• Title/Summary/Keyword: Direct PCR (dPCR)

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Effect of magnesium and calcium phosphate coatings on osteoblastic responses to the titanium surface

  • Park, Ki-Deog;Lee, Bo-Ah;Piao, Xing-Hui;Lee, Kyung-Ku;Park, Sang-Won;Oh, Hee-Kyun;Kim, Young-Joon;Park, Hong-Ju
    • The Journal of Advanced Prosthodontics
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    • v.5 no.4
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    • pp.402-408
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    • 2013
  • PURPOSE. The aim of this study was to evaluate the surface properties and in vitro bioactivity to osteoblasts of magnesium and magnesium-hydroxyapatite coated titanium. MATERIALS AND METHODS. Themagnesium (Mg) and magnesium-hydroxyapatite (Mg-HA) coatings on titanium (Ti) substrates were prepared by radio frequency (RF) and direct current (DC) magnetron sputtering.The samples were divided into non-coated smooth Ti (Ti-S group), Mg coatinggroup (Ti-Mg group), and Mg-HA coating group (Ti-MgHA group).The surface properties were evaluated using scanning electron microscopy (SEM) and X-ray photoelectron spectroscopy (XPS). The surface roughness was evaluated by atomic force microscopy (AFM). Cell adhesion, cell proliferation and alkaline phosphatase (ALP) activity were evaluated using MC3T3-E1 cells. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed. RESULTS. Cross-sectional SEM images showed that Mg and Mg-HA depositionson titanium substrates were performed successfully. The surface roughness appeared to be similaramong the three groups. Ti-MgHA and Ti-Mg group had improved cellular responses with regard to the proliferation, alkaline phosphatase (ALP) activity, and bone-associated markers, such as bone sialoprotein (BSP) and osteocalcin (OCN) mRNA compared to those of Ti-S group. However, the differences between Ti-Mg group and Ti-MgHA group were not significant, in spite of the tendency of higher proliferation, ALP activity and BSP expression in Ti-MgHA group. CONCLUSION. Mg and Mg-HAcoatings could stimulate the differentiation into osteoblastic MC3T3-E1 cells, potentially contributing to rapid osseointegration.

Korean Red Ginseng-intake has Definite Clinical Usefulness and causes Nef Gene Variation including High Frequency of Deletion

  • Cho Young Keol;Lee Hee Kyung;Ahn Sun Hee;Lee Hee Jung;Nam Ki Yeul
    • Proceedings of the Ginseng society Conference
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    • 2002.10a
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    • pp.185-211
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    • 2002
  • We have found many beneficial effects of the long-tenn intake of Korean red ginseng (KRG) in human immunodeficiency virus (HIV) type-I infected patients, including the maintenance of CD4+ T cell count for 10 years with KRG only and the delayed development of resistance mutation to ZDV. In this study, to investigate whether KRG-intake could affect the clinical progression and nef gene variation, we determined 200nef sequences from 70 patients. Follow-up period was $8.8{\pm}2.9$ years and annual decrease in CD4+T cell was $41{\pm}57/ul.$ Nested polymerase chain reaction (PCR) and direct sequencing were perfonned with peripheral blood mononuclear cells (PBMC) obtained at times during the study period. First, there was a significant correlation between survival duration and duration of KRG-intake $(36.8{\pm}38$ months)(P=0.000). There were significant correlations between the last NefProg score and CD4+ T cell count (r= 0.208, P<0.05) and annual decrease in CD4+ T cell count (r =0.346, P<0.01) in 70 patients. In addition, there were significant correlations between KRG-intake and annual decrease (r= 0.323, P<0.01), and the CD4+ T cell count itself (r=0.229, p<0.05). Furthennore, there was also a mild significance between the NefProg score and the duration of KRG-intake in only SP and RP (n=30, r=-0.281, P=0.067). In addition, we detected various defects in 21 patients $(30.0\%),$ not including 5 premature stop codons. Ten $(12.5\%)$ patients showed repeated deletion of an amino acid. Four of 10 patients were gross deletions and they were treated with KRG for more than 20 months. The number of patients with repeated gross deletions was significantly higher in the order of slow progressors $(18\%)$, typical progressors($3\%$), and rapid progressors($0\%$) (P<0.05). We also observed that long-tenn intake of KRG might make the change from A or D to T at position 54 and decrease NefProg score. Taken together, our results show clear evidence that the long-term intake of KRG has effects on nef gene variation as well as definite clinical usefulness.

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1828G>A polymorphism of the UDP-glucuronosyltransferase gene (UGT1A1) for neonatal hyperbilirubinemia in Koreans (한국인 신생아 황달에서 UGT1A1 유전자의 1828G>A 단일염기다형성에 관한 연구)

  • Kim, Ja Young;Kim, Mi Yeoun;Kim, Ji Sook;Kim, Eun Ryoung;Yoon, Seo Hyun;Lee, Hee Jae;Chung, Joo Ho
    • Clinical and Experimental Pediatrics
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    • v.49 no.1
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    • pp.34-39
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    • 2006
  • Purpose : The incidence of neonatal hyperbilirubinemia is twice as high in East Asians as in Caucasians. However, its metabolic basis has not been clearly explained. The UDP-glucuronosyltransferase gene(UGT1A1) mutation was found to be a risk factor of neonatal hyperbilirubinemia. We studied whether neonatal hyperbilirubinemia is associated with the 1828G>A(rs 10929303) polymorphism of the UGT1A1 gene, which encodes for a key enzyme of bilirubin metabolism. Methods : The genomic DNA was isolated from 80 Korean full term neonates who had greater than a 12 mg/dL level of serum bilirubin with no obvious cause, and the genomic DNA was also isolated from 164 Korean neonates of the control population. We studied a single nucleotide polymorphism (SNP) of 1828G>A in the untranslated region of the UGT1A1 gene by direct sequencing. Results : Three of the 80 neonates with a serum bilirubin level above 12 mg/dL had homozygous mutations and 10 of the 80 neonates with a serum bilirubin level above 12 mg/dL had heterozygous mutations. Thirteen of the 164 neonates of the control group had homozygous mutations and 16 neonates of the control group had heterozygous mutations. The allele frequency of 1828G>A polymorphism of UGT1A1 in the hyperbilirubinemia group was 10.0 percent, which was not significantly different from the allele frequency of 12.8 percent in the control group. Conclusion : In this study, the 1828G>A polymorphism of the UGT1A1 gene was detected in the Korean neonates with neonatal hyperbilirubinemia. Our results indicated that this SNP is not associated with the prevalence of hyperbilirubinemia in Koreans.

The relationship between Gly71Arg and TATA box polymorphism of GT1A1 gene and prolonged hyperbilirubinemia of breast milk feeding infant in Korean (지속성 고빌리루빈혈증과 연관된 모유 황달에서 UGT1A1(Gly71Arg, TATA box) 다형성에 대한 연구)

  • Lee, Jae Myoung;Han, Young Ji;Kim, Ji Sook;Kim, Eun Ryoung
    • Clinical and Experimental Pediatrics
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    • v.51 no.2
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    • pp.150-155
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    • 2008
  • Purpose : It has been known that breast milk cause prolonged unconjugated hyperbilirubinemia. UGT1A1 is a important gene of uridine diphosphate glucuronosyltransferase (UGT) which has a major role of bilirubin metabolism. These findings suggest that there is a relationship between UGT1A1 gene mutation and prolonged jaundice of breast feeding infant. The aim of study was to investigate whether a polymorphism of the UGT1A1 gene exist in prolonged hyperbilirubinemia of breast milk feeding Korean infant. Methods : The genomic DNA was isolated from 50 full term Korean neonates, who had greater than a 10 mg/dL of serem bilirubin after 2 weeks of birth with no significant cause, and the other genomic DNA was isolated from 162 full term Korean neonates of the control population. Both group fed breast milk. We performed direct sequencing of TATA box and Gly71Arg polymorphism of the UGT1A1 gene. Results : Two of the 50 neonates with hyperbilirubinemia had AA polymorphism, and 40 had GA polymorphism. Five of the 129 neonates of the control group had AA polymorphism, and 4 had GA polymorphism. The allele frequency of G>A polymorphism in the hyperbilirubinemia group was 44.0%; it was significantly higher than 5.4% of the control group. TATA box polymorpism was not different both group significantly. Conclusion : Our result indicated that Gly71Arg polymorphism is associated with the prolonged hyperbilirubinemia of breast milk-feeding infant in Korean, while TATA box polymorphism is not associated with the prolonged hyperbilirubinemia of breast milk-feeding infant in Korean.

The Clinical Features of Chronic Neonatal Hepatitis: Non-familial, Non-metabolic and Non-A, B, C Viral Hepatitis (만성 신생아 간염의 임상적 고찰: 비-가족형, 비-대사성, 비-A, B, C형 바이러스성 신생아 간염)

  • Park, Ji Ae;Lee, Chang Hun;Park, Jae Hong
    • Pediatric Gastroenterology, Hepatology & Nutrition
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    • v.9 no.2
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    • pp.242-248
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    • 2006
  • Purpose: Neonatal hepatitis is the major cause of neonatal cholestasis and may be divided into infectious, metabolic, genetic, and idiopathic neonatal hepatitis. Non-familial, non-metabolic, and non-A, B, C viral neonatal hepatitis is known to have made satisfactory progress, but little is known about its chronic clinical features. Methods: Clinical and histological assessments were carried out in 34 cases with chronic neonatal hepatitis [elevated serum alanine aminotrasferase (ALT) level for more than 6 months] except for A, B, C viral hepatitis, metabolic, or genetic neonatal hepatitis, who were admitted to the Department of Pediatrics, Pusan National University Hospital, from January 1998 to January 2004. Results: Males were more common (70%). Jaundice (100%) and hepatomegaly (44%) were frequent manifestations. Peak serum ALT levels were most commonly below 300 IU/L in 41.2% of patients and peak serum direct bilirubin levels were most commonly between 1.0~5.0 mg/dL in 50% of patients. Ten cases (34%) of 29 patients had positive serum cytomegalovirus (CMV) IgM or urine CMV polymerase chain reaction. Serum ALT level was normalized within 1 year in 11 (37.9%) of 29 cases, and within 2 years in 9 (69.2%) of 13 cases. Serum ALT level was elevated persistently over 2 years in four (30.7%) of 13 cases. Histologic findings such as portal or periportal activity, lobular necrosis, portal or periportal fibrosis were more severe in patients with persistent ALT elevation over 2 years than in those showing normalization of ALT within 2 years (p>0.05). Conclusion: When the elevation of ALT level sustains over 1 year in non-familiar, non-metabolic, non-A, B, C viral neonatal hepatitis, an assessment of the severity of liver injury and a careful monitoring about chronic liver disease may be required.

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Hsp90 Inhibitor Induces Cell Cycle Arrest and Apoptosis of Early Embryos and Primary Cells in Pigs

  • Son, Myeong-Ju;Park, Jin-Mo;Min, Sung-Hun;Hong, Joo-Hee;Park, Hum-Dai;Koo, Deog-Bon
    • Reproductive and Developmental Biology
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    • v.35 no.1
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    • pp.33-45
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    • 2011
  • Heat shock protein 90 (Hsp90) is ATPase-directed molecular chaperon and affects survival of cancer cell. Inhibitory effect of Hsp90 by inducing cell cycle arrest and apoptosis in the cancer cell was reported. However, its role during oocyte maturation and early embryo development is very insufficient. In this study, we traced the effects of Hsp90 inhibitor, 17-allylamino-17-demethoxygeldanamycin (17-AAG), on meiotic maturation and early embryonic development in pigs. We also investigated several indicators of developmental potential, including structural integrity, gene expression (Hsp90-, cell cycle-, and apoptosis-related genes), and apoptosis, which are affected by 17-AAG. Then, we examined the roles of Hsp90 inhibitor on viability of primary cells in pigs. Porcine oocytes were cultured in the NCSU-23 medium with or without 17-AAG for 44 h. The proportion of GV arrested oocytes was significantly different between the 17-AAG treated and untreated group (78.2 vs 34.8%, p<0.05). After completion of meiotic maturation, the proportion of MII oocytes was lower in the 17-AAG treated group than in the control group (27.9 vs 71.0%, p<0.05). After IVF, the percentage of penetrated oocytes was significantly lower in the 17-AAG treated group (25.2%), resulting in lower normal pronucleus formation (2PN of 14.6%). Therefore, the inhibition of meiotic progression by Hsp90 inhibitor played a critical role in fertilization status. Porcine embryo were cultured in the PZM-3 medium with or without 17-AAG for 6 days. In result, significant differences in developmental potential were detected between the embryos that were cultured with or without 17-AAG. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) showed that the number of containing fragmented DNA at the blastocyst stage increased in the 17-AAG treated group compared with control (7.5 vs 4.4, respectively). Blastocysts that developed in the 17-AAG treated group had low structural integrity and high apoptotic nuclei than those of the untreated control, resulting in decrease the embryonic qualities of preimplantation porcine blastocysts. The mRNA expressions of cell cycle-related genes were down-regulated in the 17-AAG treated group compared with control. Also, the expression of the pro-apoptotic gene Bax increased in 17-AAG treated group, whereas expression of the anti-apoptotic gene Bel-XL decreased. However, the expression of ER stress-related genes did not changed by 17-AAG. Cultured pESF cells were treated with or without 17-AAG and used for MIT assay. The results showed that viability of pESF cells were decreased by treatment of 17-AAG ($2{\mu}M$) for 24 hr. These results indicated that 17-AAG decreased cell proliferation and increased cell death. Expression patterns Hsp90 complex genes (Hsp70 and p23), cell cycle-related genes (cdc2 and cdc25c) and apoptosis-related genes (Bax and Bcl-XL) were significantly changed by using RT-PCR analysis. The spliced form of pXbp-1 product (pXbp-1s) was detected in the tunicamycin (TM) treated cells, but it is not detected in 17-AAG treated cells. In conclusion, Hsp90 appears to play a direct role in porcine early embryo developmental competence including structural integrity of blastocysts. Also, these results indicate that Hsp90 is closely associated with cell cycle- and apoptosis-related genes expression in developing porcine embryos.