• Development and Reproduction
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• v.21 no.2
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• pp.157-165
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• 2017

One of the reasons to causing blood coagulation in the tissue of xenografted organs was known to incompatibility of the blood coagulation and anti-coagulation regulatory system between TG pigs and primates. Thus, overexpression of human CD73 (hCD73) in the pig endothelial cells is considered as a method to reduce coagulopathy after pig-to-non-human-primate xenotransplantation. This study was performed to produce and breed transgenic pigs expressing hCD73 for the studies immune rejection responses and could provide a successful application of xenotransplantation. The transgenic cells were constructed an hCD73 expression vector under control porcine Icam2 promoter (pIcam2-hCD73) and established donor cell lines expressing hCD73. The numbers of transferred reconstructed embryos were $127{\pm}18.9$. The pregnancy and delivery rate of surrogates were 8/18 (44%) and 3/18 (16%). The total number of delivered cloned pigs were 10 (2 alive, 7 mummy, and 1 died after birth). Among them, three live hCD73-pigs were successfully delivered by Caesarean section, but one was dead after birth. The two hCD73 TG cloned pigs had normal reproductive ability. They mated with wild type (WT) MGH (Massachusetts General Hospital) female sows and produced totally 16 piglets. Among them, 5 piglets were identified as hCD73 TG pigs. In conclusion, we successfully generated the hCD73 transgenic cloned pigs and produced their litters by natural mating. It can be possible to use a mate for the production of multiple transgenic pigs such as ${\alpha}-1,3-galactosyltransferase$ knock-out /hCD46 for xenotransplantation.

• Journal of Embryo Transfer
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• v.31 no.3
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• pp.161-168
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• 2016

Nucleotide metabolism in endothelium is variable between different species. Recent studies demonstrated that this variability could contribute coagulation dysfunction, even though organs of the alpha 1,3-galactosyltransferase gene knockout pig were transplanted into the primate. CD73 (ecto-5'-nucelotidase) is an enzyme at cell surface catalyzing the hydrolysis of adenosine triphosphate to adenosine, which plays role on a substance for anti-inflammatory and anti-coagulant. Thus, overexpression of CD73 in endothelial cells of the pig is considered as an approach to reduce coagulopathy. In this study, we constructed a human CD73 expression vector under control of porcine Icam2 promoter (pIcam2-hCD73), which is expressed specifically at endothelial cells, and of CMV promoter as a control (CMV-CD73). First, we transfected the CMV-CD73 vector into HEK293 cells, and then confirmed CD73 expression at cell surface by flow cytometry analysis. Next, we transfected the pIcma2-CD73 and CMV-CD73 vectors into primary porcine fibroblasts and endothelial cells. Consequence was that the pIcma2-CD73 vector was expressed only at the porcine endothelial cells, meaning that the pIcam2 promoter lead to endothelial cell-specific expression of CD73 in vitro. Finally, we nucleofected the pIcam2-hCD73 vector into passage 3 fibroblasts, and enforced hygromycin selection of 400mg/ml. We were able to obtain forty three colonies harboring pIcam2-CD73 to provide donor cells for transgenic cloned porcine production.

• Journal of Embryo Transfer
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• v.33 no.3
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• pp.129-137
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• 2018

Acute vascular rejection has been known as a main barrier occurring in a xenograted tissue of alpha 1,3-galactosyltransferase knock-out (GalT KO) pig into a non-human primate (NHP). Adenosine which is a final metabolite following sequential hydrolysis of nucleotide by ecto-nucleotidases such as CD39 and CD73, act as a regulator of coagulation, and inflammation. Thus xenotransplantation of CD39 and CD73 expressing pig under the GalT KO background could lead to enhanced survival of recipient NHP. We constructed a human CD39 and CD73 expression cassette designed for endothelial cell-specific expression using porcine Icam2 promoter (pIcam2-hCD39/hCD73). We performed isolation of endothelial cells (pAEC) from aorta of 4 week-old GalT KO and membrane cofactor protein expressing pig ($GalT^{-MCP/-MCP}$). We were able to verify that isolated cells were endothelial-like cells using immunofluorescence staining analysis with von Willebrand factor antibody, which is well known as an endothelial maker, and tubal formation assay. To find optimal condition for efficient transfection into pAEC, we performed transfection with GFP expression vector using four programs of nucleofection, M-003, U-023, W-023 and Y-022. We were able find that the program W-023 was optimal for pAEC with regard to viability and transfection efficiency by flow cytometry and fluorescent microscopy analyses. Finally, we were able to obtain $GalT^{-MCP/-MCP}/CD39/CD73$ pAEC expressing CD39 and CD73 at levels of 33.3% and 26.8%, respectively. We suggested that pACE isolated from $GalT^{-MCP/-MCP}$ pig might be provided as a basic resource to understand biochemical and molecular mechanisms of the rejections and as an alternative donor cells to generate $GalT^{-MCP/-MCP}/CD39/CD73$ pig expressing CD39 and CD73 at endothelial cells.

• Analytical Science and Technology
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• v.15 no.2
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• pp.163-171
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• 2002

The adsorption characteristics of Cd(II) and Pb(II) ions has been studied by using chitin as an adsorbent. The pure chitin was obtained from the extraction of red-crab shell dumped by fish factory. Adsorption kinetics of Cd(II) and Pb(II) ions on the chitin reached at the maximum adsorption within two minutes. Adsorbed amounts of heavy metals were pH 7.0>10.5>3.5 in the following order. Adsorption ratio by chitin was 21${\sim}$99% for Cd(II) ion and 24${\sim}$95% for Pb(II) ion. Recovery ratio of Cd(II) ion on the chitin was 22${\sim}$53%, and that of Pb(II) ion was 22${\sim}$73%. The adsorption behavior of these heavy metals was explained well by Freundlich adsorption isotherm.

• Journal of Embryo Transfer
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• v.32 no.3
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• pp.227-233
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• 2017

Pig has been known to be one of the most feasible animals as a bioreactor to produce pharmaceuticals in milk and as a mediator in xenotransplantation research. Previously, we generated transgenic pigs for both purposes, which were expressing Factor 8, vWF, hTPA, and hEPO in milk, along with expression of MCP at GalT gene locus ($GalT^{-MCP/-MCP}$) as well as expressing MCP at GalT gene loci with CD73 expression ($GalT^{-MCP/+}/CD73$). In this study, we performed comparative analyses of sperm parameters between wild type male (WT) pig and those transgenic males to examine the effects of transgenes integrated into the pigs on motility, morphology, viability, and acrosome integrity of the spermatozoa. Our results showed that the rates of actively motile spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 85.0%, 83.3%, 82.5%, 83.3%, 82.5%, 77.5%, and 78.7%, respectively. Whereas, the rates of morphologically normal spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 90.0%, 80.0%, 80.0%, 83.3%, 85.0%, 91.8%, and 80.8%, respectively. In addition, the viability in spermatozoa of WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 93.9%, 82.4%, 89.9%, 83.9%, 87.4%, 92.8%, and 83.6%, respectively. The rates of spermatozoa with normal acrosome integrity in WT, Factor 8, vWF, hTPA, hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs were 98.1%, 98.6%, 98.6%, 98.7%, 98.1%, 99.5%, and 95.1%, respectively. There were no significant differences in motility, morphology, viability, and acrosome integrity of the spermatozoa among WT, Factor 8, vWF, hTPA, and hEPO, $GalT^{-MCP/+}/CD73$, and $GalT^{-MCP/-MCP}$ pigs. These mean that neither random integration nor targeted integration of the transgene into chromosome of pig effect on characteristics of spermatozoa. Ultimately, the transgenic male pigs subjected in this study could apply to propagate their progenies for production of human therapeutic proteins and advancing the xenotransplantation research.

• Korean Journal of Crystallography
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• v.12 no.4
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• pp.207-211
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• 2001

The crystal structure of the title complex, Cd(SCN)₂{CH/sub 6/H/sub 10/(NH₂)₂}₂(1) has been analyzed by X-ray single crystallography. The complex 1 crystallizes in the monoclinic system P2₁/ space group with a=11.842(2), b=7.926(2), c=11.291(2) Å, β=106.73(3)°V=1014.8(4)Ų, Z=2, R₁=0.0518 and ωR₂=0.1315 for 1775 independent reflections. The central Cd(II) atom of this com-plex has a slightly distorted octahedral coordination geometry, with the 1,2-Diaminocyclohexane ligands functioning as an N,N'-bidentate and the thiocyanate ligands bonding through the sulfur atom in a trans arrangement.

• Korean Journal of Environmental Biology
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• v.18 no.2
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• pp.269-277
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• 2000

When Persicaria thunbergii and Rumex crispus were treated with Cd($NO_3$)$_2$ and Pb($NO_3$)$_2$ of 5 or 10 mM for 5 days, the amount of bioaccumulation of $Pb^{2+}$ in the leaf of P. thunbergii was 2.87-8.08$\mu\textrm{g}$/g and that of $Cd^{2+}$ was 0.82-2.79$\mu\textrm{g}$/g. In the case of P. thunbergii, the concentration of $Pb^{2+}$ in the leaf was higher than that of $Cd^{2+}$. On the other hand, in R. crispus, the concentration of $Cd^{2+}$ and $Pb^{2+}$ were similar as follows ; 1.49$\mu\textrm{g}$/g in $Cd^{2+}$ 5mM, 2.90$\mu\textrm{g}$/g in Cd2+ 10mM, 1.83$\mu\textrm{g}$/g in $Pb^{2+}$ 5mM and 2.73$\mu\textrm{g}$/g in $Pb^{2+}$ 10mM. The remaining rate of heavy metals and the variation of pH in the cultured soil decreased as compared with control (100 % and pH 6.48) after 5 days as follows; to 77.l% and pH 6.39 in $Cd^{2+}$ 5mM, 90.2% and pH 5.79 in $Cd^{2+}$ 10 mM, 81.1% and pH 6.00 in $Pb^{2+}$ 5mM, and 85.7% and pH 5.80 in $Pb^{2+}$ 10 mM. The result of size exclusion chromatography, several phytochelatins were seperated from the extract of the leaf of both plants treated with heavy metals. The molecular mass of these phytochelatins were estimated as follows; in the case of P. thunbergii, about 4,300-8,600 da by $Cd^{2+}$ and about 3,200-9,700 da by $Pb^{2+}$, and in R. crispus, about 4,300 da by $Cd^{2+}$ and about 3,200-7,500 da by $Pb^{2+}$. In addition, $A_{254}$ of these phytochelatins were higher than $A_{280}$. [Phytochelatin, Persicaria thunbergii, Rumex crispus]

• Mycobiology
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• v.45 no.2
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• pp.73-83
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• 2017

The ability of dead cells of endophytic Drechslera hawaiiensis of Morus alba L. grown in heavy metals habitats for bioremoval of cadmium ($Cd^{2+}$), copper ($Cu^{2+}$), and lead ($Pb^{2+}$) in aqueous solution was evaluated under different conditions. Whereas the highest extent of $Cd^{2+}$ and $Cu^{2+}$ removal and uptake occurred at pH 8 as well as $Pb^{2+}$ occurred at neutral pH (6-7) after equilibrium time 10 min. Initial concentration 30 mg/L of $Cd^{+2}$ for 10 min contact time and 50 to 90 mg/L of $Pb^{2+}$ and $Cu^{2+}$ supported the highest biosorption after optimal contact time of 30 min achieved with biomass dose equal to 5 mg of dried died biomass of D. hawaiiensis. The maximum removal of $Cd^{2+}$, $Cu^{2+}$, and $Pb^{2+}$ equal to 100%, 100%, and 99.6% with uptake capacity estimated to be 0.28, 2.33, and 9.63 mg/g from real industrial wastewater, respectively were achieved within 3 hr contact time at pH 7.0, 7.0, and 6.0, respectively by using the dead biomass of D. hawaiiensis compared to 94.7%, 98%, and 99.26% removal with uptake equal to 0.264, 2.3, and 9.58 mg/g of $Cd^{2+}$, $Cu^{2+}$, and $Pb^{2+}$, respectively with the living cells of the strain under the same conditions. The biosorbent was analyzed by Fourier Transformer Infrared Spectroscopy (FT-IR) analysis to identify the various functional groups contributing in the sorption process. From FT-IR spectra analysis, hydroxyl and amides were the major functional groups contributed in biosorption process. It was concluded that endophytic D. hawaiiensis biomass can be used potentially as biosorbent for removing $Cd^{2+}$, $Cu^{2+}$, and $Pb^{2+}$ in aqueous solutions.

• Analytical Science and Technology
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• v.15 no.2
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• pp.115-119
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• 2002

The formation of inclusion complexes between cyclodextrin(CD) and Congo red was studied by spectrophotometric methods at various temperatures. The cavity sizes are 0.49 nm, 0.62 nm for $\alpha$-and $\beta$-CD, respectively. Therefore, $\alpha$-CD was not found to form an inclusion complex with Congo red due to steric hinderance. In the $\beta$-CD use two $\beta$-CD molecules formed an inclusion complex with one molecule of Congo red, from the slope of the S-shaped curve increased. Two prominent isosbestic points appear at 346 nm and 478 nm. The formation constants were decreased with the increasing temperatures, due to low binding energy between $\beta$-CD and Congo red. The thermodynamic parameters were calculated from the plot of $lnK_f$ vs 1/T. The $\Delta$H, $\Delta$S and $\Delta$G were -50.73 kJ/mol, $-108.96J/K{\cdot}mol$ and -18.26 kJ/mol, respectively.

• Journal of Korean Society of Environmental Engineers
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• v.36 no.12
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• pp.851-857
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• 2014

Treatability of heavy metals in marine sediments contaminated with mainly organic matter was investigated on the basis of washing technology using oxidizers and surfactants. Sediment samples were collected at N area which expected for remediation project of contaminated marine sediment. For additives, hydrogen peroxide ($H_2O_2$) and Tween-80 were used at oxidizer and nonionic surfactant, respectively. In experiments, sediments was mixed with sea water at the ratio of 1 : 3 than $H_2O_2$ (1 M, 3 M, 4 M, 5 M) and Tween-80 (0.05%) were added. Samples were gathered at following reaction time (10, 20, 30, 40, 50, 60, 70, 80 min and 24 h). Total Organic Carbon (TOC) was 55.2% at the conditions of 5 M $H_2O_2+0.05%$ Tween-80 24 h. Hence total heavy metals were Cu 29.5%, Zn 42.3%, Cd 73.0% and bioavailable heavy metals were Cu 60.0%, Zn 77.7%, Cd 90.2% at the conditions of 5 M $H_2O_2+0.05%$ Tween-80 10 min. The correlations for between bioavailable metals (Cu, Zn, Cd) and TOC were significant (Cu, Zn, Cd; $r^2=0.94$, 0.85, 0.69, respectively).