bluuebunny/crossref_metadata_2025 huggingface dataset:
Dynamics of hepatitis C virus NS5A quasispecies during interferon and ribavirin therapy in responder and non-responder patients with genotype 1b chronic hepatitis C
Authors: Puig-Basagoiti Francesc , Forns Xavier , Furčić Ivana , Ampurdanés Sergi , Giménez-Barcons Mireia , Franco Sandra , Sánchez-Tapias José M. , Saiz Juan-Carlos
Published: March 2005

The quasispecies nature of hepatitis C virus (HCV) may have important implications concerning resistance to antiviral agents. To determine whether HCV NS5A quasispecies composition and dynamics are related to responsiveness to combined interferon (IFN) and ribavirin therapy, extensive sequence analyses of cloned RT-PCR amplification products of HCV-1b NS5A quasispecies of sequential isolates from 15 treated (nine sustained responders and six non-responders) and three untreated patients were performed. Accumulation of mutations in NS5A during therapy was relatively frequent in the V3 domain, but unusual elsewhere. Amino acid changes were the result of the imposition of minor variants that were already present before treatment and always occurred within the first week of therapy. Before treatment, the complexity and diversity of quasispecies were lower in isolates from responders than in those from non-responders, particularly in the V3 domain, where differences in nucleotide entropy (0·35 vs 0·64,P=0·003), genetic distance (0·0145 vs 0·0302,P=0·05) and non-synonymous substitutions (0·0102 vs 0·0203,P=0·036) were statistically significant. These differences became more apparent during treatment, because complexity and diversity remained stable or tended to increase in non-responders, whereas they tended to decrease in responders. These observations suggest that the composition and dynamics of HCV NS5A quasispecies, particularly in the V3 domain, may play a role in the response to combined IFN/ribavirin therapy.
Dengue virus-induced apoptosis in hepatic cells is partly mediated by Apo2 ligand/tumour necrosis factor-related apoptosis-inducing ligand
Authors: Matsuda Takehiro , Almasan Alex , Tomita Mariko , Tamaki Kazumi , Saito Mika , Tadano Masayuki , Yagita Hideo , Ohta Takao , Mori Naoki
Published: March 2005

Although hepatic injury is reported in cases with dengue haemorrhagic fever and dengue shock syndrome, its mechanism remains poorly understood. Several findings suggest that dengue virus (DEN) induces apoptosis of hepatocytesin vivo. In this work, DEN type 2 (DEN-2) strain NGC was shown to induce apoptosis in the hepatic cell line HepG2, and infection of HepG2 cells was found to induce Apo2 ligand (Apo2L, also known as tumour necrosis factor-related apoptosis-inducing ligand or TRAIL) expression. Furthermore, Apo2L/TRAIL induced apoptosis in HepG2 cells, which expressed the Apo2L/TRAIL receptor DR5/TRAIL-R2 on their surface. Analysis of the Apo2L/TRAIL promoter revealed that this gene was activated by DEN-2 infection, whose responsive element was overlapping NF-κB- and Sp1-binding sites located at nt −75 to −65. The proteasome inhibitorN-acetyl-l-leucinyl-l-leucinyl-l-norleucinal (LLnL) inhibited Apo2L/TRAIL mRNA expression, and LLnL and anti-Apo2L/TRAIL antibody inhibited DEN-2-induced apoptosis. It was proposed that DEN infection promotes apoptosis partly through the induction of Apo2L/TRAIL expression.
Minute virus of mice small non-structural protein NS2 localizes within, but is not required for the formation of, Smn-associated autonomous parvovirus-associated replication bodies
Authors: Young Philip J. , Newman Ann , Jensen Klaus T. , Burger Lisa R. , Pintel David J. , Lorson Christian L.
Published: March 2005

The non-structural proteins NS1 and NS2 of the parvovirus minute virus of mice (MVM) are required for efficient virus replication. It has previously been shown that NS1 and NS2 interact and colocalize with the survival motor neuron (Smn) gene product in novel nuclear structures that are formed late in infection, termed Smn-associated APAR (autonomous parvovirus-associated replication) bodies (SAABs). It is not clear what molecular viral intermediate(s) contribute to SAAB formation. The current results address the role of NS2 in SAAB formation. In highly synchronized wild-type MVM infection of murine A92L cells, NS2 colocalizes with Smn and other SAAB constituents. An MVM mutant that does not produce NS2 still generates SAABS, albeit with a temporal delay. The lag in SAAB formation seen in the absence of NS2 is probably related to the temporal delay in virus replication, suggesting that, whilst NS2 is required for efficient viral infection, it is dispensable for SAAB formation.
Identification and functional analysis of VP3, the guanylyltransferase of Banna virus (genus Seadornavirus, family Reoviridae)
Authors: Jaafar Fauziah Mohd , Attoui Houssam , Mertens Peter P. C. , de Micco Philippe , de Lamballerie Xavier
Published: March 2005

Banna virus(BAV) particles contain seven structural proteins: VP4 and VP9 form an outer-capsid layer, whilst the virus core contains three major proteins (VP2, VP8 and VP10) and two minor proteins (VP1 and VP3). Sequence analysis showed that VP3 contains motifs [Kx(I/V/L)S] and (HxnH) that have previously been identified in the guanylyltransferases of other reoviruses. Incubation of purified BAV-Ch core particles with [α-32P]GTP resulted in exclusive covalent labelling of VP3, demonstrating autoguanylation activity (which is considered indicative of guanylyltransferase activity). Recombinant VP3 prepared in a cell-free expression system was also guanylated under similar reaction conditions, and products were synthesized (in the presence of non-radiolabelled GDP) that co-migrated with GMP, GDP and GpppG during TLC. This reaction, which required magnesium ions for optimum activity, demonstrates that VP3 possesses nucleoside triphosphatase (GTPase) activity and is the BAV guanylyltransferase (RNA ‘capping’ enzyme).
Inhibition of Marburg virus protein expression and viral release by RNA interference
Authors: Fowler Trent , Bamberg Sandra , Möller Peggy , Klenk Hans-Dieter , Meyer Thomas F. , Becker Stephan , Rudel Thomas
Published: March 2005

High mortality rates and lack of an available vaccine against Marburg haemorrhagic fever (MHF) highlight the need for a defensive therapy against MHF and greater knowledge of the causative agent, the Marburg virus (MARV). Here, RNA interference (RNAi) is employed to destroy MARV transcripts, disrupting replication and allowing analysis of various roles of MARV proteins. Small interfering RNAs (siRNAs) homologous to three MARV transcripts (NP, VP35 and VP30) were co-transfected into cells with plasmids encoding the corresponding nucleocapsid proteins. The resulting decrease in MARV nucleocapsid-protein levels was shown to be specific, as siRNA that was not homologous to the MARV genome did not decrease the levels of viral nucleocapsid proteins. Additionally, transcript levels of double-stranded RNA (dsRNA)-sensor proteins, the dsRNA-activated protein kinase and 2′,5′-oligoadenylate synthetase 1 remained unchanged, suggesting that the decrease in viral proteins was not a result of activation of the antiviral properties of the interferon system. Subsequently, siRNAs were shown to reduce intracellular viral proteins in MARV-infected cells and viral material released into the medium. Targeted reduction of VP30 downregulated the intracellular levels of all other viral proteins, suggesting that VP30 plays an essential role for transcription/replication. The efficient reduction of MARV replication also suggests that RNAi may provide an agent against MHF.
An investigation of the therapeutic value of vaccinia-immune IgG in a mouse pneumonia model
Authors: Law Mansun , Pütz Mike M. , Smith Geoffrey L.
Published: March 2005

Vaccinia-immune globulin (VIG) was used to treat severe complications of smallpox vaccination, but its use was controversial because it resolved disease in only some clinical cases. VIG is a pool of hyperimmune sera collected from individuals with a high neutralizing titre against the intracellular mature form (IMV) of vaccinia virus (VACV), but activity against the extracellular enveloped form (EEV) was often not considered. Here, the efficacy of anti-VACV antibodies (Abs) in protecting mice from intranasal infection with the VACV strain Western Reserve (WR) was evaluated. Mice were immunized passively with hyperimmune rabbit Abs (IgG) generated against inactivated IMV or produced following infection by VACV; subsequently, animals were challenged with VACV WR. The results demonstrated that: (i) good protection requires Abs to EEV in addition to IMV; (ii) Abs were effective when given before or up to 4 days after infection; and (iii) protection of mice from VACV WR correlated with a reduction of virus replication in lungs, but not in brain. In agreement with studies conducted before smallpox was eradicated and recent studies using EEV antigens for immunization, this study reiterates the importance of anti-EEV Abs in protecting against orthopoxvirus infection and illustrates the need to evaluate both anti-IMV and anti-EEV neutralizing Abs in VIG.
Murine gammaherpesvirus-68 ORF28 encodes a non-essential virion glycoprotein
Authors: May Janet S. , Coleman Heather M. , Boname Jessica M. , Stevenson Philip G.
Published: March 2005

Murine gammaherpesvirus-68 (MHV-68) ORF28 is a gammaherpesvirus-specific gene of unknown function. Analysis of epitope-tagged ORF28 protein indicated that it was membrane-associated and incorporated into virions inN-glycosylated,O-glycosylated and unglycosylated forms. The extensive glycosylation of the small ORF28 extracellular domain – most forms of the protein appeared to be mainly carbohydrate by weight – suggested that a major function of ORF28 is to attach a variety of glycans to the virion surface. MHV-68 lacking ORF28 showed normal lytic replicationin vitroandin vivoand normal latency establishment. MHV-68 ORF28 therefore encodes a small, membrane-bound and extensively glycosylated virion protein, whose function is entirely dispensable for normal, single-cycle host colonization.
Pathogenesis of Hong Kong H5N1 influenza virus NS gene reassortants in mice: the role of cytokines and B- and T-cell responses
Authors: Lipatov Aleksandr S. , Andreansky Samita , Webby Richard J. , Hulse Diane J. , Rehg Jerold E. , Krauss Scott , Perez Daniel R. , Doherty Peter C. , Webster Robert G. , Sangster Mark Y.
Published: March 2005

The severity of disease caused in humans by H5N1 influenza viruses remains unexplained. The NS gene of Hong Kong H5N1/97 viruses was shown to contribute to high pathogenicity of reassortants in a pig model. However, the molecular pathogenesis and host immune response underlying this phenomenon remain unclear. Here, in a mouse model, H1N1 A/Puerto Rico/8/34 (PR/8) reassortants that contained the H5N1/97 NS gene, the H5N1/01 NS gene, or an altered H5N1/97 NS gene encoding a Glu92→Asp substitution in NS1 was studied. The pathogenicity of reassortant viruses, the induction of cytokines and chemokine CXCL1 (KC) in the lungs and specific B- and T-cell responses was characterized. In mice infected with reassortant virus containing the H5N1/97 NS gene, the mouse lethal dose (50 %) and lung virus titres were similar to those of PR/8, which is highly pathogenic to mice. This reassortant virus required two more days than PR/8 to be cleared from the lungs of infected mice. Reassortants containing the altered H5N1/97 NS gene or the H5N1/01 NS gene demonstrated attenuated pathogenicity and lower lung titres in mice. Specific B- and T-cell responses were consistent with viral pathogenicity and did not explain the delayed clearance of the H5N1/97 NS reassortant. The reassortant induced elevated pulmonary concentrations of the inflammatory cytokines IL1α, IL1β, IL6, IFN-γand chemokine KC, and decreased concentrations of the anti-inflammatory cytokine IL10. This cytokine imbalance is reminiscent of the clinical findings in two humans who died of H5N1/97 infection and may explain the unusual severity of the disease.
Differential effects of R5 and X4 human immunodeficiency virus type 1 infection on CD4+ cell proliferation and activation
Authors: Locher Christopher P. , Witt Stephanie A. , Kassel Rachel , Dowell Noah L. , Fujimura Sue , Levy Jay A.
Published: March 2005

Human immunodeficiency virus type 1 (HIV-1) isolates can be distinguished by their chemokine coreceptor usage. Non-syncytium-inducing (NSI), macrophage-tropic viruses utilize CCR5 and are called R5 viruses; syncytium-inducing (SI) isolates use CXCR4 and are known as X4 viruses. R5 and X4 HIV isolates are both transmitted but, in most cases, R5 viruses predominate in the blood prior to the development of AIDS-related pathogenesis. The reason for the selective growth of the R5 strain is not known, but could reflect a replication advantage of R5 viruses over X4 viruses in CD4+cells. To explore this possibility, eight phenotypically distinct viruses were used to infect CD4+cells and cellular proliferation and activation were evaluated. In unstimulated CD4+cells, R5 virus isolates increased the level of cell activation compared with X4 virus isolates and uninfected control cells. In CD4+cells that were stimulated with interleukin 2, both R5 and X4 viruses were found to decrease the level of cell proliferation and reduce the majority of the activation markers studied when compared with uninfected control CD4+cells from the same donors. However, although equal amounts of CD4+cells were infected, R5 virus-infected CD4+cells showed a two- to fourfold increase in cellular proliferation over X4 viruses, as measured by [3H]thymidine incorporation (P=0·001) and nuclear expression of Ki67 (P=0·001). In addition, a larger proportion of CD4+T cells infected with R5 viruses had significantly higher levels of activation-marker expression (e.g. CD25, CD71 and HLA-DR) than CD4+T lymphocytes infected with X4 viruses (P<0·02). Taken together, these results indicate that CD4+cells infected with R5 virus isolates may have a selective advantage over X4 virus-infected CD4+T cells for survival and, hence, virus spread.
Determination of phosphorylated residues from human respiratory syncytial virus P protein that are dynamically dephosphorylated by cellular phosphatases: a possible role for serine 54
Authors: Asenjo Ana , Rodríguez Lorena , Villanueva Nieves
Published: March 2005

The 241 aa human respiratory synctyial virus (HRSV) Long strain P protein is phosphorylated at serines 116, 117 and/or 119, and 232. Phosphates added to these residues have slow turnover and can be detected in the absence of protein phosphatase inhibition. Inhibition of phosphatases PP1 and PP2A increases the level of phosphorylation at serines 116, 117 and/or 119, suggesting a more rapid turnover for phosphates added to these residues compared to that of S232. High-turnover phosphorylation is detected in the P-protein NH2-terminal region, mainly at S54 and, to a lesser extent, at S39, in the Long strain. When the P protein bears the T46I substitution (in the remaining HRSV strains), phosphates are added to S30, S39, S45 and S54. Phosphatase PP1 removes phosphate at residues in the central part of the P-protein molecule, whereas those in the NH2-terminal region are removed by phosphatase PP2A. The significance of the phosphorylation of the NH2-terminal region residues for some P-protein functions was studied. The results indicated that this modification is not essential for P-protein oligomerization or for its role in viral RNA synthesis. Nonetheless, dephosphorylation at S54 could facilitate P–M protein interactions that probably occur during the egress of viral particles.
Identification of functional domains within the bICP0 protein encoded by bovine herpesvirus 1
Authors: Zhang Yange , Zhou Joe , Jones Clinton
Published: March 2005

It is believed that the bICP0 protein encoded by bovine herpesvirus 1 (BoHV-1) stimulates productive infection by activating viral gene expression. Like the other ICP0-like proteins encoded byalphaherpesvirinaesubfamily members, bICP0 contains a zinc RING finger near its amino terminus. The zinc RING finger of bICP0 activates viral transcription, stimulates productive infection, and is toxic to certain cell types. Apart from the zinc RING finger, bICP0 possesses little similarity to the herpes simplex virus type 1 ICP0 protein making it difficult to predict what regions of bICP0 are important. To begin to identify bICP0 functional domains that are not part of the zinc RING finger, a panel of transposon insertion mutants that span bICP0 was developed. A large domain spanning aa 78–256, and a separate domain that is at or near aa 457 was necessary for efficient transactivation of a simple promoter. Transposon insertion at aa 91 impaired bICP0 protein stability in transfected cells. Insertion of transposons into the acidic domain of bICP0 had little or no effect on transactivation of a simple promoter or protein expression suggesting this region does not play a major role in activating gene expression. Sequences near the C terminus (aa 607–676) contain a functional nuclear localization signal. Collectively, these studies indicated that bICP0 contains several important functional domains: (i) the zinc RING finger, (ii) two separate domains that activate transcription, and (iii) a C-terminal nuclear localization signal that is also necessary for efficient transactivation.
Epitope mapping of human respiratory syncytial virus 22K transcription antitermination factor: role of N-terminal sequences in protein folding
Authors: García-Barreno Blanca , Steel John , Payá Monica , Martínez-Sobrido Luis , Delgado Teresa , Yeo Robert P. , Melero José A.
Published: March 2005

The reactivity of a panel of 12 monoclonal antibodies raised against the human respiratory syncytial virus 22 kDa (22K) protein was tested by Western blotting with a set of 22K deletion mutants. The results obtained identified sequences in the C-terminal half of the 22K polypeptide required for integrity of most antibody epitopes, except for epitope 112, which was lost in mutants with short N-terminal deletions. This antibody, in contrast to the others, failed to immunoprecipitate the native 22K protein, indicating that the N terminus of this protein is buried in the native molecule and exposed only under the denaturing conditions of Western blotting. In addition, N-terminal deletions that abolished reactivity with monoclonal antibody 112 also inhibited phosphorylation of the 22K protein previously identified at Ser-58 and Ser-61, suggesting that the N terminus is important in regulating the 22K protein phosphorylation status, most likely as a result of its requirement for protein folding.
Perturbation of epidermal growth factor receptor complex formation and Ras signalling in cells harbouring the hepatitis C virus subgenomic replicon
Authors: Macdonald Andrew , Chan Julia Ka Yu , Harris Mark
Published: March 2005

Hepatitis C virus non-structural NS5A protein inhibits epidermal growth factor (EGF)-stimulated activation of the Ras–ERK mitogen-activated protein kinase pathway at a point upstream of Ras activation. To determine the mechanism of this inhibition, the events occurring between the EGF receptor and Ras in Huh-7 cells harbouring the HCV subgenomic replicon were investigated. It was shown that, following EGF stimulation, these cells exhibited decreased EGF receptor tyrosine phosphorylation, aberrant recruitment of the adaptor proteins ShcA and Grb2 to the EGF receptor, reduced phosphorylation of ShcA and reduced Ras activation in comparison with control cells. These data are consistent with effects of NS5A and/or other components of the replicon on multiple events occurring upstream of Ras.
Hepatitis C virus (HCV) NS5A protein downregulates HCV IRES-dependent translation
Authors: Kalliampakou Katerina I. , Kalamvoki Maria , Mavromara Penelope
Published: March 2005

Translation of the hepatitis C virus (HCV) polyprotein is mediated by an internal ribosome entry site (IRES) that is located mainly within the 5′ non-translated region of the viral genome. In this study, the effect of the HCV non-structural 5A (NS5A) protein on the HCV IRES-dependent translation was investigated by using a transient transfection system. Three different cell lines (HepG2, WRL-68 and BHK-21) were co-transfected with a plasmid vector containing a bicistronic transcript carrying the chloramphenicol acetyltransferase (CAT) and the firefly luciferase genes separated by the HCV IRES sequences, and an expression vector producing the NS5A protein. Here, it was shown that the HCV NS5A protein inhibited HCV IRES-dependent translation in a dose-dependent manner. In contrast, NS5A had no detectable effect on cap-dependent translation of the upstream gene (CAT) nor on translation from another viral IRES. Further analysis using deleted forms of the NS5A protein revealed that a region of about 120 aa located just upstream of the nuclear localization signal of the protein is critical for this suppression. Overall, these results suggest that HCV NS5A protein negatively modulates the HCV IRES activity in a specific manner.
The Plowright vaccine strain of Rinderpest virus has attenuating mutations in most genes
Authors: Baron M. D. , Banyard A. C. , Parida S. , Barrett T.
Published: March 2005

The currently used vaccine strain of Rinderpest virus was derived by serial passage of the highly virulent Kabete ‘O’ strain (KO). A full-length cDNA copy of the KO strain was made from which a virus identical in pathogenicity to the wild-type virus was rescued. A series of chimeric viruses was prepared in which the coding sequences for the N, P, F, H or L proteins were replaced with the corresponding sequences from the vaccine strain. The KO-based virus with the vaccine strain H gene and that with the carboxy-terminal half of the L gene replaced with the corresponding sequence from the vaccine strain retained all or almost all of the virulence of the original KO virus. Animals infected with the KO-based virus containing the vaccine strain N, P or F gene, or the amino-terminal half of the L gene, developed high and prolonged pyrexia and leukopenia, but with reduced or absent lesions and other clinical signs; although partially attenuated, none was nearly as attenuated as the vaccine strain itself. These data indicate that the high attenuation and stability of the current vaccine are due to the accumulation of a number of separate mutations, none of which is itself so sufficiently debilitating that there is strong selective pressure in favour of the revertant.
A new intertype recombinant between genotypes C and D of hepatitis B virus identified in China
Authors: Wang Zhanhui , Liu Zhihua , Zeng Guobing , Wen Shujuan , Qi Yipeng , Ma Shiwu , Naoumov Nikolai V. , Hou Jinlin
Published: March 2005

Hepatitis B virus (HBV) genotypes have a characteristic geographical distribution. More than 90 % of chronic HBV patients in China are infected with genotypes B or C. Here, eight HBV isolates that were initially classified as genotype D by PCR-restriction fragment length polymorphism analysis were analysed in detail. The complete HBV genome was sequenced and compared with 32 sequences retrieved from GenBank, representing HBV genotypes A–G. Phylogenetic analysis of the S gene (nt 10–800) classified all eight isolates as genotype D. However, phylogenetic analyses of nt 800–10 and the open reading frames (ORFs) of the precore/core and X genes classified all eight isolates as genotype C. This discordance between phylogenetic trees reconstructed on different ORFs suggested that intertype recombination has occurred in all eight isolates. By using the simplot program, the site of recombination with genotype D was located in the preS2/S region, spanning nt 10–799 in seven of eight isolates and nt 10–1499 in the other isolate. These results demonstrate that intertype recombination should be considered as a type of variation that increases the genetic diversity of HBV. Hybrids of different HBV genotypes might exhibit specific virological properties and their significance in the diagnosis and management of chronic hepatitis B deserves further investigation.
Natural isolates of Brome mosaic virus with the ability to move from cell to cell independently of coat protein
Authors: Takeda Atsushi , Nakamura Wakako , Sasaki Nobumitsu , Goto Kaku , Kaido Masanori , Okuno Tetsuro , Mise Kazuyuki
Published: March 2005

Brome mosaic virus(BMV) requires encapsidation-competent coat protein (CP) for cell-to-cell movement and the 3a movement protein (MP) is involved in determining the CP requirement for BMV movement. However, these conclusions have been drawn by using BMV strain M1 (BMV-M1) and a related strain. Here, the ability of the MPs of five other natural BMV strains to mediate the movement of BMV-M1 in the absence of CP was tested. The MP of BMV M2 strain (BMV-M2) efficiently mediated the movement of CP-deficient BMV-M1 and the MPs of two other strains functioned similarly to some extent. Furthermore, BMV-M2 itself moved between cells independently of CP, demonstrating that BMV-M1 and -M2 use different movement modes. Reassortment between CP-deficient BMV-M1 and -M2 showed the involvement of RNA3 in determining the CP requirement for cell-to-cell movement and the involvement of RNAs 1 and 2 in movement efficiency and symptom induction in the absence of CP. Spontaneous BMV MP mutants generatedin plantathat exhibited CP-independent movement were also isolated and analysed. Comparison of the nucleotide differences of the MP genes of BMV-M1, the natural strains and mutants capable of CP-independent movement, together with further mutational analysis of BMV-M1 MP, revealed that single amino acid differences at the C terminus of MP are sufficient to alter the requirement for CP in the movement of BMV-M1. Based on these findings, a possible virus strategy in which a movement mode is selected in plant viruses to optimize viral infectivity in plants is discussed.
‘Rescue’ of mini-genomic constructs and viruses by combinations of morbillivirus N, P and L proteins
Authors: Brown D. D. , Collins F. M. , Duprex W. P. , Baron M. D. , Barrett T. , Rima B. K.
Published: March 2005

Chloramphenicol acetyltransferase (CAT)-expressing negative-sense mini-genomic constructs of measles virus (MV) and rinderpest virus (RPV) were rescued by standard technology with helper plasmids expressing the nucleocapsid (N), phospho- (P) and large (L) proteins of MV, canine distemper virus (CDV) or RPV in order to determine whether the proteins of different viruses can function together. Homogeneous sets consisting of N, P and L plasmids derived from one virus were able to generate reporter gene expression from either mini-genomic construct. Heterogeneous sets of proteins from different viruses were not functional, with the exception that a low level of activity was obtained when MV N and P protein were combined with RPV L protein in the rescue of the MV mini-genomic construct, or CDV N was combined with RPV P and L in the rescue of the RPV mini-genome. However, only homogeneous sets of plasmids were able to rescue infectious virus from full-length anti-genome-expressing plasmids.
Expression of a Toxoneuron nigriceps polydnavirus-encoded protein causes apoptosis-like programmed cell death in lepidopteran insect cells
Authors: Lapointe Renée , Wilson Rebecca , Vilaplana Lluïsa , O'Reilly David R. , Falabella Patrizia , Douris Vassilis , Bernier-Cardou Michèle , Pennacchio Francesco , Iatrou Kostas , Malva Carla , Olszewski Julie A.
Published: March 2005

The polydnavirusToxoneuron nigricepsbracovirus (TnBV) is an obligate symbiont associated with the braconid waspT. nigriceps, a parasitoid ofHeliothis virescenslarvae. Previously, to identify polydnavirus genes that allow parasitization by altering the host immune and endocrine systems, expression patterns of TnBV genes from parasitizedH. virescenslarvae were analysed and cDNAs were obtained. To study the function of the protein from one such cDNA, TnBV1, overexpression of the protein was attempted by using the baculovirusAutographa californicamulticapsid nucleopolyhedrovirus. Recovery of stable recombinant virus was unsuccessful, with the exception of recombinants with deletions/mutations within theTnBV1gene. It was hypothesized that TnBV1 expression was cytotoxic to theSpodoptera frugiperda(Sf21) insect cells that were used to produce the recombinants. Therefore, the Bac-to-Bac system was used to create recombinant baculoviruses maintained inEscherichia coliexpressing either TnBV1 (Ac-TnBV1) or an initiator-methionine mutant [Ac-TnBV1(ATG−)]. Microscopy revealed substantial cell death of Sf21 and High Five cells from 48 h post-infection with Ac-TnBV1, but not with the Ac-TnBV1(ATG−) recombinant virus. Ac-TnBV1-infected Sf21 cells, but not those with parental virus infection, showed an increased caspase-3-like protease activity, as well as increased terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling (TUNEL) for breaks in host genomic DNA. Although indicative of apoptosis, blebbing and apoptotic bodies were not observed in infected cells. Transiently expressing TnBV1 alone caused TUNEL staining in High Five cells. These data suggest that TnBV1 expression alone can induce apoptosis-like programmed cell death in two insect cell lines. Injection of Ac-TnBV1 budded virus, compared with parental virus, did not result in an alteration of virulence inH. virescenslarvae.
Analysis of the floral transcriptome uncovers new regulators of organ determination and gene families related to flower organ differentiation inGerbera hybrida(Asteraceae)
Authors: Laitinen Roosa A.E. , Immanen Juha , Auvinen Petri , Rudd Stephen , Alatalo Edward , Paulin Lars , Ainasoja Miia , Kotilainen Mika , Koskela Satu , Teeri Teemu H. , Elomaa Paula
Published: March 2005

Development of composite inflorescences in the plant family Asteraceae has features that cannot be studied in the traditional model plants for flower development. InGerbera hybrida, inflorescences are composed of morphologically different types of flowers tightly packed into a flower head (capitulum). Individual floral organs such as pappus bristles (sepals) are developmentally specialized, stamens are aborted in marginal flowers, petals and anthers are fused structures, and ovaries are located inferior to other floral organs. These specific features have made gerbera a rewarding target of comparative studies. Here we report the analysis of a gerbera EST database containing 16,994 cDNA sequences. Comparison of the sequences with all plant peptide sequences revealed 1656 unique sequences for gerbera not identified elsewhere within the plant kingdom. Based on the EST database, we constructed a cDNA microarray containing 9000 probes and have utilized it in identification of flower-specific genes and abundantly expressed marker genes for flower scape, pappus, stamen, and petal development. Our analysis revealed several regulatory genes with putative functions in flower-organ development. We were also able to associate a number of abundantly and specifically expressed genes with flower-organ differentiation. Gerbera is an outcrossing species, for which genetic approaches to gene discovery are not readily amenable. However, reverse genetics with the help of gene transfer has been very informative. We demonstrate here the usability of the gerbera microarray as a reliable new tool for identifying novel genes related to specific biological questions and for large-scale gene expression analysis.
Disclosing hidden transcripts: Mouse natural sense–antisense transcripts tend to be poly(A) negative and nuclear localized
Authors: Kiyosawa Hidenori , Mise Nathan , Iwase Shigeru , Hayashizaki Yoshihide , Abe Kuniya
Published: March 2005

Genome-wide in silico analysis identified thousands of natural sense–antisense transcript (SAT) pairs in the mouse transcriptome. We investigated their expression using strand-specific oligo-microarray that distinguishes expression of sense and antisense RNA from 1947 SAT pairs. The majority of the predicted SATs are expressed at various steady-state levels in various tissues, and cluster analysis of the array data demonstrated that the ratio of sense and antisense expression for some of the SATs fluctuated markedly among these tissues, while the rest was unchanged. Surprisingly, further analyses indicated that vast amounts of multiple-sized transcripts are expressed from the SAT loci, which tended to be poly(A) negative, and nuclear localized. The tendency that the SATs are often not polyadenylated is conserved, even in the randomly chosen SAT genes in the plant Arabidopsis thaliana. Such common characteristics imply general roles of the SATs in regulation of gene expression.
Art, dreams and active imagination:  A post‐Jungian approach to transference and the image
Authors: Schaverien Joy
Published: March 2005

Abstract:  The term active imagination is sometimes applied rather uncritically to describe all forms of creative activity that take place in depth psychology. Whilst there are many forms of expression that evoke or are evoked by active imagination, they cannot automatically be classed as active imagination. In this article investigation of visualized mental imagery, dreams and art reveals three distinct forms of image‐based psychological activity. Integrated and mediated within the transference and countertransference dynamic, it is proposed that the engagement in active imagination reflects and is influenced by the transference. Distinctions between sign and symbol, simple and big dreams as well as diagrammatic and embodied imagery clarify the differences. Examples from clinical practice demonstrate each mode in action within the analytic frame.
The place of the 17th century in Jung's encounter with China
Authors: Cambray Joe
Published: March 2005

Abstract:  After recounting several dreams and related alchemical interests of Jung's tied to the 17th century, a contextualizing look at select scientific and philosophical developments of that century is presented. Several precursors of the contemporary debates on the mind/body relation are noted, with special reference to the work of Antonio Damasio. This in turn leads to a reconsideration of the work of the 17th century polymath Gottfried Wilhelm Leibniz, which Jung read as a major precursor to his formulation of synchronicity (via Leibniz's concept of ‘pre‐established harmony’). Leibniz was the first philosopher to articulate the mind/body relationship in terms of supervenience, sharing an accord with those contemporary philosophers and scientists who see the mind as being an emergent property of the body‐brain. Similarly, these ideas are also consistent with a reformulation of synchronicity in terms of emergence.

Tracing Leibniz's interest in China reveals another set of links to Jung and to emergentism. Jung's use of Taoist concepts in developing the synchronicity principle is well known. According to scholars, Leibniz was the first major Western intellect to study the I‐Ching, through the assistance of a Jesuit missionary in Beijing, Fr. Joachim Bouvet. Some details of the Leibniz‐Bouvet correspondence are discussed here. Despite Helmut Wilhelm's presenting aspects of this correspondence at an Eranos conference, Jung does not appear to have integrated it into his writing on synchronicity—a possible reason for this omission is suggested.
Synchronicity and the I Ching: Jung, Pauli, and the Chinese Woman
Authors: Zabriskie Beverley
Published: March 2005

Abstract:  The capacity of the human mind to discover and invent both imagistic analogies and mathematical structures to represent reality is strikingly juxtaposed in the ancient Chinese text of the I Ching. Its emphasis on containing all sorts of opposites and its plastic appeal to multi‐valenced experience has kept it alive through millennia and across cultures. Jung was introduced to its Taoist wisdom by the Sinologist Richard Wilhelm. The Nobel Laureate quantum physicist Wolfgang Pauli became familiar with its philosophy and mathematics through his reading of Schopenhauer and Leibniz. In their correspondence about the nature of the unconscious and synchronicity, Pauli and Jung also exchanged their musings on Pauli's dreams of a Chinese woman, her role in his psyche and his scientific theories1.
The I Ching and the psyche‐body connection
Authors: Ma Shirley S. Y.
Published: March 2005

Abstract: Carl G. Jung's fateful meeting with Richard Wilhelm in 1929 has helped to build a bridge of depth psychological understanding between the East and the West. When Jung emerged from his ‘confrontation with the unconscious’, he felt validated by Wilhelm in his discovery of the healing power of medieval alchemical symbolism for the European psyche. Analytical psychology however offers a scientific, psychological understanding of Chinese wisdom as contained in the I Ching and Taoist alchemy.

The Taoist alchemical tradition (also known as the Inner Elixir tradition of which ‘The Secret of the Golden Flower’ is a sample text) is based on the premise that psychological experience of the Tao can be achieved through mental and physiological means such as breathing and meditative techniques, gymnastics, dietary regimens such as fasting, consumption of medicinal herbs and minerals, and special sexual practices. This tradition incorporates the I Ching and traditional Chinese medicine in the alchemical opus.

Taoist alchemy assumes the primacy of the physical body in the process of self‐realization. The psychological and cosmic forces of the trigrams of the I Ching are stored in the internal organs of the body and are the basic material for the experience of Tao. The internal organs are the foundation of the material and subtle bodies and through cultivation, the body becomes spiritualized as the spirits are embodied. The body as a reflection of the entire cosmos becomes the residence of the gods.

The realization of a new consciousness is symbolized by the hexagram Fu, meaning rebirth. The Chinese notion of Tao coincides with Jung's postulation of the unus mundus, the unity of existence which underlies the duality of psyche and matter, the psycho‐physical background of existence. In this light, in the world of inner experience, East and West follow similar paths symbolically.
Presence of RHD in serologically D–, C/E+ individuals: a European multicenter study
Authors: Gassner Christoph , Doescher Andrea , Drnovsek Tadeja Dovc , Rozman Primoz , Eicher Nicole I. , Legler Tobias J. , Lukin Sergey , Garritsen Henk , Kleinrath Thomas , Egger Bernd , Ehling Rainer , Körmöczi Günther F. , Kilga‐Nogler Susanne , Schoenitzer Diether , Petershofen Eduard K.
Published: March 2005

BACKGROUND: RHD blood group alleles with reduced or absent antigen expression are a clinically significant and heterogeneous group.

Study design and methods: To detail population genetics data on apparently D– individuals in central Europe, a six‐center study was performed with participants from Austria, Germany, Slovenia, Switzerland, and Russia. A total of 1700 serologically D– samples, positive for C and/or E, were investigated.

RESULTS: Observed unexpressed RHD alleles were 59 RHD‐CE‐D+ hybrid alleles, 9 apparently regular RHD, 1 new RHD(Y401X); DELs were 8 RHD(M295I), 6 RHD(IVS3+1G>A), and 1 new RHD(X418L); and weakly expressed RHDs were 2 weak D type 5, 1 weak D type 1, 1 RHD category VI type 1, and 1 novel weak D type 26. Although weak D type 26 was shown to have one of the lowest D antigen densities ever observed, it gave rise to anti‐D immunization in a transfused D– individual.

CONCLUSION: The relative occurrence of RHD among serologically D– samples, positive for C and/or E, differed significantly in the investigated central European regions. Considering the growing use of molecular typing techniques, correct identification of blood group alleles with scarce or missing antigen expression is of utmost clinical importance and requires reliable population‐based frequency data.
Stored platelets contain residual amounts of tissue factor: evidence from studies on platelet concentrates stored for prolonged periods
Authors: Pérez‐Pujol Silvia , Aras Omer , Lozano Miguel , Cocking‐Johnson Debra , Key Nigel S. , White James G. , McCullough Jeffrey , Escolar Giné
Published: March 2005

BACKGROUND: The advent of new strategies for pathogen reduction has raised the question of whether platelet (PLT) concentrates (PCs) exposed to longer periods of storage retain adequate hemostatic function.

STUDY DESIGN AND METHODS: The effects of prolonged storage on adhesive and procoagulant functions of PLTs in PCs have been analyzed. The ability of PLTs to interact with surfaces was assessed by en face electron microscopy. Exposure of anionic phospholipids or tissue factor (TF) antigen on PLTs was assessed by flow cytometry and by immunocytochemical methods at the ultrastructural level. Studies were performed in six different PCs followed 0, 3, 5, 7, and 11 days of storage.

RESULTS: A progressive impairment of PLT‐adhesive functions was observed after 5 days of storage. A progressive increase in expression of anionic phospholipids and development of procoagulant activity (PCA) measured by a modified prothrombin time (mPT) was observed along the storage. Incubation of PLTs with a specific anti‐TF resulted in prolongation of the mPT by approximately 10 to 15 percent. Flow cytometry revealed minimal TF expression at later storage times. Immunocytochemical studies showed minimal TF labeling when studies were performed on PLT whole mounts. Labeling was markedly improved when PLTs were previously exposed to sonication.

CONCLUSION: Prolonged storage of PCs was associated with decreased PLT‐adhesive capacities and enhanced PCA. Current preparation procedures and storage media have important limitations for preserving PCs for longer than 1 week. PLTs in PCs retain residual amounts of TF as assessed by immunocytochemical and functional assays. The origin and hemostatic significance of this TF should be investigated further.
A dose‐response analysis of lenograstim plus dexamethasone for neutrophil mobilization and collection
Authors: Heuft Hans‐Gert , Goudeva Lilia , Pulver Nicole , Grigull Lorenz , Schwella Nimrod , Blasczyk Rainer
Published: March 2005

BACKGROUND: The objective was to evaluate the dose‐response relationship of lenograstim plus dexamethasone for neutrophil mobilization and collection.

STUDY DESIGN AND METHODS: In a prospective study, 260 healthy volunteers received oral dexamethasone (8 mg) plus a single subcutaneous injection of glycosylated granulocyte–colony‐stimulating factor (G–CSF; lenograstim) at medians of 1.5 (1.0‐2.3) µg per kg (n = 43), 3 (2.4‐4.1) µg per kg (n = 73), 6 (4.3‐7.9) µg per kg (n = 123), and 12 (8.2‐17.2) µg per kg (n = 21) and underwent neutrophil collections with a polymorphonucleated neutrophil (PMN) program. White blood cell (WBC) counts and PMN mobilization and collection results were compared, and the severity and clinical significance of donor adverse reactions were evaluated. Fifty‐two neutropenic patients (29 children, 23 adults) underwent 271 neutrophil transfusions (GTXs) every other day to maintain WBC levels continuously above 0.5 × 109 per L.

RESULTS: Within the dose range 1.5, 3, and 6 µg per kg, each doubling step was associated with a 10 to 15 percent PMN increase in peripheral blood up to 32.8 (19.1‐49.2) × 109 per L (6 µg/kg; p ≤ 0.00032) as well as in the neutrophil concentrate up to 79 (34‐150) × 109 per U (6 µg/kg; p ≤ 0.00042). A further doubling to 12 µg per kg achieved neither better mobilization nor better apheresis results. The rate of clinically important adverse reactions increased already with the 6 µg per kg mobilization step. The GTX resulted in median peak WBC increments to 3.8 (0.4‐18.2) × 109 per L (children) and 1.6 (0.3‐9.4) × 109 per L (adults), but in adults the WBC threshold of 0.5 × 109 per L was not continuously exceeded.

CONCLUSIONS: The most effective dose‐response ratio for PMN mobilization was demonstrated in the 6 µg per kg lenograstim group. In neutropenic adults, GTX treatment on an every‐other‐day schedule may be ineffective.
Massive antenatal fetomaternal hemorrhage: evidence for long‐term survival of fetal red blood cells
Authors: Dziegiel Morten H. , Koldkjær Ole , Berkowicz Adela
Published: March 2005

BACKGROUND: Massive fetomaternal hemorrhage (FMH) can lead to life‐threatening anemia. Quantification based on flow cytometry with anti‐hemoglobin F (HbF) is applicable in all cases but underestimation of large fetal bleeds has been reported. A large FMH from an ABO‐compatible fetus allows an estimation of the life span of fetal red blood cells (RBCs) in the maternal circulation.

CASE REPORT: The mother went to the obstetrician twice antepartum owing to symptoms assumed to be preeclampsia; that, however, was not found. She later delivered by cesarean section owing to diminished fetal movements. No fetal weight gain was observed during the last 2 weeks of pregnancy.

STUDY DESIGN AND METHODS: Fetal RBCs were quantified by flow cytometry with anti‐HbF, anti‐Fya, anti‐s, and anti‐Jkb on a regular basis.

RESULTS: The infant had anemia at delivery and an FMH was determined to be 314 ± 17 mL (± SD) of whole blood. It is assumed that the two antenatal visits were associated with the FMH. Postpartum follow‐up showed that fetal RBCs in the maternal circulation were detectable with anti‐HbF up to 119 days. Quantification by flow cytometry based on anti‐HbF was in agreement with quantification based on anti‐Fya, anti‐s, and anti‐Jkb, although they were less sensitive.

CONCLUSION: ABO‐compatible fetal RBCs from an FMH had a life span in the maternal circulation close to that of adult RBCs.
Detection of West Nile virus RNA and antibody in frozen plasma components from a voluntary market withdrawal during the 2002 peak epidemic
Authors: Tobler L.H. , Bianco C. , Glynn S.A. , Schreiber G.B. , Dille B.J. , Prince H.E. , Lanciotti R.S. , Linnen J.M. , Gallarda J. , Shyamala V. , Smith D. , Kleinman S.H. , Busch M.P.
Published: March 2005

BACKGROUND: The US West Nile virus (WNV) epidemic in the summer and fall of 2002 included the first documented cases of transfusion‐transmitted WNV infection. In December 2002, the FDA supported a voluntary market withdrawal by the blood banking community of frozen blood components collected in WNV high‐activity areas. At the time, the prevalence of viremia and serologic markers for WNV in the blood supply was undefined.

STUDY DESIGN AND METHODS: In collaboration with America's Blood Centers, 1468 frozen plasma components (of approx. 60,000 frozen units voluntarily withdrawn from the market) were selectively retrieved from the peak epidemic regions and season (June 23, 2002‐September 28, 2002). These units were unlinked, subaliquoted, and tested by WNV enzyme immunoassays (EIAs; Focus Technologies and Abbott Laboratories) and nucleic acid amplification tests (NATs; Gen‐Probe Inc. and Roche Molecular Systems).

RESULTS: Of the 1468 EIA results from Abbott and Focus, 7 were anti‐immunoglobulin M (IgM)‐ and anti‐immunoglobulin G (IgG)‐reactive by both assays, 8 and 1 were IgM‐only‐reactive, and 8 and 23 were IgG‐only‐reactive, respectively. NAT by Gen‐Probe and Roche Molecular Systems yielded one RNA‐positive, antibody‐negative unit containing approximately 440 RNA copies per mL. An additional 10‐fold replicate NAT testing by Gen‐Probe on 14 of 15 IgM‐reactive specimens yielded 2 additional IgM‐ and IgG‐reactive units with low‐level viremia (i.e., 7/10 and 2/10 replicates tested reactive).

CONCLUSION: The prevalence of acute (RNA‐positive) and recent (IgM‐seroreactive) WNV infections indicates that transfusion risk in high‐risk areas could have been considerable and that voluntary market withdrawal of frozen components likely averted some WNV transfusion transmissions. The existence of very‐low‐level viremic units raises concerns, because WNV minipool NAT screening will miss such units and individual NAT may not completely correct this situation.