Immobilization of foreign protein into polyhedra of Bombyx mori nucleopolyhedrovirus (BmNPV).

In the late phase of Bombyx mori nucleopolyhedrovirus (BmNPV) infection, a large amount of polyhedra appear in the infected cell nucleolus, these polyhedra being dense protein crystals protecting the incorporated virions from the harsh environment. To investigate whether the foreign protein could be immobilized into the polyhedra of BmNPV, two recombinant baculoviruses were generated by a novel BmNPV polyhedrin-plus (polh(+)) Bac-to-Bac system, designated as vBmBac(polh(+))-enhanced green fluorescent protein (EGFP) and vBmBac(polh(+))-LacZ, which can express the polyhedrin and foreign protein simultaneously. Light microscopy analysis showed that all viruses produced polyhedra of normal appearance. Green fluorescence can be apparently detected on the surface of the vBmBac(polh(+))-EGFP polyhedra, but not the BmNPV polyhedra. Fluorescence analysis and anti-desiccation testing confirmed that EGFP was embedded in the polyhedra. As expected, the vBmBac(polh(+))-LacZ polyhedra contained an amount of LacZ and had a higher ß-galactosidase activity. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting were also performed to verify if the foreign proteins were immobilized into polyhedra. This study provides a new inspiration for efficient preservation of useful proteins and development of new pesticides with toxic proteins.

Ghrelin modulates insulin sensitivity and tau phosphorylation in high glucose-induced hippocampal neurons.

Ghrelin, a 28-amino acid brain-gut peptide expressed in periphery tissues and the central nervous system, has been demonstrated to increase insulin sensitivity in adipocytes. Recent data have indicated that insulin resistance exists in the brain and is related to Alzheimer's Disease (AD). The aim of this study was to investigate whether ghrelin increased high glucose-induced hippocampal neuron insulin sensitivity, and further modulated tau phosphorylation. Hippocampal neurons were cultured in concentrations of 25 mM and 75 mM glucose. The effect of ghrelin on hippocampal neuronal insulin sensitivity was detected by [(3)H]-2-deoxy-D-glucose uptake. The expression of Akt, glycogen synthase kinase-3beta (GSK-3beta) and tau phosphorylation was determined via Western blotting. Culturation in 75 mM glucose resulted in decreased neuronal glucose uptake and an increase in the level of tau phosphorylation at Ser 199. In neurons treated with ghrelin for 1 h, neuronal glucose uptake was increased and tau hyperphosphorylation was improved. Ghrelin activated Akt and GSK-3beta phosphorylation, whereas phosphatidylinositol 3-kinase (PI3-K) inhibitor wortmannin eliminated ghrelin's effect on neuronal glucose uptake and tau phosphorylation. This study demonstrated that ghrelin increased insulin-stimulated neuronal glucose uptake in 25 mM or 75 mM glucose, raised insulin sensitivity, improved insulin resistance and decreased tau abnormal phosphorylation via the PI3-K/Akt-GSK pathway. Ghrelin is a potential new medicine in the treatment of AD.

[A novel application of baculovirus in mammalian gene therapy].

Baculovirus has been widely used for the production of recombinant proteins in insect cells. The extremely high yield by baculovirus-infected insect cells or larvae makes it an attractive tool for pharmaceutical protein production. Since the finding that baculovirus can efficiently transduce mammalian cells, the applications of baculovirus have been greatly expanded. Although AcMNPV (Autographa californica multiple NPV) fails to replicate in vertebrate cells, it does express alien genes with levels of expression that are dependent on the strength of the promoter used to drive transcription of the foreign gene. Following these findings, subsequent studies have rapidly expanded the list of permissive cells that include cell lines originating from human, rodent, porcine, bovine and even fish sources. Many tries have been done to study the mechanism of baxulovirus entry into mammalian cells, but the events responsible for virus uptake and detailed mechanisms of intracellular movement and nuclear entry of the virus are still largely unknown. The application of modified baculoviruses for in vivo gene delivery has also been demonstrated. In contrast to other commonly used viral vectors, baculoviruses have the unique property of being incapable of initiating a replication cycle and producing infectious virus in mammalian cells. The AcMNPV genome is large, thus rendering the virus flexibility to carry multiple genes or large inserts. The recombinant viruses can be readily constructed and produced to high titers simply by infecting insect cells, initiate little to none microscopically observable cytopathic effects on mammalian cells and have a good biosafety profile. These attributes will undoubtedly lead to the increased application and continued development of this system for efficient gene delivery into mammalian cells.

Genetic variation at AFLPs for the Dipterocarpaceae and its relation to molecular phylogenies and taxonomic subdivisions.

Genetic differentiation was investigated among 54 Indonesian species of Dipterocarpaceae, a dominant tree family in Asian tropical rainforests, using amplified fragment length polymorphism markers. The tree developed from the resultant unweighted pair group method using arithmetic averages clearly separated all investigated dipterocarps into two major groups that corresponded to tribe Dipterocarpeae and tribe Shoreae, respectively. These results are in accordance with the topology of molecular phylogenetic trees derived from PCR-restriction fragment length polymorphism analysis of chloroplast DNA and generally support the traditional taxonomic assessments. The possibility of interspecific hybridization is also discussed.

[The inhibitory effect of vascular endothelial growth factor antisense oligonucleotides in angiogenesis of thyroid carcinoma].

OBJECTIVE: To investigate the inhibitive effect of antisense oligonucleotide (ASODN) on vascular endothelial growth factor (VEGF) expression and endothelial cell growth in thyroid carcinoma. METHODS: Targeted ASODN of VEGF was designed and synthesized, then transfected to TT (medullary thyroid carcinoma) cell line and the culture supernatant was collected in which ECV304 (endothelial cell line) was seeded. At the same time positive control [sense oligonucleotides (SODN) group] and normal control were set for comparison. Cell growth condition was observed under microscope. RT-PCR and immuocytochemistry were used for detection of VEGF mRNA and protein expression in TT cells. MTT assay was used for cell growth inhibition ratio (IR) of TT and ECV304 cells, flow cytometry (FCM) for apoptotic index (AI) of ECV304 cells and acridine orange/ethidium bromide (AO/EB) staining for apoptotic morphology of ECV304 cells. RESULTS: As compared with positive and normal control groups, VEGF mRNA and protein expressions in TT cells of ASODN transfection groups were obviously decreased (P < 0.01). Cell growth was not influenced apparently in ECV304 cells with direct ASODN administration, but ECV304 cell growth in ASODN conditioned medium was significantly inhibited and IR (0.21 +/- 0.03, 0.31 +/- 0.01, 0.42 +/- 0.22) was significantly higher than that of SODN group (0.05 +/- 0.03, P < 0.01), with the presence of apparent apoptosis. The effect mentioned above was in a dose-dependent manner. CONCLUSION: ASODN can suppress endothelial cell growth and inhibit tumor angiogenesis possibly by specifically blocking VEGF expression in thyroid carcinoma.

Targeting of human aFGF gene into silkworm, Bombyx mori L. through homologous recombination.

The long-arm and short-arm genes of fibroin light chain (L-chain) of silkworm, Bombyx Mori L., and the gene of human acidic fibroblast growth factor were cloned respectively and subsequently inserted into a transfer vector pVL1392 used as a tool to target the L-chain region of the silkworm genome. Genomic DNA from their offsprings was extracted and the expected targeting was detected using polymerase chain reaction and DNA sequencing, as well as protein analysis. The results showed that positive events occurred and that the FGF gene was integrated into the L-chain locus through homologous recombination.