Bistinospinosides A and B, Dimeric Clerodane Diterpene Glycosides from <i>Tinospora sagittata</i>

Two dimeric clerodane diterpene glycosides, namely, bistinospinosides A (<b>1</b>) and B (<b>2</b>), were isolated from the roots of <i>Tinospora sagittata</i>. Their structures were elucidated by extensive spectroscopic data interpretation. The compounds feature an unusual 1,4-epoxycyclohexane ring in their structures and may be biosynthetically constructed via an intermolecular Diels–Alder [4+2] cycloaddition from the corresponding clerodane diterpene. The compounds were evaluated in a nitric oxide inhibitory assay using J774.1 macrophage-like cells

Nucleotide distances between the Guilin HBV recombinant and other reference genotype strains^*.

<p>The reference genotype strains used in the table are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084005#pone-0084005-g001" target="_blank">Figure 1C</a>.</p

Minor Type IV Collagen α5 Chain Promotes Cancer Progression through Discoidin Domain Receptor-1

<div><p>Type IV collagens (Col IV), components of basement membrane, are essential in the maintenance of tissue integrity and proper function. Alteration of Col IV is related to developmental defects and diseases, including cancer. Col IV α chains form α1α1α2, α3α4α5 and α5α5α6 protomers that further form collagen networks. Despite knowledge on the functions of major Col IV (α1α1α2), little is known whether minor Col IV (α3α4α5 and α5α5α6) plays a role in cancer. It also remains to be elucidated whether major and minor Col IV are functionally redundant. We show that minor Col IV α5 chain is indispensable in cancer development by using α5(IV)-deficient mouse model. Ablation of α5(IV) significantly impeded the development of Kras^G12D-driven lung cancer without affecting major Col IV expression. Epithelial α5(IV) supports cancer cell proliferation, while endothelial α5(IV) is essential for efficient tumor angiogenesis. α5(IV), but not α1(IV), ablation impaired expression of non-integrin collagen receptor discoidin domain receptor-1 (DDR1) and downstream ERK activation in lung cancer cells and endothelial cells. Knockdown of DDR1 in lung cancer cells and endothelial cells phenocopied the cells deficient of α5(IV). Constitutively active DDR1 or MEK1 rescued the defects of α5(IV)-ablated cells. Thus, minor Col IV α5(IV) chain supports lung cancer progression via DDR1-mediated cancer cell autonomous and non-autonomous mechanisms. Minor Col IV can not be functionally compensated by abundant major Col IV.</p></div

Figure 1. Phylogenetic analysis of the 14 clones derived from the Guilin recombinant compared with reference strains.

<p>GenBank accession numbers and clone numbers are showed on each tree, and the genotype is indicated after every accession number. Bootstrap values are showed at each nodes, and only bootstrap values of >70% are indicated. Phylogenetic trees comparing the 14 clones with 34 reference strains representing genotypes A–H, were constructed based on the S gene (A), pre-S region (B) and whole genomes (C). And the phylogenetic tree was constructed by comparing the Guilin recombinant with the Vietnam, Thailand and Long An recombinants as well as HBV C subgenotype based on the whole genomes (D).</p

The breakpoint positions and the arrangements of the genotype fragments of the Guilin, Vietnam, Long An and Thailand recombinants.

<p>The breakpoint positions and the arrangements of the genotype fragments of the Guilin, Vietnam, Long An and Thailand recombinants.</p

Bootscan analysis demonstrating the complex recombination among genotypes A, C and G in the Guilin recombinant.

<p>The isolate from the female patient (A) and the isolate from the male patient (B) were subjected to bootscan analysis over the complete genome using the SIMPLOT program with a 500 bp window size, 10 bp step size and 100 bootstrap replicates, using gap-stripped alignments and neighbor-joining analysis, and were compared with three representative HBV genotypes: A (GenBank accession no. AB126580), C (GenBank accession no. AB050018) and G (GenBank accession no. AB064310). Woolly monkey was a known out-group (GenBank accession no. AF046996). Analysis was stared from nt 2700.</p

α5(IV) is expressed in endothelial cells and required for endothelial cell proliferation and tubulogenesis.

<p>(A) Immunofluorescent staining on lung sections shows partial colocalization of α5(IV) and CD31. Scale bar: 25μm. (B) RT-PCR analysis of α5(IV) knockdown efficiency in HMEC-1 cells. (C and D) Knockdown of α5(IV) impaired proliferation (C) and migration (D) of HMEC-1 cells, determined by BrdU incorporation and modified Boyden chamber assays, respectively. Data are presented as mean ± SD. ***P < 0.001. (E) In vitro tubulogenesis of HMEC-1 cells expressing control (sh<i>Scram</i>) or <i>COL4A5</i> (sh<i>COL4A5</i>) shRNAs. Scale bar: 200 μm.</p

DDR1 is required for ERK activation, cell proliferation and migration in lung cancer cells.

<p>(A) Western blot analyses of phosphorylation levels of ERK, Akt, FAK and Src in A549 cells with DDR1 knockdown. (B-D) Knockdown of DDR1 in A549 cells significantly impaired cell proliferation (B), migration (C), and anchorage-independent cell growth (D). Data are presented as mean ± SD. **P < 0.01, ***P < 0.001.</p

DDR1 is downregulated in α5(IV)-knockdown cells.

<p>(A) Western blot analyses of DDR1 expression in α5(IV)- or α1(IV)-knockdown A549 cells. (B and C) DDR1 staining on lung sections from <i>Col4a5</i>^+/Y and <i>Col4a5</i>^{<i>LacZ</i>/Y} mice (B) or lung tumor sections from Kras/α5 WT and Kras/α5 KO mice (C). Scale bars: 200 μm. (D) Quantitative RT-PCR analysis of DDR1 expression in A549 cells expressing control (sh<i>Scram</i>) or <i>COL4A5</i> (sh<i>COL4A5</i>) shRNAs. (E and F) A549 cells expressing control (sh<i>Scram</i>) or <i>COL4A5</i> (sh<i>COL4A5</i>) shRNAs were treated with or without 100 μg/mL cycloheximide (CHX) for 0.5, 1, 2 or 4 hours. (E) DDR1 protein levels were analyzed by western blot. (F) Relative protein levels of DDR1 after cycloheximide treatment in (E). (G) Western blot analysis of DDR1 expression in A549 cells expressing control (sh<i>Scram</i>) or <i>COL4A5</i> (sh<i>COL4A5</i>) shRNAs treated with or without 10 μM proteasome inhibitor MG132 for 4 hours or 50 mM lysosome inhibitor NH₄Cl for 6 hours. (H) A549 cells expressing control (sh<i>Scram</i>) or <i>COL4A5</i> (sh<i>COL4A5</i>) shRNAs were treated with 10 μM proteasome inhibitor MG132 for 4 hours. DDR1 was immunoprecipitated and ubiquitination levels were detected.</p

α5(IV), but not α1(IV), deficiency results in impaired activation of ERK.

<p>(A) Western blot analyses of phosphorylation levels of ERK, Akt, FAK and Src and α1(IV), α2(IV) and α5(IV) expression in α5(IV)- or α1(IV)-knockdown A549 cells. (B) Western blot analyses of phosphorylation levels of ERK in α5(IV)-knockdown A549 cells expressing constitutively active MEK1. (C-E) Expression of constitutively active MEK1 in α5(IV)-knockdown A549 cells rescued the defects in cell proliferation (C), migration (D), and anchorage-independent cell growth (E). Data are presented as mean ± SD. **P < 0.01.</p