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91.
Analysis of conditional paralytic mutants in Drosophila sarco-endoplasmic reticulum calcium ATPase reveals novel mechanisms for regulating membrane excitability 下载免费PDF全文
Sanyal S Consoulas C Kuromi H Basole A Mukai L Kidokoro Y Krishnan KS Ramaswami M 《Genetics》2005,169(2):737-750
Individual contributions made by different calcium release and sequestration mechanisms to various aspects of excitable cell physiology are incompletely understood. SERCA, a sarco-endoplasmic reticulum calcium ATPase, being the main agent for calcium uptake into the ER, plays a central role in this process. By isolation and extensive characterization of conditional mutations in the Drosophila SERCA gene, we describe novel roles of this key protein in neuromuscular physiology and enable a genetic analysis of SERCA function. At motor nerve terminals, SERCA inhibition retards calcium sequestration and reduces the amplitude of evoked excitatory junctional currents. This suggests a direct contribution of store-derived calcium in determining the quantal content of evoked release. Conditional paralysis of SERCA mutants is also marked by prolonged neural activity-driven muscle contraction, thus reflecting the phylogenetically conserved role of SERCA in terminating contraction. Further analysis of ionic currents from mutants uncovers SERCA-dependent mechanisms regulating voltage-gated calcium channels and calcium-activated potassium channels that together control muscle excitability. Finally, our identification of dominant loss-of-function mutations in SERCA indicates novel intra- and intermolecular interactions for SERCA in vivo, overlooked by current structural models. 相似文献
92.
Yoshioka Y Tsutsumi Y Ikemizu S Yamamoto Y Shibata H Nishibata T Mukai Y Okamoto T Taniai M Kawamura M Abe Y Nakagawa S Nagata S Yamagata Y Mayumi T 《Biochemical and biophysical research communications》2004,315(4):808-814
Recently, we created a lysine-deficient mutant tumor necrosis factor-alpha [mTNF-alpha-Lys(-)] with full bioactivity in vitro compared with wild-type TNF-alpha (wTNF-alpha), and site-specific PEGylation of mTNF-alpha-Lys(-) was found to selectively enhance its in vivo antitumor activity. In this study, we attempted to optimize this PEGylation of mTNF-alpha-Lys(-) to further improve its therapeutic potency. mTNF-alpha-Lys(-) was site-specifically modified at its N-terminus with linear polyethylene glycol (LPEG) or branched PEG (BPEG). While randomly mono-PEGylated wTNF-alpha (ran-LPEG5K-wTNF-alpha) with 5 kDa of LPEG (LPEG5K) had about only 4% in vitro bioactivity of wTNF-alpha, mono-PEGylated mTNF-alpha-Lys(-) [sp-PEG-mTNF-alpha-Lys(-)] with LPEG5K, LPEG20K, BPEG10K, and BPEG40K had 82%, 58%, 93%, and 65% bioactivities of mTNF-alpha-Lys(-), respectively. sp-LPEG-mTNF-alpha-Lys(-) and sp-BPEG10K-mTNF-alpha-Lys(-) had much superior antitumor activity to those of both unmodified TNF-alphas and ran-LPEG5K-wTNF-alpha, though sp-BPEG40K-mTNF-alpha-Lys(-) did not show in vivo antitumor activity. Thus, the molecular shape and weight of PEG may strongly influence the in vivo antitumor activity of sp-PEG-mTNF-alpha-Lys(-). 相似文献
93.
Mukai T Maeda Y Tamura T Matsuoka M Tsukamoto Y Makino M 《Journal of immunology (Baltimore, Md. : 1950)》2010,185(10):6234-6243
To activate naive T cells convincingly using Mycobacterium bovis bacillus Calmette-Guérin (BCG), recombinant BCG (BCG-D70M) that was deficient in urease, expressed with gene encoding the fusion of BCG-derived heat shock protein (HSP) 70 and Mycobacterium leprae-derived major membrane protein (MMP)-II, one of the immunodominant Ags of M. leprae, was newly constructed. BCG-D70M was more potent in activation of both CD4(+) and CD8(+) subsets of naive T cells than recombinant BCGs including urease-deficient BCG and BCG-70M secreting HSP70-MMP-II fusion protein. BCG-D70M efficiently activated dendritic cells (DCs) to induce cytokine production and phenotypic changes and activated CD4(+) T cells even when macrophages were used as APCs. The activation of both subsets of T cells was MHC and CD86 dependent. Pretreatment of DCs with chloroquine inhibited both surface expression of MMP-II on DCs and the activation of T cells by BCG-D70M-infected APCs. The naive CD8(+) T cell activation was inhibited by treatment of DCs with brefeldin A and lactacystin so that the T cell was activated by TAP- and proteosome-dependent cytosolic cross-priming pathway. From naive CD8(+) T cells, effector T cells producing perforin and memory T cells having migration markers were produced by BCG-D70M stimulation. BCG-D70M primary infection in C57BL/6 mice produced T cells responsive to in vitro secondary stimulation with MMP-II and HSP70 and more efficiently inhibited the multiplication of subsequently challenged M. leprae than vector control BCG. These results indicate that the triple combination of HSP70, MMP-II, and urease depletion may provide a useful tool for inducing better activation of naive T cells. 相似文献
94.
Yoshida K Kim WS Kinehara M Mukai R Ashida H Ikeda H Fujita Y Krishnan HB 《Bioscience, biotechnology, and biochemistry》2006,70(12):2957-2964
Sinorhizobium fredii USDA191 is a Gram-negative bacterium capable of forming nitrogen-fixing nodules on soybean roots. The USDA191 idhA gene encoding myo-inositol dehydrogenase, an enzyme necessary for myo-inositol utilization, is known to be involved in competitive nodulation and nitrogen fixation. In Bacillus subtilis, myo-inositol dehydrogenase catalyzes the first step of the myo-inositol catabolic pathway. Recently iolE was identified as the gene encoding 2-keto-myo-inositol dehydratase, which catalyzes the second step in the pathway. Here we report the presence of 2-keto-myo-inositol dehydratase activity in free-living USDA191 cells cultured in a medium containing myo-inositol. An iolE ortholog was cloned from USDA191. USDA191 iolE was expressed in Escherichia coli as a His(6)-tag fusion and purified to exhibit 2-keto-myo-inositol dehydratase activity. Inactivation of USDA191 iolE led to defective myo-inositol utilization. USDA191 iolE partially complemented a B. subtilis iolE deficient mutant. These results suggest that S. fredii USDA191 utilizes a myo-inositol catabolic pathway, analogous to that of B. subtilis, involving at least idhA and iolE. 相似文献
95.
Higashimoto K Soejima H Saito T Okumura K Mukai T 《Cytogenetic and genome research》2006,113(1-4):306-312
Human chromosomal region 11p15.5, which is homologous to mouse chromosome region 7F5, is a well-known imprinted region. The CDKN1C/KCNQ1OT1 imprinted domain, which is one of two imprinted domains at 11p15.5, includes nine imprinted genes regulated by an imprinting center (IC). The CDKN1C/KCNQ1OT1 IC is a differentially methylated region of KCNQ1OT1(KCNQ1OT-DMR) with DNA methylation on the maternal allele and no methylation on the paternal allele. CDKN1C (alias p57KIP2), an imprinted gene with maternal expression, encoding a cyclin-dependent kinase inhibitor, is a critical gene within the CDKN1C/KCNQ1OT1 domain. In Beckwith-Wiedemann syndrome (BWS), approximately 50% of patients show loss of DNA methylation accompanied by loss of histone H3 Lys9 dimethylation on maternal KCNQ1OT-DMR, namely an imprinting disruption, leading to diminished expression of CDKN1C. In cancer, at least three molecular mechanisms--imprinting disruption, aberrant DNA methylations at the CDKN1C promoter, and loss of heterozygosity (LOH) of the maternal allele--are seen and all three result in diminished expression of CDKN1C. Imprinting disruption of the CDKN1C/KCNQ1OT1 domain is involved in the development of both BWS and cancer and it changes the maternal epigenotype to the paternal type, leading to diminished CDKN1C expression. In this review, we describe recent advances in epigenetic control of the CDKN1C/KCNQ1OT1 imprinted domain in both humans and mice. 相似文献
96.
Shobayashi M Mukai N Iwashita K Hiraga Y Iefuji H 《Applied microbiology and biotechnology》2006,69(6):704-710
S-Adenosylmethionine (SAM) is an important metabolite that participates in many reactions as a methyl group donor in all organisms,
and has attracted much interest in clinical research because of its potential to improve many diseases, such as depression,
liver disease, and osteoarthritis. Because of these potential applications, a more efficient means is needed to produce SAM.
Accordingly, we developed a positive selection method to isolate SAM-accumulating yeast in this study. In Saccharomyces cerevisiae, one of the main reactions consuming SAM is thought to be the methylation reaction in the biosynthesis of ergosterol that
is catalyzed by Erg6p. Mutants with deficiencies in ergosterol biosynthesis may accumulate SAM as a result of the reduction
of SAM consumption in ergosterol biosynthesis. We have applied this method to isolate SAM-accumulating yeasts with nystatin,
which has been used to select mutants with deficiencies in ergosterol biosynthesis. SAM-accumulating mutants from S. cerevisiae K-9 and X2180-1A were efficiently isolated through this method. These mutants accumulated 1.7–5.5 times more SAM than their
parental strains. NMR and GC-MS analyses suggested that two mutants from K-9 have a mutation in the erg4 gene, and erg4 disruptants from laboratory strains also accumulated more SAM than their parental strains. These results indicate that mutants
having mutations in the genes for enzymes that act downstream of Erg6p in ergosterol biosynthesis are effective in accumulating
SAM. 相似文献
97.
Murakami G Tsurugizawa T Hatanaka Y Komatsuzaki Y Tanabe N Mukai H Hojo Y Kominami S Yamazaki T Kimoto T Kawato S 《Biochemical and biophysical research communications》2006,351(2):553-558
Modulation of hippocampal synaptic plasticity by estrogen has been attracting much attention. Here, we demonstrated the rapid effect of 17beta-estradiol on the density and morphology of spines in the stratum oriens (s.o., basal side) and in the stratum lacunosum-moleculare (s.l.m., apical side) by imaging Lucifer Yellow-injected CA1 neurons in adult male rat hippocampal slices, because spines in s.o. and s.l.m. have been poorly understood as compared with spines in the stratum radiatum. The application of 1nM estradiol-induced a rapid increase in the density of spines of pyramidal neurons within 2h. This increase by estradiol was blocked by Erk MAP kinase inhibitor and estrogen receptor inhibitor in both regions. Effect of blockade by agonists of AMPA receptors and NMDA receptors was different between s.o. and s.l.m. In both regions, ERalpha agonist PPT induced the same enhancing effect of spinogenesis as that induced by estradiol. 相似文献
98.
The Genetic Structure of Natural Populations of DROSOPHILA MELANOGASTER Xiii. Further Studies on Linkage Disequilibrium 下载免费PDF全文
The Raleigh, North Carolina, population of Drosophila melanogaster was examined for linkage disequilibrium in 1974, several years after previous analyses in 1968, 1969, and 1970. alphaglycerol-3-phosphate dehydrogenase-1 (alphaGpdh-1), malate dehydrogenase-1 (Mdh-1), alcohol dehydrogenase (Adh), and hexokinase-C (Hex-C, tentative name, F. M. Johnson, unpublished; position determined by the present authors to be 2-74.5) were assayed for 617 second chromosomes, and esterase-C (Est-C) and octanol dehydrogenase (Odh) were assayed for 526 third chromosomes. In addition, two polymorphic inversions in the second chromosomes [In(2L)t and In(2R)NS] were examined, and the following findings were obtained: (1) No linkage disequilibrium between isozyme genes was detected. Significant linkage disequilibria were found only between the polymorphic inversions and isozyme genes [In(2L)t vs. Adh, and In(2R)NS vs. Hex-C]. Significant disequilibrium was not detected between In(2L)t and alphaGpdh-1, which is included in the inversion, but a tendency toward disequilibrium was consistently found from 1968 to 1974. The frequency of two-strand double crossovers within inversion In(2L)t involving a single crossover on each side of alphaGpdh-1 was estimated to be 0.00022. Thus, the consistent but not significant linkage disequilibrium between the two factors can be explained by recombination after the inversion occurred. (2) Previously existing linkage disequilibrium between Adh and In(2R)NS (the distance is about 30 cM, but the effective recombination value is about 1.75%) was found to have disappeared. (3) No higher-order linkage disequilibrium was detected. (4) Linkage disequilibrium between Odh and Est-C (the distance of which was estimated to be 0.0058 +/- 0.002) could not be detected (chi(2) (df=1) = 0.9).-From the above results, it was concluded that linkage disequilibria among isozyme genes are very rare in D. melanogaster, so that the Franklin-Lewontin model (Franklin and Lewontin 1970) is not applicable to these genes. The linkage disequilibria between some isozyme genes and polymorphic inversions may be explained by founder effect. 相似文献
99.
Twenty different natural populations of Drosophila melanogaster were sampled to determine the frequencies of inversions. Based on their frequencies and geographical distributions, the inversions could be classified as follows: (1) Common cosmopolitan inversions that are present in many populations in frequencies exceeding five percent and that may exhibit frequency clines over large geographical regions; (2) Rare cosmopolitan inversions that occur throughout the species range but usually at frequencies below five percent and that may be absent in many populations; (3) Recurrent endemic inversions that are found in several adjacent populations in frequencies usually not exceeding one or two percent; and (4) Unique endemic inversions that are found only among the progeny of a single individual and that may represent one aspect of the syndrome termed "hybrid dysgenesis". Four common cosmopolitan inversions that exhibit highly significant clines in populations in the eastern United States are In(2L)t, In(2R)NS, In(3L)P and In(3R)P. 相似文献
100.