bluuebunny/crossref_metadata_2025 huggingface dataset:
Subunit organization of the abaloneHaliotis tuberculatahemocyanin type 2 (HtH2), and the cDNA sequence encoding its functional units d, e, f, g and h
Authors: Lieb Bernhard , Altenhein Benjamin , Lehnert Rolf , Gebauer Wolfgang , Markl Jürgen
Published: March 2003

We have developed a HPLC procedure to isolate the two different hemocyanin types (HtH1 and HtH2) of the European abaloneHaliotis tuberculata. On the basis of limited proteolytic cleavage, two‐dimensional immunoelectrophoresis, PAGE, N‐terminal protein sequencing and cDNA sequencing, we have identified eight different 40–60‐kDa functional units (FUs) in HtH2, termed HtH2‐a to HtH2‐h, and determined their linear arrangement within the elongated 400‐kDa subunit. From aHaliotiscDNA library, we have isolated and sequenced a cDNA clone which encodes the five C‐terminal FUs d, e, f, g and h of HtH2. As shown by multiple sequence alignments, defg of HtH2 correspond structurally to defg fromOctopus dofleinihemocyanin. HtH2‐e is the first FU of a gastropod hemocyanin to be sequenced. The newHaliotishemocyanin sequences are compared to their counterparts inOctopus,Helix pomatiaand HtH1 (from the latter, the sequences of FU‐f, FU‐g and FU‐h have recently been determined) and discussed in relation to the recent 2.3 ÅX‐ray structure of FU‐g fromOctopushemocyanin and the 15 Å three‐dimensional reconstruction of theMegathura crenulatahemocyanin didecamer from electron micrographs. This data allows, for the first time, an insight into the evolution of the two functionally different hemocyanin isoforms found in marine gastropods. It appears that they evolved several hundred million years ago within the Prosobranchia, after separation of the latter from the branch leading to the Pulmonata. Moreover, as a structural explanation for the inefficiency of the type 1 hemocyanin to form multidecamersin vivo, the additional N‐glycosylation sites in HtH1 compared to HtH2 are discussed.
Iodomycin and iodipine, a structural analogue of azidopine, bind to a common domain in hamster P‐glycoprotein
Authors: Demmer Annette , Andreae Susanne , Thole Hubert , Tümmler Burkhard
Published: March 2003

Both the overexpression of P‐glycoprotein and the broad range of substrates of this ATP‐binding cassette (ABC) transporter induce the phenomenon of multidrug resistance, one major cause of the failure of cancer chemotherapy in humans. This study reports that [125I]iodipine, a structural analogue of the 1,4‐dihydropyridine azidopine, shares a common binding site with iodomycin, a Bolton–Hunter derivative of the anthracycline daunomycin. This binding site is different from that described for iodoarylazidoprazosin, which is presumed to share a common binding site with azidopine. Edman sequencing revealed that [125I]iodipine had photolabelled the same peptide as iodomycin and spans the primary sequence of hamster isoform pgp1 from amino acid 230 to amino acid 312.
A stable cold folding intermediate of rabbit muscle D‐glyceraldehyde 3‐phosphate dehydrogenase
Authors: Zhang Nian‐xiang , Wang Chih‐chen
Published: March 2003

With decreasing temperature the reactivation yield of denatured d‐glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) upon dilution increases but the reactivation rate decreases. Neither reactivation nor aggregation during refolding can be detected at 4 °C in 48 h, and at 3 °C even in 6 days. However, the reactivation takes place once the temperature is raised with little decrease of the yield after incubation for 6 days at 3 °C. A cold folding intermediate forms in a burst phase of refolding at 4 °C as shown by a fast change of the intrinsic fluorescence followed by further conformational adjustment to a stable state in about 1 h. The stable folding intermediate has been characterized to be a dimer of partially folded GAPDH subunit with secondary structure between that of the native and denatured enzymes, a hydrophobic cluster not found in either the native or the denatured state, and an active site similar to but different from that of the native state. Chaperonin 60 (GroEL) binds with all intermediates formed at 4 °C, but the intermediates formed at the early folding stage reactivate with higher yield than those formed after conformational adjustment when dissociated from GroEL in the presence of ATP and further folded and assembled into the native tetramer.
Thapsigargin directly induces the mitochondrial permeability transition
Authors: Korge Paavo , Weiss James N.
Published: March 2003

High concentrations of thapsigargin (TG) have been used to study the process of necrotic cell death, which involves mitochondria in the cell rapidly undergoing the mitochondrial permeability transition (MPT). We therefore investigated the effects of TG on MPT in isolated liver and heart mitochondria. Using a matrix swelling assay in combination with a novel enzymatic method based on inner membrane permeability to citrate synthase substrates, TG induced MPT in a concentration‐dependent manner, independent of extramitochondrial [Ca2+] and inhibitable by cyclosporin A. Evidence from alamethicin‐permeabilized mitochondria suggests that TG induces MPT by causing Ca2+ release from mitochondrial matrix Ca2+‐binding sites. These findings suggest that the MPT‐inducing effect of TG may contribute to its pro‐necrotic and pro‐apoptotic effects in various cell types.
Mitochondria and cell death
Authors: Bernardi Paolo , Scorrano Luca , Colonna Raffaele , Petronilli Valeria , Di Lisa Fabio
Published: March 2003

Mitochondria are involved in cell death for reasons that go beyond ATP supply. A recent advance has been the discovery that mitochondria contain and release proteins that are involved in the apoptotic cascade, like cytochrome c and apoptosis inducing factor. The involvement of mitochondria in cell death, and its being cause or consequence, remain issues that are extremely complex to address in situ. The response of mitochondria may critically depend on the type of stimulus, on its intensity, and on the specific mitochondrial function that has been primarily perturbed. On the other hand, the outcome also depends on the integration of mitochondrial responses that cannot be dissected easily. Here, we try to identify the mechanistic aspects of mitochondrial involvement in cell death as can be derived from our current understanding of mitochondrial physiology, with special emphasis on the permeability transition and its consequences (like onset of swelling, cytochrome c release and respiratory inhibition); and to critically evaluate methods that are widely used to monitor mitochondrial function in situ.
Type I β‐turn conformation is important for biological activity of the melanocyte‐stimulating hormone analogues
Authors: Li Song‐Zhe , Lee Jung‐Hoon , Lee Weontae , Yoon Chang‐Ju , Baik Ja‐Hyun , Lim Sung‐Kil
Published: March 2003

In order to define which structure of α‐melanocyte‐stimulating hormone (MSH) analogues plays a critical role for ligand–receptor interaction and selectivity, we analysed receptor‐binding and cAMP‐generating activity in Chinese hamster ovary cell lines stably transfected with rMC3R and hMC4R, as well as the NMR structures of chemically synthesized α‐MSH analogues. Compared with [Ahx4]α‐MSH, the linear MTII designated as α‐MSH‐ND revealed a preference for the MC4R, whereas its IC50 and EC50 values were comparable to those of MTII reported previously. Truncation of Ahx4 and Asp5 of α‐MSH‐ND remarkably decreased the receptor‐binding and cAMP‐generating activity. Meanwhile, maximum cAMP‐generating activity was observed at a higher concentration (10−5 m) of α‐MSH‐ND(6–10), and MC4R preference was changed into MC3R preference. In contrast, [Gln6]α‐MSH‐ND(6–10) lost its cAMP‐generating activity almost completely, even though it bound to both receptors. Whereas the solution conformation of α‐MSH‐ND revealed a stable type I β‐turn structure, [Gln6]α‐MSH‐ND(6–10) revealed a tight γ‐turn composed of Gln6‐d‐Phe7‐Arg8. Replacement of the His6 residue of α‐MSH‐ND by Gln, Asn, Arg or Lys decreased not only the receptor binding, but also the cAMP‐generating activity in both the MC3R and the MC4R. The structure of [Gln6]α‐MSH‐ND exhibited a stable type I′β‐turn comprising Asp5, Gln6, d‐Phe7 and Arg8. [Lys6]α‐MSH‐ND showed a greatly reduced binding affinity and cAMP‐generating activity with the loss of MC4R selectivity. In NMR studies, [Lys6]α‐MSH‐ND also demonstrated a γ‐turn conformation around Lys6‐dPhe7‐Arg8. From the above results, we conclude that a type I β‐turn conformation comprising the residues Asp5‐His6‐(d‐Phe7)‐Arg8 was important for receptor binding and activation, as well as the selectivity of MSH analogues.
Impact of C5‐cytosine methylation on the solution structure of d(GAAAACGTTTTC)2
Authors: Marcourt Laurence , Cordier Christine , Couesnon Thierry , Dodin Guy
Published: March 2003

The solution structures of d(GAAAACGTTTTC)2 and of its methylated derivative d(GAAAAMe5CGTTTTC)2 have been determined by NMR and molecular modelling in order to examine the impact of cytosine methylation on the central CpG conformation. Detailed 1H NMR and 31P NMR investigation of the two oligomers includes quantitative NOESY, 2D homonuclear Hartmann–Hahn spectroscopy, double‐quantum‐filtered COSY and heteronuclear 1H‐31P correlation. Back‐calculations of NOESY spectra and simulations of double‐quantum‐filtered COSY patterns were performed to gain accurate information on interproton distances and sugar phase angles. Molecular models under experimental constraints were generated by energy minimization by means of the molecular mechanics program jumna. The morass software was used to iteratively refine the structures obtained. After methylation, the oligomer still has a B‐DNA conformation. However, there are differences in the structural parameters and the thermal stability as compared to the unmethylated molecule. Careful structural analysis shows that after methylation CpG departs from the usual conformation observed in other ACGT tetramers with different surroundings. Subtle displacements of bases, sugars and backbone imposed by the steric interaction of the two methyl groups inside the major groove are accompanied by severe pinching of the minor groove at the C‐G residues.
Phosphatidic acid, a key intermediate in lipid metabolism
Authors: Athenstaedt Karin , Daum Günther
Published: March 2003

Phosphatidic acid (PtdOH) is a key intermediate in glycerolipid biosynthesis. Two different pathways are known for de novo formation of this compound, namely (a) the Gro3P (glycerol 3‐phosphate) pathway, and (b) the GrnP (dihydroxyacetone phosphate) pathway. Whereas the former route of PtdOH synthesis is present in bacteria and all types of eukaryotes, the GrnP pathway is restricted to yeast and mammalian cells. In this review article, we describe the enzymes catalyzing de novo formation of PtdOH, their properties and their occurrence in different cell types and organelles. Much attention has recently been paid to the subcellular localization of enzymes involved in the biosynthesis of PtdOH. In all eukaryotic cells, microsomes (ER) harbour the complete set of enzymes catalyzing these pathways and are thus the usual organelle for PtdOH formation. In contrast, the contribution of mitochondria to PtdOH synthesis is restricted to certain enzymes and depends on the cell type. In addition, chloroplasts of plants, lipid particles of the yeast, and peroxisomes of mammalian cells are significantly involved in PtdOH biosynthesis. Redundant systems of acyltransferases, the interplay of organelles, regulation of the pathway on the compartmental level, and finally the contribution of alternative pathways (phosphorylation of diacylglycerol and cleavage of phospholipids by phospholipases) to PtdOH biosynthesis appear to be required for the balanced formation of this important lipid intermediate. Dysfunction of enzymes involved in PtdOH synthesis can result in severe defects of various cellular processes. In this context, the possible physiological role(s) of PtdOH and its related metabolites, lysophosphatidic acid and diacylglycerol, will be discussed.
NMR structure of the (1–51) N‐terminal domain of the HIV‐1 regulatory protein Vpr
Authors: Wecker K. , Roques B. P.
Published: March 2003

The human immunodeficiency virus type 1 (HIV‐1) genome encodes a highly conserved 16 kDa regulatory gene product, Vpr (viral protein of regulation, 96 amino acid residues), which is incorporated into virions, in quantities equivalent to those of the viral Gag proteins. In the infected cells, Vpr is believed to function in the early phase of HIV‐1 replication, including nuclear migration of preintegration complex, transcription of the provirus genome and viral multiplication by blocking cells in the G2 phase. Vpr has a critical role in long‐term AIDS disease by inducing infection in nondividing cells such as monocytes and macrophages. Mutations have suggested that the N‐terminal domain of Vpr encompassing the first 40 residues could be required for nuclear localization, packaging into virions and binding of transcription factor (TFIIB, Sp1), viral proteins (p6) and cellular proteins (RIP1, UNG, karyopherins). To gain insight into the structure–function relationship of Vpr, (1–51)Vpr was synthesized and its structure analyzed by circular dichroism and two‐dimensional 1H NMR in aqueous trifluoroethanol (30%) solution and refined by restrained molecular dynamics. The structure is characterized by three turns around the first three prolines, Pro5, Pro10, Pro14, followed by a long amphipathic α helix‐turn‐α helix (Asp17–Ile46) motif ended by a turn extending from Tyr47 to Thr49. The α helix‐turn‐α helix motif and the amphipathic helix are well known for being implicated in protein–protein or protein–nucleic acid interaction. Therefore structural characteristics of the (1–51) N‐terminal fragment of Vpr could explain why this region of Vpr plays a role in several biological functions of this protein.
Identification of a CK2 phosphorylation site in mdm2
Authors: Götz Claudia , Kartarius Sabine , Scholtes Petra , Nastainczyk Wolfgang , Mathias Montenarh
Published: March 2003

Mdm2 is a cellular oncoprotein the most obvious function of which is the down‐regulation of the growth suppressor protein p53. It represents a highly phosphorylated protein but only little is yet known about the sites phosphorylated in vivo, the kinases that are responsible for the phosphorylation or the functional relevance of the phosphorylation status. Recently, we have shown that mdm2 is a good substrate for protein kinase CK2 at least in vitro. Computer analysis of the primary amino acid sequence of mdm2 revealed 19 putative CK2 phosphorylation sites. By using deletion mutants of mdm2 and a peptide library we identified the serine residue at position 269 which lies within a canonical CK2 consensus sequence (EGQELSDEDDE) as the most important CK2 phosphorylation site. Moreover, by using the mdm2 S269A mutant for in vitro phosphorylation assays this site was shown to be phosphorylated by CK2. Binding studies revealed that phosphorylation of mdm2 at S269 does not have any influence on the binding of p53 to mdm2.
Independent in vitro assembly of all three major morphological parts of the 30S ribosomal subunit of Thermus thermophilus
Authors: Agalarov Sultan C. , Selivanova Olga M. , Zheleznyakova Elena N. , Zheleznaya Ludmila A. , Matvienko Nicholas I. , Spirin Alexander S.
Published: March 2003

Fragments of the 16S rRNA of Thermus thermophilus representing the 3′ domain (nucleotides 890–1515) and the 5′ domain (nucleotides 1–539) have been prepared by transcription in vitro. Incubation of these fragments with total 30S ribosomal proteins of T. thermophilus resulted in formation of specific RNPs. The particle assembled on the 3′ RNA domain contained seven proteins corresponding to Escherichia coli ribosomal proteins S3, S7, S9, S10, S13, S14, and S19. All of them have previously been shown to interact with the 3′ domain of the 16S RNA and to be localized in the head of the 30S ribosomal subunit. The particle formed on the 5′ RNA domain contained five ribosomal proteins corresponding to E. coli proteins S4, S12, S17, S16, and S20. These proteins are known to be localized in the main part of the body of the 30S subunit. Both types of particle were compact and had sedimentation coefficients of 15.5 S and 13 S, respectively. Together with our recent demonstration of the reconstitution of the RNA particle representing the platform of the T. thermophilus 30S ribosomal subunit [Agalarov, S.C., Zheleznyakova, E.N., Selivanova, O.M., Zheleznaya, L.A., Matvienko, N.I., Vasiliev, V.D. & Spirin, A.S. (1998) Proc. Natl Acad. Sci. USA95, 999–1003], these experiments establish that all three main structural lobes of the small ribosomal subunit can be reconstituted independently of each other and prepared in the individual state.
Biotransformations of putative phytoecdysteroid biosynthetic precursors in tissue cultures of Polypodium vulgare
Authors: Reixach Natàlia , Lafont René , Camps Francisco , Casas Josefina
Published: March 2003

Incubation of calli and prothalli of Polypodium vulgare with different tritium‐labelled ecdysteroids has led to modification of some previous assumptions about the biosynthesis of ecdysteroids in plants. Thus, 25‐deoxy‐20‐hydroxyecdysone was transformed efficiently in both tissues into 20‐hydroxyecdysone (20E), but no 25‐deoxyecdysteroids such as pterosterone and inokosterone were formed. Likewise, incubation of 2‐deoxyecdysone (2dE) produced exclusively ecdysone (E) and 20E, indicating a high 2‐hydroxylase activity in both tissues, despite calli not producing phytoecdysteroids. This 2‐hydroxylation was also evident in the transformation of 2,22‐dideoxyecdysone (2,22dE) into 22‐deoxyecdysone (22dE). Different ecdysteroids that do not occur in P. vulgare were formed in the incubation of 3‐dehydro‐2,22,25‐trideoxyecdysone (3D2,22,25dE) by 3α‐reduction and 3β‐reduction and 25‐hydroxylation processes. The fact that 22,25‐dideoxyecdysone and 22dE were the only 2‐hydroxylated products formed in this case suggests that only compounds bearing a 3β‐hydroxyl group are substrates for the 2‐hydroxylase. Surprisingly, 22‐hydroxylation was never observed with either 2,22dE or 3D2,22,25dE, raising the possibility that it could occur at an early step in the biosynthetic pathway. In this respect, labelled 22R‐hydroxycholesterol was efficiently converted into E and 20E, whereas 22S‐hydroxycholesterol was not transformed into ecdysteroids, because of its unsuitable configuration at C22. Finally, the conversion of 25‐hydroxycholesterol into E and 20E was greatly enhanced after thermal treatment of prothalli which induces the release of previously stored ecdysteroids. Thus, P. vulgare prothalli and calli appear to be particularly suitable models for the study of ecdysteroid biosynthesis and its regulation in plants.
2′‐Carboxy‐D‐arabitinol 1‐phosphate protects ribulose 1,5‐bisphosphate carboxylase/oxygenase against proteolytic breakdown
Authors: Khan Shahnaz , Andralojc P. John , Lea Peter J. , Parry Martin A. J.
Published: March 2003

Trypsin‐catalysed cleavage of purified ribulose 1,5‐bisphosphate carboxylase/oxygenase (Rubisco) and the resultant irreversible loss of carboxylase activity were prevented by prior incubation with the naturally occurring nocturnal Rubisco inhibitor 2′‐carboxy‐d‐arabitinol 1‐phosphate (CA1P), as well as with ribulose 1,5‐bisphosphate (RuBP), Mg2+ and CO2. CA1P also protected Rubisco from loss of activity caused by carboxypeptidase A. When similar experiments were carried out using soluble chloroplast proteases, CA1P was again able to protect Rubisco against proteolytic degradation and the consequent irreversible loss of catalytic activity. Thus, CA1P prevents the proteolytic breakdown of Rubisco by endogenous and exogenous proteases. In this way, CA1P may affect the amounts of Rubisco protein available for photosynthetic CO2 assimilation. Rubisco turnover (in the presence of RuBP, Mg2+ and CO2) may confer similar protection against proteases in the light.
Activation of human neutrophils by a synthetic anti‐microbial peptide, KLKLLLLLKLK‐NH2, via cell surface calreticulin
Authors: Cho Jang‐Hyun , Homma Ko‐ichi , Kanegasaki Shiro , Natori Shunji
Published: March 2003

We previously reported that a synthetic anti‐bacterial peptide, KLKLLLLLKLK‐NH2 (L5), showed significant chemotherapeutic activity in methicillin‐resistant Staphylococcus aureus‐infected mice, and its ability to activate human neutrophils was related to its chemotherapeutic activity. In this study, we found that activation of neutrophils by L5 was inhibited by pertussis toxin, suggesting that GTP‐binding protein (G‐protein) participates in this process. We isolated an L5‐binding protein, which turned out to be human calreticulin, with a molecular mass of 60 kDa from neutrophil membranes. From experiments using an anti‐calreticulin antibody, we proposed that calreticulin is partly localized on the surface of neutrophils, and L5‐bound calreticulin transmits a signal into cells via G‐protein to activate neutrophils to generate superoxide anion.
3,4‐Dihydrocoumarin hydrolase with haloperoxidase activity from Acinetobacter calcoaceticus F46
Authors: Kataoka Michihiko , Honda Kohsuke , Shimizu Sakayu
Published: March 2003

A novel lactonohydrolase, an enzyme that catalyzes the hydrolysis of 3,4‐dihydrocoumarin, was purified 375‐fold to apparent homogeneity, with a 22.7% overall recovery, from Acinetobacter calcoaceticus F46, which was isolated as a fluorene‐assimilating micro‐organism. The molecular mass of the native enzyme, as estimated by high‐performance gel‐permeation chromatography, is 56 kDa, and the subunit molecular mass is 30 kDa. The enzyme specifically hydrolyzes 3,4‐dihydrocoumarin, and the Km and Vmax for 3,4‐dihydrocoumarin are 0.806 mm and 4760 U·mg−1, respectively. The N‐terminal and internal amino acid sequences of the enzyme show high similarity to those of bacterial non‐heme haloperoxidases. The enzyme exhibits brominating activity with monochlorodimedon in the presence of H2O2 and 3,4‐dihydrocoumarin or an organic acid, such as acetate and n‐butyrate.
The multisubunit chloroplast RNA polymerase A from mustard (Sinapis alba L.)
Authors: Pfannschmidt Thomas , Ogrzewalla Karsten , Baginsky Sacha , Sickmann Albert , Meyer Helmut E. , Link Gerhard
Published: March 2003

We previously identified two multisubunit plastid RNA polymerases termed A and B. The B enzyme has a bacterial‐type polypeptide composition and is sensitive to the prokaryotic transcription inhibitor rifampicin (Rif); the A enzyme has a more complex subunit structure and is Rif‐resistant. Here we report results of N‐terminal sequencing and MS carried out with the A enzyme, which establish that the latter contains rpo gene products and is structurally related to the B enzyme. Furthermore, evidence is provided that the A enzyme can be converted into a Rif‐sensitive enzyme form in a phosphorylation‐dependent manner in vitro by a treatment that results in depletion of a β‐like subunit. Database searches using sequence information derived from additional polypeptides that are present in purified A preparations revealed sequence similarity with chloroplast proteins involved in RNA processing and redox control. This proteomics approach thus points to the complexity of the chloroplast transcription apparatus and its interconnections with post‐transcriptional and signalling mechanisms.
Reactive oxygen species released from granulocytes stimulate 5‐lipoxygenase activity in a B‐lymphocytic cell line
Authors: Werz Oliver , Szellas Dagmar , Steinhilber Dieter
Published: March 2003

B‐lymphocytes express 5‐lipoxygenase (5‐LO) protein but cellular leukotriene production is suppressed by selenium‐dependent peroxidases. Thus it was of interest to check whether reactive oxygen species (ROS) which are released under inflammatory conditions can stimulate B‐lymphocyte 5‐LO and counteract peroxidase‐mediated suppression of cellular 5‐LO activity. It was found that 5‐LO in the Epstein–Barr virus‐transformed B‐lymphocytic cell line BL41‐E95‐A is activated by addition of hydrogen peroxide or xanthine/xanthine oxidase and after increasing the oxidative state of the cell by azodicarboxylic acid bis(dimethylamide). Generation of endogenous ROS from mitochondria by antimycin A also lead to a threefold upregulation of 5‐LO activity in B‐cells.

There was almost no detectable endogenous superoxide formation in BL41‐E95‐A cells after stimulation with 4β‐phorbol 12‐myristate 13‐acetate. Co‐incubation experiments with BL41‐E95‐A cells and granulocytes demonstrated that granulocyte‐derived ROS can activate B‐lymphocyte 5‐LO. Addition of superoxide dismutase and/or catalase to the B‐lymphocyte/granulocyte co‐incubations and to B‐lymphocyte homogenates revealed that the 5‐LO activation is due to the superoxide‐derived release of hydroperoxides or hydrogen peroxide from granulocytes.

The data suggest that ROS formation plays an important role in the regulation of cellular 5‐LO activity in B‐lymphocytes. As leukotrienes affect B‐cell functions like cell proliferation, activation and maturation, this finding provides a new link between the formation of ROS and the regulation of immune responses.
Differences in the skin peptides of the male and female Australian tree frog Litoria splendida
Authors: Wabnitz Paul A. , Bowie John H. , Tyler Michael J. , Wallace John C. , Smith Ben P.
Published: March 2003

The skin secretions of female and male Litoria splendida have been monitored monthly over a three‐year period using HPLC and electrospray mass spectrometry. Two minor peptides are present only in the skin secretion of the male. The first of these is the female‐attracting aquatic male sex pheromone that we have named splendipherin, a 25 amino acid peptide (GLVSSIGKALGGLLADVVKSKGQPA‐OH). This pheromone constitutes about 1% of the total skin peptides during the breeding season (January to March), dropping to about 0.1% during the period June to November. Splendipherin attracts the female in water at a concentration of 10−11–10−9 m, and is species specific. The second peptide is a wide‐spectrum antibiotic of the caerin 1 group, a 25 residue peptide (GLLSVLGSVAKHVLPHVVPVIAEKL‐NH2) named caerin 1.10. The neuropeptides of L. splendida are also seasonally variable, the change identical for both the female and male. During the period October to March, the sole neuropeptide present in skin secretions is caerulein [pEQDY(SO3)TGWMDF‐NH2]; this is active on smooth muscle and is also an analgaesic. During the southern winter (June to September), more than half of the caerulein is hydrolysed to [pEQDYTGWMDF‐NH2], a peptide that shows no smooth muscle activity. In place of caerulein, a new peptide, Phe8 caerulein [pEQDY(SO3)TGWFDF‐NH2], becomes a major component of the skin secretion. Perhaps this seasonal change is involved in thermoregulation, that is, with the initiation and maintenance of the inactive (hibernation) phase of the animal.
Viresin
Authors: Chung Kyung T. , Ourth Donald D.
Published: March 2003

Immune hemolymph was collected from fifth instar larvae and 1‐day‐old pupae of Heliothis virescens after injection of prepupae with live Enterobacter cloacae. Induction of antibacterial activity against Escherichia coli K12 D31 was 7.5 times greater in pupal than in larval immune hemolymph. Lysozyme activity of immune pupal hemolymph against Micrococcus lysodeikticus was 11 times greater when compared with lysozyme activity of immune larval hemolymph. Early pupal immune response with regard to antibacterial activity was much greater than larval immune response in H. virescens. Normal pupal hemolymph showed an increase in antibacterial activity and lysozyme that was induced during metamorphosis. Antibacterial protein was isolated together with lysozyme by gel filtration chromatography and then separated from lysozyme by sequential electrophoresis with a native acid gel and SDS gel. Molecular mass of antibacterial protein was estimated to be 12 kDa. The N‐terminal amino acid sequence of 12‐kDa protein was different from those of antibacterial molecules found in other insects and has not been identified before. A sample containing 12‐kDa protein was negative for immunoblotting with anti‐synthetic cecropin B antibody. We have named the novel 12‐kDa antibacterial protein viresin. Viresin showed antibacterial activity against several Gram‐negative bacteria including E. cloacae but not against Gram‐positive bacteria.
An immunoreceptor tyrosine‐based inhibitory motif, with serine at site Y‐2, binds SH2‐domain‐containing phosphatases
Authors: Philosof‐Oppenheimer Rachel , Hampe Christiane S. , Schlessinger Karni , Fridkin Mati , Pecht Israel
Published: March 2003

Clustering of the mast cell function‐associated antigen by its specific monoclonal antibody (G63) inhibits the FcεRI‐mediated secretory response. The cytosolic tail of the mast cell function‐associated antigen contains a SIYSTL stretch, a potential immunoreceptor tyrosine‐based inhibition motif. To investigate the possible functional role of this sequence, as well as identify potential intracellular proteins that interact with it, peptides corresponding to residues 4–12 of the mast cell function‐associated antigen's N‐terminal cytoplasmic domain, containing the above motif, were synthesized and used in affinity chromatography of mast cell lysates. Both tyrosyl phosphorylated and thiophosphorylated mast cell function‐associated antigen peptides bound the src homology domain 2 (SH2)‐containing tyrosine phosphatases‐1 (SHP‐1), ‐2 (SHP‐2) and inositol 5′‐phosphatase (SHIP), though with different efficiencies. Neither the nonphosphorylated peptide nor its tyrosyl phosphorylated reversed sequence peptide bound any of these phosphatases. Point mutation analysis of mast cell function‐associated antigen pITIM binding requirements demonstrated that for SHP‐2 association the amino acid residue at position Y‐2 is not restricted to the hydrophobic isoleucine or valine. Glycine and other amino acids with hydrophilic residues, such as serine and threonine, at this position also maintain this binding capacity, whereas alanine and acidic residues abolish it. In contrast, SHP‐1 binding was maintained only when serine was substituted by valine, suggesting that the Y‐2 position provides selectivity for peptide binding to SH2 domains of SHP‐1 and SHP‐2. These results were corroborated by surface plasmon resonance measurements of the interaction between tyrosyl phosphorylated mast cell function‐associated antigen peptide and recombinant soluble SH2 domains of SHP‐1, SHP‐2 and SHIP, suggesting that the associations observed in the cell lysates may be direct. Taken together these results clearly indicate that the SIYSTL motif present in mast cell function‐associated antigen's cytosolic tail exhibits characteristic features of an immunoreceptor tyrosine‐based inhibition motif, suggesting it is a new member of the growing diverse family of immunoreceptor tyrosine‐based inhibition motif‐containing receptors.
The role of mitochondria in the regulation of calcium influx into Jurkat cells
Authors: Makowska Agnieszka , Zabłocki Krzysztof , Duszyński Jerzy
Published: March 2003

In electrically nonexcitable cells the activity of the plasma membrane calcium channels is controlled by events occurring in mitochondria, as well as in the lumen of the endoplasmic reticulum. Thapsigargin, a specific inhibitor of endoplasmic reticulum Ca2+‐ATPase, produces the release of calcium from the endoplasmic reticulum and thus, activation of store‐operated calcium channels in the plasma membrane. However, thapsigargin failed to produce significant activation of the channels in Jurkat cells that had been pretreated with mitochondria‐directed agents: an uncoupler (carbonyl cyanide m‐chlorophenylhydrazone) and oligomycin. This is in spite of the fact that Jurkat cells pretreated with carbonyl cyanide m‐chlorophenylhydrazone plus oligomycin are otherwise energetically competent, due to a high rate of glycolysis and the inhibition of mitochondrial F1Fo‐ATPase by oligomycin. The pool of intracellular ATP was found not to be influenced by the pretreatments of cells with oligomycin or with oligomycin plus carbonyl cyanide m‐chlorophenylhydrazone. In the control cells, we found that the ATP pool amounted to 23.2 ± 1.9 nmoles per 107 cells (n = 4). In cells pretreated with oligomycin the level of ATP was 21.8 ± 1.9 nmoles per 107 cells (n = 4), and in cells pretreated with both oligomycin and an uncoupler the level of ATP was 22.1 ± 0.2 nmoles per 107 cells (n = 3). Moreover, in cells pretreated with oligomycin plus carbonyl cyanide m‐chlorophenylhydrazone and suspended in a nominally calcium‐free medium, thapsigargin produces transient increases in cytosolic calcium identical to those in the control cells. Thus, this pretreatment does not modify either the content of intracellular calcium stores and/or the activity of calcium ATPase in the plasma membrane. Similar results were obtained when Jurkat cells were challenged by myxothiazol, a potent inhibitor of mitochondrial cytochrome bc1 oxidoreductase. Thapsigargin, although producing calcium release from intracellular stores, was ineffective in triggering the activation of calcium channels in the plasma membrane in the case of cells pretreated with myxothiazol and oligomycin. Our results suggest that coupled mitochondria participate directly in the control of calcium channel activity in the plasma membrane of Jurkat cells. When the mitochondrial protonmotive force is collapsed, either by carbonyl cyanide m‐chlorophenylhydrazone or myxothiazol, the channel remains inactive even under conditions of empty intracellular calcium stores.
Insulin stimulates NHE1 activity by sequential activation of phosphatidylinositol 3‐kinase and protein kinase C ζ in human erythrocytes
Authors: Sauvage Monique , Mazière Pierre , Fathallah Hassana , Giraud Françoise
Published: March 2003

The signaling cascade linking insulin receptor stimulation to the activation of Na/H exchanger (NHE) was investigated in human erythrocytes, a simple cell model expressing the NHE1 isoform and protein kinase C (PKC) α and ζ isoforms only. Our results demonstrate the presence of phosphatidylinositol (PtdIns) 3‐kinase in these cells and its activation by insulin. With a similar time‐course, insulin also promoted both the translocation and activation of PKC ζ, but had no effect on PKC α. Inhibition of PtdIns 3‐kinase with wortmannin prevented the activation of PKC ζ by insulin. Stimulation of NHE1 was observed after 10 min of insulin treatment and persisted for at least 60 min. This effect was totally abolished by wortmannin or GF 109203X, an inhibitor of all PKC isoforms, but not by Gö 6976, a specific inhibitor of conventional and novel PKCs (e.g. PKC α). These data indicate that PKC ζ activation is mediated by a PtdIns 3‐kinase‐dependent mechanism and that NHE1 stimulation involves the sequential activation of PtdIns 3‐kinase and PKC ζ. In addition, insulin stimulation of NHE1 occurred without altering the phosphorylation state of the exchanger, suggesting that the phosphorylation of an ancillary protein by PKC ζ would be responsible for activation of the transporter.
A class of zinc fingers involved in protein–protein interactions
Authors: Matthews Jacqueline M. , Kowalski Kasper , Liew Chu Kong , Sharpe Belinda K. , Fox Archa H. , Crossley Merlin , Mackay Joel P.
Published: March 2003

Zinc fingers (ZnFs) are extremely common protein domains. Several classes of ZnFs are distinguished by the nature and spacing of their zinc‐coordinating residues. While the structure and function of some ZnFs are well characterized, many others have been identified only through their amino acid sequence. A number of proteins contain a conserved C‐X2‐C‐X12‐H‐X1–5‐C sequence, which is similar to the spacing observed for the ‘classic’ CCHH ZnFs. Although these domains have been implicated in protein–protein (and not protein–nucleic acid) interactions, nothing is known about their structure or function at a molecular level. Here, we address this problem through the expression and biophysical characterization of several CCHC‐type zinc fingers from the erythroid transcription factor FOG and the related Drosophila protein U‐shaped. Each of these domains does indeed fold in a zinc‐dependent fashion, coordinating the metal in a tetrahedral manner through the sidechains of one histidine and three cysteine residues, and forming extremely thermostable structures. Analysis of CD spectra suggests an overall fold similar to that of the CCHH fingers, and indeed a point mutant of FOG‐F1 in which the final cysteine residue is replaced by histidine remains capable of folding. However, the CCHC (as opposed to CCHH) motif is a prerequisite for GATA‐1 binding activity, demonstrating that CCHC and CCHH topologies are not interchangeable. This demonstration that members of a structurally distinct subclass of genuine zinc finger domains are involved in the mediation of protein–protein interactions has implications for the prediction of protein function from nucleotide sequences.
The N‐terminal β‐barrel structure of lipid body lipoxygenase mediates its binding to liposomes and lipid bodies
Authors: May Christian , Höhne Michaela , Gnau Petra , Schwennesen Karsten , Kindl Helmut
Published: March 2003

Phospholipase A2 and a particular isoform of lipoxygenase are synthesized and transferred to lipid bodies during the stage of triacylglycerol mobilization in germinating cucumber seedlings. Lipid body lipoxygenase (LBLOX) is post‐translationally transported to lipid bodies without proteolytic modification. Fractionation of homogenates from cucumber cotyledons or transgenic tobacco leaves expressing LBLOX showed that a small but significant amount was detectable in the microsomal fraction. A β‐barrel‐forming N‐terminal domain in the structure of LBLOX, as deduced from sequence data, was shown to be crucial for selective intracellular transport from the cytosol to lipid bodies. Although a specific signal sequence for targeting protein domains to the lipid bodies could not be established, it was evident that the β‐barrel represents a membrane‐binding domain that is functionally comparable with the C2 domains of mammalian phospholipases. The intact β‐barrel of LBLOX was demonstrated to be sufficient to target in vitro a fusion protein of LBLOX β‐barrel with glutathione S‐transferase (GST) to lipid bodies. In addition, binding experiments on liposomes using lipoxygenase isoforms, LBLOX deletions and the GST‐fusion protein confirmed the role of the β‐barrel as the membrane‐targeting domain. In this respect, the cucumber LBLOX differs from cytosolic isoforms in cucumber and from the soybean LOX‐1. When the β‐barrel of LBLOX was destroyed by insertion of an additional peptide sequence, its ability to target proteins to membranes was abolished.
Positive and negative elements modulate the promoter of the human liver‐specific α2‐HS‐glycoprotein gene
Authors: Banine Fatima , Gangneux Christophe , Mercier Louis , Le Cam Alphonse , Salier Jean‐Philippe
Published: March 2003

The human α2‐HS‐glycoprotein (AHSG) and the 63‐kDa rat phosphoprotein (pp63) are homologous plasma proteins that belong to the fetuin family. AHSG and pp63 are involved in important functions such as inhibition of insulin receptor tyrosine kinase activity, inhibition of protease activities, and regulation of calcium metabolism and osteogenesis. Studies of the AHSG proximal promoter performed in vitro in rat and human cells indicate that several NF‐1 and C/EBP binding sites exert a positive effect on its transcriptional activity. However, until now, no distal elements have been examined in this gene, in either species. We report that the human AHSG gene promoter acts in a liver‐specific manner and is further controlled by three distal, 5′‐flanking elements. The negative elements III and I are, respectively, located 5′ and 3′ of the positive element II. All three elements require the natural context of the human AHSG gene to fully exert their negative or positive effect. Element I harbours a single binding site for NF‐1. This nuclear factor thus appears to be able to up‐ or downregulate the AHSG gene depending on the site it binds to. Elements I, II and possibly III are absent in the rodent Ahsg gene encoding pp63.
Trypanosoma brucei contains a 2,3‐bisphosphoglycerate independent phosphoglycerate mutase
Authors: Chevalier Nathalie , Rigden Daniel J. , Van Roy Joris , Opperdoes Fred R. , Michels Paul A. M.
Published: March 2003

Assays of phosphoglycerate mutase (PGAM) activity in lysates of bloodstream form Trypanosoma brucei appeared not to require exogenous 2,3‐bisphosphoglycerate, thus suggesting that this protist contains an enzyme belonging to the class of cofactor‐independent PGAMs. A gene encoding a polypeptide with motifs characteristic for this class of enzymes was cloned. The predicted T. brucei PGAM polypeptide contains 549 amino acids, with Mr 60 557 and pI 5.5. Comparison with 15 cofactor‐independent PGAM sequences available in databases showed that the amino‐acid sequence of the trypanosome enzyme has 59–62% identity with plant PGAMs and 29–35% with eubacterial enzymes. A low 28% identity was observed with the only available invertebrate sequence. The trypanosome enzyme has been expressed in Escherichia coli, purified to homogeneity and subjected to preliminary kinetic analysis. Previous studies have shown that cofactor‐dependent and ‐independent PGAMs are not homologous. It has been inferred that the cofactor‐independent PGAMs are in fact homologous to a family of metalloenzymes containing alkaline phosphatases and sulphatases. Prediction of the secondary structure of T. brucei PGAM and threading the sequence into the known crystal structure of E. coli alkaline phosphatase (AP) confirmed this homology, despite the very low sequence identity. Generally, a good match between predicted (PGAM) and actual (AP) secondary structure elements was observed. In contrast to trypanosomes, glycolysis in all vertebrates involves a cofactor‐dependent PGAM. The presence of distinct nonhomologous PGAMs in the parasite and its human host offers great potential for the design of selective inhibitors which could form leads for new trypanocidal drugs.
Ribonucleases expressed by human pancreatic adenocarcinoma cell lines
Authors: Fernández‐Salas Ester , Peracaula Rosa , Frazier Marsha L. , de Llorens Rafael
Published: March 2003

Human ribonucleases have been considered as a possible tumor marker for pancreatic cancer, and elevated serum levels of ribonuclease activity in patients with pancreatic cancer have been reported by many authors. The reason for this elevation is unknown. In this study, we demonstrate that human pancreatic adenocarcinoma cell lines synthesize and secrete different ribonucleases. We isolated and characterized human pancreatic, or secretory, ribonuclease (RNase 1) from the conditioned media of the human pancreatic adenocarcinoma cell lines Capan‐1, MDAPanc‐3, IBF‐CP3 and Panc‐1, and the ampullary adenocarcinoma cell line MDAAmp‐7, which represent a wide range of differentiation stages. Only one of these cell lines, Panc‐1, produces significant amounts of nonsecretory ribonuclease. We then established a purification procedure for both secretory and nonsecretory ribonucleases, consisting of concentration of the supernatant by tangential filtration, anion‐exchange and cation‐exchange liquid chromatography and C4 RP‐HPLC. Ribonuclease activity fractions were monitored using both the spectrophotometric and negative‐staining zymogram techniques. The results of N‐terminal sequence analysis, kinetic analysis and endoglycosidase digestion studies indicate that the main ribonuclease secreted by all the cell lines is the secretory‐type ribonuclease and that it is composed of several differently N‐glycosylated forms. Northern blot analyses confirm that some of the cell lines express secretory ribonuclease mRNA. The mRNA levels produced by Panc‐1 and MDAPanc‐28 are too low to be detected. Similar levels of expression of nonsecretory ribonuclease are found by Northern blot analysis in all the cell lines except Panc‐1, which expresses higher levels. Here, we describe, for the first time, that several human pancreatic cancer cell lines with different degrees of differentiation express and secrete ribonucleases. This fact indicates that one origin of the elevated serum RNase levels in patients with pancreatic cancer are tumor cells. Analysis of the oligosaccharide moiety of the RNase 1 secreted by Capan‐1 shows that it is highly glycosylated and its N‐glycan chains are significantly different from that of the RNase 1 produced by normal pancreas. These results renew the possibility of using human serum RNase 1 determination as a tumor marker.
Structural characterization of the outer core and the O‐chain linkage region of lipopolysaccharide from Pseudomonas aeruginosa serotype O5
Authors: Sadovskaya Irina , Brisson Jean‐Robert , Thibault Pierre , Richards James C , Lam Joseph S , Altman Eleonora
Published: March 2003

The point of attachment of the O‐chain in the outer core region of Pseudomonas aeruginosa serotype O5 lipopolysaccharide (LPS) was determined following a detailed analysis of the extended core oligosaccharide, containing one trisaccharide O‐chain repeating unit, present in both the wild‐type strain PAO1 and O‐chain deficient mutant strains AK1401 and PAO‐rfc. The structure of the extended core oligosaccharide was determined by various mass spectrometric methods as well as one‐dimensional and two‐dimensional NMR spectroscopy. Furthermore, the one‐dimensional analogues of NOESY and TOCSY experiments were applied to confirm the structure of the outer core region in the O‐chain polysaccharide. In both the extended core oligosaccharide and the core of the smooth LPS, a loss of one of the β‐glucosyl residues and the translocation of the α‐rhamnosyl residue, followed by the attachment of the first O‐chain repeating unit was observed. This process is complicated and could involve two distinct rhamnosyltransferases, one with α‐1,6‐linkage specificity and another with α‐1,3‐linkage specificity. It is also plausible that an α‐1,3 rhamnosyltransferase facilitates the addition of the ‘new’α‐rhamnosyl residue that will act as a receptor for the attachment of the single O‐antigen repeating unit in the LPS of the semi‐rough mutant. The 2‐amino‐2‐deoxy‐fucosyl residue of the first O‐chain repeating unit directly attached to the core was found to have a β‐anomeric configuration instead of an α configuration, characteristic for this residue as a component of the O‐chain polysaccharide. The results of this study provide the first example of the mechanistic implications of the structure of the outer core region in a fully assembled O‐chain containing LPS, differing from the O‐chain deficient rough LPS.
Nickel serves as a substrate recognition motif for the endopeptidase involved in hydrogenase maturation
Authors: Theodoratou Ekaterini , Paschos Athanasios , Magalon Axel , Fritsche Erich , Huber Robert , Böck August
Published: March 2003

The interaction of the hydrogenase maturation endopeptidase HycI with its substrate, the precursor of the large subunit, was studied. Replacement of conserved amino‐acid residues in HycI, which have been shown to bind a cadmium ion from the crystallization buffer in crystals of HybD (endopeptidase for hydrogenase 2), abolished or strongly reduced processing activity. Atomic absorption spectroscopy of purified HycI and HybD proteins showed the absence of nickel. In vitro processing assays showed that the reaction requires nickel to be bound to the precursor and the protease does not have a function in nickel delivery to the substrate. Radioactive labelling of cells with 63Ni, devoid of endopeptidase, resolved several forms of the precursor which are possibly intermediates in the maturation pathway. It is concluded that the endopeptidase uses the metal in the large subunit of [NiFe]‐hydrogenases as a recognition motif.