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At least 955 records · Page 53Linked to original sources
PubMed2002

Induction of proinflammatory cytokines in human macrophages by influenza A (H5N1) viruses: a mechanism for the unusual severity of human disease?

BACKGROUND: In 1997, the first documented instance of human respiratory disease and death associated with a purely avian H5N1 influenza virus resulted in an overall case-fatality rate of 33%. The biological basis for the severity of human H5N1 disease has remained unclear. We tested the hypothesis that virus-induced cytokine dysregulation has a role. METHODS: We used cDNA arrays and quantitative RT-PCR to compare the profile of cytokine gene expression induced by viruses A/HK/486/97 and A/HK/483/97 (both H5N1/97) with that of human H3N2 and H1N1 viruses in human primary monocyte-derived macrophages in vitro. Secretion of tumour necrosis factor alpha (TNF alpha) from macrophages infected with the viruses was compared by ELISA. By use of naturally occurring viral reassortants and recombinant viruses generated by reverse genetic techniques, we investigated the viral genes associated with the TNF-alpha response. FINDINGS: The H5N1/97 viruses induced much higher gene transcription of proinflammatory cytokines than did H3N2 or H1N1 viruses, particularly TNF alpha and interferon beta. The concentration of TNF-alpha protein in culture supernatants of macrophages infected with these viruses was similar to that induced by stimulation with Escherichia coli lipopolysaccharide. The non-structural (NS) gene-segment of H5N1/97 viruses contributed to the increase in TNF alpha induced by the virus. INTERPRETATION: The H5N1/97 viruses are potent inducers of proinflammatory cytokines in macrophages, the most notable being TNF alpha. This characteristic may contribute to the unusual severity of human H5N1 disease.

Cells, Cultured↗
PubMed2002

Mutants of Arabidopsis reveal many roles for membrane lipids.

Polyunsaturated acyl lipids constitute approximately 50% of the hydrophobic membrane barriers that delineate the compartments of cells. The composition of these lipids is critically important for many membrane functions and, thus, for proper growth and development of all living organisms. In the model plant Arabidopsis, the isolation of mutants with altered lipid compositions has facilitated biochemical and molecular approaches to understanding lipid metabolism and membrane biogenesis. Just as importantly, the availability of a series of plant lines with specific changes in membrane lipids have provided a new resource to study the structural and adaptive roles of lipids. Now, the sequencing of the Arabidopsis genome, and the development of reverse-genetics approaches provide the tools needed to make additional discoveries about the relationships between lipid structure and membrane function in plant cells.

Arabidopsis↗
PubMed2003

Characterization of a recombinant Newcastle disease virus expressing the green fluorescent protein.

A recombinant Newcastle disease virus (NDV) expressing the green fluorescent protein (GFP) was generated by applying reverse genetics techniques. The GFP open reading frame flanked by NDV transcription start and stop sequences was inserted between the fusion (F)- and hemagglutinin-neuraminidase genes in a full-length cDNA clone of NDV. This plasmid transcribing antigenome RNA was cotransfected with helper plasmids expressing viral nucleoprotein, phosphoprotein and large protein into cells stably expressing T7 RNA polymerase. The rescued virus was first propagated in embryonated eggs and the allantoic fluid was used to infect cells. Northern blot analysis of RNA isolated from infected cells demonstrated the proper transcription of the introduced GFP-mRNA. The appearance of GFP in live infected cells confirmed further the recovery of a recombinant NDV (rNDVGFP1) expressing the reporter gene. The expression of the heterologous gene was maintained stably for at least five passages in embryonated eggs. The replication kinetics in embryonated eggs and pathogenicity in chickens of rNDVGFP1 did not differ significantly from that of the parent virus. Using GFP autofluorescence, virus infected cells could be tracked easily in native preparations, organ explants and primary tracheal cell cultures. Taken together, these data demonstrate the use of GFP-expressing recombinant NDV for analysis of NDV dissemination and pathogenesis and indicate the potential usefulness of NDV as a vaccine vector.

Animals↗
PubMed2003

Molecular dissection of neuromuscular junction formation.

The muscle-specific kinase (MuSK) is the key component that mediates the synapse-inducing role of motoneuron-derived agrin at the neuromuscular junction. Recent reverse-genetics approaches have shed new light on the events triggered inside myotubes by activation of this kinase. Mice in which most of the intracellular domain of MuSK is replaced by a related kinase are viable. Analysis of the imperfect postsynaptic specializations of these mice has provided new insights into the complex postsynaptic differentiation process.

Agrin↗
PubMed2003

Improvements in transfection efficiency and tests of RNA interference (RNAi) approaches in the protozoan parasite Leishmania.

Approaches which eliminate mRNA expression directly are ideally suited for reverse genetics applications in eukaryotic microbes which are asexual diploids, such as the protozoan parasite Leishmania. RNA interference (RNAi) approaches have been successful in many species, including the related parasite Trypanosoma brucei. For RNAi tests in Leishmania, we developed improved protocols for transient and stable DNA transfection, attaining efficiencies of up to 25 and 3%, respectively. This facilitated RNAi tests at the alpha-tubulin locus, whose inhibition gives a strong lethal phenotype in trypanosomatids. However, transient or stable transfection of DNAs encoding mRNAs for an alpha-tubulin stem-loop construct and GFP to monitor transfection resulted in no effect on parasite morphology, growth or tubulin expression in Leishmania major or L. donovani. Transient transfection of a 24-nucleotide double-stranded alpha-tubulin siRNA also had no effect. Similar results were obtained in studies targeting an introduced GFP gene with a GFP stem-loop construct. These data suggest that typical RNAi strategies may not work effectively in Leishmania, and raise the possibility that Leishmania is naturally deficient for RNAi activity, like Saccharomyces cerevisae. The implications to parasite biology, gene amplification, and genetic analysis are discussed.

Animals↗
PubMed1998

Cloning of a trans-spliced glyceraldehyde-3-phosphate-dehydrogenase gene from the potato cyst nematode Globodera rostochiensis and expression of its putative promoter region in Caenorhabditis elegans.

Reverse genetics to determine the relative importance of individual pathogenicity factors of the potato cyst nematode Globodera rostochiensis depends, apart from an efficient transformation protocol for this obligatory plant parasite, on the availability of an efficient promoter. PCR-based cloning was used to isolate a cDNA encoding glyceraldehyde-3-phosphate-dehydrogenase (GAPDH, a crucial enzyme in glycolysis and gluconeogenesis; this gene was designated gpd) and its 5'-flanking region. The cDNA includes 1047 nucleotides encoding an open reading frame that shows high homology with GAPDHs from Caenorhabditis elegans and other species. Analysis of the 745 bp 5'-flanking region of the gpd gene showed no homology with a similar region in C. elegans. In this region several eukaryotic promoter elements are present. 5' Rapid amplification of cDNA ends revealed this gene was trans-spliced with a SL1 spliced leader. The 5'-flanking region of the gpd gene was fused to green fluorescent protein reporter gene and microinjected into the gonads of C. elegans. Green fluorescent protein expression, under the transcriptional control of the 5'-flanking region of gpd, was mainly observed in body wall muscles of transgenic animals. This putative promoter region of GAPDH could be a valuable tool to drive gene expression in transgenic G. rostochiensis and other related plant-parasitic nematode species.

Amino Acid Sequence↗
PubMed1997

Integration of foreign DNA and its consequences in mammalian systems.

The insertion of foreign DNA into the genomes of established cells and organisms and the consequences of this integration event are of significance for viral oncology, reverse genetics, transgenic organisms, human somatic gene therapy and evolution. This review summarizes recent experimental findings and focuses on the alteration of cellular DNA methylation at regions remote from the site of insertion. We also discuss experimental data demonstrating that foreign DNA ingested by mice is not completely degraded in their gastrointestinal tracts; fragments of this DNA have been found to be covalently linked to DNA with 70% homology to the mouse IgE receptor gene.

Animals↗
PubMed1998

Negative-strand RNA viruses: applications to biotechnology.

The establishment of reverse-genetics techniques to manipulate the genome of negative-strand RNA viruses has contributed enormously to a better understanding of the replication mechanisms and pathogenicity of this group of viruses. The generation of recombinant viruses bearing specific mutations in the coding and noncoding regions of their genomic RNAs now allows the functions in the replicative cycle of specific RNA regions and protein domains of these viruses to be studied. In addition, recombinant negative-strand RNA viruses can now be designed to have specific properties that make them attractive biotechnological tools.

Animals↗
PubMed2002

Abeta-degrading endopeptidase, neprilysin, in mouse brain: synaptic and axonal localization inversely correlating with Abeta pathology.

Metabolism of amyloid-beta peptide (Abeta) is closely associated with the pathology and etiology of Alzheimer's disease (AD). Since neprilysin is the only rate-limiting catabolic peptidase proven by reverse genetics to participate in Abeta metabolism in vivo, we performed detailed immunohistochemical analysis of neprilysin in mouse brain using neprilysin-deficient mice as a negative control. The aim was to assess, at both the cellular and subcellular levels, where Abeta undergoes neprilysin-dependent degradation in the brain and how neprilysin localization relates to Abeta pathology in amyloid precursor protein (APP)-transgenic mice. In hippocampus, neprilysin was present in the stratum pyramidale and stratum lacunosum-moleculare of the CA1-3 fields and the molecular layer of the dentate gyrus. Confocal double immunofluorescence analyses revealed the subcellular localization of neprilysin along axons and at synapses. This observation suggests that after synthesis in the soma, neprilysin, a type II membrane-associated protein, is axonally transported to the terminals, where Abeta degradation is likely to take place. Among various cell types, GABAergic and metabotropic glutamate 2/3 receptor-positive neurons but not catecholaminergic or cholinergic neurons, expressed neprilysin in hippocampus and neocortex, implying the presence of a cell type-specific mechanism that regulates neprilysin gene expression. As expected, Abeta deposition correlated inversely with neprilysin expression in TgCRND8 APP-transgenic mice. These observations not only support the notion that neprilysin functions as a major Abeta-degrading enzyme in the brain but also suggest that down-regulation of neprilysin activity, which may be caused by aging, is likely to elevate local concentrations of Abeta at and around neuronal synapses.

Alzheimer Disease↗
PubMed2003

Functional analysis of expressed peptides that bind yeast STE proteins.

Peptides are potentially useful for target validation and other reverse genetic applications. For instance, if a specific protein is susceptible to peptide inhibition, it may have a higher probability of being vulnerable to small molecules. We used the yeast two-hybrid technique to identify and study peptide binders for three yeast proteins involved in pheromone response: Ste11p, Ste18p, and Ste50p. A subset of peptide binders was shown to inhibit pheromone response in cells using two different functional assays. In addition, we utilized a variant of the yeast two-hybrid method to examine relative binding affinities based on competitive interactions in yeast. Our results suggest that binding affinity and inhibitory potency of peptides do not correlate perfectly and that peptide-protein interactions can be complex and unpredictable. Taken together these results suggest that while peptides are useful as in vivo inhibitors of protein function, caution must be exercised when choosing peptides for further studies and when inferring affinities from expression phenotypes.

GTP-Binding Protein gamma Subunits↗
PubMed2001

Rescue of viral haemorrhagic septicaemia virus minigenomes by helper virus.

A mammalian expression vector containing the bacterial chloramphenicol acetyltransferase (CAT) gene was used to demonstrate that CAT could be successfully used as a reporter system in fish cells growing at low temperatures. We then constructed a viral haemorrhagic septicaemia virus (VHSV) minigenome by cloning the CAT reporter gene between the viral leader and trailer sequences. This construct was used in transfection experiments with helper VHSV to demonstrate that the minigenome can be encapsidated and transcribed by helper virus proteins. In addition, passaging of viruses collected from cells expressing the minigenome showed that the minigenome was being packaged and replicated in the presence of helper virus. These experiments provide the initiating steps for a reverse genetics system for VHSV.

Animals↗
PubMed2002

Synthesis of influenza virus: new impetus from an old enzyme, RNA polymerase I.

Reverse genetics systems, i.e., systems for the generation of virus entirely from cloned cDNA, have been established for most nonsegmented negative-sense RNA viruses. In contrast, the generation of influenza A viruses (whose genome is composed of eight segments of negative-sense RNA) was not possible until 1999, likely due to the inherent technical difficulties of providing all eight viral RNAs as well as the four viral proteins required for replication and transcription. In 1999, we (Neumann et al., 1999, Proc. Natl. Acad. Sci. USA 96, 9345-9350) and others (Fodor et al., 1999, J. Virol. 73, 9679-9682) demonstrated the generation of influenza A virus from plasmids, relying on the cellular enzyme RNA polymerase I for the synthesis of influenza viral RNAs. In this review, we provide background on RNA polymerase I transcription and discuss its use for the generation of influenza virus from cloned cDNAs.

Animals↗
PubMed2002

Molecular characterization of an isolate from the 1989/90 epizootic of Ebola virus Reston among macaques imported into the United States.

We have determined the entire genomic sequence of the Pennsylvania strain, which was isolated along with the Virginia strain during the emergence of Ebola virus Reston in 1989/90 in the United States. Thus, either the Pennsylvania or Virginia strain, neither of which had been previously molecularly characterized, can be considered as the prototype for Ebola virus Reston. Comparative analysis showed a high degree of homology to the concomitantly analyzed and recently published Philippine strain of EBOV Reston from 1996 (Ikegami et al., Arch. Virol., 146 (2001) 2021). In comparison to EBOV Zaire, strain Mayinga, conservation could be found within the open reading frames, the 3' leader and 5' trailer region and the transcriptional signals, whereas the non-coding and intergenic regions did not show any homology. This clearly supports that EBOV Reston is a distinct species within the genus Ebola-like virus but which seems to be similar to other members with respect to transcription and replication strategies. The sequence determination provides the basis for the development of a reverse genetics system for Ebola virus Reston, which is needed to study differences in pathogenicity among filoviruses.

Amino Acid Sequence↗
PubMed2002

Replication and transcription of viral RNAs by recombinant L proteins of New Jersey serotype of vesicular stomatitis virus.

The large (L) protein of vesicular stomatitis virus (VSV), catalytic subunit of RNA-dependent RNA polymerase is responsible for the transcription and replication of VSV. The L protein of the Indiana serotype of VSV (VSV(Ind)) has previously been cloned and expressed, and used in the reverse genetics of VSV(Ind). However, the cDNA clones expressing functional L proteins of the VSV(NJ) serotype were not available. It was necessary to obtain functional clones of the New Jersey serotype of VSV (VSV(NJ)) in order to study homologous viral interference. Here we report the cDNA cloning, expression, and functional analyses of L proteins from both the Hazelhurst subtype and Concan subtype of VSV(NJ). The analysis of the expressed L proteins for the transcription and replication of VSV demonstrate that both VSV(NJ) L clones express functional RNA-dependent RNA polymerase.

Amino Acid Sequence↗
PubMed1997

Biologic effects of introducing additional basic amino acid residues into the hemagglutinin cleavage site of a virulent avian influenza virus.

We mutated the virulent avian influenza virus A/turkey/Ontario/7732/66 (H5N9)[Q-R-R-R-K-K-R\G at the hemagglutinin (HA) cleavage site] to create a mutant, R(MO-0), with additional basic residues at this site (Q-R-R-R-R-R-K-K-R\G) by reverse genetics. When tested in chicken embryo fibroblast culture, this mutant showed reduced HA cleavability compared to that of the wild-type virus, but its plaque size was not appreciably altered. Virulence of the R(MO-0) virus in chickens was lower than that of the wild-type virus. These findings indicate that addition of excessive basic residues to an optimal recognition sequence for HA cleavage enzymes at the cleavage site is deleterious for HA cleavability. Previously, we showed that a mutant containing the suboptimal HA cleavage site sequence for cleavage enzyme recognition also had reduced HA cleavability and virulence compared to the wild-type virus. We conclude that the data presented here further substantiate our belief that the level of HA cleavability correlates with the degree of virulence when all other genetic characteristics are considered equal, irrespective of the mechanisms by which HA cleavability is reduced.

Administration, Intranasal↗
PubMed2001

Broadcast interference - functional genomics.

Four recent papers mark a major shift in functional genomic analysis for multicellular organisms. RNA-mediated interference was applied to inactivate individual genes systematically on a genomic scale. These studies subjected a third of the genes in the genome of Caenorhabditis elegans to reverse genetic analysis.

Animals↗
PubMed2002

Functional genomics: the coming of age for Tetrahymena thermophila.

Over the past decade, researchers have manipulated the unique biology of Tetrahymena thermophila to generate a premier experimental organism for functional genomic analysis. A diverse array of DNA transformation methods have spearheaded in vivo strategies for discovering and dissecting universal eukaryotic processes, such as telomere addition and chromatin remodeling. Compartmentalization of this protist's genome into two functionally distinct nuclei - the silent 'germline' micronucleus and the transcriptionally active macronucleus - provides a powerful means for controlling the expression of transgenes. Heterokaryons that silently harbor homozygous recessive mutations (including lethal ones) in the germline have been exploited. The coupling of forward and reverse genetic approaches with genomics-based methods for gene discovery presents a bright future for research in this rising model eukaryote.

Animals↗
PubMed2003

Chloroplast research in the genomic age.

Chloroplast research takes significant advantage of genomics and genome sequencing, and a new picture is emerging of how the chloroplast functions and communicates with other cellular compartments. In terms of evolution, it is now known that only a fraction of the many proteins of cyanobacterial origin were rerouted to higher plant plastids. Reverse genetics and novel mutant screens are providing a growing catalogue of chloroplast protein-function relationships, and the characterization of plastid-to-nucleus signalling mutants reveals cell-organelle interactions. Recent advances in transcriptomics and proteomics of the chloroplast make this organelle one of the best understood of all plant cell compartments.

Arabidopsis↗