• Animal Bioscience
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• v.37 no.7
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• pp.1156-1167
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• 2024

Objective: MicroRNAs (miRNAs) are endogenous non-coding RNAs that can play a role in the post-transcriptional regulation of mammalian preadipocyte differentiation. However, the precise functional mechanism of its regulation of fat metabolism is not fully understood. Methods: We identified bta-miR-365-3p, which specifically targets the 3' untranslated region (3'UTR) of the FK506-binding protein 5 (FKBP5), and verified its mechanisms for regulating expression and involvement in adipogenesis. Results: In this study, we found that the overexpression of bta-miR-365-3p significantly decreased the lipid accumulation and triglyceride content in the adipocytes. Compared to inhibiting bta-miR-36 5-3p group, overexpression of bta-miR-365-3p can inhibit the expression of adipocyte differentiation-related genes C/EBPα and PPARγ. The dual-luciferase reporter system further validated the targeting relationship between bta-miR-365-3p and FKBP5. FKBP5 mRNA and protein expression were detected by quantitative real-time polymerase chain reaction and Western blot. Overexpression of bta-miR-365-3p significantly down-regulated FKBP5 expression, while inhibition of bta-miR-365-3p showed the opposite, indicating that bta-miR-365-3p negatively regulates FKBP5. Adenosine 5'-monophosphate (AMP)-activated protein kinase/mammalian target of rapamycin (AMPK/mTOR) signaling pathway is closely related to the regulation of cell growth and is involved in the development of bovine adipocytes. In this study, overexpression of bta-miR-365-3p significantly inhibited mRNA and protein expression of AMPK, mTOR, and SREBP1 genes, while the inhibition of bta-miR-365-3p expression was contrary to these results. Overexpression of FKBP5 significantly upregulated AMPK, mTOR, and SREBP1 gene expression, while inhibition of FKBP5 expression was contrary to the above experimental results. Conclusion: In conclusion, these results indicate that bta-miR-365-3p may be involved in the AMPK/mTOR signaling pathway in regulating Yanbian yellow cattle preadipocytes differentiation by targeting the FKBP5 gene.

• The Korean Journal of Nuclear Medicine
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• v.38 no.3
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• pp.253-258
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• 2004

Purpose: Chitosan has been studied as a non-viral gene delivery vector, drug delivery carrier, metal chelator, food additive, and radiopharmaceutical, among other things. Recently, galactose-graft chitosan was studied as a non-viral gene and drug delivery vector to target hepatocytes. The aim of this study was to investigate the usefulness of nuclear imaging for in vivo evaluation of targeting the hepatocyte by galactose grafting. Methods and Materials: Galactosyl methylated chitosan (GMC) was produced by methylation to lactobionic acid coupled chitosan. Cytotoxicity of $^{99m}Tc$-GMC was determined by MTT assay. Rabbits were injected via their auricular vein with $^{99m}Tc$-GMC and $^{99m}Tc$-methylated chitosan (MC), the latter of which does not contain a galactose group, and images were acquired with a gamma camera equipped with a parallel hole collimator. The composition of the galactose group in galactosylated chitosan (GC), as well as the tri-, di-, or mono-methylation of GMC, was confirmed by NMR spectroscopy. Results: The results of MTT assay indicated that $^{99m}Tc$-GMC was non-toxic. $^{99m}Tc$-GMC specifically accumulated in the liver within 10 minutes of injection and maintained high hepatic uptake. In contrast, $^{99m}Tc$-MC showed faint liver uptake. $^{99m}Tc$-GMC scintigraphy of rabbits showed that the galactose ligand principally targeted the liver while the chitosan functionalities led to excretion through the urinary system. Conclusion: Bioconjugation with a specific ligand endows some degree of targetability to an administered molecule or drug, as in the case of galactose for hepatocyte in vivo, and evaluating said targetabililty is a clear example of the great benefit proffered by nuclear imaging.

• Journal of The Korean Society of Inherited Metabolic disease
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• v.17 no.2
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• pp.48-54
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• 2017

Purpose: Fukuyama congenital muscular dystrophy (FCMD) is a rare, autosomal-recessive disorder characterized by early-onset hypotonia associated with brain malformations in dystroglycanopathy. Although the wide spectrum of congenital muscular dystrophies causes difficulty in diagnosis, correlating the genotype with the clinical phenotype can help diagnose FCMD. Here, we evaluated the correlation of targeted molecular genetic analysis of FKTN gene mutation with the FCMD phenotype. Methods: This study was conducted retrospectively with 9 subjects. Inclusion criteria included clinical symptoms characterized by early-onset hypotonia with magnetic resonance imaging (MRI) featuring brain malformations. FKTN gene-alteration analysis was performed using various FKTN gene-analysis methods, including sequencing. Results: Among the 9 subjects studied, 4 (44.4%) were male and 5 (55.6%) were female. The median age of onset of the first symptom was 3.1 months. The first symptom was a delayed milestone in 6 cases (66.7%). All 9 subjects (100%) presented with early-onset hypotonia and global delayed development. All subjects presented with cortical malformation in their brain MRIs. Of the 9 subjects, 6 subjects had previously undergone muscle biopsy and 4 cases (4/6; 66.7%) showed dystrophic or myopathic features. Pathogenic mutations causing FCMD were identified in 3 cases. Conclusions: In this study, all 3 subjects with FKTN mutations showed important MRI findings (pachygyria and cerebellar dysplasia). These data suggest that patients with characteristic phenotypes who show pachygyria and cerebellar abnormalities in brain MRIs may have a high probability of being diagnosed with FCMD.

• The korean journal of orthodontics
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• v.36 no.4
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• pp.284-294
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• 2006

Objective: Activating mutations in the fibroblast growth factor receptor-2 (FGFR2) have been shown to cause syndromic craniosynostosis such as Apert and Crouzon syndromes. The purpose of this pilot study was to investigate the resultant phenotypes induced by the two distinctive bone-targeted gene constructs of FGFR2, Pro253Arg and Cys278Phe, corresponding to human Apert and Crouzon syndromes respectively. Methods: Wild type and a transgenic mouse model with normal FGFR2 were used as controls to examine the validity of the microinjection. Micro-CT and morphometric analysis on the skull revealed the following results. Results: Both Apert and Crouzon mutants of FGFR2 induced fusion of calvarial sutures and anteroposteriorly constricted facial dimension, with anterior crossbite present only in Apert mice. Apert mice differed from Crouzon mice and transgenic mice with normal FGFR2 in the anterior cranial base flexure and calvarial flexure angle which implies a possible difference in the pathogenesis of the two mutations. In contrast, the transgenic mice with normal FGFR2 displayed normal craniofacial phenotype. Conclusion: Apert and Crouzon mutations appear to lead to genotype-specific phenotypes, possibly causing the distinctive sites and sequence of synostosis in the calvaria and cranial base. The exact function of the altered FGFR2 at each suture needs further investigation.

• Ultrasonography
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• v.32 no.1
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• pp.59-65
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• 2013

Purpose: The purpose of this study is to establish the methodology regarding synthesis of ultrasound contrast agent imaging, and to evaluate the characteristics of the synthesized ultrasound contrast agents, including size or degradation interval and image quality. Materials and Methods: The ultrasound contrast agent, composed of liposome and SF6, was synthesized from the mixture solution of $21{\mu}mol$ DPPC (1, 2-Dihexadecanoyl-sn-glycero-3-phosphocholine, $C_{40}H_{80}NO_8P$), $9{\mu}mol$ cholesterol, $1.9{\mu}mol$ of DCP (Dihexadecylphosphate, $[CH_3(CH_2)_{15}O]_2P(O)OH$), and chloroform. After evaporation in a warm water bath and drying during a period of 12-24 hours, the contrast agent was synthesized by the sonication process by addition of buffer and SF6 gas. The size of the contrast agent was controlled by use of either extruder or sonication methods. After synthesis of contrast agents, analysis of the size distribution of the bubbles was performed using dynamic light scattering measurement methods. The degradation curve was also evaluated by changes in the number of contrast agents via light microscopy immediate, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, and 84 hours after synthesis. For evaluation of the role as an US contrast agent, the echogenicity of the synthesized microbubble was compared with commercially available microbubbles (SonoVue, Bracco, Milan, Italy) using a clinical ultrasound machine and phantom. Results: The contrast agents were synthesized successfully using an evaporation-drying-sonication method. The majority of bubbles showed a mean size of 154.2 nanometers, and they showed marked degradation 24 hours after synthesis. ANOVA test revealed a significant difference among SonoVue, synthesized contrast agent, and saline (p < 0.001). Although no significant difference was observed between SonoVue and the synthesized contrast agent, difference in echogenicity was observed between synthesized contrast agent and saline (p < 0.01). Conclusion: We could synthesize ultrasound contrast agents using an evaporation-drying-sonication method. On the basis of these results, many prospective types of research, such as anticancer drug delivery, gene delivery, including siRNA or microRNA, targeted molecular imaging, and targeted therapy can be performed.

• Journal of Ginseng Research
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• v.44 no.3
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• pp.475-482
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• 2020

Background: Active natural ingredients, especially small molecules, have recently received wide attention as modifiers used to treat neurodegenerative disease by promoting neurogenic regeneration of neural stem cell (NSC) in situ. 20(S)-protopanaxadiol (PPD), one of the bioactive ingredients in ginseng, possesses neuroprotective properties. However, the effect of PPD on NSC proliferation and differentiation and its mechanism of action are incompletely understood. Methods: In this study, we investigated the impact of PPD on NSC proliferation and neuronal lineage differentiation through activation of the Wnt/glycogen synthase kinase (GSK)-3β/β-catenin pathway. NSC migration and proliferation were investigated by neurosphere assay, Cell Counting Kit-8 assay, and EdU assay. NSC differentiation was analyzed by Western blot and immunofluorescence staining. Involvement of the Wnt/GSK3β/β-catenin pathway was examined by molecular simulation and Western blot and verified using gene transfection. Results: PPD significantly promoted neural migration and induced a significant increase in NSC proliferation in a time- and dose-dependent manner. Furthermore, a remarkable increase in anti-microtubule-associated protein 2 expression and decrease in nestin protein expression were induced by PPD. During the differentiation process, PPD targeted and stimulated the phosphorylation of GSK-3β at Ser9 and the active forms of β-catenin, resulting in activation of the Wnt/GSK-3β/β-catenin pathway. Transfection of NSCs with a constitutively active GSK-3β mutant at S9A significantly hampered the proliferation and neural differentiation mediated by PPD. Conclusion: PPD promotes NSC proliferation and neural differentiation in vitro via activation of the Wnt/GSK-3β/β-catenin pathway by targeting GSK-3β, potentially having great significance for the treatment of neurodegenerative diseases.

• Maxillofacial Plastic and Reconstructive Surgery
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• v.27 no.3
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• pp.205-217
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• 2005

Distraction osteogenesis is a technique of lengthening bone including soft tissue by gradual separation of surgically divided bone surfaces. Although the biomechanical, histological, and ultrastructural changes associated with distraction osteogenesis have been widely described, the molecular mechanisms governing the formation of new bone in distracted bone segments remain largely unclear. However, such information has significant clinical implications because it may enable targeted therapeutic manipulations designed to accelerate osseous regeneration. The purpose of this study was to evaluate the expression of TGF-$\beta$1, IGF-I and bFGF in distraction osteogenesis according to different distraction rates in a rabbit's mandible. When twenty-four adult rabbits underwent open osteotomy between the premolar and mental foramen, an external bilateral distraction device was applied. Latency was allowed for five days before distraction. Three different distraction rates were 0.7 mm/day (A, n=8), 1.4 mm/day (B, n=8) and 2.4 mm/day (C, n=8). The distraction device was activated with the same distraction rhythms of twice a day until 4.9 mm (A & B group) and 8.4 mm (C group) length gains was achieved. The animals were sacrificed at postoperative 3, 7, 14 and 28 days. The bony specimens were stained with H&E for histologic examination, and RT-PCR analysis was done for the identification of the expression of TGF-$\beta$1, IGF-I and bFGF. The results obtained from this study were as follows : The 0.7 mm/day and 1.4 mm/day distraction rate groups were shown to improve regenerative bone formation on radiographic and histologic examination. Also, TGF-$\beta$1, IGF-I and bFGF expression increased in the 0.7 mm/day and 1.4 mm/day distraction rate groups. But the 2.4 mm/day distraction rate group specimen was different with adjacent normal bone and hardly expressed of growth factors. These findings suggest that improved new bone formation in the 0.7 mm/day and 1.4 mm/day distraction rates is associated with enhanced expression of TGF-$\beta$1, IGF-I and bFGF by mechanical tension stress. Additionally, the 0.7 mm/day and 1.4 mm/day distraction rate groups were significantly different from the 2.4 mm/day distraction rate group in the expression of growth factors. According to the above results, it seems possible to apply a distraction rate of up to 1.4 mm/day a day in rabbit's mandible. And further studies are needed to evaluate growth factors of TGF-$\beta$1 and IGF-I, which are excellent in expression.

• Journal of Microbiology and Biotechnology
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• v.11 no.6
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• pp.915-921
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• 2001

Phytase improves the bioavailability of phytate phosphorus in plant foods to humans and animals, and reduces the phosphorus pollution of animal waste. In order to express a high level of fungal phytase in Saccharomyces cerevisiae, various expression vectors were constructed with different combinations of promoters, translation enhancers, signal peptides, and terminator. Three different promoters fused to the phytase gene (phyA) from Aspergillus niger were tested: a galactokinase (GAL1) promoter, glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter, and yeast hybrid ADH2-GPD promoter consisting of alcohol dehydrogenase II (ADH2) and a GPD promoter. The signal peptides of phytase, glucose oxidase (GO), and rice amylase 1A(RAmy1A) were included. Plus, the translation enhancers of the ${\Omega}$ sequence and UTR70 from the tobacco mosaic virus (TMV) and spinach, respectively, were also tested. Among the recombinant vectors, pGphyA06 containing the GPD promoter, the ${\Omega}$ sequence, RAmy1A, and GAL7 terminator expressed the highest phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase activity in a culture filtrate, which was estimated at 20 IU/ml. An intracellular localization of the expressed phytase was also performed by inserting an endoplasmic reticulum (ER) retention signal, KDEL sequence, into the C-terminus of the phytase within the vector pHphyA-6. It appeared that the KDEL sequence directed most of the early expression of phytase into the intracellular compartment yet more than $60\%$ of the total phytase activity was still retained within the cell even after the prolonged (>3 days) incubation of the transformant. However, the intracellular enzyme activity of the transformant without a KDEL sequence was as high as that of the extracellular one, thereby strongly suggesting that the secretion of phytase in S. cerevisiae appeared to be the rate-limiting step for the expression of a large amount of extracellular recombinant phytase, when compared with other yeasts.

• Korean Journal of Microbiology
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• v.42 no.1
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• pp.19-25
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• 2006

This study was undertaken to develop PCR primers for the identification and detection of Streptococcus mutans (by)using species-specific forward and universal reverse primers. These primers targeted the variable regions of the 16S ribosomal RNA coding gene (rDNA). The primer specificity was tested against 11S. mutans strains and 10 different species (22 strains) of oral bacteria. The primer sensitivity was determined by testing serial dilutions of the purified genomic DNA of S. mutans ATCC $25175^T$. The data showed that species-specific amplicons were obtained from all the S. mutans strains tested, which was not observed in the other species. The direct and nested PCR could detect as little as 2 pg and 2 fg of the chromosomal DNA from S. mutans ATCC $25175^T$, respectively. This shows that the PCR primers are highly sensitive and applicable to the detection and identification of S. mutans.

• BMB Reports
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• v.42 no.1
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• pp.59-64
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• 2009

Hepatitis B virus (HBV) infection is highly prevalent worldwide. The major challenge for current antiviral treatment is the elevated drug resistance that occurs via rapid viral mutagenesis. In this study, we developed AAV vectors to simultaneously deliver two or three shRNAs targeting different HBV-related genes. These vectors showed markedly better antiviral effects than ones that delivered a single shRNA in vitro. A dual shRNA expression vector (AAV-157i/1694i), which simultaneously expressed two shRNAs targeted the S and X genes of HBV, reduced HBsAg, HBeAg and HBV DNA levels by $87{\pm}4$, $80.3{\pm}2.6$ and $86.2{\pm}7%$ respectively, eight days post-transduction. In a mouse model of prophylactic treatment, HBsAg and HBeAg were reduced to undetectable levels and the serum HBV DNA level was reduced by at least 100 fold. These results indicate that AAV-157i/1694i generates potent anti-HBV effects and that the strategy of constructing multi-shRNA expression vectors may lead to enhanced anti-HBV efficacy and overcome the evading mechanism of the virus and thus the development of drug resistance.