bluuebunny/crossref_metadata_2025 huggingface dataset:
DNase I hypersensitive sites in the 5′ flanking region of the human plasminogen activator inhibitor type 2 (PAI‐2) gene are associated with basal and tumor necrosis factor‐α‐induced transcription in monocytes
Authors: Mahony Donna , Stringer Brett W. , Dickinson Joanne L. , Antalis Toni M.
Published: March 2003

The plasminogen activator inhibitor type 2 (PAI‐2) gene encodes a serine proteinase inhibitor (serpin) which is rapidly induced in response to the inflammatory cytokine, tumour necrosis factor‐α (TNFα) in monocytes and macrophages. As an initial step towards understanding the molecular mechanisms underlying PAI‐2 gene regulation in monocytes, we report here the analysis of the chromatin structure of 9.6 kb of 5′ flanking region of the human PAI‐2 gene for potential cis‐acting regulatory regions using DNase I hypersensitivity mapping. Sites sensitive to DNase I were mapped in two monocytic cell lines representative of early monocytic differentiation; U937 cells, which synthesise low constitutive levels of PAI‐2 that were induced following treatment with TNFα, and a MonoMac6 cell line which did not synthesise PAI‐2 even after treatment with TNFα. Six DNase I hypersensitive sites (DHS) were identified; three upstream of the transcription initiation site (DH1, DH2, DH3) and three downstream of the transcription initiation site which were contained within intron A (DH4, DH5) and the exon 2/intron B junction (DH6). Among these, one distally located DH site (DH2) disappeared in both cell lines following treatment with TNFα. Two DH sites (DH1, DH6) were absent in PAI‐2‐producing U937 cells, but were present in MonoMac6 cells, which did not produce PAI‐2, indicating the possible involvement of negative regulatory elements in the suppression of PAI‐2 gene expression. The results demonstrate the involvement of chromatin structure in transcriptional responsiveness of the PAI‐2 gene promoter and identify several loci which may be key control regions for PAI‐2 gene transcription.
The interleukin‐4 site‐2 epitope determining binding of the common receptor γ chain
Authors: Letzelter Felix , Wang Yonghong , Sebald Walter
Published: March 2003

Human IL‐4 (IL‐4), one of the small four‐helix‐bundle cytokines, uses the specific IL‐4 receptor α chain together with a promiscuous subunit, the common γ chain (γc) for transmembrane signaling. The ligand‐binding properties of γc, which are presently poorly understood, were analysed by biosensor techniques employing recombinant ectodomains of γc and α receptor chains (IL4‐BP). The formation of a ternary complex between solute γc ectodomain and IL‐4 saturated IL4‐BP could be established to exhibit a high dissociation constant Kd = 3 μM and a short half life τ1/2 = 7 s. This binding affinity resulted to the major part from the interaction of γc ectodomain with IL‐4 and not from a direct contact of the ectodomains, since binding between solute γc ectodomain and IL‐4 could be established (Kd about 150 μM), whereas no binding was found between the γc ectodomain and IL4‐BP in the absence of IL‐4. The IL‐4 epitope involved in γc ectodomain interaction (site 2) was identified by means of an alanine‐scanning mutational approach. The IL‐4 site 2 comprised residues I11 and N15 on helix A together with Y124 on helix D as major binding determinants. The IL‐4 alanine variants at site 2 generally showed only moderate defects in biological activity. Even the most affected variant [A124]IL‐4 retained a partial agonist activity of about 50 % during a T‐cell proliferation assay. The dosis leading to a half‐maximal response (EC50) was not altered by site‐2 substitutions. The present results are in accordance with a two‐step‐dimerisation mechanism for IL‐4 receptor activation, where solute IL‐4 at physiological concentrations binds first via the high‐affinity site 1 to the α chain only, since the affinity of IL‐4 site 2 for γc is too low. This site‐2 affinity seems to be sufficient, however, to promote, in a second step, a productive association of γc to an IL‐4/α chain complex in the membrane.
Protein kinase Cμ downregulation of tumor‐necrosis‐factor‐induced apoptosis correlates with enhanced expression of nuclear‐factor‐κB‐dependent protective genes
Authors: Johannes Franz‐Josef , Horn Judith , Link Gisela , Haas Elvira , Siemienski Katrin , Wajant Harald , Pfizenmaier Klaus
Published: March 2003

Protein kinase Cμ (PKCμ) represents a new subtype of the PKC family characterized by the presence of a pleckstrin homology (PH) domain and an amino‐terminal hydrophobic region. In order to analyse the potential role of PKCμ in signal‐transduction pathways, stable PKCμ transfectants were established with human and murine cell lines. All transfectants showed a reduced sensitivity to tumor‐necrosis‐factor (TNF)‐induced apoptosis, which correlated with the amount of transgene expressed and with an enhanced basal transcription rate of NF‐κB‐driven genes including the inhibitor of apoptosis protein 2 (cIAP2) and TNF‐receptor‐associated protein 1 (TRAF1). Sensitivity to apoptosis induced by the lipid mediator ceramide was unchanged in PKCμ transfectants. In support of a PKCμ action on NF‐κB, we show enhancement and downregulation of TNF‐induced expression of a NF‐κB‐dependent reporter gene by transient overexpression of wild‐type and kinase‐negative mutants of PKCμ, respectively. Interestingly, no significant changes were found in an electrophoretic mobility shift assay, indicative of PKCμ action downstream of IκB degradation, probably by modulation of the transactivation capacity of NF‐κB. The dominant negative action of the kinase‐negative mutant further suggest a regulatory role of PKCμ for NF‐κB‐dependent gene expression.
Immunological, enzymatic and biochemical studies of uroporphyrinogen III‐synthase deficiency in 20 patients with congenital erythropoietic porphyria
Authors: Freesemann Anne Georga , Gross Ulrich , Bensidhoum Moad , de Verneuil Hubert , Doss Manfred Otto
Published: March 2003

Congenital erythropoietic porphyria (CEP), a rare autosomal recessive inborn error of heme biosynthesis, results from reduced activity of uroporphyrinogen III synthase (URO‐III‐S) leading to an excessive production and accumulation of porphyrins. Various clinical and biochemical observations point to a relationship between degree of disease expression and metabolic disturbance. We investigated 20 patients with early onset of clinical symptoms of CEP and, additionally, the relatives of six patients. CEP was confirmed by porphyrinemia and porphyrinuria with dominance of uroporphyrin and its isomer I. The investigation of the immunological nature of the defective URO‐III‐S gene from unrelated patients with unknown mutations was possible thanks to an antibody against the human enzyme. URO‐III‐S concentration in erythrocytes was determined by ELISA. No signal was achieved when assaying nonimmune serum by ELISA, whereas there was a positive reaction with the serum after immunisation. Furthermore, specificity of immune sera is demonstrated by immunoprecipitation of URO‐III‐S activity which caused a 33 % reduction of enzyme activity. Normal levels of immunoreactive enzyme protein 100 ± 10 % of control (x¯ ± SD, n = 12) with a reduced specific activity 15 ± 8.5 % (x¯ ± SD, n = 12) were found in erythrocytes from all patients, with the exception of a girl with a remarkably high enzyme concentration of 149 % of controls and a very low specific activity of 4 %. In consequence, all patients had cross‐reacting immunological material (CRIM)‐positive mutations. CRIM‐ratios varied between 3.2 and 24.5. The CRIM‐positive nature of the gene defect indicated that the mutations altered the activity of URO‐III‐S. The different CRIM ratios implied the presence of various mutations, which is further evidence for the heterogeneity in the genetic defect found in CEP. URO‐III‐S activity was determined in erythrocyte lysates by a coupled enzyme assay. Erythrocyte URO‐III‐S activities of patients were reduced to 4−33 % of the normal value (1.72 ± 0.14 pkat/mg protein). An increase of urinary coproporphyrin isomer I (40−61 %, norm = 17−31 %) and a halved URO‐III‐S activity can serve as a biochemical test for asymptomatic heterozygous gene carriers of CEP.
Nonenzymatic palmitoylation at Cys 3 causes extra‐activation of the α‐subunit of the stimulatory GTP‐binding protein Gs
Authors: Mollner Stefan , Ferreira Patrik , Beck Krista , Pfeuffer Thomas
Published: March 2003

Treatment of crude stimulatory GTP‐binding protein of adenylyl cyclase (Gs) from turkey erythrocyte membranes with hydroxylamine results in twofold enhancement of adenylyl cyclase activity following reconstitution with adenylyl cyclase type V expressed in Spodoptera frugiperda cells (Sf9) cells. Enhancement by hydroxylamine of immunoaffinity purified Gs was still 1.5‐fold, while that of Gs purified according to the multiple‐step procedure by Northup, J. K., Sternweis, P. C., Smigel, M. D., Schleifer, L. S., Ross, E. M. & Gilman, A. G. (Proc. Natl Acad. Sci. USA 78, 6516−6520, 1980) was close to unity. The α‐subunit of the stimulatory GTP‐binding protein expressed in Escherichia coli (rαs), likewise, failed to show an effect by hydroxylamine. Surprisingly, guanosine 5′‐O‐(3‐thiotriphosphate) (GTP[S])‐liganded rαs, treated with palmitoyl‐CoA for 14 h at 4 +C resulted in sixfold enhancement of reconstitutive activity. In contrast, that of the GDP‐liganded rαsβγ heterotrimer was not improved by palmitoylation and consecutive activation with GTP[S], although incorporation of [3H]palmitate into momomer and heterotrimer was identical. While adenylyl cyclase type‐V activity reconstituted by rαs  GTP[S] was not influenced by βγ‐subunits, that activated by palmitoylated rαs  GTP[S] was considerably inhibited, suggesting a higher affinity of palmitoylated rαs for βγ‐subunits. On treatment of either form with the proteinase Lys‐C, less than 25 % of the label was found in a stable Mr 38 000 fragment with intact C terminus, but lacking the N‐terminal portion. Absence of the latter did not affect activation by rαs, but caused a > 90 % loss of extra‐activation by palmitoylated rαs. The results also indicate that nonenzymatic, much in the same way as physiological enzymatic, palmitoylation of αs occurs predominantly on Cys 3.
Caspase 3 specifically cleaves p21WAF1/CIP1 in the earlier stage of apoptosis in SK‐HEP‐1 human hepatoma cells
Authors: Park Jeong Ae , Kim Kyu‐Won , Kim Shin Il , Lee Seung Ki
Published: March 2003

We report here that p21WAF1/CIP1, an inhibitor of cyclin kinases, underwent proteolytic processing into a smaller fragment, p14, in the early stage of apoptosis in SK‐HEP‐1 cells. Apoptosis was induced by either staurosporine or ginsenoside Rh2, a ginseng saponin with a dammarane skeleton. Proteolytic processing was the result of caspase‐3 activity, which accompanied the early changes in cell morphology and DNA fragmentation. p21WAF1/CIP1 translated in vitro was cleaved into a p14 fragment when incubated with cell extracts obtained from either ginsenoside Rh2‐treated or staurosporine‐treated cells. Cleavage was equally inhibited in both cases by adding Ac‐DEVD‐CHO, a specific caspase‐3 inhibitor, but not by Ac‐YVAD‐CHO, a specific caspase‐1 inhibitor. Similarly, p21WAF1/CIP1 was efficiently cleaved by recombinant caspase‐3, overexpressed in Escherichia coli. Moreover, the endogenous p21WAF1/CIP1 of untreated cell extracts was also cleaved by recombinant caspase 3, as measured by immunoblotting. Mutation analysis allowed identification of two caspase‐3 cleavage sites, DHVD112/L and SMTD149/F, which are located within or near the interaction domains for cyclins, Cdks, and proliferating cell nuclear antigen (PCNA). Taken together, these results show that ginsenoside Rh2 and staurosporine increase caspase‐3 activity, which in turn directly cleaves p21WAF1/CIP1 during the early stages of apoptosis. We propose that proteolytic cleavage of p21WAF1/CIP1 is a functionally relevant event that allows release of the cyclin/Cdk complex from the p21WAF1/CIP1 inhibitor, resulting in the elevated levels of cyclin/Cdk kinase activity seen in the earlier stage of apoptosis.
O‐Demethylase from Acetobacterium dehalogenans
Authors: Kaufmann Franz , Wohlfarth Gert , Diekert Gabriele
Published: March 2003

The ether‐cleaving O‐demethylase from the strictly anaerobic homoacetogen Acetobacterium dehalogenans catalyses the methyltransfer from 4‐hydroxy‐3‐methoxy‐benzoate (vanillate) to tetrahydrofolate. In the first step a vanillate :corrinoid protein methyltransferase (methyltransferase I) mediates the methylation of a 25‐kDa corrinoid protein with the cofactor reduced to cob(I)alamin. The methyl group is then transferred to tetrahydrofolate by the action of a methylcorrinoid protein :tetrahydrofolate methyltransferase (methyltransferase II). Using primers derived from the amino‐terminal sequences of the corrinoid protein and the vanillate :corrinoid protein methyltransferase (methyltransferase I), a 723‐bp fragment was amplified by PCR, which contained the gene odmA encoding the corrinoid protein of O‐demethylase. Downstream of odmA, part of the odmB gene encoding methyltransferase I was identified. The amino acid sequence deduced from odmA showed about 60 % similarity to the cobalamin‐binding domain of methionine synthase from Escherichia coli (MetH) and to corrinoid proteins of methyltransferase systems involved in methanogenesis from methanol and methylamines. The sequence contained the DXHXXG consensus sequence typical for displacement of the dimethylbenzimidazole base of the corrinoid cofactor by a histidine from the protein. Heterologous expression of odmA in E. coli yielded a colourless, oxygen‐insensitive apoprotein, which was able to bind one mol cobalamin or methylcobalamin/mol protein. Both of these reconstituted forms of the protein were active in the overall O‐demethylation reaction. OdmA reconstituted with hydroxocobalamin and reduced by titanium(III) citrate to the cob(I)alamin form was methylated with vanillate by methyltransferase I in an irreversible reaction. Methylcobalamin carrying OdmA served as methyl group donor for the methylation of tetrahydrofolate by methyltransferase II. This reaction was found to be reversible, since methyltransferase II also catalysed the methylation of cob(I)alamin containing OdmA with methyltetrahydrofolate.
Preparation of recombinant tissue inhibitor of metalloproteinases‐1 (TIMP‐1) in high yield and identification of a hydrophobic surface feature
Authors: Hodges Deborah J. , Reid David G. , Rowan Andrew D. , Clark Ian M. , Cawston Timothy E.
Published: March 2003

The work presented here describes an effective method for refolding recombinant tissue inhibitor of metalloproteinases‐1 (TIMP‐1), a 21‐kDa protein with six disulphide bonds. A yield of 30 mg TIMP‐1/l culture medium was obtained from a high level bacterial expression system, using a slow removal of denaturant in the presence of 0.5 M guanidine and a suitable redox buffer. This protein is identical to the wild‐type species when specific activity and secondary structure (by CD) are compared. The fluorescent, hydrophobic compound 8‐anilino 1‐naphthalene sulphonate (ANS) was used to quantify hydrophobic binding sites on the surface of both wild‐type and recombinant TIMP‐1. The wild‐type protein has 1 binding site with a mean Kd of 1.3 mM and the recombinant protein has 1.5 binding sites with a mean Kd of 0.39 mM. The presence of surface hydrophobic residues is confirmed by selective broadening of ethyl and aromatic signals in the 1H‐NMR spectrum on the addition of the paramagnetic probe 4‐hydroxy‐2,2,6,6‐tetramethylpiperidinyl‐N‐oxy, OH‐TEMPO, to wild‐type TIMP‐1. When wild‐type TIMP‐1 is incubated with the N‐terminal fragment of human fibroblast collagenase prior to the addition of ANS, the number of binding sites in the system decreases to 0.5 with a Kd of 0.15 mM.
Differences in in vivo acceptor specificity of two galactosyltransferases, the gmh3+ and gma12+ gene products from Schizosaccharomyces pombe
Authors: Yoko‐o Takehiko , Roy Samir Kumar , Jigami Yoshifumi
Published: March 2003

In the fission yeast Schizosaccharomyces pombe, gmh1+, gmh2+ and gmh3+ genes encode α‐1,2‐galactosyltransferase homologues. In an in vitro galactosyltransferase assay, the gmh3+ gene product showed galactose transfer activity toward α‐methyl‐
D‐mannoside as an acceptor. The disruption of gma12+, the major galactosyltransferase gene [Chappell, T. G., Hajibagheri, M. A. N., Ayscough, K., Pierce, M. & Warren, G. (1994) Mol. Biol. Cell 5, 519−528], and of gmh3+ in S. pombe caused decreases in the total remaining galactosyltransferase activity and cell surface galactose content. Disruption of gma12+ and gmh3+ also caused an increase in electrophoretic mobility of acid phosphatase, indicating their involvement in the galactosylation of cell surface glycoproteins. The gmh3Δgma12Δ double mutant cells had more severe galactose‐less phenotypes than single gene mutant cells. HPLC analysis of O‐linked mannoprotein oligosaccharides from wild‐type and disrupted strains revealed that the gma12+ gene product is responsible for the galactosylation of O‐linked oligosaccharide, whereas gmh3+ has no involvement in the process. In contrast, both the gmh3+ and gma12+ gene products are involved in the galactosylation of the N‐linked core oligosaccharide Man9GlcNAc2. From these results, it is evident that there are some functional differences between the enzymes in the process of galactosylation. It appears that the gmh3+ gene product transfers galactose to N‐linked oligosaccharide, while the gma12+ gene product transfers galactose to both N‐linked and O‐linked oligosaccharides.
Lyotropic‐salt‐induced changes in monomer/dimer/tetramer association equilibrium of formyltransferase from the hyperthermophilic Methanopyrus kandleri in relation to the activity and thermostability of the enzyme
Authors: Shima Seigo , Tziatzios Christos , Schubert Dieter , Fukada Harumi , Takahashi Katsutada , Ermler Ulrich , Thauer Rudolf K.
Published: March 2003

Formyltransferase from Methanopyrus kandleri is composed of only one type of subunits of molecular mass 32 kDa. The enzyme requires the presence of lyotropic salts for activity and thermostability. We report here that the enzyme is in a monomer/dimer/tetramer association equilibrium, the association constant being affected by lyotropic salts. At 0.01 M K2HPO4/KH2PO4, pH 7.2, the enzyme (0.4 mg/ml) was mainly present in a monomeric form. Upon increase of the phosphate concentration, the concentration of the dimer increased up to a phosphate concentration of 0.6 M, then decrease at the expense of tetramer formation up to a phosphate concentration of 1.0 M. The specific activity at 4 +C increased from < 0.1 U/mg at 0.01 M, over 1.5 U/mg at 0.6 M to 3.6 U/mg at 1.0 M. Similar results were obtained with ammonium sulfate as lyotropic salt. The findings indicate that both oligomerization and activity increase with increasing salt concentrations, suggesting that there is a causal connection. To determine this, we exploited the observation that oligomer formation was not induced by the weak lyotropic salt NaCl up to a concentration of 1.5 M and that the dissociation of the dimer into the monomer at 4 +C proceeded very slowly (50 % in approximately 6 h). This allowed us to study the effect of NaCl on the activity of the oligomers at NaCl concentrations not sufficient to induce oligomerization. At 4 +C, the activity of the oligomers increased from 0.3 U/mg at 0.25 M NaCl to 3.4 U/mg at 1.0 M NaCl. At these NaCl concentrations, the monomers were inactive. The findings indicate that oligomerization is a prerequisite for enzyme activity in the presence of NaCl. The salt‐dependent induction of oligomerization was parallelled by an increase in thermostability; strong lyotropic salts conferred thermostability at much lower concentrations than the weak lyotropic NaCl.
MtDNA mutations associated with sideroblastic anaemia cause a defect of mitochondrial cytochrome c oxidase
Authors: Bröker Sabine , Meunier Brigitte , Rich Peter , Gattermann Norbert , Hofhaus Götz
Published: March 2003

We have recently described heteroplasmic mutations of mitochondrial DNA in patients suffering from sideroblastic anaemia. The mutations change conserved residues I280 and M273 in subunit I of cytochrome oxidase, the terminal enzyme of the mitochondrial respiratory chain. As a step towards elucidating the pathogenic mechanism, we studied the biochemical consequences of the mutations by transferring mtDNA from these patients' platelets into a permanent human cell line lacking a mitochondrial genome. Mutation‐induced changes of the enzyme and the energy metabolism of the cells were characterised in the transmitochondrial cell lines. One of the mutations resulted in a decreased cellular concentration of the enzyme and a corresponding decrease in activity. The second mutation changed the structure around the binuclear centre and forced the cells to rely more strongly on glycolysis.
The N‐terminal region of non‐Aβ component of Alzheimer's Disease amyloid is responsible for its tendency to assume β‐sheet and aggregate to form fibrils
Authors: El‐Agnaf Omar M. A. , Bodles Angela M. , Guthrie David J. S. , Harriott Patrick , Irvine G. Brent
Published: March 2003

Examination of the N‐terminal sequence of non‐Aβ component of Alzheimer's Disease amyloid (NAC) revealed a degree of similarity to regions crucial for aggregation and toxicity of three other amyloidogenic proteins, namely amyloid β peptide (Aβ), prion protein (PrP) and islet amyloid polypeptide (IAPP), leading us to believe that this might be the part of the molecule responsible for causing aggregation. Secondary structure prediction analysis of NAC indicated that the N‐terminal half was likely to form a β‐structure whereas the C‐terminal half was likely to form an α‐helix. NAC in solution altered from random coil to β‐sheet structure upon ageing, a process that has previously been shown to lead to fibril formation. To delineate the region of NAC responsible for aggregation we synthesised two fragments, NAC‐(1−18)‐peptide and NAC‐(19−35)‐peptide, and examined their physicochemical properties. Upon incubation, solutions of NAC‐(1−18)‐peptide became congophilic and aggregated to form fibrils of diameter 5−10 nm, whereas NAC‐(19−35)‐peptide did not bind Congo Red and remained in solution. Circular dichroism spectroscopy was used to study the secondary structure of NAC and the two fragments. In trifluoroethanol/water mixtures, NAC and NAC‐(19−35)‐peptide adopted α‐helical structure but NAC‐(1−18)‐peptide did not. NAC‐(1−18)‐peptide and NAC formed β‐sheet in acetonitrile/water mixtures more readily than did NAC‐(19−35)‐peptide. CD spectra of NAC or NAC‐(1−18)‐peptide in aqueous solution indicate the formation of β‐sheet on ageing. We propose that the N‐terminal region of NAC is the principal determinant of aggregation. Our results indicate that NAC resembles Aβ, and other amyloidogenic proteins, in that aggregation is dependent upon β‐sheet development. These results lend support to a role for NAC in the development of neurodegenerative disease.
Membrane topology of the myelin/oligodendrocyte glycoprotein
Authors: della Gaspera Bruno , Pham‐Dinh Danielle , Roussel Guy , Nussbaum Jean‐Louis , Dautigny André
Published: March 2003

Myelin/oligodendrocyte glycoprotein (MOG), a specific component of the mammalian central nervous system, is located on the surface of the oligodendrocyte plasma membrane and the outermost lamellae of mature myelin; it is expressed during the latter steps of myelinogenesis. It has been shown that MOG may play a pathological role in autoimmune demyelinating diseases of the central nervous system, although its physiological function remains unknown. MOG is an integral membrane glycoprotein with an extracellular immunoglobulin‐like domain and two hydrophobic segments which were predicted to be membrane‐spanning on the basis of hydropathy analysis. As a first step in elucidation of MOG function, we have investigated its membrane topology, combining immunofluorescence studies on cultured oligodendrocytes and MOG‐transfected Chinese hamster ovary cells with biochemical analyses, including in vitro translation, membrane insertion and protease‐digestion assays. Our results indicate that the C‐terminal tail of MOG is located into the cytoplasm, and that only the first hydrophobic region of MOG spans the membrane whereas the second hydrophobic region appears to be semi‐embedded in the lipid bilayer, lying partially buried in the membrane with its N‐terminal and C‐terminal boundaries facing the cytoplasm.
The deoxyribonuclease activity attributed to ribosome‐inactivating proteins is due to contamination
Authors: Day Philip J. , Lord J. Michael , Roberts Lynne M.
Published: March 2003

The mode of action of ribosome‐inactivating proteins (RIPs) has, for many years, been considered to be depurination of a specific adenyl residue of ribosomal RNA, resulting in inhibition of protein synthesis. Recently, this view has been challenged by the observation that many RIP preparations have significant DNase activity in addition to their N‐glycosidase activity. In this study, we have investigated the putative DNase activity of two RIPs, ricin and pokeweed antiviral protein (PAP), and show that, in both cases, the DNase activity is due to the presence of contaminating nucleases. The N‐glycosidase and DNase activities of PAP were separately and specifically inactivated by chemical modification and heat. Gel filtration of ricin allowed physical separation of the two activities. Furthermore, neither recombinant PAP nor recombinant ricin A‐chain purified from Escherichia coli displayed DNase activity.
Inactivation of isocitrate dehydrogenase kinase/phosphatase by 5′‐[p‐(fluorosulfonyl)benzoyl]adenosine is not due to the labeling of the invariant lysine residue found in the protein kinase family
Authors: Oudot Christelle , Jault Jean‐Michel , Jaquinod Michel , Negre Didier , Prost Jean‐François , Cozzone Alain J. , Cortay Jean‐Claude
Published: March 2003

The ATPase activity of Escherichia coli isocitrate dehydrogenase kinase/phosphatase was rapidly lost after prior incubation with the ATP analogue 5′‐[p‐(fluorosulfonyl)benzoyl]adenosine (FSBA). This inactivation was prevented by the presence of either 5 mM ATP or 5 mM ADP plus Mg2+, while it could be fully reversed by subsequent addition of dithiothreitol, thereby indicating the involvement of cysteine residue(s) in this process. About 2 mol [3H]FSBA/mol IDHK/P were bound during the time course of the inactivation. However, this binding was not significantly modified by either prior incubation with ATP or subsequent addition of dithiothreitol. This suggested that FSBA‐mediated inactivation of isocitrate dehydrogenase kinase/phosphatase occurred via the formation of a disulfide bond. Accordingly, mass spectral analysis revealed that on addition of FSBA, a disulfide bond was formed between residues Cys356 and Cys523. The mutation Cys356Ser renders the enzyme insensitive to FSBA treatment indicating that Cys356 is the primary target for this analogue. However, the Cys523Ser mutant was still inactivated by FSBA and mass spectral analysis showed that this was due to the formation of a new disulfide bond between Cys356 and Cys480.
Structure of the O polysaccharide and immunochemical relationships between the lipopolysaccharides of Pseudomonas syringae pathovar tomato and pathovar maculicola
Authors: Knirel Yuriy A. , Ovod Vladimir V. , Zdorovenko Galina M. , Gvozdyak Rostislav I. , Krohn Kai J.
Published: March 2003

O polysaccharides (OPS) of the lipopolysaccharides (LPS) of Pseudomonas syringae pathovar tomato GSPB 483 and pathovar maculicola IMV 381 were studied by 1H‐NMR and 13C‐NMR spectroscopy, including two‐dimensional COSY, rotating‐frame NOE spectroscopy (ROESY), and H‐detected 1H,13C heteronuclear multiple‐quantum coherence (HMQC) experiments. The OPS from both strains were shown to be chemically identical. Two structurally different types of repeating units (O repeats 1 and 2) were elucidated in each OPS. The minor O repeat is a pentasaccharide having the structure 2. The same structure has been reported earlier for some other OPS of P. syringae. The major O repeat is a hexasaccharide with the novel structure 1 which is distinguished from 2 by the presence of the second lateral residue of 3‐acetamido‐3,6‐dideoxy‐
D‐galactose (
D‐Fuc3NAc) and by a different position of substitution of one of the
L‐rhamnose (Rha) residues in the backbone.

α‐ D‐Fucp3NAc
α‐
D‐Fucp3NAc
1
1
↓↓
3
3
←2)‐α‐
L‐Rhap‐(1←2)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←1

α‐ D‐Fucp3NAc
1
↓
3
←2)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←2

Structural and serological diversity of OPS of strains belonging to P. syringae pv. tomato and pv. maculicola and phylogenetic relatedness of the strains are discussed.
Selective coupling of STAT factors to the mouse prolactin receptor
Authors: Mayr Stefan , Welte Thomas , Windegger Michaela , Lechner Judith , May Petra , Heinrich Peter C. , Horn Friedemann , Doppler Wolfgang
Published: March 2003

Prolactin has been reported to induce distinct sets of signal transducers and activators of transcription (STAT) in a cell‐type‐specific fashion. In the mammary epithelium, although STAT1, STAT3, STAT5A, STAT5B and STAT6 are present in a latent form, only STAT5A and STAT5B are activated. This selective activation of STAT5 by prolactin was also observed in COS‐7 cells cotransfected with the long form of the mouse prolactin receptor (PRL‐R) and expression vectors for STAT1, STAT3, STAT5 and STAT6. Mutated PRL‐Rs and chimeric erythropoietin/gp130 (EPO/gp130) receptors with a tyrosine‐containing motif attached at the carboxy terminus were employed to determine the sites in the PRL‐R required for the specific activation of STAT5. The experiments revealed the importance of two motifs containing Y477 and Y578 in the PRL‐R. When linked to the EPO/gp130 receptor, these sequences were sufficient to specifically induce DNA binding of STAT5 and to activate transcription from the β‐casein gene promoter. By contrast, only weakly they induced DNA binding of STAT6 and STAT3 and did not induce STAT1. A synthetic nonapeptide with phosphorylated Y477 was able to disrupt STAT5 DNA binding in vitro. Our results define structural domains within the carboxy terminus of the PRL‐R which recruit STAT5 for signalling and which are capable of distinguishing STAT5 from other STAT proteins. The activity of STAT5 forms with deletions of the carboxy terminus was induced more strongly than that of their full‐length counterparts 2 min after activation of the PRL‐R. This effect was not dependent on the presence of Y477 and Y578 in the PRL‐R, indicating that facilitated activation of short STAT5 isoforms relies on mechanisms other than increased coupling to specific regions of the PRL‐R.
Purification and cDNA cloning of cytokinin‐specific binding protein from mung bean (Vigna radiata)
Authors: Fujimoto Yasuyuki , Nagata Ryuji , Fukasawa Hiroshi , Yano Keiichi , Azuma Masaki , Iida Akihiro , Sugimoto Seiji , Shudo Koichi , Hashimoto Yuichi
Published: March 2003

Synthetic urea derivatives such as N‐phenyl‐N′‐(4‐pyridyl)urea (4PU) and N‐(2‐chloro‐4‐pyridyl)‐N′‐phenylurea (4PU30) have strong cytokinin activities. Using tritiated 4PU30 as a probe, we previously established the presence of a cytokinin‐specific binding protein (CSBP) of high affinity (Ka for 4PU30 = 4×1010 M−1) in the soluble fraction of etiolated mung bean seedlings [Nagata, R., Kawachi, E., Hashimoto, Y. & Shudo, K. (1993) Biochem. Biophys. Res. Commun. 191, 543−549]. In this report, we purified CSBP by the use of 4PU‐Sepharose 4B, an affinity gel liganded with 4PU. We determined partial amino acid sequences of CSBP and isolated its cDNA by reverse‐transcription (RT) PCR. The cDNA encoded a protein with a calculated molecular mass of 17 kDa. A data base homology search revealed that CSBP is a novel member of a major pollen allergen/pathogenesis‐related protein family. Recombinant CSBP was expressed in Escherichia coli and was confirmed to bind specifically to cytokinins.
Crystals of a 1 : 1 complex between human interleukin‐4 and the extracellular domain of its receptor α chain
Authors: Hage Thorsten , Reinemer Peter , Sebald Walter
Published: March 2003

The specific high‐affinity binding of interleukin‐4 (IL‐4) to its receptor α chain is the crucial primary event during IL‐4 signalling. Single crystals, suitable for high resolution diffraction studies, have been obtained from a complex between IL‐4 and the ectodomain of the receptor α chain, also called IL‐4‐binding protein (IL‐4BP). The orthorhombic crystals are in spacegroup P212121 with cell constants a = 5.038 nm, b = 6.841 nm, c = 10.95 nm and diffract to a resolution of at least 0.25 nm when exposed to synchrotron radiation. The volume of the unit cell suggests the presence of a 1 : 1 IL‐4/IL‐4BP complex and HPLC analysis of the crystals confirmed that IL‐4 and IL‐4BP were present in equimolar amounts. An IL‐4 variant comprising a total of four methionine residues was generated, labelled with selenomethionine and crystallised in complex with IL‐4BP. The crystals are isomorphous to that of the complex with normal IL‐4 and therefore can be used to solve the crystallographic phase problem by the method of multiple anomalous diffraction (MAD). The crystal structure of the IL‐4/IL‐4BP complex will help to understand how IL‐4 and other helical cytokines bind and activate their cognate receptor.
Recombinant homo‐ and hetero‐oligomers of an ultrastable chaperonin from the archaeon Pyrodictium occultum show chaperone activity in vitro
Authors: Minuth T. , Frey G. , Lindner P. , Rachel R. , Stetter K. O. , Jaenicke R.
Published: March 2003

The archaeon Pyrodictium occultum is one of the most thermophilic organisms presently known. Previous experiments provided support for the significant contribution of a high‐molecular‐mass protein complex to the extreme thermotolerance of P. occultum. This protein complex, the ’thermosome', is composed of two subunits, α and β, which form a hexadecameric double ring complex. In order to obtain the thermosome in amounts sufficient for structural and functional investigations, we produced the two subunits jointly and separately in Escherichia coli BL21(DE3). In all three cases, we isolated soluble, high‐molecular‐mass double‐ring complexes from E. coli BL21(DE3). On electron micrographs, the recombinant complexes were indistinguishable from each other and from the natural thermosome. To characterize the quaternary structure of the recombinant particles, we used native gel electrophoresis, analytical gel filtration, and analytical ultracentrifugation. Spectral analysis, using absorption, fluorescence emission and far‐UV circular dichroism spectroscopy were applied to compare the three recombinant protein complexes with the natural thermosome from P. occultum. All three recombinant complex species exhibit ATPase activity. Furthermore, we could demonstrate that the recombinant complexes slow down the aggregation of citrate synthase, alcohol dehydrogenase, and insulin. Thus, we conclude that the recombinant protein complexes exhibit a chaperone‐like activity, interacting with non‐native proteins; they do so at temperatures far below the lower physiological limit of growth.
The StrQ protein encoded in the gene cluster for 5′‐hydroxystreptomycin of Streptomyces glaucescens GLA.0 is a α‐	D‐glucose‐1‐phosphate cytidylyltransferase (CDP‐	D‐glucose synthase)
Authors: Beyer Stefan , Mayer Gerd , Piepersberg Wolfgang
Published: March 2003

The gene strQ was identified as the last gene of a putative transcription unit, strB1FGHPQ, located in the gene cluster for the production of 5′‐hydroxy‐streptomycin (OH‐Sm) in Streptomyces glaucescens GLA.0. [In contrast, the corresponding operon in the str/sts‐gene cluster of the Sm‐producer Streptomyces griseus, strB1FGHIK, differs in the two distal genes; Mansouri, K. & Piepersberg, W. (1991) Mol. Gen. Genet. 228, 459−469.] The deduced StrQ protein exhibited similarities to members of the enzyme family of hexose‐1‐phosphate nucleotidylyltransferases (NDP‐hexose synthases or pyrophosphorylases), with the strongest similarity to the subfamily of α‐
D‐glucose‐1‐phosphate cytidylyltransferases (CDP‐
D‐glucose synthases). The StrQ protein was heterologously expressed in Escherichia coli. The purified protein revealed an enzyme activity of that of a CDP‐
D‐glucose synthase and a substrate specificity restricted to CTP and α‐
D‐glucose 1‐phosphate. The Km and Vmax values determined for CTP are 44 μM and 920 μM and for α‐
D‐glucose 1‐phosphate 195 μM and 1.06 mM, respectively. The CDP‐
D‐glucose synthase activity was also detected in cells of S. glaucescens under the conditions of antibiotic production, but was absent from cells of the streptomycin producer S. griseus N2‐3‐11. Also, the genomes of several strains of S. griseus did not seem to possess strQ‐related genes. In contrast, hybridisation experiments indicated that genes homologous to strQ were probably present in various other actinomycetes producing aminoglycosides. A possible function of the StrQ protein in the OH‐Sm biosynthetic pathway of GLA.0 is discussed.
Inhibition of membrane‐bound electron transport of the methanogenic archaeon Methanosarcina mazei Gö1 by diphenyleneiodonium
Authors: Brodersen Jens , Bäumer , Sebastian , Abken , Hans‐Jörg , Gottschalk , Gerhard , Deppenmeier Uwe
Published: March 2003

The proton translocating electron transport systems (F420H2:heterodisulfide oxidoreductase and H2:heterodisulfide oxidoreductase) of Methanosarcina mazei Gö1 were inhibited by diphenyleneiodonium chloride (DPI) indicated by IC50 values of 20 nmol DPI·mg–1 protein and 45 nmol DPI·mg–1 protein, respectively. These effects are due to a complex interaction of DPI with key enzymes of the electron transport chains. It was found that 2‐hydroxyphenazine‐dependent reactions as catalyzed by F420‐nonreducing hydrogenase, F420H2 dehydrogenase and heterodisulfide reductase were inhibited. Interestingly, the H2‐dependent methylviologen reduction and the heterodisulfide reduction by reduced methylviologen as catalyzed by the hydrogenase and the heterodisulfide reductase present in washed membranes were unaffected by DPI, respectively. Analysis of the redox behavior of membrane‐bound cytochromes indicated that DPI inhibited CoB‐S‐S‐CoM‐dependent oxidation of reduced cytochromes and H2‐dependent cytochrome reduction. Membrane‐bound and purified F420H2 dehydrogenase were inhibited by DPI irrespectively whether methylviologen + metronidazole or 2‐hydroxyphenazine were used as electron acceptors. Detailed examination of 2‐hydroxy‐phenazine‐dependent F420H2‐oxidation revealed that DPI is a competitive inhibitor of the enzyme, indicated by the Km value for 2‐hydroxyphenazine, which increased from 35 µm to 100 µm in the presence of DPI. As DPI and phenazines are structurally similar with respect to their planar configuration we assume that the inhibitor is able to bind to positions where interaction between phenazines and components of the electron transport systems take place. Thus, electron transfer from reduced 2‐hydroxyphenazine to cytochrome b2 as part of the heterodisulfide reductase and from H2 to cytochrome b1 as subunit of the membrane‐bound hydrogenase is affected in the presence of DPI. In case of the F420H2 dehydrogenase electron transport from FAD or from FeS centers to 2‐hydroxyphenazine is inhibited.
Biosynthesis of scarab beetle pheromones
Authors: Leal Walter Soares , Zarbin Paulo H. G. , Wojtasek Hubert , Ferreira José Tércio
Published: March 2003

Chemical communication in scarab beetles (Coleoptera: Scarabaeidae) is achieved with a wide variety of pheromones, but one typical structure is the γ‐lactone having a long unsaturated hydrocarbon chain. Several species utilize (R,Z)‐5‐(–)‐(oct‐1‐enyl)‐oxacyclopentan‐2‐one (buibuilactone), (R,Z)‐5‐(–)‐(dec‐1‐enyl)‐oxacyclopentan‐2‐one and (S,Z)‐5‐(+)‐(dec‐1‐enyl)‐oxacyclopentan‐2‐one [(R)‐japonilure and (S)‐japonilure]. Using deuterated precursors, we have demonstrated that these compounds are biosynthesized from fatty acids. (9,10‐d4)‐Palmitic acid, (9,10‐d4)‐stearic acid, (9,10‐d2)‐palmitoleic acid, (9,10‐d2)‐oleic acid, (9,10‐d2)‐8‐hydroxypalmitoleic acid and (9,10‐d2)‐8‐hydroxyoleic acid were readily incorporated by female Anomala cuprea into the pheromone molecules, while (Z)‐(5,6‐d2)‐5‐dodecenoic acid and (Z)‐(5,6‐d2)‐5‐tetradecenoic acid were not. Therefore, the reaction pathway starts from saturated fatty acids, involves their desaturation, followed by 8‐hydroxylation, chain shortening and cyclization. The products obtained from racemic (9,10‐d2)‐8‐hydroxypalmitoleic acid and (9,10‐d2)‐8‐hydroxyoleic acid were also racemic, implying that the steps following hydroxylation were not stereospecific. Perdeuterated palmitic acid was applied to disclose the mechanism of the unique hydroxylation reaction. Retention of all deuterium atoms implied that this reaction was a direct process mediated by a specific fatty acid hydroxylase, and preceding desaturation or epoxidation was not involved.
Primary structure and possible functions of a trypsin inhibitor of Bombyx mori
Authors: Kurioka Akira , Yamazaki , Masayoshi , Hirano Hisashi
Published: March 2003

A protein with a low molecular mass of 6027 was purified from cocoon shell of silkworm, Bombyx mori. Two‐dimensional polyacrylamide gel electrophoresis (2D/PAGE) resolved this protein into a single spot with pI 4.3 and Mr 6000. Amino acid sequence analysis revealed that this protein consists of 55 amino acids, six of these being cysteine residues and is highly homologous to bovine pancreatic trypsin inhibitor‐type inhibitors. The 6‐kDa protein is heat stable and acid stable and inhibits bovine trypsin by forming a low‐dissociation complex with trypsin in a 1 : 1 molar ratio (Ki = 2.8 × 10–10), but does not α‐chymotrypsin. This cocoon shell‐associated trypsin inhibitor (CSTI) was thus concluded to belong to the bovine pancreatic trypsin inhibitor class. CSTI was developmentally regulated in the silk gland at the final stage of larval growth, and its specific distribution in the middle silk gland, an organ in which silk proteins are stored during the final larval instar, occurred before the onset of spinning. This inhibitor protects the tryptic degradation of fibroin light (L) chain in vitro. These results suggest that this trypsin inhibitor may play an important part on regulating proteolytic activity in the silk gland or protecting silk proteins from degradation during histolysis.
Oligomerization of the 17‐kDa peptide‐binding domain of the molecular chaperone HSC70
Authors: Fouchaq Benoît , Benaroudj Nadia , Ebel Christine , Ladjimi Moncef M.
Published: March 2003

Crystallographic and biochemical studies have indicated that the peptide‐binding site of the molecular chaperone HSC70 is located in a small subdomain comprising a β‐sheet motif followed by a helical region, and there is some evidence of the involvement of this site in oligomerization of the protein.

 To determine the structure of this subdomain in solution and examine its involvement in oligomerization of HSC70, a 17‐kDa protein (residues 385–540 of HSC70) consisting mainly of the peptide‐binding site was constructed and analyzed for oligomerization properties. This small domain was found to bind peptides and to form oligomers in solution, probably tetramers, which dissociated into monomers on peptide binding in a manner comparable with that observed for the whole protein. Furthermore, in the 60‐kDa fragment of HSC70, which is composed of the 17‐kDa domain and the 44‐kDa ATPase domain, not only were the oligomerization properties conserved, but dissociation of multimeric species into monomers on ATP binding also occurred and peptide stimulation of ATPase activity was restored. These results indicate that the isolated 17‐kDa peptide‐binding domain is necessary and sufficient for oligomerization of the whole protein, suggesting that the peptide‐binding site may be involved in the oligomerization process.
Secretion of human meprin from intestinal epithelial cells depends on differential expression of the α and β subunits
Authors: Lottaz Daniel , Hahn Dagmar , Müller Stefan , Müller Christoph , Sterchi Erwin E.
Published: March 2003

Human meprin (N‐benzoyl‐l‐tyrosyl‐p‐aminobenzoic acid hydrolase, EC 3.4.24.18), an astacin‐type metalloprotease, is expressed by intestinal epithelial cells as a dimeric protein complex of α and β subunits. In transfected cells, intracellular proteolytic removal of the membrane anchor from the α subunit results in its secretion, while the β subunit and α/β heterodimers are retained at the cell membrane. We investigated the consequence of differential intracellular processing of α and β subunits in the human small and large intestine using subunit‐specific immunohistochemistry, in situ hybridization and biosynthetic studies in organ culture. In the ileum, both subunits localize to the brush‐border membrane of villus enterocytes. In contrast, the β subunit is not expressed in the colon, which leads to the secretion of the α subunit. We conclude that differential expression of meprin α and β subunits is a unique means of targeting the proteolytic activity of the α subunit either to the brush‐border membrane in the ileum or to the lumen in the colon, suggesting dual functions of cell‐associated and luminal meprin. Meprin α and β subunits are also coexpressed in distinct lamina propria leukocytes, suggesting an additional role for this protease in leukocyte function in the intestinal mucosa.
Acceleration of Ca2+ ionophore‐induced arachidonic acid liberation by thrombin without the proteolytic action toward the receptor in human platelets
Authors: Akiba Satoshi , Kawauchi Takashi , Sato Takashi
Published: March 2003

We investigated the regulation of arachidonic acid liberation catalyzed by group‐IV cytosolic phospholipase A2 (cPLA2) in human platelets upon stimulation with thrombin through interaction with protease‐activated receptor‐1 (PAR‐1) or glycoprotein Ib. Leupeptin, a protease inhibitor, completely inhibited thrombin‐induced arachidonic acid liberation and Ca2+ mobilization, with inhibition of its protease activity. However, preincubation with thrombin in the presence of leupeptin potentiated Ca2+ ionophore‐induced arachidonic acid liberation. The preincubation did not affect the intracellular Ca2+ level or cPLA2 activity in response to ionomycin. Human leukocyte elastase, which cleaves glycoprotein Ib, did not inhibit the enhancement of arachidonic acid liberation by thrombin in the presence of leupeptin. However, the effect of thrombin with leupeptin was abolished by a peptide corresponding to residues 54–65 of hirudin (hirudin peptide), which impairs the binding of thrombin to PAR‐1. Furthermore, Phe‐Pro‐Arg chloromethyl ketone (PPACK)‐thrombin, which binds to platelets but has no protease activity, also enhanced Ca2+ ionophore‐induced arachidonic acid liberation. In contrast, trypsin with leupeptin did not mimic the effect of thrombin with leupeptin, and furthermore trypsin‐induced arachidonic acid liberation was insensitive to hirudin peptide. On the basis of the present results, we suggest that thrombin may accelerate cPLA2‐catalyzed arachidonic acid liberation through non‐proteolytic action toward PAR‐1 but not toward glycoprotein Ib in co‐operation with the proteolytic action leading to Ca2+ mobilization.