bluuebunny/crossref_metadata_2025 huggingface dataset:
Pxl1p, a Paxillin-like Protein inSaccharomyces cerevisiae, May Coordinate Cdc42p and Rho1p Functions during Polarized Growth
Authors: Gao Xiang-Dong , Caviston Juliane P. , Tcheperegine Serguei E. , Bi Erfei
Published: June 2004

Rho-family GTPases Cdc42p and Rho1p play critical roles in the budding process of the yeast Saccharomyces cerevisiae. However, it is not clear how the functions of these GTPases are coordinated temporally and spatially during this process. Based on its ability to suppress cdc42-Ts mutants when overexpressed, a novel gene PXL1 was identified. Pxl1p resembles mammalian paxillin, which is involved in integrating various signaling events at focal adhesion. Both proteins share amino acid sequence homology and structural organization. When expressed in yeast, chicken paxillin localizes to the sites of polarized growth as Pxl1p does. In addition, the LIM domains in both proteins are the primary determinant for targeting the proteins to the cortical sites in their native cells. These data strongly suggest that Pxl1p is the “ancient paxillin” in yeast. Deletion of PXL1 does not produce any obvious phenotype. However, Pxl1p directly binds to Rho1p-GDP in vitro, and inhibits the growth of rho1-2 and rho1-3 mutants in a dosage-dependent manner. The opposite effects of overexpressed Pxl1p on cdc42 and rho1 mutants suggest that the functions of Cdc42p and Rho1p may be coordinately regulated during budding and that Pxl1p may be involved in this coordination.
The Cochaperone HspBP1 Inhibits the CHIP Ubiquitin Ligase and Stimulates the Maturation of the Cystic Fibrosis Transmembrane Conductance Regulator
Authors: Alberti Simon , Böhse Karsten , Arndt Verena , Schmitz Anton , Höhfeld Jörg
Published: June 2004

The CHIP ubiquitin ligase turns molecular chaperones into protein degradation factors. CHIP associates with the chaperones Hsc70 and Hsp90 during the regulation of signaling pathways and during protein quality control, and directs chaperone-bound clients to the proteasome for degradation. Obviously, this destructive activity should be carefully controlled. Here, we identify the cochaperone HspBP1 as an inhibitor of CHIP. HspBP1 attenuates the ubiquitin ligase activity of CHIP when complexed with Hsc70. As a consequence, HspBP1 interferes with the CHIP-induced degradation of immature forms of the cystic fibrosis transmembrane conductance regulator (CFTR) and stimulates CFTR maturation. Our data reveal a novel regulatory mechanism that determines folding and degradation activities of molecular chaperones.
Arrival, Reversal, and Departure of Neurofilaments at the Tips of Growing Axons
Authors: Uchida Atsuko , Brown Anthony
Published: June 2004

We have investigated the movement of green fluorescent protein-tagged neurofilaments at the distal ends of growing axons by using time-lapse fluorescence imaging. The filaments moved in a rapid, infrequent, and asynchronous manner in either an anterograde or retrograde direction (60% anterograde, 40% retrograde). Most of the anterograde filaments entered the growth cone and most of the retrograde filaments originated in the growth cone. In a small number of cases we were able to observe neurofilaments reverse direction, and all of these reversals occurred in or close to the growth cone. We conclude that neurofilament polymers are delivered rapidly and infrequently to the tips of growing axons and that some of these polymers reverse direction in the growth cone and move back into the axon. We propose that 1) growth cones are a preferential site of neurofilament reversal in distal axons, 2) most retrograde neurofilaments in distal axons originate by reversal of anterograde filaments in the growth cone, 3) those anterograde filaments that do not reverse direction are recruited to form the neurofilament cytoskeleton of the newly forming axon, and 4) the net delivery of neurofilament polymers to growth cones may be controlled by regulating the reversal frequency.
Antagonistic Roles of ESCRT and Vps Class C/HOPS Complexes in the Recycling of Yeast Membrane Proteins
Authors: Bugnicourt Amandine , Froissard Marine , Sereti Kostianna , Ulrich Helle D. , Haguenauer-Tsapis Rosine , Galan Jean-Marc
Published: June 2004

In Saccharomyces cerevisiae, deficiencies in the ESCRT machinery trigger the mistargeting of endocytic and biosynthetic ubiquitinated cargoes to the limiting membrane of the vacuole. Surprisingly, impairment of this machinery also leads to the accumulation of various receptors and transporters at the plasma membrane in both yeast and higher eukaryotes. Using the well-characterized yeast endocytic cargo uracil permease (Fur4p), we show here that the apparent stabilization of the permease at the plasma membrane in ESCRT mutants results from an efficient recycling of the protein. Whereas several proteins as well as internalized dyes are known to be recycled in yeast, little is known about the machinery and molecular mechanisms involved. The SNARE protein Snc1p is the only cargo for which the recycling pathway is well characterized. Unlike Snc1p, endocytosed Fur4p did not pass through the Golgi apparatus en route to the plasma membrane. Although ubiquitination of Fur4p is required for its internalization, deubiquitination is not required for its recycling. In an attempt to identify actors in this new recycling pathway, we found an unexpected phenotype associated with loss of function of the Vps class C complex: cells defective for this complex are impaired for recycling of Fur4p, Snc1p, and the lipophilic dye FM4-64. Genetic analyses indicated that these phenotypes were due to the functioning of the Vps class C complex in trafficking both to and from the late endosomal compartment.
Protective lactogenic immunity conferred by an edible peptide vaccine to bovine rotavirus produced in transgenic plants
Authors: Wigdorovitz Andrés , Mozgovoj Marina , Santos María J. Dus , Parreño Viviana , Gómez Cristina , Pérez-Filgueira Daniel M. , Trono Karina G. , Ríos Raúl D. , Franzone Pascual M. , Fernández Fernando , Carrillo Consuelo , Babiuk Lorne A. , Escribano José M. , Borca Manuel V.
Published: June 2004

Vaccines produced in transgenic plants constitute a promising alternative to conventional immunogens, presenting the possibility of stimulating secretory and systemic immunity against enteric pathogens when administered orally. Protection against enteric pathogens affecting newborn animals requires, in most cases, the stimulation of lactogenic immunity. Here, the group presents the development of an experimental immunogen based on expression of an immunorelevant peptide, eBRV4, of the VP4 protein of bovine rotavirus (BRV), which has been described as harbouring at least one neutralizing epitope as well as being responsible for the adsorption of the virus to epithelial cells. The eBRV4 epitope was efficiently expressed in transgenic alfalfa as a translational fusion protein with the highly stable reporter enzyme β-glucuronidase (βGUS), which served as a carrier, stabilized the synthesized peptide and facilitated screening for the higher expression levels in plants. Correlation of expression of the eBRV4 epitope in plants with those presenting the highest βGUS activities was confirmed by a Western blot assay specific for the BRV peptide. The eBRV4 epitope expressed in plants was effective in inducing an anti-rotavirus antibody response in adult female mice when administered either intraperitoneally or orally and, more importantly, suckling mice born from immunized female mice were protected against oral challenge with virulent rotavirus. These results demonstrate the feasibility of inducing lactogenic immunity against an enteric pathogen using an edible vaccine produced in transgenic plants.
Feline immunodeficiency virus subtypes A, B and C and intersubtype recombinants in Ontario, Canada
Authors: Reggeti F. , Bienzle D.
Published: June 2004

Knowledge of the geographical distribution of feline immunodeficiency virus (FIV) subtypes is important for understanding different disease courses and for vaccine design. Intersubtype recombination may develop in areas where more than one subtype is prevalent and has the potential to create new transmittable variants with novel pathogenic properties. In this study, 40 FIV-positive DNA samples were classified by sequence analysis of the LTR–gag region. Phylogenetic analysis indicated that 32 Canadian FIV isolates clustered with previously identified subtypes A, B and C and that subtype A was most frequent in Ontario. Four strains with inconsistent clade assignment were further analysed by sequencing of the env–LTR regions. Comparisons of phylogenetic trees constructed from the two different regions of the genome and analysis of similarities to reference sequences yielded classification of three samples as A/B and one as A/C intersubtype recombinants. Although the A/B recombinant samples were obtained from unrelated cats in geographically disparate regions, a common breakpoint was consistently identified within gag. In addition, there was no evidence of co-infection with parental strains of subtypes A and B as indicated by PCR-based limiting dilution assays, although these assays allowed for the identification of two different recombinant viruses co-existing in one sample. Both sequences contained the same breakpoint. These findings suggested that a new circulating recombinant FIV may be enzootic in Ontario.
Characterization of in vitro and in vivo phenotypes of poliovirus type 1 mutants with reduced viral protein synthesis activity
Authors: Arita Minetaro , Shimizu Hiroyuki , Miyamura Tatsuo
Published: June 2004

Sabin vaccine strains of poliovirus (PV) contain major attenuation determinants in the internal ribosomal entry site (IRES), an area that directs viral protein synthesis. To examine the effect of reduced viral protein synthesis on PV neurovirulence, spacer sequences, consisting of short open reading frames of different lengths, were introduced between the IRES and the initiation codon of viral polyprotein, resulting in PV mutants with reduced viral protein synthesis. These PV mutants had a viral protein synthesis activity 8·8–55 % of that of the parental Mahoney strain as measured in HeLa S3 cells. Only viruses with more than 28 % of the wild-type activity had intact spacer sequences following plaque purification. Mutants with 17 % or 21 % of the wild-type activity were unstable and a mutant with 8·8 % was lethal. The neurovirulence of PV mutants was evaluated in transgenic mice carrying the human PV receptor gene. In this test, mutants with more than 28 % of the wild-type activity remained neurovirulent, while a mutant with 17 % of wild-type activity exhibited a partially attenuated phenotype. This mutant stably replicated in the spinal cord; however, the stability was severely affected during the course of virus infection from the cerebrum to the spinal cord. These results suggest that reduced viral protein synthesis activity as measured in cultured cells (17–55 % of the wild-type activity) is not the main determinant of PV attenuation.
Strand transfer to the 5′ part of a tRNA as a mechanism for retrovirus patch-repair recombination in vivo
Authors: Carrasco María L. , Duch Mogens , Pedersen Finn Skou
Published: June 2004

By screening for marker-cassette deletion mutants of a murine leukaemia virus-based replication-competent vector, two occurrences of tRNA sequence patch insertions were identified. In one of the cases, 28 nucleotides from the 5′ end of tRNALys4 were inserted in the plus-strand orientation, which points to a novel strand-transfer mechanism to tRNAs during reverse transcriptase-mediated retroviral recombination.
Downregulation of the cellular adhesion molecule Thy-1 (CD90) by cytomegalovirus infection of human fibroblasts
Authors: Leis Martina , Marschall Manfred , Stamminger Thomas
Published: June 2004

The deregulation of cellular adhesion molecules by human cytomegalovirus (HCMV) appears to be correlated with the development of vascular disease. In this study, it was investigated whether the expression of Thy-1 (CD90), a member of the immunoglobulin superfamily of adhesion molecules with constitutive expression on fibroblast cells, is modulated following infection with HCMV. It was observed that Thy-1 cell surface expression decreased significantly during the course of infection. Addition of neutralizing antibodies, as well as UV inactivation of virus, prevented Thy-1 downregulation. In contrast, inhibition of virus replication by cidofovir did not alter Thy-1 regulation by HCMV, indicating that immediate-early (IE) and/or early (E) gene products are responsible. Interestingly, after infection of fibroblasts with a recombinant GFP-expressing virus, infected as well as non-infected cells showed a reduced Thy-1 cell surface expression. From these findings, it is concluded that IE or E gene products of HCMV induce a so far unidentified soluble factor that mediates Thy-1 downregulation.
An important determinant of the ability of Turnip mosaic virus to infect Brassica spp. and/or Raphanus sativus is in its P3 protein
Authors: Suehiro Noriko , Natsuaki Tomohide , Watanabe Tomoko , Okuda Seiichi
Published: June 2004

Turnip mosaic virus(TuMV, genusPotyvirus, familyPotyviridae) infects mainly cruciferous plants. Isolates Tu-3 and Tu-2R1 of TuMV exhibit different infection phenotypes in cabbage (Brassica oleraceaL.) and Japanese radish (Raphanus sativusL.). Infectious full-length cDNA clones, pTuC and pTuR1, were constructed from isolates Tu-3 and Tu-2R1, respectively. Progeny virus derived from infections with pTuC induced systemic chlorotic and ringspot symptoms in infected cabbage, but no systemic infection in radish. Virus derived from plants infected with pTuR1 induced a mild chlorotic mottle in cabbage and infected radish systemically to induce mosaic symptoms. By exchanging genome fragments between the two virus isolates, the P3-coding region was shown to be responsible for systemic infection by TuMV and the symptoms it induces in cabbage and radish. Moreover, exchanges of smaller parts of the P3 region resulted in recombinants that induced complex infection phenotypes, especially the combination of pTuC-derived N-terminal sequence and pTuR1-derived C-terminal sequence. Analysis by tissue immunoblotting of the inoculated leaves showed that the distributions of P3-chimeric viruses differed from those of the parents, and that the origin of the P3 components affected not only virus accumulation, but also long-distance movement. These results suggest that the P3 protein is an important factor in the infection cycle of TuMV and in determining the host range of this and perhaps other potyviruses.
The rat cytomegalovirus homologue of parvoviral rep genes, r127, encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication
Authors: van Cleef Koen W. R. , Scaf Wendy M. A. , Maes Karen , Kaptein Suzanne J. F. , Beuken Erik , Beisser Patrick S. , Stassen Frank R. M. , Grauls Gert E. L. M. , Bruggeman Cathrien A. , Vink Cornelis
Published: June 2004

An intriguing feature of the rat cytomegalovirus (RCMV) genome is open reading frame (ORF) r127, which shows similarity to the rep genes of parvoviruses as well as the U94 genes of human herpesvirus type 6A (HHV-6A) and 6B (HHV-6B). Counterparts of these genes have not been found in other herpesviruses. Here, it is shown that the r127 gene is transcribed during the early and late phases of virus replication in vitro as an unspliced 1·1 kb transcript containing the complete r127 ORF. Transcripts of r127 were also detected in various organs of RCMV-infected rats at 1 week post-infection (p.i.), but only in the salivary gland at 4 months p.i. Using rabbit polyclonal antibodies raised against the r127-encoded protein (pr127), pr127 was found to be expressed as early as 12 h p.i. within the nuclei of RCMV-infected cells in vitro. Expression of pr127 was also observed within the nuclei of cells in various organs of RCMV-infected rats at 3 weeks p.i. Moreover, pr127 was demonstrated to bind single- as well as double-stranded DNA. Finally, an RCMV r127 deletion mutant (RCMVΔr127) was generated, in which the r127 ORF was disrupted. This deletion mutant, however, was shown to replicate with a similar efficiency as wild-type RCMV (wt RCMV), both in vitro and in vivo. Taken together, it is concluded that the RCMV r127 gene encodes a nuclear protein with single- and double-stranded DNA-binding activity that is dispensable for virus replication, not only in vitro, but also during the acute phase of infection in vivo.
Human immunodeficiency virus 1 downregulates cell surface expression of the non-classical major histocompatibility class I molecule HLA-G1
Authors: Derrien Muriel , Pizzato Nathalie , Dolcini Guillermina , Menu Elisabeth , Chaouat Gérard , Lenfant Françoise , Barré-Sinoussi Françoise , Bouteiller Philippe Le
Published: June 2004

Human immunodeficiency virus 1 (HIV-1) downregulates cell surface expression of HLA-A and HLA-B but not HLA-C or HLA-E to ultimately escape immune defences. Here, it is shown that cell surface expression of the non-classical HLA-G1 is also downregulated by HIV-1, by using co-transfection experiments and infection with cell-free HIV-1 of HLA-G1-expressing U87 glioma cells or macrophages in primary culture. Moreover, co-transfection experiments using proviruses deleted in eitherneforvpuor plasmids encoding HIV-1 Nef and Vpu mixed together with a HLA-G1-expressing construct demonstrated that HLA-G1 downregulation is Nef-independent and Vpu-dependent, contrasting with the Nef- and Vpu-dependent HLA-A2 downregulation. Together, these results show that the decrease of HLA-A2 and HLA-G1 caused by HIV-1 occurs through distinct mechanisms.
Inhibition of influenza virus matrix (M1) protein expression and virus replication by U6 promoter-driven and lentivirus-mediated delivery of siRNA
Authors: Hui Eric Ka-Wai , Yap Ee Ming , An Dong Sung , Chen Irvin S. Y. , Nayak Debi P.
Published: June 2004

Small interfering RNA (siRNA)-induced RNA degradation has been used recently as an antivirus agent to inhibit specific virus replication. This report shows that 21 nt duplexes of siRNA of the influenza virus M gene can cause specific inhibition of influenza virus matrix (M1) protein expression in transfected 293T cells. Furthermore, it is shown that a lentivirus vector can be used to effectively deliver M gene siRNAs into Madin–Darby canine kidney cells and can cause specific inhibition of M1 protein expression and influenza virus replication. Therefore, lentivirus-mediated delivery of siRNA and gene silencing can be used in studying the specific functions of virus genes in replication and may have a potential therapeutic application.
Analysis of the RNA of Potato yellow vein virus: evidence for a tripartite genome and conserved 3′-terminal structures among members of the genus Crinivirus
Authors: Livieratos I. C. , Eliasco E. , Müller G. , Olsthoorn R. C. L. , Salazar L. F. , Pleij C. W. A. , Coutts R. H. A.
Published: June 2004

Double-stranded RNA preparations produced from potato plants graft-inoculated with a Peruvian isolate of Potato yellow vein virus (PYVV; genus Crinivirus, family Closteroviridae) contain five RNA species denoted RNA 1, RNA 2, RNA 3, x and y of approximately 8, 5·3, 3·8, 2·0 and 1·8 kbp, respectively. The complete nucleotide sequences of PYVV RNAs 1, 2 and 3 and Northern hybridization analysis showed that PYVV RNA 1 contained the replication module and an additional open reading frame (p7), while two distinct species, RNAs 2 and 3, contain the Closteroviridae hallmark gene array. Pairwise comparisons and phylogeny of genome-encoded proteins showed that PYVV shares significant homology with other criniviruses but is most closely related to the Trialeurodes vaporariorum-vectored Cucumber yellows virus. Secondary structure prediction of the 3′-untranslated regions of all three PYVV RNAs revealed four conserved stem–loop structures and a 3′-terminal pseudoknot structure, also predicted for all fully characterized members of the genus Crinivirus and some members of the genera Closterovirus and Ampelovirus.
Tax protein of human T-cell leukaemia virus type 1 induces interleukin 17 gene expression in T cells
Authors: Dodon Madeleine Duc , Li Zhenlin , Hamaia Samir , Gazzolo Louis
Published: June 2004

Tax protein of human T-cell leukaemia virus type 1 (HTLV-1) induces the expression of several cellular genes that are involved in T cell activation and proliferation. In this study, it was observed that Tax upregulated the expression of human interleukin 17 (IL17), a cytokine mainly produced by activated CD4+memory T cells. Indeed, IL17 mRNA was highly expressed in HTLV-1-infected T cells as well as in Tax-expressing Jurkat T cells, whereas it was not detectable in HTLV-1-negative T cell lines. The clinical relevance of these observations was further demonstrated by quantitative assessment of IL17 expression in lymphocytes isolated from one HTLV-1-infected patient. To define the transcriptional activation of the IL17 gene by Tax, the 5′-flanking region of this gene was cloned and a reporter gene analysis performed. The presence of a Tax-responsive region spanning 614 bp upstream of the initiation start site was identified, in HeLa as well as in Jurkat cells, stimulated with phorbol myristate acetate and Ca2+ionophore. Finally, Tax mutants were used to show that the transcriptional activation of the IL17 promoter by Tax was dependent on the CREB/ATF pathway. As IL17 upregulates the expression of several pro-inflammatory cytokines, these observations provide new insights into the involvement of the Tax protein in the pathophysiology of HTLV-1-associated inflammatory disorders.
Full-length open reading frame of a recombinant hepatitis C virus strain from St Petersburg: proposed mechanism for its formation
Authors: Kalinina Olga , Norder Helene , Magnius Lars O.
Published: June 2004

The full-length ORFs for the hepatitis C virus recombinant RF1_2k/1b (N687) and the non-recombinant 1b strain N589 were sequenced. A single recombination point was found and the sizes of the genes (C, E1, E2, p7, NS2, NS3, NS4 and NS5) were according to the parental subtypes. The PKR-eIF2α phosphorylation site homology domain sequence of the E2 protein was identical to those of genotype 2 strains, while the IFN-α-sensitivity-determining region of the NS5A protein was identical to those of interferon-resistant 1b strains. For the parental strains, two hairpin structures, HS1 and HS2, were predicted for the plus-strand up- and downstream of the crossover site, which were not present in the recombinant strain. HS2 shared similarity with the motif1 hairpin of turnip crinkle virus RNA that binds to the RNA-dependent RNA polymerase and facilitates 3′-terminal extension during recombination. This study suggests that RF1_2k/1b has emerged by homologous recombination during minus-strand synthesis via template switching because of constraints imposed by the HS1 hairpin of the 3′-parental genome.
The X protein of Borna disease virus regulates viral polymerase activity through interaction with the P protein
Authors: Poenisch Marion , Unterstab Gunhild , Wolff Thorsten , Staeheli Peter , Schneider Urs
Published: June 2004

Borna disease virus polymerase activity is negatively regulated by the viral X protein. Using a virus minireplicon system it was found that all X mutants that no longer interacted with the viral P protein failed to exhibit significant inhibitory activity. The action of X could further be neutralized by expression of a P fragment that contained the X interaction domain but lacked all domains known to mediate interaction with other viral proteins. X thus appears to regulate the activity of the Borna disease virus polymerase by targeting the polymerase cofactor P.
Complete nucleotide sequence and genome organization of Grapevine leafroll-associated virus 3, type member of the genus Ampelovirus
Authors: Ling Kai-Shu , Zhu Hai-Ying , Gonsalves Dennis
Published: June 2004

This study reports on the complete genome sequence of Grapevine leafroll-associated virus 3, the type member of the genus Ampelovirus. The genome is 17 919 nt in size and contains 13 open reading frames (ORFs). Previously, the sequence of 13 154 nt of the 3′-terminal of the genome was reported. The newly sequenced portion contains a 158 nt 5′ UTR, a single papain-like protease and a methyltransferase-like (MT) domain. ORF1a encodes a large polypeptide with a molecular mass of 245 kDa. With a predicted +1 frameshift, the large fusion protein generated from ORF1a/1b would produce a 306 kDa polypeptide. Phylogenetic analysis using MT domains further supports the creation of the genus Ampelovirus for mealy-bug-transmitted viruses in the family Closteroviridae.
A novel protein expression strategy using recombinant bovine respiratory syncytial virus (BRSV): modifications of the peptide sequence between the two furin cleavage sites of the BRSV fusion protein yield secreted proteins, but affect processing and function of the BRSV fusion protein
Authors: König Patricia , Giesow Katrin , Schuldt Kathrin , Buchholz Ursula J. , Keil Günther M.
Published: June 2004

The bovine respiratory syncytial virus (BRSV) fusion (F) protein is cleaved at two furin cleavage sites, which results in generation of the disulfide-linked F1 and F2 subunits and release of an intervening peptide of 27 aa (pep27). A series of mutated open reading frames encoding F proteins that lacked the entire pep27, that contained an arbitrarily chosen 23 aa sequence instead of pep27 or in which pep27 was replaced by the amino acid sequences for the bovine cytokines interleukin 2 (boIL2), interleukin 4 (boIL4) or gamma interferon (boIFN-γ) was constructed. Transient expression experiments revealed that the sequence of the intervening peptide influenced intracellular transport, maturation of the F protein and F-mediated syncytium formation. Expression of boIL2, boIL4 or boIFN-γ in place of pep27 resulted in secretion of the cytokines into the culture medium. All mutated F proteins except the boIFN-γ-containing variant could be expressed by and were functional for recombinant BRSV. Characterization of the cell culture properties of the recombinants demonstrated that the amino acid sequence between the two furin cleavage sites affected entry into target cells, direct spreading of virions from cell to cell and virus growth. Secretion of boIL2 and boIL4 into the medium of cells infected with the respective recombinants demonstrated that the F protein can be used to express secreted heterologous bioactive peptides or (glyco)proteins, which might be of interest for the development of novel RSV vaccines.
Prion protein gene polymorphisms in healthy and scrapie-affected Spanish sheep
Authors: Acín Cristina , Martín-Burriel Inmaculada , Goldmann Wilfred , Lyahyai Jaber , Monzón Marta , Bolea Rosa , Smith Allister , Rodellar Clementina , Badiola Juan José , Zaragoza Pilar
Published: June 2004

The Rasa Aragonesa sheep is the second most important Spanish breed after the Merino breed. Reported here is the prion protein (PrP) haplotype frequency distribution for scrapie-related codons (136, 154 and 171) and a sequencing study of the complete PrP gene open reading frame for this breed and six other closely related breeds. The study includes four scrapie-affected sheep flocks belonging to Rasa Aragonesa and Rasa Navarra breeds. Thirty-eight scrapie-affected sheep, 502 healthy sheep from scrapie-affected flocks and 905 sheep from a breed survey were genotyped. The most frequent PrP haplotype in both scrapie and healthy flocks was ARQ, which was found at significantly higher frequency in scrapie-affected sheep. The susceptibility-associated VRQ haplotype was found at low frequencies in six out of eight breeds, but was not present in the 38 scrapie-affected sheep. The resistance-associated ARR haplotype was found in all breeds except one (Ojinegra) at frequencies ⩾14 %. Fourteen amino acid polymorphisms were detected in these Spanish sheep, including the known amino acid substitutions at codons 112, 136, 141, 143, 154, 171 and 176, and new polymorphisms at codons 101 (Q→R), 151 (R→G), 151 (R→H), 172 (Y→D) and 175 (Q→E). Most of the novel polymorphic codons show frequencies lower than 5 %.
Internal cleavage of the human cytomegalovirus UL37 immediate-early glycoprotein and divergent trafficking of its proteolytic fragments
Authors: Mavinakere Manohara S. , Colberg-Poley Anamaris M.
Published: June 2004

The human cytomegalovirus UL37 gene encodes at least three isoforms, which share N-terminal UL37 exon 1 (UL37x1) sequences. UL37 proteins traffic dually into the endoplasmic reticulum (ER) and to mitochondria. Trafficking of the UL37 glycoprotein (gpUL37) in relation to its post-translational processing was investigated. gpUL37 is internally cleaved in the ER and its products traffic differentially. Its C-terminal fragment (UL37COOH) is ER-localized and N-glycosylated. Unlike conventional ER signal sequences, its N-terminal () fragment is stable and traffics to mitochondria. Inhibition of N-glycosylation did not block pUL37 cleavage and dramatically decreased the levels of but not of UL37COOH. pUL37M, which differs from gpUL37 by the lack of residues 178–262 and hence the UL37x3 consensus signal peptidase cleavage site, traffics into the ER and mitochondria, but is neither cleaved nor N-glycosylated. This finding of a relationship between ER processing and mitochondrial importation of UL37 proteins is unique for herpesvirus proteins.
Experience‐dependent neuronal specialization and functional organization in the central auditory area of a songbird
Authors: Cousillas Hugo , Richard Jean‐Pierre , Mathelier Maryvonne , Henry Laurence , George Isabelle , Hausberger Martine
Published: June 2004

The effect of early experience on brain development was investigated in the central auditory area of a songbird, the field L complex, which is analogous to the mammalian auditory cortex. Multi‐unit recordings of auditory responses in the field L complex of adult starlings raised without any experience of adult song during development provide strong evidence of developmental plasticity both in the neuronal responses and in the functional organization of this area. Across the entire area, experimental birds, separated from adults from the age of 1 week old until they were 2 years old, had a much larger number of neurons that responded to all the stimuli than did control birds. The well‐known tonotopy demonstrated in adult wild birds using the same procedure was altered. This study is the first to bring evidence of developmental plasticity in the organization of the central auditory areas in songbirds. These results are discussed in relation to other reports on effects of early experience on brain development.
Allele‐dependent changes of olivocerebellar circuit properties in the absence of the voltage‐gated potassium channels Kv3.1 and Kv3.3
Authors: McMahon Anne , Fowler Stephen C. , Perney Teresa M. , Akemann Walther , Knöpfel Thomas , Joho Rolf H.
Published: June 2004

Double‐mutant mice (DKO) lacking the two voltage‐gated K+ channels Kv3.1 and Kv3.3 display a series of phenotypic alterations that include ataxia, myoclonus, tremor and alcohol hypersensitivity. The prominent cerebellar expression of mRNAs encoding Kv3.1 and Kv3.3 subunits raised the question as to whether altered electrical activity resulting from the lack of these K+ channels might be related to the dramatic motor changes. We used the tremorogenic agent harmaline to probe mutant mice lacking different K+ channel alleles for altered olivocerebellar circuit properties. Harmaline induced the characteristic 13‐Hz tremor in wildtype mice (WT); however, no tremor was observed in DKO suggesting that the ensemble properties of the olivocerebellar circuitry are altered in the absence of Kv3.1 and Kv3.3 subunits. Harmaline induced tremor in Kv3.1‐single mutants, but it was of smaller amplitude and at a lower frequency indicating the participation of Kv3.1 subunits in normal olivocerebellar system function. In contrast, harmaline tremor was virtually absent in Kv3.3‐single mutants indicating an essential role for Kv3.3 subunits in tremor induction by harmaline. Immunohistochemical staining for Kv3.3 showed clear expression in the somata and proximal dendrites of Purkinje cells and in their axonal projections to the deep cerebellar nuclei (DCN). In DCN, both Kv3.1 and Kv3.3 subunits are expressed. Action potential duration is increased by ≈ 100% in Purkinje cells from Kv3.3‐single mutants compared to WT or Kv3.1‐single mutants. We conclude that Kv3.3 channel subunits are essential for the olivocerebellar system to generate and sustain normal harmaline tremor whereas Kv3.1 subunits influence tremor amplitude and frequency.
Neonatal methamphetamine administration induces region‐specific long‐term neuronal morphological changes in the rat hippocampus, nucleus accumbens and parietal cortex
Authors: Williams Michael T. , Brown Russell W. , Vorhees Charles V.
Published: June 2004

Previous studies have demonstrated that rats exposed to methamphetamine (MA) during the neonatal period display deficits in spatial learning and memory. The underlying correlates are unknown; therefore, this study was devised to determine whether neuronal changes occur in the dentate gyrus (DG), nucleus accumbens (NAcc) and cortex of adult rats exposed to 10 mg/kg MA administered four times daily from P11–20 using Golgi–Cox staining [Gibb, R. & Kolb, B. (1998) J. Neurosci. Meth., 79, 1–4]. The DG and NAcc demonstrated a decrease in the number of spines per neuron and the NAcc showed an associated decrease in dendritic length. Selective changes in cortex were observed because increased dendritic length in the parietal cortex occurred with no change in the number of spines, and no differences were noted for either dendritic length or spines in the medial frontal cortex. The data suggest a potential cause for the learning and memory deficits induced by neonatal MA exposure; however, the underlying mechanism that produces these neuronal changes is unknown.
Status epilepticus in immature rats leads to behavioural and cognitive impairment and epileptogenesis
Authors: Kubová Hana , Mareš Pavel , Suchomelová Lucie , Brožek Gustav , Druga Rastislav , Pitkänen Asla
Published: June 2004

It remains under dispute whether status epilepticus (SE) in the perinatal period or early childhood or the underlying neuropathology is the cause of functional impairment later in life. The present study examined whether SE induced by LiCl–pilocarpine in normal immature brain (at the age of 12 or 25 days; P12 or P25) causes cognitive decline and epileptogenesis, and the data were compared to those of rats undergoing SE as adults. Rats in the P12 group had impaired memory (repeated exposure to open‐field paradigm) and emotional behaviour (lower proportion of open‐arm entries and higher incidence of risk assessment period in elevated plus‐maze) when assessed 3 months after SE, although not as severe as in the older age groups. Importantly, video‐electroencephalography monitoring 3 months after SE demonstrated that 25% of rats in the P12 and 50% in P25 group developed spontaneous seizures. Only nonconvulsive seizures (ictal activity in hippocampus accompanied by automatisms) were recorded in the P12 group whereas rats in the P25 group exhibited clonic convulsions. The present findings indicate that SE is harmful to the immature brain as early as P12, which might be compared with early infancy in humans.
Continuous cytosine‐b‐D‐arabinofuranoside infusion reduces ectopic granule cells in adult rat hippocampus with attenuation of spontaneous recurrent seizures following pilocarpine‐induced status epilepticus
Authors: Jung Keun‐Hwa , Chu Kon , Kim Manho , Jeong Sang‐Wuk , Song Young‐Mok , Lee Soon‐Tae , Kim Jin‐Young , Lee Sang Kun , Roh Jae‐Kyu
Published: June 2004

Brief or prolonged seizures induce various patterns of plasticity. Axonal or dendritic remodelling and development of ectopic granule cells have been described in the hilus and molecular layer of the adult rodent hippocampus. Hippocampal cell proliferation also occurs after seizures. However, whether the seizure‐induced cell proliferation plays a pathological or reparative role in the epileptic brain is unknown. In this study, we attempted to suppress the seizure‐induced cell proliferation with the antimitotic agent cytosine‐b‐D‐arabinofuranoside (Ara‐C) and to examine the development of spontaneous recurrent seizures (SRS). Experimental status epilepticus was induced with pilocarpine, and Ara‐C or vehicle alone was infused continuously with an osmotic minipump. SRS were video‐monitored. BrdU immunohistochemistry was used for the spatial and temporal analysis of hippocampal cell proliferation, and double labelling with NeuN, calbindin and GFAP antibodies was performed for the differentiation of BrdU‐positive cells. Timm staining was also performed for evaluation of mossy fibre sprouting (MFS). With continuous Ara‐C infusion, the likelihood of developing SRS was decreased and, during the latent period, the development of ectopic granule cells in the hilus and new glia in the CA1 area was reduced when compared with the vehicle‐infused group, while MFS was not altered. The results suggest that the hippocampal cell proliferation plays a pro‐epileptogenic role rather than a compensatory role, and that the epileptogenic process may be associated with the generation of new glia in the CA1 area and/or new neurons in the dentate gyrus, particularly the ectopically located hilar granule cells.
The LIM homeobox gene, L3/Lhx8, is necessary for proper development of basal forebrain cholinergic neurons
Authors: Mori Tetsuji , Yuxing Zhang , Takaki Hiromi , Takeuchi Mayumi , Iseki Ken , Hagino Seita , Kitanaka Jun‐ichi , Takemura Motohiko , Misawa Hidemi , Ikawa Masahito , Okabe Masaru , Wanaka Akio
Published: June 2004

Basal forebrain cholinergic neurons (BFCNs) are involved in cognitive functions such as learning and memory, and are affected in several neurodegenerative diseases (e.g. Alzheimer's disease). Despite their importance, the molecular mechanisms of their development are not fully elucidated. A recent report demonstrated that some BFCNs in adult rat are positive for L3/Lhx8, a LIM homeobox transcription factor. To examine the function of L3/Lhx8 in the development of BFCNs, we generated L3/Lhx8 gene‐disrupted mice. In these mice, cells expressing cholinergic neuron markers, such as choline acetyltransferase, vesicular acetylcholine transporter and p75 low‐affinity NGF receptor, were markedly reduced in the basal forebrain, whereas other cholinergic neurons including brain stem and spinal motor neurons expressed the markers. Neurotransmitter phenotypes other than cholinergic in the basal forebrain appeared intact. From these results, we suggested that L3/Lhx8 has a pivotal and specific role in the development and/or maintenance of BFCNs.
β‐Endorphin elevations in the ventral tegmental area regulate the discriminative effects of Δ‐9‐tetrahydrocannabinol
Authors: Solinas M. , Zangen A. , Thiriet N. , Goldberg S. R.
Published: June 2004

β‐Endorphin is an endogenous opioid that produces behavioral effects similar to heroin and morphine and is released in the nucleus accumbens by cocaine, amphetamine and ethanol, suggesting a general involvement in the reinforcing effects of abused drugs. Here we show that, in rats, Δ‐9‐tetrahydrocannabinol (THC), the main psychoactive ingredient in cannabis, produces large increases in extracellular levels of β‐endorphin in the ventral tegmental area and lesser increases in the shell of the nucleus accumbens. We then used a two‐lever choice THC‐discrimination procedure to investigate whether THC‐induced changes in endogenous levels of β‐endorphin regulate the discriminative effects of THC. In rats that had learned to discriminate injections of THC from injections of vehicle, the opioid agonist morphine did not produce THC‐like discriminative effects but markedly potentiated discrimination of THC. Conversely, the opioid antagonist naloxone reduced the discriminative effects of THC. Bilateral microinjections of β‐endorphin directly into the ventral tegmental area, but not into the shell of the nucleus accumbens, markedly potentiated the discriminative effects of ineffective threshold doses of THC but had no effect when given alone. This potentiation was blocked by naloxone. Together these results indicate that certain psychotropic effects of THC related to drug abuse liability are regulated by THC‐induced elevations in extracellular β‐endorphin levels in brain areas involved in opiate reward and reinforcement processes.
S1P and LPA trigger Schwann cell actin changes and migration
Authors: Barber Siân C. , Mellor Harry , Gampel Alex , Scolding Neil J.
Published: June 2004

The processes by which a Schwann cell (SC) migrates towards, wraps around and, in some cases, myelinates an axon are incompletely understood. The complex morphological rearrangements involved in these events require fundamental changes in the actin cytoskeleton. Sphingosine 1‐phosphate (S1P) and lysophosphatidic acid (LPA) are two modulators of the actin cytoskeleton, and receptors for these signalling lipids are expressed on SCs at the time of differentiation. Previous work has revealed a role for LPA in SC survival, morphology and differentiation, but the effects of S1P have received less attention. Here we show that S1P and LPA both cause major rearrangements to the actin cytoskeleton in primary rat SCs and the SCL4.1/F7 rat SC line. S1P and LPA caused formation of lamellipodia and a circular geodesic actin network. We also show that S1P and LPA increased cell migration. The small GTPases RhoA and Rac1 were both activated by S1P/LPA treatment, but the actin rearrangements were dependent on Rac1 and not RhoA. These effects of S1P/LPA could be mimicked by SCL4.1/F7 cell‐conditioned medium, which was found to contain S1P. Reduction in cellular synthesis of S1P by adding the sphingosine kinase inhibitor dimethyl sphingosine during medium conditioning reduced the ability of conditioned medium to cause actin rearrangements. These results support a role for S1P as an autocrine signal regulating the actin cytoskeleton during Schwann cell development.
Brain‐derived neurotrophic factor signaling modifies hippocampal gene expression during epileptogenesis in transgenic mice
Authors: Lähteinen Sari , Pitkänen Asla , Knuuttila Juha , Törönen Petri , Castrén Eero
Published: June 2004

Brain‐derived neurotrophic factor (BDNF) regulates neuronal survival, differentiation and plasticity. It has been shown to promote epileptogenesis and transgenic mice with decreased and increased BDNF signaling show opposite alterations in epileptogenesis. However, the mechanisms of BDNF action are largely unknown. We studied the gene expression changes 12 days after kainic acid‐induced status epilepticus in transgenic mice overexpressing either the functional BDNF receptor trkB or a dominant‐negative truncated trkB. Epileptogenesis produced marked changes in expression of 27 of 1090 genes. Cluster analysis revealed BDNF signalling‐mediated regulation of functional gene classes involved in cellular transport, DNA repair and cell death, including kinesin motor kinesin family member 3A involved in cellular transport. Furthermore, the expression of cytoskeletal and extracellular matrix components, such as tissue inhibitor of metalloproteinase 2 was altered, emphasizing the importance of intracellular transport and interplay between neurons and glia during epileptogenesis. Finally, mice overexpressing the dominant‐negative trkB, which were previously shown to have reduced epileptogenesis, showed a decrease in mRNAs of several growth‐associated genes, including growth‐associated protein 43. Our data suggest that BDNF signaling may partly mediate the development of epilepsy and propose that regrowth or repair processes initiated by status epilepticus and promoted by BDNF signaling may not be as advantageous as previously thought.
Clock gene mRNA and protein rhythms in the pineal gland of mice
Authors: Karolczak Magdalena , Burbach Guido J. , Sties Gabriele , Korf Horst‐Werner , Stehle Jörg H.
Published: June 2004

In vertebrates, the rhythmic transcription of clock genes, regulated by their own gene products, provides the basis for self‐sustaining circadian clockworks. These endogenous clocks are lost in most mammalian tissues, but not in the central pacemaker of the hypothalamic suprachiasmatic nucleus (SCN). An interesting model system to understand this phylogenetic shift in function of clock gene products is the rodent pineal gland, as its intrinsic clockwork was replaced during evolution by an input‐dependent oscillator. By means of immunohistochemistry, immunoblotting and real time PCR, we investigated the day/night expression profiles of all major clock genes and their products in the pineal gland of one melatonin‐proficient and one melatonin‐deficient mouse strain. All clockwork elements known to be indispensable for a sustained rhythm generation in the SCN were also found in the pineal organ of both mouse strains. Only mPer1 mRNA and PER1 protein accumulation coincides with timecourses of many other pineal genes and their products, which are cyclicAMP inducible. Here, presented data together with the known mechanisms for regulation of the mPer1 gene in the rodent pineal gland forward the idea that in this tissue PER1 may have a trigger function for initiating the cycles of the clockwork's transcriptional/translational feedback loops.
Control of the temporal fidelity of synaptic transmission by a presynaptic high voltage‐activated transient K+ current
Authors: D'Incamps B. Lamotte , Hess D. , El Manira A.
Published: June 2004

The type of K+ channels controlling the waveform of the presynaptic spike and synaptic transmission were examined in the lamprey spinal cord. Reticulospinal neuron somata displayed a transient K+ current with a high voltage‐activation and inactivation. This current was selectively blocked by catechol at 100 µm. Reticulospinal axons also displayed a high voltage‐activated fast K+ current sensitive to catechol. The function of this presynaptic high voltage‐activated fast K+ current in controlling synaptic transmission was investigated by using paired intracellular recordings from reticulospinal axons and their targets. Blockade of this current by catechol (100 µm) prolonged the presynaptic spike elicited by a single stimulus leading to a potentiation of the postsynaptic EPSP. Calcium imaging of reticulospinal axons showed an increase in presynaptic calcium transients after blockade of the presynaptic K+ current by catechol. During high frequency firing, catechol revealed an activity‐dependent decrease in the spike duration, which resulted in a depression of synaptic transmission. These results suggest that the presynaptic high voltage‐activated transient K+ current acts to optimize the temporal fidelity of synaptic transmission by minimizing activity‐dependent changes in the presynaptic spike waveform and calcium dynamics.
Effects of visual contrast on visual evoked potentials and Doppler signal
Authors: Zaletel Marjan , Strucl Martin , Pogacnik Tomaz , Zvan Bojana
Published: June 2004

We studied visually evoked cerebral blood flow responses (VEFR) and visual evoked potentials (VEP) to different visual contrasts and analysed the relationship between them. The records were made from 35 healthy volunteers aged 38.6 ± 10.1 years. The stimulus was a black‐and‐white checkerboard with visual contrasts (VC) of 1%, 10% and 100%. The VEFR were measured in the posterior cerebral artery using transcranial Doppler, and the VEP were recorded from the occipital leads. We found the relationship between visual contrast and VEFR (r = 0.79, P < 0.01) as well as between visual contrast and VEP (r = 0.71, P < 0.01). We also found moderate association between the VEP and the VEFR (r = 0.69, P < 0.01). The analysis of the regression slopes between two different age subgroups (P < 0.01) did not show a significant difference (P = 0.020). We concluded that a simultaneous recording of VEFR and VEP to visual contrasts could allow an assessment of neurovascular coupling in humans.