馬千里日記考(2)

　1920年から24年にいたる時期は、中国社会にとって「国民革命への過渡期」であると考えられている。そうであるならば、政治の場ではなおも軍閥支配が続くこの間も、社会の底流では人々の意識の変革に向けて様々な運動が続けられていたということができよう。とりわけ五四運動以後に際立ってきた近代的マスメディアの盛行が、そうした意識変革に与えた影響は無視し難いものであった。1920年に馬千里が中心となってすすめた『新民意報』の発刊は、まさにそうした運動のひとつであったということができる。本稿は、その発刊から停刊に至る間の同報について「馬千里日記」をもとに明らかにしたものである。そこでは専らこの新聞を対象として、その経営について、論説について、副刊について、更にはこの新聞が当時の社会にどのような影響を与えたかについて論述した。また馬千里周辺の若い知識人たちが、それぞれに、その後どのような方向に進んだかについても言及した

馬千里日記考(1)

　中華民国前期は、歴史の形成に当って中間的知識層の担った役割が大きい時代であった。ここに取り上げる馬千里もそうした知識層の一人に数えてよいであろう。彼はその生涯のほとんどを天津で過ごし、主としてこの地の教育にあたった。だが、教育者であると同時に、彼の45年の生涯を大きく彩るものは、「救国」をめざす社会的活動であった。1919年の五四運動がその契機となるものであったといえよう。本稿はこの点を中心に述べ、特に国民大会に注目している。こうした馬千里の生涯が明らかになるのは、彼が成人して以来23年間日記を書き続けたからである。近代中国の激動の時代を生きた馬千里など若い知識入群像の動向を知るためにも、このR記は貴重である。そこで本稿は、この日記が現在に伝えられた経緯を述べるとともに、この日記がもつ中国近代史における資料としての価値や可能性についても言及した

Expression and characterization of human factor IX. Factor IXthr-397 and factor IXval-397.

Factor IXLong Beach has a single amino acid substitution at 397 (Ile to Thr) in the catalytic domain which results in severe hemophilia B. Recent investigations have shown that the substitution of threonine for isoleucine at 397 may affect a part of the macromolecular substrate binding site. Because threonine has a hydroxyl group in its side chain, it is possible that this hydroxyl group makes new hydrogen bonds and disturbs the substrate binding site. We used three techniques: molecular biology, which includes site-directed mutagenesis and recombinant protein expression in tissue culture; computer-aided kinetic data analysis; and molecular modeling to study this mutation site. We have produced two mutant factor IX molecules that have isoleucine 397 replaced by valine or threonine. Factor IXwild type and the two mutants (factor IXVal and factor IXThr) were expressed in human kidney cells and purified using a conformation-specific monoclonal antibody column. After the activation by factor XIa, these three molecules were able to bind p-aminobenzamidine and increase its fluorescence intensity in a similar manner. Factor IXVal and factor IXwild type had indistinguishable activities in an activated partial thromboplastin time (aPTT) assay and similar kinetic parameters with factor X as a substrate. Factor IXThr had only 5% clotting activity compared with normal factor IX, a slightly lower Km and significantly reduced kcat, using factor X as a substrate. We developed energy-refined (AMBER v.3.1) computer models of the three factor IX molecules based on previous work. Three factor IXa models (Ile, Val, or Thr at 397) with a fragment of the factor X activation site were used to predict the effect of the mutation at 397 and evaluate the significance of the new hydrogen bond thought to form between the side chain hydroxyl group of threonine 397 and the carbonyl oxygen of tryptophan 385. This new hydrogen bond would affect the position of an amide proton of adjacent glycine 386 which has been proposed to make a hydrogen bond with a backbone carbonyl oxygen of the P3 residue of factor X. In addition to the new hydrogen bond, there is significant movement in the side chain of tryptophan 385 between the factor IXawild type-factor X model and the factor IXaThr-factor X model that could interfere with substrate binding. This movement could be caused by the change in the molecular volume, the orientation of the side chain at 397, and the new hydrogen bond

Changing Residue 338 in Human Factor IX from Arginine to Alanine Causes an Increase in Catalytic Activity

This study was designed to identify functionally important factor IX (FIX) residues. Using recombinant techniques and cell culture, we produced a mutant FIX with arginine at 338 changed to alanine (R338A-FIX). This molecule had approximately 3 times greater clotting activity than that of wild type FIX (wt-FIX) in the activated partial thromboplastin assay. R338A-FIX reacted normally with a panel of three FIX specific monoclonal antibodies and migrated on sodium dodecyl sulfate-polyacrylamide gels indistinguishably from wt-FIX. Using functional assays, we determined that R338A-FIXa's Kd for factor VIIIa (FVIIIa) was similar to that of wt-FIXa. Our kinetic analysis, using factor X as substrate, indicated that the mutation's major effects were a 3-fold increase in kcat and a 2-fold decrease in Km both manifested only in the presence of FVIIIa. R338A-FIXa's increased catalytic efficiency did not result from ablation of a thrombin sensitive site, reported to occur at arginine 338, since in our assays the thrombin inhibitor, hirudin, had no effect on activity of either wt-FIXa or R338A-FIXa. R338A-FIXa and wt-FIXa had equal activity, with or without FVIIIa, toward the synthetic substrate, methylsulfonyl-D-cyclohexylglycyl-arginine-p-nitroanilide. Interestingly, R338A-FIXa had reduced affinity for heparin. Therefore, we propose that R338A-FIXa's increased activity is not due to an allosteric effect on the active site, but that the Arg-338 residue is part of an exosite that binds both factor X and the mucopolysaccharide, heparin

Aptamer beacons for the direct detection of proteins." Anal Biochem 294(2

We have designed a new class of molecules, which we term aptamer beacons, for detecting a wide range of ligands. Similar to molecular beacons, aptamer beacons can adopt two or more conformations, one of which allows ligand binding. A fluorescence-quenching pair is used to report changes in conformation induced by ligand binding. An anti-thrombin aptamer was engineered into an aptamer beacon by adding nucleotides to the 5-end which are complementary to nucleotides at the 3-end of the aptamer. In the absence of thrombin, the added nucleotides will form a duplex with the 3-end, forcing the aptamer beacon into a stem-loop structure. In the presence of thrombin, the aptamer beacon forms the ligand-binding structure. This conformational change causes a change in the distance between a fluorophore attached to the 5-end and a quencher attached to the 3-end. Aptamer beacon can be a sensitive tool for detecting proteins and other chemical compounds