Congenital biliary atresia is an incurable disease of newborn infants, of unknown genetic causes, that results in congenital deformation of the gallbladder and biliary duct system. Here, we show that ...during mouse organogenesis, insufficient SOX17 expression in the gallbladder and bile duct epithelia results in congenital biliary atresia and subsequent acute 'embryonic hepatitis', leading to perinatal death in ~95% of the Sox17 heterozygote neonates in C57BL/6 (B6) background mice. During gallbladder and bile duct development, Sox17 was expressed at the distal edge of the gallbladder primordium. In the Sox17(+/-) B6 embryos, gallbladder epithelia were hypoplastic, and some were detached from the luminal wall, leading to bile duct stenosis or atresia. The shredding of the gallbladder epithelia is probably caused by cell-autonomous defects in proliferation and maintenance of the Sox17(+/-) gallbladder/bile duct epithelia. Our results suggest that Sox17 plays a dosage-dependent function in the morphogenesis and maturation of gallbladder and bile duct epithelia during the late-organogenic stages, highlighting a novel entry point to the understanding of the etiology and pathogenesis of human congenital biliary atresia.
We previously reported that sevoflurane anesthesia reversibly suppresses the expression of the clock gene, Period2 (Per2), in the mouse suprachiasmatic nucleus (SCN). However, the molecular ...mechanisms underlying this suppression remain unclear. In this study, we examined the possibility that sevoflurane suppresses Per2 expression via epigenetic modification of the Per2 promoter.
Mice were anesthetized with a gas mixture of 2.5% sevoflurane/40% oxygen at a 6 L/min flow for 1 or 4 h. After termination, brains were removed and samples of SCN tissue were derived from frozen brain sections. Chromatin immunoprecipitation (ChIP) assays using anti-acetylated-histone antibodies were performed to investigate the effects of sevoflurane on histone acetylation of the Per2 promoter. Interaction between the E'-box (a cis-element in the Per2 promoter) and CLOCK (the Clock gene product) was also assessed by a ChIP assay using an anti-CLOCK antibody. The SCN concentration of nicotinamide adenine dinucleotide (NAD(+)), a CLOCK regulator, was assessed by liquid chromatography-mass spectrometry.
Acetylation of histone H4 in the proximal region of the Per2 promoter was significantly reduced by sevoflurane. This change in the epigenetic profile of the Per2 gene was observed prior to suppression of Per2 expression. Simultaneously, a reduction in the CLOCK-E'-box interaction in the Per2 promoter was observed. Sevoflurane treatment did not affect the concentration of NAD(+) in the SCN.
Independent of NAD(+) concentration in the SCN, sevoflurane decreases CLOCK binding to the Per2 promoter E'-box motif, reducing histone acetylation and leading to suppression of Per2 expression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Abstract
Background
Ames test is used worldwide for detecting the bacterial mutagenicity of chemicals
.
In silico analyses of bacterial mutagenicity have recently gained acceptance by regulatory ...agencies; however, current in silico models for prediction remain to be improved. The Japan Pharmaceutical Manufacturers Association (JPMA) organized a task force in 2017 in which eight Japanese pharmaceutical companies had participated. The purpose of this task force was to disclose a piece of pharmaceutical companies’ proprietary Ames test data.
Results
Ames test data for 99 chemicals of various chemical classes were collected for disclosure in this study. These chemicals are related to the manufacturing process of pharmaceutical drugs, including reagents, synthetic intermediates, and drug substances. The structure-activity (mutagenicity) relationships are discussed in relation to structural alerts for each chemical class. In addition, in silico analyses of these chemicals were conducted using a knowledge-based model of Derek Nexus (Derek) and a statistics-based model (GT1_BMUT module) of CASE Ultra. To calculate the effectiveness of these models, 89 chemicals for Derek and 54 chemicals for CASE Ultra were selected; major exclusions were the salt form of four chemicals that were tested both in the salt and free forms for both models, and 35 chemicals called “known” positives or negatives for CASE Ultra. For Derek, the sensitivity, specificity, and accuracy were 65% (15/23), 71% (47/66), and 70% (62/89), respectively. The sensitivity, specificity, and accuracy were 50% (6/12), 60% (25/42), and 57% (31/54) for CASE Ultra, respectively. The ratio of overall disagreement between the CASE Ultra “known” positives/negatives and the actual test results was 11% (4/35). In this study, 19 out of 28 mutagens (68%) were detected with TA100 and/or TA98, and 9 out of 28 mutagens (32%) were detected with either TA1535, TA1537, WP2
uvrA,
or their combination.
Conclusion
The Ames test data presented here will help avoid duplicated Ames testing in some cases, support duplicate testing in other cases, improve in silico models, and enhance our understanding of the mechanisms of mutagenesis.
Molecular multilayers were fabricated using a Ru complex containing Fe cations on an indium tin oxide surface to control the properties of the Ru-complex multilayers such as the multilayer ...orientation and the electron transport. The Ru-complex multilayer films containing Fe cations were thicker than those containing Zr cations, which have been used previously. The electron transport properties of the multilayers containing Fe cations were evaluated. Solid-state sandwich cell measurements showed that the Ru-complex multilayer films containing Fe cations exhibited increased electron transport with a lower transport coefficient β of 0.01 Å(-1), whereas those that contain Zr cations have β ∼ 0.07 Å(-1). Thus, Fe cations are effective in obtaining thicker Ru-complex layers with increased electron transport abilities.
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Sequential assembly of layers containing redox-active dinuclear Ru complexes that differ in their redox potential on ITO substrates afforded a series of layered structures with ...different potential gradients. The Ru complexes Ru-NP and Ru-CP contain tetrapodal phosphonate anchors and bridging 2,3,5,6-tetra(2-pyridyl)pyrazine or 1,2,4,5-tetra(2-pyridyl)benzene ligands, respectively. Oxidation potentials of E1/2=+0.83 and +1.04V vs. Fc+/Fc (Ru-NP) and E1/2=−0.37 and 0.09V (Ru-CP) were observed for the Ru(II)-Ru(II)/Ru(II)-Ru(III) and Ru(II)-Ru(III)/Ru(III)-Ru(III) couples. As the Ru-NP and Ru-CP units contain several accessible redox-active electronic states, electron transfer (ET) blockage and mediation occurred at the Ru-NP/Ru-CP heterojunction on account of the large potential gradient. An examination of the electrochemical behavior of the multilayer diblock ITO||(Ru-CP)n|(Ru-NP)n (n=1–5) heterofilms revealed that in ITO||(Ru-CP)4|(Ru-NP)4, the Ru(III)-Ru(III)/Ru(III)-Ru(IV) couple is involved in the electron mediation pathway on the Ru-NP/Ru-CP heterojunction. In the case of the multilayer triblock ITO||(Ru-NP)n|(Ru-CP)n|(Ru-NP)n (n=4) heterofilms, two catalytic peaks were observed, indicating that the inner and outer Ru-NP/Ru-CP junctions display two different catalytic ET processes. In the alternating Ru-NP/Ru-CP multilayer heterofilms, the ET mediation processes are governed by the sequence order of the Ru-NP/Ru-CP heterojunction attached to the ITO electrode. In multilayer homo- and heterofilms, the dependence of the ET distance on the layer number is discussed on the basis of potential-step chronoamperometry (PSCA) results. In the multilayer Ru-NP and Ru-CP homofilms, relatively low β values were observed, while in the multilayer heterofilms, the ET rate of the catalytic wave was not affected by the number of layers, as the energy gap between the Ru-NP/Ru-CP units is the rate-determining factor. Therefore, the molecular sequence in such multilayer heterofilms, composed of layers containing Ru-NP or Ru-CP complexes, plays an important role.
A 38-year-old man was referred to our hospital for sudden onset of intermittent claudication (IC) with decreased ankle-brachial pressure index (ABI) of 0.64. Enhanced computed tomography (CT) showed ...cystic adventitial disease of the right popliteal artery with no arterial stenosis. He was observed carefully because of symptom regression and non-stenosed popliteal artery. One week later, however, severe IC with no arterial pulsation relapsed. Enhanced CT revealed severe stenosis of the affected popliteal artery with increased adventitial cyst. He received continuous intravenous administration of heparin (10,000 units/day) and the symptom disappeared the next day with normal ABI of 1.0. Considering the high risk of progression to critical limb ischemia, we performed surgical intervention including excision of the affected popliteal artery and interposition with an autologous saphenous vein graft. Neither leakage of the mucinous contents from the adventitial cyst nor connection between the adventitial cyst and the knee joint was recognized intraoperatively. He had no recurrence of the symptom thereafter.
To investigate the reported discrepancy regarding the immunohistochemical expression of kisspeptin neurons, we produced a new antibody against synthetic peptide containing the same amino acid ...residual sequence as rat kisspeptin10. Although the antibody showed cross-reactivities against neurons other than kisspeptin neurons, these cross-reactivities were excluded by preabsorption with neuropeptide FF (NPFF). Immunohistochemistry using the antibody preabsorbed with NPFF showed specific kisspeptin immunoreactivities (IRs) in the arcuate nucleus (Arc), the inner layer of the median eminence, and the infundibulum in the rat hypothalamus. IRs were more intense in the adult female rats than in the males. This sexual dimorphism became observable at the 7th day after birth. These IRs intensified with age. Ovariectomy enhanced the IRs in the Arc in the female rats. In contrast, regarding IRs in the anteroventral periventricular nucleus (AVPV), only a few immunoreactive fibers were detected in the adult rats. We applied this antibody for the investigation of the interaction between kisspeptin fibers and gonadotropin-releasing hormone (GnRH) neurons. No direct morphological interaction between the cell bodies of GnRH neurons and kisspeptin fibers was observed in the medial preoptic area. Many projections of kisspeptin fibers were found close to the GnRH fibers in the lateral part of the median eminence. However, we did not observe any direct contact between kisspeptin fibers and the GnRH fibers. These results suggest that kisspeptin neurons regulate GnRH neurons not via the synaptic contact but via another information transmission process that is not synapse-dependent, such as volume transmission.
Elevation of intracellular Ca2+ concentration (Ca2+i) activates Ca2+/calmodulin-dependent kinases (CaMK) and promotes gene transcription. This signaling pathway is referred to as ...excitation–transcription (E-T) coupling. Although vascular myocytes can exhibit E-T coupling, the molecular mechanisms and physiological/pathological roles are unknown. Multiscale analysis spanning from single molecules to whole organisms has revealed essential steps in mouse vascular myocyte E-T coupling. Upon a depolarizing stimulus, Ca2+ influx through Cav1.2 voltage-dependent Ca2+ channels activates CaMKK2 and CaMK1a, resulting in intranuclear CREB phosphorylation. Within caveolae, the formation of a molecular complex of Cav1.2/CaMKK2/CaMK1a is promoted in vascular myocytes. Live imaging using a genetically encoded Ca2+ indicator revealed direct activation of CaMKK2 by Ca2+ influx through Cav1.2 localized to caveolae. CaMK1a is phosphorylated by CaMKK2 at caveolae and translocated to the nucleus upon membrane depolarization. In addition, sustained depolarization of a mesenteric artery preparation induced genes related to chemotaxis, leukocyte adhesion, and inflammation, and these changes were reversed by inhibitors of Cav1.2, CaMKK2, and CaMK, or disruption of caveolae. In the context of pathophysiology, when the mesenteric artery was loaded by high pressure in vivo, we observed CREB phosphorylation in myocytes, macrophage accumulation at adventitia, and an increase in thickness and cross-sectional area of the tunica media. These changes were reduced in caveolin1-knockout mice or in mice treated with the CaMKK2 inhibitor STO609. In summary, E-T coupling depends on Cav1.2/CaMKK2/CaMK1a localized to caveolae, and this complex converts Ca2+i changes into gene transcription. This ultimately leads to macrophage accumulation and media remodeling for adaptation to increased circumferential stretch.
New dinuclear ruthenium or osmium complexes with cyclometalated bonds in either tridentate bridging (BL) or ancillary ligands (L), (L)M(BL)M(L) (where M = Ru, Os; L = ...bis(N-methylbenzimidazolyl)pyridine, -benzene; BL= tetrapyridylpyrazine (tppz), -benzene (tpb)), were synthesized, and their mixed-valence-state characteristics were investigated. All of the complexes showed successive one-electron redox processes, each of which correspond to M(II/III) (M = Ru, Os) or ligand reduction waves. In addition, an M(III/IV) couple was observed in cyclometalated M2(bis(benzimidazolyl)benzene)2(BL) complexes (M = Ru, Os). Effects of the cyclometalated bonds on the redox behaviors and the accessibility to the mixed-valence M(II)–M(III) dinuclear complexes are discussed. Introduction of a cyclometalated bond induced a large negative potential shift in the redox potentials of dinuclear ruthenium and osmium complexes, depending on either bridging or ancillary sites of the cyclometalated bonds: the change falls within the range of −1.0 to −1.2 V for the bridging sites and −0.65 to −0.7 V for the ancillary ones. This large negative potential shift arises from the strong electron-donating property of the phenyl anion in a metal–C bond. Replacing the ruthenium by osmium in the dinuclear complexes with the same bridging ligand results in an increase of the potential separation (ΔE(1)) and the comproportionation constant (K com) of the mixed-valence complexes having the tppz bridging ligand (ΔE(1) and K com values: Os > Ru); however, complexes having the tpb bridging ligand showed the opposite trend (ΔE(1) and K com: Os < Ru). In addition to the results of EPR and DFT calculation, it was found that the orbital energy levels of the central metal ion (namely, either Ru or Os) in the mixed-valence complex determines the degree of orbital mixing between metal dπ orbitals and bridging-ligand π or π* orbitals, which leads to either hole- or electron-transfer exchange mechanisms.
Cystine is formed from two molecules of the cysteine under oxidized conditions, but is reversibly converted to cysteine by reduction. Growth of Escherichia coli is retarded in the presence of excess ...cystine. Transcriptome analysis showed 11 up-regulated and 26 down-regulated genes upon exposure to excess cystine. The reporter assay confirmed regulation by cystine of the expression of one up-regulated membrane gene, yijE, and two down-regulated membrane genes, yhdT and yihN. In order to identify the as yet unidentified gene encoding cystine efflux transporter, the putative cystine efflux candidate, yijE gene, was over-expressed. Expression of the yijE gene suppressed the slow growth of E. coli in the presence of high concentration of extracellular cystine. In good agreement, the knock-out of yijE gene increased the sensibility to cystine. These observations altogether imply that the yijE gene is involved in response to cystine in E. coli.
The Escherichia coli yijE gene, induced by cystine, is involved in response to excess cystine.
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