Wollamides: Antimycobacterial Cyclic Hexapeptides from an Australian Soil <i>Streptomyces</i>

A soil <i>Streptomyces</i> nov. sp. (MST-115088) isolated from semiarid terrain near Wollogorang Station, Queensland, returned two known and two new examples of a rare class of cyclic hexapeptide, desotamides A and B (<b>1</b> and <b>2</b>) and E and F (<b>3</b> and <b>4</b>), respectively, together with two new d-Orn homologues, wollamides A and B (<b>5</b> and <b>6</b>). Structures were assigned by detailed spectroscopic and C₃ Marfey’s analysis. The desotamides/wollamides exhibit growth inhibitory activity against Gram-positive bacteria (IC₅₀ 0.6–7 μM) and are noncytotoxic to mammalian cells (IC₅₀ >30 μM). The wollamides exhibit antimycobacterial activity (IC₅₀ 2.8 and 3.1 μM), including reduction in the intracellular mycobacterial survival in murine bone marrow-derived macrophages

Aranciamycins I and J, Antimycobacterial Anthracyclines from an Australian Marine-Derived <i>Streptomyces</i> sp.

Chemical analysis of an Australian marine-derived <i>Streptomyces</i> sp. (CMB-M0150) yielded two new anthracycline antibiotics, aranciamycins I (<b>1</b>) and J (<b>2</b>), as well as the previously reported aranciamycin A (<b>3</b>) and aranciamycin (<b>4</b>). The aranciamycins <b>1</b>–<b>4</b>, identified by detailed spectroscopic analysis, were noncytotoxic when tested against selected Gram-negative bacteria and fungi (IC₅₀ >30 μM) and exhibited moderate and selective cytotoxicity against Gram-positive bacteria (IC₅₀ >1.1 μM) and a panel of human cancer cell lines (IC₅₀ > 7.5 μM). Significantly, <b>1</b>–<b>4</b> were cytotoxic (IC₅₀ 0.7–1.7 μM) against the <i>Mycobacterium tuberculosis</i> surrogate <i>M. bovis</i> bacille Calmette-Guérin

Library clone insert size and GFP fluorescence in virus-making cells.

<p>A: scatter-plot of cloned insert size and total well GFP fluorescence in transfected HEK293T cells. B: frequency distribution histogram of clones in different insert size category.</p

Knockdown of ApoL1 in Zebrafish Larvae Affects the Glomerular Filtration Barrier and the Expression of Nephrin

<div><p>APOL1, a secreted high-density lipoprotein, is expressed in different human tissues. Genetic variants of <i>APOL1</i> are described to be associated with the development of end stage renal diseases in African Americans. In human kidney, APOL1 is mainly expressed in podocytes that are responsible for proper blood filtration. Since mice do not express ApoL1, the zebrafish is an ideal model to study the role of ApoL1. Injection of morpholinos against zApoL1 into zebrafish eggs and larvae, respectively, induces severe edema indicating a leakage of the filtration barrier. This was demonstrated in zApoL1 knockdown larvae by intravascular injection of fluorescently-labeled 10- and 500-kDa dextrans and by clearance of the vitamin D-binding protein from the circulation. Immunohistochemistry and RT-PCR revealed the reduction of nephrin, a podocyte-specific protein essential for blood filtration. Coinjection of human nephrin mRNA rescued the zApoL1 knockdown induced phenotype. Reduced APOL1 and nephrin levels were also found in biopsies of patients suffering from end stage renal diseases. Our results demonstrate that zApoL1 is essential for proper blood filtration in the zebrafish glomerulus and that zApoL1 affects the expression of nephrin.</p></div

Genes producing very high or very low viral titer.

<p>Genes producing very high or very low viral titer.</p

Comparison of target cell transduction rate and GFP fluorescence of corresponding virus-producing wells.

<p>A,C – target cell transduction rates; B,D – GFP fluorescence of virus producing wells. Bars in A and B represent mean of 4 wells derived from independent single colony isolates of the gene-expressing plasmids (x-axis). Genes underlined in A, produced high viral titer in bulk well experiments (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051733#pone-0051733-g001" target="_blank">Figures 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051733#pone-0051733-g002" target="_blank">2</a>.), the rest were low titer producers. Error bars = SD. Data for graphs A and C were obtained by high-content imaging of transduced cells, while data for graphs B and D were obtained by scanning the transfected cells using a fluorescence plate reader. C and D: Mean and SD for high and low titer well data represented in A and B. In all cell lines mean for combined low titer genes was significantly different from the mean for high titer wells (Aspin-Welch test, P value: HEK293T = 4.70 E−11; HACAT = 5.68 E−23; MDA-MB468 = 2.32 E−36; WMM1175 = 7.29 E−12; MCF10A  = 2.98 E−21).</p

zApoL1 KD induces pericardial edema formation.

<p>Zebrafish larvae (4 dpf) developed edema (arrow in A) after the KD of zApoL1 in contrast to CtrlMO and untreated larvae (B, C). Paraffin tissue sections of the larvae showed enlarged Bowman’s space (arrows in D) due to the knockdown of zApoL1 compared to CtrlMO and untreated larvae (E, F). g—glomerulus, m—myotome, i—intestine, y—yolk sac. Panel G shows that more than 70% of the larvae developed edema in response to KD of zApoL1. Data are means±SD of 4 experiments on a total of 100 larvae. Scale bars represent 500 μm (A-C) and 10 μm (D-F). We have used t-test for statistical analysis.</p

Mollemycin A: An Antimalarial and Antibacterial Glyco-hexadepsipeptide-polyketide from an Australian Marine-Derived <i>Streptomyces</i> sp. (CMB-M0244)

A marine-derived <i>Streptomyces</i> sp. (CMB-M0244) isolated from a sediment collected off South Molle Island, Queensland, produced mollemycin A (<b>1</b>) as a new first in class glyco-hexadepsipeptide-polyketide. The structure of <b>1</b> was assigned by detailed spectroscopic analysis, supported by chemical derivatization and degradation, and C₃ Marfey’s analysis. Mollemycin A (<b>1</b>) exhibits exceptionally potent and selective growth inhibitory activity against Gram-positive and Gram-negative bacteria (IC₅₀ 10–50 nM) and drug-sensitive (3D7; IC₅₀ 7 nM) and multidrug-resistant (Dd2; IC₅₀ 9 nM) clones of the malaria parasite <i>Plasmodium falciparum</i>

APOL1 and nephrin expression in kidney biopsies.

<p>Tissue sections of biopsies of patients suffering from membranous glomerulonephritis (MGN) and focal segmental glomerulosclerosis (FSGS) stained with antibodies against APOL1 and nephrin showed a marked decrease of APOL1 (A, I) as well as nephrin (B, J) in podocytes. Scale bars represent 20 μm.</p

Effect of low virus titer-producing genes on viability of the transfected cells.

<p>HEK293T cells were transfected with gene (x-axis) expressing plasmids either alone or with viral packaging vector day after seeding. 21 h after transfection, medium was replaced with PBS containing Hoechst33342 and propidium iodide (PI), which stain DNA and dying cells respectively. Cells were scanned in blue (Hoechst, A) red (PI, B) and green (GFP, C) channels and number of objects determined in each channel. A and B represent independent object counts, data in C are expressed as proportion of GFP positive cells in the Hoechst stained population. A - Significantly reduced number of surviving cells in the well compared to untreated wells (CELLS): lipofectamine alone (MOCK) (P = 0.034); MOCK with packaging plasmids (P = 7.5E−10); all others (P≤1.1E−05) B - Significantly increased PI positive cells compared to vector: expression plasmid only BCL2L1 (P = 6.88E−05), C3ORF1(P = 0.03), MTCH1(P = 4.66E−05), PNMAL2 (P = 0.01) P values determined using Aspin-Welch test; Bars = mean of 4 wells, error bars = SD, CELLS-untreated well, MOCK-wells where expression plasmid has been omitted. AWAT2, and vector were high virus titer producing in previous experiments.</p