[Detection of microvasculature in rat hind limb using synchrotron radiation].

OBJECTIVE: To detect deep-level microvascular structure in rat hind limb by synchrotron radiation and microangiographic technique. METHODS: Microangiography in vivo and ex vivo was performed by synchrotron radiation based absorption imaging and phase contrast imaging, with omnipaque and barium sulfate solution as contrast media, respectively, and synchrotron radiation-based micro-computed tomography (SRmCT) was also performed to reveal three-dimensional morphology of the blood vessel in rat hind limb. RESULTS: Using microangiographic technique in vivo and in vitro (with barium sulfate), blood vessels in the rat limb muscle could be visualized with high resolution, and the fourth branches of iliac artery in rat hind limb could be detected with the minimum visualized blood vessels about 40 µm and 9 µm in diameter, respectively. In addition, the vascular network could be defined and analyzed at the micrometer scale from the 3D renderings of limb vessel as shown by SRmCT. CONCLUSION: Synchrotron radiation-based microangiography and SRmCT thus provided a practical and effective means to observe the microvasculature of rat hindlimb, which might be useful in assessment of angiogenesis in lower limbs.

Functional endothelial cells derived from embryonic stem cells labeled with HIV transactivator peptide-conjugated superparamagnetic nanoparticles.

BACKGROUND: The development of regenerative therapies using derivatives of embryonic stem (ES) cells would be facilitated by a non-invasive method to monitor transplanted cells in vivo, for example, magnetic resonance imaging of cells labeled with superparamagnetic iron oxide (SPIO) nanoparticles. Although ES cells have been labeled with SPIO particles, the potential adverse effects of the label have not been fully examined. The objective of this study was to determine whether SPIO labeling affects murine ES cell viability, proliferation, or ability to differentiate into functional endothelial cells (ECs). METHODS: Cross-linked iron oxide (CLIO, an SPIO) was conjugated with human immunodeficiency virus transactivator of transcription (HIV-Tat) peptides, and murine ES cells were labeled with either CLIO-Tat, CLIO, or HIV-Tat. After labeling, ES cells were cultured for 4 days and Flk-1(+) ES cells identified and sorted by immunocytochemistry and fluorescence activated cell sorting (FACS). Flk-1(+) cells were replated on fibronectin-coated dishes, and ECs were obtained by culturing these for 4 weeks in endothelial cell growth medium supplemented with vascular endothelial growth factor (VEGF). ES cell viability was determined using trypan blue exclusion, and the proportion of SPIO(+) cells was evaluated using Prussian blue staining and transmission electron microscopy. After differentiation, the behavior and phenotype of ECs were analyzed by reverse transcription-polymerase chain reaction, flow cytometry, immunocytochemistry, DiI-labeled acetylated low-density lipoprotein (AcLDL) uptake, and Matrigel tube formation assay. RESULTS: CLIO-Tat was a highly effective label for ES cells, with > 96% of cells incorporating the particles, and it did not alter the viability of the labeled cells. ECs derived from CLIO-Tat(+) ES cells were very similar to murine aortic ECs in their morphology, expression of endothelial cell markers, ability to form vascular-like channels, and scavenging of AcLDL from the culture medium. CONCLUSIONS: CLIO-Tat is a highly effective label for ES cells and does not adversely affect cell viability, differentiation, or behavior. CLIO-Tat could be a useful marker for the non-invasive monitoring of transplanted stem cells.

Polymorphisms in MMP-9 and TIMP-2 in Chinese patients with varicose veins.

BACKGROUND: Varicose veins (VVs), a common vascular disease, are functionally characterized by dilation and tortuosity and are widely prevalent in the adult population. The pathophysiology and molecular mechanism of VVs are still unclear. A genetic risk for VVs has been demonstrated, although no genetic variant pertaining to VVs has been identified. Matrix metalloproteinases (MMPs) and their endogenous tissue inhibitors (TIMPs), which can prevent excessive extracellular matrix (ECM) degradation, greatly impact vascular remodeling and may play a vital role in patients with VVs. We evaluated a potential association between polymorphisms in the promoters of MMP-9 and TIMP-2 and the risk for VVs in the Chinese population. MATERIALS AND METHODS: Genotyping of the promoter region polymorphisms -1562C/T in MMP-9 and -418G/C in TIMP-2 was performed with PCR and restriction fragment length polymorphism (PCR-RFLP) assays with a group of 60 patients with VVs and 60 healthy controls. Purified PCR products were sequenced. RESULTS: A significant correlation was found between patients with VVs and controls at -1562C/T in MMP-9. The TIMP-2 gene polymorphism -418G/C was also associated with VVs. CONCLUSIONS: Our results suggest that polymorphisms in the promoter region of MMP-9 and TIMP-2 are associated with VVs in the Chinese population.

A novel economic method for high throughput production of recombinant baculovirus by infecting insect cells with Bacmid-containing diminopimelate-auxotrophic Escherichia coli.

Developing cost-effective methods for high throughput production of recombinant baculoviruses in insect cells is very challenging, because the baculovirus DNA preparation and the following transfection procedure are labour-intensive and time consuming. We developed a new method of introducing recombinant Bacmid DNA from bacteria into insect cells simply using invasive diaminopimelate (DAP) auxotrophic Escherichia coli to infectSpodoptera frugiperda 9 cells. The E. coli cells with recombinant Bacmids enter insect cells with the help of the invasion factor from Yersinia pseudotubercolusis. Without DAP in medium, the cell wall of DAP auxotrophic E. coli cannot be synthesized so that the bacterial cell will disrupt and release recombinant Bacmid. The released Bacmids will generate infective recombinant baculovirus particles in insect cells. We combined this E. coli invasion method with the zero background transposition system to generate recombinant baculovirus in a rapid and simple way. Without preparation and purification of recombinant Bacmids from E. coli and the labour-intensive and complex transfection procedure, this transfection reagent free method enables a convenient and economic high throughput production of recombinant baculoviruses.

[Screening for peptides of anti-rotavirus by phage-displayed technique].

In this study, a 15-mer phage display peptide library was employed to pan against human rotavirus immobilized on solid phase. 4 different peptides were selected and could bind with rotavirus particles specifically. Plaque reduction neutralization test and MTT analysis results indicated that 3 of the peptides can inhibit rotavirus infecting in vitro. A peptide which sequence is QSNPIHIITNTRNHP showed the best efficiency--93% neutralization infectivity. Two other peptides, A and B, showed 40% and 50% neutralization infectivity respectively. Amino sequence analysis results indicate the 3 peptides containing 2 conserved motifs: SNPIHII and NIP. No putative trypsin hydrolysis site was found in C peptide, however, 4 and 3 potential sites were found in A and B peptides respectively. Using trypsin inhibitor, both A and B peptides showed the similar antiviral effect as that of C peptide. It suggests that the intactness of the 2 conserved motifs play an important role in counteracting virus infection. According to the results of this study, peptide C is hopeful to be exploited as an antiviral peptide drug.

A highly efficient method for the generation of a recombinant Bombyx mori nuclear-polyhedrosis-virus Bacmid and large-scale expression of foreign proteins in silkworm (B. mori) larvae.

In the post-genomic era, one of the challenges and a source of competition is the development of high-throughput, large-scale and low-cost eukaryotic cDNA cloning and expression systems. The baculovirus expression system is the most popular one and plays an important role in the high-level expression of eukaryotic proteins. In the present study, a convenient, rapid and highly efficient method for the construction of recombinant BmNPV (Bombyx mori nuclear polyhedrosis virus)-Bacmid vector (BmBacmid) for low-cost protein expression in silkworm (B. mori) larvae was established by using the MAGIC (mating-assisted genetically integrated cloning) strategy. By simply mixing the donor bacteria strain containing the constructed donor vector pCTdual harbouring foreign genes and the recipient strain containing modified BmBacmid, 99.8% positive recombinant BmNPV-Bacmids were obtained. Reporter genes egfp (enhanced green fluorescent protein gene) and DsRed (Discosoma sp. red fluorescent protein gene) and target gene man (beta-mannanase gene) encoding beta-mannanase were expressed in the silkworm larvae of B. mori at high level by injection of recombinant BmBacmid DNA directly with the standard calcium phosphate transfection procedure. The possibility of constructing a high-quality baculovirus cDNA library by transferring an ordinal plasmid cDNA library into the recipient BmBacmid in Escherichia coli was explored.