bluuebunny/crossref_metadata_2025 huggingface dataset:
Isolation of avian infectious bronchitis coronavirus from domestic peafowl (Pavo cristatus) and teal (Anas)
Authors: Liu Shengwang , Chen Jianfei , Chen Jinding , Kong Xiangang , Shao Yuhao , Han Zongxi , Feng Li , Cai Xuehui , Gu Shoulin , Liu Ming
Published: February 2005

Coronavirus-like viruses, designated peafowl/China/LKQ3/2003 (pf/CH/LKQ3/03) and teal/China/LDT3/2003 (tl/CH/LDT3/03), were isolated from a peafowl and a teal during virological surveillance in Guangdong province, China. Partial genomic sequence analysis showed that these isolates had the S–3–M–5–N gene order that is typical of avian coronaviruses. The spike, membrane and nucleocapsid protein genes of pf/CH/LKQ3/03 had >99 % identity to those of the avian infectious bronchitis coronavirus H120 vaccine strain (Massachusetts serotype) and other Massachusetts serotype isolates. Furthermore, when pf/CH/LKQ3/03 was inoculated experimentally into chickens (specific-pathogen-free), no disease signs were apparent. tl/CH/LDT3/03 had a spike protein gene with 95 % identity to that of a Chinese infectious bronchitis virus (IBV) isolate, although more extensive sequencing revealed the possibility that this strain may have undergone recombination. When inoculated into chickens, tl/CH/LDT3/03 resulted in the death of birds from nephritis. Taken together, this information suggests that pf/CH/LKQ3/03 might be a revertant, attenuated vaccine IBV strain, whereas tl/CH/LDT3/03 is a nephropathogenic field IBV strain, generated through recombination. The replication and non-pathogenic nature of IBV in domestic peafowl and teal under field conditions raises questions as to the role of these hosts as carriers of IBV and the potential that they may have to transmit virus to susceptible chicken populations.
Real-time reverse transcriptase PCR for the endogenous koala retrovirus reveals an association between plasma viral load and neoplastic disease in koalas
Authors: Tarlinton Rachael , Meers Joanne , Hanger Jon , Young Paul
Published: February 2005

Koala retrovirus (KoRV) is a newly described endogenous retrovirus and is unusual in that inserts comprise a full-length replication competent genome. As koalas are known to suffer from an extremely high incidence of leukaemia/lymphoma, the association between this retrovirus and disease in koalas was examined. Using quantitative real-time reverse transcriptase PCR it was demonstrated that KoRV RNA levels in plasma are significantly increased in animals suffering from leukaemia or lymphoma when compared with healthy animals. Increased levels of KoRV were also seen for animals with clinical chlamydiosis. A significant positive association between viral RNA levels and age was also demonstrated. Real-time PCR demonstrated as much as 5 log variation in KoRV proviral DNA levels in genomic DNA extracted from whole blood from different animals. Taken together these data indicate that KoRV is an active endogenous retrovirus and suggests that it may be causally linked to neoplastic disease in koalas.
Transcription of the murine gammaherpesvirus 68 ORF73 from promoters in the viral terminal repeats
Authors: Coleman Heather M. , Efstathiou Stacey , Stevenson Philip G.
Published: February 2005

Gammaherpesviruses persist as latent episomes in a dynamic lymphocyte pool. The regulated production of an episome maintenance protein is therefore crucial to their survival. The transcription initiation site of the murine gammaherpesvirus 68 episome maintenance protein, ORF73, was mapped to the viral terminal repeats, more than 10 kb distant from the open reading frame (ORF) itself. A 5′ non-coding exon in the terminal repeats was spliced to the right end of the viral unique sequence, and then across ORFs 75a, 75b, 75c and 74 to ORF73. The right-hand portion of a single repeat unit was sufficient for constitutive promoter activity. The unique left end of the viral genome further enhanced ORF73 transcription. This, together with the large size of the predominant ORF73 mRNA, suggested that transcription initiates in distal repeat units and then splices between repeats to generate an extensive 5′ untranslated region. A second promoter in the left-hand portion of the proximal terminal repeat unit generated a transcript which overlapped that of ORF73, but failed to splice to the ORF73 coding exon and so transcribed ORF75a. In distal repeat copies, however, transcription from this promoter would enter the next repeat unit to become an ORF73 mRNA. There was a third promoter just upstream of ORF73 itself. These data indicate that ORF73 transcription is highly complex, and support the idea that the terminal repeats of gamma-2-herpesviruses constitute a vital component of episomal persistence.
Nef expressed from human immunodeficiency virus type 1 extrachromosomal DNA downregulates CD4 on primary CD4+ T lymphocytes: implications for integrase inhibitors
Authors: Gillim-Ross Laura , Cara Andrea , Klotman Mary E.
Published: February 2005

Recently developed integrase inhibitors targeting the HIV-1 integrase (IN) protein block integration of HIV DNA in the target cell, preventing subsequent virus replication. In the absence of integration, viral DNA is shunted towards the formation of extrachromosomal DNA (E-DNA). Although HIV-1 E-DNA does not support productive replication, it is transcriptionally active and produces viral proteins. However, the significance of E-DNA in virus replication and pathogenesis is poorly understood. In this study, the functional activity of the HIV-1 Nef protein expressed in the absence of viral integration was analysed. Using both a recombinant HIV-1 IN defective virus and a diketo acid IN inhibitor, evidence was provided showing that Nef expressed from E-DNA downregulates CD4 surface expression on primary CD4+ T lymphocytes. These results suggest that proteins expressed in the absence of integration may have potential clinical consequences, an issue that should be further explored with the introduction of IN inhibitors.
Pig anelloviruses are highly prevalent in swine herds in France
Authors: Bigarré L. , Beven V. , de Boisséson C. , Grasland B. , Rose N. , Biagini P. , Jestin A.
Published: February 2005

A survey of anelloviruses in swine herds from Britanny, France, is reported. By using PCR targeted to the conserved untranslated region, prevalences of 93 and 73 % were found among 15 herds and 33 animals, respectively. The lung was the organ found to be positive most frequently among the five organs tested from 32 animals. The highest identity levels of our nucleotide sequences were found with pig isolates from Japan and with an isolate from Tupaia belangeri. Interestingly, when aligning all available swine isolates from France and Japan, at least two phylogenetic groups were identified, each one containing clones from France and Japan. Some animals carried clones from both groups, demonstrating intra-individual variability. Despite the putative harmlessness of anelloviruses, the potential inoculum carried by pigs must be further evaluated as a sanitary threat.
Induction of mucosal and systemic immune response by single-dose oral immunization with biodegradable microparticles containing DNA encoding HBsAg
Authors: He Xiao-Wen , Wang Fang , Jiang Lei , Li Jun , Liu Shan-kui , Xiao Zhen-Yu , Jin Xiao-Qin , Zhang Ya-Nan , He Ying , Li Kai , Guo Ying-Jun , Sun Shu-Han
Published: February 2005

The purpose of this work was to assess the ability of plasmid DNA encoding hepatitis B virus (HBV) HBsAg encapsulated in poly(dl-lactide-co-glycolic acid) (PLGA) microparticles to induce local and systemic HBsAg-specific immunity following a single dose of oral immunization. RT-PCR analysis demonstrated prolonged transcription of plasmid DNA, consistent with the sustained expression and presentation of target antigen observed by confocal laser scanning microscopy, in gut-associated lymphocyte tissue (GALT) from mice immunized orally with plasmid DNA encapsulated into PLGA microparticles. Oral administration of PLGA-DNA microparticles induced a long-lasting and stable antigen-specific antibody response, both serum total antibody and intestinal IgA, in BALB/c mice. Mice immunized orally exhibited antigen-specific gamma interferon production and cytotoxic T lymphocyte responses in spleen and GALT after restimulationin vitrowith HBsAg or tumour cells stably expressing HBsAg. In contrast, naked DNA vaccines given by intramuscular injection induced only systemic cellular and humoral responses to HBsAg, which were much lower than the responses elicited by oral DNA encapsulated in PLGA microparticles at equivalent doses. The results are encouraging with regard to obtaining good compliance and vaccination coverage with candidate plasmid DNA vaccines, especially in developing countries.
Genetic diversity of a natural population of Cucurbit yellow stunting disorder virus
Authors: Marco C. F. , Aranda M. A.
Published: February 2005

An analysis of nucleotide sequences in five coding and one non-coding genomic regions of 35Cucurbit yellow stunting disorder virus(CYSDV) isolates collected on a local scale over an 8 year period is reported here. In total, 2277 nt were sequenced for each isolate, representing about 13 % of the complete virus genome. Mean nucleotide diversity for the whole population in synonymous positions in the coding regions was 0·00068, whilst in the 5′ untranslated region (5′ UTR) of genomic RNA2, it was 0·00074; both of these values are very small, compared with estimates of nucleotide diversity for populations of other plant viruses. Nucleotide diversity was also determined independently for each of the ORFs and for the 5′ UTR of RNA2; the data showed that variability is not distributed evenly among the different regions of the viral genome, with the coat protein gene showing more diversity than the other four coding regions that were analysed. However, the low variability found precluded any inference of selection differences among gene regions. On the other hand, no evidence of selection associated with host adaptation was found. In contrast, at least a single amino acid change in the coat protein appears to have been selected with time.
VaZyMolO: a tool to define and classify modularity in viral proteins
Authors: Ferron François , Rancurel Corinne , Longhi Sonia , Cambillau Christian , Henrissat Bernard , Canard Bruno
Published: February 2005

Viral structural genomic projects aim at unveiling the function of unknown viral proteins by employing high-throughput approaches to determine their 3D structure and to identify their function through fold-homology studies. The ‘viral enzyme module localization’ (VaZyMolO) tool has been developed, which aims at defining viral protein modules that might be expressed in a soluble and functionally active form, thereby identifying candidates for crystallization studies. VaZyMolO includes 114 complete viral genome sequences of both negative- and positive-sense, single-stranded RNA viruses available from NCBI. In VaZyMolO, a module is defined as a structural and/or functional unit. Modules were first identified by homology search and then validated by the convergence of results from sequence composition analysis, motif search, transmembrane region search and domain definitions, as found in the literature. The public interface of VaZyMolO, which is accessible from http://www.vazymolo.org, allows comparison of a query sequence to all VaZyMolO modules of known function.
Hepatitis C virus and hepatitis B virus bind to heparin: purification of largely IgG-free virions from infected plasma by heparin chromatography
Authors: Zahn Astrid , Allain Jean-Pierre
Published: February 2005

Binding to heparin of hepatitis C virus (HCV) and hepatitis B virus (HBV) from chronic carriers was investigated. Eighty per cent of HCV RNA from an agammaglobulinaemic patient (IgG-free virus) was retained on immobilized heparin and eluted with ⩾0·4 M NaCl, in contrast to ∼20 % from immunocompetent chronic carriers (with ⩽8 % IgG-free virus). Increased binding to heparin of the HCV fraction that was not retained by a protein G column suggested that antibodies complexed to the virions partially inhibited the interaction. A higher proportion (15–80 %) of HBV from chronic carriers bound to heparin and eluted with ⩾0·4 M NaCl. After washing of the heparin columns with 0·3 M NaCl, <1 % of total plasma proteins co-eluted with HCV or HBV. By this one-step heparin chromatography, without ultracentrifugation, IgG-free HCV and IgG-free HBV were preferentially purified from human plasma by 1000-fold and greater than 500-fold, respectively. Following assessment with an anti-E2 envelope protein antibody, the amount of immunoprecipitated HCV particles after heparin purification was similar to that in the original plasma, suggesting that undamaged virions were purified. This was further supported by heparin-purified HCV binding to lymphocyte cell lines in a dose-dependent manner. Intact HBV particles were detected by electron microscopy. It was concluded that HCV and HBV from chronically infected patients bind to heparin, the closest homologue of liver heparan sulfate, and that heparin chromatography is an efficient and gentle method for purifying these viruses from human plasma. In the absence of cell-culture systems or alternative robust purification methods, heparin chromatography may help greatly in binding and infectivity studies.
African swine fever virus infection disrupts centrosome assembly and function
Authors: Jouvenet Nolwenn , Wileman Thomas
Published: February 2005

African swine fever virus (ASFV) is a large, enveloped DNA virus that assembles in perinuclear sites located close to the centrosome. It is reported here that the microtubule network becomes disorganized soon after the onset of viral DNA replication and formation of assembly sites. ASFV infection resulted in loss of γ-tubulin and pericentrin at the centrosome; this was due to protein relocalization, but not degradation. ASFV infection also inhibited the ability of the centrosome to nucleate microtubules. The reorganization of microtubules seen in ASFV-infected cells may therefore be mediated by γ-tubulin and pericentrin redistribution, and consequent disruption of centrosome assembly and function.
Mitogen-induced upregulation of hepatitis C virus expression in human lymphoid cells
Authors: Pham Tram N. Q. , MacParland Sonya A. , Coffin Carla S. , Lee Samuel S. , Bursey Ford R. , Michalak Tomasz I.
Published: February 2005

Considering growing evidence indicating that hepatitis C virus (HCV) replicates in lymphoid cells, establishment of a reliable and sensitive method for detection of HCV in these cells may provide means for monitoring the infection and the efficacy of sterilizing antiviral therapy. In this study, conditions forex vivoaugmentation and detection of the HCV genome in peripheral blood mononuclear cells (PBMCs) from patients with chronic hepatitis C (CHC) or after a sustained virological response (SVR) to antiviral treatment were assessed. Following stimulation with combinations of mitogens and/or cytokines, PBMCs and, in certain cases, affinity-purified T and B cells were examined for HCV positive- and negative-strand RNA by using RT-PCR followed by nucleic acid hybridization, while the presence of viral NS3 protein was determined by flow cytometry. HCV RNA augmentation was assessed by quantification of Southern and dot-blot hybridization signals. The results showed that treatment of peripheral lymphoid cells with mitogens stimulating T- and B-cell proliferation and with cytokines supporting their growth significantly increased HCV RNA detection in patients with both CHC and SVR. This enhancement was up to 100-fold for the HCV genome and fivefold for the NS3 protein compared with untreated cells. In conclusion, HCV RNA can be readily detected in circulating lymphoid cells in progressing hepatitis C and following SVR afterex vivocell stimulation. As such, this method offers a new investigative tool to study HCV lymphotropism and to monitor virus presence during the course of HCV infection.
Analysis of tombusvirus revertants to identify essential amino acid residues within RNA-dependent RNA polymerase motifs
Authors: Boonrod K. , Chotewutmontri S. , Galetzka D. , Krczal G.
Published: February 2005

The RNA-dependent RNA polymerase (RdRp) of Tomato bushy stunt virus (TBSV) contains an arginine- and proline-rich (RPR) motif. This motif functions as an RNA-binding domain and is essential for tombusvirus replication. A mutant carrying three arginine substitutions in this motif rendered the virus unable to replicate in Nicotiana benthamiana plants and protoplasts. When the replicase function was provided in trans, by expressing the TBSV RdRp in N. benthamiana plants, an infectious variant could be isolated. Sequence analysis showed that only the substituted glycine residue (position 216) had reverted to arginine; all other substitutions remained unchanged. This finding suggested that strong selection pressure is active to maintain necessary sequences of the viral RdRp and that the analysis of revertants may help to identify essential viral functions.
Herpes simplex virus type 2 membrane protein UL56 associates with the kinesin motor protein KIF1A
Authors: Koshizuka Tetsuo , Kawaguchi Yasushi , Nishiyama Yukihiro
Published: February 2005

The herpes simplex virus UL56 gene product is a C-terminal-anchored, type II membrane protein of unknown function. UL56 was found to interact with KIF1A, a member of the kinesin-3 family, in a yeast two-hybrid screen and a GST pull-down assay. KIF1A mediates the transport of synaptic vesicle precursors and is essential for the function and viability of neurons. When overexpressed, KIF1A co-localized with full-sized UL56, but no clear co-localization was observed when co-expressed with the UL56 mutant protein lacking its C-terminal transmembrane domain (TMD). Although the C-terminal TMD was not essential for the interaction with KIF1A in the yeast two-hybrid screen and GST pull-down assays, these results indicate that the C-terminal TMD, as well as aa 69–217, of UL56 are important for the interaction with KIF1A in vivo. The hypothesis that the UL56 protein affects vesicular trafficking in infected cells, potentially by acting as a receptor for motor proteins in neurons, is discussed.
Contamination of a specific-pathogen-free rat breeding colony with Human parainfluenzavirus type 3
Authors: Miyata Hironori , Kanazawa Tamotsu , Shibuya Kazumoto , Hino Shigeo
Published: February 2005

Routine antibody surveillance for Sendai virus in a breeding colony suggested viral invasion into laboratory rats. A more specific haemagglutination-inhibition test implied that the agent was related closely to Human parainfluenza virus type 3 (hPIV3), rather than Sendai virus. To isolate this virus, Vero cells were inoculated with lung homogenates of 30 young animals from the colony. One of the cultures became positive at the second passage by RT-PCR directed to the hPIV3 NP and L genes. Cytopathic effect with cell fusion was observed at the third passage. The HN gene of this virus (KK24) had >93 % similarity to those of other hPIV3 isolates, suggesting a human origin of KK24. Experimental intranasal inoculation of KK24 into SD rats showed virus replication in the lungs at 3–5 days post-infection (p.i.). Pathological examination of the lungs at day 5 p.i. indicated a moderate detachment, degradation and apoptosis of bronchial epitheliocytes with peribronchial mononuclear infiltrations. At day 7 p.i., these changes became less prominent, and no lesions were apparent at day 10 p.i. or later. The infected rats seroconverted at day 7 p.i. On the contrary, none of the 30 experimentally infected ICR mice showed any pathological lesions in their lungs, despite seroconversion at 7 days p.i. These results suggest that hPIV3 can invade rat colonies and has a moderate and transient pathogenicity in rats. This is the first report of non-experimental hPIV3 infection in laboratory rats, unexpectedly detected by antibody screening for Sendai virus.
An intact RBR-binding motif is not required for infectivity of Maize streak virus in cereals, but is required for invasion of mesophyll cells
Authors: McGivern David R. , Findlay Kim C. , Montague Nicholas P. , Boulton Margaret I.
Published: February 2005

The replication-associated protein (RepA) of Maize streak virus interacts in yeast with retinoblastoma-related protein (RBR), the negative regulator of cell-cycle progression. This may allow geminiviruses to subvert cell-cycle control to provide an environment that is suitable for viral DNA replication. To determine the importance of this interaction for MSV infection, the RBR-binding motif, LxCxE, was mutated to IxCxE or LxCxK. Whilst RBR binding in yeast could not be detected for the LxCxK mutant, the IxCxE protein retained limited binding activity. Both mutants were able to replicate in maize cultures and to infect maize plants. However, whereas the wild-type virus invaded mesophyll cells of mature leaves, the LxCxK mutant was restricted to the vasculature, which is invaded prior to leaf maturity. Mature leaves contain high levels of RBR and it is suggested that the MSV RepA–RBR interaction is essential only in tissues with high levels of active RBR.
A three-nucleotide mutation altering the Maize streak virus Rep pRBR-interaction motif reduces symptom severity in maize and partially reverts at high frequency without restoring pRBR–Rep binding
Authors: Shepherd Dionne N. , Martin Darren P. , McGivern David R. , Boulton Margaret I. , Thomson Jennifer A. , Rybicki Edward P.
Published: February 2005

Geminivirus infectivity is thought to depend on interactions between the virus replication-associated proteins Rep or RepA and host retinoblastoma-related proteins (pRBR), which control cell-cycle progression. It was determined that the substitution of two amino acids in the Maize streak virus (MSV) RepA pRBR-interaction motif (LLCNE to LLCLK) abolished detectable RepA–pRBR interaction in yeast without abolishing infectivity in maize. Although the mutant virus was infectious in maize, it induced less severe symptoms than the wild-type virus. Sequence analysis of progeny viral DNA isolated from infected maize enabled detection of a high-frequency single-nucleotide reversion of C(601)A in the 3 nt mutated sequence of the Rep gene. Although it did not restore RepA–pRBR interaction in yeast, sequence-specific PCR showed that, in five out of eight plants, the C(601)A reversion appeared by day 10 post-inoculation. In all plants, the C(601)A revertant eventually completely replaced the original mutant population, indicating a high selection pressure for the single-nucleotide reversion. Apart from potentially revealing an alternative or possibly additional function for the stretch of DNA that encodes the apparently non-essential pRBR-interaction motif of MSV Rep, the consistent emergence and eventual dominance of the C(601)A revertant population might provide a useful tool for investigating aspects of MSV biology, such as replication, mutation and evolution rates, and complex population phenomena, such as competition between quasispecies and population turnover.
Experimental infection of sheep with ovine herpesvirus 2 via aerosolization of nasal secretions
Authors: Taus Naomi S. , Traul Donald L. , Oaks J. Lindsay , Crawford Timothy B. , Lewis Gregory S. , Li Hong
Published: February 2005

Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever in clinically susceptible ruminants, including cattle, bison and deer. Studies of OvHV-2 have been hampered by the lack of an in vitro propagation system. Here, the use of nasal secretions collected from OvHV-2-infected sheep experiencing intense virus shedding episodes as a source of infectious virus for experimental animal infections was examined. OvHV-2 uninfected sheep were nebulized with nasal secretions containing approximately 108 to 101 copies of OvHV-2 DNA. The time to detectable viral DNA in peripheral blood leukocytes (7–12 days post-infection) and virus-specific antibody in plasma (9–32 days post-infection) varied with the dose of inocula administered. Here, the use of nasal secretions as a source of infectious OvHV-2 was defined and the minimum infectious dose of a pool of nasal secretions that can be used in further studies of viral pathogenesis and vaccine development was determined.
The herpesvirus saimiri Rta gene autostimulates via binding to a non-consensus response element
Authors: Walters Matthew S. , Hall Kersten T. , Whitehouse Adrian
Published: February 2005

Herpesvirus saimiri ORF 50a protein expression is sufficient to reactivate the entire lytic-replication cycle. ORF 50a functions as a sequence-specific transactivator that is capable of activating delayed-early gene expression via direct binding to an ORF 50 response element (RE) within the respective promoter. Here, it is shown that ORF 50a is capable of transactivating its own promoter. Deletion analysis of the ORF 50a promoter showed that the ORF 50-responsive element is contained within an 80 bp fragment, situated 293–373 bp from the transcription initiation site. Gel-retardation analysis further mapped the RE to a 34 bp fragment that was able to confer ORF 50 responsiveness to an enhancerless SV40 minimal promoter. Sequence analysis showed that this RE has no direct similarity to previously identified ORF 50 REs. Therefore, it is concluded that ORF 50a is capable of stimulating its own promoter via a novel RE.
Characterization of genotype H hepatitis B virus strain identified for the first time from a Japanese blood donor by nucleic acid amplification test
Authors: Ohnuma Hitoshi , Yoshikawa Akira , Mizoguchi Hideaki , Okamoto Hiroaki ,   the JRC NAT Screening Research Group
Published: February 2005

The Japanese Red Cross has been conducting a nucleic acid amplification test (NAT) screening for hepatitis B virus (HBV), hepatitis C virus and human immunodeficiency virus 1 among blood donors since July 1 1999. The first case of HBV genotype H was found and reported in Japan. Serological markers of HBV were not detected in this NAT-positive donation. It may be that the positive donation was in the serological window period at the early stage of infection. The complete genome of 3215 nt was sequenced, and the sequence had 99·3 % homology with the strain from Los Angeles, USA (LSA2523). Here, a leucine zipper motif was found in the region of the HBV surface antigen conserved through genotypes A–H.
Analysis of the processing and transmembrane topology of the E2p7 protein of hepatitis C virus
Authors: Isherwood Beverley J. , Patel Arvind H.
Published: February 2005

Hepatitis C virus C, E1, E2 and p7 proteins are cleaved from a viral polyprotein by host signal peptidases. Cleavage at the E2/p7 site is incomplete in genotype 1a strain H (resulting in E2, p7 and E2p7 species), although it has been reported to be more efficient in genotype 1b strain BK. Here, the proteolytic processing and transmembrane topology of genotype 1a strain H77c p7 was investigated when expressed in the context of E2p7. Partial processing was seen at the E2/p7 site in mammalian cells, the efficiency of which improved in the presence of nucleotide sequences downstream of p7. In insect cells, no processing at the E2/p7 site occurred and the uncleaved E2p7 species was incorporated into virus-like particles when expressed in the context of CE1E2p7c-myc. E2p7c-myc formed a heterodimer with E1, indicating that, like the well-characterized E1–E2 complex, the E1–E2p7 heterodimer may also play a functional role in virus replication. Comparison of the p7 signal peptide sequences of strains BK and H77c revealed 3 aa differences (positions 720, 733 and 742). Mutational analysis showed that the V720L change in the H77c sequence substantially increased processivity at the E2/p7 site. The p7 protein adopts a double membrane-spanning topology with both its N and C termini orientated luminally in the endoplasmic reticulum. The transmembrane topology of E2p7 species was examined by two independent means. In both cases, the C terminus of p7 in E2p7 was found to be cytoplasmically orientated, indicating that p7 adopts a dual transmembrane topology.
Structure–Function Analysis of Cf-9, a Receptor-Like Protein with Extracytoplasmic Leucine-Rich Repeatsw⃞
Authors: van der Hoorn Renier A.L. , Wulff Brande B.H. , Rivas Susana , Durrant Marcus C. , van der Ploeg Anke , de Wit Pierre J.G.M. , Jones Jonathan D.G.
Published: February 2005

The tomato (Lycopersicon pimpinellifolium) resistance protein Cf-9 belongs to a large class of plant proteins with extracytoplasmic Leu-rich repeats (eLRRs). eLRR proteins play key roles in plant defense and development, mainly as receptor-like proteins or receptor-like kinases, conferring recognition of various pathogen molecules and plant hormones. We report here a large-scale structure–function analysis of an eLRR protein. A total of 66 site-directed mutants of Cf-9 were analyzed for activity in Avr9 recognition and for protein stability and the results interpreted with the help of a homology model of the Cf-9 structure. Conserved Trp and Cys pairs in the N-terminal LRR-flanking domain appear to be important for Cf-9 activity and are probably exposed at the putative concave inner surface of the Cf-9 protein, where recognition specificity also resides. Removal of each of the 22 putative N-linked glycosylation sites (PGS) revealed that many PGSs contribute to Cf-9 activity and that the PGSs in the putative α-helices of the LRR modules are essential. Immunoblot analysis and mass spectrometry showed that all but one of the PGSs are N-glycosylated. Introduction of glycosylation at the putative concave β-sheet surface blocks Cf-9 activity, in some cases probably by disturbing specific recognition, and in another case by steric hindrance with existing N-glycans. The glycosylation pattern and several other features are conserved in other eLRR proteins, where similar mutations show similar phenotypes.
RIN13 Is a Positive Regulator of the Plant Disease Resistance Protein RPM1
Authors: Al-Daoude Antonious , de Torres Zabala Marta , Ko Jong-Hyun , Grant Murray
Published: February 2005

The RPM1 protein confers resistance to Pseudomonas syringae pv tomato DC3000 expressing either of the Type III effector proteins AvrRpm1 or AvrB. Here, we describe the isolation and functional characterization of RPM1 Interacting Protein 13 (RIN13), a resistance protein interactor shown to positively enhance resistance function. Ectopic expression of RIN13 (RIN13s) enhanced bacterial restriction mechanisms but paradoxically abolished the normally rapid hypersensitive response (HR) controlled by RPM1. In contrast with wild-type plants, leaves expressing RIN13s did not undergo electrolyte leakage or accumulate H2O2 after bacterial delivery of AvrRpm1. Overexpression of RIN13 also altered the transcription profile observed during a normal HR. By contrast, RIN13 knockout plants had the same ion leakage signatures and HR timing of wild-type plants in response to DC3000(avrRpm1) but failed to suppress bacterial growth. The modified phenotypes seen in the RIN13s/as plants were specific to recognition of AvrRpm1 or AvrB, and wild-type responses were observed after challenge with other incompatible pathogens or the virulent DC3000 isolate. Our results suggest that cell death is not necessary to confer resistance, and engineering enhanced resistance without activation of programmed cell death is a real possibility.
Role of β-Oxidation in Jasmonate Biosynthesis and Systemic Wound Signaling in Tomato
Authors: Li Chuanyou , Schilmiller Anthony L. , Liu Guanghui , Lee Gyu In , Jayanty Sastry , Sageman Carolyn , Vrebalov Julia , Giovannoni James J. , Yagi Kaori , Kobayashi Yuichi , Howe Gregg A.
Published: February 2005

Jasmonic acid (JA) is a lipid-derived signal that regulates plant defense responses to biotic stress. Here, we report the characterization of a JA-deficient mutant of tomato (Lycopersicon esculentum) that lacks local and systemic expression of defensive proteinase inhibitors (PIs) in response to wounding. Map-based cloning studies demonstrated that this phenotype results from loss of function of an acyl-CoA oxidase (ACX1A) that catalyzes the first step in the peroxisomal β-oxidation stage of JA biosynthesis. Recombinant ACX1A exhibited a preference for C12 and C14 straight-chain acyl-CoAs and also was active in the metabolism of C18 cyclopentanoid-CoA precursors of JA. The overall growth, development, and reproduction of acx1 plants were similar to wild-type plants. However, the mutant was compromised in its defense against tobacco hornworm (Manduca sexta) attack. Grafting experiments showed that loss of ACX1A function disrupts the production of the transmissible signal for wound-induced PI expression but does not affect the recognition of this signal in undamaged responding leaves. We conclude that ACX1A is essential for the β-oxidation stage of JA biosynthesis and that JA or its derivatives is required both for antiherbivore resistance and the production of the systemic wound signal. These findings support a role for peroxisomes in the production of lipid-based signaling molecules that promote systemic defense responses.
Identification and Dynamics of Two Classes of Aurora-Like Kinases in Arabidopsis and Other Plants
Authors: Demidov Dmitri , Van Damme Daniël , Geelen Danny , Blattner Frank R. , Houben Andreas
Published: February 2005

Aurora-like kinases play key roles in chromosome segregation and cytokinesis in yeast, plant, and animal systems. Here, we characterize three Arabidopsis thaliana protein kinases, designated AtAurora1, AtAurora2, and AtAurora3, which share high amino acid identities with the Ser/Thr kinase domain of yeast Ipl1 and animal Auroras. Structure and expression of AtAurora1 and AtAurora2 suggest that these genes arose by a recent gene duplication, whereas the diversification of plant α and β Aurora kinases predates the origin of land plants. The transcripts and proteins of all three kinases are most abundant in tissues containing dividing cells. Intracellular localization of green fluorescent protein–tagged AtAuroras revealed an AtAurora-type specific association mainly with dynamic mitotic structures, such as microtubule spindles and centromeres, and with the emerging cell plate of dividing tobacco (Nicotiana tabacum) BY-2 cells. Immunolabeling using AtAurora antibodies yielded specific signals at the centromeres that are coincident with histone H3 that is phosphorylated at Ser position10 during mitosis. An in vitro kinase assay demonstrated that AtAurora1 preferentially phosphorylates histone H3 at Ser 10 but not at Ser 28 or Thr 3, 11, and 32. The phylogenetic analysis of available Aurora sequences from different eukaryotic origins suggests that, although a plant Aurora gene has been duplicated early in the evolution of plants, the paralogs nevertheless maintained a role in cell cycle–related signal transduction pathways.
Dissecting the β-Aminobutyric Acid–Induced Priming Phenomenon in Arabidopsis
Authors: Ton Jurriaan , Jakab Gabor , Toquin Valérie , Flors Victor , Iavicoli Annalisa , Maeder Muriel N. , Métraux Jean-Pierre , Mauch-Mani Brigitte
Published: February 2005

Plants treated with the nonprotein amino acid β-aminobutyric acid (BABA) develop an enhanced capacity to resist biotic and abiotic stresses. This BABA-induced resistance (BABA-IR) is associated with an augmented capacity to express basal defense responses, a phenomenon known as priming. Based on the observation that high amounts of BABA induce sterility in Arabidopsis thaliana, a mutagenesis screen was performed to select mutants impaired in BABA-induced sterility (ibs). Here, we report the isolation and subsequent characterization of three T-DNA–tagged ibs mutants. Mutant ibs1 is affected in a cyclin-dependent kinase–like protein, and ibs2 is defective in AtSAC1b encoding a polyphosphoinositide phosphatase. Mutant ibs3 is affected in the regulation of the ABA1 gene encoding the abscisic acid (ABA) biosynthetic enzyme zeaxanthin epoxidase. To elucidate the function of the three IBS genes in plant resistance, the mutants were tested for BABA-IR against the bacterium Pseudomonas syringae pv tomato, the oomycete Hyaloperonospora parasitica, and BABA-induced tolerance to salt. All three ibs mutants were compromised in BABA-IR against H. parasitica, although to a different extent. Whereas ibs1 was reduced in priming for salicylate (SA)-dependent trailing necrosis, mutants ibs2 and ibs3 were affected in the priming for callose deposition. Only ibs1 failed to express BABA-IR against P. syringae, which coincided with a defect in priming for SA-inducible PR-1 gene expression. By contrast, ibs2 and ibs3 showed reduced BABA-induced tolerance to salt, which correlated with an affected priming for ABA-inducible gene expression. For all three ibs alleles, the defects in BABA-induced sterility and BABA-induced protection against P. syringae, H. parasitica, and salt could be confirmed in independent mutants. The data presented here introduce three novel regulatory genes involved in priming for different defense responses.
Dermatitis in a printed‐circuit board manufacturing facility
Authors: Rischitelli Gary
Published: February 2005

Dermatitis complaints are frequently encountered among workers in the microelectronics industry and have been associated with a variety of physical, chemical and psychosocial causes. Employees working in the silk‐screening and kiln‐drying areas of a printed‐circuit board manufacturer complained of recurrent itching and rashes. This case report describes those symptoms and the results of a brief survey of the affected employees, which provided useful data regarding the likely aetiology. A brief epidemiological survey instrument was used to gain information regarding the pattern, duration and frequency of employee symptoms. Data from the survey were used to identify seasonal variations in symptoms that correlated with specific environmental factors in the plant. A brief epidemiological investigation using a self‐reported symptom survey was an efficient tool to catalogue the character, pattern, duration and frequency of complaints that correlated with specific environmental factors in the plant (temperature and low humidity), which were likely causes of the outbreak.
A Giant Dissecting Aneurysm of Ascending Aorta
Authors: Baysan Oben , Erinc Kursad , Uzun Mehmet , Yokusoglu Mehmet , Genc Celal , Karaeren Hayrettin , Isik Ersoy
Published: February 2005

Aortic aneurysm is a serious clinical challenge for the cardiologist. Aneurysm expansion frequently associated with significant dissection and rupture risk. Currently available diagnostic modalities make earlier diagnosis and therapy possible hence giant aneurysm with dissection is relatively rare. In this case report, we present a patient with giant aortic aneurysm with dissection.
Preventing youth access to alcohol: outcomes from a multi‐community time‐series trial*
Authors: Wagenaar Alexander C. , Toomey Traci L. , Erickson Darin J.
Published: February 2005

Aims/intervention  The Complying with the Minimum Drinking Age project (CMDA) is a community trial designed to test effects of two interventions designed to reduce alcohol sales to minors: (1) training for management of retail alcohol establishments and (2) enforcement checks of alcohol establishments.

Design  CMDA is a multi‐community time‐series quasi‐experimental trial with a nested cohort design.

Setting/participants  CMDA was implemented in 20 cities in four geographic areas in the US Midwest.

Measurements  The core outcome, propensity for alcohol sales to minors, was directly tested with research staff who attempted to purchase alcohol without showing age identification using a standardized protocol in 602 on‐premise and 340 off‐premise alcohol establishments. Data were collected every other week in all communities over 4 years. Mixed‐model regression and Box–Jenkins time‐series analyses were used to assess short‐ and long‐term establishment‐specific and general community‐level effects of the two interventions.

Findings  Effects of the training intervention were mixed. Specific deterrent effects were observed for enforcement checks, with an immediate 17% reduction in likelihood of sales to minors. These effects decayed entirely within 3 months in off‐premise establishments and to an 8.2% reduction in on‐premise establishments.

Conclusions  Enforcement checks prevent alcohol sales to minors. At the intensity levels tested, enforcement primarily affected specific establishments checked, with limited diffusion to the whole community. Finally, most of the enforcement effect decayed within 3 months, suggesting that a regular schedule of enforcement is necessary to maintain deterrence.
Should nortriptyline be used as a first‐line aid to help smokers quit? Results from a systematic review and meta‐analysis
Authors: Wagena E. J. , Knipschild P. , Zeegers M. P. A.
Published: February 2005

Objectives  The objective of this paper is to evaluate the efficacy of nortriptyline for smoking cessation compared to placebo and bupropion sustained release.

Data sources  Randomized trials were identified by (1) checking electronic and (2) online publicly accessible registers of clinical trials; (3) searching references of identified studies and screening abstract books of conferences and symposia, and (4) personal communication with the first authors of identified papers.

Review methods  We included randomized trials in which nortriptyline was compared to placebo or bupropion hydrochloride SR. The main clinical outcome measure was (at least) 6‐month prolonged abstinence, confirmed with a biochemical test. To investigate the efficacy of nortriptyline in time, we calculated the percentage of smokers who relapsed in time.

Results  We identified five randomized trials, including 861 smokers. Compared to placebo medication, nortriptyline resulted in significantly higher prolonged abstinence rates after at least 6 months [relative risk (RR) = 2.4, 95% CI 1.7–3.6; RD = 0.11, 95% CI 0.07–0.15]. The difference in efficacy between nortriptyline and placebo was highest in the first months after the target quit date. However, the number of people who remained abstinent decreased substantially and significantly faster over time in the nortriptyline group. Although bupropion resulted in higher abstinence rates compared with nortriptyline, the difference was not statistically significant (RR = 1.7, 95% CI 0.7–4.1).

Conclusion  This systematic review and meta‐analysis shows that the use of nortriptyline for smoking cessation resulted in higher prolonged abstinence rates after at least 6 months compared to placebo treatment. Furthermore, the use of nortriptyline for smoking cessation is well tolerated and safe. As a result, we believe health care professionals should be recommended to prescribe nortriptyline as a first‐line therapy for smoking cessation, also because of the much lower cost of nortriptyline compared to bupropion SR.
Demographic variation and biomass allocation of Agropyron cristatum grown on steppe and dune sites in the Hunshandake Desert, North China
Authors: Wang R. Z.
Published: February 2005

Demographic and biomass allocation patterns of Agropyron cristatum were measured on steppe and dune sites in the Hunshandake desert of North China in 2001 and 2002. Total plant population, reproductive shoot densities and its differentiation rates were significantly higher in the steppe sites in both years. Plant heights for both vegetative and reproductive shoots were greater in the year with the higher rainfall. The dune sites had a higher biomass allocation to vegetative shoots and roots, while the steppe sites had a higher biomass allocation to reproductive shoots and seed production. It is suggested that the population demography and biomass allocation of the species responded to the differences in the soil variables in the steppe and dune sites.
Response of herbage regrowth and water‐soluble carbohydrate concentration of ryegrass species to defoliation practices when grown in a Mediterranean environment
Authors: Callow M. N. , Michell P. , Baker J. E. , Cocks P. S. , Hough G.M.
Published: February 2005

Three experiments were conducted to determine the association between leaf number per tiller at defoliation, water‐soluble carbohydrate (WSC) concentration and herbage mass of juvenile ryegrass plants when grown in a Mediterranean environment. Seedlings of ryegrass were grown in nursery pots arranged side‐by‐side and located outside in the open‐air to simulate a mini‐sward in Experiments 1 and 2, and a mixture of annual ryegrass and subterranean clover (Trifolium subterraneum L.) was grown in a small plot field study in Experiment 3. Swards were defoliated mechanically with the onset of defoliation commencing within 28 d of germination. Frequency of defoliation ranged from one to nine leaves per tiller, whilst defoliation height ranged from 30 mm of pseudostem height that removed all leaf laminae in Experiment 1, to 50 mm of pseudostem height with some leaf laminae remaining post‐defoliation in Experiments 2 and 3.

A positive relationship between herbage mass of ryegrass, WSC concentration and leaf number per tiller at defoliation was demonstrated in all experiments. In Experiment 1, the herbage mass of leaf, pseudostem and roots of tillers defoliated at one leaf per tiller was reduced to 0·10, 0·09 and 0·06 of those tillers defoliated less frequently at six leaves per tiller. However, the reduction in herbage mass from frequent defoliation was less severe in Experiment 2 and coincided with a 0·20 reduction in WSC concentration of pseudostem compared with 0·80 measured during Experiment 1. In Experiment 3, the highest harvested herbage mass of ryegrass occurred when defoliation was nine leaves per tiller. Although the harvested herbage from this sward contained senescent herbage, the in vitro dry‐matter digestibility of the harvested herbage did not differ significantly compared with the remaining treatments that had been defoliated more frequently.

Leaf numbers of newly germinated ryegrass tillers in a Mediterranean environment were positively associated with WSC concentration of pseudostem and herbage mass. A minimum period of two to three leaf appearances was required to restore WSC concentrations to levels measured prior to defoliation thereby avoiding a significant reduction in herbage mass. However, maximum herbage mass of a mixed sward containing ryegrass and subterranean clover was achieved when defoliation was delayed to nine leaves per tiller.