bluuebunny/crossref_metadata_2025 huggingface dataset:
Differences in substrate specificities between cysteine protease CPB isoforms of Leishmania mexicana are mediated by a few amino acid changes
Authors: Juliano Maria A. , Brooks Darren R. , Selzer Paul M. , Pandolfo Hector L. , Judice Wagner A. S. , Juliano Luiz , Meldal Morten , Sanderson Sanya J. , Mottram Jeremy C. , Coombs Graham H.
Published: September 2004

The CPB genes of the protozoan parasite Leishmania mexicana encode stage‐regulated cathepsin L‐like cysteine proteases that are important virulence factors and are in a tandem array of 19 genes. In this study, we have compared the substrate preferences of two CPB isoforms, CPB2.8 and CPB3, and a H84Y mutant of the latter enzyme, to analyse the roles played by the few amino acid differences between the isoenzymes in determining substrate specificity. CPB3 differs from CPB2.8 at just three residues (N60D, D61N and D64S) in the mature domain. The H84Y mutation mimics an additional change present in another isoenzyme, CPB18. The active recombinant CPB isoenzymes and mutant were produced using Escherichia coli and the S1‐S3 and S1′‐S3′ subsite specificities determined using a series of fluorogenic peptide derivatives in which substitutions were made on positions P3 to P3′ by natural amino acids. Carboxydipeptidase activities of CPB3 and H84Y were also observed using the peptide Abz‐FRAK(Dnp)‐OH and some of its analogues. The kinetic parameters of hydrolysis by CPB3, H84Y and CPB2.8 of the synthetic substrates indicates that the specificity of S3 to S3′ subsites is influenced greatly by the modifications at amino acids 60, 61, 64 and 84. Particularly noteworthy was the large preference for Pro in the P2′ position for the hydrolytic activity of CPB3, which may be relevant to a role in the activation mechanism of the L. mexicana CPBs.
The HNF1β transcription factor has several domains involved in nephrogenesis and partially rescues Pax8/lim1‐induced kidney malformations
Authors: Wu Guizhi , Bohn Silvia , Ryffel Gerhart U.
Published: September 2004

The tissue‐specific transcription factors HNF1α and HNF1β are closely related homeodomain proteins conserved in vertebrate evolution. Heterozygous mutations in human HNF1β but not in HNF1α genes are associated with kidney malformations. Overexpression of HNF1β in Xenopus embryos leads to defective pronephros development, while HNF1α has no effect. We have defined the regions responsible for this functional difference between HNF1β and HNF1α in transfected HeLa cells as well as in injected Xenopus embryos. Using domain swapping experiments, we located a nuclear localization signal in the POUH domain of HNF1β, and showed that the POUS and POUH domains of HNF1β mediate a high transactivation potential in transfected cells. In injected Xenopus embryos three HNF1β domains are involved in nephrogenesis. These include the dimerization domain, the 26 amino acid segment specific for splice variant A as well as the POUH domain. As HNF1β together with Pax8 and lim1 constitute the earliest regulators in the pronephric anlage, it is possible that they cooperate during early nephrogenesis. We have shown here that HNF1β can overcome the enlargement and the induction of an ectopic pronephros mediated by overexpression of Pax8 and lim1. However, the phenotype induced by Pax8 and lim1 overexpression and characterized by cyst‐like structures and thickening of the pronephric tubules was not altered by HNF1β overexpression. Taken together, HNF1β acts antagonistically to Pax8 and lim1 in only some processes during nephrogenesis, and a simple antagonistic relationship does not completely describe the functions of these genes. We conclude that HNF1β has some distinct morphogenetic properties during nephrogenesis.
Integrin receptor specificity for human red cell ICAM‐4 ligand
Authors: Hermand Patricia , Gane Pierre , Callebaut Isabelle , Kieffer Nelly , Cartron Jean‐Pierre , Bailly Pascal
Published: September 2004

The red cell intercellular adhesion molecule‐4 (ICAM‐4) binds to different members of the integrin receptor families. To better define the ICAM‐4 integrin receptor specificity, cell transfectants individually expressing various integrins were used to demonstrate that αLβ2, αMβ2, and αIIbβ3 (activated) bind specifically and dose dependently to the recombinant ICAM‐4‐Fc protein. We also show that cell surface ICAM‐4 interacts with the cell surface αVβ3 integrin. In addition, using a α4β1 cell transfectant and β2 integrin‐deficient LAD cells, we show here that ICAM‐4 failed to interact with α4β1 even after α4β1 activation by phorbol ester or with the monoclonal antibody TS2/16 (+ Mn2+). ICAM‐4 amino acids that are critical for αIIbβ3 and αVβ3 interaction were identified by domain deletion analysis, site‐directed mutagenesis and synthetic peptide inhibition. Our results provide evidence that the β3 integrin binding sites encompass the first and second Ig‐like domains of ICAM‐4. However, while the αIIbβ3 contact site comprises the ABED face of domain D1 with an extension in the C′‐E loop of domain D2, the αVβ3 contact site comprises residues on both faces of D1 and in the C′‐E loop of D2. These data, together with our previous results, demonstrate that different integrins bind to different but partly overlapping sites on ICAM‐4, and that ICAM‐4 may accommodate multiple integrin receptors present on leukocytes, platelets and endothelial cells.
RNA helicase A interacts with nuclear factor κB p65 and functions as a transcriptional coactivator
Authors: Tetsuka Toshifumi , Uranishi Hiroaki , Sanda Takaomi , Asamitsu Kaori , Yang Jiang‐Ping , Wong‐Staal Flossie , Okamoto Takashi
Published: September 2004

RNA helicase A (RHA), a member of DNA and RNA helicase family containing ATPase activity, is involved in many steps of gene expression such as transcription and mRNA export. RHA has been reported to bind directly to the transcriptional coactivator, CREB‐binding protein, and the tumor suppressor protein, BRCA1, and links them to RNA Polymerase II holoenzyme complex. Using yeast two‐hybrid screening, we have identified RHA as an interacting molecule of the p65 subunit of nuclear factor κB (NF‐κB). The interaction between p65 and RHA was confirmed by glutathione‐S transferase pull‐down assay in vitro, and by co‐immunoprecipitation assay in vivo. In transient transfection assays, RHA enhanced NF‐κB dependent reporter gene expression induced by p65, tumor necrosis factor‐α, or NF‐κB inducing kinase. The mutant form of RHA lacking ATP‐binding activity inhibited NF‐κB dependent reporter gene expression induced by these activators. Moreover, depletion of RHA using short interfering RNA reduced the NF‐κB dependent transactivation. These data suggest that RHA is an essential component of the transactivation complex by mediating the transcriptional activity of NF‐κB.
Expression in yeast of a novel phospholipase A1 cDNA from Arabidopsis thaliana
Authors: Noiriel Alexandre , Benveniste Pierre , Banas Antoni , Stymne Sten , Bouvier‐Navé Pierrette
Published: September 2004

During a search for cDNAs encoding plant sterol acyltransferases, we isolated four full‐length cDNAs from Arabidopsis thaliana that encode proteins with substantial identity with animal lecithin : cholesterol acyltransferases (LCATs). The expression of one of these cDNAs, AtLCAT3 (At3g03310), in various yeast strains resulted in the doubling of the triacylglycerol content. Furthermore, a complete lipid analysis of the transformed wild‐type yeast showed that its phospholipid content was lower than that of the control (void plasmid‐transformed) yeast whereas lysophospholipids and free fatty acids increased. When microsomes from the AtLCAT3‐transformed yeast were incubated with di‐[1‐14C]oleyl phosphatidylcholine, both the lysophospholipid and free fatty acid fractions were highly and similarly labelled, whereas the same incubation with microsomes from the control yeast produced a negligible labelling of these fractions. Moreover when microsomes from AtLCAT3‐transformed yeast were incubated with either sn‐1‐ or sn‐2‐[1‐14C]acyl phosphatidylcholine, the distribution of the labelling between the free fatty acid and the lysophosphatidylcholine fractions strongly suggested a phospholipase A1 activity for AtLCAT3. The sn‐1 specificity of this phospholipase was confirmed by gas chromatography analysis of the hydrolysis of 1‐myristoyl, 2‐oleyl phosphatidylcholine. Phosphatidylethanolamine and phosphatidic acid were shown to be also hydrolysed by AtLCAT3, although less efficiently than phosphatidylcholine. Lysophospatidylcholine was a weak substrate whereas tripalmitoylglycerol and cholesteryl oleate were not hydrolysed at all. This novel A. thaliana phospholipase A1 shows optimal activity at pH 6–6.5 and 60–65 °C and appears to be unaffected by Ca2+. Its sequence is unrelated to all other known phospholipases. Further studies are in progress to elucidate its physiological role.
Identification of intracellular target proteins of the calcium‐signaling protein S100A12
Authors: Hatakeyama Takashi , Okada Miki , Shimamoto Seiko , Kubota Yasuo , Kobayashi Ryoji
Published: September 2004

In this report, we have focused our attention on identifying intracellular mammalian proteins that bind S100A12 in a Ca2+‐dependent manner. Using S100A12 affinity chromatography, we have identified cytosolic NADP+‐dependent isocitrate dehydrogenase (IDH), fructose‐1,6‐bisphosphate aldolase A (aldolase), glyceraldehyde‐3‐phosphate dehydrogenese (GAPDH), annexin V, S100A9, and S100A12 itself as S100A12‐binding proteins. Immunoprecipitation experiments indicated the formation of stable complexes between S100A12 and IDH, aldolase, GAPDH, annexin V and S100A9 in vivo. Surface plasmon resonance analysis showed that the binding to S100A12, of S100A12, S100A9 and annexin V, was strictly Ca2+‐dependent, whereas that of GAPDH and IDH was only weakly Ca2+‐dependent. To localize the site of S100A12 interaction, we examined the binding of a series of C‐terminal truncation mutants to the S100A12‐immobilized sensor chip. The results indicated that the S100A12‐binding site on S100A12 itself is located at the C‐terminus (residues 87–92). However, cross‐linking experiments with the truncation mutants indicated that residues 87–92 were not essential for S100A12 dimerization. Thus, the interaction between S100A12 and S100A9 or immobilized S100A12 should not be viewed as a typical S100 homo‐ or heterodimerization model. Ca2+‐dependent affinity chromatography revealed that C‐terminal residues 75–92 are not necessary for the interaction of S100A12 with IDH, aldolase, GAPDH and annexin V. To analyze the functional properties of S100A12, we studied its action in protein folding reactions in vitro. The thermal aggregation of IDH or GAPDH was facilitated by S100A12 in the absence of Ca2+, whereas in the presence of Ca2+ the protein suppressed the aggregation of aldolase to less than 50%. These results suggest that S100A12 may have a chaperone/antichaperone‐like function which is Ca2+‐dependent.
Structured DNA promotes phosphorylation of p53 by DNA‐dependent protein kinase at serine 9 and threonine 18
Authors: Soubeyrand Sébastien , Schild‐Poulter Caroline , Haché Robert J. G.
Published: September 2004

Phosphorylation at multiple sites within the N‐terminus of p53 promotes its dissociation from hdm2/mdm2 and stimulates its transcriptional regulatory potential. The large phosphoinositide 3‐kinase‐like kinases ataxia telangiectasia mutated gene product and the ataxia telangectasia and RAD‐3‐related kinase promote phosphorylation of human p53 at Ser15 and Ser20, and are required for the activation of p53 following DNA damage. DNA‐dependent protein kinase (DNA‐PK) is another large phosphoinositide 3‐kinase‐like kinase with the potential to phosphorylate p53 at Ser15, and has been proposed to enhance phosphorylation of these sites in vivo. Moreover, recent studies support a role for DNA‐PK in the regulation of p53‐mediated apoptosis. We have shown previously that colocalization of p53 and DNA‐PK to structured single‐stranded DNA dramatically enhances the potential for p53 phosphorylation by DNA‐PK. We report here the identification of p53 phosphorylation at two novel sites for DNA‐PK, Thr18 and Ser9. Colocalization of p53 and DNA‐PK on structured DNA was required for efficient phosphorylation of p53 at multiple sites, while specific recognition of Ser9 and Thr18 appeared to be dependent upon additional determinants of p53 beyond the N‐terminal 65 amino acids. Our results suggest a role for DNA‐PK in the modulation of p53 activity resultant from the convergence of p53 and DNA‐PK on structured DNA.
Irregular dimerization of guanylate cyclase‐activating protein 1 mutants causes loss of target activation
Authors: Hwang Ji‐Young , Schlesinger Ramona , Koch Karl‐Wilhelm
Published: September 2004

Guanylate cyclase‐activating proteins (GCAPs) are neuronal calcium sensors that activate membrane bound guanylate cyclases (EC 4.6.1.2.) of vertebrate photoreceptor cells when cytoplasmic Ca2+ decreases during illumination. GCAPs contain four EF‐hand Ca2+‐binding motifs, but the first EF‐hand is nonfunctional. It was concluded that for GCAP‐2, the loss of Ca2+‐binding ability of EF‐hand 1 resulted in a region that is crucial for targeting guanylate cyclase [Ermilov, A.N., Olshevskaya, E.V. & Dizhoor, A.M. (2001) J. Biol. Chem.276, 48143–48148]. In this study we tested the consequences of mutations in EF‐hand 1 of GCAP‐1 with respect to Ca2+ binding, Ca2+‐induced conformational changes and target activation. When the nonfunctional first EF‐hand in GCAP‐1 is replaced by a functional EF‐hand the chimeric mutant CaM–GCAP‐1 bound four Ca2+ and showed similar Ca2+‐dependent changes in tryptophan fluorescence as the wild‐type. CaM–GCAP‐1 neither activated nor interacted with guanylate cyclase. Size exclusion chromatography revealed that the mutant tended to form inactive dimers instead of active monomers like the wild‐type. Critical amino acids in EF‐hand 1 of GCAP‐1 are cysteine at position 29 and proline at position 30, as changing these to glycine was sufficient to cause loss of target activation without a loss of Ca2+‐induced conformational changes. The latter mutation also promoted dimerization of the protein. Our results show that EF‐hand 1 in wild‐type GCAP‐1 is critical for providing the correct conformation for target activation.
Escherichia coli cyclophilin B binds a highly distorted form of trans‐prolyl peptide isomer
Authors: Konno Michiko , Sano Yumi , Okudaira Kayoko , Kawaguchi Yoko , Yamagishi‐Ohmori Yoko , Fushinobu Shinya , Matsuzawa Hiroshi
Published: September 2004

Cyclophilins facilitate the peptidyl‐prolyl isomerization of a trans‐isomer to a cis‐isomer in the refolding process of unfolded proteins to recover the natural folding state with cis‐proline conformation. To date, only short peptides with a cis‐form proline have been observed in complexes of human and Escherichia coli proteins of cyclophilin A, which is present in cytoplasm. The crystal structures analyzed in this study show two complexes in which peptides having a trans‐form proline, i.e. succinyl‐Ala‐trans‐Pro‐Ala‐p‐nitroanilide and acetyl‐Ala‐Ala‐trans‐Pro‐Ala‐amidomethylcoumarin, are bound on a K163T mutant of Escherichia coli cyclophilin B, the preprotein of which has a signal sequence. Comparison with cis‐form peptides bound to cyclophilin A reveals that in any case the proline ring is inserted into the hydrophobic pocket and a hydrogen bond between CO of Pro and Nη2 of Arg is formed to fix the peptide. On the other hand, in the cis‐isomer, the formation of two hydrogen bonds of NH and CO of Ala preceding Pro with the protein fixes the peptide, whereas in the trans‐isomer formation of a hydrogen bond between CO preceding Ala‐Pro and His47 Nε2 via a mediating water molecule allows the large distortion in the orientation of Ala of Ala‐Pro. Although loss of double bond character of the amide bond of Ala‐Pro is essential to the isomerization pathway occurring by rotating around its bond, these peptides have forms impossible to undergo proton transfer from the guanidyl group of Arg to the prolyl N atom, which induces loss of double bond character.
Effects of Climatic Factors Prevailing Prior to Flowering or Subsequent to Boll Setting on Flower and Boll Production and Retention of Cotton in Egypt
Authors: Sawan Z. M. , Hanna L. I. , McCuistion W. L.
Published: September 2004

This study was conducted to investigate the nature of the effects of climatic factors prevailing prior to flowering or subsequent to boll setting on flower and boll production and retention in Egyptian cotton (Gossypium barbadense L.). Two uniform field trials, using the cotton cv. Giza 75, were carried out in 1992 and 1993 at the Agricultural Research Centre, Giza, Egypt. Randomly chosen plants were used to record daily numbers of flowers and bolls during the production stage (68 days in the first season and 62 days in the second season). The daily records of the climatic factors (air temperature, temperature magnitude, evaporation, surface soil temperature, sunshine duration and humidity) were recorded during the entire period of production and the 15 days preceding or succeeding flower opening. The effects of climatic factors on flower and boll production were quantified in non‐limiting management techniques. Relationships in the form of simple and multiple correlations were computed between climatic factors and flower and boll production and retention. The data indicate that evaporation, minimum humidity and sunshine duration were the most effective climatic factors during preceding and succeeding periods on fruit production and retention. The relationship between climatic factors preceding flowering and cotton production generally exceeded the relationship between the factors during the entire and latter periods of production stage. The result was a negative correlation between either evaporation or sunshine duration with flower and boll formation. In addition, a positive correlation was evident between minimum humidity and flower and boll production. The data indicate that low evaporation rate, reduced sunshine duration and high humidity would enhance flower and boll formation. Accordingly, the deleterious effects of climatic factors on cotton production could be minimized through applying appropriate production practices, which would control and adjust the impact of these factors, and this could lead to an important improvement in cotton yield in Egypt.
Nitrogen Fertilization on Rye Pasture: Effect on Forage Chemical Composition, Voluntary Intake, Digestibility and Rumen Degradation
Authors: Ferri C. M. , Stritzler N. P. , Pagella J. H.
Published: September 2004

The effects of nitrogen (N) fertilization of a rye pasture (Secale cereale L.) on the chemical composition of forage and its utilization in vivo and in situ were investigated in winter (W) and early spring (S). Half of the paddock to be assayed was spread with 93 kg urea–N ha−1 (treatment F), and the other half was not fertilized (treatment NF). Each experiment lasted for 17 days; apparent dry matter digestibility in vivo, voluntary dry matter intake (DMI) and digestible DMI were measured on nine rams per treatment. Two rumen cannulated animals per treatment were under the same feeding regime, and used to collect samples of rumen contents. The concentrations of ammonia (NH3) and short‐chain fatty acids and pH were measured in these samples. The offered forage was also incubated in the rumen of three Holstein–Friesian steers to estimate its degradation parameters in situ. Fertilization with N increased total N and decreased dry matter (DM) and non‐structural carbohydrate concentrations in both periods. DMI was reduced by fertilization. The rumen concentrations of NH3 were higher in F than in NF in both periods. In both periods, the effective DM degradability of forage was higher in F than in NF. N fertilization affects chemical composition, voluntary intake and rumen digestion of forage from rye pasture.
Effect of Timing and Nitrogen Fertilizer Application on Winter Oilseed Rape (Brassica napus L.). I. Growth Dynamics and Seed Yield
Authors: Barłóg P. , Grzebisz W.
Published: September 2004

The field experiments conducted on the grey‐brown podzolic soil in the four growing seasons (1998–2001) at Krzeslice Farm, central‐western Poland comprised seven fertilization variants: 80NF + 80CAN; 80CAN + 80CAN; 80AN + 80AN; 80NF + 50CAN + 30CN; 80CAN + 50CAN +30CN; 80AN + 50AN + 30CN (where NF – nitrofos NPK; CAN – calcium‐ammonium nitrate; AN – ammonium nitrate; CN – calcium nitrate) and control (without N) applied in split rates at the beginning of spring regrowth (80 kg N ha−1), stem elongation (80 or 50) and flower buds visible stages (30). The yielding effect of tested fertilization variants was significant in comparison with the control (2.24 t ha−1). The highest mean seed yield (3.64 t ha−1) was collected from 80AN + 80AN and 80CAN + 80CAN variants. Mean values of 4 years indicate that the second N rate division (80 + 50 + 30) decreased yield, although not significantly in comparison with these two N treatments. Plants grown on these treatments have developed different patterns of growth to yield the seeds. These patterns were characterized by very high crop growth rate during flowering (above 21 g m−2 day−1) and negative at maturation (down to −2.5 g m−2 day−1). Plants fertilized with ammonium nitrate (80AN + 80AN) reached maximum growth rate earlier (65 days), which lasted longer (20 days) than plants fertilized with calcium‐ammonium nitrate (71  days lasting 17.5 days). Plants grown on the control treatment reached the highest crop growth rate within 79 days (14.8 g m−2 day−1), which lasted 15 days.
Effect of Timing and Nitrogen Fertilizer Application on Winter Oilseed Rape (Brassica napus L.). II. Nitrogen Uptake Dynamics and Fertilizer Efficiency
Authors: Barłóg P. , Grzebisz W.
Published: September 2004

Field experiments were carried out on grey‐brown podzolic soil in the four consecutive growing seasons (1998–2001) at Krzeslice Farm, central‐western Poland. The effect of seven N fertilization treatments (in kg N ha−1): 80NF + 80CAN; 80NF + 50CAN + 30CN; 80CAN + 80CAN; 80CAN + 80CAN + 30CN; 80AN + 80AN; 80AN + 50AN + 30CN, where, NF – nitrofos NPK, CAN – calcium‐ammonium nitrate, AN – ammonium nitrate, CN – calcium nitrate and control (without N) on N uptake dynamics and N efficiency was studied. Mineral fertilizers were applied at the start of spring regrowth, beginning of stem elongation and at the flower‐bud‐visibility stage. The study revealed two distinct strategies of oilseed rape plants’ adaptation to timing and N fertilizer application sequences. Both strategies based on nitrogen uptake rate (NUR), were analysed at different plant growth stages. Ammonium nitrate (AN) applied in the two‐split system gave the highest NUR (387 mg m−2 day−1) during stem elongation (for comparison, a value of 166 mg m−2 day−1 was obtained in the control). In the case of calcium‐ammonium nitrate (CAN), a moderate level of NUR was obtained (304 mg m−2 day−1) but N uptake lasted 12 days longer compared with the AN treatment. Hence, N accumulation in leaves at the end of flowering explained about 81 % of yield variability. The second adaptation strategy was attributed to the three‐split N treatment. Plants fertilized with AN and CAN fertilizers showed an inconsistent pattern of NUR with time. Nitrogen accumulation in stems at the beginning of maturity, explained 69 % of yield variability. Nitrogen‐use efficiency did not show any response to N treatments.
Chronic stress attenuates GABAergic inhibition and alters gene expression of parvocellular neurons in rat hypothalamus
Authors: Verkuyl J. Martin , Hemby Scott E. , Joëls Marian
Published: September 2004

Chronic stress causes disinhibition of the hypothalamus–pituitary–adrenal axis. Consequently, the brain is overexposed to glucocorticoids which in humans may precipitate stress‐related disorders, e.g. depression. The hypothalamus–pituitary–adrenal activity is strongly regulated by GABAergic input to parvocellular neurons in the hypothalamic paraventricular nucleus. We here report a reduced frequency of miniature inhibitory postsynaptic currents (mIPSCs) in parvocellular neurons of rats exposed to 3 weeks of unpredictable stress. The mIPSC amplitude and kinetic properties were unchanged, pointing to a presynaptic change caused by chronic stress. Because paired‐pulse inhibition was unaffected by chronic stress, the number of functional GABAergic synaptic contacts rather than the release probability seems to be reduced after chronic stress. Linearly amplified RNA from postsynaptic cells was hybridized with multiple cDNA clones of interest, including most GABAA receptor subunits. In agreement with the electrophysiological observations, relative expression of the prevalent GABAAα1, α3, γ1 and γ2 receptor subunits, which largely contribute to the recorded responses, was not altered after chronic stress. However, expression of the extra‐synaptic GABAAα5 subunit, earlier linked to depression in humans, and of the δ receptor subunit were found to be significantly changed. In conclusion, chronic stress leads to presynaptic functional alterations in GABAergic input to the paraventricular nucleus which could contribute to the observed disinhibition of the hypothalamus–pituitary–adrenal axis; additionally other aspects of GABAergic transmission may also be changed due to transcriptional regulation of specific receptor subunits in the parvocellular neurons.
Axon behaviour at Schwann cell – astrocyte boundaries: manipulation of axon signalling pathways and the neural adhesion molecule L1 can enable axons to cross
Authors: Adcock Kathryn H. , Brown David J. , Shearer Morven C. , Shewan Derryck , Schachner Melitta , Smith George M. , Geller Herbert M. , Fawcett James W.
Published: September 2004

Axon regeneration in vivo is blocked at boundaries between Schwann cells and astrocytes, such as occur at the dorsal root entry zone and around peripheral nerve or Schwann cell grafts. We have created a tissue culture model of these boundaries in Schwann cell – astrocyte monolayer co‐cultures. Axon behaviour resembles that in vivo, with axons showing a strong preference for Schwann cells over astrocytes. At boundaries between the two cell types, axons growing on astrocytes cross readily onto Schwann cells, but only 15% of axons growing on Schwann cells are able to cross onto astrocytes. Treatment with chondroitinase or chlorate to reduce inhibition by proteoglycans did not change this behaviour. The neural adhesion molecule L1 is present on Schwann cells and not astrocytes, and manipulation of L1 by application of an antibody, L1‐Fc in solution, or adenoviral transduction of L1 into astrocytes increased the proportion of axons able to cross onto astrocytes to 40–50%. Elevating cAMP levels increased crossing from Schwann cells onto astrocytes in live and fixed cultures, and had a co‐operative effect with NT‐3 but not with NGF. Inactivation of Rho with a cell‐permeant form of C3 exoenzyme also increased crossing from Schwann cells to astrocytes. Our experiments indicate that the preference of axons for Schwann cells is largely mediated by the presence of L1 on Schwann cells but not astrocytes, and that manipulation of growth cone signalling pathways can allow axons to disregard boundaries between the two cell types.
Mu‐opioid receptor knockout mice show diminished food‐anticipatory activity
Authors: Kas Martien J. H. , Van Den Bos Ruud , Baars Annemarie M. , Lubbers Marianne , Lesscher Heidi M. B. , Hillebrand Jacquelien J. G. , Schuller Alwin G. , Pintar John E. , Spruijt Berry M.
Published: September 2004

We have previously suggested that during or prior to activation of anticipatory behaviour to a coming reward, µ‐opioid receptors are activated. To test this hypothesis schedule induced food‐anticipatory activity in µ‐opioid receptor knockout mice was measured using running wheels. We hypothesized that µ‐knockout mice show little food‐anticipatory activity. In wildtype mice we observed that food‐anticipatory activity increased proportional to reduced food intake levels during daily scheduled food access, and thus reflects the animal's physiological need for food. µ‐Knockout mice do not adjust their schedule induced running wheel behaviour prior to and during feeding time in the same way as wildtype mice; rather than showing more running wheel activity before than during feeding, they showed an equal amount of activity before and during feeding. As food‐anticipatory activity is dependent on the mesolimbic dopamine system and µ‐opioid receptors regulate dopaminergic activity, these data suggest a change in the dopamine system's activity in µ‐knockout mice. As we observed that µ‐knockout mice tended to show a stronger locomotor activity response than wildtype mice to the indirect dopamine agonistd‐amphetamine, it appears that the dopaminergic systemper seis intact and sensitive to activation. We found no differences in the expression of pro‐opiomelanocortin, a precursor of endogenous endorphin, in the arcuate nucleus between µ‐knockout mice and wildtype mice during restricted feeding, showing that the µ‐opioid receptor does not regulate endogenous endorphin levels. These data overall suggest a role for µ‐opioid receptors in adapting reward related behaviour to the requirements of the environment.
Lesions of the Edinger–Westphal nucleus in C57BL/6J mice disrupt ethanol‐induced hypothermia and ethanol consumption
Authors: Bachtell Ryan K. , Weitemier Adam Z. , Ryabinin Andrey E.
Published: September 2004

The Edinger–Westphal nucleus (EW) is a brain region that has recently been implicated as an important novel neural target for ethanol. Thus, the EW is the only brain region consistently showing elevated c‐Fos expression following both voluntary and involuntary ethanol administration. Ethanol‐induced c‐Fos expression in the EW has been shown to occur in urocortin I‐positive neurons. Moreover, previous reports using several genetic models have demonstrated that differences in the EW urocortin I system are correlated with ethanol‐mediated behaviours such as ethanol‐induced hypothermia and ethanol consumption. The aim of this study was to confirm these relationships using a more direct strategy. Thus, ethanol responses were measured following electrolytic lesions of the EW in male C57BL/6J mice. Both EW‐lesioned and sham‐operated animals were tested for several ethanol sensitivity measures and ethanol consumption in a two‐bottle choice test. The results show that lesions of the EW significantly disrupted ethanol‐induced hypothermia, while having no effect on pupillary dilation, locomotor activity or ethanol‐induced sedation. In addition, EW‐lesioned animals showed significantly lower ethanol preference and total ethanol dose consumed in the two‐bottle choice test. EW‐lesioned animals also consumed less sucrose than sham‐operated animals, but did not have altered preferences for sucrose or quinine in a two‐bottle choice test. These data support previously observed genetic correlations between EW urocortin I expression and both ethanol‐induced hypothermia and ethanol consumption. Taken together, the findings suggest that the EW may function as a sensor for ethanol, which can influence ethanol consumption and preference.
Beneficial effects of enriched environment on adolescent rats from stressed pregnancies
Authors: Laviola Giovanni , Rea Monica , Morley‐Fletcher Sara , Di Carlo Simonetta , Bacosi Antonella , De Simone Roberta , Bertini Mario , Pacifici Roberta
Published: September 2004

The capacity of an early environmental intervention to normalize the behavioural and immunological dysfunctions produced by a stressed pregnancy was investigated. Pregnant Sprague‐Dawley rats underwent three 45‐min sessions per day of prenatal restraint stress (PS) on gestation days 11–21, and their offspring were assigned to either an enriched‐environment or standard living cages throughout adolescence [postnatal days (pnd) 22–43]. Juvenile rats from stressed pregnancies had a prominent depression of affiliative/playful behaviour and of basal circulating CD4 T lymphocytes, CD8 T lymphocytes and T4/T8 ratio. They also showed increased emotionality and spleen and brain frontal cortex levels of pro‐inflammatory interleoukin‐1β (IL‐1β) cytokine. A more marked response to cyclophosphamide (CPA: two 2 mg/kg IP injections) induced immunosuppression was also found in prenatal stressed rats. Enriched housing increased the amount of time adolescent PS rats spent in positive species‐typical behaviours (i.e. play behaviour), reduced emotionality and reverted most of immunological alterations. In addition to its effects in PS rats, enriched housing increased anti‐inflammatory IL‐2 and reduced pro‐inflammatory IL‐1β production by activated splenocytes, also producing a marked alleviation of CPA‐induced immune depression. In the brain, enriched housing increased IL‐1β values in hypothalamus, while slightly normalizing these values in the frontal cortex from PS rats. This is a first indication that an environmental intervention, such as enriched housing, during adolescence can beneficially affect basal immune parameters and rats response to both early stress and drug‐induced immunosuppression.
Unique accumulation of neuropeptides in an insect: FMRFamide‐related peptides in the cockroach, Periplaneta americana
Authors: Predel R. , Neupert S. , Wicher D. , Gundel M. , Roth S. , Derst C.
Published: September 2004

FMRFamides belong to the most extensively studied neuropeptides in invertebrates and exhibit diverse physiological effects on different target organs, such as muscles, intestine and the nervous system. This study on the American cockroach confirms for the first time that extended FMRFamides occur in non‐dipteran insects. By means of tandem mass spectrometry, these neuropeptides were structurally elucidated, and sequence information was used for subsequent cloning of the cockroach FMRFamide gene. This precursor gene encodes for 24 putative peptides and shows sufficient similarity with the Drosophila FMRFamide gene. Of the 24 peptides, 23 were detected by mass spectrometric methods; it is the highest number of neuropeptide forms shown to be expressed from a single precursor in any insect. The expression was traced back to single neurons in the thoracic ganglia. The unique accumulation of these FMRFamide‐related peptides in thoracic perisympathetic organs provides the definite evidence for a tagma‐specific distribution of peptidergic neurohormones in neurohaemal release sites of the insect CNS. Excitatory effects of the cockroach FMRFamides were observed on antenna–heart preparations. In addition, the newly described FMRFamides reduce the spike frequency of dorsal‐unpaired median neurons and reduce the intracellular calcium concentration, which may affect the peripheral release of the biogenic amine octopamine.
SNAP‐25 in hippocampal CA1 region is involved in memory consolidation
Authors: Hou Qiuling , Gao Xiang , Zhang Xuehan , Kong Lingwei , Wang Xinming , Bian Wei , Tu Yanyang , Jin Meilei , Zhao Guoping , Li Baoming , Jing Naihe , Yu Lei
Published: September 2004

As a synaptosomal protein, SNAP‐25 plays a role in a number of neuronal functions including axonal growth, dendrite formation, fusion of synaptic vesicles with membrane and the expression of long‐term potentiation (LTP) in the hippocampus. Using a learning/memory behavior screening, we identified SNAP‐25 as one of the differentially expressed genes in the hippocampus upon behavioral training. The inhibition of SNAP‐25 with intracerebroventricular antisense oligonucleotide caused a deficit in long‐ but not short‐term memory for step‐down inhibitory avoidance. Intra‐CA1 infusion of the SNAP‐25 antisense oligonucleotide impaired long‐term contextual fear memory and spatial memory and interfered with the LTP of synaptic transmission in the CA1 region. The inhibitory effect on LTP was not mediated by a pre‐synaptic mechanism because paired pulse facilitation of synaptic transmission was not affected after administration of the antisense oligonucleotide. Together, the results suggest that SNAP‐25 in the CA1 region is involved in memory consolidation.
Monocarboxylate transport inhibition alters retinal function and cellular amino acid levels
Authors: Bui Bang V. , Vingrys Algis J. , Wellard John W. , Kalloniatis Michael
Published: September 2004

We assessed the effect of the in vivo application of monocarboxylate transport inhibitors on retinal function and amino acid immunocytochemistry. We wanted to determine the impact that altered aerobic metabolite availability has on retinal function and the characteristics of amino acid shunting into metabolic pools. Electroretinograms were collected from anaesthetized rats at various times after intravitreal injection of the monocarboxylate transport inhibitors α‐cyano‐4‐hydroxycinnamate (4‐CIN; 2 µL, 0.1–10 mm) or p‐(dipropylsulphamoyl)benzoic acid (probenecid; 1–10 mm). Changes in retinal function were compared with quantitative amino acid immunocytochemical changes in retinas harvested 20 and 40 min after either 4‐CIN or vehicle treatment. The injection of 4‐CIN resulted in a dose‐dependent reduction of the ON‐bipolar cell P2 wave amplitude (20–80%) and delay in its implicit time. The phototransduction sensitivity was mildly reduced whereas the ON‐bipolar cell P2 sensitivity was unaffected. Probenecid induced functional changes similar to those observed with 4‐CIN. We also mapped the amino acid alterations within specific cell classes induced by 4‐CIN application. All neurones displayed a reduced glutamate content averaging 48%; reduced GABA (31%) and glycine (28%) were found within amacrine cells and glutamine was reduced in all cell classes except photoreceptor and Müller cells. All cell classes in the retina demonstrated increases in aspartate (57%), whereas leucine (24%) and ornithine (21%) were only significantly increased in photoreceptor and bipolar cells. The reduction in glutamate immunolabelling in specific retinal cell classes was mirrored by an increase in aspartate levels at these locations. In addition, attenuated glutamine immunolabelling also closely matched the spatial pattern observed for glutamate. Our immunocytochemical analysis provides evidence that monocarboxylate transport inhibition induces a shift in the equilibrium of glutamate transamination reactions involving aspartate throughout the retina whereas photoreceptor and bipolar cells also use glutamate transamination reactions involving ornithine and leucine. The distribution pattern of glutamine secondary to monocarboxylate inhibition suggests that this amino acid is a major precursor for glutamate throughout the retina.
Adrenoceptor subtype‐specific acceleration of the hypoxic depression of excitatory synaptic transmission in area CA1 of the rat hippocampus
Authors: Pearson Tim , Frenguelli Bruno G.
Published: September 2004

The depression of excitatory synaptic transmission by hypoxia in area CA1 of the hippocampus is largely dependent upon the activation of adenosine A1 receptors on presynaptic glutamatergic terminals. As well as adenosine, norepinephrine levels increase in the hypoxic/ischemic hippocampus. We sought to determine the influence of α‐ and β‐adrenoceptor (AR) activation on the hypoxic depression of synaptic transmission utilizing electrophysiological, pharmacological and adenosine sensor techniques. Norepinephrine depressed synaptic transmission and significantly accelerated the hypoxic depression of synaptic transmission. The α‐AR agonist 6‐fluoronorepinephrine mimicked both of these effects whilst the α2‐AR antagonist yohimbine, but not the α1‐AR antagonist urapidil, prevented the actions of 6‐fluoronorepinephrine. In contrast, the β‐AR agonist isoproterenol enhanced synaptic transmission and only accelerated the hypoxic depression of transmission in hypoxia‐conditioned slices in which the hypoxic release of adenosine is reduced. The effects of isoproterenol were blocked by the non‐selective β‐AR antagonist propranolol and the selective β1‐AR antagonist betaxolol. Using an enzyme‐based adenosine sensor we observed that the application of the β‐AR agonist resulted in increased extracellular adenosine during repeated hypoxia. Our results suggest that α2‐AR activation facilitates the hypoxic depression of synaptic transmission probably via the known α2‐AR‐mediated inhibition of presynaptic calcium channels whereas β1‐AR activation does so via increased extracellular adenosine and greater activation of inhibitory adenosine A1 receptors.
Developmental expression of the Ca2+‐binding proteins calretinin and parvalbumin at the calyx of Held of rats and mice
Authors: Felmy Felix , Schneggenburger Ralf
Published: September 2004

Ca2+‐binding proteins of the EF‐hand family are widely expressed in the CNS, and contribute to intracellular Ca2+ buffering in neurons. In nerve terminals, Ca2+‐binding proteins are likely to regulate transmitter release probability and synaptic short‐term‐plasticity. Here, we investigated the developmental expression pattern of calretinin and parvalbumin at a large excitatory synapse, the calyx of Held in the medial nucleus of the trapezoid body (MNTB) of rats and mice. We used two‐colour immunofluorescence imaging with primary antibodies detecting one of the Ca2+‐binding proteins, and a presynaptic marker protein, Rab‐3A. Calretinin was found in nerve terminals of the calyx of Held, but not in postsynaptic principal cells. The presynaptic density of Calretinin staining, and the degree of colocalization with Rab‐3A increased during postnatal development (P6–P31). Surprisingly, not all calyces of Held expressed calretinin. In rats, calretinin‐containing calyces were irregularly interspersed with calretinin‐negative calyces, whereas in mice, calretinin‐positive calyces were preferentially located in the lateral portion of the MNTB. The percentage of calretinin‐positive calyces increased during development, to about 75% and 20% at P30 in rats and in mice, respectively. Parvalbumin was present in the presynaptic calyces of Held and in the nerve fibres entering the MNTB, as well as in the somata of the MNTB principal neurons. An up‐regulation of calretinin and parvalbumin in calyces of Held probably increases the presynaptic Ca2+ buffering strength during postnatal development, but the unexpected heterogeneity of calretinin expression might cause differences in Ca2+ signalling and transmitter release probability between calyces of Held.
Reduced plasticity of cortical whisker representation in adult tenascin‐C‐deficient mice after vibrissectomy
Authors: Cybulska‐Klosowicz Anita , Zakrzewska Renata , Pyza Elzbieta , Kossut Malgorzata , Schachner Melitta
Published: September 2004

The effect of the extracellular matrix recognition molecule tenascin‐C on cerebral plasticity induced by vibrissectomy was investigated with 2‐deoxyglucose (2DG) brain mapping in tenascin‐C‐deficient mice. Unilateral vibrissectomy sparing row C of vibrissae was performed in young adult mice. Two months later, cortical representations of spared row C vibrissae and control row C on the other side of the snout were visualized by [14C]2DG autoradiography. In both wild‐type and tenascin‐C‐deficient mice, cortical representation of the spared row was expanded in all layers of the barrel cortex. However, the effect was significantly more extensive in wild‐type animals than in the mutant. Elimination of tenascin‐C by genetic manipulation thus reduces the effect of vibrissectomy observed in the somatosensory cortex. No increase in number of fibres in the vibrissal nerve of spared vibrissae was seen, and occurrence of additional nerve to the spared follicle was very rare. Thus, in tenascin‐C‐deficient mice functional plasticity seems to be impaired within the CNS.
The remyelinating potential andin vitrodifferentiation of MOG‐expressing oligodendrocyte precursors isolated from the adult rat CNS
Authors: Crang A. J. , Gilson J. M. , Li W.‐W. , Blakemore W. F.
Published: September 2004

There is a long‐standing controversy as to whether oligodendrocytes may be capable of cell division and thus contribute to remyelination. We recently published evidence that a subpopulation of myelin oligodendrocyte glycoprotein (MOG)‐expressing cells in the adult rat spinal cord co‐expressed molecules previously considered to be restricted to oligodendrocyte progenitors [G. Liet al. (2002)Brain Pathol., 12, 463–471]. To further investigate the properties of MOG‐expressing cells, anti‐MOG‐immunosorted cells were grown in culture and transplanted into acute demyelinating lesions. The immunosorting protocol yielded a cell preparation in which over 98% of the viable cells showed anti‐MOG‐ and O1‐immunoreactivity; 12–15% of the anti‐MOG‐immunosorted cells co‐expressed platelet‐derived growth factor alpha receptor (PDGFRα) or the A2B5‐epitope. When cultured in serum‐free medium containing EGF and FGF‐2, 15–18% of the anti‐MOG‐immunosorted cells lost anti‐MOG‐ and O1‐immunoreactivity and underwent cell division. On removal of these growth factors, cells differentiated into oligodendrocytes, or astrocytes and Schwann cells when the differentiation medium contained BMPs. Transplantation of anti‐MOG‐immunosorted cells into areas of acute demyelination immediately after isolation resulted in the generation of remyelinating oligodendrocytes and Schwann cells. Our studies indicate that the adult rat CNS contains a significant number of oligodendrocyte precursors that express MOG and galactocerebroside, molecules previously considered restricted to mature oligodendrocytes. This may explain why myelin‐bearing oligodendrocytes were considered capable of generating remyelinating cells. Our study also provides evidence that the adult oligodendrocyte progenitor can be considered as a source of the Schwann cells that remyelinate demyelinated CNS axons following concurrent destruction of oligodendrocytes and astrocytes.
Microstimulation of V1 input layers disrupts the selection and detection of visual targets by monkeys
Authors: Slocum Warren M. , Tehovnik Edward J.
Published: September 2004

Electrical microstimulation delivered to primary visual cortex (V1) concurrently with the presentation of visual targets interferes with the selection of these targets. To determine the source of this interference, we stimulated the visual input layers of V1 as rhesus monkeys generated saccadic eye movements to visual targets presented at and outside the receptive field of the stimulated neurons. Columns of cells in V1 innervated by the left and right eye are segregated according to eye dominance, such that cells within a column respond best to visual stimuli presented to the ocular dominant eye. Interference was maximal when targets were presented to the ocular dominant eye, moderate when presented to the ocular inferior eye, and negligible when presented to both eyes. Thus, electrical microstimulation of the visual input layers of V1 disrupts the flow of visual information along the geniculostriate pathway. Knowing how electrical stimulation of V1 affects visual behaviour is necessary when using monkeys to develop a visual prosthesis for the blind.