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91.
92.
Ishii S Chang HH Kawasaki K Yasuda K Wu HL Garman SC Fan JQ 《The Biochemical journal》2007,406(2):285-295
Fabry disease is a lysosomal storage disorder caused by the deficiency of alpha-Gal A (alpha-galactosidase A) activity. In order to understand the molecular mechanism underlying alpha-Gal A deficiency in Fabry disease patients with residual enzyme activity, enzymes with different missense mutations were purified from transfected COS-7 cells and the biochemical properties were characterized. The mutant enzymes detected in variant patients (A20P, E66Q, M72V, I91T, R112H, F113L, N215S, Q279E, M296I, M296V and R301Q), and those found mostly in mild classic patients (A97V, A156V, L166V and R356W) appeared to have normal K(m) and V(max) values. The degradation of all mutants (except E59K) was partially inhibited by treatment with kifunensine, a selective inhibitor of ER (endoplasmic reticulum) alpha-mannosidase I. Metabolic labelling and subcellular fractionation studies in COS-7 cells expressing the L166V and R301Q alpha-Gal A mutants indicated that the mutant protein was retained in the ER and degraded without processing. Addition of DGJ (1-deoxygalactonojirimycin) to the culture medium of COS-7 cells transfected with a large set of missense mutant alpha-Gal A cDNAs effectively increased both enzyme activity and protein yield. DGJ was capable of normalizing intracellular processing of mutant alpha-Gal A found in both classic (L166V) and variant (R301Q) Fabry disease patients. In addition, the residual enzyme activity in fibroblasts or lymphoblasts from both classic and variant hemizygous Fabry disease patients carrying a variety of missense mutations could be substantially increased by cultivation of the cells with DGJ. These results indicate that a large proportion of mutant enzymes in patients with residual enzyme activity are kinetically active. Excessive degradation in the ER could be responsible for the deficiency of enzyme activity in vivo, and the DGJ approach may be broadly applicable to Fabry disease patients with missense mutations. 相似文献
93.
Miyako Kusano Atsushi Fukushima Henning Redestig Makoto Kobayashi Hitomi Otsuki Hitoshi Onouchi Satoshi Naito Masami Yokota Hirai Kazuki Saito 《Amino acids》2010,39(4):1013-1021
Methionine (Met) is an essential amino acid for all organisms. In plants, Met also functions as a precursor of plant hormones,
polyamines, and defense metabolites. The regulatory mechanism of Met biosynthesis is highly complex and, despite its great
importance, remains unclear. To investigate how accumulation of Met influences metabolism as a whole in Arabidopsis, three methionine over-accumulation (mto) mutants were examined using a gas chromatography–mass spectrometry-based metabolomics approach. Multivariate statistical
analyses of the three mto mutants (mto1, mto2, and mto3) revealed distinct metabolomic phenotypes. Orthogonal projection to latent structures–discriminant analysis highlighted discriminative
metabolites contributing to the separation of each mutant and the corresponding control samples. Though Met accumulation in
mto1 had no dramatic effect on other metabolic pathways except for the aspartate family, metabolite profiles of mto2 and mto3 indicated that several extensive pathways were affected in addition to over-accumulation of Met. The pronounced changes
in metabolic pathways in both mto2 and mto3 were associated with polyamines. The findings suggest that our metabolomics approach not only can reveal the impact of Met
over-accumulation on metabolism, but also may provide clues to identify crucial pathways for regulation of metabolism in plants. 相似文献
94.
Toxicity of free proline revealed in an arabidopsis T-DNA-tagged mutant deficient in proline dehydrogenase 总被引:5,自引:0,他引:5
The toxicity of proline (Pro) to plant growth has raised questions despite its protective functions in response to environmental stresses. To evaluate Pro toxicity, we isolated an Arabidopsis T-DNA-tagged mutant, pdh, that had a defect in Pro dehydrogenase (AtProDH), which catalyzes the first step of Pro catabolism. The pdh mutant showed hypersensitivity to exogenous application of < or =10 mM L-Pro, at which wild-type plants grew normally. A dose-dependent increase in internal free Pro accumulation was observed in pdh plants during external Pro supply. These results do not just prove the toxicity of Pro, but also suggest that AtProDH is the only enzyme acting as a functional ProDH in ARABIDOPSIS: To further analyze the targets of Pro toxicity, we compared the expression of thousands of genes by pdh plants with that by wild-type plants by cDNA microarray analysis. Most genes were unaffected. Here we demonstrate Pro toxicity by using the pdh mutant and discuss a cause-and-effect action between an excess of free Pro and growth inhibition in ARABIDOPSIS. 相似文献
95.
'Working' cardiomyocytes exhibiting plateau action potentials from human placenta-derived extraembryonic mesodermal cells 总被引:3,自引:0,他引:3
Okamoto K Miyoshi S Toyoda M Hida N Ikegami Y Makino H Nishiyama N Tsuji H Cui CH Segawa K Uyama T Kami D Miyado K Asada H Matsumoto K Saito H Yoshimura Y Ogawa S Aeba R Yozu R Umezawa A 《Experimental cell research》2007,313(12):2550-2562
The clinical application of cell transplantation for severe heart failure is a promising strategy to improve impaired cardiac function. Recently, an array of cell types, including bone marrow cells, endothelial progenitors, mesenchymal stem cells, resident cardiac stem cells, and embryonic stem cells, have become important candidates for cell sources for cardiac repair. In the present study, we focused on the placenta as a cell source. Cells from the chorionic plate in the fetal portion of the human placenta were obtained after delivery by the primary culture method, and the cells generated in this study had the Y sex chromosome, indicating that the cells were derived from the fetus. The cells potentially expressed 'working' cardiomyocyte-specific genes such as cardiac myosin heavy chain 7beta, atrial myosin light chain, cardiac alpha-actin by gene chip analysis, and Csx/Nkx2.5, GATA4 by RT-PCR, cardiac troponin-I and connexin 43 by immunohistochemistry. These cells were able to differentiate into cardiomyocytes. Cardiac troponin-I and connexin 43 displayed a discontinuous pattern of localization at intercellular contact sites after cardiomyogenic differentiation, suggesting that the chorionic mesoderm contained a large number of cells with cardiomyogenic potential. The cells began spontaneously beating 3 days after co-cultivation with murine fetal cardiomyocytes and the frequency of beating cells reached a maximum on day 10. The contraction of the cardiomyocytes was rhythmical and synchronous, suggesting the presence of electrical communication between the cells. Placenta-derived human fetal cells may be useful for patients who cannot supply bone marrow cells but want to receive stem cell-based cardiac therapy. 相似文献
96.
Satoh M Andoh Y Clingan CS Ogura H Fujii S Eshima K Nakayama T Taniguchi M Hirata N Ishimori N Tsutsui H Onoé K Iwabuchi K 《PloS one》2012,7(2):e30568
The progression of obesity is accompanied by a chronic inflammatory process that involves both innate and acquired immunity. Natural killer T (NKT) cells recognize lipid antigens and are also distributed in adipose tissue. To examine the involvement of NKT cells in the development of obesity, C57BL/6 mice (wild type; WT), and two NKT-cell-deficient strains, Jα18(-/-) mice that lack the type I subset and CD1d(-/-) mice that lack both the type I and II subsets, were fed a high fat diet (HFD). CD1d(-/-) mice gained the least body weight with the least weight in perigonadal and brown adipose tissue as well as in the liver, compared to WT or Jα18(-/-) mice fed an HFD. Histologically, CD1d(-/-) mice had significantly smaller adipocytes and developed significantly milder hepatosteatosis than WT or Jα18(-/-) mice. The number of NK1.1(+)TCRβ(+) cells in adipose tissue increased when WT mice were fed an HFD and were mostly invariant Vα14Jα18-negative. CD11b(+) macrophages (Mφ) were another major subset of cells in adipose tissue infiltrates, and they were divided into F4/80(high) and F4/80(low) cells. The F4/80(low)-Mφ subset in adipose tissue was increased in CD1d(-/-) mice, and this population likely played an anti-inflammatory role. Glucose intolerance and insulin resistance in CD1d(-/-) mice were not aggravated as in WT or Jα18(-/-) mice fed an HFD, likely due to a lower grade of inflammation and adiposity. Collectively, our findings provide evidence that type II NKT cells initiate inflammation in the liver and adipose tissue and exacerbate the course of obesity that leads to insulin resistance. 相似文献
97.
Satoshi Mitsuda Shigeyasu Nabeshima Hideo Hirohara 《Applied microbiology and biotechnology》1989,31(4):334-337
Summary Characteristics of the enantioselective hydrolysis of the acetic ester of 4-hydroxy-3-methyl-2-(2-propynyl)-2-cyclopentenone (HMPC) by Arthrobacter lipase were investigated in a water/oil biphasic reaction mixture. Kinetic studies revealed that the strict enantioselectivity was entirely due to a difference in the catalytic constants for the enantiomeric substrates and that (S)-HMPC acetate acted as a competitive inhibitor. The comparison of enantioselectivity for the acetates of HMPC analogues indicated that hydrophobic substituents in the HMPC molecule were essential for the strict enantioselectivity.Biological preparation of an optically active alcohol. Part II 相似文献
98.
The replication of positive-strand RNA viruses involves not only viral proteins but also multiple cellular proteins and intracellular membranes. In both plant cells and the yeast Saccharomyces cerevisiae, brome mosaic virus (BMV), a member of the alphavirus-like superfamily, replicates its RNA in endoplasmic reticulum (ER)-associated complexes containing viral 1a and 2a proteins. Prior to negative-strand RNA synthesis, 1a localizes to ER membranes and recruits both positive-strand BMV RNA templates and the polymerase-like 2a protein to ER membranes. Here, we show that BMV RNA replication in S. cerevisiae is markedly inhibited by a mutation in the host YDJ1 gene, which encodes a chaperone Ydj1p related to Escherichia coli DnaJ. In the ydj1 mutant, negative-strand RNA accumulation was inhibited even though 1a protein associated with membranes and the positive-strand RNA3 replication template and 2a protein were recruited to membranes as in wild-type cells. In addition, we found that in ydj1 mutant cells but not wild-type cells, a fraction of 2a protein accumulated in a membrane-free but insoluble, rapidly sedimenting form. These and other results show that Ydj1p is involved in forming BMV replication complexes active in negative-strand RNA synthesis and suggest that a chaperone system involving Ydj1p participates in 2a protein folding or assembly into the active replication complex. 相似文献
99.
Yamazaki S Iwamoto R Saeki K Asakura M Takashima S Yamazaki A Kimura R Mizushima H Moribe H Higashiyama S Endoh M Kaneda Y Takagi S Itami S Takeda N Yamada G Mekada E 《The Journal of cell biology》2003,163(3):469-475
Heparin-binding EGF-like growth factor (HB-EGF) is first synthesized as a membrane-anchored form (proHB-EGF), and its soluble form (sHB-EGF) is released by ectodomain shedding from proHB-EGF. To examine the significance of proHB-EGF processing in vivo, we generated mutant mice by targeted gene replacement, expressing either an uncleavable form (HBuc) or a transmembrane domain-truncated form (HBdeltatm) of the molecule. HB(uc/uc) mice developed severe heart failure and enlarged heart valves, phenotypes similar to those in proHB-EGF null mice. On the other hand, mice carrying HBdeltatm exhibited severe hyperplasia in both skin and heart. These results indicate that ectodomain shedding of proHB-EGF is essential for HB-EGF function in vivo, and that this process requires strict control. 相似文献
100.