bluuebunny/crossref_metadata_2025 huggingface dataset:
Homocysteine decreases endothelin‐1 production by cultured human endothelial cells
Authors: Demuth Karine , Atger Véronique , Borderie Didier , Benoit Marie‐Odile , Sauvaget Dominique , Lotersztajn Sophie , Moatti Nicole
Published: March 2003

Hyperhomocysteinemia is believed to be responsible for the development of vascular disease via several mechanisms, including the impairment of endothelial‐cell functionality. In‐vitro studies have demonstrated that homocysteine decreases the production or bioavailability of vasodilator autacoids, such as prostacyclin and NO. Here, we show that the treatment of human endothelial cells with noncytotoxic homocysteine concentrations leads to a dose‐dependent decrease in both the secretion of the vasoconstrictor agent endothelin‐1 (ET‐1) and the level of its mRNA. Homocysteine had an inhibitory effect at pathophysiological (0.1 and 0.5 mmol·L−1) and pharmacological noncytotoxic (1.0 and 2.0 mmol·L−1) concentrations. Mean percentage variation from control for ET‐1 production was −36.2 ± 18.9% for 0.5 mmol·L−1 homocysteine and −41.5 ± 26.8% for 1.0 mmol·L−1 homocysteine, after incubation for 8 h. Mean percentage variation from control for steady‐state mRNA was −17.3 ± 7.1% for 0.5 mmol·L−1 homocysteine and −46.0 ± 10.1 for 1.0 mmol·L−1 homocysteine, after an incubation time of 2 h. ET‐1 production was also reduced by incubation with various other thiol compounds containing free thiol groups, but not by incubation with thiol compounds with no free thiol group. Co‐incubation of cells with homocysteine and the sulfhydryl inhibitor N‐ethylmaleimide prevented the effect of homocysteine on ET‐1 production, confirming a sulfhydryl‐dependent mechanism. Based on the reciprocal feedback mechanism controlling the synthesis of vasoactive mediators, these preliminary data suggest a mechanism by which homocysteine may selectively impair endothelium‐dependent vasodilation by primary inhibition of ET‐1 production.
Presence of asparagine‐linked N‐acetylglucosamine and chitobiose in Pyrus pyrifolia S‐RNases associated with gametophytic self‐incompatibility
Authors: Ishimizu Takeshi , Mitsukami Yoshiro , Shinkawa Toyohide , Natsuka Shunji , Hase Sumihiro , Miyagi Masaru , Sakiyama Fumio , Norioka Shigemi
Published: March 2003

S‐RNases encoded by the S‐locus of rosaceous and solanaceous plants discriminate between the S‐alleles of pollen in gametophytic self‐incompatibility reactions, but it is not clear how. We report the structures of N‐glycans attached to each of the N‐glycosylation sites of seven S‐RNases in Pyrus pyrifolia of the Rosaceae. The structures were identified by chromatographic analysis of pyridylaminated sugar chains prepared from S4‐RNase and by liquid chromatography/electrospray ionization‐mass spectrometric analysis of the protease digests of reduced and S‐carboxymethylated S‐RNases. S4‐RNase carries various types of sugar chains, including plant‐specific ones with β1→2‐linked xylose and α1→3‐linked fucose residues. More than 70% of the total N‐glycans of S4‐RNase are, however, an N‐acetylglucosamine or a chitobiose (GlcNAcβ1→4GlcNAc), which has not been found naturally. The N‐acetylglucosamine and chitobiose are mainly present at the N‐glycosylation sites within the putative recognition sites of the S‐RNase, suggesting that these sugar chains may interact with pollen S‐product(s).
Introduction of a C‐terminal aromatic sequence from snake venom phospholipases A2 into the porcine pancreatic isozyme dramatically changes the interfacial kinetics
Authors: Janssen Marcel J. W. , Burghout Pieter J. , Verheij Hubertus M. , Slotboom Arend J. , Egmond Maarten R.
Published: March 2003

Porcine pancreatic phospholipase A2 (PLA2) was modified by single and multiple site‐directed mutations at sites thought to be involved in interfacial binding. Charged and polar residues in the C‐terminal region were replaced by aromatic residues on the basis of an analogy with snake venom PLA2s, which display high affinity for a zwitterionic interface. The PLA2 variants constructed were N117W, N117W/D119Y and K116Y/N117W/D119Y. Titration with micelles of a zwitterionic substrate suggests that the variants N117W and K116Y/N117W/D119Y possess improved ability to bind to the micellar substrate interface, relative to the wild‐type enzyme. Improved interfacial binding was confirmed by direct binding studies with micelles of a zwitterionic substrate analogue, indicating up to five times higher affinity for both variants. Interfacial binding is not improved for the variant N117W/D119Y. Maximal enzyme velocities (

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) with the zwitterionic substrate were between 25 and 75% of that of the wild‐type enzyme. However, competitive inhibition and direct binding studies with a strong inhibitor revealed that the affinity for substrate present at the interface (Km*) is perturbed by the mutations made. For the variant N117W, the slight decrease observed in

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is most likely made up of a 24‐fold reduction in catalytic turnover (kcat) and 18‐fold improved substrate binding (Km*).
Proteome analysis of heat shock protein expression in Bradyrhizobium japonicum
Authors: Münchbach Martin , Dainese Paola , Staudenmann Werner , Narberhaus Franz , James Peter
Published: March 2003

A set of 19 heat shock proteins (Hsp) was observed – by subtractive two‐dimensional gel electrophoresis – to be induced when Bradyrhizobium japonicum, the nitrogen‐fixing root‐nodule symbiont of soybean, was temperature up‐shifted from 28 °C to 43 °C. Up‐regulated protein spots were excised from multiple two‐dimensional gels. The proteins were concentrated using a funnel‐gel device before being blotted onto poly(vinylidene difluoride) membranes for digestion with trypsin before MS and tandem MS analysis or for Edman sequence determination. Five proteins in the range 8–20 kDa were identified as the small Hsp (sHsp; HspB, C, D, E and H) and three others showed strong sequence similarity to the sHsp family. Two other low molecular mass proteins corresponded to GroES1 and GroES2, and five novel proteins were found. Four proteins of ≈ 60 kDa were identified as GroEL2, GroEL4, and GroEL5 and DnaK. An analysis of the heat shock induction of DnaK, of four of the most strongly induced GroESL proteins and six of the sHsp revealed that the proteins could be placed into four distinct regulatory groups based on the kinetics of protein appearance.
Class II DNA photolyase from Arabidopsis thaliana contains FAD as a cofactor
Authors: Kleiner Oliver , Butenandt Jens , Carell Thomas , Batschauer Alfred
Published: March 2003

The major UV‐B photoproduct in DNA is the cyclobutane pyrimidine dimer (CPD). CPD‐photolyases repair this DNA damage by a light‐driven electron transfer. The chromophores of the class II CPD‐photolyase from Arabidopsis thaliana, which was cloned recently [Taylor, R., Tobin, A. & Bray, C. (1996) Plant Physiol. 112, 862; Ahmad, M., Jarillo, J.A., Klimczak, L.J., Landry, L.G., Peng, T., Last, R.L. & Cashmore, A.R. (1997) Plant Cell9, 199–207], have not been characterized so far. Here we report on the overexpression of the Arabidopsis CPD photolyase in Escherichia coli as a 6 × His‐tag fusion protein, its purification and the analysis of the chromophore composition and enzymatic activity. Like class I photolyase, the Arabidopsis enzyme contains FAD but a second chromophore was not detectable. Despite the lack of a second chromophore the purified enzyme has photoreactivating activity.
Fibrinolytic properties of activated FXII
Authors: Braat Ellen A. M. , Dooijewaard Gerard , Rijken Dingeman C.
Published: March 2003

Activated factor XII (FXIIa), the initiator of the contact activation system, has been shown to activate plasminogen in a purified system. However, the quantitative role of FXIIa as a plasminogen activator in contact activation‐dependent fibrinolysis in plasma is still unclear. In this study, the plasminogen activator (PA) activity of FXIIa was examined both in a purified system and in a dextran sulfate euglobulin fraction of plasma by measuring fibrinolysis in a fibrin microtiter plate assay. FXIIa was found to have low PA activity in a purified system. Dextran sulfate potentiated the PA activity of FXIIa about sixfold, but had no effect on the PA activity of smaller fragments of FXIIa, missing the binding domain for negatively charged surfaces. The addition of small amounts of factor XII (FXII) to FXII‐deficient plasma induced a large increase in contact activation‐dependent PA activity, as measured in a dextran sulfate euglobulin fraction, which may be ascribed to FXII‐dependent activation of plasminogen activators like prekallikrein. When more FXII was added, PA activity continued to increase but to a lesser extent. In normal plasma, the addition of FXII also resulted in an increase of contact activation‐dependent PA activity. These findings suggested a significant contribution of FXIIa as a direct plasminogen activator. Indeed, at least 20% of contact activation‐dependent PA activity could be extracted from a dextran sulfate euglobulin fraction prepared from normal plasma by immunodepletion of FXIIa and therefore be ascribed to direct PA activity of FXIIa. PA activity of endogenous FXIIa immunoadsorped from plasma could only be detected in the presence of dextran sulfate. From these results it is concluded that FXIIa can contribute significantly to fibrinolysis as a plasminogen activator in the presence of a potentiating surface.
Differential effect of pressure and temperature on the catalytic behaviour of wild‐type human butyrylcholinesterase and its D70G mutant
Authors: Weingand‐Ziadé Alexandra , Renault Frédérique , Masson Patrick
Published: March 2003

The combined action of temperature (10–35°C) and pressure (0.001–2 kbar) on the catalytic activity of wild‐type human butyrylcholinesterase (BuChE) and its D70G mutant was investigated at pH 7.0 using butyrylthiocholine as the substrate. The residue D70, located at the mouth of the active site gorge, is an essential component of the peripheral substrate binding site of BuChE. Results showed a break in Arrhenius plots of wild‐type BuChE (at Tt ≈ 22 °C) whatever the pressure (dTt/dP = 1.6 ± 1.5 °C·kbar−1), whereas no break was observed in Arrhenius plots of the D70G mutant. These results suggested a temperature‐induced conformational change of the wild‐type BuChE which did not occur for the D70G mutant. For the wild‐type BuChE, at around a pressure of 1 kbar, an intermediate state, whose affinity for substrate was increased, appeared. This intermediate state was not seen for the mutant enzyme. The wild‐type BuChE remained active up to a pressure of 2 kbar whatever the temperature, whereas the D70G mutant was found to be more sensitive to pressure inactivation (at pressures higher than 1.5 kbar the mutant enzyme lost its activity at temperatures lower than 25 °C). The results indicate that the residue D70 controls the conformational plasticity of the active site gorge of BuChE, and is involved in regulation of the catalytic activity as a function of temperature.
DNA bending and sequence‐dependent backbone conformation
Authors: Ojha Rejadra P. , Dhingra Madan M. , Sarma Mukti H. , Shibata Masayuki , Farrar Margaret , Turner Christopher J. , Sarma Ramaswamy H.
Published: March 2003

Although DNA bending plays a crucial role in several biological processes, very little is known experimentally about the relationship between sugar phosphate conformation and sequence directed bending. In this paper, we determine the coupling constants for a nonselfcomplementary 11‐mer A‐tract DNA duplex from 2D NMR experiments and along each chain of the duplex, we report the sugar pucker, torsional preferences and conformational averaging about the C3′‐O3′, C4′‐C5′ and C5′‐O5′ bonds for each nucleotide. The A‐tract exists as an equilibrium blend of canonical B‐form and noncanonical B‐form in which the exocyclic C4′‐C5′ bond is intransconformation as in the original Watson‐Crick model [Crick, F.H.C. & Watson, J.D. (1954)Proc. Roy. Soc. (London),A223, 80–96]. Thetransconformation at the C4′‐C5′ can increase the interphosphate distance and lead to local unwinding of the duplex and rolling of the base pair into the major groove. This will create a kink or hinge. At the 3′‐end of the A‐tract in the purine‐thymine step, the duplex is compressed by the presence of a junction between A and B forms of DNA exclusively in one strand, with consequent reduction of the phosphate‐phosphate distance. The coupling constant data seriously disagree with the A‐tract DNA bending model of Crothers [Koo, H.‐S., Wu, H.‐M. & Crothers, D.M. (1986)Nature320, 501–506], but is in agreement with the finding of Leroyet al. [Leroy, J.‐L., Charretier, E., Kochoyan, M. & Gueron, M. (1988)Biochemistry27, 8894–8898] that the structure that drives bending in the A‐tract is locally different from B‐DNA. Structural distortions are extremely localized with little or no propagation. It is likely that transcription factor proteins recognize these preexisting deformations in the free DNA itself and mold it into the matrix of the protein.
The multifunctional oxidase activity of ceruloplasmin as revealed by anion binding studies
Authors: Musci Giovanni , Bellenchi Gian C. , Calabrese Lilia
Published: March 2003

The effect of multiple binding of azide, N3−, on the structural and functional properties of ceruloplasmin (CP) has been reinvestigated by means of both spectroscopic and enzymatic techniques. High affinity binding of the anion to human CP resulted in a dramatic increase of the absorbance at 610 nm and in a concomitant decrease of the optical density at 330 nm. The oxidase activity toward Fe(II) was essentially unaffected, while turnover parameters versus nonferrous substrates dramatically changed, with an ≈ 100‐fold enhancement of the kcat/Km parameter. Chloride at physiological concentration proved to behave very similarly to N3− bound with high affinity, in that it not only induced the spectroscopic changes previously interpreted in terms of an intramolecular electron transfer from reduced type 1 to type 3 copper ions [Musci, G., Bonaccorsi di Patti, M.C. & Calabrese, L. (1995) J. Protein Chem. 14, 611–617], but it also enhanced some 60‐fold the kcat/Km value. A different behavior was observed with chicken CP, where a decrease at 330 nm occurred without a concomitant modification at 603 nm. The chicken enzyme was less sensitive also in terms of enzymatic activity, which was nearly unchanged in the presence of either high affinity N3− or Cl−. At higher N3− concentrations, optical changes of both human and chicken CP were mainly focussed on the appearance of ligand‐to‐metal charge transfer bands below 500 nm, and the anion behaved as an inhibitor of the oxidase activity versus Fe(II) as well as noniron substrates. The well known bleaching of the blue chromophore could be observed, at neutral pH, only at very high N3−/CP ratios.

The data presented in this paper are consistent with a mechanism of structural and functional modulation of CP by anions, that would be able to dictate the substrate specificity of the cuproprotein, and suggest the possibility that CP may act in vivo as a multifunctional oxidase.
Direct interaction of EEA1 with Rab5b
Authors: Callaghan Judy , Nixon Susan , Bucci Cecilia , Toh Ban‐Hock , Stenmark Harald
Published: March 2003

The early endosomal autoantigen EEA1 is essential for early endosomal membrane fusion. It binds to endosomes via a C‐terminal domain (EEA1‐CT). To identify proteins interacting with EEA1‐CT, we screened a human brain library in the yeast two‐hybrid system. Fourteen clones reacted strongly with EEA1‐CT. Sequencing of these clones revealed that they all contained the ORF of the small GTPase, Rab5b. Further two‐hybrid analysis suggested that Rab5b also interacts with the N‐terminus of EEA1 (EEA1‐NT). The interaction of both EEA1‐CT and EEA1‐NT with Rab5b was confirmed biochemically, and was found to be GTP dependent. Confocal immunofluorescence microscopy indicated that EEA1 colocalizes with Rab5b on early endosomes. Although EEA1‐CT and EEA1‐NT interacted strongly with wild‐type Rab5b in the two‐hybrid system, we detected no interaction with wild‐type Rab5a, even though GTPase‐deficient mutants of both Rab5a and Rab5b interacted equally well with EEA1. This difference could not be explained by differences in intrinsic GTPase activities, as these were found to be very similar. Instead, we speculate that yeast may contain a GTPase‐activating protein (GAP) activity that stimulates Rab5a but not Rab5b. In contrast, pig brain cytosol was found to contain a GAP activity that stimulates the GTPase activity of Rab5b in preference to that of Rab5a. These data provide evidence that EEA1 interacts with both Rab5a and Rab5b, and that the GTPase activities of the two proteins are differentially regulated in vivo.
Why reversing the sequence of the α domain of human metallothionein‐2 does not change its metal‐binding and folding characteristics
Authors: Pan Peter Kuan‐yeu , Zheng Z. F. , Lyu P. C. , Huang P. C.
Published: March 2003

A novel peptide, the backward reading sequence of human metallothionein‐2 α domain, was synthesized and its chemical and spectroscopic properties analyzed. This folded retro‐α domain was able to bind Cd(II) in identical stoichiometries with the chemically synthesized α domain of metallothionein‐2. Nearly identical to the α domain, Cd‐binding retro‐α domain showed a characteristic ultraviolet absorption spectrum with a shoulder at 245–250 nm (due to cadmium–thiolate charge transfer), and the absorption shoulder was abolished by acidification [suggesting mercaptide bonding between Cd(II) and the cysteine residues]. Similar metal‐binding capabilities between α domain and retro‐α domain were observed also by pH titration and in the reaction with the sulfhydryl reagent 5,5′‐dithiobis(2‐nitrobenzoic acid). A two‐state cooperativity of the metal‐cluster formation was observed spectroscopically in the titration of the retro‐α domain, indicating that the retro‐protein is foldable. In contrast to other proteins, our results indicate that the reversion of the amino acid sequence for the α domain does not change its foldability and metal‐binding capacity, suggesting that the order of its sequence is not critical to the formation of a critical metal–tetrathiolate nucleus. However, CD spectra of the Cd‐binding α domain and retro‐α domain showed that the reversal direction of the domain sequence backbone significantly affects the formation of structure even when it is foldable.
Purification and biochemical characterization of a novel cysteine protease of Entamoeba histolytica
Authors: Spinella Sandrine , Levavasseur Etienne , Petek Farhettin , Rigothier Marie C.
Published: March 2003

Cysteine proteases are important virulence factors of Entamoeba histolytica, the causative agent of amoebiasis. A novel cysteine protease from parasite extracts was purified 15‐fold by a procedure including concanavalin A–Sepharose, hydroxylapatite and DEAE–Sepharose chromatography. The purification resulted in the obtainment of an homogeneous protein with a molecular mass of 66 kDa on native PAGE. In 10% SDS/PAGE, three bands of 60, 54 and 50 kDa were evident. Each of the three specific mouse antisera raised against these proteins showed cross‐reactivity with the three bands obtained from the purified eluate. The N‐terminal sequencing of the first 10 amino acids from the three proteins showed 100% identity. These results support the hypothesis of a common precursor for the 60, 54 and 50‐kDa proteins. Protease activity of the purified enzyme was demonstrated by electrophoresis in a gelatine‐acrylamide copolymerized gel. Its activity was quantified by cleaving a synthetic fluorogenic peptide substrate such as N‐carbobenzyloxy‐arginyl‐arginyl‐7‐amido‐4‐methylcoumarin. The optimum pH for the protease activity was 6.5; however, enzymatic activity was observed between pH 5 and pH 7.5. Typical of cysteine proteases, the enzyme was inhibited by 4‐[(2S,3S)‐carboxyoxiran‐2‐ylcarbonyl‐l‐leucylamido]butylguanidine and iodoacetamide, and activated by free sulfhydryl groups. The cellular location of the enzyme was examined on trophozoites before and after contact with red blood cells using indirect immunofluorescence and cellular fractionation. The 60‐kDa cysteine protease translocated to the amoebic surface upon the interaction of trophozoites with red blood cells. This result provided evidence for participation of the 60‐kDa protease in erythrophagocytosis.
Tissue variant effects of heme inhibitors on the mouse cytochrome c oxidase gene expression and catalytic activity of the enzyme complex
Authors: Vijayasarathy C. , Damle Shirish , Lenka Nibedita , Avadhani Narayan G.
Published: March 2003

The in vivo effects of heme biosynthesis inhibitors, succinylacetone and CoCl2 on the cytochrome c oxidase (COX) gene expression and enzyme activity in different mouse tissues were investigated. Succinylacetone and CoCl2 showed tissue‐specific differences in their ability to modulate heme aa3 content. A single dose of succinylacetone treatment for 8 h reduced the heme aa3 content of kidney mitochondria with no effect on the liver. CoCl2 treatment for 8 h, however, selectively affected the heme aa3 level in the liver. Reduced mitochondrial heme aa3 with both treatments was accompanied by ≈ 50% reduced, mitochondrial genome‐encoded COX I and II mRNAs and nuclear genome‐encoded COX Vb mRNAs, but no change in COX IV mRNA level. Use of isolated mouse liver and brain mitochondrial systems showed a 50–80% reduction in mitochondrial transcription and translation rates in heme‐depleted tissues. Blue native gel electrophoresis followed by immunoblot analysis showed that the complex from heme‐depleted tissues contained a 30–50% reduction in levels of subunits I, IV, Vb and near normal levels of subunit VIc, indicating altered subunit content. Treatment of submitochondrial particles with protein kinase A and ATP resulted in partial dissociation of COX, suggesting a mechanistic basis for the reduced subunit content of the complex from heme‐depleted tissues. Surprisingly, the enzyme from heme‐depleted tissues showed twofold to fourfold higher turnover rates for cytochrome c oxidation, suggesting alterations in the kinetic characteristics of the enzyme following heme reduction. This is probably the first evidence that the tissue heme level regulates not only the mammalian COX gene expression, but also the catalytic activity of the enzyme, probably by affecting its stability.
Membrane transport and in vitro metabolism of the Ras cascade messenger, glycerophosphoinositol 4‐phosphate
Authors: Berrie Christopher P. , Iurisci Cristiano , Corda Daniela
Published: March 2003

The glycerophosphoinositols, phosphoinositide metabolites formed by Ras‐dependent activation of phospholipase A2 and a lysophospholipase, have been proposed to be markers of Ras‐induced cell transformation. These compounds can have important cellular effects; GroPIns4P is an inhibitor of G protein‐stimulated adenylate cyclase and is transiently produced in several cell types after growth factor receptor stimulation of phosphatidylinositol 3‐kinase and the small G protein Rac, indicating the importance of defining further its cellular actions and metabolism. We show here that, in postnuclear membranes from Swiss 3T3 cells, there is no high‐affinity ‘receptor’ binding of GroPIns4P. Instead, possibly through the interaction with a transporter, GroPIns4P rapidly equilibrates between medium and cell cytosol, and, at higher concentrations, can concentrate in the cell cytosol. GroPIns4P can be dephosphorylated to GroPIns in vitro by an enzyme that is membrane‐associated, Ca2+‐dependent, GroPIns4P‐selective and has a specific pH profile. Under in vitro phosphorylating conditions, there is production of GroPIns(4,5)P2 and other inositol phosphates. As these in vitro enzyme activities do not fully correlate with the in vivo handling of GroPIns4P, the intracellular GroPIns4P levels may be controlled by its direct physical removal from the cells.
The presence of the alternatively spliced A2 cassette in the vacuolar H+‐ATPase subunit A prevents assembly of the V1 catalytic domain
Authors: Hernando Natividad , David Pe’er , Tarsio Maureen , Bartkiewicz Marcjanna , Horne William C. , Kane Patricia M. , Baron Roland
Published: March 2003

Vacuolar ATPases (V‐ATPases) are multisubunit enzymes that couple the hydrolysis of ATP to the transport of H+ across membranes, and thus acidify several intracellular compartments and some extracellular spaces. Despite the high degree of genetic and pharmacological homogeneity of V‐ATPases, cells differentially modulate the lumenal pH of organelles and, in some cells, V‐ATPases are selectively targetted to the plasma membrane. Although the mechanisms underlying such differences are not known, the subunit isoform composition of V‐ATPases could contribute to altered assembly, targeting or activity. We previously identified an alternatively spliced variant of the chicken A subunit in which a 30 amino acid cassette (A1) containing the Walker consensus sequence for ATP binding is replaced by a 24 amino acid cassette (A2) that lacks this feature. We have examined the ability of chimeric yeast/chicken A subunits containing either the A1 or the A2 cassette to restore the V‐ATPase activity of yeast that lack the A subunit. The A1‐containing chimeric subunit, but not the chimera that contains the A2 cassette, partially restores the ability of the mutated yeast to grow at neutral pH. Both chimeric proteins are expressed, although at lower levels than the similarly transfected yeast A subunit. The A2‐containing subunit fails to associate with the vacuolar membrane or support the assembly of V‐ATPase complexes. Thus, the substitution of the A1 sequence by A2 not only removes the Walker nucleotide binding sequence but also compromises the ability of the A subunit to assemble with other V‐ATPase subunits.
Decisive structural determinants for the interaction of proline derivatives with the intestinal H+/peptide symporter
Authors: Brandsch Matthias , Knütter Ilka , Thunecke Frank , Hartrodt Bianka , Born Ilona , Börner Volker , Hirche Frank , Fischer Gunter , Neubert Klaus
Published: March 2003

To elucidate the decisive structural factors relevant for dipeptide–carrier interaction, the affinity of short amide and imide derivatives for the intestinal H+/peptide symporter (PEPT1) was investigated by measuring their ability to inhibit Gly‐Sar transport in Caco‐2 cells. Dipeptides with proline or alanine in the C‐terminal position displayed affinity constants (Ki) of 0.15–1.2 mm and 0.08–9.5 mm, respectively. There was no clear relationship between hydrophobicity, size or ionization status of the N‐terminal amino acid and the affinity of the dipeptides. However, analyzing the individual peptide bond conformations of Xaa‐Pro dipeptides, a striking correlation between the cis/trans ratios (trans contents 24–70%) and the affinity constants was observed. After correcting the Ki values for the incompetent cis isomers, the Ki corr values of most dipeptides were in a small range of 0.1–0.16 mm. This result revealed the decisive role of peptide bond conformation even for a transport protein that is quite promiscuous in substrate translocation. When measuring affinity constants of Xaa‐Pro and Xaa‐Sar dipeptides, the cis/trans ratios cannot be ignored. Lower affinities of Lys‐Pro, Arg‐Pro and Pro‐Pro indicate that additional molecular factors affect their binding at PEPT1. The Ki values obtained for the corresponding Xaa‐Ala dipeptides support this conclusion.

Potential substrates or inhibitors of peptide transport were found among Xaa‐piperidides and Xaa‐thiazolidides. Dipeptides with N‐terminal proline displayed a very diverse affinity profile. However, in contrast to current knowledge, several Pro‐Xaa dipeptides such as Pro‐Leu, Pro‐Tyr and Pro‐Pro are recognized by PEPT1 with appreciable affinities. Binding seems mainly determined by the hydrophobicity of the C‐terminal amino acid and the rigidity of the structure.
Ribosomal gene disruption in the extreme thermophile Thermus thermophilus HB8
Authors: Simitsopoulou Maria , Avila Horacio , Franceschi François
Published: March 2003

S17 is a primary rRNA‐binding protein which has been implicated in ribosome assembly and translational fidelity. We describe the generation and biochemical characterization of an S17 minus ribosomal mutant, a ribosomal protein‐lacking mutant obtained in Thermus thermophilus HB8. The S17 mutant was obtained by insertional inactivation of the target gene with the kanamycin adenyl transferase (kat) gene, making use of a Thermus–Escherichia shuttle vector and the natural ability of Thermus to transform. In the final construct used to transform Thermus cells, the S17 coding region was replaced with the kat gene cloned in‐frame with the first three amino acids of S17. Hence, in vivo transcription of the kat gene was under the control of the ribosomal operon promoter. As in Escherichia coli, the Thermus S17 mutant exhibited a temperature‐sensitive phenotype. Two‐dimensional PAGE, Western blot, and ELISA confirmed the absence of S17 from the mutant ribosomes. Sucrose‐gradient profiles of mutant cells showed a clear separation and normal proportions of 50S and 30S subunits and a normal ratio between them. In addition, the S17 mutant showed the presence of a 20S peak representing assembly‐defective particles. The successful re‐incorporation of protein S17 into the mutant ribosomes was demonstrated when reconstitution with isolated S17 was performed at 60 °C.
Reaction of neuronal nitric oxide synthase with the nitric oxide spin‐trapping agent, iron complexed with N‐dithiocarboxysarcosine
Authors: Yoneyama Hirohito , Kosaka Hiroaki , Ohnishi Taira , Kawazoe Takeshi , Mizoguchi Kazuhiro , Ichikawa Yoshiyuki
Published: March 2003

A water‐soluble iron complex with N‐dithiocarboxysarcosine (Fe–DTCS) has been developed as an ESR spin‐trapping agent for NO and successfully applied to ESR imaging of endogenous NO production in mice. We attempted to measure NO produced by purified neuronal NO synthase (nNOS) by this method, but could not detect NO. We speculated that Fe–DTCS inhibits NOS activity. In fact, it markedly inhibited NOS activity with an IC50 value of 9.7 ± 0.7 µm in the citrulline‐formation assay. DTCS alone did not inhibit the activity. An iron complex with N‐methyl‐d‐glucamine dithiocarbamate, a similar spin‐trapping agent for NO, also inhibited the activity, with an IC50 value of 25.1 ± 2.9 µm. Fe–DTCS suppressed cytochrome c and ferricyanide reductase activities of nNOS, and markedly increased nNOS‐mediated NADPH oxidation. Concomitantly, it accelerated oxygen consumption caused by activated nNOS. These results suggest that the ESR spin‐trapping agent Fe–DTCS inhibits NO synthesis by interfering with the physiological electron flow from NADPH to nNOS heme iron.
Glucuronic acid directly linked to galacturonic acid in the rhamnogalacturonan backbone of beet pectins
Authors: Renard Catherine M. G. C. , Crépeau Marie‐Jeanne , Thibault Jean‐François
Published: March 2003

Sugar‐beet pulp was de‐esterified and submitted to 72 h hydrolysis by 0.1 m HCl at 80 °C. Oligomers containing a single glucuronic acid (GlcA) moiety in addition to n( 2) repeats of the dimer →4)‐α‐d‐GalpA‐(1→2)‐α‐l‐Rhap‐(1→ were isolated from the hydrolysate by ion‐exchange and gel‐permeation. Glycosyl linkage composition analysis and 1H NMR studies indicated that the GlcA was attached to O‐3 of a galacturonic acid (GalA) residue, as shown for the two pentamers β‐d‐GlcpA‐(1→3)‐α‐d‐GalpA‐(1→2)‐α‐l‐Rhap‐(1→4)‐α‐d‐GalpA‐(1→2)‐l‐Rhap and α‐d‐GalpA‐(1→2)‐α‐l‐Rhap‐(1→4)‐[β‐d‐GlcpA‐(1→3)]‐α‐d‐GalpA‐(1→2)‐l‐Rhap. Substitution by GlcA was estimated as occurring on one GalA residue out of 72 in the rhamnogalacturonan fraction of the backbone of beet pectins.
Role of the N‐terminal region of ribosomal protein S7 in its interaction with 16S rRNA which binds to the concavity formed by the β‐ribbon arm and the α‐helix
Authors: Miyamoto Atsushi , Usui Mitsuhiro , Yamasaki Nobuyuki , Yamada Naotaka , Kuwano Eiichi , Tanaka Isao , Kimura Makoto
Published: March 2003

The ribosomal protein S7, a primary 16S rRNA‐binding protein, plays an essential role in stabilizing the 3′ major domain of 16S rRNA and also in feedback regulation of the str operon, as a translational repressor. We examined amino acid residues in ribosomal protein S7 from Bacillus stearothermophilus (BstS7) that are essential for 16S rRNA binding. Truncation of the N‐terminal 10 residues of BstS7 abolished its binding to 16S rRNA, whereas removal of the C‐terminal eight residues had no effect on the binding activity. Subsequently, we used site‐directed mutagenesis to identify essential basic residues in the N‐terminal region for 16S rRNA binding. Mutation of Arg3 and Lys8 significantly weakened the binding activity, and a smaller decrease in binding activity was observed with Arg2 and Arg9 mutations. These observations indicate that N‐terminal basic residues, especially Arg3 and Lys8, play a crucial role as positively charged recognition groups for the negatively charged phosphate backbone of 16S rRNA. In addition, the mutagenesis study showed that Arg75, Arg78, Arg94, and Arg101, which are located in a concavity formed by the β‐ribbon arm and the α‐helix (α4), individually make only a small contribution to 16S rRNA binding, but together probably form a positively charged binding site for 16S rRNA. With regard to aromatic residues, Tyr84 on the tip of the β‐ribbon arm was found to be involved in 16S rRNA binding, whereas the conserved aromatic residues Trp102 and Tyr106 in the concavity had little effect. We then probed the 16S rRNA‐binding site(s) for the N‐terminal region of S7 with iron tethered to the mutant of BstS7 containing a single cysteine residue at position 4. The N‐terminal region of S7 is placed in close proximity to helix 43 in the 16S rRNA. Probing also revealed additional cleavages between nucleotides 1397 and 1438, near the P‐site region in 16S rRNA. This finding is consistent with a three‐dimensional model of 16S rRNA that shows close proximity of helix 43 to the P‐site during three‐dimensional folding.
A proton‐NMR investigation of the fully reduced cytochrome c7 from Desulfuromonas acetoxidans
Authors: Assfalg Michael , Banci Lucia , Bertini Ivano , Bruschi Mireille , Giudici‐Orticoni Marie T. , Turano Paola
Published: March 2003

The solution structure via 1H NMR of the fully reduced form of cytochrome c7 has been obtained. The protein sample was kept reduced by addition of catalytic amounts of Desulfovibrio gigas iron hydrogenase in H2 atmosphere after it had been checked that the presence of the hydrogenase did not affect the NMR spectrum. A final family of 35 conformers with rmsd values with respect to the mean structure of 8.7 ± 1.5 nm and 12.4 ± 1.3 nm for the backbone and heavy atoms, respectively, was obtained. A highly disordered loop involving residues 54–61 is present. If this loop is ignored, the rmsd values are 6.2 ± 1.1 nm and 10.2 ± 1.0 nm for the backbone and heavy atoms, respectively, which represent a reasonable resolution.

The structure was analyzed and compared with the already available structure of the fully oxidized protein. Within the indetermination of the two solution structures, the result for the two redox forms is quite similar, confirming the special structural features of the three‐heme cluster. A useful comparison can be made with the available crystal structures of cytochromes c3, which appear to be highly homologous except for the presence of a further heme. Finally, an analysis of the factors affecting the reduction potentials of the heme irons was performed, revealing the importance of net charges in differentiating the reduction potential when the other parameters are kept constant.
Molecular cloning and characterization of a hemolymph clottable protein from tiger shrimp (Penaeus monodon)
Authors: Yeh Maw‐Sheng , Huang Chang‐Jen , Leu Jiann‐Horng , Lee Yuan C. , Tsai Inn‐Ho
Published: March 2003

To investigate the coagulation system in crustacean decapoda, a homodimeric glycoprotein of 380 kDa was purified from the hemolymph of tiger shrimp (Penaeus monodon) by sequential DEAE anion exchange chromatography. The purified protein was coagulated by the shrimp hemocyte transglutaminase in the presence of Ca2+. The clottable protein contains 44% α helices and 26% β sheets as determined by circular dichroism spectra. Its conformation is stable in buffer of pH 4–9. To solve its primary structure, partial sequences of the purified polypeptides from cyanogen bromide cleavage and endopeptidase digestion were also determined. A shrimp cDNA expression library was constructed. By combination with antibody screening, reverse transcriptase PCR using degenerate primers from determined amino acid sequences and cDNA library screening with digoxigenin‐labeled DNA probes, the entire cDNA of 6124 bp was obtained. This cDNA encodes a protein of 1670 amino acids, including a 14‐amino acid signal peptide. With four potential N‐glycosylation sites, the clottable protein was found to contain 3.8% high‐mannose glycan; and Man8GlcNAc and Man9GlcNAc were released upon endo‐β‐N‐acetylglucosaminidase hydrolysis. Upon conducting a protein sequence database survey, the shrimp clottable protein shows 36% identities to the crayfish clotting protein and lower similarities to members of insect vitellogenins, apolipoprotein B and mammalian von Willebrand factor. Notably, a region rich in Gln residues, a polyGln motif and five Ser‐Lys‐Thr‐Ser repeats are present in the shrimp protein, suggesting this protein might be a transglutaminase substrate. Northern blot analysis revealed that the clottable protein is expressed in most of the shrimp tissues but not in the mature hemocytes.
Sphingomyelinase treatment of rat hepatocytes inhibits cell‐swelling‐stimulated glycogen synthesis by causing cell shrinkage
Authors: van Sluijters Daphne A. , van Woerkom George M. , Aerts Johannes M. F. G. , Meijer Alfred J.
Published: March 2003

Breakdown of plasma‐membrane sphingomyelin caused by TNF‐α is known to inhibit glucose metabolism and insulin signalling in muscle and fat cells. In hepatocytes, conversion of glucose to glycogen is strongly activated by amino acid‐induced cell swelling. In order to find out whether breakdown of plasma‐membrane sphingomyelin also inhibits this insulin‐independent process, the effect of addition of sphingomyelinase was studied in rat hepatocytes.

Sphingomyelinase (but not ceramide) inhibited glycogen synthesis, caused cell shrinkage, decreased the activity of glycogen synthase a, but had no effect on phosphorylase a. Cell integrity was not affected by sphingomyelinase addition as gluconeogenesis and the intracellular concentration of ATP were unchanged. As a control, glycogen synthesis was studied in HepG2 cells. In these cells, the basal rate of glycogen production was high, could not be stimulated by amino acids, nor be inhibited by sphingomyelinase.

Regarding the mechanism responsible for the inhibition of glycogen synthase a, sphingomyelinase did not affect amino acid‐induced, PtdIns 3‐kinase‐dependent, phosphorylation of p70S6 kinase, but caused an increase in intracellular chloride, which is known to inhibit glycogen synthase phosphatase.

It is concluded that the decrease in cell volume, following the breakdown of sphingomyelin in the plasma membrane of the hepatocyte, may contribute to the abnormal metabolism of glucose when TNF‐α levels are high.
Synthesis and hydrolysis of ATP and the phosphate–ATP exchange reaction in soluble mitochondrial F1 in the presence of dimethylsulfoxide
Authors: Tuena de Gómez‐Puyou Marietta , Pérez‐Hernández Gerardo , Gómez‐Puyou Armando
Published: March 2003

In medium containing 40% dimethylsulfoxide, soluble F1 catalyzes the hydrolysis of ATP introduced at concentrations lower than that of the enzyme [Al‐Shawi, M.K. & Senior, A.E. (1992), Biochemistry31, 886–891]. At this concentration of dimethylsulfoxide, soluble F1 also catalyzes the spontaneous synthesis of a tightly bound ATP to a level of approximately 0.15 mol per mol F1[Gómez‐Puyou, A., Tuena de Gómez‐Puyou, M. & de Meis, L. (1986) Eur. J. Biochem. 159, 133–140]. The mechanisms that allow soluble F1 to carry out these apparently opposing reactions were studied. The rate of hydrolysis of ATP bound to F1 under uni‐site conditions and that of synthesis of ATP were markedly similar, indicating that the two ATP molecules lie in equivalent high affinity catalytic sites. The number of enzyme molecules that have ATP at the high affinity catalytic site under conditions of synthesis or uni‐site hydrolysis is less than the total number of enzyme molecules. Therefore, it was hypothesized that when the enzyme was treated with dimethylsulfoxide, a fraction of the F1 population carried out synthesis and another hydrolysis. Indeed, measurements of the two reactions under identical conditions showed that different fractions of the F1 population carried out simultaneously synthesis and hydrolysis of ATP. The reactions continued until an equilibrium level between F1·ADP + Pi ⇄ F1·ATP was established. At equilibrium, about 15% of the enzyme population was in the form F1·ATP. The ΔG° of the reaction with 0.54 µm F1, 2 mm Pi and 10 mm Mg2+ at pH 6.8 was −2.7 kcal·mol−1 in favor of F1·ATP. The ΔG° of the reaction did not exhibit important variations with Pi concentration; thus, the reaction was in thermodynamic equilibrium. In contrast, ΔG° became significantly less negative as the concentration of dimethylsulfoxide was decreased. In water, the reaction was far to the left. The equilibrium constant of the reaction diminished linearly with an increase in water activity. The effect of solvent is fully reversible. In comparison to other enzymes, F1 seems unique in that solvent controls the equilibrium that exists within an enzyme population. This results from the effect of solvent on the partition of Pi between the catalytic site and the medium, and the large energetic barrier that prevents release of ATP from the catalytic site. In the presence of dimethylsulfoxide and Pi, ATP is continuously hydrolyzed and synthesized with formation and uptake of Pi from the medium. This process is essentially an exchange reaction analogous to the phosphate–ATP exchange reaction that is catalyzed by the ATP synthase in coupled energy transducing membranes.
Generation of epitope‐specific antibodies to rat GHBP in the sheep using an interspecies switching strategy involving site‐directed mutagenesis of ovine GHBP
Authors: Shand John H. , Allan Gordon J. , Beattie James , Flint David J.
Published: March 2003

Site‐directed antibodies to the growth hormone receptor could be potentially useful as growth hormone mimics but, in previous attempts, we found that antisera generated using peptides derived from growth hormone receptor sequences failed to recognize the intact protein. As an alternative approach to this problem, we have now adopted a strategy of epitope‐switching between rat and ovine growth hormone receptors to produce rat epitopes in the correct structural context. Using site‐directed mutagenesis, we altered the two dominant linear epitopes in the ovine growth hormone binding protein to the analogous sequences in rat growth hormone binding protein. Site A, between Thr28 and Leu34, is equivalent to epitope 1 in ovine growth hormone binding protein and site B, between Ser121 and Asp124, corresponds to epitope 5. The wild‐type ovine growth hormone binding protein and the two mutant proteins were bacterially expressed, refolded and, following purification by metal‐chelate affinity chromatography, used to raise antisera in sheep. We showed using RIA, in which wild‐type ovine growth hormone binding protein acted as a competitor for the binding of rat growth hormone binding protein, that only the site A mutant protein elicited a specific anti‐rat growth hormone binding protein response. This was confirmed in subsequent RIA studies using the antiserum to the site A mutant protein in which only peptides corresponding to the site A sequences in mutant ovine growth hormone binding protein and rat growth hormone binding protein, but not that in wild‐type ovine growth hormone binding protein, were able to act as competitors for rat growth hormone binding protein. Antibodies specific for rat growth hormone binding protein could be separated from the antiserum to the site A mutant protein by means of affinity chromatography using immobilized wild‐type ovine growth hormone binding protein to remove antibodies which cross‐reacted with the ovine protein. The work lays the foundations for further studies in which the biological effects of these antibody fractions will be investigated and demonstrates an approach with general applicability in the production of antibodies directed towards specific epitopes on protein molecules.
Implications of hemolin glycosylation and Ca2+‐binding on homophilic and cellular interactions
Authors: Bettencourt Raul , Gunne Hans , Gastinel Louis , Steiner Håkan , Faye Ingrid
Published: March 2003

Insects are useful models for the study of innate immune mechanisms because of their lack of antibodies and receptors involved in adaptive immune response. Nevertheless, hemolin cloned from moths is a soluble and membrane associated Ig‐related molecule that is up‐regulated during immune response [Lanz‐Mendoza, H. & Faye, I. (1999) Dev. Comp. Immunol.23, 359–374]. The hemolin monomeric form has four, pair‐wise, interacting Ig‐domains, forming a strongly bent horseshoe structure [Su, X.‐D., Gastinel, L.N., Vaughn, D.E., Faye, I., Poon, P. & Bjorkman, P. (1998) Science281, 991–995]. To elucidate the nature of its homophilic and cellular interactions, the glycosylation and Ca2+‐binding properties of hemolin were investigated. We used Hyalophora cecropia hemolin isolated from hemolymph of bacteria‐injected pupae, or produced as a recombinant protein in a baculovirus/insect cell system. Both types of hemolin contain N‐acetylglucosamine and probably sialic acid, as indicated by peptide:N‐glycosidase F and neuraminidase digestion and glycosylation detection by Western‐blotting analysis. The N‐acetylglucosamine residues on hemolin were confirmed with the use of specific lectins. In addition, hemolin was shown to specifically bind calcium when spotted onto nitrocellulose and treated as for 45Ca2+ autoradiography. Earlier studies demonstrated that hemolin can bind to hemocytes and this was tested for its dependence on calcium and carbohydrates, using hemolin‐coated fluorescent microspheres. A greater level of attachment of microspheres occurred in the presence of calcium than if calcium was absent. Furthermore, this binding was inhibited by EGTA and N‐acetylglucosamine or N‐acetylneuraminic acid, implying that carbohydrates and calcium are crucial factors in homophilic binding and cell‐adhesion events mediated by this Ig‐superfamily molecule.