Showing papers by "David A. Jackson published in 2008"

The multifunctional NS1 protein of influenza A viruses.

Abstract: The non-structural (NS1) protein of influenza A viruses is a non-essential virulence factor that has multiple accessory functions during viral infection. In recent years, the major role ascribed to NS1 has been its inhibition of host immune responses, especially the limitation of both interferon (IFN) production and the antiviral effects of IFN-induced proteins, such as dsRNA-dependent protein kinase R (PKR) and 2'5'-oligoadenylate synthetase (OAS)/RNase L. However, it is clear that NS1 also acts directly to modulate other important aspects of the virus replication cycle, including viral RNA replication, viral protein synthesis, and general host-cell physiology. Here, we review the current literature on this remarkably multifunctional viral protein. In the first part of this article, we summarize the basic biochemistry of NS1, in particular its synthesis, structure, and intracellular localization. We then discuss the various roles NS1 has in regulating viral replication mechanisms, host innate/adaptive immune responses, and cellular signalling pathways. We focus on the NS1-RNA and NS1-protein interactions that are fundamental to these processes, and highlight apparent strain-specific ways in which different NS1 proteins may act. In this regard, the contributions of certain NS1 functions to the pathogenicity of human and animal influenza A viruses are also discussed. Finally, we outline practical applications that future studies on NS1 may lead to, including the rational design and manufacture of influenza vaccines, the development of novel antiviral drugs, and the use of oncolytic influenza A viruses as potential anti-cancer agents.

A new influenza virus virulence determinant: The NS1 protein four C-terminal residues modulate pathogenicity

Abstract: The virulence of influenza virus is a multigenic trait. One determinant of virulence is the multifunctional NS1 protein that functions in several ways to defeat the cellular innate immune response. Recent large-scale genome sequence analysis of avian influenza virus isolates indicated that four C-terminal residues of the NS1 protein is a PDZ ligand domain of the X-S/T-X-V type and it was speculated that it may represent a virulence determinant. To test this hypothesis, by using mice as a model system, the four C-terminal amino acid residues of a number of influenza virus strains were engineered into the A/WSN/33 virus NS1 protein by reverse genetics and the pathogenicity of the viruses determined. Viruses containing NS1 sequences from the 1918 H1N1 and H5N1 highly pathogenic avian influenza (HPAI) viruses demonstrated increased virulence in infected mice compared with wt A/WSN/33 virus, as characterized by rapid loss of body weight, decreased survival time, and decreased mean lethal dose. Histopathological analysis of infected mouse lung tissues demonstrated severe alveolitis, hemorrhaging, and spread of the virus throughout the entire lung. The increase in pathogenicity was not caused by the overproduction of IFN, suggesting the NS1 protein C terminus may interact with PDZ-binding protein(s) and modulate pathogenicity through alternative mechanisms.

The Influenza Virus M2 Protein Cytoplasmic Tail Interacts with the M1 Protein and Influences Virus Assembly at the Site of Virus Budding

Abstract: The cytoplasmic tail of the influenza A virus M2 proton-selective ion channel has been shown to be important for virus replication. Previous analysis of M2 cytoplasmic tail truncation mutants demonstrated a defect in incorporation of viral RNA (vRNA) into virions, suggesting a role for M2 in the recruitment of M1-vRNA complexes. To further characterize the effect of the M2 cytoplasmic tail mutations on virus assembly and budding, we constructed a series of alanine substitution mutants of M2 with mutations in the cytoplasmic tail, from residues 71 to 97. Mutant proteins M2-Mut1 and M2-Mut2, with mutations of residues 71 to 73 and 74 to 76, respectively, appeared to have the greatest effect on virus-like particle and virus budding, showing a defect in M1 incorporation. Mutant viruses containing M2-Mut1 and M2-Mut2 failed to replicate in multistep growth analyses on wild-type (wt) MDCK cells and were able to form plaques only on MDCK cells stably expressing wt M2 protein. Compared to wt M2 protein, M2-Mut1 and M2-Mut2 were unable to efficiently coimmunoprecipitate with M1. Furthermore, statistical analysis of planar sheets of membrane from cells infected by virus containing M2-Mut1 revealed a reduction in M1-hemagglutinin (HA) and M2-HA clustering as well as a severe loss of clustering between M1 and M2. These results suggest an essential, direct interaction between the cytoplasmic tail of M2 and M1 that promotes the recruitment of the internal viral proteins and vRNA to the plasma membrane for efficient virus assembly to occur.

sparse inflorescence1 encodes a monocot-specific YUCCA-like gene required for vegetative and reproductive development in maize.

Abstract: The plant growth hormone auxin plays a critical role in the initiation of lateral organs and meristems. Here, we identify and characterize a mutant, sparse inflorescence1 (spi1), which has defects in the initiation of axillary meristems and lateral organs during vegetative and inflorescence development in maize. Positional cloning shows that spi1 encodes a flavin monooxygenase similar to the YUCCA (YUC) genes of Arabidopsis, which are involved in local auxin biosynthesis in various plant tissues. In Arabidopsis, loss of function of single members of the YUC family has no obvious effect, but in maize the mutation of a single yuc locus causes severe developmental defects. Phylogenetic analysis of the different members of the YUC family in moss, monocot, and eudicot species shows that there have been independent expansions of the family in monocots and eudicots. spi1 belongs to a monocot-specific clade, within which the role of individual YUC genes has diversified. These observations, together with expression and functional data, suggest that spi1 has evolved a dominant role in auxin biosynthesis that is essential for normal maize inflorescence development. Analysis of the interaction between spi1 and genes regulating auxin transport indicate that auxin transport and biosynthesis function synergistically to regulate the formation of axillary meristems and lateral organs in maize.

The Relationship between Auxin Transport and Maize Branching

Abstract: Maize (Zea mays) plants make different types of vegetative or reproductive branches during development. Branches develop from axillary meristems produced on the flanks of the vegetative or inflorescence shoot apical meristem. Among these branches are the spikelets, short grass-specific structures, produced by determinate axillary spikelet-pair and spikelet meristems. We investigated the mechanism of branching in maize by making transgenic plants expressing a native expressed endogenous auxin efflux transporter (ZmPIN1a) fused to yellow fluorescent protein and a synthetic auxin-responsive promoter (DR5rev) driving red fluorescent protein. By imaging these plants, we found that all maize branching events during vegetative and reproductive development appear to be regulated by the creation of auxin response maxima through the activity of polar auxin transporters. We also found that the auxin transporter ZmPIN1a is functional, as it can rescue the polar auxin transport defects of the Arabidopsis (Arabidopsis thaliana) pin1-3 mutant. Based on this and on the groundbreaking analysis in Arabidopsis and other species, we conclude that branching mechanisms are conserved and can, in addition, explain the formation of axillary meristems (spikelet-pair and spikelet meristems) that are unique to grasses. We also found that BARREN STALK1 is required for the creation of auxin response maxima at the flanks of the inflorescence meristem, suggesting a role in the initiation of polar auxin transport for axillary meristem formation. Based on our results, we propose a general model for branching during maize inflorescence development.

The Relationship between Auxin Transport and Maize Branching 1(C)(W)(OA)

Abstract: Maize (Zea mays) plants make different types of vegetative or reproductive branches during development. Branches develop from axillary meristems produced on the flanks of the vegetative or inflorescence shoot apical meristem. Among these branches are the spikelets, short grass-specific structures, produced by determinate axillary spikelet-pair and spikelet meristems. We investigated the mechanism of branching in maize by making transgenic plants expressing a native expressed endogenous auxin efflux transporter (ZmPIN1a) fused to yellow fluorescent protein and a synthetic auxin-responsive promoter (DR5rev) driving red fluorescent protein. By imaging these plants, we found that all maize branching events during vegetative and reproductive development appear to be regulated by the creation of auxin response maxima through the activity of polar auxin transporters. We also found that the auxin transporter ZmPIN1a is functional, as it can rescue the polar auxin transport defects of the Arabidopsis (Arabidopsis thaliana) pin1-3 mutant. Based on this and on the groundbreaking analysis in Arabidopsis and other species, we conclude that branching mechanisms are conserved and can, in addition, explain the formation of axillary meristems (spikelet-pair and spikelet meristems) that are unique to grasses. We also found that BARREN STALK1 is required for the creation of auxin response maxima at the flanks of the inflorescence meristem, suggesting a role in the initiation of polar auxin transport for axillary meristem formation. Based on our results, we propose a general model for branching during maize inflorescence development. Two major stem cell systems, the shoot and root apical meristems, form the apical-basal axis of plant growth. Secondary axes of growth are established by axillary meristems that are responsible for the formation of branches and flowers. The plant hormone auxin

Renal cells activate the platelet receptor CLEC-2 through podoplanin

Abstract: We have recently shown that the C-type lectin-like receptor, CLEC-2, is expressed on platelets and that it mediates powerful platelet aggregation by the snake venom toxin rhodocytin. In addition, we have provided indirect evidence for an endogenous ligand for CLEC-2 in renal cells expressing HIV-1. This putative ligand facilitates transmission of HIV through its incorporation into the viral envelope and binding to CLEC-2 on platelets. The aim of the present study was to identify the ligand on these cells which binds to CLEC-2 on platelets. Recombinant CLEC-2 exhibits specific binding to HEK-293T (human embryonic kidney) cells in which the HIV can be grown. Furthermore, HEK-293T cells activate both platelets and CLEC-2-transfected DT-40 B-cells. The transmembrane protein podoplanin was identified on HEK-293T cells and was demonstrated to mediate both binding of HEK-293T cells to CLEC-2 and HEK-293T cell activation of CLEC-2-transfected DT-40 B-cells. Podoplanin is expressed on renal cells (podocytes). Furthermore, a direct interaction between CLEC-2 and podoplanin was confirmed using surface plasmon resonance and was shown to be independent of glycosylation of CLEC-2. The interaction has an affinity of 24.5+/-3.7 microM. The present study identifies podoplanin as a ligand for CLEC-2 on renal cells.

A Novel Gene Expression Profile in Lymphatics Associated with Tumor Growth and Nodal Metastasis

Abstract: Invasion of lymphatic vessels is a key step in the metastasis of primary tumors to draining lymph nodes. Although the process is enhanced by tumor lymphangiogenesis, it is unclear whether this is a consequence of increased lymphatic vessel number, altered lymphatic vessel properties, or both. Here we have addressed the question by comparing the RNA profiles of primary lymphatic endothelial cells (LEC) isolated from the vasculature of normal tissue and from highly metastatic T-241/vascular endothelial growth factor (VEGF)-C fibrosarcomas implanted in C57BL/6 mice. Our findings reveal significant differences in expression of some 792 genes (i.e., >or=2-fold up- or down-regulated, P

Cell traffic and the lymphatic endothelium.

Abstract: The principal immune function of the afferent lymphatics is to bear antigen and leukocytes from peripheral tissues to the draining lymph nodes. Recent research has shown that passage of leukocytes into the afferent lymphatic capillaries is far from an indolent process; rather it is carefully orchestrated by an array of adhesion molecules, as well as by chemokines and their receptors. Here we review the current knowledge of leukocyte trans-lymphatic endothelial migration and its role in the development of an immune response.

The influenza A virus spliced messenger RNA M mRNA3 is not required for viral replication in tissue culture.

Abstract: Influenza A virus genome RNA segment 7 encodes three known mRNAs, two of which, M2 mRNA and M mRNA3, are derived by alternative splicing of the primary collinear mRNA transcript using alternative 5′ splice sites. The function of M mRNA3 is currently unknown, therefore we attempted to determine whether it is essential for virus replication. Recombinant viruses unable to produce M mRNA3 and/or M2 mRNA were created by mutating the shared 3′ splice site. Growth of the mutant viruses in M2-expressing MDCK cells was not significantly affected by the lack of M mRNA3. During the course of a wild-type virus infection, levels of M mRNA3 began to decrease while those of M2 mRNA increased, which may indicate a potential mechanism of alternative splicing control. These data suggest that neither M mRNA3 nor any potential protein product are essential for influenza virus replication in tissue culture.

Dual functions of the KNOTTED1 homeodomain: sequence-specific DNA binding and regulation of cell-to-cell transport.

Abstract: Homeodomain proteins are well-characterized developmental regulators that control expression of target genes through sequence-specific DNA binding. The homeodomain forms a trihelical structure, with the third helix conferring specific interactions with the DNA major groove. A specific class of plant homeodomain proteins, called KNOX [KNOTTED1 (KN1)-like homeobox], also has the ability to signal between cells by directly trafficking through intercellular channels called plasmodesmata. Trafficking is mediated by a signal that is also contained within the homeodomain. Movement protein binding protein 2C was identified as a protein that interacts with the KN1 homeodomain and regulates the cell-to-cell trafficking of KN1 by sequestering the protein on microtubules. Therefore, KN1 has multiple potential cellular addresses, each of which is conferred by its homeodomain.

Severe sequence-specific toxicity when capecitabine is given after Fluorouracil and leucovorin.

Abstract: Purpose Options for single-agent fluoropyrimidine adjuvant therapy after bowel cancer resection include intravenous fluorouracil with leucovorin (FU/LV) or oral capecitabine. These treatments have similar efficacy but differ in convenience and toxicity. We therefore wished to compare their overall acceptability to patients. Patients and Methods Patients scheduled for adjuvant single-agent fluoropyrimidine therapy were randomly assigned to receive once-weekly FU/LV (425 mg/m2 FU, 45 mg LV) for 6 weeks, followed by two 3-week cycles of capecitabine (1,250 mg/m2 twice daily, days 1 through 14), or the same treatments but in reverse order. After 12 weeks, the patients were asked which treatment they preferred, and received the preferred treatment for an additional 12 weeks. The primary end point was patient preference. Results After 40 of the planned 74 patients had been randomly assigned, real-time adverse event monitoring led to early trial closure because of excess sequence-specific toxicity. Eleven of 14 ...

Absence of lymphatics at the bone-implant interface: implications for periprosthetic osteolysis.

Abstract: Background Wear particles, found at the bone-implant interface surrounding a loose prosthesis, are commonly phagocytosed by macrophages. Wear particles and wear particle-containing macrophages are also found in regional lymph nodes draining arthroplasty tissues. The means by which wear particles are transported from arthroplasty tissues to lymph nodes is uncertain, as the presence or absence of lymphatic vessels in periprosthetic tissues has not been established.Methods We determined immunophenotypic expression of LYVE-1 and podoplanin, two highly specific lymphatic endothelial cell markers, in the hip arthroplasty pseudocapsule surrounding the false joint and the bone-implant interface of the femoral and acetabular pseu-domembrane.Results LYVE-1+/podoplanin+ lymphatic vessels were not identified in the pseudomembrane but were found in the pseudocapsule. Normal bone did not contain lymphatic vessels.Interpretation Our findings suggest that the wear particles shed at the bone-implant interface are not tran...

Intratumoural lymphatics in benign and malignant soft tissue tumours.

Abstract: Soft tissue sarcomas do not generally metastasise via lymphatics, and the presence or absence of lymphatic vessels within sarcomas and benign soft tissue tumours is not known. In this study, we determined whether lymphatic vessels were present in a wide range of benign and malignant soft tissue lesions by examining intratumoural expression of the lymphatic endothelial cell markers, Lyve-1 and podoplanin. Intratumoural Lyve-1+/podoplanin+ lymphatics were not identified in sarcomas apart from all cases of epithelioid sarcoma (a tumour which is known to metastasise to lymph nodes) and a few cases of leiomyosarcoma, rhabdomyosarcoma and synovial sarcoma. Intratumoural lymphatics were also absent in most benign soft tissue tumours. Reparative and inflammatory soft tissue lesions contained lymphatics, as did all (pseudosarcomatous) proliferative myofibroblastic lesions including nodular, proliferative and ischaemic fasciitis, elastofibroma, nuchal fibroma and deep fibromatosis. Our results show that most soft tissue sarcomas do not contain intratumoural lymphatics, a finding which is consistent with the infrequent finding of sarcoma metastasis to lymph nodes. In contrast to fibrosarcoma and a number of other malignant spindle cell tumours, proliferative fibroblastic/myofibroblastic lesions of soft tissue contain intralesional lymphatic vessels.

Optical system with potential for remote health monitoring of subsea machinery

Abstract: A prototype fibre optic sensing system is described with potential to remotely monitor the condition of 3 phase variable frequency sub-sea motors and electric submersible pumps. An indication that the integrity of a powerful electric motor may be compromised can be gained by spectral analysis of the stators drive current, the relative phases of the currents, the measurement of vibration at specific locations on the motor and the temperature of the bearings. The optical interrogation system is based on an imbalanced Mach Zehnder fibre interferometer, illuminated with a broad band source with FBG based current and vibration sensors. Signals from sensors operating at an effective distance of 7 km have been demonstrated.

High temperature sensors exploiting low coherence signal recovery

Abstract: High temperature probes are described, one with an operating temperature ≥1000°C based on a sapphire etalon interrogated by low coherence interferometry, the second a miniature Fabry-Perot maximum operational temperature 700°C interrogated by a tuneable laser capable of linear and sinusoidal scan rates of 2 kHz and 20kHz repectively.

High temperature Fabry-Perot probe interrogated with tunable fibre ring laser

Abstract: A high temperature fibre-optic based probe is described based on a miniature Fabry-Perot with a maximum operating temperature of ~700degC limited by the gold coated fibre transceiver link connecting the optical source to the probe. It was interrogated by a tunable fibre laser which could be linearly or sinusoidally tuned at scanning rates of 2 and 20 kHz, respectively. The transfer function of the system is in the form of channelled spectra rather than the usual interference fringes observed for interferometric sensors. The periodicity of the channelled spectra fringes is governed by the Fabry-Perot free spectral range which is related to the inverse of the probe temperature.

A swept source OCT at 1300 nm with angular compounding for art and archaeological conservation

Abstract: We have assembled a swept source optical coherence tomography (OCT) system for use in Art & Archaeological Conservation. The system is illuminated by an in-house swept source with a central wavelength of 1300 nm. This system includes dedicated optics to allow for angular averaging for speckle noise reduction. We demonstrate that the averaging produces significant improvements in speckle SNR in art and archaeological type objects and allows easier identification of features. We further demonstrate that there is practically no loss of axial resolution.

High temperature fibre optic based sensor interrogated by a fast tuneable ring laser

Abstract: A High temperature probe, based on a miniature fibre optic Fabry-Perot, operational temperature 700degC set by the maximum temperature of the gold plated optical fibre transceiver link used is presented The probe is interrogated by a tuneable laser at 1300 nm, line width ~90 nm, with a second stage booster amplifier giving a maximum quasi-static output power of ~27 mW Scanning rates of 1 kHz and 20 kHz for linear and sinusoidal scans respectively