bluuebunny/crossref_metadata_2025 huggingface dataset:
A spectroscopic study of the reaction of NAMI, a novel ruthenium(III)anti‐neoplastic complex, with bovine serum albumin
Authors: Messori Luigi , Orioli Pierluigi , Vullo Daniela , Alessio Enzo , Iengo Elisabetta
Published: March 2003

The reaction of Na[transRuCl4Me2SO(Im)] (NAMI; where Im is imidazole), a novel anti‐neoplastic ruthenium(III) complex, with BSA, was studied in detail by various physico‐chemical techniques. It is shown that NAMI, following chloride hydrolysis, binds bovine serum albumin tightly; spectrophotometric and atomic absorption data point out that up to five ruthenium ions are bound per albumin molecule when BSA is incubated for 24 h with an eightfold excess of NAMI. CD and electronic absorption results show that the various ruthenium centers bound to albumin exhibit well distinct spectroscopic features. The first ruthenium equivalent produces a characteristic positive CD band at 415 nm whereas the following NAMI equivalents produce less specific and less marked spectral effects. At high NAMI/BSA molar ratios a broad negative CD band develops at 590 nm. Evidence is provided that the bound ruthenium centers remain in the oxidation state +3. By analogy with the case of transferrins it is proposed that the BSA‐bound ruthenium ions are ligated to surface histidines of the protein; results from chemical modification experiments with diethylpyrocarbonate seem to favor this view. Spectral patterns similar to those shown by NAMI are observed when BSA is reacted with two strictly related ruthenium(III) complexes Na[transRuCl4(Me2SO)2] and H(Im)[transRuCl4(Im)2] (ICR), implying a similar mechanism of interaction in all cases. It is suggested that the described NAMI‐BSA adducts may formin vivoand may be relevant for the biological properties of this complex; alternatively NAMI/BSA adducts may be tested as specific carriers of the ruthenium complex to cancer cells. Implications of these findings for the mechanism of action of NAMI and of related ruthenium(III) complexes are discussed.
31P NMR analysis of the DNA conformation induced by protein binding
Authors: Castagné Claire , Murphy Elizabeth C. , Gronenborn Angela M. , Delepierre Muriel
Published: March 2003

Complexes of the HMG box protein SRY with two duplexes of 8 and 14 base pairs have been studied by 31P NMR and complete assignment of all phosphorus signals of the bound DNA duplexes are presented. While for the free DNA, all 31P signals display limited spectral dispersion (< 0.8 p.p.m.) for the bound duplexes, 31P resonances are spread over 2 p.p.m. Based on the previously published 3D structure of hSRY‐HMG, with the 8 mer it is demonstrated that the upfield shifted resonances correspond to the site of partial intercalation of an isoleucine side chain into the DNA. Moreover, the observation of significant difference in linewidths between the two duplexes allows to estimate lifetime of the complexes from 31P–31P 2D exchange experiments.
Amino‐acid sequence and glycan structures of cysteine proteases with proline specificity from ginger rhizome Zingiber officinale
Authors: Choi Kyung H. , Laursen Richard A.
Published: March 2003

The ginger proteases (GP‐I and GP‐II), isolated from the ginger rhizome Zingiber officinale, have an unusual substrate specificity preference for cleaving peptides with a proline residue at the P2 position. The complete amino‐acid sequence of GP‐II, a glycoprotein containing 221 amino acids, and about 98% that of GP‐I have been determined. Both proteases, which are 82% similar, have cysteine residues at positions 27 and histidines at position 161, corresponding to the essential cysteine–histidine diads found in the papain family of cysteine proteases, and six corresponding cysteine residues that form the three invariant disulfide linkages seen in this family of proteins. The sequence homology with other members (papain, bromelain, actinidin, protease omega, etc.) of this family is ≈ 50%. GP‐II has two predicted glycosylation sites at Asn99 and Asn156. Analyisis by electrospray and collision‐induced dissociation MS showed that both sites were occupied by the glycans (Man)3(Xyl)1(Fuc)1(GlcNAc)2 and (Man)3(Xyl)1(Fuc)1(GlcNAc)3, in a ratio of ≈ 7 : 1. Both glycans are xylose containing biantennary complex types that share the common core structural unit, Man1→6(Man1→3) (Xyl1→2)Man1→4GlcNAc1→4(Fuc1→3)GlcNAc for the major form, with an additional N‐acetylglucosamine residue being linked, in the minor form, to one of the terminal mannose units of the core structure.
Suramin blocks nucleotide triphosphate binding to ribosomal protein L3 from Trypanoplasma borreli
Authors: Avliyakulov Nuraly K. , Lukeš Julius , Kajava Andrey V. , Liedberg Bo , Lundström Ingemar , Svensson Samuel P. S.
Published: March 2003

Ribosomal protein L3 (L3) has been demonstrated to participate in formation of the peptidyltransferase center and is essential for its catalytic activity. In the present study we show that L3 is able to bind nucleotide triphosphates with high and specific affinity in vitro. L3 was serendipitously identified by screening of a genomic phage library from a primitive kinetoplastid flagellate Trypanoplasma borreli with the ATPase domain of the topoisomerase II gene as a probe. The cloned gene was overexpressed and purified as a his‐tag fusion protein in E. coli. Radioligand binding experiments, using [γ‐35S]ATP, showed that L3 is able to bind ATP but also GTP and UTP with similar high affinity (IC50 50–100 nm), while it has no ATPase activity. Furthermore, we showed that L3 has more than 500‐fold higher affinity for nucleotide triphosphates compared to the corresponding nucleotide monophosphates and diphosphates. Molecular genetic and biochemical analyses allowed us to localize the NTP binding domain of L3 to the N‐terminal 296 residues. Suramin, a polysulfonated naphthylamine derivative of urea, known for its chemotherapeutic effects completely inhibited the binding of [γ‐35S]ATP at subclinical levels. Results obtained with surface plasmon resonance technology showed that suramin both forms weak multimolecular complexes with L3 and binds strongly to L3 in nearly stoichiometric amounts.
A second functional Δ5 fatty acid desaturase in the cellular slime mould Dictyostelium discoideum
Authors: Saito Tamao , Morio Takahiro , Ochiai Hiroshi
Published: March 2003

A cDNA with homology to fatty acid desaturases was selected by searching the cDNA data bank of Dictyostelium discoideum (http://www.csm.biol.tsukuba.ac.jp/cDNAproject.html) with conserved histidine box motifs. Using this sequence, genomic DNA encoding the Δ5 desaturase was amplified from the genomic DNA of D. discoideum, and its desaturase activity was confirmed by the overexpression mutation in D. discoideum and the gain‐of‐function mutation in yeast. The cloned cDNA is 1565 nucleotides in length, and the deduced amino‐acid sequence comprised 467 amino‐acid residues containing an N‐terminal cytochrome b5 domain that shared 43% identity with cytochrome b5 of Oryza sativa. The whole sequence was 42% identical to the Δ5 desaturase of Mortierella alpina. This desaturase is a novel member of the cytochrome b5‐containing Δ5 fatty acid desaturase. As we have already reported one other Δ5 desaturase in Dictyostelium, this organism is the first to be confirmed as having two functional Δ5 fatty acid desaturase genes. The substrate specificities of the two functional Δ5 desaturases of D. discoideum were also examined.
Negative feedback and ultrasensitivity can bring about oscillations in the mitogen‐activated protein kinase cascades
Authors: Kholodenko Boris N.
Published: March 2003

Functional organization of signal transduction into protein phosphorylation cascades, such as the mitogen‐activated protein kinase (MAPK) cascades, greatly enhances the sensitivity of cellular targets to external stimuli. The sensitivity increases multiplicatively with the number of cascade levels, so that a tiny change in a stimulus results in a large change in the response, the phenomenon referred to as ultrasensitivity. In a variety of cell types, the MAPK cascades are imbedded in long feedback loops, positive or negative, depending on whether the terminal kinase stimulates or inhibits the activation of the initial level. Here we demonstrate that a negative feedback loop combined with intrinsic ultrasensitivity of the MAPK cascade can bring about sustained oscillations in MAPK phosphorylation. Based on recent kinetic data on the MAPK cascades, we predict that the period of oscillations can range from minutes to hours. The phosphorylation level can vary between the base level and almost 100% of the total protein. The oscillations of the phosphorylation cascades and slow protein diffusion in the cytoplasm can lead to intracellular waves of phospho‐proteins.
Characterization of potato proteinase inhibitor II reactive site mutants
Authors: Beekwilder Jules , Schipper Bert , Bakker Petra , Bosch Dirk , Jongsma Maarten
Published: March 2003

Potato proteinase inhibitor II (PI‐2) is composed of two sequence repeats. It contains two reactive site domains. We developed an improved protocol for the production of PI‐2 using the yeast Pichia pastoris as the expression host. We then assessed the role of its two reactive sites in the inhibition of trypsin and chymotrypsin by mutating each of the two reactive sites in various ways. From these studies it appears that the second reactive site strongly inhibits both trypsin (Ki = 0.4 nm) and chymotrypsin (Ki = 0.9 nm), and is quite robust towards mutations at positions P2 or P1′. In contrast, the first reactive site inhibits only chymotrypsin (Ki = 2 nm), and this activity is very sensitive to mutations. Remarkably, replacing the reactive site amino acids of domain I with those of domain II did not result in inhibitory activities similar to domain II. The fitness for protein engineering of each domain is discussed.
Effect of streptozotocin‐induced diabetes on rat liver Na+/K+‐ATPase
Authors: Sennoune Souad , Gerbi Alain , Duran Marie‐Josée , Grillasca Joel‐Paul , Compe Emmanuel , Pierre Sandrine , Planells Richard , Bourdeaux Madeleine , Vague Philippe , Pieroni Gerard , Maixent Jean‐Michel
Published: March 2003

Na+/K+‐ATPase during diabetes may be regulated by synthesis of its α and β subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin‐induced diabetes on liver Na+/K+‐ATPase. We then evaluated whether fish oil treatment prevented the diabetes‐induced changes. Diabetes mellitus induced an increased Na+/K+‐ATPase activity and an enhanced expression of the β1 subunit; there was no change in the amount of the α1 and β3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No α2 andα3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes‐induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+‐ATPase activity can be associated with the enhanced expression of the β1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high‐affinity site for ouabain (IC50 = 10−7 m) could be explained by the presence of (αβ)2 diprotomeric structure of Na+/K+‐ATPase or an as yet unknown α subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.
Structure–function relationships in the carboxylic‐ester‐hydrolase superfamily
Authors: Smialowski‐Fléter Sylvie , Moulin André , Villard Claude , Puigserver Antoine
Published: March 2003

CNBr fragments from porcine intestinal glycerol‐ester hydrolase were separated by SDS/PAGE under reducing and nonreducing conditions, and their amino‐acid sequences were analysed. Two intra‐chain disulfide bridges were identified, namely Cys70–Cys99 (loop A) and Cys256–Cys267 (loop B). As the Cys71 sulfhydryl group could not be alkylated with iodoacetamide, it is suggested that the residue is blocked rather than being present in the free form. The two disulfide bridges of intestinal glycerol‐ester hydrolase are present in the cholinesterase family, although the enzyme showed only about 35% identity with these proteins. Furthermore, the finding that glycerol‐ester hydrolase was partly inactivated under reducing conditions suggests that one or both disulfide bridges are important for the enzyme conformation. Lastly, glycerol‐ester hydrolase was also found to hydrolyse cholinergic substrates, although residues Trp86 and Asp74 which are considered to be the main constituents of the ‘anionic’ subsite responsible for substrate binding in cholinesterases were absent from loop A. Other amino‐acid residues in the glycerol‐ester hydrolase may therefore be responsible for the binding of cholinergic substrates to the enzyme.
Pressure denaturation of β‐lactoglobulin
Authors: Botelho Michelle M. , Valente‐Mesquita Vera L. , Oliveira Kesley M. G. , Polikarpov Igor , Ferreira Sérgio T.
Published: March 2003

β‐Lactoglobulin, the main whey protein in bovine milk, exists in several isoforms of which the most abundant are isoforms A and B. We have previously reported the denaturation of β‐lactoglobulin A by hydrostatic pressure [Valente‐Mesquita, V.L., Botelho, M.M. & Ferreira, S.T. (1998) Biophys. J.75, 471–476]. Here, we compare the pressure stabilities of isoforms A and B. These isoforms differ by two amino‐acid substitutions: Asp64 and Val118 in isoform A are replaced by glycine and alanine, respectively, in isoform B. Replacement of the buried Val118 residue by the smaller alanine side‐chain is not accompanied by significant structural rearrangements of the neighbouring polypeptide chain and creates a cavity in the core of β‐lactoglobulin. Pressure denaturation experiments revealed different stabilities of the two isoforms. Standard volume changes (ΔVunf) of – 49 ± 8 mL·mol−1 and −75 ± 3 mL·mol−1, and unfolding free energy changes (ΔGunf) of 8.5 ± 1.3 kJ·mol−1 and 11.3 ± 0.4 kJ·mol−1 were obtained for isoforms A and B, respectively. The volume occupied by the two methyl groups of Val118 removed in the V118A substitution is ≈ 40 Å3 per monomer of β‐lactoglobulin, in excellent agreement with the experimentally measured difference in ΔVunf for the two isoforms (ΔΔVunf = 26 mL·mol−1, corresponding to ≈ 43 Å3 per monomer). Thus, the existence of a core cavity in β‐lactoglobulin B may explain its enhanced pressure sensitivity relative to β‐lactoglobulin A. β‐Lactoglobulin undergoes a reversible pH‐induced conformational change around pH 7, known as the Tanford transition. We have compared the pressure denaturation of β‐lactoglobulin A at pH 7 and 8. Unfolding free energy changes of 8.5 ± 1.3 and 8.3 ± 0.3 kJ·mol−1 were obtained at pH 7 and 8, respectively, showing that the thermodynamic stability of β‐lactoglobulin is identical at these pH values. Interestingly, ΔVunf was dependent on pH, and varied from −49 ± 8 mL·mol−1 to −68 ± 2 mL·mol−1 at pH 7 and 8, respectively. The large increase in ΔVunf at pH 8 relative to pH 7 appears to be associated with an overall expansion of the protein structure and could explain the increased pressure sensitivity of β‐lactoglobulin at alkaline pH.
Utilization of leucine and acetate as carbon sources for sterol and fatty acid biosynthesis by Old and New World Leishmania species, Endotrypanum monterogeii and Trypanosoma cruzi
Authors: Ginger Michael L. , Prescott Mark C. , Reynolds David G. , Chance Michael L. , Goad L. John
Published: March 2003

The relative roles of acetate and leucine in the provision of a carbon source for fatty acid and sterol biosynthesis in several trypanosomatid species were investigated using 14C‐ and 13C‐labelled acetate, glucose and leucine as substrates. Promastigotes of Leishmania species synthesized a large proportion of their sterol from leucine. L. major (LV39), L. amazonensis and L. mexicana were the most efficient utilizers of leucine, producing at least 70–77% of their sterol from leucine; L. braziliensis, L. donovani and L. tropica apparently produced less sterol from leucine (23–36%) and L. major (LV561), L. adleri and L. panamamensis were intermediate, utilizing leucine to provide 51–58% of their sterol. In all the cases the balance of the sterol produced was apparently synthesized from carbon arising from acetate. The related trypanosomatid Endotrypanum monterogeii also produced a large amount (77%) of its sterol from leucine rather than acetate. By contrast Trypanosoma cruzi elaborated only 8% of its sterol from leucine and used acetate far more effectively than the Leishmania species for sterol biosynthesis. The fatty acid moieties of the triacylglycerols and phospholipids were produced from acetate. Leucine was also incorporated into the fatty acids to varying extents in the different organisms showing that leucine can also be metabolized in trypanosomatids to generate acetyl‐CoA.
Uncoupling proteins 2 and 3 interact with members of the 14.3.3 family
Authors: Pierrat Benoit , Ito Moriko , Hinz Willy , Simonen Marjo , Erdmann Dirk , Chiesi Michele , Heim Jutta
Published: March 2003

Uncoupling proteins (UCPs) are members of the superfamily of the mitochondrial anion carrier proteins (MATP). Localized in the inner membrane of the organelle, they are postulated to be regulators of mitochondrial uncoupling. UCP2 and 3 may play an important role in the regulation of thermogenesis and, thus, on the resting metabolic rate in humans. To identify interacting proteins that may be involved in the regulation of the activity of UCPs, the yeast two‐hybrid system was applied. Segments of hUCP2 containing the hydrophilic loops facing the intermembrane space, or combinations of these, were used to screen an adipocyte activation domain (AD) fusion library. The 14.3.3 protein isoforms θ, β, ζ were identified as possible interacting partners of hUCP2. Screening of a human skeletal muscle AD fusion library, on the other hand, yielded several clones all of them encoding the γ isoform of the 14.3.3 family. Mapping experiments further revealed that all these 14.3.3 proteins interact specifically with the C‐terminal intermembrane space domain of both hUCP2 and hUCP3 whereas no interactions could be detected with the C‐terminal part of hUCP1. Direct interaction between UCP3 and 14.3.3 θ could be demonstrated after in vitro translation by coimmunoprecipitation. When coexpressed in a heterologous yeast system, 14.3.3 proteins potentiated the inhibitory effect of UCP3 overexpression on cell growth. These findings suggest that 14.3.3 proteins could be involved in the targeting of UCPs to the mitochondria.
ARP3β, the gene encoding a new human actin‐related protein, is alternatively spliced and predominantly expressed in brain neuronal cells
Authors: Jay Philippe , Bergé‐Lefranc Jean‐Louis , Massacrier Annick , Roessler Erich , Wallis Deeann , Muenke Maximilian , Gastaldi Marguerite , Taviaux Sylvie , Cau Pierre , Berta Philippe
Published: March 2003

A cDNA encoding a new human actin‐related protein (ARP) was cloned. The corresponding protein is highly conserved with the previously described ARP3 protein, suggesting that it represents a second isoform of the human ARP3 subfamily. This new actin‐related protein was subsequently named ARP3β and represents the second example of multiple isoforms of an actin‐related protein in a single organism. The ARP3β gene was mapped to chromosome band 7q34, centromeric to Sonic Hedgehog. Gene structure analysis revealed that at least part of the observed ARP3β mRNA heterogeneity is caused by alternative splicing resulting in exon skipping. Transcripts produced after exon 2 skipping are predicted to encode truncated products, whose functionality is still unclear. An ARP3β pseudogene was detected on chromosome 2p11 by database searching. Several ARP3β mRNA species were detected by Northern blotting and their abundance varied importantly among tissues: the highest expression levels were detected in fetal and adult brain, whereas lower levels were observed in liver, muscle and pancreas. In contrast, ARP3 mRNAs were detected in all tissues tested. Using in situ hybridization, the expression of ARP3β in brain was shown to be restricted to neurons and epithelial cells from choroid plexus. This suggests a specific function for ARP3β in the physiology of the development and/or maintenance of distinct subsets of nerve cells.
Identification of a novel nuclear factor‐kappaB sequence involved in expression of urokinase‐type plasminogen activator receptor
Authors: Wang Yao , Dang Jinjun , Wang Heng , Allgayer Heike , Murrell George A. C. , Boyd Douglas
Published: March 2003

We have previously defined the promoter of human urokinase‐type plasminogen activator receptor (uPAR) gene in a 188‐bp fragment between bases −141 and +47 relative to the translation start site. Here, we report that a novel nuclear factor‐kappaB (NF‐κB)‐like sequence (5′‐GGGAGGAGTC‐3′) at −45 is located in the uPAR promoter and one of the two DNase I‐protected regions, region I between bases −51 and −30. This NF‐κB‐like motif differs at positions 7–9 from the decameric consensus sequences of NF‐κB (5′‐GGGRNNYYCC‐3′ where R indicates A or G, Y indicates C or T, and N indicates any nucleotide) and at positions 1 and 7–9 from the κB‐like motifs (5′‐HGGARNYYCC‐3′ where H indicates A, C, or T, R indicates A or G, Y indicates C or T, and N indicates any nucleotide). Nuclear extracts from HCT116 cells contain proteins that specifically bind to the NF‐κB‐like site at position −45. Mutation of the NF‐κB‐like motif decreased the binding of transcription factor NF‐κB and reduced the uPAR promoter activity in comparison with the wild‐type sequences. Co‐transfection with a dominant negative I‐κB kinase‐2 expression vector reduced uPAR promoter activity by 65–75%. These results demonstrate that a previously uncharacterized NF‐κB motif is required for uPAR promoter activity.
Molecular cloning and functional expression of triterpene synthases from pea (Pisum sativum)
Authors: Morita Masayo , Shibuya Masaaki , Kushiro Tetsuo , Masuda Kazuo , Ebizuka Yutaka
Published: March 2003

Ursane type triterpene is one of the most widespread triterpene aglycones found in plants, together with oleanane type, and these two types often occur together in the same plant. Pisum sativum is known to produce both types of triterpenes. Homology based PCRs with degenerate primers designed from the conserved sequences found in the known β‐amyrin synthases have resulted in cloning of two triterpene synthase cDNAs from immature seeds of P. sativum. They show high sequence identities to each other (78%) and also to the known β‐amyrin synthases (70–90%). ORFs of the full‐length clones named as PSY (2277 bp, codes for 759 amino acids) and PSM (2295 bp, codes for 765 amino acids) were ligated into the yeast expression vector pYES2 under the control of GAL1 promoter. Heterologous expression in yeast revealed PSY to be a P. sativumβ‐amyrin synthase. Surprisingly, however, PSM turned out to be a novel mixed amyrin synthase producing both α‐ and β‐amyrin. Several minor triterpenes were also identified as the PSM byproducts. The presence of such multifunctional triterpene synthase would account for the co‐occurence of ursane and oleanane type triterpenes in plants.
Kinetic properties of the 2‐oxoglutarate dehydrogenase complex from Azotobacter vinelandii
Authors: Bunik Victoria , Westphal Adrie H. , de Kok Aart
Published: March 2003

The 2‐oxoglutarate dehydrogenase complex was purified from Azotobacter vinelandii. The complex consists of three components, 2‐oxoglutarate dehydrogenase/decarboxylase (E1o), lipoate succinyltransferase (E2o) and lipoamide dehydrogenase (E3). Upon purification, the E3 component dissociates partially from the complex. From reconstitution experiments, the Kd for E3 was found to be 26 nm, about 30 times higher than that for the pyruvate dehydrogenase complex. The Km values for the substrates 2‐oxoglutarate, CoA and NAD+ were found to be 0.15, 0.014 and 0.17 mm, respectively. The system has a high specificity for 2‐oxoglutarate, which is determined by the action of both E1o and E2o. Above 4 mm substrate inhibition is observed.

From steady‐state inhibition experiments with substrate analogs, two substrate‐binding modes are revealed at different degrees of saturation of the enzyme with 2‐oxoglutarate. At low substrate concentrations (10−6 to 10−5 m), the binding mainly depends on the interaction of the enzyme with the substrate carboxyl groups. At a higher degree of substrate saturation (10−4 to 10−3 m) the relative contribution of the 2‐oxo group in the binding increases.

A kinetic analysis points to a single binding site for a substrate analog under steady state conditions. Saturation of this site with an analog indicates that two kinetically different complexes are formed with 2‐oxoglutarate in the course of catalysis. From competition studies with analogs it is concluded that one of these complexes is formed at the site that is sterically identical to the substrate inhibition site.

The data obtained are represented by a minimal scheme that considers formation of a precatalytic complex SE between the substrate and E1o before the catalytic complex ES, in which the substrate is added to the thiamin diphosphate cofactor, is formed. The incorrect orientation of the substrate molecule in SE or the occupation of this site by analogs is supposed to cause substrate or analog inhibition, respectively.
Isolation and characterization of RNA aptamers specific for the hepatitis C virus nonstructural protein 3 protease
Authors: Fukuda Kotaro , Vishnuvardhan Daesety , Sekiya Satoru , Hwang Joonsung , Kakiuchi Nobuko , Taira Kazunari , Shimotohno Kunitada , Kumar Penmetcha K. R. , Nishikawa Satoshi
Published: March 2003

Nonstructural protein 3 (NS3) from hepatitis C virus (HCV) is a serine protease that provides an essential function in maturation of the virus by cleaving the nonstructural regions of the viral polyprotein. The goal of this work was to isolate RNA aptamers that bind specifically to the NS3 protease active site in the truncated polypeptide ΔNS3. RNA aptamers were selected in vitro by systematic evolution of ligands by exponential enrichment (SELEX). The RNA pool for SELEX had a 30‐nucleotide randomized core region. After nine selection cycles, a pool of ΔNS3‐specific RNA aptamers were obtained. This RNA pool included 45 clones that divided into three main classes (G9‐I, II and III). These classes include the conserved sequence GA(A/U)UGGGAC. These aptamers bind to ΔNS3 with a binding constant of about 10 nm and inhibit approximately 90% of the protease activity of ΔNS3 and MBP‐NS3 (full‐length of NS3 fused with maltose binding protein). In addition, these aptamers inhibited approximately 70% of the MBP‐NS3 protease activity in the presence of the NS4A peptide P41. G9‐I aptamer appeared to be a noncompetitive inhibitor for ΔNS3 with a Ki ≈ 100 nm in the presence of P41. These results suggest that the pool of selected aptamers have potential as anti‐HCV compounds. Mutational analysis of the G9‐I aptamer demonstrated that the sequences required for protease inhibition are in stem I, stem III and loop III of the aptamer. These regions include the conserved sequence GA(A/U)UGGGAC.
Molecular characterization of the thermosensitive E1 ubiquitin‐activating enzyme cell mutant A31N‐ts20
Authors: Salvat Catherine , Acquaviva Claire , Scheffner Martin , Robbins Ian , Piechaczyk Marc , Jariel‐Encontre Isabelle
Published: March 2003

According to our current knowledge, protein ubiquitination involves three steps: activation of ubiquitin through formation of an energy‐rich bond with an E1 ubiquitin‐activating enzyme; and transfer of activated ubiquitin onto E2 ubiquitin‐conjugating enzymes, which, in turn, alone, or in combination with E3 ubiquitin–protein ligase enzymes, transfer ubiquitin onto target proteins. A31N‐ts20 cells are mouse embryo fibroblasts, thermosensitive for E1. We show here that: (a) the enzymatic activity of the enzyme is heat‐inactivatable in vitro; and (b) a major mechanism responsible for E1 inactivation in vivo consists of accelerated destruction. Surprisingly, a >90% reduction in E1 abundance little alters the formation of the bulk of protein–ubiquitin conjugates when A31N‐ts20 cells are grown at the nonpermissive temperature, indicating that cautious interpretation of results is required when studying ubiquitination of specific substrates using this cell line. Surprisingly, our data also indicate that, in vivo, ubiquitination of the various protein substrates in A31N‐ts20 cells requires different amounts of E1, indicating that this mutant cell line can be used for unveiling the existence of differences in the intimate mechanisms responsible for the ubiquitination of the various cell proteins in vivo, and for providing criteria of reliability when developing in vitro ubiquitination assays for specific proteins.
Modified amino acids and peptides as substrates for the intestinal peptide transporter PepT1
Authors: Meredith David , Temple Catherine S. , Guha Nishan , Sword Corinna J. , Boyd C. A. Richard , Collier Ian D. , Morgan Keith M. , Bailey Patrick D.
Published: March 2003

The binding affinities of a number of amino‐acid and peptide derivatives by the mammalian intestinal peptide transporter PepT1 were investigated, using the Xenopus laevis expression system. A series of blocked amino acids, namely N‐acetyl‐Phe (Ac‐Phe), phe‐amide (Phe‐NH2), N‐acetyl‐Phe‐amide (Ac‐Phe‐NH2) and the parent compound Phe, was compared for efficacy in inhibiting the uptake of the peptide [3H]‐d‐Phe‐l‐Gln. In an equivalent set of experiments, the blocked peptides Ac‐Phe‐Tyr, Phe‐Tyr‐NH2 and Ac‐Phe‐Tyr‐NH2 were compared with the parent compound Phe‐Tyr. Comparing amino acids and derivatives, only Ac‐Phe was an effective inhibitor of peptide uptake (Ki = 1.81 ± 0.37 mm). Ac‐Phe‐NH2 had a very weak interaction with PepT1 (Ki = 16.8 ± 5.64 mm); neither Phe nor Phe‐NH2 interacted with PepT1 with measurable affinity. With the dipeptide and derivatives, unsurprisingly the highest affinity interaction was with Phe‐Tyr (Ki = 0.10 ± 0.04 mm). The blocked C‐terminal peptide Phe‐Tyr‐NH2 also interacted with PepT1 with a relatively high affinity (Ki = 0.94 ± 0.38 mm). Both Ac‐Phe‐Tyr and Ac‐Phe‐Tyr‐NH2 interacted weakly with PepT1 (Ki = 8.41 ± 0.11 and 9.97 ± 4.01 mm, respectively). The results suggest that the N‐terminus is the primary binding site for both dipeptides and tripeptides. Additional experiments with four stereoisomers of Ala‐Ala‐Ala support this conclusion, and lead us to propose that a histidine residue is involved in binding the C‐terminus of dipeptides. In addition, a substrate binding model for PepT1 is proposed.
Expanding baculovirus surface display
Authors: Ernst Wolfgang J. , Spenger Alexandra , Toellner Lars , Katinger Hermann , Grabherr Reingard M.
Published: March 2003

To create a tool for eukaryotic surface display, this approach is aimed at demonstrating a direct modification of the native envelope protein gp64 of Autographa californica NPV without disturbing viral infectivity. Short affinity‐tag peptides, the biotin mimic streptagII, and the gp41 amino‐acid motif ELDKWA of HIV‐1, specific for the human monoclonal antibody 2F5, were engineered into the baculovirus major coat protein gp64 and presented on the viral surface. Two different streptag peptides were inserted at the naturally occurring NotI site at amino‐acid 278 of gp64. Additionally, the ten‐amino‐acid peptide GG‐ELDKWA‐GG, containing the epitope of mAb 2F5, was introduced into gp64 envelope protein at the same position. In all cases we were able to propagate viable virus‐achieving infectious titers in the range of wild‐type AcMNPV. Streptag and ELDKWA‐epitope surface localization on purified virus particles was demonstrated by flow cytometry and Western blot analysis. We could also show selective retention of mutant viruses by specific interaction between chimeric virions and their target counterparts, recognizing the epitope or the streptag peptide in the viral envelope. These data provide evidence that altering the surface properties of the baculovirus virion could be of value in improving baculovirus display technology and developing new applications.
Properties of three isoforms of the 116‐kDa subunit of vacuolar H+‐ATPase from a single vertebrate species
Authors: Mattsson Jan P. , Li Xinji , Peng Sheng‐Bin , Nilsson Fredrik , Andersen Per , Lundberg Lennart G. , Stone Dennis K. , Keeling David J.
Published: March 2003

Vacuolar H+‐ATPases (V‐ATPases) are involved in a wide variety of essential cellular processes. An unresolved question is how the cell regulates the activity of these proton pumps and their targeting to distinct cellular compartments. There is growing evidence for the presence of subunit diversity amongst V‐pumps, particularly regarding the 116‐kDa subunit (called the a subunit). We have cloned and characterized three isoforms (a1, a2 and a3) of this subunit from chicken. The amino‐acid sequences of these homologues are approximately 50% similar and their nucleotide differences indicate that they are products of distinct genes. The levels of mRNA expression of these isoforms was quantified by ribonuclease protection analysis. The a1 and a2 isoforms have a similar tissue distribution, with the highest level of mRNA expression in brain, an intermediate level in kidney and relatively low levels in liver and bone. In contrast, the highest level of expression of the a3 isoform is in bone and liver, with a moderate level in kidney, and the lowest level in brain. An antibody against the a1 isoform reacted with a 116 kDa protein in a brain V‐ATPase preparation that was not detected in bone or liver V‐ATPase preparations, whereas an antibody against the a3 isoform reacted with a 116‐kDa peptide in bone and liver, but not brain V‐ATPases preparations. The bone and brain V‐ATPases showed differential sensitivity to the inhibitors bafilomycin and (2Z,4E)‐5‐(5,6‐dichloro‐2‐indolyl)‐2‐methoxy‐N‐[4‐(2,2,6,6‐tetramethyl)piperidinyl]‐2,4‐pentadienamide. Thus, this work demonstrates the presence of structurally and functionally distinct V‐ATPases in a single vertebrate species.
Involvement of Gln937 of Streptococcus downei GTF‐I glucansucrase in transition‐state stabilization
Authors: Monchois Vincent , Vignon Michel , Escalier Pierre‐Claude , Svensson Birte , Russell Roy R. B.
Published: March 2003

Multiple alignment of deduced amino‐acid sequences of glucansucrases (glucosyltransferases and dextransucrases) from oral streptococci and Leuconostoc mesenteroides has shown them to share a well‐conserved catalytic domain. A portion of this domain displays homology to members of the α‐amylase family (glycoside hydrolase family 13), which all have a (β/α)8 barrel structure. In the glucansucrases, however, the α‐helix and β‐strand elements are circularly permuted with respect to the order in family 13. Previous work has shown that amino‐acid residues contributing to the active site of glucansucrases are situated in structural elements that align with those of family 13. In α‐amylase and cyclodextrin glucanotransferase, a histidine residue has been identified that acts to stabilize the transition state, and a histidine is conserved at the corresponding position in all other members of family 13. In all the glucansucrases, however, the aligned position is occupied by glutamine. Mutants of glucosyltransferase I were constructed in which this glutamine, Gln937, was changed to histidine, glutamic acid, aspartic acid, asparagine or alanine. The effects on specific activity, ability to form glucan and ability to transfer glucose to a maltose acceptor were examined. Only histidine could substitute for glutamine and maintain Michaelis–Menten kinetics, albeit at a greatly reduced kcat, showing that Gln937 plays a functionally equivalent role to the histidine in family 13. This provides additional evidence in support of the proposed alignment of the (β/α)8 barrel structures. Mutation at position 937 altered the acceptor reaction with maltose, and resulted in the synthesis of novel gluco‐oligosaccharides in which α1,3‐linked glucosyl units are joined sequentially to maltose.
Kinetics of tryptophan oxidation in plasma lipoproteins by myeloperoxidase‐generated HOCl
Authors: Jerlich Andreas , Hammel Michal , Nigon Fabienne , Chapman M. John , Schaur R. Jörg
Published: March 2003

The relative susceptibility of the apoprotein components of human lipoproteins [high‐density lipoprotein (HDL) and low‐density lipoprotein (LDL)] and their subclasses to oxidation by the myeloperoxidase/H2O2/Cl− system in vitro was studied by measuring the decrease in rate of tryptophan fluorescence. Whereas the lipoprotein‐modification rate showed a saturation type of dependence on the concentration of myeloperoxidase, a biphasic dependence on the concentration of the lipoproteins was found. High concentrations of H2O2 were also found to inhibit tryptophan oxidation in LDL but to a lesser extent in HDL. The optimal rate of LDL and HDL modification was observed at pH 6.0. HDL was modified much more rapidly than LDL, which may be due to differences in size and different relative contents of protein and lipids per particle. No differences in rates of modification of LDL subclasses were observed, when the assays were standardized to equal LDL protein concentrations, but, when standardized to equal particle mass, an optimum at subclass 8 was found, which is probably due to differences in apolipoprotein B‐100 conformation. It was concluded that HDL may have a beneficial effect in retarding LDL modification in inflammatory processes.
Transition metal‐mediated glycoxidation accelerates cross‐linking of β‐amyloid peptide
Authors: Loske Claudia , Gerdemann Andrea , Schepl Walter , Wycislo Matthias , Schinzel Reinhard , Palm Dieter , Riederer Peter , Münch Gerald
Published: March 2003

β‐Amyloid deposits, hallmarks of Alzheimer's disease, contain both sugar‐derived ‘advanced glycation end products’ (AGEs) and copper and iron ions. Our in vitro experiments using synthetic β‐amyloid peptide and glucose or fructose show that formation of covalently cross‐linked high‐molecular‐mass β‐amyloid peptide oligomers is accelerated by micromolar amounts of copper (Cu+, Cu2+) and iron (Fe2+, Fe3+) ions. Formation of these covalent AGE cross‐links can be inhibited by capping agents of amino groups, redox‐inactive metal chelators and antioxidants, suggesting that these drugs may be able to slow down the formation of insoluble β‐amyloid deposits in vivo and possibly the progression of Alzheimer's disease.
Treatment of cells with the polyamine analog N1,N11‐diethylnorspermine retards S phase progression within one cell cycle
Authors: Alm Kersti , Berntsson Pia S. H. , Kramer Debora L. , Porter Carl W. , Oredsson Stina M.
Published: March 2003

When Chinese hamster ovary cells were seeded in the presence of the spermine analog N1,N11‐diethylnorspermine (DENSPM), cell proliferation ceased; this was clearly apparent by cell counting 2 days after seeding the cells. However, 1 day after seeding there was a slight difference in cell number between control and DENSPM‐treated cultures. To investigate the reason for this easily surpassed slight difference, we used a sensitive bromodeoxyuridine/flow cytometry method. Cell cycle kinetics were studied during the first cell cycle after seeding cells in the absence or presence of DENSPM. Our results show that DENSPM treatment did not affect the progression of the cells through G1 or the first G1/S transition that took place after seeding the cells. The first cell cycle effect was a delay in S phase as shown by an increase in the DNA synthesis time. The following G2/M transition was not affected by DENSPM treatment. DENSPM treatment inhibited the transient increases in putrescine, spermidine, and spermine pools that took place within 24 h after seeding. Thus, in conclusion, the first cell cycle phase affected by the inhibition of polyamine biosynthesis caused by DENSPM was the S phase. Prolongation of the other cell cycle phases occurred at later time points, and the G1 phase was affected before the G2/M phase.
Modulation of molecular mechanisms involved in protein synthesis machinery as a new tool for the control of cell proliferation
Authors: Caraglia Michele , Budillon Alfredo , Vitale Giovanni , Lupoli Giovanni , Tagliaferri Pierosandro , Abbruzzese Alberto
Published: March 2003

In the past years, the attention of scientists has focused mainly on the study of the genetic information and alterations that regulate eukaryotic cell proliferation and that lead to neoplastic transformation. All therapeutic strategies against cancer are, to date, directed at DNA either with cytotoxic drugs or gene therapy. Little or no interest has been aroused by protein synthesis mechanisms. However, an increasing body of data is emerging about the involvement of translational processes and factors in control of cell proliferation, indicating that protein synthesis can be an additional target for anticancer strategies. In this paper we review the novel insights on the biochemical and molecular events leading to protein biosynthesis and we describe their involvement in cell proliferation and tumorigenesis. A possible mechanistic explanation is given by the interactions that occur between protein synthesis machinery and the proliferative signal transduction pathways and that are therefore suitable targets for indirect modulation of protein synthesis. We briefly describe the molecular tools used to block protein synthesis and the attempts made at increasing their efficacy. Finally, we propose a new multimodal strategy against cancer based on the simultaneous intervention on protein synthesis and signal transduction.
Mechanism of reaction of myeloperoxidase with hydrogen peroxide and chloride ion
Authors: Furtmüller Paul G. , Obinger Christian , Hsuanyu Yuchiong , Dunford H. Brian
Published: March 2003

The reaction of myeloperoxidase compound I (MPO‐I) with chloride ion is widely assumed to produce the bacterial killing agent after phagocytosis. Two values of the rate constant for this important reaction have been published previously: 4.7 × 106 m−1·s−1 measured at 25 °C [Marquez, L.A. and Dunford, H.B. (1995) J. Biol. Chem.270, 30434–30440], and 2.5 × 104 m−1·s−1 at 15 °C [Furtmüller, P.G., Burner, U. & Obinger, C. (1998) Biochemistry37, 17923–17930]. The present paper is the result of a collaboration of the two groups to resolve the discrepancy in the rate constants. It was found that the rate constant for the reaction of compound I, generated from myeloperoxidase (MPO) and excess hydrogen peroxide with chloride, decreased with increasing chloride concentration. The rate constant published in 1995 was measured over a lower chloride concentration range; the 1998 rate constant at a higher range. Therefore the observed conversion of compound I to native enzyme in the presence of hydrogen peroxide and chloride ion cannot be attributed solely to the single elementary reaction MPO‐I + Cl− → MPO + HOCl. The simplest mechanism for the overall reaction which fit the experimental data is the following:

image

image

image

where MPO‐I–Cl− is a chlorinating intermediate. We can now say that the 1995 rate constant is k2 and the corresponding reaction is rate‐controlling at low [Cl−]. At high [Cl−], the reaction with rate constant k3 is rate controlling. The 1998 rate constant for high [Cl−] is a composite rate constant, approximated by k2k3/k−2. Values of k1 and k−1 are known from the literature. Results of this study yielded k2 = 2.2 × 106 m−1·s−1, k−2 = 1.9 × 105 s−1 and k3 = 5.2 × 104 s−1. Essentially identical results were obtained using human myeloperoxidase and beef spleen myeloperoxidase.
Kiwi protein inhibitor of pectin methylesterase
Authors: Camardella Laura , Carratore Vito , Ciardiello Maria A. , Servillo Luigi , Balestrieri Ciro , Giovane Alfonso
Published: March 2003

A protein acting as a powerful inhibitor of plant pectin methylesterase was isolated from kiwi (Actinidia chinensis) fruit. The complete amino‐acid sequence of the pectin methylesterase inhibitor (PMEI) was determined by direct protein analysis. The sequence comprises 152 amino‐acid residues, accounting for a molecular mass of 16 277 Da. The far‐UV CD spectrum indicated a predominant alpha‐helix conformation in the secondary structure. The protein has five cysteine residues but neither tryptophan nor methionine. Analysis of fragments obtained after digestion of the protein alkylated without previous reduction identified two disulfide bridges connecting Cys9 with Cys18, and Cys74 with Cys114; Cys140 bears a free thiol group. A database search pointed out a similarity between PMEI and plant invertase inhibitors. In particular, the four Cys residues, which in PMEI are involved in the disulfide bridges, are conserved. This allows us to infer that also in the homologous proteins, whose primary structure was deduced only by cDNA sequencing, those cysteine residues are engaged in two disulfide bridges, and constitute a common structural motif. The comparison of the sequence of these inhibitors confirms the existence of a novel class of proteins with moderate but significant sequence conservation, comprising plant proteins acting as inhibitors of sugar metabolism enzymes, and probably involved in various steps of plant development.
Interaction of the neurotoxic and nontoxic secretory phospholipases A2 with the crotoxin inhibitor from Crotalus serum
Authors: Faure Grazyna , Villela Christina , Perales Jonas , Bon Cassian
Published: March 2003

Crotalus durissus terrificus snakes possess a protein in their blood, named crotoxin inhibitor from Crotalus serum (CICS), which protects them against crotoxin, the main toxin of their venom. CICS neutralizes the lethal potency of crotoxin and inhibits its phospholipase A2 (PLA2) activity. The aim of the present study is to investigate the specificity of CICS towards snake venom neurotoxic PLA2s (β‐neurotoxins) and nontoxic mammalian PLA2s. This investigation shows that CICS does not affect the enzymatic activity of pancreatic and nonpancreatic PLA2s, bee venom PLA2 and Elapidaeβ‐neurotoxins but strongly inhibits the PLA2 activity of Viperidaeβ‐neurotoxins. Surface plasmon resonance and PAGE studies further demonstrated that CICS makes complexes with monomeric and multimeric Viperidaeβ‐neurotoxins but does not interact with nontoxic PLA2s. In the case of dimeric β‐neurotoxins from Viperidae venoms (crotoxin, Mojave toxin and CbICbII), which are made by the noncovalent association of a PLA2 with a nonenzymatic subunit, CICS does not react with the noncatalytic subunit, instead it binds tightly to the PLA2 subunit and induces the dissociation of the heterocomplex. In vitro assays performed with Torpedo synaptosomes showed a protective action of CICS against Viperidaeβ‐neurotoxins but not against other PLA2 neurotoxins, on primary and evoked liberation of acetylcholine. In conclusion, CICS is a specific PLA2 inhibitor of the β‐neurotoxins from the Viperidae family.