bluuebunny/crossref_metadata_2025 huggingface dataset:
H19 sense and antisense transgenes modify insulin‐like growth factor‐II mRNA levels
Authors: Wilkin Françoise , Paquette Jean , Ledru Elisabeth , Mamelin Catherine , Pollak Michael , Deal Cheri L.
Published: March 2003

The oppositely‐imprinted genes insulin‐like growth factor‐II (IGF2) and H19, a putative tumor suppressor, often show coordinate, reciprocal regulation and are believed to play a role in carcinogenesis. To explore the possible interactions between these genes, we stably transfected diHepG2 cells with a plasmid containing either the sense or the antisense H19 cDNA sequences and verified their expression by Northern analysis and by RNase protection analysis. Levels of H19, IGF2 and γ‐actin mRNA were quantified by competitive RT‐PCR analysis. Although H19 sense transgene overexpression (n = 24 clones) did not decrease the low, basal levels of IGF2 mRNA compared to control cells, levels of IGF2 mRNA were positively correlated with the levels of H19 antisense mRNA (P < 0.0001, n = 40 clones). Furthermore, the increase in IGF2 mRNA level was accompanied by an elevation of IGF‐II peptide in conditioned media. To see if H19 mRNA had a specific effect on transcription, we also performed transient transfections with reporter gene constructs containing IGF2 promoter 3 in the presence of sense or antisense H19 cDNA sequences under control of a cytomegalovirus promoter. We show a lower reporter gene activity from reporter gene constructs in the presence of sense H19 cDNA than from those with antisense or neomycin. Our results suggest that H19 participates in the repression of IGF2, at least in part through effects on IGF2 transcription, an effect which may contribute to its action as a tumor suppressor.
Benign prostatic hyperplasia‐associated prostate‐specific antigen (BPSA) shows unique immunoreactivity with anti‐PSA monoclonal antibodies
Authors: Wang Tang J. , Slawin Kevin M. , Rittenhouse Harry G. , Millar Lisa S. , Mikolajczyk Stephen D.
Published: March 2003

We previously identified a modified molecular form of prostate‐specific antigen that is significantly elevated in the nodular transition zone tissue of prostates with benign prostatic hyperplasia. This prostate‐specific antigen form, designated BPSA, is inactive and contains clipped polypeptide bonds at amino‐acid residues Lys145–146 and Lys182–183. BPSA is not elevated in prostate cancer tissues and may therefore be a prostate‐specific antigen marker to better discriminate benign prostatic hyperplasia from early prostate cancer. In this work we characterize the immunoreactivity of BPSA in competition assays with prostate‐specific antigen using anti‐prostate‐specific antigen mAb recognizing six different epitopes on the prostate‐specific antigen molecule. One mAb showed > 50% loss of immunoreactivtiy with BPSA compared with prostate‐specific antigen, while the binding of two mAbs was largely unaffected and three mAbs had intermediate reactivity. BPSA purified from prostate tissue and seminal plasma, as well as BPSA generated in vitro by mild trypsin‐treatment were found to have a similar pattern of reactivity to the six mAbs. However, other forms of inactive seminal plasma prostate‐specific antigen, either intact or clipped at Lys145 only, had immunoreactivity similar to total prostate‐specific antigen. These results demonstrate that BPSA has unique immunological properties from other forms of prostate‐specific antigen, which should allow the development of BPSA‐specific mAbs for the study of benign prostatic hyperplasia. Measurement of BPSA levels in the serum may help discriminate benign prostatic hyperplasia from early prostate cancer.
The structure of the carbohydrate backbone of the lipopolysaccharides from Bordetella hinzii and Bordetella bronchiseptica
Authors: Vinogradov Evgeny
Published: March 2003

The structure of the core‐lipid A region of the lipopolysaccharides from Bordetella hinzii and Bordetella bronchiseptica has been analyzed. Lipopolysaccharides were deacylated using strong alkaline hydrolysis, the products were separated by high performance anion‐exchange chromatography and analyzed by NMR and mass spectrometry. The following structure of the products can be deduced from the experimental results:

where for the product from Bordetella hinzii N = H, R = H, β‐FucN4N‐ or partially N‐acetylated Sug‐(1–3)‐β‐FucN4N and for the product from Bordetella bronchiseptica N = α‐Hep, R = H, β‐FucN4N, β‐FucN4NMe or partially N‐acetylated Sug‐(1–3)‐β‐FucN4N or Sug‐(1–3)‐β‐FucN4NMe; Sug = 2,3‐diamino‐2,3,4‐trideoxy‐hex‐4‐enuronopyranosyl.
The antibiotic and anticancer active aurein peptides from the Australian Bell Frogs Litoria aurea and Litoria raniformis
Authors: Rozek Tomas , Wegener Kate L. , Bowie John H. , Olver Ian N. , Carver John A. , Wallace John C. , Tyler Michael J.
Published: March 2003

Seventeen aurein peptides are present in the secretion from the granular dorsal glands of the Green and Golden Bell Frog Litoria aurea, and 16 from the corresponding secretion of the related Southern Bell Frog L. raniformis. Ten of these peptides are common to both species. Thirteen of the aurein peptides show wide‐spectrum antibiotic and anticancer activity. These peptides are named in three groups (aureins 1–3) according to their sequences. Amongst the more active peptides are aurein 1.2 (GLFDIIKKIAESF‐NH2), aurein 2.2 (GLFDIVKKVVGALGSL‐NH2) and aurein 3.1 (GLFDIVKKIAGHIAGSI‐NH2). Both L. aurea and L. raniformis have endoproteases that deactivate the major membrane‐active aurein peptides by removing residues from both the N‐ and C‐termini of the peptides. The most abundant degradation products have two residues missing from the N‐terminal end of the peptide. The solution structure of the basic peptide, aurein 1.2, has been determined by NMR spectroscopy to be an amphipathic α‐helix with well‐defined hydrophilic and hydrophobic regions. Certain of the aurein peptides (e.g. aureins 1.2 and 3.1) show anticancer activity in the NCI test regime, with LC50 values in the 10−5−10−4 m range. The aurein 1 peptides have only 13 amino‐acid residues: these are the smallest antibiotic and anticancer active peptides yet reported from an anuran. The longer aurein 4 and 5 peptides, e.g. aurein 4.1 (GLIQTIKEKLKELAGGLVTGIQS‐OH) and aurein 5.1 (GLLDIVTGLLGNLIVDVLKPKTPAS‐OH) show neither antibacterial nor anticancer activity.
Transgalactosylation by thermostable β‐glycosidases from Pyrococcus furiosus and Sulfolobus solfataricus
Authors: Petzelbauer Inge , Reiter Andreas , Splechtna Barbara , Kosma Paul , Nidetzky Bernd
Published: March 2003

The hyperthermostable β‐glycosidases from the Archaea Sulfolobus solfataricus (SsβGly) and Pyrococcus furiosus (CelB) hydrolyse β‐glycosides of d‐glucose or d‐galactose with relaxed specificities pertaining to the nature of the leaving group and the glycosidic linkage. To determine how specificity is manifested under conditions of kinetically controlled transgalactosylation, the major transfer products formed during the hydrolysis of lactose by these enzymes have been identified, and their appearance and degradation have been determined in dependence of the degree of substrate conversion. CelB and SsβGly show a marked preference for making new β(1→3) and β(1→6) glycosidic bonds by intermolecular as well as intramolecular transfer reactions. The intramolecular galactosyl transfer of CelB, relative to glycosidic‐bond cleavage and release of glucose, is about 2.2 times that of SsβGly and yields β‐d‐Galp‐(1→6)‐d‐Glc and β‐d‐Galp‐(1→3)‐d‐Glc in a molar ratio of ≈ 1 : 2. The partitioning of galactosylated SsβGly between reaction with sugars [kNu (m−1·s−1)] and reaction with water [kwater (s−1)] is about twice that of CelB. It gives a mixture of linear β‐d‐glycosides, chiefly trisaccharides at early reaction times, in which the prevailing new glycosidic bonds are β(1→6) and β(1→3) for the reactions catalysed by SsβGly and CelB, respectively. The accumulation of β‐d‐Galp‐(1→6)‐d‐Glc at the end of lactose hydrolysis reflects a 3–10‐fold specificity of both enzymes for the hydrolysis of β(1→3) over β(1→6) linked glucosides. Galactosyl transfer from SsβGly or CelB to d‐glucose occurs with partitioning ratios, kNu/kwater, which are seven and > 170 times those for the reactions of the galactosylated enzymes with 1‐propanol and 2‐propanol, respectively. Therefore, the binding interactions with nucleophiles contribute chiefly to formation of new β‐glycosides during lactose conversion. Likewise, noncovalent interactions with the glucose leaving group govern the catalytic efficiencies for the hydrolysis of lactose by both enzymes. They are almost fully expressed in the rate‐limiting first‐order rate constant for the galactosyl transfer from the substrate to the enzyme and lead to a positive deviation by ≈ 2.5 log10 units from structure–reactivity correlations based on the pKa of the leaving group.
Early activation of lipoxygenase in lentil (Lens culinaris) root protoplasts by oxidative stress induces programmed cell death
Authors: Maccarrone Mauro , Van Zadelhoff Guus , Veldink Gerrit A. , Vliegenthart Johannes F. G. , Finazzi‐Agrò Alessandro
Published: March 2003

Oxidative stress caused by hydrogen peroxide (H2O2) triggers the hypersensitive response of plants to pathogens. Here, short pulses of H2O2 are shown to cause death of lentil (Lens culinaris) root protoplasts. Dead cells showed DNA fragmentation and ladder formation, typical hallmarks of apoptosis (programmed cell death). DNA damage was evident 12 h after the H2O2 pulse and reached a maximum 12 h later. The commitment of cells to apoptosis caused by H2O2 was characterized by an early increase of lipoxygenase activity, of ultraweak luminescence and of membrane lipid peroxidation, which reached 720, 350 and 300% of controls, respectively, at 6 h after H2O2 treatment. Increased lipoxygenase activity was paralleled by an increase of its protein and mRNA level. Lipoxygenase inhibitors nordihydroguaiaretic acid, eicosatetraynoic acid and plamitoyl ascorbate prevented H2O2‐induced DNA fragmentation and ultraweak luminescence, only when added together with H2O2, but not when added 8 h afterwards. Inhibitory anti‐lipoxygenase monoclonal antibodies, introduced into the protoplasts by electroporation, protected cells against H2O2‐induced apoptosis. On the other hand, lentil lipoxygenase products 9‐ and 13‐hydroperoxy‐octadecadienoic acids and their reduced alcohol derivatives were able to force the protoplasts into apoptosis. Altogether, these findings suggest that early activation of lipoxygenase is a key element in the execution of apoptosis induced by oxidative stress in plant cells, in a way surprisingly similar to that observed in animal cells.
Synthetic substrates and inhibitors of β‐poly(L‐malate)‐hydrolase (polymalatase)
Authors: Gasslmaier Bernd , Krell Christoph M. , Seebach Dieter , Holler Eggehard
Published: March 2003

Polymalatase from Physarum polycephalum calalysed the hydrolysis of β‐poly[l‐malate] and of the synthetic compounds β‐di(l‐malate), β‐tetra(l‐malate), β‐tetra(l‐malate) β‐propylester, and l‐malate β‐methylester. Cyclic β‐tri(l‐malate), cyclic β‐tetra(l‐malate), and d‐malate β‐methylester were not cleaved, but were competitive inhibitors. The O‐terminal acetate of β‐tetra(l‐malate) was neither a substrate nor an inhibitor. l‐Malate was liberated; the Km,Ki and Vmax values were measured. The appearance of comparable amounts of β‐tri(l‐malate), and β‐di(l‐malate) during the cleavage of β‐tetra(l‐malate) indicated a distributive mechanism for small substrates. The accumulation of a series of oligomers, peaking with the 11‐mer and 12‐mer in the absence of higher intermediates, indicated that the depolymerization of β‐poly(l‐malate) was processive. The results indicate that β‐poly(l‐malate) is anchored at its OH‐terminus by the highly specific binding of the penultimate malyl residue. The malyl moieties beyond 12 residues downstream from the OH‐terminus extend into a diffuse second, electrostatic binding site. The catalytic site joins the first binding site, accounting for the cleavage of the polymer into malate residues. It is proposed that the enzyme does not dissociate from β‐poly(l‐malate) during hydrolysis, when both sites are filled with the polymer. When only the first binding site is filled, the reaction partitions at each oligomer between hydrolysis and dissociation.
A novel hemocyte‐specific membrane protein of Sarcophaga (flesh fly)
Authors: Hori Shohei , Kobayashi Ayako , Natori Shunji
Published: March 2003

Extensive tissue remodeling takes place during metamorphosis of holometabolous insects. It has been shown that hemocytes play crucial roles in the recognition and elimination of apoptotic cells and larval tissue fragments produced during metamorphosis. We report the immunoaffinity purification, cDNA cloning, and characterization of a prepupal hemocyte membrane protein of Sarcophaga (flesh fly) with a molecular mass of 120 kDa. This protein is a novel type I transmembrane protein with 18 repeats of an epidermal growth factor‐like domain in the predicted extracellular region. Expression of the protein was restricted exclusively to prepupal hemocytes. This protein is suggested to be a scavenger receptor for tissue remodeling.
A novel type of carbohydrate–protein linkage region in the tyrosine‐bound S‐layer glycan of Thermoanaerobacterium thermosaccharolyticum D120‐70
Authors: Schäffer Christina , Dietrich Katharina , Unger Bernd , Scheberl Andrea , Rainey Fred A. , Kählig Hanspeter , Messner Paul
Published: March 2003

The surface‐layer (S‐layer) protein of Thermoanaerobacterium thermosaccharolyticum D120‐70 contains glycosidically linked glycan chains with the repeating unit structure →4)[α‐d‐Galp‐(1→2)]‐α‐l‐Rhap‐(1→3)[β‐d‐Glcp‐(1→6)]‐β‐d‐Manp‐(1→4)‐α‐l‐Rhap‐(1→3)‐α‐d‐Glcp‐(1→. After proteolytic degradation of the S‐layer glycoprotein, three glycopeptide pools were isolated, which were analyzed for their carbohydrate and amino‐acid compositions. In all three pools, tyrosine was identified as the amino‐acid constituent, and the carbohydrate compositions corresponded to the above structure. Native polysaccharide PAGE showed the specific heterogeneity of each pool. For examination of the carbohydrate–protein linkage region, the S‐layer glycan chain was partially hydrolyzed with trifluoroacetic acid. 1D and 2D NMR spectroscopy, including a novel diffusion‐edited difference experiment, showed the O‐glycosidic linkage region β‐d‐glucopyranose→O‐tyrosine. No evidence was found of additional sugars originating from a putative core region between the glycan repeating units and the S‐layer polypeptide. For the determination of chain‐length variability in the S‐layer glycan, the different glycopeptide pools were investigated by matrix‐assisted laser desorption ionization‐time of flight mass spectrometry, revealing that the degree of polymerization of the S‐layer glycan repeats varied between three and 10. All masses were assigned to multiples of the repeating units plus the peptide portion. This result implies that no core structure is present and thus supports the data from the NMR spectroscopy analyses. This is the first observation of a bacterial S‐layer glycan without a core region connecting the carbohydrate moiety with the polypeptide portion.
Molecular characterization of dipeptidyl peptidase activity in serum
Authors: Durinx Christine , Lambeir Anne‐Marie , Bosmans Eugène , Falmagne Jean‐Bernard , Berghmans Raf , Haemers Achiel , Scharpé Simon , De Meester Ingrid
Published: March 2003

Dipeptidyl peptidase IV (DPPIV, EC 3.4.14.5) is a serine type protease with an important modulatory activity on a number of chemokines, neuropeptides and peptide hormones. It is also known as CD26 or adenosine deaminase (ADA; EC 3.5.4.4) binding protein. DPPIV has been demonstrated on the plasmamembranes of T cells and activated natural killer or B cells as well as on a number of endothelial and differentiated epithelial cells. A soluble form of CD26/DPPIV has been described in serum. Over the past few years, several related enzymes with similar dipeptidyl peptidase activity have been discovered, raising questions on the molecular origin(s) of serum dipeptidyl peptidase activity. Among them attractin, the human orthologue of the mouse mahogany protein, was postulated to be responsible for the majority of the DPPIV‐like activity in serum. Using ADA‐affinity chromatography, it is shown here that 95% of the serum dipeptidyl peptidase activity is associated with a protein with ADA‐binding properties. The natural protein was purified in milligram quantities, allowing molecular characterization (N‐terminal sequence, glycosylation type, CD‐spectrum, pH and thermal stability) and comparison with CD26/DPPIV from other sources. The purified serum enzyme was confirmed as CD26.
Thermodynamic analysis of helix‐engineered forms of the activation domain of human procarboxypeptidase A2
Authors: Fernández Ana M. , Villegas Virtudes , Martínez Jose C. , van Nuland Nico A. J. , Conejero‐Lara Francisco , Avilés Francesc X. , Serrano Luis , Filimonov Vladimir V. , Mateo Pedro L.
Published: March 2003

Thermodynamic characterization of the activation domain of human procarboxypeptidase A2, ADA2h, and its helix‐engineered mutants was carried out by differential scanning calorimetry. The mutants were engineered by changing residues in the exposed face of the two α helices in order to increase their stability. At neutral and alkaline pH the three mutants, α‐helix 1 (M1), α‐helix 2 (M2) and α‐helix 1 and α‐helix 2 (DM), were more stable than the wild‐type domain, in the order DM, M2, M1 and wild‐type. Under these conditions the CD and NMR spectra of all the variants are very similar, indicating that this increase in stability is not the result of gross structural changes. Calorimetric analysis shows that the stabilizing effect of mutating the water‐exposed surfaces of the helices seems to be mainly entropic, because the mutations do not change the enthalpy or the increase in heat capacity of denaturation. The unfolding behavior of all variants changes under acidic conditions: whereas wild‐type and M1 have a strong tendency to aggregate, giving rise to a β conformation upon unfolding, M2 and DM unfold reversibly, M2 being more stable than DM. CD and NMR experiments at pH 3.0 suggest that a region involving residues of the second and third β strands as well as part of α‐helix 1 changes its conformation. It seems that the enhanced stability of the altered conformation of M2 and DM reduces the aggregation tendency of ADA2h at acidic pH.
Caspase‐3 mediated cleavage of HsRad51 at an unconventional site
Authors: Flygare Jenny , Hellgren Dennis , Wennborg Anders
Published: March 2003

The human recombinase HsRad51 is cleaved during apoptosis. We have earlier observed cleavage of the 41‐kDa full‐length protein into a 33‐kDa product in apoptotic Jurkat cells and in in vitro translated HsRad51 after treatment with activated S‐100 extract. In this study, site‐directed mutagenesis was used for mapping of the cleavage site to AQVD274 ↓ G, which does not correspond to a conventional caspase cleavage site. The absence of HsRad51 cleavage in staurosporine‐treated apoptotic MCF‐7 cells, which lack caspase‐3, indicates that caspase‐3 is essential for HsRad51 cleavage in vivo. Cleavage into the 33‐kDa fragment was generated by recombinant caspase‐3 and ‐7 in in vitro translated wild type HsRad51, but not in the HsRad51 AQVE274 ↓ G mutant. Similarly, HsRad51 of Jurkat cell extracts was cleaved into the 33‐kDa product by recombinant caspase‐3, whereas caspase‐7 failed to cleave endogenous HsRad51. The cleavage of in vitro translated wild type and AQVE274 ↓ G mutant HsRad51 as well as of endogenous HsRad51 also gave rise to a smaller fragment, which corresponds in size to a recently reported DVLD187 ↓ N HsRad51 cleavage product. In Jurkat cell extracts, the AQVD274 ↓ G and DVLD187 ↓ N cleavage products of HsRad51 appeared at equal concentrations of caspase‐3. Moreover both fragments were generated by induction of apoptosis in MDA‐MB 157 cells with staurosporine and in Jurkat cells with camptothecin. Thus, two sites in the HsRad51 sequence are targets for caspase cleavage both in vitro and in vivo.
Tumor necrosis factor‐α mRNA remains unstable and hypoadenylated upon stimulation of macrophages by lipopolysaccharides
Authors: Mijatovic Tatjana , Houzet Laurent , Defrance Patrick , Droogmans Louis , Huez Georges , Kruys Véronique
Published: March 2003

TNF‐α gene expression is regulated at transcriptional and post‐transcriptional levels in mouse macrophages. The post‐transcriptional regulation is mediated by the AU‐rich element (ARE) located in the TNF‐α mRNA 3′ untranslated region (UTR), which controls its translation and stability. In resting macrophages, the ARE represses TNF‐α mRNA translation. Activation of macrophages with various agents [for example lipopolysaccharide (LPS), viruses] results in translational derepression, leading to the production of high levels of TNF‐α. TNF‐α ARE has also been shown to confer mRNA instability as its deletion from the mouse genome leads to an increase in the TNF‐α mRNA half‐life [Kontoyiannis, D., Pasparakis, M., Pizzaro, T., Cominelli, F. & Kollias, G. (1999) Immunity10, 387–398]. In this study, we measured the half‐life as well as the poly(A) tail length of TNF‐α mRNA in the course of macrophage activation by LPS. We report that TNF‐α mRNA is short lived even in conditions of maximal TNF‐α synthesis. Moreover, TNF‐α mRNA is hypoadenylated in a constitutive manner. These results reveal that TNF‐α mRNA rapid turnover does not constitute a regulatory step of TNF‐α biosynthesis in macrophages and that TNF‐α mRNA translational activation upon LPS stimulation is not accompanied by a change of poly(A) tail length.
Structural domains involved in the RNA folding activity of RNA helicase II/Gu protein
Authors: Valdez Benigno C.
Published: March 2003

RNA helicase II/Gu (RH II/Gu) is a nucleolar protein that unwinds dsRNA in a 5′ to 3′ direction, and introduces a secondary structure into a ssRNA. The helicase domain is at the N‐terminal three‐quarters of the molecule and the foldase domain is at the C‐terminal quarter. The RNA folding activity of RH II/Gu is not a mere artifact of its binding to RNA. This study narrows down the RNA foldase domain to amino acids 749–801 at the C‐terminus of the protein. Dissection of this region by deletion and site‐directed mutagenesis shows that the four FRGQR repeats, as well as the C‐terminal end bind RNA independently. These juxtaposed subdomains are both important for the RNA foldase activity of RH II/Gu. Mutation of either repeat 2 or repeat 4, or simultaneous mutation of Lys792, Arg793 and Lys797 at the C‐terminal end of RH II/Gu to alanines inhibits RNA foldase activity. The last 17 amino acids of RH II/Gu can be replaced by an RNA binding motif from nucleolar protein p120 without a deleterious effect on its foldase activity. A model is proposed to explain how RH II/Gu binds and folds an RNA substrate.
Simple formal kinetics for the reversible uptake of molecular hydrogen by [Ni–Fe] hydrogenase from Desulfovibrio gigas
Authors: De Lacey Antonio L. , Moiroux Jacques , Bourdillon Christian
Published: March 2003

Enzymatic electrocatalysis, triggered and monitored by means of cyclic voltammetry, enabled us to achieve quantitative analysis of the kinetics of the hydrogenase catalyzed process, in the 7.8–10.0 pH range, in the presence of an electrochemically generated redox mediator. The quantitative analysis can be carried out by use of a quite simple SRC model. The simplicity of the SRC model is compatible with the existence of multiple redox microstates, which can be combined in a potential adjustable triangular mechanism consisting of three catalytic cycles, which are formally identical from the kinetic point of view. The steps involved in the kinetic control of the reversible process are H2 uptake or production at the Ni–Fe catalytic site and the intermolecular electron transfer between the mediator and the distal [4Fe−4S] cluster. The related rate constants have been determined. For the two accompanying intramolecular electron transfers which proceed at equilibrium, the equilibrium constants were found to be in very good agreement with previously published data.
Identification of IκBα as a substrate of Fas‐associated phosphatase‐1
Authors: Nakai Yuji , Irie Shinji , Sato Taka‐Aki
Published: March 2003

Fas (APO‐1/CD95), a member of the tumor necrosis factor receptor (TNFR)/nerve growth factor receptor (NGFR) superfamily, is a cell‐surface molecule that induces apoptosis upon activation. Fas‐associated phosphatase‐1 (FAP‐1) is a 250‐kDa protein tyrosine phosphatase (PTP) that is associated with the negative regulatory domain of Fas (C‐terminal 15 amino acids). Human tumor cell lines become resistant to Fas‐mediated apoptosis when transfected with FAP‐1, indicating that FAP‐1 functions as a negative regulator in Fas‐mediated death signaling. However, the mechanisms by which FAP‐1 inhibits apoptosis are still unclear. In order to determine how FAP‐1 affects the signaling mediated by Fas, we set out to identify substrates of FAP‐1. Toward this end, we prepared synthetic proteins with either the catalytic domain of FAP‐1 (C‐terminal 399 amino acids) or its inactive form (Cys2408→Ser) fused to glutathione‐S‐transferase (GST). Using an in vitro dephosphorylation reaction, we found that FAP‐1 dephosphorylates IκBα. Furthermore, a substrate trapping mutant was found to bind tyrosine‐phosphorylated IκBα. Taken together, our data confirm that IκBα is a substrate of FAP‐1.
Functional characterization of isoschizomeric His‐Cys box homing endonucleases from Naegleria
Authors: Elde Morten , Willassen Nils P. , Johansen Steinar
Published: March 2003

Several species within the amoeboflagellate genus Naegleria harbor an optional ORF containing group I introns in their nuclear small subunit ribosomal DNA. The different ORFs encode homing endonucleases with 65 to 95% identity at the amino‐acid level. I‐NjaI, I‐NanI and I‐NitI, from introns in Naegleria jamiesoni, N. andersoni and N. italica, respectively, were analyzed in more detail and found to be isoschizomeric endonucleases that recognize and cleave an approximal 19‐bp partially symmetrical sequence, creating a pentanucleotide 3′ overhang upon cleavage. The optimal conditions for cleavage activity with respect to temperature, pH, salt and divalent metal ions were investigated. The optimal cleavage temperature for all three endonucleases was found to be 37 °C and the activity was dependent on the concentration of NaCl with an optimum at 200 m
m. Divalent metal ions, primarily Mg2+, are essential for Naegleria endonuclease activity. Whereas both Mn2+ and Ca2+ could substitute for Mg2+, but with a slower cleavage rate, Zn2+ was unable to support cleavage. Interestingly, the pH dependence of DNA cleavage was found to vary significantly between the I‐NitI and I‐NjaI/I‐NanI endonucleases with optimal pH values at 6.5 and 9, respectively. Site‐directed mutagenesis of conserved I‐NjaI residues strongly supports the hypothesis that Naegleria homing endonucleases share a similar zinc‐binding structure and active site with the His‐Cys box homing endonuclease I‐PpoI.
Pyrokinin neuropeptides in a crustacean
Authors: Torfs Pieter , Nieto Julia , Cerstiaens Anja , Boon Doris , Baggerman Geert , Poulos Constantine , Waelkens Etienne , Derua Rita , Calderón Jorge , De Loof Arnold , Schoofs Liliane
Published: March 2003

Identification of substances able to elicit physiological or behavioural processes that are related to reproduction would greatly contribute to the domestication of commercially important crustaceans that do not reproduce easily in captivity. Crustaceans are thought to release urine signals used for chemical communication involved in courtship behaviour. In contrast to insects, very little is known about the endocrinological processes underlying this phenomenon. Therefore, an extract of 3500 central nervous systems of female white shrimp Penaeus vannamei was screened for myotropic activity in order to purify pyrokinin‐like peptides that belong to the pyrokinin/PBAN neuropeptide family. Members of this family regulate reproductive processes in insects, including pheromone biosynthesis. Purification of these pyrokinins was achieved by a combination of reversed‐phase and normal‐phase chromatography. Subsequent characterization by mass spectrometry, Edman degradation and peptide synthesis resulted in the elucidation of two novel peptides. Pev‐PK 1 has the primary sequence DFAFSPRL‐NH2 and a second peptide (Pev‐PK 2) is characterized as the nonapeptide ADFAFNPRL‐NH2. Pev‐PK 1 contains the typical FXPRL‐NH2 (X = G, S, T or V) C‐terminal sequence that characterizes members of the versatile pyrokinin/PBAN family. Pev‐PK 2 displays an Asn residue at the variable X position of the core pyrokinin sequence. These crustacean pyrokinins are the first to be found in a noninsect. The synthetic peptides display myotropic activity on the Leucophaea maderae as well as on the Astacus leptodactylus hindgut.
The stability and folding process of amyloidogenic mutant human lysozymes
Authors: Takano Kazufumi , Funahashi Jun , Yutani Katsuhide
Published: March 2003

Amyloid deposits are frequently formed by mutant proteins that have a lower stability than the wild‐type proteins. Some reports, however, have shown that mutant‐induced thermodynamic destabilization is not always a general mechanism of amyloid formation. To obtain a better understanding of the mechanism of amyloid fibril formation, we show in this study that equilibrium and kinetic refolding–unfolding reaction experiments with two amyloidogenic mutant human lysozymes (I56T and D67H) yield folding pathways that can be drawn as Gibbs energy diagrams. The equilibrium stabilities between the native and denatured states of both mutant proteins were decreased, but the degrees of instability were different. The Gibbs energy diagrams of the folding process reveal that the Gibbs energy change between the native and folding intermediate states was similar for both proteins, and also that the activation Gibbs energy change from the native state to the transition state decreased. Our results confirm that the tendency to favor the intermediate of denaturation facilitates amyloid formation by the mutant human lysozymes more than equilibrium destabilization between the native and completely denatured states does.
UV‐A‐related alterations of young and adult lens water‐insoluble α‐crystallin, plasma membranous and cytoskeletal proteins
Authors: Weinreb O. , Dovrat A. , Dunia I. , Benedetti E. L. , Bloemendal H.
Published: March 2003

The damaging effects of UV‐A irradiation on lens water‐insoluble α‐crystallin, plasma membranous and cytoskeletal proteins derived from bovine lenses were studied. Young and adult bovine lenses were kept viable for 2 months in organ culture. After 24 h of incubation they were irradiated, and analyses of the proteins by one‐dimensional and two‐dimensional gel electrophoresis followed by Western blotting were carried out at several time intervals. RNA isolation, PCR and Northern blotting were also performed. We identified age‐related changes in water‐insoluble α‐crystallin, the major membrane protein MP26 and the cytoskeletal proteins vimentin, phakinin and actin between control and UV‐irradiated lenses. It appeared that adult lenses are more susceptible to UV light than young lenses, and protein modification occurred more frequently in adult lenses. UV‐A irradiation affects not only the cytoskeletal structure, as deduced by the abnormal arrangement of actin in the fiber cells, but also leads to degradation of actin mRNA. Furthermore, analysis of the expression of hsp25 and hsp70 revealed some alteration in the protein pattern of adult lenses. We suggest that degradation of the cytoskeletal proteins following irradiation is due to, at least in part, the decreased protective ability of heat shock proteins upon aging.
Aldehyde dehydrogenase
Authors: Hempel John , Kuo Ingrid , Perozich John , Wang Bi‐Cheng , Lindahl Ronald , Nicholas Hugh
Published: March 2003

Alignment of all known, diverse members of the aldehyde dehydrogenase (ALDH) extended family revealed only two strictly conserved, nonglycine residues, a glutamate and a phenylalanine residue. Both occur in one of the highly conserved ‘motif’ segments and both occupy strategic locations in the tertiary structure at the bottom of the catalytic funnel. In class 3 ALDH, these are Glu333 and Phe335. In addition, Asp247, which is not highly conserved but is characteristic of class 3 ALDHs, hydrogen bonds the main chain between Glu333 and Phe335. These three residues were mutated conservatively. Michaelis constants determined for both NAD/propanal and NADP/benzaldehyde substrate pairs show all three residues to be crucial to effective catalysis, and suggest that the hydrogen bond to Asp247 is a key element in maintaining precise geometry of key elements at the active site.
Identification of phospholipids as new components that assist in the in vitro trimerization of a bacterial pore protein
Authors: de Cock Hans , Pasveer Maurice , Tommassen Jan , Bouveret Emmanuelle
Published: March 2003

The in vitro trimerization of folded monomers of the bacterial pore protein PhoE, into its native‐like, heat‐ and SDS‐stable form requires incubations with isolated cell envelopes and Triton X‐100. The possibility that membranes could be isolated that are enriched in assembly factors required for assembly of the pore protein was now investigated. Fractionation of total cell envelopes of Escherichia coli via various techniques indeed revealed the existence of membrane fractions with different capacities to support assembly in vitro. Fractions containing mainly inner membrane vesicles supported the formation of trimers that were associated with these membrane vesicles. However, only a proportion of these trimers were heat‐ and SDS‐stable and these were formed with slow kinetics. In contrast, fractions containing mainly outer membrane vesicles supported formation of high amounts of heat‐stable trimers with fast kinetics. We identified phospholipids as active assembly components in these membranes that support trimerization of folded monomers in a process with similar characteristics as observed with inner membrane vesicles. Furthermore, phospholipids strongly stimulate the kinetics of trimerization and increase the final yield of heat‐stable trimers in the context of outer membranes. We propose that lipopolysaccharides stabilize the assembly competent state of folded monomers as a lipochaperone. Phospholipids are involved in converting the folded monomer into new assembly competent intermediate with a short half‐life that will form heat‐stable trimers most efficiently in the context of outer membrane vesicles. These results provide biochemical evidence for the involvement of different lipidic components at distinct stages of the porin assembly process.
The protein kinase Cdk5
Authors: Maccioni Ricardo B. , Otth Carola , Concha Ilona I. , Muñoz Juan P.
Published: March 2003

A set of different protein kinases have been involved in tau phosphorylations, including glycogen synthase kinase 3β (GSK3β), MARK kinase, MAP kinase, the cyclin‐dependent kinase 5 (Cdk5) system and others. The latter system include the catalytic component Cdk5 and the regulatory proteins p35, p25 and p39. Cdk5 and its neuron‐specific activator p35 are essential molecules for neuronal migration and for the laminar configuration of the cerebral cortex. Recent evidence that the Cdk5/p35 complex concentrates at the leading edge of axonal growth cones, together with the involvement of this system in the phosphorylation of neuronal microtubule‐asociated proteins (MAPs), provide further support to the role of this protein kinase in regulating axonal extension in developing brain neurons. Although the aminoacid sequence of p35 has little similarity with those of normal cyclins, studies have shown that its activation domain may adopt a conformation of the cyclin‐folded structure. The computed structure for Cdk5 is compatible with experimental data obtained from studies on the Cdk5/p35 complex, and has allowed predictions on the protein interacting domains. This enzyme exhibits a wide cell distribution, even though a regulated Cdk5 activity has been shown only in neuronal cells. Cdk5 has been characterized as a proline‐directed Ser/Thr protein kinase, that contributes to phosphorylation of human tau on Ser202, Thr205, Ser235 and Ser404. Cdk5 is active in postmitiotic neurons, and it has been implicated in cytoskeleton assembly and its organization during axonal growth. In addition to tau and other MAPs, Cdk5 phosphorylates the high molecular weight neurofilament proteins at their C‐terminal domain. Moreover, nestin, a protein that regulates cytoskeleton organization of neuronal and muscular cells during development of early embryos, and several other regulatory proteins appear to be substrates of Cdk5 and are phosphorylated by this kinase. Studies also suggest, that in addition to Cdk5 involvement in neuronal differentiation, its activity is induced during myogenesis, however, the mechanisms of how this activity is regulated during muscular differentiation has not yet been elucidated. Recent studies have shown that the β‐amyloid peptide (Aβ) induces a deregulation of Cdk5 in cultured brain cells, and raises the question on the possible roles of this tau‐phosphorylating protein kinase in the sequence of molecular events leading to neuronal death triggered by Aβ. In this context, there are evidence that Cdk5 is involved in tau hyperphosphorylation promoted by Aβ in its fibrillary form. Cdk5 inhibitors protect hippocampal neurons against both tau anomalous phosphorylations and neuronal death. The links between the studies on the Cdk5/p35 system in normal neurogenesis and its claimed participation in neurodegeneration, provide the framework to understand the regulatory relevance of this kinase system, and changes in its regulation that may be implicated in disturbances such as those occurring in Alzheimer disease.
SUT1 is a putative Zn[II]2Cys6‐transcription factor whose upregulation enhances both sterol uptake and synthesis in aerobically growing Saccharomyces cerevisiae cells
Authors: Ness Frédérique , Bourot Stéphane , Régnacq Matthieu , Spagnoli Roberto , Bergès Thierry , Karst Francis
Published: March 2003

Budding yeast Saccharomyces cerevisiae is a facultative anaerobe whose growth upon oxygen starvation depends on its capacity to import exogenously supplied sterols, whereas the cells are not permeable to these molecules when grown aerobically. Few genes have been identified as being involved in sterol uptake. A higher SUT1 gene dosage leads to a modest, but significant, increase in sterol uptake under aerobic conditions. Based on sequence and physiological data, SUT1 is a hypoxic gene negatively regulated when the cells are grown in the presence of oxygen. We replaced the SUT1 promoter with the constitutive PMA1 gene promoter in order to enhance its transcription. We observed that sterol uptake was then comparable with that obtained with a sterol importing hem1 mutant, although the heme status of the strain was not modified in a process which still occurs when the cells are not growing. Unexpectedly, SUT1 constitutive expression led to a parallel significant increase in endogenous sterol biosynthesis. Moreover, here we present new data showing that the structurally related YPR009 gene (SUT2) is a functional homologue of SUT1, and that both gene products may represent two novel yeast regulatory proteins involved in sterol uptake.
Contributions of the substrate‐binding arginine residues to maleate‐induced closure of the active site of Escherichia coli aspartate aminotransferase
Authors: Matharu Annelise , Hayashi Hideyuki , Kagamiyama Hiroyuki , Maras Bruno , John Robert A.
Published: March 2003

Crystallography shows that aspartate aminotransferase binds dicarboxylate substrate analogues by bonds to Arg292 and Arg386, respectively [Jager, J, Moser, M. Sauder, U. & Jansonius, J. N. (1994) J. Mol. Biol., 239, 285–305]. The contribution of each interaction to the conformational change that the enzyme undergoes when it binds ligands via these residues, is assessed by probing mutant forms of the enzyme lacking either or both arginines. The probes used are NaH3BCN which reduces the cofactor imine, the reactive substrate analogue, cysteine sulfinate and proteolysis by trypsin. The unreactive substrate analogue, maleate, is used to induce closure. Each single mutant reacted only 2.5‐fold more slowly with NaH3BCN than the wild‐type indicating that charge repulsion by the arginines contributes little to maintaining the open conformation. Maleate lowered the rate of reduction of the wild‐type enzyme more than 300‐fold but had little effect on the reaction of the mutant enzymes indicating that the ability of this dicarboxylate analogue to bridge the arginines precisely makes the major contribution to closure. The R292L mutant reacted 20 times more rapidly with cysteine sulfinate than R386L but 5 × 104 times more slowly than the wild‐type enzyme, consistent with the proposal that enzyme’s catalytic abilities are not developed unless closure is induced by bridging of the arginines. Proteolysis of the mutants with trypsin showed that, in the wild‐type enzyme, the bonds most susceptible to trypsin are those contributed by Arg292 and Arg386. Proteolysis of the next most susceptible bond, at Arg25 in the double mutant, was protected by maleate demonstrating the presence of an additional site on the enzyme for binding dicarboxylates.
Kinetics of membrane lysis by custom lytic peptides and peptide orientations in membrane
Authors: Chen H. M. , Clayton A. H. A. , Wang W. , Sawyer W. H.
Published: March 2003

To aid the development of custom peptide antibiotics, a kinetic study of membrane lysis by cecropin B (CB) and its analogs, cecropin B1 (CB1) and cecropin B3 (CB3) was carried out to determine the mechanism by which these peptides disrupt the bilayer structure of liposomes of defined composition. Disruption of the phospholipid bilayer was determined by a fluorescence assay involving the use of dithionite to quench the fluorescence of lipids labeled with N‐7‐nitro‐2,1,3‐benzoxadiazol‐4‐yl. Lytic peptides caused the disruption of liposomes to occur in two kinetic steps. For liposomes composed of mixtures of phosphatidylcholine and phosphatidic acid, the time constants for each kinetic step were shorter for CB and CB1 than for CB3. Oriented circular dichroism experiments showed that the peptides could exist in at least two different membrane‐associated states that differed primarily in the orientation of the helical segments with respect to the bilayer surface. The results are discussed in terms of kinetic mechanisms of membrane lysis. The mode of actions of these peptides used for the interpretation of their kinetic mechanisms were supported by surface plasmon resonance experiments including or excluding the pore‐forming activities.
Phenylmethanesulfonyl fluoride inactivates an archaeal superoxide dismutase by chemical modification of a specific tyrosine residue
Authors: De Vendittis Emmanuele , Ursby Thomas , Rullo Rosario , Gogliettino Maria Angela , Masullo Mariorosario , Bocchini Vincenzo
Published: March 2003

The gene encoding the superoxide dismutase from the hyperthermophilic archaeon Sulfolobus solfataricus (SsSOD) was cloned and sequenced and its expression in Escherichia coli obtained. The chemicophysical properties of the recombinant SsSOD were identical with those of the native enzyme. The recombinant SsSOD possessed a covalent modification of Tyr41, already observed in native SsSOD [Ursby, T., Adinolfi, B.S., Al‐Karadaghi, S., De Vendittis, E. & Bocchini, V. (1999) J. Mol. Biol.286, 189–205]. HPLC analysis of SsSOD samples prepared from cells treated or not with phenylmethanesulfonyl fluoride (PhCH2SO2F), a protease inhibitor routinely added during the preparation of cell‐free extracts, showed that the modification was caused by PhCH2SO2F. Refinement of the crystal model of SsSOD confirmed that a phenylmethanesulfonyl moiety was attached to the hydroxy group of Tyr41. PhCH2SO2F behaved as an irreversible inactivator of SsSOD; in fact, the specific activity of both native and recombinant enzyme decreased as the percentage of modification increased. The covalent modification caused by PhCH2SO2F reinforced the heat stability of SsSOD. These results show that Tyr41 plays an important role in the enzyme activity and the maintenance of the structural architecture of SsSOD.
Incomplete refolding of a fragment of the N‐terminal domain of pig muscle 3‐phosphoglycerate kinase that lacks a subdomain
Authors: Szilágyi Andrea N. , Kotova Nina V. , Semisotnov Gennady V. , Vas Mária
Published: March 2003

Refolding of pig muscle 3‐phosphoglycerate kinase (PGK) from a mixture of its complementary proteolytic fragments that did not correspond to the individual domains resulted in a high degree of reactivation [Vas, M., Sinev, M.A., Kotova, N. & Semisotnov, G.V. (1990) Eur. J. Biochem.189, 575–579]. An independent refolding of the 27.7 kDa C‐terminal proteolytic fragment (which encompasses the whole C domain) has been noted, but the refolding ability of the 16.8‐kDa N‐terminal proteolytic fragment, which lacks a single subdomain from the N domain, remained to be seen. Here the refolding processes of the isolated fragments are compared. Within the first few seconds of initiation of refolding, pulse‐proteolysis experiments show the formation of a structure with moderate protease resistance for both fragments. This structure, however, remains unchanged upon further incubation of the N‐terminal fragment, whereas refolding of the C‐terminal fragment continues as detected by a further increase in proteolytic resistance. The non‐native character of the folding intermediate of the N fragment is indicated by the elevated fluorescence intensity of the bound hydrophobic probe 8‐anilino‐1‐naphtalene sulphonate. Its CD spectrum shows the formation of secondary structure distinct from the native one. The noncooperative phase‐transition observed in microcalorimetry indicates the absence of a rigid tertiary structure, in contrast with the refolded C‐terminal fragment for which a cooperative transition is seen. Size‐exclusion chromatography supported the globular character of the intermediate, and showed its propensity to form dimers. No binding of the substrate, 3‐phosphoglycerate (3‐PGri), to the isolated N‐terminal fragment, could be detected but the presence of the complementary C‐terminal fragment led to restoration of the substrate binding ability of the N domain. Thus, refolding of the isolated N‐terminal fragment yields a highly flexible, globular, potentially productive intermediate with non‐native secondary structure and highly exposed hydrophobic clusters, which favour dimerization.