OEOA was retained for a longer period in mouse blood plasma than OA.

<p>MS-MS product ion mass spectra of OEOA (A) and OA (B). (C) K562 cells were treated with OA or OEOA (1 µM) for 6 h. Culture media and cell lysates were collected at indicated time points for HPLC-MS/MS analysis. Data was represented as mean ± SEM in cell lysate compared to total exposure, n = 2 (* <i>p</i><0.05). (D) Concentrations of OA and OEOA in mouse plasma after intraperitoneal injection. Blood was collected from mice at different time points after single administration of OA and OEOA. Data was represented as mean ± SEM, n = 2 animals per time point (* <i>p</i><0.05).</p

OEOA attenuated phosphorylation of Rb protein in K562 and Jurket cells.

<p>(A) K562 and Jurket cells were treated with OEOA (0.1–10 µM) for 2 days, and the cell lysates were subjected to Western blot analysis for p-Rb and Rb. Actin served as an equal loading control. Histograms in (B) show the relative expression of p-Rb (normalized to actin) as compared to the vehicle-treated cells. Results were representative blots from three separate experiments, (* <i>p</i><0.05).</p

OEOA promoted erythroid differentiation in K562 cells.

<p>K562 cells were treated with OEOA (0.1–10 µM) for 2 days. Total RNA was reverse transcribed and subjected to real time-PCR analysis with primers specific to <i>γ-globin</i> (A) and <i>cd41b</i> (B), respectively. <i>hprt1</i> served as an internal housekeeping gene control. Data were expressed as fold change to the control cells as mean ± SEM of three independent experiments (* <i>p</i><0.05). (C) K562 cells were treated with OEOA (0.1–10 µM) for 2 days. Western blot analysis of Bcr-Abl and Erk1/2 was performed. Actin served as an equal loading control. Histograms on the right show the relative expression of various proteins (normalized to actin) as compared to the control cells. Results were representative blots from three separate experiments, (* <i>p</i><0.05).</p

OEOA inhibited cell proliferation in leukemia cell lines.

<p>Different cell lines were treated with various concentrations of OEOA or OA for 2 days. Cell growth was measured by MTT assay: (A) K562, (B) HEL, (C) Jurket (D) HEKneo, and (E) HepG2, MCF<b>-</b>7 and HeLa cells. Data are mean ± SEM of three independent experiments (* <i>p</i><0.05).</p

OEOA did not induce cell death in K562 and HEL cells.

<p>K562 (A & C) and HEL (B & D) cells were treated with OEOA (1 µM) for 6 days. Cell viability was measured by trypan blue exclusion as described in Materials and Methods. Data are mean ± SEM of three independent experiments (* <i>p</i><0.05).</p

OEOA induced G1 cell cycle arrest in K562 cells.

<p>(A) Cells were incubated with OEOA (1 or 10 µM) for 24 h. The distribution of cell cycle was examined by PI staining method. The table summarized the distribution of cells in OEOA-treated or control cells. Data represented mean ± SEM of three independent experiments (* <i>p</i><0.05). (B) K562 cells were cultured in the presence of OEOA (1 or 10 µM) for 2 days. Total proteins were collected for Western blot analysis to detect the expression of p27, Cdk4, Cdk6, Cyclin D1, Cyclin E and RAMP. Actin served as an equal loading control. Histograms on the right show the relative expression of various proteins (normalized to actin) as compared to the control cells. Results were representative blots from three separate experiments, (* <i>p</i><0.05).</p

Diarylheptanoids from Rhizomes of Alpinia officinarum Inhibit Aggregation of α‑Synuclein

Two new diarylheptanoids, alpinin A (<b>1</b>) and alpinin B (<b>2</b>), together with 18 known diarylheptanoids (<b>3</b>–<b>20</b>), were isolated from the rhizomes of Alpinia officinarum. Their structures were elucidated by comprehensive spectroscopic analysis, including high-resolution mass spectrometry, infrared spectroscopy, and one- and two-dimensional nuclear magnetic resonance spectroscopy. Structurally, alpinin A is a new member of the small family of oxa-bridged diarylheptanoids and contains the characteristic 2,6-<i>cis</i>-configured tetrahydropyran motif (C₁–C₅ oxa bridge). The absolute configuration of alpinin A was confirmed by asymmetric total synthesis of the enantiomer (<i>ent</i>-<b>1</b>), corroborating the assignment of the molecular structure. The absolute configuration of alpinin B was determined on the basis of the analysis of the circular dichroism exciton chirality spectrum. We evaluated the inhibitory activity of all isolated diarylheptanoids against α-synuclein aggregation at 10 μM. Alpinins A and B significantly inhibited α-synuclein aggregation by 66 and 67%, respectively

p35 interacts with NIF-1.

<p>(A) Mouse NIF-1 encodes a 1,291-amino acid protein and contains 6 zinc finger domains, an LXXLL domain, and a leucine zipper-like motif. (B & C) Mapping of interaction domains between NIF-1 and p35. Yeast was co-transformed with different domains of p35 and NIF-1. +, strong interaction; −, absence of interaction. (B) The C-terminal region of NIF-1 (amino acids 1,066–1,291) containing the 6^th zinc finger domain, leucine zipper-like motif, and short C-terminus was sufficient to interact with p35. (C) The N-terminal region of p35 (corresponding to the p10 fragment) was required for the interaction between p35 and NIF-1. p10 comprises the 98 N-terminal amino acids of p35, while p25 contains the C-terminal region of p35. (D & E) Direct interaction between NIF-1 and p35. Recombinant GST fusion proteins encoding different regions of NIF-1 were incubated with lysate prepared from p35-overexpressing COS-7 cells (D) or recombinant p35 protein (E). The bound proteins were pulled down by glutathione-Sepharose and analyzed by western blot analysis (Lysate, as an input control). Bottom panel: Coomassie-stained gel. (F) Association of p35 with NIF-1 in mammalian cells. COS-7 cells were transiently transfected with NIF-1 and p35. Cell lysate was immunoprecipitated (IP) with p35 or NIF-1 antibody as indicated and subjected to western blot analysis. Rabbit normal IgG (IgG) was used as a negative control.</p

Dependence of p35 nuclear export on its NES.

<p>(A) Consensus sequence of the NES on p35. The functional NES comprises a core of closely spaced leucine residues or other hydrophobic amino acids. The critical hydrophobic residues in the putative NES of p35 are underlined. An NES mutant of p35, p35NES, was generated by mutating the 3 conserved hydrophobic residues (i.e., leucine 226, 227, and 230) to alanine. (B) The NES of p35 is required to mediate the nuclear export of NIF-1. COS-7 cells were co-transfected with HA-NIF-1 and p35WT or p35NES and subsequently stained with HA antibody. The results of the quantitative analysis represent the mean ± SEM of 3 replicates (***<i>p</i> <0.05, one-way ANOVA followed by the Student–Newman–Keuls test). (C) Mutating the NES of p35 increased the population of Neuro-2A cells containing nuclear p35. Neuro-2A cells were transfected with p35WT-GFP or p35NES-GFP and subsequently differentiated by RA. The cells were stained with p35 antibody. Representative fluorescent images depicting the localization of p35WT and p35NES (indicated by the p35 staining and GFP expression). (D) Quantitative analysis of cells with nuclear p35. Cells with nuclear p35 were counted if the GFP signal in the nucleus was>75% than that of the cytoplasm. Results represent the mean ± SEM of 3 replicates (***<i>p</i> <0.05, Student's <i>t</i>-test). (E) LMB treatment caused the accumulation of p35 in the nucleus of cortical neurons. Cultured cortical neurons were treated with LMB for 1 h, and subcellular fractionation was performed. Western blot analysis of p35 and Cdk5. Total: protein extracted from the same batch of neurons using RIPA.</p

Two pairs of unusual melibiose and raffinose esters from <i>Scrophularia ningpoensis</i>

<p>A pair of unusual melibiose esters (<b>1<i>α</i></b>/<b>1<i>β</i></b>) and a pair of unusual raffinose esters (<b>2<i>α</i>/2<i>β</i></b>), were isolated from <i>Scrophularia ningpoensis</i>. Structures of them were established by detailed spectroscopic analyses to be 6-<i>O</i>-(<i>E</i>)-cinnamoyl-<i>α</i>-d-galactopyranosyl-(1→6)-<i>α</i>(<i>β</i>)-d-glucopyranose (<b>1<i>α</i></b>/<b>1<i>β</i></b>) and 6-<i>O</i>-(<i>E</i>)/(<i>Z</i>)-cinnamoyl-<i>α</i>-d-galactopyranosyl-(1→6)-<i>α</i>-d-glucopyranosyl-(1→2)-<i>β</i>-d-fructofuranose (<b>2<i>α</i>/2<i>β</i></b>), respectively. All these compounds were evaluated for antifouling activity against the settlement of <i>Balanus amphitrite</i> larvae, along with the cytotoxic effect against the proliferation of HeLa cell lines.</p