bluuebunny/crossref_metadata_2025 huggingface dataset:
Metabolic regulation of the trehalose content of vegetative yeast
Authors: Winkler Karl , Kienle Iris , Burgert Markus , Wagner Jean-Claude , Holzer Helmut
Published: July 2002

We have investigated the mechanism by which heat shock conditions lead to a reversible accumulation of trehalose in growing yeast. When cells of S. cerevisiae MI growing exponentially at 30°C were shifted to 45°C for 20 min, or to 39°C for 40 min, the concentration of trehalose increased by about 25‐fold; an effect reversed upon lowering the temperature to 30°C. This was compared to the more than 50‐fold rise in trehalose levels obtained upon transition from the exponential to the stationary growth phase. Whereas the latter was paralleled by a 12‐fold increase in the activity of trehalose‐6‐phosphate synthase, no significant change in the activities of trehalose‐synthesizing and ‐degrading enzymes was measured under heat shock conditions. Accordingly, cycloheximide did not prevent the heat‐induced accumulation of trehalose. However, the concentrations of the substrates for trehalose‐6‐phosphate synthase, i.e. glucose‐6‐phosphate and UDP‐glucose, were found to rise during heat shock by about 5–10‐fold. Since the elevated levels of both sugars are still well below the K
m‐values determined for trehalose‐6‐phosphate synthase in vitro, they are likely to contribute to the increase in trehalose under heat shock conditions. A similar increase in the steady‐state levels was obtained for other intermediates of the glycolytic pathway between glucose and triosephosphate, including ATP. This suggests that temperature‐dependent changes in the kinetic parameters of glycolytic enzymes vary in steady‐state levels of intermediates of sugar metabolism, including an increase of those that are required for trehalose synthesis. Trehalose, glucose‐6‐phosphate, UDP‐glucose, and ATP, were all found to increase during the 40 min heat treatment at 39°C. Since this also occurs in a mutant lacking the heat shock‐induced protein HSP104 (Δhsp104), this protein cannot be involved in the accumulation of trehalose under these heat shock conditions. However, mutant Δhsp104, in contrast to the parental wild‐type, was sensitive towards a 20 min incubation at 50°C. Since this mutant also accumulated normal levels of trehalose, we conclude that HSP104 function, and not the accumulation of trehalose, protects S. cerevisiae from the damage caused by a 50°C treatment.
Identification and partial purification of GTPase‐activating proteins from yeast and mammalian cells that preferentially act on Ypt1/Rab1 proteins
Authors: Tan Tjie J. , Vollmer Petra , Gallwitz Dieter
Published: July 2002

Two GTPase‐activating proteins of apparent molecular mass of 100 kDa and 30 kDa have been partially purified from porcine liver cytosol usinig mammalian Ypt1/Rab1 protein as substrate. Both proteins act most efficiently on Ypt1/Rab1 p, but are inactive with H‐Ras p21. From the budding yeast Saccharomyces cerevisiae, a cytosolic 40 kDa yptGAP was partially purified. It accelerates the intrinsic GTPase activity of wild‐type Yptlp but not of H‐Ras p21 or a mutant ypt1p with an animo acid substitution of the effector domain which renders the protein functionally inactive in yeast cells.
Characterization of Lex, Ley and A Ley antigen determinants in KDN‐containing O‐linked glycan chains from Pleurodeles waltlii jelly coat eggs
Authors: Strecker Gérard , Wieruszeski Jean-Michel , Michalski Jean-Claude , Alonso Catherine , Boilly Benoni , Montreuil Jean
Published: July 2002

Novel acidic oligosaccharides were isolated in large amounts by reductive alkaline treatment of the jelly coat of Pleurodeles waltlii (Michah) eggs. The oligosaccharides were found to contain the newly described KDN as acidic monosaccharide and possess either the Lex, Ley and A Ley antigenic determinants. Occurrence of Lex and Ley determinants previously recognized as tumor‐associated antigen (TAA) demonstrates that mucins of lower animals may represent a rich and easily available source for preparing TAA. Moreover, it reinforces the hypothesis according to which TAA are evolution markers.
Structure of the guinea pig neutrophil cationic peptide gene
Authors: Nagaoka Isao , Someya Akimasa , Iwabuchi Kazuhisa , Yamashita Tatsuhisa
Published: July 2002

Guinea pig neutrophils contain the antimicrobial cationic peptides GNCP‐1 and GNCP‐2 in the granules. In this study, the GNCP gene was isolated, and the structure was characterized. Using cDNA probes, one phage clone was isolated from a guinea pig genomic library. The gene spanned > 3 kb, and comprised three exons and two introns. Sequence analysis revealed that the gene encoded GNCP‐2. Exon 1 mainly coded for the 5′ untranslated region, exon 2 coded for the prepro‐peptide region of GNCP‐2, and exon 3 coded for the mature peptide region of GNCP‐2 and the 3′ untranslated region. Primer extension analysis indicated that the transcription initiation site was located to a thymidine residue, 93 bp upstream of the ATG initiation codon or GNCP‐2 mRNA. A possible TATA box was located 24 bp upstream or the transcription start site. Interestingly, the pyrimidine‐rich sequences identified in the promoter regions of the human neutrophil elastase and myeloperoxidase genes were also found in the 5′ flanking region of the GNCP‐2 gene.
Identification of DEBS 1, DEBS 2 and DEBS 3, the multienzyme polypeptides of the erythromycin‐producing polyketide synthase from Saccharopolyspora erythraea
Authors: Caffrey Patrick , Bevitt Debra J. , Staunton James , Leadlay Peter F.
Published: July 2002

The ery A region of the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea has previously been shown to contain three large open reading frames (ORFs) that encode the components of 6‐deoxyerythronolide B synthase (DEBS). Polyclonal antibodies were raised against recombinant proteins obtained by overexpression of 3′ regions of the ORF2 and ORF3 genes. In Western blotting experiments, each antiserum reacted strongly with a different high molecular weight protein in extracts of erythromycin‐producing S. erythraea cells. These putative DEBS 2 and DEBS 3 proteins were purified and subjected to N‐terminal sequence analysis. The protein sequences were entirely consistent with the translation start sites predicted from the DNA sequences of ORFs 2 and 3. A third high molecular weight protein co‐purified with DEBS 2 and DEBS 3 and had an N‐terminal sequence that matched a protein sequence translated from the DNA sequence some 155 base pairs upstream from the previously proposed start codon of ORF1.
Spleen protein tyrosine kinases TPK‐IIB and CSK display different immunoreactivity and opposite specificities toward c‐src‐derived peptides
Authors: Brunati Anna Maria , Allee Guillaume , Marin Oriano , Donella-Deana Arianna , Cesaro Luca , Bougeret Cecile , Fagard Remi , Benarous Richard , Fischer Siegmund , Pinna Lorenzo A.
Published: July 2002

Polyclonal antibodies have been raised against two synthetic peptides reproducing the 48–64 and 353–369 sequences of CSK, a protein tyrosine kinase implicated in the down‐regulation of src‐related protein kinases. Both antibodies specifically recognize recombinant CSK and a CSK‐related 49 kDa protein tyrosine kinase present in spleen but they do not cross‐react with purified TPK‐IIB, a spleen protein tyrosine kinase sharing with CSK catalytic activity toward src kinases and incapability to autophosphorylate. CSK and TPK‐IIB once resolved from each other by heparin‐Sepharose affinity chromatography, display opposite specificities toward synthetic peptides reproducing the sequences around the main phosphoacceptor residues of pp6Ocsrc
, namely Tyr‐416 and Tyr‐527. These data support the view that TPK‐IIB and CSK may exert opposite effects on the activity of src‐related protein tyrosine kinases.
Effects of fatty acid oxidation on glucose utilization by isolated hepatocytes
Authors: Berry Michael N. , Phillips John W. , Henly Debra C. , Clark Dallas G.
Published: July 2002

We have studied the inhibitory action of long‐ and short‐chain fatty acids on hepatic glucose utilization in hepatocytes isolated from fasted rats. The rates of hepatic glucose phosphorylation and glycolysis were determined from the tritiated products of [2‐3H] and [6‐3H]glucose metabolism, respectively. The difference between these was taken as an estimate of the ‘cycling’ between glucose and glucose‐6‐phosphate. In the presence of 40 mM glucose this cycling was estimated at 0.68 μ;molmin/g wet wt. Glucose phosphorylation was unaffected during palmitate and hexanoate oxidation to ketone bodies but glycolysis was inhibited. The rate of glucose cycling was increased during this phase to 1.25 μmol/min > g. Following the complete metabolism of the fatty acids, glycolysis was reinstated and cycling rates returned to control levels. Hepatic glucose cycling appears to be an important component of the glucose/fatty acid cycle.
Bovine inositol monophosphatase: proteolysis and structural studies
Authors: Greasley P.J. , Gore M.G. , Rees-Milton K.J. , Ragan C.I.
Published: July 2002

Bovine brain inositol monophosphatase is inactivated when trypsin catalyses the cleavage of a single peptide bond between Lys‐36 and Ser‐37. This proteolysis is closely followed by cleavage at two other sites in the protein between Lys‐78 and Ser‐79 and between Lys‐156 and Ser‐157 suggesting that all of these sites are exposed in the native conformation of the protein. All of these residues are predicted to lie at the ends of α helices. The most susceptible bond (Lys‐36‐Ser‐37) is predicted to lie in a highly flexible region of the protein. Circular dichroism studies suggest that approximately 40% of the secondary structure of this protein is helical which is similar to that predicted by the algorithm of Gamier et al. [(1978) J. Mol. Biol. 120, 97‐120].
A role for iron in transcriptional activation by FNR
Authors: Green Jeffrey , Guest John R.
Published: July 2002

FNR is a transcriptional regulator which controls the expression of target genes in response to anoxia in Escherichia coli. The mechanism by which FNR senses and responds to anaerobiosis is unknown but indirect evidence suggests that an iron cofactor is involved. Using KMnO4 as a probe for DNA melting at active promoters, footprinting studies have now shown that the ferrous iron chelator, ferrozine, inhibits open complex formation in vivo, and that FNR with a high iron‐content is essential for open complex formation in vitro. Since open complex formation is an essential pre‐requisite for transcription, it is concluded that transcriptional activation by FNR is mediated by a ferrous iron cofactor.
Kinetic evidence that His‐711 of neutral endopeptidase 24.11 is involved in stabilization of the transition state
Authors: Dion Natalie , Le Moual Hervé , Crine Philippe , Boileau Guy
Published: July 2002

Neutral endopeptidase 24.11 (EC 3.4.24.11; NEP) is a membrane‐bound Zn‐metalloendopeptidase with a catalytic activity and a specificity very similar to that of thermolysin, a bacterial zinc‐endoprotease. NEP can be inactivated by reaction with diethylpyrocarbonate, due to the modification of a histidine residue present in the active site of the enzyme. This histidine residue was proposed to be analogous to His231 in thermolysin, which is involved in the stabilization of the tetrahedral intermediate during the transition state. Using site‐directed mutagenesis of the cDNA encoding rabbit NEP, we have created two mutants of NEP where His711 was replaced by either Gln or Phe (NEP‐Gln711 and NEP‐Phe711). Determination of kinetic parameters showed that both mutants had K
m values very similar to that of the non‐mutated enzyme but that their k
cat values were 25‐fold lower. The calculated difference in free energy needed to form the transition state complex was increased by 2.2 kcal/mol for both mutants. These observations strongly suggest that His711 is involved in the stabilization of the transition state by forming an hydrogen bond with the oxyanion of the tetrahedral intermediate.
The vanadium chloroperoxidase from the fungus, Curvularia inaequalis
Authors: van Schijndel J.W.P.M. , Simons L.H. , Vollenbroek E.G.M. , Wever R.
Published: July 2002

The binding of vanadate to the novel vanadium chloroperoxidase from C. inaequalis was investigated. Reconstitution experiments of apochloroperoxidase by vanadate at different pH values showed that in the pH 6–7 range an acid/base group is present which affects the binding of the vanadate. It is proposed that this group is a histidine. This hypothesis was tested by specifically modifying this residue using diethylpyrocarbonate. In the apo‐enzyme 9 histidines were modified, whereas in the holo‐enzyme 6 histidines were modified. Modification with diethylpyrocarbonate had no effect on the chlorinating activity of the holo‐enzyme, but when the apo‐enzyme was modified the reactivation by vanadate was strongly inhibited. We conclude that histidine in the active site of chloroperoxidase is involved in the binding of vanadate.
Immunohistochemical analysis of rat S‐adenosylmethionine synthetase isozymes in developmental liver
Authors: Horikawa Saburo , Ozasa Hisashi , Ota Kazuo , Tsukada Kinji
Published: July 2002

Mammalian S‐adenosylmethionine (AdoMet) synthetase exists as two isozymes, liver‐type and kidney(non‐hepatic)‐type enzymes. The developmental expression of these two isozyme proteins has been investigated in rat liver using immunohistochemical techniques. The liver‐type AdoMet synthetase is expressed only in adult liver, but not in fetal liver. On the other hand, the kidney‐type AdoMet synthetase is predominantly expressed in fetal liver and faintly detected in adult liver. It was also found that both isozymes were localized to the hepatocytes of rat liver. These results clearly show that AdoMet synthetase isozymes are developmentally regulated within hepatocytes. In addition, in rat kidney we have shown that the kidney‐type AdoMet synthetase is predominantly localized to the distal tubule.
Cooperative homodimeric hemoglobin from Scapharca inaequivalvis
Authors: Gambacurta Alessandra , Piro Maria Cristina , Ascoli Franca
Published: July 2002

The overexpression of the fully functional, cooperative homodimeric hemoglobin of the bivalve mollusc. Scapharca inaequivalvis, has been accomplished in E. coli from its cDNA. The latter was isolated by PCR amplification of total RNA and sequenced. The cDNA‐derived sequence differed by a single amino acid when compared to that previously obtained from purified protein. Interest in this hemoglobin resides in the unique assemblage of the two identical subunits, with the heme groups facing each other in the inside of the molecule, opposite to that occurring in vertebrate hemoglobins. The results presented here are the basis for future studies of structure/function relationships by site directed mutagenesis.
Protein‐radical enzymes
Authors: Pedersen Jens Z. , Finazzi-Agrò Alessandro
Published: July 2002

Protein‐radical enzymes use a free radical located on an intrinsic amino acid residue as a cofactor. The amino acid involved can be a tyrosine (ribonucleotide reductase, photosystem II, prostaglandin H synthase), a modified tyrosine (amine oxidase, galactose oxidase), a tryptophan (cytochrome c peroxidase), a modified tryptophan (methylamine dehydrogenase) or a glycine (ribonucleotide reduetase, pyruvate formate lyase). The mechanistic role of these radicals appears to be that of a one‐electron gate, allowing the separation of single reducing equivalents in time and space.
N‐terminal amino acid sequences of the subunits of the Na+‐translocating F1F0 ATPase from Propionigenium modestum
Authors: Gerike Ursula , Dimroth Peter
Published: July 2002

We report here the N‐terminal protein sequences of the subunits of the ATPase from Propionigenium modestum. Subunits c, b, δ, α and β start with an N‐terminal methionine residue, the γ and ε subunits have an alanine N‐terminus, from which N‐formylmethionine was hydrolyzed by posttranslational modification, and subunit a contains a blocked N‐terminus. Each of the N‐terminal sequences exactly matches a portion of the DNA sequence in the gene encoding the respective subunit protein on the unc operon. Thus, the exact translational start for each subunit protein can be identified and the primary structures of the protein transcripts can be clearly defined. Based on these data the putative size of the open reading frame that was envisaged from the DNA sequence had to be revised for the α and δ subunits.
Anionic phospholipids and protein translocation
Authors: de Kruijff Ben
Published: July 2002

Anionic phospholipids determine, in diverse ways, the membrane interaction of proteins involved in or undergoing membrane insertion or translocation. How these lipids modulate protein localization, organization, folding and membrane insertion is herein summarized and generalized, leading to a proposal for the function of anionic lipids in cellular transport of newly synthesized proteins.
Enzyme‐aided construction of medium‐sized alditols of complete O‐linked saccharides
Authors: Maaheimo Hannu , Penttilä Leena , Renkonen Ossi
Published: July 2002

We have constructed by enzyme‐aided in vitro synthesis a hexasaccharide alditol Galβ1—4GlcNAcβ1—6Galβ1—4GlcNAcβ1—6(Galβ1—3)GalNAc‐ol and shown that it resists the action of endo‐β‐galactosidase from Bacteroides fragilis under conditions where a related pentasaccharide alditol, GlcNAcβ1—3Galβ1—4GlcNAcβ1—6(Galβ1—3)GalNAc‐ol, was completely cleaved. Together with earlier results from this laboratory, our present data imply that endo‐β‐galactosidase from B. fragilis, apparently, can be used to distinguish between GlcNAcβ1—6Gal and GlcNAcβ1—3Gal units within linear backbone sequences of all known types of oligo‐(N‐acetyllactosamino)glycans.
Interleukin‐8 processing by neutrophil elastase, cathepsin G and proteinase‐3
Authors: Padrines Marc , Wolf Marlene , Walz Alfred , Baggiolini Marco
Published: July 2002

Activated neutrophils secrete two forms of IL‐8 with 77 and 72 amino acids, IL‐8(77) and IL‐8(72), along with proteinases that could process these cytokines. Significant conversion of IL‐8(77) to more potent, N‐terminally truncated forms was observed upon incubation with neutrophil granule lysates and purified proteinase‐3. IL‐8(72) was considerably more resistant to proteolytic processing than IL‐8(77). The present observations indicate that neutrophil proteinases released in inflamed tissues convert IL‐8 to more active forms and therefore tend to conserve or enhance, rather than decrease IL‐8 activity.
Substrate recognition by casein kinase‐II: The role of histidine‐160
Authors: Dobrowolska Grazyna , Meggio Flavio , Marin Oriano , Lozeman Fred J. , Li Dongxia , Pinna Lorenzo A. , Krebs Edwin G.
Published: July 2002

Casein kinase‐II (CK‐II) belongs to the protein kinases recognizing serine/threonine in proximity to acidic residues in protein substrates. Crystallography and mutagenesis studies on the cAMP‐dependent protein kinase (PKA) disclosed that glutamic acid‐170 (E170), is important for interaction of substrates with the enzyme. At a position corresponding to E170 in PKA most Ser/Thr kinases have an aspartic or glutamic acid, while CK‐II has a histidine residue (H160). In order to examine the relevance of this substitution for CK‐II substrate specificity, a mutant of the catalytic α subunit (H160D), in which H160 was changed to aspartic acid, was made. Our results show that H160 is not primarily involved in canonical substrate recognition, but does interact with an acidic residue located at position −2 with respect to the target Ser/Thr.
Comparison of the binding sites of plant ferredoxin for two ferredoxin‐dependent enzymes
Authors: De Pascalis A.R. , Schürmann P. , Bosshard H.R.
Published: July 2002

Differential chemical modification of acidic residues was used to map the binding site of plant ferredoxin (Fd) for the chloroplast enzyme ferredoxin:thioredoxin reductase (FTR). Binding of FTR to Fd inhibits chemical modification of Fd residues D34, D65, E92, E93, E94 and C‐terminal A97. The binding site demarcated by these residues differs from that for ferredoxin:NADP+ reductase (FNR). The FTR site includes C‐terminal residues but not helix 24–31, which is part of the FNR site. Both sites enclose the [2Fe‐2S] cluster.
Bacterial and plant‐produced scFv proteins have similar antigen‐binding properties
Authors: Bruyns Anne-Marie , De Jaeger Geert , De Neve Myriam , De Wilde Chris , Van Montagu Marc , Depicker Ann
Published: July 2002

A gene encoding a single‐chain variable (scFv) antibody fragment was expressed as a cytoplasmic and endoplasmic reticulum‐targeted protein in transgenic tobacco plants. In both cases, the scFv accumulated up to 0.01% of total soluble protein (TSP). The same scFv fragment was also produced in the periplasm of Escherichia coli. Measurement of the affinity by ELISA indicates that the affinity of the bacterially made scFv is about 80‐fold lower than that of the parental Fab fragment. The results suggest that the affinity of the plant‐produced scFv fragments is reduced to a similar extent, implying that all the plant‐produced scFv fragments are antigen binding.
Possible role of immune surveillance at the initial phase of metastasis produced by B16BL6 melanoma cells
Authors: Kikkawa Hironori , Imafuku Hidetoshi , Tsukada Hideo , Oku Naoto
Published: July 2002

The relationship among the real‐time trafficking of lung metastatic B16BL6 cells, metastatic potential, and the injected number of the cells was examined, since the smaller the number of tumor cells injected, the more clearly the immune defense may be observed. When 1×106 or 1×105 B16BL6 cells were injected into mice via the tail vein, both numbers of cells accumulated in the lung at a similar rate: there was an approximately 10‐fold difference in the number of accumulated cells between the two doses. Elimination from the lung was not dependent on the cell number but on the proportion of accumulated cells. However, the injection of 1×104 cells resulted in lung accumulation less than one‐tenth of that obtained with 1×105 cell injection. Metastasis was observed when 1×105 or 1×106 B16BL6 cells were injected, but not after injection of 1×104 cells. To clarify the roles of the immune defense system at the initial phase of metastasis, we challenged macrophage‐depleted mice with 1×104 tumor cells. Treatment of mice with 2‐chloroadenosine prior to the tumor cell challenge cancelled the suppression of not only metastasis but also the lung accumulation. Furthermore, the administration of 2‐chloroadenosine following the tumor cell challenge had little effect on the metastatic potential. These results suggest that the immune surveillance whose action was obvious at the low dose of challenged tumor cells functions strongly at the initial phase but not at the advanced stages of the metastatic process, and that macrophages play an important role in the suppression of metastasis.
Identification of two proteins, S14 and UIP1, that interact with UCH37
Authors: Li Tianwei , Duan Wei , Yang Hongyuan , Lee Mui-Khin , Bte Mustafa Fatimah , Lee Boon-Heng , Teo Tian-Seng
Published: July 2002

By the use of the yeast two‐hybrid screen we have identified two proteins that interacted with UCH37: S14, which is a subunit of PA700 and a novel protein, UIP1 (UCH37 interacting protein 1). The interaction of UCH37 with S14 or UIP1 was confirmed by in vitro binding assay and in vivo co‐immunoprecipitation analysis. The C‐terminal extension of UCH37 is essential for interaction with S14 or UIP1 as shown by the yeast two‐hybrid assay and the in vitro binding assay. Furthermore, UIP1 blocked the interaction between UCH37 and S14 in vitro.
ESR studies on reaction of saccharide with the free radicals generated from the xanthine oxidase/hypoxanthine system containing iron
Authors: Luo Gui-min , Qi Dan-hua , Zheng Ying-guang , Mu Ying , Yan Gang-ling , Yang Tong-shu , Shen Jia-Cong
Published: July 2002

The free radicals generated from the iron containing system of xanthine oxidase and hypoxanthine (Fe‐XO/HX) were directly detected by using spin trapping. It was found that not only superoxide anion (O2

−) and hydroxyl radical (OH), but also alkyl or alkoxyl radicals (R) were formed when saccharides such as glucose, fructose and sucrose were added into the Fe‐XO/HX system. The generated amount of R was dependent on the kind and concentration of saccharides added into the Fe‐XO/HX system and no R were detected in the absence of saccharides, indicating that there is an interaction between the saccharide molecules and the free radicals generated from the Fe‐XO/HX system and saccharide molecules are essential for generating R in the Fe‐XO/HX system. It is expected that the toxicity of R would be greater than of hydrophilic O2

− and OH because they are liposoluble and their lives are longer and the active sites of biomolecules are closely related with lipophilic phase, thus they can damage cells more seriously than O2

− and OH. The R generated from the saccharide containing Fe‐XO/HX can be effectively scavenged by selenium containing abzyme (Se‐abzyme), indicating Se‐abzyme is a promising antioxidant.
In search of a function for the TIS21/PC3/BTG1/TOB family
Authors: Matsuda Satoru , Rouault Jean-Pierre , Magaud Jean-Pierre , Berthet Cyril
Published: July 2002

The Btg family of anti‐proliferative gene products includes Pc3/Tis21/Btg2, Btg1, Tob, Tob2, Ana/Btg3, Pc3k and others. These proteins are characterized by similarities in their amino‐terminal region: the Btg1 homology domain. However, the pleiotropic nature of these family proteins has been observed and no common physiological function among family members was suggested from the history of their identification. Recent progress in the search for Btg family functions has come from the analysis of cell regulation and of cell differentiation. It is now emerging that every member of this family has a potential to regulate cell growth. We would like to propose here to use a nomenclature APRO as a new term for the family.
Identification of essential acidic residues of outer membrane protease OmpT supports a novel active site
Authors: Kramer R.Arjen , Vandeputte-Rutten Lucy , de Roon Gerard Jan , Gros Piet , Dekker Niek , Egmond Maarten R.
Published: July 2002

Escherichia coli outer membrane protease OmpT has previously been classified as a serine protease with Ser99 and His212 as active site residues. The recently solved X‐ray structure of the enzyme was inconsistent with this classification, and the involvement of a nucleophilic water molecule was proposed. Here, we substituted all conserved aspartate and glutamate residues by alanines and measured the residual enzymatic activities of the variants. Our results support the involvement of a nucleophilic water molecule that is activated by the Asp210/His212 catalytic dyad. Activity is also strongly dependent on Asp83 and Asp85. Both may function in binding of the water molecule and/or oxyanion stabilization. The proposed mechanism implies a novel proteolytic catalytic site.
Characterisation of S‐nitrosohaemoglobin by mass spectrometry
Authors: Ferranti Pasquale , Malorni Antonio , Mamone Gianfranco , Sannolo Nicola , Marino Gennaro
Published: July 2002

Recent studies have demonstrated the biological importance of the interaction of S‐nitrosothiols, which can be considered as nitric oxide (NO) protein donors, especially haemoglobin, at the level of Cys residues. It was recently proposed that S‐nitrosohaemoglobin is formed within red blood cells and serves as a regulatory function. In human haemoglobin the α‐subunit contains one Cys residue and the β‐subunit contains two Cys residues, one of which (β‐Cys93) is highly reactive and conserved among species, although its function has remained unknown. Electrospray ionization mass spectrometry was used to monitor the results of exposure of haemolysates to S‐nitrosocysteine under different conditions and thus addressed some aspects of NO–haemoglobin interaction. When an equimolar ratio of S‐nitrosothiol was added to haemoglobin, only a single NO molecule was added. Peptide mapping by liquid chromatography–mass spectrometry located the nitrosyl group at the level of β‐Cys93 demonstrating that this was the preferred site of formation of S‐nitrosohaemoglobin. The present data also suggest that electrospray mass spectrometry can allow quantification and characterisation of S‐nitrosoproteins in blood.
A structure prediction for the ligand‐binding region of the integrin β subunit: evidence for the presence of a von Willebrand factor A domain
Authors: Tuckwell Danny S , Humphries Martin J
Published: July 2002

The integrins are a family of cell surface receptors that mediate biologically important adhesive interactions. Integrin‐ligand binding has been extensively studied because of the potential for the development of anti‐adhesive therapies, but the molecular basis of this interaction is still poorly understood. A conserved region near the N‐terminus of the β subunit appears to be of particular importance in ligand binding, but to date this domain has not been expressed in isolation. As a prelude to expression and potential structure determination, we have performed a detailed structure prediction for this region. Primary, secondary and tertiary structure analyses indicate that the region folds into a von Willebrand factor A‐domain, thereby potentially placing a previously characterised module at the centre of a key functional region.
Adsorption to silica nanoparticles of human carbonic anhydrase II and truncated forms induce a molten‐globule‐like structure
Authors: Billsten Peter , Freskgård Per-Ola , Carlsson Uno , Jonsson Bengt-Harald , Elwing Hans
Published: July 2002

Human carbonic anhydrase II pseudo‐wild type (HCAIIpwt) and two truncated variants were adsorbed to ≈9 nm silica nanoparticles. Ellipsometry was used as an indirect measure of protein adsorption. The structural changes of adsorbed proteins were investigated with the use of circular dichroism (CD), intrinsic fluorescence, ANS binding ability and inhibitor binding capacity. It was found that the variants that were truncated at positions 5 and 17 in the N‐terminal end attain a molten‐globule‐like state after interaction with the silica nanoparticles. In contrast, the more stable HCAIIpwt retained most of its native structure after 24 h adsorption to silica nanoparticles. The result suggests that surface induced unfolding may give rise to intermediates similar to those for unfolding induced by, for example GuHCl. Thus, the intermediate observed has some features of the molten globule.
Specific cation binding site in mammalian cytochrome oxidase
Authors: Kirichenko Anna , Vygodina Tatiana , Mkrtchyan Hratchik M , Konstantinov Alexander
Published: July 2002

Calcium ion binds reversibly with cytochrome c oxidase from beef heart mitochondria (K
d∼2 μM) shifting α‐ and γ‐absorption bands of heme a to the red. Two sodium ions compete with one Ca2+ for the binding site with an average dissociation constant ∼3.6 mM. The Ca2+‐induced spectral shift of heme a is specific for mammalian cytochrome c oxidase and is not observed in bacterial or yeast aa
3 oxidases although the Ca2+‐binding site has been revealed in the bacterial enzyme [Ostermeier, C., Harrenga, A., Ermler, U. and Michel, H. (1997) Proc. Natl. Acad. Sci. USA 94, 10547–10553]. As His‐59 and Gln‐63 involved in Ca2+ binding with Subunit I of P. denitrificans oxidase are not conserved in bovine oxidase, these residues have to be substituted by alternative ligands in mammalian enzyme, which is indeed the case as shown by refined structure of bovine heart cytochrome oxidase (S. Yoshikawa, personal communication). We propose that it is interaction of Ca2+ with the species‐specific ligand(s) in bovine oxidase that accounts for perturbation of heme a. The Ca2+/Na+‐binding site may be functionally associated with the exit part of `pore B' proton channel in subunit I of mammalian cytochrome c oxidase.
Induction of the mitogen‐activated protein kinase phosphatase MKP3 by nerve growth factor in differentiating PC12
Authors: Camps Montserrat , Chabert Christian , Muda Marco , Boschert Ursula , Gillieron Corine , Arkinstall Steve
Published: July 2002

In PC12 sympathetic neurons activation and nuclear translocation of ERK family MAP kinases plays an essential role in processes underlying nerve growth factor (NGF)‐dependent differentiation. We have recently cloned MKP‐3 as a novel dual specificity phosphatase displaying selectivity towards inactivation of the ERK1 and ERK2 MAP kinases. Here we report that in PC12 cells, MKP‐3 undergoes powerful and specific up‐regulation by NGF while a number of mitogens and cellular stresses are ineffective. NGF‐stimulated MKP‐3 expression appears after 1 h, is maximal at 3 h, and is sustained for 5 days. This coincides with a critical period of neurite outgrowth and terminal differentiation. Consistent with a role mediating inhibition of PC12 cell MAP kinases, NGF‐stimulated ERK2 activation was suppressed considerably following pretreatment with fibroblast growth factor and 9‐cis‐retinal, two additional differentiation factors found to induce powerfully MKP‐3 expression. Given the clear cytosolic localization of MKP3 in PC12 cells and sympathetic neurons, these results suggest a critical role for inactivating ERK MAP kinases in non‐nuclear compartments during essential stages of NGF‐mediated PC12 differentiation.
Interaction of the N‐terminal domain of MukB with the bacterial tubulin homologue FtsZ
Authors: Lockhart Andrew , Kendrick-Jones John
Published: July 2002

The MukB protein is involved in the process of chromosome partition in Escherichia coli and has a domain structure reminiscent of the eukaryotic motor proteins kinesin and myosin. This has led to the suggestion that MukB may function as a motor protein in vivo. In order to test this idea we have recombinantly expressed the N‐terminal domain of MukB (residues 1–342) as a poly‐His tagged fusion protein for biochemical characterisation. The purified protein (Muk342) is monomeric and has low basal Mg‐ATPase (1.23 min−1) and Mg‐GTPase (0.17 min−1) activities. Muk342 binds with high affinity to the prokaryotic tubulin homologue FtsZ and we have evidence that FtsZ can stimulate nucleotide turnover by Muk342. These properties are consistent with MukB functioning as a motor protein using FtsZ as a track or anchor for generating force within E. coli.
Oxidative DNA damage induced by homogentisic acid, a tyrosine metabolite
Authors: Hiraku Yusuke , Yamasaki Masayoshi , Kawanishi Shosuke
Published: July 2002

We examined the mechanism of DNA damage induced by a mutagenic tyrosine metabolite, homogentisic acid (HGA), using 32P‐5′‐end‐labeled DNA fragments obtained from the human p53 tumor suppressor gene. HGA caused DNA damage in the presence of Cu(II), particularly at thymine and cytosine residues. Catalase and bathocuproine inhibited the DNA damage, suggesting the involvement of H2O2 and Cu(I). The formation of 8‐oxo‐7,8‐dihydro‐2′‐deoxyguanosine by HGA increased depending on HGA concentration in the presence of Cu(II). It is concluded that H2O2 is generated during Cu(II)‐catalyzed HGA autoxidation and reacts with Cu(I) to form the Cu(I)‐peroxide complex, capable of causing oxidative DNA damage.
Different patterns of IL‐1β secretion, adhesion molecule expression and apoptosis induction in human endothelial cells treated with 7α‐, 7β‐hydroxycholesterol, or 7‐ketocholesterol
Authors: Lemaire Stéphanie , Lizard Gérard , Monier Serge , Miguet Carole , Gueldry Serge , Volot Fabienne , Gambert Philippe , Néel Dominique
Published: July 2002

Among oxysterols oxidized at C7 (7α‐, 7β‐hydroxycholesterol, and 7‐ketocholesterol), 7β‐hydroxycholesterol and 7‐ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)‐1β and/or tumor necrosis factor (TNF)‐α secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule‐1 (ICAM‐1), vascular cell adhesion molecule‐1 (VCAM‐1), and E‐selectin) on human umbilical venous endothelial cells (HUVECs). Only 7β‐hydroxycholesterol and 7‐ketocholesterol were potent inducers of apoptosis and of IL‐1β secretion. TNF‐α secretion was never detected. Depending on the oxysterol considered, various levels of ICAM‐1, VCAM‐1 and E‐selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the α‐ or β‐hydroxyl radical position plays a key role in apoptosis and IL‐1β secretion.
Alcoholysis reactions from starch with α‐amylases
Authors: Santamaría R.I , Del Río G , Saab G , Rodríguez M.E , Soberón X , López-Munguía A
Published: July 2002

The ability of α‐amylases from different sources to carry out reactions of alcoholysis was studied using methanol as substrate. It was found that while the enzymes from Aspergillus niger and Aspergillus oryzae, two well‐studied saccharifying amylases, are capable of alcoholysis reactions, the classical bacterial liquefying α‐amylases from Bacillus licheniformis and Bacillus stearothermophilus are not. The effect of starch and methanol concentration, temperature and pH on the synthesis of glucosides with α‐amylase from A. niger was studied. Although methanol may inactivate α‐amylase, a 90% substrate relative conversion can be obtained in 20% methanol at a high starch concentration (15% w/v) due to a stabilizing effect of starch on the enzyme. As the products of alcoholysis are a series of methyl‐oligosaccharides, from methyl‐glucoside to methyl‐hexomaltoside, alcoholysis was indirectly quantified by high performance liquid chromatography analysis of the total methyl‐glucoside produced after the addition of glucoamylase to the α‐amylase reaction products. More alcoholysis was obtained from intact soluble starch than with maltodextrins or pre‐hydrolyzed starch. The biotechnological implications of using starch as substrate for the production of alkyl‐glucosides is analyzed in the context of these results.
Targeted gene delivery into α9β1‐integrin‐displaying cells by a synthetic peptide
Authors: Schneider Holm , Harbottle Richard P. , Yokosaki Yasuyuki , Jost Philipp , Coutelle Charles
Published: July 2002

We have investigated the usefulness of two small synthetic peptides comprising either a linear or a cyclic PLAEIDGIEL domain and a DNA‐binding moiety of 16 lysine residues to mediate gene transfer selectively into α9β1‐integrin‐displaying cells. Such specific gene delivery could only be achieved with the peptide containing the cyclic PLAEIDGIEL domain. However, inclusion of the cationic liposome LipofectAMINE into the peptide/DNA complexes resulted for both peptides in efficient gene transfer with significant targeting specificity. Naturally, the integrin α9β1 is present only in a few highly specialised tissues and abundant throughout the human airway epithelia in vivo. Targeting gene vectors to this integrin therefore appears a useful approach to gene therapy of lung diseases such as cystic fibrosis. As the integrin α9β1 is associated with tissue differentiation during foetal development and may cause resurgence of the foetal phenotype in colon cancers, such vectors may also be applicable for prenatal and cancer gene therapy.
The effect of exercise on systemic and bone concentrations of growth factors in rats
Authors: Bravenboer Nathalie , Engelbregt Mia J. T. , Visser Nanette A. , Popp‐Snijders Corrie , Lips Paul
Published: July 2002

Exercise can prevent bone loss and increase bone density. Growth factors such as insulin‐like growth factor‐I (IGF‐I) and transforming growth factor β (TGFβ) are thought to be involved in the local response to mechanical loading, resulting in bone remodelling. We tested the effect of additional weight bearing during exercise on the systemic response of IGF‐I and local bone response of IGF‐I and TGFβ.

Thirty‐four female Wistar rats (aged 3 months, weight 226.9 ± 20.2 g) were randomly divided in four groups: group 1 baseline controls; group 2 sedentary controls; group 3 ran 15 min a day on a motor‐driven exercise belt; group 4 ran 15 min a day with a backpack containing 40 g. The animals ran 5 days a week, for 6 weeks, with an average velocity of 16.6 ± 4.4 m/min, and a slope of 5° uphill.

The serum growth hormone (GH) concentration was significantly higher in the running rats (group 3, P = 0.009) than in the sedentary controls (group 2). The IGF‐I and IGF binding protein 3 (IGFBP3) levels in serum and the IGF‐I levels in liver were similar in all groups. In the tibia no significant differences were observed in IGF‐I, IGFBP3 and TGFβ concentration. In the humerus, the IGF‐I concentration was lower in the running rats (group 3) than in the sedentary controls (P = 0.04), but it was higher in the rats that ran with additional weight than in those without (P = 0.009). The TGFβ concentration in the humerus was lower in both group 3 (P = 0.001) and 4 (P = 0.03) than in the sedentary controls.

The effects in bone caused by mechanical stimulation cannot be explained by changes in serum IGF‐I and IGF‐I produced in the liver. The concentrations of IGF‐I and TGFβ in bone appeared to be modulated by running exercise. © 2001 Orthopaedic Research Society. Punlished by Elsevier Science Ltd. All rights reserved.
Domain Wall Motion in Magnetic Nanostrips
Authors: Alejos Oscar , Raposo Víctor , Martínez Eduardo
Published: April 2020

Domain walls (DWs) are the transition regions between two magnetic domains. These objects have been very relevant during the last decade, not only due to their intrinsic interest in the development of novel spintronic devices but also because of their fundamental interest. The study of DW has been linked to the research on novel spin–orbit coupling phenomena, such as the Dzyaloshinskii–Moriya interaction and the spin Hall effect. DWs can be nucleated in ferromagnetic (FM) nanostrips and can be driven by conventional magnetic fields and spin currents due to the injection of electrical pulses, which make them very promising for technological applications of recording and logic devices. In this article, based on full micromagnetic simulations supported by extended one‐dimensional models, we describe the static and dynamic properties of DWs in thin FM and ferrimagnetic wires with perpendicular magnetic anisotropy. This article aims to provide a fundamental theoretical description of the fundaments of DWs, and the numerical tools and models that allow describing the DW dynamics in previous and future experimental setups.
Polymers and Nanostructured Materials for Drug Nanoparticles, Bioimaging, and Cell Delivery
Authors: Fryer Claudia , Yang Yonghong , Wang Tom , Murray Patricia , Zhang Haifei
Published: April 2020

Polymers can be designed, synthesized, and modified for a wide range of applications. The use of biopolymers and nanostructured materials (either made of polymers or fabricated with polymers) has been extensively investigated for biomedical and biological applications. In this article, we describe the use of polymers for the preparation of poorly water‐soluble drug nanoparticles (covering top‐down and bottom‐up approaches, selection of stabilizers, and encapsulation materials), bioimaging (covering polymers, small molecules, nanoparticles for various imaging techniques including X‐ray radiography, positron emission tomography, magnetic resonance imaging, ultrasonography, fluorescent imaging, photoacoustic imaging, and multimodal imaging), and cell encapsulation and cell delivery (covering the basics, important parameters, and applications). We finish the article with perspectives in these exciting research areas. This article is written with the basics and recent progress in the relevant research areas and thus is suitable for a broad readership.
Continuous Intravenous Anakinra Infusion to Calm the Cytokine Storm in Macrophage Activation Syndrome
Authors: Monteagudo Luke Adam , Boothby Aaron , Gertner Elie
Published: April 2020

Objective

The objective of this study was to report the benefit of a therapeutic approach consisting of intravenous (IV) continuous anakinra (recombinant human interleukin‐1 receptor antagonist) infusions in treating severely ill adult patients with secondary hemophagocytic lymphohistiocytosis or macrophage activation syndrome (MAS).

Methods

A retrospective chart review of five patients treated at Regions Hospital from 2016 to 2019 was conducted. Demographic, clinical, and laboratory characteristics and outcomes were recorded.

Results

Continuous IV anakinra infusions up to 2400 mg/d resulted in rapid serologic, then clinical response in 4 of 5 severely ill patients who were refractory to all other therapies, including subcutaneous anakinra. Subsequently, 3 of 5 patients have been maintained on anakinra or canakinumab, with no recurrence of MAS.

Conclusion

Continuous infusion of IV anakinra may result in rapid serologic and subsequent clinical improvement in adult patients with MAS. This method for treating cytokine storm should be considered in the current COVID‐19 pandemic in the subgroup of patients with severe disease who have a cytokine storm presentation.
Outcome of patients with Fanconi anemia developing myelodysplasia and acute leukemia who received allogeneic hematopoietic stem cell transplantation: A retrospective analysis on behalf of EBMT group
Authors: Giardino Stefano , de Latour Regis P. , Aljurf Mahmoud , Eikema Dirk‐Jan , Bosman Paul , Bertrand Yves , Tbakhi Abdelghani , Holter Wolfgang , Bornhäuser Martin , Rössig Claudia , Burkhardt Birgit , Zecca Marco , Afanasyev Boris , Michel Gerard , Ganser Arnold , Alseraihy Amal , Ayas Mouhab , Uckan‐Cetinkaya Duygu , Bruno Benedicte , Patrick Katharine , Bader Peter , Itälä‐Remes Maija , Rocha Vanderson , Jubert Charlotte , Diaz Miguel A. , Shaw Peter J. , Junior Luiz G. D. , Locatelli Franco , Kröger Nicolaus , Faraci Maura , Pierri Filomena , Lanino Edoardo , Miano Maurizio , Risitano Antonio , Robin Marie , Dufour Carlo ,   for the Severe Aplastic Anemia and Chronic Malignancies Working Parties of European Blood and Marrow Transplantation group
Published: April 2020

Allogeneic hematopoietic stem cell transplantation (allo‐HSCT) is curative for bone marrow failure in patients with Fanconi anemia (FA), but the presence of a malignant transformation is associated with a poor prognosis and the management of these patients is still challenging. We analyzed outcome of 74 FA patients with a diagnosis of myelodysplastic syndrome (n = 35), acute leukemia (n = 35) or with cytogenetic abnormalities (n = 4), who underwent allo‐HSCT from 1999 to 2016 in EBMT network. Type of diagnosis, pre‐HSCT cytoreductive therapies and related toxicities, disease status pre‐HSCT, donor type, and conditioning regimen were considered as main variables potentially influencing outcome. The 5‐year OS and EFS were 42% (30‐53%) and 39% (27‐51%), respectively. Patients transplanted in CR showed better OS compared with those transplanted in presence of an active malignant disease (OS:71%[48‐95] vs 37% [24‐50],P = .04), while none of the other variables considered had an impact. Twenty‐two patients received pre‐HSCT cytoreduction and 9/22 showed a grade 3‐4 toxicity, without any lethal event or negative influence on survival after HSCT(OS:toxicity pre‐HSCT 48% [20‐75%] vs no‐toxicity 51% [25‐78%],P = .98). The cumulative incidence of day‐100 grade II‐IV a‐GvHD and of 5‐year c‐GvHD were 38% (26‐50%) and 40% (28‐52%). Non‐relapse‐related mortality and incidence of relapse at 5‐years were 40% (29‐52%) and 21% (11‐30%) respectively, without any significant impact of the tested variables. Causes of death were transplant‐related events in most patients (34 out of the 42 deaths, 81%). This analysis confirms the poor outcome of transformed FA patients and identifies the importance of achieving CR pre‐HSCT, suggesting that, in a newly diagnosed transformed FA patient, a cytoreductive approach pre‐HSCT should be considered if a donor have been secured.