Masakatsu Izaki

Detection of serine proteinase inhibitors in human cornified cells

Abstract A screening test for serine proteinase inhibitors revealed trypsin and urokinase inhibitors in the extract of human cornified cells. No inhibition for α-chymotrypsin, thrombin or plasmin was detected. Characterization of the inhibitors separated with a Sephacryl S-200 gel column demonstrated that: 1) trypsin inhibitor with a molecular weight of 45,000 was labile to heat, acid and alkali and showed temporary inhibition, and 2) urokinase inhibitor with a molecular weight of 35,000 was found relatively stable and exhibited time dependent inhibition. Both were distinct from a known thiol proteinase inhibitor which showed high stability and immediate inhibition. Regulatory roles of serine proteinase inhibitors are postulated.

Light and Electron Microscopic Immunohistochemistry of Solar Elastosis: A Study with Cutis Rhomboidalis Nuchae

Biopsied skin specimens from 10 males with cutis rhomboidalis nuchae, 50–83 years in age, as well as age‐matched non‐sun‐exposed skin and young normal skin specimens were subjected to a light and electron microscopic immunohistochemical study using anti‐elastin polyclonal antibody and anti‐microfibril HB‐8 monoclonal antibody. Conventional histochemical and electron microscopic techniques were also used.

Purification of epidermal plasminogen activator inhibitor

A plasminogen activator inhibitor was purified from human cornified cell extract by DEAE‐Sepharose, Sephacryl S‐200, and high‐performance liquid chromatographies on hydroxyapatite HPHT and anion‐exchanger Mono Q at pH 7.2 and 8.0. The purified inhibitor showed M r 43000 and pI 5.2. 50% inhibition of fibrinolytic activity (1.5 IU) of urokinase and tissue‐type plasminogen activator was attained by 0.60 ng and 11.0 ng purified inhibitor respectively. Synthetic substrate assay demonstrated slow tight‐binding inhibition to both urokinase and tissue‐type plasminogen activator. The inhibitor did not inactivate plasmin, thrombin, glandular kallikrein or trypsin.

Fibrin and collagen deposition and fibroblasts proliferation in granuloma of murine leprosy. Comparison of two mouse strains with different immune reactions.

Comparative immunofluorescence study with murine lepromas induced in C57BL/6NJcl (immunologically high responder) and CBA/N (low responder) mouse strains revealed that fibrin formation was associated with cellmediated immune resistance against invasive bacilli. Histochemistry on paraffin sections further elucidated fibroblast proliferation and formation of collagen fibers following fibrin deposition only in murine lepromas with positive host reactions.

Elastase activity in granulomatous inflammation in experimental murine leprosy

Proteolytic activity for [3H]elastin, pyro-Glu-Pro-Val-pNA(S-2484), and Suc-(Ala)3-pNA(AAApNA) was demonstrated in the bound fraction extracted with 2 M KSCN + 0.1% Triton X-100 from hypersensitivity-type murine lepromas in C57BL/6N mice, while elastase-inhibitor activity was separately observed in the soluble fraction extracted with a Tris-saline buffer. Sephacryl S-200 gel chromatography showed a peak of elastolytic activity with approximately 20,000 in molecular weight. The following DEAE-Sepharose chromatography demonstrated three fractions of elastolytic activity (E-I, II, III). The inhibitory profile showed that E-I is a thiol proteinase, while E-II and E-III belong to serine proteinase-type elastases. Both E-II and E-III showed different properties with neutrophil elastase or elastase secreted from cultured macrophages, but identical characteristics to membrane bound-type elastase of monocytes. A lower level of elastolytic activity was detected in the bound fraction of nonhypersensitivity-type murine lepromas in CBA/N mice, suggesting a more involvement of membrane bound-type elastase from monocytes/macrophages during the tissue remodelings of hypersensitivity-type granulomas.

Ultracytochemical and Light Microscopic Histochemical Studies in Murine Leprosy

我々は,系統の異なるマウスを用いて,二極型の鼠らいを作製し,それぞれの病型におけるマクロファージの中のライソゾームの変化に関する研究を行った。ハワイ株鼠らい菌(Mycobacterium lepraemurium)1×107をC57BL/6N, CBA/J, C57BL/6N(nu/+).およびC57 BL/6N (nu/nu)マウスの胸骨部皮下に接種した。生じた肉芽腫性病変の凍結切片上でGomoriのazo-dye methodにより酸フォスファターゼおよびアルカリフォスファターゼを組織化学的に検索した。主として酸フォスファターゼをマーカーとしてライソゾームを電顕的レベルで検索することができた。その結果,鼠らい菌に対して抵抗性を有する系統のマウスで見られる,免疫学的に活性化されたマクロファージ中では,ライソゾームの構造が保持され,酸フォスファターゼ活性が,ライソゾーム内に局在して見られた。このような所見は,ヒトのT型(類結核型)で観察された所見と同一であった。しかしながら,免疫学的な機能に異常のある系統のマウスに出現したマクロファージ中では,ライソゾームの形態が破壊され,膜構造が失われ,酸フォスファターゼ活性が細胞質内にび慢性に出現するのが見られた。この所見は,やはりヒトで,L型(らい腫らい)で観察された結果と同一の成績であった。ライソゾームの形態と機能に関しては,鼠らいはヒトのらいを研究するための,良い実験モデルになりうることが示唆された。