Template properties of mutagenic cytosine analogues in reverse transcription

We have studied the mutagenic properties of ribonucleotide analogues by reverse transcription to understand their potential as antiretroviral agents by mutagenesis of the viral genome. The templating properties of nucleotide analogues including 6-(β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c](1,2)oxazin-7-one, N(4)-hydroxycytidine, N(4)-methoxycytidine, N(4)-methylcytidine and 4-semicarbazidocytidine, which have been reported to exhibit ambiguous base pairing properties, were examined. We have synthesized RNA templates using T3 RNA polymerase, and investigated the specificity of the incorporation of deoxyribonucleoside triphosphates opposite these cytidine analogues in RNA by HIV and AMV reverse transcriptases. Except for N(4)-methylcytidine, both enzymes incorporated both dAMP and dGMP opposite these analogues in RNA. This indicates that they would be highly mutagenic if present in viral RNA. To study the basis of the differences among the analogues in the incorporation ratios of dAMP to dGMP, we have carried out kinetic analysis of incorporation opposite the analogues at a defined position in RNA templates. In addition, we examined whether the triphosphates of these analogues were incorporated competitively into RNA by human RNA polymerase II. Our present data supports the view that these cytidine analogues are mutagenic when incorporated into RNA, and that they may therefore be considered as candidates for antiviral agents by causing mutations to the retroviral genome

<所内学術研究成果報告>H. 「環境保全・地球環境温暖化防止をターゲットとする新パルプ資源ケナフの栽培と利用に関する研究」

本研究は, エコマテリアルとしての非木材繊維資源に最も適切である一年生植物ケナフ(Hibiscus cannabinus L.)の栽培とその利用を目的に, 1993年より開始した研究である。従来の成果は, すでに本年報1992,\u2794,\u2795,\u2796,\u2797,\u2798,および\u2799年に報告した。特に従来のケナフ栽培の成果の総決算として, 1998年より平塚市および平塚ケナフ普及協会との共同研究が行われてきた。特に, 平塚市では休耕田対策としてケナフの栽培を推奨し, 現在, 栽培したケナフのパルプ化と紙製造を行って市政に還元している。この現状はさらに展開し, 平塚市のみならず日本全国にその輪が広がり大きな活動となっている。これらの栽培や利用は最も基礎的な指導と, より学術的な研究成果の提供が常に必要であり, この点を最も重要な課題としている。さらに, 環境教育に対する展開を学校, 公民館などを中心に行い, 2000年度は, 平塚キャンパスで市内6小学校の生徒28名のケナフ教育を行った。まお, 研究室内では, 栽培研究の他に, a)種子の発芽阻害実験, b)海水による阻害実験, c)生長に伴うクロロフィル量および水分量の測定実験, d)光合成測定実験, e)花の成分(色素)研究, f)葉など各器官の成分研究などを行っている。取り扱った種類も, ローゼル(H. sabdariffa L.)類も加えると30種に近い

Error Analysis of Dopamine D2 Receptor Occupancy Study with Agonist Ligand [11c]MNPA

Objectives: Occupancy of dopamine D2 receptors by antipsychotic drugs can be estimated from reduction in the observed binding potential (BPND). Because BPND varies widely in occupancy studies, accuracy of the measurement of wide range BPND should be confirmed. The purpose of this study is to investigate errors in quantitative analysis for estimating dopamine D2 receptor occupancy by antipsychotics with agonist ligand [11C]MNPA which has high affinity and selectivity to dopamine D2 receptors1.\nMethods: Simulated TACs of [11C]MNPA with several noise levels were generated to investigate the bias and variation of parameter estimates caused by the statistical noise for non-linear least square (NLS) fitting and a simplified reference tissue model (SRTM) methods. A dynamic tracer concentrations was consisted of three target conditions (K1=0.44, k2=0.067, k3=0.02, 0.1 or 0.2, k4=0.18), and a reference (K1=0.44, k2=0.067) with a dynamic frame (20sec * 9, 1min * 5, 2min * 4, 4min * 11, 5min * 6, totally 90min) and a measured input function from human study1. Three true BPND values were assumed; 1.111 (baseline), 0.556 (occupancy = 50%) and 0.111 (90%). Then Gaussian noise was added at the noise level 1%, 3%, 5%, 7% and 10%, and five hundred noisy data sets were generated for each2. The reliability of BPND estimated by NLS and a SRTM and the calculated occupancy, dependency of scan durations (32, 44, 60, 75 and 90min) for SRTM were investigated. \nResults: For NLS and SRTM methods, the bias of estimated BPND values became larger as the noise level increased and these bias of BPND were larger than those of occupancy (Fig. 1A and 1B). In the case of small BPND, the bias became larger. For SRTM method, reliable and unbiased occupancy estimates of [11C]MNPA could be obtained by 60 min with the relative standard deviation remaining less than 10%. However, shorten scan duration depredate the quantification of very small binding potential (Fig1C).\nConclusions: Dopamine D2 receptor occupancy by antipsychotics can be estimated precisely by SRTM method with an optimal scan duration with [11C]MNPA. \nReference: [1] Otsuka T, 2008, Neuroimage, 41, suppl. 2, T134. [2] Ikoma Y, 2008, Neuroimage, 47, 43-50.[pic_01]Fig. 1 (A) Noise level dependency: Bias of BP and Occupancy estimated by NLS, (B) estimated by SRTM, (C) Scan Duration Dependency: Bias of BP and Occupancy at a 3% noise level estimated by SRTM.Brain\u2709 & BrainPET\u270

Quantative analysis of dopaine D2 receptor binding in human brain using PET with a agonist redioligand[11C]MNPA

Intrroduction:It has been establishied in vitro that dopamine D2 receptor could be classified into two inrweconvertible affinity states to natural agonist dopamine,the high-and low-affinity states.Dopamine D2 receotir binding in vivo has wildly been measured by PET with the use of antagonist radioligands,such as[11C]racloprode,however,the high-ans low-ahhinity states of dopamine D2 receptors can be distinguished by the antagonist radiologands.(R)-2-11CH3O-N-n-propylnorapomorphine([11C]MNPA)has recentaly been devdeloped as a potent new agonist for in vivo imaging of the high-affinity state of dopamine D2 receptors[1]. In presentstudy,the kinetics of[11C]MNPA in the without an arterial input function wewe alos validated.Neuroreceptor Mapping 200