FAM83H mutations cause ADHCAI and alter intracellular protein localization.

Lee, S-K,Lee, K-E,Jeong, T-S,Hwang, Y-H,Kim, S,Hu, J C-C,Simmer, J P,Kim, J-W Journal of Dental Research, Inc 2011 Journal of dental research Vol.90 No.3

<P>Mutations in a family with sequence similarity 83 member H (FAM83H) cause autosomal-dominant hypocalcification amelogenesis imperfecta (ADH CAI). All FAM83H ADHCAI-causing mutations terminate translation or shift the reading frame within the specific exon 5 segment that encodes from Ser(287) to Glu(694). Mutations near Glu(694) cause a milder, more localized phenotype. We identified disease-causing FAM83H mutations in two families with ADHCAI: family 1 (g.3115C>T, c.1993 C>T, p.Q665X) and family 2 (g.3151C>T, c.2029 C>T, p.Q677X). We also tested the hypothesis that truncation mutations alter the intracellular localization of FAM83H. Wild-type FAM83H and p.E694X mutant FAM83H fused to green fluorescent protein (GFP) localized in the cytoplasm of HEK293T cells, but the mutant FAM83H proteins (p.R325X, p.W460X, and p.Q677X) fused to GFP localized mainly in the nucleus with slight expression in the cytoplasm. We conclude that nuclear targeting of the truncated FAM83H protein contributes to the severe, generalized enamel phenotype.</P>

Cytoskeletal Patterns, In Vitro Maturation and Parthenogenetic Development of Rabbit GV Oocytes

Ju, J.C.,Chen, T.H.,Tseng, J.K.,Tsay, C.,Yeh, S.P.,Chou, P.C.,Chen, C.H.,Liu, C.T. Asian Australasian Association of Animal Productio 2002 Animal Bioscience Vol.15 No.12

The purposes of this study were to optimize the in vitro maturation (IVM) and culture (IVC) systems of rabbit oocytes. Cytoskeletal structures in the germinal vesicle stage (GV) and during IVM are also investigated. Ovaries were transported from local slaughterhouses and the cumulus-oocyte complexes (COCs) were collected from ovarian follicles (${\geq}1mm$). COCs were randomly allocated to TCM199-based medium ($T_1$, TCM-199) supplemented with $NaHCO_3$, glucose, sodium pyruvate and FSH ($T_2$), $T_2+E_2+LH$ ($T_3$), $T_3+FBS$ ($T_4$), or $T_1+E_2+LH+FSH+FBS$ ($T_5$), for IVM. In Experiment 1, COCs were retrieved from the follicles and 51 GV oocytes were fixed in the fixative (MTSB-XF) for nuclear and cytoplasmic examinations. In Experiment 2, progressive changes of both the nucleus and the cytoskeleton were examined at 0, 6, 16, and 20 h after IVM. Maturation (MR) and developmental rates were assessed in Experiment 3. Cytoplasmic microtubules (MT) were clearly observed in rabbit GV oocytes. To our knowledge, this is the first report that describes the appearance of MT structures in the GV stage ooplasm. Tremendous variations in cytoskeletal alterations were observed among treatments with the exception of the vitelline ring (VR), which is constantly visible and unchanged during maturation. Germinal vesicle breakdown (GVBD) does not occur at 6 h after onset of maturation culture. When the oocytes for IVM were collected within 2 h, results from Experiment 3 showed that rates of nuclear maturation were 42, 8, 42, 37 and 65% at 16 h of IVM for $T_1$ through $T_5$, respectively, in which $T_1$, $T_4$ and $T_5$ had significantly greater MR than those in other groups (p<0.05). Morula/blastocyst development after parthenogenetic activation ranged from 20 to 63% with significantly greater rates in $T_3$, $T_4$ and $T_5$ (p<0.05). These results suggested that oocytes recovered from slaughterhouse ovaries can be matured and parthenogenetically activated in vitro, but the MR remained low in this study. Addition of $E_2$ and LH in the medium may be beneficial for cytoplasmic maturation, but FBS exerts a nega- tive role in the subsequent development of parthenogenetic embryos when energy substrates are provided in the IVC media. More studies are required for improving the MR and further development of the GV stage rabbit oocytes.

Programmed cell death ligand 1 alleviates psoriatic inflammation by suppressing IL-17A production from programmed cell death 1-high T cells

Kim, J.H.,Choi, Y.J.,Lee, B.H.,Song, M.Y.,Ban, C.Y.,Kim, J.,Park, J.,Kim, S.E.,Kim, T.G.,Park, S.H.,Kim, H.P.,Sung, Y.C.,Kim, S.C.,Shin, E.C. Mosby 2016 The journal of allergy and clinical immunology Vol.137 No.5

<P>Background: Psoriasis is one of the most common chronic inflammatory diseases of the skin. Recently, IL-17-producing T cells have been shown to play a critical role in psoriatic inflammation. Programmed cell death 1 (PD-1) is a coinhibitory receptor expressed on T cells in various chronic inflammatory diseases; however, the expression and function of PD-1 during psoriatic inflammation have not previously been characterized. Objective: We examined PD-1 expression on IL-17A-producing T cells from imiquimod-treated mice and patients with psoriasis. Additionally, we investigated the therapeutic effect of recombinant programmed cell death ligand 1 (PD-L1) protein on imiquimod-induced psoriatic inflammation. Methods: PD-1 expression on IL-17A-producing gamma delta T cells from imiquimod-treated mice was examined by means of multicolor flow cytometric analysis. In the psoriatic skin of patients, PD-1 and IL-17A expression was analyzed by using immunofluorescence. The therapeutic effect of PD-L1-Fc fusion protein (PD-L1-Fc) was assessed in imiquimod-treated mice ex vivo and in vivo. Results: During imiquimod-induced psoriatic inflammation, PD-1 is overexpressed on CD27(-)V gamma 1(-) gamma delta T cells. Furthermore, PD-1 expression on IL-17A(+) T cells was confirmed in psoriatic skin tissues from patients and imiquimod-treated mice. In the CD27(-)V gamma 1(-) gamma delta T-cell population, V gamma 4(-) gamma delta T cells with V gamma 6 mRNA expression showed a high level of PD-1 expression. Furthermore, these PD-1(hi)V gamma 4(-)(V gamma 6(+)) gamma delta Tcells were specialized for anti-CD3-induced IL-17A production, which was inhibited by PD-L1-Fc treatment. In imiquimod-treated mice PD-L1-Fc reduced psoriatic inflammation when given alone and enhanced the therapeutic effect of anti-p40 when given in combination. Conclusion: PD-1 is overexpressed in IL-17A-producing T cells in both imiquimod-treated mice and patients with psoriasis. Moreover, recombinant PD-L1-Fc alleviates psoriatic inflammation in imiquimod-treated mice.</P>

Serine palmitoyltransferase inhibitor myriocin induces growth inhibition of B16F10 melanoma cells through G₂/M phase arrest

Lee, Y.‐,S.,Choi, K.‐,M.,Choi, M.‐,H.,Ji, S.‐,Y.,Lee, S.,Sin, D.‐,M.,Oh, K.‐,W.,Lee, Y.‐,M.,Hong, J.‐,T.,Yun, Y.‐,P.,Yoo, H.‐,S. Blackwell Publishing Ltd 2011 Cell proliferation Vol.44 No.4

<P><B>Abstract</B></P><P><B>Objectives: </B> Melanoma is the most aggressive form of skin cancer, and it resists chemotherapy. Candidate drugs for effective anti‐cancer treatment have been sought from natural resources. Here, we have investigated anti‐proliferative activity of myriocin, serine palmitoyltransferase inhibitor, in the <I>de novo</I> sphingolipid pathway, and its mechanism in B16F10 melanoma cells.</P><P><B>Material and methods: </B> We assessed cell population growth by measuring cell numbers, DNA synthesis, cell cycle progression, and expression of cell cycle regulatory proteins. Ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate levels were analysed by HPLC.</P><P><B>Results: </B> Myriocin inhibited proliferation of melanoma cells and induced cell cycle arrest in the G<SUB>2</SUB>/M phase. Expressions of cdc25C, cyclin B1 and cdc2 were decreased in the cells after exposure to myriocin, while expression of p53 and p21<SUP>waf1/cip1</SUP> was increased. Levels of ceramide, sphingomyelin, sphingosine and sphingosine‐1‐phosphate in myriocin‐treated cells after 24 h were reduced by approximately 86%, 57%, 75% and 38%, respectively, compared to levels in control cells.</P><P><B>Conclusions: </B> Our results suggest that inhibition of sphingolipid synthesis by myriocin in melanoma cells may inhibit expression of cdc25C or activate expression of p53 and p21<SUP>waf1/cip1</SUP>, followed by inhibition of cyclin B1 and cdc2, resulting in G<SUB>2</SUB>/M arrest of the cell cycle and cell population growth inhibition. Thus, modulation of sphingolipid metabolism by myriocin may be a potential target of mechanism‐based therapy for this type of skin cancer.</P>

Phase II study of S-1 combined with oxaliplatin as therapy for patients with metastatic biliary tract cancer: influence of the <i>CYP2A6</i> polymorphism on pharmacokinetics and clinical activity

Kim, K-p,Jang, G,Hong, Y S,Lim, H-S,Bae, K-s,Kim, H-S,Lee, S S,Shin, J-G,Lee, J-L,Ryu, M-H,Chang, H-M,Kang, Y-K,Kim, T W Nature Publishing Group 2011 The British journal of cancer Vol.104 No.4

<P><B>Background:</B></P><P>Advanced biliary cancer is often treated with fluoropyrimidine-based chemotherapy. In this study, we evaluated the efficacy and tolerability of a combination of S-1, an oral fluoropyrimidine prodrug, and oxaliplatin in patients with metastatic biliary cancer.</P><P><B>Methods:</B></P><P>Patients with histologically confirmed metastatic biliary cancer and no history of radiotherapy or chemotherapy were enrolled. Oxaliplatin was administered intravenously (130 mg m<SUP>−2</SUP>), followed by 14-day administration of oral S-1 (40 mg m<SUP>−2</SUP> twice daily) with a subsequent 7-day rest period every 21 days. Pharmacokinetic analysis of S-1 was performed at cycle 1. Patients were genotyped for <I>CYP2A6</I> polymorphisms (<SUP>*</SUP>1, <SUP>*</SUP>4, <SUP>*</SUP>7, <SUP>*</SUP>9 or <SUP>*</SUP>10), and pharmacokinetic and clinical parameters compared according to the <I>CYP2A6</I> genotype.</P><P><B>Results:</B></P><P>In total, 49 patients were evaluated, who received a median of four cycles. The overall response rate was 24.5%. Median progression-free and overall survival was 3.7 and 8.7 months, respectively. The most common haematological grade 3 out of 4 toxicity was neutropenia (14%), while non-hematological grade 3 out of 4 toxicities included anorexia (14%), nausea (12%), asthenia (10%), vomiting (10%), and diarrhoea (4%). Biotransformation of S-1 (AUC<SUB>0−24 h</SUB> of 5-fluorouracil/AUC<SUB>0−24 h</SUB> of tegafur) was 1.85-fold higher for the <I>*1/*1</I> group than for the other groups (90% confidence interval 1.37–2.49). Diarrhoea (<I>P</I>=0.0740), neutropenia (<I>P</I>=0.396), and clinical efficacy (response rate, <I>P</I>=0.583; PFS, <I>P</I>=0.916) were not significantly associated with <I>CYP2A6</I> genotype, despite differences in 5-FU exposure.</P><P><B>Conclusion:</B></P><P>The combination of S-1 and oxaliplatin appears to be active and well tolerated in patients with metastatic biliary cancer, and thus is feasible as a therapeutic modality. <I>CYP2A6</I> genotypes are associated with differences in the biotransformation of S-1. However, the impact of the <I>CYP2A6</I> polymorphism on variations in clinical efficacy or toxicity requires further evaluation.</P>

Foxp3 is a key downstream regulator of p53-mediated cellular senescence

Kim, J-E,Shin, J-S,Moon, J-H,Hong, S-W,Jung, D-J,Kim, J H,Hwang, I-Y,Shin, Y J,Gong, E-Y,Lee, D H,Kim, S-M,Lee, E Y,Kim, Y S,Kim, D,Hur, D,Kim, T W,Kim, K-p,Jin, D-H,Lee, W-J Macmillan Publishers Limited 2017 Oncogene Vol.36 No.2

<P>The downstream events and target genes of p53 in the process of senescence are not fully understood. Here, we report a novel function of the forkhead transcription factor Foxp3, which is a key player in mediating T-cell inhibitory functions, in p53-mediated cellular senescence. The overexpression of Foxp3 in mouse embryonic fibroblasts (MEFs) accelerates senescence, whereas Foxp3 knockdown leads to escape from p53-mediated senescence in p53-expressing MEFs. Consistent with these results, Foxp3 expression resulted in the induction of senescence in epithelial cancer cells, including MCF7 and HCT116 cells. Foxp3 overexpression also increased the intracellular levels of reactive oxygen species (ROS). The ROS inhibitor N-acetyl-L-cysteine rescued cells from Foxp3-expression-induced senescence. Furthermore, the elevated ROS levels that accompanied Foxp3 overexpression were paralleled by an increase in p21 expression. Knockdown of p21 in Foxp3-expressing MEFs abrogated the Foxp3-dependent increase in ROS levels, indicating that Foxp3 acts through the induction of p21 and the subsequent ROS elevation to trigger senescence. Collectively, these results suggest that Foxp3 is a downstream target of p53 that is sufficient to induce p21 expression, ROS production and p53-mediated senescence.</P>

Report of the CCQM-K97: measurement of arsenobetaine standard solution and arsenobetaine content in fish tissue (tunafish)

Ma, L D,Wang, J,WEI, C,Kuroiwa, T,Narukawa, T,Ito, N,HIOKI, A,CHIBA, K,Yim, Y H,Lee, K S,Lim, Y R,Turk, G C,Davis, C W,Mester, Z,Yang, L,McCooeye, M,Maxwell, P,Cankur, O,Tokman, N,Coskun, F G BUREAU INTERNATIONAL DES POIDS ET MESURES 2017 METROLOGIA -BERLIN- Vol.54 No.-

<P></P> <P>The CCQM-K97 key comparison was organized by the inorganic analysis working group (IAWG) of CCQM as a follow-up to completed pilot study CCQM-P96 and P96.1 to test the abilities of the national metrology institutes to accurately quantitate the mass fraction of arsenobetaine (AsB) in standard solution and in fish tissue. A pilot study CCQM-P133 was parallelized with this key comparison. National Institute of Metrology (NIM), China and National Metrology Institute of Japan (NMIJ) acted as the coordinating laboratories.</P> <P>Six NMIs participated in CCQM-K97 and two institutes participated in CCQM-P133, and all of them submitted the results. Some NMIs submitted more than one results by different methods. The results were in excellent agreement with each other, and obviously better than those of previous P96 and P96.1. Therefore the calibrant which each NMI used was comparable. It shows that the capabilities of some of the participants have been improved after the previous pilot studies.</P> <H2>Main text</H2> <P> To reach the main text of this paper, click on <A HREF='http://www.bipm.org/utils/common/pdf/final_reports/QM/K97/CCQM-K97.pdf'>Final Report</A>. Note that this text is that which appears in Appendix B of the BIPM key comparison database <A HREF='http://kcdb.bipm.org/'>kcdb.bipm.org/</A>.</P> <P>The final report has been peer-reviewed and approved for publication by the CCQM, according to the provisions of the CIPM Mutual Recognition Arrangement (CIPM MRA).</P>

15-lipoxygenase metabolites play an important role in the development of a T-helper type 1 allergic inflammation induced by double-stranded RNA

Jeon, S. G.,Moon, H.-G.,Kim, Y.-S.,Choi, J.-P.,Shin, T.-S.,Hong, S.-W.,Tae, Y.-M.,Kim, S.-H.,Zhu, Z.,Gho, Y. S.,Kim, Y.-K. Blackwell Publishing Ltd 2009 Clinical and experimental allergy Vol.39 No.6

<P>Summary</P><P>Background</P><P>We recently demonstrated that the T-helper type 1 (Th1) immune response plays an important role in the development of non-eosinophilic inflammation induced by airway exposure of an allergen plus double-stranded RNA (dsRNA). However, the role of lipoxygenase (LO) metabolites in the development of Th1 inflammation is poorly understood.</P><P>Objective</P><P>To evaluate the role of LO metabolites in the development of Th1 inflammation induced by sensitization with an allergen plus dsRNA.</P><P>Methods</P><P>A Th2-allergic inflammation mouse model was created by an intraperitoneal injection of lipopolysaccharide-depleted ovalbumin (OVA, 75 μg) and alum (2 mg) twice, and the Th1 model was created by intranasal application of OVA (75 μg) and synthetic dsRNA [10 μg of poly(I : C)] four times, followed by an intranasal challenge with 50 μg of OVA four times. The role of LO metabolites was evaluated using two approaches: a transgenic approach using 5-LO<SUP>−/−</SUP> and 15-LO<SUP>−/−</SUP> mice, and a pharmacological approach using inhibitors of cysteinyl leucotriene receptor-1 (cysLTR1), LTB4 receptor (BLT1), and 15-LO.</P><P>Results</P><P>We found that the Th1-allergic inflammation induced by OVA+dsRNA sensitization was similar between 5-LO<SUP>−/−</SUP> and wild-type (WT) control mice, although Th2 inflammation induced by sensitization with OVA+alum was reduced in the former group. In addition, dsRNA-induced Th1 allergic inflammation, which is associated with down-regulation of 15-hydroxyeicosateraenoic acids production, was not affected by treatment with cysLTR1 or BLT1 inhibitors, whereas it was significantly lower in 12/15-LO<SUP>−/−</SUP> mice compared with WT control mice. Moreover, dsRNA-induced allergic inflammation and the recruitment of T cells following an allergen challenge were significantly inhibited by treatment with a specific 15-LO inhibitor (PD146176).</P><P>Conclusion</P><P>15-LO metabolites appear to be important mediators in the development of Th1-allergic inflammation induced by sensitization with an allergen plus dsRNA. Our findings suggest that the 15-LO pathway is a novel therapeutic target for the treatment of virus-associated asthma characterized by Th1 inflammation.</P>

Molecular identification and functional delineation of a glutathione reductase homolog from disk abalone (<i>Haliotis discus discus</i>): Insights as a potent player in host antioxidant defense

Herath, H.M.L.P.B.,Wickramasinghe, P.D.S.U.,Bathige, S.D.N.K.,Jayasooriya, R.G.P.T.,Kim, Gi-Young,Park, Myoung Ae,Kim, Chul,Lee, Jehee Elsevier 2017 FISH AND SHELLFISH IMMUNOLOGY Vol.60 No.-

<P><B>Abstract</B></P> <P>Glutathione reductase (GSR) is an enzyme that catalyzes the biochemical conversion of oxidized glutathione (GSSG) into the reduced form (GSH). Since the ratio between the two forms of glutathione (GSH/GSSG) is important for the optimal function of GSH to act as an antioxidant against H<SUB>2</SUB>O<SUB>2</SUB>, the contribution of GSR as an enzymatic regulatory agent to maintain the proper ratio is essential. Abalones are marine mollusks that frequently encounter environmental factors that can trigger the overproduction of reactive oxygen species (ROS) such as H<SUB>2</SUB>O<SUB>2</SUB>. Therefore, we conducted the current study to reveal the molecular and functional properties of a GSR homolog in the disk abalone, <I>Haliotis discus discus</I>. The identified cDNA sequence (2325 bp) has a 1356 bp long open reading frame (ORF), coding for a 909 bp long amino acid sequence, which harbors a pyridine nucleotide-disulfide oxidoreductase domain (171–246 aa), a pyridine nucleotide-disulfide oxidoreductase dimerization domain, and a NAD(P)(+)-binding Rossmann fold superfamily signature domain. Four functional residues: the FAD binding site, glutathione binding site, NADPH binding motif, and assembly domain were identified to be conserved among the other species. The recombinant abalone GSR (rAbGSR) exhibited detectable activity in a standard glutathione reductase activity assay. The optimum pH and optimal temperature for the reaction were found to be 7.0 and 50 °C, respectively, while the ionic strength of the medium had no effect. The enzymatic reaction was vastly inhibited by Cu<SUP>+2</SUP> and Cd<SUP>+2</SUP> ions. A considerable effect of cellular protection was detected with a disk diffusion assay conducted with rAbGSR. Moreover, an MTT assay and flow cytometry confirmed the significance of the protective role of rAbGSR in cell function. Furthermore, <I>AbGSR</I> was found to be ubiquitously distributed in different types of abalone tissues. <I>AbGSR</I> mRNA expression was significantly upregulated in response to three immune challenges: <I>Vibrio parahaemolyticus</I>, <I>Listeria monocytogenes</I>, and lipopolysaccharide (LPS), thus indicating its possible involvement in host defense mechanisms during pathogenic infections. Taken together, the results of the current study suggest that AbGSR plays an important role in antioxidant-mediated host defense mechanisms and also provide insights into the immunological contribution of AbGSR.</P> <P><B>Highlights</B></P> <P> <UL> <LI> We identified a glutathione reductase homolog (AbGSR) from disk abalone. </LI> <LI> AbGSR resembled functionally important domain architecture of GSR family. </LI> <LI> Recombinant AbGSR confirmed its biochemical properties via enzymatic assays. </LI> <LI> First functional antioxidant properties assessment of a molluscan GSR. </LI> <LI> <I>AbGSR</I> expression was modulated upon induced pathogen stress in gill and hemocytes. </LI> </UL> </P>

Morphological and Molecular Phylogenetic Position of Prorocentrum micans sensu stricto and Description of Prorocentrum koreanum sp. nov. from Southern Coastal Waters in Korea and Japan

Han, M.S.,Wang, P.,Kim, J.H.,Cho, S.Y.,Park, B.S.,Kim, J.H.,Katano, T.,Kim, B.H. G. Fischer 2016 PROTIST -JENA- Vol.167 No.1

Prorocentrum micans is an extremely variable dinoflagellate species, with many different local forms reported worldwide. Because of this morphological diversity, it is important to establish whether these various forms belong to P. micans sensu stricto. For this study, P. micans-like specimens were isolated from several localities in the southern coastal waters of Korea and Japan. The morphological characteristics and the molecular signatures of P. micans were re-examined. Moreover, a new Prorocentrum species, Prorocentrum koreanum sp. nov. was established through detailed light microscopy and scanning electron microscopy observations. Examination of the periflagellar platelets revealed that P. koreanum sp. nov. differs from P. micans. Furthermore, P. koreanum and P. micans exhibited different distribution patterns of trichocyst pores. Through molecular phylogeny analysis of small subunit (SSU) rRNA, internal transcribed spacer (ITS), and large subunit (LSU) rRNA sequence, we found P. koreanum to be more closely related to P. mexicanum and P. rhathymum than to P. micans. Additionally, ITS2 compensatory base changes also provide strong evidence to support P. koreanum and P. micans being separate species.