Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

The process of sexual reproduction in eukaryotes starts when gametes from two different sexes encounter each other. Paramecium, a unicellular eukaryote, undergoes conjugation and uses a gametic nucleus to enter the sexual reproductive process. The molecules responsible for recognizing mating partners, hypothetically called mating-type substances, are still unclear. We have identified an O3-type mating substance polypeptide and its gene sequence using protein chemistry, molecular genetics, immunofluorescence, RNA interference, and microinjection. The O3-type substance is a polypeptide found in the ciliary membranes, located from the head to the ventral side of cells. The O3-type substance has a kinase-like domain in its N-terminal part located outside the cell and four EF-hand motifs that bind calcium ions in its C-terminal part located inside the cell. RNA interference and immunofluorescence revealed that this polypeptide positively correlated with the expression of mating reactivity. Microinjection of an expression vector incorporating the O3Pc-MSP gene (Oms3) induced additional O3 mating type in the recipient clones of different mating types or syngen. Phylogenetic analysis indicates that this gene is widely present in eukaryotes and exhibits high homology among closely related species. The O3Pc-MSP (Oms3) gene had nine silent mutations compared to the complementary mating type of the E3 homologue gene.

DOI： 10.3390/microorganisms12030588

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Fertilization-initiated development and adult-onset aging are standard features in the life history of eukaryotes. In Paramecium, the number of cell divisions after the birth of a new generation is an essential parameter of sexual phase transition and aging. However, the gene driving this process and its evolutionary origin have not yet been elucidated. Here we report several critical outcomes obtained by molecular genetics, immunofluorescence microscopy, transformation by microinjection, and enzymological analysis. The cloned immaturin gene induces sexual rejuvenation in both mature and senescent cells by microinjection. The immaturin gene originated from proteobacteria’s glutathione-S-transferase (GST) gene. However, immaturin has been shown to lose GST activity and instead acquire nuclease activity. In vitro substrates for immaturin-nuclease are single- and double-stranded DNA, linear and circular DNA, and single-stranded viral genome RNA such as coronavirus. Anti-immaturin antibodies have shown that the subcellular localizations of immaturin are the macronucleus, cytoplasm, cell surface area, and cilia. The phase transition of sexuality is related to a decrease in the intracellular abundance of immaturin. We propose that sexual maturation and rejuvenation is a process programmed by the immaturin gene, and the sexual function of each age is defined by both the abundance and the intracellular localization mode of the immaturin-nuclease.

DOI： 10.3390/microorganisms11010082

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elmer Press, Inc.  

DOI： 10.14740/jocmr4623

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Peroxisome proliferator-activated receptor (PPAR) γ1, a nuclear receptor, is abundant in the murine placenta during the late stage of pregnancy (E15-E16), although its functional roles remain unclear. PPARγ1 is encoded by two splicing isoforms, namely Pparγ1canonical and Pparγ1sv, and its embryonic loss leads to early (E10) embryonic lethality. Thus, we generated knockout (KO) mice that carried only one of the isoforms to obtain a milder phenotype. Pparγ1sv-KO mice were viable and fertile, whereas Pparγ1canonical-KO mice failed to recover around the weaning age. Pparγ1canonical-KO embryos developed normally up to 15.5 dpc, followed by growth delays after that. The junctional zone of Pparγ1canonical-KO placentas severely infiltrated the labyrinth, and maternal blood sinuses were dilated. In the wild-type, PPARγ1 was highly expressed in sinusoidal trophoblast giant cells (S-TGCs), peaking at 15.5 dpc. Pparγ1canonical-KO abolished PPARγ1 expression in S-TGCs. Notably, the S-TGCs had unusually enlarged nuclei and often occupied maternal vascular spaces, disturbing the organization of the fine labyrinth structure. Gene expression analyses of Pparγ1canonical-KO placentas indicated enhanced S-phase cell cycle signatures. EdU-positive S-TGCs in Pparγ1canonical-KO placentas were greater in number than those in wild-type placentas, suggesting that the cells continued to endoreplicate in the mutant placentas. These results indicate that PPARγ1, a known cell cycle arrest mediator, is involved in the transition of TGCs undergoing endocycling to the terminal differentiation stage in the placentas. Therefore, PPARγ1 deficiency, induced through genetic manipulation, leads to placental insufficiency.

DOI： 10.1016/j.ydbio.2021.07.003

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Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1) protein, which mediates intracellular cholesterol trafficking from the brush border membrane to the endoplasmic reticulum, where chylomicron assembly takes place in enterocytes or in the intestinal absorptive epithelial cells. Cholesterol is a minor lipid constituent of chylomicrons; however, whether or not a shortage of cholesterol attenuates chylomicron assembly is unknown. The aim of this study was to examine the effect of ezetimibe, a potent NPC1L1 inhibitor, on trans-epithelial lipid transport, and chylomicron assembly and secretion in enterocytes. Caco-2 cells, an absorptive epithelial model, grown onto culture inserts were given lipid micelles from the apical side, and chylomicron-like TAG-rich lipoprotein secreted basolaterally were analyzed after a 24-h incubation period in the presence of ezetimibe up to 50 μM. The secretion of lipoprotein and apolipoprotein B48 were reduced by adding ezetimibe (30% and 34%, respectively). Although ezetimibe allowed the cells to take up cholesterol normally, the esterification was abolished. Meanwhile, oleic acid esterification was unaffected. Moreover, ezetimibe activated sterol regulatory element-binding protein 2 by approximately 1.5-fold. These results suggest that ezetimibe limited cellular cholesterol mobilization required for lipoprotein assembly. In such conditions, large lipid droplet formation in Caco-2 cells and the enterocytes in mice were induced, implying that unprocessed TAG was sheltered in these compartments. Although ezetimibe did not reduce the post-prandial lipid surge appreciably in triolein-infused mice, the results of the present study indicated that pharmacological actions of ezetimibe may participate in a novel regulatory mechanism for the efficient chylomicron assembly and secretion.

DOI： 10.1016/j.bbalip.2020.158808

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The nuclear receptor peroxisome proliferator-activated receptor (PPAR)γ has been implicated in the pathogenesis of various human diseases including fatty liver. Although nuclear translocation of PPARγ plays an important role in PPARγ signaling, details of the translocation mechanisms have not been elucidated. Here we demonstrate that PPARγ2 translocates to the nucleus and activates signal transduction through H2O2-dependent formation of a PPARγ2 and transportin (Tnpo)1 complex via redox-sensitive disulfide bonds between cysteine (Cys)176 and Cys180 of the former and Cys512 of the latter. Using hepatocyte cultures and mouse models, we show that cytosolic H2O2/Tnpo1-dependent nuclear translocation enhances the amount of DNA-bound PPARγ and downstream signaling, leading to triglyceride accumulation in hepatocytes and liver. These findings expand our understanding of the mechanism underlying the nuclear translocation of PPARγ, and suggest that the PPARγ and Tnpo1 complex and surrounding redox environment are potential therapeutic targets in the treatment of PPARγ-related diseases.

DOI： 10.1016/j.freeradbiomed.2020.06.005

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A 72-year-old man with a 10-year history of coronary heart disease started evolocumab treatment once a month after developing excess myalgia due to therapy with a 3-hydroxy-methylglutaryl CoA reductase inhibitor. No side effects such as myalgia symptoms had been reported during the first 14 months of evolocumab treatment; however, he suddenly presented with acute severe thrombocytopenia following the 14th treatment. His platelet count continued to decrease to a nadir of 1,000/μL. His platelet-associated immunoglobulin G level had elevated to 790 ng/107 cells. He started receiving a combination of steroid therapy, high-dose immunoglobulin therapy, and platelet transfusions, but the first-line therapy was ineffective. He was subsequently treated with a thrombopoietin receptor agonist, and his platelet count recovered to 250,000/μL.

DOI： 10.1155/2020/3281626

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Publishing type：Research paper (scientific journal)   Publisher：Springer Science and Business Media LLC  

DOI： 10.1186/s12974-019-1632-z

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Other Link： http://link.springer.com/article/10.1186/s12974-019-1632-z/fulltext.html

DOI： 10.3164/jcbn.17-116

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We surveyed genome sequences from the basidiomycetous mushroom Coprinopsis cinerea and isolated a cDNA homologous to CMKA, a calmodulin-dependent protein kinase (CaMK) in Aspergillus nidulans. We designated this sequence, encoding 580 amino acids with a molecular weight of 63,987, as CoPK02. CoPK02 possessed twelve subdomains specific to protein kinases and exhibited 43, 35, 40% identity with rat CaMKI, CaMKII, CaMKIV, respectively, and 40% identity with CoPK12, one of the CaMK orthologs in C. cinerea. CoPK02 showed significant autophosphorylation activity and phosphorylated exogenous proteins in the presence of Ca2+/CaM. By the CaM-overlay assay we confirmed that the C-terminal sequence (Trp346-Arg358) was the calmodulin-binding site, and that the binding of Ca2+/CaM to CoPK02 was reduced by the autophosphorylation of CoPK02. Since CoPK02 evolved in a different clade from CoPK12, and showed different gene expression compared to that of CoPK32, which is homologous to mitogen-activated protein kinase-activated protein kinase, CoPK02 and CoPK12 might cooperatively regulate Ca2+-signaling in C. cinerea.

DOI： 10.1080/09168451.2018.1462692

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：Medical Association of Nippon Medical School  

Background: The antidiabetic drug teneligliptin is a novel dipeptidyl peptidase-4 (DPP-4) inhibitor with a thiazolidine-specific structure. This study aimed to investigate whether teneligliptin can activate PPARγ directly and/or indirectly in cell-based assays. Methods: Promoter assays using the reporter construct driven under the control of the SV40 promoter and the PPAR response element (PPRE) were performed. Luciferase activity was measured after a 3-day incubation of vector-transduced cells with various concentrations of teneligliptin. Results: Treatment of the cells with 50 μM teneligliptin significantly transactivated a reporter gene. The presence of the PPARγ antagonist, GW9662, did not affect the activation of PPRE-reporter expression by teneligliptin. Conclusion: We found that teneligliptin could increase PPARγ activity in cell-based assays irrespective of the PPARγ ligand-binding domain.

DOI： 10.1272/jnms.2018_85-15

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Within the calanoid copepods, the bioluminescent species comprise 5-59% of the abundance and 10-15% of the biomass in the world's oceans. Most of the luminous species belong to the superfamily Augaptiloidea. The composition of bioluminescent species within the calanoid copepods shows latitudinal patterns; 5-25% of total calanoid copepods are found in high-latitude oceans, while 34-59% are in low-latitude oceans, reflecting a prey-predator relationship. Bioluminescent species of calanoid copepods are able to produce the light-emitting substrate coelenterazine. It is then transferred to higher predators through the food chain, and might be used for bioluminescence in other luminous organisms. A notable feature of copepod bioluminescence is the secreted-type, and its major function may be as an antipredatory response or a defensive behavior. Identification of more than 20 luciferase genes from calanoid copepods has revealed the highly conserved sequences of those genes. This leads us to the speculation that the genes for luciferase within the group of calanoid copepods have evolved independently of comparable genes outside of this group. We discuss here the ecological and biological functions of copepod bioluminescence, the significant diversity in luminous intensity, which might be evolutionarily relevant to their motility and habitat depth, and the promising future directions of bioluminescence studies.

DOI： 10.1093/plankt/fbx016

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Ezetimibe inhibits Niemann-Pick C1-like 1 (NPC1L1), an apical membrane cholesterol transporter of enterocytes, thereby reduces intestinal cholesterol absorption. This treatment also increases extrahepatic reverse cholesterol transport via an undefined mechanism. To explore this, we employed a trans-intestinal cholesterol efflux (TICE) assay, which directly detects circulation-to-intestinal lumen H-3-cholesterol transit in a cannulated jejunal segment, and found an increase of TICE by 45%. To examine whether such increase in efflux occurs at the intestinal brush border membrane(BBM)-level, we performed luminal perfusion assays, similar to TICE but the jejunal wall was labelled with orally-given H-3-cholesterol, and determined elevated BBM-to-lumen cholesterol efflux by 3.5-fold with ezetimibe. Such increased efflux probably promotes circulation-to-lumen cholesterol transit eventually; thus increases TICE. Next, we wondered how inhibition of NPC1L1, an influx transporter, resulted in increased efflux. When we traced orally-given H-3-cholesterol in mice, we found that lumen-to-BBM H-3-cholesterol transit was rapid and less sensitive to ezetimibe treatment. Comparison of the efflux and fractional cholesterol absorption revealed an inverse correlation, indicating the efflux as an opposite-regulatory factor for cholesterol absorption efficiency and counteracting to the naturally-occurring rapid cholesterol influx to the BBM. These suggest that the ezetimibe-stimulated increased efflux is crucial in reducing cholesterol absorption. Ezetimibe-induced increase in cholesterol efflux was approximately 2.5-fold greater in mice having endogenous ATP-binding cassette G5/G8 heterodimer, the major sterol efflux transporter of enterocytes, than the knockout counterparts, suggesting that the heterodimer confers additional rapid BBM-to-lumen cholesterol efflux in response to NPC1L1 inhibition. The observed framework for intestinal cholesterol fluxes may provide ways to modulate the flux to dispose of endogenous cholesterol efficiently for therapeutic purposes.

DOI： 10.1371/journal.pone.0152207

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Secreted luciferases isolated from copepod crustaceans are frequently used for nondisruptive reporter-gene assays, such as the continuous, automated and/or high-throughput monitoring of gene expression in living cells. All known copepod luciferases share highly conserved amino acid residues in two similar, repeated domains in the sequence. The similarity in the domains are ideal nature for designing PCR primers to amplify cDNA fragments of unidentified copepod luciferases from bioluminescent copepod crustaceans. Here, we introduce how to establish a cDNA encoding novel copepod luciferases from a copepod specimen by PCR with degenerated primers.

DOI： 10.1007/978-1-4939-3813-1_3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC INTERNAL MEDICINE  

We herein present a 60-year-old man with adrenocortical carcinoma who had gynecomastia. An endocrinological examination revealed increased levels of serum estradiol and dehydroepiandrosterone-sulfate ( DHEA-S) and reduced levels of free testosterone. Magnetic resonance imaging showed an adrenal tumor with heterogeneous intensity. Iodine-131 adosterol scintigraphy showed an increased uptake at the same site. Because feminizing adrenocortical carcinoma was suspected, right adrenalectomy was performed; the pathological diagnosis was adrenocortical carcinoma. The results of immunostaining indicated a virilizing tumor. Aromatase activity was identified on RT-PCR. As disorganized steroidogenesis is pathologically present in adrenocortical carcinoma, this diagnosis should be made with caution.

DOI： 10.2169/internalmedicine.55.5912

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Here, we describe the isolation of two nickel-induced genes in Paramecium caudatum, NCI16 and PcGST1, by subtractive hybridization. NCI16 encoded a predicted four-transmembrane domain protein (similar to 16 kDa) of unknown function, and PcGST1 encoded glutathione S-transferase (GST; similar to 25 kDa) with GST and glutathione peroxidase (GPx) activities. Exposing cells to cobalt chloride also caused the moderate upregulation of NCI16 and PcGST1 mRNAs. Both nickel sulfate and cobalt chloride dose dependently induced NCI16 and PcGST1 mRNAs, but with different profiles. Nickel treatment caused a continuous increase in PcGST1 and NCI16 mRNA levels for up to 3 and 6 days, respectively, and a notable increase in H2O2 concentrations in P. caudatum. NCI16 expression was significantly enhanced by incubating cells with H2O2, implying that NCI16 induction in the presence of nickel ions is caused by reactive oxygen species (ROS). On the other hand, PcGST1 was highly induced by the antioxidant tertbutylhydroquinone (tBHQ) but not by H2O2, suggesting that different mechanisms mediate the induction of NCI16 and PcGST1. We introduced a luciferase reporter vector with an similar to 0.42-kb putative PcGST1 promoter into cells and then exposed the transformants to nickel sulfate. This resulted in significant luciferase upregulation, indicating that the putative PcGST1 promoter contains a nickel-responsive element. Our nickel-inducible system also may be applicable to the efficient expression of proteins that are toxic to host cells or require temporal control.

DOI： 10.1128/EC.00112-14

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：2  

We recently reported the cDNA sequences of 11 copepod luciferases from the superfamily Augaptiloidea in the order Calanoida. They were classified into two groups, Metridinidae and Heterorhabdidae/Lucicutiidae families, by phylogenetic analyses. To elucidate the evolutionary processes, we have now further isolated 12 copepod luciferases from Augaptiloidea species ( Metridia asymmetrica, Metridia curticauda, Pleuromamma scutullata, Pleuromamma xiphias, Lucicutia ovaliformis and Heterorhabdus tanneri). Codon-based synonymous/nonsynonymous tests of positive selection for 25 identified copepod luciferases suggested that positive Darwinian selection operated in the evolution of Heterorhabdidae luciferases, whereas two types of Metridinidae luciferases had diversified via neutral mechanism. By in silico analysis of the decoded amino acid sequences of 25 copepod luciferases, we inferred two protein sequences as ancestral copepod luciferases. They were expressed in HEK293 cells where they exhibited notable luciferase activity both in intracellular lysates and cultured media, indicating that the luciferase activity was established before evolutionary diversification of these copepod species. © 2013 Elsevier B.V.

DOI： 10.1016/j.gene.2013.07.011

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate expression of a number of genes associated with the cellular differentiation and development. Here, we show the abundant and ubiquitous expression of a newly identified splicing variant of mouse Ppar gamma (Ppar gamma 1sv) that encodes Ppar gamma 1 protein, and its importance in adipogenesis. The novel splicing variant has a unique 5'-UTR sequence, relative to those of Ppar gamma 1 and Ppar gamma 2 mRNAs, indicating the presence of a novel transcriptional initiation site and promoter for Ppar gamma expression. Ppar gamma 1sv was highly expressed in the white and brown adipose tissues at levels comparable to Ppar gamma 2. Ppar gamma 1sv was synergistically up-regulated with Ppar gamma 2 during adipocyte differentiation of 3T3-L1 cells and mouse primary cultured preadipocytes. Inhibition of Ppar gamma 1sv by specific siRNAs completely abolished the induced adipogenesis in 3T3-L1 cells. C/EBP beta and C/EBP delta activated both the Ppar gamma 1sv and Ppar gamma 2 promoters in 3T3-L1 preadipocytes. These findings suggest that Ppar gamma 1sv and Ppar gamma 2 synergistically regulate the early stage of the adipocyte differentiation.

DOI： 10.1371/journal.pone.006558

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Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) and its nuclear homolog CaMKP-N (PPM1E) are Ser/Thr protein phosphatases that belong to the PPM family. CaMKP-N is expressed in the brain and undergoes proteolytic processing to yield a C-terminally truncated form. The physiological significance of this processing, however, is not fully understood. Using a wheat-embryo cell-free protein expression system, we prepared human CaMKP-N (hCaMKP-N(WT)) and the truncated form, hCaMKP-N(1-559), to compare their enzymatic properties using a phosphopeptide substrate. The hCaMKP-N(1-559) exhibited a much higher V max value than the hCaMKP-N(WT) did, suggesting that the processing may be a regulatory mechanism to generate a more active species. The active form, hCaMKP-N(1-559), showed Mn2+ or Mg2+-dependent phosphatase activity with a strong preference for phospho-Thr residues and was severely inhibited by NaF, but not by okadaic acid, calyculin A, or 1-amino-8-naphthol-2,4-disulfonic acid, a specific inhibitor of CaMKP. It could bind to postsynaptic density and dephosphorylate the autophosphorylated Ca2+/calmodulin-dependent protein kinase II. Furthermore, it was inactivated by H2O2 treatment, and the inactivation was completely reversed by treatment with DTT, implying that this process is reversibly regulated by oxidation/reduction. The truncated CaMKP-N may play an important physiological role in neuronal cells. © 2013 Atsuhiko Ishida et al.

DOI： 10.1155/2013/134813

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Copepods are the dominant taxa in zooplankton communities of the ocean worldwide. Although bioluminescence of certain copepods has been known for more than a 100 years, there is very limited information about the structure and evolutionary history of copepod luciferase genes. Here, we report the cDNA sequences of 11 copepod luciferases isolated from the superfamily Augaptiloidea in the order Calanoida. Highly conserved amino acid residues in two similar repeat sequences were confirmed by the multiple alignment of all known copepod luciferases. Copepod luciferases were classified into two groups of Metridinidae and Heterorhabdidae/Lucicutiidae families based on phylogenetic analyses, with confirmation of the interrelationships within the Calanoida using 18S ribosomal DNA sequences. The large diversity in the specific activity of planktonic homogenates and copepod luciferases that we were able to express in mammalian cultured cells illustrates the importance of bioluminescence as a protective function against predators. We also discuss the relationship between the evolution of copepod bioluminescence and the aspects of their ecological characteristics, such as swimming activity and vertical habitat.

DOI： 10.1093/molbev/mss009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER CHEMICAL SOC  

Cortisol is a classical biomarker for the stress levels of human beings. We fabricated highly sensitive bioluminescent probes for salivary cortisol. The following strategies were contrived in the molecular design. Gaussia princeps luciferase (GLuc) was dissected into two fragments, between which an N-terminal-extended ligand binding domain of glucocorticoid receptor (GR HLBD), named Simgr4, was inserted. First, this unique single-chain probe was then situated downstream of a glucocorticoid response element (GRE) promoter in a reporter-gene system for constructing two ON-OFF switches for cortisol. Second, a circularly permutated (CP) variant of Simgr4 was formulated. The reporter-gene system exerted an improved signal-to-background (S/B) ratio of 8.5 to cortisol. Furthermore, a circularly permutated (CP) variant of Simgr4 exerted a 10 x enhanced detection limit to cortisol and a long dynamic range from 10(-9) to 10(-6) M cortisol, covering all of the normal clinical ranges of serum, urine, and saliva. This optimized probe successfully determined daily fluctuations of salivary cortisol and the correlations with those by ELISA. This study is the first to investigate the contribution of the HLBD of a nuclear receptor and multiple ON-OFF switches for molecular probes and salivary cortisols.

DOI： 10.1021/bc260220k

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-BLACKWELL PUBLISHING, INC  

The fluorescence excitation and emission maxima of a GFP-like protein from the marine copepod Chiridius poppei (CpYGFP) show a significant red shift (lambda(ex) = 509 nm, lambda(em) = 517 nm) compared with those of GFP from Aequorea victoria (avGFP) and other GFP-like proteins from marine copepods. We performed crystallographic and biochemical studies to understand why this shift occurs in CpYGFP. The structure of CpYGFP showed that the imidazole side chain of His52 is involved in stacking on the phenol moiety of the chromophore. We investigated the potential role of His52 in causing the red-shifted spectral properties by performing mutational analyses of H52T, H52D and H52F. The emission wavelengths of H52T and H52D were blue-shifted and that of H52F was red-shifted relative to the wild type. Comparison of its structure of another copepod GFP (ppluGFP2) having an emission maximum at 502 nm showed that the imidazole ring of His54 (corresponding to His52 in CpYGFP) is flipped out of the stacking position with the chromophore. These findings suggest that pi-pi stacking interaction between His52 and the phenol moiety of the chromophore is the likely cause of the red-shift in light emission.

DOI： 10.1111/j.1365-2443.2009.01305.x

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We cloned two forms of the secreted and thermostable luciferase genes, MpLuc1 and MpLuc2, from the marine copepod, Metridia pacifica. The 840-bp MpLuc1 cDNA comprised a 630-bp open reading frame encoding a 210-amino acid polypeptide (22.7 kDa). MpLuc1 had the closest homology with Metridia longa luciferase. The 753-bp MpLuc2 cDNA consisted of a 567-bp open reading frame (20.3 kDa), and it had the closest homology with Gaussia princeps luciferase. Single-specimen genomic PCR confirmed the presence of two luciferase genes in M. pacifica, and single-specimen RT-PCR revealed that both luciferase mRNAs were expressed. Both MpLuc1 and MpLuc2 (MpLucs) specifically reacted with the substrate coelenterazine producing identical bioluminescent spectra (lambda max, 485 nm), but with different kinetics. Adding salt such as MgCl2 and CaCl2 to the reaction mixture significantly enhanced MpLuc1 and MpLuc2 activities. Wild-type MpLucs were remarkably thermostable; MpLuc1 retained about 60% of the original activity even after incubation at 90 degrees C for 30 min. MpLucs expressed in NIH-3T3 and HeLa cells were largely secreted into the culture medium. Continuous monitoring of secreted MpLuc1 driven by the c-fos promoter demonstrated the potential usefulness of MpLuc1 in nondisruptive reporter assays. (c) 2008 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gene.2008.07.041

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Authorship：Lead author   Language：English   Publisher：Japan Society of Protistology  

Linear DNA molecules injected into the macronucleus of Paramecium caudatum became integrated into genomic DNA. The plasmid harboring fusion gene of P. caudatum histone H2B and a yellow fluorescent protein (YFP) named PcVenus was linearized and microinjected into the macronucleus of P. caudatum. Southern blots of total cellular DNA from three fluorescence-positive transformant clones probed with the PcVenus sequence revealed that the injected DNA had become randomly inserted into the chromosome. Free linear monomers or multimers of injected DNA molecules as seen in P. primaurelia and P. tetraurelia were not evident even though the plasmid possessed extant telomeric repeats at its both extremities. We also cloned fragments containing the integration site of genomic DNA from transformant cells by plasmid rescue. Sequence analysis of the flanking DNA confirmed random insertion of the linear plasmid into the chromosomal DNA with extant telomere repeats at the fusion junction. Therefore, P. caudatum maintains introduced DNA in a unique manner by non-homologous or illegitimate, rather than homologous recombination.

DOI： 10.18980/jjprotozool.41.2_159

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Other Link： http://id.ndl.go.jp/bib/9796684

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Cytoplasmic exchange between conjugating cells of Paramecium caudatum has been implicated by mating experiments using wild-type and behavioral mutant cells. To observe macromolecular transport between mating cells, we cloned and expressed the P caudatum histone H2B gene as a fusion protein attached to an enhanced yellow fluorescent protein (YFP) named PcVenus. Significant fluorescent signals derived from histone H2B-PeVenus were detected throughout the macro- and micronuclei of transformant cells after microinjection of the expression vector. The normal growth and high mating reactivity of the transformants indicated that H2B-PcVenus functioned normally. Seven hours after a transformant cell expressing histone 1-1213-PeVenus was mated with an untransformed complementary mating-type cell, fluorescence derived from histone H2B-PcVenus was emitted from the macronuclei of the untransformed cell. About 48 h later, the fluorescent signal was detected not only in the macro- and micronuclei of untransformed cells but also in the macronuclear ardagen of both mating cells. This suggests that conjugant cells share parental histones during meiosis and subsequent DNA rearrangement. Single-cell RT-PCR analysis demonstrated the presence of H2B-PcVenus mRNA in untransformed cells 15 and 24 h after conjugation. We concluded that at least the mRNA of histone H2B-PcVenus was transferred from the transformed, to the untransformed cell during conjugation. (c) 2007 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gene.2007.02.013

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A crustacean gene, encoding for a new class of GFP-like protein, has been isolated from a cDNA library of the deep-sea (benthic) copepod crustacean, Chiridius poppei, by expression cloning. The cDNA library was constructed in a pBluescript II vector and screened using a non-UV transilluminator, obtaining a positive clone. The clone consisted of a 781 -bp fragment of cDNA with a 660-bp open reading frame, which encoded for a 219-amino acid polypeptide with a calculated molecular mass of 24.7 kDa. The protein was overexpressed in Escherichia coli, purified to homogeneity by anion-exchange and size-exclusion chromatographies. The protein, CpYGFP, had excitation and emission maxima at 507 and 517 nm, respectively. CpYGFP existed as a dimer in solution and could be expressed either alone or as a fusion protein in HeLa cells. Dual labeling experiments carried out with CpYGFP-actin and DsRed2-Nuc demonstrated the usefulness of CpYGFP as a reporter in the subcellular localization of actin. (c) 2006 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.gene.2005.11.031

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We have used cultured human mammary epithelial cells (HMEC) and breast tumor-derived lines to gain information on defects that occur during breast cancer progression. HMEC immortalized by a variety of agents (the chemical carcinogen benzo(a)pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked upregulation (10-15 fold) of the telomere-binding protein, TRF2. Upregulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite lifespan and immortal HMEC. TRF2 protein was not upregulated by the oncogenic agents alone in the absence of immortalization, nor by expression of exogenously introduced hTERT genes. We found TRF2 levels to be at least twofold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. The dispersed distribution of TRF2 throughout the nuclei in some immortalized and tumor-derived cells indicated that not all the TRF2 was associated with telomeres in these cells. The process responsible for accumulation of TRF2 in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells' ability to maintain an indefinite lifespan.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS INC ELSEVIER SCIENCE  

Gradient elution chromatography of recombinant apoaequorin carried out in the presence of Ca2+ revealed two isoforms of apoaequorin, reduced and oxidized, whereas in the presence of EDTA 3 isoforms were observed. In a regeneration mixture of apoaequorin, coelenterazine, EDTA, and 2-mercaptoethanol, four isoforms were obtained, of which only one, aequorin, gave light with Ca2+. A method is described for the preparation of highly pure aequorin. The aequorin was stable in solution for approximately 10 days at 4 degreesC and pH 7.6, and then it gradually lost activity with a half-life of about 20 days until it was almost completely inactive on day 30. (C) 2003 Elsevier Science (USA). All rights reserved.

DOI： 10.1016/S1046-5928(03)00186-4

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

We have developed a novel expression vector, pTub-tel3, for transformation in Paramecium caudatum. The vector was constructed by cloning P. caudatum alpha-tubulin 5' and 3' non-coding regions. To examine transformation with the pTub-tel3 construct, we chose the green fluorescent protein (GFP) as a selection marker. When a linearized pTub-tel3 vector containing a GFP open reading frame was injected into the macronucleus, the GFP transcript was expressed in many clones whereas protein expression was detected only after extensive optimization of original GFP codons. GFP-derived fluorescence was distributed throughout the nuclei and cytoplasm except for contractile and food vacuoles. Upon continuous cell division, notable heterogeneity of GFP fluorescence among descendants from the same transformant has emerged. This expression vector can be applied to the analysis of protein trafficking and localization in addition to exogenous gene expression in P. caudatum. (C) 2002 Published by Elsevier Science B,V.

DOI： 10.1016/S0378-1119(01)00886-1

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Paramecium caudatum has a sexually immature period that lasts for about 60 fissions. To examine the possibility that telomere length is one of the determining factors of the duration of immaturity, we cloned the telomerase reverse transcriptase (TERT) gene from P. candatum, and analyzed its expression levels at mRNA, telomerase activity, and telomere length during the course of clonal division. Paramecium TERT (Pc_TERT) cDNA encodes a basic protein of 107 kDa that harbors conserved RT motifs, T motif, CP motif, and N motif. Pc_TERT mRNA is expressed at very low levels only detectable by RT-PCR, but constitutively, during immature and mature periods, exhibiting abundant telomerase activity. No clear phase shift in Pc_TERT expression, telomerase activity, or telomere length was observed at the point of maturation in P, caudatum. Instead, the telomere elongates successively as cells divide in P. caudatum, although a close species, P. tetraurelia, was reported to keep the length constant. We discuss possible mechanisms for the expression of sexual activity associated with telomere length in P. caudatum. (C) 2001 Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0378-1119(01)00337-7

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

A recent notion, that a variety of toxicants causing necrosis can lead to apoptosis as well, has been demonstrated with cultured cells, but not with in an vivo system. In the present study, we examined the induction of both apoptosis and necrosis in the kidneys of Wistar rats exposed to mercuric chloride (HgCl(2)). A single injection of HgCl(2), to rats at a dose of 4 mg/kg resulted in an increase in the renal DNA fragmentation evaluated as an occurrence of apoptosis, prior to urinary excretion of alkaline phosphatase (ALP) and renal morphological changes assessed as necrotic phenomena. The mercury-promoted DNA fragmentation was induced in a dose-dependent manner. Terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining and morphological observation of the nuclei revealed that apoptotic cells caused by HgCl(2) were predominantly found in the proximal tubules, but not in the distal tubules, glomeruli or medullary tubules. When we confirmed the proximal tubular-selective apoptosis by inorganic mercury with a combined technique of TUNEL staining with synchrotron radiation X-ray fluorescence (SR-XRF) imaging, it was shown that the apoptotic cells localized in the proximal tubules did contain higher level of mercury. Thus these results indicate that the proximal tubular cells-dominant site-specific distribution of mercury appears to be associated with induction of renal apoptosis and necrosis. (C) 1999 Published by Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S1382-6689(99)00012-5

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Language：Japanese   Publisher：Japan Society for Occupational Health  

DOI： 10.1539/sangyoeisei.KJ00001990620

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Language：English   Publishing type：Research paper (scientific journal)  

DOI： 10.1248/jhs1956.44.P27

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DOI： 10.1248/jhs1956.44.P35

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Authorship：Lead author   Language：Japanese  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：JAPAN SOC OCCUPATIONAL HEALTH  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

The gene (termed daa) encoding N-acyl-D-aspartate (D-Asp) amidohydrolase (D-AAase) from the Alcaligenes xylosoxydans subsp. xylosoxydans (Alcaligenes A-6) was cloned in Escherichia coli (E. cell) JM109. The daa gene consists of 1,494 nucleotides and encodes 498 amino acid residues. The molecular weight of D-AAase was calculated to be 53,581. The N-terminal amino acid sequence (NH2-TDRSTLDDAP-) predicted by the nucleotide sequence matched exactly those of the purified D-AAase from both Alcaligenes A-6 and cloned E. coli, with the exception of the removal of the N-terminal methionine processed after translation. A comparison of the amino acid sequence of D-AAase with that of D-aminoacylase from Alcaligenes A-6 showed high overall homology (56%), D-AAase from Alcaligenes A-6 showed 25 similar to 29% homology with Bacillus stearothermophilus, porcine, and human L-aminoacylases. The daa was highly expressed in E. coli, and the recombinant enzyme was purified to homogeneity with 17.8% yield.

DOI： 10.1016/0922-338X(95)94197-Y

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

DOI： 10.14952/SEIKAGAKU.2015.870138

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Language：Japanese   Publisher：(株)メディカルレビュー社  

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J-GLOBAL

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J-GLOBAL

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Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.47.231

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Language：English   Presentation type：Poster presentation  

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Language：Japanese   Presentation type：Oral presentation (invited, special)  

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