bluuebunny/crossref_metadata_2025 huggingface dataset:
Lipid droplet and milk lipid globule membrane associated placental protein 17b (PP17b) is involved in apoptotic and differentiation processes of human epithelial cervical carcinoma cells
Authors: Than Nandor G. , Sumegi Balazs , Bellyei Szabolcs , Berki Timea , Szekeres Gyorgy , Janaky Tamas , Szigeti Andras , Bohn Hans , Than Gabor N.
Published: December 2004

The intracellular role of placental protein 17b (PP17b)/TIP47 has been controversial, because it is considered to be a protein required for mannose 6‐phosphate receptor transport from endosome to trans‐Golgi as well as a neutral lipid droplet‐associated protein. The similarity between the amino acid sequences of PP17 variants, adipophilin and perilipins, and between their gene structures indicate that PP17b as well as other alternatively spliced PP17 variants belong to the lipid storage droplet protein family, containing also some differentiation factors. Using a specific antibody, PP17b was detected in lipid droplet fractions and co‐localized with neutral lipid droplets stained by Nile red, and fluorescently labelled PP17 antibody in HeLa cells with confocal microscopy. PP17b was also detected in milk, associated to milk lipid globule membranes. Cytostatic agents induced apoptosis and PP17b synthesis in HeLa cells, which was significantly inhibited by protein kinase C (PKC) inhibitor, indicating the involvement of NF‐κB and AP‐1 transcription factors in this process, while protein kinase A (PKA) inhibitor had only a modest inhibitory effect. Cell differentiation induced by dibutyryl cyclic AMP or phorbol myristate acetate also increased PP17b synthesis, demonstrating its strong involvement in cell differentiation. PP17b synthesis was higher in M than in G0/G1 phases in control, apoptotic and differentiated cells. This data shows that PP17b is a neutral lipid droplet‐associated protein, and its expression is regulated by PKC‐ and PKA‐dependent pathways.
A possible physiological function and the tertiary structure of a 4‐kDa peptide in legumes
Authors: Yamazaki Toshimasa , Takaoka Motoko , Katoh Etsuko , Hanada Kazuki , Sakita Masashi , Sakata Kyoko , Nishiuchi Yuji , Hirano Hisashi
Published: December 2004

Previously, we isolated a 4‐kDa peptide capable of binding to a 43‐kDa receptor‐like protein and stimulating protein kinase activity of the 43‐kDa protein in soybean. Both of them were found to localize in the plasma membranes and cell walls. Here, we report the physiological effects of 4‐kDa peptide expressed transiently in the cultured carrot and bird's‐foot trefoil cells transfected with pBI 121 plasmid containing the 4‐kDa peptide gene. At early developmental stage, the transgenic callus grew rapidly compared to the wild callus in both species. Cell proliferation of in vitro cultured nonembryogenic carrot callus was apparently affected with the 4‐kDa peptide in the medium. Complementary DNAs encoding the 4‐kDa peptide from mung bean and azuki bean were cloned by PCR and sequenced. The amino‐acid sequences deduced from the nucleotide sequences are homologous among legume species, particularly, the sites of cysteine residues are highly conserved. This conserved sequence reflects the importance of intradisulfide bonds required for the 4‐kDa peptide to perform its function. Three dimensional structure of the 4‐kDa peptide determined by NMR spectroscopy suggests that this peptide is a T‐knot scaffold containing three β‐strands, and the specific binding activity to the 43‐kDa protein and stimulatory effect on the protein phosphorylation could be attributed to the spatial arrangements of hydrophobic residues at the solvent‐exposed surface of two‐stranded β‐sheet of 4‐kDa peptide. The importance of these residues for the 4‐kDa peptide to bind to the 43‐kDa protein was indicated by site‐directed mutagenesis. These results suggest that the 4‐kDa peptide is a hormone‐like peptide and the 43‐kDa protein is involved in cellular signal transduction of the peptide.
Mechanisms of accumulation of arachidonate in phosphatidylinositol in yellowtail
Authors: Tanaka Tamotsu , Iwawaki Dai , Sakamoto Masahiro , Takai Yoshimichi , Morishige Jun‐ichi , Murakami Kaoru , Satouchi Kiyoshi
Published: December 2004

It is known that phosphatidylinositol (PtdIns) contains abundant arachidonate and is composed mainly of 1‐stearoyl‐2‐arachidonoyl species in mammals. We investigated if this characteristic of PtdIns applies to the PtdIns from yellowtail (Seriola quinqueradiata), a marine fish. In common with phosphatidylcholine (PtdCho), phosphatidylethanolamine (PtdEtn) and phosphatidylserine (PtdSer) from brain, heart, liver, spleen, kidney and ovary, the predominant polyunsaturated fatty acid was docosahexaenoic acid, and levels of arachidonic acid were less than 4.5% (PtdCho), 7.5% (PtdEtn) and 3.0% (PtdSer) in these tissues. In striking contrast, arachidonic acid made up 17.6%, 31.8%, 27.8%, 26.1%, 25.4% and 33.5% of the fatty acid composition of PtdIns from brain, heart, liver, spleen, kidney and ovary, respectively. The most abundant molecular species of PtdIns in all these tissues was 1‐stearoyl‐2‐arachidonoyl. Assay of acyltransferase in liver microsomes of yellowtail showed that arachidonic acid was incorporated into PtdIns more effectively than docosahexaenoic acid and that the latter inhibited incorporation of arachidonic acid into PtdCho without inhibiting the utilization of arachidonic acid for PtdIns. This effect of docosahexaenoic acid was not observed in similar experiments using rat liver microsomes and is thought to contribute to the exclusive utilization of arachidonic acid for acylation to PtdIns in yellowtail. Inositolphospholipids and their hydrolysates are known to act as signaling molecules in cells. The conserved hydrophobic structure of PtdIns (the 1‐stearoyl‐2‐arachidonoyl moiety) may have physiological significance not only in mammals but also in fish.
Mutational analysis of plasminogen activator inhibitor‐1
Authors: Wind Troels , Jensen Jan K. , Dupont Daniel M. , Kulig Paulina , Andreasen Peter A.
Published: December 2004

The serpin plasminogen activator inhibitor‐1 (PAI‐1) is a fast and specific inhibitor of the plasminogen activating serine proteases tissue‐type and urokinase‐type plasminogen activator and, as such, an important regulator in turnover of extracellular matrix and in fibrinolysis. PAI‐1 spontaneously loses its antiproteolytic activity by inserting its reactive centre loop (RCL) as strand 4 in β‐sheet A, thereby converting to the so‐called latent state. We have investigated the importance of the amino acid sequence of α‐helix F (hF) and the connecting loop to s3A (hF/s3A‐loop) for the rate of latency transition. We grafted regions of the hF/s3A‐loop from antithrombin III and α1‐protease inhibitor onto PAI‐1, creating eight variants, and found that one of these reversions towards the serpin consensus decreased the rate of latency transition. We prepared 28 PAI‐1 variants with individual residues in hF and β‐sheet A replaced by an alanine. We found that mutating serpin consensus residues always had functional consequences whereas mutating nonconserved residues only had so in one case. Two variants had low but stable inhibitory activity and a pronounced tendency towards substrate behaviour, suggesting that insertion of the RCL is held back during latency transition as well as during complex formation with target proteases. The data presented identify new determinants of PAI‐1 latency transition and provide general insight into the characteristic loop–sheet interactions in serpins.
H2O2, but not menadione, provokes a decrease in the ATP and an increase in the inosine levels in Saccharomyces cerevisiae
Authors: Osorio Hugo , Carvalho Elisabete , del Valle Mercedes , Günther Sillero María A. , Moradas‐Ferreira Pedro , Sillero Antonio
Published: December 2004

When Saccharomyces cerevisiae cells, grown in galactose, glucose or mannose, were treated with 1.5 mm hydrogen peroxide (H2O2) for 30 min, an important decrease in the ATP, and a less extensive decrease in the GTP, CTP, UTP and ADP‐ribose levels was estimated. Concomitantly a net increase in the inosine levels was observed. Treatment with 83 mm menadione promoted the appearance of a compound similar to adenosine but no appreciable changes in the nucleotide content of yeast cells, grown either in glucose or galactose.

Changes in the specific activities of the enzymes involved in the pathway from ATP to inosine, in yeast extracts from (un)treated cells, could not explain the effect of H2O2 on the levels of ATP and inosine. Application of a mathematical model of differential equations previously developed in this laboratory pointed to a potential inhibition of glycolysis as the main reason for that effect. This theoretical consideration was reinforced both by the lack of an appreciable effect of 1.5 mm (or even higher concentrations) H2O2 on yeast grown in the presence of ethanol or glycerol, and by the observed inhibition of the synthesis of ethanol promoted by H2O2. Normal values for the adenylic charge, ATP and inosine levels were reached at 5, 30 and 120 min, respectively, after removal of H2O2 from the culture medium. The strong decrease in the ATP level upon H2O2 treatment is an important factor to be considered for understanding the response of yeast, and probably other cell types, to oxidative stress.
Deadenylation of interferon‐β mRNA is mediated by both the AU‐rich element in the 3′‐untranslated region and an instability sequence in the coding region
Authors: Pasté Muriel , Huez Georges , Kruys Véronique
Published: December 2004

Viral infection of fibroblastic and endothelial cells leads to the transient synthesis of interferon‐β (IFN‐β). The down‐regulation of IFN‐β synthesis after infection results both from transcriptional repression of the IFN‐β gene and rapid degradation of mRNA. As with many cytokine mRNAs, IFN‐β mRNA contains an AU‐rich element (ARE) in its 3′‐untranslated region (UTR). AREs are known to mediate mRNA deadenylation and destabilization. Depending on the class of ARE, deadenylation was shown to occur through synchronous or asynchronous mechanisms. In this study, we analysed IFN‐β mRNA deadenylation in natural conditions of IFN‐β synthesis, e.g., after viral infection. We show that human IFN‐β mRNA follows an asynchronous deadenylation pathway typical of a mRNA containing a class II ARE. A deletion analysis of the IFN‐β natural transcript demonstrates that poly(A) shortening can be mediated by the ARE but also by a 32 nucleotide‐sequence located in the coding region, that was identified previously as an instability determinant. In fact, these elements are able to act independently as both of them have to be removed to abrogate mRNA deadenylation. Our data also indicate that deadenylation occurs independently of mRNA translation. Moreover, we show that deadenylation of IFN‐β mRNA is not under the control of viral infection as IFN‐β mRNA derived from a constitutively expressed gene cassette is deadenylated in absence of viral infection. Finally, an unidentified nuclear event appears to be a prerequisite for IFN‐β mRNA deadenylation as IFN‐β mRNA introduced directly into the cytoplasm does not undergo deadenylation. In conclusion, our study demonstrates that IFN‐β mRNA poly(A) shortening is under the control of two cis‐acting elements recruiting a deadenylating machinery whose activity is independent of translation and viral infection but might require a nuclear event.
Structure of the polysaccharide chain of the lipopolysaccharide from Flexibacter maritimus
Authors: Vinogradov Evgeny , MacLean Leann L. , Crump Elizabeth M. , Perry Malcolm B. , Kay William W.
Published: December 2004

Flexibacter maritimus, a Gram‐negative bacterium, is a fish pathogen responsible for disease in finfish species and a cause of cutaneous erosion disease in sea‐caged salmonids. For the development of serology based diagnostics, protective vaccines, and a study of pathogenesis, the structural analysis of the lipopolysaccharide (LPS) produced by the bacterium has been undertaken. We now report that an acidic O‐specific polysaccharide, obtained by mild acid degradation of the F. maritimus LPS was found to be composed of a disaccharide repeating unit built of 2‐acetamido‐3‐O‐acetyl‐4‐[(S)‐2‐hydroxyglutar‐5‐ylamido]‐2,4,6‐trideoxy‐β‐glucose and 5‐acetamido‐7‐[(S)‐3‐hydroxybutyramido]‐8‐amino‐3,5,7,8,9‐pentadeoxynonulopyranosonic acid (Sug) having the structure:

The configuration of the C‐2–C‐7 fragment of the latter monosaccharide (B) was assigned β‐manno; however, the configuration at C‐8 could not be established. NMR data indicate that the two monosaccharides have opposite absolute configurations. The repeating unit includes a linkage via a (S)‐2‐hydroxyglutaric acid residue, reported here for the first time as a component of a bacterial polysaccharide. The LPS was also found to contain a minor amount of a disaccharide β‐Sug‐(2‐3)‐l‐Rha, isolated from the products of the acidic methanolysis of the LPS.
Tetranectin binds hepatocyte growth factor and tissue‐type plasminogen activator
Authors: Westergaard Uffe B. , Andersen Mikkel H. , Heegaard Christian W. , Fedosov Sergey N. , Petersen Torben E.
Published: December 2004

In the search for new ligands for the plasminogen kringle 4 binding‐protein tetranectin, it has been found by ligand blot analysis and ELISA that tetranectin specifically bound to the plasminogen‐like hepatocyte growth factor and tissue‐type plasminogen activator. The dissociation constants of these complexes were found to be within the same order of magnitude as the one for the plasminogen‐tetranectin complex. The study also revealed that tetranectin did not interact with the kindred proteins: macrophage‐stimulating protein, urokinase‐type plasminogen activator and prothrombin. In order to examine the function of tetranectin, a kinetic analysis of the tPA‐catalysed plasminogen activation was performed. The kinetic parameters of the tetranectin‐stimulated enhancement of tPA were comparable to fibrinogen fragments, which are so far the best inducer of tPA‐catalysed plasminogen activation. The enhanced activation was suggested to be caused by tetranectin's ability to bind and accumulate tPA in an active conformation.
Cytochrome P460 of Nitrosomonas europaea
Authors: Bergmann David J. , Hooper Alan B.
Published: December 2004

The heme of cytochrome P460 of Nitrosomonas europaea, which is covalently crosslinked to two cysteines of the polypeptide as with all c‐type cytochromes, has an additional novel covalent crosslink to lysine 70 of the polypeptide [Arciero, D.M. & Hooper, A.B. (1997) FEBS Lett.410, 457–460]. The protein can catalyze the oxidation of hydroxylamine. The gene for this protein, cyp, was expressed in Pseudomonas aeruginosa strain PAO lacI, resulting in formation of a holo‐cytochrome P460 which closely resembled native cytochrome P460 purified from N. europaea in its UV‐visible spectroscopic, ligand binding and catalytic properties. Mutant versions of cytochrome P460 of N. europaea in which Lys70 70 was replaced by Arg, Ala, or Tyr, retained ligand‐binding ability but lost catalytic ability and differed in optical spectra which, instead, closely resembled those of cytochromes c′. Tryptic fragments containing the c‐heme joined only by two thioether linkages were observed by MALDI‐TOF for the mutant cytochromes P460 K70R and K70A but not in wild‐type cytochrome P460, consistent with the structural modification of the c‐heme only in the wild‐type cytochrome. The present observations support the hypothesized evolutionary relationship between cytochromes P460 and cytochromes c′ in N. europaea and M. capsulatus[Bergmann, D.J., Zahn, J.A., & DiSpirito, A.A. (2000) Arch. Microbiol. 173, 29–34], confirm the importance of a heme‐crosslink to the spectroscopic properties and catalysis and suggest that the crosslink might form auto‐catalytically.
O‐MACS, a novel member of the medium‐chain acyl‐CoA synthetase family, specifically expressed in the olfactory epithelium in a zone‐specific manner
Authors: Oka Yuichiro , Kobayakawa Ko , Nishizumi Hirofumi , Miyamichi Kazunari , Hirose Satoshi , Tsuboi Akio , Sakano Hitoshi
Published: December 2004

In rodents, the olfactory epithelium (OE) can be divided into four topographically distinct zones, and each member of the odorant receptor (OR) gene family is expressed only in one particular zone. To study the functional significance of the zonal structure of the OE, we searched for genes expressed in a zone‐specific manner by using the differential display method. Among the clones isolated from the rat OE, we characterized a novel olfactory protein termed O‐MACS, a member of the medium‐chain acyl‐CoA synthetase family. The o‐macs gene encodes a protein of 580 amino acids, sharing 56–63% identity with other MACS family proteins. RT‐PCR analysis demonstrated that the o‐macs gene is expressed only in the OE, unlike other MACS family genes. In situ hybridization revealed that the o‐macs transcripts are present in the neuronal cell layer of olfactory sensory neurons (OSNs) as well as in the supporting and basal cell layers in the most dorso‐medial area (zone 1) of the OE. Developmental analysis revealed that the o‐macs gene is already expressed on embryonic day 11.5, before the onset of the OR gene expression, in a restricted area within the rat olfactory placode. Recombinant O‐MACS protein tagged with c‐Myc and His6 demonstrated an acyl‐CoA synthetase activity for fatty acid activation, and protein localization to mitochondria like other MACS family proteins. The present study indicates that this novel protein may play important roles in processing odorants in a zone‐specific manner, or the zonal patterning of the OE during development.
Initiation of JC virus DNA replication in vitro by human and mouse DNA polymerase α‐primase
Authors: Smith Richard W. P. , Nasheuer Heinz‐Peter
Published: December 2004

Host species specificity of the polyomaviruses simian virus 40 (SV40) and mouse polyomavirus (PyV) has been shown to be determined by the host DNA polymerase α‐primase complex involved in the initiation of both viral and host DNA replication. Here we demonstrate that DNA replication of the related human pathogenic polyomavirus JC virus (JCV) can be supported in vitro by DNA polymerase α‐primase of either human or murine origin indicating that the mechanism of its strict species specificity differs from that of SV40 and PyV. Our results indicate that this may be due to differences in the interaction of JCV and SV40 large T antigens with the DNA replication initiation complex.
Antimicrobial peptides from hylid and ranin frogs originated from a 150‐million‐year‐old ancestral precursor with a conserved signal peptide but a hypermutable antimicrobial domain
Authors: Vanhoye Damien , Bruston Francine , Nicolas Pierre , Amiche Mohamed
Published: December 2004

The dermal glands of frogs produce antimicrobial peptides that protect the skin against noxious microorganisms and assist in wound repair. The sequences of these peptides are very dissimilar, both within and between species, so that the 5000 living anuran frogs may produce ≈ 100 000 different antimicrobial peptides. The antimicrobial peptides of South American hylid frogs are derived from precursors, the preprodermaseptins, whose signal peptides and intervening sequences are remarkably conserved, but their C‐terminal domains are markedly diverse, resulting in mature peptides with different lengths, sequences and antimicrobial spectra. We have used the extreme conservation in the preproregion of preprodermaseptin transcripts to identify new members of this family in Australian and South American hylids. All these peptides are cationic, amphipathic and α‐helical. They killed a broad spectrum of microorganisms and acted in synergy. 42 preprodermaseptin gene sequences from 10 species of hylid and ranin frogs were analyzed in the context of their phylogeny and biogeography and of geophysical models for the fragmentation of Gondwana to examine the strategy that these frogs have evolved to generate an enormous array of peptide antibiotics. The hyperdivergence of modern antimicrobial peptides and the number of peptides per species result from repeated duplications of a ≈ 150‐million‐year‐old ancestral gene and accelerated mutations of the mature peptide domain, probably involving a mutagenic, error‐prone, DNA polymerase similar to Escherichia coli Pol V. The presence of antimicrobial peptides with such different structures and spectra of action represents the successful evolution of multidrug defense by providing frogs with maximum protection against infectious microbes and minimizing the chance of microorganisms developing resistance to individual peptides. The hypermutation of the antimicrobial domain by a targeted mutagenic polymerase that can generate many sequence changes in a few steps may have a selective survival value when frogs colonizing a new ecological niche encounter different microbial predators.
Chick embryo anchored alkaline phosphatase and mineralization process in vitro
Authors: Hamade Eva , Azzar Gérard , Radisson Jacqueline , Buchet René , Roux Bernard
Published: December 2004

Bone alkaline phosphatase with glycolipid anchor (GPI‐bALP) from chick embryo femurs in a medium without exogenous inorganic phosphate, but containing calcium and GPI‐bALP substrates, served as in vitro model of mineral formation. The mineralization process was initiated by the formation of inorganic phosphate, arising from the hydrolysis of a substrate by GPI‐bALP. Several mineralization media containing different substrates were analysed after an incubation time ranging from 1.5 h to 144 h. The measurements of Ca/Pi ratio and infrared spectra permitted us to follow the presence of amorphous and noncrystalline structures, while the analysis of X‐ray diffraction data allowed us to obtain the stoichiometry of crystals. The hydrolysis of phosphocreatine, glucose 1‐phosphate, glucose 6‐phosphate, glucose 1,6‐bisphosphate by GPI‐bALP produced hydroxyapatite in a manner similar to that of β‐glycerophosphate. Several distinct steps in the mineral formation were observed. Amorphous calcium phosphate was present at the onset of the mineral formation, then poorly formed hydroxyapatite crystalline structures were observed, followed by the presence of hydroxyapatite crystals after 6–12 h incubation time. However, the hydrolysis of either ATP or ADP, catalysed by GPI‐bALP in calcium‐containing medium, did not lead to the formation of any hydroxyapatite crystals, even after 144 h incubation time, when hydrolysis of both nucleotides was completed. In contrast, the hydrolysis of AMP by GPI‐bALP led to the appearance of hydroxyapatite crystals after 12 h incubation time. The hydroxyapatite formation depends not only on the ability of GPI‐bALP to hydrolyze the organic phosphate but also on the nature of substrates affecting the nucleation process or producing inhibitors of the mineralization.
Expression, purification and characterization of the second Kunitz‐type protease inhibitor domain of the human WFIKKN protein
Authors: Nagy Alinda , Trexler Mária , Patthy László
Published: December 2004

Recently we have described a novel secreted protein (the WFIKKN protein) that consists of multiple types of protease inhibitory modules, including two tandem Kunitz‐type protease inhibitor‐domains. On the basis of its homologies we have suggested that the WFIKKN protein is a multivalent protease inhibitor that may control the action of different proteases. In the present work we have expressed the second Kunitz‐type protease inhibitor domain of the human protein WFIKKN in Escherichia coli, purified it by affinity chromatography on trypsin‐Sepharose and its structure was characterized by CD spectroscopy. The recombinant protein was found to inhibit trypsin (Ki = 9.6 nm), but chymotrypsin, elastase, plasmin, pancreatic kallikrein, lung tryptase, plasma kallikrein, thrombin, urokinase or tissue plasminogen activator were not inhibited by the recombinant protein even at 1 μm concentration. In view of the marked trypsin‐specificity of the inhibitor it is suggested that its physiological target may be trypsin.
Alternagin‐C, a nonRGD‐disintegrin, induces neutrophil migration via integrin signaling
Authors: Mariano‐Oliveira Andréa , Coelho Ana Lúcia J. , Terruggi Cristina H. B. , Selistre‐de‐Araújo Heloísa S. , Barja‐Fidalgo Christina , De Freitas Marta S.
Published: December 2004

Recently, a new protein containing a disintegrin domain, alternagin‐C (Alt‐C), was purified from Bothrops alternatus venom. Unlike other disintegrins, in Alt‐C an ECD amino acid mogif takes the place of the RGD sequence. Most disintegrins contain an RGD/KGD sequence and are very potent inhibitors of platelet aggregation, as well as other cell interactions with the extracellular matrix, including tumor cell metastasis and angiogenesis. The present study investigated the effects of Alt‐C on human neutrophil chemotaxis in vitro and the activation of integrin‐mediated pathways. Alt‐C showed a potent chemotactic effect for human neutrophils when compared to N‐formyl‐methionyl‐leucyl‐phenylalanine peptide (fMLP), a classic chemotactic agent. Moreover, preincubation of neutrophils with Alt‐C significantly inhibited chemotaxis toward fMLP and itself. In addition, a peptide containing an ECD sequence presented a chemotactic activity and significantly inhibited chemotaxis induced by Alt‐C and fMLP. A significant increase of F‐actin content was observed in cells treated with Alt‐C, showing that the chemotactic activity of Alt‐C on neutrophils is driven by actin cytoskeleton dynamic changes. Futhermore, this protein was able to induce an increase of phosphotyrosine content triggering focal adhesion kinase activation and its association with phosphatidylinositol 3‐kinase. Alt‐C was also able to induce a significant increase in extracellular signal‐regulated kinase 2 nuclear translocation. The chemotactic activity of Alt‐C was partially inhibited by LY294002, a specific phosphatidylinositol 3‐kinase inhibitor, and by PD98056, a Map kinase kinase inhibitor. These findings suggest that Alt‐C can trigger human neutrophil chemotaxis modulated by intracellular signals characteristic of integrin‐activated pathways and that these effects could be related to the ECD mogif present in disintegrin‐like domain.
Perturbation of folding and reassociation of lactate dehydrogenase by proline and trimethylamine oxide
Authors: Chilson Oscar P. , Chilson Anne E.
Published: December 2004

Investigations of protein–solute interactions typically show that osmolytes favor native conformations. In this study, the effects of representative compatible and counteracting osmolytes on the reactivation of lactate dehydrogenase from two different conformational states were explored. Contrary to expectations, proline and trimethylamine oxide inhibited both the initial time course and the extent of reactivation of lactate dehydrogenase from bovine heart following denaturation in guanidine hydrochloride, as well as following inactivation at pH 2.3. Reactivation of acid‐dissociated porcine heart lactate dehydrogenase was inhibited by both proline and trimethylamine oxide (2 m). In all instances, trimethylamine oxide was the more effective inhibitor of reactivation. Analysis of the catalytic properties of the reactivating enzyme provided evidence that the molecular species that was enzymatically active during the initial stages of reactivation of acid‐inactivated porcine heart lactate dehydrogenase reflects a non‐native conformation. Proline and trimethylamine oxide stabilize polypeptides through exclusion from the polypeptide backbone; the inhibition of renaturation/reassociation described here is probably due to attenuation of this stabilizing influence through favorable interactions of the osmolytes with sidechains of residues that lie at the interfaces of the monomers and dimers that associate to form the active tetramer. In addition, these osmolytes may stabilize non‐native intermediates in the folding pathway. The high viscosity of solutions containing more than 3 m proline was a major factor in the inhibition of reassociation of acid‐dissociated porcine heart lactate dehydrogenase as well as other viscosity‐dependent transformations that may occur during reactivation following unfolding in guanidine hydrochloride.
Role of conformational flexibility for enzymatic activity in NADH oxidase from Thermus thermophilus
Authors: Žoldák Gabriel , Šut'ák Róbert , Antalík Marián , Sprinzl Mathias , Sedlák Erik
Published: December 2004

NADH oxidase from Thermus thermophilus is a homodimer with an unknown physiological function. As is typical for an enzyme isolated from a thermophile, the catalytic rate, kcat, is low at low temperatures and increases with temperature, achieving an optimum at the physiological temperature of the organism, i.e. at ≈ 70 °C for T. thermophilus. At low temperatures, the kcat of several enzymes from thermophilic and mesophilic organisms can be increased by chaotropic agents. The catalytic rate of NADH oxidase increases in the presence of urea. At concentrations of 1.0–1.3 m urea it reaches 250% of the activity in the absence of urea, at 20 °C. At higher urea concentrations the enzyme activity is inhibited. The urea‐dependent activity changes correlate with changes in the fluorescence intensity of Trp47, which is located in the active site of the enzyme. Both fluorescence and circular dichroism measurements indicate that the activation by chaotropic agents involves local environmental changes accompanied by increased dynamics in the active site of the enzyme. This is not related to the global structure of NADH oxidase. The presence of an aromatic amino acid interacting with the flavin cofactor is common to numerous flavin‐dependent oxidases. A comparison of the crystal structure with the activation thermodynamic parameters, ΔH* and TΔS*, obtained from the temperature dependence of kcat, suggests that Trp47 interacts with a water molecule and the isoalloxazine flavin ring. The present investigation suggests a model that explains the role of the homodimeric structure of NADH oxidase.
DNA sequence specificity of triplex‐binding ligands
Authors: Keppler Melanie D. , James Peter L. , Neidle Stephen , Brown Tom , Fox Keith R.
Published: December 2004

We have examined the ability of naphthylquinoline, a 2,7‐disubstituted anthraquinone and BePI, a benzo[e]pyridoindole derivative, to stabilize parallel DNA triplexes of different base composition. Fluorescence melting studies, with both inter‐ and intramolecular triplexes, show that all three ligands stabilize triplexes that contain blocks of TAT triplets. Naphthylquinoline has no effect on triplexes formed with third strands composed of (TC)n or (CCT)n, but stabilizes triplexes that contain (TTC)n. In contrast, BePI slightly destabilizes the triplexes that are formed at (TC)n (CCT)n and (TTC)n. 2,7‐Anthraquinone stabilizes (TC)n (CCT)n and (TTC)n, although it has the greatest effect on the latter. DNase I footprinting studies confirm that triplexes formed with (CCT)n are stabilized by the 2,7‐disubstituted amidoanthraquinone but not by naphthylquinoline. Both ligands stabilize the triplex formed with (CCTT)n and neither affects the complex with (CT)n. We suggest that BePI and naphthylquinoline can only bind between adjacent TAT triplets, while the anthraquinone has a broader sequence of selectivity. These differences may be attributed to the presence (naphthylquinoline and BePI) or absence (anthraquinone) of a positive charge on the aromatic portion of the ligand, which prevents intercalation adjacent to C+GC triplets. The most stable structures are formed when the stacked rings (bases or ligand) alternate between charged and uncharged species. Triplexes containing alternating C+GC and TAT triplets are not stabilized by ligands as they would interrupt the alternating pattern of charged and uncharged residues.
Social Change Affects Vocal Structure in a Callitrichid Primate (Callithrix kuhlii)
Authors: Rukstalis Michael , Fite Jeffrey E. , French Jeffrey A.
Published: December 2004

Vocal plasticity in the face of changing social context is well‐documented in passerine birds, but the degree to which changes in social environment affect the vocal structure of non‐human primates is a topic which has remained largely unexplored. We assessed whether modification of social environments, in this case the presence of marmosets in neighboring cages, influenced the vocal morphology of phee calls, which possess ‘signature’‐like features, in Wied's black tufted‐ear marmosets (Callithrix kuhlii). Individual phee calls were obtained over a period of 8 wk from 11 animals maintained in rooms with stable social environments. After this baseline phase, seven marmosets were housed for an additional 8 wk in rooms that contained cages with unfamiliar marmosets, while four marmosets maintained the same neighbors as the first phase. Calls were digitized and both frequency and temporal parameters were measured. Multivariate discriminant function analyses (DFA) generated from vocalizations collected in the first phase produced functions that accurately classified calls to the correct individual, showing that calls had significant individual distinctiveness. DFAs generated from vocalizations in the first phase of the study continued to show high classification accuracy for marmosets in a stable social environment, but DFAs from the first phase were significantly less likely to correctly classify vocalizations in marmosets that were housed next to novel conspecifics. These data show that phee calls, which have signature‐like properties in marmosets, can be modified by changes in social context. The results suggest a degree of plasticity in vocal signals that is rare among non‐human primates.
The Causes of Resource Monopolization: Interaction Between Resource Dispersion and Mode of Competition
Authors: Weir Laura K. , Grant James W. A.
Published: December 2004

The degree of monopolization of resources is thought to be higher in groups that compete by interference than by exploitation. However, the monopolization of resources will presumably depend on (1) whether the dispersion of resources is economically defendable, and (2) whether some competitors have the ability to defend these resource distributions and hence capitalize on this potential. We tested for an interaction between the effects of temporal resource dispersion and aggressiveness on the degree of resource monopolization in a foraging system. Two species of fish differing markedly in aggressiveness (high: convict cichlids, Archocentrus nigrofasciatus; low: goldfish, Carrasius auratus) were allowed to compete intra‐specifically in groups of four for food that was either potentially defendable (arrived asynchronously) or not (arrived synchronously). As predicted, the monopolization of food, measured as the coefficient of variation of food eaten within groups, was significantly higher in the defendable than in the undefendable treatment for convict cichlids but not for goldfish. However, the monopolization of food was higher in the non‐aggressive goldfish than in the aggressive convict cichlids. Future studies should quantify and compare the monopolization in species that compete primarily via scramble competition to those that use primarily resource defence.
The bark beetle Pityogenes chalcographus (L.) (Scolytidae) in living trees: reproductive success, tree mortality and interaction with Ips typographus
Authors: Hedgren P. O.
Published: December 2004

Abstract:  Tree‐killing ability of Pityogenes chalcographus was experimentally investigated by baiting living spruce Picea abies with synthetic aggregation pheromone, thereby exposing them to beetle attack. Reproductive success was estimated by rearing beetles from stem sections taken from killed trees. The presence of Ips typographus on several killed trees allowed a comparison of reproductive success in the presence and absence of this species. Furthermore, the possibility that breeding material colonized by P. chalcographus may induce subsequent attacks by I. typographus on nearby trees was also studied. The tree killing ability of P. chalcographus was low as only 8% of the baited trees, attacked by P. chalcographus alone, were killed. Tree death was strongly related to the presence of I. typographus, which is in line with the association with aggressive wood‐living fungi in this species. The reproductive success was low, with on average less than one daughter beetle produced per mother beetle. The success was significantly higher in trees also inhabited by I. typographus. Tree‐parts colonized by P. chalcographus induced almost no attacks by I. typographus on nearby trees.
Significance of flower exploding pollination on the reproduction of the Scotch broom, Cytisus scoparius (Leguminosae)
Authors: Suzuki Nobuhiko
Published: December 2004

The significance of single flower visits by pollinators on the reproductive success of the Scotch broom, Cytisus scoparius, with explosive flowers was investigated. Unexploded flowers (intact flowers) produced no fruit, implying that autonomous selfing and apomixis do not occur and that the explosion of flowers by insects is necessary for the fertilization of flowers. The fruit set in the natural condition was intermediate between that in artificial selfing or exploding and that in artificial outcrossing. Seed set and the ratio of seeds to ovules did not differ significantly between the natural condition and artificial outcrossing. The fruit set by a single flower visit from Apis mellifera was similar to natural conditions. Apis mellifera was the most frequent visitor and the number of flowers tripped open per hour was the highest among all the flower visitors. Approximately 75% of pollen on a flower disappeared in a single flower visit by A. mellifera. The small amount of pollen remaining on already‐exploded flowers appears to be the reason why A. mellifera do not frequently visit already‐exploded flowers. The higher seed set by outcrossing was believed to contribute to the seed set in both natural conditions and single flower visits by A. mellifera, and did not differ between them. Therefore, at least in Japan, A. mellifera appears to be the most important pollinator for C. scoparius, resulting in a high outcrossing rate with each single flower visit.
Beyond the 12 lead: Review of the use of additional leads for the early electrocardiographic diagnosis of acute myocardial infarction
Authors: Carley Simon D
Published: December 2004

Despite known limitations, the standard 12 lead ECG is the principal risk stratification device for patients presenting with chest pain to the ED. However, it has a sensitivity of less than 60% for MI. One reason for this is that the standard placement of chest leads fails to interrogate many areas of the myocardium. Various workers have addressed this problem through the use of additional leads or body surface mapping. Additional leads on the posterior and right thoracic surface have been shown to give additional information, which may be important to the emergency physician. This review demonstrates the need for additional leads in the acute setting and makes recommendations about the utility of using additional leads in the ED.
Ultrasound for primary confirmation of mechanical capture in emergency transcutaneous pacing
Authors: Tam Michael MK
Published: December 2004

Clinical confirmation of mechanical capture in emergency transcutaneous pacing is sometimes difficult because of weak pulse volume or interference from skeletal muscle contractions. This report is of a case of using bedside ultrasound for primary confirmation of emergency transcutaneous pacemaker capture, and for differentiating between causes of persistent hypotension despite successful pacing. The technique of ultrasound‐guided transcutaneous pacing is described.
Topography of cocaine‐induced gene regulation in the rat striatum: relationship to cortical inputs and role of behavioural context
Authors: Willuhn Ingo , Sun Weiwen , Steiner Heinz
Published: December 2004

Psychostimulants alter gene expression in projection neurons of the striatum, and such neuroplasticity is implicated in drug addiction and dependence. Evidence indicates that excitatory inputs from the cortex and thalamus are critical for these molecular changes. In the present study, we determined the topography of cocaine‐induced changes in gene expression in the rat striatum and investigated whether these molecular alterations are associated with particular cortical inputs. Acute induction of c‐fos (by 25 mg/kg of cocaine), and the c‐fos response and dynorphin expression after repeated cocaine treatment (25 mg/kg, 4 days) were assessed as examples for short‐term and longer‐term molecular changes, respectively. In addition, we examined whether these molecular effects were influenced by the behaviour performed during cocaine action (running‐wheel training vs. open field). Our results demonstrate that the overall topography of cocaine‐induced gene regulation in the striatum is remarkably stable. Both acute and longer‐term molecular changes were maximal in caudal dorsal striatal sectors that receive convergent inputs from the medial agranular and the sensorimotor cortex. In contrast, relatively minor or no effects were found in rostral and ventral striatal sectors. However, running‐wheel training under the influence of cocaine enhanced the c‐fos response to a subsequent cocaine challenge selectively in parts of the caudal sensorimotor striatum. These results indicate that cocaine produces molecular adaptations preferentially in cortico‐basal ganglia circuits through the sensorimotor striatum, and that some of these neuronal changes are influenced by the behaviour performed during drug exposure.
Mechanisms underlying the noradrenergic modulation of longitudinal coordination during swimming in Xenopus laevis tadpoles
Authors: Merrywest Simon D. , McDearmid Jonathan R. , Kjaerulff Ole , Kiehn Ole , Sillar Keith T.
Published: December 2004

Noradrenaline (NA) is a potent modulator of locomotion in many vertebrate nervous systems. When Xenopus tadpoles swim, waves of motor neuron activity alternate across the body and propagate along it with a brief rostro–caudal delay (RC‐delay) between segments. We have now investigated the mechanisms underlying the reduction of RC‐delay s by NA. When recording from motor neurons caudal to the twelfth postotic cleft, the mid‐cycle inhibition was weak and sometimes absent, compared to more rostral locations. NA enhanced and even unmasked inhibition in these caudal neurons and enhanced inhibition in rostral neurons, but to a lesser extent. Consequently, the relative increase in the amplitude of the inhibition was greater in caudal neurons, thus reducing the RC‐inhibitory gradient. We next investigated whether NA might affect the electrical properties of neurons, such that enhanced inhibition under NA might promote postinhibitory rebound firing. The synaptic inputs during swimming were simulated using a sustained positive current, superimposed upon which were brief negative currents. When these conditions were held constant NA enhanced the probability of rebound firing – indicating a direct effect on membrane properties – in addition to any indirect effect of enhanced inhibition. We propose that NA preferentially enhances weak caudal inhibition, reducing the inhibitory gradient along the cord. This effect on inhibitory synaptic transmission, comprising parallel pre‐ and postsynaptic components, will preferentially facilitate rebound firing in caudal neurons, advancing their firing relative to more rostral neurons, whilst additionally increasing the networks ability to sustain the longer cycle periods under NA.
Regulation of RGS proteins by chronic morphine in rat locus coeruleus
Authors: Gold Stephen J. , Han Ming‐Hu , Herman Amy E. , Ni Yan G. , Pudiak Cindy M. , Aghajanian George K. , Liu Rong‐Jian , Potts Bryan W. , Mumby Susanne M. , Nestler Eric J.
Published: December 2004

The present study explored a possible role for RGS (regulators of G protein signalling) proteins in the long term actions of morphine in the locus coeruleus (LC), a brainstem region implicated in opiate physical dependence and withdrawal. Morphine influences LC neurons through activation of µ‐opioid receptors, which, being Gi/o‐linked, would be expected to be modulated by RGS proteins. We focused on several RGS subtypes that are known to be expressed in this brain region. Levels of mRNAs encoding RGS2, ‐3, ‐4, ‐5, ‐7, ‐8 and ‐11 are unchanged following chronic morphine, but RGS2 and −4 mRNA levels are increased 2–3‐fold 6 h following precipitation of opiate withdrawal. The increases in RGS2 and −4 mRNA peak after 6 h of withdrawal and return to control levels by 24 h. Immunoblot analysis of RGS4 revealed a striking divergence between mRNA and protein responses in LC: protein levels are elevated twofold following chronic morphine and decrease to control values by 6 h of withdrawal. In contrast, levels of RGS7 and −11 proteins, the only other subtypes for which antibodies are available, were not altered by these treatments. Intracellular application of wild‐type RGS4, but not a GTPase accelerating‐deficient mutant of RGS4, into LC neurons diminished electrophysiological responses to morphine. The observed subtype‐ and time‐specific regulation of RGS4 protein and mRNA, and the diminished morphine‐induced currents in the presence of elevated RGS4 protein levels, indicate that morphine induction of RGS4 could contribute to aspects of opiate tolerance and dependence displayed by LC neurons.
Somatic and visceral afferents to the ‘vasodepressor region’ of the caudal midline medulla in the rat
Authors: Potas Jason R. , Keay Kevin A. , Henderson Luke A. , Bandler Richard
Published: December 2004

Previous research has found that the integrity of a restricted region of the caudal midline medulla (including caudal portions of nucleus raphé obscurus and nucleus raphé pallidus) was critical for vasodepression (hypotension, bradycardia, decreased cardiac contractility) evoked either by haemorrhage or deep pain. In this anatomical tracing study we found that the vasodepressor part of the caudal midline medulla (CMM) receives inputs arising from spinal cord, spinal trigeminal nucleus (SpV) and nucleus of the solitary tract (NTS). Specifically: (i) a spinal–CMM projection arises from neurons of the deep dorsal horn, medial ventral horn and lamina X at all spinal segmental levels, with approximately 60% of the projection originating from the upper cervical spinal cord (C1–C4); (ii) a SpV–CMM projection arises primarily from neurons at the transition between subnucleus caudalis and subnucleus interpolaris; (iii) a NTS–CMM projection arises primarily from neurons in ventrolateral and medial subnuclei. In combination, the specific spinal, SpV and NTS regions which project to the CMM receive the complete range of somatic and visceral afferents known to trigger vasodepression. The role(s) of each specific projection is discussed.
Syntaxin I modulation of presynaptic calcium channel inactivation revealed by botulinum toxin C1
Authors: Stanley Elise F.
Published: December 2004

The chick ciliary ganglion calyx‐type nerve terminal was used to examine voltage‐sensitive inactivation of presynaptic N‐type Ca2+ channels and to test if this inactivation is modulated by the transmitter release‐associated protein syntaxin I. We tested the role of this protein with botulinum toxin C1 (BtC1) which cleaves syntaxin I close to its membrane anchor. The presynaptic Ca2+ current inactivated as two distinct populations with ∼75% inactivating at a depolarized potential, V1/2∼−15 mV, with the remainder inactivating at ∼−75 mV. BtC1 had no detectable effect on the latter component but resulted in a ∼7 mV positive shift in the V1/2 of the −15 mV inactivating component. These results confirm that the bulk of presynaptic N‐type Ca2+ channels are in general resistant to voltage dependent inactivation and provide the first direct evidence that the physiological properties of presynaptic nerve terminal Ca2+ channels are subject to modulation by release site‐associated proteins.