bluuebunny/crossref_metadata_2025 huggingface dataset:
Electron-lucent inclusion bodies are structures specialized for aphid transmission of cauliflower mosaic virus
Authors: Khelifa Mounia , Journou Sandra , Krishnan Kalpana , Gargani Daniel , Espérandieu Pascal , Blanc Stéphane , Drucker Martin
Published: September 2007

Cauliflower mosaic virus (CaMV) is transmitted by aphids. For acquisition by the vector, a transmissible complex must form, composed of the virus particle, the viral coat-associated protein P3 and the helper protein P2. However, the components of the transmissible complex are largely separated in infected plant cells: most P3 virions are confined in electron-dense inclusion bodies, whereas P2 is sequestered in electron-lucent inclusion bodies (elIBs). This spatial separation controls virus acquisition by favouring the binding of virus-free P2 to the vector first, rendering the vector competent for later uptake of P3 virions. Consequently, sequential acquisition of virus from different cells or tissues is possible, with important implications for the biology of CaMV transmission. CaMV strains Campbell and CM1841 contain a single amino acid mutation (G94R) in the helper protein P2, rendering them non-transmissible from plant to plant. However, the mutant P2-94 protein supports aphid transmission when expressed heterologously and supplied to P3–CaMV complexes in vitro. The non-transmissibility of P2-94 was re-examined in vivo and it is shown here that the non-transmissibility of this P2 mutant is not due to low accumulation levels in infected plants, as suggested previously, but more specifically to the failure to form elIBs within infected plant cells. This demonstrates that elIBs are complex viral structures specialized for aphid transmission and suggests that viral inclusion bodies other than viral factories, most often considered as ‘garbage cans’, can in fact exhibit specific functions.
Molecular mechanisms of reversion to the ts+ (non-temperature-sensitive) phenotype of influenza A cold-adapted (ca) virus strains
Authors: Tsfasman T. M. , Markushin S. G. , Akopova I. I. , Ghendon Y. Z.
Published: September 2007

A ts+ ca− (non-temperature-sensitive, non-cold-adapted) revertant of the A/Leningrad/134/47/57 ca strain influenza virus [A/Leningrad/134/47/ts+18/1957(H2N2)], obtained in our previous study, lost phenotypic manifestation of ts mutations by the PB2, NP and NS genes, although, according to sequencing data, it acquired only two true reversions of a mutation in the PB2 and PB1 genes. Direct sequencing showed the appearance of 27 additional mutations (13 coding) in the genes encoding the PB2, PB1, PA, NP, M and NS proteins of the revertant, along with the above-mentioned two true reversions. We conjecture that some of these mutations suppressed phenotypic manifestation of ts mutations in the NS and NP genes.
Discovery of herpesviruses in bats
Authors: Wibbelt Gudrun , Kurth Andreas , Yasmum Nezlisah , Bannert Michael , Nagel Sabine , Nitsche Andreas , Ehlers Bernhard
Published: September 2007

Seven novel gammaherpesviruses (GHV) and one novel betaherpesvirus were discovered in seven different European bat species (order Chiroptera, family Vespertilionidae) with a pan-herpesvirus PCR assay, targeting the DNA polymerase (DPOL) gene. The sequences of six bat GHV were similarly related to members of the gammaherpesvirus genera Percavirus and Rhadinovirus. The seventh GHV was related to the porcine lymphotropic herpesvirus 1 (genus Macavirus). The betaherpesvirus appeared to be a distant relative of human cytomegalovirus. For three bat GHV a 3.6 kbp locus was amplified and sequenced, spanning part of the glycoprotein B gene and the majority of the DPOL gene. In phylogenetic analysis, the three bat GHV formed a separate clade with similar distance to the Percavirus and Rhadinovirus clades. These novel viruses are the first herpesviruses to be described in bats.
A monopartite begomovirus-associated DNA β satellite substitutes for the DNA B of a bipartite begomovirus to permit systemic infection
Authors: Saeed Muhammad , Zafar Yusuf , Randles John W. , Rezaian M. Ali
Published: September 2007

DNA β is a circular single-stranded satellite DNA which co-infects with certain monopartite helper begomoviruses to cause economically important diseases, such as cotton leaf curl disease (CLCuD). DNA β encodes a single protein, βC1. Tomato leaf curl New Delhi virus (ToLCNDV) is a bipartite begomovirus in which both DNA A and DNA B are required for systemic infection. Inoculation of tomato plants with ToLCNDV DNA A alone induced local but not systemic infection, whereas co-inoculation with DNA A and the DNA β associated with CLCuD resulted in systemic infection. DNA β containing a disrupted βC1 open reading frame (ORF) did not mobilize DNA A systemically. Co-inoculation of plants with DNA A and a construct of the βC1 ORF, under the control of the cauliflower mosaic virus 35S promoter, resulted in the systemic movement of DNA A. In inoculated tobacco and onion epidermal cells, βC1 fused to GFP was localized at the cell periphery in association with punctate bodies, around and within the cell nucleus and with the endoplasmic reticulum. It is concluded that heterologous βC1 protein can replace the movement function of the DNA B of a bipartite begomovirus. Evidence is also provided that tomato leaf curl virus-encoded C4 protein confers the same movement function to ToLCNDV DNA A. The intracellular distribution of βC1 is consistent with the hypothesis that it has a role in transporting the DNA A from the nuclear site of replication to the plasmodesmatal exit sites of the infected cell.
Increased human immunodeficiency virus type 1 Env expression and antibody induction using an enhanced alphavirus vector
Authors: Forsell Mattias N. E. , McInerney Gerald M. , Dosenovic Pia , Hidmark Åsa S. , Eriksson Christopher , Liljeström Peter , Grundner Christoph , Karlsson Hedestam Gunilla B.
Published: September 2007

Viral vectors encoding heterologous vaccine antigens are potent inducers of cellular immune responses, but they are generally less efficient at stimulating humoral immunity. To improve the induction of antibody responses by Semliki Forest virus-based vaccines, a vector encoding a translation-enhancer element and a novel internal signal sequence for increased expression and secretion of soluble antigens was designed. Approximately tenfold more human immunodeficiency virus type 1 gp120 was secreted into culture supernatants of infected cells using the enhanced vector compared with the parental vector. This translated into a significant increase in gp120-specific antibodies in immunized mice, suggesting that antigen-expression levels from the parental vector are limiting for induction of antibody responses. These data encourage the use of the enhanced vector for elicitation of immune responses against heterologous antigens during vaccination.
Comparison of the antiviral potentials among the pseudorabies-resistant transgenes encoding different soluble forms of porcine nectin-1 in transgenic mice
Authors: Ono Etsuro , Tomioka Yukiko , Watanabe Yuki , Amagai Keiko , Morimatsu Masami , Shinya Kyoko , Cherel Pierre
Published: September 2007

Nectin-1 is an alphaherpesvirus receptor that binds to virion glycoprotein D by the first immunoglobulin (Ig)-like domain. The possibility of making animals resistant to pseudorabies virus (PRV) infection has been investigated by generating transgenic mice expressing soluble forms of porcine nectin-1. Previously, transgenic mice were generated that expressed a fusion protein made of the entire ectodomain of nectin-1 fused to the Fc portion of human IgG, or the first Ig-like domain fused to the Fc portion of porcine IgG. Here, the contribution of the second and third Ig-like domains of nectin-1 was analysed by generating transgenic mice expressing the entire ectodomain of nectin-1 fused to the porcine Fc portion. Transgenic mice expressing each of three different fusion proteins were challenged with PRV for comparison of their resistance. Altogether, mice transgenic for a chimera that carried the entire ectodomain were more resistant than those transgenic for a chimera that carried the first Ig-like domain.
Rubella virus-induced superinfection exclusion studied in cells with persisting replicons
Authors: Claus Claudia , Tzeng Wen-Pin , Liebert Uwe G. , Frey Teryl K.
Published: September 2007

For the first time, homologous superinfection exclusion was documented for rubella virus (RUB) by using Vero cells harbouring persisting RUB replicons. Infection with wild-type RUB was reduced by tenfold, whereas Sindbis virus infection was unaffected. Replication following infection with packaged replicons and transfection with replicon transcripts was also restricted in these cells, indicating that restriction occurred after penetration and entry. Translation of such ‘supertransfecting’ replicon transcripts was not impaired, but no accumulation of supertransfecting replicon RNA could be detected. We tested the hypothesis favoured in the related alphaviruses that superinfection exclusion is mediated by cleavage of the incoming non-structural precursor by the pre-existing non-structural (NS) protease, resulting in an inhibition of minus-strand RNA synthesis. However, cleavage of a precursor translated from a supertransfecting replicon transcript with an NS protease catalytic-site mutation was not detected and the event in the replication cycle at which superinfection exclusion is executed remains to be elucidated.
Measles virus minigenomes encoding two autofluorescent proteins reveal cell-to-cell variation in reporter expression dependent on viral sequences between the transcription units
Authors: Rennick Linda J. , Duprex W. Paul , Rima Bert K.
Published: September 2007

Transcription from morbillivirus genomes commences at a single promoter in the 3′ non-coding terminus, with the six genes being transcribed sequentially. The 3′ and 5′ untranslated regions (UTRs) of the genes (mRNA sense), together with the intergenic trinucleotide spacer, comprise the non-coding sequences (NCS) of the virus and contain the conserved gene end and gene start signals, respectively. Bicistronic minigenomes containing transcription units (TUs) encoding autofluorescent reporter proteins separated by measles virus (MV) NCS were used to give a direct estimation of gene expression in single, living cells by assessing the relative amounts of each fluorescent protein in each cell. Initially, five minigenomes containing each of the MV NCS were generated. Assays were developed to determine the amount of each fluorescent protein in cells at both cell population and single-cell levels. This revealed significant variations in gene expression between cells expressing the same NCS-containing minigenome. The minigenome containing the M/F NCS produced significantly lower amounts of fluorescent protein from the second TU (TU2), compared with the other minigenomes. A minigenome with a truncated F 5′ UTR had increased expression from TU2. This UTR is 524 nt longer than the other MV 5′ UTRs. Insertions into the 5′ UTR of the enhanced green fluorescent protein gene in the minigenome containing the N/P NCS showed that specific sequences, rather than just the additional length of F 5′ UTR, govern this decreased expression from TU2.
Effects of vpu start-codon mutations on human immunodeficiency virus type 1 replication in macrophages
Authors: Richards Kathryn H. , Clapham Paul R.
Published: September 2007

The human immunodeficiency virus type 1 (HIV-1) vpu protein increases the release of virus particles from infected cells. Mutations that abrogate vpu function have a profound effect on HIV-1 replication in primary macrophage cultures. About 1.24 % of primary isolates in the HIV databases have vpu start-codon mutations. In addition, the envelope of the AD8 isolate was reported to compensate for the lack of vpu, whilst the YU-2 virus (cloned directly from the brain tissue of an infected individual) is macrophage-tropic, despite having a vpu start-codon mutation. These observations raise the possibility that envelopes evolve to compensate for the loss of vpu function in vivo. Chimeric vpu
+ and vpu
− replication-competent clones were constructed that contained the envelopes of SF162, AD8 or YU-2. Macrophages were infected with these chimeras and virus release was measured over time by a reverse transcriptase ELISA. It was found that vpu-deficient chimeras carrying AD8 and YU-2 envelopes were consistently released at lower levels than their wild-type (wt) vpu counterparts, indicating that these envelopes did not compensate for the lack of vpu. Non-chimeric vpu
+ and vpu
− AD8 and YU-2 followed similar patterns, although replication by vpu-deficient AD8 was variable, with virion release reaching 60 % of that recorded for AD8 with a wt vpu. In summary, no evidence was found that the AD8 or YU-2 envelopes can compensate for the lack of vpu for replication in macrophages.
A cucumber mosaic virus mutant lacking the 2b counter-defence protein gene provides protection against wild-type strains
Authors: Ziebell Heiko , Payne Tina , Berry James O. , Walsh John A. , Carr John P.
Published: September 2007

Several plant virus mutants, in which genes encoding silencing suppressor proteins have been deleted, are known to induce systemic or localized RNA silencing against themselves and other RNA molecules containing homologous sequences. Thus, it is thought that many cases of cross-protection, in which infection with a mild or asymptomatic virus mutant protects plants against challenge infection with closely related virulent viruses, can be explained by RNA silencing. We found that a cucumber mosaic virus (CMV) mutant of the subgroup IA strain Fny (Fny-CMVΔ2b), which cannot express the 2b silencing suppressor protein, cross-protects tobacco (Nicotiana tabacum) andNicotiana benthamianaplants against disease induction by wild-type Fny-CMV. However, protection is most effective only if inoculation with Fny-CMVΔ2b and challenge inoculation with wild-type CMV occurs on the same leaf. Unexpectedly, Fny-CMVΔ2b also protected plants against infection with TC-CMV, a subgroup II strain that is not closely related to Fny-CMV. Additionally,in situhybridization revealed that Fny-CMVΔ2b and Fny-CMV can co-exist in the same tissues but these tissues contain zones of Fny-CMVΔ2b-infected host cells from which Fny-CMV appears to be excluded. Taken together, it appears unlikely that cross-protection by Fny-CMVΔ2b occurs by induction of systemic RNA silencing against itself and homologous RNA sequences in wild-type CMV. It is more likely that protection occurs through either induction of very highly localized RNA silencing, or by competition between strains for host cells or resources.
A feline immunodeficiency virus vif-deletion mutant remains attenuated upon infection of newborn kittens
Authors: Shen Xiaoying , Leutenegger Christian M. , Stefano Cole Kelly , Pedersen Niels C. , Sparger Ellen E.
Published: September 2007

This report characterizes lentivirus attenuation associated with a vif mutation by inoculation of newborn kittens with a vif-deleted feline immunodeficiency virus provirus plasmid (FIV-pPPRΔvif). Virus in peripheral blood, antiviral antibody or CD4 T-cell count alterations were not detected in kittens inoculated with FIV-pPPRΔvif plasmid, with the exception of one kitten that demonstrated FIV Gag antibody production at 42 weeks after inoculation. In contrast, wild-type FIV-pPPR-infected kittens were viraemic, seropositive and exhibited a decrease in the CD4 T-cell subset in peripheral blood. Interestingly, FIV-specific T-cell proliferative responses detected at 32 and 36 weeks after infection were comparable for both FIV-pPPRΔvif- and wild-type FIV-pPPR-inoculated kittens and suggested the possibility of a discreet tissue reservoir supporting sustained FIV-pPPRΔvif expression or replication. Overall, these findings confirmed that the severe virus attenuation for both replication and pathogenicity exhibited by a vif-deleted FIV mutant is similar for both neonatal and adult hosts.
mgpB and mgpC sequence diversity in Mycoplasma genitalium is generated by segmental reciprocal recombination with repetitive chromosomal sequences
Authors: Iverson‐Cabral Stefanie L. , Astete Sabina G. , Cohen Craig R. , Totten Patricia A.
Published: September 2007

Mycoplasma genitalium is associated with sexually transmitted infections in men and women that, if untreated, can persist, suggesting that mechanism(s) exist to facilitate immune evasion. Approximately 4% of the limited M. genitalium genome contains repeat sequences termed MgPar regions that have homology to mgpB and mgpC, which encode antigenic proteins associated with attachment. We have previously shown that mgpB sequences vary within a single strain of M. genitalium in a pattern consistent with recombination between mgpB and MgPar sequences (Iverson‐Cabral et al.). In the current study, we show that mgpC heterogeneity similarly occurs within the type strain, G‐37T, cultured in vitro and among cervical specimens collected from a persistently infected woman. In all cases, alternative mgpC sequences are indicative of recombination with MgPar regions. Additionally, the isolation of single‐colony M. genitalium clonal variants containing alternative mgpB or mgpC sequences allowed us to demonstrate that mgpB and mgpC heterogeneity is associated with corresponding changes within donor MgPar regions, consistent with reciprocal recombination. Better‐defined systems of antigenic variation are typically mediated by unidirectional gene conversion, so the generation of genetic diversity observed in M. genitalium by the mutual exchange of sequences makes this organism unique among bacterial pathogens.
Identification of the genes directly controlled by the response regulator CiaR in Streptococcus pneumoniae: five out of 15 promoters drive expression of small non‐coding RNAs
Authors: Halfmann Alexander , Kovács Márta , Hakenbeck Regine , Brückner Reinhold
Published: September 2007

The two‐component regulatory system CiaRH of Streptococcus pneumoniae has been implicated in β‐lactam resistance, maintenance of cell integrity, competence and virulence, but the genes that are regulated directly by the system have not been defined. Using transcriptional mapping, in vitro CiaR binding, and in vivo analysis of CiaR‐mediated regulation, 15 promoters were identified to be directly controlled by the response regulator CiaR. A direct repeat, TTTAAG‐N5‐TTTAAG, was found to be essential for CiaR binding and regulation. It is present, either completely or with subtle changes, in all promoter regions. Fourteen promoters of the regulon are activated by CiaR, and one was found to be controlled negatively. The genes that are transcribed from these promoters included ciaRH, loci that are predicted to be involved in the modification of teichoic acids (lic), in sugar metabolism (mal, man), stress response (htrA), chromosome segregation (parB), protease maturation (ppmA) and unknown functions. Remarkably, the five strongest promoters of the CiaR regulon drive expression of small RNAs. These small RNAs, designated csRNAs for cia‐dependent small RNAs, are non‐coding, between 87 and 151 nt in size, and show a high degree of similarity to each other. The analysis of deletion mutants in the csRNA genes revealed that csRNA4 and csRNA5 affect stationary‐phase autolysis. The identification of five small non‐coding regulatory RNAs opens new perspectives to approach the physiological role of the CiaRH two‐component regulatory system.
Role of the Escherichia coli SbmA in the antimicrobial activity of proline‐rich peptides
Authors: Mattiuzzo Maura , Bandiera Antonella , Gennaro Renato , Benincasa Monica , Pacor Sabrina , Antcheva Nikolinka , Scocchi Marco
Published: September 2007

In contrast to many antimicrobial peptides, members of the proline‐rich group of antimicrobial peptides inactivate Gram‐negative bacteria by a non‐lytic mechanism. Several lines of evidence indicate that they are internalized into bacteria and their activity mediated by interaction with unknown cellular components. With the aim of identifying such interactors, we selected mutagenized Escherichia coli clones resistant to the proline‐rich Bac7(1–35) peptide and analysed genes responsible for conferring resistance, whose products may thus be involved in the peptide's mode of action. We isolated a number of genomic regions bearing such genes, and one in particular coding for SbmA, an inner membrane protein predicted to be part of an ABC transporter. An E. coli strain carrying a point mutation in sbmA, as well as other sbmA‐null mutants, in fact showed resistance to several proline‐rich peptides but not to representative membranolytic peptides. Use of fluorescently labelled Bac7(1–35) confirmed that resistance correlated with a decreased ability to internalize the peptide, suggesting that a bacterial protein, SbmA, is necessary for the transport of, and for susceptibility to, proline‐rich antimicrobial peptides of eukaryotic origin.
Genome‐wide analysis of T‐DNA integration into the chromosomes ofMagnaporthe oryzae
Authors: Choi Jaehyuk , Park Jongsun , Jeon Junhyun , Chi Myoung‐Hwan , Goh Jaeduk , Yoo Sung‐Yong , Park Jaejin , Jung Kyongyong , Kim Hyojeong , Park Sook‐Young , Rho Hee‐Sool , Kim Soonok , Kim Byeong Ryun , Han Seong‐Sook , Kang Seogchan , Lee Yong‐Hwan
Published: September 2007

Agrobacteriumtumefaciens‐mediatedtransformation (ATMT) has become a prevalent tool for functional genomics of fungi, but our understanding of T‐DNA integration into the fungal genome remains limited relative to that in plants. Using a model plant‐pathogenic fungus,Magnaporthe oryzae, here we report the most comprehensive analysis of T‐DNA integration events in fungi and the development of an informatics infrastructure, termed aT‐DNAanalysisplatform (TAP). We identified a total of 1110T‐DNA‐taggedlocations (TTLs) and processed the resulting data via TAP. Analysis of the TTLs showed that T‐DNA integration was biased among chromosomes and preferred the promoter region of genes. In addition, irregular patterns of T‐DNA integration, such as chromosomal rearrangement and readthrough of plasmid vectors, were also observed, showing that T‐DNA integration patterns into the fungal genome are as diverse as those of their plant counterparts. However, overall the observed junction structures between T‐DNA borders and flanking genomic DNA sequences revealed that T‐DNA integration into the fungal genome was more canonical than those observed in plants. Our results support the potential of ATMT as a tool for functional genomics of fungi and show that the TAP is an effective informatics platform for handling data from large‐scale insertional mutagenesis.
The role of iron in Mycobacterium smegmatis biofilm formation: the exochelin siderophore is essential in limiting iron conditions for biofilm formation but not for planktonic growth
Authors: Ojha Anil , Hatfull Graham F.
Published: September 2007

Many species of mycobacteria form structured biofilm communities at liquid–air interfaces and on solid surfaces. Full development of Mycobacterium smegmatis biofilms requires addition of supplemental iron above 1 μM ferrous sulphate, although addition of iron is not needed for planktonic growth. Microarray analysis of the M. smegmatis transcriptome shows that iron‐responsive genes – especially those involved in siderophore synthesis and iron uptake – are strongly induced during biofilm formation reflecting a response to iron deprivation, even when 2 μM iron is present. The acquisition of iron under these conditions is specifically dependent on the exochelin synthesis and uptake pathways, and the strong defect of an iron–exochelin uptake mutant suggests a regulatory role of iron in the transition to biofilm growth. In contrast, although the expression of mycobactin and iron ABC transport operons is highly upregulated during biofilm formation, mutants in these systems form normal biofilms in low‐iron (2 μM) conditions. A close correlation between iron availability and matrix‐associated fatty acids implies a possible metabolic role in the late stages of biofilm maturation, in addition to the early regulatory role. M. smegmatis surface motility is similarly dependent on iron availability, requiring both supplemental iron and the exochelin pathway to acquire it.
Aerobic TMAO respiration in Escherichia coli
Authors: Ansaldi Mireille , Théraulaz Laurence , Baraquet Claudine , Panis Gaël , Méjean Vincent
Published: September 2007

In the absence of oxygen, Escherichia coli can use alternative exogenous electron acceptors, including trimethylamine oxide (TMAO), to generate energy. In this study, we showed that in contrast to the other anaerobic respiratory systems, the TMAO reductase (Tor) system was expressed during both aerobiosis and anaerobiosis. By using a torA–lacZ fusion and quantitative reverse transcription polymerase chain reaction, we established that the torCAD operon encoding the Tor system was induced in the presence of TMAO mainly during exponential phase, and that optimal induction required a certain level of DNA supercoiling. We also showed that the presence of oxygen prevented neither the biogenesis of the Tor system nor the reduction of TMAO. The physiological role of TMAO reduction during aerobiosis has not been yet established, but our experiments suggest that alkaline TMA production could enhance the growth conditions by increasing the pH of the culture.
A biosynthetic gene cluster for a secreted cellobiose lipid with antifungal activity from Ustilago maydis
Authors: Teichmann Beate , Linne Uwe , Hewald Sandra , Marahiel Mohamed A. , Bölker Michael
Published: September 2007

The phytopathogenic basidiomycetous fungus Ustilago maydis secretes large amounts of the glycolipid biosurfactant ustilagic acid (UA). UA consists of 15,16‐dihydroxypalmitic or 2,15,16‐trihydroxypalmitic acid, which is O‐glycosidically linked to cellobiose at its terminal hydroxyl group. In addition, the cellobiose moiety is acetylated and acylated with a short‐chain hydroxy fatty acid. We have identified a 58 kb spanning gene cluster that contains 12 open reading frames coding for most, if not all, enzymes needed for UA biosynthesis. Using a combination of genetic and mass spectrometric analysis we were able to assign functional roles to three of the proteins encoded by the gene cluster. This allowed us to propose a biosynthesis route for UA. The Ahd1 protein belongs to the family of non‐haem diiron reductases and is required for α‐hydroxylation of palmitic acid. Two P450 monooxygenases, Cyp1 and Cyp2, catalyse terminal and subterminal hydroxylation of palmitic acid. We could demonstrate that infection of tomato leaves by the plant pathogenic fungus Botrytis cinerea is prevented by co‐inoculation with wild‐type U. maydis sporidia. U. maydis mutants defective in UA biosynthesis were unable to inhibit B. cinerea infection indicating that UA secretion is critical for antagonistic activity.
Increased SA in NPR1‐silenced plants antagonizes JA and JA‐dependent direct and indirect defenses in herbivore‐attacked Nicotiana attenuata in nature
Authors: Rayapuram Cbgowda , Baldwin Ian T.
Published: September 2007

The phytohormone jasmonic acid (JA) is known to mediate herbivore resistance, while salicylic acid (SA) and non‐expressor of PR‐1 (NPR1) mediate pathogen resistance in many plants. Herbivore attack on Nicotiana attenuata elicits increases in JA and JA‐mediated defenses, but also increases SA levels and Na‐NPR1 transcripts from the plant’s single genomic copy. SA treatment of wild‐type plants increases Na‐NPR1 and Na‐PR1 transcripts. Plants silenced in NPR1 accumulation by RNAi (ir‐npr1) are highly susceptible to herbivore and pathogen attack when planted in their native habitat in Utah. They are also impaired in their ability to attract Geocorus pallens predators, due to their decreased ability to release cis‐α‐bergamotene, a JA‐elicited volatile ‘alarm call’. In the glasshouse, Spodoptera exigua larvae grew better on ir‐npr1 plants, which had low levels of JA, JA–isoleucine/leucine, lipoxygenase‐3 (LOX3) transcripts and JA‐elicited direct defense metabolites (nicotine, caffeoyl putrescine and rutin), but high levels of SA and isochorismate synthase (ICS) transcripts, suggesting de novo biosynthesis of SA. A microarray analysis revealed downregulation of many JA‐elicited genes and upregulation of SA biosynthetic genes. JA treatment restored nicotine levels and resistance to S. exigua in ir‐npr1 plants. We conclude that, during herbivore attack, NPR1 negatively regulates SA production, allowing the unfettered elicitation of JA‐mediated defenses; when NPR1 is silenced, the elicited increases in SA production antagonize JA and JA‐related defenses, making the plants susceptible to herbivores.
Metabolic engineering of morphinan alkaloids by over‐expression and RNAi suppression of salutaridinol 7‐O‐acetyltransferase in opium poppy
Authors: Allen Robert S. , Miller James A. C. , Chitty Julie A. , Fist Anthony J. , Gerlach Wayne L. , Larkin Philip J.
Published: September 2007

We demonstrate that both over‐expression and suppression of the gene encoding the morphinan pathway enzyme salutaridinol 7‐O‐acetyltransferase (SalAT) in opium poppy affects the alkaloid products that accumulate. Over‐expression of the gene in most of the transgenic events resulted in an increase in capsule morphine, codeine and thebaine on a dry‐weight basis. The transgenic line with the highest alkaloid content had 41%, 37% and 42% greater total alkaloids than the control in three independent trials over 3 years. DNA‐encoded hairpin RNA‐mediated suppression of SalAT resulted in the novel accumulation of the alkaloid salutaridine at up to 23% of total alkaloid; this alkaloid is not detectable in the parental genotype. Salutaridine is not the substrate of SalAT but the substrate of the previous enzyme in the pathway, salutaridine reductase. RNA transcript analysis of 16 primary T0 transformants and their segregating T1 progeny revealed an average reduction in SalAT transcript to about 12% of the control. Reduction in SalAT transcript was evident in both leaves and latex. Reverse transcriptase PCR and high‐performance liquid chromatography analyses confirmed cosegregation of the expressed transgene with the salutaridine accumulating phenotype.
Role of IFN‐γ in an experimental murine model of West Nile virus‐induced seizures
Authors: Getts Daniel R. , Matsumoto Izuru , Müller Marcus , Getts Meghann Teague , Radford Jane , Shrestha Bimmi , Campbell Iain L. , King Nicholas J. C.
Published: September 2007

Seizures are a major complication of viral encephalitis. However, the mechanisms of seizure‐associated neuronal dysfunction remain poorly understood. We report that intranasal inoculation with West Nile virus (WNV) (Sarafend) causes limbic seizures in C57BL/6 mice, but not in interferon (IFN)‐γ‐deficient (IFN‐γ−/−) mice. Both strains showed similar levels of virus in the brain, as well as similar concentrations of the cytokines, tumor necrosis factor and interleukin‐6, both of which can alter neuronal excitability. Experiments in chimeric IFN‐γ−/−mice reconstituted with IFN‐γ‐producing leukocytes showed that IFN‐γ is not required during central nervous system infection for limbic seizure development, suggesting a role for IFN‐γ in the developing brain. This was supported responses to pentylenetetrazole, kainic acid (KA), andN‐methyl‐d‐aspartate (NMDA). Both strains of mice exhibited similar behavior after pentylenetetrazole challenge. However, while NMDA and KA treatment resulted in characteristic seizures in C57BL/6 mice, these responses were diminished (NMDA treatment) or absent (KA treatment) in IFN‐γ−/−mice. Furthermore, NMDA‐receptor blockade with MK‐801 in WNV‐infected C57BL/6 mice abrogated seizures and prolonged survival. Our data show that IFN‐γ plays an important role in the development of the excitatory seizure pathways in the brain and that these cascades become pathogenic in encephalitic WNV infection.
Important differences between human and mouse APOE gene promoters: limitation of mouse APOE model in studying Alzheimer’s disease
Authors: Maloney Bryan , Ge Yuan‐Wen , Alley George M. , Lahiri Debomoy K.
Published: September 2007

Apolipoprotein E (ApoE), encoded by the apolipoprotein E gene (APOE), plays an important role in the pathogenesis of Alzheimer’s disease (AD). The APOE ε4 variant is strongly associated with AD. APOE promoter polymorphisms have also been reported to associate with higher AD risk. Mouse models of APOE expression have long been used to study the pathogenesis of AD. Elucidating the role of the APOE gene in AD requires understanding of how its regulation differs between mouse and human APOE genes, and how the differences influence AD risk. We compared the structure and function of both the human APOE gene promoter (hAPOEP) and mouse APOE gene promoter (mAPOEP) regions. Homology is less than 40% at 180 bp or more upstream of the two species’ transcription start site (TSS, +1). Functional analysis revealed both similarities and important differences between the two sequences, significantly affected by human versus rodent cell line origin. We likewise probed nuclear extracts from several cell lines of different origins (astrocytic, glial, and neuronal) and mouse brain with specific hAPOEP and mAPOEP fragments. Each fragment shared DNA–protein interactions with the other but, notably, also bound distinct factors, demonstrated by gel shift and southwestern analyses. We determined possible identities for these distinct factors. These results suggest that regulation of mouse and human APOE genes may be sufficiently unique to justify the use of both the human APOE promoter sequence in transgenic rodent models and non‐rodent AD models for studying factors involved in AD pathogenesis.
Protection of dopaminergic neurons by electroconvulsive shock in an animal model of Parkinson’s disease
Authors: Anastasia Agustín , De Erausquin Gabriel A. , Wojnacki José , Mascó Daniel H.
Published: September 2007

Electroconvulsive shock (ECS) improves motor function in Parkinson’s disease. In rats, ECS stimulates the expression of various factors some of which have been proposed to exert neuroprotective actions. We have investigated the effects of ECS on 6‐hydroxydopamine (6‐OHDA)‐injected rats. Three weeks after a unilateral administration of 6‐OHDA, 85–95% nigral dopaminergic neurons are lost. Chronic ECS prevented this cell loss, protect the nigrostriatal pathway (assessed by FloroGold retrograde labeling) and reduce motor impairment in 6‐OHDA‐treated animals. Injection of 6‐OHDA caused loss of expression of glial cell‐line derived neurotrophic factor (GDNF) in the substantia nigra. Chronic ECS completely prevented this loss of GDNF expression in 6‐OHDA‐treated animals. We also found that protected dopaminergic neurons co‐express GDNF receptor proteins. These results strongly suggest that endogenous changes in GDNF expression may participate in the neuroprotective mechanism of ECS against 6‐OHDA induced toxicity.
Post‐synaptic density perturbs insulin‐induced Kv1.3 channel modulation via a clustering mechanism involving the SH3 domain
Authors: Marks D. R. , Fadool D. A.
Published: September 2007

The olfactory bulb (OB) contains the highest concentration of the insulin receptor (IR) kinase in the central nervous system; however, its functional role and modulation in this region remains poorly understood. IR kinase contains a number of proline‐rich motifs, making it an excellent candidate for modulation by SH3 domain‐containing adaptor proteins. Kv1.3, a voltage‐gated Shaker potassium channel and tyrosine phosphorylation substrate of IR kinase, contains several proline‐rich sequences and a canonical post‐synaptic density 95 (PSD‐95)/discs large/zO‐1 domain (PDZ) recognition motif common to most Shaker family members. We sought to determine if a functional relationship existed between Kv1.3, IR kinase, and the SH3/PDZ adaptor protein PSD‐95. Through patch‐clamp electrophysiology, immunochemistry, and co‐immunoprecipitation, we found that while Kv1.3 and PSD‐95 alone interact via the canonical C‐terminal PDZ recognition motif of the channel, this molecular site of interaction acts to cluster the channels but the PSD‐95 SH3‐guanylate kinase domain functionally modulates Kv1.3 activity via two proline‐rich domains in its N‐ and C‐terminal. Therefore, these data suggest that adaptor domains responsible for ion‐channel clustering and functional modulation are not necessarily coupled. Moreover, IR kinase and Kv1.3 can only be co‐immunoprecipitated in the presence of PSD‐95 as the adapting linker. Functionally, insulin‐dependent Kv1.3 phosphorylation that causes channel current suppression is blocked via interaction with the PSD‐95 SH3‐guanylate kinase domain. Because all the three proteins co‐localize in multiple lamina of the OB that are known to be rich in synaptic connections, membrane excitability and synaptic transmission at critical locations in the OB have the capacity to be finely regulated.
Combinatorial co‐expression of pheromone receptors, V2Rs
Authors: Silvotti Lucia , Moiani Arianna , Gatti Rita , Tirindelli Roberto
Published: September 2007

Basal neurons of the vomeronasal organ of the mouse express a superfamily of about 120 pheromone receptors, named V2Rs, that are grouped in four families, A, B, C, and D, according to sequence homology. Family‐A, ‐B, and ‐D V2Rs are expressed as one receptor gene per cell, but we previously reported their co‐expression with family‐C V2Rs. Here, we show that basal neurons can be further grouped according to the combinatorial expression of different V2Rs. Altogether, these findings suggest that in each basal neuron a transcriptional program is active for expressing a combination of two compatible receptors and for excluding, at the same time, the expression of all other V2Rs. Further analyses revealed non‐random combinations of co‐expression between family‐C V2Rs and genes of the class Ib major histocompatibility complex. Thus, each basal neuron of the vomeronasal organ represents a highly qualified sensory unit for detecting very specific combinations of pheromonal cues.
Reorganization of prion protein membrane environment during low potassium‐induced apoptosis in primary rat cerebellar neurons
Authors: Rivaroli Anna , Prioni Simona , Loberto Nicoletta , Bettiga Arianna , Chigorno Vanna , Prinetti Alessandro , Sonnino Sandro
Published: September 2007

We studied the changes occurring in the membrane environment of prion protein (PrP) during apoptosis induced by low potassium in primary rat cerebellar neurons. Ceramide levels increased during apoptosis‐inducing treatment, being doubled with respect to time‐matched controls after 24 h. Sphingomyelin levels were parallely decreased, while cholesterol and ganglioside contents were not affected. Changes in ceramide and sphingomyelin composition were exclusively restricted to a detergent‐resistant membrane fraction. The pro‐apoptotic treatment was accompanied by the down‐regulation of PrP and of the non‐receptor kinase Fyn. The levels of PrP and Fyn were correspondingly reduced in the detergent‐resistant membrane fraction. In control cells, the membrane microenvironment separated by immunoprecipitation with anti‐PrP antibody contained 80% of the detergent‐resistant PrP and 35% and 38% of the sphingolipids and cholesterol respectively. Upon low potassium treatment, 20% of the PrP originally present in the detergent‐resistant fraction was immunoprecipitated, together with 19% of sphingolipids and 22% of cholesterol. Thus, PrP in the immunoprecipitate from apoptotic cells was ninefold less than in control ones, while sphingolipids and cholesterol were about 50% with respect to controls cells. The molar ratio between cholesterol, sphingomyelin and ceramide was 15 : 6 : 1 in the PrP‐rich environment from control neurons, and 6 : 2 : 1 in that from apoptotic cells.
Identification of NCAM‐binding peptides promoting neurite outgrowth via a heterotrimeric G‐protein‐coupled pathway
Authors: Hansen Raino Kristian , Christensen Claus , Korshunova Irina , Kriebel Martin , Burkarth Nadine , Kiselyov Vladislav V. , Olsen Marianne , Østergaard Søren , Holm Arne , Volkmer Hansjürgen , Walmod Peter S. , Berezin Vladimir , Bock Elisabeth
Published: September 2007

A combinatorial library of undecapeptides was produced and utilized for the isolation of peptide binding to the fibronectin type 3 modules (F3I–F3II) of the neural cell adhesion molecule (NCAM). The isolated peptides were sequenced and produced as dendrimers. Two of the peptides (denoted ENFIN2 and ENFIN11) were confirmed to bind to F3I–F3II of NCAM by surface plasmon resonance. The peptides induced neurite outgrowth in primary cerebellar neurons and PC12E2 cells, but had no apparent neuroprotective properties. NCAM is known to activate different intracellular pathways, including signaling through the fibroblast growth factor receptor, the Src‐related non‐receptor tyrosine kinase Fyn, and heterotrimeric G‐proteins. Interestingly, neurite outgrowth stimulated by ENFIN2 and ENFIN11 was independent of signaling through fibroblast growth factor receptor and Fyn, but could be inhibited with pertussis toxin, an inhibitor of certain heterotrimeric G‐proteins. Neurite outgrowth induced by trans‐homophilic NCAM was unaffected by the peptides, whereas knockdown of NCAM completely abrogated ENFIN2‐ and ENFIN11‐induced neuritogenesis. These observations suggest that ENFIN2 and ENFIN11 induce neurite outgrowth in an NCAM‐dependent manner through G‐protein‐coupled signal transduction pathways. Thus, ENFIN2 and ENFIN11 may be valuable for exploring this particular type of NCAM‐mediated signaling.
The extracellular matrix in multiple sclerosis pathology
Authors: Van Horssen Jack , Dijkstra Christine D. , De Vries Helga E.
Published: September 2007

The extracellular matrix (ECM) is a substrate upon which cells migrate, proliferate and differentiate. It is involved in the maintenance of cytoarchitecture, regulation of homeostasis, and it influences interactions between cells and molecules via specific receptors. Although a substantial body of knowledge has accumulated concerning the role of the ECM in peripheral tissues, little is known of the structure and function of the ECM in the CNS. However, marked changes in the expression of ECM constituents have been documented in various neurological disorders, including multiple sclerosis. This review focuses on the structure and function of the ECM in the CNS and in particular on the occurrence and involvement of ECM changes in the pathology of multiple sclerosis. Increased knowledge of the expression and functional role of ECM proteins in the CNS can lead to a better understanding of complex neurobiological processes both under normal as well as pathological conditions.
Human embryonic stem cell‐derived neural precursors as a continuous, stable, and on‐demand source for human dopamine neurons
Authors: Ko Ji‐Yun , Park Chang‐Hwan , Koh Hyun‐Chul , Cho Youl‐Hee , Kyhm Jee‐Hong , Kim Young‐Soo , Lee Inchul , Lee Yong‐Sung , Lee Sang‐Hun
Published: September 2007

Human embryonic stem (hES) cells can be guided to differentiate into ventral midbrain‐type neural precursor (NP) cells that proliferate in vitro by specific mitogens. We investigated the potential of these NP cells derived from hES cells (hES‐NP) for the large‐scale generation of human dopamine (DA) neurons for functional analyses and therapeutic applications. To address this, hES‐NP cells were expanded in vitro for 1.5 months with six passages, and their proliferation and differentiation properties determined over the NP passages. Interestingly, the total hES‐NP cell number was increased by > 2 × 104‐folds over the in vitro period without alteration of phenotypic gene expression. They also sustained their differentiation capacity toward neuronal cells, exhibiting in vitro pre‐synaptic DA neuronal functionality. Furthermore, the hES‐NP cells can be cryopreserved without losing their proliferative and developmental potential. Upon transplantation into a Parkinson’s disease rat model, the multi‐passaged hES‐NP cells survived, integrated into the host striatum, and differentiated toward the neuronal cells expressing DA phenotypes. A significant reduction in the amphetamine‐induced rotation score of Parkinson’s disease rats was observed by the cell transplantation. Taken together, these findings indicate that hES‐NP cell expansion is exploitable for a large‐scale generation of experimental and transplantable DA neurons of human‐origin.
Peptide transporter 2 (PEPT2) expression in brain protects against 5‐aminolevulinic acid neurotoxicity
Authors: Hu Yongjun , Shen Hong , Keep Richard F. , Smith David E.
Published: September 2007

The proton‐coupled oligopeptide transporter PEPT2 (or SLC15A2) is the major protein involved in the reclamation of peptide‐bound amino acids and peptide‐like drugs in kidney. PEPT2 is also important in effluxing peptides and peptidomimetics from CSF at the choroid plexus, thereby limiting their exposure in brain. In this study, we report a neuroprotective role for PEPT2 in modulating the toxicity of a heme precursor, 5‐aminolevulinic acid (5‐ALA). Our findings demonstrate that in PEPT2‐deficient mice, 5‐ALA administration results in reduced survivability, a worsening of neuromuscular dysfunction, and CSF concentrations of substrate that are 8–30 times higher than that in wild‐type control animals. The ability of PEPT2 to limit 5‐ALA exposure in CSF suggests that it may also have relevance as a secondary genetic modifier of conditions (such as acute hepatic porphyrias and lead poisoning) in which 5‐ALA metabolism is altered and in which 5‐ALA toxicity is important.
Spinal cord mitochondria display lower calcium retention capacity compared with brain mitochondria without inherent differences in sensitivity to cyclophilin D inhibition
Authors: Morota Saori , Hansson Magnus J. , Ishii Nagao , Kudo Yoshihisa , Elmér Eskil , Uchino Hiroyuki
Published: September 2007

The mitochondrial permeability transition (mPT) is a potential pathogenic mechanism in neurodegeneration. Varying sensitivity to calcium‐induced mPT has been demonstrated for regions within the CNS possibly correlating with vulnerability following insults. The spinal cord is selectively vulnerable in e.g. amyotrophic lateral sclerosis and increased mPT sensitivity of mitochondria derived from the spinal cord has previously been demonstrated. In this study, we introduce whole‐body hypothermia prior to removal of CNS tissue to minimize the effects of differential tissue extraction prior to isolation of spinal cord and cortical brain mitochondria. Spinal cord mitochondria were able to retain considerably less calcium when administered as continuous infusion, which was not related to a general increased sensitivity of the mPT to calcium, its desensitization to calcium by the cyclophilin D inhibitor cyclosporin‐A, or to differences in respiratory parameters. Spinal cord mitochondria maintained a higher concentration of extramitochondrial calcium during infusion than brain mitochondria possibly related to an increased set‐point concentration for calcium uptake. A hampered transport and retention capacity of calcium may translate into an increased susceptibility of the spinal cord to neurodegenerative processes involving calcium‐mediated damage.