• Title/Summary/Keyword: Replication Protein A

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Recombinant Adenoviral Vector Containing Tumor-Specific L-Plastin Promoter Fused to Cytosine Deaminase Gene as a Transcription Unit: Generation and Functional Test

  • Chung, In-Jae;Deisseroth, Albert-B.
    • Archives of Pharmacal Research
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    • v.27 no.6
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    • pp.633-639
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    • 2004
  • The expression of therapeutic transgenes in recombinant adenoviral vectors is a major cause of toxicity in dividing cancer cells as well as non dividing normal cells. To solve the problem of toxicity to normal cells, we have reported on a recombinant adenoviral vector system (AdLP-) in which the expression of the transgene is directed by the tumor-specific L-plastin promoter (LP) (Chung et al., 1999). The object of this study was to generate a recombinant adenoviral vector system which would generate tumor cell specific expression of cytosine deaminase (CD) gene. We report the construction of a replication-incompetent adenoviral vector in which CD is driven by the L-plastin promoter (AdLPCD). Infection of 293 cells by AdLPCD generated the functional CD protein as measured by HPLC analysis for the conversion of 5-Fluorocy-tosine (5-FC) to 5-Fluorouracil (5-FU). HPLC analysis in conjunction with counting radioactivity for [6-$^3$H]-5FC and [6-$^3$H]-5FU demonstrated vector dose-dependent conversion of 5-FC to 5-FU in AdLPCD infected ovarian cancer cells. The results from present and previous studies(Peng et al., 2001; Akbulut et al., 2003) suggest that the use of the AdLPCD/5-FC system may be of value in the treatment of cancer including microscopic ovarian cancer in the peritoneal cavity.

Plasma Surface Modification of Patterned Polyurethane Acrylate (PUA) Film for Biomedical Applications

  • Yun, Young-Shik;Kang, Eun-Hye;Yun, In-Sik;Kim, Yong-Oock;Yeo, Jong-Souk
    • Proceedings of the Korean Vacuum Society Conference
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    • 2015.08a
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    • pp.223.2-223.2
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    • 2015
  • Polyurethane acrylate (PUA) has been introduced to utilize as a mold material for sub-100 nm lithography as it provides advantages of stiffness for nanostructure formation, short curing time, flexibility for large area replication and transparency for relevant biomedical applications. Due to the ability to fabricate nanostructures on PUA, there have been many efforts to mimic extracellular matrix (ECM) using PUA especially in a field of tissue engineering. It has been demonstrated that PUA is useful for investigating the nanoscale-topographical effects on cell behavior in vitro such as cell attachment, spreading on a substrate, proliferation, and stem cell fate with various types of nanostructures. In this study, we have conducted surface modification of PUA films with micro/nanostructures on their surfaces using plasma treatment. In general, it is widely known that the plasma treated surface increases cell attachment as well as adsorption of ECM materials such as fibronectin, collagen and gelatin. Effect of plasma treatment on PUA especially with surface of micro/nanostructures needs to be understood further for its biomedical applications. We have evaluated the modified PUA film as a culture platform using adipose derived stem cells. Then, the behavior of stem cells and the level of adsorbed protein have been analyzed.

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3',5'-Cyclic Adenosine Monophosphate (cAMP) as a Signal and a Regulatory Compound in Bacterial Cells (원핵세포에서 신호물질 및 조절인자로서의 3',5'-Cyclic Adenosine Monophosphate의 역할)

  • Chun, Se-Jin;Seok, Young-Jae;Lee, Kyu-Ho
    • Microbiology and Biotechnology Letters
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    • v.34 no.4
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    • pp.289-298
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    • 2006
  • 3',5'-cyclic adenosine monophosphate (cAMP) is an important molecule, which mediates diverse cellular processes. For example, it is involved in regulation of sugar uptake/catabolism, DNA replication, cell division, and motility in various acterial species. In addition, cAMP is one of the critical regulators for syntheses of virulence factors in many pathogenic bacteria. It is believed that cAMP acts as a signal for environmental changes as well as a regulatory factor for gene expressions. Therefore, intracellular concentration of cAMP is finely modulated by according to its rates of synthesis (by adenylate cyclase), excretion, and degradation (by cAMP phosphodiesterase). In the present review, we discuss the bacterial physiological characteristics governed by CAMP and the molecular mechanisms for gene regulation by cAMP. Furthermore, the effect of cAMP on phosphotransferase system is addressed.

Growth Performance and Digestive Characteristics of Starry Flounder Platichthys stellatus on the Moist and Extruded Pellets (습사료와 부상사료에 대한 강도다리(Platichthys stellatus)의 성장과 소화 특성)

  • Kim, Pyong Kih
    • Korean Journal of Fisheries and Aquatic Sciences
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    • v.45 no.6
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    • pp.679-685
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    • 2012
  • A study was carried out to observe the effects of feed types on the growth, feed preference, and enteric feed transition rate of juvenile starry flounder, Platichthys stellatus for 45 days. Fifty fish (avg. 135 g) were stocked each in replication, and fed a commercial extruded pellet diet (EP, 45% protein) and a moist pellet diet (MP, 65% raw mackerel+35% feed powder in wet basis), respectively. The MP presented the higher performance than that of the EP on the feed efficiency ($68.3{\pm}0.9%$ for EP and $92.3{\pm}4.3%$ for MP) and the specific growth rate ($1.07{\pm}0.07$ for the EP and $1.20{\pm}0.05%$ for the MP). In contrast, the EP showed the higher feed preference in terms of the daily feed intake ($1.57{\pm}0.08%$ for the EP and $1.30{\pm}0.01$ for the MP) and the ad libitum feeding rate after a fast of 72 hours (1.73% for the EP and 1.35% for the MP). The feed transition rate through intestinal canals decreased exponentially in both the EP and the MP, showing the faster transition rate with the EP. In the result, starry flounder appeared to have the better feed preference to the EP, but have the higher feed efficiency and growth performance to the MP.

Molecular Miology of the Poliovirus (폴리오바이러스의 분자생물학)

  • 최원상
    • Journal of Life Science
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    • v.7 no.4
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    • pp.392-401
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    • 1997
  • The poliovirus is a small, and non-enveloped virus. The RNA genome of poliovirus is continuous, linear, and has a single open reading frame. This polyprotein precursor is cleaved proteolytically to yield mature products. Most of the cleavages occur by viral protease. The mature proteins derived from the P1 polyprotein precursor are the structural components of the viral capsid. The initial cleavage by 2A protease is indirectly involved in the cleavage of a cellular protein p220, a subunit of the eukaryotic translation initiation factor 4F. This cleavage leads to the shut-off of cap-dependent host cell translation, and allows poliovirus to utilize the host cell machinery exclusively for translation its own RNA, which is initiated by internal ribosome entry via a cap-independent mechanism. The functional role of the 2B, 2C and 2BC proteins are not much known. 2B, 2C, 2BC and 3CD proteins are involved in the replication complex of virus induced vesicles. All newly synthesized viral RNAs are linked with VPg. VPg is a 22 amino acid polypeptide which is derived from 3AB. The 3C and 3CD are protease and process most of the cleavage sites of the polyprotein precursor. The 3C protein is also involved in inhibition of RNA polymerase II and III mediated transcription by converting host transcription factor to an inactive form. The 3D is the RNA dependent RNA polymerase. It is known that poliovirus replication follows the general pattern of positive strand RNA virus. Plus strand RNA is transcribed into complementary minus strand RNA that, in turn, is transcribed for the synthesis of plus strand RNA is transcribed into complementary minus strand RNA that, in turn, is transcribed for the synthesis of plus strand RNA strands. Poliovirus RNA synthesis occurs in a membranous environment but how the template RNA and proteins required for RNA replication assemble in the membrane is not much known. The RNA requirements for the encapsidation of the poliovirus genome (packaging signal) are totally unknown. The poliovirus infection cycle lasts approximately 6 hours.

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Experimental Infection of Different Tomato Genotypes with Tomato mosaic virus Led to a Low Viral Population Heterogeneity in the Capsid Protein Encoding Region

  • Sihelska, Nina;Vozarova, Zuzana;Predajna, Lukas;Soltys, Katarina;Hudcovicova, Martina;Mihalik, Daniel;Kraic, Jan;Mrkvova, Michaela;Kudela, Otakar;Glasa, Miroslav
    • The Plant Pathology Journal
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    • v.33 no.5
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    • pp.508-513
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    • 2017
  • The complete genome sequence of a Slovak SL-1 isolate of Tomato mosaic virus (ToMV) was determined from the next generation sequencing (NGS) data, further confirming a limited sequence divergence in this tobamovirus species. Tomato genotypes Monalbo, Mobaci and Moperou, respectively carrying the susceptible tm-2 allele or the Tm-1 and Tm-2 resistant alleles, were tested for their susceptibility to ToMV SL-1. Although the three tomato genotypes accumulated ToMV SL-1 to similar amounts as judged by semiquantitative DAS-ELISA, they showed variations in the rate of infection and symptomatology. Possible differences in the intra-isolate variability and polymorphism between viral populations propagating in these tomato genotypes were evaluated by analysis of the capsid protein (CP) encoding region. Irrespective of genotype infected, the intra-isolate haplotype structure showed the presence of the same highly dominant CP sequence and the low level of population diversity (0.08-0.19%). Our results suggest that ToMV CP encoding sequence is relatively stable in the viral population during its replication in vivo and provides further demonstration that RNA viruses may show high sequence stability, probably as a result of purifying selection.

Cytotoxic T Lymphocytes Elicited by Dendritic Cell-Targeted Delivery of Human Papillomavirus Type-16 E6/E7 Fusion Gene Exert Lethal Effects on CaSki Cells

  • Wu, Xiang-Mei;Liu, Xing;Jiao, Qing-Fang;Fu, Shao-Yue;Bu, You-Quan;Song, Fang-Zhou;Yi, Fa-Ping
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.6
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    • pp.2447-2451
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    • 2014
  • Human papillomavirus (HPV) is the primary etiologic agent of cervical cancer. Consideration of safety and non human leukocyte antigen restriction, protein vaccine has become the most likely form of HPV therapeutic vaccine, although none have so far been reported as effective. Since tumor cells consistently express the two proteins E6 and E7, most therapeutic vaccines target one or both of them. In this study, we fabricated DC vaccines by transducing replication-defective recombinant adenoviruses expressing E6/E7 fusion gene of HPV-16, to investigate the lethal effects of specific cytotoxic T lymphocytes (CTL) against CaSki cells in vitro. Mouse immature dendritic cells (DC) were generated from bone marrow, and transfected with pAd-E6/E7 to prepare a DC vaccine and to induce specific CTL. The surface expression of CD40, CD68, MHC II and CD11c was assessed by flow cytometry (FCM), and the lethal effects of CTL against CaSki cells were determined by DAPI, FCM and CCK-8 methods. Immature mouse DC was successfully transfected by pAd-E6/E7 in vitro, and the transfecting efficiency was 40%-50%. A DC vaccine was successfully prepared and was used to induce specific CTL. Experimental results showed that the percentage of apoptosis and killing rate of CaSki cells were significantly increased by coculturing with the specific CTL (p <0.05). These results illustrated that a DC vaccine modified by HPV-16 E6/E7 gene can induce apoptosis of CaSki cells by inducing CTL, which may be used as a new strategy for biological treatment of cervical cancer.

Use of Tannery Wastes in the Diet of Broiler

  • Alam, M.J.;Amin, M.R.;Samad, M.A.;Islam, M.A.;Wadud, M.A.
    • Asian-Australasian Journal of Animal Sciences
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    • v.15 no.12
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    • pp.1773-1775
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    • 2002
  • Tannery waste contained 90.93% DM, 77.02% CP, 0.77% CF, 2.83% EE, 7.19% ash and 3,450 kcal ME/kg DM. A total of 144 day-old broiler chicks were divided into three dietary groups; $D_1$ (Containing 10% protein concentrate-PC), $D_2$ (Containing 5% PC+5% tannery waste-TW) and $D_3$ (Containing 10% TW) having 3 replicates of 16 chicks in each. The birds were fed broiler starter diet containing 22% CP, 3,000 kcal ME/kg and broiler finisher diet containing 21% CP, 3,100 kcal ME/kg up to 42 days of age, and meat yield traits were measured from the representative birds from each replication to asses the feasibility of using tannery waste in the diet of broiler. Feed intake, live weight, feed conversion efficiency and livability did not differ between diets (p>0.05) but the cost of production and profitability differed significantly (p<0.001). Profitability of D1, D2, and D3 diets were 2.98, 9.90 and 14.04 Taka/kg respectively. Diets did not affect on meat yield traits (p>0.05), except gizzard, shank and feather weight (p<0.01). Gizzard and shank weigh were improved with increasing level of tannery wastes in the diet, hence tannery waste can be used without any harmful effect in the broiler diet.

Effect of combinatorial bone morphogenetic protein 2 and bone morphogenetic protein 7 gene delivery on osteoblastic differentiation

  • Bae, Young;Kim, Kyoung-Hwa;Kim, Su-Hwan;Lee, Chul-Woo;Koo, Ki-Tae;Kim, Tae-Il;Seol, Yang-Jo;Ku, Young;Rhyu, In-Chul;Chung, Chong-Pyoung;Lee, Yong-Moo
    • Journal of Periodontal and Implant Science
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    • v.39 no.sup2
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    • pp.279-286
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    • 2009
  • Purpose: Gene therapy (ex vivo) has recently been used as a means of delivering bone morphogenetic proteins (BMPs) to sites of tissue regeneration. In the present study, we investigated the effect of co-transduction of adenoviruses expressing BMP-2 and BMP-7 on osteogenesisof C2C12 cells in vitro. Methods: A replication-defective human adenovirus 5 (Ad5) containing a cDNA for BMPs in the E1 region of the virus (Ad5BMP-2 and Ad5BMP-7) was constructed by in vivo homologous recombination. Functional activity of Ad5BMP-2 and Ad5BMP-7 were evaluated in mouse stromal cells (W20-17cells). C2C12 cells are transduced with various MOI (multiplicity of infection) of Ad5BMP-2 and Ad5BMP-7 to assess most effective and stable titer. Based on this result, C2C12 cells were transduced with Ad5BMP-2 and Ad5BMP-7 alone or by combination. BMPs expression, alkaline phosphatase (ALPase) activity, cell proliferation, and mineralization were assessed. Results: Ad5BMP-2 and Ad5BMP-7 are successfully transduced to W20-17 cells, and secreted BMPs stimulated cell differentiation. Also, C2C12 cells transduced with Ad5BMPs showed expression of BMPs and increased ALPaseactivity. In all groups, cell proliferation was observed over times. At 7days, cells co-transduced with Ad5BMP-2 and Ad5BMP-7 showed lower proliferation than the others. C2C12 cells co-transduced with Ad5BMP-2 and Ad5BMP-7 had greater ALPaseactivity than that would be predicted if effect of individual Ad5BMPs were additive. Little mineralized nodule formation was detected in cells transduced with individual Ad5BMPs. In contrast, Ad5BMP-2 and Ad5BMP-7 combination stimulated mineralization after culturing for 10 days in mineralizing medium. Conclusions: Present study demonstrated that adenoviruses expressing BMPs gene successfully produced BMPs protein and these BMPs stimulated cells to be differentiated into osteoblastic cells. In addition, the osteogenic activity of Ad5BMPs can be synergistically increased by co-transduction of cells with Ad5BMP-2 and Ad5BMP-7.

Menstrual Discomfort and Dietary Habits in Adolescents (여중생의 월경 불편감과 식습관 관련 특성)

  • Hong Kyung-ja;Kim Hae-won;Ahn Hye-young
    • Child Health Nursing Research
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    • v.11 no.3
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    • pp.330-339
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    • 2005
  • Purpose: This study was done to identify menstrual discomfort and dietary habits, and factors related to the menstrual discomforts. Method: Participants were 320 female middle school students in G city. The Menstrual Discomfort Questionaire(MDQ) and dietary habit lists were used as tools. Results: Factors related to MDQ were found to be the VAS scores (r=.361, p=.002), a mount of menstrual bleeding (r=.131, p=.019), height (r=.134, p=.016), adequacy of meal time (t=7.19, p=.008), consumption of milk & milk products (F=3.20, p=.042) and, hot, salty & irritant foods (f=8.01, p=.000), eating more than 3 kinds of side-dishes with each meal (F=8.32, p=.000), and various protein foods (F=5.15, p=.006). In stepwise regression, 4 variables (VAS scores, height, hot, salty & irritant foods, more than 3 kinds of side-dishes with each meal) explained $20.3\%$ of the variance in the total MDQ scores. Conclusion: Reduction of hot, salty & irritant foods and having more than 3 side-dishes with each meal would be effective in menstrual discomfort management. As well, good and, balanced dietary habits should be encouraged for early adolescent girls. To keep up with the ideal dietary habits, nutritional education & counseling should be continued. In a further study, a replication study with larger samples and more specified nutritional assessment are recommended.

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