bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural heterogeneity in the O polysaccharide of Pseudomonas syringae pv. coriandricola GSPB 2028 (NCPPB 3780, W‐43)
Authors: Knirel Yuriy A. , Ovod Vladimir V. , Paramonov Nikolay A. , Krohn Kai J.
Published: March 2003

The O polysaccharide (OPS) of the lipopolysaccharide of Pseudomonas syringae pv. coriandricola GSPB 2028 (NCPPB 3780, W‐43) was studied by Smith degradation and 1H‐NMR and 13C‐NMR spectroscopy, including two‐dimensional COSY, TOCSY, NOESY, and H‐detected 1H,13C heteronuclear multiple‐quantum coherence (HMQC) experiments. The OPS was shown to consist of pentasaccharide O repeats of two types both containing four
L‐rhamnose and one 3‐acetamido‐3,6‐dideoxy‐
D‐galactose (
D‐Fuc3NAc) residue. Structure 1 of the major O repeat which had been established earlier [Das, S., Ramm, M., Kochanowski, H. & Basu, S. (1994) J. Bacteriol. 176, 6550−6557], was confirmed by our data, and a new structure 2 was elucidated for the minor O repeat and found to differ from the structure 1 only in the position of substitution of one of the rhamnose residues in the main chain.

α‐ D‐Fucp3NAc

1

↓

2

←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

↓

2

←3)‐α‐ L‐Rhap‐(1←2)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←3)‐α‐
L‐Rhap‐(1←

2

A role of structural and immunochemical features of the LPS for defining the taxonomical position of the bacterium studied is discussed.
Purification, cDNA cloning and expression of human NG,NG‐dimethylarginine dimethylaminohydrolase
Authors: Kimoto Masumi , Miyatake Satoko , Sasagawa Takayo , Yamashita Hiromi , Okita Misako , Oka Tatsuzou , Ogawa Tadashi , Tsuji Hideaki
Published: March 2003

cDNA encoding NG,NG‐dimethylarginine dimethylaminohydrolase from rat kidney had been cloned [Kimoto, M., Sasakawa, T., Tsuji, H., Miyatake, S., Oka, T., Nio, N. & Ogawa, T. (1997) Biochim. Biophys. Acta 1337, 6−10]. The enzyme hydrolyzes NG,NG‐dimethyl‐
L‐arginine and NG‐monomethyl‐
L‐arginine, which are known as endogenous inhibitors for the nitric oxide‐generating system. In the present study, human NG,NG‐dimethylarginine dimethylaminohydrolase has been purified to homogeneity from liver and characterized. The cDNA clone encoding human NG,NG‐dimethylarginine dimethylaminohydrolase was isolated from a human kidney λgt10 library using a probe prepared from a plasmid containing the entire coding region of rat NG,NG‐dimethylarginine dimethylaminohydrolase. Its open reading frame encoded a protein of 285 amino acids with a molecular mass of 31 121 Da. The deduced amino acid sequence exhibits 93 % identity with that of rat. The cDNA was expressed as a fusion protein in Escherichia coli and the recombinant protein exhibited enzyme activity which is the same as that of natural enzyme.
An apyrase from Mimosa pudica contains N5,N10‐methenyl tetrahydrofolate and is stimulated by light
Authors: Ghosh Rinku , Biswas Susweta , Roy Siddhartha
Published: March 2003

An apyrase (NTP/NDPase) implicated in the response of Mimosa pudica to stimuli, such as touch, has been cloned, sequenced and expressed in Escherichia coli. While purifying and characterizing this enzyme, it was observed that a chromophore is associated with it, having absorption in the ultraviolet‐A/blue region of the spectrum. The absorbance maximum of the chromophore, purified from the enzyme complex by gel filtration and HPLC, is around 350 nm. The chromophore has been identified as N5,N10‐methenyl tetrahydrofolate (MTHF) by comparing the excitation and emission spectra of synthetic MTHF and the isolated cofactor, and by reconstitution of the enzyme complex with synthetic MTHF. Upon excitation with light (350 nm), an increase of apyrase activity was observed in the purified or reconstituted holoenzyme but not in the apoenzyme. The wavelength dependence of the light stimulation matched well with the fluorescence excitation spectra of the cofactor, MTHF. Possible implications of the results for signal transduction in M. pudica have been discussed.
The role of the fourth extracellular domain of the rat corticotropin‐releasing factor receptor type 1 in ligand binding
Authors: Sydow Sabine , Flaccus Andrea , Fischer André , Spiess Joachim
Published: March 2003

The role of the fourth extracellular loop (e4) of rat corticotropin‐releasing factor (CRF) receptor, type 1, in ligand binding was investigated using chimeric receptor molecules. e4 of CRF receptor, type 1, was replaced by the corresponding domains of two other G protein‐coupled receptors, the rat glucagon receptor or the human pituitary adenylate cyclase activating polypeptide (PACAP) receptor. Both chimeras were transported properly to the cell membranes of transfected chinese hamster ovary cells as indicated by immunocytochemical analysis. Ovine CRF (oCRF) was bound specifically, but with low affinity (Kd = 2–5 µm). Cyclic AMP was not accumulated intracellularly in response to increasing concentrations of oCRF. Based on these data, it is concluded that e4 of rat CRF receptor, type 1, is involved in ligand binding. To confirm the importance of e4 in binding CRF, three negatively charged amino acids of e4, Glu336, Asp337 and Glu338, were replaced by Gln, Asn and Gln, respectively. No effect on ligand binding and cyclic AMP accumulation was observed (Kd = 5 nm; EC50 = 1.5 nm). However, when Tyr346, Phe347 and Asn348 of e4 were changed to three alanine residues, ligand binding affinity as well as efficacy in cyclic AMP accumulation were significantly decreased (Kd = 64 nm; EC50 = 32 nm).
Desulfovibrio gigas neelaredoxin
Authors: Silva Gabriela , Oliveira Solange , Gomes Cláudio M. , Pacheco Isabel , Liu Ming Y. , Xavier António V. , Teixeira Miguel , LeGall Jean , Rodrigues‐Pousada Claudina
Published: March 2003

Neelaredoxin, a small non‐heme blue iron protein from the sulfate‐reducing bacterium Desulfovibrio gigas[Chen, L., Sharma, P., LeGall, J., Mariano, A.M., Teixeira M. and Xavier, A.V. (1994) Eur. J. Biochem.226, 613–618] is shown to be encoded by a polycistronic unit which contains two additional open reading frames (ORF‐1 and ORF‐2) coding for chemotaxis‐like proteins. ORF‐1 has domains highly homologous with those structurally and functionally important in methyl‐accepting chemotaxis proteins, including two putative transmembrane helices, potential methylation sites and the interaction domain with CheW proteins. Interestingly, ORF‐2 encodes a protein having homologies with CheW proteins.

 Neelaredoxin is also shown to have significant superoxide dismutase activity (1200 U·mg–1), making it a novel type of iron superoxide dismutase. Analysis of genomic data shows that neelaredoxin‐like putative polypeptides are present in strict anaerobic archaea, suggesting that this is a primordial superoxide dismutase.

 The three proteins encoded in this operon may be involved in the oxygen‐sensing mechanisms of this anaerobic bacterium, indicating a possible transcriptional mechanism to sense and respond to potential stress agents.
Crystal state and solution conformation of the B blood group trisaccharide α‐l‐Fucp‐(1→2)‐[α‐d‐Galp]‐(1→3)]‐β‐d‐Galp‐OCH3
Authors: Otter Albin , Lemieux Raymond U. , Ball Richard G. , Venot André P. , Hindsgaul Ole , Bundle David R.
Published: March 2003

The crystal structure of the human B blood group related trisaccharide α‐l‐Fucp‐(1→2)‐[α‐d‐Galp]‐(1→3)‐β‐d‐Galp‐OCH3 (1) has been determined. The solution structure of 1 was studied by two‐dimensional NMR techniques at 600 MHz in D2O solution and the conformational properties were analyzed in terms of the torsional angles φH and ψH, derived from 3JCH coupling constants, and 10 inter‐residue proton–proton distances. 3JCH could be accurately measured by a recently introduced two‐dimensional heteronuclear correlation experiment (EXSIDE). The nuclear Overhauser enhancement‐derived distances and the calculated torsion angles were compared with the same information available from the crystal structure. The agreement is excellent, indicating that the trisaccharide adopts a restricted conformation in solution, which was also predicted by the Hard Sphere Exo‐Anomeric forcefield. The data of 1 are complemented by NMR studies of the closely related α‐l‐Fucp‐(1→2)‐[6‐deoxy‐α‐d‐Galp]‐(1→3)‐β‐d‐GalpO‐(CH2)7CH3 trisaccharide (2).
Pre‐steady‐state kinetics of the reactions of [NiFe]‐hydrogenase from Chromatium vinosum with H2 and CO
Authors: Happe Randolph P. , Roseboom Winfried , Albracht Simon P. J.
Published: March 2003

Results are presented of the first rapid‐mixing/rapid‐freezing studies with a [NiFe]‐hydrogenase. The enzyme from Chromatium vinosum was used. In particular the reactions of active enzyme with H2 and CO were monitored. The conversion from fully reduced, active hydrogenase (Nia‐SR state) to the Nia‐C* state was completed in less than 8 ms, a rate consistent with the H2‐evolution activity of the enzyme. The reaction of CO with fully reduced enzyme was followed from 8 to 200 ms. The Nia‐SR state did not react with CO. It was discovered, contrary to expectations, that the Nia‐C* state did not react with CO when reactions were performed in the dark. When H2 was replaced by CO, a Nia‐C* EPR signal appeared within 11 ms; this was also the case when H2 was replaced by Ar. With CO, however, the Nia‐C* state decayed within 40 ms, due to the generation of the Nia‐S·CO state (the EPR‐silent state of the enzyme with bound CO). The Nia‐C* state, induced after 11 ms by replacing H2 by CO in the dark, could be converted, in the frozen enzyme, into the EPR‐detectable state with CO bound to nickel (Nia*·CO) by illumination at 30 K (evoking the Nia‐L* state), followed by dark adaptation at 200 K. This can be explained by assuming that the Nia‐C* state represents a formally trivalent state of nickel, which is unable to bind CO, whereas nickel in the Nia‐L* and the Nia*·CO states is formally monovalent.
ATP and phosphate reciprocally affect subunit association of human recombinant High Km 5′‐nucleotidase
Authors: Spychala Jozef , Chen Valerie , Oka Jun , Mitchell Beverly S.
Published: March 2003

IMP‐specific, High Km 5′‐nucleotidase ( EC 3.1.3.5) is an ubiquitous enzyme, the activity of which is highly regulated by substrate, ATP, and inorganic phosphate. The
cDNA encoding this enzyme has recently been cloned and found to contain a unique stretch of nine glutamic and four aspartic acid residues at the C‐terminus. To study the effects of this acidic tail, and of ATP and inorganic phosphate on enzyme function, we generated several structural modifications of the 5′‐nucleotidase cDNA, expressed the corresponding proteins in Escherichia coli and compared their molecular and kinetic properties. As with the enzyme purified from human placenta, all recombinant proteins were activated by ATP and inhibited by inorganic phosphate. Although the S0.5‐values were higher, the specific activities of the purified protein variants (except that truncated at the C‐terminus) were similar. The molecular mass of the full‐length enzyme subunit has been estimated at 57.3 kDa and the molecular mass of the native protein, as determined by gel‐filtration chromatography, was estimated to be 195 kDa. Increasing the concentration of NaCl to 0.3 m promoted oligomerization of the protein and the formation of aggregates of 332 kDa. ATP induced further oligomerization to 715 kDa, while inorganic phosphate reduced the estimated molecular mass to 226 kDa. In contrast to the truncation of 30 amino acids at the N‐terminus, which did not alter enzyme properties, the removal of the polyglutamic/aspartic acid tail of 13 residues at the C‐terminus caused profound kinetic and structural changes, including a 29‐fold decrease in specific activity and a significant increase in the sensitivity to inhibition by inorganic phosphate in the presence of AMP. Structurally, there was a dramatic loss of the ability to form oligomers at physiological salt concentration which was only partially restored by the addition of NaCl or ATP. These data suggest an important function of the polyglutamic acid tract in the process of association and dissociation of 5′‐nucleotidase subunits.
Allosteric modulation of BPTI interaction with human α‐ and ζ‐thrombin
Authors: De Cristofaro Raimondo , Landolfi Raffaele
Published: March 2003

In this study, thrombin interaction with the basic pancreatic trypsin inhibitor (BPTI) was investigated in the presence of different allosteric modulators of thrombin, that is the C‐terminal hirudin peptide 54–65 (Hir54‐‐65), a recombinant thrombomodulin form (TMEGF4–6) and Na+. BPTI binding to α‐thrombin is positively linked to Na+. Under low sodium concentration (5 m
m Na+) the BPTI affinity for α‐thrombin was roughly threefold lower than in the presence of 150 m
m sodium (Ki = 320 µm vs. 100 µm). The hirudin fragment, which binds to the fibrinogen recognition site (FRS) of thrombin, induced a progressive and saturable decrease (3.6‐fold) of α‐thrombin affinity for BPTI, whereas the thrombomodulin peptide, which binds to a more extended region of FRS, caused a 5.5‐fold increase of the enzyme affinity for the inhibitor. The opposite effect exerted by Hir54‐‐65 and TMEGF4–6 was also observed for BPTI interaction with ζ‐thrombin, in which the amidic bond between W148 and T149 is cleaved. However, in this case the effect by Hir54‐‐65 and TMEGF4–6, although qualitatively similar to that observed with α‐thrombin, had a smaller magnitude. Thrombin hydrolysis of Protein C was also differently affected by Hir54‐‐65 and TMEGF4–6 peptides. While the latter enhanced the Protein C activation, the former caused a reduction of both α‐ and ζ‐thrombin kcat/Km′ for Protein C cleavage. These results showed that (a) Na+ facilitates BPTI interaction with thrombin; (b) Hir54‐‐65 and TMEGF4–6, though sharing in part the same binding site at the thrombin FRS, can affect in opposite way thrombin’s interaction with BPTI and Protein C; (c) such findings along with the results obtained with ζ‐thrombin might be explained by admitting that the thermodynamic linkage between FRS and the critical W60‐loop is also controlled by ligation and/or conformational state of the W148 insertion loop.
Purification of a new intestinal anti‐proliferative factor from normal human small intestine
Authors: Lavagna Christian , Poirée Jean‐Claude , Fournel Sophie , Rampal Patrick
Published: March 2003

Previous studies suggest that intestinal cell proliferation may be controlled by endogenous mitosis inhibitors. We describe here the isolation of a protein named intestinal anti‐proliferative factor (IAF) from human small intestine. Successive DEAE anion exchange, isoelectric focusing and gel filtration chromatographies led to a purified anti‐proliferative protein fraction used to produce antibodies. Using these antibodies as affinity chromatography ligand, IAF was purified from human small intestine cytosolic fraction. IAF was a potent inhibitor of adenocarcinoma colon cells (HT‐29 D4 line) DNA synthesis and proliferation with 50% inhibition observed at picomolar concentrations. Analyzed on SDS/PAGE under reducing conditions, this protein migrates with an apparent molecular mass of 120 kDa and amino acid sequence of two internal peptides displays no homology with another listed protein. Cell cycle studies showed that the growth inhibitory effect was maximal between mid G1 and early S phases. Moreover, flow cytometry studies demonstrated that IAF inhibited the progression of HT‐29 D4 cells from G1 to S phase. Northern blot analysis using a dipeptidyl peptidase IV probe revealed that the growth arrest mediated by IAF was not linked to differentiation processes. By Western blotting with polyclonal antibodies against IAF, we found that this protein was not detected in differentiated colonic carcinoma. Our results suggest that IAF might regulate intestinal cell proliferation.
The cell surface expression level of the human interleukin‐5 receptor α subunit determines the agonistic/antagonistic balance of the human interleukin‐5 E13Q mutein
Authors: Van Ostade Xaveer , Van Der Heyden José , Verhee Annick , Vandekerckhove Joël , Tavernier Jan
Published: March 2003

The human interleukin‐5 (IL‐5) receptor consists of an α‐chain that specifically binds the ligand with intermediate affinity, and a βc‐chain, that associates with the IL‐5/IL‐5Rα complex, leading to a high‐affinity, signal transducing receptor complex. Structure‐function studies showed that modification of the putative βc‐chain binding site in IL‐5 (E13Q mutein) converted the molecule into an antagonist. However, analysis of the effect of this mutant IL‐5 on COS‐1 cells transfected with both receptor subunits, did not show reduced interaction with the βc subunit [Tavernier, J., Tuypens, T., Verhee, A., Plaetinck, G., Devos, R., Van der Heyden, J., Guisez, Y. & Oefner, C. (1995) Proc. Natl Acad. Sci. USA89, 7041–7045]. To gain more insight into the mechanism of IL‐5 antagonism by E13Q, we tested its biological activity on two FDC‐P1 subclones that express clearly different numbers of α‐subunits yet an almost constant number of murine βc‐subunits. Here we show that E13Q has a biological activity comparable to wild‐type IL‐5 only when a high number of α‐chains is present on the cells. Confirming the critical role of the IL5Rα cell‐surface expression level, treatment with suboptimal doses of a neutralising anti‐IL‐5Rα antibody results in reduced activity of the mutant but not of wild‐type IL‐5.
Interactions of the Streptomyces lividans initiator protein DnaA with its target
Authors: Majka Jerzy , Jakimowicz Dagmara , Messer Walter , Schrempf Hildgund , Lisowski Marek , Zakrzewska‐Czerwińska Jolanta
Published: March 2003

The Streptomyces lividans DnaA protein (73 kDa) consists, like other bacterial DnaA proteins, of four domains; it binds to 19 DnaA boxes in the complex oriC region. The S. lividans DnaA protein differs from others in that it contains an additional stretch of 120 predominantly acidic amino acids within domain II. Interactions between the DnaA protein and the two DnaA boxes derived from the promoter region of the S. lividans dnaA gene were analysed in vitro using three independent methods: Dnase‐I‐footprinting experiments, mobility‐shift assay and surface plasmon resonance (SPR). The Dnase‐I‐footprinting analysis showed that the wild‐type DnaA protein binds to both DnaA boxes. Thus, as in Escherichia coli and Bacillus subtilis, the S. lividans dnaA gene may be autoregulated. SPR analysis showed that the affinity of the DnaA protein for a DNA fragment containing both DnaA boxes from the dnaA promoter region (KD = 1.25 nm) is 10 times higher than its affinity for the single ‘strong’ DnaA box (KD = 12.0 nm). The mobility‐shift assay suggests the presence of at least two classes of complex containing different numbers of bound DnaA molecules. The above data reveal that the DnaA protein binds to the two DnaA boxes in a cooperative manner. To deduce structural features of the Streptomyces domain II of DnaA protein, the amino acid DnaA sequences of three Streptomyces species were compared. However, according to the secondary structure prediction, Streptomyces domain II does not contain any common relevant secondary structural element(s). It can be assumed that domain II of DnaA protein can play a role as a flexible protein spacer between the N‐terminal domain I and the highly conserved C‐terminal part of DnaA protein containing ATP‐binding domain III and DNA‐binding domain IV.
Structural elucidation of a novel exopolysaccharide produced by a mucoid clinical isolate of Burkholderia cepacia
Authors: Cérantola Stéphane , Lemassu‐Jacquier Anne , Montrozier Henri
Published: March 2003

The structure of the exopolysaccharide (EPS) produced by a clinical isolate of Burkholderia cepacia isolated from a patient with fibrocystic lung disease has been investigated. By means of methylation analyses, carboxyl reduction, partial depolymerization by fuming HCl and chemical degradations such as Smith degradation, lithium‐ethylenediamine degradation and β‐elimination, supported by GC/MS and NMR spectroscopic analyses, the repeat unit of the EPS has been identified and was shown to correspond to the acidic branched heptasaccharide with the following structure:

This partially acetylated acidic polymer, distinguished by the presence of the less usual d‐isomer of rhamnose and of a trisubstituted glucuronic acid residue, could represent the main EPS produced by this bacterial species.
Use of doxycycline‐controlled gene expression to reversibly alter milk‐protein composition in transgenic mice
Authors: Soulier Solange , Stinnakre Marie‐Georges , Lepourry Laurence , Mercier Jean‐Claude , Vilotte Jean‐Luc
Published: March 2003

A reverse tetracycline transactivator‐encoding cDNA under the control of the mammary specific β‐lactoglobulin promoter was linked to a bovine α‐lactalbumin transcription unit driven by a reverse tetracycline‐controlled transactivator/doxycycline‐inducible human cytomegalovirus promoter. The construct was microinjected into eggs from α‐lactalbumin‐deficient mice. These mice produce a highly viscous lactose‐free milk and have a shortened lactation period. Mice from three out of the nine transgenic lines investigated expressed reverse tetracycline‐controlled transactivator mRNA in their lactating mammary glands at levels detectable by Northern analysis. Following doxycycline addition to the drinking water, lactation was fully restored in animals from the three lines. Doxycycline removal resulted in a reversal of phenotype. The observed mammary‐specific and high expression of the doxycycline inducible reporter gene (up to 5.2 mg of recombinant α‐lactalbumin·mL−1of milk, i.e. up to 13‐fold induction) opens up exciting prospects to use the tetracycline system to study the development and functioning of the mammary gland, and to control the production level of active pharmaceutical proteins in the milk of transgenic animals.
A novel form of human neuropsin, a brain‐related serine protease, is generated by alternative splicing and is expressed preferentially in human adult brain
Authors: Mitsui Shinichi , Tsuruoka Nobuo , Yamashiro Kyoko , Nakazato Hiroshi , Yamaguchi Nozomi
Published: March 2003

We have cloned cDNAs encoding two isoforms of a human novel serine protease. They encoded sequences of 260 and 305 amino acids, and both showed significant homology to mouse neuropsin. Mouse neuropsin has been reported to be involved in hippocampal plasticity, therefore we designated the proteins as type 1 and type 2 neuropsin, respectively. The amino acid sequences of the two types of human neuropsin were identical, except that type 2 carried an insert of 45 amino acids at the C‐terminus of the leader sequence. The essential three amino acids in the active site triad, His, Asp, and Ser, and the single putative N‐glycosylation site were conserved in human and mouse neuropsin. Sequence analysis of the 946 bp genomic DNA spanning the region encoding the insertion sequence revealed that two isoforms were generated in human brain by alternative splicing. However, the mouse genomic sequence did not conserve the 3′ acceptor consensus sequence at the corresponding position, suggesting that type 2 neuropsin was a species‐specific splice variant. When the open reading frames of human neuropsin were expressed in insect cells, both types of neuropsin were detected in the conditioned media by western blot analysis using anti‐human neuropsin serum. Northern blot hybridization and reverse transcription‐polymerase chain reaction showed predominant expression of type 1 neuropsin in pancreas. Type 2 neuropsin was preferentially expressed in human adult brain and hippocampus, although both types were expressed in fetal brain and placenta in comparable amounts. Dot blot hybridization showed that neuropsin was expressed in various regions of adult brain, including the hippocampus and cerebral cortex, and also in various fetal tissues. These results suggest that human type 2 neuropsin may be important to the adult brain plasticity, although both types may be necessary for the development of the nervous system.
Mitogen‐activated protein kinase (p38‐, JNK‐, ERK‐) activation pattern induced by extracellular and intracellular singlet oxygen and UVA
Authors: Klotz Lars‐Oliver , Pellieux Corinne , Briviba Karlis , Pierlot Christel , Aubry Jean‐Marie , Sies Helmut
Published: March 2003

Ultraviolet A (UVA; 320–400 nm) radiation in human skin fibroblasts induces a pattern of mitogen‐activated protein kinase (MAPK) activation consisting of a rapid and transient induction of p38 and c‐Jun‐N‐terminal kinase (JNK) activity but not extracellular signal‐regulated kinases (ERK). UVA activation of p38 can be inhibited by the singlet oxygen (1O2) quenchers azide and imidazole, but not by the hydroxyl radical scavengers mannitol or dimethylsulfoxide, pointing to the involvement of 1O2. The same effect has been shown for JNK. Like UVA, 1O2 generated intracellularly upon photoexcitation of Rose Bengal activates p38 and JNK but not ERK. p38 and JNK activation was also elicited by chemiexcitation for the intracellular generation of 1O2 by the lipophilic 1,4‐endoperoxide of N,N′‐di(2,3‐dihydroxypropyl)‐1,4‐naphthalene dipropionamide. In contrast, extracellular generation of 1O2, by irradiation of Rose Bengal immobilized on agarose beads or by chemiexcitation employing the hydrophilic 1,4‐endoperoxide of disodium 3,3′‐(1,4‐naphthylidene) dipropionate, was ineffective in activating p38 or JNK. These data suggest that the activation of p38 and JNK by 1O2 occurs only when the electronically excited molecule is generated intracellularly.
The structures of the carbohydrate backbones of the lipopolysaccharides from Escherichia coli rough mutants F470 (R1 core type) and F576 (R2 core type)
Authors: Vinogradov Evgeny V. , van der Drift Koen , Thomas‐Oates Jane E. , Meshkov Sergey , Brade Helmut , Holst Otto
Published: March 2003

The lipopolysaccharides (LPS) from Escherichia coli rough mutant strains F470 (R1 core type) and F576 (R2 core type) were deacylated yielding in each case a mixture of oligosaccharides with one predominant product which was isolated using high‐performance anion‐exchange chromatography. In addition, one oligosaccharide present in minor quantities was isolated from LPS of E. coli strain F576 (R2 core type). The structures of the oligosaccharides were determined by chemical analyses and NMR spectroscopic experiments. Furthermore, de‐O‐acylated and dephosphorylated LPS preparations were investigated by fast‐atom bombardment and collision induced dissociation tandem mass spectrometry. The combined data allow us to deduce the following carbohydrate backbones of the E. coli R1 and R2 core types which share the following structure (Scheme 1):

but differ in the substituents R1 and R2 which for the R1 core type are predominantly:

and to a minor extent:

and for the R2 core type predominantly:

and to a minor extent:

in which all sugars are d‐pyranoses (l,d‐Hep, lglycerodmanno‐heptopyranose; P, phosphate).
The identity determinants required for the discrimination between tRNAGlu and tRNAAsp by glutamyl‐tRNA synthetase from Escherichia coli
Authors: Sekine Shun‐ichi , Nureki Osamu , Tateno Masaru , Yokoyama Shigeyuki
Published: March 2003

We previously elucidated the major determinant set for Escherichia coli tRNAGlu identity (U34, U35, C36, A37, G1•C72, U2•A71, U11•A24, U13•G22••Α46, and Δ47) and showed that the set is sufficient to switch the identity of tRNAGln to Glu [Sekine, S., Nureki, O., Sakamoto, K., Niimi, T., Tateno, M., Go, M., Kohno, T., Brisson, A., Lapointe, J. & Yokoyama, S. (1996) J. Mol. Biol.256, 685–700]. In the present study, we attempted to switch the identity of tRNAAsp, which has a sequence similar to that of tRNAGlu, and consequently possesses many nucleotide residues corresponding to the Glu identity determinants (U35, C36, A37, G1•C72, and U11•A24). A simple transplantation of the rest of the major determinants (U34, U2•A71, U13•G22••Α46, and Δ47) to the framework of tRNAAsp did not result in a sufficient switch of the tRNAAsp identity to Glu. To confer an optimal glutamate accepting activity to tRNAAsp, two other elements, C4•G69 in the middle of the acceptor stem and C12•G23••C9 in the augmented D helix, were required. Consistently, the two base pairs, C4•G69 and C12•G23, in tRNAGlu had been shown to exist in the interface with glutamyl‐tRNA synthetase (GluRS) by phosphate‐group footprinting. We also found the two elements in the framework of tRNAGln, and determined that their contributions successfully changed the identity of tRNAGln to Glu in the previous study. By the identity‐determinant set (C4•G69 and C12•G23••C9 in addition to U34, U35, C36, A37, G1•C72, U2•A71, U11•A24, U13•G22••Α46, and Δ47) the activity of GluRS was optimized and efficient discrimination from the noncognate tRNAs was achieved.
Nonselective coupling of the human µ‐opioid receptor to multiple inhibitory G‐protein isoforms
Authors: Gaibelet Gérald , Meilhoc Eliane , Riond Joëlle , Saves Isabelle , Exner Torsten , Liaubet Laurence , Nürnberg Bernd , Masson Jean‐Michel , Emorine Laurent J.
Published: March 2003

The human µ‐opioid receptor was expressed in Saccharomyces cerevisiae. Binding of [3H]diprenorphine to yeast spheroplasts was specific and saturable (Kd = 1 nm, Bmax = 0.2–1 pmol·mg−1 of membrane proteins). Inhibition of [3H]diprenorphine binding by antagonists and agonists with varying opioid selectivities (µ, δ and κ) occurred with the same order of potency as in mammalian tissues. Affinities of antagonists were the same with yeast spheroplasts as in reference tissues whereas those of agonists, except etorphine and buprenorphine, were 10‐fold to 100‐fold lower. Addition of heterotrimeric Gi,o‐proteins purified from bovine brain shifted the µ‐opioid receptor into a high‐affinity state for agonists. Using individually purified Gα‐subunits re‐associated with βγ‐dimers, we showed that αo1, αo2, αi1, αi2 and αi3 reconstituted high‐affinity agonist binding with equal efficiency. This suggests that the structural determinants of the µ‐opioid receptor responsible for G‐protein coupling are not able to confer a high degree of specificity towards any member of the Gi,o family. The selective effects of opioid observed in specialized tissues upon opioid stimulation may be a result of regulation of G‐protein activity by cell‐specific factors which should conveniently be analysed using the reconstitution assay described here.
Conformational variation of the central CG site in d(ATGACGTCAT)2 and d(GAAAACGTTTTC)2
Authors: Cordier Christine , Marcourt Laurence , Petitjean Michel , Dodin Guy
Published: March 2003

The determination of the solution structure of two self‐complementary oligomers d(ATGACGTCAT)2 (CG10) and d(GAAAACGTTTTC)2 (CG12), both containing the 5′‐pur‐ACGT‐pyr‐3′ sequence, is reported. The impact of the base context on the conformation of the central CpG site has been examined by a combined approach of: (a) 2D 1H‐NMR and 31P‐NMR; (b) molecular mechanics under experimental constraints; (c) back‐calculations of NOESY spectra and iterative refinements of distances; and (d) 3D‐homology search of the central tetrad ACGT within the complete oligonucleotides. A full NMR study of each fragment is achieved by means of standard 2D experiments: NOESY, 2D homonuclear Hartmann–Hahn spectroscopy, double‐quantum‐filtered COSY and heteronuclear 1H‐31P correlation. Sugar phase angle, ε–ζ difference angle and NOE‐derived distances are input as experimental constraints to generate molecular models by energy minimization with the help of jumna. The morass program is used to iteratively refine the structures obtained. The similarity of the two ACGTs within the whole oligonucleotides is investigated. Both the decamer and the dodecamer adopt a B‐like DNA conformation. However, the helical parameters within this conformational type are significantly different in CG12 and CG10. The central CpG step conformation is not locked by its nearest environment (5′A and 3′T) as seen from the structural analysis of ACGT in the two molecules. In CG12, despite the presence of runs of A‐T pairs, CpG presents a high twist of 43° and a sugar phase at the guanine of about 180°, previously observed in other ACGT‐containing‐oligomers. Conversely, ACGT in CG10 exhibits strong inclinations, positive rolls, a flat profile of sugar phase, twist and glycosidic angles, as a result of the nucleotide sequence extending beyond the tetrad. The structural specificity of CG10 and its flexibility (as reflected by its energy) are tentatively related to the process of recognition of the cyclic AMP response element by its cognate protein.
Manipulating the coordination mumber of the ferric iron within the cambialistic superoxide dismutase of Propionibacterium shermanii by changing the pH‐value
Authors: Schmidt Marius
Published: March 2003

The structure of the Propionibacterium freudenreichii subspecies shermanii superoxide dismutase (SOD) was determined at various pH values. As a comparison, the structure of the fluoride coordinated SOD was solved. The SOD crystallizes at pH 6.1 in the space group C2221 with two subunits, A and B, in the asymmetric unit. An increase of the pH value changes the cell parameters slightly but not the symmetry of the crystals. The overall structure of the SOD remains a compact tetrameter and is comparable to that at pH 6.1 no matter whether the pH increases or fluoride is added. At values above pH 7.4, an additional hydroxide ion can bind to the active center. Its position is similar to the binding site of the fluoride. The coordination number changes from five to six if the pH increases or fluoride is added. The binding behavior of the hydroxide ion is different for subunit A and B. Structures at different pH‐values are comparable with models derived by spectroscopic methods. The influence of temperature on the binding properties of the hydroxide ion was investigated using analysis of an X‐ray structure solved at pH 8.1 and 140 K. Compared to the structure at room temperature, the structural changes are observable but remain small. The consequences of hydroxide binding to the iron are discussed.
Reaction mechanism of phosphoglucosamine mutase from Escherichia coli
Authors: Jolly Laure , Ferrari Paul , Blanot Didier , van Heijenoort Jean , Fassy Florence , Mengin‐Lecreulx Dominique
Published: March 2003

The phosphoglucosamine mutase (GlmM) from Escherichia coli, specifically required for the interconversion of glucosamine‐6‐phosphate and glucosamine‐1‐phosphate (an essential step in the pathway for cell‐wall peptidoglycan and lipopolysaccharide biosyntheses) was purified to homogeneity and its kinetic properties were investigated. The enzyme was active in a phosphorylated form and catalysed its reaction according to a classical ping‐pong bi‐bi mechanism. The dephosphorylated and phosphorylated forms of GlmM could be separated by HPLC and coupled MS showed that only one phosphate was covalently linked to the active site of the enzyme. The site of phosphorylation was clearly identified as Ser102 in the 445‐amino acid polypeptide. GlmM was also capable of catalysing the interconversion of glucose‐1‐phosphate and glucose‐6‐phosphate isomers, although at a much lower (1400‐fold) rate. Interestingly, the mutational change of the Ser100 to a threonine residue resulted in a 20‐fold increase of the nonspecific phosphoglucomutase activity of GlmM, suggesting that the presence of either a serine or a threonine at this position in the consensus sequence of hexosephosphate mutases could be one of the factors that determines the specificity of these enzymes for either sugar‐phosphate or amino sugar‐phosphate substrates.
Identification of AtPIS, a phosphatidylinositol synthase from Arabidopsis
Authors: Collin Sylvie , Justin Anne‐Marie , Cantrel Catherine , Arondel Vincent , Kader Jean‐Claude
Published: March 2003

Phosphatidylinositol synthase is the enzyme responsible for the synthesis of phosphatidylinositol, a key phospholipid component of all eukaryotic membranes and the precursor of messenger molecules involved in signal transduction pathways for calcium‐dependent responses in the cell. Using the amino acid sequence of the yeast enzyme as a probe, we identified an Arabidopsis expressed sequence tag potentially encoding the plant enzyme. Sequencing the entire cDNA confirmed the homology between the two proteins. Functional assays, performed by overexpression of the plant cDNA in Escherichia coli, a bacteria which lacks phosphatidylinositol and phosphatidylinositol synthase activity, showed that the plant protein induced the accumulation of phosphatidylinositol in the bacterial cells. Analysis of the enzymatic activity in vitro showed that synthesis of phosphatidylinositol occurs when CDP‐diacylglycerol and myo‐inositol only are provided as substrates, that it requires manganese or magnesium ions for activity, and that it is at least in part located to the bacterial membrane fraction. These data allowed us to conclude that the Arabidopsis cDNA codes for a phosphatidylinositol synthase. A single AtPIS genetic locus was found, which we mapped to Arabidopsis chromosome 1.
Characterization of specific binding sites for a mitogenic sulfated peptide, phytosulfokine‐α, in the plasma‐membrane fraction derived from Oryza sativa L.
Authors: Matsubayashi Yoshikatsu , Sakagami Youji
Published: March 2003

Treatment of rice cells with an endogenous mitogenic peptide, phytosulfokine‐α (PSK‐α), results in cell proliferation. In the present study, [3H]PSK‐α prepared by catalytic reduction of a PSK‐α analog containing tetradehydroisoleucine was employed to identify putative PSK‐α target molecules on rice plasma membranes. Membrane binding of the ligand was found to be saturable, reversible and pH dependent. Scatchard analysis demonstrated the existence of both high‐ and low‐affinity binding sites with Kd values of 1.4 nm and 27 nm, respectively. Competition studies with [3H]PSK‐α and several PSK‐α analogs showed that displacing activity closely corresponds to the ability to induce cell proliferation. The properties of the binding sites distributed on plasma membranes are consistent with the function of PSK‐α receptors in activating a cascade of molecular events involved in plant cell proliferation.
Sequencing and characterization of the citrus weevil, Diaprepes abbreviatus, trypsin cDNA
Authors: Yan Xin‐Hua , De Bondt Hendrik L. , Powell Charles C. , Bullock Robert C. , Borovsky Dov
Published: March 2003

Trypsin mRNA from the citrus weevil, Diaprepes abbreviatus, was reverse transcribed and amplified by PCR. A cDNA species of 513 bp was cloned and sequenced. The 3′ and 5′ ends of the gene (262 bp and 237 bp, respectively) were amplified by rapid amplification of cDNA ends, cloned and sequenced. The deduced sequence of the trypsin cDNA (860 bp) encodes for 250 amino acids including 11 amino acids of activation and signal peptides and exhibited 16.8% identity to trypsin genes of selected Lepidoptera and Diptera. A three‐dimensional model of Diaprepes trypsin contained two domains of β‐barrel sheets as has been found in Drosophila and Neobellieria. The catalytic active site is composed of the canonical triad of His41, Asp92 and Ser185 and a specificity pocket occupied by Asp179 with maximal activity at pH 10.4. Southern blot analysis indicated that at least two copies of the gene are encoded by Diaprepes midgut. Northern blot analysis detected a single RNA band below 1.35 kb at different larval ages (28–100 days old). The message increased with age and was most abundant at 100 days. Trypsin activity, on the other hand, reached a peak at 50 days and fell rapidly afterwards indicating that the trypsin message is probably regulated translationally. Feeding of soybean trypsin inhibitor and Aedes aegypti trypsin modulating oostatic factor affected trypsin activity and trypsin biosynthesis, respectively. These results indicate that Diaprepes regulates trypsin biosynthesis with a trypsin modulating oostatic factor‐like signal.
Hydrolysis by somatic angiotensin‐I converting enzyme of basic dipeptides from a cholecystokinin/gastrin and a LH‐RH peptide extended at the C‐terminus with Gly‐Arg/Lys‐Arg, but not from diarginyl insulin
Authors: Isaac R Elwyn , Michaud Annie , Keen Jeff N , Williams Tracy A , Coates David , Wetsel William C , Corvol Pierre
Published: March 2003

Endoproteolytic cleavage of protein prohormones often generates intermediates extended at the C‐terminus by Arg‐Arg or Lys‐Arg, the removal of which by a carboxypeptidase (CPE) is normally an important step in the maturation of many peptide hormones. Recent studies in mice that lack CP activity indicate the existence of alternative tissue or plasma enzymes capable of removing C‐terminal basic residues from prohormone intermediates. Using inhibitors of angiotensin I‐converting enzyme (ACE) and CP, we show that both these enzymes in mouse serum can remove the basic amino acids from the C‐terminus of CCK5‐GRR and LH‐RH‐GKR, but only CP is responsible for converting diarginyl insulin to insulin. ACE activity removes C‐terminal dipeptides to generate the Gly‐extended peptides, whereas CP hydrolysis gives rise to CCK5‐GR and LH‐RH‐GK, both of which are susceptible to the dipeptidyl carboxypeptidase activity of ACE. Somatic ACE has two similar protein domains (the N‐domain and the C‐domain), each with an active site that can display different substrate specificities. CCK5‐GRR is a high‐affinity substrate for both the N‐domain and C‐domain active sites of human sACE (Km of 9.4 µm and 9.0 µm, respectively) with the N‐domain showing greater efficiency (kcat : Km ratio of 2.6 in favour of the N‐domain). We conclude that somatic forms of ACE should be considered as alternatives to CPs for the removal of basic residues from some Arg/Lys‐extended peptides.
Relationship of membrane sidedness to the effects of the lipophosphoglycan of Leishmania donovani on the fusion of influenza virus
Authors: Razinkov Vladimir , Martin Isabelle , Turco Salvatore J. , Cohen Fredric S. , Ruysschaert Jean‐Marie , Epand Richard M.
Published: March 2003

Cells expressing the influenza hemagglutinin protein were fused to planar lipid bilayers containing the viral receptor GD1a at pH 5.0. An amphiphile known to alter membrane properties is lipophosphoglycan (LPG). This glycoconjugate was added from aqueous solution to either the cis or the trans monolayer to examine its effects on the fusion process. LPG markedly inhibited the formation of fusion pores when present in the cis monolayer but LPG in the trans monolayer had no effect on the parameters of pore formation or on the properties of the pores. The N‐terminal segment of the HA2 subunit of the influenza hemagglutinin protein is important for membrane fusion. The effect of LPG on the conformation and membrane insertion of a synthetic 20‐amino‐acid peptide, corresponding to the influenza fusion peptide, was examined at pH 5.0 by attenuated total reflection Fourier transform infrared spectroscopy and by the fluorescence properties of the Trp residues of this peptide. It was found that cis LPG did not prevent insertion of the peptide into the membrane but it did alter the conformation of the membrane‐inserted peptide from α‐helix to β‐structure. The β‐structure was oriented along the bilayer normal. The effect of cis LPG on the conformation of the fusion peptide probably contributes to the observed inhibition of pore formation and lipid mixing. In contrast, trans LPG has no effect on the conformation or angle of membrane insertion of the peptide, nor does it affect pore formation by HA‐expressing cells. The ineffectiveness of trans LPG, despite it having strong positive curvature‐promoting properties, may be a consequence of the size of this amphiphile being too large to enter a fusion pore.
The inhibitory properties and primary structure of a novel serine proteinase inhibitor from the fruiting body of the basidiomycete, Lentinus edodes
Authors: Odani Shoji , Tominaga Kei , Kondou Satomi , Hori Hiroshi , Koide Takehiko , Hara Saburo , Isemura Mamoru , Tsunasawa Susumu
Published: March 2003

A novel proteinase inhibitor, Lentinus proteinase inhibitor, has been purified from the fruiting bodies of the edible mushroom, Lentinus edodes, by buffer extraction and affinity chromatography on immobilized anhydrotrypsin. The protein simultaneously inhibits bovine β‐trypsin and α‐chymotrypsin at independent sites, with apparent dissociation constants of 3.5 × 10−10 m and 4 × 10−8 m, respectively. The purified protein is eluted as two well‐separated peaks on reversed‐phase HPLC, one of which is inhibitory‐active and the other inactive, and they are interconvertible under folding/unfolding conditions. Among the mammalian and microbial serine proteinases examined, including human enzymes of blood coagulation and fibrinolysis, activated factor XI was inhibited by the Lentinus proteinase inhibitor. Chemical modification studies suggest involvement of one or more arginine residues in the inhibition of trypsin. The complete primary structure composed of 142 amino acids with an acetylated N‐terminus was determined by protein analysis. The theoretical molecular mass (15999.2) from the sequence is close to the experimental value of 15999.61 ± 0.61 determined by mass spectrometry. Although there are no apparently homologous proteinase inhibitors in the protein database, there is a rather striking similarity to the propeptide segment of a microbial serine proteinase, as well as to the N‐terminal region of the mature enzyme.
A CD9, αIIbβ3, integrin‐associated protein, and GPIb/V/IX complex on the surface of human platelets is influenced by αIIbβ3 conformational states
Authors: Longhurst Celia M. , White Melanie M. , Wilkinson Deborah A. , Jennings Lisa K.
Published: March 2003

A noncovalently associated complex comprising of CD9, the fibrinogen (Fg) receptor αIIbβ3, integrin‐associated protein (IAP), and glycoprotein (GP) Ib/V/IX complex was isolated from Chaps‐solubilized human platelets. The CD9 complex was immunoprecipitated by mAbs specific for CD9 (mAb7), IAP (BRIC126), GPIb (SZ1), GPIX (GR‐P), β3 (AP3) and αIIb (C3). Additionally, the association between CD9 and αIIbβ3 was demonstrated by ELISA. In this system, CD9 did not bind to vitronectin receptor (αvβ3) suggesting that CD9/αIIbβ3 association was αIIb‐subunit or αIIbβ3‐complex dependent. D3, an αIIbβ3‐activating mAb that is also an anti‐LIBS (ligand‐induced binding site), immunoprecipitated primarily αIIbβ3 with GPIb and IAP. CD9 was not detected in D3 immunoprecipitates. D3 binding induced platelet aggregation via direct αIIbβ3 activation and was upregulated by the αIIbβ3 antagonist eptifibatide. In contrast, AP3 and C3 exhibited neither effect. In addition, D3 also inhibited whole blood clot retraction, in contrast to AP3 and C3, suggesting that conformational constraints on αIIbβ3 by D3 binding not only influenced the CD9 complex but also affected αIIbβ3 post receptor occupancy events. The CD9 complex was immunoprecipitated in the presence of eptifibatide, demonstrating that αIIbβ3 receptor occupancy was not sufficient to cause complex dissociation. CD9 complex isolation was also independent of platelet activation, although a twofold increase in the quantity of CD9 complex was seen after platelet activation by α‐thrombin in the presence of CaCl2 compared with that present in EDTA. Stirred platelets showed fibrinogen‐mediated aggregation by α‐thrombin in the presence of CaCl2 but not with EDTA, suggesting that fibrinogen crosslinking of CD9 complexes via αIIbβ3 could be partially responsible for this increase. These findings imply that the platelet CD9 complex is independent of platelet activation although it is dependent upon the conformation state of αIIbβ3.