• Title/Summary/Keyword: PCR additive

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In vitro Methanogenesis, Microbial Profile and Fermentation of Green Forages with Buffalo Rumen Liquor as Influenced by 2-Bromoethanesulphonic Acid

  • Agarwal, Neeta;Kamra, D.N.;Chatterjee, P.N.;Kumar, Ravindra;Chaudhary, L.C.
    • Asian-Australasian Journal of Animal Sciences
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    • v.21 no.6
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    • pp.818-823
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    • 2008
  • The interaction of fibre degrading microbes and methanogens was studied using two forages, lucerne (Medicago sativa) hay and maize (Zea mays) hay, as substrate and 2-bromoethanesulphonic acid (BES) as an additive in an in vitro gas production test. Gas and methane production (ml/g dry matter) were significantly higher (p<0.05) on lucerne as compared to maize hay. Inclusion of BES in the incubation medium significantly suppressed methane emission irrespective of substrate. The population density of total bacteria, fungi, Ruminococcus flavefaciens and Fibrobacter succinogenes was higher, whereas that of methanogens was lower with maize hay as compared to lucerne as substrate. BES suppressed methanogen population by 7 fold on lucerene and by 8.5 fold on maize at 24 h incubation as estimated by real time-PCR. This suppression was accompanied by almost complete (>98% of control) inhibition of methanogenesis. The proportion of acetate decreased, whereas that of propionate increased significantly by inclusion of BES, resulting in narrowing of acetate to propionate ratio. In vitro true digestibility (IVTD) of lucerne was significantly higher as compared to maize but BES inclusion did not affect IVTD.

Association between PCR-RFLP Polymorphism of the Fifth Intron in Lipoprotein Lipase Gene and Productive Traits in Pig Resource Family

  • Zhang, B.Z.;Lei, M.G.;Deng, C.Y.;Xiong, Y.H.;Zuo, B.;Li, F.E.
    • Asian-Australasian Journal of Animal Sciences
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    • v.18 no.4
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    • pp.458-462
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    • 2005
  • The study was aimed at detecting polymorphism of the fifth intron in lipoprotein lipase (LPL) gene and analyzing association between the polymorphism and productive traits. A pair of primers was designed for amplifying the fifth intron. Sequence analysis indicated that a G1171C substitution existed in Large White breed. The mutation was detected by PCR-AfaI-RFLP. Polymorphism analysis in a pig resource family showed that there existed significant effects on carcass and meat quality traits. Thoraxwaist fat thickness of BB genotype was significantly higher (14.2%, p<0.05) than that of AA on carcass traits, while BB genotype was significantly lower (3.6% p<0.01, 4.1% p<0.01; 2.3% p<0.01, 1.9% p<0.01; 1.8% p<0.01, 1.4% p<0.05) than AA and AB genotype in pH of m. Longissimus Dorsi (LD), m. Biceps Femoris (BF), m. Semipinali Capitis (SC). The allelic frequencies were also significantly different between indigenous Chinese breeds and exotic breeds. Data analyzed revealed that the mutation locus affected production traits mostly by additive effects. Based on these results, it is necessary to do more studies on LPL gene before making the LPL locus into the application of marker-assisted selection (MAS) programs.

Expression of Human Lactoferrin in Pichia pastoris (Pichia pastoris에서 Human Lactoferrin의 발현)

  • 임소용;주인선;윤동훈;성창근
    • Journal of the Korean Society of Food Science and Nutrition
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    • v.26 no.4
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    • pp.669-674
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    • 1997
  • This study was attempted to express human lactoferrin gene that has importance as a functional additive in food industry. Lactoferrin has distinctive antibacterial properties. Also, a number of phy-siological roles have been postulated for the lactoferrin in the modulation of immune and inflamatory responses and as a growth factor. Since it did not show feasible growth inhibition by antimicrobial test against HLF, Pichia pastoris was selected the best lactoferrin expression host. HLF expression plasmid pHIL-SI was integrated into the genomic DNA of P. pastoris GSl15. The integration was confirmed not only with 2.4Kb fragment of HLF gene by PCR(polymerase chain reaction) product, but also with same size of specific signal by southern blotting. Among the various pichica transformants, the JY-1 cell showed a positive response for the expression of HLF by the immunoblotting anaysis. The recombinant HLF protein was started to be secreated at 48hr of culture and reached at the highest secreation level at 96hr.

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Quantitative Real-Time PCR of Porcine Parvovirus as a Model Virus for Cleaning Validation of Chromatography during Manufacture of Plasma Derivatives (혈장분획제제 제조공정에서 크로마토그래피 세척 검증을 위한 모델바이러스로서의 Porcine Parvovirus 정량)

  • Kil Tae Gun;Kim Won Jung;Lee Dong Hyuk;Kang Yong;Sung Hark Mo;Yoo Si Hyung;Park Sue-Nie;Kim In Seop
    • Korean Journal of Microbiology
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    • v.41 no.3
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    • pp.216-224
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    • 2005
  • Chromatography has now been used successfully to provide the requisite purity for human plasma-derived biop-harmaceuticals such as coagulation factors and immunoglobulins. Recently, increasing attention has been focused on establishing efficient cleaning procedures to prevent potential contamination by microorganisms as well as carry-over contamination from batch to batch. The purpose of present study was to develop a cleaning validation system for the assurance of virus removal and/or inactivation during chromatography process. In order to establish an assay system for the validation of virus clearance during chromatography cleaning process, a quantitative real-time PCR method for porcine parvovirus(PPV) was developed, since PPV, a model virus for human parvovirus B19, has a high resistance to a range of physico-chemical treatment. Specific primers for amplification of PPV DNA was selected, and PPV DNA was quantified by use of SYBR Green I. The sensitivity of the assay was calculated to be 1.5 $TCID_{50}/ml$. The established real-time PCR assay was successfully applied to the validation of PPV removal and cleaning during SP-Sepharose cation chromatography for thrombin purification and Q-Sepharose anion chromatography for factor VIII purification. The comparative results obtained by real-time PCR assay and infectivity titrations suggested that the real-time PCR assay could be a useful method for chromatography cleaning validation and that it could have an additive effect on the interpretation and evaluation of virus clearance during the virus removal process.

Effects of Dietary Allium fistulosum L. and Tannic Acid on in vitro Ruminal Fermentation Characteristics and Methane Emission (국내산 파(Allium fistulosum L.)와 탄닌산을 이용한 사료첨가제가 in vitro 반추위 발효성상과 메탄 발생에 미치는 영향)

  • Lee, Shin-Ja;Eom, Jun-Sik;Kim, Hyun-Sang;Kim, Hyeong-Suk;Lee, Sung-Sill
    • Korean Journal of Organic Agriculture
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    • v.26 no.4
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    • pp.775-787
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    • 2018
  • This study was conducted to investigate for the natural methane emission inhibitor as a feed additive no adversely effect on rumen fermentation. Five different Control (Wheat barn (0.05 g), MRA(Methane Reduction Additive)-1 (Allium fistulosum L. (0.05 g)), MRA-2 (Sodium Lauryl Sulfate (0.025 g) + Wheat barn (0.025 g) mixed), MRA-3 (Sodium Dodecyl Sulfate (0.025 g) + Wheat barn (0.025 g) mixed), and MRA-4 (Allium fistulosum L. (0.02 g) + Tannic acid (0.02 g) + Wheat barn (0.01 g) mixed) contents were used to perform 3, 6, 9, 12, 24 and 48 h incubation for in vitro fermentation. Ruminal pH values were ranged within normal ruminal microbial fermentation. Dry matter digestibility was not significantly different across the treatments during the whole fermentation time. Also, the result of microbial growth had no adversely effect on during the whole fermentation time. At 24 h, methane emission was significantly lower (P<0.05) than all treatments except to MRA-1. Especially, MRA-4 carbon dioxide emission was significantly lower (P<0.05) than control at 9, 24 and 48 h incubation. In addition MRA-4 propionate concentration was significantly higher (P<0.05) than control at 24 h incubation. The result of RT-PCR Ciliate-associated methanogens were significantly lower (P<0.05) at MRA-1, MRA-3 and MRA-4 than control at 24 h incubation. Based on the present results, MRA-4 could be suggestible methane emission inhibitor as a natural feed additive.

Antiproliferative Properties of Methanolic Extract of Nigella sativa against the MDA-MB-231 Cancer Cell Line

  • Dilshad, Ahmad;Abulkhair, Omalkhair;Nemenqani, Dalal;Tamimi, Waleed
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.11
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    • pp.5839-5842
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    • 2012
  • Breast cancer is the most commonly diagnosed cancer in women in the world and is one of the leading causes of death due to cancer. Health benefits have been linked to additive and synergistic combinations of phytochemicals in fruits and vegetables. Nigella sativa has been shown to possess anti-carcinogenic activity, inhibiting growth of several cancer cell lines in vitro. However, the molecular mechanisms of the anti-cancer properties of Nigella sativa phytochemical extracts have not been completely understood. Our data showed that Nigella sativa extracts significantly inhibited human breast cancer MDA-MB-231 cell proliferation at doses of $2.5-5{\mu}g/mL$ (P<0.05). Apoptotic induction in MDA-MB-231 cells was observed in a dose-dependent manner after exposure to Nigella sativa extracts for 48 h. Real time PCR and flow cytometry analyses suggested that Nigella sativa extracts possess the ability to suppress the proliferation of human breast cancer cells through induction of apoptosis.

FSHR Gene Mutation and Its Effect on Litter Size in Pigs

  • Zhang, Shujun;Xiong, Yuanzhu;Den, Changyan;Xiao, Senmu;Xu, Jianxiang;Xia, Yu;Liu, Xiaohua;Wang, Chunfang;Sun, Shulin
    • Asian-Australasian Journal of Animal Sciences
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    • v.17 no.2
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    • pp.179-182
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    • 2004
  • The polymorphism of the locus FSHRB in intron6-Exon7 of FSHR gene was investigated by PCR-RFLPs in Erhualian, Large White and Landrace${\times}$Large White; The association of polymorphism and litter size was analyzed by using SAS. The results showed that 1) the polymorphism of the locus FSHRB was significantly associated with litter size; 2) the total born number (TBN) and number born alive (NBA) of the sows with BB genotype were increased (p<0.05) with additive effects of 1.02-1.42 and 1.04-1.27 pigs per litter, respectively; 3) among the sows with genotype AA, AB or BB, there was an insignificant difference in born weight and weaning weight. This gene may be an effective potential tool used in conjunction with traditional selection methods.

Frequencies, Inheritance of Porcine FSH-${\beta}$ Retroposon and its Association with Reproductive Traits

  • Li, Feng'e;Xiong, Yuanzhu;Deng, Changyan;Jiang, Siwen;Zheng, Rong
    • Asian-Australasian Journal of Animal Sciences
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    • v.15 no.2
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    • pp.179-183
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    • 2002
  • The fragment in intron I of FSH-${\beta}$ gene was amplified by PCR. According to the polymorphism, we analyzed the distribution of FSH-${\beta}$ retroposon in different pig breeds; its inheritance pattern in Large White${\times}$Meishan reference family; and the association of FSH-${\beta}$ retroposon with litter size, female reproductive organs measurement, ultrasonic backfat and other traits. The results showed that almost each Chinese indigenous pig had the retroposon, while foreign pig breeds rarely had; the frequencies of porcine FSH-${\beta}$ retroposon were strongly associated with breeds (p<0.01); the pattern of inheritance was consistent with Mendelian fashion; total number born (TNB) and number born alive (NBA) were increased per FSH-${\beta}$ retroposon (p<0.01) with additive effects of 1.2-1.8 and 1.4-1.8 pigs/litter, respectively; between the FSH-${\beta}$ retroposon carriers and non-carriers, there was an insignificant difference in the measurement of female reproductive organs, body weight at birth, backfat thickness, loin meat height, lean meat percentage, teat number, days to 100 kg, and average daily gain.

Effect of Single Nucleotide Polymorphism of Endothelial Differentiation G-Protein Coupled Receptor 1 (EDG1) Gene on Marbling Score in Hanwoo

  • Shin, Sung-Chul;Chung, Eui-Ryong
    • Food Science of Animal Resources
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    • v.32 no.6
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    • pp.776-782
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    • 2012
  • Marbling (intramuscular fat) is the most economically important meat quality trait in Hanwoo (Korean cattle). The endothelial differentiation G-protein coupled receptor 1 (EDG1) gene, involved in blood vessel formation, is located within the genomic region of a quantitative trait locus (QTL) for marbling on bovine chromosome 3. Thus, the EDG1 gene can be considered as a positional and functional candidate gene for meat quality in beef cattle. This study aimed to identify single nucleotide polymorphisms (SNPs) in the EDG1 gene and to evaluate their associations with carcass traits in Hanwoo population. We have sequenced a fragment of 5'-UTR of the EDG1 gene and identified one SNP. Genotyping of the g.166A>G SNP marker was carried out using PCR-RFLP analysis in 309 Hanwoo steers in order to evaluate their association with carcass traits. The g.166A>G SNP marker showed a significant effect on the marbling score. Animals with the GG genotype had higher marbling score compared with AA and AG genotypes (p<0.05). This SNP marker also showed a significant additive effects for the marbling score (p<0.05). These results suggest that the EDG1 gene can be used as a molecular marker for DNA marker-assisted selection in order to increase the levels of the marbling score in Hanwoo.

Anti-inflammatory Activities Verification of Vaccinum oldhami Fruit Ethanol Extracts on RAW 264.7 (RAW 264.7을 이용한 정금나무 열매(Vaccinum oldhami fruit)의 항염증 효과)

  • Lee, Jin-Young;Joo, Da-Hye;Yoo, Dan-Hee;Chae, Jung-Woo
    • Journal of Life Science
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    • v.27 no.4
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    • pp.417-422
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    • 2017
  • The purpose of this study was to investigate the role of the Vaccinum oldhami fruit extract as a cosmetic additive. As a result of having macrophage (RAW 264.7) measured a cell toxicity effects of 70% ethanol extract from Vaccinum oldhami fruit, it shown 118% with toxicity at $500{\mu}g/ml$ concentration. In nitric oxide synthesis inhibition effect, 70% ethanol extracts from Vaccinum oldhami fruit shown 47.3% at $1,000{\mu}g/ml$ concentration. The iNOS, COX-2 protein expression inhibitory effect by western blot of 70% ethanol extract from Vaccinum oldhami fruit was decreased by 36.13%, 29.61% at $500{\mu}g/ml$ concentration. And iNOS, COX-2 mRNA expression inhibitory effect by reverse-transcription-PCR of 70% ethanol extract from Vaccinum oldhami fruit was decreased by 62.25%, 90.07% at $500{\mu}g/ml$ concentration. All these finding that extract from Vaccinum oldhami fruit could prove that their have effects anti-inflammatory efficacy. And extract from Vaccinum oldhami fruit has potential as a cosmetic ingredients.