• Title, Summary, Keyword: Reverse pharmacology

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A Simple Method for Elimination of False Positive Results in RT-PCR

  • Martel, Fatima;Grundemann, Dirk;Schomig, Edgar
    • BMB Reports
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    • v.35 no.2
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    • pp.248-250
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    • 2002
  • Discrimination between the amplification of mRNA and contaminating genomic DNA is a common problem when performing a reverse transcriptase-polymerase chain reaction (RT-PCR). Even after treatment of the samples with DNAse, it is possible that negative controls (samples in which no reverse transcriptase was added) will give positive results. This indicates that there was amplification of DNA, which was not generated during the reverse transcriptase step. The possibility exists that Taq DNA polymerase acts as a reverse transcriptase, generating cDNA from RNA during the PCR step. In order to test this hypothesis, we incubated samples with a DNAse-free RNAse after the cDNA synthesis. Comparison of the results that were obtained from these samples (incubated with or without DNAse-free RNAse) confirms that the reverse transcriptase activity of Taq DNA polymerase I is a possible source of false positive results when performing RT-PCR from intronless genes. Moreover, we describe here a simple and rapid method to overcome the false positive results that originate by this activity of Taq polymerase.

HIV-1 reverse transcriptase 및 protease의 유전자 cloning

  • 최관용
    • Proceedings of the Korean Society of Applied Pharmacology
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    • pp.81-81
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    • 1993
  • Reverse transcriptase가 유도되는 것을 SDS-PAGE 및 효소활성 분석으로 확인할 수 있었으며 효소의 정제를 위한 예비실험으로 ammonium sulfate 및 DEAE cellulose ion exchange chromatorgraphy를 시행하였을 때 specific activity의 증가를 보여주므로서 reverse transcriptase의 분자클로닝 후 발현된 효소가 활성을 갖고 있음을 알 수 있었다. pMAL; cRI 재조합 유전자에 있는 protease의 유전자 발현을 유도했을 때 E. coli세포의 성장에 toxic하게 작용함을 관찰할 수 있었다. 이는 발현된 protease가 E. coil의 번식에 영향을 주는 것으로 믿어지며 Protease의 유전자 발현을 위한 host의 선택, inducer의 첨가시간 등 실험조건의 확립이 필요할 것으로 사료된다.

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Minimal Amount of Insulin Can Reverse Diabetic Heart Function: Sarcoplasmic Reticulum $Ca^{2+}$ Transport and Phospholamban Protein Expression

  • Kim, Hae-Won;Cho, Yong-Sun;Lee, Yun-Song;Lee, Eun-Hee;Lee, Hee-Ran
    • The Korean Journal of Physiology and Pharmacology
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    • v.3 no.2
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    • pp.175-182
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    • 1999
  • In the present study, the underlying mechanisms for diabetic functional derangement and insulin effect on diabetic cardiomyopathy were investigated with respect to sarcoplasmic reticulum (SR) $Ca^{2+}-ATPase$ and phospholamban at the transcriptional and translational levels. The maximal $Ca^{2+}$ uptake and the affinity of $Ca^{2+}-ATPase$ for $Ca^{2+}$ were decreased in streptozotocin-induced diabetic rat cardiac SR, however, even minimal amount of insulin could reverse both parameters. Levels of both mRNA and protein of phospholamban were significantly increased in diabetic rat hearts, whereas the mRNA and protein levels of SR $Ca^{2+}-ATPase$ were significantly decreased. In case of phospholamban, insulin treatment reverses these parameters to normal levels. Minimal amount of insulin could reverse the protein levels; however, it could not reverse the mRNA level of SR $Ca^{2+}-ATPase$ at all. Thus, the decreased SR $Ca^{2+}$ uptake appear to be largely attributed to the decreased SR $Ca^{2+}-ATPase$ level, which is further impaired due to the inhibition by the increased level of phospholamban. These results indicate that insulin is involved in the control of intracellular $Ca^{2+}$ in the cardiomyocyte through multiple target proteins via multiple mechanisms for the decrease in the mRNA for both SR $Ca^{2+}-ATPase$ and phospholamban which are unknown and needs further study.

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Friend Virus 를 이용한 Reverse Transcriptase 억제제의 생체제의 약효검색

  • 안형수;황인숙;이상준;김동섭;허인회
    • Proceedings of the Korean Society of Applied Pharmacology
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    • pp.278-278
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    • 1994
  • HIV와 유사하게 retro virus의 일종인 Friend virus of Anemia strain (FVA)을 BALB/c mice 에게 감염시 reverse transcriptase 의 활성에 의해 비장의 erythroid progenitor cell 에서 증식되므로서 비장의 비대 및 빈혈을 초래하게 된다. 이를 지표로 하여 reverse transcriptase 억제작용을 지닌 항 virus 약물을 천연성분으로 부터 검색하고자 하였다. 우선, 대조약물로 사용한 기존의 항 AIDS 약물인 zidovudine (AZT) 을 FVA 가 감염된 BAB/c mice 에게 18일간 투여시 (약 100 mg/kg/day, p.o. ) 대조군에 비해 비장의 비대 및 reverse transcriptase 활성이 90% 이상 억제되었으며, 이들의 혈청을 정상 BALB/c mice 에게 재투여시에도 유사한 결과를 나타내어 reverse transcriptase 를 억제하므로서 항 virus 작용을 나타낸다는 것을 입증하였다. 그러나, 혈액중 Hemoglobin등 빈혈의 지수는 대조군과 유사한 수치를 나타내므로서 정상으로 회복되지를 못하였다. 이것은 zidovudine 자체가 지니는 골수억제에 의한 부작용 때문인 것으로 고려된다.

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Inhibition of glycine on morphine-induced hyperactivity, reverse tolerance and postsynaptic dopamine receptor supersensitivity in mice

  • Shin, Kyung-Wook;Baik, Jong-Won;Hong, Jin-Tae;Shin, Im-Chul;Oh, Ki-Wan
    • Proceedings of the PSK Conference
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    • pp.152.2-153
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    • 2003
  • We examined the effects of glycine on the morphine-induced hyperactivity, reverse tolerance and postsynaptic dopamine receptor supersensitivity in mice. A single administration of morphine (10 mg $kg^{-1}$ s.c.) induced hyperactivity as measured in mice. The morphine-induced hyperactivity aws inhibited dose-dependently by the pretreatment with glycine(100, 200 and 400 mg $kg^{-1}$ i.p.). (omitted)

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HIV-1 RT (reverse transcriptase) 저해제에 대한 내성 발현 기전

  • 임광진
    • Proceedings of the Korean Society of Applied Pharmacology
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    • pp.67-69
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    • 1995
  • reverse transcription은 AIDS를 일으킨다고 알려진 바이러스인 HIV-1의 번식에는 필수적이나 인체 세포에는 필수적이 아니기에 이 단계를 표적으로 하는 AIDS 치료제가 우선적으로 개발되었다. 그 단계에 필요한 효소가 바이러스에 의해 만들어진 RT이며 이 효소의 작용을 저해하는 nucleoside 유도체들인 AZT, DDC, DDI 들이 현재 AIDS 환자의 치료에 사용되고 있다. 이들 nucleoside 유도체들은 세포안으로 들어가 triphosphate 형태로 변화된 후 dNTP와 상경적으로 경쟁하며 합성 중인 바이러스의 DNA에 들어가 DNA의 합성을 정지시켜 바이러스의 증식을 억제한다. 그러나, 이들 nucleoside 유도체들은 치료용량에서 심한 독성을 나타낼 뿐만 아니라 장기 투여시 내성을 나타내는 바이러스가 생겨나 AIDS의 치료를 불가능하게 하고 있다.

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Identification of Lys49-PLA2 from crude venom of Crotalus atrox as a human neutrophil-calcium modulating protein

  • Sultan, Md. Tipu;Li, Hong-Mei;Lee, Yong Zu;Lim, Soon Sung;Song, Dong-Keun
    • The Korean Journal of Physiology and Pharmacology
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    • v.20 no.2
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    • pp.177-183
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    • 2016
  • We fortuitously observed a human neutrophil intracellular free-calcium concentration ($[Ca^{2+}]_i$) increasing activity in the commercially available phosphodiesterase I (PDE I), which is actually dried crude venom of Crotalus atrox. As this activity was not observed with another commercially available pure PDE I, we tried to find out the causative molecule(s) present in 'crude' PDE, and identified Lys49-phospholipase A2 (Lys49-PLA2 or K49-PLA2), a catalytically inactive protein which belongs to the phospholipase A2 family, by activity-driven three HPLC (reverse phase, size exclusion, reverse phase) steps followed by SDS-PAGE and LC-MS/MS. K49-PLA2 induced $Ca^{2+}$ influx in human neutrophils without any cytotoxic effect. Two calcium channel inhibitors, 2-aminoetoxydiphenyl borate (2-APB) ($30{\mu}M$) and SKF-96365 ($20{\mu}M$) significantly inhibited K49-PLA2-induced $[Ca^{2+}]_i$ increase. These results suggest that K49-PLA2 modulates $[Ca^{2+}]_i$ in human neutrophils via 2-APB- and SKF-96365-sensitive calcium channels without causing membrane disruption.

FVA에 감염된 BALB/c mice를 이용한 생체내 항 AIDS 약물의 약효검색

  • 안형수;이상준;김동섭;허인회
    • Proceedings of the Korean Society of Applied Pharmacology
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    • pp.79-79
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    • 1993
  • BALB/C mice 에게 FVA를 감염시킨 후, zidobudine을 1 mg/ml 의 농도로 식수에 용해하여 18일 동안 자유롭게 섭취 시켰을때 (150-200 mg/kg/day) 비장비대가 90% 억제되었고, 혈액 중 reverse transcriptase 활성은 85%로 억제효과를 나타내었으며, 빈혈지수는 회복되는 경향을 나타내었다. 한편, ddI 는 AZT와 동일 용량에서 비장비대는 17%, 혈액 중 reverse transcriptase 억제효과는 34% 억제효과를 나타내었다. 반면에 ddC는 단독투여시 비장비대를 38% 억제하였으며, interferon $\alpha$와 병용투여시 83%의 억제효과를 나타내었다. 이상은 FVA 감염에 의한 mice의 비장비대를 지표로 하여 reverse transcriptase의 억제효과를 지닌 항 AIDS약물의 약효검색법으로 활용할 수 있음을 제시해준다.

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Cloning, Expression and Purification of HIV-1 Reverse Transcriptase

  • Goo, Jae-Hwan;Park, Kwan-Yong
    • Proceedings of the Korean Society of Applied Pharmacology
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    • pp.76-76
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    • 1995
  • Virus-encoded HIV-1 reverse transcriptase (RTase) is one of the major targets for the development of drugs for HIV-1 since it is an essential enzyme-for the replication cycle of HIV-1. We cloned the entire reverse trancriptase gene into an inducible expression vector with tac promotor= RTase was stably overexpressed and induced by IPTG and the highly-expressed RTase was purified partially by use of DEAE cellulose and Mono Q column. The partially purified enzyme (663kDa, 51kDa) as exhibited by SDS-PAGE showed the high specific activity (16,570U/mg) when the assay for the RTase activity was carried out using $^3$H-dTTP and poly(rA): oligo(dT)12-18 as the substrate.

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PKC-Independent Stimulation of Cardiac $Na^+/Ca^{2+}$ Exchanger by Staurosporine

  • Kang, Tong-Mook
    • The Korean Journal of Physiology and Pharmacology
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    • v.12 no.5
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    • pp.259-265
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    • 2008
  • $[Ca^{2+}]_i$ transients by reverse mode of cardiac $Na^+/Ca^{2+}$ exchanger (NCX1) were recorded in fura-2 loaded BHK cells with stable expression of NCX1. Repeated stimulation of reverse NCX1 produced a long-lasting decrease of $Ca^{2+}$ transients ('rundown'). Rundown of NCX1 was independent of membrane $PIP_2$ depletion. Although the activation of protein kinase C (PKC) was observed during the $Ca^{2+}$ transients, neither a selective PKC inhibitor (calphostin C) nor a PKC activator (PMA) changed the degrees of rundown. By comparison, a non-specific PKC inhibitor, staurosporine (STS), reversed rundown in a dose-dependent and reversible manner. The action of STS was unaffected by pretreatment of the cells with calphostin C, PMA, or forskolin. Taken together, the results suggest that the stimulation of reverse NCX1 by STS is independent of PKC and/or PKA inhibition.