The Prooxidant Action of Dietary Antioxidants Leading to Cellular DNA Breakage and Anticancer Effects: Implications for Chemotherapeutic Action Against Cancer

Plant-derived dietary antioxidants have attracted considerable interest in recent past for their ability to induce apoptosis and regression of tumors in animal models. While it is believed that the antioxidant properties of these agents may contribute to lowering the risk of cancer induction by impeding oxidative injury to DNA, it could not account for apoptosis induction and chemotherapeutic observations. In this article, we show that dietary antioxidants can alternatively switch to a prooxidant action in the presence of transition metals such as copper. Such a prooxidant action leads to strand breaks in cellular DNA and growth inhibition in cancer cells. Further, the cellular DNA breakage and anticancer effects were found to be significantly enhanced in the presence of copper ions. Moreover, inhibition of antioxidant-induced DNA strand breaks and oxidative stress by Cu(I)-specific chelators bathocuproine and neocuproine demonstrated the role of endogenous copper in the induction of the prooxidant mechanism. Since it is well established that tissue, cellular, and serum copper levels are considerably elevated in various malignancies, such a prooxidant cytotoxic mechanism better explains the anticancer activity of dietary antioxidants against cancer cells.     

Survivability of parthenogenetic embryos following in vivo transfer in naturally synchronized Capra hircus

The present study was conducted to see the in vivo developmental potency of caprine parthenogenetic embryos generated in a modified way. The good quality caprine oocytes were matured in presence of cytochalasin B (CCB) and then activated by 7% ethanol followed by 2 mM 6-dimethyl amino purine (6-DMAP) and embryo development was recorded. Early stage parthenogenetic embryos (two to four cells) were surgically transferred in recipients (10). The pregnancy diagnosis was done by nonreturn to oestrus, ultrasonography (USG), and progesterone estimation. The levels of progesterone were above normal values (1 ng/ml) of pregnancy and fall below the level of pregnancy just before retuned to oestrus. Progesterone profile revealed that out of ten recipients (G1–G10), four goats (G1, G2, G3, and G5) returned to oestrus after 43?±?7.29 (Mean?±?SE) d of embryo transfer and six goats (G4, G6, G7, G8, G9, and G10) did not return to cycle even after 70 d of embryo transfer. In three recipients (G4, G5, and G6), the USG on day 40 revealed that there was fluid filled uterine body with solid fetus-like structure. These might be dead fetus and had started resorption. The progesterone profile also corroborated the assumption of pregnancy in these animals. Authors believe that this may be the first report on in vivo diploid parthenogenetic embryo development in caprine species.     

Isolation of Messenger Ribonucleic Acid for Myosin Heavy Chain

A chicken embryonic polysome fraction that contains 50–60 monoribosomes and synthesizes the heavy chains of myosin is separated from other polysomes of smaller sizes by centrifugation through two cycles of discontinuous and continuous sucrose gradients. The unique properties of the polyadenylic acid segment present at the 3′-end of eukaryotic messenger RNA (mRNA) were used to purify the mRNA for myosin heavy chain from the phenol-extracted total RNA obtained from this polysome fraction. The total RNA was filtered thro ugh millipore filters resulting in partition of the riboscmal RNA (rRNA) and mRNA species. This millipore-bound RNA fraction, which consists of the mRNA and some ribosomal RNAs, was eluted from the filters with sodium dodecyl sulfate (SDS). Subsequent chromatography of this fraction on a cellulose column gave two well-separated peaks: an unadsorbed peak of ribosomal RNAs which was eluted with buffers of high ionic strength and an adsorbed peak of mRNA which was eluted only with a buffer of low ionic strength. Polyacrylamide gel electrophoresis of the mRNA peak fraction showed a single band with no detectable amounts of other RNAs, the mRNA migrating slower than 28S rRNA. The product of in vitro translation of the purified mRNA using a homologous cell-free system was identified as the myosin heavy chain by the following criteria: coprecipitation with carrier myosin at low ionic strength; elution properties on DEAE-cellulose column; and comigration with the heavy chain in polyacrylamide gel electrophoresis. In order to demonstrate the fidelity of translation of the mRNA, ¹⁴C-labeled products of the in vitro translation were copurified with unlabeled myosin heavy chains added as a carrier. The mixture of polypeptides was then cleaved with CNBr and the resulting peptides were separated by molecular sieving. The correlation between the radioactivity and the UV absorbance in the separated peptides indicates that total synthesis of the myosin heavy chain was achieved.     

An Antioxidant Extract of Tropical Lichen,Parmotrema reticulatum,Induces Cell Cycle Arrest and Apoptosis in Breast Carcinoma Cell Line MCF-7

This report highlights the phytochemical analysis, antioxidant potential and anticancer activity against breast carcinoma of 70% methanolic extract of lichen, Parmotrema reticulatum (PRME). Phytochemical analysis of PRME confirms the presence of various phytoconstituents like alkaloids, carbohydrates, flavonoids, glycosides, phenols, saponins, tannins, anthraquinones, and ascorbic acid; among which alkaloids, phenols and flavonoids are found in abundant amount. High performance liquid chromatography (HPLC) analysis of PRME revealed the presence of catechin, purpurin, tannic acid and reserpine. Antioxidant activity was evaluated by nine separate methods. PRME showed excellent hydroxyl and hypochlorous radical scavenging as well as moderate DPPH, superoxide, singlet oxygen, nitric oxide and peroxynitrite scavenging activity. Cytotoxicity of PRME was tested against breast carcinoma (MCF-7), lung carcinoma (A549) and normal lung fibroblast (WI-38) using WST-1 method. PRME was found cytotoxic against MCF-7 cells with an IC₅₀ value 130.03±3.11 µg/ml while negligible cytotoxicity was observed on A549 and WI-38 cells. Further flow cytometric study showed that PRME halted the MCF-7 cells in S and G2/M phases and induces apoptosis in dose as well as time dependent manner. Cell cycle arrest was associated with downregulation of cyclin B1, Cdk-2 and Cdc25C as well as slight decrease in the expression of Cdk-1 and cyclin A1 with subsequent upregulation of p53 and p21. Moreover PRME induced Bax and inhibited Bcl-2 expression, which results in increasing Bax/Bcl-2 ratio and activation of caspase cascade. This ultimately leads to PARP degradation and induces apoptosis in MCF-7 cells. It can be hypothesised from the current study that the antioxidant and anticancer potential of the PRME may reside in the phytoconstitutents present in it and therefore, PRME may be used as a possible source of natural antioxidant that may be developed to an anticancer agent.     

Stabilizing Exposure of Conserved Epitopes by Structure Guided Insertion of Disulfide Bond in HIV-1 Envelope Glycoprotein

Entry of HIV-1 into target cells requires binding of the viral envelope glycoprotein (Env) to cellular receptors and subsequent conformational changes that culminates in fusion of viral and target cell membranes. Recent structural information has revealed that these conformational transitions are regulated by three conserved but potentially flexible layers stacked between the receptor-binding domain (gp120) and the fusion arm (gp41) of Env. We hypothesized that artificial insertion of a covalent bond will ‘snap’ Env into a conformation that is less mobile and stably expose conserved sites. Therefore, we analyzed the interface between these gp120 layers (layers 1, 2 and 3) and identified residues that may form disulfide bonds when substituted with cysteines. We subsequently probed the structures of the resultant mutant gp120 proteins by assaying their binding to a variety of ligands using Surface Plasmon Resonance (SPR) assay. We found that a single disulfide bond strategically inserted between the highly conserved layers 1 and 2 (C65-C115) is able to ‘lock’ gp120 in a CD4 receptor bound conformation (in the absence of CD4), as indicated by the lower dissociation constant (Kd) for the CD4-induced (CD4i) epitope binding 17b antibody. When disulfide-stabilized monomeric (gp120) and trimeric (gp140) Envs were used to immunize rabbits, they were found to elicit a higher proportion of antibodies directed against both CD4i and CD4 binding site epitopes than the wild-type proteins. These results demonstrate that structure-guided stabilization of inter-layer interactions within HIV-1 Env can be used to expose conserved epitopes and potentially overcome the sequence diversity of these molecules.     

INPPO Actions and Recognition as a Driving Force for Progress in Plant Proteomics: Change of Guard,INPPO Update,and Upcoming Activities

The International Plant Proteomics Organization (INPPO) is a non‐profit organization whose members are scientists involved or interested in plant proteomics. Since the publication of the first INPPO highlights in 2012, continued progress on many of the organization's mandates/goals has been achieved. Two major events are emphasized in this second INPPO highlights. First, the change of guard at the top, passing of the baton from Dominique Job, INPPO founding President to Ganesh Kumar Agrawal as the incoming President. Ganesh K. Agrawal, along with Dominique Job and Randeep Rakwal initiated the INPPO. Second, the most recent INPPO achievements and future targets, mainly the organization of first the INPPO World Congress in 2014, tentatively planned for Hamburg (Germany), are mentioned.     

TGFβ mediated LINC00273 upregulation sponges mir200a-3p and promotes invasion and metastasis by activating ZEB1

Transforming growth factor β (TGFβ) is a prominent cytokine that promotes tumor progression by activating epithelial-to-mesenchymal transition (EMT). This study indicated that TGFβ exerted metastasis by inducing zinc finger E-box binding homeobox 1 (ZEB1) and a long noncoding RNA, LINC00273, expressions in A549 cells. Knocking down LINC00273 diminished TGFβ induced ZEB1 expression as well as metastasis. Mechanistically, LINC00273 acted as a molecular sponge of microRNA (miR)-200a-3p which liberate ZEB1 to perform its prometastatic functions. LINC00273 knockdown and miR200a3p mimic transfection of A549 cells were used for validating the link between TGFβ and LINC00273 induced metastasis. RNA pulldown and luciferase assay were performed to establish mir200a-3p-LINC00273 interaction. High expressions of LINC00273, TGFβ, and ZEB1 with concurrent low miR200a-3p expression had been verified in vivo and in patient samples. Overall, LINC00273 promoted TGFβ-induced lung cancer EMT through miR-200a-3p/ZEB1 feedback loop and may serve as a potential target for therapeutic intervention in lung cancer metastasis.