bluuebunny/crossref_metadata_2025 huggingface dataset:
Functional organization of the major late transcriptional unit of canine adenovirus type 2
Authors: Szelechowski Marion , Fournier Annie , Richardson Jennifer , Eloit Marc , Klonjkowski Bernard
Published: April 2009

Vectors derived from canine adenovirus type 2 (CAV-2) are attractive candidates for gene therapy and live recombinant vaccines. CAV-2 vectors described thus far have been generated by modifying the virus genome, most notably early regions 1 and 3 or the fiber gene. Modification of these genes was underpinned by previous descriptions of their mRNA and protein-coding sequences. Similarly, the construction of new CAV-2 vectors bearing changes in other genomic regions, in particular many of those expressed late in the viral cycle, will require prior characterization of the corresponding transcriptional units. In this study, we provide a detailed description of the late transcriptional organization of the CAV-2 genome. We examined the major late transcription unit (MLTU) and determined its six families of mRNAs controlled by the putative major late promoter (MLP). All mRNAs expressed from the MLTU had a common non-coding tripartite leader (224 nt) at their 5′ end. In transient transfection assays, the predicted MLP sequence was able to direct luciferase gene expression and the TPL sequence yielded a higher amount of transgene product. Identification of viral transcriptional products following in vitro infection confirmed most of the predicted protein-coding regions that were deduced from computer analysis of the CAV-2 genome. These findings contribute to a better understanding of gene expression in CAV-2 and lay the foundation required for genetic modifications aimed at vector optimization.
MicroRNAs 221 and 222 target p27Kip1 in Marek's disease virus-transformed tumour cell line MSB-1
Authors: Lambeth Luke S. , Yao Yongxiu , Smith Lorraine P. , Zhao Yuguang , Nair Venugopal
Published: April 2009

MicroRNAs (miRNAs) are a class of short RNAs that function as post-transcriptional suppressors of protein expression and are involved in a variety of biological processes, including oncogenesis. Several recent studies have implicated the involvement of miR-221 and miR-222 in tumorigenesis as these miRNAs are upregulated in a number of cancers and affect the expression of cell cycle regulatory proteins such as the cyclin-dependent kinase (cdk) inhibitor p27Kip1. Marek's disease virus (MDV) is a highly oncogenic herpesvirus that affects poultry, causing acute neoplastic disease with lymphomatous lesions in several organs. MDV-encoded oncogenes such as Meq are directly implicated in the neoplastic transformation of T cells and have been well studied. More recently, however, the involvement of both host and virus-encoded miRNAs in the induction of MD lymphomas is being increasingly recognized. We analysed the miRNA expression profiles in the MDV-transformed lymphoblastoid cell line MSB-1 and found that endogenous miRNAs miR-221 and miR-222 were significantly upregulated. Demonstration of the conserved binding sites for these miRNAs in the chicken p27Kip1 3′-untranslated region sequence and the repression of luciferase activity of reporter constructs indicated that miR-221 and miR-222 target p27Kip1 in these cells. We also found that overexpression of miR-221 and miR-222 decreased p27Kip1 levels and that treatment with retrovirally expressed antagomiRs partially alleviated this suppression. These data show that an oncogenic herpesvirus, as in the case of many cancers, can exploit the miRNA machinery for suppressing cell cycle regulatory molecules such as p27Kip1 in the induction and progression of T-cell lymphomas.
Human cytomegalovirus US9 protein contains an N-terminal signal sequence and a C-terminal mitochondrial localization domain, and does not alter cellular sensitivity to apoptosis
Authors: Mandic Lana , Miller Matthew S. , Coulter Corinne , Munshaw Brian , Hertel Laura
Published: April 2009

The human cytomegalovirus (CMV) US2–US11 genomic region contains a cluster of genes whose products interfere with antigen presentation by the major histocompatibility complex (MHC) proteins. Although included in this cluster, the US9 gene encodes a glycoprotein that does not affect MHC activity and whose function is still largely uncharacterized. An in silico analysis of the US9 amino-acid sequence uncovered the presence of an N-terminal signal sequence (SS) and a C-terminal transmembrane domain containing the specific hallmarks of known mitochondrial localization sequences (MLS). Expression of full-length US9 and of US9 deletion mutants fused to GFP revealed that the N-terminal SS mediates US9 targeting to the endoplasmic reticulum (ER) and that the C-terminal MLS is both necessary and sufficient to direct US9 to mitochondria in the absence of a functional SS. This dual localization suggested a possible role for US9 in protection from apoptosis triggered by ER-to-mitochondria signalling. Fibroblasts infected with the US2–US11 deletion mutant virus RV798 or with the parental strain AD169varATCC were equally susceptible to death triggered by exposure to tumour necrosis factor (TNF)-α, tunicamycin, thapsigargin, brefeldin A, lonidamine and carbonyl cyanide m-chloro phenyl hydrazone, but were 1.6-fold more sensitive to apoptosis induced by hygromycin B. Expression of US9 in human embryonic kidney 293T cells or in fibroblasts, however, did not protect cells from hygromycin B-mediated death. Together, these results classify US9 as the first CMV-encoded protein to contain an N-terminal SS and a C-terminal MLS, and suggest a completely novel role for this protein during infection.
A multipartite single-stranded negative-sense RNA virus is the putative agent of fig mosaic disease
Authors: Elbeaino Toufic , Digiaro Michele , Alabdullah Abdulkader , De Stradis Angelo , Minafra Angelantonio , Mielke Nicole , Castellano Maria Antonietta , Martelli Giovanni P.
Published: April 2009

Several dsRNA bands (approx. 0.6–7 kbp in size) were recovered from tissues of mosaic-diseased fig seedlings which contained the enveloped round structures known as double membrane bodies (DMBs). blast analysis of a 4353 and a 1120 nt sequence from the two largest RNA segments showed homology with the polymerase and the putative glycoprotein precursor genes of negative-sense single-stranded RNA viruses of the family Bunyaviridae. Negative- and positive-sense riboprobes designed from both RNA segments hybridized to two bands of approximately 7 and 2.3 kbp in Northern blots of dsRNAs. Thus, these segments were identified as putative RNA-1 and RNA-2 of a novel virus for which the name fig mosaic virus (FMV) is proposed. Identity levels of predicted amino acids of the protein encoded by FMV RNA-1 with those of species of the family Bunyaviridae and European mountain ash ringspot-associated virus (EMERaV) were 28 and 54 %, respectively. RNA-2 showed 38 % identity at the amino acid level only with EMARaV. RNA-1 segment contained five conserved motifs (A–E) and an endonucleolytic centre of comparable genes of L RNA of bunyaviruses and EMARaV RNA-1. In a phylogenetic tree constructed with RdRp sequences, EMARaV grouped with FMV in a clade distinct from those of all bunyavirus genera. The consistent association of DMBs with mosaic symptoms and the results of molecular investigations strongly indicate that DMBs are particles of FMV, the aetiological agent of fig mosaic disease.
Glycoprotein L sets the neutralization profile of murid herpesvirus 4
Authors: Gillet Laurent , Alenquer Marta , Glauser Daniel L. , Colaco Susanna , May Janet S. , Stevenson Philip G.
Published: April 2009

Antibodies readily neutralize acute, epidemic viruses, but are less effective against more indolent pathogens such as herpesviruses. Murid herpesvirus 4 (MuHV-4) provides an accessible model for tracking the fate of antibody-exposed gammaherpesvirus virions. Glycoprotein L (gL) plays a central role in MuHV-4 entry: it allows gH to bind heparan sulfate and regulates fusion-associated conformation changes in gH and gB. However, gL is non-essential: heparan sulfate binding can also occur via gp70, and the gB–gH complex alone seems to be sufficient for membrane fusion. Here, we investigated how gL affects the susceptibility of MuHV-4 to neutralization. Immune sera neutralized gL− virions more readily than gL+ virions, chiefly because heparan sulfate binding now depended on gp70 and was therefore easier to block. However, there were also post-binding effects. First, the downstream, gL-independent conformation of gH became a neutralization target; gL normally prevents this by holding gH in an antigenically distinct heterodimer until after endocytosis. Second, gL− virions were more vulnerable to gB-directed neutralization. This covered multiple epitopes and thus seemed to reflect a general opening up of the gH–gB entry complex, which gL again normally restricts to late endosomes. gL therefore limits MuHV-4 neutralization by providing redundancy in cell binding and by keeping key elements of the virion fusion machinery hidden until after endocytosis.
Infectious bursal disease virus persistently infects bursal B-lymphoid DT40 cells
Authors: Delgui Laura , González Dolores , Rodríguez José F.
Published: April 2009

Infectious bursal disease virus (IBDV), an important avian pathogen, exhibits a specific tropism for immature B-lymphocyte populations. We have investigated the ability of IBDV to replicate in chicken B-lymphoid DT40 cells, a tumour cell line derived from the bursa of Fabricius of a chicken infected with avian leukosis virus. Our results show that IBDV persistently infects DT40 cells. Establishment of the persistent infection is associated with an extensive remodelling of the hypervariable region of the VP2 capsid polypeptide, accumulating 14 amino acid changes during the first 60 days of the persistent infection. The amino acid sequence of the non-structural VP5 polypeptide, involved in virus dissemination, is not altered during the persistent infection. Results described in this report constitute the first demonstration of the ability of IBDV to establish a persistent infection in vitro.
Novel anellovirus discovered from a mortality event of captive California sea lions
Authors: Ng Terry Fei Fan , Suedmeyer Wm Kirk , Wheeler Elizabeth , Gulland Frances , Breitbart Mya
Published: April 2009

A viral metagenomic study was performed to investigate potential viral pathogens associated with a mortality event of three captive California sea lions (Zalophus californianus). This study identified a novel California sea lion anellovirus (ZcAV), with 35 % amino acid identity in the ORF1 region to feline anelloviruses. The double-stranded replicative form of ZcAV was detected in lung tissue, suggesting that ZcAV replicates in sea lion lungs. Specific PCR revealed the presence of ZcAV in the lung tissue of all three sea lions involved in the mortality event, but not in three other sea lions from the same zoo. In addition, ZcAV was detected at low frequency (11 %) in the lungs of wild sea lions. The higher prevalence of ZcAV and presence of the double-stranded replicative form in the lungs of sea lions from the mortality event suggest that ZcAV was associated with the death of these animals.
Expression and processing of human immunodeficiency virus type 1 gp160 using the vesicular stomatitis virus New Jersey serotype vector system
Authors: Wu Kunyu , Kim Gyoung Nyoun , Kang C. Yong
Published: April 2009

The Indiana serotype of vesicular stomatitis virus (VSVIND), but not the New Jersey serotype (VSVNJ), has been widely used as a gene expression vector. In terms of prime–boost-based vaccine strategies, it would be desirable to use two different VSV serotypes to avoid immunity against the priming viral vector. Here, we report that we have applied the VSVNJvector system for expression of theenvgene of human immunodeficiency virus type 1 (HIV-1). The HIV-1envgene was inserted into the VSVNJvector system at two different sites: between the P and M genes (NP-gp160-MGL) and between the G and L genes (NPMG-gp160-L). The HIV-1envgene product, gp160, was efficiently expressed and processed in cells infected with either of these two recombinant VSV–HIV-1gp160viruses. In this study, we have investigated the applicability of the VSVNJvector system for foreign gene expression.
New inhibitors of prion replication that target the amyloid precursor
Authors: Charvériat Mathieu , Reboul Marlène , Wang Qian , Picoli Christèle , Lenuzza Natacha , Montagnac Alain , Nhiri Naima , Jacquet Eric , Guéritte Françoise , Lallemand Jean-Yves , Deslys Jean-Philippe , Mouthon Franck
Published: April 2009

At present, there is no effective therapy for any of the neurodegenerative amyloidoses, despite renewed efforts to identify compounds active against the various implicated pathogenetic molecules. We have screened a library of 2960 natural and synthetic compounds in two cell lines chronically infected with mouse prions, and have identified eight new inhibitors of prion replication in vitro. They belong to two distinct chemical families that have not previously been recognised as effective in the field of transmissible spongiform encephalopathies: seven are 3-aminosteroids and one is a derivative of erythromycin A with an oxime functionality. Our results suggest that these aminosteroids inhibit prion replication by triggering a common target, possibly implicated in the regulatory pathways of cellular prion protein metabolism. Furthermore, using a quantitative approach for the study of protein stability, it was shown that the erythromycin A derivative altered prion protein stability by direct interaction. Such direct targeting of this amyloid precursor might provide new clues for the understanding of prion diseases and, more importantly, help to define new molecules that are active against prion diseases.
A truncated T antigen expressed from an alternatively spliced BK virus early mRNA
Authors: Abend Johanna R. , Joseph Amy E. , Das Dweepanita , Campbell-Cecen Deniz B. , Imperiale Michael J.
Published: April 2009

The early region of BK virus (BKV) is known to encode two well-characterized tumour (T) antigens, large T antigen (TAg) and small T antigen (tAg). In this study, we provide evidence of a third early BKV mRNA that codes for an additional early region product with an apparent molecular mass of 17–20 kDa. This truncated form of TAg (truncTAg) is expressed from an alternatively spliced mRNA that is derived from the excision of a second intron from the mRNA encoding TAg. The first 133 aa of truncTAg are identical to those of TAg but the additional splice results in translation from a different reading frame, adding three new amino acids before reaching a stop codon. TruncTAg is expressed in both BKV-transformed and lytically infected cells and it is found to be primarily localized to the nucleus. The function of BKV truncTAg is likely to be relevant to transformation, similar to the additional T antigens of simian virus 40, JC virus and mouse polyomavirus.
Investigation of a role for lysine residues in non-structural proteins 2 and 2/3 of the hepatitis C virus for their degradation and virus assembly
Authors: Welbourn Sarah , Jirasko Vlastimil , Breton Valérie , Reiss Simon , Penin Francois , Bartenschlager Ralf , Pause Arnim
Published: April 2009

It has been demonstrated that both uncleaved, enzymitically inactive NS2/3 and cleaved NS2 proteins are rapidly degraded upon expression in cells, phenomena described to be blocked by the addition of proteasome inhibitors. As this degradation and its regulation potentially constitute an important strategy of the hepatitis C virus (HCV) to regulate the levels of its non-structural proteins, we further investigated the turnover of these proteins in relevant RNA replication systems. A lysine-mutagenesis approach was used in an effort to prevent protein degradation and determine any effect on various steps of the viral replication cycle. We show that, while NS2-lysine mutagenesis of protease-inactive NS2/3 results in a partial stabilization of this protein, the increased NS2/3 levels do not rescue the inability of NS2/3 protease inactive replicons to replicate, suggesting that uncleaved NS2/3 is unable to functionally replace NS3 in RNA replication. Furthermore, we show that the cleaved NS2 protein is rapidly degraded in several transient and stable RNA replicon systems and that NS2 from several different genotypes also has a short half-life, highlighting the potential importance of the regulation of NS2 levels for the viral life cycle. However, in contrast to uncleaved NS2/3, neither ubiquitin nor proteasomal degradation appear to be significantly involved in NS2 degradation. Finally, although NS2 lysine-to-arginine mutagenesis does not affect this protein's levels in a JFH-1 cell culture infection system, several of these residues are identified to be involved in virion assembly, further substantiating the importance of regions of this protein for production of infectious virus.
Differential Cannabinoid Receptor Expression during Reactive Gliosis: a Possible Implication for a Nonpsychotropic Neuroprotection
Authors: De Filippis Daniele , Steardo Antonio , D'Amico Alessandra , Scuderi Caterina , Cipriano Mariateresa , Esposito Giuseppe , Iuvone Teresa
Published: April 2009

Activated microglia and astrocytes produce a large number of inflammatory and neurotoxic substances in various brain pathologies, above all during neurodegenerative disorders. In the search for new neuroprotective compounds, interest has turned to marijuana derivatives, since in severalin vitro,in vivo, and clinical studies, they have shown a great ability to control neuroinflammation.

Despite the emerging evidence regarding pharmacological activities of cannabinoids, their effective introduction into clinical therapy still remains controversial and strongly limited by their unavoidable psychotropicity. Since the psychotropic effect of cannabinoids is generally linked to the activation of the CB1 receptor on neurons, the aim of our review is to clarify the function of the two cannabinoid receptors on glial cells and the differential role played by them, highlighting the emerging evidence of a CB2-mediated control of neuroinflammation that could liberate cannabinoids from the slavery of their central side effects.
Reviewing the current classification of inhibitor of growth family proteins
Authors: Unoki Motoko , Kumamoto Kensuke , Takenoshita Seiichi , Harris Curtis C.
Published: April 2009

Inhibitor of growth (ING) family proteins have been defined as candidate tumor suppressors for more than a decade. Recent emerging results using siRNA and knockout mice are expanding the previous understanding of this protein family. The results of ING1 knockout mouse experiments revealed that ING1 has a protective effect on apoptosis. Our recent results showed that ING2 is overexpressed in colorectal cancer, and induces colon cancer cell invasion through an MMP13‐dependent pathway. Knockdown of ING2 by siRNA induces premature senescence in normal human fibroblast cells, and apoptosis or cell cycle arrest in various adherent cancer cells. Taken together, these results suggest that ING2 may also have roles in cancer progression and/or malignant transformation under some conditions. Additionally, knockdown of ING4 and ING5 by siRNA shows an inhibitory effect on the transition from G2/M to G1 phase and DNA replication, respectively, suggesting that these proteins may play roles during cell proliferation in some context. ING family proteins may play dual roles, similar to transforming growth factor‐β, which has tumor suppressor‐like functions in normal epithelium and also oncogenic functions in invasive metastatic cancers. In the present article, we briefly review ING history and propose a possible interpretation of discrepancies between past and recent data. (Cancer Sci 2009; 100: 1173–1179)
GATA2‐dependent and region‐specific regulation of Gata2 transcription in the mouse midbrain
Authors: Nozawa Daisuke , Suzuki Norio , Kobayashi‐Osaki Maki , Pan Xiaoqing , Engel James Douglas , Yamamoto Masayuki
Published: April 2009

Transcription factor GATA2 is expressed in numerous mammalian tissues, including neural, hematopoietic, cardiovascular and urogenital systems, and yet it plays important roles in the regulation of tissue‐restricted gene expression. The Gata2 gene itself is also under stringent tissue‐specific control and multiple cis‐regulatory domains have been identified in the Gata2 locus. In this study we sought out and then examined in detail the domains that regulate Gata2 in the midbrain. We identified two discrete domains in the Gata2 promoter that direct midbrain expression; these distal 5H and proximal 2H regulatory domains are located 3.0 and 1.9 kbp, respectively, upstream of the transcriptional initiation site. Importantly, both domains contain GATA factor binding sites. Our analyses further revealed that GATA2 is essential for Gata2 gene expression in the midbrain, whereas GATA3 is not. Both the 2H and 5H domains have the independent ability to activate Gata2 gene expression in the midbrain superior colliculus, whereas the distal‐5H domain is additionally capable of activating Gata2 transcription in the inferior colliculus. These results demonstrate that two distinct regulatory domains contribute to the Gata2 gene expression in the mouse midbrain and that Gata2 midbrain transcription is under positive autoregulation.
Expression cloning of Xenopus zygote arrest 2 (Xzar2) as a novel epidermalization‐promoting factor in early embryos of Xenopus laevis
Authors: Nakajima Yuka , Okamoto Harumasa , Kubo Tai
Published: April 2009

In vertebrates, BMPs are known to induce epidermal fate at the expense of neural fate. To further explore the molecular mechanisms of epidermal differentiation, we have developed an expression cloning system for isolating cDNAs that encode intrinsic proteins with epidermal‐inducing activity. Under our conditions, 92.5% of the dissociated animal cap cells treated with the conditioned medium from H2O‐injected control oocytes differentiated into neural tissue, which developed neural fibers and expressed a neural marker (NCAM). In contrast, when dissociated animal cap cells were treated with the supernatant collected from the culture of BMP‐4 mRNA‐injected oocytes, the microcultures differentiated into epidermal tissue, which developed cilium. The cells expressed an epidermal marker (keratin), but not NCAM. Using the dissociated animal cap cells in a functional screening system, we cloned a cDNA encoding a novel polypeptide, Xenopus zygote arrest 2 (Xzar2). Over‐expression of Xzar2 caused anterior defects and suppressed expressions of the neural markers. The epidermalization‐promoting activity of Xzar2 was substantially not affected by over‐expression of the BMP signaling antagonists Smad6 and 7, and a dominant negative receptor for BMP (tBR). Our results suggest that Xzar2 is involved in epidermal fate determination mainly through signaling pathways distinct from that of BMP‐Smad during early embryogenesis.
Atg17 recruits Atg9 to organize the pre‐autophagosomal structure
Authors: Sekito Takayuki , Kawamata Tomoko , Ichikawa Rie , Suzuki Kuninori , Ohsumi Yoshinori
Published: April 2009

Autophagy is a degradation system of cytoplasmic proteins and organelles via formation of double‐membrane vesicles called autophagosomes. In the yeast Saccharomyces cerevisiae, autophagosomes are formed via the pre‐autophagosomal structure (PAS) in a manner dependent on Atg proteins. Under nutrient‐rich condition, Atg9 is recruited to the PAS by binding to Atg11 for the Cvt pathway. However, because Atg9 is recruited to the PAS in atg11Δ cells in starved condition and autophagy is induced, autophagy‐specific mechanism for the Atg9 recruitment to the PAS has been assumed. Here, we demonstrate that, in autophagy‐inducing condition, Atg9 is recruited to the PAS in a manner dependent on Atg17. Atg9 physically interacts with Atg17 in the presence of rapamycin. This interaction requires Atg1, a protein kinase essential for autophagy. Consistently, the Atg17‐dependent PAS localization of Atg9 requires Atg1. However, its kinase activity is dispensable for this process. It rather regulates the equilibrium of assembly and disassembly of Atg9 at the PAS.
A sensitive loop‐mediated isothermal amplification (LAMP) method for detection of Renibacterium salmoninarum, causative agent of bacterial kidney disease in salmonids
Authors: Gahlawat S K , Ellis A E , Collet B
Published: April 2009

Loop‐mediated isothermal amplification (LAMP) is a novel technique for nucleic acid amplification with high specificity, sensitivity and rapidity and does not require expensive equipment or reagents. In the present study, we developed and evaluated a LAMP method for the rapid detection of Renibacterium salmoninarum causing the bacterial kidney disease in salmonids. This method was more sensitive than quantitative real‐time polymerase chain reaction (qPCR). Using DNA template extracted from cultured R. salmoninarum, the LAMP method gave an amplification signal from template diluted to 10−8 while the limit of detection of qPCR was10−7. The LAMP method was also highly specific and did not amplify DNA purified from five other Gram‐positive and ‐negative bacterial fish pathogens. The method also worked well using extracts of macrophages infected with R. salmoninarum and kidney material from rainbow trout, which were positive for R. salmoninarum by qPCR and crude R. salmoninarum culture. There was some evidence for inhibitors of the LAMP reaction in the kidney samples, which was overcome by diluting the sample.
Two pathogens of Greenshell™ mussel larvae, Perna canaliculus: Vibrio splendidus and a V. coralliilyticus/neptunius‐like isolate
Authors: Kesarcodi‐Watson A , Kaspar H , Lategan M J , Gibson L
Published: April 2009

Bacterial pathogens of Greenshell™ mussel (GSM) larvae can cause batch losses during hatchery production. Twenty‐two isolates were screened using a larval bioassay. Two strains were identified as potential pathogens. Phenotypic identification of these strains revealed two non‐reactive Gram‐negative, oxidase positive rods. Sequencing of the 16S rRNA gene identified Vibrio splendidus and a V. coralliilyticus/neptunius‐like isolate as pathogens of GSM larvae, with an ability to cause 83% and 75% larval mortality in vitro, respectively, at a concentration of 102 CFU mL−1. Histopathology indicated that the route of infection was via the digestive system. Using healthy larvae as target hosts, Koch’s postulates were confirmed for the two isolates. This is the first report on pathogens of GSM larvae.
Differential Effects of Restricted Versus Unlimited High‐Fat Feeding in Rats on Fat Mass, Plasma Hormones and Brain Appetite Regulators
Authors: Shiraev T. , Chen H. , Morris M. J.
Published: April 2009

The rapid rise in obesity has been linked to altered food consumption patterns. There is increasing evidence that, in addition to total energy intake, the macronutrient composition of the diet may influence the development of obesity. The present study aimed to examine the impact of high dietary fat content, under both isocaloric and hypercaloric conditions, compared with a low fat diet, on adiposity, glucose and lipid metabolism, and brain appetite regulators in rats. Male Sprague–Dawley rats were exposed to one of three diets: control (14% fat), ad lib high‐fat palatable (HFD, 35% fat) or high‐fat palatable restricted (HFD‐R, matched to the energy intake of control) and were killed in the fasting state 11 weeks later. Body weight was increased by 28% in unrestricted HFD fed rats, with an almost tripling of caloric intake and fat mass (P < 0.001) and double the plasma triglycerides of controls. Glucose intolerance and increased insulin levels were observed. HFD‐R animals calorie matched to control had double their fat mass, plasma insulin and triglycerides (P < 0.05). Only ad lib consumption of the HFD increased the hypothalamic mRNA expression of the appetite‐regulating peptides, neuropeptide Y and pro‐opiomelanocortin. Although restricted consumption of palatable HFD had no significant impact on hypothalamic appetite regulators or body weight, it increased adiposity and circulating triglycerides, suggesting that the proportion of dietary fat, independent of caloric intake, affects fat deposition and the metabolic profile.
Divergent Regulation of Hypothalamic Neuropeptide Y and Agouti‐Related Protein by Photoperiod in F344 rats With Differential Food Intake and Growth
Authors: Ross A. W. , Johnson C. E. , Bell L. M. , Reilly L. , Duncan J. S. , Barrett P. , Heideman P. D. , Morgan P. J.
Published: April 2009

Hypothalamic genes involved in food intake and growth regulation were studied in F344 rats in response to photoperiod. Two sub‐strains were identified: F344/NHsd (F344/N) and F344/NCrHsd (F344/NCr); sensitive and relatively insensitive to photoperiod respectively. In F344/N rats, marked, but opposite, changes in the genes for neuropeptide Y (NPY) (+97.5%) and agouti‐related protein (AgRP) (−39.3%) expression in the arcuate nucleus were observed in response to short (8 : 16 h light/dark cycle, SD) relative to long (16 : 8 h light/dark cycle, LD) day photoperiods. Changes were associated with both reduced food intake and growth. Expression of the genes for cocaine and amphetamine‐regulated transcript (CART) and pro‐opiomelanocortin (POMC) in the arcuate nucleus was unchanged by photoperiod. POMC in the ependymal layer around the third ventricle was markedly inhibited by SD. Parallel decreases in the genes for growth hormone‐releasing hormone (GHRH) and somatostatin (Somatostatin) mRNA in the arcuate nucleus and Somatostatin in the periventricular nucleus were observed in SD. Serum levels of insulin‐like growth factor (IGF)‐1 and insulin were lower in F344/N rats in SD, whereas neither leptin nor corticosterone levels were affected. By contrast, F344/NCr rats that show only minor food intake and growth rate changes showed minimal responses in these genes and hormones. Thus, NPY/AgRP neurones may be pivotal to the photoperiodic regulation of food intake and growth. Potentially, the SD increase in NPY expression may inhibit growth by decreasing GHRH and Somatostatin expression, whereas the decrease in AgRP expression probably leads to reduced food intake. The present study reveals an atypical and divergent regulation of NPY and AgRP, which may relate to their separate roles with respect to growth and food intake, respectively.
Recruitment of CD8+ T cells expressing granzyme A is associated with lesion progression in human cutaneous leishmaniasis
Authors: FARIA D. R. , SOUZA P. E. A. , DURÃES F. V. , CARVALHO E. M. , GOLLOB K. J. , MACHADO P. R. , DUTRA W. O.
Published: April 2009

Human infection with Leishmania braziliensis leads to the establishment of cutaneous leishmaniasis (CL), characterized by the appearance of skin lesions that progress from nonulcerated to ulcerated forms. Our goal was to characterize the immunological kinetics associated with this progression, comparing the cellular composition, cytokines and granzyme expression between lesions of patients with early (E‐CL) and late stages (L‐CL) of CL. Histopathological analysis showed that lesions from L‐CL had more exuberant inflammatory infiltrate as compared to E‐CL. Although E‐CL and L‐CL lesions were predominantly mononuclear, lesions from E‐CL patients presented higher neutrophil and eosinophil counts than L‐CL. While percentages of CD4+ and of CD68+ cells were slightly higher in L‐CL, a fivefold increase of CD8+ cells was observed in L‐CL, as compared to E‐CL. Moreover, CD8+ T‐cells from L‐CL expressed significantly higher levels of granzyme A than E‐CL. Interestingly, granzyme A expression was positively correlated with intensity of the inflammatory infiltrate in L‐CL but not E‐CL. Lastly, percentages of IFN‐γ+ and IL‐10+ cells were higher in L‐CL as compared to E‐CL, with CD4+ T‐cells and CD68+ monocytes as the main sources of these cytokines, respectively. These results suggest that recruitment of CD8+ granzyme A+ T cells is involved in lesion progression in human CL.
Relative imbalance between T regulatory cells and activated T cells in mice with differential morbidity in chronic Schistosoma mansoni infections
Authors: WATANABE K. , CARTER J. M. , NEELY‐BURNAM M. , COLLEY D. G.
Published: April 2009

Chronic (20‐week) Schistosoma mansoni infections of CBA/J male mice present as two distinct forms of morbidity. Most mice develop moderate splenomegaly syndrome (MSS) resembling the intestinal form of chronic human schistosomiasis mansoni, while approximately 20% of mice develop hypersplenomegaly syndrome (HSS), more consistent with the severe hepatosplenic form of chronic human schistosomiasis mansoni. Here, we report the relative proportions of natural T regulatory cells (Treg) and activated CD4+ T cells (Tact) for both splenic and granulomatous cell populations of MSS and HSS mice. Proportions of both Treg and Tact are greater in HSS than MSS mice. However, the ratios of Treg to Tact in both splenic and granulomatous cell populations from MSS mice are significantly higher than those of HSS mice. For both HSS and MSS mice, in vitro proliferation of their CD3+ splenic cells induced by soluble egg antigens is inversely correlated with the ratio of Treg to Tact. Also, spleen or granuloma cells from MSS mice produced higher mean levels of IFN‐γ than those from HSS mice. Differential IFN‐γ productive capacities dictated by Treg : Tact ratios may contribute to the development of differential morbidities in this model of chronic experimental schistosomiasis mansoni.
Variation in the gene encoding acetolactate‐synthase in Lolium species and proactive detection of mutant, herbicide‐resistant alleles
Authors: DÉLYE C , BOUCANSAUD K , PERNIN F , LE CORRE V
Published: April 2009

Lolium species (ryegrasses) are genetically highly variable plants that are both forage crops and major weeds across the globe. As weeds, they rapidly evolve resistance under the selective pressure of acetolactate‐synthase (ALS) inhibitors, the most resistance‐prone herbicide group. Quick and accurate diagnosis is therefore of importance to prevent resistance spread in ryegrass. To develop proactive molecular tools for the detection of mutant, resistant ALS alleles, we assessed variation in the ryegrass ALS gene. Sequencing the full 1929‐bp ALS coding sequence in 59 plants from six distant locations revealed a total of 208 polymorphic nucleotide positions (one every 9.3 nucleotides). The heterogeneous distribution of synonymous and non‐synonymous substitutions along the ALS coding sequence suggested that nucleotide variation of ALS is shaped by purifying and background selection. Using regions of the ALS coding sequence with a low number of polymorphic nucleotide sites, five derived cleaved amplified polymorphic sequence (dCAPS) assays were developed targeting codons crucial for herbicide sensitivity. These enabled the first detection in ryegrass of a Pro‐197‐Thr substitution that confers herbicide resistance. Most assays could also be used to genotype Festuca and Vulpia plants. These dCAPS assays should prove powerful tools for both resistance diagnosis and population genetics studies.
Factors affecting the efficacy of Orobanche cumana chemical control in sunflower
Authors: EIZENBERG H , HERSHENHORN J , EPHRATH J E
Published: April 2009

Broomrapes (Orobanche spp.) are root parasites, causing severe damage to vegetable and field crops. Their early in situ detection for efficient control is crucial but difficult, because of their sub‐surface development. Minirhizotron technology enables the in situ observation of host–parasite interactions. The initial underground development of Orobanche cumana on sunflower roots and its control with imazapic were investigated. Imazapic (2.4 g active ingredient ha−1) was applied post‐emergence on sunflower at the eight‐true‐leaf stage, when O. cumana attachments were first observed. No viable or new attachments were detected on roots of the treated sunflower after herbicide application. However, within the same time frame, the already existing O. cumana attachments in the non‐treated controls continued to develop and new attachments appeared. Orobanche cumana shoot emergence was correlated with growing day‐degrees (GDD, d °C). This correlation was quantified by a four‐parameter logistic equation. Control efficacy was affected by depth and number of parasite attachments per minirhizotron camera frame: it was highest in the topmost layer, and decreased with depth and number of attachments. The results obtained in this study emphasise the need to develop a decision support system based on GDD that should be validated under long‐term field conditions.
A novel duplex SSP–PCR typing method for KIR gene profiling
Authors: Ashouri E. , Ghaderi A. , Reed E. F. , Rajalingam R.
Published: April 2009

Killer‐cell immunoglobulin‐like receptors (KIR) control the function of natural killer cells. The number and type of KIR genes are substantially variable among individuals. Sequence‐specific primer–directed polymerase chain reaction (SSP‐PCR) based genotyping is the most commonly used method to assess the KIR gene content. However, it requires a minimum of 16 gene‐specific amplifications and often yields false‐negative results. Herein, we describe the development of a simple and efficient duplex SSP‐PCR assay to identify the presence and absence of 16 KIR genes. This system further distinguishes subsets of KIR2DS4 and KIR3DP1 alleles. The assay was subjected to a blind validation using a panel of 78 reference DNA standards from the UCLA KIR Exchange Program, which showed 100% specificity and accuracy. Compared with the conventional SSP typing methods, the present method is an accurate, simple, cost‐effective and labor‐saving KIR genotyping method for high volume testing.
Selection for verticillium wilt resistance in potato breeding populations derived from four cross types
Authors: Bae J. , Tai G. , Jansky S. H.
Published: April 2009

Verticillium wilt (VW) is one of the important yield‐limiting diseases for potato production. To develop resistant clones, the potential for early generation selection was studied using three basic selection methods, individual, family, and within family selection, for two clonal generations. A total of 152 clones were derived from four cross types (2x × 2x, 2x × 4x, 4x × 2x and 4x × 4x). Clones were evaluated for maturity, symptom expression, yield and stem colonization in replicated trials. Heritability and selection response for the traits were estimated for each selection method. Direct selection in the second clonal generation and individual selection showed more gain than that from other methods. Both 2x × 2x and 4x × 2x families were higher yielding and had lower stem colonization scores than 2x × 4x and 4x × 4x crosses. Therefore, 2x × 2x or 4x × 2x crosses between carefully chosen parents with high yield and VW resistance may produce offspring with superior performance.
S‐RNase genotyping and incompatibility group assignment by PCR and pollination experiments in Japanese plum
Authors: Guerra M. E. , Rodrigo J. , López‐Corrales M. , Wünsch A.
Published: April 2009

Most Japanese plum‐type cultivars are self‐incompatible and cross pollination is necessary to ensure fruit set. In this study, the S‐RNase genotype and the incompatibility group of 68 Japanese plum‐type cultivars were determined by PCR amplification of the S‐RNase gene. The S‐RNase genotype of 50 cultivars is first reported here and five new Japanese plum S‐RNase alleles (So, Sp, Sq, Sr, Ss) were identified. The results obtained, together with information compiled from previous studies, allowed describing 12 new incompatibility groups (VIII–XIX). The self‐incompatibility of several cultivars and the cross‐compatibility among different incompatibility groups were verified by self‐ and cross‐pollination experiments followed by observation of pollen tube growth. Five cultivars behaved as self‐compatible, but two of them do not have the Se allele, which has been correlated with self‐compatibility. Thus, additional sources of self‐compatibility different from Se appear to be involved in Japanese plum self‐compatibility.
Development and mapping of gene‐tagged SNP markers in laccases of maize (Zea mays L.)
Authors: Andersen J. R. , Asp T. , Lu Y. C. , Kloiber‐Maitz M. , Ouzonova M. , Luebberstedt T.
Published: April 2009

Laccases, EC 1.10.3.2 or p‐diphenol : dioxygen oxidoreductases, have been proposed to be involved in the oxidative polymerization of monolignols into lignins in plants. While 17 laccases have been identified in Arabidopsis, only five (ZmLac1–5) have so far been identified in maize. By a bioinformatic approach, 14 putative laccases were identified in maize. One putative laccase was identical to ZmLac1, while five were highly homologous to either ZmLac4 or ZmLac5. Sequence alignment of allelic sequences enabled the development of TaqMan single nucleotide polymorphism (SNP) markers for nine putative laccases. Four of these gene‐tagged SNP markers were validated in a doubled‐haploid mapping population of 140 individuals, mapping these loci to chromosomes 1, 2, 3 and 7, respectively.
Evaluation of resistance to melon yellow spot virus in a cucumber germplasm collection
Authors: Sugiyama M. , Okuda M. , Sakata Y.
Published: April 2009

Melon yellow spot virus (MYSV), a member of the genus Tospovirus, is a serious thrips‐transmitted virus of cucurbits in Japan. Resistant cultivars provide an effective means for reducing the impact of the disease; however, no MYSV‐resistant cucumber has been reported. Susceptibilities of 398 cucumber accessions originating from 26 countries were evaluated by mechanical inoculation of MYSV. Thirteen accessions from South Asia, South East Asia and unknown origin had low disease severity indices (DSIs), and 10 of them showed no necrotic lesions on inoculated leaves. No or little positive reaction was detected by DAS‐ELISA analysis of inoculated leaves of these accessions. 27028930 showed resistance to a melon isolate (MYSV‐S) of MYSV, and ‘Yamakyuri‐1’ showed moderate resistance to a cucumber isolate (FuCu05P).
Endo‐sponge assisted treatment of anastomotic leakage following colorectal surgery
Authors: Riss S. , Stift A. , Meier M. , Haiden E. , Grünberger T. , Bergmann M.
Published: April 2009

Aim  Endo‐sponge assisted treatment (endo‐sponge) represents a novel approach to treat patients with anastomotic dehiscence following anterior resection for rectal cancer. Yet, limited data are available to predict success, compatibility with radiotherapy and/or chemotherapy as well as acceptance by the patients.

Method  Between September 2007 and June 2008, nine patients suffering from anastomotic leakage after anterior rectal resection (n = 6) or suffering from leakage of rectal stump following Hartmann’s procedure (n = 3) were treated by endo‐sponge. We recorded clinical outcome and patient’s comfort using a 10‐point visual analogue scale (VAS).

Results  Median time of endo‐sponge treatment was 3 weeks (range: 2–8). There were no minor or major complications. In 6 (66.6%) patients, the anastomotic leakage healed successfully. Three patients showed no response and needed further surgical intervention. The lack of success was due to complexity of the leakages, which comprised either more than 270° of the circumference or consisted of two distant fistulas. Formation of granulation tissue was unaffected by chemotherapy. For the question ‘alteration in daily life activity’, a median score of 5 (range: 1–9) was found. Measuring ‘pain sensation’ during endo‐sponge treatment patients scored a median of 3 (range: 0–6).

Conclusions  Endo‐sponge treatment can be recommended as an alternative approach to treat pelvic sepsis following anastomotic dehiscence or rectal stump insufficiency. Extended leakages should be treated by different approaches having little probability of successful healing, but can lead to discomfort for the patient. Radiochemotherapy does not cause a problem for application of the endo‐sponge.
Open vs laparoscopic repair of full‐thickness rectal prolapse: a re‐meta‐analysis
Authors: Sajid M. S. , Siddiqui M. R. S. , Baig M. K.
Published: April 2009

Objective  A re‐meta‐analysis of available data within the published literature comparing laparoscopic rectopexy (LR) with open repair (OR).

Method  We searched MEDLINE, EMBASE, CINAHL, PubMed and the Cochrane databases from January 1990 to October 2008. We searched the following MESH terms: ‘laparoscopy’, ‘prolapse’ and ‘rectal prolapse’. We used the following text words: ‘rectopexy’, ‘haemorrhoids’, ‘minimally invasive’ and ‘keyhole surgery’. The bibliography of selected trials and a Cochrane review was scrutinized and relevant references obtained. Selected trials were analysed to conduct a meta‐analysis.

Results  Twelve comparative studies on 688 patients qualified for the review. There were 330 patients in LR group and 358 in the OR group. LR takes longer to perform compared with OR. This difference was statistically significant [random effects model: standardized mean difference (SMD) 1.63, 95% CI (1.14–2.12), z = 6.56, P < 0.001]. There was a significant reduction in hospital stay between LR vs OR [random effects model: SMD −1.75, 95% CI (−2.45 to −1.05), z = −4.90, P < 0.001]. There was no statistical difference relating to morbidity, constipation, incontinence or mortality between the two groups.

Conclusion  laparoscopic rectopexy is a safe and effective modality and is comparable to OR, however, there is still a paucity of randomized controlled trials within the literature regarding this subject. Until these trials are conducted, we would advise caution in deriving absolute conclusions.
Seasonal variation in short‐term mortality after surgery for colorectal cancer?
Authors: Iversen L. H. , Nielsen H. , Pedersen L. , Harling H. , Laurberg S.
Published: April 2009

Objective  Comorbidity has a major impact on short‐term and long‐term survival of colorectal cancer (CRC) and many CRC patients suffer from comorbidities. Mortality rates for comorbidities like cardio‐respiratory diseases exhibit distinct seasonal variations with highest rates in the winter. Therefore, we hypothesized some seasonal variation in 30‐day mortality after surgery for CRC as well.

Method  In a nationwide study, we examined the seasonal pattern in 30‐day mortality after surgery for CRC from 1996 to 2006. We identified 33 556 CRC patients in the Danish hospital discharge registries. Monthly 30‐day mortality rates were calculated and we constructed a fitted curve of the monthly mortality rates using a periodic regression model. We stratified the analyses for tumour site, urgency of surgery for colon cancer and the level of comorbidity based on American Society of Anaesthesiologists (ASA) score.

Results  The overall 30‐day mortality was 8.7% [95% confidence interval 8.4–9.0%). Significant seasonal variation in monthly 30‐day mortality could not be identified. For colon cancer, a nonsignificant increase was seen in July. An even higher increase in July was observed for CRC patients with moderate or severe comorbidity (ASA score ≥ III), but was also nonsignificant.

Conclusion  Although comorbidity is a well‐known negative predictor of short‐term survival of CRC, monthly 30‐day mortality after surgery for CRC did not exhibit seasonal variation like that observed for comorbid conditions such as cardio‐respiratory diseases.