A proteomic view on the developmental transfer of homologous 30 kDa lipoproteins from peripheral fat body to perivisceral fat body via hemolymph in silkworm, Bombyx mori

<p>Abstract</p> <p>Background</p> <p>A group of abundant proteins of ~30 kDa is synthesized in silkworm larval peripheral fat body (PPFB) tissues and transported into the open circulatory system (hemolymph) in a time-depended fashion to be eventually stored as granules in the pupal perivisceral fat body (PVFB) tissues for adult development during the non-feeding stage. These proteins have been shown to act anti-apoptotic besides being assigned roles in embryogenesis and defense. However, detailed protein structural information for individual PPFB and PVFB tissues during larval and pupal developmental stages is still missing. Gel electrophoresis and chromatography were used to separate the 30 kDa proteins from both PPFB and PVFB as well as hemolymph total proteomes. Mass spectrometry (MS) was employed to elucidate individual protein sequences. Furthermore, 30 kDa proteins were purified and biochemically characterized.</p> <p>Results</p> <p>One- and two-dimensional gel electrophoresis (1/2D-PAGE) was used to visualize the relative changes of abundance of the 30 kDa proteins in PPFB and PVFB as well as hemolymph from day 1 of V instar larval stage to day 6 of pupal stage. Their concentrations were markedly increased in hemolymph and PVFB up to the first two days of pupal development and these proteins were consumed during development of the adult insect. Typically, three protein bands were observed (~29, 30, 31 kDa) in 1D-PAGE, which were subjected to MS-based protein identification along with spots excised from 2D-gels run for those proteomes. Gas phase fragmentation was used to generate peptide sequence information, which was matched to the available nucleotide data pool of more than ten highly homologous insect 30 kDa lipoproteins. Phylogenetic and similarity analyses of those sequences were performed to assist in the assignment of experimentally identified peptides to known sequences. Lipoproteins LP1 to LP5 and L301/302 could be matched to peptides extracted from all bands suggesting the presence of full length and truncated or modified protein forms in all of them. The individual variants could not be easily separated by classical means of purification such as 2D-PAGE because of their high similarity. They even seemed to aggregate as was indicated by native gel electrophoresis. Multistep chromatographic procedures eventually allowed purification of an LP3-like protein. The protein responded to lipoprotein-specific staining.</p> <p>Conclusions</p> <p>In <it>B. mori </it>larvae and pupae, 30 kDa lipoproteins LP1 to LP5 and L301/302 were detected in PPFB and PVFB tissue as well as in hemolymph. The concentration of these proteins changed progressively during development from their synthesis in PPFB, transport in hemolymph to storage in PVFB. While the 30 kDa proteins could be reproducibly separated in three bands electrophoretically, the exact nature of the individual protein forms present in those bands remained partially ambiguous. The amino acid sequences of all known 30 kDa proteins showed very high homology. High-resolution separation techniques will be necessary before MS and other structural analysis can shed more light on the complexity of the 30 kDa subproteome in <it>B. mori</it>. A first attempt to that end allowed isolation of a <it>B. mori </it>LP3-like protein, the complete structure, properties and function of which will now be elucidated in detail.</p

Ongoing geographical spread of Tomato yellow leaf curl virus

Tomato yellow leaf curl virus (TYLCV) seriously impacts tomato production throughout tropical and sub-tropical regions of the world. It has a broad geographical distribution and continues to spread to new regions in the Indian and Pacific Oceans including Australia, New Caledonia and Mauritius. We undertook a temporally-scaled, phylogeographic analysis of all publicly available, full genome sequences of TYLCV, together with 70 new genome sequences from Australia, Iran and Mauritius. This revealed that whereas epidemics in Australia and China likely originated through multiple independent viral introductions from the East-Asian region around Japan and Korea, the New Caledonian epidemic was seeded by a variant from the Western Mediterranean region and the Mauritian epidemic by a variant from the neighbouring island of Reunion. Finally, we show that inter-continental scale movements of TYLCV to East Asia have, at least temporarily, ceased, whereas long-distance movements to the Americas and Australia are probably still ongoing

Synthesis of Novel Temozolomide-Fatty Acid Imide Hybrid Compounds for the Chemotherapeutic Treatment of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is an aggressive form of brain cancer that originates from glial cells, which make up the supportive tissue surrounding neurons. Temozolomide (TMZ) is the current chemotherapeutic drug administered to treat GBM as it works to inhibit the growth of the cancer cells. This research study focuses on developing a method for synthesizing novel hybrid compounds that combines TMZ with various fatty acids known to have anticancer properties, forming a series of imide compounds with potential chemotherapeutic effects. Once the novel hybrid compounds are successfully synthesized, they will be tested for their anticancer properties on glioblastoma cells

Vitellogenin from the Silkworm, <i>Bombyx mori</i>: An Effective Anti-Bacterial Agent

<div><p>Silkworm, <i>Bombyx mori</i>, vitellogenin (Vg) was isolated from perivisceral fat body of day 3 of pupa. Both Vg subunits were co-purified as verified by mass spectrometry and immunoblot. Purified Vg responded to specific tests for major posttranslational modifications on native gels indicating its nature as lipo-glyco-phosphoprotein. The Vg fraction had strong antibacterial activity against Gram negative bacterium <i>Escherichia coli</i> and Gram positive bacterium <i>Bacillus subtilis</i>. Microscopic images showed binding of Vg to bacterial cells and their destruction. When infected silkworm larvae were treated with purified Vg they survived the full life cycle in contrast to untreated animals. This result showed that Vg has the ability to inhibit the proliferation of bacteria in the silkworm fluid system without disturbing the regular metabolism of the host.</p></div

Scanning electron micrographs showing <i>E. coli</i> cells (top 4) and <i>B. subtilis</i> incubated with a) control, b) Tris citrate buffer, c) BSA, d) purified Vg.

<p>Arrows point to changes on the cell walls. The scale bars correspond to 1 µm unless stated otherwise.</p

Binding of FITC-labeled Vg to <i>E. coli</i> (top) and <i>B. subtilis</i>.

<p>After washing with 25mM Tris buffer, the microbes were applied to microscope slides and observed under an Olympus fluorescence microscope. Images from the BSA control were completely black. Scale bars 40µm. For brightfield images see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0073005#pone.0073005.s003" target="_blank">Figure S3</a>.</p

Therapeutic effect of Vg protein on silkworm (n = 75, Table S4) infected with <i>E. coli</i> and <i>B. subtilis</i> (in percent survival for two days).

<p>200 µg of purified Vg was injected into infected silkworms for treatment. The healthy control group received a slit in the middle appendage with a sterile needle but no other treatment. Images were taken after 2 days. 2D-gel images (pH 3–10, MW 10–200kDa) of the respective hemolymph proteome are depicted on top.</p

Sensitivity assay to examine the antibacterial activity of purified Vg protein against <i>E. coli</i> a) and <i>B. subtilis</i> b) bacteria.

<p>Ampicillin served as reference.</p