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71.
Fukasawa A Nagashima T Aoyama T Fukuda N Matsuda H Ueno T Sugiyama H Nagase H Matsumoto Y 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》2007,859(2):272-275
A simple and sensitive high-performance liquid chromatography (HPLC) method utilizing UV detection was developed for the determination of plasma pyrrole (Py)-imidazole (Im) polyamides in rats and applied to the pharmacokinetic study of compounds. After deproteinization of plasma with methanol, Py-Im polyamides were analyzed with a reversed-phase TSK-GEL ODS-80TM (4.6 mmx15.0 cm TOSOH Co., Japan) column maintained at 40 degrees C. The mobile phase solvent A was 0.1% acetic acid and the solvent B was HPLC-grade acetonitrile (0-10 min, A: 100-20%, B: 0-80% linear gradient; 10-15 min, A: 40%, B: 60%). The flow rate was 1.0 ml/min. The detection wavelength was set at 310 nm. The method was used to determine the plasma concentration time profiles of Py-Im polyamides after intravenous injection. 相似文献
72.
Takahashi Yoshihiro Khairun Hisam Bin Nasir Akiko Ito Hiroyuki Kanzaki Hideo Matsumura Hiromasa Saitoh Shizuko Fujisawa Sophien Kamoun Ryohei Terauchi 《Plant signaling & behavior》2007,2(5):396-398
A high-throughput in planta overexpression screen of a Nicotiana benthamiana cDNA library identified a mitogen activated protein kinase kinase (MAPKK), NbMKK1, as a potent inducer of hypersensitive response (HR)-like cell death. NbMKK1-mediated cell death was attenuated in plants whereby expression of NbSIPK, an ortholog of tobacco SIPK and Arabidopsis AtMPK6, was knocked down by virus-induced gene silencing (VIGS), suggesting that NbMKK1 functions upstream of NbSIPK. In accordance with this result, NbMKK1 phosphorylated NbSIPK in vitro, and furthermore NbMKK1 and NbSIPK physically interacted in yeast two-hybrid assay. VIGS of NbMKK1 in N. benthamiana resulted in a delay of Phytophthora infestans INF1 elicitin-mediated HR as well as in the reduction of resistance against a non-host pathogen Pseudomonas cichorii. Our data of NbMKK1, together with that of LeMKK4,1 demonstrate the presence of a novel defense signaling pathway involving NbMKK1/LeMKK4 and SIPK.Key Words: MAPK, defense, cell death, in planta screenMitogen activated protein kinase (MAPK) cascades are highly conserved signaling pathways in eukaryotes, comprising three tiered classes of protein kinase, MAPKKK (MAPKK kinase), MAPKK and MAPK, that sequentially relay phosphorylation signals.2 The Arabidopsis genome carries genes for 20 MAPKs, 10 MAPKKs3 and more than 25 MAPKKKs.4 In plants, MAPK signaling is known to function in various biotic4,5 and abiotic6 stress responses and cytokinesis.7 In defense signaling, extensive research has been carried out for two tobacco MAPKs, SIPK8 (salicylic-acid-induced protein kinase; hereafter designated as NtSIPK) and WIPK9 (wound-induced protein kinase = NtWIPK), and their orthologs in Arabidopsis10 (AtMPK6 and ATMPK3, respectively), partly because kinase activities of these two MAPKs are easy to detect by an in gel kinase assay using myeline basic protein (MBP) as substrate.11 Both NtSIPK and NtWIPK are activated by the interaction between host resistance (R)- gene and cognate avirulence gene of pathogen11,12 and elicitor perception by host cells.13,14 Shuqun Zhang and his group showed that an upstream kinase of both NtSIPK and NtWIPK is NtMEK2.15 Transient overexpression of constitutively active NtMEK2 caused phosphorylation of NtSIPK and NtWIPK, resulting in rapid HR-like cell death in tobacco leaves.15 Later, the same lab showed that overexpression of NtSIPK alone also caused HR-like cell death.16 The downstream target proteins of NtSIPK and AtMPK6 are being identified and include 1-aminocyclopropane-1-carboxylic acid sythase-6 (ACS-6).17,18 Although recent studies identified another MAPK cascade (NtMEK1 → Ntf6) involved in defense responses19,20 we can still say that the current research focus of MAPK defense signaling centers around the cascade comprising [NtMEK2→ NtSIPK/NtWIPK→ target proteins] of tobacco and its orthologous pathways in other plant species.In an effort to search for plant genes involved in HR-like cell death, we have been employing a high-throughput in planta expression screen of N. benthamiana cDNA libraries. In this experimental system, a cDNA library was made in a binary potato virus X (PVX)-based expression vector pSfinx.21 The cDNA library was transferred to Agrobacterium tumefaciens, and 40,000 of the bacterial colonies were individually inoculated by toothpicks onto leaf blades of N. benthamiana leaves. The phenotype around the inoculated site was observed 1–2 weeks following the inoculation. This rapid screen identified 30 cDNAs that caused cell death after overexpression, including genes coding for ubiquitin proteins, RNA recognition motif (RRM) containing proteins, a class II ethylene-responsive element binding factor (EREBP)-like protein22 and a MAPKK protein (this work). Such an in planta screening technique has been used before for the isolation of fungal21 and oomycete23,24 elicitors and necrosis inducing genes, but not for isolation of plant genes. Overexpression screening of cDNA libraries is a common practice in prokaryotes, yeast and amimal cells,25,26 so it is a surprise that this approach has not been systematically applied in plants. Given its throughput, we propose that this virus-based transient overexpression system is a highly efficient way to isolate novel plant genes by functional screen.27 Since overexpression frequently causes non-specific perturbation of signaling, genes identified by overexpression should be further validated by loss-of-function assays, for instance, VIGS.28Overexpression of the identified MAPKK gene, NbMKK1, triggered a rapid generation of H2O2, followed by HR-like cell death in N. benthamiana leaves (this work). NbMKK1-GFP fusion protein overexpression also caused cell death, and curiously NbMKK1-GFP was shown to localize consistently in the nucleus. Sequence comparison classified NbMKK1 to the Group D of MAPKKs about which little information is available. So far, a MAPKK, LeMKK4, from tomato belonging to the Group D MAPKKs, was shown to cause cell death after overexpression.1 Based on amino acid sequence similarity and phylogenetic analyses, LeMKK4 and NbMKK1 seem to be orthologs. To see whether NbMKK1 transduces signals through SIPK and WIPK, we performed NbMKK1 overexpression in N. benthamiana plants whereby the expression of either NbSIPK or NbWIPK (WIPK ortholog in N. benthamiana) was silenced by VIGS. NbMKK1 did not induce cell death in NbSIPK-silenced plants, suggesting that the NbMKK1 cell death signal is transmitted through NbSIPK. Indeed, NbMKK1 phosphorylated NbSIPK in vitro, and NbMKK1 and NbSIPK physically interacted in yeast two-hybrid assay. These results suggest that NbMKK1 interacts with NbSIPK, most probably with its N-terminal docking domain, and phosphorylates NbSIPK in vivo to transduce the cell death signal downstream.NbMKK1 exhibits constitutive expression in leaves. To determine the function of NbMKK1 in defense, we silenced NbMKK1 by VIGS, and such plants were challenged with Phytophthora infestans INF1 elicitin29 and Pseudomonas cichorii, a non-host pathogen. INF1-mediated HR cell death was remarkably delayed in NbMKK1-silenced plants. Likewise, plant defense against P. cichorii was compromised in NbMKK1-silenced plants. These results indicate that NbMKK1 is an important component of signaling of INF1-mediated HR and non-host resistance to P. cichorii.Together, our analyses of NbMKK1 and independent work from Greg Martin''s lab on LeMKK41 suggest that a Group D MAPKK, NbMKK1/LeMKK4, functions upstream of SIPK and transduces defense signals in these solanaceous plants (Fig. 1). In plants as well as in other eukaryotes, it is common that kinases have multiple partners. The work on these kinases fits this concept. A single MAPK (e.g., SIPK) is phosphorylated by multiple MAPKKs (e.g., NtMEK2 and NbMKK1), and a single MAPKK (e.g., NtMEK2) can phosphorylate multiple MAPKs (e.g., NtSIPK and NtWIPK).Open in a separate windowFigure 1Defense signaling through NbMKK1/LeMKK4. Two defense signal pathways involving NtMEK2 (indicated as MEK2) → WIPK/SIPK and NtMEK1(indicated as MEK1) → Ntf6 are well documented. By our and Pedley and Martin''s1 works, another novel MAPKK, NbMKK1/LeMKK4 was demonstrated to participate in defense signaling by phosphorylation of SIPK. 相似文献
73.
Tanida Kotomi Shimada Mihoko Khor Seik-Soon Toyoda Hiromi Kato Kayoko Kotorii Nozomu Kotorii Tatayu Ariyoshi Yu Kato Takao Hiejima Hiroshi Ozone Motohiro Uchimura Naohisa Ikegami Azusa Kume Kazuhiko Kanbayashi Takashi Imanishi Aya Kamei Yuichi Hida Akiko Wada Yamato Kuroda Kenji Miyamoto Masayuki Hirata Koichi Takami Masanori Yamada Naoto Okawa Masako Omata Naoto Kondo Hideaki Kodama Tohru Inoue Yuichi Mishima Kazuo Honda Makoto Tokunaga Katsushi Miyagawa Taku 《Sleep and biological rhythms》2022,20(1):137-148
Sleep and Biological Rhythms - Idiopathic hypersomnia (IH) is a rare sleep disorder characterized by excessive daytime sleepiness, great difficulty upon awakening, and prolonged sleep time. In... 相似文献
74.
Miyata Seiko Otake Hironao Fujishiro Hiroshige Iwamoto Kunihiro Noda Akiko Sone Michihiko Ozaki Norio 《Sleep and biological rhythms》2022,20(3):403-411
Sleep and Biological Rhythms - Sleep problems and obstructive sleep apnea (OSA) increase with age and disturb life in old age. Positional therapy is one option to treat OSA, but the differences in... 相似文献
75.
Hideharu Abe Akiko Sakurai Arisa Ochi 《Biochemical and biophysical research communications》2019,508(2):654-659
The glomerulus is a network of capillaries known as a tuft, located at the beginning of a nephron in the kidney. Here we describe a novel method for the induction of a macroscopically visible three-dimensional glomerulus-like sphere (GLS). This procedure did not require any additional cytokines and completed the formation of spheres within 24?h. After the formation was complete, GLS maintained a steady state for at least five days without proliferation and without a decrease in viability. Therefore, this procedure assists various assays for a prolong period of time. Overall, our protocol allows for a very simple mixing of cells from different sources to obtain fine-grained and highly dispersed GLSs. The kidney filtration barrier is a unique structure characterized by a complex three-dimensional framework of podocytes and endothelial cells. GLS exhibited the induction of many podocyte-specific gene profiles similar to those in adult human kidneys, suggesting that the sphere formation process is important for the maturation of podocytes. Focal segmental glomerulosclerosis (FSGS) is one of the major causes of steroid-resistant nephrotic syndrome, and some circulating permeability factors in the patient's serum FSGS have been implicated in the pathogenesis of the disease. Serum from patients with FSGS induced the collapse of GLS, which imitates the appearance of glomerulosclerosis in patients. In conclusion, the investigation and use of GLS may provide a novel method to elucidate the molecular mechanisms underlying complicated and unexplained events in glomeruli in a similar condition in adult kidneys. 相似文献
76.
The cDNA sequence coding for the coat protein of cucumber mosaic virus (Japanese Y strain) was cloned, and its nucleotide sequence was determined. The sequence contains an open reading frame that encodes the coat protein composed of 218 amino acids. The nucleotide and deduced amino acid sequences of the coat protein of this strain were compared with those of the Q strain; the homologies of the sequences were 78% and 81%, respectively. Further study of the sequences gave an insight into the genome organization and the molecular features of the coat protein. The coding region can be divided into three characteristic regions. The N-terminal region has conserved features in the positively charged structure, the hydropathy pattern and the predicted secondary structure, although the amino acid sequence is varied mainly due to frameshift mutations. It is noteworthy that the positions of arginine residues in this region are highly conserved. Both the nucleotide and amino acid sequences of the central region are well conserved. The amino acid sequence of the C-terminal region is not conserved, because of frameshift mutations, however, the total number of amino acids is conserved. The nucleotide sequence of the 3'-noncoding region is divergent, but it could form a tRNA-like structure similar to those reported for other viruses. Detailed investigation suggests that the Y and Q strains are evolutionarily distant. 相似文献
77.
RFLPs of cpDNA were examined for 18 species ofAster, six species ofKalimeris, two species ofMiyamayomena and one species and one variety ofHeteropappus from Japan, using 16 restriction endonucleases. Approximately 275 restriction sites were surveyed, and a total of 74 restriction
site mutations was detected, and 31 of these were phylogenetically informative. Sixteen most parsimonious trees constructed
from Wagner parsimony analysis indicated the polyphyly ofKalimeris andMiyamayomena sensu Kitamura;K. miqueliana belongs to a different clade from the remaining species ofKalimeris, and two species ofMiyamayomena did not make a single clade. This result suggests that the shortening or loss of pappus have happened parallelly in different
evolutionary lineages. We must be careful to assess the pappus character in taxonomy and phylogeny, and it is desirable to
examine their phylogenetic relationships using a molecular data. 相似文献
78.
Xu J Zheng SL Komiya A Mychaleckyj JC Isaacs SD Chang B Turner AR Ewing CM Wiley KE Hawkins GA Bleecker ER Walsh PC Meyers DA Isaacs WB 《American journal of human genetics》2003,72(1):208-212
Rare germline mutations of macrophage scavenger receptor 1 (MSR1) gene were reported to be associated with prostate cancer risk in families with hereditary prostate cancer (HPC) and in patients with non-HPC (Xu et al. 2002). To further evaluate the role of MSR1 in prostate cancer susceptibility, at Johns Hopkins Hospital, we studied five common variants of MSR1 in 301 patients with non-HPC who underwent prostate cancer treatment and in 250 control subjects who participated in prostate cancer-screening programs and had normal digital rectal examination and PSA levels (<4 ng/ml). Significantly different allele frequencies between case subjects and control subjects were observed for each of the five variants (P value range.01-.04). Haplotype analyses provided consistent findings, with a significant difference in the haplotype frequencies from a global score test (P=.01). Because the haplotype that is associated with the increased risk for prostate cancer did not harbor any of the known rare mutations, it appears that the observed association of common variants and prostate cancer risk are independent of the effect of the known rare mutations. These results consistently suggest that MSR1 may play an important role in prostate carcinogenesis. 相似文献
79.
Sakamoto K Hayashi A Sakamoto A Kiga D Nakayama H Soma A Kobayashi T Kitabatake M Takio K Saito K Shirouzu M Hirao I Yokoyama S 《Nucleic acids research》2002,30(21):4692-4699
A suppressor tRNA(Tyr) and mutant tyrosyl-tRNA synthetase (TyrRS) pair was developed to incorporate 3-iodo-L-tyrosine into proteins in mammalian cells. First, the Escherichia coli suppressor tRNA(Tyr) gene was mutated, at three positions in the D arm, to generate the internal promoter for expression. However, this tRNA, together with the cognate TyrRS, failed to exhibit suppressor activity in mammalian cells. Then, we found that amber suppression can occur with the heterologous pair of E.coli TyrRS and Bacillus stearothermophilus suppressor tRNA(Tyr), which naturally contains the promoter sequence. Furthermore, the efficiency of this suppression was significantly improved when the suppressor tRNA was expressed from a gene cluster, in which the tRNA gene was tandemly repeated nine times in the same direction. For incorporation of 3-iodo-L-tyrosine, its specific E.coli TyrRS variant, TyrRS(V37C195), which we recently created, was expressed in mammalian cells, together with the B.stearothermophilus suppressor tRNA(Tyr), while 3-iodo-L-tyrosine was supplied in the growth medium. 3-Iodo-L-tyrosine was thus incorporated into the proteins at amber positions, with an occupancy of >95%. Finally, we demonstrated conditional 3-iodo-L-tyrosine incorporation, regulated by inducible expression of the TyrRS(V37C195) gene from a tetracycline-regulated promoter. 相似文献
80.
Nanako Katagiri Takeo Shobuike Bin Chang Akiko Kukita Hiroshi Miyamoto 《Microbes and infection / Institut Pasteur》2012,14(13):1123-1132
Human nucleotide oligomerization domain-like receptor family apoptosis inhibitory protein (NAIP) prevents apoptosis by inhibiting caspase-3, -7, and -9. Four functional Naip exist in the murine genome, each of which is equally similar to human NAIP. Among them, Naip5 induces pyroptosis by promoting caspase-1 activation in response to Legionella pneumophila infection in macrophages. However, the contribution of human NAIP to this response is unclear. To investigate the role of human NAIP in macrophage survival, we stably expressed human NAIP in RAW264.7 macrophages. Human NAIP inhibited camptothecin-induced apoptosis in macrophages; however, it promoted cytotoxicity in L. pneumophila-infected cells. This cytotoxicity was associated with caspase-1. In addition, human NAIP restricted the intracellular growth of L. pneumophila. L. pneumophila flagellin was required for cytotoxicity, caspase-1 activation, and restriction of intracellular bacterial growth. Expression of murine Naip5 produced comparable results. These data indicate that human NAIP regulates the host response to L. pneumophila infection in a manner similar to that of murine Naip5 and that human NAIP and murine Naip5 regulate cell survival by inhibiting apoptosis or by promoting pyroptosis in response to specific cellular signals. 相似文献