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31.
Masaharu Sakagami Yukiteru Ouji Norikazu Kawai Masayasu Misu Masahide Yoshikawa Tadashi Kitahara 《Biochemistry and Biophysics Reports》2019
Vestibular hair cells (V–HCs) in the inner ear have important roles and various functions. When V–HCs are damaged, crippling symptoms, such as vertigo, visual field oscillation, and imbalance, are often seen. Recently, several studies have reported differentiation of embryonic stem (ES) cells, as pluripotent stem cells, to HCs, though a method for producing V–HCs has yet to be established. In the present study, we used vestibular cell conditioned medium (V-CM) and effectively induced ES cells to differentiate into V–HCs. Expressions of V-HC-related markers (Math1, Myosin6, Brn3c, Dnah5) were significantly increased in ES cells cultured in V-CM for 2 weeks, while those were not observed in ES cells cultured without V-CM. On the other hand, the cochlear HC-related marker Lmod3 was either not detected or detected only faintly in those cells when cultured in V-CM. Our results demonstrate that V-CM has an ability to specifically induce differentiation of ES cells into V–HCs. 相似文献
32.
Akihiro Kinoshita Tomoo Tamura Chiharu Aoki Tohru Nakanishi Shizuo Sobue Fujio Suzuki Kojiro Takahashi Masaharu Takigawa 《Cell biology international》1995,19(8):647-654
Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with 125 I-ET-1, two major bands of 43 kDa and 46 kDa were separated by SDS-PAGE. Scatchard analysis demonstrated two classes of ET receptors with Kd values of 1 × 10?10 M and 5 × 10?9 M. The numbers of high- and low- affinity receptors were 1 × 104 and 2 × 105 per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-β1, IL-1β, RA and EGF. ET-1 stimulated DNA synthesis in cultured rabbit chondrocytes. ET-1 also stimulated calcium incorporation through the cell membrane of chondrocytes. These findings indicate that ET-1 has a physiological effect on chondrocytes via its receptors on the cells. 相似文献
33.
34.
Association of a regulatory gene, slyA with a mouse virulence of Salmonella serovar Choleraesuis 总被引:1,自引:0,他引:1
Kaneko A Mita M Sekiya K Matsui H Kawahara K Danbara H 《Microbiology and immunology》2002,46(2):109-113
The influence of slyA gene, originally found in Salmonella serovar Typhimurium as a regulatory gene for the expression of virulence genes, on a mouse virulence of S. serovar Choleraesuis was investigated by using an slyA-defective mutant. The defective mutant was constructed by the insertion of a kanamycin-resistance gene (aph) into the cloned slyA gene, and the homologous recombination with the intact slyA gene on the chromosome. The mutant strain showed the LD50 value for BALB/c mouse approximately 10(5) higher than that of the parent strain. The increase of the LD50 value was the same order as that shown by the mutation of the slyA gene of S. serovar Typhimurium, although LD50 of the wild-type strain of S. serovar Choleraesuis was 40-fold higher than that of S. serovar Typhimurium. The time course of infection observed in the mice organs also proved the clear difference of the virulence between the parent and the mutant strains. These results suggested that the slyA gene product functions as a virulence-associated regulator also in S. serovar Choleraesuis. 相似文献
35.
Hiroo Horinishi Shuichi Hashizume Masaharu Seguchi Kazuko Takahashi 《Biochemical and biophysical research communications》1975,67(3):1136-1143
A soluble fraction from Escherichia coli B was found to incorporate methionine into 95°C CCl3COOH-insoluble fraction. The incorporation required methionyl-tRNA synthetase, methionine tRNA, ATP, Mg2+ and bovine milk casein. The casein could be replaced by arginylated bovine serum albumin and arginylated bovine α-lactalbumin. A mixture of 19 amino acids other than methionine and GTP had no effect on the incorporation. KCl was rather inhibitory. Puromycin, RNase A and trypsin inhibited the incorporation, while DNase I did not. The soluble fraction also incorporated the methionyl moiety of methionyl-tRNA. This incorporation was not affected by the addition of free methionine. 相似文献
36.
Taiji Asami Naoki Nishizawa Yoshihiro Ishibashi Kimiko Nishibori Masaharu Nakayama Yasuko Horikoshi Shin-ichi Matsumoto Masashi Yamaguchi Hirokazu Matsumoto Naoki Tarui Tetsuya Ohtaki Chieko Kitada 《Bioorganic & medicinal chemistry letters》2012,22(20):6391-6396
Metastin/kisspeptin, a 54-amino acid peptide, is the ligand of the G-protein-coupled receptor KISS1R which plays a key role in pathways that regulate reproduction and cell migration in many endocrine and gonadal tissues. The N-terminally truncated decapeptide, metastin(45–54), has 3–10 times higher receptor affinity and intracellular calcium ion-mobilizing activity but is rapidly inactivated in serum. In this study we designed and synthesized stable KISS1R agonistic decapeptide analogs with selected substitutions at positions 47, 50, and 51. Replacement of glycine with azaglycine (azaGly) in which the α-carbon is replaced with a nitrogen atom at position 51 improved the stability of amide bonds between Phe50-Gly51 and Gly51-Leu52 as determined by in vitro mouse serum stability studies. Substitution for tryptophan at position 47 with other amino acids such as serine, threonine, β-(3-pyridyl)alanine, and d-tryptophan (d-Trp), produced analogs that were highly stable in mouse serum. d-Trp47 analog 13 showed not only high metabolic stability but also excellent KISS1R agonistic activity. Other labile peptides may have increased serum stability using amino acid substitution. 相似文献
37.
Futami J Nukui E Maeda T Kosaka M Tada H Seno M Yamada H 《Journal of biochemistry》2002,132(2):223-228
Cationization of a protein is considered to be a powerful strategy for internalizing a functional protein into cells. Cationized proteins appear to adsorb to the cell surface by electrostatic interactions, then enter the cell in a receptor- and transporter-independent fashion. Thus, in principle, all cell types appear to take up cationized proteins. Since ribonucleases (RNases) have a latent cytotoxic potential, cationized RNases could be useful cancer chemotherapeutics. In this study, we investigated the effect of the degree of cationization on the cytotoxicity of RNase A by modifying carboxyl groups with ethylenediamine. We found that there is an optimum degree of modification for cytotoxicity, in which 5 to 7 out of 11 carboxyl groups in RNase A are modified, toward MCF-7 and 3T3-SV-40 cells. More interestingly, the cytotoxicity of cationized RNase As correlates well with the value of [RNase activity] x [estimated concentration of RNase free from RNase inhibitor], mimicking the practical enzymatic activity of cationized RNase As in cytosol. The results indicate that cationization of a protein to an optimum level is important for maintaining protein function in the cytosol. Sophisticated protein cationization techniques will help to advance protein transduction technology. 相似文献
38.
39.
Sanai M Endo S Matsunaga T Ishikura S Tajima K El-Kabbani O Hara A 《Archives of biochemistry and biophysics》2007,464(1):122-129
Mammalian 3α-hydroxysteroid dehydrogenases (3α-HSDs) have been divided into two types: Cytosolic NADP(H)-dependent 3α-HSDs belonging to the aldo-keto reductase family, and mitochondrial and microsomal NAD+-dependent 3α-HSDs belonging to the short-chain dehydrogenase/reductase family. In this study, we characterized a rat aldo-keto reductase (AKR1C17), whose functions are unknown. The recombinant AKR1C17 efficiently oxidized 3α-hydroxysteroids and bile acids using NAD+ as the preferred coenzyme at an optimal pH of 7.4-9.5, and was inhibited by ketamine and organic anions. The mRNA for AKR1C17 was detected specifically in rat kidney, where the enzyme was more highly expressed as a cytosolic protein than NADP(H)-dependent 3α-HSD (AKR1C9). Thus, AKR1C17 represents a novel NAD+-dependent type of cytosolic 3α-HSD with unique inhibitor sensitivity and tissue distribution. In addition, the replacement of Gln270 and Glu276 of AKR1C17 with the corresponding residues of NADP(H)-dependent 3α-HSD resulted in a switch in favor of NADP+ specificity, suggesting their key roles in coenzyme specificity. 相似文献
40.
Fujita S Ohnishi T Watanabe B Yokota T Takatsuto S Fujioka S Yoshida S Sakata K Mizutani M 《The Plant journal : for cell and molecular biology》2006,45(5):765-774
Arabidopsis dwf4 is a brassinosteroid (BR)-deficient mutant, and the DWF4 gene encodes a cytochrome P450, CYP90B1. We report the catalytic activity and substrate specificity of CYP90B1. Recombinant CYP90B1 was produced in Escherichia coli, and CYP90B1 activity was measured in an in vitro assay reconstituted with NADPH-cytochrome P450 reductase. CYP90B1 converted campestanol (CN) to 6-deoxocathasterone, confirming that CYP90B1 is a steroid C-22 hydroxylase. The substrate specificity of CYP90B1 indicated that sterols with a double bond at positions C-5 and C-6 are preferred substrates compared with stanols, which have no double bond at the position. In particular, the catalytic efficiency (k(cat)/K(m)) of CYP90B1 for campesterol (CR) was 325 times greater than that for CN. As CR is more abundant than CN in planta, the results suggest that C-22 hydroxylation of CR before C-5alpha reduction is the main route of BR biosynthetic pathway, which contrasts with the generally accepted route via CN. In addition, CYP90B1 showed C-22 hydroxylation activity toward various C(27-29) sterols. Cholesterol (C27 sterol) is the best substrate, followed by CR (C28 sterol), whereas sitosterol (C29 sterol) is a poor substrate, suggesting that the substrate preference of CYP90B1 may explain the discrepancy between the in planta abundance of C27/C28/C29 sterols and C27/C28/C29 BRs. 相似文献