bluuebunny/crossref_metadata_2025 huggingface dataset:
‘Antifreeze’ glycoproteins from polar fish
Authors: Harding Margaret M. , Anderberg Pia I. , Haymet A. D. J.
Published: December 2004

Antifreeze glycoproteins (AFGPs) constitute the major fraction of protein in the blood serum of Antarctic notothenioids and Arctic cod. Each AFGP consists of a varying number of repeating units of (Ala‐Ala‐Thr)n, with minor sequence variations, and the disaccharide β‐d‐galactosyl‐(1→3)‐α‐N‐acetyl‐d‐galactosamine joined as a glycoside to the hydroxyl oxygen of the Thr residues. These compounds allow the fish to survive in subzero ice‐laden polar oceans by kinetically depressing the temperature at which ice grows in a noncolligative manner. In contrast to the more widely studied antifreeze proteins, little is known about the mechanism of ice growth inhibition by AFGPs, and there is no definitive model that explains their properties. This review summarizes the structural and physical properties of AFGPs and advances in the last decade that now provide opportunities for further research in this field.

High field NMR spectroscopy and molecular dynamics studies have shown that AFGPs are largely unstructured in aqueous solution. While standard carbohydrate degradation studies confirm the requirement of some of the sugar hydroxyls for antifreeze activity, the importance of following structural elements has not been established: (a) the number of hydroxyls required, (b) the stereochemistry of the sugar hydroxyls (i.e. the requirement of galactose as the sugar), (c) the acetamido group on the first galactose sugar, (d) the stereochemistry of the β‐glycosidic linkage between the two sugars and the α‐glycosidic linkage to Thr, (e) the requirement of a disaccharide for activity, and (f) the Ala and Thr residues in the polypeptide backbone. The recent successful synthesis of small AFGPs using solution methods and solid‐phase chemistry provides the opportunity to perform key structure‐activity studies that would clarify the important residues and functional groups required for activity.

Genetic studies have shown that the AFGPs present in the two geographically and phylogenetically distinct Antarctic notothenioids and Arctic cod have evolved independently, in a rare example of convergent molecular evolution. The AFGPs exhibit concentration dependent thermal hysteresis with maximum hysteresis (1.2 °C at 40 mg·mL−1) observed with the higher molecular mass glycoproteins. The ability to modify the rate and shape of crystal growth and protect cellular membranes during lipid‐phase transitions have resulted in identification of a number of potential applications of AFGPs as food additives, and in the cryopreservation and hypothermal storage of cells and tissues.
Surface polysaccharide involvement in establishing the rhizobium–legume symbiosis
Authors: Fraysse Nicolas , Couderc François , Poinsot Véréna
Published: December 2004

When the rhizosphere is nitrogen‐starved, legumes and rhizobia (soil bacteria) enter into a symbiosis that enables the fixation of atmospheric dinitrogen. This implies a complex chemical dialogue between partners and drastic changes on both plant roots and bacteria. Several recent works pointed out the importance of rhizobial surface polysaccharides in the establishing of the highly specific symbiosis between symbionts. Exopolysaccharides appear to be essential for the early infection process. Lipopolysaccharides exhibit specific roles in the later stages of the nodulation processes such as the penetration of the infection thread into the cortical cells or the setting up of the nitrogen‐fixing phenotype. More generally, even if active at different steps of the establishing of the symbiosis, all the polysaccharide classes seem to be involved in complex processes of plant defense inhibition that allow plant root invasion. Their chemistry is important for structural recognition as well as for physico‐chemical properties.
Three‐dimensional structures of thermophilic β‐1,4‐xylanases from Chaetomium thermophilum and Nonomuraea flexuosa
Authors: Hakulinen Nina , Turunen Ossi , Jänis Janne , Leisola Matti , Rouvinen Juha
Published: December 2004

The crystal structures of thermophilic xylanases from Chaetomium thermophilum and Nonomuraea flexuosa were determined at 1.75 and 2.1 Å resolution, respectively. Both enzymes have the overall fold typical to family 11 xylanases with two highly twisted β‐sheets forming a large cleft. The comparison of 12 crystal structures of family 11 xylanases from both mesophilic and thermophilic organisms showed that the structures of different xylanases are very similar. The sequence identity differences correlated well with the structural differences. Several minor modifications appeared to be responsible for the increased thermal stability of family 11 xylanases: (a) higher Thr : Ser ratio (b) increased number of charged residues, especially Arg, resulting in enhanced polar interactions, and (c) improved stabilization of secondary structures involved the higher number of residues in the β‐strands and stabilization of the α‐helix region. Some members of family 11 xylanases have a unique strategy to improve their stability, such as a higher number of ion pairs or aromatic residues on protein surface, a more compact structure, a tighter packing, and insertions at some regions resulting in enhanced interactions.
Connection of transport and sensing by UhpC, the sensor for external glucose‐6‐phosphate in Escherichia coli
Authors: Schwöppe Christian , Winkler Herbert H. , Neuhaus H. Ekkehard
Published: December 2004

UhpC is a membrane‐bound sensor protein in Escherichia coli required for recognizing external glucose‐6‐phosphate (Glc6P) and induction of the transport protein UhpT. Recently, it was shown that UhpC is also able to transport Glc6P. In this study we investigated whether these transport and sensing activities are obligatorily coupled in UhpC. We expressed a His‐UhpC protein in a UhpC‐deficient E. coli strain and verified that this construct does not alter the basic biochemical properties of the Glc6P sensor system. The effects of arginine replacements, mutations of the central loop, and introduction of a salt bridge in UhpC on transport and sensing were compared. The exchanges R46C, R266C and R149C moderately affected transport by UhpC but strongly decreased the sensing ability. This suggested that the affinity for Glc6P as a transported substrate is uncoupled in UhpC from its affinity for Glc6P as an inducer. Four of the 11 arginine mutants showed a constitutive phenotype but had near wild‐type transport activity suggesting that Glc6P can be transported by a molecule locked in the inducing conformation. Introduction of an intrahelical salt bridge increased the transport activity of UhpC but abolished sensing. Three conserved residues from the central loop were mutated and although none of these showed transport, one exhibited increased affinity for sensing. Taken together, these data show that transport by UhpC is not required for sensing, that conserved arginine residues are important for sensing and not for transport, and that residues located in the central hydrophilic loop are critical for transport and for sensing.
Assessment of porcine and human 16‐ene‐synthase, a third activity of P450c17, in the formation of an androstenol precursor
Authors: Soucy Penny , Lacoste Lucille , Luu‐The Van
Published: December 2004

Recently, we have shown that the biosynthesis of androstenol, a potential endogenous ligand for the orphan receptors constitutive androstane receptor and pregnane‐X‐receptor, requires the presence of enzymes of the steroidogenic pathway, such as 3β‐hydroxysteroid dehydrogenase, 5α‐reductase and 3α‐hydroxysteroid dehydrogenase. In this report, we examine at the molecular level whether the enzyme 17α‐hydroxylase/17,20‐lyase (P450c17), which possesses dual 17α‐hydroxylase and 17,20‐lyase activities and catalyzes the production of precursors for glucocorticoids and sex steroids, is also able to catalyze the formation of a third class of active steroids, 16‐ene steroids (including androstenol). The role of components of the P450 complex is also assessed. We transfected human embryonic kidney (HEK‐293) cells with various amounts of vectors expressing P450c17, NADPH‐cytochrome P450 reductase, and cytochrome b5. Our results showed that P450c17 possesses a 16‐ene‐synthase activity able to transform pregnenolone into 5,16‐androstadien‐3β‐ol, without the formation of the precursor 17‐hydroxypregnenolone. Cytochrome b5 has a much stronger effect on the 16‐ene‐synthase activity than on the 17α‐hydroxylase/17,20‐lyase activities. On the other hand, P450reductase has a drastic effect on the latter, but a negligible one on 5,16‐androstadien‐3β‐ol synthesis. Our results therefore demonstrate that human P450c17, as other enzymes of the classical steroidogenic pathway, is involved in the biosynthetic pathway leading to the formation of androstenol.
Purification of a plant nucleotide pyrophosphatase as a protein that interferes with nitrate reductase and glutamine synthetase assays
Authors: Moorhead Greg B. G. , Meek Sarah E. M. , Douglas Pauline , Bridges Dave , Smith Catherine S. , Morrice Nick , MacKintosh Carol
Published: December 2004

An activity that inhibited both glutamine synthetase (GS) and nitrate reductase (NR) was highly purified from cauliflower (Brassica oleracea var. botrytis) extracts. The final preparation contained an acyl‐CoA oxidase and a second protein of the plant nucleotide pyrophosphatase family. This preparation hydrolysed NADH, ATP and FAD to generate AMP and was inhibited by fluoride, Cu2+, Zn2+ and Ni2+. The purified fraction had no effect on the activity of NR when reduced methylviologen was used as electron donor instead of NADH; and inhibited the oxidation of NADH by both spinach NR and an Escherichia coli extract in a time‐dependent manner. The apparent inhibition of GS and NR and the ability of ATP and AMP to relieve the inhibition of NR can therefore be explained by hydrolysis of nucleotide substrates by the nucleotide pyrophosphatase. We have no evidence that the nucleotide pyrophosphatase is a specific physiological regulator of NR and GS, but suggest that nucleotide pyrophosphatase activity may underlie some confusion in the literature about the effects of nucleotides and protein factors on NR and GS in vitro.
Preventing nondesired RNA‐primed RNA extension catalyzed by T7 RNA polymerase
Authors: Nacheva Genoveva A. , Berzal‐Herranz Alfredo
Published: December 2004

The transcription patterns of 64 linear double stranded DNA templates obtained with T7 RNA polymerase were investigated. These templates consisted of 17 nucleotide‐long sequences under the control of the minimal bacteriophage T7 promoter and represented all possible combinations of nucleotides at positions +8, +10 and +11. Two clearly distinct types of template were identified, which produced the range of transcription patterns observed: (a) those that yielded 17‐nucleotide‐long RNA as the only detectable run‐off product (only 15% of the total), and (b) templates that in addition to the expected full‐length RNA, produced other products longer than 17 nucleotides. Self‐complementarity analysis of the expected run‐off transcripts showed that those obtained from the first type of template were able to form stable intermolecular duplexes with non‐base‐paired 3′‐ends. However, the second type of template yielded RNAs able to generate energetically favorable intermolecular duplexes with 3′‐end complementarity, therefore yielding an RNA‐primed RNA‐template. The gel‐purified 17‐nucleotide‐long RNAs transcribed from the latter yielded longer products when incubated under in vitro transcription conditions in the absence of a DNA template. No extension was observed when assaying the 17‐nucleotide RNA products resulting from the first type of template. We observed that just a single nucleotide change within the DNA template could convert the RNA product from an RNA‐primed template into a nonextendible dimer thus leading to a drastic switch of the 17‐nucleotide product yield from less than 10% to 100%. Further, two type B DNA templates were extended by two nucleotides at the 3′‐end, to produce RNA transcripts theoretically unable to form 3′‐end base‐paired duplexes. The full‐length products of these modified DNA templates were found to be nonextendible by T7 RNA polymerase under the standard in vitro transcription conditions.
The nucleotide‐binding domains of P‐glycoprotein
Authors: Berridge Georgina , Walker Jennifer A. , Callaghan Richard , Kerr Ian D.
Published: December 2004

The two nucleotide‐binding domains (NBDs) of a number of ATP‐binding cassette (ABC) transporters have been shown to be functionally dissimilar, playing different roles in the transport process. A high degree of co‐operativity has been determined for the NBDs of the human multidrug transporter, P‐glycoprotein. However, the issue of functional symmetry in P‐glycoprotein remains contentious. To address this, the NBDs of P‐glycoprotein were expressed and purified to 95% homogeneity, as fusions to maltose‐binding protein. The NBDs were engineered to contain a single cysteine residue in the Walker‐A homology motif. Reactivity of this cysteine residue was demonstrated by specific, time‐dependent, covalent labelling with N‐ethylmaleimide. No differences in the rates of labelling of the two NBDs were observed. The relative affinity of binding to each NBD was determined for a number of nucleotides by measuring their ability to effect a reduction in N‐ethylmaleimide labelling. In general, nucleotides bound identically to the two NBDs, suggesting that there is little asymmetry in the initial step of the transport cycle, namely the recognition and binding of nucleotide. Any observed functional asymmetry in the intact transporter presumably reflects different rates of hydrolysis at the two NBDs or interdomain communications.
A dimer of the FeS cluster biosynthesis protein IscA from cyanobacteria binds a [2Fe2S] cluster between two protomers and transfers it to [2Fe2S] and [4Fe4S] apo proteins
Authors: Wollenberg Markus , Berndt Carsten , Bill Eckhard , Schwenn Jens D. , Seidler Andreas
Published: December 2004

Two proteins with similarity to IscA are encoded in the genome of the cyanobacterium Synechocystis PCC 6803. One of them, the product of slr1417 which accounts for 0.025% of the total soluble protein of Synechocystis was over‐expressed in E. coli and purified. The purified protein was found to be mainly dimeric and did not contain any cofactor. Incubation with iron ions, cysteine and Synechocystis IscS led to the formation of one [2Fe2S] cluster at an IscA dimer as demonstrated (by the binding of about one iron and one sulfide ion per IscA monomer) by UV/Vis, EPR and Mössbauer spectroscopy. Mössbauer spectroscopy further indicated that the FeS cluster was bound by four cysteine residues. Site‐directed mutagenesis revealed that of the five cysteine residues only C110 and C112 were involved in cluster binding. It was therefore concluded that the [2Fe2S] cluster is located between the two protomers of the IscA dimer and ligated by C110 and C112 of both protomers. The cluster could be transferred to apo ferredoxin, a [2Fe2S] protein, with a half‐time of 10 min. Surprisingly, incubation of cluster‐containing IscA with apo adenosine 5′‐phosphosulfate reductase led to a reactivation of the enzyme which requires the presence of a [4Fe4S] cluster. This demonstrates that it is possible to build [4Fe4S] clusters from [2Fe2S] units.
Characterization of ICAM‐4 binding to the I domains of the CD11a/CD18 and CD11b/CD18 leukocyte integrins
Authors: Ihanus Eveliina , Uotila Liisa , Toivanen Anne , Stefanidakis Michael , Bailly Pascal , Cartron Jean‐Pierre , Gahmberg Carl G
Published: December 2004

Intercellular adhesion molecule‐4 (ICAM‐4, LW blood group antigen), a member of the immunoglobulin superfamily expressed on red cells, has been reported to bind to CD11a/CD18 and CD11b/CD18 leukocyte integrins. The location of the ICAM‐4 binding sites on CD11a/CD18 and CD11b/CD18 are not known. CD11/CD18 integrin I domains have been found to act as major binding sites for physiological ligands and a negatively charged glutamic acid in ICAMs is considered important for binding. ICAM‐4 lacks such a residue, which is replaced by an arginine. However, we demonstrate here that ICAM‐4 in red cells and transfected fibroblasts interacts specifically with the I domains of CD11a/CD18 and CD11b/CD18 integrins. The binding was inhibited by anti‐I domain and anti‐ICAM‐4 antibodies and it was dependent on divalent cations. Interestingly, ICAM‐4 negative red cells were still able to bind to the CD11b/CD18 I domain but the binding of these cells to the CD11a/CD18 I domain was clearly reduced. Using a solid phase assay, we were able to show that isolated I domains directly and specifically bind to purified recombinant ICAM‐4 in a cation dependent manner. Competition experiments indicated that the binding sites in ICAM‐4 for the CD11a and CD11b I domains are different. However, the ICAM‐4 binding region in both I domains seems to overlap with the regions recognized by the ICAM‐1 and ICAM‐2. Thus we have established that the I domains contain an ICAM‐4 binding region in CD11a/CD18 and CD11b/CD18 leukocyte integrins.
Argininosuccinate synthetase from the urea cycle to the citrulline–NO cycle
Authors: Husson Annie , Brasse‐Lagnel Carole , Fairand Alain , Renouf Sylvie , Lavoinne Alain
Published: December 2004

Argininosuccinate synthetase (ASS, EC 6.3.4.5) catalyses the condensation of citrulline and aspartate to form argininosuccinate, the immediate precursor of arginine. First identified in the liver as the limiting enzyme of the urea cycle, ASS is now recognized as a ubiquitous enzyme in mammalian tissues. Indeed, discovery of the citrulline–NO cycle has increased interest in this enzyme that was found to represent a potential limiting step in NO synthesis. Depending on arginine utilization, location and regulation of ASS are quite different. In the liver, where arginine is hydrolyzed to form urea and ornithine, the ASS gene is highly expressed, and hormones and nutrients constitute the major regulating factors: (a) glucocorticoids, glucagon and insulin, particularly, control the expression of this gene both during development and adult life; (b) dietary protein intake stimulates ASS gene expression, with a particular efficiency of specific amino acids like glutamine. In contrast, in NO‐producing cells, where arginine is the direct substrate in the NO synthesis, ASS gene is expressed at a low level and in this way, proinflammatory signals constitute the main factors of regulation of the gene expression. In most cases, regulation of ASS gene expression is exerted at a transcriptional level, but molecular mechanisms are still poorly understood.
Open questions in ferredoxin‐NADP+ reductase catalytic mechanism
Authors: Carrillo Néstor , Ceccarelli Eduardo A.
Published: December 2004

Ferredoxin (flavodoxin)‐NADP(H) reductases (FNR) are ubiquitous flavoenzymes that deliver NADPH or low potential one‐electron donors (ferredoxin, flavodoxin) to redox‐based metabolisms in plastids, mitochondria and bacteria. The plant‐type reductase is also the basic prototype for one of the major families of flavin‐containing electron transferases that display common functional and structural properties. Many aspects of FNR biochemistry have been extensively characterized in recent years using a combination of site‐directed mutagenesis, steady‐state and transient kinetic experiments, spectroscopy and X‐ray crystallography. Despite these considerable advances, various key features in the enzymology of these important reductases remain yet to be explained in molecular terms. This article reviews the current status of these open questions. Measurements of electron transfer rates and binding equilibria indicate that NADP(H) and ferredoxin interactions with FNR result in a reciprocal decrease of affinity, and that this induced‐fit step is a mandatory requisite for catalytic turnover. However, the expected conformational movements are not apparent in the reported atomic structures of these flavoenzymes in the free state or in complex with their substrates. The overall reaction catalysed by FNR is freely reversible, but the pathways leading to NADP+ or ferredoxin reduction proceed through entirely different kinetic mechanisms. Also, the reductases isolated from various sources undergo inactivating denaturation on exposure to NADPH and other electron donors that reduce the FAD prosthetic group, a phenomenon that might have profound consequences for FNR function in vivo. The mechanisms underlying this reductive inhibition are so far unknown. Finally, we provide here a rationale to interpret FNR evolution in terms of catalytic efficiency. Using the formalism of the Albery–Knowles theory, we identified which parameter(s) have to be modified to make these reductases even more proficient under a variety of conditions, natural or artificial. Flavoenzymes with FNR activity catalyse a number of reactions with potential importance for biotechnological processes, so that modification of their catalytic competence is relevant on both scientific and technical grounds.
Interaction of the P‐type cardiotoxin with phospholipid membranes
Authors: Dubovskii Peter V. , Lesovoy Dmitry M. , Dubinnyi Maxim A. , Utkin Yuri N. , Arseniev Alexander S.
Published: December 2004

The cardiotoxin (cytotoxin II, or CTII) isolated from cobra snake (Naja oxiana) venom is a 60‐residue basic membrane‐active protein featuring three‐finger beta sheet fold. To assess possible modes of CTII/membrane interaction 31P‐ and 1H‐NMR spectroscopy was used to study binding of the toxin and its effect onto multilamellar vesicles (MLV) composed of either zwitterionic or anionic phospholipid, dipalmitoylglycerophosphocholine (Pam2Gro‐PCho) or dipalmitoylglycerophosphoglycerol (Pam2Gro‐PGro), respectively. The analysis of 1H‐NMR linewidths of the toxin and 31P‐NMR spectral lineshapes of the phospholipid as a function of temperature, lipid‐to‐protein ratios, and pH values showed that at least three distinct modes of CTII interaction with membranes exist: (a) nonpenetrating mode; in the gel state of the negatively charged MLV the toxin is bound to the surface electrostatically; the binding to Pam2Gro‐PCho membranes was not observed; (b) penetrating mode; hydrophobic interactions develop due to penetration of the toxin into Pam2Gro‐PGro membranes in the liquid‐crystalline state; it is presumed that in this mode CTII is located at the membrane/water interface deepening the side‐chains of hydrophobic residues at the tips of the loops 1–3 down to the boundary between the glycerol and acyl regions of the bilayer; (c) the penetrating mode gives way to isotropic phase, stoichiometrically well‐defined CTII/phospholipid complexes at CTII/lipid ratio exceeding a threshold value which was found to depend at physiological pH values upon ionization of the imidazole ring of His31. Biological implications of the observed modes of the toxin–membrane interactions are discussed.
Photodynamic treatment and H2O2‐induced oxidative stress result in different patterns of cellular protein oxidation
Authors: Sakharov Dmitri V. , Bunschoten Anton , van Weelden Huib , Wirtz Karel W. A.
Published: December 2004

Photodynamic treatment (PDT) is an emerging therapeutic procedure for the management of cancer, based on the use of photosensitizers, compounds that generate highly reactive oxygen species (ROS) on irradiation with visible light. The ROS generated may oxidize a variety of biomolecules within the cell, loaded with a photosensitizer. The high reactivity of these ROS restricts their radius of action to 5–20 nm from the site of their generation. We studied oxidation of intracellular proteins during PDT using the ROS‐sensitive probe acetyl‐tyramine‐fluorescein (acetylTyr‐Fluo). This probe labels cellular proteins, which become oxidized at tyrosine residues under the conditions of oxidative stress in a reaction similar to dityrosine formation. The fluorescein‐labeled proteins can be visualized after gel electrophoresis and subsequent Western blotting using the antibody against fluorescein. We found that PDT of rat or human fibroblasts, loaded with the photosensitizer Hypocrellin A, resulted in labeling of a set of intracellular proteins that was different from that observed on treatment of the cells with H2O2. This difference in labeling patterns was confirmed by 2D electrophoresis, showing that a limited, yet distinctly different, set of proteins is oxidized under either condition of oxidative stress. By matching the Western blot with the silver‐stained protein map, we infer that α‐tubulin and β‐tubulin are targets of PDT‐induced protein oxidation. H2O2 treatment resulted in labeling of endoplasmic reticulum proteins. Under conditions in which the extent of protein oxidation was comparable, PDT caused massive apoptosis, whereas H2O2 treatment had no effect on cell survival. This suggests that the oxidative stress generated by PDT with Hypocrellin A activates apoptotic pathways, which are insensitive to H2O2 treatment. We hypothesize that the pattern of protein oxidation observed with Hypocrellin A reflects the intracellular localization of the photosensitizer. The application of acetylTyr‐Fluo may be useful for characterizing protein targets of oxidation by PDT with various photosensitizers.
The astacin protein family inCaenorhabditis elegans
Authors: Möhrlen Frank , Hutter Harald , Zwilling Robert
Published: December 2004

In the nematodeCaenorhabditis elegans, 40 genes code for astacin‐like proteins (nematode astacins, NAS). The astacins are metalloproteases present in bacteria, invertebrates and vertebrates and serve a variety of physiological functions like digestion, hatching, peptide processing, morphogenesis and pattern formation. With the exception of one distorted pseudogene, all the otherC. elegansastacins are expressed and are evidently functional. For 13 genes we found splicing patterns differing from the Genefinder predictions in WormBase, sometimes markedly. The GFP expression pattern for NAS‐4 shows a specific localization in anterior pharynx cells and in the whole digestive tract (as the secreted form). In contrast, NAS‐7 is found in the head of adult hermaphrodites, but not in pharynx cells or in the lumen of the digestive tract. In embryos, NAS‐7 fluorescence becomes detectable just before hatching. InC. elegansastacins, three basic structural and functional moieties can be discerned: a prepro portion, the central catalytic chain and long C‐terminal extensions with presumably regulatory functions. Within the regulatory moiety, EFG‐like, CUB, SXC, and TSP‐1 domains can be distinguished. Based on structural differences of the regulatory unit we established six NAS subgroups, which seemingly represented different functional and evolutionary clusters. This pattern deduced exclusively from the domain arrangement in the regulatory moiety is perfectly reflected in an evolutionary tree constructed solely from amino acid sequence information of the catalytic chain. Related catalytic chains tend to have related regulatory extensions. The notable gene, NAS‐39 shows a striking resemblance to human BMP‐1 and the tolloids.
De novo RNA synthesis by a recombinant classical swine fever virus RNA‐dependent RNA polymerase
Authors: Yi Guang‐Hui , Zhang Chu‐Yu , Cao Sheng , Wu Hai‐Xiang , Wang Yi
Published: December 2004

Classical swine fever virus nonstructural protein 5B (NS5B) encodes an RNA‐dependent RNA polymerase, a key enzyme of the viral replication complex. To better understand the initiation of viral RNA synthesis and to establish an in vitro replication system, a recombinant NS5B protein, lacking the C‐terminal 24‐amino acid hydrophobic domain, was expressed in Escherichia coli. The truncated fusion protein (NS5BΔ24) was purified on a Ni‐chelating HisTrap affinity column and demonstrated to initiate either plus‐ or minus‐strand viral RNA synthesis de novo in a primer‐independent manner but not by terminal nucleotidyle transferase activity. De novo RNA synthesis represented the preferred mechanism for initiation of classical swine fever virus RNA synthesis by RNA‐dependent RNA polymerase in vitro. Both Mg2+ and Mn2+ supported de novo initiation, however, RNA synthesis was more efficient in the presence of Mn2+ than in the presence of Mg2+. De novo initiation of RNA synthesis was stimulated by preincubation with 0.5 mm GTP, and a 3′‐terminal cytidylate on the viral RNA template was preferred for de novo initiation. Furthermore, the purified protein was also shown, by North‐Western blot analysis, to specifically interact with the 3′‐end of both plus‐ and minus‐strand viral RNA templates.
A 49 kDa microtubule cross‐linking protein from Artemia franciscana is a coenzyme A‐transferase
Authors: Oulton Mindy M. , Amons Reinout , Liang Ping , MacRae Thomas H.
Published: December 2004

Embryos and larvae of the brine shrimp, Artemia franciscana, were shown previously to possess a protein, now termed p49, which cross‐links microtubules in vitro. Molecular characteristics of p49 were described, but the protein's identity and its role in the cell were not determined. Degenerate oligonucleotide primers designed on the basis of peptide sequence obtained by Edman degradation during this study were used to generate p49 cDNAs by RT‐PCR and these were cloned and sequenced. Comparison with archived sequences revealed that the deduced amino acid sequence of p49 resembled the Drosophila gene product CG7920, as well as related proteins encoded in the genomes of Anopheles and Caenorhabditis. Similar proteins exist in several bacteria but no evident homologues were found in vertebrates and plants, and only very distant homologues resided in yeast. When evolutionary relationships were compared, p49 and the homologues from Drosophila, Anopheles and Caenorhabditis formed a distinct subcluster within phylogenetic trees. Additionally, the predicted secondary structures of p49, 4‐hydroxybutyrate CoA‐transferase from Clostridium aminobutyricum and glutaconate CoA‐transferase from Acidaminococcus fermentans were similar and the enzymes may possess related catalytic mechanisms. The purified Artemia protein exhibited 4‐hydroxybutyrate CoA‐transferase activity, thereby establishing p49 as the first crustacean CoA‐transferase to be characterized. Probing of Western blots with an antibody against p49 revealed a cross‐reactive protein in Drosophila that associated with microtubules, but to a lesser extent than did p49 from Artemia.
The Chorus Song of Cooperatively Breeding Laughing Kookaburras (Coraciiformes, Halcyonidae: Dacelo novaeguineae): Characterization and Comparison Among Groups
Authors: Baker Myron C.
Published: December 2004

I studied vocalizations of laughing kookaburras in Western Australia by sampling the laugh‐song choruses of eight different groups and the isolated vocalizations of four individuals of this cooperatively breeding species. These data provided a description of the acoustic structure of vocal elements of the laugh song and a between‐group comparison of laugh choruses. I identified six different categories of syllables: some syllable types appear graded with modal forms predominating. Group choruses were produced by several birds vocalizing simultaneously, usually following initiation by a single bird producing one of two typical introductory sets of syllable repetitions. Statistical analyses of samples of mid‐chorus vocalizations of kookaburra groups revealed that the samples from each of the eight groups clustered in principal coordinate space and the group clusters segregated from each other to a significant degree. Linear discriminant analysis assigned 24 of the 25 samples to their correct groups. These results suggest that there is group‐specific vocal signature information in the laugh chorus. The within‐group similarity and between‐group differences may result from heritable variation or from imitation learning. Observations of the contexts of the laugh chorus vocalization supported the interpretations of others that the chorus song is involved in group advertisement of territory occupancy and in defense of the communal borders.
The Costs of Neighbors for a Territorial Dragonfly, Perithemis tenera
Authors: Eason Perri K. , Switzer Paul V.
Published: December 2004

Past researchers have often considered neighbors to be beneficial to territorial residents, particularly compared with non‐neighbor conspecific competitors. However, neighbors have the potential to be costly to residents in terms of both defensive costs and lost resources. In this study, we assessed the relative costs of defending a mating territory against neighbors and non‐neighbors for the dragonfly Perithemis tenera, comparing across males with different numbers of contiguous neighbors; we also examined the possibility that the presence of contiguous neighbors might reduce the detection of potential mates. When neighbors were present, residents experienced a greater total number of intrusions by males; this increase in intrusions was due to higher numbers of intrusions by neighbors, as the number of intrusions by non‐neighbor males did not differ. Residents with immediately adjacent neighbors also made more sorties toward neighbors than did residents whose nearest neighbors’ territories were not immediately adjacent. Interestingly, although the number of visits by females did not vary with the presence of neighbors, residents with neighbors made fewer sorties toward females than did residents without neighbors. Our results suggest that defensive costs increased when neighbors were present, that residents with neighbors may have missed opportunities to acquire mates, and thus that living with neighbors can be costly in this species.
Assessment of within‐population genetic structure in Quercus crispula and Q. dentata by amplified fragment length polymorphism analysis
Authors: Ishida Takahide A. , Kimura Masahito T.
Published: December 2004

The spatial genetic structure was assessed by amplified fragment length polymorphism (AFLP) technique for Quercus crispula Blume (37 individuals) and Q. dentata Thunberg (54 individuals) growing along a 550‐m transect in Ishikari, Hokkaido, northern Japan. The simple Mantel test revealed significant negative correlation between genetic similarity and geographic distance in Q. dentata and negative but insignificant correlation in Q. crispula. Spatial autocorrelation analysis revealed that Mantel's r generally decreased from positive to negative values with the increase of spatial distance in both oak species with significant deviation from zero for the 50–100‐m (positive) and 250–300‐m classes (negative) in Q. dentata. Thus, significant spatial genetic structure was revealed at least in Q. dentata
Normal levels of immunocompetence in possums (Trichosurus vulpecula) exposed to different laboratory housing conditions post capture
Authors: Begg Douglas , Kemp Roslyn , Griffin Frank
Published: December 2004

Specific and non‐specific immunological tests were used to monitor aspects of the immune response in captive possums. The tests included total and differential white blood cell counts, lymphocyte transformation assay, and enzyme linked immunosorbant assay. The level of free cortisol present in possum plasma samples was evaluated as an endocrine marker for stress. Four different housing conditions were used to test whether stress could be managed or avoided in captive animals. Animals were caged individually or as groups in pens. Bacille Calmette‐Gurein (BCG) and tetanus toxoid immunization was used to evoke primary cell mediated and antibody responses in test animals. The results indicated that there was no significant difference in immunological responses or endocrine parameters in animals held under any of the housing conditions. The results infer that wild possums adapt quickly post‐capture to novel housing conditions and produce representative patterns of immunity when held in housing conditions and fed ad libitum.
Rapid downregulation of antigen processing enzymes in ex vivo generated human monocyte derived dendritic cells occur endogenously in extended cultures
Authors: Ishri RK , Menzies Scott , Hersey Peter , Halliday Gary M
Published: December 2004

Dendritic cells, the most potent antigen presenting cells, have been shown in murine models to induce immune responses against many antigens. Their role in the initiation of antitumour immunity has received enormous attention. Their ability to process and present antigen is dependent on their state of maturation. This study examines the activity of human monocyte‐derived dendritic cells at two different time points and the corresponding changes in the proteolytic enzyme activity. Dendritic cells were produced from peripheral blood mononuclear cells of normal volunteers. Plastic adherent cells were cultured for 5 or 7 days with recombinant human (rh)GM‐CSF and rhIL‐4. Flow cytometry showed that day 5 dendritic cells (DC) were less mature than day 7 DC as indicated by the expression of CD1a, CD11c, CD14, CD80, CD83, CD86 and MHC‐II. Proteolytic activity of the enzymes cathepsin C and cathepsin G and phagocytosis of particulate antigens also showed significant differences between d5 dendritic cells and d7 dendritic cells. Allogeneic costimulatory activity of d7 dendritic cells was also significantly increased. Induction of immunity requires active presentation of antigens by antigen processing cells on their MHC‐I and/or MHC‐II molecules. Study of peptide carriers and peptide precursor molecules showed a significant decrease in CLIP levels in the day 7 DC, suggesting their decreased ability to process antigens but no difference in their ability to load MHC‐II molecules. These findings indicate that the length of time in culture, in the absence of exogenous maturation − inducing stimuli affects dendritic cell maturation. Intracellular enzymatic activities of dendritic cells also changed rapidly with small changes in phenotype.
The usefulness of a modified adult protocol for the clearance of paediatric cervical spine injury in the emergency department
Authors: Browne Gary J , Lam Lawrence T , Barker Ruth A
Published: December 2004

Objective: To determine if the use of a modified adult protocol that uses cervical spine imaging on presentation for the assessment of cervical spine injury in children improves clinical outcome.

Methods: This is a case series study on all consecutive trauma patients presenting from April to July 2000 inclusive to the ED of a major paediatric trauma hospital. Children presenting to the ED with potential cervical spine injury (CSI) were identified using standard selection criteria. Patient demographics, mechanism of injury, method and time of presentation, associated injuries, radiological investigation and clinical outcome were recorded. The major outcome measures for this study were: time to clearance of the cervical spine, length of stay in the ED and admission to an in‐hospital bed. Data were analysed for compliance to the protocol, this being the standard assessment pathway of cervical spine clearance used by our trauma service.

Results: The trauma registry identified 1721 trauma presentations during the 4‐month study period; 208 presentations representing 200 children with potential CSI were entered into the study. Males represented 72.5% of the study population, having a mean age of 8.32 years, although 29% were less than 5 years of age. The majority of presentations (69%) occurred outside of normal working hours. In 17.8% of cases the cervical spine was cleared based on clinical assessment alone, half less than 5 years of age. Compliance to the protocol occurred in 78% of presentations. However, when examined by age group, children 5 years of age or above were 1.5 times more likely to comply with the protocol as compared with younger children. Adequate plain imaging was not obtained in 18% of presentations, this group almost exclusively less than 5 years of age. There were no missed injuries and no short or long‐term neurological sequelae reported during this study. There were no differences in time to clearance, length of stay and admission rate between compliant and non‐compliant groups.

Conclusions: Modified adult protocols for cervical spine clearance offer guidance in managing the majority of children suffering blunt trauma. However, we recommend caution in rigidly applying such protocols, especially to children of young age.
Atypical presentation of intramedullary spinal cord lesion
Authors: H Kwik Michele , Barakate Michael S , Wong Shen , Tan Lisa
Published: December 2004

We report a patient who presented with atypical clinical manifestations including worsening abdominal pain from an intramedullary spinal cord lesion. It is important to consider non‐abdominal causes of abdominal pain for patients with an atypical presentation. The described case demonstrates the challenges facing the physician with the early diagnosis of acute abdominal pain. Spinal cord lesions, although uncommon, remain a potentially disabling and life‐threatening cause of abdominal pain.
BDNF mRNA expression in rat hippocampus and prefrontal cortex: effects of neonatal ventral hippocampal damage and antipsychotic drugs
Authors: Lipska Barbara K. , Khaing Zin Z. , Weickert Cynthia Shannon , Weinberger Daniel R.
Published: December 2004

Brain‐derived neurotrophic factor (BDNF) plays an important role in development, synapse remodelling and responses to stress and injury. Its abnormal expression has been implicated in schizophrenia, a neuropsychiatric disorder in which abnormal neural development of the hippocampus and prefrontal cortex has been postulated. To clarify the effects of antipsychotic drugs used in the therapy of schizophrenia on BDNF mRNA, we studied its expression in rats treated with clozapine and haloperidol and in rats with neonatal lesions of the ventral hippocampus, used as an animal model of schizophrenia. Both antipsychotic drugs reduced BDNF expression in the hippocampus of control rats, but did not significantly lower its expression in the prefrontal cortex. The neonatal hippocampal lesion itself suppressed BDNF mRNA expression in the dentate gyrus and tended to reduce its expression in the prefrontal cortex. These results indicate that, unlike antidepressants, antipsychotics down‐regulate BDNF mRNA, and suggest that their therapeutic properties are not mediated by stimulation of this neurotrophin. To the extent that the lesioned rat models some pathophysiological aspects of schizophrenia, our data suggest that a neurodevelopmental insult might suppress expression of the neurotrophin in certain brain regions.
α2A and α2C‐adrenoceptor regulation in the brain: α2A changes persist after chronic stress
Authors: Flügge G. , Van Kampen M. , Meyer H. , Fuchs E.
Published: December 2004

Stress‐induced activation of the central nervous noradrenergic system has been suspected to induce depressive disorders. As episodes of depression often occur some time after a stress experience we investigated whether stress‐induced changes in the α2‐adrenoceptor (α2‐AR) system persist throughout a post‐stress recovery period. Brains of male tree shrews were analysed after 44 days of chronic psychosocial stress and after a subsequent 10‐day recovery period. Expression of RNA for α2A and α2C‐adrenoceptors was quantified by in situ hybridization, and receptor binding was determined by in vitro receptor autoradiography. Activities of the sympathetic nervous system and of the hypothalamo–pituitary–adrenal axis were increased during chronic stress but normalized during recovery. α2A‐AR RNA in the glutamatergic neurons of the lateral reticular nucleus was elevated significantly after stress and after recovery (by 29% and 17%). In the dorsal motor nucleus of the vagus, subtype A expression was enhanced after recovery (by 33%). In the locus coeruleus, subtype A autoreceptor expression was not changed significantly. Subtype C expression in the caudate nucleus and putamen was elevated by stress (by 5 and 4%, respectively) but normalized during recovery. Quantification of 3H‐RX821002 binding revealed receptor upregulation during stress and/or recovery. Our data therefore show: (i) that chronic psychosocial stress differentially regulates expression of α2‐adrenoceptor subtypes A and C; (ii) that subtype A heteroreceptor expression is persistently upregulated whereas (iii), subtype C upregulation is only transient. The present findings coincide with post mortem studies in depressed patients revealing upregulation of α2A‐ARs.
Delayed peripheral nerve regeneration and central nervous system collateral sprouting in leucocyte common antigen‐related protein tyrosine phosphatase‐deficient mice
Authors: Van der Zee C. E. E. M. , Man T. Y. , Van Lieshout E. M. M. , Van der Heijden I. , Van Bree M. , Hendriks W. J. A. J.
Published: December 2004

Cell adhesion molecule‐like receptor‐type protein tyrosine phosphatases have been shown to be important for neurite outgrowth and neural development in several animal models. We have previously reported that in leucocyte common antigen‐related (LAR) phosphatase deficient (LAR‐ΔP) mice the number and size of basal forebrain cholinergic neurons, and their innervation of the hippocampal area, is reduced. In this study we compared the sprouting response of LAR‐deficient and wildtype neurons in a peripheral and a central nervous system lesion model. Following sciatic nerve crush lesion, LAR‐ΔP mice showed a delayed recovery of sensory, but not of motor, nerve function. In line with this, neurofilament‐200 immunostaining revealed a significant reduction in the number of newly outgrowing nerve sprouts in LAR‐ΔP animals. Morphometric analysis indicated decreased axonal areas in regenerating LAR‐ΔP nerves when compared to wildtypes. Nonlesioned nerves in wildtype and LAR‐ΔP mice did not differ regarding myelin and axon areas. Entorhinal cortex lesion resulted in collateral sprouting of septohippocampal cholinergic fibres into the dentate gyrus outer molecular layer in both genotype groups. However, LAR‐ΔP mice demonstrated less increase in acetylcholinesterase density and fibre number at several time points following the lesion, indicating a delayed collateral sprouting response. Interestingly, a lesion‐induced reduction in number of (septo‐entorhinal) basal forebrain choline acetyltransferase‐positive neurons occurred in both groups, whereas in LAR‐ΔP mice the average cell body size was reduced as well. Thus, regenerative and collateral sprouting is significantly delayed in LAR‐deficient mice, reflecting an important facilitative role for LAR in peripheral and central nervous system axonal outgrowth.
Kinetic properties of human AMPA‐type glutamate receptors expressed in HEK293 cells
Authors: Grosskreutz Julian , Zoerner Antje , Schlesinger Friedrich , Krampfl Klaus , Dengler Reinhard , Bufler Johannes
Published: December 2004

AMPA‐type glutamate receptors (AMPAR) display a high variability in functional properties, which determine the time course of excitatory postsynaptic potentials. They are assembled as tetramers of GluR subunits 1–4 of different splice variants and nuclear edited isoforms. Presently, the kinetics of activation, desensitization and recovery from desensitization of human AMPARs (GluR1, 3 and 4 flip and flop, and GluR2 flip and flop in R and G edited forms, respectively) transiently expressed in HEK293 cells were studied with patch‐clamp techniques and ultra fast agonist application. Activation time constants were identical for all receptors (0.13 ms). The GluR2 flip G variant showed the slowest desensitization (10.8 ms), GluR4 flip the fastest (1.6 ms). Recovery from desensitization varied between 3.1 ms (GluR4 flip) and 178 ms (GluR1 flip). To determine functional interactions between subunits in heteromeric receptors the GluR1 flip and the GluR2 flip R were coexpressed. The time constant of desensitization increased linearly from 2.5 ms (GluR1 flip homomers) to 6.8 ms (GluR2 flip R homomers) with the amount of GluR2 flip R cDNA transfected. Recovery followed a monoexponential time course and had a time constant of 178 ms in GluR1 flip homomeric expression. In all GluR1 flip/GluR2 flip heteromers and in GluR2 flip R homomers desensitization recovered with a time constant of ≈50 ms. Thus, subunit interaction seems likely during recovery but not desensitization. Both parameters might influence the ability of AMPA receptors to mediate glutamate induced chronic excitotoxicity in neurodegenerative diseases.