bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression and distribution of penaeidin antimicrobial peptides are regulated by haemocyte reactions in microbial challenged shrimp
Authors: Muñoz Marcelo , Vandenbulcke Franck , Saulnier Denis , Bachère Evelyne
Published: March 2003

Penaeidins are a family of antimicrobial peptides constitutively produced and stored in the haemocytes of penaeid shrimp. In response to microbial stimulation, they are released into the blood circulation and they further attach to shrimp cuticle surfaces through a chitin‐binding property. In the present paper, we have analysed their expression, regulation and distribution in shrimp tissues in response to experimental microbial challenge. We have shown that penaeidin mRNA and protein are restricted to granular haemocytes and that their expression and distribution are regulated through dramatic changes in haemocyte populations, both circulating and infiltrating shrimp tissues. Two distinct phases in the immune reactions were evidenced: (a) a migration of haemocytes towards the infection site within the first 12 h following microbial injection, with a local and massive release of peptides; (b) the appearance into the blood circulation and tissues of a haemocyte population displaying increased penaeidin‐transcriptional activity, which may correspond to a systemic reaction involving haemocyte proliferation process. Finally, in vitro confrontation of haemocytes and bacteria revealed that penaeidins are released from granular haemocytes by a novel phenomenon of intracellular degranulation, probably followed by the lysis of the cells. Furthermore, penaeidins were shown covering bacterial surfaces suggesting that the peptides could be involved in opsonic activity. Penaeidin‐positive bacteria were observed to be phagocytosed mainly by hyaline cells, a population that does not express penaeidins.
Expression of uncoupling protein‐3 in subsarcolemmal and intermyofibrillar mitochondria of various mouse muscle types and its modulation by fasting
Authors: Jimenez Maria , Yvon Cedric , Lehr Lorenz , Léger Bertrand , Keller Patrick , Russell Aaron , Kuhne Françoise , Flandin Pierre , Giacobino Jean‐Paul , Muzzin Patrick
Published: March 2003

Uncoupling protein‐3 (UCP3) is a mitochondrial inner‐membrane protein abundantly expressed in rodent and human skeletal muscle which may be involved in energy dissipation. Many studies have been performed on the metabolic regulation of UCP3 mRNA level, but little is known about UCP3 expression at the protein level. Two populations of mitochondria have been described in skeletal muscle, subsarcolemmal (SS) and intermyofibrillar (IMF), which differ in their intracellular localization and possibly also their metabolic role. To examine if UCP3 is differentially expressed in these two populations and in different mouse muscle types, we developed a new protocol for isolation of SS and IMF mitochondria and carefully validated a new UCP3 antibody. The data show that the density of UCP3 is higher in the mitochondria of glycolytic muscles (tibialis anterior and gastrocnemius) than in those of oxidative muscle (soleus). They also show that SS mitochondria contain more UCP3 per mg of protein than IMF mitochondria. Taken together, these results suggest that oxidative muscle and the mitochondria most closely associated with myofibrils are most efficient at producing ATP. We then determined the effect of a 24‐h fast, which greatly increases UCP3 mRNA (16.4‐fold) in muscle, on UCP3 protein expression in gastrocnemius mitochondria. We found that fasting moderately increases (1.5‐fold) or does not change UCP3 protein in gastrocnemius SS or IMF mitochondria, respectively. These results show that modulation of UCP3 expression at the mRNA level does not necessarily result in similar changes at the protein level and indicate that UCP3 density in SS and IMF mitochondria can be differently affected by metabolic changes.
Monoterpene biosynthesis in lemon (Citrus limon)
Authors: Lücker Joost , El Tamer Mazen K. , Schwab Wilfried , Verstappen Francel W. A. , van der Plas Linus H. W. , Bouwmeester Harro J. , Verhoeven Harrie A.
Published: March 2003

Citrus limon possesses a high content and large variety of monoterpenoids, especially in the glands of the fruit flavedo. The genes responsible for the production of these monoterpenes have never been isolated. By applying a random sequencing approach to a cDNA library from mRNA isolated from the peel of young developing fruit, four monoterpene synthase cDNAs were isolated that appear to be new members of the previously reported tpsb family. Based on sequence homology and phylogenetic analysis, these sequences cluster in two separate groups. All four cDNAs could be functionally expressed in Escherichia coli after removal of their plastid targeting signals. The main products of the enzymes in assays with geranyl diphosphate as substrate were (+)‐limonene (two cDNAs) (–)‐β‐pinene and γ‐terpinene. All enzymes exhibited a pH optimum around 7; addition of Mn2+ as bivalent metal ion cofactor resulted in higher activity than Mg2+, with an optimum concentration of 0.6 mm. Km values ranged from 0.7 to 3.1 µm. The four enzymes account for the production of 10 out of the 17 monoterpene skeletons commonly observed in lemon peel oil, corresponding to more than 90% of the main components present.
Structure, mechanism and function of prenyltransferases
Authors: Liang Po‐Huang , Ko Tzu‐Ping , Wang Andrew H.‐J
Published: March 2003

In this review, we summarize recent progress in studying three main classes of prenyltransferases: (a) isoprenyl pyrophosphate synthases (IPPSs), which catalyze chain elongation of allylic pyrophosphate substrates via consecutive condensation reactions with isopentenyl pyrophosphate (IPP) to generate linear polymers with defined chain lengths; (b) protein prenyltransferases, which catalyze the transfer of an isoprenyl pyrophosphate (e.g. farnesyl pyrophosphate) to a protein or a peptide; (c) prenyltransferases, which catalyze the cyclization of isoprenyl pyrophosphates. The prenyltransferase products are widely distributed in nature and serve a variety of important biological functions. The catalytic mechanism deduced from the 3D structure and other biochemical studies of these prenyltransferases as well as how the protein functions are related to their reaction mechanism and structure are discussed. In the IPPS reaction, we focus on the mechanism that controls product chain length and the reaction kinetics of IPP condensation in the cis‐type and trans‐type enzymes. For protein prenyltransferases, the structures of Ras farnesyltransferase and Rab geranylgeranyltransferase are used to elucidate the reaction mechanism of this group of enzymes. For the enzymes involved in cyclic terpene biosynthesis, the structures and mechanisms of squalene cyclase, 5‐epi‐aristolochene synthase, pentalenene synthase, and trichodiene synthase are summarized.
SF2/ASF protein inhibits camptothecin‐induced DNA cleavage by human topoisomerase I
Authors: Kowalska‐Loth Barbara , Girstun Agnieszka , Piekiełko Agnieszka , Staroń Krzysztof
Published: March 2003

A splicing factor SF2/ASF is a natural substrate for the kinase activity of human topoisomerase I. This study demonstrates that SF2/ASF inhibits DNA cleavage by human topoisomerase I induced by the anti‐cancer agent camptothecin. The inhibition is independent of the phosphorylation status of SF2/ASF. We show that the inhibition did not result from binding of SF2/ASF to DNA that would hinder interactions between topoisomerase I and DNA. Neither it was a consequence of a loss of sensitivity of the enzyme to camptothecin. We provide evidence pointing to reduced formation of the cleavable complex in the presence of SF2/ASF as a primary reason for the inhibition. This effect of SF2/ASF is reflected by inhibition of DNA relaxation catalysed by topoisomerase I.
Identification and structural characterization of a sialylated lacto‐N‐neotetraose structure in the lipopolysaccharide of Haemophilus influenzae
Authors: Cox Andrew D. , Hood Derek W. , Martin Adele , Makepeace Katherine M. , Deadman Mary E. , Li Jianjun , Brisson Jean‐Robert , Moxon E. Richard , Richards James C.
Published: March 2003

A sialylated lacto‐N‐neotetraose (Sial‐lNnT) structural unit was identified and structurally characterized in the lipopolysaccharide (LPS) from the genome‐sequenced strain Road (RM118) of the human pathogen Haemophilus influenzae grown in the presence of sialic acid. A combination of molecular genetics, MS and NMR spectroscopy techniques showed that this structural unit extended from the proximal heptose residue of the inner core region of the LPS molecule. The structure of the Sial‐lNnT unit was identical to that found in meningococcal LPS, but glycoforms containing truncations of the Sial‐lNnT unit, comprising fewer residues than the complete oligosaccharide component, were not detected. The finding of sialylated glycoforms that were either fully extended or absent suggests a novel biosynthetic feature for adding the terminal tetrasaccharide unit of the Sial‐lNnT to the glycose acceptor at the proximal inner core heptose.
Cloning and functional characterization of Phaeodactylum tricornutum front‐end desaturases involved in eicosapentaenoic acid biosynthesis
Authors: Domergue Frédéric , Lerchl Jens , Zähringer Ulrich , Heinz Ernst
Published: March 2003

Phaeodactylum tricornutum is an unicellular silica‐less diatom in which eicosapentaenoic acid accumulates up to 30% of the total fatty acids. This marine diatom was used for cloning genes encoding fatty acid desaturases involved in eicosapentaenoic acid biosynthesis. Using a combination of PCR, mass sequencing and library screening, the coding sequences of two desaturases were identified. Both protein sequences contained a cytochrome b5 domain fused to the N‐terminus and the three histidine clusters common to all front‐end fatty acid desaturases. The full length clones were expressed in Saccharomyces cerevisiae and characterized as Δ5‐ and Δ6‐fatty acid desaturases. The substrate specificity of each enzyme was determined and confirmed their involvement in eicosapentaenoic acid biosynthesis. Using both desaturases in combination with the Δ6‐specific elongase from Physcomitrella patens, the biosynthetic pathways of arachidonic and eicosapentaenoic acid were reconstituted in yeast. These reconstitutions indicated that these two desaturases functioned in the ω3‐ and ω6‐pathways, in good agreement with both routes coexisting in Phaeodactylum tricornutum. Interestingly, when the substrate selectivity of each enzyme was determined, both desaturases converted the ω3‐ and ω6‐fatty acids with similar efficiencies, indicating that none of them was specific for either the ω3‐ or the ω6‐pathway. To our knowledge, this is the first report describing the isolation and biochemical characterization of fatty acid desaturases from diatoms.
Identification of crucial residues for the antibacterial activity of the proline‐rich peptide, pyrrhocoricin
Authors: Kragol Goran , Hoffmann Ralf , Chattergoon Michael A. , Lovas Sandor , Cudic Mare , Bulet Philippe , Condie Barry A. , Rosengren K. Johan , Montaner Luis J. , Otvos Laszlo
Published: March 2003

Members of the proline‐rich antibacterial peptide family, pyrrhocoricin, apidaecin and drosocin appear to kill responsive bacterial species by binding to the multihelical lid region of the bacterial DnaK protein. Pyrrhocoricin, the most potent among these peptides, is nontoxic to healthy mice, and can protect these animals from bacterial challenge. A structure–antibacterial activity study of pyrrhocoricin against Escherichia coli and Agrobacterium tumefaciens identified the N‐terminal half, residues 2–10, the region responsible for inhibition of the ATPase activity, as the fragment that contains the active segment. While fluorescein‐labeled versions of the native peptides entered E. coli cells, deletion of the C‐terminal half of pyrrhocoricin significantly reduced the peptide's ability to enter bacterial or mammalian cells. These findings highlighted pyrrhocoricin's suitability for combating intracellular pathogens and raised the possibility that the proline‐rich antibacterial peptides can deliver drug leads into mammalian cells. By observing strong relationships between the binding to a synthetic fragment of the target protein and antibacterial activities of pyrrhocoricin analogs modified at strategic positions, we further verified that DnaK was the bacterial target macromolecule. Inaddition, the antimicrobial activity spectrum of native pyrrhocoricin against 11 bacterial and fungal strains and the binding of labeled pyrrhocoricin to synthetic DnaK D‐E helix fragments of the appropriate species could be correlated. Mutational analysis on a synthetic E. coli DnaK fragment identified a possible binding surface for pyrrhocoricin.
The lectin from leaves of Japanese cycad, Cycas revoluta Thunb. (gymnosperm) is a member of the jacalin‐related family
Authors: Yagi Fumio , Iwaya Toshinobu , Haraguchi Tomokazu , Goldstein Irwin J.
Published: March 2003

A novel lectin was isolated from leaves of the Japanese cycad, Cycas revoluta Thunb. (gymnosperm), and its characteristics including amino acid composition, molecular mass, carbohydrate binding specificity and partial amino acid sequences were examined. The inhibition analysis of hemagglutinating activity with various sugars showed that the lectin has a carbohydrate‐binding specificity similar to those of mannose recognizing, jacalin‐related lectins. Partial amino acid sequences of the lysylendopeptic peptides shows that the lectin might have a repeating structure and belong to the jacalin‐related lectin family.
The soluble form of the membrane‐bound transferrin homologue, melanotransferrin, inefficiently donates iron to cells via nonspecific internalization and degradation of the protein
Authors: Food Michael R. , Sekyere Eric O. , Richardson Des R.
Published: March 2003

Melanotransferrin (MTf) is a membrane‐bound transferrin (Tf) homologue found particularly in melanoma cells. Apart from membrane‐bound MTf, a soluble form of the molecule (sMTf) has been identified in vitro[Food, M.R., Rothenberger, S., Gabathuler, R., Haidl, I.D., Reid, G. & Jefferies, W.A. (1994) J. Biol. Chem.269, 3034–3040] and in vivo in Alzheimer's disease. However, nothing is known about the function of sMTf or its role in Fe uptake. In this study, sMTf labelled with 59Fe and 125I was used to examine its ability to donate 59Fe to SK‐Mel‐28 melanoma cells and other cell types. sMTf donated 59Fe to cells at 14% of the rate of Tf. Analysis of sMTf binding showed that unlike Tf, sMTf did not bind to a saturable Tf‐binding site. Studies with Chinese hamster ovary cells with and without specific Tf receptors showed that unlike Tf, sMTf did not donate its 59Fe via these pathways. This was confirmed by experiments using lysosomotropic agents that markedly reduced 59Fe uptake from Tf, but had far less effect on 59Fe uptake from sMTf. In addition, an excess of 56Fe‐labelled Tf or sMTf had no effect on 125I‐labelled sMTf uptake, suggesting a nonspecific interaction of sMTf with cells. Protein‐free 125I determinations demonstrated that in contrast with Tf, sMTf was markedly degraded. We suggest that unlike the binding of Tf to specific receptors, sMTf was donating Fe to cells via an inefficient mechanism involving nonspecific internalization and subsequent degradation.
Chaperone and antichaperone activities of trigger factor
Authors: Huang Guo‐Chang , Chen Jia‐Jia , Liu Chuan‐Peng , Zhou Jun–Mei
Published: March 2003

Reduced denatured lysozyme tends to aggregate at neutral pH and competition between productive folding and aggregation substantially reduces the efficiency of refolding. Trigger factor, a folding catalyst and chaperone can, depending on the concentration of trigger factor and the solution conditions, cause either a substantial increase (chaperone activity) or a substantial decrease (antichaperone activity) in the recovery of native lysozyme as compared with spontaneous refolding. When trigger factor is working as a chaperone, the reactivation rates of lysozyme are decelerated and aggregation decreases with increasing trigger factor concentrations. Under conditions where antichaperone activity of trigger factor dominates, the reactivation rates of lysozyme are accelerated and aggregation is increased. Trigger factor and lysozyme were both released from the aggregates on re‐solubilization with urea indicating that trigger factor participates directly in aggregate formation and is incorporated into the aggregates. The apparently dual effect of trigger factor toward refolding of lysozyme is a consequence of the peptide binding ability and may be important in regulation of protein biosynthesis.
A single charged surface residue modifies the activity of ikitoxin, a beta‐type Na+ channel toxin from Parabuthus transvaalicus
Authors: Inceoglu A. Bora , Hayashida Yuki , Lango Jozsef , Ishida Andrew T. , Hammock Bruce D.
Published: March 2003

We previously purified and characterized a peptide toxin, birtoxin, from the South African scorpion Parabuthus transvaalicus. Birtoxin is a 58‐residue, long chain neurotoxin that has a unique three disulfide‐bridged structure. Here we report the isolation and characterization of ikitoxin, a peptide toxin with a single residue difference, and a markedly reduced biological activity, from birtoxin. Bioassays on mice showed that high doses of ikitoxin induce unprovoked jumps, whereas birtoxin induces jumps at a 1000‐fold lower concentration. Both toxins are active against mice when administered intracerebroventricularly. Mass determination indicated an apparent mass of 6615 Da for ikitoxin vs. 6543 Da for birtoxin. Amino acid sequence determination revealed that the amino‐acid sequence of ikitoxin differs from birtoxin by a single residue change from glycine to glutamic acid at position 23, consistent with the apparent mass difference of 72 Da. This single‐residue difference renders ikitoxin much less effective in producing the same behavioral effect as low concentrations of birtoxin. Electrophysiological measurements showed that birtoxin and ikitoxin can be classified as beta group toxins for voltage‐gated Na+ channels of central neurons. It is our conclusion that the N‐terminal loop preceding the α‐helix in scorpion toxins is one of the determinative domains in the interaction of toxins with the target ion channel.
Purification and kinetic analysis of the two recombinant arogenate dehydrogenase isoforms of Arabidopsis thaliana
Authors: Rippert Pascal , Matringe Michel
Published: March 2003

The present study reports the first purification and kinetic characterization of two plant arogenate dehydrogenases (EC 1.3.1.43), an enzyme that catalyses the oxidative decarboxylation of arogenate into tyrosine in presence of NADP. The two Arabidopsis thaliana arogenate dehydrogenases TyrAAT1 and TyrAAT2 were overproduced in Escherichia coli and purified to homogeneity. Biochemical comparison of the two forms revealed that at low substrate concentration TyrAAT1 is four times more efficient in catalyzing the arogenate dehydrogenase reaction than TyrAAT2. Moreover, TyrAAT2 presents a weak prephenate dehydrogenase activity whereas TyrAAT1 does not. The mechanism of the dehydrogenase reaction catalyzed by these two forms has been investigated using steady‐state kinetics. For both enzymes, steady‐state velocity patterns are consistent with a rapid equilibrium, random mechanism in which two dead‐end complexes, E–NADPH–arogenate and E–NADP–tyrosine, are formed.
Steady‐state kinetics of the glutaminase reaction of CTP synthase from Lactococcus lactis
Authors: Willemoës Martin , Sigurskjold Bent W
Published: March 2003

CTP synthase catalyzes the reaction glutamine + UTP + ATP → glutamate + CTP + ADP + Pi. The rate of the reaction is greatly enhanced by the allosteric activator GTP. We have studied the glutaminase half‐reaction of CTP synthase from Lactococcus lactis and its response to the allosteric activator GTP and nucleotides that bind to theactive site. In contrast to what has been found for the Escherichia coli enzyme, GTP activation of the L. lactis enzyme did not result in similar kcat values for the glutaminase activity and glutamine hydrolysis coupled to CTP synthesis. GTP activation of the glutaminase reaction never reached the levels of GTP‐activated CTP synthesis, not even when the active site was saturated with UTP and the nonhydrolyzeable ATP‐binding analog adenosine 5′‐[γ‐thio]triphosphate. Furthermore, under conditions where the rate of glutamine hydrolysis exceeded that of CTP synthesis, GTP would stimulate CTP synthesis. These results indicate that the L. lactis enzyme differs significantly from the E. coli enzyme. For the E. coli enzyme, activation by GTP was found to stimulate glutamine hydrolysis and CTP synthesis to the same extent, suggesting that the major function of GTP binding is to activate the chemical steps of glutamine hydrolysis. An alternative mechanism for the action of GTP on L. lactis CTP synthase is suggested. Here the binding of GTP to the allosteric site promotes coordination of the phosphorylation of UTP and hydrolysis of glutamine for optimal efficiency in CTP synthesis rather than just acting to increase the rate of glutamine hydrolysis itself.
Identification and characterization of thioredoxin and thioredoxin reductase from Aeropyrum pernix K1
Authors: Jeon Sung‐Jong , Ishikawa Kazuhiko
Published: March 2003

We have identified and characterized a thermostable thioredoxin system in the aerobic hyperthermophilic archaeon Aeropyrum pernix K1. The gene (Accession no. APE0641) of A. pernix encoding a 37 kDa protein contains a redox active site motif (CPHC) but its N‐terminal extension region (about 200 residues) shows no homology within the genome database. A second gene (Accession no. APE1061) has high homology to thioredoxin reductase and encodes a 37 kDa protein with the active site motif (CSVC), and binding sites for FAD and NADPH. We cloned the two genes and expressed both proteins in E. coli. It was observed that the recombinant proteins could act as an NADPH‐dependent protein disulfide reductase system in the insulin reduction. In addition, the APE0641 protein and thioredoxin reductase from E. coli could also catalyze the disulfide reduction. These indicated that APE1061 and APE0641 express thioredoxin (ApTrx) and thioredoxin reductase (ApTR) of A. pernix, respectively. ApTR is expressed as an active homodimeric flavoprotein in the E. coli system. The optimum temperature was above 90 °C, and the half‐life of heat inactivation was about 4 min at 110 °C. The heat stability of ApTR was enhanced in the presence of excess FAD. ApTR could reduce both thioredoxins from A. pernix and E. coli and showed a similar molar specific activity for both proteins. The standard state redox potential of ApTrx was about −262 mV, which was slightly higher than that of Trx from E. coli (−270 mV). These results indicate that a lower redox potential of thioredoxin is not necessary for keeping catalytic disulfide bonds reduced and thereby coping with oxidative stress in an aerobic hyperthermophilic archaea. Furthermore, the thioredoxin system of aerobic hyperthermophilic archaea is biochemically close to that of the bacteria.
Extra terminal residues have a profound effect on the folding and solubility of a Plasmodium falciparum sexual stage‐specific protein over‐expressed in Escherichia coli
Authors: Sati Sushil Prasad , Singh Saurabh Kumar , Kumar Nirbhay , Sharma Amit
Published: March 2003

The presence of extra N‐ and C‐ terminal residues can play a major role in the stability, solubility and yield of recombinant proteins. Pfg27 is a 27K soluble protein that is essential for sexual development in Plasmodium falciparum. It was over‐expressed using the pMAL‐p2 vector as a fusion protein with the maltose binding protein. Six different constructs were made and each of the fusion proteins were expressed and purified. Our results show that the fusion proteins were labile and only partially soluble in five of the constructs resulting in very poor yields. Intriguingly, in the sixth construct, the yield of soluble fusion protein with an extended carboxyl terminus of 17 residues was several fold higher. Various constructs with either N‐terminal or smaller C‐terminal extensions failed to produce any soluble fusion protein. Furthermore, all five constructs produced Pfg27 that precipitated after protease cleavage from its fusion partner. The sixth construct, which produced soluble protein in high yields, also gave highly stable and soluble Pfg27 after cleavage of the fusion. These results indicate that extra amino acid residues at the termini of over‐expressed proteins can have a significant effect on the folding of proteins expressed in E. coli. Our data suggest the potential for development of a novel methodology, which will entail construction of fusion proteins with maltose binding protein as a chaperone on the N‐terminus and a C‐terminal ‘solubilization tag’. This system may allow large‐scale production of those proteins that have a tendency to misfold during expression.
Biosynthesis of vitamin B2
Authors: Kaiser Johannes , Schramek Nicholas , Eberhardt Sabine , Püttmer Stefanie , Schuster Michael , Bacher Adelbert
Published: March 2003

GTP cyclohydrolase II catalyzes the hydrolytic release of formate and pyrophosphate from GTP producing 2,5‐diamino‐6‐ribosylamino‐4(3H)‐pyrimidinone 5′‐phosphate, the first committed intermediate in the biosynthesis of riboflavin. The enzyme was shown to contain one zinc ion per subunit. Replacement of cysteine residue 54, 65 or 67 with serine resulted in proteins devoid of bound zinc and unable to release formate from the imidazole ring of GTP or from the intermediate analog, 2‐amino‐5‐formylamino‐6‐ribosylamino‐4(3H)‐pyrimidinone 5′‐triphosphate. However, the mutant proteins retained the capacity to release pyrophosphate from GTP and from the formamide‐type intermediate analog. The data suggest that the enzyme catalyzes an ordered reaction in which the hydrolytic release of pyrophosphate precedes the hydrolytic attack of the imidazole ring. Ring opening and formate release are both dependent on a zinc ion acting as a Lewis acid, which activates the two water molecules involved in the sequential hydrolysis of two carbon–nitrogen bonds.
Equilibrium unfolding and conformational plasticity of troponin I and T
Authors: Martins Samantha M. , Chapeaurouge Alex , Ferreira Sérgio T.
Published: March 2003

The structures and stabilities of recombinant chicken muscle troponin I (TnI) and T (TnT) were investigated by a combination of bis‐ANS binding and equilibrium unfolding studies. Unlike most folded proteins, isolated TnI and TnT bind the hydrophobic fluorescent probe bis‐ANS, indicating the existence of solvent‐exposed hydrophobic domains in their structures. Bis‐ANS binding to binary or ternary mixtures of TnI, TnT and troponin C (TnC) in solution is significantly lower than binding to the isolated subunits, which can be explained by burial of previously exposed hydrophobic domains upon association of the subunits to form the native troponin complex. Equilibrium unfolding studies of TnT and TnI by guanidine hydrochloride and urea monitored by changes in far‐UV CD and bis‐ANS fluorescence revealed noncooperative folding transitions for both proteins and the existence of partially folded intermediate states. Taken together, these results indicate that isolated TnI and TnT are partially unstructured proteins, and suggest that conformational plasticity of the isolated subunits may play an important role in macromolecular recognition for the assembly of the troponin complex.
Requirement of caspase and p38MAPK activation in zinc‐induced apoptosis in human leukemia HL‐60 cells
Authors: Kondoh Masuo , Tasaki Emi , Araragi Saeko , Takiguchi Masufumi , Higashimoto Minoru , Watanabe Yoshiteru , Sato Masao
Published: March 2003

Zinc (Zn), an endogenous regulator of apoptosis, and has abilities both to induce apoptosis and inhibit the induction of apoptosis via the modulation of caspase activity. Due to the multifunctions of Zn, the intracellular Zn level is strictly regulated by a complex system in physiological and pathological conditions. The commitment of Zn to the regulation of apoptosis is not fully understood. In the present study, we investigated the role of intracellular Zn level in the induction of apoptosis in human leukemia cells (HL‐60 cells) using a Zn ionophore [pyrithione (Py)]. Treatment of HL‐60 cells with Zn for 6 h in the presence of Py (1 µm) exhibited cytotoxicity in a Zn dose‐dependent manner (25–200 µm). Necrotic cells, assayed by trypan blue permeability, increased in number in a Zn dose‐dependent fashion (50–100 µm), but the appearance of apoptotic cells, assayed by formation of a DNA ladder and terminal deoxynucleotidyltransferase‐mediated dUTP‐biotin nick end‐labeling method, peaked at 25 µm, suggesting the dependence of intracellular Zn level on the execution of apoptosis. In fact, treatment with Py resulted in increases in intracellular Zn levels, and N,N,N′,N′‐tetrakis (2‐pyridylmethyl)ethylenediamine, a cell‐permeable Zn chelator, inhibited DNA ladder formation induced by Py/Zn treatment (1 µm Py and 25 µm Zn). Py/Zn treatment activated the caspases, as assessed by the proteolysis of poly(ADP‐ribose) polymerase (PARP), which is a substrate of caspase, and activated p38 mitogen‐activated protein kinase (p38MAPK), which is a transducer of apoptotic stimuli to the apparatus of the apoptosis execution. Z‐Asp‐CH2‐DCB, a broad‐spectrum inhibitor of caspase, attenuated proteolysis of PARP and DNA ladder formation by Py/Zn, indicating that apoptosis induced by Py/Zn is mediated by caspase activation. The p38MAPK‐specific inhibitor SB203580 also inhibited induction of apoptosis by Py/Zn. Although SB203580 suppressed the proteolysis of PARP, Z‐Asp‐CH2‐DCB did not inhibit the phosphorylation of p38MAPK, raising the possibility that apoptosis triggered by Py/Zn might be mediated by the p38MAPK/caspase pathway.
Oxygen‐dependent regulation of hypoxia‐inducible factors by prolyl and asparaginyl hydroxylation
Authors: Lando David , Gorman Jeffrey J. , Whitelaw Murray L. , Peet Daniel J.
Published: March 2003

To sustain life mammals have an absolute and continual requirement for oxygen, which is necessary to produce energy for normal cell survival and growth. Hence, maintaining oxygen homeostasis is a critical requirement and mammals have evolved a wide range of cellular and physiological responses to adapt to changes in oxygen availability. In the past few years it has become evident that the transcriptional protein complex hypoxia‐inducible factor (HIF) is a key regulator of these processes. In this review we will focus on the way oxygen availability regulates HIF proteins and in particular we will discuss the way oxygen‐dependent hydroxylation of specific amino acid residues has been demonstrated to regulate HIF function at the level of both protein stability and transcriptional potency.
TheN‐acetylglutamate synthase/N‐acetylglutamate kinase metabolon ofSaccharomyces cerevisiaeallows co‐ordinated feedback regulation of the first two steps in arginine biosynthesis
Authors: Pauwels Katia , Abadjieva Agnes , Hilven Pierre , Stankiewicz Anna , Crabeel Marjolaine
Published: March 2003

InSaccharomyces cerevisiae, which uses the nonlinear pathway of arginine biosynthesis, the first two enzymes,N‐acetylglutamate synthase (NAGS) andN‐acetylglutamate kinase (NAGK), are controlled by feedback inhibition. We have previously shown that NAGS and NAGK associate in a complex, essential to synthase activity and protein level [Abadjieva, A., Pauwels, K., Hilven, P. & Crabeel, M. (2001)J. Biol. Chem.276, 42869–42880].

The NAGKs of ascomycetes possess, in addition to the catalytic domain that is shared by all other NAGKs and whose structure has been determined, a C‐terminal domain of unknown function and structure. Exploring the role of these two domains in the synthase/kinase interaction, we demonstrate that the ascomycete‐specific domain is required to maintain synthase activity and protein level.

Previous results had suggested a participation of the third enzyme of the pathway,N‐acetylglutamylphosphate reductase, in the metabolon. Here, genetic analyses conducted in yeast at physiological level, or in a heterologous background, clearly demonstrate that the reductase is dispensable for synthase activity and protein level.

Most importantly, we show that the arginine feedback regulation of the NAGS and NAGK enzymes is mutually interdependent. First, the kinase becomes less sensitive to arginine feedback inhibition in the absence of the synthase. Second, and as inNeurospora crassa, in a yeast kinase mutant resistant to arginine feedback inhibition, the synthase becomes feedback resistant concomitantly.

We conclude that the NAGS/NAGK metabolon promotes the co‐ordination of the catalytic activities and feedback regulation of the first two, flux controlling, enzymes of the arginine pathway.
A general pattern for substrate recognition by P‐glycoprotein
Authors: Seelig Anna
Published: March 2003

P‐glycoprotein actively transports a wide variety of chemically diverse compounds out of the cell. Based on a comparison of a hundred compounds previously tested as P‐glycoprotein substrates, we suggest that a set of well‐defined structural elements is required for an interaction with P‐glycoprotein. The recognition elements are formed by two (type I unit) or three electron donor groups (type II unit) with a fixed spatial separation. Type I units consist of two electron donor groups with a spatial separation of 2.5 ± 0.3 Å. Type II units contain either two electron donor groups with a spatial separation of 4.6 ± 0.6 Å or three electron donor groups with a spatial separation of the outer two groups of 4.6 ± 0.6 Å. All molecules that contain at least one type I or one type II unit are predicted to be P‐glycoprotein substrates. The binding to P‐glycoprotein increases with the strength and the number of electron donor or hydrogen bonding acceptor groups forming the type I and type II units. Correspondingly, a high percentage of amino acids with hydrogen bonding donor side chains is found in the transmembrane sequences of P‐glycoprotein relevant for substrate interaction. Molecules that minimally contain one type II unit are predicted to be inducers of P‐glycoprotein over‐expression.
Neutral N‐glycans in adult rat brain tissue
Authors: Chen Yi‐Ju , Wing David R. , Guile Geoffrey R. , Dwek Raymond A. , Harvey David J. , Zamze Susanne
Published: March 2003

Oligosaccharides expressed on cell surface and extracellular matrix glycoconjugates are potentially of crucial importance in determining many cell interactions. The complexity of cellular organisation of the brain and suggested involvement of N‐glycosylation in neural development, make this an ideal system to study the potential role of glycosylation in tissue development, maintenance and function. Neural tissues are known to contain some highly unusual glycan structures but the structures expressed in neural tissue have not as yet been studied systematically. As a first initiative to assess the type of N‐glycosylation occurring in neural tissue, we have characterised all of the major neutral N‐linked oligosaccharides expressed in adult rat using a combination of matrix‐assisted laser‐desorption ionisation mass spectrometry, exoglycosidase sequencing combined with normal‐phase HPLC, and two‐dimensional HPLC mapping. Oligomannosidic glycans, Man(9−5)GlcNAc2, constituted approximately 15 % of the total brain N‐glycan pool. The other neutral N‐glycan components consisted of a series of diantennary structures (6.5 %), (2,6)‐branched triantennary glycans (1 %) and hybrid structures (3 %). Both the complex and hybrid N‐glycans were characterised by the presence of outer‐arm α(1,3)‐fucosylation (forming the Lewisx determinant), α(1,6)‐core fucosylation and a bisecting GlcNAc residue. Some of these are unusual or novel structures not having been reported elsewhere. A large proportion of the diantennary N‐glycans either lacked Gal residues entirely or were unsubstituted on one Man residue of the trimannosyl core, notably the Man α(1,3)‐arm. This isomeric form is indicative of the action of a novel β‐hexosaminidase activity and suggests a modification in the classical biosynthetic pathway for N‐linked oligosaccharides. Furthermore, expression of large amounts of oligomannosidic glycans is not usually associated with tissue glycoproteins and suggests a possible involvement of these structures in neural cell interactions.
Characterization of the microheterogeneities of PIXY321, a genetically engineered granulocyte‐macrophage colony‐stimulating factor/interleukin‐3 fusion protein expressed in yeast
Authors: Balland Alain , Krasts Dace A. , Hoch Kayt L. , Gerhart Mary J. , Stremler Kay E. , Waugh Steve M.
Published: March 2003

PIXY321, a human cytokine analog genetically engineered by the fusion of granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) and interleukin‐3 (IL‐3), was expressed in yeast under the control of the alcohol dehydrogenase 2 (ADH2) promoter and the α‐mating factor expression system. To provide the material necessary for the evaluation of PIXY321 in clinical trials, the production was scaled up to the 1200‐l scale and the PIXY321 molecule isolated by four successive steps of ion‐exchange chromatography. Multiple heterogeneities, due to the presence of different patterns of glycosylation as well as multiple amino acid sequences at both N and C termini, were characterized on the purified molecule using complementary analytical techniques including electrophoresis, liquid chromatography and electrospray mass spectrometry. Four different N‐terminal sequences were identified but simplified to a reproducible ratio of two sequences, the mature form and a form starting at Ala3, by adjustment of the process conditions. Molecules lacking 1−6 residues at the C‐terminus were identified and their relative frequencies quantified. Amino acid modifications, such as three oxidized Met residues at positions 79, 141 and 187 and one deamidated Asn residue at position 176, were detected at low level. Microheterogeneities in glycosylation were characterized on four different sites, one located in the GM‐CSF portion and three in the IL‐3 portion of the molecule. The sites were shown to be differentially occupied and to carry 0−10 mannose residues according to their location in the sequence. Precise measurement of the heterogeneities at the molecular level were used to tune the process conditions and ensure reproducibility of the clinical product between lots.
Mutational analysis of disulfide bonds in the trypsin‐reactive subdomain of a Bowman‐Birk‐type inhibitor of trypsin and chymotrypsin
Authors: Philipp Sabine , Kim Young‐Mi , Dürr Ingolf , Wenzl Gabriele , Vogt Michael , Flecker Peter
Published: March 2003

It is widely believed that protein folding is a hierarchical process proceeding from secondary structure via subdomains and domains towards the complete tertiary structure. Accordingly, protein subdomains should behave as independent folding units. However, this prediction would underestimate the well‐established structural significance of tertiary context and domain interfaces in proteins. The principal objective of this work was to distinguish between autonomous and cooperative refolding of protein subdomains by means of mutational analysis. The double‐headed Bowman‐Birk inhibitor of trypsin and chymotrypsin of known crystal structure was selected for study. The relative orientation of the two subdomains is stabilized by intramolecular and water‐mediated hydrogen bonds and close ion pairs across a polar domain interface. The binary arrangement of a trypsin‐reactive and a chymotrypsin‐reactive subdomain facilitates the distinction of local and global irregularities in the mutants of this protein by means of functional assays.

The functional consequences of five replacements in the S‐S bond framework of the trypsin‐reactive subdomain are analyzed in the present report. The mutants were subjected to refolding experiments in a refolding buffer and on trypsin‐Sepharose as a template with complementary structure leading into a fully active state. The stability of the variants was assessed by means of subsequent equilibration experiments in solution. The mutants may be grouped into the following two classes : the class‐I mutations located within β‐strand A are characterized by a breakdown of the trypsin‐ and the chymotrypsin‐reactive subdomain upon refolding in solution and a complicated behavior in the equilibration experiments; by contrast, the Class‐II mutations (β‐strand B) display rather local perturbations and a reversible return to the initial ratio of the two subdomains. This points to a significance of polar interactions connecting the β‐strand A of the trypsin‐reactive with the chymotrypsin‐reactive subdomain. In conclusion, the polar domain interface appears as a major refolding unit of the Bowman‐Birk inhibitor.
A gene encoding a vicilin‐like protein is specifically expressed in fern spores
Authors: Shutov Andrei D. , Braun Holger , Chesnokov Yuriy V. , Bäumlein Helmut
Published: March 2003

The isolation and characterisation of a cDNA coding for a vicilin‐like protein of the fern Matteuccia struthiopteris is described. The corresponding gene is specifically expressed during late stages of spore development. Extensive sequence comparisons suggest that the fern protein can be considered as a molecular missing link between single‐domain germin/spherulin‐like proteins and two‐domain seed storage globulins of gymnosperms and angiosperms. Further, evidence is provided for the existence of a superfamily of structurally related, functionally different proteins which includes storage globulins of the vicilin and legumin families, a membrane‐associated sucrose‐binding protein of soybean, a Forssman antigen‐binding lectin of velvet bean, the precursor of the vacuolar membrane bound proteins MP27/MP32 of pumpkin, the embryogenesis‐specific protein Gea8 of carrot, the fern‐spore‐specific protein described here as well as the functionally diverse family of germins/germin‐like proteins and the spherulins of myxomycetes. We propose that seed storage globulins of spermatophytes evolved from desiccation‐related single‐domain proteins of prokaryotes via a duplicated two‐domain ancestor that is best represented by the extant fern spore‐specific vicilin‐like protein.
Enzymic confirmation of reactions involved in routes to astaxanthin formation, elucidated using a direct substrate in vitro assay
Authors: Fraser Paul D. , Shimada Hiroshi , Misawa Norihiko
Published: March 2003

An in vitro assay procedure for the carotenoid (β‐ionone ring) 3,3′‐hydroxylase and 4,4′‐oxygenase has been developed that enables efficient conversion of non‐radiolabeled carotenoid substrates added directly into aqueous solution. The following enzymic conversions were demonstrated and apparent kinetic constants (Vmax, Km, and specificity constants) obtained : (a) 3,3′‐hydroxylase (from Agrobacterium aurantiacum and Alcaligenes sp. strain PC‐1) converted phoenicoxanthin (adonirubin) to astaxanthin, 3‐hydroxyechinenone to 4‐ketozeaxanthin (adonixanthin), 3′‐hydroxyechinenone to 4‐ketozeaxanthin, as well as echinenone to 4‐ketozeaxanthin via 3‐ and 3′‐hydroxyechinenone; (b) 4,4′‐Oxygenase (from A. aurantiacum, Alcaligenes sp. strain PC‐1 and Haematococcus pluvialis) converted 4‐ketozeaxanthin to astaxanthin, 3‐hydroxyechinenone to phoenicoxanthin, 3′‐hydroxyechinenone to phoenicoxanthin, and echinenone to canthaxanthin. Determination of substrate specifities allowed assessment of biosynthetic routes to astaxanthin formation and demonstrated that pathways via mono‐hydroxylated and ketolated products are enzymically feasible.
Inter‐α‐trypsin inhibitor proteoglycan family
Authors: Bost Frédéric , Diarra‐Mehrpour Maryam , Martin Jean‐Pierre
Published: March 2003

Extracellular matrix (ECM) is composed of several macromolecules associated in a complex network. This structure allows cells to adhere, migrate and interact. Hyaluronic acid (HA) is a glycosaminoglycan (GAG) and a major representative of ECM. HA‐binding proteins such as CD44, aggrecan, and versican, have been implicated in structuring the ECM by stabilizing large macromolecular aggregates. They also play an important role in tumor metastasis and cell motility.

Recently, further HA‐binding proteins were identified : the inter‐α‐trypsin inhibitor(ITI)‐related proteins. ITI is a glycoprotein composed of three polypeptides : two heavy chains (HC1 and HC2) and one light chain (bikunin). Bikunin confers the protease‐inhibitor function. The heavy chains' function was unknown. Recent studies have shown that HC1 and HC2 are linked in vivo and in vitro to hyaluronic acid. This linkage greatly improves extracellular matrix stability. It also demonstrates that ITI‐related proteins might be considered as HA‐binding proteins (HABP).

The ITI related proteins are composed of four polypeptides (HC1, HC2, HC3 and the bikunin) encoded by four genes H1, H2, H3 and L. Unlike the majority of plasma protein a non‐disulfide covalent linkage exists between heavy chains and bikunin.

This review presents the recent progress concerning the interactions between ITI and ECM showing that ITI‐related proteins are HABP members. We will focus on the heavy chain linkage with HA, which represents the demonstration of covalent binding between proteins and HA.
Selection of a RNA aptamer that binds to human activated protein C and inhibits its protease function
Authors: Gal Sang Wan , Amontov Sergey , Urvil Petri Tapani , Vishnuvardhan Daesety , Nishikawa Fumiko , Kumar Penmetcha K. R. , Nishikawa Satoshi
Published: March 2003

A high‐affinity RNA aptamer to human activated protein C (APC) was selected from a pool of random sequences using in vitro selection. Activated protein C, a trypsin‐like serine protease plays an important role along with thrombin as a regulator in blood clotting cascade. After seven rounds of selection and amplification, a single predominant nucleic acid sequence APC‐167, a 167‐base oligonucleotide with a random sequence core of 120 bases, was obtained. The selected aptamer did not bind to thrombin or factor Xa and thus demonstrated specificity to APC. Furthermore, this aptamer was a non‐competitive inhibitor to the cleavage reaction of a fluorogenic substrate catalyzed by APC. The inhibition constant (Ki) of APC‐167 was 83 nM. The 99‐base oligonucleotide (APC‐99) derived from APC‐167 by deleting both primer binding sites, was also found to inhibit APC strongly (Ki = 137 nM). Two stem‐loop structures and at least one G U wobble base pair in the stem were elucidated as important structural motifs for binding.
Structure and cross‐reactivity of the O‐specific polysaccharide of Proteus penneri strain 26, another neutral Proteus O‐antigen containing 2‐acetamido‐2,6‐dideoxy‐	L‐glucose (N‐acetyl‐	L‐quinovosamine)
Authors: Shashkov Alexander S. , Arbatsky Nikolay P. , Widmalm Göran , Knirel Yuriy A. , Zych Krystyna , Sidorczyk Zygmunt
Published: March 2003

A neutral O‐specific polysaccharide obtained from the lipopolysaccharide of Proteus penneri strain 26 was studied using sugar analysis and 1H and 13C NMR spectroscopy, including two‐dimensional NMR techniques. The following structure of the trisaccharide repeating unit was established : ←6)‐α‐
D‐GlcpNAc‐(1←3)‐α‐
L‐QuipNAc‐(1←3)‐α‐
D‐GlcpNAc‐(1← where
L‐QuiNAc is 2‐acetamido‐2,6‐dideoxy‐
L‐glucose (N‐acetyl‐
L‐quinovosamine). Cross‐reactivity of the Proteus penneri 26 anti‐O serum with other strains of P. penneri isolated in Poland and USA and one strain of P. vulgaris is discussed.