Dual function of a bumblebee (Bombus ignitus) serine protease inhibitor that acts as a microbicidal peptide and anti-fibrinolytic venom toxin.

In bee venoms, low-molecular-weight peptides, including serine protease inhibitors (SPIs), exhibit multifunctional activities. Although SPIs in bee venoms are relatively well known, those that function in both the body and secreted venom of bees are not well-characterized. In this study, we identified a bumblebee (Bombus ignitus) SPI (BiSPI) that displays microbicidal and anti-fibrinolytic activities. BiSPI was found to consist of a trypsin inhibitor-like domain containing a P1 site and ten cysteine residues. We observed that the BiSPI gene was ubiquitously transcribed in the body, including the venom glands. In correlation, the BiSPI protein was detected both in the body and secreted venom by using an antibody against a recombinant BiSPI peptide produced in baculovirus-infected insect cells. Recombinant BiSPI exhibited inhibitory activity against trypsin but not chymotrypsin and inhibited microbial serine proteases and plasmin but not elastase or thrombin. Moreover, recombinant BiSPI recognized carbohydrates and bound to fungi and gram-negative and gram-positive bacteria. Consistent with these properties, recombinant BiSPI exhibited microbicidal activities against bacteria and fungi through induction of structural damage by binding to the microbial surfaces. Additionally, recombinant BiSPI inhibited the plasmin-mediated degradation of human fibrin and was thus concluded to exhibit anti-fibrinolytic activity. Moreover, the peptide showed no effect on hemolysis. These findings demonstrate the dual function of BiSPI, which acts as a microbicidal peptide and anti-fibrinolytic venom toxin.

Real-time in vivo monitoring of viable stem cells implanted on biocompatible scaffolds.

PURPOSE: Three-dimensional fibrous scaffolds provide an environment that enhances transplanted stem cell survival in vivo and facilitates imaging their localization, viability, and growth in vivo. To assess transplanted stem cell viability on biocompatible polymer scaffolds in vivo, we developed in vivo imaging systems for evaluation of implanted viable neural stem cells (NSC) and mesenchymal stem cells (MSC) on scaffolds using luciferase or sodium/iodide symporter (NIS) genes. METHODS: Firefly luciferase stably expressing-C6 cell was established (C6-Fluc). The human neural stem cell, F3, was infected with adenoviral vector carrying luciferase gene (F3-Fluc) and MSC expressing NIS controlled by ubiquitin C promoter using lentiviral vector was established by treating blasticidine for 2 weeks (MSC-NIS). Chitosan and poly L-lactic acid (PLLA) scaffolds were used for in vivo image. In vivo expression of luciferase and human NIS was examined by bioluminescence image or (99m)Tc-pertechnetate gamma camera image, respectively. The cell/scaffold complex was implanted into subcutaneous or abdominal area of BALB/C nude mouse. For quantitative evaluation of cell viability, regions of interest were drawn on (99m)Tc-pertechnetate scintigraphy by manual. RESULTS: The gradual increase of luciferase activity was observed in C6-Fluc seeded with chitosan according to the increase in the number of cells. C6-Fluc/chitosan complex subcutaneously implanted into nude mice showed longitudinal bioluminescence image until 34 days. Luciferase image of abdominal-injected C6-Fluc/PLLA complex was saturated in only 14 days, showing great cell growth due to abundant nutrients. F3 cells showed well-incorporated pattern with fibrous chitosan scaffold using scanning electron microscopy. F3 infected with Ad-Fluc showed >100-fold higher luciferase activity than luciferase activity in F3. Cell-number-dependent increase of luciferase activity was shown in F3-Fluc seeded on chitosan. F3-Fluc incorporation into chitosan after abdominal injection was clearly visible on bioluminescence image up to 11 days. Radionuclide imaging showed higher uptake by MSC-NIS on PLLA scaffolds than by MSC-NIS not seeded on a scaffold. Quantitative data showed significantly better survival of MSC-NIS on PLLA scaffolds than without scaffold at 72 h post-implantation, which concurred with histologic findings. CONCLUSION: These results suggest that NSC-Fluc and MSC-NIS cells incorporated within polymer scaffolds can be monitored on a long-term basis by serial in vivo imaging. We believe that a biocompatible scaffold-based imaging system could be used to assess stem cell viabilities in a non-invasive way to aid the development of regenerative therapeutics.

Development of a sodium/iodide symporter (NIS)-transgenic mouse for imaging of cardiomyocyte-specific reporter gene expression.

UNLABELLED: Development of a small animal imaging system for differentiated cell-specific reporter gene expression will enable us to image cellular differentiation in vivo. In this study, we developed a sodium/iodide symporter (NIS)-transgenic mouse in which NIS is constitutively expressed as an imaging reporter gene only in cardiomyocytes. METHODS: To express NIS gene in cardiomyocytes, alpha-myosin heavy chain (alpha-MHC)-NIS was constructed and used for the production of NIS-transgenic mice. Twelve lines of positive founder were obtained. The adequacy of the transgenic mouse model was tested by in vivo scintigraphy, microPET, and a biodistribution study. RESULTS: The myocardium of transgenic mice showed rapid and intense uptake of 131I, which was much higher than that of the thyroid, and also showed long retention by gamma-camera pinhole imaging. The relative uptake ratio of the heart of transgenic mice was 4.6 +/- 1.5, which was 3.8 +/- 1.2 times higher than that of control wild-type mice. The uptake of the heart was completely blocked by oral administration of KClO4, an NIS inhibitor. The heart of transgenic mouse was also clearly and intensely visualized on microPET using 124I. Biodistribution data of these mice showed the uptake of 40-160 %ID/g (percentage injected dose per gram of tissue) of (99m)Tc-pertechnetate in the heart compared with 40-60 %ID/g in the stomach, respectively. NIS expression in the myocardium was confirmed by immunohistochemistry using a NIS-specific antibody. CONCLUSION: We developed a transgenic mouse model to image cardiomyocytes with a gamma-camera and microPET using an alpha-MHC promoter and NIS. The transgenic mouse can be used as an imaging model for cardiomyocyte-specific reporter gene expression and cellular differentiation into cardiomyocytes after cardiac stem or progenitor cell transplantation.

Reversing the silencing of reporter sodium/iodide symporter transgene for stem cell tracking.

UNLABELLED: To track neural stem cells transfected with reporter gene, perpetual stem cell transgene expression is required. Referring to the knowledge about epigenetic modulation, we succeeded in reversing the silencing of sodium/sodide symporter (hNIS) transgenes transfected into human neural stem (HB1.F3) cells. METHODS: hNIS and hygromycin resistance gene were linked with IRES (Internal Ribosome Entry Site) under control of cytomegalovirus promoter, and this construct was transfected into HB1.F3 cells to yield the F3-NIS cell lines. hNIS transgene expression was examined by (125)I uptake and reverse transcriptase polymerase chain reaction (RT-PCR). The iodide uptake of F3-NIS III cells was initially higher by up to 12.9-fold than that of nontransfected HB1.F3 cells. However, repeated passage gradually silenced hNIS expression. The recovery of hNIS transgene expression by demethylating agent (5-azacytidine) or histone deacetylase inhibitor (trichostatin A; TSA) treatment was investigated. RESULTS: As hNIS transgene was gradually silenced in F3-NIS III cells, after the eighth passage its iodide uptake was 1.9-fold higher than that of nontransfected HB1.F3 cells. 5-azacytidine treatment (up to 40 micromol/L) for 24 h in F3-NIS III cells increased iodide uptake and hNIS messenger RNA (mRNA) 1.8- and 1.9-fold versus nontreated F3-NIS cells, respectively. Moreover, after TSA treatment (up to 62.5 nmol/L) for 24 h, iodide uptake and hNIS mRNA in F3-NIS III cells increased 36- and 1.9-fold versus nontreated F3-NIS III cells, respectively. The synergistic effect of demethylation and histone deacetylation inhibition was significant at high-dose 5-azacytidine and low-dose of TSA treatment. After treating F3-NIS III cells in vitro for 24 h with 62.5 nmol/L TSA, the cells were implanted into BALB/c nude mice. The TSA-treated F3-NIS III cells were clearly visible on gamma-camera imaging using (99m)Tc-pertechnetate as compared with F3-NIS III cells not treated with TSA. CONCLUSION: These results suggest that 2 well-known mechanisms of epigenetic modulation synergistically are involved in silencing reporter hNIS transgene in a neural stem cell line. Transgene silencing was reversed using demethylation and histone deacetylation inhibition. We conclude that silenced reporter transgenes once successfully expressed in stem cells might be awakened by pharmacologic treatment before infusion to track stem cells in vivo.