Novel propanamide analogue and antiproliferative diketopiperazines from mangrove <i>Streptomyces</i> sp. Q24

<p>A new propanamide analogue (<b>1</b>), along with one known alkaloid (<b>2</b>) and four known diketopiperazines (<b>3</b>–<b>6</b>), was isolated from a cultured broth of the actinomycete <i>Streptomyces</i> sp. Q24 that was obtained from a sample of mangrove soil. The structures of these isolates were characterised as 3-acetylamino-<i>N</i>-2-thienyl-propanamide (<b>1</b>), <i>N</i>-acetyltryptamine (<b>2</b>), cyclo-(l-phenylalanine-l-4-hydroxyproline) (<b>3</b>), cyclo-(l-leucine-l-4-hydroxyproline) (<b>4</b>), cyclo-(l-phenylalanine-d-4-hydroxyproline) (<b>5</b>) and cyclo-(l-leucine-l-proline) (<b>6</b>) based on their NMR and HRESIMS data as well as optical rotation. Three diketopiperazines (<b>3</b>, <b>4</b>, <b>6</b>) showed activity in inhibiting the proliferation of human glioma U87-MG and U251 cells. This type of the new propanamide analogue (<b>1</b>) is first found from a nature source and the antiproliferative property of these three diketopiperazines against glioma cells is also reported herein for the first time.</p

Plasma HDL fractions associated with β-COP.

<p>Lipoprotein fractions from normal human serum were isolated by FPLC, and equal volumes from each FPLC fraction were subjected to SDS PAGE and assayed for β-COP by Western blotting analysis. The β-COP positive fractions were shown in the inset. The Western blots were repeated three times using plasma samples from three healthy donors (biological replicates = 3).</p

Proteins identified in apoA-1 conditioned media by proteomics.

<p>Proteins identified in apoA-1 conditioned media by proteomics.</p

Cellular cholesterol localization with ADFP as an indicator in β-COP shRNA transduced macrophages observed by a confocal laser scanning microscopy.

<p>Typical images from each group are shown here. Additional cell images are available in the Support Information Files. (A) Non cholesterol-loading THP-1 macrophages, showing background fluorescence. Relative fluorescence units measured are shown in Fig 2H, BG (background) column. (B) THP-1 macrophages loaded with cholesterol, showing intense punctate fluorescence. A closer view of the cell indicated by the arrow is shown in Fig 2F. Higher fluorescence intensity is also shown in Fig 2H, NC column. (C) THP-1 macrophages loaded with cholesterol and subsequently incubated with apoA-1, with nearly invisible fluorescence and a low level of quantification in Fig 2H, PC column. (D) THP-1 macrophages transduced with β-COP shRNA, loaded with cholesterol and then treated with apoA-1. Vesicles with intense fluorescence are clearly visible. A closer view of the cell indicated by an arrow is shown in Fig 2G, displaying vesicles in size from 500 to 1000 nm in diameter. High fluorescence levels are also reflected in Fig 2H, the β-COP column. (E) THP-1 macrophages transduced by the scrambled shRNA, loaded with cholesterol and then treated with apoA-1, no visible vesicles and also low in fluorescence in Fig 2H, the scrambled column. (H) Relative fluorescence intensity in each condition. *** and ###, P<0.001, compared to BG, PC and scrambled groups. Error bars represent mean ± SEM from 80–100 cells (16 to 20 cells from each experiment, repeated five times).</p

Effect of β-COP shRNA on the designated protein expression and cholesterol efflux of THP-1 macrophages.

<p>(A) β-COP mRNA levels determined by qRT-PCR. Experimental conditions are indicated in the x-axis labeling: NC refers to negative control, PC means positive control, and the β-COP and scrambled labels indicate the corresponding shRNA treatment. Minus signs indicate no shRNA transduced or apoA-1 incubation, while plus signs designate β-COP shRNA, scrambled shRNA or apoA-1 treatment. The same labels were also used in the entire study where applicable. ***, P<0.001, compared to all. (B) β-COP protein levels determined by Western blotting. ***, P<0.001, compared to all. (C) ApoA-1 mediated cholesterol efflux. The cells were simultaneously loaded with 50 μg/ml of acetylated LDL and 0.2 μCi [³H] cholesterol. ApoA-1 mediated [³H] cholesterol efflux was determined as Method. ***, P<0.001, compared to the PC and scrambled shRNA transduced groups. (D) ADPF protein levels determined by Western blotting. ***, P<0.001, compared to the NC and β-COP shRNA transduced groups. ###, P<0.001, compared to the NC and β-COP shRNA transduced group. Data were the mean of five repeated experiments ± SEM (n = 5).</p

Translocation of β-COP to the cell membrane induced by apoA-1.

<p>(A) Increased β-COP on the cell membrane by apoA-1 is shown by Western blotting (left top panel). Membrane P4HB, due to its small molecular weight, served as a loading control in the same blotting (right upper panel). No detectable cytosolic GAPDH marker in the membrane (left lower panel), while it is abundant in the cytosolic fraction (right lower panel). (B) No β-COP translocation to the cell membrane on Tangier fibroblasts treated by apoA-1. Membrane marker P4HB is equal in each lane (top right panel) and no detection of cytosolic protein marker GAPDH in the membrane fraction (bottom left panel). Two Tangier fibroblasts cell lines from different patients were used (biological replicates = 2). (C) Increased β-COP translocation to the membrane by apoA-1 in the live cell surface-labeled assay (left panel). Total β-COP is equal in all lysate samples, indicating no alteration of β-COP expression and turnover by apoA-1 incubation. Total β-COP level also served as a control for equal amount protein used in immunoprecipitation (right panel). (D) β-COP appeared on the culture media. β-COP in the media was detected by immunoblot analysis. Minus signs indicated no treatment, while plus signs indicated cholesterol loading or apoA-1 incubation. The experiments were repeated four times.</p

Colocalization of β-COP and apoA-1 or apoE on the cell surface observed by immunogold electron microscopy.

<p>β-COP is indicated by large gold particles (18 nm, open arrows) while apoA-1 and apoE are represented by small gold particles (12 nm). (A) No detection of β-COP on fibroblast without addition of apoA-1 to the culture medium during incubation. (B) ApoA-1 and β-COP colocalization on protrusion complex on the fibroblast surface with incubation of 10 μg/ml apoA-1 in the medium. (C) ApoE and β-COP colocolization on the fibroblast surface following incubation of 10 μg/ml apoE. (D) ApoA-1 and β-COP colocalization on protrusion complex on the THP-1 macrophage surface with incubation of 10 μg/ml apoA-1. (E) ApoE and β-COP colocalization on protrusion complex on THP-1 macrophage surface following incubation of 10 μg/ml apoE. (F) No protrusion complex on cell surface on two different Tangier fibroblast cell lines after treatment with apoA-1 or apoE. The experiments were repeated four times.</p

Antiglioma pseurotin A from marine <i>Bacillus</i> sp. FS8D regulating tumour metabolic enzymes

<p>Pseurotin A was isolated from a culture of marine <i>Bacillus</i> sp. FS8D and showed to be active against the proliferation of four different glioma cells with IC₅₀ values of 0.51–29.3 μM. It has been found that pseurotin A downregulated the expression of tumour glycolytic enzymes pyruvate kinase M2 (PKM2) and lactate dehydrogenase 5 (LDH5) and upregulated the expression of pyruvate dehydrogenase beta (PDHB), adenosine triphosphate synthase beta (ATPB) and cytochrome C (Cyto-C), the important regulators for tricarboxylic acid cycle and oxidative phosphorylation. The data suggested that targeting multiple metabolic enzymes might be one of the antiglioma mechanisms of pseurotin A.</p

New streptophenazines from marine <i>Streptomyces</i> sp. 182SMLY

<p>Six phenazines including three new ones were isolated from the culture of a marine actinomycete <i>Streptomyces</i> sp. 182SMLY. Based on the analyses of NMR, HRESIMS, optical rotation value, and CD data, the structures of these isolated compounds were determined as new phenazines of (-)-streptophenazines M–O and known phenazines of 1-carbomethoxyphenazine and (-)-streptophenazines A and B. (-)-Streptophenazine B showed activity in suppressing the growth of methicillin-resistant <i>Staphylococcus aureus</i> with MIC value of 4.2 μg/mL.</p