• Journal of Microbiology
• /
• v.37 no.1
• /
• pp.21-26
• /
• 1999

Two novel calcium-binding proteins, named CAB-I and CAB-II, have been isolated from Streptomyces coelicolor. Purification of the calcium-binding proteins involved heat treatment, fractionation with ammonium sulfate, acid treatment, anion exchange and hydrophobic interaction column chromatography, FPLC gel filtration, and preparative isoelectric focusing. A chelex competitive assay and 45Ca autoradiography verified the calcium-binding ability of the proteins. The major band CAB-II has an apparent molecular weight of 26,000 determined by SDS-polyacrylamide gel electrophoresis and 340,000 determined by gel filtration. The isoelectric point of this molecule showed the acidic nature of the molecule. N-terminal amino acid sequence analysis shows homology to rat Ca2+/calmodulin-dependent protein kinase-II (CAB-II) and yeast phosphoprotein phosphatase (CAB-I).

• Bulletin of the Korean Chemical Society
• /
• v.33 no.11
• /
• pp.3665-3670
• /
• 2012

Metal-p-$NH_2$-Bn-DOTA (paraammionobenzyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid: ABDOTA) complex was synthesized and purified for bio-tagging to quantify biological target materials using laser ablation (LA)-ICP-MS. Since the preparation of a pure and stable tagging complex is the key procedure for quantification, magnetic particles were used to purify the synthesized metal-ABDOTA complex. The magnetic particles immobilized with the complex attracted to a permanent magnet, resulting in fast separation from free un-reacted metal ions in solution. Gd ions formed the metal-complex with a higher yield of 64.3% (${\pm}3.9%$ relative standard deviation (RSD)) than Y ions, 52.3% (${\pm}2.5%$ RSD), in the pH range 4-7. The complex bound to the magnetic particles was released by treatment with a strong base, of which the recovery was 81.7%. As a reference, a solid phase extraction (SPE) column packed with Chelex-100 resin was employed for separation under similar conditions and produced comparable results. The tagging technique complemented polydimethylsiloxane (PDMS) microarray chip sampling in LA-ICP-MS, allowing determination of small sample volumes at high throughputs. For application, immunoglobulin G (IgG) was immobilized on the pillars of PDMS microarray chips and then tagged with the prepared Gd complex. IgG could then be determined through measurement of Gd by LA-ICP-MS. A detection limit of 1.61 ng/mL (${\pm}0.75%$ RSD) for Gd was obtained.

• Journal of Nutrition and Health
• /
• v.53 no.6
• /
• pp.563-569
• /
• 2020

Purpose: The vascular smooth muscle cells (VSMCs) in mature animals have implicated to play a major role in the progression of cardiovascular diseases such as atherosclerosis. This study aimed at optimizing the protocol in culturing primary VSMCs (pVSMCs) from rat thoracic aorta and investigating the effect of cellular zinc (Zn) deficiency on cell proliferation of the isolated pVSMCs. Methods: The thoracic aorta from 7-month-old Sprague Dawley rats was isolated, minced and digested by the enzymatic process of collagenase I and elastase, and then inoculated with the culture Dulbecco Modified Eagle Medium (DMEM) at 37℃ in an incubator. The primary cell culture morphology was observed using phase-contrast microscopy and cellular Zn was depleted using Chelex-100 resin (extracellular zinc depletion only) or 3 µM N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN) (extracellular and intracellular zinc depletion). Western blot analysis was used for the detection of SM22α and calponin as smooth muscle cell marker proteins and von Willebrand factor as endothelial cell marker protein to detect the culture purity. Cell proliferation by Zn depletion (1 day) was measured by MTT assay. Results: A primary culture protocol for pVSMCs from rat thoracic aorta was developed and optimized. Isolated cultures exhibited hill and valley morphology as the major characteristics of pVSMCs and expressed the smooth muscle cell protein markers, SM22α and calponin, while the endothelial marker von Willebrand factor was hardly detected. Zn deprivation for 1 day culture decreased rat primary vascular smooth muscle cell proliferation and this pattern was more prominent under severe Zn depletion (3 µM TPEN), while less prominent under mild Zn depletion (Chelexing). Conclusion: Our results suggest that cellular Zn deprivation decreased pVSMC proliferation and this may be involved in phenotypic modulation of pVSMC in the aorta.

• Journal of Radiopharmaceuticals and Molecular Probes
• /
• v.7 no.2
• /
• pp.119-125
• /
• 2021

⁸²Sr for ⁸²Rb generator was produced through the irradiation of the proton beam on the ^nat.RbCI target at the target irradiation facility installed at the end of the Rl-dedicated beamline of the 100 MeV proton linear accelerator of KOMAC (Korea Multi-purpose Accelerator Complex). The average current of the proton beam was 1.2 µA for irradiation time of 150 min. For the separation and purification of the ⁸²Sr from ^nat.RbCI irradiated, Chelex-100 resin was used. The activities of ⁸²Sr in the irradiated ^nat.RbCI target solution and after purification were 45.29 µCi and 43.4 µCi, respectively. The separation and purification yield was 95.8%. As an adsorbent to be filled in the generator for ⁸²Sr adsorption hydrous tin oxide was selected. The adsorption yield of ⁸²Sr into the generator adsorbent was > 99 %, and the total amount of ⁸²Sr adsorbed to the generator was 21.6 µCi as of the day of the ⁸²Rb elution experiment. When the elution amount was 22 mL, the maximum⁸²Rb elution yield was 93.3%, and the elution yield increased as the flow rate increased. After the eluted ⁸²Rb was filled in the correction phantom of the small PET for animals, a PET image was taken. The image scan time was set to 5 min, and the phantom PET image was successfully obtained. As results of impurity analysis on eluted ⁸²Rb using ICP-MS, ^nat.Rb stable isotopes that compete in vivo of ⁸²Rb were identified as undetected levels and were determined to be No-Carrier-Added (NCA).