Helix12-Stabilization Antagonist of Vitamin D Receptor

To develop strong vitamin D receptor (VDR) antagonists and reveal their antagonistic mechanism, we designed and synthesized vitamin D analogues with bulky side chains based on the “active antagonist” concept in which antagonist prevents helix 12 (H12) folding. Of the synthesized analogues, compounds <b>3a</b> and <b>3b</b> showed strong antagonistic activity. Dynamic hydrogen/deuterium exchange coupled with mass spectrometry (HDX-MS) and static X-ray crystal structure analyses indicated that compound <b>3a</b> stabilizes H11–H12 but displaces H6–H7 so that <b>3a</b> is a novel rather than “active” or “passive” type of antagonist. We classified <b>3a</b> as a third type of antagonist and called it “H11–H12 stabilization antagonist”. HDX-MS analysis indicated that antagonist <b>3b</b> is an “active” antagonist. To date there are no reports relating to nuclear receptor antagonist that strongly stabilizes H12. In this study, we found first VDR antagonist that stabilizes H12 and we showed that antagonistic mechanism is diverse depending on each antagonist structure. Additionally, HDX-MS was proven to be very useful for investigations of protein structure alterations resulting from ligand binding

Is a euryhaline fugu species very close to and suitable for studying osmoregulation-4

<p><b>Copyright information:</b></p><p>Taken from "is a euryhaline fugu species very close to and suitable for studying osmoregulation"</p><p>BMC Physiology 2005;5():18-18.</p><p>Published online 20 Dec 2005</p><p>PMCID:PMC1351200.</p><p>Copyright © 2005 Kato et al; licensee BioMed Central Ltd.</p>ies were used for the analyses. Bootstrap values from 2,000 times replications are indicated at major nodes. Bars indicate 5% replacement of a nucleotide per site. Accession numbers were as follows: , AB199318; , AB199319; , AB199320; , AB199321; , AB199322; , AB199323; , CR688806; , NC_004387; , NC_001717; , J01415; , J01420; and , X14848

Is a euryhaline fugu species very close to and suitable for studying osmoregulation-3

<p><b>Copyright information:</b></p><p>Taken from "is a euryhaline fugu species very close to and suitable for studying osmoregulation"</p><p>BMC Physiology 2005;5():18-18.</p><p>Published online 20 Dec 2005</p><p>PMCID:PMC1351200.</p><p>Copyright © 2005 Kato et al; licensee BioMed Central Ltd.</p>ematoxylin and eosin and examined for abundance of glomeruli. All the other species, , , , and , also have glomerulous nephron (data not shown). . Higher magnification view of the glomeruli of FW-acclimated indicated by a box in A. . Higher magnification views of the glomeruli of SW-acclimated indicated by boxes in B. . Paraffin-embedded sections of the kidneys of indicated species were stained with anti-Na-K-ATPase (NKA) antibody (green) and Hoechst 33342 (red). NKA antibody strongly stained basolateral surface of proximal segment (p) and entire cell of distal segment (d). , , and showed similar result to (data not shown). . Frozen sections of the kidneys of were stained with anti-NKA antibody (red) and Alaxa Fluor 488-labeled phalloidin (green). Phalloidin binds to actin filaments, and strongly stains a well-developed apical brush border of proximal segments. . Proximal segment of the nephron of . . Distal segment of the nephron of . All scale bars represent 50 μm

Is a euryhaline fugu species very close to and suitable for studying osmoregulation-1

<p><b>Copyright information:</b></p><p>Taken from "is a euryhaline fugu species very close to and suitable for studying osmoregulation"</p><p>BMC Physiology 2005;5():18-18.</p><p>Published online 20 Dec 2005</p><p>PMCID:PMC1351200.</p><p>Copyright © 2005 Kato et al; licensee BioMed Central Ltd.</p>n = 18 for FW; , n = 35 for FW and n = 36 for BW; , n = 10 for FW and n = 6 for BW; , n = 32 for FW and n = 10 for BW; , n = 26 for FW and n = 11 for BW; and , n = 6 for FW

Is a euryhaline fugu species very close to and suitable for studying osmoregulation-0

<p><b>Copyright information:</b></p><p>Taken from "is a euryhaline fugu species very close to and suitable for studying osmoregulation"</p><p>BMC Physiology 2005;5():18-18.</p><p>Published online 20 Dec 2005</p><p>PMCID:PMC1351200.</p><p>Copyright © 2005 Kato et al; licensee BioMed Central Ltd.</p

Vitamin D Analogues with a <i>p</i>‑Hydroxyphenyl Group at the C25 Position: Crystal Structure of Vitamin D Receptor Ligand-Binding Domain Complexed with the Ligand Explains the Mechanism Underlying Full Antagonistic Action

Vitamin D receptor (VDR) antagonists can be classified into two categories: the first category of VDR antagonists, which do not stabilize the helix 11–12, and the second category of antagonists, which destabilize the helix 6–7 region. To elucidate the mechanism underlying the first category antagonists by using the crystal structure, we designed and synthesized several VDR ligands with a <i>p</i>-hydroxyphenyl group at the C25-position. Of these, 22<i>S</i>-butyl-25-carbonyl analogue <b>5b</b> and 25-di-<i>p</i>-hydoroxyphenyl analogues <b>6a</b>,<b>b</b> showed strong antagonistic activity. We succeeded in cocrystallizing the ligand-binding domain of VDR complexed with <b>5b</b> and found that the structure showed an alternative conformation of the helix 11–12 that explained the mechanism of the first category antagonists. Taking the present and previous studies together, we could elucidate the mechanisms underlying first and second categories antagonists based on individual crystal structures. This study provides significant insights into antagonism against not only VDR but also nuclear receptors

Identification of the Histidine Residue in Vitamin D Receptor That Covalently Binds to Electrophilic Ligands

We designed and synthesized vitamin D analogues with an electrophile as covalent modifiers for the vitamin D receptor (VDR). Novel vitamin D analogues <b>1</b>–<b>4</b> have an electrophilic enone group at the side chain for conjugate addition to His301 or His393 in the VDR. All compounds showed specific VDR-binding potency and agonistic activity. Covalent bond formations of <b>1</b>–<b>4</b> with the ligand-binding domain (LBD) of VDR were evaluated by electrospray ionization mass spectrometry. All compounds were shown to covalently bind to the VDR-LBD, and the abundance of VDR-LBD corresponding conjugate adducts of <b>1</b>–<b>4</b> increased with incubation time. Enone compounds <b>1</b> and <b>2</b> showed higher reactivity than the ene-ynone <b>3</b> and dienone <b>4</b> compounds. Furthermore, we successfully obtained cocrystals of VDR-LBD with analogues <b>1</b>–<b>4</b>. X-ray crystallographic analysis showed a covalent bond with His301 in VDR-LBD. We successfully synthesized vitamin D analogues that form a covalent bond with VDR-LBD

Identification of the Histidine Residue in Vitamin D Receptor That Covalently Binds to Electrophilic Ligands

We designed and synthesized vitamin D analogues with an electrophile as covalent modifiers for the vitamin D receptor (VDR). Novel vitamin D analogues <b>1</b>–<b>4</b> have an electrophilic enone group at the side chain for conjugate addition to His301 or His393 in the VDR. All compounds showed specific VDR-binding potency and agonistic activity. Covalent bond formations of <b>1</b>–<b>4</b> with the ligand-binding domain (LBD) of VDR were evaluated by electrospray ionization mass spectrometry. All compounds were shown to covalently bind to the VDR-LBD, and the abundance of VDR-LBD corresponding conjugate adducts of <b>1</b>–<b>4</b> increased with incubation time. Enone compounds <b>1</b> and <b>2</b> showed higher reactivity than the ene-ynone <b>3</b> and dienone <b>4</b> compounds. Furthermore, we successfully obtained cocrystals of VDR-LBD with analogues <b>1</b>–<b>4</b>. X-ray crystallographic analysis showed a covalent bond with His301 in VDR-LBD. We successfully synthesized vitamin D analogues that form a covalent bond with VDR-LBD

Roles of MCT1b and MCT4b in the fugu swimbladder.

<p>A, A model of lactic acid transfer mediated by MCT1b and MCT4b. In this model, the presence of MCT1b in tightly sealed arterial capillaries of the rete mirabile allows back-diffusion of lactate from the venous side to the arterial side (box ii), despite the presence of tight junctions that are essential for the efficient delivery of glucose to gas gland cells (box i), by preventing its shunt diffusion directly to the venous side (box iv). The expression of MCT1b and MCT4b in arterial capillaries and gas gland cells was demonstrated in this study. A loose (leaky) association between venous capillary endothelial cells and a tight (impermeable) adhesion between arterial capillary endothelial cells were demonstrated by Wagner <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034579#pone.0034579-Wagner1" target="_blank">[25]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034579#pone.0034579-Wagner2" target="_blank">[26]</a>. Gas gland cells' glucose requirement and lactic acid secretion have been demonstrated by others <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034579#pone.0034579-Pelster2" target="_blank">[14]</a>. B, A model of the rete mirabile assuming free permeability between arterial and venous capillaries. In the absence of restricted permeability (tight junctions), delivery of glucose to gas gland cells becomes inefficient (box iii).</p

Synteny analysis of <i>MCT</i> and <i>SMCT</i> gene families.

<p>Synteny of neighboring genes of MCTs and SMCTs in the genome databases of human (Hsap), chicken (Ggal), <i>X. tropicalis</i> (Xtro), zebrafish (Drer), medaka (Olat), stickleback (Gacu), <i>Tetraodon</i> (Tnig), and fugu (Trub) are shown. chr, chromosome; gr, group; sc, scaffold; and uctg, ultracontig.</p