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141.
Crystal structure of tetrameric homoisocitrate dehydrogenase from an extreme thermophile, Thermus thermophilus: involvement of hydrophobic dimer-dimer interaction in extremely high thermotolerance
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Miyazaki J Asada K Fushinobu S Kuzuyama T Nishiyama M 《Journal of bacteriology》2005,187(19):6779-6788
The crystal structure of homoisocitrate dehydrogenase involved in lysine biosynthesis from Thermus thermophilus (TtHICDH) was determined at 1.85-A resolution. Arg85, which was shown to be a determinant for substrate specificity in our previous study, is positioned close to the putative substrate binding site and interacts with Glu122. Glu122 is highly conserved in the equivalent position in the primary sequence of ICDH and archaeal 3-isopropylmalate dehydrogenase (IPMDH) but interacts with main- and side-chain atoms in the same domain in those paralogs. In addition, a conserved Tyr residue (Tyr125 in TtHICDH) which extends its side chain toward a substrate and thus has a catalytic function in the related beta-decarboxylating dehydrogenases, is flipped out of the substrate-binding site. These results suggest the possibility that the conformation of the region containing Glu122-Tyr125 is changed upon substrate binding in TtHICDH. The crystal structure of TtHICDH also reveals that the arm region is involved in tetramer formation via hydrophobic interactions and might be responsible for the high thermotolerance. Mutation of Val135, located in the dimer-dimer interface and involved in the hydrophobic interaction, to Met alters the enzyme to a dimer (probably due to steric perturbation) and markedly decreases the thermal inactivation temperature. Both the crystal structure and the mutation analysis indicate that tetramer formation is involved in the extremely high thermotolerance of TtHICDH. 相似文献
142.
A polysaccharide deacetylase homologue, PdaA, in Bacillus subtilis acts as an N-acetylmuramic acid deacetylase in vitro
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A polysaccharide deacetylase homologue, PdaA, was determined to act as an N-acetylmuramic acid deacetylase in vitro. Histidine-tagged truncated PdaA (with the putative signal sequence removed) was overexpressed in Escherichia coli cells and purified. Measurement of deacetylase activity showed that PdaA could deacetylate peptidoglycan treated with N-acetylmuramoyl-L-alanine amidase CwlH but could not deacetylate peptidoglycan treated with or without DL-endopeptidase LytF (CwlE). Reverse-phase high-performance liquid chromatography and mass spectrometry (MS) and MS-MS analyses indicated that PdaA could deacetylate the N-acetylmuramic acid residues of purified glycan strands derived from Bacillus subtilis peptidoglycan. 相似文献
143.
144.
Nagaoka Y Otsu K Okada F Sato K Ohba Y Kotani N Fujii J 《Biochemical and biophysical research communications》2005,331(1):215-223
Singlet oxygen is a causal factor in light-induced skin photoaging and the cytotoxic process of tumor cells in photodynamic chemotherapy. To develop a better understanding of the functional consequences of protein modification by singlet oxygen, the effects of naphthalene endoperoxide on lysosomal protease, cathepsin, were examined. When the soluble fraction of normal human fetal skin fibroblast cells was treated with the endoperoxide, the activities of cysteine proteases, cathepsins B and L/S, were inhibited, but that of aspartate protease, cathepsin D/E, was not. The reduction of the endoperoxide-treated soluble fractions by treatment with dithiothreitol barely recovered the activities. Cathepsin B, purified from normal human liver, exhibited similar profiles to that in cytosol. These data suggest that singlet oxygen oxidatively modifies an amino acid residue essential for catalysis and consequently results in the irreversible inactivation of cysteine protease-type cathepsin. 相似文献
145.
Eguchi J Kuwashima N Hatano M Nishimura F Dusak JE Storkus WJ Okada H 《Journal of immunology (Baltimore, Md. : 1950)》2005,174(11):7194-7201
We previously demonstrated that IL-4 gene-transfected glioma cell vaccines induce effective therapeutic immunity in preclinical glioma models, and have initiated phase I trials of these vaccines in patients with malignant gliomas. To gain additional mechanistic insight into the efficacy of this approach, we have treated mice bearing the MCA205 (H-2(b)) or CMS-4 (H-2(d)) sarcomas. IL-12/23 p40(-/-) and IFN-gamma(-/-) mice, which were able to reject the initial inoculation of IL-4 expressing tumors, failed to mount a sustained systemic response against parental (nontransfected) tumor cells. Paracrine production of IL-4 in vaccine sites promoted the accumulation and maturation of IL-12p70-secreting tumor-infiltrating dendritic cells (TIDCs). Adoptive transfer of TIDCs isolated from vaccinated wild-type, but not IL-12/23 p40(-/-), mice were capable of promoting tumor-specific CTL responses in syngeneic recipient animals. Interestingly, both STAT4(-/-) and STAT6(-/-) mice failed to reject IL-4-transfected tumors in concert with the reduced capacity of TIDCs to produce IL-12p70 and to promote specific antitumor CTL reactivity. These results suggest that vaccines consisting of tumor cells engineered to produce the type 2 cytokine, IL-4, critically depend on type 1 immunity for their observed therapeutic efficacy. 相似文献
146.
Appican produced by rat C6 glioma cells, the chondroitin sulfate (CS) proteoglycan form of the amyloid precursor protein, contains an E disaccharide, -GlcUA-GalNAc(4,6-O-disulfate)-, in its CS chain. In this study, the appican CS chain from rat C6 glioma cells was shown to specifically bind several growth/differentiation factors including midkine (MK) and pleiotrophin (PTN). In contrast, the appican CS from SH-SY5Y neuroblastoma cells contained no E disaccharide and showed no binding to either MK or PTN. These findings indicate that the E motif is essential in the interaction of the appican CS chain with growth/differentiation factors, and suggest that glial appican may mediate the regulation of neuronal cell adhesion and migration and/or neurite outgrowth. 相似文献
147.
Ji G Feldman ME Deng KY Greene KS Wilson J Lee JC Johnston RC Rishniw M Tallini Y Zhang J Wier WG Blaustein MP Xin HB Nakai J Kotlikoff MI 《The Journal of biological chemistry》2004,279(20):21461-21468
Genetically encoded signaling proteins provide remarkable opportunities to design and target the expression of molecules that can be used to report critical cellular events in vivo, thereby markedly extending the scope and physiological relevance of studies of cell function. Here we report the development of a transgenic mouse expressing such a reporter and its use to examine postsynaptic signaling in smooth muscle. The circularly permutated, Ca2+-sensing molecule G-CaMP (Nakai, J., Ohkura, M., and Imoto, K. (2001) Nat. Biotechnol. 19, 137-141) was expressed in vascular and non-vascular smooth muscle and functioned as a lineage-specific intracellular Ca2+ reporter. Detrusor tissue from these mice was used to identify two separate types of postsynaptic Ca2+ signals, mediated by distinct neurotransmitters. Intrinsic nerve stimulation evoked rapid, whole-cell Ca2+ transients, or "Ca2+ flashes," and slowly propagating Ca2+ waves. We show that Ca2+ flashes occur through P2X receptor stimulation and ryanodine receptor-mediated Ca2+ release, whereas Ca2+ waves arise from muscarinic receptor stimulation and inositol trisphosphate-mediated Ca2+ release. The distinct ionotropic and metabotropic postsynaptic Ca2+ signals are related at the level of Ca2+ release. Importantly, individual myocytes are capable of both postsynaptic responses, and a transition between Ca2+ -induced Ca2+ release and inositol trisphosphate waves occurs at higher synaptic inputs. Ca2+ signaling mice should provide significant advantages in the study of processive biological signaling. 相似文献
148.
Large-scale analysis of protein-protein interaction sites is especially needed in the postgenomic era. The combination of affinity labeling with mass spectrometry is a potentially useful high-throughput screening method for this purpose. However, reagents in current use are not ideal as some cause damage to the target molecule and others have poor solubility in physiologic aqueous buffers. In this paper, we describe a novel affinity labeling reagent, acyl adenylate, which is highly soluble in aqueous solutions and reacts in a pH-dependent manner. The adenylate of deoxycholic acid reacts with amino groups on the side chain of a lysine residue and at the N-terminus of proteins/peptides. The reactivity and stability of this reagent were investigated, and it was confirmed that, after formation of a reversible ligand-protein complex under weakly acidic conditions, derivatization with acyl adenylate occurred at the target site under weakly alkaline condition. We further demonstrated the utility of this reagent for affinity labeling using a monoclonal antibody with high affinity for deoxycholic acid. Competitive ELISA indicated that deoxycholic acid was labeled around the antibody ligand binding site, thus enabling the structural elucidation of the ligand-protein interaction. In addition, LC/ESI-MS/MS analysis of the labeled peptide obtained by enzymatic digestion and affinity extraction allowed the identification of the structure surrounding the antigen binding site. 相似文献
149.
Niwa K Takano R Obata Y Hiura H Komiyama J Ogawa H Kono T 《Biology of reproduction》2004,71(5):1560-1567
Mouse parthenotes result in embryonic death before 10 days of gestation, but parthenogenetic embryos (ng/fg PE) that contain haploid sets of genomes from nongrowing (ng) oocytes derived from newborn fetuses and fully grown (fg) oocytes derived from adults can develop into 13.5-day-old fetuses. This prolonged development is due to a lack of genomic imprinting in ng oocytes. Here, we show maternal genomes of oocytes derived from ng/fg PE are competent to support normal development. After 28 days of culture, the ovaries from ng/fg PE grew as well as the controls, forming vesicular follicles with follicular antrums. The oocytes collected from the developed follicles were the same size as those of the controls. To determine whether maternal primary imprinting had been established in the oocytes derived from ng/fg PE, we examined the DNA methylation status in differentially methylated regions of three imprinted genes, Igf2r, Lit1, and H19. The results showed that maternal-specific modifications were imposed in the oocytes derived from ng/fg PE. Further, to assess nuclear competence to support development, we constructed matured oocytes containing a haploid genome derived from ng/fg PE oocytes by serial nuclear transfer. After in vitro fertilization and culture and embryo transplantation into recipients, two live pups were obtained. One developed normally to a fertile adult. These results revealed that oocytes derived from ng/fg PE can be normally imprinted during oogenesis and acquire competence to participate in development as female genomes. 相似文献
150.
Taurine prevents the ischemia-induced apoptosis in cultured neonatal rat cardiomyocytes through Akt/caspase-9 pathway 总被引:7,自引:0,他引:7
Takatani T Takahashi K Uozumi Y Matsuda T Ito T Schaffer SW Fujio Y Azuma J 《Biochemical and biophysical research communications》2004,316(2):484-489
Activated Akt kinase has been proposed as a central role in suppressing apoptosis by modulating the activities of Bcl-2 family proteins and/or caspase-9. To study the mechanism underlying the anti-apoptotic effect of taurine, the interaction between taurine and Akt/caspase-9 pathway was examined using a simulated ischemia model with cultured rat neonatal cardiomyocytes sealed in closed flasks. Taurine (20mM) treatment attenuated simulated ischemia-induced decline in the activity of Akt. Although taurine treatment had no effect on the expression of Bcl-2 in mitochondria and the level of cytosolic cytochrome c, it inhibited ischemia-induced cleavage of caspases 9 and 3. Moreover, adenovirus transfer of the dominant negative form of Akt objected taurine-mediated anti-apoptotic effects, cancelling the suppression of caspase-9 and caspase-3 activities by taurine. These findings provide the first evidence that taurine inhibits ischemia-induced apoptosis in cardiac myocytes with the increase in Akt activities, by inactivating caspase-9. 相似文献