A Diastereoselective Cyclic Imine Cycloaddition Strategy To Access Polyhydroxylated Indolizidine Skeleton: Concise Syntheses of (+)-/(−)-Lentiginosines and (−)-2-<i>epi</i>-Steviamine

We describe in this paper the development of a novel diastereoselective cyclic imine cycloaddition strategy to access the polyhydroxylated indolizidine skeleton and its application in the concise syntheses of (+)-/(−)-lentiginosines and (−)-2-<i>epi</i>-steviamine

Preparation of Zeolite NaA Membranes on Macroporous Alumina Supports by Secondary Growth of Gel Layers

Zeolite NaA membranes are synthesized on tubular α-Al₂O₃ supports by the secondary growth of gel layers. The gel layer is prepared by wetting–rubbing hydrogel with a composition similar to the secondary synthesis solution <i>x</i>Na₂O:2SiO₂:Al₂O₃:150H₂O. The hydrogel’s good uniformity makes it easier to gain a uniform gel layer. Zeolite NaA membranes are characterized by SEM and pervaporation separation of a 90 wt % ethanol/water mixture at 75 °C. The effects of the pretreatment time, pretreatment temperature, gel loading, and Na₂O/Al₂O₃ ratio of the hydrogel on properties of zeolite NaA membranes are investigated. It is found that zeolite NaA membranes with high separation factors up to 10 000 can be obtained by secondary growth of gel layers. The optimal pretreatment conditions of the hydrogel are as follows: pretreatment temperature, 50 °C; pretreatment time, ≥18 h; and gel loading, 0.6–0.9 mg/cm². The optimal composition of the hydrogel is 2.2Na₂O:2SiO₂:Al₂O₃:150H₂O, which is the same as the synthesis solution. Like crystal seeds, the gel layer improves the zeolite membrane formation on the support surface, and the role of the gel layer becomes more significant with greater amounts of crystals

Three new complexes based on methyl-pyrimidine-2-thione: <i>in situ</i> transformation, crystal structures and properties

<p>Three complexes based on methyl-pyrimidine-2-thione, 5-methyl-5,8,9,10-tetrahydro-5,9methanopyrimido[5,4-e][1, 3]diazocine-2,7-dithiol (mtmdd) (<b>1</b>), and Ni(mpymt)₂(N₂H₄)·H₂O (<b>2</b>) and [Cd(mpymt)₂]_∞ (<b>3</b>) have been synthesized under hydrothermal conditions and characterized by elemental analysis, infrared (IR) spectroscopy, thermal gravimetric analysis, powder X-ray diffraction, and single-crystal X-ray diffraction. Structural analysis reveals that <b>1</b> is generated from an <i>in situ</i> translation of mpymt. In <b>2</b>, two Ni(mpymt)₂ fragments were joined into a dinuclear complex by two NH₂–NH₂ molecules. Compound <b>3</b> is a two-dimensional Cd coordination polymer constructed by Cd₂(mpymt)₂ SBUs and mpymt anions. Variable-temperature magnetic susceptibility measurement of <b>2</b> revealed strong antiferromagnetic interactions between nickel magnetic centers. The fluorescent properties of <b>1</b> and <b>3</b> were investigated in the solid state.</p

A thiosemicarbazone copper(II) complex as a potential anticancer agent

<div><p>The preparation and the structure of a copper(II) complex, [Cu(4ML)Cl] (<b>1</b>) (H4ML = 2-acetylpyridine-4-methylthiosemicarbazone), are described. Complex <b>1</b> crystallizes in a monoclinic <i>P2</i>_<i>1</i><i>/c</i> space group with <i>a</i> = 7.977(2) Å, <i>b</i> = 15.824(5) Å, <i>c</i> = 9.126(2) Å, <i>α</i> = <i>γ</i> = 90°, <i>β</i> = 91.974(2)°, <i>V</i> = 1151.26(5) Å³, <i>Z</i> = 4, <i>F</i>(0 0 0) = 620. According to X-ray crystallographic studies, each Cu(II) ion lies in a square planar coordination geometry based on the 4ML⁻ and Cl⁻ ligands. The complex displayed excellent inhibitory activity against various tumor cells (HeLa, HepG-2 and SGC-7901), offering lower IC₅₀ value of 3.2 ± 0.7 μM than cisplatin (10 ± 2 μM) on HeLa cells at 48 h. Complex <b>1</b> could significantly suppress HeLa cell viability in a dose-dependent manner. Flow cytometric analysis showed that <b>1</b> induced HeLa cell apoptosis, which might be associated with cell cycle arrests at S and G2 phases. Consistent with results of DNA cleavage experiments, comet assay indicated that <b>1</b> caused severe DNA fragmentation. The production of ROS was elevated with increasing concentration of <b>1</b>, suggesting that <b>1</b> was capable of promoting HeLa cell apoptosis through an oxidative DNA damage pathway.</p></div

Five metal imidazole dicarboxylate-based compounds comprising M₃(MIDC)₂ entities (M = Zn²⁺, Co²⁺, Mn²⁺): syntheses, structures and properties

<div><p>Five metal imidazole dicarboxylate-based compounds, {[Zn₃(MIDC)₂(4,4′-bipy)₃](4,4′-bipy)·8H₂O}_n (<b>1</b>), {[Co₃(MIDC)₂(4,4′-bipy)₃](4,4′-bipy)·6H₂O}_n (<b>2</b>), {[Co₃(MIDC)₂(py)₂(H₂O)₂]}_n (<b>3</b>), {[Mn₆(MIDC)₄(py)₅(H₂O)₄]}_n (<b>4</b>), and {[Mn₃(MIDC)₂(Phen)₃(H₂O)₂]}_n (<b>5</b>) (H₃MIDC = 2-methyl-1H-imidazole-4,5-dicarboxylic acid; 4,4′-bipy = 4,4′-bipyridine; py = pyridine; Phen = 1,10-phenanthroline), have been synthesized under hydrothermal conditions and characterized by elemental analyses, IR spectroscopy, thermogravimetric analysis, and single-crystal X-ray diffraction. We control the coordination modes of H₃MIDC via hydrazine and obtained a series of coordination compounds containing honeycomb-like [M₃(MIDC)₂]_n layers. We also investigated the effects of different neutral terminal or bridging ligands on [M₃(MIDC)₂]_n layers. Coplanar [M₃(MIDC)₂]_n layers and 4,4-bipy were used to construct 3-D frameworks of <b>1</b> and <b>2</b>. Puckered [M₃(MIDC)₂]_n layers were found in <b>3–5</b>; <b>4</b> is the first [M₃(L)₂]_n layer structure with two crests and troughs during each period (L = imidazole-4,5-dicarboxylic acid or its analog). Compound <b>5</b> is the first puckered [M₃(L)₂]_n layer structure decorated by chelating neutral ligands. Compound <b>1</b> exhibits weak blue photoluminescence in the solid state at room temperature. Variable-temperature magnetic susceptibility measurements of <b>2–5</b> indicate strong antiferromagnetic interactions.</p></div

Ubiquitin Ligase gp78 Targets Unglycosylated Prion Protein PrP for Ubiquitylation and Degradation

<div><p>Prion protein PrP is a central player in several devastating neurodegenerative disorders, including mad cow disease and Creutzfeltd-Jacob disease. Conformational alteration of PrP into an aggregation-prone infectious form PrP^Sc can trigger pathogenic events. How levels of PrP are regulated is poorly understood. Human PrP is known to be degraded by the proteasome, but the specific proteolytic pathway responsible for PrP destruction remains elusive. Here, we demonstrate that the ubiquitin ligase gp78, known for its role in protein quality control, is critical for unglycosylated PrP ubiquitylation and degradation. Furthermore, C-terminal sequences of PrP protein are crucial for its ubiquitylation and degradation. Our study reveals the first ubiquitin ligase specifically involved in prion protein PrP degradation and PrP sequences crucial for its turnover. Our data may lead to a new avenue to control PrP level and pathogenesis.</p></div

gp78 specifically interacts with PrP.

<p>(<b>A</b>) Co-immunoprecipitation analysis of interactions between gp78 and ugPrP. HEK293 cells were transfected with myc-tagged gp78 and/or ugPrP as indicated. Proteins were extracted and immunoprecipitated with beads coated with PrP antibody 3F4. Immunoprecipitates were separated on SDS-PAGE, and probed with anti-myc (top panel) or anti-PrP (middle panel). The amounts of myc-gp78 in cell extracts were evaluated and presented in bottom panel. (<b>B</b>) HsHrd1 does not bind ugPrP. Proteins were extracted from cells expressing myc-tagged HsHrd1 and ugPrP. The indicated immunoprecipitations and immunoblottings were carried out as described above in (A). (<b>C</b>) gp78 binds unglycosylated PrP preferentially. HEK293 cells were transfected with plasmids expressing myc-gp78 and wild-type PrP or ugPrP (last lane). Immunoprecipiation was carried out using anti-myc beads, and later eluted with myc peptides. Western blotting was done as described above. Only unglycosylated g0 form of PrP (lane 3, top panel), which migrated at the same position as ugPrP control (lane 4), was detected in myc-gp78 immunoprecipitation. (<b>D</b>) Endogenous gp78 interacts with PrP. HEK293 cells were transfected with the plasmid expressing wild-type PrP or ugPrP. Cell extracts were subjected to immunoprecipitation with beads coated with PrP antibody and immunoblotting with gp78 antibody.</p

gp78 promotes ugPrP ubiquitylation and degradation.

<p>(<b>A</b>) gp78 knockdown efficiency in HEK293 cells. HEK293 cells were transfected with the gp78 shRNA or control plasmid. Cell extracts were subjected to immunoblotting analysis to determine the levels of gp78 and actin (loading control). Knockdown efficiency (27.4%) was indicated underneath the panel. (<b>B</b>) Effect of gp78 knockdown on ugPrP turnover. Degradation kinetics of ugPrP was assessed in gp78 knockdown or control HEK293 cells. Actin is shown as a loading control. Protein stability assay via cycloheximide chase was done as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092290#pone-0092290-g001" target="_blank">Figure 1</a>. (<b>C</b>) gp78 deficient MEFs. gp78 expression was evaluated by western blotting in gp78^−/− and control MEFs. (<b>D</b>) ugPrP degradation is impaired in gp78^−/− MEFs. gp78^−/− and control MEFs were transfected with the plasmid encoding ugPrP. ugPrP turnover was evaluated by cycloheximde chase as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092290#pone-0092290-g001" target="_blank">Figure 1</a>. (<b>E</b>) gp78 is important for ugPrP ubiquitylation. gp78^−/− and control MEFs expressing ugPrP were treated with MG132 for 6 h and then lysed, and later subjected to immunoprecipitation with PrP antibody. The immunoprecipitates were analyzed by western blotting with Ub antibody and 3F4 for ugPrP (upper panels). The amount of actin in cell extracts was assessed by immunoblotting with actin antibody (bottom panel).</p

PrP degradation involves the proteasome.

<p>(<b>A</b>) PrP is degraded by the proteasome. HEK293 cells transfected with PrP were treated with or without the inhibitor of the proteasome MG132, cycloheximde was added to shut off protein synthesis and start the chase. Indicated times were taken and processed for immunoblotting with antibody recognizing PrP (3F4) or actin control. The arrows indicate PrP proteins attached with two (g2), one (g1) or no (g0) glycan. As some signals were saturated in the cycloheximide chase results presented (top panel), a lower exposure of the blot was included (middle panel). (<b>B</b>) Quantitation of the data in A. Three different glyco-forms of PrP were analyzed separately as indicated using ImageQuant software. We chose non-saturated bands for quantitation and normalized with the loading control actin. Whereas the bands for g1 species were quantified using the blot more heavily exposed (top panel), the g0 and g2 species were quantified with the lighter blot (middle panel). The experiments were done at least three times, and the average values with standard deviation are shown. (<b>C</b>) ugPrP turnover requires the proteasome. The plasmid expressing ugPrP (N181Q and N197Q) devoid of glycosylation was transfected into HEK293 cells. ugPrP stability in the presence or absence of MG132 was conducted as described in (A). (<b>D</b>) Quantitation of the data in C from 3 experiments.</p

The C-terminal sequences are critical for ugPrP ubiquitylation and turnover.

<p>(<b>A</b>) Domain structure of PrP and various deletion mutants constructed. Four domains (i.e, ssER, OR, HC, GPI) are indicated. NMR analysis demonstrated disorder structure for the N-terminal segment and globular structure for the C-terminal half of PrP. The deleted portions are shown as dashed lines. (<b>B</b>) The effects of various deletions on ugPrP stability. Degradation kinetics of wild-type ugPrP and mutants (left panels) in HEK293 cells was assayed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092290#pone-0092290-g001" target="_blank">Figure 1</a>. Actin serves as the loading control (right panels). (<b>C</b>) The C-terminal deletions maintain efficient gp78-binding. The interaction between gp78 and and ugPrP derivatives was assessed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092290#pone-0092290-g002" target="_blank">Figure 2A</a>. (<b>D</b>) Deletions of C-terminal sequences impair ugPrP ubiquitylation. The ubiquitylation pattern of ugPrP derivatives in HEK293 cells was determined as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0092290#pone-0092290-g003" target="_blank">Figure 3E</a> after MG132 treatment.</p