Proteomic Analysis of Growth Phase-Dependent Expression of Legionella pneumophila Proteins Which Involves Regulation of Bacterial Virulence Traits

Legionella pneumophila, which is a causative pathogen of Legionnaires' disease, expresses its virulent traits in response to growth conditions. In particular, it is known to become virulent at a post-exponential phase in vitro culture. In this study, we performed a proteomic analysis of differences in expression between the exponential phase and post-exponential phase to identify candidates associated with L. pneumophila virulence using 2-Dimentional Fluorescence Difference Gel Electrophoresis (2D-DIGE) combined with Matrix-Assisted Laser Desorption/Ionization–Mass Spectrometry (MALDI-TOF-MS). Of 68 identified proteins that significantly differed in expression between the two growth phases, 64 were up-regulated at a post-exponential phase. The up-regulated proteins included enzymes related to glycolysis, ketone body biogenesis and poly-3-hydroxybutyrate (PHB) biogenesis, suggesting that L. pneumophila may utilize sugars and lipids as energy sources, when amino acids become scarce. Proteins related to motility (flagella components and twitching motility-associated proteins) were also up-regulated, predicting that they enhance infectivity of the bacteria in host cells under certain conditions. Furthermore, 9 up-regulated proteins of unknown function were found. Two of them were identified as novel bacterial factors associated with hemolysis of sheep red blood cells (SRBCs). Another 2 were found to be translocated into macrophages via the Icm/Dot type IV secretion apparatus as effector candidates in a reporter assay with Bordetella pertussis adenylate cyclase. The study will be helpful for virulent analysis of L. pneumophila from the viewpoint of physiological or metabolic modulation dependent on growth phase

Sequestration and Oxidation of Cr(III) by Fungal Mn Oxides with Mn(II) Oxidizing Activity

Biogenic manganese oxides (BMOs) have gained increasing attention for environmental application because of their sequestration and oxidizing abilities for various elements. Oxidation and sequestration of Cr(III) by BMOs, however, still remain unknown. We prepared BMOs in liquid cultures of Acremonium strictum strain KR21-2, and subsequently conducted single or repeated treatment experiments in Cr(NO3)3 at pH 6.0. Under aerobic conditions, newly formed BMOs exhibited a rapid production of Cr(VI) without a significant release of Mn(II), demonstrating that newly formed BMO mediates a catalytic oxidation of Cr(III) with a self-regeneration step of reduced Mn. In anaerobic solution, newly formed BMOs showed a cessation of Cr(III) oxidation in the early stage of the reaction, and subsequently had a much smaller Cr(VI) production with significant release of reduced Mn(II). Extraordinary sequestration of Cr(III) was observed during the repeated treatments under anaerobic conditions. Anaerobically sequestered Cr(III) was readily converted to Cr(VI) when the conditions became aerobic, which suggests that the surface passivation is responsible for the anaerobic cessation of Cr(III) oxidation. The results presented herein increase our understanding of the roles of BMO in Cr(III) oxidation and sequestration processes in potential application of BMOs towards the remediation of Cr(III)/Cr(VI) in contaminated sites

RXR Partial Agonist Produced by Side Chain Repositioning of Alkoxy RXR Full Agonist Retains Antitype 2 Diabetes Activity without the Adverse Effects

We previously reported RXR partial agonist CBt-PMN (1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1<i>H</i>-benzotriazole-5-carboxylic acid: <b>5</b>, EC₅₀ = 143 nM, <i>E</i>_max = 75%), which showed a potent glucose-lowering effect without causing serious adverse effects. However, it remains important to elucidate the structural requirements for RXR efficacy and the glucose-lowering effect because RXR-permissive heterodimers such as PPAR/RXR or LXR/RXR are reported to be activated differently depending upon the chemical structure of RXR agonists. In this work, we show that an RXR partial agonist, NEt-4IB (6-[ethyl-(4-isobutoxy-3-isopropylphenyl)­amino]­pyridine-3-carboxylic acid: <b>8b</b>, EC₅₀ = 169 nM, <i>E</i>_max = 55%), can be obtained simply by repositioning the side chains (interchanging the isobutoxy and isopropoxy groups) at the hydrophobic moiety of the RXR full agonist NEt-3IB (6-[ethyl-(3-isobutoxy-4-isopropylphenyl)­amino]­pyridine-3-carboxylic acid: <b>7b</b>, EC₅₀ = 19 nM). NEt-4IB (<b>8b</b>) showed antitype 2 diabetes activity without the above side effects upon repeated oral administration to mice at 10 mg/kg/day, similarly to <b>5</b>

Mechanism of Retinoid X Receptor Partial Agonistic Action of 1‑(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)‑1<i>H</i>‑benzotriazole-5-carboxylic Acid and Structural Development To Increase Potency

We have reported that retinoid X receptor (RXR) partial agonist 1-(3,5,5,8,8-pentamethyl-5,6,7,8-tetrahydro-2-naphthyl)-1<i>H</i>-benzotriazole-5-carboxylic acid (CBt-PMN, <b>4a</b>) shows a significant antidiabetes effect in the KK-A^y type 2 diabetes model mice, with reduced side effects compared to RXR full agonists. To elucidate the mechanism of the RXR partial agonist activity of <b>4a</b>, we synthesized derivatives of <b>4a</b>, evaluated their RXR agonist activity, and performed structure–activity relationship analysis. Reporter gene assay revealed that though <b>6b</b>, which possesses an amino group at the 2-position of 5-carboxybenzimidazole, showed RXR full-agonist activity, compounds <b>6d</b> and <b>6e</b>, which possess an oxygen atom and a sulfur atom at the corresponding position, respectively, showed weak RXR agonist activity. On the other hand, <b>6c</b>, which has a trifluoromethyl group at the corresponding position, acts as an RXR partial agonist, having similar <i>E</i>_max (67 ± 2%) and lower EC₅₀ (15 ± 0 nM) compared to those of <b>4a</b> (<i>E</i>_max = 75 ± 4%, EC₅₀ = 143 ± 2 nM). A fluorescence polarization assay of cofactor recruitment confirmed that fluorescein-labeled D22 coactivator peptide was less efficiently recruited to RXR by <b>4a</b> and <b>6c</b> than by LGD1069 (<b>1</b>), a known RXR full agonist. Electrostatic potential field calculations and computational docking studies suggested that full agonists show an electrostatic attraction, which stabilizes the holo structure and favors coactivator recruitment, between the side chain at the benzimidazole 2-position and the α-carbonyl oxygen of asparagine-306 in helix 4 (H4) of the RXR receptor. However, RXR partial agonists <b>4a</b> and <b>6c</b> lack this interaction. Like <b>4a</b>, <b>6c</b> showed a significant antidiabetes effect in KK-A^y type 2 diabetes model mice with reduced levels of the side effects associated with RXR full agonists. These findings should aid the design of new RXR partial agonists as antitype 2 diabetes drug candidates