , Navdaeva Vera , Haenni Simon , Erni Bernhard
Published: March 2003

Thirteen glucose analogues bearing electrophilic groups were synthesized (five of them for the first time) and screened as inhibitors of the glucose transporter (EIIGlc) of the Escherichia coli phosphoenolpyruvate–sugar phosphotransferase system (PTS). 2′,3′‐Epoxypropyl β‐d‐glucopyranoside (3a) is an inhibitor and also a pseudosubstrate. Five analogues are inhibitors of nonvectorial Glc phosphorylation by EIIGlc but not pseudosubstrates. They are selective for EIIGlc as demonstrated by comparison with EIIMan, another Glc‐specific but structurally different transporter. 3a is the only analogue that inhibits EIIGlc by binding to the high‐affinity cytoplasmic binding site and also strongly inhibits sugar uptake mediated by this transporter. The most potent inhibitor in vitro, methyl 6,7‐anhydro‐d,l‐glycero‐α‐d‐gluco‐heptopyranoside (1d), preferentially interacts with the low‐affinity cytoplasmic site but only weakly inhibits Glc uptake. Binding and/or phosphorylation from the cytoplasmic side of EIIGlc is more permissive than sugar binding and/or translocation of substrates via the periplasmic site. EIIGlc is rapidly inactivated by the 6‐O‐bromoacetyl esters of methyl α‐d‐glucopyranoside (1a) and methyl α‐d‐mannopyranoside (1c), methyl 6‐deoxy‐6‐isothiocyanato‐α‐d‐glucopyranoside (1e), β‐d‐glucopyranosyl isothiocyanate (3c) and β‐d‐glucopyranosyl phenyl isothiocyanate (3d). Phosphorylation of EIIGlc protects, indicating that inactivation occurs by alkylation of Cys421. Glc does not protect, but sensitizes EIIGlc for inactivation by 1e and 3d, which is interpreted as the effect of glucose‐induced conformational changes in the dimeric transporter. Glc also sensitizes EIIGlc for inactivation by 1a and 1c of uptake by starved cells. This indicates that Cys421 which is located on the cytoplasmic domain of EIIGlc becomes transiently accessible to substrate analogues on the periplasmic side of the transporter.
Dnmt3a and Dnmt1 functionally cooperate during de novo methylation of DNA
Authors: Fatemi Mehrnaz , Hermann Andrea , Gowher Humaira , Jeltsch Albert
Published: March 2003

Dnmt3a is a de novo DNA methyltransferase that modifies unmethylated DNA. In contrast Dnmt1 shows high preference for hemimethylated DNA. However, Dnmt1 can be activated for the methylation of unmodified DNA. We show here that the Dnmt3a and Dnmt1 DNA methyltransferases functionally cooperate in de novo methylation of DNA, because a fivefold stimulation of methylation activity is observed if both enzymes are present. Stimulation is observed if Dnmt3a is used before Dnmt1, but not if incubation with Dnmt1 precedes Dnmt3a, demonstrating that methylation of the DNA by Dnmt3a stimulates Dnmt1 and that no physical interaction of Dnmt1 and Dnmt3a is required. If Dnmt1 and Dnmt3a were incubated together a slightly increased stimulation is observed that could be due to a direct interaction of these enzymes. In addition, we show that Dnmt1 is stimulated for methylation of unmodified DNA if the DNA already carries some methyl groups. We conclude that after initiation of de novo methylation of DNA by Dnmt3a, Dnmt1 becomes activated by the pre‐existing methyl groups and further methylates the DNA. Our data suggest that Dnmt1 also has a role in de novo methylation of DNA. This model agrees with the biochemical properties of these enzymes and provides a mechanistic basis for the functional cooperation of different DNA MTases in de novo methylation of DNA that has also been observed in vivo.
Permeability transition‐independent release of mitochondrial cytochrome c induced by valinomycin
Authors: Shinohara Yasuo , Almofti Mohamad Radwan , Yamamoto Takenori , Ishida Taro , Kita Fumiyo , Kanzaki Hideki , Ohnishi Masakatsu , Yamashita Kikuji , Shimizu Shigeomi , Terada Hiroshi
Published: March 2003

To examine whether valinomycin induces a mitochondrial permeability transition (PT), we investigated its effects on mitochondrial functions under various conditions. The acceleration of mitochondrial respiration and swelling, induced by valinomycin, were found to be insensitive to inhibitors of the ordinary PT, indicating that valinomycin does not induce the ordinary PT. Results of experiments using mitochondria isolated from transgenic mice expressing human bcl‐2 also supported this conclusion. Furthermore, evidence for induction of PT pores by valinomycin was not obtained by either electron microscopic analysis of mitochondrial configurations or by measurement of the permeability of the inner mitochondrial membrane by use of polyethylene glycol. However, valinomycin did induce a significant release of cytochrome c, and thus it may be a nice tool to study the processes of mitochondrial cytochrome c release.
Identification of a set of genes involved in the biosynthesis of the aminonucleoside moiety of antibiotic A201A from Streptomyces capreolus
Authors: Saugar Irene , Sanz Eloísa , Rubio Miguel Ángel , Espinosa Juan Carlos , Jiménez Antonio
Published: March 2003

A novel cosmid (pABC6.5) whose DNA insert from Streptomyces capreolus, the A201A antibiotic producer, overlaps the inserts of the previously reported pCAR11 and pCAR13 cosmids, has been isolated. These two latter cosmids were known to contain the aminonucleoside antibiotic A201A resistance determinants ard2 and ard1, respectively. Together, these three cosmids have permitted the identification of a DNA stretch of 19 kb between ard1 and ard2, which should comprise a large region of a putative A201A biosynthetic (ata) gene cluster. The sequence of the 7 kb upstream of ard1 towards ard2 reveals seven consecutive open reading frames: ataP3, ataP5, ataP4, ataP10, ataP7, ata12 and ataPKS1. Except for the last two, their deduced products present high similarities to an identical number of counterparts from the pur cluster of Streptomyces alboniger that were either known or proposed to be implicated in the biosynthesis of the N6,N6‐dimethyl‐3′‐amino‐3′‐deoxyadenosine moiety of puromycin. Because A201A contains this chemical moiety, these ataP genes are most likely implicated in its biosynthesis. Accordingly, the ataP4, ataP5 and ataP10 genes complemented specific puromycin nonproducing Δpur4, Δpur5 and Δpur10 mutants of S. alboniger, respectively. Amino acid sequence comparisons suggest that ata12 and ataPKS1 could be implicated in the biosynthesis of the d‐rhamnose and α‐p‐coumaric acid moieties of A201A. Further sequencing of 2 kb of DNA downstream of ard1 has disclosed a region which might contain one end of the ata cluster.
Membrane embedded location of Na+or H+binding sites on the rotor ring of F1F0ATP synthases
Authors: von Ballmoos Christoph , Meier Thomas , Dimroth Peter
Published: March 2003

Recent crosslinking studies indicated the localization of the coupling ion binding site in the Na+‐translocating F1F0ATP synthase ofIlyobacter tartaricuswithin the hydrophobic part of the bilayer. Similarly, a membrane embedded H+‐binding site is accepted for the H+‐translocating F1F0ATP synthase ofEscherichia coli. For a more definite analysis, we performed parallax analysis of fluorescence quenching with ATP synthases from bothI. tartaricusandE. coli. Both ATP synthases were specifically labelled at their c subunit sites withN‐cyclohexyl‐N′‐(1‐pyrenyl)carbodiimide, a fluorescent analogue of dicyclohexylcarbodiimide and the enzymes were reconstituted into proteoliposomes. Using either soluble quenchers or spinlabelled phospholipids, we observed a deeply membrane embedded binding site, which was quantitatively determined forI. tartaricusandE. colito be 1.3 ± 2.4 Å and 1.8 ± 2.8 Å from the bilayer center apart, respectively. These data show a conserved topology among enzymes of different species. We further demonstrated the direct accessibility for Na+ions to the binding sites in the reconstitutedI. tartaricusc11oligomer in the absence of any other subunits, pointing to intrinsic rotor channels. The common membrane embedded location of the binding site of ATP synthases suggest a common mechanism for ion transfer across the membrane.
Solution structure of the Alzheimer amyloid β‐peptide (1–42) in an apolar microenvironment
Authors: Crescenzi Orlando , Tomaselli Simona , Guerrini Remo , Salvadori Severo , D'Ursi Anna M. , Temussi Piero Andrea , Picone Delia
Published: March 2003

The major components of neuritic plaques found in Alzheimer disease (AD) are peptides known as amyloid β‐peptides (Aβ), which derive from the proteolitic cleavage of the amyloid precursor proteins. In vitro Aβ may undergo a conformational transition from a soluble form to aggregated, fibrillary β‐sheet structures, which seem to be neurotoxic. Alternatively, it has been suggested that an α‐helical form can be involved in a process of membrane poration, which would then trigger cellular death.

Conformational studies on these peptides in aqueous solution are complicated by their tendency to aggregate, and only recently NMR structures of Aβ‐(1–40) and Aβ‐(1–42) have been determined in aqueous trifluoroethanol or in SDS micelles. All these studies hint to the presence of two helical regions, connected through a flexible kink, but it proved difficult to determine the length and position of the helical stretches with accuracy and, most of all, to ascertain whether the kink region has a preferred conformation. In the search for a medium which could allow a more accurate structure determination, we performed an exhaustive solvent scan that showed a high propensity of Aβ‐(1–42) to adopt helical conformations in aqueous solutions of fluorinated alcohols. The 3D NMR structure of Aβ‐(1–42) shows two helical regions encompassing residues 8–25 and 28–38, connected by a regular type I β‐turn. The surprising similarity of this structure, as well as the sequence of the C‐terminal moiety, with those of the fusion domain of influenza hemagglutinin suggests a direct mechanism of neurotoxicity.
Amino acids 3–13 and amino acids in and flanking the 23FxxLF27 motif modulate the interaction between the N‐terminal and ligand‐binding domain of the androgen receptor
Authors: Steketee Karine , Berrevoets Cor A , Dubbink Hendrikus J. , Doesburg Paul , Hersmus Remko , Brinkmann Albert O. , Trapman Jan
Published: March 2003

The N‐terminal domain (NTD) and the ligand‐binding domain (LBD) of the androgen receptor (AR) exhibit a ligand–dependent interaction (N/C interaction). Amino acids 3–36 in the NTD (AR3−36) play a dominant role in this interaction. Previously, it has been shown that a ΦxxΦΦ motif in AR3−36, 23FxxLF27, is essential for LBD interaction. We demonstrate in the current study that AR3−36 can be subdivided into two functionally distinct fragments: AR3−13 and AR16−36. AR3−13 does not directly interact with the AR LBD, but rather contributes to the transactivation function of the AR.NTD‐AR.LBD complex. AR16−36, encompassing the 23FxxLF27 motif, is predicted to fold into a long amphipathic α‐helix. A second ΦxxΦΦ candidate protein interaction motif within the helical structure, 30VREVI34, shows no affinity to the LBD. Within AR16−36, amino acid residues in and flanking the 23FxxLF27 motif are demonstrated to modulate N/C interaction. Substitution of Q24 and N25 by alanine residues enhances N/C interaction. Substitution of amino acids flanking the 23FxxLF27 motif by alanines are inhibitory to LBD interaction.
Enzymic properties of recombinant BACE2
Authors: Kim Yong‐Tae , Downs Deborah , Wu Shili , Dashti Azar , Pan Yujun , Zhai Peng , Wang Xinjuan , Zhang Xuejun C. , Lin Xinli
Published: March 2003

BACE2 (Memapsin 1) is a membrane‐bound aspartic protease that is highly homologous with BACE1 (Memapsin 2). While BACE1 processes the amyloid precursor protein (APP) at a key step in generating the β‐amyloid peptide and presumably causes Alzheimer's disease (AD), BACE2 has not been demonstrated to be directly involved in APP processing, and its physiological functions remain to be determined. In vivo, BACE2 is expressed as a precursor protein containing pre‐, pro‐, protease, transmembrane, and cytosolic domains/peptides. To determine the enzymatic properties of BACE2, two variants of its pro‐protease domain, pro‐BACE2‐T1 (PB2‐T1) and pro‐BACE2‐T2 (PB2‐T2), were constructed. They have been expressed in Escherichia coli as inclusion bodies, refolded and purified. These two recombinant proteins have the same N terminus but differ at their C‐terminal ends: PB2‐T1 ends at Pro466, on the boundary of the postulated transmembrane domain, and PB2‐T2 ends at Ser431, close to the homologous ends of other aspartic proteases such as pepsin. While PB2‐T1 shares similar substrate specificities with BACE1 and other ‘general’ aspartic proteases, the specificity of PB2‐T2 is more constrained, apparently preferring to cleave at the NH2‐terminal side of paired basic residues. Unlike other ‘typical’ aspartic proteases, which are active only under acidic conditions, the recombinant BACE2, PB2‐T1, was active at a broad pH range. In addition, pro‐BACE2 can be processed at its in vivo maturation site by BACE1.
Monitoring the structural consequences of Phe12→d‐Phe and Leu15→Aib substitution in human/rat corticotropin releasing hormone
Authors: Spyroulias Georgios A. , Papazacharias Spyridon , Pairas George , Cordopatis Paul
Published: March 2003

A new human/rat CRH analogue has been synthesized using the Fmoc/tBu solid‐phase synthetic protocol. The sequence of the new peptide differs from the original in two positions, 12 and 15, at which the native amino acids l‐phenylalanine 12 and l‐leucine 15 have been replaced by the nonprotein amino acids d‐phenylalanine and α‐aminoisobutyric acid (Aib), respectively. The high resolution three‐dimensional solution structure of [d‐Phe12, Aib15]CRH has been determined by 688 distance constraints (656 meaningful NOE and 32 H‐bonds distance limits) and 21 angle constraints. A family of 40 energy‐minimized conformers was obtained with average rmsd of 0.39 ± 0.16 Å and 0.99 ± 0.13 Å for backbone and heavy atoms, respectively, and distance penalty functions of 0.42 ± 0.03 Å2. The NMR data acquired in a solvent system of water/trifluoroethanol (34%/66%, v/v) revealed that this 41‐polypeptide adopts an almost linear helical structure in solution with helical content which reaches an 84% of the residues. Structural analysis confirmed the existence of two helical peptide fragments. The first was comprised of residues Ile6–Arg16 and the second of residues Glu20–Ile40, forming an angle of 34.2°. The structural differences with respect to the native peptide have been identified in the region d‐Phe12–Glu20 where double substitution at positions 12 and 15 seems to perturb the elements of the native 35‐residue helix. These structural rearrangements promote non‐native intramolecular interactions in the region of the molecule between either the hydrophobic side‐chains of d‐Phe12, Aib15 and Leu18, or the charged groups of the residue pairs Arg16–Glu20 and His13–Glu17 being responsible for changes in hormonal functionality. This CRH analogue currently exhibits lack of any activity.
Thermodynamic and kinetic characterization of trihaem cytochrome c3 from Desulfuromonas acetoxidans
Authors: Correia Ilídio J. , Paquete Catarina M. , Louro Ricardo O. , Catarino Teresa , Turner David L. , Xavier António V.
Published: March 2003

Trihaem cytochrome c3 (also known as cytochrome c551.5 and cytochrome c7) is isolated from the periplasmic space of Desulfuromonas acetoxidans, a sulfur‐reducing bacterium. Thermodynamic and kinetic data for the trihaem cytochrome c3 are presented and discussed in the context of the possible physiological implications of its functional properties with respect to the natural habitat of D. acetoxidans, namely as a symbiont with green sulfur bacteria working as a mini‐sulfuretum. The thermodynamic properties were determined through the fit of redox titration data, followed by NMR and visible spectroscopy, to a model of four functional centres that describes the network of cooperativities between the three haems and one protolytic centre. The kinetics of trihaem cytochrome c3 reduction by sodium dithionite were studied using the stopped‐flow technique and the data were fitted to a kinetic model that makes use of the thermodynamic properties to obtain the rate constants of the individual haems. This analysis indicates that the electrons enter the cytochrome mainly via haem I. The reduction potentials of the haems in this cytochrome show little variation with pH within the physiological range, and the kinetic studies show that the rates of reduction are also independent of pH in the range studied. Thus, although the trihaem cytochrome c3 is readily reduced by hydrogenases from Desulfovibrio sp. and its haem core is similar to that of the homologous tetrahaem cytochromes c3, its physico‐chemical properties are quite different, which suggests that these multihaem cytochromes with similar structures perform different functions.
Regulation of mammalian translation factors by nutrients
Authors: Proud Christopher G.
Published: March 2003

Protein synthesis requires both amino acids, as precursors, and a substantial amount of metabolic energy. It is well established that starvation or lack of nutrients impairs protein synthesis in mammalian cells and tissues. Branched chain amino acids are particularly effective in promoting protein synthesis. Recent work has revealed important new information about the mechanisms involved in these effects. A number of components of the translational machinery are regulated through signalling events that require the mammalian target of rapamycin, mTOR. These include translational repressor proteins (eukaryotic initiation factor 4E‐binding proteins, 4E‐BPs) and protein kinases that act upon the small ribosomal subunit (S6 kinases). Amino acids, especially leucine, positively regulate mTOR signalling thereby relieving inhibition of translation by 4E‐BPs and activating the S6 kinases, which can also regulate translation elongation. However, the molecular mechanisms by which amino acids modulate mTOR signalling remain unclear. Protein synthesis requires a high proportion of the cell's metabolic energy, and recent work has revealed that metabolic energy, or fuels such as glucose, also regulate targets of the mTOR pathway. Amino acids and glucose modulate a further important regulatory step in translation initiation, the activity of the guanine nucleotide‐exchange factor eIF2B. eIF2B controls the recruitment of the initiator methionyl‐tRNA to the ribosome and is activated by insulin. However, in the absence of glucose or amino acids, insulin no longer activates eIF2B. Since control of eIF2B is independent of mTOR, these data indicate the operation of additional, and so far unknown, regulatory mechanisms that control eIF2B activity.
Optimizing the delivery systems of chimeric RNA·DNA oligonucleotides
Authors: Liang Li , Liu De‐Pei , Liang Chih‐Chuan
Published: March 2003

Special oligonucleotides for targeted gene correction have attracted increasing attention recently, one of which is the chimeric RNA·DNA oligonucleotide (RDO) system. RDOs for targeted gene correction were first designed in 1996, and are typically 68 nucleotides in length including continuous RNA and DNA sequences (RNA is 2′‐O‐methyl‐modified). They have a 25 bp double stranded region homologous to the targeted gene, two hairpin ends of T loop and a 5 bp GC clamp, that give the molecule much greater stability [Fig. 1]. One mismatch site in the middle of the double‐stranded region is designed for targeted gene therapy. RDOs have been used recently for targeted gene correction of point mutations both in vitro and in vivo, but many problems must be solved before clinical application. One of the solutions is to optimize the delivery vectors for RDOs. To date, few RDO delivery systems have been used. Therefore, new vectors should be tried for RDO transfer, such as the use of nanoparticles. Additionally, different kinds of modifications should be applied to RDO carrier systems to increase the total correction efficiency in vivo. Only with the development of delivery systems can RDOs be used for gene therapy, and successfully applied to functional genomics.

Diagrammatic structure of RDO. RDOs are typically 68 nucleotides in length, including continuous 2′‐O‐methyl‐modified RNA and DNA sequences, a 25 bp double‐stranded region homologous to the targeted gene, two hairpin ends of T loop and a 5 bp GC clamp, which give the molecule greater stability. One mismatch site in the middle of the double‐stranded region is designed for targeted gene therapy.

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ER–resident chaperone interactions with recombinant antibodies in transgenic plants
Authors: Nuttall James , Vine Nicholas , Hadlington Jane L. , Drake Pascal , Frigerio Lorenzo , Ma Julian K.‐C.
Published: March 2003

In this study, we demonstrate that the folding and assembly of IgG in transgenic tobacco plants is orchestrated by BiP (binding protein), an endoplasmic reticulum resident chaperone. Expression of BiP and calreticulin was examined in transgenic tobacco plants that express immunoglobulin chains, either singly or in combination to form IgG antibody. BiP mRNA expression was lowest in wild‐type nontransformed plants and those that expressed immunoglobulin light chain alone. Higher mRNA levels were detected in plants expressing fully assembled immunoglobulin (light and heavy chains), and the most abundant levels of RNA transcript were found in those plants that expressed immunoglobulin heavy chain alone. Estimation of total BiP demonstrated a similar pattern, with the highest levels detected in plants expressing immunoglobulin heavy chain alone. Immunoprecipitation studies demonstrated that BiP was associated with immunoglobulin chains extracted from protoplast lysates, but not from secreted fluids. Again, most BiP was coprecipitated from plants expressing heavy chain only and those that produced full length IgG. The binding of BiP to Ig heavy chains was ATP‐sensitive. Co‐expression of heavy and light chain resulted in IgG assembly and displacement of BiP from the heavy chain as the amount of light chain increased. Although calreticulin mRNA and total protein levels varied in a similar manner to those of BiP in the transgenic plants, there was no evidence for association between calreticulin and Ig chains, by coimmunoprecipitation. The results indicate that BiP, but not calreticulin, takes part in immunoglobulin folding and assembly in transgenic plants.
Convulxin induces platelet shape change through myosin light chain kinase and Rho kinase
Authors: Riondino Silvia , Gazzaniga Pier P. , Pulcinelli Fabio M.
Published: March 2003

Once platelets are activated, the first event to occur is a rapid change in shape, associated with Ca2+/calmodulin‐dependent myosin light chain (MLC) phosphorylation and with Rho kinase activation. The purpose of this study was to investigate which is the biochemical pathway that leads to platelet shape change in response to convulxin, a selective GpVI activator, and to verify whether MLC phosphorylation is essential for this process. The inhibition of the Ca2+‐dependent pathway by means of the Ca2+ chelator BAPTA, the Ca2+/calmodulin inhibitor W‐7 or the cAMP enhancing drug iloprost reduced about 50% of platelet shape change in response to convulxin. The treatment with either the Rho kinase inhibitors Y27632 or HA 1077 had no effect on platelet shape change induced by convulxin. When both Ca2+/calmodulin‐dependent and Rho kinase‐dependent pathways were concomitantly inhibited by the combined use of Y27632 plus BAPTA, W‐7 or iloprost, platelet shape change was completely abolished. Our findings suggest that convulxin‐induced platelet shape change occurs via both pathways, the Ca2+/calmodulin‐dependent, which appears to be more important, and the Rho kinase‐dependent one. The pattern of MLC phosphorylation was not modified by Rho kinase inhibitors. Conversely, the inhibition of the Ca2+‐dependent pathway caused a strong reduction of MLC phosphorylation in BAPTA‐treated platelets, and a total inhibition in W‐7 or iloprost‐treated platelets. Our results demonstrate that following Rho kinase‐dependent pathway platelet shape change can occur without the involvement of MLC phosphorylation.
The membrane‐bound [NiFe]‐hydrogenase (Ech) from Methanosarcina barkeri: unusual properties of the iron‐sulphur clusters
Authors: Kurkin Sergei , Meuer Jörn , Koch Jürgen , Hedderich Reiner , Albracht Simon P. J.
Published: March 2003

The purified membrane‐bound [NiFe]‐hydrogenase from Methanosarcina barkeri was studied with electron paramagnetic resonance (EPR) focusing on the properties of the iron–sulphur clusters. The EPR spectra showed signals from three different [4Fe−4S] clusters. Two of the clusters could be reduced under 101 kPa of H2, whereas the third cluster was only partially reduced. Magnetic interaction of one of the clusters with an unpaired electron localized on the Ni–Fe site indicated that this was the proximal cluster as found in all [NiFe]‐hydrogenases. Hence, this cluster was assigned to be located in the EchC subunit. The other two clusters could therefore be assigned to be bound to the EchF subunit, which has two conserved four‐Cys motifs for the binding of a [4Fe−4S] cluster. Redox titrations at different pH values demonstrated that the proximal cluster and one of the clusters in the EchF subunit had a pH‐dependent midpoint potential. The possible relevance of these properties for the function of this proton‐pumping [NiFe]‐hydrogenase is discussed.
O‐GalNAc incorporation into a cluster acceptor site of three consecutive threonines
Authors: Takeuchi Hideyuki , Kato Kentaro , Hassan Helle , Clausen Henrik , Irimura Tatsuro
Published: March 2003

O‐Glycosylation of three consecutive Thr residues in a fluorescein‐conjugated peptide PTTTPLK − which mimics a portion of mucin 2 − by four isozymes of UDP‐N‐acetylgalactosaminyltransferases (pp‐GalNAc‐T1, T2, T3, or T4) was investigated. Partially glycosylated versions of this peptide, PT*TTPLK, PTTT*PLK, PT*TT*PLK, PTT*T*PLK, PT*°TTPLK, and PTTT*°PLK (*, N‐acetylgalactosamine; °, galactose), were also tested. The products were separated by RP‐HPLC and characterized by MALDI‐TOF MS and peptide sequencing. The first and the third Thr residues act as the peptide's initial glycosylation sites for pp‐GalNAc‐T4, which were different from the sites for pp‐GalNAc‐T1 and T2 (the first Thr residue) or T3 (the third Thr residue) shown in our previous report. All pp‐GalNAc‐T isozymes tested exhibited distinct specificities toward glycopeptides. The most notable findings were: (a) prior incorporation of an N‐acetylgalactosamine residue at the third Thr greatly enhanced N‐acetylgalactosamine incorporation into the other Thr residues when pp‐GalNAc‐T2, T3, or T4 were used; (b) the enhancing effect of the N‐acetylgalactosamine residue on the third Thr was completely abrogated by galactosylation of this N‐acetylgalactosamine; (c) prior incorporation of an N‐acetylgalactosamine at the first Thr did not have any enhancing effect; (d) pp‐GalNAc‐T2 was unique as it transferred N‐acetylgalactosamine into the second Thr residue only when N‐acetylgalactosamine was attached to the third one.
Heterologous expression and folding analysis of a β‐tubulin isotype from the Antarctic ciliate Euplotes focardii
Authors: Pucciarelli Sandra , Miceli Cristina , Melki Ronald
Published: March 2003

Mammalian tubulins and actins attain their native conformation following interactions with CCT (the cytosolic chaperonin containing t‐complex polypeptide 1). To study the β‐tubulin folding in lower eukaryotes, an isotype of β‐tubulin (β‐T1) from the Antarctic ciliate Euplotes focardii, was expressed in Escherichia coli. Folding analysis was performed by incubation of the 35S‐labeled, denatured β‐T1 in the presence, or absence, of purified rabbit CCT and cofactor A, a polypeptide that stabilizes folded monomeric β‐tubulin. We show for the first time in protozoa that β‐tubulin folding is assisted by CCT and requires cofactor A. In addition, we observed that E. focardiiβ‐T1 competes with human β5 tubulin isotype for binding to CCT. The affinity of CCT to E. focardiiβ‐T1 and β5 tubulin are compared. Finally, the mitochondrial chaperonin mt‐cpn60 binds to β‐T1 but is unable to release it in a native or quasi‐native state.
Elucidation of the role of fructose 2,6‐bisphosphate in the regulation of glucose fluxes in mice using in vivo13C NMR measurements of hepatic carbohydrate metabolism
Authors: Choi In‐Young , Wu Chaodong , Okar David A. , Lange Alex J. , Gruetter Rolf
Published: March 2003

Fructose 2,6‐bisphosphate (Fru‐2,6‐P2) plays an important role in the regulation of major carbohydrate fluxes as both allosteric activator and inhibitor of target enzymes. To examine the role of Fru‐2,6‐P2 in the regulation of hepatic carbohydrate metabolism in vivo, Fru‐2,6‐P2 levels were elevated in ADM mice with adenovirus‐mediated overexpression of a double mutant bifunctional enzyme, 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase (n = 6), in comparison to normal control mice (control, n = 6). The rates of hepatic glycogen synthesis in the ADM and control mouse liver in vivo were measured using new advances in 13C NMR including 3D localization in conjunction with [1‐13C]glucose infusion. In addition to glycogen C1, the C6 and C2–C5 signals were measured simultaneously for the first time in vivo, which provide the basis for the estimation of direct and indirect synthesis of glycogen in the liver. The rate of label incorporation into glycogen C1 was not different between the control and ADM group, whereas the rate of label incorporation into glycogen C6 signals was in the ADM group 5.6 ± 0.5 µmol·g−1·h−1, which was higher than that of the control group of 3.7 ± 0.5 µmol·g−1·h−1 (P < 0.02). The rates of net glycogen synthesis, determined by the glycogen C2–C5 signal changes, were twofold higher in the ADM group (P = 0.04). The results provide direct invivo evidence that the effects of elevated Fru‐2,6‐P2 levels in the liver include increased glycogen storage through indirect synthesis of glycogen. These observations provide a key to understanding the mechanisms by which elevated hepatic Fru‐2,6‐P2 levels promote reduced hepatic glucose production and lower blood glucose in diabetes mellitus.
Mixed lineage kinase LZK and antioxidant protein‐1 activate NF‐κB synergistically
Authors: Masaki Megumi , Ikeda Atsushi , Shiraki Eriko , Oka Shogo , Kawasaki Toshisuke
Published: March 2003

Leucine zipper‐bearing kinase (LZK) is a novel member of the mixed lineage kinase (MLK) family [Sakuma, H., Ikeda, A., Oka, S., Kozutsumi, Y., Zanetta, J. P., and Kawasaki, T. (1997) J. Biol. Chem.272, 28622–28629]. We have previously shown that LZK activates the c‐Jun‐NH2 terminal kinase (JNK) pathway, but not the extracellular signal‐related kinase (ERK) pathway, by acting as a mitogen‐activated protein kinase kinase kinase (MAPKKK) [Ikeda, A., Hasegawa, K., Masaki, M., Moriguchi, T., Nishida, E., Kozutsumi, Y., Oka, S., and Kawasaki, T. (2001) J. Biochem.130, 773–781]. However, the mode of activation of LZK remains largely unknown. By means of a yeast two‐hybrid screening system, we have identified a molecule localized to mitochondria, antioxidant protein‐1 (AOP‐1), that binds to LZK and which acts as a modulator of LZK activity. Recently, several MAPKKKs involved in the JNK pathway, such as MEKK1, TAK1 and MLK3, were shown, using over‐expression assay systems, to activate a transcription factor, NF‐κB, through activation of the IKK complex. Using similar assay systems, we demonstrated that LZK activated NF‐κB‐dependent transcription through IKK activation only weakly, but this was reproducible, and that AOP‐1 enhanced the LZK‐induced NF‐κB activation. We also provided evidence that LZK was associated directly with the IKK complex through the kinase domain, and that AOP‐1 was recruited to the IKK complex through the binding to LZK.
The effect of ST2 gene product on anchorage‐independent growth of a glioblastoma cell line, T98G
Authors: Haga Yasushi , Yanagisawa Ken , Ohto‐Ozaki Hiromi , Tominaga Shin‐ichi , Masuzawa Toshio , Iwahana Hiroyuki
Published: March 2003

The ST2 gene, which is specifically induced by growth stimulation in fibroblasts, encodes interleukin‐1 receptor‐related proteins and is widely expressed in hematopoietic, helper T, and various cancer cells. However, the physiological as well as pathological functions of the ST2 gene products are not yet fully understood. In this study, we analyzed the expression of the ST2 gene in human glioma cell lines and human brain tumor samples with real‐time polymerase chain reaction method, the results of which revealed that the expression level of the ST2 gene in glioma cell lines and glioblastoma samples is significantly lower than that in a fibroblastic cell line, TM12, and benign brain tumors, suggesting the reverse relationship between malignancy and ST2 expression. As we could not detect the soluble ST2 protein in the culture fluid of the T98G glioblastic cell line by ELISA, we established stable transformants of T98G that continuously produce and secrete the ST2 protein, in order to study the effect of the ST2 protein on malignancy. Although we could not detect a remarkable difference in proliferation between transformants and control cells in conventional tissue culture dishes, the efficiency of colony formation in soft agar was significantly decreased in the case of cells that continuously produce the ST2 protein. Furthermore, inhibition of colony formation in soft agar was observed in wild‐type T98G cells when purified soluble ST2 protein was added to the culture, in a dose‐dependent manner. Taken together, the results suggest that the expression of ST2 suppressed the anchorage‐independent growth and malignancy.
Genomic structure, expression and characterization of a STAT5 homologue from pufferfish (Tetraodon fluviatilis)
Authors: Sung Shu‐Chiun , Fan Ting‐Jia , Chou Chih‐Ming , Leu Jiann‐Horng , Hsu Ya‐Li , Chen Shui‐Tsung , Hsieh Yueh‐Chun , Huang Chang‐Jen
Published: March 2003

The STAT5 (signal transducer and activator of transcription 5) gene was isolated and characterized from a round‐spotted pufferfish genomic library. This gene is composed of 19 exons spanning 11 kb. The full‐length cDNA of Tetraodon fluviatilis STAT5 (TfSTAT5) contains 2461 bp and encodes a protein of 785 amino acid residues. From the amino acid sequence comparison, TfSTAT5 is most similar to mouse STAT5a and STAT5b with an overall identity of 76% and 78%, respectively, and has < 35% identity with other mammalian STATs. The exon/intron junctions of the TfSTAT5 gene were almost identical to those of mouse STAT5a and STAT5b genes, indicating that these genes are highly conserved at the levels of amino acid sequence and genomic structure. To understand better the biochemical properties of TfSTAT5, a chimeric STAT5 was generated by fusion of the kinase‐catalytic domain of carp Janus kinase 1 (JAK1) to the C‐terminal end of TfSTAT5. The fusion protein was expressed and tyrosine‐phosphorylated by its kinase domain. The fusion protein exhibits specific DNA‐binding and transactivation potential toward an artificial fish promoter as well as authentic mammalian promoters such as the β‐casein promoter and cytokine inducible SH2 containing protein (CIS) promoter when expressed in both fish and mammalian cells. However, TfSTAT5 could not induce the transcription of β‐casein promoter via rat prolactin and Nb2 prolactin receptor. To our knowledge, this is the first report describing detailed biochemical characterization of a STAT protein from fish.
Plasminogen activator inhibitor type‐1 inhibits insulin signaling by competing with αvβ3 integrin for vitronectin binding
Authors: López‐Alemany Roser , Redondo Juan M. , Nagamine Yoshikuni , Muñoz‐Cánoves Pura
Published: March 2003

Functional cooperation between integrins and growth factor receptors has been reported for several systems, one of which is the modulation of insulin signaling by αvβ3 integrin. Plasminogen activator inhibitor type‐1 (PAI‐1), competes with αvβ3 integrin for vitronectin (VN) binding. Here we report that PAI‐1, in a VN‐dependent manner, prevents the cooperation of αvβ3 integrin with insulin signaling in NIH3T3 fibroblasts, resulting in a decrease in insulin‐induced protein kinase B (PKB) phosphorylation, vascular endothelial growth factor (VEGF) expression and cell migration. Insulin‐induced HUVEC migration and angiotube formation was also enhanced in the presence of VN and this enhancement is inhibited by PAI‐1. By using specific PAI‐1 mutants with either VN binding or plasminogen activator (PA) inhibiting activities ablated, we have shown that the PAI‐1‐mediated interference with insulin signaling occurs through its direct interaction with VN, and not through its PA neutralizing activity. Moreover, using cells deficient for uPA receptor (uPAR) we have demonstrated that the inhibition of PAI‐1 on insulin signaling is independent of uPAR‐VN binding. These results constitute the first demonstration of the interaction of PAI‐1 with the insulin response.
Choline increases serum insulin in rat when injected intraperitoneally and augments basal and stimulated aceylcholine release from the rat minced pancreas in vitro
Authors: Ilcol Yesim Ozarda , Gurun M. Sibel , Taga Yavuz , Ulus Ismail H.
Published: March 2003

Intraperitoneal injection of choline (30–90 mg·kg−1) produced a dose‐dependent increase in serum insulin, glucose and choline levels in rats. The increase in serum insulin induced by choline (90 mg·kg−1) was blocked by pretreatment with the muscarinic acetylcholine receptor antagonists, atropine (2 mg·kg−1), pirenzepine (2 mg·kg−1) and 4‐diphenylacetoxy‐N‐methylpiperidine (2 mg·kg−1) or the ganglionic nicotinic receptor antagonist, hexamethonium (15 mg·kg−1). The effect of choline on serum insulin and glucose was enhanced by oral glucose administration (3 g·kg−1). Choline administration was associated with a significant (P < 0.001) increase in the acetylcholine content of pancreatic tissue. Choline (10–130 µm) increased basal and stimulated acetylcholine release but failed to evoke insulin release from the minced pancreas at considerably higher concentrations (0.1–10 mm). Hemicholium‐3, a choline uptake inhibitor, attenuated the increase in acetylcholine release induced by choline augmentation. Choline (1–32 mm) inhibited [3H]quinuclidinyl benzilate binding to the muscarinic receptors in the pancreatic homogenates. These data show that choline, a precursor of the neurotransmitter acetylcholine, increases serum insulin by indirectly stimulating peripheral acetylcholine receptors through the enhancement of acetylcholine synthesis and release.