Historical Memory and Literary Writing of Pearl

departmental bulletin pape

ゲンチョ チョウセイ ノウカン ハンノウ ケンサ ノ リンショウテキ ケンキュウ 5ネンカン 273レイ ニ ツイテノ ケントウ

聴性脳幹反応(Auditory Brainstem Response,以下ABRと略す)は音刺激により誘発される聴覚伝導路由来の脳波であり,1970年にJewettらにより報告されて以来,他覚的聴力検査及び脳幹部の神経学的検査として臨床的に広く活用されている。我々は3kHzサイン波一波のクリックを刺激音とするABRを施行しているが,今回1985年から1989年までの5年間に施行した273例について検討した。症例は男性147例,女性126例であり,年齢的に純音聴力検査が不可能である0-4歳の幼児に多く施行した。幼児に対し施行した聴力検査症例152例のうち,85例に主訴となった聴力障害以外の何等かの基礎疾患があり,その半数が閾値上昇を示した。これらの疾患を有する症例では,難聴の合併率が高いばかりでなく,脳幹部を始めとする中枢神経系に異常を持つことがあるため,ABRがスクリーニング検査として非常に有用であり,早期及び経時的に施行することが必要であると思われた。また聴神経腫瘍を主とする小脳橋角部腫瘍の診断にあたっては,ABRのV波潜時の延長所見の検索は極めて有用であり,診断学的に高い特異性と感受性を有する検査手段のひとつとみなされる。さらに,他覚的聴力検査としてABRの記録は,機能性難聴の確定診断にも不可欠である事が確かめられた。journal articl

Measurement of the e+e-→D(*)+D(*)- cross sections

journal articl

Discrimination of Hairpin Polyamides with an α-Substituted-γ-aminobutyric Acid as a 5‘-TG-3‘ Reader in DNA Minor Groove

Pyrrole-imidazole (Py-Im) polyamides containing stereospecifically α-amino- or α-hydroxyl-substituted γ-aminobutyric acid as a 5‘-TG-3‘ recognition element were synthesized by machine-assisted Fmoc solid-phase synthesis. Their binding properties to predetermined DNA sequences containing a core binding site of 5‘-TGCNCA-3‘/3‘-ACGN‘GT-5‘ (N·N‘ = A·T, T·A, G·C, and C·G) were then systematically studied by surface plasmon resonance (SPR). SPR results revealed that the pairing of stereospecifically α-amino-/α-hydroxyl-substituted γ-aminobutyric acids, (R or S)-α,γ-diaminobutyric acid (γRN or γSN) and (R or S)-α-hydroxyl-γ-aminobutyric acid (γRO or γSO), side-by-side with β-alanine (β) in such polyamides significantly influenced the DNA binding affinity and recognition specificity of hairpin polyamides in the DNA minor groove compared with β/β, β/γ, and γ/β pairings. More importantly, the polyamide Ac-Im-γSO-ImPy-γ-ImPyβPy-β-Dp (β/γSO) favorably binds to a hairpin DNA containing a core binding site of 5‘-TGCNCA-3‘/3‘-ACGN‘GT-5‘ (N·N‘ = A·T) with dissociation equilibrium constant (KD) of 1.9 × 10-7 M over N·N‘ = T·A with KD = 3.7 × 10-6 M, with a 19-fold specificity. By contrast, Ac-Im-γSN-ImPy-γ-ImPyβPy-β-Dp (β/γSN) binds to the above sequence with N·N‘ = A·T with KD = 8.7 × 10-7 M over N·N‘ = T·A with KD = 8.4 × 10-6 M, with a 9.6-fold specificity. The results also show that the stereochemistry of the α-substituent, as well as the α-substituent itself may greatly alter binding affinity and recognition selectivity of hairpin polyamides to different DNA sequences. Further, we carried out molecular modeling studies on the binding by an energy minimization method, suggesting that α-hydroxyl is very close to N3 of the 3‘-terminal G to induce the formation of hydrogen bonding between hydroxyl and N3 in the recognition event of the polyamide Ac-Im-γSO-ImPy-γ-ImPyβPy-β-Dp (β/γSO) to 5‘-TGCNCA-3‘/3‘-ACGN‘GT-5‘ (N·N‘ = A·T). Therefore, SPR assays and molecular modeling studies collectively suggest that the (S)-α-hydroxyl-γ-aminobutyric acid (γSO) may act as a 5‘-TG-3‘ recognition unit

Synthesis of Pyrrole–Imidazole Polyamide <i>seco</i>-1-Chloromethyl-5-hydroxy-1,2-dihydro‑3<i>H</i>‑benz[<i>e</i>]indole Conjugates with a Vinyl Linker Recognizing a 7 bp DNA Sequence

Convergent synthetic routes for <i>N</i>-methylpyrrole (P) and <i>N</i>-methylimidazole (I) <i>seco</i>-1-chloromethyl-5-hydroxy-1,2-dihydro-3<i>H</i>-benz­[<i>e</i>]­indole (CBI) conjugates with a vinyl linker were developed. New hairpin polyamide–<i>seco</i>-CBI conjugates, compounds <b>16</b>–<b>19</b>, were synthesized, and their DNA sequence-specific alkylating activities were evaluated via high-resolution denaturing gel electrophoresis and high-performance liquid chromatography (HPLC) product analysis. The new synthetic route for PI conjugates with a vinyl linker consisted of the introduction of a vinylpyrrole unit (<b>8</b>–<b>11</b>) into the C terminal of a PI polyamide synthesized by (fluorenylmethoxy)­carbonyl solid-phase peptide synthesis (SPPS), followed by liquid-phase coupling with <i>seco</i>-CBI. The yield of the conjugates was significantly improved compared with that of the method reported previously, which allows us to synthesize various substituted conjugates containing a vinyl linker. Conjugates <b>16</b>–<b>19</b> were designed to investigate the substituent effect of the vinyl linker, and conjugate <b>16</b><i><b>S</b></i> was synthesized to evaluate the reactivity between racemic and <i>S</i> enantiomers of the <i>seco</i>-CBI derivative. The results of high-resolution denaturing gel electrophoresis using 208 bp DNA fragments indicated that alkylation by compounds <b>16</b> and <b>17</b>, in which the H of the vinyl linker of compound <b>16</b> was replaced with F, occurred predominantly at the A of the 5′-TTTGTCA-3′ sequence at nanomolar concentrations. In clear contrast, compounds <b>18</b> and <b>19</b>, which were methyl or Br derivatives of compound <b>16</b>, did not exhibit any DNA alkylating activity. Moreover, HPLC product analysis using synthetic oligonucleotides demonstrated that alkylation occurred between the N3 of the adenine of the oligomer and the cyclopropane ring of <b>16</b><i><b>S</b></i>. Density functional calculation of substituted vinylpyrrole <i>seco</i>-CBI units indicated that methyl and Br substituents led to a significantly distorted geometry of the vinyl group with the pyrrole ring compared with H and F derivatives. Molecular modeling studies offered the additional information that steric hindrance reduced the DNA alkylating activity of these derivatives

C−H to N Substitution Dramatically Alters the Sequence-Specific DNA Alkylation, Cytotoxicity, and Expression of Human Cancer Cell Lines

We designed and synthesized sequence-specific alkylating conjugates 1 and 2, which selectively alkylate matched sequences at nanomolar concentrations. Conjugates 1 and 2 differ only in that the C−H is substituted by an N in the second ring, which precisely recognizes and effectively alkylates DNA according to the recognition rule of Py-Im polyamides. We investigated sequence-specific DNA alkylation, cytotoxicity in 39 human cancer cell lines, and the effect on expression levels in cancer cell lines by Py-Im conjugates 1 and 2. The COMPARE analysis of the mean graphs showed that conjugates 1 and 2 did not correlate well with each other (r = 0.65) despite having a common DNA alkylating mechanism (purine N3 alkylation). Array-based gene expression analysis demonstrated that there are several oppositely regulated genes. The results suggest the intriguing possibility that DNA alkylating agents recognizing longer base-pair sequences may provide a promising approach for developing new types of antigene agents

Enantioselective DNA Alkylation by a Pyrrole−Imidazole <i>S</i>-CBI Conjugate

Conjugates 12S and 12R of N-methylpyrrole (Py)−N-methylimidazole (Im) seven-ringed hairpin polyamide with both enantiomers of 1,2,9,9a-tetrahydrocyclopropa[1,2-c]benz[1,2-e]indol-4-one (CBI) were synthesized, and their DNA alkylating activity was examined. High-resolution denaturing gel electrophoresis revealed that 12S selectively and efficiently alkylated at one match sequence, 5‘-TGACCA-3‘, in 450-bp DNA fragments. The selectivity and efficiency of the DNA alkylation by 12S were higher than those of the corresponding cyclopropapyrroloindole (CPI) conjugate, 11. In sharp contrast, another enantiomer, 12R, showed very weak DNA alkylating activity. Product analysis of the synthetic decanucleotide confirmed that the alkylating activity of 12S was comparable with 11 and that 12S had a significantly higher reactivity than 12R. The enantioselective reactivity of 12S and 12R is assumed to be due to the location of the alkylating cyclopropane ring of the CBI unit in the minor groove of the DNA duplex. Since the CBI unit can be synthesized from commercially available 1,3-naphthalenediol, the present results open up the possibility of large-scale synthesis of alkylating Py−Im polyamides for facilitating their use in future animal studies

Sequence-Specific DNA Interstrand Cross-Linking by Imidazole−Pyrrole CPI Conjugate

Sequence-Specific DNA Interstrand Cross-Linking by Imidazole−Pyrrole CPI Conjugat

Efficient Generation of 2‘-Deoxyuridin-5-yl at 5‘-(G/C)AA^XU^XU-3‘ (X = Br, I) Sequences in Duplex DNA under UV Irradiation

The photoreactivity of 5-halouracil-containing DNA was investigated using 450 base pair DNA fragments under 302 nm irradiation. Heat-dependent cleavage selectively occurs at 5‘-(G/C)AAXUXU-3‘ and 5‘-(G/C)AXUXU-3‘ (X = Br, I) sequences in double-stranded DNA. HPLC product analysis indicated that 2‘-deoxyribonolactone residues are effectively generated at these sequences. These observations will be useful in studying the molecular basis of the sequence-dependent DNA-damaging process in UV-irradiated 5-halouracil-containing DNA