bluuebunny/crossref_metadata_2025 huggingface dataset:
Biotinylation in the hyperthermophile Aquifex aeolicus
Authors: Clarke David J. , Coulson Joseph , Baillie Ranald , Campopiano Dominic J.
Published: December 2004

Biotin protein ligase (BPL) catalyses the biotinylation of the biotin carboxyl carrier protein (BCCP) subunit of acetyl CoA carboxylase and this post‐translational modification of a single lysine residue is exceptionally specific. The exact details of the protein–protein interactions involved are unclear as a BPL:BCCP complex has not yet been isolated. Moreover, detailed information is lacking on the composition, biosynthesis and role of fatty acids in hyperthermophilic organisms. We have cloned, overexpressed and purified recombinant BPL and the biotinyl domain of BCCP (BCCPΔ67) from the extreme hyperthermophile Aquifex aeolicus. In vitro assays have demonstrated that BPL catalyses biotinylation of lysine 117 on BCCPΔ67 at temperatures of up to 70 °C. Limited proteolysis of BPL with trypsin and chymotrypsin revealed a single protease‐sensitive site located 44 residues from the N‐terminus. This site is adjacent to the predicted substrate‐binding site and proteolysis of BPL is significantly reduced in the presence of MgATP and biotin. Chemical crosslinking with 1‐ethyl‐3‐(dimethylamino‐propyl)‐carbodiimide (EDC) allowed the isolation of a BPL:apo‐BCCPΔ67 complex. Furthermore, this complex was also formed between BPL and a BCCPΔ67 mutant lacking the lysine residue (BCCPΔ67 K117L) however, complex formation was considerably reduced using holo‐BCCPΔ67. These observations provide evidence that addition of the biotin prosthetic group reduces the ability of BCCPΔ67 to heterodimerize with BPL, and emphasizes that a network of interactions between residues on both proteins mediates protein recognition.
The swinging movement of the distal histidine residue and the autoxidation reaction for midge larval hemoglobins
Authors: Kamimura Satoshi , Matsuoka Ariki , Imai Kiyohiro , Shikama Keiji
Published: December 2004

Some insects have a globin exclusively in their fast‐growing larval stage. This is the case in the 4th‐instar larva of Tokunagayusurika akamusi, a common midge found in Japan. In the polymorphic hemoglobin comprised of 11 separable components, hemoglobin VII (Ta‐VII Hb) was of particular interest. When its ferric met‐form was exposed to pH 5.0 from 7.2, the distal histidine was found to swing away from the E7 position. As a result, the iron(III) was converted from a hexacoordinate to a pentacoordinate form by a concomitant loss of the axial water ligand. The corresponding spectral changes in the Soret band were therefore followed by stopped‐flow and rapid‐scan techniques, and the observed first‐order rate constants of kout = 25 s−1 and kin = 128 s−1 were obtained for the outward and inward movements, respectively, of the distal histidine residue in 0.1 m buffer at 25 °C. For O2 affinity, Ta‐VII Hb showed a value of P50 = 1.7 Torr at pH 7.4, accompanied with a remarkable Bohr effect (δH+ = −0.58) almost equal to that of mammalian hemoglobins. We have also investigated the stability property of Ta‐VII HbO2 in terms of the autoxidation rate over a wide range of pH from 4 to 11. The resulting pH‐dependence curve was compared with those of another component Ta‐V HbO2 and sperm whale MbO2, and described based on a nucleophilic displacement mechanism. In light of the O2 binding affinity, Bohr effect and considerable stability of the bound O2 against acidic autoxidation, we conclude that T. akamusi Hb VII can play an important role in O2 transport and storage as the major component in the larval hemolymph.
Oxygen control of nif gene expression in Klebsiella pneumoniae depends on NifL reduction at the cytoplasmic membrane by electrons derived from the reduced quinone pool
Authors: Grabbe Roman , Schmitz Ruth A.
Published: December 2004

In Klebsiella pneumoniae, the flavoprotein, NifL regulates NifA mediated transcriptional activation of the N2‐fixation (nif) genes in response to molecular O2 and ammonium. We investigated the influence of membrane‐bound oxidoreductases on nif‐regulation by biochemical analysis of purified NifL and by monitoring NifA‐mediated expression of nifH′‐′lacZ reporter fusions in different mutant backgrounds. NifL‐bound FAD‐cofactor was reduced by NADH only in the presence of a redox‐mediator or inside‐out vesicles derived from anaerobically grown K. pneumoniae cells, indicating that in vivo NifL is reduced by electrons derived from membrane‐bound oxidoreductases of the anaerobic respiratory chain. This mechanism is further supported by three lines of evidence: First, K. pneumoniae strains carrying null mutations of fdnG or nuoCD showed significantly reduced nif‐induction under derepressing conditions, indicating that NifL inhibition of NifA was not relieved in the absence of formate dehydrogenase‐N or NADH:ubiquinone oxidoreductase. The same effect was observed in a heterologous Escherichia coli system carrying a ndh null allele (coding for NADH dehydrogenaseII). Second, studying nif‐induction in K. pneumoniae revealed that during anaerobic growth in glycerol, under nitrogen‐limitation, the presence of the terminal electron acceptor nitrate resulted in a significant decrease of nif‐induction. The final line of evidence is that reduced quinone derivatives, dimethylnaphthoquinol and menadiol, are able to transfer electrons to the FAD‐moiety of purified NifL. On the basis of these data, we postulate that under anaerobic and nitrogen‐limited conditions, NifL inhibition of NifA activity is relieved by reduction of the FAD‐cofactor by electrons derived from the reduced quinone pool, generated by anaerobic respiration, that favours membrane association of NifL. We further hypothesize that the quinol/quinone ratio is important for providing the signal to NifL.
Mapping of the epitope of a monoclonal antibody protecting plasminogen activator inhibitor‐1 against inactivating agents
Authors: Bødker Julie S. , Wind Troels , Jensen Jan K. , Hansen Martin , Pedersen Katrine E. , Andreasen Peter A.
Published: December 2004

Plasminogen activator inhibitor‐1 (PAI‐1) belongs to the serpin family of serine proteinase inhibitors. Serpins inhibit their target proteinases by an ester bond being formed between the active site serine of the proteinase and the P1 residue of the reactive centre loop (RCL) of the serpin, followed by insertion of the RCL into β‐sheet A of the serpin. Concomitantly, there are conformational changes in the flexible joint region lateral to β‐sheet A. We have now, by site‐directed mutagenesis, mapped the epitope for a monoclonal antibody, which protects the inhibitory activity of PAI‐1 against inactivation by a variety of agents acting on β‐sheet A and the flexible joint region. Curiously, the epitope is localized in α‐helix C and the loop connecting α‐helix I and β‐strand 5A, on the side of PAI‐1 opposite to β‐sheet A and distantly from the flexible joint region. By a combination of site‐directed mutagenesis and antibody protection against an inactivating organochemical ligand, we were able to identify a residue involved in conferring the antibody‐induced conformational change from the epitope to the rest of the molecule. We have thus provided evidence for communication between secondary structural elements not previously known to interact in serpins.
Optimization of P1–P3 groups in symmetric and asymmetric HIV‐1 protease inhibitors
Authors: Andersson Hans O. , Fridborg Kerstin , Löwgren Seved , Alterman Mathias , Mühlman Anna , Björsne Magnus , Garg Neeraj , Kvarnström Ingmar , Schaal Wesley , Classon Björn , Karlén Anders , Danielsson U. Helena , Ahlsén Göran , Nillroth Ullrika , Vrang Lotta , Öberg Bo , Samuelsson Bertil , Hallberg Anders , Unge Torsten
Published: December 2004

HIV‐1 protease is an important target for treatment of AIDS, and efficient drugs have been developed. However, the resistance and negative side effects of the current drugs has necessitated the development of new compounds with different binding patterns. In this study, nine C‐terminally duplicated HIV‐1 protease inhibitors were cocrystallised with the enzyme, the crystal structures analysed at 1.8–2.3 Å resolution, and the inhibitory activity of the compounds characterized in order to evaluate the effects of the individual modifications. These compounds comprise two central hydroxy groups that mimic the geminal hydroxy groups of a cleavage‐reaction intermediate. One of the hydroxy groups is located between the δ‐oxygen atoms of the two catalytic aspartic acid residues, and the other in the gauche position relative to the first. The asymmetric binding of the two central inhibitory hydroxyls induced a small deviation from exact C2 symmetry in the whole enzyme–inhibitor complex. The study shows that the protease molecule could accommodate its structure to different sizes of the P2/P2′ groups. The structural alterations were, however, relatively conservative and limited. The binding capacity of the S3/S3′ sites was exploited by elongation of the compounds with groups in the P3/P3′ positions or by extension of the P1/P1′ groups. Furthermore, water molecules were shown to be important binding links between the protease and the inhibitors. This study produced a number of inhibitors with Ki values in the 100 picomolar range.
Identification of a novel inner‐core oligosaccharide structure in Neisseria meningitidis lipopolysaccharide
Authors: Cox Andrew D. , Wright J. Claire , Gidney Margaret A. J. , Lacelle Suzanne , Plested Joyce S. , Martin Adele , Moxon E. Richard , Richards James C.
Published: December 2004

The structure of the lipopolysaccharide (LPS) from three Neisseria meningitidis strains was elucidated. These strains were nonreactive with mAbs that recognize common inner‐core epitopes from meningococcal LPS. It is well established that the inner core of meningococcal LPS consists of a diheptosyl‐N‐acetylglucosamine unit, in which the distal heptose unit (Hep II) can carry PEtn at the 3 or 6 position or not at all, and the proximal heptose residue (Hep I) is substituted at the 4 position by a glucose residue. Additional substitution at the 3 position of Hep II with a glucose residue is also a common structural feature in some strains. The structures of the O‐deacylated LPSs and core oligosaccharides of the three chosen strains were deduced by a combination of monosaccharide analysis, NMR spectroscopy and MS. These analyses revealed the presence of a structure not previously identified in meningococcal LPS, in which an additional β‐configured glucose residue was found to substitute Hep I at the 2 position. This provided the structural basis for the nonreactivity of LPS with these mAbs. The determination of this novel structural feature identified a further degree of variability within the inner‐core oligosaccharide of meningococcal LPS which may contribute to the interaction of meningococcal strains with their host.
Reaction of human UMP‐CMP kinase with natural and analog substrates
Authors: Pasti Claudia , Gallois‐Montbrun Sarah , Munier‐Lehmann Hélène , Veron Michel , Gilles Anne‐Marie , Deville‐Bonne Dominique
Published: December 2004

UMP‐CMP kinase catalyses an important step in the phosphorylation of UTP, CTP and dCTP. It is also involved in the necessary phosphorylation by cellular kinases of nucleoside analogs used in antiviral therapies. The reactivity of human UMP‐CMP kinase towards natural substrates and nucleotide analogs was reexamined. The expression of the recombinant enzyme and conditions for stability of the enzyme were improved. Substrate inhibition was observed for UMP and CMP at concentrations higher than 0.2 mm, but not for dCMP. The antiviral analog l‐3TCMP was found to be an efficient substrate phosphorylated into l‐3TCDP by human UMP‐CMP kinase. However, in the reverse reaction, the enzyme did not catalyse the addition of the third phosphate to l‐3TCDP, which was rather an inhibitor. By molecular modelling, l‐3TCMP was built in the active site of the enzyme from Dictyostelium. Human UMP‐CMP kinase has a relaxed enantiospecificity for the nucleoside monophosphate acceptor site, but it is restricted to d‐nucleotides at the donor site.
Regulated interaction of endothelin B receptor with caveolin‐1
Authors: Yamaguchi Tomohiro , Murata Yasunobu , Fujiyoshi Yoshinori , Doi Tomoko
Published: December 2004

The peptide hormone endothelin transmits various signals through G protein‐coupled receptors, the endothelin type A (ETAR) and B (ETBR) receptors. Caveolae are specialized lipid rafts containing polymerized caveolins. We examined the interaction of ETBR with caveolin‐1, expressed in Sf9, COS‐1, and HEK293 cells, and its effects on the subcellular distribution and the signal transduction of ETBR. ETBR formed a complex with caveolin‐1 in cells in which these two proteins were coexpressed and in the mixture after purification and reconstitution (as examined by immunoprecipitation) suggesting the direct binding of ETBR with caveolin‐1. The complex formed efficiently only when the ETBR was ligand‐free or bound to an antagonist, RES‐701‐1, whereas the addition of ET‐1 or another antagonist, BQ788, dissociated the complex, suggesting the structural recognition of ETBR by caveolin‐1. In contrast, the ETAR bound to caveolin‐1 regardless of ligand binding. Caveolin‐1 utilized its scaffolding domain (residues 82–101) and the C‐terminal domain (residues 136–178) to bind to ETBR, as for other signalling molecules. Furthermore, the amount of ETBR localized in caveolae increased significantly with the expression of caveolin‐1 and decreased with the addition of ET‐1. The disruption of caveolae by filipin reduced the ET‐1‐derived phosphorylation of ERK1/2. These results suggest the possibility that the binding to caveolin‐1 retains the ligand‐free ETBR in caveolae and regulates the ET signal.
Sp1 and Sp3 are involved in up‐regulation of human deoxyribonuclease II transcription during differentiation of HL‐60 cells
Authors: Chou San‐Fang , Chen Hui‐Ling , Lu Shao‐Chun
Published: December 2004

Expression of DNase II in macrophages is potentially crucially important in the removal of unwanted DNA. We have previously shown that DNase II expression is up‐regulated at the transcriptional level during the phorbol 12‐myristate‐13‐acetate (PMA)‐induced differentiation of HL‐60 and THP‐1 cells. In this study, we investigated the cis‐regulatory elements and transcription factors involved in this process in HL‐60 cells. cis‐Regulatory elements in the DNase II promoter were located by 5′ deletion and site‐directed mutagenesis of promoter‐luciferase constructs and transient transfection of HL‐60 cells. Furthermore, the binding proteins were identified by electrophoretic mobility shift assay (EMSA) in the presence of specific antibodies. In the DNase II promoter, 249 base pairs upstream of the transcription start site were essential for maximal promoter activity in both untreated and PMA‐treated HL‐60 cells and, within this region, three Sp1 and Sp3 binding sites were identified as essential for transcriptional regulation and PMA induction. Western blot analysis showed that PMA treatment resulted in increased levels of Sp1 and Sp3 proteins. Furthermore, cotransfection analysis in Drosophila SL2 cells showed that Sp1 was more potent than Sp3 in activating the DNase II promoter. We therefore conclude that Sp1 and/or Sp3 are involved in the up‐regulation of DNase II expression during the differentiation of HL‐60 cells.
Complex II from phototrophic purple bacterium Rhodoferax fermentans displays rhodoquinol‐fumarate reductase activity
Authors: Miyadera Hiroko , Hiraishi Akira , Miyoshi Hideto , Sakamoto Kimitoshi , Mineki Reiko , Murayama Kimie , Nagashima Kenji V. P. , Matsuura Katsumi , Kojima Somei , Kita Kiyoshi
Published: December 2004

It has long been accepted that bacterial quinol‐fumarate reductase (QFR) generally uses a low‐redox‐potential naphthoquinone, menaquinone (MK), as the electron donor, whereas mitochondrial QFR from facultative and anaerobic eukaryotes uses a low‐redox‐potential benzoquinone, rhodoquinone (RQ), as the substrate. In the present study, we purified novel complex II from the RQ‐containing phototrophic purple bacterium, Rhodoferax fermentans that exhibited high rhodoquinol‐fumarate reductase activity in addition to succinate‐ubiquinone reductase activity. SDS/PAGE indicated that the purified R. fermentans complex II comprises four subunits of 64.0, 28.6, 18.7 and 17.5 kDa and contains 1.3 nmol heme per mg protein. Phylogenetic analysis and comparison of the deduced amino acid sequences of R. fermentans complex II with pro/eukaryotic complex II indicate that the structure and the evolutional origins of R. fermentans complex II are closer to bacterial SQR than to mitochondrial rhodoquinol‐fumarate reductase. The results strongly indicate that R. fermentans complex II and mitochondrial QFR might have evolved independently, although they both utilize RQ for fumarate reduction.
The GxxxG motif of the transmembrane domain of subunit e is involved in the dimerization/oligomerization of the yeast ATP synthase complex in the mitochondrial membrane
Authors: Arselin Geneviève , Giraud Marie‐France , Dautant Alain , Vaillier Jacques , Brèthes Daniel , Coulary‐Salin Bénédicte , Schaeffer Jacques , Velours Jean
Published: December 2004

A conserved putative dimerization GxxxG motif located in the unique membrane‐spanning segment of the ATP synthase subunit e was altered in yeast both by insertion of an alanine residue and by replacement of glycine by leucine residues. These alterations led to the loss of subunit g and the loss of dimeric and oligomeric forms of the yeast ATP synthase. Furthermore, as in null mutants devoid of either subunit e or subunit g, mitochondria displayed anomalous morphologies with onion‐like structures. By taking advantage of the presence of the endogenous cysteine 28 residue in the wild‐type subunit e, disulfide bond formation between subunits e in intact mitochondria was found to increase the stability of an oligomeric structure of the ATP synthase in digitonin extracts. The data show the involvement of the dimerization motif of subunit e in the formation of supramolecular structures of mitochondrial ATP synthases and are in favour of the existence in the inner mitochondrial membrane of associations of ATP synthases whose masses are higher than those of ATP synthase dimers.
Dynamic reorganization of the motor domain of myosin subfragment 1 in different nucleotide states
Authors: Bódis Emőke , Szarka Krisztina , Nyitrai Miklós , Somogyi Béla
Published: December 2004

Atomic models of the myosin motor domain with different bound nucleotides have revealed the open and closed conformations of the switch 2 element [Geeves, M.A. & Holmes, K.C. (1999) Annu. Rev. Biochem.68, 687–728]. The two conformations are in dynamic equilibrium, which is controlled by the bound nucleotide. In the present work we attempted to characterize the flexibility of the motor domain in the open and closed conformations in rabbit skeletal myosin subfragment 1. Three residues (Ser181, Lys553 and Cys707) were labelled with fluorophores and the probes idengified three fluorescence resonance energy transfer pairs. The effect of ADP, ADP.BeFx, ADP.AlF–4 and ADP.Vi on the conformation of the motor domain was shown by applying temperature‐dependent fluorescence resonance energy transfer methods. The 50 kDa lower domain was found to maintain substantial rigidity in both the open and closed conformations to provide the structural basis of the interaction of myosin with actin. The flexibility of the 50 kDa upper domain was high in the open conformation and further increased in the closed conformation. The converter region of subfragment 1 became more rigid during the open‐to‐closed transition, the conformational change of which can provide the mechanical basis of the energy transduction from the nucleotide‐binding pocket to the light‐chain‐binding domain.
Play Behavior in Juvenile Golden Lion Tamarins (Callitrichidae: Primates): Organization in Relation to Costs
Authors: De Oliveira Cláudia R. , Ruiz‐Miranda Carlos R. , Kleiman Devra G. , Beck Benjamin B.
Published: December 2004

Play is a complex behavior that is widespread among vertebrates. Despite the potential benefits for the development of social, cognitive and motor skills, play behavior has costs: energy expenditure, and the risks of injury and predation. As the fitness benefits of play are presumably to be gained as adults, we expect young animals to minimize its immediate costs. Here, we describe play behavior in golden lion tamarins (GLTs) (Leontopithecus rosalia) and test cost‐minimizing hypotheses. We collected data on play behavior and adult vigilance in nine groups (four wild and five supplemented, the latter deriving from a reintroduction) of wild‐living lion tamarins (16 infants) in Brazil. Results showed that play occupied 3.8% of the activity budget, occurred during the hottest times of the day, and was characterized by being mainly social, involving partners of different ages, and sometimes different species. We found no differences in playing time between wild and supplemented groups. The lion tamarins played less in ‘dangerous’ substrates (canopy branches and forest floor), and more in ‘safe’ substrates (large branches and vine tangles), and favored playing in the center of the group over the periphery. The reproductive animals were vigilant during 78% of the time the infants were playing, and were mostly oriented away from the playing animals. The observed organization of play behavior suggests that juvenile GLTs minimize the risk of predation and accidental injuries, but do not seem to minimize energy expenditure and the risks of social injuries. The social‐cognitive benefits acquired from experience of older partners during play may overcome the costs of the latter behavior, and because little time was spent in play, the energetic costs may be inconsequential.
The Grooming Invitation Dance of the Honey Bee
Authors: Land Benjamin B. , Seeley Thomas D.
Published: December 2004

The grooming invitation dance is a striking behavior in honey bee colonies that has not been extensively studied. The objectives of this study were (1) to describe the dance through video analysis, (2) to test the functional hypothesis that it is a grooming solicitation signal, and (3) to analyze the stimuli that cause its production. A worker bee producing the grooming invitation dance stands stationary and vibrates her whole body from side‐to‐side at a frequency of 4.2 ± 0.2 Hz for 9.3 ± 1.0 s. Sometimes the bee mixes bouts of body vibration with brief bouts of self‐grooming (average duration = 1.4 s). Bees that perform the grooming invitation dance have a far higher probability of being quickly groomed by a nest mate than do bees that do not perform the dance. Bees that had chalk dust puffed onto the bases of their wings produced significantly more grooming invitation dances than did control bees that received only puffs of air. This shows that it may be the accumulation of small particles at the bases of the wings that normally triggers the dance. We suggest that the evolutionary origin of this signal is self‐grooming behavior.
Genetic diversity measures of the bovine Alberes breed using microsatellites: variability among herds and types of coat colour*
Authors: Casellas J. , Jiménez N. , Fina M. , Tarrés J. , Sánchez A. , Piedrafita J.
Published: December 2004

The Alberes population is a native bovine breed of Catalonia with an unclear origin, which historically some authors have assumed as being composed of two different colour varieties (black and fawn). Sixteen microsatellite loci were analysed, all of them included in the AIRE2066 European Concerted Action list. Overall expected and observed heterozygosities reached values of 0.649 and 0.662, respectively. Genetic differences among black and fawn varieties were not significant (FST = 0.007), and therefore the population is a single variety with a great colour gradation. On the contrary, we detected significant genetic differences among herds (FST = 0.026; p < 0.001), showing a genetic heterogeneity over short geographical distances. The number of migrants per generation among pairs of herds oscillates between 1.46 (Roig and Freixe herds) and 5.62 (Castanyers and Roig herds). Moreover, inbreeding and bottleneck situations can be rejected. The Alberes breed has been grouped within the Cantabrian trunk, closely related to the Asturiana de la Montaña and Alistana breeds, although some other breeds may also have influenced the population along its history.
Troop size, home range area and seasonal range use of the Japanese macaque in the Northern Japan Alps
Authors: Izumiyama Shigeyuki , Mochizuki Takashi , Shiraishi Toshiaki
Published: December 2004

Home range area and habitat use of the wild Japanese macaque (Macaca fuscata) were investigated between elevations of 600 m and 3000 m in the Northern Japan Alps, in areas where there is great variation in vegetation type. A total of 22 troops were located either visually or by radiotracking from December 1996 to February 2000. The average troop size was 48.4 individuals, the average ranging area (65% probability distribution calculated using the adaptive kernel method) was 3.7 km2 and the average elevation of each troop varied from 740 m to 1458 m. Two troop types were distinguished according to their habitat use: (i) ‘rural’ type groups frequently utilized cultivated land (n = 12); and (ii) ‘natural’ type groups never utilized cultivated land (n = 10). Natural groups inhabited higher elevation areas and showed a greater dependency on deciduous broad‐leaved forests during all seasons. In contrast, rural groups utilized the larch plantation more in spring, and the red pine forest in summer. Rural groups were observed on cultivated lands more often from summer to winter than in spring. The size of the rural groups was twice as large as that of the natural groups. Troop size and home range area showed significant correlation within each group type. The per capita home range area of rural groups was smaller than that of natural groups. These results suggest that per capita home range area is negatively correlated with habitat quality.
Five‐year study on the autumn food habits of the Asiatic black bear in relation to nut production
Authors: Hashimoto Yukihiko , Kaji Mikio , Sawada Haruo , Takatsuki Seiki
Published: December 2004

We studied the autumn food habits of the Asiatic black bear (Ursus thibetanus) from 1993 to 1997, based on 202 fecal samples in the Chichibu Mountains, central Japan. Nuts occupied the highest proportions of autumn foods (59.9–85.8% important values). Although the proportion of nuts of Quercus crispula, Fagus crenata and Fagus japonica varied greatly between the years, acorns of Q. crispula were most prevalent in four of the five years. We also determined the relative nut production of these three species by counting the number of nuts or cupules on the ground. Black bears consumed the nuts according to their relative availabilities. Nuts of Q. crispula appeared to be the most important food because: (i) these nuts were eaten in the highest proportion in four of the five study years; and (ii) even in poor years the bears consumed acorns of Q. crispula, whereas nuts of Fagus spp. were not consumed. We discuss the significance of alternative foods for black bears in relation to food tree diversity in the forest and the necessity for long‐term studies examining the food habits of Asiatic black bears.
Effects of climate on the radial growth of tree species in the upper and lower distribution limits of an altitudinal ecotone on Mount Norikura, central Japan
Authors: Takahashi Koichi , Azuma Hiroto , Yasue Koh
Published: December 2004

Tree‐ring width chronologies were developed for Abies veitchii, Betula ermanii and Betula platyphylla var. japonica in their altitudinal ecotone (approximately 1600 m a.s.l.) on Mount Norikura, central Japan, to determine what climatic conditions affect the growth of tree species in the upper and lower distribution limits of an altitudinal ecotone. This altitude was the lower distribution limit for A. veitchii and B. ermanii in the subalpine zone, and was the upper distribution limit for B. platyphylla var. japonica in the montane zone on Mount Norikura. Tree‐ring widths of the two Betula species and A. veitchii were positively correlated with the August precipitation of the current and previous years, respectively. Precipitation in August (the hottest month) was reduced compared with other months during summer. Tree‐ring width of B. platyphylla var. japonica showed no correlation with temperatures in any month in its upper distribution limit. In contrast, tree‐ring widths of B. ermanii and A. veitchii were negatively correlated with the August temperatures of the current and previous years, respectively, at the lower distribution limit of these species. Therefore, the two Betula species and A. veitchii responded to climatic conditions of the current and previous years, respectively. The present study also suggests that a water deficit in August reduces growth of these three species in this altitudinal ecotone, irrespective of the upper or lower distribution limits, and that a high August temperature is more detrimental to the growth of A. veitchii and B. ermanii in their lower distribution limits. Thus, the three species with different altitudinal distributions examined in the present study responded differently to climatic conditions in this altitudinal ecotone on Mount Norikura.
Forest structure and tree species distribution in relation to topography‐mediated heterogeneity of soil nitrogen and light at the forest floor
Authors: Tateno Ryunosuke , Takeda Hiroshi
Published: December 2004

We investigated the patterns of soil nitrogen (N) and forest floor light availability, forest structure and tree species distribution along a topographic gradient on a 200‐m mountain slope in a cool‐temperate deciduous broad‐leaved forest in Japan. Rates of soil N mineralization and nitrification decreased from lower to upper slope positions, revealing that N availability decreased up the slope. Maximum tree height and above‐ground biomass were greater on the lower than the upper parts of the slope. Canopy openness, an index of light availability at the forest floor, increased up the slope. Tree species could be placed into three groups according to their distribution patterns on the slope. ‘Ridge’ and ‘valley’ species were distributed on the upper and lower parts of the slope, respectively, whereas ‘uniform’ species were distributed over the entire slope. Topography‐mediated resource gradients of soil N and light may be important determinants of species distribution patterns and forest regeneration, and the results of this study imply that the determinants of the regeneration process differ between the lower and upper parts of a slope. The former may be relatively light limited and the latter may be soil N limited. Valley species may have a greater ability to compete for light, whereas ridge species have a greater ability to compete for soil N. The broad distribution of uniform species probably reflects an ability to effectively compete for both light and soil N.
Proinflammatory effects of copper deficiency on neutrophils and lung endothelial cells
Authors: Lominadze David , Saari Jack T , Percival Susan S , Schuschke Dale A
Published: December 2004

Dietary copper deficiency increases the accumulation of circulating neutrophils in the rat lung microcirculation. This process includes neutrophil adhesion to, migration along, and emigration though the vascular endothelium. The current study was designed to examine the role of copper in each of these steps. Neutrophils were isolated from rats fed either a copper‐adequate (CuA, 6.1 µg Cu/g diet) or copper‐deficient diet (CuD, 0.3 µg Cu/g diet) for 4 weeks. First, transient and firm adhesion of neutrophils to P‐selectin in a flow chamber showed there were more adhered CuD neutrophils than CuA ones. This effect is probably caused by the increased expression of CD11b that was observed in the current study. Second, the evaluation of neutrophil migration under agarose showed that the CuD neutrophils moved farther than the CuA group in response to IL‐8 but not fMLP; this suggests an increased sensitivity to a CD11/CD18‐independent signalling pathway. Third, the contractile mechanism of endothelial cells was studied. Elevated F‐actin formation in Cu‐chelated lung microvascular endothelial cells suggests that neutrophil emigration may be promoted by enhanced cytoskeletal reorganization of the endothelium during copper deficiency. Combined, these results support the theory that dietary copper deficiency has proinflammatory effects on both neutrophils and the microvascular endothelium that promote neutrophil−endothelial interactions.
Developmental changes in calbindin‐D28k and calretinin expression in the mouse suprachiasmatic nucleus
Authors: Ikeda Masayuki , Allen Charles N.
Published: December 2004

The hypothalamic suprachiasmatic nucleus, the primary circadian pacemaker in mammals, and the retinohypothalamic tract, the retinal afferent fibres to the suprachiasmatic nucleus, both mature during early postnatal life. The establishment of circadian rhythms is thought to depend on input from the retina, but the mechanism remains unknown. Here we examined developmental changes in the expression of the Ca2+‐binding proteins calbindin‐D28k and calretinin in the mouse hypothalamus. Robust calbindin‐D28k immunoreactivity was observed in the dorsomedial suprachiasmatic nucleus and the supraoptic nucleus in neonatal mice (postnatal day 3). The calbindin‐D28k immunoreactivity decreased significantly in the suprachiasmatic nucleus but not in the supraoptic nucleus during postnatal days 9–15, when retinohypothalamic tract projections to the suprachiasmatic nucleus are completed. Calretinin immunoreactivity was low in the neonatal suprachiasmatic nucleus and increased with development in the ventrolateral suprachiasmatic nucleus, in parallel with the developmental reduction of calbindin‐D28k immunoreactivity observed in the dorsomedial suprachiasmatic nucleus. Developmentally stable calretinin immunoreactivity was also observed in retinohypothalamic tract fibres. Organotypic slice cultures of the suprachiasmatic nucleus were prepared from postnatal day 3 mice to examine the effect of the absence of retinohypothalamic tract inputs on developmental changes in calbindin‐D28k and calretinin expression. After 12 days in vitro, the cultured suprachiasmatic nucleus slices exhibited dense calbindin‐D28k immunoreactivity similar to neonatal mice, and calretinin immunoreactivity in the ventrolateral suprachiasmatic nucleus similar to young adult mice. These results demonstrate a developmental reduction in calbindin‐D28k expression that paralleled retinohypothalamic tract formation and a developmental increase in calretinin expression that is independent of retinohypothalamic tract connections to suprachiasmatic nucleus neurons.
The integration of cortical and behavioural dynamics during initial learning of a motor task
Authors: Serrien Deborah J. , Brown Peter
Published: December 2004

Here we test the hypothesis that frequency and topographically specific changes in the strength of functional cortico–cortical coupling occur during the acquisition of a completely new task. To this end we studied the behavioural and cortical dynamics of a bimanual multifrequency coordination pattern during which one hand moved at twice the frequency of the other hand. This pattern represents a noninherent assignment and necessitates training before appropriate interlimb decoupling takes place. Results showed that acquisition of the multifrequency task was associated with an improved behavioural output that matched specific changes in the electroencephalogram dynamics. In particular, practice of the coordination pattern was accompanied by a decrease in coherence between the primary sensorimotor regions, and over the midline area in the alpha and beta bands, respectively, along with an increase in functional interhemispheric coupling between the prefrontal areas in the gamma band. These data suggest that the strength of cortico–cortical connectivity is adaptively modified across regions and across frequencies during early learning as the functional couplings are created and optimized for the purpose of movement execution.
Forebrain astroglial plasticity is induced following withdrawal from repeated cocaine administration
Authors: Bowers M. Scott , Kalivas Peter W.
Published: December 2004

Astrocytes actively participate in synaptic plasticity and respond to insult or metabotropic glutamate receptor activation with increased expression of the intermediate filament glial fibrillary acidic protein (GFAP). Extended withdrawal from repeated cocaine administration induces many forms of neuroplasticity. The present study with rats utilized a 3‐week withdrawal period from daily cocaine administration (i.p.; 7 days) to investigate whether astrocytes participate in cocaine‐mediated plasticity observed in brain nuclei associated with addiction. Following the 3‐week withdrawal period, immunoblotting revealed increased GFAP expression in the prefrontal cortex (PFC) and in the shell and core compartments of the nucleus accumbens (NAshell and NAcore). Upregulation of GFAP did not occur in the striatum or in any brain region tested following shorter withdrawal times from repeated cocaine (24 h or 1 week) or following 2‐h withdrawal from an acute cocaine injection (30 mg/kg i.p.). However, GFAP expression increased following a 3‐week withdrawal from a single cocaine injection selectively in the NAshell. Cell counts revealed that astrocyte cell number increased only in the NAcore while immunoblots of a marker for immature or reactive astrocytes, vimentin, showed an increase only in the PFC following the 3‐week withdrawal. Taken together, these results suggest that altered intermediate filament expression within forebrain astrocytes may be a significant part of the plasticity occurring during withdrawal from repeated cocaine. Furthermore, the increase in GFAP may arise from regionally distinct mechanisms, with the NAcore relying more on cell proliferation while the PFC relies on a larger reactive astrocyte population.
Mu‐ and delta‐opioid receptor antagonists decrease proliferation and increase neurogenesis in cultures of rat adult hippocampal progenitors
Authors: Persson Anders I. , Thorlin Thorleif , Bull Cecilia , Zarnegar Parisa , Ekman Rolf , Terenius Lars , Eriksson Peter S.
Published: December 2004

Opioids have previously been shown to affect proliferation and differentiation in various neural cell types. In the present study, cultured rat adult hippocampal progenitors (AHPs) were shown to release β‐endorphin. Membrane preparations of AHPs were found to bind [125I]β‐endorphin, and immunoreactivity for mu‐ and delta‐opioid receptors (MORs and DORs), but not for kappa‐opioid receptors (KORs), was found on cells in culture. Both DNA content and [3H]thymidine incorporation were reduced after a 48‐h incubation with 100 µm naloxone, 10 µm naltrindole or 10 µmβ‐funaltrexamine, but not nor‐binaltorphimine, suggesting proliferative actions of endogenous opioids against MORs and DORs on AHPs. Furthermore, analysis of gene and protein expression after incubation with MOR and DOR antagonists for 48 h using RT‐PCR and Western blotting suggested decreased signalling through the mitogen‐activated protein kinase (MAPK) pathway and lowered levels of genes and proteins that are important in cell cycling. Cultures were incubated with naloxone (10 or 100 µm) for 10 days to study the effects on differentiation. This resulted in an approximately threefold increase in neurogenesis, a threefold decrease in astrogliogenesis and a 50% decrease in oligodendrogenesis. In conclusion, this study suggests that reduced signalling through MORs and DORs decreases proliferation in rat AHPs, increases the number of in vitro‐generated neurons and reduces the number of astrocytes and oligodendrocytes in culture.
Analysis of differential gene expression supports a role for amyloid precursor protein and a protein kinase C substrate (MARCKS) in long‐term memory
Authors: Solomonia R. O. , Morgan K. , Kotorashvili A. , McCabe B. J. , Jackson A. P. , Horn G.
Published: December 2004

Previous work has identified the intermediate and medial part of the hyperstriatum ventrale (IMHV) as a region of the chick brain storing information acquired through the learning process of imprinting. We have examined in this brain region changes in expression of candidate genes involved in memory. Chicks were exposed to a rotating red box and the strength of their preference for it, a measure of learning, determined. Brain samples were removed ≈24 h after training. Candidate genes whose expressions were different in IMHV samples derived from strongly imprinted chicks relative to those from chicks showing little or no learning were identified using subtractive hybridization. The translation products of two candidate genes were investigated further in samples from the left and right IMHV and from two other brain regions not previously implicated in imprinting, the left and right posterior neostriatum. One of the proteins was the amyloid precursor protein (APP), the other was myristoylated alanine rich C kinase substrate (MARCKS). In the left IMHV the levels of the two proteins increased with the strength of learning. The effects in the right IMHV were not significantly different from those in the left. There were no effects of learning in the posterior neostriatum. This is the first study to relate changes in the amounts of MARCKS and APP proteins to the strength of learning in a brain region known to be a memory store and demonstrates that the systematic identification of protein molecules involved in memory formation is possible.
D2 Dopamine receptor blockade results in sprouting of DA axons in the intact animal but prevents sprouting following nigral lesions
Authors: Tripanichkul W. , Stanic D. , Drago J. , Finkelstein D. I. , Horne M. K.
Published: December 2004

Recently it was demonstrated that sprouting of dopaminergic neurons and a microglial and astrocyte response follows both partial lesions of the substantia nigra pars compacta and blockade of the D2 dopamine receptor. We therefore studied the effects of the combination of these two treatments (lesioning and D2 dopamine receptor blockade). Haloperidol administration caused a 57% increase in dopaminergic terminal tree size (measured as terminal density per substantia nigra pars compacta neuron) and an increase of glia in the striatum. Following small to medium nigral lesions (less than 60%), terminal tree size increased by 51% on average and returned density of dopaminergic terminals to normal. In contrast, administration of haloperidol for 16 weeks following lesioning resulted in reduced dopaminergic terminal density and terminal tree size (13%), consistent with absent or impaired sprouting. Glial cell numbers increased but were less than with lesions alone. When haloperidol was administered after the striatum had been reinnervated through sprouting (16–32 weeks after lesioning), terminal tree size increased up to 150%, similar to the effect of haloperidol in normal animals. By examining the effect of administering haloperidol at varying times following a lesion, we concluded that a switch in the effect of D2 dopamine receptor blockade occurred after dopaminergic synapses began to form in the striatum. We postulate that when synapses are present, D2 dopamine receptor blockade results in increased terminal density, whereas prior to synapse formation D2 dopamine receptor blockade causes attenuation of a sprouting response. We speculate that D2 dopamine receptors located on growth cones ‘push’ neurites toward their targets, and blockade of these receptors could lead to attenuation of sprouting.
NMDA receptor‐mediated extracellular adenosine accumulation in rat forebrain neurons in culture is associated with inhibition of adenosine kinase
Authors: Lu Yin , Chung Hye Joo , Li Ya , Rosenberg Paul A.
Published: December 2004

The effect of N‐methyl‐d‐aspartate (NMDA) on regulation of extracellular adenosine was investigated in rat forebrain neurons in culture. NMDA evoked accumulation of extracellular adenosine with an EC50 value of 4.8 ± 1.2 µm. The effect of NMDA was blocked by (+)‐5‐methyl‐10,11‐dihydro‐5H‐dibenzo [a, d] cyclohepten‐5,10‐imine hydrogen maleate indicating that NMDA receptor activation was involved. The NMDA effect was also blocked by chelation of extracellular Ca2+ indicating that influx of calcium was required. The nitric oxide–cyclic GMP signalling pathway was not involved, as nitric oxide synthase inhibitors were unable to block, and cGMP analogs were unable to mimic, the effect of NMDA. The source for extracellular adenosine was likely to be intracellular adenosine as the ecto‐5′‐nucleotidase inhibitor αβ‐methylene‐ADP was unable to block the effect of NMDA. One possible cause of intracellular adenosine accumulation might be NMDA receptor‐mediated inhibition of mitochondrial function and ATP hydrolysis. We found that NMDA caused a concentration dependent depletion of intracellular ATP with an EC50 value of 21 ± 8 µm. NMDA also caused a significant decrease in adenosine kinase activity, assayed by two different methods. Consistent with the hypothesis that inhibition of adenosine kinase is sufficient to cause an increase in extracellular adenosine, inhibition of adenosine kinase by 5′‐iodotubercidin resulted in elevation of extracellular adenosine. However, in the presence of a concentration of 5′‐iodotubercidin that inhibited over 90% of adenosine kinase activity, exposure to NMDA still caused adenosine accumulation. These studies suggest that several possible mechanisms are likely to be involved in NMDA‐evoked extracellular adenosine accumulation.
The ability of atypical antipsychotic drugs vs. haloperidol to protect PC12 cells against MPP+‐induced apoptosis
Authors: Qing Hong , Xu Haiyun , Wei Zelan , Gibson Kurt , Li Xin‐Min
Published: December 2004

The present study examined the effects of the atypical antipsychotic drugs clozapine, olanzapine, quetiapine and risperidone, on N‐methyl‐4‐phenylpyridinium ion‐induced apoptosis and DNA damage in PC12 cells, and explored the molecular mechanisms underlying these effects. Haloperidol, a typical antipsychotic drug, was used for comparison. Exposure of PC12 cells to 50 µm N‐methyl‐4‐phenylpyridinium ion for 24 h resulted in a 35–45% loss of cells in culture. Pretreatment with the aforementioned atypical antipsychotic drugs significantly reduced the N‐methyl‐4‐phenylpyridinium ion‐induced cell loss, whereas haloperidol (10–100 µm) did not have this protective effect. Hoechst 33258 staining revealed the apoptotic nuclear features of the N‐methyl‐4‐phenylpyridinium ion‐induced cell death, and showed that the atypical antipsychotic drugs, but not haloperidol, effectively prevented PC12 cells from this N‐methyl‐4‐phenylpyridinium ion‐induced apoptosis. DNA fragmentation assays further confirmed the N‐methyl‐4‐phenylpyridinium ion‐induced nuclear fragmentation. Pretreatment with the atypical antipsychotic drugs completely prevented this nuclear fragmentation, whereas haloperidol only partially prevented it. In vitro oligonucleotide assays indicated an activation of a specific glycosylase that recognizes and cleaves bases (at the 8‐hydroxyl‐2‐deoxyguanine site) that were damaged by N‐methyl‐4‐phenylpyridinium ion. Pretreatment with the atypical antipsychotic drugs more effectively attenuated this N‐methyl‐4‐phenylpyridinium ion‐induced activation than did haloperidol. Northern blot analyses showed that the atypical antipsychotic drugs, but not haloperidol, blocked the N‐methyl‐4‐phenylpyridinium ion‐induced substantial increase of copper/zinc superoxide dismutase mRNA in PC12 cells. Atypical antipsychotic drugs slightly up‐regulated the expression of copper/zinc superoxide dismutase mRNA, whereas haloperidol strongly increased the expression of copper/zinc superoxide dismutase mRNA. These data may account for the different therapeutic effects and side‐effect profiles of typical and atypical antipsychotic drugs in schizophrenia.