Postmenopausal osteoporosis (PMOP) is a common systemic skeletal disease characterized by reduced bone mass and microarchitecture deterioration. Although differentially expressed SOX5 has been found in bone marrow from ovariectomized mice, its role in osteogenic differentiation in human mesenchymal stem cells (hMSCs) from bone marrow in PMOP remains unknown. In this study, we investigated the biological function of SOX5 and explore its molecular mechanism in hMSCs from patients with PMOP. Our findings showed that the mRNA and protein expression levels of SOX5 were upregulated in hMSCs isolated from bone marrow samples of PMOP patients. We also found that SOX5 overexpression decreased the alkaline phosphatase (ALP) activity and the gene expression of osteoblast markers including Collagen I, Runx2 and Osterix, which were increased by SOX5 knockdown using RNA interference. Furthermore, $TNF-{\alpha}$ notably upregulated the SOX5 mRNA expression level, and SOX5 knockdown reversed the effect of $TNF-{\alpha}$ on osteogenic differentiation of hMSCs. In addition, SOX5 overexpression increased Kruppel-like factor 4 (KLF4) gene expression, which was decreased by SOX5 silencing. KLF4 knockdown abrogated the suppressive effect of SOX5 overexpression on osteogenic differentiation of hMSCs. Taken together, our results indicated that $TNF-{\alpha}$-induced SOX5 upregulation inhibited osteogenic differentiation of hMSCs through KLF4 signal pathway, suggesting that SOX5 might be a novel therapeutic target for PMOP treatment.
The present investigation tested the hypothesis that the activation of protein kinase G (PKG) leads to a phosphorylation of $Ca^{2+}-activated$ potassium channel $(K_{Ca}\;channel)$ and is involved in the activation of $K_{Ca}$ channel activity in cerebral arterial smooth muscle cells of the rabbit. Single-channel currents were recorded in cell-attached and inside-out patch configurations of patch-clamp techniques. Both molsidomine derivative 3-morpholinosydnonimine-N-ethylcarbamide $(SIN-1,\;50\;{\mu}M)$ and 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate $(8-pCPT-cGMP,\;100\;{\mu}M),$ a membrane-permeable analogue of cGMP, increased the $K_{Ca}$ channel activity in the cell-attached patch configuration, and the effect was removed upon washout of the drugs. In inside-out patches, single-channel current amplitude was not changed by SIN-1 and 8-pCPT-cGMP. Application of ATP $(100\;{\mu}M),$ cGMP $(100\;{\mu}M),$ ATP+cGMP $(100\;{\mu}M\;each),$ PKG $(5\;U/{\mu}l),$ ATP $(100\;{\mu}M)+PKG\;(5\;U/{\mu}l),$ or cGMP $(100\;{\mu}M)+PKG\;(5\;U/{\mu}l)$ did not increase the channel activity. ATP $(100\;{\mu}M)+cGMP\;(100\;{\mu}M)+PKG\;(5\;U/{\mu}l)$ added directly to the intracellular phase of inside-out patches increased the channel activity with no changes in the conductance. The heat-inactivated PKG had no effect on the channel activity, and the effect of PKG was inhibited by 8-(4-Chlorophenylthio)-guanosine-3',5'-cyclic monophosphate, Rp-isomer $(Rp-pCPT-cGMP,\;100\;{\mu}M),$ a potent inhibitor of PKG or protein phosphatase 2A (PP2A, 1 U/ml). In the presence of okadaic acid (OA, 5 nM), PP2A had no effect on the channel activity. The $K_{Ca}$ channel activity spontaneously decayed to the control level upon washout of ATP, cGMP and PKG, and this was prevented by OA (5 nM) in the medium. These results suggest that the PKG-mediated phosphorylations of $K_{Ca}$ channels, or some associated proteins in the membrane patch increase the activity of the $K_{Ca}$ channel, and the activation may be associated with the vasodilating action.
Bisphenol A (BPA), an environmental endocrine disrupter, enters the human body continuously in food and drink. Young children are likely to be more vulnerable than adults to chemical exposure due to the immaturities of their organ systems, rapid physical development, and higher ventilation, metabolic rates, and activity levels. The direct effect of BPA on peripheral tissue might also be of importance to the development of insulin resistance. However, the influence that BPA has on insulin signaling molecules in skeletal muscle has not been previously investigated. In this study, we examined the effect of BPA on fasting blood glucose (FBG) in post-weaned Wistar rats and on insulin signaling proteins in C2C12 skeletal muscle cells. Subsequently, we investigated the effects of BPA on insulin-mediated Akt phosphorylation in C2C12 myotubes. In rats, BPA treatment (0.1-1,000 ng/mL for 24 hours) resulted in the increase of FBG and plasma insulin levels, and reduced insulin-mediated Akt phosphorylation. Furthermore, the mRNA expression of insulin receptor (IR) was decreased after 24 hours of BPA treatment in C2C12 cells in a dose-dependent manner, whereas the mRNA levels of other insulin signaling proteins, including insulin receptor substrate-1 (IRS-1) and 5'-AMP-dependent protein kinase (AMPK), were unaffected. Treatment with BPA increased GLUT4 expression and protein tyrosine phosphatase 1B (PTP1B) activity in C2C12 myotubes, but not in protein levels. We conclude that exposure to BPA can induce insulin resistance by decreasing IR gene expression, which is followed by a decrease in insulin- mediated Akt activation and increased PTP1B activity.
Park, Mee-Jung-;Lee, Sang-Ho-;Park, Doo-Soon-;Cho, Tai-Soon;Lee, Sun-Mee-
한국응용약물학회:학술대회논문집
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한국응용약물학회 1994년도 춘계학술대회 and 제3회 신약개발 연구발표회
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pp.340-340
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1994
Since total hepatic ischemia(IS) occurs with transplantation, there has been interest in evaluating hepatic function after ischemia and subsequent reflow of blood. Four groups of animals were studied: group 1 (sham), group 2 (30mins IS), group 3 (60mins IS), and g.cup 4 (90mins IS). Serum transaminase(STA), wet weight-to-dry weight ratio(W/D), lipid peroxides(LPO), glucose-6-phosphatase(G-6-Pase) activity, Na$\^$+//K$\^$+/-ATPase(ATPase) activity were measured at 1, 5 and 24hrs after hepatic ischemia. Significant changes occurred between 1 and 5hrs of reperfusion. STA was 3579${\pm}$401, 4593${\pm}$675 and 6348${\pm}$808 U/L in group 2, 3 and 4 respectively. These changes were ischemic time-dependent manner. W/D in group 3 and 4 were significantly increased than that in sham group at all time points measured. In sham group, the level of LPO in the liver microsome remained constant at approximately 0. 5nmole MDA formed/mg protein througllout the experiment, In all ischemic groups on the other hand, the level of LPO started to increase at ischemia and markedly increased at all reperfusion period. Similar to STA, these changes were also dependent on duration of ischemia. Although G-6-Pase activity remained unchanged in both group 2 and group 3 until 5hrs of reperfusion, marked decrease in G-6-Pase activity was observed at grcup 4. ATPase activity was significantly decreased at 1, 5 and 24 hrs of reperfusion in group 3, whereas it was not changed in group 2. Furthermore, ATPase activity in group 4 started to decrease at ischemia and markedly decreased for entire reperfusion period. These data suggest that severity of hepatocellular injury is associated with period of ischemia as well as period of reperfusion.
A 2-year-old 4.0-kg female Shih Tzu with history of hematemesis and melena was referred to Veterinary Medical Teaching Hospital, Seoul national University for further evaluation and treatment. During physical examination, the dog revealed mild depression, dry mucous membrane and abdominal pain. Hematologic values were normal and serum chemical values showed increased serum bile acid (53.47 umol/l, preprandial), fasting serum ammonia concentration (184 g/dl), alanine transferase (98 U/L), alkaline phosphatase (871 U/L) and gamma glutamyl transpeptidase (21 U/L), and decreased blood urea nitrogen (4 mg/dl), total protein (4.1 g/dl) and albumin (1.2 g/dl). Microhepatica was shown in abdominal radiography. During the ultrasound examination, dilated tortuous vein communicating with caudal vena cava ws observed near the stomach. Intraoperative jejunal vein portography was performed during laparotomy to confirm the location and size of shunt vessel. According to history taking, physical examination, hematologic and serum chemical examination and radiographic study, it was diagnosed as single extrahepatic portosystemic shunt. The anomalous vessel (7 mm, o.d.) that enter the caudal vena cava from the left gastric vein, near the level of the diaphragm, was identified. A Ameroid constrictor (5 mm, i.d.) was applied to the shunting vessel near the caudal vena cava. Hematologic and serum chemical values recovered gradually and were revealed normal values 4 months after surgery. Four month after surgery serum bile acids concentrations were 0.56 $\mu$mol/l (preprandial) and 18.45 umol/l (postprandial). Abdominal radiograph showed normal gastric axis and it revealed normal size of the liver. Fine texture and increased echogenecity of liver and enlargement of portal vein were shown in ultrasonography. Single extrahepatic portosystemic shunt might be treated surgically using Ameroid constrictor.
The purpose of the present study is to examine the effect of cell proliferation and alkaline phosphatase activity in osteoblastic cells and to compare the bone healing ability of rat calvarial defects between the control group and the safflower seed extract treated group. Osteoblastic cells were obtained from calvariae of a fetal rat. Cells were cultured containing DMEM and safflower seed extract ($10^{-6}g/ml$, $10^{-3}g/ml$) at $37^{\circ}$ with 5% $CO_2$ in 100% humidity for 3 days. MTT was performed to examine the viability of the cells, and alkaline phosphatase activity was analyzed to examine the mineralization in vitro. Rat calvarial defects($5{\times}5mm$) in 250g Sprague-Dawly were made using round bur. Rats were administrated with safflower seed extract(0.35g/kg/day) for experimental periods. Calvarial defects were studied histopathologically and immunohistochemically at 1,4, and 8 weeks. High concentration group($10^{-3}g/ml$) of safflower seed extract significantly increased in the cell proliferation and alkaline phos phatase synthesis in osteoblastic cells. The infiltration of inflammatory cells and osteoclastic activities were decreased in the safflower seed extract treated group as compared with control group. Bone maturation was accelerated in the safflower seed extract treated group as compared to control group. No difference in osteoinductive process was observed between the control and the safflower seed extract treated group. Immunohistochemical observation revealed that protein expression of TGF-$\beta$and osteonectin during early healing phase in the safflower seed extract treated group was slightly increased as compared to control group. These results indicate that safflower seed extract promotes the healing process in bony defect of rat calvariae, and retains a potential applicability as an adjuvant therapeutic modality for regeneration of periodontal bony defect.
홍삼 사포닌분획(SF)투여 시 TCDD(2,3,7,8-tetracholorodibenzo-${\rho}$-dioxin)투여(TT)에 의해 감소했던 웅성 기니피그의 체중과 간, 콩팥, 지라, 고환의 무게가 유의하게 증가하였다(p>0.01). SF투여 시 TCDD투여에 의해 감소했던 헤마토크릿 값, 적혈구 및 혈소판의 수, 아밀라아제 및 lactate dehydrogenase의 활성도, 요산, 총단백질 및 알부민의 양은 증가했고, 증가했던 백혈구의 수, 중성지질, 총콜레스테롤(TC), 혈당, 저밀도지단백질-콜레스테롤, 고밀도지단백질-콜레스테롤, 크레아티닌, 혈중 요소질소, 칼슘 및 인의 양과 creatinine kinase, aspartate aminotransferase, alanine aminotransferase, alkaline phosphatase의 활성은 감소하였다. 그리고 적혈구, 백혈구, 혈소판, 혈당, TC, 칼슘 및 알부민을 제외한 모든 지표들은 통계학적 유의성을 보였다(p>0.01). 이상의 결과로부터 SF는 TCDD에 의해 유도된 기니피그의 급성독성을 완화시켜 주는 효과가 있음을 알 수 있었다.
Bone morphogenetic proteins(BMPs) are a group of transforming growth factor beta(TGF-${\beta}$)-related factors and multifunctional proteins, especially the only known biologic factors capable of inducing endochondral bone formation at an extraskeletal site. This study was performed to investigate the effect of the partially purified porcine BMP(pBMP) at an ectopic site. PBMP was partially purified from porcine bone matrix and its activity was monitored by an in vivo bioassay. The purification method utilized extraction of the bone-inducing activity with 4M guanidine, followed by chromatography on heparin-Sepharose. Active fractions were assayed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. And the fractions were reconstituted with inactive insoluble collagenous bone matrix from rats, acid soluble type I collagen from rat tail and chondroitin-6-sulfate sodium salt and implanted into the pectroralis muscle pouches of Sprague-Dawley rats. And the carrier complex was implanted on the opposite side as control. The rats were sacrificed at the day of 1st, 3rd, 5th, 7th, 11th, 14th and 21st after implantation and examined histologically, radiologically and biochemically. And alkaline phosphatase activity and calcium content were used as indices of bone formation. The results were as follows ; 1. Active fractions were localized in a zone between 31 and 40 KDa on SDS-PAGE. 2. The implanted 3.0mg of the partially purified pBMP induced cartilage and bone in the muscle tissue of rats through an endochondral ossification process. 3. Inactive insoluble bone matrix, type I collagen and chondroitin-6-sulfate have functioned as carriers for pBMP, but revealed some foreign body reactions. 4. Soft X-ray didn't reveal significant change between the experimental and the control group. 5. The alkaline phosphatase activities in the experimental group of 5th, 7th, 11th, 14th and 21st were increased significantly compared with control (p<0.01) with the peak in the group of 11th day. 6. With time, the calcium content of the experimental group increased. And the calcium contents in the experimental group of 11th, 14th and 21st were increased significantly compared with control (p<0.01).
Brain, heart, liver, lung, kidney and thymus etc. 12 organs were removed and homogenized from Dawley-Sprague rats after suffocation. After fractionation of the tissue cytosols, enzymatic activities of the key enzymes in metabolic inositol phosphates cycle, PLC, IPSK and Ins(1,4,5) P35-phosphatase, were measured respectively. Hybridoma monoclones producing anti-lP3K murine monoclonal antibodies were obtained by the fusion of SP2/Ag 0-14 and spleen cells of mouse immunized with purified 53KDa IPSK, screening and cloning procedures. 18 cloned hybridoma cells were obtained, background due to nonspecific binding was very low with 10 clones. These Abs were purified from ascitic fluids by using affi-gel 15, and determined subtype of Abs. When immunoreactivities for rat tissues IP3K were exercised by adding the mixed Abs of 19Gl and 19G2b, they showed an overall similarity with noncompetitive inhibition. Brain tissue has high sensitivity for anti-lP3K Ab, whereas heart tissue has very low activity. In kinetic parameters Km value was 1.58 mM and Vmx value was 5.41umol/min/ml, respectively Only one form of 40 KDa IPSK was detected in heart tissues, however rat brain contains at least three immunologically distinct IP3K (53, 51 and 40 KDa) in western blot analysis. Of them 53 KDa protein was major enzyme in enzymatic activity. Northern blot analysis with 32P-labeled CDNA probe which encodes 1.8 Kb IPSK gene was performed. These results suggest that IPSK are regulated at transcriptional level during rat tissue development.
The effect of ginseng administration on the patients of acute viral (B type) hepatitis has been oberved and the results were as follows. The albumin/globulin ratio of the ginseng administered group has significantly improved 4 weeks after admission while that of control group has not been improved suggesting that the ginseng might be effective in improving the protein metabolism. The thymol turbidity test again gave a similar result. Recovery of the disorder of bilirubin metabolism was also accelerated in the ginseng administered group compared with control group. The raised bilirubin value of the former returnedto the normal value 2 week after admission while that of the latter reached to normal 4-5 weeks after admission. However no significant difference of the bilirubin level between ginseng treated and non-treated groups could be observed. Cholesterol metabolism is also stimulated in ginseng administered group. The lowered cholesterol level of the ginseng group returned to normal 3-4 weeks after admission while that of latter reached to normal 5-6 weeks after admission. The raised S-GOT and S-GPT levels of the ginseng treated group returned to the normal value 3-4 weeks after admission while those of control group rehimed to normal in 5 weeks after admission suggesting that the ginseng improved impaired liver function. The improvement of the raised transaminase level seemed to be accelerate6 by the ginseng administration, however, no significant difference of the transaminase level between the ginseng treated and non-treated group could be observed. A significant effect of ginseng on the raised alkaline phosphatase level was observed. From the above results, it seemed that ginseng might stimulate the improvement of the disturbance of liver function, particularly at the early phase of its development of acute liver disease suggesting that panax ginseng might play a significant role in preventing the disease developing to be chronic.
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