Synthesis of the C4-<i>Epi</i>-Lomaiviticin B Core Reveals Subtle Stereoelectronic Effects

An efficient synthesis of the C4-<i>epi</i>-lomaiviticin B core is reported. The synthesis features a diastereoselective anionic formal furan Diels–Alder reaction and a stereoselective oxidative enolate dimerization. During the investigation, subtle yet critical stereoelectronic effects imparted by the C4-stereocenter were observed

Summary diagram of alteration of hematopoiesis, UPR signaling and apoptosis in <i>Grp78</i> conditional knockout in the hematopoietic system.

<p>GRP78 depletion in the hematopoietic system leads to altered hematopoiesis, activated UPR signaling and enhanced apoptosis. Open arrows represent an increased level and closed arrows represent a decreased level.</p

Acute Inducible Ablation of GRP78 Reveals Its Role in Hematopoietic Stem Cell Survival, Lymphogenesis and Regulation of Stress Signaling

<div><p>GRP78, a master regulator of the unfolded protein response (UPR) and cell signaling, is required for inner cell mass survival during early embryonic development. However, little is known about its role in adult hematopoietic stem cells (HSCs) and hematopoiesis. Here we generated a conditional knockout mouse model that acutely deletes <em>Grp78</em> in the adult hematopoietic system. Acute GRP78 ablation resulted in a significant reduction of HSCs, common lymphoid and myeloid progenitors, and lymphoid cell populations in the mutant mice. The GRP78-null induced reduction of the HSC pool could be attributed to increased apoptosis. Chimeric mice with <em>Grp78</em> deletion only in the hematopoietic cells also showed a loss of HSCs and lymphopenia, suggesting a cell intrinsic effect. Analysis of GRP78 deficient bone marrow (BM) cells showed constitutive activation of all the major UPR signaling pathways, including activation of eIF2α, ATF6, <em>xbp-1</em> splicing, as well as caspase activation. A multiplex cytokine assay further revealed alteration in select cytokine and chemokine serum levels in the mutant mice. Collectively, these studies demonstrate that GRP78 plays a pleiotropic role in BM cells and contributes to HSC survival and the maintenance of the lymphoid lineage.</p> </div

GRP78 deficiency in the BM reduced HSC-enriched population through increased cell death.

<p>A: Representative flow cytometric analysis with BM cells using Lin, c-Kit, Sca-1 and CD34. B: ?Left) Quantitation of flow cytometric analysis of Lin^-c-Kit⁺Sca-1⁺CD34⁻ (LT-HSC) and Lin^-c-Kit⁺Sca-1⁺CD34⁺ (ST-HSC) populations in the BM (n = 6 for <i>78^f/f</i>, n = 6 for <i>c78^f/f</i>). (Right) Quantitation of flow cytometric analysis of HSC-enriched LSK population in the BM (n = 12 for <i>78^f/f</i>, n = 16 for <i>c78^f/f</i>, n = 4 for <i>c78^f/+</i>). C: Total BM cell number from <i>78^f/f</i> (n = 11) and <i>c78^f/f</i> (n = 16) mice. D: Quantitation of LSK percentage and total BM cell number from <i>78^f/f</i>(t) and <i>c78^f/f</i>(t) mice (n = 3 for each analysis). E: (Left) Representative flow cytometric analysis of apoptotic LSK cells using Annexin V and 7-AAD. (Right) Summary of apoptotic LSK cells (Annexin V⁺7-AAD^–) (n = 5 for <i>78^f/f</i>, n = 6 for <i>c78^f/f</i>). F: (Left) Representative flow cytometric analysis of LSK cell cycle status by Hoechst and Pyronin Y staining. (Right) Summary of cell cycle distribution of LSK cells from <i>78^f/f</i> (n = 4) and <i>c78^f/f</i> (n = 4) mice. All data are presented as mean ± s.e (*P<0.05, **P<0.01, Student’s <i>t</i> test).</p

Deletion of GRP78 in the hematopoietic system leads to altered hematopoiesis.

<p>A: Quantitation of flow cytometric analysis of lymphoid and myeloid progenitors including common lymphoid progenitor (CLP), common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP) and megakaryocyte-erythroid progenitor (MEP) from <i>78^f/f</i> (n = 6) and <i>c78^f/f</i> (n = 6) mice. B: Quantitation of flow cytometric analysis of lymphoid and myeloid progenitors including common lymphoid progenitor (CLP), common myeloid progenitor (CMP), granulocyte-monocyte progenitor (GMP) and megakaryocyte-erythroid progenitor (MEP) from <i>78^f/f</i>(t) (n = 3) and <i>c78^f/f</i>(t) (n = 3) mice. C: Quantitation of lymphoid and myeloid cells from flow cytometric analysis with BM cells in <i>78^f/f</i> and <i>c78^f/f</i> mice using lineage markers B220, CD3, Gr-1 and Mac-1 (n = 4 for each genotype). D: Quantitation of lymphoid and myeloid cells from flow cytometric analysis with BM cells in <i>78^f/f</i>(t) and <i>c78^f/f</i>(t) mice using lineage markers B220, CD3, Gr-1 and Mac-1 (n = 3 for each genotype). All data are presented as mean ± s.e (*P<0.05, **P<0.01, ***P<0.001, Student’s <i>t</i> test).</p

Differential cytokine and chemokine expression in serum of GRP78 deficient mice.

<p>(Upper, middle, lower) bar graphs demonstrating the differential expression of cytokines and chemokines in the serum of <i>78^f/f</i> (n = 3) and <i>c78^f/f</i> (n = 3) mice. The ones that exhibit major difference between the <i>c78^f/f</i> and <i>78^f/f</i> mice are circled and bold highlighted. The data is presented as mean ± s.e. The P values are indicated (***P<0.001, Student’s <i>t</i> test).</p

Knockout of GRP78 in BM cells activates UPR signaling pathways.

<p>A: Western blot results using BM cell lysates (n = 3 for each genotype) for detection of GRP78, phospho-eIF2α, total eIF2α, CHOP, ATF6 (p50), ATF6 (p90), calreticulin, pro-caspase-7 intermediate, cleaved caspase-7 and β-actin. B: (Upper panel) RT-PCR results for detection of <i>xbp-1</i> spliced [<i>xbp-1(s)</i>], <i>xbp-1</i> unspliced [<i>xbp-1(u)</i>] and <i>β-actin</i> mRNA levels from BM cells of <i>78^f/f</i> and <i>c78^f/f</i> mice (n = 3 for each genotype). The PCR image was inverted for better clarity. (Lower panel) Quantitation of the ratio of <i>xbp-1(s)</i> to <i>xbp-1(u)</i>. The average ratio of <i>xbp-1(s)</i>/<i>xbp-1(u)</i> in <i>78^f/f</i> was set as 1. The data is presented as mean ± s.e. (*P<0.05, Student’s <i>t</i> test).</p

GRP78 deficiency leads to lymphopenia.

<p>A: GRP78 expression in WT BM subpopulations. (Left) <i>Grp78</i> mRNA expression in WT BM subpopulations measured by quantitative real-time PCR. The experiments were performed in duplicates; each replicate contains pooled BM from two WT mice. (Right) GRP78 expression in LSKCD34⁻ and LSKCD34⁺ subpopulations in WT mice (n = 4) measured by flow cytometry. The bar graph represents the medium intensities of GRP78 staining with LSK cells set as 1. B: (Upper) Representative PCR genotyping results from <i>78^f/f</i> and <i>c78^f/f</i> BM 6 days post completion of pI.pC treatment. (Lower) Western blot results for detection of GRP78 protein level in the BM performed in duplicates. C: Organ size and morphology from mice of the indicated genotypes. Arrows on top of the heart indicate thymus. D: Quantitation of the thymus cellularity (n = 4 for <i>78^f/f</i>, n = 4 for <i>c78^f/f</i>) and spleen weight (n = 14 for <i>78^f/f</i>, n = 20 for <i>c78^f/f</i>). E: H&E staining of paraffin sections of thymus and spleen of <i>78^f/f</i> and <i>c78^f/f</i> mice. Arrows indicate megakaryocytes in the spleen. The scale bar represents 200 µm in thymus and 20 µm in spleen. F: Peripheral lymphocyte count using complete blood count analysis with tail peripheral blood from (Left) <i>78^f/f</i> (n = 12), <i>c78^f/f</i> (n = 16) mice and (Right) <i>78^f/f</i>(t) (n = 3), <i>c78^f/f</i>(t) (n = 3) chimeric mice. All data are presented as mean ± s.e (**P<0.01, ***P<0.001, Student’s <i>t</i> test).</p

A Unified Strategy for the Synthesis of 7‑Membered-Ring-Containing <i>Lycopodium</i> Alkaloids

A unique subset of the <i>Lyco­podium</i> alkaloid natural products share a 7-membered-ring substructure and may potentially arise from a common biosynthetic precursor. To both explore and exploit these structural relationships, we sought to develop a unified biosynthetically inspired strategy to efficiently access these complex polycyclic alkaloids through the use of a cascade sequence. In pursuit of these goals, the first total synthesis of (+)-fastigiatine (<b>2</b>) was accomplished via a series of cascade reactions; we describe herein a full account of our efforts. Insight from these endeavors led to critical modifications of our synthetic strategy, which enabled the first total syntheses of (−)-himeradine A (<b>1</b>), (−)-lyco­pecurine (<b>3</b>), and (−)-dehydro­lyco­pecurine (<b>4</b>), as well as the syntheses of (+)-lyco­nadin A (<b>5</b>) and (−)-lyco­nadin B (<b>6</b>). Our approach features a diastereo­selective one-pot sequence for constructing the common 7-membered-ring core system, followed by either a biomimetic transannular Mannich reaction to access himeradine A (<b>1</b>), lyco­pecurine (<b>3</b>), and dehydro­lyco­pecurine (<b>4</b>) or an imine reduction for lyco­nadins A (<b>5</b>) and B (<b>6</b>). This strategy may potentially enable access to all 7-membered-ring-containing <i>Lyco­podium</i> alkaloids and provides additional insight into their biosynthetic origin

Active promotion of GRP78 to the cell surface in cancer cells resistant to therapy.

<p>(A) Validation of Tam resistant phenotype of the MCF7L-TamR cells. MCF7L-parental (P) and -TamR cells were pre-starved in phenol-red free (PRF) medium containing 5% CS-FBS for 5 d before subjected to estrogen (E2) (1 nM) or Tam (100 nM) treatment for 8 d. Cells were seeded in quadruplicates in 96-well plate and cell numbers were counted by an in situ cell cytometer. At day 8, cells were stained by methylene blue as shown in the lower panel. Error bars represent standard deviation; *P<0.001, two-sided t test, cell growth under Tam vs. E2. (B) Immunoblots of MEF cells using MAb159, with β-actin as loading control. Left lane showed MEF lysates from <i>Grp78 floxed/floxed</i> mice. Right lane showed abolishment of the GRP78 band after infection with adenovirus expressing the Cre-recombinase to knockout the <i>Grp78 floxed/floxed</i> alleles. (C) Representative Western blots for enhanced sGRP78 level in resistant cancer cells. Parental (P) and TamR derivatives of the human breast cancer cell models of MCF7L and MCF7/HER2-18, as well as the parental androgen sensitive LNCaP cell line and the androgen-independent C4-2B cells were subjected to biotinylation and NeutrAvidin agarose pull-down to enrich for cell surface protein. Cell surface GRP78 (sGRP78) and total intracellular GRP78 (tGRP78) in the cell lysate were probed by Western blot. The amount of total lysate was 10% of the amount used for the avidin pull-down. β-actin served as loading control for tGRP78, while membrane protein, EphB4, or Na, K-ATPase α1 (NKA α1) served as loading control of cell surface proteins in breast or prostate cancer cells (PCa), respectively. The experiments were repeated twice. The protein bands were quantitated and the relative levels of tGRP78 in the parental and resistant cell lines were normalized against β-actin, and sGRP78 level are normalized against EphB4 or NKA α1, respectively, which are shown by mean ± standard deviation (S.D.) in the graph below. The levels in parental cell lines and in androgen sensitive cell line, LNCaP are set as 1.</p