円偏光二色性による正常及び異常ヘモグロビンのアロステリック遷移の研究

金沢大学医学部研究課題/領域番号:X00210----477971研究期間(年度):1979出典：「円偏光二色性による正常及び異常ヘモグロビンのアロステリック遷移の研究」研究成果報告書　課題番号X00210----477971（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-X00210----477971/）を加工して作

Hbの内在性トリプトファン蛍光をプローブとしたアロステリック構造変化の動力学

金沢大学医学部研究課題/領域番号:57570798, 研究期間(年度):1982 – 1983出典：研究課題「Hbの内在性トリプトファン蛍光をプローブとしたアロステリック構造変化の動力学」課題番号57570798（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-57570798/）を加工して作

His C2プロトンの重水素交換反応の速度論的解析によるHbのR【double arrow】T遷移の研究

金沢大学医学部研究課題/領域番号:56771080, 研究期間(年度):1981出典：「His C2プロトンの重水素交換反応の速度論的解析によるHbのR【double arrow】T遷移の研究」研究成果報告書　課題番号56771080（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-56771080/）を加工して作

Quantitative evaluation for the role of β146 His and β143 His residues in the Bohr effect of human hemoglobin in the presence of 0.1 M chloride ion

Two different methods were used to determine the number of Bohr protons released upon oxygenation of human hemoglobin (Hb A) and Hb A lacking β146 His (des-His Hb A) at the pH ranging from pH 5.0 to 9.0 in the presence of 0.1 M Cl- at 25 °C. One is the direct differential titration method, the other is based on the measurement of oxygen affinity as a function of pH. The results obtained for Hb A or des-His Hb A with two methods were completely mutually consistent. The number of Bohr protons released from des-His Hb A upon oxygenation at pH 7.5 was about 44% less than that from Hb A, while at pH 5.5 the number of Bohr protons taken up by des-His Hb A was 20% greater than that by Hb A. The differences in the number of Bohr protons between Hb A and des-His Hb A could not be simply ascribed to the lack of β146 His from Hb A. The pK(a) values, which were determined by the deuterium exchange method using 1H NMR, were 8.0 for β146 His of deoxy-Hb A and 6.5 for that of CO Hb A, while those of β143 His were 5.2 for deoxy-Hb A and 6.0 for CO Hb A. From these pK(a) values, in addition to those of α1 Val proposed for the modified CO and deoxy-Hb A with carbamylated β chains by Van Beek and de Bruin, it became evident that almost all (about 92%) of the alkaline Bohr protons released upon oxygenation of Hb A in the presence of 0.1 M Cl- could be acountd for by the protons from these 2 residues, although the involvement of other histidine residues could not be denied. About half the acid Bohr protons from Hb A, which corresponds to the higher pH part (above pH 5.0) of the acid Bohr effect, could be explained by the involvement of β143 His residue. The residual acid Bohr effect in the more acidic pH region was presumably contributed by an amino acid residue with pK(a) values of 4.05 and 5.95 for the deoxy- and CO Hb A, respectively, although the amino acid residue was unspecified. In des-His Hb A, however, the acid and alkaline Bohr effects could not be satisfactorily explained if the pK(a) values of β143 His and α1 Val remained unchanged in comparison with those of Hb A and moreover the involvement of other residues in the Bohr effect was not taken into account. These results indicate that removal of β-carboxyl terminal histidine may cause considerably large perturbations to the tertiary structure of the subunit and/or to the deoxy-quaternary structure of des-His Hb A, so that its acid and alkaline Bohr effects may be altered more extensively than would be expected by deletion of only 1 residue

Induction of programmed cell death (apoptosis) by influenza virus infection in tissue culture cells

The process of cell death caused by influenza virus infection in cultured MDCK and HeLa cells was analysed. This infection gave rise to nuclear fragmentation and chromatin condensation accompanied by chromosomal DNA fragmentation into oligonucleosomes. Chromosomal DNA fragmentation progressed concomitantly with cell lysis of MDCK cells and HeLa cells, producing high and low yields of virus particles, respectively, indicating that the extent of cell lysis was not proportional to the virus production. The endonuclease inhibitor zinc blocked DNA fragmentation in MDCK cells. Cycloheximide inhibited DNA fragmentation as well as cell lysis. Inhibition occurred when the drug was added to the medium within 2 h after infection but not efficiently at 4 h or later. Infection induced the Fas Ag gene, which encodes a possible apoptosis-mediating molecule, in the early infectious stage followed by the expression of Fas Ag on the cell surface. These results suggested that influenza virus infection causes apoptotic death of cultured cells, and their fate might be determined at an early stage of the infection by induction of an apoptotic gene

The relationship between the PD-L1 expression of surgically resected and fine-needle aspiration specimens for patients with pancreatic cancer

BACKGROUND: Recently, therapeutic antibodies against programmed death-1/programmed death-ligand 1 (PD-1/PD-L1) have shown promising clinical results for several solid tumors, including pancreatic cancer. In this study, we evaluated the relationship between the PD-L1 expression of surgical resected and fine-needle aspiration (FNA) specimens for patients with pancreatic cancer. METHODS: Of 121 patients who underwent endoscopic ultrasound-guided (EUS)-FNA before surgery for pancreatic cancer in an academic center, the 94 (78%) with adequate FNA specimens for a histological evaluation were retrospectively analyzed. All the patients had undergone upfront surgery without any chemotherapy or radiotherapy. We performed immunohistochemistry (IHC) staining to investigate the PD-L1 expression in both resected and FNA specimens. The positive-stained cells were counted, and their percentage was used for the investigation. RESULTS: Of the 94 patients, 16 (17%) and 11 (10%) were defined as positive on resected cancer specimens using cutoff points of 5% and 10% positively stained cancer cell counts, respectively. The concordance rates for the positive frequency of PD-L1 expression between resected and FNA specimens were 44% (7/16) and 55% (6/11) when the positivity was set to ≥ 5% and ≥ 10%, respectively. The concordance rates for the negative frequency of PD-L1 expression between two specimens were 97% (76/78) and 99% (82/83) when the positivity was set to ≥ 5% and ≥ 10%, respectively. CONCLUSIONS: Approximately, half of the patients with PD-L1 expression positive and almost all the patients with PD-L1 expression negative could be diagnosed on FNA specimens

血色素の分光学: 共鳴ラマン分光と蛍光ラベルエフェクターによる協同作用の研究

血色素(Hb)の協同的酸素結合の立体化学的機構のあらましについては比較的よく実証されてきた。しかし、酸素親和性の調節機構やR→T四次構造変化のトリが一機構については、未だに論争があり、実験的にも証明されていないことが多い。そこで、我々は【α_1】【β_2】サブユニット界面にアミノ酸置換の起った異常Hbを用いた。これらの異常Hbはこの界面の相互作用の不完全さ故に、様々な程度の機能異常を示すことが期待される。従って、異常Hbの機能と構造の違いとを比較・検討することにより、酸素親和性の調節機構や四次構造変化の引き金となる構造因子が明らかになると考えられる。そこで、まず、正常及び異常Hbの酸素平衡曲線を種々の条件下で測定し、アロステリック・パラメータを決定した。次に、ヘム近傍の構造については共鳴ラマン分光法で、アロステリック・エフェクター結合部位の構造は蛍光ラベル・エフェクターを用いて検討し、以下の結果が得られた。 1)R状態の酸素解離定数はHbの種類によらず、ほぼ一定であったが、T状態の酸素解離定数(【K_T】)は異常Hbの種類により大きく異っていた。このことは、異常Hbの機能異常はデオキシ型T構造の多様性にあることを示唆している。 2)異常Hbのヘム鉄-近位ヒスチジン結合の伸縮振動に由来するラマン線のみが、測定条件や機能異常の程度に対応して変化した。しかも、この振動数変化は異常Hbの【K_T】の違いと相関した。 3)蛍光ラベル-有機リン酸や平衡ゲルロ過法の結果から予想に反して、異常Hbの有機リン酸(アロステリック・エフェクター)の結合部位の構造は正常Hbとよく似ていることがわかった。以上の結果から、異常Hbの機能異常の原因は、デオキシ型T構造の酸素親和性の多様性にあること、又、この酸素親和性を調節する構造因子の1つが、ヘム鉄と近位ヒスチジン間の結合の強さであることが、今回の研究より明らかとなった。The outline of the stereochemical mechanism of the cooperative oxygen binding of hemoglobin (Hb) has been well documented. However, the modulation mechanism of oxygen affinity and the trigger mechanism for the R-T structural transition remain unproven experimentally. To resolve these problems we have been investigated the structural and functional relationships of abnormal Hbs with an amino acid substitution at the contact. The aim of this study is to point out the structural element(s) essential for allosteric oxygen binding by comparing the structural alterations with functional ones of various abnormal Hbs.Analysis of Oxygen equilibrium curves for normal and abnormal Hbs in terms of the two-state allosteric model suggested that the abnormal allosteric oxygen binding of Hb is due to altered molecular properties of the deoxy-T state. To clarify the molecular basis of this situation, the resonance Raman spectra in the low-frequency region of deoxy Hbs and the binding of fluorescence-labelled allosteric effector (MANT-ATP) to deoxy Hbs were examined.The high-affinity deoxy Hb gave the Fe-His stretching Raman line at 220-220 similar to that of the isolated chains, while the low-affinity one gave the Raman line at 214-216 . However, deoxy Hbs with intermediate levels of oxygen affinity unexpectedly exhibited the Raman line at intermediate frequencies. Furthermore, this varied frequencies of Fe-His stretching Raman lines for various Hbs was found to correlate well with varied oxygen affinities of deoxy-T state. In contrast, the MANT-ATP binding experiments showed that the stucture of the organic phosphate binding site of abnormal Hbs is almost identical to that of Hb A.Based on the above results, we concluded that the Fe-His bond presumably comprises an important part of the regulation mechanism for hemoglobin oxygen affinity and quaternary structural changes.研究課題/領域番号:60480134, 研究期間(年度):1985–1986出典：「血色素の分光学: 共鳴ラマン分光と蛍光ラベルエフェクターによる協同作用の研究」研究成果報告書　課題番号60480134 (KAKEN：科学研究費助成事業データベース（国立情報学研究所）)　　　本文データは著者版報告書より作

The activation of Proteinase-Activated Receptor-1 (PAR1) mediates gastric cancer cell proliferation and invasion

<p>Abstract</p> <p>Background</p> <p>In addition to regulating platelet function, the G protein-coupled sub-family member Proteinase-activated receptor-1 (PAR1) has a proposed role in the development of various cancers, but its exact role and mechanism of action in the invasion, metastasis, and proliferation process in gastric cancer have yet to be completely elucidated. Here, we analyzed the relationship between PAR1 activation, proliferation, invasion, and the signaling pathways downstream of PAR1 activation in gastric cancer.</p> <p>Methods</p> <p>We established a PAR1 stably transfected MKN45 human gastric cancer cell line (MKN45/PAR1) and performed cell proliferation and invasion assays employing this cell line and MKN28 cell line exposed to PAR1 agonists (α-thrombin and TFLLR-NH₂). We also quantified NF-κB activation by electrophoretic mobility shift assay (EMSA) and the level of Tenascin-C (TN-C) expression in conditioned medium by ELISA of MKN45/PAR1 following administration of α-thrombin. A high molecular weight concentrate was derived from the resultant conditioned medium and subsequent cultures of MKN45/PAR1 and MKN28 were exposed to the resultant concentrate either in the presence or absence of TN-C-neutralizing antibody. Lysates of these subsequent cells were probed to quantify levels of phospholyrated Epidermal Growth Factor Receptor (EGFR).</p> <p>Result</p> <p>PAR1 in both PAR1/MKN45 and MKN28 was activated by PAR1 agonists, resulting in cell proliferation and matrigel invasion. We have shown that activation of NF-κB and EGFR phosphorylation initially were triggered by the activation of PAR1 with α-thrombin. Quantitative PCR and Western blot assay revealed up-regulation of mRNA and protein expression of NF-κB target genes, especially TN-C, a potential EGFR activator. The suppressed level of phosphorylated EGFR, observed in cells exposed to concentrate of conditioned medium in the presence of TN-C-neutralizing antibody, identifies TN-C as a putative autocrine stimulatory factor of EGFR possibly involved in the sustained PAR1 activation responses observed.</p> <p>Conclusion</p> <p>Our data indicate that in gastric carcinoma cells, PAR1 activation can trigger an array of responses that would promote tumor cell growth and invasion. Over expression of NF-κB, EGFR, and TN-C, are among the effects of PAR1 activation and TN-C induces EGFR activation in an autocrine manner. Thus, PAR1 is a potentially important therapeutic target for the treatment of gastric cancer.</p

Shigeru Matsukawa standing in front of his barrack.

Photo of Shigeru Matsukawa during his service with the 442nd Battalion in Italy during World War II, 1945