15 research outputs found
Substituent Effects on Core Structures and Heterogeneous Catalytic Activities of Mn<sup>III</sup>(μ-O)<sub>2</sub>Mn<sup>IV</sup> Dimers with 2,2′:6′,2″-Terpyridine Derivative Ligands for Water Oxidation
[(OH<sub>2</sub>)Â(R-terpy)ÂMnÂ(μ-O)<sub>2</sub>MnÂ(R-terpy)Â(OH<sub>2</sub>) ]<sup>3+</sup> (R-terpy = 4′-substituted 2,2′:6′,2″-terpyridine,
R = butoxy (BuO), propoxy (PrO), ethoxy (EtO), methoxy (MeO), methyl
(Me), methylthio (MeS), chloro (Cl)) have been synthesized as a functional
oxygen-evolving complex (OEC) model and characterized by UV–vis
and IR spectroscopic, X-ray crystallographic, magnetometric, and electrochemical
techniques. The UV–vis spectra of derivatives in water were
hardly influenced by the 4′-substituent variation. X-ray crystallographic
data showed that Mn centers in the Mn<sup>III</sup>(μ-O)<sub>2</sub>Mn<sup>IV</sup> cores for derivatives with R = H, MeS, Me,
EtO, and BuO are crystallographically indistinguishable, whereas the
derivatives with R = MeO and PrO gave the significantly distinguishable
Mn centers in the cores. The indistinguishable Mn centers could be
caused by rapid electron exchange between the Mn centers to result
in the delocalized MnÂ(μ-O)<sub>2</sub>Mn core. The exchange
integral values (<i>J</i> = −196 to −178 cm<sup>–1</sup>) for delocalized cores were lower than that (<i>J</i> = −163 to −161 cm<sup>–1</sup>) for
localized cores, though the Mn···Mn distances are nearly
the same (2.707–2.750 Å). The half wave potential (<i>E</i><sub>1/2</sub>) of a Mn<sup>III</sup>–Mn<sup>IV</sup>/Mn<sup>IV</sup>–Mn<sup>IV</sup> pair of the derivatives decreased
with an increase of the electron-donating ability of the substituted
groups for the delocalized core, but it deviated from the correlation
for the localized cores. The catalytic activities of the derivatives
on mica for heterogeneous water oxidation were remarkably changed
by the substituted groups. The second order rate constant (<i>k</i><sub>2</sub>/mol<sup>–1</sup> s<sup>–1</sup>) for O<sub>2</sub> evolution was indicated to be correlated to <i>E</i><sub>1/2</sub> of a Mn<sup>III</sup>–Mn<sup>IV</sup>/Mn<sup>IV</sup>–Mn<sup>IV</sup> pair; <i>k</i><sub>2</sub> increased by a factor of 29 as <i>E</i><sub>1/2</sub> increased by 28 mV
Substituent Effects on Core Structures and Heterogeneous Catalytic Activities of Mn<sup>III</sup>(μ-O)<sub>2</sub>Mn<sup>IV</sup> Dimers with 2,2′:6′,2″-Terpyridine Derivative Ligands for Water Oxidation
[(OH<sub>2</sub>)Â(R-terpy)ÂMnÂ(μ-O)<sub>2</sub>MnÂ(R-terpy)Â(OH<sub>2</sub>) ]<sup>3+</sup> (R-terpy = 4′-substituted 2,2′:6′,2″-terpyridine,
R = butoxy (BuO), propoxy (PrO), ethoxy (EtO), methoxy (MeO), methyl
(Me), methylthio (MeS), chloro (Cl)) have been synthesized as a functional
oxygen-evolving complex (OEC) model and characterized by UV–vis
and IR spectroscopic, X-ray crystallographic, magnetometric, and electrochemical
techniques. The UV–vis spectra of derivatives in water were
hardly influenced by the 4′-substituent variation. X-ray crystallographic
data showed that Mn centers in the Mn<sup>III</sup>(μ-O)<sub>2</sub>Mn<sup>IV</sup> cores for derivatives with R = H, MeS, Me,
EtO, and BuO are crystallographically indistinguishable, whereas the
derivatives with R = MeO and PrO gave the significantly distinguishable
Mn centers in the cores. The indistinguishable Mn centers could be
caused by rapid electron exchange between the Mn centers to result
in the delocalized MnÂ(μ-O)<sub>2</sub>Mn core. The exchange
integral values (<i>J</i> = −196 to −178 cm<sup>–1</sup>) for delocalized cores were lower than that (<i>J</i> = −163 to −161 cm<sup>–1</sup>) for
localized cores, though the Mn···Mn distances are nearly
the same (2.707–2.750 Å). The half wave potential (<i>E</i><sub>1/2</sub>) of a Mn<sup>III</sup>–Mn<sup>IV</sup>/Mn<sup>IV</sup>–Mn<sup>IV</sup> pair of the derivatives decreased
with an increase of the electron-donating ability of the substituted
groups for the delocalized core, but it deviated from the correlation
for the localized cores. The catalytic activities of the derivatives
on mica for heterogeneous water oxidation were remarkably changed
by the substituted groups. The second order rate constant (<i>k</i><sub>2</sub>/mol<sup>–1</sup> s<sup>–1</sup>) for O<sub>2</sub> evolution was indicated to be correlated to <i>E</i><sub>1/2</sub> of a Mn<sup>III</sup>–Mn<sup>IV</sup>/Mn<sup>IV</sup>–Mn<sup>IV</sup> pair; <i>k</i><sub>2</sub> increased by a factor of 29 as <i>E</i><sub>1/2</sub> increased by 28 mV
Rab39a Interacts with Phosphatidylinositol 3-Kinase and Negatively Regulates Autophagy Induced by Lipopolysaccharide Stimulation in Macrophages
<div><p>Rab39a has pleiotropic functions in phagosome maturation, inflammatory activation and neuritogenesis. Here, we characterized Rab39a function in membrane trafficking of phagocytosis and autophagy induction in macrophages. Rab39a localized to the periphery of LAMP2-positive vesicles and showed the similar kinetics on the phagosome to that of LAMP1. The depletion of Rab39a did not influence the localization of LAMP2 to the phagosome, but it augments the autophagosome formation and LC3 processing by lipopolysaccharide (LPS) stimulation. The augmentation of autophagosome formation in Rab39a-knockdown macrophages was suppressed by Atg5 depletion or an inhibitor for phosphatidylinostol 3-kinase (PI3K). Immunoprecipitation analysis revealed that Rab39a interacts with PI3K and that the amino acid residues from 34<sup>th</sup> to 41<sup>st</sup> in Rab39a were indispensable for this interaction. These results suggest that Rab39a negatively regulates the LPS-induced autophagy in macrophages.</p> </div
Augmentation of autophagy induced by LPS in Rab39a-KD macrophages.
<p>(A) Immunostaining analysis of LPS-induced autophagy in Rab39a-KD macrophages. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with LPS for 24 h and immunostained with anti-LC3 antibody. An arrowhead indicates an LC3-dot fluorescence. (B) Proportion of Raw264.7 macrophages with LC3-dots induced by LPS stimulation for 24 h. Data represent the mean and SD of three or four independent experiments. *<i>p</i> < 0.05 (unpaired Student’s <i>t</i>-test). (C) Immunoblot analysis of LC3 processing in macrophages treated with LPS. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with LPS for 24 h and subjected to immunoblot analysis using indicated antibodies. (D) Autophagic flux in Rab39a-KD macrophages treated with LPS. Raw264.7 macrophages treated with control or Rab39a siRNA were treated with LPS in the absence or presence of protease inhibitors, E64d and pepstatin A, for 24 h. Cell lysates were subjected to immunoblot analysis using indicated antibodies.</p
Involvement of Caspase-1 and classical autophagy pathway in autophagy induced by LPS
<p>(A) Autophagy induced by LSP in Caspase-1-KD macrophages. The proportion of macrophages with LC3-dot (upper panel) and immunoblot analysis of LC3 processing (lower panel) are shown. (B) Autophagy induced by LSP in Atg5-KD macrophages. The proportion of macrophages with LC3-dot (upper panel) and immunoblot analysis of LC3 processing (lower panel) are shown. (C) Autophagy induced by LPS in the presence of a PI3K inhibitor, 3-MA. Transfected macrophages were stimulated by LPS in the presence or absence of 3-MA at 10 mM for 24 h. The proportion of macrophages with LC3-dot (upper panel) and immunoblot analysis of LC3 processing (lower panel) are shown. Data represent the mean and SD of three or four independent experiments. *<i>p</i> < 0.05 (unpaired Student’s <i>t</i>-test).</p
Suppression of LPS-induced autophagosome formation by Rab39a overexpression.
<p>(A, B) Raw264.7 macrophages expressing EGFP (A) or EGFP-Rab39a (B) were treated with LPS and immunostained with anti-LC3 antibody. (C) Proportion of macrophages with LC3-dot expressing EGFP or EGFP-Rab39a induced by LPS stimulation for 24 h. Data represent the mean and SD of three independent experiments. *<i>p</i> < 0.05 (unpaired Student’s <i>t</i>-test).</p
Autophagy induced by TLR2 or TLR7/8 ligand in Rab39a-KD macrophages.
<p>(A) Immunostaining analysis of Rab39a-KD macrophages stimulated by TLR2 or TLR7/8 ligand. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with TLR2 ligand Pam3CSK4 at 10 ng/ml or TLR7 ligand R848 at 1 μg/ml for 24 h and immunostained with anti-LC3 antibody. (B) Proportion of Raw264.7 macrophages with LC3-dots induced by Pam3CSK4 or R848 for 24 h. (C) Immunoblot analysis of LC3 processing in macrophages treated with Pam3CSK4 or R848. Raw264.7 macrophages transfected with control or Rab39a siRNA were treated with Pam3CSK4 or R848 for 24 h and subjected to immunoblot analysis using indicated antibodies. Data represent the mean and SD of three or four independent experiments. *<i>p</i> < 0.05 (unpaired Student’s <i>t</i>-test).</p
Interaction of Rab39a with Beclin1.
<p>(A) HEK293T cells were transfected with plasmids for Myc-Beclin1, Myc-Vps34, Myc-UVRAG or Myc-Atg14L and EGFP-Rab39a or EGFP. Whole cell lysates (WCL) were used for immunoprecipitation (IP) with anti-GFP antibody, followed by immunoblot analysis (IB) with anti-Myc antibody. For detection of input, aliquots of 15 μg of WCL were used. (B) ClustalW alignment of the amino acid sequences of Rab39a, Rab39a_M1 and Rab39b is shown. (C) HEK293T cells were transfected with plasmids for Myc-Beclin1 and EGFP-Rab39a, EGFP-Rab39a_M1, EGFP-Rab39b or EGFP. WCL were used for IP with anti-GFP antibody followed by IB with anti-Myc antibody. For detection of input, aliquots of 15 μg of WCL were used.</p
Beclin1-dot formation in LPS-induced autophagy.
<p>(A) Control or Rab39a-KD macrophages were treated with LPS for 12 h and immunostained with anti-Beclin1 antibody. An arrowhead indicates a Beclin1-dot. (B) The proportion of control or Rab39a-KD macrophages with Beclin-dot in LPS-induced autophagy is shown. Data represent the mean and SD of three independent experiments. (C) Localization of Beclin1-dot. Rab39a-KD macrophages were treated wtih LPS for 12 h and immunostained with anti-Beclin1 (red) and anti-ubiquitin (green) antibodies.</p
Localization of Rab39a in macrophages.
<p>(A) Immunostaining of Raw264.7 macrophages expressing EGFP-Rab39a with anti-LAMP2 antibody. (B) Representative sequence images from FRAP analysis of EGFP-Rab39a on latex bead-containing phagosomes. The region marked by a broken-line circle was photobleached at 4 sec, and the recovery of fluorescence was monitored. (C) Temporal changes in fluorescence intensities on the bleached phagosomes. The relative intensity was defined as the ratio of fluorescence intensity at each time point to that at 0 sec. Data represent means and standard errors of means (n=10).</p