Ruowen Ge

Crystal structure of an avian influenza polymerase PA(N) reveals an endonuclease active site

The heterotrimeric influenza virus polymerase, containing the PA, PB1 and PB2 proteins, catalyses viral RNA replication and transcription in the nucleus of infected cells. PB1 holds the polymerase active site and reportedly harbours endonuclease activity, whereas PB2 is responsible for cap binding. The PA amino terminus is understood to be the major functional part of the PA protein and has been implicated in several roles, including endonuclease and protease activities as well as viral RNA/complementary RNA promoter binding. Here we report the 2.2 ångström (Å) crystal structure of the N-terminal 197 residues of PA, termed PAN, from an avian influenza H5N1 virus. The PAN structure has an α/β architecture and reveals a bound magnesium ion coordinated by a motif similar to the (P)DXN(D/E)XK motif characteristic of many endonucleases. Structural comparisons and mutagenesis analysis of the motif identified in PAN provide further evidence that PAN holds an endonuclease active site. Furthermore, functional analysis with in vivo ribonucleoprotein reconstitution and direct in vitro endonuclease assays strongly suggest that PAN holds the endonuclease active site and has critical roles in endonuclease activity of the influenza virus polymerase, rather than PB1. The high conservation of this endonuclease active site among influenza strains indicates that PAN is an important target for the design of new anti-influenza therapeutics.

Crystal structure of the polymerase PA C –PB1 N complex from an avian influenza H5N1 virus

The recent emergence of highly pathogenic avian influenza A virus strains with subtype H5N1 pose a global threat to human health. Elucidation of the underlying mechanisms of viral replication is critical for development of anti-influenza virus drugs. The influenza RNA-dependent RNA polymerase (RdRp) heterotrimer has crucial roles in viral RNA replication and transcription. It contains three proteins: PA, PB1 and PB2. PB1 harbours polymerase and endonuclease activities and PB2 is responsible for cap binding; PA is implicated in RNA replication and proteolytic activity, although its function is less clearly defined. Here we report the 2.9 ångström structure of avian H5N1 influenza A virus PA (PAC, residues 257–716) in complex with the PA-binding region of PB1 (PB1N, residues 1–25). PAC has a fold resembling a dragon’s head with PB1N clamped into its open ‘jaws’. PB1N is a known inhibitor that blocks assembly of the polymerase heterotrimer and abolishes viral replication. Our structure provides details for the binding of PB1N to PAC at the atomic level, demonstrating a potential target for novel anti-influenza therapeutics. We also discuss a potential nucleotide binding site and the roles of some known residues involved in polymerase activity. Furthermore, to explore the role of PA in viral replication and transcription, we propose a model for the influenza RdRp heterotrimer by comparing PAC with the λ3 reovirus polymerase structure, and docking the PAC structure into an available low resolution electron microscopy map.

The role of Vascular Endothelial Growth Factor (VEGF) in Vasculogenesis, Angiogenesis, and Hematopoiesis in Zebrafish development

Vascular endothelial growth factor (VEGF, VEGF-A), a selective mitogen for endothelial cells is a critical factor for vascular development. Two isoforms that differ in the presence of exons 6 and 7, Vegf(165) and Vegf(121), are the dominant forms expressed in zebrafish embryo. Simultaneous overexpression of both isoforms in the embryo results in increased production of flk1, tie1, scl, and gata1 transcripts, indicating a stimulation of both endothelial and hematopoietic lineages. We also demonstrate that vegf can stimulate hematopoiesis in zebrafish by promoting the formation of terminally differentiated red blood cells. Simultaneous overexpression of both isoforms also causes ectopic vasculature and blood cells in many of the injected embryos as well as pericardial edema in later stage embryos. Overexpression of vegf also resulted in earlier onset of flk1, tie1, scl, and gata1 expression in the embryo, indicating a possible role of vegf in stimulating the differentiation of both vascular and hematopoietic lineages. Co-injection of RNAs for both isoforms results in increased expression of three of these markers over and above that observed when either RNA is singly injected and analysis of vegf expression in the notochord mutants no tail and floating head suggests that the notochord patterns the formation of the dorsal aorta by stimulating adjacent somite cells to express vegf, which in turn functions as a signal in dorsal aorta patterning. Finally, studies of vegf expression in cloche mutant indicate that vegf expression is generally independent of cloche function. These results show that in the zebrafish embryo, vegf can not only stimulate endothelial cell differentiation but also hematopoiesis. Moreover, these effects are most dramatic when both vegf isoforms are co-expressed, indicating a synergistic effect of the expression of the two forms of the VEGF protein.

CLONING AND CHARACTERIZATION OF VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) FROM ZEBRAFISH, DANIO RERIO

We have cloned and sequenced a zebrafish (Danio rerio) Vascular Endothelial Growth Factor (vegf) cDNA. It encodes a precursor protein of 188 amino acids with a putative 23 amino acids signal peptide. Sequence comparison analysis indicates that the zebrafish vegf cDNA corresponds to the human VEGF165 isoform and shows about 52% identity to human VEGF165 at the amino acid level. A 2.8 kb vegf message RNA was detected in adult zebrafish by Northern blot analysis. Expression of vegf165 is also detected by RT-PCR in adult fish and throughout the zebrafish embryonic development. Whole mount in situ hybridization of zebrafish embryos indicates strong expression in four areas of the 18-19 h post-fertilization (hpf) embryo: within the anterior central nervous system in the prospective optic stalk, in mesoderm overlapping the bilaterally located merging heart fields, in mesoderm underlying and flanking the hindbrain posterior to rhombomere 4, and in medial regions of the somites. The study of vegf function in zebrafish embryonic vascular development will contribute to our understanding of the mechanisms of vertebrate endothelial cell differentiation and vasculature formation.

A micromanipulation system with dynamic force-feedback for automatic batch microinjection

In this paper, we report the development of a prototype micromanipulation system for automatic batch microinjection of zebrafish embryos. Such automatic batch processing is made possible by (i) the development of a machine vision algorithm to identify the number of embryos in a batch and to locate the centerline of each embryo, (ii) the integration of a piezoresistive micro-force sensor with a micropipette to measure the penetration force of the embryo in real time and (iii) the synthesis of a position control with dynamic force feedback by exploiting the characteristics of the force profile associated with the microinjection process. The effectiveness of this prototype micromanipulation system has been demonstrated in an experiment. The experimental results demonstrate that the technique of position control with dynamic penetration-force feedback is practicable for automatic batch microinjection applications.

Histone deacetylase 3 (hdac3) is specifically required for liver development in zebrafish

Histone deacetylases (HDACs) are key transcription regulators that function by deacetylating histones/transcription factors and modifying chromatin structure. In this work, we showed that chemical inhibition of HDACs by valproic acid (VPA) led to impaired liver development in zebrafish mainly by inhibiting specification, budding, and differentiation. Formation of exocrine pancreas but not endocrine pancreas was also inhibited. The liver defects induced by VPA correlate with suppressed total HDAC enzymatic activity, but are independent of angiogenesis inhibition. Gene knockdown by morpholino demonstrated that hdac3 is specifically required for liver formation while hdac1 is more globally required for multiple development processes in zebrafish including liver/exocrine pancreas formation. Furthermore, overexpression of hdac3 but not hdac1 partially rescued VPA induced small liver. One mechanism by which hdac3 regulates zebrafish liver growth is through inhibiting growth differentiation factor 11 (gdf11), a unique target of hdac3 and a member of the transforming growth factor beta family. Simultaneous overexpression or morpholino knockdown showed that hdac3 and gdf11 function antagonistically in zebrafish liver development. These results revealed a novel and specific role of hdac3 in liver development and the distinct functions between hdac1 and hdac3 in zebrafish embryonic development.

Peptides derived from human decorin leucine-rich repeat 5 inhibit angiogenesis.

Excessive angiogenesis is involved in many human diseases, and inhibiting angiogenesis is an important area of drug development. There have been conflicting reports as to whether decorin could function as an angiogenic inhibitor when used as an extracellular soluble factor. In this study, we demonstrated that not only purified decorin but also the 26-residue leucine-rich repeat 5 (LRR5) of decorin core protein functions as angiogenesis inhibitor by inhibiting both vascular endothelial growth factor (VEGF) and basic fibroblast growth factor-induced angiogenesis. Peptide LRR5 inhibited angiogenesis through multiple mechanisms, including inhibiting VEGF-stimulated endothelial cell (EC) migration, tube formation on Matrigel, cell attachment to fibronectin, as well as induction of EC apoptosis without significantly affecting their proliferation. We further demonstrated that different subregions of LRR5 inhibited different aspects of angiogenesis, with the middle region (LRR5M, 12 residues) inhibiting endothelial cell tube formation up to 1000 times more potently than LRR5. Although the C-terminal region (LRR5C) potently inhibited VEGF-stimulated endothelial cell migration, the N-terminal region (LRR5N) is as active as LRR5 in inhibiting endothelial cell attachment to fibronectin. Although both LRR5M and LRR5N induced EC apoptosis dose-dependently similar to LRR5 through a caspase-dependent pathway, LRR5C has no such function. We further showed that the inhibition of tube formation by LRR5 and LRR5M is linked with their ability to suppress VEGF-induced focal adhesion kinase phosphorylation and the assembly of focal adhesions and actin stress fibers in ECs, but not their ability to interfere with endothelial cell attachment to the matrix. Circular dichroism studies revealed that LRR5 undergoes an inter-conversion between 310 helix and β-sheet structure in solution, a characteristic potentially important for its anti-angiogenic activity. Peptide LRR5 and its derivatives are therefore novel angiogenesis inhibitors that may serve as prototypes for further development into anti-angiogenic drugs.

Transplantation of Nanoparticle Transfected Skeletal Myoblasts Overexpressing Vascular Endothelial Growth Factor-165 for Cardiac Repair

Background— We investigated the feasibility and efficacy of polyethylenimine (PEI) based human vascular endothelial growth factor-165 (hVEGF165) gene transfer into human skeletal myoblasts (HSM) for cell based delivery to the infarcted myocardium. Methods and Results— Based on optimized transfection procedure using enhanced green fluorescent protein (pEGFP), HSM were transfected with plasmid-hVEGF165 (phVEGF165) carried by PEI (PEI- phVEGF165) nanoparticles. The transfected HSM were characterized for transfection and expression of hVEGF165 in vitro and transplanted into rat heart model of acute myocardial infarction (AMI): group-1=DMEM injection, group-2= HSM transplantation, group-3= PEI-phVEGF165–transfected HSM (PEI-phVEGF165 myoblast) transplantation. A total of 48 rats received cyclosporine injection from 3 days before and until 4 weeks after cell transplantation. Echocardiography was performed to assess the heart function. Animals were sacrificed for molecular and histological studies on the heart tissue at 4 weeks after treatment. Based on optimized transfection conditions, transfected HSM expressed hVEGF165 for 18 days with >90% cell viability in vitro. Apoptotic index was reduced in group-2 and group-3 as compared with group-1. Blood vessel density (×400) by immunostaining for PECAM-1 in group-3 was significantly higher (P=0.043 for both) as compared with group-1 and group-2 at 4 weeks. Regional blood flow (ml/min/g) in the left ventricular anterior wall was higher in group-3 (P=0.043 for both) as compared with group-1 and group-2. Improved ejection fraction was achieved in group-3 (58.44±4.92%) as compared with group-1 (P=0.004). Conclusion— PEI nanoparticle mediated hVEGF165 gene transfer into HSM is feasible and safe. It may serve as a novel and efficient alternative for angiomyogenesis in cardiac repair.

fgfr3 and regionalization of anterior neural tube in zebrafish.

Here we describe the isolation of the zebrafish fgfr3 gene, its structure and chromosomal location. Expression in wild type embryos occurs in the axial mesoderm, the diencephalon, the anterior hindbrain and the anterior spinal cord. In the hindbrain, a differential expression of fgfr3 was detected at several levels of intensity, with the highest expression in the posterior rhombomere 1 that is morphologically distinct from the anterior part, which develops into the cerebellum. Further, analysis of fgfr3 expression in mutants deficient in the formation of the midbrain-hindbrain boundary (MHB), noi(-/-) and ace(-/-), demonstrated that in the absence of Pax2.1 and FGF8 activity, the expression domains of FGFR3 expand into the MHB, tegmentum, cerebellum and optic tectum, which are the affected structures in these mutants.

Inhibition of angiopoietin-1 expression in tumor cells by an antisense RNA approach inhibited xenograft tumor growth in immunodeficient mice.

Angiopoietin‐1 (Ang1) is an angiogenic growth factor that functions through activation of its endothelium‐specific tyrosine kinase receptor Tie2; it mediates the interaction between endothelial and surrounding cells to promote the remodeling, maturation and stabilization of blood vessels. Although Ang1 is expressed constitutively in many adult tissues, its role in tumor growth and metastasis is not clear. Here we describe experiments in which Ang1 expression was inhibited in HeLa cells by an antisense RNA approach. The modified HeLa cells produced significantly less Ang1 protein both in cultured cells and in tumors formed when these cells were injected into immunodeficient mice. The Ang1 antisense tumors grew much more slowly, with significantly reduced tumor angiogenesis compared with control tumors. Furthermore, they also had substantially increased tumor cell apoptosis and decreased tumor necrosis. Our results indicate that the perturbation of Ang1 expression in tumors could be an effective method to control tumor growth by inhibiting tumor angiogenesis and that antisense RNA is an efficient way to inhibit Ang1 protein production in tumor cells.