Umeji Murakami

Highly-expressed S100A3, a calcium-binding protein, in human hair cuticle

Analyses on sodium dodecyl sulfate-polyacrylamide gel electrophoreses showed that the human hair cuticle extracts mainly consist of a 7-kDa component and keratin proteins. The S-carboxymethylation of the cuticle extracts made the 7-kDa band shift to the 15-kDa position. After electroblotting of the S-carboxymethyl derivative, the membrane pieces carrying the 15-kDa band were treated with trypsin and the released peptides were separated by reverse-phased HPLC. Amino acid sequence analyses revealed that the peptides corresponded to the partial sequences deduced from human genome coding for S100A3, a cysteine-rich calcium binding protein. The anti S100A3 serum, prepared by immunizing a synthetic peptide antigen, reacted with the 7-kDa and 15-kDa bands in immunoblotting analyses. Immunofluorescence microscopy showed intense labeling to the cuticular layer with the anti S100A3 serum. These results indicated that S100A3 was highly expressed in the human hair cuticle.

Purification and characterization of a myosin-cleaving protease from rat heart myofibrils

A proteolytic enzyme, which causes the limited degradation of cardiac myosin, was purified from rat heart myofibrils. The purified enzyme (a myosin-cleaving protease) was apparently homogeneous by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Autolysis of the purified enzyme was observed at neutral pH without high concentration of CaCl2. The molecular weight was estimated to be 26 000-27 000. The enzyme was active against casein, N-acetyl-L-tyrosine ethyl ester and N-glutaryl-L-phenylalanine-4-nitroanilide (Glu-Phe-NAn), but less active with N-benzoyl-DL-arginine-4-nitroanilide. Optimum pH values for the enzyme were 9.0 for casein and 8.4 for Glu-Phe-NAn. Caseinolytic activity of the enzyme was completely inhibited with phenylmethylsulfonyl fluoride and diisopropylphosphofluoride and partially inhibited with L-1-tosyl-L-phenylalanine chloromethyl ketone (Tos-PheCH2Cl) and soybean trypsin inhibitor. Tos-LysCH2Cl had no effect. Sulfhydryl reagents, metal-chelating agents and metal ions except for Zn2+ had little or no effect on the activity. Degradation of cardiac myosin with the enzyme produced two fragments having molecular weights of 130 000 and 94 000, accompanied by the disappearance of myosin heavy chain and light chain 2. Myosin degradation with the enzyme was more restrictive than with chymotrypsin.

Coexistence of Fast and Slow Myosin Isozymes in Human External Urethral Sphincter. A Preliminary Report

Human external urethral sphincter muscle was isolated for biochemical research. Myosin samples were prepared and pyrophosphate gel electrophoresis and two-dimensional electrophoresis were performed. Assignment in pyrophosphate gel of myosin isozymes of human external urethral sphincter was carried out by making comparisons with those from human rectus abdominis muscle. Myosin light chains in human external urethral sphincter were identified using two-dimensional electrophoresis. Human external urethral sphincter, similar to other voluntary muscles, is composed of fast and slow twitch muscle fibers, since fast and slow myosin components were shown in both pyrophosphate gel and two-dimensional electrophoretograms.

Safety of the Immunization with Hair Keratin as an Antigen Using Oil-Adjuvant to Cows and their Calves, and Usage of the Carcass as Edible Meat

乳牛に対する,毛髪ケラチン抗原とオイルアジュバントによる免疫の安全性を確認するため,免疫後の乳牛の体温と注射局所の状態,および免疫牛が出産した子牛の生時体重,疾病発生率,および発育成績を非免疫牛を対照に調査した.また,免疫牛の食肉の安全性について,胸垂皮下投与の有効性を調べた.12頭のホルスタイン雌牛を尻部筋肉注射区と胸垂皮下注射区の2区にランダムに配置し,4週間隔で3回毛髪ケラチン50mg/2.5mlとオイルアジュバントの等量(2.5ml)混和乳化液を投与した.初回免疫後1週間は毎日体温を測定し,その後6週間まで毎週1度注射局所の状態を観察した.初回免疫から10週間後に各区2頭をと殺し,枝肉の瑕疵の有無を調べた.免疫牛の産子への影響を調べるため,免疫牛および非免疫牛の産子各々18頭および20頭について,生時体重,疾病発生率,および発育成績を3年間にわたり比較した.両区とも初回免疫後の体温については免疫の影響は見られなかった.初回免疫1週間後には注射局所に腫脹が認められ,少なくとも6週後まで存続した.これらの腫脹をと畜検査で調べたところ,尻部筋肉注射区は注射局所の腫脹硬結が尻部の筋肉組織に存在し,瑕疵となっていた.一方,胸垂皮下注射区は注射局所に腫脹硬結が見られたが,それらは胸垂の脂肪組織に留まり,瑕疵はなかった.免疫子牛と非免疫子牛の平均生時体重,疾病発生率,発育成績には有意の差は認められなかった.