• Journal of Microbiology and Biotechnology
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• v.31 no.1
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• pp.163-170
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• 2021

Enzyme replacement therapy for lysosomal storage diseases usually requires recombinant enzymes containing mannose-6-phosphate (M6P) glycans for cellular uptake and lysosomal targeting. For the first time, a strategy is established here for the in vitro mannosyl-phosphorylation of high-mannose type N-glycans that utilizes a recombinant Mnn14 protein derived from Saccharomyces cerevisiae. Among a series of N-terminal- or C-terminal-deleted recombinant Mnn14 proteins expressed in Pichia pastoris, rMnn1477-935 with deletion of N-terminal 76 amino acids spanning the transmembrane domain (46 amino acids) and part of the stem region (30 amino acids), showed the highest level of mannosyl-phosphorylation activity. The optimum reaction conditions for rMnn1477-935 were determined through enzyme assays with a high-mannose type N-glycan (Man8GlcNAc2) as a substrate. In addition, rMnn1477-935 was shown to mannosyl-phosphorylate high-mannose type N-glycans (Man7-9GlcNAc2) on recombinant human lysosomal alpha-glucosidase (rhGAA) with remarkably high efficiency. Moreover, the majority of the resulting mannosyl-phosphorylated glycans were bis-form which can be converted to bis-phosphorylated M6P glycans having a superior lysosomal targeting capability. An in vitro N-glycan mannosyl-phosphorylation reaction using rMnn1477-935 will provide a flexible and straightforward method to increase the M6P glycan content for the generation of "Biobetter" therapeutic enzymes.

• Journal of Veterinary Science
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• v.23 no.3
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• pp.48.1-48.12
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• 2022

Background: The poor intracellular concentration of enrofloxacin might lead to treatment failure of cow mastitis caused by Staphylococcus aureus small colony variants (SASCVs). Objectives: In this study, enrofloxacin composite nanogels were developed to increase the intracellular therapeutic drug concentrations and enhance the efficacy of enrofloxacin against cow mastitis caused by intracellular SASCVs. Methods: Enrofloxacin composite nanogels were formulated by an electrostatic interaction between gelatin (positive charge) and sodium alginate (SA; negative charge) with the help of CaCl₂ (ionic crosslinkers) and optimized by a single factor test using the particle diameter, zeta potential (ZP), polydispersity index (PDI), loading capacity (LC), and encapsulation efficiency (EE) as indexes. The formation mechanism, structural characteristics, bioadhesion ability, cellular uptake, and the antibacterial activity of the enrofloxacin composite nanogels against intracellular SASCVs strain were studied systematically. Results: The optimized formulation was comprised of 10 mg/mL (gelatin), 5 mg/mL (SA), and 0.25 mg/mL (CaCl₂). The size, LC, EE, PDI, and ZP of the optimized enrofloxacin composite nanogels were 323.2 ± 4.3 nm, 15.4% ± 0.2%, 69.6% ± 1.3%, 0.11 ± 0.02, and -34.4 ± 0.8 mV, respectively. Transmission electron microscopy showed that the enrofloxacin composite nanogels were spherical with a smooth surface and good particle size distributions. In addition, the enrofloxacin composite nanogels could enhance the bioadhesion capacity of enrofloxacin for the SASCVs strain by adhesive studies. The minimum inhibitory concentration, minimum bactericidal concentration, minimum biofilm inhibitory concentration, and minimum biofilm eradication concentration were 2, 4, 4, and 8 ㎍/mL, respectively. The killing rate curve had a concentration-dependent bactericidal effect as increasing drug concentrations induced swifter and more radical killing effects. Conclusions: This study provides a good tendency for developing enrofloxacin composite nanogels for treating cow mastitis caused by intracellular SASCVs and other intracellular bacterial infections.

• Journal of Radiopharmaceuticals and Molecular Probes
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• v.6 no.2
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• pp.92-101
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• 2020

The cyclic Arg-Gly-Asp (cRGD) peptide is well-known as a binding molecule to the integrin α_vβ₃ receptor which is highly expressed on activated endothelial cells and new blood vessels in tumors. Although numerous results have been reported by the usage of cRGD peptide-based ligands for cancer diagnosis and therapy, the distinct mechanisms, and functions of cRGD-integrin binding to cancer cells are still being investigated. In this study, we evaluated the internalization efficacy of different types of cRGD peptides (monomer, dimer and tetramer form) in integrin α_vβ₃ overexpressing cancer cells. Western blot and flow cytometric analysis showed U87MG expresses highly integrin α_vβ₃, whereas CT-26 does not show integrin α_vβ₃ expression. Cytotoxicity assay indicated that all cRGD peptides (0-200 µM) had at least 70-80% of viability in U87MG cells. Fluorescence images showed cRGD dimer peptides have the highest cellular internalization compare to cRGD monomer and cRGD tetramer peptides. Additionally, transmission electron microscope results clearly visualized the endocytic internalization of integrin α_vβ₃ receptors and correlated with confocal microscopic results. These results support the rationale for the use of cRGD dimer peptides for imaging, diagnosis, or therapy of integrin α_vβ₃-rich glioblastoma.

• Journal of Ginseng Research
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• v.47 no.3
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• pp.420-428
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• 2023

Background: Ginsenoside F2 (GF2), a minor component of Panax ginseng, has been reported to possess a wide variety of pharmacological activities. However, its effects on glucose metabolism have not yet been reported. Here, we investigated the underlying signaling pathways involved in its effects on hepatic glucose. Methods: HepG2 cells were used to establish insulin-resistant (IR) model and treated with GF2. Cell viability and glucose uptake-related genes were also examined by real-time PCR and immunoblots. Results: Cell viability assays showed that GF2 up to 50 μM did not affect normal and IR-HepG2 cell viability. GF2 reduced oxidative stress by inhibiting phosphorylation of the mitogen-activated protein kinases (MAPK) signaling components such as c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase 1/2 (ERK1/2), and p38 MAPK, and reducing the nuclear translocation of NF-κB. Furthermore, GF2 activated PI3K/AKT signaling, upregulated the levels of glucose transporter 2 (GLUT-2) and GLUT-4 in IR-HepG2 cells, and promoted glucose absorption. At the same time, GF2 reduced phosphoenolpyruvate carboxykinase and glucose-6-phosphatase expression as well as inhibiting gluconeogenesis. Conclusion: Overall, GF2 improved glucose metabolism disorders by reducing cellular oxidative stress in IR-HepG2 cells via MAPK signaling, participating in the PI3K/AKT/GSK-3β signaling pathway, promoting glycogen synthesis, and inhibiting gluconeogenesis.

• Nuclear Engineering and Technology
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• v.56 no.5
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• pp.1803-1812
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• 2024

The attractive properties of gadolinium-based nanoparticles as a positive contrast agent for magnetic resonance imaging (MRI) have piqued the interest of both researchers and clinicians. Nonetheless, due to the biotoxicity of gadolinium (III) ions' free radicals, there is a need to address this issue. Therefore, this research aimed to develop a biocompatible, dispersible, stable, hydrophilic, and less toxic cellulose nanocrystals/gadolinium oxide nanocomposite as contrast agent properties for MRI purposes. This study aimed to synthesize gadolinium oxide nanoparticles coated with cellulose nanocrystals with polyethylene glycol and sodium hydroxide (CNCs-PEG/NaOH)/Gd₂O₃ using the gamma irradiation method to reduce the particle size. The results showed that using a gamma irradiation dose of 10 kGy, quasi-spherical morphology with a size of approximately 5.5 ± 0.65 nm could be produced. Furthermore, the cytocompatibility of (CNCs-PEG/NaOH)/Gd₂O₃ nanocomposite synthesized was assessed through MTT assay tests on Hep G2 cells, which demonstrated good cytocompatibility without any cytotoxic effects within a concentration range of (10 ㎍/mL - 150 ㎍/mL) and had sufficient cellular uptake. Moreover, the T₁-weighted MRI of (CNCs-PEG/NaOH)/Gd₂O₃ nanocomposite revealed promising results as a positive contrast agent. It is envisaged that the gamma irradiation method is promising in synthesizing (CNCs-PEG/NaOH)/Gd₂O₃ nanocomposite with nanoscale for different applications, especially in the radiotherapy field.

• The Korean Journal of Nuclear Medicine
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• v.37 no.6
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• pp.382-392
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• 2003

Purpose: Uptakes of Tc-99m MIBI (MIBI) and Tc-99m tetrofosmin (tetrofosmin) in human non-small cell lung cancer A549, multidrug-resistance associated protein (MRP) expressing cell, were investigated in vitro and in vivo. Materials and Methods: Western blot analysis and immunohistochemistry were used for detection of MRP in A549 cells with anti-MRPr1 antibody. Cellular uptakes of two tracers were evaluated at $100{\mu}M$ of verapamil (Vrp), $50{\mu}M$ of cyclosporin A (CsA) and $25{\mu}M$ of butoxysulfoximide (BSO) after incubation with MIBI and tetrofosmin for 30 and 50 min at $37^{\circ}C$, using single cell suspensions at $1{\times}10^6cells/ml$. Radioactivities in supernatants and pellets were measured with gamma well counter. A549 cells were inoculated in each flanks of 24 nude mice. Group 1 (Gr1) and Gr3 mice were treated with only MIBI or tetrofosmin, and Gr2 and Gr4 mice were treated with 70mg/kg of CsA i.p. for 1 hour before injection of 370KBq of MIBI or tetrofosmin. Mice were sacrificed at 10, 60 and 240 min. Radioactivities of organs and tumors were expressed as percentage injected dose per gram of tissue (%ID/gm). Results: Western blot analysis of the A549 cells detected expression of MRPr1 (190 kDa) and immunohistochemical staining of tumor tissue for MRPr1 revealed brownish staining in cell membrane but not P-gp. Upon incubating A549 cells for 60 min with MIBI and tetrofosmin, cellular uptake of MIBI was higher than that of tetrofosmin. Coincubation with modulators resulted in an increase in cellular uptakes of MIBI and tetrofosmin. Percentage increase of MIBI was higher than that of tetrofosmin with Vrp by 623% and 427%, CsA by 753% and 629% and BSO by 219% and 140%, respectively. There was no significant difference in tumoral uptakes of MIBI and tetrofosmin between Gr1 and Gr3. Percentage increases in MIBI (114% at 10 min, 257% at 60 min, 396% at 240 min) and tetrofosmin uptake (110% at 10 min, 205% at 60 min, 410% at 240 min) were progressively higher by the time up to 240 min with CsA. Conclusion: These results indicate that MIBI and tetrofosmin are suitable tracers for imaging MRP-mediated drug resistance in A549 tumors. MIBI may be a better tracer than tetrofosmin for evaluating MRP reversal effect of modulators.

• The Korean Journal of Nuclear Medicine
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• v.32 no.2
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• pp.121-128
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• 1998

Purpose: $Na^+-K^+$ ATPase activity has been estimated by the degree of inhibition of cation transport by cardiac glycosides (ouabain) using Rb-86 as a substrate. The biological characteristisc of T1-201 is known to be similar to those of potassium as a transport substrate in the presence of glucose, insulin or phobol myristate acetate (PMA). The purpose of this study was to measure ouabain sensitive $Na^+-K^+$ ATPase activity using T1-201 and compare with that using Rb-86. Materials and Methods: Smooth muscle cells isolated from rat aorta or human placental umbilical artery were cultured, and used to measure cellular $Na^+-K^+$ ATPase activity. $Na^+-K^+$ ATPase activity was measured as a percentage decrease in cellular uptake of T1-201 or Rb-86 by ouabain under the presence of glucose, insulin or PMA in media. Results: $Na^+-K^+$ ATPase activity measured with T1-201, as a transport substrate, was not different from those measured with Rb-86 in rat or human smooth muscle cell preparation. Incubation with high concentration glucose resulted in about 30% decrease in enzyme activity. In contrast, insulin or PMA resulted in 10-70% or 28% increases from baseline activity, respectively. Conclusion: These results suggests that 71-201 could replace Rb-86 in measurement of ouabain sensitive $Na^+-K^+$ ATPase activity in vitro. High level of glucose concentration decreased cellular $Na^+-K^+$ ATPase activity, but insulin or PMA increased it.

• Nuclear Medicine and Molecular Imaging
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• v.40 no.5
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• pp.263-270
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• 2006

Purpose: Several radioisotope-labeled thymidine derivatives such as $[^{11}C]$thymidine was developed to demonstrate cell proliferation in tumor. But it is difficult to track metabolism with $[^{11}C]$thymidine due to rapid in vivo degradation and its short physical half-life. 3'-$[^{18}F]$fluoro-3'-deoxythymidine ($[^{18}F]$FLT) was reported to have the longer half life of fluorine-18 and the lack of metabolic degradation in vivo. Here, we described the synthesis of the 3'-$[^{18}F]$fluoro-3'-deoxythymidine ($[^{18}F]$FLT) and compared with $([^{18}F]FET)\;and\;([^{18}F]FDG)$ in cultured 9L cell and obtained the biodistribution and PET image in 9L tumor hearing rats. Material and Methods: For the synthesis of $[^{18}F]$FLT, 3-N-tert-butoxycarbonyl-(5'-O-(4,4'-dimet hoxytriphenylmethyl)-2'-deoxy-3'-O-(4-nitrobenzenesulfonyl)-${\beta}$-D-threopentofuranosyl)thymine was used as a FLT precursor, on which the tert-butyloxycarbonyl group was introduced to protect N3-position and nitrobenzenesulfonyl group. Radiolabeling of nosyl substitued precursor with $^{18}F$ was performed in acetonitrile at $120^{\circ}C$ and deproteced with 0.5 N HCI. The cell uptake was measured in cultured 9L glioma cell. The biodistribution was evaluated in 9L tumor bearing rats after intravenous injection at 10 min, 30 min, 60 min and 120 min and obtained PET image 60 minutes after injection. Results: The radiochemical yield was about 20-30% and radiochemical purity was more than 95% after HPLC purification. Cellular uptake of $[^{18}F]$FLT was increased as time elapsed. At 120 min post-injection, the ratios of tumor/blood, tumor/muscle and tumor/brain were $1.61{\pm}0.34,\;1.70{\pm}0.30\;and\;9.33{\pm}2.22$, respectively. The 9L tumor was well visualized at 60 min post injection in PET image. Conclusion: The uptake of $[^{18}F]$FLT in tumor was higher than in normal brain and PET image of $[^{18}F]$FLT was acceptable. These results suggest the possibility of $[^{18}F]$FLT at an imaging agent for brain tumor.

• Asian-Australasian Journal of Animal Sciences
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• v.29 no.5
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• pp.652-658
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• 2016

Twenty $Pelibuey{\times}Katahdin$ ewes ($35{\pm}2.3kg$) were used to determine the effects of the consumption of standardized plant extract containing a mixture of quaternary benzophenanthridine alkaloids and protopine alkaloids (QBA+PA) on growth performance, dietary energetics, visceral mass, and ruminal epithelial health in heat-stressed ewes fed with a high-energy corn-based diet. The basal diet (13.9% crude protein and 2.09 Mcal of net energy [NE] of maintenance/kg of dry matter) contained 49.7% starch and 15.3% neutral detergent fiber. Source of QBA+PA was Sangrovit RS (SANG) which contains 3 g of quaternary benzophenathridine and protopine alkaloids per kg of product. Treatments consisted of a daily consumption of 0 or 0.5 g SANG/ewe. Ewes were grouped by weight and assigned to 10 pens (5 pens/treatment), with two ewes per pen. The experimental period lasted 70 days. The mean temperature humidity index during the course of this experiment was $81.7{\pm}1.0$ (severe heat stress). There were no treatment effects on water intake. Dry matter intake was not affected (p = 0.70) by treatments, but the group fed SANG had a numerically (11.2%) higher gain in comparison to the control group, SANG improved gain efficiency (8.3%, p = 0.04), dietary NE (5.2%, p<0.01) and the observed-to-expected NE (5.9%, p<0.01). Supplemental SANG did not affect ($p{\geq}0.12$) carcass characteristics, chemical composition of shoulder, and organ weights (g/kg empty body weight) of stomach complex, intestines, and heart/lung. Supplemental SANG decreased liver weight (10.3%, p = 0.02) and increased visceral fat (16.9%, p = 0.02). Rumen epithelium of ewes fed SANG had lower scores for cellular dropsical degeneration (2.08 vs 2.34, p = 0.02), parakeratosis (1.30 vs 1.82, p = 0.03) and neutrophil infiltration (2.08 vs 2.86, p = 0.05) than controls. It is concluded that SANG supplementation helped ameliorate the negative effects of severe heat on growth performance of feedlot ewes fed high-energy corn-based diets. Improvement in energetic efficiency may have been mediated, in part, by anti-inflammatory effects of supplemental SANG and corresponding enhancement of nutrient uptake.

• The Korean Journal of Nuclear Medicine
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• v.23 no.1
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• pp.41-48
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• 1989

$^{99m}Tc-Pertechnetate\;(TcO_4^-)$ is concentrated by the stomach after intravenous injection, allowing the detection of ectopic gastric mucosa. It has been used to develop a noninvasive test of gastric secretion. However the cellular site of concentration is still controversial, that is whether mucin-secreting epithelial cell or acid-secreting parietal cell. This study is planned to investigate the effects of cimetidine and gastric acidity on the retention of $TcO_4^-$ in the gastric wall of the rat. Also we further attempted to clarify the uptake and secreting cell of $TcO_4^-$ in the gastric mucosa. One hundred rats were divided into two groups, preliminary (40 rats) and main examination group (60 rats). Preliminary examination group was composed of fasting group (20 rats) for the detection of the time for reaching stable $TcO_4^-$ retention ratio in gastric wall and post-prandial group (20 rats) for the detection of the time for reaching the maximal gastric acidity. Main examination group was composed of fasting group (30 rats), which was subdivided into control group (10 rats), cimetidine group (10rats), $Mylanta^{(R)}$ group (10 rats) and post?prandial group (30 rats), which was subaivided into 90 min group (10 rats), 90 min cimetidine group (10 rats), and 120 min group (10 rats). Retention ratio (%) of $TcO_4$ in the gastric wall and the pH of the gastric contents were measured in the extracted stomach of the six groups. Gastric wall retention ratio of $TcO_4^-$ was calculated by the gastric wall radioactivity (cpm) divided by total gastric radioactivity (cpm) at 30 mins after intravenous injection of 0.4 mCi of $TcO_4^-$. The results were as follows: 1) The time required for reaching stable $TcO_4$ retention ratio and the lowest gastric PH were 30 min and 90 min, respectively. 2) In the fasting group, the gastric wall retention ratio of $TcO_4^-$ was significantly increased in the cimetidine group, compared with the control group (P < 0.01). However there was no significant difference between the control and $Mylanta^{(R)}$ group 3) The $TcO_4^-$ retention ratios of 90 min and 120 min groups were lower than that of the fasting control group (p < 0.05), either. After administration of cimetidine, the retention ratio was significantly increased in 90 min group (p < 0.01). 4) While $TcO_4^-$ retention ratio and gastric pH were well correlated in the post-prandial 120 min group (r=0.7112, p<0.05), in the post-prandial 90 min and 90 min cimetidine groups correlated poorly. However, there was no correlation in the three fasting groups at all. Referring the above results, we infer that $TcO_4^-$ is secreted into the gastric lumen by both parietal and non-parietal cells, with dominant non-parietal $TcO_4^-$ secretion in the fasting state and dominant parietal $TcO_4^-$ secretion in the stimulated state.