• Journal of Acupuncture Research
• /
• v.31 no.1
• /
• pp.7-21
• /
• 2014

Objectives : To observe the regenerative effects of indirect moxibustion, a traditional Korean medical treatment on skeletal muscles using mouse model of skeletal muscle adiposity. Methods : Twenty seven ICR male mice were randomly assigned into Intact control(n=3), glycerol treatment together without moxibustion(n=12), and glycerol treatment together with moxibustion (n=12) groups. Mice of glycerol treatment groups were injected with 50 ${\mu}l$ DW(distilled water) containing 50 % of glycerol into the two tibialis anterior. After injection, moxibustion was applied at 'Shenshu'($BL_{23}$) and 'Zusanli'($ST_{36}$) acupoints three times per each session, every days for twelve days(total 12 treatments). Phospho-Erk1/2, Myostatin protein levels were analyzed by western blotting and immunofluo-rescence staining techniques for tissues of the tibialis anterior muscle. Smad, phospho-Smad were analyzed by immunofluorescence staining. Results : 1. Histological analysis of sections from injected TA muscles showed that glycerol induced rapidly muscle necrosis, with a maximum at day 3. 6 days and 9 days after injection, muscle was regenerating. 2. According to western blotting and immunofluorescence staining, phospho-Erk1/2 protein signals in glycerol treatment with moxibustion group were stronger compared to Intact and glycerol treatment without moxibustion group. 3. According to western blotting and immunofluorescence staining, myostatin protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group. 4. According to immunofluorescence staining, Smad protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group. 5. According to immunofluorescence staining, phospho-Smad protein signals in glycerol treatment without moxibustion group were stronger compared to Intact and glycerol treatment with moxibustion group. Conclusions : These results confirm that indirect moxibustion of 'Shenshu'($BL_{23}$) and 'Zusanli'($ST_{36}$) influences muscle regeneration in mouse models of skeletal muscle adiposity. Further discussion, and the establishment of moxibustion mechanism will prompt clinical application of moxibustion.

• The Korean Journal of Zoology
• /
• v.38 no.4
• /
• pp.490-497
• /
• 1995

In vitro tissue culture of fat body of Hyphantria cunea in the medium containing [35S]-methionine reveaied that storage protein-i (SP-1) is taken up into fat body of prepupae and 1-day-old pupae. Using Western blotting and ligand binding method, we were able to identify the protein band of the SP-1 receptor protein. For the partial purification, the membrane proteins of fat body cells were solubilixed with 1% Triton X-1OO and applied to anion exchange chromatography. The results revealed the molecular weight of the receptor protein to be about 80 kl)a in SDSPAGE, and the P1 was estimated to be about 6.1. The mobility of the receptor protein in 8D8-PAGE was highly dependent on both temperature during electrophoresls and the condition of samples whether they were in reducing or nonreducing.

• The Korean Journal of Physiology and Pharmacology
• /
• v.16 no.3
• /
• pp.153-158
• /
• 2012

Cellular effects of ethanol in YD-15 tongue carcinoma cells were assessed by MTT assay, caspase activity assay, Western blotting and flow cytometry. Ethanol inhibited the growth and proliferation of YD-15 cells in a dose- and time-dependent manner in an MTT assay. The effects of ethanol on cell cycle control at low percent range of ethanol concentration (0 to 1.5%), the condition not inducing YD-15 cell death, was investigated after exposing cells to alcohol for a certain period of time. Western blotting on the expression of cell cycle inhibitors showed that p21 and p27 was up-regulated as ethanol concentration increases from 0 to 1.5% whilst the cell cycle regulators, cdk1, cdk2, and cdk4 as well as Cyclin A, Cyclin B1 and Cyclin E1, were gradually down-regulated. Flow cytometric analysis of cell cycle distribution revealed that YD-15 cells exposed to 1.5% ethanol for 24 h was mainly arrested at G2/M phase. However, ethanol induced apoptosis in YD-15 cells exposed to 2.5% or higher percent of ethanol. The cleaved PARP, a marker of caspase-3 mediated apoptosis, and the activation of caspase-3 and -7 were detected by caspase activity assay or Western blotting. Our results suggest that ethanol elicits inhibitory effect on the growth and proliferation of YD-15 tongue carcinoma cells by mediating cell cycle arrest at G2/M at low concentration range and ultimately induces apoptosis under the condition of high concentration.

• Journal of Life Science
• /
• v.22 no.3
• /
• pp.318-323
• /
• 2012

This study involved observation of the inhibitory effect of 70% EtOH and water extracts from Doinseunggitang on melanin synthesis, tyrosinase activity, and western blotting using B16F10 melanoma cells. Doinseunggitang extracts inhibited melanin synthesis and tyrosinase activity in a dependent manner. As a result, it was found that Doinseunggitang 70% EtOH extracts inhibit melanin synthesis and tyrosinase activity, respectively, by 40% and 51%. In addition, western blotting analysis showed that 70% EtOH extracts inhibited tyrosinase, MITF, TRP-1, and TRP-2 expression. These results show that 70% ethanol extracts of Doinseunggitang could be developed as a skin whitening material in cosmetics.

• Molecules and Cells
• /
• v.46 no.9
• /
• pp.538-544
• /
• 2023

The formation of uniform vitreous ice is a crucial step in the preparation of samples for cryogenic electron microscopy (cryo-EM). Despite the rapid technological progress in EM, controlling the thickness of vitreous ice on sample grids with reproducibility remains a major obstacle to obtaining high-quality data in cryo-EM imaging. The commonly employed classical blotting process faces the problem of excess water that cannot be absorbed by the filter paper, resulting in the formation of thick and heterogeneous ice. In this study, we propose a novel approach that combines the recently developed nanowire self-wicking technique with the classical blotting method to effectively control the thickness and homogeneity of vitrified ice. With simple procedures, we generated a copper oxide spike (COS) grid by inducing COSs on commercially available copper grids, which can effectively remove excess water during the blotting procedure without damaging the holey carbon membrane. The ice thickness could be controlled with good reproducibility compared to non-oxidized grids. Incorporated into other EM techniques, our new modification method is an effective option for obtaining high-quality data during cryo-EM imaging.

• Asian-Australasian Journal of Animal Sciences
• /
• v.23 no.1
• /
• pp.33-40
• /
• 2010

In this study, the production of transgenic goats using sperm to integrate exogenous DNA and artificial insemination (AI) was carried out and the technical protocols for sperm-mediated gene transfer (SMGT) in the goat were optimized. The standard sperm parameters and the ability to bind foreign genes were assessed to select suitable sperm donor bucks. A total of 134 oestrous does were divided into 4 groups and inseminated using different methods and sperm numbers. The does of Groups I to III were inseminated with fresh semen ($1-2\times10^{7}$ and $10^{6}$ sperm) or frozen-thawed semen ($10^{6}$ sperm), respectively, through conventional intra-cervical AI, and the does of Group IV with frozen-thawed semen ($10^{6}$ sperm) through intrauterine AI. Total genomic DNAs were extracted from ear biopsies of the offspring. The presence of $pEGFP-N_{1}$ DNA was screened by PCR and then by Southern blotting analysis. A total of 76 live kids were produced and 8 kids were tested transgene positive on the basis of agarose gel electrophoresis of the PCR-amplified fragment. Southern blotting analysis of the samples showed 5 positive kids. A transgenic ratio of 10.53% was detected using PCR and 6.58% using Southern blotting. The positive kid rate assayed by PCR and Southern blotting of frozen-thawed goat semen was 3.61% and 9.27% higher than that of untreated semen. The results show that transgenic goats can be produced efficiently by the method of artificial insemination using sperm cells to integrate the exogenous DNA and intrauterine insemination allowed low numbers of DNA-transfected spermatozoa to be used, with satisfactory fertility.

• Clinical and Experimental Reproductive Medicine
• /
• v.33 no.4
• /
• pp.253-263
• /
• 2006

Objective: Identification of the regulatory mechanism for arrest and initiation of primordial follicular growth is crucial for female fertility. Previously, we found 15 expressed sequence tags (ESTs) that were specifically abundant in the day-S-subtracted cDNA library and that the B357 clone was novel. The present study was conducted to obtain the whole sequence of the novel gene including B357 and to characterize its mRNA and protein expression in mouse ovary and testis. Methods: The extended sequence of the 2,965-bp cDNA fragment for the clone B357 was named ${\underline{5}}-{\underline{d}}ay-{\underline{o}}vary-{\underline{s}}pecific\;gene-{\underline{1}}$ (5DOS1) and submitted to GenBank (accession number ${\underline{AY751521}}$). Expression of 5DOS1 was characterized in both female and male gonads at various developmental stages by Northern blotting, real-time RT-PCR, in situ hybridization, Western blotting, and immunohistochemistry. Results: The 5DOS1 transcript was highly expressed in the adult testis, brain, and muscle as compared to the other tissues. In the ovary, the 5DOS1 transcript was detected in all oocytes from primordial to antral follicles, and highly expressed at day 5 after birth and decreased thereafter. In contrast, expression of 5DOS1 showed a gradual increase during testicular development and its expression was limited to various stages of male germ cells except spermatogonia. Conclusions: This is the first report on the expression and characterization of the 5DOS1 gene in the mouse gonads. Further functional analysis of the 5DOS1 protein will be required to predict its role in gametogenesis.

• Journal of Genetic Medicine
• /
• v.10 no.1
• /
• pp.33-37
• /
• 2013

Purpose: Myotonic dystrophy 1 (DM1, OMIM 160900) is an autosomal-dominant muscular disorder caused by an expansion of CTG repeats in the 3' UTR of the DMPK gene. Variable expansions of CTG repeats preclude the accurate determination of repeat size. We tried to show the clinical and analytical validity of the application of Southern blotting after long-range PCR was demonstrated in Korean DM1 patients. Materials and Methods: The Southern blotting of long-range PCR was applied to 1,231 cases with clinical suspicion of DM1, between 2000 and 2011. PCR was performed using genomic DNA with forward 5'-CAGTTCACAACCGCTCCGAGC-3' and reverse 5'-CGTGGAGGATGGAACACGGAC-3' primers. Subsequently, the PCR fragments were subjected to gel electrophoresis, capillary transfer to a nylon membrane, hybridization with a labeled (CAG)10 probe. The correlation between clinical manifestations and the CTG repeat expansions were analyzed. Results: Among a total of 1,231 tested cases, 642 individuals were diagnosed with DM1 and the range of the detected expansion was 50 to 2,500 repeats; fourteen cases with mild DM1 ($75{\pm}14$ repeats), 602 cases with classical DM1 ($314{\pm}143$ repeats), and 26 cases with congenital DM1 ($1,219{\pm}402$ repeats). The positive and negative predictive values were 100%. The age at test requested and the CTG repeat numbers were inversely correlated (R=-0.444, P<0.01). Conclusion: This study indicates that Southern blotting after long-range PCR is a reliable diagnostic method DM1.

• Asian Pacific Journal of Cancer Prevention
• /
• v.16 no.3
• /
• pp.941-945
• /
• 2015

Semaphoring is a transmembrane receptor which participates in many cytokine-mediated signal pathways that are closely related to the angiogenesis, occurrence and development of carcinoma. The present study was designed to access the effect of mono-antibody (mAb) guided radioimmunotherapy (RIT) on skin carcinoma and investigate the potential mechanisms. Semaphoring mAb was acquired from mice (Balb/c), purified with rProtein A column; purity, concentration and activity were tested with SDS-PAGE and indirect ELISA; specificity and expression on the cutanuem carcinoma line and tissue were tested by Western blotting; morphology change was assessed by microscopy. MTT assay and colony inhibition tests were carried out to test the influence on the proliferation of tumor cells; Western blotting was also carried out for expression of apoptosis-associated (caspase-3, Bax, Bcl-2) and proliferation-related (PI3K, p-Akt, Akt, p-ERK1/2, ERK1/2) proteins and analyse the change in signal pathways (PI3K/Akt and MEK/ERK). The purity of purified semaphorin mAb was 96.5% and the titer is about $1{\times}10^6$. Western blotting showed semaphoring mAb to have specifically binding stripes with semaphoring b1b2 protein, B16F10, and A431 cells at 39KDa, 100KDa and 130KDa, respectively. Positive expression was detected both in cutanuem carcinoma line and tissue and it mostly located in cell membranes. MMT assay revealed dose-relate and time-relate inhibitory effect of semaphorin mAb on A431 and B16F10. Colony inhibition tests also showed dose-relate inhibitory effects. Western blotting demonstrated the expression of apoptosis and proliferation-related protein and changes in signal pathway. In conclusion, we demonstrated that semaphorin is highly expressed on the tumor cell-surfaces and RIT with semaphorin mAb has effect in i nhibiting proliferation and accelerating apoptosis of tumor cells.

• Clinical and Experimental Pediatrics
• /
• v.49 no.2
• /
• pp.198-202
• /
• 2006

Purpose : Fas is a cell surface receptor that transduces apoptotic death signals. Interaction of extracelluar domain of Fas with Fas ligand(FasL) triggers the apoptotic process in many diseases. We investigated the expression of Fas and FasL in the hippocampus of 7-day-old newborn rat brains following hypoxia-ischemia injury. Methods : The 7-days-old newborn rats were exposed to 8 percent oxygen for two hours after the ligation of right common carotid arteries. The newborn rats were killed and their brains were removed at 12, 14 and 48 hours after hypoxic-ischemic injury. The expressions of Fas and FasL of the right hippocampus were observed by western blotting and immunofluorescent staining. Results : Fas and FasL were strongly expressed in the right hippocampus ipsilateral to the ligation of the common carotid artery by western blotting at 12 hours following hypoxic-ischemic injury, and then slowly decreased. The immunofluorescent expressions of Fas and FasL strongly increased in the CA1 area of the right hippocampus at 12 and 24 hours following hypoxic-ischemic injury. The immunofluorescent expression of Fas decreased at 48 hours, but the expression of FasL persisted strongly at 48 hours following hypoxic-ischemic injury. Conclusion : The interaction of Fas with FasL on the cell surface may be involved in neuronal injury following hypoxic-ischemic injury in the developing brain.